# High sensitivity detection of Hepatitis B virus RNA based on 3D-DNA nanomachine and protein nanopore sensing

## TL;DR

A new method detects Hepatitis B virus RNA in blood with high accuracy using DNA nanomachines and nanopore sensing, offering a faster and more sensitive alternative to traditional tests.

## Contribution

A novel amplification-free HBV RNA detection method using a 3D DNA nanomachine and alpha-hemolysin nanopore sensing is introduced.

## Key findings

- The method achieved a detection limit of 12.5 fM and 97% accuracy compared to RT-qPCR.
- It showed 100% accuracy in determining treatment continuation or drug discontinuation in 18 clinical cases.
- The method effectively distinguishes target RNA from interfering nucleic acids in clinical serum samples.

## Abstract

Serum Hepatitis B virus (HBV) RNA serves as a non-invasive biomarker for monitoring chronic hepatitis B and guiding treatment decisions. However, current diagnostic tests relying on Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) require centralized facilities and tedious operational steps, and there is currently no standardized HBV RNA detection method. Herein, we describe the rapid amplification-free detection of HBV RNA in clinical serum samples via a nanopore sensing method. Our method involves the design of a DNA nanomachine capable of translation of each target HBV RNA into a large amount of 7-nt single-strand DNA (ssDNA) as signal reporters that produce blockage features when passing through an alpha-hemolysin (α-HL) nanopore, the target can be quantified by identifying the frequency of this blockage occurrence. We demonstrate that this unique nanopore sensing feature not only distinguishes Signal Report (SR) DNA from interfering nucleic acids in clinical serum samples but also ensures a high assay sensitivity with a limit-of-detection (LOD) at 12.5 fM. The clinical potential of this method was validated through testing serum samples from 26 HBV-infected-patients and 7 healthy-participants. When compared with the RT-qPCR method, it showed only one false result, achieving an accuracy rate of 97.0%. Additionally, testing results for 18 cases during the treatment period demonstrated that our method’s conclusions on continued treatment and drug discontinuation were highly consistent with clinical diagnoses, with an accuracy rate of 100%. Therefore, the method developed in this study expands the clinical applications of 3D DNA nanomachine and nanopore sensing technology in clinical detection.

The online version contains supplementary material available at 10.1186/s43556-025-00282-7.