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为了建立一种急性心肌梗死(acute myocardial infarction,AMI)生物标志物的高通量悬浮芯片检测新技术,本研究将肌钙蛋白Ⅰ(cardiac troponinⅠ,c TnⅠ)、肌酸激酶同工酶(creatine kinase isoenzyme,CK-MB)和肌红蛋白(myoglobin MYO)在悬浮芯片荧光编码微球上进行偶联制备成探针微球,在液相反应体系中,探针微球上结合的AMI生物标志物与样品中相应的标志物共同竞争液相中各自特异性的生物素化单抗,反应后结合生物素化二抗,再结合荧光报告分子后上机检测,获得中位荧光强度值(mean fluorescent intensity,MFI),绘制出3种AMI生物标志物检测的标准曲线,确定了建立的AMI生物标志物的高通量悬浮芯片检测新技术的最低检出限(limit of detection,LOD)、特异性、测量区间、准确度等性能指标,并与实际的患者血清和正常健康人血清中上AMI标志物进行检测。对c TnⅠ、CK-MBⅠ和MYO的LOD为0.002、0.050和0.038μg/L,标准曲线的测量区间在45个数量级,方程决定系数R2>0.99;其他蛋白标志物所获得的MFI值同空白对照比均未有明显变化。3种AMI标志物检测的变异系数在2.50%8.68%之间,相对标准偏差在2.50%7.90%之间,均小于10%。因此,AMI生物标志物的高通量悬浮芯片检测技术可完全可以满足临床检测的需要,该操作简单,灵敏快速,成本低廉,为临床AMI的快速诊断提供了新方法。
Abstract：To establish a new technology for the detection of acute myocardial infarction(AMI)biomarkers by high-throughput suspension array,this study made cardiac troponinⅠ(cTnⅠ),creatine kinase isoenzyme(CK-MB)and myoglobin(MYO)coupled on the fluorescent encoded microspheres of suspension array for the preparation of probe microspheres.In liquid phase reaction system,there were simultaneous competitions for respective biotinylated antibody between the AMI biomarkers combined on the probe microspheres and corresponding biomarkers in the samples.After the reaction,the biotinylated second antibody and the fluorescent report molecule were combined and the mean fluorescent intensity(MFI)was able to be detected.Three of the standard curves of the AMI biomarkers detection were plotted.The performance indicators of the new technology for the detection of AMI biomarkers by high-throughput suspension array,such as limit of detection(LOD),specificity,test range and accuracy,etc.were determined.AMI markers were detected in serum samples from the AMI patients and the normal healthy persons.LODs of c TnⅠ,CK-MB and MYO were 0.027,0.050 and 0.038μg/L and the detection ranges of the standard curves were 45 orders of magnitude,the coefficient of determination of the equations,R2>0.99.There were no significant variations of the obtained MFI values among other protein markers compared with the blank control.The variation coefficients of the detection of the three AMI markers were between 2.50%8.68%,and the relative standard deviations were between 2.50%7.90%,which were all less than 10%.Thus,high-throughput suspension array for the detection of AMI biomarkers could completely satisfy the needs of clinical detection.The operation was simple with charaters of sensitivity and rapidity,which might be at low cost but provided a new method for the rapid diagnosis of clinical AMI.
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基本信息:
DOI：10.13417/j.gab.036.005014
中图分类号:R446.1;R542.22
引用信息:
[1]郑凤娇,郑小玲,刘楠.血清样本急性心肌梗死生物标志物的高通量悬浮芯片检测技术研究[J].基因组学与应用生物学,2017,36(12):5014-5020.DOI:10.13417/j.gab.036.005014.
基金信息:
广东省自然科学基金(No.2016A030313138)资助
2017-10-13
2017-10-13
2017-10-13