Low-Cost and Highly Sensitive Immunosensing Platform for Aflatoxins

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Low-cost and highly sensitive immunosensing platform for aflatoxins using one-step competitive displacement reaction mode and portable glucometer-based detection Dianping Tang, Youxiu Lin, Qian Zhou, Yuping Lin, Peiwu Li, Reinhard Niessner, and Dietmar Knopp Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac503616d • Publication Date (Web): 20 Oct 2014 Downloaded from http://pubs.acs.org on October 21, 2014

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Analytical Chemistry

Low-Cost and Highly Sensitive Immunosensing Platform for Aflatoxins Using One-Step Competitive Displacement Reaction Mode and Portable Glucometer-Based Detection Dianping Tang,*,† Youxiu Lin,† Qian Zhou,† Yuping Lin,† Peiwu Li,ǁ Reinhard Niessner,‡ and Dietmar Knopp*,‡

†

Key Laboratory of Analysis and Detection for Food Safety (MOE & Fujian Province), Institute of Nanomedicine

and Nanobiosensing, Department of Chemistry, Fuzhou University, Fuzhou 350108, People's Republic of China

‡

Chair for Analytical Chemistry, Institute of Hydrochemistry, Technische Universität München, Marchioninistrasse 17, D-81377 München, Germany

‖

Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Wuhan 430062, People's Republic of China

CORRESPONDING AUTHOR INFORMATION Phone: +86-591-2286 6125; fax: +86-591-2286 6135; e-mail: [email protected] (D. Tang) Phone: +49-89-2180 78252; fax: +49-89-2180 78255; e-mail: [email protected] (D. Knopp)

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ABSTRACT: Aflatoxins are highly toxic secondary metabolites produced by a number of different fungi, and present in a wide range of food and feed commodities. Herein we designed a simple and low-cost immunosensing platform for highly sensitive detection of mycotoxins (aflatoxin B1, AFB1, used as a model) on polyethyleneimine (PEI)-coated mesoporous silica nanocontainers (PEI-MSN). The assay was carried out by using a portable personal glucometer (PGM) as the readout based on a competitive displacement reaction mode between target AFB1 and its pseudo hapten (PEI-MSN) for monoclonal anti-AFB1 antibody (mAb). To construct such an assay protocol, two nanostructures including mAb-labeled gold nanoparticle (mAb-AuNP) and PEI-MSN were initially synthesized, and then numerous glucose molecules were gated into the pores based on the interaction between negatively charged mAb-AuNP and positively charged PEI-MSN. In the presence of target AFB1, a competitive-type displacement reaction was implemented between mAb-AuNP and PEI-MSN by target AFB1 through the specific antigen-antibody reaction. Accompanying the reaction, target AFB1 could displace the mAb-AuNP from the surface of PEI-MSN, resulting in the release of the loading glucose from the pores due to the gate open. The released glucose molecules could be quantitatively determined by using a portable PGM. Under the optimal conditions, the PGM signal increased with the increment of AFB1 concentration in the range from 0.01 to 15 μg/kg (ppb) with a detection limit (LOD) of 0.005 μg/kg (5 ppt) at the 3sblank criterion. The selectivity and precision were acceptable. Importantly, the methodology was further validated for assaying naturally contaminated or spiked blank peanut samples, and consistent results between the PGM-based immunoassay and the referenced enzyme-linked immunosorbent assay (ELISA) were obtained. Therefore, the developed immunoassay provides a promising approach for rapid screening of organic pollutants because it is simple, low-cost, sensitive, specific, and without the need of multiple separation and washing steps.

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Mycotoxins, as the toxic secondary metabolites produced by various mold species, contaminate many agricultural commodities in the field or during storage.1,2 Several of these compounds are carcinogenic, mutagenic, and immunosuppressive posing a significant threat to human and animal health through ingestion.3 Among these mycotoxins, aflatoxins have assumed significance because of their deleterious effects on human beings, poultry and livestock. Typically, only four, aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2) are naturally found in a wide variety of foodstuffs, species and medicinal plants.4,5 The order of toxicity, AFB1 > AFB2 > AFG1 > AFG2, indicates that AFB1 is the most potentially toxic.6 In 2006, the European Union (EU) has set the legal limit for sum of aflatoxins to 4 μg/kg for aflatoxins and to 2 µg/kg for AFB1.7 This has prompted adoption of regulatory limits in several countries, which, in turn, requires the development of validated official analytical methods for rapid and cost-effective screening of AFB1 on a large scale. To meet the regulatory levels fixed by the EU or other international organizations, as well as to assess the toxicological risk for humans and animals, various analytical methods and strategies have been applied for the detection of AFB1, e.g., thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), gas chromatography (GC), liquid chromatography-mass spectrometry (LC-MS), enzyme-linked immunosorbent assay (ELISA), direct fluorimetry, fluorescence polarization, and various biosensors/lateral flow devices (LFD).8-13 However, the chromatographic methods not only require expensive instruments and skilled operators, but also complicated sample pretreatment (such as immunoaffinity or solid phase extraction columns),14,15 thus limiting their application, especially in the developing countries. In contrast, the immunoreaction-based microarray chips usually require an expensive robot for spotting probes and a confocal fluorescence scanner or a CCD-camera for obtaining the detectable signal,16-18 which can hamper their broad application. Although TLC with either visual assessment or instrumental densitometry is routinely applied in many laboratories in the developing world, they often give false positive results around the cutoff value owing to the poor visible color.19,20 Thus, it is still a challenge to exploit new approaches that can improve the simplicity, selectivity and sensitivity of these analytical methods. The rapidly emerging research field of nanotechnology provides excitingly new possibilities for advanced development of new analytical methods.21-23 One major merit of using nanotechnology 3



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is that one can control and tailor their properties in a very predictable manner to meet the needs of specific applications.24,25 Recently, target-induced displacement reaction strategy attracts substantial research interests in the analytical fields. Turega et al. established a new fluorescence displacement assay to quantify guest binding based on size and shape criteria for guest binding inside the cavity of an octanuclear cubic coordination cage.26 Zhou et al. developed a simple and efficient approach to purify DNA-invertase conjugates from reaction mixture via a biotin displacement strategy to release desthiobiotinylated DNA-invertase conjugates from streptavidin-coated magnetic beads.27 Unfortunately, most of the present displacement reaction systems were applied to detect nucleic acids, and a few reports were focused on the antigen-antibody reaction.28,29 One possible reason was ascribed to difficult exploiting the weak-affinity pseudo hapten with target antibody. Inspiringly, Wu[29] and Gao[28] found that polyethyleneimine (PEI)-functionalized microspheres could be used as the pseudo hapten for the antibody reaction. Relative to the specific antigen-antibody reaction, the pseudo hapten-antibody interaction was inherently weak, thereby resulting in the progression of target-induced displacement immunoreaction. Another important issue for development of the displacement reaction mode is to adopt a simple and sensitive signal-transduction method. The personal glucometer (PGM) is currently one of the most widely used diagnostic devices in the world because of its portable size, easy operation, low cost and reliable quantitative results.30,31 Glucose is necessary for the development of PGM-based assay method.32 Recently, Lerbret et al. found a strong preferential interaction of glucose molecules with the pore wall of mesoporous silica, which could be induced significant concentration gradients within the pore.33 In this case, the loading glucose molecules could be closed into the pores by using an external gate. Recent reports on the design of capped and gated mesoporous silica nanocontainer (MSN) have shown promise in the generation of controlled release systems, e.g., by using nanoparticles, macrocyclic compounds, dendrimers, and biomacromolecules as the blocking caps to control opening/closing of pore entrances of MSN.34-37 Accordingly, the pores of MSN were capped with the gate units that locked the substrate in the pores and allowed the stimuli-responsive unlocking of the gates and the cargo release.38 In this

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regard, our motivation in this work is to utilize PEI-coated MSN loaded with glucose as the pseudo hapten for the development of PGM-based immunoassay. To this end, we report a simple, low cost and portable immunoassay protocol for rapid screening of small molecular AFB1 based on target-induced release of glucose from the PEI-coated MSN by coupling a competitive-type displacement reaction mode with a general PGM readout, without the need of multiple separation and washing steps. Based on the electrostatic attraction, monoclonal anti-AFB1 antibody-labeled gold nanoparticles (mAb-AuNP) with negative charge (as the molecular gate) are initially coated onto the surface of PEI-functionalized MSN, which can gate the glucose molecules within the pores. Upon target AFB1 introduction, a specific competitive-type displacement reaction for mAb-AuNP is carried out between PEI-MSN and target AFB1, thus resulting in the dissociation of mAb-AuNP from PEI-MSN. Thereafter, the previously loaded glucose molecules can be released from the pores and monitored by using an external PGM. By evaluation of the PGM signal the concentration of target AFB1 in the sample can be calculated.

■EXPERIMENTAL SECTION Material and Reagent. Monoclonal anti-AFB1 antibody (mAb 62, clone 2B7) was prepared and characterized in our laboratory as described.39 AFB1 standards with various concentrations were purchased from Express Technol. Co. Ltd. (Beijing, China). Polyethyleneimine (PEI, branched, MW 600, 99 wt %) was purchased from Alfa Aesar®. Tetraethoxysilane (TEOS) and sodium citrate tribasic hydrate were obtained from Sigma (USA). HAuCl4·4H2O was purchased from Sinopharm Chem. Re. Co., Ltd. (Shanghai, China). Cetyltrimethylammonium bromide (CTAB) was products of Beijing Dingguo Biotechnol. Co. Ltd. (Beijing, China). All other reagents were of analytical grade and were used without further purification. Ultrapure water obtained from a Millipore water purification system (18.2 MΩ cm-1, Milli-Q, Merck Millipore, Darmstadt, Germany) was used in all runs. Personal glucometer (PGM) was purchased from Roche (Accu-Chem® Active, Selangor Darul Ehsan, Malaysia). PGM buffer (pH 7.3) was prepared with 72.9 mM Na2HPO4, 27.1 mM NaH2PO4, 50 mM NaCl and 5 mM MgCl2.

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Food Samples and Extraction Procedure. Peanut matrix for further addition calibration was prepared as follows: 5.0 g of ground blank peanut sample was milled first with a small Midea Food Mixer (BP252AG, Guangdong, China) and then extracted with MeOH/water (37.5 mL, 80:20, v/v) under gentle stirring for 1 h followed by filtration. 20 mL of the extract was diluted into 60 mL of water (20%, v/v, final content of MeOH). Finally, calibration solutions were prepared by spiking aliquots of an AFB1 stock solution (acetonitrile as solvent) into different volumes of diluted extract. Contaminated peanut samples were made available as slurry (150% of water, v/w). A portion of 7.5 g of slurry was extracted with 18 mL of MeOH for 1 h followed by filtration. To 5.0 mL of extract, 15.0 mL of PGM buffer was added to obtain a final concentration of 20% (v/v) MeOH. Preparation of Nanogold-Labeled Anti-AFB1 Antibody (mAb-AuNP). Gold colloids (AuNP) with 16 nm in diameter were prepared according to our previous paper.40 AuNP-labeled anti-AFB1 antibody was synthesized through the interaction between cysteine or NH3+-lysine residues of proteins and AuNP.41 The labeling process was simply summarized as follows: (i) gold colloids (C[Au] = 24 nM) were adjusted to pH 9 by using Na2CO3; (ii) 200 μL of 40 μg/mL anti-AFB1 in PGM buffer was injected to 10 mL of the resulting gold colloids, and incubated for 12 h at 4 ºC; and (iii) the resultant mixture was centrifuged for 30 min (14,000g) at 4 °C, and washed twice with PGM buffer. Finally, the soft sediment (i.e. mAb-AuNP) was suspended in 1.0 mL of PGM buffer containing 1.0 wt % BSA, and stored at 4 °C. Synthesis of Mesoporous Silica Nanoparticles and Surface Coating with PEI. Mesoporous silica nanoparticles (MSN) were prepared according to the literature.42 Briefly, 0.5 g of CTAB was initially dissolved in 200 mL of distilled water, and then 1.75 mL of sodium hydroxide (2.0 M) was slowly added to CTAB solution with stirring for 20 min at 80 ºC. Afterwards, 2.5 mL of TEOS was dropped into the above solution, and vigorously stirred for 2 h until a white precipitate was obtained. Following that, the prepared product was filtered, washed with distilled water and methanol, and dried in air. To remove the CTAB, MSN was refluxed for 10 h in a solution composed of HCl (37%, 1.5 mL) and methanol (75 mL), and then washed with distilled water and methanol. The resulting MSN was dried for 4 h at 60 ºC in vacuum to remove the remaining solvent from the pores.

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Next, the PEI-coated MSN (PEI-MSN) was synthesized consulting to the previous report.43 200 mg of MSN and 300 mg of PEI were mixed in 40 mL of ultrapure water. The suspension was stirred overnight at room temperature. During this process, the positively charged PEI molecules were adsorbed onto the negatively charged MSN, thus resulting in the formation of positively charged PEI-MSN. Following that, the mixture was centrifuged, washed and dried to get the PEI-MSN. The content of nitrogen in the PEI-MSN was determined to be 4.47% by the element analysis. Glucose Loading into PEI-MSN with mAb-AuNP Capping. The loading of glucose molecules into the PEI-MSN was performed according to the literature with minor modification.33 Initially, 10 mg of PEI-MSN was suspended into 1.0 mL of PGM buffer containing 1.0 M glucose, and then the mixture was gently shaken overnight at room temperature. During this process, glucose molecules were entered into the pores of the MSN.33,44,45 After that, 500 μL of mAb-AuNP (C[Au] = 24 nM) was added into the suspension, and incubated for 6 h at 4 ºC with gently stirring. As a result, the negatively charged mAb-AuNP was attached onto the positively charged PEI-MSN, and capped on the pores. The mixture was centrifuged for 5 min at 5,000g and washed with PGM buffer. Finally, the obtained PEI-MSN-mAb-AuNP loaded with glucose (designated as MSN-mAb-AuNP) was suspended into 500 μL of PGM buffer, and used for the detection of target AFB1. Immunoassay Procedure and PGM Measurement. 10 μL of the above prepared MSN-mAb-AuNP suspension was initially injected into a 200-μL PCR tube, and AFB1 standards/samples with various concentrations were added into the tube. The tubes were shaken occasionally during the reaction at room temperature. During this process, AFB1 triggered the MSN-mAb-AuNP complex and displaced the mAb-AuNP particles from the PEI-MSN probe because of the specific antigen-antibody reaction. The use of the specific monoclonal antibody was conducive for the opening of the molecular gate, thereby resulting in the release of the entrapped glucose molecules from the pores. After incubation for 20 min, a 3-μL aliquot of the supernatant was removed for glucose measurement using the commercially available PGM. The obtained PGM signal was registered as the immunosensing signal in dependence on the concentration of target analyte AFB1. All measurements were carried out at room temperature (25 ± 1.0 ºC). All data obtained were based on three parallel measurements. Scheme 1 represents the fabrication process and measurement principle of PGM-based immunoassay. 7



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Safety. Aflatoxins are powerful hepatotoxins and carcinogens, so great care should be taken to avoid personal exposure and potential laboratory contamination. All items coming in contact with aflatoxins (glassware, vials, tubes, etc.) were immersed in a 10% bleach solution for 1-2 h before they were discarded. Pure aflatoxin standard was handled in a hood with extreme caution.

■RESULTS AND DISCUSSION Design Strategy of PGM-Based Immunosensing. To develop a user-friendly and sensitive immunoassay protocol, an alternative immunosensing strategy that does not require multiple sample separation and washing steps would be advantageous. In this work, the target analyte AFB1 is determined using a one-step immunoreaction format based on the competition between PEI-MSN and target analyte AFB1 for mAb-AuNP. The as-synthesized PEI-MSN is employed as a pseudo hapten for the reaction with mAb-AuNP by electrostatic interaction. The PEI, as a cationic polymer containing numerous repeating units of an amine and two methylene groups, is usually utilized for cationizing various proteins/nanostructures due to its low toxicity.46,47 Because of the high charge density of branched PEI (one unit of positive charge in a mass unit of 43), functionalization of only a few PEI molecules with the MSN introduces a large number of positive charges for the reaction with negatively charged species.48,49 Typically, antibodies (as one kind of proteins) have a net electrical charge polarity in aqueous solution, which depends on the isoelectric points of the species and the ionic composition of the solution. By tuning and controlling the pH of the solution, the antibodies can exhibit the expected charges. To this regard, the positively charged PEI-MSN can be reactive for negatively charged mAb-AuNP. Accompanying the assembly between PEI-MSN and mAb-AuNP, the loading glucose molecules can be gated into the pores. Upon target AFB1 introduction (one kind of neutral molecules), the specific antigen-antibody reaction between AFB1 and the gold-labeled mAb can cause the detachment of the latter from PEI-MSN, thereby resulting in the opening of the pores. In this case, the loaded glucose molecules can release out of the MSN and can be determined using a portable glucometer. The obtained PGM signal is directly proportional to the concentration of target AFB1 in the sample

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Characterization of PGM-Based Immunosensing Platform. To realize our strategy for the formation of MSN-mAb-AuNP assemblies based on the electrostatic attraction, one precondition is to synthesize the positively charged PEI-MSN and the negatively charged mAb-AuNP. To this end, we used microelectrophoresis (Microelectrophoresis Apparatus Mk II, Rank Brothers Ltd, England) to characterize the functionalized nanostructures (Figure 1A). As shown from column 'a' in Figure 1A, the zeta potential of the as-synthesized MSN was -5.7 mV, which derived from –OH groups on the surface of MSN. Significantly, a positive zeta potential (+20.9 mV, column 'b') was achieved when PEI was coated on the MSN (i.e. PEI-MSN), indicating that branched PEI molecules were assembled onto the MSN. In contrast, the unmodified AuNP exhibited a negative zeta potential (-8.4 mV, column 'c'), which originated from the negatively charged citrate ions (adsorbed on the AuNP) during the synthesis. Meanwhile, we also observed that the as-prepared mAb-AuNP in PGM buffer had a more negative zeta potential (-18.6 mV, column 'd') relative to AuNP alone. The reason might be the fact that the isoelectric point of anti-AFB1 antibody was lower than pH 7.3, thus displaying a negatively charged species. Thus, the successful preparation of PEI-MSN and mAb-AuNP with the desired species could provide a chance for the development of PGM-based immunoassay. However, an additional question has to be answered, i.e., whether the negatively charged mAb-AuNP can be readily assembled onto the positively charged PEI-MSN by the electrostatic forces. To verify this issue, we used transmission electron microscopy (TEM, H-7650, Hitachi Instruments, Tokyo, Japan) and dynamic light scattering (DLS, Zetasizer Nano S90, Malvern, London, UK) to investigate the PEI-MSN and mAb-AuNP before and after the reaction. Figure 1B shows typical TEM image of the as-synthesized PEI-MSN, and the mean size was ~180 nm. The pore size distribution curve displayed a narrow size distribution of the pores with a Barrett-Joyner-Halenda average pore diameter of 2.3 nm (data not shown). Moreover, the size of PEI-MSN (~180 nm) was almost identical to the DLS result (Figure 1D). PEI-MSN and mAb-AuNP were homogeneously dispersed in PGM buffer without aggregation, as indicated from the DLS analysis (Figure 1D). More favorably, many mAb-AuNP were assembled onto the surface of PEI-MSN when they were (Figure 1C), and the mean size was increased to ~245 nm (Figure 1D). The results also indicated that mAb-AuNP could be conjugated onto the PEI-MSN through 9



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electrostatic attraction. That is to say, the as-synthesized PEI-MSN could be used as a pseudo hapten for the interaction with mAb immobilized on AuNP. Elucidation of PGM-Based Immunosensing Method for Target AFB1. In the present work, the obtainable signal derived from glucose molecules released from pores of PEI-MSN in the presence of target analyte. Logically, another two questions arise as to whether (i) small glucose molecules can be gated within the pores through the mAb-AuNP, and (ii) target AFB1 can trigger the release of glucose from the pores. To clarify this point, the as-prepared MSN-mAb-AuNP suspension was monitored by using an external PGM in the absence and presence of target AFB1. The PGM signal was collected and registered intermittently (every 5 minutes). As seen from curve 'a' in Figure 2A, the PGM signal was almost unchanged over 60 min in the absence of target AFB1, indicating that glucose molecules could be gated firmly into the pores through the adsorbed negatively charged mAb-AuNP. Upon target AFB1 introduction, however, the PGM signal increased with the increment of incubation time, and the steady-state signal was reached at ~20 min (curve 'b' in Figure 2A). The results also suggested that (i) it took some time for the competitive displacement of mAb-AuNP from PEI-MSN by the target AFB1, (ii) target AFB1 could induce the release of glucose from the MSN-mAb-AuNP, and (iii) the competitive-type displacement reaction could be rapidly implemented within 20 min. To avoid possible error resulting from different additions of samples, all PGM signals obtained in this work were recorded at ~20 min after addition of the samples. To further demonstrate the reliability of PGM-based immunosensing strategy, several control tests were carried out in detail with and without PEI on MSN or mAb on AuNP because the as-synthesized MSN and AuNP had distinct charges themselves. As seen from columns 'a' and 'b' in Figure 2B, when pure MSN, glucose and mAb-AuNP were mixed, no significant PGM signals (≈ control test) in the presence of 1.0 μg/kg AFB1 were obtained, regardless of stationary reaction (column 'a') or with vigorous agitation (column 'b'). This indicated that glucose could not be gated into the pores of MSN without PEI. Undoubtedly, the negatively charged AuNP could also be assembled on the positively charged PEI-MSN. For this reason, pure AuNP was used for reaction with PEI-MSN in the presence of glucose. The as-prepared nanocomposites were employed for the detection of 1.0 μg/kg AFB1. Almost no PGM signal was observed after stationary incubation for 60 min (column 'c'), suggesting that the pores at the MSN-PEI-AuNP could not be opened by target 10


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AFB1 without target antibody (mAb). When the formed nanocomposites were vigorously stirred for 60 min, however, a relatively high PGM signal was achieved, as seen from column 'd' in Figure 2B. The reason might be most likely explained by the fact that the AuNP assembled by electrostatic attraction on the PEI-MSN were detached during the vigorously stirring, thereby resulting in the release of a few glucose molecules. The results from columns 'c' and 'd' indicated that pure AuNP could gate the glucose within the pores of PEI-MSN, but it could not be displaced by target AFB1. Vice versa, no detectable signal was achieved when PEI and mAb were simultaneously absent (column 'e'). As shown from column 'f', a strong PGM signal could be achieved when PEI-MSN and mAb-AuNP were used as the immunosensing probes. Based on these results, the preliminary conclusion could be drawn that the designed MSN-mAb-AuNP could be utilized for the detection of target AFB1. Optimization of Assay Conditions. To efficiently adsorb the positively charged mAb-AuNP on the PEI-MSN (but not to hinder the loading of glucose molecules into its pores), the concentration of PEI for the preparation of PEI-MSN should be very crucial. Although the high-concentration branched PEI facilitates the attachment of mAb-AuNP, it is not conducive for the loading and release of glucose molecules from the pores. On the opposite, low-concentration branched PEI might result in the leakage of glucose from the pores because of the attachment of too few mAb-AuNP. As shown from Figure 3A, an optimal PGM signal could be obtained when 300 mg of PEI and 200 mg of MSN (i.e., 3 : 2 mass ratio for WPEI : WMSN) in 40 mL of ultrapure water were employed for the preparation of PEI-MSN. By the same token, the labeling amount of mAb on the AuNP also greatly affects the sensitivity of PGM-based immunoassay. High loading amount of mAb might cause more mAb binding sites unoccupied on the MSN-mAb-AuNP, while a low mAb load might decrease the probability of the antigen-antibody reaction, thus resulting in the low detectable signal. Figure 3B displays the effect of different-concentration mAb on the PGM signal. A strong PGM signal was acquired at 200 μL of 40 μg/mL anti-AFB1 containing 10 mL of gold colloids (C[Au] = 24 nM). Thus, this condition was utilized for the preparation of mAb-AuNP.

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Another important parameter for the preparation of immunosensing probes was the ratio of PEI-MSN and mAb-AuNP. On the one hand, it should ensure that the pores could be completely gated by mAb-AuNP to prevent the leakage of glucose molecules. On the other hand, the large aggregation of PEI-MSN and mAb-AuNP through layer-by-layer assembly of nanoparticles should be avoided. As seen from Figure 3C, the PGM signal increased with the decreasing amount of mAb-AuNP, and decreased when the PEI-MSN and mAb-AuNP volume ratio exceeded 2 : 1. The reason for the decrease at the high-concentration mAb-AuNP might be attributed to the fact that too many free antibodies were exposed on the MSN-mAb-AuNP. Therefore, the volume ratio 2 : 1 of PEI-MSN (10 mg/mL) and mAb-AuNP (C[Au] = 24 nM) (v/v) was used for the preparation of MSN-mAb-AuNP in this study. Analytical Performance of PGM-Based Immunoassay toward Target AFB1. Under optimal conditions, the analytical properties of the developed immunosensing format were investigated. The detection was carried out by using an external PGM after target AFB1 reacted with MSN-mAb-AuNP for 20 min in PGM buffer at room temperature. As seen from Figure 4A, the PGM signal increased with the increasing AFB1 concentration in the sample. A linear dependence between PGM signal (mM) and AFB1 level (μg/kg) could be achieved in the dynamic range from 0.01 to 15 μg/kg (ppb). The regression equation could be fitted to y (mM) = 1.9488 × C[AFB1] + 0.5714 (μg/kg, R2 = 0.9978, n = 21). The limit of detection (LOD) could be estimated to 0.005 μg/kg (5 ppt) at a signal-to-noise ratio of 3σ (where σ is the standard deviation of a blank solution, n = 13). Apparently, the LOD of PGM-based immunoassay toward target analyte AFB1 was comparable with commercialized AFB1 ELISA kits obtained from different companies (e.g. Quicking Biotech: 100 ppt; MaxSignal®: 50 ppt; MyBioSource: 250 ppt; Diagnostic Automation: 5 ppt). Due to the legal limit of AFB1 (< 2 μg/kg), the PGM-based immunoassay could completely meet the requirement of AFB1 monitoring in foodstuff. Next, we determined the reproducibility and precision of PGM-based immunoassay by using different batches of MSN-mAb-AuNP for the analysis of three AFB1 samples with different levels. As shown from Table 1, the relative standard deviation (RSD) by using the same-batch MSN-mAb-AuNP was 2.7 – 9.2% (n = 5), while that of the inter-assay with use of different batches

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was 4.2 – 12.5% (n = 5). The results point out the importance of batch-wise preparation and use of MSN-mAb-AuNP. Further, the specificity of PGM-based immunoassay was monitored by using AFB2, AFG1, AFG2, and two protein antigens alpha-fetoprotein (AFP) and thyroid-stimulating hormone (TSH). As seen from Figure 4B, AFG1, AFG2, AFP and TSH alone or in combination with the target did not cause the significant increase in the PGM signal relative to the control test. However, the relatively strong PGM signal toward AFB2 sample could be also easily explained by the fact that the used AFB1 antibody has a high cross-reactivity for AFB2, as described in our previous report.39 Thus, the specificity of PGM-based immunoassay was acceptable. Evaluation of Real Samples. The feasibility of applying the PGM-based immunoassay to assess aflatoxin levels in a complex matrix was also monitored. Initially, we spiked AFB1 standards with random concentrations into blank peanut homogenate, followed by a centrifugation and extraction procedure. Finally, these samples were measured by the PGM-based immunoassay and the commercialized MaxSignal® AFB1 ELISA kit, respectively. Further, we also used the developed PGM-based immunoassay to analyze the naturally contaminated peanut samples by using the same method (Note: These samples purchased from the local supermarket were set for various times under dull and wet conditions to provoke growth of fungi and achieve a high-concentration of AFB1 prior to measurement). The aflatoxin content obtained by the PGM-based immunoassay was calculated according to the above-mentioned regression equation (y = 1.9488 × C[AFB1] + 0.5714). The results are summarized in Table 2. As seen from this table, all RSD values were lower than 9%, indicating that the PGM-based immunoassay could be utilized for quantitative monitoring of target AFB1 in real samples. ■ CONCLUSIONS In this work, we designed a novel immunosensing protocol for rapid and sensitive monitoring of the mycotoxin, aflatoxin B1 by using a portable personal glucometer. Compared with conventional competitive-type or sandwich-type immunoassay formats, the PGM-based immunoassay strategy abdicates secondary detection antibodies or enzyme-antibody bioconjugates. More importantly, 13



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the competitive-type displacement immunosensing method can be successfully carried out as one-step incubation reaction with target analyte, and does not need multiple separation and washing steps, thus constituting a rapid and cost-effective bioanalytical method. Although the present work focused on target AFB1, it can be supposed that the immunosensing strategy can be easily extended to monitor other biotoxins and biomolecules using the appropriate antibodies for the preparation of mAb-AuNP bioconjugates.

■AUTHOR INFORMATION Corresponding Author *Phone: +49-89-2180 78252; fax: +49-89-2180 78255; e-mail: [email protected] (D. Knopp) Phone: +86-591-2286 6125; fax: +86-591-2286 6135; e-mail: [email protected] (D. Tang)

■ACKNOWLEDGEMENT Support by the National Natural Science Foundation of China (grant nos. 41176079 & 21475025), the National Science Foundation of Fujian Province (grant no. 2014J0105), the Program for Changjiang Scholars and Innovative Research Team in University (grant no. IRT1116), and the Alexander von Humboldt-Foundation of Germany is gratefully acknowledged.

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Table 1. Reproducibility and Precision of PGM-Based Immunoassay for Target AFB1 C[AFB1], a

type

intra-assay

inter-assay

a

assay no.; PGM signal (mM)

RSD

μg/kg

1

2

3

4

5

(%, n = 5)

0.05

0.7

0.7

0.6

0.6

0.6

8.6

0.5

1.6

1.4

1.7

1.8

1.6

9.2

5

9.1

9.6

9.7

9.4

9.2

2.7

0.05

0.5

0.5

0.6

0.6

0.5

10.1

0.5

1.8

1.5

1.4

1.7

1.9

12.5

5

8.7

9.6

9.4

9.2

8.8

4.2

While the intra-assay tests were carried out with the same-batch of MSN-mAb-AuNP, different MSN-mAb-AuNP batches were

used for the inter-assay tests.

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Table 2. Results Obtained by the PGM-Based Immunoassay and Commercial AFB1 ELISA Kit (MaxSignal AFB1 ELISA) for Spiked or Naturally Contaminated Peanut Samples method; concentration (μk/kg, n = 3) type

spiked

a

no.

PGM-based immunoassay

AFB1 ELISA kit

RSD (%)

1

0.23 ± 0.03

0.25 ± 0.01

5.9

2

0.67 ± 0.12

0.61 ± 0.13

6.6

3

1.17 ± 0.23

1.32 ± 0.18

8.5

4

3.57 ± 0.45

3.44 ± 0.53

2.6

5

8.91 ± 1.1

9.05 ± 0.91

1.1

1

17.8 ± 1.34

19.2 ± 1.56

5.4

2

2.65 ± 0.35

2.44 ± 0.41

5.8

3

7.87 ± 1.32

8.21 ± 1.57

3.0

4

4.56 ± 0.98

4.32 ± 0.51

3.8

5

6.81 ± 1.18

6.59 ± 1.39

2.3

peanut

naturally contaminated peanut

a

Each sample was determined in triplicate, and the high-concentration AFB1 sample was assayed with an appropriate dilution.

19



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■FIGURE LEGENDS Scheme 1. Schematic illustration of PGM-based immunosensing protocol using mAb-AuNP-gated PEI-MSN loading with glucose. The competitive-type displacement reaction mode between pseudo hapten PEI-MSN and target analyte AFB1 for the gold-labelled antibody is accompanied by analyte concentration-depended glucose release, as detected by a portable glucometer. Figure 1. (A) Zeta potentials of (a) the as-prepared MSN, (b) PEI-functionalized MSN, (c) the as-synthesized AuNP and (d) mAb-conjugated AuNP; (B,C) TEM images of (B) PEI-MSN and (C) MSN-AuNP; and (D) DLS data of PEI-MSN and MSN-mAb-AuNP.

Figure 2. (A) PGM signal response of the prepared MSN-mAb-AuNP immunosensing probe relative to reaction time in the (a) absence and (b) presence of 1.0 μg/kg target AFB1, and (B) PGM response of the immunosensing probes obtained using different preparations of MSN-assemblies and 1.0 μg/kg target AFB1: (a) (MSN + mAb-AuNP + glucose) after gentle shaking, (b) (MSN + mAb-AuNP + glucose) after heavy shaking, (c) (PEI-MSN + AuNP + glucose) after gentle shaking, (d) (PEI-MSN + AuNP + glucose) after heavy shaking, (e) MSN + AuNP + glucose, and (f) PEI-MSN + mAb-AuNP + glucose (Note: The nanocomposites, formed under different conditions, were centrifuged and collected for the followed detection of target analyte). Figure 3. Dependence of PGM signal toward 1.0 µg/kg target AFB1 on (A) mass ratio of PEI and MSN in 40 mL of ultrapure water for preparation of PEI-MSN, (B) volume of 40 µg/mL anti-AFB1 and 10 mL of gold colloids (C[Au] = 24 nM) for preparation of mAb-AuNP, and (C) volume ratio of PEI-MSN (10 mg/mL) and mAb-AuNP (C[Au] = 24 nM) for preparation of MSN-mAb-AuNP. Figure 4. (A) Calibration curve of PGM-based immunosensing platform toward AFB1 standards with different concentrations (PGM signal vs. AFB1 level), and (B) PGM responses of the developed immunoassay against target AFB1 (1.0 µg/kg), AFB2 (1.0 µg/kg), AFG1 (1.0 µg/kg), AFG2 (1.0 µg/kg), AFP (20 ng/mL), and TSH (20 ng/mL). Each data point represents the average value obtained from three different measurements. The errors bars represent the 95% confidence interval of the mean for the PGM signal.

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Scheme 1

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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■ FOR ONLY TOC

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Low-Cost and Highly Sensitive Immunosensing Platform for Aflatoxins

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