Development of Immunoenzyme Assay of Herbicide Acetochlor and Its Application to Soil Testing with Comparison of Sample Preparation Techniques

Abstract

Acetochlor is a selective herbicide affecting weeds of cereal plants. Its analysis in soils allows accessing their suitability for crops and risks of contamination of agricultural products. The aim of this study was to develop a microplate enzyme immunoassay for the determination of acetochlor in soil extracts. For the development, rabbit antibodies specific to acetochlor were obtained by immunization with a conjugate of carrier protein with a derivative of acetochlor with mercaptopropionic acid. Another derivative with mercaptosuccinic acid was applied for immobilization on the solid phase. In the study, organic extracts have been obtained from soil varying solvents and their ratios, and using QuEChERS protocol. The extracts have been tested to estimate residual influences of the sample matrix. Optimal conditions for the immunoassay were selected, appropriate sample preparation techniques, and the composition of the medium for competitive immune interaction. The most effective approach involved dichloromethane extraction, followed by careful evaporation and subsequent reconstitution of the dry residue in a 10 mM phosphate-buffer solution supplemented with 0.1% gelatin. The resulting analytical system exhibited a detection limit of 59.4 ng/mL for acetochlor, with a working range spanning from 112 to 965 ng/mL. Taking into account the soil sample preparation, the LOD was estimated as 0.3 µg/g with the working range from 0.66 to 5.7 µg/g of soil. Analysis of prepared extracts from gray forest soil demonstrated a revealing of acetochlor between 74% and 124%.

1. Introduction

The use of pesticides in agriculture has undergone changes in recent years [1]. On the one hand, the information about the impact of pesticides on living organisms and the establishment of regulatory demands limits their use. On the other hand, violation of the legislative framework by unscrupulous farmers and earlier active application of pesticides lead to the need to control their content [1,2]. The determination of pesticides in soil is a necessary condition for assessing the suitability of agricultural soils for farming. Recent studies have shown that even soils used in organic farming are contaminated with substances that do not decompose for a long time [2,3,4]. In the connection with this, a large number of so-called organic products are contaminated with pesticides [5]. In this context, the main meaning of the concept of organic farming is lost, and the efficiency of soils use is determined by their primary analysis for toxic substance content [6]. Note also that only a small fraction of the pesticides used reaches the target organism, and the rest become environmental contaminants through leaching, wind erosion, evaporation, etc. [7,8].

Acetochlor is a representative of lactam herbicides, applied before emergence mainly in fields with corn, sorghum, and soybeans for weed control [9,10,11]. Previous studies of acetochlor’s toxicity to animals, plants, and ecotoxicology have confirmed its toxic properties, and the results are summarized in an official document [12]. Acetochlor exhibits moderate acute toxicity and causes skin irritation upon contact. An increased incidence of tumors in mammals has been noted, but no mutagenic effect has been confirmed in vivo experiments. In nature, acetochlor exhibits moderate stability and mobility in the upper soil layers and sediment systems, as do its toxicologically significant metabolites. Acetochlor also permeates water, posing a hazard to herbivorous birds in corn fields during the post-emergence period. It is also highly toxic to all groups of aquatic organisms [11,12,13]. Its action is associated with absorption by primary roots and disruption of protein biosynthesis in annual weeds [14]. The main toxic effects of this xenobiotic on living organisms are cardiovascular disorders, neurotoxicity, negative impact on the reproductive system, and immunodeficiency states [15,16,17]. Some studies show that the yield of some crops is reduced when acetochlor is used [18]. At the same time, most of the substance penetrates into surrounding water bodies due to the migration of acetochlor from soils with rainwater [8,19]. Oliveira et al. [20] showed that the distribution of acetochlor in the soil did not change over the course of two years. Due to the generalization of toxicity data, acetochlor was banned for use in EU countries [21]. However, it is used in the US, so it enters other countries with products imported from the US [22]. In this regard, acetochlor should be controlled in soil, water, and agricultural products [23].

The main methods used for the determination of acetochlor and related compounds are chromatographic with mass spectrometric detection [24,25]. However, despite their high accuracy and reproducibility, there are also disadvantages—requirements for highly qualified personnel, significant duration of one analysis, impossibility of conducting screening studies using a large number of samples and the need for additional purification of the resulting extracts. An alternative solution for screening is immunochemical analysis, which is based on the antigen–antibody interaction, and there are a large number of commercial test systems used in medicine, veterinary medicine, and environmental monitoring, which have proven their practical applicability and effectiveness [26,27].

Soil is a complex multi-component system, which, in addition to inorganic salts, sand and clay particles, contains a large number of organic compounds, which are humic substances, lignin, flavonoids, lipids, etc. [28,29,30]. There are various methods for extracting pesticides from soil, which promote maximum dissolution and extraction of the target analyte and destruction of the analyte-sample component bonds [31,32,33,34,35]. However, when extracting pesticides from complex samples, co-extraction and release of accompanying hydrophobic compounds, including other toxicants and dyes, occurs [31,36]. To assess the influence of the residual effect of the sample matrix, and to select the optimal acetochlor extraction technique, this work compares the data obtained from the analysis of soil extracts. QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) techniques are promising based on the literature data [37,38], and therefore these options were used for acetochlor extraction in raw investigation. This technique is described as suitable for different pesticides included acetochlor [39,40,41,42].

The aim of this work is to develop an enzyme immunoassay for the determination of acetochlor in soil with the selection of optimal conditions for detecting the analyte in resulting extracts. The significance and novelty of this study lie in the following. Soils are complex systems, and extraction of the target compound (acetochlor) followed by transfer of the analyte into an aqueous-organic phase can affect its recognition by antibodies. While most studies consider either the extraction of a pesticide/pesticide group from a large sample set using a single method, or a comparison of extraction techniques for chromatographic analysis, the peculiarity of this study was the study of the residual influence of the sample matrix in different extracts from soil samples under heterologous immunoassay conditions. Therefore, the primary objective of this study is to investigate the residual influence of sample matrix during acetochlor extraction using different methods, compare QuEChERS extraction techniques in this context, and select a method that allows for the reliable detection of acetochlor in gray forest soil samples.

2. Materials and Methods

2.1. Materials and Components

Acetochlor (90% min by assay) from LeapChem (Hangzhou, China) was used in the work. Stock solutions of acetochlor (12 mg/mL) were prepared in dimethyl sul-foxide (ACS reagent, ≥99.9%) and stored at 4 °C. To obtain the conjugate of carrier protein with acetochlor (hapten–protein conjugate), bovine serum albumin (BSA, ≥98%), soybean trypsin inhibitor (SIT, BioReagent, suitable for cell culture), N,N’-dicyclohexylcarbodiimide (EDC, ≥97.0% (T), for peptide synthesis), N-hydroxysuccinimide (NHS, ≥97.0% (T), for peptide synthesis), 2-mercaptosuccinic acid (MSA, 97%) and 3-mercaptopropionic acid (MPA, ≥99.0% (HPLC)), as well as methanol (United States Pharmacopeia (USP) Reference Standard) from Sigma-Aldrich (St. Louis, MO, USA) were used. Transparent 96-well microplates from Costar 9018 (Corning Costar, Murphysboro, IL, USA) were used for the ELISA. Tween-20 (for molecular Biology, Sigma-Aldrich, St. Louis, MO, USA) and sulfuric acid (Khimmed, Moscow, Russia), anti-species antibodies (sheep immunoglobulins binding rabbit immunoglobu-lins) labeled with horseradish peroxidase (IMTEK, Moscow, Russia), commercial solution of 3,3’,5,5’-tetramethylbenzidine substrate with hydrogen peroxide (Immunotech, Moscow, Russia) were used. All auxiliary reagents (salts, acids, alkalis and organic solvents) were of analytical or chemical grade (Khimmed, Moscow, Russia). Deionized water Milli Q (resistance 18 MOhm cm), obtained using the Simplicity system (Millipore, Cleveland, OH, USA), was used to prepare aqueous solutions and buffers. Phosphate buffer with salt (PB) in tablets was purchased from EcoLab (St. Petersburg, Russia). After dissolving tablets 10 mM PB with 0.1 M NaCl, pH 7.4 was obtained. For extraction, QuEChERS extraction kits manufactured by Copure (Biocomma, Shenzhen, China) were used. Incomplete and com-plete Freund’s adjuvants used for immunization were from InvivoGen (Vista Sorrento Pkwy, San Diego, CA, USA).

The absorption spectra of proteins and their conjugates were measured in cuvettes using a Biochrom Libra S80 spectrophotometer (Biochrom, Cambridge, UK), and in microplate wells using a Zenyth 3100 multifunctional reader (Anthos Instrument, Wals, Austria).

2.2. Methods

2.2.1. Preparation of a Conjugate of Acetochlor with a Carrier Protein

A derivative of acetochlor with 3-mercaptopropionic acid (ACMPA) was synthesized by the reaction of acetochlor with mercaptopropionic acid according to the reaction described in [43]. Method details are described in the Supplement Information.

The hapten–protein conjugates were obtained by the activated ether method according to the procedure described in [44] with modifications. A total of 15 mg ACMPA was dissolved in 100 μL DMSO, 400 μL of a solution containing 62 mg NHS and 104 mg EDC was added, and carboxyl groups were activated for 3 h. In the case of acetochlor with 2-mercaptosuccinic acid (ACMSA) derivative, 12.5 mg NHS and 25 mg EDC were used per 7 mg of the acetochlor derivative.

A total of 20 mg BSA or SIT were dissolved in 1500 μL of water. Then 300 μL of the C or ACMSA solution with activators (NHS and EDC) was added to 750 μL of the protein solution, 750 μL of Milli Q water was added, and the mixture was left with shaking for 3 h at 4 °C. The pH of the reaction mixture was then adjusted to 11.0 with 10 M NaOH and reaction mixtures left overnight at 4 °C. The resulting conjugate preparations were purified by dialysis against 10 mM PB for 48 h at 4 °C. After purification of the conjugates through a filter with a pore diameter of 0.22 μm, absorption spectra of the preparations were obtained. Aliquots of 50–100 μL of the conjugates were stored and stored at –20 °C.

2.2.2. Rabbit Immunization and Preparation of Antisera

The use of animals was carried out in accordance with Directive 2010/63/EU (dated 22 September 2010). Immunization of rabbits was carried out in accordance with the legislation of the Russian Federation and was approved by the Local Ethics Committee at the Research Center of Biotechnology of the Russian Academy of Sciences, and the investigation was performed according to the ethical guidelines (protocol 08 dated 25 October 2024). Female gray chinchilla rabbits (3 kg) were obtained from the Manikhino laboratory animal nursery (Moscow region, Russia). For the first subcutaneous injection, hapten–protein conjugate was diluted in 0.9% sodium chloride solution with equal volumes of complete Freund’s adjuvant (650/650 µL), and mixed well to achieve an emulsion. The resulting concentration was 1.0 mg/mL for protein. For the second injection, 0.5 mg of conjugate was diluted in saline and incomplete Freund’s adjuvant in equal volumes (500/500 µL). The emulsion was administered intramuscularly and subcutaneously every two weeks. Blood was collected into vacuum tubes one week after the dose was administered (on the 35th, 49th, 63rd, and 77th days (preparations 1–4 of antisera) from the ear vein. Blood samples were left to stand for 1 h at +4 °C and then centrifuged for 15 min at +4 °C in 6000× g mode. The antisera were aliquoted into 1 mL, labeled, and stored at −20 °C.

2.2.3. Competitive Enzyme Immunoassay of Acetochlor

The BSA-ACMSA conjugate (1.0 μg/mL in PB) was adsorbed into the wells of the microplate overnight at 4 °C. The microplate was then washed three times with PBS containing 0.05% Tween-80 (PBST). Then, an antiserum dilution was varied from 1:100 to 1:1,000,000, and incubated for 1 h at 37 °C. After washing with PBST, anti-species antibodies conjugated with horseradish peroxidase were added (the dilution of the commercial preparation was 1:3000). After washing (three times with PBST and once with distilled water), the activity of the enzyme label bound to the carrier was determined by adding 100 μL of the substrate solution (commercial TMB + H₂O₂ solution). The reaction was stopped after 15 min by adding 0.1 M H₂SO₄. Optical density was measured at 450 nm and a dependence of optical density on antiserum dilution was plotted using Origin 9.0 software (OriginLab, Wellesley, MA, USA). The antiserum dilution corresponding to OD450 = 1.0 was selected for competitive ELISA.

For the competitive assay, after the immobilization and washing stage of the microplate, 50 μL of acetochlor solutions in the appropriate medium (concentrations in the range of 20,000–0.12 and 0 ng/mL) and 50 μL of antiserum (the final chosen dilution was 1:300,000) were added to the wells. The microplate was then incubated for 1 h at 37 °C. The procedures were then carried out as described above. The principle of competitive interaction between the immobilized hapten (in the wells of the microplate) and the free analyte (in the sample) for the binding sites of specific antibodies is shown in Scheme 1.

2.2.4. Preparation of Samples for ELISA

The topsoil (soil depth: 0–20 cm) samples from corn fields were collected for analysis (Vladimir Opolye, Russia). Standard samples of gray forest soil characteristic of this region (Vladimir Opolye) were purchased from the Pryanishnikov Institute of Agrochemistry. Also, a commercial universal peat soil for seedlings was purchased (Fornosovo, Russia, www.nevatorf.ru (accessed on 30 October 2025)). This soil for vegetable, fruit, and berry crops has the following characteristics: total nitrogen—250 mg/L, phosphorus—280 mg/L, potassium—400 mg/L, pH not less than 5.5, humidity—not more than 65%. The samples were dried and sieved through a 2 mm mesh as described in [45]. The samples were initially dried at RT in open vials to remove excess moisture, ground in an agate mortar and pestle, crushing organic inclusions, then 1 g of soil was weighed. Subsequently, solid-phase extraction was carried out as described below. A total of 15 mL of organic extractant (dichloromethane or hexane) and weighed portions of salts for extraction by the QuEChERS method were added to the sample (

Table 1. The composition of salts when using various extraction schemes, as well as numbering in order of application.

Option No.	Title	Salt Composition per One Sample Extraction (50 mL Tube)
1	QuEChERS AOAC	6 g MgSO4, 1.5 g sodium acetate
2	QuEChERS EN	4 g MgSO4, 1 g NaCl, 1 g trisodium citrate dehydrate, 0.5 g disodium hydrogen citrate sesquihydrate
3	QuEChERS modified	4 g MgSO4 anhydrous, 1 g NaCl
4	DCM extraction	Pure extractant without salts

). The salt composition is provided in Table 1. Acetochlor extraction was carried out by shaking on a vortex for 1 h, and then the vials with samples were centrifuged for 15 min at 4 °C and 6500× g. After that, 10 mL of the organic supernatant layer was collected. Decreased volume of extractant (10 mL) was collected to avoid re-suspension of the sediment and the capture of components of the extracted sample. Then 5 mL of organic extract was transferred into Eppendorf tubes, which were then evaporated to dryness. Evaporation of the extracts was carried out at room temperature under a hood without heating, naturally due to the volatility of the solvent. The dry residue was re-dissolved in 1 mL 10 mM PBS with 20% content of methanol in buffer or in a methanol–water mixture (1:4 vol/vol). The solutions were used undiluted for ELISA.

2.2.5. Processing of Enzyme Immunoassay Results

The parameters of the competitive curves as dependencies of optical density at 450 nm from acetochlor concentrations were determined based on the four-parameter sigmoid equation y = (A1 − A2)/(1 + (x/x0)^p) + A2. The values of IC₁₀, IC₂₀, IC₅₀, and IC₈₀ were calculated as concentrations inhibiting the analytical signal by 10, 20, 50, and 80%, respectively. The IC₁₀ value was estimated as the detection limit, IC₂₀–IC₈₀—the working range of the determined concentrations as was shown in works [46,47,48].

3. Results and Discussion

3.1. Production and Characterization of Immunoreagents

Acetochlor is a low-molecular aromatic organic compound that does not have a carboxyl group accessible for modification. Figure 1 demonstrates the chemical structures of native acetochlor and its derivatives. Acetochlor derivatives with mercaptopropionic (MPA) and mercaptosuccinic (MSA) acids were obtained as described in [43,44] to obtain hapten–protein preparations.

Conjugation of resulting derivatives of acetochlor—ACMSA and ACMPA with BSA and STI was carried out using the activated ester method, and the resulting preparations were characterized by UV–visible spectrophotometry (Figure 2). The conjugates spectra were compared with the spectra of native proteins. As can be seen from Figure 1, the conjugates are spectrally different from the original proteins, the absorption maxima are shifted from 280 nm towards 265 nm, corresponding the absorption of native acetochlor [49]. These spectra indicate the successful synthesis of hapten–protein preparations. Both conjugates with ACMSA showed pronounced aggregation during the synthesis process, which affected the shape of the absorption spectra (Figure 2).

Both STI-ACMPA and STI-ACMSA were used to immunize animals to obtain specific antibodies and to evaluate the effect of the mercapto derivative on the specificity of the resulting antibodies. Since ACMPA has three carbon atoms in the mercaptopropionic acid residue, and ACMSA has four, as well as an additional carboxyl group, it was interesting to compare obtained conjugate–antiserum/antibody combinations. STI conjugates were used to immunize animals to obtain specific antibodies. The resulting BSA conjugates were applied for immobilization in the microplate wells.

Series of antiserum preparations were obtained from four blood collections. All of them were tested in ELISA with heterologous conjugates, namely, titration of antiserum and further competitive interaction with acetochlor in buffer were carried out. The results are presented in Table S1. As a result of testing, a pair of hapten–protein and antiserum preparations was identified, which demonstrated the highest sensitivity of acetochlor determination. As can be seen from the table, the best analytical characteristics are achieved with a combination of immobilized BSA-ACMSA and antiserum against STI-ACMPA.

Figure 3a shows the results of titration of antiserum containing antibodies to STI-ACMPA on the BSA-ACMSA conjugate. The competitive curve is presented in Figure 3b.

Based on the achieved results, heterologous conditions were chosen-conjugate for the immobilization of the BSA-ACMSA, antiserum to STI-ACMPA. As can be seen, the differences are not only in carrier proteins, but also in the original derivatives. Previous studies have demonstrated an increase in sensitivity and the preferred use of heterologous scheme in competitive immunoassays using heterologous conjugates. For example, a study by Moshcheva et al. [50] demonstrated a more than 1000-fold increase in assay sensitivity when choosing heterologous conjugates. One explanation for this increase in sensitivity, in addition to differences in hapten structure, was the different hapten loading on the carrier protein surface. The authors also screened antisera at different times to identify the most effective combinations of immunoreactants. The work of Wang et al. [51] showed the importance of another moment—the position of the carboxyl group of the hapten and the level of its exposure, as well as the overall distribution of electron density in the hapten molecule due to the presence of various functional groups. Returning to Figure 1 and analyzing the structures of the acetochlor derivatives used to obtain hapten–protein conjugates, we can see the differences in the side chains structure, i.e., three and four carbon atoms for ACMPA and ACMSA, respectively. Despite the presence of four carbon atoms in the ACMSA derivative, this additional atom acts as an additional carboxyl group. This functional group can interact with the nearby amino group of the protein during the synthesis of hapten–protein preparations, thereby ensuring a rigid position of the hapten and reducing the number of hapten molecules on the BSA surface. At the same time, the ACMPA core will be mobile and further from the protein surface. These differences may provide the variations in the recognition of immobilized hapten and free acetochlor in the sample, making the BSA-ACMSA conjugate a preferable reagent for interaction with antibodies. This combination is described for the first time, since both immunogen and antigen, immobilized at the phase, were previously used in the work, only with ACMPA derivative [44]. The IC₁₀ value as low as 7 ng/mL was achieved using antiserum obtained in the 4th blooding by the end of immunization. The chosen combination of reagents was used for further study. The specificity of the antibody in antiserum was assessed by ELISA testing of other pesticides instead of acetochlor as competitors—butachlor, propachlor, metolachlor, atrazine, imidaclopride, fipronil, parathion-metyl. Among them, only butachlor showed cross-reactivity as low as 6.9% and propachlor 6.5%. Other pesticides showed no cross-interactions, and the level of cross-reactivity was less than 0.1%.

3.2. Application of Different Extraction Techniques of Acetochlor from Soil Samples

The assay can be transformed from model solutions to real samples only when the maximum revealing of the analyte is ensured. Extraction of acetochlor and satellite compounds from soil is traditionally carried out with organic solvents by the solid–liquid extraction method and, more recently, by the QuEChERS method [37,38]. Table S2 presents the example options from the literature closest to this development for extracting pesticides from soil, including acetochlor, and provides features of each technique. The examples provided in Table S2 demonstrate the absence of a unified methodology and allow for optimization of sample preparation options for development tasks. That is why it was decided to test different extraction methods using commercial QuEChERS pouches and modified versions as preparation of salt mixture and direct extraction without salt application (Table 1).

In this work, comparison of various extraction techniques was carried out using a commercial soil for vegetable seedlings purchased in a local store. This product is universal, contains all the components of soil for agricultural work. Various extractions were tested, including DCM as an extractant, the composition of salt mixtures during solid-phase extraction using the QuEChERS method with modifications. Extraction was carried out for an hour, which exceeds the extraction time carried out by the traditional QuEChERS method, which will allow achieving maximum extraction from such a complex object as soil.

It was noted that turbid supernatants are formed, the organic layer is poorly separated during extraction by the AOAC method (option 1 in Table 1). The sediment is slightly stirred up and salts are captured when collecting the upper organic layer. This may negatively affect further analysis. In addition, the capture of salts in the organic layer, its subsequent drying and re-dissolution of the dry residue will cause salt to enter the buffer solution. This contamination of media with salt excess can negatively affect the results of the antigen–antibody interaction. Therefore, after collecting the dichloromethane layer, additional centrifugation was required to precipitate randomly captured salts and soil components and exclude them from organic extract. Such observations were not related to the choice of solvent.

Unlike AOAC extraction, the supernatant was easily collected after centrifugation when using the QuEChERS EN method (option 2, Table 1). The supernatant was colored from straw yellow to light ocher. In the third option voluminous black inclusions were observed on the surface of the organic layer, which indicated swelling of the peat in dichloromethane [52,53]. These inclusions were visible to the naked eye after centrifugation of the sediments of soil components and a mixture of MgSO₄ and NaCl only. However, re-precipitation was required in neither the second, nor the third case, and the organic layer was easily collected. The last, fourth option of extraction without salts demonstrated incomplete sedimentation of soli components and mechanical inclusions, and therefore.

The literature contains different extraction methods for soil and agricultural samples [35,41,47,54], and the extraction procedures differ significantly. For example, the recommended additive at the second stage of extract purification silica modified with an ethylenediamine-N-propyl phase (PSA (Primary Secondary Amine) with C₁₈) is not always used, but it allows obtaining satisfied recoveries, for example, 71–120%, as shown in the work [55]. The revealing depends, among other things, on the chemical composition of the soil and the presence of various types of interactions with its components [56]. The selection of the main organic solvent is carried out based on the solubility of the detected analyte and accompanying components of the sample. In the case of developing immunoassays, the residual effect of matrix components and compatibility with the immune components of the system are also important.

Dichloromethane (DCM) as extractant was used in this work. Traditionally, acetonitrile has been used for extraction by the QuEChERS technique [32], but typically the solvent is selected based on the solubility of the target analyte. Bodur et al. [57] showed that extraction with DCM demonstrated satisfactory recoveries of 81–120% confirmed by GC–MS. DCM subsequently volatilizes quite easily, which is preferable for subsequent re-dissolution of sample components and immunoassay development. Therefore, DCM was used in this work.

The soil is able to absorb the solvent partially, therefore 66% of the organic layer (10 mL instead of added 15 mL) was sampled for analysis. Subsequently, and after additional centrifugation of the extracts obtained by the AOAC method, only 5 mL of each extract were used for evaporation, with the corresponding mathematical recalculation of the acetochlor content in the samples. All extracts were collected and evaporated to dryness for further analysis using the developed ELISA. Since dichloromethane is immiscible with water, it cannot be used as an organic phase introduced into the antigen–antibody interaction system. Therefore, performing the immunoassay requires complete removal of the organic solvent followed by transfer of the analyte to a medium compatible with antibodies. Evaporation was performed without heating to avoid losses, and the dichloromethane was completely evaporated.

3.3. Selection of ELISA Conditions for Soil Extracts

The main difficulty in developing and using immunoassays for detecting hydrophobic pesticides such as acetochlor is the need to evaporate the resulting extract in the organic phase and transfer the target analyte into a medium compatible with immunoreagents. Two approaches were used here—transfer into a buffer containing a water-miscible organic solvent, as well as re-dissolution in a water–organic mixture. The use of buffer without organic solvent can lead to losses of the analyte [58].

The main requirements for such a solvent is mixing with water without limitation without denaturation of antibodies. Methanol, ethanol, and acetonitrile were considered in previous investigation on butachlor [59]. In this case, methanol was considered as the most optimal solvent at a concentration of no more than 30% to avoid inactivation of antibodies [59,60]. This approach has been used in our previous works with the analysis of extracts containing hydrophobic compounds [58,59]. Initially, PBS with 20% methanol was tested to re-dissolve dry residue. The calibration curve for of acetochlor was plotted using the same composition of the medium for the correct estimation of the results. A calibration curve using PBS with 20% methanol as a medium allowed for the detection limit 21.6 ng/mL and the working range from 61.8 to 2250 ng/mL.

Some artifacts associated with a high background signal were observed during the analysis of the obtained extracts. In this case, we are talking about the background signal that is obtained with complete inhibition of antibodies by the free analyte (Scheme 1). That is the signal at the maximum concentration of acetochlor in the wells of the microplate in competitive ELISA. However, the optical density of the sample and the pure buffer solution with the same acetochlor concentration did not match. The background signal in the wells when using a buffer solution with 20% methanol was 0.14, while in the case of samples this value increased to 0.19–0.34 optical units (Figure 4), which subsequently affected the results of the added-found experiments. The discrepancy between the optical densities of the sample and the standard led to false negative results and a significant underestimation (at least 5 times) of the acetochlor content in the samples. Because extrapolation of the optical density to the calibration curve will correspond to a point with a reduced acetochlor content. This is why it is so important to reduce the background signal in ELISA as much as possible.

These observations confirmed the need for additional refinement of the system and replacement of the environment. New assay conditions were tested to reduce the non-specific signal by using a buffer solution containing gelatin instead of Tween-80. The introduction of a protein component in the working buffer solution reduces non-specific adsorption of the reaction mixture components. Thus, replacing PB with 0.1% Tween-80 by PB with 0.1% gelatin as a medium for diluting the antiserum together with replacing PBS with 20% methanol with a methanol–water mixture with a ratio of 1:4 (vol/vol) allowed us to level out the level of non-specific signal in the wells of the microplate (Figure 5). The methanol concentration in the mixture was 20%. When adding an antiserum solution in a buffer without methanol, the sample was diluted by half, so the total volumetric methanol concentration in the microplate well was 10%. Such methanol content preserves antibody activity (does not inhibit them) while promoting complete dissolution of acetochlor from the samples. The choice of working buffer was performed based on experience with surface-active substances that have a tendency to non-specific adsorption on the surface [38,61]. Replacing the analysis medium from PB with methanol to PB with gelatin led to a change in the analytical characteristics of the test system (Figure 5, for comparison). Thus, the detection limit was 59.4 ng/mL, and the working range was from 111.7 to 965 ng/mL. The use of pure soil extract with the addition of acetochlor as a sample demonstrated similar analytical characteristics—the detection limit was 36.2 ng/mL and the working range from 78 to 1070 ng/mL with a background signal value of 0.07 (for a buffer solution with gelatin, this parameter was 0.06). It is worth noting that the change in conditions had a dramatic effect on the results of acetochlor detection, making it possible to reduce the background signal regardless of the type of soil (soil #1 and soil #2, commercial and from the field, respectively).

Thus, the choice of analysis conditions made it possible to minimize the non-specific signal and approach the analysis of extracts obtained in different ways using the ELISA method. Finally, the optimal buffer for the analysis was chosen as PBS with 0.1% gelatin at the competitive stage. A water–methanol mixture with a ratio of 1:4 (vol/vol) was used to re-dissolve the dry residues after extract evaporation.

3.4. Analysis of the Obtained Extracts by the ELISA

The resulting spiked soil extracts were tested under optimal conditions.

Table 2. Results of analysis of soil samples extracts prepared by different techniques with added acetochlor (n = 2) by extrapolation of optical density data in microplate wells to a calibration curve obtained in a buffer with 0.1% gelatin.

Option	Soil Extraction Technique	Added, ng/mL	Detected, ng/mL	Detected, %
1	QuEChERS AOAC	750	314.4 ± 39.9	41.9 ± 12.7
		250	106.7 ± 6.9	42.7 ± 6.5
		90	43.9 ± 4.5	48.8 ± 10.3
2	QuEChERS EN	750	664.2 ± 21.9	88.6 ± 3.3
		250	273.6 ± 19.9	110.3 ± 7.3
		90	105.7 ± 9.9	117.4 ± 9.4
3	QuEChERS modified	750	513.3 ± 12.3	81.7 ± 2.4
		250	192.1 ± 14.2	76.8 ± 7.4
		90	69.6 ± 0.7	77.33 ± 1.0
4	DCM only	750	652.5 ± 39.2	87 ± 6.0
		250	158.0 ± 22.1	63.2 ± 14
		90	74.7 ± 6.3	83 ± 8.5

presents the data obtained in the “added-found” experiments. Also, the features observed during the preparation of the extracts were noted and added in Table 2. Comparison of different extraction techniques in ELISA showed that it is not possible to completely eliminate the matrix effect in some cases. However, when developing the ELISA, we took into account the features of the matrices so that we could reliably determine acetochlor in samples. Thus, when using the solid-phase extraction system for the QuEChERS AOAC method, rather low values of acetochlor content are observed (less than 50%), which was not resolved by replacing the competitive analysis medium with a buffer with gelatin. Perhaps, this technique requires repeated extraction or a change in the extraction conditions. Satisfactory results were obtained for other extraction methods, which confirmed their efficiency.

Each of the considered options for extracting acetochlor from soil had its own advantages and disadvantages for ELISA purposes. Let us summarize all the observations and results for each of them. Option 1 was characterized by the speed of extraction, however, there were light stirring of salts, impossibility of collecting organic supernatant without capturing the salt layer. As a result, the obtained extract required for repeated sedimentation to sample the organic supernatant. Option 2 allowed for rapid extraction and good sample precipitation, but gave the colored organic layer. However, this observation did not have any negative influences on the immunoassay results. Option 3 gave incomplete sedimentation of salts and sample components but rapid extraction. Option 4 did not require additional investments, but provided incomplete sedimentation of the sample as well as separation of the organic layer. Thus, after comparing four extraction methods using different techniques for extracting acetochlor from soil, the following conclusion can be made. The method that allows for the most complete extraction of the sample from a soil sample is option 2 (QuEChERS EN). It should be noted that this method minimizes the residual influence of the sample matrix and extraction components, which proved decisive in choosing the sample preparation technique. The method using individual salts (option 3, QuEChERS modified) ranks second, as it allows for the detection of acetochlor within acceptable limits, taking into account the analytical error. Unfortunately, option 1 (QuEChERS AOAC) has proven unsuitable for use in immunoassay conditions.

Gray forest loamy soils are typical of the Vladimir Opolie region (links) [62,63]. The collected samples are representative of this type. The soil samples were characterized using parameters previously demonstrated for other soils in the study [45]. The characteristics of the soil samples from the fields are presented in Table S3 in the Supplement Information. The selected option (QuEChERS EN) was used to test soil samples after adding acetochlor. The soil extracts were analyzed under the same conditions used under optimization. Acetochlor was extracted from spiked gray forest loam soil samples, as well as to a peat-containing soil. The results for detecting acetochlor in the resulting extracts are presented in

Table 3. Results of analysis of soil samples from field with added acetochlor (n = 2) prepared by QuEChERS EN by extrapolation of optical density data in microplate wells to a calibration curve obtained in a buffer with 0.1% gelatin.

Soil Sample	Added, µg/g of Soil	Detected, µg/g	Detected, %
1	4.5	3.9 ± 0.13	86.7 ± 3.3
	1.5	1.59 ± 0.12	106 ± 7.5
	0.5	0.62 ± 0.06	124 ± 9.7
2	4.5	3.35 ± 0.21	74.4 ± 6.3
	1.5	1.24 ± 0.11	82.6 ± 8.9
	0.5	0.57 ± 0.07	114 ± 12.3
3	4.5	5.2 ± 0.38	115.6 ± 7.3
	1.5	1.4 ± 0.04	93.3 ± 2.9
	0.5	0.43 ± 0.04	86 ± 9.3

.

It is evident that the results are satisfactory, with between 74% and 124% of the added amount recovered despite the complexity of the samples and testing method. Similar results for the determination of acetochlor in other soil samples are presented in the works [42,64] and in Table S2. Thus, recoveries in soil samples ranged from 90 to 104% when the pesticide was introduced into the soil in an amount of 1–100 μg/kg in the work of Wang et al. [42]. Typically, analytical conditions are selected such that the testing result can detect the pesticide recoveries in the range of at least 80–120% [64], which is consistent with our data. The obtained results were recalculated in accordance with the sample preparation technique. When the soil extract after QuEChERS EN extraction method is evaporated and then dissolved in 1000 µL of the methanol–water mixture (1:4 vol/vol), the concentration step followed dilution is performed with the possibility of the determination of acetochlor by the developed immunoassay. The LOD was estimated as 0.3 µg/g with the working range from 0.66 to 5.7 µg/g of soil.

4. Conclusions

The development of immunoassay test systems requires the production of specific immunoreagents, such as hapten–protein conjugates and specific antibodies. It depends on the structure of the original hapten, the animal’s immune response to antibody production, and the assay conditions. Optimization of the immunoassay included the selection of a medium that would minimize the matrix effect of sample extract and extrapolate the data obtained from analyzing the extracts to a calibration curve. This work is focused, in general, on the characteristics of the matrix and its residual influence on the results of acetochlor determination by the immuno method.

This paper shows how replacing a 20% methanol buffer with a 0.1% gelatin buffer affects the analytical characteristics of the system, including the background signal level. The method of sample preparation is of great importance. The use of solid-phase extraction systems for these purposes is justified, since it reduces the effect of components co-extracted together with the analyte or included during the collection of the supernatant organic layer. The best effects were achieved when using QuEChERS EN extraction systems containing MgSO₄, NaCl, trisodium citrate dehydrate, and disodium hydrogen citrate sesquihydrate. The use of this extraction technique allows reducing the non-specific background, removing the interfering effect of sample components that are extracted together with acetochlor, increasing the degree of its detection. The developed system has prospects for application in the analysis of gray forest loamy soil samples for acetochlor content, and the discovered patterns can be used to develop analytical test systems for the analysis of low-molecular toxic analytes in complex samples.