Multifunctional Peroxidase-Encapsulated Nanoliposomes: Bioetching

Research Article Cite This: ACS Appl. Mater. Interfaces 2019, 11, 23832−23839

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Multifunctional Peroxidase-Encapsulated Nanoliposomes: Bioetching-Induced Photoelectrometric and Colorimetric Immunoassay for Broad-Spectrum Detection of Ochratoxins Jie Wei,†,‡ Huaming Chen,† Haihang Chen,† Yunyan Cui,† Aori Qileng,† Weiwei Qin,*,† Weipeng Liu,† and Yingju Liu*,† Department of Applied Chemistry, College of Materials and Energy and ‡The Guangdong Provincial Key Laboratory of Food Quality and Safety, College of Food Science, South China Agricultural University, Guangzhou 510642, China

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†

S Supporting Information *

ABSTRACT: In this study, a versatile dual-modal readout immunoassay platform was achieved for sensitive and broadspectrum detection of ochratoxins based on the photocurrent response of flexible CdS/ZnO nanorod arrays/reduced graphene oxide and the localized surface plasmon resonance (LSPR) peak shift of Au nanobipyramids (Au NBPs). By using nanoliposomes as the vehicle to carry the secondary antibody and encapsulate horseradish peroxidase (HRP), the photocurrent change and the peak shift can be greatly amplified. The reaction mechanism was investigated in detail, indicating that HRP can trigger enzymatic bioetching in the presence of H2O2. In the photoelectrochemical detection, the oxidized HRP can etch CdS on the photoelectrode, resulting in the photocurrent change, while in the colorimetric detection, HRP can oxidize H2O2 to produce hydroxyl radicals that can etch Au NBPs to form multiple color changes and LSPR shifts. Compared with the common single-modal immunoassay for ochratoxins, such dual-modal immunoassay is more precise and reliable, owing to the completely independent signal conversion and transmission mechanism. Therefore, we hope that this accurate, simple, and visualized strategy may create a new avenue and provide innovative inspiration for food analysis. KEYWORDS: ochratoxins, dual modality, photoelectrochemistry, colorimetry, immunosensor, broad spectrum

1. INTRODUCTION Ochratoxins including ochratoxins A−C (OTA, OTB, and OTC, respectively) are generated by various Aspergillus and Penicillium fungi.1 These toxins can be found in foods, agricultural by-products, and feedstuffs such as grains, beans, nuts, cocoa, dried fruits, and fermented drinks.2,3 Especially, OTA, due to its chemical and thermal stabilities, not only brings economic losses to agriculture but also poses a threat to human and animal health since it can induce multiple toxicity effects on kidney, liver, and immune modulatory system.4,5 The World Health Organization (WHO) has established 100 ng/kg of body weight as the tolerable weekly intake.6 As analogues, OTB and OTC are the dechlorinated derivative and ethyl esterification of OTA, respectively,7 but they usually coexist with OTA and possess a similar or even higher cytotoxicity, immunotoxicity, or genotoxicity.8 Therefore, the simultaneous determination of three ochratoxins is important for food safety and human health. At present, various traditional analytical methods are used to detect ochratoxins, such as thin-layer chromatography,9 highperformance liquid chromatography,10 liquid chromatography−mass spectrometry,11 and enzyme-linked immunosorbent assay (ELISA).12,13 However, these methods were usually © 2019 American Chemical Society

restricted in actual applications, owing to the inaccuracy, timeconsuming, low sensitivity, expensive equipment, or professional operation. Recently, photoelectrochemical (PEC) detection as a rapid technique has been extensively explored.14−17 Compared with traditional ELISA, the PEC immunoassay possesses low detection limit and high detection sensitivity due to different forms of energy and total separation of excitation source (light) and detection signal (photocurrent).18 The relatively simple and cheap instruments are more convenient for miniaturization. In addition, we have found that the antibody produced from OTB-ovalbumin (antibody) with OTB-bovine serum albumin (antigen) possessed excellent sensitivity and broad specificity to ochratoxins, while the simultaneous determination of these ochratoxins was realized by the broad-spectrum immunorecognition of the corresponding antigens and antibody.19 Even if the PEC immunoassay has some advantages, it is still difficult to meet the increasing test demand if only the singlemodality immunoassay is used. Therefore, more scholars began Received: March 7, 2019 Accepted: June 17, 2019 Published: June 17, 2019 23832

DOI: 10.1021/acsami.9b04136 ACS Appl. Mater. Interfaces 2019, 11, 23832−23839

Research Article

ACS Applied Materials & Interfaces Scheme 1. Schematic Diagram of the Construction of a Dual-Modality Immunosensor

Biochemical Co. Ltd., Shanghai. Graphite flakes were purchased from Tianjin Alfa Aesar Chemical Co., Ltd. Horseradish peroxidase (HRP) was from Sangon Biological Engineering Technology & Services Co., Ltd., Shanghai. The antigen of ochratoxins (1 mg/mL) and corresponding antibody (Ab1, 1 mg/mL) were from the College of Food Sciences, South China Agricultural University, Guangzhou. Secondary goat antirabbit antibody (Ab2, 1 mg/mL) was purchased from Santa Cruz. OTA, OTB, and OTC were from Puhuashi Technology Company, Beijing. Phosphate-buffered saline (PBS) of different pH values was prepared by mixing 1/15 M stock solutions of KH2PO4 and Na2HPO4 at specific ratios. In addition, the washing buffer in the immunoassay was 0.5% Tween 20 in 0.01 M PBS (PBST, 0.01 M and pH 7.4). Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were recorded using FEI TEM (Tecnai 12, Holland) and Hitachi FE-SEM (S-4800, Japan). X-ray powder diffraction (XRD) was performed with a X-ray diffractometer (D/ max-IIIA, Japan), while the absorbance was scanned from 300 to 900 nm on a UV−vis spectrophotometer (UV2550, Shimadzu, Japan) at about 25 °C. The photocurrent was measured by a three-electrodebased electrochemical workstation (CHI 660D, Chenhua Instruments Co., Ltd., Shanghai) with a photoelectrochemical system (PEAC 200A, Ida, China) utilizing LED as the illuminant source (20 mW cm−2), where CdS/ZnO NRs/rGO was used as the working electrode, with the Ag/AgCl electrode and platinum sheet electrode as the reference electrode and counter electrode, respectively. The electrolyte was PBS (pH 7.4, 1/15 M) containing 0.1 M AA. 2.2. Synthesis of CdS/ZnO NRs/rGO. Graphene oxide (GO) was prepared from graphite flakes by a modified Hummer’s method. Specifically, 160 mL of H2SO4 was added to a dry flask, followed by the slow addition of 4 g of graphite flakes and 14 g of KMnO4 under gentle stirring in an ice bath. The mixture was continuously stirred at 35 °C for 24 h and diluted with 400 mL of ice water. Then, H2O2 was added until the mixture was no longer discolored and had no gas evolution. After stirring for another 2 h, the soluble inorganic impurities and ions were removed by centrifugation at 5000 rpm. After washing with 300 mL of HCl solution (1 M) three times, neutralizing and dialyzing for 1 week, the precipitates were finally stored in water or ethanol. Then, the GO solution in water or in ethanol was equally mixed, followed by immersing the zinc foil into this mixture for interfacial gelation. After 3 h, the zinc foil was taken out, washed, and immersed in water for 30 min to remove physically adsorbed GO platelets. After freeze-drying, the rGO film was peeled off followed by immersing in the mixture of zinc nitrate (0.04 M) and methenamine (0.04 M) at 95 °C for 5 h to form the ZnO NRs/rGO film. Finally, the film was immersed in a mixture of Cd(NO3)2 (10 mM) and thioacetamide (10 mM) at 40 °C for 40 min and then annealed at 550 °C for 2 h to form the CdS/ZnO NRs/rGO film. 2.3. Preparation of HRP-Encapsulated Liposome−Ab2 Conjugate. The HRP-encapsulated liposome was synthesized according to the thin lipid film hydrated method.33 Briefly, a mixture containing cholesterol, DPPE, and DPPC (molar ratio at 10:1:10, 30 mg) was dissolved in 4 mL of methanol and chloroform (1:6, v/v). Then, it was transferred to a round flask for rotary evaporation at 45

to construct dual-readout biosensors based on different signal conversion modes and transmission mechanisms,20−23 where the two signal paths are completely independent. The interference between them can be hardly found; thus, the errors caused by detection condition and personnel operation can be partly corrected. Recently, the colorimetric detection based on the localized surface plasmon resonance (LSPR) properties of noble metal nanoparticles has attracted considerable attention in the field of on-site detection and preliminary screening.24−26 Particularly, the morphology of noble metal nanoparticles can be adjusted to display multiple colors when absorbing and scattering visible light.27 Au nanobipyramids (Au NBPs) with sharp edges are expected to be ideal colorful chromogenic substrates due to their high sensitivity to refractive index.28 Compared with the traditional monochromic intensity change, the colorimetry based on a polychromatic display is more conducive to identify by naked eyes. Therefore, a dual-modality immunoassay platform from colorimetry with PEC can achieve more precise and reliable detection, owing to the completely independent signal conversion and transmission mechanism. Furthermore, the substrate of the PEC immunosensor can influence its efficiency. Graphene has been widely used in water splitting,29,30 lithium-ion batteries,31 and supercapacitors32 because of its laminated structure, mechanical properties, and conductivity. In this work, a flexible reduced graphene oxide (rGO) film was employed as the substrate for the in situ growth of CdS/ZnO nanorod arrays (NRs). Due to its high photoelectric conversion efficiency, it was used as the photoelectrode for PEC detection. Then, nanoliposomes, as the label carrier, encapsulate a large amount of HRP to link PEC immunoassay and colorimetric detection, where CdS/ ZnO NRs/rGO can be irreversibly bioetched by the oxidized HRP to produce the photocurrent change, and Au NBPs can be bioetched by the hydroxyl radicals into different sizes and shapes to exhibit a series of colors and LSPR band shifts. Therefore, the dual-modality readout immunoassay platform with PEC and colorimetric detection was realized.

2. EXPERIMENTAL SECTION 2.1. Materials and Instruments. Methenamine, thioacetamide, sodium citrate, ascorbic acid (AA), Triton X-100, AgNO3, NH3·H2O (25%), H2O2 (30%), NaBH4, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and cholesterol were from Aladdin Biological Technology Co. Ltd., Beijing. Hexadecyl trimethyl ammonium bromide (CTAB) and Zn(NO3)2 were purchased from Adamas Reagent Co., Ltd., Shanghai. Tween 20 and dopamine hydrochloride were purchased from Acros Organics, Shanghai. Chloroauric acid and hexadecyl trimethyl ammonium chloride (CTAC) were from Macklin 23833


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Figure 1. (A, B) SEM image of rGO, (C, D) ZnO NRs/rGO, and (E, F) CdS/ZnO NRs/rGO. (G) Elemental mapping of CdS/ZnO NRs/rGO. (H) XRD of (a) rGO, (b) ZnO NRs/rGO, and (c) CdS/ZnO NRs/rGO. (I) UV−vis spectra of (a) rGO, (b) ZnO NRs/rGO, and (c) CdS/ZnO NRs/rGO. The insets are the photographs of rGO (black), ZnO NRs/rGO (white), and CdS/ZnO NRs/rGO (yellow). °C to form the uniform lipid film, followed by the addition of 2 mL of PBS (0.01 M) containing 2.5 mg/mL HRP. After incubation at 35 °C for 2 h, the mixture was sonicated for 5 min on an ice bath. Finally, the liposomes were centrifuged and washed to remove the unencapsulated HRP. The HRP-encapsulated liposome was conjugated with Ab2 via glutaraldehyde coupling method. Specifically, 2 mL of the liposomes was dropwise added into a 1 mL of 2.5% glutaraldehyde solution under gentle stirring for 60 min. The excess glutaraldehyde solution was disposed by dialysis in PBS overnight. Subsequently, 50 μL of 1 mg/mL Ab2 was added and incubated for 12 h at 4 °C. After centrifugation and washing, the HRP-encapsulated liposome−Ab2 conjugate was stored at 4 °C. 2.4. Preparation of Au NBPs. Au NBPs were synthesized by a modified seed-mediated growth method.34 Briefly, the gold seeds were prepared by fast reduction with freshly prepared NaBH4 (25 mM, 0.25 mL) in a 10 mL solution including HAuCl4 (0.25 mM), CTAC (50 mM), and citric acid (5 mM). Then, the solution was heated at 80 °C for 1.5 h, where its color changed from brown to red, representing the formation of gold seeds. Finally, 1.25 mL of such seeds was injected into the solution containing CTAB (100 mL, 100 mM), HCl (2 mL, 1 M), HAuCl4 (5 mL, 10 mM), AgNO3 (1 mL, 10 mM), and AA (0.8 mL, 100 mM), reacted for 2 h at 30 °C, and purified. 2.5. Fabrication of the Dual-Modality Immunosensor. The platform of this dual-modality readout immunoassay is shown in Scheme 1. The wells were incubated with 50 μL of dopamine (1 mg/ mL) for 0.5 h at 37 °C. After drying, 20 μL of the ochratoxin antigen (10 μg/mL) and 20 μL of the blocking solution were successively added. By using the competitive immunoassay, 20 μL of the mixture containing different concentrations of ochratoxins A−C and specific volumes of Ab1 (5 μg/mL) were added to each well. Afterward, 20 μL of the HRP-encapsulated liposome−Ab2 suspension was dropped. (Note: all above steps were incubated at 37 °C for 60 min, and each well was washed with PBST). Successively, the liposomes were dissolved by adding 10 μL of 10 mg/mL Triton X-100 to release the encapsulated HRP. Finally, 300 μL of H2O2 (1 M) was added into the microplate and reacted at 37 °C for 15 min. For PEC detection, 200 μL of the resulted solution was used to etch CdS/ZnO NRs/rGO. In addition, the remaining 100 μL of enzymatic hydrolysate was transferred into the mixture containing 10 μL of HCl (1 M) and

100 μL of Au NBPs. After etching for 10 min at 50 °C, the absorption spectra of the mixture were measured in the range of 300−900 nm for colorimetric detection.

3. RESULTS AND DISCUSSION 3.1. Characterization of CdS/ZnO NRs/rGO. In PEC detection, the photoelectrical materials were mostly deposited on FTO. Here, a flexible rGO film was used as the substrate to immobilize PEC materials. The photograph of such a black film can be found in Figure S1A, implying that the size can be adjusted by the Zn foil. In addition, Figure 1A,B shows the cross-sectional SEM of this film at different scales, while the thickness of the whole rGO film was about 300 μm (Figure 1A). In this film, many graphene sheets were accumulated in a quasi-parallel manner to form a three-dimensional porous morphology (Figure 1B), providing large surface areas and facilitating rapid electron transfer. After the growth of ZnO NRs, the photograph of such a gray film can be found in Figure S1B, while the SEM images of ZnO NRs showed that they scattered on the rGO film with high-density coverage (Figure 1C). As shown in Figure 1D, uniform sizes and ordered shapes with inherent hexagonal apex diameters of about 250−400 nm can be observed. In addition, the ZnO NRs exhibited a smooth surface morphology. After the deposition of CdS, a clear yellow can be observed on the film (Figure S1C), while in Figure 1E,F, the solid ZnO NRs became hollow and tubular due to the Kirkendall effect from different diffusion rates of Cd and Zn,35 suggesting the deposition of CdS. This structure of nanotubes with a large surface-to-volume ratio was more conductive to transfer photoinduced electrons. The elemental mappings of CdS/ZnO NRs/rGO are shown in Figure 1G, revealing that Zn, O, C, Cd, and S were uniformly distributed. The crystalline structure of the samples was measured using X-ray diffraction analysis. As shown in Figure 1H (curve a), the formation of rGO was proven by the diffraction peak at 23.9° (JCPDS no. 74-2328). The characteristic diffraction peaks were found at 31.7, 34.4, 36.2, 47.5, 56.5, 23834


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with the number of lipid molecules in one liposome (N). Assuming that the HRP concentration in the liposome as the initial concentration, the number of HRP molecules encapsulated per liposome can be estimated as 894, which was large enough to amplify the signal. Since the original concentration of Ab2 to prepare HRP-liposome−Ab2 was about 16.7 μg/mL, the number of Ab2 on each liposome can be calculated to be 37. Therefore, the HRP-encapsulated liposome−Ab2 conjugate can be used for the signal amplification.

and 62.8°, assigning to (100), (002), (101), (102), (110), and (103) of ZnO with the hexagonal structure (JCPDS no. 361451) (curve b). Some new characteristic diffraction peaks of 24.8, 26.5, 28.2, 43.7, and 52.0° were indexed to (100), (002), (101), (110), and (112) of CdS (JCPDS no. 41-1049), indicating the successful decoration of CdS on ZnO NRs (curve c). The optical properties of such composites were studied by UV−vis spectrophotometry in the range of 250− 800 nm. As shown in Figure 1I, the rGO showed full absorption in the UV−vis region (curve a), while the absorption spectra of ZnO NRs had a distinct absorption edge around 400 nm with an estimated band gap of 3.1 eV (curve b). After combining with CdS (curve c), the spectrum of CdS/ZnO NRs/rGO moved toward a long-wavelength region, showing absorption edges around 570 nm with the band gap of 2.2 eV. Thus, the introduction of CdS can improve the absorption of ZnO in a long-wavelength region and enhance the photoelectric properties. 3.2. Characterization of HRP-Encapsulated Liposome−Ab2 Conjugate. The TEM image (Figure 2A)

A = A1p1 + A 2 p2 + A3p3 N=

4π 2 [r + (r − t )2 ] A

(1)

(2)

In this dual-modal immunosensor, the HRP-encapsulated liposome−Ab2 conjugate plays an important role to achieve signal generation. The rapid release of HRP from liposomes can shorten the reaction time and increase the signal response. Thus, it is important to explore whether Triton X-100 could easily break the liposomes to release the HRP without changing the enzyme activity. As shown in Figure 2C, after the injection of Triton X-100, the photocurrent change increased dramatically and reached the maximum in 10 min, which was attributed to the corrosion of the as-released HRP on the photoelectric substrate. However, the signal was not obvious without the surfactant, indicating that the liposome can be rapidly dissolved by Triton X-100. To explore the signal amplification of HRP-encapsulated liposome, a comparative experiment was designed with different labels. As shown in Figure 2D, the photocurrent of the CdS/ZnO NRs/rGO electrode was about 179 μA, and the LSPR peak position of Au NBPs was around 780 nm (histogram a). After incubation with conventional HRP−Ab2 (histogram b), the photocurrent and LSPR peak position presented a slight decline and blue shift, respectively, which were due to the etching of CdS and Au NBPs by the HRPinduced catalysis reaction. However, when the immunosensor was incubated with the HRP-encapsulated liposome−Ab2 conjugate (histogram c), the photocurrent and LSPR peak were remarkably reduced to 46 μA and 550 nm, respectively, suggesting that the introduction of liposomes can encapsulate more HRP for enzyme-induced biological etching so as to amplify the response signal remarkably and improve the sensor performance effectively. 3.3. Mechanism of the Dual-Modality Readout Immunosensor. Scheme 1 shows an illustration of the dual-modality readout immunoassay platform with PEC and colorimetric analysis for ochratoxins A−C. Briefly, dopamine with excellent adhesion was used to immobilize the antigen in the 96-well microplate, followed by the addition of the mixture with different concentrations of ochratoxins and a constant antibody. Therefore, the immovable antigen and removable ochratoxins could competitively combine with Ab1. Then, the liposome was used as the vehicle to conjugate with more Ab2 and encapsulate more HRP for the signal amplification. After that, the surfactant was added to dissolve liposomes rapidly, and HRP was released. Such an as-released HRP can react with H2O2; thus, CdS, the important component of the photoelectrode, was corroded. In addition, the hydroxyl radicals produced by HRP catalytic oxidation of H2O2 can bioetch Au NBPs to form different sizes and shapes, showing a vivid color change and shift of the LSPR peak.

Figure 2. (A) TEM image of HRP-liposome. (B) Size distribution of HRP-liposome (black) and HRP-liposome−Ab2 (green). (C) Photocurrent variation in the presence of a surfactant (red) and without a surfactant (blue). (D) Photocurrent (blue) and LSPR response (green) of different immunosensors for (a) uncorroded electrode, (b) HRP−Ab2, and (c) HRP-liposome−Ab2.

showed that the HRP-encapsulated liposomes were spherical or quasi-spherical. In Figure 2B, the average diameter was detected as about 168 nm by dynamic light scattering (DLS). After binding with Ab2, it changed to 210 nm, proving that Ab2 was successfully modified on the HRP-encapsulated liposome. According to eq 1, the average head group surface area in one lipid molecule (A) was estimated to be 0.45 nm2/lipid, where A1, A2, and A3 were 19, 71, and 41 Å2 for cholesterol, DPPC, and DPPE, respectively,36 and p1, p2, and p3 were the mole fractions of these three substances from the molar ratio of 10:10:1. According to eq 2, assuming liposomes as a monolayer with a thickness (t) of 4 nm37 and the liposome radius (r) was 84 nm from the DLS, the number of lipid molecules in one liposome (N) was calculated to be 380,000. Then, the concentration of the liposomes was estimated to be 4.2 × 1013/mL by dividing the total lipid molecule number 23835


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ACS Applied Materials & Interfaces As for the PEC detection (Figure 3A), CdS/ZnO NRs were modified on the rGO film as a novel flexible photoelectrode to

replace traditional FTO or ITO electrodes. Specifically, due to the high electron mobility, the introduction of the 3D rGO film provided a rapid charge channel. Besides, the configurations of ZnO and CdS were propitious to separate and transfer charge. Therefore, the recombination of photogenerated electron−hole pairs was limited. When the CdS/ ZnO NRs/rGO was irradiated, both CdS and ZnO semiconductors were activated to produce photogenerated electrons, while the photoinduced electron−hole pairs were formed by the electron transfer from the valence bands (VB) to conduction bands (CB). Due to the negative potential, photoelectrons can rapidly inject from the CB of CdS into ZnO then transfer to rGO to generate the photocurrent. In this band gap alignment, the coexistence of two semiconductors CdS and ZnO could improve charge separation. Meanwhile, the excited holes at the VB of ZnO NRs can transfer back to the VB of CdS. In the presence of a hole-trapping reagent of AA, the photocorrosion of CdS/ZnO NRs/rGO could be effectively avoided, thus ensuring a stable and efficient photocurrent. More importantly, CdS could be irreversibly bioetched by HRP-induced enzymatic catalysis in the presence of H2O2.38,39 After that, a weak photoelectric conversion was obtained since the ZnO NRs can only adsorb UV light. Therefore, the photocurrent intensity of CdS/ZnO NRs/rGO decreased with the amount of HRP, achieving the “signal-off” PEC detection. In addition, HRP can react with H2O2 to catalyze the oxidation of sulfur ions in CdS to sulfate anions according to eqs 3−5:40

Figure 3. (A) Schematic diagram of the response mechanism of dualmodality PEC and colorimetric immunosensor. (B) TEM graphs of Au NBPs with etching times of (a) 0, (b) 3, (c) 5, and (d) 10 min with HRP. (C) Corresponding UV−vis spectra of Au NBPs with different etching times. The insets in (C) were the relative photographs of (a)− (d).

−

CdS → Cd2 + + S2

(3)

HRP(red) + H 2O2 → HRP(ox) + H 2O

(4)

Figure 4. (A−C) Photocurrent response and (D−F) UV−vis spectra for OTA, OTB, and OTC at different concentrations of 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, and 0 μg/L. (G) Calibration curve based on the photocurrent response and (H) UV−vis spectra. (I) Universal calibration curve of ochratoxins. The insets were the photographs of corresponding immunosensors of OTA, OTB, and OTC at 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5 μg/L (from left to right). 23836


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equation of OTC was Δλ = 139.1 + 43.0[log COTC (μg/L)] with an LOD of 1.99 ng/L (r2 = 0.996, S/N = 3). Normally, OTA, OTB, and OTC coexist in contaminated food. Therefore, it is important to detect these ochratoxins simultaneously. It is simpler and more convenient to obtain the concentration of ochratoxins by establishing one universal calibration equation than three respective equations of OTA, OTB, and OTC. In a previous work, the OTA calibration plot from ELISA was utilized to detect all ochratoxins including OTA, OTB, and OTC.19 However, the immunosensor constructed with broad-spectrum antibodies has different responses to OTA, OTB, and OTC in equal concentrations. Herein, the average response of three calibration curves was used to construct a universal standard curve. As shown in Figure 4I, this immunosensor presented the same linear detection range from 1 ng/L to 5 μg/L for both PEC and colorimetric readouts. The calibration equations of PEC and colorimetric detections were ΔΙ =84.1 + 21.7 log C (μg/L) and Δλ = 152.8 + 46.9 log C (μg/L), respectively. In addition, the corresponding LODs of ochratoxins were calculated to be 0.7 ng/L (r2 = 0.996) and 1.7 ng/L (r2 = 0.985) with a signalto-noise ratio of 3. Compared with the previous work, (Table S1), this immunosensor exhibited a comparable or lower detection limit. Specially, due to the use of broad-spectrum immunologic recognition molecules, simultaneous detection of three ochratoxins (OTA, OTB, and OTC) can be achieved, while most of the reported methods can only detect OTA. In addition, this dual-modality readout immunosensor can provide more visual, sensitive, and accurate results. Benefiting from the different mechanisms and relatively independent signal transduction, there is no interference between these two signal routes in the analysis. Moreover, the visual color display is conducive to realize on-site detection. To assess the practicability and reliability of this dualmodality immunosensor in an actual analysis, the recovery test was implemented to monitor ochratoxins in the lake water from the South China Agricultural University. The sample was filtered to remove insoluble impurities, and then 0.001, 0.01, and 0.1 μg/L standard ochratoxins were added. In Figure 5A− C, the recoveries of OTA, OTB, and OTC were 95−112%,

−

HRP(ox) + S2 → HRP(red) + SO4 2

(5)

As for the colorimetric detection, Au NBPs can be also bioetched. Briefly, H2O2 can be degraded into hydroxyl radicals (·OH), a kind of the strongest oxidants, which can etch Au NBPs to form the vivid color change from brown to pink, thus corresponding to an LSPR shift.41,42 In Figure 3B(a), Au NBPs have a standard bipyramid shape with a brown color (Figure 3C(a)). After 3 min of reaction, both ends of Au NBPs were etched, and the color turned to gray green (Figure 3B(b),C(b)). After a longer time, the two tips of Au NBPs were completely etched and the structures have an olivelike shape, while the solution turned to bluish green (Figure 3B(c),C(c)). With continuous etching, Au NBPs were eventually etched into a quasi-spherical structure, and the solution changed into pink (Figure 3B(d),C(d)). Correspondingly, the UV−vis spectrum of Au NBPs showed two emission peaks at 780 and 516 nm, corresponding to the longitudinal plasmon peak and transverse plasmon peak, respectively, (Figure 3C(a)). During the etching process, with the change of morphology and color of Au NBPs, the LSPR peaks were blueshifted (curve b to d). Consequently, the construction of a dual-readout immunosensor based on PEC and colorimetric detection was realized. 3.4. Signal Response of Dual-Modality Readout Immunoassay. Under optimum conditions (Supporting Information, Figure S2), the fabricated dual-modality readout immunosensor was introduced to the broad-spectrum detection of three ochratoxins based on the competitive method. In PEC detection (Figure 4A−C), when the concentrations of OTA, OTB, and OTC increased, a lower amount of the liposome−Ab2 conjugate can be immobilized, thereby releasing less HRP for enzymatic bioetching of CdS and a corresponding stronger photocurrent. As shown in Figure 4G, a linear relationship between photocurrent change (ΔI) and the logarithm of ochratoxin concentrations can be found between 1 ng/L and 5 μg/L. Among this, ΔI can be calculated by subtracting I0 from I, where I was the photocurrent in the presence of ochratoxins, and I0 was the photocurrent without ochratoxins. Specifically, the calibration equation of OTA was ΔΙ = 86.8 + 22.8[log COTA (μg/L)] with a detection limit (LOD) of 0.79 ng/L (r2 = 0.973, signal-tonoise ratio (S/N) = 3), while the calibration equation of OTB was ΔΙ = 93.8 + 24.0[log COTB (μg/L)] with an LOD of 0.57 ng/L (r2 = 0.992, S/N = 3). In addition, the calibration equation of OTC was ΔΙ = 71.2 + 18.1[log COTC (μg/L)] with an LOD of 0.89 ng/L (r2 = 0.992, S/N = 3). Meanwhile, the colorimetric detection was implemented according to the etching degree of Au NBPs by recording the color change and LSPR peak shifting. In Figure 4D−F, with the decrease in the concentration of ochratoxins, the progressive blue shift of the peak position and series of distinct color transitions (brown → gray → green → blue → purple → pink) can be observed. As shown in Figure 4H, the calibration plot presented a linear relationship between Δλ and the logarithm of ochratoxin concentrations in the range from 1 ng/L to 5 μg/L. The Δλ represents the shift value of the LSPR peak position, which can reflect the index of the morphological change of Au NBPs. The calibration equation of OTA was Δλ = 167.3 + 50.9[log COTA (μg/L)] with an LOD of 1.46 ng/L (r2 = 0.992, S/N = 3), while the calibration equation of OTB was Δλ = 164.6 + 48.9[log COTB (μg/L)] with an LOD of 1.28 ng/L (r2 = 0.979, S/N = 3). In addition, the calibration

Figure 5. (A−C) Recoveries of the detection of (A) OTA, (B) OTB, and (C) OTC in the water sample. (D) Recoveries of OTA, OTB, and OTC calculated by the universal calibration equation at 2.5 ng/L. 23837


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(2017A030313077), the National Key Research and Development Program of China (SQ2017YFC160089), the Program for the Top Young Innovative Talents of Guangdong Province (2016TQ03N305), and the Foundation for High-Level Talents in South China Agricultural University. The authors thank Dr. Yongming Zhong of South China Agricultural University for the discussion on the characterization of materials.

92−122%, and 91−117%, respectively. This satisfactory result showed that the detection method can basically meet the requirement of real samples. As shown in Figure 5D, the recovery rates of OTA, OTB, and OTC estimated by the universal calibration equation were 136, 112, and 82% in PEC detection and 112, 130, and 88% in colorimetry, respectively. These results were still acceptable because the universal calibration equation facilitated simultaneous estimation of three ochratoxin concentrations without the need to detect OTA, OTB, and OTC separately; thus, the testing time was shortened, and the operation steps were simplified. Therefore, this novel dual-modality immunoassay platform with excellent sensitivity and accuracy may possess tremendous potential for actual application.

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4. CONCLUSIONS In this work, a dual-modality readout immunoassay platform for ochratoxin detection was constructed by integrating PEC and colorimetric detections based on different conversion mechanisms and signal transmissions. Comparing with traditional single modality assay, this immunosensor displayed a more accurate and reliable outcome. These outstanding properties can be attributed to following aspects. (i) The introduction of liposomes can effectively amplify detection signals by encapsulating large amounts of HRP and loading more Ab2, while the existence of HRP is the premise of biological etching. (ii) The multifunctional HRP can induce enzymatic catalytic reaction in the presence of H2O2, where the HRP(ox) can irreversibly etch CdS to change the photocurrent, and hydroxyl radicals can etch Au NBPs to produce color changes and LSPR peak position shifts. Especially, the introduction of colorimetry makes the detection easier to observe with naked eyes. Therefore, this construction strategy has great commercial application prospects since it cannot only realize the broad-spectrum detection of ochratoxins but also extend to other food contaminant analyses by simply changing the corresponding conjugated immune molecules.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.9b04136. Photographs of the flexible rGO film, rGO/ZnO NRs and CdS/ZnO (NRs)/(rGO), optimization of detection conditions, comparison of available biosensors for analysis of ochratoxins, recoveries results from the asprepared immunosensor for OTA, OTB, and OTC, recoveries results of OTA, OTB, and OTC by the universal calibration equation at 2.5 ng/L (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (W.Q.). *E-mail: [email protected]. Fax: 86-20-85285026. Phone: 86-20-85280319 (Y.L.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Scientific Foundation of China (21874048, 21705167, and 21705051), the Scientific Foundation of Guangdong Province 23838


Research Article


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