Ultrasensitive Electrochemiluminescent Competitive Immunoassay for β-Adrenergic Agonist Salbutamol Based on Quantum Dots and Enzymatic Amplification

Cadmium chloride (CdCl₂•2.5H₂O, 99%), thioglycollic acid (TGA), isopropyl alcohol (99.7%), Se powder (99.95%), sodium borohydride (NaBH₄, 96%), monopotassium phosphate (KH₂PO₄, 99%), disodium hydrogen phosphate dodecahydrate (Na₂HPO₄•12H₂O, 99%), potassium chloride (KCl, 99.5%), Tween 20, and ethanol (95%) were all purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Aluminum oxide polishing powder (Al₂O₃, 1.0, 0.3 and 0.05 um, 99%) was purchased from Tianjin Aidahengsheng Technology Co., Ltd (Tianjin, China). Hydroquinone (HQ), chitosan, Tris(hydroxymethyl) aminomethane, 25% glutaraldehyde aqueous solution, succinic anhydride and isopropyl alcohol were purchased from Sinopharm Chemical Reagent Co., Ltd. HRP labeled goat against rabbit IgG (HRP-GaRIgG) was purchased from Zhongshan Gold Bridge Biotechnology Co., Ltd (Beijing China). Ethyl-3-(dimethyl aminopropyl) carbodiimide (EDC, 98%), N-hydroxysuccinimide (NHS, 97%) and bovine serum albumin (BSA, 98%) were purchased from Fluka. Ovalbumin (OVA, 99%) was purchased from Sigma-Aldrich Co. Ltd (USA). Salbutamol (SAL), clenbuterol (CLB), ractopamine (RAC) and phenylethanolamine A (PA) were obtained from National Institutes for Food and Drug Control (Beijing, China). Phosphate buffered saline (PBS) prepared by Na₂HPO₄•12H₂O, KH₂PO₄ and NaCl was used throughout the whole work. All other reagents and chemicals were commercially available and of analytical reagent grade. All aqueous solutions were prepared with sub-boiling doubly distilled water.

The electrochemical measurement for ECL was carried out on a MPI-A multifunctional electrochemical and chemiluminescent analytical system (Xi An Remax Electronic Science & Technology Co., Ltd, Xi'An, China). The experiment applied a conventional three-electrode system with a modified glassy carbon electrode (GCE, 3 mm diameter) as working, a platinum wire as auxiliary, and an Ag/AgCl (saturated KCl solution) as reference electrodes. Cathodic potential in a range from 0 to −1.3 V was applied to the GCE using the cyclic voltammetric (CV) technique, while the ECL emission was recorded. The photomultiplier tube (detection range from 300 to 800 nm) was biased at −750 V. UV-vis absorption spectrum was recorded with Agilent 8453 UV-vis photospectrometer (Agilent Co. America). The High Resolution Transmission Electron Microscope (HRTEM) images were obtained from Tecnai G2 F20 S-TWIN 200 KV (FEI Co. U.S.A). Electrochemical impedance spectroscopy (EIS) was carried out with a RST electrochemical working station (Suzhou Risetest Instrument Co., Ltd, China), using the same three-electrode system as that in the ECL detection.

According to previously reported methods for preparing CdSe QDs^33,34 with a slight modification, water-soluble CdSe QDs used the thioglycolic acid (TGA) as a stabilizer agent were prepared. 49.6 mg CdCl₂•2.5H₂O was dissolved in 50 mL doubly distilled water, then 37.26 μL TGA was added under stirring. The pH of the solution was adjusted to 11 by dropwise addition of 3 mol/L NaOH. After the solution was bubbled with highly pure N₂ for 30 min, 2 mL of 50 mM NaHSe, prepared through reducing Se powder by NaBH₄, was injected into the mixture to obtain a clear yellow solution of CdSe QDs precursors. Then the NaHSe and cadmium solution were refluxed for 8 hours at 100°C to form a clear orange solution named TGA-Capped CdSe QDs. The obtained QDs solution kept at 4°C could be stable for 3 months.

Salbutamol antibody and salbutamol antigen used in this experiment were prepared as follows:¹⁴ 120 mg salbutamol dissolved in 10 mL anhydrous ethanol, and 61 mg succinic anhydride was added under stirring at room temperature for 5 hours, the obtained white suspension was centrifuged (10000 rpm, 15 min) and washed with ethanol for three times. After the precipitate dried at vacuum under 50°C for 5 h, the obtained white salbutamol derivatives was added to EDC/NHS, which led to the final molar ratio of SAL:EDC:NHS at 1: 1:1. After mixing at room temperature for 24 h, the carrier proteins (BSA/OVA) was slowly added under stirring for 24 hours. The above prepared solution was dialyzed with 0.01 mol/L (NH₄)₂CO₃ solution for four days at 4°C, which need regularly replacing the dialysate. Finally, the salbutamol-protein conjugates were lyophilized and stored at −40°C before use. The prepared salbutamol-BSA and salbutamol-OVA were respectively served as immunogen and coating-antigen to establish indirect competitive immunoassay.

The preparation of the polyclonal antibodies (pAb) against salbutamol was described in our previous literature.¹⁴ Polyclonal antibodies were derived from the antisera of adult New Zealand rabbits immunized with immunogen, and stored at −60°C before use. The above animal experiments have been approved by the institutional committees, and were performed in compliance with the relevant laws and institutional guidelines.

The bare glassy carbon electrode was polished using Al₂O₃ powder and rinsed with doubly-distilled water (Scheme 1A). 100 μL of quantum dots concentrated by centrifugation (8000 rpm, 10 min) was added on the surface of the polished glassy carbon electrode (Scheme 1B). After drying the electrode in air, 10 μL of 0.025% chitosan solution was coated on the QDs film for fixing QDs on the electrode surface (Scheme 1C). 8 μL of 2% glutaraldehyde was added to activate the chitosan film for 2 h at room temperature, then 8 μL SAL coating antigen incubated overnight in the refrigerator at 4°C. 8 μL BSA was coated on the electrode and incubated for 1 h at room temperature in order to block the excessive nonspecific binding sites (Scheme 1D). The electrode was thoroughly washed with PBS (0.01 mol/L, pH = 7.4) and dried at room temperature.

Scheme 1. Construction of immunosensor GCE (A), GCE/QDs (B), GCE/QDs/chitosan (C), GCE/QDs/chitosan-coating antigen/BSA (D), and ECL detection without (E) and with (F) the enzymatic amplification.

5 μL standard solutions of SAL with different concentrations were mixed with 5 μL the SAL-antibody to obtain the incubation solutions. 10 μL mixed solution was dropped on the electrode surface at room temperature for 1 h and then was washed thoroughly with streams of PBS and dried at room temperature. The standard solution of SAL in the incubation solution and the coating antigen would compete with the limited binding sites of the SAL-antibody to obtain the immunocomplex (Scheme 1E). 8 μL HRP-GaRIgG was coated on the electrode surface at room temperature for 1h and then was washed thoroughly with streams of PBS. Following that, the electrode was scanned from 0 to −1.3 V in Tris-HCl buffer (0.1 mol/L pH = 9.0) in presence of 0.5 mmol/L HQ (Scheme 1F).

The above-prepared CdSe QDs were diluted twice with doubly-distilled water, and measured by ultraviolet absorption spectrum. Fig. 1A showed that the UV-vis absorption peak of CdSe QDs was at 525 nm, and size of the QDs can be calculated according to the empirical formula:^31,35 D = (9.8127 × 10⁻⁷) λ³ − (1.7147 × 10⁻³) λ² + 1.0064 λ − 194.84, where λ is the QDs position of the first absorption peak, D is the diameter of QDs. The diameter of CdSe QDs was calculated to be 2.90 nm. From the HRTEM images (as shown in Fig. 1B), we could see that TGA-Capped CdSe QDs presented a relatively uniform sphere with a narrow size distribution, and the size of the CdSe QDs was approximately 3–4 nm.

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The modification of the electrode surface in the preparation process of the immunesensors was characterized by Electrochemical Impedance Spectroscopy (EIS).³⁶ As shown in Fig. 2, the semicircle diameter corresponds to the electron-transfer resistance (R_et) in EIS. The bare GCE (a) showed a small semicircle diameter, which certified that the bare GCE had a weak resistive force. The CdSe QDs/GCE (b) had a bigger semicircle diameter than the bare GCE, which indicated that CdSe QDs blocked the electron transfer and resulted in the increase of Ret. After coating chitosan, the semicircle diameter of chitosan/CdSe QDs/GCE (c) increased larger than CdSe QDs/GCE, therefore the film layer of chitosan would hinder the transfer of electrons. Similarly, coating antigen and BSA could both form the additional barrier to prevent the electron-transfer kinetics of the redox probe to the electrode surface (curve d, e). These results in the whole EIS experiment demonstrated that the sensor was successfully fabricated according to the procedures described in Scheme 1.

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Fig. 3A showed the cyclic voltammograms (CVs) of detection electrode in oxygen-saturated Tris-HCl buffer (pH = 9.0) in presence of 0.5 mmol/L HQ. We found two reduction peaks at about −0.71 V and −0.91 V, similar to that of CdTe QDs.³⁷ The peak at −0.71 V was due to the reduction of the dissolved O₂ to H₂O₂, and H₂O₂ was frequently used as co-reactant of cathodic ECL of CdSe QDs. During the cathodic potential scan, the H₂O₂ reacted with the electron-injected QD^−• formed at −0.91 V to produce excited QDs^*, which was unstable, and quickly returned to the ground state. Then a strong cathodic ECL emission could be observed with an emission peak at −1.16 V (Fig. 3B). Based on the above discussions and the previous reports,^19,37 the possible ECL mechanisms could be expressed as follows:

We all know that the enzymatic substrate HRP-GaRIgG can make the consumption of H₂O₂ in presence of HQ (Eq. 5), which resulted in a reduction of the amount of H₂O₂, and ultimately led to the ECL signal of CdSe QDs decreased.

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In order to get the maximum sensitivity for the detection of SAL, the amounts of antibody and coating antigen were optimized. In this study, we tested the effect of different concentrations (0–35 μg/mL) of coating antigen on the ECL quenching efficiency (Fig. 4A). It was obvious that the ECL quenching increased with the increasing amount of coating antigen, which contributed to the increasing of the quantity of SAL-antibody and HRP-GaRIgG conjugated on the surface of the electrode. Fig. 4A showed that when the concentration of coating antigen exceeded 25 μg/mL, the ECL quenching slowly increased and tended a plateau. Thus, 25 μg/mL was chosen as the best concentration of the coating antigen for immunosensor fabrication. In a similar way, we tested the ECL quenching efficiency with the different concentrations (0–25 μg/mL) of SAL-antibody. When the concentration was beyond 20 μg/mL, the ECL quenching increased slowly (Fig. 4B). Therefore, 20 μg/mL SAL-antibody was added to the incubation solution for immunosensor fabrication.

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Various concentrations of standard SAL solutions were tested under the optimal concentration of the coated antigen (25 μg/mL) and antibody (20 μg/mL) (Fig. 5A). With the increasing concentration of the standard solution of SAL, the ECL intensity significantly enhanced. In Fig. 5B, the linear regression equation was Y = 3127X + 2887.86, with the regression coefficient R = 0.9924, where Y was the ECL intensity and X was the logarithm of the concentration of SAL (log [SAL]). The linear response range of the sensor for the SAL was from 0.1 to 1000 ng/mL and the detection limit was 0.0084 ng/mL (S/N = 3). Compared with the previous literatures for detecting SAL, this work has a wider linear range and a lower LOD (LOD = 8.4 pg/mL), indicating that the proposed immunosensor has an excellent analytical performance.

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In order to prove the specificity of the proposed immunosensor, we added the common β-adrenergic agonists including clenbuterol (CLB), ractopamine (RAC), phenylethanolamine A (PA) as the interfering substances into the incubation solution with 100 ng/mL. As shown in Fig. 6A, the cross-reactivities of these three interfering substances were almost negligible, demonstrating that this immunosensor had the excellent specificity for the determination of SAL.

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The reproducibility and stability of the proposed immunosensor were investigated with 1.0 ng/mL and 10 ng/mL SAL standard solution. Fig. 6B shows the ECL intensity of this biosensor under continuous scanning for 7 cycles, the relative standard deviation (RSD) was in the range of 1.35%-8.37%. The coincident signals displayed an excellent reliability, stability and acceptable fabrication reproducibility.

The feasibility of the proposed method was investigated by detecting actual samples including pork and pork liver (randomly collected from the market in Suzhou). Sample handling process was as follows:³⁸ First, 2 g of the actual sample (pork or pork liver) was homogenized for half an hour, followed by the addition of 60 mL 0.01 mol/L HCl solutions and then stored the sample into the refrigerator at 4°C overnight. Next day, the supernatant of prepared sample was diluted 1000-fold with double distilled water to detect by our immunosensors. Before detecting, the diluted supernatant was filtered through a 0.45 μm membrane to remove some insolubles. No residual of SAL were detected, therefore they could be used as blank samples. Then three different concentrations of SAL standard solution incorporated in actual samples with 1 ng/mL, 10 ng/mL and 100 ng/mL, respectively. As shown in Table I, the recovery rates of the actual samples with adding standard solution of SAL were in the range of 84%–114%, suggesting that the acceptable accuracy of the immunosensor for the detection of SAL in actual samples.