Trends in Analytical Chemistry 172 (2024) 117573

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Trends in Analytical Chemistry

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

Nanozyme-enhanced paper-based biosensor technologies

Anupriya Baranwal a, Ravi Shukla a, b, **, Vipul Bansal a, *
a
Sir Ian Potter NanoBioSensing Facility, NanoBiotechnology Research Laboratory, School of Science, RMIT University, Melbourne, VIC, 3000, Australia
b
Centre for Advanced Materials & Industrial Chemistry, RMIT University, Melbourne, VIC, 3000, Australia

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

Keywords: Nanozymes, enzyme-mimetic nanomaterials that combine features of nanoscale material with the catalytic
Nanozyme properties of enzymes, have undergone rapid development in recent years. While enzymes have remained in­
Enzyme-mimic tegral to biosensor technologies for the past several decades, the emerging potential of nanozymes to overcome
On-site detection
the low stability and high production costs of biological enzymes has turned them into promising biosensor
Food safety
Environmental analysis
candidates. Nanozymes have already been explored for a myriad of biosensing applications. However, only a
Health monitoring small subset of these efforts has resulted in devices and platforms that are potentially suitable for on-site/point-
Disease diagnosis of-care monitoring. To promote efforts in this area, the review first provides a concise overview of different
Point-of-care devices paper-based technologies, their architecture and functionality, in a language conducive to the nanozyme
POCD research community. The review, then, critically discusses the progress made in the integration of nanozymes in
biosensor platforms for on-site detection of food contaminants, environmental pollutants, and disease bio­
markers. The challenges associated with the transition of solution-based nanozyme biosensors to relevant devices
and technologies are highlighted, and strategies to overcome those challenges are proposed. We envision that
this review could narrow the gap impeding the translation of nanozyme-based biosensor technologies from
laboratories to real-world scenarios by encouraging consolidated cross-disciplinary and trans-sectoral efforts.

context of biosensor technologies, the most exploited group of nano­

zymes belongs to oxidoreductases that include peroxidase (POX), oxi­
1. Introduction dase (OX), catalase (CAT), superoxide dismutase (SOD) and laccase
(LAC) enzymes. Among these, the POX- and OX-mimics are more
Natural enzymes have traditionally been at the forefront of biosensor commonly employed for the development of nanozyme biosensors,
technologies due to their high target specificity. However, their inherent perhaps due to their high similarity with the horseradish peroxidase
shortcomings, such as low stability under ambient conditions, special (HRP), which has remained the workhorse of biochemical assays over
storage requirements, and exhaustive yet expensive production and the past several decades. In addition to their enzyme-mimicking char­
purification steps, have led to the development of various enzyme- acteristics, many of these nanozymes exhibit additional electrical,
mimic platforms [1]. Among these, nanozymes have garnered incred­ magnetic, and optical properties, which can facilitate the integration
ible attention over the past decade due to their high stability, ease of and automation of relevant procedures like magnetic target separation
modification, low production cost, and better shelf-life than natural and detection with higher accuracy and fewer preparatory steps [2,12,
enzymes [2]. Nanozymes can be commonly classified as nanomaterials 13].
that show enzyme-mimicking catalytic activity [3,4]. So far, hundreds of As such, over the last decade, the field of nanozymes has undergone
nanomaterials, including but not limited to metal and metal oxides, remarkable growth, leading to the development of ultra-sensitive, high-
carbon-based nanomaterials, and metal-organic frameworks (MOFs), performance biosensing platforms for applications across multiple
have been studied for enzyme-mimicking activities [5–10]. As critically fields, including medicine [12,14], environmental monitoring [11,15],
reviewed in one of our recent studies, out of the six key classes of natural food safety [16–18], and agriculture [7,19]. We will refer to them as
enzymes, the nanozyme activity of inorganic materials is predominantly nanozyme biosensors (NzBS) in this review. Considering the rising de­
restricted to two enzyme classes: oxidoreductases (EC1) and hydrolases mand for biosensors in these fields, the development of
(EC3) [11]. The current review of the field further reveals that in the

* Corresponding author.
** Corresponding author.
E-mail addresses: ravi.shukla@rmit.edu.au (R. Shukla), vipul.bansal@rmit.edu.au (V. Bansal).

https://doi.org/10.1016/j.trac.2024.117573
Received 2 October 2023; Received in revised form 8 January 2024; Accepted 1 February 2024
Available online 9 February 2024
0165-9936/© 2024 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
A. Baranwal et al. Trends in Analytical Chemistry 172 (2024) 117573

Abbreviations: Ig Immunoglobulin
L. monocytogenes Listeria monocytogenes
Ab1 Primary antibody LAC Laccase
Ab2 Secondary antibody LAMP Loop-mediated isothermal amplification
Ac Acetochlor LC Liquid chromatography
AcA Acetic acid LC-MS Liquid chromatography-mass spectrometry
Ach Acetylcholine LFA Lateral flow assay
AChE Acetylcholinesterase LFD Lateral flow device
A. flavus Aspergillus flavus LFTS Lateral flow test strip
Ag+ Silver ion LOD Limit of detection
ACC-hNF Acetylcholinesterase/Choline oxidase/Copper phosphate mAb Monoclonal antibody
hybrid nanoflower mGCN modified graphitic carbon nitride
ATCh Acetylthiocholine iodide Mn–Cu NFs Manganese dioxide–copper phosphate hybrid
Au NP Gold nanoparticle nanoflowers
ATPS Aqueous two-phase solution MnO2 NF Manganese dioxide nanoflower
AuNC Gold nanocluster mPB NPs magnetic Prussian blue nanoparticles
BSA Bovine serum albumin MPL Maximum permissible limit
CA Catechol MRE Molecular recognition element
CA125 Cancer antigen 125 NC Nitrocellulose
CAPEX Capital expenditure NE Norepinephrine
CAT Catalase Ni(OH)2–Pt Platinum nanoparticle-modified 2D Ni(OH)2 nanosheet
CFU Colony forming unit NzBS Nanozyme biosensors
Ch Choline OPEX Operating expenditure
Chl Chlorpyrifos OPP Organophosphorus pesticide
ChO Choline oxidase OX Oxidase
C. jejuni Campylobacter jejuni p53 Tumour protein 53
CL Chemiluminescence p24 HIV protein capsid
CLB Clenbuterol pAb Polyclonal antibody
CN− Cyanide ion PAD Paper-based analytical device
CoOxH-GO Cobalt hydroxide/oxide-modified graphene oxide PAH Persistent aromatic hydrocarbon
Co-m-CeO2 Cobalt-doped mesoporous cerium oxide Pb+2 Lead ion
COVID-19 Coronavirus disease 2019 PBS Phosphate buffer saline
Cr+4 Chromium ion PCR Polymerase chain reaction
CS-MoSe2 NS Chitosan-functionalized MoSe2 nanosheets PDA Polydopamine
Cu NPs Copper nanoparticles Pd NPs/meso-C Palladium nanoparticle dispersed on mesoporous
DA Dopamine carbon
DAB 3,3′-diaminobenzidine Pd–Pt NPs Palladium-platinum nanoparticles
DFA Diagnostics For All PFAS Perfluoroalkyl and polyfluoroalkyl substance
DIA Dipstick immunoassay PGA-Fe/CS Iron-site-containing poly-γ-glutamic acid/chitosan
E. coli O157:H7 Escherichia coli O157:H7 PNC Polyacrylic acid-coated nanoceria
Ep Epinephrine POC Point-of-care
E. sakazakii Enterobacter sakazakii POX Peroxidase
ECL Electrochemiluminescence PSA Prostate-specific antigen
ELISA Enzyme-linked immunosorbent assay AuNR@Pt Platinum-coated gold nanorods
EOPO Poly(ethylene glycol-ran-propylene glycol) RAC Ractopamine
EPA U.S. Environmental Protection Agency RAT Rapid antigen test
FBP Foodborne pathogen RPA Recombinase polymerase amplification
Fp Fenpropathrin S. aureus Staphylococcus aureus
GBM Glioblastoma Multiforme SARS-CoV-2 Severe Acute Respiratory Syndrome Coronavirus 2
g-C3N4/BiFeO3 Graphitic carbon nitride/bismuth ferrite SERS Surface-enhanced Raman scattering
GC-MS Gas chromatography-mass spectrometry SOD Superoxide dismutase
GLIF Group Legible Immunohaemetology Format SPE Screen-printed electrode
GOx Glucose oxidase SPR Surface plasmon resonance
GSH Glutathione TMB 3,3′,5,5′-tetramethylbenzidine
H2O2 Hydrogen peroxide TMZ Temozolomide
HACCP Hazard Analysis Critical Control Points UA Uric acid
hCG human chorionic gonadotropin WHO World Health Organization
Hg+2 Mercury ion ZIF-8/GO Zeolitic imidazolate frameworks-8/graphene oxide
HIV Human immunodeficiency virus μPAD Microfluidic paper-based analytical device
HPLC High-performance liquid chromatography 2D Two-dimensional
HPLC-MS High-performance liquid chromatography-mass 3D Three-dimensional
spectrometry 4-AP 4-aminoantipyrine
HRP Horseradish peroxidase 4-MBA 4-mercaptobutyramidine
ICP-MS Inductively coupled plasma mass spectrometry 8-OHdG 8-hydroxy-2′-deoxyguanosine

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on-site/point-of-care (POC) detection platforms that adhere to the [35–38]. Importantly, most of these reviews do not provide a compre­
World Health Organization’s (WHO) ASSURED (affordable, sensitive, hensive understanding of different device format, their basic structural
specific, user-friendly, rapid and robust, equipment-free, and delivered and functional components, and rarely offer insight into nanozyme
to end-users) guidelines is of supreme importance. To this end, extensive integration in these platforms. Since nanozymes have positioned them­
efforts have been made to develop nanobiosensing platforms that can selves as important candidates for integration into the real-world bio­
perform efficiently not only in research laboratories but also in-field, sensing devices, a review focussing on their capabilities for on-site/POC
in-clinic, and on-site. monitoring will help in advancing the field. A recent review reflects
Numerous reviews, focussing on different aspects of nanozymes, upon this aspect; however, without focussing on device design, which
have been published in recent years, reflecting the intense interest in this remains a key challenge for analytical chemists [39]. With this rationale,
area. These reviews can be classified into three major categories. The the present review provides an interdisciplinary and intersectoral
first category addresses the fundamentals of nanozymes, including but perspective on the integration of nanozymes in paper-based biosensing
not limited to types of enzyme-mimicking activities, fabrication strate­ platforms for on-site detection and POC monitoring. The review paints a
gies, approaches to enhance their properties and various applications [2, comprehensive picture of the latest developments, technical break­
3,20–23]. Reviews in the second category focus on NzBS in specific throughs, and unresolved challenges associated with the development of
fields, typically a specific aspect of food [16,24–29], health [30,31], or portable NzBS. Finally, the review provides insights into directions to be
the environment [11,15,32–34]. The third category includes reviews on undertaken in future research endeavours. It is anticipated that this
nanozymes covering some aspect of devices, but their discussion mostly review will encourage consolidated efforts across multiple disciplines
revolves around nanozymes types, examples, and fabrication strategies and will inspire exciting innovations in this emerging yet promising

Fig. 1. Schematic illustration of nanozyme biosensors (NzBS) and their readouts in facilitating on-site/POC monitoring of food safety and quality, environmental
pollutants, and human health.

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field. Fig. 1 illustrates different NzBS platforms and their possible their performance, (iii) Since nanozymes can offer higher thermal and
readouts that could be employed for monitoring of food contaminants, ambient stability over natural enzymes, their incorporation in
environmental pollutants, and health and disease biomarkers. paper-based devices can provide a significant opportunity by simpli­
fying the logistics of low-temperature storage and transportation,
2. General approaches for the development of NzBS for real- reducing costs, and making this technology more widely available,
world applications especially to users in resource-limited settings, and (iv) Incorporating
nanozymes into paper-based devices is relatively simple, which is
Paper-based sensing devices have emerged as one of the most crucial for the widespread adoption of these biosensors, especially in
influential low-cost technologies in recent human history. These can be settings where advanced infrastructure may be limited. These attributes
built into different formats, including lateral flow devices (LFDs), dip­ collectively make NzBS promising tools for a wide range of applications,
sticks, and microfluidic paper-based analytical devices (μPADs) [40]. particularly in fields such as healthcare, environmental monitoring, and
Recently, the incorporation of nanozymes in these paper-based devices food safety. In the following section, we present a general idea of
has gained enormous attention due to their tremendous potential in different paper-based systems to provide a basic understanding to the
enhancing these devices at various fronts viz. (i) Nanozymes can amplify readers.
the signal output of paper-based biosensors, significantly improving
their sensitivity. This is crucial for the detection of low concentrations of 2.1. Lateral flow devices (LFDs)
analytes, making them suitable for applications where high sensitivity is
essential, (ii) Nanozymes exhibit robust stability, ensuring the longevity LFDs are simple, inexpensive, paper-based analytical devices pri­
of the biosensor devices. This stability is especially advantageous for marily designed for the rapid detection of target analytes in an on-site
on-site applications in remote settings where the biosensors may be setting without requiring expensive or sophisticated equipment. These
subjected to variable environmental factors which may compromise are also commonly referred to as lateral flow assays (LFAs) or lateral

Fig. 2. Schematic illustration showing typical paper-based devices outlining their components, formats, and types of signal readouts, including (A) LFD, (B) dipstick, and (C)
μPAD. Both LFD and dipstick can be designed in sandwich and competitive assay formats. The presence of analyte in a sandwich format generates the signal at both test and
control lines, while in a competitive format the signal is seen only at the control line.

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flow test strips (LFTS). A brief description of the different components of are indicative of a positive result. If the target analyte is absent from the
an LFD, along with a range of sensing readouts possible by LFDs, is sample, the visual signal is observed at both the test and control lines. In
discussed below, while a comprehensive discussion on these can be an alternative competitive LFD layout, instead of labelled Ab1, a
found elsewhere [41,42]. labelled-analyte analogue can be incorporated on the conjugate pad,
while unlabelled Ab1 is pre-immobilized on both the test line and con­
2.1.1. LFD components and formats trol line. On loading a sample containing the target analyte, the labelled
A typical LFD consists of four major components – a sample pad, a analogue on the conjugation pad is hydrated and moves along the test
conjugate pad, a nitrocellulose (NC) membrane, and an absorption pad strip. At the test line, the target analyte competes with the labelled
(Fig. 2A). The sample pad is usually made of cellulose or glass fibre and analogue for binding to Ab1, thereby preventing the binding of the
is the component where the sample is loaded to start the assay. Its main labelled analogue to Ab1, resulting in no signal. This labelled analogue
function is to ensure continuous and uniform distribution of samples to subsequently reaches the control line and interacts with Ab1, producing
the downstream components of the LFD. Occasionally, a sample pad is a visual signal. In the absence of a target, the labelled analogue interacts
also designed for sample pre-treatment. The conjugation pad is placed with Ab1 at both the test and control lines, thus producing visual signals
next to the sample pad and is used for manual or automated deposition at both lines. Nanozymes have been incorporated in both formats of LFA,
of biorecognition molecules and tracers (referred to as molecular as will be discussed through examples throughout this review.
recognition elements – MREs in this review). These MREs can sometimes
be pre-labelled depending on the choice of the sensing output. It is 2.1.2. Types of LFD signal readouts
essential for the conjugate pad to instantly release the MREs upon Conventionally, LFDs enable unaided naked-eye detection of the
contacting the moving sample. This control is achieved by the choice of target analyte by generating visual signals at the test and/or control
material used in the conjugate pad, which, in turn, affects the sensitivity lines. While simple, rapid, and equipment-free, most of these LFDs
of the assay. The common materials used for conjugate pads include provide only qualitative information and exhibit rather low sensitivity.
glass fibre, cellulose, and polyester. The conjugate pad is followed by a Thus, they are suitable for those applications where a positive or
test pad typically made of NC membrane along which the sample is negative response is adequate to meet the requirements. These include
transported via. capillary action. The NC membrane is used to dispense pregnancy tests and infectious diseases, such as rapid diagnostic tests
components along the test and control lines. The visual appearance of developed for malaria [48] and COVID-19 [49]. For other niche appli­
these lines confirms the presence or absence of the target analyte. Non- cations, such as biomarker analysis and detection of food contaminants,
specific adsorption at the test and control lines and the wicking rate of LFDs with higher sensitivities and quantitative readouts can aid in early
NC membranes can considerably affect the sensitivity of the test. The disease diagnosis, preventing industries from incurring economic losses
absorption pad is the last component in an LFD, which is placed at the due to contaminated food recalls, and protecting individuals from
end of the strip to wick excess sample and prevent it from flowing associated health risks. To meet this requirement, LFDs with different
backwards. All these LFD components are assembled onto an adhesive signal readouts such as spectroscopic, electrochemical, magnetic, and
backing card, which does not participate in the assay and serves only as a photothermal, have been reported [41,50,51].
supporting platform. LFDs with spectroscopic (or optical) readouts can be categorised into
An LFD can be designed in either a sandwich or a competitive assay colorimetric, fluorescence, chemiluminescence (CL), surface plasmon
format. A sandwich format is generally used for larger analytes as they resonance (SPR), and surface-enhanced Raman scattering (SERS) sen­
tend to possess multiple binding sites, such as the viral protein p24 sors [52]. Colorimetric remains the most popular mode of the LFD signal
antigen for HIV detection [43] and the whole bacterial cell [44]. In this readout, as it has already seen success for qualitative POC applications.
format, an analyte-specific labelled primary antibody (Ab1) is tempo­ In the quantitative context, it captures the color produced at the test and
rarily incorporated on the conjugate pad, which enables them to control lines by recoding the image through an image sensor and
descend readily when exposed to the sample. An unlabelled version of calculating the color intensity or optical density by an image processor
Ab1 is permanently immobilized on the test line, while the control line is to determine analyte concentration [53,54]. Smartphones can be
immobilized with a secondary antibody (Ab2) that can recognise Ab1. deployed to record, analyze, and transfer visual signals, making them
During the assay, the sample migrates from the sample pad to the con­ suitable for POC diagnosis [40,51]. Despite these merits, colorimetric
jugate pad, where the labelled Ab1 captures the analyte and forms a assays suffer from low sensitivity for trace analytes. Fluorometric assays
complex. This labelled complex is then captured by pre-immobilized are inherently more sensitive than colorimetric assays, resulting in the
Ab1 at the test line, producing a visual signal proportional to the ana­ development of fluorescence-based LFDs for the sensitive detection of
lyte concentration. Excess labelled Ab1 that did not form complex with target analytes. In general, fluorescence assays monitor target-induced
the analyte flows through the test line to get captured by Ab2 at the changes in the physicochemical properties of fluorophores, such as in­
control line. The appearance of a visual signal at both lines is indicative tensity, lifetime, and polarization to enable quantitative detection [52].
of a positive result, while in the absence of analyte, a signal appears only However, the use of traditional fluorescent dyes and tags may lead to
on the control line. photobleaching and chemical degradation. In such instances, CL-based
Conversely, the competitive assay format is generally used for LFDs can be developed, as CL reaction does not undergo photo­
smaller molecular analytes as they tend to possess a single binding site, bleaching and offers merits, including but not limited to, high sensi­
such as pesticides [45,46] and veterinary drugs [47]. This format typi­ tivity, low background signal, real-time monitoring, prolonged substrate
cally has two layouts. In the first layout, the labelled Ab1 is incorporated stability, and quantitative analysis. The CL-based assays are designed to
on the conjugate pad. The test and control lines are pre-immobilized emit light which is captured and measured to determine the presence or
with the analyte or its analogue and Ab2, respectively. On being absence of the target analyte. SPR-based LFDs offer the prospects of
exposed to a sample containing the target analyte, the labelled Ab1 rapid, real-time detection of analytes in a label-free environment with
binds with the analyte and forms a complex. This labelled Ab1-analyte high sensitivity and specificity. These LFDs measure changes in the
complex then moves towards the NC membrane, where it competes refractive index associated with the binding of the target analyte to the
with the analyte analogue bound on the test line, leaving either no or immobilized MRE [55]. SERS-based assays employ the amplification of
only a few analogue molecules to be replaced. As a result, no change in electromagnetic fields generated by the excitation of localized surface
the visual signal is observed at the test line in an analyte-positive sam­ plasmons and allow highly sensitive detection of analytes in low sample
ple. The Ab1-analyte complex is subsequently captured by Ab2 at the volume.
control line thereby producing a visual signal. The lack of a visual signal Electrochemical LFDs rely on measuring changes in the electro­
at the test line, and the appearance of a visual signal at the control line chemical signal (e.g., current, potential, or resistance) before and after

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the interaction of analytes with MREs. Conventional electrochemical to make the sample flow across channels [62]. They can be designed in
LFDs use paper-based test strips armed with multiple electrodes and two- (2D) and three-dimensional (3D) formats by patterning the paper.
electroactive tracers for the generation of charged species in response to For designing 2D μPADs, techniques like wax dipping, cutting, plasma
the analyte-MRE interaction. Considering several electrochemical etching, photolithography, inkjet etching, etc. are used to pattern
detection technologies have already found their way into mainstream physical or chemical hydrophobic boundaries onto the paper.
consumer applications (e.g., finger-prick glucose sensors), the techno­ Conversely, 3D μPADs are designed by folding and stacking paper to
logical translation of electrochemical LFDs might be easier than other control the sample flow for analyte detection [61,63]. Like LFDs and
technologies. We see this as one of the key attractive features of the dipsticks, reagents can be pre-loaded onto the paper and functional
electrochemical LFDs. Recent advancements in photolithography and bio/chemical molecules can be pre-immobilized by using surface
screen-printed technologies have significantly contributed to further modification techniques such as physical adsorption, carrier-mediated
reducing the cost of electrode incorporation in LFDs. Despite this, the deposition, and chemical coupling [64]. Both 2D and 3D μPADs can
development of electrochemical LFDs continues to be challenging and also be used for filtering samples, performing chromatographic separa­
expensive, mostly due to the challenges associated with the reproduc­ tions, and evaluating biochemical reactions. Overall, like LFDs and
ibility of signals and the requirement of an inexpensive electrochemical dipsticks, μPADs offer a simple and portable design, inexpensive
reader to be associated with a standalone LFD. These requirements manufacturing, rapid response, and user-friendliness. In addition, they
collectively hinder their on-site application in resource-limited settings. also offer facile multiplexed analysis, which, while feasible with LFDs
The development of better-performing LFDs has seen ongoing efforts. and dipsticks, is more conveniently achieved with μPADs. Owing to such
These include the incorporation of multi-functional tracers in LFDs, merits, the use of μPADs has been widely explored in food safety testing,
which can produce multiple readouts simultaneously (e.g., colorimetric environmental monitoring, and clinical diagnosis by employing optical,
and electrochemical [56], colorimetric and magnetic [57], colorimetric electrochemical, electrochemiluminescence (ECL), and
and SERS [58], or fluorescence, photothermal, and electrochemical photo-electrochemical modes of readouts [65,66]. While the μPAD
[59]). Innovations in this area have rendered LFDs with enhanced per­ technology has some obvious advantages, several concerns continue to
formance, including improved signal-to-noise ratio, accuracy, and pre­ persist, such as (i) inefficient sample delivery due to evaporation and
cision by combining the merits of different transducers. These retention, (ii) coffee ring effect caused by drying of the solutes around
multi-modal LFDs are gaining slow, yet desired attention. the edges of the test zone, (iii) reagent shelf-life and device stability, (iv)
batch-to-batch variations, and (v) challenges associated with multi­
2.2. Dipsticks plexing and real-sample analysis in complex matrices requiring
pre-treatment [67,68]. Considering these issues, limited commercial
Dipsticks are simple, portable, user-friendly paper-based devices that success has been made. This includes a GLIF™ (Group Legible Immu­
offer rapid detection opportunities within a low-cost manufacturing nohaemetology Format) device for Blood Group analysis [69] and
framework. These devices use a simplified format of LFD by removing low-cost μPAD platforms under development by Diagnostics For All
certain components of a typical LFD discussed in the previous section (DFA) to monitor liver function, child nutrition, immunity, nucleic acid
(Fig. 2B). For instance, both the sample pad and conjugate pad can be detection, and ascertain cattle heat [70].
removed, and the paper strip can be directly immersed in the sample
solution. One of the common examples is pH test strips, designed by 3. Deployment of NzBS for monitoring food safety and quality
immobilizing a combination of acid-alkali indicators onto the paper strip
to generate semi-quantitative visual signals that can be compared to a Food safety and security, i.e., access to safe food for everyone across
reference color-chart provided with the strips [40]. Similarly, urine test the globe, is one of the most fundamental needs of our society [71].
strips contain specific reagents that can react with the targeted urinary Despite ongoing economic development across the globe, several parts
compounds (e.g., glucose, ketone bodies) to produce a characteristic of the world continue to face challenges in reliable access to food, thus
color, with a readout within 1–2 min. Despite being rapid and raising the question of food security. While the green revolution and
user-friendly, the simplified design of conventional dipsticks often re­ globalization have supported food security to a reasonable extent, the
sults in poor sensitivity and specificity. An alternate version of dipstick importance of food safety is also becoming exceedingly important. The
can be employed to perform dipstick immunoassays (DIAs). These DIAs presence of contaminants, whether intentional or accidental, not only
often contain a simple pad comprised of either NC or nylon membrane threaten human health but also cause enormous economic loss to food
that contains a target-specific capture antibody. Upon contacting the industries. Contaminated food can result in over 250 diseases by acting
sample, once the target analyte binds to the dipstick, it is exposed to an as a pathogen transmission agent [71,72]. Food safety regulations have
enzyme-labelled detection antibody, which upon subsequent exposure been put in place by regulatory agencies worldwide to prevent in­
to the chromogenic substrate of the enzyme, produces a signal. Like dividuals from health complications and risks associated with food
LFDs, dipsticks can also be developed in both sandwich and competitive contaminants, such as pathogens, mycotoxins, allergens, and pesticides.
formats [60]; however, their application is mostly limited to detecting This has led to food industries incorporating Hurdle Technology, and
analytes in large sample volumes. Hazard Analysis Critical Control Points (HACCP) systems in their food
quality assurance program to regularly check for pathogen contamina­
2.3. Microfluidic paper-based analytical device (μPAD) tion [73]. To achieve this, methods that can detect food contaminants
rapidly with high precision, excellent sensitivity, and reliability are
μPADs hold great potential in POC diagnostics due to their ability to required.
combine the advantageous features of a paper with that of a microfluidic Over the years, various methods, including but not limited to high-
platform. Typically, μPADs consist of filter paper patterned by wax to performance liquid chromatography (HPLC), HPLC-mass spectrometry
create a sample loading zone, hydrophilic channels, and hydrophobic (HPLC-MS), gas chromatography-mass spectrometry (GC-MS), induc­
barrier (Fig. 2C). In most μPADs, sample flow is achieved by capillary tively coupled plasma mass spectrometry (ICP-MS) and real-time poly­
force across the hydrophilic channel. They are different from LFDs and merase chain reaction (PCR) have been established to detect food
dipsticks in the context that the creation of microfluidic channels re­ contaminants [16]. These methods enable analyte detection with high
quires more precise printing of hydrophobic (typically wax) and hy­ precision, sensitivity, and reliability. However, these traditional
drophilic regions on the paper [61]. However, these printing processes methods are complex, labour-intensive, time-consuming, and require
are now well established, and despite this requirement, μPADs are expensive and sophisticated instruments to be operated by trained
highly cost-effective, as no pump or external source of energy is required personnel in designated laboratories. The high capital and operating

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expenditure (CAPEX and OPEX) requirements of these technologies also industries are always on the lookout for technologies that can be
limit their wider adoption in food industries, which already suffer from incorporated into various stages of the food chain to ensure the delivery
low-margin cost pressures [71]. Overall, these techniques fail to meet of safe and reliable food to consumers [71].
the demand for rapid, inexpensive on-site detection of large food sam­ Biosensors have emerged as a suitable alternative and in some cases
ples, especially in resource-limited settings [74,75]. Thus, food as a complementary technique to the aforementioned methods for rapid,

Fig. 3. Schematic illustration of sandwich LFDs incorporating nanozymes for the detection of foodborne pathogens. (A) Pd–Pt NPs for the detection of E. coli O157:
H7. Adapted with permission from Ref. [44] ©2018 American Dairy Science Association; (B) Pd–Pt NPs for the simultaneous detection of S. enteritidis and E. coli
O157:H7. Adapted with permission from Ref. [84] © 2017 American Chemical Society; and (C) Fe3O4 NPs for the selective detection of live L. monocytogenes [86]
and E. sakazakii [87].

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sensitive, and inexpensive detection of food contaminants [76,77]. production of color only at the control line, indicating the absence of
Further, the incorporation of nanozymes in biosensor platforms to pro­ bacteria. In this LFD assay, the intensity of the blue color was found to
duce NzBS is reported to offer additional merits such as prolonged sta­ directly correlate with bacterial concentration. Owing to the high
bility, and label-free detection, while also offering the prospects for POX-mimic activity of Pd–Pt NPs, they could oxidise TMB even at trace
further reduction of the cost of these technologies [78]. The last few bacterial concentrations producing a visual signal. As a result, the
years have seen a surge in reports on NzBS for the detection of food developed LFD exhibited 111-fold higher sensitivity than that of con­
contaminants, as captured in recent reviews [16,26,79]. While encour­ ventional (nanozyme-free) colloidal gold LFD and allowed E. coli O157:
aging, the NzBS for the detection of food safety and quality in the context H7 detection with a limit of detection (LOD) of 87 and 9 × 102 CFU mL− 1
of a POC device has been barely discussed in previous reviews. Since a in the phosphate buffer saline (PBS) and milk, respectively. The speci­
thorough understanding of the developments taking place towards ficity analysis against eleven non-target bacteria suggested excellent
real-world translation of biosensor technologies is critical, we have specificity of this LFD towards E. coli O157:H7. The colorimetric
compiled the following section to selectively discuss some of the response time was 4 min in the PBS; however, the response time in milk
important examples of portable NzBS for the POC detection of various was not indicated, while the authors did acknowledge that a longer
food contaminants and captured others in Supplementary Table 1. response time was required in milk due to interference from other
substrates.
3.1. Foodborne pathogens (FBPs) While the detection of E. coli O157:H7 alone is useful, there have also
been reports of detecting E. coli O157:H7 along with other pathogens. A
FBPs are biological contaminants that compromise food quality and dual-LFD was reported by incorporating mesoporous Pd–Pt NPs as a
make it unsafe for human consumption. The ability of FBPs to remain nanozyme for simultaneous detection of Salmonella enteritidis and E. coli
dormant in food matrices over extended periods until they encounter an O157:H7 (Fig. 3B) [84]. The working principle of the device was like the
appropriate human or animal host makes them a real threat to food one discussed above, but the use of a parallel design strategy in the
safety. Over the years, natural enzyme-based immunoassays and sensors biosensor platform prevented cross-interference between S. enteritidis
have been used for the detection of FBPs. However, in the recent past, and E. coli O157:H7, enabling the respective LODs of ~20 and ~34 CFU
nanozymes have started to replace natural enzymes in these platforms mL− 1. The incorporation of POX-mimic Pd–Pt NPs ensured naked-eye
by offering the merits of rapid detection at low cost, and improved detection of pathogens by enhancing the colorimetric signal. The esti­
sensitivity and stability under harsh environmental conditions. Of more mated recoveries ranging from 91.4 to 117.0 % in milk and ice cream
than 200 known foodborne diseases, some of the key FBP outbreaks samples suggested the suitability of this assay in practical scenarios. This
include Escherichia coli O157:H7, Listeria monocytogenes, Staphylococcus dual-LFD was also integrated with a smartphone device to expedite its
aureus, and Salmonella spp. [80]. Considering the complexity of the food applicability in food traceability.
chain from cultivation, through harvesting, refining, and trading to L. monocytogenes, another common FBP, is associated with foodborne
preparing it for human consumption; FBPs may enter the chain at any listeriosis in humans [85]. The pathogen exhibits high resilience over
point. Unfortunately, effective measures to control these pathogens from wide temperatures and pH ranges and can grow even in refrigerated
affecting raw agricultural produce and seafood still do not exist. As a foods. Prompted by the need to detect live L. monocytogenes rapidly with
result, their presence can be merely reduced to make these foods safe for high sensitivity, a sandwich LFD was developed by combining Fe3O4 NPs
consumers in the raw, unprocessed state; however, not entirely averted as a POX-mimic with the propidium monoazide-based loop-mediated
or eliminated [81]. The maximum permissible limit (MPL) for different isothermal amplification (LAMP-PMA) reaction products (Fig. 3C) [86].
FBPs varies across jurisdictions, and the presence of some FBPs is more In this approach, the differentiation between live and dead bacteria was
relaxed than others depending on the food type and their level of mediated by the PMA dye. This dye can selectively permeate across the
infectivity [82,83]. Further, the MPL for the same FBP in a single dead bacterial membrane and insert into their DNA; however, has no
jurisdiction can also vary remarkably depending on the type of food (e. such effect on live bacteria due to their intact membranes. Consequently,
g., dairy, ready-to-eat food, meat, seafood, infant powder, leafy vege­ only the DNA from live cells in the target hlyA gene region could be
tables, fresh produce, poultry, etc.). Thus, while developing nano­ amplified by the subsequent LAMP reaction. For the development of the
biosensors, their MPLs should be taken into consideration for specific LFD, the conjugate pad was pre-loaded with Fe3O4 NPs
foods. The NzBS reported so far for POC detection of FBPs are discussed modified-anti-biotin Ab (signal probe) along with FITC- and
further. biotin-labelled LAMP products. On the NC membrane, anti-FITC Ab and
A sandwich LFD for the detection of E. coli O157:H7 was proposed by goat anti-mouse IgG were loaded to form test and control lines,
incorporating bimetallic palladium-platinum nanoparticles (Pd–Pt NPs) respectively. When the L. monocytogenes was present in the sample, the
as a POX-mimic (Fig. 3A) [44]. The assay was designed by signal probe interacted with the biotin-labelled end of the LAMP prod­
pre-immobilizing Pd–Pt NPs-labelled anti-E. coli monoclonal antibody uct, forming a conjugate complex. This complex migrated along the NC
(Pd–Pt NPs-labelled mAb) as a ‘signal probe’ on the conjugate pad; membrane and was captured by the anti-FITC antibodies at the test line,
anti-E. coli polyclonal antibody (anti-E. coli pAb) as a ‘capture probe’ on forming a sandwich immune-complex. Any excess unbound nanozyme
the test line; and goat anti-mouse immunoglobulin G (goat-anti-mouse probes leaving the test line were captured at the control line by the goat
IgG) on the control line. During analysis, the sample containing E. coli anti-mouse IgG. A colorimetric signal at both test and control lines
migrates from the sample pad to the conjugate pad by capillary action, appeared due to nanozyme-mediated oxidation of the chromogenic
where it interacts with Pd–Pt NPs-labelled mAb to form a complex. As substrates (3,3′-diaminobenzidine – DAB and H2O2). The practical
this complex migrates further on the NC membrane, it binds to the feasibility of the LFD was validated by detecting L. monocytogenes bio­
anti-E. coli-pAb on the test line, such that E. coli is now sandwiched film formation on stainless steel and lettuce surfaces with LODs of 11.6
between these two antibodies. Since Pd–Pt NPs-labelled mAb are used in and 10.2 CFU mL− 1, respectively.
excess on the conjugate pad, only some of these mAb bind to the E. coli Enterobacter sakazakii is another important FBP that is known to
and the remaining free Pd–Pt NPs-labelled mAb migrate to the control affect infants and neonates by causing meningoencephalitis, necrotizing
line to interact with goat-anti-mouse IgG. In the next step, when the enterocolitis, meningitis, and sepsis [88,89]. Selective colorimetric
colorimetric POX substrates (3,3′,5,5′-tetramethylbenzidine (TMB) and detection of live E. sakazakii was demonstrated by developing a
H2O2) are introduced to the sample pad, they migrate and form a stable continual cascade sandwich LFD (Fig. 3C) [87]. To achieve a distinction
blue product at the test and control lines where Pd–Pt NPs are already between live and dead bacteria, a similar strategy as discussed above for
present. If the E. coli O157:H7 is not present in the sample, Pd–Pt L. monocytogenes was employed. The authors used the LAMP process
NPs-labelled mAb does not conjugate to the test line, which results in the along with PMA treatment, which allowed selective amplification of the

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A. Baranwal et al. Trends in Analytical Chemistry 172 (2024) 117573

DNA from the live bacteria. Afterwards, Fe3O4 NPs-labelled anti-biotin via. food and/or water can cause debilitating conditions such as car­
Ab was used as a POX-mimic for signal amplification on the LFD to diovascular, neurological, and reproductive disorders, as well as kidney
enable the detection of DNA from live E. sakazakii. The developed dysfunction, and cognitive decline [97,98]. A nanozyme-based colori­
platform showed a LOD of 10 CFU mL− 1 while exhibiting high selectivity metric sensor for the detection of mercury ions (Hg+2) was proposed
against fifteen non-E. sakazakii strains. The reported sensitivity on this [99], in which chitosan-functionalized MoSe2 nanosheets (CS–MoSe2
LFD was claimed to be 10 times higher than that typically achieved by NS) were assessed as both POX and OX mimetic systems (Fig. 4A). In the
real-time PCR (100 CFU mL− 1) [90]. When the nanozyme strip was absence of Hg+2, the CS-MoSe2 NS exhibited poor POX- and OX-like
tested to detect E. sakazakii in spiked infant powder formulation, re­ activities. However, in its presence, the catalytic activities were
coveries ranging from 95.3 to 108.7% were observed. notably improved due to in situ reduction of chitosan-captured Hg+2 to
Campylobacter jejuni is one of the most common and frequent causes Hg0 on the nanozyme surface. This led to the rapid oxidation of TMB to a
of acute foodborne gastroenteritis in humans. Given its prevalence in the persistent blue product, which suggested a positive correlation between
gastrointestinal tracts of poultry, their products are often implicated as TMB oxidation and Hg+2 ion concentration. Furthermore, a portable
the primary source of human infection [91]. In the last decade alone, the sensor for the POC detection of Hg+2 was proposed by transferring the
number of C. jejuni infections has grown tremendously making it a major assay onto an absorbent pad and employing a smartphone device to
threat to public health and economy. In view of this, a sandwich LFD was analyze the changes in the colorimetric signal. When evaluated as a
developed by incorporating POX-mimic platinum-coated gold nanorods POX-mimic platform in the presence of H2O2, the developed sensor
(AuNR@Pt) and Raman reporter 4-mercaptobutyramidine (4-MBA) for could detect Hg+2 ions within a linear range of 25 nM–2.5 μM with the
rapid and sensitive detection of C. jejuni [92]. The components of the calculated LOD of 8.4 nM; while in the absence of H2O2, as OX-mimic,
fabricated LFD were similar to a conventional LFD; however, without the sensor achieved a linear range of 0.1–4.0 μM with the calculated
distinct test and control lines. The sensing mechanism relied on the LOD of 27 nM. The pad sensor also displayed good selectivity towards
binding of C. jejuni to the signal probe (anti-C. jejuni Hg+2 ions when evaluated against other common cations. The authors
mAb-4-MBA/AuNR@Pt) to form an immunocomplex, which was then further assessed the portable PAD sensor in enriched water and serum
captured by the anti-C. jejuni pAb to form a sandwich complex. Conse­ samples for Hg+2 detection. The ability of this sensor to detect Hg+2
quently, a colorimetric response was generated by AuNR@Pt-mediated below the MPL of 2 ppb or 9.97 nM set by the U.S. Environmental
rapid oxidation of POX substrates (TMB + H2O2) enabling a LOD of Protection Agency (EPA) guidelines in potable water [100] suggests its
75 CFU mL− 1. Next, the 4-MBA in the sandwich immunocomplex was potential application in on-site monitoring.
excited with a 785 nm laser to produce a SERS spectrum, thereby Pesticides play a crucial role in minimising the damage caused by
resulting in a LOD of 50 CFU mL− 1. Notably, under both colorimetric various pests and pathogens. However, residual pesticides could accu­
and SERS readouts, a concentration-dependent response corresponding mulate in soil, air, water, and eventually in food, adversely impacting
to linear ranges of 102–106 CFU mL− 1 and 102–5 × 106 CFU mL− 1, human, animal, and environmental health [101,102]. Therefore, the
respectively were obtained. The practical application of the proposed need for rapid, reliable, and inexpensive POC pesticide detection plat­
assay was further validated in milk samples and recoveries ranging from forms has grown incessantly. A competitive two-way LFD was proposed
89.3 to 107.6% were obtained. by employing Pt NPs-modified 2D Ni(OH)2 nanosheets (Ni(OH)2–Pt) as a
In addition to bacteria, fungi play a crucial role in food spoilage and POX-mimic for simultaneous detection of herbicide (acetochlor – Ac)
pose potential threats to human health. Among the 1.5 million identified and insecticide (fenpropathrin – Fp) (Fig. 4B) [46]. The synergistic effect
fungal species, approximately 300 have been reported to induce health between the 2D nanosheets and Pt NPs improved the binding energy for
complications, ranging from minor allergic reactions to severe life- the absorption of the reaction intermediate, thereby enhancing the
threatening infections [93]. A notable fungus in this regard is Asper­ catalytic activity of the Ni(OH)2–Pt nanozyme. Cross-reactivity between
gillus flavus, known for causing infections in tree nuts, grains, cereals, targets during simultaneous detection was eliminated by adopting a
spices, and oil seeds, and producing highly toxic aflatoxins, which can two-way design in the LFD platform. In the absence of the target analytes
induce carcinogenicity, nephrotoxicity, neurotoxicity, and hepatotox­ (negative sample), anti-Ac/anti-Fp-modified Ni(OH)2–Pt conjugates
icity under severe infections [94]. Driven by the need for accurate and bind with their corresponding target analogues at the test line to form a
sensitive early A. flavus detection, a sandwich LFD was developed by black band. The excess unbound anti-Ac/anti-Fp-modified Ni(OH)2–Pt
combining colorimetric and photothermal modes of readout [95]. The conjugates leaving the test line are captured by goat anti-mouse IgG at
proposed LFD incorporated a conjugate complex containing POX-mimic the control line to form additional black bands. On exposing the test line
Cu NPs, photothermal polydopamine (PDA), and rabbit pAb as a to POX substrates (TMB + H2O2), the black bands turned to deep blue
dual-signal probe (Cu NPs-PDA@pAb), whereas nanobody (instead of due to the POX-mimic activity of Ni(OH)2–Pt nanozyme. On the other
mAb) acted as a capture probe. A nanobody is a small recombinant hand, in the presence of target analytes (positive sample), a competition
antibody that possesses only heavy-chain peptides of a typical antibody between target analytes vs. their analogues occurs, leaving fewer or no
and exhibits high selectivity and stability compared to its natural anti­ anti-Ac/anti-Fp-modified Ni(OH)2–Pt conjugates free to bind at the test
body counterpart [96]. In the presence of A. flavus, a colorimetric (blue) line. Consequently, inconspicuous, or faint blue bands are observed
signal was observed due to the oxidation of POX substrates (TMB + suggesting an inverse correlation between Ac and Fp concentrations and
H2O2). The blue color intensity directly correlated with the concentra­ TMB oxidation. By employing the two-way LFD platform, the authors
tion of the A. flavus biomass and resulted in a LOD of 0.45 ng mL− 1. The could simultaneously detect Ac and Fp with LODs of 0.63 ng mL− 1 and
sensitivity was further augmented by exposing the test line to an 808 nm 0.24 ng mL− 1, respectively. Interference studies in the presence of
near-infrared laser. Consequently, A. flavus concentration-dependent chlorpyrifos, diazinon, malathion, atrazine, and glyphosate suggested
increase in temperature was observed, which led to an improved LOD excellent specificity of this two-way LFD towards target analytes. The
of 0.22 ng mL− 1. Notably, the proposed method exhibited 19- (colori­ practical implication of the portable two-way sensor was also evaluated
metric) and 40-times (photothermal) higher sensitivity compared to in real food and water samples with estimated recoveries ranging from
conventional LFDs. Furthermore, the practical applicability of the LFD 97.1 to 111.5%. We note that both these chemical residues (Ac and Fp)
was assessed by investigating spiked peanut and maize samples. The have an organophosphorus structure and considering the wide appli­
estimated recoveries in these food samples ranged from 89.9 to 109%. cability of this group of chemicals in agriculture, we have elaborated on
different approaches for their detection in a later section focussed on
3.2. Heavy metal ions and pesticides environmental monitoring (Section 4.2)

Prolonged exposure to toxic heavy metal ions, even in trace amounts

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A. Baranwal et al. Trends in Analytical Chemistry 172 (2024) 117573

Fig. 4. Schematic illustration of paper-based devices incorporating nanozymes for the detection of foodborne ionic and molecular contaminants. (A) a
paper PAD incorporating chitosan functionalized MoSe2 nanosheet for detection of Hg+2 ions [99]; (B) a two-way sandwich LFD incorporating Pt NPs-modified Ni
(OH)2 nanosheets for the simultaneous detection of two organophosphorus pesticides [46]; and (C) a one-way competitive LFD incorporating a magnetic Prussian
blue (mPB) nanozyme for the simultaneous detection of two veterinary drugs [47]. (For interpretation of the references to color in this figure legend, the reader is
referred to the Web version of this article.)

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A. Baranwal et al. Trends in Analytical Chemistry 172 (2024) 117573

3.3. Other food contaminants issues, rapid, real-time detection of such contaminants with high
sensitivity and selectivity is highly imperative. Various techniques like
Other potential food contaminants include veterinary drugs (e.g., GC-MS, liquid chromatography (LC), LC-MS, and HPLC have been
clenbuterol – CLB and ractopamine – RAC) and small molecules (H2O2) traditionally employed [109], and continue to be predominantly
that are used either for improved agricultural yields or preservation of employed for detecting such environmental pollutants. Despite being
food and can find their way into consumer food. The accumulation of widely regarded as the ‘gold standard,’ these techniques suffer from
veterinary drug residues in livestock products may lead to skin allergies, numerous limitations, including extensive analysis time, expensive
anaphylactic shock, heart palpitations, tremors, and microbial resis­ instrumentation, and dependence on technical expertise, as discussed in
tance in consumers [98,103]. A one-way LFD was developed for the the previous section. These limitations have made them inadequate for
simultaneous detection of CLB and RAC by employing magnetic Prussian applications requiring continuous monitoring and on-site testing. In
blue nanoparticles (mPB NPs) as a POX mimic (Fig. 4C) [47]. The mPB view of this, NzBS have emerged as excellent alternatives to these con­
NPs coated with target-specific antibodies acted as bifunctional signal ventional techniques by offering advantages of rapid analysis, low cost,
tags, ensuring high precision and wide linear range during dual-mode portability, and prolonged stability in resource-limited settings.
readout. The developed assay could detect CLB and RAC with LODs of Although the application of NzBS for environmental monitoring is
0.2 and 0.12 ng mL− 1 and linear ranges of 0.15–12 and 0.5–6 ng mL− 1, relatively new, numerous reports exploring their potential have been
respectively. Interference studies conducted in the presence of strepto­ published in recent years [11,15,32]. In this section, we will focus on
mycin, 3-hydroxytyramine hydrochloride, 17β-estradiol, and 3-ami­ some of the salient examples of NzBS that have demonstrated potential
no-2-oxazolidinone suggested that the proposed LFD exhibits for the POC detection of environmental contaminants, while the
satisfactory specificity. The potential practical utility of this LFD was remaining are discussed in Supplementary Table 2.
analyzed by detecting CLB and RAC in pork and mutton samples,
revealing estimated recoveries ranging from 84.0 to 119.9%. 4.1. Heavy metals and other toxic species
The excessive use of small molecules like H2O2 can compromise food
and milk quality by making it unsafe for human consumption [104,105]. Several heavy metals and other toxic species are released into the
The POX-like activity of chitosan was explored on a PAD platform for environment through natural and anthropogenic activities, which can
colorimetric detection of H2O2 [106]. In the presence of H2O2, chitosan pose serious health challenges. For instance, cyanide (CN− ) is a
could oxidise TMB into a blue product, whose intensity was proportional contaminant that is extremely toxic to both vertebrates and in­
to H2O2 concentration. Consequently, authors could detect H2O2 with a vertebrates. Cyanides are widely employed in industries for organic
calculated LOD of 1.55 μM and a linear range of 10 μM–10 mM. Another synthesis and metallurgy, which might cause the accidental release of
report investigated the POX-like activity of Pd NPs dispersed on meso­ CN− into the environment. Oral consumption of CN− can prove lethal
porous carbon (Pd NPs/meso-C) in a paper-based colorimetric sensor for with an average estimated dose of 1.4 mg kg− 1 body weight and cause
the detection of H2O2 [107]. This chemosensor enabled visual detection death within minutes [110]. To avoid such complications, accurate
of H2O2 within a concentration range of 0–250 μM with a LOD of ~1 μM. detection of CN− in environmental samples is important. Given this, a
The practical implication of the proposed sensor was validated by nylon membrane paper-based sensor was developed by incorporating
detecting H2O2 concentration in milk samples with estimated recoveries amorphous cobalt hydroxide/oxide-modified graphene oxide
ranging from 100.9 to 109.7%. (CoOxH-GO) material as a POX-mimic (Fig. 5A) [111]. In the presence of
Overall, it is commendable to see the progress made by NzBS within a H2O2, the nanozyme could oxidise a nearly colorless POX substrate
short timeframe for monitoring food safety; however, most of these Amplex red to a pink resorufin product. The sensing mechanism
strategies rely on colorimetric detection. While this lends ease of signal involved inhibition of POX-mimic activity of nanozyme by CN− , which
detection, the colorimetric analysis may, at times, pose limitations in appeared as a reduction in the pink color intensity of the membrane
monitoring often colored food analytes that can mask visual signals. This corresponding to an indirect correlation to CN− concentration. The
interference may affect visual signal interpretation and, at times, lead to proposed sensor showed a LOD of 100 nM with a linear operational
inaccurate analysis. Considering that food matrices are complex, contain range of 100 nM–100 μM while exhibiting good selectivity for CN− ions
interfering molecules, and often have unevenly distributed analytes in against other anions. The sensor could also detect CN− in water and
trace amounts [71], it is important to combine different modes of sig­ laboratory waste samples with high sensitivity. Interestingly, although
nalling to enable the reliable detection of food contaminants. The ex­ the authors employed Amplex red as a colorimetric substrate in this
amples illustrated above suggest that the use of NzBS for on-site study, the same substrate can also act as a fluorogenic substrate, which
detection of analytes relevant to food safety is restricted to a few key could have potentially resulted in higher sensitivity. However, it could
pathogens, heavy metal ions, pesticides, antibiotics, and small mole­ have perhaps compromised the ability to detect CN− ions by unaided
cules. However, the list of important food analytes is rather large, which eyes.
remains to be detected by NzBS. Important considerations such as as­ Another heavy metal with high toxicity is mercury, as Hg+2 ions can
pects of their geographical traceability, authenticity, food content origin cause severe damage to the human immune system and organs. Its
for their genetic modification, presence of allergens, shelf life, etc., can accumulation, even at low concentrations, can result in disorders like
all be potentially addressed by developing appropriate NzBS. However, Minamata disease and Hunter-Russell syndrome, and cause permanent
the experimental realisation of relevant POC devices remains to be seen. damage to the nervous system and kidney [112]. Prompted by the acute
toxicity of Hg+2 ions, a μPAD was developed by incorporating Au NPs as
4. Deployment of NzBS for environmental monitoring POX-mimic [113]. In the presence of Hg+2 ions, an Au–Hg alloy was
formed, which improved the POX-like activity of Au NPs. Consequently,
Extensive anthropogenic activities, along with unavoidable natural intense blue spots were observed on the μPAD due to enhanced oxida­
events, have led to the contamination of our environments with a wide tion of POX substrates (TMB + H2O2). The colorimetric signal was
range of toxic compounds, turning environmental toxicity issues into an dependent on Hg+2 ion concentration, implying a direct correlation
alarming global concern [108]. Rapid advances in industrial and agri­ between metal ion concentration and signal intensity. The authors could
cultural activities have resulted in increased environmental contami­ achieve a linear range of 0.1–200 ng and a calculated LOD of 0.06 ng in a
nation. The presence of toxic contaminants such as heavy metals, single drop (2 μL containing 0.4 ng Hg) of the test sample. The μPAD also
pesticides, dyes, drugs, pathogens, gases, and persistent organic hy­ demonstrated excellent selectivity towards Hg+2 when tested against
drocarbons, whether natural or man-made, not only threatens human interfering cations. The practical implication of the proposed sensor was
health but also endangers environmental security [109]. Owing to these validated by detecting Hg+2 ions in tap water samples with estimated

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A. Baranwal et al. Trends in Analytical Chemistry 172 (2024) 117573

Fig. 5. Schematic illustration of portable platforms incorporating nanozymes for the detection of environmental contaminants. (A) CoOxH-GO nanohybrid
incorporating a nylon membrane sensor for detection of CN− ions [111]; (B) AChE and ChO containing inorganic-organic hybrid nanoflowers for dual-mode col­
orimetric/electrochemical detection of paraoxon pesticide. In figure, I: TMB; II: stop solution; III: acetylcholine; and IV: ACC-hNFs loading zone. Adapted with
permission from Ref. [56] © 2019 Elsevier B.V.; and (C) MnO2 NFs-incorporating LFD for dual-mode colorimetric/chemiluminescent detection of chlorpyrifos
(Chl) [119].

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A. Baranwal et al. Trends in Analytical Chemistry 172 (2024) 117573

recoveries ranging from 84.4 to 112.9%. with a key difference that instead of two OPPs (Ac and Fp) simulta­
Furthermore, inadvertent silver (Ag+) ion exposure can cause neously detected by OPP-specific Abs in the previous case in a two-way
neurotoxicity and damage the liver and kidney [114,115]. To detect Ag+ mobility format, only one OPP (Chl) was detected in this case in a
ions, a colorimetric sensor was developed by utilizing zeolitic imida­ one-way mobility format. The strategy also differs in the choice of
zolate frameworks-8/graphene oxide (ZIF-8/GO) as a POX-mimic [116]. nanozyme from Ni(OH)2–Pt in the previous case to MnO2 in the current
The working principle of this sensor relied on the Ag+ ion-triggered case, but both are POX-mimics. During Chl detection, the MnO2 NFs
POX-mimic activity of ZIF-8/GO, producing a colorimetric response labelled with anti-Chl-mAb (MnO2-anti-Chl-mAb) acted as a dual signal
proportional to Ag+ concentration. The sensor showed a LOD of 1.43 nM probe. The sensing mechanism involved competitive binding of
and high selectivity against other metal ions. The practical applicability MnO2-anti-Chl-mAb to the Chl-BSA pre-immobilized on the test line vs.
of this sensor was assessed by utilizing ZIF-8/GO-modified filter papers Chl present in the sample, such that in the presence of Chl, the Ab sites of
for detecting Ag+ in river water and human serum. Estimated recoveries the MnO2-Ab complex are blocked, and it is not able to bind to the test
of 98.3–103.1% in river water and 97.8–102.3% in human serum re­ line. Due to this competitive format, the Chl concentration appears
flects the potential of the proposed sensor for real-world applications. inversely related to the brown intensity of MnO2 at the test line. While
the color was easily conspicuous to the unaided eye at high Chl con­
4.2. Organophosphorus pesticides (OPPs) centrations, the trace concentrations were not. This challenge was
overcome by a complementary approach, in which the POX-mimic ac­
OPPs are among the most commonly used pesticides due to their tivity of MnO2 NFs was utilized to promote the oxidation of a chemi­
wide utility as fungicides, insecticides, herbicides, helminthicides, and luminescent substrate, luminol, in the presence of H2O2. This allowed
nematicides [102]. Exposure to OPPs, even in trace amounts, can cause quantitative detection of lower concentrations of Chl with a calculated
serious health complications, such as nerve damage, cardiovascular LOD of 0.033 ng mL− 1 and a linear detection range of 0.1–50 ng mL− 1.
disorders, and cancers [16,117]. This makes precise monitoring of OPP Further, the potential practical applicability of this LFD assay was tested
levels in soil, water, and food products crucial to warrant food and by monitoring Chl in environmental water samples and traditional
ecosystem safety. Among various NzBS reported for OPPs [7,11,19], a Chinese medicines with estimated recoveries ranging from 90 to 120%.
notable example is a portable colorimetric PAD sensor developed by Based on a similar strategy, another dual-mode LFD was developed,
combining acetylcholinesterase (AChE) enzyme with γ-MnOOH nano­ but by employing a graphitic carbon nitride/bismuth ferrite (g-C3N4/
wires as a degradable OX-mimic nanozyme [118]. This sensor was BiFeO3) nanocomposite as a POX-mimic, and investigating simultaneous
developed based on the idea that OPPs tend to act as AChE inhibitors. In detection of an OPP (e.g., Chl) and a carbamate (e.g., carbaryl) pesticide
the absence of OPPs, AChE remains active and can break down ace­ [45]. This strategy is similar to that illustrated in Fig. 4C, with a key
tylthiocholine iodide (ATCh) to produce thiocholine, which can disin­ difference that instead of two veterinary drugs detected in the previous
tegrate γ-MnOOH nanowires. This leads to the loss of their OX-like case, two pesticides were detected in this case using Chl- and
activity towards TMB oxidation, preventing formation of the blue carbaryl-specific Abs. Further, while the employed nanozyme is
product. However, in the presence of OPP, the oxidation of TMB takes different in the two cases, both are POX-mimic. Under optimal condi­
place, such that the intensity of the blue signal is proportional to the OPP tions, this NzBS could achieve a LOD of 0.033 ng mL− 1 within 10 min for
concentration. The concept was demonstrated for omethoate and both pesticides and analyze them in lake water and traditional Chinese
dichlorvos, the two important OPPs with respective LODs of 10 and 3 ng medicine samples with recoveries ranging from 80 to 119% (Chl) and
mL− 1 achieved using this sensor. While the underlying principle of this 90–118% (carbaryl). Overall, the high sensitivity and selectivity of the
sensor does not allow differentiation between various OPPs, the sensor reported NzBS, as well as their ability to operate in a range of envi­
could detect OPPs with recoveries of 96.2–104.5% and 93.1–105.4% in ronmental samples, point towards their promising future for on-site
mouse serum and Chinese cabbage, respectively. environmental monitoring. However, it is also notable that the range
In another example of an AChE-based sensor for OPP detection, AchE of pollutants studied by NzBS is still very limited, with a key focus so far
was combined with the choline oxidase (ChO) enzyme to develop POX- on certain pesticides and heavy metal ions. The range of environmental
mimic copper phosphate hybrid nanoflowers (ACC-hNFs) via. protein- pollutants of high concern is rather large, including persistent aromatic
biomimetic mineralization [56] (Fig. 5B). In this case, in the absence hydrocarbons (PAHs), perfluoroalkyl and polyfluoroalkyl substances
of an OPP, AchE acts on its substrate (acetylcholine) to produce choline, (PFAS), and cocktails of chemical residues from industrial effluents that
on which ChO acts to produce H2O2, which can then oxidise TMB to a continue to bioaccumulate in our environment [120].
blue product due to the POX-mimic activity of hNFs. Further inclusion of As discussed above, the studies performed to date provide proof-of-
an acidic solution in this strategy converts this blue charge transfer concept validation for the high potential of NzBS in environmental
complex of TMB to a further oxidized yellow diimine product, which, detection, and the field is becoming ripe to implement this emerging
due to its higher extinction coefficient, offers better sensitivity for sensor technology to evaluate its performance for other environmental
colorimetric detection. However, in the presence of paraoxon, an OPP, analytes of high concern. We also noticed that studies in this area,
the tandem reaction was blocked due to the inhibition of AChE, and the mostly focus on spiked samples. This is a significant step forward from
colorimetric signal intensity was reduced. The authors combined this assessing the sensor performance in standard buffers and supports the
colorimetric assay with a screen-printed electrode (SPE) to enhance the robustness of the developed sensors. To advance the field, we recom­
sensitivity by achieving a dual mode of detection. This lab-on-paper mend future studies also consider studying samples collected from the
NzBS could achieve a remarkably lower LOD of 6 fg mL− 1 while main­ contaminated sites without spiking them and cross validating the sensor
taining excellent selectivity for paraoxon. The practical implication of performance with traditional analytical tools. Further, efforts are also
the proposed NzBS was validated by detecting paraoxon in real envi­ recommended to translate the proof-of-concept prototypes into com­
ronmental (e.g., tap and river water) and agricultural (e.g., rice and mercial platforms and technologies.
apple) samples with estimated recoveries ranging from 82.6 to 112.1%.
However, as in the previous study, the use of AChE does not allow this 5. Deployment of NzBS for health monitoring and disease
sensing strategy to be selective for a specific OPP. diagnosis
Another NzBS for OPP (chlorpyrifos – Chl) detection that addresses
the challenge of selectivity among different OPPs utilized an anti-Chl While biosensors have seen some progress towards their applicability
monoclonal antibody (anti-Chl-mAb) in combination with a POX- in food and environmental analysis, they have transformed the field of
mimic manganese dioxide nanoflowers (MnO2 NFs) in an LFD plat­ medical diagnostics. For instance, pregnancy detection strips, which are
form [119] (Fig. 5C). This strategy is similar to that illustrated in Fig. 4B, simple LFDs, have been in extensive consumer use for more than a

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A. Baranwal et al. Trends in Analytical Chemistry 172 (2024) 117573

decade. The recent emergence of COVID-19 also saw heavy reliance on individual with poor glycemic control. Thus, a sensor that can provide
LFD-based rapid antigen test (RAT) kits for the POC detection of even a step-resolution of 0.1 mM is good enough to detect glucose levels.
different strains of human coronaviruses (severe acute respiratory syn­ Similarly, the concentration of the human chorionic gonadotropin
drome coronavirus 2 – SARS-CoV-2) [121]. These LFDs not only rapidly (hCG), the pregnancy hormone, increases exponentially during the first
analyze target molecules in low sample volume with high precision but trimester of pregnancy (doubling every 48 h) with a typical concentra­
also enable the POC detection of important biomarkers in real time. In tion of 20–50 mIU mL− 1 in urine four weeks post-conception, while the
terms of volumes, electrochemical glucose monitoring strips and color­ basal concentration of hCG is less than 5 mIU mL− 1. With such high
imetric pregnancy strips are the most successful POC detection platforms concentrations and a large step-increase in hCG concentrations, it has
that have captured most of the biosensor market. In retrospect, these are become possible to estimate the status of pregnancies at least qualita­
rather easier targets to detect, as their relative concentrations in the tively at home using simple urine LFDs. Rapid technological advances in
respective biological fluids are rather high. For instance, the typical POC devices have now diverted the focus to detect other important
fasting blood glucose concentration in a healthy individual is around clinical analytes that are present in lower concentrations and are
5–6 mM [6,122], which can increase to over 10 mM in a diabetic therefore more challenging to detect in a POC manner. As reflected in

Fig. 6. Schematic illustration of paper-based devices incorporating nanozymes for the detection of clinically important analytes. (A) an advanced dipstick allowing
automated target pre-concentration and signal enhancement for the detection of E. coli O157:H7. Adapted with permission from Ref. [123]. © 2019, American
Chemical Society; (B) an advanced μPAD enabling on-device filtration of whole blood for direct detection of uric acid. Adapted with permission from Ref. [133]. ©
2020 Elsevier B. V.; and (C) an aptamer-based LFD for colorimetric detection of CA125, an ovarian cancer biomarker [146].

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A. Baranwal et al. Trends in Analytical Chemistry 172 (2024) 117573

previous sections on food and environmental analysis, nanozymes can instruments, and time-consuming protocols makes it inapt for rapid
assist in improving the sensitivity of POC detection technologies. In view screening and POC testing. Given this, the development of simple, rapid,
of this, the following section discusses the recent examples that show the reliable, and user-friendly test kits continues to be highly desirable for
integration of nanozymes in biosensor platforms for POC applications. A SARS-CoV-2 diagnosis. One such test kit was developed by incorporating
thorough review of the field suggests that the NzBS in the field of health POX-mimic Co–Fe@hemin in a CL-based LFD setup for the detection of
monitoring and human disease diagnostics have been more extensively SARS-CoV-2 spike antigen [129]. A standard sandwich LFD format as
explored than those in the field of food safety and environmental previously discussed in section 2.1.1 was employed, in which
monitoring. Some salient examples are discussed below, while an Co–Fe@hemin-labelled anti-spike antibodies were incorporated in the
extensive list is captured in Supplementary Table 3. conjugate pad as the signal probe. In the presence of SARS-CoV-2 spike
antigen, a dark brown color was observed both at the test and control
5.1. Pathogens lines due to the accumulation of signal probe. Although the color was
easily visible to the unaided eye at high spike antigen concentrations,
Infectious diseases are among the leading causes of mortality and the trace concentrations were not. This challenge was overcome by
morbidity in developing and underdeveloped countries. They are caused utilizing the POX-mimic activity of Co–Fe@hemin that promoted the
by pathogenic microorganisms such as bacteria, viruses, fungi, or par­ oxidation of a CL substrate luminol in the presence of H2O2 and NaOH.
asites. The mortality rates associated with infectious diseases could be This allowed quantitative detection of lower concentrations of spike
reduced to a great extent, provided they are detected in early stages as antigen with a calculated LOD of 0.1 ng mL− 1 and a linear detection
this will allow timely implementation of intervention strategies, better range of 0.2–100 ng mL− 1. While the developed test strip exhibited high
patient management, and efficient prevention of outbreaks. Though, sensitivity and specificity towards viral spike antigen in standard buffer,
currently available molecular diagnostic approaches are suitable for no such validation was made in real clinical samples making it difficult
sensitive and reliable detection; they are hardly suitable for POC ap­ to draw any conclusion on its potential in POC testing.
plications. Conversely, while POC devices such as LFDs offer low cost
and user-friendliness, these suffer from poor sensitivity. Biomarker 5.2. Small molecules
preconcentration and detection signal enhancement are commonly
adopted approaches to enhance their sensitivity. Albeit effective, these Several small molecules in our body act as important biomarkers of
approaches require multi-step processes, which can then compromise high clinical relevance [37]. These include sugars and their in­
their application for unskilled workers in POC settings, a key attractive termediates (e.g., glucose), natural waste products (e.g., uric acid),
feature of LFDs. Efforts have been made to overcome such challenges by neurotransmitters (e.g., norepinephrine), amino acids (e.g., gluta­
attaining both target analyte pre-concentration and signal enhancement thione), lipids (e.g., cholesterol), vitamins (e.g., ascorbic acid) and other
onto the paper device itself. In one such attempt, an advanced format of metabolites (e.g., lactate). Additionally, small molecules such as pesti­
a DIA was produced by incorporating a thick fiberglass paper (3D wick) cides and toxic metal species may also find their way into our bodies
as a replacement for the standard sample pad. This assisted in the pre- from inadvertent exposure [36]. Therefore, precise monitoring of small
concentration of E. coli O157:H7, facilitating its rapid and sensitive molecules in various body fluids is important for the effective diagnosis
detection [123] (Fig. 6A). The platform utilized Pt–Au NPs as and management of various diseases and disorders. In view of this, a
POX-mimic nanozyme, while standard sets of antibodies, as typically portable glucose sensor was developed by immobilizing hybrid conju­
used at test and control lines were used. The key distinguishing feature gate of modified graphitic carbon nitride (mGCN), chitin, and acetic acid
of this platform was the use of an aqueous two-phase solution containing (AcA) onto a filter paper with the help of poly(vinyl alcohol) hydrogel
poly(ethylene glycol-ran-propylene glycol) (EOPO) and sodium citrate. [130]. The hybrid material offered bifunctional nanozyme activity with
The EOPO-poor and EOPO-rich macroscopic phases acted as the leading mGCN mimicking the activity of glucose oxidase and chitin-AcA
and lagging phases when 3D wick was dipped in this solution containing mimicking POX activity. The working principle behind glucose sensing
the POX substrates (TMB + H2O2) and the target bacteria. The E. coli was based on mGCN-mediated oxidation of glucose to gluconic acid and
bacteria in the leading phase first interact with signal probes (Pt–Au H2O2, which was subsequently decomposed by chitin-AcA to mediate
NPs) at the conjugate pad and eventually bind with capture probes at the concurrent oxidation of the TMB substrate. The biosensor could detect
test line to form a sandwich immunocomplex. This results in red/pink glucose within the range of 25–1000 mg dL− 1 in standard buffers while
color at both the test and control lines in the presence of bacteria. displaying high specificity towards glucose. The potential practical
Subsequently, the lagging phase containing POX substrates reaches the applicability of this strategy was demonstrated by detecting glucose in
test and control lines, where the POX-mimic activity of Pt–Au NPs human serum and urine and comparing its performance against a
converts TMB into a deep blue product, thereby enhancing the visual commercial glucometer. In another recent study, a foldable μPAD
signal. This advanced dipstick showed a 30-fold enhancement over a incorporating POX-mimic single iron-site-containing poly-γ-glutamic
conventional LFD with a LOD of 3.3 × 104 CFU mL− 1 for E. coli O157:H7. acid/chitosan hydrogel (PGA-Fe/CS) was designed for colorimetric
This platform could also detect E. coli O157:H7 in SurineTM (a urine detection of glucose [131]. The μPAD consisted of two pads, where GOx
surrogate) samples within 30 min with a LOD of 105 CFU mL− 1. incorporating ‘pad A’ served as the sample loading pad and ‘pad B’
Apart from bacteria, several pathogenic viruses have been associated containing PGA-Fe/CS nanozymes and TMB substrate acted as the
with infectious diseases. One such pathogen is the SARS-CoV-2, which detection pad. Under optimized conditions, the detection of glucose was
caused the global pandemic of COVID-19. Considering the serious health mediated in two simple steps: (i) generation of H2O2 on ‘pad A’ by
concerns associated with SARS-CoV-2 infection, its rapid early-stage GOx-mediated glucose oxidation (pH 7) and (ii) utilization of generated
detection with high accuracy is extremely important [124]. However, H2O2 on ‘pad B’ upon folding by the POX-mimic nanozyme for TMB
SARS-CoV-2 infections are generally asymptomatic, especially at the oxidation (pH 3.5) to generate a blue product. The intensity of blue
early stages (incubation period) of infection; and if symptoms are pre­ product directly correlated with glucose concentration, thereby result­
sented, these are often non-specific, including fever, cough, sore throat, ing in a LOD of 15.4 μM and a linear detection range of 0.2–1.5 mM. By
headache, nausea, loss of taste/smell, shortness of breath, fatigue, employing the developed μPAD, authors could reliably detect glucose in
muscle/body aches, etc. making precise detection of SARS-CoV-2 chal­ spiked serum samples with estimated recoveries ranging from 102.5 to
lenging [125,126]. As seen in the case of other viral infections, reverse 108.3%. While the proposed platform could successfully mediate
transcription PCR (RT-PCR) has proven to be the gold standard tech­ glucose detection, its requirement for two different pH for optimal
nique in detecting SARS-CoV-2 infection [127,128]. But its requirement performance may pose challenges in remote POC applications.
for a designated biosafety laboratory, skilled personnel, expensive Uric acid (UA) is a small molecule that is produced by the

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A. Baranwal et al. Trends in Analytical Chemistry 172 (2024) 117573

mammalian liver as an end-product of purine metabolism. Abnormal UA mesoporous cerium oxide (Co-m-CeO2) as POX-mimic for the simulta­
concentrations can cause numerous health complications, including neous detection of glucose, galactose, cholesterol, choline, and acetyl­
hyperuricemia, gout, UA stones, chronic kidney disease, and cardio­ choline [141]. Five enzymes including GOx, galactose oxidase,
vascular disorders [132]. A portable μPAD was developed by utilizing cholesterol oxidase, ChO, and a mixture of AchE and ChO were indi­
POX-mimic mesoporous Prussian blue NPs for on-site detection of UA in vidually immobilized in Co-m-CeO2 pores to generate five different
whole blood samples (Fig. 6B) [133]. The sensing mechanism involved enzyme@Co-m-CeO2 composites. These composites along with the TMB
UA-mediated reduction of oxidized blue TMB to a colourless TMB. A key substrate were then individually integrated into six detection zones with
distinguishing feature of this μPAD design is that coating with high ionic the first being the control zone and the remaining five being the corre­
strength solutions of certain regions of this device provided spatial sponding target analyte zone. In the presence of target analytes,
control over the sample transport, which allowed on-device removal of immobilized enzymes catalyzed the oxidation of their respective targets
erythrocytes and haemachrome prior to analysis. The sensor could to produce H2O2, which subsequently activated the POX-mimic activity
detect UA in whole blood samples without any pre-treatment in the of Co-m-CeO2 to produce blue color by TMB substrate oxidation. Under
linear range of 1.5–8.5 mg dL− 1. This μPAD device showed comparable optimized conditions, the developed μPAD could rapidly (20 min) detect
performance to with BeneCheck-PLUSTM multi-monitoring system. glucose, galactose, cholesterol, choline, and acetylcholine simulta­
Owing to these unique features, this portable μPAD could offer good neously with respective LODs of 7, 20, 8, 7, and 11 μM. The practical
prospects as a POC device. application of the proposed multiplex sensor was further validated by
Norepinephrine (NE) is one of the crucial neurotransmitters in the analysing target analytes in spiked serum samples. The estimated re­
mammalian central nervous system that regulates a wide range of coveries ranging from 98.8 to 102.3% not only affirmed the reliability
physiological and homeostatic functions. Imbalance in the optimal and accuracy of the developed platform but also indicated its potential
concentration of NE has been associated with numerous clinical condi­ in multiplexed POC monitoring.
tions including Parkinson’s disease, ganglia neuroblastoma, depression, In line with the need for multiplex detection, another colorimetric
and attention deficit hyperactivity disorder [134]. To monitor NE, μPAD was developed by incorporating LAC-mimicking manganese
electrochemical and colorimetric biosensors were developed by utilizing dioxide–copper phosphate hybrid nanoflowers (Mn–Cu NFs) for simul­
OX-mimic polyacrylic acid-coated nanoceria (PNC) [135]. The colori­ taneous detection of phenolic neurotransmitters dopamine (DA) and
metric sensing was achieved on a μPAD through PNC-mediated oxida­ epinephrine (Ep) [142]. LAC belongs to the oxidoreductase family of
tion of TMB to a blue product which was reduced back to its colorless enzymes and catalyzes oxidation of aromatic compounds (largely phe­
form in the presence of NE. The sensor revealed a LOD of 863 nM and nols) and reduction of molecular oxygen to generate water. To enable
could operate in the linear range of 1–25 μM. To further improve the the detection of both neurotransmitters on a single device, authors
sensitivity of this colorimetric μPAD, an electrochemical system was divided the μPAD into two sections, each carrying three detection zones,
developed. By measuring the reduction current produced during the one semi-circular sample loading zone, and one control zone. The
electrochemical reduction of oxidized TMB, a LOD of 66 nM and a linear detection zones for DA were pre-immobilized with both 4-aminoantipyr­
range of 0.1–300 μM could be achieved. The electrochemical platform ine (4-AP) and Mn–Cu NFs nanozyme; however, only nanozymes were
also exhibited high selectivity towards NE in the presence of interfering incorporated in Ep detection zones. The sensing mechanism relied on the
ions and molecules, while showing estimated recoveries of 93.4–108% nanozyme-mediated oxidation of neurotransmitters in their respective
in human plasma. Notably, despite achieving better sensitivity over detection zones and generated distinct visual responses – violet for DA
colorimetric PAD, the electrochemical platform lacked a portable setup, and orange for Ep. The intensity of the visual signal positively correlated
which may make it incompatible with in-field applications. with neurotransmitter concentration thereby resulting in calculated
Glutathione (GSH), another important small molecule, protects cells LODs of 54 nM and 34.5 nM for DA and Ep, respectively and a linear
from oxidative stress by maintaining a redox balance both inside and detection range of 0.5–50 μM for both neurotransmitters. The practical
outside the cell. However, high GSH levels may also lead to enhanced application of the proposed device was further corroborated in two-fold
drug resistance against anticancer drugs, like temozolomide (TMZ) and diluted spiked human serum samples. Estimated recoveries ranging from
cisplatin by overcoming attacks from oxidative agents [136–138]. 97.2 to 103.9% and low coefficients of variation (2.7–4.8%) indicated
Therefore, among many applications, precise monitoring of intracellular that the developed μPAD could be potentially employed for POC
GSH concentration could help predict drug resistance and subsequently application.
improve the treatment efficiency. Consistent with this need, an
SPR-based μPAD was developed by integrating gold nanoclusters 5.3. Macromolecules
(AuNCs) as a POX-mimic and gold salt as the detection reagent [139].
The GSH sensing mechanism relied on the AuNCs’ ability to reduce gold Biomacromolecules such as proteins, glycoproteins, antibodies, en­
salt into SPR-active Au NPs. In the presence of GSH, distinct colored zymes, and nucleic acids, are among the most commonly studied bio­
patterns corresponding to varying concentrations of GSH were observed markers for a range of diseases and metabolic disorders [143].
on the μPAD. A linear detection range of 25–500 μM with the calculated Immunoassays, including enzyme-linked immunosorbent assays
LOD of 9.8 μM for GSH could be achieved. The ability of these μPADs to (ELISA), are among the most popular tools for detecting macromolecular
detect intracellular GSH levels in the TMZ-resistant glioblastoma mul­ biomarkers due to their high convenience and reasonable specificity.
tiforme (GBM) cells was demonstrated post cell-lysis. A positive corre­ Several other approaches like immunohistochemistry, PCR, SPR, and
lation between GSH level and TMZ resistance in GBM cells was noted SERS have also been used to monitor macromolecular biomarkers [144].
with estimated recoveries of 99.6–104.7%, suggesting the potential Most of these approaches lack portability and rely on technical exper­
applicability of this SPR-based μPAD in facilitating cancer precision tise. Therefore, the development of macromolecular diagnostic methods
therapy. that are rapid, inexpensive, sensitive, and can offer on-site detection has
While single biomarker detection is suitable for cases where the as­ become increasingly essential. A sandwich-format LFD was proposed for
sociation of a specific biomarker with a disease is well-established, the detection of tumour protein 53 (p53) while incorporating
complex diseases with heterogeneous etiologies require simultaneous POX-mimic mesoporous Pt–Pd NPs [145]. p53 is a regulatory protein
measurement of multiple heterogeneous factors with diverse molecular that prevents cells from proliferating in an uncontrolled manner, and
profiles [140]. Multiplex detection techniques can capture the dynamic therefore acts as a tumour suppressor. A standard LFD fabrication
and interconnected nature of biomolecular interactions by concurrently strategy as discussed in section 2.1.1 was followed, and the production
analyzing multiple biomarkers in a single sample. In one such example, a of a blue colour at the test and control line due to the POX-mediated
colorimetric μPAD was developed by integrating cobalt-doped oxidation of TMB allowed detection of p53. A portable strip reader

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A. Baranwal et al. Trends in Analytical Chemistry 172 (2024) 117573

was used to convert the intensity of the visual signal at the test line into (vii) nanomaterial functionalization strategies, (viii) the transducer
quantitative information, revealing a 0.1–10 ng mL− 1 linear range of platform, and (ix) the target analyte. Clearly, the advancements ach­
detection and LOD of 0.05 ng mL− 1. Analysis of clinical samples sug­ ieved in the field within a short span are exemplary; yet several chal­
gested that this device could analyze p53 concentration without sig­ lenges and obstacles remain unaddressed, hampering the exploitation of
nificant difference from commercial ELISA kit. the full potential of nanozymes in biosensors for real-world applications.
In another study, a competitive LFD was proposed by utilizing POX- Firstly, many of the nanozyme-based sensing concepts have
mimic AuNPs for the detection of an ovarian cancer biomarker, cancer remained within the confines of academic laboratories, as these
antigen 125 (CA125) (Fig. 6C) [146]. The LFD fabrication strategy was solution-based detection strategies have not necessarily been converted
largely similar to the competitive-format LFD discussed in section 2.1.1, into devices and technologies suitable for on-site operation. Considering
with a key difference being the use of a CA125-specific aptamer as a the lack of focus in this area, this review was drafted to capture the ef­
capture probe. Notably, all other examples illustrated in this review forts made towards the exploitation of nanozymes for developing on-site
have utilized target-specific antibodies as the capture probe, while only detection technologies and biosensor platforms. As reflected from this
a limited examples in the literature have utilized aptamers for this review, only a limited success has hitherto been achieved. Therefore, a
purpose on an LFD device. The POX substrate employed in this study was key question that must be asked is – what has stopped the conversion of
DAB, while most other studies employ TMB. This LFD could detect solution-based NzBS strategies into portable platforms that can be
CA125 antigen with a LOD of 5.21 U mL− 1 and a linear range of 7.5–200 deployed on-site in industries and clinics? We believe a major limiting
U mL− 1 within 20 min. The CA125 antigen could also be detected in factor is a knowledge gap that the nanozyme community involved in
human serum with results comparable to other established immunoas­ developing these biosensors faces. Most nanozyme researchers are ma­
says and commercial kits. terials and analytical chemists, who may have a limited understanding
In another interesting study, an LFD was developed that could detect of device fabrication strategies, which may be a rather simple task for
both macromolecule (prostate-specific antigen – PSA) and small mole­ engineering colleagues. Instead, the nanozyme biosensor community
cule (8-hydroxy-2′-deoxyguanosine – 8-OHdG) by employing a com­ has deep expertise in materials synthesis and bioconjugation chemistry,
mercial glucometer [147]. While the developed platform did not utilize forming the backbone of most advancements seen in the field to date.
a nanozyme activity for the signal enhancement, the strategy followed is Having felt that bridging this knowledge gap between the science and
worth briefly mentioning, as the developed LFD already utilized Au NPs, engineering disciplines is critical to advancing NzBS to the next level, in
which may (i) be potentially modified to act as a nanozyme and (ii) serve this review, we have attempted to provide easy-to-follow design prin­
as an interesting platform for the detection of multiple biomolecules. ciples for biosensor devices and platforms. We hope that the readers of
The LFD fabrication strategy was largely like the LFDs discussed in this review will be able to appreciate the basic components and design
earlier sections with key a difference coming from MRE and sensing aspects of paper-based devices, which will then create synergies to
formats: ‘sandwich’ for PSA and ‘competitive’ for 8-OHdG. enhance multidisciplinary and multi-sectoral collaborations.
While the growth of NzBS in the health sector is certainly in a better Furthermore, it is evident from this review that even though some of
place than food and environmental sector, most reported platforms are the NzBS have been transitioned from the solution phase to a device
still in the proof-of-concept stage and often detect single analytes. format, none of these have been translated into products and technol­
Reliable detection of complex diseases and disorders often requires in­ ogies. This could be attributed to both technical and commercial rea­
formation about simultaneous changes in the concentration of multiple sons. It is not the intent of this review to extensively discuss commercial
analytes. The need for multiplex analysis in clinical samples could see challenges associated with the translation of NzBS technologies, as these
the integration of advanced data analysis techniques to interpret com­ challenges are typically not unique to NzBS and are equally encountered
plex data and generate meaningful information. By leveraging the power by other biosensors and medical devices/technologies. Briefly, to ensure
of integrated data analysis coupled with machine learning techniques, the simplicity and affordability of these devices, various factors need to
researchers could extract valuable insights from complex datasets and be considered: (i) The choice of raw materials significantly impacts the
develop biosensors with improved selectivity and reliability [148,149]. overall cost of paper-based sensors. Opting for low-cost, readily avail­
Furthermore, machine learning algorithms could be employed to able materials without compromising performance is essential. Re­
enhance the adaptability and performance of paper-based biosensors, searchers have explored a variety of paper types, such as filter paper,
making them well-suited for a wide range of applications in diagnostics, cellulose-based materials, and even recycled paper, to reduce material
personalized medicine, and beyond. costs. Moreover, the selection of affordable conductive inks, polymers,
and recognition elements plays a pivotal role in achieving cost-
6. Challenges and future perspectives effectiveness, (ii) Complex designs often translate to increased
manufacturing costs. Keeping the sensor design simple and eliminating
Ever since the discovery of enzyme-mimicking properties of nano­ unnecessary components not only enhances the ease of fabrication but
materials, research in the field of nanozymes has garnered phenomenal also reduces the overall cost. Striking a balance between functionality
interest due to their excellent catalytic properties and higher stability and simplicity is crucial for achieving affordability, (iii) The fabrication
under harsh conditions compared to natural enzymes and other enzyme- process plays a crucial role in determining the overall cost of paper-
mimics. This has paved the way for significant advancements in tailoring based sensors. Utilizing simple and scalable techniques, such as screen
different nanomaterials as nanozymes and employing them for a range printing, inkjet printing, and wax printing, can substantially reduce
of biological applications. The merits of low cost, high stability, easy production costs. These methods not only require minimal equipment
manipulation, and scalable production have made nanozymes promising but also allow for large-scale production, making them economically
surrogates for natural enzymes in biosensor platforms. This has led to a viable for mass deployment., and (iv) Integration with commonly
burgeoning list of nanozyme-based sensing strategies that have been available technologies, such as smartphones or inexpensive handheld
explored for the detection of analytes relevant to food safety and quality, readers, can eliminate the need for specialized, costly equipment and
environmental pollution, and diseases and disorders. Rich diversity is subsequently reduce the cost of paper-based devices. While significant
offered by these NzBS platforms in the form of (i) the methodology of advances have been made in enhancing the affordability of paper-based
detection (e.g., sandwich and competitive formats), (ii) the readout sensors, challenges persist. Ensuring robustness, reproducibility, and
mode (single and dual), (iii) the device type (LFD, PADs, microPADs, sensitivity without compromising cost-effectiveness remains a balance
and dipsticks), (iv) different nanomaterials (metal, metal oxide, bime­ that biosensor researchers and industries must strike. Careful consider­
tallic, etc.), (v) the type of enzyme-mimic activity (peroxidase, oxidase, ation of these factors can pave the way for the widespread adoption of
etc.), (vi) device fabrication strategy (single, parallel, and two-way), paper-based NzBS, enabling their application in diverse fields and

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A. Baranwal et al. Trends in Analytical Chemistry 172 (2024) 117573

addressing pressing global challenges. reported in this paper.

Importantly, in the context of technical challenges, we would like to
highlight a few aspects that need concerted efforts – some related to Data availability
fundamental science aspects to be led by academic researchers and
others that are applied in nature, in collaboration with relevant in­ No data was used for the research described in the article.
dustries. The first of these relates to the catalytic efficiency of nano­
zymes, as higher catalytic activity is expected to result in improved Acknowledgements
sensitivity and faster sensor response. While studies have reported
nanozymes with catalytic efficiencies equivalent to their natural coun­ A.B. acknowledges the Australian government and RMIT University
terparts, most nanozymes show significantly inferior catalytic perfor­ for the RTP Stipend Scholarship towards a PhD degree. V.B. acknowl­
mance. Efforts are required to further enhance the catalytic activity of edges the Australian Research Council for funding support through an
nanozymes by deploying both experimental and computational material ARC-Discovery project (DP2301201650). Authors acknowledge the Ian
design principles. A thorough understanding of parameters that under­ Potter Foundation for establishing the Sir Ian Potter NanoBioSensing
pin the catalytic efficiencies of nanozymes will lead to rational design Facility at RMIT University which continues to enable us to develop
strategies for nanozymes suitable for the development of translatable next-generation detection technologies.
NzBS.
The next aspect relates to the performance of nanozymes in the Appendix A. Supplementary data
complex matrix in which the analyte is present. While a nanozyme may
show exceptional stability and activity under controlled conditions, Supplementary data to this article can be found online at https://doi.
these nanomaterials may undergo aggregation and decomposition in a org/10.1016/j.trac.2024.117573.
complex sample matrix, which can deteriorate their activity. Even if the
stability of the nanozyme is preserved in complex matrices, the com­
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