催化共振散射光谱法测定α-淀粉酶、过氧化氢和葡萄糖
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摘要
第一部分:绪论
介绍了共振散射技术理论。综述了近年来共振散射技术在分析化学中的应用,介绍了α-淀粉酶、过氧化氢和葡萄糖的分析进展。
第二部分:可溶性淀粉的共振散射光谱研究及其在α-淀粉酶活力测定中的应用
在0.8mg/ml聚丙烯酰胺(PAM)存在条件下,可获得较稳定的平均粒径为317nm的淀粉微粒。它在423nm处有一较强的共振散射峰。在50℃,pH为5.3的磷酸盐缓冲溶液中,淀粉微粒被α-淀粉酶(α-amylase,AMS)催化水解成小分子,导致423nm处共振散射峰强度降低。在选定条件下,AMS酶活力浓度在0.00835～0.501μg/ml(或0.00138-0.0827U/ml)范围内与共振散射强度降低值△I呈现良好的线性关系,其线性回归方程为△I=155C+13,相关系数R=0.9857。本法应用于人体唾液α-淀粉酶活力测定,结果满意。
第三部分:HRP酶催化-阳离子表面活性剂缔合微粒共振散射光谱法测定痕量过氧化氢
在醋酸缓冲溶液中,辣根过氧化物酶(HRP)可以催化H2O2与过量的I-反应生成I3-,I3-分别与阳离子表面活性剂TDMBA、DDAC、CTAB、ODC、CPCM、DPC和TBAB等形成缔合物微粒TDMBA-I3、DDAC-I3、CTAB-I3、ODC-I3、CPCM-I3、DPC-I3和TBAB-I3,该缔合物微粒均在460nm处有一个共振散射峰。过氧化氢的浓度在0.17～172.8、0.432～172.8、1.728～259.2、1.728～86.4、1.728～216、0.864～259.2和0.864～86.4×10-7mol/L范围内分别与各体系在460nm处的共振光谱强度呈线性关系。其检出限分别为0.086、0.34、1.72、0.46、0.36、0.64和0.44×10-7mol/L。本法应用于过氧化氢测定,结果满意。
第四部分:双酶催化-阳离子表面活性剂缔合微粒共振散射光谱法测定痕量葡萄糖
在醋酸缓冲溶液中,葡萄糖(G)在葡萄糖氧化酶(GOD)的作用下生成葡糖糖酸和H2O2,HRP可以催化H2O2与过量的I-反应生成I3-,I3-分别与阳离子表面活性剂十四烷基苄基二甲基氯化铵(TDMBA)、氯代十六烷基吡啶(CPCM)和十二烷基苄基二甲基氯化铵(DDAC)形成缔合物微粒TDMBA-I3、CPCM-I3和DDAC-I3,该缔合物微粒分别在460nm、540nm和430nm处有一个共振散射峰。对于TDMBA、CPCM和DDAC体系,葡萄糖的浓度分别在0.2～20×10-7mol/L、0.2～60×10-7mol/L和0.2～40×10-7mol/L范围内与其在最强的共振散射峰处的共振散射强度呈线性关系,其检出限分别为0.0856×10-7mol/L、0.118×10-7mol/L和0.096×10-7mol/L。将TDMAB体系应用于人血清中葡萄糖的测定,结果满意。
Part I Introduction
Summarized the application of resonance scattering technology in analytical chemistry. The application of resonance scattering technology in analytical chemistry in resent years was summarized. The analytical progress ofα-amylase, H2O2 and glucose were introduced. Two hundred and forty-three references were quoted.
Part II Resonance scattering spectra of starch and its application to assay ofα-amylase activity
In presence of 0.8mg/mL polyacrylamide(PAM), starch forms stable microparticles that the average diameter is about 317nm. It exhibits a strong resonance scattering peak at 423nm. In pH=5.3 phosphate buffer solution at 50℃, starch microparticles is catalyzed and hydrolysised into small molecules byα-amylase, which leads the resonance scattering intensity at 423nm to decrease greatly. Under proper experimental conditions, the decreased resonance scattering intensity is well linear to the concentration ofα-amylase in the range of 0.00835~0.501μg/ml (or 0.00138~0.0827U/ml), and the regression equation and the correlation coefficient are△I = 155C + 13 and 0.9857, respectively. The method is used to the determination ofα-amylase activity in human saliva, with satisfactory results.
Part III HRP catalytic resonance scattering spectral determination of trace hydrogen peroxide using cationic Surfactant association particle
In acetic acid buffer solution, horseradish peroxidase (HRP) may catalyze H2O2 oxidation of excess I- to form I3-, the I3- combined with cationic surfactant (CS), such as tetradecyl dimethylbenzyl ammonium chloride (TDMAB), dodecyl dimethylbenzyl ammonium chloride(DDAC), cetyltrimethyl ammonium bromide (CTAB), dimethyl benzyl ammonium chloride(ODAC), cetylpyridinium chloride (CPCM), chlorinated dodecyl pyridine(DPC) and terabutyl ammonium iodide(TBAB) to produce association microparticles TDMBA-I3、D DAC-I3、CTAB-I3、ODC-I3、CPCM-I3、DPC-I3 and TBAB-I3, which exhibit the strongest resonance scattering peak at 460 nm, the hydrogen peroxide concentration in the range of 0.17～172.8、0.432～172.8、1.728～259.2、1.728～86.4、1.728～216、0.864～259.2 and 0.864～86.4×10-7mol?L-1, was linear to the intensity at 460 nm,with a detection limit of 0.086、0.34、1.72、0.46、0.36、0.64 and 0.44×10-7mol?L-1 respectively. The method is used to the determination of hydrogen peroxide in real samples, with satisfactory results.
Part IV A highly sensitive and selective resonance scattering spectral probe for glucose based on GOD and HRP catalytic effects
In acetic acid buffer solution, glucose (G) can be oxidized to gluconic acid and H2O2 in the presence of glucose oxidase (GOD). The H2O2 oxidized excess I- catalytically to form I3- in the presence of horseradish peroxidase (HRP). The I3- combined with cationic surfactant (CS) such as tetradecyl dimethylbenzyl ammonium chloride (TDMAB), cetylpyridinium chloride(CPCM) and dodecyl dimethylbenzyl ammonium chloride (DDAC) to produce TDMBA-I3, CPCM-I3 and DDAC-I3 association particles that exhibited the strongest resonance scattering (RS) peak at 460 nm, 540 nm and 430 nm, respectively. The G concentration in the range of 0.2~20×10-7 mol/L, 0.2~60×10-7 mol/L and 0.2~40×10-7 mol/L was linear to their RS intensity at the strongest RS wavelength, with a detection limit of 8.6×10-9 mol/L, 1.2×10-8 mol/L and 9.6×10-9 mol/L respectively. The TDMAB catalytic RS method was used to the determination of glucose in human serum, with satisfactory results.
介绍了共振散射技术理论。综述了近年来共振散射技术在分析化学中的应用,介绍了α-淀粉酶、过氧化氢和葡萄糖的分析进展。
第二部分:可溶性淀粉的共振散射光谱研究及其在α-淀粉酶活力测定中的应用
在0.8mg/ml聚丙烯酰胺(PAM)存在条件下,可获得较稳定的平均粒径为317nm的淀粉微粒。它在423nm处有一较强的共振散射峰。在50℃,pH为5.3的磷酸盐缓冲溶液中,淀粉微粒被α-淀粉酶(α-amylase,AMS)催化水解成小分子,导致423nm处共振散射峰强度降低。在选定条件下,AMS酶活力浓度在0.00835～0.501μg/ml(或0.00138-0.0827U/ml)范围内与共振散射强度降低值△I呈现良好的线性关系,其线性回归方程为△I=155C+13,相关系数R=0.9857。本法应用于人体唾液α-淀粉酶活力测定,结果满意。
第三部分:HRP酶催化-阳离子表面活性剂缔合微粒共振散射光谱法测定痕量过氧化氢
在醋酸缓冲溶液中,辣根过氧化物酶(HRP)可以催化H2O2与过量的I-反应生成I3-,I3-分别与阳离子表面活性剂TDMBA、DDAC、CTAB、ODC、CPCM、DPC和TBAB等形成缔合物微粒TDMBA-I3、DDAC-I3、CTAB-I3、ODC-I3、CPCM-I3、DPC-I3和TBAB-I3,该缔合物微粒均在460nm处有一个共振散射峰。过氧化氢的浓度在0.17～172.8、0.432～172.8、1.728～259.2、1.728～86.4、1.728～216、0.864～259.2和0.864～86.4×10-7mol/L范围内分别与各体系在460nm处的共振光谱强度呈线性关系。其检出限分别为0.086、0.34、1.72、0.46、0.36、0.64和0.44×10-7mol/L。本法应用于过氧化氢测定,结果满意。
第四部分:双酶催化-阳离子表面活性剂缔合微粒共振散射光谱法测定痕量葡萄糖
在醋酸缓冲溶液中,葡萄糖(G)在葡萄糖氧化酶(GOD)的作用下生成葡糖糖酸和H2O2,HRP可以催化H2O2与过量的I-反应生成I3-,I3-分别与阳离子表面活性剂十四烷基苄基二甲基氯化铵(TDMBA)、氯代十六烷基吡啶(CPCM)和十二烷基苄基二甲基氯化铵(DDAC)形成缔合物微粒TDMBA-I3、CPCM-I3和DDAC-I3,该缔合物微粒分别在460nm、540nm和430nm处有一个共振散射峰。对于TDMBA、CPCM和DDAC体系,葡萄糖的浓度分别在0.2～20×10-7mol/L、0.2～60×10-7mol/L和0.2～40×10-7mol/L范围内与其在最强的共振散射峰处的共振散射强度呈线性关系,其检出限分别为0.0856×10-7mol/L、0.118×10-7mol/L和0.096×10-7mol/L。将TDMAB体系应用于人血清中葡萄糖的测定,结果满意。
Part I Introduction
Summarized the application of resonance scattering technology in analytical chemistry. The application of resonance scattering technology in analytical chemistry in resent years was summarized. The analytical progress ofα-amylase, H2O2 and glucose were introduced. Two hundred and forty-three references were quoted.
Part II Resonance scattering spectra of starch and its application to assay ofα-amylase activity
In presence of 0.8mg/mL polyacrylamide(PAM), starch forms stable microparticles that the average diameter is about 317nm. It exhibits a strong resonance scattering peak at 423nm. In pH=5.3 phosphate buffer solution at 50℃, starch microparticles is catalyzed and hydrolysised into small molecules byα-amylase, which leads the resonance scattering intensity at 423nm to decrease greatly. Under proper experimental conditions, the decreased resonance scattering intensity is well linear to the concentration ofα-amylase in the range of 0.00835~0.501μg/ml (or 0.00138~0.0827U/ml), and the regression equation and the correlation coefficient are△I = 155C + 13 and 0.9857, respectively. The method is used to the determination ofα-amylase activity in human saliva, with satisfactory results.
Part III HRP catalytic resonance scattering spectral determination of trace hydrogen peroxide using cationic Surfactant association particle
In acetic acid buffer solution, horseradish peroxidase (HRP) may catalyze H2O2 oxidation of excess I- to form I3-, the I3- combined with cationic surfactant (CS), such as tetradecyl dimethylbenzyl ammonium chloride (TDMAB), dodecyl dimethylbenzyl ammonium chloride(DDAC), cetyltrimethyl ammonium bromide (CTAB), dimethyl benzyl ammonium chloride(ODAC), cetylpyridinium chloride (CPCM), chlorinated dodecyl pyridine(DPC) and terabutyl ammonium iodide(TBAB) to produce association microparticles TDMBA-I3、D DAC-I3、CTAB-I3、ODC-I3、CPCM-I3、DPC-I3 and TBAB-I3, which exhibit the strongest resonance scattering peak at 460 nm, the hydrogen peroxide concentration in the range of 0.17～172.8、0.432～172.8、1.728～259.2、1.728～86.4、1.728～216、0.864～259.2 and 0.864～86.4×10-7mol?L-1, was linear to the intensity at 460 nm,with a detection limit of 0.086、0.34、1.72、0.46、0.36、0.64 and 0.44×10-7mol?L-1 respectively. The method is used to the determination of hydrogen peroxide in real samples, with satisfactory results.
Part IV A highly sensitive and selective resonance scattering spectral probe for glucose based on GOD and HRP catalytic effects
In acetic acid buffer solution, glucose (G) can be oxidized to gluconic acid and H2O2 in the presence of glucose oxidase (GOD). The H2O2 oxidized excess I- catalytically to form I3- in the presence of horseradish peroxidase (HRP). The I3- combined with cationic surfactant (CS) such as tetradecyl dimethylbenzyl ammonium chloride (TDMAB), cetylpyridinium chloride(CPCM) and dodecyl dimethylbenzyl ammonium chloride (DDAC) to produce TDMBA-I3, CPCM-I3 and DDAC-I3 association particles that exhibited the strongest resonance scattering (RS) peak at 460 nm, 540 nm and 430 nm, respectively. The G concentration in the range of 0.2~20×10-7 mol/L, 0.2~60×10-7 mol/L and 0.2~40×10-7 mol/L was linear to their RS intensity at the strongest RS wavelength, with a detection limit of 8.6×10-9 mol/L, 1.2×10-8 mol/L and 9.6×10-9 mol/L respectively. The TDMAB catalytic RS method was used to the determination of glucose in human serum, with satisfactory results.
引文
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[16] J. Y. Wang, Z. L. Zhao. Enzymatic determination of glucose by optical-fiber sensor sequential injection renewable surface spectrophotometry. Chemical Research in Chinese Universities,2006,22(3):287-291.
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