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. 2020 Mar 5;5(10):5548–5555. doi: 10.1021/acsomega.0c00249

Detection of Six β-Agonists by Three Multiresidue Immunosensors Based on an Anti-bovine Serum Albumin-Ractopamine-Clenbuterol-Salbutamol Antibody

Chenxi Gu †, Pengfei Ren †, Fan Zhang †, Guozheng Zhao ‡,*, Jian Shen †, Bo Zhao †,*
PMCID: PMC7081638  PMID: 32201848

Abstract

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According to an indirect competitive immunoassay, six β-agonists (clenbuterol (CL), salbutamol (SAL), ractopamine (RAC), terbutaline (TER), mabuterol (MAB), and tulobuterol (TUL)) were detected by three novel multiresidue immunosensors on the basis of the successful preparation of bovine serum albumin (BSA)-RAC-CL-SAL multideterminant antigen and anti-BSA-RAC-CL-SAL antibody. A new strategy was reported to detect six β-agonists by combining nanotechnology, electrochemical detection, and specific immune technology. At the same concentration, the amperometric response for detection of six β-agonists was in a sequence of GCE/GNP/SAL > GCE/GNP/RAC > GCE/GNP/CL. Detection limits of six β-agonists show that the multiresidue detection performance of the GCE/GNP/RAC immunosensor is better than those of GCE/GNP/SAL and GCE/GNP/CL immunosensors. Three immunosensors manifest superior properties with a wide linear range, low detection limit, excellent reproducibility, and stability. The proposed GCE/GNP/RAC immunosensor displays high accuracy and can be effectively used for real sample detection.

1. Introduction

β-agonists are the derivatives of phenylethanolamines, which are the generic term for a class of β-receptor stimulants.1−3 After being eaten by animals, they promote protein synthesis and inhibit fat production during metabolism,4,5 which increases the feed conversion rate, growth rate, and lean meat rate by more than 10%.6,7 If people eat the meat containing β-agonists, there will be palpitations, muscle fibrillation, hand shaking, dizziness, fatigue, and other abnormal conditions, especially for patients with hypertension, heart disease, hyperthyroidism, and prostatic hypertrophy.8,9 Chromosome aberration and malignant tumors may occur if consumed for a long time.10,11 Therefore, a highly sensitive multiresidue analysis method for rapid detection of multiple β-agonists is urgently needed in the field of food safety.

At present, the commonly used detection methods can be divided into chromatographic analysis, electrochemical analysis, and immunoassay.12−14 The shortcomings of the existing detection methods for β-agonists are mainly manifested in the following: (1) Chromatographic analysis is the main method for the determination of veterinary drug residues of β-agonists, but it is not suitable for primary screening because of its high cost and complex sample pretreatment. (2) The weak selectivity of electrochemical analysis limits its application in practical samples with complex composition. (3) Due to its specificity, the traditional enzyme-linked immunoassay cannot simultaneously detect and rapidly screen a variety of β-agonists, which is easy to miss. (4) The maximum standard of β-agonists in food is low in many countries, which requires high sensitivity of rapid detection methods. The existing detection technology is not suitable for high throughput rapid screening because of its limitations.

Electrochemical immunosensors are a new class of sensors that combine highly sensitive electrochemical sensing technology with highly specific immunological techniques.15−17 Based on the specific binding of the antigen and antibody, the electrochemical immunosensor analysis can detect the change of the antigen–antibody conjugate and convert the nonelectric signal into an electrical signal18,19 so as to realize the correlation between the electric signal and the concentration of the substance to be tested, which is a relatively advanced detection method and has broad application prospects in food, environment, and other fields.20−22 Poo-arporn et al. prepared a disposable electrochemical sensor for determination of RAC with a detection limit of 13 nM (S/N = 3).23 A disposable electroanalytical device for the competitive enzyme-linked immunosorbent assay of phenylethanolamine A (PA) was reported by Deng et al. with a detection range of 0.005–60 ng·mL–1 and a detection limit of 2.6 pg·mL–1.24 Kang et al. developed a facile and sensitive method for the detection of CL using a personal glucose meter, suggesting a good linear correlation with the logarithm of the CL concentrations in the range of 0.1–100 ng·mL–1.25 A novel ultrasensitive competition-type electrochemiluminescent immunosensor was developed for detecting brombuterol with a low detection limit of 0.3 pg·mL–1 and a wide linear range from 0.001 to 500 ng·mL–1.26 Inspired by the merits of electrochemical immunosensors, after the successful preparation of the BSA-RAC-CL-SAL multideterminant antigen and anti-BSA-RAC-CL-SAL antibody, three multiresidue electrochemical immunosensors were constructed in this work to detect six β-agonists CL, SAL, RAC, TER, MAB, and TUL with a new strategy involving nanotechnology, electrochemical detection, and specific immune technology.

2. Results and Discussion

2.1. Identification of BSA-RAC-CL-SAL

The UV–vis absorption spectra of 0.2 mg·mL–1 SAL, 1.0 mg·mL–1 BSA-RAC-CL, and 1.0 mg·mL–1 BSA-RAC-CL-SAL are shown in Figure 1. From Figure 1, the UV–vis absorption curves of BSA-RAC-CL and BSA-RAC-CL-SAL are obviously different in the wavelength range of more than 280 nm. The maximum absorption wavelengths of BSA-RAC-CL and BSA-RAC-CL-SAL are 274 and 281 nm, respectively, and the maximum absorption peak of BSA-RAC-CL-SAL moves to a long-wavelength region, which proves that BSA-RAC-CL is successfully coupled with SAL.

Figure 1.

Figure 1

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UV–vis absorption spectra of (A) 0.2 mg·mL–1 SAL, 1.0 mg·mL–1 BSA-RAC-CL, and 1.0 mg·mL–1 BSA-RAC-CL-SAL, (B) graphene/RAC complex, (C) graphene/CL complex, and (D) graphene/SAL complex.

2.2. UV–vis Characterization of the Graphene/β-Agonist (RAC, CL, or SAL) Complex

In order to study the interaction between graphene and β-agonist (RAC, CL, or SAL) on the surface of electrodes, ultraviolet–visible spectrum (UV–vis) analysis was performed on the β-agonist (RAC, CL, or SAL) standard, graphene, and graphene/β-agonist (RAC, CL, or SAL) complex. The results are shown in Figure 1B–D. A clean quartz slide was taken to simulate the process of electrode surface modification. The slide surface was dripped with 100 μL of graphene suspension and 50 μL of 0.2 g·L–1 standard solution of β-agonist (RAC, CL, or SAL) (the ratio of graphene and standard solution was the same as that of electrode modification). The slide was dried at 37 °C and then dispersed uniformly in redistilled water.

From Figure 1B, there are a wide range of characteristic absorption peaks at 266.8 nm for GNP and two characteristic absorption peaks at 221.6 and 273.2 nm for the standard solution of RAC. The graphene/RAC complex showed a weak absorption peak at 225.1 nm, which shifted to the right in comparison with the characteristic absorption peak at 221.6 nm of RAC. Compared with the GNP absorption curve, the absorbance of the graphene/RAC complex increased gradually in the range of 216.0–266.8 nm, mainly due to the interaction between GNP and RAC. The absorption peak of RAC at 273.2 nm is combined with that of GNP at 266.8 nm, which results in the ultraviolet absorption peak of the GNP/RAC complex appearing at 269.5 nm, which is close to half of the sum of two independent absorption peaks (270.0 nm). The absorption peaks of GNP/RAC at 225.1 and 269.5 nm show that RAC has been bound to the surface of GNP.

Figure 1C shows that there are characteristic absorption peaks at 210.0, 241.6, and 294.5 nm for CL standard solution, and there is no coincidence with the characteristic peaks of GNP (266.8 nm). For the GNP/CL complex, there are CL absorption peaks at 210 and 241.6 nm in the GNP/CL curve. Due to the interaction between GNP and CL, there is a gentle absorption peak in the range of 266.8–294.5 nm, which is a composite peak of CL and GNP. It is proven that CL has been modified on the surface of GNP.

As shown in Figure 1D, there are characteristic absorption peaks at 223.6 and 275 nm for SAL standard solution. The GNP/SAL complex shows a weak absorption peak at 223.6 nm, which is also the absorption peak of SAL. The absorption peak of SAL at 275 nm is close to that of GNP at 266.8 nm, and the two absorption peaks compounded with each other. As a result, the ultraviolet absorption peak of GNP/SAL appears at 271.9 nm, which basically coincides with half of the sum of the two independent absorption peaks (270.9 nm). The absorption peaks of GNP/SAL at 223.6 and 271.9 nm prove that the GNP/SAL complex formed.

2.3. Detection of Six β-Agonists by the Graphene/CL Multiresidue Electrochemical Immunosensor

2.3.1. Characterization

Figure 2A shows the CV curves for GCE, GCE/GNP/CL, and anti-BSA-RAC-CL-SAL antibody immunization. From Figure 2A, the CV curve of GCE indicates a pair of stable and obvious [Fe(CN)6]3–/[Fe(CN)6]4– redox peaks. The redox current response of the electrode GCE/GNP/CL is much stronger than that of GCE, which indicates that graphene has excellent electrochemical properties and greatly improves the electron transfer rate on the surface of the electrodes. After incubation of the GCE/GNP/CL electrochemical immunosensor and anti-BSA-RAC-CL-SAL antibody, the peak current decreased significantly due to the specific bonding interaction between CL and anti-BSA-RAC-CL-SAL antibody, indicating that the anti-BSA-RAC-CL-SAL antibody hindered the transmission of electrons.

Figure 2.

Figure 2

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(A) CV curves for (a) GCE, (b) GCE/GNP/CL, and (c) anti-BSA-RAC-CL-SAL antibody immunization. (B) EIS curves for (a) GCE, (b) GCE/GNP, (c) GCE/GNP/CL, (d) GCE/GNP/CL/OVA, and (e) anti-BSA-RAC-CL-SAL antibody immunization. Effect of (C) anti-BSA-RAC-CL-SAL antibody volume and (D) incubation time on the DPV peak current.

Figure 2B describes the EIS for GCE, GCE/GNP, GCE/GNP/CL, GCE/GNP/CL/OVA, and anti-BSA-RAC-CL-SAL antibody immunization. The electron transfer resistance on the electrode surface is evaluated generally by its semicircular diameter in the high-frequency zone. The larger the semicircular diameter, the more difficult the superficial electron transfer and the higher the charge transfer resistance. As shown in Figure 2B, the initial value of Z′ of GCE is 88 Ω, and the impedance value is 50 Ω. The Z′ initial value of the graphene-modified electrode (curve b) is 68 Ω, which moves to the left compared with that of GCE (88 Ω). It is because the impedance value is almost zero due to the change of the electrode material, which shows that the electron transfer rate is very fast; that is, graphene can effectively improve the electron transfer on the electrode surface. The impedance values of GCE/GNP/CL (curve c), GCE/GNP/CL/OVA (curve d), and anti-BSA-RAC-CL-SAL antibody immunization (curve e) are 307, 507, and 782 Ω, respectively, which indicates that the specific immune reaction between the anti-BSA-RAC-CL-SAL antibody and electrochemical immunosensor has a great influence on the electronic conduction of the electrode surface. The specific binding of the anti-BSA-RAC-CL-SAL antibody to the electrode surface seriously hinders the transmission of electrons, thus proving the reliability of the specific detection of the graphene/CL electrochemical immunosensor.

2.3.2. Optimization of Conditions

The conditions involving the anti-BSA-RAC-CL-SAL antibody volume and incubation time were optimized at 37 °C as shown in Figure 2C,D before detecting six β-agonists. As depicted in Figure 2C, the peak current of DPV decreases with the increase of anti-BSA-RAC-CL-SAL antibody volume in the range of 2–8 μL and then tends to be stable, which indicates that the specific binding between the anti-BSA-RAC-CL-SAL antibody and electrochemical immunosensor was saturated. Therefore, the optimal volume of the anti-BSA-RAC-CL-SAL antibody was identified to be 8 μL. As shown in Figure 2D, it is noticeable that the peak current of the graphene/CL electrochemical immunosensor decreases gradually as the incubation time increases to 40 min due to the specific immune reaction between the anti-BSA-RAC-CL-SAL antibody and immunosensor. After 40 min, the current value achieves a plateau, indicating that the immune reaction was basically completed within 40 min. Thus, 40 min was the best incubation time.

2.3.3. Detection of Six β-Agonists

Figure 3 records the performance of the GCE/GNP/CL electrochemical immunosensor by DPVs corresponding to different concentrations of β-agonists. As demonstrated in Figure 3, after the first incubation, the peak current of DPV (curve b) of the GCE/GNP/CL immunosensor was lower than that of the GCE/GNP/CL (curve a), and then the peak currents of DPV (curves c, d, and e) of the GCE/GNP/CL decreased steadily after incubation with a series of different concentrations. Due to the graphene sheet prepared by liquid-phase exfoliation with a large area, more CL can be combined by π–π interaction and van der Waals force, and then more anti-BSA-RAC-CL-SAL antibodies can be specifically bound to the surface of the electrode after the first incubation. As a result, the effective area of the electrode is greatly reduced, which hinders the transmission of electrons and is consistent with the results from EIS analysis. In addition, with the decrease in the concentration of the β-agonist, the peak current of DPV decreases gradually due to the competitive principle of the GCE/GNP/CL. The peak current of DPV is proportional to the concentration of the β-agonist.

Figure 3.

Figure 3

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(A) CL detected by the GCE/GNP/CL electrochemical immunosensor (a, b, c, d, e, f, g, h, i, j, and k corresponding to GCE/GNP/CL, concentrations of 5000, 4000, 3000, 2000, 1000, 500, 100, 10, 1, and 0 ng·mL–1, respectively). (B) SAL detected by the GCE/GNP/CL electrochemical immunosensor (a, b, c, d, e, f, g, h, i, and j corresponding to GCE/GNP/CL, concentrations of 5000, 4000, 3000, 2000, 50, 10, 5, 1, and 0 ng·mL–1, respectively). (C) RAC detected by the GCE/GNP/CL electrochemical immunosensor (a, b, c, d, e, f, g, h, i, j, k, and l corresponding to GCE/GNP/CL, concentrations of 5000, 4000, 3000, 2000, 1000, 100, 50, 10, 5, 1, and 0 ng·mL–1, respectively). (D) TER detected by the GCE/GNP/CL electrochemical immunosensor (a, b, c, d, e, f, g, h, i, j, k, l, and m corresponding to GCE/GNP/CL, concentrations of 7000, 5000, 3000, 2000, 1000, 500, 100, 50, 10, 5, 1, and 0 ng·mL–1, respectively). (E) MAB detected by the GCE/GNP/CL electrochemical immunosensor (a, b, c, d, e, f, g, h, i, j, k, and l corresponding to GCE/GNP/CL, concentrations of 7000, 5000, 4000, 2000, 1000, 500, 100, 50, 10, 1, and 0 ng·mL–1, respectively). (F) TUL detected by the GCE/GNP/CL electrochemical immunosensor (a, b, c, d, e, f, g, h, i, j, k, and l corresponding to GCE/GNP/CL, concentrations of 7000, 5000, 4000, 3000, 2000, 1000, 500, 100, 50, 10, and 0 ng·mL–1, respectively).

The GCE/GNP/CL immunosensor was used to detect six standard solutions of β-agonists as shown in Table 1. From Table 1, the best linear ranges were TER and MAB followed by TUL, CL, SAL, and RAC. The lowest detection limit was CL followed by SAL, TER, TUL, MAB, and RAC. The anti-BSA-RAC-CL-SAL antibody is a polyclonal antibody, and the structures of CL and SAL are similar to those of TER, MAB, and TUL, which enables the GCE/GNP/CL immunosensor to multiresidue detect six β-agonists. The detection results of six standard solutions of β-agonists show differences in the linear range and detection limit. The factors affecting the detection of the immunosensor mainly include the following aspects: (1) The selectivity of the polyclonal anti-BSA-RAC-CL-SAL antibody is relatively weak. (2) The relative competitive strength of CL fixed on the surface of the immunosensor is different from those of β-agonists in incubation solution. (3) Large changes in β-agonist concentration lead to an imbalance of competition. (4) The immunosensor interface may affect the binding rate of the anti-BSA-RAC-CL-SAL antibody with CL on the electrode surface.

Table 1. Detection Results of Six β-Agonists by the GCE/GNP/CL Immunosensor.
β-agonist linear range (ng·mL–1) correlation coefficient detection limit (ng·mL–1)
CL 1–5000 0.992 0.1
SAL 1–5000 0.990 0.2
RAC 1–5000 0.991 0.4
TER 1–7000 0.990 0.2
MAB 1–7000 0.991 0.3
TUL 10–7000 0.992 0.2
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2.3.4. Reproducibility and Stability

To measure the reproducibility of the GCE/GNP/CL immunosensor, six concentrations of SAL were detected three times, and the relative standard deviations of the GCE/GNP/CL immunosensor were 0.67, 0.71, 0.35, 0.43, 0.52, and 0.46%, corresponding to the SAL concentrations of 0.5, 1, 5, 10, 50, and 2000 ng·mL–1, respectively, demonstrating that the prepared immunosensor has satisfactory reproducibility. In addition, the immunosensor was stored at 4 °C, and the current response was measured three times a day for 7 consecutive days. The relative standard deviation was less than 9%. In conclusion, the GCE/GNP/CL immunosensor has a good reproducibility and stability in a relatively long time.

2.4. Detection of Six β-Agonists by the Graphene/RAC Multiresidue Electrochemical Immunosensor

2.4.1. Detection of Six β-Agonists

The GCE/GNP/RAC immunosensor was used to detect six standard solutions of β-agonists as shown in Figure S5 and Table 2. The widest linear range is RAC and MAB followed by CL, TER, TUL, and SAL. As shown in Figure 3 and Figures S3 and S5, at the same concentration, the amperometric response for detection of six β-agonists was in a sequence of GCE/GNP/SAL > GCE/GNP/RAC > GCE/GNP/CL. From Table 1, Table S2, and Table 2, it can be seen that there is only one β-agonist (CL) with a detection limit of 0.1 ng·mL–1 in Table 1, four β-agonists (CL, SAL, RAC, and TUL) with a detection limit of 0.1 ng·mL–1 in Table S2, and six β-agonists (CL, SAL, RAC, TER, MAB, and TUL) with a detection limit of 0.1 ng·mL–1 in Table 2, which shows that the multiresidue detection performance of the GCE/GNP/RAC immunosensor is better than those of GCE/GNP/SAL and GCE/GNP/CL immunosensors. The three multiresidue immunosensors based on the anti-BSA-RAC-CL-SAL antibody manifest outstanding performance with a wide linear range and low detection limit, compared with the previous different material-modified electrodes for RAC detection, for example, Au/OMC/GCE (detection limit 1.49 ng·mL–1),27 3D MnO2/RGO@nickel foam (detection limit 3.92 ng·mL–1),28 ATONPs/CNTs/GCE (detection limit 1.11 ng·mL–1),29 and MCF/CPE (detection limit 3.38 ng·mL–1),30 which can be ascribed to the high specific area of the GCE/GNP/β-agonist (RAC, CL, or SAL) caused by the nanoscale effect of the graphene, increasing the loading capacity of the β-agonists.

Table 2. Detection Results of Six β-Agonists by the GCE/GNP/RAC Immunosensor.
β-agonist linear range (ng·mL–1) correlation coefficient detection limit (ng·mL–1)
CL 5–7000 0.992 0.1
SAL 50–5000 0.989 0.1
RAC 1–7000 0.988 0.1
TER 10–5000 0.990 0.1
MAB 1–7000 0.996 0.1
TUL 10–5000 0.993 0.1
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2.4.2. Analysis of Real Samples

Detection of real samples is a key step to evaluate the practical application ability of the immunosensor. The GCE/GNP/RAC immunosensor is selected to detect CL and RAC in real samples. The pig lean, fat, or liver sample was crushed and weighed (1 g) into a 10 mL centrifuge tube. A standard solution of CL or RAC was added to the sample by a standard addition method. Then 3 mL of acetonitrile–acetone extraction solution (volume ratio 1:1) was added. The mixture was sonicated and centrifuged for 20 and 5 min, respectively. The supernatant was transferred into test tube, dried under nitrogen, and then mixed with 1 mL of PBS (pH = 7.4). The extracts were dissolved and stored at 4 °C until detection. The detection results of CL and RAC in real samples by the GCE/GNP/RAC multiresidue immunosensor are shown in Tables 3 and 4, respectively.

Table 3. Detection Results of CL in Real Samples by the GCE/GNP/RAC Immunosensor.
real sample amount added (ng·mL–1) amount measured (ng·mL–1) RSD (%) recovery (%)
pig lean 500 497.2 479.7 474.3 2.3 96.7
2000 1972.9 1990.9 1975.4 0.5 99.0
6000 5869.3 5998.3 6114.4 2.5 99.9
pig fat 500 459.1 563.7 567.8 13.2 106.0
2000 1965.7 2238.7 1834.3 11.6 100.6
6000 4821.8 4828.3 4831.5 0.1 80.5
pig liver 500 473.7 475.1 550.8 9.1 100.0
2000 1925.5 2020.8 1881.7 3.2 97.1
6000 5716.4 5790.6 5845.4 1.0 96.4
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Table 4. Detection Results of RAC in Real Samples by the GCE/GNP/RAC Immunosensor.
real sample amount added (ng·mL–1) amount measured (ng·mL–1) RSD (%) recovery (%)
pig lean 500 517.5 490.1 421.7 9.5 95.3
2000 1947.3 2009.1 2067.4 3.0 100.4
6000 5857.2 5919.5 5940.3 0.9 98.4
pig fat 500 548.4 490.9 510.1 6.3 103.3
2000 2084.8 1872.2 1783.1 8.7 95.7
6000 5175.8 5271.4 5325.5 1.5 87.6
pig liver 500 486.7 488.5 487.4 0.2 97.5
2000 2314.6 1680.2 2088.3 14.6 101.4
6000 5545.4 6143.7 5522.4 5.6 95.6
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From Table 3, the average recoveries of pig lean, fat, and liver samples are 96.4–106.0% except the recovery (80.5%) of a sample, and the relative standard deviations are 0.1–13.2%. In addition, from Table 4, the average recoveries of pig lean, fat, and liver samples are 95.3–103.3% except the recovery (87.6%) of a sample, and the relative standard deviations are 0.2–14.6%. Therefore, the constructed immunosensor has high accuracy and can be effectively used for real sample detection.

3. Conclusions

In this paper, based on the preparation of a BSA-RAC-CL-SAL multideterminant antigen, the anti-BSA-RAC-CL-SAL antibody was obtained successfully by immunization, serum collection, and purification and preservation. Using an indirect competitive immunoassay, three novel graphene/β-agonist (RAC, CL, or SAL) multiresidue electrochemical immunosensors were fabricated by nanotechnology, electrochemical detection, and specific immune technology to accomplish the detection of six β-agonists (CL, SAL, RAC, TER, MAB, and TUL). With the decrease in the concentration of the β-agonist, the peak current of DPV decreases gradually due to the competitive principle of the graphene/β-agonist (RAC, CL, or SAL) electrochemical immunosensor, where the peak current of DPV is proportional to the concentration of β-agonist. The amperometric response for detection of six β-agonists was in a sequence of GCE/GNP/SAL > GCE/GNP/RAC > GCE/GNP/CL with the same concentration, and the detection performance of the GCE/GNP/RAC immunosensor is better than those of GCE/GNP/SAL and GCE/GNP/CL immunosensors. The three multiresidue immunosensors display attractive performance with a wide linear range and low detection limit, which can be ascribed to the high specific area of the GCE/GNP/β-agonist (RAC, CL, or SAL) caused by the nanoscale effect of the graphene, increasing the loading capacity of the β-agonists. The constructed immunosensor exhibits high accuracy and can be applied to the detection of real samples.

4. Experimental Section

4.1. Reagents

RAC, CL, SAL, TER, MAB, and TUL were purchased from National Institutes for Food and Drug Control. Ractopamine hydrochloride, clenbuterol hydrochloride, salbutamol sulfate, and Freund’s complete and incomplete adjuvants were supplied by Sigma-Aldrich. Ovalbumin (OVA), glutaric anhydride, tributylamine, N,N-dimethylformamide (DMF), 1,4-dioxane, isobutyl chloroformate, ammonium sulfate, barium chloride, potassium ferricyanide K3[Fe(CN)6], and potassium ferrocyanide K4[Fe(CN)6] were obtained from Sinopharm Chemical Reagent Co., Ltd. Phosphate buffer solution (PBS, 0.1 M, pH = 7.4) was prepared from potassium dihydrogen phosphate and sodium hydroxide. All other chemicals and reagents were of analytical grade. Redistilled water was used in all of the experiments. Pig lean, fat, and liver samples were purchased from a local supermarket.

4.2. Apparatus

Electrochemical measurements were performed by using a CHI600E electrochemical workstation (Shanghai Chenhua Apparatus Co., Ltd., China). A three-electrode system was used with platinum (Pt) wire, Ag/AgCl, and glassy carbon as the counter electrode, reference electrode, and working electrode, respectively. The UV absorption spectrum was measured by using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., USA).

4.3. Preparation of the BSA-RAC-CL-SAL Multideterminant Antigen

Salbutamol sulfate (143.6 mg, 0.6 mmol) was dissolved in 6 mL of anhydrous methanol and the solvent was evaporated under pressure to obtain a yellow oily salbutamol base. Then the white solid was obtained by centrifugation after dissolving in 12 mL of anhydrous ethanol, adding 68 mg (0.6 mmol) of glutaric anhydride, and stirring at room temperature for 6 h.

The white solid was dissolved in mixed solvents (4 mL DMF and 4 mL 1,4-dioxane), and 78.6 μL of tributylamine (0.3 mmol) was added, stirred in an ice bath for 10 min, added with isobutyl chloroformate (43.2 mL, 0.3 mmol), and stirred at room temperature for 1 h. The above solution was added drop by drop to 10 mL of cold PBS solution containing 50 mg of BSA-RAC-CL and reacted overnight at room temperature. Finally, the BSA-RAC-CL-SAL multideterminant antigen was obtained by dialysis in PBS solution at 4 °C for three days and changing dialysate twice a day.

4.4. Preparation of the Anti-BSA-RAC-CL-SAL Antibody

4.4.1. Immunization

2 mL of 1 mg·mL–1 BSA-RAC-CL-SAL multideterminant antigen was mixed with the same amount of Freund’s complete adjuvant and emulsified. Each rabbit was vaccinated with 0.25 mg of above immunogen. Then the Freund’s incomplete adjuvant was mixed with 2 mL of 1 mg·mL–1 BSA-RAC-CL-SAL multideterminant antigen and emulsified. The emulsification method, immune site, and immune dose were the same as before, and the immunization was strengthened every 2 weeks. Protocols for the animal studies were approved by the Regulations of Experimental Animal Administration, State Committee of Science and Technology of the People’s Republic of China.

4.4.2. Serum Collection

After three booster immunizations, 10 mL of venous blood was collected. Standing at room temperature for 30 min and then standing at 4 °C for 30 min, the supernatant was collected by centrifuging at 3000 rpm for 30 min.

4.4.3. Purification and Preservation

10 mL of normal saline was added to 10 mL of serum, and then 5 mL of (NH4)2SO4 saturated solution was added drop by drop, mixed fully for 30 min, and then centrifuged at 3000 rpm for 20 min to remove the precipitate. 30 mL of ammonium sulfate saturated solution was added to the supernatant, fully mixed, and kept for 30 min. The supernatant was removed by centrifugation for 20 min at 3000 rpm. The precipitation was dissolved in 20 mL of normal saline, added with 10 mL of ammonium sulfate saturated solution, and mixed fully for 30 min. The supernatant was removed by centrifugation for 20 min at 3000 rpm.

The precipitate was dissolved in 10 mL of normal saline and dialyzed overnight in pure water and then dialyzed in normal saline at 4 °C for 24 h, changing the dialysate three times. The SO42– was examined with 1% barium chloride solution until there was no SO42– in the dialysate. The dialysate was centrifuged, and the supernatant was taken and stored at low temperature.

4.5. Construction of Three Graphene/β-Agonist (RAC, CL, or SAL) Multiresidue Electrochemical Immunosensors

A bare glassy carbon electrode (GCE) was polished with 0.05 μm alumina powder and sonicated in ethanol/distilled water (volume ratio 1:1) and distilled water consecutively for 30 s and then dried at room temperature. The treated GCE was coated with 4 μL of graphene suspension (GNP)31 and 2 μL of 0.2 g·L–1 RAC, CL, or SAL standard solution. The graphene and β-agonist (RAC, CL, or SAL) were fully combined and fixed on the GCE surface at 37 °C overnight. Furthermore, the modified electrode was incubated in 0.05% ovalbumin solution to block the active sites on graphene that were not bound by RAC, CL, or SAL. Finally, the graphene/β-agonist (RAC, CL, or SAL) multiresidue electrochemical immunosensor was prepared by washing the modified electrode with PBS buffer solution (0.1 M, pH 7.4).

4.6. Electrochemical Measurements

In order to characterize the changes of electrochemical behavior on the surface of electrodes, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to determine the responses of electrodes at different stages during the preparation and detection of immunosensors, including the bare GCE, graphene-modified electrode, β-agonist hapten-modified electrode, and electrode after the antibody immune response. The CV curve was measured with the scanning range of −0.2 to 0.8 V and scanning rate of 100 mv·s–1 in PBS buffer solution (0.1 M, pH 7.4) of 2 mM K3[Fe(CN)6]/K4[Fe(CN)6]. The EIS spectrum was determined with scanning frequencies of 0.01–106 Hz and pulse amplitude of 5 mV·s–1 in 0.1 M KCl, 5 mM K3[Fe(CN)6], and 5 mM K4[Fe(CN)6] solution.

4.7. Optimization of Conditions

Optimizing the amount of anti-BSA-RAC-CL-SAL antibody in incubation solution: In 50 μL of incubation solution (anti-BSA-RAC-CL-SAL antibody and PBS), antibodies were added with volumes of 2, 4, 6, 8, 10, and 12 μL. The immunosensor was immersed in incubation solution for 30 min. After incubation, the electrode surface was washed with PBS and analyzed by DPV.

Optimizing the incubation time: The optimum volume of anti-BSA-RAC-CL-SAL antibody was added to the incubation solution (anti-BSA-RAC-CL-SAL antibody and PBS) with a total volume of 50 μL. The electrode was incubated, and the peak current of DPV was measured per 5 min.

4.8. Detection of β-Agonists

In order to investigate the multiresidue detection performance, the graphene/β-agonist (RAC, CL, or SAL) immunosensor was applied to the quantitative DPV detection of RAC, CL, SAL, TER, MAB, and TUL β-agonist standard solutions. Under the optimum conditions, the immunosensor was placed in 50 μL of a series of β-agonist standard solutions (β-agonist, quantitative antibody, and PBS) (1–7000 ng·mL–1) and incubated at 37 °C for the optimal time. After incubation, the graphene/β-agonist (RAC, CL, or SAL) immunosensor was washed with PBS and put into PBS buffer solution (0.1 M, pH 7.4) of 2 mM K3[Fe(CN)6]/K2[Fe(CN)6]. The peak current difference of DPV (ΔI = Ix – I0, where Ix is the sample detection corresponding peak current and I0 is the blank sample detection corresponding peak current) was used to establish a linear relationship with the sample concentration. The voltage range was −0.2 to 0.5 V, and the pulse amplitude was 50 mV. The preparation and detection process of the multiresidue immunosensor based on the anti-BSA-RAC-CL-SAL antibody are described in Scheme 1.

Scheme 1. Schematic Description of the Preparation and Detection Process of the Multiresidue Immunosensor.

Scheme 1

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Acknowledgments

This work was supported by Linfen Key Research and Development Projects (Social Development) (No. 1909), Cultivation Plan of Young Scientific Researchers in Higher Education Institutions of Shanxi Province, Scientific and Technological Innovation Programs of Higher Education Institutions in Shanxi (No. 2019L0469), Technology Support Program of Science and Technology Department of Jiangsu Province (BE2015703), Huaian Key Research and Development Program (HAS201619), and the 1331 Engineering of Shanxi Province.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c00249.

  • Supplementary details on the preparation of the BSA-RAC and BSA-RAC-CL, identification of BSA-RAC and BSA-RAC-CL, detection of six β-agonists by graphene/SAL multiresidue electrochemical immunosensor (characterization, optimization of conditions, detection of six β-agonists, reproducibility, and stability), and detection of six β-agonists by graphene/RAC multiresidue electrochemical immunosensor (characterization, optimization of conditions, detection of six β-agonists, reproducibility, and stability) (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao0c00249_si_001.pdf (840.6KB, pdf)

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