Author:

Abstract:

New technologies like DNA biosensors and microarrays are important routine tools in genetic screening. However large amounts of DNA are required for reliable and fast readout. Furthermore solid phase DNA amplification allows improving detection limits in DNA biosensors. Many of the solid phase PCR methods involve gold surfaces and gold nanoparticles (Au NP), because of their plasmon qualities. However uncontrolled molecular interactions on the gold surface hampers the efficiency of the amplification reaction. Even with the use of spacers which allow to decrease steric hindrance, amplification is suboptimal. Recently researchers showed that the cause of this inhibition can be found in nonspecific interaction of Taq polymerase with the Au NP surface. Use of surface covering molecules such as BSA improved the reaction efficiency. However BSA cannot be used in high concentrations because of enzyme inhibition and disturbance of optical readout. In an attempt to improve solid phase PCR, we tested a surface functionalization to eliminate the enzyme interaction with the gold surface. We used a hydroxyl-terminated hepta- (ethylene glycol) undecane thiol (PEO-alkanethiol) as a backfilling agent to optimize the orientation of the DNA on the surface and to remove non-covalent bound DNA from the surface. The Poly-Ethylene-Oxide groups of the molecule are protein repulsive and together with the non-charged OH groups, non-specific interactions are minimized. We tested this principle on Au NPs. Therefore DNA functionalized Au NPs with a Forward and Reverse primer for the amplification of an 80bp oligonucleotide were backfilled with the PEO-alkanethiol. Real-time PCR of a dilution curve in a Rotor Gene device (Qiagen, USA) allowed us to compare relatively the efficiency of the amplification. With PEO-alkanethiol we reached an amplification efficiency of more than 50% which is significantly better than MCH backfilled Au NPs which only could reach an efficiency of maximum 30%. Future research will be directed at the application of backfilling agents in a solid phase DNA Fibre Optic SPR biosensor for detection of Salmonella spp.