Label-free highly sensitive detection of DNA approximate length and concentration by impedimetric CRISPR-dCas9 based biosensor technology

Abstract

In this study, we designed a CRISPR-dCas9-based biosensor with potential clinical use in glioblastoma subtype discrimination through detection of isocitrate dehydrogenase R132H (IDH) mutation status. The electrode was modified to detect mutant DNA cysteamine (Cys), PAMAM, dCas9 and sgRNA for R132H mutations, respectively. The biosensor system we proposed was able not only to detect mutant DNA, but also to measure the approximate length of DNA. Therefore, it can be considered that the biosensor technology that we developed is novel in the field of DNA biosensors. Another superior capability of the biosensor system is that it can simultaneously measure DNA concentration by electrochemical impedance spectroscopy and DNA length by capacitive detection, which lowers the concentration-based false-positive signals. The calibration range was obtained between 100 fM and 1000 fM, LOD and LOQ were also calculated as 33.96 fM and 102.91 fM respectively. Moreover, thanks to the sensitivity of the capacitive detection, the biosensor was able to discriminate the same EIS signals of the 200 bp and 250 fM concentration data and 1000 bp and 50 fM concentration data. In conclusion, the biosensor was capable of detect target DNA and DNA length, simultaneously in minutes.