新型流动注射化学发光生物传感器的研制
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摘要
本论文主要是将流动注射化学发光分析技术(FI-CL)与纳米材料相结合构筑生物传感器,提高传感器的灵敏度、选择性、分析速度和自动化程度,对不同的目标分析物进行了测定,为疾病的临床早期诊断提供了新的检测检验方法。本文着重进行了以下几方面的研究:
1.研制了一种新型的化学发光双酶传感器,采用流动注射分析方法快速检测样品中胆固醇的含量。该传感器首先将金纳米粒子(GNPs)包覆在介孔SBA-15上制备金/SBA-15复合材料,然后用硅烷化试剂将金/SBA-15固定在预处理的玻璃微珠上,用于吸附固定胆固醇氧化酶(COD)和辣根过氧化物酶(HRP),构建基于介孔通道的双酶生物传感器,用于胆固醇的测定。胆固醇在COD的催化下生成H2O2,H2O2在HRP的催化作用下与鲁米诺发生反应,并产生化学发光信号。以介孔材料作为酶的固定载体,将介孔双酶催化反应通道引入流动注射化学发光反应中,增加了化学发光反应的接触面积,从而使发光效率大大提高。另外,介孔SBA-15与金纳米粒子的结合也使酶的固定效率大大提高,双酶传感器检测胆固醇的线性范围是1×10-6 ~ 1×10-3mol/L,检测限为5×10-7 mol/L(3σ)。将该方法用于人体血清样品中胆固醇的测定,结果准确可靠。
2.研制了一种用于葡萄糖快速检测的流动注射化学发光双酶传感器。该传感器将掺杂金纳米粒子的壳聚糖膜包覆在硅烷化试剂预处理的玻璃微珠上,用于吸附固定葡萄糖氧化酶(GOD)和辣根过氧化物酶。葡萄糖在GOD的催化下发生氧化反应生成H2O2,生成的H2O2在HRP的催化作用下与鲁米诺发生反应,并产生化学发光信号。实验表明,壳聚糖中掺杂的GNPs不仅能够有效的吸附酶分子并保持其生物活性,还对Luminol-H2O2-HRP化学发光体系具有增敏作用。通过化学发光光谱和紫外光谱表征,详细研究了固定化GNPs增强Luminol-H2O2-HRP体系的化学发光机理。在优化的实验条件下,该传感器对葡萄糖检测的线性范围为0.01~6.0mmol/L,检测限为5.0μmol/L(3σ)。将该方法用于临床血清样品中葡萄糖含量的测定,获得了满意的结果。
3.结合流动注射化学发光分析技术,发展了一种新型的同时检测两组分肿瘤标志物的免疫分析方法。该法以分别包被了甲胎蛋白单克隆抗体(AFP)和癌胚抗原单克隆抗体(CEA)的微孔板作为免疫反应器,基于夹心式的免疫反应方式,结合流动注射化学发光分析技术,设计了一种简便的、可实现两组分甚至多组分免疫分析物同时检测的新型可移动式免疫流通检测池。该免疫反应体系中以HRP作为酶标二抗的标记物,结合Luminol-H2O2-HRP化学发光体系实现对酶标记物的检测,通过控制移动式免疫流通检测池在检测窗口的位置来实现对两组分肿瘤标志物的同时免疫检测。在最佳检测条件下,得到AFP的线性范围为1.25~50ng/mL,检测限为1.06ng/mL(3σ);CEA的线性范围为1.25~40ng/mL,检测限为1.00ng/mL(3σ)。该免疫分析法已实现了两组分免疫分析物的同时检测,通过进一步优化实验方案,有望实现多组分免疫分析体系的自动化检测,使对肿瘤标志物的检测更加快速、简便。
4.在酸性介质中,高锰酸钾能氧化穿心莲内酯发生化学发光反应,而甲醛的存在可使发光强度增强,在体系中加入表面活性剂十六烷基三甲基溴化铵可以进一步增强该体系的化学发光强度。据此建立了一种流动注射化学发光测定穿心莲内酯的新方法。在优化的实验条件下,该方法对穿心莲内酯的检测线性范围为5.0×10-6~2.5×10-4g/mL,检出限为1.0×10-6g/mL,相对标准偏差为0.9%(n=12,c=1.0×10-5g/mL)。样品测量通量为150/小时。该法用于药物中穿心莲内酯含量的测定,结果令人满意。同时,结合荧光光谱和化学发光光谱表征技术,对高锰酸钾-甲醛-穿心莲内酯体系化学发光反应机理进行了进一步探讨。该方法的建立为穿心莲内酯的快速测定提供了一种简便、快速、高效的分析方法。
The main idea of the research was to develop the sensitive biosensors based onthe combination of flow injection chemiluminescence (FI-CL) and some novelnanoparticles. It provided the alternate ways for the clinical diagnoses of manydiseasesatearlystage.Theexperimentalprotocolscouldbesummarized asfollows:
1.Anovelflowinjectionchemiluminescence bienzyme-channelingbiosensorwasdeveloped for the determination of cholesterol. The biosensor was constructed byentrappingcholesterol oxidase (COD) and horseradish peroxidase (HRP) in thehybridmaterialswhich was synthesized bygold nanoparticles andmesopores silica structuresSBA-15 (GNPs/SBA-15). In the presence of cholesterol, the enzymatic reaction ofCOD-cholesterol-dissolved oxygen system generated hydrogen peroxide in thebienzyme-entrapped mesopores, which was immediately catalyzed by the sameentrapped HRP to lead to a sensitive and fast CL response of Luminol-H2O2-HRP.With well-ordered hexagonal mesoporous silica structures, SBA-15 mesoporousmaterials could not only adsorb COD and HRP firmly, but also enlarge CL reactioninterface because of the large specific surface area. The doped gold nanoparticles inSBA-15increasedtheabsorptionefficiencyofenzymemoleculeslargelyandhelpedtokeep bioactivity of enzyme molecules. Under the optimized experimental conditions,the detection limit of cholesterol was down to 5×10?7 mol/L (3σ) with a very widelinear range from 1.0×10?6 to 1×10?3 mol/L. The method has been satisfactorilyappliedtothedeterminationofthe cholesterollevel inserum samples.Theconstructedbienzyme channeling biosensor provided a strategy for CL detection of oxidasesubstratesbyusingthemesoporous materials.
2. Bienzymatic biosensor for the determination of glucose by flow injectionchemiluminescence detection was proposed. Hybrids of Gold nanoparticles (GNPs) and chitosan were chosen as the immobilization matrix of glucose oxidase (GOD) andhorseradish peroxidase to fabricate the biosensors with silane-pretreated glassmicrobeads. After the enzyme catalyzing oxidation of glucose in GOD biosensor, theproduced H2O2 flowed into HRP biosensor to react with luminol. The doped GNPs inchitosan were found to enhance the classical CL reaction of Luminol-H2O2-HRP. TheCLenhancement was investigated indetail byCLandUV-visiblespectrum.Undertheoptimizedexperimental conditions, glucosecouldbe determinedinalinear rangefrom0.01 to 6.0 mmol/L with a detection limit of 5.0μmol/L at 3σ. The accuracy of theproposed method was examined by detecting the glucose level in four clinical serumsamples from the hospital. The proposed method provides a new alternative todetermineglucose.
3. A novel flow-through chemiluminescent immunosensor was designed for thesimultaneous detection ofα-fetoprotein (AFP) and carcinoembryonic antigen (CEA).Two transparent microtubes immobilized with monoclonal antibodies were set in amobile flow cell to fabricate the multi-analyte immunoassay channels. Based on asandwich immunoassay format, two mixtures of the sample antigens andcorresponding horseradish peroxidase labeled antibodies were introduced into thechannels for on-line incubation. Upon injection of luminol and H2O2, the different CLsignals from the two channels were sequentially detected by moving the handle of themobile flow cell. Under the optimal experimental conditions, AFP and CEA could bedetected inthelinear ranges of1.25 ~50 and 1.25~40ng/mLwithdetectionlimits of1.06 and 1.0 ng/mL, respectively. The accuracy of the proposed system was assessedby human serum samples. The novel multi-analyte strategy provided an automated,simple,andlow-costapproachwithoutusingofexpensivearraydetector.
4. Asimple flow injection chemiluminescence procedure for the determination ofandrographolide (AD) was proposed. The method was based on the CL emittingreaction between andrographolide and potassium permanganate in acidic medium withformaldehyde as the CL enhancer. The surfactant of cetyl-trimethyl ammoniumbromide (CTAB) could further enhance the signal of the above CLsystem. Under theoptimized experimental conditions, andrographolide could be determined in a linearrange from 5.0×10-6 to 2.5×10-4 g/mLwith a correlation coefficient of 0.999 (n = 7)and a detection limit of 1.0×10-6 g/mL. The relative standard deviation of theproposed method was calculated as 0.9% from 12 repetitive injections of 1.0×10-5g/mL andrographolide. The sample throughput was 150/h. The method has been satisfactorily applied to the determination of andrographolide in pharmaceuticalformulations. The result was shown that there was no obvious interference from thecommon medicaments. In addition, the CL reaction mechanism of KMnO4-HCHO-ADsystem was also explored by the fluorescence and CL spectrum. The proposed methodprovided an alternative strategy for the rapid determination of andrographolide.
1.研制了一种新型的化学发光双酶传感器,采用流动注射分析方法快速检测样品中胆固醇的含量。该传感器首先将金纳米粒子(GNPs)包覆在介孔SBA-15上制备金/SBA-15复合材料,然后用硅烷化试剂将金/SBA-15固定在预处理的玻璃微珠上,用于吸附固定胆固醇氧化酶(COD)和辣根过氧化物酶(HRP),构建基于介孔通道的双酶生物传感器,用于胆固醇的测定。胆固醇在COD的催化下生成H2O2,H2O2在HRP的催化作用下与鲁米诺发生反应,并产生化学发光信号。以介孔材料作为酶的固定载体,将介孔双酶催化反应通道引入流动注射化学发光反应中,增加了化学发光反应的接触面积,从而使发光效率大大提高。另外,介孔SBA-15与金纳米粒子的结合也使酶的固定效率大大提高,双酶传感器检测胆固醇的线性范围是1×10-6 ~ 1×10-3mol/L,检测限为5×10-7 mol/L(3σ)。将该方法用于人体血清样品中胆固醇的测定,结果准确可靠。
2.研制了一种用于葡萄糖快速检测的流动注射化学发光双酶传感器。该传感器将掺杂金纳米粒子的壳聚糖膜包覆在硅烷化试剂预处理的玻璃微珠上,用于吸附固定葡萄糖氧化酶(GOD)和辣根过氧化物酶。葡萄糖在GOD的催化下发生氧化反应生成H2O2,生成的H2O2在HRP的催化作用下与鲁米诺发生反应,并产生化学发光信号。实验表明,壳聚糖中掺杂的GNPs不仅能够有效的吸附酶分子并保持其生物活性,还对Luminol-H2O2-HRP化学发光体系具有增敏作用。通过化学发光光谱和紫外光谱表征,详细研究了固定化GNPs增强Luminol-H2O2-HRP体系的化学发光机理。在优化的实验条件下,该传感器对葡萄糖检测的线性范围为0.01~6.0mmol/L,检测限为5.0μmol/L(3σ)。将该方法用于临床血清样品中葡萄糖含量的测定,获得了满意的结果。
3.结合流动注射化学发光分析技术,发展了一种新型的同时检测两组分肿瘤标志物的免疫分析方法。该法以分别包被了甲胎蛋白单克隆抗体(AFP)和癌胚抗原单克隆抗体(CEA)的微孔板作为免疫反应器,基于夹心式的免疫反应方式,结合流动注射化学发光分析技术,设计了一种简便的、可实现两组分甚至多组分免疫分析物同时检测的新型可移动式免疫流通检测池。该免疫反应体系中以HRP作为酶标二抗的标记物,结合Luminol-H2O2-HRP化学发光体系实现对酶标记物的检测,通过控制移动式免疫流通检测池在检测窗口的位置来实现对两组分肿瘤标志物的同时免疫检测。在最佳检测条件下,得到AFP的线性范围为1.25~50ng/mL,检测限为1.06ng/mL(3σ);CEA的线性范围为1.25~40ng/mL,检测限为1.00ng/mL(3σ)。该免疫分析法已实现了两组分免疫分析物的同时检测,通过进一步优化实验方案,有望实现多组分免疫分析体系的自动化检测,使对肿瘤标志物的检测更加快速、简便。
4.在酸性介质中,高锰酸钾能氧化穿心莲内酯发生化学发光反应,而甲醛的存在可使发光强度增强,在体系中加入表面活性剂十六烷基三甲基溴化铵可以进一步增强该体系的化学发光强度。据此建立了一种流动注射化学发光测定穿心莲内酯的新方法。在优化的实验条件下,该方法对穿心莲内酯的检测线性范围为5.0×10-6~2.5×10-4g/mL,检出限为1.0×10-6g/mL,相对标准偏差为0.9%(n=12,c=1.0×10-5g/mL)。样品测量通量为150/小时。该法用于药物中穿心莲内酯含量的测定,结果令人满意。同时,结合荧光光谱和化学发光光谱表征技术,对高锰酸钾-甲醛-穿心莲内酯体系化学发光反应机理进行了进一步探讨。该方法的建立为穿心莲内酯的快速测定提供了一种简便、快速、高效的分析方法。
The main idea of the research was to develop the sensitive biosensors based onthe combination of flow injection chemiluminescence (FI-CL) and some novelnanoparticles. It provided the alternate ways for the clinical diagnoses of manydiseasesatearlystage.Theexperimentalprotocolscouldbesummarized asfollows:
1.Anovelflowinjectionchemiluminescence bienzyme-channelingbiosensorwasdeveloped for the determination of cholesterol. The biosensor was constructed byentrappingcholesterol oxidase (COD) and horseradish peroxidase (HRP) in thehybridmaterialswhich was synthesized bygold nanoparticles andmesopores silica structuresSBA-15 (GNPs/SBA-15). In the presence of cholesterol, the enzymatic reaction ofCOD-cholesterol-dissolved oxygen system generated hydrogen peroxide in thebienzyme-entrapped mesopores, which was immediately catalyzed by the sameentrapped HRP to lead to a sensitive and fast CL response of Luminol-H2O2-HRP.With well-ordered hexagonal mesoporous silica structures, SBA-15 mesoporousmaterials could not only adsorb COD and HRP firmly, but also enlarge CL reactioninterface because of the large specific surface area. The doped gold nanoparticles inSBA-15increasedtheabsorptionefficiencyofenzymemoleculeslargelyandhelpedtokeep bioactivity of enzyme molecules. Under the optimized experimental conditions,the detection limit of cholesterol was down to 5×10?7 mol/L (3σ) with a very widelinear range from 1.0×10?6 to 1×10?3 mol/L. The method has been satisfactorilyappliedtothedeterminationofthe cholesterollevel inserum samples.Theconstructedbienzyme channeling biosensor provided a strategy for CL detection of oxidasesubstratesbyusingthemesoporous materials.
2. Bienzymatic biosensor for the determination of glucose by flow injectionchemiluminescence detection was proposed. Hybrids of Gold nanoparticles (GNPs) and chitosan were chosen as the immobilization matrix of glucose oxidase (GOD) andhorseradish peroxidase to fabricate the biosensors with silane-pretreated glassmicrobeads. After the enzyme catalyzing oxidation of glucose in GOD biosensor, theproduced H2O2 flowed into HRP biosensor to react with luminol. The doped GNPs inchitosan were found to enhance the classical CL reaction of Luminol-H2O2-HRP. TheCLenhancement was investigated indetail byCLandUV-visiblespectrum.Undertheoptimizedexperimental conditions, glucosecouldbe determinedinalinear rangefrom0.01 to 6.0 mmol/L with a detection limit of 5.0μmol/L at 3σ. The accuracy of theproposed method was examined by detecting the glucose level in four clinical serumsamples from the hospital. The proposed method provides a new alternative todetermineglucose.
3. A novel flow-through chemiluminescent immunosensor was designed for thesimultaneous detection ofα-fetoprotein (AFP) and carcinoembryonic antigen (CEA).Two transparent microtubes immobilized with monoclonal antibodies were set in amobile flow cell to fabricate the multi-analyte immunoassay channels. Based on asandwich immunoassay format, two mixtures of the sample antigens andcorresponding horseradish peroxidase labeled antibodies were introduced into thechannels for on-line incubation. Upon injection of luminol and H2O2, the different CLsignals from the two channels were sequentially detected by moving the handle of themobile flow cell. Under the optimal experimental conditions, AFP and CEA could bedetected inthelinear ranges of1.25 ~50 and 1.25~40ng/mLwithdetectionlimits of1.06 and 1.0 ng/mL, respectively. The accuracy of the proposed system was assessedby human serum samples. The novel multi-analyte strategy provided an automated,simple,andlow-costapproachwithoutusingofexpensivearraydetector.
4. Asimple flow injection chemiluminescence procedure for the determination ofandrographolide (AD) was proposed. The method was based on the CL emittingreaction between andrographolide and potassium permanganate in acidic medium withformaldehyde as the CL enhancer. The surfactant of cetyl-trimethyl ammoniumbromide (CTAB) could further enhance the signal of the above CLsystem. Under theoptimized experimental conditions, andrographolide could be determined in a linearrange from 5.0×10-6 to 2.5×10-4 g/mLwith a correlation coefficient of 0.999 (n = 7)and a detection limit of 1.0×10-6 g/mL. The relative standard deviation of theproposed method was calculated as 0.9% from 12 repetitive injections of 1.0×10-5g/mL andrographolide. The sample throughput was 150/h. The method has been satisfactorily applied to the determination of andrographolide in pharmaceuticalformulations. The result was shown that there was no obvious interference from thecommon medicaments. In addition, the CL reaction mechanism of KMnO4-HCHO-ADsystem was also explored by the fluorescence and CL spectrum. The proposed methodprovided an alternative strategy for the rapid determination of andrographolide.
引文
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