Development of dCas9 Modified Impedimetric Biosensor for Detection of Isocitrate Dehydrogenase IDH1 Mutations Used as a Diagnostic and Prognostic Biomarker in Glioblastomas
Project no. 22230 · Status: Kapanmış · 2020
Summary
Glioblastoma (GBM, WHO stage IV astrocytoma) is the most common and most aggressive type of malignant brain tumor. Glioblastomas are divided into two subtypes, which are histologically indistinguishable from each other, as primary GBM (WHO, IDH wild type) and secondary GBM (WHO, IDH mutant). The prognosis and response to temozolomide and radiotherapy in patients with different types varies considerably. The most common genetic anomaly that distinguishes primary and secondary GBM is somatic single nucleotide mutations in the R132 codon of the isocitrate dehydrogenase (IDH1) gene. In this study, we designed a CRISPR-dCas9-based biosensor to detect the isocitrate dehydrogenase (IDH) missense mutation in the R132 codon. The electrode was modified with cystamine (Cys), PAMAM, dCas9/sgRNA to detect the mutant sequence. The biosensor we developed was able to detect DNA with high sensitivity within minutes. Another innovation of the biosensor system we have developed is that it can not only detect mutant DNAs, but also measure the approximate DNA length. For this reason, it can be thought that the biosensor technology we developed is new in the field of DNA biosensors. Another capability of the biosensor system is that it can simultaneously measure DNA concentration by electrochemical impedance spectroscopy and DNA length with capacitive detection, which reduces concentration-based false positive signals. The calibration range was obtained between 100 fM and 1000 fM, and the LOD and LOQ were calculated as 33.96 fM and 102.91 fM, respectively. Also, thanks to its capacitive detection sensitivity, it can distinguish the same EIS signals of 200 bp and 250 fM concentration data, and 1000 bp and 50 fM concentration data. As a result, the developed biosensor was able to detect target mutant DNA, DNA length and DNA concentration simultaneously with high sensitivity within minutes. Keywords: biosensor, chronoimpedance, electrochemical impedance spectroscopy, capacitance, CRISPR, dCas9