Food and Chemical Toxicology 168 (2022) 113307

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Food and Chemical Toxicology

journal homepage: www.elsevier.com/locate/foodchemtox

Biosensor for heavy metals detection in wastewater: A review

Karthik Velusamy a, Selvakumar Periyasamy b, P. Senthil Kumar c, d, *, Gayathri Rangasamy e,
J. Mercy Nisha Pauline a, Pradeep Ramaraju a, Sneka Mohanasundaram a, Dai-Viet Nguyen Vo f, **
a
Department of Industrial Biotechnology, Government College of Technology, Coimbatore, 641013, India
b
Department of Chemical Engineering, School of Mechanical, Chemical and Materials Engineering, Adama Science and Technology University, Adama, 1888, Ethiopia
c
Department of Chemical Engineering, Sri Sivasubramaniya Nadar College of Engineering, Chennai, 603 110, India
d
Centre of Excellence in Water Research (CEWAR), Sri Sivasubramaniya Nadar College of Engineering, Chennai, 603 110, India
e
Department of Sustainable Engineering, Institute of Biotechnology, Saveetha School of Engineering, SIMATS, Chennai, 602105, India
f
Institute of Environmental Sciences, Nguyen Tat Thanh University, Ho Chi Minh City, Viet Nam

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

Handling Editor: Dr. Jose Luis Domingo Pollution due to heavy metals is a global issue in recent years. Initially, there were fewer contaminants, which
has increased exponentially owing to rapid industrialization and various anthropogenic activities. Toxicity due to
Keywords: heavy metals causes a lot of health problems and organ system failure in human beings. It also affects other forms
Wastewater of living beings such as plants, animals and even the microbiota. This has been reported by various press reports
Heavy metals
and research findings. In this review, the production of heavy metals, associated effects on the environment and
Biosensor
the technologies employed for detecting these heavy metals are comprehensively discussed. The analytical in­
Pollutants detection
Reproducibility struments, including biosensors, have been found to be more beneficial than other techniques. Biosensor exhibits
numerous special features, such as reproducibility, reusability, linearity, sensitivity, selectivity, and stability.
Over the last three years, biosensors have also had a detection limit of 65.36 ng/mL for heavy metals. The design
of biosensors, features and types were also explained in detail. The limit of detection for the heavy metals in
wastewater using biosensors was also included with recent references up to the last five years.

2018). The level of toxicity compared among the heavy metals is as

1. Introduction
The earth is composed of four different systems such as geosphere,
atmosphere, biosphere and hydrosphere. These spheres are rich in
minerals and nutrients. More than 35 metals have been identified in the
form of ores. Among these, 23 metals possess a high specific density
greater than 5 g/cm3 and an atomic weight greater than 40.04, implying
that such metals are five times denser than water and toxic to the
environment (Duffus, 2002; Li et al., 2017). Hence, these 23 metals have
been classified as heavy metals, which include bismuth, tin, thallium,
gold, arsenic, cerium, gallium, mercury, chromium, cobalt, antimony,
copper, iron, cadmium, lead, manganese, nickel, silver, uranium, tellu­
rium, platinum, vanadium and zinc. All over the world, heavy metal
pollution is increasing daily and is introduced into the environment by
various activities. Some pollutants are released from fertilizers used in
agriculture, metals and ores from mining and effluent from a myriad of
industries (Rajesh Banu et al., 2021; Karthik et al., 2021a). Hence, the
upper crust of the earth is rich in heavy metals (Masindi and Muedi,
follows: Co < Al < Cr < Pb < Ni < Zn < Cu < Cd < Hg (Mansourri and
Madani, 2016). Mercury, lead and cadmium have received more atten­
tion in recent decades as the side effects caused by these metals are fatal.
Heavy metals routes exist within organisms, such as the entry of heavy
metals into plants via the root system via soil and water, resulting in
bioaccumulation. In animals, it can enter through food and water and it
is called biomagnification. In humans, heavy metals can enter through
dermal exposure, inhalation of metal-containing fumes, and the
gastrointestinal tract, but they only through selective and non-selective
channels as the heavy metals cannot pass through the lipid membrane
(Jan et al., 2015).
Some metals help organisms in their metabolism, but excessive
intake of metals causes some types of ailments. The disease mechanisms
primarily involve free radical release, cell injury, protein conforma­
tional change, enzyme inhibition, neuron damage, promoting apoptosis,
inhibition of neurotransmitters, and Deoxyribonucleic acid (DNA)
destruction (Mohod and Dhote, 2013). Various technologies have
emerged to detect the concentration and the toxicity exhibited by these

* Corresponding author. Department of Chemical Engineering, Sri Sivasubramaniya Nadar College of Engineering, Chennai, 603 110, India.
** Corresponding author.
E-mail addresses: senthilkumarp@ssn.edu.in (P.S. Kumar), vo.nguyen.dai.viet@gmail.com (D.-V. Nguyen Vo).

https://doi.org/10.1016/j.fct.2022.113307
Received 7 May 2022; Received in revised form 1 July 2022; Accepted 13 July 2022
Available online 30 July 2022
0278-6915/© 2022 Elsevier Ltd. All rights reserved.
K. Velusamy et al.
List of abbreviations
CO Carbon monoxide
Al Aluminium
Pb Lead
Cr Chromium
Cd Cadmium
Ni Nickel
Hg Mercury
Zn Zinc
Mg Magnesium
Cu Copper
Fe Iron
Ag Silver
DNA Deoxy Ribonucleic Acid
IUPAC International Union of Pure Applied Chemistry
MCL Most hazardous metal level
heavy metals (Sithara et al., 2017; Karthik et al., 2021b; Velusamy et al.,
2021a, 2022a; Asaithambi et al., 2020). It includes electrothermal
atomic absorption spectrometry, inductively coupled plasma-optical
emission spectrometry, high-pressure liquid chromatography induc­
tively coupled plasma-mass spectrometry, cold vapor atomic absorption
spectrometry, atomic absorption spectrometry, hydride generation
atomic absorption spectrometry and flame atomic absorption spec­
trometry (Arjomandi and Shirkhanloo, 2019). Compared to these tools,
biosensors have major advantages in terms of application, cost, versa­
tility, and are robust. It detects the signal produced by the interaction
between the analyte and biological components, which is proportional
to the heavy metal concentration (Hassani et al., 2020). According to the
International Union of Pure and Applied Chemistry (IUPAC), a biosensor
is a self-integrated device capable of providing certain information
quantitatively or semi-quantitatively by using the biological compo­
nents that directly contact the transducer elements (Pearson et al.,
2000). It detects heavy metals, and provides the level of toxicity and
concentration of heavy metals available in the water. Working princi­
ples, merits and demerits for heavy metal detection using different
technology are shown in Table 1.
From the above-mentioned Table, it is evident that the biosensor has
more advantages for heavy metal detection in soil and wastewater. In
this review, the design, features, and the role of biosensors in heavy
metal detection are briefly discussed.
2. Source of heavy metals in wastewater
Heavy metals are present in the soil, water, air and food in the form
of hydroxides, oxides, sulphate, phosphates, silicates and organic com­
pounds. Heavy metals are introduced into the environment mainly by
natural and anthropogenic processes (Karthik et al., 2022).
2.1. Natural process
The emission of metals occurs naturally in the environment in the
form of volcanic eruptions, rock weathering, biogenic sources, forest
fires, biogenic processes, marine salt and so on. These weathering pro­
cesses lead to the release of metals from their endemic spheres to the
ecospheres. Natural disasters, climate change, soil matrix, hyporheic
exchange and geological elements all have an impact on the quality of
ground water and surface water. Earthquakes, hurricanes, tornadoes,
floods and tsunamis are examples of natural disasters. Flooding is a
major source of heavy metal contamination in water bodies (Akhtar
et al., 2021). Climate change affects not only hydrological and ecolog­
ical systems but also the economy and life. Lead, cadmium, chromium,
2
Food and Chemical Toxicology 168 (2022) 113307
TMT 2,4,6-trimercapto-1,3,5-triazine
LOD Limit of detection
SELEX Systemic Evolution of Ligands by Exponential Enrichment
QCM Quartz Crystal Microbalance
LCD Liquid Crystal Display
MIP Molecularly Imprinted Polymet
SMFC Sediment Microbial Fuel Cell
DMFC Direct Methanol Fuel Cell
CMFC Cube Microbial Fuel Cell
GWA- Guanine nano wire-based Amplification
GNP Gold Nano Particle
GOD Glucose Oxidase
ssDNA Single Stranded Deoxy Ribonucleic Acid
ppm Parts per million
dsDNA Double Stranded Deoxy Ribonucleic Acid
pM Pico Molar
mM Milli Molar
zinc, nickel and copper are the most prevalent heavy metals (Engwa
et al., 2019). However, this is less harmful compared to the emission by
the anthropogenic process.
2.2. Anthropogenic process
This is the major source causing greater pollution by heavy metal
contamination in our ecosystem. The heavy metals are introduced into
the environment mainly by i) deposition of atmospheric particulates, e.
g., fuel combustion; ii) disposal of sewage sludge and effluents, e.g.,
industries, emissions from mining, and automobiles; iii) the heavy
metals from soil and air are also finally discharged into the waterbodies
through rainfall. When exposed to sunlight, the heavy metals present in
water vapor mixes with air which reaches the earth during precipitation
and gets deposited in soil and the metals runoff from soil into water­
bodies like rivers, seas and lakes. Recent data analysis suggests that
globally 20 ha of arable land were irrigated with such heavy metal
contaminated wastewater (Wuana and Okieimen, 2011; Mohamed et al.,
2022; Velusamy et al., 2022a,b). The total concentration of heavy metals
present in various waterbodies during the period between 2010 and
2017 is compared with the World Health Organization (WHO) and
United States Environment Protection Agency (US EPA) prescribed
standards are shown in Table 2.
Natural and anthropogenic processes are two significant sources of
heavy metals. Volcanic eruptions and forest fires were natural processes,
but anthropogenic processes such as fertilizer used in agriculture, un­
derground metal mining and industrial machinery production also result
in the release of heavy metals in wastewater, which will then be dis­
charged into water bodies such as rivers, lakes and seas.
The rising level of heavy metal concentration in the environment was
caused due to urbanization and rapid industrialization. A decrease in
population density leads to a decrease in heavy metal discharge. Hence
the heavy metals contamination is directly proportional to the popula­
tion (Verma and Dwivedi, 2013). The overall process of heavy metal
discharge to the water bodies from various sources is clearly shown in
Fig. 1.
2.3. Mass balance equation
Mass balance equation for the heavy metals in wastewater is given
by: (Wuana and Okieimen, 2011).
Mtotal = (Mi + Maf + Ms + Mm + Mu + Mip + Mow)-(Mvp + Msd)
Mtotal - Total mass of heavy metals in wastewater.
K. Velusamy et al. Food and Chemical Toxicology 168 (2022) 113307

Table 1 Table 2
Principles, advantages and disadvantages of technologies for detecting heavy Comparison of heavy metal concentration present in water bodies with World
metals in wastewater. Health Organizations and United States Environmental Protection Agency
Detection Principle Advantages Disadvantages Reference
standards.
technology S. Metal 2010 2017 Standard Reference
No.
Enzyme Heavy metals High Detection of Tolun Mean (μg/ Mean (μg/ WHO US
method bound with sensitivity single heavy et al. L) L) EPA
sulfhydryl or metal ion is (2012)
carbonyl groups hard. 1 Cr 138.0 413.27 50 100 Qiaoqiao et al.
in the active Water samples (2020),
center of the only detectable. Kumar et al.
enzyme. It (2019)
causes changes 2 Mn 518.0 2562.15 100 50 Qiaoqiao et al.
in structural (2020),
and property Kumar et al.
properties. It (2019)
gives the 3 Fe 1479.55 1654.05 300 300 Qiaoqiao et al.
quantitative (2020),
relationship Kumar et al.
between (2019)
changes in 4 Co 27.07 3994.82 – – Qiaoqiao et al.
enzymes and (2020),
the Kumar et al.
concentration (2019)
of heavy metals 5 Ni 80.99 945.86 20 – Qiaoqiao et al.
Test strip The detection of It is low-cost The LOD is Wang (2020),
heavy metals is and easy to relatively high. et al. Kumar et al.
performed by a perform (2020) (2019)
chromophore 6 Cu 1198.89 537.87 200 200 Qiaoqiao et al.
Immunoassay The primary Quickly The Han et al. (2020),
antibody- detectable preparation of (2019) Kumar et al.
antigen More monoclonal (2019)
complex, and sensitivity antibody for 7 Zn 1180.13 723.11 1000 1000 Qiaoqiao et al.
secondary and more metal ion is (2020),
antibody are selectivity very difficult. Kumar et al.
tested for It is difficult to (2019)
sensitivity and produce poly- 8 As 317.74 3981.78 10 10 Qiaoqiao et al.
selectivity by clonal antibody (2020),
coupling with for the Kumar et al.
heavy metals detection of (2019)
and studying its heavy metals. 9 Cd 25.33 180.88 3 5 Qiaoqiao et al.
affinity (2020),
Biosensor Using biological Limit of Ability of anti- Arlett Kumar et al.
substances such detection is interference is et al. (2019)
as DNA low poor. (2011) 10 Hg 15.93 1.01 1 2 Qiaoqiao et al.
enzymes, Time (2020),
proteins, and response is Kumar et al.
antibodies to short (2019)
detect heavy Low cost, WHO – World Health Organization, USEPA – United States Environmental
metals more
Protection Agency.
sensitive,
Cr – Chromium, Mn – Magnesium, Fe – Iron, Co – cobalt, Ni – Nickel, Cu -
and more
specificity Copper, Zn – Zinc.
It can be As – Arsenic, Cd – Cadmium, Hg – Mercury.
used for the
continuous
detecting
signal in
real-time

Mi - Mass of heavy metals from various industries.

Maf - Mass of heavy metals from agriculture through fertilizer.
Ms - Mass of heavy metals from soil.
Mm-Mass of heavy metals from mining.
Mu-Mass of heavy metals from urban areas.
Mip - Mass of heavy metals as inorganic pollutants.
Mow - Mass of heavy metals as organic wastes.
Mvp - Mass of heavy metals that are vaporized.
Msd - Mass of heavy metals are deposited in the soil during runoff.
Fig. 1. Various sources of heavy metals by natural and anthropo­
3. Environmental effects of heavy metals genic processes.

Water is said to be the life and blood of the biosphere. However,

many contaminants in the form of heavy metals, organic pollutants and

3
K. Velusamy et al. Food and Chemical Toxicology 168 (2022) 113307

inorganic pollutants are discharged into water and dissolved. Among
these contaminants, heavy metal contamination plays a critical role in
causing adverse effects on plants, animals and humans (Rezania et al.,
2016; Kandasamy et al., 2022). Heavy metal contamination problems
are being raised all over the world due to bio-accumulation, bio-­
magnification and changes in the food chain. Heavy metal content in the
river bed is greater than in the river upstream (Kumar et al., 2013;
Karthik et al., 2021c). The procedure developed to analyze the hazard­
ous nature of heavy metals in the living system consists of three char­
acteristics: persistence, bio-accumulation and toxicity. The heavy
metals, which are both persistent and bio-accumulated are more toxic
(DeForest et al., 2007; Velusamy et al., 2021b; Karthik et al., 2020). To
calculate bio-accumulation, the formula used is shown below.
Bio accumulation factor = Cplant/Csoil
many animals. Heavy metals bind to the gills, liver, kidneys, and other
tissues like skin and muscles. This causes side effects on humans when
consuming these fish that are grown in heavy metal contaminated water
(Mansouri et al., 2012). Microorganisms are also harmed when high
amounts of trace metals enter the aquatic environment. There are
several methods to determine the bioaccumulation of metals (Igiri et al.,
2018). The process of accumulating heavy metals in aquatic organisms is
defined as bioaccumulation. There are several methods to determine the
bioaccumulation of metals. For example, in the 1980s, the free ion ac­
tivity model (FIAM), the biotic ligand model (BLM), and later the sub­
cellular partitioning model (SPM) were established (Yousafzai et al.,
2017).
3.3. Effects on plants

where Cplant: metal concentration in plant.
Csoil: metal concentration in soil.
3.1. Essential and non-essential heavy metals
Heavy metals were classified into essential and non-essential heavy
metals based on their activity in living organisms. Classification is
detailed in Table 3.
Essential heavy metals also when it exceeds the threshold level, are
detrimental to humans, plants, animals and even micro-organisms. It
affects the metabolism, growth, reproduction and survival of the or­
ganism. The heavy metals accumulated in living organisms get trans­
ferred from one food chain to another as they are persistent. These heavy
metals enter organisms directly from abiotic environments such as soil,
water, and food. Compared to soil and food, water, heavy metals are
more soluble in water (Li et al., 2022).
3.2. Effects on marine animals
The deposition of heavy metals in fish depends upon the fish’s
characteristics external environmental factors and the concentration of
heavy metals present in water. These heavy metals are toxic and
persistent in small amounts, causing oxidative activity in marine ani­
mals. So, this is highly significant in terms of ecotoxicology (Jaiswal
et al., 2018). External factors include bio-availability and water prop­
erties. Initially, heavy metals cause the evolution of new enzymes that
change the metabolism of fish which leads to toxicity, tissue death, and
necrosis (Balali-Mood et al., 2021). These heavy metal contaminants
affect marine animal reproduction and have led to the extinction of
In plants, heavy metals enter the root system through water from the
soil (Peralta-Videa et al., 2009). In the aquatic environment, all parts of
the plants contain these heavy metals as the plant is fully submerged in
water. These heavy metals result in nonspecific stress responses, re­
ductions in seed germination and seed development (Nagajyoti et al.,
2010). This leads to ill effects on photosynthesis, photophosphorylation,
CO2 fixation, electron transport system and Calvin cycle. Some of the
physical symptoms due to heavy metal exposure are chlorotic and
necrotic leaf spots. To avoid these types of contamination, plants bind
with phytochelatins, glutathione and metallothionein (Manara, 2012).
3.4. Effects on animals and insects
In animals, the heavy metals intake through the food chain leads to
modification in lifestyle characteristics and genetic variation of toler­
ance. The larval stage of insects and reptiles are more sensitive to heavy
metals. It also affects the development of eggs (Yedjou et al., 2012).
Metal contamination experiences the risk of extinction of organisms in
the environment (Posthuma and Van Straalen, 1993). For adaptation,
the animal’s mechanism includes detoxification, compartmentation,
avoidance behavior and excretion. The effect of heavy metals in higher
organisms is similar to the effects in humans (Yedjou et al., 2012).
3.5. Effects on humans
In humans, discovering the toxicity levels of heavy metals is
complicated. The toxicity depends on several factors, including route of
exposure, dosage, specification of heavy metals, genetic background,
age group, gender and nutritional status. The metals which affect
humans and their modes of entry are shown in Table 4.
4. Design and features of biosensor

Table 3 In recent decades, biosensor research has experienced rapid growth.

Differences between the essential and non-essential heavy metals.
S. Essential heavy metals
No.
1. These heavy metals help in
day-to-day life of humans,
plants and other living
organisms.
E.g., Mg, Cu, Zn, and Fe.
2. These metals produce
disease only at a higher
concentration. At trace level,
it functions as metabolic
pathways.
3. Larger intake of heavy
metals result in serious
health conditions.
Mg – Magnesium, Cu – Copper, Zn – Zinc, Fe – Iron, Hg – Mercury, Pb – Lead, Cd
– Cadmium.
Non-essential heavy metals
The heavy metals that do not
possess any known biological
roles in our environment are
called biologically non-
essential heavy metals.
E.g., Hg, Pb, and Cd.
These metals are very toxic in
nature, even in lower
concentrations.
These metals can cause
disease even if they are
ingested in a small quantity.
A biosensor is a class of analytical instruments that possess a hybrid of
Reference physical and chemical reaction sensing techniques (Buenger et al.,
2012). Biosensor is a detecting device that uses both biological com­
Ali et al. ponents and a physiochemical transducer. It monitors, reads and con­
(2019) verts biological responses into a physical, chemical or electrical signal.
Biosensors can be applied to various samples like body fluids, cell cul­
tures and environmental contaminants. The human nose is the best
example of a biosensor, which is very sensitive and selective. The nano
Ali et al. technology was also used to build the biosensor.
(2019)
4.1. Design of biosensor
There are two main fundamental operating principles in biosensors:
"biological recognition" and "sensing" (Tamboli et al., 2016). Compo­
nents of a biosensor with different types of bio-receptors and transducer
combined with signal processing unit and output are shown in Fig. 2.
According to the IUPAC definition, there are three main and basic

4
K. Velusamy et al. Food and Chemical Toxicology 168 (2022) 113307

Table 4 it is identified in biochemical pathways and purified for biosensor ap­

The entry and toxicity of heavy metals in humans with the most hazardous metal plications. Once the target sequences of DNA have been identified, those
contamination level (MCL). DNA sequences are artificially developed and immobilized on the sur­
Entry Toxicity Hazardous Reference face of a bioreceptor and then used as the bio-recognition element
Metal metal level (Dauphin-Ducharme and Plaxco, 2016). The sample component con­
(mg/L) tains a bio-sensitive layer. It binds with bio-receptors which is bound
Arsenic Ingestion, Visceral cancer, 0.05 Gunatilake covalently with the transducer to detect the target analyte by producing
inhalation and vascular disease, (2015) some signals.
dermal brain damage
The forms of interactions between the bio-receptor and target ana­
absorption
Cadmium Food, cigarette Kidney damage, 0.01 Gunatilake lyte are antibody-antigen interactions, enzymatic interactions, nucleic
smoking headache, (2015), acid interactions and interactions using synthetic bio-receptors. En­
diarrhoea, lungs Clemens zymes, detect analytes using enzymatic interaction and antibodies
damage et al. (2013) detect analytes using antibody-antigen interactions depending on the
Chromium Dermal Liver damage, 0.05 Gunatilake
contact, insomnia (2015),
applications. The construction mechanism of bio-receptors is achieved
inhalation ATSDR. by the adsorption or immobilization of these biological elements on the
(2012) surface of the bio-receptor. Those immobilizations/adsorptions include
Mercury Respiration, Circulatory 0.000003 Gunatilake entrapment, adsorption, covalent bonding, micro-encapsulation, and
bloodstream system, nervous (2015),
cross-linking (Sassolas et al., 2012). The advantages of immobilizing
system, DNA and Park and
chromosomal Zheng et al. biological components on the biosensors are as follows: i) It helps to
damage (2012) monitor the presence of an analyte in liquid sample, e.g., biological
Lead Exposure Kidneys and 0.006 Gidlow fluids and environmental samples; ii) Reusable; iii) The advanced
pathway, circulatory (2015) bio-recognition unit improves the sensitivity, selectivity and perfor­
ingestion and system, stomach
mance of biosensors; iv) Techniques for immobilization are simple and
inhalation ache, diarrhoea
adaptable.
DNA – Deoxyribonucleic acid.
4.1.2. Transducer
elements in biosensors bio-receptors (biorecognition system), trans­ The chemical reaction between the bio-receptor and target analyte
ducer, microelectronics (signal processing system) and display (Nigam produces various changes, such as changes in pH, light, heat generation,
and Shukla, 2015). or mass changes. The physical elements that recognize such phenomena
are called transducers. It converts one form of signal to another
4.1.1. Bio-receptors detectable form. The transducer is classified into many types based on its
A bio-receptor is an external component that contacts the analyte mode of operation such as optical transducers, electrochemical trans­
directly and serves as a recognition system. The main function of these ducers, piezoelectric and thermal transducers (Rebollar-Pérez et al.,
bio-receptors is to capture the analyte with high selectivity and high 2016).
sensitivity. Common bio-receptors used recently are enzymes, whole An optical transducer is widely used in optical biosensors (Dai and
cells, antibodies, aptamers and DNA (Kim et al., 2016). Choi, 2013) which produces light, luminescence, fluorescence, and
Enzymes can achieve analyte specificity with their three- refractive index as a result of the biochemical reaction. Optical trans­
dimensional structures by binding cavities using electrostatic forces, ducer based on surface plasmon resonance transducer is used. In this
hydrogen bonding and other non-covalent interactions that result in the type of transducer in which electromagnetic waves are used for analyte
recognition of analytes. Additionally, it is a fast-acting, highly specific detection. It can detect the analyte by immobilizing the bio-component
biological component that binds to a substrate or analyte through the (Monosik et al., 2012). The phenomena that can be detected using the
catalytic effect of a particular reaction. It converts the analyte into a optical transducer are reflectance, absorbance, chemiluminescence,
measurable product for analysis. bioluminescence, refractive index, luminescence and light scattering.
Aptamers are single-stranded oligonucleotides designed using a An electrochemical transducer (amperometry, conductometric,
combinatorial selection process called Systemic Evolution of Ligands by potentiometric, chronoamperometric, field-effect transistors) is used to
Exponential Enrichment (SELEX) (Kang et al., 2018). Aptamer searches detect electrical signals produced as a result of the interaction between
for oligonucleotide sequences randomly from the library that has high bioreceptor and an analyte. There are many bio-recognition systems
binding affinities between the aptamers and target analyte. It is basically used in this type, but it predominantly involves enzymes. A reference
a protein with three-dimensional structure that occurs naturally and also

Fig. 2. General components of a biosensor.

5
K. Velusamy et al.
electrode along with another electrode is required to maintain a stable
potential in the electrolyte solution and a working electrode is required
for detection. Among other types of transducers, it has a few advantages,
such as low cost, easy handling and easy to construct. Thermal trans­
ducers (thermistor and pyroelectric) quantify the thermal energy
evolved or absorbed by the analyte during the reaction with the bio-
receptor (Monosik et al., 2012). The thermal transducer is also widely
used for various analyses. In the same way, electrodes were screen
printed using egg white/ZnO rice structured composite to detect heavy
metals.
A piezoelectric transducer (mass-sensitive quartz crystal microbal­
ance) is more sensitive to mass, density or viscosity and it works on its
relationship between the mass that is adsorbed on the crystal surface and
the resonant frequency of the oscillating piezoelectric crystal (Marrazza,
2014). QCM is widely used in piezoelectric transducers. It is fabricated
with a thin plate made of quartz fitted with electrodes made up of metals
attached to each side. The biological entity is immobilized to develop a
reaction between the analyte and bio-receptor. An analyte with a con­
stant flow rate is applied to the immobilized ligand. The amounts of
analyte and bio-receptor interactions are directly proportional to each
other. It helps to detect and quantify the concentration of the analyte
present in the solution. The signal generated by these transducers is in an
analogous form that cannot be read directly. So, microelectronics or
signal conditioning units are used to convert the signal from the trans­
ducer into a readable format.
4.1.3. The signal conditioning unit
The signal conditioning unit consists of a complex electronic circuit
that quantifies or converts the transducer signal into a readable format.
It can convert the signal of various low-pass, high-pass and notch filters
from analog form to digital form (Karunakaran et al., 2015). The display
units finally quantify the processed units.
4.1.4. Display units
Display units include a user interpretation system. There are many
types of hardware used as display units in biosensors: computers, liquid
crystal displays (LCD) and printers (which give a pictorial or graphical
representation of the signal that is measured). It hosts software that
helps to provide the biosensor results in a user-friendly manner. These
display units vary according to the user’s output format requirements.
For example, the data at the end may be numeric, table, graphics or an
image.
1) Analyte - required compound to detect; 2) Bioreceptor - consist of
antibody/Enzyme/DNA/whole cell which is immobilized on the bio­
receptor; 3) Transducer - to detect the changes when bioreceptor in­
teracts with the required analyte; 4) Processor - converts the signal to
readable format; 5) Display - helps to visualize the results.
4.2. Features of biosensor
The design gives the intended purpose of its application. Even so,
some of the features will define the performance of the biosensor. Every
biosensor possesses some static and dynamic attributes. Here, different
features of a biosensor which help to detect the heavy metals in
wastewater are shown in Fig. 3.
Biosensors are highly selective and sensitive in nature, producing a
precise signal proportional to the concentration of the analyte. Few
biosensors are highly reusable, reproducible and stable.
4.2.1. Selectivity
Selectivity serves as a major feature in the biosensor. It has the ability
to bind with the specific target and produce a readable signal. Some­
times a false-positive result may occur, which means the target sample is
absent in the solution, but it detects other than the required target as a
positive result. Such results are generated due to the poor selectivity of
some biosensors. An example of selectivity is the antigen-antibody
6
Food and Chemical Toxicology 168 (2022) 113307
Fig. 3. Features of biosensors.
interaction. Naturally available bio-recognition elements such as en­
zymes and antibodies show some good specificity and selectivity. Some
bio-recognition elements are artificially produced, such as aptamers,
molecularly imprinted polymers (MIPs), and enzymes.
Antibodies achieve selectivity by forming a Y-shaped conformation
with their binding domains (Gilles-Gonzalez and Gonzalez, 2005). En­
zymes have binding pockets with hydrogen bonding to achieve analyte
specificity. It also contains electrostatic bio-recognition patterns. The
enzyme and antibody are highly selective bio-recognition elements.
Their biological role is the specific reason for their success in other
physiological and immunological processes (Price, 2001). The nucleic
acid or aptamer-based bio-recognition elements are recognized by using
non-specific electrostatic interactions, developed by the negative charge
in DNA that competes with analytes. Such interactions can be reduced
by using some peptide nucleic acids (Ferapontova, 2018). Aptamers are
oligonucleotide subunits which cause non-specific binding with heavy
metals. This is counteracted by post-synthesis chemical modification of
aptamer, which reduces the non-specific binding (Yao et al., 2010).
MIPs are synthetic modalities and beneficial bio-recognition ele­
ments. The only drawback is poor biosensor selectivity resulting in non-
specific binding of analytes due to the heterogeneous interactions within
the binding cavity (Wackerlig and Schirhagl, 2016). This type of
non-specific binding is more common. By using polymer cross-linking,
the non-specific binding can be reduced and the binding cavity can be
protected (Whitcombe et al., 2011). However, in this case, extensive
cross-linking creates a highly dense polymer construction leading to the
permanent entrapment of the bio analyte template and reducing the
diffusion of bio analyte through the construction (Verheyen et al.,
2011). In addition to MIPs, nanozymes as synthetic modalities are now
being validated in complex bio-fluid matrices (Zhou et al., 2017).
4.2.2. Sensitivity
Sensitivity is considered an important feature of biosensors. This
defines the relationship between the bio analyte concentration and the
signal intensity of the transducer. In other words, the ability of a
biosensor to detect the analyte even at a very low concentration is
defined as sensitivity. The detection limit range is from nanogram per
milliliter to femtogram per milliliter, which means that the biosensor is
more sensitive. Many publications give the least possible detection limit,
but increased sensitivity within the relevant physiological concentration
ranges is required (Saha et al., 2012). The biosensor sensitivity and the
range are calculated using the available number of binding sites per
surface area, the dissociation constant, equilibrium, and steric hin­
drances. The availability of analyte binding sites depends on the
increased surface loading density of the bio-recognition element. Based
on the attribute of sensitivity, the best recognition element is an aptamer
owing to its small size, measuring only 1–2 nm in size. Hence, aptamers
have a denser surface area with more binding sites per unit surface area
(Crivianu-Gaita and Thompson, 2016). For antibodies, the surface
loading influences the sensitivity and is caused by the steric hindrances
K. Velusamy et al.
from the bio-recognition elements adjacent to it, which cause the
conformational changes that result in the binding site being inaccessible.
Similarly, in nucleic acids as bio-recognition elements, the sensitivity
range is constrained due to steric hindrances, which can be overcome by
using locked nucleic acids (Kang et al., 2018). The range, sensitivity and
response time of biosensor depends on the depth and surface structure
density of bioreceptor (Zhou et al., 2017). In addition, if the receptor
surface is thicker per unit area, then it increases the binding site of
biorecognition element.
4.2.3. Linearity
It denotes the accuracy of the obtained output within a working
range in which the measured signal is directly proportional to the con­
centration of an analyte in the sample. The equation y = mx + c gives the
biosensor linearity where y is the output signal, m is the biosensor
sensitivity and c is the analyte concentration. Linearity depends on the
biosensor resolution and analyte concentration. Biosensor resolution is
defined as the extent of change in analyte concentration that is required
to produce a signal. If the dynamic range or linearity is higher, then the
detection of high substrate concentration will be good.
4.2.4. Reproducibility
Reproducibility is defined as the ability of the biosensor to fabricate
many identical biosensors and generate identical signals to a target
analyte. In other words, it is the ability of biosensors to create an
identical response for many experimental set-ups (Nikhil et al., 2016).
The precision and accuracy of the biosensor contribute to its reproduc­
ibility. Precision represents the sensor’s ability to give the same result
every time. To improve reproducibility, the element structure of
bio-recognition and production process must be understood. It can also
be limited through the development process, e.g., recognition element
construction (batch-to-batch variability) and surface construction vari­
ability. Antibody and enzyme-based biosensors were mostly available in
the market. These biosensors are carefully controlled and calibrated to
produce precise results. The antibodies and the enzyme are very sensi­
tive to pH and temperature variations, making it difficult for industrial
production, especially in low-resource regions (Turner, 2013). The
aptamers have highly reproducible biosensors and are an ideal
bio-recognition element (Iliuk et al., 2011). The aptamer chemical
synthesis is well defined in the production of highly robust
bio-recognition elements. Post-synthesis modifications enhance the
stability, reproducibility and decrease non-specific binding of the
aptamers (Justino et al., 2015). The production process of cost-effective
MIP is simple and lacks back-to-back variations, resulting in highly
reproducible biosensors (Borje and Andrew, 2012). Such reproducible
signal gives a more reliable and interference-resistant biosensor
response.
4.2.5. Reusability
Reusability is defined as a biosensor’s ability to be used multiple
times (Ferreira et al., 2017). Reusable biosensors are more efficient in
increasing biosensor technology accessibilities in low-resource regions.
In these biosensors, the target bio analyte dissociates from the
bio-receptor to make the binding site available for the consequent
binding. The forces that occur between the analyte and bio-recognition
elements play a crucial role in the regeneration and reusability of the
biosensor. In the case of enzymes, regeneration does not require any
extra mechanisms as the enzymes are not used up and remain unaltered
during the catalytic reactions. Hence, the binding sites are preserved,
and the activity remains intact. This type of regeneration is also called
passive regeneration (Zhao et al., 2017). Antibody, aptamer, or DNA
also serve as reusable biorecognition elements. Thermodynamic forces
in the form of enthalpy and entropy in bio-recognition elements domi­
nate the affinity-based interaction. Enthalpy dominates
charge-mediated interactions, whereas entropy dominates hydrophobic
interactions developed due to the solvent. Hence, dissociating the
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Food and Chemical Toxicology 168 (2022) 113307
analyte using the solvent helps to reuse the biosensor. For example, the
amino acid charge will differ according to the solvent pH, placing these
biosensors at different pH, which disrupts the protein-based in­
teractions. It helps to remove the analyte and then use the biosensor
again. The main purpose of recreation is the bio-recognition element
binding site is to control the biological elements’ integrity (Goode et al.,
2015).
4.2.6. Stability
Stability is another one of the features that determine the perfor­
mance of biosensors. It is defined as the degree of capability to handle a
moderate disturbance developing inside and outside the bio-sensing
system, which can drift due to this disturbance. This could also cause
an error in measured concentration and affects accuracy and precision.
Stability plays an important role in applications that require long in­
cubation steps or continuous monitoring. The transducer and electronics
influence stability because they are temperature-sensitive. To ensure a
stable response, appropriate tuning of sensor is required. The factor that
affects the stability are bio-receptor affinity in which the large number
of analytes bounds towards the bio-receptor and bio-receptor degrada­
tion in which the bio-components will degrade over a period of time.
5. Biosensor for heavy metals detection in wastewater
In wastewater, heavy metal pollution is caused by agricultural
pollution, fertilizer, mining and volcanic eruptions. There are different
methods for heavy metal detection in wastewater. Among the detection
methods, biosensors have more advantages in detection and quantifi­
cation. This biosensor is classified according to its in-built components,
such as transducers and bio-receptors. The brief classification of the
biosensor is shown in Fig. 4.
A bioreceptor is classified as enzyme-based (protein-based, or
antibody-based) and whole-cell (natural cell or genetically engineered
cell). Transducers are classified as electrochemical, optical, piezoelectric
and thermal.
5.1. Biosensor classification based on bio-receptor
5.1.1. Enzyme based biosensor
In these types of bio-receptors, enzymes are mostly used as the bio-
recognition element because of their advantages, such as being rapid,
simple and more selective for the detection of heavy metals. When used
in on-site analysis at low concentrations, these enzyme-based biosensors
are expensive (Ghica et al., 2013). According to the monitoring mode,
the analyte is differentiated into two types: direct and indirect. Direct
mode is defined as monitoring the concentration of analytes or products
formed during the enzymatic reaction. This direct method is simple,
portable and continuously operational, but the disadvantage is that it
can only be used for a limited number of environmental pollutants due
to its specificity. Then the indirect mode is defined as monitoring the
enzyme inhibition of an analyte. It works by analyzing the enzyme in­
hibitors (Rebollar-Pérez et al., 2016). In addition, this is the major
principle of the enzyme-based biosensor (Guascito et al., 2008). The
enzyme inhibition activity that develops in the presence of heavy metals
was observed. The complete mechanism of heavy metal detection by
using the enzymes as bio-receptors is shown in Fig. 5. The trace metals
that have to be detected are inherent parts of the enzyme structure, such
as metalloproteins, that are needed for enzyme activity. Usually, in an
enzyme-based biosensor, the target metal ions react with the thiol group
of the sulfhydryl group present in the enzymatic structure, which causes
conformational changes and affects the catalytic activity. These
continuous changes in the enzyme activity can be read as a signal. (Vel.
Krawczyk et al., 2000). Some of the enzymes such as glucose oxidase
(GOD), phosphatase, urease, glutathione-S-transferase, lactate dehy­
drogenase, alkaline phosphate, acid invertase are used to detect heavy
metals such as cadmium, copper, lead, zinc and mercury. Among these
K. Velusamy et al. Food and Chemical Toxicology 168 (2022) 113307

Fig. 4. Classification of biosensor based on bioreceptor and transducer.

Fig. 5. Mechanism of an enzyme-based biosensor that aids in the detection of heavy metals.

enzymes, glucose oxidase can be used in a single enzyme format for
detecting the concentration of different types of heavy metals. It helps to
detect Ag+, Cu2+, Pb2+, Cd2+, Cr3+, Co2+, Fe3+, Ni2+ and Hg2+ (Mehta
et al., 2016). By immobilizing the glucose oxidase on an electro poly­
merized platinum electrode using ferrocene, Cr6+ can be quantified up
to 9.6 μM. Horseradish peroxidase is also used to detect Cr3+ and Cr6+. It
is immobilized on a neutral red modified carbon electrode. The limit of
detection is achieved at 0.09 and 1.15 μM for Cr6+ and Cr3+, respectively
(Attar et al., 2014). Similarly, multiple enzyme prototyping has been
developed to achieve increased biosensor sensitivity. For example,
glucose oxidase combined with the invertase efficiently detects Hg2+,
Ag+, Pb2+ and Cd2+ with a detection limit of 0.5 nM. Mutarotase has
been coupled with this complex to detect the concentration of Hg2+ and
Ag+ by using the conductometric thin-film interdigitated ceramic
transduction platform (Soldatkin et al., 2012).
Phosphatase, invertase and other enzymes were produced in bacteria
and immobilized in the receptor. When heavy metals bind to these en­
zymes, they limit their function, resulting in the production of the bio­
logical signal that is detected by the transducer. The signal processing
unit was used to convert these signals into readable data. Entrapment is
used to immobilize enzymes, with covalent binding and microencap­
sulation being the most commonly used methods.

8
K. Velusamy et al.
Urease is another enzyme that is mostly used for heavy metal
detection. In the water sample, urease cross-linked microelectrodes are
used to detect various heavy metals such as mercury, cobalt, cadmium
and lead. Sol-gel entrapped urease on the electrodes was used to detect
Cd2+ and Pb2+ metal ions with a detection limit of 10 ppm. Inactivation-
based oxidases and dehydrogenases in the presence of metal ions’ help
to quantify the Cd2+, Hg2+, Pb2+ and Cu2+. Alkaline phosphatase
enzyme immobilized on gold electrodes helps detect Cd2+, Ni2+, Zn2+
and Pb2+ with a detection limit of 0.5 ppm, 5 ppm, 2 ppm and 40 ppm
(Berezhetskyy et al., 2008). This method has the advantages of stability
up to 10 h and storage stability at 40 ◦ C (Amine et al., 2014). Hence, the
overall advantages of the enzyme-based biosensors are that it allows
rapid and selective determination of the metals, but the disadvantages
are that it is not cost-efficient for the purification of an enzyme. Another
one is its vulnerability when exposed to the extracellular environment.
Many problems are solved by using the immobilized enzyme however,
multiple detection is unstable and different enzymes have different
optimal conditions on a single platform, which makes it difficult to
verify (Mehta et al., 2016).
5.1.2. Antibody based biosensor
An antibody, otherwise known as immunoglobulin (Ig), creates an
immune response against antigen in our body. Antibody is Y-shaped and
is produced by one of the types of white blood cells, B Lymphocytes. It is
used as bio-recognition element in the biosensor to recognize the spe­
cific metals present in the sample. A tight bond formed between the
antigen-forming complexes and the antibody in the process of recogni­
tion of metals present in the given sample. The detection mechanism of
heavy metals by using the antibody-based biosensor is shown in Fig. 6.
These antibodies are classified into five groups on the basis of its func­
tion and structure. Other than antibodies, monoclonal antibodies,
polyclonal antibodies and recombinant antibodies are also widely used
as bio-recognition elements for heavy metals.
The major advantages of using the antibody-based biosensor are that
ease of operation, cost-efficiency, and portability. The monoclonal an­
tibodies were explored for detecting mercury, and this detection ach­
ieved a range of 0.02–300 ppm. For detecting the cadmium, the metal
ion that first interacted with the keyhole limper hemocyanin carrier with
Fig. 6. Mechanism of antibody-based biosensors for detecting heavy metals.
9
Food and Chemical Toxicology 168 (2022) 113307
the isothiocyanobenzyl ethylenediamine N, N, N, N′ -tetra acetic acid,
which is the bio-functional chelator (Zhu et al., 2007). The
metal-specific antibodies were immobilized using various substrates
that increased the scope of their potential for applying biosensors (Lin
and Chung, 2008). For the detection of lead, a surface plasmon
resonance-based sensor was used and achieved detection of 0.27 ppm.
The main benefits of using this antibody-based biosensor are its repro­
ducibility. The true activity of the biosensor was regained up to 92.7%
for 35 days. Some studies also reported that the immobilized antibody is
more sensitive (10-1000-fold) than the soluble antibody. Different
methods were used for the immobilization of antibodies onto the
biosensor, such as covalent immobilization, non-covalent binding and
coupling by affinity interaction (Sharma et al., 2016). The factors that
affect antibody-based biosensor activity include pH, temperature, and
ionic strength (Dzantiev and Zherdev, 2013).
Antibodies such as monoclonal antibody, polyclonal antibody and
recombinant antibody were prepared and immobilized on the receptor.
When the analyte is added, it selectively binds to the required probe,
such as antibody-antigen interactions at the epitope of the antibody.
These interactions were detected and the signal was guided by a trans­
ducer and converted into a readable format using a signal processing
unit.
5.1.3. Whole cell-based biosensor
A whole cell-based biosensor was described a decade ago. It has been
widely used to detect metal ions since it was developed. Both prokary­
otic and eukaryotic cells are used in whole cell-based biosensors. The
cells used as the bio-recognition elements are microorganisms, algae,
plant cells, protozoa and fungi. These are all natural or maybe recom­
binant (Gu et al., 2004; Acha et al., 2010). The whole cell-based
biosensor works on the principle of detecting the concentration of
required metal ions. It is used to determine the functionality, toxicity
and genotoxicity. Natural cell receptors specifically bind to heavy metals
with proteins of non-catalytic or non-immunogenic origin (Rodri­
guez-Mozaz et al., 2004). In whole-cell-based biosensors, heavy metal
sensing is mediated by metalloproteins such as ArsR, AntR, and CueR. It
can work as a transcriptional factor and adjust the downstream
expression of genes in the presence of metal ions. Microbes cannot
K. Velusamy et al.
survive in an environment containing heavy metals, so some strategies
have been used to adapt these microbes to such a hostile environment. 1)
Decellularization by the removal of cell membrane 2) Metal transport
across the cell from the bulk solution 3) Metal sequestration is per­
formed in extracellular media to prevent it from entering the cell 4)
Chemical transformation is reduced in the sample, resulting in less
toxicity to the cells. To modulate the sensitivity and selectivity of the
whole cell-based biosensor, genetically and biochemically engineered
host Escherichia coli and other bacterial cells are used (Yoon et al., 2018).
The Znt-operon and the egfp operon show a selective response to cad­
mium and mercury due to the Zntr regulatory protein. The toxicity of
Cd2+ and Cu2+ was detected using cyanobacterial cells (Anabaena Tor­
ulosa) (Shing et al., 2008). Based on fluorescence detection of Cu2+, Pb2+
and Cd2+, A. torulosa cells entrapped within cellulose membranes ach­
ieved concentrations of 2.5–10.0, 0.5–5.0, and 0.5–10.0 μg/L, with an
overall reproducibility of 6.35% (Wong et al., 2013). Other microbes
like Tetraselmis chuii, circinella and rhodotorula mucilaginosa have also
been used to detect copper with a 0.46 nM detection limit, Chlorella sp.,
Phormidium sp. for Pb2+ and Hg2+, Acidthiobacillus ferrooxidans for Cr2+
and Bacillus sphaericus for Ni2+ are some metal-detecting organisms
(Singh and Mittal, 2012). The microbe S. aureus is used to detect Pb2+,
Cd2+ and Sb3+ up to the detection limits of 10, 33 and 1 nM, respec­
tively. The microbe B. subtilis has the ability to detect heavy metals such
as Cd2+, Zn2+, Sb3+ and Sn2+ with a detection limit of 3.3 nM, 1, 33 nM,
40 nM and 100 nM, respectively. The various mechanisms that take
place inside the whole cell immobilized for the detection of heavy metals
are shown in Fig. 7. The general methods that are carried out to
immobilize the microbes on the transducer or bio-receptor include both
physical and chemical methods, e.g., adsorption or cross-linking
entrapment. Adsorption is the main method that affects the bio­
sensor’s stability and sensitivity. A yeast biosensor is mostly used, which
is more specific for some metal targets (Jarque et al., 2016). The whole
cell-based biosensor has the advantages of being simple and low-cost. It
is also used in many targets for the detection of various other pollutants.
Isolation and purification of the cell is easy compared to the
Fig. 7. Working of whole cells biosensor.
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Food and Chemical Toxicology 168 (2022) 113307
enzyme-based biosensor, and it is also easy to regenerate the cells to
grow insitu. Despite the advantages, some disadvantages are slow
response, less sensitivity, poor selectivity and determining
bio-availability is difficult due to the larger size of pollutants (Carlson
et al., 2012).
Whole cells (natural or genetic) are cultured from bacteria or other
organisms. When heavy metals are added, they first penetrate the cell
membrane and begin to interact with regulatory proteins via DNA. It
leads to transcription and translation and produces reporter proteins.
The transducer receives the signal and converts it into a readable format.
5.1.4. DNA-based biosensor
In the last few years, DNA-based biosensors have been increasing for
detecting and monitoring environmental heavy metal pollution because
many metals show more affinity toward DNA. They can selectively
interact with nucleic acids. A DNA-based biosensor is otherwise known
as a nucleic acid biosensor. Interactions of heavy metals and DNA leads
to heavy metal detection in wastewater, as shown in Fig. 8. The DNA and
metal ion interactions are considered important because those in­
teractions may cause adverse effects and damage the replication and
transcription of the DNA, leading to mutations and the tumorigenesis.
The basic principle of DNA-based biosensors is the hybridization of
complementary strands of DNA. In nucleic acid, there are four potential
bindings for the metal ions, which helps in the detection of heavy metals
more easily. Immobilized double-stranded DNA helps to detect heavy
metals such as Cd2+, Pb2+ and Ni2+. Future, it will be able to quantify the
heavy metal As5+. Single-stranded DNA, on the other hand, aids in the
detection of Cd2+ with a detection limit of 10 pM (Wong et al., 2007). A
DNA-based biosensor is classified into two groups: Geno sensor and
aptasensors. In a genosensor, probe selection and immobilization are the
key factors. But in aptasensor, the DNA that has been selected plays the
role of high-specific recognition element of heavy metals. In an
aptamer-based biosensor, the nucleic acid sequence is selected randomly
based on the high detection ability using Systematic Evolution of Li­
gands by Exponential Enrichment (Malekzad et al., 2017). Production of
K. Velusamy et al.
Fig. 8. Illustration of aDNA based biosensor.
these aptamers is very easy (Pashazadeh et al., 2017). To detect Pb2+,
Sorba and colleagues designed a gold nanoparticle-based calorimetric
aptasensor, which gives a detection limit of up to 98.7 pM. In the
application of aptasensor for the detection of lead in the sample, in the
absence of lead, it remains red and in the presence of lead, it turns red to
blue. The range of 0.01–1 mM Pb2+ ion concentration is quantified with
a high coefficient of determination (Radhakrishnan and Kumar, 2022).
The DNA is extracted and when it interacts with the heavy metals, it
produces the modified protein. This leads to the detection of the fluc­
tuation by the transducer and it is converted to readable format by using
the signal processing unit. Then it is viewed in the light emitting diode
display.
The advantages of the aptamers are that they are always stable, even
in unsuitable environmental conditions, including pH and temperature.
The advantages include easy to synthesize and modify, smaller size and
long storage and shelf life. It is also cheaper with good chemical stability
is good (Omidinia et al., 2014). DNA can also serve as an enzyme to
detect heavy metals in the form of DNAzyme. It is created artificially
using oligonucleotide sequences. The presence of heavy metal ions is
detected by using the cleavage of ribonucleic acids by DNAzymes in
DNAzymes. Heavy metals such as mercury, copper, silver and lead can
be detected and quantified using these DNAzymes (Mazumdar et al.,
2010). Detection of Pb2+ occurs by developing the turn-on fluorescent
sensor using the graphene oxide DNAzyme which gives a detection limit
of 300 pM (Zhao et al., 2011). In the same way, guanine rich enhancer
sequence DNAzyme is used in a cytometric method for selective Pb2+
detection (Nie et al., 2012). Detection of Cu2+, Pb2+ and Hg2+ ions is
done by using the G-quadruplex DNAzyme. The main advantages of
DNAzyme are that it can be synthesized economically in an in-vitro se­
lection process with high selectivity and high sensitivity.
5.2. Biosensor according to transducer
5.2.1. Electrochemical biosensor
For monitoring, a heavy metal enzyme-based electrochemical
biosensor is used. The enzymes that are widely used are glucose oxidase,
urease and peroxidase (Dong et al., 2018; Suresh et al., 2022). Aptamers
provide a strong and stable interaction with the metals, detecting heavy
metals within very low limits (Zhang et al., 2018). There are many new
electrochemical biosensors that have been developed to detect heavy
metals. They are sediment microbial fuel cells, single chamber batch,
more microbial fuel cells and direct methanol fuel cells (DMFC) (Zhao
et al., 2018). The above-mentioned biosensor is based on the voltage
signal change in the immobilized electrogenic activity of bacteria. This
11
Food and Chemical Toxicology 168 (2022) 113307
biosensor has a low detection limit. Hence, it has been introduced as a
sensor to monitor wastewater quality (Tao et al., 2017). The design and
components of an electrochemical biosensor for the detection of heavy
metals is shown in Fig. 9. The nanomaterials are integrated with the
electrochemical biosensor to improve the performance. For example,
guanine nano wire-based amplification method (GWA) is used in the
quantification of Hg2+ in industrial waste and it has more advantages
and provides a picomolar detection limit.
In this type, the biological component is immobilized on the current
electrode, and the nanomaterial is adsorbed on the working electrode
and then the reference electrode helps to detect the target. When the
analyte interacts with the biological component, the transducer induces
the signal and converts it into a readable format.
The bio-sensing process of this quantification is in the form of
creating guanine wire structures in the formation of strong and stable T-
Hg2+-T in the presence of Hg2+ mismatches. A multi-walled carbon
nanotube electrode with peptide is used to detect multiple heavy metals
such as Cd2+ and Hg2+. These electrochemical biosensors are further
divided into subclasses based on their measured signal; they are
potentiometric, amperometric and conductometric biosensors. Potenti­
ometric biosensors: in this biosensor, an ion-selective electrode is used,
which is placed on top of an ion-selective membrane which helps to
recognize target ions present in the sample. Amperometric biosensors:
biosensor-similar to the potentiometric. The major advantages are that it
has high sensitivity and is faster when compared to potentiometric.
However, the disadvantage is being poor selectivity (Martinkova et al.,
2017). By using the biological components integrated with the elec­
trode, the detection and quantification are done by measuring the
electrical conductivity that occurred in the sample due to the
biochemical reaction (Dzyadevych and Jaffrezic-Renault, 2014). Ac­
cording to the World Health Organization, an electrochemical biosensor
provides a better result in the detection of heavy metals with acceptable
limit values.
5.2.2. Optical biosensor
Some biosensors were designed to emit bioluminescence signals in
the presence of low concentrations of heavy metals (Martin-Betancor
et al., 2015). Such transducer detects the optical changes when the
binding occurs between the analyte and the bio-receptor. Materials and
design of an optical biosensor for the detection of heavy metals in
wastewater is shown in Fig. 10. This biosensor can analyze the affinity
and the catalytic reaction. The change in absorbance or fluorescence in
the sensing element occurs, which results in changes in the refractive
index between the samples of multiple densities. By using the various
K. Velusamy et al. Food and Chemical Toxicology 168 (2022) 113307

Fig. 9. Working mechanism of electrochemical biosensors.

Fig. 10. Working of optical biosensors.

monitoring wavelengths, it can detect multiple analytes. optical changes and the transducer produces a signal and converts it into
Biological components are immobilized to surface plasmon reso­ output.
nance and imaging surface plasmon resonance, interferometers, reso­ The optic probes can transmit signals accompanied by changes in
nators and ratings. When it interacts with the analyte, it shows some polarity, wavelength, wave propagation, distribution of the spectrum or

12
K. Velusamy et al. Food and Chemical Toxicology 168 (2022) 113307

changes in the intensity of the light occur (Peltomaa et al., 2018). To Table 5
detect or monitor chromium in wastewater, enzyme-based optical bio­ Biosensors and their specification for detecting heavy metals.
sensors with specific optical properties and a porous silicon structure Biosensor Specification Heavy Detection Reference
were used (Biswas et al., 2017). Urease and acid phosphates were used metal limit
in the past as optical biosensors to detect or trace heavy metals (Tagad Electrochemical Detect the heavy Cd2+, 10− 8 Storey et al.
et al., 2016). The calorimetric bioactive paper-based biosensor based on metals by using Pb2+ -10− 11M (2011)
beta-galactosidase was created to detect Ag+, Ni2+ and Cr4+. In the the changes in Goriushkina
optical configuration, the biosensor can be internal or external. The current between et al. (2009)
the working and
closed incident light wave allows the waves to interact with the target reference
internally. But in external, the light wave directly passes through the electrodes
sample and provides the signal. These biosensors are classified into Optical When the target Ag+, 2.1 nM Sun et al.
several types such as absorption-based biosensors; they allow one to and the analyte Ni2+, (2019)
interact, optical Cr4+
detect the target analyte by using different concentrations. Surface
changes occur,
plasmon resonance biosensor: it relies on optical techniques to detect which leads to
dielectric and metal ion interactions. The detection is by excitation of heavy metal
the metal ions from the target analyte, which results in electromagnetic detection
plasmons. Fluorescence-based optical biosensor: directly detects the Piezoelectrical Due to the Hg2+, 5.40 nM Skladal (2016)
adsorption or Pb2+
target ion using changes in the frequency of electromagnetic radiation desorption
emitted by the metal ion. Luminescence-based optical biosensor: this occurring by the
biosensor is based on bioluminescence, in which the atom or molecules analyte, this
being triggered results in an exothermic chemical reaction which re­ results in
changes in the
leases minimum heat on returning to the ground state, then it is released.
current
By using this chemical reaction, the detection of heavy metals is carried generation,
out (Asal et al., 2018). The advantages of the optical biosensors are helping to
minimum instrumentation, simplicity and insensitivity to electromag­ monitor and
netic interference. detect heavy
metals
1
Voltametric Measuring the Cd, Zn 0.4pgML− Sangamithirai
5.2.3. Piezoelectric biosensor current while the et al. (2018)
The transducer is integrated with a piezoelectric material as the bio- oxidation or
recognition element. Bio-molecular interaction is measured by the reduction
process occurs
change in the mass of the analyte. The molecule which is required to
when the
detect is directly attached to the piezoelectric surface (Pohanka, 2018). interaction
For precise detection of Hg2+, a piezoelectric nano biosensor measured between the
by crystal micro-balance monitoring technology. When a mechanical analyte and the
force is applied, an electrical signal is produced (Pohanka, 2017). Nat­ transducer
occurs
ural and synthetic materials such as quartz crystal exhibit piezoelectric
Calorimetric While the Cd 0.77 μM Radhakrishnan
effects. It has the advantages of easy availability, stability in the aqueous reaction happens et al. (2014)
solution and high-temperature resistance. between the
sample and the
biorecognition
5.2.4. Voltametric biosensor
elements, the
A voltametric biosensor works under the principle of measuring the change in optical
current produced during the oxidation or reduction process that occurs density occurs,
during the interaction between an analyte and the transducer (Goyal which results in
et al., 2010). In this biosensor, the working electrode is applied at a the heavy metal
detection
constant potential and the reference electrode helps measure the current
Enzyme based The enzymatic Hg2+, 0.5–40 Mehta et al.
that is directly proportional to the bio-catalytic layer formation or the reaction or the Ag+, ppm (2016)
electroactive species concentrations (Sangamithirai et al., 2018). inhibition of Cu2+,
enzyme Cd2+,
reactions in the Pb2+,
5.2.5. Calorimetric biosensor
target analyte or Cr3+,
Calorimetric biosensor is also called a thermal biosensor. The sample is used to Fe3+,
working principle of this type of biosensor is that the transducer moni­ detect heavy Co2+,
tors the interactions between an analyte and a bio-receptor resulting in metals Ni2+,
temperature changes which is correlated to the analyte concentration. In Cro2-
4
Antibody based The detection Hg, Pb 0.27 ppm Balahura et al.
other words, it works under the changes in enthalpy during the inter­
method is (2019)
action between the bio-receptor and the analyte. In this device, therm­ amplification
istors or thermopiles are used (Perumal and Hashim, 2014). The and detection of
advantages of the calorimetric biosensor are that it detects reactants the antibody-
without labeling, frequent recalibration is not required and no distur­ antigen
interaction
bance is caused by the optical and electrochemical properties of the Whole cell It detects the bio- Cu2+, 1 μM-100 Wong et al.
sample (Mohanty and Kougianos, 2006). The types of biosensors, its based availability Pb2+, μM (2013)
specification, detection of heavy metals and detecting limit is consoli­ concentration of Cd2+,
dated in Table 5. the metal ions Cr2+,
that it requires Hg2+
Cd2+– Cadmium ion, Pb2+– Lead ion, Ag+– Silver ion, Ni2+– Nickel
and quantifies
ion, Cr2+– Chromium ion, Hg2+– Mercury ion, Zn – Zinc, Cu2+– Copper the toxicity
ion, Fe3+– Iron, Co2+– Cobalt ion, Cro – Chromium (II) oxide. (continued on next page)

13
K. Velusamy et al.
Table 5 (continued )
Biosensor Specification Heavy Detection Reference
metal limit
DNA based Interactions Pb2+, 300pM Nie et al. (2012)
between the Cd2+,
nucleic acid and Ni2+,
the target metal Cu2+,
ions are detected Hg2+
6. Role of nano technology in preparation of biosensor
Due to the vast application of nano technology, it is used in the field
of nanoelectronics, catalysis, magnetic data storage, structural compo­
nents, biomaterials, and biosensors, nanomaterials and is currently
experiencing rapid development (Sagar et al., 2020). The utilization of
NPs, nanotubes, nanowires, etc., as biosensor in diagnostic devices is
being investigated by many researchers across the world (Hussain et al.,
2021). Novel and smart biodevices which can detect minute concen­
trations of the desired analyte are emerging as a result of advancements
in the characterisation of nanomaterials. Nanomaterials are also being
coupled with other polymers and organic materials to form nano­
composites which is also a forerunner in the development of sensors. In
the design of biosensors, nanomaterials are serve as transducer mate­
rials. Surface-to-volume ratio, chemical activity, mechanical strength,
electrocatalytic characteristics, and diffusivity is improved multifolds in
a nanostructured materials (Khand et al., 2021). The high performance
of a biosensor is also significantly influenced by nanomaterials which
are used to detect biomolecules including DNA, bacteria, viruses, and
other types. The standardisation of the immobilization technique that
can be used to knit together a biomolecule to a nanomaterial is a
remarkable problem to work upon (Supriya et al., 2021).
7. Future prospects for biosensors
The past decade has witnessed the development of biosensors
tremendously as a technology to detect environmental contaminants,
especially heavy metals. Several research groups across the globe are
focusing on developing new designs and fabrication techniques for
biosensors which can be used specifically against a contaminant or any
other biomolecule. The biosensors possess several advantages such as
robustness, cost-effectiveness, rapidity, selectivity and enhanced sensi­
tivity. Although it has some drawbacks, such as the effects of pH vari­
ation, temperature, and the need for sample preparation, it finctions on
par with other types of sensors. However, its potential cannot be limited
to detect heavy metals in waste water alone. In the forecoming years,
coupling the nanomaterials with suitable materials with superior optical
and electrochemical properties will enhance the surface area which will
lead to the development of better sensors. The properties of conducting
polymers, carbon nanotubes, graphene, quantum dots and magnetic
nanoparticles has to be explored. For the real-time monitoring of envi­
ronmental pollutants, body fluids and food pathogens, wireless
networking-equipped and global positioning system (GPS) and Internet
of Things (IoT) enabled biosensors can be developed, and this will
expand the research prospects in this field. Modern discoveries include
fluorescence cumulative optical-based biosensors and coupling of
genetically engineered microbial biosensors for heavy metal detection.
These biosensors are emerging to work under vast range of conditions.
Biosensor can also be used for heavy metal detection in food as well
alongside the environmental samples. For environmental and agricul­
tural food pollution detection, DNA-based biosensors are also mostly
used. The key aspects of biosensors that could still be improved includes
the reduction of cost, increasing sensitivity and selectivity and
improving multi-target detection. The design and fabrication techniques
has to be further standardized and optimized to further refine the
product design.
14
Food and Chemical Toxicology 168 (2022) 113307
8. Conclusion
This review describes the various technologies and role of biosensors
in detecting heavy metals in wastewater. The detection limits of various
recent technologies and biosensors were discussed. Biosensors are more
efficient compared to other technologies on the basis of the advantages,
disadvantages and limits of biosensor detection. It also has the special
features of r-usability, reproducibility, sensitivity and selectivity. In real-
time scenario, the cost of implementing a biosensor is low compared to
other technologies. Even though it has some better features for detecting
heavy metals, some parameters like cost minimization still needs
improvement. The difficulties associated with the technological shift
from academics to industries also must be addressed. The pollution of
the environment with heavy metals and other substances is increasing
rapidly, so it is not sufficient to use conventional analytical techniques
for monitoring. So, it is vital to work on improving the existing tech­
niques or develop new techniques to detect this pollution efficiently.
Biosensors can address the demerits of conventional methods. It can be
used to detect metal ions from water and any other matrix by using
appropriate materials. The possibility of coupling biosensor with other
analytical techniques must be explored and applied for better outcomes.
CRediT authorship contribution statement
Karthik Velusamy: Writing – review & editing, Conceptualization,
Data curation. Selvakumar Periyasamy: Writing – review & editing,
Conceptualization, Data curation. P. Senthil Kumar: Conceptualiza­
tion, Validation, and, Supervision. Gayathri Rangasamy: Visualization,
Data curation, and, Formal analysis. J. Mercy Nisha Pauline: Visuali­
zation, Data curation, and, Formal analysis. Pradeep Ramaraju: Visu­
alization, Data curation, and, Formal analysis. Sneka
Mohanasundaram: Visualization, Data curation, and, Formal analysis.
Dai-Viet Nguyen Vo: Conceptualization, Validation, and, Supervision.
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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