biomedicines

Review
Aptamer-Based Biosensors for the Colorimetric Detection of
Blood Biomarkers: Paving the Way to Clinical Laboratory Testing
Anna Davydova * and Mariya Vorobyeva

Institute of Chemical Biology and Fundamental Medicine SB RAS, Akad. Lavrentiev Ave., 8,
630090 Novosibirsk, Russia; kuzn@niboch.nsc.ru
* Correspondence: anna.davydova@niboch.nsc.ru; Tel.: +7-383-363-5129

Abstract: Clinical diagnostics for human diseases rely largely on enzyme immunoassays for the
detection of blood biomarkers. Nevertheless, antibody-based test systems have a number of short-
comings that have stimulated a search for alternative diagnostic assays. Oligonucleotide aptamers
are now considered as promising molecular recognizing elements for biosensors (aptasensors) due to
their high affinity and specificity of target binding. At the moment, a huge variety of aptasensors
have been engineered for the detection of various analytes, especially disease biomarkers. However,
despite their great potential and excellent characteristics in model systems, only a few of these
aptamer-based assays have been translated into practice as diagnostic kits. Here, we will review
the current progress in the engineering of aptamer-based colorimetric assays as the most suitable
format for clinical lab diagnostics. In particular, we will focus on aptasensors for the detection of
blood biomarkers of cardiovascular, malignant, and neurodegenerative diseases along with common
inflammation biomarkers. We will also analyze the main obstacles that have to be overcome before
aptamer test systems can become tantamount to ELISA for clinical diagnosis purposes.

Keywords: aptasensors; colorimetric detection; blood biomarkers; point-of-care testing

Citation: Davydova, A.; Vorobyeva,
M. Aptamer-Based Biosensors for the
Colorimetric Detection of Blood
Biomarkers: Paving the Way to
1. Introduction
Clinical Laboratory Testing. Clinical diagnostics for infectious, oncological, autoimmune, and other diseases rely
Biomedicines 2022, 10, 1606. https:// on test systems based on the specific molecular recognition of certain disease biomarkers
doi.org/10.3390/biomedicines10071606 in patients’ blood. A great majority of diagnostic systems employ antibodies as analyte-
Academic Editor: Natalia Komarova
recognizing elements. The wide repertoire of specific antibodies, high sensitivity of the
assays, and availability of commercial diagnostic kits with straightforward, standardized
Received: 1 June 2022 protocols made ELISA a method of choice for measuring blood biomarkers. However,
Accepted: 4 July 2022 ELISA has several shortcomings that originate from the intrinsic properties of antibodies.
Published: 6 July 2022 Using antibodies requires strict storage and delivery conditions for diagnostic kits. Batch-
Publisher’s Note: MDPI stays neutral to-batch variations between different lots of the same antibody or differences in the affinity
with regard to jurisdictional claims in and specificity of antibodies for the same antigen made by different vendors can affect the
published maps and institutional affil- accuracy and reproducibility of the detection. The latter problem becomes especially acute
iations. in long-term studies.
At the same time, nucleic acid aptamers—short DNA or RNA fragments that bind
specified molecular targets due to a unique spatial structure—represent a prospective
alternative for protein antibodies (Table 1). Owing to their chemical nature, aptamers
Copyright: © 2022 by the authors. are stable to thermal denaturation, possess a much longer shelf-life, and have no strict
Licensee MDPI, Basel, Switzerland.
requirements for delivery and storage. The standard chemical synthesis of oligonucleotide
This article is an open access article
aptamers guarantees minimal batch-to-batch variations. Furthermore, the in vitro selection
distributed under the terms and
of aptamers takes place on a lab bench and does not require the immunization of animals;
conditions of the Creative Commons
therefore, aptamers can be readily selected even for non-immunogenic or toxic targets.
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).

Biomedicines 2022, 10, 1606. https://doi.org/10.3390/biomedicines10071606 https://www.mdpi.com/journal/biomedicines

Biomedicines 2022, 10, 1606 2 of 23

Table 1. Comparison of aptamers and monoclonal antibodies.

Aptamers Monoclonal Antibodies

Hybridoma technology, including
Selection method In vitro selection
immunization of animals
Synthesis method Chemical or enzymatic synthesis Produced using cell cultures
Cannot be obtained for
Limitations imposed on the
No limitations non-immunogenic
target molecules
or toxic substances
Affinity Kd ≈ 0.1–100 nM Kd ≈ 0.1–100 nM
Specificity High High
Irreversible denaturation
Can renaturate after heat treatment after heat treatment
Stability
Stable during long-term storage Very sensitive to delivery and
storage conditions
Immunogenicity Not shown High
Possibility of chemical modification Wide Limited

Currently, a large number of aptamer-based analytical systems (aptasensors) have

been proposed for food safety, environmental monitoring, and the diagnosis of various
diseases [1–9]. The relative ease of the chemical modification of aptamers and their com-
patibility with different biosensor platforms has provided a wide spectrum of detection
systems, from portable devices to very complex sensors. The overwhelming majority of
them are aptasensors with optical (colorimetric, fluorescent, or luminescent) [10,11] and
electrochemical types of detection [12,13]. It should be noted that aptasensors utilizing
fluorescent and electrochemical detection usually possess a high sensitivity and selectiv-
ity, but often need additional sample pre-processing, specialized equipment, and highly
qualified personnel.
Nevertheless, very few of these aptamer-based test systems have found practical
applications in real clinical laboratories. In our opinion, this may be because the wide
potential diversity of aptamer-compatible biosensor platforms led to the dissipation of
research efforts. In contrast, the characteristics of antibodies impose a greater number of
restrictions. This factor limits a choice of variants for diagnostic test systems and allows
for more in-depth concentration on each of them, which ultimately leads to practical
use. Moreover, aptamer-based tests often represent quite sophisticated systems of an
unconventional format, with equipment and protocols that are unusual for a clinical
laboratory. Therefore, they are poorly perceived by the medical community, who are the
end users of any diagnostic assay.
In the context of clinical diagnostics, colorimetric aptasensors have attracted particular
attention. They require only a standard spectrophotometer or colorimeter, which is routine
for any clinical lab, and imply typical ELISA protocols. At the moment, there are several
aptamer-based commercially available diagnostic kits for the colorimetric detection of indi-
vidual biomarkers in biological samples [14,15]. In our opinion, colorimetric aptasensors
seem to be the most prospective candidates for routine laboratory diagnostics.
In this review, we will focus on a critical analysis of the currently developed aptamer-
based colorimetric test systems, including their characteristics, limitations, and future
prospects. Since blood biomarkers are of the utmost importance in clinical diagnostics
and the monitoring of different diseases, we narrowed down the topic of this review to
colorimetric aptasensors for the detection of blood biomarkers.

2. Aptamer-Based Biosensors: General Principles of Detection

Colorimetric aptasensors fall into several groups based on their principle of colori-
metric signal generation. Here, we will list the most common types of detection employed
 Biomedicines 2022, 10, x FOR PEER REVIEW 3 of 24

2. Aptamer‐Based Biosensors: General Principles of Detection

Biomedicines 2022, 10, 1606 3 of 23
Colorimetric aptasensors fall into several groups based on their principle of colori‐
metric signal generation. Here, we will list the most common types of detection employed
in colorimetric aptasensors, which will be further discussed below. The first group of ap‐
in colorimetric aptasensors,
tasensors relies which provided
on the color change will be further discussedand
by the dispersion below. The first
aggregation group of
of AuNPs
aptasensors
in the presence of different salts (Figure 1). Unmodified AuNPs tend to aggregate in salt‐ of
relies on the color change provided by the dispersion and aggregation
AuNPs in the solutions,
containing presence of different
causing red salts (Figure
to blue color 1). Unmodified
changes. AuNPs tend
The non‐specific to aggregate
absorption of
in salt-containing solutions, causing red to blue color changes. The non-specific
polyanionic aptamers prevents the aggregation of AuNPs, and the color remains red. absorption
This
of polyanionic aptamers
type of detection is veryprevents thethe
simple, and aggregation
result can beof inspected
AuNPs, and thenaked
by the color eye.
remains red.
At the
This
same time, the different components of biological fluids (proteins, salts, etc.) could hinderAt
type of detection is very simple, and the result can be inspected by the naked eye.
thethe
same time, the different components
dispersion/aggregation of biological
of AuNPs, resulting fluids
in a lower (proteins,
sensitivity andsalts, etc.) could
selectivity of
hinder the dispersion/aggregation of AuNPs, resulting in a lower sensitivity and selectivity
detection.
of detection.

Figure
Figure 1. Colorimetric
1. Colorimetric detectionusing
detection usingthe
thedispersion/aggregation
dispersion/aggregation ofofgold
goldnanoparticles
nanoparticles (AuNPs)
(AuNPs)in in
salted
salted solution.
solution. UnmodifiedAuNPs
Unmodified AuNPsaggregate
aggregate in
in the
the salt‐containing
salt-containingsolution,
solution,turning a red
turning colored
a red colored
solution into a blue solution. The non‐specific absorption of nucleic acids prevents the aggregation
solution into a blue solution. The non-specific absorption of nucleic acids prevents the aggregation of
of AuNPs, and the solution remains red.
AuNPs, and the solution remains red.
The other group of colorimetric aptasensors generates an analytical signal through
The other group of colorimetric aptasensors generates an analytical signal through an
an enzymatic reaction. Horseradish peroxidase and alkaline phosphatase are typically
enzymatic reaction. Horseradish peroxidase and alkaline phosphatase are typically used
used in such aptasensors (Figure 2A). This type of test system provides a high sensitivity
in such aptasensors
and selectivity and(Figure 2A). This
is also fully type ofwith
compatible testELISA
systemprotocols
provides a high
and sensitivity
equipment. How‐and
selectivity
ever, theand is also fully
properties compatible
of protein with
enzymes canELISA
changeprotocols and equipment.
due to denaturation duringHowever,
storage orthe
properties of protein enzymes can change due to denaturation during storage
batch‐to‐batch variation, and this affects the reproducibility of the results. or batch-to-
batch variation, and this affects the reproducibility of the results.
Recently, non-covalent complexes of hemin with quadruplex-forming DNA and differ-
ent nanomaterials were proposed as peroxidase-mimicking non-protein analogs (Figure 2B).
This approach allows for the creation of more cost-effective and stable aptasensors.
 Biomedicines
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10, 1606 4 of 424
of 23

Figure
Figure 2. General
2. General scheme
scheme ofof detection
detection forfor a colorimetricaptasensor
a colorimetric aptasensorwith
withperoxidase
peroxidase(A)
(A)ororperoxidase-
peroxi‐
dase‐like (B) generation of the analytical signal. First, a biotinylated aptamer forms a complex with
like (B) generation of the analytical signal. First, a biotinylated aptamer forms a complex with the
the analyte in a microplate well; then, streptavidin‐conjugated peroxidase binds biotin. Next, perox‐
analyte in a microplate well; then, streptavidin-conjugated peroxidase binds biotin. Next, peroxidase
idase (A) or a peroxidase‐mimicking analog (B) oxidizes the chromogenic substrate, turning a col‐
(A)orless
or a peroxidase-mimicking analog (B) oxidizes the chromogenic substrate, turning a colorless
solution into a colored solution.
solution into a colored solution.
Recently, non‐covalent complexes of hemin with quadruplex‐forming DNA and dif‐
3. ferent
Aptasensors for Biomarker
nanomaterials Detection
were proposed as peroxidase‐mimicking non‐protein analogs
In the2B).
(Figure present
This review,
approachweallows
concentrated on the detailed
for the creation of moreanalysis of colorimetric
cost‐effective aptasen-
and stable ap‐
sors for the detection of biomarkers of various diseases, including oncological, cardiovascu-
tasensors.
lar, neurodegenerative, autoimmune, and inflammatory pathologies. When analyzing this
3. Aptasensors
data, for Biomarker
we paid particular Detection
attention to the sensitivity of the test systems (limit of detection)
and their selectivity (the ability to discriminate
In the present review, we concentrated molecules
on the detailedsimilar
analysistoofthe target). Table
colorimetric ap‐ 2
summarizes the characteristics of the published aptamer-based colorimetric test
tasensors for the detection of biomarkers of various diseases, including oncological, car‐systems.
diovascular, neurodegenerative, autoimmune, and inflammatory pathologies. When ana‐
lyzing
Table this data, we
2. Colorimetric paid particular
aptasensors for theattention
detectiontoofthe sensitivity
disease of the test systems (limit of
biomarkers.
detection) and their selectivity (the ability to discriminate molecules similar to the target).
Target Type of the Aptamer
Table Limit
2 summarizes the of Detectionof the published Selectivity
characteristics Ref.
aptamer‐based colorimetric test
systems.
DNA 0.3 pM [16]
DNA2. Colorimetric aptasensors
Table 18.0 for
nMthe detection of disease biomarkers.
[17]
VEGF165 DNA 2.6 nM
Target Type of the Aptamer Limit of Detection Selectivity Ref.
DNA 0.11 nM
DNA 0.3 pM [16] [18]
DNA
DNA 0.13 nM
18.0 nM [19]
[17]
VEGF165 DNA
DNA 2.6 nM [20]
DNA
DNA 0.11 nM
160 exosome/mL [18] [21]
DNA 0.13 nM [19]
DNA 7700 exosome/mL [22]
DNA [20]
CD63 DNA 5.2 × 105 exosome/mL [23]
CD63 DNA 160 exosome/mL [21]
DNA
DNA 13.5 × 105 exosome/mL
7700 exosome/mL [22] [24]
DNA 1.4 × 104 exosome/mL [25]
DNA Fibrocyte exosomes [26]
Biomedicines 2022, 10, 1606 5 of 23

Table 2. Cont.

Target Type of the Aptamer Limit of Detection Selectivity Ref.

DNA 83 nM [27]
DNA 0.09 µg/mL [28]
MUC1
Exosomes from normal liver
DNA 3.94 × 105 exosome/mL [29]
cells (L-02)
DNA 16.7 pM [30]
CEA DNA 2.2 pM [31]
DNA 5.5 pM [32]
PSA DNA 0.7 pM [33]
HER2 DNA 10 nM/20 nM (LFA) [34]
DKK1 DNA 2.3 pM [35]
RNA 1 pM [36]
Epinephrine, norepinephrine,
51 nM (RNA) 3-methoxytyramine,
RNA and DNA [37]
0.5 nM (DNA) 3,4-dihydroxyphenylacetic acid,
and homovanillic acid
3,4-dihydroxyphenylalanine,
catechol,
DNA 0.36 µM 3,4-dihydroxyphenylacetic acid, [38]
homovanillic acid, epinephrine,
and ascorbic acid
Dopamine
Hydroquinone, glucose, ascorbic
acid, L-phenylalanine,
0.14 µM (colorimetry) L-tryptophan, uric acid,
DNA [39]
78.7 nM (fluorescence) norepinephrine,
5-hydroxytryptamine, and
3,4-dihydroxyphenylalanine
0.6 µM (colorimetry)
DNA [40]
3.3 nM (fluorescence)
Cortisol, epinephrine
DNA ~0.3 µM [41]
norepinephrine, and serotonin
α-Syn oligomers DNA 10 nM [42]
DNA 150 nM [43]
Cortisol DNA 0.7 mM [44]
DNA 2.8 nM [45]
DNA 0.07 pM [46]
CRP
DNA 10 nM [47]
sIL-2Ra DNA 1 nM [48]
IL-6 DNA [49]
HNE DNA 0.4 pM [50]
Troponin T DNA 3.13 nM [51]
Troponin I DNA 0.5 pM [52]
HIF-1α DNA 2 fM [53]
Thrombospondin-1 DNA 7 fM [54]
DNA 0.1 mM [55]
HbA1c DNA [56]
20 -F-RNA [57]
Biomedicines 2022, 10, 1606 6 of 23

Table 2. Cont.

Target Type of the Aptamer Limit of Detection Selectivity Ref.

DNA 2.6 pM [58]
Insulin
DNA 0.2 pM [59]
DNA 3.7 nM [60]
RBP4
DNA 50 fM [61]
DNA 1 nM [60]
Vaspin
DNA 0.1 nM [62]
Visfatin DNA 0.4 nM [60]

3.1. Cancer
3.1.1. VEGF
Vascular endothelial growth factor (VEGF) is a signaling protein secreted by both nor-
mal endothelial and cancer cells that plays an important role in angiogenesis regulation [63].
It is now considered as an important biomarker for cancer [64], neurodegenerative diseases
(Alzheimer’s disease, Parkinson’s disease, etc.) [65,66], rheumatoid arthritis [67,68], and
psoriasis [69].
The selection of VEGF-binding DNA aptamers has been shown to result in quadruplex-
forming aptamers with a high affinity for their molecular target [70,71]. J. Dong used a
VEGF-specific DNA aptamer for colorimetric microplate sandwich-type detection [16]. In
the first step, a recombinant VEGF protein was immobilized in microplate wells, with the
subsequent addition of the biotinylated aptamer. Horseradish peroxidase conjugated with
streptavidin was used for aptamer–VEGF complex visualization. Free VEGF in the analyzed
samples bound to the aptamers in the microplate well, displacing the pre-immobilized
VEGF. Surface-unbound aptamers were then washed out, and the colorimetric signal
decreased with the rise in VEGF concentration. The limit of detection for the developed
assay was 0.3 pM in buffer solution. This aptasensor was also used for VEGF detection in
human serum samples. The obtained results agreed with the reference chemiluminescent
ELISA results. Notably, this aptasensor allowed for VEGF detection in serum samples
without any preliminary manipulations (filtration, precipitation, etc.), providing fast and
simple detection.
In general, G-quadruplex structures can bind the hemin molecule, and this complex
can oxidize a chromogenic substrate in the presence of hydrogen peroxide, thus mimicking
horseradish peroxidase activity. This feature of quadruplex-forming aptamers was shown
to be useful for the chemiluminescent detection of VEGF [17]. After target binding, the
aptamer forms an active quadruplex structure and then binds hemin; the resulting complex
catalyzes the oxidation of the substrate (luminol) in the presence of hydrogen peroxide
(Figure 3). In this study, the intensity of the luminescent signal linearly increased with the
rise in VEGF concentration in solution. The developed aptasensor had a high sensitivity
(the detection limit was 18 nM or 684 ng/mL); however, in the absence of a target, a rather
high nonspecific signal appeared due to the spontaneous quadruplex formation. After
dividing the aptamer into two separate oligonucleotides, the active quadruplex structure
formed only in the presence of VEGF, which significantly reduced the nonspecific signal.
As a result, the detection limit was lowered to 2.6 nM.
Wu et al. proposed a VEGF-specific aptasensor based on the color change in a colloid
solution of AuNPs [18]. The authors designed an aptazyme consisting of the VEGF-specific
aptamer and a DNAzyme connected by a short nucleotide sequence. Without a protein
target, the DNAzyme and aptamer form a hairpin that prevents DNA substrate cleavage.
Uncleaved DNA hybridizes with short complementary oligonucleotides on the surface of
the AuNPs, thus inducing particle aggregation and a color change from red to blue. In the
presence of VEGF, both the aptamer and DNAzyme are restored their active conformation.
Biomedicines 2022, 10, 1606 7 of 23

The selective cleavage of the substrate by the DNAzyme prevents the aggregation of AuNPs,
Biomedicines 2022, 10, x FOR PEER REVIEW 7 of 24
and the solution remains red. The developed aptasensor was shown to detect 0.1 to 100 nM
of VEGF in a buffer solution. The results of analysis in 1% spiked serum samples showed
good agreement with VEGF detection in a model buffer solution, thus demonstrating the
principal applicability of the assay for real clinical samples.

Figure 3. Chemiluminescent VEGF detection based on the peroxidase-mimicking activity of the hemin
and G-quadruplex aptamer complex
Figure 3. Chemiluminescent VEGF[17]. Targetbased
detection binding induces
on the quadruplex structure
peroxidase‐mimicking formation
activity of the he‐in
the VEGF aptamer.
min and The resulting
G‐quadruplex aptamer–target
aptamer complex [17]. complex binds hemin
Target binding inducesand catalyzesstructure
quadruplex the oxidation
for‐
mationin
of luminol in the
the presence
VEGF aptamer. The resulting
of hydrogen aptamer–target complex binds hemin and catalyzes the
peroxide.
oxidation of luminol in the presence of hydrogen peroxide.
C. Chang [19] et al. proposed an even more sensitive AuNP-based VEGF detection
assay with Wusignal
et al. proposed a VEGF‐specific
amplification, which takes aptasensor based
less than on thefor
an hour color
thechange
analysisin aand
colloid
does
solution of AuNPs [18]. The authors designed an aptazyme consisting
not recruit any enzymatic reactions. The system includes aptamer-containing hairpin of the VEGF‐spe‐
DNA, cifictwo
aptamer
DNAand a DNAzyme
substrates, and connected by a short
two auxiliary DNAnucleotide
fragments. sequence.
WithoutWithout a pro‐
the target, all
tein target, the DNAzyme and aptamer form a hairpin that prevents
the DNA molecules form “closed” intramolecular structures, but not intermolecular DNA substrate cleav‐
com-
age.Along
plexes. Uncleaved
withDNA this, hybridizes
the auxiliarywith shortfragments
DNA complementary oligonucleotides
are adsorbed on the sur‐
by the AuNPs, and
face of the AuNPs, thus inducing particle aggregation and a color change
the dispersed nanoparticles give a red color to the solution. In contrast, the addition offrom red to blue.
In the presence of VEGF, both the aptamer and DNAzyme are restored their active con‐
VEGF switches the aptamer to an active structure, which leads to the reorganization of the
formation. The selective cleavage of the substrate by the DNAzyme prevents the aggrega‐
hairpin DNA. “Opened” hairpin DNA, in turn, forms a duplex with the DNA substrate
tion of AuNPs, and the solution remains red. The developed aptasensor was shown to
and initiates a nonlinear chain reaction, producing the dendrimer-like structure containing
detect 0.1 to 100 nM of VEGF in a buffer solution. The results of analysis in 1% spiked
auxiliary DNA fragments (Figure 4). The poor adsorption of DNA dendrimers on AuNPs
serum samples showed good agreement with VEGF detection in a model buffer solution,
leads to an aggregation of nanoparticles and a red–blue color change. The detection limit
thus demonstrating the principal applicability of the assay for real clinical samples.
for REVIEW
Biomedicines 2022, 10, x FOR PEER this assay was [19]
C. Chang 0.13 et nMal.(10 ng/mL).
proposed anThe
evenstabilization
more sensitiveof AuNPs by additional
AuNP‐based oligonu-
VEGF detection8 of 24
cleotides prevented their aggregation in the absence of VEGF and further
assay with signal amplification, which takes less than an hour for the analysis and does improved the
limitnot
of detection to 185 pM (5 ng/mL). The developed assay was successfully
recruit any enzymatic reactions. The system includes aptamer‐containing hairpin tested on
VEGF-spiked samples of diluted (2.5%) blood serum.
DNA, two DNA substrates, and two auxiliary DNA fragments. Without the target, all the
DNA molecules form “closed” intramolecular structures, but not intermolecular com‐
plexes. Along with this, the auxiliary DNA fragments are adsorbed by the AuNPs, and
the dispersed nanoparticles give a red color to the solution. In contrast, the addition of
VEGF switches the aptamer to an active structure, which leads to the reorganization of
the hairpin DNA. “Opened” hairpin DNA, in turn, forms a duplex with the DNA sub‐
strate and initiates a nonlinear chain reaction, producing the dendrimer‐like structure con‐
taining auxiliary DNA fragments (Figure 4). The poor adsorption of DNA dendrimers on
AuNPs leads to an aggregation of nanoparticles and a red–blue color change. The detec‐
tion limit for this assay was 0.13 nM (10 ng/mL). The stabilization of AuNPs by additional
oligonucleotides prevented their aggregation in the absence of VEGF and further im‐
proved the limit of detection to 185 pM (5 ng/mL). The developed assay was successfully
tested on VEGF‐spiked samples of diluted (2.5%) blood serum.

4. AuNP-based
FigureFigure 4. AuNP‐based aptasensor
aptasensorfor
forVEGF detectionwith
VEGF detection withsignal
signal amplification
amplification proposed
proposed by
by C.C.
C.C. Chang [19]. The aptasensor consists of aptamer-containing hairpin DNA, two
Chang [19]. The aptasensor consists of aptamer‐containing hairpin DNA, two DNA substrates, andDNA substrates,
and two
twoauxiliary
auxiliaryDNADNA fragments. Withoutthe
fragments. Without thetarget,
target,the
thesingle‐stranded
single-stranded auxiliary
auxiliary DNA DNA fragments
fragments are
adsorbed on the AuNPs, preventing their aggregation and giving a red color to
are adsorbed on the AuNPs, preventing their aggregation and giving a red color to the solution. The the solution. The
addition
addition of VEGFof VEGF switches
switches the aptamer
the aptamer to anto an active
active structure,
structure, whichwhich
leadsleads to reorganization
to the the reorganization of
of the
the hairpin DNA. “Opened” hairpin DNA, in turn, forms a duplex with the DNA substrate and
hairpin DNA. “Opened” hairpin DNA, in turn, forms a duplex with the DNA substrate and initiates
initiates a nonlinear chain reaction involving the auxiliary DNA fragments. The resulting den‐
a nonlinear chainstructure
drimer‐like reactionisinvolving the auxiliary
poorly adsorbed DNA fragments.
on the AuNPs, The resulting
and their aggregation dendrimer-like
causes a red to blue
structure is poorly
color change. adsorbed on the AuNPs, and their aggregation causes a red to blue color change.

3.1.2. CD63 as an Exosome Surface Protein

The glycoprotein CD63, a member of the tetraspanin family, is exposed on exosome
membranes in different amounts depending on the cell type. Exosomes, in turn, are now
considered as biomarkers of oncological diseases [72]. A DNA aptamer selected by Base
Biomedicines 2022, 10, 1606 8 of 23

3.1.2. CD63 as an Exosome Surface Protein

The glycoprotein CD63, a member of the tetraspanin family, is exposed on exosome
membranes in different amounts depending on the cell type. Exosomes, in turn, are now
considered as biomarkers of oncological diseases [72]. A DNA aptamer selected by Base
Pair Biotechnologies, Inc. was used as a recognition element in different biosensors for
the detection of CD63-positive exosomes. For example, Y. Jiang et al. [20] developed a
colorimetric aptasensor based on the dispersion/aggregation of AuNPs, that discriminated
between exosomes with different CD63 content. The authors also proposed a panel of
aptamers for the simultaneous detection of several protein markers for the more accurate
identification of exosomes.
Zhang et al. employed the same aptamer for the multicolor detection of exosomes [21].
First, they loaded the surface of streptavidin magnetic beads with a biotinylated aptamer for
the specific capture of CD63-positive exosomes. Second, a cholesterol-modified DNA probe
was embedded into the lipid bilayer of exosomes, exposing the single-stranded “sticky” end
to trigger a chain hybridization reaction with the biotinylated DNA oligonucleotides H1 and
H2. Then, H1 and H2 formed complexes with streptavidin-conjugated alkaline phosphatase.
The enzyme dephosphorylated ascorbic acid phosphate in a silver salt solution, which,
in turn, led to the deposition of a silver shell on the surface of the Au nanorods (AuNRs)
and a resulting multicolor change (Figure 5). The aptasensor allowed for the detection
of exosomes from MCF-7 cell cultures in the range of 1400 to 280,000 particles/mL, with
Biomedicines 2022, 10, x FOR PEER the limit of detection determined as 160 particles/mL. This method was also well
REVIEW 9 ofsuited
24
for detecting exosomes from colorectal and breast cancer cell cultures, which proved
its universality.

Figure
Figure 5.5.Multicolor
Multicoloraptamer-based
aptamer‐based system
system for
forCD63‐positive
CD63-positiveexosome
exosome detection [21].
detection Exosomes
[21]. Exosomesare are
captured by CD63‐specific aptamers immobilized on magnetic beads. Then, a cholesterol‐modified
captured by CD63-specific aptamers immobilized on magnetic beads. Then, a cholesterol-modified
DNA anchor embeds into the lipid bilayer of exosomes, with the ssDNA “sticky” end exposed to
DNA anchor embeds into the lipid bilayer of exosomes, with the ssDNA “sticky” end exposed to
trigger a chain hybridization reaction with the biotinylated oligonucleotides H1 and H2. Next, H1
trigger
and H2a chain hybridization
bind with reaction withalkaline
streptavidin‐conjugated the biotinylated oligonucleotides
phosphatase. H1 and of
The dephosphorylation H2. Next, H1
ascorbic
and H2phosphate
acid bind withinstreptavidin-conjugated
silver salt solution leadsalkaline phosphatase.
to the deposition of a The dephosphorylation
silver shell on the surfaceofofascorbic
the
AuNRs
acid and a resulting
phosphate in silvermulticolor change.
salt solution leads to the deposition of a silver shell on the surface of the
AuNRs and a resulting multicolor change.
Another enzyme‐recruiting colorimetric aptasensor for exosomes was proposed in
[22]. First, exosomes
Another were covalently
enzyme-recruiting immobilized
colorimetric on the
aptasensor forsurface
exosomesof aldehyde latex mi‐
was proposed in [22].
crobeads. Then, a biotinylated CD63‐specific aptamer was added to the suspension.
First, exosomes were covalently immobilized on the surface of aldehyde latex microbeads. For
an analytical
Then, signal,CD63-specific
a biotinylated the authors used an HRP–streptavidin
aptamer was added to theconjugate and colorless
suspension. dopa‐
For an analytical
mine solution; thus, colored polydopamine was deposited on the exosome
signal, the authors used an HRP–streptavidin conjugate and colorless dopamine solution;surface. The
limit of detection of the assay was 7700 particles/mL. Similar aptasensors were
thus, colored polydopamine was deposited on the exosome surface. The limit of detection developed
offor
theHER2
assayandwasαvβ6
7700integrin detection.Similar aptasensors were developed for HER2 and
particles/mL.
Peroxidase‐mimicking
αvβ6 integrin detection. nanomaterials represent a promising alternative to natural
protein enzymes in aptamer‐based assays.
Peroxidase-mimicking nanomaterials represent For example, Y. Xia et al.
a promising [23] used to
alternative single‐
natural
walled carbon nanotubes (SWCNTs) with peroxidase‐like activity. In the absence of a tar‐
protein enzymes in aptamer-based assays. For example, Y. Xia et al. [23] used single-walled
get, the CD63‐specific aptamers are adsorbed on the SWCNT surface, enhancing their cat‐
carbon nanotubes (SWCNTs) with peroxidase-like activity. In the absence of a target, the
alytic activity and colored product formation. In the presence of exosomes, the aptamers
dissociate from SWCNTs, thus decreasing their catalytic activity. The detection limit of
the assay was 5.2 × 105 particles/mL. This method showed good agreement with commer‐
cial immunoassays in the analysis of serum samples from healthy donors and patients
with breast cancer. A similar detection system developed in [24] recruits carbon nitride
nanosheets as peroxidase mimics. The aptasensor distinguished exosomes from cancer
Biomedicines 2022, 10, 1606 9 of 23

CD63-specific aptamers are adsorbed on the SWCNT surface, enhancing their catalytic
activity and colored product formation. In the presence of exosomes, the aptamers dissoci-
ate from SWCNTs, thus decreasing their catalytic activity. The detection limit of the assay
was 5.2 × 105 particles/mL. This method showed good agreement with commercial im-
munoassays in the analysis of serum samples from healthy donors and patients with breast
cancer. A similar detection system developed in [24] recruits carbon nitride nanosheets
as peroxidase mimics. The aptasensor distinguished exosomes from cancer and normal
cell cultures and allowed for quantitative exosome detection in blood serum samples from
breast cancer patients with a limit of detection of 13.5 × 105 particles/mL.
Lateral flow assays for exosome detection have been constructed based on CD63-
specific aptamers and nanomaterials. In [25], an anchor DNA conjugated with Au@Pd
nanopopcorn captured exosomes from a solution (Figure 6A). Then, the exosome com-
plexes were visualized using aptamer-containing nanoflowers immobilized at the test
line. Specific aptamer binding with CD63 on the exosomes’ membranes caused them to
become concentrated at the test line, and subsequent laser irradiation generated a thermal
signal and produced a characteristic black band. The limit of detection for the assay was
1.4 × 104 particles/mL, comparable with that of fluorescent and electrochemical assays.
Biomedicines 2022, 10, x FOR PEER REVIEW 10 of 24
The sensitivity of detection significantly decreased in spiked serum, but the dilution of
serum samples (by 10 times) improved the sensitivity.

Figure 6. Lateral flow assays for CD63-positive exosomes. (A) Au@Pd nanoparticle-based aptasensor
proposed
Figure 6.in Lateral
[25]. An anchor
flow DNA
assays fragment conjugated
for CD63‐positive exosomes.with(A)Au@Pd
Au@Pdnanopopcorn formsaptasen‐
nanoparticle‐based a complex
with
sor proposed in [25]. An anchor DNA fragment conjugated with Au@Pd nanopopcorn formsat
exosomes. Nanoflower-modified CD63 aptamers provide exosome concentration the test
a com‐
line. Subsequent laser irradiation generates a thermal signal and produces
plex with exosomes. Nanoflower‐modified CD63 aptamers provide exosome concentration at the a characteristic black
test line. Subsequent laser irradiation generates a thermal signal and produces a characteristic
band at the test line. (B) AuNP-based aptasensor developed in [26]. Without exosomes, the aptamer black
band at the
conjugated testAuNP
with line. (B) AuNP‐based
binds aptasensor developed
to a complementary DNA fragment in [26].
atWithout exosomes,
the test line, the aptamer
producing a colored
conjugated with AuNP binds to a complementary DNA fragment at the test line, producing a col‐
band due to the accumulation of AuNPs. In the presence of exosomes, the AuNP-modified aptamer
ored band due to the accumulation of AuNPs. In the presence of exosomes, the AuNP‐modified
binds CD63binds
aptamer on the exosome
CD63 on thesurface
exosome and the test
surface andline
theremains colorless.
test line remains colorless.

YuYu
et et
al.al.[26]
[26]developed
developed another aptamer-based
another aptamer‐based lateral
lateral flow
flow assay
assay for exosome
for exosome detec‐de-
tection. Without exosomes, a CD63-specific aptamer conjugated with AuNPs binds
tion. Without exosomes, a CD63‐specific aptamer conjugated with AuNPs binds to a com‐ to a
plementary DNA fragment at the test line pad, producing a colored band due to the accu‐
mulation of AuNPs in this region. Otherwise, in the presence of exosomes, the aptamer
binds CD63 on the exosome surface, and no coloring of the test line is observed (Figure 6B).
This assay allowed the authors to discriminate between exosomes isolated from a non‐small
cell lung cancer cell culture and exosomes from a culture of fibrocytes. We should note,
Biomedicines 2022, 10, 1606 10 of 23

complementary DNA fragment at the test line pad, producing a colored band due to the
accumulation of AuNPs in this region. Otherwise, in the presence of exosomes, the aptamer
binds CD63 on the exosome surface, and no coloring of the test line is observed (Figure 6B).
This assay allowed the authors to discriminate between exosomes isolated from a non-small
cell lung cancer cell culture and exosomes from a culture of fibrocytes. We should note,
however, that zero signal in the presence of the analyte seems to be a serious disadvantage
of the assay, making it prone to false positive results.

3.1.3. Mucin-1
The transmembrane mucin glycoprotein MUC1, which is overexpressed in cancer
cells, serves as a biomarker for most of the adenocarcinomas, as well as lung cancer, breast
cancer, etc. [73,74].
C. Ferreira et al. selected a DNA aptamer that binds the MUC1 recombinant protein
with high affinity [27]. The MUC1-5TR-1 aptamer was used as a capture probe for an
ELISA-like colorimetric sandwich test system with a limit of detection of 1 µg/mL.
S. Liu et al. developed an aptazyme-based assay for MUC1 detection [28]. They com-
bined a MUC1-specific aptamer and peroxidase-mimicking DNAzyme in one aptazyme
molecule. In the absence of the analyte, the aptamer is bound to the complementary DNA
immobilized on the magnetic beads and therefore can be discarded from the solution
after magnetic separation. In the presence of MUC1, the aptamer forms an active struc-
ture, which induces a reorganization of the whole aptazyme molecule. As a result, the
DNAzyme restores its catalytical activity and oxidizes a chromogenic substrate. The limit
of detection for this aptasensor was about 5 nM, both in a model buffer solution and in 10%
human serum.
Y. Zhou et al. used an aptazyme-based approach for MUC1 detection on the surface
of exosomes [29]; the whole analysis took less than an hour. The limit of detection of the
developed aptasensor was 3.94 × 105 particles/mL.

3.1.4. Carcinoembryonic Antigen

The glycoprotein carcinoembryonic antigen (CEA) is one of the most widely used
biomarkers for gastrointestinal, breast, and cervical cancer [75,76].
C. Luo et al. [30] developed an aptasensor for CEA detection based on the disper-
sion/aggregation of AuNPs (red–blue color change). The limit of detection was 3 ng/mL
in a model buffer solution. The aptasensor was tested for CEA detection in spiked samples
of diluted (2%) blood serum.
K. Liang et al. also employed AuNPs in their CEA detection system with signal am-
plification [31]. Without CEA, an aptamer forms a duplex with complementary DNA in
solution, thus blocking DNA adsorption on AuNPs. In the presence of CEA, complemen-
tary DNA dissociates from the aptamer and induces rolling circle amplification, resulting in
the adsorption of single-stranded DNA fragments onto AuNPs. Measuring the absorbance
ratio at 660 and 520 nm allows one to determine the CEA concentration. The limit of
detection for the proposed system was 2 pM.
N. Shahbazi et al. [32] developed a homogenic assay for CEA detection with an
aptazyme made of a CEA-specific aptamer and two-component quadruplex-forming
DNAzyme. Two DNAzyme fragments were connected via a linker sequence comple-
mentary to the aptamer fragment. In the absence of CEA, the aptamer–linker complex
inactivates the DNAzyme module. After CEA addition, the aptamer dissociates from the
linker, and the DNAzyme acquires an active conformation and oxidates the chromogenic
substrate. The limit of detection was 5.5 pM (1 ng/mL), and the aptazyme was successfully
used for CEA detection in saliva samples from healthy donors.

3.1.5. Other Cancer Biomarkers

Prostate-specific antigen (PSA) is a glycoprotein that is normally present in the blood
at a very low level (0.5–2 ng/mL). An increased PSA level (4–10 mg/mL) could indicate
Biomedicines 2022, 10, 1606 11 of 23

prostate cancer [15]. Shayesteh et al. [33] developed an AuNP-based aptasensor for colori-
metric PSA detection. Their assay detected PSA in the physiological range of concentrations,
and the limit of detection was as low as 20 pg/mL. This method also allowed for PSA
detection in spiked samples of diluted blood serum.
The transmembrane protein HER2 (human epidermal growth factor receptor type 2) is
a member of the tyrosine protein kinase family. Increased HER2 expression is characteristic
of lung, breast, and ovarian cancers [77]. A DNA aptamer was used for the development of
colorimetric systems for HER2 detection both in solution and in LFA format [34]. Homoge-
neous HER2 detection based on the dispersion/aggregation of AuNPs in salted solution
had a limit of detection of 10 nM in diluted (10%) serum samples. In LFA format, without a
protein, the biotinylated aptamer forms a nanocomplex with AuNPs that is captured by
streptavidin at the test line, resulting in the visualization of red dots. In addition, negatively
charged AuNPs are trapped by a positively charged polymer at the control line, producing
red coloring. In the presence of HER2, the aptamer dissociates from the AuNPs and forms
a specific complex with the protein; hence, the red dots at the test line disappear, while the
control line color remains the same. The limit of detection for this assay was 20 nM.
Y. Zhou et al. selected DNA aptamers that specifically bind Dickkopf-1 protein (DKK1),
a WNT pathway antagonist. Increased levels of DKK-1 are typical for many types of
oncological diseases [35]. The TD10 aptamer with the highest affinity was used in an ELISA-
like microplate colorimetric sandwich system. The aptamer was covalently immobilized on
a microplate for DKK1 capture. Anti-DKK1 antibodies served as a reporter probe to recruit
streptavidin-conjugated horseradish peroxidase. The developed assay was used for DKK1
detection with concentrations ranging from 62.5 to 4000 pg/mL. The aptasensor system
was successfully applied for DKK1 detection using blood serum samples from patients with
colorectal cancer. The obtained results were in good agreement with the results obtained
using a commercial ELISA kit.

3.2. Neurodegenerative Diseases

3.2.1. Dopamine
Dopamine is a small organic molecule with a molecular weight of 189 Da. As a
member of the catecholamine family of neurotransmitters, it has various functions in
the central nervous system. Changes in dopamine levels can cause neurodegenerative
pathologies, such as Parkinson’s and Alzheimer’s diseases [78]. C. Mannironi et al. selected
a dopamine-binding 67 nt RNA aptamer with a KD of 1.6 µM [79]. This aptamer was used
in a competitive colorimetric assay for dopamine detection [36], which was analogous to
competitive ELISA. The developed method was used for dopamine detection in spiked
samples of diluted serum (10%) after filtration through a dialysis membrane (3 kDa). The
limit of detection was 1 pM, which is about 1000 times more sensitive than ELISA.
R. Walsh et al. converted a dopamine-binding RNA aptamer into DNA form [80]. The
DNA homolog retained the ability to bind dopamine with high affinity and selectivity. The
limit of competitive colorimetric detection based on the DNA aptamer was 3.2 pM, close
to that of RNA aptamers [37]. The DNA aptamer was used as a recognizing element in a
colorimetric aptasensor based on the dispersion/aggregation of AuNPs in salt solution [38].
This aptasensor provided dopamine detection for concentrations ranging from 0.54 to
5.4 µM, and the limit of detection was 0.36 µM.
Y. Zhang et al. used a DNA aptamer for bimodal dopamine detection [39]. Free
fluorescein-conjugated aptamers in solution bind to gold nanoparticles and block their
aggregation, while the particles themselves act as a fluorescence quencher for the fluorescein
residue. In the absence of dopamine, fluorescently-labeled aptamers bind to AuNPs, which
quench their fluorescence. In the presence of dopamine, the aptamers dissociate from the
AuNPs, which leads to their aggregation and an increase in fluorescence intensity. The
limit of detection was 140 nM in colorimetric mode and 78.7 nM in fluorescent mode.
The proposed aptasensor was well suited for quantitative dopamine detection in spiked
serum samples.
Biomedicines 2022, 10, 1606 12 of 23

Another bimodal aptasensor for dopamine detection was developed in [40]. The DNA
aptamer immobilized on the nanochip captures dopamine from solution, and a subsequent
alkaline treatment results in dopamine oxidation and the formation of a colored product.
The limit of detection was 0.6 µM. To further improve the sensitivity of the assay, the
authors used fluorescent Au nanoclusters. In this method, a product of dopamine oxidation
quenches the fluorescence of the Au nanoclusters. The limit of detection for the fluorescent
assay was 3.3 nM. The principal applicability of the developed aptasensor was shown for
dopamine detection in model biological samples (artificial cerebrospinal fluid and fetal
bovine serum).
N. Nakatsuka et al. [81] performed an alternative selection of dopamine-binding
Biomedicines 2022, 10, x FOR PEER REVIEW DNA
13 of 24
aptamers. Their aptamer was used in a lateral flow assay to measure dopamine in urine [41].
In the absence of dopamine, the aptamer hybridizes with a complementary DNA fragment
immobilized on AuNPs. The resulting complex binds to DNA at the control zone, forming
control zone, forming a red line (zone “C” in Figure 7). In the presence of dopamine,
a red line (zone “C” in Figure 7). In the presence of dopamine, AuNP-modified DNA
AuNP‐modified DNA dissociates from the aptamer and forms a complementary complex
dissociates from
with another thegiving
DNA, aptamer and
a red lineforms
in the atest
complementary
zone (zone “T” in complex with
Figure 7). Theanother
developedDNA,
giving
aptasensor was applied for dopamine detection in urine samples within clinically relevantwas
a red line in the test zone (zone “T” in Figure 7). The developed aptasensor
applied for dopamine
concentration detection
ranges (2.6–3.2 μM inor
urine samples
500–600 within
ng/mL). It isclinically relevant
worth noting that concentration
the whole
ranges (2.6–3.2 µM or 500–600
assay only took about 15 min. ng/mL). It is worth noting that the whole assay only took
about 15 min.

Figure7.7.Lateral
Figure Lateralflow
flowassay
assayfor
fordopamine
dopaminedetection
detectionproposed
proposedinin[41].
[41].Without
Withoutdopamine,
dopamine,the
theaptamer
ap‐
tamer forms a red‐colored complex with AuNP‐modified DNA1, which is trapped by DNA2 at the
forms a red-colored complex with AuNP-modified DNA1, which is trapped by DNA2 at the control
control line. In the presence of dopamine, the aptamer dissociates from AuNP‐modified DNA1, and
line. In the
duplex presencebetween
formation of dopamine,
DNA1 andthe aptamer dissociates
DNA3 provides from AuNP-modified
red coloring at the test line. DNA1, and duplex
formation between DNA1 and DNA3 provides red coloring at the test line.
3.2.2. Other Biomarkers for Neurodegenerative Diseases
3.2.2. Other Biomarkers for Neurodegenerative Diseases
Alpha‐synuclein (α‐syn) belongs to a group of proteins found in nerve tissue. The α‐
Alpha-synuclein (α-syn) belongs to a group of proteins found in nerve tissue. The
syn protein can form soluble oligomers, the increased content of which has been found in
α-syn protein can form
the cerebrospinal fluid soluble
and blood oligomers,
plasma ofthe increased
patients withcontent of which
Parkinson’s has[82,83].
disease been found
K.
inTsukakoshi
the cerebrospinal fluid and blood plasma of patients with Parkinson’s disease
et al. [84] generated a DNA aptamer that selectively binds α‐syn oligomers. [82,83].
K.K.Tsukakoshi et al.
Sun et al. [42] [84] generated
employed a DNA
this aptamer to aptamer that selectively
create a AuNP‐based binds α-syn
colorimetric oligomers.
aptasensor;
K.however,
Sun et al.its[42] employed this aptamer to create a AuNP-based colorimetric aptasensor;
application for α‐syn oligomer detection in real samples was restricted due
however, its application
to the non‐selective for α-synofoligomer
aggregation the AuNPsdetection
in serum.in real samples was restricted due to
the non-selective aggregation of the AuNPs in serum.
3.3. Stress‐Related Disease
3.3. Stress-Related Disease
Cortisol, a glucocorticoid hormone, participates in various physiological processes.
It isCortisol, a glucocorticoid
considered as a biomarkerhormone, participates
of stress [85]; in various
elevated cortisol physiological
levels processes.
are characteristic of
It stress‐related
is consideredconditions,
as a biomarker of stress
including [85];
chronic elevated
fatigue cortisol
syndrome, levels are bipolar
depression, characteristic
disor‐ of
der, and post‐traumatic stress disorder [86,87].
J. Martin et al. selected a cortisol‐binding DNA aptamer and used it as a recognizing
element for a AuNP‐based colorimetric aptasensor [43]. The developed aptasensor al‐
lowed for the detection of physiological concentrations of cortisol (from 150 to 600 nM).
A similar aptasensor was developed by X. Bao et al. [44]. However, it was much less sen‐
Biomedicines 2022, 10, 1606 13 of 23

stress-related conditions, including chronic fatigue syndrome, depression, bipolar disorder,

and post-traumatic stress disorder [86,87].
J. Martin et al. selected a cortisol-binding DNA aptamer and used it as a recognizing
element for a AuNP-based colorimetric aptasensor [43]. The developed aptasensor allowed
for the detection of physiological concentrations of cortisol (from 150 to 600 nM). A similar
aptasensor was developed by X. Bao et al. [44]. However, it was much less sensitive, and
the limit of detection was only 690 µM (0.25 mg/mL).
The same DNA aptamer served as a component of a lateral flow assay for
Biomedicines 2022, 10, x FOR PEER REVIEW 14 cortisol
of 24
detection [45]. In the absence of cortisol, the aptamer adsorbs on AuNPs and blocks their
interaction with cysteamine on the membrane at the test line. In the presence of cortisol,
the aptamer does not bind with AuNPs; instead, they interact with cysteamine, producing
a red line in the test zone (Figure 8). In contrast to most LFAs, the developed aptasensor
a red
lacksline in the line
a control test on
zone
the(Figure 8).The
test strip. In contrast
assay wastoused
mostfor
LFAs, thedetection
cortisol developed aptasensor
in artificial
lacks a control line on the test strip. The assay was used
sweat samples, with a limit of detection of 2.8 nM (1 ng/mL). for cortisol detection in artificial
sweat samples, with a limit of detection of 2.8 nM (1 ng/mL).

Figure
Figure8.8.Aptamer-based
Aptamer‐basedlaterallateral flow assay
assay for
forcortisol
cortisoldetection
detectiondeveloped
developed in in [45].
[45]. In the
In the presence
presence
of of cortisol,the
cortisol, theaptamer
aptamerbinds
binds toto its
its target, while
while unbound
unboundAuNPs
AuNPsinteract with
interact withmembrane‐bound
membrane-bound
cysteamine,
cysteamine, resultingininred
resulting redline
lineformation.
formation.Without
Withoutcortisol,
cortisol,the
theaptamer
aptamerisisadsorbed
adsorbedon onAuNPs
AuNPsand
and prevents their interaction with cysteamine; thus, the membrane remains colorless.
prevents their interaction with cysteamine; thus, the membrane remains colorless.

3.4.Cardiovascular
3.4. CardiovascularDiseases
Diseases
Cardiactroponins,
Cardiac troponins,troponin
troponinIIand
and troponin
troponin T, T, are
are validated
validated biomarkers
biomarkersofofcardiovas‐
cardiovascu-
cular diseases, including myocardial infarction [88].
lar diseases, including myocardial infarction [88]. F. Torrini et al. F. Torrini et selected
al. selected
DNA DNA ap‐
aptamers
tamers for troponin T and employed them in colorimetric assay [51],
for troponin T and employed them in colorimetric assay [51], both in direct and sandwich both in direct and
sandwich
formats. In formats.
the direct Inanalysis,
the directthe
analysis,
biotinylated aptamer aptamer
the biotinylated was added wasinto
added into micro‐
microplate wells
plate wells with immobilized troponin T. Then, aptamer–protein complexes were visual‐
with immobilized troponin T. Then, aptamer–protein complexes were visualized using
ized using HRP–streptavidin conjugates. In sandwich format, the immobilized aptamer
HRP–streptavidin conjugates. In sandwich format, the immobilized aptamer captures the
captures the analyte from the sample, while the second aptamer acts as a reporter. In un‐
analyte from the sample, while the second aptamer acts as a reporter. In undiluted serum
diluted serum samples, the limit of detection was 3.42 nM for direct analysis and 3.13 nM
samples, the limit of detection was 3.42 nM for direct analysis and 3.13 nM for sandwich
for sandwich format. The authors emphasized that despite the close values of the detec‐
format. The authors emphasized that despite the close values of the detection limits, the
tion limits, the sandwich assay seemed to be more promising since it provided a better
sandwich assay seemed
specific/nonspecific to be
signal more promising since it provided a better specific/nonspecific
ratio.
signal A.ratio.
Sinha et al., obtained a troponin I‐specific aptamer using the on‐a‐chip SELEX
A. Sinha
method [89].et al. aptamer
This obtainedacted a troponin I-specific aptamer
as the analyte‐capturing using
element of athe on-a-chip SELEX
chemiluminescent
method [89]. This aptamer acted as the analyte-capturing element
microchip aptasensor [52]. Primary troponin‐specific antibodies and peroxidase‐conju‐ of a chemiluminescent
microchip aptasensor
gated secondary [52]. Primary
antibodies were usedtroponin-specific
for visualization. antibodies
The limitand peroxidase-conjugated
of detection was 0.5 pM,
secondary antibodies were
which is comparable used for visualization.
to commercial The limit
ELISA kits (12.5–40 pM orof detection was 0.5
300–1000 ng/L). ThepM, which
assay
is allowed
comparable to commercial
for troponin ELISA
I detection kits (12.5–40
in blood pM or 300–1000
serum samples ng/L).
from patients The
with assay allowed
cardiovascu‐
forlartroponin
diseasesIand detection
from ain blood donor.
healthy serum samples from patients with cardiovascular diseases
and from a healthy
Protein donor.
HIF‐1α, which controls oxygen transport, represents a potential biomarker of
Protein HIF-1α,
myocardial infarctionwhich
[90]. controls
Q. Wangoxygen transport,
et al. [53] employed represents a potential biomarker
the AuNP‐conjugated aptamer of
as a reporter
myocardial probe and
infarction developed
[90]. Q. Wang a sandwich‐type assay for
et al. [53] employed thethe detection of HIF‐1α
AuNP-conjugated on
aptamer
asexosomes
a reporterformed afterdeveloped
probe and myocardiala infarction.
sandwich-type Microplate‐immobilized
assay for the detection HIF‐1α‐specific
of HIF-1α on
antibodies
exosomes captureafter
formed the myocardial
exosomes in infarction.
the wells, while the peroxidase‐like activity
Microplate-immobilized of the
HIF-1α-specific
AuNP–aptamer conjugate provides the generation of an analytical signal. The limit of de‐
tection was 7 fM (0.2 ng/L) in a model buffer solution. The assay was applied for the de‐
tection of HIF‐1α‐positive exosomes in blood serum samples from model animals with
myocardial infarction.
Thrombospondin‐1 is a member of a family of secreted extracellular matrix proteins
Biomedicines 2022, 10, 1606 14 of 23

antibodies capture the exosomes in the wells, while the peroxidase-like activity of the
AuNP–aptamer conjugate provides the generation of an analytical signal. The limit of
detection was 7 fM (0.2 ng/L) in a model buffer solution. The assay was applied for the
detection of HIF-1α-positive exosomes in blood serum samples from model animals with
myocardial infarction.
Thrombospondin-1 is a member of a family of secreted extracellular matrix proteins
Biomedicines 2022, 10, x FOR PEER REVIEW 15 of 24
that play an important role in cell adhesion, migration and proliferation, angiogenesis,
inflammation, atherosclerosis, and thrombosis [91,92]. A specific DNA aptamer was se-
lected and used for the colorimetric detection of thrombospondin-1 in [54]. The aptamer,
immobilized
immobilized on magnetic
on magnetic beads,forms
beads, formsaacomplementary
complementary complex
complexwith a biotinylated
with ol‐
a biotinylated
igonucleotide. In the absence of the target, the streptavidin–HRP conjugate binds
oligonucleotide. In the absence of the target, the streptavidin–HRP conjugate binds to the to the
magnetic
magnetic beads
beads duedueto to biotin–streptavidininteractions.
biotin–streptavidin interactions. As
Asthe target
the protein
target displaces
protein the
displaces
biotinylated oligonucleotide from the complex with the bead‐bound aptamer,
the biotinylated oligonucleotide from the complex with the bead-bound aptamer, the per- the peroxi‐
dase conjugate cannot bind with the beads, which, in turn, leads to a decrease in the col‐
oxidase conjugate cannot bind with the beads, which, in turn, leads to a decrease in the
orimetric signal (Figure 9). The limit of detection was 7 fM in a model buffer solution. The
colorimetric signal (Figure 9). The limit of detection was 7 fM in a model buffer solution.
assay allowed for the measurement of thrombospondin‐1 in blood serum samples from
The assay allowed for the measurement of thrombospondin-1 in blood serum samples from
patients with atherosclerosis and healthy donors.
patients with atherosclerosis and healthy donors.

Figure 9. Aptamer-based
Figure system
9. Aptamer‐based for for
system thrombospondin-1
thrombospondin‐1 detection
detectiondeveloped
developedby byK.
K.JiJi et
et al.
al. [54]. The
The
aptamer on magnetic beads forms a duplex with biotinylated DNA that binds the streptavidin–HRP
aptamer on magnetic beads forms a duplex with biotinylated DNA that binds the streptavidin–
HRP conjugate.
conjugate.HRP oxidizes
HRP the chromogenic
oxidizes substrate
the chromogenic and generates
substrate a colorimetric
and generates signal. Throbmos‐
a colorimetric signal.
pondin‐1 displaces biotinylated DNA from the complex with the bead‐bound aptamer. The peroxi‐
Throbmospondin-1 displaces biotinylated DNA from the complex with the bead-bound aptamer.
dase conjugate cannot bind with the beads, which leads to a decrease in the colorimetric signal in‐
The peroxidase
tensity. conjugate cannot bind with the beads, which leads to a decrease in the colorimetric
signal intensity.
3.5. Other Diseases (Inflammation, Diabetes, etc.)
3.5. Other Diseases (Inflammation, Diabetes, etc.)
3.5.1. C‐Reactive Protein
3.5.1. C-Reactive Protein
C‐reactive protein (CRP) is a general inflammatory biomarker for a wide spectrum
C-reactive protein (CRP) is a general inflammatory biomarker for a wide spectrum of
of diseases, including cardiovascular [93] and rheumatic disorders [94]. A CRP‐specific
diseases, including cardiovascular [93] and rheumatic disorders [94]. A CRP-specific DNA
DNA aptamer was selected by B. Wu et al., for an SPR‐based detection system [95]. Alt‐
aptamer
houghwasthe
selected
assay wasby B.very
Wu sensitive
et al. for (limit
an SPR-based detection
of detection of 10 pM system [95]. Although
in model buffer), SPRthe
assayanalysis
was very is not a common method for routine clinical diagnostics. The same aptamera
sensitive (limit of detection of 10 pM in model buffer), SPR analysis is not
common method for routine
served as a reporter probeclinical diagnostics.
for colorimetric The same
sandwich type aptamer
detectionserved
in [46].as
A aconjugate
reporter
probeoffor
thecolorimetric
CRP‐specificsandwich type detection
ligand citicoline with BSAin [46]. A the
provided conjugate
selectiveofcapture
the CRP-specific
of CRP in
ligand citicolinewells.
microplate withABSA provided the selective
peroxidase‐mimicking capture of complex
AuNP–aptamer CRP in microplate
provided CRP wells. A
visu‐
peroxidase-mimicking
alization. The limit AuNP–aptamer
of detection for the complex
proposed provided
assay wasCRP as visualization.
low as 0.07 pM.The Thelimit of
devel‐
detection
opedfor the proposed
aptasensor allowed assayforwas as low as 0.07 of
the measurement pM.CRPTheindeveloped
blood samplesaptasensor allowed
from rats with
for the measurement
acute myocardial of CRP in blood
infarction. samples
The results werefrom rats agreement
in good with acutewithmyocardial infarction.
those obtained us‐
The results were inELISA
ing a standard good agreement
kit. with those obtained using a standard ELISA kit.
M. António
M. Antónioet al.et
[47]
al.developed
[47] developedanother AuNP-based
another AuNP‐based colorimetric aptasensor
colorimetric for CRP
aptasensor for
CRP detection.
detection. Without aWithout a target protein,
target protein, the aptamer
the aptamer interacts
interacts with AuNPs
with AuNPs and prevents
and prevents their
their aggregation
aggregation in saltThe
in salt solution. solution. Theof
addition addition
CRP leadsof CRP
to theleads to the formation
formation of an ap‐
of an aptamer–CRP
tamer–CRP complex and the aggregation of AuNPs, resulting in a
complex and the aggregation of AuNPs, resulting in a color change. The limit of detectioncolor change. The limit
of detection was 10 nM in the model buffer solution. However, the presence of serum
albumin, even at a concentration 10‐fold lower than that in blood (≥3 g/L), inhibited the
CRP‐specific dispersion/aggregation of AuNPs, which resulted in a very low sensitivity
of the assay, making it inapplicable for biological samples.
Biomedicines 2022, 10, 1606 15 of 23

was 10 nM in the model buffer solution. However, the presence of serum albumin, even
at a concentration 10-fold lower than that in blood (≥3 g/L), inhibited the CRP-specific
dispersion/aggregation of AuNPs, which resulted in a very low sensitivity of the assay,
making it inapplicable for biological samples.

3.5.2. Interleukins and Their Receptors

The soluble form of the α-subunit of the IL-2 receptor, sIL-2Ra, is found at elevated
levels in the sera of subjects suffering from various inflammatory processes, including
autoimmune, oncological, and infectious diseases [96]. J. Jeon et al. [48] proposed a colori-
metric aptasensor based on an sIL-2Rα-specific DNA aptamer. In the absence of sIL-2Rα,
the aptamer–AuNP complex has an increased negative charge that attracts a positively
charged substrate, orthophenylenediamine. Due to the peroxidase-like activity of AuNPs,
substrate oxidation results in the development of a brown color. In the presence of sIL-2Rα,
the aptamer dissociates from the AuNPs, thus decreasing the negative charge on the AuNPs,
which is followed by the fading of the brown color. The developed method allowed for an
express analysis (about 25 min) to be performed with a limit of detection of 1 nM, both in a
model buffer solution and diluted serum samples.
Interleukin-6 (IL-6) is a cytokine involved in the immune response in various inflam-
matory diseases, as well as in the regulation of metabolic and regenerative processes [97].
A pair of DNA aptamers that bind different epitopes of murine IL-6 were used for a colori-
metric assay based on the AuNP dispersion/aggregation effect [49]. The assay provided
IL-6 detection for concentrations ranging from 1 to 125 µg/mL and took as little as 5 min
for signal generation. However, this test system selectively detects only murine IL-6 and is
not suited for human IL-6.

3.5.3. Human Neutrophile Elastase

Human neutrophil elastase (HNE) belongs to the class of serine proteases and partici-
pates in the immune response to various pathogens. Changes in HNE expression can lead
to the development of acute respiratory distress syndrome, chronic obstructive pulmonary
disease, cystic fibrosis, acute lung injury, arthritis, emphysema, and atherosclerosis [98]. An
HNE-specific DNA aptamer [99] was applied for colorimetric detection using the intrinsic
enzymatic activity of HNE for the generation of an analytical signal [50]. The aptamer, im-
mobilized on a solid support (magnetic particles or microplate wells), captures HNE from
solution. The selective cleavage of the peptide substrate by HNE results in the generation
of a colored product. The limit of detection in a model solution was 0.4 pM. However, the
components of the biological samples significantly inhibited elastase activity and decreased
the sensitivity of detection. While in model HNE-spiked samples, this problem was solved
via the heat inactivation of inhibitors; the applicability of the assay for real clinical samples
remains questionable.

3.5.4. Biomarkers of Diabetes

Diabetes mellitus is a group of endocrine pathologies characterized by elevated blood
glucose levels. Diabetes-related complications include cardiovascular diseases, renal fail-
ure, blindness, and foot/leg amputation [100]. The level of glycated hemoglobin HbA1c
in blood provides an accurate estimation of average blood glucose for the preceding
2–3 months [101]. H. Lin et al. selected an HbA1c-specific DNA aptamer using SELEX on
microchips [102]. This aptamer served as a selective capture probe for a chemiluminescent
aptamer-antibody sandwich assay [55]. The aptasensor showed a limit of detection of
0.1 mM in diluted blood samples and allowed for the analysis to be performed in 25 min
in automatic mode. As a further optimization of the assay, J. Li et al. [56] replaced the
reporter anti-HbA1c antibody with a second DNA aptamer. The results for blood HbA1c
measured by the developed aptasensor were in good agreement with those obtained using
the reference HPLC method.
Biomedicines 2022, 10, 1606 16 of 23

Different research groups [103–105] have performed alternative selections of hemoglobin-

Biomedicines 2022, 10, x FOR PEER REVIEW 17 of 24
binding DNA aptamers. However, these aptamers have been further used as biospecific
elements for electrochemical, SPR, and fluorescent aptasensors with quite complicated analyt-
ical schemes and equipment [104,106–109], which can hardly be applied in routine clinical
Measuring the key diabetic hormone insulin also provides important information for
lab practice.
theMeasuring
diagnosticstheandkeymanagement of diabetes.
diabetic hormone A. Rafati
insulin et al. [58]important
also provides applied a information
quadruplex‐ for
forming DNA aptamer for colorimetric insulin detection. The biotinylated
the diagnostics and management of diabetes. A. Rafati et al. [58] applied a quadruplex- aptamer was
immobilized on a streptavidin magnetic bead/DNA nanotube composite.
forming DNA aptamer for colorimetric insulin detection. The biotinylated aptamer was In the presence
of insulin, the
immobilized onaptamer forms the
a streptavidin quadruplex
magnetic structure
bead/DNA that binds
nanotube hemin forInthe
composite. theperoxi‐
presence
dase‐like oxidation of a chromogenic substrate. The limit of detection for the assay was
of insulin, the aptamer forms the quadruplex structure that binds hemin for the peroxidase-
2.6 pM, which is comparable to an ELISA kit (42 pM).
like oxidation of a chromogenic substrate. The limit of detection for the assay was 2.6 pM,
The same DNA aptamer was used in another assay for insulin detection in serum
which is comparable to an ELISA kit (42 pM).
samples from patients with diabetes [59]. A thiol‐modified aptamer was covalently im‐
The same DNA aptamer was used in another assay for insulin detection in serum
mobilized on golden nanorods (AuNRs) possessing peroxidase‐mimicking activity. In the
samples from patients with diabetes [59]. A thiol-modified aptamer was covalently im-
presence of insulin, the aptamer–insulin complex inhibits the catalytic activity of AuNRs
mobilized on golden nanorods (AuNRs) possessing peroxidase-mimicking activity. In the
(Figure 10), while in the absence of the analyte, peroxidase‐like oxidation provides a col‐
presence of insulin,
orimetric signal. The the aptamer–insulin
limit of detection forcomplex
the assay inhibits the in
was 0.2 pM catalytic activity of
serum samples. ForAuNRs
the
(Figure 10), while in the absence of the analyte, peroxidase-like oxidation
simultaneous detection of insulin and glucose, the analytical system was supplied with provides a colori-
metric
glucosesignal. TheAfter
oxidase. limitenzymatic
of detection for the
glucose assay was
oxidation, 0.2 pM peroxide
hydrogen in serumaccumulates
samples. For in the
simultaneous detection of insulin and glucose, the analytical system was
the solution and participates in the AuNR‐catalyzed oxidation of the substrate. The au‐ supplied with
glucose
thors suggested this binary aptasensor could be particularly useful for the differential di‐ the
oxidase. After enzymatic glucose oxidation, hydrogen peroxide accumulates in
solution
agnosisand participates
of type 1 and typein2 the AuNR-catalyzed oxidation of the substrate. The authors
diabetes.
suggested this binary aptasensor could be particularly useful for the differential diagnosis
of type 1 and type 2 diabetes.

Figure
Figure 10.Insulin
10. Insulindetection
detection by aptasensor
aptasensorbased
basedon onAuAunanorods with
nanorods peroxidase‐like
with activity
peroxidase-like [59].[59].
activity
Without
Without insulin,AuNRs
insulin, AuNRscatalyze
catalyze the
the oxidation
oxidationofofthe
thechromogenic
chromogenicsubstrate (TMB)
substrate in ain
(TMB) peroxidase‐
a peroxidase-
like manner, resulting in a color change in the solution. Insulin binds with the aptamer on the
like manner, resulting in a color change in the solution. Insulin binds with the aptamer on the AuNRs
AuNRs and inhibits the oxidation of TMB.
and inhibits the oxidation of TMB.
Adipokines, peptide hormones produced by adipose tissue, are considered as poten‐
Adipokines, peptide hormones produced by adipose tissue, are considered as potential
tial biomarkers of obesity and diabetes. Lee et al. [60] developed an aptamer‐based micro‐
biomarkers of obesity and diabetes. Lee et al. [60] developed an aptamer-based microplate
plate detection system for vaspin, visfatin, and retinol‐binding protein 4 (RBP4). Adi‐
detection system for vaspin, visfatin, and retinol-binding protein 4 (RBP4). Adipokine-
pokine‐specific DNA aptamers immobilized in microplate wells were used for analyte
specific DNA aptamers immobilized in microplate wells were used for analyte capture,
capture, while specific antibodies were used as reporter components. The limits of detec‐
while
tion specific
were 3.7antibodies
nM for RBP4, were usedfor
1 nM asvaspin,
reporter
andcomponents. The limits
0.4 nM visfatin, both inofmodel
detection were
buffer
3.7solution
nM forand
RBP4, 1 nM for vaspin, and
in diluted serum samples. 0.4 nM visfatin, both in model buffer solution and in
dilutedR.serum samples.
Torabi et al. [61] developed a chemiluminescent assay for RBP4 based on a specific
DNA R. Torabi et The
aptamer. al. [61] developed
immobilized a chemiluminescent
RBP4 assay
aptamer selectively for RBP4
captured based on
the analyte a specific
from so‐
DNA aptamer.
lution. The immobilized
The complexes RBP4 aptamer
were then visualized selectively
using anti‐RBP4 capturedconjugated
antibodies the analyte from
with
covalently crosslinked luminol‐modified AuNPs (Figure 11). The limit of detection for the
Biomedicines 2022, 10, 1606 17 of 23
Biomedicines
Biomedicines 2022, 10, x FOR PEER2022, 10, x FOR PEER REVIEW
REVIEW 18 of 24 18 of 2

solution. The complexes were then visualized using anti-RBP4 antibodies conjugated with
covalently
assay was 50 fMassay
crosslinked was
(1 pg/mL) 50infM (1 pg/mL)
luminol-modified
a model in asolution.
AuNPs
buffer model buffer
(Figure solution.
The11). The
The limit
aptasensor ofaptasensor
was detection
also was also applied
for the
applied
for thewas
assay for
measurement the measurement of
of RBP4 in aserum
50 fM (1 pg/mL) model RBP4 in
samples serum
bufferfrom samples
patients
solution. from
Thewith patients
diabeteswas
aptasensor with diabetes
and also
healthy and healthy
applied
donors,
for donors,
and the resultsof
the measurement and the
agreed results
RBP4quite agreed
well with
in serum quite well
thosefrom
samples with
obtained those
using
patients obtained using
a commercial
with a commercial
healthy ELISA
ELISA
diabetes and
kit.
donors, kit.
and the results agreed quite well with those obtained using a commercial ELISA kit.

Figure
Figure 11. Chemiluminescent
11. Chemiluminescent detection
detection of of
Figure 11. Chemiluminescent RBP4
RBP4 proposed
detection
proposed ofin in [61].
RBP4
[61]. RBP4-specific
proposed in [61].
RBP4‐specific aptamer captures
RBP4‐specific
aptamer capturesaptamer capture
the analyte
analyte in
inthethe
the analyte inwell.
microplate
microplate the The
well.microplate
The well. Thecomplexes
aptamer–RBP4
aptamer–RBP4 aptamer–RBP4
complexes complexes
are viaare
visualized
are visualized visualized
via ofvia conjugates
conjugates
conjugates of o
anti-RBP4 anti‐RBP4
anti‐RBP4 antibodies
antibodies with antibodies
withcovalently
covalently with covalently
crosslinked crosslinked luminol‐modified
luminol‐modified
crosslinked luminol-modified AuNPs.
AuNPs. AuNPs.

A
A pair
pairof DNAA
ofDNA pair of DNA
aptamers
aptamers aptamers
recognizing
recognizing recognizing
thethe
different the different
epitopes
different of epitopes
of vaspin
epitopes of
werewere
vaspin usedvaspin
for were
used for used fo
detection in
detection detection
in lateral
lateral flow in lateral
flow assay
assay [62].flow
[62]. Theassay
The first [62]. The first aptamer—conjugated
firstaptamer—conjugated
aptamer—conjugated with
with AuNPs—servedwith AuNPs—served
AuNPs—served as
aasreporter
a reporter as while
probe, a reporter
probe, while
thetheprobe,
second
second while the second aptamer—immobilized
aptamer—immobilized
aptamer—immobilized onon a test
a test on a testvaspin
line—captured
line—captured line—captured
vaspinthe
from from thevaspin
solutionsolution from
(Figure the solution
(Figure
12). (Figure
12). Without
Without 12).
vaspin,
vaspin, theWithout
the vaspin, theconjugate
AuNP–aptamer
AuNP–aptamer AuNP–aptamer bindsconjugate
conjugatebinds with bind
with with
complementary complementary
DNA at the DNA
control at
line,the control
forming a line,
red forming
color. In a
the
complementary DNA at the control line, forming a red color. In the presence of the analyte, red color.
presence In
of the
the presence of th
analyte, the analyte,
vaspin‐bound the vaspin‐bound
AuNP–aptamer AuNP–aptamer
conjugate passesconjugate
the passes
control
the vaspin-bound AuNP–aptamer conjugate passes the control line and stops in the test line the
andcontrol
stops line
in and stops in
the test zone, the test
where zone,binds
vaspin wheretovaspin
the bindsaptamer.
second to the second
The aptamer.
limit of The limit
detection forofthedetection for th
zone, where vaspin binds to the second aptamer. The limit of detection for the proposed
proposed proposed
LFA was LFA wasboth
about in 0.1 nM, both in the model buffer solution and serum samples.
LFA was about 0.1 about
nM, both0.1 nM,
in the model thebuffer
model buffer
solution solution and serum
and serum samples.samples.

Figure 12. Lateral flow assay for vaspin detection based on two aptamers [62]. The complex of vaspin
Figure
and AuNP-modifiedFigure
12. Lateral flow 12. Lateral
assay
aptamer for flow
vaspin
1 binds assay
with for vaspin
detection
aptamer based detection
on
2 at the twoline,
test based
aptamers on two
[62].
resulting aptamers
inThe
an complex
increase [62]. The complex o
inofAuNP
vaspin and vaspin and aptamer
AuNP‐modified AuNP‐modified
1 binds aptamer
with 1 binds
aptamer 2 with
at the aptamer
test line, 2 at the test
resulting in line,
an resulting in an increas
increase
concentration and red coloring. In the absence of vaspin, AuNP-aptamer 1 binds the complementary
DNA and provides red coloring at the control zone.
Biomedicines 2022, 10, 1606 18 of 23

4. Challenges and Future Directions

An analysis of the up-to-date literature shows quite a large variety of aptamer-based
assays for the colorimetric detection of disease biomarkers in blood. The majority of these
works are the ‘proof-of-principle’ type, which develop a general scheme of the aptasensor
and pay less attention to its routine use for the analyses of clinical samples. In our opinion,
for the successful translation of aptamer-based tests to clinical diagnostics, we first need a
unified, generally accepted methodology for characterizing aptamer-based assays in terms
of their sensitivity and selectivity/specificity. In addition, the current design of aptamer-
based detection systems often does not account for interfering substances in biological
fluids. As a consequence, the analytical characteristics of the assay may worsen from model
analyte solutions to real clinical samples.
We propose here the following criteria for the development of aptamer-based colori-
metric assays:
• The detection method must be simple and compatible with the standard equipment,
consumables, and protocols in clinical diagnostic laboratories;
• The possible adverse effects of interfering substances in clinical samples (proteins, salts,
small molecules, etc.) must be evaluated during the engineering of detection systems;
• If the assay protocol includes the pre-processing of the samples, this step should be
properly optimized and described in the protocol in full detail.
It is worth mentioning as a separate point the design of sandwich-type colorimetric
aptasensors. Test systems of this type provide especially high specificity and selectivity,
but tend to rely on aptamer/antibody pairs. Surely, the displacement of even one antibody
by an aptamer would improve the reproducibility, stability, and cost of the test system.
However, in the further development of aptamer-based diagnostics, we must look toward
aptamer/aptamer sandwich-type assays, which are completely antibody free. This task
requires novel, robust techniques for the selection of pairs of aptamers for different epitopes
of the same analyte.

5. Conclusions
The chemical nature of oligonucleotide aptamers and their stability and flexibility in
assay design make them unprecedentedly useful for engineering reliable and cost-effective
test systems. We are certain that the systematic, rational design of aptasensors and the
creation of unified criteria for their validation will significantly broaden their area of
application in clinical diagnostics and will make aptamer-based assays as routine as PCR
or ELISA.

Author Contributions: Conceptualization, M.V. and A.D.; writing—original draft preparation, A.D.;
writing—review and editing, M.V.; visualization, M.V. and A.D. All authors have read and agreed to
the published version of the manuscript.
Funding: This research was funded by a joint grant from the Russian Science Foundation and the
Government of the Novosibirsk Region, project number 22-15-20050.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The figures were created with Biorender.com.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.
Biomedicines 2022, 10, 1606 19 of 23

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