A Biotin Streptavidin Module for Signal Amplification in ... · Biomedicals (USA); the STR sulfate...

International Journal of Applied Engineering Research ISSN 0973-4562 Volume 12, Number 23 (2017) pp. 13552-13564

© Research India Publications. http://www.ripublication.com

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A Biotin–Streptavidin Module for Signal Amplification in

Immunochromatographic Analysis to Control Antibiotic Levels

O.D. Hendrickson, A.E. Urusov, D.A. Kuznetsova, A.V. Zherdev and B.B. Dzantiev*

A.N. Bach Institute of Biochemistry, Federal Research Center «Fundamentals of Biotechnology» of the Russian Academy of Sciences,

Leninsky prospect 33, 119071, Moscow, Russia. * Corresponding author

Orcid: 0000-0003-3799-9383

Abstract

In this study, we proposed a new amplification scheme to

improve the sensitivity of immunochromatographic analysis

(ICA) and maintain high-intensity coloration. It was based on

the indirect introduction of a label into immune complexes

formed via a biotin–streptavidin module. The registered signal

(test zone coloration) was amplified by multiplying the label

binding. During the assay, biotinylated antibodies interacted

with colored conjugates of streptavidin and gold nanoparticles

(GNPs), and the addition of a biotinylated protein promoted

aggregation of the label. In the developed assay, the detection

limit of streptomycin (STR) was 30 ng/mL; the achieved

characteristics exceeded those of standard competitive ICA

using antibodies linked directly to a label. Because of the

universal nature of the proposed amplification scheme, it

could be applied to other compounds, including antibiotics

from different chemical classes. Such a sensitive ICA

amplification scheme could also be considered an efficient

screening tool for antibiotics in medical monitoring and food

safety.

Keywords: immunochromatography, amplification, biotin–

streptavidin module, antibiotics, nanoparticles aggregation

INTRODUCTION

Antibiotics are not only considered efficient therapeutic

preparations but also important risk factors in human health.

Resistance to antibiotics is one of the most serious health

concerns related to the uncontrolled consumption of

antibiotics. In addition, changes in intestinal microflora cause

disbacteriosis, leading to the emergence of allergic diseases,

the inhibition of vitamin synthesis, and the propagation of

pathogenic microbes in organs and tissues [1]. Thus, efficient

analytical tools to screen for antibiotic levels are in high

demand, both for making therapeutic decisions and ensuring

food safety.

Various physicochemical and biochemical approaches are

used for antibiotic detection [2-4]. Among the latter,

immunochemical methods are often used, due to their

simplicity and sensitivity, and that they can be utilized

without complex, high-cost equipment [5-7].

Immunochromatographic analysis most closely meets the

demands of rapid and accurate monitoring [8]. This assay

(also known as lateral flow immunoassay) uses the contact

between a multi-membrane test-strip with immobilized

reactants and a test sample. After this contact, the reagents

move along the test strip, resulting in specific interactions and

the formation of colored zones. Furthermore, ICA can be

applied without the use of additional equipment and reagents.

However, ICA is typically less sensitive than other

immunochemical methods [9]. Thus, new assay formats are

necessary for the ICA of compounds (including antibiotics)

that should be controlled in samples containing extremely low

antibiotics’ concentration.

The immunochromatographic assays used for low molecular

weight compounds traditionally include analyte-specific

antibodies directly linked to a colored marker, gold

nanoparticles [8]. Reducing the concentration of antibody–

GNP conjugate for the purpose of lowering the detection limit

decreases the concentration of colored markers and the

magnitude of the detected signal, thus reducing sensitivity. An

indirect introduction of the marker into detectable specific

immune complexes (so-called indirect labeling) may solve

this problem [10-12].

In this study, we proposed a new type of competitive ICA that

indirectly introduces a label via the streptavidin–biotin

module and the subsequent aggregation of GNPs. The biotin–

streptavidin system is characterized by its unique high affinity

(Kd = 10-15 М) and specificity of interaction [13]. The binding

of streptavidin–biotin modules with any label (fluorophore,

enzyme, or nanoparticle) is often used to introduce markers in

immunoassays [14, 15]. The detected signal was amplified

after multiple interactions (each streptavidin molecule binds

four molecules of biotin). Thus, applying this ICA

amplification scheme may ensure high specificity, rapid

interaction, and resistance to changes in temperature, pH, and

denaturizing agents.

In the proposed assay, conjugates of GNPs with streptavidin,

and biotinylated monoclonal antibodies and bovine serum

albumin (BSA) were used instead of the traditional conjugates

of specific antibodies with GNPs. These structures formed

large complexes in the analytic zone of the test strip:

mailto:[email protected]



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biotinylated antibodies bound to streptavidin–GNPs

conjugates and BSA–biotin aggregated several markers. Such

multiple interactions led to the appearance of a test line with

high color intensity; thereby allowing for a lower detection

limit.

This study investigated streptomycin (STR) which belongs to

the group of aminoglycosides used to treat a number of

bacterial infections in cattle, such as tuberculosis,

endocarditis, brucellosis, plague, tularemia, and fever [16-18].

However, STR is known to promote the growth of

microorganisms resistant to antibiotics [18, 19], which means

there is an urgent need to control STR. Several studies have

focused on STR detection by immunoassay techniques,

including immunochromatography [20-22]. However, they

were based on traditional ICA formats and did not

demonstrate the high sensitivity required for medical

monitoring. In contrast, the universal character of the

proposed amplification scheme allows for ICA to be applied

to other antibiotics, as well as other low molecular weight

analytes.

EXPERIMENTAL

Materials, reagents, and equipment

The BSA, 3,3’,5,5’-tetramethylbenzidine, chloroauric acid,

Triton X-100, dimethyl sulfoxide (DMSO), cyanuric chloride,

and triethylamine were from Sigma-Aldrich (USA); the

biotin-N-hydroxysuccinimide ester was from ICN

Biomedicals (USA); the STR sulfate was from AppliChem

(Germany); and the recombinant STR was from IMTEK

(Russia). The monoclonal antibodies against STR were

obtained from the All-Russian Center of Molecular

Diagnostics and Therapy (Moscow, Russia), the goat anti-

mouse (GAM) polyclonal antibodies and peroxidase-labeled

anti-mouse immunoglobulins were obtained from the

Gamaleya Institute of Microbiology and Epidemiology

(Moscow, Russia). The purity of all other reagents (salts,

acids, alkalis, and organic solvents) was of analytical grade or

higher. Milli-Q deionized water (Millipore, USA) was used to

prepare the solutions. Costar 9018 transparent microplates

(Corning, USA) were used for the enzyme-linked

immunosorbent assay (ELISA). Optical density was measured

using a microplate photometer Zenyth 3100 (Anthos Labtec

Instruments, Austria).

The preparation of the GNPs

GNPs with an average diameter of 30 nm were synthesized

according to [23]. Briefly, 0.5 mL of a 1% water solution of

HAuCl4 was added to 48.75 mL of water. The mixture was

brought to the boil, and then 0.75 mL of a 1% sodium citrate

solution was added. After boiling for 30 min, the preparation

was cooled. The obtained GNPs were stored at 4∘C.

The characterization of GNPs by transmission electron

microscopy (TEM)

The aliquot of the GNP preparation was dropped onto a

formvar film-coated grid and studied by transmission electron

microscopy (TEM) on a JEM-100C microscope (Jeol, Japan)

at an accelerating voltage of 80 kV. The obtained images were

analyzed using the ImageTool program (UTHSCSA, USA).

The selection of protein concentration for conjugation with

GNPs

For conjugation with GNPs, 100 µL of aqueous solutions of

anti-STR antibodies or streptavidin (at concentrations from 5

to 200 µg/mL) were mixed with 1 mL of the GNPs (OD520 =

1) and incubated for 10 min at room temperature while

stirring. Then, 0.1 mL of a 10% NaCl solution was added and

incubated for 10 min at room temperature while stirring.

Finally, the optical density at 580 nm was measured.

The immobilization of the anti-STR antibodies and

streptavidin on GNPs

The pH of the GNPs solution was adjusted to 8.5–9.0 with

potassium carbonate. Then, anti-STR antibodies or

straptavidin (5 and 20 μg per mL of the GNPs solution,

respectively) were diluted in 10 mM Tris buffer, pH 8.5, were

added. The resulting mixture was incubated for 45 min at

room temperature, followed by the addition of a 10% aqueous

solution of BSA (VGNPs:Vprotein = 40:1) and 15 min of vigorous

stirring. The GNPs were pelleted by centrifugation at 15 000 g

for 15 min at 4°C. The supernatant was removed, and the

residue was dissolved in 10 mM Tris buffer, pH 8.5, with 1%

BSA and 1% sucrose (TBSA) and subjected to centrifugation

in the same environment. The resulting sediment was

dissolved in TBSA containing 0.05% sodium azide and stored

at 4°C.

The synthesis of the STR–BSA conjugate

STR was coupled with BSA in 10:1 molar ratio according to

[24] with modifications. Cyanuric chloride (9 mg) was

dissolved in 1 mL of acetone, cooled to 4oC, and added

dropwise while stirring to a cold solution of STR (36 mg, 25

µmol) in 1 mL of 50 mM carbonate buffer, pH 9.6 (CB),

containing 50 µL of triethylamine. The reaction mixture was

incubated at 4oC for 1 h. The activated STR was diluted by

CB:acetone:triethylamine, 30:20:1, to obtain a 0.05 µmol

concentration of the activated STR. Then, 1 mL of the

resulting solution was added dropwise with vigorous stirring

to a solution of BSA (34 mg, 0.5 µmol) in 4 mL of CB and

incubated for 1 h at room temperature. After that, the reaction

mixture was dialyzed against 50 mM potassium phosphate



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buffer, pH 7.4, containing 100 mM NaCl (phosphate-buffered

saline, PBS). The conjugates were stored at -20oC.

The biotinylation of the anti-STR antibodies and BSA

Both monoclonal antibodies to STR and BSA were

biotinylated according to a previously described method [25].

A solution of activated biotin ester in DMSO was added to a

solution of protein in PBS, to achieve a protein:biotin molar

ratio of 1:20 or 1:8 for BSA and antibodies, respectively. The

reaction was carried out for 2 h at room temperature while

stirring. Then, the reaction mixture was dialyzed against PBS

for 16 h at 4°C. After dialysis, the conjugates were separated

by centrifugation for 15 min at 14 000 g in centrifuge tubes

(Corning, USA) with cellulose acetate filters (0.45 μm pore

diameter). The biotinylated antibodies and BSA were stored at

4°C.

The production of the immunochromatographic assays

MdiEasypack membrane kits (Advanced Microdevices, India)

were used to manufacture the immunochromatographic test

kits. They included a plastic support, a working nitrocellulose

membrane CNPC with a 15 µm pore size, a GFB-R4

separation membrane, and an AP045 adsorption membrane.

The reagents were immobilized on the membranes using an

IsoFlow automated dispenser (Imagene Technology, USA) at

a rate of 0.1 µL per mm. After dispensing, the membranes

were dried at room temperature for at least 20 h.

The working and terminal absorbent pads were fixed on a

plastic pad. After the assembly of the membrane components,

the obtained sheets were cut with an Index Cutter-1 (A-Point

Technologies, USA) into test strips of 3.5 mm in width and

stored at 20–22°C in a sealed package containing silica gel.

Both for the standard and amplification ICA schemes, the

analytical zone of the test strips was formed by the STR–BSA

conjugate (1 mg/mL in PBS). To form the control zone, GAM

antibodies were immobilized on the working membrane at a

concentration of 0.25 µg/mL.

The microplate ELISA

The STR–BSA conjugate (100 μL, 1 μg/mL) in PBS was

adsorbed in microplate wells at 4°С overnight. Then, the

microplate was washed four times with PBS containing 0.05%

Triton Х-100 (PBST). After that, 50 μL of STR solution in

PBST was added to the wells (at concentrations from 3 pg/mL

to 2 µg/mL), followed by the addition of 50 μL of specific

antibodies (500 ng/mL in PBST) to each well. The mixtures

were incubated for 1 h at 37°С. After washing the microplate

with PBST, 100 μL of peroxidase-labeled anti-mouse

immunoglobulins (1:3 000 dilution in PBST) was added to the

wells, and the plate was incubated for 1 h at 37°С. The

microplate was washed (three times with PBST and once with

distilled water). To measure the activity of the formed

immune complexes, 100 μL of a 0.4 mM solution of 3,3’,5,5’-

tetramethylbenzidine in 40 mM Na-citrate buffer, рН 4.0,

containing 3 mM of Н2О2 was added to the wells and

incubated for 15 min at room temperature. The reaction was

stopped by the addition of 50 μL of 1 М H2SO4, and the

optical density was recorded at 450 nm.

The standard ICA scheme

For the standard ICA scheme, 50 μL of a STR dilution (at

concentrations from 0.01 ng/mL to 10 µg/mL) and 50 μL of

specific antibodies at concentrations of 400 ng/mL were

mixed, and the test strips were immersed in these solutions.

After 20 min of incubation, the strips were scanned for

detection. For statistical processing, all the measurements

were performed in triplicate.

The ICA amplification scheme

For the ICA amplification scheme, 100 μL of a STR dilution

(at concentrations from 0.01 ng/mL to 10 µg/mL in PBST or

spiked milk samples), 50 μL of biotinylated antibodies at a

concentration of 100 ng/mL, 25 μL of biotinylated BSA at a

concentration of 12.5 ng/mL, and 25 μL of streptavidin–GNPs

conjugate at a dilution corresponding to OD520 = 0.03 were

mixed. The test strips were immersed in these mixtures and

incubated for 20 min. Then, detection was performed as

described above. For statistical processing, all the

measurements were performed in triplicate.

In the preliminary experiments, the concentrations of the

reagents varied from 3–400 ng/mL for biotinylated antibodies

and 0.1–800 ng/mL for biotinylated BSA. The dilutions of

streptavidin–GNPs conjugate varied to obtain OD520 = 0.004–

2.

The registration of the ICA data

The binding in the test zones was recorded with the use of a

CanoScan LiDE 90 scanner (Canon, Japan), with a resolution

of 600 dpi without correcting for contrast or color, followed

by digital processing of the images with TotalLab software

(Nonlinear Dynamics, UK). This program was used to

determine the spot boundaries, add the intensities of all pixels

belonging to a particular unit, and normalize the sums to the

spot surface area, thereby representing color intensity in

relative units.

Processing the ELISA and ICA data

The plots of optical density or color intensity (y) versus the

analyte concentrations in the samples (x) were fitted to a four-

parameter logistic function using Origin 7.5 software

(OriginLab, USA):



13555

p

x

x

AAAy

0

212

1

,

where A1 was the maximum signal value, A2 was the

minimum signal value, p was the slope of the calibration

curve, and x0 was the antigen concentration causing 50%

inhibition of the STR–BSA binding (IC50).

The antigen concentration resulting in 10% inhibition (IC10)

was calculated from this fitting and then regarded as the

instrumental detection limit of the assay [26].

The visual limit of detection in the ICA was interpreted as the

minimal analyte concentration that caused the complete

absence of coloration in the test zone.

The preparation of the spiked milk samples

The milk samples were purchased from the local store. A

standard solution of STR (1 mg/mL) was added to undiluted

and 5- and 10-fold diluted milk samples to obtain a row of

STR dilutions in the range of 0.01 ng/mL – 10 µg/mL. The

obtained spiked samples were thoroughly mixed.

RESULTS AND DISCUSSION

The immunochromatographic detection of low molecular

weight compounds is usually based on competitive

interactions. In the standard scheme, the antigens in the

sample and that immobilized in the working membrane of the

test strip competitively interact with specific antibodies

labeled with colored markers, GNPs. The lower the analyte

content in the sample, the more labeled antibodies bind with

antigens that are immobilized in the analytic zone, increasing

the color intensity of the test line. Reducing the concentration

of specific antibodies is among the different approaches for

decreasing the detection limit of ICA. In such cases, even

small amounts of antigens in the test samples prevent the

binding of immunoglobulins in the test zones. However, the

decrease of antibody–marker conjugates’ concentration also

reduced the color intensity of the test line, which decreased

the signal intensity and, thus, assay reliability.

To solve this problem, we proposed a new format of

competitive ICA based on the indirect introduction of a label

via the streptavidin–biotin module. Signal amplification was

achieved via binding multiplication and the aggregation of

GNPs.

The synthesis and characterization of the immunoreagents

To develop the amplification scheme, we first obtained and

characterized its components. GNPs were synthesized

according to the standard protocol by the reduction of HAuO4

with sodium citrate [23]. We used TEM to determine the

dimensional characteristics and aggregation state of

nanoparticles, which indicated that the obtained sol consisted

of homogeneous non-aggregated individual particles ca. 30

nm in diameter and spherical in shape (Fig. 1).

Figure 1: A microphotograph of GNPs obtained by TEM.



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The obtained GNPs were conjugated with anti-STR

monoclonal antibodies and streptavidin, as recommended in

[24]. Preliminary experiments were performed to determine

the optimal loading of the immobilized proteins on GNP

surfaces. The colloidal aggregation test allowed us to study

GNP stabilization according to the concentration of protein

absorbed upon the addition of salt. As a result of this test, we

obtained flocculation curves (the plots of the optical density at

580 nm versus protein concentrations; data not shown).

According to these dependencies, we selected the protein

concentrations that best stabilized the conjugates. Therefore,

for further conjugations, anti-STR antibodies and streptavidin

were taken at 5 and 20 µg per mL of GNPs, respectively. The

functional properties of specific antibodies conjugated to

GNPs were confirmed by binding with STR–BSA

immobilized on the membrane of a test strip.

The competitive ELISA of STR

The immunoreagents were characterized by ELISA. Under the

selected ELISA regime, namely, the STR–BSA conjugate

immobilized at a concentration of 1 μg/mL and specific

antibodies at a concentration of 500 ng/mL, STR was

determined with a detection limit of 20 ng/mL (Fig. 2).

The standard ICA scheme

During the standard ICA, the test strip was immersed in a

solution containing an analyte and conjugates of anti-STR

antibodies with GNPs. STR in the sample reacted with the

active sites of the antibodies, thereby preventing them from

interacting with the STR–BSA adsorbed in the test zone. The

excess conjugate bound anti-species antibodies in the control

zone (Fig. 3A). Therefore, we observed an inverse

dependence of color intensity in the test zone and STR

concentration in the sample.

1 10 100 1000

0,10

0,15

0,20

0,25

0,30

0,35

OD

45

0

[STR], ng/mL

Figure 2: The competitive curve for ELISA of STR.



13557

A

B

Figure 3: The schemes for standard ICA (A) and ICA with amplification (B).

The calibration curve for the standard ICA of STR and the

coloration of their respective test strips is shown in Fig. 4. The

visual limit of STR detection was 3.3 µg/mL and IC10 was 100

ng/mL. Although the sensitivity of the assay satisfied the

maximum permitted levels of STR in food (200 ng/g

according to Commission Regulation [EC] No. 1881/2016),

improving its analytical characteristics seems to be quite

important, taking into account the possible pre-treatment of

the detected probes.



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А

0,01 0,1 1 10 100 1000 10000

0

5

10

15

20

25

30

35

40

45

50

55

L

ine i

nte

ncit

y. a

rb

. u

nit

s

[STR], ng/mL

В

10000 3333 1111 370.4 123.5 41.2 13.7 0.01

ng/mL

Figure 4: The calibration curve of STR for standard ICA (A) and the appearance of the test strips (B).

The ICA amplification scheme

In this study, we proposed an ICA amplification scheme to

solve the problem of the contradiction between high assay

sensitivity and low signal intensity in the standard ICA

scheme. We suggested an indirect introduction of the label to

simultaneously increase the amount of marker and reduce the

concentration of specific antibodies. A variation of this

scheme was developed previously using anti-species

antibodies conjugated with GNPs [10, 11]. In the current

study, the indirect introduction of the label was performed by

the streptavidin–biotin module: streptavidin was conjugated

with GNPs, and biotin was conjugated with antibodies.

Furthermore, biotinylated BSA was added to the system to

ensure the binding of the GNPs conjugate with streptavidin,

thus creating an aggregation processes in the analytic zone

and changing the antibody:marker ratio. The scheme of this

format is demonstrated in Fig. 3B.

The conditions for assembling specific complexes in the ICA

amplification scheme were optimized at zero antigen

concentration. The optimization of the streptavidin–GNPs

concentration was carried out by varying its dilution in the

range corresponding to OD520 = 0.002–2. In this case, only

biotinylated antibodies at a concentration of 200 ng/mL were

used. The obtained concentration dependence (Fig. 5)



13559

demonstrated that the maximal signal intensity was observed

at the conjugate concentration corresponding to OD520 = 0.02.

Therefore, a dilution of the streptavidin–GNPs conjugate to

OD520 = 0.03 seemed to be optimal and was used for further

optimization experiments.

1E-4 1E-3 0,01

0

5

10

15

20

25

30

35

40

45

50L

ine i

nte

nsi

ty,

arb

. u

nit

s

OD 520 of streptavidin-GNPs

Figure 5: The dependence of the test zone coloration on the dilution of streptavidin–GNP conjugate.

The concentration of biotinylated antibodies was also

optimized. For this purpose, assays were carried out with

different amounts of antibodies (3–400 ng/mL) and fixed

concentrations of streptavidin–GNPs and BSA–biotin

conjugates (corresponding to OD520 = 0.03 and 60 ng/mL,

respectively). The dependence of the test zone coloration on

the concentration of biotinylated antibodies is presented in

Fig. 6. Because the signals were of the highest intensity at a

concentration of 200 ng/mL, this value was selected.

10 100

0

5

10

15

20

25

30

35

40

45

50

Lin

e i

nte

nsi

ty. a

rb

. u

nit

s

[Biotinylated antibodies], ng/mL

Figure 6: The dependence of the test zone coloration on the concentration of biotinylated antibodies.



13560

The optimal concentration of biotinylated BSA in the reaction

media was found to be ca. 8–30 ng/mL to ensure high

analytical signals. Adding more biotinylated BSA resulted in a

sharp decrease in the test zone coloration (Fig. 7).

0,1 1 10 100

0

5

10

15

20

25

30

35

40

45

50

Lin

e i

nte

nsi

ty, a

rb

. u

nit

s

[Biotinylated BSA], ng/mL

Figure 7: The dependence of the test zone coloration on the concentration of biotinylated BSA.

After preliminary determining the reagents’ concentrations,

we carried out a two-factor optimization of biotinylated BSA

and antibody concentrations. For a fixed amount of a GNPs

conjugate (OD520 = 0.03), the BSA–biotin and antibody–biotin

concentrations were varied in wider ranges (0.1–800 and 25–

400 ng/mL, respectively). Satisfactory color intensity was

achieved when using BSA–biotin and antibody–biotin at

concentrations of 12.5 and 100 ng/mL, respectively. Even

though the latter was lower than what we determined in the

preliminary optimization experiment (200 ng/mL), thus

slightly lowering the intensity of the band color, this

concentration allowed us using half as much reagent.

During final step of the optimization, the streptavidin–GNPs

conjugate was applied in different dilutions (corresponding to

OD520 = 2–0.004) at the aforementioned concentrations of all

other components. The dependence is shown in Fig. 8.

0,01 0,1 1

0

10

20

30

40

50

60

70

Lin

e in

ten

sity

, a

rb.

un

its

OD 520 of streptavidin-GNPs

Figure 8: The dependence of the test zone coloration on the dilution of the streptavidin–GNPs conjugate.



13561

Fig. 8 shows that the dependence of test zone coloration on

the concentration has a bell-shaped shape; the earlier selected

value (OD520 = 0.03) lay in the high-signal range. This low

concentration significantly reduced the consumption of

colloidal gold.

Finally, the optimum combination of test zone coloration and

the assay sensitivity was achieved using biotinylated

antibodies and BSA at concentrations of 100 and 12.5 ng/mL,

respectively, and streptavidin–GNPs conjugate at the dilution

corresponding to OD520 = 0.03.

Fig. 9 demonstrates the appearance of the test strips and the

STR calibration curve. The visual detection limit was 370

ng/mL, and IC10 was 30 ng/mL. The estimated visual

detection limit and IC10 values were 10-fold and 3-fold higher,

respectively, than those obtained as a result of the standard

ICA scheme.

A

0,01 0,1 1 10 100 1000 10000

0

5

10

15

20

25

30

35

40

45

50

Lin

e i

nte

nsi

ty, a

rb

.un

its

[STR], ng/mL

B

10000 3333 1111 370.4 123.5 41.2 13.7 0.01

ng/mL

Figure 9: The calibration curve of STR for ICA in the amplification scheme (A) and the appearance of the test strips (B).

Testing the ICA amplification scheme on food samples

The developed amplification scheme was tested for STR

detection in milk samples. In the first case, the milk samples

were tested directly, without sample preparation or dilution. In

the second case, the milk samples were diluted 5- and 10-fold

by PBST, spiked with various concentrations of STR, and

then used in the ICA amplification scheme. The appearance of



13562

the test strips after the assays is shown in Fig. 10. For the

undiluted samples, the liquid did not reach the absorption

membrane and no bands were formed on the working

membrane. For the diluted samples, the membranes looked

similar to those observed for STR detection in the buffer.

10000

3333

1111

370

124

41,2

13,7

0.0

1

10000

3333

1111

370

124

41.2

13.7

0.0

1

10000

3333

1111

370

123

41.2

13.7

0.0

1

ng/mL

Undiluted milk 5-fold diluted milk 10-fold diluted milk

Figure 10: The appearance of the test strips after the detection of STR in milk samples in the ICA amplification scheme.

Fig. 11 shows the competitive curves of STR in 5- and 10-fold

diluted milk samples. The detection limits for STR detection

did not differ reliably from those obtained as a result of the

assay in PBST (IC10 = 30 ng/mL). The biological matrix of

the probe did not influence the immune interactions, showing

no significant shift in the amplitude of the analytical signals.

Therefore, a pre-treatment of the tested samples, which

requires no organic solvents or complex manipulations, can

precede the ICA amplification scheme. Taking into account

the maximum permitted levels of STR in food (200 ng/g), the

ICA amplification scheme is recommended as a sensitive and

rapid method to screen for STR contamination.

0,01 0,1 1 10 100 1000 10000

0

10

20

30

40

50

60

b

Lin

e i

nte

nsi

ty, a

rb

. u

nit

s

[STR], ng/mL

a

Figure 11: The calibration curves of STR for the ICA amplification scheme in 5-fold (a) and 10-fold (b) diluted milk.



13563

CONCLUSION

The results confirmed the efficiency of the proposed ICA

amplification scheme to reach a lower detection limit. It

showed a 10-fold increase in sensitivity compared to the

standard ICA scheme. Furthermore, the universal nature of the

proposed amplification scheme could be applied to the

immunodetection of antibiotics from other classes (i.e.,

tetracyclines and beta-lactams) with similar effects on signal

enhancement, shifting the calibration curves for lower analyte

concentrations and, thus, providing efficient tools for rapid

screening in food safety and medical monitoring.

ACKNOWLEDGMENTS

The authors are grateful to Prof. P.G. Sveshnikov (All-

Russian Center of Molecular Diagnostics and Therapy,

Moscow, Russia) for providing monoclonal antibodies against

STR, and S.M. Pridvorova (Federal Research Center

«Fundamentals of Biotechnology», Moscow, Russia) for the

TEM studies.

This study was financially supported by the Ministry of

Science and Education of the Russian Federation (grant

agreement No. 14.613.21.0061 on 17.07.2017, unique

identifier RFMEFI61317X0061).

LIST OF SYMBOLS AND ABBREVIATIONS

BSA – bovine serum albumin

CB – carbonate buffer

DMSO – dimethyl sulfoxide

ELISA – enzyme-linked immunosorbent assay

GAM – goat anti-mouse

GNPs – gold nanoparticles

ICA – immunochromatographic analysis

PBS – phosphate-buffered saline

PBST – PBS containing 0.05% Triton X-100

STR – streptomycin

TBSA – Tris buffer containing BSA and sucrose

TEM – transmission electron microscopy

°C

h

IC10

IC50

min

mL

mol

μg

µg/mL

µL

ng/g

ng/mL

nm

pg/mL

REFERENCES

[1] Mikkelsen, K.H., Allin, K.H., and Knop, F.K., 2016,

“Effect of antibiotics on gut microbiota, glucose

metabolism and body weight regulation: a review of

the literature”, Diabetes Obes. Metab., 18(5): pp.

444-453.

[2] Chen, T., Cheng, G., Ahmed, S., Wang, Y., Wang,

X., Hao, H., and Yuan, Z., 2017, “New

methodologies in screening of antibiotic residues in

animal-derived foods: biosensors”, Talanta, 175, pp.

435-442.

[3] Greno, M., Castro-Puyana, M., Garcia, M. A.,

Marina, M. L., 2017, “Analysis of antibiotics by CE

and CEC and their use as chiral selectors: an update”.

Electrophoresis. DOI: 10.1002/elps.201700306.

[4] Gaudin, V., 2017, “Advances in biosensor

development for the screening of antibiotic residues

in food products of animal origin - a comprehensive

review”. Biosens. Bioelectron., 90, pp. 363-377.

[5] Raeisossadati, M.J., Danesh, N.M., Borna, F.,

Gholamzad, M., Ramezani, M., Abnous, K., and

Taghdisi, S.M., 2016, “Lateral flow based

immunobiosensors for detection of food

contaminants”. Biosens Bioelectron., 86, pp. 235-

246.

[6] Hao, N., and Wang, K., 2016, “Recent development

of electrochemiluminescence sensors for food

analysis”. Anal. Bioanal. Chem., 408(25), pp. 7035-

7048.

[7] Dmitrienko, S.G., Kochuk, E.V., Apyari, V.V.,

Tolmacheva, V.V., and Zolotov, Y.A., 2014, “Recent

advances in sample preparation techniques and

methods of sulfonamides detection – a review”,

Anal. Chim. Acta, 850, pp. 6-25.

[8] Dzantiev, B.B., Byzova, N.A., Urusov, A.E., and

Zherdev, A.V., 2014, “Immunochromatographic

methods in food analysis”, TRAC – Trends in Anal.

Chem.,. 55, pp. 81-93.



13564

[9] Babington, R., Matas, S., Marco, M. P., and Galve,

R., 2012, “Current bioanalytical methods for

detection of penicillins”. Anal. Bioanal. Chem.,

403(6), pp. 1549-1566.

[10] Urusov, A.E., Petrakova, A.V., Zherdev, A.V., and

Dzantiev, B.B., 2016, "Multistage in one touch"

design with a universal labelling conjugate for high-

sensitive lateral flow immunoassays”. Biosens.

Bioelectron., 86, pp. 575-579.

[11] Urusov, A.E., Zherdev, A.V., and Dzantiev, B.B.,

2014, “Use of gold nanoparticle-labeled secondary

antibodies to improve the sensitivity of an

immunochromatographic assay for aflatoxin B1”,

Microchim. Acta, 181(15), pp. 1939-1946.

[12] Taranova N.A., Urusov A.E., Sadykhov E.G.,

Zherdev A.V., and Dzantiev B.B., 2017,

“Bifunctional gold nanoparticles as agglomeration

enhancing tool for high sensitive lateral flow test: a

case study with procalcitonin”, Microchim. Acta,

184(10), pp. 4189-4195.

[13] Green, N.M., 1975, “Avidin”, Adv. Protein Chem.,

29, pp. 85-133.

[14] Wu, J., Chen, Y., Yang, M., Wang, Y., Zhang, C.,

Sun, J., Xie, M., and Jiang, X., 2017, “Streptavidin-

biotin-peroxidase nanocomplex-amplified

microfluidics immunoassays for simultaneous

detection of inflammatory biomarkers”. Anal. Chim.

Acta, 982, pp. 138-147.

[15] You, Y., Lim, S., Hahn, J., Choi, Y.J., and

Gunasekaran, S., 2018, “Bifunctional linker-based

immunosensing for rapid and visible detection of

bacteria in real matrices”. Biosens. Bioelectron., 100,

pp. 389-395.

[16] Boisset, S., Caspar, Y., Sutera, V., and Maurin, M.,

2014, “New therapeutic approaches for treatment of

tularaemia: a review”. Front. Cell Infect. Microbiol,

4, p. 40.

[17] Riva, M.A., 2014, “From milk to rifampicin and back

again: history of failures and successes in the

treatment for tuberculosis”. J. Antibiot. (Tokyo),

67(9), pp. 661-665.

[18] Chandrika, N.T., and Garneau-Tsodikova, S., 2016,

“A review of patents (2011-2015) towards combating

resistance to and toxicity of aminoglycosides”,

MedСhemСomm., 7(1), pp. 50-68.

[19] Takahashi, Y., and Igarashi, M., “Destination of

aminoglycoside antibiotics in the 'post-antibiotic

era'”. J. Antibiot. (Tokyo), 2017. DOI:

10.1038/JA.2017.117.

[20] Song, E., Yu, M., Wang, Y., Hu, W., Cheng, D.,

Swihart, M.T., and Song, Y., 2015, “Multi-color

quantum dot-based fluorescence immunoassay array

for simultaneous visual detection of multiple

antibiotic residues in milk”, Biosens. Bioelectron.,

72, pp. 320-325.

[21] Taranova, N.A., Berlina, A.N., Zherdev, A.V.,

Dzantiev, B.B., 2015, “'Traffic light'

immunochromatographic test based on multicolor

quantum dots for the simultaneous detection of

several antibiotics in milk”, Biosens. Bioelectron.,

63, pp. 255-261.

[22] Byzova, N.A., Zvereva, E.A., Zherdev, A.V.,

Eremin, S.A., Sveshnikov, P.G., and Dzantiev, B.B.,

2011, “Pretreatment-free immunochromatographic

assay for the detection of streptomycin and its

application to the control of milk and dairy

products”. Anal. Chim. Acta, 701(2), pp. 209-217.

[23] Byzova, N.A., Zvereva, E.A., Zherdev, A.V.,

Eremin, S.A., and Dzantiev, B.B., 2010, “Rapid

pretreatment-free immunochromatographic assay of

chloramphenicol in milk”. Talanta, 81(3), pp. 843-

848.

[24] Abuknesha, R.A., and Luk, C., 2005, “Enzyme

immunoassays for the analysis of streptomycin in

milk, serum and water: development and assessment

of a polyclonal antiserum and assay procedures using

novel streptomycin derivatives”, Analyst, 130(6), pp.

964-970.

[25] Bayer, E.A., and Wilchek, M., 1990, “Protein

biotinylation”. Methods Enzymol., 184, pp. 138-160.

[26] Sittampalam, G.S., Smith, W.C., Miyakawa, T.W.,

Smith, D.R., and Mcmorris, C., 1996, “Application

of experimental design techniques to optimize a

competitive ELISA”. J. Immunol. Methods, 190(2),

pp. 151-161.

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