摘要
生物活性分子的固定是研制生物传感器的关键环节,寻找简便有效的固定方法以及研制具有高生物兼容性的固定材料,对改善生物传感性能至关重要。随着纳米技术的迅速发展,各种纳米材料已广泛用于生物传感器的研制。制备多功能纳米复合材料,并藉此研制具有高灵敏度、高选择性的生物传感方法备受关注。本文中我们制备了几种金属／金属氧化物-聚合物-酶纳米复合材料,并成功用于高敏葡萄糖生物传感和凝血酶适配体传感研究。主要工作如下：
1.对电化学生物传感器的分类、生物活性物质的固定方法、纳米材料的分类和特性及其在生物传感中的应用进行了简要综述,并在此基础上提出了本论文的基本构思。
2.采用一锅原位化学氧化法在含葡萄糖氧化酶(GOx)、Fe3O4-Au纳米复合材料和1,6-己二硫醇(HDT)的水相悬浊液中,通过对苯醌(BQ)的氧化作用制备了Fe3O4-Au-poly(HDT) (PHDT)-GOx新型磁性聚合物生物纳米复合材料(MPBNCs),可高活性、高负载量地固定GOx。采用透射电子显微镜、扫描电子显微镜和紫外可见分光光度法表征了所制MPBNCs。采用简便有效的磁性分离/固定法将MPBNCs固定于金磁电极上,所制备的葡萄糖生物传感器灵敏度高(110μA cm-2mM-1),检测限低(0.33μM,S/N=3),响应时间短(<5s),抗干扰能力强,稳定性好。该生物传感器的性能优于常规电聚合法和化学预氧化/电聚合单体法所制备的传感器。
3.采用一锅原位化学氧化法制备聚多巴胺(PDA)-GOx纳米复合物,通过生化原位合成法在所制PDA-GOx纳米复合物表面修饰金纳米粒子(AuNPs),再藉AuNPs的表面催化作用,以抗坏血酸(AA)作还原剂还原H2PtCl6为铂纳米粒子(PtNPs),制备了新型双金属聚合物生物纳米复合材料：PDA-GOx-AuNPs-PtNPs纳米复合物。将此复合物滴干在金电极表面制备了灵敏度高(195μAcm-2mM-1)、检测限低(0.21 gM,S/N=3)、响应时间短(<5 s)、抗干扰能力强和稳定性好的葡萄糖安培传感器。
4.利用原位化学氧化/生物化学合成法制备PDA-GOx-AuNPs纳米复合物,将巯基化凝血酶适配体组装固定于该复合物修饰的金电极表面,研制了凝血酶适配体传感器。其基本检测原理是基于凝血酶与其适配体的特异性结合可抑制该GOx电极的响应电流。该传感器的线性范围宽(1-40 nM),检测限低(0.125 nM,S/N=3),响应时间短(<10s)。
The immobilization of biomolecules is the key step in constructing a biosensor. Immobilization of biomolecules with effective methods and highly biocompatible materials is essential to improve the biosensing performance. With the rapid development of nanotechnology, a variety of nanomaterials have been used to construct various biosensors. Preparation of multifunctional nanocomposite materials is important to develop a biosensor with high sensitivity and selectivity. In this thesis, we prepared several metal (or metal oxide)-polymer-enzyme nanocomposite materials for high performance glucose biosensing and thrombin aptasensing. The main contents are as follows,
1. The classification of electrochemical biosensors, the immobilization method of the biomolecules, the classification and characteristics of nano-materials, as well as their application in biosensing, have been reviewed.
2. One-pot chemical oxidation of 1,6-hexanedithiol (HDT) in its aqueous suspension containing glucose oxidase (GOx) and Fe3O4-Au nanocomposites by 1,4-benzoquinone yields novel Fe3O4-Au-poly(HDT) (PHDT)-GOx magnetic polymeric bionanocomposites (MPBNCs) with GOx immobilized at high load and high activity. Transmission/scanning electron microscopy and UV-Vis spectrophotometry are used to characterize the prepared MPBNCs. A Fe3O4-Au-PHDT-GOx/Au electrode has been prepared by facile and efficient magnetism separation/immobilization of the MPBNCs onto an Au magnetism-electrode for biosensing of glucose, which exhibits high detection sensitivity (110μA cm-2 mM-1), low detection limit (0.33μM, S/N= 3), rapid response time (< 5 s), and excellent anti-interferent ability and stability. The biosensor performs better than those based on the existing protocols of conventional electropolymerization and chemical preoxidation/electropolymerization of monomer.
3. Novel bimetallic polymeric bionanocomposites (PBNCs) of polydopamine (PDA), AuNPs-PtNPs bimetallic nanoparticles were prepared. First, We prepared PDA-GOx nanocomposites with high-load/activity GOx entrapped via one-pot chemical protocol, namely, mixing biogenic dopamine (DA) as a monomer, GOx as a model molecule, and BQ as an oxidizing reagent to trigger DA polymerization to allow one-pot chemical synthesis; second, Au nanoparticles (AuNPs) was generated via in situ biochemical generation protocol, yielded PDA-GOx-AuNPs nanocomposites; the last, we synthesized the PDA-GOx-AuNPs-PtNPs nanocomposites by H2PtCl6 reduction onto the AuNPs surfaces with the AuNPs of PDA-GOx-AuNPs as the seed and ascorbic acid as the reductant. The PBNCs based biosensor via dip-dry method exhibits high detection sensitivity (195μA cm-2 mM-1), low detection limit (0.21μM, S/N= 3), rapid response time (< 5 s), and excellent anti-interferent ability and stability.
4. A thrombin aptasensor was prepared by immobilizing a thiolated thrombin aptamer (TBA) onto the electrode modified with a PDA-GOx-AuNPs nanocomposite material prepared via in situ chemical/biochemical protocol. The measurement of the aptasensor was based on the inhibition of the amperometric response of the GOx-based electrode originated from the specific thrombin-aptamer binding. The aptasensor exhibits wide linear detection range (1-40 nM), low detection limit (0.125 nM, S/N=3), rapid response time (< 10 s), and excellent anti-interferent ability.
引文
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