Automated and label-free detection of HIV DNA via digital microfluidics-chemiluminescence analysis

Automated and sensitive detection of nucleic acid is crucial for reliable and safe disease diagnostics because the minimum manual operation can effectively avoid sample contamination and infection risk. Herein, a sensitive, label-free and automated method was developed for detecting trace DNA by integrating digital microfluidics (DMF) with chemiluminescence (CL) detection. DMF can automatically manipulate discrete droplets. Taking advantage of sensitive and simple CL detection, DMF-CL integration can automatically and sensitively detect HIV DNA via the preset pathway. To further improve the sensitivity, on-chip exponential amplification reaction (EXPAR) was explored to produce large quantities of double-stranded DNA (dsDNA) in the presence of HIV DNA. Then, SYBR Green I (SG) intercalated into dsDNA to generate singlet oxygen upon light irradiation, which in situ oxidized potassium ferrocyanide into potassium ferricyanide. Finally, the CL reaction of potassium ferricyanide and luminol ensured the label-free, reliable and specific detection of HIV DNA in the range of 10 pM to 2 nM. Saving time-consuming and laborious manual operations, the DMF-CL integration greatly reduced the reagent consumption and avoided the contamination of DNA sample as well. Therefore, DMF-CL offers a promising and useful technology for developing automated and reliable biosensing and bioanalysis.