玉米赤霉烯酮的表面等离子体共振芯片无标记快速测定

Label-free and Rapid Determination of Zearalenone by Surface Plasmon Resonance Biochip

玉米赤霉烯酮(Zearalenone,ZEN)是农产品中一种常见的真菌毒素,严重危害食品安全。现有ZEN检测方法存在操作复杂、仪器贵重、样品通常需要标记等问题。基于自行设计的便携式表面等离子体共振(Surface plasmon resonance,SPR)生物传感器,制备了用于ZEN检测的生物芯片,提出直接检测法和抑制检测法。实验结果表明,直接检测法适用于ZEN抗体筛选与免疫动力学基础研究;抑制检测法的检测限小于2 ng/m L,完成一个样品检测仅需6 min,可用于痕量ZEN毒素小分子的快速检测。基于表面等离子体共振的ZEN检测方法简单,无需标记,仪器便携,成本低,可用于现场快速检测。

Zearalenone（ ZEN） is a common fungal toxin in agricultural products. The existing detection methods of ZEN are complicated,with precious instrument,and the samples usually need to be marked.Based on the self-developed surface plasmon resonance（ SPR） biosensor,the biochip for ZEN detection was prepared and the direct detection method and inhibition detection method were proposed. The preparation process of the biochip was as follow： modifying the gold film by self-assembled monolayer（ SAM） technique; activating the gold film surface with N-ethyl-N＇-（ 3-dimethylaminopropyl）carbodiimide（ EDC） solution; fixing the ZEN-BSA on the surface of the gold film as the biological probe; inactivating the remaining ester bond with ethanolamine. Direct detection method was to detect the ZEN antibody directly by using the biochip; and the standard curve of direct detection of ZEN antibody in the concentration range of 10 ～ 50 μg/m L was established. In the inhibition detection method,the ZEN toxin and ZEN antibody with different concentrations were mixed and then flowed into the chip surface.The ZEN small molecules in the sample would inhibit the binding between antibody and the biological probe（ ZEN-BSA） on the chip surface. The concentration of ZEN small molecule could be calculated according to the inverse relationship between the ZEN small molecule concentration and the changes of response value. The standard curve of inhibition detection method for ZEN toxin small molecule in the concentration range of 0 ～ 32 ng/m L was established. The experimental results showed that the direct detection method was suitable for screening of ZEN antibody and basic research of immune kinetics; inthe concentration range of 10 ～ 50 μg/m L,the relationship between SPR response value and ZEN concentration was basically linear; and the standard curve equation was： f（ x） = 0. 002 5 x-0. 477 0,R2= 0. 995 2. At least 50 sets of samples can be detected continuously. After being used for more than50 sets of samples,the response value of SPR was decreased,which indicated the probe that was fixed on the surface of the biochip was damaged. The chip regeneration can be achieved after HCl was flowed into the chip surface,and the sensor chip can be used continually after re-assembly and fixation of the biological probe. The detection limit of the inhibition detection method was up to 2 ng/m L; the standard deviation of the repeated tests（ 5 times） for each concentration was less than 8%; and the detection of a single sample only costed 6 min. The inhibition detection method could be used for rapid detection of trace ZEN toxin small molecule. The detection method of ZEN based on the surface plasmon resonance was simple. The samples did not need to be marked,which could avoid the environment pollution. The developed device was portable with low cost,as the cost of the entire SPR system was no more than30 000 yuan which was far below the price of commercial SPR instrument,and the cost of each biochip was less than 200 yuan; the device was stable and portable,its size and weight were like the host of personal computer. The biochip preparation process and the immune response detection process could be monitored in real time. The device and method could be expected to be used for on-site rapid detection of ZEN,thus can effectively prevent ZEN from harming food safety.