乳制品中三聚氰胺检测新方法研究
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摘要
三聚氰胺是一种用途广泛的化工原料,分子中含氮量达66.6%,一些不法企业将其添加到牛奶、乳制品、饲料中,造成粗蛋白质虚高的假相,不仅对乳制品、养殖业等产业产生不利影响,对人类健康也构成严重威胁。2007年美国的“宠物粮”风波和2008年我国的“三鹿牌婴幼儿奶粉重大食品安全事故”给中国企业和行业的形象造成了极为不利的影响,给受害家庭造成严重伤害。因此,研制新的方便、准确、灵敏、快速、适用于现场监测的三聚氰胺分析方法,特别是痕量水平的三聚氰胺的分析方法是当前分析化学领域的重要研究课题,也是分析工作者面临的巨大挑战。本课题基于三聚氰胺的物理与化学性质,分别建立了分光光度法和电化学传感器法检测乳制品中三聚氰胺的新方法。
     (1)建立了pH渐变光谱-秩消失因子分析法测定牛奶中三聚氰胺的新方法。在本方法中,通过记录多个pH点溶液的吸收光谱,获得酸度-光谱双线性数据矩阵,再应用秩消失因子分析法计算牛奶中三聚氰胺的浓度。应用本方法对牛奶中三聚氰胺进行了测定,最优条件下,校正试样和混合牛奶试样的检测线性范围分别为0.04 ~ 4.0μg/mL和0.04 ~ 3.5μg/mL,检测限达12 ng/mL,对三个加标牛奶样品平行测定三次的回收率为98.06~100.43%,相对标准偏差为0.27~6.15%,相对预测偏差和均方根预测偏差分别为0.91%和0.0151。该法简单、便捷,可用于未知复杂基质中三聚氰胺的检测。
     (2)研制了一种新型简易的三聚氰胺传感器,这种传感器以电聚合对氨基苯甲酸分子印迹聚合物(MIP)为识别元件。在模板分子三聚氰胺存在下,将对氨基苯甲酸通过电位循环扫描的方式电沉积到玻碳电极上制得对氨基苯甲酸分子印迹聚合物薄膜。用电化学阻抗谱(EIS)和循环伏安法(CV)表征了该修饰电极的表面特征。用差分脉冲伏安法(DPV)来验证铁氰化物的氧化峰电流在分子印迹传感器上的变化。研究和优化了影响对氨基苯甲酸分子印迹聚合物性能的几个重要参数。在最佳实验条件下,铁氰化物的氧化电流相对变化量与三聚氰胺的浓度呈线性关系,线性范围为4.0μmol/L~0.45 mmol/L,线性相关系数为0.9992。检出限为0.36μmol/L(S/N = 3)。该传感器用于牛奶中三聚氰胺的检测,具有较高的选择性,灵敏度和可重复性。研究结果表明,用分子印迹技术制备无电活性物质的电化学传感器是可行的。
     (3)以没食子酸和邻苯二胺为混合功能单体,制备了没食子酸-邻苯二胺共聚物修饰的三聚氰胺分子印迹电极。以0.1 mol/L NaOH的50%(v/v)乙腈-水溶液为模板洗脱液,以铁氰化物为电化学探针,考察了分子印迹聚合物与三聚氰胺之间的相互作用。循环伏安法(CV),电化学阻抗谱(EIS)和扫描电子显微镜(SEM)的表征结果说明,三聚氰胺分子凭借与印迹膜之间的氢键相互作用、π-π堆积作用和静电相互作用等,占据了印迹通道,阻碍了探针分子进入电极表面,从而使铁氰化物的氧化-还原峰电流降低。基于这一原理,我们用方波伏安法(SWV)考察了含有不同浓度三聚氰胺的铁氰化物溶液在印迹电极和非印迹电极上的伏安行为,建立了以没食子酸和邻苯二胺为混合功能单体的分子印迹传感器测定三聚氰胺的方法。在最佳实验条件下, SWV测定三聚氰胺的线性范围为10.0 nmol/L ~ 0.1μmol/L和10.0μmol/L ~ 100.0μmol/L,检出限为1.6 nmol/L(S/N = 3)。本法简便快捷,用于乳制品中三聚氰胺的检测,方法精密度(RSD)小于4%,回收率大于90%。没食子酸和邻苯二胺共聚物修饰的三聚氰胺分子印迹电极具有响应时间短,使用方便,廉价和制作简便等优点。
Melamine (1,3,5-triazine-2,4,6-triamine), a kind of triazine analogue with three amino groups, is an industrial chemical used in the production of melamine-formaldehyde resins. Because it contains a substantial amount of nitrogen (66% by mass), it has been used as a filler for protein-rich diets by unethical manufacturers. This was mirrored in the pet food incident in early 2007 and the milk products scandal recently when melamine was added to raw materials to obtain high protein contents. Melamine has a low oral acute toxicity, but the chronic administration of high concentrations can induce renal pathology and even death, especially in babies and children. Melamine in combination with cyanuric acid is able to form insoluble melamine cyanurate crystals in the kidney, which causes renal failure. Therefore, a sensitive and reliable method is urgently needed for the determination of melamine in food and, particularly, in dairy products for children. In this paper, the quantitative determination of melamine in milk products using spectrophotometric method and electrochemical sensor were constructed, respectively, according to the physical and chemical properties of melamine.
     (1) A new spectrophotometric method has been developed in this paper to determine melamine in milk by applying rank annihilation factor analysis (RAFA) based on pH gradual change-UV spectral data (pH-spectra). In the proposed method, the spectra of the sample solutions at different pH data points were recorded and the pH-spectra bilinear data matrix was generated. Based on these data, the RAFA analysis was then applied to calculate the concentration of melamine in milk. The experiments have been conducted and the results were satisfactory. Under the optimized conditions, linearity of the proposed method was in the range of 0.04–4.0μg/mL for calibration samples, and 0.04~3.5μg/mL for the mixed solution of melamine with the background milk components. The limit of detection (LOD) was 12 ng/mL. The relative predictive error (RPEs) and root mean square error of prediction (RMSEP) of applying RAFA were 0.91% and 0.0151, respectively.
     (2) A novel and simple sensor is developed in this paper for melamine detection, which is based on an electropolymerized molecularly imprinted polymer (MIP) of para-aminobenzoic acid (pABA). The poly(para-aminobenzoic acid) (P-pABA) film was deposited in a pABA solution by potentiodynamic cycling of potential with and without the template (melamine) on a glassy carbon electrode. The surface feature of the modified electrode was characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The molecular imprinted sensor was tested by differential pulse voltammetry (DPV) to verify the changes in redox peak currents of hexacyanoferrate. Several important parameters controlling the performance of the P-pABA were investigated and optimized. In the optimal conditions, the relative redox peak currents of hexacyanoferrate were linear with the concentration of melamine ranged from 4.0μmol/L to 0.45 mmol/L, with a linear correlation coefficient of 0.9992. The detection limit was 0.36μmol/L (S/N = 3). The MIP sensor was successfully applied to the determination of melamine in milk products and showed high selectivity, sensitivity, and reproducibility. The results of this research demonstrate that it is feasible to use the molecular imprinting methodology when preparing sensing devices for analytes that are electrochemically inactive.
     (3) A novel and simple electrochemical method is developed based on molecularly imprinted polymer (MIP) with dual functional monomers to determine total content of melamine in milk products. With gallic acid (trihydroxybenzoic acid, GA) and o-phenylenediamine (o-PD) as dual-functional monomers, melamine as a template, MIP film on a glassy carbon electrode was constructed as melamine sensor. The template can be quickly removed by 0.1 mol/L NaOH solution (50% (v/v) acetonitrile as solvent). The electrochemical probe of hexacyanoferrate was used to investigate the interactions between MIP and melamine. Results of cyclic voltammetries (CV), electrochemical impedance spectrometry (EIS) and scanning electron microscopy (SEM) proved that melamine might interact with MIP mainly through electrostatic and hydrogen-bonding interactions. The interactions between MIP and melamine cause to the decrease in the peak currents of hexacyanoferrate, which could be used for electrochemical sensing of melamine. The voltammetric behaviour of hexacyanoferrate solutions containing different concentrations of melamine on MIP and non-molecularly imprinted polymer (NIP) sensors was investigated by square wave voltammetry (SWV). Several important parameters controlling the performance of the MIP electrode were investigated and optimized. In the optimal conditions, the sensor responded sensitively to melamine from 10.0 nmol/L to 0.1μmol/L, and from 10.0μmol/L to 100μmol/L. The detection limit on the basis of a signal to noise ratio of 3 was as low as 1.6 nmol/L. The proposed method is simple and rapid. The same method was applied successfully to the determination of melamine in milk products, and method precision (RSD<4%) and recoveries (>90%) were satisfactory. The poly(gallic acid-o-phenylenediamine) (P(GA-o-PD)) electrodes have the merit of low response time, convenient and low-cost for determination of melamine, and are disposable and simple to construct.
引文
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