基于聚合物固定生物识别分子和纳米增敏新法的安培生物传感研究
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摘要
生物传感分析是将生物活性材料(酶、抗体／抗原、核酸、适体等)的分子识别功能与物理化学传感换能技术有机结合的现代分析化学分支,具有灵敏度高、选择性好、操作便利、分析速度快、成本低、易于实现实时监测等特点,在复杂环境乃至活体分析中也不乏其应用实例。自第一支生物传感器在20世纪60年代诞生以来,生物传感分析领域发展迅速,备受学术界和产业界关注。有效固定生物识别材料是研制生物传感器的关键步骤之一,采用化学聚合法或电化学聚合法制备生物兼容性聚合物生物敏感材料一直是固定生物识别分子的重要方法。近年来,随着纳米科技的蓬勃发展,纳米生物传感迅速成为生物传感分析领域的国际前沿和热点。本学位论文中,我们简要综述了电化学生物传感器的近期进展,以及聚合物和纳米材料在生物传感领域的应用,并针对基于聚合物固定生物识别分子和纳米增敏新法的生物传感分析开展了系列研究工作,主要内容如下：
1.将化学／电化学聚合法有机结合,提出了在含酶和单体悬浊液／水溶液中一锅法化学预氧化—电聚合单体(CPEM)的新方法用于高效固定酶,藉此研制了高敏安培生物传感器。考察了1,4-苯二硫酚(BDT)、1,6-已二硫醇(HDT)、邻苯二胺、邻氨基酚和吡咯等单体；铁氰化钾和对苯醌两种预氧化剂；葡萄糖氧化酶(GOx)和碱性磷酸酶两种酶及其生物传感器。代表性实验步骤如下：在BDT单体和酶的水相悬浊液中加入预氧化剂K3Fe(CN)6引发化学氧化聚合,得到包埋有大量高活性酶分子的BDT寡聚体—酶复合物,再电氧化聚合该复合物和BDT单体,最终制得高性能酶膜。与常规电聚合法(CEP)相比,CPEM法所制葡萄糖安培生物传感器的灵敏度提高至32.4倍。通过电化学石英晶体微天平(EQCM)和紫外—可见光谱法的定量测试,发现采用CPEM法可显著提高酶膜中酶的负载量和比活性。
2.以酶生H2O2(EG-H2O2)为预氧化剂,提出了高效固定酶的一锅法生化预氧化—电聚合单体(BPEM)新方法,藉此研制了高敏葡萄糖安培生物传感器。基本实验步骤如下：在含GOx和2,5-二巯基-1,3,4-噻二唑(DMcT)的水相悬浊液中加入葡萄糖,酶生H2O2使DMcT氧化聚合,生成DMcT寡聚体—GOx复合物,再电氧化聚合该复合物和DMcT单体,可在电极表面制得高性能酶膜。与CEP法比,该法所制传感器的灵敏度提高至119倍,检测下限降低了2个数量级。发现BPEM法所制酶电极的灵敏度比基于外加H2O2的CPEM法更高,原因可能为生化预氧化聚合／包埋作用主要发生在酶分子附近(反应临近性),故酶负载量更大。采用EQCM监测了电极修饰过程,发现DMcT聚合膜可被电还原而从电极上脱落,这有利于电极基底的电化学再生。
3.采用多巴胺(DA)为单体、HAuCl4或H2PtCl6为预氧化剂,通过CPEM法固定GOx和半乳糖氧化酶,制备了新型聚合物生物纳米复合物(PBNCs):聚多巴胺(PDA)-酶-纳米金(或铂)(AuNPs或PtNPs)复合物,籍此研制了高敏葡萄糖和半乳糖安培生物传感器。PDA基质具有优异的吸附性能和生物相容性,有利于高效固定酶和纳米增敏。与CEP法相比,基于该PBNCs的酶电极更灵敏,金和镀铂金电极上对葡萄糖的检测灵敏度分别达99和129μA cm-2 mmol-1L。基于PBNCs的第二代安培传感器亦有优异性能。
4.提出了化学氧化聚合-磁性分离／固定新方法用于高效固定酶,藉此研制了高敏葡萄糖安培生物传感器。先采用化学共沉淀法合成了Fe3O4@Au核壳型磁性复合纳米粒子,然后据化学氧化聚合法制得聚1,6-已二硫醇(PHDT)-GOx-Fe3O4@Au纳米复合物,并借助磁场将该复合物分离／固定在磁性电极表面。所制酶电极对葡萄糖的检测灵敏度达110μA cm-2 mmol-1L,检测限为0.3μmol L-1。该法简便、省时、高效。
5.提出采用新型HDT电聚合膜固定AuNPs、实现纳米增敏压电免疫传感的新方法。通过EQCM等技术研究了HDT电聚合过程和机理。结合理论分析和实验结果,定量考察了AuNPs在HDT电聚合膜上的覆盖度和纳米增敏效应。比较研究了羊抗人免疫球蛋白G (anti-hIgG)的吸附,及后续的与人免疫球蛋白G (hIgG)的免疫反应。与常规HDT自组装单层修饰电极相比,基于HDT电聚合膜的修饰电极可更有效地固定抗体、性能更稳定、制备时间更短。新型硫醇聚合膜材料有望作为硫醇自组装单层的有益补充而在生物传感研究中广泛应用。
6.基于化学氧化聚合法制备了免疫PBNCs:PDA-PtNPs-anti-hIgG复合物,该复合物免疫亲合性高并可高效催化H202电还原,籍此研制了高敏三明治型安培免疫传感器。该传感器可检测低至0.018 ngmL-1的hIgG,具有良好的重现性、稳定性、再生性和特异性,可用于临床人血清样品中目标物检测。
7.基于化学氧化法制备了PDA-PtNPs-GOx纳米复合物,然后借助葡萄糖存在下酶生H2O2生化还原HAuCl4,在纳米复合物表面原位生成AuNPs用于高效吸附固定抗体及酶标GOx,制得GOxads/anti-hIgG/AuNPs/PDA-PtNPs-GOx生物纳米复合物,籍此研制了高敏三明治型电化学免疫传感器。该法优势在于能在该生物纳米复合物表面和内部均高效固定GOx酶标,有效地提高了酶标的负载量及活性。以醌／氢醌为媒介体,实现了超敏免疫信号的输出,所制免疫传感器可检测低至2 pg mL-1的hIgG。
8.将AuNPs/PDA-PtNPs-GOx纳米复合物滴干修饰于金电极表面,共价固定巯基化适体以特异结合凝血酶,以GOx标记物的催化电流为信号输出,研制了“信号衰减”型电化学适体传感器。凝血酶与适体结合后产生传质阻力,可显著抑制标记酶的催化反应速度,降低酶生H2O2氧化电流。所制适体传感器可检测低至0.1 nmolL-1的凝血酶。
9.结合磁分离／富集技术,提出了以适体缠绕的单壁碳纳米管(SWCNTs)为放大平台的新型三明治型适体安培传感器。以凝血酶适体Ⅰ修饰的磁性纳米粒子、凝血酶、凝血酶适体Ⅱ缠绕的SWCNTs构建三明治型复合物,借助磁场将其分离并富集至磁性金电极上,通过亚甲基蓝分子对SWCNTs上缠绕的适体Ⅱ的位置取代作用,成功分离得到亚甲基蓝/SWCNTs修饰的金电极。以亚甲基蓝的脉冲伏安峰电流为分析信号,所制适体传感器可检测低至3 pmol L-1的凝血酶,优于其它类似传感器。
By combining the recognition ability of bioactive species (enzyme, antibody/antigen, nucleic acid, aptamer, etc.) and the physical/chemical transducer, biosensing analysis has become one of the most important branch of modern analytical chemistry. Biosensor is well acknowledged as a device that can detect the target with high selectivity, time/cost-effectiveness, and operation facility, being competent for working in complex samples, on-line monitoring, and even in vivo analysis. Since the inception of the first biosensor in 1960s, biosensing research has gained an explosive development and received wide academic and industrial attention. The effective immobilization of biorecognition materials is regarded as one of the key steps to construct a biosensor, and the use of chemical or electrochemical polymerization to prepare biocompatible polymeric biosensing films is always one of the most important methods to immobilize biorecognition molecules. Recently, along with the explosive development of nanoscience and nanotechnology, nanobiosensing has become the frontier and focus of this field. In this dissertation, the recent advances of electrochemical biosensors as well as the biosensing applications of polymers and nanomaterials are reviewed, and a series of biosensing studies are conducted based on novel methods for polymeric immobilization of biorecognition molecules and for nano enhancement, as summarized below.
1. Combining the chemical polymerization and electrochemical polymerization, we propose the one-pot chemical preoxidation and electropolymerization of monomer (CPEM) in enzyme-containing aqueous suspensions (or solutions) as a universal strategy for high-activity and high-load immobilization of enzymes to construct amperometric biosensors. We investigated the monomer of 1,4-benzenedithiol (BDT),1,6-hexanedithiol (HDT), o-phenylenediamine, o-aminophenol or pyrrole, the preoxidant of K3Fe(CN)6 or p-benzoquinone, and the enzyme of glucose oxidase (GOx) or alkaline phosphatase (AP) to develop GOx-based glucose biosensors or AP-based disodium phenyl phosphate biosensors. The typical experimental procedures are as follows. Into an aqueous suspension of BDT and GOx, the preoxidant of K3Fe(CN)6 was added to obtain BDT oligomer-enzyme composite; then high-performance enzyme film was gained by co-electrodeposition of these composites and poly(1,4-benzenedithiol). Compared with the biosensor based on protocol of conventional electropolymerization (CEP), the CPEM-based glucose biosensor presented a 32.4-fold sensitivity. We also found that the load and activity of the immobilized enzymes was largely improved, as estimated by electrochemical quartz crystal microbalance (EQCM) and UV-vis spectrophotometry.
2. We propose the biochemical preoxidation and electropolymerization of monomer (BPEM) using enzyme generated-H2O2 (EG-H2O2) as the preoxidant to immobilize enzyme with high load and activity, thus to construct a high-performance amperometric glucose biosensor. Basic experimental procedures are as follows. In an aqueous suspension of 2,5-dimercapto-1,3,4-thiadiazole (DMcT) and GOx, glucose was added, and the EG-H2O2 biochemically oxidized DMcT to DMcT oligomer (DMcTO), which entrapped GOx and yielded DMcTO-GOx composites, followed by co-electrodeposition of these composites and DMcT polymer to form a high performance enzyme film. Compared with the CEP protocol, the BPEM-based glucose biosensor presented 119-fold sensitivity, as well as a detection limit of 2 orders lower. We found that the performance of BPEM-based biosensor was superior to that based on the CPEM protocol using externally-added H2O2 as the preoxidant, which may be ascribed to the improved proximity of biochemical preoxidation and thus an increased enzyme load. We also found that DMcT polymer could be cathodically detached from the electrode surface, favorably enabling the electrochemical regeneration of the electrode substrate, as monitored by EQCM.
3. We propose the preparation of novel poly dopamine (PDA)-enzyme-metallic nanoparticles nanocomposites using the CPEM protocol, with dopamine (DA) as the monomer and HAuCl4 or H2PtCl6 as the preoxidant, to immobilize GOx (or galactose oxidase) for fabricating high performance glucose (or galactose) biosensors. The PDA exhibited excellent adsorbability and biocompatibility, being favorable for highly efficient immobilization of enzyme molecules and nano enhancement. Compared with the CEP protocol, the PBNCs-based biosensor showed obviously improved sensitivity of 99μA cm-2 mmol-1 L and 129μA cm-2 mmol-1 L for glucose at bare Au and platinized Au electrode, respectively. Also, the prepared PBNCs electrode is able to work well in the second generation biosensing mode.
4. We propose a novel protocol of chemical polymerization with magnetic separation/immobilization for high-sensitivity amperometric glucose biosensing. We prepared Fe3O4@Au core-shell hybrid magnetic nanoparticles (MNPs) using the method of chemical co-precipitation, then synthesized poly(1,6-hexanedithiol) (PHDT)-GOx-Fe3O4@Au nanocomposites using chemical polymerization, which were magnetically separated and immobilized on the magnetism electrode. This protocol was simple, time-saving, and highly efficient. The glucose biosensor showed a high detection sensitivity of 110μA cm-2 mmol-1L, detection limit of 0.3μmol L-1, S/N=3).
5. We propose electrosynthesized PHDT as a new matrix to immobilize AuNPs and anti-body for performance-enhanced piezoelectric immunosensing. Using the EQCM, we investigated the process and mechanism for HDT electropolymerzation, and estimated the coverage of AuNPs and the nano enhancement effect theoretically and experimentally. We comparatively examined the adsorption of anti-human immunoglobulin G (anti-hIgG) and the subsequent immunoreaction with immunoglobulin G (hIgG). Compared with the electrode modified with HDT self-assembled monolayer (SAM), the PHDT modified electrode showed better stability and can be constructed more time-effectively, demonstrating that the novel thiol-polymer materials may become a useful alternative to thiol-based SAM and find wide biosensing applications.
6. Novel PDA-PtNPs-anti-hIgG nanocomposites were prepared using chemical synthesis, which present satisfactory immuno-recognition efficiency and strong catalysis ability to electrochemical reduction of H2O2 for high performance sandwich-type amperometric immunosensing. The immunosensor has a limit of detection of 0.018 ng mL-1, as well as good reproducibility, stability, regeneration ability, specificity, and satisfactory feasibility for assay in clinical human serum samples.
7. Chemical oxidation was used to prepare PDA-PtNPs-GOx nanocomposites, on the surface of which AuNPs were biochemically prepared from reduction of HAuCl4 by the entrapped GOx-generated H2O2 to effectively immobilize antibody and GOx, yielding GOxads/antibody/AuNPs/PDA-PtNPs-GOx PBNCs for high performance sandwich-type amperometric immunosensing. The thus-prepared PBNCs feature high load and activity of the enzyme label immobilized both in the interior and on the surface for ultrasensitive signal readout. Using p-benzoquinone/hydroquinone as the mediator, the proposed immunosensor could detect the target antigen of hIgG down to a concentration of 2 pg mL-
8. The AuNPs/PDA-PtNPs-GOx nanocomposites were cast onto an Au electrode to covalently bind a thiolated aptamer for thrombin, and a "signal-off'electrochemical aptasensor was thus constructed based on signal output of the GOx label. The binding of thrombin with the captured aptamer obviously blocked the mass-transfer inside the film on the electrode, leading to the suppression of the enzymatic reaction on the electrode and the decrease of the oxidation current of EG-H2O2. The thus-prepared aptasensor could detect thrombin down to a concentration of 0.1 nmol L/-1.
9. We propose a novel sandwich-type amperometric aptasensor using aptamer-wrapped single-walled carbon nanotubes (SWCNTs) as an amplification platform and the magnetic separation/immobilization technique to effectively improve the sensitivity. In a suspension containing Fe3O4 MNPs modified with thrombin aptamerⅠ(aptamer@MNPs) and SWCNTs modified with thrombin aptamerⅡ(aptamer@SWCNTs), the target thrombin was added to yield the sandwich-type composites of aptamer I@MNPs-thrombin-aptamerⅡ@SWCNTs. The composites were magnetically separated and collected onto the surface of a magnetism Au electrode, methylene blue was added to detach the aptamerⅡfrom SWCNTs via its stronger interaction with SWCNTs than aptamerⅡdoes, and the methylene blue-adsorbed SWCNTs modified electrode was obtained after magnetically removing the aptamer I@MNPs-thrombin-aptamerⅡcomposites. Based on the differential pulse voltammetric detection of methylene blue, the thus-prepared aptasensor could detect thrombin down to a concentration of 3 pmol L-1.
1.将化学／电化学聚合法有机结合,提出了在含酶和单体悬浊液／水溶液中一锅法化学预氧化—电聚合单体(CPEM)的新方法用于高效固定酶,藉此研制了高敏安培生物传感器。考察了1,4-苯二硫酚(BDT)、1,6-已二硫醇(HDT)、邻苯二胺、邻氨基酚和吡咯等单体；铁氰化钾和对苯醌两种预氧化剂；葡萄糖氧化酶(GOx)和碱性磷酸酶两种酶及其生物传感器。代表性实验步骤如下：在BDT单体和酶的水相悬浊液中加入预氧化剂K3Fe(CN)6引发化学氧化聚合,得到包埋有大量高活性酶分子的BDT寡聚体—酶复合物,再电氧化聚合该复合物和BDT单体,最终制得高性能酶膜。与常规电聚合法(CEP)相比,CPEM法所制葡萄糖安培生物传感器的灵敏度提高至32.4倍。通过电化学石英晶体微天平(EQCM)和紫外—可见光谱法的定量测试,发现采用CPEM法可显著提高酶膜中酶的负载量和比活性。
2.以酶生H2O2(EG-H2O2)为预氧化剂,提出了高效固定酶的一锅法生化预氧化—电聚合单体(BPEM)新方法,藉此研制了高敏葡萄糖安培生物传感器。基本实验步骤如下：在含GOx和2,5-二巯基-1,3,4-噻二唑(DMcT)的水相悬浊液中加入葡萄糖,酶生H2O2使DMcT氧化聚合,生成DMcT寡聚体—GOx复合物,再电氧化聚合该复合物和DMcT单体,可在电极表面制得高性能酶膜。与CEP法比,该法所制传感器的灵敏度提高至119倍,检测下限降低了2个数量级。发现BPEM法所制酶电极的灵敏度比基于外加H2O2的CPEM法更高,原因可能为生化预氧化聚合／包埋作用主要发生在酶分子附近(反应临近性),故酶负载量更大。采用EQCM监测了电极修饰过程,发现DMcT聚合膜可被电还原而从电极上脱落,这有利于电极基底的电化学再生。
3.采用多巴胺(DA)为单体、HAuCl4或H2PtCl6为预氧化剂,通过CPEM法固定GOx和半乳糖氧化酶,制备了新型聚合物生物纳米复合物(PBNCs):聚多巴胺(PDA)-酶-纳米金(或铂)(AuNPs或PtNPs)复合物,籍此研制了高敏葡萄糖和半乳糖安培生物传感器。PDA基质具有优异的吸附性能和生物相容性,有利于高效固定酶和纳米增敏。与CEP法相比,基于该PBNCs的酶电极更灵敏,金和镀铂金电极上对葡萄糖的检测灵敏度分别达99和129μA cm-2 mmol-1L。基于PBNCs的第二代安培传感器亦有优异性能。
4.提出了化学氧化聚合-磁性分离／固定新方法用于高效固定酶,藉此研制了高敏葡萄糖安培生物传感器。先采用化学共沉淀法合成了Fe3O4@Au核壳型磁性复合纳米粒子,然后据化学氧化聚合法制得聚1,6-已二硫醇(PHDT)-GOx-Fe3O4@Au纳米复合物,并借助磁场将该复合物分离／固定在磁性电极表面。所制酶电极对葡萄糖的检测灵敏度达110μA cm-2 mmol-1L,检测限为0.3μmol L-1。该法简便、省时、高效。
5.提出采用新型HDT电聚合膜固定AuNPs、实现纳米增敏压电免疫传感的新方法。通过EQCM等技术研究了HDT电聚合过程和机理。结合理论分析和实验结果,定量考察了AuNPs在HDT电聚合膜上的覆盖度和纳米增敏效应。比较研究了羊抗人免疫球蛋白G (anti-hIgG)的吸附,及后续的与人免疫球蛋白G (hIgG)的免疫反应。与常规HDT自组装单层修饰电极相比,基于HDT电聚合膜的修饰电极可更有效地固定抗体、性能更稳定、制备时间更短。新型硫醇聚合膜材料有望作为硫醇自组装单层的有益补充而在生物传感研究中广泛应用。
6.基于化学氧化聚合法制备了免疫PBNCs:PDA-PtNPs-anti-hIgG复合物,该复合物免疫亲合性高并可高效催化H202电还原,籍此研制了高敏三明治型安培免疫传感器。该传感器可检测低至0.018 ngmL-1的hIgG,具有良好的重现性、稳定性、再生性和特异性,可用于临床人血清样品中目标物检测。
7.基于化学氧化法制备了PDA-PtNPs-GOx纳米复合物,然后借助葡萄糖存在下酶生H2O2生化还原HAuCl4,在纳米复合物表面原位生成AuNPs用于高效吸附固定抗体及酶标GOx,制得GOxads/anti-hIgG/AuNPs/PDA-PtNPs-GOx生物纳米复合物,籍此研制了高敏三明治型电化学免疫传感器。该法优势在于能在该生物纳米复合物表面和内部均高效固定GOx酶标,有效地提高了酶标的负载量及活性。以醌／氢醌为媒介体,实现了超敏免疫信号的输出,所制免疫传感器可检测低至2 pg mL-1的hIgG。
8.将AuNPs/PDA-PtNPs-GOx纳米复合物滴干修饰于金电极表面,共价固定巯基化适体以特异结合凝血酶,以GOx标记物的催化电流为信号输出,研制了“信号衰减”型电化学适体传感器。凝血酶与适体结合后产生传质阻力,可显著抑制标记酶的催化反应速度,降低酶生H2O2氧化电流。所制适体传感器可检测低至0.1 nmolL-1的凝血酶。
9.结合磁分离／富集技术,提出了以适体缠绕的单壁碳纳米管(SWCNTs)为放大平台的新型三明治型适体安培传感器。以凝血酶适体Ⅰ修饰的磁性纳米粒子、凝血酶、凝血酶适体Ⅱ缠绕的SWCNTs构建三明治型复合物,借助磁场将其分离并富集至磁性金电极上,通过亚甲基蓝分子对SWCNTs上缠绕的适体Ⅱ的位置取代作用,成功分离得到亚甲基蓝/SWCNTs修饰的金电极。以亚甲基蓝的脉冲伏安峰电流为分析信号,所制适体传感器可检测低至3 pmol L-1的凝血酶,优于其它类似传感器。
By combining the recognition ability of bioactive species (enzyme, antibody/antigen, nucleic acid, aptamer, etc.) and the physical/chemical transducer, biosensing analysis has become one of the most important branch of modern analytical chemistry. Biosensor is well acknowledged as a device that can detect the target with high selectivity, time/cost-effectiveness, and operation facility, being competent for working in complex samples, on-line monitoring, and even in vivo analysis. Since the inception of the first biosensor in 1960s, biosensing research has gained an explosive development and received wide academic and industrial attention. The effective immobilization of biorecognition materials is regarded as one of the key steps to construct a biosensor, and the use of chemical or electrochemical polymerization to prepare biocompatible polymeric biosensing films is always one of the most important methods to immobilize biorecognition molecules. Recently, along with the explosive development of nanoscience and nanotechnology, nanobiosensing has become the frontier and focus of this field. In this dissertation, the recent advances of electrochemical biosensors as well as the biosensing applications of polymers and nanomaterials are reviewed, and a series of biosensing studies are conducted based on novel methods for polymeric immobilization of biorecognition molecules and for nano enhancement, as summarized below.
1. Combining the chemical polymerization and electrochemical polymerization, we propose the one-pot chemical preoxidation and electropolymerization of monomer (CPEM) in enzyme-containing aqueous suspensions (or solutions) as a universal strategy for high-activity and high-load immobilization of enzymes to construct amperometric biosensors. We investigated the monomer of 1,4-benzenedithiol (BDT),1,6-hexanedithiol (HDT), o-phenylenediamine, o-aminophenol or pyrrole, the preoxidant of K3Fe(CN)6 or p-benzoquinone, and the enzyme of glucose oxidase (GOx) or alkaline phosphatase (AP) to develop GOx-based glucose biosensors or AP-based disodium phenyl phosphate biosensors. The typical experimental procedures are as follows. Into an aqueous suspension of BDT and GOx, the preoxidant of K3Fe(CN)6 was added to obtain BDT oligomer-enzyme composite; then high-performance enzyme film was gained by co-electrodeposition of these composites and poly(1,4-benzenedithiol). Compared with the biosensor based on protocol of conventional electropolymerization (CEP), the CPEM-based glucose biosensor presented a 32.4-fold sensitivity. We also found that the load and activity of the immobilized enzymes was largely improved, as estimated by electrochemical quartz crystal microbalance (EQCM) and UV-vis spectrophotometry.
2. We propose the biochemical preoxidation and electropolymerization of monomer (BPEM) using enzyme generated-H2O2 (EG-H2O2) as the preoxidant to immobilize enzyme with high load and activity, thus to construct a high-performance amperometric glucose biosensor. Basic experimental procedures are as follows. In an aqueous suspension of 2,5-dimercapto-1,3,4-thiadiazole (DMcT) and GOx, glucose was added, and the EG-H2O2 biochemically oxidized DMcT to DMcT oligomer (DMcTO), which entrapped GOx and yielded DMcTO-GOx composites, followed by co-electrodeposition of these composites and DMcT polymer to form a high performance enzyme film. Compared with the CEP protocol, the BPEM-based glucose biosensor presented 119-fold sensitivity, as well as a detection limit of 2 orders lower. We found that the performance of BPEM-based biosensor was superior to that based on the CPEM protocol using externally-added H2O2 as the preoxidant, which may be ascribed to the improved proximity of biochemical preoxidation and thus an increased enzyme load. We also found that DMcT polymer could be cathodically detached from the electrode surface, favorably enabling the electrochemical regeneration of the electrode substrate, as monitored by EQCM.
3. We propose the preparation of novel poly dopamine (PDA)-enzyme-metallic nanoparticles nanocomposites using the CPEM protocol, with dopamine (DA) as the monomer and HAuCl4 or H2PtCl6 as the preoxidant, to immobilize GOx (or galactose oxidase) for fabricating high performance glucose (or galactose) biosensors. The PDA exhibited excellent adsorbability and biocompatibility, being favorable for highly efficient immobilization of enzyme molecules and nano enhancement. Compared with the CEP protocol, the PBNCs-based biosensor showed obviously improved sensitivity of 99μA cm-2 mmol-1 L and 129μA cm-2 mmol-1 L for glucose at bare Au and platinized Au electrode, respectively. Also, the prepared PBNCs electrode is able to work well in the second generation biosensing mode.
4. We propose a novel protocol of chemical polymerization with magnetic separation/immobilization for high-sensitivity amperometric glucose biosensing. We prepared Fe3O4@Au core-shell hybrid magnetic nanoparticles (MNPs) using the method of chemical co-precipitation, then synthesized poly(1,6-hexanedithiol) (PHDT)-GOx-Fe3O4@Au nanocomposites using chemical polymerization, which were magnetically separated and immobilized on the magnetism electrode. This protocol was simple, time-saving, and highly efficient. The glucose biosensor showed a high detection sensitivity of 110μA cm-2 mmol-1L, detection limit of 0.3μmol L-1, S/N=3).
5. We propose electrosynthesized PHDT as a new matrix to immobilize AuNPs and anti-body for performance-enhanced piezoelectric immunosensing. Using the EQCM, we investigated the process and mechanism for HDT electropolymerzation, and estimated the coverage of AuNPs and the nano enhancement effect theoretically and experimentally. We comparatively examined the adsorption of anti-human immunoglobulin G (anti-hIgG) and the subsequent immunoreaction with immunoglobulin G (hIgG). Compared with the electrode modified with HDT self-assembled monolayer (SAM), the PHDT modified electrode showed better stability and can be constructed more time-effectively, demonstrating that the novel thiol-polymer materials may become a useful alternative to thiol-based SAM and find wide biosensing applications.
6. Novel PDA-PtNPs-anti-hIgG nanocomposites were prepared using chemical synthesis, which present satisfactory immuno-recognition efficiency and strong catalysis ability to electrochemical reduction of H2O2 for high performance sandwich-type amperometric immunosensing. The immunosensor has a limit of detection of 0.018 ng mL-1, as well as good reproducibility, stability, regeneration ability, specificity, and satisfactory feasibility for assay in clinical human serum samples.
7. Chemical oxidation was used to prepare PDA-PtNPs-GOx nanocomposites, on the surface of which AuNPs were biochemically prepared from reduction of HAuCl4 by the entrapped GOx-generated H2O2 to effectively immobilize antibody and GOx, yielding GOxads/antibody/AuNPs/PDA-PtNPs-GOx PBNCs for high performance sandwich-type amperometric immunosensing. The thus-prepared PBNCs feature high load and activity of the enzyme label immobilized both in the interior and on the surface for ultrasensitive signal readout. Using p-benzoquinone/hydroquinone as the mediator, the proposed immunosensor could detect the target antigen of hIgG down to a concentration of 2 pg mL-
8. The AuNPs/PDA-PtNPs-GOx nanocomposites were cast onto an Au electrode to covalently bind a thiolated aptamer for thrombin, and a "signal-off'electrochemical aptasensor was thus constructed based on signal output of the GOx label. The binding of thrombin with the captured aptamer obviously blocked the mass-transfer inside the film on the electrode, leading to the suppression of the enzymatic reaction on the electrode and the decrease of the oxidation current of EG-H2O2. The thus-prepared aptasensor could detect thrombin down to a concentration of 0.1 nmol L/-1.
9. We propose a novel sandwich-type amperometric aptasensor using aptamer-wrapped single-walled carbon nanotubes (SWCNTs) as an amplification platform and the magnetic separation/immobilization technique to effectively improve the sensitivity. In a suspension containing Fe3O4 MNPs modified with thrombin aptamerⅠ(aptamer@MNPs) and SWCNTs modified with thrombin aptamerⅡ(aptamer@SWCNTs), the target thrombin was added to yield the sandwich-type composites of aptamer I@MNPs-thrombin-aptamerⅡ@SWCNTs. The composites were magnetically separated and collected onto the surface of a magnetism Au electrode, methylene blue was added to detach the aptamerⅡfrom SWCNTs via its stronger interaction with SWCNTs than aptamerⅡdoes, and the methylene blue-adsorbed SWCNTs modified electrode was obtained after magnetically removing the aptamer I@MNPs-thrombin-aptamerⅡcomposites. Based on the differential pulse voltammetric detection of methylene blue, the thus-prepared aptasensor could detect thrombin down to a concentration of 3 pmol L-1.
引文
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