molecules

Article
The Development of Aptamer-Based Gold Nanoparticle Lateral
Flow Test Strips for the Detection of SARS-CoV-2 S Proteins on
the Surface of Cold-Chain Food Packaging
Xiaotong Li 1 , Jiachen Wang 1 , Ge Yang 2 , Xiaona Fang 3 , Lianhui Zhao 1 , Zhaofeng Luo 4, * and Yiyang Dong 1, *

1 Laboratory of Food Safety and Risk Assessment, College of Life Science and Technology, Beijing University of
Chemical Technology, Beijing 100029, China; 2021210678@buct.edu.cn (X.L.); 2021201147@buct.edu.cn (J.W.);
2020400269@buct.edu.cn (L.Z.)
2 CAMS Key Laboratory of Antiviral Drug Research, Beijing Key Laboratory of Antimicrobial Agents, NHC
Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of
Medical Sciences and Peking Union Medical College, Beijing 100050, China; yangge@imb.cams.cn
3 Department of Basic Medicine, Anhui Medical College, Hefei 230601, China; memoryna@mail.ustc.edu.cn
4 Key Laboratory of Zhejiang Province for Aptamers and Theragnostic, Aptamer Selection Center,
Hangzhou Institute of Medicine (HIM), Chinese Academy of Sciences, Hangzhou 310022, China
* Correspondence: lzf@ucas.edu.cn (Z.L.); yydong@mail.buct.edu.cn (Y.D.)

Abstract: The COVID-19 pandemic over recent years has shown a great need for the rapid, low-cost,
and on-site detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this
study, an aptamer-based colloidal gold nanoparticle lateral flow test strip was well developed to
realize the visual detection of wild-type SARS-CoV-2 spike proteins (SPs) and multiple variants.
Under the optimal reaction conditions, a low detection limit of SARS-CoV-2 S proteins of 0.68 nM
was acquired, and the actual detection recovery was 83.3% to 108.8% for real-world samples. This
suggests a potential tool for the prompt detection of SARS-CoV-2 with good sensitivity and accuracy,
and a new method for the development of alternative antibody test strips for the detection of other
viral targets.

Citation: Li, X.; Wang, J.; Yang, G.;

Keywords: SARS-CoV-2; S protein; COVID-19; lateral flow test strips; AuNPs; aptamer
Fang, X.; Zhao, L.; Luo, Z.; Dong, Y.
The Development of Aptamer-Based
Gold Nanoparticle Lateral Flow Test
Strips for the Detection of SARS-CoV-2
S Proteins on the Surface of Cold-Chain 1. Introduction
Food Packaging. Molecules 2024, 29, SARS-CoV-2 is a human-infecting β-coronavirus that was found in Wuhan in De-
1776. https://doi.org/10.3390/ cember 2019 [1]. The disease caused by its infection was initially named 2019-nCoV by
molecules29081776 the World Health Organization (WHO) on 12 January 2020, and then formally named
Academic Editor: Borislav Angelov
COVID-19, or novel coronavirus pneumonia, on 1 February [2]. According to the WHO,
more than 770 million cases have been reported, with 7 million reported deaths [3]. In this
Received: 17 February 2024 outbreak, SARS-CoV-2 from an unknown animal source, possibly a seafood market, might
Revised: 8 April 2024 have crossed the species barrier to infect humans [4]. Although the likelihood of food-to-
Accepted: 9 April 2024
human transmission is considered lower than other ways, such as respiratory droplets
Published: 13 April 2024
and cs, it should not be neglected as a risk factor given the large volumes of refrigerated
foods being transported across different countries and regions [5]. SARS-CoV-2 virions
adhering to solid surfaces are reported to be stable, with a viability up to longer than 72 h
Copyright: © 2024 by the authors.
(on plastic) [6]. Therefore, sensitive, fast, and low-cost SARS-CoV-2 detection is urgently
Licensee MDPI, Basel, Switzerland.
needed for cold-chain foods.
This article is an open access article SARS-CoV-2 is a spherical or pleomorphic enveloped particle containing single-
distributed under the terms and stranded (positive-sense) RNA associated with a nucleoprotein within a capsid comprised
conditions of the Creative Commons of matrix protein [7]. The four main structural proteins encoded by COVID-19 are envelope
Attribution (CC BY) license (https:// protein (E protein), spike protein (S protein), membrane protein (M protein), and nucleo-
creativecommons.org/licenses/by/ capsid protein (N protein) [8]. These four proteins are important for the viral infection of
4.0/). cells and replication and transcription [7,9]. Among them, S protein is a protruding protein

Molecules 2024, 29, 1776. https://doi.org/10.3390/molecules29081776 https://www.mdpi.com/journal/molecules

Molecules 2024, 29, 1776 2 of 17

widely distributed on the surface of the viral envelope that mainly mediates the fusion of
the viral and host cell membranes [10]. It consists of two subunits, S1 and S2: S1 mainly
contains the receptor binding domain (RBD), which is responsible for recognizing cellular
receptors [11]. A recent study has shown that N-protein-based assays are more sensitive
for detecting SARS-CoV-2 infection, while S-protein-based assays are more specific [12],
and thus, S proteins are equally valuable as assay targets and can be used as biomarkers in
antigen detection.
Currently, there are various detection methods for SARS-CoV-2, which are broadly
classified into three categories: (1) Nucleic acid detection [13], including fluorescence
quantitative PCR [14], microfluidic chip [15], Isothermal amplification technique [16], etc.
Real-time fluorescence quantitative PCR (RT-qPCR) is the method for detecting viral RNA in
samples from the upper respiratory tract, featuring high specificity, high sensitivity, and pre-
infection detectability [13]. Since the outbreak of the epidemic, nucleic acid testing has been
used as the “gold standard” for the diagnosis of SARS-CoV-2. (2) Serological testing [17]
is a method for detecting the presence of SARS-CoV-2-specific antibodies (IgM/IgG) in
a patient’s blood. (3) Antigen detection [18] is a method that utilizes specific monoclonal
antibodies prepared against the antigenic proteins of SARS-CoV-2 to detect the intrinsic
components of the virus, such as the N and S proteins of SARS-CoV-2, using immunofluidic
chromatography [19], enzyme-linked immunosorbent assays [20], chemiluminescence [21],
and other methods. One of them, the colloidal gold immunochromatographic assay [22], is
widely utilized due to its simplicity, speed, and low cost for quick detection in food safety
and public healthcare.
Lateral flow assays (LFAs) [19] are one type of diagnostic scheme that can provide
rapid, point-of-care results. Aptamers are single-strand nucleic acid sequences that fold
into secondary structures and have target binding affinities on par with antibodies [23].
As chemically synthesized agents, aptamers are also produced more affordably and re-
producibly than antibodies [24], and also have other multifarious advantages such as
good specificity, long-term stability during storage, ease to produce, liable modification,
and meritable flexibility in different sensing formats, making them well suited for rapid
virus detection. Currently, the application of the combination of aptamers and lateral flow
assays to the development of target identification has yielded many results [25–27]. DNA
aptamers against SARS-CoV-2 S protein have been successfully identified using an in vitro
iterative library selection process called the systematic evolution of ligands by exponential
enrichment (SELEX) [28,29].
The market for new crown assays, dominated by PCR, is indeed dominating at present.
However, traditional nucleic acid detection methods, although accurate, are expensive
in terms of instrumentation and require specialized equipment and personnel, which
may be limiting in remote areas or emergency situations [30]. In contrast, aptamer test
strip technology is simple to operate, has a short detection time, provides an intuitive
visualization of results, and costs less than $1 per test to manufacture, which provides the
advantages of accurate, portable, and efficient detection [27]. These features have allowed
the test strip method to fill a market gap in certain specific scenarios and applications [31].
Especially in the early stages of the outbreak, due to the lack of supply of nucleic acid test
kits and limitations in testing capacity, the test strip method played an important role as a
complementary testing tool.
Secondly, aptamer test strip technology has a wide range of application areas [32],
which can be used not only for clinical testing in medical institutions, but also in scenarios
such as rapid screening in public places and personal self-testing, further expanding its
market space. With the advancement of science and technology and in-depth research, test
strip methods are being improved and optimized, and commercial applications will become
more widespread. The investment and support from various parties such as governments,
medical institutions, and enterprises for the new crown testing technology will also further
boost the market. Innovations in technology offer more possibilities for the development of
test strip methods in the new crown testing market [33].
 Molecules2024,
Molecules 2024,29,
29,x xFOR
FORPEER
PEERREVIEW
REVIEW 3 3ofof1717

alsofurther
also furtherboost
boostthethemarket.
market.Innovations
Innovationsinintechnology
technologyoffer
offermore
morepossibilities
possibilitiesfor
forthe
the
Molecules 2024, 29, 1776 3 of 17
development of test strip methods in the new crown testing
development of test strip methods in the new crown testing market [33]. market [33].
InInthis
thiswork,
work,thethe“universal”
“universal”aptamer
aptamerMSA52
MSA52was waschosen
chosenasasthe
thedetection
detectionaptamer,
aptamer,
whichshowed
which showedgenerally
generallyhighhighaffinity
affinityfor
forwild-type
wild-typeSARS-CoV-2
SARS-CoV-2spike spikeproteins,
proteins,asaswell
well
In this work, the “universal” aptamer MSA52 was chosen as the detection aptamer,
asasAlpha,
Alpha, Beta,
Beta, Gamma,
Gamma, Epsilon,Kappa,
Epsilon, Kappa, Delta,
Delta, andOmicron
and Omicron proteins,
proteins, withKD
with KDvalues
values
which showed
ranging from22totogenerally highAaffinity for wild-type SARS-CoV-2 spikebased
proteins, as well
ranging from 1010nMnM[29].
[29]. lateralflow
A lateral flow chromatography
chromatography teststrip
test stripbased on onnucleic
nucleic
as Alpha,
acidaptamers Beta,
aptamersusing Gamma,
usingnanogold Epsilon, Kappa,
nanogoldasasaachromogenic Delta,
chromogenicagent and Omicron
agentwas proteins,
wassuccessfully with
successfullydeveloped,KD
developed, values
an
acid an
ranging
aptamer from 2 to
engineering 10 nM [29].
approach A lateral
was flow
used tochromatography
select a capture test strip
aptamer based
with on
highnucleic
binding acid
aptamer engineering approach was used to select a capture aptamer with high binding
aptamers using nanogold as a chromogenic agent was successfully developed, an aptamer
affinity,and
affinity, andthe
theapplication
applicationofofthetheoptimized
optimizedconditions
conditionswas wassuccessfully
successfullyvalidated
validated forthethe
engineering approach was used to select a capture aptamer with high bindingforaffinity,
on-sitescreening
on-site screeningofofSARS-CoV-2
SARS-CoV-2SP SPinincold-chain
cold-chainfood
foodpackages,
packages,both
bothvisually
visuallyand
andquan-
quan-
and the application of the optimized conditions was successfully validated for the on-site
titatively.
titatively.
screening of SARS-CoV-2 SP in cold-chain food packages, both visually and quantitatively.

2.2.Results
2.Results
Results
2.1. Characterization
2.1.2.1. ofofAuNPs
Characterization
Characterization ofAuNPs
AuNPsand
and Determination
and ofofCoupling
Determination
Determination ofCoupling
CouplingConditions
Conditions
Conditions
The
The The approximate
approximate
approximate binding
binding
binding state
state ofofgold
state ofgold
gold nanoparticles
nanoparticles
nanoparticles (AuNPs)
(AuNPs)
(AuNPs) and
and and nucleic
nucleic
nucleic acid
acidacidap-
ap- ap-
tamers
tamers
tamers can
cancan be
bebe determined
determined
determined by transmission
bybytransmission electron microscopy.
transmissionelectron microscopy.
microscopy. The The results
The results of
results of the the trans-
the transmis-
trans-
mission
mission electron
sion electron
electron microscopy
microscopy
microscopy characterization
characterization
characterization show show
show(Figure(Figure 1a,b)the
1a,b)1a,b)
(Figure that that
that AuNPs theAuNPs
the AuNPs are
are spherical
are
spherical
in shape,
spherical ininwith
shape,
shape, withaarelatively
a relatively
with relatively
homogeneous homogeneous
homogeneous particle
particleparticle
size (aboutsize
size 20 (about
nm), 20
(about 20nm),
and nm),
are andandare
well arewell
well
dispersed.
dispersed.
From Figure
dispersed. From
From Figure
1c,Figure
it can 1c,1c,seen
be ititcan
can
that
bebeseen
seen
the AuNPsthatthe
that the AuNPs
after
AuNPscouplingafter
after coupling
maintain
coupling maintain
amaintain
regular aaregular
regular
arrangement
arrangement
of certain of
gaps certain
between gaps
them, between
and no them,
dispersionand no
or dispersion
condensation
arrangement of certain gaps between them, and no dispersion or condensation occurs. or condensation
occurs. The occurs.
comparison
The
of comparison
the before and of the
after before
pictures and after
shows pictures
that the shows
nucleic that
acid
The comparison of the before and after pictures shows that the nucleic acid aptamer and the
aptamernucleic
and acid
the aptamer
gold and
nanoparti-
the
the gold
cles
gold form nanoparticles
a stable complex
nanoparticles forma[34].
form astable
stable complex[34].
In complex
addition, [34].In
after Inaddition,
the addition,
aptamerafterafterthe
binds the aptamerthe
toaptamer
AuNPs, binds
binds toto
AuNPs
become
AuNPs,
AuNPs, the larger,
the AuNPs
AuNPs and the absorption
become
become larger,and
larger, wavelength
and theabsorption
the of thewavelength
absorption AuNP–aptamer
wavelengthofofthe isAuNP–aptamer
the slightly shifted to
AuNP–aptamer
isisthe right shifted
slightly
slightly under
shiftedUV totospectrophotometer
theright
the rightunderunderUV characterization
UV spectrophotometer
spectrophotometer [35]. As shown in Figure[35].
characterization
characterization 2, the
[35].As UV
As
absorption
shown in peak
Figure of
2, bare
the UV AuNPs is
absorption at 520
peak nm,ofand
bare that of
AuNPs
shown in Figure 2, the UV absorption peak of bare AuNPs is at 520 nm, and that of the theis AuNP–aptamer
at 520 nm, and is at
that 525
of nm,
the
with a rightward
AuNP–aptamer
AuNP–aptamer shift
isisatat 525 ofnm,
525nm, 5 nm with
with between
aarightward the two.
rightward shiftofof55nm
shift nmbetween
betweenthe thetwo.
two.

(a)
(a) (b)
(b) (c)
(c)
Figure
Figure 1.1.TEM
Figure 1.TEM
TEM test
test results.
results.
test (a)(a)
(a)
results. AuNPs;
AuNPs; (b)(b)
(b)
AuNPs; AuNPs;
AuNPs; (c)AuNP–aptamer.
(c)
AuNPs; AuNP–aptamer.
(c) AuNP–aptamer.

520nm
520 nm
525nm
525 nm

Figure
Figure 2.2.The
Figure 2.The
UV
TheUV absorption
absorption
UV spectra
spectra
absorption ofofAuNPs
spectra ofAuNPs and
and
AuNPs AuNP–aptamer
AuNP–aptamer
and conjugation.
conjugation.
AuNP–aptamer conjugation.
 Molecules 2024, 29, x FOR PEER REVIEW 4 of 17
Molecules 2024, 29, x FOR PEER REVIEW 4 of 17

Molecules 2024, 29, 1776 4 of 17

The determination of the aptamer concentration in the coupling solution is crucial to
the experimental
The determinationresults. The aptamer
of the aptamer added in in
concentration thethe
process
coupling of making
solutionthe coupling
is crucial to solu-
tion was
the experimental selected from
The results. the aptamer
The aptamer
determination MSA52,
of theadded with
in the
aptamer a high-affinity
process of making
concentration specific recognition
the coupling
in the coupling solu-ability
solution is crucial
for SARS-CoV-2
tion was to
selected S protein,
from the aptamer
the experimental published
results.MSA52, by Zhang Zifeng
with a high-affinity
The aptamer added in the et al. [29].
specific
process The coupled
recognition
of making aptamer
ability
the coupling
concentration
solution Swas
for SARS-CoV-2 gradients
selected
protein, were
fromset
published toaptamer
theby 0.5 µM, MSA52,
Zhang 1 µM, 2etwith
Zifeng µM, 3a µM,
al. [29]. 4 µM,
high-affinity
The and
coupled 5aptamer
µM,recognition
specific and the
optimal
abilityamount
concentration gradients ofwere
aptamer
for SARS-CoV-2 added
0.5 µM,was
set Stoprotein, determined
published
1 µM, 2 µM, to be
by3Zhang
µM, 3 µM
Zifeng
4 µM, andetby determining
5al.
µM,[29].
andThe the
the coupled
ODaptamer
optimal T/ODC through
amount an immunochromatographic
concentration
of aptamer gradients
added was were set to reader
determined 0.5 beC10066-10
toµM, 31 µM,
µM 2by (Figure
µM, 3 µM, 3).4 µM,
determining theand 5 µM,
ODT/ODand the optimal
C through amount of aptamer added
an immunochromatographic was C10066-10
reader determined(Figureto be 33).
µM by determining the
ODT /ODC through an immunochromatographic reader C10066-10 (Figure 3).

(a) (b)
Figure 3. Effect of (a)aptamer concentration on test strip results. (a) Physical(b) diagram of coupling so-
lution at
Figure 3. Figure different
Effect of3.aptameraptamer concentrations: from left to right, aptamer concentrations were 0.5 µM,
Effect ofconcentration on test stripon
aptamer concentration results. (a) Physical
test strip results. diagram of coupling
(a) Physical diagramso-
of coupling
lution1atµM, 2 µM, aptamer
different 3 µM, 4 µM, and 5 µM. (b) Absorbance values of test strips measured
solution at different aptamer concentrations: from left to right, aptamer concentrationsµM,
concentrations: from left to right, aptamer concentrations by0.5
were the immuno-
were 0.5 µM,
1 µM, chromatographic
2 µM, 3 µM,
1 µM, 2 µM,4 µM,reader
3 µM,and 5atµM.
4 µM,
different
and(b)
aptamer concentrations.
Absorbance
5 µM. values of
(b) Absorbance test strips
values of testmeasured by theby
strips measured immuno-
the immunochro-
chromatographic reader at different aptamer concentrations.
2.2.matographic
Optimizationreader at Strip
of Test different aptamer concentrations.
Conditions
2.2. Optimization
2.2.1. of Test Strip
2.2.Optimization
Optimization of
of SAConditions
Test Concentrations
Strip Conditionsand Molar Ratios of SA to Biotin-DNAT
2.2.1.
2.2.1. OptimizationOptimization
of SA of SA
Concentrations
The text line (T line) signal Concentrations
valueand is Molar
lowerand Molar
Ratios
than ofRatios
that SA to of
of the SA to line
Biotin-DNAT
control Biotin-DNAT
(C line) when
the streptavidin
The text The
line text (SA)
(T line) concentrations
linesignal
(T line) signal
value of thethan
value
is lower isTlower
andthatCthan
lines
of are
that
the equal
of
control from
theline (Cthe
control experimental
line
line) (C line) when
when
results,
the which may
streptavidin be
(SA) because the
concentrations complementary
of the T and binding
C lines
the streptavidin (SA) concentrations of the T and C lines are equal from the experimental capacity
are equal of
frompoly the T-poly C of
experimental
results, which may be because the complementary binding capacity of poly T-poly C of So,C of
the C line
results, is stronger
which may thanbe that of
because a certain
the segment
complementary of the DNA
binding strand
capacity of the
of aptamer.
poly T-poly
the Cwe investigated
linethe C line is than
is stronger the that
concentration
stronger than
of aT-linear
that ofsegment
a certain certainofstreptavidin
segment
the DNAofstrand (0.2DNA
the mg/mL,
of thestrand 0.4 of
aptamer. mg/mL,
the 0.6
So, aptamer.
So,
we investigated we
mg/mL), fixed investigated
thethe the
intensity of of
concentration concentration
C-linear
T-linear of T-linear
streptavidin
streptavidin streptavidin
at 0.2
(0.2mg/mL,
mg/mL,and (0.2 mg/mL,
0.4also examined
mg/mL, 0.4 mg/mL,
0.6 the
mg/mL), 0.6
different mg/mL),
fixed molar fixed of
ratios
the intensity the
ofSA intensity
C-linear of C-linear
to Biotin-DNA
streptavidin streptavidin
T (2:1,
at 1:1, at1:6)
1:2, 1:4,
0.2 mg/mL, 0.2 mg/mL,
and for
also and also
favorable
examined probeexamined
the fixa-
the different molar ratios of SA to Biotin-DNA (2:1, 1:1, 1:2,
different molar ratios of SA to Biotin-DNAT (2:1, 1:1, 1:2, 1:4, 1:6) for favorable probe fixa- line,
tion on nitrocellulose (NC) membranes. The higher T the concentration1:4, 1:6)
of for
SA favorable
in the T probe
the fixation
higher on
the nitrocellulose
signal level, and(NC)
the membranes.
best color The
development higher
tion on nitrocellulose (NC) membranes. The higher the concentration of SA in the T line, the
was concentration
achieved at an of
SA SA in
concen-the T
line,
tration the
of 0.4higher
mg/mL the signal
(Figure level,
4a) with anda the
molar best color
ratio of development
1:4
the higher the signal level, and the best color development was achieved at an SA concen- (Figure 4b). was
Under achieved
these at an
condi- SA
tration concentration
tions,
of 0.4the absorbance
mg/mL of
(Figure0.4 mg/mL
of 4a)
the with (Figure
test strips
a molar was4a) with a
satisfied
ratio molar
of 1:4and ratio of
a comparable
(Figure 1:4 (Figure
4b). Underdegree 4b). Under
of color de-
these condi- these
theconditions,
tions,velopment
absorbance the
of the ofTabsorbance
andtest
the C lines ofwas
strips the test strips was
wasreached.
satisfied and asatisfied and adegree
comparable comparableof color degree
de- of color
development of the T
velopment of the T and C lines was reached. and C lines was reached.

(a) (b)
(a)
Figure 4. Cont. (b)
Molecules 2024, 29, x FOR PEER REVIEW 5 of 17
Molecules 2024, 29, 1776 5 of 17

(c) (d)
Figure 4. Optimization
Figure of testofstrip
4. Optimization testconditions. (a) Absorbance
strip conditions. values ofvalues
(a) Absorbance test strips measured
of test by the
strips measured
immunochromatographic reader at different SA concentrations. (b) Absorbance values
by the immunochromatographic reader at different SA concentrations. (b) Absorbance values of test strips of
measured by the immunochromatographic reader at different aptamer concentrations
test strips measured by the immunochromatographic reader at different aptamer concentrations atat different
ratios of SA to C- and T-line aptamers. (c) Absorbance values of test strips measured by the immu-
different ratios of SA to C- and T-line aptamers. (c) Absorbance values of test strips measured by the
nochromatographic reader with different NC membrane types. (d) Absorbance values of test strips
immunochromatographic reader with different NC membrane types. (d) Absorbance values of test
measured by the immunochromatographic reader with different binding buffers.
strips measured by the immunochromatographic reader with different binding buffers.
2.2.2. Optimization
2.2.2. Optimizationof NC Membranes
of NC Membranes
Here, we we
Here, choose thethe
choose four models
four of NC
models of NCmembranes:
membranes:BSK95, BSK110,
BSK95, BSK140,
BSK110, BSK140,andand
BSK160. As shown in the data of Figure 4c, the T/C values of BSK95 and BSK140
BSK160. As shown in the data of Figure 4c, the T/C values of BSK95 and BSK140 were were
closest to one
closest andand
to one thethe
absorbance of BSK140
absorbance of BSK140waswas
higher. As As
higher. a result, BSK140
a result, is the
BSK140 most
is the most
suitable for this experiment.
suitable for this experiment.

2.2.3. Optimization
2.2.3. of Running
Optimization Buffer
of Running Buffer
In this study,
In this the signal
study, values
the signal of theofreactions
values under under
the reactions three buffers (PBS, HEPES,
three buffers and
(PBS, HEPES,
water)
and were
water) compared (Figure 4d),
were compared in which
(Figure 4d), in thewhich
HEPES thebuffer
HEPEShadbuffer
the best
hadcolor devel-
the best color
development
opment response.response. It is basically
It is basically consistentconsistent
with thewith thecomposition
buffer buffer composition
used in usedthe ap-in the
aptamer
tamer literature
literature [29].specific
[29]. The The specific compositions
compositions of theofthree
the three buffers
buffers werewere
set upsetasupfollows:
as follows:
(1) (1)
50 50
mM mM of of HEPES,
HEPES, 1%1% sucrose,
sucrose, 0.1%
0.1% Tween-20,
Tween-20, 2.52.5 mmol/L
mmol/L of of CaCl
CaCl 2 , 2.5
2, 2.5 mmol/L
mmol/L of of
MgClMgCl , 6 mmol/L
2, 6 2mmol/L of KCl,
of KCl, 150 mmol/L
150 mmol/L of NaCl,of NaCl,
and 1% and 1%(2)
BSA; BSA; (2) ultrapure
ultrapure water, 1% water,
su- 1%
sucrose,
crose, 0.1% Tween-20,
0.1% Tween-20, 2.5 mmol/L
2.5 mmol/L of CaCl of2,CaCl 2 , 2.5 mmol/L
2.5 mmol/L of MgClof2,MgCl 2 , 6 mmol/L
6 mmol/L of KCl, of150KCl,
mmol/L of NaCl, and 1% BSA; and (3) 10 mM of PBS, 1% sucrose, 0.1% Tween-20, and 1%and
150 mmol/L of NaCl, and 1% BSA; and (3) 10 mM of PBS, 1% sucrose, 0.1% Tween-20,
BSA.1% BSA.

2.3.2.3. An Aptamer
An Aptamer Engineering
Engineering Approach
Approach to Selecting
to Selecting T-Line
T-Line Complementary
Complementary Sequences
Sequences
FromFrom
the the analysis
analysis of aptamer
of aptamer andand protein
protein binding
binding sites,
sites, DT80DT80
andand
DT82DT82
are are on the
on the
extended sequence of the aptamer poly T [36] (Table 1). Therefore, the binding
extended sequence of the aptamer poly T [36] (Table 1). Therefore, the binding sites were sites were
identified as DA20 (Figure 5a) and DT66 (Figure 5b). The lower the binding energy of the
identified as DA20 (Figure 5a) and DT66 (Figure 5b). The lower the binding energy of the
site, the more stable the binding, which means that the binding effect is the best at DA20.
site, the more stable the binding, which means that the binding effect is the best at DA20.
After that, the aptamer sequence where DA20 is located was selected and extended by
After that, the aptamer sequence where DA20 is located was selected and extended by 10
10 nt, 15 nt, and 20 nt, and the complementary sequences of the resulting aptamers were
nt, 15 nt, and 20 nt, and the complementary sequences of the resulting aptamers were
MSA10, MSA15, and MSA20, in order.
MSA10, MSA15, and MSA20, in order.
Based on the MOE molecule docking results (Figure 5 and Table 1) and the selected
Table 1. Aptamer and protein binding sites.
aptamers as described in the original literature, it was finally decided to retain the four
truncated
Numberaptamers, MSA52-10,
Energy AptamerMSA52-15, MSA52-20,
Binding Site Proteinand MSA52-22
Binding Site (Table
Aptamer2).Sequence
In the
presence1of 500 ng/mL−15.89of SP, MSA52-10
DA20 manifested a high inhibition
Lys921 rate and good
DT18-DT22color
rendering 2 effect (Figure
−0.9 5c,d). DT66 Gln935 DC65-DG71
3 −0.72 DT80 Lys285 DT77-DT84
4 −0.64 DT82 Lys278 DT77-DT84
 MSA-10 Biotin-ACGCCAAGGA
MSA-15 Biotin-ACGCCAAGGAGATGC
MSA-20 Biotin-ACGCCAAGGAGATGCTTCGC
MSA-22 Biotin-CGCCAGGCCCGGAGCCAAACCC
Molecules 2024, 29, 1776 6 of 17
Control-line DNA Biotin-AAAAAAAAAA

(a) (b)

(c) (d)

(e)
Figure
Figure 5. Molecular 5. Molecular
docking docking and aptamer-complementary
and aptamer-complementary chain element(a)optimization.
chain element optimization. DA20 (a)
docking site and its aptamer sequence. (b) DA66 docking site and its aptamer
docking site and its aptamer sequence. (b) DA66 docking site and its aptamer sequence. (c) Ab- sequence. (c) A
ance values of test strips measured by the immunochromatographic reader
sorbance values of test strips measured by the immunochromatographic reader with different with different ap
complementary sequences with blank conditions. (d) Absorbance values of test strips measu
aptamer-complementary sequences with blank conditions. (d) Absorbance values of test strips
measured by immunochromatography with different aptamer-complementary sequences with SP
500ppb conditions. (e) MOE molecular docking result.

Based on the MOE molecule docking results (Figure 5 and Table 1) and the selected
aptamers as described in the original literature, it was finally decided to retain the four
truncated aptamers, MSA52-10, MSA52-15, MSA52-20, and MSA52-22 (Table 2). In the
presence of 500 ng/mL of SP, MSA52-10 manifested a high inhibition rate and good color
rendering effect (Figure 5c,d).

Table 2. Truncated aptamer sequences and C-Line complementary sequence.

Name Sequences (5′ -3′ )

MSA-10 Biotin-ACGCCAAGGA
MSA-15 Biotin-ACGCCAAGGAGATGC
MSA-20 Biotin-ACGCCAAGGAGATGCTTCGC
MSA-22 Biotin-CGCCAGGCCCGGAGCCAAACCC
Control-line DNA Biotin-AAAAAAAAAA
 2.4. Optimization of Detection Conditions
Optimization of sample addition: The total spiked volume was set to 50 µL, and the
Molecules 2024, 29, 1776
spiked volume of nanogold–aptamer coupling (1 µL, 2 µL, 3 µL, 4 µL, 5 µL) was opti-
7 of 17
mized. With the increment of the spiking volume, the color of the T and C lines progres-
sively deepened, and ODT/ODC gradually increased. Comparing the blank and spiking
experiments (Figureof6),
2.4. Optimization the color
Detection change was most pronounced at a spiking volume of 2
Conditions
µL, and the highest inhibition
Optimization of samplerate was observed
addition: The totalatspiked
this time.
volume was set to 50 µL, and
the spiked volume of nanogold–aptamer coupling (1strip
The detection time was optimized: after the test µL, 2was
µL, added
3 µL, 4 toµL,the sample,
5 µL) was its
ODT/OD C value With
optimized. was read every 1 min
the increment from
of the 1 min
spiking until the
volume, the25th
colormin, and
of the the results
T and C lines ob-
tained are shown deepened,
progressively in Figure 6d.
andCalculation of the inhibition
ODT /ODC gradually rate
increased. yielded an
Comparing theoptimal detec-
blank and
spiking experiments (Figure
tion time range of 8 min–15 min. 6), the color change was most pronounced at a spiking volume
of 2 µL, and the highest inhibition rate was observed at this time.

(a) (b) (c) (d)

Figure 6. Optimization
Figure of of
6. Optimization detection
detectionconditions.
conditions. (a) (a)T/C
T/Cand
andinhibition
inhibition ratio
ratio withwith different
different amounts
amounts of
of coupling solution added. (b) Test strip experiments with different coupling solution
coupling solution added. (b) Test strip experiments with different coupling solution spiking amounts spiking
amountsunder under
blank blank conditions,
conditions, from leftfrom left 1toµL,
to right: right:
2 µL,13µL,
µL, 42 µL,
µL,and
3 µL, 4 µL,
5 µL. andstrip
(c) Test 5 µL. (c) Test strip
experiments
experiments with different coupling solution spiking amounts under 500ppb SP
with different coupling solution spiking amounts under 500ppb SP conditions, from left to right: conditions, from
left to1 right: 1 µL, 2 µL, 3 µL, 4 µL, and 5 µL. (d) T/C and inhibition rate
µL, 2 µL, 3 µL, 4 µL, and 5 µL. (d) T/C and inhibition rate change over time. change over time.

2.5. Quantitative Detection

The detection timeofwas
SP optimized:
by Test Strips
after the test strip was added to the sample, its
ODT /ODC value was read every 1 min from 1 min until the 25th min, and the results
Test strips capable of visualizing and detecting SARS-CoV-2 S protein were con-
obtained are shown in Figure 6d. Calculation of the inhibition rate yielded an optimal
structed by incubating different concentrations of SP (0 ng/mL, 100 ng/mL, 300 ng/mL,
detection time range of 8 min–15 min.
600 ng/mL, 800 ng/mL, and 1000 ng/mL) and AuNP–aptamer for 30 min and then per-
forming spiking reactions
2.5. Quantitative Detectionfor quantitative
of SP by Test Stripscalibration (Figure 7). The higher the concen-
tration ofTestSP,strips
the lighter the color of the area
capable of visualizing and detectingdelineated by the STprotein
SARS-CoV-2 line, and itsconstructed
were visual detec-
tion by
range is 0.1 µg/mL–1
incubating µg/mL. The T/C
different concentrations of SPratio had a100
(0 ng/mL, good linear
ng/mL, connection
300 ng/mL, 600with
ng/mL,the SP
800 ng/mL,yand
concentration. 1000 ng/mL)+and
= −0.000408x 0.70AuNP–aptamer
(R2 = 0.97) was for the
30 min and equation
linear then performing
at thespiking
detection
timereactions
of 15 min,forand
quantitative calibration
the detection (Figureof
sensitivity 7).the
Theinitially
higher the concentration
constructed stripofwas
SP, the
calcu-
lighter the color of the area delineated by the T line, and its visual
lated to be LOD = 91.2 ng/mL (≈0.68 nM) based on a 3sigma/k scheme, where sigma refers detection range is
0.1 µg/mL–1 µg/mL. The T/C ratio had a good linear connection with the SP concentra-
to the standard deviation of the blank control and k refers to the slope of the linear equa-
tion. y = −0.000408x + 0.70 (R2 = 0.97) was the linear equation at the detection time of
tion.15 This
min, and the detection sensitivity ofwe
suggests that the test strips thedeveloped can measure
initially constructed SP calculated
strip was at low levelsto beand
fulfillLOD
the =requirements
91.2 ng/mL (≈ of0.68
thenM)
national
based SPon detection
a 3 sigma/k standard. Although
scheme, where therefers
sigma sensitivity
to the of
the test strips prepared in this work is lower than other methods (Table 3),
standard deviation of the blank control and k refers to the slope of the linear equation. Thisthe efficiency
and convenience
suggests that theof the
test test strip
strips method incan
we developed detecting
measureand reading
SP at results
low levels on thethe
and fulfill basis
re- of
quirements
adequate visualofdetection
the nationalareSPirreplaceable.
detection standard. Although the sensitivity of the test strips
prepared in this work is lower than other methods (Table 3), the efficiency and convenience
of the test strip method in detecting and reading results on the basis of adequate visual
detection are irreplaceable.
olecules 2024, 29, x FOR PEER REVIEW 8 o

Molecules 2024, 29, 1776 8 of 17

(a) (b)
Figure 7. Quantitative detection of SP. (a) Standard curve. (b) Calibration curve and color d
ment of test strips established at 15 min detection time, from left to right: 0 ng/mL, 100 ng/
ng/mL, 600 ng/mL, 800 ng/mL, and 1000 ng/mL.
(a) (b)
Table 3. Detection of SARS-CoV-2 by strip method.
Figure 7. Quantitative
Figure 7. Quantitative detection
detection of SP.
SP.(a)
(a)Standard
Standard curve.
curve. (b) Calibration
(b) Calibration curve
curve and and
color color deve
devel-
Target ment of test strips
opment of test established
strips
Detection at 15 min detection time, from LOD
established
Range at 15 min detection time, from left left to right: 0 ng/mL, 100 ng/mL,
to right: 0 ng/mL, 100 ng/mL,
Refere
300 600
ng/mL, ng/mL, 600 ng/mL,
ng/mL, 800 ng/mL,
800 ng/mL, and and
10001000 ng/mL.
ng/mL.
S Protein / 100 pM [37
N Protein Table 3. 0.1–500 ng/mL
Detection of SARS-CoV-2 by strip
Table 3. Detection of SARS-CoV-2 by strip method.
method. 0.1–0.5 ng/mL [38
N gene 0.25–100 copies/mL
Target Detection Range 0.25
LOD copy/mL Reference [39
TargetIgG Detection Range
10 ng/mL–100 µ g/mL /
S Protein
LOD4 pM
100 ng/mL [37]
Reference [40
S Protein
SARS-CoV-2 Virus N Protein /
0–50 ng/mL 0.1–500 ng/mL 100
0.1–0.5 pM
ng/mL
10 ng/mL [38] [37] [41
N gene 0.25–100 copies/mL 0.25 copy/mL [39]
N Protein
S Protein 0.1–500
100 ng/mL
IgGng/mL–100010ng/mL ng/mL–100 µg/mL
0.1–0.5 ng/mL
491.2
ng/mLng/mL [40]
[38]
This w
N gene 0.25–100Virus
SARS-CoV-2 copies/mL 0–50 ng/mL 0.2510copy/mL
ng/mL [41] [39]
S Protein 100 ng/mL–1000 ng/mL
IgG 2.6. 10 ng/mL–100
Specificity Test µ g/mL 491.2 ng/mL
ng/mL This work
[40]
SARS-CoV-2 Virus 0–50 ng/mL protein, HCoV-OC43-RBD
HCoV-229E-RBD 10 ng/mL
protein, RSV-F protein, human [41] I
2.6. Specificity Test
S Protein 100BSA,
tein, ng/mL–1000
and SARS-CoV-2ng/mL SP were selected91.2 ng/mL the specificity contrast
to perform This work tes
HCoV-229E-RBD protein, HCoV-OC43-RBD protein, RSV-F protein, human IgG pro-
the
tein,concentration
BSA, and SARS-CoV-2 condition
SP wereof 1000 ng/mL.
selected As shown
to perform in Figure
the specificity 8, the
contrast test color
under of the T
2.6. the
Specificity Test
the concentration condition of 1000 ng/mL. As shown in Figure 8, the color
S-protein group showed a significant decrease, and the rest of the absorbance of the T line of
the S-protein group showed a significant decrease, and the rest of the absorbance had no
significant
HCoV-229E-RBD change protein,
and wasHCoV-OC43-RBD
close to that of theprotein,
blank control group, which
RSV-F protein, humanindicat
IgG p
significant change and was close to that of the blank control group, which indicated that
tein,the
BSA,
the kithad
kit hadgood
and good detection
SARS-CoV-2
detection SP specificity.
were selected to perform the specificity contrast test un
specificity.
the concentration condition of 1000 ng/mL. As shown in Figure 8, the color of the T lin
the S-protein group showed a significant decrease, and the rest of the absorbance had
significant change and was close to that of the blank control group, which indicated t
the kit had good detection specificity.

Figure 8. Results of a selectivity study of nucleic acid aptamer test strips for SP detection.
Figure 8. Results of a selectivity study of nucleic acid aptamer test strips for SP detection.
2.7. Reproducibility Evaluation
2.7. Reproducibility Evaluation
In order to explore the inter-batch and intra-batch precision of the aptamer-based test
strips prepared by this method, experimental verification was carried out. Three different
In order to explore the inter-batch and intra-batch precision of the aptamer-ba
batches of test strips prepared by this method were randomly selected, and three different
Figure 8.
stripsResults
concentrations ofofa Sselectivity
prepared by study
this method,
protein (0.25 ofexperimental
µg/mL, nucleic acid aptamer
0.5 µg/mL, test
1verification
µg/mL) strips
werewas
usedfor SPsampling
detection.
carried
for out. Three d
batches of test strips prepared by this method were randomly selected, and
experiments. Each concentration in each batch was measured in parallel three times. The three d
2.7. concentrations
Reproducibility Evaluation
of S protein (0.25 µg/mL, 0.5 µg/mL, 1 µg/mL) were used for samp
periments.
In order to Each
exploreconcentration in each
the inter-batch batch was precision
and intra-batch measuredofinthe
parallel three tim
aptamer-based
strips prepared
mean value by
( Xthis method, experimental
), standard verification
deviation (SD), was carried
and coefficient out. Three
of variation (CV differ
= (SD
batches of test strips prepared by this method were randomly selected, and
100%) were calculated (Table 4). In general, a CV within 15% is considered to mthree differ
 precision requirements of the test strip. It was proven that the CV of the test stri
pared in the experiment was less than 15%, and the reproducibility was good.
Molecules 2024, 29, 1776 9 of 17
Table 4. Precision evaluation results of the test strip.

Batch mean value(μg/mL)

SP Concentration X of T/C SD (CV = (SD/X) × 100%)CV (%
(X), standard deviation (SD), and coefficient of variation
were calculated (Table 4). In general, a CV within 15% is considered to meet the precision
1
requirements of the test strip. 0.2717 0.0398
It was proven that the CV of the test strips prepared in the 14.64
1 0.5
experiment was less than 15%,0.4413 0.0565
and the reproducibility was good. 12.81
0.25 0.5780 0.0851 14.73
Table 4. Precision evaluation results of the test strip.
1 0.3519 0.0416 11.82
2 0.5Batch SP Concentration (µg/mL)
0.5032 X of T/C 0.0669SD CV (%)
13.30
1 0.2717 0.0398 14.64
0.25 0.6602 0.0873 13.23
1 0.5 0.4413 0.0565 12.81
1 0.3122
0.25 0.5780 0.0396
0.0851 14.73 12.70
3 0.5 0.4710
1 0.3519 0.0696
0.0416 11.82 14.78
0.5 0.5032 0.0669 13.30
0.25 2 0.6535
0.25 0.6602
0.0967
0.0873 13.23
14.80
1 0.3119
1 0.3122
0.0403
0.0396 12.70
13.05
Intra-batch 0.5 3 0.4718
0.5 0.4710 0.0643
0.0696 14.78 13.63
0.25 0.6535 0.0967 14.80
0.25 0.6305 0.0897 14.25
1 0.3119 0.0403 13.05
Intra-batch 0.5 0.4718 0.0643 13.63
2.8. Stability Assessment 0.25 0.6305 0.0897 14.25

To evaluate the stability of the prepared test strips, the same batch of freshly pr
2.8. Stability Assessment
test strips was stored at room temperature (25 °C) and taken out on the 1st, 2nd,
To evaluate the stability of the prepared test strips, the same batch of freshly prepared
and 16th was
test strips days to detect
stored at roomdifferent
temperature concentrations
(25 ◦ C) and takenofoutS proteins
on the 1st,(1000 ng/mL,
2nd, 4th, 8th 500
and 250 ng/mL). The results showed that the T/C values of the test
and 16th days to detect different concentrations of S proteins (1000 ng/mL, 500 ng/mL, strips increased
and 250 ng/mL).
(within 15%) on The
theresults showed
second daythat
andthethen
T/C values of the test
stabilized, andstrips
the increased
test strips slightly
were still e
(within 15%) on the second day and then stabilized, and the test strips
in detecting the three concentrations of S proteins (Figure 9), which indicates were still effective in that t
detecting the three concentrations of S proteins (Figure 9), which indicates that the prepared
pared testhave
test strips strips havedegree
a certain a certain degree
of stability of stability
at room at room temperature.
temperature.

Figure 9. Stability evaluation result.

Figure 9. Stability evaluation result.
2.9. Recovery Assay
2.9. Recovery
The aboveAssay
test strip was applied to the testing of frozen solutions on the surface of
cold-chain food packaging
The above withwas
test strip the addition
appliedofto
S-protein standard
the testing of solution
frozen (0, 200, 400, 600,
solutions on the su
800, 900, 1000 ng/mL), and the recoveries obtained ranged from 83.3% to 108.8% (Table 5)
cold-chain
with relativefood packaging
standard with
deviations theofaddition
(RSDs) of S-protein
2.3% to 6.2%. standard
The results solution
showed that the (0, 2
600,
lateral chromatographic test strips had high accuracy and reliability when applied to the to 108.
800, 900, 1000 ng/mL), and the recoveries obtained ranged from 83.3%
ble 5) with
detection of Srelative
protein instandard deviations (RSDs) of 2.3% to 6.2%. The results show
real samples.
the lateral chromatographic test strips had high accuracy and reliability when app
the detection of S protein in real samples.
Molecules 2024, 29, x FOR PEER REVIEW 10 of 17

Molecules 2024, 29, 1776 10 of 17

Table 5. Results of spiked recovery experiments of new crown S protein in cold-chain food pouch
samples (n = 3).
Table 5. Results of spiked recovery experiments of new crown S protein in cold-chain food pouch
Concentration of(n = 3).
samples Test Strip Concen-
Sample Detection Result Recovery Rate (%) RSD (%)
SP (ng/mL) tration (ng/mL)
0 Negative TestUndetected
Strip
Concentration Undetected
Detection Undetected
Recovery
Sample Concentration RSD (%)
200 of SP (ng/mL)
Positive Result
166.7 83.3 Rate (%) 2.6
Cold-chain food (ng/mL)
400 Positive 435.2 108.8 2.3
packaging bags— 0 Negative Undetected Undetected Undetected
600 Cold-chain Positive 200 571.7
Positive 166.795.2 83.3 5.8 2.6
tap water rinsing
800 food packagingPositive 400 752.4
Positive 435.294.1 108.8 6.2 2.3
(1 mL)
900 bags—tap Positive 600 Positive
860.2 571.795.6 95.2 4.6 5.8
1000 water rinsing Positive 800 Positive
968.1 752.496.8 94.1 3.0 6.2
(1 mL) 900 Positive 860.2 95.6 4.6
1000 Positive 968.1 96.8 3.0
3. Discussion
In this experiment, the principle of competition method was designed as shown in
3. Discussion
Figure 10, which is essentially a competitive interaction between the aptamer-complemen-
In this experiment, the principle of competition method was designed as shown in
tary chain on the T line and the SP for AuNP–aptamer conjugates. The paper strip was
Figure 10, which is essentially a competitive interaction between the aptamer-complementary
formed by using AuNPs as a color developer, an aptamer coupled with AuNPs as the
chain on the T line and the SP for AuNP–aptamer conjugates. The paper strip was formed
recognition element, and the aptamer-complementary chain as the capture probe. One
by using AuNPs as a color developer, an aptamer coupled with AuNPs as the recognition
end of the aptamer that binds to the S protein modifies sulfhydryl groups and couples
element, and the aptamer-complementary chain as the capture probe. One end of the ap-
AuNPs, and the other end adds 10 T bases (ploy T) to form a AuNP–aptamer-poly T com-
tamer that binds to the S protein modifies sulfhydryl groups and couples AuNPs, and the
plex.
otherTheendsample
adds 10solution
T bases flows
(ploy T)along the NC
to form membrane by capillary
a AuNP–aptamer-poly action.The
T complex. In the ab-
sample
sence of SP in the sample solution, the aptamers in complexes will pair
solution flows along the NC membrane by capillary action. In the absence of SP in the with the comple-
mentary pairing of
sample solution, thethe T line, in
aptamers while the DNA
complexes C (poly A) immobilized in the C line binds
will pair with the complementary pairing of the
complementarily to poly T, causing two red
T line, while the DNAC (poly A) immobilized in the bands toCappear; when
line binds SP exists in the to
complementarily sample,
poly T,
the AuNP–aptamers in the sample binds to the SP, thus weakening
causing two red bands to appear; when SP exists in the sample, the AuNP–aptamers the binding force of
in the
the complementary sequences on the T line, and the color of the test
sample binds to the SP, thus weakening the binding force of the complementary sequences line will become
lighter.
on the As the concentration
T line, and the color of of the
the test
SP isline
larger,
will the colorlighter.
become of the TAs
line
thebecomes lighter of
concentration until
the
itSP
disappears.
is larger, the color of the T line becomes lighter until it disappears.

Figure 10. Schematic representation of SP detection via Apt-LFA. (a) Structure of LFA strip. (b) Nega-
Figure 10. Schematic representation of SP detection via Apt-LFA. (a) Structure of LFA strip. (b) Neg-
tive result
ative result of
of Apt-LFA
Apt-LFA (without
(without SP).
SP). (c)
(c) Positive
Positive result
result of
of Apt-LFA
Apt-LFA (with
(with SP).
SP).
The effect of AuNP–aptamers is critical to the sensitivity of the assay. Due to electro-
The effect of AuNP–aptamers is critical to the sensitivity of the assay. Due to electro-
static repulsion, colloidal gold produced by the reduction method of trisodium citrate is
static repulsion, colloidal gold produced by the reduction method of trisodium citrate is
negatively charged by the encapsulation of trinitrate citrate anions and is distributed in an
negatively charged by the encapsulation of trinitrate citrate anions and is distributed in
aqueous solution [42]. It is well known that, owing to the negatively charged colloidal, gold
an aqueous solution [42]. It is well known that, owing to the negatively charged colloidal,
particles can be sheltered by Na+ , which causes the colloidal gold particles to combine due
gold
to hydrophobic interactions and van, which
particles can be sheltered by Na + causes
der Waals the colloidal
forces. gold should
The aptamer particlesbetocovalently
combine
due
joined to the AuNPs through Au-S to create functionalized AuNPs. In terms of be
to hydrophobic interactions and van der Waals forces. The aptamer should thecova-
three
lently
coupling methods (salt aging [35], freezing [43], and low pH), the solution coupled of
joined to the AuNPs through Au-S to create functionalized AuNPs. In terms bythe
the
three coupling
traditional methods
salt-aging (salt aging
method [35], freezing
is relatively [43], and low
time-consuming, butpH), the solution
its coupled coupled
solution is the
by the stable
most traditional salt-aging
and can method
be left for at leastisone
relatively
month.time-consuming, but its coupled
In the coupling process, too littlesolution
aptamer
addition is not enough to protect the nanogold. Too much aptamer addition will result in
Molecules 2024, 29, 1776 11 of 17

too much free aptamer in the coupling solution, reducing the binding of the aptamer of
the coupled AuNPs to the S protein, so that, even after the addition of the protein, there is
still a free aptamer binding to the complementary chain. Therefore, the optimal amount of
aptamer to be added was determined by the state of the coupling solution and the color
development of the test strip.
In addition, another decisive factor is the choice of T-line aptamer-complementary
chains. The aptamers screened from nucleic acid libraries generally have a length of
~80 bases, but the region that exerts target binding is only 10–15 nucleotides [44]. If the
selected complementary chain is not its active site for protein binding, the complementary
chain will still be complementary to the aptamer, even if the SP binds to the aptamer
after adding the SP, resulting in a decrease in sensitivity, which is not conducive to the
experiment. Therefore, MOE molecular docking was utilized to simulate effective binding
sites and simplify the selection process of complementary sequences. On this basis, different
complementary chain lengths were set to obtain the best reaction result. Finally, we decided
to retain the four truncated aptamers, MSA52-10, MSA52-15, MSA52-20, and MSA52-22.
Then, the blank and spiking experiments were carried out, respectively, in which the best
color development and the highest inhibition rate were found to be for MSA52-10. This
may be due to the fact that the base that is not at the effective site will still bind to the
complementary chain, even if the aptamer and SP successfully bind. In this way, we can
save costs and improve the sensitivity of the experiment.
Then, the conditions of the test strip were optimized. The coupling status of SA
with biotin-modified probes not only impacts the amount of probe immobilization on
NC membranes but also influences the binding efficiency of probes to nucleic acids [45].
The higher the SA concentration, the more aptamers are bound. However, too much SA
will cover the aptamers, resulting in insufficient aptamers. Nitrocellulose membrane is
the largest pad in the test strip, which is decisive for the sensitivity of the test strip [46].
Different NC membrane types have different pore sizes and chromatographic properties
with different flow rates. In addition, the running buffer is vital for the spatial structure
of the aptamer and directly contributes to the binding of the aptamer to the target [47].
Excessive addition of AuNP–aptamers will interfere with the experimental detection limit,
and the addition of too little sample is not enough to develop the color. Finally, an effective
detection time frame is the most important. We select the best reaction conditions by color
rendering and absorbance.
In this study, a competitive transverse flow test strip with AuNPs as the marker
was successfully established, which uses high-affinity aptamer-immobilized AuNPs for
the detection of SARS-CoV-2 S proteins in cold-chain foods. y = −0.000408x + 0.70
(R2 = 0.97) was the linear equation, and the detection sensitivity was calculated to be
LOD = 91.2 ng/mL (≈0.68 nM). Meanwhile, we examined the detection specificity of the
aptamer test strip method and verified the analytical accuracy, stability, and practical
application performance of this lateral chromatography test strip.
In this paper, we constructed an innovative assay kit to target multiple variants of
SARS-CoV-2 SP using aptamer technology. To date, several studies have successfully
detected SARS-CoV-2 SPs (Table 6). Compared with the LFA detection methods in other
studies, this study obtained high detection sensitivity through molecular docking, the
selection of complementary sequences of nucleic acid aptamers with high affinity, and
the optimization of experimental conditions. And compared to other methods, although
the detection limit of LFA detection methods is usually lower, the test strips have certain
advantages in rapid detection and other aspects.
In the field of novel coronavirus detection, although PCR has long been considered the
gold standard, the expensive specialized equipment required and the specialized operators
limit its convenient application in many scenarios. In contrast, the LFA method can be used
not only outside the laboratory, but even by patients themselves at home, and its long shelf
life and lack of need for special storage conditions make it particularly suitable for use
in developing countries, small outpatient care centers, and remote areas and battlefields.
Molecules 2024, 29, 1776 12 of 17

The test strips provide results in less than 20 min and cost less than USD 1 per test to
manufacture in our hands. By enabling more frequent home testing, more readily available
diagnostic tools (e.g., test strips) may reduce the burden of virus epidemics on the health
care system.

Table 6. Detection of SARS-CoV-2 S protein by biosensors.

Method Target Detection Range LOD Reference

E-AB S Protein (S1) 0.001–1000 fg/mL 1 ag/mL [48]
SPR S Protein (S1) 1–100 nM 0.26 nM [49]
Fluorescence (FL) S Protein 10 fg/mL–10 ng/mL 7.8 fg/mL [50]
PEC S Protein 75 fg/mL–150 pg/mL 1.22 fg/mL [51]
S Protein (RBD) 62.5–4000 ng/mL 62.5 ng/mL
LFA [52]
S Protein (S1) 250–4000 ng/mL 250 ng/mL
LSPR S Protein (RBD) 2.03–9420 pM 0.83 pM [53]

Our research presents a test strip constructed using the spike protein as the detec-
tion target, which has certain potential advantages and application possibilities for virus
detection in real clinical samples. The spike protein is the main surface protein of the
SARS-CoV-2 virus. Compared to PCR detection and test strip methods targeting the nucle-
ocapsid protein, the method developed in this research does not require virus lysis, thereby
saving time and operational steps, and reducing the complexity of sample processing. In
addition, the use of complementary strands as the test line not only reduces the cost but also
ensures the reproducibility of the assay. Therefore, test strips based on the spike protein
offer advantages such as rapidity, convenience, and non-invasiveness, facilitating rapid
screening and diagnosis.
In conclusion, this study successfully applied the aptamer-based competitive method
to SP detection, which not only improves the sensitivity and efficiency of new coronavirus
detection, but also reduces the cost, provides an auxiliary that complements PCR detec-
tion, provides a new method and idea for virus detection, shows a convenient and fast
application prospect in cold-chain food detection, and provides a new method for the de-
velopment of alternative antibody detection reagents. After that, we can also try to develop
multi-mode aptamer detection or multi-target detection, so as to develop a multi-functional
test strip method for virus detection.

4. Materials and Methods

4.1. Reagents and Materials
The recombinant baculovirus-produced SARS-CoV-2 S protein (40589-V08B1) was ob-
tained from Beijing Yiqiao Shenzhou Technology Co., Ltd. (Beijing, China). The sequence of
aptamer binding to the SARS-CoV-2 S protein is 5′ -SH-TTTTTTACGCCAAGGTGTCACTC
CGTAGGGTTGGCTCCGGGCCTGGCGTCGGTCGCGAAGCATCTCCTTGGCGTTTTTTT
TTT-3′ , and the probe sequence of line C is biotin-AAAAAAAAAA. The other aptamer
sequences are shown in Table 2. Aptamers and DNA probes were commercially synthesized
and purified by Sangon Biotech Co., Ltd. (Shanghai, China). Streptavidin (SA) and bovine
serum albumin (BSA) were ordered from Sigma-Aldrich (Saint Louis, MO, USA). Phosphate-
buffered saline (PBS, PH 7.4) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
(HEPES) were purchased from Beijing Solarbio Company (Beijing, China). HAuCl4 ·4H2 O,
Proclin300, Tris(2-carboxyethyl)phosphine (TCEP), Tween-20, and sucrose were purchased
from China Pharmaceutical Group Co., Ltd. (Beijing, China). Trisodium citrate and other
reagents were purchased from Beijing Chemical Works. All inorganic chemicals and or-
ganic solvents were of at least analytical grade and buffer solutions were prepared with
ultrapure water.
Plastic adhesive backing (6 × 30 cm), sample pads (8975), and absorbent pads (S270)
were purchased from Hang Zhou Bulus Trading Co., Ltd. (Hangzhou, China), and the
nitrocellulose (NC) membrane was obtained from Shenzhen Baisui Kang Industrial Co.,
Molecules 2024, 29, 1776 13 of 17

Ltd. (Shenzhen, China). The NC membrane types and parameters are shown in Table 7.
All plates were dispensed by an IsoFlow Dispenser (Imagine Technology, Wilmington,
DE, USA) and then cut into strips for experiments using a programmable strip cutter
(HGS201, AUTOKUN, Hangzhou, China). The signal values of the test strips were read
by an immunochromatographic reader, the C10066-10 (Hamamatsu Corporation, Hama-
matsu, Japan).

Table 7. The NC membrane types and parameters.

Name Speed (s/4 cm) Diameter (µm)

BSK95 100 ± 20 12–15
BSK110 120 ± 30 8–12
BSK140 140 ± 30 5–8
BSK160 160 ± 30 4–6

4.2. Preparation of Gold Nanoparticles

According to previous reports [54], AuNPs were prepared using a modified citric acid
reduction method. Initially, 1 mL of a 1% HAuCl4 aqueous solution was added to 100 mL
of ultrapure water in a 250 mL conical flask wrapped in tinfoil, stirred (300 rpm), and
heated to boiling. Then, 2 mL of a 1% sodium citrate solution was quickly added while the
rotational speed was adjusted to 350 rpm and maintained for 1 min. The solution continued
to be heated for 14 min with stirring (300 rpm) until the color changed to burgundy, after
which it was cooled to room temperature. The nanoparticles were stored at 4 ◦ C away from
light and were used at least 24 h after synthesis was completed.

4.3. Conjugation of Aptamer and Nanogolds

The conventional method for aptamer attachment on nanogold particles was typically
performed by using a salt-aging process [35]: 50 µL of 5′ -sulfide aptamer (3 µM) was
activated by 1 µL of TCEP (10 mM) for 1 h at room temperature. During this time, 1 mL of
the nanogold solution prepared above was concentrated 10-fold and the pH was reduced
to 3.0. After mixing, the mixture was incubated for 2 h, and then 2 M NaCl was added to a
final concentration of 75 mM over a 6 h period. Finally, it was stored at 4 ◦ C for over 6 h
until use.

4.4. Selection of T-Line Complementary Sequences

T-line complementary sequences were selected as per the original literature [29] and
MOE molecular docking results. Secondary and tertiary structures of the aptamer were
predicted by the DNA Fold Web server [55] and the 3dRNA/DNA Web server [56], and
the structure of the S protein was obtained from PubChem [57]. The MOE was utilized to
dock the aptamer to the SP to obtain the optimal binding site. All complementary chain
sequences are shown in Table 2.

4.5. Pre-Treatment of Test Strips

The sample pad, NC membrane, absorbent pad, and PVC plastic adhesive backing are
the four material elements that compose the aptamer-based lateral flow test strip [19]. To
load the probe in the detection region, streptavidin (SA) was used as the connector between
the probe and the membrane because of the adsorption of proteins by the nitrocellulose
membrane. Utilizing biotin- and SA-specific binding properties, the probe, one end of
which was modified with biotin and complementary to the AuNP–aptamer coupler, was
immobilized onto the NC membrane. Fixed SA with a 1:4 molar ratio of aptamer, a T-
line SA concentration of 0.4 mg/mL, and a C-line SA concentration of 0.4 mg/mL were
incubated with biotin-DNAT and biotin-DNAC , respectively, for 2 h to form SA-biotin-DNA
complexes. The tubes were then washed three times for 30 min each with ultrafiltration
centrifuge tubes (30 kD) to remove excess probe. The remaining solution was added to
HEPES, and the final volume was the same as the original solution volume.
Molecules 2024, 29, 1776 14 of 17

The sample pads need to be immersed in HEPES containing 1% BSA, 0.25% Tween-20,
and 1% sucrose, dried at 37 ◦ C, and stored in a cool, ventilated area to minimize non-specific
adsorption between SA and AuNPs.

4.6. Assembly of Test Strips

The NC membrane, sample pad, and absorbent pad were sequentially laminated at
2 mm and pasted onto a plastic backing plate. Then, the T-line and C-line solutions were
sprayed onto the NC membrane using an IsoFlow Dispenser and dried at 37 ◦ C for 30 min.
The T-line SA concentration was 0.4 mg/mL and the ratio of SA to biotinized aptamer
(DNAT ) was 1:4. The concentration of SA in line C was 0.2 mg/mL and the ratio of SA to
biotinylated aptamer (DNAC ) was 1:4. The test strip plate was cut into 4 cm strips using
the HGS201 programmable strip cutter and stored in a self-sealing bag with desiccant.

4.7. Sample Test and Evaluation Methods

In this work, we used commercially available frozen solutions from frozen food
packages as samples to verify the accuracy and reliability of aptamer-based lateral flow
test strips. The ice on the surface of frozen food packages was melted at room temperature
and mixed with different concentrations of SP to prepare sample solutions with different
SP concentrations. Then, 2 µL of successfully coupled AuNP–aptamer, 28 µL of binding
buffer, and 20 µL of sample solution were taken and incubated for half an hour and then
added dropwise into the sample pad of the test strip. Ten minutes later, the absorbance
of the T and C lines by immunochromatography was scanned and recorded with an
immunochromatographic reader, the C10066-10.
The inhibition rate is introduced to evaluate the optimization results of various prop-
erties throughout this study and is calculated as follows [58]:
T0 T
C0 − C
Inhibition = T0
C0

T0 and C0 represent the absorbance of the T and C lines when there is no SP in the
sample, while T and C represent the absorbance of the T and C lines when the sample has
different S proteins. The absorbance ratio of T and C lines versus concentration was used
for quantitative analysis and a standard calibration curve was plotted.

Author Contributions: Conceptualization, Z.L. and Y.D.; methodology, X.L. and J.W.; investigation
X.L., J.W., G.Y. and X.F.; resources, Z.L., Y.D., G.Y. and X.F.; validation, X.L., J.W., X.F., G.Y. and
L.Z.; writing—original draft preparation, X.L.; writing—review and editing, X.L., Y.D. and L.Z.;
supervision, J.W., L.Z. and Z.L. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was supported by the Beijing University of Chemical Technology—China–
Japan Friendship Hospital Biomedical Transformation Engineering Research Center Joint Project
(grant no. RZ2020-02) and the National Key Research and Development Program of China (grant no.
2016YFF0203703).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: All data can be easily obtained and linked in the respective sections.
Conflicts of Interest: The authors declare no conflicts 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.
Molecules 2024, 29, 1776 15 of 17

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