Ultrasensitive electrochemical detection of mRNA using branched DNA amplifiers

We describe here an ultrasensitive electrochemical detection of messenger RNA (mRNA) protocol without RNA purification and reverse transcription-polymerase chain reaction (RT-PCR) amplification. The new mRNA electrical detection capability is coupled to the amplification feature of branched DNA (bDNA) technology and with the magnetic beads (MB) based electrochemical bioassay. After sandwich-type hybridization reactions among the DNA probes modified magnetic beads, mRNA target and bDNA amplifiers, numerous alkaline phosphatase tracers were captured to the MB surface. The quantification of mRNA was realized by square wave voltammetric detection of the enzymatic product with a disposable screen printed electrode. The proof of concept was demonstrated with human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) mRNA. The parameters (e.g., amount of DNA probes coated magnetic beads, substrate concentration and enzymatic reaction time) that govern the sensitivity of the assay were optimized. The voltammetric response is highly linear over the range of 1.35–216 pM mRNA, and the limit of detection is estimated to be 0.68 pM (3.4 attomoles in 5 μL of sample). Such bDNA and magnetic bead based electrochemical bioassay shows great promise for simple, cost-effective and quantitative gene expression analysis.