阻抗编码微球液态生物芯片系统及其在检验医学中应用
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摘要
目的：为研制和开发不同阻抗编码微球液态生物芯片及其检测装置提供实验基础并进行科学论证。研究和探讨不同粒径阻抗编码免疫诊断微球定量分析技术及其在检验医学中应用。
方法和结果：1）推导球、包括微球阻抗理论计算公式，提出微球阻抗理论计算值与微球材料电阻率呈正比，与球粒径呈反比。2）制备三种不同粒径羧基化聚苯乙烯阻抗微球并进行表征分析，结果发现，微球阻抗稳定性保持至少1年，不同粒径和浓度的微球在溶液中沉降速度与微球粒径和微球浓度呈正比。3）借助血球计数仪可区分不同粒径阻抗编码微球；参考库尔特电阻抗原理，研制微球阻抗检测和区分技术和装置，并对三种不同粒径阻抗编码羧基化聚苯乙烯微球进行微孔两端电压变化信号检测，结果发现，阻抗编码微球粒径越大，微孔两端电压变化信号越强，为不同粒径微球阻抗编码技术提供可行性和科学性实验依据。4）将人甲胎蛋白（AFP）单克隆抗体，人胰岛素（INS）单克隆抗体和人肿瘤坏死因子α（TNFα）单克隆抗体分别标记在2微米，5微米和10微米粒径阻抗编码微球表面，借助流式细胞仪的微球表面荧光强度定量检测功能，建立不同粒径阻抗编码微球分别定量检测不同待测抗原方法和技术，并与酶联免疫吸附实验（ELISA）技术进行方法学性能比较，结果发现，不同粒径阻抗编码微球定量检测技术在检测重复性，检测准确性，最低检测限，和线性范围等方面均优于ELISA技术。5）用量子点标记的亲和素（B-QD）和生物素化的抗抗体（AAb-A）微球表面量子点荧光显示系统(AAb-AB-QD)取代传统的FITC标记的抗抗体微球表面荧光强度显示系统(AAb-FITC)，结果发现，检测同样浓度胰岛素微球表面量子点平均荧光强度（MFI）明显高于微球表面FITC平均荧光强度，变异系数也大于FITC荧光显示系统。6）采用三种不同粒径阻抗编码免疫诊断微球定量检测技术，分别定量检测临床确诊的肝癌患者血清AFP，2型糖尿病患者血清INS和创伤MODS患者血清TNFα等水平，结果表明均明显高于其正常对照组。
结论：不同粒径微球阻抗编码技术具有可行性和科学性，不同粒径阻抗编码免疫诊断微球定量检测待测抗原技术在方法学性能上优于ELISA技术，并可应用于临床检验诊断。阻抗编码微球液态生物芯片及其检测装置具有可行性和科学性，值得深入研究。
Objective:To research and develop different impedance encoded microspheres for liquid biochip and detection device to provide the experimental basis and scientific proof, we have conducted research and discussion of different impedance coding microsphere quantitative detection technology and its application in laboratory medicine.Methods and results:1) To put forward the theoretical values and microspheres impedance theory formula and the microsphere impedance resistivity is inversely proportional to their sizes.2) Preparing three kinds of different particle diameters of carboxyl polystyrene microspheres and impedance characterization analysis, it was found, microspheres impedance stability can maintain in more than1year, with different particle size and concentration of particles in solution settling velocity and particle size and concentration is proportional to the microspheres.3) With the assistance of blood counting instrument to distinguish between the different particle sizes of impedance encoded microspheres and reference to Kurt theory of electrical impedance, we have originated detecting and distinguishing technology and device. Three different particle sizes microspheres was detected for microporous ends voltage variation signal, as impedance coding microsphere size bigger, microporous both ends of the voltage variation signal is stronger, for different size impedance microsphere coding technology to provide a practical and scientific experimental basis.4) The human alpha fetal protein (AFP) monoclonal antibody, human insulin (INS) monoclonal antibodies and human tumor necrosis factor alpha (TNFa) monoclonal antibodies were marked at2,5and10micron diameter impedance coding microsphere surface. By surface fluorescence intensity quantitative detection function in flow cytometry, we set different diameter impedance coding microsphere quantitative detection of the antigen and enzyme linked immunosorbent assay (ELISA) technology methodology for performance comparison. Impedance coding microsphere quantitative detection technique is better than that of ELISA technology in the detection of repetitiveness, accuracy, lowest limit, linear range and etc..5) Using quantum dot labeled streptavidin (B-QD) and biotinylated antibody (AAb-A) microsphere surface Quantum Dots Fluorescent Display System (AAb-AB-QD) to replace the traditional FITC labeled antibodies microsphere surface fluorescence intensity display system (AAb-FITC), it was found detecting with the same concentration of insulin microsphere surface quantum dot fluorescence intensity (MFI) was significantly higher than that of the microsphere surface FITC average fluorescence intensity, coefficient of variation is greater than FITC fluorescent display system.6)Using three different sizes impedance encoding immune diagnose microballoons quantitative detection technology, respectively, for quantitative detection of clinically diagnosed serum AFP in patients with hepatocellular carcinoma, serum insulin in patients with type2diabetes mellitus and serum TNFa level in patients with MODS, the results show that were significantly higher than those in the normal control group. Conclusion:Different diameter impedance microsphere coding technology has feasibility and scientificity. It outperforms the ELISA technology in methodology of detecting antigen in quantity, and can be practice preliminary application in clinical diagnosis. Different diameter impedance encoded microspheres liquid biological chip and detection device has the feasibility and scientificity, worthy of further study.
方法和结果：1）推导球、包括微球阻抗理论计算公式，提出微球阻抗理论计算值与微球材料电阻率呈正比，与球粒径呈反比。2）制备三种不同粒径羧基化聚苯乙烯阻抗微球并进行表征分析，结果发现，微球阻抗稳定性保持至少1年，不同粒径和浓度的微球在溶液中沉降速度与微球粒径和微球浓度呈正比。3）借助血球计数仪可区分不同粒径阻抗编码微球；参考库尔特电阻抗原理，研制微球阻抗检测和区分技术和装置，并对三种不同粒径阻抗编码羧基化聚苯乙烯微球进行微孔两端电压变化信号检测，结果发现，阻抗编码微球粒径越大，微孔两端电压变化信号越强，为不同粒径微球阻抗编码技术提供可行性和科学性实验依据。4）将人甲胎蛋白（AFP）单克隆抗体，人胰岛素（INS）单克隆抗体和人肿瘤坏死因子α（TNFα）单克隆抗体分别标记在2微米，5微米和10微米粒径阻抗编码微球表面，借助流式细胞仪的微球表面荧光强度定量检测功能，建立不同粒径阻抗编码微球分别定量检测不同待测抗原方法和技术，并与酶联免疫吸附实验（ELISA）技术进行方法学性能比较，结果发现，不同粒径阻抗编码微球定量检测技术在检测重复性，检测准确性，最低检测限，和线性范围等方面均优于ELISA技术。5）用量子点标记的亲和素（B-QD）和生物素化的抗抗体（AAb-A）微球表面量子点荧光显示系统(AAb-AB-QD)取代传统的FITC标记的抗抗体微球表面荧光强度显示系统(AAb-FITC)，结果发现，检测同样浓度胰岛素微球表面量子点平均荧光强度（MFI）明显高于微球表面FITC平均荧光强度，变异系数也大于FITC荧光显示系统。6）采用三种不同粒径阻抗编码免疫诊断微球定量检测技术，分别定量检测临床确诊的肝癌患者血清AFP，2型糖尿病患者血清INS和创伤MODS患者血清TNFα等水平，结果表明均明显高于其正常对照组。
结论：不同粒径微球阻抗编码技术具有可行性和科学性，不同粒径阻抗编码免疫诊断微球定量检测待测抗原技术在方法学性能上优于ELISA技术，并可应用于临床检验诊断。阻抗编码微球液态生物芯片及其检测装置具有可行性和科学性，值得深入研究。
Objective:To research and develop different impedance encoded microspheres for liquid biochip and detection device to provide the experimental basis and scientific proof, we have conducted research and discussion of different impedance coding microsphere quantitative detection technology and its application in laboratory medicine.Methods and results:1) To put forward the theoretical values and microspheres impedance theory formula and the microsphere impedance resistivity is inversely proportional to their sizes.2) Preparing three kinds of different particle diameters of carboxyl polystyrene microspheres and impedance characterization analysis, it was found, microspheres impedance stability can maintain in more than1year, with different particle size and concentration of particles in solution settling velocity and particle size and concentration is proportional to the microspheres.3) With the assistance of blood counting instrument to distinguish between the different particle sizes of impedance encoded microspheres and reference to Kurt theory of electrical impedance, we have originated detecting and distinguishing technology and device. Three different particle sizes microspheres was detected for microporous ends voltage variation signal, as impedance coding microsphere size bigger, microporous both ends of the voltage variation signal is stronger, for different size impedance microsphere coding technology to provide a practical and scientific experimental basis.4) The human alpha fetal protein (AFP) monoclonal antibody, human insulin (INS) monoclonal antibodies and human tumor necrosis factor alpha (TNFa) monoclonal antibodies were marked at2,5and10micron diameter impedance coding microsphere surface. By surface fluorescence intensity quantitative detection function in flow cytometry, we set different diameter impedance coding microsphere quantitative detection of the antigen and enzyme linked immunosorbent assay (ELISA) technology methodology for performance comparison. Impedance coding microsphere quantitative detection technique is better than that of ELISA technology in the detection of repetitiveness, accuracy, lowest limit, linear range and etc..5) Using quantum dot labeled streptavidin (B-QD) and biotinylated antibody (AAb-A) microsphere surface Quantum Dots Fluorescent Display System (AAb-AB-QD) to replace the traditional FITC labeled antibodies microsphere surface fluorescence intensity display system (AAb-FITC), it was found detecting with the same concentration of insulin microsphere surface quantum dot fluorescence intensity (MFI) was significantly higher than that of the microsphere surface FITC average fluorescence intensity, coefficient of variation is greater than FITC fluorescent display system.6)Using three different sizes impedance encoding immune diagnose microballoons quantitative detection technology, respectively, for quantitative detection of clinically diagnosed serum AFP in patients with hepatocellular carcinoma, serum insulin in patients with type2diabetes mellitus and serum TNFa level in patients with MODS, the results show that were significantly higher than those in the normal control group. Conclusion:Different diameter impedance microsphere coding technology has feasibility and scientificity. It outperforms the ELISA technology in methodology of detecting antigen in quantity, and can be practice preliminary application in clinical diagnosis. Different diameter impedance encoded microspheres liquid biological chip and detection device has the feasibility and scientificity, worthy of further study.
引文
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