sensitive rapid immunochromatographic detection …sensitive rapid immunochromatographic detection...
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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].
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
516 Nadezhda A. Taranova et al.
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.
Sensitive Rapid Immunochromatographic Detection of the Herbicide Atrazine 517
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.
518 Nadezhda A. Taranova et al.
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
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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
Sensitive Rapid Immunochromatographic Detection of the Herbicide Atrazine 519
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.
520 Nadezhda A. Taranova et al.
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
С (Atrazine), ng/mL
(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
С (Atrazine), ng/mL
(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
С (Atrazine), ng/mL
(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.
Sensitive Rapid Immunochromatographic Detection of the Herbicide Atrazine 521
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.
522 Nadezhda A. Taranova et al.
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
Sensitive Rapid Immunochromatographic Detection of the Herbicide Atrazine 523
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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