微流控蛋白印迹杂交与传统Western blot对目的蛋白的检测效果比较

Comparison of the effects of microfluidic paper-based immunoassay and traditional Western blot on the detection of target proteins

目的借助微流控蛋白印迹实验实现对目的蛋白省时、省力和省试剂的定性以及相对定量分析。方法选取O6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)蛋白为目的蛋白,选用高表达MGMT蛋白的MCF7细胞为细胞模型,用传统蛋白印迹杂交和纸芯片蛋白印迹杂交实验法对经过药物MGMT阻断剂(lomeguatrib)处理后MCF7细胞的MGMT蛋白表达量做定性以及相对定量分析。结果纸芯片蛋白印迹杂交可以成功进行MGMT的定性、相对定量分析,而且上样检测量可低至10~25 pg。这相比于传统Western blot的上样检测蛋白检测限1~5 ng,提高了3个数量级。整个操作流程只需要1 h,所用试剂量少,实验成本更低。而且,纸芯片的多通道构造可以实现如同反向蛋白杂交分析(RPPA)一样的,对组织蛋白表达量进行高通量的系统检测。结论微流控蛋白印迹杂交相较于传统Western blot可以检测到更微量级的蛋白,节省试剂和样品,省时和省力,并可进行高通量的定性以及相对定量分析。

Objective To achieve the goal of the qualitatively and relatively quantitatively analyze of the target protein in a time-saving,labor-saving and reagents-saving way by the microfluidic paper-based immunoassay. Methods We chose MGMT as the target protein and compared the qualitative and semi-quantitative results of the MGMT expression in the MCF7 cells which was treated with MGMT inhibitor,lomeguatrib,in both the traditional Western blot and the paper chip immunoassay. Results Microfluidic paper-based immunoassay can make the qualitative and relative quantitative detection on protein expression. Compared to the sensitivity of 1-5 ng of the traditional Western blot,the microfluidic paper-based immunoassay could detect as low as 10-25 pg of the protein. The sensitivity could be improved by 3 orders of magnitude. The entire operational duration took only 1 hour with less costed reagents being consumed. It maked the high-throughput protein detection as sensitive as the reverse phase protein assays（ RPPA） does. Conclusions The paper chip immunoassay could be performed qualitatively and semi-quantitatively to detect protein expression,and is more effective than that of traditional Western blot.