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International Journal of Applied Engineering Research ISSN 0973-4562 Volume 12, Number 23 (2017) pp. 13847-13860
© Research India Publications. http://www.ripublication.com
13847
Synthesis of the Most Effective Streptavidin Conjugates with Small Gold
Nanoparticles for Indirect Labeling in Lateral Flow Assay
Alexey V. Samokhvalov1,$, Shyatesa C. Razo1,2, Irina V. Safenkova1, Elvira S. Slutskaya1,
Svetlana M. Pridvorova1, Anatoly V. Zherdev1, Boris B. Dzantiev1*
1A.N. Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Leninsky prospect 33, Moscow 119071, Russia.
2Agricultural-Technological Institute, Peoples’ Friendship University of Russia, Mikluho-Maklaya street 8/2, Moscow 117198, Russia.
*Correspondence1Orcid: 0000-0002-3621-4321
Abstract
The use of gold nanoparticles (GNPs) with a small size
(diameter <10 nm) for the immobilization of biomolecules
requires the small area of each nanoparticle and the large
curvature of the surface to be taken into account. The aim of
this study is to determine the optimal conditions for the
biomolecules’ immobilization using small GNPs synthesized in
different ways. The GNPs were synthesized by the two most
typical methods: using either sodium borohydride or a
combination of sodium citrate and tannic acid as reducing
agents. The average diameters were determined to be equal to
4.4 ± 0.6 and 6.9 ± 1.5 nm by transmission electron microscopy,
respectively. Based on these nanoparticles, 24 conjugates were
synthesized and characterized at different pH (6.5, 7.4, and 9.0)
and different concentrations of immobilized protein
(streptavidin with concentrations of 20, 40, 60, and 80 μg/mL).
Depending on the synthesis conditions, polydispersity indexes
and formed precipitates varied, and the hydrodynamic
diameters of the conjugates varied from 1570 to 15 nm. For
both of the GNPs preparations, the conjugates that were most
monodisperse, stable, and without aggregate formation were
obtained at pH 9.0 with streptavidin concentrations of 40
(GNPs by sodium citrate/tannic acid) and 80 μg/mL (sodium
borohydride). The binding properties of the all streptavidin–
GNP conjugates synthesized at pH 9.0 were tested using lateral
flow tests. All conjugates showed high binding properties up to
0.004 optical densities for conjugate λmax. This study has great
potential to improve the synthesis of the conjugates with small
GNPs for immunoassay applications.
Keywords: conjugates with gold nanoparticles; gold
nanoparticles; lateral flow tests; streptavidin; small
nanoparticles.
INTRODUCTION
Gold nanoparticles (GNPs) and their conjugates are some of the
most studied nanomaterials, with promising applications in
many immunoanalytical systems for medicine and agriculture
[1, 2]. As in most of these applications, the monodispersity of
GNPs is a desirable feature; moreover, synthesis should result
in controlled NP size, distribution, shape, and stability. To date,
chemical reduction is still the most common strategy for the
synthesis of GNPs, most probably because of the simplicity of
the method and apparatus required [3, 4]. A variety of reducing
(for example, borohydrides, sodium citrate, aminoboranes,
hydrazine, and formaldehyde) and stabilizing agents
(hydrazine, ascorbic acid, cetyltrimethylammonium bromide,
poly[vinyl alcohol], poly[vinylpyrrolidone], poly[ethylene
glycol], sodium citrate, and tannic acid) can be used to
synthesize GNPs [5–10]. Related to this, the second important
point is the coating of nanoparticles with stabilizing molecules
that further influence the immobilization of target biomolecules
(antibodies, enzymes, streptavidin, etc.). The method of GNP
synthesis affects the surface features, which can affect the
immobilization of biomolecules [11, 12]. For small GNPs
(diameter <10 nm), these are particularly relevant issues related
to the small area of each nanoparticle and the large curvature of
the surface [13, 14]. In these terms, uniform, spherical, small
GNPs can only be obtained using a limited number of methods.
Under these conditions, the number of syntheses of small GNPs
deserving attention is limited.
One of the most common syntheses for obtaining monodisperse
and stable small GNPs (5–10 nm diameter) is a chemical
synthesis with tannic acid and sodium citrate as both reducing
and stabilizing agents [15, 16]. The combined use of sodium
citrate and tannic acid enables control over the nucleation,
growth, and stabilization processes, which leads to
reproducible monodisperse GNPs. Ranoszek-Soliwoda et al.
showed that the formation of a sodium citrate–tannic acid
complex acts as a reducing agent in the synthesis of NPs [17].
As a result, NPs are stabilized by the oxidation products of the
complex [17]. The second typical synthesis for obtaining small
GNPs is based on sodium borohydride reduction [18–20].
Although the preparation of GNPs by both chemical reductions
can probably make a stable and reproducible colloid containing
spherical GNPs, the surface coverage of the nanoparticles is
different. This factor is of great importance for synthesizing
conjugate biomolecules immobilized on the GNP surface by
nondirectional and noncovalent physical adsorption [12, 21]. It
is well known that the main active forces of noncovalent
immobilization are donor–acceptor interactions involving SH-
International Journal of Applied Engineering Research ISSN 0973-4562 Volume 12, Number 23 (2017) pp. 13847-13860
© Research India Publications. http://www.ripublication.com
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groups of molecules, hydrophobic interactions, and Coulomb
interactions between NH2 groups of lysine and ions on the
surfaces of GNPs [22, 23].
In this paper, we present the comparison of the conditions for
the synthesis of GNP conjugates synthesized by reduction with
the sodium citrate/tannic acid and the sodium borohydride
methods. We synthesized the GNP conjugates under different
conditions, and experimentally defined that the pH affects the
characteristics of the resulting conjugates in different ways. For
immobilization to the GNP surface, we chose streptavidin,
which forms a high-affinity strong noncovalent interaction with
biotin [24, 25]. We used a lateral flow assay (LFA), the most
common method using GNPs as a colored label, to find the
effective streptavidin–GNP conjugates for improving the
sensitivity of the assay. Streptavidin–GNP conjugates can be
used to detect, localize, or quantify the binding of biotinylated
molecules in the test zone of lateral flow tests. The
streptavidin–GNP conjugate is a very effective tool as a signal
amplifier and for increasing sensitivity, particularly for the
indirect labeling in LFA [26–28]. Targeting molecules need not
be directly modified with GNPs, only biotinylated so that they
are able to interact with streptavidin–GNP conjugates. The
formation of the GNP–streptavidin complexes with
biotinylated antibodies in the test zone of the lateral flow test
strips leads to an increase in the number of bound GNP labels
and improves the sensitivity of LFA.
MATERIALS AND METHODS
Reagents
Polyclonal antibodies (fraction of IgG) specific to plant virus
were used for biotinylation and testing streptavidin–GNP
conjugates. Streptavidin from Streptomyces avidinii was
purchased from Imtech (Russia). Tetrachloroauric acid, tannic
acid, and sodium borohydride were purchased from Fluka
(Taufkirchen, Germany). Tris(hydroxymethyl)aminomethane,
nitric acid (70% w/w, purified by redistillation), bovine serum
albumin (BSA), biotin amidohexanoyl-6-aminohexanoic acid
N-hydroxysuccinimide ester, and sodium citrate were
purchased from Sigma-Aldrich (USA). The Au standard for
inductively coupled plasma mass spectrometry (ICP-MS) (999
± 2 mg/mL in 5% HCl [w/w]) came from Fluka, Switzerland;
ICP-MS internal standards came from Bruker Daltonics
Chemical Analysis, USA; hydrochloric acid (38% w/w, ultra-
pure) came from Reactiv-component, Russia. All acids, alkali,
salts, and solvents were purchased from Khimmed (Moscow,
Russia). The compounds used in buffer solutions were of
analytical or chemical grade. Solutions were prepared using
water deionized by a Milli-Q system produced by Millipore
(USA). Lateral flow test strips were fabricated using plastic
supports with a nitrocellulose membrane (CNPC 12) and
absorbent pads (AP045) produced by Advanced Microdevices
(India).
Synthesis of gold nanoparticles using sodium citrate/tannic
acid
GNPs were prepared according to the protocol described by
Hermanson [29] with modification. For the synthesis of 100
mL of GNPs, 1 mL of 1% aqueous solution of tetrachloroauric
acid was added to 79 mL of deionized water, then heated to 60
ºC. Simultaneously, aqueous solutions of 1% sodium citrate (4
mL), 1% tannic acid (0.5 mL), 25 mM potassium carbonate (0.5
mL), and deionized water (15 mL) were mixed and also heated
to 60 ºC. Then, two solutions at 60 ºC were mixed and refluxed
with slight stirring until the tetrachloroauric acid had been
completely reduced and a stable colloidal solution had formed.
The solution was then cooled to room temperature.
Synthesis of gold nanoparticles using sodium borohydride
GNPs were prepared according to the protocol described by
Dykman [30] with modification. Aqueous solutions of 30 mM
EDTA (0.5 mL) and potassium carbonate (0.2 mL) were added
to an Erlenmeyer flask with deionized water (48.7 mL). The
mixture was cooled to 4 °C and all further processes were
carried out at this temperature. Then, 1% (w/v) aqueous
solution of tetrachloroauric acid (0.5 mL) was added with
intense stirring. Following this, 0.5% (w/v) aqueous solution of
sodium borohydride (125 µL) was quickly added to the flask.
The reaction mixture was incubated for the night at 4−6 °C with
intense stirring.
Synthesis of gold nanoparticle conjugates
Synthesis of streptavidin–GNP conjugates was carried out as
described by Hermanson [29] with modification. The
streptavidin conjugates were synthetized with both kinds of
GNPs at pH 6.5, 7.4, and 9.0. To adjust the pH of GNPs, 0.2 M
potassium carbonate solution or 0.02 M HCl were used. To
determine the minimal stabilizing concentration of the
streptavidin for the conjugation (flocculation method), the
obtained GNP solution (1.0 mL portions) was added to
solutions (0.1 mL) of streptavidin in concentrations from 1 to
250 μg/mL. The mixture was incubated at room temperature for
10 min. Then we added a 10% NaCl solution (0.1 mL) to each
sample, stirred the mixtures for 10 min, and measured OD580,
plotting its dependence in the presence of 10% NaCl against the
streptavidin concentration (flocculation curves). The
concentration corresponding to the beginning of the plateau in
the flocculation curve was determined, and a concentration
exceeding this value by 10–15% was chosen for streptavidin
conjugation.
To synthesize streptavidin–GNP conjugates, streptavidin was
added to GNPs (at pH 6.5, 7.4, and 9.0) to the final
concentration of 20, 40, 60, and 80 μg/mL. The mixture was
incubated without stirring at 20−22 °C for 30 min. Then, BSA
was added to each sample to the final concentration of 100
μg/mL. The separation of unbound streptavidin was carried out
International Journal of Applied Engineering Research ISSN 0973-4562 Volume 12, Number 23 (2017) pp. 13847-13860
© Research India Publications. http://www.ripublication.com
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using Beckman Coulter Optima L-100XP ultracentrifuge
(USA) at 45,000 g for 25 min. After the removal of the
supernatant, the precipitate was resuspended in 10 mM Tris-
HCl buffer, pH 8.5. The spectra of the GNPs and their
conjugates were recorded with a Biochrom Libra S60 double
beam spectrophotometer (Biochrom, UK).
Transmission electron microscopy
The size and shape of GNPs were determined using
transmission electron microscopy (TEM) (Jeol CX-100
electron microscope, Japan; accelerating voltage 80 kV;
magnification of 3,300,000–25,000,000x). The GNPs samples
for TEM measurements were dropped onto carbon-coated
copper grids (300 mesh, Pelco International, USA) coated with
polyvinyl formal support film. The obtained images of GNPs
and their conjugates were digitalized with Image Tool software
(University of Texas Health Science Center in San Antonio,
USA). To approximate TEM data about the distribution of
GNPs’ diameters, one-peak Gauss approximation was used.
The fitting was calculated by OriginPro 9.0 software (Origin
Lab, USA).
Dynamic light scattering and electrophoretic light
scattering
All experiments were performed with a Zetasizer Nano ZSP
(Malvern Instruments, UK), which features a 4 mW He–Ne
laser (633 nm). The temperature was stabilized at 25°C, and the
scattering angle was 173°. All measurements were performed
at least 80 times.
A disposable cell (DTS0012; Malvern Instruments, UK) was
used for dynamic light scattering (DLS) measurements. The
hydrodynamic sizes of GNPs and streptavidin–GNP conjugates
were measured by DLS. The polydispersity index (PdI) was
calculated as: (standard deviation/mean value)2.
For determining the streptavidin isoelectric point (pI), zeta
potentials of the streptavidin were measured at different pH
(4.0, 5.0, 6.0, and 7.0) using electrophoretic light scattering
(ELS) according to the Malvern Instruments recommendation
[31]. For ELS measurements, samples were loaded into a
disposable clear zeta cell (DTS1060; Malvern Instruments,
UK). Statistical processing was performed using Malvern
software ver. 7.11.
Measurements of Au by inductively coupled plasma mass
spectrometry
To estimate of GNPs and conjugate amounts in the solutions,
Au concentrations were obtained by ICP-MS. The ICP-MS
measurements were carried out with a quadrupole ICP-MS
instrument Aurora M90 (Bruker Corp., USA) equipped with a
MicroMist low-flow nebulizer. A series of Au standard
solutions (0.1–5.0 ppb in 1% HCl [v/v]) were prepared before
each experiment. Scandium was used as the internal standard,
eliminating the fluctuations coming from the measuring
conditions. All samples were prepared in triplicate. Quantum
software (Bruker Corp., version v.3.1) was used for data
collection and processing. The measurements and calculations
were carried out as described previously by Byzova et al. [31,
32].
The ICP-MS measurements were carried out on equipment at
the Shared Access Equipment Centre “Industrial
Biotechnology” of the Federal Research Center “Fundamentals
of Biotechnology” of the Russian Academy of Sciences
(Moscow, Russia).
Biotinylation of antibodies
For testing streptavidin–GNP conjugates, polyclonal antibodies
were biotinylated as described by Hermanson [29]. The
antibodies were dialyzed against a 1,000-fold volume of 50
mM potassium phosphate, pH 7.4, 0.14 M NaCl (PBS), for at
least 14 hours at 4 °C. Then, biotin amidohexanoyl-6-
aminohexanoic acid N-hydroxysuccinimide ester (10 mg/mL in
dimethyl sulfoxide) was added to the antibodies in a 20:1 molar
ratio. The mixture was incubated for 1 hour at room
temperature under intensive stirring and then dialyzed against
PBS overnight.
Preparation of lateral flow test strips
A binding zone was formed on the nitrocellulose membrane
CNPC 12 using biotinylated antibodies. We dispensed
biotinylated antibodies (1 mg/mL) at 0.15 µL per mm
membrane width in PBS containing 5% glycerol [33] using
IsoFlow dispenser (Imagene Technology, USA). The
nitrocellulose membranes were dried at 37 °C for 8 h.
Following this, we attached adsorbed pads AP045 to plastic
supports with the nitrocellulose membranes. After assembling
the membranes, we used an Index Cutter-1 (Gibbstown, NJ,
USA) to cut multimembrane composites into 3.5 mm-wide
strips and used an FR-900 continuous band sealer (Wenzhou
Dingli Packing Machinery, China) to seal the strips
hermetically into bags composed of laminated aluminum foil
with silica gel as a desiccant. We carried out the cutting and
packing at 20–22 °C in a separate room with a relative humidity
of no more than 30%.
Lateral flow assay and data processing
The synthesized streptavidin–GNP conjugates were adjusted to
the same optical density at λmax using 10 mM Tris-HCl buffer,
pH 8.5. Then, PBS with 0.05% Triton X-100 (PBST) was
added to final optical density (λmax) of conjugates
corresponding 0.2, 0.07, 0.02, and 0.004. The test strips were
dipped into the obtained conjugate solutions. Ten minutes after
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© Research India Publications. http://www.ripublication.com
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beginning the assay, the result was visually checked, a digital
image of the test strips was obtained with a CanoScan LiDE 90
scanner, and the integrated intensities of the color in the binding
zones were calculated as described previously by Safenkova et
al. [34].
RESULTS AND DISCUSSION
Characterization of gold nanoparticles
GNPs synthesized by reduction with sodium citrate/tannic acid
(SC–TA) were of an average diameter of 4.4 ± 0.6 nm (n =
133), and the form factor (ratio of maximum and minimum
axes) was 1.1 ± 0.05 (Figure 1-A) by TEM. GNPs synthesized
by reduction with sodium borohydride (BH) were of an average
diameter of 6.9 ± 1.5 nm (n = 205), and the form factor was 1.1
± 0.1 (Figure 1-B) by TEM. Thus, the obtained sizes were
close, taking into account standard deviations. The TEM
provided data regarding the true dimensions of GNP cores,
facilitating their characterization. However, differences
between the two methods of synthesis were determined, among
other things, by the surface features of the nanoparticles, which
are better characterized by the DLS.
The colloidal state and the hydrodynamic size of GNPs in the
case of all samples were measured and calculated using DLS
(Figure 2). According to DLS, the synthesized GNPs were
monodispersed and their average diameter was 8.8 ± 1.8 and
5.7 ± 1.1 nm. Because the hydrated shell around the GNP
contributed to the data of DLS measurements, the average
hydrodynamic diameter of GNPs SC–TA was larger than GNPs
BH. For both GNPs, polydispersities were closer: a PdI of
0.248 for GNPs SC–TA and 0.217 for GNPs BH. Here and
further to evaluate the PdI values, we propose using the
manufacturer's recommendation [35], which indicates a PdI
smaller than 0.05 (rarely seen) for highly monodisperse
standards, and a PdI greater than 0.7 for samples with a very
broad size distribution.
To further assess the amounts of GNPs and their conjugates and
to evaluate the changes occurring during the synthesis of
conjugates, GNP solutions were characterized with respect to
their absorbance by UV–VIS spectrophotometry. UV–Vis
spectroscopy is an simple method for detecting GNPs because
GNPs exhibit a characteristic absorption peak at about 520 nm,
which is attributed to surface plasmon excitation due to the
combined vibration of free electrons in resonance with the light
wave [36]. The absorption band maximum for GNPs SC–TA
and GNPs BH was observed at λ = 521 nm and λ = 507 nm,
respectively. Figure 3 shows the UV–Vis absorption spectra of
GNPs synthesized using a mixture of both SC–TA and BH.
A summary of GNP characterizations is shown in Table 1. As
can be seen, GNPs of homogenous shape and size were
obtained in syntheses carried out using both reducing agents.
Figure 1: TEM images and size distribution histograms of GNPs synthesized using a mixture of sodium citrate and tannic acid
(A) and sodium borohydride (B).
International Journal of Applied Engineering Research ISSN 0973-4562 Volume 12, Number 23 (2017) pp. 13847-13860
© Research India Publications. http://www.ripublication.com
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Figure 2: DLS size distribution histograms of GNPs synthesized using a mixture of sodium citrate
and tannic acid (A) and sodium borohydride (B).
Figure 3: UV–Vis spectra of GNPs. Curves 1 and 2 represent the spectra of GNPs synthesized using a mixture
of sodium citrate and tannic acid, and sodium borohydride, respectively.
International Journal of Applied Engineering Research ISSN 0973-4562 Volume 12, Number 23 (2017) pp. 13847-13860
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Table 1: The overall results for the syntheses of GNPs using a combination of sodium citrate/tannic acid, and sodium
borohydride.
Reagent TEM diameter, nm Form factor DLS diameter, nm PdI λmax, nm
Sodium citrate / tannic acid mixture 4.4 ± 0.6 1.1 ± 0.05 8.8 ± 1.8 0.248 521
Sodium borohydride 6.9 ± 1.5 1.1 ± 0.10 5.7 ± 1.1 0.217 507
Choice of conditions for conjugate synthesis
The main factors for effective conjugation by nondirectional
and noncovalent immobilization by physical adsorption are the
conjugation pH and concentration of biomolecules
(streptavidin). Native streptavidin from Streptomyces avidinii used for conjugates' syntheses has no carbohydrate and has an
acidic isoelectric point [24]. The exact value of pI was found to
be equal to 5.2 by ELS measuring zeta potentials of the
streptavidin at different pH. Due to the symmetric tetrameric
structure of the streptavidin [24], the orientation of streptavidin
upon immobilization is minimally responsible for the
efficiency of the biotin-binding. These circumstances
determine the choice of the pH range. The classical
recommendation regarding the functionalization of GNPs
argues that the pH of the medium should be at least 0.5 units
higher than the pI of the immobilized protein [37], at which
point the transition of the protein from the dissolved to the
aggregated state is energetically favorable. Therefore, the
conjugation was performed at three pH values (6.5, 7.4, and
9.0).
The choice of streptavidin concentration was associated with
full coverage of the GNP surface. For the starting point, we
used a typical flocculation method determining the minimal
stabilizing concentration of streptavidin (see Materials and
methods, section 2.4). This means that the addition of 10%
NaCl solution results in changes in the electric double layer of
GNP and a shift in the equilibrium between the electrostatic
repulsion and attraction of particles [38] and, correspondingly,
the flocculation of the GNPs and the change in the solution
color. The aggregation of GNPs in the presence of excess salt
(10% NaCl) will stop if the GNP surface is covered with
streptavidin in large part. An absorption change at the
wavelength of 580 nm (Figure 4) indirectly indicates the
presence of aggregates in the preparation of GNPs. However,
the typical flocculation view was observed only for GNPs BH
(Figure 4-B). For GNPs SC–TA, no change in color or
corresponding change of OD580 were observed (Figure 4-A).
The reason is probably associated with good stabilization of the
GNPs’ surface by the complex of oxidized products of SC–TA
[17]. Obviously, the flocculation method is not very
informative for GNPs synthesized with SC–TA.
Figure 4: Flocculation curves of streptavidin’s immobilization
on the GNP’s surface. Dependencies of the absorbance at 580
nm in the presence of excess salt (10% NaCl) for GNPs
synthesized using a mixture of sodium citrate and tannic acid
(A), and sodium borohydride (B) after the addition of different
concentrations of the streptavidin. Curves 1, 2, and 3 represent
the plots for the used pH 6.5 (A), 7.4 (B), and 9.0 (C),
respectively.
The range of concentration dependence, starting from a
concentration of 15 μg/mL (point on plateau curve) for GNPs
BH at pH 9.0 (see Figure 4-B), corresponds to the stabilization
of the GNP by streptavidin. With the addition of excess salt,
conjugates synthesized at lower streptavidin concentrations
International Journal of Applied Engineering Research ISSN 0973-4562 Volume 12, Number 23 (2017) pp. 13847-13860
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were not stable. At the same time, the outlet to the plateau did
not occur at pH 6.5 and 7.4 (see Figure 4-B). Taking into
account the diameter of streptavidin (5 nm [39, 40]) and the
average size of the nanoparticles determined by TEM (see
section 3.1), approximately two to three molecules of
streptavidin can be placed on one nanoparticle. This means that
at 100% streptavidin binding to the GNP surface, all GNPs in
the solution would be streptavidin covered even at 15 μg/mL.
However, the percentage of streptavidin bonded to the surface
of GNP depends on the equilibrium constants and curves in
Figure 4-B, confirming the pH effect on the immobilization.
For this reason, we synthesized conjugates with GNP BH at pH
6.5, 7.4, and 9.0 using streptavidin with concentrations more
than that of the flocculation point (20, 40, 60, and 80 μg/mL).
For GNP SC–TA, the same synthesis conditions were chosen.
Characterization of streptavidin–gold nanoparticle
conjugates
Each of the 24 conjugates (12 GNP SC–TA and 12 GNP BH)
were characterized before ultracentrifugation by dynamic light
scattering. A summary of the data is shown in Table 2. For GNP
SC–TA, precipitate formation and high PdI were observed for
all streptavidin concentrations at pH 6.5 (see Table 2). At pH
7.4 these effects were less pronounced, and at high
concentrations of streptavidin, the precipitate did not fall out,
and the agglomerates formed became smaller. At pH 9.0, large
aggregates were absent, and with an increase in streptavidin
concentration from 20 to 80 μg/mL, a small decrease in
hydrodynamic diameters from 25 (98.3%) to 15.6 nm (99.1%)
occurred. The same effect was observed for GNP BH (see
Table 2). Some difference in the synthesis of conjugates
appeared at pH 7.4, at which conjugates with GNP BH had a
lower polydispersity. The resulting distributions of the
hydrodynamic diameters for streptavidin–GNP SC–TA (Figure
5-A, B, C) and streptavidin–GNP BH (Figure 6-A, B, C) well
confirm the values presented in Table 2. The formation of
aggregates under different conditions was visualized using
TEM. Figure 7-A, B and C present the typical images of
conjugate aggregates with different sizes, and Figure 7-D
presents the streptavidin–GNP conjugate of the monodisperse
state according DLS.
Table 2: Properties of the streptavidin–GNP conjugates obtained under different conditions.
Conditions for conjugate
synthesis
Streptavidin
concentration,
µg/mL
DLS diameter (%
volume), nm
PdI Precipitate
Sodium citrate /
tannic acid mixture
pH 6.5 20 1570 (100%) 0.316 yes
40 1990 (37.4%)
1020 (45.4%)
257 (1%)
0.507 yes
60 1727 (26.3%) 264.9 (31%)
48 (25.4%)
0.422 yes
80 44.5 (77.3%)
131.8 (22.7%)
0.269 no
pH 7.4 20 283.9 (100%) 0.254 yes
40 143.5 (63.8%)
36.8 (36.2%)
0.197 no
60 30.6 (82%)
13 (13.5%)
0.268 no
80 28.9 (93.1%) 0.296 no
pH 9.0 20 25 (98.3%) 0.207 no
40 17.8 (87.8%)
8.3 (10.6%)
0.306 no
60 16.5 (98%) 0.308 no
80 15.6 (99.1%) 0.304 no
Sodium borohydride pH 6.5 20 39 (87.7%) 0.242 no
40 1305 (100%) 0.442 yes
60 1001 (72.6%) 0.437 yes
80 26.2 (98.5%) 0.263 no
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pH 7.4 20 23.2 (82.4%)
9.7 (17.6%)
0.276 no
40 57.7 (93.2%) 0.266 no
60 46.5 (94%) 0.246 no
80 26.2 (98.5%) 0.263 no
pH 9.0 20 8.3 (28%)
16.4 (70.7%)
0.366 no
40 21.2 (96.8%) 0.285 no
60 18.3 (97.4%) 0.34 no
80 16.9 (99.1%) 0.303 no
Figure 5: DLS data of the distribution of hydrodynamic diameters of streptavidin–GNP conjugates
(with GNP SC–TA) before ultracentrifugation at pH 6.5 (A), 7.4 (B), and 9.0 (C) and after ultracentrifugation at pH 9.0 (D).
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Figure 6. DLS data of the distribution of hydrodynamic diameters of streptavidin–GNP conjugates (with GNP BH) before
ultracentrifugation at pH 6.5 (A), 7.4 (B), and 9.0 (C) and after ultracentrifugation at pH 7.4 (D), 9.0 (E).
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For ultracentrifugation, all conjugates of streptavidin–GNP
SC–TA at pH 9.0 and all streptavidin–GNP BH conjugates at
pH 7.4 and 9.0 were chosen to separate unbound streptavidin
molecules. After ultracentrifugation, the distribution of the
hydrodynamic diameters of streptavidin–GNP conjugates
showed more polydispersity for pH 7.4 (see Figure 6-D).
Therefore, the conjugates that were most monodisperse, stable,
and without aggregate formation were obtained at pH 9.0 for
both of the GNP preparations. Conjugates with streptavidin
concentrations of 40 (GNP SC–TA) and 80 μg/mL (GNP BH)
have the narrowest distributions (see Figures 5-D and 6-E,
respectively).
Biotin binding properties of streptavidin–gold nanoparticle
conjugates in lateral flow assay
The conjugates (pH 9.0) synthesized at different streptavidin
concentrations were compared according to biotin binding
properties in LFA. In LFA with GNP label, the detectable
signal is the color in the binding zone; it is affected by the
affinity of the conjugate and the optical properties of the
conjugate with GNP. To correctly compare conjugate binding
properties, optical properties were studied with the absorption
spectra of the conjugates (Figure 8). As can be seen in Figure
8, the maxima of the absorption bands were close between
different conjugates and shifted to longer wavelengths: λ =
519–523 nm for GNP SC–TA, 513–516 nm for GNP BH.
However, the peaks of the conjugates differed. Using the ICP-
MS (see Materials and methods, section 2.7), we confirmed that
the optical densities of the conjugates correlate with the Au
concentration (Table 3), which means that differences in peaks
are most likely associated with losses of the conjugate during
the ultracentrifugation.
A B
C D
Figure 7: TEM images of streptavidin–GNP conjugates before ultracentrifugation: (A) GNP SC–TA and 60 µg/mL of
streptavidin at pH 6.5; (B) GNP BH and 60 µg/mL of streptavidin at pH 6.5; (C) GNP SC–TA and 80 µg/mL of streptavidin at pH
7.4; and (C) GNP SC–TA and 80 µg/mL of streptavidin at pH 9.0.
International Journal of Applied Engineering Research ISSN 0973-4562 Volume 12, Number 23 (2017) pp. 13847-13860
© Research India Publications. http://www.ripublication.com
13857
Table 3: Primary data of ICP-MS measurements carried out with a quadrupole ICP-MS instrument
Aurora M90 (Bruker Corp., USA).
Parameters
OD (λmax) Au concentration,
µg/mL
GNP concentration, nM
GNPs with sodium citrate / tannic acid mixture 0.83 14.2 28
GNPs sodium borohydride 0.67 11.5 6
Streptavidin–GNP conjugate at pH 9.0 Parameters
Reducing agent Streptavidin
concentration, µg/mL
OD (λmax) Au concentration,
µg/mL
GNP concentration, nM
Sodium citrate /
tannic acid mixture
20 0.65 8.8 17
40 0.53 8.2 16
60 0.63 9.0 18
80 0.50 7.9 15
Sodium borohydride 20 0.44 7 3
40 0.35 6.5 3
60 0.35 5.9 3
80 0.49 7.5 4
Figure 8. Absorption spectra of streptavidin–GNP conjugates after ultracentrifugation at pH 9.0.
All conjugates showed staining in the binding zones and high
binding properties up to 0.004 optical densities at λmax (Figure
9). Differences in the color intensity in the binding zones were
very weak at high conjugate concentrations and slightly more
intense for 40 and 60 µg/mL of streptavidin at low conjugate
concentrations (0.02, 0.004 of optical density) for GNP SC–TA
(see Figure 9-F, E). However, at large concentrations (0.2, 0.07
of optical density), the binding zones at 20 and 80 µg/mL of
streptavidin were blurred and wide for both kinds of GNP (see
Figure 9-A, B). Therefore, the most effective streptavidin–GNP
conjugates for LFA with indirect labeling were synthesized
using pH 9.0 and 40–60 µg/mL of streptavidin.
International Journal of Applied Engineering Research ISSN 0973-4562 Volume 12, Number 23 (2017) pp. 13847-13860
© Research India Publications. http://www.ripublication.com
13858
A B
C D
Figure 9. Lateral flow assay. A, B, C, D: Test strips after analysis with conjugates at different optical densities (0.2, 0.07, 0.02,
0.004, respectively). For test strips (1) GNP SC–TA and 20 µg/mL of streptavidin; (2) GNP BH, 20 µg/mL; (3) GNP SC–TA, 40
µg/mL; (4) GNP BH, 40 µg/mL; (5) GNP SC–TA, 60 µg/mL; (6) GNP BH, 60 µg/mL; (7) GNP SC–TA, 80 µg/mL; and (8) GNP
BH, 80 µg/mL. E, F: Dependences of the color intensity in the test zone of the test strip on the optical density of conjugates with
GNP SC–TA (E) or with GNP BH (F). For 20 (1), 40 (2), 60 (3), and 80 (4) µg/mL of streptavidin.
CONCLUSION
The results confirm that through the use of both reagents—SC–
TA and BH—is it possible to obtain monodisperse, spherical
GNPs. The optimal conditions of the conjugate synthesis did
not differ for small GNPs synthesized by different methods.
The paper shows that the flocculation method is not informative
for the choice of the synthesis conditions for GNP SC–TA. In
carrying out a flocculation experiment, the nanoparticles
obtained by reduction with BH showed stability at three
different pH (6.5, 7.4, 9.0), but the characteristics of the
conjugates by the DLS method showed significant differences.
For the synthesis of monodisperse and stable conjugates of
International Journal of Applied Engineering Research ISSN 0973-4562 Volume 12, Number 23 (2017) pp. 13847-13860
© Research India Publications. http://www.ripublication.com
13859
small GNPs (irrespective of the synthesis method) with
streptavidin (pI = 5.2), a pH 9.0 is optimal. TEM and DLS data
confirmed the presence of aggregates for both types of GNP in
the synthesis under conditions of pH 6.5 and 7.4. The binding
properties of the all streptavidin–GNP conjugates synthesized
at pH 9.0 were high. In LFA, conjugates showed binding up to
0.004 optical densities (λmax). Thus, for LFA with indirect
labeling and small GNP, we recommend synthesizing the
streptavidin–GNP conjugates using pH 9.0 and 40–60 µg/mL
of streptavidin.
ACKNOWLEDGMENTS
This work was financially supported by the Ministry of
Education and Science of the Russian Federation within the
framework of the Federal Targeted Program “Research and
Development in Priority Areas of Science and Technology
Complex of Russia for 2014–2020” (Agreement from
September 26, 2017, no. 14.607.21.0184, unique project
identifier no. RFMEFI60717X0184).
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