Sandwich structure electrochemical assay for single stranded DNA detection

A novel DNA sequence biosensor that utilizes enzyme-labeled probes and electrochemical detection via an interdigitated array (IDA) electrode on a microfluidic chip is presented. Sandwich structure DNA sequence recognition is performed by immobilizing a single stranded DNA (ssDNA) on the microchannel surface (primary probe) that is complementary to a portion of the target, followed by hybridization with target ssDNA and a second ssDNA (secondary DNA probe), which is complementary to another region of the target and is conjugated with β-Galactosidase (β-Gal). Surface confined β-Gal is detected by introducing its substrate, 4-aminophenyl-D galactopyranoside (PAPG), into the system; β-Gal cleaves PAPG into the electrochemical active compound, p-amino phenol (PAP), which is detected by a Au IDA in a microfluidic channel. The signal is amplified by cycling its reduced and oxidized form between the interleaved anode and cathode. On the chips which are integrated in 60 μm high channels, the oxidation currents show linear correlation to the immobilized target DNA concentration between 0 and 100 nM. Currents collected on chips where 0 and 0.5 nM target DNA is immobilized are significantly different. Similarly, on the chips integrated in the 35 μm channels where target DNA is not introduced to form the sandwich (negative control), the oxidation current shows non differentiable results whether PAPG is present or absent in buffer solution. On the contrary, on the chip where 0.1 nM target DNA hybridized with probes, the oxidation current is significantly higher when the buffer contains PAPG. As the enzymatic reaction time increases from 15 to 30 seconds, the signal difference between PAPG and buffer solution almost doubled. By reducing the channel height from 60 to 35 μm, the total assay time is reduced from 80 minutes to 35 minutes.