Flow chemiluminescence sensor for determination of clenbuterol based on molecularly imprinted polymer

In this paper, a novel flow chemiluminescence (CL) clenbuterol sensor based on molecularly imprinted polymer (MIP) on line enrichment nanogram clenbuterol and chemiluminescence reaction of potassium permanganate and formaldehyde in the polyphosphate enhanced by clenbuterol. Clenbuterol in the urine was selectively adsorbed on the clenbuterol-imprinted polymer, which was packed into the flow cell. The formaldehyde and the polyphosphate with potassium permanganate flowed through the flow cell and reacted with the on line adsorbed clenbuterol and produced strong CL. The results show that the sensor was reversible. The CL intensity was linear with clenbuterol concentration from 1.0 × 10⁻⁹ g/mL to 5.0 × 10⁻⁸ g/mL. The detection limit was 3.0 × 10⁻¹⁰ g/mL. The R.S.D. for ng/mL clenbuterol was less than 5% (n = 3). The present method offered a high selectivity and sensitivity that made the quantitative analysis of trace clenbuterol (ng/mL) in the animal urine sample.     

多壁碳纳米管-分子印迹传感器测定盐酸克伦特罗

结合碳纳米材料和分子印迹技术,建立了以K3[Fe(CN)6]为探针测定盐酸克伦特罗的方法。以邻苯二胺为功能单体,盐酸克伦特罗为模板,采用电化学聚合法在多壁碳纳米管修饰电极表面制备了分子印迹薄膜。用乙腈水溶液可快速去除模板,得到多壁碳纳米管-分子印迹传感器。用循环伏安法、交流阻抗法和石英晶体微天平技术对印迹膜进行了表征,膜厚为12.3 nm。K3[Fe(CN)6]的相对峰电流与盐酸克伦特罗的浓度在4.0×10-8～6.6×10-6 mol/L范围内呈线性关系,检测限为8.1×10-9 mol/L。选择性实验表明传感器对结构类似物具有较强的抗干扰能力。此传感器可用于猪肉中盐酸克伦特罗的测定,加标回收率为101.3%～107.9%。     

气相色谱-质谱法测定动物组织中盐酸克伦特罗残留量不确定度影响因素的研究

对动物性食品中盐酸克伦特罗残留量测定过程中可能影响到结果质量的因素进行了研究。研究中使用有代表性的实验数据,建立数学模型,通过不确定度来源分析建立了该方法不确定度的评定方法。结果为根据标准溶液浓度与标准曲线回归得出的样品溶液的浓度对该项检测的不确定度贡献最大,其次衍生液体积和检测重复性对不确定度也有一定的贡献,而用电子天平称重引起的不确定度基本可以忽略。     

Quantum dots based electrochemiluminescent immunosensor by coupling enzymatic amplification for ultrasensitive detection of clenbuterol

An ultrasensitive electrochemiluminescence (ECL) immunosensor based on CdSe quantum dots (QDs) has been designed for the detection of clenbuterol. The immunosensor was fabricated by layer by layer and characterized with atomic force microscopic images (AFM) and electrochemical impedance spectra (EIS). In oxygen-saturated pH = 9.0 Tris-HCl buffer, a strong ECL emission of QDs could be observed during the cathodic process due to the H₂O₂ product from electrochemical reduction of dissolved oxygen. Upon the formation of immunocomplex, the second antibody labeled with horseradish peroxidase was simply immobilized on the electrode surface. The ECL emission decreased since steric hindrance of the immunocomplex slowed down the electron-transfer speed of dissolved oxygen, and also could be greatly amplified by an enzymatic cycle to consume the self-produced coreactant. Using clenbuterol as model analyte, the ECL intensity was determined by the concentration of competitive immunoassay of clenbuterol with a wide calibration in the range of 0.05 ng mL⁻¹ to 1000 ng mL⁻¹, and a low detection limit was 0.02 ng mL⁻¹. The immunosensor shows good stability and fabrication reproducibility. It was applied to detecting practical samples with the satisfactory results. This immunosensing strategy opens a new avenue for detection of residue and application of QDs in ECL biosensing.     

液相芯片技术同时检测莱克多巴胺、盐酸克伦特罗和沙丁胺醇

建立了一种同时检测兽药莱克多巴胺(RAC)、克伦特罗(CL)、沙丁胺醇(SAL)的多残留的新方法.采用液相悬浮芯片技术,基于间接竞争法的原理,将3种兽药抗原分别偶联到不同的荧光微球上作为探针,以藻红蛋白(PE)荧光标记二抗为信号,通过优化实验条件,分别建立3种兽药的标准曲线.基于特异性识别实验,建立3种兽药同时检测的标准曲线.结果表明,同时检测的3条曲线均呈现良好的线性相关,线性相关系数(R2)均大于0.99,RAC,CL和SAL的检测范围分别为1～ 500 μg/L,0.1～500μg/L,1～100μg/L,检出限分别为0.68,0.095和0.88 μg/L.与其它结构类似物的交叉反应率均低于1.5％,实际样品中的加标回收率令人满意.     

An immunoassay based on lab-on-a-chip for simultaneous and sensitive detection of clenbuterol and ractopamine

Both clenbuterol (CLB) and ractopamine (RAC) are β-adrenergic agonists. After long-term excessive intake, there will be adverse reactions such as headache, chest tightness, limb numbness, and serious life-threatening. Simultaneous detection of CLB and RAC in related samples is of great importance for human health. In this work, we outline a microfluidics-based indirect competitive immunoassay (MICI) system that can sensitively detect residual CLB and RAC in pork, swine blood and swine urine. The rapid detection of multiple samples can be achieved in one chip, which greatly improves the detection efficiency. This method has good stability and reproducibility and the microfluidic chips are easy to manufacture. The linear ranges for CLB and RAC detection by MICI are 0.1–2.5 ng/mL and 0.1–5 ng/mL, and the limits of detection (LODs) are 0.094 ng/mL and 0.091 ng/mL, respectively. This straightforward and portable immunoassay system provides a good platform for rapid detection of harmful substances in food samples.     

Capillary Electrophoresis Coupled with Electrochemiluminescence for the Facile Separation and Determination of Salbutamol and Clenbuterol in Urine

A capillary electrophoresis coupled with tris(2,2′‐bipyridyl) ruthenium(II) (Ru(bpy)₃²⁺) electrochemiluminescence detection system was developed to determine salbutamol and clenbuterol in urine. Some factors that affected the performances of separation and detection were investigated. Under the optimized conditions, one single quantitative analysis of salbutamol and clenbuterol was achieved at a separation voltage of 15 kV within 9 min, and the LODs (S/N=3) and LOQs (S/N=10) of salbutamol and clenbuterol were 8.43×10^?8 mol/L, 2.61×10^?7 mol/L and 2.73×10^?7 mol/L, 8.21×10^?7 mol/L, respectively. The recovery obtained from the analysis of spiked urine samples was between 88.6 % and 104.7 % with RSDs lower than 6.70 %. The method was successfully applied to determine salbutamol and clenbuterol in urine samples.     

分子印迹电位型传感器快速检测猪尿液中的克伦特罗

以盐酸克伦特罗为模板分子,采用沉淀聚合法合成了克伦特罗的分子印迹聚合物,并以其为离子载体,制得分子印迹聚合物膜克伦特罗离子选择性电极。在最优实验条件下,电极对克伦特罗阳离子的检出限可达7.0×10-8mol/L,线性范围为1.0×10-7～1.0×10-4mol/L,能斯特斜率为55.7 mV/decade。此电极具有优越的选择性、快速的响应时间以及良好的稳定性;已成功应用于实际猪尿样品中克伦特罗的测定,加标回收率为98%～107%,检测时间小于3 min。     

基于离子液体修饰碳纳米管电极的碱性磷酸酶电化学检测

利用自制的离子液体修饰碳纳米管电极(CNTs-ILE),在对CNTs-ILE的特性及对碱性磷酸酶(AP)酶促反应的底物磷酸对硝基苯酯(PNPP)和产物对硝基苯酚(PNP)进行电化学研究的基础上,采用安培法对酶促反应进程实现了持续动态的检测.实验表明,CNTs-ILE的循环伏安特性优于传统玻碳电极,且它在检测PNP时呈现出良好的抗钝化特性.当AP浓度范围在1.0～100 U/L时,电流大小与AP的浓度呈良好的线性关系.本方法可快速检测AP浓度,检测时间在20 min内.CNTs-ILE具有良好的抗钝化、操作简单及制作成本低等特点,在碱性磷酸酶的检测领域中有望得到进一步发展.在传统ELISA基础上,利用CNTs-ILE对具体样本最终的AP酶促反应产物进行电化学检测,通过峰值电流大小确定抗原浓度,为检测以AP为标记酶的抗原和抗体提供了理论依据.     

Determination of Salbutamol,Clenbuterol, and Brombuterol in Urine by a Highly Sensitive Chemiluminescence Enzyme Immunoassay

A salbutamol-bovine serum albumin and a salbutamol-ovalbumin coating antigen were synthesized, and six New Zealand white rabbits were treated with the immunogen to obtain polyclonal antibodies to develop a rapid, sensitive, and indirect chemiluminescent enzyme immunoassay (ciCLEIA) for the analysis of swine and bovine urine. The prepared antibodies showed high cross-reactivities with clenbuterol (139.6%) and brombuterol (225%). Under the optimized conditions, the linear dynamic range and the limit of detection (LOD) for salbutamol were from 0.007 to 0.17 µg L^?1 and 0.003 µg L^?1, with a correlation coefficient of 0.9965 and a half maximum inhibition concentration of 0.028 µg L^?1. Recoveries for salbutamol, clenbuterol, and brombuterol were from 78.8% to 119.0% with intra-assay and inter-assay coefficients of variation less than 13.9% and 19.7%, respectively. The reported ciCLEIA was about 10-fold more sensitive for salbutamol and 20-fold more sensitive for clenbuterol compared to conventional methods. This study showed that ciCLEIA was a reliable, convenient, and sensitive method for the simultaneous determination of salbutamol, clenbuterol, and brombuterol in swine and bovine urine.