Enhancement of Aptamer Microarray Sensitivity through Spacer Optimization and Avidity Effect
Enhancement of Aptamer Microarray Sensitivity through Spacer Optimization and Avidity Effect
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DOI：
10.1021/ac801285a
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发表时间：
2009-03-01
影响因子：
7.4
通讯作者：
Chen, Lin-Chi
中科院分区：
文献类型：
--
作者：
Lao, Yeh-Hsing;Peck, Konan;Chen, Lin-Chi
本研究旨在利用适体微阵列技术对复杂生物基质中的靶蛋白进行超灵敏检测。两个广泛研究的适体（HTQ和HTDQ）结合凝血酶不同的表位被选择用于微阵列研究。虽然HTQ和HTDQ具有纳摩尔到单摩尔的亲和性，但研究发现，当直接应用适体时，在血清总蛋白（STP）过量10倍或100倍（w/w）的情况下，很难检测到每升凝血酶中的几纳摩尔。通过研究十二烷基（12-碳）和低聚脱氧胸腺嘧啶（oligo(dT)）间隔剂，我们观察到两种间隔剂都能增强微阵列信号响应，但低聚（dT）间隔剂明显优于十二烷基。此外，我们发现两个不同的适体（HTQ和HTDQ）共印的微阵列点像二价分子结构一样起作用，并表现出贪婪效应。通过寡聚（dT）间隔剂和贪婪效应的协同作用，可以在10%未标记血清或超过1万倍标记血清总蛋白的情况下检测到皮摩尔范围的凝血酶。与使用单个适配体而不使用间隔剂相比，它对应于100-1000倍的灵敏度增强。
This work aims for ultrasensitive detection of target proteins in complex biological matrixes based on aptamer microarrays. Two extensively studied aptamers (HTQ and HTDQ) that bind distinct epitopes of thrombin are chosen for the microarray study. Although HTQ and HTDQ have nanomolar to sulmanomolar affinities, it is found that either aptamer when applied directly has difficulty in detecting a few nanomoles per liter thrombin in the presence of a 10- or 100-fold (w/w) excess of serum total protein (STP). By investigating dodecyl (12-carbon) and oligodeoxythymidine (oligo(dT)) spacers, we observe both spacers enhance the microarray signal response, but oligo(dT) is strikingly better than dodecyl. Moreover, we discover that a microarray spot coprinted with the two distinct aptamers (HTQ and HTDQ) functions like a bivalent molecular construct and exhibits an avidity effect. With the synergy of oligo(dT) spacers and the avidity effect, detection of picomolar-range thrombin in the presence of either 10% unlabeled serum or a 10 000-fold excess of labeled serum total protein is achieved. It corresponds to a 100-1000-fold sensitivity enhancement as compared to using an individual aptamer without a spacer.