CRISPR-dCas9 powered impedimetric biosensor for label-free detection of circulating tumor DNAs

• Published in: Analytica chimica acta 2020-07, Vol.1121, p.35-41
• Main Authors: Uygun, Zihni Onur, Yeniay, Levent, Gi̇rgi̇n Sağın, Ferhan
• Format: Article
• Language: English
• Subjects: Biosensing Techniques - methods; Cancer diagnosis; Chronoimpedance; Circulating tumor DNA; Circulating Tumor DNA - blood; Class I Phosphatidylinositol 3-Kinases - genetics; CRISPR-Cas Systems - genetics; CRISPR/Cas9 technology; Deactivated Cas9; Dielectric Spectroscopy; Electrochemical impedance spectroscopy; Electrodes; Graphite - chemistry; Humans; Impedimetric biosensor; Label-free analysis; Limit of Detection; Liquid biopsy; Reproducibility of Results; RNA, Guide, CRISPR-Cas Systems - metabolism
• ISSN: 0003-2670; 1873-4324
• DOI: 10.1016/j.aca.2020.04.009

Label-free biosensors which can be integrated into lab-on-a-chip platforms have the advantage of using small volumes for rapid and inexpensive measurements contrary to label-based technologies which are often more costly and time-consuming. In this study, graphene oxide screen printed electrodes (GPHOXE) were modified by deactivated Cas9 (dCas9) proteins and synthetic guide RNA (sgRNA) as the biorecognition receptor for label-free detection of circulating tumor DNAs (ctDNA). This was achieved by detection of a tumor related mutation (PIK3CA exon 9 mutation) via sequence-specific recognition followed by electrochemical impedance spectroscopy (EIS) analysis. The biosensor showed high specificity as there was no impedance signal for other ctDNA sequences, even the single nucleotide mismatch. dCas9-sgRNA modified biosensor demonstrated linear detection limits between 2 and 20 nM for 120 bp ctDNA’s in 40 s. The calibration curve showed good linearity, LOD was calculated as 0.65 nM and LOQ was calculated as 1.92 nM. Selectivity and repeatability studies were carried out in real blood samples and the recovery was higher than 96%. In conclusion, dCas9-sgRNA was effectively immobilized and optimized on GPHOXE as the selective biorecognition receptor of this ultrafast impedimetric biosensor. The CRISPR-dCas9 powered impedimetric system showed good selectivity, high repeatability and good recovery properties. This is the first literature to report the use of CRISPR/Cas technology as a label-free tool that can be used in an impedimetric system for detection of ctDNA’s. [Display omitted] •First CRISPR/dCas biosensor for label-free circulating tumor DNA (ctDNA) detection.•dCas9-sgRNA covalently immobilized on graphene oxide screen printed electrodes.•Rapid, selective and accurate detection by electrochemical impedance spectroscopy.•Promising form of liquid biopsy with CRISPR/Cas9 powered biosensor system for ctDNA.