MicroRNA (miRNA) profile-based point-of-care (POC) diagnostic methods have attracted considerable attention. In our laboratory, singleplex miRNA detection on a power-free poly(dimethylsiloxane) (PDMS) microfluidic chip with laminar flow-assisted dendritic amplification (LFDA) has been developed. In this study, to obtain the miRNA profile and to improve the reliability of the diagnosis, multiplex miRNA detection on the same system is demonstrated without compromising any advantages of the singleplex miRNA detection. The limit of detection (LOD) was at the femto-to picomolar level and the assay time was 20 min. The sensitivity, rapidity, and portability of the microfluidic chip are adequate for POC diagnosis.

We report on a detection method for methylated DNA on a microfluidic chip, which needs no external power for fluid pumping. The methylated DNA was sandwiched by immobilized probe DNA and an anti-methylcytosine antibody. The fluorescence signal was amplified by our original amplification technology. The detection method was first optimized using a 22-mer DNA sequence, then further validated using a 60-mer DNA sequence adapted from the SEPT9 gene. We were able to detect the methylated 60-mer DNA at 0.4 nM within 18 min.

MicroRNAs (miRNAs) are attracting considerable attention as potential biomarkers for the early diagnosis of cancer. We have been developing a detection method for miRNAs on a microfluidic chip with external-power-free fluid pumping and enzyme-free amplification. The assay is completed within 20 min. Here, we describe the specificity of this miRNA detection method. First, the specificity against mismatched sequences was investigated. The nonspecific detection of a 2-nucleotide mismatched sequence was negligible, while that of a 1-nucleotide mismatched sequence was observed to a reasonable extent. Next, the disturbance in mature miRNA detection by existence of its precursor miRNA was evaluated. One precursor miRNA out of four tested showed significant nonspecific responses at 1 nM or higher concentrations. However, those responses were much lower than that of the target mature miRNA at 0.1 nM. Finally, we tried to detect three endogenous miRNAs, which are known to be potential cancer biomarkers, in human leucocyte total RNA. The measured concentraions of these miRNAs agreed well with those obtained by quantitative reverse transcription polymerase chain reaction. These results indicate that the on-chip miRNA detection method has good specificity, which is promising for applications to real biological samples.