基于纳米金组合电极的HIV p24微流控安培免疫传感芯片研究

Micro Fluidic Amperometric Immunosensor Chip for HIV-p24 Antigen Based on Gold Nanoensemble Electrode

研制了以聚碳酸酯(PC)为基底,以表面修饰了人免疫缺陷病毒(HIV)核心抗原p24单克隆一级抗体(Rp24I)的纳米金组合电极(GNEE)为工作电极的微流控安培免疫传感芯片,并应用于p24抗原(简称p24)实时分析。检测原理是基于夹心免疫分析法,在电压驱动和流动条件下,一次性加入p24样品和纳米金胶标记的p24二级抗体(Rp24Ⅱ-Au,金胶直径为50～100nm)溶液,利用不同物质因受电场影响而在微管道中的迁移速率不同,使得p24抗原和Rp24Ⅱ-Au依次到达GNEE电极,与其表面的Rp24I生成三明治型免疫复合物(Rp24I/p24/Rp24Ⅱ-Au)。接着以方波溶出伏安法(SWSV)将复合物上的金胶粒子溶出,由于溶出电流大小与被测定p24呈正比关系,从而获得p24的测定校正曲线。在pH6.2的磷酸缓冲液(PBS)中p24检测时间小于2min,线性范围为1～500ng/L(R2=0.9975),检测限为0.25ng/L。该芯片集成了加样、分离和检测系统,简化了分析步骤,缩短了检测时间,灵敏度达1μA.ng-1.mL,明显高于传统酶联免疫吸附试验(ELISA)方法,对艾滋病的早期诊断和大范围筛查具有应用价值。

An electrochemical detection biosensor chip for the analyzing HIV p24 antigen using gold nanoen- semble electrode（GNEE） modified by the first monoclonal antibody of p24 antigen（Rp24 I） and microfluidic control system has been developed（Rp24 I/GNEE）. The detection principle is based on double sandwich immunoassay method. Firstly, the first monoclonal antibody of p24 antigen（Rp24 I） was fixed on GNEE surface through chemical derivation method, then when the serum containing p24 antigen passes through the modified electrode, p24 can combine on its surface, furthermore adding the secondary p24 antibody marked by nano-gold （Rp24 II-Au, the diameter of particles is between 5-10 nm） and incubation to form sandwich compound （Rp24 I/p24/Rp24 II-Au）. Then the square wave stripping voltammetry（SWSV）will be employed to dissolve the nano gold particles marked on the secondary p24 antibody to solution. The dissolve current was in directly proportional with the contention of p24, which can be employed for quantitative determination of p24. Under optimal experimental condition of pH 6. 2 PBS, p24 can be detected within 2 minutes and a calibration curve with one linear range from 1 to 500 ng/L （R2 =0. 9975）and a detection limit of 0. 25 ng/L for HIV p24 determination is obtained. The biosensor’s sensitivity can reach to lμA ·ng^-1 · mL in which GNEE used as a working electrode has greatly increased the surface area of electrodes. The biosensor can obviously improve the p24 detective sensitivity reaching to 1μA·ng^-1·mL. The biochip can simplify the adding, separating, and determination procedure within one step which has useful for early diagnosis of AIDS and large-scale screening test.