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

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.

Non-invasive cortisol detection in saliva by using molecularly cortisol imprinted fullerene-acrylamide modified screen printed electrodes.

In this study, we developed an impedimetric sensor system by using molecularly cortisol imprinted acrylamide polymers on fullerene modified carbon electrode to detect cortisol in real saliva samples. The polymer layer was formed on fullerene modified screen printed carbon electrodes with the ratio of 1:2 (cortisol:acrylamide) and ammonium persulfate (APS) was used as the initiator of polymerization. The sensor surface was investigated by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and Scanning Electron Microscopy (SEM). Performances of the electrode were determined as follows; calibration curve was calculated between 0.5 nM and 64 nM, R2 = 0.9939, LOD and LOQ were 0.14 nM and 0.44 nM, respectively. Real human saliva samples were obtained from 15 participants between 25 and 32 ages regardless of gender. The samples, which were collected at least 30 min after waking up and the new sensor method analysis were compared tandem mass spectroscopy coupled liquid chromatography system (LC-MS/MS). The sensor analysis showed a significant correlation with LC-MS/MS results (R2 = 0.9727).