Development of novel biomimetic enzyme-linked immunosorbent assay method based on Au@SiO₂ nanozyme labelling for the detection of sulfadiazine

ABSTRACT

In the current study, a rapid and novel biomimetic nano-enzyme-linked immunosorbent assay has been established for the detection of sulfadiazine (SDZ) using Au@SiO₂ nanoparticles labelling as a marker. In this system, gold nanoparticles exhibited intrinsic peroxidase and activity with excellent water-solubility, and the SiO₂ nanoparticles as ideal carries possessed a large surface area and ease of surface functionalization. The molecularly imprinted polymer films were selected as bionic antibody because of its advantages of selective recognition and reusability. Combine the above advantages, the method exhibited good stability, recognition capability and sensitivity. Under the optimal condition, the limit of detection of the immunosorbent assay was 0.2 mg/L. Beef sample spiked with SDZ was analysed, and the recoveries were in the 78.00% to 90.96% range. The assay could be used to detect SDZ residues in pork and chicken samples, and the results were not significantly different from those obtained by high-performance liquid chromatography.

KEYWORDS:

• Nanozyme
• Au@SiO2 nanoparticles
• molecularly imprinted polymer film
• biomimetic immunosorbent assay
• sulfadiazine

Introduction

Sulfonamides (SAs) are commonly used in veterinary drugs. Sulfadiazine (SDZ) belongs to the SA class of antibacterial compounds. However, misuse can lead to residues in food animals and the environment. Excessive SDZ in food and the environment can have harmful effects, such as anaphylactic reactions (Hiba, Carine, Haifa, Ryszard, & Farouk, 2016), poisoning (Khalili, Soudbakhsh, & Talasaz, 2011), kidney failure or urinary stones (Dognon, Degand, Douny, Delahaut, Igout, Dahouda, Youssao, & Scippo, 2018). A rapid and sensitive analytical approach for the determination of SDZ is urgently needed.

Various researches for detecting of SDZ have been reported, including gas chromatography (GC) (Santos & Ramos, 2016), high-performance liquid chromatography (HPLC) (Vosough, Onilghi, & Salemi, 2016) and liquid chromatography coupled to mass spectrometry (Danezis, Anagnostopoulos, Liapis, & Koupparis, 2016; Jansomboon, Boontanon, Boontanon, Polprasert, & Da, 2016; Robert, Brasseur, Dubois, Delahaut, & Gillard, 2016). However, these methods are tedious and time-consuming. Therefore, they are not suitable for the quick on-site testing. Immunosorbent assays represent an alternative method widely used to detect the SDZ, with high sensitivity and cost effectiveness (Chu, Xu, He, Wang, & Lu, 2011). Enzyme-linked immunosorbent assay (ELISA) is a common approach to detect veterinary drug residues in foods because of its high sensitivity, simple operation and economic benefit (Liu et al., 2012; Shen et al., 2011; Song, Wang, Zhang, & Wang, 2011; Sun & Zhuang, 2015). However, this approach still has some shortcomings concerning the required biological antibodies such as low stability, a short lifetime and lack of reusability; therefore, the immunosorbent assay should be developed and optimized in the detection of SDZ (Jiang et al., 2013).

Molecular imprinting technology has been widely used in bionic immunoassay because of its ability for synthesizing artificial antibodies (Wang, Jiang, Ju, Qiao, & Xu, 2017). Molecularly imprinted polymers (MIPs) have the advantages of simple fabrication, stability and reusability, and have been widely applied in veterinary drug analysis (Lata, Sharma, Naik, Raiput, & Mann, 2015; Li et al., 2016; Shi et al., 2011; Tang et al., 2017). In recent years, remarkable progress in developing artificial antibodies for MIP-coated 96-well microplates has been achieved(Bi & Liu, 2014), and many biomimetic enzyme-linked immunosorbent assays (BELISA) have been developed, which have enhanced identification capabilities and decreased costs (Tang et al., 2017; Zhang, Cui, Han, Yu, & Shi, 2017). However, natural enzymes are macromolecules and have structural shielding effects, which limit its application in the immunosorbent assay (Jiang, Wu, Xu, Qiao, & Xu, 2017). Therefore, in the biomimetic immunosorbent assay, a new marker to replace the enzyme is necessary.

Artificial enzyme mimics have been attracting increasing attention over the past few years (Wiester, Ulmann, & Mirkin, 2011). Recently, gold nanoparticles (AuNPs) were reported to exhibit peroxidase enzyme-like properties, and they have been applied in bionics, biosensings and biomedicines (Deng et al., 2014; Lin, Ren, & Qu, 2014; Shah, Purohit, Singh, Karakoti, & Singh, 2015). The appearance and recent progress of nanotechnology provides new opportunities for the development of nanoparticles, which have stable and high catalytic activity (Yamada et al., 2011). Silica has been shown to be a multifunctional biomaterial with its good biocompatibility, which can directly improve the stability of nanoparticles in complex biological systems. SiO₂ nanoparticles are ideal carriers which provide as large surface area and ease of surface functionalization (Huang et al., 2014; Mao, Bao, Han, Li, & Niu, 2012; Shao et al., 2014). More importantly, these nanomaterials are much smaller than natural enzyme and therefore have potential as a marker for the BELISA method.

In this research, we reported a sensitive direct competitive BELISA method for the detection of SDZ. The assay used the MIP film as a bionic antibody for recognition and Au@SiO₂ nanostructures labelled as a marker takes the place of horseradish peroxidase (HRP). These factors were optimized and the peroxidase activity of the nanostructures was evaluated.

Material and methods

Material and reagents

Pork, beef and chicken samples were obtained from a supermarket (Taian, Shandong) in May 2019.

The SDZ ( >  98%) was purchased from the Shandong Xiya chemical industry Co., Ltd. (Linyi, China). Sodium borohydride (NaBH₄) was purchased from Tianjin Kemiou Chemical Reagent Co., Ltd. (Tianjin, China). Cetyltrimethylammonium bromide (CTAB), 3,3’,5,5’-tetramethylbenzidine (TMB) and chlorauric acid (HAuCl₄·3H₂O) were obtained from Beijing Biotopped Science & Technology Co., Ltd. (Beijing, China). Ethylsilicate (TEOS) was from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). 3-aminopropyl triethoxysilane (APTES) was obtained from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Methacrylic acid (MAA), 2,2-azobisisobutyronitrile (AIBN), methanol, acetic acid and acetonitrile were from Tianjin Chemical Reagent Factory (Tianjin, China). MAA was vacuum distilled and AIBN was recrystallized before use. Other materials and solutions are included in the supplementary material.

Apparatus

Microlon 96-well plates (Costar^®, Corning) were purchased from Beijing Biolead Biology Sci & Tech Co., Ltd. (Beijing, China). The 96-well plates were washed in a ST-36wet microplate washer (Kehua Bio-engineering Co., Ltd., Shanghai, China). Immunoassay absorbance was read with a Multimode Plate Reader (Molecular Devices, Sunnyvale, CA). The wavelength was set in a dual-wavelength mode (450–650 nm).

Synthesis of Au nanorods (NRs)

Au NRs were synthesized by a seed-mediated method (He et al., 2011). First, 7.5 mL of CTAB (0.1 M) aqueous solution was mixed with 100 μL of HAuCl₄ (24 mM), and then 1.8 mL of deionized water was added to dilute the mixture. After that, 0.6 mL of ice-cold NaBH₄ (0.01M) was added quickly. The solution was stirred by stirred magnetically for 3 min, the colour of the solution changed from bright yellow to brown, which indicated that the Au seeds were formed. A 240 μL of the Au seed was added to the preparation growth solution to incubate at 28°C for 12 h, and then the Au NRs were purified by centrifugation (12,000 rpm, 10 min) twice. The sediment was redispersed in double distilled water.

The details of preparing the growth solution are mentioned in supplementary material.

Synthesis of Au@SiO₂ NRs

All reactions were conducted in a temperature-controlled water bath at 30°C. Firstly, 10 mL of the Au NRs was centrifugated (6500 rpm/min, 10 min) twice and was then dissolved in double distilled water. The precipitate was collected and dissolved in 10 mL of deionized water. Then, the pH of the above solution was adjusted to around 10.6 using a small quantity of NaOH (0.1 M) solution. The mixture was stirred magnetically for 20 min, and then three 10 μL of TEOS ethanol solution (20% v/v) were added at 30-min intervals by stirred magnetically. Finally, this mixed solution was reacted at 30°C for 24 h.

Surface modification of Au@SiO₂ NRs

First, 20 mL of Au@SiO₂ was centrifuged (6500 rpm/min, 10 min), and then successively dispersed in ethanol, ethyl acetate and toluene solution. Thus, 5 mL of Au@SiO₂ toluene solution was obtained. Finally, 20 μL of APTES was added, and then the mixture solution was treated with ultrasound for 2 h, before two washes in ethyl acetate and ethanol.

Preparation of the nanozyme conjugate

Conjugation of the SDZ antigen and the Au@SiO₂ NRs was performed as follows: 5 mL of Au@SiO₂ NRs and 10 μL of glutaraldehyde (25% aqueous solution) were added to a reaction vessel. After stirring by magnetically stirred for 1 h, an equimolar mass of SDZ solution was added, then the solution was stirred for another 1 h. Finally, these products were washed to remove unreacted materials and preserved at 4°C.

Synthesis of MIP film

The MIP and non-molecularly imprinted polymer (NIP) films were prepared on the surface of 96-well plates. The preparation process is described in supplementary material.

Direct competitive BELISA procedure

The details of the procedure are introduced in supplementary material and Scheme 1.

Scheme 1. Schematic representation of the BELISA procedure.

Sample preparation

To determine the feasibility of the experiment, a beef sample was assessed, which was determined that the beef samples did not contain SDZ through HPLC. Briefly, the beef sample was minced, and 5.0 g was placed in a 50 mL container. After 1.0 mL of a SDZ standard solution (0.50, 2.50, 12.50 mg/L) were spiked, the mixtures were reacted at 4°C for 24 h. After that, the mixtures were extracted using 20 mL of ethyl acetate twice. The resulting extracts were collected and the fluid was evaporated. Then 1 mL of methanol was added to redissolve the solids. The liquid product was analysed by the BELISA procedure after filtering with a 0.22-μm membrane.

To detect SDZ residues, pork and chicken samples were prepared described above, without the addition of a SDZ standard. The extract was studied by the BELISA procedure, and the content of SDZ was determined.

Results and discussions

Characterization of Au@SiO₂ nanostructures

The absorption spectra of the Au NRs and Au@SiO₂ nanoparticles were assessed through UV-vis-NIR (Figure S2). The Au NRs had an absorption peak at 810 nm, which indicated that the Au NRs were synthesized successfully. Au NRs were coated with mesoporous silica to form core–shell Au@SiO₂ nanostructures. After coating with SiO₂ nanoparticles, the band was red-shifted to 906 nm, which indicated that Au NRs@SiO₂ nanostructures were formed.

Characterization of the imprinted film

The FT-IR spectra of the imprinted film after extraction (a), the imprinted film before extraction (b), the non-imprinted film (c) and SDZ (d) are compared in Figure 1. The SDZ stretching peak near 3381 and 3453 cm⁻¹, indicating an N-H stretching vibration, and the peak near 1108 cm⁻¹ were assigned to S = O group. For the MIP film before extraction, the features around 1105 cm⁻¹ was identified as an S = O stretching vibrations. The stretch shift (S = O) might be caused by the oxygen atoms of S = O in SDZ combining with the hydrogen atoms of –OH in MAA to form hydrogen bonds. However, the S = O stretching vibration peak was not found in the imprinted film after extraction indicating that the SDZ was extracted completely. These results demonstrated that the MIP film was prepared successfully.

Figure 1. The FT–IR spectra of the imprinted film after extraction (a), imprinted film before extraction (b), non-imprinted film (c) and sulfadiazine (d).

Thermo-gravimetric analysis (TGA)

A TGA curve of the imprinted film is depicted in Figure S3. When the temperature was between 0°C and 370°C, there was no significant reduction in the mass of MIP film. When the temperature exceeded 370°C, the mass decrease sharply. This result showed that the imprinted film has good thermal stability.

Static adsorption experiment

The adsorption capacity is an important property for the imprinted film. A static adsorption experiment was performed in a concentration range of 100 mg/L to 500 mg/L. As shown in Figure S4, the adsorbability of the MIP film and the NIP film increased with increasing SDZ concentrations. The MIP film exhibited a better adsorption ability for SDZ than the NIP film. The adsorption capacity of the MIP film (18.38 μg/well) was more than 1.6 times that of the NIP film (11.53 μg/well) at a 300 mg/L concentration.

Optimization of the BELISA conditions

The sensitive can be improved by optimizing the conditions of BELISA procedure, including the reaction solution. The recognition capability and selectivity of the MIP film towards the SDZ were influenced deeply by the solvent used for the preparation of the standard solution and samples, hence it influenced the accuracy of the BELISA procedure. Methanol, ethyl alcohol and acetonitrile were studied for use as preparation solutions. As shown in Figure 2, the results indicated that when methanol was used the inhibition ratio and the sensitivity of the method were better than those with other solutions. Therefore, methanol used throughout the experiment as the preparation solution.

Figure 2. Effect of different solutions on the BELISA method.

Selectivity of the BELISA assay

The selectivity of MIP and NIP films were investigated (Figure 3) using SDZ standard solution with 0.0512–160 mg/L concentrations in methanol. In contrast, the inhibition rate of the MIP film was higher at the same concentration of SDZ. From the standard curves, when the inhibition ratio was 50% and the SDZ concentration of the imprinted film was 32 mg/L, while that of the NIP film was 163 mg/L. Above results demonstrated that the MIP film had better recognition capability and higher sensitive for SDZ than the NIP film.

Figure 3. Standard BELISA competition curves for sulfadiazine with concentrations of 0.0512–160 mg/L in methanol.

To evaluate the specificity of the BELISA method, two structural analogues of SDZ (sulfathiazole and sulfamethazine) were applied to the competitive assay. Standard curves are established in Figure 4. The cross-reactivity (CR) was calculated as the percentage between the IC₅₀ values for SDZ and that for other interfering compound using the following the equation:$\text{\%}\mathrm{C}\mathrm{R}=\left\{\mathrm{I}{\mathrm{C}}_{50}\left(\mathrm{s}\mathrm{u}\mathrm{l}\mathrm{f}\mathrm{a}\mathrm{d}\mathrm{i}\mathrm{a}\mathrm{z}\mathrm{i}\mathrm{n}\mathrm{e}\right)/\mathrm{I}{\mathrm{C}}_{50}\left(\mathrm{s}\mathrm{t}\mathrm{r}\mathrm{u}\mathrm{c}\mathrm{t}\mathrm{u}\mathrm{r}\mathrm{a}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{a}\mathrm{l}\mathrm{o}\mathrm{g}\mathrm{u}\mathrm{e}\right)\right\}\times 100\text{\%}$

The analysis is presented in Table 1. The cross-reactivity values for sulfamethazine and sulfathiazole were 19.7% and 30.6%, respectively, which demonstrated that the MIP film had a lower absorption towards structural analogues than the SDZ. The results determined that the MIP film has advantages to be used as artificial antibodies in the BELISA method.

Figure 4. Cross-reactivity of three kinds of antibiotics.

Table 1. The cross-reactivity (CR) ratio of three kinds of antibiotics.

Antibiotics	Antibiotics structure	IC50 (mg/L)	CR (%)
Sulfadiazine		30	100
Sulfamethazine		152	19.7
Sulfathiazole		98	30.6

Parameters of the BELISA method

The standard curve was assessed by SDZ in methanol under optimal conditions. The limit of detection (LOD, IC₁₅) of the method was 0.2 mg/L (40 μg/kg). According to the Ministry of Agriculture of the People’s Republic of China (NY 5070–2002), the maximum allowable residue limit of SDZ is 100 μg/kg. Therefore, the developed BELISA method can effectively applied to the determination of SDZ in food.

Accuracy of the BELISA assay

The accuracy and sensitivity of the proposed method were evaluated. The beef sample spiked with sulafdiazine at different levels of 0.5, 2.5 and 12.5 mg/L were assessed using the developed BELISA method. The results are analysed in Table 2. Good recoveries of 78.00%–90.96% were achieved.

Table 2. The recoveries of spiked sulfadiazine in beef samples by determination using the BELISA method.

Sample	Sample content (mg/L)	Spiked level (mg/L)	Analysis result (mg/L, ±SD)	Recovery (%, ±RSD)
Beef	0.13	0.50	0.52 ± 0.01	78.00 ± 2.56
		2.50	2.20 ± 0.40	82.80 ± 1.81
		12.50	11.50 ± 0.50	90.96 ± 4.35

To validate the applicability of the BELISA method, pork and chicken samples were analysed by HPLC. The SDZ in the pork and chicken samples at levels of 3.56 ± 0.03 μg/mL and 5.88 ± 0.02 μg/mL, respectively, as assessed through HPLC. The resulting concentrations obtained by BELISA method were 3.11 ± 0.02 μg/mL and 5.66 ± 0.05 μg/mL. There was no significant difference between the BELISA method and HPLC method (P > 0.05). Above data verified that the BELISA techniques have good accuracy for the detection of SDZ in foods.

Strengths and advantages of the BELISA method

In this research, a BELISA method has been established for the determination of SDZ using Au@Pt@SiO₂ nanozyme as a marker. The BELISA method is simple and no large equipments are required compared with conventional methods. Furthermore, the imprinted film is more stable than the biological antibody and can be reused. Therefore, the BELISA method has a low cost in detecting SDZ residues.

Conclusions

In this study, using a synthetic enzyme in place of a natural enzyme as a label, a rapid BELISA method to detect SDZ residues was invented. The assay investigated the stability and ease of preparation of these novel nanoparticles. The proposed method has potential applications in antibiotic residue analysis in agricultural and food products.

Ethical approval

This article does not contain any studies with animals performed by any of the authors.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

Dr. Zhixiang Xu has received research grants from the National Natural Science Foundation of China (grant number 31972149) and Key R & D Project of Jining (grant number 2019NYNS002).