11 (2022) 100201

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Biosensors and Bioelectronics: X

journal homepage: www.journals.elsevier.com/biosensors-and-bioelectronics-x

Next generation biosensors employing molecularly imprinted polymers as

sensing elements for in vitro diagnostics
Soumya Rajpal , Prashant Mishra *
Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, New Delhi, 110016, India

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

Keywords: Biosensors for in vitro diagnostics (IVD) have huge potential and are likely to play a significant role in advanced
Molecularly imprinted polymers as well as in the low resource healthcare setups. With time, these devices are gaining traction in cardiac diag­
Biosensors nosis, cancer, autoimmune diseases and pathogenic microbial infections. Periodical advancements in nano­
In vitro diagnostics
materials employed in biosensors have led to miniaturization and improved characteristics of both plasmonic and
Microbial detection
Virus detection
electrical sensors. Mostly antibodies are employed in the development of IVD however, there are challenges due
Nanomaterials to the fragile nature of these biological molecules that adversely affects the storage and shelf life of biosensors.
Ongoing developments in the synthesis of artificial antibodies or MIPs that offer improved stability, selectivity
and multiplexing ability facilitates their application in biosensors for IVD. In addition, versatility and facile
synthesis protocols of MIPs enable their rapid integration into mass manufacturing systems. Here, we discuss the
changing dynamics of IVD enabled by MIP based biosensors. We will mainly focus on microbes and clinically
relevant biomarkers that have been targeted for infectious disease detection and different imprinting strategies
relevant to them. The biosensor platforms employing MIPs for their POC and IVD potential for biological samples
have been discussed. The commercial potential of MIPs which has translated into 8 commercial platforms so far
including IVD have been delineated. To improve the performance of MIPs in general, the computationally aided
MIP design that is actively focused to address some of the challenges associated with the translation of MIPs for
various applications including IVD have been discussed.

1. Introduction 2020; Singh et al., 2019). Instantaneous clinical translation of biosensors

In existence for the past six decades and with continuous research
and development thereon, molecularly imprinted polymers (MIPs)
showcase a remarkable presence in the field of separation science for
applications in food, biomedical and environmental analysis. The
biomedical field, in particular, is centered on accurate diagnostics that
further enables better therapeutics available to mankind. For various
diseases, IVD currently target the detection of different biomarkers
(qualitative and quantitative) by employing immunoassays like ELISA,
lateral flow assays and nucleic acid amplification tests such as RT-PCR.
These conventional yet standard methods require experienced personnel
for handling, sophisticated laboratory settings and lack point-of-care
(POC) applicability, especially needed in resource-limited areas. State-
of-the-art transducer platforms and novel nanomaterials have signifi­
cantly benefitted the biosensor research, especially in the domain of
bacterial and viral detection, with their increased sensitivity, short assay
times and/or high portability (Gahlaut et al., 2019, 2020; Moudgil et al.,
is however limited when the intrinsic biorecognition components are
mainly natural molecules like antibodies, enzymes and nucleic acids
(Díaz-Amaya et al., 2019; Guillem et al., 2021; Zhao et al., 2020b). As
natural biomolecules are essentially developed inside
temperature-controlled biological systems, their performance is subject
to experimental and storage conditions for stable sensing outside the
biological systems.
Synthetic recognition elements most relevant for biomedical appli­
cations have changed the dynamics of biosensor development, in vitro
assays, imaging and even so, therapeutics. In-vitro diagnostics (IVD)
developed using MIPs can benefit from the versatility, fast response,
accurate identification, ease of development and stability of the bio­
assays. Currently, biosensor research is actively focused on obtaining a
high-end performance and MIPs step in as excellent alternatives to
biomolecules like antibodies and aptamers with tailorable characteris­
tics for easy functionalization, broad temperature range compatibility,
storage and performance stability. MIPs have been able to establish

* Corresponding author.
E-mail address: pmishra@dbeb.iitd.ac.in (P. Mishra).

https://doi.org/10.1016/j.biosx.2022.100201
Received 31 May 2022; Received in revised form 10 July 2022; Accepted 11 July 2022
Available online 21 July 2022
2590-1370/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
S. Rajpal and P. Mishra Biosensors and Bioelectronics: X 11 (2022) 100201

Fig. 1. Schematics of biosensor development for various bio-analytes employing molecularly imprinted polymers (MIPs) as bio-recognition elements. For disease
detection, biomarkers including small molecules, toxins, proteins (surface or secreted), or even whole cells can be targeted. MIPs developed specific for whole cells
like bacteria are termed as cell imprinted polymers (CIPs) and those specific to viruses are called virus imprinted polymers (VIPs).

Fig. 2. Timeline showing different imprinting processes and strategies that have enabled the evolution of the currently used advanced “MIPs” (Below the line) (Chen
et al., 2016). Timeline also marks the commercial platforms that have emerged to market MIPs for applications in diagnostics and other applications (Above the line).

themselves as potential substitutes of antibodies in ELISA, biosensors
based on optical and electrochemical sensing and are even readily
adopted as standalone formulations for infectious disease detection.
Schematics of biosensor development for various bio-analytes employ­
ing molecularly imprinted polymers (MIPs) as bio-recognition elements
is depicted in Fig. 1.
Molecular imprinting is the phenomenon to create specific adsorp­
tion characteristics in a polymer matrix for any target molecule that can
range from a small molecules (Goyal et al., 2019; Korposh et al., 2014;
Rajpal et al., 2021; Wei et al., 2007), proteins (Bhakta et al., 2015;
Bonini et al., 2007; Sullivan et al., 2019; Sun et al., 2019a) and even
whole microorganisms like viruses and bacteria (Altintas et al., 2015;
Gast et al., 2019; Heidt et al., 2019; Schirhagl et al., 2012; Tai et al.,
2005). The synthesis of MIPs is homologous to antibody development
and involves appropriate monomers and cross-linkers with functional­
ities mimicking amino acids while incorporating recognition sites. The
fundamental process involves a target template dissolved in a suitable
solvent (porogen), combined with functional monomers in a so-called
‘pre-polymerization mixture’. Following this, compatible cross-linkers
and initiators are added to induce the polymerization reaction in the
mixture. The type of reactants employed can either enable non-covalent
bond formation like H- bonds, hydrophobic bonds, van der Waals in­
teractions or covalent bonds. Finally, a dissolving agent is added to
appropriately cleave the template that is intrinsically bound to the
polymer matrix. The cavities created post-removal resemble the shape
and size and most-importantly, the rebinding potential specifically to
the original template. This description represents the traditional syn­
thesis process known as bulk imprinting where the template is trapped in
the entire polymer and post-extraction the imprinted sites are present
throughout the polymer volume (for example: MIPs for 3-oxo-C12AHL
(Ma et al., 2018)). When the template is large (like proteins), the low
accessibility of the mild extraction agents deep within the polymer,
poses a challenge for its complete removal. Bulk polymerization consists
of several methods like precipitation polymerization where the
pre-polymerization mixture is soluble in the given solvent, but the
resulting polymer is not, and this leads to precipitation as soon as the
polymer is formed. Another approach called high dilution polymerization
employs a solvent which is compatible with the polymer. A very dilute
monomer solution is considered that allows highly hydrated micro- or
nanogel formation. The resulting MIP nanoparticles are analogous to the
natural antibodies with an average size of ~14 nm and molecular weight
of 100 kDa. Emulsion polymerization is also a very commonly known
method and generally takes the form of either oil-in-water or
water-in-oil phase system. The choice of hydrophilic or hydrophobic
monomers in accordance with the solvent is very critical. Moreover, the
complete removal of surfactants and stabilizers is problematic during
the washing steps.
Over the recent decades, the MIP synthesis process has evolved and
diversified to obtain improved performance and template-focused
polymer design (Fig. 2). For example, processes like surface imprinting
allows the imprinted sites to be uniquely confined on the surface
allowing easy extraction and rebinding. This allows reduced steric

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Fig. 3. (a) Schematic representation of three types of direct micro-contact imprinting (1) stamp imprinting, (2) Film imprinting and (3) Sacrificial layer method. (b)
Preparation of artificial stamps for indirect micro-contact imprinting. Adapted from (Jia et al., 2018), Copyright 2018, Elsevier.

hindrance and easy diffusion through the polymer material (Yilmaz
et al., 2000). An extension to this process is solid phase synthesis where
the template is immobilized on a surface and the monomers polymerize
around it. Post-imprinting, the nanogels are obtained in aqueous or
organic solvents depending on the polymerization agents used. Pilet­
sky’s group has optimized the protocol for template specific use (Can­
farotta et al., 2016). Here the template is immobilized on activated glass
beads using surface reactive groups, mostly amino and carboxyl groups.
The pre-polymerization mixture is composed of functional monomers
like N-isopropylacrylamide (NIPAM), N-tert butyl acrylamide (TBAM)
and acrylic acid (AA) keeping the total concentration less than 1%. The
crosslinker is N,N′ -methylene bis (acrylamide) (BIS) (2% mol) is used
during the polymerization. The washing step is crucially divided into
two steps that separates the low affinity polymers and unreacted
monomers from the high affinity MIPs using a temperature-based
elution. The temperatures can go up to 50–70 ◦ C to separate MIPs
thus rendering the process unfavorable for thermosensitive proteins.
Additional limitations leading to MIP heterogeneity are caused by the
non-oriented immobilization of the protein due to the conventional
amino and carboxyl-based surface chemistries employed. This has been
overcome with the use of affinity ligands, for example glass beads
functionalized with inhibitors specific to enzymes or metal-chelate

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Table 1 silico screening and simulated design (elaborated in later sections).

List of different clinically relevant biomarkers for which MIPs have been Consider the case of IVD for pathogen detection which usually target
developed to indicate the presence of specific diseases. a protein that is either secreted or expressed on the surface of the
Biomarker Disease membrane. For these biomarkers, an epitope or a distinctive sequence of
Cardiac troponin T (cTnT), Cardiac Cardiac disease
amino acids unique to the protein structure can be selected for tem­
troponin I (cTnI), Myoglobin, plating MIPs, hence corresponding to ‘antibody mimetics’. Epitopes are
Heart-fatty acid binding protein, realistically profiting as it is feasible to obtain synthetic peptides suffi­
Angiotensin (II) ciently than (rare) proteins and additional non-specific binding sites in
C-reactive protein (CRP), Sepsis
the polymer matrix caused by whole proteins is no longer a problem
Interleukin-1β (IL-1β), Interleukin
8 (IL-8), Lactate (Rachkov and Minoura, 2001).
Prostate Specific Antigen (PSA) Prostate cancer Cancer The versatility of MIPs comes from its ability to imprint molecules up
Biomarker to few angstroms to large cells up to microns. When the template is a
Pro-gastrin-releasing-peptide (Pro Small cell lung cancer bacterial cell, for example, there are two commonly used techniques
GRP), Neuron specific enolase biomarkers
(NSE)
employed namely cell mediated lithography and microcontact
Cancer antigen 15–3 (CA 15–3), Breast cancer stamping.
Human epidermal growth factor biomarkers Cell mediated lithography is done like emulsion polymerization
receptor – extracellular domain where a biphasic system involves cells arranged on the aqueous-organic
(HER2-ECD)
interfaces and imprinting is performed to generate polymer particles
Carcinoembryonic antigen (CEA) Colorectal cancer
biomarker with cavities complementary to the shape and size of the cell. Micro­
Alpha fetoprotein (AFP) Hepatic cancer contact stamping is analogous to soft lithography where the cells are
biomarker immobilized on surface and conformal stamps are created on a polymer
Cancer antigen-125 (CA-125) Ovarian cancer surface like PDMS. In this way, shape and size selective recognition sites
biomarker
are formed. Cells can be stamped in different forms like direct or indirect
Galectin-3, Hyaluronan-linked Miscellaneous cancer
protein 1, Kininogen fragments biomarkers microcontact imprinting or sacrificial layer method imprinting as sum­
(K-2209 & K-1944), Regenerating marized in schematic representation given in Fig. 3. However, the large
Protein 1 B, Vascular endothelial dimensions and enormous surface chemistry makes the imprinting
growth factor (VEGF) nEpidermal
process highly complex. Moreover, handling pathogens requires so­
growth factor receptor (EGFR)
Metallothionein phisticated laboratory conditions and poses a risk of infections. In the
Dopamine neurotrophic factor Neurological diseases biomarkers next sections, we discuss numerous examples of templating bio­
protein, β -Amyloid-42 molecules and cells relevant to pathogen detection for diverse biosensor
Transferrin (TrF) Plasma iron levels biomarker applications.
Cytochrome c, Lysozyme Metabolic disorders biomarkers
Oxytocin Autism Miscellaneous
β 2-microglobulin Many diseases biomarkers 2. MIPs developed for relevant biomarkers
Serum C-terminal telopeptide of Bone loss
type I collagen The U.S. FDA encourages the use of biomarkers in medical product
Pepsin Gastric reflux
design and suggests the success of personalized medicine be funda­
Non-structural protein 1, Heat- Dengue fever viral
denatured non-structural protein infection mentally dependent on biomarker diagnosis. From the perspective of
1 pathogen infections, biomarkers are biochemical indicators that are
Interleukin-2 Inflammations and either associated with the pathogen or are ‘host-derived’ that originate
cancers inside the human body in response to the infection (Mayeux, 2004).
Leukotriene-4 & insulin
Fibrinopeptide B Venous
The structural and chemical profile of a pathogen can range from
thromboembolism small molecules like signaling molecules and metabolites, proteins like
Ovalbumin Allergy surface proteins or secretary proteins and/other membrane components.
Cystatin C Chronic renal diseases A classic example of a molecular marker is N-acyl homoserine lactones
Chymotrypsinogen Acute kidney failure
(AHL) specific to Gram-negative bacteria. AHLs are quorum sensing
2-Aminoadipic Acid Diabetes
molecule that characterize the virulence and biofilm forming status of
the bacteria. Jiang and coworkers have employed magnetic molecularly
complex to bind His tagged proteins (Dinc et al., 2016; Mourão et al., imprinted polymers (MMIPs) prepared using methacrylic acid (MAA) as
2017; Xu et al., 2016, 2017). The approach is commonly employed by monomer, to capture and detect AHLs (Jiang et al., 2016). The study
Haupt’s group who further improved the selection of monomers like reports a unique method of dummy imprinting by using a rationally
4-acrylamidophenyl(amino)-methaniminium acetate (AB) or 2-(tri­ designed analogue template 2,5-dimethyl-4-hydroxy-3(2H)-furanone
fluoromethyl)acrylic acid (TFMAA), N-phenylacrylamide (PAA) in (DMHF) for templating MIPs. As the name suggests, dummy imprinting
accordance with the protein composition dominated by acidic, basic or employs templates that are similar in size, shape and functionality to the
benzenic groups, respectively. target molecule and are useful alternatives especially when obtaining
It is pertinent to note that inherent compositional and structural the original template is costly. As an integral biofilm component, the
diversity of proteins comprising monomeric units in the form of 20 capture of AHLs using MIPs have shown the attenuation of biofilm in
amino acids in their sequence is acknowledged during antibody devel­ another report (Piletska et al., 2011). 3-oxo-C12-AHL sequestering MIPs
opment and MIP design should similarly observe a larger library of specific to P. aeruginosa biofilms were prepared with itaconic acid
functional monomers to effectively incorporate equivalent specificity. monomer and resulted in ~80% reduction of biofilm components
The process looks straightforward when the template is a small mole­ including bacterial cells. This demonstrates additional application of
cule, however, the protein-macromolecular interactions are highly MIPs in coatings for medical devices.
ubiquitous in nature. With the increase in template size and complexity, Cell membrane components like lipopolysaccharides (LPS) with its
the imprinting process requires a more stringent design. The MIP specificity to Gram-negative bacterial cell wall can also be a direct in­
research community has recently adopted an epitope imprinting process dicator of disease severity (Shelburne et al., 1993). LPS as a template
biomimicking the natural antibody selection that is further guided by in was used to imprint nanoparticles for specific detection of P. aeruginosa
(Long et al., 2016). The amphiphilic nature of LPS was employed in

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Fig. 4. Examples of pseudo-assays and biomimetic strategies employing MIPs for analysis. Reprinted and modified with permission from (Chen et al., 2018).
Copyright 2018, American Chemical Society.

Fig. 5. COVID-19 diagnostics principle by nconNP based MIP sensor analyzing the samples prepared from nasopharyngeal swab specimens of patients. Reprinted
with permission from (Raziq et al., 2021), Copyright 2021, Elsevier.

inverse emulsion polymerization resulting in imprinted cavities specific
for the hydrophilic domain of LPS. The bacterial capture could be done
in vivo shown by specific accumulation of nanoparticles at the infection
site.
Microbial toxins also characterize as crucial biomarkers and while
they significantly damage the host tissue, the concentration is predictive
of the degree and stage of infection. A recent study reported the detec­
tion and selective capture of pyocyanin toxin specifically secreted by
P. aeruginosa (Rajpal et al., 2021). The unique polymeric material is
biocompatible with reference to its performance in physiologically

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S. Rajpal and P. Mishra
Fig. 6. Types and examples of commonly used label-free sensors used in MIP research employing different methods like physical deposition, covalent attachment,
and in-situ polymerization for MIP integration into sensors. Reprinted with permission of (Ayankojo et al., 2022), Copyright 2022, MDPI.
relevant biological media and is developed in a core-shell type format of
magnetic MIPs that allows selective capture in a short time. These
standalone MIPs as precision biomarker tests for pyocyanin can be
directly applied into IVD thereby, improving the clinical decision
making.
Toxins can also be proteinaceous in nature and can occur in the
secreted form or integrated with pathogen structure. A typical example
is S. aureus specific enterotoxin called Protein A that exists as a mem­
brane component but is also secreted in the exponential phase of the
bacterial cycle. Therefore, its detection can mark the active phase of
infection and has been targeted to develop MIPs for clinical detection of
S. aureus (Khan et al., 2016). The protein was imprinted using
electro-polymerization employing 3-aminophenol as the monomer and
the detection was done using cyclic voltammetry. This simple fabrica­
tion with MIPs allowed to achieve a detection limit of ~17 nM with high
specificity compared to similar proteins.
Similarly for viral detection, surface proteins correlate with the viral
load. In one example, a viral hexon protein - part of the human adeno­
virus type 5 (hAdV5) icosahedral capsid has been imprinted to develop
selective MIPs polymerized using silane-based monomers like TEOS,
APTES and APTMS (Gast et al., 2019). This study uniquely circumvents
the handling risk of pathogens by using surface protein components
instead of whole viruses and serves as a quasi-epitope imprinting strat­
egy that in turn, improves the MIP performance.
In addition to pathogen derived biomarkers, infection is also indi­
cated by the human body through expression of different proteins. For
example, in case of dengue infection, the virus infected cells display NS1
protein on the surface. This protein was considered as a target template
and a 15-mer peptide sequence was employed in an epitope imprinting
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Biosensors and Bioelectronics: X 11 (2022) 100201
strategy with acrylamide monomers to develop MIPs functionalized
QCM sensors (Tai et al., 2005). The system is ideal as an in vitro cellular
assay for quantitatively recognizing proteins. The comparable perfor­
mance of the MIPs with monoclonal antibodies shown here presents a
tremendous potential for clinical translation of such MIP sensors.
One very regularly assayed inflammatory marker in IVD is C-reactive
protein (CRP), whose levels differentially mark bacterial and viral in­
fections (Sasaki et al., 2002). Therefore, Kim and coworkers imprinted
CRP and the imprinted films show a comparable performance to
anti-CRP antibodies (Kim et al., 2011). Biomarkers hold a lot of signif­
icance in estimating various diseased conditions, surely not limited to
sepsis. Currently, more than 100 publications highlight MIPs developed
for relevant biomarkers for diseases like cancer, cardiac diseases etc
listed in Table 1 and have been summarized from the extensive review
by Crapnell et al. (2020) and Mostafa et al. (2021).
3. MIPs for IVD assays development
3.1. MIPs based biosensors
The design of IVD require consideration of some important factors to
enable high diagnostic potential including high signal to noise ratio,
high sensitivity for low abundance biomarker analysis (useful for early-
stage diagnosis), miniaturized setup to require (a) little instrumentation
and lab-space (b) low sample volumes; straightforward testing with
minimal sample processing required, minimal susceptibility to sample
matrix interference.
The integration of artificial biorecognition elements or MIPs into
biosensor readout platforms have enabled their commercial viability.
S. Rajpal and P. Mishra Biosensors and Bioelectronics: X 11 (2022) 100201

Table 2 polyvinylpyrrolidone (PVP), dimethylaminoethyl methacrylate

Different types of biosensors employing MIPs for infectious disease detection in (DMAEMA) and polyamine combined with common cross-linkers like
real biological samples that indicates their in-vitro diagnostic (IVD) potential. TRIM, EGDMA and divinylbenzene (DVB) (Crapnell et al., 2019). These
Target analyte/ Sensor type LOD Detection Reference sensors commonly employ carbon nanotubes, conductive polymers like
microbe in polypyrrole and metallic nanoparticles. Imprinted carbon nano-tube tips
biological with human papillomavirus derived E7 protein were employed to
sample
demonstrate ~10 pg per liter sensitivity using electrochemical imped­
Peptide epitope QCM 1.39 ng/ Human Gupta et al. ance spectroscopy (Cai et al., 2010). The ultrasensitive detection was
specific to mL blood (2018)
obtained with an architectural improvement where the protein was
Neisseria samples
meningitidis electropolymerized into a non-conducting polymer nanocoating based
Peptide epitope QCM 0.161 Human Kushwaha on polyphenol. In this case, the high-signal-to-noise recordings enabled
specific to nM blood et al. (2019) by the uniquely imprinted sensor and selectivity compared to similar
Mycobacterium samples proteins (E6) proves itself for high clinical utility.
leprae
Escherichia coli HTM 2 × 104 Urine Heidt et al.
During the current pandemic, MIPs were used to develop a portable
CFU/mL (2019) diagnostic platform based on electrochemical sensing for the detection
Escherichia coli HTM 1.01 × Urine Steen of SARS-CoV-2 nucleoprotein (ncovNP) (Raziq et al., 2021). This plat­
104 Redeker form demonstrated fM level sensitivity and robust performance in the
CFU/mL et al.
clinical samples of the nasopharynx swabs of patients (Fig. 5).
(2017b)
Escherichia coli SPR 0.57 Urine Özgür et al. Proof-of-concept presented in such studies validates the commercial
CFU/mL (2020) potential of MIPs.
Acinetobacter Electrochemical 30 CFU/ Human Roushani
baumannii mL blood et al. (2020) 3.1.2. Piezoelectric sensors
samples
IVD are benefited with sensors offering less detection time. Gram-
Peptide epitope of QCM 0.50 Human Harijan
Salmonella typhi (ng/ml) blood et al. (2022) negative bacteria imprinted polypyrrole (PPy) based QCM sensor has
invasive protein samples demonstrated detection within 3 min (Tokonami et al., 2014). This was
D (SipD) uniquely combined with dielectrophoresis to concentrate the bacteria in
Hepatitis B surface SPR 208.22 Human Uzun et al.
the surface vicinity of the chip. PPy can allow excellent memory of the
antibody mIU/mL serum (2009)
(HBsAb) target analyte which has been exploited to generate specific bacterial
Hepatitis A virus Fluorescence 8.6 pM Human Yang et al. memory that do not bind with non-selective similar shaped bacteria like
serum (2017) A. calcoaceticus, E. coli, and S. marcescens.
Hepatitis A virus Resonance light 0.1 Diluted Luo et al.
scattering (RLS) pmol/L human (2020)
3.1.3. Optical sensors
serum
Japanese Fluorescence 0.32 nM Diluted He et al. Optical sensors most popularly employ surface plasmon resonance
encephalitis human (2016) for the signal readout. The analyte binding on the imprinted layer in­
virus serum duces a resonance shift caused by electron density changes at the surface
Japanese Fluorescence 9.6 pM Diluted Liang et al.
of the chip. A highly sensitive imprinted sensor has been reported for the
encephalitis human (2016)
virus serum
detection of secreted bacterial factor (RoxP) (Ertürk et al., 2018). The
Peptide epitope of QCM ~ μM Human Tai et al. amount of this protein biomarker directly correlates to different skin
Dengue virus serum (2006) pathologies. They used microcontact printing to imprint tyramine layers
protein on gold electrodes to develop the SPR-MIP sensor. Proof-of-concept
nonstructural
studies on skin swaps were at par with the state-of-the art ELISA as­
protein (NS1)
HIV p24 protein Electrochemical 0.083 Human Ma et al. says and detection were achieved in sub nanomolar range with a high
pg/mL serum (2017) affinity for RoxP (Kd ~3.3 nM).
SARS-CoV-2 whole Electrochemical 4.9 Saliva (el Sharif Real time microbial identification has also been established
virus log10 et al., 2022)
employing similar microcontact imprinting technology. Both SPR and
pfu/mL
QCM based sensors were used to imprint E. coli using a synthetic amino
acid, N-methacryloyl l-histidine methylester (MAH) monomers that
Further improvement in MIP synthesis guided with computational form similar recognition as offered by natural antibodies yet are
design and miniaturization of transducer platforms has accelerated the chemically and physically stable (Yilmaz et al., 2015). These most
development of advanced MIP sensors that are comparatively robust, commonly used sensor types are summarized in Fig. 6.
stable and present a large-scale manufacturing potential. After estab­
lishing their potential with electrochemical sensors, optical readout 3.1.4. Thermal readout methods
methods (like surface plasmon resonance (SPR) based), piezoelectric MIP sensors based on heat-transfer methods (HTM) have advantages
(like quartz crystal microbalance (QCM)) and thermal methods, they over the other methods by virtue of relative simplicity, versatility and
have been further tested with other commercially interesting platforms straightforward data interpretation with no requirement of sensing
like microfluidic based analytical systems, lateral flow lab-on-chip as­ hardware (Dar et al., 2020; van Grinsven et al., 2014; 2016). Addi­
says etc. We discuss some of the milestones achieved in the field of tionally, they allow easy miniaturization that makes IVD applicable for
pathogen diagnostics and explore their potential for POC applications. POC applications. Grinsven et al., developed HTM based sensor platform
Some examples of pseudo-assays and biomimetic strategies employing with bacterial imprinted polyurethane-coated aluminum chips (van
MIPs for analysis are illustrated in Fig. 4. Grinsven et al., 2016). The respective chips imprinted with E. coli and
S. aureus distinguished the Gram-negative from Gram-positive bacteria
3.1.1. Electrochemical sensors and also differentiated live from dead cells. A follow up-study estab­
The surface imprinted polymers (SIPs) for electrochemical bio­ lished the excellent IVD potential of the sensor for urinary tract infection
sensors are generally prepared using free-radical polymerization and/ (UTI) detection (Heidt et al., 2019). Urine samples spiked with bacteria
sol gel process. Some electropolymerizable monomers include MAA, were assayed to obtain the dynamic range of detection of 104–105
bacteria per mL which related to the cut-off value for UTI diagnosis.

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Fig. 7. Schematic illustration of the proposed bio­

mimetic immuno-like membranes with aligned mo­
lecular nanocavities for point-of-care protein
biosensing. (a) Immuno-like membrane in micro­
fluidic biochips, (b) separation and sensing proced­
ures, (b-1) loading of human serum samples into the
microfluidic biochips and capturing CRPs from
human serum samples by the immuno-like mem­
brane, (b-2) loading of SDS and releasing of CRPs
from the immuno-like membrane, and (b-3) delivery
of SDS with CRPs to the electrodes for electronic
sensing. Reprinted with permission from (Hong et al.,
2013), Copyright 2013, Elsevier.

Table 3
Different types of nanoparticles used as core-shell format with MIP surfaces to target infectious disease detection.
Type of Advantages Disadvantages Application
nanoparticle

Quantum Dots Non-destructive operation mode, high signal output, Heavy metal content used CIPs for Listeria monocytogenes (Zhao et al., 2019), MIPs for
tunable spectral properties, photostable, chemically inert, during fabrication (example: AHL detection (Cui et al., 2020), CIPs for Escherichia coli
low cytotoxicity CdSe) can be toxic O157:H7 (Chen et al., 2017), Hepatitis A virus and Hepatitis B
virus (Luo et al., 2019c)
Gold nanoparticles Enhanced plasmonic characteristics, high electronic Toxic for in vivo applications MIPs for E. coli detection (Gür et al., 2019; Shan et al., 2018),
(AuNPs) conductivity, thiol chemistry for molecular binding DPA imprinted AuNPs for B. cereus spores (Gültekin et al.,
2009), Hydrogel based MIPs for capture of Influenza A virus (
Randriantsilefisoa et al., 2020)
Silver Enhanced plasmonic characteristics, low cost, high Toxic for in vivo applications AgNPs embedded MIPs with antibacterial activity (Gong
nanoparticles electronic conductivity et al., 2021)
(AgNPs)
Carbon Good electrical, mechanical, optical properties. Mostly Low water solubility Multi-walled carbon nanotubes (MWCNT) combining MIPs
nanomaterials used for fabricating electrochemical sensors and aptamers for Hepatitis C virus antigen (Ghanbari and
(CNMs) Roushani, 2018), MWCNT for HIV-p24 detection (Ma et al.,
2017)
Magnetic Easy isolation and concentration, good electric High aggregation and MIPs capturing pyocyanin for P.aeruginosa detection (Rajpal
nanoparticles conductivity, facile fabrication, high surface area polydispersity, low stability in et al., 2021), MIPs for SARS-CoV-2 specific peptide detection
(MNPs) long term (Fresco-Cala et al., 2021), VIPs for Japanese encephalitis
virus (He et al., 2016; Luo et al., 2019b), MMIPs for AHL
detection (Jiang et al., 2016), CIPs for S. aureus detection (
Bezdekova et al., 2020)
Silica Chemically stable, biocompatible, tunable pore size and Slight aggregation and MIPs for dipicolinic acid (DPA) to detect bacterial sporulation
nanoparticles well defined-structural characteristics, surface reactivity polydispersity events (Smith et al., 2011), MIPs with hybrid core of
(SiNPs) and easy of functionalization, biodegradability, water CdTe/CdS quantum dot (QD)-based silica nanoparticles for
soluble hepatitis A virus (HAV) detection (Luo et al., 2019a), MIPs for
viral hexon protein specific to human Adenovirus type 5
(hAdV5) (Gast et al., 2019), MIPs for SARS-CoV-2 specific
peptide detection (Batista et al., 2022)

HTM methods can however be limited by the intrinsic noise of the (Cornelis et al., 2019; Steen Redeker et al., 2017a). Cornelis and co­
thermal resistance signal, therefore increasing the response time may workers could achieve an LOD of 100 CFU/mL even in complex samples
affect the sensitivity of the system. Various improvements to the sensing by employing a planar meander element as a replacement of the basic
platforms have been introduced to enhance the sensitivity factor heating unit (Cornelis et al., 2019).

8
S. Rajpal and P. Mishra
Fig. 8. “Antibody” Mimics (S-Ab) as Bioorthorgonal Catalysts Shape-Selectively Recognized the Template Bacteria (S. aureus) Based on Shape Memory Ability and
Catalyzed the Production of Active Antibacterial Molecules. Reprinted with permission from (Niu et al., 2021) Copyright 2021, American Chemical Society.
More examples employing the electrochemical, piezoelectric, optical
and thermal readout methods with application in real biological samples
that indicates their in-vitro diagnostic (IVD) potential are summarised in
Table 2.
3.1.5. Microfluidics
POC applications can be enabled by the unification of MIPs with
microfluidics systems, the latter having tremendous advantages to offer,
namely minimized reagent and sample consumption, multiplexing
technology, miniaturization and automation. In a typical example, CRP-
imprinted nanocavities were integrated with microfluidics system for
POC applications (Hong et al., 2013). These imprinted ‘immuno-like
membranes’ showed similar performance to antibody-CRP interactions
with easy and rapid detection (~110 s) in complex biofluids like serum
(Fig. 7).
The benefits of MIPs and microfluidics are currently being combined
with paper-based sensing to enable pump-free sample transportation
which would reduce the cost of the sensor to achieve affordable di­
agnostics. Sun and coworkers published about ultrasensitive detection of
glycoproteins which are important clinical biomarkers for human dis­
eases (Sun et al., 2019b). They developed a boronate affinity sandwich
assay by growing gold nanorods on paper cellulosic fiber matrices and
combined it with signal tags to use for electrochemical assays. These
could detect the target ovalbumin (OVA) glycoprotein in a linear range
of 1 pg/mL to 1000 ng/mL with an LOD of 0.87 pg/mL.
3.2. Standalone MIP assays based on nanomaterials
Similar to antibodies, MIPs can be manipulated and modified to
exhibit unique characteristics to mark the binding events. For example,
fluorescent tagging can be integrated with polymers using fluorescent
monomers or using nanoparticle cores such as quantum dots (QDs) that
can offer detection at extremely low analyte concentrations. Similarly,
chromophores/dyes can be used for ease of data reading as in lateral
flow rapid test strips that directly translates to POC applications. Other
nanomaterials in accordance to the specific application have been
incorporated with MIPs like gold nanoparticles (AuNPs), silver nano­
particles (AgNPs), carbon nanomaterials (CNMs), Magnetic nano­
particles (MNPs), silica nanoparticles (SiNPs) and titanium dioxide
nanomaterials (TNMs) summarized along with their advantages and
disadvantages in Table 3.
The most common format in which nanomaterial-MIP conjugates is
prepared is the core-shell morphology where some carrier nanoparticle
with a MIP envelope contains the bound template. After polymerization
is complete and the template is extracted via elution, the analyte specific
cavities are generated. These nanocomposites can also be applied to
9
Biosensors and Bioelectronics: X 11 (2022) 100201
sandwich immunoassays for IVD applications. Other strategies deposit
MIP nanomaterials onto cantilever or electrode substrates to enable
selective analyte binding. Conductive nanomaterials combined with
MIPs have become a popular choice to synergistically improve biosensor
efficacy by reducing the latter’s insulating nature and mitigating the
former’s inherent dispersal and toxicity challenges (Denmark et al.,
2020).
4. MIP potential for in vivo applications: Imaging and
theranostics
MIPs showcase enormous potential for application in in vitro and in
vivo bioimaging and theranostics. It has a well-established portfolio in
the field of cancer research (Bhakta and Mishra, 2021; Haupt et al.,
2020). For in vivo applications, MIPs are designed to possess a high
imprinting factor to present higher selectivity. This is important as the in
vivo systems contain a highly complex proteome and a plethora of small
molecules and metabolites in circulation, that means, substantially
increased amount of interference. Therefore, NIP is expected to have
negligible binding. The dissociation constant (KD) in a nanomolar range
which is a representative of higher binding affinity is another important
factor and must be tested in both buffer and physiological relevant
media. The size of the MIP nanoparticles should be preferably below
100 nm and the polymeric material must go through biocompatibility
studies like testing in cell cultures, serum and blood.
The biotargeting and bioimaging potential of MIPs has been
demonstrated for a number of cancer biomarkers like hyaluronic acid
(Kunath et al., 2015), monosaccharides like sialic acid, fucose, mannose
etc. A targeted drug delivery of doxorubicin to breast cancer cell lines
with epidermal growth factor receptor (EGFR) using MIP-NPs reported
by Canfarotta et al., is proof-of-concept for the theranostic potential of
MIPs (Canfarotta et al., 2018).
This concept has been extended to viral and bacterial infections also.
Recently, bacteria imprinted bio-orthogonal catalysts for anti-bacterial
drug action against E. coli and S. aureus has been reported (Niu et al.,
2021) (Fig. 8). This is an example of targeted drug delivery based on
copper catalysis and the polymer-based bio-othogonal nanocatalysts
(E-Ab and S-Ab) resemble the shape and size of the two bacteria
respectively. The in situ and in vivo studies show the copper containing
MIPs attach on the specific bacterial surface that mediates the drug
activation and specific inhibition at the target site. In another example
dealing with drug resistance in bacteria, thermosensitive hydrogel
imprinted with β-lactamase(a bacterial enzyme) was used to retain the
efficacy of the antibiotic drugs (Li et al., 2016). The biocompatible
hydrogel was also tested for its action in vivo on wounds infected with
methicillin-resistant Staphylococcus aureus (MRSA) bacteria.
S. Rajpal and P. Mishra
Table 4
Type of monomers that can be employed for development of multi-functional
and/or stimuli responsive MIPs (Chen et al., 2015, 2016; Sajini and Mathew,
2021).
Monomer
•N-isopropylacrylamide (NIPAAm)
•2-acrylamide-2-methyl propane sulfonic acid
(AMPS)
•N-tert-butyl acrylamide (TBAm)
•Butyl methacrylate
Azobenzene based monomers:
•4-[(4-methacryloyloxy) phenylazo] benzoic
acid (MPABA)
•4-((4-methacryloyloxy)-phenylazo) pyridine
•4-[(4-methacryloyloxy) phenylazo]
benzenesulfonic acid (MAPASA)
•p-phenylazoacrylanilide (PhAAAn)
•4-{4-[2,6-bis(n-butyl-amino) pyridine-4-yl]-
phenylazo}-phenyl methacrylate (FM)
• 5-[(4-(methacryloyloxy) phenyl) diazenyl]
isophthalic acid (MAPDIA)
•2(S)-(4-isobutylphenyl) propyloxy-40-
[(triisopro-poxysilyl) propyloxy] azobenzene
(BPPO-AZO-TPPSP)
•(4-methacryloyloxy) nonafluoroazobenzene
(MANFAB)
•Spiropyran methacrylate (SPMA)
•1,8-Naphthalimide dye
•Dansyl-modified β-cyclodextrin
•4-Methylamino-N-allylnaphthal-imide (4-
MAANI)
•Vinyl-substituted zinc (ii) protoporphyrin
(ZnPP)
•Dansyl methacrylate
•(2-Hydroxyethyl anthrancene-9-carboxylate)
methacrylate (AnHEMA)
•4-Dimethylamino-N-allylnaphthalimide (F1);
4-Piperazinyl-N-allylnaphthalile (F2)
•Nitrobenzoxadiazole (NBD)
•Methacrylic acid (MAA)
•Acrylic acid (AA)
•2-(Diethylamino) ethyl methacrylate
(DEAEMA)
•2-(Dimethylamino) ethyl methacrylate
(DMAEMA)
•Hydroxyethyl methacrylate (HEMA)
•4-Vinyl pyridine (4-VPY)
•4-Vinylphenylboronic acid (p-VPBA)
•1-Phenyl-3-methyl-4-methacryloyl-5-
pyrazolone (PMMP)
•Dexamethasone-21 phosphate disodium (DXP)
•N-(3-(dimethylamino)propyl] methacrylamide
(DMAPMA)
•Phenyltriethoxysilane (PTES)
•Diisobutyldimethoxysilane (DIDMS)
•Methyltrimethoxysilane (MTMS)
•Isobutyltriethoxysilane (IBTES)
•Ureidopropyltrimethoxysilane (UPTMS)
•Trimethoxy (octyl) silane (TMOS)
•(2-Cyanoethyl) triethoxysilane (CETES)
•Tetraethoxysilane (TEOS)
•3-Aminopropyltrimethoxysilane (APTMS)
•3-Aminopropyltriethoxysilane (APTES)
•3-Mercaptopropyltrimethoxysilane(MPTMS)
•Methylvinyldiethoxysilane
•3-Methylacryloxyprolyl trimethoxy silane
•Glycidoxypropyltrimethoxysilane
•Dopamine
•Aniline
•Anthranilic acid
•Itaconic acid
•Lactic acid (lactide)
•Glycolic acid (glycolide)
•4-(Aminomethyl) benzoic acid (MABA)
•p-Aminobenzamidine (PAB)
Function
Thermo-responsive
Photo-responsive (Stimuli based
switching between trans and cis
isomeric forms)
Photo-responsive (Stimuli based
changes in ring structure (open
and closed)
Fluorescence based
pH responsive
Salt-responsive
Silica based monomers (sol gel
process) (Batista et al., 2022;
Bhakta et al., 2015)
Bio-derived similar monomers (
Gagliardi et al., 2017; Gu et al.,
2013; M. Zhao et al., 2020a)
10
Biosensors and Bioelectronics: X 11 (2022) 100201
Table 4 (continued )
Monomer Function
•N-(3-aminopropyl) methacrylamide Others commonly used functional
•N-hydroxy methylacrylamide monomers(Fresco-Cala et al.,
•Hydroxyethyl acrylate 2021; Khan et al., 2012)
•Glycidyl methacrylate
•Methyl methacrylate
•Aminophenyl boronic acid (APBA)
•2-Vinyl pyridine
•Allylamine
•1-Vinylimidazole
•Styrene
•4-Vinyl benzoic acid
•Pyrrole
•o-Phenylenediamine
•N-phenylacrylamide (PAM)
•Butyl acrylate
•Tertbutyl acrylate
•Trifluoromethyl acrylic acid
•Acrylamide
•Methacrylamide
•N-methacryloyl-l-alanine methyl ester (MA-L- Ji et al. (2016)
Ala-OMe)
N-isopropylacrylamide (NIPAAm) was used as the monomer to develop
a temperature responsive polymer. This served a second role as the
smart polymer could be reversibly activated by a temperature stimulus
to release the β-lactamase that can later degrade the residual antibiotics.
Similarly, MIPs have been developed as antivirals with therapeutic
potential. In a first-of-its-kind report of MIPs as anti-viral antibody
mimics synthesized using mini-emulsion polymerization against the
enteric phages of the Leviviridae family, they demonstrate excellent
reduction of viral infection in in situ plaque assays (Sankarakumar and
Tong, 2013) The whole virus imprinted particles have shown a rapid
phage titer reduction kinetics, reaching an equilibrium after only 3 h of
contact time. However, it was noted that the rapid mutability of viruses
sometimes renders the epitope imprinted polymers non-effective and
therefore, whole virus imprints perform better as antiviral agents.
The versatile and smart-polymer design, biocompatibility and easy
fabrication enables the economical scale up of MIPs for pharmaceutical
use. These examples of anti-bacterial and anti-viral action MIPs are just
the gateways to an entirely new field of pharmaceutical research and
will eventually lead to development of novel therapeutics.
5. Improved MIP performance with artificial design
The increasing number of ‘MIP’ publications about artificial anti­
bodies developed for a plethora of biomarkers and pathogens have not
shown its readiness for clinical translation. Generally, polymer matrices
have an inherent adsorbing nature therefore, weak interactions with
non-specific templates are unavoidable but can nevertheless be mini­
mized (Belbruno, 2019). Hence, MIPs with high imprinting factors are
desired where the non-imprinted ‘control’ polymer exhibits extremely
low binding capacity.
A diverse pool of monomers and polymerizable compounds is
referred for the imprint development however, due to the intervention
of computational screening approaches the conventional trial-and-error
method is being ruled out. Multi-component systems can be modeled
with a variety of methods including quantum mechanics (QM), molec­
ular mechanics (MM) and molecular dynamics (MD) (Suryana et al.,
2021). Most of these methods are targeted to elucidate the
pre-polymerization interactome between the monomers and template.
The strength of binding and type of non-covalent bonds including
hydrogen bonding, electrostatic interactions and hydrophobic bonds
established in this system has a direct impact on the selective nature of
the imprinted cavities. Molecular dynamics simulation is the next
crucial method that allows for artificial prediction of the effect of sol­
vent, time of polymerization and other conditions intrinsic to MIP
S. Rajpal and P. Mishra
Fig. 9. A graphical summary of the computational approach to rational selection of a monomer for protein-imprinted film synthesis. GScore indicates Glide empirical
scoring function; MD, molecular docking; QCC, quantum chemical calculation. Reprinted with permission from (Boroznjak et al., 2017) Copyright 2017, Wiley.
synthesis. Ab initio approaches (QM) have been used by several re­
searchers to determine the optimal template to functional monomer
ratio (He et al., 2015; Saad et al., 2015; Tadi and Motghare, 2013).
While QM can help to decipher the electronic structure of a less complex
system, it becomes challenging and computationally expensive when the
template is as large as peptides and proteins. As an alternative, MM
involves molecular docking that is routinely used to study drug-protein
interactions and has now been extended to analyze monomer-template
binding energies. Combinatorial screening combined with in silico
studies and experimental analysis is practically necessary and is widely
accepted for effective polymer design. A library of commonly used
monomers has been discussed in Table 4 for development of MIPs
applicable in biosensors. Selection of biocompatible functional mono­
mers that is of particular therapeutic interest can also be empowered by
molecular modelling.
The electrostatic, steric and H-bond enthalpies in a sampled space
are calculated to estimate the binding free energy of the monomers and
this forms the basis of the scoring functions employed in MM-based
programs. Pan and coworkers reported the computational docking of
acrylamide monomers and S. aureus protein A to analyze multi-point
hydrogen bonds that essentially drive the MIP-protein binding along
with other weak interactions like electrostatic and hydrophobic bonding
(Pan et al., 2009). Artificial receptors have also been tailor made for
immunoglobulin G (IgG), an intrinsic component of the immune reac­
tion and commonly employed in ELISA kits. The functional monomer
selection amongst m-phenylenediamine (mPD), dopamine, and 3,4-eth­
ylenedioxythiophene was based on molecular docking and quantum
chemical calculations using the glide docking tool (Boroznjak et al.,
2017) (Fig. 9). Unlike the drug-screening studies targeted at a single
protein pocket, this study establishes the importance of monomers
interacting uniformly over the whole protein surface that is essential to
fabricate a specific cavity in MIPs. The functional monomer mPD could
predictively show multiple H-bond interactions with the entire surface
of the protein and therefore demonstrated higher binding affinity in the
experiments compared to the other monomers.
During the COVID-19 pandemic, multiple research facilities have
developed virus-affinity MIPs guided by in silico predictions. Parisi et al.,
have employed molecular docking using Autodock tools based on the
11
Biosensors and Bioelectronics: X 11 (2022) 100201
AMBER force field (Parisi et al., 2021). This has allowed to screen the
functional monomers like acrylamide, acrylic acid, N-iso­
propylacrylamide (NIPAm), N-tert-butylacrylamide (TBAm) and
N-(3-aminopropyl)methacrylamide hydrochloride (NAPMA) for ratio­
nally imprinting spike protein of the virus, a surface protein of interest
for many COVID based immunoassays. In vitro studies confirmed the
viral binding and an additional inhibition effect of the MIPs which find
direct application in theranostics. Similar computational tools have also
been used to screen from a larger library of around 20 monomers to
select optimally binding monomers with a peptide epitope sequence as a
smaller template compared to the whole spike protein (Batista et al.,
2022; Fresco-Cala et al., 2021). Cubuk et al. has further studied more
epitopes along with molecular dynamics simulations using GROMOS
force field to predictively analyze the stability of monomer binding over
a time period (Cubuk et al., 2021).
The molecular simulations are performed considering the thermo­
dynamic factors controlling molecular interactions in imprinted systems
that is represented as (Equation (1))
∑
ΔGbind = ΔGt+r + ΔGr + ΔGh + ΔGvib + ΔGp + ΔGconf + ΔGvdW
(1)
where the Gibbs free energy change for complex formation (ΔGbind) is
the combined energy changes associated with the loss of translational
and rotational freedom (ΔGt + r), restriction of rotors upon complexation
(ΔGr), hydrophobic interactions (ΔGh), residual soft vibrational modes
∑
(ΔGvib), the sum of interacting polar group contributions ( ΔGp),
adverse conformational changes (ΔGconf) and unfavorable van der Waals
interactions (ΔGvdW) (Nicholls et al., 2021; Williams et al., 1991, 2004).
6. Commercialization of MIPs
The success in the development of MIPs at the laboratory scale has
motivated the commercial adoption of this technology, while facilitating
its mass-production for applications in the field of not only diagnostics
but also separation science and therapeutics. Around seven companies
now show a full market presence which are summarized Table 5:
Among these, MIP diagnostics Ltd. which started as a spin off
S. Rajpal and P. Mishra Biosensors and Bioelectronics: X 11 (2022) 100201

Table 5 that aims at label-free detection of the target molecules. These de­
The commercial success of MIPs demonstrated by eight established companies. velopments not only direct the replacement of antibodies in assays but
Name of the Co-founders and Company MIP solutions offered also, devising novel assay formats for biomarker sensing and pathogen
company collaborators origin detection. The customized designing and the mass-manufacturing of
1. MIP Borje Sellergren, 1999 in Solid phase extraction MIPs with batch-to-batch uniformity offered by this company presents a
Technologies Klaus Mosbach Sweden; materials for industry, promising future for IVD. Parallelly, companies like Sixth wave have
Collaborators: becomes a Purification of pesticides also demonstrated their success in development of novel ‘AMIPs’ for
● FeF chemicals subsidiary of and toxins from food, COVID detection. These facets point us to a conclusion that highlight the
(a subsidiary of Biotage AB analysis of NSAIDs in
Novo Nordisk) from 2010 water, large scale
fast R&D and commercialization potential of the MIP technology. Sixth
● France based protein purification, wave has combined these novelty MIPs into strip-based tests, breath
Sanofi-Aventis selective separation of analyzers, masks, HVAC filters and applied for its patent. Additionally,
● Supelco, a biopharmaceuticals and companies like Semorex (Israel) also specialize in development of MIPs
division of Sigma peptides
for disease identification like cancer, celiac disease, inflammatory bowel
Aldrich
2. Semorex Bernard S. Green 2001 in Israel MIPs for healthcare syndrome. A typical example is presented in the patented MIPs specific
(www.se industry: targeted for bile acids like deoxycholic acid (DCA) that can directly aid in di­
morex.com) therapeutics for cancer, agnostics and therapeutics for medical conditions like gallstones, colo­
celiac disease, obesity rectal cancer and inflammation.
and inflammatory bowel
disease; detection of
chemical warfare agents 7. Summary and conclusion
like sarin and soman
(organophosphate nerve The advent of high-throughput signal readout methods, advanced
gases)
nanomaterials and miniaturized system design and employment of in
3. Polyintell Karsten Haupt 2004 in MIPs for specific target
SAS France analytes for food safety, silico design for MIPs have enhanced the potential for next-generation
(Affinisep) pharmaceutical IVD for point-of-care devices. Point-of-care application with multi­
(www.affini industry, clinical plexing capability is a major target to facilitate access to diagnostics in
sep.com) diagnosis, environment
resource-constrained areas. Here we summarize the proficiency of MIPs
and doping tests
4. Mipsalus Nicolas O. Krogh, 2006 in MIPs for sensing and
in all these domains. The previous sections establish the sensing per­
APS (www. Klaus Gregorius Denmark pharmaceutical formance of MIPs that functionally mimic natural antibodies and yet are
mipsalus. applications to treat easy to develop with high thermal and chemical stability. The sensitivity
dk/) (rare) metabolic diseases of fM level is readily achievable with excellent performance in complex
like phenylketonuria
sample matrices. MIPs have shown versatile applications in various
5. Nanomyp Jorge Fernando, 2011 in Spain MIPs for SPE cartridges;
(www.na Fernandez as controlled release sensing platforms and have been integrated with piezoelectric, optical,
nomyp.com) Sanchez systems for drugs, thermal as well as electrochemical sensors along with microfluidic de­
● Spin off from antioxidants and vice. Moreover, conventional 2D MIP biosensors are changing to cutting-
University of antimicrobials; quality edge 3D bioMEMs such as MIP electronic tongues and MIP handheld
Granada control for packaging of
food products; as part of
analyzers (Hong et al., 2016; Huynh and Kutner, 2015). Functionaliza­
optical sensing platform tion of MIPs in the core-shell nanoparticle format or as films on sensor
6. Ligar Nigel Slaughter 2011 in New MIPs for purification of surfaces is uncomplicated and does not require critical handling and
Polymers ● Spin off from Zealand cannabinoid extracts; storage conditions like conventional biomolecules. However, the fast
(www.ligar. Waikato Institute removal of contaminants
commercialization of these sensors is subject to selection of a suitable
nz) of Technology from wine, water oils
(WinTec) and etc; Extraction of sensor platform and imprinting method. The optimization of the readout
University of valuable bioactive method and MIP development must be simultaneously scrutinized ac­
Waikato molecules from juices cording to the target analyte, which adds to the complexity and some­
Collaborators and plant products times, the cost of the technology. We have already discussed alternate
● Amber
Purification Ltd
strategies that work standalone for binding event analysis. MIPs coupled
● Te Whāi Ao Ltd with fluorophores, chromophores and tunable nanomaterials are
(as ‘The Refinery’) evolving to overcome such design complexities of MIPs based assay. For
7. Sixth Wave Jonathan 2013 in North MIPs for military and example, QDs are the most commonly employed that can generate
(www.sixt Gluckman America mining applications;
multiplexed detection systems, followed by magnetic nanoparticles that
hwave.com) extraction of
cannabinoids; gold enable easy sample purification. MIP-based magnetic transduction nano
mining; rapid viral biosensors are highly desirable and invite magnetics researchers to offer
detection (product line - innovative platforms to facilitate advanced IVD development (Li et al.,
‘AMIPs’) 2019).
8. MIP Sergey Piletsky 2015 in ‘nanoMIPs’ for in vitro
diagnostics Spin off from Leicester diagnostics
Obtaining MIPs with high homogeneity and reproducibility during
(www. University of scale up can be challenging as is the case with monoclonal antibodies.
mip-dx.com) Leicester The current solution offered by the solid phase synthesis strategy
devised by Canfarotta and coworkers is highly promising to limit the
batch variations but employs a limited set of monomers for synthesis
company from the University of Leicester, has particular focus for
(Canfarotta et al., 2016). For every target molecule especially proteins
development of MIPs for the IVD applications. The unique biocompat­
and large cells, the intrinsic functional diversity must be considered to
ible, high affinity ‘nanoMIPs’ have been employed for the detection of
enable rational selection from a variety of compatible monomers.
SARS-CoV-2 in a recent collaboration with StreamBio. The design
Computer-aided MIP design is paving the way for fast selection of
combines magnetic nanoparticles, high affinity MIPs, fluorescent
polymerization agents and conditions. This can directly reduce the
tagging along with lateral flow assay format to develop a rapid (10 min)
screening time of multiple experimental polymer combinations and
diagnostic test applicable to the current pandemic as PoC diagnostics.
allow rapid and guided design. Further adjustments in the NIP fabrica­
MIP diagnostics also offer a universal electrochemical sensor platform
tion such as the use of analog templates as opposed to no template, are

12
S. Rajpal and P. Mishra
required for appropriate MIP comparison (Ndunda, 2020).
Nevertheless, rapid progression of synthesis strategies has brilliantly
enabled the clinical utility of MIP based sensors and the proof-of-concept
established in various studies demonstrate full applicability and high
biocompatibility in physiological samples. However, extensive studies at
a large scale are required to enhance the commercialization in the
domain of IVD and even more, for theranostics. As drug delivery agents,
the toxicity and pharmacokinetics have to be thoroughly analyzed to
produce the best MIP performance in varied cellular environment. The
existing market players are delivering MIP solutions designed with
utmost precision and potentially changing the dynamics of existing
immuno and other classical antibody-based assays.
8. Future perspectives
The current developments in MIP research projects a reduction in the
gap between laboratory scale production and large-scale manufacturing
to enhance the commercial presence of MIPs. Low cost of MIP with high
binding affinities and additional smart properties like magnetic nature,
temperature sensitivity, and pH sensitivity is likely to increase their
applications in IVD. The increasing interest and identification of more
biomarkers for disease detection will improve the portfolio of MIPs
which are presently limited to ‘model’ analytes. With the advantage of
multiplexing that MIPs offer, it is anticipated to majorly capture and
innovate the industry of POC devices, offering multiple molecular
testing in a single bedside device. Numerous applications like the
detection of quorum sensing molecules can be explored only by MIPs
because of its intrinsic nature of being able to imprint any target
molecule, big or small. This is envisioned to unfold numerous possibil­
ities in the field of diagnostics that will directly upgrade therapeutics.
Though molecular imprinting technology is known since long for small
molecules and commercialized as well, developing MIPs for macro­
molecules like proteins including several biomarkers is still in its in­
fancy. The major drawback in the development of MIPs for biomarkers is
either nonavailability or high cost of template. In this regard, epitope
imprinting combined with computational approach for the selection of
monomers have paved way to more versatile and economic approach for
MIPs synthesis for biomarkers which is key to the development of IVD.
Further solid phase imprinting and production of biomimetic MIP films
on sensing elements have enormous potential for the development of
new generation IVD.
CRediT authorship contribution statement
Soumya Rajpal: Writing – original draft. Prashant Mishra:
Conceptualization, Writing – original draft, Writing – review & editing,
Visualization, Supervision, Funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgement
Prashant Mishra would like to acknowledge grants received from
Department of Science and Technology (DST), Government of India
(DST/NM/NNetRA/2018 (G)-IITD) and Ministry of Electronics and In­
formation Technology, Government of India (5(1) 2017-NANO).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.biosx.2022.100201.
13
Biosensors and Bioelectronics: X 11 (2022) 100201
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