A simple label-free photoelectrochemical immunosensor for highly sensitive detection of aflatoxin B₁ based on CdS–Fe₃O₄ magnetic nanocomposites

Abstract

A simple label-free photoelectrochemical (PEC) platform was developed based on magnetic CdS–Fe₃O₄ nanocomposites and used for detection of aflatoxin B₁ (AFB₁). CdS quantum dots (QDs) were successfully assembled on mesoporous Fe₃O₄ NPs via covalent binding and employed as the photoactive materials on the surface of screen-printed electrode (SPE). H₂O₂ was employed as a sacrificial electron donor for scavenge photo-generated holes in the valence band of CdS QDs. Anti-aflatoxin B₁ (anti-AFB₁) was conjugated onto CdS–Fe₃O₄ nanocomposites modified SPE by using the classic EDC coupling reactions between the carboxy group (–COOH) groups on the surfaces of the TGA capped CdS QDs and the amino group (–NH₂) groups of the antibody (Ab). The concentrations of AFB₁ were measured through the decreased photocurrent resulting from the increased steric hindrances due to the formation of the immunocomplex. Under the optimal conditions, the PEC biosensor for AFB₁ determination exhibited a linear range from 0.01 ng mL⁻¹ to 80 ng mL⁻¹ with a detection limit of 5.0 pg mL⁻¹. Besides, the PEC biosensor has been applied in the corn samples detection. The proposed PEC biosensor is simple, sensitive, fast and stable, which opens up a new promising PEC platform for other small molecules analysis.

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1. Introduction

Aflatoxins produced by Aspergillus flavus and Aspergillus parasiticus are toxic secondary metabolites and generally recognised as potent carcinogens, mutagens and teratogens.^1–3 Aflatoxins have major health and economic problems worldwide. Among the aflatoxins, aflatoxin B₁ (AFB₁) is one of the most toxic compound and identified as group I carcinogens by the International Agency for Research on Cancer of the World Health Organisation (WHO).⁴ In generally, AFB₁ could be detected in agricultural commodities, such as corn, peanuts and feedstuffs. There has been an increasing interest to develop simple and sensitive analytical method for detection of aflatoxin B₁.^5–7

There are some methods developed for the detection of AFB₁, such as high-performance liquid chromatography (HPLC)^8,9 and enzyme-linked of immunosorbent assay (ELISA).^10,11 Although the good sensitivity can be achieved, these methods are laborious, expensive and time-consuming.¹² Photoelectrochemical (PEC) analysis is a newly emerged yet dynamically developing technique and has attracted substantial attention.^13–16 In the PEC detection process, light is used as the excitation source and the generated photocurrent is used as the detection signal, which is just the reverse process of electrochemiluminescence (ECL). Because of the separation of excitation source and detection signal, the PEC analysis possesses higher sensitivity than the conventional electrochemical analytical method¹⁷ and the use of electronic detection makes the PEC instrument simple and low-cost.¹⁸ During the past decade, PEC analysis have been applied in the determination of DNA damage,¹⁹ DNA,²⁰ proteins²¹ and small molecules.²² In this work, a simple PEC immunosensor was first developed for fast detection of AFB₁.

Semiconductor quantum dots (QDs) have shown great promising application prospect due to its high fluorescence quantum yields, stability against photobleaching and size-controlled luminescence properties.^22–24 As a result, QDs exhibit a wide range of electrical and optical properties and can be used for various applications such as light-emitting diodes, solar cells, lasers and transistors.²⁵ CdS QDs has received much attention because of the ideal band gap (2.25 eV), and it has appeared as an eminent material for PEC analysis.^13,25,26 Mesoporous Fe₃O₄ NPs has well magnetic properties, good hydrophilicity, large surface areas and high internal pore surface area, which have great potential for much load of CdS QDs. Hence, considering the above aspects, magnetic CdS–Fe₃O₄ nanocomposites could be an ideal material for the PEC tests.

Herein, we developed a simple label-free PEC immunosensor for AFB₁ detection based on magnetic CdS–Fe₃O₄ nanocomposites, as shown in Scheme 1. CdS QDs were employed as the photoactive materials and loaded on the surface of mesoporous Fe₃O₄ nanoparticles (NPs) through covalent binding between carboxyl groups (–COOH) and amido groups (–NH₂). The magnetic CdS–Fe₃O₄ nanocomposites were then employed for antibody immobilization. After that, the obtained Ab–CdS–Fe₃O₄ bioconjugates were assembled on the screen-printed electrode (SPE) and then BSA was then applied to block the unbound active sites. AFB₁ was detected via specific affinity interactions between antibody and antigen. The photocurrent decreased with the increase of AFB₁ concentration. Besides, H₂O₂ was employed as a sacrificial electron donor for scavenge photo-generated holes in the valence band of CdS QDs, therefore leading an enhanced photocurrent. The established PEC immunosensor provides a simple, fast and sensitive strategy for detection of AFB₁ and has applied in corn samples analysis. In addition, it can provide a general approach in the detection of other hazardous substances in food.

Scheme 1 Fabrication process of the PEC immunosensor.

2. Experimental section

2.1. Materials and reagents

Cadmium chloride (CdCl₂·2.5H₂O), sodium sulfide (Na₂S·9H₂O), ferric chloride (FeCl₃·6H₂O) and sodium hydroxide (NaOH) were purchased from Sinopharm Chemical Reagent Shanghai Co., Ltd. Thioglycolic acid (TGA) was obtained from Tianjin Kermel Chemical Reagent Co. N-Hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were obtained from Aladdin Reagent Database Inc. (Shanghai, China). AFB₁ and anti-AFB₁ were purchased from Saijie Biotechnology Co., Ltd. (Shanghai, China). Bovine serum albumin (BSA) were obtained from Sigma-Aldrich (Beijing, China). All other chemicals were of analytical reagent grade and used without further purification. Phosphate buffer solution (PBS, pH 7.4) which prepared from disodium hydrogen phosphate (Na₂HPO₄·12H₂O, 0.1 mol L⁻¹) and potassium phosphate monobasic (KH₂PO₄, 0.1 mol L⁻¹) was used for preparation of the antibody, antigen and washing buffer solution. All aqueous solutions were prepared using ultrapure water (Milli-Q, Millipore).

2.2. Apparatus

Scanning electron microscope (SEM) image and energy dispersive spectrometer (EDS) were obtained using a field emission SEM (Zeiss, Germany). Transmission electron microscope (TEM) image was obtained from an H-800 microscope (Hitachi, Japan). All PEC measurements were carried out with a home-built PEC system. A common low-cost 30 W LED lamp was used as irradiation source. Photocurrent was measured on a CHI 760D electrochemical workstation (Chenhua Instrument Shanghai Co., Ltd., China) using a three-electrode system containing a carbon working electrode, a carbon counter electrode and a Ag/AgCl reference electrode.

2.3. Synthesis of CdS QDs

In this work, the utilized CdS QDs was synthesized according to the previous report with a slight modification.²⁵ Firstly, 250 μL of TGA was added to 50 mL of 1.0 × 10⁻² mol L⁻¹ CdCl₂ aqueous solution and N₂ was bubbled throughout the solution for 30 min to remove the dissolved O₂. During this period, 1.0 mol L⁻¹ NaOH was added to adjust the above solution to the desired pH value of 11. Then, 5.5 mL of 0.1 mol L⁻¹ Na₂S aqueous solution was injected into this solution to obtain TGA-capped water-soluble CdS QDs. After that, the mixture was refluxed under N₂ atmosphere for 4 h. All the finally obtained TGA-capped CdS QDs was diluted with the same volume of water and stored in a refrigerator at 4 °C for future use.

2.4. Synthesis of CdS–Fe₃O₄ nanocomposites

The monodisperse Fe₃O₄ NPs with mesoporous structure were prepared based on the previously reported literature.²⁷ FeCl₃·6H₂O (1 g) was dissolved in ethylene glycol (20 mL) to form a clear solution, followed by the addition of NaAc (3 g) and ethylenediamine (10 mL). The mixture was stirred vigorously for 30 min and then sealed in a Teflon-lined stainless-steel autoclave. The autoclave was heated to and maintained at 200 °C for 8 h, and allowed to cool to room temperature. The black products were washed several times with ultrapure water and dried under vacuum.

To obtain the CdS–Fe₃O₄ nanocomposites, 2 mL of TGA-capped CdS QDs solution was mixed with EDC/NHS solution containing 1 mL of 0.5 mol L⁻¹ of EDC and 1 mL 0.1 mol L⁻¹ of NHS. 1 mg of monodisperse Fe₃O₄ NPs was then added into the above solution and oscillated about 30 min. After that, the obtained solution was treated with magnetic separation and washed several times with ultrapure water.

2.5. Preparation of Ab–CdS–Fe₃O₄ bioconjugates

The CdS–Fe₃O₄ nanocomposites were pretreated with EDC/NHS solution to activate carboxyl. Then, 1 mL of 10 μg mL⁻¹ anti-AFB₁ solution was added into 1 mL of pretreated CdS–Fe₃O₄ nanocomposites and incubated 24 h under shaking at 4 °C. The resulting bioconjugates of Ab–CdS–Fe₃O₄ were obtained under an external magnetic field and redispersed in 1 mL of PBS (pH 7.4, 0.1 mol L⁻¹). Finally, the prepared Ab–CdS–Fe₃O₄ bioconjugates were stored in a refrigerator at 4 °C for the future use.

2.6. Fabrication of the label-free PEC immunosensor

The SPE were first washed with ultrapure water and ethanol, respectively. 4 μL of the Ab–CdS–Fe₃O₄ bioconjugates was dropped on the surface of SPE. After drying in air, the modified electrode was washed with the washing buffer thoroughly and then 4 μL of BSA was incubated on the modified electrode for 1 h at 4 °C to block non-specific binding sites followed by washing with washing buffer. Next, 4 μL of AFB₁ solution with different concentrations was dropped onto the above electrodes and incubated for 1 h at 4 °C followed by washing with washing buffer. The resulting SPE electrodes were finally applied in PEC measurement.

2.7. PEC detection

The PEC detection was carried out in PBS (pH 8.0, 0.1 mol L⁻¹) containing 0.1 mol L⁻¹ KCl and 7 mmol L⁻¹ H₂O₂ which served as a sacrificial electron donor during the photocurrent measurement. All PEC measurements were carried out with a home-built PEC system. A common low-cost 30 W LED lamp was used as excitation light and was switched on and off every 10 s.

3. Results and discussion

3.1. Characterization of CdS–Fe₃O₄ nanocomposites and mesoporous Fe₃O₄ NPs

SEM was used as a characterization technique to record the morphology of CdS–Fe₃O₄ nanocomposites. As shown in Fig. 1A, the as-prepared CdS–Fe₃O₄ nanocomposites own monodisperse size with an average diameter of 60–70 nm and the corresponding EDX spectrum (Fig. 1B) confirm that CdS was successfully coupled with Fe₃O₄ NPs. Fig. 1C presents the TEM of the Fe₃O₄ NPs. It can be seen that the Fe₃O₄ NPs have an average size of about 70 nm. Moreover, the mesoporous structures of these Fe₃O₄ NPs with the porous size about 10 nm provide the possibility for the load of CdS QDs. Fig. 1D shows the TEM image of CdS QDs, and the diameter of CdS QDs was about 4–5 nm. As shown in Fig. 1E, the HR-TEM image of CdS–Fe₃O₄ nanocomposites could further prove that CdS QDs was successfully loaded on the Fe₃O₄ NPs.

Fig. 1 (A) SEM image and (B) EDS image of CdS–Fe₃O₄ nanocomposites. (C) TEM image of mesoporous Fe₃O₄ NPs and CdS QDs (D). (E) HR-TEM image of CdS–Fe₃O₄ nanocomposites.

3.2. Characterization of the fabricated PEC immunosensor

EIS was an effective method for characterizing the interface properties of electrodes and applied to monitor the fabrication procedure of the immunosensor. Fig. 2A showed the Nyquist plot of SPE with different modification process using [Fe(CN)₆]^3−/4− as redox probe. The concentration of AFB₁ used in EIS test was 10 ng mL⁻¹. The impedance spectrum includes a semicircle and a linear part. The electron-transfer resistance (R_et) which equals the semicircle diameter, reflects the restricted diffusion of the redox probe through the multilayer system related directly to film permeability. For the bare SPE, it exhibited a very small semicircle diameter (curve a), suggesting a diffusional limiting step of the electrochemical process. After modified with CdS–Fe₃O₄–Ab on the SPE electrode, the resistance R_et increased obviously (curve b) indicating that the modified layers hindered the access of the redox probe to the SPE surface. The resistance R_et increased after the BSA blocking (curve c) and subsequent stepwise immobilization of Ag (curve d) because of the insulating effect of the proteins, indicating the successful immobilization of every step.

Fig. 2 (A) EIS and (B) corresponding photocurrent responses of the electrodes: (a) SPE (b) SPE/CdS–Fe₃O₄–Ab (c) SPE/CdS–Fe₃O₄–Ab/BSA (d) SPE/CdS–Fe₃O₄–Ab/BSA/AFB₁. Inset: photocurrent responses of SPE/Fe₃O₄–Ab (a) and SPE/CdS–Fe₃O₄–Ab (b). The EIS spectra was obtained in 2.5 mmol L⁻¹ [Fe(CN)₆]^3−/4− solution containing 0.10 mol L⁻¹ KCl. The PEC tests were performed in 0.1 mol L⁻¹ PBS (pH = 8.0) containing 0.1 mol L⁻¹ KCl and 7 mmol L⁻¹ H₂O₂ with 0 V applied potential.

The fabrication procedure of the PEC immunosensor could also be monitored by photocurrent responses, as shown in Fig. 2B. After CdS–Fe₃O₄–Ab was modified onto a bare SPE electrode (curve b), the photocurrent intensity increased properly, and as shown in Fig. 2B (inset), the photocurrent intensity was significantly enhanced after the load of CdS QDs thanks to the effect of CdS QDs. Subsequently, the photocurrent intensity decreased gradually after immobilization of BSA (curve c) and antigen (curve d). This could be attributed to the fact that the immobilization of these protein materials on the SPE/CdS–Fe₃O₄–Ab electrode partly obstructed H₂O₂ to the surface of CdS for reaction with the photogenerated holes. Therefore, the photocurrent responses also proved the successful fabrication of the immunosensor.

3.3. Optimization of experimental conditions for PEC detection

The effects of experimental conditions, such as pH and the concentration of H₂O₂, on the analytical performance of the above immunosensor were investigated. Fig. 3A showed the influence of pH on the photocurrent responses of the immunosensor, which was operated in PBS with different pH values ranging from 6.5 to 8.5. The maximum photocurrent response was achieved at pH 8.0, thus 8.0 was selected as the optimum pH value in subsequent PEC tests. The concentration of H₂O₂ also has significant effect on the sensing performance. As shown in the Fig. 3B, with the increase of concentration of H₂O₂, the photocurrent intensity increased and reached a plateau at 7 mmol L⁻¹. Thus, 7 mmol L⁻¹ H₂O₂ was selected as the optimal concentration of the sacrificial electron donor during the PEC tests.

Fig. 3 Effects of pH (A) and H₂O₂ concentration (B) on the photocurrent response of the immunosensor.

3.4. PEC detection of aflatoxin B₁

The photocurrent response was directly related to the concentration of AFB₁. Hence, the concentration of AFB₁ could be detected by monitoring the photocurrent signal of immunosensor. The photocurrent decreases with the increase of the concentration of AFB₁. As shown in Fig. 4, the decrement of the photocurrent was proportional to the logarithm of the concentration of the AFB₁ ranging from 0.01 ng mL⁻¹ to 80 ng mL⁻¹ with a correlation coefficient of 0.9899, and the limit of detection (LOD) was 5 pg mL⁻¹ (S/N = 3). It was comparable and even better than those of many reported immunoassay methods for AFB₁, such as optical biosensing approach,²⁸ electrochemical immunoassay²⁹ and electrochemical impedance spectroscopy method,³⁰ which is a promising potential method in the fast detection of other significant proteins in food.

Fig. 4 Effect of different AFB₁ concentrations on the differential photocurrent responses, ΔI = I₀ − I, I₀ was the photocurrent of the immunosensor without AFB₁ immobilization and I was the photocurrent of the immunosensor. The PEC tests were performed in 0.1 mol L⁻¹ PBS (pH = 8.0) containing 0.1 mol L⁻¹ KCl and 7 mmol L⁻¹ H₂O₂ with 0 V applied potential.

3.5. Stability, reproducibility and selectivity

The stability of the immunosensor was evaluated. After the immunosensor was stored at 4 °C in a refrigerator for 2 weeks, 91% of its initial response was obtained for the detection of 0.1 ng mL⁻¹ AFB₁, indicating the good storage stability of the proposed sensor. The photocurrent responses were also recorded under several on/off irradiation cycles. As shown in Fig. 5A, the photocurrent response displayed no obvious change, indicating the stable readout for signal collection.

Fig. 5 (A) Time-based photocurrent response of the immunosensor under several on/off irradiation cycles. (B) The responses of interference (a) 0.1 ng mL⁻¹ AFB₁ (b) 0.1 ng mL⁻¹ AFB₁ + 10 ng mL⁻¹ DON (c) 0.1 ng mL⁻¹ AFB₁ + 10 ng mL⁻¹ OTA (d) 0.1 ng mL⁻¹ AFB₁ + 10 ng mL⁻¹ ZON.

The reproducibility of the immunosensor was evaluated by both intra-assay and inter-assay relative standard deviation (RSD). Analyzed from the experimental results, the intra-assay RSDs were 4.3%, 3.6% and 3.2% towards 0.1, 1, and 10 ng mL⁻¹ of AFB₁. The inter-assay RSDs of 5.7%, 5.3%, and 4.8% were obtained by measuring the same samples with five electrodes prepared independently at the identical experimental conditions. The results suggested the good precision and acceptable reproducibility of this biosensor.

The selectivity was assessed by using the deoxynivalenol (DON), ochratoxin (OTA), zearalenone (ZON) as interfering agents. It was evaluated by measuring the photocurrent response of 0.1 ng mL⁻¹ AFB₁ solution containing 10 ng mL⁻¹ of interfering substance, respectively. As shown in Fig. 5B, these interfering agents could not cause an obvious signal change, which indicated the satisfactory selectivity for AFB₁ detection.

3.6. Real sample analysis

In order to validate the feasibility of the PEC immunosensor, it was used for corn samples detection. As shown in Table 1, different concentrations of AFB₁ (1.00, 5.0, and 10.0 ng mL⁻¹) were added into corn samples, the average recoveries of the immunosensor were in the range of 99.2–101.4% and the relative standard deviation (RSD) was 2.3–4.1%. Hence, the immunosensor can be practically used as a quantitative method for AFB₁ detection in corn samples.

Table 1 The results of the AFB₁ determination in corn samples

Sample (ng mL^−1)	Addition content (ng mL^−1)	Detection content (ng mL^−1)	RSD (%)	Recovery (%)
0.24	1.00	1.31, 1.27, 1.19, 1.21, 1.29	4.1	101.4
	5.00	5.18, 5.37, 4.97, 5.38, 5.21	3.2	99.6
	10.00	10.15, 10.39, 9.97, 10.41, 9.89	2.3	99.2

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4. Conclusions

In this study, a simple label-free PEC immunosensor was established based on magnetic CdS–Fe₃O₄ nanocomposites for the detection of AFB₁. CdS QDs were assembled on the mesoporous Fe₃O₄ NPs and then employed as the photoactive materials. H₂O₂ was used as a sacrificial electron donor for scavenging the photo-generated holes in the valence band of CdS QDs and resulted in the enhancement of the photocurrent. This immunoassay exhibited a low detection limit of 5 pg mL⁻¹ as well as a wide linear range from 0.01 ng mL⁻¹ to 80 ng mL⁻¹ for AFB₁. Besides, the proposed biosensor has been applied in corn samples detection successfully. This strategy was simple, fast, cost-effective and ultrasensitive, which opens a new perspective for other small molecules analysis.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (nos 21175057, 21375047 and 21377046), the Science and Technology Plan Project of Jinan (no. 201307010) and QW thanks the Special Foundation for Taishan Scholar Professorship of Shandong Province and University of Jinan (no. ts20130937).

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