Real-time analysis of PCR inhibition on microfluidic materials

In order to clarify the suitability of different materials for development of microchip format PCR chambers, a real-time polymerase chain reaction (PCR) analysis was performed. The analysis was demonstrated on a modified 264 bp (base pair) PCR template fragment originating from the human GAPD gene, using SYBR Green® chemistry on different types of oxidized silicon, native silicon and other surfaces of interest. The PCR reactions with the test surfaces were studied to determine the inhibitory potential of the surfaces by comparing them to the reference reactions without the test surfaces. The analysis included the final level of fluorescence, the detection threshold delay, the amplification efficiency and the melting point analysis. Minor inhibition effects were observed for native silicon and SF6 etched surface without oxidation, while fluorocarbon-coated silicon showed a strong inhibition of PCR. Chemical, physical, as-deposited and thermal silicon dioxide surfaces on silicon resulted only in a delayed detection threshold, showing some level of inhibition during the early part of PCR. The chemically oxidized and the plasma oxidized silicon and silica surfaces were also analyzed with X-ray photoelectron spectrometer equipment showing the adsorption of DNA and the used polymerase. The effect of bovine serum albumine and poly-vinyl pyrrolidone in alleviating the inhibition was observed to be insignificant. Also the effect of starting amount of the template DNA was minimal, suggesting that the adsorption of DNA was not important for the observed inhibition.