Immobilization-free electrochemical detection of isothermal primer generation-rolling circle amplification for simple and sensitive DNA analysis

Abstract

Electrochemical deoxyribonucleic acid (DNA) sensors have been widely studied in recent decades. Their simplicity, portability, and low cost are conductive to point-of-care and on-site applications. Typically, an electrode surface is immobilized with a capture probe, to which a complementary target sequence is hybridized, giving rise to a change in the electrochemical readout. Undesirably, the immobilization process usually requires the use of functionalized probe and is time-consuming. Besides, the hybridization of the target to the electrode-bound probe is much less efficient than solution-phase hybridization due to steric hindrance. In view of these issues, there has been an increasing interest in immobilization-free approach. Recent attempts have been particularly devoted to its coupling with enzymatic DNA amplification so as to achieve high sensitivity. One major challenge is to keep the entire assay components and procedures simple. In this study, a new immobilization-free electrochemical detection scheme coupled with isothermal DNA amplification reaction (primer generationrolling circle amplification, PG-RCA) was developed. A redox indicator (methylene blue (MB)) and indium tin oxide (ITO) electrode were utilized for simple DNA detection. In the absence of the target, MB can freely diffuse to the ITO electrode to effect charge transfer and be detected by differential pulse voltammetry (DPV). While in the presence of the target, PG-RCA at an operating temperature of 37 {493}C produces in an exponential manner single-stranded amplicons, which forms complex with MB through intercalative binding. The complex has a lower DPV current than the free MB as a result of slower diffusion to and electrostatic repulsion with the electrode. Results show that MB is compatible with PG-RCA and RCA reaction. The amplification power of PG-RCA is stronger than RCA. The electrochemical and fluorescence results also show that this new DNA detection method is specific and sensitive (with a detection limit of 100 fM) with linear working range from 100 fM to 1 nM. Reproducible measurements can be performed by simple electrode washing step. With the simple instrumentation and assay protocol, this new immobilization-free electrochemical detection platform could allow DNA analysis to be performed in a decentralized setting.