Label-free electrochemical DNA biosensor array for simultaneous detection of the HIV-1 and HIV-2 oligonucleotides incorporating different hairpin-DNA probes and redox indicator

A label-free electrochemical DNA biosensor array was developed as a model system for simultaneous detection of multiplexed DNAs using microlitres of sample. A novel multi-electrode array was comprised of six gold working electrodes and a gold auxiliary electrode, which were fabricated by gold sputtering technology, and a printed Ag/AgCl reference electrode was fabricated by screen-printing technology. The DNA biosensor array for simultaneous detection of the human immunodeficiency virus (HIV) oligonucleotide sequences, HIV-1 and HIV-2, was fabricated in sequence by self-assembling each of two kinds of thiolated hairpin-DNA probes onto the surfaces of the corresponding three working electrodes, respectively. The hybridization events were monitored by square wave voltammetry using methylene blue (MB) as a hybridization redox indicator. The oxidation currents of MB accumulated on the array decreased with increasing the concentration of HIVs due to higher affinity of MB for single strand rather than double strands of DNA. Under the optimized conditions, the peak currents were linear over ranges from 20 to 100 nM for HIV-1 and HIV-2, with the same detection limits of 0.1 nM (S/N = 3), respectively. The biosensor array showed a good specificity without the obvious cross-interference. Furthermore, single-base mutation oligonucleotides and random oligonucleotides can be easily discriminated from complementary target DNAs. This work demonstrates that different hairpin-DNA probes can be used to design the label-free electrochemical biosensor array for simultaneous detection of multiplexed DNA sequences for various clinical applications.