Synthesis of the Most Effective Streptavidin Conjugates ... · Synthesis of gold nanoparticle conjugates . Synthesis of streptavidin–GNP conjugates was carried out as described

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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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-



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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



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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



13850

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).



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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.



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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



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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.



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.



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



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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Documents

Synthesis of the Most Effective Streptavidin Conjugates ... · Synthesis of gold nanoparticle conjugates . Synthesis of streptavidin–GNP conjugates was carried out as described