sensitive rapid immunochromatographic detection …sensitive rapid immunochromatographic detection...

International Journal of Applied Chemistry.

ISSN 0973-1792 Volume 12, Number 4 (2016) pp. 513-525

© Research India Publications

http://www.ripublication.com

Sensitive Rapid Immunochromatographic Detection

of the Herbicide Atrazine: Comparison of Labels and

Assay Formats

Nadezhda A. Taranova1, Anastasiya A. Semeykina1, Enrika Andzeviciute2,

Anatoly V. Zherdev1, and Boris B. Dzantiev1

1A.N. Bach Institute of Biochemistry, Research Center of Biotechnology of the

Russian Academy of Sciences, Leninsky prospect 33, 119071 Moscow, Russia. 2Department of Analytical and Environmental Chemistry, Faculty of Chemistry,

Vilnius University, Naugarduko Street 24, LT-03225 Vilnius, Lithuania.

Corresponding author: Boris B. Dzantiev

Abstract

Immunochromatographic methods for determination of the herbicide atrazine

have been comparatively characterized. The pre-incubation of the sample and

labelled antibody allowed decreasing a visual detection limit in 10 times as

compared with a standard immunochromatographic test-system based on gold

nanoparticles. By this way, up to 5 ng/mL of atrazine can be detected. Use of

silver staining cannot improve the assay. Using alternative labels (coloured latex

particles) reduced the detection limit to 2 ng/mL, the lowest value from the

compared variants.

Keywords: immunochromatography; test strips; signal enhancement;

nanoparticles; herbicide; atrazine

1. INTRODUCTION

Interest in food quality control has grown significantly in recent years. This has been

caused by an expansion of the list of controlled compounds, changes in the maximum

permissible levels for a number of contaminants, as well as by active public interest in

food safety issues and the possibilities of its rapid confirmation [1].

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514 Nadezhda A. Taranova et al.

To date, the most effective analytical approach that combines high performance and

speed is immunochromatography. In carrying out immunochromatography, the contact

of the test-strip with the sample initiates all subsequent interactions between the analyte

and immunoreactants without requiring additional reagents or operator involvement.

Due to this, the testing may be implemented directly at the sampling place for 5 to 15

minutes, and the subsequent decision may be made promptly on the basis of the assay

result [2]. These advantages have led to successful commercialization and widespread

use of immunochromatography in different fields of medical diagnostics [3-5].

In recent years, immunochromatography has also been used increasingly in the control

of food quality as a means of monitoring all stages of the processing chain "from farm

to fork". Immunochromatographic methods are applied actively for different classes of

toxic contaminants in food products [1]. The current state of these methods’

developments is summarized in a number of special reviews focused on the detection

of mycotoxins [6], phytotoxins [6, 7], infectious agents [8], food allergens [9], etc.

Herbicides are an important class of contaminant, for which rapid control tools are

required. The use of herbicides can increase crop yields and reduce labour costs for

their cultivation. However, they convey toxic effects through food to humans and

animals [10, 11]. In this connection, there is a need to monitor herbicides in soil, water,

agricultural products and foodstuffs. Modern screening methods should provide a

highly sensitive and rapid non-laboratory determination of herbicides in a variety of

complex matrices.

The herbicide atrazine was selected as an investigational antigen. It is widely used to

control weeds and can easily get into food. The negative effect of atrazine on humans

and animals is determined mainly by its influence on endocrine system [12, 13]. The

maximum residue level of atrazine in water is 10 ng/g [12]. Rapid determination of

atrazine in liquid samples (drinking water, juice, milk) is important because these

liquids have no need for complex sample preparation and results can be obtained in a

minimum amount of time after the sampling.

The aim of this work was to develop a simple, rapid, and highly sensitive

immunochromatographic analysis of atrazine. Currently a number of solutions in

immunochromatography provide increased sensitivity. Unfortunately, existing

publications are focused on assessment of individual solutions, whereas the comparison

of different variants is practically absent in the literature [14, 15]. In this regard, this

paper discusses common format of immunochromatography and three variants for

reducing the detection limit of immunochromatographic assay; these are based on the

use of:

1) The aggregation of silver ions on the surface of gold nanoparticle (GNPs) labels,

2) alternative label – latex particles (LPs), and

3) separating of the specific and detecting reactions by labelling of anti-species

antibodies with GNPs and the sample pre-incubation with native specific

antibodies.

Sensitive Rapid Immunochromatographic Detection of the Herbicide Atrazine 515

2. MATERIALS AND METHODS

2.1. Reagents and Materials

Anti-atrazine rabbit antiserum was obtained earlier at the A. N. Bach Institute of

Biochemistry (Moscow, Russia), as described in [16]. Sheep anti-rabbit

immunoglobulins were from Imtek (Moscow, Russia). Goat anti-rabbit

immunoglobulin G (GARI) were from Arista Biologicals (Allentown, USA).

Gold chloride, N-(4-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC),

N-hydroxysulfosuccinimide (NHS), N-hydroxysulfosuccinimide (sulfo-NHS) and

N,N′-dimethylformamide (DMFA) were from Fluka (St. Louis, USA). Non-ionic

detergent Tween 20 and bovine serum albumin (BSA) were from Sigma-Aldrich (St.

Louis, USA). Blue latex particles were from Magsphere (Pasadena, USA).

CNPF-backed nitrocellulose membranes and pre-treated TYPEGBF-R7L sample pads

were from Advanced Microdevices (Ambala Cantt, India). CFSP223000 adsorption

pads, and fiberglass macroporous CFCP203000 conjugate pads were from Millipore

(Billerica, USA).

Amicon Ultra 100 kDa centrifugal filter units were from Millipore (Billerica, USA).

All solutions for syntheses were prepared using purified water obtained using Milli-Q

system (Millipore, Billerica, USA).

2.2.Methods

2.2.1. Syntheses of herbicide–protein conjugates

The conjugate of atrazine and bovine serum albumin (Atr–BSA) was synthesized by

the carbodiimide method described in our earlier work [16]. The atrazine carboxylate

derivative (N-(6-(N-isopropylamino)-2-chloro-1,3,5-triazin-4-yl)-6-aminocaproic

acid) (0.05 mmol) was diluted in 0.5 ml of dimethylformamide, then 0.1 mmol N-

hydroxysuccinimide and 0.1 mmol 1-ethyl-3(3-dimethylaminopropyl)carbodiimide

were added, and the mixture was stirred for 2 h at room temperature. Then, the solution

of the activated hapten was cooled to +4°C and added to the cooled protein solution (4

mg BSA) in 0.5 ml of 0.02 M Na-carbonate buffer, pH 9.5, containing the same volume

of dimethylformamide. The resultant mixture was incubated with stirring for 1 h at

room temperature and for 16 h at +4°C. The conjugates were separated from low

molecular weight compounds by gel-filtration on Sephadex G-25 (Pharmacia, 1 × 20

cm column, in PBS) and/or by dialysis.

2.2.2. Preparation of particle–antibody conjugates

GNPs were prepared by citrate reduction of gold salt, as described in [17]. Antibodies

(GARI and anti-atrazine ones) were immobilized on GNPs by physical adsorption,

according to [18, 19]. Potassium carbonate solution (0.1 M) was added to the GNP

solution (D520 = 1.0) until it reached pH 8.5, and then the antiserum was added to a

concentration of 50 µg/mL (basing on a total content of IgG in antisera) and GARI

solution at a concentration of 8 µg/mL. The mixture was incubated for 30 min with


stirring at room temperature. Then, the BSA solution (10%) was added to a final

concentration of 0.25%.

The LP–antibody conjugate was prepared according to [20] by covalent immobilization

through carboxyl groups. The molar LP:IgG ratio was 1:90. The concentration of NHS

was 9.2 mM. The mixture was incubated for 2 h with stirring at room temperature. The

solution was blocked with 10% BSA, and then activators were removed by

centrifugation.

The diameter of GNPs, according to transmission electron microscopy, was 25±3 nm,

LPs – 340±10 nm.

2.2.3. Preparation of immunochromatographic test strips

Reagents were applied onto membranes using an IsoFlow dispenser (Imagene

Technology, Lebanon, USA). Conjugate GNP was dispensed with an optical density of

4.0 (wavelength 520 nm) and conjugate LP was dispensed with a concentration of 0.5%;

the conjugate load was 32 μL per 1 cm of strip width. The test zone was formed by the

Atrazine–BSA conjugate (2.0 mg/mL), and the control zone was formed by GARI (0.25

mg/mL in PBS). Two microliters of both reactants were applied per 1 cm of strip width.

All concentrations and dispensing modes were chosen based on the earlier data [21].

After dispensing reagents and assembling the membrane components, the lists were cut

into strips 3.5 mm wide using an Index Cutter-1 (A-Point Technologies, Gibbstown,

USA). Each test strip was 75 mm in length. Each test strip was packaged in laminated

aluminium foil using a FR-900 mini-conveyor (Wenzhou Dingli Packing Machinery,

Wenzhou, Zhejiang, China), using pre-packaged silica gel (0.5 g) as desiccant. Splitting

and packing were carried out at 20–22ºC in a special room with relative humidity under

30%.

2.2.4. Preparation of immunochromatographic test strips with silver–enhanced

staining [14]

Two sheets of PT-R7 membrane were washed three times with distilled water and dried

at 60°C for at least 20 h. One sheet was impregnated with a 0.3% aqueous solution of

silver lactate, and the other was impregnated with a 3.0% hydroquinone solution in 0.5

M citrate buffer, pH 4.0; the membranes were dried in the dark at room temperature for

at least 20 h and cut into strips of 20 × 3.5 mm in size.

Membranes impregnated with silver lactate and hydroquinone were sequentially

applied to the working membrane of a test strip containing gold-stained zones after the

immunoassay and moistened with 100 μL distilled water.

2.2.5. Immunochromatographic assay

The assay was carried out at room temperature. The test strip was vertically submerged

into a sample for 10 min. Afterwards, the assay results were recorded.

2.2.6. Immunochromatographic assay with silver–enhanced staining

The assay was carried out under the same conditions (see 2.2.5). After 10 minutes of

incubation, membranes containing silver lactate and hydroquinone were removed, and

the assay results were recorded.


2.2.7. Immunochromatographic assay with separated specific and detection stages

The assay was carried out at room temperature. The samples were prepared in a

microwell plate by mixing 25 μl of 0.04–10 ng/mL serial dilutions with 25 μl of anti-

atrazine antiserum (1:500) in a 50 mM phosphate buffer, pH 7.4, containing 0.1 M NaCl

and 0.05% Triton Х-100 (PBST). Each test-strip was vertically immersed in the sample

solution and incubated for 5 min. The test-strip was then washed by immersing it in

PBST for 5 min, and incubated for 5 min in a solution of GNP–GARI (D580 = 2.0 in

Tris-buffer, pH 7.5, containing 1% BSA, 0.25% Tween-20, 1% sucrose and 0.1 sodium

azide). After 5 min of washing by immersion in PBST, the test-strip was scanned.

2.2.8. Immunochromatographic data processing

Digital images of binding zones on the working membrane of the test strip were

registered with the CanoScan LiDE 90 scanner. The colour intensity of the membrane

zones was calculated with TotalLab v2.01 software as described in [22] (TotalLab,

Newcastle upon Tyne, UK).

The general form of the equation of the calibration curve is:

Y = {(A – D)/[1 + (x/C)B]} + D,

where A is the asymptotic maximum; B is the point of inflection of the curve in the

semi-logarithmic coordinates; C is the concentration of the analyte at the inflection

point of the curve; and D is the asymptotic minimum (intensity of the background

signal) [22].

The limit of detection (LOD) was calculated as the concentration yielding a signal

reducing three times the standard deviation of the background signal for the sample

without atrazine.

3. RESULTS

3.1. Development and characterization of a standard immunochromatographic

system

First, a standard immunochromatographic system using antiserum as a capture reactant

for determining atrazine was developed. The following parameters were varied for

selection of the optimal completion of the test system:

- concentration of antiserum;

- concentration of the conjugate Atr-BSA applied in the test zone;

- quantity of the detecting conjugate between GNPs and specific antiserum;

- composition of the reaction medium.

The antiserum concentration (calculated on the base of total protein amount) in the

course of the synthesis of the conjugates ranged from 10 to 50 µg/mL. Excess of

antisera (50 µg/mL) was selected due to a large number of ballast proteins like albumins

and nonspecific immunoglobulins.


The concentration of the Atr-BSA conjugate in the test zone was varied from 0.5 to 2.0

mg/mL. Maximum colour intensities and the minimum relative errors (<12.5%) were

reached for the conjugate concentration equal to 2 mg/mL.

An optimum working nitrocellulose membrane with a pore size of 8 μm allows for

testing non-viscid samples [19, 23].

With the use of the selected completion of the test system, we obtained the calibration

curve that is shown in Fig. 1.

0,01 0,1 1 10

0

2000

4000

6000

8000

10000

12000

14000

Co

lou

r in

ten

cit

y,

RU

C (Atrazine), ng/mL

50 17 5.5 1.8 0.6 0.2 0.07 0

С (Atrazine), ng/mL

(A) (B)

Figure 1: The calibration curve and the appearance of test strips for standard

immunochromatographic system for detection of atrazine

The test system is characterized by the visual detection limit of 50 ng/mL, and

colorimetric detection limit of 0.2 ng/mL. Relative error of atrazine determination in

the working range of 1‒100 ng/mL does not exceed 10.9%. However, this test system

is characterized by a significant background colouration and low colour intensity of the

test zone, and the limit of detection is high.

3.2. The development of enhanced immunochromatographic test systems

The known methods for sensitivity-increasing of immunochromatographic test systems

are based on the increase in the colour intensity of the test zone. We have compared the

most widely used of such methods. Three variants of test systems for atrazine have been

developed:

A - test system based on the GNPs with silver-enhanced staining;

B - test system using latex particles as labels;

C - test system with pre-incubation of the sample with native specific antibodies

and the labelling of anti-species antibodies.

Variant A is based on the aggregation of silver ions on the GNPs surface. It is widely

used in electron microscopy [24, 25]. According to the existing publications, this


method allows for reducing the limit of detection of immunochromatographic assays

from 10 to25 times [26, 27].

Variant B is based on the change of colloidal label. Thus, the replacement of GNPs to

fluorescent markers reduces the detection limit by more than 20 times [28]. The use of

carbon nanotubes can reduce the detection limit by 50 times due to the greater contrast

of this label [29]. At the same time, coloured latex particles (cheap and mass-produced)

have great practical potential and are not characterized in comparative terms.

Variant C allows increasing the time of analyte reaction with antibodies. It is noted [30]

that, because of this, the detection limit decreases by approximately 10 times.

For each of these variants, optimization of immunochromatography conditions was

conducted by the same way as it was done (and given above) for the standard analysis.

Table 1 shows selected parameters of each of the test systems.

Table 1. Optimal configurations of the enhanced immunochromatographic systems

for atrazine detection

Parameter Detection system

A B C

Working membrane CNPF 8 µm CNPF 8 µm CNPF 8 µm

С (Atr-BSA), mg/mL 2 2 0.25

A520 (GNP-antibody) 4.0 - 2.0

C (LP-antibody), % - 0.1 -

Antiserum dilution 1:8,000 1:3,000 1:1,000

3.3. Comparison of the analytical characteristics of the enhanced

immunochromatographic test systems

Based on the obtained data, immunochromatographic systems for atrazine detection

have been made and tested. Fig. 1 shows the calibration curves and the appearance of

strips for different variants of enhanced analysis.


0,01 0,1 1 10

0

10000

20000

30000

40000

50000

60000

Co

lou

r in

ten

sit

y, R

U

C (Atrazine). ng/mL

50 17 5.5 1.8 0.6 0.2 0.07 0


(A)

0.01 0.1 1 10

0

2000

4000

6000

8000

10000

12000

14000

Co

lou

r in

ten

sit

y,

RU

C (Atrazine), ng/mL

50 17 5.5 1.8 0.6 0.2 0.07 0


(B)

0,01 0,1 1 10

0

1000

2000

3000

4000

5000

6000

7000

Co

lou

r in

ten

sit

y, R

U

C (Atrazine), ng/mL

Function = Logistic

A1 = 6288,76357, A2 = 49,20066

x0 = 0,30825, p = 1,44694

EC20 = 0,11825, EC50 = 0,30825

EC80 = 0,80351

9 3 1 0.3 0.1 0.04 0.01 0


(C)

Figure 2: Calibration curves (A, C, E) and appearance (B, D, F) of the enhanced

immunochromatographic test-systems for atrazine detection: A, B – test-system with

silver–enhanced staining; C, D – test-system based on LPs; E, F – test-system with pre-

incubation.


A four-parameter sigmoidal function was used to approximate the calibration curves of

the competitive immunoassay as recommended in [22]. The coefficient values of

approximating curves are shown in Table 2. The relative error of quantitative detection

is less than 12.5%, except for the system with silver–enhanced staining, for which it is

increased to 20‒25%.

Table 2. The coefficients of the equation approximating Y = {(A – D)/[1 + (x/C)B]}+D

for calibration curves of the developed test systems to atrazine

Coefficients

R2 A B C D

Standard 12637.8 0.71 0.99 99.0 0.999

Enhanced variant A 58078.6 1.1 1.8 6893.7 0.992

Enhanced variant B 12816.7 0.9 0.2 997.0 0.977

Enhanced variant C 6320.5 1.4 0.3 -0.9 0.998

Visual and colorimetric detection limits (DL) using the developed test systems were

evaluated. The corresponding parameters are summarized in Table 3.

Table 3. Visual and colorimetric limits of atrazine detection using the developed

test systems

Visual DL, ng/mL Colorimetric DL, ng/mL

Standard 50 0.2

Enhanced variant A 20 0.5

Enhanced variant B 2 0.02

Enhanced variant C 5 0.08

3.4. Analyte recovery in the juice samples

The evaluation of the atrazine detection efficiency for real food matrixes was

implemented in additional experiments. Repeated measurements were carried out to

determine inter-assay variability.

Standard immunochromatographic system and enhanced system based on blue LPs as

labels were tested using commercial apple juice diluted twice with PBST with different

concentrations of alled atrazine. As can be seen from Table 4, for test-system based on

LPs high recovery (95–103%) of the target antigen was achieved for all samples. The

recovery for standard immunochromatographic system is worse (90-110%) due to low

colour intensity.


Table 4. Recovery (R) levels for two developed test systems for atrazine to be

detected in apple juice samples (n=5).

Added, ng/mL Found, ng/mL R, %

Standard 0.5 0.45±0.15 90±29

1 0.9±0.3 92±27

10 11±2 110±22

Enhanced variant B 0.1 0.09±0.01 95±14

0.5 0.51±0.04 103±7

1 1.03±0.09 102±8

4. DISCUSSION

A standard immunochromatographic system using GNPs as a label allows one to

determine high concentrations of atrazine (≥50 ng/mL for visual detection). It is

characterized by a high background value and low colour intensity of the test zone.

Enhancement based on the aggregation of silver ions reduces the visual detection limit

by 2.5 times. But because of high standard deviation, colorimetric DL increases by 2.5

times. Furthermore, the use of auxiliary fluids and additional steps leads to

complications and an increase in assay time. The main disadvantage of a silver–

enhanced staining is the interference effect of chloride ions, which limits the scope of

tests that use chloride-free aqueous solutions [14].

The second method, based on the label change to blue LPs, reduces the visual DL by

25 times, and the colorimetric DL by 10 times. This effect is provided by a large number

of chromogen per one detectable immune complex. For LPs the label size may be

increased to hundreds of nanometres, whereas in the case of GNPs a similar growth is

accompanied by aggregation of particles and requires additional measures to prevent it.

As a result, an optimal size of GNPs for immunochromatography accords to their

diameter in the range of 30‒40 nm [2].

The third variant of sensitivity increase is based on the indirect labelling of specific

antibodies. Earlier it was successfully implemented for competitive

immunochromatographic analysis [31, 32]. Pre-incubation of the sample with free

specific antibodies (antiserum agaist atrazine) and a further manifestation of the formed

immune complexes by the universal conjugate GNP–GARI make it possible to achieve

a visual DL of 5 ng/mL, which is 10 times lower than the DL of the standard test system,

but about 2.5 times greater compared with the test system based on the LPs.

5. CONCLUSIONS

The implemented comparative characteristics of different ways to reduce the detection

limit in competitive immunochromatographic analysis using the same specific reagents

gives evidence of the prospects for the use of alternative labels and indirect labelling of

specific antibodies. For the object being studied, atrazine herbicide, the best results

were obtained using latex particles as the label. It is substantially related to the large

size of these particles as compared with gold nanoparticles, the traditional


immunochromatographic labels. Signal enhancement method based on the use of silver

salts did not shown efficacy in the case of competitive assay. Significant variation in

repeated measurements prevents reduction of the limit of detection. Probably the main

area of application of this enhancement is sandwich immunochromatography with a

direct relationship between the analyte concentration and recorded colour intensity.

The established patterns can be used for the development of rapid, highly sensitive test

systems for the detection of compounds from different classes.

ACKNOWLEDGEMENTS

This investigation was financially supported by the Ministry of Education and Science

of the Russian Federation in the frame of the State Targeted Program “Research and

Development in Priority Areas of Development of the Russian Scientific and

Technological Complex for 2014–2020” (contract 14.613.21.0040 from November 11,

2015; unique identifier of applied research: RFMEFI61315X0040).

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