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Dec 30, 2015 - Nanospherical Brush as Catalase Container for Enhancing the. Detection Sensitivity of Competitive Plasmonic ELISA. Xiaolin Huang,. †,...
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Nanospherical Brush as Catalase Container for Enhancing the Detection Sensitivity of Competitive Plasmonic ELISA Xiaolin Huang,†,§ Rui Chen,†,‡,§ Hengyi Xu,*,† Weihua Lai,† and Yonghua Xiong*,†,‡,§ †

State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, P. R. China College of Life Science, Nanchang University, Nanchang 330031, P. R. China § Jiangxi-OAI Joint Research Institute, Nanchang University, Nanchang 330047, P. R. China ‡

S Supporting Information *

ABSTRACT: Plasmonic enzyme-linked immunosorbent assay (pELISA) based on catalase (CAT)-mediated gold nanoparticle growth shows great potential for the determination of disease-related biomarkers at ultralow concentrations by using sandwich formats. However, the relatively low sensitivity of this strategy using competitive formats limits its adoption for hapten detection. Herein, we present an improved competitive pELISA for ultrasensitive detection of ochratoxin A (OTA), where silica nanoparticles carrying poly(acrylic acid) brushes (SiO2@PAA) were used to decrease the affinity of competing antigens to anti-OTA monoclonal antibodies and amplify the signal as a “CAT container” (SiO2@PAA@CAT). The developed competitive pELISA exhibits extremely high sensitivity for OTA with detection limits of 10−18 and 5 × 10−20 g/mL by the naked eye and microplate reader, respectively. These values are at least 7 orders of magnitude lower than that of competitive CAT-based pELISA (10−11 g/mL by the naked eye) and 8 orders of magnitude lower than that of horseradish peroxidase-based conventional ELISA (10−11 g/mL by the microplate reader), respectively. Reliability and robustness of the proposed method were evaluated using actual agricultural products and human serum samples. This study demonstrated the potential of this modified method in practical applications involving the ultrasensitive detection of mycotoxins or other haptens.

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pELISAs usually suffer from several potential shortcomings in practical applications, including unstable aggregation of AuNPs and narrow linear detection ranges.21,25,26 Stevens et al. reported a sandwich colorimetric pELISA based on catalase (CAT)-mediated AuNP growth for determination of ultralow concentrations of PSA and HIV-1 capsid antigen p24.27 In this experiment, hydrogen peroxide (H2O2) plays a key role in regulating the kinetics of AuNP growth to obtain blue- or redcolored solutions because the state of AuNP growth is extremely sensitive to H2O2 concentration. Spherical AuNPs were obtained, and the solution appeared red when the H2O2 concentration was 120 μM; whereas, the obtained AuNPs aggregated and solution was blue when the H2O2 concentration slightly decreased to 119.95 μM.28 Thus, in sandwich pELISA, only an ultralow amount of CAT-labeled antibodies can induce sufficient changes in H2O2 concentration (approximately 0.05 μM) to produce significant tonality changes, thereby largely enhancing detection sensitivity. The limit of detection (LOD) of sandwich pELISA for PSA and p24 antigen reached 10−18 g/ mL under the naked eye.27 Similar results were obtained by

nzyme-linked immunosorbent assay (ELISA) is a wellknown and powerful immunoassay platform1 that has been widely used in food quality control, 2,3 environmental monitoring,4,5 and clinical diagnosis.6,7 Nevertheless, conventional ELISA employing enzyme catalysis substrates that generate colored molecules as signal reporters has relatively low sensitivity, ranging from μg/mL to ng/mL.8−10 Various novel signal transduction mechanisms, such as electrochemistry,11 chemiluminiscence,12 electrochemiluminescence,13 fluorescence,14 time-resolved fluorescence,15 and Raman dyes,16 have been used to improve the sensitivity of conventional ELISA. However, specialized equipment is necessary to detect ultralow concentrations of analytes using the above-mentioned methods, which may limit their widespread utilization in ultrasensitive biosensing. Gold nanoparticles (AuNPs) exhibit an ultrahigh extinction coefficient of surface plasmon resonance absorption.17,18 AuNPs have shown great potential as excellent signal reporters in detection of trace amounts of certain target analytes in immunoassays.17−19 Plasmonic ELISA (pELISA) based on enzyme-induced aggregation or growth of AuNPs has gained considerable attention because of its high sensitivity, low cost, and sufficient signal intensity for naked-eye detection.20−24 Nonetheless, most of the reported AuNP aggregation-based © XXXX American Chemical Society

Received: November 29, 2015 Accepted: December 30, 2015

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Scheme 1. (A) Schematic of SiO2@PAA@CAT@OTA Preparation and (B) Schematic Diagram of the Proposed Quantitative Immunoassay Based on CAT-Catalyzed Growth of AuNPs

loading of the competing antigen or lowering the affinity of the competing antigen to the antibody can increase the sensitivity of competitive ELISA. Compared with sandwich ELISA, we speculate that SiO2@PAA as enzyme container could exhibit a greater potential in improving sensitivity of competitive pELISA because SiO2@PAA can be not only served as traditional enzyme container to generate a signal amplification but also used as a regulator to adjust the binding ability between competitive antigens and antibodies because of its relatively greater volume weight. To confirm this concept, in the present work, OTA was selected as model analyte, which is mainly produced by several Aspergillus and Penicillium, and is classified as a Group 2B carcinogen by the International Agency for Research on Cancer because of its potential nephrotoxic, teratogenic, and immunosuppressive activities.34,35 To maximize the sensitivity of competitive pELISA for OTA detection, SiO2@PAA was synthesized and used as a “CAT container” (SiO2@PAA@CAT) to generate a signal amplification for pELISA. Meanwhile, SiO2@PAA@CAT@OTA with a lower affinity to anti-OTA monoclonal antibodies (anti-OTA mAbs) was prepared by adjusting the coupling ratio of the OTA and CAT of SiO2@PAA@CAT (Scheme 1). The resultant pELISA using SiO2@PAA@CAT@OTA as competing antigen exhibited excellent sensitivity for the determination of OTA, with LODs of 10−18 and 5 × 10−20 g/mL by the naked eye and microplate reader, respectively. These LODs are at least 7 orders of magnitude lower than that of competitive CAT-based pELISA (by the naked eye) and 8 orders of magnitude lower than that of HRP-based conventional ELISA (by the microplate reader), respectively.

Battaglia et al. for detection of HIV-1 protein gp120 at an ultralow concentration of 10−17 g/mL.28 Conversely, in competitive pELISA, sufficient level of CAT-labeled antigens bound on the antibody-coated microplate when no target exists in the sample; the solution appeared colorless or blue-colored because of the large amount of H2O2 consumption. To generate a significant red color, a large number of analytes are needed to compete with CAT-labeled antigens, thereby deteriorating the detection sensitivity of competitive pELISA. As expected, Peng et al. reported a competitive pELISA using CAT−methyltestosterone (MT) conjugate as competing antigens for detection of MT, in which the LOD only reached up to 10−11 g/mL under the naked eye.29 A similar result was also observed in our preliminary experiment on detection of ochratoxin A (OTA) by competitive pELISA (Figure S13). The above results indicate that the detection sensitivity of colorimetric pELISA based on CAT-induced AuNP growth for detection of hapten with competitive immunoassays is considerably lower than the sensitivity obtained for determination of disease-related biomarkers with sandwich immunoassays. Recently, polymer-coated silica nanoparticles (SiO2) as an alternative of conventional nanoparticles for enzyme container have been widely used as a novel signal amplification method for developing an ultrasensitive immunoassay because of their high enzyme loading and high enzyme activity.30,31 For example, Qu et al. used silica nanoparticles carrying poly(acrylic acid) brushes (SiO2@PAA) as horseradish peroxidase (HRP) container to enhance the detection sensitivity of conventional sandwich ELISA by approximately 267-fold.31 In competitive ELISA, detection sensitivity is related to the signal reporters, as well as the relative affinity of antibodies for competitor conjugates and analytes.32,33 In theory, increasing the enzyme B

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EXPERIMENTAL SECTION Materials. 1-Ethyl-3-[3-(dimethylamino)propyl] carbodiimide (EDC, cat. no. 39311), N,N′-dicyclohexylcarbodiimide (DCC, cat. no. D80002), N-hydroxysulfosuccinimide (NHSS, cat. no. 56485), N-hydroxysuccinimide (NHS, cat. no. 130672), bovine liver CAT (cat. no. C1345), bovine serum albumin (BSA, cat. no. A4737), HAuCl4 (cat. no. 520918), H2O2 (cat. no. 349887), and MES (cat. no. M3671) were purchased from Sigma−Aldrich Chemical Co. (St. Louis, MO). Mouse antiOTA mAbs were obtained from Jiangxi Zodolabs Biotech Corp. (Jiangxi, China). All other reagents were analytical grade and purchased from Sinopharm Chemical Corp. (Shanghai, China). The chemicals and materials were used without further purification. 96-Well polystyrene microtiter plates were obtained from Costar Inc. (Cambridge, MA). Immobilization of CAT on SiO2@PAA. CAT was covalently immobilized on SiO2@PAA using the active ester method after electrostatic entrapment. In brief, 1.0 mg of SiO2@PAA were suspended and mixed with 0.5 mL of 50 mM phosphate buffer solution (PBS, pH = 7.0) containing a final concentration of 4.5 mg/mL CAT at ambient temperature for 1 h. After removing the excess CAT by centrifugation and washing with PBS, the electrostatic absorption of CAT was converted into a covalent bond by adding 0.5 mL of 50 mM EDC. The conjugation was allowed to proceed at ambient temperature for 3 h. After centrifugation and discarding the supernatant, the obtained SiO2@PAA@CAT conjugates were washed with PBS (containing 0.05% Tween-20) and stored in PBS (containing 0.1% BSA) at 4 °C with a concentration of 1.0 mg/mL. The SiO2@PAA@CAT complex was characterized by UV−vis spectroscopy (Ultrospec 4300 pro UV/visible spectrophotometer, Amersham Pharmacia Biotech), Fourier transform infrared spectroscopy (Nicolet 5700 FTIR spectrometer, Thermo Fisher Scientific, Inc., Waltham, MA), dynamic light scattering (DLS, Zeta Sizer Nano ZS90, Malvern Instruments Ltd., Worcestershire, U.K.), and transmission electron microscopy (TEM, JEOL JEM-2100, JEOL Ltd., Tokyo, Japan). The binding capacity of CAT was determined by indirect measurement of unbound CAT residues in the supernatant with NanoDrop 2000 (Thermo Fisher Scientific). The activities of free and immobilized CAT were determined by measuring the decrease in absorbance at 240 nm because of H2O2 consumption as previously described.36 Preparation of SiO2@PAA@CAT@OTA. The SiO2@ PAA@CAT@OTA conjugates were prepared by covalently attaching the carboxyl group of OTA to the amino group of SiO2@PAA@CAT in the presence of DCC/NHS, as previously described.37 In brief, the carboxyl group of OTA was activated by suspending OTA, DCC, and NHS in 250 μL of DMF at a mole ratio of 1:5:5. After stirring in the dark at room temperature for 2 h, the activation efficiency of the carboxyl group was tested using thin layer chromatography.38 The activated solution was centrifuged at 10000 rpm for 15 min to remove excess DCC and then added into SiO2@PAA@CAT solution with mole ratios of OTA and CAT to SiO2@PAA@ CAT at 0.1:1, 1:1, and 10:1, respectively. After stirring overnight in the dark, the solution was centrifuged at 10000 rpm for 10 min to remove excess reagents and then washed once with PBS. The precipitates were resuspended in PBS (containing 0.01% BSA) with a concentration of 1.0 mg/mL and stored at 4 °C until further use. For comparison, HRPOTA and CAT-OTA conjugates were also prepared by adding

the activated OTA solution into free CAT or HRP solution with mole ratios of OTA to CAT or HRP at 5:1. After stirring overnight in the dark at room temperature, the reaction solution was dialyzed against 0.01 M PBS (pH 7.0) for 72 h at 4 °C. The extent of conjugation between SiO2@PAA@CAT or CAT and OTA was characterized using UV−vis spectroscopy. Antibody−antigen Binding Affinity Measurements. The binding kinetics was evaluated by biolayer interferometry (BLI) using a BLItz System (ForteBio, Menlo Park, CA). Briefly, anti-OTA mAbs were preimmobilized on the tip surface of Anti-Mouse IgG Fc Capture biosensors (ForteBio) at 25.0 μg/mL in PBS for 600 s. Unbound anti-OTA mAbs were removed from the surface of the sensors by incubation in PBS for 60 s. SiO2@PAA@[email protected]:1, SiO2@PAA@CAT@ OTA1:1, SiO2@PAA@CAT@OTA10:1, CAT-OTA, or HRPOTA at four different concentrations were allowed to bind to the anti-OTA mAbs on the surface for 200 s. Then, the SiO2@ PAA@[email protected]:1, SiO2@PAA@CAT@OTA1:1, SiO2@ PAA@CAT@OTA10:1, CAT-OTA, or HRP-OTA was allowed to dissociate by incubation of the sensors in PBS for 120 s. Association/dissociation kinetics were globally fit using the built-in BLItz software to a 1:1 binding model to calculate the kon and koff. KD was calculated as the kon divided by the koff. All sensorgrams are shown in the Supporting Information. Procedure of Direct Competitive pELISA for OTA. 96Well polystyrene microtiter plates were coated with 100 μL of anti-OTA mAbs diluted to 1:40000 in PBS at 4 °C overnight. After washing the plates for three times with PBST (PBS, pH 7.4, 0.01 M, containing 0.05% Tween 20), the plates were blocked with blocking buffer (1 mg/mL of BSA in PBS) at 37 °C for 1 h. Afterward, the plates were washed three times with PBST. Subsequently, 50 μL/well of SiO2@PAA@CAT@ OTA1:1 diluted in PBS was added and incubated with 50 μL/ well of OTA standards to a desired final concentration by diluting a stock solution with PBS containing 5% methanol. After 1 h at 37 °C, the unbound content was discarded, followed by washing of the microplate three times with PBST, twice with PBS, and once with deionized water. Then, 100 μL of 240 μM H2O2 in MES buffer (1 mM, pH 6.5) per well was added into the microplate. After 30 min, 100 μL of freshly prepared HAuCl4 (0.2 mM) in MES buffer was added to each well. After incubating for 10 min, the reaction was stopped by adding 50 μL of 100 μM glutathione to each well.39 Finally, the absorbance at 562 nm and photographs of the solutions were recorded with a microplate reader and a Sony DSC-HX300 digital camera, respectively. The CAT- or HRP-based ELISA was compared with the SiO2@PAA@CAT-based immunoassay. Detailed procedures of CAT-based and HRP-based ELISA are provided in the Supporting Information. Sample Preparation. Samples of corn, wheat, rice, and coffee collected from a local grocery store were finely ground and stored in the freezer at −20 °C before analysis. OTA-free blood samples were provided by the First Affiliated Hospital of Nanchang University. Blood samples were centrifuged at 1500g, and frozen serum was collected in plastic cryotubes and stored in the laboratory at −20 °C until analysis. Samples for pELISA were prepared using the OTA ELISA kit according to the manufacturer’s instructions. Briefly, 1 g or 1 mL of each sample was added into a 10 mL centrifuge tube and extracted with 5 mL of 50% (v/v) methanol in PBS by vigorous shaking for 10 min. Then, the mixture was centrifuged at 8000g for 20 min, and the supernatant was stored for OTA analysis. C

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Figure 1. (A) SiO2@PAA before and after immobilization of CAT. The color change of nanoparticles and their response to centrifugation indicate the success of CAT immobilization. Free CAT cannot be separated by centrifugation. (B) UV−vis spectra of free CAT, SiO2@PAA, and SiO2@ PAA@CAT. (C) FTIR spectra of CAT, SiO2@PAA, and SiO2@PAA@CAT. (D) Hydrodynamic diameter of SiO2@PAA and SiO2@PAA@CAT. (E) TEM image of SiO2@PAA@CAT clearly showing the core−shell structure of SiO2@PAA@CAT.

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RESULTS AND DISCUSSION Synthesis and Characterization SiO2@PAA@CAT. SiO2 nanoparticles were initially prepared to fabricate SiO2@PAA based on a previous report by Stöber et al.40 The as-prepared SiO2 nanoparticles were monodispersed with an average size of 80.1 ± 1.2 nm. The SiO2@PAA was synthesized according to a surface-initiated reversible addition−fragmentation chain transfer (RAFT) polymerization route.41 The detailed synthesis and characterization can be found in the Figures S1−S6. Afterward, the “CAT container” was obtained by covalently coupling the amino group of the CAT enzyme with the carboxyl group of SiO2@PAA using the active ester method after electrostatic entrapment.42 Three key factors that influenced the enzyme loadings and activities including the pH value of coupling buffer, EDC concentration, and contents of CAT were investigated and optimized as shown in Figure S7. The resultant SiO2@PAA@CAT appeared in a characteristic light brown color of CAT and can be visually detected with the naked eye (Figure 1A). The UV−vis spectrum of SiO2@PAA@ CAT showed a characteristic absorption peak at 406 nm from the Soret iron(III) heme structure of CAT (Figure 1B).43 FTIR analysis of SiO2@PAA@CAT showed characteristic absorption peaks corresponding to protein amide bands I (1644 cm−1) and II (1533 cm−1). By contrast, the SiO2@PAA alone displayed characteristic absorption peaks of carboxylic acid at 1273.13 cm−1 (C−O stretch), 1718.35 cm−1 (CO stretch), and 2933.14 cm−1 (O−H stretch) without the amide II band signal

(Figure 1C). Dynamic light scattering (DLS) analysis showed that the average hydrodynamic diameter of SiO2@PAA@CAT slightly increased from 189.6 nm (SiO2@PAA) to 220.2 nm (Figure 1D). Furthermore, the zeta-potential largely increased from −42 to −12.85 mV. Moreover, TEM images (Figure 1E) of SiO2@PAA@CAT after background staining with phosphotungstic acid show that the SiO2@PAA@CAT complex holds a clear core−shell structure with a uniform size distribution. In this structure, an 80 nm silica core was surrounded by a PAA corona with an average dry thickness of 47.2 ± 3.7 nm (n = 50). The above results demonstrated that the CAT was successfully covalently attached onto the surface of the SiO2@ PAA. Under the optimized coupling conditions, the resultant SiO2@PAA@CAT exhibits a very high enzyme loading level at 440.5 ± 8.4 μg CAT per mg of SiO2@PAA. This loading level corresponds to each SiO2@PAA@CAT containing 1900 CAT molecules determined according to the previously reported method.31 Moreover, the catalytic properties of the SiO2@ PAA@CAT complex, including specific activities, Km values (Michaelis−Menten constant), and Vmax values (maximum reaction rate) were determined by measuring the decrease in absorbance at 240 nm according to H2O2 consumption, as described by Feng et al.36 The results in Table S1 indicate that the SiO2@PAA@CAT has a lower Vmax and higher Km values than free CAT. This finding may be explained by the complex three-dimensional structure of PAA brushes, which hinders the D

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principle of competitive ELISA, the competitive antigen with lower affinity for anti-OTA mAbs improves the sensitivity of competitive ELISA. By developing CAT-based direct competitive pELISA, we found that the LOD (10−11 and 10−14 g/ mL by the naked eye and microplate reader, respectively) was lowered by 3 orders of magnitude as compared with HRPbased conventional ELISA (10−11 g/mL by the microplate reader). Moreover, we also analyzed the binding of anti-OTA mAbs to three different SiO2@PAA@CAT@OTA conjugates. The KD values of SiO2@PAA@CAT@OTA10:1, SiO2@PAA@ CAT@OTA1:1, and SiO2@PAA@[email protected]:1 are also shown in Table 1. Among these values, the KD of SiO2@ PAA@CAT@OTA1:1 for anti-OTA mAbs was 3.4 × 10−5 M, which is about 17-fold and 262-fold higher than those of CATOTA and HRP-OTA for anti-OTA mAbs, respectively. To achieve a better analytical performance in the detection of ultralow concentrations of OTA, we first selected SiO2@PAA@ CAT@OTA1:1 conjugates as competitive antigens for the development of a direct competitive pELISA. The concentrations of anti-OTA mAbs and SiO2@PAA@CAT@OTA1:1 were optimized by checkerboard titration. The results shown in Figure S12 and Table S2 indicate that the optimum working conditions are 40000-fold diluted anti-OTA mAbs (1 mg/mL) and 50-fold diluted SiO2@PAA@CAT@OTA1:1 (1 mg/mL). Under these conditions, the solution in the highlighted well was basically colorless with an absorbance of 0.083 ± 0.001. Under the developed optimum conditions, the competitive standard curves of the proposed method were determined by diluting different concentrations (from 0 to 10−12 g/mL) of the OTA standard in 5% methanol−PBS. Figure 2A displays the notable

accessibility of H2O2 molecules to the enzyme active sites and thus reduces the catalytic reaction rate of CAT on SiO2@PAA. However, the specific activities of CAT on SiO2@PAA were retained by approximately 52.2% compared with those of free CAT. In brief, the resultant SiO2@PAA@CAT, which served as a “CAT container,” exhibited an adequate CAT loading capability and enzyme activity for signal amplification in the pELISA. Development of Direct Competitive pELISA. In competitive pELISA formats, the SiO2@PAA@CAT and antiOTA mAb conjugates or SiO2@PAA@CAT and OTA conjugates are essential detection components for indirect or direct competitive pELISA. First, we prepared the SiO2@ PAA@CAT@anti-OTA mAb conjugate according to the EDC/ NHSS method as previously described.44 However, the enzymatic activity of the resultant SiO2@PAA@CAT@ antiOTA mAb conjugates sharply declined by more than 70% after anti-OTA mAb immobilization (Figure S10). A possible reason is that the anti-OTA mAbs on the surface of SiO2@PAA@CAT blocked the enzyme active sites.45 Using the SiO2@PAA@ CAT@anti-OTA mAb conjugates as enzyme labels in an indirect competitive pELISA, the LOD for OTA detection only reached 10−13 g/mL (Figure S11). To retain a higher enzymatic activity of SiO2@PAA@CAT for enhancing the detection sensitivity, we proposed covalent coupling of the carboxylic group of OTA with the amino group of SiO2@PAA@CAT to form SiO2@PAA@CAT@OTA conjugates in the presence of DCC/NHS.37 The enzymatic activity of SiO2@PAA@CAT@ OTA conjugates exhibits a negligible change (Figure S10). To investigate the effect of the coupling ratio of OTA and SiO2@ PAA@CAT on the sensitivity of the competitive pELISA, we synthesized three different SiO2@PAA@CAT@OTA conjugates as competing antigens by controlling the mole ratio of OTA and CAT of SiO2@PAA@CAT at 0.1:1, 1:1, and 10:1, respectively. For comparison, HRP-OTA and CAT-OTA conjugates were also synthesized using the same method. The relative affinity of competitor antigens for antibodies is a key parameter that influences the sensitivity of competitive ELISA. Thus, the binding kinetics of anti-OTA mAbs for the three different SiO2@PAA@CAT@OTA conjugates, HRPOTA, and CAT-OTA were evaluated by BLI analysis using a BLItz System.46,47 The results of the anti-OTA mAb binding studies for different competing antigens, particularly the association rates (kon), dissociation rates (koff), and the equilibrium dissociation constants (KD), are summarized in Table 1. The KD of HRP-OTA for anti-OTA mAbs was 1.3 ×

Figure 2. Quantitative immunoassay of OTA in spiked PBS containing 5% methanol with different concentrations of OTA. (A) Naked-eye detection of OTA with different concentrations in 5% methanol−PBS. (B) Graph showing the absorbance of the solutions at 562 nm compared with the concentration of OTA. Each value represents the mean of four independent experiments (n = 4).

Table 1. Characterization of KD Values for Anti-OTA mAbs and Different OTA Antigen Using the BLItz® System type SiO2@PAA@[email protected]:1 SiO2@PAA@CAT@OTA1:1 SiO2@PAA@CAT@OTA10:1 CAT-OTA HRP-OTA

KD (M) 1.8 3.4 5.5 2.0 1.3

× × × × ×

10−4 10−5 10−6 10−6 10−7

kon (1/Ms) 1.7 1.6 1.2 1.4 1.0

× × × × ×

102 103 104 104 105

koff (1/s) 3.1 5.5 6.9 2.8 1.4

× × × × ×

10−2 10−2 10−2 10−2 10−2

color change of the AuNP solutions from red to blue to colorless with decreasing OTA concentration, which can be easily discriminated with the naked eye. The LOD of the naked eye, defined as the lowest concentration of OTA that produced a red-colored AuNP solution, was 10−18 g/mL. We also analyzed the OTA concentration-dependent absorbance change with the microplate reader. In Figure 2B, the optical density of AuNP solution at 562 nm (OD562) decreased from 0.227 ± 0.007 to 0.101 ± 0.011 with the decrease in OTA concentrations from 10−18 g/mL to 10−20 g/mL and then

10−7 M, and the LOD of the HRP-based conventional competitive ELISA only reached up to 10−11 g/mL. The KD of CAT-OTA for anti-OTA mAbs was 2.0 × 10−6 M, which was approximately 10-fold higher than that of HRP-OTA for antiOTA mAbs, suggesting a weaker binding ability between CATOTA and anti-OTA mAbs. In accordance with the basic E

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Figure 3. Specificity experiment for 1 ng/mL OTA, DON, ZEN, AFB1, and FB1. Each value represents the mean of three independent experiments (n = 3).

simultaneously analyzed with conventional ELISA and our proposed approach. As shown in Table S5 and S6, the average recovery from our proposed method ranged from 80.9% to 124.0%, with a coefficient of variation (CV) ranging from 8.75% to 16.31%. On the other hand, the average recovery from conventional ELISA ranged from 81% to 118%, with a CV ranging from 10.09% to 14.30%. The results indicate that the newly developed pELISA is comparable with conventional ELISA, and no significant difference in the quantitative determination of OTA was noted between the two methods (p > 0.05). In addition, these results show that the AuNP growth-based immunoassay can be used for detecting ultralow concentrations of OTA in actual food or serum samples. The ultrahigh sensitivity of our method allows the repeated dilution of complex food or serum samples, which largely reduces the matrix interference from background substances in the sample.

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CONCLUSIONS

In this work, we designed a new nanocomposite and CAT complex (SiO2@PAA@CAT@OTA1:1) that serves as a competing antigen in the detection of ultralow concentrations of OTA with the use of competitive pELISA. The novel competing antigen exhibited dual roles for improving the sensitivity of competitive pELISA, including increasing the CAT loading capacity by PAA brushes and decreasing the affinity of competing antigen for anti-OTA mAbs by coupling reasonable amounts of OTA onto large SiO2@PAA@CAT nanocomposites. The improved competitive pELISA exhibited ultrahigh sensitivity in detecting OTA molecules at LODs of 10−18 and 5 × 10−20 g/mL by the naked eye and microplate reader, respectively. These LODs are at least 7 orders of magnitude lower than that of competitive CAT-based pELISA (by the naked eye) and 8 orders of magnitude lower than HRPbased conventional ELISA (by the microplate reader), respectively. The reliability and robustness of the proposed method were also tested using actual food and serum samples. In brief, the developed competitive pELISA provides promising and versatile opportunities for the detection of other toxins and haptens at several molecular levels. F

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(4) Brena, B. M.; Arellano, L.; Rufo, C.; Last, M. S.; Montaño, J.; Cerni, E. E.; Gonzalez-Sapienza, G.; Last, J. A. Environ. Sci. Technol. 2005, 39, 3896−3903. (5) Wang, L.; Chen, W.; Xu, D.; Shim, B. S.; Zhu, Y.; Sun, F.; Liu, L.; Peng, C.; Jin, Z.; Xu, C.; et al. Nano Lett. 2009, 9, 4147−4152. (6) Liu, D.; Huang, X.; Wang, Z.; Jin, A.; Sun, X.; Zhu, L.; Wang, F.; Ma, Y.; Niu, G.; Hight Walker, A. R.; et al. ACS Nano 2013, 7, 5568− 5576. (7) Ambrosi, A.; Airò, F.; Merkoçi, A. Anal. Chem. 2010, 82, 1151− 1156. (8) de la Rica, R.; Stevens, M. M. Nat. Protoc. 2013, 8, 1759−1764. (9) Lai, W.; Tang, D.; Zhuang, J.; Chen, G.; Yang, H. Anal. Chem. 2014, 86, 5061−5068. (10) Vashist, S. K.; Schneider, E. M.; Luong, J. H. Biosens. Bioelectron. 2015, 67, 73−78. (11) Chikkaveeraiah, B. V.; Bhirde, A. A.; Morgan, N. Y.; Eden, H. S.; Chen, X. ACS Nano 2012, 6, 6546−6561. (12) Wang, S.; Ge, L.; Song, X.; Yu, J.; Ge, S.; Huang, J.; Zeng, F. Biosens. Bioelectron. 2012, 31, 212−218. (13) Zamolo, V. A.; Valenti, G.; Venturelli, E.; Chaloin, O.; Marcaccio, M.; Boscolo, S.; Castagnola, V.; Sosa, S.; Berti, F.; Fontanive, G.; et al. ACS Nano 2012, 6, 7989−7997. (14) Maeda, Y.; Smith, B.; Agre, P.; Knepper, M. J. Clin. Invest. 1995, 95, 422−428. (15) Papanastasiou-Diamandi, A.; Christopoulos, T. K.; Diamandis, E. P. Clin. Chem. 1992, 38, 545−548. (16) Guarrotxena, N.; Liu, B.; Fabris, L.; Bazan, G. C. Adv. Mater. 2010, 22, 4954−4958. (17) Shen, W.; Deng, H.; Gao, Z. J. Am. Chem. Soc. 2012, 134, 14678−14681. (18) Guo, L.; Xu, Y.; Ferhan, A. R.; Chen, G.; Kim, D.-H. J. Am. Chem. Soc. 2013, 135, 12338−12345. (19) Yang, X.; Yu, Y.; Gao, Z. ACS Nano 2014, 8, 4902−4907. (20) Howes, P. D.; Rana, S.; Stevens, M. M. Chem. Soc. Rev. 2014, 43, 3835−3853. (21) Zhou, C.-H.; Zhao, J.-Y.; Pang, D.-W.; Zhang, Z.-L. Anal. Chem. 2014, 86, 2752−2759. (22) Lin, M.; Pei, H.; Yang, F.; Fan, C.; Zuo, X. Adv. Mater. 2013, 25, 3490−3496. (23) Huang, Z.; Wang, H.; Yang, W. ACS Appl. Mater. Interfaces 2015, 7, 8990−8998. (24) Liu, D.; Wang, Z.; Jin, A.; Huang, X.; Sun, X.; Wang, F.; Yan, Q.; Ge, S.; Xia, N.; Niu, G.; et al. Angew. Chem., Int. Ed. 2013, 52, 14065− 14069. (25) Liang, J.; Yao, C.; Li, X.; Wu, Z.; Huang, C.; Fu, Q.; Lan, C.; Cao, D.; Tang, Y. Biosens. Bioelectron. 2015, 69, 128−134. (26) Liu, D.; Yang, J.; Wang, H.-F.; Wang, Z.; Huang, X.; Wang, Z.; Niu, G.; Hight Walker, A.; Chen, X. Anal. Chem. 2014, 86, 5800− 5806. (27) de La Rica, R.; Stevens, M. M. Nat. Nanotechnol. 2012, 7, 821− 824. (28) Cecchin, D.; de La Rica, R.; Bain, R.; Finnis, M.; Stevens, M.; Battaglia, G. Nanoscale 2014, 6, 9559−9562. (29) Peng, C.; Duan, X.; Khamba, G. W.; Xie, Z. Anal. Methods 2014, 6, 9616−9621. (30) Zhao, Y.; Zheng, Y.; Kong, R.; Xia, L.; Qu, F. Biosens. Bioelectron. 2016, 75, 383−388. (31) Qu, Z.; Xu, H.; Xu, P.; Chen, K.; Mu, R.; Fu, J.; Gu, H. Anal. Chem. 2014, 86, 9367−9371. (32) Robert, E. C. Hapten Versus Competitor Design Strategies for Immunoassay Development. In Immunoanalysis of Agrochemicals: Emerging Technologies; Nelson, J. O., Karu, A. E., Wong, R. B., Eds.; ACS Symposium Series 586; American Chemical Society: Washington, DC, 1995; pp 140−152. (33) Li, K.; Chen, R.; Zhao, B.; Liu, M.; Karu, A. E.; Roberts, V. A.; Li, Q. X. Anal. Chem. 1999, 71, 302−309. (34) Liu, X.; Xu, Y.; Wan, D.; Xiong, Y.; He, Z.; Wang, X.; Gee, S. J.; Ryu, D.; Hammock, B. D. Anal. Chem. 2015, 87, 1387−1394.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b04518. Details about the following are available: synthesis and characterization of SiO2@PAA, SiO2@PAA@CAT, SiO2@PAA@CAT@OTA, and CAT-OTA (Figure S1− S9 and Table S1); change of the CAT activity of SiO2@ PAA@CAT after anti-OTA mAb and OTA immobilization (Figure S10); SiO2@PAA@CAT@anti-OTA mAb conjugate-based pELISA for OTA detection (Figure S11); checkerboard method for anti-OTA mAbs and SiO2@PAA@CAT@OTA (Figure S12 and Table S2); CAT-based pELISA for OTA detection (Figure S13); HRP-based conventrational ELISA for OTA detection (Figure S14); SiO2@PAA@[email protected]:1-based pELISA for OTA detection (Figure S15); SiO2@PAA@ CAT@OTA 10:1-based pELISA for OTA detection (Figure S16); additional pictures from four independent experiments for SiO2@PAA@CAT@OTA1:1-based pELISA (Figure S17); antibody−antigen binding affinity analysis (Figure S18); error analysis (Table S3); application of Poisson-Binomial model in the interpretation of single molecule detection of plasmonic ELISA (Table S4); and recoveries of OTA spiked to practical samples detected by pELISA and conventional ELISA (Table S5 and S6) (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Address: State Key Laboratory of Food Science and Technology, Nanchang University, 235 Nanjing East Road, Nanchang 330047, P.R. China. E-mail: [email protected] and [email protected]. Tel: +0086-791-8830-4447, ext 9512. Fax: +0086-791-8830-4400. *Address: State Key Laboratory of Food Science and Technology, and Jiangxi-OAI Joint Research Institute, Nanchang University, 235 Nanjing East Road, Nanchang 330047, P.R. China. E-mail: [email protected]. Tel: +0086-791-8833-4578. Fax: +0086-791-8833-3708. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by the National Basic Research Program of China (2013CB127804), Training Plan for the Main Subject of Academic Leaders of Jiangxi Province (20142BCB22004), Training Plan for the Young Scientist (Jinggang Star) of Jiangxi Province (20142BCB23004), and the Innovation Fund Designated for Graduate Students of Nanchang University (cx2015107).

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REFERENCES

(1) Xianyu, Y.; Wang, Z.; Jiang, X. ACS Nano 2014, 8, 12741−12747. (2) Asensio, L.; González, I.; García, T.; Martín, R. Food Control 2008, 19, 1−8. (3) Szakal, C.; Roberts, S. M.; Westerhoff, P.; Bartholomaeus, A.; Buck, N.; Illuminato, I.; Canady, R.; Rogers, M. ACS Nano 2014, 8, 3128−3135. G

DOI: 10.1021/acs.analchem.5b04518 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry (35) Liu, X.; Xu, Y.; Xiong, Y.; Tu, Z.; Li, Y.; He, Z.; Qiu, Y.; Fu, J.; Gee, S. J.; Hammock, B. D. Anal. Chem. 2014, 86, 7471−7477. (36) Feng, Q.; Hou, D.; Zhao, Y.; Xu, T.; Menkhaus, T. J.; Fong, H. ACS Appl. Mater. Interfaces 2014, 6, 20958−20967. (37) Kawamura, O.; Sato, S.; Kajii, H.; Nagayama, S.; Ohtani, K.; Chiba, J.; Ueno, Y. Toxicon 1989, 27, 887−897. (38) Sherma, J. J. Chromatogr. A 2000, 880, 129−147. (39) Gobbo, P.; Biondi, M. J.; Feld, J. J.; Workentin, M. S. J. Mater. Chem. B 2013, 1, 4048−4051. (40) Stöber, W.; Fink, A.; Bohn, E. J. Colloid Interface Sci. 1968, 26, 62−69. (41) Qu, Z.; Hu, F.; Chen, K.; Duan, Z.; Gu, H.; Xu, H. J. Colloid Interface Sci. 2013, 398, 82−87. (42) Qu, Z.; Chen, K.; Gu, H.; Xu, H. Bioconjugate Chem. 2014, 25, 370−378. (43) Periasamy, A. P.; Ho, Y.-H.; Chen, S.-M. Biosens. Bioelectron. 2011, 29, 151−158. (44) Huang, X.; Aguilar, Z. P.; Li, H.; Lai, W.; Wei, H.; Xu, H.; Xiong, Y. Anal. Chem. 2013, 85, 5120−5128. (45) Hoffman, A. S.; Stayton, P. S. Prog. Polym. Sci. 2007, 32, 922− 932. (46) Ekiert, D. C.; Friesen, R. H.; Bhabha, G.; Kwaks, T.; Jongeneelen, M.; Yu, W.; Ophorst, C.; Cox, F.; Korse, H. J.; Brandenburg, B.; et al. Science 2011, 333, 843−850. (47) Friesen, R. H.; Lee, P. S.; Stoop, E. J.; Hoffman, R. M.; Ekiert, D. C.; Bhabha, G.; Yu, W.; Juraszek, J.; Koudstaal, W.; Jongeneelen, M. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 445−450. (48) Zaied, C.; Abid, S.; Zorgui, L.; Bouaziz, C.; Chouchane, S.; Jomaa, M.; Bacha, H. Food Control 2009, 20, 218−222.

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DOI: 10.1021/acs.analchem.5b04518 Anal. Chem. XXXX, XXX, XXX−XXX