A DNA biochip for on-the-spot multiplexed pathogen identification

Yeung, Siu-Wai; Lee, Thomas Ming-Hung; Cai, Hong; Hsing, I-Ming

The single microchamber design poses particular challenges to the electrochemical platform used for the sequence-specific PCR amplicons detection. Addressability and compatibility are two important considerations regarding immobilization chemistry for the oligonucleotide detection capture probes. For a multiplexed assay, it is necessary to individually modify the detection platform so that each individual electrode in an electrode array has a specific capture probe. When using either high temperature or ultra-violet glue to seal the microchamber, it is recommended that immobilization should be carried out after the siliconÀglass bonding process so as to prevent damage to the capture probes. In doing so, the more common chemical attachment (spotting) method cannot be used because all the active electrode surfaces are embedded within the same microchamber and they would receive identical modifications. One simple way to achieve site-specific probe immobilization onto individual electrode surfaces can Figure 4 . A schematic representation of the assay protocol in the siliconÀglass microchamber. The three main steps were (A) sample preparation: thermal cell lysis and magnetic particle-based isolation of specific genomic DNAs; (B) target DNA amplification: generation of single-stranded rich amplicons by asymmetric PCR; (C) product detection: gold nanoparticle labeling, electrocatalytic silver deposition, and electrochemical silver dissolution. be achieved by electrochemical copolymerization of pyrrole and pyrroleÀoligonucleotide (11) . Figure 2 illustrates the strategy to immobilize different capture probes onto each individual electrode. A solution of pyrrole and oligonucleotide 1 bearing a pyrrole group is introduced into the microchamber. When a cyclic voltammetric scan is applied to electrode 1, with other electrodes disconnected or grounded, oligonucleotide 1 is selectively deposited on this particular electrode. Then, the microchamber is washed with water to ensure there is no pyrroleÀoligonucleotide 1 monomer is left. This procedure is repeated for the other electrodes with different pyrroleÀoligonucleotide polymerization solutions. In our model system with two target analytes and four working electrodes, the capture probes specific to E.coli and B.subtilis are immobilized in duplicate. Before proceeding to the complete analytical protocol, the ability of these immobilized capture probes to recognize their complementary targets should be tested. Figure 3 shows the fluorescence images of the four functionalized electrodes (A and D: B.subtilis probe; B and C: E.coli probe) exposed to a sample containing a fluorescently-labeled sequence complementary to the E.coli probe. It is clear that electrodes B and C exhibit much higher fluorescence intensity than electrodes A and D, indicating the highly specific probe immobilization as well as hybridization offered by the electrochemical pyrrole-based attachment chemistry. Another criterion for the selection of immobilization method is the compatibility with other processes, in particular the PCR. Due to the fact that the detection electrodes are within the reaction chamber, the linkage between the immobilized capture probe and electrode surface must be strong enough to survive through the thermal cycling process (especially the high denaturation temperature). Moreover, the detector surface should interact only with the specific amplicon but not with other components employed in the assay protoco