CRISPR/Cas13a powered electrochemical microfluidic biosensor for nucleic acid amplification-free miRNA diagnostics

Abstract

Non-coding small RNAs, such as microRNAs, are becoming the biomarkers of choice for multiple diseases in clinical diagnostics. A dysregulation of these microRNAs can be associated to many different diseases, such as cancer, dementia or cardiovascular conditions. The key for an effective treatment is an accurate initial diagnosis at an early stage, improving the patient's survival chances. Here, we introduce a CRISPR/Cas13a powered microfluidic, integrated electrochemical biosensor for the on-site detection of microRNAs. Through this unique combination, the quantification of the potential tumor markers microRNA miR-19b and miR-20a has been realized without any nucleic acid amplification. With a readout time of 9 minutes and an overall process time of less than 4 hours, a limit of detection of 10 pM was achieved, using a measuring volume of less than 0.6 µl. Furthermore, we demonstrate the feasibility of our versatile sensor platform to detect miR-19b in serum samples of children, suffering from brain cancer. The validation of our results with a standard qRT-PCR method shows the ability of our system to be a low-cost and target amplification-free tool for nucleic acid based diagnostics.MicroRNAs (miRNAs) are composed of 18-25 nucleotides in their mature form and play an essential role as regulators of gene expression in many biological processes by binding to specific messenger RNA targets and promoting their degradation or translational inhibition 1,2 . In recent years, the popularity of miRNAs in research has been constantly growing, as the presence or dysregulation of distinct miRNAs can indicate specific medical conditions. For instance, in cancer research the up or down regulation of miRNA expression levels has been linked to certain cancer types, including lung cancer or brain tumors. This offers new possibilities for the detection and monitoring of such diseases, which is why miRNAs will continue to come in the focus of clinical diagnostics 3-5 .

A Critical Review of Electrochemical Glucose Sensing: Evolution of Biosensor Platforms Based on Advanced Nanosystems

“…Since many glucose oxidase-based electrochemical biosensors have been used as idealized model systems for the fabrication of diverse sensing platforms, they have opened up the possibility of their use in a new generation of (integrated) electrochemical biosensors for the detection of several other analytes. For example, a combination of an electrochemical glucose biosensor with advanced DNA technologies on a small microfluidic device has been developed at low cost in order to detect miRNAs [ 134 ].…”

Multifunctional microfluidic chip for cancer diagnosis and treatment

“…Based on this characteristic, Cas13a can be used to amplify nucleic acid signals without the synthetic nucleic acid amplification steps [102]. On microfluidic chips, the enzyme Cas13a, the targetspecific CRISPR RNA (crRNA) and the labeled reported RNA (reRNA) combined with an electrochemical biosensor was used to detect miRNA in the serum samples of patients who suffered from brain tumors [103]. When the serum sample contained target miRNA, the Cas13a/ crRNA complex caused reRNA collateral cleavage due to the "collateral activity".…”

“…When the serum sample contained target miRNA, the Cas13a/ crRNA complex caused reRNA collateral cleavage due to the "collateral activity". The change of reRNA was detected by the electrochemical biosensors and the current signal readout is inversely proportional to the concentration of miRNA in the serum sample [103]. It is worth mentioning that the detection time of this method is short (less than 4 h) and the needed sample volume is less than 0.6 μL.…”

Emerging diagnostic tools for detection of COVID-19 and perspective

“…Another perspective to be explored is pairing CRISPR-Cas with electrochemical based sensing as it can help in achieving even lower limit of detection. Can Dincer et.al, reported (CRISPR)/Cas13a-powered microfluidic based electrochemical sensor for detection of cancer reaching a limit of 10 pM on site (Bruch et al 2019 ). Such a system should be made available at this crucial time in detection of COVID-19.…”