Novel Electrochemical Immunoassay for Quantitative Monitoring of

Sep 2, 2013 - Novel Electrochemical Immunoassay for Quantitative Monitoring of Biotoxin Using Target-Responsive Cargo Release from Mesoporous Silica ...
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Novel Electrochemical Immunoassay for Quantitative Monitoring of Biotoxin Using Target-Responsive Cargo Release from Mesoporous Silica Nanocontainers Bing Zhang,† Bingqian Liu,† Jiayao Liao,‡ Guonan Chen,† and Dianping Tang*,† †

Key Laboratory of Analysis and Detection for Food Safety (Ministry of Education & Fujian Province), Department of Chemistry, Fuzhou University, Fuzhou 350108, People’s Republic of China ‡ Key Laboratory on Luminescence and Real-Time Analysis (Ministry of Education), College of Chemistry, Southwest University, Chongqing 400715, People’s Republic of China ABSTRACT: A novel homogeneous immunoassay protocol was designed for quantitative monitoring of small molecular biotoxin (brevetoxin B, PbTx-2, as a model) by using targetresponsive cargo release from polystyrene microsphere-gated mesoporous silica nanocontainer (MSN). Initially, monoclonal mouse anti-PbTx-2 capture antibody was covalently conjugated onto the surface of MSN (mAb-MSN), and the electroactive cargo (methylene blue, MB) was then trapped in the pores of mAb-MSN by using aminated polystyrene microspheres (APSM) based on the electrostatic interaction. Upon addition of target PbTx-2, the positively charged APSM was displaced from the negatively charged mAb-MSN because of the specific antigen−antibody reaction. Thereafter, the molecular gate was opened, and the trapped methylene blue was released from the pores. The released methylene blue could be monitored by using a square wave voltammetry (SWV) in a homemade microelectrochemical detection cell. Under optimal conditions, the SWV peak current increased with the increasing of PbTx-2 concentration in the range from 0.01 to 3.5 ng mL−1 with a detection limit (LOD) of 6 pg mL−1 PbTx-2 at the 3Sblank criterion. Intra- and interassay coefficients of variation with identical batches were ≤6% and 9.5%, respectively. The specificity and sample matrix interfering effects were acceptable. The analysis in 12 spiked seafood samples showed good accordance between results obtained by the developed immunoassay and a commercialized enzyme-linked immunosorbent assay (ELISA) method. Importantly, the target-responsive controlled release system-based electrochemical immunoassay (CRECIA) offers a promising scheme for the development of advanced homogeneous immunoassay without the sample separation and washing procedure.

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specific binding members separately labeled with an acridanbased chemiluminescent compound and a peroxidase.11 Kreisig et al. presented a homogeneous fluorescence-based immunoassay by using a phosphorylation-specific antibody-labeled acceptor and the corresponding peptide probe with a donor fluorophore.12 Among these methods, the assays usually required proprietary or specialty fluorescent labels or enzyme labels. Unfavorably, these labels may result in the production of background issues due to autofluorescence of biological samples or interfering compounds in some cases. In contrast with optical detection methods, the electrochemical detection method holds great potential as the next-generation detection strategy because of its high sensitivity, simple instrumentation, and excellent compatibility with miniaturization technologies.13−15 Recently, Hu and colleagues developed a separation-free, electrochemical proximity immunoassay format with direct readout that is amenable to highly sensitive and selective quantitation of a wide variety of target proteins.16 The

he important role of small molecular detection in the fields of medical diagnostics, drug discovery, environmental monitoring, and food safety has driven the everincreasing demand for developing simple, sensitive, highly selective, and cost-effective biosensors.1,2 One approach has been to devise so-called homogeneous immunoassay formats where no separation of a detectable specific binding member is needed.3 The scheme depends on devising a detection principle that is modulated and either turned on or turned off as a result of the binding reaction.4 Usually, the homogeneous immunoassay involves in the immobilization of the biomolecules on the nano/microbeads and takes place in the solution, thus allowing the integration of multiple liquid handling processes.5 In contrast, heterogeneous immunoassay usually requires a step of separating antigen or antibody from the samples and multistep washing.6 Hence, exploring a new homogeneous immunoassay without the need of sample separation and washing procedure would be advantageous. Nowadays, most homogeneous immunoassays are usually implemented by using fluorescence or chemiluminescence.7−10 Akhavan-Tafti and co-workers reported a homogeneous chemiluminescent immunoassay method featuring the use of © 2013 American Chemical Society

Received: July 1, 2013 Accepted: September 2, 2013 Published: September 2, 2013 9245

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from a Millipore water purification system (≥18 MΩ, Milli-Q, Millipore) was used in all runs. Phosphate-buffered saline (PBS, 0.1 M) solution with various pH values was prepared by mixing K2HPO4 and KH2PO4, and 0.1 M KCl was used as the supporting electrolyte. Synthesis of Antibody-Conjugated Mesoporous Silica Nanoparticle (mAb-MSN). Prior to bioconjugation, mesoporous silica nanoparticle (MSN) with 150 nm in diameter was synthesized according to our previous report.29 Briefly, 0.25 g of CTAB was initially dissolved in 120 mL of distilled water, and then 875 μL of sodium hydroxide (2.0 M) was injected into CTAB solution with stirring for 20 min at 80 °C. Afterward, 1.25 mL of TEOS was dropped into the above solution, and vigorously stirred for 2 h until white precipitates were obtained. Following that, the obtained product was filtered, washed with distilled water and methanol, and dried in air. Subsequently, 0.164 g of the precipitates was refluxed for 10 h in a mixture containing 1.5 mL of HCl (37%) and 75 mL of methanol to remove the surfactant template. The formed MSN was filtered, washed with distilled water and methanol, and dried for 4 h at 60 °C. Next, the as-prepared MSN was used for the conjugation of mAb by using the glutaraldehyde, similar to our recent report.30 Fifty mg of dried MSN was dispersed in 1 mL of anhydrous ethanol by ultrasonication for 2 h, and then 50 μL APTES was added into the mixture with stirring for 6 h at room temperature (RT). The aminated MSN was formed and separated by centrifugation. Subsequently, the resultant particles were redispersed in 1 mL of anhydrous ethanol containing 300 μL of glutaraldehyde (25 wt %), and continuously stirred for another 6 h at RT. After centrifugation, the glutaraldehyde-functionalized MSN was dispersed in 1 mL of PBS (pH 7.4). Then, 200 μL of the modified MSN suspension was added to 300 μL of carbonate buffer (pH 9.6) containing anti-PbTx-2 antibody (0.2 mg mL−1), and shaken overnight at 4 °C. Following that, 10 μL of 10 wt % BSA in pH 7.4 PBS was injected the suspension, and incubated for 2 h at 4 °C to block the possible residual sites on the MSN. To reduce the resultant Schiff bases and any excess aldehydes, 50 μL of 25 mg mL−1 sodium cyanoborohydride was added to the suspension, and incubated for 1 h at 4 °C. Afterward, the mixture was collected by centrifugation. The obtained pellet (designated as mAb-MSN, C[MSN] ≈ 10 mg mL−1) was resuspended into 1.0 mL of PBS (pH 6.5), and stored at 4 °C for further use. Loading of Methylene Blue in mAb-MSN. The loading of MB in the mAb-MSN was prepared consulting to the literature.26 Briefly, 60 mg of MB aqueous solution was initially added into the prepared-above mAb-MSN (1 mL, C[MSN] ≈ 10 mg mL−1), and then the resulting mixture was gently shaken overnight at 4 °C. During this process, partial MB molecules were stirred into the pores of MSN. After the suspension was filtered, the obtained mAb-MSN loading with MB (designated as MB-mAb-MSN) was added into 1.0 mL of PBS (pH 6.5) containing 30 mg mL−1 APSM, and incubated for 6 h at 4 °C with gentle shaking. As a result, the positively charged APSM was adsorbed on the surface of the negatively charged mAbMSN, and capped on the pores. The APSM-capped mAb-MSN nanocontainer was filtered and washed several times using pH 6.5 PBS until a low background signal was achieved to remove any physically adsorbed MB on the surface of MSN. Finally, the APSM-capped MB-mAb-MSN (designated as MSN-APSM)

electrochemical signal mainly derived from the labeled electroactive species methylene blue. Despite many advances in this field, there is still the quest for new schemes and strategies to simplify the assay procedure of homogeneous immunoassays. Mesoporous silica (MSN) attracts substantial research interests due to its nontoxic nature, high surface area, large pore volume, tunable pore size and chemically modifiable surface, which allows the encapsulation of substrates in the pores.17−19 Recent reports on the design of capped and gated mesoporous silica derivatives have shown promise in the generation of controlled-release systems, for example, by using inorganic nanoparticles, polymers, and larger supramolecular assemblies as the blocking caps to control opening/closing of pore entrances of mesoporous silica.20−23 However, such controlled release system was usually employed for drug delivery.24,25 To the best of our knowledge, there is no report focusing on target-responsive controlled cargo release from mesoporous silica for the development of electrochemical homogeneous immunoassays until now. One of the major bottlenecks is the readout method. Favorably, the Willner group recently reported a new method for the encapsulation of electroactive species (methylene blue and thionine) in the pores of mesoporous silica by means of functional nanostructures consisting of the Mg2+- or Zn2+-dependent DNAzyme sequence.26 The embedding cargo could be released from the mesoporous silica in the presence of target, which was monitored by fluorescence. We reasoned that it might be generally useful for other types of highly sensitive and selective assays if such assays could be extended to detect species other than fluorescence assay. Brevetoxin B (PbTx-2), as a neurotoxin produced by algae, can cause intoxication and even mortality through consumption of brevetoxin-contaminated shellfish, and affect respiratory irritation through aerosol exposure at coastal areas.27,28 Herein, we report the proof-of-concept of a controlled release systembased homogeneous electrochemical immunoassay for quantitative detection of PbTx-2 (as a model analyte). The assay is carried out based on target-responsive controlled release of methylene blue from polystyrene microsphere-gated mesoporous silica nanocontainer. Initially, methylene blue is loaded into the pores of monoclonal mouse anti-PbTx-2 antibodyfunctionalized mesoporous silica, and the pores are then capped with aminated polystyrene microspheres. Upon target introduction, the molecular gate is opened, resulting in the cargo release from the pores. The released methylene blue can be quantitatively monitored by a voltammetry without the need of sample separation and washing procedure.

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EXPERIMENTAL SECTION Materials and Reagents. Individual standard stock samples of brevetoxin B (PbTx-2, with a neutral charge) were purchased from Express Technol. Co., Ltd. (Beijing, China). Monoclonal mouse anti-PbTx-2 antibody (mAb) was provided by Jiangnan University (Wuxi, China). Glutaraldehyde, methylene blue (MB), cetyltrimethylammonium bromide (CTAB), and tetraethoxysilane (TEOS) were purchased from Sinopharm Chem. Re. Co., Ltd. (Shanghai, China). Aminated polystyrene microspheres (APSM, 25 nm in diameter) were obtained from Phosphorex, Inc. (U.S.A.). 3-Aminopropyltriethoxysilane (APTES) was purchased from Sigma-Aldrich (U.S.A.). All other reagents were of analytical grade and were used without further purification. Ultrapure water obtained 9246

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Scheme 1. Schematic Illustration of the CRECIA-Based Electrochemical Immunoassay Based on Target-Responsive Controlled Release of Methylene Blue (MB) from Aminated Polystyrene Microspheres (APSM)-Gated Antibody-Functionalized Mesoporous Silica Nanocontainer (MSN)a

The molecular gate was closed based on the electrostatic interaction between the positively change −NH3+ groups on the APSM and the negatively charged antibody on the MSN. a

was redispersed in 1.0-mL PBS (pH 6.5) (C[MSN] ≈ 10 mg mL−1) and used for the detection of PbTx-2. Immunoassay Protocol and Voltammetric Measurement. To monitor the release process of methylene blue from mesoporous silica nanocontainer in the presence of target PbTx-2, 50 μL of the prepared-above MSM-APSM suspension was initially diluted with 450 μL of PBS (pH 6.5) in a 1.5-mL centrifugal tube. Then, 50 μL of various-concentration PbTx-2 standards/samples were added to the tube. The tubes were shaken occasionally during the reaction at RT. During this process, target PbTx-2 triggered the MSN-APSM, and displaced the APSM from the MSN-APSM because of the specific antigen−antibody reaction, thereby resulting in the release of methylene blue from the nanocontainers. After incubation for 80 min at RT, 300 μL of the resulting supernatant was transferred into a homemade microelectrochemical detection cell for voltammetric measurement. Electrochemical measurements were carried out with a microAutoLab Type III system (Eco Chemie, The Netherlands). The detection system comprised an indium−tin oxide (ITO, 5 wt % In2O3 + SnO2) working electrode, a platinum wire as auxiliary electrode, and an Ag/AgCl reference electrode. The electrochemical signal was recorded and collected in pH 6.5 PBS by using square wave voltammetry (SWV) from −400 to 0 mV (vs Ag/AgCl; amplitude = 25 mV; frequency = 15 Hz; increase E = 4 mV). All electrochemical measurements were done in an unstirred electrochemical cell at room temperature (25 ± 1.0 °C). All data were obtained with three measurements each in parallel. Scheme 1 represents the assay protocol of target-responsive controlled release system for electrochemical immunoassay of PbTx-2.

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nanostructures. With target introduction, a competitive-type displacement reaction is implemented between APSM and target PbTx-2 for anti-PbTx-2 antibody binding on the MSN because of the specific antigen−antibody reaction. The displaced APSM affords the open of molecular gate, resulting in the release of methylene blue from the pores. The released methylene blue can give rise to the increase of electrochemical signal. The amount of the increased current directly depends on the concentration of target analyte in the sample. To construct such a controlled release system-based electrochemical immunoassay (CRECIA), we design an inhibition assay protocol based on target-responsive controlled release of methylene blue from mesoporous silica nanocontainers. Monoclonal mouse anti-PbTx-2 capture antibody is conjugated covalently onto the outer surface of MSN by APTES and glutaraldehyde. The conjugation was easily implemented, and similar reports have been described in our previous papers.33,34 Aminated polystyrene microspheres with good biocompatibility and water solubility have been found to have numerous applications in scientific research, diagnostics, electronics, pharmaceuticals, and nanotechnology. Since the conjugated anti-PbTx-2 antibody on the MSN is a kind of proteins, the isoelectric point (Ip) is about 5.7 obtained by isoelectric focusing (IEF) electrophoresis. In pH 6.5 PBS, the as-prepared mAb-MSN is negatively charged, which can adsorb the positively charged APSM, thus resulting in the close of nanocontainers. In this case, methylene blue is blocked into the pores. Vice versa, the release of methylene blue from MSNAPSM can be also triggered by challenge with target PbTx-2. Design of electrostatic interaction-based immunoassay system was also reported recently by the Yan’s group.35 Initially, we used transmission electron microscope (TEM) to investigate the feasibility of our design. Figure 1a shows the TEM image of the aminated mesoporous silica with an average size of 150 nm. The aminated MSN has a BET surface area of 956 m2 g−1 and an average BJH pore size of 3.2 nm (Figure 2a). The average size of APSM was 25 nm, as shown in Figure 1b. After mixing with mAb-MSN and APSM together, many APSM spheres were attached on the surface of mAb-MSN (Figure 1c), indicating that MSN-APSM could be formed through the electrostatic interaction. More significantly, when 0.5 ng mL−1 PbTx-2 was added into the MSN-APSM system, partial APSM spheres were dissociated from the MSN-APSM (Figure 1d). The electrostatic interaction between the positively changed −NH3+ groups on the APSM (ξ potential = +23.12 mV) and

RESULTS AND DISCUSSION

Construction and Characterization of Controlled Release System-Based Electrochemical Immunoassay (CRECIA). Silica nanostructures as a kind of semiconductor nanomaterials usually have weak electrochemical activity, while methylene blue as a good redox indicator has been widely used in the electroanalytical chemistry.31,32 Mesoporous silica could be utilized as the building block for the encapsulation of methylene blue. With the formation of capped and gated mesoporous silica derivatives, methylene blue is firmly blocked into the pores. In the absence of target, the entrapped methylene blue in the MSN exhibits very weak electrochemical behavior owing to the blockade of semiconductor silica 9247

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the MSN acted as a gate framework. Without the immobilized mAb around the pores, the pores could not be blocked. So, various mAb concentrations were used for construction of MSN-APSM. To ensure the adequate dissociation of APSM, high concentration of PbTx-2 (10 ng mL−1) was employed for the release of methylene blue. As seen from Figure 3A, the electrochemical signal increased with the increasing mAb concentration, and tended to level off at ≥0.12 mg mL−1 mAb in 500-μL glutaraldehyde-functionalized MSN solution (C[MSN] = 20 mg mL−1). To save the cost, 0.12 mg mL−1 mAb was used for preparation of mAb-MSN in this work. The effect of entrapped amount of MB in the mAb-MSN on the current response of the developed immunoassay was also investigated since the electrochemical signal mainly originated from MB. If the entrapped amount of MB is too small, the released MB is not conducive for the production of electrochemical signal. By using 0.5 ng mL−1 PbTx-2 as an example, we investigated the effect of various MB concentrations on the electrochemical signal of the developed CRECIA. As indicated from Figure 3B, the strong currents could be achieved when the concentration of MB was higher than 55 mg mL−1 MB in the mAb-MSN (C[MSN] ≈ 10 mg mL−1). The results also revealed that the selected amount of MB should be ≥55 mg mL−1. Considering the viscosity effect of using high-concentration MB, 60 mg mL−1 MB should be preferable. By the same token, dependence of electrochemical signal on the concentration of APSM (as the molecular gate) was also monitored (0.5 ng mL−1 PbTx-2 used in this case). The reason might be the fact that the entrapment of MB in the pores was fulfilled by APSM. As shown from Figure 3C, when the concentration of APSM was lower than 30 mg mL−1 in MBmAb-MSN (C[MSN] ≈ 10 mg mL−1), the electrochemical signal was relatively weak. The reason might be the fact that the addition of the low-concentration APSM in the MB-mAb-MSN could cause the aggregation of MSN-APSM (Figure 2b−e). Therefore, 60 mg mL−1 MB and 30 mg mL−1 APSM were utilized for preparation of MSN-APSM at acceptable throughput. The release time of MB from the MSN-APSM directly affected the electrochemical signal of the developed CRECIA. During this process, the antigen−antibody reaction and the release of MB from the pores were simultaneously implemented. The molecular gates were switched on by the specific antigen−antibody reaction. Figure 3D shows the effect of different release times on the current of the CRECIA toward

Figure 1. TEM images of (a) mesoporous silica nanoparticles, (c) the formed MSN-APSM and (d) probe “c” after incubation for 80 min at RT with 0.5 ng mL−1 PbTx-2, and (b) SEM image of aminated polystyrene microspheres.

the negatively charged antibody on the MSN (ξ potential = −16.58 mV) in pH 6.5 PBS should be responsible for the formation of the inhibition immunosensing probe MSN-PSMS (ξ potential = +3.14 mV), indicating the electrostatic nature of the interaction between APSM and mAb-MSN.36 The results revealed that the developed CRECIA could be preliminarily applied for the detection of target PbTx-2. Another question arises as to whether the addition of APSM affected the dispersity of mAb-MSN. We used dynamic light scattering (DLS) to investigate the size distribution of mAbMSN after reaction with various-concentration APSM. As seen from Figure 2b and Figure 2c, the low-concentration APSM resulted in the formation of large-sized nanocomposites. We suspected the reason might be attributed to the fact that one APSM particle at the low concentration could adsorb more mAb-MSN particles than that of high-concentration APSM. To avoid the issue, the high-concentration APSM should be necessary during the preparation of MSN-APSM. Optimization of Experimental Conditions. To achieve an optimal analytical performance of the developed CRECIA, the amount of MB/mAb/APSM applied for preparation of MSN-APSM and the release time of MB should be optimized. During the preparation of MSN-APSM, the conjugated mAb on

Figure 2. (a) Nitrogen adsorption−desorption isotherm of mesoporous silica nanoparticles measured at 78 K (inset shows pore size distribution), and DLS data of mAb-MSN after reaction with (b) 10 mg mL−1 APSM and (c) 40 mg mL−1 APSM, respectively. 9248

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Figure 3. Effect of (A) mAb concentration, (B) MB concentration, (C) APSM concentration, (D) the release time of MB, and (F) pH of PBS on SWV peak current of the developed CRECIA and (E) dynamic analysis of cargo release from (a) the APSM-MSN and (b) the uncapped mAb-MSN without being triggered by PbTx-2.

Figure 4. (a) Typical SWV response curves of the CRECIA-based immunoassays for different-concentration target PbTx-2 standards and (b) SWV peak currents versus various target PbTx-2 levels. Potential scanning was from −400 mV to 0 mV (vs Ag/AgCl, amplitude = 25 mV, frequency = 15 Hz, increase E = 4 mV). Measurements were performed in pH 6.5 PBS. Each data point represents the average value obtained from three different measurements. The error bars represent the 95% confidence interval of the mean for y-axis currents.

0.5 ng mL−1 PbTx-2. A maximum current was obtained after 80 min. Too longer time did not obviously change the electrochemical signal. Hence, 80 min was chosen for the release of MB. For control test, dynamic analysis of cargo release from the uncapped MB-mAb-MSN and the APSMcapped MB-mAb-MSN without being triggered by PbTx-2 was also investigated (Figure 3E). As shown from curve a in Figure 3E, the currents of using the uncapped MB-mAb-MSN increased with the increasing reaction time, and then tended to equilibrium after 50 min. The results indicated that the cargo release from the uncapped MB-mAb-MSN (curve a in Figure 3E) was faster than that of from APSM-MSN with being triggered by PbTx-2 (Figure 3D). Importantly, the currents of by using APSM-MSN without being triggered by PbTx-2 were not nearly changed with the increasing time, suggesting that the

capping strategy based on the electrostatic interaction was successful with good efficiency (curve b in Figure 3E). Since the adsorption of APSM on the mAb-MSN is based on the electrostatic interaction, the pH of the reaction solution should be important for the analytical performance of the CRECIA, especially at the three points: below, at, above the isoelectric point (Ip ≈ 5.7) of anti-PbTx-2. In this case, MSNAPSM was initially prepared in pH 6.5 PBS system, and then the as-prepared MSN-APSM was redispersed into various-pH PBS solutions in the absence of target PbTx-2. The resulting supernatant was monitored by the mentioned-above method. As indicated from Figure 3F, the currents almost tended to level off when pH of PBS was higher than 5.7. In contrast, the currents increased with the decreasing pH of PBS from 5.7 to 3.5. The reason might be most likely a consequence of the fact that partial MSN-APSM conjugates were separated each other 9249

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Figure 5. (a) Specificity of the CRECIA-based immunoassay toward PbTx-1, PbTx-2, PbTx-3, ODA, AFB1, and MCLR (0.5 ng mL−1 used in this case). (Note: These interfering agents had a neutral charge.) (b) Sample matrix interfering effect.

the incubation solution. As seen in Figure 5a, the developed CRECIA exhibited a high cross-reactivity (CR) of >95% for PbTx-1 and PbTx-3, while no false compliant results were obtained for ODA, AFB1, and MCLR. The false positive results for PbTx-1 and PbTx-3, however, are easily explained by the fact that the used anti-PbTx-2 antibody has a CR of 97.45% and 120.63% for PbTx-1 and PbTx-3, respectively.37 Therefore, the developed CRECIA possessed an acceptable selectivity. To investigate the interfering effects of sample matrix components on the analytical properties of the developed CRECIA, several possible components in the seawater, such as Na+, Ca2+, Mg2+, Cl−, SO42−, HCO3−, and F−, were added into the incubation solution containing 0.5 ng mL−1 PbTx-2, respectively. As shown in Figure 5b, these interfering ions did not almost affect the significant change in the electrochemical signal relative to target PbTx-2 alone. The high specificity and anti-interference of the developed CRECIA might be ascribed to the specific antigen−antibody reaction and the strong electrostatic interaction between mAb-MSN and APSM. Analysis of Real Sample. To monitor the possible application of the developed CRECIA for real samples, PbTx2 standards with various concentrations were spiked into three types of seafood samples, such as Sinonovavula constricta, Musculista senhousia, and Tegillarca granosa, respectively, according to our previous reports.38,39 The as-prepared specimens were measured by using the CRECIA and commercialized PbTx-2 ELISA kit (Abraxis LLC, Warminster, U.S.A.) (ultraviolet detector) as a reference method. The assay results using these two methods are listed in Table 1. Statistical comparison of the experimental results with those of ELISA was also performed using a t-test for comparison of means by the application of an F-test and linear regression analysis between two methods. As shown in Table 1, the slope and intercept for the regression equation were close to the ideal unity 1 and 0. Meanwhile, the texp values in all cases were less than tcrit (tcrit[4,0.05] = 2.77). The results revealed a good accordance between both analytical methods, thereby could be regarded as an optional scheme for quantitative determination of PbTx-2.

owing to the formation of mAb-MSN with negative charge or without charge when pH of PBS was ≤5.7, thereby resulting in the release of the entrapped methylene blue from the pores. Considering the bioactivity of the conjugated anti-PbTx-2 and the capping efficiency of mAb-MSN toward MB by APSM, however, pH 6.5 of PBS was used as the reaction solution for the detection of PbTx-2. Dose−Response Curve of the Developed CRECIA. Under the optimal conditions, the developed CRECIA was employed for quantifying PbTx-2 standards with various concentrations based on target-responsive controlled release of MB from APSM-gated mAb-MSN nanocontainers. The assay was carried out in pH 6.5 PBS after incubation target PbTx-2 with MSN-APSM for 80 min at RT. As shown in Figure 4a, the electrochemical signals increased with the increasing PbTx-2 concentrations. A linear dependence between the peak current and the concentration of PbTx-2 was obtained in the range from 10 pg mL−1 to 3.5 ng mL−1 with a low detection limit of 6 pg mL−1 PbTx-2 estimated at a signal-to-noise ration of 3σ (where σ is the standard deviation of a blank solution, n = 11) (Figure 4b). Although our designed system has not yet been optimized for maximum efficiency, the LOD of the developed CRECIA was obviously lower than that of commercialized PbTx-2 ELISA kit (LOD: 0.05 ng mL−1, Abraxis LLC, USA). When the level of PbTx-2 in the sample is more than 3.5 ng mL−1, an appropriate dilution is preferable. More inspiringly, the developed CRECIA did not require sample separation and a complicated wash procedure. Reproducibility, Specificity, and Stability of the Developed CRECIA. To monitor the precision of determinations, we repeatedly assayed high-middle-low 3 PbTx-2 concentrations, using identical batches of MSN-APSM throughout. Experimental results indicated that the coefficients of variation (CVs) of the intra-assay between 5 runs were 5.9%, 3.2%, and 4.8% for 0.05, 0.5, and 3 ng mL−1 PbTx-2, respectively, whereas the CVs of the interassay with various batches were 6.7%, 9.4%, and 8.3% for PbTx-2 toward the mentioned-above targets. The low CVs indicated the possibility of MSN-APSM batch preparation. When the as-prepared MSNAPSM was not in use, it was stored in pH 6.5 PBS at 4 °C. No obvious change in the electrochemical signal was observed after storage for 18 days but a 5% decrease of the current was noticed at 24th day. Further, the specificity of the developed CRECIA was also investigated by spiking PbTx-1, PbTx-2, PbTx-3, okadaic acid (ODA), aflatoxin B1 (AFB1), and microcystin-LR (MCLR) in

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CONCLUSIONS In summary, we for the first time demonstrate the ability of homogeneous electrochemical immunoassay for directly quantitative detection of small molecular biotoxins (PbTx-2 as a model), by using target-responsive controlled release of guest molecules (methylene blue) from polystyrene micro9250

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Control During Aquatic Product Processing) (2012BAD29B06) is gratefully acknowledged.

Table 1. Comparison of the Assay Results for Real Samples Using the developed CRECIA and the Commercialized ELISA Method

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method; concentration (mean ± SD, ng mL−1, n = 3)a sample 1 2 3 4 5 6 7 8 9 10 11 12

b

CRECIA

ELISA

texp

c 0.065 ± 0.019 0.89 ± 0.13 2.78 ± 0.39

0.079 ± 0.015 0.72 ± 0.11 2.34 ± 0.42

1.01 1.73 1.33

0.09 ± 0.01 0.93 ± 0.21 2.57 ± 0.26

0.066 ± 0.012 1.22 ± 0.19 2.83 ± 0.22

2.66 1.77 1.32

0.058 ± 0.009 1.12 ± 0.12 2.71 ± 0.17

0.064 ± 0.01 0.92 ± 0.25 2.95 ± 0.09

0.77 1.25 2.16

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a

The regression equation (linear) for these data is as follows: y = 1.0132x + 0.0064 (R2 = 0.9971, n = 27; x axis, by CRECIA; y axis, by ELISA). bSamples 1−4, 5−8, and 9−12 were used as S. constricta, M. senhousia, and T. granosa supernatants as matrices, respectively. Samples 1, 5, and 9 were the unspiked specimens. These samples were made available as the extract supernatant using the sample preparation. cNot detected. The minimum concentration of PbTx-2 detectable by ELISA is typically 0.05 ng mL−1.

sphere-gated antibody-functionalized mesoporous silica nanocontainers. The electrochemical signal is amplified by the released electroactive species, methylene blue, from the semiconductor nanostructures in the presence of target PbTx2. Compared with conventional homogeneous immunoassays and our most recent report,39 highlight of the developed immunoassay is simple, enzyme-free, label-free, and userfriendly without the need of sample separation and washing. In addition, the precision, reproducibility, and specificity of the homogeneous immunoassay are acceptable. More importantly, the developed CRECIA method does not require sophisticated instruments, and can be utilized by the public for quantitative detection of other small molecular toxins by controlling the used target antibody, thereby representing a versatile detection method.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Phone: +86-591-2286 6125. Fax: +86-591-2286 6135. E-mail: [email protected]. Author Contributions

Zhang, Liu, and Liao contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Support by the National Natural Science Foundation of China (41176079, 41076059, 21075019), the National “973” Basic Research Program of China (2010CB732403), the Doctoral Program of Higher Education of China (20103514120003), the National Science Foundation of Fujian Province (2011J06003), the Program for Changjiang Scholars and Innovative Research Team in University (IRT1116), and the National Key Technologies R&D Program of China during the 12th FiveYear Plan Period (Key Technology of Quality and Safety 9251

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Analytical Chemistry

Article

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dx.doi.org/10.1021/ac4019878 | Anal. Chem. 2013, 85, 9245−9252