An isothermal, non-enzymatic, and dual-amplified fluorescent sensor for highly sensitive DNA detection

Idorenyin Iwe,Zhigang Li

doi:10.1515/revac-2021-0140

Abstract

Abstract Sensitive DNA assays are of importance in life science and biomedical engineering, but they are heavily dependent on thermal cycling programs or enzyme-assisted schemes, which require the utilization of bulky devices and costly reagents. To circumvent such requirements, we developed an isothermal enzyme-free DNA sensing method with dual-stage signal amplification ability based on the coupling use of catalytic hairpin assembly (CHA) and Mg2+-dependent deoxyribozyme (DNAzyme). In this study, the sensing system involves a set of hairpin DNA probes for CHA (ensuring the first stage of signal amplification) as well as ribonucleobase-modified molecular beacons that serve as activatable substrates for DNAzymes (warranting the second stage of signal amplification). An experimentally determined detection limit of about 0.5 pM is achieved with a good linear range from 0.5 to 10 pM. The results from spiked fetal bovine serum samples further confirm the reliability for practical applications. The non-thermal cycling, enzyme-free, and dual-amplified features make it a powerful sensing tool for effective nucleic acid assay in a variety of biomedical applications.

Highlights

Sensitive DNA assays are of importance in life science and biomedical engineering, but they are heavily dependent on thermal cycling programs or enzymeassisted schemes, which require the utilization of bulky devices and costly reagents
We present a simple and robust isothermal sensing method for the detection of target DNA with a dual-stage amplification capability, which is realized by the coupling use of both catalytic hairpin assembly (CHA) and Mg2+-dependent DNAzyme
It is worth noting that the signal ratio of the proposed system with/without Mg2+ is similar to the signal ratio of the DNAzyme system with/without Mg2+, which strongly agrees with the design concept (Figure 2a)

Summary

Introduction

Abstract: Sensitive DNA assays are of importance in life science and biomedical engineering, but they are heavily dependent on thermal cycling programs or enzymeassisted schemes, which require the utilization of bulky devices and costly reagents. To circumvent such requirements, we developed an isothermal enzyme-free DNA sensing method with dual-stage signal amplification ability based on the coupling use of catalytic hairpin assembly (CHA) and Mg2+-dependent deoxyribozyme (DNAzyme). The non-thermal cycling, enzyme-free, and dualamplified features make it a powerful sensing tool for effective nucleic acid assay in a variety of biomedical applications

Methods

Results

Conclusion