青霉素G的金标记免疫共振散射光谱分析
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摘要
第一部份:绪论
综述了金纳米微粒的制备、表征及其在生化分析中的应用;综述了近年来共振散射技术在生化分析中的应用。介绍了青霉素G的分析进展。
第二部份:痕量青霉素的金纳米标记免疫共振散射光谱快速检测
在pH8.0条件下,用粒径为9.0 nm的金纳米微粒标记兔抗青霉素获得青霉素金标免疫探针。在pH5.4磷酸氢二钠-柠檬酸缓冲溶液及聚乙二醇存在下,金标记兔抗青霉素与青霉素G产生特异性结合,并生成胶体金免疫复合物。在PEG作用下胶体金免疫复合物发生聚集,导致体系在580 nm处的共振散射峰增强。青霉素G浓度在7.5 ng.mL-1-1.65×103 ng.mL-1范围内与580 nm处的共振散射光强度增加值呈线性,方法的检出限为0.070 ng.mL-1。该法用于定量分析牛奶中的青霉素,简便快速,结果令人满意。
第三部份:痕量青霉素G的免疫纳米金催化共振散射光谱检测
小粒径的金、银和免疫纳米金粒子对氯金酸-盐酸羟胺这一反应具有较强的催化作用。基于金纳米微粒在580 nm处的共振散射效应及免疫纳米金的催化作用,可用它做检测技术研究该纳米反应在生化分析中的应用。我们用粒径为9.0 nm的金纳米微粒标记兔抗青霉素抗体。在pH5.4的柠檬酸-磷酸氢二钠缓冲溶液中,青霉素G与金标兔抗青霉素发生特异性结合生成胶体金免疫复合物,离心分离。以适量金标兔抗青霉素的上层清液做晶种,加入pH3.36盐酸-柠檬酸钠缓冲溶液、40μg.mL-1氯金酸和21.6μg.mL-1盐酸羟胺,利用金纳米粒子表面发生的自催化反应使晶种的粒径增大,在580 nm处共振散射强度增强。随着青霉素G浓度C(0.15-225 ng.mL-1)的增大,上层清液中免疫纳米金微粒数量降低,I580 nm值随之降低。△I580 nm与C成线性关系,其回归方程为△I580 nm=0.28C+5.16,检出限为0. 050 ng. mL-1。该法用于牛奶中青霉素G的检测,结果较好,其回收率在101.6-120.3%之间。
第四部份:免疫纳米金催化Cu(II)-葡萄糖反应的共振散射光谱研究及其分析应用
小粒径的金纳米粒子对酒石酸钾钠铜络合物-葡萄糖这一微粒反应具有较强的催化作用,其产物氧化亚铜-金复合纳米微粒在608 nm处存在较强的共振散射效应。将纳米金标记技术、共振散射光谱检测技术和纳米金催化作用有机地结合,可建立高灵敏、高选择性的生化分析新方法。用粒径为9.0 nm的金纳米微粒标记兔抗青霉素G抗体(RAPG)可获得青霉素G(PG)的光谱探针(AuRAPG)。在pH5.2的柠檬酸-磷酸氢二钠缓冲溶液中,PG与AuRAPG发生特异性结合生成胶体金免疫复合物,离心分离。取适量上层清液做晶种,在0.010 g.mL-1氢氧化钠-0.86 mg.mL-1硫酸铜-0.080 mg.mL-1的葡萄糖-水浴70℃条件下反应,反应形成粒径较大的氧化亚铜-金复合微粒,导致608 nm处共振散射强度I608 nm增强。随着PG浓度CPG(0.090-21.6 ng.mL-1)增大,上层清液中免疫纳米金微粒数量降低,I608 nm值降低。△I608 nm降低值与CPG成线性关系,其回归方程为△I608 nm=3.86C+1.1,检出限为0. 010 ng.mL-1。该法用于牛奶中PG的检测,结果较好,其回收率在100.6-109.2%之间。
PartⅠIntrodction
The preparation and identification of gold nanoparticles, the application of gold-labeled technology in biochemical analysis and the application of resonance scattering technology in analytical chemistry were introduced. The analytical progress of penicillin G was also reviewed.
PartⅡCombination nanogold resonance scattering effect with immunoreaction for assay of trace penicillin G
A new immunonanogold resonance scattering spectral assay was developed for the sensitive determination of penicillin G in milk sample, based on resonance scattering (RS) effect of the nanogold at 580 nm and the nanoglold labeled immunoreactions in the presence of pH 5.4 phosphate citric acid buffer solutions and the polythylene glycol (PEG). The resonance scattering intensity at 580 nm (I580 nm) enhanced greatly with penicillin G. The enhanced RS intensity△I580 nm was linear to the penicillin G concentration in the range of 7.5-1.7×103 ng.mL-1, with a detection limit of 0.78 ng.mL-1. The results indicate that the immunonanogold labeled RS spectral assay has a high specificity and good sensitivity for quantitative determination of penicillin G in raw milk sample.
PartⅢImmunonanogold Probe as Catalyst for HAuCl4-NH2OH Particle Reaction and Its Application to Resonance Scattering Assay of Trace Penicillin G
Both nanogold and nanosilver exhibit catalytic activity toward the HAuCl4-NH2OH particle reaction. Based on the catalytic effect of nanogold on the redox reaction and the gold particle resonance scattering (RS) effect at 580 nm, RS can be used to monitor the nanocatalytic reaction and utilized in bioanalytical assay. As a model, we used nanogold in size of 9 nm to label goat anti-rabbit penicillin G to obtain an immunonanogold probe for penicillin G. The immunoreactions between rabbit anti-penicillin G antibody labeled nanogold and penicillin G took place in pH 5.4 phosphate-citric acid buffer solutions, and after centrifugation, the supernatant of the nanogold-labeled immunoreaction was used as nanocatalyst for the redox reaction. After the immunonanogold catalytic reaction, the resonance scattering intensity enhanced at 580 nm (I580 nm). With the addition of penicillin G, the concentration of immunonanogold in the supernatant reduced linearly, which leaded the I580 nm to decrease accordingly. Under the optimal conditions, the decreased intensity ?I580 nm was inversely proportional to the penicillin G concentration in the range of 0.15-225 ng.mL-1, with a detection limit of 0.05 ng.mL-1 penicillin G. These results indicate that the immunonanogold-catalytic resonance scattering spectral assay offers good results for quantitative determination of penicillin G in raw milk sample, with the recovery between 101.6% and 120.3%.
PartⅣ
As the seeds, small size nanogold can initiate the particle reaction between Cu (II) and glucose. Based on the (Au) nucleus (Cu2O) shell complex nanoparticles resonance scattering (RS) effect at 608 nm, RS can be used to monitor the particle reaction and utilized in bioanalytical assay. As a model, we used nanogold in size of 9 nm to label rabbit anti-penicillin G (RAPG) to obtain an immunonanogold probe for penicillin G (PG). The immunoreactions between rabbit anti-penicillin G antibody labeled nanogold (AuRAPG) and PG took place in pH 5.2 phosphate-citric acid buffer solutions, and after centrifugation, the supernatant of the immunoreaction products were used as seeds for the reaction between Cu (II) and glucose. Then, the RS intensity enhanced at 608 nm (I608 nm). With the addition of PG, the concentration of AuRAPG in the supernatant reduced linearly, which leaded the I608 nm to decrease accordingly. Under the optimal conditions, the decreased RS intensity was inversely proportional to the PG concentration in the range of 0.09-21.6 ng.mL-1, with a detection limit of 0.01 ng.mL-1 PG. It is shown that the data produced here can be used to determine trace PG in raw milk sample, with the recovery between 100.6% and 109.2%.
综述了金纳米微粒的制备、表征及其在生化分析中的应用;综述了近年来共振散射技术在生化分析中的应用。介绍了青霉素G的分析进展。
第二部份:痕量青霉素的金纳米标记免疫共振散射光谱快速检测
在pH8.0条件下,用粒径为9.0 nm的金纳米微粒标记兔抗青霉素获得青霉素金标免疫探针。在pH5.4磷酸氢二钠-柠檬酸缓冲溶液及聚乙二醇存在下,金标记兔抗青霉素与青霉素G产生特异性结合,并生成胶体金免疫复合物。在PEG作用下胶体金免疫复合物发生聚集,导致体系在580 nm处的共振散射峰增强。青霉素G浓度在7.5 ng.mL-1-1.65×103 ng.mL-1范围内与580 nm处的共振散射光强度增加值呈线性,方法的检出限为0.070 ng.mL-1。该法用于定量分析牛奶中的青霉素,简便快速,结果令人满意。
第三部份:痕量青霉素G的免疫纳米金催化共振散射光谱检测
小粒径的金、银和免疫纳米金粒子对氯金酸-盐酸羟胺这一反应具有较强的催化作用。基于金纳米微粒在580 nm处的共振散射效应及免疫纳米金的催化作用,可用它做检测技术研究该纳米反应在生化分析中的应用。我们用粒径为9.0 nm的金纳米微粒标记兔抗青霉素抗体。在pH5.4的柠檬酸-磷酸氢二钠缓冲溶液中,青霉素G与金标兔抗青霉素发生特异性结合生成胶体金免疫复合物,离心分离。以适量金标兔抗青霉素的上层清液做晶种,加入pH3.36盐酸-柠檬酸钠缓冲溶液、40μg.mL-1氯金酸和21.6μg.mL-1盐酸羟胺,利用金纳米粒子表面发生的自催化反应使晶种的粒径增大,在580 nm处共振散射强度增强。随着青霉素G浓度C(0.15-225 ng.mL-1)的增大,上层清液中免疫纳米金微粒数量降低,I580 nm值随之降低。△I580 nm与C成线性关系,其回归方程为△I580 nm=0.28C+5.16,检出限为0. 050 ng. mL-1。该法用于牛奶中青霉素G的检测,结果较好,其回收率在101.6-120.3%之间。
第四部份:免疫纳米金催化Cu(II)-葡萄糖反应的共振散射光谱研究及其分析应用
小粒径的金纳米粒子对酒石酸钾钠铜络合物-葡萄糖这一微粒反应具有较强的催化作用,其产物氧化亚铜-金复合纳米微粒在608 nm处存在较强的共振散射效应。将纳米金标记技术、共振散射光谱检测技术和纳米金催化作用有机地结合,可建立高灵敏、高选择性的生化分析新方法。用粒径为9.0 nm的金纳米微粒标记兔抗青霉素G抗体(RAPG)可获得青霉素G(PG)的光谱探针(AuRAPG)。在pH5.2的柠檬酸-磷酸氢二钠缓冲溶液中,PG与AuRAPG发生特异性结合生成胶体金免疫复合物,离心分离。取适量上层清液做晶种,在0.010 g.mL-1氢氧化钠-0.86 mg.mL-1硫酸铜-0.080 mg.mL-1的葡萄糖-水浴70℃条件下反应,反应形成粒径较大的氧化亚铜-金复合微粒,导致608 nm处共振散射强度I608 nm增强。随着PG浓度CPG(0.090-21.6 ng.mL-1)增大,上层清液中免疫纳米金微粒数量降低,I608 nm值降低。△I608 nm降低值与CPG成线性关系,其回归方程为△I608 nm=3.86C+1.1,检出限为0. 010 ng.mL-1。该法用于牛奶中PG的检测,结果较好,其回收率在100.6-109.2%之间。
PartⅠIntrodction
The preparation and identification of gold nanoparticles, the application of gold-labeled technology in biochemical analysis and the application of resonance scattering technology in analytical chemistry were introduced. The analytical progress of penicillin G was also reviewed.
PartⅡCombination nanogold resonance scattering effect with immunoreaction for assay of trace penicillin G
A new immunonanogold resonance scattering spectral assay was developed for the sensitive determination of penicillin G in milk sample, based on resonance scattering (RS) effect of the nanogold at 580 nm and the nanoglold labeled immunoreactions in the presence of pH 5.4 phosphate citric acid buffer solutions and the polythylene glycol (PEG). The resonance scattering intensity at 580 nm (I580 nm) enhanced greatly with penicillin G. The enhanced RS intensity△I580 nm was linear to the penicillin G concentration in the range of 7.5-1.7×103 ng.mL-1, with a detection limit of 0.78 ng.mL-1. The results indicate that the immunonanogold labeled RS spectral assay has a high specificity and good sensitivity for quantitative determination of penicillin G in raw milk sample.
PartⅢImmunonanogold Probe as Catalyst for HAuCl4-NH2OH Particle Reaction and Its Application to Resonance Scattering Assay of Trace Penicillin G
Both nanogold and nanosilver exhibit catalytic activity toward the HAuCl4-NH2OH particle reaction. Based on the catalytic effect of nanogold on the redox reaction and the gold particle resonance scattering (RS) effect at 580 nm, RS can be used to monitor the nanocatalytic reaction and utilized in bioanalytical assay. As a model, we used nanogold in size of 9 nm to label goat anti-rabbit penicillin G to obtain an immunonanogold probe for penicillin G. The immunoreactions between rabbit anti-penicillin G antibody labeled nanogold and penicillin G took place in pH 5.4 phosphate-citric acid buffer solutions, and after centrifugation, the supernatant of the nanogold-labeled immunoreaction was used as nanocatalyst for the redox reaction. After the immunonanogold catalytic reaction, the resonance scattering intensity enhanced at 580 nm (I580 nm). With the addition of penicillin G, the concentration of immunonanogold in the supernatant reduced linearly, which leaded the I580 nm to decrease accordingly. Under the optimal conditions, the decreased intensity ?I580 nm was inversely proportional to the penicillin G concentration in the range of 0.15-225 ng.mL-1, with a detection limit of 0.05 ng.mL-1 penicillin G. These results indicate that the immunonanogold-catalytic resonance scattering spectral assay offers good results for quantitative determination of penicillin G in raw milk sample, with the recovery between 101.6% and 120.3%.
PartⅣ
As the seeds, small size nanogold can initiate the particle reaction between Cu (II) and glucose. Based on the (Au) nucleus (Cu2O) shell complex nanoparticles resonance scattering (RS) effect at 608 nm, RS can be used to monitor the particle reaction and utilized in bioanalytical assay. As a model, we used nanogold in size of 9 nm to label rabbit anti-penicillin G (RAPG) to obtain an immunonanogold probe for penicillin G (PG). The immunoreactions between rabbit anti-penicillin G antibody labeled nanogold (AuRAPG) and PG took place in pH 5.2 phosphate-citric acid buffer solutions, and after centrifugation, the supernatant of the immunoreaction products were used as seeds for the reaction between Cu (II) and glucose. Then, the RS intensity enhanced at 608 nm (I608 nm). With the addition of PG, the concentration of AuRAPG in the supernatant reduced linearly, which leaded the I608 nm to decrease accordingly. Under the optimal conditions, the decreased RS intensity was inversely proportional to the PG concentration in the range of 0.09-21.6 ng.mL-1, with a detection limit of 0.01 ng.mL-1 PG. It is shown that the data produced here can be used to determine trace PG in raw milk sample, with the recovery between 100.6% and 109.2%.
引文
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