Ultra-sensitive analyte detection through kinetic proofreading

现有技术的免疫吸附测定具有差的检测限，这使得病理状态的早期检测更加困难。除了低灵敏度之外，由于非特异性抗体-抗原相互作用，测量蛋白质还受到低特异性的阻碍。我建议将联合收割机DNA纳米技术与动力学校正方法相结合，用于超灵敏、无背景的分析物检测。在这样的实验中，短的寡核苷酸会将分析物产生的记录转化为可检测的微观结构。聚合将由单分子DNA折纸种子引发。因此，可以将测定的灵敏度推向单分子水平。除了有希望的高灵敏度之外，还应该通过动力学校正步骤来保证高特异性。我预见随着时间的推移，通过破坏种子来过滤假阳性信号。但是，通过在分析物的存在下稳定种子，只有真阳性才能在一定时间的校对中存活下来。我计划从DNA检测开始测试这个概念。特别短的寡核苷酸如siRNA由于其长度目前难以检测。然后，我将通过估计模型蛋白质的检测限来扩展对蛋白质的测定。

Current state of the art immunosorbent assays have poor detection limits which makes the early detection of a pathological state more difficult. In addition to low sensitivity, measuring proteins is hampered by low specificity due to unspecific antibody-antigen interactions. I propose to combine DNA nanotechnology with a kinetic proofreading approach for ultrasensitive, background free analyte detection. In such an essay, short oligonucleotides would polymerize and thereby transduce a record created by the analyte into detectable, microscopic structures. The polymerization would be initiated by single molecule DNA origami seeds. Thus, the assay’s sensitivity could be pushed towards the single molecule level. Besides the promising high degree in sensitivity, high specificity should be guaranteed by a kinetic proofreading step. I foresee to filter out false-positive signals by destroying the seeds over time. But, by stabilizing the seed in the presence of an analyte, only true-positives would survive a certain time of proofreading. I plan to test the concept starting with the detection of DNA. Especially short oligonucleotides like siRNA are currently difficult to detect because of its length. Then, I will expand the assay towards proteins by estimating a model protein’s limit of detection.