Satterfield Brent C, West Jay A A, Caplan Michael R
Harrington Department of Bioengineering, Arizona State University Tempe, AZ, USA.
Nucleic Acids Res. 2007;35(10):e76. doi: 10.1093/nar/gkm113. Epub 2007 May 21.
The majority of efforts to increase specificity or sensitivity in biosensors result in trade-offs with little to no gain in overall accuracy. This is because a biosensor cannot be more accurate than the affinity interaction it is based on. Accordingly, we have developed a new class of reagents based on mathematical principles of cooperativity to enhance the accuracy of the affinity interaction. Tentacle probes (TPs) have a hairpin structure similar to molecular beacons (MBs) for enhanced specificity, but are modified by the addition of a capture probe for increased kinetics and affinity. They produce kinetic rate constants up to 200-fold faster than MB with corresponding stem strengths. Concentration-independent specificity was observed with no false positives at up to 1 mM concentrations of variant analyte. In contrast, MBs were concentration dependent and experienced false positives above 3.88 muM of variant analyte. The fast kinetics of this label-free reagent may prove important for extraction efficiency, hence sensitivity and detection time, in microfluidic assays. The concentration-independent specificity of TPs may prove extremely useful in assays where starting concentrations and purities are unknown as would be the case in bioterror or clinical point of care diagnostics.
大多数提高生物传感器特异性或灵敏度的努力都会导致权衡，整体准确性几乎没有提高或根本没有提高。这是因为生物传感器的准确性不会超过其基于的亲和相互作用。因此，我们基于协同性的数学原理开发了一类新型试剂，以提高亲和相互作用的准确性。触手探针（TPs）具有与分子信标（MBs）相似的发夹结构以提高特异性，但通过添加捕获探针进行修饰以提高动力学和亲和力。它们产生的动力学速率常数比具有相应茎强度的MB快200倍。在高达1 mM浓度的变异分析物中观察到与浓度无关的特异性，没有假阳性。相比之下，MBs是浓度依赖性的，在变异分析物浓度高于3.88 μM时会出现假阳性。这种无标记试剂的快速动力学可能对微流控分析中的提取效率很重要，从而对灵敏度和检测时间也很重要。TPs与浓度无关的特异性在起始浓度和纯度未知的分析中可能非常有用，生物恐怖或临床即时诊断中就是这种情况。
