DNA Nanosieve-Based Regenerative Electrochemical Biosensor Utilizing Nucleic Acid Flexibility for Accurate Allele Typing in Clinical Samples

Herein, an interface-based DNA nanosieve that has the ability to differentiate ssDNA from dsDNA has been demonstrated for the first time. The DNA nanosieve could be readily built through thiol-DNA's self-assembly on the gold electrode surface, and its cavity size was tunable by varying the concentration of thiol-DNAs. Electrochemical chronocoulometry using [Ru(NH3)(6)](3+) as redox revealed that the average probe-to-probe separation in the 1 mu M thiol-DNA-modified gold electrode was 10.6 +/- 0.3 nm so that the rigid dsDNA with a length of similar to 17 nm could not permeate the nanosieve, whereas the randomly coiled ssDNA could enter it due to its high flexibility, which has been demonstrated by square wave voltammetry and methylene blue labels through an upside-down hybridization format. After combining the transiently binding characteristic of a short DNA duplex and introducing a regenerative probe (the counterpart of ssDNA), a highly reproducible nanosieve-based E-DNA model was obtained with a relative standard deviation (RSD) as low as 2.7% over seven cycles. Finally, we built a regenerative nanosieve-based E-DNA sensor using a ligation cycle reaction as an ssDNA amplification strategy and realized one-sensor-based continuous measurement to multiple clinical samples with excellent allele-typing performance. This work holds great potential in low-cost and high-throughput analysis between biosensors and biochips and also opens up a new avenue in nucleic acid flexibility-based DNA materials for future applications in DNA origami and molecular logic gates.

Automated summary

A novel interface-based DNA nanosieve capable of differentiating single-stranded DNA from double-stranded DNA was demonstrated for the first time. This nanosieve was used to construct a highly reproducible E-DNA model and sensor for continuous measurement of multiple clinical samples with excellent allele-typing performance. The work shows potential for low-cost, high-throughput analysis and opens up new avenues in DNA origami and molecular logic gates research.