Amplification-free DNA Sensor for the One-Step Detection of the Hepatitis B Virus Using an Automated Paper-Based Lateral Flow Electrochemical Device

Until now, an electrochemical lateral flow assay (eLFA) capable of detecting nucleic acids has remained a challenge and has been scarcely explored because of its complicated multistep nature. Here, we report an automated paper-based eLFA device for the quantitative detection of the hepatitis B virus (HBV)-the major cause of liver cirrhosis and hepatocellular carcinoma (HCC). Using a time-delayed microfluidic strategy fabricated on paper, an automated and precisely sequenced solution transfer was enabled by single sample loading. A gold metallization strategy was employed for the signal-on electrochemical detection of the target DNA. Furthermore, a pyrrolidinyl peptide nucleic acid (so-called acpcPNA) was used as a probe in this study because it offers higher specificity and yields lower background currents than those of traditional probes. Under optimal conditions, a broad dynamic range (10 pM to 2 mu M) with an excellent detection limit (down to 7.23 pM) was achieved. The overall operation can be completed within 7 min of sample loading. The proposed sensor was successfully applied in HBV DNA detection in sera from patients without any amplification step (e.g., PCR) required, thus simplifying the operation further. Additionally, the results obtained from this present device are in accordance with the standard real-time PCR, thus supporting the accuracy of the method.

Automated summary

An automated paper-based eLFA device was developed for quantitative detection of hepatitis B virus (HBV) using a time-delayed microfluidic strategy and gold metallization for signal-on electrochemical detection of target DNA. The use of pyrrolidinyl peptide nucleic acid as a probe provided higher specificity and lower background currents, achieving a broad dynamic range and excellent detection limit. The device simplifies the operation for HBV DNA detection without the need for amplification steps, while producing results consistent with standard real-time PCR.