Highly selective sensing and measurement of microRNA-541 based on its sequence-specific digestion by the restriction enzyme Hinf1

Abstract

In this study, a genosensor is introduced to detect microRNA-541 through an enzymatic digestion method and using a restriction enzyme (RE). Hinf1 is a type of RE which can cut the double helix DNA at specific sequences. The hybridization event and the corresponding enzymatic reactions are studied through guanine signal tracing on a pencil graphite electrode modified with graphene quantum dots (GQDs/PGE). The stages of fabricating the electrode are monitored by atomic force microscopy, and its electrochemical behavior is studied by cyclic voltammetry. The results indicate that the guanine current response of a 25-mer oligonucleotide of 7-guanine immobilized on the electrode surface decreases after hybridization despite an increase in the number of the guanine bases. Also, after enzyme treatment, the current decreases further due to the separation of a number of guanine bases from ds-DNA. A comparison of the analytical parameters of the proposed method with those of the conventional guanine oxidation method indicates that the linear concentration range in the proposed method, i.e. 1.0 fM to 1.0 nM, is lower than that in the conventional method, i.e. 10.0 pM–1.0 μM. On the basis of these findings, it is concluded that the use of Hinf1 enzyme makes it possible to measure microRNA at a femtomolar level. The selectivity of the designed biosensor has been proved using a non-complementary sequence with a one-base mismatch in the recognition site, rather than a complementary sequence. Finally, the proposed genosensor can be satisfactorily applied to measure microRNA-541 in human plasma samples.