Impedimetric CRISPR-dCas9 Based Biosensor System for Sickle Cell Anemia Mutation

Abstract

Sickle cell anemia is one of the single point mutation diseases with symptoms such as stroke, lethargy, chronic anemia, and increased mortality, and it causes red blood cells to become sickle-shaped. In the study, a biosensor system was developed to detect this mutation quickly and cost-effectively. This biosensor system was prepared by forming a SAM layer with 4-Aminothiophenol (4-ATP) on the gold electrode, and coating it with amino graphene. It was then modified with SG-RNA with the sequence of the target mutation after CRISPR-dCas9 immobilization. The nanomaterial used in the preparation of the biosensor increased the sensitivity of the method by increasing the surface area. The biosensor prepared in this way was optimized and made to perform DNA analysis. As a measurement method, electrochemical impedance spectroscopy (EIS) was used. Electrochemical measurements were carried out in 50 mM pH 7.0 phosphate buffer solution, which includes 5 mM Fe(CN)64- /3- and 10 mM KCl, as redox probe solution by CV and EIS in this redox probe solution. EIS parameters were 10,000–0.05 Hz frequency, 10 mV AC and 180 mV DC potentials, and CV parameters were between - 0.2 to 0.5 V potential, 100 mV/s scan rate for 5 cycles. The DNA measurement time of the biosensor system was determined by the chronoimpedance measurements taken by applying a frequency of 500 Hz under 200 mV DC current. Measurement time of the biosensor was found to be 100 seconds. With the CRISPR-Cas9 based electrochemical biosensor system, which gives faster results compared to the measurement methods in the literature, a linear measurement between 40 pM and 1000 pM with a length of 400 base pairs was taken.