A competitive microfluidic immunological clenbuterol analysis using a microELISA system

Abstract

We present a novel method to analyze clenbuterol based on a competitive microfluidic immunoassay scheme with a micro-ELISA system, and obtain a limit of detection that is less than 0.1 ng ml⁻¹ with a quantitative working range of 0.1 ng ml⁻¹ to 27.0 ng ml⁻¹. The approach was envisaged to be a promising method for efficient onsite clenbuterol control with good sensitivity and portability.

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Clenbuterol (CLB) is a β₂-adrenergic agonist, which is frequently administered in human and veterinary medicine for treatment of pulmonary disease.^1,2 In recent years, CLB has been widely reported to be illegally used in food producing animals due to the efficacy of growth promotion. Since CLB has resulted in several food safety incidents in some developing countries, routine monitoring of CLB abuse becomes exceedingly important for public health. At present, some technologies have been developed to screen CLB, for instance, capillary electrophoresis (CE),^3,4 gas chromatography-mass spectroscopy (GC-MS),⁵ liquid chromatography-mass spectroscopy (LC-MS),⁶ enzyme-linked immunoassay (ELISA)⁷ and so forth. However, these technologies can not always meet the requirements for efficient CLB control in terms of reagent availability, method sensitivity, assay promptness, and/or field adaptability. At present, there are limited reports about CLB analysis using microfluidic immunoassay system, although the experiment relating to the determination of CLB has been performed with a high throughput microfluidic immunoassay system using a complicated confocal laser induced fluorescence (LIF) scanner, which is obviously not suitable for onsite assay.⁸

We hereby develop a competitive microfluidic immunoassay method to analyze CLB as schematically shown in Fig. 1 using microELISA system as previously reported.^9,10 Firstly, the polystyrene microbeads (diameter, 45 μm) were pretreated and coated with the CLB coupled bovine serum albumin (CLB–BSA) through carbodiimide as catalyzer, then 8 μl microbeads solution were introduced to the reaction channel in microchip at a speed of 100 μl min⁻¹, and the excess microbeads were flushed out at a speed of 400 μl min⁻¹; secondly, 12 μl premixed CLB and mourine CLB-antibody solution were sucked into the channel at a speed of 50 μl min⁻¹, where CLB–BSA and CLB combined with the CLB-antibody competitively during the premixed solution flowed at a speed of 10 μl min⁻¹ constantly, followed by the CLB-antibody combined with CLB was washed out; thirdly, 14 μl horseradish peroxidase labelled goat anti-CLB-antibody conjugate solution were injected at the same speed of premixed CLB and mourine CLB-antibody solution and conjugated with CLB-antibody; at last, 20 μl 3,3′,5,5′- tetramethylbenzidine (TMB) with H₂O₂ solution were injected at a speed of 20 μl min⁻¹ and constantly flowed at a speed of 5 μl min⁻¹ for chromogenesis and detection. The system carrier solution was phosphate buffer saline (PBS, pH 7.4) containing 0.2% bovine serum albumin (BSA) and 0.05% tween-20. The whole experiments were completed under the room temperature (ca. 20 °C) and the screening program ended in approximately ten minutes.

Fig. 1 The competitive clenbuterol immunoassay scheme.

The glass microchip (3 × 7 cm) with reaction channel (width, 200 μm; depth, 200 μm) was shown in Fig. 2. CLB–BSA incubated polystyrene microbeads, CLB-antibody, and other reagents were pushed into the channel through port (a) by a programmed syringe pump. Microbeads were trapped and retained in the reaction channel by a dam (e) with a 20 μm slit while excessive microbeads were flushed out through port (b), and fluids can flow through the dam slit from port (a) to port (c) freely. The port (c) and the port (b) were both waste fluid outlets. The miniaturized thermal lens microscopic (TLM) probe (d) was integrated onto the microchip and placed 2.0 cm downstream to the dam of the microchip to detect the chromophoric signal of the substrate catalyzed by horseradish peroxidase labeled goat anti-CLB-antibody.¹¹ The results were analyzed by a lab-made software package. Although 0.2% BSA was used in carrier solution to block non-specific adsorption in the reaction channel, some residual substances might have a negative impact on the system. Hence, the flow path needed to be purged with pure water for about five minutes after each assay.

Fig. 2 Diagram of microchip with microbeads and TLM probe.

To test method utility, 12 μl negative, positive CLB-antibody or blank solution and 14 μl anti-CLB-antibody solution were introduced to the microchip sequentially to competitively react with CLB–BSA immobilized on the microbeads at a speed of 10 μl min⁻¹ each time. After stopping for 20 seconds in this program, the reaction products was pushed from reaction channel for detection, and the reaction products were identified as TLM peaks, while their concentration is quantified by the peak height. The detection signals were acquired in only one minute by micro-ELISA as shown in Fig. 3. The intensity of the resulting signals was inversely related or inhibited to the concentration of CLB in the PBS solution since the strategy of the test is based on the indirect competitive immunoassay scheme. Comparing with negative sample (zero inhibition) and the system carrier solution as blank (100% inhibition), the positive sample has an intermediate signal intensity, which demonstrates that an analyte existence incurred inhibition occurs and the method of competitive microfluidic immunological CLB analysis is feasible.

Fig. 3 Spectrum of thermal lens microscopic signal versus elapsed assay time. The red line, the black line, the blue line denotes negative, positive, and blank results, respectively.

In order to further investigate the quantitative capability of the method, serial standard CLB solutions with different concentrations (0.01, 0.1, 0.3, 1.0, 3.0, 9.0, 27.0 ng ml⁻¹) were applied for CLB analysis using microELISA system. The inhibition rate, which can reflect analytical sensitivity, is defined in eqn (1).

I₀—signal intensity of negative sample (zero inhibition); I₁₀₀—signal intensity of blank (100% inhibition); I_s—signal intensity of the sample.

As shown in Fig. 4, the inhibition rate correlated to the logarithm of CLB concentration with values of 48.32%, 27.36%, 46.86%, 54.15%, 74.59%, 80.21% and 82.84%, respectively. The inhibition rate attained its maximum value of 82.84% when the concentration of CLB was 27.0 ng ml⁻¹, and its minimum value of 27.36% when the concentration of CLB was 0.1 ng ml⁻¹, while the inhibition versus the logarithm of CLB concentration ranging from 0.1 ng ml⁻¹ to 27 ng ml⁻¹ was obvious. The inhibition rate was fitted to a sigmoid curve with the empirical equation as in eqn (2):

where y (%) was the inhibition rate corresponding to the concentration of positive sample, x was the logarithm value of CLB concentration, A₁ = 33.778 ± 12.1, A₂ = 83.307 ± 8.02, x₀ = 0.0699 ± 0.25, dx = 0.3144 ± 0.25. The resultant correlation coefficient was 0.97573, the standard deviation was presented as the error bars, and the error bar was relatively larger when the concentration of CLB was 0.01 ng ml⁻¹. It is demonstrated that reliable quantitative immunoassay using microELISA system for CLB analysis from 0.1 to 27.0 ng ml⁻¹ is applicable, and the limit of detection (LOD) was evaluated to be less than 0.1 ng ml⁻¹ according to the criterion that the inhibition rate was lower than 10%, which showed a competitive microfluidic immunological clenbuterol analysis using microELISA system was more sensitive compared with other methods. For example, the LOD of an immunochromatographic assay for detection of clenbuterol was 0.22 ng ml⁻¹,¹² the LOD of using capillary electrophoresis with laser induced fluorescence for detection of clenbuterol was 0.7 ng ml⁻¹.¹³

Fig. 4 Fitting chart of inhibition rate versus CLB concentration.

Moreover, the specificity of CLB-antibody was confirmed by ELISA which was illustrated in Table 1. The combination of CLB-antibody and CLB had a high specificity (100%), while other CLB analogues were of low reactivity (<10%). Comparing with other methods such as ELISA, CE or LC-MS, the method of competitive immunological CLB analysis using microELISA system had many advantages (e.g., the rapid detection process, the automatic test operation, the small sample volume), and the polystyrene microbeads with larger specific surface area were meritorious for CLB–BSA immobilization comparing with direct analyte incubation using conventional microtitre plates,^14,15 thus, the more binding sites furnished by the microbeads benefited the process of immuno-reaction and analytical sensitivity in this study.

Table 1 The verification of the specificity of CLB-antibody

β2- Agonists	Cross-reactivity (%)
Clenbuterol	100%
Salbutamol	0%
Ractopamine	0%
Tulobuterol	7%
Bambuterol	5%
Terbutaline	<1%
Cimaterol	<1%

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In conclusion, we have developed a novel method for competitive immunological CLB analysis using microELISA system, the inhibition positively correlates to the concentration of the CLB, a limit of detection that is less than 0.1 ng ml⁻¹ and a quantitative working range of 0.1 ng ml⁻¹ to 27.0 ng ml⁻¹ are acquired. The method is thus envisaged to be a promising method for onsite screening of CLB and other small molecular food contaminants in the future.

Acknowledgements

The authors thank Beijing Kwinbon Biotechnology Co., Ltd for providing monoclonal CLB antibody in this research.

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