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Dopamine-Loaded Liposomes for in-Situ Amplified Photoelectrochemical Immunoassay of AFB1 to Enhance Photocurrent of Mn2+-Doped Zn3(OH)2V2O7 Nanobelts Youxiu Lin, Qian Zhou, and Dianping Tang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03451 • Publication Date (Web): 06 Oct 2017 Downloaded from http://pubs.acs.org on October 7, 2017

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Analytical Chemistry

Dopamine-Loaded Liposomes for in-Situ Amplified Photoelectrochemical Immunoassay of AFB1 to Enhance Photocurrent of Mn2+-Doped Zn3(OH)2V2O7 Nanobelts Youxiu Lin, Qian Zhou, and Dianping Tang*

Key Laboratory of Analytical Science of Food Safety and Biology (MOE & Fujian Province), Collaborative Innovation Center of Detection Technology for Haixi Food Safety and Products (Fujian Province), State Key Laboratory of Photocatalysis on Energy and Environment, Department of Chemistry, Fuzhou University, Fuzhou 350108, People's Republic of China

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

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ABSTRACT: A novel signal-amplified strategy based on dopamine-loaded liposome (DLL) was developed for competitive-type non-enzymatic photoelectrochemical (PEC) immunoassay of smallmolecular aflatoxin B1 (AFB1) in foodstuff, using Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts. The signal was amplified by high-loaded capacity of liposome and high-efficient dopamine molecule to enhance photocurrent of Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts. The loaded dopamine in the liposome was used as an electron donor to scavenge the hole and inhibit the charge recombination. To design such an immunoassay system, AFB1-bovine serum albumin (AFB1-BSA) conjugate was covalently bound with the multifunctional liposome via the cross-linkage glutaraldehyde, whereas monoclonal anti-AFB1 antibody was labeled onto magnetic bead by typical carbodiimide coupling. Upon addition of target AFB1, a competitive immunoreaction was carried out between the analyte and the AFB1-BSA-DLL for the conjugated antibody on the magnetic bead. Followed by magnetic separation, the carried DLL on the magnetic beads was lysed by using Triton X-100 to release the encapsulated dopamine. The as-produced dopamine (as an elector donor) increased the photocurrent of the Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts. The photocurrent depended on the as-released amount of the dopamine. The change in the photocurrent enhanced with the increasing AFB1 concentration. Under the optimal conditions, Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts exhibited good photoelectrochemical responses for the detection of AFB1, and allowed the detection of AFB1 at a concentration as low as 0.3 pg mL-1 within a linear range from 0.5 pg mL-1 to 10 ng mL-1. Importantly, this system provided an ideal PEC immune sensing platform based on Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts and the high-loaded liposome for the detection of small molecules.

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Aflatoxin B1 (AFB1, one of the most potent carinogens, mutagens and teratogens) has extensively existed in many agricultural commodities (e.g., corn, peanut, cotton, seed and bean).1-3 Nowadays, various methods and schemes have been reported for the quantitative/quanlitative screening of mycotoxins. Recently, photoelectrochemical (PEC) assay gains the great attention in this field due to its cheap instruments, simple operation, high sensitivity and low background signal.4-7 To develop the assay system, the light-based excitation source and the electricity-based readout signal should be necessary.8 Typically, there are at least two critical aspects for obtaining low limits of quantification and detection: i) the photoactive materials to generate the photogenerated charge which can rapidly separate to form the electron/hole (e-/h+) pair and produce the photocurrent under illumination; and ii) the biomolecules to influence the photocurrent by involving in the oxidation/reduction reaction at the PEC electrode surface.9 Therefore, choice of suitable photoactive materials and design of the signal-amplified system are crucial to improve the sensitivity of PEC detection methods. It is generally accepted that a favorable photoactive material has high visible light harvesting capability to enhance the light absorption, and possesses the fast charge transport to separate the electron/hole (e−/h+) pair and suppress the charge recombination.10-12 The rapid emergence of low-dimensional organic-inorganic intercalation vanadium oxide-based nanostructures opens a new horizon for the development of high-efficiency photoactive materials owing to easy synthesis, abundant source and high chemical stability.13-15 Zn3(OH)2V2O7•2H2O is an important transition metal vanadate with a wide band gap of 3.4 eV for the generation of the photocurrent under UV light. Meanwhile, V's 3d orbital is lower than other first transition metals in energy, which easily lowers the conduction band energy level. In addition, Zn3(OH)2V2O7•2H2O has another two unique advantages: i) to easily extend the band edge (site) adsorption to visible light region, and ii) to effectively separate the electron/hole (e-/h+) pair and reduce the charge recombination.16-18 Certainly, one disadvantage of using Zn3(OH)2V2O7•2H2O lies in the weak absorption to the visible light because of its large band gap. However, this phenomenon can be improved through controlling their morphology/crystal with various synthetic methods, or doping other elements to adjust the band gap.19 Nanobelts with single, highly crystalline anatase phase and ordered array are beneficial to augment light absorption, and provide a direct electrical pathway to effectively reduce the recombination of charge and hole.20-22 By doping a few transition metal ions, the nanohybrids can generate a midgap state below the conduction band edge, and cause the electron excitation into 3



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midgap state, thereby extending the absorption wavelength to enhance the absorption of the visible light. Furthermore, the resulting midgap state traps the photogenerated charge to reduce the recombination of the photogenerated electron−hole pairs. To this end, our motivation is synthesizing Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts for construction of photoactive materials in this work. Another important issue for development of high-efficiency PEC sensing system is to amplify the detectable signal by adopting innovative and powerful signal-generation tags.23 Liposomes with highly versatile lipid bilayer structure can form the enclosed vesicles with high carrying capacity to encapsulate various signaling tracers (e.g., enzymes, dyes, fluorescence molecules, quantum dots, and electroactive compounds).24,25 Lin's group devised a portable immunosensing system with the glucometer readout by encapsulating glucose molecules into liposomes.26 Zarghami's group utilized dopamine-loaded liposomes to detect the activity of telomerase by monitoring voltammetric signal of the released dopamine.27 Just as the high-loading ability, liposomes can be used as the carriers for the signal amplification. Favorably, we recently found that dopamine molecules could be used as the electron donors to enhance the photocurrent of Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts. By scavenging holes and inhibiting the process of charge recombination,28 dopamine-loaded liposomes (DLL) can be utilized for the signal amplification of the PEC-based sensing system on Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts. Based on the above-mentioned advantages, herein we report the proof-of-concept of novel and feasible PEC immunosensing protocol for the detection of small-molecular aflatoxin B1 (Scheme 1). The competitive immunoreaction is first executed on anti-AFB1 antibody-conjugated magnetic bead by using AFB1-BSA-labeled DLL (dopamine-loaded liposome) as the competitor. Thereafter, the carried liposomes onto magnetic beads accompanying the antigen-antibody reaction are lysed with the aid of Triton X-100. Upon the released dopamine introduction, the photocurrent is detected on Mn2+-doped Zn3(OH)2V2O7•2H2O ultra-thin nanobelts. Use of low-dimensional organic-inorganic intercalation vanadium oxide-based nanostructures is expected to enhance the sensitivity of PEC immunoassay by coupling with the dopamine-loaded liposome. The aim of this study is to set up a novel in-situ signal-amplified PEC immunosensing system for the quantitative screening of the low-concentration mycotoxins in foodstuff.

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Scheme 1 Schematic illustration of Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts-based photoelectrochemical (PEC) immunoassay for aflatoxin B1 (AFB1) on monoclonal anti-AFB1 antibody-modified magnetic bead (mAb-MB) by using dopamine (DA)-loaded liposome (DLL) labeled with AFB1-BSA conjugate: (A) competitive-type immune reaction and (B) photocurrent measurement.

■ EXPERIMENTAL SECTION Preparation of Mn2+-Doped Zn3(OH)2V2O7•2H2O Nanobelts. The as-prepared nanobelts were synthesized by a simple one-step hydrothermal method similar to the literature.29,30 Initially, 74 mg of NH4VO3 was added into hot deionized water (44 mL, 80 °C) under vigorous stirring to obtain a pale yellow suspension. Then, 190 mg of Zn(NO3)2·2H2O and the different-amount MnSO4 [0.004, 0.008, 0.012, 0.016 and 0.04 g for 0.5%, 1%, 1.5%, 2% and 5% Mn2+-doped Zn3(OH)2V2O7•2H2O, respectively] were thrown into the resulting solution under the same conditions. Following that, the resultant suspension was transferred into 100-mL Teflon autoclave and heated for 12 h at 180 °C. 5



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Afterwards, the precipitate was collected and washed with deionized water/ethanol for several times. Finally, the product was dried in an oven at 60 °C for further use. Conjugation of AFB1-BSA with Dopamine-Loaded Liposome (AFB1-BSA-DLL). Prior to experiment, dopamine-loaded liposome (DLL) was prepared via the reversed-phase evaporation method (Please see the detailed experimental process in the Supporting Information).31,32 Following that, the as-prepared DLL was used for the labeling of AFB1-BSA conjugate by the cross-linkage glutaraldehyde.33 1.0 mL of the above-prepared DLL suspension was dropped into glutaraldehyde aqueous solution (0.4 mL, 25 wt%) under gentle stirring (60 min at 20 °C). Afterwards, the resulting solution was disposed by dialysis in phosphate buffer (pH 7.4) at 4 °C to remove excess reagents including glutaraldehyde. Subsequently, AFB1-BSA conjugates (300 µL, 0.5 mg mL−1) were added into the mixture, and gently shaken overnight on a shaker at 4 °C. Excess AFB1-BSA conjugates were removed by ultrafiltration. Finally, the obtained AFB1-BSA-DLL was dispersed into PBS (1.0 mL, pH 7.0) containing 1.0 wt% BSA and stored at 4 °C when not in use. Immunoreaction Protocol and Photocurrent Measurement. Scheme 1 represents schematic illustration of Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts-based PEC immunosensing system for target AFB1. A split-type photoelectrochemical immunosensing platform was utilized for the assay development similar to our previous report.34 In this system, Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts were modified onto the Fluorine-doped tin oxide (FTO) electrode, while monoclonal anti-AFB1 antibodies (mAb) were covalently conjugated onto the carboxylated magnetic beads (MB) by the carbodiimide coupling (Please see preparation procedure in the Supporting Information).35 The immunoreaction was initially carried out in a 500-µL centrifugal tube containing 100 µL of the above-prepared MB-mAb. After magnetic separation, 50 µL of AFB1 standard/sample and 50 µL of AFB1-BSA-DLL (constant concentration prepared above) were simultaneously added into the tube, and reacted for 60 min at 37°C on a shaker. The resulting suspensions was magnetically separated and washed with pH 7.0 PBS. After that, Triton X-100 (100 µL, 10 mg mL-1) solution was injected into the tube to rupture the conjugated liposomes on magnetic beads. Following that, the resulting product containing the as-released dopamine from the liposomes was transferred into the detection cell for photoelectrochemical measurement by using Mn2+-doped Zn3(OH)2V2O7•2H2O nanobeltsmodified FTO electrode (Please see the measurement process in the Supporting Information).

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■ RESULTS AND DISCUSSION Design of Mn2+-Doped Zn3(OH)2V2O7•2H2O Nanobelts-Based PEC Immunoassay. In this work, magnetic controlled immunoreaction with a competitive-type assay format is implemented in a centrifugal tube between target AFB1 and AFB1-BSA-DLL for the labeled monoclonal anti-AFB1 antibody on magnetic beads. One major advantage of using magnetic controlled immunosensing protocol is that it enables the rapid separation and purification of bionanocomposites in an external magnetic field after synthesis/reaction for each step. Use of liposomes with high loaded capacity is expected to increase the carried dopamine, while utilization of Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts is to improve the photoelectrochemical properties of nanobelts. After formation of immunocomplexes, the dopamine-loaded liposomes accompanying magnetic beads are initially dissolved by the added Triton X-100. Thereafter, the as-released dopamine molecules (as the electron donors) enhance the photocurrent of Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts through scavenging holes because of suitable energy level. In this case, the photocurrent is doubly amplified to enhance the sensitivity of PEC immunoassay. Characterization of Mn2+-Doped Zn3(OH)2V2O7•2H2O Nanobelts. Figure 1A shows typical transmission electron microscope (TEM; H-7650, Hitachi Instruments, Tokyo, Japan) image of the Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts. The morphology of the as-synthesized nanostructures was belt-like and ultra-thin with an average size of 800 × 40 nm (length × width), indicating that the nanocrystals were neither nanospheres nor nanotubes. Such an ultra-thin structure could efficiently increase the transmittance of light to enhance the photocurrent. Logically, two puzzling questions arise as to whether i) the doping Mn2+ ions caused the change of crystal phase, and ii) Mn2+ ions were doped into Zn3(OH)2V2O7•2H2O nanobelts. To clarify these issue, we used X-ray diffraction pattern (XRD; PANalytical X'Pet spectrometer) (Figure 1B) and X-ray photoelectron spectroscopy (XPS; Thermo Fisher Scientific, Model Escalab 250 spectrometer) (Figure 1C-D) to characterize the as-prepared nanostructures. As control test, Zn3(OH)2V2O7•2H2O without the doping Mn2+ ions were determined by using XRD. As indicated from curve 'a' in Figure 1B, the narrow XRD lines of this sample fitted exactly the hexagonal phase of Zn3(OH)2V2O7•2H2O (JCPDS no. 50-0570). Upon doping of Mn2+ ions, XRD patterns were almost in accordance with that of Zn3(OH)2V2O7•2H2O alone, and no trace of manganese related phases were observed except for the whole slight shift to

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lower angles (Figure 1B, curve 'b'), indicating that the doping of Mn2+ ions did not cause significant change in the crystal phase.36,37 Figure 1C-D gives the XPS data analysis of the as-synthesized nanostructures. Obviously, the characteristic peaks for Zn, V and Mn elements existed in the nanocrystals (Figure 1C). Two strong characteristic peaks at 1045.2 and 1022.2 eV belonged to Zn 2p1/2 and Zn 2p3/2, respectively, while the peaks located at 525.1 and 517.5 eV assigned to V 2p1/2 and V 2p3/2, respectively, which were the characteristic peaks of V5+ oxidation state.38-39 Furthermore, two very small characteristic peaks at 653 and 642.1 eV for Mn 2p1/2 and Mn 2p3/2 could be simultaneously appeared (Figure 1D). The reason on the very small signals was ascribed to the relative low-amount Mn (1.0%) in the starting target material.40 These results revealed that Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts were successfully synthesized by using our design.

Figure 1. (A) TEM image, (B-b) XRD patterns and (C) XPS analyses of Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts [notes: (B-a) XRD patterns of Zn3(OH)2V2O7•2H2O and (D) XPS spectra of Mn 2p].

Characterization of Dopamine-Loaded Liposome. As described above, the dopamine-loaded liposome is used as the enhancers to increase the photocurrent of Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts. Figure 2A shows TEM image of the as-prepared DLL. The dopamine loaded liposome exhibited a perfect spherical shape with a thin shell and no fracture of the capsule wall. Such a 8


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typical vesicle structure with a uniform shape could be observed from the insets of Figure 2A. To further verify that dopamine molecules were encapsulated into the liposomes, we used UV−vis absorption spectra (Tecan Infinite 200 Pro, TECAN, Switzerland) to investigate the different components (Figure 2B). No characteristic absorption peak was achieved at liposomes alone (curve 'a'), while only one strong absorption peak at ~280 nm was appeared at pure dopamine (curve 'b'). After formation of DLL, the characteristic peak of dopamine molecules could be observed at ~280 nm (curve 'c'), indicating that dopamine was doped to the liposomes. Based on the above-synthesized DLL and the nanobelts, a precondition for development of PEC sensor lies in the fact whether the loaded dopamine can enhance the photocurrent of Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts. As shown in Figure 2C, almost no photocurrent was achieved at bare FTO electrode (curve 'a') and a relatively weak photocurrent was observed at Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts/FTO (curve 'b'). Upon addition of dopamine, a strong photocurrent significantly appeared in Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts/FTO (curve 'd'), however, no obvious change of the photocurrent in bare FTO electrode (curve 'c'). The reason might be attributed to the fact that dopamine was used as a good electron donor to scavenge the holes of Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts, thus, the process of charge recombination was inhibited to improve the intensity of photocurrent. Based on the above-mentioned results, we might make a conclusion that dopamine could enhance the photocurrent of Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts. For the development of DLL-based PEC immunosensing platform, another key question arises as to whether Triton X-100 could readily resolve the liposomes and release the dopamine. To clarify this issue, we investigated the change of photocurrent before and after adding Triton X-100 under different conditions. As seen from histograms 'a-b' in Figure 2D, the photocurrents of Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts-modified FTO electrode were not almost changed in the absence and presence of Triton X-100 when using AFB1-BSA and AFB1-BSA-lipsomes as the competitors in the competitive immunoassay format. When AFB1-BSA-DLL was present in this system, significantly, the photocurrents largely increased upon addition of Triton X-100 (histogram 'c'). The increase in the photocurrent was attributed to the as-released dopamine from the liposomes. These results also demonstrated that only the free dopamine could enhance the photocurrent in this system and other substance could not disturb the experimental phenomenon.

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Figure 2. (A) TEM image of dopamine-loaded liposome [insets: (top) the corresponding photograph and (bottom) magnification image]; (B) UV−vis absorption spectra of (a) liposome, (b) dopamine and (c) dopamine-loaded liposome; (C) photocurrent responses of (a,c) bare FTO electrode and (b,d) Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts-modified FTO electrode before (a,b) and after (c,d) adding dopamine; and (D) the photocurrents of the different signal labels (a) AFB1-BSA (b) AFB1-BSA-liposome (c) AFB1-BSA-DLL in the absence and presence of Triton X-100 (note: 0.1 ng mL-1 AFB1 used in this case).

Optimization of Experimental Conditions. To increase the photocurrents of the Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts, the doped Mn2+ in the Zn3(OH)2V2O7•2H2O should be optimized. As shown in Figure 3A, a maximum photocurrent change (∆I = Itarget − Ibackground, where Ibackground and Itarget were the photocurrent before and after reaction with target AFB1, respectively) could be acquired at a 1.0% doped ratio of Mn2+ by using 0.1 ng mL-1 AFB1 as an example. It was attributed to the fact that crystal defects could influence the charge carrier dynamics of Zn3(OH)2V2O7•2H2O, including excitation, separation, trap and transfer. Simultaneously, the doped Mn2+ could be acted as the initial charge carrier acceptor to reduce the electron-hole recombination and promote interfacial charge transfer from the excited Zn3(OH)2V2O7•2H2O nanobelts to FTO electrode. However, the 10


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change of photocurrent toward target decreased after 1.0% doped ratio, which might be attributed to the too many Mn2+ ions would form a new electron−hole recombination central. Thus, 1.0% doped ratio of Mn2+ was adopted for the preparation of Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts. Also, the pH of detection solution and immunoreaction time also affect the sensitivity of PEC immunoassay. To investigate the pH influence, the photocurrents were measured within the range of 5.5–8.5. An optimal photocurrent signal was obtained at pH 7.0 (Figure 3B, curve 'a'). Curve 'b' in Figure 3B gives the effect of different immunoreaction time on the photocurrent of the PEC immunoassay. The photocurrents of the Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts/FTO increased with the increasing reaction time and tended to level off after 35 min. A longer reaction time did not significantly improve the photocurrent signal. To minimize the detection time, 35 min was selected as the incubation time for antigen−antibody reaction. Hence, pH 7.0 and 35 min were chosen for the pH of detection solution and incubation time of antigen−antibody reaction.

Figure 3. The effects of (A) Mn2+-doped ratio in Zn3(OH)2V2O7•2H2O, (B-a) pH of detection solution and (B-b) incubation time for the antigen-antibody reaction on the photocurrent of the PEC immunoassay (note: 0.1 ng mL-1 AFB1 used in this case).

Analytical Performance of Photoelectrochemical Immunoassay. To evaluate the detectability of the developed PEC immunoassay, AFB1 standards with different concentrations were tested by Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts-based PEC sensor accompanying the signal-amplified strategy of DLL under optimal conditions. The photocurrents of the developed PEC immunoassay toward a series of different-concentration AFB1 standards are depicted in Figure 4A. Owing to the

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high-efficiency dopamine toward the photocurrent of Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts photoactive materials, the photocurrents decreased with the increasing AFB1 concentration. A linear correlation between photocurrents and the logarithm of AFB1 concentrations was observed in the dynamic range from 0.5 pg mL-1 to 10 ng mL-1 with a limit of detection (LOD) of 0.3 pg mL-1 at signal-to-noise ratio of 3 (Figure 4B). The linear regression equation could be fitted to y (nA) = 641.0 - 398.48 × LogC[AFB1] (ng mL-1, R2 = 0.9979, n = 8). Obviously, the lowest detection concentration of our strategy was smaller than those of rapid AFB1 ELISA kit (0.2 ng mL-1; Cat# SE120001, Sigma) and AFB1 low matrix ELISA kit (0.02 ng mL-1; Cat# SE120002, Sigma).

Figure 4. (A) Photocurrents of Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts-based PEC immunoassay toward target AFB1 standards with different concentrations, (B) the relationship between the peak current value and AFB1 concentration (inset: Linear calibration plot for AFB1), (C) the stability of the Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts-modified FTO electrode and (D) the specificity of the developed PEC immunoassay toward AFB1 (0.1 ng mL-1), AFB2 (10.0 ng mL-1), AFG1 (10.0 ng mL-1), AFG2 (10.0 ng mL-1), OTA (10.0 ng mL-1) and OA (10.0 ng mL-1) (note: Each data point represents the average value obtained from three different measurements).

As the signal-generation tag, the stability of Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts-coated 12


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FTO electrode was also very crucial during the measurement. As shown in Figure 4C, the produced photocurrents on the modified electrode were almost the same (RSD = 0.54%) in 12 'on-off' runs by controlling the light irradiation switch. Hence, the stability of Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts-modified FTO electrode was satisfactory. The selectivity of the developed PEC immunoassay based on Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts was studied against non-target analytes such as AFB2, AFG1, AFG2, ochratoxin A (OTA) and okadaic acid (OA). Relative to the background signal (i.e., blank sample), target AFB1 and non-target AFB2 decreased the photocurrents of Mn2+-doped Zn3(OH)2V2O7•2H2O-modified FTO electrode, whereas other non-target analytes including AFG1, AFG2, OTA and OA did not cause the significant change in the photocurrent, regardless of whether they coexisted or not (Figure 4D). Nevertheless, the cross-reactivity of our system with non-target AFB2 might be mostly likely as a consequence of the similar molecular structure with target AFB1. Considering this concern, the specificity of nanobelts-based PEC immunoassay was acceptable. Monitoring of AFB1 in Real Samples. To investigate the accuracy of our newly developed strategy toward the real sample evaluation, Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts-based PEC immunoassay was utilized for the quantitative screening of 12 peanut samples including 6 naturally contaminated and 6 spiked samples (note: Peanut samples and extraction procedure were similar to our previous report,41 as described in the Supporting Information). The AFB1 concentrations in the cases were calculated on the basis of regression equation in Figure 4B. As a reference, the results were compared with those obtained from commercial AFB1 ELISA kit (Sensitivity: 5.0 pg mL-1; Diagnostic Automation Inc.). As indicated from Table 1, all texp values were lower than tcrit (tcrit[0.05,4] = 2.77). Moreover, the regression equation based on the average values between two methods could be fixed to y = 1.0386x - 0.1324 (r = 0.9964, n = 12, x-axis: PEC immunosensor; y-axis: ELISA kit). Therefore, the accuracy of Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts-based PEC immunoassay was in accordance with commercial AFB1 ELISA kit for the real sample analysis. ■ CONCLUSIONS In summary, this work demonstrated a novel PEC immunosensing protocol for the detection of small-molecular mycotoxins (AFB1 used in this case) by coupling with dopamine-loaded liposome

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and Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts for the signal amplification. Experimental results revealed that the released dopamine from the liposomes after the immunoreaction could enhance the photocurrent of Mn2+-doped Zn3(OH)2V2O7•2H2O nanobelts. Compared with conventional enzyme immunoassays with photoelectrochemical measurement, introduction of dopamine-loaded liposome could efficiently avoid the participation of natural enzymes (e.g., HRP and ALP). Moreover, the whole assay time including the immunoreaction and PEC measurement was

Dopamine-Loaded Liposomes for in-Situ Amplified ... - ACS Publications

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