biosensors

Article
Development of a Rapid Gold Nanoparticle-Based Lateral Flow
Immunoassay for the Detection of Dengue Virus
Cynthia Martinez-Liu 1 , Carlos Machain-Williams 2 , Natalia Martinez-Acuña 1 , Sonia Lozano-Sepulveda 1 ,
Kame Galan-Huerta 1 , Daniel Arellanos-Soto 1 , Mayra Meléndez-Villanueva 1 , Diana Ávalos-Nolazco 1 ,
Katya Pérez-Ibarra 1 , Sergio Galindo-Rodríguez 3 , Aurora de Jesús Garza-Juarez 1
and Ana María Rivas-Estilla 1, *

1 Department of Biochemistry and Molecular Medicine, Hospital Universitario “Dr. Jose E. Gonzalez”,
Autonomous University of Nuevo Leon, Monterrey 64460, Nuevo León, Mexico; [email protected] (C.M.-L.);
[email protected] (N.M.-A.); [email protected] (S.L.-S.);
[email protected] (K.G.-H.); [email protected] (D.A.-S.);
[email protected] (M.M.-V.); [email protected] (D.Á.-N.);
[email protected] (K.P.-I.); [email protected] (A.d.J.G.-J.)
2 Laboratory of Arbovirology, Centro de Investigaciones Regionales “Dr. Hideyo Noguchi”,
Universidad Autónoma de Yucatan, Merida 97000, Yucatan, Mexico; [email protected]
3 Department of Chemistry, Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León,
Monterrey 66450, Nuevo León, Mexico; [email protected]
* Correspondence: [email protected]; Tel.: +52-81-8333-7747

Abstract: Flavivirus detection in humans and mosquito reservoirs has been an important issue
Citation: Martinez-Liu, C.; since it can cause a variety of illnesses and could represent a health problem in geographical zones
Machain-Williams, C.;
where the vector is endemic. In this work, we designed and characterized a biosensor based on
Martinez-Acuña, N.;
gold nanoparticles (AuNPs) and antibody 4G2 for the detection of dengue virus (DENV) in vitro,
Lozano-Sepulveda, S.;
obtaining different conjugates (with different antibody concentrations). The AuNP–4G2 conjugates at
Galan-Huerta, K.; Arellanos-Soto, D.;
concentrations of 1, 3, and 6 µg/mL presented an increase in the average hydrodynamic diameter
Meléndez-Villanueva, M.;
Ávalos-Nolazco, D.; Pérez-Ibarra, K.;
compared to the naked AuNPs. Also, as part of the characterization, differences in the UV-Vis
Galindo-Rodríguez, S.; et al. absorbance spectrum and electrophoretic migration were observed between the conjugated AuNPs
Development of a Rapid Gold (with BSA or antibody) and naked AuNPs. Additionally, we used this biosensor (AuNP–4G2
Nanoparticle-Based Lateral Flow conjugate with 3 µg/mL antibody) in the assembly of a competitive lateral flow assay (LFA) for the
Immunoassay for the Detection of development of an alternative test to detect the flavivirus envelope protein in isolated DENV samples
Dengue Virus. Biosensors 2022, 12, as a future tool for dengue detection (and other flaviviruses) in the mosquito vector (Aedes aegypti) for
495. https://doi.org/10.3390/ the identification of epidemic risk regions. Functionality tests were performed using Dengue virus
bios12070495 2 isolated solution (TCID50 /mL = 4.58 × 103 ) as a positive sample and PBS buffer as a negative control.
Received: 29 April 2022 The results showed that it is possible to detect Dengue virus in vitro with this gold nanoparticle-based
Accepted: 4 July 2022 lateral flow assay with an estimated detection limit of 5.12 × 102 PFU. We suggest that this biosensor
Published: 7 July 2022 could be used as an additional detection tool by coupling it to different point-of-care tests (POCT) for
Publisher’s Note: MDPI stays neutral
the easy detection of other flaviviruses.
with regard to jurisdictional claims in
published maps and institutional affil- Keywords: dengue virus; gold nanoparticle; lateral flow assay; biosensor; immunochromatographic
iations. strip; point-of-care test (POCT); rapid test

Copyright: © 2022 by the authors. 1. Introduction

Licensee MDPI, Basel, Switzerland.
Dengue is a mosquito-transmitted virus and the leading cause of arthropod-borne
This article is an open access article
viral disease worldwide. The dengue virus (DENV) causes a febrile disease with severe
distributed under the terms and
muscle spasms and joint pain. Although most cases are asymptomatic, severe cases and
conditions of the Creative Commons
death can occur [1].
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
In the Americas, dengue incidence has increased from 1.5 million cumulative cases
4.0/).
in the 1980s to 16.2 million in the decade between 2010 and 2019. The year 2013 was

Biosensors 2022, 12, 495. https://doi.org/10.3390/bios12070495 https://www.mdpi.com/journal/biosensors

Biosensors 2022, 12, 495 2 of 19

an epidemic year with 2 million cases recorded; 37,692 were severe and 1280 deaths
occurred. In 2019, more than 3.1 million cases were registered, with 28,000 severe cases and
1534 deaths [2].
Aedes aegypti is the mosquito vector responsible for dengue propagation and is widely
distributed in the Americas [3]. Therefore, approximately 500 million people are at risk of
dengue infection [2], powered by the recent propagation of Zika in 2013 [4] and Chikun-
gunya in 2015 [5]. This mosquito is also responsible for the transmission of other viruses
such as Yellow Fever virus (YFV) and West Nile virus (WNV) and is considered the most
important vector of human pathogen transmission [6].
Without any efficacious and fully licensed vaccines or treatment available against
these diseases, vector control remains the immediate alternative to reduce or prevent
transmission. Current national protocols in Mexico for vector control and transmission
reduction include larvae control, entomological surveillance, and neighborhood spraying.
Entomological surveillance incorporates molecular methods [7], usually quantitative real-
time-PCR (RT-qPCR), for the identification, quantification, and genotyping of DENV and
other arboviruses in mosquito populations [8–12]. However, this process requires a large
logistic workflow from the collection of mosquitos around households to sample processing,
nucleic acid isolation, and virus detection in authorized centers, requiring specialized
reagents and equipment, well-trained personnel, and time.
Several tools have been designed as point-of-care tests (POCT) to improve current
strategies even when no appropriate equipment or laboratory installations exist. POCT are
performed outside of a laboratory, use mobile devices, and results are available in minutes.
POCT developed for the detection of mosquito-borne viruses include the identification
of proteins and viral RNA using gold nanoparticle biosensors [13,14], loop-mediated
isothermal amplification (LAMP) [15,16], and most recently a portable device based on
real-time PCR [17].
Among the different types of POCT, lateral flow assays (LFAs) are the simplest as
they are easy to use without the need for specialized equipment. LFAs are paper-based
chromatographic tests in which detection reagents are preloaded and activated when the
sample is added, obtaining results in approximately 15 min. LFAs use gold nanoparticles
(AuNPs) as labeling molecules that are functionalized with biological recognition elements
to form specific biosensors that capture the antigens of interest to carry out the detection.
There are different design formats of LFAs; the selected format depends on the reagents
that are available and the size and concentration of the analyte. Most LFAs are designed
in a sandwich format using two antibodies that bind to different sites on the antigen to
be detected (a capture antibody and an antibody immobilized in the solid phase of LFAs).
In the presence of the analyte, two signals will appear in the test and control zone. In the
competitive format, target analytes bind to reporters (recognition-labeling molecules) and
block these reporters, preventing their binding to the ligand immobilized in the solid phase
of LFAs. In the presence of the analyte in the sample, a single signal is shown (in the control
zone) indicating that the analyte has bound to the reporter and formed a complex that
binds to the secondary antibody immobilized in the solid phase of the LFA. This type of
format is used when the analyte is small, the amount of analyte in the sample is very low,
and/or the analyte has a single epitope for detection, which makes it impossible to use
two antibodies in LFAs [18].
Recently, authors have reported the development of LFAs for virus detection based on
the use of monoclonal antibodies and different labeling molecules such as gold nanoparti-
cles and coated beads. These LFAs have been used for the identification of DENV and other
flaviviruses in patient sera and isolated virus samples in order to improve the monitoring
of these viruses [19,20].
In this work, we design and characterize a biosensor based on AuNPs and a commer-
cial antibody commonly used in clinical diagnostics [21–23] (4G2 antibody) that specifically
recognizes the domain II of protein E of the flavivirus group (including dengue virus, West
Nile virus, Japanese encephalitis, Yellow Fever virus, Zika virus, etc.). In addition, we use
Biosensors 2022, 12, 495 3 of 19

this biosensor in the assembly of a competitive LFA for the development of an alternative
test to detect the flavivirus envelope protein in isolated DENV samples as a future tool for
dengue virus detection (and other flaviviruses) in the mosquito vector in order to identify
epidemic risk regions. Also, we discuss the challenges and achievements reached during
the design and testing of the biosensor and the competitive LFA.

2. Materials and Methods

2.1. Reagents
Citrate-coated spherical Gold Nanoparticles were purchased from Nanopartz
(cat. A11-60-CIT-DIH), sucrose from Fermont (cat. PQ07642), and Tris from Biorad (cat. 161-0719).
Bovine serum albumin (BSA), sodium azide (cat. 26628-22-8), sodium phosphate dibasic
heptahydrate (Na2 HPO4 ·7H2O) (cat. 7782-85-6), sodium dihydrogen phosphate monohy-
drate (NaH2 PO4 ·H2 O) (cat.10049-21-5), sodium tetraborate decahydrate (Na2 B4 O7 ·10H2 O)
(cat. 1303-96-4), triton X-100 (cat. 9002-93-1), sodium phosphate dibasic (Na2 HPO4 )
(cat. 10890), polyvinyl alcohol (PVA) (cat. 341584-500g), polyvinylpolypyrrolidone (PVP)
(cat. 25249-54-1), casein (cat. C6554), and tween 20 (cat. 9005-64-5) were purchased
from Sigma-Aldrich.
Antibody against the flavivirus group, antigen 4G2, was purchased from Novus
Biologicals (cat. NBP2-52709-0.2 mg) and goat anti-mouse IgG antibody was purchased
from Abcam (cat. Ab6708). Absorbent pad (cat. 07.623.30), backing card (cat. 07.615.40),
nitrocellulose membrane (NC) (cat. 07.626.10), sample pad (cat. 07.622.30), and conjugation
pad (cat. 07.614.30) were obtained from Claremont Bio.

2.2. Dengue Virus Propagation

DENV was isolated from viremic patient serum. The viremic patient was sampled
under routine care at Linares General Hospital; located in Linares County in Nuevo Leon,
Mexico. The patient sought care in August 2012. The virus obtained from the sample was
isolated and propagated in C6/36 cells, sequenced by the Sanger platform, and identified
with the following access number in Genbank: KM279427.1. Viruses were propagated
on C6/36 cells in Leibovitz L-15 media, supplemented with fetal bovine serum (5–10%),
incubated at room temperature (25–28 ◦ C) without a CO2 atmosphere in closed culture
bottles, and purified by a traditional gradient-free ultracentrifugation protocol in a sucrose
solution followed by selective virion precipitation as previously reported [24]. Viral isolates
were titrated using the protocol for the formation of lytic foci or plaques in Huh-7 cells,
stained with Naphtol blue-black dye and Median Tissue Culture Infectious Dose (TCID50 ),
and the plaque-forming unit per mL (PFU/mL) was calculated by viral titration [25];
Equations and calculations can be viewed in Supplementary Information S1. Aliquots of
viral stock were stored at −80 ◦ C until use.

2.3. Preparation of Biosensor (Antibody-Conjugated AuNPs)

For the formation of the biosensor, the AuNPs were conjugated with the antibody
4G2 (cat. NBP2-52709-0.2 mg), a commercial antibody that specifically recognizes domain
II of protein E of the flavivirus group (including dengue virus, West Nile virus, Japanese
encephalitis, Yellow Fever virus, Zika virus, etc.). For conjugation, the pH of the AuNPs was
adjusted to 6 by adding 0.1 M borate solution (pH 6), then, different final concentrations of
antibody 4G2 (0, 3, and 6 µg/mL) were added into the AuNP colloidal solution (1 mL) with
OD 1 (concentration of 0.05 mg/mL of AuNPs, according to the manufacturer). The mixture
was incubated at 4◦ C for 40 min using a rotator. Following incubation, a conjugation
solution (sucrose 2.1 M, BSA 0.1%, sodium azide 1%) was added and incubated at 4 ◦ C for
10 min in the rotator. Then, 20 µL of 10% BSA was added and the mix was incubated for
10 min at 4 ◦ C with shaking. The mixture of antibody-conjugated AuNPs was centrifuged at
2500× g for 15 min. The supernatant was discarded and the pellet was resuspended in
250 µL of storage solution (0.1 M sucrose, 0.1% BSA, and 0.01% sodium azide), concentrating
the amount of AuNPs 4 times. The AuNP solution was stored at 4 ◦ C until use.
Biosensors 2022, 12, 495 4 of 19

2.4. Optimization of the Conjugation of Nanoparticles and GAT Value Calculation

To optimize the conjugation of antibodies on the surface of the AuNPs, the pH of the
resuspended solution was changed before the addition of the antibody. To adjust the pH of
the nanoparticle solution, a 0.1 M borate solution was used at a different pH (5.0–10.0). The
stability of the conjugates at different pH values was measured by the gold aggregation test
(GAT), modifying the absorbances used in the equation reported by Chamorro et al. [26]
and adjusting the presented absorbance values for the 60 nm AuNPs. An amount of 100 µL
of nanoparticles was placed in a 96-well plate, then 20 µL of 0.1 M borate buffer of each pH
was added. An amount of 10 µL of the antibody (to a final concentration of 10 µg/mL) was
added to the nanoparticles and incubated for 30 min under shaking at 4 ◦ C. Each sample
was prepared in triplicate and the absorbance value was measured at 534 and 600 nm in
a spectrophotometer. Subsequently, a 20% NaCl solution was added to each well, incubated
for 10 min at room temperature, and the absorbance at 534 and 600 nm was measured. The
values were used to calculate the GAT value using the following equation, identifying the
least stable conjugate as the one with the highest GAT value:

UAbsChange = [UAbs534 − UAbs600]Before NaCl addition − [UAbs534 − UAbs600]After NaCl addition (1)

2.5. Characterization of AuNPs and the Biosensor AuNP–4G2

For the characterization of AuNPs and conjugates, an electrophoretic mobility test,
colloidal gold aggregation test (GAT), and ultraviolet–visible absorption spectroscopy (UV–
Vis) were performed and physicochemical parameters were determined (hydrodynamic
diameter, ζ-potential, and polydispersity index) by dynamic light scattering (DLS) [27].
For the electrophoretic mobility test, 100 µL of the nanoparticles or the conjugates
were centrifuged at 2500× g for 15 min at 4 ◦ C. The pellet was recovered and resuspended
in 20 µL of 0.1 M borate buffer pH 8 and mixed with the loading buffer (Tris HCl pH 8, 40%
glycerol, and bromophenol blue) and loaded in 1% agarose gel (0.5X TBE). The gel was run
in 0.5% TBE buffer for 1 h at 150 V and 200 mA.
To identify the resistance to aggregation of the nanoparticles and conjugates due
to antibody/albumin adsorption, the gold aggregation test (GAT) was performed as
mentioned above.
Ultraviolet−visible spectra of the AuNPs and AuNP conjugates were measured with
a spectrometer Microplate reader Cytation 3 (Biotek Instruments). In a 96-well plate, 100 µL
of the AuNPs or conjugates (24 h after conjugation) were placed into and read by the
equipment. The samples were read in triplicate in the spectrophotometer in a range of
450−700 nm wavelength every 2 nm and the averages were plotted.
Hydrodynamic size, ζ-Potential, and polydispersity index of AuNPs and conjugates
were determined using a DLS Zetasizer Nanoseries (Marvern instruments, Malvern, UK)
with a 4 mW laser (633 nm) at 25 ◦ C. To prepare the samples, 100 µL of the AuNPs and
conjugates (24 h after being prepared in storage solution) were placed in a cell (quartz to
measure size or disposable capillary cell for ζ-potential), 900 µL of deionized water was
added, and the samples were read by the equipment. Each measurement consisted of
20 runs and samples were analyzed in triplicate.

2.6. Preparation and Assembly of the Lateral Flow Assays

In this work, we developed an LFA in a competitive format in which the presence
of the analyte (the virus antigen) from the sample “competes” to bind to the biosensor
with the analyte present in the test zone of the detection window. The signal in the test
zone indicates the absence of the target analyte in the sample, whereas the signal in the
control zone indicates that the biosensor flowed correctly through the LFA. Therefore, the
design allows two signals to be observed when the test is “negative” for the presence of
viral antigen, whereas one signal (only in the control zone) is observed when the test is
“positive”, indicating the presence of the viral antigen in the sample. Due to the AuNPs
 Biosensors 2022, 12, 495 5 of 19

Biosensors 2022, 12, x FOR PEER REVIEW 6 of 21

used in the biosensor preparation, the signals are visible to the naked eye as reddish
spots (Figure 1).

Figure 1. Scheme of the competitive lateral flow assay designed in this study for the detection of
flaviviruses. (A) On the strip, the sample flows by capillarity to the different components, reacting
Figure
with the1. biosensor
Scheme ofonthethecompetitive lateral
conjugate pad flow assay
producing designed
a signal on thein this study
detection for the detection
membrane. (B) If the of
flaviviruses.
sample does(A) notOn the strip,
contain the sample
flaviviruses, the flows by capillarity
biosensor flows to the to the different
detection components,
membrane, reacting
binding to
with the biosensor
the analyte presenton
in the
theconjugate
test zone pad producing
(DENV) and thea secondary
signal on the detection
antibody membrane.
against (B) Ifinthe
IgG present
sample doeszone,
the control not contain flaviviruses,
generating the(negative
two signals biosensortest).
flows toIfthe
(C) thedetection membrane,
sample contains binding to
flaviviruses, thethe
analyte present in the test zone (DENV) and the secondary antibody against IgG present
biosensor–analyte complex is formed, which flows to the detection membrane, generating signals
in the con-
trol zone, generating two signals (negative test). (C) If the sample contains flaviviruses, the biosen-
only in the control zone (positive test). In both cases, the signal from the control zone must be
sor–analyte complex is formed, which flows to the detection membrane, generating signals only in
observed to confirm correct operation of the LFA.
the control zone (positive test). In both cases, the signal from the control zone must be observed to
confirmThecorrect operation
LFA consists ofoffour
the components
LFA. pretreated and mounted together on an adhesive
backing card: a sample pad, a conjugation pad, a nitrocellulose membrane (detection
2.8. Statisticaland
membrane), Analysis
an absorbent pad. The performance diagram and the components of the
LFA We compared
are shown the AuNP
in Figure control
1A. Each and conjugates
component usingseparately
was prepared one-wayasanalysis
followsof variance
and then
(ANOVA)
mounted on andanTukey’s
adhesivemultiple-comparison post-test. Differences between groups were
backing card before use.
The sample
significant at a ppad
value of 0.05. Statistical
was previously treated with a solution
analyses andofgraphics
0.1 M sodium
weretetraborate
made with
decahydrate,
GraphPad 0.05%
Prism 6.0 triton, and 0.5%
(GraphPad BSA adjusted
Software, Inc., San to pH 8CA,
Diego, for USA).
5 min in rotary shaking,
allowed to dry for 2 h at 37 ◦ C, and stored at 4 ◦ C until use.
3. Results
In this work, we designed a competitive POC detection system formed by a biosensor
based on AuNPs coupled to specific antibodies against protein E present in the Flaviviri-
dae family that detects the antigen forming a AuNP–antibody–antigen complex and the
Biosensors 2022, 12, 495 6 of 19

The fiberglass conjugation pad was treated with a solution of 0.05 M dibasic sodium
phosphate, 0.05% triton, 0.5% BSA, and 0.5% polyvinyl alcohol pH 7.4 for 5 min under
stirring and allowed to dry at 37 ◦ C for 2 h. Subsequently, the AuNP–4G2 (3 µg/mL)
conjugates were applied to the conjugation pad until saturated and allowed to dry for
30 min at 37 ◦ C.
For the detection membrane, a nitrocellulose membrane (Nitrocellulose FF 170) was
used. Solutions were prepared as follows: DENV antigen undiluted and goat anti-mouse
IgG secondary antibody were diluted separately with 1:1 in a 1% methanol–PBS solution.
DENV antigen solution was immobilized on the membrane as a dot in the test zone,
whereas the secondary antibody solution was placed as a dot in the control zone. Both
were allowed to dry at room temperature for 10 min and subsequently, the membrane
was blocked with a TBS solution with 0.015% casein, 0.3% polyvinylpyrrolidone, and
0.001% tween 20 pH 8 for 10 min under stirring at room temperature and allowed to dry at
room temperature.
The cellulose absorption pad had no treatment. Each of the dry components was cut
into 10 cm rectangles, assembled on the adhesive backing card, and placed in the Matrix
1201 membrane cutter from Kinematic Automation to form the strips. The LFAs were
stored at 20–22 ◦ C in sealed packages containing silica gel.

2.7. Performance Test of the LFA Using AuNP–4G2 Biosensor

To determine the in vitro functionality of the LFA, PBS buffer 10 mM NaCl was
used as a negative control and the DENV-isolated solution at a TCID50 /mL = 4.58 × 103
(3.2 × 103 PFU/mL) as a positive sample. Before adding the sample, the LFAs were allowed
to dry for 10 min at room temperature and then 160 µL (5.12 × 102 PFU) of the DENV-
positive sample was placed on the sample pad and allowed to migrate through the strip by
capillarity. After 10 min, the results were observed in the detection window. According to
the design of the LFA, two reddish spots (one in the test zone and the other in the control
zone) were interpreted as negative (see Figure 1B) and the LFAs with only one reddish dot
(in the control zone) were interpreted as positive (see Figure 1C).

2.8. Statistical Analysis

We compared the AuNP control and conjugates using one-way analysis of variance
(ANOVA) and Tukey’s multiple-comparison post-test. Differences between groups were
significant at a p value of <0.05. Statistical analyses and graphics were made with GraphPad
Prism 6.0 (GraphPad Software, Inc., San Diego, CA, USA).

3. Results
In this work, we designed a competitive POC detection system formed by a biosensor
based on AuNPs coupled to specific antibodies against protein E present in the Flaviviri-
dae family that detects the antigen forming a AuNP–antibody–antigen complex and the
assembly of different components that allow the flow and visible signal detection in the
LFA in 10 min. With no antigen in the analyzed sample, the biosensor is free to bind to
the antigen present on the detection membrane, specifically in the test zone. In addition,
as a test control, a secondary antibody (goat anti-mouse IgG antibody) that binds to the
primary antibody (antibody against flavivirus group antigen 4G2) used in the biosensor
was added to the control zone of the detection membrane to ensure that the AuNPs are
coated with the detection antibody and have also migrated correctly through the LFA.
This work is divided into three main parts: first the preparation and characterization
of a biosensor that recognizes the antigen (DENV), second, the design and assembly of the
LFA, and third, the assessment of the functionality and the optimization of the LFA.
 was added to the control zone of the detection membrane to ensure that the AuNPs are
coated with the detection antibody and have also migrated correctly through the LFA.
This work is divided into three main parts: first the preparation and characterization
of a biosensor that recognizes the antigen (DENV), second, the design and assembly of the
LFA, and third, the assessment of the functionality and the optimization of the LFA.
Biosensors 2022, 12, 495 7 of 19
3.1. Preparation and Characterization of the Biosensor
To prepare our biosensor, citrate-coated AuNPs of 60 nm were conjugated by adsorp-
3.1. with
tion Preparation and Characterization
a 4G2 antibody. of the Biosensor
First, we determined the optimal conditions for the conjugation
process. For this purpose, the pH of the nanoparticle
To prepare our biosensor, citrate-coated AuNPs of solution
60 nm was
wereadjusted
conjugated from by5adsorp-
to 10.
After that, aAuNPs
tion with were conjugated
4G2 antibody. First, we with a final antibody
determined the optimal concentration
conditions of
for10the
µ g/mL (max-
conjugation
imum
process. concentration of antibody).
For this purpose, the pH of The theoptimal pH forsolution
nanoparticle conjugation was determined
was adjusted from 5 toby 10.
measuring the aggregation
After that, AuNPs capability
were conjugated with ofathe
finalconjugated AuNPs. EachofAuNP–antibody
antibody concentration 10 µg/mL (max-
conjugate was incubated
imum concentration with a 20%The
of antibody). NaCl solution
optimal pH where the saline environment
for conjugation was determined gener-by
measuring
ates negativethe aggregation
charges capability
in the medium andofincreases
the conjugated AuNPs. between
the interactions Each AuNP–antibody
AuNPs with
conjugatecharges
negative was incubated
availablewith a 20%surface
on their NaCl solution where thecovered
(not completely saline environment
by the antibody), generates
re-
negative
sulting in charges in the medium
the aggregation and increases
of the AuNPs and the theformation
interactions between AuNPs
of agglomerates. with
If the nega-
conju-
tive charges
gation available
conditions on their
did not allowsurface (not completely
the antibodies to bindcovered by the antibody),
to the surface of the AuNPs resulting
and
in theitaggregation
cover completely, of the AuNPs
aggregates willand the formation
be generated in theofpresence
agglomerates. If the
of sodium conjugation
chloride. The
conditionsofdid
formation notaggregates
these allow the antibodies
was measured to bind to the
in each surfacetested
condition of the during
AuNPsthe and cover it
conjuga-
completely,
tion process by aggregates
the gold will be generated
aggregation in the presence
test (GAT). of sodium
In this test, chloride.the
they measured The formation
absorbance
of these aggregates was measured in each condition tested
values at 534 and 600 nm before and after the addition of NaCl to the AuNP solutionduring the conjugation process
(at
different pH values) after conjugation with the antibody. The values were used to calcu-at
by the gold aggregation test (GAT). In this test, they measured the absorbance values
534the
late andnumber
600 nm of before
GATs and afterthe
from theformula
addition of NaCl toby
established theChamorro
AuNP solution (at different
et al. [26]; the lower pH
values)
the valueafter conjugation
of the GAT number, with the
the antibody.
better theThe values
coating of were used to of
the surfaces calculate the number
the nanoparticles
of GATs
with from the formula
the antibodies, established
indicating by Chamorro
the best condition et al. [26]; the lower the value of the
for conjugation.
GATFor number,
the 60 nm citrate-coated AuNPs used, pH 6 wasnanoparticles
the better the coating of the surfaces of the determined as with
thethe
best antibodies,
for con-
indicating the best condition for conjugation.
jugation with the 4G2 antibody, showing a minimum aggregation value of 0.0715 com-
paredFor theother
to the 60 nmvalues
citrate-coated
obtainedAuNPs used,which
at pH 5–10, pH 6 was were determined as the
between 0.077 andbest for conju-
0.213 (Fig-
gation
ure 2). with the 4G2 antibody, showing a minimum aggregation value of 0.0715 compared
to the other values obtained at pH 5–10, which were between 0.077 and 0.213 (Figure 2).

OptimizationofofpH
Figure2.2.Optimization
Figure pHconditions
conditionsfor
forthe
theconjugation
conjugationofofantibodies
antibodieson
onthe
thesurface
surfaceofofAuNPs.
AuNPs.
For the 60 nm citrate-coated AuNPs used, the optimal pH of the conjugation was determined
For the 60 nm citrate-coated AuNPs used, the optimal pH of the conjugation was determined using using
the gold aggregation test (GAT) to establish at which pH the minimum value was obtained (less
aggregation, greater stability), using a maximum concentration of 4G2 antibody (10 µg/mL). The
results indicated a minimum aggregation value of 0.0715 at pH 6 compared to the other values
reported using a pH from 5 to 10 for AuNP–4G2, whereas the naked AuNPs presented aggregation
in the presence of salts at any pH tested.

Once the best conditions for conjugation have been determined, the concentration
of antibody used for conjugation was optimized using 1, 3, and 6 µg/mL to coat 1 mL of
citrate-coated gold nanoparticles (OD 1). As a conjugation control, BSA was used to coat
the AuNPs (AuNP–BSA). The conjugates obtained (AuNP–4G2), the control (AuNP–BSA),
and the unconjugated AuNPs (AuNP CIT) were characterized to identify the minimum
concentration of antibody for coating the nanoparticle surface that generates the stability of
the AuNPs in a colloidal state. Various techniques were used to characterize the conjugates
 the AuNPs (AuNP–BSA). The conjugates obtained (AuNP–4G2), the control (AuNP–
BSA), and the unconjugated AuNPs (AuNP CIT) were characterized to identify the mini-
mum concentration of antibody for coating the nanoparticle surface that generates the
stability of the AuNPs in a colloidal state. Various techniques were used to characterize
Biosensors 2022, 12, 495
the conjugates and evaluate their properties. First, the absorbance spectrum 8band of 19
of the
surface plasmon of the AuNPs and conjugates was determined by a spectral scan per-
formed in the UV-visible range of 450 to 700 nm by spectrometry (see Figure 3). The UV-
Visand
spectrum
evaluateof theproperties.
their AuNP CITs First,(as
thepurchased) shows aband
absorbance spectrum spectral band with
of the surface a maximum
plasmon of
absorbance
the AuNPspeak at 534 nm,
and conjugates waswhereas the by
determined AuNP–4G2 (theperformed
a spectral scan three conjugates) and AuNP–
in the UV-visible
BSA presented
range of 450 toa 700
maximum absorbance(see
nm by spectrometry peak at 536
Figure 3). nm. This shift
The UV-Vis in theofvalue
spectrum of the max-
the AuNP
imum peak indicated the configuration of the AuNP surface, going from a barenm,
CITs (as purchased) shows a spectral band with a maximum absorbance peak at 534 state to a
whereas
coated state.the AuNP–4G2 (the three conjugates) and AuNP–BSA presented a maximum
absorbance peak at 536 nm. This shift in the value of the maximum peak indicated the
configuration of the AuNP surface, going from a bare state to a coated state.

Figure
Figure 3. Characterization of
3. Characterization of AuNPs
AuNPs and andAuNP–4G2
AuNP–4G2 conjugates. UV-Vis
conjugates. spectra
UV-Vis of AuNPs
spectra with
of AuNPs with
citrate without treatments (AuNP CIT), AuNP–BSA conjugates (control), AuNPs
citrate without treatments (AuNP CIT), AuNP–BSA conjugates (control), AuNPs conjugated with conjugated with
different
different concentrationsofofanti-flavivirus
concentrations anti-flavivirus antibody
antibodyofof1, 1,
3, 3,
and 6 µg/mL
and 6 µ g/mL(AuNP–4G2
(AuNP–4G2 conjugates).
conjugates). A
shiftAin
shift
theinmaximum
the maximum peak
peak absorbance of
absorbance of the
thespectrum
spectrum was observed
was in the
observed inAuNP–4G2 conjugates
the AuNP–4G2 conjugates
at different antibody concentrations and in the AuNP–BSA conjugates compared
at different antibody concentrations and in the AuNP–BSA conjugates compared to the naked to the naked AuNPs
(the maximum
AuNPs (the maximumabsorbance peaks arepeaks
absorbance indicated
are by the dotted
indicated bylines).
the dotted lines).
To corroborate the state of aggregation of the AuNP–4G2 conjugates, the GAT assay
Toperformed.
was corroborateThethe stateshowed
results of aggregation of theAuNPs
that the naked AuNP–4G2(AuNPconjugates, the GAT assay
CIT) in the presence
wasofperformed. Thebuffer
PBS and borate results
pHshowed that
6 reported the naked
a high AuNPs
aggregation (AuNP
number CIT) whereas
(<0.159), in the presence
the of
PBSAuNP–4G2
and borate buffer pH
conjugates 6 reported
presented valuesa lower
high than
aggregation number
0.02, indicating (0.159),
their stabilitywhereas
even the
in the presence
AuNP–4G2 of a high
conjugates concentration
presented valuesoflower
salts (see
thanFigure 4). Among their
0.02, indicating the AuNP–4G2
stability even in
conjugates, it was observed that AuNP–4G2 conjugated with 3 µg/mL presented
the presence of a high concentration of salts (see Figure 4). Among the AuNP–4G2 a lower conju-
aggregation value compared to AuNPs without coating or conjugated with 1 µg/mL of
gates, it was observed that AuNP–4G2 conjugated with 3 µ g/mL presented a lower
the antibody. Additionally, no major difference was observed between the aggregation
value of the AuNP–4G2 conjugates with 3 and 6 µg/mL, so it was decided to work with a
concentration of 3 µg/mL to conjugate the AuNPs and obtain the biosensor that was used
in the LFA for the detection of flaviviruses.
For the physicochemical characterization, dynamic light scattering (DLS) was used
to determine the hydrodynamic size, ζ-potential, and state of aggregation of the AuNPs
and conjugates. The values of each measured parameter can be seen in Table S2 in the
Supplementary Materials.
 aggregation value compared to AuNPs without coating or conjugated with 1 µ g/mL of
the antibody. Additionally, no major difference was observed between the aggregation
value of the AuNP–4G2 conjugates with 3 and 6 µ g/mL, so it was decided to work with a
Biosensors 2022, 12, 495 concentration of 3 µ g/mL to conjugate the AuNPs and obtain the biosensor that was used 9 of 19
in the LFA for the detection of flaviviruses.

Figure 4. Determination of the aggregation value of the AuNPs and conjugates. The aggregation
Figure 4. Determination
value was of the the
calculated using aggregation value of thetest
gold aggregation AuNPs
(GAT). andAll
conjugates. The aggregation
the conjugates presented values
value was calculated using the gold aggregation test (GAT). All the conjugates presented values
lower than those reported in the naked AuNP indicating the presence of the surface coverage of the
lower than those reported in the naked AuNP indicating the presence of the surface coverage of the
AuNPby
AuNP bythetheantibodies,
antibodies, protecting
protecting the AuNP
the AuNP from from the formation
the formation of conglomerates
of conglomerates and preserving
and preserving
stabilityininthe
stability the colloidal
colloidal state.
state. p  0.001,
(*** (*** p < 0.001,  0.0001,
**** p**** p < 0.0001,
n = 3). n = 3).

Thethe
For results obtained showed
physicochemical that the AuNP
characterization, dynamic CITlight
presented an (DLS)
scattering averagewashydrodynamic
used
diameter
to determine of the
67.69, whereas thesize,
hydrodynamic control AuNP–BSA
ζ-potential, and
and state ofthe AuNP–4G2
aggregation at AuNPs
of the concentrations
and
of 1,conjugates. The values
3, and 6 µg/mL of eachan
presented measured
increaseparameter can behydrodynamic
in the average seen in Table S1diameter
in the (size)
Supplementary Materials.
of 103.5, 107.87, 117.20, and 103.63, respectively. The increase in the size of the conjugates
and The
the results
control obtained showed
indicates that the AuNP
the formation CIT AuNP–antibody
of the presented an average hydrodynamic
complex. However, the
diameter of 67.69, whereas the control AuNP–BSA and
maximum increase in the AuNP size was observed in the AuNP–4G2 the AuNP–4G2 at concentrations
conjugate at an
of 1, 3, and 6 µ g/mL presented an increase in the average hydrodynamic diameter (size)
antibody concentration of 3 µg/mL, indicating that this concentration is the optimal one
of 103.5, 107.87, 117.20, and 103.63, respectively. The increase in the size of the conjugates
for the saturation of the nanoparticle surface (see Figure 5a).
and the control indicates the formation of the AuNP–antibody complex. However, the
On the other hand, the measured polydispersity value was less than 0.2, which indi-
maximum increase in the AuNP size was observed in the AuNP–4G2 conjugate at an an-
cated that
tibody the AuNP–4G2
concentration and indicating
of 3 µ g/mL, the control were
that thismonodisperse
concentration isinthea optimal
colloidal state
one for and did
not present aggregation, which is ideal for use
the saturation of the nanoparticle surface (see Figure 5a). in the detection of the analyte (Figure 5b).
When
On analyzing
the other themeasured
hand, the ζ-potential values obtained,
polydispersity value wasweless
observed
than 0.2,that
whichtheindi-
AuNP CIT
(naked)
Biosensors 2022, 12, x FOR PEER REVIEW
cated thatpresented
the AuNP–4G2a value the−
and of 34.9 mV,
control werea monodisperse
lower value than the 4G2–AuNP
in a colloidal conjugates,
state and did 10 of 21
indicating
not the successful
present aggregation, deposition
which is ideal forofuse
antibodies on theof
in the detection surface of the
the analyte nanoparticle
(Figure 5b). and
preserving stability (Figure
When analyzing 5c). values obtained, we observed that the AuNP CIT
the ζ-potential
(naked) presented a value of -34.9 mV, a lower value than the 4G2–AuNP conjugates, in-
dicating the successful deposition of antibodies on the surface of the nanoparticle and
preserving stability (Figure 5c).

(a)
Figure 5. Cont.
Biosensors 2022, 12, 495 10 of 19

(a)

(b)

(c)
Figure
Figure5.5.Physicochemical
Physicochemical analysis of AuNPs
analysis of AuNPsandand
conjugates by DLS.
conjugates (a) Hydrodynamic
by DLS. diameter
(a) Hydrodynamic diameter
(size),
(size),(b)
(b)polydispersity
polydispersityindex, and
index, (c)(c)
and ζ-potential values
ζ-potential of the
values naked
of the AuNPs
naked (AuNP
AuNPs CIT),CIT),
(AuNP control
control
(AuNP–BSA), and conjugates (AuNP–4G2) (* p  0.05, *** p  0.001, **** p  0.0001, n = 3).
(AuNP–BSA), and conjugates (AuNP–4G2) (* p < 0.05, *** p < 0.001, **** p < 0.0001, n = 3).

Biosensors 2022, 12, x FOR PEER REVIEW As

Ascomplementary
complementarytests forfor
tests thethe
characterization of the
characterization AuNPs
of the and and
AuNPs AuNP–4G2 con-
AuNP–4G2 con-
11 of 21
jugates,
jugates,the
theelectrophoretic
electrophoretic mobility testtest
mobility andand
thethe
gold aggregation
gold test test
aggregation werewere
performed.
performed.
According to the results observed in Figure 6, the AuNPs performed
According to the results observed in Figure 6, the AuNPs performed a delaya delay in mobility
in mobil-
when they they
ity when were were
coatedcoated
with 3with
and 63 µand
g/mL of antibody
6 µg/mL comparedcompared
of antibody to the controls
to the(BSA,
controls
without antibody). This change in mobility suggests a larger size of the conjugated AuNP–
(BSA, without antibody). This change in mobility suggests a larger size of the conjugated
4G2 due to slower migration.
AuNP–4G2 due to slower migration.

Figure 6. Electrophoretic mobility shift assay for AuNP–BSA

AuNP–BSA and and AuNP–4G2
AuNP–4G2 conjugates.
conjugates. Agarose
gel electrophoresis was used to identify the migration differences of each conjugate of AuNPs with
increasing concentrations
increasing concentrations of
of anti-flavivirus
anti-flavivirus antibodies
antibodies (0,
(0, 3,
3, and
and 66 µg/mL).
µ g/mL).

3.2. Lateral Flow Assay Design

The LFA designed in this work was based on the competition of the antigen present
in the tested sample with the antigen anchored to the nitrocellulose membrane by binding
Biosensors 2022, 12, 495 11 of 19

3.2. Lateral Flow Assay Design

The LFA designed in this work was based on the competition of the antigen present in
the tested sample with the antigen anchored to the nitrocellulose membrane by binding to
the biosensor for the detection of flaviviruses. In the presence of flaviviruses, the biosensor
forms a complex with the antigen present in the sample and is unable to bind to the
previously immobilized antigen on the strip, but it can bind to the secondary antibody
(into the control zone), observed by a single red dot. In the absence of flaviviruses in the
Biosensors 2022, 12, x FOR PEER REVIEW 12 of
sample, the biosensor binds to the test zone and the control zone, resulting in a negative
test showing two red dots (see Figure 7).

Molecular interactions for DENV detection by a lateral flow test

Figureinteractions
Figure 7. Molecular 7. Molecularinvolved
interactions involved
in the possibleinresults
the possible results
obtained obtained
in the LFA. The in the LFA. The detect
detection
window is made windowup ofisthe
made
test up
zoneof and
the test
the zone and
control the control
zone. zone.the
In the first, In DENV
the first,isthe DENV is immobiliz
immobilized
whereas in the second, the secondary anti-IgG antibody is immobilized. When placing the samp
whereas in the second, the secondary anti-IgG antibody is immobilized. When placing the sample,
the flavivirus present interacts with the biosensor forming a complex, which prevents it from join
the flavivirus present interacts with the biosensor forming a complex, which prevents it from joining
the test zone but is recognized by the control zone, obtaining a positive result (a signal). If the sam
the test zone but
doesis recognized
not presentby thethe control
antigen tozone, obtaining
be detected, theabiosensor
positive result
flows(atosignal). If the sample
the detection window and for
does not present the antigen to be detected, the biosensor flows to the detection
a complex with the virus present in the test zone, also binding to the antibody window andinforms
the control zo
a complex with the virusa present
obtaining negativein the test
result (twozone, also Results
signals). bindingwillto the antibody
be null when inthethe
testcontrol
zone haszone,
no signal, in
cating that
obtaining a negative the (two
result biosensor did not
signals). flow properly
Results on the
will be null LFA.
when the test zone has no signal,
indicating that the biosensor did not flow properly on the LFA.
To optimize the LFA, different measurements of the materials that compose it we
To optimize
tested. InLFA,
the different
addition, measurements
the overlap distanceofbetween
the materials that compose
the materials it were to obtain
was modified
tested. In addition, the in
better flow overlap distance
the strip betweenimprove
and therefore the materials was modified
the formation to obtain
of complexes for detectio
a better flow Finally,
in the strip
the and
exacttherefore improve
measurements of the
the formation
LFA were of complexesasfor
determined detection.
shown in Figure 8A
Finally, the exact measurements
Additionally, of the LFA were
it was established determined
that the minimumas shownofinsample
volume Figureto8A,B.
obtain a res
was 160 µ L.
Biosensors 2022, 12, 495 12 of 19

Biosensors 2022, 12, x FOR PEER REVIEW 13 of

Additionally, it was established that the minimum volume of sample to obtain a result
was 160 µL.

Figure 8. Design of the lateral flow assay for the detection of flaviviruses. (A) From left to right,
the strip is composed of a sample pad, conjugate pad, detection membrane (with a test zone and
a control zone), andFigure
absorbent pad.ofEach
8. Design of the flow
the lateral overlapping
assay forcomponents
the detectionasofshown in the (A)
flaviviruses. image
From have
left to right, t
specific measurements
stripthat allow theofflow
is composed in thepad,
a sample stripconjugate
for the formation of biosensor–virus
pad, detection membrane (with complexes
a test zone and a co
trol detection
for detection. (B) The zone), andmembrane
absorbent pad. Each ofbythe
is formed theoverlapping components
test zone and as zone,
the control shownwhere
in the image ha
specific measurements that allow the flow in the strip for the formation of biosensor–virus co
each one has a defined size that makes it possible to observe the results with the naked eye and the
plexes for detection. (B) The detection membrane is formed by the test zone and the control zo
separation betweenwhere
them,each
withonea constant measure, ensures the results of both zones do not overlap.
has a defined size that makes it possible to observe the results with the naked e
and the separation between them, with a constant measure, ensures the results of both zones do n
3.3. Performance and Reproducibility Tests of Lateral Flow Assay
overlap.
To test the functionality of the LFAs, 160 µL of each sample (DENV isolated as a positive
sample and PBS buffer as a negative
3.3. Performance sample) was used.
and Reproducibility Tests ofSamples (positive
Lateral Flow Assayor negative) were
placed on the sampleTo padtestofthe
thefunctionality
LFA and allowed to run160
of the LFAs, at room
µ L oftemperature
each sample and(DENV afterisolated as
10 min, the results were observed and photographic evidence was taken. The results
positive sample and PBS buffer as a negative sample) was used. Samples (positive or ne can be
observed in Figure 9. In
ative) theplaced
were LFAs on where positive
the sample pad samples were
of the LFA analyzed,
and allowed to a single signaltemperatu
run at room
was observed inand the after
control area,the
10 min, indicating the union
results were observed of the
andvirus with theevidence
photographic AuNP–4G2 was taken. T
conjugates (forming a complex),
results and preventing
can be observed in Figure 9.the AuNP–4G2
In the LFAs where conjugate
positive from binding
samples were analyzed
to the immobilized singlevirus in was
signal the observed
test zone.in Furthermore,
the control area,a indicating
signal was theobserved in the
union of the virus with t
control zone where the conjugates (forming a complex with the antigen or not) bound to conjuga
AuNP–4G2 conjugates (forming a complex), and preventing the AuNP–4G2
the immobilized from bindingantibody.
anti-mouse to the immobilized virus inthe
The LFAs where thenegative
test zone. Furthermore,
samples a signal was o
were placed
served in the control zone where the conjugates (forming
(which contained only PBS buffer) showed two signals in the detection window (in the a complex with the antigen
test area and the control area), one indicating the binding of the AuNP–4G2 conjugate negative
not) bound to the immobilized anti-mouse antibody. The LFAs where the to sa
ples were placed (which contained only PBS buffer) showed
the immobilized virus in the test zone and the other the recognition of the conjugate to the two signals in the detecti
window (in
immobilized anti-mouse the testinarea
antibody theand the control
control zone. area), one indicating the binding of the AuN
4G2 conjugate to the immobilized
Fifteen LFAs from the same batch were serially virus in the test
tested zone anddetection
to observe the other the recognition
repro-
the conjugate to the immobilized anti-mouse antibody
ducibility under the same conditions. Figure 10A shows the representative samples from in the control zone.
the same batch testing negative samples that did not have DENV (only PBS buffer 10 mM
NaCl). It was observed that the PBS buffer 10 mM NaCl was optimal to use as a run buffer
for this test because none of its components generated false negatives or caused aggregation
of the conjugates. Figure 10B shows the representative samples of the batch of LFAs tested
with PBS and DENV where a single dot (in the control zone) corresponding to a positive
result was observed. The consistency of the results showed that the tests from the same
batch had the same characteristics and could detect the virus in vitro.
Biosensors 2022, 12, 495 13 of 19
Biosensors 2022, 12, x FOR PEER REVIEW 14 of 21

Figure 9.
Figure 9. Functionality Functionality
test testflow
of the lateral of the lateral
assay forflow
theassay for theofdetection
detection of flaviviruses.
flaviviruses. PBS withPBSDENVwith DENV
(+) and PBS (−) were used for the LFA functionality test. The biosensor placed on the conjugate pad,
(+) and PBS (−) were used for the LFA functionality test. The biosensor placed on the conjugate pad,
upon contact with the sample, was able to recognize the virus, form a complex, and not bind to the
upon contact with the sample,
analyte (DENV)was able
bound to to
therecognize
membranethe virus,
in the testform
zone,aproducing
complex,aand notred
visible bind to the
signal in the con-
Biosensors 2022, 12, x FOR PEER REVIEW 15 o
analyte (DENV) bound to by
trol zone thebinding
membrane in biosensor
to the the test zone, producing
antibody 4G2 witha visible red signal in the control
the membrane-attached IgG secondary
antibody.
zone by binding to the biosensor antibody 4G2 with the membrane-attached IgG secondary antibody.

Fifteen LFAs from the same batch were serially tested to observe detection reproduc-
ibility under the same conditions. Figure 10A shows the representative samples from the
same batch testing negative samples that did not have DENV (only PBS buffer 10 mM
NaCl). It was observed that the PBS buffer 10 mM NaCl was optimal to use as a run buffer
for this test because none of its components generated false negatives or caused aggrega-
tion of the conjugates. Figure 10B shows the representative samples of the batch of LFAs
tested with PBS and DENV where a single dot (in the control zone) corresponding to a
positive result was observed. The consistency of the results showed that the tests from the
same batch had the same characteristics and could detect the virus in vitro.

Figure
Figure 10. Functional 10. Functional
reproducibility test reproducibility testassay
of the lateral flow of theforlateral flow assay for
the identification the identification of
of flaviviruses.
viviruses. The tests were carried out with the same batch of tests
The tests were carried out with the same batch of tests assembled under the same conditions assembled under the same con
using
tions using PBS buffer for the negative sample or DENV for the positive sample (160 µ L total v
PBS buffer for the negative sample or DENV for the positive sample (160 µL total volume). (A) In
ume). (A) In the LFAs where negative samples (only with PBS buffer) were placed, two red d
the LFAs where negative
(signals)samples (only with
were observed PBS buffer)
indicating were placed,
the absence of DENV. two(B)red
In dots (signals)
the LFAs where were
positive samp
observed indicating the absence of DENV. (B) In the LFAs where positive samples were
were placed (PBS and DENV), a single signal was observed in the control zone where the DENV placed
(PBS and DENV), athe sample
single “competed”
signal was observedwith the virus
in the immobilized
control in thethe
zone where LFADENV
to bindinto
thethe biosensor. No f
sample
positives or negatives were observed in any of the tests performed.
“competed” with the virus immobilized in the LFA to bind to the biosensor. No false positives or
negatives were observed in any of the tests performed.
4. Discussion
Surveillance of flavivirus outbreaks has been of utmost importance due to the sev
ity of the clinical presentation that can occur during acute infection in humans and ot
vertebrates. In addition, the epidemic potential of flaviviruses is increased by the g
graphic expansion of the vectors. The creation of ideal habitats for their reproduction c
Biosensors 2022, 12, 495 14 of 19

4. Discussion
Surveillance of flavivirus outbreaks has been of utmost importance due to the severity
of the clinical presentation that can occur during acute infection in humans and other
vertebrates. In addition, the epidemic potential of flaviviruses is increased by the geo-
graphic expansion of the vectors. The creation of ideal habitats for their reproduction
constantly threatens public health and highlights the need for monitoring tools for the pre-
vention and containment of outbreaks. In response to this need, in this work, we proposed
a fast, portable, and low-cost tool for the identification of DENV and other flaviviruses
using a POC test that uses a biosensor composed of gold nanoparticles and antibodies
for detection.
This biosensor was made by functionalizing 60 nm of spherical gold nanoparticles
with anti-flavivirus antibodies using passive adsorption conjugation. The size and shape of
the AuNPs are crucial in determining the sensitivity of the detection. In terms of size, it has
been reported that AuNPs with sizes between 20 and 40 nm are the most used for detection
in LFAs [28,29]. However, a recent study in 2017 by Zhan et al. [30] compared AuNPs of
40, 60, and 100 nm, and showed that they were able to improve analytical sensitivity by
up to 256 times when 60 and 100 nm AuNPs were used for the formation of the biosensor
with antibodies. This is because the larger the size, the greater the surface area available for
binding to antibodies, improving the detection of the analyte. Other authors have used and
evaluated the functionality of the 40–60 nm AuNPs for the development of biosensors for
use in LFAs [31,32].
Also, in terms of shape, spherical AuNPs have been widely used in the detection of
analytes in LFA tests due to their unique optical and physicochemical properties, high
biocompatibility, tunable monolayer, controlled dispersion, high surface area for function-
alization with detection elements, low toxicity, high stability, and their characteristic red
color that allows straightforward detection of complex formations without the need for
extra equipment.
On the other hand, the antibody used for the formation of the biosensor (4G2 antibody)
has been widely used in flavivirus detection assays and has recently been used as a control
in dengue virus neutralization assays [33] and Zika immunodetection [34]. Its binding
specificity to protein E of viruses of the flaviviridae family such as dengue (the 4 serotypes
including serotype 2 used in this work) [35,36], Zika [37], West Nile virus [38], and tick-
borne encephalitis virus (TBEV) [39] has been reported by multiple authors over time.
To ensure the functionalization of AuNPs, the conjugates (4G2–AuNP) were subjected
to various physicochemical characterization tests. The results were compared with the
naked nanoparticles (AuNP CIT), obtaining differences between the properties of the
conjugates with respect to the unconjugated AuNPs to establish a pattern of conjugation
control. First, the optimal conditions for the formation of the AuNP–antibody conjugates
were determined using a set concentration of anti-flavivirus 4G2 antibody (10 µg/mL),
varying the pH of the AuNP solution just before placing the antibody during the protocol
of conjugation. In previous studies, it has been reported that the pH during conjugation
determines the binding orientation of the antibodies in the AuNPs and can influence the
optimal coating of their surface [40].
Subsequently, the stability of each conjugate formed at different pH values was tested
using the GAT. The optimal pH for these conjugates was determined to be 6, where a lower
aggregation of the AuNPs was observed in the presence of salts. The correct coating of
the AuNPs ensures their monodisperse state that favors the uptake of the antigen by the
biosensor and the formation of individual AuNP–antibody–antigen complexes, which can
be used in an LFA or other detection systems.
Although using a high concentration of antibodies stabilizes the nanoparticles and
increases the probability of the correct orientation of the antibodies on the surface, it has
been reported that it can also cause the inaccessibility of the antibodies due to the effects of
superposition and, therefore, could compromise its ability to detect the antigen [41].
Biosensors 2022, 12, 495 15 of 19

Therefore, once the conjugation protocol was optimized, the optimal concentration of
the antibody required for biosensor formation was determined. For this, three different
concentrations of the antibody (1, 3, and 6 µg/mL) were tested for the formation of the
conjugates. Each conjugate was characterized and its stability was analyzed by physico-
chemical parameters and its resistance to aggregation by the GAT test.
It was observed that the three AuNP–4G2 conjugates in the spectral scan UV-visible
range presented a shift in the maximum absorbance peak compared to the naked AuNPs.
This shift in the maximum absorbance peak of the conjugates has been previously re-
ported as being indicative of the adsorption of the antibodies on the surface of the AuNP
forming a monolayer [42].
Additionally, the hydrodynamic diameter of the conjugates was measured by DLS.
An increase in the hydrodynamic diameter (relative size) proportional to the increase in the
antibody used for conjugation was observed, indicating that at a higher concentration of the
antibody, the formation of the monolayer in the AuNP increased. However, when 6 µg/mL
of the antibody was added, a decrease in the hydrodynamic diameter was observed. These
results may indicate the supersaturation of antibodies on the surface of the AuNP where at
higher concentrations of the antibody there is competition or steric hindrance to binding
to the surface.
Other parameters such as the ζ-potential and polydispersity index of the conjugates
were compared between conjugates and with the naked AuNPs. It was observed that the
increase in the ζ-potential measured in the AuNPs coincides with the addition of antibodies
on the surface of the AuNP when forming the conjugates, suggesting that the negative
charge that the AuNP surface possesses decreased as the AuNPs formed ionic bonds
with the antibody.
The values obtained in the physicochemical parameters showed that the three conju-
gates presented similar characteristics, so in the last differentiation step, an aggregation
test was performed. This test measured the stability of the conjugates in saline solution
(20% NaCl) where a considerable difference was not observed between the stability of the
conjugate with 3 and 6 µg/mL (GAT values less than 0.02), indicating that from 3 µg/mL,
the surface of the AuNPs has an antibody monolayer that prevents aggregation. These tests
have been performed by other authors in order to determine the minimum concentration
of antibodies for the coating of AuNPs [43,44]. Therefore, the biosensor that was used to
assemble the LFA was 3 µg/mL.
The LFA reported in this work was assembled with a biosensor using a competitive
format to identify dengue (and other flaviviruses). This format is based on the fact that the
antigen immobilized that is present in the detection zone “competes” with the antigen of
the sample to bind to the biosensor. However, initially, we designed both formats, direct
or sandwich-type and competitive type using the same biosensor, where under similar
conditions we did not achieve the detection of the analyte using the sandwich-type. It has
been reported that sandwich-type LFAs are typically used for larger analytes with multiple
antigenic sites using different antibodies in the design [45].
The first LFA design was a sandwich-type using the same antibody both for the
functionalization of the AuNPs and the elements immobilized on the detection membrane.
In the functionality tests performed, we could not observe any signal in the test zone when
we added DENV to the carrier solution (PBS) but a signal was observed in the control
zone, indicating that the sample migrated correctly through the LFA. In order for the LFA
to be functional and detect the analyte, we changed the treatments of each pad hoping
that the detection would improve; however, after several optimization attempts, there was
no positive result. There are multiple works where the sandwich format is used for viral
detection; however, in the detection system reported in this work it did not work.
Then, with the same biosensor and the DENV as an immobilized analyte in the test
zone, an LFA with a competitive format was designed. Subsequently, the LFA was tested
in vitro to determine its functionality with DENV samples and the carrier solution (PBS).
It was observed that in a batch of LFAs when the DENV positive sample was placed in
Biosensors 2022, 12, 495 16 of 19

the test, a signal was generated in the detection window, demonstrating that the virus was
binding to the biosensor and preventing binding to the virus immobilized in the membrane.
The LFAs where only the carrier solution was tested, showed two signals indicating that the
biosensor bound to the antigen immobilized on the membrane, therefore indicating a true
negative. The LFA designed with this AuNP-based biosensor allowed the detection of
DENV in vitro; however, it is worth mentioning that this test can detect various flaviviruses
due to the nature of the antibody (anti-flaviviruses 4G2) used for biosensor formation, so it
could be included as an accessible option for flaviviruses monitoring.
In this work, the biosensor that we used in the LFA contained ~0.2 mg/mL of gold
nanoparticles. It has been reported that the concentration of AuNPs can enhance the
detection of the analyte in the LFA. However, a recent study published by Khlebtsov et al.
in 2019 [46] indicates that although it is true that the signal obtained in the test zone
decreases as the number of applied particles decreases, the amount of the signal observed
in the test zone and the LFA limit of detection (LOD) are mainly determined by the light
absorption of the particles used, which also depends on its size and shape.
The results found in this work offer this LFA as an alternative method for the detection
of pathogens from the transmitter vector. In 2018, Basso et al. [47] generated a similar DENV
detection system (serotypes 1–4) using a biosensor with antibody-coated nanoparticles,
determining the presence of the virus through the physicochemical changes observed in
the nanoparticle. Despite its advantages, the disadvantage of this method is the specialized
equipment required for the analysis of each sample, making it not feasible as a field test
compared to our portable test for which reading equipment is not necessary.
Other methods based on biosensors composed of AuNPs coated with other molecules,
such as DNAzymes [48], have been used for the identification of arboviruses in the trans-
mitting mosquito; however, these are usually complex to produce (and therefore expensive)
and require an environment favorable for the development of the reaction, which makes it
difficult to handle in an uncontrolled environment.
Regarding the detection limit, the authors usually report this parameter in different
units of measurement, obtaining the value through a calibration curve. Here, we used an
antigen solution based on the complete virus that was quantified by the TCID50 method,
whereas most other detection systems use purified protein or genome copies. Under the
reported conditions, our system has an estimated detection limit of 5.12 × 102 PFU (see
Figure 10). This estimated value was based on the performance of the test when we used
diluted virus samples, showing that the LFA worked only when we added the undiluted
sample (see Supplementary Material SI). Testing the LFA with 1:10 dilutions produces
a negative result, suggesting that the tested antigen concentrations are near the limit of
detection of the test.
Compared to other novel proposed systems, we have a comparable limit of detection
to other antibody-based tests, such as observed in the Tamiflu-resistant Influenza virus
detection system against neuraminidase with 5 × 102 PFU [49] or the SARS-CoV-2 magnetic
lateral flow system against the spike protein that showed a limit of detection (at the naked
eye) of 6 × 102 PFU [50]. As expected, the limit of detection of antibody-based systems
is lower than that of nucleic acid amplification-based tests, which can reach a limit of
detection of 2 × 102 PFU [51].
The advantages of our test with a lateral flow format are its ease of use and minimal
sample-handling steps, which are favorable for the environment. The functionality test
showed that the results are not modified over time, preserving the signal in the detection
window, which can be useful for capturing the results after the test. This LFA was designed
to monitor flaviviruses in mosquito samples in order to identify areas of epidemic risk.
The naked-eye readings will allow monitoring without equipment, making this method
accessible for low-income regions.
We consider this prototype to be an advancement in the design of an accessible and
easy-to-use LFA system for the detection of dengue virus and other flaviviruses but it is
necessary to include other studies to determine its sensitivity and specificity. Functionality
Biosensors 2022, 12, 495 17 of 19

tests of the LFA with mosquito samples and the optimization and improvement of this LFA
are required in order to use this LFA as a vector-monitoring tool. Among the improvements
and optimizations of the design, different antibodies could be tested to determine if the
sandwich format can improve the detection limit and the performance of the LFA.

5. Conclusions
In this study, a biosensor based on antibodies coupled to gold nanoparticles was
developed and characterized for the detection of DENV, with the potential to detect other
flaviviruses. The AuNP–4G2 biosensor showed physicochemical characteristics, which
distinguishes it from naked AuNPs. Additionally, with this biosensor, a functional compet-
itive LFA was designed and tested. The total time of the operation of this screening test,
from the application of the sample on the LFA to obtaining the results, was a maximum of
10 min. The LFA functionality results showed that it is possible to detect DENV in vitro
with this test.
Due to its performance and response times, this test proved to be a fast, easy, reliable,
and low-cost alternative design for a point-of-care test for the monitoring of flaviviruses
in areas endemic to the vector, both for early detection in mosquitoes and containment of
possible outbreaks. This test prototype is expected to be used for the design of a commercial
device for the rapid, sensitive, and specific mass screening of different samples for the
accurate detection of DENV and other different flaviviruses, in order to increase primary
prevention to avoid diseases caused by these viruses and decrease the consequences for
vulnerable populations.

Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/bios12070495/s1. Table S1: Equations and calculations to determine
the TCID50 and PFU. Table S1 shows data for titration of virus stocks; Table S2: Determination of
the hydrodynamic diameter (relative size), ζ-potential, and distribution (polydispersity index) of the
AuNP conjugates.
Author Contributions: Conceptualization, C.M.-W.; Data curation, N.M.-A.; Formal analysis, C.M.-L.
and K.G.-H.; Funding acquisition, A.M.R.-E.; Investigation, C.M.-L.; Methodology, C.M.-L., N.M.-
A., S.L.-S., M.M.-V. and S.G.-R.; Project administration, A.M.R.-E.; Resources, C.M.-W., K.G.-H.
and S.G.-R.; Software, D.Á.-N.; Supervision, N.M.-A., S.L.-S. and A.M.R.-E.; Visualization, C.M.-
L.; Writing—review and editing, C.M.-L., N.M.-A., S.L.-S., D.A.-S., M.M.-V., K.P.-I., A.d.J.G.-J. and
A.M.R.-E. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the CONACYT, as part of the national laboratories program
with grant number 315848. The researchers involved did not receive any other specific compensation
from funding agencies in the public, commercial, or non-profit sectors.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: We acknowledge CONACYT for the grant awarded with the number 700317. We
acknowledge Natalia Martinez for her support with the design of the figures presented in this article
and to the Medical Virology Research and Innovation Center (CIIVIM) belonging to the Autonomous
University of Nuevo León for the support provided.
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.

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