Sensors and Actuators Reports 4 (2022) 100078

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Colorimetric Visual Sensors for Point-of-needs Testing

Sadagopan Krishnan *, Zia ul Quasim Syed
Department of Chemistry, Oklahoma State University, Stillwater, Oklahoma 74078, United States

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

Keyswords: The concept of color in chemistry can take us back to when the separation of color-based natural products, dyes,
Point-of-needs and other chemical compounds led to the technique called chromatography. The fundamental color-based
Visual sensors chemical separation opened the modern ultra-speed, ultra-resolution chromatographic methods used for all
Color
analytical purposes in chemistry, biology, nanotechnology, and materials science today. The same color property
Real samples
Molecules
of molecules either directly available or that can be created through indirect secondary chemical and
Biomarkers biochemical reactions have led to the development of colorimetric sensors. More than ever before the need for
user-friendly molecular diagnostics and on-site chemical analysis has been well recognized for their lifesaving
roles in the pandemic era. The objective of this review article is to share some representative recent de­
velopments on colorimetric approaches for molecular diagnostics of relevance to viruses, food safety, disease
diagnostics and management, and environmental analysis. Attention to the purpose of the sensor designs,
rationale, and mechanistic aspects of the approaches are covered, and the demonstration of applicability for real
sample analysis in each cited literature report is reviewed. Our scientific community should consider mandating
sensor research reports to explicitly disclose the following key sensor attributes right in the abstract paragraph:
(i) real sample evaluations, (ii) sample pretreatments, sample matrix dilutions, and their durations, (iii) dif­
ferences in the analytical performance of isolated samples and complex real sample matrices, and (iv) robust
independent validation/correlation methods to assess the fit-for-purpose of the sensor.

1. Introduction assays, immunohistochemistry, and enzyme-linked immunosorbent as­

The pandemic world has realized the compelling need for accessible,
affordable, and user-friendly instrument-free molecular diagnostics for a
vast majority of the global resource-limited settings. Negative COVID
testing results have become a mandatory document to travel anywhere
nowadays (specifically, real-time reverse transcription-PCR (RT-PCR) is
the only accepted method at present). Moreover, even before the
pandemic situation, colorimetric sensors as biomedical and environ­
mental tools were recognized to play crucial roles in human life. [1–3]
Colorimetric sensors that are easy to use, portable, instrument-free, low
cost, and offer sensitive and selective detection toward various analytes
are required for point-of-need and point-of-care applications. [4. 5. 6. 7]
Colorimetric sensors are classified under optical sensors that display a
single-, dual-, or multi-color change in the presence of target molecules
being detected[4. 8. 9. 10]. Addressing the emerging colorimetric
instrument-free sensors is the overarching goal of this review article.
Traditional molecular biology, biochemistry, nucleic acids, protein, and
immunology-based assays (e.g., polymerase chain reaction, radioactive
says) are valuable tools for laboratory-based testing for their accuracies
and reliable measurements. [11] However, the utilization and depen­
dence of such methodologies for large-scale population-based screening
are not feasible. Traditional nucleic acid and protein biological assays
involve time-consuming, technically challenging procedures that only
trained, educated professionals can perform, and they do require so­
phisticated expensive laboratory settings. Such methods are out of
reach, and/or not implementable for use by any literate people, and not
accessible for a vast majority of the global population living in a
resource-limited economy [12]. Thus, modern science and technology
advancements have not fulfilled the equal access of tools to every human
on the planet, but rather to those living in economically sound
geographical locations, the so-called developed countries. Hence, new
user-friendly, cost-effective, instrument-free molecular detection ap­
proaches, such as colorimetric assays and sensors, for everyone on the
planet offer a huge positive impact on human life in this pandemic era
and intellectually strengthen the whole healthy undeniably connected
world. In addition to human health-related accessible and point-of-need

* Corresponding author.
E-mail address: gopan.krishnan@okstate.edu (S. Krishnan).

https://doi.org/10.1016/j.snr.2022.100078
Received 19 October 2021; Received in revised form 18 January 2022; Accepted 21 January 2022
Available online 2 February 2022
2666-0539/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/).
S. Krishnan and Z.Q. Syed
molecular diagnostics, other equally important areas include food and
agriculture quality and safety, environmental analysis, and
human-caused, politically driven unpleasant biosecurity and biothreat
issues that require large-scale rapid on-site molecular diagnostics suit­
able for both resource-limited and economically sound settings.
A recent review covered the pros and cons of several instrument-free
nanoparticles-based strategies for glucose detection in most biological
fluids (blood, urine, saliva, sweat, tears, and interstitial fluid)
concluding with feasibilities for potential non-invasive point-of-care
tests based on a colorimetric nanosensor, and thus improve life quality
and healthcare [13]. Physiological and diabetic relevant glucose con­
centrations are in millimolar levels with the highest being present in
blood and interstitial fluids (minimally invasive collection, but still
discomforting and painful for routine intraday measurements); and,
several orders of magnitude lower in sweat, saliva, and tears (non-in­
vasive collection, painless, simplicity, and stress-free, but analytically
challenging for reliable detection due to variations in the sample com­
positions around the day and sources of artifacts in the samples)[14].
Unlike millimolar glucose, most environmental, pathogenic, food, and
clinical biomarkers are present at ultra-low concentrations in biofluids
(e.g., fasting insulin, cancer markers, viruses, small molecules, nucleic
acids, proteins, miRNA, RNA, receptors, and infectious disease bio­
markers) making their colorimetric visual detection challenging to
achieve at a point-of-need.
Another review discussed the recent advances in point-of-care
nucleic acid extraction technologies for rapid diagnosis of human and
plant diseases at the genomic level for the identification of disease-
causing pathogens and their pathogenesis [15]. This review covered
the extraction and isolation aspects of high-quality nucleic acid from
various complex raw samples such as human blood, saliva, sputum,
nasal swabs, urine, and plant tissues. A recent book chapter (Book title:
Nanomaterials Design for Sensing Applications) discussed colorimetric
sensors and sensor arrays with topics covering receptor specificity,
immobilization strategies for sensing molecules on a solid support, vi­
sual output, and interpretation and analysis [16]. In addition, the uses of
various nanomaterials (e.g., carbon dots, metal nanoparticles, hybrid
nanomaterials, nanozymes with catalytic properties, and nanorods) as
reporters have been extensively discussed in the literature.
When it comes to instrument-free miniaturized portable detectors,
microfluidic paper-based analytical devices (µPADs) cannot be missed
out [17–20]. The µPADs feature a hydrophilic paper patterned with
well-defined, millimeter-sized channels, and surrounded by a hydro­
phobic polymer suitable for large-scale point-of-care applications due to
low cost, paper availability, lightweight, and portability[21]. The paper
sensors require minimal reagents, less or no energy consumption when
utilized with the capillary action, and by exploiting the paper porosity
and built-in surface chemical functionalities, the desired bioconjugation
of selective receptors to target molecules can be achieved. Additionally,
when incorporated into a 3D format, reagent diffusion issues can be
overcome, and multiplex assaying becomes feasible with better control
over the fluid flow. In particular, the widely used later flow assays
involve portable handy sensors that are simple to operate and detect
results rapidly [22]. Two, three, and multi-line later flow assays to
measure more than one analyte have been achieved. [10,23] However,
some limitations of later flow assays that are being actively addressed by
researchers include reproducibility issues, sample pretreatment re­
quirements, cross-reactivity issues affecting selective analyte detection,
and are mostly limited to qualitative/semi-quantitative applications.
[24]
The development of colorimetric instrument-free approaches has
advanced significantly with the gold nanoparticles that possess sensitive
plasmonic properties in combination with color changes. Compared to
the traditional low extinction coefficient organic dyes-based sensors,
noble metal nanoparticles possess stronger plasmonic extinction in the
visible light region and offer ultra-high sensitivity [25]. In this regard,
several nanoparticle-aggregation-based visual colorimetric sensor
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Sensors and Actuators Reports 4 (2022) 100078
strategies are reported in the literature. Challenges with such sensors
include the uncontrollable nature of the nanoparticles aggregation and
self-aggregation in complex samples that can act as sources of high
background signals and false-positive results. Hence,
non-aggregation-based gold nanorods-etched plasmonic colorimetric
sensors that utilize alterations in the aspect ratio yielding distinct visual
multicolor responses toward most target analytes have been devised
[26–28]. Thus, tuning the chemical, physical, electrical, gravimetric,
and optical properties of various nanomaterials allowed the develop­
ment of several new generations of sensors. In the same way, certain
supramolecular low molecular weight gels[29] offered visual sensing of
various chemical analytes and toxic ions.
In the subsequent sections, we will cover various colorimetric visual
sensor approaches and a wide variety of target analytes detected re­
ported in the past five years, and how researchers are constantly tackling
new limitations in each strategy developed. This article should not be
seen as an extensive review of all reported articles in the field, but rather
focused on highlighting representative studies and the basis of the sensor
principles and designs in each case to meet the intended sensing ob­
jectives. We additionally discuss the performance of the sensors in
complex real samples either pre-treated, diluted, or used as such with no
pretreatments to understand the robustness of the designed sensors.
Fig. 1 illustrates the number of published documents over the past 44
years, which indicates ever-growing attention and intellectual efforts by
scientists around the world working in the field of colorimetric sensors.
2. Various diverse target analytes
2.1. Biomarkers
A biomarker is broadly classified as “A defining characteristic that is
measured as an indicator of normal biological processes, pathogenic
processes or responses to an exposure or intervention.”[30,31]. In this
review article, we focus on molecular markers present in various clinical
biofluids that indicate an abnormal condition at elevated or diminished
levels depending on the up and down regulation biochemical pathways
involved, respectively.
Viruses. A piezoresistive (a strain-induced change in electrical
resistance) wireless biosensor [32] incorporating clustered regularly
interspaced short palindromic repeats (CRISPR)-Cas12a technology has
been reported for the detection of human papillomavirus (HPV)-DNA. In
this sensor, binding of the HPV-DNA to a guide RNA induced a
Cas12a-mediated non-specific cleavage of the biotin/thiol-modified
linker DNA that connected Au@Pt (Au core and surface Pt) nano­
particles and streptavidin-coated magnetic bead. The freed and sepa­
rated Au@Pt nanoparticles efficiently catalyzed the conversion of H2O2
to O2, and increased the current of a flexible interdigitated
electrodes-modified piezoresistive block made of abrasive paper-molded
microstructure polydimethylsiloxane and MXene (Ti3C2Tx)-PEDOT:
PSS film; here, PEDOT: PSS is poly(3, 4-ethylenedioxythiophene): poly
(styrenesulfonate), and MXene is two-dimensional transition-metal
carbonitrides (Tx refers to surface termination groups such as -F, -O, and
-OH). However, the solution composition of the detected HPV concen­
trations (logarithmic concentration, 1.0–1000 nM range with a limit of
detection of ~ 534 pM) was difficult to trace in the article as well as in
the supporting information.
Freezing-based labeling of DNA-gold nanoparticles probes with
increased poly(A)-base numbers enabled the design of single step,
convenient, and relatively cheaper probes [33]. The authors found that
approximately 10 A bases were required at the sequence ends, and the
formation of secondary DNA structures would shield the exposed A
bases that can result in an inefficient labeling outcome. Demonstration
of the method using three probes, DNA-AuNP, RNA-AuNP, and
DNA-enzyme-AuNP was reported, and the method involved a single
mixing step without the standard tedious thiol modification procedure.
This poly(A)-rich approach facilitated advancing CRISPR-based
S. Krishnan and Z.Q. Syed
Fig. 1. Documents versus year reproduced from the search term “colorimetric sensors” used in Scopus, the Elsevier’s abstract and citation database. Copyrights
Elsevier B.V.
diagnostics via user-friendly colorimetric, fluorescence, and lateral flow
detection strategies. Electricity and instrument-free detection of HIV
type 1(HIV-1) p24 antigen at as low as 0.03 ng/mL has been reported
based on a 3D origami μPAD [34]. This report eliminated the need for
external instrumentation in μPAD approaches. Colorimetric
enzyme-linked immunosorbent assay methodology was utilized in this
μPAD test for on-site detection and use in resource-limited settings.
Fig. 2 and Table 1 illustrate some representative colorimetric detection
approaches for various viruses and virus-like particles detection with
TMB/peroxidase system using various nanoparticles.
miRNA biomarkers. A capillary self-driven complementary DNA
hydrogel film sensor allowed sensitive detection of miRNA targets via
direct hybridization [39]. During the hybridization with the target
miRNA in solution, the permeability of the DNA hydrogel film increased
and thus created a self-driven capillary action. Such naturally driven
physical phenomenon, when exploited in sensors, eliminates the need
for an external source for sample analysis, and additionally offers sen­
sitive detection, in this case, the miRNAs hybridization with the DNA
probes in the DNA hydrogel. A simple measurement of the sample so­
lution flow-through distance in the capillary tube was accomplished.
Moreover, as low as 20 nL sample was found to be sufficient, reducing
the assay cost further. Clinical diagnosis, as well as point-of-care tests in
resource-limited settings, are the anticipated applications.
Fluorescence turn-on detection of biothiols by an indicator-
displacement assay. Glutathione (GSH), cysteine (Cys), and homo­
cysteine (Hcy) are biothiols that are key biomarkers of many life para­
lyzing diseases (e.g., renal failure, liver damage, and Alzheimer’s).
While instrument-based high-performance liquid chromatography,
fluorescence, mass spectrometry, Raman analysis, electrochemical,
capillary electrophoresis, and colorimetric methods were available for
most analyses, instrument-free high sensitivity visual color sensors are
unmatchable to address any global health crisis. To address this need, an
indicator displacement assay as a fluorescence turn-on sensor has been
designed for the detection of the biothiols in biological fluids [40]. The
working principle of this sensor involves a competitive binding of the
biothiol marker and murexide indicator dye towards Hg2+ ions. Inter­
estingly, the biothiols exhibited a stronger binding affinity with Hg2+
ions than the murexide dye. As a result, the quenched fluorescence
emission of the murexide dye by the Hg2+ ion is recovered in the pres­
ence of added biothiols for naked-eye detection as the output utilizing a
UV irradiation excitation source. In particular, the biothiols showed
severalfold greater fluorescence recoveries than other amino acids
tested, suggesting a dominant selectivity of the competitive assay for
biothiols. The linear detection ranges were 0.1–40 μM for GSH, 0.5–30
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Sensors and Actuators Reports 4 (2022) 100078
μM for Cys, and 0.5–50 μM for Hcy. The applicability of the sensor for
analyzing pretreated real human urine (100-fold diluted) and serum
(500-fold diluted) samples was demonstrated with a satisfactory sample
recovery (>94% in most cases).
2.2. Ions
It is important to measure both toxic ions to know their dangerously
abnormal/exposure levels as well as biologically crucial metal ions to
know their healthy levels and deficiency. For these purposes, it would be
advantageous to measure the desired metal ion concentrations in
various biofluids at the point of need to save the waiting time of testing
results from a test laboratory and thus take immediate lifesaving mea­
sures. Similarly, the detection of some non-metallic compounds, oxides,
and anions also holds practical significance as illustrated below for ni­
trite, chloride, bromate, and other chemical and biochemical
compounds.
Hg2þ ion detection using a fluorescent electrospun fibrous
membrane. To overcome the sensitivity limitations of a portable, low-
cost, and convenient paper-based platform, a fluorescent electrospun
fibrous membrane was designed for detection of Hg2+ contamination in
less than a minute and down to a sub-ppb level (0.309 ppb, i.e., 0.697
nM, much lower than the regulatory permitted maximum 2 ppb mercury
level in drinking water)[41]. In these paper strips, a novel
mercury-favored organic dye was immobilized on a cellulose acetate
/polycaprolactone electrospun fiber that exhibited the property of
fluorescence resonance energy transfer signals. The designed
instrument-free visual readout of Hg2+ level-dependent fluorescence
enhancement was demonstrated in Hg2+-spiked drinking water and skin
whitening serum that was digested with 65 % nitric acid and diluted
with ultrapure water.
Cd2þ ion detection. Glutathione modified gold nanoparticles
deposited on a polyethylene terephthalate sensor strip allowed real-
time/on-site colorimetric detection of Cd2+ ions in drinking water, by
turning the water into red [42]. In contrast, the approach of aggregation
of glutathione modified gold nanoparticles suspension in water samples
containing Cd2+ turned the water blue. The limit of detection of this
Cd2+ sensor strip was 18.8 nM, which is less than the US Environmental
Protection Agency permitted maximum contamination level of 44.5 nM
Cd2+ in potable water, thus making the sensor practically relevant.
Using the sensor strip, on-site detection of Cd2+ in real tap water samples
was demonstrated. Another colorimetric fluorescent sensor design
involved mixing of an orange emission glutathione-stabilized gold
nanoclusters with a blue emission ethylenediamine functionalized
S. Krishnan and Z.Q. Syed Sensors and Actuators Reports 4 (2022) 100078

Fig. 2. 3,3′ ,5,5′ -Tetramethylbenzidine/ horseradish peroxidase (TMB/HRP) system. 1) Sandwich immunoassay for the detection of the respiratory virus using
chromogen polymer shell with TMB nanoparticles. Reproduced with permission from [ref 35]. Copyrights published by Royal Society of Chemistry 2021. Colori­
metric assays, although lack sensitivity and require amplification, are simplistic compared to signal quenching associated chemiluminescence and fluorescent
processes. To overcome these issues, a sandwich immunoassay with peroxidase stacked on gold nanoparticles encapsulated in liposomes with high peroxidase activity
has been shown to yield signal amplification. Poly (lactide-co-glycolide) (PLGA) is a biocompatible carrier used for encapsulating TMB conjugated with antibodies for
capturing the respiratory virus. Signal amplification was achieved through co-precipitation; upon addition of dimethyl sulfoxide, the PLGA layer dissolves and
releases TMB-nanoparticles which are oxidized by the self-fabrication of copper nano-flowers to develop a blue color confirming the presence of a virus. 2) Detection
of norovirus using vanadium pentoxide nanoparticles conjugated with liposomes a dual-modality sensor which uses UV–Vis spectrometer for optical response and
electrochemical redox detection with the help of TMB. Reproduced with permission from [ref 36]. Copyrights 2020 published by Elsevier B.V. Single-mode
colorimetric sensors suffer from sensitivity issues due to low selectivity, the inadequate concentration of analytes, and the complexities of real sample matrices
containing several interfering or nonspecific pools of molecules causing sensor fouling. Such drawbacks can be overcome using dual- or multi-modality detection
features. Vanadium pentoxide (VONP), a robust nanozyme, was encapsulated inside the liposomes. Magnetic nanoparticles and liposomes are conjugated with
anti-norovirus antibodies which can recognize norovirus. Nanoconjugates of VONP-liposome-norovirus-magnetic nanoparticles are separated magnetically upon
interaction, further treated with triton x surfactant to release encapsulated VONP. In the absence of virus-like particles, insufficient oxidation of TMB was observed
that did not generate a noticeable color product. The developed sensor showed fg/mL detection levels. 3) Influenza virus detection using a one-step synthesis of gold
nanoparticles coated on carbon nanotubes. Reproduced with permission from [ref 37]. Copyrights 2016 published by Elsevier B.V. In this study, two nanomaterials
were combined. Gold nanoparticles were introduced on the surface of carbon nanotubes in a single-step synthesis making the process environmentally friendly and
inexpensive and has been successfully applied for the detection of the influenza virus. 4) Colorimetric detection of hepatitis virus by catalytic oxidation of TMB by
silver deposited gold nanoparticles. Reproduced with permission from [ref 38]. Copyrights published by Elsevier B.V. Gold-silver core-shell nanoparticles were
developed for the detection of the hepatitis virus. In the presence of silver and hydroquinone, the chromogen substrate, TMB, was oxidized, and the presence of
immunocomplex causes color development. The proposed immunoassay was shown to be highly specific and sensitive with a detection limit of 10 pg/mL.

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S. Krishnan and Z.Q. Syed
Table 1
Representative comparison of literature reports on various colorimetric virus sensors.
Analyte
Avian influenza A (H7N9)
i) Influenza A virus surface
protein (H3N2)ii)
Hepatitis A virus (HAV)
(DNA)
Human immunodeficiency
virus type 1 (HIV-1)
(DNA)
Maize chlorotic mottle
virus (MCMV) (DNA)
Influenza virus (HN)
(Nucleotide)
i) Influenza A virus (H1N1)
ii) Influenza A virus
(H3N2)
Influenza A virus
Influenza A virus
Human immunodeficiency
virus (HIV)
Bovine viral diarrhea virus
(BVDV) (RNA)
i) Middle east respiratory
syndrome (MERS) ii)
Mycobacterium
tuberculosis (MTB) iii)
Human papillomavirus
(HPV)
Human papillomavirus
(HPV) DNA
Norovirus like particles
(NoV-LPs)
Antibodies of i) Hepatitis C
core antigen virus (HCV)
ii) Hepatitis B surface
antigen virus (HBV)
Kaposi`s sarcoma
associated herpesvirus
(KSHV)& bacillary
angiomatosis (BA)
Influenza viruses (HN)
Hepatitis C Virus(HCV)
Avian Influenza virus
(H7N9)
RNA of Rift valley fever
virus (RFVR)
Antibodies of i) Influenza
ii) Human
immunodeficiency virus
iii) Dengue
Human papillomavirus like
particles (HPV16)(DNA)
Human norovirus (HuNoV
I & II)(RNA)
Sensors and Actuators Reports 4 (2022) 100078
Approach Detection label use Sample matrix Limit of detection Linear range Ref.
Colorimetric Streptavidin-biotin/ i) 0.1 M PBS bufferii) 5 Visible: 25 pg/ Visible: 50 to 150 pg/ [107]
sandwich Alkaline phosphatase (ALP) Spiked H7N9 sample in mLInstrument: 1.25 pg/ mLInstrument: 5 to 50
immunoassay normal serum (0 – 160 mL pg/mL
pg/mL)
Direct colorimetric Aggregation of gold 10 mM Tris–HCl buffer 11.16 µg/mL 0 to 240 µg/mL [108]
assay nanoparticles (AuNPs)
Invader assisted Streptavidin- biotin i) Buffer ii)Serum 1 fM 100 to 102 fM [109]
enzyme linked
immunosorbent assay
(iaELISA)
Surface enhanced SERS nano tags/ 10-fold dilution of 0.24 pg/mL 8 to 64 pg/mL [110]
Raman scattering- Streptavidin-biotin Oligonucleotides in
Lateral flow assay buffer
(SERS-LFA)
Asymmetric Aggregation of gold RNA extract in buffer 23.9 pg/µL 23.9 ng/µL to 23.9 pg/ [111]
polymerase chain nanoparticles (AuNPs) µL
reaction colorimetric
assay
Colorimetric assay Carbon nanotubes/Gold Diluted human serum 3.4 plaque forming 10 to 5 × 104 PFU/mL [37]
3,3´,5,5´- with PBS (Phosphate units/mL (PFU/mL)
tetramethylbenzidine (TMB) buffer saline) (1 µg/mL)
Modified enzyme Gold nanoparticles/ 3,3´,5,5´- Diluted with PBS 10.79 pg/mL 10 pg/mL to 10 µg/mL [112]
linked tetramethylbenzidine (TMB)
immunosorbent assay
(ELISA)
Surface enhanced SERS nano tags Diluted with 0.1x PBS 1.9 × 104 plaque forming 0 to 1 × 106 PFU/mL [113]
raman scattering units/mL (PFU/mL)
Lateral flow assay
(SERS-LFA)
One step polymerase Green fluorescence label DNA extracted from 2 aM Not mentioned [114]
chain reaction (PCR) blood (5000 copies/mL)
Direct colorimetric Aggregation of gold RNA extract fromi) 80 TCID50/mL (Tissue 5 to 80 TCID50/mL [115]
nanoparticles Bovine bloodii) Human culture infectious dose)
BloodBuffer
Fluorescent & Aggregation of silver Synthetic DNA in 0.1 M MERS: 1.53 nMMTB: 20 to 1000 nM50 to [116]
colorimetric nanoparticles (AgNPs) PBS. 1.27 nMHPV: 1.03 nM 2500 nM20 to 2500
nM
Interdigitated Aggregation of gold DNA isolated from 30 nM Not mentioned [117]
electrode sensor nanoparticles (AuNPs) serum diluted in
deionized distilled water
Colorimetric Graphene-gold Diluted human serum 92.7 pg/mL 100 pg/mL to 10 µg/ [118]
immunoassay nanoparticles/ 3,3´,5,5´- mL
tetramethylbenzidine (TMB)
Wave guide-mode Horseradish peroxidase Undiluted human blood 0.03 nM 1 nM to 10 nM [119]
sensor (WM) (HRP)
Surface enhanced Streptavidin-Biotin DNA oligonucleotide in KSHV: 0.043 pMBA: 0 to 20 pM [120]
raman scattering- buffer 0.074 pM
lateral flow assay.
(SERS-LFA)
Colorimetric assay Aggregation of glycan Genes obtained from 8 Hemagglutinin units Not mentioned [121]
functionalized gold clinical isolates of virus (HA)
nanoparticles
13 13 9
Enzyme mediated Streptavidin-Biotin Human serum 10− g/mL 10− to 10− g/mL [122]
colorimetric assay
Colorimetric 4-aminoantipyrine (4-AAP)/ Diluted chicken serum 3.5 pg/mL 0.01 to 50 ng/mL [123]
immunoassay hemin samples
Colorimetric assay Aggregation of gold 10-fold diluted serum 10 RNA copies/assay 100 to 105 RNA copies [124]
nanoparticles
Paper based Bioluminescence resonance i) Antibody spiked HIV: 2.8 nM H1N1: 7.1 1 to 100 nM [60]
colorimetric energy transfer offurimazine porcine serumii) Whole nM DEN: 19.3 nM
blood
Direct colorimetric Aggregation of gold Oligonucleotides in 10 ng/mL 10 to 103 ng/mL [125]
assay nanoparticles buffer
1 4
Horseradish 2,2′ -Azino-bis(3- i) 10 times diluted PCR i) Diluted: 10 copies/mL 1 to 10 copies/mL [126]
peroxidase integrated ethylbenzthiazoline-6- bufferii) 20 times diluted ii) Pancreatic: 11.27
pancreatic tissue sample copies/mL
(continued on next page)
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S. Krishnan and Z.Q. Syed Sensors and Actuators Reports 4 (2022) 100078

Table 1 (continued )
Analyte Approach Detection label use Sample matrix Limit of detection Linear range Ref.

polymerase chain sulfonic acid)/ Horseradish spiked with diluted

reaction peroxidase (ABTS/ HRP) virus.
Urinary biomarkers of Colorimetric sensor Digital imaging Undiluted urine samples Highest sensitivity: Not mentioned [127]
tuberculosis array (CSA) 79.5%
Hepatitis C virus (RNA) Direct colorimetric Salt-induced aggregation of 1x Hybridization buffer 5.6 ng/µL or 500 nM 370 nM to 3 µM [128]
biosensing gold nanoparticles (AuNPs)
i) Influenza (H1N1) (RNA) Loop mediated Hydroxynaphthol blue i) Inactivated viral i) 3.2 × 10− 5 Hem i) 3.2 × 100 to 3.2 × [129]
ii) Staphylococcus isothermal (HNB) strains. (10-fold dilution agglutination units 10− 5 HAU for H1N1ii)
aureus (DNA) amplification (LAMP) 32 HAU stock)ii) (HAU) per reaction for 3.2 × 100 to 3.2 × 104
Cultured bacterial H1N1 viruses and 3 × CFU for 101 to 10− 4
strains(10 times diluted 100ii) Colony forming staphylococcus aureus
samples of 3 × 105 CFU/ units (CFU) per reaction
µL stock) for bacteria.
Middle East respiratory Direct colorimetric Salt induced aggregation of Synthetic 1 picomole/µL Not mentioned [130]
syndrome coronavirus assay double-stranded DNA oligonucleotides in
(MERS-CoV) (ds DNA) (dsDNA) self-assembly buffer
shielded gold nanoparticle
1 picomole/µL
Influenza A virus (H1N1) Paper based Polydiaceytlene (PDA) i) Buffer diluted viral 5 × 103 TCID50 viruses 5 × 103 to 5 × 105 [62]
Dengue virus & colorimetric immobilized/ strains. (Tissue culture infectious TCID50 [131]
Trypanosoma brucei Bioactive cellulose polyvinylidene fluoride ii) 1% nasal fluid spiked dose) Not mentioned [132]
Influenza (H1N1) microparticles for the (PVDF with viral strains 100 nM 0 to 104 PFU/mL in [133]
Human norovirus colorimetric detection ZZ–CBM3 (carbohydrate Diluted oligonucleotides i) EIS PBS [134]
(AG3 capsid specific Sandwich binding molecule): Biotin i) 0.1 M Buffer 3.3 plaque forming units 0 to 104 PFU/mL in [135]
aptamer) immunoassay. labelled gold nanoparticles. ii) 80% spiked saliva PFU/mL (PBS) spiked saliva [136]
Human Confirmed by Horseradish peroxidase i) Buffer. 4.7 PFU/mL (saliva) 20 to 103 viruses per [36]
immunodeficiency virus electrochemical (HRP/TMB) ii) Human Serum ii) Colorimetric assay [137]
(HIV) DNA impedance Horseradish peroxidase iii) Shellfish 1.34 PFU/mL (PBS) 10− 14 M to 10− 9 M [63]
Dengue virus spectroscopy(EIS) (HRP/TMB) iv) MS2 phage 2.27 PFU/mL (saliva) 4.9 nM to 9.3 nM [106]
serotypes (DENV 1, 2, 3, Colorimetric 2,2′ -Azino-bis(3- (bacteriophage) 3 viruses/assay 105 to 109 DNA copies [138]
and 4) nanozyme ethylbenzthiazoline-6- i)Oligonucleotides in 25 Or 30 virus/mL i) Electrochemical: 10 [139]
Porcine circovirus 2 aptasensor sulfonic acid)/ Horseradish mM Tris buffer 100 fM in Buffer fg/mL to 10 pg/mL [140]
(DNA) G3/hemin based peroxidase ii) 10 times diluted DENV-1 8.8 nM, DENV- ii) Colorimetric: 1 pg/ [141]
Norovirus like particles Isothermal (ABTS/ HRP) serum 2: 4.9 nM, DENV-3: 9.3 mL to 100 ng/mL [142]
Rabies virus exponential 2,2′ -Azino-bis(3- Prototype strains in nM, and DENV-4: 5.1 nM 0.1 to 6 nM for [143]
(DNA) amplification reaction ethylbenzthiazoline-6- buffer Cocktail LOD: 36.7 nM, Fluorescent. [35]
Zika virus strains (EXPAR) sulfonic acid)/ Horseradish DNA extract from serum 42.6 nM, 35.2 nM and 0.5 to 60 nM for [144]
Human G4/Hemin peroxidase samples diluted with 37.1 nM for the addition Colorimetric [145]
immunodeficiency virus Direct colorimetric (ABTS/ HRP) buffer of DENV-1TS, DENV- Not mentioned
(HIV) DNA sensing Aggregation of gold i) 10% Human Serum 2TS, DENV-3TS and 10 to 60 nM
Influenza Virus strains Electrochemical and nanoparticles (AuNPs) ii) 50% Human Serum DENV-4TS (TS: Not mentioned
(H1 to H16) colorimetric V2O5 nanoparticles- iii) 100% Human Serum Stereotype) 2.3 ng/µL to 23.3 ng/
Largemouth bass virus combined dual encapsulated liposomes Diluted with DI water 7.8 × 109 DNA copies/ µL
DNA. modality read out (VONP-LPs) i) Buffer mL 1 to 103 nM
Human papilloma virus sensor Horseradish peroxidase ii) Spiked cerebrospinal i) Electrochemical: 4.1 10 to 1500 virus
(HPV) DNA Dual mode (HRP/TMB fluid fg/ mL. particles/mL
Pseudo coronavirus fluorescent and Glucose oxidase/ HRP- TMB i) 100 times diluted ii) Colorimetric: 0.34 pg/ RSVA: 5.2 nM to 52 aM
spike protein colorimetric Single nucleotide specific using DNase free water. mL RSVB: 910 pM to 91
RNA templates of Fluorescent programmable ii) Whole blood Fluorescent - 15 pM aM
Respiratory syncytial and colorimetric riboregulators using Reverse i) 100 times diluted Colorimetric - 60 pM Not mentioned
virus A & B Ratiometric pH transcriptase recombinase serum. 260aM in clinical 10 fg/mL to 10 ng/mL
(RSVA & RSVB) sensitive direct polymerase amplification ii) 1% Diluted serum. samples 101–104 plaque
RNA of coronavirus. colorimetric assay (RT-RPA) 2-fold dilution with 10 7.8 nM or approx. 4.72 forming units/mL
Middle East respiratory Direct Visual Inhibition of Ag+ to urease mM PBS × 1012 copies/mL 104–106 copies/ml
syndrome (MERS) Colorimetric using phenol red as an Extracted DNA were 10- Visual: 2.3 ng 160 fM to 1 nM
i) Influenza DNA Direct Colorimetric indicator fold diluted Spectral: 1.2 ng
ii) Coronavirus spike assay Aggregation of gold i) Synthetic 6.99 ng/µL
protein Direct colorimetric nanoparticles (AuNPs) oligonucleotides in 1 nM
Envelope protein of zika assay using LFA Aggregation of gold Buffer. Colorimetric - 1 ng/mL
virus Direct colorimetric nanoparticles (AuNPs) ii) Clinical samples (10, SERS - 4pg/mL
Sars-CoV2 virus specific assay and SERS based Salt aggregation of gold 100, 1000 times diluted) 5.2 aM - RSVA
genes Paper based & direct nanoparticles (AuNPs) Pseudo coronavirus 91 aM - RSVB
visual colorimetric Aggregation of AuNPs using protein in buffer. 20 aM – Coronavirus
assay based on antispike antibody modified 10-fold dilution in buffer 200 aM- MERS
Nucleic acid surface. 1:5 diluted human saliva Influenza - 32.37 fg/mL
sequence-based RNA toehold switch sensor. with 2X saliva Influenza 10% spiked
amplification Lac Z hydrolyzed stabilization solution serum - 54.97 fg/mL
(NASBA) chromogenic substrates (1:1) Coronavirus- 143 fg/mL
Paper based Toehold switch sensor i) Buffer. 6.3 × 104 copies/mL
Sandwich immunoa amplified using Nucleic acid ii) 10% spiked human (Single site probe)
ssay sequence-based serum 0.58 pM- Color
Colorimetric binding amplification (NASBA) i) 1:10 Diluted serum 2.17 pM- SERS
assay & a label free Copper nanoflowers with 10 mM PBS. 1.11 pM-Fluorescence
electrochemical (CuNFs) with ii) 1:1 Diluted urine with (Four site probe)
impedance sensor TMB-NPs encapsulated in 10 mM PBS 160 fM, 259 fM & 395 fM
(EIS) poly lactic co glycolic acid
(continued on next page)

6
S. Krishnan and Z.Q. Syed
Table 1 (continued )
Analyte Approach Detection label use
Colorimetric, (PLGA)
SERS, High affinity antibody
Fluorescence memetic called biotinylated
clamp peptides (HRP/TMB)
Saline induced self-
aggregation of AuNPs
graphene oxide. In this sensor, the introduction of Cu2+ ions quenched
the orange emission of gold nanoclusters making the blue emission a
background reference [43]. In the presence of Cd2+ ions, aggregation of
the Cu2+- glutathione-stabilized gold nanoclusters induced the emission
of orange fluorescence with the associated change in color of the solu­
tion from blue to red. This fluorescent paper strip approach offered a
limit of detection of 33.3 nM Cd2+ in solution, and upon integration with
a smartphone platform, as low as 100 nM Cd2+ in rice samples was
detected. Food safety and environmental protection are related appli­
cation areas for the detection of toxic levels of metal ions present as
contaminations.
Pb2þ ion detection. Exposure to toxic levels of lead in the envi­
ronment has been a growing problem in developing countries due to
expanding industrialization and outsourcing by developed countries.
Hence, methods that are instrument-free, sensitive, and selective are
valuable for lead detection. Gold nanoparticles based colorimetric
detection of lead required specific surface modifications and incubation
times. To make such assays rapid, the use of synthesized gold nano­
particles without surface modifications was found to offer selective
Pb2+detection instantly from a red to blue color change with a limit of
detection of 18 μM, and naked-eye detection of 60 μM with a limit of
quantification of 53.5 μM [44]. For other metal ions, such a color change
was not observed to be prominent. Stronger interactions between the
Pb2+ ion and gold nanoparticles over the tested other metal ions and the
resulting elevated aggregation of the nanoparticles were proposed to be
the underlying mechanistic causes of selectivity for Pb2+.
Visual detection of Fe2þ ions. Ferrous and ferric ions are crucial
markers of many biological processes, diseases, and hence accurately
measuring their concentrations is significant in human health di­
agnostics. Chemically bonded 1,10-phenanthroline-5-amine onto cellu­
lose acetate by using 4,4′ -methylene diphenyl diisocyanate as a cross-
linker facilitated a cellulose-based fluorescent sensor for a highly se­
lective and visual rapid detection of Fe2+ ions with a detection limit of
50 ppb. In the fluorescence mode, the detection limit reported was 2.6
ppb [45].
Cl¡ ion and thiol-containing drug detection via oxidative
etching of silver nanoparticles. A distance-based paper sensor for a
simple, inexpensive, instrument-free, and portable determination of
chloride ions was accomplished through an oxidative etching of silver
nanoparticles forming AgCl in the presence of Cl− and H2O2. The
resulting band length of the white precipitate of AgCl was utilized for a
distance-based detection output in proportion to Cl− concentrations
[46]. In this sensor, the peroxide reacts with silver nanoparticles in the
channel of the paper device, and the Cl− ions present in the analytical
sample forms a white precipitate of AgCl. A working concentration
range of 25–1000 mg/L with the naked eye detection limit of 2 mg/L
(0.08 μg) was attained with good reproducibility (relative standard
deviation of < 4.51% for n = 10 measurements) without the use of any
external instrumentation and the performance metrics matched the
traditional tests of real water samples. Similar oxidative etching of silver
nanocrystals allowed control over the morphology change of the nano­
material from a triangular prism shape to a disk shape. In the presence of
trace levels of halides as mediators, the etching of silver nanocrystals
caused a change of blue to yellow color. This property was used to detect
a captopril drug that inhibits the oxidative etching of silver nanocrystals,
via the formation of gold-thiol bonding, in a concentration-dependent
Sensors and Actuators Reports 4 (2022) 100078
Sample matrix Limit of detection Linear range Ref.
Diluted RNA buffer (1
pM to 10− 2 pM)
manner, and the associated reversal of a range of color changes from
yellow to blue [47].
Controlling nanoparticle aggregation for selective toxic ions
detection. In many colorimetric platforms, the nonspecific aggregation
of gold nanoparticles caused by changes in ionic strength (salt concen­
tration) influenced the sensor performance. A colorimetric sensor with
aptamer-functionalized microparticles was identified to have more
tolerance to aggregation upon increasing the ionic strength. This prop­
erty was favorably used to enhance the detection performance of
analytical targets as a cost-effective, instrument-free, portable, and
reliable aptasensor for multiplex colorimetric detection [48]. This study
described a well-controlled fabrication process of the device within ±1
standard deviation (SD) (relative SD = 5.69%, n = 40) and utilized a
salt-induced aggregation mechanism for highly stable multilayered label
particles. DNA aptamers as capture biomolecules and polyethyleneimine
as the encapsulating agent allowed for a sensitive and highly specific
colorimetric multiplexed detection of Hg2+ and As3+ ions in the pres­
ence of other possible interfering ions and with a limit of detection of 1
ppm for each ion.
Picomolar visual detection of mercury and silver ions: Pro­
grammable nanoprobes. As per the environmental protection agency
(EPA) regulation, the maximum permissible level of inorganic mercury
in drinking water is 10 nM (2 ppb). To achieve ultra-sensitive colori­
metric detection, nanotechnology strategies to detect picomolar mer­
cury levels have been developed. One such approach was based on a
hybridization chain reaction combined with plasmonic gold nano­
particles that enabled an ultra-low colorimetric detection of 2 ppt (10
pM) of mercury and/or silver ions with an estimated cost of less than a
penny per test [49]. Application of the approach for mercury detection
in water, soil, and urine samples was demonstrated involving the assay
duration of about four hours and with a color change duration of only a
few minutes.
Smartphone-assisted paper-based sensing of cyanide in drink­
ing water. Using an ordinary filter paper as a durable solid support,
impregnation of europium tetrakis dibenzoylmethide triethylammo­
nium and gold nanoparticles yielded a fluorescence turn-on cyanide
assay in aqueous media [50]. In this method, the cyanide detection was
based on a fluorescence recovery from the dissolution of gold nano­
particles combined with the ligand exchange of the triethylamine groups
of the europium compound with the cyanide group of the target mole­
cule to attain an increased fluorescence signal output. This paper plat­
form featured naked eye distinguishable color transition in the cyanide
concentration range of 10− 2 to 10− 12 M. The authors developed an
image processing algorithm applicable to Android smartphones for
relating the color changes to the corresponding CN− concentration and
thus a quantitative cyanide detector to assess water quality and safety at
off-field conditions was demonstrated.
Nitrite detection by gold nanorods etching. Nitrite can cause
irreversible oxidation of hemoglobin and interfere with the oxygen
transport system, and nitrites can convert secondary amines and amides
in the stomach to carcinogenic nitrosamines. Hence, nitrite detection in
food products is important. Exploiting the wavelength shifts in the
longitudinal localized surface plasmonic resonance of gold nanorods
upon etching (aspect ratio change) by use of certain chemical reagents
(called additives), a colorimetric nitrite assay with ultrahigh sensitivity
over the conventional Griess assay has been reported [51]. Specifically,
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S. Krishnan and Z.Q. Syed Sensors and Actuators Reports 4 (2022) 100078

NH4Br and HCl were used in this study with the proposed mechanism of
a change in chemical equilibrium that promotes the etching of gold
atoms at the gold nanorod ends. As low as 1 nM of nitrite caused a Δλ
change of 3 nm, and the method offered a dynamic range of 0.016–8.0
μM. Successful application of the method for determining nitrate content
in water and processed food samples, spinach, and rose petals was
demonstrated.
BrO3- ion detection using silver hexagonal nanoprisms. BrO3- is
a potential human carcinogen and often formed from the ozonation
process with Br-. A distance-based paper analytical device with silver
hexagonal nanoprisms allowed highly sensitive and selective determi­
nation of both Br- and BrO3- based on a pink color change to yellow and
by measuring the length of the yellow color band [52]. The color change
was attributed to the oxidative reaction of the nanoprisms in the pres­
ence of Br- and BrO3- forming silver bromide and silver nanospheres
(yellow color). Dynamic ranges of 25 μg L− 1 to 2 mg L− 1 for Br-, and
from 0.5 to 50 μg L− 1 for BrO3- were obtained. The naked-eye detection
limits reported were 10 and 0.5 μg L− 1 for Br- and BrO3-, respectively.
Real samples applicability using drinking water and pretreated samples
of rice and flour was demonstrated with a good sample recovery.
2.3. Other chemical and biochemical compounds in various real sample
matrices
Below we cover a diverse range of chemicals and biochemicals and
the sensing principles devised for their detection in practical sample
matrices. Table 2 presents a representative collection of literature re­
ports on non-virulent analyte detection by colorimetric assays.
H2O2 detection using hydrogel combined plasmonic gold
nanobipyramids. In this work, the sensor response mechanism was
based on H2O2 guided etching of gold nanobipyramids that facilitated a
plasmon-mediated color change of an agarose hydrogel-doped gold
nanobipyramids and were filled in a plexiglass tube [53]. The detection
was based on a height readout of color in proportion to the concentra­
tion of H2O2. The agarose hydrogel acted as a support material for
loading the gold nanobipyramids with stability and additionally pro­
moted the separation process of complex samples. Linear relationships
in four ranges of H2O2 concentrations were demonstrated, 20− 100 μM,
200− 1000 μM, 1− 10 mM, and 10− 100 mM, respectively. The method
eliminated the need for expensive and sophisticated detection in­
struments and circumvented the issue of nanoparticles aggregation.
Diluted (50-fold or higher) contact lens solution and hair dye as real
samples were tested to offer satisfactory results.
Bilirubin detection in urine, serum, and blood. The normal bili­
rubin levels in the serum of healthy people are in the range of 5.0− 20.0
µM [54]. Both elevated and deficient concentrations of bilirubin in
human serum are not good as they indicate various disorders [55]; [56].
Hence, it is necessary to monitor the bilirubin levels in the blood.
Ultrasound-assisted decomposition of 4-cyano-4′ -pentylbiphenyl liquid
crystalline molecules on a paper strip substrate by hydroxyl radicals,
that were generated from an oxidase enzymatic reaction of the analyte,
allowed highly sensitive colorimetric visualization of bilirubin in

Table 2
Representative listing of non-virulent analyte detection by colorimetric assays.
Analyte Approach Detection label use Sample matrix Limit of detection Linear range Ref.

Terpenes Rapid colorimetric assay Malachite green Buffer 3 µM 0 to 0.15 µM [146]

Proteins Luminescence Europium based metal chelate Buffer 4 ng 16 to 250 ng [147]
Campylobacter Visual colorimetric / Chromogenic agar Buffer. Milk: 1 × 102 CFU/mL 10[2] to 108 CFU/mL [148]
species microfluidic Guinea Green B 10 times diluted whole Raw chicken: 1 × 104 10− 3 to 1 µg/mL [149]
Cholinestérase Colorimetric dip stick assay Dopamine milk. CFU/mL 2 to 15 µM [150]
inhibitors Visual colorimetric/ UV Raw chicken meat 10− 3 µg/mL
Sulfide ion Visible Buffer Buffer: 0.03 µM
Buffer. Serum: 16.3 µM
Fetal bovine serum. Spiked serum: 5 µM
Spiked serum

Sweat pH & lactate Visual colorimetric Methyl orange & bromocresol Buffer. 10 mM 0 to 25 mM [151]
Cadmium (Cd2+) Visual detection green Human volunteers 1.12 µg/L 2 – 20 µg/L [152]
Aptamer functionalized gold Buffer
nanoparticles

Cyanide (C1 & C2) Colorimetric & fluorogenic Diaminomalonitrile HEPES buffer 0.45 µM C1: 4.12 to 35 µM [153]
C-reactive protein Colorimetric Citrate capped gold nanoparticles Buffer. 1.2 µg/mL C2: 3.25 to 35 µM [154]
Mercury ion Visual colorimetric Ligand immobilized conjugate 100 times diluted 0.26 µg/L 0.889 to 20.7 ug/mL [155]
Palladium ion nanomaterial nanomaterial urine 1.17 µg/L 0.002 to 0.1 mg/L [156]
Copper ion Visual colorimetric Modified mesoporous silica Water 0.37 µg/L 0.002 to 0.1 mg/L [157]
Mercury ion nanomaterial Ligand supported mesoporous Water 53 nM 0.002 to 0.1 mg/L [158]
Chromium ion Visual colorimetric silica Water Sewage water: 0.4 nM 0 to 10 µM [159]
Lead ion nanomaterial/ Optical Rhodamine B Spiked samples. Tap water: 0.4 nM 20 to 100 nM [160]
Mercury ion Visual colorimetric Gold-Silver bimetallic Real water samples 2.89 nM 0 to 200 nM [161]
Iron ion Colorimetric/ UV Visible nanoparticles Sewage water. 3.5 nM. 100 nM to 4 µM [162]
Glutathione spectroscopy Dual emission carbon dots Tap water Undetected in real Fluorometry: 0.09 to [163]
Mercury ion Colorimetric paper-based/ 3,3, 5,5 -tetramethylbenzidine Spiked water. samples 0.3 µM [164]
luminescence (TMB) Tap water. Fluorometry: 0.09 µM Colorimetry: none
Visual colorimetry Doped carbon dots Lake water Colorimetry: 0.3 µM 0.9 to 30 µM
Colorimetric/ Fluorescent 3,3, 5,5 -tetramethylbenzidine Buffer. 84 nM 0.1 to 700 nM
Colorimetry (TMB) Tap water. 0.05 nM
Colorimetry Silver/Copper nanocluster River water.
Fetal bovine serum.
Normal human serum
Buffer.
100 times diluted
urine and serum
Spiked buffer.
Human serum
Spiked water.
Real

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biological samples [57]. Here, the bilirubin substrate was used as a
model system to represent the oxidase enzymatic reactions. The analysis
involved measuring the length of color on the paper strips dipped into a
bilirubin solution. A linear detection range of 2.0 to 30.0 pM bilirubin
with the limit of detection of 0.80 pM, high reproducibility with a
relative standard deviation of < 3.50%, and sample recoveries of spiked
bilirubin in human urine and serum samples in the range of
99.09–107.89% were obtained. In one other report, integration of a
lateral-flow blood plasma separation strip with a color generating
detection endpoint was designed as an equipment-free approach to

Fig. 3. Paper based analytical devices for naked eye colorimetric virus detection: 1) Paper-based antibody detection using bioluminescent switching sensor proteins.
Reproduced with permission from [ref. 60]. Copyrights 2018 Published by Wiley-VCH Verlag GmbH & Co. Paper-based fluorescence analytical devices (PAD) are
cost-effective, easy to use and transport, thus making them as effective tools for point of care analysis (POC). However, they suffer from auto-fluorescence, lower
sensitivity, and liquid sample handling issues. Bioluminescence resonance energy transfer (BRET) assay is independent of an external light source. The shorter optical
length of the paper helped in suppressing the bioluminescence stimulus generation from blood. The absence of background interferences and direct scattering of light
by antibodies in plasma makes this method an easy to use a device with a higher sensitivity and specificity. 2) Schematic illustration of the principle of
surface-enhanced Raman spectroscopy (SERS) using a lateral flow stick, and detection of human immunodeficiency virus (HIV) DNA. Reproduced with permission
from [ref. 61]. Copyright 2015 Elsevier B.V. Lateral flow sticks (LF) are good analytical tools for rapid analysis with low interference, but they are challenged with
detecting low analyte concentrations. Gold nanoparticles when combined with LF facilitate signal amplification. However, they still lack specificity and sensitivity.
SERS was shown to overcome such shortcomings. By using a SERS tag or Raman reporter such as malachite green isothiocyanate (MGITC) functionalized gold
nanoparticles, a strong Raman effect can be created to attain high sensitivity with a small use of a sample. This is a one-of-a-kind sensor used for human immu­
nodeficiency virus (HIV) DNA detection with a limit of detection of 0.24 pg/mL. 3) Colorimetric detection of the influenza-A virus using PVDF-PDA membrane.
Reproduced with permission from, [62]. Copyright 2019 Elsevier B.V. Polydiaceytlene (PDA) are polyacetylene type conducting polymers. Conformational changes
in the structure of PDA due to external binding, pH, and thermal changes make them change their color from blue to red. PDA immobilized on polyvinylidene fluoride
(PVDF) membrane was used to detect the influenza virus. When the concentration of the virus increased the color of the PDA layer deepened making it a potential
POC device. However, tremendous control over the PDA layer exposure must be ensured to eliminate artifacts stimuli from the environment and thus avoid creating
false-positive color changes. 4) Precise and programmable detection of mutations using ultra-specific riboregulators on a paper substrate. Reproduced with
permission from, [63]. Copyright 2020 Elsevier. Distinctive changes in the genetic material are vital keys for evolution. These changes are taking place at the RNA
transcription level which alters the cellular roles ultimately supporting the viruses to mutate rapidly. Such mutations are detrimental to the current regime of
treatments for several diseases. Hence, identifying and analyzing such mutations are of paramount interest for combating viral diseases. These changes are usually
monitored by molecular probes, but they are expensive to afford in developing countries. Single nucleotide-specific programmable riboregulators (SNIPRs) have been
claimed to be a powerful tool sensitive enough to detect such programmable mutations. SNIPR integrated with paper-based devices enabled detection of cancer
mutations directly from whole blood in a cost-effective manner with high sensitivity and specificity.

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S. Krishnan and Z.Q. Syed
detect two hepatobiliary disease-associated markers, total bilirubin, and
direct bilirubin present in whole blood [58]. Application to clinical
samples was demonstrated. Advantages of the method were claimed to
be the ease of portability (< 40 g), low cost (< $5), rapidity (< 5 min),
instrument-free, high sensitivity (< 1 μM), and accuracy (89.5 % for
total bilirubin, 94.7 % for direct bilirubin).
Caffeine detection in food and biological matrices. Caffeine-
induced removal of a nano zinc compound from the surface of 5, 10,
15, 20-tetra(4-pyridyl)-21H-23H-porphine-CdTe quantum dots led to a
recovery of fluorescence property of the quantum dots [59]. Electro­
static attraction and nitrogen/zinc coordination were proposed to be the
underlying mechanistic factors. These molecular properties enabled a
visual paper-based sensor with a dynamic range of 50 pM to 3 nM, and a
detection limit of 15.3 pM caffeine. Applicability for real samples such as
tea water, cell culture fluid, newborn bovine serum, and human plasma
was reported. Fig. 3 illustrates some representative paper-based color­
imetric devices for various protein and nucleotide biomarkers targets.
Colorimetric organophosphorus pesticide detection. Organo­
phosphorus pesticides are the most used pesticides in the United States
(~ 80 million pounds used annually) mostly for agricultural and to some
extent for residential purposes. These pesticides are neurotoxicants and
their contamination of food, water, and surfaces during human con­
sumption can cause severe health problems. Hence, the detection of
organophosphates has gained considerable significance recently.
Coupling aggregation-induced emission nanoparticles (PTDNPs-0.10)
and two-dimension MnO2 nanoflakes (2D-MnNFs) offered an
instrument-free naked-eye visual detection of organophosphorus pesti­
cide on a smartphone [64]. In this fluorescent paper analytical device, a
PTDNP-MnNFs composite was made based on electrostatic interactions
between 2D-MnNFs and PTDNPs-0.10, and the fluorescence emission of
PTDNPs-0.10 was quenched through a fluorescence resonance energy
transfer process. Exploiting the enzymatic activity suppression of
acetylcholinesterase in the presence of organophosphorus pesticide, and
variations in the fluorescence intensity, a detection limit of 0.027 ng/mL
were reported. In the same sensor, by measuring the variations in
brightness intensity, and by using a smartphone, a detection limit of
0.73 ng/mL was attained.
Quantum dot to quantum dot fluorescence resonance energy
transfer for pH sensing. pH measurements are important in several
biochemical tests, industry processes, biomedical and life sciences, and
environmental monitoring. While traditional pH meters are routinely
used, enabling a precise determination of pH values as an instrument-
free approach is advantageous for resource-limited settings such as in
the form of a high-resolution pH paper. Despite yielding a greater
sensitivity than absorbance-based sensors, fluorescence detection in
several approaches required an excitation source. To overcome this
bottleneck and make the fluorescence sensing instrument-free for visual
naked eye analysis, quantum dots-based sensors have been developed.
One recent CdTe quantum dots and nanozinc paper-based sensor offered
visual detection of three carbamate pesticides (metolcarb, carbofuran,
and carbaryl). Demonstrations of application for real food matrices such
as apple, cabbage, and tea water were shown [65]. In another pH sensor
report, the fluorescence resonance energy transfer process[66] between
two quantum dots was created by designing a brighter donor than the
acceptor quantum dot through concerted compositional and morpho­
logical choices. This approach is particularly useful when the quantum
dot acceptor emission intensity is inherently lower than that of the
donor.
Phenanthroline-based visual and precise detection of pH
values. Improving the resolution of pH paper-based detection would
allow highly accurate measurements for on-site applications. One report
utilized an intramolecular charge transfer process to construct a
cellulose-based superior pH sensor. This sensor combined a phenan­
throline moiety chromophore with a urea group as a bridge to offer a
highly sensitive, pH-responsive, and extended conjugation structure
[67]. Precise and visual determination of pH values under extreme
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Sensors and Actuators Reports 4 (2022) 100078
acidic and strong basic environments were demonstrated. Under visible
light mode, the sensor material could readily discriminate pH values of
14.0, 13.0, 12.0, and 11.0 and distinguish pH 2.0 from 1.0 by the naked
eye. While on a fluorescent mode, the sensor could offer more accurate
pH values in the range of 11.6–13.2 at an increment level of 0.2–0.4
magnitudes accompanied by a highly contrasting color change covering
extreme acidity and strong basicity ranges of pH values with high
precision.
Transcription factor-based fluorescent bead assay for visual
antibiotic sensing. Utilization of allosteric transcription factors (TFs)
as biorecognition elements for in vitro sensing and exploiting the dif­
ferential binding affinity between a TF and its cognate DNA in connec­
tion with the proportionate response to an analyte target (termed as TF-
DNA-analyte systems), a modular bead-based biosensor was designed
[68]. In this approach, the DNA-functionalized beads displayed the
advantages of efficient mixing and spatial separation, and the TF-labeled
semiconductor quantum dots functioned as the source of bright fluo­
rescent indicators. Fluorescent levels of the TF-DNA bound form onto
the bead and that of TF unbound states were differentiated. Application
of the method for antibiotic tetracycline exhibited a nanomolar sensi­
tivity with visual instrument-free detection of the bead fluorescence
within 5 minutes of analyte addition.
Oxidative etching of silver nanoparticles for glucose detection
on a paper-based microfluidic device. Connecting the H2O2 produc­
tion from glucose oxidation, catalyzed by the enzyme glucose oxidase,
with oxidative etching of silver nanoparticles resulted in the discolor­
ation of purple color as the sensor signal output in proportion to the
target glucose concentration [69]. The described sensing approach was
distance-based glucose detection on paper-based microfluidics and
involved a biocompatible nanocellulose film immobilized with the
enzyme, and the color disappearance was attributed to the morpholog­
ical transformation of etched silver nanoparticles. The assay duration
was 40 min with a naked-eye detection limit of 0.1 mM glucose.
Demonstration of the sensor for glucose spiked artificial urine samples
and control urine samples displayed sample recoveries in the practically
desirable range of 96–100%. To overcome the invasive, discomfort, and
anxiety causing blood glucose measurements by the fingerpick
approach, non-invasive colorimetric hyperglycemia sensing in saliva
based on plasmonic multibranched gold nanostructures that change
their shape and color for naked-eye detection has been reported with a
limit of detection 0.4 mg/dL [70].
Paper-based immunosensor for naked eye milk quality test.
Alkaline phosphatase (ALP) is a metalloprotein biomarker that can
indicate liver disease and bone disorders [71]. ALP is also naturally
present in raw milk (at ~191.0 U/mL ALP), and the absence of ALP
serves as a crucial indicator for confirming the appropriate pasteuriza­
tion process of milk that removes any pathogenic bacterial contamina­
tions. While traditionally used colorimetric approaches suffered from
sensitivity, new instrument-free approaches with improved sensitivity
are constantly evolving. One such report was based on a paper-based
ALP-antibody biosensor that yielded a blue-green precipitate as an
analytical signal resulting from the catalytic activity of ALP with 5-bro­
mo-4-chloro 3-indolyl phosphate [72]. The authors devised a digital
image colorimetry analysis of the blue-green complex captured using a
smartphone camera. A wide dynamic range from 10 to 1000 U/mL with
a detection limit of 0.87 (± 0.07) U/mL ALP was obtained. Applicability
to detect ALP in commercial and raw milk samples was demonstrated.
Negligible interferences were found from the common co-existing milk
components and thus confirming the excellent selectivity of the method
arising from the innate high affinity antibody-ALP binding interaction
and the additional enzymatic substrate selective reaction involved.
Wearable paper microfluidic sensor for salivary glucose moni­
toring. Paper-based wearable sensors are very attractive materials due
to their lightweight, low-cost, simplicity, and if engineered to offer high
sensitivity and low detection limits, they can be instrument-free diag­
nostic approaches [73–76]. One such sensor type was a microfluidic
S. Krishnan and Z.Q. Syed
paper-based device with a wearable feature when integrated into a sil­
icone mouthguard using a 3D-printed holder [77]. The limits of detec­
tion achieved for glucose and nitrite ions were 27 μM and 7 μM,
respectively. The dynamic ranges were 0 to 2.0 mM and 0 to 400 μM for
glucose and nitrite, respectively. This sensor was successfully demon­
strated for monitoring salivary glucose concentration after consumption
of chocolate. In addition, application of the method was shown for a real
patient saliva analysis to measure the diabetic’s relevant higher glucose
concentration and in patients diagnosed with periodontitis displaying
elevated nitrite concentrations than normal.
3D paper-based device for silver ion detection with a personal
glucose meter. Design of a 3D origami microfluidic paper-based
analytical device with a nanoporous membrane featured an analyte-
triggered self-growing of silver nanoparticles to block the membrane’s
pores in situ for rapid and efficient signal amplification by a handheld
personal glucose meter. This device utilized the biocatalytic reaction
between glucose oxidase and glucose to accomplish sensitive and
portable biosensing of silver ions [78]. Specific detection of the analyte
with a limit of detection of as low as ~58.1 pM (3σ) Ag+ ion allowed the
approach as one of the most sensitive Ag+ assays. Satisfactory sample
recovery was attained upon analyzing various real water examples such
as tap water, drinking water, pond water, and soil water, and thus
support feasibility for practical usability.
Multimodule split aptamer constructs for visual detection of
cocaine and cathinones. Although colorimetric aptamer-based sensors
offer the advantages of on-site and point-of-care options, the bottleneck
of achieving naked-eye detection is encountered by poor signal ampli­
fication during the recognition of target analytes. To address this
problem, researchers reported a generalizable strategy that involved the
engineering of novel multimodule split DNA constructs called CBSA­
zymes [79]. These constructs utilize a cooperative binding split aptamer
as a highly target-responsive bioreceptor and a split DNAzyme as an
efficient detection signal reporter. In the presence of the target analyte,
the CBSAzymes come together to form a complex and catalyze the
oxidation of a given color-forming substrate (e.g., 2,2′ -azino-bis(3-eth­
ylbenzthiazoline)-6-sulfonic acid yields a dark green color within 5
min). While in the absence of a target analyte, the two fragments of the
CBSAzymes remain separated. Such CBSAzymes strategy facilitates
sensitive and visual detection of small molecules that often give small
detection signal changes compared to their larger counterparts. The
authors designed a cocaine-binding CBSAzyme for naked-eye detection
of cocaine at concentrations as low as 10 μM. To demonstrate the generic
applicability, the authors developed additional designs of methyl­
enedioxypyrovalerone (MDPV)-binding CBSAzyme for visual detection
of MDPV, and 10 other synthetic cathinones at low micromolar con­
centration detection and thus applicability for biological samples.
Thus, the design of cooperative binding split aptamers (CBSA) con­
taining two target-binding domains was found to offer an enhanced
target response when compared to single-domain split aptamers. In this
study, between the two target-binding domains, a duplex C3 spacer
abasic site was introduced, and the EATR signal amplification was
attained through an exonuclease III’s apurinic endonuclease activity.
Detection of dehydroisoandrosterone-3-sulfate binding to the selectively
designed CBSA at a 100-fold greater target sensitivity over a non-EATR-
based assay was demonstrated, with a detection limit of 1 μM in 50%
urine. Moreover, EATR-mediated aggregation of CBSA-modified gold
nanoparticles was utilized for visual detection of micromolar concen­
trations of cocaine as an instrument-free colorimetric assay. The versa­
tility of the approach for the detection of broader small-molecule targets
of relevance to various fields was suggested to be possible.
Nanoceria based paper analytical device for colorimetric anti­
oxidant activity. A portable thermometer-shaped paper analytical de­
vice has been designed for rapid, instrument-free determination of
antioxidant activity [80]. In the presence of antioxidants in food, the
detection channel deposited with nanoceria undergoes a partial reduc­
tion of cerium ions from Ce4+ to Ce3+ forming highly reactive oxidation
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Sensors and Actuators Reports 4 (2022) 100078
products. Ceria-antioxidant complexes are formed through a charge
transfer process from the binding of the oxidation products and/or the
parent antioxidant compounds to the hydroxyl-rich ceria nanoparticles,
causing a yellow to brown color change. Measuring the distance of the
brown color on the detection channel, using an integrated ruler, corre­
sponded to the extent of antioxidant activity, and hence the approach
did not require any instrument for detection. However, the selectivity of
the assay that simply relies on a non-specific electrostatic interaction of
the analyte with the ceria nanoparticle would be prone to interferences
from analogous molecules to the analyte. Antioxidant assays performed
for 11 tea samples using the nanoceria paper device correlated with the
traditional assays at 95% confidence level with high recovery and sensor
stability over 50 days when stored at ambient and low-temperature
conditions (6 and –20 ◦ C), thus paving the way for a cheap and
portable screening of antioxidant activity in real samples.
Visual detection of azodicarbonamide procarcinogen in flour:
(a) Colorimetric acidic and basic conditions. Flour products consti­
tute a large portion of our daily diet. To improve the gluten and
whiteness of flour, additives such as azodicarbonamide (ADA) are
added. ADA, historically used as a foaming agent of rubber articles, has
now been commonly utilized in the flour industry as a bleaching agent,
gluten fortifier, or dough conditioner. ADA is a procarcinogen, which
means by itself holds low toxicity effects but when converted to certain
metabolites such as biurea (BIU) and semicarbazide (SEM), under high-
temperature conditions during baking, they may become carcinogenic.
Hence, there are regulations set on the maximum permissible level of
ADA in flour products (45 mg/kg (ppm)) and some countries have even
banned the use of ADA in flour. Hence, the determination of ADA levels
in flour has practical significance. For the visual detection and quanti­
tation of carcinogenic semicarbazide hydrochloride in commercial flour
products, colorimetric reactions with potassium ferrocyanide (blue-
colored product, pH 6.0) and phosphomolybdic acid (brownish-green
colored product, pH 9.0) were designed [81]. As low as 10 ppm and 2.0
ppm concentrations within 2− 4 min assay duration were detected by the
two described reactions, respectively, and the results were compared to
measurements by spectrophotometry. Extracts from commercial bread
products were successfully tested with a relative standard deviation of <
3 % and a sample recovery of > 94%. (b) Anti-aggregation of gold
nanoparticles. So far, we looked at several sensor principles based on
analyte presence-induced aggregation of nanoparticles. Alternatively,
anti-aggregation of nanoparticles was also utilized as the sensor working
principle, which is discussed here. To overcome the sophisti­
cated/expensive instrument needs and time-consuming pretreatment
steps, a rapid colorimetric method has been developed for visual
detection of ADA in flour by utilizing glutathione (GSH)-induced gold
nanoparticles aggregation via an Au-SH covalent network structure
formation helping a color change process [82]. In the presence of ADA,
the favorable coupling of each pair of GSH molecules cause a decrease in
the number of freely available initial − SH groups of GSH, and this, in
turn, diminishes the aggregation of gold nanoparticles that was origi­
nally induced by the uncoupled free GSH molecules through Au-thiol
bonding. This field-portable colorimetric sensor allowed detection of
as low as 0.33 μM ADA (38.3 ppb) by the naked eye and 0.23 μM (26.7
ppb) ADA by spectrophotometry within a 2 h duration. For ADA
detection in a flour sample, a satisfactory sample recovery in the range
of 91− 104% with good reproducibility was observed (relative standard
deviation was < 6%). The measured visual detection limit of the ADA
sensor is lower than the regulatory permitted ADA levels in flour (i.e.,
45 mg/kg) making the approach promising for on-site detection of ADA
in flour products.
Naked eye detection of dopamine by a ratiometric fluorescent
probe. Dopamine is a neurotransmitter. Under alkaline conditions,
dopamine quenches the fluorescence of thioglycolic acid-functionalized
orange, fluorescent cadmium telluride quantum dots but not the amino-
functionalized blue, fluorescent carbon nanodots. Based on this prop­
erty, a ratiometric fluorescence sensor for selective detection of
S. Krishnan and Z.Q. Syed
dopamine using a mixture of the described two fluorescent materials has
been devised [83]. In this sensor, variations in the dual emission in­
tensity ratios between the two fluorescent probes with dopamine con­
centration displayed a continuous color change from pink to purple to
blue that was noticeable to the naked eye. Dopamine in water and urine
samples were successfully measured with a detection limit of 1.3 μM,
and to justify the practical utility, the authors designed a ratiometric
fluorescent probe-agarose hydrogel that offered a visual detection.
pH indicator and acid additive enabled base gaseous analytes
colorimetric sensing. For visual detection of gaseous analytes with
basicity such as ammonia, a vertically aligned array configuration was
fabricated by drop-casting a dye solution consisting of a pH indicator, an
acid compound, and a hydrophilic polyethylene glycol on a poly­
acrylonitrile nanofiber mat [84]. The type of pH Indicator used, and the
amount of citric acid added facilitated control over the detection
sensitivity output. Upon exposure to ammonia gas, prominent coloration
was observed in the range of 0–1 ppm for bromophenol blue, 0–5 ppm
for bromocresol green, and 10–100 ppm for chlorophenol red indicators.
Increasing the concentration of citric acid additive neutralized a greater
number of the base molecules and this diminished the color change
between the pH indicator and basic analytes. The number of coloration
elements observed in the strip device was related to the analyte con­
centration and the total number of sensing elements to the resolution of
the method.
Colorimetric contact lens moisture and pressure sensor. To
mitigate the requirement of complex electronics and circuits in contact
lens sensors, an instrument-free detection of pathologically relevant
signals of eye diseases has been reported [85]. The structurally colored
contact lens sensor with a tunable color can directly identify changes in
moisture and pressure of relevance to xerophthalmia and glaucoma
diagnosis, respectively. The structurally colored contact lens sensor was
made from a biocompatible hydrogel without the addition of any
chemical pigments and thus offering superior biosafety and wearing
comfort for wearable applications. The application area of this sensor is
point-of-care ophthalmic health monitoring.
Color to a thermal signal readout concept. To mitigate the low
resolution and boundary issues of yes or no decision-based colorimetric
sensors, conversion of the color signal into a thermal signal readout was
attempted [86]. This sensor utilized the well-known blue color readout
of the oxidized 3,3′ ,5,5′ -tetramethylbenzidine (TMBox) substrate with
silver ions (Ag+) as the starting step. Upon reduction of the blue colored
TMBox into colorless TMB in the presence of the target alkaline phos­
phatase, which catalyzed the hydrolysis of ascorbic acid phosphate into
ascorbic acid, the discoloration of blue color could be linked to give a
highly sensitive near-infrared laser-driven photothermal response,
measured by a common thermometer useful in low-resource settings.
2.4. Colorimetric polymerase chain reaction
RT-PCR has been the gold standard (due to high sensitivity and
specificity) for infectious disease diagnosis including COVID-19. How­
ever, RT-PCR is very resource-demanding, laboratory-based, and re­
quires highly trained personnel, special facilities, and high-cost
instrumentation. Hence, a vast majority of the globe does not have ac­
cess to RT-PCR as evident from the ongoing delta/omicron variants in
the present pandemic outbreak and time delays in getting test results in
resource-limited countries that accelerated the virus spread. As a result,
there has been increasing attention to developing semi-instrumentation
and visual colorimetric-based PCR detection approaches to derive cost-
effective user-friendly PCR methods for viruses and other target mole­
cules. One such method is called the reverse transcription loop-mediated
isothermal amplification (RT-LAMP) [87], a nucleic acid amplification
approach for detecting infectious diseases within an hour holding great
potential for point-of-care applications [88–90]. Several excellent recent
reviews on advancing the LAMP technique appeared in the literature
[91–93], so in this article, we aim to outline this technique briefly and
12
Sensors and Actuators Reports 4 (2022) 100078
bring this accounted for with other colorimetric sensors reviewed here.
In the LAMP method, two inner and two outer primers are used to
recognize multiple distinct regions of the target nucleotide molecules
adding high specificity. In addition, two extra loop primers are used to
enhance amplification and detection (Fig. 4). RT-LAMP assay results can
be made visual colorimetric through DNA probe designs with gold,
magnetic, and other metallic nanoparticles, the use of fluorescent dyes
and pH indicators, and gel electrophoresis with UV detection [94–98]
(Fig. 5). Despite several promising innovative advancements and dem­
onstrations made on the LAMP method, challenges such as sensitivity
issues related to false-negative detection, lack of usability with un­
treated samples, contamination issues at the point-of-care sites causing
false positives, and the associated assay reliability are existing bottle­
necks [99]. A closed tube detection system has been devised to avoid
contaminations after amplification steps among other avenues for
overcoming limitations toward point-of-care LAMP applications
[100–102].
2.5. Visual detection of influenza viruses by LAMP-nanoparticles sensor
Multiplex challenges in differentiating LAMP products derived from
multiple target nucleic acids and the requirement of specialized in­
struments have limited the on-site application of multiplex LAMP. One
integrated assay approach called the multiplex reverse transcription
LAMP featuring a cascade invasive reaction using nanoparticles
(abbreviated as mRT-LAMP-CIRN) mitigated the prior limitations of
multiplexing and dependence on instruments [103]. In this report, three
subtypes of influenza viruses such as A/H1N1pdm09, A/H3, and influ­
enza B, were detected with 98.3% sensitivity (related to true positive
rate) and 100% specificity. Out of 35 clinical specimens that showed
positive identification of the influenza A/H1N1pdm09 on the gold
standard real-time RT-PCR, 34 of them agreed with the
mRT-LAMP-CIRN results. The disagreement in the result of one of the
specimens was attributed to the stringent recognition need of eight re­
gions of the target sequence by the six primers used in the mRT-LAMP
reaction, in contrast to the involvement of only three regions of the
target sequence in the real-time RT-PCR technique. In this regard, mu­
tations can often evade the target sequence from positive identification.
The designed mRT-LAMP-CIRN was cost-effective and instrument-free
for on-site use offering analytical sensitivities of 101 copies of RNA for
both A/H1N1pdm09 and A/H3, and 102 copies of RNA for influenza B.
Thus, sensitive, and convenient user-friendly colorimetric methodolo­
gies for the detection of viruses are significant to containing the spread
of the virus and avoiding any future pandemic devastations around the
globe. Fig. 6 illustrates the colorimetric detection of organophosphorus
and antioxidants using different forms of gold nanomaterial.
Fig. 7 represents the catalytic hairpin assembly used for the ratio­
metric detection of HIV gene using phenol red as the indicator. Table 3
presents a representative collection of literature reports on colorimetric
assay kits that are either in the clinical or production stage.
3. Summary and prospects
This article aims to offer some insights into recent instrument-free
colorimetric visual sensors developed for a vast variety of target mole­
cules to solve problems in various diverse fields. The authors made every
attempt to address the principle and purpose of the sensor designs dis­
cussed, conceptual ideas, the significance of target analytes, mechanistic
aspects, analytical features, and demonstration of applicability for real
samples. Based on the discussion and representative illustrations of
several published articles, we can understand and appreciate the
tremendous advancements made in designing visual colorimetric sen­
sors applicable for the detection and quantification of a large diverse
group of analytes.
Overcoming the traditional sensitivity, selectivity and ultra-low
detection issues, minimization and elimination of special
S. Krishnan and Z.Q. Syed Sensors and Actuators Reports 4 (2022) 100078

Fig. 4. Loop mediated isothermal amplification procedure for viral genetic material detection: 1) HRPzyme assisted PCR amplification for the detection of SARS-
CoV-2 infection. Reproduced with permission from 37. Copyrights reserved by Elsevier. 2) Colorimetric detection of SARS-CoV-2 with improved loop-mediated
isothermal amplification. Reproduced with permission from 38. Copyrights reserved with Nature Communications. 3) Highly sensitive colorimetric detection of
SARS-CoV-2 RNA using RT-LAMP. Reproduced with permission from 39 Copyrights reserved by Elsevier.

Fig. 5. A universal polymerase chain reaction. Reproduced with

permission from [ref 36]. Published by Wiley. The PCR approach
published in this report was a one-pot single-step method for rapid
detection without losing sensitivity. The principle of this asym­
metric PCR is the use of a universal tag and a forward primer; the
primer is depleted during the amplification process thus leading to
the assembly of a universal tag close to the end of amplification.
The colorimetric signal is generated by aggregation of gold nano­
particles which contains complementary oligonucleotides; when
complimentary pairs come together the nanoparticles aggregate,
and a color change is induced. The method reserves its distinctive
nature by preventing nonspecific binding from the universal tag,
single-stranded asymmetric amplification, and size of the
amplicons.

13
S. Krishnan and Z.Q. Syed Sensors and Actuators Reports 4 (2022) 100078

Fig. 6. Enzyme-linked immunosorbent assay (ELISA) and etching of gold nanorods. 1) Reproduced with permission from ref [104], copyright reserved by Elsevier.
Traditional methods of detection of organophosphorus is through either chromatography or ELISA, both methods are sensitive and accurate. However, they are
expensive and involve time-consuming procedures, an alternative method to overcome these fallbacks is the use of noble metals with colorimetric analysis. One of the
shortcomings of certain colorimetric assays is the single-color-based signal intensity changes for a visual distinction of concentration levels. In this study, using gold
nanorods, a multicolor sensor approach was developed, which was an etching based or growth-based mechanism of gold nanorods that featured a responsive swing of
the localized surface plasmon resonance (LSPR) of the nanorods to display a multicolor response upon the conversion of 3,3′ ,5,5′ -tetramethylbenzidine (TMB) to
TMB2+ catalyzed by horseradish peroxidase (HRP) in proportion to the analyte concentration. 2) and 3) Reproduced with permission from ref. [105]. Copyrights
reserved with American Chemical Society. Antioxidants are free radical scavengers in the human body, and they are responsible for protecting the body from various
diseases, disorders, and invaders. Conventional methods of detection include chromatography and electrophoresis. These techniques lack multi-analyte detection.
Therefore, the referenced study was aimed at addressing the multi-analyte detection issue with gold nanorods and gold nano-bipyramids, and upon altering the
aspect ratio of the gold nanomaterial, a band of color responses was observed as shown. The intensity of the color change relied on the extent of etching of the surface
of gold, which enabled a naked eye detection in proportion to the concentration of antioxidants. A statistical model known as the linear discriminant analysis was
used to investigate responsive changes in the matrix and enable distinguishing various antioxidants.

instrumentation needs have largely been addressed. Progress to apply
the sensors for complex real samples with satisfactory to excellent
sample recoveries have been demonstrated. Incorporation of fluores­
cence emission-based visual detection approaches, displacement assays,
and FRET-based detection outputs to overcome the colorimetric sensi­
tivity limitations are discussed. Still, fluorescent methods in most cases
required an excitation source to initiate the process, while the resulting
visible color emissions allowed naked-eye detection. When it comes to
complex samples, pretreatment and/or extensive sample dilutions are
found to be necessary for most sensors to minimize non-specific sensor
surface fouling and/or signal interferences. Overcoming and mitigating
the pretreatment needs would offer more practical advantages and fill
the gap from being laboratory-based and technically challenging assays
to becoming point-of-need tests.
Multiplex approaches have only seen a tip of an iceberg, and there
exists a vast knowledge gap that needs to be explored to improve the
probability of affirmative identification from a colorimetric sensor test
of a pool of key molecular targets, toxins, pathogens, and biomarkers

14
S. Krishnan and Z.Q. Syed Sensors and Actuators Reports 4 (2022) 100078

Fig. 7. Colorimetric detection of human immunodeficiency virus

(HIV) gene using the ratiometric method. Reproduced with
permission from [ref 106]. Copyrights reserved by Elsevier B.V.
2020. In this study, a cost-effective, instrument-free approach was
designed to respond to pH changes without requiring any chro­
mogens. It is well established that silver can coordinate with
cytosine-cytosine (C-C) on the catalytic hairpin. Urease is reactive
to silver, which inhibits the enzyme’s activity. Urease contains
nickel active binding sites, which can catalyze urea to carbon di­
oxide and ammonia. When a complementary target nucleic acid
approaches hairpin 1, the hairpin sequence rearranges to form
hairpin 2 releasing silver ions, which inhibit the urease activity
and a color signal is generated. The color change depended on the
concentration of silver, a lower concentration was unable to inhibit
urease and that displayed a pink color readout.

Table 3
Representation of various colorimetric assay kits either in the clinical or production stage.
Analyte Approach Method Limit of detection in Linear range Ref.
human/ clinical

Uric acid Enzymatic Absorbance based using a 12.11 µmol/L 12.11 -1550.05 [165]
Arsenic Silver diethyldithiocarbamate microplate reader 4 µg/L µmol/L [166]
(1→3)-β-D-glucan marker method Colorimetric 120 pg/mL 0- 468 µg/L [167]
fungal disease 96 well plate. Photometer 127 µmol/L NA [168]
Phenylalanine Enzymatic Visual 99.2 % accuracy 152 – 1210 µmol/ [169]
Mycobacterium species Reverse hybridization Lateral flow assay L
NA

over a one molecular target assay. Wearable and continuous visual
sensors for real-time analyte monitoring is one other emerging fasci­
nating direction. With the pandemic being around us for quite a while
now, the forward-looking sensor research direction must prioritize the
most essential lifesaving needs accessible for the entire global popula­
tion regarding instrument-free sensors, water/air quality monitoring,
food safety, biosafety, chemical, and biological threats, and environ­
mental analysis.
Declaration of Competing Interest
There are no conflicts to declare.
Acknowledgements
We are pleased to present this article for the special issue honoring
SK’s research mentor, Prof. Jim Rusling, University of Connecticut,
Storrs, CT, USA. Funding support from the Oklahoma Center for the
Advancement of Science and Technology program (HR21–013), the
College of Arts and Sciences Research Program, and the Vice President
for Research, Oklahoma State University, are greatly acknowledged.
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