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

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

Ten microliters of avidin-coated magnetic particles (3.0 mm, VMS-30-10, Spherotech, Libertyville, IL, USA) were washed with an equal volume of saline/sodium citrate buffer (SSC, 150 mM NaCl/15 mM sodium citrate, pH 7.0). After centrifugation and pipetting, the supernatant was removed and the magnetic particles were incubated with 10 mL of 10 nM biotinylated genome capture probe overnight at room temperature. Oligonucleotide sequence of the genome capture probe for E.coli was 5 0 -biotin-GACAAGAAAATC-TCCAACATCC-3 0 while that for B.subtilis was 5 0 -biotin-CCAGTTTCCAATGACCCTCCCC-3 0 . The capture probe functionalized magnetic particles were finally washed with 10 mL of the SSC buffer, resuspended in 10 mL of the SSC buffer, and stored at 4 C. Genome isolation. The sample containing E.coli or B.subtilis or both (1 mL), which was cultured in Luria-Bertaini broth overnight at 37 C, was mixed with 1 mL each of the biotinylated genome capture probe (1 nM) for E.coli and B.subtilis along with 7 mL of the SSC buffer. The mixture was injected into the reaction chamber and sealed with Bostik's Blu-Tack. Then, the silicon-glass device was placed in a plexiglass holder with contact pins for electrical connections to the heater and temperature sensors ( Figure 1C ). The chamber was maintained at 90 C for 5 min to lyse the cell and at the same time denature the genomic DNAs. After that, the temperature was cooled to 50 C and held for 10 min to allow specific hybridization between the denatured genomic DNAs and capture probes. Subsequently, 1 mL of the functionalized magnetic particles were added into the chamber and incubated for 10 min to capture the specific genomes onto the magnetic particles. Finally, SSC buffer was used to remove any unwanted materials with an external magnet to keep the particles within the microchamber. Electrochemical amplicon detection. After the asymmetric PCR, the solution was allowed to stand at room temperature for 1 h. Unhybridized amplicons were washed away with the SSC buffer. Gold nanoparticle label was bound to the hybridized amplicons by exposing the electrode to a stre-ptavidinÀgold nanoparticle (5 nm) solution (the stock was diluted 10 times with 0.05 M 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid/0.2 M NaCl) for 30 min at room temperature. The unbound gold nanoparticles were removed by flushing the microchamber with phosphatebuffered nitrate solution (0.3 M NaNO 3 /10 mM sodium phosphate, pH 7.0). Electrocatalytic silver deposition onto the hybrid-bound gold nanoparticles was then achieved by applying a potential of À0.18 V in a silver nitrate solution (1 mM AgNO 3 /1 M KNO 3 ) for 20 s. Finally, the amount of deposited silver was determined by measuring the oxidative silver dissolution response with an applied anodic current of 10 mA in the same silver nitrate solution, and the time to reach a potential of +0.65 V was taken as the signal.