Sensors and Actuators Reports 5 (2023) 100159

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Nanosensor platforms for detection of milk adulterants

Himshweta a, Minni Singh b, *
a
Biosensor Development Group, Department of Biotechnology and Food Technology, Punjabi University Patiala, Punjab, 147002, India
b
Functional Food and Nanotechnology Group, Department of Biotechnology and Food Technology, Punjabi University Patiala, Punjab, 147002, India

A R T I C L E I N F O A B S T R A C T

Keywords: Milk is a substrate for adulteration, and it is crucial to identify adulterants, and their quantities in milk products.
Nanomaterials In order to safeguard consumers from fake products and health risks, authenticity of milk products is in high
Electrochemical nanosensors demand. Analytical tools that are comprehensive, quick, and sensitive are necessary to monitor milk quality and
Optical nanosensors
screen for any undesirable substances. The traditional methods for the detection of adulterants in milk products
Milk products
Adulterations
are experiencing more difficulties due to their poor selectivity, sensitivity, and unsuitability for complex
Adulterants matrices. The researchers are paying close attention to nanomaterial based sensing systems, which are seen to be
among the most effective alternatives to the traditional approaches. This review focuses on optical and elec­
trochemical nanosensors including mechanisms and performances, in relation to their benefits, drawbacks, and
applicability to milk products. It also addresses new developments in the field of nanoemsembles platforms for
the rapid detection of adulterants in dairy products. A section of particular interest is the detection of milk
authenticity, as it is known that milk from different animal species are intermixed.

1. Introduction techniques are available to store milk for future use.

Food product adulteration is quickly increasing, posing a threat to
consumer safety and the shelf life of products. Nowadays, food adul­
teration has become widespread in numerous food items, including
milk, meat, cereals, packaged foods, honey, vegetable oils, fruit juices,
and alcoholic drinks [1]. Adulteration of food is a well-known issue in
the food processing industry. To address this, the food standards and
safety regulatory authority has established standard rules for all food
products under various acts and laws in the majority of the world’s
nations. These rules are meant to verify the genuineness of the product
and ensure its safety. Unfortunately, companies often adulterate food
with less expensive substitutes to maximize their profits. Milk and dairy
products have become a staple food in the global diet because they are
essential for creating and maintaining a balanced diet for individuals of
all ages [2]. Since milk is a good source of high-quality proteins, lipids,
vitamins, and minerals, it contains casein and whey proteins that are
crucial for human nutrition and the necessary supply of amino acids.
Milk is easily absorbed and digestible, making it particularly crucial for
newborns, nursing mothers, kids, and the elderly. Additionally, milk
proteins provide the amino acids required for both adults and children’s
normal growth [3]. Cheese and butter are examples of dairy products
that have historically been consumed in large quantities. Many
Due to the growing demand for fresh, genuine, and flavourful
products among consumers, the dairy business is rapidly expanding.
Milk and dairy products are more in demand and more expensive than
other food items in the market. Unfortunately, milk is easily adulterated
to reduce costs or extend the shelf life of dairy products. Milk adulter­
ation is defined as intentional reduction of milk quality through
blending or the addition of inferior substances prior to sale. Due to their
great nutritional value, widespread production and consumption, both
raw milk and lacteous derivatives are susceptible to adulteration
through the addition of foreign chemicals or cheap additives [4]. The
dairy industry is predicted to experience steady growth until 2025, with
annual increases of 2.1% for whole milk powder and 2.0% for skim milk
powder, in addition to 1.7% for butter and 1.4% for cheese, respectively
[5]. Adulteration of milk occurs primarily through the addition of less
expensive substitutes to increase its fat and protein content, as well as
volume and shelf life [6]. Since the presence of adulterants may cause
major health problems, it is imperative to properly evaluate the quality
of milk and dairy products both before and after packaging. Milk and
other dairy products are commonly adulterated with melamine, urea,
formaldehyde, starch, hydrogen peroxide (H2O2), vegetable oil, water,
and detergents [7–10] as illustrated in Fig. 1.
In 2011, Spink and Moyer [11] noted that the Food and Drug

* Corresponding author.
E-mail addresses: himshweta85@gmail.com (Himshweta), minnisingh@pbi.ac.in (M. Singh).

https://doi.org/10.1016/j.snr.2023.100159
Received 31 October 2022; Received in revised form 30 April 2023; Accepted 11 May 2023
Available online 12 May 2023
2666-0539/© 2023 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
Himshweta and M. Singh
Administration had classified economically motivated adulteration
(EMA) as a sub-category of food that poses threat to public health on a
global scale. EMA is defined as the fraudulent act of intentionally
substituting or adding materials to a product to enhance its perceived
value or reduce its manufacturing costs for economic gain [11]. There­
fore, it is important to have appropriate procedures for distinguishing
between adulterated and original milk to offer consumers with pure and
wholesome milk, both from the dairy sector’s perspective but also for
the benefit of consumers [12]. From an economic standpoint, milk
adulteration has become a prevalent practice, challenging every aspect
of the dairy industry worldwide. The 2008 Chinese milk scandal, which
highlighted the melamine adulteration in infant formula, brought the
issue to the forefront. It served as an eye opener for regulatory bodies
worldwide, prompting them to establish rigorous framework guidelines
to combat food adulteration. However, despite these efforts, milk fraud
continues to persist and remains a significant problem. This is due to the
lack of the effective and prompt detection technology for milk
adulteration.
Various analytical techniques have been developed to address the
detrimental effects of food adulterants in dairy products and foods.
Several chromatographic techniques, including High Performance
Liquid Chromatography (HPLC), Ultra High Performance Liquid Chro­
matography/Tandem mass spectrometry (UPLC/MS/MS) and gas chro­
matography/mass spectrometry (GC/MS), as well as spectroscopic
techniques such as Fourier transform near-IR (FT-NIR) spectroscopy,
mid infrared (MIR), ultraviolet–visible (UV– Vis), and Raman spectros­
copy, are among them [13–17]. While these chromatographic and mass
spectrometric methods are characterized by outstanding sensitivity and
precision, they are costly, complex to operate, require qualified opera­
tors, and are not suitable for field applications. The surge in the number
of publications on the identification of adulterants in dairy products
underscores the global importance of this issue. Therefore, it has become
essential to develop cost-effective and real-time detection probes.
Among the various types of probes available these probes,
nanomaterial-based sensors are highly valued for their potential appli­
cations in food and dairy products. They are preferred due to their facile
synthesis, large surface area, easy surface functionalization, higher
sensitivity and stability make them ideal candidate for detection.
Additionally, the nanomaterials exhibit unique optical, electrical, and
magnetic properties can be employed for the analytical applications
such as extraction, sample preparation and preconcentration of various
Fig. 1. Adulterants in milk.
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analytes present in food, biological and environmental samples [18,19].
Nanomaterials can be integrated with specific analytical methods to
monitor analytes effectively.
In recent years, sensor based approaches have emerged as a prom­
ising solution to overcome the limitations of instrumental analysis and
conventional methods. Although these techniques are useful for trace
analysis and offer the advantages of cost effectiveness, minimum sample
requirement, sensitivity, and portability, their stability must be
improved in some cases before they can be effectively applied in the
commercial market. The development of portable nanosensors for the
detection of adulterants without sample preparation or pre-treatment
has gained significant attention in recent years. Hence, a holistic re­
view of nanomaterial-based sensors is necessary to guide future research
towards the highly efficient and rapid detection of adulterants in dairy
products. In this review we survey the nanoensembles based platforms
for detection of dairy adulterants, specifically those using metallic
nanoparticles, aptamers, quantum dots, carbon dots, nanocomposite,
nanocrystals, carbon nanotubes, nanowires, molecularly imprinted
polymers as shown in Fig. 2. Based on the advanced functions and
synthesis of nanomaterials integrated optical and electrochemical
nanosensors are discussed in detail.
2. Nanosensors for monitoring dairy adulterants
Milk and dairy products are staple foods consumed worldwide on
daily basis [20]. Nascimento et al. classified the milk adulterants into
four types of adulterations: 1) with nitrogen contents, 2) fat content of
milk and addition of detergents, 3) with water and other substances, 4)
to enhance the shelf life of dairy product [4]. Economically motivated
adulteration, which involves the addition of vegetable protein, milk
from various species, water, and whey, is among the most common
forms of milk adulteration [21,22] although, these adulterations do not
result in serious health issues. Although, some milk adulterants such as
urea, melamine, formalin, detergents, ammonium sulphate, benzoic
acid, salicylic acid, H2O2, and sugars, can pose serious health risks and
should not be ignored [23]. From an economic perspective, adulterating
milk with a mixture of substances can be harmful. While adding water to
milk does not pose any health risks as long as the water is not polluted, it
does lower the nutritional value of milk. The quality of milk is deter­
mined by various factors, including the total Solid-Not-Fat (SNF) con­
tent. Adulterants are often added to increase the SNF concentration, but
Himshweta and M. Singh
Fig. 2. Nanomaterials employed as platforms for sensing adulterants.
this changes the natural purity and quality of the milk [23].
In addition to the concerns over food adulteration, there are specific
adulterants that are particularly worrisome. One such adulterant is
melamine (1, 3, 5-triazine-2, 4, 6-triamine), an organic compound that
has been widely used in the manufacturing industry. Due to the presence
of exocyclic amino groups (-NH2) and three endocyclic hybrid nitrogen
rings in melamine it act as nucleophilic reagents [24]. The chemical
industry utilizes melamine, formaldehyde and additives to synthesize
melamine resin, a synthetic polymer used in the production of home
appliances, plastics, board or wood coatings, and adhesives. It is also
used in inks, pesticides and fertilizers. However, this compound is often
misused by being added to various food products to enhance their pro­
tein levels due to its high nitrogen content, which makes up 66% of its
total mass [25]. The nitrogen-rich substance, melamine, has been added
to food and milk to falsely elevate the protein content. However, mel­
amine is a poisonous compound, and consuming excessive amounts of it
in food or milk can lead to kidney and bladder issues, as well as death.
Consequently, the detection of melamine in food and milk is highly
desirable, and various types of detection techniques have been devel­
oped for this purpose. Melamine gained notoriety as a food fraud in
China in 2008 when it was illegally added to milk and milk-based
products, leading to kidney damage and even death in many infants.
The US Food and Drug Administration (FDA) have set the limit for
melamine in infant formula at 1 mg/kg body weight, while the limit for
other dairy products is 2.5 mg/kg [26,27]. Nonetheless, the World
Health Organization (WHO) has also established a daily limit for mel­
amine intake of 0.2 mg/kg of body weight [21,28]. Since the 2008
incident in China, melamine has been found in a wide variety of
milk-based products, with reported melamine levels ranging from 0.09
to 6200 mg/kg. In addition to melamine, a number of other chemical
compounds such as urea, amidinourea, ammeline, cyanamide, dicyan­
diamide, 3-aminotriazole, 4-aminotriazole, guanidine, biuret, triuret,
and cyromazine have been used as adulterants to increase the nitrogen
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Sensors and Actuators Reports 5 (2023) 100159
content of milk [21].Although melamine has low acute toxicity, its
intake above the established limit can result in renal failure due to the
formation of insoluble cyanurate crystals in the kidney [29].
Adulteration of milk and dairy products with ammonium sulfate is a
common practice to increase the protein content artificially. Adding
ammonium sulfate to milk can also help in retaining the density of
diluted milk while raising the lactometer reading, making it appear
richer in quality [30]. However, this practice can be hazardous to human
health as consuming excessive amounts of ammonium sulfate can lead to
various health issues like respiratory distress, gastrointestinal irritation,
and even death in severe cases. Furthermore, ammonium sulfate adul­
teration can also affect the quality and texture of the dairy products,
leading to a decline in their nutritional value. The use of preservatives
like formalin, salicylic acid, benzoic acid, and H2O2 in dairy products is a
common adulteration practice [22]. These chemicals can extend the
shelf life of milk by preventing spoilage and delaying the onset of mi­
crobial growth. However, their prolonged consumption can lead to
various health hazards such as cancer, asthma, skin allergies, and
digestive disorders. Formalin, a strong disinfectant, is particularly
harmful and can cause severe health issues such as liver and kidney
damage. Similarly, salicylic acid and benzoic acid can cause irritation
and inflammation of the digestive system, while H2O2 can cause respi­
ratory distress and chemical burns on the skin [22]. Due to the high cost
of milk fat, some milk and dairy product producers opt to remove it and
replace it with non-dairy fats like vegetable oil for financial reasons
[31]. To emulsify and dissolve the oil, detergents are added to the milk.
However, the presence of peroxides and detergents in milk can both
contribute to gastrointestinal issues that can lead to gastritis and
inflammation of the intestines. Excessive amounts of starch in milk can
cause diarrhoea due to the actions of undigested starch in the colon.
However, stored starch in the body might pose a danger to individuals
with diabetes [32].
Urea is a common adulterant that is added to milk to increase its
Himshweta and M. Singh
nitrogen content and thereby make it appear more protein-rich. Urea is a
naturally occurring nitrogenous compound found in urine as a metabolic
by-product. It affects the kidney as a large portion of urea content is
removed by them. Carbamide, another name for urea, can be produced
synthetically. It serves as both a fertiliser and a raw material for the
production of plastics. Carbon dioxide and liquid ammonia are used to
prepare urea in large quantities. These two substances are combined at
high pressure and temperature to form ammonium carbamate. At low
pressure, ammonium carbamate breaks down to form urea and water.
Urea can also be used to produce barbiturates. However, when used to
adulterate milk, it has a number of negative effects on humans and other
milk consumers. Addition of urea increases the nitrogen content in milk.
The significant portion of non-protein nitrogen in milk is contributed by
urea, which is a main component of milk.
The Food Safety and Standards Authority of India (FSSAI) act of 2006
and the Prevention of Food Adulteration Act (PFA) rules of 1955 state
that the upper allowable limit of urea in milk is 15.39 µM [33]. Urea can
have harmful effects on human health beyond the threshold, leading to
cancer, indigestion, ulcers, urinary tract blockage, and renal failure.
Urea can contaminate milk in two ways: by intentionally addition of
more urea and by addition of synthetic milk to pure milk. Therefore,
regular checking of urea in milk quantitatively is crucial and can be
useful in certain ways since, above the permissible limit, it can be
harmful to humans [32].
Furthermore, the use of additives such as carbonates and bi­
carbonates in dairy products can also have adverse effects. Research has
shown that these additives can interfere with hormone signalling that
controls growth and reproduction [22,23]. As such, monitoring the
concentration of urea in milk and limiting the use of additives are both
critical for ensuring the safety and quality of dairy products for human
consumption. Currently, the detection of adulterants can only be carried
out on small sample sizes [34], and this can only be achieved by
high-throughput methods in centralized laboratories. However, this is a
expensive exercise in terms of both time and resources. Therefore, the
need for rapid on-site detection methods is essential.
Emerging nanosensor technologies have been increasingly applied to
satisfy the growing demands of different areas. With the advancements
in nanotechnology and nanoscience, the nanosensor technology has
flourished even further. Moreover, recent developments in nano­
materials and nanotechnology demonstrated their potential for devel­
oping novel detection methods. Nanomaterials find applications in a
wide range of fields due to their simple synthesis, facile surface chem­
istry, and low cost. They are often used as labels and reporters owing to
their unique properties, which can be utilized to design novel nano­
sensors. Nanosensors are highly sophisticated, accurate, and sensitive
systems that can identify one or more specific physical or chemical
processes based on a specific signal. Compared to traditional methods,
these nanometer-scale sensors offer significant improvements in speed,
selectivity, and sensitivity [35]. Nanosensors are usually constructed by
combining a receptor with a transducer. The receptor can be made of
any organic or inorganic material that interacts with the analyte or its
derivatives. On the other hand, the transducer’s function is to converts
the response into a measurable signal, such as electrical, electro­
chemical, or optical signals. It acts as a sensing platform and is often
made of nanomaterials such as carbon nanotubes, quantum dots, gra­
phene oxide, metallic gold, iron oxide particles, and electrospun poly­
meric nanofibers. The applications of nanomaterials in sensors and
biosensors increase the efficiency and sensitivity of assays [35]. The
unique properties of nanomaterials, such as their flexible shape,
improved optical, mechanical, electrical, and thermal characteristics,
high specific surface area, and nanoconfinement, significantly enhance
the transducing ability of signals [36]. When evaluating nanosensors,
several key factors are considered, including their selectivity, which
describes how well the system can distinguish the analyte from other
sample components. Nanosensors are classified based on the transducer
mechanism used for generating output signals, such as electrical,
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Sensors and Actuators Reports 5 (2023) 100159
electrochemical, thermal, mechanical, optical, gravimetric, piezoelec­
tric, and magnetic [37,38]. Electrochemical nanosensors rely on the
principle of electrochemistry. By using nanomaterial on carbon based
electrode or glassy carbon electrode, they can measure the electro­
chemical reaction with analyte, either by formation or utilising ions or
electrons with higher efficiency and selectivity. Optical nanosensors use
nanomaterials to detect and analyze light, allowing them to detect and
measure a range of physical, chemical, and biological parameters. Op­
tical nanosensors are small analytical device that detects an analyte by
measuring changes in optical signals using optical transducers with the
enhanced optical properties of nanomaterials such as metallic nano­
particles, up-conversion nanoparticles, carbon nanotubes and quantum
dots [36,39]. Fig. 3 is a classification of nanosensors used for detection
of milk adulterants based on transducers used- optical and
electrochemical.
2.1. Optical nanosensors
Optical nanosensors produce the optically generated light after the
analyte binds to the receptor. The light is then measured by using
various optical transducers such as absorbance, fluorescence, polar­
isation, colorimetric, chemiluminescence, phosphorescence, biolumi­
nescence, and surface plasmon resonance (SPR) [40]. There are two
types of optical nanosensors: label-free and label-based. In the label-free
method, the analytes contact with the transducer directly results in the
detection of a signal. Label-based nanosensors, in contrast, use a label to
generate an optical signal through colorimetric, fluorescent, Localised
Surface Plasmon Resonance (LSPR), Surface Enhanced Raman Scat­
tering (SERS) or luminescent techniques [41,42]. Table 1 summarizes
the different nanomaterials based optical sensors, their target analyte,
detection method and limit of detection. Optical nanosensors offers
significant advantages over traditional analytical methods as they
enable the direct and real time detection of various biological and
chemical compounds without the need for labels. Their benefits include
excellent sensitivity due to the nanoscale size of the sensor, which allows
for the detection of even small amounts of analytes. They are
cost-effective and highly specific, enabling the detection of a specific
target analyte while minimizing false positives [41]. The following
sections address several types of optical sensors used for milk adulter­
ants analysis, including chemiluminescence, SERS, fluorescence, color­
imetric and LSPR nanosensors.
2.1.1. Chemilumniscence sensors
In chemilumniscence (CL) method, a chemical compound gets elec­
tronically excited and returning to its ground state, which results in the
spontaneous generation of light. This phenomenon leads to emission of
light and provides certain advantages, like low background noise, a
broad liner range, higher sensitivity, cost effectiveness and controlled
emission rate. In contrast to fluorescence, CL generates energy through
chemical reactions. Two distinct mechanisms govern the two types of
CL. These are: direct CL and indirect CL. Direct CL occurs when a
molecule undergoes a chemical reaction and produces light as a result.
This process involves the direct conversion of chemical energy into light
without the need for an external energy source.Here, the excited state of
the molecule is generated by the energy released during the chemical
reaction, and the excess energy is then emitted as light. This type of CL is
commonly observed in reactions involving highly reactive species such
as radicals and excited states of molecules. Examples of direct CL include
the reaction of luminol with hydrogen peroxide and the reaction of
fireflies with oxygen. These two extensively studied luminogenic sub­
stances are susceptible to oxidation by H2O2 and superoxide radical
anion (O•–2 ). The changes in the chemical structure of the chemiexcited
intermediates produced by these molecules result in the emission of light
[43]. During the indirect CL, the transfer of energy occurs when a
chemiexcited molecule (as energy donor, D) transfers energy to a second
molecule (as acceptor, E), which becomes further excited during the
Himshweta and M. Singh
Fig. 3. Classification of nanosensors used for detection of milk adulterants based on transducers used- optical and electrochemical.
process.
The process of indirect charge transfer in CL involves the transfer of
energy, leading to the excitation of a fluorophore or photosensitizer. The
excited molecule can then act independently. If molecular oxygen (O2) is
present, the photosensitizer can produce reactive oxygen species (ROS)
1
such as O•–2 , hydroxyl radicals (HO ), or O2 and the reactivity of these
•
species with biomolecules spans a wide spectrum [43]. There are three
types of CL based on their energy source: 1) ordinary CL occurs when a
chemical reaction produces excited-state species that emit light as they
return to their ground state. This type of chemiluminescence can be seen
in a variety of chemical reactions, such as the reaction between luminol
and hydrogen peroxidase, 2) biochemiluminescence (BCL) which in­
volves the use of enzymes to produce luminescence. BCL is a type of
indirect luminescence, and it can be further classified based on the type
of enzyme used, and 3) electrochemiluminescence (ECL) involves the
use of an electrochemical reaction to produce luminescence. CL gener­
ally involves multiple oxidation reactions to produce an excited state,
followed by transitioning to the ground state and emitting light. In this
process, organic compounds like lumino, lucigenin, lophine, and oxalate
peroxide esters are often used [44]. One of the key approaches in the
area of optical sensing for melamine, identified by researchers, is the use
of CL-based sensors, which is particularly fascinating.
Melamine is a harmful substance that can cause kidney damage and
other serious health problems, especially in infants and young children.
However, its presence in food has become a serious public health
concern due to its toxic properties. Detecting melamine is also important
for maintaining consumer confidence in the safety and quality of the
food supply. Melamine is not an approved food additive and is not safe
for human consumption. Therefore, it is essential to identify and prevent
its presence in food products. Therefore, detection and prevention of
food adulterants are crucial for protecting public health and ensuring
that consumers can trust the food products they purchase. Du and co-
workers studied a turn-on chemiluminescence resonance energy trans­
fer (CRET) method for melamine detection by reacting bis (2, 4, 6-tri­
chlorophenyl) oxalate, H2O2, fluorescein as a donor and dispersed
gold nanoparticles as an acceptor. The presence of gold nanoparticles
significantly reduced the CL signal of the bis (2, 4, 6-trichlorophenyl)
oxalate- H2O2-fluorescein reaction. This is due to the absorption band
of the dispersed gold nanoparticles overlaps with the CL spectrum.
Melamine causes gold nanoparticles to aggregate, which leads to a
redshift in the absorption band of the dispersed gold nanoparticles. As a
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Sensors and Actuators Reports 5 (2023) 100159
result, the absorption band of aggregated gold nanoparticles does not
overlap with CL spectrum. This prevents the CRET, and restores the
signal. This enables the determination of melamine with a detection
limit of 3 × 10− 7 µM in adulterated milk samples [45].
Electrochemiluminescence (ECL) is a process that involves the
emission of light as a result of a chemical reaction that occurs on an
electrode surface. In ECL, an electrochemical reaction produces excited
species that can emit light as they return to their ground state. ECL is
similar to other types of luminescence, such as fluorescence and phos­
phorescence, but it differs in that it requires an electrochemical reaction
to produce light. Chen and co-workers developed an ECL sensor for
melamine detection in milk sample. The sensor uses a composite film
modified electrode consisting of Ru(bpy)2+3 doped silica nanoparticles
(RUDS)/carboxylic acid functionalized multi-walled carbon nanotubes
(CMWCNTs)/Nafion. The CMWCNTs promote higher electron transfer
and electrical conductivity in the composite film. Moreover, CMWCNTs
increase the availability of more Ru(bpy)2+ 3 for ECL reaction, therefore
enhancing the ECL intensity. The relative ECL signal was found to be
proportional to the melamine concentration in the range of 5.0 × 10− 7 to
0.1 µM, with a detection limit of 1.0 × 10− 7 µM [46]. Building upon this
work, a simple label-free aptamer-DNAzyme coupled nanosensors was
developed for the detection of melamine in raw milk. Aptamers are short
single-stranded DNA molecules that can bind to a specific target mole­
cule with high affinity and specificity. DNAzymes, on the other hand, are
synthetic DNA molecules that can catalyze specific chemical reactions in
a manner similar to enzymes. Aptamers and DNAzymes can be combined
to create a new type of biosensor, called an "aptazyme" or "apta­
mer-DNAzyme coupled sensor". In these sensors, the aptamer is used as
the recognition element to selectively capture the target molecule, while
the DNAzyme is used as the signal transducer to generate a detectable
signal. In order to develop an aptamer-DNAzyme conjugated sensor,
suitable aptamers were first identified using microarray analysis, and
then the aptamer properties were optimised to lower background noise
and improve performance. This was achieved by increasing the
signal-to-noise (S/N) ratio of the screened sensor. The screening process
involved microarray-based screening and CL detection of the melamine.
The sensor showed maximum signal intensity at 2 £ 103 µM melamine
(S/N ratio, of 20.2) in raw milk [47].
H2O2 is a chemical compound that is commonly used as a disinfec­
tant, bleaching agent, and oxidizer. It is also found in trace amounts in
nature, including in milk. When hydrogen peroxide is present in milk, it
Himshweta and M. Singh Sensors and Actuators Reports 5 (2023) 100159

Table 1
Optical nanosensors for the detection of adulterants in dairy products.
Sample Nanomaterial Target analyte Detection method LOD (µM) References
3
Raw milk Aptamer-DNAzyme Melamine Chemiluminescence 2 £ 10 [47]
Milk AuNPs Melamine CRET 3 × 10− 7 [45]
11
Milk samples Single-stranded DNA ssDNA/g-C3N4 H2O2 Electrochemiluminescence 3.3 × 10− [51]
nanosheets hybrid
Milk Acid-modified silver nanoparticles (SAA- Melamine Colorimetric 0.01 [116]
AgNPs)
3
Milk Triton X-100-AuNPs Melamine Colorimetric with smartphone 5 × 10− [126]
Milk Gold nanoparticles suspended in Tollen’s Formalin Colorimetric NA [138]
reagent
Milk AuNPs Anionic detergents Colorimetric SDBS- 66 and commercial [123]
ADs- 319
Milk Iron doped copper tin hydroxide CuSn H2O2 Colorimetric 9.49 [130]
(OH)6 microspheres
Fresh milk Copper phosphate modified by hydrophilic H2O2 Colorimetric 79 [134]
phytic-acid
Dairy milk products Zero valent manganese nanoparticles (ZV- H2O2 Colorimetric 0.2 [133]
Mn NPs)
Milk sample FePt-Au Ternary Metallic Nanoparticles H2O2 Colorimetric 12.33 [129]
with Au(I)
Milk Optical fiber -based localized surface Melamine Colorimetric 0.033 [145]
plasmon resonance of AuNPs
Milk AgNPs Melamine Colorimetric 0.032 [121]
Milk samples AgNPs using cellulose nanowhiskers H2O2 Colorimetric 14 and [131]
(CNW) 112
Liquid milk samples Acacia Gum–Stabilised Silver H2O2 Colorimetric - [144]
Nanoparticles
Cow milk Molybdenum disulfide quantum dots Formaldehyde Colorimetric NA [137]
(MoS2 QDs)
Milk Microfluidic paper-based analytical Urea and H2O2 Colorimetric Urea and H2O2 were 2.4 × [139]
devices (mPADs) 103 and 100
Milk Novel pincer zinc complex stabilized gold Melamine Colorimetric 0.019 [128]
nanoparticles
Pasteurized milk β-Cyclodextrin-functionalized silver Melamine Colorimetric 4.98
[111]
samples nanoparticles
14
Pre-treated milk Citrate-capped gold nanoparticles (AuNPs Melamine Colorimetric 2.37 × 10 [118]
samples
− 3
Processed raw milk Asymmetrically modified gold Melamine Colorimetric 1.05 × 10 [127]
nanoparticles
Liquid milk AuNPs Melamine Colorimetric 0.238 [119]
Raw milk AuNPs Melamine Colorimetric 0.56 [110]
Milk Sodium D-gluconate stabilised silver Melamine Colorimetric 0.5 [120]
nanoparticles
Milk Label-free Ag NPs Melamine Colorimetric 2.32 × 103 [108]
Milk H2O2–Au nanoparticles Melamine Colorimetric 400 [117]
Milk AuNPs-Aptamer Melamine Colorimetric 11.89 and 3.9 [114]
Milk AgNPs Melamine Colorimetric 0.01 [24]
Raw milk AgNPs Melamine Colorimetric 3.96 [109]
Raw milk and milk Au-NP-H2O2-TMB Melamine Colorimetric 2 £ 10− 6 [113]
powder
Milk AgNPs Melamine Colorimetric 0.07 [124]
Milk Ag-rutin, Ag-curcumin Melamine Colorimetric 0.079 and 1.9 [125]
Cow milk Lab-made apps (PhotoMetrix®, and Starch, H2O2, and NaClO Colorimetric coupled to NA [132]
RedGIM®) Smartphone Image Analysis
Milk Zirconium-based metal-organic Melamine Fluorescence 0.09 [99]
frameworks composite
Zr-based MOFs
Raw milk AuNPs-CdS QDs system Melamine Fluorescence 3.17 [88]
Cow milk and infant Glutathione stabilized gold nanoclusters Melamine Fluorescence 28.2 [91]
formula (AuNCs)
Pure milk and milk Dual emissive fluorescent silica Melamine Fluorescence 0.018 [101]
powder nanohybrid
4
Milk powder Acrylate citric acid (ACA) based polymeric melamine Fluorescence 2.32 × 10− [107]
membrane sensor
4
Dairy product AuNPs on the CsPbBr3 NCs@BaSO4 Melamine Fluorescence 4.2 × 10− [96]
samples
Milk Ds DNA template copper nanoparticles Melamine Fluorescence 0.79 [95]
Milk Graphene Quantum Dots Melamine Fluorescence 0.12 [100]
Raw milk AgNPs Melamine Fluorescence 0.03 [92]
3
Milk Au@Carbon quantum dots Melamine Fluorescence and Smartphone 4 × 10− [94]
nanocomposites
Milk Graphene oxide (GO) with 2,7 H2O2 Fluorescence 1 × 107 [122]
-dichlorfluorescein diacetate (DCFH-DA)
Milk CdS QDs Melamine Fluorescence 1.11 [103]
(continued on next page)

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Himshweta and M. Singh Sensors and Actuators Reports 5 (2023) 100159

Table 1 (continued )
Sample Nanomaterial Target analyte Detection method LOD (µM) References

Milk Fluorescent copper nanoparticles Melamine Fluorescence 0.79 [97]

templated by dsDNA
Raw milk and milk Aminofunctionalized carbon dots (C-dots) Melamine FRET 0.036 [89]
powder and gold nanoparticles (AuNPs)
Raw milk Polydopamine-glutathione nanoparticles Melamine FRET 0.023 [93]
(PDA-GNPs) and AgNPs
3
Milk Protoporphyrin IX (PpIX) and Graphene Melamine FRET 3.6 × 10− [102]
quantum dots (GQDs)
Raw milk AuNPs Melamine FRET 0.018 [90]
Milk ZnO nanorods Urea UV-Visible 1.185 × 103 [140]
Diluted milk Silver nanocubes Formaldehyde UV-Visible 12.7 [146]
Milk Urease/ZnFe2O4 nanocomposites Urea UV-Visible 10 [112]
Milk AgNPs Urea UV-Visible Capped nanoparticles 3.23 [141]
and uncapped nanoparticles
5.02
Mozzarella and feta Monolithically integrated silicon chips Bovine milk UV-Visible 0.5 and 0.25% (w/w) [211]
cheese
Milk samples Optical fibre tip coated with the Formaldehyde UV-Visible 6.7 and 16.6 [136]
polyoxometalate salt
4 4
Milk Janus-type Au-mesoporous silica Urea UV-Visible 2.35 × 10 and 4.35 × 10 [106]
nanoparticles functionalized with a
fluorescent-oligonucleotide
Infant formula Chondroitin Sulfate-Reduced Gold Melamine UV-Visible 0.1 [57]
Nanoparticles
Yogurt samples Gold nanoparticles conjugated to Bovine species in sheep- UV-Visible 1.6 × 10− 9 for PCR product [208]
streptavidin (SA-AuNPs) and goat-derived products specific to cow and goat, and
(yogurt) 3.1 × 10− 9 for sheep-
specific PCR product
Liquid milk Cysteamine functionalized core-shelled Sodium thiocyanate and SERS Sodium thiocynate- 0.37; [79]
nanoparticles benzoic acid benzoic acid- 80.25
Milk Silver-coated gold nanoparticles Urea and ammonium SERS Urea- 830 and ammonium [78]
(Au@AgNPs) sulfate sulphate- 0.38
- Ag metasurface on SiO2-Au-coated silicon Melamine SERS 0.001 [85]
(Ag/SiO2/Au/Si)
Infant formula AgNPs Melamine SERS 79.28 [80]
Milk Fe/N-codoped carbon dots (CDFeN) Urea FL, SERS, and RRS 0.001 [83]
Milk Silver nanosol (AgNP) Urea SERS 4.92 × 10− 3 [75]
- Au nanostructured gold-coated ITO Melamine SERS and electrochemical 1.18 £ 10− 6, 1.89 £ 10− 10
[63]
Au nanostructures on ZnO NWs-coated CF and 5.74 £ 10− 5
Liquid infant Ag micro- and nanostructures Melamine SERS 39.64 [54]
formula
Skimmed milk Spherical magnetic-core gold shell Melamine SERS 390 [53]
nanoparticles (AuNPs) and rod-shaped
gold nanoparticles (nanorods)
Milk powder, infant Citrate coated gold nanoparticles (Au NPs) Melamine SERS 0.79 for lactose and milk [55]
formula, lactose, powder; and 1.59 for infant
whey protein formula
Milk AgNPs Melamine SERS and Colorimetric 0.4 [82]
Milk AgNPs Melamine SERS 5.15 and 7.92 (instrument [77]
detection limit)
Infant formula milk Silver film over nanospheres (AgFON) Melamine SERS 0.79 [76]
powder solution
6
Milk Ag nanorods (NRs) Melamine SERS 0.001 and 1.0 £ 10− [72]
Milk Au@SiO2 NPs Melamine SERS 7.92 [67]
Milk AgNPs Melamine SERS 2.1 [71]
Milk Portable pipette-Ag-NPs-basil-seed Melamine SERS 0.85 [70]
Milk and milk Cyclodextrin-decorated silver Melamine SERS 2.37 × 104 [58]
powder nanoparticles CD-AgNPs
Milk Silver nanostructures (AgNS) Melamine and urea SERS 1.69 × 103 for melamine; [84]
480 for urea
2% reduced fat raw AuNPs Melamine SERS and Colorimetric 1.98 [61]
milk
Milk liquid and AgNPs Melamine SERS 15.9 [62]
powder samples
Liquid milk AuNPs Melamine SERS 1.34 [60]
Milk powder Silver nanoparticles coated amino Melamine SERS 0.019 [56]
modified polystyrene microspheres (PS-
NH2/Ag NPs)
Milk AgNPs with MIP Melamine SERS 16.5 [68]
Milk Gold Nanoparticles namely Gold Nano Melamine SERS 0.87 [74]
Spheres (GNSs), Gold Nanorods (GNRs)
and Triangular Gold Nanoprisms (GNPrs)
on porous monolith scaffold
6
Raw milk and milk Silver nanoparticles and covalently bonded Melamine SERS 1.0 × 10− [69]
powder 2-nitro-5-mercaptobenzoic acids (TNBs)
(continued on next page)

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Himshweta and M. Singh Sensors and Actuators Reports 5 (2023) 100159

Table 1 (continued )
Sample Nanomaterial Target analyte Detection method LOD (µM) References
6
Milk Poly-thymine (T) aptamer to the gold Melamine SERS 7.93 × 10− [65]
nanoparticles thymine-rich aptamers were
modified on AuNPs
Milk Au-Capped Si Nanopillar Melamine SERS 0.079 in PBS and 2.37 in [81]
milk
Cow, camel, goat, Cit-AgNP Fat, casein, urea, and SEIRA and ANN Fat -1.07 × 104 [213]
buffalo, and lactose of cow, camel, goat, Casein -2.681
infants’ formula buffalo, and infants’ Lactose -2.30
powdered milk formula powdered milk Urea -349.65

Abbreviation:Gold nanoparticles- AuNPs; AgNPs-silver nanoparticles; gold-silicon oxide nanoparticles- Au@SiO2 NPs; Gold nanoparticle-hydrogen peroxide-3,3′ ,5,5′ -
Tetramethylbenzidine- Au-NP-H2O2-(TMB); sodium hypochlorite- NaClO; barium sulfate-coated perovskite nanocrystals-(CsPbBr3NCs@BaSO4); zinc ferrite- urease/
ZnFe2O4; Surface-enhanced infrared absorption spectroscopy- SEIRA; Artificial neural networking –ANN, SERS-surface enhanced Raman scattering;FL-fluoresence;
RRS- Resonance Rayleigh scattering; Fluorescence resonance energy transfer-FRET; Sodium dodecyl benzene sulfonate –SDBS; Anionic detergents-AD; Chem­
iluminescence resonance energy transfer- CRET; Phosphate buffer saline –PBS; MOF-metal organic framework; Molecularly imprinted polymers-MIP; citrate silver
nanoparticles- Cit-AgNP; Iron tin oxide- ITO; ZnO-Zinc oxide; CdS QDs- cadmium sulphide quantum dots.

can react with enzymes called catalases to produce water and oxygen
gas. This reaction is important because it helps to protect the milk from
spoilage by harmful microorganisms and ensuring the safety of the milk.
While it is naturally present in milk in trace amounts, adding it as a
preservative requires strict regulation and adherence to guidelines to
ensure that it is safe for human consumption. However, excessive pro­
duction of H2O2 can lead to various diseases such as cancer and Alz­
heimer’s disease [48]. Therefore, it is important to accurately detect and
monitor H2O2 levels in various food and environmental samples. An ECL
sensor for H2O2 with an extremely low detection limit has been pro­
duced using a hybrid of polypyrrole-dodesylbenzen sulfate-cerium oxide
nanocomposite (PPy-DBS-CeO2) on a platinum (Pt) electrode. The
addition of CeO2 nanoparticles to the composite matrix improved the
ECL signal. The sensor demonstrated linear correlation between the
signal and the logarithm of the H2O2 concentration ranging from 5 ×
10− 10 to 50 µM (R2= 0.9922) and exhibited a very low detection limit of
1.2 × 10− 10 µM. Moreover, the sensor showed good stability and
reproducibility making it useful for detecting H2O2 in milk and water
samples [49]. Another simple and inexpensive approach for the detec­
tion of H2O2 concentrations in semi-fat milk samples, involves the use of
a hydroxyethyl cellulose sensitive membrane and a photodetector to
measure CL. The reaction between luminol and H2O2 is catalysed by
cobalt hydroxide, resulting in the detection of H2O2 in milk at concen­
tration of 852.6 µM [50]. However, the detection limit of this method is
relatively high, which may limit its usefulness in certain applications.
H2O2 is commonly used as a fungicide in food and as a disinfectant
and antiseptic additive in food. However, for humans, it can be carci­
nogenic, accelerate ageing, and leads to cardiovascular disease. Here, a
novel non-enzymatic ECL sensor using single-stranded DNA (ssDNA)/g-
C3N4 nanosheets (NS) was developed to address the demand for rapid
and accurate detection of H2O2 in food. The g-C3N4 NS and ssDNA so­
lution were mixed to synthesize ssDNA/g-C3N4 NS hybrid. To increase
the luminescence signal, the ssDNA/g-C3N4 NS was modified on a glassy
carbon electrode. This modification catalyzes the ECL reaction of
luminol- H2O2. The suggested sensor displayed great sensitivity across
wide dynamic range of 1 × 10− 10 to 1 × 104 µM with lowest detection
limit of 3.3 × 10− 11 µM for H2O2 [51]. This detection limit is substan­
tially lower than that achieved with previous methods. Using this
technique, milk samples can respond to H2O2 in just one min. Therefore,
this method stands out as one of the best ECL based sensing technique for
detection of H2O2 in food stuffs.
2.1.2. Surface enhanced Raman scattering sensors
Surface Enhanced Raman Scattering (SERS) is a laser based sensing
technique used to amplify the weak Raman signals generated by mole­
cules on a metallic surface. The amplification is achieved by the inter­
action between the laser light and the surface plasmons of the metal,
which can enhance the Raman signal up to several orders of magnitude.
This method is highly sensitive for increase of Raman signal intensities
by enhancement factor of 10-11 folds [34]. The SERS sensor exhibited
significant enhancement by utilizing the plasmonic properties of metal
nanoparticles. The intensity enhancement of the SERS signal can reach
up to 14 folds [52]. Moreover, this method is suitable for label-free
identification of biochemical and chemical compounds. Probes in this
technique are metallic nanoparticles either in colloidal form or tagged/
encapsulated with Raman active molecules. In last few years, different
types of nanomaterials have been used as substrates for signals
enhancement in SERS technique for detection of food adulterants.
Nanoparticles can significantly enhance the signal intensities and
therefore widely used to increase the detection limit. Laser light excites
the nanoparticles, driving the surface charge and creating a highly
localized plasmonic-enhanced field. When an analyte is absorbed or
positioned closer to this field, a greater enhancement in the Raman
spectra is observed [34]. So, SERS property of nanoparticles is used for
developing sensing assay for the determination of dairy adulterants.
Melamine adulteration in food and pharmaceutical products is a
serious problem, and there is an increasing need to safeguard the public
from exposure to such adulterated items. To address this concern, one
effective way is to develop a portable technique for on-site and fast
testing to reduce the risk of adulterated items. For the quick detection of
melamine, a novel approach based on surface-enhanced Raman spec­
troscopy has been devised. In order to establish a complex with the
melamine molecules, spherical magnetic-core gold shell nanoparticles
(AuNPs) and rod-shaped AuNPs (nanorods) tagged with Raman-active
molecules were used. 5, 5′ -Dithiobis (2-nitrobenzoic acid) was selected
as the Raman active molecule because it can easily form a self-assembled
monolayer (SAM) on the surface of a gold nanoparticle and exhibits high
Raman scattering due to the symmetric NO2 stretch. A detection limit of
390 µM for milk samples was achieved. Analysis was completed just in
15 minutes [53]. Galvanic displacement reaction of copper (Cu) by sil­
ver (Ag) is also a good way to develop portable sensing platform for
melamine detection. The low cost and highly portable surfaces of Cu
tape and Cu coin, with silver nanostructures were used to develop the
sensor. AgNPs as SERS substrates were able to detect 39.64 µM of mel­
amine in baby formula milk within 15 minutes [54].
The colorimetric and Raman responses of citrate coated AuNPs were
examined. The addition of isopropanol (IPA) results in agglomerations
of AuNPs, which increases the melamine-specific Raman signal and
remove matrix interference for substrate synthesis. Through the AuNPs
agglomeration, Raman signal for melamine was enhanced by 5 folds,
resulting in melamine detection at 0.79 µM for lactose and milk powder,
while for infant formula it was 1.59 µM [55]. Thus, this portable SERS
sensor is a useful tool for on- site analysis of melamine adulteration in
various food and pharmaceutical matrices, such as milk powder, infant
formula, lactose, povidone, whey protein, wheat bran, and wheat
gluten.

8
Himshweta and M. Singh
A highly active SERS-coated amino modified polystyrene micro­
sphere (PS-NH2/Ag NPs) with homogeneous surface morphology has
been produced. Nanojets are used for non-destructive analysis of analyte
adsorbed on the smooth gold or glass surface. Due to efficient coupling
of surface plasmonic resonance, a 10 folds increase in Raman signal
intensities was observed between PS-NH2/Ag NPs and gold surface
compared to glass surface. These microsphere SERS substrates were able
to detect 2-mercaptopyridine as low as 0.001 µM concentrations. In
order to create SERS encoded PS-NH2/Ag NPs microspheres, four
distinct thiol compounds have been successfully used as tags. A linear
correlation was found between SERS intensity and melamine concen­
trations (0.1-10 µM) with detection limit of 0.019 µM [56]. This detec­
tion limit is significantly lower than the standard set by US Food Drug
Administration (FDA) of 0.02 µM. This research has shown promising
results in the development of gold nanoparticles synthesized by
glycosaminoglycan and chondroitin sulphate as reducing agents for the
detection of melamine in baby formula. Chondroitin sulfate and Au3+
were mixed together and heated. The conversion yield of Au3+ to Au0
was determined to be 80.1% using inductively coupled plasma-atomic
emission spectroscopy. This analytical technique is widely utilized for
the precise quantification of metal ions and has proven to be a reliable
method for the determination of the yield of Au3+ to Au0. The high
conversion yield observed in this study indicates the efficiency of the
reduction process and suggests that the chondroitin sulfate-reduced gold
nanoparticles could be a promising tool for various applications,
including the detection of melamine in milk products. As the concen­
tration of melamine in the gold nanoparticle solution increased, the
characteristic surface plasmon resonance band of gold nanoparticles at
530 nm decreased, and a new peak emerged at 620 nm indicating the
formation of larger gold-melamine complexes.This red shift in the
absorbance spectrum indicates a change in the morphology and size of
the gold nanoparticles, which is likely due to the interaction between
melamine and the gold nanoparticles. The observed changes in the
absorbance spectrum suggest that the absorbance ratio (A620/A530) can
be utilized for rapid detection of melamine even at lower concentrations.
The suggested method can measure melamine spiked in baby formula up
to 0.1 µM within 13 min [57]. Melamine nanosensing using chondroitin
sulfate-reduced gold nanoparticles might be a potential technology for
rapid on-site melamine screening of milk products.
Conventional silver mirror reaction with α-cyclodextrin (CD), is used
to synthesize AgNPs embellished with CD. In order to identify melamine,
CD-AgNPs are used as SERS substrate. The 704 cm− 1 intensity of the
Raman band is used for detection of melamine in milk and milk powder.
The electromagnetic field and the chemical or electronic enhancement
effect of AgNPs on the triazine ring of melamine, exhibit a 28 cm− 1 red
shift of the melamine peak at 704 cm− 1 compared to the solid melamine
peak. Using CD-AgNPs as the SERS substrate instead of traditional
AgNPs significantly enhances the sensitivity of the method, providing an
enhancement in scattering efficiency by a factor of up to ~6 folds. So, a
detection limit of 2.37 × 104 µM, was achieved for the detection of
melamine in milk and milk powder [58]. Whereas, the detection limit of
traditional AgNPs was 3.96 µM with enhancement of 5 folds [59]. This
improvement in sensitivity is due to the unique surface properties of
CD-AgNPs, provides a stronger electromagnetic field for SERS detection.
The interaction between AuNPs and analyte allows the quick detection
of melamine in liquid milk using SERS method. This interaction causes
the AuNPs aggregation which triggers SERS "hot spots" that significantly
enhance the melamine signals in the Raman spectrum. The detection
limit of sensor is 1.34 µM and it takes 30 min for detection [60].
Although this work is useful for melamine detection in milk matrices as
per European law standards, further improvements are required to
reduce the analysis time of the procedure.
Lang’s research group combines two approaches for detection of
melamine in milk: AuNPs with colorimetric and SERS based detection.
Rapid screening was obtained through colorimetric technique, which
has the advantage of AuNPs colour transition from red to blue or purple
9
Sensors and Actuators Reports 5 (2023) 100159
when interacting with melamine. However, in the presence of inter­
fering substances, the colorimetric approach generates imprecise and
falsely positive quantitative signals. The SERS approach, which can
directly use the AuNPs from the colorimetric method, was used as a
quick validation tool to minimise these effect. Three sizes of AuNPs (15,
30, and 50 nm) were investigated to maximise the integration of these
two methods. The 30 nm Au NPs were found to be the optimal size for
both the colorimetric and SERS approaches based on detection limit and
the ability to quantify melamine. The proposed colorimetric-SERS assay
has a detection limit of 1.98 µM in milk, and it takes approximately 20
min, making it a rapid and sensitive method for the detection of analytes
in milk samples [61].
SERS technique is broadly used to identify the adulterations in dairy
products. Following the dilution of milk with varying volumes of water
the AgNPs coated on a cylindrical support could detect 5 µL of diluted
sample, without any drying steps. The outcomes revealed that, in the
absence of dilution, the potent matrix effect of the milk sample entirely
suppressed the melamine peaks. This phenomenon may be attributed to
either the robust affinity of milk constituents to occupy the active sites of
AgNPs or the obstruction of laser penetration by the opaque milk solu­
tion. Moreover, a detection limit of 0.019 µM was obtained for melamine
in water. In addition, the dilution approach is effectively used to mini­
mize the impact of matrix effects from original samples for the detection
of melamine in both liquid and powder forms of milk. It is noteworthy
that these findings suggest a dilution factor of 75 can significantly
diminish the matrix effect, though with a slight variation in detection
sensitivity. In real sample, the detection limit is increased to 15.9 µM,
which is near to the permitted levels as specified in milk products [62].
This work describes the development of disposable and portable
conductive substrates for dual detection of organic toxicants using SERS
and electrochemistry. Gold nanostructures were electrodeposited on
three substrates bare indium tin oxide (ITO) electrode, ITO coated with
plane gold and carbon fibre (CF) covered with ZnO nanowires (ZnO
NWs) and their sensitivities were improved by adding a gold layer and
ZnO nanowires. The substrates bare indium tin oxide (ITO) electrode,
ITO coated with plane gold and carbon fibre (CF) covered with ZnO NWs
showed good SERS signal reproducibility and were able to detect mel­
amine at low concentrations 1.18 × 10− 6, 1.89 × 10− 10, and 5.74 × 10− 5
µM, respectively [63].
Aptamers are oligonucleotides (single stranded) bind the target
moiety with higher specificity and affinity. These aptamers are
commonly screened using a combinatorial technique called systematic
evolution of ligands by exponential enrichment. Aptamers offer several
advantages over antibodies, such as their ease of synthesis, desirable
storage properties, and the ability to be easily modified. Due to these
inherent advantages, aptamers are widely used as recognition elements
in biosensor construction and are highly effective in detecting a diverse
range of substances, including metal ions, organic molecules, peptides,
proteins, and even cells [64] . An aptamer-modified SERS nanosensor
and an oligonucleotide chip, has been created to detect trace amounts of
melamine in milk. The Raman tag and poly-thymine (T) aptamer are
attached to the surface of gold nanoparticles in order to produce the
SERS nanosensor. The study resulted in the formation of a structure
known as "T-M-T" (thymine-melamine-thymine), which involved the
creation of multiple hydrogen bonds between molecules of melamine
and thymine. During the measurement, as the concentration of mel­
amine increases, the intensity of Raman signal also increases. Therefore,
able to determine the melamine with detection limit of 7.93 × 10− 6 µM
which is much lower than the safety standard [65]. Although the mel­
amine detection using this method showed excellent sensitivity
compared to previous methods, and requires 2 h incubation time to
achieve maximum SERS intensity.
According to time-dependent density functional theory, the presence
of quantum effects like electron tunneling across the gap between
nanoparticles would result in a significant reduction of electromagnetic
field enhancements at very short distances [66]. Gold nanoparticles
Himshweta and M. Singh
have been reported to affect the Raman signal due to their localized
surface plasmon resonance (LSPR) properties. The interaction of the
incident light with the gold nanoparticles generates strong electromag­
netic fields at their surface, leading to enhanced Raman scattering of
nearby molecules [67]. However, this effect can be both constructive
and destructive depending on the size and shape of the nanoparticles
and the distance between the nanoparticle and the Raman-active
molecule. To minimize the distortion in Raman signals that arise from
charge transfer and electronic tunneling effects, SiO2 shell-isolated gold
nanoparticles (Au@SiO2NPs) were utilized for signal enhancement
instead of conventional Au NPs. When the Au@SiO2 NPs colloid is
combined with the melamine solution or the melamine-treated milk,
aggregation start occurring. In contrast to aggregated AuNPs, the
aggregated Au@SiO2 NPs on a Cu substrate are capable of enhancing the
Raman signals of melamine without distortion, by using the surface
plasma effect. The time required for detection is 15 minutes with a
detection limit of less than 7.92 µM [67]. Due to simplicity in operation,
this method can be applied for rapid detection of melamine in milk,
moreover the Au@SiO2 NPs have demonstrated stability in the envi­
ronment for >2 months.
Molecularly imprinted polymers (MIPs) synthesized through the
imprinting method, have the advantages of higher specificity, stability
and selectivity for target analyte. MIPs are widely used as recognition
element in sensing platforms to enhance the selectivity of sensors. Hu
and co-workers designed a AgNPs combined with MIPs-Raman spec­
troscopic-active substrate (MIPs-SERS) for melamine detection. MIPs
were synthesized by bulk polymerization, using melamine as the (tem­
plate), methacrylic acid (functional monomer), ethylene glycol dime­
thacrylate (cross-linking agent), and 2, 2′ -azobisisobutyronitrile
(initiator). The specific affinity of MIPs-AgNPs for melamine and the
quick adsorption equilibration rate were confirmed by static and kinetic
adsorption experiments. The melamine levels in tap water and skim milk
is determined through SERS spectra. The detection limit for melamine in
tap water was found to be 1.9 µM, while skim milk had a detection limit
of 16.5 µM [68]. The method requires 26 min for melamine detection. To
make this technique more appealing to food industry professionals and
government laboratories, the sample pre-treatment procedures can be
simplified by modifying the synthesis of these conjugated MIPs-AgNPs
or incorporating more effective SERS-active substrates.
A SERS-based approach for the detection of melamine is described,
which utilizes the charge-transfer phenomena caused by multi-hydrogen
bonding. In this method, a silver-2-nitro-5-mercaptobenzoic acid (Ag-
TNBs) is used as the melamine detecting probe. This approach depends
on the formation of multiple hydrogen bonds between TNB and mel­
amine, which result significant variations in the Raman frequency.
Additionally, it shows great selectivity for melamine when exposed to
other potentially interfering milk components. The limit of detection is
1.0 × 10− 6 µM, which is significantly lower than the levels approved by
the Food and Drug Administration (FDA) and the USA [69].An inte­
grated portable system has been developed using a transfer pipette for
flow injection, using a SERS sensor based on basil seeds and a plastic
container. Using the transfer pipette, a tiny amount of liquid sample is
put onto the dry basil-seed based SERS sensor. The dried basil seed can
function as a sponge to hold the liquid sample, which allows the plas­
monic AgNPs to remain in close contact with the analyte-containing
liquid sample and increase the sensitivity of the instrument. The trans­
fer pipette will subsequently discharge the surplus liquid sample from
the plastic container, leaving the basil-seed-based SERS substrate
exposed to the air. This process avoids the laborious task of removing
melamine from milk and reduces interference from the opaque liquid
sample on the SERS signal. The pipette-basil-seed based SERS system can
be used to detect melamine in milk with a detection limit of 0.85 μM and
methyl parathion in orange juice with a detection limit 0.1 μM [70]. A
portable pipette-Ag-NPs@basil-seed device have many advantages such
as low-cost, safe, disposable, and usable, making it deployable in the
field for the detection of food additive and pollutants.
10
Sensors and Actuators Reports 5 (2023) 100159
Once again, in 2017, a portable SERS based array was developed
using AgNPs coated on surface of cellulose paper. The paper was treated
with hexadecenyl succinic anhydride to provide a hydrophobic top
layer, which can concentrate hydrophobic analyte like melamine by
preventing the mostly aqueous sample from spreading across the paper,
thus enhancing the detection sensitivity. Afterward, a SERS sensor array
has been used to produce patterns with AgNPs on hydrophobic paper
using the pen-writing method. The end product array exhibits excep­
tional Raman scattering amplification, as shown for the fluorescent dye
Rhodamine 6G, with a limit of detection 8.0 × 10− 5 µM without fluo­
rescence interference. The pen written array can detect 2.1 µM of mel­
amine in milk and yogurt [71].
A unique one-dimensional silver nanostructure has been designed for
melamine detection, using the inorganic polymer silver cyanide (AgCN).
The high-yielding Ag nanorods (NRs) are developed from well-known
–[Ag-CN]- chains of polymeric AgCN at room temperature. This hap­
pens due to the colour shift from yellow to orange, to red and ultimately
to green. The as-synthesised Ag NRs preserve the parental 1D shape of
AgCN. As a result, SERS is effectively conducted using the Ag NRs to
detect melamine, down to picomolar levels. The stronger SERS signal
observed at 1327 cm− 1 for lower melamine concentrations of 0.001 and
1.0 £ 10− 6 µM indicates that the stretching of C-N (carbon-nitrogen)
bond of melamine preferentially interacts with the surface of silver ions.
However, the presence of the ’-NH2’ group and ring ’N’ on the melamine
molecule, attached to the AgNPs surface, suggests an adsorptive
behaviour at higher concentration range 1 × 103 to 0.1 µM [72]. Thus,
the melamine binding to the Ag surface supports its fluxional (non-rigid
molecules undergo dynamics in which their atoms change positions at
symmetry-equivalent angles) behaviour.
Hui and co-workers developed a simple and cost-effective assay for
melamine detection using Ag nanoparticles as a SERS substrate. The
nanoparticles were prepared by salt reduction in an aqueous solution at
room temperature without the surfactant addition. The effect of
different morphologies of AgNPs on the SERS enhancement factor for
melamine was checked. The enhancement factor (EF) factor reached
nearly 106 for highly surface-roughened particles [73]. Although, mel­
amine was successfully measured using a SERS-based assay. The linear
ranges and limits were not clearly defined. Hence, more efforts are
required for melamine detection.
Gold nano spheres embedded monolith conjugates are capable of fast
detection of melamine traces in milk. To produce the monolith, a silicon
wafer covered with gold was used for the polymerization of glycidyl
methacrylate (GMA), ethylene dimethacrylate (EDMA), cyclohexanol
(porogen), and 2, 2-dimethoxy-2-phenylacetophenone (a photo-
initiator). Three different types of gold nanoparticles, including Gold
Nano Spheres (GNSs), Gold Nanorods (GNRs), and Triangular Gold
Nanoprisms (GNPrs), were mounted on monolithic surfaces and exam­
ined using the signal molecule Rhodamine to determine the impact of
the monolith on SERS signal activity (R6G). The maximum enhancement
factor was calculated for the three shapes incorporated with monoliths
to monitor their Raman enhancement efficiency. Gold Nano Spheres
integrated into a GMA-EDMA Monolith Sensor (GNS@GEMS) were
proposed for melamine sensing in commercial milk, since GNSs were the
most effective of the three morphologies for the detection of R6G. The
limit of detection for the assay was found to be 0.87 µM and was ach­
ieved within 10 min. This approach complies with the FDA tolerance
limit in the USA and China 7.29 μM [74].
Active silver nanosol (AgNP) synthesised from AgNO3 and sodium
citrate as a reducing agent was used as a substrate for Surface enhanced
Raman scattering (SERS). In the presence of AgNPs substrate, urea in­
teracts with dimethylglyoxime to create 4, 5-dimethyl-2-imidazole ke­
tone, resulting in a significant increase in the intensity of the SERS peak
observed at 1320 cm− 1. A linear increase in SERS intensity was found
with increasing urea concentration from 0.008 to 0.825 µM with a
detection limit of 0.005 µM and a recovery rate of 97.4-101% [75].
Using the SERS effect of silver film over nanospheres (AgFON), a direct
Himshweta and M. Singh
approach for detecting melamine in newborn formula milk powder so­
lution was developed. Vacuum magnetron sputtering and dip-coating
techniques were used to create AgFON. Without any pre-treatments
steps, the SERS spectra of melamine in a newborn formula milk pow­
der solution were directly detected using AgFON as a substrate. The
electric field distribution of AgFON’s was modelled using a finite dif­
ference time domain solution, which demonstrated that the high-density
hot spots of the substrate were responsible for the significant increase in
Raman signals. The silver film over nanospheres showed good
enhancement ability and good stability [76].
A hydrophobic-SERS based direct-droplet platform for melamine
detection was developed using functionalized commercially available
filter paper and AgNPs. This innovative SERS substrate not only meets
the requirements for easy and large-scale preparation but also enables
direct droplet detection with a reusable property [77]. Additionally, the
technique was effectively used to find melamine in milk at a concen­
tration of 5.15 µM after dilution, with an instrument detection limit
(IDL) of just 7.92 µM in a linear range from 7.92 – 0.792 × 104 µM.
Furthermore, this SERS substrate could be effectively used in other areas
such as pesticide (thiram) and dye (malachite green) detection in real
environment.
Urea and ammonium sulfate are commonly used in food adulteration
to increase the nitrogen content of foods such as milk, which is often
used as a measure of protein content. Urea is used to artificially increase
the protein content of milk, while ammonium sulfate is used to dilute
milk and increase its volume. Both of these practices are illegal and can
have negative health consequences for consumers. Detection methods
such as surface-enhanced Raman scattering (SERS) can help identify the
presence of these adulterants in food products. In the current study, level
of urea and ammonium sulphate in milk was simultaneously determined
using SERS based on AuNPs and silver-coated gold nanoparticles
(Au@AgNPs). Ammonium sulphate at a concentration of 0.38 µM and
urea up to 830 µM can be detected using AuNPs and bimetallic core
shelled nanoparticles in combination with the coffee ring effect [78].
According to this study, SERS based on the coffee ring effect has the
potential to be further utilised for identifying other hazardous and
prohibited adulterants in milk and milk products. Therefore, bi-metallic
nanoparticles can be used for real time examination of milk adulterants.
SERS also offers quick detection of sodium thiocyanate (STC) and ben­
zoic acid (BA) preservatives in liquid milk using by cysteamine func­
tionalized core shelled nanoparticle (Au@Ag-CysNPs). STC
concentrations from 6.17 to 120 µM and BA concentrations from 120 to
1.97 µM in milk samples were detectable using a spectrum covering the
350-2350 cm− 1 region. The results demonstrated that Au@Ag-CysNPs
could detect STC up to 0.37 µM in the milk sample, with a limit of
quantification 0.48 µM and a coefficient of determination (R2) of
0.9833. Up to 80.25 µM could be detected for BA with a quantification
limit of 83.52 µM and R2 value of 0.9903 [79].
Given their low cost, high productivity, and outstanding detection
capability, SERS-active substrates are widely employed in food safety
and environmental monitoring. A high performance three-dimensional
(3D) plasmonic structure has been developed as a SERS substrate for
melamine detection. A 3D biomimetic SERS substrate has been created
by depositing AgNPs onto cicada wing bioscaffold arrays using laser
molecular beam epitaxy. This approach allowed for the synthesis of high
density of hotspots by depositing a large number of nanoparticles, with
an average diameters of 10 nm and nanogaps of sub-10 nm onto the
surface of chitin nanopillars. The constructed 3D Ag/cicada SERS sub­
strate shows 0.1 µM limit of detection for Rhodamine 6G, a higher
enhancement factor of 1.09 × 105, and outstanding signal uniformity of
6.8%. Furthermore, the molecular fingerprints of melamine in baby
formula has 79.28 µM limit of detection [80].
A proposed approach for the real-time assay of melamine involves
the application of a highly uniform gold (Au)-capped Si nanopillar as a
SERS substrate in combination with electrochemistry. This
electrochemical-SERS platform act as a single chip, offering higher
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Sensors and Actuators Reports 5 (2023) 100159
sensitivity, real-time calibration of SERS assays, and reusability of SERS
chips. The detection limit achieved were 0.079 µM in phosphate buffer
saline (PBS) and 2.37 µM in milk, and found below the maximum
permitted levels [81]. It was observed that electrochemically active
analyte-surface interaction significantly improves detection sensitivity.
A dual-mode readout sensing system that combines colorimetric
analysis with SERS can effectively distinguish between actual and false-
positive signals of melamine in milk. The colorimetry analysis enables
the plasmonic nanoparticles to change colour in the presence of mel­
amine. The silver colloids, with a diameter of 20 nm, proved to be
effective for both colorimetric and SERS techniques. However, the
presence of interfering substances may cause false-positive results when
using the colorimetric approach. This limitation can be overcome with
SERS spectroscopy, which direct and unaltered acquisition of signature
spectra from the same plasmonic nanoparticles utilised in the colori­
metric test. The limit of detection for melamine in milk was 0.4 µM [82].
Therefore, the colorimetric and SERS approach not only enables quick
preliminary screening of melamine with the naked eyes but also lowers
false-positive signals significantly via surface selection methods.
Accurate and selective detection of urea in dairy products is essential
for maintaining quality control and promoting health applications. To
achieve ultra trace urea assay, a unique aptamer (Apt) turn-on tri-mode
approach was developed. The Fe/N-codoped carbon dots (CDFeN) were
produced hydrothermally using ammonium ferric citrate as a precursor.
The oxidation of 3, 3′ , 5, 5′ -tetramethylbenzidine (TMB) by H2O2 is
strongly catalysed by CDFeN, resulting in the formation of an oxidised
tetramethylbenzidine (TMBox) probe with surface-enhanced Raman
scattering, resonance Rayleigh scattering, and fluorescence (SERS, RRS,
and FL) signals at 1,598 cm− 1, 370 nm, and 405 nm, respectively. The
urea aptamer can inhibit the indicator reaction, thereby reducing the tri-
signals. While, the addition of urea activates the indicator reaction,
resulting in a linear increase in SERS/RRS/FL intensity, with the
detection limit for fluorescence and SERS were 0.005 µM and 0.001 µM,
respectively [83]. Moreover, the catalytic Apt-CDFeN platform can also
detect trace amount of adenosine triphosphate and estradiol. This
method is simple in operation and demonstrates higher sensitivity for
urea detection than previously reported SERS, fluorescence and elec­
trochemical methods.
To create silver nanostructures (AgNS) on filter paper: P1, 0.01; P2,
0.1; and P3, and 0.2 M of AgNO3 (silver nitrate) for SERS substrates were
used. The in-situ one-step silver mirror reaction (SMR) was used for
detection of melamine and urea. The AgNS has grown in a
concentration-dependent manner, leading to the development of com­
plex structures. The limit of detection for melamine and urea in milk
samples was found to be 0.169 × 104 and 480 µM, respectively [84]. As a
result, the paper-based SERS substrate can identify on-site chemical
contaminants in water and milk samples, exhibiting good selectivity and
stability for upto 4 months.
SERS is widely recognised as an ultrasensitive method for detecting
trace molecules. However, creating a sensitive and stable SERS substrate
remains a significant challenge for practical applications. In this study, a
hexagonally packed silver metasurface on SiO2-Au-coated silicon (Ag/
SiO2/Au/Si) substrate that act as metal–insulator–metal (MIM) SERS
substrate has been developed. The etching process of a self-assembled
nanosphere mask alters the geometry of Ag metasurface on the SiO2/
Au/Si substrate, which is responsible for wavelength matching of optical
absorbance and excitation laser wavelength between Ag/SiO2/Au/Si
substrate. Additionally, an enhancement of Raman signals was
observed. The SERS performance for melamine was significantly
improved by spin-coating a thin layer of graphene oxide on the sub­
strate, greatly increase the enhancement factor of 1.1 × 105 and a limit
of detection of 0.001 µM [85].
2.1.3. Fluorescence sensors
Fluorescence assays use several analytical parameters such as, the
intensity, wavelength, fluorescence lifetime, and fluorescence
Himshweta and M. Singh
anisotropy of emission. However, various factors like changes in pH,
charge, polarity, or viscosity can interfere with the driving signals.
Fluorophores such as organic dyes, carbon and graphene quantum dots
(CDs and GrQDs), and semiconductor quantum dots (QDs) are used in
fluorescent sensors. These sensors have gained popularity due to simple,
accessible, highly sensitive, high-output, fast response, and minimal
background noise. The limitation of these methods is that they require
specific instrumentation, which may not be economically feasible [86].
Fluorescence sensors exhibit a phenomenon known as fluorescence
resonance energy transfer (FRET). This non-radiative process occurs
during energy transfer between donor molecule in an excited state to an
acceptor in a ground state by dipole-dipole interactions [87]. This
method has been widely used in various fluorescence applications such
as medical diagnostics, optical imaging and sensing. The FRET system is
based on three features: the orientation of donor and acceptor; the dis­
tance between acceptor and donor; the extent of spectral overlap be­
tween the emission (donor) and absorption (acceptor) spectrum.
Quantum dots, organic dyes and metal nanoparticles act as fluorescent
probes for the detection of milk adulterants [64]. Gold nanoparticles
exhibit greater quenching efficiency, stable and tunable optical prop­
erties, as well as greater energy transfer distance than the Forster dis­
tance (R0). In recent times, FRET assays have commonly been applied for
detection of analytes that can bind onto the surface of gold nano­
particles. When gold nanoparticles are functionalized with specific
molecules, they can act as energy acceptors and can be used to modulate
the FRET signal. This makes FRET assays highly sensitive and specific for
detecting the binding of analytes to the gold nanoparticle surface.
Additionally, FRET assays can be designed to detect a wide range of
analytes.
2.1.3.1. Metal nanoparticles based sensor. Nanoparticles are often used
for the fluorometric detection of melamine in milk samples in two ways:
they are used as direct fluorescent probes or are attach/immobilize with
antibodies/aptamers. The inner filter effect (IFE) of gold nanoparticles
(AuNPs) on the fluorescence of CdS quantum dots (QDs) can be used as
fluorescence labels for detection of melamine in raw milk. The L-
cysteine (L-Cys)-capped CdS QDs fluorescence is noticeably reduced by
AuNPs through IFE in the presence of citrate-stabilized AuNPs. When
melamine is added, the fluorescence of the AuNPs-CdS QDs system is
restored because melamine causes AuNPs to aggregate and decrease in
plasmon absorption at 522 nm, which "turned on" the CdS QDs IFE-
fluorescence. The detection limit of 0.13 µM was obtained, which is
substantially lower than the detection limit of HPLC method 3.17 µM
coupled with a UV detector [88]. In this work, a FRET system between
AuNPs and amino-functionalized carbon dots (C-dots) has been devel­
oped. Here, AuNPs serve as energy acceptors while C-dots serve as en­
ergy donors. Melamine is identified using the fluorescence intensity of
C-dots under optimum experimental conditions with a detection limit of
0.036 µM and a response time of 5 min [89].
The electrostatic interactions between AuNPs (negatively charged as
acceptor) and upconversion nanoparticles (UCNPs positively charged as
donor) lead to the aggregation of AuNPs in the presence of melamine.
This enhances the fluorescence intensity of UCNPs. Under optimum
conditions, melamine can be detected with detection limit of 0.018 µM,
in 12 min of incubation time in raw milk [90]. Compared to earlier
studies, this approach shown significant benefits, such as low cost,
simple operation and efficiently avoid the background interference.
Gold nanoclusters (AuNCs) offer attractive features due to their
distinct electrical and optical characteristics, such as fluorescence,
tunable size, surface chemistry and aggregation state. As a result, AuNCs
have potential applications in various fields, including biosensors, bio­
imaging, nanobiotechnology, medicine delivery, and diagnostics. A
novel nanosensor made up of glutathione stabilised AuNCs to detect
melamine in cow milk has been developed. The hydrogen bond in
melamine and AuNCs causes the nanoclusters to aggregate. In the
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Sensors and Actuators Reports 5 (2023) 100159
presence of melamine, the fluorescence intensity increases at 430 nm,
leading to “turn-on” effect. However, the induced aggregation and
subsequent emission quenching decrease the intensity to 610 nm.
Despite the presence of interfering molecules such as alanine, glycine,
glucose and cysteine, the current probe successfully detected 28.2 µM
melamine in cow milk [91]. Carbon dots can act as reducing and sta­
bilising agent for silver ions nanoparticles, enabling them to be com­
plexed with silver nanoparticles as a fluorescent probe for the detection
of melamine in raw milk. The intrinsic fluorescence emission of C-dots is
dramatically decreased by melamine, as the silver ions interact with
nitrogen atoms and triazine groups of melamine. The visual detection of
melamine by UV-Vis absorption and fluorescent method has a low
detection limit of 0.03 µM, as well as good selectivity [92].
Polydopamine-glutathione nanoparticles (PDA-GNPs) can be used as
reducing agent for the synthesis of silver nanoparticles (AgNPs). Here
the PDA-GNPs act as energy donor, while the AgNPs serve as acceptor
molecule. Upon interaction with melamine and Ag (I), the fluorescence
of PDA-GNPs is reduced, leading to the formation of Ag (I)-melamine
complex. This complex prevents the synthesis of AgNPs but enhances the
fluorescence intensity. The fluorescence enhancement efficiency ex­
hibits a detection limit of 0.023 µM for melamine sensing. The suggested
approach is simple, time-saving, low-cost, and has been successfully
employed to detect melamine in real milk products [93]. Melamine in
milk can be evaluated visually using fluorescent gold
nanoparticles-carbon quantum dots nanocomposites (Au-CQDs). With
an increase in melamine content, the Au-CQD fluorescent emission can
be improved achieving a limit of detection 0.0036 µM. The fluorescence
standard array and smartphone are used to visually determine the
approximate concentration of melamine adulteration in milk samples
[94]. However, rapid detection using ds DNA templated copper nano­
particles, requires specialised detectors such as photomultiplier tubes
due to the weak fluorescence emission. This restricts their applications
for in situ analyte detection, which uses cost effective and more sensitive
detectors. This paper reports the increase in fluorescence properties of ds
DNA templated copper nanoparticles by using a mixture of sodium
ascorbate and Taq buffer. The fluorescence signal intensity of ds
DNA-templated copper nanoparticles increases 11 times, and the
maximum fluorescence signal is reached 4 times faster with this method.
The method shows detection limit 0.79 µM for melamine in milk sam­
ples. Compared to the conventional approach, the fluorescence
enhancement technique also yields 2.95 times higher sensitivity for
melamine detection [95]. Barium sulfate-coated CsPbBr3 perovskite
nanocrystals (CsPbBr3 NCs@BaSO4) are highly stable and exhibit
greater florescence in aqueous buffer. An ultrasensitive fluorescence
nanosensors was constructed for turn-on detection of melamine, using
the CsPbBr3 NCs@BaSO4 as signal readout and the modulation of the
inner filter effect of citrate-protected AuNPs. The fluorescence of the
CsPbBr3 NCs@BaSO4 was significantly reduced by the AuNPs due to the
inner filter effect. Melamine-induced aggregation of the AuNPs leads to
a reduction in the inner filter effect, allowing for the recovery of fluo­
rescence in CsPbBr3 NCs@BaSO4 and a detection limit of 4.2 × 10− 4 µM
in dairy product samples was achieved [96]. This strategy can be used to
indirectly detection of non-fluorescent species by using perovskite NCs
as signal readouts.
Fluorescent dsDNA templated copper nanoparticles have gained
attention due to their wide range of applications in detecting various
analytes. A portable handheld dsDNA templated copper nanoparticles
based fluorometer is a preferable choice over the conventional methods,
as it can detect analytes in lower concentrations. This device uses an
Ultraviolet Light-emitting diode (UV LED) for illumination and a posi­
tive intrinsic negative (PIN) photodiode for detection, both of which are
low-cost and readily available components used in the construction of a
portable fluorometer. The devised system can detect melamine in milk
samples up to a concentration of 0.79 µM [97]. To determine the pres­
ence of melamine or melamine-Cu(II) in milk products, the AgNC53-­
based "label-free" fluorescence technology is utilized. A modified
Himshweta and M. Singh
C3A-rich aptamer as a template is used to synthesize a novel luminous
DNA silver hydrocolloid (AgNC53), for detecting melamine and
metal-melamine species in milk products. The emission of AgNC53 at
617 nm is sensitive to both melamine-Cu (II) and melamine, but it does
not respond to free Cu (II) ions. The AgNC53 is used to precisely detect
the melamine concentration in milk, with a detection limit of 0.027 µM
[98]. Metal organic framework (MOFs)-based sensors have expanded
the applicability of nanomaterials in melamine detection for the first
time, surpassing previously reported carbon dots, quantum dots, metal
nanoclusters, nanocomposites, and other fluorescence sensors. Specif­
ically, a zirconium-based metal–organic frameworks (Zr-based MOFs) is
coated by tris (2, 2-bipyridyl) ruthenium (II) chloride hexahydrate ([Ru
(bpy)3]2+) composite, termed as UiO-66-NH2@Ru, was synthesized and
evaluated for its ability to detect melamine using a ratiometric fluores­
cence technique. The solvothermal approach was used to synthesise
UiO-66-NH2@Ru, which showed dual emission signals at 445 and 595
nm upon a single excitation at 350 nm. The probe demonstrated a
detection limit of 0.09 µM. The synthetic UiO-66-NH2@Ru probe
quickly determined the presence of melamine in milk powder with great
sensitivity and selectivity [99]. This work indicates the potential of
Zr-based MOFs as a substrate to encapsulate fluorescent dye for devel­
oping ratiometric sensors for the detection of melamine.
2.1.3.2. Quantum dots based sensors. Quantum dots, such as ZnS and Cd,
are semiconductor nanomaterials that exhibit unique fluorescent prop­
erties, such as higher quantum yield, size dependent spectrum, broad
excitation and narrow emission range, longer fluorescence time and
higher photostability. Due to these features, quantum dots are prefer­
ably used as fluorescent probes for sensing target analytes. Different
reports have been published on the monitoring of analytes with fluo­
rescent probes, because they strongly enhance or quench the fluores­
cence intensity according to presence or absence of analyte. Based on
charge transfer quenching of the fluorescence for graphene quantum
dots (GQDs) in the presence of Hg2+, a fast fluorescence detection device
for melamine has been proposed. The synthesised GQDs have mostly
aromatic sp2 domains and were strongly luminous. Melamine interacts
with mercury through nitrogen atoms in its amine and triazine groups,
bringing more Hg2+ to the surface of GQDs, hence reducing the GQDs
fluorescence. The quenching occurs due to the charge transfer from the
GQDs to Hg2+, with melamine acting as the linking agent. This sensing
method has potential applications since the melamine showed a detec­
tion limit of 0.12 μM, which is significantly below the regulation
threshold. The device also exhibited high melamine selectivity in the
presence of potential interferences [100].
There is a high demand for the development of a fluorescent
nanoprobe that allows for high-sensitivity and direct visualisation
enabling rapid analysis. For designing this fluorescent silica nanohybrid,
a unique assembly approach for implanting hydrophobic green and red
quantum dots (QDs) by thiol-metal coordination into silica scaffold was
devised. This nanohybrid exhibited well-preserved fluorescence char­
acteristic of original QDs, as well as robust optical/colloid stability
during alkylsilane mediated phase transition and outer silica shell
development. Combining spectra from two-colour fluorescent nano­
hybrid with analyte-specific gold nanoparticles resulted in the devel­
opment of an inner filter-based nanoprobe for visual measurement of
melamine [101]. Graphene quantum dots (GQDs) are an essential
member of the fluorescent compounds that exhibit great photostability,
anti-photobleaching, scintillation, strong biocompatibility and low
toxicity. In this work, a fluorescence resonance energy transfer (FRET)
biosensor based on protoporphyrin IX (PpIX) and GQDs was created for
melamine detection. PpIX forms bond on the surface of GQDs to create
self-assembled nanosensors, and the FRET process occurred between
GQDs and PpIX. However, in the presence of melamine, no FRET occurs.
Based on the fluorescence intensity ratio of PpIX and GQDs, the FRET
system could detect 3.6 × 10− 3 µM melamine in milk samples [102].
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Üzek et al. found that coating cadmium sulphide quantum dots (CdS
QDs) on molecularly imprinted shell by mini-emulsion polymerisation
produced specific recognition sites on the surface of nanocomposite for
melamine detection, greatly lowering the detection limit to 1.11 µM.
The same nanocomposites also showed excellent reusability perfor­
mance for 20 cycles. So, the nanosensor technology is a good alternative
to existing melamine detection technologies [103].
2.1.3.3. Dye based sensors. Over the last decade, self-assembled fluo­
rescent nanoparticles based on small-molecule organic dyes have gained
prominence in imaging and sensing applications. This is mainly because
they can combine the spectral characteristics, tunability, and biocom­
patibility of organic fluorophores with the brightness, chemical, and
colloidal stability of inorganic materials. The abundant flexibility of dye-
based nanomaterials allows for the creation of distinctive characteris­
tics, ranging from small molecular aggregations to complex core-shell
nanoarchitectures with hyperbranched polymers. However, the syn­
thesis of efficient organic dye based nanoparticles, fluorescent molecules
typically do not perform well, even in highly concentrated solutions, or
much less in aggregates. They are particularly susceptible to self-
quenching due to processes like reabsorption and energy transmission
[104]. Recently, AuNPs-based fluorescence resonance energy transfer
(FRET) assays have been developed for the study of small organic
compounds, such as melamine, which have a propensity to bond to the
AuNPs surface and interact with dyes to adsorb onto gold surfaces. It has
been suggested that dyes (such as fluorescein, rhodamine B, etc.) can be
electrostatically deposited onto the surface of raw or functionalized
AuNPs. However, this leads to a significant reduction in fluorescence
through FRET between the dyes and the AuNPs. Due to the competitive
adsorption of dyes and analytes on AuNPs surface, the fluorescence of
the dyes would reappear in the presence of targets [64].
In this study, single-stranded DNA was used as a matrix for the
synthesis of blue-emitting silver nanoclusters (AgNCs) via ambient hy­
drothermal process. Sodium tetrahydridoborate (NaBH4) served as the
reducing agent, whereas DNA was used as a stabiliser or coating agent.
The interaction between Rhodamine 6G (Rh6G) and the synthesized
DNA-AgNCs resulted in the formation of a self-assembled complex of
DNA-AgNC and Rh6G. Fluorescence-resonance-energy transfer (FRET)
from AgNCs (donor) to Rh6G simultaneously reduced the fluorescence
emission of AgNCs (acceptor). However, the addition of melamine
caused the self-assembled complex to break down, resulting in an anti-
FRET effect. This caused the emission recovery of AgNCs and fluores­
cence reduction of Rh6G in the DNA-AgNC-Rh6G complex. A good
linear relationship between ratiometric fluorescence intensities against
melamine concentration from 0.1-10 µM, with a detection limit of 0.025
µM was achieved. Ratiometric fluorescence intensity IAgNCs/IRh6G
response to melamine were selective relative to interfering molecules.
Based on the DNA-AgNC-Rh6G complex, a unique dual-emitting ratio­
metric fluorescence sensor for melamine has been developed. Specif­
ically, this sensor can visually detect melamine fluorescence in both
aqueous solutions and on wet filter paper [105].
For the designing of optical probes, a unique bio-inspired nano ar­
chitectonics methodology has been used. It is based on nanodevices that
combine an enzyme receptor subunit, a signalling subunit, and a
mechanism of communication between the two locations, based on the
synthesis of chemical messengers by the enzymatic subunit to detach
reporter molecules from the silica surface. An Alexa Fluor 647-labeled
oligonucleotide-based urea nanosensor has been created using enzyme
functionalized Janus gold mesoporous silica nanoparticles (Au-MSNPs).
The urease enzyme is grafted onto gold face, while silica face is Janus
AuMSNPs functionalized with amino groups to which the labelled
oligonucleotide binds electrostatically. The enzyme-mediated hydroly­
sis of urea to ammonia and subsequent deprotonation of amino groups
on the silica face enable the nanodevice to release fluorescent oligonu­
cleotide. This simple nano device has been used to detect urea in human
Himshweta and M. Singh
blood samples and to identify adulterated milk using fluorimetry [106].
With a wide variety of enzymes and reporter species to choose from, this
concept can be applied to the design of various optical probes, making it
a suitable method for food and biochemical analysis.
Radical polymerization is a process used to synthesize polymers,
which involves the formation of free radicals that initiate and propagate
the polymerization reaction. This type of polymerization typically oc­
curs under high temperatures and pressures, and uses monomers that
contain a double bond, such as vinyl monomers. The applications of
radical polymerization are vast and varied, including the production of
plastics, adhesives, coatings, and composites. One example of the
application of radical polymerization is the construction of a melamine-
selective acrylate citric acid (ACA) based polymeric membrane sensor.
This sensor displayed a focused fluorescence reaction to melamine (Ex/
Em = 388/425 nm) with a response time of about 1 minute. The limit of
detection and limit of quantitation were determined to be 2.32 × 10− 4
and 8 × 10− 4 µM, respectively [107]. This demonstrates how radical
polymerization can be used to create innovative sensors with specific
target recognition, making it a valuable tool in various fields, including
food and environmental monitoring.
2.1.4. Colorimetric sensors
Colorimetry is a simple and cost effective method of visual detection
that has received significant attention due to its simplicity in operation,
equipment, and higher sensitivity compared to methods. Label-free
detection of melamine can be achieved using AgNPs which aggregate
in the presence of melamine, resulting in a yellow-to-red colour shift.
Melamine concentrations in raw milk can be detected by naked eyes or a
UV-Vis spectrophotometer. The detection limit for melamine is 2.3 ×
10− 3 µM (3s) [108]. Such colorimetric sensors are termed as “turn off”
sensors because melamine leads to nanoparticles aggregation, which
change the signal intensity and therefore can be easily visualised. Later,
a new method for detecting melamine at room temperature using
interference biosynthesis of silver nanoparticles was developed. The
presence of melamine inhibits or partially synthesizes nanoparticles,
leading to a colorimetric change that is detected spectrally. The limit of
detection was 3.96 µM in raw milk. The sensing was performed with
caffeic acid as a reducing agent, and the role of caffeic acid in interfer­
ence biosynthesis-based sensing was confirmed [109].
Gold nanoparticles (AuNPs) possess unique physical and chemical
properties such as easily-tunable optoelectronic potentials, outstanding
biocompatibility, high extinction coefficient, and exceptional adapt­
ability for multi-functionalization. AuNPs are ideal option for the con­
struction of detecting sensors due to these distinctive characteristics.
AuNPs-based colorimetric assays are valuable because the molecular
recognition events can easily be converted into colour changes that
result from the inter-particle plasmon coupling during AuNPs aggrega­
tion (red-to-purple or blue) or re-dispersion of AuNPs aggregate (purple-
to-red). Unmodified AuNPs can detect melamine in raw milk, and the
colour shift from red to blue (or purple) can be seen by naked eye
without specialised apparatus. The limit of detection for melamine in
raw milk is 0.56 µM [110].
In addition to silver nanoparticles based sensors, β–cyclodextrin
functionalized silver nanoparticles, were used as probes for colorimetric
sensing of melamine. The monodispersed and spherical-shaped silver
nanoparticles had an average particle size of 10 nm, and the addition of
melamine cause nanoparticle aggregation. The hydrophobic cavities of
β-cyclodextrin-functionalized silver nanoparticles facilitated host-guest
interactions with melamine, resulting in efficient detection of mel­
amine at a concentration of 4.98 µM by the colorimetric reaction in
pasteurized milk samples [111]. Although, this method can be used for
real time monitoring of melamine, it requires pre-treatment steps that
increase the analysis time.
This method offers a unique economical approach for designing
bioactive enzyme-zinc ferrite (urease/ZnFe2O4) nanocomposites in a
single pot and single step by UV-Vis method. Urease molecules are
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immobilised by original ZnFe2O4 nanoparticles via electrostatic
adsorption and covalent conjugation during ZnFe2O4 growth. As a
result, the bioactive urease/ZnFe2O4 nanoparticle composites are
formed. This approach is anticipated to give the nanomaterials with new
features for various intriguing applications. The urea calibration curve
in milk shows a linear response of 4 × 103 to 8.0 × 104 µM [112].
Although, the method is simple in operation, its sensitivity has not been
measured, making it unsuitable for real time monitoring of urea in milk.
The peroxidase-like activity of bare AuNPs can be increased by the
addition of melamine, which causes the AuNPs to aggregate in presence
of melamine. The bare AuNPs help to catalyse 3, 3′ , 5, 5′ -tetramethly­
benzidine (TMB)-H2O2 reaction, causing the significant enhancement in
absorbance for colorimetric reaction. Melamine in milk and milk pow­
der were detected using UV- Vis spectroscopy with a detection limit of
2.0 × 10− 6 µM and with naked-eye detection at 5.0 × 105 µM was found
in raw milk and milk powder [113]. Furthermore, the proposed method
may improve the peroxidase-like activity of Au NPs, which could pro­
vide an idea for developing effective nanomaterials-based mimetic en­
zymes, and applications for Au NPs in different fields.
To detect melamine, an aptamer is used as a recognition element and
AuNPs as a colour indicator, allowing for on-site detection using naked
eyes or a UV-Vis spectrometer. The suggested approach was used to
detect melamine in whole milk, with a detection limit of 11.89 µM using
naked eyes and 3.9 µM using a UV-Vis spectrometer [114]. Similarly,
AgNPs were successfully synthesized from leaves extract of Jatropha
gossypifolia. These biofunctionalised AgNPs in size range between 18-30
nm, act as a colorimetric probe and detect melamine up to 2 µM in raw
milk [115].
Many articles have been reported for melamine detection, but only
handful methods shown sensitivities below 0.05 µM. One such method
involves the use of sulfanilic acid-modified silver nanoparticles (SAA-
AgNPs), for melamine detection in milk samples. The interaction of
melamine exocyclic amine group with SAA causes fast aggregation of
SAA-AgNPs, accompanied by a visible colour shift with naked-eye,
resulting in quantification of melamine using a simple UV-visible spec­
trometer. This method has a detection limit of 0.01 µM, which is below
the safety limits [116]. Another simple method, using H2O2-Au nano­
particles based technique is studied for melamine detection in dairy
products using naked eye or with a UV-Vis spectrometer. Melamine can
be detected as 400 µM by visually and UV-Vis spectrometer [117].
Citrate-capped AuNPs of different sizes 15 nm (AuNPs-I), 30 nm (AuN­
Ps-II), and 40 nm (AuNPs-III) are effective for melamine detection. In the
presence of melamine, the AuNPs aggregated in aqueous solution,
causing a visible colour shift from red to blue. This colour change caused
the absorption peak for AuNPs-I, AuNPs-II, and AuNPs-III to move from
527 nm, 526 nm, and 525 nm to 638 nm, 626 nm, and 680 nm,
respectively. The AuNPs-II showed lowest detection limit for melamine
as 0.0237 µM, AuNPs-I (0.0337 µM) and AuNPs-III (0.0897 µM),
respectively. Therefore, AuNPs-II was selected for on-site melamine
detection in pre-treated milk samples [118].
Xin and co-workers designed simple field-portable colorimetric
technique to detect melamine in liquid milk. Methanobactin (Mb) has
the potential to reduce Au (III) to Au (0) and hence enable the Au-NPs
synthesis. However, the interaction of melamine with oxazolone ring
of Mb, interferes the Au-NPs synthesis process. Melamine also promotes
the aggregation of Au-NPs, enabling detection of melamine in liquid
milk using naked eyes with a detection limit of 5.56 µM. The detection
limit is further improved to 0.24 µM sing the UV-Vis method [119]. In
the presence of melamine, the sodium D-gluconate stabilised silver
nanoparticles agglomerate due to hydrogen bonding between sodium
D-gluconate and melamine, resulting in a colour shift. UV-Visible
spectroscopy is used to monitor the aggregation-induced colour shift
from yellow to red. The detection limit is determined to be 0.5 µM, and it
is also used for real-time sample analysis [120]. In another study, a
colorimetric method based on silver nanoparticles has been proposed for
the detection of melamine in milk. This approach is based on the idea
Himshweta and M. Singh
that melamine induces silver nanoparticles to aggregate, resulting in a
sharp shift from yellow to red. By analysing the silver nanoparticles
absorption spectra with an UV-Vis spectrometer, it is possible to mea­
sure the amount of melamine present in an adulterated sample. The
current colorimetric approach, uses silver nanoparticles of 35 nm, and
can detect melamine of 0.32 µM concentration [121].
Using graphene oxide (GO) and 2,7 -dichlorfluorescein diacetate
(DCFH-DA), Nanda and co-workers devised an analytical technique for
measuring H2O2 in milk. Under ultraviolet illumination at 365 nm, the
method involves placing 1000 µL aliquots of 10-fold diluted samples of
high and low-fat milk onto 100 µL of GO and 100 µL of 100 M DCFH-DA
produced green colour. The suggested approach shown detection limit
of 1 × 107 µM and acceptable sample recovery values [122]. Due to the
potential risk it poses to the public health, the detection of H2O2 in milk
is a significant concern. Current methods for detecting H2O2 are time
consuming and require specialised lab equipments. Chemical assays, on
the other hand, are sensitive enough to identify a broad linear range of
H2O2 concentration. The detection sensitivity of the system can be
improved by optimizing experimental conditions and integrating
microfluidic devices, so that the H2O2 can be detected as each event
occurs. Although it may be possible to detect H2O2 with the naked eye,
but it is inherently challenging to precisely quantify its concentration.
Recent years have witnessed a rise in the instances of economically
motivated adulteration (EMA) of milk and newborn milk formula with
toxic substances such urea, melamine and detergents. Typically, deter­
gent is utilized to mix low-cost non-dairy fats to create synthetic milk.
Anionic detergents (ADs) are frequently employed for this purpose. The
interaction of detergents with cell membrane proteins leads to the for­
mation of micelles, which can cause damage to the cell membrane. As a
result, toxic effects such as acute renal failure, cardiac dysfunction,
hemolysis, ventricular tachycardia, and coagulation dysfunctions can
occur [123]. Thus it is important to detect any adulterations in food
products for safety purposes. A new method has been developed for
detecting ADs in milk, which utilizes the optical and spectral charac­
teristics of gold nanoparticles (AuNPs). In the presence of AD, the salt
induced aggregation of AuNPs is prevented as AD shields the gold
nanoparticles and produces a red shift in surface plasmon resonance
absorption. The technique is unique for AD detection and remains un­
affected by other adulterants. The limit of detection for ADs such as
sodium dodecylbenzenesulfonate (SDBS) and commercial ADs was
found 66 µM and 319 µM, respectively, in milk [123].
The synthesis of stable colloidal silver nanoparticles with a mean
diameter of 14.0±2.7 nm can be achieved using sodium borohydride as
the reducing agent without the use of a stabilising agent. The spectro­
photometric analysis at 475 nm revealed a linear range of melamine
concentrations from 0.26 to 11.89 µM with a limit of detection of 0.07
µM [124].
Melamine toxicity has gained worldwide attention as it causes renal
failure and mortality in people. A colorimetric detection of melamine in
milk samples has been developed by in-situ production of AgNPs from
tannic acid. Melamine can be selectively detected in vitro at concen­
trations ranging from 0.05 to 1.4 μM with a limit of detection 0.01 μM,
which is lower than the melamine safety criterion of 7.29 μM [24]. The
current approach is highly selective, with no noticeable interference
from other chemicals such as urea, glucose, glycine and ascorbic acid.
This approach does not require organic co-solvents, enzymatic pro­
cesses, light sensitive dye molecules, and specialised equipment, which
addresses some limitations of other traditional methods.
The green method based colorimetric sensing of melamine, bio­
polyphenols (rutin and curcumin) were used as reducing and stabilising
agents for synthesizing AgNPs. However, the presence of melamine in
the reaction medium prevents the nanoparticle synthesis. This may be
due to the interaction of melamine with Ag+ ions and biopolyphenols. A
pale red coloured solution was produced, due to the formation of
aggregated AgNP masses at low melamine concentrations. However, at
high melamine concentrations, a colourless solution was produced,
15
Sensors and Actuators Reports 5 (2023) 100159
indicating a disruption in the synthesis of AgNPs. The sensor shows limit
of detection for melamine 0.079 µM and 1.9 µM by Ag-rutin and Ag-
curcumin, respectively. The suggested sensor was successfully used to
identify melamine in raw milk samples [125]. Negatively charged cit­
rate ions create an electrostatic coating on AuNPs, which keeps them
stable and dispersed. Citrate-capped AuNPs modified with Triton X-100
become more resistant to the effects of high ionic strength and a wide pH
range. Aggregation leads to colour shift from wine red to blue and a new
absorption peak at 630 nm. Hence, Triton X-100-AuNPs can detect
melamine at concentrations as low as 0.005 µM. Additionally, by con­
necting with a smartphone, a paper-based quantitative detection system
using this colorimetric probe is possible [126].
Asymmetrically modified large sized AuNPs are synthesized for the
detection of melamine in raw milk. By increasing the size of AuNPs
(from 13 to 42 nm), the sensitivity of colorimetric detection of melamine
by citrate-stabilized AuNPs can be greatly enhanced (limit of detection
by visual observation: from 1.0 × 106 to 0.001 µM). However, AuNPs are
unstable in solution, and the accuracy of aggregation-based colorimetric
detection method is greatly reduced, due to the quick evaporation of
solution colour, induced by the precipitation of AuNPs aggregates after
adding melamine. To overcome this limitation, polyethylene glycol
(PEG as stabilising agent) is added on the surface of AuNPs, to stabilise
the AuNPs. As a result, the addition of melamine to asymmetrically
PEGylated AuNPs causes a colour shift from purple to blue, and colour
change can last for a long period. This is primarily due to the formation
of stable oligomers of AuNPs, which uses the asymmetrical modification
of AuNPs with the PEG chain. The method significantly improves the
long-term stability and detection range, from 0.001 to 1 × 103 µM, in
processed milk, providing a new route for real-time sample detection
[127].
AuNPs stabilised by an unsymmetrical terpyridyl zinc complex,
linked to thymine fragment at one terminal and a quaternary ammo­
nium salt at the other end, provide a colorimetric based visual detection
of melamine in raw milk. Even in the absence of melamine or in the
presence of trace amounts of melamine, a clear colour change from red
to blue was observed with naked eye. The change in AuNP aggregation,
attributed to the selective binding of the thymine fragment and mel­
amine via hydrogen bonding interactions, led to detection limit of 0.019
µM for melamine [128].
In this study, iron platinum-gold (FePt- Au) nanocubes were used as
seeds to create FePt-Au ternary metallic hybrid nanoparticles (FePt-Au
HNPs), which were subsequently mixed with Au(I) precursor using the
hydrothermal method. The FePt-Au HNPs also had outstanding
peroxidase-like activity, which quickly catalyse the oxidation of sub­
strate 3,3′ ,5,5′ tetramethylbenzidine (TMB) to produce a blue colour
that became visible to the naked eye after only 30 seconds. Notably, the
colour response occurred instantly as a result of rapid transfer of elec­
trons between the substrate and H2O2, facilitated by FePt-Au HNPs. A
visual colorimetric sensor for ultrafast detection of H2O2, based on the
catalytic mechanism of fast electron transfer and the intrinsic
peroxidase-like activity of FePt-Au HNP. This sensor had a large dy­
namic range of 20–700 μM. Furthermore, the limit of detection of 12.33
μM was successfully applied to detect H2O2 in milk samples [129]. Liu
and co-workers developed iron-doped copper tin hydroxide (CuSn(OH)6
(Fe/CuSn(OH)6) microspheres using the co-precipitation method. For
the first time, they were selected as a nanoenzyme to detect H2O2, by
employing the peroxidase substrate 3, 3′ , 5, 5′ -tetramethylbenzidine
(TMB), which is catalytically oxidised by H2O2. Fe/CuSn(OH)6 micro­
spheres exhibit high peroxidase-like activity. Colourless TMB may un­
dergo a blue colour shift to oxidised TMB (oxTMB) during the oxidation
process. The Fe/CuSn(OH)6 microspheres have a strong peroxidase
mimicking activity, which allowed researchers to effectively build a
novel and sensitive H2O2 colorimetric detection method. Experimental
findings showed that the Fe/CuSn(OH)6 microspheres nanoenzyme not
only exhibited higher peroxidase-like activity than horseradish peroxi­
dase but also enabled quick detection of H2O2 in the broad range (30 -1
Himshweta and M. Singh
× 103 μM), with a detection limit of 9.49 μM (S/N = 3). Therefore, the
qualitative detection of H2O2 by this approach is a reliable method
[130].
In later study, a method was developed for creating AgNPs based
hybrid material consisting of cellulose nanowhiskers and nano­
structured polysaccharide, as a colorimetric probe for H2O2 detection.
These findings showed that AgNPs frequently undergo catalytic break­
down when exposed to H2O2, resulting in a decrease in AgNPs at 410 nm
in proportion to H2O2 concentration. The limits of detection were found
to be 0.014 μM and 112 μM, respectively, and were linearly dependent
on H2O2 concentration in the ranges of 0.01-30 μM and 60-600 μM
[131].
The H2O2 sensor is easy to use and suitable for real sample analysis,
even in the presence of other interfering compounds. The detection of
milk adulterants using a colorimetric approach and smartphone image
analysis is discussed here. This method involves finding starch, sodium
hypochlorite, and H2O2 in milk by observing colour difference for each
substance. Utilizing lab-created software (PhotoMetrix® and RedGIM®)
and the histograms of the red, green, and blue images, the partial least
squares regression was used to analyse the photos. The smartphone
camera is used to automatically calculate and process the image histo­
grams. The image histograms are handled within the app and auto­
matically calculated using the smartphone camera. The outcomes have
demonstrated its capacity to estimate the concentration of the three
adulterants, highlighting the potential of using digital photos and
smartphone apps in conjunction with chemometric techniques. This
technique offers a quick, inexpensive, and portable solution to measure
adulterants in cow milk [132]. The colorimetric sensor is made of
nanoparticles that replace the naturally occurring enzyme-based sensor.
The zero valent nanoparticles have transformed the field of optical
sensing, particularly because they are simple to prepare, easy surface
modification, and readily shifted electron. Zero valent manganese
nanoparticles (ZV-Mn NPs) are synthesized using an easy approach, and
they are then treated with a novel kind of ionic liquid (IL). The absor­
bance peak is also seen at 652 nm wavelength. The quick electron
transfer mechanism between the substrate and H2O2 was found to be the
reason for increased catalytic activity of ZV-Mn NPs. By coating of IL on
ZV-Mn NPs, a linear range of 10-280 μM was achieved and a detection
limit of 0.2 μM was possible. The H2O2 sensor was successfully applied
to dairy milk products, demonstrating its effectiveness [133].
Hydrophilic phytic acid PA/Cu3(PO4)2.3H2O was used to modify the
Cu3(PO4)2 .3H2O by a simple co-precipitation process. The
Cu3(PO4)2.3H2O and PA/Cu3(PO4)2.3H2O were shown to have
peroxidase-like activity, and peroxidase catalyse the substrate 3,3,5,5-
tetramethylbenzidine in the presence of H2O2. Since the catalytic ac­
tivity of PA/Cu3(PO4)2.3H2O is approximately four times more than that
of Cu3(PO4)2.3H2O, this suggests that the addition of PA can increase the
peroxidase activity. A colorimetric approach using PA/Cu3(PO4)2.3H2O
electrode for the detection of H2O2 was developed with a linear range of
100- 5 × 103 μM and a detection limit of 79 μM. Finally, the detection of
H2O2 in milk samples proved the applicability of the PA/
Cu3(PO4)2.3H2O designed electrode. The suggested method is built on
inorganic materials with improved peroxidase activity, which helps in
the detection of H2O2[134].
Formaldehyde is a chemical compound that can be used to adulterate
dairy products. This harmful substance is added to milk to prevent
spoilage and extend its shelf life. However, the addition of formaldehyde
to dairy products can have severe health implications, such as respira­
tory issues, irritation of the eyes, throat, and skin, and even cancer.
Previous reports on formaldehyde detection in milk have utilized
extrinsic fiber optic devices [135]. However, that detection relies on
refractive index alterations, which are seldom selective. To address this
issue, an optical sensor was designed for the detection of formaldehyde.
The sensor consists of an optical fibre and open tip that is coated with the
decamolybdodivanadophosphate salt [(C4H9)4N]4 H[PMo10V2O40],
which is insoluble in water and shows changes in UV-Vis spectrum upon
16
Sensors and Actuators Reports 5 (2023) 100159
contact with formaldehyde. The sensor’s limit of detection for formal­
dehyde was 6.7 µM and the limit of quantification was 19.9 µM. These
values were lower than the permissible limit for food ingestion, while
being close to the values obtained through standard spectrophotometric
method (6.7 µM and 16.6 µM, respectively). The sensor was successfully
tested for formaldehyde measurement in milk, and the results were
statistically similar (α = 0.05) to the traditional method of formaldehyde
analysis [136]. For at least eight days, the sensor has maintained its
sensitivity.
Another study introduces a novel colorimetric sensor array that uses
a combination of organic chemicals and molybdenum disulfide quantum
dots (MoS2 QDs). Aldehydes and ketones, which are oxygen-
functionalized volatile substances, are particularly and more strongly
favoured by MoS2 QDs. Using Linear Discriminate Analysis (LDA), this
newly constructed sensor array is initially employed to classify eight
distinct aldehydes and ketones. During the training and prediction
phases, the classification accuracy of 96% and 83% were attained
respectively. Next, formaldehyde in milk samples is semi-quantitatively
and quantitatively analysed using the newly invented colorimetric
sensor array. The addition of formaldehyde to milk adulterates it and
extends its shelf life. Formaldehyde was added to samples of cow milk
that were obtained directly from dairy farmers and supermarkets, and
the concentration of formaldehyde ranged from 33.3 to 832.5 µM. The
partial least squares regression (PLS-R) approach was used to examine
the sensor array’s response to these samples, and calibrate for formal­
dehyde concentration. Formaldehyde in cow milk can be directly ana­
lysed using the suggested sensor without any sample pre-treatment step
[137].
Despite formalin being illegal in the majority of nations for use in
milk or any other food product, it is still used since it may significantly
extend food’s shelf life and its easy availability. However, formalin is a
toxic substance that can cause serious health problems when consumed
in high amounts. The ingestion of formalin-adulterated milk can lead to
health issues such as nausea, vomiting, abdominal pain, diarrhoea, and
even death in severe cases. Formalin can also cause long-term health
problems such as cancer and reproductive disorders. In a related study,
the addition of a small amount of gold nanoparticle solution to Tollen’s
reagent improved its abilities, and caused a perceptible shift in colour
due to the presence of trace amounts of formalin in the solution. Various
milk samples were prepared with varying amounts of formalin in order
to develop this novel approach, and a colour shift was clearly seen
following the reaction with gold nanoparticles in Tollen’s reagent. The
UV-Visible Spectrophotometry was used to visualize the production of
gold core, silver shell particles. [138]. This study offers a simple,
affordable method for visually detecting formalin adulteration in milk,
which could be used for quality control and subsequently developed for
use in testing other food products. However, the detection limit of
method is not clearly defined.
This work describes the simultaneous colorimetric detection of urea,
H2O2, and pH in cow milk samples using alizarin and bromothymol blue
as indicator in microfluidic paper-based analytical devices (µPADs).
Colorimetric examination was carried out using digital photographs
taken by an office scanner. The quantity of 0.5 and 10 mL chromogenic
and sample solution was added into the detection and sampling zones,
respectively. The detection limits for urea and H2O2 were found to be
2.4 × 103 and 100 µM, respectively. Colorimetric assays for pH mea­
surements enabled monitoring of solution pH with a resolution of 0.25
units. The µPADs was used to identify target adulterants with good
repeatability (RSD # 6.0%) and accuracy (91-102%) rate [139]. The
proposed technique was successfully used for analyzing suspected
adulterants in sixteen milk samples, which were examined without any
pre-treatment steps. Based on the findings, µPADs with colorimetric
sensing is useful tool for fast screening of suspected adulterants in milk.
Urea is commonly employed for adulterating milk to increase the
solid-not-fat value (SNF), nitrogen content, and richness. However,
adulteration in milk has been a prevalent practise for economic gain,
Himshweta and M. Singh
which is immoral and harmful to health. The current work describes the
detection of urea, a frequent milk adulterant, using a fibre optic sensor
based on Lossy mode resonance (LMR). The ZnO nanorods structure was
formed on the fiber-optic probe using a hydrothermal technique, and an
extra enzymatic layer of urease was coated to detect the presence of urea
in milk. Due to the extra urease layer, this sensor is selective in detecting
the urea concentration in milk sample. A change in the refractive index
is monitored, as the urea content in milk increases from 5.0 × 104 to 8.0
× 105 µM, resulting in redshifts in the peak wavelengths of absorbance
spectra. The performance characteristics of the proposed sensor were
estimated, using these peak shifts, and an 8-fold increase in sensitivity to
the non-enzymatic sensor was found [140]. The sensor has 1.18 £ 103
µM detection limit and quick response time of 5-7 s. Moreover this
label-free and low cost sensor can be used for the pathophysiological
examination of urea range in human. The identification of urea in milk is
crucial in the dairy sector for the quality control operations. The amount
of urea in milk was measured using silver nanoparticles (both uncapped
and citrate-capped) by UV-Vis spectroscopy. In presence of urea the
nanoparticles change their colour from yellow to blue. The proposed
model was capable of measuring urea concentrations of 3.23 and 5.02
µM (between 6 mg/dL and 90 mg/dL) in milk. It was verified by
para-dimethylaminobenzaldehyde (DMAB) method of Food Safety and
Standards Authority of India (FSSAI). This proposed study can help
small dairy firms evaluate both qualitatively and quantitatively the urea
concentration in milk [141]. The gold nanoparticles have been exten­
sively researched for their potential in nanosensing applications. Their
unique features, including aggregation, fluorescence quenching, wide
absorption at the surface plasmon band, and biocompatibility, make
them promising candidates for ensuring food safety. The gold nano­
particles may replace the present conventional approach for detecting
pollutants, pesticides, heavy metals, adulterants, and other contami­
nants that pose significant danger to food safety [142].
2.1.5. Localised surface plasmon resonance sensor
The surface plasmon resonance (SPR) occurs in materials, those have
negative real and small positive imaginary dielectric constants. In
resonance, the electromagnetic fields stimulate surface conduction
electrons to oscillate coherently [143]. The SPR technique measures
materials’ adsorption onto the surface of gold (Au) or silver (Ag) or
metal nanoparticles. The oscillation of free electrons on the surface of
the dielectric/metal, produces the charge density of surface plasmon
polaritons (dipole-photon hybrid particles). Incident beam properties
changes due to minute fluctuations in the refractive index such as phase,
wavelength, angle, and SPR excitation. Moreover, the sensing ability of
SPR can be enhanced by adding metal or magnetic nanoparticles and
carbon nanostructures, due to their higher sensitivity, label-free and
multiplexing ability [38]. There is a resonant oscillation of conduction
electrons on the surface of noble metal nanoparticles, which creates
unique optical properties. This phenomenon is known as localized sur­
face plasmon resonance (LSPR). Researchers have revealed novel ap­
plications that utilize LSPR and enable precise design and manipulation
of metallic structures on the nanoscale.
The objective of this study was to develop eco-friendly silver nano­
particles (SNPs) stabilised by acacia gum, which could be used as a LSPR
colorimeter sensor for the detection of reactive oxygen species like
H2O2. Inorganic precursor silver nitrate solution (AgNO3), white sugar
(reducing agent) and Acacia gum (stabilising agent) were used for the
silver nanoparticles synthesis. Upon the addition of H2O2 solution in
Acacia gum-capped SNP dispersion, a change in the LSPR band was
observed at 407 nm, which facilitated the plasmon colorimetric sensing
of H2O2. After the addition of H2O2 solution, the Acacia gum-stabilized
SNPs eventually changed from yellow to transparent, with a notable
change in the LSPR absorbance. This happened due to SNPs aggregation
after the addition of H2O2 solution. This potential of the designed SNPs
can detect the presence of H2O2 in liquid milk sample [144]. Although,
the method is simple, cost effective and can be applied as a colour
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Sensors and Actuators Reports 5 (2023) 100159
indicator, but the detection limit for H2O2 sensing in liquid milk is
missing.
The colorimetric detection of melamine in liquid milk samples by
unmodified AuNPs using LSPR has been proposed. The biosensing sys­
tem includes an optical attenuator, three different types of 600 m pre­
mium optical fibres with Sub Miniature version A (SMA905)
connections, a broadband light source covering the spectral range of 200
nm to 1700 nm, and a small spectrometer with a linear charge coupled
device (CCD) array. Its operation is based on the fact that the addition of
melamine in Au NPs solution causes a colour change from wine-red to
blue due to the inter-particle coupling effects, which also results changes
in the wavelength and absorbance in the LSPR band [145]. The LSPR
biosensing device showed a linear response for melamine concentration
from 0.1 to 0.9 μM, with a detection limit of 0.033 μM in liquid milk
samples.
The Ag nanocubes were fabricated using a sodium sulphide and
solvothermal technique. During the synthesis process, all edge-length
nanocubes (32 and 44 nm) were produced at 145 and 155◦ C, respec­
tively. The refractive index sensitivity of nanocubes was examined using
a glucose aqueous solution, and the colloidal dispersion of nanocubes
has a refractive index sensitivity of 161 nm per RIU (refractive index
unit). The sensitivity of the LSPR chip created by immobilising nano­
cubes on the (3- aminopropyl) trimethoxysilane modified glass coverslip
was found to be 116 nm per RIU. Additionally, formaldehyde detection
in water and milk samples was accomplished using nanocubes with edge
lengths of 44 nm. The interaction between the aryl amine of 4-amino­
thiophenol immobilised on the nanocubes and the electrophilic carbon
atom of the formaldehyde was used to detect formaldehyde, with a
detection limit of 37.96 µM. The formaldehyde sensitivity in water and
diluted milk was 0.62 and 0.29 nm µM− 1, respectively [146].
2.2. Electrochemical nanosensors
Due to their portability, affordable price, and simple instrumenta­
tion, electrochemical nanosensors are extensively used for the detection
of milk adulterants, as summarised in Table 2. These sensors offer
numerous benefits applicable to majority of analytical chemistry fields,
including excellent sensitivity, selectivity, and extraordinary speed. To
create electrochemical sensors, several electrochemical techniques have
been investigated, with cyclic voltammetry (CV), popular due to its
ability to understand electrode processes and redox mechanisms. Square
wave voltammetry (SWV) and differential pulse voltammetry (DPV) are
very helpful in identifying small levels of electroactive molecules [147].
Electrochemical impedance spectroscopy (EIS) is an effective method for
studying a wide range of physiochemical processes both in solid and
solutions phases. It has many uses in the development of batteries,
sensors, physical electrochemistry and can provide details on reaction
parameters, corrosion rates, the porosity and coating of electrode sur­
faces, mass transfer, and interfacial capacitance measurements.
Another promising future technology is the inherent sensitivity and
broad linear range of the chemiluminescence method, which are com­
bined with electrochemistry to create electrochemiluminescent (ECL)
tests. Electrochemical sensors are very adaptable and have a quick
response time. Generally nanoparticles like gold nanoparticles (AuNPs),
silver nanoparticles (AgNPs), cerium dioxide (CeO2) and magnetic
nanoparticles are used to modify the working electrodes because of their
distinct electrical, chemical and physical features. The output-electrical
signals of electrochemical sensors are altered when targets molecules
react with electrode-immobilized components. Electrochemical sensors
can depend on several electrical characteristics and are divided into
potentiometric, amperometric, impedimetric, conductometric and vol­
tammetric techniques [147]. However electrochemical sensors have
some limitations such as electrode fouling and charging which results in
poor repeatability of the assay, but these limitations can be overcome by
the applications of nanomaterials.
Himshweta and M. Singh Sensors and Actuators Reports 5 (2023) 100159

Table 2
Electrochemical nanosensors for the detection of adulterants in dairy products.
Sample Nanomaterial Target analyte Detection method LOD (µM) References
− 5
Infant formula Molecularly Imprinted Conducting Melamine Voltammetric 1.72 × 10 [162]
Polymer Composite
− 7
Milk sample Polymeric nanoparticles decorated Melamine EIS 5.6 × 10 [150]
multi-walled carbon nanotubes
3
Liquid milk, Au electrode modified with MIP poly (2- Melamine EIS 3 × 10− [149]
yogurt, mercaptobenzimidazole) (PMBI)
powder milk
8
Milk Ionic liquid/zinc oxide nanoparticles/ Melamine Voltammetric 9.6 × 10− [156]
chitosan/gold electrode
− 6
Milk Au@PANI Melamine Voltammetric 1.39 × 10 [158]
Milk sample Overoxidized Poly-(para–aminophenol) Melamine Voltammetric and 0.34 [163]
film on glassy carbon electrode (GCE/Ox- EIS
PPAP)
Milk OMC/GCE Melamine Voltammetric 2.0 × 10− 3 [161]
Milk samples (Co-ZIF)- (NiMWs) Urea Voltammetric 0.3 [165]
Cow milk Zinc oxide nanospheres modified Pt Melamine and urea Voltammetric 3 × 10− 6 and 1 × 10− 6
for melamine and [166]
electrode urea
Milk Nickel sulfide/graphene oxide on glassy Urea EIS ~3.79 [154]
carbon electrode (NiS/GO/MGCE)
Milk Conductive 2D Metal-organic Urea Amperometric 414, 6.188 and 0.471 [172]
Framework (Co, NiCo, Ni) Nanosheets
Milk Graphene–Polyaniline composite Urea Voltammetric 5.88 [164]
Milk Polyaniline (PANI) and graphitized Urea Amperometric 1165.5 and 832.5 [178]
nanodiamond (GND) nanocomposite
3
Milk NF/Ag-N-SWCNTs/GCE Urea Voltammetric 4.7 × 10− [168]
Milk GNPlt-CNT Urea Amperometric NA [175]
Milk Graphene nanoplatelet/graphitized Urea Conductometric 83.25 [176]
nanodiamond-based nanocomposite
Milk Electrospun nanofibers modified with Urea EIS 1.83 [167]
zinc oxide nanoparticles
Milk Fe3O4/MWCNT/PANI-Nafion Urea Voltammetric, EIS 67 [177]
nanocomposite and amperometric
Skimmed and Voltammetric electronic tongue of Formaldehyde, urea, melamine Voltammetric 1 × 104, 4.16 × 103, and 950 for [155]
semi- platinum, gold, and copper formaldehyde, urea, and melamine
skimmed
milk sample
Milk AuNPs@PPy composites Formaldehyde Voltammetric and 400 [171]
EIS
− 3
Milk CuO nanoparticles Glucose, H2O2 and Amperometric 6.8 × 10 for glucose; [191]
formaldehyde hydrogen peroxide- 0.034; and for
formaldehyde- 0.026
Milk MnO2/graphene/carbon nanotubes H2O2 Amperometric 0.1 [180]
Milk samples GO-ESM H2O2 Amperometric 0.031 [181]
Milk Hybrid nano-interface of iron oxide H2O2 Amperometric 3.7 × 10− 3 [182]
nanoparticles and carbon nanotubes
Milk samples Nafion and bimetallic nanoparticles H2O2 Amperometric 0.7 µA [185]
reduced graphene oxide modified glassy
electrode (Nf/Pd@Ag/rGO-NH2/GC)
Milk samples N-doped carbon nanofibers (PtNi/ H2O2 Amperometric 0.0375 [186]
NCNFs)
Milk samples L-proline assisted silver nanoparticles H2O2 Voltammetric 0.05 [169]
Milk samples Ferumoxytol and reduced graphene H2O2 Amperometric ~0.38 [188]
oxide decorated with platinum
nanoparticles
Milk samples Monodisperse palladium nanoparticles H2O2 Amperometric 75 [179]
Milk sample Carbon paste electrode with CuFe2O4 H2O2 Voltammetric 0.064 [160]
nanoparticle
Real milk Titanium dioxide conjugated gold H2O2 EIS 10 [153]
sample nanoparticles
Milk sample CuONPs H2O2 Voltammetric 0.03 [170]
Fresh liquid Cu- MOFs H2O2 Amperometric 0.35 [48]
milk samples
Skimmed milk Graphene/Ag nanoparticles H2O2 Amperometric 7.9 [187]
and whole
milk
Milk Samples CuMn2O4 Spinel Nanoflakes/GCE H2O2 Amperometric 0.012 [199]
Bovine milk PEDOT:PSS-rGO-AuNPs H2O2 Amperometric 0.08 [184]
samples
Milk samples PB nanoparticles doped (PEDOT) H2O2 Amperometric 0.16 [183]
Raw Cow Milk AuPt NPs H2O2 Voltammetric 4.8 in UHT milk (ultra-high temperature [173]
and UHT processing);
milk 2.5 in raw cow milk sample
Milk Ruthenium nanoparticles decorated on H2O2 Amperometric 0.468 [189]
nitride carbon
(continued on next page)

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Himshweta and M. Singh Sensors and Actuators Reports 5 (2023) 100159

Table 2 (continued )
Sample Nanomaterial Target analyte Detection method LOD (µM) References

Milk PAMAM dendrimer encapsulated with H2O2 Amperometric 2.7 × 104 [192]
gold nanoparticles
Milk samples Carbon nanotubes (CNTs), reduced H2O2 Amperometric ~ 0.31 [193]
graphene oxide (RGO) and platinum
nanoparticles (Pt NPs) nanocomposite
Milk Pt/rGO-CNT hybrid H2O2 Amperometric 1.5 × 104 [195]
Milk sample GL-VS2 nanostructures H2O2 Amperometric 0.026 [197]
Raw milk Novel polypyrrole-graphene oxide-gold H2O2 Amperometric 5 [200]
samples nanocomposite
Milk PtNiCo-NPs H2O2 Amperometric 0.37 [196]
Milk AgNPs-Zn-MOF H2O2 Amperometric 0.067 [157]
Milk Porous carbon nanofibers embedded H2O2 Amperometric 10 [194]
with cobalt nanoparticles
Milk samples Copper Oxide Nanoparticle H2O2 Voltammetric 0.21 [159]
Milk samples Cu/Cu2O nanocomposites H2O2 Amperometric 0.04 [190]
Milk samples Fe-Cu (LDH)/ Fe3O4 nanoparticles H2O2 Voltammetric 0.6 [174]
Milk Cu2O/Ag nanocomposites H2O2 Amperometric 0.2 [198]
Milk Pt/teflon/SiO2/Si biosensor device Melamine, starch, urea, EIS 1.54 × 103 -urea, 6.22-melamine, 1.43 × [152]
allantoin, cyanuric acid, 10− 3-starch, 4.59- allantoin, 3.87-
benzoic acid, ammonium cyanuric acid, 7.28- benzoic Acid, 13.4-
sulphate and sodium ammonium sulphate, and sodium
bicarbonate bicarbonate-26.19
Cow Milk Lab on chip device Si-SiO2 Pt electrode Soap EIS ≥3.16 [151]

Abbreviations: Electrochemical impedence spectroscopy- EIS; gold polyaniline nanocomposites- Au@PANI; nafion- single-walled carbon nanotubes (SWCNT’s)
decorated with silver nanoparticles (Ag-NPs) modified glassy carbon electrode-NF/Ag-N-SWCNTs/GCE; graphene nanoplatelets-carbon nanotubes (GNPlts-CNT);
ferric oxide multiwall carbon nanotubes polyaniline- (Fe3O4/MWCNT/PANI-Nafion) nanocomposite; MOFs-metal organic framework; cuprous manganite/glass
carbon electrode-CuMn2O4/GCE; polypyrrole reduced gold nanoparticles- AuNPs@PPy composites Amorphous carbon nanoparticles-CNPs; Cuprous oxide/silver
nanocomposites-Cu2O/Ag; Copper oxide nanoparticles on carbon cloth -CuO NPs/CC; Silver nanoparticles-zinc-metal organic framework -AgNPs-Zn-MOF; Copper/
cuprous oxide nanocomposites-Cu/Cu2O; Fe-Cu layered double hydroxide magnetic nanoparticles-(LDH)/Fe3O4; platinum nickel cobalt nanoparticles- PtNiCo-NPs;
Grass-like vanadium disulfide nanostructures-GL-VS2; platinum/reduced graphene oxide carbon nanotubes- Pt/rGO-CNT hybrid; Viologen-terminated polyamido­
amine –PAMAM; bimetallic gold-platinum nanoparticles -AuPt NPs; Graphene-based ternary nanocomposite of poly (3,4-ethylenedioxythiophene) polystyrene sul­
fonate, reduced graphene oxide and gold nanoparticles (PEDOT:PSS-rGO-AuNPs); Prussian blue nanoparticles doped poly(3,4-ethylenedioxythiophene) -PB-PEDOT;
Copper oxide nanoparticles –CuONPs; Graphene oxide doped eggshell membrane -GO-ESM; Ordered mesoporous carbon/glassy carbon electrode -OMC/GCE; Cobalt
zeolitic imidazolate framework (Co-ZIF)-nickel microwires (NiMWs).

2.2.1. Impedimetric sensors First, a poly (acrylic acid-co-(7-(4-vinylbenzyloxy)-4-methyl coumarin)-

One of the most significant electrochemical methods is electro­
chemical impedance spectroscopy (EIS), which measures the impedance
in a circuit in ohms (as a resistance unit). EIS has several advantages
over other electrochemical techniques. It is a stable method that uses
small signal analysis, can perform signal relaxations over a very wide
range of applied frequency, using commercially available electro­
chemical working stations [148]. The EIS can analyse the mass-transfer,
charge-transfer, and diffusion processes, and can investigate the
intrinsic characteristics of materials or particular processes that may
affect the conductance, resistance, or capacitance of an electrochemical
system. Innovative electrode designs using nanomaterials can be engi­
neered to functionalize the transducer surface, and receptors or recog­
nition elements can be tethered to design electrochemical sensors.
In the current study, molecularly imprinted (MIP) impedimetric
sensor for detecting melamine was introduced. The gold (Au) electrode
was replaced with MIP poly (2-mercaptobenzimidazole) (PMBI) and
synthesized by electrochemical polymerization of 2-MBI with cyclic
voltammetry (CV) in the presence of the template (melamine) molecule.
The π-donor-acceptor relationship between melamine and PMBI is the
primary propelling factor behind recognition. The sensor shown detec­
tion limit of 0.003 µM in liquid milk, yogurt and milk powder samples
[149]. This sensor is useful for monitoring analytes that are not elec­
trochemically active in food matrices.
The multi-walled carbon nanotubes (MWCNTs) and nanosized
molecularly imprinted polymeric (MIP) nanoparticles (NPs) improve the
electrode surface area and provide a high binding capacity for mel­
amine. Additionally, the MWCNTs in MIP-MWCNTs nanohybrids act as
"electronic bridges" to speed up the flow of electrons inside the MIP film.
In this work, melamine was used as a template, and self-assembled
polymeric nanoparticles were used to decorate MWCNTs, resulting in
molecularly imprinted nanohybrids with "necklace-like" nanostructures.
co-ethylhexyl acrylate) amphiphilic copolymer (poly (AA-co-VMc-co-
EHA), PAVE) with photosensitive coumarin units was created. Then, the
photosensitive (MIP-MWCNTs) with "necklace-like" structures were
created by co-assembling the PAVE copolymers and MWCNTs in the
presence of template molecules. After using the MIP-MWCNTs nano­
hybrids to modify electrode surface and photo-polymerizing the nano­
hybrids coumarin units, a complex film with network architecture was
created. The melamine MIP sensor was successfully created by
electrolysis-based extraction of melamine molecules with detection limit
5.6 × 10− 6 µM for melamine in milk samples, milk powder, infant for­
mula, and animal feed [150]. Moreover, the MIP sensor was found stable
for 30 days.
Adding pulverized soap to milk is a common method of milk adul­
teration [151]. The soap is used to increase the volume of the milk and
make it appear thicker and creamier than it actually is. However, this
practice is highly unethical and poses serious health risks to consumers.
When soap is added to milk, it can cause gastrointestinal problems such
as nausea, vomiting, and diarrhoea. In addition, it can also lead to skin
rashes and allergic reactions in some people. Moreover, the long-term
consumption of adulterated milk can have adverse effects on health
and nutrition. Electrical impedance spectroscopy (EIS) can be used to
detect and quantify soap adulterants in cow milk. It offers a quick,
cost-efficient, and simple platform for monitoring the milk quality. This
study examined variations in impedance, capacitance, conductance, and
current for soap adulteration in milk at concentrations of 0.1%, 0.3%,
0.5%, 0.7%, and 0.9% (w/w). It has been observed that with increased
in soap concentration in the milk, capacitance, conductance, and current
was increased, while impedance decreased. The coefficient of sensitivity
for the milk samples containing soap was determined and explained in
terms of the conductance values [151]. The EIS and current measure­
ments, can detect the concentration of ≥ 3.16 µM soap in milk samples.

19
Himshweta and M. Singh
The current method uses EIS to demonstrate a unique diagrammatic
method for the identification and measurement of polar and non-polar/
ionic adulterants in milk. To conduct this study, regulated amount of
commonly used milk adulterants, such as melamine, starch, urea,
allantoin, cyanuric acid, benzoic acid, ammonium sulphate, and sodium
bicarbonate, are added to a sample of pure milk. The mixture of silicon
dioxide (SiO2) and Teflon layer thicknesses are tuned to produce the
proper hydrophobicity/dielectricity for the liquid milk samples during
the synthesis of a platinum Teflon silicon dioxide silicon (Pt/Teflon/
SiO2/Si) sensor. When non-polar/ionic adulterants are added under
controlled conditions between 0.5 and 9 mg/g, the electrical impedance,
capacitance, and conductance for polar samples change in the opposite
direction from those of non-polar/ionic adulterants [152]. The detection
limit for different adulterants were calculated for polar adulterants such
as urea (1.54 × 103 µM), melamine (6.22 µM), starch (1.43 × 10− 3),
allantoin (4.59 µM), cyanuric acid (3.87 µM); for non-polar/ionic adu­
letrants, benzoic acid (7.28 µM), ammonium sulphate (13.4 µM) and
sodium bicarbonate (26.19 µM), respectively. All the adulterants were
identified within 15 s of time.
The main contribution of this study is the development of an
impedimetric biosensor for H2O2 detection, employing gold nano­
particles (AuNP) trapped within aminated titanium dioxide (TiO2)
particles. To modify the AuNPs trapped inside TiO2, an amide bond was
formed between the carboxyl end terminal of Hb and the amine terminal
from TiAu-APTS. Hemoglobin (Hb), which has peroxidase-like activity,
was used as the bioreceptor for sensing of H2O2 in solution. Therefore,
Hb was covalently bonded to the altered TiO2 surface. The biosensor had
a wide linear range response between 100 and 1.5 × 104 µM, and a
detection limit of 10 µM was achieved using an alternate impedance
approach [153]. It is also interesting that no redox mediators were used
in the development of the impedimetric biosensor. In this method, nickel
sulphide/graphene oxide NiS/GO was created using the superficial hy­
drothermal method and used to modify a glassy carbon electrode
(NiS/GO/MGCE). The modified electrode showed a limit of detection
3.79 µM for urea in water using electrochemical and electrochemical
impedance spectroscopy modes, with the electrode process being
diffusion-controlled [154].
2.2.2. Voltammetric sensors
Voltammetry entails measuring the current in relation to the applied
potential. This method is based on the measurement of the output cur­
rent and the constantly changing applied potential differential. A vol­
tammogram is a depiction of potential versus current, which can provide
information about the kinetics and process of electrochemical reactions
[38].Voltammetry sensors are widely used for food quality evaluation
and detecting pesticides, adulterants, vitamins, heavy metals and
pharmaceuticals analytes. The advantages of using these nanosensors
include higher sensitivity and its capability for analysing two or more
analytes in a single sample through quantitative analysis methods.
The voltammetric electronic tongue is used to detect and distinguish
hazardous compounds purposely added to milk to extend its shelf life or
increase protein content. The electronic tongue comprises three active
electrodes made of platinum, gold, and copper. The measuring princi­
ples entailed extracting data from cyclic voltammograms obtained in
pure and contaminated milk. The electrode response was investigated
using electrochemical quartz crystal microbalance to understand the
mechanism by which the tongue could detect between the samples. The
main mechanisms are electrochemical formation and dissolution of ox­
ides and the reduction of a copper-melamine ionic pair on the surface of
the copper electrode. Furthermore, the electronic tongue was capable of
detecting adulterations in several types of milk (whole, skimmed, and
semi-skimmed) and milk from various brands. For formaldehyde, urea,
and melamine, the concentration of adulterant was found 1 × 104, 4.16
× 10− 3, and 950 µM, respectively [155].
Melamine is added to food as an adulterant with the intention of
inflating the total protein. Illicit adulteration of baby milk powder with
20
Sensors and Actuators Reports 5 (2023) 100159
melamine has led chronic renal and urinary tract failure. For the
detection of melamine, a number of analytical techniques have been
developed. One unique electrochemical technique uses modified gold
electrodes, zinc oxide nanoparticles (ZnONPs), and ionic liquids (1-
ethyl-3-methylimidazolium trifluoromethanesulfonate [EMIM][Otf])
(AuE). Methylene blue was utilised as a redox indicator to increase
electron transport in electrochemical cells. In the presence of melamine,
the electrochemical behaviour of the modified gold electrode was
checked by cyclic voltammetry and differential pulse voltammetry with
a detection limit of 9.6 × 10− 8 µM. The suggested approach is fast and
simple for analysing the melamine level in milk powder products [156].
The selective and accurate measurement of H2O2 at physiological
pH, using an electrode modified with silver nanoparticles-zinc-metal
organic framework (AgNPs-Zn-MOF) was described. Drop casting was
used to modify the AgNPs-Zn-MOF on the glass carbon (GC) electrode.
Cyclic voltammetry (CV) of the AgNPs-ZnMOF modified electrode
showed a high anodic peak at +0.20 V, corresponding to the oxidation of
Ag0 to Ag+, and a subsequent reduction peak at -0.50 V, corresponding
to the reduction of Ag+ to Ag0. No reaction was seen at GC, 3 GC/Zn-
MOF, and GC/TA-AgNPs modified electrode, it displays a significant
decrease in peak for H2O2 at -0.67 V with a current of -21 µA. The
current response of H2O2 rise linearly from 1 to 5 × 103 µM, with a
correlation coefficient of 0.9915 and a limit of detection of 0.067 µM (S/
N=3). The proposed method was used to detect H2O2 in milk, human
urine, and blood serum samples [157]. Another study utilized gold and
polyaniline composites (Au@PANI) deposited on the surface of glass
carbon electrode to enhance the sensitivity and intensity of signals for
the detection of melamine. Melamine form hydrogen bonds with
Au@PANI composite, this helps in the detection of melamine. Electro­
chemical analysis by cyclic voltammetry and differential pulse voltam­
metry presents a detection limit of 1.39 × 10− 6 µM for melamine [158]
as shown in Table 2. The sensor was found stable for up to 4 weeks.
Kamyabi & Hajari have successfully developed an electrochemical
sensor for H2O2 detection by electrodepositing copper oxide nano­
particles on the activated pencil graphite electrodes. The modified
electrode (CuO/APGE), significantly reduces the over potential of H2O2
oxidation upon the addition of H2O2 with a starting potential of around
+0.05 V (vs. Ag/AgCl). A quick chemical reaction at electrode surface
occurs between H2O2 and CuO transform CuII to CuI and finally H2O2 to
oxygen. As CuI is released, it is electro-oxidized again to CuII and CuO,
continues to catalyse the reaction. The electrocatalytic response of this
sensor had a detection limit of 0.21µM (signal/noise = 3) and was
proportional to the H2O2 concentration. A low detection limit and a
strong signal were displayed by the H2O2 sensor [159]. In this study, a
highly sensitive voltammetric sensor, made of CuFe2O4 nanoparticles
(RGO/CuFe2O4/CPE) was developed for the detection of H2O2. The
modified electrode displayed a quick amperometric response of <2s at
pH 5, good linear range of 2 to 200 μM, and low detection limit of 0.52
μM for the determination of H2O2. Additionally, H2O2 peak current for
differential pulse voltammetry (DPV) increased linearly in the concen­
tration ranges from 2 to 10 μM and 10 to 103 μM, and the detection limit
for H2O2 is 0.064 μM. The developed sensor was successfully used for the
measurement of H2O2 in biological and pharmaceutical samples, such as
milk, green tea, hair dye cream, and mouthwash solution [160]. Guo and
co-workers synthesized ordered mesoporous carbon/glassy carbon
electrode (OMC/GCE) for melamine detection in milk samples. They
proposed an electroactive Cu-melamine complex that can detect mel­
amine by converting it from a non-electroactive melamine to electro­
active state in presence of copper ions in 0.1 M (pH 9.0) borate buffer
containing 20% methanol (v/v) as the supporting electrolyte on an an
OMC modified electrode. For surface characterisation of modified
electrode the cyclic voltammetry (CV) has been used. The anodic peak of
Cu-melamine complex is linear with a detection limit of 2.0 × 10− 3 µM
(based on S/N = 3) for melamine. The proposed sensor has been suc­
cessfully applied for melamine detection in milk products [161].
This works describes a quick and sensitive electrochemical sensor
Himshweta and M. Singh
based on a molecularly imprinted composite thin-film, developed by in-
situ co-electropolymerization of aniline and acrylic acid with melamine
as a template. The carboxylic group from acrylic acid and amine from
aniline interact non-covalently with the template, which enhances the
sensitivity of sensor. Electrochemical measurements by cyclic voltam­
metry showed a limit of detection of 1.72 × 10− 5 μM for melamine in
infant formula and raw milk [162]. The findings showed that the peak
current remained 90% of its initial current for 20 days. The electro­
chemical measurement of melamine can be done by electro­
polymerization as a preconcentration method. Here, melamine is
preconcentrated on glassy carbon electrode modified with over oxidized
poly-(para-aminophenol) film (GCE/Ox-PPAP) by potentiodynamic
method. During the electrochemical reaction, non-electroactive mel­
amine was changed into electroactive poly- (melamine) (PMel). The
preconcentrated melamine was measured by using square wave vol­
tammetry (SWV) as the electro-analytical signal. The GCE/Ox-PPAP
sensor displayed good calibration sensitivity of 4.718 μA L μM, and a
limit of detection 0.34 μM (S/N = 3) in milk samples [163].
Detection of urea is critical in various industries such as food, dairy,
and environmental monitoring. Traditional methods for urea detection
are either costly and require specialised apparatus, or employ an enzy­
matic approach. Graphene-polyaniline (Gr-PANi) based electrochemical
sensor for non-enzymatic urea detection is proposed. Using the cyclic
voltammetry (CV) approach, a Gr-PANi composite was created by
electro-depositing PANi on the surface of a Gr modified glass carbon
electrode (GCE). The current response of the urea sensor was found to be
4.74 times larger than a pure PANi-based sensor, and 67.2 times greater
than that of a pure Gr-based sensor. The modified sensor has limit of
detection of 5.88 µM, in milk samples and remains stable for 10 days.
The linear range is 1 × 103 to 8 × 103 µM, which covers the physiological
range of urea content in human blood. The limit of detection was found
to be 140 µM found in plasma and milk [164]. The peak current depends
upon the analyte concentration which is helpful for urea detection. This
Gr-PANi composite-based urea sensor is a simple, low-cost, and
non-enzymatic method which has a wide range of applications. Urea
plays crucial biological function in metabolism and helping to determine
protein levels in the human body. Exceeding its prescribed limit can
cause heart failure, dehydration, cachexia, and liver failure, making
precise urea measurement crucial for diagnosis and treatment. In this
study, cobalt zeolitic imidazolate framework (Co-ZIF)-nickel microwires
(NiMWs) were produced using a simple one-pot solvothermal approach.
The sensor demonstrated a wide range determination of urea from 0.5 to
500 µM with limit of detection of 0.3µM. The sensor was successfully
employed to quantify urea in human biological fluid and milk samples
[165].
In humans, adulteration of cow milk with melamine and urea causes
indigestion, acidity, ulcers, and kidney stones. A highly sensitive
acetylcholinesterase cyclic voltammetric biosensor based on zinc oxide
nanospheres modified platinum (Pt) electrode was successfully devel­
oped for simultaneous detection of melamine and urea in cow milk
sample. The bioelectrode showed 100% permeability for melamine and
urea, which improved the selectivity of sensor. The platinum zinc oxide
acetylcholinesterase chitosan (Pt/ZnO/AChE/Chitosan) bioelectrode
can detect melamine and urea up to 0.001 to 0.02 µM range, with
detection limit of 3 × 10− 6 and 1 × 10− 6 µM, respectively. The sensor
revealed high recovery (99.96-102.22%), making it suitable for mel­
amine and urea detection in cow milk samples [166]. The current
density tended to be nearly constant and maintained 94% of its consis­
tency even after 24 days.
It is necessary to use analytical methods to assess dairy products,
particularly milk, to ensure food safety against adulteration. Urea is one
of the compounds that might be used to adulterate milk, and this raises
serious concerns owing to its negative health consequences on con­
sumers. A biosensing platform was created for electrochemical detection
of urea. Polymeric electrospun nanofibers of polyamide 6 (PA6) and
polypyrrole (PPy) were used to create the sensing platform, and
21
Sensors and Actuators Reports 5 (2023) 100159
modified with zinc oxide nanoparticles (ZnO). The fluorine doped tin
oxide FTO/PA6/PPy/ZnO/urease electrode has great sensitivity for urea
detection with a limit of detection of 1.83 µM. The obtained results
shown the potential of electrospun nanofiber-based electrodes for
detection of adulterants in dairy products [167].
The enzyme-based techniques have certain drawbacks such as high
manufacturing costs and low stability, which has increased interest in
the construction of non-enzymatic sensors. In this study, a one-step
thermal reduction approach was used, using melamine as the nitrogen
source, to produce and use single-walled carbon nanotubes (SWCNT’s)
decorated with silver nanoparticles (Ag-NPs). The SWCNT’s character­
istics were noticeably changes by the nitrogen doping, and it showed a
greater affinity for Ag-NPs. The Ag-N-SWCNTs nanocomposite had
higher catalytic activity than N-SWCNTs and pristine-SWCNTs due to
the integration of the large surface area and electrical characteristics of
N-SWCNTs with Ag-NPs. The glassy carbon electrode (GCE) is modified
with Ag-N-SWCNTs and a layer of Nafion (Nf) was used to construct a
non-enzymatic electrochemical urea sensor. The sensor had a low limit
of detection of 4.7 × 10− 3 and an improved sensitivity of 141.44 μA
mM− 1 cm− 2 for the detection of urea, with ~ 3 s response time [168]. As
a result, this sensor may be used to detect urea in a variety of samples
from the food, fertiliser, and environmental industries. Additionally, the
modified electrode demonstrated good stability up to 150 min under
ambient conditions with no decrease in activity or storage.
Using a one-step voltammetry technique, L-proline associated silver
nanoparticles were electrodeposited on a glassy carbon electrode. L-
proline, acting as a stabiliser, plays a crucial role in reducing the rate of
metallic silver oxidation and preventing aggregations. More signifi­
cantly, L-proline resulted in a 200 mV change in potential towards a less
negative potential and an approximately two fold increase in sensitivity.
This low-cost, simple sensor is free from the many drawbacks of enzyme
or noble based sensors and offers a good sensing performance for the
electrochemical detection of H2O2. Compared to silver nanoparticles
without L-proline, the resulting L-proline-stabilized silver nanoparticles
showed a lower detection limit of 0.05 μM, a broader detection linear
range from 0.1 to 5.14 × 103 μM, and higher selectivity in the presence
of interfering species like ascorbic acid, uric acid, L-glucose, glycine, and
lactose for H2O2 detection. The fast electron transfer kinetics of the
metallic silver (Ag0) in L-proline stabilised silver nanoparticles
contribute to the outstanding H2O2 electrocatalytic properties. Addi­
tionally, the sensor was successfully used to detect H2O2 in milk samples
[169]. The copper oxide nanoparticles and polyalizarin yellow R onto a
glassy carbon electrode (PAYR/CuONPs/ GCE sensor showed excellent
properties for detecting and measuring H2O2, with a linear range of
0.1-140 μM, a sensitivity of 1.4154 μAcm− 2 μM− 1, and a detection limit
of 0.03 μM using the DPV technique [170].
For the detection of formaldehyde, an electrochemical sensor based
on polypyrrole (PPy)-reduced gold was constructed. To get PPy/GCE,
the pyrrole was potentiostatically transformed onto a glassy carbon
electrode. Following over potential treatment of the polypyrrole, AuNPs
were coated onto the PPy/GCE. Due to high conductivity and
outstanding catalytic activity, the PPy/GCE modified by AuNPs
demonstrated a massive current response. The formaldehyde detection
sensor was built using AuNPs/PPy/GCE, and the quantitative determi­
nation of formaldehyde was accomplished using DPV. The sensor can
detect formaldehyde in pure water with a detection limit of 20 μM, while
the milk sample shown detection limit of 400 μM. The electrochemical
sensor provides good selectivity, repeatability, and stability for form­
aldehyde detection [171].
2D metal-organic frameworks (MOFs) offer certain advantages for
electrocatalysis due to their higher mass transfer rate, larger surface area
and faster charge transfer. So, proposed method utilizes three conduc­
tive 2D MOFs based on metal carbonyl complex (Ni, NiCo, and Co).
Cyclic voltammetry and the current-time techniques were used to
examine electrochemical oxidation and detection. Non-enzymatic Ni-,
NiCo-, and Co-MOF sensors shown higher urea catalytic activity. Among
Himshweta and M. Singh
them, broader linear range (0.5-832.5 μM) is exhibited by Ni-MOF, a
detection limit of 0.471 μM, and a faster reaction time compared to
NiCo- and Co-MOF. Additionally, Ni- and NiCo-MOF modified elec­
trodes were successfully used to identify milk samples [172].
The adulteration of milk with H2O2 has a long history and is widely
known throughout the world. However, the quick breakdown of H2O2 in
raw cow milk makes its measurement very difficult. In this study, a non-
enzymatic electrochemical sensor has been developed for spotting H2O2
in adulterated milk. The bimetallic gold-platinum nanoparticles (AuPt
NPs) were employed as electrocatalysts to specifically increase the
electrochemical signal of H2O2. The screen printed carbon electrode
(SPCE) modified with the AuPt alloy NPs presents good electrocatalytic
activity toward electro-reduction of H2O2 in both Ultra-high tempera­
ture (UTH) and raw milk samples. The sensor showed a sensitivity of
152.9 µA mM− 1 cm− 2 and low limit of detection of 4.8 µM. Application
of this sensor and portable electrochemical analyzer-simulator is shown
by analysing a sample of raw cow milk for H2O2 adulteration. The sensor
showed sensitivity of 155.5 µA mM− 1 cm− 2, a detection limit of 2.5 µM,
and the linear response was found in the range of 2.5 to 5 × 103 µM
[173]. The synergistic electrocatalytic effect of bimetallic AuPt NPs on
SPCE surface is responsible for the high sensing efficiency. This sensor
offers a highly sensitive and alternative way for on-site, real-time
monitoring of H2O2 adulteration in raw milk.
The objective of this study was to create a cutting-edge sensing
platform for measuring H2O2 in milk samples. The proposed sensor was
created on glassy carbon electrode that was enriched with iron copper
(Fe-Cu) layered double hydroxide magnetic nanoparticles
(LDH@Fe3O4/GCE). The modified FeCu-LDH@Fe3O4/GCE electrode
exhibited high cathodic peak currents and higher electrocatalytic ac­
tivity for H2O2 electroreduction. The proposed sensor showed a limit of
detection of 0.6 µM, with wide linear range of 2 to 400 µM [174]. After
ten days, the signals in H2O2 had decreased by 6%, demonstrating the
excellent stability of the sensor.
2.2.3. Amperometric sensors
An amperometric sensor detects the current response resulting from
electrochemical oxidation or reduction processes to determine the
concentration of an analyte. A constant potential is applied between the
working and reference electrodes, and the surface current on working
electrode is proportional to the analyte present in the solution. This
technique provides quick and linear reaction, making it more appro­
priate than potentiometric sensors. However, these sensors have some
drawbacks, such as weak selectivity and interference from other elec­
troactive compounds. The application of amperometric sensors began
initially in the field of clinical and diagnostic fields, but with the advent
of nanofabrication technology has made amperometric sensors more
stable, affordable, and highly sensitive. This has enabled their use in
therapeutics, food, environmental, and agricultural sectors [36,38].
The detection of urea is very important for diagnosing renal disor­
ders and milk adulteration. A low-cost urea sensing device based on
graphene nanoplatelets (GNPlts) can proficiently detect the ions
(amperometric sensing) during the hydrolysis of urea. To increase
sensitivity of sensor, the GNPlts were linked with urease. For main­
taining the graphitic activity of graphene planes, edge functionalized
GNPlts were utilised. The developed platform has high specificity and
can detect urea concentration ranging from 1.67 to 1.33 × 104 µM in just
15 seconds [175]. Furthermore, a novel direct current voltage
(IV)-based, mediator-free nanocomposite has been developed to detect
urea. Nanocomposites comprises immobilised urease enzyme, graphene
nanoplatelets, and graphitized nanodiamonds. Without any complicated
reactions, this nanocomposite is a sensitive and produces direct signal in
the form of current at 0 V. Compared to other similar methods, this
platform shown high sensitivity and a limit of detection of 832.5 µM
with 20 s response time [176]. As a result, using this nanocomposite
improves the sensing abilities and enzyme loading capacity. This work
describes an electrochemical biosensing method for urea detection in
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Sensors and Actuators Reports 5 (2023) 100159
milk samples. Ferric oxide multiwall carbon nanotubes polyaniline
(Fe3O4/MWCNT/PANI-Nafion) nanocomposite has been used to elec­
trochemically modify the glassy carbon electrode. Purified bacterial
urease enzyme was adsorbed on to the nanocomposite-modified elec­
trode using glutaraldehyde as a cross linker. Various electrochemical
methods, were applied to determine urea concentrations in buffer
media, including cyclic voltammetry (CV), differential pulse voltam­
metry (DPV), and chronoamperometry (CA), and detection limit of 67
µM was attained [177]. The created biosensor showed a fast response
time of only 3 seconds and demonstrated good storage stability for up to
60-days. In terms of fast time response this sensor is more suitable for
rapid detection of urea in milk. To detect urea without a mediator, a
nanocomposite of polyaniline (PANI) and graphitized nanodiamond
(GNDs-PANI) was created at room temperature using interfacial poly­
merization. The nanocomposite showed good linearity, with limit of
detection values of 1165.5 µM (for PANI) and 832.5 µM (for GNDs-PANI)
[178].
A chronoamperometric sensor has been developed for the detection
of H2O2, using monodisperse palladium nanoparticles. Palladium
nanoparticles of 1.8, 2.0, and 3.0 nm diameter were produced by py­
rolysis and thioether stabilised. With the help of organic linker, palla­
dium nanoparticles were immobilised on gold electrode surfaces. The
sensor showed good efficiency for detection of H2O2 in milk samples and
phosphate-buffered saline (PBS) solution, achieving the detection limits
of 45 µM and 75 µM, respectively. The lower sensitivity of sensor allows
the detection of H2O2 below the permissible upper limit [179].
A hybrid consisting of graphene, carbon nanotubes, and MnO2 was
used as the sensing component in the construction of a non-enzymatic
amperometric H2O2 sensor. The hybrid was created using a chemical
procedure in a single pot reaction. High electrocatalytic activity of the
electrode was observed due to oxidation of H2O2. The developed sensor
has a detection limit of 0.1 µM and a wide linear range of 1-1.03 × 103
µM for H2O2 in milk samples (R = 0.9998) [180]. Later, a novel elec­
trochemical bioplatform was prepared with graphene oxide doped
eggshell membrane (GO-ESM). A simple sonication process was used to
synthesize GO-ESM bioplatform using GO nano-sheets and ESM. Intro­
ducing an O-ring to the surface of the carbon ceramic electrode (CCE),
the GO- ESM electrochemistry was examined. Prussian blue (PB) was
added to the GO-ESM platform using a simple dip-coating technique.
The resulting modified electrode (PB|GO-ESM|CCE) was then employed
as a novel H2O2 electrochemical sensor. The developed sensor shown
effective amperometric response to H2O2 over a concentration range of
0.125-195 µM, and a detection limit of 0.031 µM (S/N = 3). The PB|
GO-ESM|CCE has been applied for detection of H2O2 content in milk
samples [181]. This designed bio-platform has good practical applica­
tions due to its excellent stability, affordability, nontoxic nature, elec­
trochemical properties, and biocompatibility.
Carbon nanotubes and nano iron oxide were used to create a hybrid
interface, resulting in better performance for the detection of H2O2.
Thermal co-precipitation method was used to create nano iron oxide,
and it was dissolved in a nafionic solution. Multi-walled carbon nano­
tubes were adsorbed by the catalase enzyme and added to the mixture.
The resulting solution was utilised to modify the electrode. Studies on
interference, repeatability, and stability were conducted, and yielded
positive outcomes. The limit of detection and quantification were found
as 3.7 × 10− 3 µM and 0.122 µM, respectively. This platform was suc­
cessfully applied for the detection of H2O2 in milk samples, attributed to
the convincing findings achieved regarding the biosensor’s performance
[182].
In this study, Prussian blue (PB) nanoparticles doped with poly (3,4-
ethylenedioxythiophene (PEDOT) was used to construct electrochemical
sensor for the detection of H2O2.The PEDOT/PB composite with PB
particles ensuring greater stability and increased electron transport
during electrochemical reactions. The PEDOT/PB modified electrode
revealed good catalytic activity toward the electrochemical reduction of
H2O2, and the sensor was used for the H2O2 determination in range of
Himshweta and M. Singh
0.5 to 839 μM, with a detection limit of 0.16 μM. The sensor showed
excellent conductivity, outstanding repeatability, selectivity, and long-
term stability, offering considerable potential for the designing of
H2O2 electrochemical sensors [183].
The specific optical, electrical, thermal, mechanical, and catalytic
properties of graphene-based nanocomposites make them versatile in
materials science. The current study, describes graphene-based ternary
nanocomposite made of poly (3, 4-ethylenedioxythiophene) polystyrene
sulfonate, reduced graphene oxide, and gold nanoparticles (PEDOT:PSS-
rGO-AuNPs) for the detection of H2O2. Compared to each component of
electrode, the hybrid nanocomposite showed superior electrochemical
characteristics and greater stability, demonstrating the synergistic
interaction of PEDOT, rGO, and AuNPs. Horseradish peroxidase (HRP)
was used to assemble the nanocomposite, which was produced via a
simple one-step process. The modified electrode has been used for the
amperometric detection of H2O2 and shown good linear range from 5 to
400 μM, and a low detection limit of 0.08 μM (S/N = 3). This biosensor
was stable and unresponsive to interfering compounds, and was useful
to detect H2O2 in tap water and bovine milk samples. The improved
sensing properties could be attributed to the close contact of AuNPs with
the rough surface of the PEDOT:PSS-rGO nanocomposite, which serves
as an excellent substrate for the growth of nanoparticles due to its high
electrical conductivity and wide surface area [184]. Only 5.9% of the
current signal was lost after 7 days, demonstrating the good reliability of
PEDOT:PSS-rGO-AuNPs-HRP for detecting H2O2.
In this study, nafion (Nf) and Pd@Ag bimetallic nanoparticles sup­
ported on (3-aminopropyl) triethoxysilane (APTES) functionalized
reduced graphene oxide (rGO-NH2) glassy carbon (GC) electrode were
used to construct a sensitive, selective, and stable electrochemical sensor
for the detection of H2O2. The powerful synergistic impact of Pd and Ag
nanoparticles led to a linear range for H2O2 determination of 2- 1.95 ×
104 µM, with a detection limit of 0.7 µA and sensitivity of 1307.46 µA
mM− 1 cm− 2. The results demonstrated that H2O2 in real samples can be
determined using the novel Nf/Pd@Ag/rGO-NH2/GC sensor [185].
For non-enzymatic electrochemical detection of H2O2, highly
dispersed platinum (Pt) based nanoparticles with higher electrocatalytic
selectivity and stability are highly required. In this study, the platinum
nickel (PtNi) alloy nanoparticles immersed in N-doped carbon nano­
fibers (PtNi/NCNFs) by pyrolyzing electrospun nanofiber composites
made of polyvinyl pyrrolidone and Pt/Ni-based salts. Compared to Pt/
NCNFs, Ni/NCNFs, and other PtNi/NCNFs samples, the electrochemical
tests indicated that PtNi/NCNFs (3:1) exhibited the best electrocatalytic
activity for H2O2 reduction. The proposed sensor displayed high selec­
tivity and low detection limit of 0.0375 µM. Due to the uniformity of
PtNi/NCNFs and the embedded structure, it prevents nanoparticle ag­
gregation, and showed acceptable recoveries for H2O2 detection in milk
samples [186]. After 15 days, the sensor retained its 98.5% of the initial
current response, indicating the good stability. This confirms that
embedding alloy nanoparticles into NCNFs could prevent detachment
and aggregation of PtNi alloy nanoparticles, thereby improve the sta­
bility of the nanocomposite.
Similarly a non-enzymatic electrochemical sensor based on a laser-
scribed graphene electrode embellished with silver nanoparticles
(LSG-Ag) is used to detect H2O2. This inexpensive method, uses a Digital
versatile disc (DVD) laser, enabled the direct production of graphene
oxide and reduces it into an electrochemical transducer with conductive,
light-weight, reduced, and flexible properties. The LSG-Ag sensor
demonstrated a strong catalytic reaction for H2O2 reduction at -0.5 V
and a quick amperometric reaction time of 3 s. The sensor showed a
detection limit of 7.9 µM, strong repeatability with reproducibility of 3%
and 4.5% relative standard deviation (RSD), respectively. Additionally,
the LSG-Ag retained its function after being bent continuously for 40
times, but after 80 times 13% loss in current was recorded. The devel­
opment of electrochemical systems can be facilitated by the use of laser-
induced sensors, as they offer a suitable platform for flexible, depend­
able, sensitive, and selective detection of analytes [187].
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Sensors and Actuators Reports 5 (2023) 100159
On a glassy carbon electrode (GCE), various interfaces were built
using platinum nanoparticles (Pt NPs), ferumoxytol (Fer), and reduced
graphene oxide (rGO) to create a high-performance amperometric H2O2
sensor. The composites (rGO, Fer, and Pt NPs) have a synergistic cata­
lytic effect on the various interfaces, resulting in higher electrocatalytic
activity toward the reduction of H2O2 with a low detection limit of
(~0.38 μM), a linear range (0.4-10, 7.5- 4.3 × 103, 4.9 × 103 -1.08 × 104
μM) at working voltage of +0.1 V (vs. Ag/AgCl), and high sensitivity
(340 μA mM− 1 cm− 2, n= 4). After holding for 15 days at room tem­
perature, the sensor maintained a sensitivity reaction to H2O2 of
approximately 97%. It was successfully used to measure H2O2 in milk
samples [188]. For both industrial and biological applications, the
development of high-performance platinum (Pt)-free non-enzymatic
H2O2 sensors is crucial. Here, the pyrolysis of tris (2, 2′ -bipyridyl)
ruthenium(II) chloride (TBRC) with carbon to produce ultrafine,
surface-clean, evenly dispersed ruthenium nanoparticles and nitride
carbon (Ru/NC) has been reported. To create ultrafine and highly
distributed Ru NPs (1.20 nm in diameter) on nitride carbon substrate,
the nitrogen (N)-containing Ru complex of TBRC was used as the metal
precursor. The designed Ru/NC-800 sensor showed detection limit of
0.468 µM for the non-enzymatic detection of H2O2. This study offers a
simple method for simultaneously producing ultrafine metal nano­
particles and nitride carbon for electrocatalytic non-enzymatic sensing
[189] .
To sensitively measure H2O2, copper/cuprous oxide (Cu/Cu2O)
nanocomposites were electrodeposited on a fluorine doped tin oxide
(FTO) glass substrate. The Cu/ Cu2O nanocomposites were created on
FTO using magnetic agitation and a constant current density of 0.4 mA
cm− 2.The constructed Cu/Cu2O/FTO electrode shown outstanding
electrocatalytic activity for H2O2 oxidation due to synergistic effects of
Cu and Cu2O. By using linear sweep voltammetry and amperometry, the
electrocatalytic activity of Cu/Cu2O/FTO was assessed. The designed
sensor had a linear range of 0.2-2 × 103 μM for H2O2, with a detection
limit of 0.04 μM. The electrodeposited Cu/Cu2O nanocomposites are
promising nanomaterials for electrochemical sensors, as shown by the
successful use of the proposed H2O2 sensor for the measurement of H2O2
in milk samples [190].
Researchers have placed a significant emphasis on non-enzymatic
electrochemical sensors for the simultaneous detection of glucose,
H2O2, and formaldehyde. These sensors have quick response times,
convenient operation, and excellent sensitivity. In this project, CuO
nanoparticles on carbon cloth (CuO NPs/CC) were promptly prepared
using the flame synthesis, which combines the benefits of simple oper­
ation, reduced time, and low cost. The (CuO NPs/CC) was successfully
employed as a working electrode to create a non-enzymatic electro­
chemical sensor. The results reveal that glucose has a detection limit of
6.8 × 10− 3 μM (S/N = 3) and sensitivity of 12,050 μm AM− 1 cm− 2, while
the H2O2 has a detection limit of 0.034 μM (S/N = 3) and a sensitivity of
1479 mM− 1 cm− 2. Additionally, the sensitivity of formaldehyde is 2406
μA mM− 1 cm− 2, and the detection limit is 0.026 μM (S / N = 3). It
demonstrates that flame-synthesised CuO NPs/CC electrocatalysis offers
greater opportunities for the determination of glucose, H2O2, and
formaldehyde [191].
In order to meet the requirements in the fields of biology, medicine,
food production, and the environment, it is essential to develop tech­
niques for monitoring H2O2. Here, the second (G2.0) and third genera­
tion (G3.0) poly(amidoamine) PAMAM dendrimers were designed, and
subsequently coated with gold nanoparticles to create G2.0 and G3.0
Vio-PAMAM-AuNPs based sesnor. Glassy carbon electrode (GCE) was
coated with the G2.0 and G3.0 Vio-PAMAM-AuNPs to create the cor­
responding G2.0 and G3.0 Vio-PAMAM-AuNPs/GCE modified elec­
trodes. A nonenzymatic sensor based on the G3.0 Vio-PAMAM-AuNPs/
GCE for the detection of H2O2 has been produced, which showed
outstanding electrocatalytic activity towards reduction of H2O2 and a
greater current response compared to the G2.0 Vio-PAMAM-AuNPs/
GCE. With a detection limit of 2.7 × 104 μM and high sensitivity of
Himshweta and M. Singh
202.7 µmA mM− 1 cm− 2, the designed nonenzymatic sensor has exhibi­
ted good performance towards H2O2 detection in the wide linear range
of 100 to 6.2 × 103 μM. The modified G3.0 Vio-PAMAMAuNPs/GCE
electrode with a dendritic structure contained a large number of viol­
ogen mediator and AuNPs. The sesnor demonstrated good operational
and long-time stability, and the quantification of H2O2 in real samples
was confirmed by standard method [192]. Furthermore, the perfor­
mance of the nonenzymatic sensor was monitored for 30 days in the
absence and presence of H2O2 to evaluate its long-term ability.
By combining a 3D-structured glassy carbon electrode (GCE) with
carbon nanotubes (CNTs), reduced graphene oxide (RGO), and platinum
nanoparticles (Pt NPs), an enzyme-free H2O2 electrochemical sensor was
created. The carbon-based nanocomposites (RGO/CNTs) and Pt NPs
implanted 3D interfaces work synergistically to increase conductivity
and catalytic ability, resulting in outstanding electrocatalytic properties
for H2O2 reduction. RGO/CNTs-Pt/GCE operates at a working voltage of
-0.2 V (vs. Ag/AgCl) and offers high sensitivity (347 5 μA mM− 1 cm− 2, n
= 3) with a linear range of 0.3- 18 μM and 10- 4 × 103, and a detection
limit 0.31 μM. The sensor was successfully used to identify H2O2 in milk
samples, which is significant for detecting the misuse of H2O2 in milk.
Moreover, it is interference-resistant and retains long-term stability
[193].
The High cost of precious metal electrocatalysts and in­
strumentations has limits the applications of H2O2 electrochemical
sensors. To address this issue, a highly active non-precious metal cobalt/
carbon nanocomposite electrocatalyst for effective H2O2 detection on a
variety of sensor platforms, such as screen-printed electrodes (SPEs),
free-standing film electrodes, and traditional glassy carbon electrodes
(GCEs) has been designed. The nanocomposite was produced by
carbonating electrospun polyacrylonitrile nanofibers containing ZIF-67
(zeolitic imidazole framework) particles. The resulting hollow meso­
porous carbon particles and exposed cobalt nanoparticles, are contained
in a hierarchically porous nitrogen-doped carbon nanofiber film, which
are electrically highly conductive and possess enough mechanical
strength to be used as a self-standing film. As a result, it displays
detection limit of 10 μM when evaluated using GCEs. The linear detec­
tion range increases to 5.0 × 104 μM when used as free-standing film
electrodes. Additionally, when combined with a portable potentiostat
and a cell phone, it allows for the mobile detection of H2O2 on SPEs
[194].
Here, a high surface area porous carbon hybrid made of reduced
graphene oxide nanosheets (rGO) and carbon nanotubes (CNTs) is
treated with cathodic polarisation to deposit ultralow mass loadings of
platinum (Pt) nanoparticles. With detection limit of 1 μM, linear
detection up to 1.5 × 104 μM, and a high sensitivity of 2027 μA/ mM− 1
cm− 2. The Pt/rGO-CNT hybrid exhibits good H2O2 sensing characteris­
tics compared to previoulsy reported Pt/carbon material based H2O2
sensors. This sensor can identify trace amounts of H2O2 in milk and juice
samples [195]. Non-enzymatic electrochemical sensor using Pt-based
nanoparticles was created for H2O2 detection. The nanomaterial was
synthesized on FTO (fluorine-doped tin oxide) electrode using a drop
casting process. The amperometric and cyclic voltammetric results
revealed that PtNiCo-NPs shown greater electrochemical performance
than other mono- or bimetallic materials. With a good sensitivity of
1374.4 µA mM− 1 cm− 2 at a scan rate of 20 mV s− 1 (vs. Ag/AgCl), the
catalytic performance for H2O2 sensing on PtNiCo-NPs had a wide linear
range of 5 to 1.65 × 104 μM [196]. This study presents, a novel method
for creating ternary nanomaterials for H2O2 sensing with outstanding
electrochemical properties.
Researchers aim to design novel inorganic nanomaterials for the
substantial low-level detection of food preservative compounds. In
current study, sonochemical technique was used to create a novel grass-
like vanadium disulfide (GL-VS2) without the use of surfactants or
templates. The electrocatalyst VS2 helps to reduce H2O2. In electro­
chemical experiments, the glassy carbon electrode (GL-VS2/GCE)
modified with GL-VS2 shows more electrocatalytic activity and a lower
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Sensors and Actuators Reports 5 (2023) 100159
reduction potential than unmodified GCE. The GL-VS2/GCE also dem­
onstrates a broad linear response range (0.1-260 µM), sensitivity of (0.23
µA µM− 1 cm -2), detection limit of 0.026 µM and great selectivity for
H2O2 detection. The GL-VS2/GCE electrode is suitable for H2O2 detec­
tion in milk and urine samples [197]. The GL-VS2 electrochemical sensor
maintained 97.2% of its initial current response for 50 M H2O2 in 0.1 M
NaOH solution for up to 8 days.
An easy one-step method was employed to create cuprous oxide/
silver (Cu2O/Ag) nanocomposites, which been used to build an elec­
trochemical sensor for the detection of H2O2.In this procedure, glucose
was used to convert AgNO3 and Cu (NO3)2 to Cu2O/Ag nanocomposites
in the presence of hexadecyl trimethyl ammonium bromide (CTAB).
Cu2O/Ag nanocomposites with evenly dispersed and closely coupled
Cu2O and Ag nanoparticles were produced. The Cu2O and Ag particles
had size of less than 20 nm and less than 100 nm, respectively. The
Cu2O/Ag nanocomposites-based sensor shown good performance for
H2O2 base on electrochemical measurements, with a linear range of 0.2
to 4000 µM, a high sensitivity of 87.0 µA mM− 1 cm− 2, and a detection
limit of 0.2 µM. The anti-interference capability tests have shown that
this sensor has an excellent H2O2 selectivity. Additionally, the H2O2
recovery experiments in diluted milk solution indicate that it can be
used for routine H2O2 analysis [198].
Cu-based metal organic frameworks (MOFs), have many metal sites,
high surface area, and easy synthesis, are promising features of sensing
materials. In this study, a novel electrochemical sensor for H2O2, a novel
three-dimensional (3D) flower-like Cu-MOF was synthesised. It was
coupled with ultra-thin MXene nanosheets. The structure properties of
MOFs, the nature of Cu-contained nanomaterials, high electrical con­
ductivity of MXene, and catalytic activity of Cu-MOF/MXene/GCE
during the electrocatalytic reduction of H2O2 resulting in the ultra-
sensitive detection of H2O2. Using chronoamperometry at a detection
potential of -0.35 V, the Cu-MOF/MXene/GCE demonstrated a broad
linear range from 1 to 6.129 × 103 µM, and the detection limit found to
be 0.35 µM. Due to the decreased detection potential and specific
catalysis of CuMOF to H2O2, the sensor offers strong selectivity, good
anti-interference properties. The electrochemical sensor can successfully
detect H2O2 in milk and serum samples. The suggested sensor has sub­
stantial potential for real-time measurement and monitoring of H2O2 in
foods and biological samples due to its high sensitivity, quick detection,
and simple operation [48].
Recent advances in H2O2 detection has become a promising area of
study in electrochemical sensing. This study successfully created an
enzymatic-free electrochemical sensor using cuprous manganite/glass
carbon electrode (CuMn2O4/GCE). for The sensor showed good
amperometric detection ability for H2O2 by synthesising CuMn2O4/GCE
spinel nanoflakes through coprecipitation and pyrolysis methods, and
drop coating them onto the surface of a bare glassy carbon electrode
(GCE). The CuMn2O4/GCE sensor displayed high sensitivity of 3.420
AM− 1 cm− 2 and a detection limit of 0.012 µM, compared to previously
described spinel AB2O4-based electrocatalysts in phosphate buffer so­
lution. The Spinel nanoflakes CuMn2O4 has high electrochemical ac­
tivity, porous structure and synergistic interaction between Cu and Mn,
promotes electron transfer and increase active sites are the key features
of the sensor. The presence of high levels of Cu+ and Mn3+ ions in the
nanocatalyst facilitate the capture and reduction of trace H2O2. Addi­
tionally, the CuMn2O4/GCE sensor displays an acceptable recovery in
monitoring H2O2 produced from rat blood, commercial milk, and dis­
infectants [199] .
In healthcare, processes, food, and other areas, qualitative or quan­
titative assessments of H2O2 are crucial due to its significant part in the
signalling pathways of many diseases, are extremely important. To
address this need, an electrochemical ultra-high nanocomposites made
of polypyrrole (PPy), graphene oxide (GO), and gold nanoparticles
(AuNPs) has been developed for the detection of H2O2. The sensor can
detect H2O2 at a negative potential in the range of -20 V vs. Ag/AgCl.
The created (PPy-GO-AuNPs/GCE) electrode demonstrated high
Himshweta and M. Singh
electrocatalytic activity towards H2O2. The nonenzymatic H2O2 sensor
showed a higher sensitivity of 41.35 µA/mM amperometric response,
from linear range between 2.5 × 103- 2.5 × 104 µM and detection limit
of 5 µM (S/N = 3). The nanocomposite sensor was successfully applied
for H2O2 sensing in milk sample [200].
One of the most common forms of adulteration is the replacement of
high-valued species milk with lower-cost cow milk, and this is the most
prevalent form of milk adulteration where mixing of various kinds of
milk species For example, the addition of cow’s milk to buffalo’s milk is
a common form of milk adulteration [201]. Cow’s milk may be used in
adulteration because it is less expensive and produces more milk than
buffalos. This form of adulteration must be halted because the cow’s
milk protein leads to allergy to some individuals and particularly in
children; in addition, also have some adverse effects on human [202]. As
a result, the purpose of this study is to explain the current and advanced
analytical methods used for species verification of milk and dairy
products are discussed in below section.
3. Milk adulteration with milk from different species
The simplest way to adulterate milk is through blending the milk
from other sources and different animal species. Due to genetic and non-
genetic variability, detecting adulterated milk quantitatively is signifi­
cantly more difficult [23]. Sheep, goats, and buffalo milk are more
expensive than cow’s milk, so it has been a common practice for many
years to mix sheep, goats, and buffalo’s milk with cow’s milk. The
Protected Designation of Origin (PDO) councils are highly concerned to
ensuring the quality and authenticity of the cheese they produce.
Therefore, they rely on analytical tools to verify and control any adul­
teration throughout the production process. In accordance with inter­
national standards, there are limited rapid methods that can be utilised
in the field due to their lack of verification.
Lateral flow immunochromatography is a diagnostic technique that
utilizes antibodies to detect the presence a specific protein or an antigen,
in a sample. The sample is applied to a test strip, which contains a series
of membrane layers that capture and concentrate the target substance,
producing a visible signal, such as a colored line, when it is present. This
technique is widely used in medical diagnostics, food safety, and envi­
ronmental monitoring due to its simplicity, rapidity, and low cost [203].
By applying this method, the study aimed to comply with the criteria set
by the Association of Official Analytical Chemists (AOAC), was to vali­
date a quick test using lateral flow immunochromatography for the
identification of cow’s milk from other species milk, such as buffalo’s
milk. 146 known negative samples from various animals were analysed,
and found no false-positive results. In all the batches, 0.5% of cow’s milk
adulteration was detected, confirming the threshold value set in the
probability of detection (POD) study, i.e. 1.00 (0.94–1.00). Such a level
is below the permissible limit set by European Commission Regulation
(EC) for cow’s milk in other species milk, which is 1%. This quick test for
detecting cow’s milk in other species milk has been proven to be an
appropriate method for routine analysis of milk. This type of test is
relatively simple to execute and can be done by untrained personnel
during milk collection at the farm or upon arrival at dairies.
Additionally, sensometric methods are also useful for the monitoring
of the sensory characteristics of milk samples that have been adulterated
with whey. Sensometric methods are used to evaluate and analyze
sensory data, such as data obtained from human taste panels or con­
sumer preferences. These methods aim to understand the relationships
between the sensory properties of products and their acceptability by
consumers. Examples of sensometric methods include principal
component analysis (PCA), discriminant analysis, and cluster analysis. A
sensometric approach combines quantitative descriptive analysis with
chemometric methods to identify characteristics that enable the detec­
tion of intentional adulteration of raw fluid milk with cheese whey.
Chemometric methods, on the other hand, are used to analyze chemical
data, such as data obtained from spectroscopic or chromatographic
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Sensors and Actuators Reports 5 (2023) 100159
techniques. These methods aim to understand the relationships between
chemical properties and the behavior of chemical systems. Examples of
chemometric methods include partial least squares (PLS) regression,
principal component analysis (PCA), and artificial neural networks.
Chemometric techniques in combination with descriptive sensory
testing can be applied to check the authenticity of milk due to the
presence of cheese whey [204]. However, due to inconsistent results
found during the sample analysis, the efficiency of principal component
analysis (PCA) and hierarchical cluster analysis (HCA) for the identifi­
cation of cheese whey in milk is not fully demonstrated.
Lateral flow method is useful for the identification of soymilk in
whole bovine milk. Here, soymilk protein in adulterated milk competes
soymilk protein at the test line compete for a limited number of rabbit
anti-soy protein antibodies that are attached to AuNPs. Anti-rabbit IgG
was immobilised at the control line to ensure the flow characteristics of
antibody-conjugated AuNPs. The presence of soymilk in milk is shown
by the complete absence or faint band at the test line. The detection limit
for soymilk in whole bovine milk was 1.75% (v/v), and the results were
obtained in 5 minutes. A built-in lateral flow device can be used to test
for soymilk in milk [205]. The present methods for identifying cow milk
and goat milk adulteration are time consuming and labour intensive.
However, the lateral flow immunoassay (LFIA can be used for detection
of cow milk aduletration in goat milk. By using a particular monoclonal
antibody (mAb) tagged with colloidal gold nanoparticles (GNPs) that
bind to cow milk casein, a competitive lateral flow immunoassay (LFIA)
strip was created. The detection limit for cow milk in goat milk was
found 0.07%, which offers greater sensitivity over commercial ELISA
(Enzyme linked Immunosorbent Assay) kit that have detection limit of
0.1% [206]. These findings imply that cow milk adulteration/ contam­
ination with goat milk can be monitored within 6 minutes by GNPs-LFIA
strip. Hence, strip method can be used as fast screening assay for real
time monitoring of cow’s milk adulteration in goat milk.
This paper describes the first immunoplatforms used to detect
adulteration of milk with milk or colostrum from other animals. The
electrochemical bioplatforms developed enable the accurate measure­
ment of immunoglobulin G (IgG) from cows, sheep, or goats. The process
involves sandwiching horseradish peroxidase (HRP)-conjugated and
unconjugated IgGs from each animal species onto magnetic microbeads
(MBs) used as solid supports. Amperometric transduction is achieved
using the H2O2/hydroquinone system at disposable electrodes. The
immunoplatforms provide detection limit of 0.0023, 0.0026 and 0.0020
µM, for bovine, ovine, and caprine IgGs, respectively. The bioplatforms
can be effectively used to identify the specific content of the target IgGs
in milk samples from various animals (cows, sheep, and goats) and types
(colostrum, raw, and pasteurised), without any matrix impact, only after
sample dilution. Additionally, used to find milk that had been adulter­
ated with other species milk at 0.1% level lower than those mandated by
European law (1.0%, v/v). The method relies on measuring the total
amount of the three target IgGs in raw milks and can be used to identify
milk adulterated with colostrum. The proposed method provides
essential information about the milk sample, such as its animal origin, if
it had heat treatment, and its adulteration with milk or colostrum from
other species, all within 30 min [207]. Compared to previously reported
methods, the current method is an effective procedure for detecting milk
adulteration with milk from other animals. However, its higher time
requirement and detection limits still need improvement.
Using dairy products as a paradigm, a paper-based DNA biosensor for
food authenticity was developed. DNA was extracted from milk samples,
and by specific DNA probes, species-specific DNA sequences were
amplified and recognised by the biosensor. Gold nanoparticles were
used for optical detection. Three species, namely cow, sheep, and goat
PCR product, were detected using the biosensor. The detection limit was
1.6 × 10− 9 µM for cow, and 3.1 × 10− 9 µM for sheep, respectively.
Furthermore, based on binary mixtures of cows’, ewes’ and goats’ milk
yogurts, adulteration down to 0.01% was detected, whereas ewes’ and
goats’ yogurts contained 0.01 to 5% cows’ yogurt. The method can also
Himshweta and M. Singh
be used to find the contaminants in beef, olive oil, and beans food
products [208]. The biosensor works quickly and ends within 10–15
minutes after sample application. Results are visible by the naked eye,
and require less time than the electrochemical method described earlier.
For accurate labeling and quality assessment, authenticity of cow,
goat, ewe, and buffalo milk is a prevalent problem globally. These milk
species are commonly adulterated with cow milk, for great economic
worth. Instead of DNA and protein-based techniques, non-invasive and
rapid spectroscopic methods have become increasingly popular. Syn­
chronized fluorescence spectroscopy (SFS) is considered a fast, non-
destructive, non-intrusive, and relatively economical analytical tech­
nique. The key benefit of SFS is that it can simultaneously provide in­
formation on structure and stability while analyzing fluorescent
molecules at low concentrations. During SFS, spectra are acquired by
scanning the excitation and emission monochromators simultaneously
with a specific wavelength difference (Δλ). Given its unique fingerprint
identification ability, SFS has been utilized for food sample analysis
[209]. Therefore, SFS method is used to distinguish between cow, goat,
sheep, and buffalo milk species. The method is based on variations in the
type and amounts of fluorophore compounds in various milk species. In
order to do this, SF spectra of milk species are captured in the excitation
range of 250 and 550 nm wavelength. Following the identification of the
binary mixture type (cow-cow, goat-cow, or ewe-cow), partial least
square (PLS) models have been established for quantifying cow milk in
other milk species. For goat-cow, ewe-cow, and buffalo-milk, the limit of
detection values were found to be 3.52%, 4.06%, and 3.52%, respec­
tively [209]. In spite of this, much work needs to be done in order to
reduce the detection limit of the method as per the European Commis­
sion, which is 1%. For the quick identification of cow-milk adulteration
with buffalo milk, a competitive lateral flow immunoassay utilising
amorphous carbon nanoparticles (CNPs) and non-immunoglobulin an­
tigen has been developed. Purified polyclonal antibodies were conju­
gated to CNPs and sprayed on a conjugate pad against a particular
buffalo milk protein fraction. The control line included anti-rabbit an­
tibodies produced in goats (0.5 g/cm), whereas the test line was made up
of buffalo’s skimmed milk proteins (1.6 g/cm). If there is no buffalo milk
present in the sample, a black or grey test line would be visible. Test lines
disappear when cow’s milk is 5% adulterated with buffalo milk. The test
may be conducted on hot milk samples collected at milk collection
centers [210].
Two varieties of cheese, Mozzarella di Bufala Campana and Feta,
with Protected Designation of Origin and must be regularly examined to
prevent fraudulent adulteration with bovine milk. This ensures that
consumers truly purchase these high-end goods and prevent health
problems associated with bovine milk allergy. For very first time, a
silicon-based photonic immunosensor has been used to detect the
adulteration of feta and mozzarella cheese with cow’s milk. The pho­
tonic immunosensor utilises monolithically integrated Mach-Zehnder
interferometers and corresponding light sources on a silicon chip. The
competitive immunoassay occurred in three steps, involving a reaction
with the antiserum, a biotinylated anti-rabbit IgG antibody, and strep­
tavidin. The antiserum used was a rabbit polyclonal antiserum produced
against bovine-casein. This assay greatly reduced the non-specific signal
caused by the cheese matrix, and the test was completed in just 9 mi­
nutes. Moreover, it could measure bovine cheese in mozzarella or feta as
low as 0.5 and 0.25% (w/w), respectively. Both quantification limits fall
below the European commission (EC) regulations. Moreover, the
maximum permissible content of bovine milk in mozzarella and feta
according to EC is 1% (w/w) [211]. Livas and co-workers have devel­
oped a new electrochemical immunosensing device that combines a
3D-printed micro cell, two thermoplastic electrodes and a Sb2O5/S­
nO2-modified working electrode. The evaluation of the cheese adulter­
ation, specifically the addition of cow’s milk to cheese made from
ewe/goat’s milk, shows the relevance of the immunodevice for ana­
lysing complex matrices. The device works through two steps (i)
developing a competitive immunoassay using biotinylated anti-bovine
26
Sensors and Actuators Reports 5 (2023) 100159
k-casein tagged with streptavidin-conjugated CdSe/ ZnS quantum dots
(QDs) on the surface of the screen-printed working electrode, and (ii)
stripping voltammetric based detection of released Cd(II) after the acidic
dissolution of Cd-based QDs. The detection limit of the sensor was found
to be 0.07% (v/v) cow’s milk in ewe/goat’s cheese [212] as summarised
in Table 3. The proposed sensor is an easy-to-build and ultrasensitive
immunodetection device with broad application to various QDs-based
bioassays. This is due to the synergistic combination of 3D printing,
QD labeling, and in-situ production of nanostructured sensing surface.
Milk, being one of the most significant multicomponent super foods,
requires quality control throughout the production process, from the
farm to the finished goods. Surface-Enhanced Infrared Absorption
Spectroscopy (SEIRA) is a technique that combines the principles of
surface-enhanced Raman spectroscopy (SERS) and infrared absorption
spectroscopy. It is used to enhance the sensitivity of infrared spectros­
copy for the detection of molecules adsorbed on metal surfaces, orien­
tation and conformation of molecules on the surface [213].Whereas,
artificial neural networks (ANNs) are computational models that are
designed to simulate the behaviour of biological neurons in the brain.
ANNs are composed of multiple layers of interconnected nodes, or
neurons, which can process and transmit information. So in this study, a
localised surface plasmon resonance optical sensor based on glass sub­
strate coated with citrate-capped silver nanoparticles (Cit-AgNP) was
designed for the qualitative assessment of milk. The synthesis of this
sensor required the surface modification of glass slides before coating
them with Cit-AgNPs. To qualitatively distinguish cow, camel, goat,
buffalo, and babies formula powdered milk, the SEIRA sensor connected
to an ANN has been developed. Additionally, it may be used to quantify
the major milk constituents, including fat, casein, urea, and lactose in
each kind of milk. The newly designed sensor offers a number of benefits
over the established methods of milk analysis utilising MilkoScanTM (a
device frequently used for determining the primary milk components,
milk abnormalities, and assessing the safety of milk products), ANN
enables the removal of pre-treatment processes, a small sample amount,
quick operation, and useful for on-site analysis, thus reducing waste
generation [213].
The first enzyme-free immunoassay (optical and electrochemical)
that may be employed as a detection bioplatforms of bovine IgG as goat
milk adulterant is presented here. In a competitive colorimetric micro­
plate immunoassay, Prussian blue nanoparticles (PBNPs) were utilised
as antibody catalytic labels. Due to the enhanced catalytic activity of
PBNPs compared to natural peroxidase, higher sensitivity was achieved
(0.01% cow/goat volume ratio), which is 100 times lower than the
maximum permitted by European regulation at 1% (v/v). The remark­
able stability of the antibody-PBNP bioconjugates over 4 weeks (>94%
of the original reaction) further supports the effective anchoring of the
antibodies to the surface of the PBNPs. However, for the detection of
bovine IgG, a label-free voltammetric immunoassay was created. The
redox pair of ferri-ferrocyanide and the screen-printed gold electrodes
modified with bovine IgG antibodies was used as a sensing principle.
Both immunoassays showed a selective response solely to bovine IgG,
when the selectivity of the generated immunoassays was tested using
other species milk with comparable composition [214]. Newly devel­
oped enzyme-free immunoassays have a variety of appealing features for
the identification of milk adulteration. These immunoassay are different
from conventional ones and can be applied to routine milk analysis in
quality control laboratories (optical immunoassay) or on-site check­
points (electrochemical immunoassay) using wireless electrochemical
detectors.
4. Conclusion and future outlook
Milk is considered a balanced meal for newborns and children, as
well as a good source of proteins and minerals for everyone. However, its
poor quality is becoming an increasing concern, due to the widespread
adulteration of milk with chemicals and other substances, in order to
Himshweta and M. Singh
Table 3
Adulteration of dairy products with milk of other species.
Sample Nanomaterial
Milk CNPs
Whole bovine milk Rabbit anti-soy protein antibodies conjugated
to gold nanoparticles (AuNPs)
Dairy products mAb labeled with colloidal gold nanoparticles
Ewe/goat’s cheese Streptavidin-conjugated CdSe/ ZnS quantum
dots (QDs)
Milk PBNPs
Milk with milk from HRP-MBs
other animal species
Abbreviations: LFA-Lateral flow immunoassay; carbon nanoparticles- CNPs; Monoclonal antibody –mAb;CNPs-carbon nanoparticles;; horseradish peroxidase mag­
netic beads- HRP-MBs; Ab-Prussian blue nanoparticles – PBNPs.
serve their own interests. This necessitates the development of sensitive
and rapid testing technologies for commonly used milk adulterants.
Nanotechnology is making a substantial contribution to the accurate and
sensitive identification of adulterants in milk samples, as explained in
this article.
In this review, we have summarised the nanoensembles platform as a
sensing material for the detection of illegal adulterants in dairy prod­
ucts. Nanomaterials enable the signal amplification of sensors such as
optical and electrochemical sensors, thus enhancing the methods
sensitivity in the process. It is important to note that this review focuses
on assays of milk adulterants that use nanomaterials such as nanorods,
nanoclusters, nanoparticles, carbon dots, quantum dots, nano­
composites, nanocrystals, fullernes, nanoplates, nanowires, and nano­
tubes. Furthermore, we have examined in detail various mechanisms in
adulterants detection, through optical mode of detection by fluorescence
resonance energy transfer (FRET), chemiluminescence, surface-
enhanced Raman scattering (SERS), colorimetric aggregation, inner
filter effect, and localised surface plasmon resonance (LSPR) based
nanosensors. Additionally, we also discuss the different types of elec­
trochemical nanosensors: impedimetric, voltammetric and ampero­
metric. According to research articles, gold nanoparticles and silver
nanoparticles based sensing, either by aggregation or H-bonding, seems
to be a successful strategy. On the other hand, many scientists have
developed various nanomaterials that address milk adulteration using
optical sensors including colorimetric, SERS and SPR techniques. Aside
from the approaches mentioned above, a few papers have also explored
FRET and eletrochemilumniscence, among others. The high recognition
of specific molecular targets that aptamers naturally possess may in­
crease the assay selectivity. Although, metal nanoparticle-based colori­
metric tests are quick and easy to perform, they have some drawbacks,
including unstable functionalized metal nanoparticles, poor selectivity,
and limited availability. There are some limitations of fluorescence
sensors that need to be improved by enhancing the purification method
to increase their stability, uniform optical properties, better water sol­
ubility, easy surface functionalization, and improving characterization
methods. While, electrochemical nanosensors exhibits a quick response
time, higher sensitivity and lower energy consumptions, however, some
of the adulterants are poor electroactive, which limits their utility in
electrochemical sensors. So, it is required to design nanomaterials
incorporated electrochemical sensing probes for the detection of adul­
terants. Similarly, for immunoassay nanosensors are based on antigen-
antibody interactions. The main disadvantages of this method are that
specific antibody or antigen should be produced to detect particular
antigen or antibody, and false results can be achieved due to non-specific
interactions between antigen and antibody.
Species identification in milk and dairy products has gained interest
due to its significance in food authentication, and for customer trans­
parency. So far, several methods for determining species authentication
in dairy products have been suggested. Recently, DNA based, surface-
enhanced infrared absorption spectroscopy (SEIRA) sensor with
Sensors and Actuators Reports 5 (2023) 100159
Target analyte Detection method LOD (%, v/v) References
Cow’s milk with LFA No visible test line [210]
buffalo’s milk
soymilk in milk LFA 1.75 [205]
Cow milk in goat milk LFA 0.07 [206]
Cow’s milk in ewe/ 0.07 [212]
goat’s cheese
Cow’s milk into Voltammetric ≤0.1 [214]
goat’s milk immunoassay
Colostrum Amperometric 0.0023, 0.0026 and 0.0020 µM [207]
bovine, ovine, and caprine IgGs
artificial neural networking (ANN), synchronous fluorescence spec­
troscopy (SFS), lateral flow immunoassay (LFIA) and immunoassay have
become more prevalent due to their benefits in sample preparation,
rapidity, non-destructiveness, simple performance, and on-field use
make them advantageous. However, the drawbacks of these techniques
could result in erroneous negative outcomes due to cross reactivity with
immunochemical tests, or reduced sensitivity with DNA-based tech­
niques and other methods. Moreover, the need for costly machines in
spectroscopic methods, sufficient databases, and multicomponent
analysis may prevent their application.
Considering the future prospects in the field of detecting dairy
adulteration, we highlighted the interest in creating portable, simple-to-
detect, real-time, and quick, in situ monitoring devices for large-scale
and on-field applications. Various novel nanosensors have been
proved their efficacy for the detection of milk adulterants via different
assays. Majority of assays have good linear range and very low limit of
detection, enabling the presence of adulterants at lower concentrations.
However, study should be more focused on the use of novel nano­
structures like nanocrystals, nanocubes and nanoflowers, nanowires,
nanorods, and nanotubes, since the sensing assays are very fewer for
adulterants detection in dairy products. Nanoensembles offer a break­
through in dairy adulterations assay and enable the world to sustain a
safe and healthy environment. However, their development necessitates
good optimization techniques and sophisticated instruments. Addition­
ally, the scientific community is committed to the construction of
nanoensembles based diagnostic platforms, that could transform the
present scenario of the food sectors and meet the growing demands for
food safety and security.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data Availability
Data will be made available on request.
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