An Electrolyte-Gated Transistor combined with CRISPR/Cas13a for RNA detection

Ribonucleic acids (RNA) detection is a crucial step in the identification of viral or bacterial infections in humans and animals. To date, reverse transcriptase-polymerase chain reaction (RT-PCR) remains the gold standard, but it is based on an amplification step which takes time and can induce transcription errors. However, clustered regularly interspaced short palindromic repeats linked to a Cas endoribonuclease particle (CRISPR/Cas) have recently revolutionized the recognition step of two types of RNA, i.e. the CRISPR-RNA and the target, providing a much better selectivity compared to the naked hybridization on which RT-PCR is based. In this study, we combine the high sensitivity of the CRISPR/Cas13a system with the transduction and amplification capabilities of an electrolyte-gated graphene field-effect transistor (EGGFET) for the detection of specific RNA sequences, which promise selective and sensitive detection, without PCR amplification. In these devices, fabricated on flexible plastic substrates, the active material of the transistors (reduced graphene oxide - rGO) is deposited by additive printing techniques. The rGO is then functionalized using Au nanoparticles decorated with polyU RNA strands immobilized by a thiol-gold bond. In the presence of a specific RNA sequence, the enzymatic function of the CRISPR/Cas13a complex is activated and the polyU RNA strands are cleaved from the Au nanoparticles, inducing a loss of negative charges on the rGO layer. This phenomenon leads to significant shift of the charge neutrality point (CNP) of the rGO, converted into a shift of the transistor's transfer curves. This sensing device was tested for the detection of a SARS-CoV-2 RNA sequence and showed a linear response in the range of 10-7 - 102 ng.µL-1. With the optimized device, the LOD was found to be 75´10-9 ng.mL-1, which was estimated to be around 10 fM, as expected for an amplification-free CRISPR/Cas-mediated nucleic acid sensor. The sequence of the target RNA to be detected can be adjusted by modifying the corresponding crRNA, making this sensor highly versatile and multipurpose. This work is an important cornerstone for the complete development of a point-ofcare RNA sensor. Such an RNA sensor could not only detect the presence of viruses, but could also be used to track bacterial growth in food, after infection in humans or animals, or in diseases where miRNAs are produced