![Examples of paper-based POC aptasensors: (A) Sandwich LFA using streptavidin-biotin immobilization for simultaneous capturing albumin and glycated albumin on test line [55]; (B) Working principle of cortisol LFA with an adsorption-desorption sensing mechanism, where either (i) cortisol capture is detected via AuNPs-linker hybridization on the test line (positive result), or (ii) no cortisol is captured on the test line (negative result) [53]; (C) operations steps of paper-based microfluidic integrated blood potassium concentration detection device [58].](https://www.researchgate.net/publication/371003035/figure/fig2/AS:11431281161257234@1684961568585/Examples-of-paper-based-POC-aptasensors-A-Sandwich-LFA-using-streptavidin-biotin_Q320.jpg)
![Molecular Docking results of the interaction of aptamer and (A) cortisol, (B) progesterone, and (C) testosterone [230].](https://www.researchgate.net/publication/371003035/figure/fig3/AS:11431281161266018@1684961568813/Molecular-Docking-results-of-the-interaction-of-aptamer-and-A-cortisol-B_Q320.jpg)
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Content may be subject to copyright.
Citation: Aslan, Y.; Atabay, M.;
Chowdhury, H.K.; Göktürk, I.;
Saylan, Y.; Inci, F. Aptamer-Based
Point-of-Care Devices: Emerging
Technologies and Integration of
Computational Methods. Biosensors
2023,13, 569. https://doi.org/
10.3390/bios13050569
Received: 31 March 2023
Revised: 14 May 2023
Accepted: 15 May 2023
Published: 22 May 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
biosensors
Review
Aptamer-Based Point-of-Care Devices: Emerging Technologies
and Integration of Computational Methods
Yusuf Aslan 1,2,† , Maryam Atabay 1,3 , † , Hussain Kawsar Chowdhury 1,2 , Ilgım Göktürk 1,3 ,
Ye¸seren Saylan 3and Fatih Inci 1, 2, *
1UNAM—National Nanotechnology Research Center, Bilkent University, Ankara 06800, Turkey
2Institute of Materials Science and Nanotechnology, Bilkent University, Ankara 06800, Turkey
3Department of Chemistry, Hacettepe University, Ankara 06800, Turkey
*Correspondence: finci@bilkent.edu.tr
† These authors contributed equally to this work.
Abstract:
Recent innovations in point-of-care (POC) diagnostic technologies have paved a critical
road for the improved application of biomedicine through the deployment of accurate and affordable
programs into resource-scarce settings. The utilization of antibodies as a bio-recognition element in
POC devices is currently limited due to obstacles associated with cost and production, impeding
its widespread adoption. One promising alternative, on the other hand, is aptamer integration,
i.e., short sequences of single-stranded DNA and RNA structures. The advantageous properties
of these molecules are as follows: small molecular size, amenability to chemical modification, low-
or nonimmunogenic characteristics, and their reproducibility within a short generation time. The
utilization of these aforementioned features is critical in developing sensitive and portable POC
systems. Furthermore, the deficiencies related to past experimental efforts to improve biosensor
schematics, including the design of biorecognition elements, can be tackled with the integration
of computational tools. These complementary tools enable the prediction of the reliability and
functionality of the molecular structure of aptamers. In this review, we have overviewed the usage
of aptamers in the development of novel and portable POC devices, in addition to highlighting the
insights that simulations and other computational methods can provide into the use of aptamer
modeling for POC integration.
Keywords: aptamer; aptasensor; biosensor; computational methods; nanomaterials; point-of-care
1. Introduction
The growing number of discoveries on the chemical pathways taken by diseases have
driven our recent advancements in medicine and biomedical technologies [
1
–
3
]. Tracking
biomarkers in our bodies provides us with plentiful information about a number of health
conditions, such as an indication of specific physiological conditions, the presence of a
disease, the progress of treatment, and the risk of disease development. The presence of
certain biomarkers can be recognized as a conformation of diseases, such as cancer [
4
],
cardiovascular diseases [5], neurodegenerative diseases [6], viral infections [7], and organ
injuries [
8
]. Therefore, the application of
in vitro
biomarker detection has been expanded to
many diseases; accordingly, the necessity of rapid and cost-effective solutions has increased
in our search for the early diagnosis of life-threatening diseases.
Today’s widely employed conventional biomarker quantification tools include mass
spectrometry [
9
], cell culture, polymerase chain reaction (PCR), enzyme-linked immunosor-
bent assay (ELISA), Western blot, and flow cytometry [
10
]; however, these methods are
difficult to integrate into financially-restricted and resource-scarce settings. Metamor-
phosing the current conventional practices with point-of-care (POC) systems is vital to
delivering healthcare to patients in developing countries more efficiently. Therefore, the
Biosensors 2023,13, 569. https://doi.org/10.3390/bios13050569 https://www.mdpi.com/journal/biosensors
Biosensors 2023,13, 569 2 of 34
qualifications of ideal POC devices have been standardized with a set of criteria known as
ASSURED, which stands for affordability, sensitivity, specificity, user-friendliness, rapid,
equipment-free, and deliverable [
11
,
12
]. Currently, ASSURED is evolving into a newly
suggested acronym called RE-ASSURED [
13
]. The addition of real-time connectivity, ease-
of-specimen collection, and environmentally friendly qualifications are justified by the
rapid transmission of data to individuals/patients; the increased feedback availability
for treatment monitoring, possibly over smartphones or other such mobile systems; the
provision of noninvasive sample analysis, with an increased capacity for self-testing; and a
reduction of the risks related to environmental hazards [
13
]. Concentrating this focus on
limited resources and expanding POC technologies into multiple settings for self-testing
purposes, the ideal POC device requires many qualifications, such as low cost, portability,
short turnaround time, specificity, and sensitivity at the first glance. Conventionally, anti-
bodies have been utilized as the recognition elements in POC systems in order to detect
markers in biospecimens. However, in some cases, high specificity over many targets
can be impeded due to single-point mutations or conformational isomers [
14
]. This being
the case, aptamers shine out as a strong candidate for replacing antibodies since they are
tertiary structures of relatively short nucleic acid sequences that can selectively recognize
the desired target with high affinity and specificity, as well as having the capacity to be
easily customized for such changes [
15
]. The complementary base pairs found in aptamers
tend to form secondary structures (stem ring, hairpin, spiral, pseudoknot, clover, and so
on), which can collectively combine into tertiary structures [
16
]. These structures can be
computationally predicted by the identification of Watson–Crick base pairing regions [
17
].
The properties of aptamers have been explained in more detail in the following section.
On the other hand, the utilization of simulations and other such computational methods
(e.g., Molecular Docking calculations, Molecular Dynamics (MD) simulations, Density
Functional Theory (DFT), Quantum Mechanics and Molecular Mechanics (QMMM), and
Artificial intelligence (AI) methods) to design a biosensing system for POC applications
has not been encountered frequently. Combining and validating these studies with experi-
ments would hold significant potential for aiding scientists and individuals as end-users
by predicting experimental drawbacks, thus reducing the number of false interpretations,
shortening the optimization time, and reducing both unnecessary material usage and the
associated costs. Therefore, the impact of simulations and computational methods in POC
applications is often highlighted.
In this study, we have reviewed both the significant benefits of aptamers and the
current emerging applications for aptamer-oriented POC diagnostic platforms, in addition
to discussing the integration of simulation and computational methods.
2. Aptamers
The idea for this use of single-stranded oligonucleotides (aptamers), ribonucleic acid
(RNA), or single-strand deoxyribonucleic acid (ssDNA) was initially proposed in 1990 [
18
].
Although RNA and ssDNA aptamers can have different sequences and folding patterns,
they can be designed to bind to the same target [
19
]. These single-stranded oligonucleotides
have unique tertiary structures, which allow specific interactions with target molecules.
Aptamers are generated
in vitro
through the Systematic Evolution of Ligands by Expo-
nential Enrichment (SELEX) approach [
20
,
21
], which can be conducted against different
molecules, including proteins, small compounds, cells, and nanoparticles [
18
]. The SELEX
method involves repeated rounds of selecting and amplifying target-specific nucleic acid
sequences from a large pool of random sequences. The conventional SELEX approach
includes the combination of a random nucleic acid library with the target molecules, the
removal of non-target-specific sequences, the amplification of target-specific sequences,
and the characterization of the isolated aptamers [
22
,
23
]. The whole process is repeated,
once the selected aptamers are sufficiently specific to the target and demonstrate the in-
tended levels of the enrichment. Over the last three decades, researchers have made
various updates to the traditional SELEX strategy in order to boost its efficiency and cost-
Biosensors 2023,13, 569 3 of 34
effectiveness [
24
] while, at the same time, increasing the selectivity and affinity of the
generated aptamers [
20
]. Such advances included streamlining the selection procedure,
reducing generation time, and optimizing the amplification and characterization of target-
specific aptamers. Immunoprecipitation-coupled SELEX, for instance, was developed to
enhance affinity under normal physiological scenarios [
25
], whereas the capture-SELEX
was established to improve the selection effectiveness of aptamers for unidentified small
molecular targets [
26
]. Furthermore, whole cells [
27
], tissue or organs in animals [
28
]
have also been utilized as SELEX tools for distinguishing between healthy and diseased
conditions [
29
], monitoring prognosis [
30
], and discovering new biomarkers [
31
]. Emer-
gent technologies, such as microfluidics [
32
], capillary electrophoresis [
33
], atomic force
microscopy [
34
], and several in silico approaches, have also been used to improve the
performance at the current stage of SELEX strategy.
As shown in Figure 1, hairpin, spiral, stem ring, pseudoknot, and clover are common
structures of SELEX-generated aptamers that can bind to many targets, such as, proteins,
toxins, viruses, and vitamins [
35
,
36
]. These aptamers have been studied for their applica-
tions in diagnostic and therapeutic purposes [
19
]. Owing to their high affinity and other
properties exclusive to aptamers toward the target proteins, they have been employed as
a biorecognition element in biosensors as an alternative to antibodies [
37
]. In addition,
in comparison with antibodies, aptamers have many favorable advantages: (1) They are
chemically and structurally stable. While antibodies easily undergo irreversible denat-
uration, aptamers can recover their native conformation by tuning the conditions, such
as changing the pH of the medium (slight changes), salt concentration, chelating agents,
and temperature. Aptamers are not damaged in many of these conditions, except for the
extremes of pH value, wherein the structure of the aptamer can be irreversibly damaged.
(2) Furthermore, the production of monoclonal antibodies is laborious and very expensive,
while aptamers can be synthesized in greater quantity, accuracy, and reproducibility, and at
a relatively lower cost. (3) In addition, aptamers can be chemically modified or conjugated
without sacrificing their binding affinity, whereas antibodies require stochastic modifica-
tions, which bring a possible loss in binding activity [
14
]. (4) They can be stored for a long
time without losing their activities because of thermal denaturation [
38
–
41
]. (5) Lastly,
aptamers have no or low toxicity and immunogenicity, which is crucial for
in vitro
and
in vivo
applications [
42
,
43
]. Aptamers are good candidates for biosensors that use optical,
electrochemical, and mass-based detection techniques, and are compatible with antibodies.
In addition, they have been studied for the creation of biosensors with high detection,
sensitivity, and stability [
44
]. Despite the advances in SELEX procedures, along with all the
properties and applications of the aptamers that have been considered, studies show that
some aptamers are unable to bind the target molecule [
45
]. As an example, Zong and Liu
showed that there was no specific binding in the structure of arsenic(III)-binding aptamer,
despite this structure having been used and reported on in two dozen peer-reviewed
articles, previously [
46
]. There are some new analytical techniques for a more in-depth
understanding of the aptamer-target structures. In these techniques, (a) the kinetic and
thermodynamic information of the binding event and (b) the structure of aptamer and
aptamer-target complex are investigated. In part (a), analytical methods are divided into
two groups: those methods in which interactions are studied in solution, and those meth-
ods in which aptamer-target kinetics are characterized while one of the biding structures
is immobilized. Nowadays, aptamer immobilization protocols are used for designing
electrochemical, mass-sensitive, or optical aptasensors. Part (b) is subdivided into two
types of techniques: high-resolution methods, such as nuclear magnetic resonance (NMR)
spectroscopy, X-ray crystallography, and electron microscopy (EM); and low-resolution
techniques. Low-resolution techniques that can provide information about the size and
shape of the aptamer include small-angle X-ray scattering (SAXS) and circular dichroism
(CD) spectroscopy [45].
Biosensors 2023,13, 569 4 of 34
Biosensors2023,13,xFORPEERREVIEW4of35
Figure1.Thestructuresandtargetsofaptamers.CreatedwithBioRender.com(accessedon13
March2023).
Inadditiontothesimulationandcomputationmethodsthatwerebrieflymentioned
intheprevioussection,onenovel(andunconventional)waytoselectaptamerswitha
highaffinityfortargetingmoleculesistoperformArtificialintelligence(AI)‐assistedstrat‐
egies.AI,includingmachine/deep‐learningalgorithms,isastrongandaccurateapproach
forpredictingtheinteractionbetweenaptamersandtargets.Therefore,thecombination
ofthesimulationandcomputationmethods(whicharestructure‐basedmethods)withAI
methodscouldbeapromisingapproachtopredictinginteractionsbetweenaptamersand
targetmolecules[47].
Inthenextsections,differenttypesofaptasensorswillbediscussedindetail,along
withtheirapplications.
3.AptasensorsinPOC‐BasedBiosensingPlatforms
Immunochromatographytestsarelargelyemployedformonitoringinfections,dis‐
eases,andotherhealthconditionsinPOCsettings[48,49].Thebiomoleculesusedas
probesinthePOCdeviceshaveincludedglycan,aptamers,enzymes,nucleicacids,and
antibodies[48–51].Thevastmajorityoftargetsthatcanbecapturedbyaptamersaresmall
metalionsandwholecells.Withthehelpofaptamers,medicaldevicesthatutilizeap‐
tamer‐coupledPOCfunctionsbringtheircapabilitiesfromthelabtothebed‐side,and
thusplayasignificantroleinthehealthcaresector.Aptamer‐basedbiosensorsareuseful
formanydiagnosticprocedures,includingthedetectionofillness,cancer,heartdisease,
etc.CurrenttechniquesforillnessdiagnosiswithPOChavebeendevelopedusingasim‐
plelateralflowsystem,ormodestelectricalandoptical‐basedsystems[48,51,52].Further‐
more,theintegrationofpaper,nanomaterials,portable,andsmartphonePOC‐oriented
aptamer‐basedbiosensors(aptasensors)canbedividedintovariousplatformsaccording
tothetypeoftransducingmechanisms:colorimetric,fluorescent,surfaceplasmonreso‐
nance(SPR),andelectrochemical(Table1).Whenevaluatedonthesesensingdevices,ap‐
tamersmadeagainstvariousbiomarkersshowedavarietyofdetectionlimitranges.In
thefollowingsections,theseaptasensorstrategieswillbediscussedindetail.
Figure 1.
The structures and targets of aptamers. Created with BioRender.com (accessed on 13
March 2023).
In addition to the simulation and computation methods that were briefly mentioned
in the previous section, one novel (and unconventional) way to select aptamers with a high
affinity for targeting molecules is to perform Artificial intelligence (AI)-assisted strategies.
AI, including machine/deep-learning algorithms, is a strong and accurate approach for
predicting the interaction between aptamers and targets. Therefore, the combination of
the simulation and computation methods (which are structure-based methods) with AI
methods could be a promising approach to predicting interactions between aptamers and
target molecules [47].
In the next sections, different types of aptasensors will be discussed in detail, along
with their applications.
3. Aptasensors in POC-Based Biosensing Platforms
Immunochromatography tests are largely employed for monitoring infections, dis-
eases, and other health conditions in POC settings [
48
,
49
]. The biomolecules used as
probes in the POC devices have included glycan, aptamers, enzymes, nucleic acids, and
antibodies [
48
–
51
]. The vast majority of targets that can be captured by aptamers are
small metal ions and whole cells. With the help of aptamers, medical devices that utilize
aptamer-coupled POC functions bring their capabilities from the lab to the bed-side, and
thus play a significant role in the healthcare sector. Aptamer-based biosensors are useful
for many diagnostic procedures, including the detection of illness, cancer, heart disease, etc.
Current techniques for illness diagnosis with POC have been developed using a simple
lateral flow system, or modest electrical and optical-based systems [
48
,
51
,
52
]. Furthermore,
the integration of paper, nanomaterials, portable, and smartphone POC-oriented aptamer-
based biosensors (aptasensors) can be divided into various platforms according to the type
of transducing mechanisms: colorimetric, fluorescent, surface plasmon resonance (SPR),
and electrochemical (Table 1). When evaluated on these sensing devices, aptamers made
against various biomarkers showed a variety of detection limit ranges. In the following
sections, these aptasensor strategies will be discussed in detail.
Biosensors 2023,13, 569 5 of 34
Table 1. Evaluating POC devices in terms of the target analyte, physical condition, sensing principle, and limit of detection (LOD).
Target Analyte Physical Condition Sensing Principle Limit of Detection
(LOD) Reference
Cortisol Stress Colorimetric 0.37 ng·mL−1[53]
Dopamine Alzheimer’s, Parkinson’s, and Huntington’s diseases Colorimetric 10 ng·mL−1[54]
Glycated albumin GDM Colorimetric 0.8 mg·mL−1and
1.5 mg·mL−1[55]
PDGF-BB and thrombin Tumor regions (e.g., liver, gastrointestinal tract) and hemostasis Colorimetric 1.0 nM and 1.5 nM [56]
CXCL 9 Antibody-mediated rejection of kidney transplantation Colorimetric 10 pg·mL−1[57]
K+Chronic kidney disease Colorimetric 0.01 mM [58]
HER2 Breast cancer Colorimetric 10 nM [59]
RBP4 Type 2 diabetes mellitus Colorimetric 90.76 ±2.81 nM [60]
Exosomes Leukemia Colorimetric 42 particles·µL−1[61]
IL-6 Brain injury or inflammation Colorimetric 1.95 µg·mL−1[62]
Mycobacterium tuberculosis DNA TB Colorimetric 0.28 nM [63]
PDGF-BB Tumor growth and progression Colorimetric 10 fM [64]
Cortisol Stress Fluorescent 1 nM [65]
Aβand tau protein Alzheimer Fluorescent 50 pM and 10 pM [66]
Mucin 1 Tumor Fluorescent 0.15 fg·mL−1mucin 1 or
3 CTCs·mL−1[67]
AFP Hepatocellular carcinoma Fluorescent 400 pg·mL−1[68]
Mutated BRCA-1 Breast cancer Fluorescent 0.34 fM [69]
Cortisol Stress Fluorescent 6.76 ng·mL−1[70]
Ig E Allergic disease Fluorescent 0.13 IU·mL−1[71]
PFLDH Malaria Fluorescent 18 fM (0.6 pg·mL−1)[72]
Glucose, ATP, L-Tyrosinamide,
and thrombin
Diabetes, molecular marker for cellular energy, metabolic syndrome and
melanoma, and hemostasis Fluorescent 1.1 mM, 0.1 mM, 3.5 µM and 25
nM [73]
MPT64 secreted from
Mycobacterium tuberculosis TB Electrochemical 81 pM [74]
Biosensors 2023,13, 569 6 of 34
Table 1. Cont.
Target Analyte Physical Condition Sensing Principle Limit of Detection
(LOD) Reference
YadA Diarrhea, mesenteric lymphadenitis, arthritis, and sepsis Electrochemical 7.0 ×104CFU·mL−1[75]
CRP Cerebrovascular diseases, myocardial infectious inflammation, and
cancer Electrochemical 0.44 pg·mL−1[76]
Exosomes Cancer Electrochemical 5×103particles·mL−1[77]
Thrombin Hemostasis Electrochemical 0.12 pM [78]
α-thrombin Blood clotting cascade Electrochemical 10 pM [79]
Thrombin Anticoagulation and cardiovascular disease Electrochemical 1 pM [80]
p24-HIV protein HIV Electrochemical 51.7 pg·mL−1[81]
α-Syn Parkinson’s disease Electrochemical 1×10–6 pM [82]
MCF-7 breast cancer cells Breast cancer Electrochemical 6 cells·mL−1[83]
SARS-CoV-2 COVID-19 Electrochemical 9.79 fg·mL−1[84]
AFP Liver cancer Electrochemical 0.65 pg·mL−1[85]
CEA and NSE Cancer Electrochemical 2 pg·mL−1and
10 pg·mL−1[86]
Thrombin Blood coagulation cascade SPR 1 nM [87]
Dopamine Neurological and psychiatric disorders SPR 10−13 M [88]
SARS-CoV-2 spike
glycoprotein COVID-19 SPR 36.7 nM [89]
Exosomes Breast cancer SPR 5×103exosomes·mL−1[90]
Cortisol Stress LSPR 0.1 nM [91]
Insulin Diabetes SPR 5 pM [92]
HER2 proteins Breast cancer OF-SPR 20 g·mL−1[93]
Abbreviations: CXCL 9: CXC-motif chemokine ligand 9, PDGF-BB: Platelet-derived growth factor-BB, HER2: Human epidermal growth factor receptor 2, RBP4: Retinol-binding
protein 4, IL-6: Interleukin-6, A
β
: Amyloid beta oligomer, AFP: Alpha-fetoprotein, BRCA-1: Breast cancer gene-1, Ig E: Immunoglobulin E, PFLDH: Plasmodium falciparum lactate
dehydrogenase, ATP: adenosine triphosphate, YadA: Yersinia adhesin A, CRP: C-reactive protein,
α
-Syn:
α
-Synuclein, CEA: Carcinoembryonic antigen, NSE: neuron-specific enolase,
OF-SPR: optical fiber-surface plasmon resonance, K+: Blood potassium ion, HIV: Human immunodeficiency virus, AFP: Prime alpha-fetoprotein/alpha-fetoprotein, TB: Tuberculosis,
CTC: Circulating tumor cells, GDM: Gestational diabetes mellitus, CFU: Colony-forming unit.
Biosensors 2023,13, 569 7 of 34
3.1. Colorimetric Aptasensors
Colorimetric methods provide visual recognition for aptamer-target binding, which
is either discerned with the naked eye or measured quantitatively using an optical reader.
The concentration of the detected targets is determined by any alterations in scattering
and absorption efficiencies [
94
], the refractive index of the surrounding medium [
94
],
the electromagnetic spectrum [
95
] or wavelength, and the full width at half maximum
(FWHM) values of the resonance signal [
96
]. Such methods hold great potential for on-site
diagnosis, especially in resource-limited settings, since they are cost-effective and straight-
forward [
97
]. However, the laborious fabrication process [
98
], inefficiency in detecting
multiple targets [
99
] and narrow working pH range are some of the current obstacles in col-
orimetric aptasensors [
100
]. Herein, we have classified this concept into two subcategories
according to their fabrication substrates, i.e., paper-based and ‘in solution’ colorimetric
aptamer assays.
3.1.1. Paper-Based Colorimetric Aptasensors
Paper is an excellent and cost-effective off-the-shelf material for immobilizing
biomolecules (e.g., protein, nucleic acids, aptamers, antibodies, and so on) [
101
–
103
].
Assays on paper require minimal sample volume and provide quick visual results with-
out external instruments. These assays can also be stored in ambient conditions with
temperature-stable modified aptamers [
16
]. Although paper-based biosensors have many
pros, as stated above, they also come with some limitations, including: (i) sample evap-
oration or entrapment on the paper substrate; (ii) instability, due to environmental inter-
ference factors; (iii) reader requirements for high sensitivity and quantitative results; and
(iv) low mechanical stability [
104
]. Additionally, false negative and false positive outcomes
would be possible for these platforms, due to concentrations of the target so low as to
fall below the LOD value [
105
], or from the cross-reactivity between structurally similar
targets [106], respectively.
Lateral flow assay (LFA) is a common paper-based method, allowing for on-site
biomarker detection within minutes, and using naked-eye or portable systems. LFAs
use two essential sensing strategies: (1) sandwich, and (2) competitive assays. Sandwich
assays capture targets between detection and capture bioreceptors for selective signal
generation [
107
], whereas competitive assays use target competition to detect either labelled
bioreceptors [108] or capture bioreceptors [109].
For an example of the sandwich format, an LFA strategy tracked both glycated albu-
min and albumin concentrations in serum to record the glycemic status of patients with
gestational diabetes mellitus (GDM), a glucose intolerance disorder in pregnant people [
55
].
The assay was able to detect albumin as a target molecule between AuNPs conjugated
with both detection aptamer and capture aptamer on the test line. The positive result was
discerned from the appearance of red bands on both the test line and control line. The dual
assay also used a smartphone-integrated handheld colorimetric reader for quantitatively
tracking albumin status in serum. Furthermore, the platform detected glycated albumin
and serum albumin concentrations as low as 0.8 mg
·
mL
−1
and 1.5 mg
·
mL
−1
, respectively
(Figure 2A). Another study simultaneously monitored platelet-derived growth factor-BB
(PDGF-BB) and thrombin, which are biomarkers for several tumor regions (e.g., liver and
gastrointestinal tract) and for coagulation abnormalities, respectively [
56
]. There were
two different test lines and a single control line present on the assay. AuNPs-labelled
PDGF-BB specific aptamers and AuNPs-labelled thrombin specific aptamers were utilized
as conjugated detection probes. In the presence of targets, the detection probes were sep-
arately captured in their respective test lines. The third band was used as a control line
for capturing excess detection probes. A portable colorimetric reader was also used for
determining the target concentrations. This assay provided LOD values of 1.0 nM and
1.5 nM for PDGF-BB and thrombin, respectively. By way of contrast, a competitive assay
has been reported for the detection of CA125, an ovarian cancer serum biomarker, in serum
samples [
110
]. The assay utilized the intrinsic peroxidase activity of AuNPs for catalyzing
Biosensors 2023,13, 569 8 of 34
the oxidation reaction of DAB/H
2
O
2
substrate, where the excess amount of AuNPs would
result in an enhanced color intensity, and a deficiency of AuNPs would lead to a lower
color intensity. During the assay operation, a consistent amount of AuNPs-labelled CA125
biomarkers were placed on the conjugate pad, where they competed with CA125 in the
sample to bond with the immobilized CA125 specific aptamers present on the test line.
Therefore, the concentration of the bound CA125 was inversely proportional to the color
intensity on the test line. Further, the concentrations of biotargets were determined with
grayscale intensities of test line images captured on a smartphone. The assay was able to
detect CA125 down to 5.21 U·mL−1within 20 min.
Biosensors2023,13,xFORPEERREVIEW8of35
reportedforthedetectionofCA125,anovariancancerserumbiomarker,inserumsam‐
ples[110].TheassayutilizedtheintrinsicperoxidaseactivityofAuNPsforcatalyzingthe
oxidationreactionofDAB/H
2
O
2
substrate,wheretheexcessamountofAuNPswouldre‐
sultinanenhancedcolorintensity,andadeficiencyofAuNPswouldleadtoalowercolor
intensity.Duringtheassayoperation,aconsistentamountofAuNPs‐labelledCA125bi‐
omarkerswereplacedontheconjugatepad,wheretheycompetedwithCA125inthe
sampletobondwiththeimmobilizedCA125specificaptamerspresentonthetestline.
Therefore,theconcentrationoftheboundCA125wasinverselyproportionaltothecolor
intensityonthetestline.Further,theconcentrationsofbiotargetsweredeterminedwith
grayscaleintensitiesoftestlineimagescapturedonasmartphone.Theassaywasableto
detectCA125downto5.21U∙mL
−1
within20min.
Figure2.Examplesofpaper‐basedPOCaptasensors:(A)SandwichLFAusingstreptavidin‐biotin
immobilizationforsimultaneouscapturingalbuminandglycatedalbuminontestline[55];(B)
WorkingprincipleofcortisolLFAwithanadsorption–desorptionsensingmechanism,whereeither
(i)cortisolcaptureisdetectedviaAuNPs‐linkerhybridizationonthetestline(positiveresult),or
(ii)nocortisoliscapturedonthetestline(negativeresult)[53];(C)operationsstepsofpaper‐based
microfluidicintegratedbloodpotassiumconcentrationdetectiondevice[58].
Inaddition,somestudiesweredesignedtocombineantibodiesandaptamersina
singlehybridLFA.Insuchanexample,CXC‐motifchemokineligand(CXCL)9(acritical
urinalbiomarkerforantibody‐mediatedrejectionofkidneytransplantation)wasdetected
onatrackingsandwichformatLFA[57].AntibodiesspecifictoCXCL9wereinitiallyuti‐
lizedascaptureprobesonthetestline,andthedetectionaptamersspecifictoCXCL9
wereconjugatedtoAuNPstoensuresignaltransduction.Theusageofaptamerandanti‐
bodyincombinationimprovedthebindingefficiencyofCXCL9.Further,thecaptureap‐
tamersspecifictotheAuNP‐conjugateddetectionaptamerswerealsoimmobilizedonthe
controllinetosecurethecorrectoperationoftheassay.InthepresenceofCXCL9inurine
samples,twodifferentredbandswerediscernedfordesignatingapositiveresult.Fur‐
thermore,theLODoftheassaywasreportedas10pg∙mL
−1
,andthespecificityoftheassay
wasdeterminedtobe71%bycomparingtheestimatedglomerularfiltrationrates(eGFR)
ofpatients,whichisabloodtestparameterforrenalfunction[111].
Inadditiontosandwichandcompetitiveformatassays,LFAsalsoexploitedthetar‐
get‐inducedconformationalchangeinthelabelledaptamersthroughadsorption–desorp‐
tionmechanisms[112–114].Theworkingprincipleofthisstrategyreliesontheadsorption
ofanaptameronthesurfaceofalabel,andthefollowingdesorptionoftheaptamerfrom
thelabelaftertheirconformationalchangesuponinteractionwiththetargetmolecules
[112].Intheliterature,thissensingstrategyhasalsobeencombinedwithduplexaptamers,
wherethelabelledduplexaptamersweredisassociatedfromtheircomplementary
Figure 2.
Examples of paper-based POC aptasensors: (
A
) Sandwich LFA using streptavidin-biotin im-
mobilization for simultaneous capturing albumin and glycated albumin on test line [
55
]; (
B
) Working
principle of cortisol LFA with an adsorption–desorption sensing mechanism, where either (i) cortisol
capture is detected via AuNPs-linker hybridization on the test line (positive result), or (ii) no cortisol
is captured on the test line (negative result) [
53
]; (
C
) operations steps of paper-based microfluidic
integrated blood potassium concentration detection device [58].
In addition, some studies were designed to combine antibodies and aptamers in a
single hybrid LFA. In such an example, CXC-motif chemokine ligand (CXCL) 9 (a critical
urinal biomarker for antibody-mediated rejection of kidney transplantation) was detected
on a tracking sandwich format LFA [
57
]. Antibodies specific to CXCL 9 were initially
utilized as capture probes on the test line, and the detection aptamers specific to CXCL
9 were conjugated to AuNPs to ensure signal transduction. The usage of aptamer and
antibody in combination improved the binding efficiency of CXCL 9. Further, the capture
aptamers specific to the AuNP-conjugated detection aptamers were also immobilized on
the control line to secure the correct operation of the assay. In the presence of CXCL 9 in
urine samples, two different red bands were discerned for designating a positive result.
Furthermore, the LOD of the assay was reported as 10 pg·mL−1, and the specificity of the
assay was determined to be 71% by comparing the estimated glomerular filtration rates
(eGFR) of patients, which is a blood test parameter for renal function [111].
In addition to sandwich and competitive format assays, LFAs also exploited the target-
induced conformational change in the labelled aptamers through adsorption–desorption
mechanisms [
112
–
114
]. The working principle of this strategy relies on the adsorption of an
aptamer on the surface of a label, and the following desorption of the aptamer from the label
after their conformational changes upon interaction with the target molecules [
112
]. In the
literature, this sensing strategy has also been combined with duplex aptamers, where the
labelled duplex aptamers were disassociated from their complementary aptamers via target-
aptamer binding. An example of this strategy was demonstrated for monitoring cortisol—a
stress level-associated steroid hormone—in human saliva [
53
]. In this assay, duplex DNA
was formed with the hybridization of AuNPs-conjugated linker DNA and cortisol-specific
Biosensors 2023,13, 569 9 of 34
DNA aptamer. The duplex DNA aptamer was separated from its complementary aptamer
in the presence of cortisol due to the conformational change in the aptamer, and the cortisol-
bound aptamer was released. The remaining linker-AuNP conjugation was captured using
a linker complementary strand on the test line. Furthermore, the assay was validated with
a different complementary sequence on the control line which was able to interact with
both separated and unseparated duplex DNA structures (Figure 2B). Cortisol was detected
with a LOD of 0.37 ng
·
mL
−1
with the use of this assay. The same research group have also
employed a similar duplex DNA dissociation strategy for dopamine detection in urine,
which was able to detect dopamine concentration as low as 10 ng·mL−1[54].
The integration of microfluidic platforms with disposable paper strips has also been
utilized for aptamer-based biomarker detection applications [
115
–
121
]. Recently, a low-cost
and smartphone-adapted sensing platform was introduced for measuring potassium ion
(K
+
) levels in whole blood, which is an essential parameter for chronic kidney disease [
58
].
In short, the platform comprises PMMA-sealed paper microfluidic chip and finger pumps
for transporting different reagents into the reaction chamber. A four-strand aptamer struc-
ture was utilized as a detection probe, which conformationally changed into a
G-quartet
structure upon interaction with K
+
ions. At first, the aptamers were adsorbed onto the
surface of the AuNPs via electrostatic force. Then, whole blood was introduced to the
AuNPs–aptamer complex, and the complex conformationally turned into G-quartet struc-
tures after target interaction, releasing the AuNPs due to a weakened electrostatic force.
Finally, the bare AuNPs were aggregated with NaCl introduction, and transduced a K
+
ion-specific color change (Figure 2C). The LOD of the platform was reported as 0.01 mM,
and had an affordable fabrication cost (US $0.50 per microchip).
3.1.2. In-Solution Colorimetric Aptasensors
Though in-solution colorimetric aptasensors are rapid and easy to operate, they require
a relatively large reagent volume than those of paper-based platforms [
122
]. They mostly
utilize nanoparticles or enzymes to produce a color change, relying on the concentration
of target analytes. In addition, metal nanoparticles, especially AuNPs, have an intrinsic
localized surface plasmon (LSPR) property, where the electrons are collectively oscillating
between a nanostructure and dielectric interface that is induced with the electromagnetic
interaction of incident light beam [
7
,
123
–
125
]. This phenomenon has benefited numer-
ous colorimetric sensing platforms since the absorption spectrum of these materials was
found in the visible region of the electromagnetic spectrum [
126
]. Many solution-based
aptasensors utilize the absorption–desorption mechanism of aptamers onto AuNPs due
to the changes in electrostatic interactions. A common working principle is that aptamer
functionalized AuNPs are protected against NaCl until they are subjected to a conforma-
tional change upon target binding. Aptamers are released from the surface of AuNPs due
to target–aptamer binding. Desorption of aptamers weakens the electrostatic repulsion be-
tween AuNPs and exposes the particles to negative charge neutralization (NaCl). This leads
to AuNPs aggregation which causes a color change from red to blue [
127
]. An example
of such a sensing mechanism was reported for monitoring the levels of human epidermal
growth factor receptor 2 (HER2), a potential breast cancer biomarker, in human serum [
59
].
HER2 specific aptamers were initially adsorbed on the surface of AuNPs after an incubation
step. Upon HER2 introduction into AuNPs–aptamer structures, the AuNPs were released
due to the HER2 binding induced conformational changes. Further, NaCl was applied
to the solution for aggregating bare AuNPs, leading to a HER2 concentration-dependent
color change. This assay provided a rapid and cost-effective platform for breast cancer
diagnosis with a LOD of 10 nM. A similar AuNPs aggregation strategy was introduced for
capturing retinol-binding protein 4 (RBP4), a potential biomarker for the early diagnosis of
type 2 diabetes mellitus [
60
]. This assay improved some contents and assets of conventional
RBP4 detection methods, such as ELISA and Western Blot, and reduced both the response
time (from several hours to 5 min) and LOD values (90.76
±
2.81 nM). Another study
demonstrated the detection of interleukin-6 (IL-6), a peptide used for the early diagnosis
Biosensors 2023,13, 569 10 of 34
of brain injury or inflammation, in a bed-side setting [
62
]. In this study, a sandwich type
of two complimentary aptamer-AuNPs conjugates were employed for the capture of IL-6
where each aptamer targeted a different binding location of IL-6. The color of AuNPs
changed from red to pink within 5 min of introducing the IL-6 (Figure 3A). The assay was
able to detect IL-6 down to 1.95
µ
g
·
mL
−1
within a concentration range of 3.3–125
µ
g
·
mL
−1
.
In addition to protein biomarkers, researchers have performed significant research
on the detection of extracellular vesicles, which carry crucial information in a packaged
nano- to micron-sized entities [
128
–
131
]. As an example, a platform was developed for the
detection of leukemia-derived exosomes using terminal deoxynucleotidyl transferase and
salt-induced AuNPs aggregation. Magnetic beads were used to isolate exosomes, which
were selectively captured using nucleolin–aptamer AuNPs (Figure 3B). Thereafter, the
LOD was improved to 42 particles
·µ
L
−1
using a double signal amplification method [
61
].
Despite most of the in-solution studies using a direct visual output with salt-induced
AuNPs aggregation, one study utilized the photothermal effect of AuNPs for tracking
biomarkers via temperature change. In this study, the photothermal effect of AuNPs was
used for tracking biomarkers via temperature changes for a DNA detection assay targeting
Mycobacterium tuberculosis. AuNPs that had been conjugated with ssDNA interacted
with target DNA, leading to the aggregations, which had a high photothermal effect under
a near-infrared laser. The LOD of the assay was 0.28 nM, with a response time under
40 min [
63
]. Metal nanoparticles rather than AuNPs were also indirectly employed for
provoking a target-specific color intensity [
132
]. Such a study was reported for tumor-
originated exosome detection by indirectly exploiting platinum nanoparticles (PtNPs) [
133
].
Researchers used PtNPs to reduce H
2
O
2
into O
2
, generating a flame emission that cor-
related with captured CTC-originated exosomes. Exosomes were sandwiched between
CD63-specific aptamer-functionalized MNPs and EpCAM-modified PtNPs (Figure 3C). The
number of exosomes was measured using the changes in the green intensity of the atomic
flame, with a LOD of 7.6
×
10
2
particles
·
mL
−1
within a 1 min reaction response. Silver
nanoparticles (AgNPs) have also gathered attention as a colorimetric label for aptamers
due to their availability as a cost-effective material, and their ease of functionalization [
134
].
One study developed a simple colorimetric detection method for adenosine in human urine
using AgNPs and adenosine-specific aptamers [
135
]. The aptamers prevented particle
aggregation in high NaCl concentrations and complexed with adenosine to stabilize the
particles against aggregation. The concentration of adenosine was determined by measur-
ing the dispersity and absorption intensity of the AgNPs, with a LOD of 21 nM in a linear
range of 60–280 nM. Beyond nanoparticle-induced color change, enzymes were also utilized
for colorimetric aptasensors. Such an example was reported for the capture of alkaline
phosphate (AP) isozymes (placental alkaline phosphatase (PLAP) and intestinal alkaline
phosphatase (IAP)) for the clinical diagnosis and prognosis of colorectal cancer. PLAP–IAP
isozymes were initially captured using MNPs functionalized aptamers, which had affinity
against AP heterodimers. Target isolation was obtained with the magnetic separation of
aptamer-MNPs. PLAP–IAP isozymes catalyzed a chromogen (p-nitrophenyl phosphate,
pNPP) using an enzyme–signal amplification mechanism for inducing a color change in
the presence of the target (Figure 3D). The receiver operating characteristic (ROC) analysis
of the assay demonstrated a sensitivity of 92% and the specificity as 82% compared to the
results derived from the ones in clinical practice [
136
]. The emergence of novel nanozymes,
i.e., artificial nanomaterials that exhibit physicochemical properties of natural enzymes
(e.g., Fe
3
O
4
nanoparticles), has presented attractive application alternatives to conventional
catalytic labels (e.g., horseradish peroxidase: HRP) [
137
]. A nanoenzyme-based sandwich
formatted aptamer sorbent assay (NLASA), for instance, was reported for monitoring
platelet-derived growth factor-BB (PDGF-BB) using naked eye detection [
64
]. The assay
operated similarly to a regular sandwich-based ELISA where, instead of the antibody–
enzyme complex, aptamer-functionalized Fe
3
O
4
@C nanowires were utilized as nanozymes
for catalyzing the oxidation reaction of tetramethylbenzidine. This reaction caused a color
change in the presence of PDGF-BB. The usage of single dimension nanowires eliminated
Biosensors 2023,13, 569 11 of 34
the drawbacks caused by the instability of regular peroxidase enzymes. This aptasensor
was able to detect down to 10 fM, within a working range between 10 fM to 100 fM.
Biosensors2023,13,xFORPEERREVIEW11of35
instance,wasreportedformonitoringplatelet‐derivedgrowthfactor‐BB(PDGF‐BB)using
nakedeyedetection[64].Theassayoperatedsimilarlytoaregularsandwich‐basedELISA
where,insteadoftheantibody–enzymecomplex,aptamer‐functionalizedFe
3
O
4
@Cnan‐
owireswereutilizedasnanozymesforcatalyzingtheoxidationreactionoftetra‐
methylbenzidine.ThisreactioncausedacolorchangeinthepresenceofPDGF‐BB.The
usageofsingledimensionnanowireseliminatedthedrawbackscausedbytheinstability
ofregularperoxidaseenzymes.Thisaptasensorwasabletodetectdownto10fM,within
aworkingrangebetween10fMto100fM.
Figure3.Workingprincipleschematicsofsolution‐basedPOCaptasensors:(A)Theaggregationof
AuNPs‐integratedcomplementarysandwichaptamersforIL‐6determination[62],(B)Integration
ofMNPsandAuNPstoaptamersforthetrackingoftumor‐expressedexosomes[61],(C)Visual
detectionofmagneticallyisolatedexosomesviaCu
2+
drivenatomicflameassay[133],(D)Isolation
andtrackingofCTCsviaMNPsdrivenmagneticseparationandAPcatalyzedenzymaticvisual
assay,respectively[136].
3.2.FluorescentAptasensors
Fluorescentaptasensorsarecapableofhighsensitivity(lowsignal‐to‐noiseratio)
witharapidturnaroundtime[138].Thegenerationoffluorescenceresponseisobtained
usingthemodificationofaptamerswithfluorophores,fluorescentproteins,quantumdots
(QDs),andupconversionnanoparticles(UCNPs)[139].Targetinduced‐conformational
changeoffluorescentlylabelledaptamersprovidestheopportunityfordevelopingfluo‐
rescencequenchingorfluorescenceenhancementcoupledaptasensors.Therefore,we
haveclassifiedfluorescentaptasensorsintotwosubcategoriesaccordingtotheirsensing
mechanisms,i.e.,fluorescencequenchingormetal‐enhancedfluorescence(MEForplas‐
mon‐enhancedfluorescence(PEF)).
3.2.1.FluorescenceQuenching‐BasedAptasensors
Försterresonanceenergytransfer(FRET)isanonradiativeenergytransferbetween
donorandquencher,usefulformonitoringbiorecognitionchanges[140,141].Theeffi‐
ciencyofFRETdependsonthespectraloverlap,separationdistance,andrelativeorienta‐
tion[142–144].Bymodifyingaptamerswithquenchersandfluorophores,fluorescence
quenchingcanbeusedasatoolforbiomarkertrackingbyobservingtheaptamerconfor‐
mationaldynamics.
Figure 3.
Working principle schematics of solution-based POC aptasensors: (
A
) The aggregation of
AuNPs-integrated complementary sandwich aptamers for IL-6 determination [
62
], (
B
) Integration
of MNPs and AuNPs to aptamers for the tracking of tumor-expressed exosomes [
61
], (
C
) Visual
detection of magnetically isolated exosomes via Cu2+ driven atomic flame assay [133], (D) Isolation
and tracking of CTCs via MNPs driven magnetic separation and AP catalyzed enzymatic visual assay,
respectively [136].
3.2. Fluorescent Aptasensors
Fluorescent aptasensors are capable of high sensitivity (low signal-to-noise ratio) with
a rapid turnaround time [
138
]. The generation of fluorescence response is obtained using
the modification of aptamers with fluorophores, fluorescent proteins, quantum dots (QDs),
and upconversion nanoparticles (UCNPs) [
139
]. Target induced-conformational change
of fluorescently labelled aptamers provides the opportunity for developing fluorescence
quenching or fluorescence enhancement coupled aptasensors. Therefore, we have classified
fluorescent aptasensors into two subcategories according to their sensing mechanisms,
i.e., fluorescence quenching or metal-enhanced fluorescence (MEF or plasmon-enhanced
fluorescence (PEF)).
3.2.1. Fluorescence Quenching-Based Aptasensors
Förster resonance energy transfer (FRET) is a nonradiative energy transfer
between donor and quencher, useful for monitoring biorecognition changes [
140
,
141
].
The efficiency of FRET depends on the spectral overlap, separation distance, and relative
orientation [
142
–
144
]. By modifying aptamers with quenchers and fluorophores, fluores-
cence quenching can be used as a tool for biomarker tracking by observing the aptamer
conformational dynamics.
Recently, steroid hormones have been observed to quench fluorescence signals of
QDs by means of surface adsorption, which was utilized to develop a selective cortisol
(a biomarker for numerous diseases including Cushing’s syndrome and stress disorders)
monitoring assay in saliva [
65
]. QDs that had been functionalized with MNPs were
modified with cortisol-specific aptamers for fluorescence quenching. The assay exhibited
an LOD down to 1 nM within a 20 min turnaround time. FRET was used for simultaneous
detection of the Alzheimer biomarkers A
β
and tau protein with polydopamine-capped
gold nanorods (AuNRs@PDA) and dual-color CdSe/CdS/ZnS QDs [
66
]. Aptamer-target
Biosensors 2023,13, 569 12 of 34
binding caused conformational changes and selective fluorescence recovery under single
wavelength excitation (Figure 4A). The aptasensor had LOD values of 50 pM for A
β
and
10 pM for tau, providing a simple and effective Alzheimer diagnosis platform compared
to ELISA. A wearable and paper-based FRET aptasensor was developed for monitoring
cortisol concentrations [
70
]. MoS
2
nanosheets on carboxyfluorescein-modified aptamers
formed the FRET mechanism. The hybrid 3D origami microfluidic device integrated
with a custom build smartphone-mounted detector was used to monitor fluorescence
recovery (Figure 4B). The presence of cortisol in the perspired sweat intervened with the
FRET mechanism by conformationally changing the aptamer structure. The fluorescence
intensity was recovered proportionally to the captured amount of cortisol, and the LOD
was 6.76 ng·mL−1in artificial sweat.
Biosensors2023,13,xFORPEERREVIEW12of35
Recently,steroidhormoneshavebeenobservedtoquenchfluorescencesignalsof
QDsbymeansofsurfaceadsorption,whichwasutilizedtodevelopaselectivecortisol(a
biomarkerfornumerousdiseasesincludingCushing’ssyndromeandstressdisorders)
monitoringassayinsaliva[65].QDsthathadbeenfunctionalizedwithMNPsweremod‐
ifiedwithcortisol‐specificaptamersforfluorescencequenching.Theassayexhibitedan
LODdownto1nMwithina20minturnaroundtime.FRETwasusedforsimultaneous
detectionoftheAlzheimerbiomarkersAβ andtauproteinwithpolydopamine‐capped
goldnanorods(AuNRs@PDA)anddual‐colorCdSe/CdS/ZnSQDs[66].Aptamer‐target
bindingcausedconformationalchangesandselectivefluorescencerecoveryundersingle
wavelengthexcitation(Figure4A).TheaptasensorhadLODvaluesof50pMforAβand
10pMfortau,providingasimpleandeffectiveAlzheimerdiagnosisplatformcompared
toELISA.Awearableandpaper‐basedFRETaptasensorwasdevelopedformonitoring
cortisolconcentrations[70].MoS
2
nanosheetsoncarboxyfluorescein‐modifiedaptamers
formedtheFRETmechanism.Thehybrid3Dorigamimicrofluidicdeviceintegratedwith
acustombuildsmartphone‐mounteddetectorwasusedtomonitorfluorescencerecovery
(Figure4B).ThepresenceofcortisolintheperspiredsweatintervenedwiththeFRET
mechanismbyconformationallychangingtheaptamerstructure.Thefluorescenceinten‐
sitywasrecoveredproportionallytothecapturedamountofcortisol,andtheLODwas
6.76ng∙mL
−1
inartificialsweat.
Figure4.FluorescencebiosensorsforaptamerintegratingPOC:(A)FRETassaybasedonQDsmod‐
ifiedssDNAutilizationonpolydopaminecoatedAuNRsfortheearlydiagnosisofAlzheimer[66],
(B)paper‐basedandMoS
2
inducedfluorescentquenchingassayforcortisolmonitoringfromsweat
samples[70],(C)CHAamplifiedandfluorescencequenchedassayforvisualdetectionofCTCs[67],
and(D)ahybridantibodyandaptamerassayforMEFmediatedmalariatracking[72].
Apartfromfluorescentdyes,UCNPshavegainedattentionfortheirreducedinter‐
ferenceofautofluorescenceofpapersubstratesandscatteringlightsfrombiologicalsam‐
ples[145]duetotheirabilitytoconvertmultiplelow‐energyphotonsintoasinglehigh‐
energyphotonthroughNIRexcitation[146].Forinstance,UCNP–aptamer–TAMRAwas
employedtodetectIgE,anallergicbiomarker,onapaper‐basedfluorescentassay[71].
Theaptamer’sstem–loopstructurecausedaluminescenceenergytransfer,atypeofnon‐
radiativeenergytransferphenomenonsimilartotheFRETmechanism.Theadditionof
IgEdisruptedthestructureandincreasedthedistancebetweentheUCNPsandthe
TAMRA,recoveringafluorescencesignalproportionaltothecapturedIgE,withaLOD
of0.13IU∙mL
−1
.Astudyshowedthedetectionofmucin1(asurfaceproteinofcirculating
tumorcells(CTCs))throughsimultaneousvisualandfluorescentresponses[67].Catalytic
hairpinassembly(CHA)andcationexchangereactionswerecombinedforthetransduc‐
tionofsignals(Figure4C).Themucin1‐aptamerunderwentaconformationalchangein
Figure 4.
Fluorescence biosensors for aptamer integrating POC: (
A
) FRET assay based on QDs
modified ssDNA utilization on polydopamine coated AuNRs for the early diagnosis of Alzheimer [
66
],
(
B
) paper-based and MoS
2
induced fluorescent quenching assay for cortisol monitoring from sweat
samples [
70
], (
C
) CHA amplified and fluorescence quenched assay for visual detection of CTCs [
67
],
and (D) a hybrid antibody and aptamer assay for MEF mediated malaria tracking [72].
Apart from fluorescent dyes, UCNPs have gained attention for their reduced in-
terference of autofluorescence of paper substrates and scattering lights from biological
samples [
145
] due to their ability to convert multiple low-energy photons into a single
high-energy photon through NIR excitation [
146
]. For instance, UCNP–aptamer–TAMRA
was employed to detect IgE, an allergic biomarker, on a paper-based fluorescent assay [
71
].
The aptamer’s stem–loop structure caused a luminescence energy transfer, a type of non-
radiative energy transfer phenomenon similar to the FRET mechanism. The addition
of IgE disrupted the structure and increased the distance between the UCNPs and the
TAMRA, recovering a fluorescence signal proportional to the captured IgE, with a LOD of
0.13 IU
·
mL
−1
. A study showed the detection of mucin 1 (a surface protein of circulating
tumor cells (CTCs)) through simultaneous visual and fluorescent responses [
67
]. Catalytic
hairpin assembly (CHA) and cation exchange reactions were combined for the transduction
of signals (Figure 4C). The mucin 1-aptamer underwent a conformational change in the
presence of mucin 1, and then, accordingly, released P1-DNA, which was hybridized with
Ag+-functionalized hairpin DNA. The released Ag+ ions bound to QDs, quenching their
fluorescence and transducing a visible color change proportional to the amount of mucin
1 captured. The LOD was reported as 0.15 fg
·
mL
−1
mucin 1 or 3 CTCs
·
mL
−1
. A hybrid
biosensor using both antibodies and aptamers was developed to detect alpha-fetoprotein
(AFP), a biomarker for hepatocellular carcinoma [
68
]. The biosensor utilized silica-coated
CdTe QDs, in addition to anti-AFP monoclonal antibodies conjugated to AuNPs, with the
sandwich structure bringing the AuNPs and QDs in close proximity for FRET. The LOD
was reported as 400 pg
·
mL
−1
, with a dynamic range of 0.5–45 ng
·
mL
−1
. Fluorescence
Biosensors 2023,13, 569 13 of 34
quenching was used in a hollow hydrogel microneedle biosensor for detecting glucose,
ATP, L-tyrosinemia, and thrombin in interstitial fluid [
73
]. Aptamers conjugated to a flu-
orophore and a quencher functionalized DNA competitor were used. The hybridization
was disrupted in the presence of targets, causing the fluorescence signal to be recovered.
The assay had a 2 min turnaround time, and the LOD values were reported as 1.1 mM,
0.1 mM, 3.5
µ
M, and 25 nM for glucose, ATP, L-tyrosinamide, and thrombin, respectively.
The performance of this biosensor could be improved with different fluorophores for each
aptamer probe to increase target specificity.
3.2.2. Metal-Enhanced Fluorescence-Based Aptasensors
The fluorescence signal of low-concentration biomarkers requires bulky instruments
for efficient measurements, thereby limiting their expansion into the POC and bed-side
settings [
147
]. There are many efforts to resolve such challenges through the introduc-
tion of metal-enhanced fluorescence (MEF) via metal/metal oxide nanostructures (e.g.,
nanoparticles, nanorods, nanocavities, nanoholes, planar surfaces, etc.) [
147
–
149
]. In
short, MEF is the enhancement of fluorescence due to the coupling effect between fluores-
cence excitation/emission and plasmonic resonance of surface plasmons confined to metal
nanostructures [
147
,
150
]. For an efficient MEF application, the distance between metal
nanostructures needs to be optimized (mostly between 20–50 nm for planar plasmonic
structures [
147
]), since nonradiative quenching effects (such as FRET) may dominate at
short distances (mostly between 1–10 nm [
151
]). Recently, aptamers are shown to be per-
formed on MEF biosensors due to the tunability of surface plasmon–fluorophore separation
distances with the conformational change in the aptamers [
152
]. An example of a solution-
based MEF strategy was tested in detecting mutated breast cancer gene-1 (BRCA-1), a
pathogenic variant of tumor suppressor genes [
153
], by utilizing FRET and MEF strategies
at the same platform [
69
]. Two AuNPs, different in size (60 nm and 20 nm, respectively),
were modified by combining dsDNA and ssDNA. Clustered regularly interspaced short
palindromic repeats (CRISPR) is an evolving gene editing tool that recognizes and cleaves
target nucleic acid sequences with the use of the Cas 12 protein, which sweeps around the
nucleic acid sequence and determines the target for cleavage. In the study, two AuNPs
were combined by modifying them with ssDNA and fluorescein isothiocyanate (FITC) func-
tionalized dsDNA. This interaction formed FRET, since the particle distance between FITC
and AuNPs was 2 nm only. In the presence of BRCA-1, the AuNPs–ssDNA complex was
separated from the dsDNA–AuNPs structure. The remaining complex had a 7 nm distance
between FITC and other AuNPs, which caused signal amplification with MEF. BRCA-1
was detected as low as 0.34 fM within 30 min. The utilization of CRISPR-Cas9 eliminated
the requirement for nucleic acid amplification, and also presented high sensitivity.
Recently, a hybrid antibody–aptamer integrated immunosensor and close-packed,
honeycomb-structured AuNPs nanoarrays were combined for metal-enhanced fluorescent
detection of Plasmodium falciparum lactate dehydrogenase (PfLDH), a malaria marker,
from whole blood [
72
]. The target was sandwiched between an oriented malaria antibody
and a cyanine 5 (Cy5) modified malaria aptamer for adjusting the distance between the flu-
orophore molecule and plasmonic nanoarray (10 nm) for optimized plasmonic (Figure 4D).
The assay detected malaria target concentrations down to 18 fM (0.6 pg·mL−1).
3.3. SPR-Based Aptasensors
Since surface plasmon resonance (SPR) biosensors are the most extensively studied
class of optical biosensors, they have recently attracted immense interest from the scientific
community [
132
,
154
–
160
]. Research in plasmonics has primarily concentrated on automa-
tion, the integration of SPR biosensors, and the development of complex optical transducers
based on metallic nanostructures (i.e., nanoplasmonics), which improve the sensing ca-
pabilities and facilitate its miniaturization [
161
,
162
]. This research has been motivated
by an unmet need, hoping that POC biosensors might improve and promote healthcare
globally. The research and improvement of surface biofunctionalization techniques have
Biosensors 2023,13, 569 14 of 34
also been crucial to their successful clinical application, improving the sensitivity and
selectivity required for a reliable label-free analysis. SPR and LSPR biosensors’ ease of
use, reliability, and adaptability have stimulated the development of novel biomedical
tests that allow for noninvasive, more precise, timely, and informative detection of human
diseases [
163
]. Considering the basics of SPR-type systems, there are many efforts for
adapting, designing, and improving the Kretschmann configuration, which serves as the
foundation for the construction of numerous prism-assisted biosensors [
164
]. To detect
biomolecular interactions taking place at the sensor surface, prism-coupled systems use the
SPR phenomenon, which manifests as an intensity dip in the reflected light [
163
]. SPR is a
potent and popular biological and chemical sensing technology that can follow molecule
interactions in real time [165].
Aptamer-based SPR biosensors have drawn significant attention among the various
SPR-based sensing applications due to their ease-of-use, viability, and affordability for
target detection [
166
]. Notably, because there is no space for the aptamer to bind with
another molecule, most tiny molecules only bind aptamers with the one-site binding
configuration [
167
]. The mass of the binding component causes variations in the index of
refraction at the biosensor surface, which are detectable using an SPR instrument [168].
Over the years, aptamer-based POC urinary biosensors have been established to detect
diverse urinary markers at low concentrations, including 8-OHdG [
169
], cocaine [
170
],
advanced glycation end products (AGEs) [
171
], and dopamine [
172
]. A metabolic illness
that causes hyperglycemia due to inadequate insulin production, diabetes is a complex
chronic disease. To manage blood sugar, one must inject insulin every day for the rest of
one’s life. A crucial diagnostic tool for identifying and controlling medical disorders is gly-
cosylated hemoglobin (HBA1C) which is a crucial parameter for determining blood glucose
levels [
164
]. An integrated microfluidic biosensor for testing HBA1C was created by Chang
et al. [
173
]. The technique employed nucleic-acid aptamers to detect HBA1C with high
sensitivity and high specificity. The technology reduced the risk of diabetic complications
by returning results in 25 min and costing less than conventional procedures. Accord-
ing to Duanghathaipornsuk et al., the performance of the sensing was also influenced
by the strength of the binding affinity. For the SPR sensing of hemoglobin and glycated
hemoglobin, DNA nanocages were created to increase the binding stability and strength of
the aptamers to their target proteins [
174
]. The DNA aptamer-embedded origami cage struc-
ture produced 22-fold and 9-fold increases in binding affinity and selectivity for glycated
hemoglobin, compared to the ssDNA aptamer. To detect human thrombin and vascular
endothelial growth factor (VEGF) proteins, Chen et al. built a four-chambered microfluidic
SPR biosensor based on microarrays of RNA aptamers [
175
]. The surface transcription
reaction of T7 RNA polymerase could directly and swiftly synthesize RNA aptamers in
the microfluidic format, enabling one-step multiplexed protein biosensing. Numerous
target molecules could be analyzed simultaneously using a single SPR biosensor. The
reproducibility of the sensor array had a big impact on how reliable SPR biosensors were.
Inoue et al. reported an SPR aptasensor utilizing an inkjet spotter that could accurately
regulate the position and volume of an ejected aptamer solution to overcome this issue [
87
].
Simultaneous observations of SPR signals resulting from various thrombin concentrations
were obtained using a portable multianalysis SPR aptasensor with a capillary-driven flow
chip. By eliminating manual intervention during the preparation process and utilizing the
BlockAce reagent (which was frequently used as a blocking solution with ELISA technology
for separating biomolecule spots), this method dramatically increased the reproducibility
of SPR aptasensors. Thus, the SPR aptasensor’s detection limit was comparable to that of
other SPR biosensors (1 nM). According to Dejeu et al., the analyte recognition caused by
the conformational shift of aptamers caused the negative SPR signals to be seen during the
detection of tyrosinase [
176
]. They discovered that aptamer configuration rearrangement
caused the refractive index to increase by a small molecule, and the aptamer complex to
deviate from the total of the refractive index increments of the constituent parts. These
findings offer new perspectives and suggestions for comprehending the consequences of
Biosensors 2023,13, 569 15 of 34
the refractive index increment’s nonlinearity on variations in SPR signal. In addition, Hu
et al., have created SPR biosensors for dopamine detection based on the idea of noncova-
lent aptamer immobilization by covering the surface of a gold film with a single layer of
graphene [
88
]. They demonstrated that the presence of dopamine altered the structure of
the aptamer, which was capable of amplifying surface refractive index signals at the fiber
surface. It was shown that the use of graphene as a sensing layer for SPR could be useful
for small-molecule detection. With a lower limit of detection of 10
−13
M, the aptasensor
displayed remarkable sensitivity.
Sandwich-like detection techniques for aptamers have also been developed, and they
are similar to the idea of ELISA [
177
]. The SPR plastic optical fiber (POF) system with an
aptamer-based surface has been created for SARS-CoV-2 spike glycoprotein detection [
89
].
After creating a mixed layer of gold using a mixture of PEGthiol and BiotinPEGlipo, a
streptavidin coating was applied. Then, a biotin-modified aptamer was immobilized on
the streptavidin coating. An optical POC device beneficial in the early detection of the
SARS-CoV-2 virus, the plastic OF aptasensor has a LOD of 36.7 nM. Previously, VEGF,
(chosen as a circulating protein due to its being likely linked to cancer) had been detected
in the nanomolar range using an SPR POF system by Cennamo et al. [
178
]. They had
immobilized a DNA-aptamer sequence that is unique to VEGF on the gold surface.
It is well known that the SPR signal can be noticeably amplified with electronic cou-
pling between the localized surface plasmons of AuNPs and the SP waves connected to
a gold chip [
179
,
180
]. LSPR has an impact on the optical characteristics of AuNPs, which
can be exploited to boost SPR sensing [
166
]. Using the stable chemical conjugation of
mercapto- and amino- functional groups to gold, it is simple to link biological ligands with
AuNPs. An effective aptamer-based SPR biosensor for breast cancer-derived exosomes
with dual gold nanoparticle-assisted signal amplification was established by Wang’s re-
search group [
90
]. Exosomes collided with the gold substrate that had a CD63 aptamer
immobilized on it. To create a sandwich combination of CD63 aptamer/exosome/aptamer–
T30–AuNPs, aptamer-coated T30-linked AuNPs (aptamer–T30–AuNP) were then added.
This resulted in a single AuNPs-amplified SPR response. To achieve dual-signal amplifi-
cation, the A30-coated AuNPs were ultimately inserted through the hybridization of two
complementary sequences (T30 and A30). This technique made it possible to catch exo-
somes with a LOD of 5
×
10
3
exosomes
·
mL
−1
. The same team recently unveiled a different
amplification technique utilizing AuNPs with polydopamine functionalization [
181
]. On
the polydopamine-modified AuNPs, chloroauric acid was reduced with polydopamine
molecules to produce tiny AuNPs, which further improved the SPR response. Utilizing
polydopamine-modified AuNPs made exosome detection easier than using the earlier
technique of poly(A) and T-DNA hybridization. Jo et al. developed a highly sensitive
LSPR aptasensor for the direct detection of cortisol in saliva [
91
]. With a LOD of 0.1 nM,
the LSPR aptasensor demonstrated excellent detection performance for a broad range
of cortisol concentrations ranging from 0.1–1000 nM. (Figure 5) shows the foundations
of sampling salivary cortisol and creating an easy-to-use detection method for the LSPR
aptasensor. They showed that using aptamers in LSPR may enhance the detection of
small compounds, such as cortisol, when the size of AuNPs and the immobilization of the
aptamer were adjusted.
Biosensors 2023,13, 569 16 of 34
Biosensors2023,13,xFORPEERREVIEW16of35
compounds,suchascortisol,whenthesizeofAuNPsandtheimmobilizationoftheap‐
tamerwereadjusted.
Figure5.Therepresentationoftheproceduresneededtosamplesalivacortisolanddetectsalivary
cortisolusinganLSPRaptasensor[91].
Recently,SinghdemonstratedaQD‐basedaptasensorwithaptamerfunctionality,
whichwasusedtodetectinsulinindiabetespatients’serumsamples[92].Theschematic
fortheaptamer‐functionalizedQD‐basedaptasensorfordetectingseruminsulininpa‐
tientsamplesisshownin(Figure6A).High‐molecular‐weightdendrimerswereimmobi‐
lizedonthecysteaminelayerwithlessnonspecificbinding.Thedevelopedaptasensor
wascapableofdetectingseruminsulinconcentrationsaslowas5pM,whichiscriticalfor
identifyinginsulinlevelsinchallengingclinicalsamples.Theresultingaminogroupsof
cysteamineandthePAMAMdendrimer,towhichthecarboxylatedCdSe/ZnSQDswere
bound,werejoinedbyglutaraldehyde.ThedevelopedSPRaptasensor’sschematicsare
shownin(Figure6B).Becauseitsuccessfullyassessedinsulinlevelsinpatientsamples
withgoodsensitivity,specificity,andrepeatability,thedevelopedQD‐plasmon‐con‐
nectedmicrofluidicaptasensorisfavorable.
Figure 5.
The representation of the procedures needed to sample saliva cortisol and detect salivary
cortisol using an LSPR aptasensor [91].
Recently, Singh demonstrated a QD-based aptasensor with aptamer functionality,
which was used to detect insulin in diabetes patients’ serum samples [
92
]. The schematic
for the aptamer-functionalized QD-based aptasensor for detecting serum insulin in patient
samples is shown in (Figure 6A). High-molecular-weight dendrimers were immobilized
on the cysteamine layer with less nonspecific binding. The developed aptasensor was
capable of detecting serum insulin concentrations as low as 5 pM, which is critical for
identifying insulin levels in challenging clinical samples. The resulting amino groups of
cysteamine and the PAMAM dendrimer, to which the carboxylated CdSe/ZnS QDs were
bound, were joined by glutaraldehyde. The developed SPR aptasensor’s schematics are
shown in (Figure 6B). Because it successfully assessed insulin levels in patient samples
with good sensitivity, specificity, and repeatability, the developed QD-plasmon-connected
microfluidic aptasensor is favorable.
Biosensors 2023,13, 569 17 of 34
Biosensors2023,13,xFORPEERREVIEW17of35
Figure6.SPRmicroarrayaptasensordesignforseruminsulindetection.(A)Theschematicforthe
aptamer‐functionalizedQD‐basedsensor.(B)Differenceimageandrespectivesensogramsofse‐
ruminsulin‐Abinsulin‐MNPconjugatesbindingontheimmobilizedaptamermicroarray[92].
UsingaHER2proteinbiomarker,anOF‐SPRforbreastcancerdetectionwaspub‐
lishedbyLoyezetal.[93].TospecificallydetectHER2proteins,anti‐HER2ssDNAap‐
tamersweredirectlybondedtothegoldsurface.TotargetHER2,thiolatedaptamerswere
immobilizedonthesurfaceofgold.Anti‐HER2antibodies(20g∙mL
−1
)wereusedassignal
enhancerstolowerthedevice’slimitofdetectionaftertheidentificationofHER2atlow
concentrations(Figure7A).TheamplitudespectrumthatwasreleasedfromtheOF‐SPR
setupresultedinadip‐curve,asshownin(Figure7B,C).Determinationoflowmolecular
masstargetsonOF‐SPRisachallengingtask.Asingle‐stepcis‐duplexedaptamer(cis‐DA)
andanAuNP‐integratedOF‐SPRplatformweredevelopedforsolvingthischallenge
[182].ThetargetwaschosenasssDNAforproof‐of‐conceptstudies.AuNPswerefunc‐
tionalizedwithaptamercomplementaryelements(ACE)tocreateacomplexwithlinker
DNAandaptamerconjugation.Beforethetarget’sintroduction,AuNPswerefoundnear
thesurfaceofgold‐coatedOF‐SPR.Theapplicationoftarget‐inducedACEbreakagewas
duetothehighaffinityofaptamerstowardsssDNA.Theincreasingdistancebetween
AuNPsandthefibersurfacecausedadecreaseintheSPRsignalinconjunctionwiththe
increasingssDNAconcentration.ThisOF‐SPR‐basedbiomarkercapturestrategyenabled
sixtimesthesignalamplificationcomparedtocis‐DAcomplexfreeplatformanddetected
thelowmolecularmasstarget,ssDNA,aslowas230nM.
Figure7.(A)Therepresentationofthegold‐coated,uncladfiberutilizedinasandwichconfigura‐
tionwithamplificationfromantibodiestodetectHER2moleculesusingSPR.(B)Thespectrometer
andawhitelightsourcearebothlinkedtotheopticalfiberprobe.Theinstrumentmaybeconnected
toalaptopandisportable.(C)GaussianSPRcurveachievedbythegold‐coatedOF[93].
Figure 6.
SPR microarray aptasensor design for serum insulin detection. (
A
) The schematic for the
aptamer-functionalized QD-based sensor. (
B
) Difference image and respective sensograms of serum
insulin-Abinsulin-MNP conjugates binding on the immobilized aptamer microarray [92].
Using a HER2 protein biomarker, an OF-SPR for breast cancer detection was published
by Loyez et al. [
93
]. To specifically detect HER2 proteins, anti-HER2 ssDNA aptamers were
directly bonded to the gold surface. To target HER2, thiolated aptamers were immobilized
on the surface of gold. Anti-HER2 antibodies (20 g
·
mL
−1
) were used as signal enhancers to
lower the device’s limit of detection after the identification of HER2 at low concentrations
(Figure 7A). The amplitude spectrum that was released from the OF-SPR setup resulted
in a dip-curve, as shown in (Figure 7B,C). Determination of low molecular mass targets
on OF-SPR is a challenging task. A single-step cis-duplexed aptamer (cis-DA) and an
AuNP-integrated OF-SPR platform were developed for solving this challenge [
182
]. The
target was chosen as ssDNA for proof-of-concept studies. AuNPs were functionalized
with aptamer complementary elements (ACE) to create a complex with linker DNA and
aptamer conjugation. Before the target’s introduction, AuNPs were found near the surface
of gold-coated OF-SPR. The application of target-induced ACE breakage was due to the
high affinity of aptamers towards ssDNA. The increasing distance between AuNPs and the
fiber surface caused a decrease in the SPR signal in conjunction with the increasing ssDNA
concentration. This OF-SPR-based biomarker capture strategy enabled six times the signal
amplification compared to cis-DA complex free platform and detected the low molecular
mass target, ssDNA, as low as 230 nM.
Biosensors2023,13,xFORPEERREVIEW17of35
Figure6.SPRmicroarrayaptasensordesignforseruminsulindetection.(A)Theschematicforthe
aptamer‐functionalizedQD‐basedsensor.(B)Differenceimageandrespectivesensogramsofse‐
ruminsulin‐Abinsulin‐MNPconjugatesbindingontheimmobilizedaptamermicroarray[92].
UsingaHER2proteinbiomarker,anOF‐SPRforbreastcancerdetectionwaspub‐
lishedbyLoyezetal.[93].TospecificallydetectHER2proteins,anti‐HER2ssDNAap‐
tamersweredirectlybondedtothegoldsurface.TotargetHER2,thiolatedaptamerswere
immobilizedonthesurfaceofgold.Anti‐HER2antibodies(20g∙mL
−1
)wereusedassignal
enhancerstolowerthedevice’slimitofdetectionaftertheidentificationofHER2atlow
concentrations(Figure7A).TheamplitudespectrumthatwasreleasedfromtheOF‐SPR
setupresultedinadip‐curve,asshownin(Figure7B,C).Determinationoflowmolecular
masstargetsonOF‐SPRisachallengingtask.Asingle‐stepcis‐duplexedaptamer(cis‐DA)
andanAuNP‐integratedOF‐SPRplatformweredevelopedforsolvingthischallenge
[182].ThetargetwaschosenasssDNAforproof‐of‐conceptstudies.AuNPswerefunc‐
tionalizedwithaptamercomplementaryelements(ACE)tocreateacomplexwithlinker
DNAandaptamerconjugation.Beforethetarget’sintroduction,AuNPswerefoundnear
thesurfaceofgold‐coatedOF‐SPR.Theapplicationoftarget‐inducedACEbreakagewas
duetothehighaffinityofaptamerstowardsssDNA.Theincreasingdistancebetween
AuNPsandthefibersurfacecausedadecreaseintheSPRsignalinconjunctionwiththe
increasingssDNAconcentration.ThisOF‐SPR‐basedbiomarkercapturestrategyenabled
sixtimesthesignalamplificationcomparedtocis‐DAcomplexfreeplatformanddetected
thelowmolecularmasstarget,ssDNA,aslowas230nM.
Figure7.(A)Therepresentationofthegold‐coated,uncladfiberutilizedinasandwichconfigura‐
tionwithamplificationfromantibodiestodetectHER2moleculesusingSPR.(B)Thespectrometer
andawhitelightsourcearebothlinkedtotheopticalfiberprobe.Theinstrumentmaybeconnected
toalaptopandisportable.(C)GaussianSPRcurveachievedbythegold‐coatedOF[93].
Figure 7.
(
A
) The representation of the gold-coated, unclad fiber utilized in a sandwich configuration
with amplification from antibodies to detect HER2 molecules using SPR. (
B
) The spectrometer and a
white light source are both linked to the optical fiber probe. The instrument may be connected to a
laptop and is portable. (C) Gaussian SPR curve achieved by the gold-coated OF [93].
Biosensors 2023,13, 569 18 of 34
3.4. Electrochemical Aptasensors
Electrochemical methods are advantageous because of their high sensitivity, low
detection limit, minimal time consumption, and low cost of equipment [
183
]. They have
received significant attention in developing aptamer-based biosensors due to their low-cost,
high sensitivity and selectivity, low detection limits, and the potential to develop flexible
POC systems [184–187].
Recently, significant attempts have been made to develop POC-based electrochemi-
cal aptasensors for several biomarkers. An electrochemical aptasensor, for instance, was
developed to detect a protein called MPT64 secreted from Mycobacterium tuberculosis (a Tu-
berculosis (TB) causative agent) and these biomarkers were detected using Electrochemical
Impedance Spectroscopy (EIS). Briefly, HS-(CH
2
)
6
-OP(O)
2
O-(CH
2
CH
2
O)
6
-TTTTT-aptamer
was immobilized on an Au electrode, where Au-based electrodes were used to self-assemble
thiol-based aptamers using gold-sulfur (Au-S) bonds. 6-Mercapto-1-hexanol and triethy-
lene glycol mono-11-mercaptoundecyl ether were used as the antifouling agent to avoid
nonspecific binding. The aptasensor exhibited a detection limit of 81 pM. It significantly
decreased the detection time down to 30 min compared to the detection time of both
traditional sputum microscopy and PCR (several hours or even days) [
74
]. Another elec-
trochemical aptasensor was developed using the same Au–S interaction strategy with a
screen-printed gold electrode to detect the Yersinia adhesin A (YadA) biomarker of Yersinia
enterocolitica from Yersinia species, a species capable of causing diarrhea, mesenteric lym-
phadenitis, arthritis, and bacterial blood poisoning called sepsis [
75
,
188
]. The aptamer
was selected using cell-SELEX, and likewise, the immobilization was performed through
the Au–S method on a screen-printed gold electrode. The device had a detection limit of
7.0
×
10
4
CFU
·
mL
−1
and could exhibit a linear performance range of 7.0
×
10
4
CFU
·
mL
−1
to 7.0 ×107CFU·mL−1[75].
Metal–organic frameworks (MOFs) are self-assembled organic-inorganic crystalline
nanomaterials consisting of metal ions surrounded by organic linkers [
189
]. MOFs have
a large surface area that makes them capable of adsorbing functional materials (such as
enzymes, DNA probes, and nanoparticles); the high porosity of MOFs enables them to
encapsulate signal molecules and release them to generate a strong signal response when
in contact with target materials. Moreover, they possess tunability of the framework,
which shows excellent potential in the field of biosensor development [
190
–
192
]. The use
of an electrochemical aptasensor with Zirconium-based MOFs (Zr-MOFs), as well as an
aptamer as the recognition element for exosomes, exhibited a detection range of 1.7
×
10
4
to
3.4 ×108
particles
·
mL
−1
. It could achieve a detection limit of 5
×
10
3
particles
·
mL
−1
[
77
].
C-reactive protein (CRP) is a nonspecific biomarker for cerebrovascular diseases, myocar-
dial infectious inflammation, and cancer; however, the precise measurement of CRP is
very challenging in POC settings [
193
,
194
]. An aptamer-based electrochemical biosen-
sor with enhanced signal modality was developed to detect (CRP) using a triple-step
strategy. Briefly, the probe molecule number was increased using a horseradish peroxidase-
labeled CRP antibody (HRP-Ab
CRP
) enzyme-catalyzed reaction; likewise, the use of a
Zeolitic imidazolate framework (ZIF
67
)-MOF with rhomboid dodecahedra structure was
observed to improve the specific surface area; and, carbonizing the ZIF
67
(C-ZIF
67
) increased
the conductivity. This three-step strategy enhanced the signal response of the biosensor
(Figure 8A). The developed biosensor could achieve a linear response range of 10 pg
·
mL
−1
to 10 µg·mL−1and a detection limit of 0.44 pg·mL−1[76].
Biosensors 2023,13, 569 19 of 34
Biosensors2023,13,xFORPEERREVIEW20of35
Thesensorexhibitedalinearrangefrom50fg.mL
−1
to50ng.mL
−1
andadetectionlimitof
9.79fg.mL
−1
[84].Two‐dimensional(2D)materialshavebeenreportedtoimprovetheelec‐
trodereactivityandchemicalloadingquantityontheelectrodeofbiosensorsduetotheir
electrochemicalpropertiesandhighactivesurfacearea[206].Hence,anelectrochemical
aptasensorwasdevelopedbymodifyingascreen‐printedcarbonelectrodewith2D
MoSe
2
/WSe
2
todetectabiomarkerforprimarylivercancernamedprimealpha‐fetopro‐
tein(AFP);thiswasabletoachieveadetectionlimitof0.65pg.mL
−1
(Figure8F)[85].More‐
over,theadditionofnanomaterials,suchasAuNPs,hasalsobeenattempted,andcaused
animprovementinthesignalresponse;asaresult,thedetectionlimitdecreasedfrom24
pg.mL
−1
to2.4pg.mL
−1
[207].
Figure8.(A)FabricationoftheaptasensorforCRPdetectionwithatriple‐stepsignalamplification
strategy[76].(B)Fabricationandα‐thrombindetectionusingaAu‐coatedNAAOmembraneap‐
tasensor[79].(C)Aptasensorfabricationforthedetectionofp24‐HIV[81].(D)Fabricationandα‐
SyndetectionofPPy‐COOHconductivepolymer‐basedaptasensor[82].(E)Signal‐enhancingmul‐
tiplehairpinassemblyforSARS‐CoV‐2detection[84].(F)Aptasensorfabricationwith2D
MoSe
2
/WSe
2
andAFPdetection[85].
Areagentlesselectrochemicalbiosensingmethodnamednanoscalemolecularpen‐
dulum(NMP),capableofdetectingproteinbiomarkersforcancerandCOVID‐19,has
beendevelopedbyimmobilizingantibody‐conjugatedDNAonascreen‐printedelectrode
(SPE)[208].Ferrocenewasusedastheredoxreporterinthismethodthatoxidizedatan
approximatepotentialof+400mV.However,whenthetargetwasboundbytheantibody,
thetarget‐boundantibodymovedslower,delayingtheoxidation,andthischangeinoxi‐
dationwasusedasthesensingprinciple[208,209].ThedevelopedSARS‐CoV‐2sensorfor
COVID‐19detectioncouldexhibitaLODof1fg.mL
‐1
fornucleoproteinsand20copies.mL
‐
Figure 8.
(
A
) Fabrication of the aptasensor for CRP detection with a triple-step signal amplification
strategy [
76
]. (
B
) Fabrication and
α
-thrombin detection using a Au-coated NAAO membrane ap-
tasensor [
79
]. (
C
) Aptasensor fabrication for the detection of p24-HIV [
81
]. (
D
) Fabrication and
α
-Syn
detection of PPy-COOH conductive polymer-based aptasensor [
82
]. (
E
) Signal-enhancing multiple
hairpin assembly for SARS-CoV-2 detection [
84
]. (
F
) Aptasensor fabrication with 2D MoSe
2
/WSe
2
and AFP detection [85].
Thrombin is used as the biomarker for hematological system disorders [
78
]. Human
α
-thrombin detecting aptasensor was developed based on the Au–S immobilization method
with MCH as the co-adsorbent (Figure 8B) [
79
]. The Au was coated with either nanoporous
anodized alumina or aluminum oxide (NAAO), due to its high surface-to-volume ratio,
structured pores, and high pore density [
195
]. The aptasensor exhibited selectivity towards
α
-thrombin over
γ
-thrombin and lysozyme. It could achieve a detection limit of 10 pM
with 500
µ
M concentration of human serum albumin (HSA) interfering protein, which
was better than the aptamer functionalized MoS
2
nanosheet/platinum (Pt) electrode-based
thrombin aptasensor (53 pM in 1% human serum) and then the other Au–S binding-based
aptasensors as well [
196
,
197
]. Furthermore, the implemented 4-electrode system improved
the sensor’s accuracy by eliminating the effect of the current carrying outer electrodes over
the voltage-measuring electrodes [79].
Materials such as graphene oxide (GO) and graphene have been reported to im-
prove the limit of detection and the sensitivity of electrochemical biosensors due to
their large aspect ratio and highly porous structure, respectively. A capacitive biosen-
sor with a laser-induced graphene (LIG) electrode was developed to detect thrombin using
the amide group generating 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochlo-
ride (EDC)/N-hydroxy succinimide (NHS) aptamer immobilization strategy [
78
]. In this
method, electrodes were modified with EDC/NHS mixture solution to develop carboxylate
group (-COOH) on the electrode. Consequently, the carboxylate group of the electrode
could be used to immobilize aptamers modified with amino group (-NH
2
) by creating the
covalent amide group (-CO-NH-) [
198
–
202
]. The amide bond-based immobilization has
Biosensors 2023,13, 569 20 of 34
much higher stability with no significant loss in signal after being in storage for five days,
compared to the 23–30% loss of thiolated DNA/mercaptohexanol electrode-based aptasen-
sors [
203
,
204
]. In another study, GO has been used as an enhancer in a voltammetry-based
electrochemical aptasensor for thrombin detection, using the same EDC/NHS aptamer
immobilization strategy. The aptasensor using GO as an enhancer had a range of 0.005 nM
to 50 nM with a detection limit of 1 pM [
80
]. However, the LIG-based aptasensor could
achieve a dynamic range of 0.01–1000 nM and a detection limit of 0.12 pM, which was
much higher than the GO enhancer-based aptasensor [
78
]. A screen-printed graphene
quantum dot (GQD) electrode-based electrochemical aptasensor with EDC/NHS immobi-
lized aptamers was developed for the early diagnosis of Human Immunodeficiency Virus
(HIV) through the detection of p24-HIV protein, which exhibited linear performance from
0.93 ng
·
mL
−1
to 93
µ
g
·
mL
−1
, and had a detection limit of 51.7 pg
·
mL
−1
(Figure 8C) [
81
].
The use of polypyrrole-2-carboxylic acid (PPy-COOH) has also been reported to immobilize
NH
2
-modified aptamers through amide group (-CO-NH-) generation (Figure 8D). For
detecting
α
-Synuclein (
α
-Syn), the biomarker for Parkinson’s disease, this PPy-COOH
conductive polymer-based aptasensor exhibited linear performance in a range of 1
×
10
–8
to 0.1 nM and had a detection limit of 1 ×10–6 pM [82].
A DNA tetrahedron (TDN) linked dual-aptamer (AS1411 and MUC1) based aptasen-
sor to detect the MCF-7 breast cancer cells was developed on Au electrodes based on the
Au–S approach and modified with MCH, Pt nanoparticles decorated porous coordina-
tion network-224 (PCN-224), G-quadruplex/hemin DNAzyme (GQH), and horseradish
peroxidase (HRP). The use of TDN provided the sensor with a uniform interface, using
two aptamers increased the cell culture efficiency, and using Pt/PCN-224 enhanced the
signal. Moreover, GQH and HRP increased the catalytic activity, thus improving the
aptasensor’s overall performance. The sensor exhibited a linear response range of 20 to
1
×
10
7
cells
·
mL
−1
and a detection limit of 6 cells
·
mL
−1
[
83
]. Using dual aptamers has
also been reported in the development of a sensor for odontogenic ameloblast-associated
protein (ODAM), a periodontal disease biomarker in gingival crevicular fluid (GCF) [
205
].
Avidin or streptavidin are tetrameric proteins with an affinity for biotin that have
been reported to be used to immobilize biotin-labeled aptamers. The streptavidin–biotin
immobilization was used to develop a dual-aptamer POC electrochemical aptasensor with
an enhanced signal response for detecting SARS-CoV-2, the causative agent of COVID-19.
Two aptamers bound the SARS-CoV-2 target in a sandwich assay for proximity binding,
increasing the local concentrations of DNA strands. This increased concentration resulted
in the displacement of DNA, converting the SARS-CoV-2 to output DNA that induced
a chain reaction of hybridization, creating long and linear concatemers with multiple
hairpin assemblies. These multiple hairpin assemblies were labeled with biotin, and
immobilized a substantially large amount of streptavidin–alkaline phosphatase (ST–ALP).
This large quantity of ALP could generate an enhanced signal reaction when reacting with
1-naphthol phosphate (1-NPP) through an electrochemical oxidation reaction (Figure 8E).
The sensor exhibited a linear range from 50 fg
·
mL
−1
to 50 ng
·
mL
−1
and a detection limit
of 9.79 fg
·
mL
−1
[
84
]. Two-dimensional (2D) materials have been reported to improve
the electrode reactivity and chemical loading quantity on the electrode of biosensors
due to their electrochemical properties and high active surface area [
206
]. Hence, an
electrochemical aptasensor was developed by modifying a screen-printed carbon electrode
with 2D MoSe
2
/WSe
2
to detect a biomarker for primary liver cancer named prime alpha-
fetoprotein (AFP); this was able to achieve a detection limit of 0.65 pg
·
mL
−1
(Figure 8F) [
85
].
Moreover, the addition of nanomaterials, such as AuNPs, has also been attempted, and
caused an improvement in the signal response; as a result, the detection limit decreased
from 24 pg·mL−1to 2.4 pg·mL−1[207].
A reagentless electrochemical biosensing method named nanoscale molecular pen-
dulum (NMP), capable of detecting protein biomarkers for cancer and COVID-19, has
been developed by immobilizing antibody-conjugated DNA on a screen-printed electrode
(SPE) [
208
]. Ferrocene was used as the redox reporter in this method that oxidized at an ap-
Biosensors 2023,13, 569 21 of 34
proximate potential of +400 mV. However, when the target was bound by the antibody, the
target-bound antibody moved slower, delaying the oxidation, and this change in oxidation
was used as the sensing principle [
208
,
209
]. The developed SARS-CoV-2 sensor for COVID-
19 detection could exhibit a LOD of 1 fg
·
mL
−1
for nucleoproteins and 20 copies
·
mL
−1
for
viral particles [
208
]. A paper-based electrochemical aptasensor was developed for cancer
biomarker detection, and it was capable of simultaneously measuring two biomarkers,
carcinoembryonic antigen (CEA) and neuron-specific enolase (NSE), using two sensing
electrodes. This paper-based aptasensor offered a low production cost, and could exhibit
high sensitivity, with a detection limit of 2 pg
·
mL
−1
for CEA and 10 pg
·
mL
−1
for NSE. In
addition, the aptasensor showed a maximum relative error of 7.81% for CEA and 22.43%
for NSE, when compared to commercially available systems. Furthermore, the simultane-
ous detection of multiple biomarkers showed the potential to improve the performance
accuracy of POC testing and achieve the early detection of cancer [
86
]. Methods such as
inkjet printing have also been attempted by developing carbon nanotube (CNT)-aptamer
complex ink based on the strong
π
–
π
stacking interaction of the nucleotide bases of the
ssDNA aptamer and sidewalls of the CNT. The developed aptasensor exhibited a detection
limit of 90 ng
·
mL
−1
against lysozyme with a linear range of 0 to 1.0
µ
g
·
mL
−1
[
210
]. Other
flexible platforms, such as polyester films, have also been considered for fabricating sensors
with more robust structures while keeping them flexible. A screen-printed electrochemical
strip for the early detection of Alzheimer’s disease (AD) was developed on a polyester film
by detecting the decrease in the amount of miRNA-29a. The decline of miRNA-29a levels in
brain has been reported among AD patients, which increases the BACE1 gene expression,
itself a risk factor for Alzheimer, making miRNA-29a a promising biomarker for AD. An
anti-miRNA-29a probe was used for the specific binding of the anti-miRNA-29a, which was
tagged with methylene blue (MB) as the redox mediator to generate the electrochemical
signal, and AuNPs were used to improve the signal. The sensor showed a LOD of 0.2 nM
in human serum [211].
4. Computational Approaches for Aptamer Modeling and POC Testing Integrations
After elaborating aptasensors and their properties, and considering the complexity
of the bio–nano interface in their structures, one must inquire as to which simulation and
theoretical computation methods can be used to study biosensors in a realistic and mean-
ingful way [
212
]. Due to experimental limitations at the microscopic level, the integrated
approaches that combine in silico design with experimental measurements have immense
potential to exacerbate the hurdles involved in developing biosensors [
212
,
213
]. This part
of the review presents an overview of the application of Molecular Docking calculation,
Molecular Dynamics Simulation (MD), Density Functional Theory (DFT), Quantum Me-
chanics and Molecular Mechanics (QMMM), and Artificial intelligence (AI) to study the
typical disease biomarkers, including aptamer, to design and develop biosensors [214].
4.1. Molecular Docking Calculation and Molecular Dynamics Simulation
Briefly, MD simulations predict how every atom in an aptamer, protein, or other
molecular systems moves over time, in accordance with a general model of physics [
215
].
A variety of important biomolecular processes, including ligand binding, protein folding,
and conformational changes can be captured with these simulations. MD simulations are
often applied in combination with experimental structural biology techniques including
cryo-electron microscopy, NMR, FRET, Electron Paramagnetic Resonance (EPR), and X-ray
crystallography [
216
]. In particular, aptamers have been studied in silico to enable the
identification of high-affinity aptamers for the design and development of biosensors [
215
].
For instance, the use of molecular docking calculation, MD simulation, and electrochemical
measurements identified five sensitive and selective RNA aptamers to apply for designing
an electrochemical biosensor to detect ammonium dissolved in water. They validated
their work experimentally by detecting target molecules and found that using different
aptamers led to a significant difference in the biosensor’s response [
217
]. Streptomycin is an
Biosensors 2023,13, 569 22 of 34
aminoglycoside antibiotic that is used to treatment of human and animal infections caused
by bacteria. Because of the serious side effects of this drug and finding a way to detect its
trace amount in food products and serum, Nosrati and Roushani applied molecular docking
calculations and MD simulations to predict the binding pocket of
79-mer
ssDNA aptamer in
the interaction with streptomycin. Their findings were in fair agreement with experimental
results and help to optimize aptamer efficiency in biosensing applications [
218
]. Chen et al.
proposed an optical liquid crystal (LC) biosensor (label-free, cost-effective, and aptamer-
based) for detection of insulin. They showed that their sensor is able to detect insulin
in diluted human urine and serum, and thus has a potential basis for POC testing [
219
].
Zhao et al., investigated a rapid and sensitive aptamer-based biosensor for the detection
of domoic acid (DA), an amnesic shellfish toxin produced by red tide algae known as
Pseudo-nitzschia. They explored the binding mechanism between DA and the aptamer using
a molecular docking calculation and MD simulation. In their study, the time of the process
of detection was 7 min [220].
4.2. Density Functional Theory
DFT is a mature theory that provides a reliable method for studying materials in their
crystal state, as well as their molecular structure. Various commercial and open-source
software are available to execute the DFT computations for particular systems. The combi-
nation of DFT and the designed experiment would be helpful to further our understanding
of how to design efficient, reliable, and cost-effective biosensors [
221
,
222
]. In 2020, Ouyang
et al. designed a self-powered photoelectrochemical (PEC) aptasensor for ultrasensitive
detection of Microcystin-LR (hepatotoxins released by cyanobacteria during eutrophication
process that is the most ubiquitous, plentiful, and varied congener) and they used DFT
computation to study the electron transfer path of the system. They proved that their
structure would be a good candidate in the field of sensing [
223
]. For the first time, Ouyang
et al. synthesized a 3D-printed bionic self-powered sensing device. Experimental results
and DFT computation showed that their aptasensor had high sensitivity and selectivity to
the detection of Bisphenol A (BPA) environmental toxins and active endocrine disrupters.
Their work provides a new strategy to detect BPA in food and environmental samples [
224
].
Fernandez et al., reported a disposable POC sensing platform specific for the detection of
salivary cortisol. Their biosensor used a cortisol-specific aptamer to achieve high specificity
to cortisol, and they then analyzed the activity of their biosensor using DFT computation.
The potential of their work was demonstrated by detecting the salivary cortisol variations
in five humans [
225
]. Li et al., fabricated the (BiVO4/2D-C3N4/DNA) aptamer photo-
electrochemical (PEC) biosensor. Using DFT computation, they showed that their sensor
provided an excellent detection property for Microcystin-LR molecule. In addition, by
changing the DNA aptamer, their biosensor showed a higher sensitivity for the detection
of heavy metal ions, antibiotics, and tumor markers [
226
]. Using a CD63 aptamer and
coupling DNA nanotechnology with single-atom catalysts, Zeng et al., synthesized the
single-atom biosensor. Their product served as a photoelectrochemical sensing platform to
detect biomolecules. Their study introduces new opportunities for protein diagnostics and
biosecurity [227].
4.3. Quantum Mechanics and Molecular Mechanics
Applying the QMMM methodology for studying biological systems containing several
thousands of atoms can be a promising strategy due to overcoming the cost of simula-
tion [
228
]. QMMM theory is an efficient approach to simulate physicochemical phenomena
while the modifications of electronic structures can be modeled using quantum mechanical
approaches and the environment can be approximated using classical molecular mechanical
approaches [
229
]. A combination of QMMM with the experimental result can provide us
with a possible way to study the modified systems. In 2022, for the first time, Karuppaiah
et al. reported an electrochemical cortisol aptasensor formed by conjugating methylene
blue with cortisol-specific DNA aptamers through the use of QMMM calculations and
Biosensors 2023,13, 569 23 of 34
molecular docking calculations. According to their results (Figure 9), the aptasensor mea-
sured clinically meaningful cortisol levels in human serum without any reagent [
230
]. By
using a highly controlled covalent functionalization strategy and QMMM calculation, Pur-
widyantri et al., could design ultrasensitive aptamer-based biosensors that can early detect
hepatitis C virus (HCV) core protein, facilitating the early diagnosis of HCV infections.
Their device is specific, and achieves attomolar detection of the viral protein in human
blood plasma [231].
Biosensors2023,13,xFORPEERREVIEW23of35
reagent[230].Byusingahighlycontrolledcovalentfunctionalizationstrategyand
QMMMcalculation,Purwidyantrietal.,coulddesignultrasensitiveaptamer‐basedbio‐
sensorsthatcanearlydetecthepatitisCvirus(HCV)coreprotein,facilitatingtheearly
diagnosisofHCVinfections.Theirdeviceisspecific,andachievesattomolardetectionof
theviralproteininhumanbloodplasma[231].
Figure9.MolecularDockingresultsoftheinteractionofaptamerand(A)cortisol,(B)progesterone,
and(C)testosterone[230].
4.4.ArtificialIntelligence(AI)
TheapplicationofAIandbiosensorshascausedthecross‐disciplinaryconceptofAI‐
biosensors.Inusualflexiblebioelectronicmaterials,suchastextiles,flexiblefilms,band‐
agesandpatches,playasignificantroleinAI‐biosensors[232].Intelligenthydrogelsthat
aresensitivetopH,temperature,ions,andmolecules[233],inadditiontocarbonmaterials
withsuchpropertiesaselectricalconductivityandlightweightflexibility[234],aswellas
smartpolymersthatcanchangecolor,solubility,orshapewhenaffectedbytemperature,
[235]aresomeexamplesofflexiblebioelectronicmaterialsthatwillfacilitatethefabrica‐
tionofAI‐biosensors.Furthermore,theAI‐biosensorshaverelevancetowirelessdata
communication,suchasradio‐frequencyidentification,Wi‐Fi,andBluetooth,totransmit
theinformationbetweenbiosensorsandsmartphone‐basedplatforms[232].Herein,it
shouldbementionedthatAIistheextractingknowledgefromdatawithouthumaninter‐
vention[236]andthismethodissuitableforthepredictionofmassivesequencesincom‐
parisontothestructured‐basedmethodsthatarenotproperforpredictingtheaffinityof
alargenumberofsequencestoonetargetsimultaneously[47].
5.Conclusions
Trackingbiomarkersinphysiologicalfluidsprovidesuswithawealthofinformation
regardingillnesspresence,treatmentprogress,anddiseaserisk.Inparticular,anideal
POCbiosensorholdsgreatpotentialtoprovidecriticalinformationtohealthcareprovid‐
ersandpatientsinresource‐constrainedcountries.ThetermASSURED(affordable,sen‐
sitive,specific,user‐friendly,rapid,equipment‐free,delivered)wascoinedtodefinea
flawlesstestfordevelopingcountrieswhereresourcesarelimited[13].Forinstance,Sinha
etal.developedamicrofluidicchipbiosensorwithfield‐effecttransistorsensorarrays,
usingaptamersascaptureprobes,forthedetectionofcardiovasculardiseasebiomarkers
[32].GiventhatitmeetspracticallyalloftheASSUREDstandards,thischipprovedvery
Figure 9.
Molecular Docking results of the interaction of aptamer and (
A
) cortisol, (
B
) progesterone,
and (C) testosterone [230].
4.4. Artificial Intelligence (AI)
The application of AI and biosensors has caused the cross-disciplinary concept of
AI-biosensors
. In usual flexible bioelectronic materials, such as textiles, flexible films, ban-
dages and patches, play a significant role in AI-biosensors [
232
]. Intelligent hydrogels
that are sensitive to pH, temperature, ions, and molecules [
233
], in addition to carbon
materials with such properties as electrical conductivity and light weight flexibility [
234
],
as well as smart polymers that can change color, solubility, or shape when affected by
temperature, [
235
] are some examples of flexible bioelectronic materials that will facilitate
the fabrication of AI-biosensors. Furthermore, the AI-biosensors have relevance to wireless
data communication, such as radio-frequency identification, Wi-Fi, and Bluetooth, to trans-
mit the information between biosensors and smartphone-based platforms [
232
]. Herein,
it should be mentioned that AI is the extracting knowledge from data without human
intervention [
236
] and this method is suitable for the prediction of massive sequences in
comparison to the structured-based methods that are not proper for predicting the affinity
of a large number of sequences to one target simultaneously [47].
5. Conclusions
Tracking biomarkers in physiological fluids provides us with a wealth of information
regarding illness presence, treatment progress, and disease risk. In particular, an ideal POC
biosensor holds great potential to provide critical information to healthcare providers and
patients in resource-constrained countries. The term ASSURED (affordable, sensitive, spe-
cific, user-friendly, rapid, equipment-free, delivered) was coined to define a flawless test for
developing countries where resources are limited [
13
]. For instance, Sinha et al. developed
a microfluidic chip biosensor with field-effect transistor sensor arrays, using aptamers as
capture probes, for the detection of cardiovascular disease biomarkers [
32
]. Given that it
meets practically all of the ASSURED standards, this chip proved very compatible for diag-
Biosensors 2023,13, 569 24 of 34
nostics. The device was portable and automated, minimizing human intervention, and the
chip could operate with clinical samples that had not yet been processed. Additionally, the
analysis’s sample volume was small, and the detection time was quick. Despite researchers
working hard to bring microfluidics to the market, there are very few microfluidic chips
commercially available that meet the ASSURED criteria. The market adoption of microflu-
idic technology, complicated and time-consuming governmental approval procedures,
and customer acceptance are barriers to its commercialization [
237
]. The REASSURED
requirements, where R and E stand for real-time connectivity and ease of sample collection,
will be the new standards that must be met by next-generation POC diagnostic equipment.
As shown in (Figure 10), fluorescent and electrochemical POC sensors are highly sensitive,
and their shelf life and stability are limited. Colorimetric POC sensors are easily available,
low-cost, and sensitive, but have a short shelf-life. On the other hand, SPR POC sensors
are portable, easy to operate and provide real-time analysis, but they are expensive and
require advanced devices.
Biosensors2023,13,xFORPEERREVIEW24of35
compatiblefordiagnostics.Thedevicewasportableandautomated,minimizinghuman
intervention,andthechipcouldoperatewithclinicalsamplesthathadnotyetbeenpro‐
cessed.Additionally,theanalysis’ssamplevolumewassmall,andthedetectiontimewas
quick.Despiteresearchersworkinghardtobringmicrofluidicstothemarket,thereare
veryfewmicrofluidicchipscommerciallyavailablethatmeettheASSUREDcriteria.The
marketadoptionofmicrofluidictechnology,complicatedandtime‐consuminggovern‐
mentalapprovalprocedures,andcustomeracceptancearebarrierstoitscommercializa‐
tion[237].TheREASSUREDrequirements,whereRandEstandforreal‐timeconnectivity
andeaseofsamplecollection,willbethenewstandardsthatmustbemetbynext‐gener‐
ationPOCdiagnosticequipment.Asshownin(Figure10),fluorescentandelectrochemi‐
calPOCsensorsarehighlysensitive,andtheirshelflifeandstabilityarelimited.Colori‐
metricPOCsensorsareeasilyavailable,low‐cost,andsensitive,buthaveashortshelf‐
life.Ontheotherhand,SPRPOCsensorsareportable,easytooperateandprovidereal‐
timeanalysis,buttheyareexpensiveandrequireadvanceddevices.
Figure10.Theprosandconsofaptamer‐basedPOCdevices.CreatedwithBioRender.com(ac‐
cessedon11May2023).
Antibodieshavetraditionallybeenusedastheprimaryrecognitionelementinbio‐
sensingstudies,butaptamersarebecominganalternativechoiceduetotheirhighaffinity.
Aptamerspossessseveraladvantagesoverantibodies,suchastheirabilitytorecovertheir
nativeconformationwithslightchangesinpH,saltconcentration,chelatingagents,and
temperature.Antibodies,incontrast,canundergoirreversibledenaturationwhenex‐
posedtotheseconditions,resultinginapermanentlossoftheirbiologicalactivityand
bindingability.Aptamerscanbesynthesizedinlargequantitieswithhighaccuracy,re‐
producibility,andatalowercostthanmonoclonalantibodies,whicharelaboriousand
expensivetoproduce.Unlikemodifiedantibodieswithalossofbindingactivity,ap‐
tamerscanundergochemicalmodificationswithoutsacrificingtheirbindingaffinity.Ap‐
tamersalsohaveexcellentthermalstability,whichallowsthemtowithstandroomtem‐
peratureconditionsforextendedperiodswithoutlossintheiractivity.Inaddition,ap‐
tamersarenontoxicandhavelowimmunogenicity,makingthemsuitableforbothinvitro
andinvivoapplications.Beforeaptamerscanbeemployedinclinicaltrialsormadecom‐
merciallyavailable,moreneedstobelearnedabouttheirpharmacokineticsandinterac‐
tionswiththeirtargets,astheresearchintothemisstillinitsinfancy.Inthisreview,we
Figure 10.
The pros and cons of aptamer-based POC devices. Created with BioRender.com (accessed
on 11 May 2023).
Antibodies have traditionally been used as the primary recognition element in biosens-
ing studies, but aptamers are becoming an alternative choice due to their high affinity.
Aptamers possess several advantages over antibodies, such as their ability to recover their
native conformation with slight changes in pH, salt concentration, chelating agents, and
temperature. Antibodies, in contrast, can undergo irreversible denaturation when exposed
to these conditions, resulting in a permanent loss of their biological activity and binding
ability. Aptamers can be synthesized in large quantities with high accuracy, reproducibility,
and at a lower cost than monoclonal antibodies, which are laborious and expensive to
produce. Unlike modified antibodies with a loss of binding activity, aptamers can undergo
chemical modifications without sacrificing their binding affinity. Aptamers also have ex-
cellent thermal stability, which allows them to withstand room temperature conditions
for extended periods without loss in their activity. In addition, aptamers are nontoxic and
have low immunogenicity, making them suitable for both
in vitro
and
in vivo
applications.
Before aptamers can be employed in clinical trials or made commercially available, more
needs to be learned about their pharmacokinetics and interactions with their targets, as
the research into them is still in its infancy. In this review, we have elaborated the usage
of aptamers for designing and developing novel POC biosensors, such as electrochemical,
optical, colorimetric, and fluorescent. Between these biosensors, most studies have been
carried out through optical modality including colorimetric, fluorescent, and SPR, and
Biosensors 2023,13, 569 25 of 34
followingly, electrochemical biosensors account for the second greatest number of efforts
in this area. In addition, we have mentioned the studies that use molecular docking, MD
simulation, DFT, and QMMM computation, in addition to the role and application of AI
for tackling the experimental limitations of aptamer development. It should be mentioned
that the number of studies that used the QMMM computations was low, and increasing the
number of studies on these computations can be a possible development for future studies.
Author Contributions:
Conceptualization, Writing—Original Draft, Review and Editing, Visualiza-
tion, Y.A.; Conceptualization, Writing—Original Draft, Review and Editing, M.A.; Writing—Original
Draft, Review and Editing, H.K.C.; Writing—Original Draft, Review and Editing, I.G.; Supervision,
Reviewing, Editing, Y.S.; Supervision, Reviewing, Editing, F.I. All authors have read and agreed to
the published version of the manuscript.
Funding:
Fatih Inci gratefully acknowledges the support from the Scientific and Technological Re-
search Council of Turkey (TÜB˙
ITAK), the 2232 International Fellowship for Outstanding Researchers
(Project No: 118C254), and the Turkish Academy of Sciences Outstanding Young Scientists Award
Program (TÜBA-GEB˙
IP). However, the entire responsibility of the publication/article belongs to
the owner of the publication/article. The financial support received from TÜB˙
ITAK does not mean
that the content of the publication is approved in a scientific sense by TÜB˙
ITAK. This work was
also supported by the Young Scientist Awards Program (BAGEP) award from the Science Academy.
Maryam Atabay and Ye¸seren Saylan gratefully acknowledge the support from TÜB˙
ITAK 2247-D
National Early-Stage Researchers Program (Project No: 121C226).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
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