Fluorescent and Colorimetric Dual-Readout Immunochromatographic Assay for the Detection of Phenamacril Residues in Agricultural Products

Abstract

The fungicide phenamacril has been employed to manage Fusarium and mycotoxins in crops, leading to persistent residues in the environment and plants. Detecting phenamacril is pivotal for ensuring environmental and food safety. In this study, haptens and artificial antigens were synthesized to produce antiphenamacril monoclonal antibodies (mAbs). Additionally, gold nanoparticles coated with a polydopamine shell were synthesized and conjugated with mAbs, inducing fluorescence quenching in quantum dots. Moreover, a dual-readout immunochromatographic assay that combines the positive signal from fluorescence with the negative signal from colorimetry was developed to enable sensitive and precise detection of phenamacril within 10 min, achieving detection limits of 5 ng/mL. The method’s reliability was affirmed by using spiked wheat flour samples, achieving a limit of quantitation of 0.05 mg/kg. This analytical platform demonstrates high sensitivity, outstanding accuracy, and robust tolerance to matrix effects, making it suitable for the rapid, onsite, quantitative screening of phenamacril residues.

Graphical Abstract

Introduction

Fusarium graminearum infects a wide variety of plants, causing multiple devastating diseases such as fusarium head blight and seedling blight in wheat and barley. (1) These diseases not only lead to severe yield losses but also result in grain contamination with mycotoxins produced by the fungi, posing significant health risks to humans. The mycotoxins produced by F. graminearum primarily include fumonisins, gibberellin A3, deoxynivalenol (DON), 3-acetyl-DON, 15-acetyl-DON, and nivalenol. (2) Additionally, F. graminearum is responsible for causing keratitis, onychomycosis, and cutaneous diseases in humans. (3,4) Over the past 30 years, the development of styrene acrylic imidazole fungicides has been acknowledged as a highly effective method for controlling Fusarium species infections. (5) Unfortunately, the efficacy of these fungicides has been diminishing due to increasing resistance in the pathogen populations. (6)

As an alternative, 2-cyano-3-amino-3-phenylacrylic acetate was first synthesized in China in 1998 (Figure 1) and commercialized in 2015 under the trade name phenamacril. (7) However, phenamacril has been reported to be moderately mobile in Jiangxi red soil, tending to remain in the soil phase and potentially leaching into groundwater. (8) Research by Donau et al. focused on its degradation characteristics, showing that phenamacril degrades slowly in activated sludge, which could pose a concern if the compound leaches into the surface water or groundwater or if production residues enter the wastewater system, (9) indicating a potential risk of environmental migration. Tao et al. reported on the absorption, accumulation, and metabolic characteristics of phenamacril in contaminated plants. (10) Subsequently, a temporary maximum residue limit of 0.05 mg/kg (GB 2763–2021) for wheat in China was established for phenamacril. In the current literature, extensive studies on different toxins have been continuously reported from 1966 to the present, primarily focusing on immunoassays, including enzyme-linked immunosorbent assays (ELISAs) and immunochromatographic test strips. (11–16) However, information on phenamacril has been scarce. Nonetheless, analytical testing provides essential technical support for the safety evaluation of phenamacril and can offer reference data for its safe management and usage.

Figure 1.

Chemical structure of phenamacril.

For the determination of phenamacril, Cao and Chuan-zong utilized gas chromatography (GC) to analyze the residue of JS 399–19 in wheat as it was being developed. (17) Sun et al. established a method based on UHPLC-MS/MS for analyzing phenamacril residues in flour and rice, with a limit of quantification ranging from 5 to 33 μg/kg in these samples. (18) Another method, employing GC-MS/MS, was developed by Jinchuan et al. to detect phenamacril residues in rice, wheat, sorghum, and corn, achieving a limit of quantitation (LOQ) of less than 5 μg/kg. (19) To date, no rapid immunoassay has been reported for the onsite detection of phenamacril in food safety and environmental inspections.

The immunochromatographic test strip (ITS) has become a prevalent tool for the rapid qualitative and quantitative detection of pesticide residues in agricultural commodities and environmental samples. (20–23) While most ITSs employ single-readout nanomaterials-based methods, the advent of dual-readout formats has garnered the interest of researchers due to their enhanced sensitivity, accuracy, and, in some cases, broader detection range compared to single-readout systems. (24) For instance, dr-ITSs developed for detecting chlorpyrifos residues, biomarkers, and Campylobacter jejuni—based on colorimetry-chemiluminescence, colorimetry-fluorescence, and colorimetry-SERS modes—have demonstrated superior detection sensitivity over existing single-readout methodologies. (25–27) In the realm of lateral flow assays, gold nanoparticles (Au NPs) have increasingly been recognized as ideal labels for contaminant testing, enabling qualitative discrimination through color variation on test lines. Colloidal gold immunochromatography, in particular, has been widely adopted for the economical and portable screening of hazardous substances in food and environmental contexts. Recently, the utilization of polydopamine (PDA) nanoparticles as labels has attracted researchers’ attention due to their high chemical reactivity, versatile adhesion capacity, and excellent biocompatibility. Moreover, PDA-coated Au NPs (Au@PDA) exhibit enhanced color intensity, significantly higher levels of ultraviolet–visible (UV–vis) absorption, and colloidal stability against aggregation, thereby improving detection sensitivity. (28) Additionally, Au NPs have shown remarkable efficiency in quenching fluorescence probes, facilitating the variation of signal readout for highly sensitive and quantitative analysis. (29–31)

In this study, we developed a dual-readout immunochromatographic assay (dr-ICGA) for phenamacril detection by integrating an Au@PDA probe with quantum dots (QDs) within a single ICGTS to generate dual-readout signals. This dual-readout system allows for the evaluation of the immunoassay without equipment at one sensitivity range, along with an ultrasensitive fluorescent readout on the same sample. Here, the fluorescent signal increases while the colorimetric signal decreases simultaneously in response to the analyte concentration. The antiphenamacril monoclonal antibody (mAb) was produced and conjugated to the Au@PDA, serving as a probe. In the absence of the analyte, the Au@PDA-mAb binds with the antigen immobilized on the test line, leading to a red color appearance on the test line; meanwhile, the fluorescence of the QDs on the T line is completely suppressed by the Au@PDA-mAb nanoparticles. Conversely, the presence of the analyte reduces the red color on the T line, as observed visually, and enhances fluorescence under UV excitation. This method exhibits high sensitivity and specificity and holds significant potential for onsite phenamacril detection in food. It is noteworthy that, to date, no antibody or immunoassay for phenamacril detection has been reported.

Materials and Methods

Instrumentation

Specialized instruments utilized in this study include a hypothermic high-speed centrifuge (Eppendorf, Hamburg, Germany), a spot-spray system: Bio-Dot XYZ-3050 (BIO–DOT, California, USA), a Programmable Strip Cutter ZQ2000 (Shanghai Kinbio Tech. Co., Ltd., Shanghai, China), a double-beam Lambda 25 UV/vis spectrometer (PerkinElmer, Waltham, MA, USA), a Multiscan Ascent spectrophotometer (Thermo Fisher, Vantaa, Finland), transmission electron microscopy JEM-2010 (JEOL, Tokyo, Japan), and an LC-MS/MS (Shimadzu Corporation, Kyoto, Japan)

Reagents and Materials

The reagents and materials employed include bovine serum albumin (BSA), ovalbumin (OVA), PDA hydrochloride solution, N,N-dimethylformamide (DMF), N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC), EDC·HCl, Freund’s complete and incomplete adjuvants, 50% (w/v) polyethylene glycol (PEG) solution, horseradish peroxidase (HRP)-conjugated rabbit antimouse IgG antibody, goat antimouse IgG antibody, HAuCl4, ethyl benzimidate hydrochloride (5333–86-8), cyanoacetic acid (372–09-8), 4-(dimethylamino) pyridine (1122–58-3), and dicyclohexylcarbodiimide (DCC, 538–75-0) provided by Sigma-Aldrich (MO, USA). Tert-butyl-4-hydroxybutanoate (59854–12-5) was acquired from Acmec Biochemical (Tokyo Chemical Industry, Tokyo, Japan). PEG-20000 was sourced from Aladdin Chemistry Co., Ltd. (Shanghai, China). 3,3′,5,5′-Tetramethylbenzidine (TMB) was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Standards of phenamacril, azoxystrobin, picoxystrobin, fluopimomide, and pyraclostrobin were obtained from Dr. Ehrenstorfer (Augsburg, Germany). All other reagents used were of analytical purity.

Balb/c mice were procured from Yangzhou University Medical Centre (Yangzhou, China). The mouse myeloma cell line SP 2/0 was acquired from REPROCELL Inc. (Yokohama, Japan). The HiTrap Protein G HP antibody purification column was purchased from GE Healthcare Life Sciences (Uppsala, Sweden). NUNC 96-well microplates were supplied by Thermo Fisher Scientific (Waltham, MA, USA). Nitrocellulose (NC) membrane, conjugate pad, and absorbent pad were provided by Millipore (MA, USA). PVC plates and sample pads were obtained from Shanghai Kinbio Tech. Co., Ltd. (Shanghai, China). Q2525-ZnCdSe/ZnS QDs were sourced from Wuhan Jiayuan Quantum Dots Co., Ltd. (Wuhan, China). The nine wheat flour samples were purchased from various locations, including Hema supermarket, Suguo supermarket, and a local vegetable market in Nanjing, China.

All animal experiments were conducted following the approval from the appropriate Institutional Animal Care and Use Committees.

Preparation of Haptens and Antigens

Hapten Synthesis

Hapten synthesis is depicted through a schematic in Figure S1, illustrating the production and conjugation of a hapten with a free carboxyl group, derived from ethyl benzimidate hydrochloride as the basic skeleton structure through a four-step reaction process described as follows.

Synthesis of Ethyl Benzimidate, Compound ②

Ethyl benzimidate hydrochloride (81.1 mmol, 15.02 g, Compound ①) was dissolved in 100 mL of distilled water. Subsequently, 6.812 g of NaHCO₃ (81.1 mmol) was added, and the pH was adjusted to 8. The solution was then extracted with ethyl acetate three times (200, 100, and 50 mL each time). The organic layer thus obtained was dried with MgSO₄, filtered, and concentrated to yield ethyl benzimidate (Compound ②).

Preparation of tert-Butyl 4-(2-Cyanoacetoxy) Butanoate, Compound ④

A mixture of 1.480 g of cyanoacetic acid (17.4 mmol), 1.856 g of tert-butyl 4-hydroxybutanoate (11.6 mmol, Compound ③), and 0.71 g of 4-(dimethylamino) pyridine (DMAP, 5.8 mmol) was dissolved in 30 mL of CH₂Cl₂. Following the reaction, the mixture was cooled to 0 °C. Then, 6.13 g of dicyclohexylcarbodiimide (DCC, 23.2 mmol) was added, and the mixture was stirred overnight. After filtration, the filtrate was evaporated to dryness. The residue was redissolved in 100 mL of ethyl acetate and washed sequentially with 5% NaHCO₃ (two times, 50 and 30 mL), 5% NaHSO₄ (two times, 50 mL each), and saturated NaCl solution (two times, 50 mL each). The organic layer was dried overnight with anhydrous MgSO₄, filtered, and concentrated to obtain tert-butyl 4-(2-cyanoacetoxy) butanoate (Compound ④).

Production of the tert-Butyl (E)-4-((3-Amino-2-cyano-3-phenylacryloyl)oxy) Butanoate, Compound ⑤

A mixture of 2.7 g of Compound ② (18.12 mmol), 2.63 g of Compound ④ (11.6 mmol), and 0.394 g of imidazole (IMI, 5.8 mmol) was dissolved in toluene (60 mL). The reaction was conducted in a sealed vial heated at 100 °C with stirring for 24 h. Following the reaction, 50 mL of ethyl acetate and 50 mL of water were added to separate the final product. The organic layer was washed with 5% NaHSO₄ (2 × 50 mL) and 5% NaHCO₃ (1 × 50 mL), dried over anhydrous MgSO₄, filtered, and the solvent removed under reduced pressure. After recrystallization in ethyl acetate/hexane, 1.03 g of Compound ⑤ was obtained.

Preparation of the Phenamacril Hapten, Compound ⑥

Here, 0.68 g of Compound ⑤ (2.06 mmol) was dissolved in CH₂Cl₂ (6 mL), and trifluoroacetic acid (TFA, 4 mL) was added. The reaction mixture was stirred overnight at room temperature. Toluene (8 mL) was then added and thoroughly vortexed. The terminal-protecting t-butyl ester group was selectively hydrolyzed, given the conjugated ester of phenamacril’s exceptional stability to esterases and base. The solvent was removed under reduced pressure to yield a clear, yellow, viscous product. After recrystallization in ethyl acetate/hexane, the product obtained was the phenamacril hapten (Figure 2).

Figure 2.

Chemical structure of phenamacril hapten.

Preparation of Antigens

The hapten was conjugated to BSA and OVA, respectively, to prepare the immunogen and coating antigen using the carbodiimide method. (32) Initially, 2.2 mg of the hapten was dissolved in 0.5 mL of DMF. Subsequently, 1 mg of NHS and 2.3 mg of EDC were added (with a molar ratio of hapten:NHS:EDC approximately 1:1.5:2) and stirred in darkness at room temperature for 12 h. The resulting solution was then added dropwise to a solution of BSA or OVA (13.3 mg of BSA or 9.1 mg of OVA dissolved in 1 mL of 0.1 M carbonate buffer, pH 9.6) and stirred at room temperature for an additional 9 h. The final solution obtained was dialyzed in 0.01 M PBS buffer (pH 7.4) and subsequently stored at −20 °C for further use.

Production of mAb against Phenamacril

Three 6-week-old female Balb/c mice were immunized with 50 μg of the phenamacril-BSA immunogen in Freund’s complete adjuvant via multisite subcutaneous injection. Five biweekly booster treatments followed, using the immunogen emulsified with Freund’s incomplete adjuvant and administered intraperitoneally. Tail-blood samples were collected 1 week after each booster immunization to monitor antisera, employing indirect enzyme-linked immunosorbent assays (i-ELISAs) and indirect competitive ELISAs (ic-ELISAs) to evaluate the immune responses. The mouse producing the most sensitive antibody to phenamacril was selected for cell fusion. Splenocytes were fused with SP 2/0 myeloma cells using a 50% polyethylene glycol (PEG) solution and cultured in a HAT medium. Culture supernatants were evaluated using ic-ELISAs approximately 7–9 days after cell fusion. Hybridomas showing strong analyte inhibition were subcloned by limiting dilution to establish stable monoclonal hybridoma cell lines. (33) These monoclonal hybridomas were then used for large-scale production of ascites fluid mAbs, which were collected and purified using a Protein G column.

Characterization of mAb

The binding capacity of the interaction between antigenic epitopes and an antibody, referred to as antibody affinity, was assessed. The affinity constant ($\mathrm{Ka}$) was determined using the coated antigen and mAb dilution method by ic-ELISA. The phenamacril-OVA solution was prepared at concentration gradients of 1, 0.5, and 0.25 μg/mL, while the mAb was diluted to 0.196, 0.098, 0.049, 0.0245, and 0.01225 μg/mL. A nonlinear fitting curve was calculated using a four-parameter logistic (4-PL) model in Origin 2021 software. The $\mathrm{Ka}$ value was calculated according to the formula

$$Ka=(n-1)/2\times (n\mathrm{A}\mathrm{b}1-\mathrm{A}\mathrm{b}2)\mathrm{K}\mathrm{a}=(\mathrm{n}-1)/2\times (\mathrm{n}\mathrm{A}\mathrm{b}1-\mathrm{A}\mathrm{b}2)$$ (1)

where $n$ represents the concentration ratio of phenamacril-OVA (n > 1), and Ab₁ and Ab₂ are the concentrations of mAb corresponding to half of the maximum absorbance (OD 50%) at different concentrations of the coating antigen.

The sensitivity and specificity of the mAb were evaluated by ic-ELISAs. Phenamacril standards were prepared by dilution in 10% methanol-PBS to 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, and 0 ng/mL. Next, 50 μL of the standard solutions and 50 μL of mAb solutions were added to each well for incubation. Sensitivity was expressed using the half-maximal inhibitory concentration value (IC₅₀).

Additionally, the specificity of ic-ELISA was clarified by assessing the cross-reactivity (CR) toward four structural analogues of methoxyacrylate fungicides (azoxystrobin, pyraclostrobin, fluopimomide, and picoxystrobin), calculated using the equation

$$\mathrm{C}\mathrm{R}\left(\mathrm{\%}\right)=\left[\mathrm{I}\mathrm{C}50\right(\mathrm{a}\mathrm{n}\mathrm{a}\mathrm{l}\mathrm{y}\mathrm{t}\mathrm{e})/\mathrm{I}\mathrm{C}50(\mathrm{s}\mathrm{t}\mathrm{r}\mathrm{u}\mathrm{c}\mathrm{t}\mathrm{u}\mathrm{r}\mathrm{a}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{a}\mathrm{l}\mathrm{o}\mathrm{g}\mathrm{u}\mathrm{e}\mathrm{s}\left)\right]\times 100\mathrm{\%}$$ (2)

Fabrication of Dual-Readout Immunochromatographic Test Strip

Preparation of Colloidal Gold Nanoparticles

Colloidal Au NPs were synthesized with minor modifications to previously reported methods. (34,35) The glassware utilized in the experiment was initially treated with aqua regia (V_HNO3:V_HCl = 1:3), rinsed several times with ultrapure water, and dried. A solution of 0.01% (w/w) HAuCl₄ in distilled water (100 mL) was heated to boiling. While stirring constantly, 5 mL of 1% trisodium citrate was added. The reaction mixture changed color sequentially from light yellow to blue, black, and finally to bright red within 3 min. The mixture was further boiled and stirred for an additional 10 min. The resultant colloidal gold solution was cooled and stored at 4 °C in the dark. The colloidal Au NPs were characterized by a Multiscan Ascent spectrophotometer in the range of 400–660 nm.

Preparation of Au@PDA Nanoparticles

Initially, a 5 mg/mL PDA hydrochloride solution was prepared by dissolving 500 mg of dopamine in 100 mL of Tris-HCl buffer (10 mM, pH 8.5). Then, 10 mL of the colloidal gold solution was mixed with 40 μL of 3% H₂O₂ and dispersed into 200 μL of PDA hydrochloride solution. The mixture was stirred in the dark at room temperature for 1 h, allowing the colloidal Au NPs to be coated with PDA shells. The Au@PDA core–shell nanoparticles were then isolated by centrifugation at 10,000 rpm for 30 min at 4 °C. The precipitate was resuspended in 10 mL of ultrapure water and characterized using transmission electron microscopy.

Preparation of the Au@PDA-mAb Conjugate

Following reported methods with adjustments, (36) the optimal pH and antibody amount for the Au@PDA-mAb conjugate were investigated. To optimize pH conditions, 1 mL of Au@PDA solution was placed into 2 mL tubes, and the pH was adjusted to 6, 7, and 8 using 1 M K₂CO₃ solution, respectively. Then, 25 μL of purified mAb (0.98 mg/mL) was added to each tube and mixed thoroughly for the Au@PDA-mAb probe conjugation. Next, 10 μL of 20% PEG-20000 and BSA were added for blocking. The final optimal pH condition was determined based on the performance of the Au@PDA-mAb-based ICGTSs.

To find the optimal antibody dosage for the Au@PDA conjugate, 1 mL of the Au@PDA solution (with the optimized pH) was added separately into four 2 mL Eppendorf tubes. Subsequently, 25, 30, 35, and 40 μL of mAb (0.98 mg/mL) were added to each tube, mixed thoroughly, and the coupling reaction continued for 15 min. The optimal mAb dosage was identified based on the color changes of the T and C lines when the Au@PDA-mAb probes were applied to the ICGTSs.

The Au@PDA-mAb probe was synthesized using the optimized pH and antibody dosage by adding the appropriate amount of mAb dropwise to 1 mL of Au@PDA solution and stirring for 1 h. The solution was then blocked using 10 μL of 20% PEG-20000 for 30 min and 10 μL of 20% BSA for another 30 min to block the remaining active sites on the Au@PDA NPs surface. The solution was centrifuged at 10,000 rpm for 30 min at 4 °C. The supernatant was discarded, and the pellet was resuspended in 100 μL of ultrapure water.

Preparation and Characterization of QDs-OVA

EDC·HCl (10 mg/mL, 2.4 μL) was added to 100 μL of QDs in boronic acid (800 nM, pH = 6), and the mixture was stirred in the dark for 24 h. Subsequently, OVA (30 mg/mL, 10 μL) was added dropwise, stirring continued in the dark for another 24 h. The final solution was centrifuged at 10,000 rpm for 30 min at 4 °C, and the sediment was resuspended in 100 μL of ultrapure water to obtain QDs-OVA conjugates. Zeta potential analysis was performed to estimate the surface charge of the synthesized QDs-OVA conjugates, aiding in characterizing the features of the conjugates.

Fabrication of the Fluorescent and Colorimetric Dual-Readout Immunochromatographic Ctrip

The fluorescent and colorimetric dr-ICGTS is composed of five key components, arranged from the bottom to the top, as illustrated in Figure 3: a PVC plate, sample pad, conjugate pad, NC membrane, and absorbent pad. Before fabrication, the sample pad (350 mm × 20 mm) and conjugate pad (350 mm × 10 mm) were pretreated as previously detailed. (37) The Au@PDA-mAb probe was immobilized on the conjugate pad, while the NC membrane (350 mm × 25 mm) featured a T line and a C line, coated with a mixture of phenamacril-OVA and QDs-OVA and goat antimouse IgG antibody, respectively. These lines were centrally located on the membrane, spaced 6 mm apart. Subsequently, the sample pad, conjugate pad, NC membrane, and absorbent pad (350 mm × 17 mm) were affixed onto the PVC plate (350 mm × 60 mm) to assemble the ICGTS. The completed assembly was then sectioned into strips 3.5 mm in width and stored in sealed bags with desiccant gel at room temperature until further testing.

Figure 3.

Schematic illustration of a dual-readout immunochromatographic test strip.

Optimization was performed for the quantities of phenamacril-OVA and QDs-OVA applied to the T line, utilizing the colorimetric and fluorescent intensities on the T line to assess the impact of varying application amounts on the test strip’s efficacy. The concentration of the rabbit antimouse IgG fixed on the C line was 1 mg/mL, with 1 μL of the Au@PDA-mAb probe being utilized. The phenamacril-OVA concentrations applied to the T line were 0.2, 0.4, 0.6, and 0.8 mg/mL, respectively, whereas the QDs-OVA concentrations were 60, 120, 240, and 480 nM.

To evaluate the assay’s performance, sample extracts (100 μL) were applied to the sample pad, where capillary action facilitated the movement of the solution through the strip. The presence of phenamacril in the sample led to competition with the immobilized antigen for antibody binding, resulting in a reduction in the red color intensity on the T line—a visual colorimetric signal. Simultaneously, the fluorescence of QDs on the T line was restored and observable under UV light, thus achieving a dual-readout capability. In the absence of phenamacril, the T line appeared distinctly red to the naked eye, with suppressed fluorescence; conversely, the presence of phenamacril faded the red color to light pink or caused it to vanish entirely, with a concurrent increase in fluorescence on the T line

Characterization of the Dual-Readout Immunochromatographic Strip

Phenamacril standards and analogues, spanning concentrations of 0.63–20 ng/mL, were prepared in PBS containing 10% methanol to assess the dr-ICGTS’s performance. The sensitivity of the immunoassay for phenamacril detection was determined through dual readouts of color and fluorescence. The colorimetric signal allowed for visual comparison between the T and C lines using the reduction in red color, while the fluorescent signal was assessed by the increased green fluorescence intensity on the T line under UV light.

Cross-reactivities (CRs) of the test strip toward methoxyacrylate fungicides (azoxystrobin, picoxystrobin, fluopimomide, and pyraclostrobin) were evaluated.

Method Validation and Application in Wheat Flour Sample

Nine wheat flour samples were obtained from various sources: Hema supermarket (samples 1–3), Suguo supermarket (samples 4–6), and a local market (samples 7–8). These samples were analyzed using the developed dr-ICGA and verified by LC-MS/MS. (18) For sample extraction, a 5 g sample was placed in centrifugation tubes, to which 10 mL of ultrapure water was added, followed by mixing for 2 min using a vortex mixer. Subsequently, 25 mL of acetonitrile was added, and the mixture was subjected to ultrasonic extraction for 15 min. Moreover, 3 g of NaCl was incorporated into the mixture, which underwent an additional 30 min of ultrasonic extraction before being centrifuged at 4000 rpm for 5 min. From the upper organic phase, 5 mL was collected and evaporated under a stream of nitrogen. The residue was then redissolved in 1 mL of 10% methanol in PBS, preparing it for testing. To mitigate matrix interference, extracts were diluted 10-fold with 10% methanol-PBS before analysis. The reliability of the method was verified by comparing LC-MS/MS analysis results with those obtained from the test strips.

Additionally, six 5 g samples of phenamacril-free flour (as verified by LC-MS/MS) were placed in centrifugation tubes, each receiving 10 mL of ultrapure water and mixed for 2 min using a vortex mixer. These samples were then spiked with a phenamacril standard to final concentrations of 0.20, 0.10, 0.05, 0.025, and 0.0125 mg/kg, respectively, with a control sample receiving an equivalent volume of ultrapure water (0 mg/kg). Each sample was mixed again for 5 min by vortexing. Acetonitrile was added to each tube to facilitate extraction using ultrasonication. Comparative analyses were conducted using the dr-ICGA and LC-MS/MS, performed in five and three replicates, respectively.

Results and Discussion

Characterization of Haptens and Antigens

The hapten designed to mimic phenamacril was synthesized from ethyl benzimidate hydrochloride and characterized through LC-MS and 1H NMR spectroscopy. A peak at m/z 274.2 in the mass spectrum, as depicted in Figure S2, confirmed the hapten’s presence, aligning with the expected molecular weight. The 1H NMR spectroscopy results were as follows (Figure S3): 1H NMR (400 MHz, DMSO-d6) δ 12.18 (s, 1H, –COOH), 9.23 (s, 1H, -NH), 8.97 (s, 1H, -NH), 7.64–7.46 (m, 5H, Ar–H), 4.15 (t, J = 6.5 Hz, 2H, -OCH2–), 2.32 (t, J = 7.4 Hz, 2H, –CH2CO–), and 1.85 (p, J = 6.9 Hz, 2H, –CH2). These results further confirmed the hapten’s synthesis with the proposed structure.

The immunogen and coating antigen were synthesized by conjugating the hapten to carrier proteins BSA and OVA, respectively. Ultraviolet spectrophotometric scanning demonstrated a noticeable shift in the ultraviolet absorption peaks of the immunogen (phenamacril-BSA) and coating antigen (phenamacril-OVA) compared to the peaks of BSA or OVA alone, as shown in Figure S4. These shifts indicated successful synthesis of both the immunogen and coating antigen

Generation of Specific Antiphenamacril mAb

A mouse spleen with a titer of 80,000 and a phenamacril inhibition rate of approximately 70.8% at 1 μg/mL was utilized for the fusion of hybridoma cells. Through limiting dilution cloning, a final monoclonal hybridoma cell line, E3, capable of stable mAb secretion, was derived. Post ascites fluid production and purification, the mAb was characterized by SDS-PAGE (Figure S5), displaying a light chain band at 25 kDa and a heavy chain band at 50 kDa.

Binding affinity curves for the antiphenamacril mAb were established via ic-ELISA (Figure 4). At a coating concentration of 0.25 μg/mL, the antibody concentration corresponding to 50% optical density (OD) was 2.65 × 10–10 mol/L; at 0.5 μg/mL, it was 2.85 × 10–10 mol/L; and at 1 μg/mL, it was 2.41 × 10–10 mol/L. Consequently, the affinity constant (Ka) at coating concentrations of 1 and 0.5 μg/mL phenamacril-OVA was 2.54 × 109 L/mol; at 0.5 and 0.25 μg/mL, it was 1.64 × 109 L/mol; and at 1 and 0.25 μg/mL, it was 2.15 × 109 L/mol. Averaging these three affinity constants provided Ka of 2.11 × 109 L/mol for the mAb, indicating a strong binding affinity.

Figure 4.

Binding affinity curves of antiphenamacril mAb at different concentrations of coating antigen.

A standard curve was established with phenamacril concentrations ranging from 62.5 to 1.95 ng/mL as the abscissa and the B/B0 as the ordinate (Figure 5). The logistic regression model applied was: Y = 0.08476 + (0.91997 – 0.08476)/(1 + (x/17.46289)1.2819) (3), with an R2 value of 0.9998. The IC50 reached 17.46 ng/mL with a linear response range (IC20–IC80) from 5.92 to 51.50 ng/mL. In addition, the specificity of the antibody was evaluated through its CR with four structural analogues of methoxyacrylate fungicides (azoxystrobin, pyraclostrobin, fluopimomide, and picoxystrobin), revealing negligible CR (<0.35%) towards all tested compounds (Table 1). This suggests that the synthesized antibodies exhibit a minimal preference for binding to the ethyl ester end of phenamacril.

Figure 5.

Standard curve of ic-ELISA for the determination of phenamacril (n = 3).

Table 1.

Cross-Reactivity of mAb toward Four Structural Analogues by ic-ELISAs

Compounds	Structures	IC50 (ng/mL)	Cross-reactivity (%)
Phenamacril		17.46	100
Azoxystrobin		>5,000	<0.35%
Pyraclostrobin		>5,000	<0.35%
Fluopimomide		>5,000	<0.35%
Picoxystrobin		>5,000	<0.35%

Characterization of the Synthesized Au@PDA Nanoparticles and QDs-OVA

The AuNPs exhibited a maximum UV absorbance of 0.73 at 528 nm, as shown in Figure S6A. Transmission electron microscopy (TEM) images demonstrated that the AuNPs were well dispersed with minimal Oswald ripening, having an average diameter of approximately 15 nm (Figure S6B). The colloidal gold particles were uniformly coated with a transparent and homogeneous layer of polydopamine, resulting in an average particle diameter of 20 nm (Figure S6C), indicating the successful synthesis of both AuNPs and Au@PDA nanoparticles.

The –COOH on the surface of the QDs was conjugated with the OVA containing -NH2 groups via EDC, yielding QDs-OVA. The zeta potential of the QDs shifted from–30.90 ± 0.95 mV, and the zeta potential of QDs-OVA was–24.10 ± 1.93 mV, suggesting an increase in particle size and a decrease in suspension stability, indicative of successful QDs-OVA synthesis (Figure 6).

Figure 6.

Characterization of QDs and QDs-OVA by zeta potential analysis.

Au@PDA NPs demonstrated superior mechanical and thermal stability, enhanced light scattering and near-infrared absorption capabilities, and increased resistance to acidic and alkaline conditions compared to bare AuNPs. (38,39) They also exhibited a higher photothermal conversion efficiency, improving the sensitivity of Au@PDA-based lateral flow immunoassays (LFIAs) over those based on bare AuNPs. (28) Additionally, the unique internal structure and shell passivation of the QDs contributed to their high fluorescent emission and photoluminescent quantum yield while maintaining structural stability in aqueous environments. (40) These properties of Au@PDA nanoparticles and QDs lay the foundation for achieving high sensitivity, selectivity, and interference tolerance in the developed assays.

Conjugation of the Au@PDA-mAb Probe

The optimal pH and appropriate concentration of antibody for the Au@PDA-mAb conjugate were evaluated. The findings demonstrated that at pH 7 and with 35 μL of mAb (0.98 mg/mL) (Figure 7), the Au@PDA-mAb conjugates exhibited the most pronounced color intensity on the T line. Consequently, these conditions—pH 7 and 34.3 μg of mAb—were identified as optimal for the conjugation process of the Au@PDA-mAb probe.

Figure 7.

Optimization of pH values and amount of mAb in Au@PDA-mAb conjugate.

Optimization of the Optimal Concentration of Phenamacril-OVA and QDs-OVA

As shown in Figure S7, when the concentration of phenamacril-OVA exceeded 0.6 mg/mL, both the C and T lines were distinctly visible, although the intensity of the T line’s red color did not significantly increase with rising concentrations. In the case of QDs-OVA, concentrations below 240 nM resulted in weak fluorescence, which became overly strong and necessitated more probes at concentrations above 240 nM. Therefore, the optimal concentration for phenamacril-OVA was established at 0.6 mg/mL and for QDs-OVA at 240 nM.

Performance of Dual-Readout Immunochromatographic Assay

Generally, ICGAs have primarily utilized a single-readout system, either colorimetric or fluorometric, for signal detection. However, studies suggest that these single-readout systems, particularly those relying on naked-eye identification, may not always yield reliable results due to potential interference, leading to false-positive or false-negative outcomes. (41,42) To enhance the monitoring of pesticide residues, the development of new ICGAs featuring multiple signal modalities is imperative. The dr-ICGA trials (Figure 8) demonstrated that with increasing concentrations of phenamacril, the T line’s red colorimetric signal gradually faded and ultimately vanished, while the fluorescent signal on the T line progressively intensified under UV light. This dual-signal improves naked-eye detectability and user-friendliness, especially for nonprofessionals, facilitating easier interpretation. The integration of a negative colorimetric readout with a positive fluorescent readout not only augments sensitivity but also enhances the accuracy of observations made with the naked eye. When the concentration of phenamacril is below 5 ng/mL, the T line displays a visible red band under natural light without a distinct green fluorescent band under UV light. With a gradual increase in phenamacril concentration to 5 ng/mL or higher, the red color of the T line becomes significantly lighter than that of the C line and completely vanishes under natural light, while the green fluorescence fully recovers under UV light within 10 min. Thus, the application of a dual-signal channel, employing positive signal readout in fluorescence mode and negative signal readout in colorimetric mode, demonstrates promising sensitivity and accuracy for the determination of phenamacril, with a defined limit of detection (cutoff value) of 5 ng/mL. Compared with the result from ELISA (IC50 of 17.46 ng/mL), the sensitivity of the dr-ICGA increased by more than 3.5 times. Sensitive, accurate, and portable ICGAs employing colorimetric/fluorescent/photothermal modes for dual/multisignal output have been successfully applied to quantitatively detect small and large molecule analytes in trace amounts. (41,43) Previous reports on dr-ITSs for the detection of chlorpyrifos residues, biomarkers, and C. jejuni—based on colorimetry-chemiluminescence, colorimetry-fluorescence, and colorimetry-SERS modes—with limits of detection at 0.033 ng/mL, 0.049 ng/mL, and 50 cfu/mL, respectively, have shown higher detection sensitivity than other existing single-readout-based detection methods. (25–27) A similar conclusion was reported by Chen et al., who developed a dual-readout chemiluminescent-gold lateral flow test assay for detecting tumor biomarkers with ultrahigh sensitivity, noting that the sensitivity surpassed that of ELISA, chemiluminescence-ELISA, and chemiluminescence lateral flow tests. (44) In comparison, new ICGAs with dual/multiple signals offer enhanced sensitivity, accuracy, and a broader application range, performing exceptionally in recent detection and diagnosis efforts.

Figure 8.

Sensitivity analysis of the proposed fluorescent and colorimetric dual-readout immunochromatographic strip for phenamacril detection. Phenamacril was loaded at 20, 10, 5, 2.5, 1.25, 0.63, and 0 ng/mL, respectively, in triplicate. Panel (A) indicates the result based on visual evaluation, whereas panel (B) shows the result under UV light.

The specificity results (Figure 9) indicated that the strips exhibited a strong positive reaction on the T line, observable through visual evaluation, and intense fluorescence recovery under UV light only in the presence of phenamacril. For the analogues, the fluorescence was undetectable, and the red color of the T lines remained visible. These findings suggest that the dual-readout immunochromatographic strip exhibits high selectivity in distinguishing phenamacril, with no significant cross-reactivity with the four other analogues and fungicides, thus indicating good specificity for the determination of phenamacril.

Figure 9.

Specificity of the dual-readout lateral flow test strip. 1000 ng/mL of azoxystrobin, picoxystrobin, fluopimomide, and pyraclostrobin, and 20 ng/mL of phenamacril were loaded, respectively. The left figure indicates the result based on visual evaluation, whereas the right figure shows the result under UV light.

Method Validation and Application

As illustrated in Figure 10 and Table 2, when phenamacril content in spiked wheat flour exceeds 0.05 mg/kg, the results from the dr-ICGTS are positive (±, + ). The accuracy of the immunochromatographic assay was substantiated by LC-MS/MS, (18) with phenamacril recovery ranging from 81.60 to 98.50% (Table 2). Validation using spiked wheat flour samples further affirmed the accuracy and reliability of the developed immunoassay. Additionally, the limit of quantitation of the immunoassay aligns with the testing requirements of the Chinese national standard (0.05 mg/kg in wheat flour), demonstrating its suitability for onsite testing, as the assay can be completed within 10 min.

Figure 10.

Application of the prepared immunochromatographic strip for the detection of phenamacril in wheat flour. The left half of the figure represents the immunochromatographic result of the phenamacril standards diluted in 10% of methanol-PBS, whereas the right half of the figure shows the result from the wheat extracts spiked with phenamacril standards.

Table 2.

Consistency and Comparison between dr-ICGA and LC-MS/MS for the Determination of Phenamacril in Wheat Flour^a

sample types	sample sources	sample no.	spiked level (mg/kg)	LC-MS/MS (n = 3)			dr-ICGA (n = 5)
				detected concentration (μg/kg)	recovery (%)	CV (%)	close up to left visual result (dilute 10 times)
spiked positive samples	Hema supermarket	1	0	00	ND	ND	−, −, −, −, −,
		1–1	0.0125	1.02	81.60	9.66	−, −, −, −, −,
		1–2	0.025	2.28	91.20	6.64	−, ± , −, ± , −,
		1–3	0.05	4.68	93.60	7.77	±, ± , ± , ± , ± ,
		1–4	0.1	985	98.50	3.99	±, + , + , + , + ,
		1–5	0.2	19.67	9835	1 95	+, + , + , + , + ,
blinded samples	Hema supermarket	1		ND	ND	ND	−, −, −, −, −,
		2		ND	ND	ND	−, −, −, −, −,
		3		ND	ND	ND	−, −, −, −, −,
	Suguo supermarket	4		ND	ND	ND	−, −, −, −, −,
		5		ND	ND	ND	−, −, −, −, −,
		6		ND	ND	ND	−, −, −, −, −,
	local market	7		ND	ND	ND	−, −, −, −, −,
		8		ND	ND	ND	−, −, −, −, −,
		9		ND	ND	ND	−, −, −, −, −,

^a−, negative: the concentration of phenamacril in wheat flour is <0.05 mg/kg; ±, weakly positive: the concentration of phenamacril in wheat flour is 0.05–0.1 mg/kg; +, positive: the concentration of phenamacril in wheat flour is > 0.1 mg/kg; ND, none detected.

The developed dr-ICGTS was also applied to the analysis of nine real wheat flour samples acquired from local supermarkets. The results, as detailed in Table 2, indicated that all nine samples tested negative for phenamacril, a finding corroborated by LC-MS/MS. The consistency between the outcomes of this immunoassay and LC-MS/MS underscores the robustness of the proposed methodology, suggesting its applicability for the rapid and high-throughput screening of phenamacril residues. This technique meets regulatory standards for detecting trace levels of phenamacril in environmental or food matrices. To our knowledge, this study is the first report on mAb generation and the development of fluorescent and colorimetric dual-readout immunochromatographic assays for detecting phenamacril in agricultural products. The findings demonstrate that the developed immunochromatographic test strip, leveraging both colorimetric and fluorescent signals, offers simplicity, affordability, high sensitivity, good accuracy, and matrix tolerance. This establishes its potential as an effective tool for rapid, onsite quantitative screening of phenamacril residues in various agricultural foods.