基于核酸适配体的微流控芯片酶反应器用于蛋白质的分析

An enzyme reactor based on aptamer modified microfluidic chip for protein analysis

将核酸适配体作为胰蛋白酶固定化介质,制备了一种新型的微流控芯片酶反应器,并与高效液相色谱-串联质谱联用,搭建了在线分析平台;分别使用标准蛋白及混合蛋白样品对芯片的酶解效率及联用平台的分析能力进行了初步评价。结果表明,5 ng肌红蛋白经该平台分析后肽段覆盖率可达到37%;对500 ng混合蛋白进行3次平行分析,肽段覆盖率及相对标准偏差分别为44.3%、6.5%(牛血清白蛋白),65.0%、2.7%(肌红蛋白)和62.0%、5.6%(细胞色素c);初步实验表明,该在线分析平台具有检测灵敏度高、重现性好、酶解效率高的特点,有望在蛋白质组学分析中发挥重要作用。

As a kind of recognition molecule,aptamer has been studied and applied widely in numerous science fields in recent years.Immobilized enzymatic reactor has drawn much attention because of its striking advantages,such as high digestion efficiency and ease in coupling with the separation and detection systems.In this study,a novel microfluidic enzymatic chip,which immobilized trypsin based on aptamer,was prepared and proposed.An online analysis platform,which consisted of an aptamer-based chip and high performance liquid chromatography tandem mass spectrometry,was established by using a 6-port valve and applied to protein analysis.The enzymatic capacity and stability performance of chip reactor were characterized by using mixed protein sample,which consisted of bovine serum albumin（BSA）,myoglobin（Mb） and cytochrome c（Cyt.c）.The sample digestion time of the chip reactor was about 5.76 s while 1 μL/min of flow rate was adopted;and moreover,5 ng of Mb was identified successfully with the sequence coverage of 37%.Furthermore,the sequence coverages and the relative standard deviations were 44.3% and 6.5% for BSA,65.0% and 2.7% for Mb,62.0% and 5.6% for Cyt.c respectively when 500 ng digest of mixed proteins were analyzed in three runs.According to experimental results,the online analysis platform possesses the ability of high sensitivity and good stability,which can provide a promising tool for rapid and high-throughput proteomics study in the near future.