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Feb 7, 2015 - Figure 1. Chemical structures of different marine toxins: brevetoxin A, 1; brevetoxin B, 2; brevetoxin C, 3; okadaic acid, 4; saxitoxin ...
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Novel Colorimetric Immunoassay for Ultrasensitive Monitoring of Brevetoxin B Based on Enzyme-Controlled Chemical Conversion of Sulfite to Sulfate Wenqiang Lai, Junyang Zhuang, and Dianping Tang* State Key Laboratory of Photocatalysis on Energy and Environment, Key Laboratory of Analysis and Detection for Food Safety (Fujian Province and Ministry of Education), Institute of Nanomedicine and Nanobiosensing, Department of Chemistry, Fuzhou University, Fuzhou 350108, People’s Republic of China ABSTRACT: A simple colorimetric immunoassay for quantitative monitoring of brevetoxin B on a functionalized magnetic bead by using glucose oxidase (GOx)/antibrevetoxin antibody-labeled gold nanoparticle as the signal transduction tag was developed. The assay was carried out on the basis of GOx-controlled sulfite-to-sulfate chemical conversion with a silver(I)−3,3′,5,5′tetramethylbenzidine [Ag(I)−TMB] system. Initially, the sulfite was used as an inhibitor of Ag(I) to hinder the color development of TMB due to the formation of insoluble silver sulfite. Accompanying H2O2 generation with GOx-catalyzed glucose, the sulfite was converted into the sulfate, thus resulting in the colorless-to-blue change. Under the optimal conditions, the absorbance decreased with increasing brevetoxin B from 0.5 to 200 ng/kg with a detection limit of 0.1 ng/kg (ppt). The precision and specificity were acceptable. Furthermore, the methodology gave results matching well with the referenced brevetoxin ELISA kit for monitoring of spiked Musculista senhousia samples. KEYWORDS: colorimetric immunoassay, brevetoxin B, marine biotoxin, sulfite-to-sulfate enzyme-controlled chemical conversion

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INTRODUCTION Ongoing effort has been made worldwide to expand assay development, especially immunoassays, with the aim of manufacturing portable and affordable detection devices while preserving the essential benefits in sensitivity, robustness, broad applicability, and suitability to automation.1−4 Typically, the assay is usually implemented on the basis of certain affinity ligands for specific interaction with the target molecules to trigger the signal transduction cascade.5−8 In contrast, enzymebased colorimetric immunoassay is often utilized as an optional scheme for assay development and quality control monitoring in different industries because of its simplicity, low cost, practicality, and direct rapid readout with the naked eye.9−14 Despite some advances in this field, there is still a need for more flexible, yet highly sensitive, quantitative, and easy-to-use methods to keep pace with expectations in future point-of-use testing. To achieve a user-friendly colorimetric immunoassay with sufficient sensitivity, a highly efficient and feasible color development would be advantageous and valuable, especially for the trace target analyte, because the assay is basically designed for visual inspection.15−17 Classical methods for visible color development have employed horseradish peroxidase (HRP) and alkaline phosphatase to catalyze the 3,3′,5,5′tetramethylbenzidine (TMB)−H2O2 and ascorbic acid 2phosphate−H2O2 systems, respectively.18−20 TMB, a widely used chromogenic substrate in staining procedures, is utilized as a visualizing reagent in enzyme-linked immunosorbent assays (ELISA) because it yields reaction products with high absorption coefficients and lacks carcinogenicity.21,22 Traditional TMB-based colorimetric assays were designed by using bioactive enzymes (e.g., HRP).23,24 Unfortunately, bioactive enzyme-based reaction is susceptible to interference and assay © XXXX American Chemical Society

conditions during the generation stage of visible color, for example, pH, temperature, and instability caused by structural unfolding.25 Furthermore, the colorimetric readout must be controlled and preserved free from interferences in much the same way as the antigen−antibody reaction.26 Therefore, our group designed a new colorimetric immunoassay based on the chelate reaction between squaric acid and iron(III).27 However, we recently found the natural color of iron ions sometimes leads to a false-negative result, especially during trace monitoring process. The emergence of the visible color based on the reaction between silver(I) and TMB opens a new possibility for the development of colorimetric assay.28 Our motivation in this study is to explore an enzyme-controlled colorimetric immunoassay based on the conversion of sulfite to sulfate by using glucose oxidase (GOx)-labeling detection antibody with the silver(I)−TMB system. Brevetoxin B, 2 (Figure 1), one of the most potent neurotoxins produced by the red tide organism Gymnodinium breve Davis, can cause intoxication and even mortality through consumption of contaminated shellfish and produce respiratory irritation through aerosol exposure at coastal areas.29 Thus, sensitive and specific determination of brevetoxin B would be valuable. As a demonstration of the capability of our design, we herein utilize brevetoxin B as a model analyte for the development of an ultrahigh-sensitivity colorimetric immunoassay. Received: December 24, 2014 Revised: February 6, 2015 Accepted: February 7, 2015

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DOI: 10.1021/acs.jafc.5b00425 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 1. Chemical structures of different marine toxins: brevetoxin A, 1; brevetoxin B, 2; brevetoxin C, 3; okadaic acid, 4; saxitoxin acetate, 5; dinophysistoxin-1, 6.

Figure 2. Schematic illustration of (A) GOx-mediated sulfite-to-sulfate chemical conversion for color development of the Ag(I)−TMB system and (B) Ag(I)−TMB-based colorimetric immunoassay by coupling with enzyme-controlled sulfite-to-sulfate chemical conversion.

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(Tianjin, China). The brevetoxin B ELISA kit was obtained from Abraxis LLC (Warminster, PA, USA). All other reagents were used as received without further purification. All water used was of Milli-Q ultrapure grade (Merck Millipore, Darmstadt, Germany). Different buffer solutions including pH 9.6 carbonate buffer, pH 7.0 phosphatebuffered saline (PBS, 0.5 mM), and pH 7.4 PBS were purchased from Sigma-Aldrich (Shanghai, China). Food Samples and Extraction Procedure. A Musculista senhousia matrix (5.0 g, ground blank) was first milled with a small food mixer and then extracted with aqueous dimethyl sulfoxide (20 mL, 50%, w/v). After slight stirring for 60 min, the suspension was filtered with a nylon mesh (100 nm), no. 1 filter, and a GF/B filter (Whatman, Dassel, Germany) in turn. The extract color was a slight opalescent yellow. Finally, the solution for the calibration curve was prepared by spiking brevetoxin B standards into the diluted extract with various volumes. Preparation of Brevetoxin−BSA-Conjugated Magnetic Bead and mAb/GOx-Labeled Gold Nanoparticle. Bevetoxin−BSA-

MATERIALS AND METHODS

Safety. To avoid possible exposure and potential contamination, all tools and items in contact with marine toxins should be put into a 10 wt % bleach solution for 2−3 h before they are discarded because marine toxins are powerful hepatotoxins and carcinogens. Pure marine toxin standards were handled in a hood with great caution. Materials and Reagent. Monoclonal mouse anti-brevetoxin antibody (mAb) and brevetoxin B−bovine serum albumin (brevetoxin−BSA) conjugate were gifted from the College of Chemistry of Soochow University (Suzhou, China). The standards including brevetoxins A, B, and C, 1, 2, and 3, okadaic acid, 4, saxitoxin acetate, 5, and dinophysistoxin-1, 6 (Figure 1), were purchased from Express Technology Co., Ltd. (Beijing, China). GOx and HAuCl4·4H2O were purchased from Sigma-Aldrich (Shanghai, China). Silver nitrate (AgNO3), TMB, and BSA were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Sodium sulfite (Na2SO3) and sodium sulfate (Na2SO4) were obtained from Fuchen Chemicals B

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Figure 3. UV−vis absorption spectra of (A) (a) 1.0 mM TMB and (b) 1.0 mM TMB + 2.0 mM Ag(I); (B) (a) 2.0 mM sulfite + 2.0 mM Ag(I) + 1.0 mM TMB, (b) 2.0 mM sulfate + 2.0 mM Ag(I) + 1.0 mM TMB, and (c) 2.0 mM Ag(I) + 1.0 mM TMB; and (C) (a) 1.0 mM H2O2 + 1.0 mM sulfite + 2.0 mM Ag(I) + 1.0 mM TMB and (b) 0 mM H2O2 + 1.0 mM sulfite +2.0 mM Ag(I) + 1.0 mM TMB. (Insets) Corresponding photographs. conjugated magnetic bead was prepared similarly to our previous study.27 Briefly, 3-aminopropyltriethoxysilane (30 μL, 98 wt %) was initially added into 1.0 mL of anhydrous ethanol containing 50 mg of magnetic beads (∼100 nm) (Chemical GmbH, Berlin, Germany) for reaction for 6 h at room temperature under gentle shaking on a shaker. Following that, the aminated magnetic beads, collected by an external magnet, were suspended in 1.0 mL of 15 wt % glutaraldehyde and reacted for another 6 h. Afterward, the precipitate obtained by magnetic separation was placed into brevetoxin−BSA carbonate buffer (1.0 mL, 100 μg/mL, pH 9.6) and incubated for 6 h under the same conditions. Finally, the as-prepared brevetoxin−BSA-conjugated magnetic beads (brevetoxin−magnetic bead) were magnetically separated and stored in 1.0 mL of pH 7.4 PBS containing 1.0 wt % BSA and 0.1 wt % sodium azide for further use. Gold nanoparticle (AuNP, ∼16 nm) was synthesized and functionalized with mAb/GOx according to our previous work.27 One microliter of 1.0 mg/mL mAb and 10 μL of 1.0 mg/mL GOx were simultaneously added into 1.0 mL of 0.24 μM gold colloids (pH 9.0). The resulting mixture was incubated for 12 h at 4 °C with slight shaking. Following that, the pellet (i.e., mAb−AuNP−GOx) obtained by centrifugation (14000 rpm, 20 min, 4 °C) was suspended in 1.0 mL of pH 7.4 PBS containing 1.0 wt % BSA and 0.1 wt % sodium azide. Monitoring of GOx Activity Based on Sulfite-to-Sulfate Conversion with Ag(I)−TMB System. Figure 2A displays the detection principle of GOx activity based on sulfite-to-sulfate conversion with the Ag(I)−TMB system. The assay was carried out as follows: (i) 10 μL of GOx with different concentrations (from 0 to 500 μg/mL) was added into 50 μL of 0.5 mM PBS (pH 7.0) containing 4.0 mM glucose and incubated for 30 min at 37 °C; (ii) 20 μL of 2.0 mM nitric acid and 50 μL of 1.0 mM sodium sulfite were injected into the resulting solution and reacted for 2 min at room temperature; (iii) 50 μL of 2.0 mM silver nitrate was added into the mixture and reacted for 1 min at room temperature; and (iv) 100 μL

of 1.0 mM TMB substrate solution (ethanol/buffer, 1:9, v/v) in pH 4.0 citrate acid−disodium hydrogen phosphate buffer was added and incubated for 30 min at room temperature. The resultant mixture was monitored on an SH-1000 microplate reader (Corona Electric Co., Ltd., Japan), and the absorbance was recorded at 652 nm. Colorimetric Immunoassay toward Brevetoxin B Based on Enzyme-Controlled Sulfite-to-Sulfate Chemical Conversion with the Ag(I)−TMB System. Figure 2B gives the monitoring process of colorimetric immunoassay toward target brevetoxin B based on enzyme-controlled sulfite-to-sulfate chemical conversion with the Ag(I)−TMB system. Initially, 25 μL of brevetoxin B standard/sample and 50 μL of GOx−AuNP−mAb suspension (C[Au] ≈ 0.24 μM) were added in sequence to 25 μL of brevetoxin−magnetic bead suspension (6.0 mg/mL) in a 200 μL PCR tube and incubated for 60 min at 37 °C with gentle shaking. After that, the resulting suspension was collected by using an external cylindrical magnet, 10 mm in diameter and 10 mm in depth, to produce an inhomogeneous magnetic density, 0.2 T at the surface (Shanghai Da Xue Electromagnetic Devices Co., Ltd., China), and washed with the washing buffer. Fifty microliters of 4.0 mM glucose (pH 7.0, PBS) was added to the precipitate and incubated for 30 min at 37 °C. Following that, 20 μL of 2.0 mM nitric acid, 50 μL of 1.0 mM sodium sulfite, and 50 μL of 2.0 mM silver nitrate were injected into the mixture in turn as before. Finally, 100 μL of 1.0 mM TMB substrate solution was added and incubated for 30 min for color development. Meanwhile, the absorbance was registered and recorded at 652 nm on a plate reader.

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RESULTS AND DISCUSSION Design Strategy of Ag(I)−TMB System Based on Sulfite-to-Sulfate Chemical Conversion. To develop a user-friendly and sensitive colorimetric immunoassay, a stable and highly efficient color progression would be advantageous. C

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Figure 4. (A) UV−vis absorption spectra of (a) glucose + GOx + sulfite + silver(I) + TMB, (b) glucose + sulfite + silver(I) + TMB, (c) GOx + sulfite + silver(I) + TMB, (d) glucose + GOx + silver(I) + TMB, (e) glucose + GOx + sulfite + silver(I), and (f) glucose + GOx + sulfite + TMB, respectively; (B) corresponding photographs.

Typically, the peroxidase-based H2O2−TMB system has been widely used for ELISA development. However, most systems involve low sensitivity or poor visible color and thus are unfavorable for routine use. Recent papers have shown that TMB could be used as a selective and sensitive indicator for in situ quantification of Ag(I).30,31 Addition of TMB into an Ag(I) aqueous solution could give rise to the oxidized form of TMB and Ag(0), thereby inducing a blue color and a strong characteristic absorption peak centered at 652 nm. As is wellknown, silver sulfite is insoluble in water, whereas silver sulfate is slightly soluble. However, the sulfite can be oxidized to sulfate through hydrogen peroxide under acidic condition.32 Thus, the sulfite can be used as an inhibitor of silver ion to control and tailor the color development of TMB at the visualization stage upon hydrogen peroxide introduction. Typically, hydrogen peroxide can be generated by glucose oxidase in the catalytic oxidation of β-D-glucose. In this case, H2O2-controlled chemical conversion of sulfite to sulfate can be utilized for quantitative monitoring of GOx activity in the presence of glucose. Figure 3A shows the UV−vis adsorption spectra of TMB solution in the absence and presence of 2.0 mM Ag(I). No characteristic absorption peak from 800 to 300 nm was observed at pure TMB solution (curve a). In contrast, two strong absorption peaks were simultaneously achieved after interaction of 1.0 mM TMB with 2.0 mM Ag(I) (curve b), which was ascribed to the oxidation of TMB by Ag(I). Moreover, the colorless TMB solution (Figure 3A, photograph a) exhibited an obvious blue color change in the presence of Ag(I) (Figure 3A, photograph b). Furthermore, we also investigated the dynamic response curve (absorbance vs reaction time) between TMB and Ag(I) (Figure 3A, left inset). The absorbance intensity rapidly increased with the increasing reaction time and tended to level off after 5 min. To ensure adequate reaction between Ag(I) and TMB, 30 min was used for the color development in this work. These results revealed that Ag(I) could oxidize TMB to the oxidized TMB to trigger a blue color and an absorption peak at 652 nm. The second concern in our design was whether the sulfite-tosulfate conversion could induce the progression of the Ag(I)− TMB system. To determine this point, sulfite and sulfate alone (2.0 mM) were added into the Ag(I)−TMB system, respectively. Ag(I) was initially incubated with sulfite/sulfate for 30 min at room temperature, and then TMB was added into the resulting solution. The resulting mixture was monitored using UV−vis absorption spectroscopy (Figure 3B). As seen

from curve b, introduction of sulfate into the system did not appreciably change the characteristic peak or absorbance in comparison with the Ag(I)−TMB system alone (curve c). When the sulfite was added into the Ag(I)−TMB system, significantly, the absorbance largely decreased (curve a). This was attributed to the fact that the added sulfite initially reacted with Ag(I) to form the insoluble silver sulfite (Ag2SO3) and hindered the interaction of Ag(I) with TMB. The results revealed that the sulfite could hinder the progression of the Ag(I)−TMB system, whereas the sulfate had almost no effect on the colorimetric assay of the system. Therefore, the chemical conversion of sulfite to sulfate could trigger the continuance of the Ag(I)−TMB system with the colored product in the presence of sulfite. The third important question arises as to whether hydrogen peroxide (H2O2) could cause the chemical conversion between sulfite and sulfate to complement the Ag(I)−TMB system. To demonstrate this, 50 μL of 1.0 mM sodium sulfite, 50 μL of 2.0 mM silver nitrate, and 100 μL of 1.0 mM TMB were added into acidic solution containing 1.0 mM H2O2 in turn. As shown from curve a in Figure 3C, the characteristic absorption peaks were almost the same as that of the Ag(I)−TMB system alone (Figure 3A, curve b). When 50 μL of 2.0 mM silver nitrate and 100 μL of 1.0 mM TMB were injected in sequence into 50 μL of 1.0 mM sodium sulfite, however, almost no absorption peaks were observed (Figure 3C, curve b). Meanwhile, the resulting mixture changed from blue to colorless (Figure 3C, insets). On the basis of the results in Figure 3, we deduced that the added H2O2 could convert the sulfite into the sulfate and relieve the inhibition effect of sulfite toward Ag(I), thereby resulting in increasing absorbance and colorimetric progression. Usually, H2O2 can be produced through glucose oxidase through the catalytic oxidation of glucose in the presence of oxygen. Therefore, the designed system could be utilized for monitoring the activity of GOx. Control Tests for Ag(I)−TMB System with Sulfite-toSulfate Conversion. To further investigate that the Ag(I)− TMB system could be realized by GOx-triggered production of H2O2 accompanying the chemical conversion between sulfite and sulfate, several control tests were carried out under different conditions (Figure 4). As indicated from curve a, two strong absorption peaks at 652 and 372 nm appeared when glucose, sulfite, Ag(I), and TMB were added into GOx solution in sequence. Moreover, the absorbance at 652 nm increased with increasing GOx concentration. The reason might be most D

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Figure 5. Effects of (A) silver(I) concentration and (B) sulfite concentration on the absorbance of the Ag(I)−TMB system by coupling with the sulfite-to-sulfate chemical conversion (1.0 mM TMB used in the case).

concentration and then tended to slow after 2.0 mM. To decrease the background signal and avoid excessive Ag(I), 2.0 mM Ag(I) was used for development of the colorimetric assay. Normally, the free Ag(I) in the solution can trigger TMB to promote the change in the absorbance and color development. However, the colorimetric assay can be hindered in the presence of sulfite. By using 1.0 mM TMB and 2 mM Ag(I), we also investigated the effect of sulfite concentration on the colorimetric assay. As seen from Figure 5B, the absorbance decreased with the increase in sulfite concentration and reached a minimum value at 4.0 mM. However, we found that the absorbance (i.e., the slope for the plots) changed very quickly within the low-concentration range of 0−1.0 mM, suggesting that a slight decrease in the sulfite level induced a big increase in the absorbance. Considering the subsequent reaction between sulfite and H2O2 with high sensitivity, the highconcentration sulfite should not be preferable. Therefore, 1.0 mM sulfite was chosen as the inhibitor of Ag(I) to hinder color development in this work. Monitoring for the Ag(I)−TMB System toward H2O2 and GOx. As is well-known, H2O2 can oxidize sulfite to sulfate under acidic conditions. To achieve a high sensitivity for development of the subsequent colorimetric immunoassay, the determination of low-concentration H2O2 with the system would be valuable. For this purpose, the Ag(I)−TMB system was utilized for the detection of H2O2 by using sulfite as the inhibitor. Experimental results indicated that the Ag(I)−TMB system could be used for monitoring of H2O2 concentration with a wide linear range (10 μM−1.0 mM) and a low detection limit (2.5 μM). The linear regression equation could be fitted to y = 0.0016 × C[H2O2] + 0.8218 (μM, R2 = 0.9976). Furthermore, we also investigated the Ag(I)−TMB system for the detection of GOx by using glucose as the enzymatic substrate. H2O2 was obtained through GOx by the catalytic oxidation of glucose. The absorbance linearly increased with increasing GOx concentration within the range of 0.5−20 μg/ mL. A detection limit of 0.1 μg/mL GOx could be achieved at the 3sblank criterion. The linear regression equation could be fitted to y = 0.0531 × C[GOx] + 0.7354 (μg/mL, R2 = 0.9982). Such a low detection limit provided a precondition for the development of a high-sensitivity colorimetric immunoassay. Analytical Performance of the Ag(I)−TMB-Based Colorimetric Immunoassay. Figure 2 shows the fabrication process of the colorimetric immunoassay for the detection of brevetoxin B, used as a model, based on the Ag(I)−TMB system accompanying enzyme-triggered sulfite-to-sulfate chem-

likely a consequence of the fact that Ag(I) could be released through the oxidizing reaction of enzymatic product toward sulfite to oxidize TMB into TMB(ox). When GOx (curve b) or glucose (curve c) was absent from the system, however, the absorbance greatly decreased and the color became light blue (Figure 4B, tubes b and c). The results indicated that glucose or GOx alone did not eliminate the inhibition effect of sulfite toward the Ag(I)−TMB system. However, the inhibition effect of the Ag(I)−TMB system could be relieved by sulfite regardless of the presence of glucose and GOx (Figure 4A, curve d, and Figure 4B, tube d). For comparison, we also monitored the effect of Ag(I) and TMB toward the designed system. As seen from curves e and f (Figure 4A), no absorption peak was achieved in the absence of Ag(I) or TMB even if glucose/GOx/sulfite simultaneously existed in the system. Furthermore, the resultant solution was colorless (Figure 4B, tubes e and f). These results further revealed that (i) the visible color mainly originated from the interaction between Ag(I) and TMB; (ii) sulfite could hinder the interaction of Ag(I) with TMB; (iii) H2O2 could oxidize sulfite to sulfate, which could not hinder the interaction between Ag(I) and TMB; and (iv) the colorimetric assay could be controlled by changing the GOx level. Such a cascade reaction could be used for monitoring enzymatic activity, which could be employed for colorimetric immunoassay development based on the change in the visible color by conjugating GOx with detection antibody. Optimization of Assay Conditions. To achieve a good colorimetric immunoassay with high sensitivity for subsequent work, one important precondition was that the Ag(I)−TMB system should be highly efficient toward the sulfite-to-sulfate chemical conversion in the presence of low-concentration GOx. Actually, the system mainly consisted of three steps: (i) the added glucose was oxidized to gluconic acid and H2O2 by GOx; (ii) the produced H2O2 catalytically oxidized sulfite to sulfate under the acidic condition; and (iii) the generated sulfate triggered the colored product relative to the Ag(I)−TMB system. During this process, the sulfite (SO32−) was used as an inhibitor of Ag(I) to hinder the color development of TMB owing to the formation of insoluble silver sulfite, which could be eliminated through the sulfate (SO42−). To adequately complement the catalytic efficiency of GOx, the concentrations of sulfite, Ag(I), and TMB should be optimized. First, we investigated the effect of Ag(I) on the Ag(I)−TMB system. In this case, 1.0 mM TMB was used for color development on the basis of our previous work.27 As shown in Figure 5A, the absorbance initially rapidly increased with increasing Ag(I) E

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Figure 6. (A) Absorbance intensity; (B) calibration plots of Ag(I)−TMB-based colorimetric immunoassay by coupling with enzyme-mediated sulfite-to-sulfate chemical conversion toward different concentrations of BTB standards. (Inset) Corresponding photographs.

Figure 7. (A) Reproducibility; (B) specificity of the Ag(I)−TMB-based colorimetric immunoassay by coupling with enzyme-mediated sulfite-tosulfate chemical conversion.

100 ng/kg,33 competitive ELISA = 25 mg/kg,34 LC-MS/MS = 12 μg/kg,35 immunodipstick assay = 100 ng/kg,36 glucometerbased immunoassay = 10 ng/kg).37 Due to the legal limit of brevetoxin (≤80 μg of toxin per 100 g of shellfish tissue),38,39 the Ag(I)−TMB-based colorimetric immunoassay could completely meet the requirement of brevetoxin B monitoring in seafood. Next, we investigated the reproducibility and precision of the as-prepared brevetoxin−magnetic bead and mAb−AuNP−GOx by monitoring three brevetoxin B concentrations including 0.5, 50, and 150 ng/kg, respectively. Each sample concentration was determined six times. As seen from Figure 7A, the relative standard deviations (RSD) were 9.8, 6.9, and 7.5% (n = 6) for the above-mentioned target brevetoxin B, respectively. Therefore, the reproducibility and precision of the Ag(I)−TMBbased colorimetric immunoassay were acceptable. Furthermore, the specificity and selectivity of the Ag(I)− TMB-based colorimetric immunoassay were also studied by spiking brevetoxin A, 1 (50 ng/kg), brevetoxin B, 2 (50 ng/kg), brevetoxin C, 3 (50 ng/kg), okadaic acid, 4 (50 ng/kg), saxitoxin acetate, 5 (50 ng/kg), and dinophysistoxin-1, 6 (50 ng/kg), into blank M. senhousia matrix. As shown in Figure 7B, the as-prepared brevetoxin−magnetic bead and mAb−AuNP− GOx displayed high cross-reactivity toward brevetoxins A and C, whereas no false compliant results were achieved for okadaic acid, saxitoxin acetate, and dinophysistoxin-1. The reason might be the fact that brevetoxins A and C had chemical structures similar to that of brevetoxin B.

ical conversion. The assay was carried out on brevetoxin− magnetic bead by using mAb−AuNP−GOx as the detection antibody with a competitive-type immunoassay format. Brevetoxin−magnetic beads were prepared by covalent conjugation of brevetoxin−BSA onto the aminated magnetic bead with glutaraldehyde, whereas mAb−AuNP−GOx was synthesized on the basis of the interaction between gold nanoparticles and proteins. In the presence of target brevetoxin B, the analyte competed with the immobilized brevetoxin−BSA on magnetic beads for the labeled anti-brevetoxin B antibody on the AuNP. Accompanying the immunocomplex, the GOx catalyzed glucose into gluconic acid and H2O2, and the produced H2O2 could oxidize sulfite to sulfate under acidic condition, which could make Ag(I) oxidize TMB to TMBox, thus resulting in the change of the absorbance. As shown from Figure 6A, the absorbance decreased with increasing brevetoxin B concentration in the sample. A good linear dependence between the absorbance (au) and brevetoxin B level (ng/kg) could be acquired in the dynamic range from 0.5 to 200 ng/kg. The linear regression equation could be fitted to y = −0.0062 × C[brevetoxin B] + 2.0062 (ng/kg, R2 = 0.9996, n = 18). The detection limit (LOD) was estimated to be 0.1 ng/kg (ppt) at the 3sblank criterion, whereas the limit of quantification (LOQ) was calculated to be 0.3 ng/kg according to our previous method29 (Figure 6B). To further clarify the merits of Ag(I)− TMB-based colorimetric immunoassay, the obtained LOD was compared with commercialized brevetoxin ELISA kits obtained from Abraxis LLC (LOD = 50 ng/kg) and other detection schemes (e.g., capillary electrophoresis-based immunoassay = F

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Journal of Agricultural and Food Chemistry Monitoring of M. senhousia Samples. The feasibility of applying the as-prepared brevetoxin−magnetic bead and mAb− AuNP−GOx to evaluate brevetoxin levels in a complex matrix was monitored. Initially, we spiked brevetoxin B standards at random concentrations into M. senhousia homogenate, followed by centrifugation and extraction. Finally, these samples were assayed by the Ag(I)−TMB-based colorimetric immunoassay and the commercialized Abraxis brevetoxin B ELISA kit, respectively. The content of brevetoxin B in these samples obtained by Ag(I)−TMB-based colorimetric immunoassay was calculated according to the above-fitted regression equation (y = −0.0062 × C[brevetoxin B] + 2.0062). The results are listed in Table 1. As seen from this table, all RSD values were