Covalent immobilization of oligo-DNA on a solid substrate via surface and subsurface reactions and application to quartz crystal microbalance biosensors
Two chemical reactions are developed for site specific, covalent oligo-DNA attachment to a polyethylene-co -acrylic acid (PEAA) substrate. One reaction uses 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) to activate the surface carboxylate groups on PEAA toward nucleophilic substitution. The other reaction makes use of thionyl chloride(SOCl 2 ) to convert subsurface carboxylic acid groups within the PEAA to the acid chloride. Aqueous solutions of alkyl amines and 95% ethyl alcohol will react with the acid chloride of PEAA to form amides and ester, respectively. Chemical and spectroscopic evidence, as well as contact angle measurements, show that the reactions are due to formation of acid chloride groups below the polymer's surface. To explain the film's properties, a three-layer model of its morphology is proposed. Practical application of the acid chloride film to the tethering of amino-alkyl-5 ' -modified oligo-DNA is demonstrated. A site-specific reaction tethers the oligo-DNA to the film exclusively via its aminoalkyl tail. A highly active, surface-tethered oligo-DNA results. It is also shown that a 5' -alkylamino tail on a synthetic oligo-DNA probe can be used to tether the probe to PEAA film via conventional, aqueous, carbodiimide chemistry, even at a pH below 8, where nearly all of the alkyl amine groups are protonated. Hybridization of denatured ds-DNA to the probe-modified film is not detectable using ethidium bromide staining, probably because of competition from homogeneous hybridization, but ss-DNA made by asymmetrical PCR amplification of the SLT-II gene from E. coli O157:H7 is detectable without difficulty. The ss-SLT-II gene product hybridizes at room temperature in 4 hours or less. This is one of the least complicated, and most rapid detections of a PCR product using membrane hybridization yet reported. The above methods for tethering oligo-DNA to PEAA are successfully used to make a QCM biosensor after a thin film of PEAA is applied onto the QCM electrode. It is also found that the use of PEAA as a support for DNA hybridizations gives superior results because it is virtually free of non-specific DNA adsorption and has low background fluorescence.