适配体传感法快速测定啤酒中赭曲霉毒素A

应用自组装方式，构建了金纳米粒子/核酸适体/氨基碳量子点荧光传感赭曲霉毒素A高灵敏检测方法。在pH 3.0酒石酸-HCl缓冲溶液中，巯基修饰的赭曲霉毒素A核酸适体在金纳米粒子表面自组装，形成金纳米粒子/核酸适体复合物，再在pH 7.0的磷酸盐缓冲溶液中，氨基碳量子点在金纳米粒子/核酸适体复合物上自组装，形成金纳米粒子/核酸适体/氨基碳量子点复合物荧光传感检测体系。金纳米粒子的摩尔吸光系数大、能带宽使其具有强烈的荧光猝灭功能，氨基碳量子点形成金纳米粒子/核酸适体/氨基碳量子点后发生荧光猝灭, 此时体系的荧光为背景荧光，其强度记为F₀；由于金纳米粒子/核酸适体/氨基碳量子点复合物荧光传感检测体系中核酸适体对赭曲霉毒素A具有特异性识别与结合功能，向金纳米粒子/核酸适体/氨基碳量子点复合物荧光传感检测体系溶液中加入赭曲霉毒素A后，赭曲霉毒素A则与复合物中核酸适体立即发生特异性结合并释放出氨基碳量子点，体系荧光恢复，其荧光强度记为F。依据体系荧光强度的变化(F-F₀)与赭曲霉毒素A浓度之间的关系，建立赭曲霉毒素A核酸适体荧光传感检测方法。研究了金纳米粒子和核酸适体摩尔比、孵化时间、pH等因素对传感器性能的影响，确定了最优条件为金纳米粒子∶核酸适体t为1∶190、孵化时间为6 min、pH 7.0时；在最优条件下，赭曲霉毒素A浓度在0.005～1.00 ng·mL-1范围与体系荧光强度变化呈良好线性关系，线性回归方程为：F-F₀=6.499+211.6c(c为赭曲霉毒素A的浓度，单位：ng·mL-1)，相关系数r为0.995 5，按3倍标准差与工作曲线的斜率的比值(3σ/k)计算，得检测限为3 pg·mL-1。在实际样品中的回收率在93.3%～108.9%，相对标准偏差小于5%，能满足啤酒样品中赭曲霉毒素A快速检测要求。对13个市售啤酒样品进行检测，其中6个样品检出赭曲霉毒素A，污染率为46.15%。受污染样品的赭曲霉毒素A含量在0.008～0.63 ng·mL-1范围。该荧光传感法检测赭曲霉毒素A具有灵敏性好、特异性高、常见真菌毒素无干扰、方法简单、快速，便于大众化推广应用的优点。

A Novel Aptasensors Assay for Fast Detection of Ochratoxin A in Beer

In this paper, a novel highly sensitive fluorescent aptasensor was constructed and used to detect ochratoxin A based on gold nanoparticles/aptamer/amino-functioned carbon quantum dots by using self-assembly. Gold nanoparticles/aptamer/amino-functioned carbon quantum dots were prepared as following. First, thiol-modified aptamer was attached to the surface of the gold nanoparticles in pH 3.0 tartaric acid-HCl buffer to form gold nanoparticles/aptamer by assembling. Second, amino- functioned carbon quantum dots were added to the gold nanoparticles/aptamer dispersion to form gold nanoparticles/aptamer/amino-functioned carbon quantum dots under electrostatic interactions in phosphate buffer solution (pH 7.0), by removing the excessive amino-functioned carbon quantum dots with centrifugation. The fluorescence of the amino-functioned carbon quantum dots was efficiently quenched by the gold nanoparticles, which are excellent quencher for fluorescence sensing as they have very high molar extinction coefficients and broad energy bandwidth. The fluorescent intensity of the quenched system was background fluorescence (F₀). When ochratoxin A was addition to the fluorescence quenched system, the specific reaction between aptamer in the nanocompostes and ochratoxin A took place, simultaneously, amino-functioned carbon quantum dots were released, and a turn on amino-functioned carbon quantum dots fluorescence signal (F) was detected. The emission intensity increase (F-F₀) could be used for the quantification of the amount of ochratoxin A in samples. The influence facts on the sensor performance were investigated including the molar ratio of gold nanoparticles and Apt, pH and incubation time. The optimum conditions were gold nanoparticles∶aptamer=1∶190 in molar ratio, pH 7.0 and incubation time was 6 minutes. Under the optimum conditions, a linear fluorescence signal response to ochratoxin A concentration was over a wide ochratoxin A concentration range of 0.005～1.00 ng·mL-1. The linear regression equation is: F-F₀=6.499+211.6 c(ng·mL-1), linear correlation coefficient is: r=0.995 5 with a diction limit of 3 pg·mL-1 according 3σ/k (σ: relative standard deviations, k: slope of the working curve). The recovery was between 93.3%～108.9% in real samples, and the relative standard deviation was less than 5%. The proposed method was employed to detect ochratoxin A in beer samples, the results showed that ochratoxin A was found in 6 of 13 beer samples, with a positive rate of 46.15%. The concentration of ochratoxin A was in the range of 0.008～0.63 ng·mL-1. The fluorescent apasensor method used to detect ochratoxin A has the advantages of highly sensitive, highly specific, without interference of common mycotoxins, simple, very fast, convenient for popularization and application.