基于核酸适配体-多功能胶体晶体编码微球液相芯片多元检测谷物中真菌毒素新体系研究

For enrichment, purification, fast, low-cost detection of multiplex mycotoxins, multifunctional colloidal crystal microspheres which had different pore size, material composition, magnetism and optical properties were fabricated by self-assembly of nano-SiO2, nano-Fe3O4, gel and chemical etching techniques. The surface modification and immobilization methods of mycotoxin aptamer on the microsphere surface were studied. The enrichment and purification conditions for the typical mycotoxins in grains were researched by the microspheres encoded by their reflection peak positions. Based on the specific reaction between the mycotoxin and its aptamer, the new multiplex detection system on the surface of multifunctional colloidal crystal microspheres was established by designing the mycotoxin aptamer and its anti-aptamer. The influence factors on detection signals were researched, which included enrichment, purification,concentrations of aptamer and anti-mycotoxin aptamer, oligonucleotide length of anti-mycotoxin aptamer, hybridization conditions, pore size and material composition of microspheres. The sensitivity, recovery ratio, stability, repeatability and specificity of the new detection system were studied. The application of the established assay method for multiplex mycotoxins in grains was performed and the results were compared with that of HPLC method. The new enrichment, purification and detection system for multiplex mycotoxins in grains was established by aptamer-multifunctional colloidal crystal microsphere suspension array.

针对真菌毒素富集、净化、快速、低成本多元检测问题，通过纳米SiO2颗粒，Fe3O4颗粒共组装、凝胶及刻蚀技术等制备不同孔径及材料组成具有磁性和光学特性的多功能胶体晶体微球。以这些反射峰编码的微球为液相芯片载体，以谷物中典型真菌毒素为对象，研究微球表面修饰和核酸适配体（aptamer）的固定方法，研究应用该微球富集和净化谷物样品中真菌毒素的条件。设计真菌毒素aptamer和其互补链，构建基于aptamer作用原理的该微球真菌毒素液相芯片检测新方法。研究净化前后、aptamer互补链碱基长度、aptamer和互补链浓度比、杂交条件、不同孔径及材料对检测信号的影响，研究该检测方法的灵敏度，回收率，稳定性、重复性和特异性。在实际试样检测中和传统的高效液相色谱(HPLC)方法进行比较研究，建立基于aptamer-多功能胶体晶体微球编码载体富集、净化以及快速检测谷物中多元真菌毒素液相芯片新技术体系。

针对谷物中的真菌毒素样品前处理，多元检测问题，通过微流控自组装技术设计和建立了基于磁性光子晶体微球-核酸适配体的样品富集、净化前处理真菌毒素技术和方法，富集回收效率达80%，该技术方法可以替代免疫亲和柱样品富集、净化体系，大大降低了检测分析成本。设计和建立了基于光子晶体微球编码的核酸适配体高通量同时检测谷物中AFB1，FB1，OTA的新型液相生物芯片荧光技术和方法。检测灵敏度达到了pg/g，检测线性范围在2-3个数量级之间，仅仅需要2μL试剂/样品。该检测技术方法在检测灵敏度、速度、成本、高通量方面明显优于免疫分析检测方法和技术。解决了同时检测多种真菌毒素信号相互干扰、检测灵敏度低、通量低、成本高等问题。为将新一代液相生物芯片技术推广应用到一线提供了理论基础。建立了基于G-四联体-核酸适配体以及竞争适配体高通量检测OTA的单颗粒光子晶体微球高通量化学发光技术和方法。解决了当前基于G-四联体-核酸适配体化学发光方法检测通量低，试剂用量大，背景值高，信号干扰大的问题。通过电化学方法和凝胶方法制备了二氧化钛修饰的三维多孔硅表面，在其表面构建和设计了基于核酸适配体检测多元真菌毒素的微阵列生物芯片高通量分析技术和方法，克服了多孔硅表面的不稳定性，和普通二维平面芯片载体相比，该方法具有巨大的内表面积，可以固定更多的生物探针分子，提高信噪比，检测灵敏度，线性检测范围。建立了基于稳定的光学非标记多孔硅在线检测技术方法，为真菌毒素的非标记检测提供了简单、快速、灵敏和低成本光学在线检测平台。该非标记检测技术明显优于SPR技术和方法。