Ag/Au nanoparticle-based electrochemical detection of DNA : its application in bioaerosols recognition

The detection of airborne microorganisms has received considerable attention in the area of air quality control especially after the recent outbreak of SARS and bird flu in Hong Kong. Traditional detections involving culture techniques and microscopic examinations are tedious and time-consuming. Molecular analysis in particular DNA-based identification, together with the microfabrication techniques, offers the possibility to perform bioaerosol recognition in microfabricated devices for on-site and real time monitoring. A microdevice containing a silicon substrate with heaters and temperature sensors for rapid thermal cycling of DNA amplification and electrochemical-based detection on an indium tin oxide (ITO) substrate has been established for this purpose. In this work, various strategies have been investigated to enhance the performance of silver-on-gold indicator-based electrochemical detection and subsequent multiple species identification. First the signal amplification by multiple-labeled target sequence with nanoparticle tags and electroless silver enhancement were established. Background reduction by selecting appropriate detection supports such as electroconducting polymer poly(2-aminobenzonic acid) for amino-modified probe immobilization has been studied. Another approach is to implement the enhanced hybridization of DNA targets with peptide nucleic acid probes (a DNA analog) on a cationic electroconducting polymer polypyrrole substrate. Neutrally charged PNA probes, when hybridizing with DNA in liquid phase, can be electrostatically attracted to the polypyrrole surface. Sensitive signal amplification by novel silver electrodeposition on gold nanoparticles is employed to further reduce the background noise caused by eletroless silver enhancement technique. Finally the identification of two bioaerosol pathogens - Stachybotrys chartarum (fungi) and Bacillus subtilis (bacteria) is successfully demonstrated in the microfabricated device. Six ITO-based sensing electrodes on the microfabricated chip can be individually modified by the "voltagel-dependent" electropolymerization process. Genome DNAs of one or multiple pathogens of air samples are amplified by multiplex PCR. Hybridization of the amplified PCR products and DNA probes immobilized on the selected electrodes are monitored by the aforementioned Ag-Au electrochemistry-based approach. Utilizing Ag electrodeposition protocol to reduce the background noise and multiple biotinylated nano-gold reporters to enhance the detection signal, a reliable and sensitive detection of multiple bioaerosol mixtures is proven possible