Journal: Biomedical microdevices

Article Title: A plastic, disposable microfluidic flow cell for coupled on-chip PCR and microarray detection of infectious agents

doi: 10.1007/s10544-011-9584-9

Figure Lengend Snippet: Comparison of dN20 nonsense signals after different washing conditions using the Wound v2.0 array. Six flow cells with a pipettor using two different volumes of 1X SSPE and 25 μL of water versus the gold standard of hybridizing in Frame-Seals and washing arrays in a 1X SSPE microarray bath followed by a water rinse. The flow cells undergo an on-chip amplification procedure whereas the Frame Seals are used only for hybridization using amplified product from a standard PCR tube. The Frame Seals are subsequently removed in order to directly expose the array to the wash buffers in the bath, a water rinse, and air drying. The flow cells are dried per the protocol described in the experimental section and imaged with an Aurora Port Array 5000 for 0.2 seconds. The input sample is 104 genomic copies of S. pyogenes for both the flow cells and the Frame Seals. The nonsense spots are identified with Spotfinder, and the integral fluorescent intensity of each of 4 nonsense spots minus the local background is averaged and plotted along with error bars, which represent the minimum and maximum values. The nonsense signals are inversely proportional to the effectiveness of washing because the dN20 nonsense probes fluoresce due to non-specific binding with labeled product.

Article Snippet: A 15 μL master mix is prepared containing final concentrations of 1X PCR buffer (HotstarTaq Plus, Qiagen), 2 mM MgCl 2 , 1.2 mg/mL BSA, 200 μM dNTPs, 10% (w/v) formamide, 2.4U Taq DNA Polymerase, and 0.03-1 μM of each primer set.

Techniques: Comparison, Microarray, Amplification, Hybridization, Binding Assay, Labeling

Journal: Biomedical microdevices

Article Title: A plastic, disposable microfluidic flow cell for coupled on-chip PCR and microarray detection of infectious agents

doi: 10.1007/s10544-011-9584-9

Figure Lengend Snippet: Image of an MRSA v4.0 array through the film of a completely-intact plastic flow cell following on-chip amplification of 105 genomic copies of MRSA using PCR primers for mecA and tufA. The flow cell was washed with 35μl of 1X SSPE without implementing a drying protocol, and imaged for 0.5 sec with a GenePix 4000B. Colored circles correspond to different probes: Green, positive control Cy3 labeled oligos; purple, mecA gene, which confers methicillin resistance; blue, tufA gene found in S. Aureus strains; orange, tufA gene found in all Staph strains; red, nonsense spots that serve as a control for washing.

Article Snippet: A 15 μL master mix is prepared containing final concentrations of 1X PCR buffer (HotstarTaq Plus, Qiagen), 2 mM MgCl 2 , 1.2 mg/mL BSA, 200 μM dNTPs, 10% (w/v) formamide, 2.4U Taq DNA Polymerase, and 0.03-1 μM of each primer set.

Techniques: Amplification, Positive Control, Labeling, Control