Quantitation of surface coverage of oligonucleotides bound to chip surfaces: a fluorescence-based approach using alkaline phosphatase digestion

Silanized chip surfaces provide a reliable substrate for immobilization of oligonucleotides. The ability for rapid and sensitive detection of oligonucleotide surface coverage on these chips is crucial for their wide and effective applications in biotechnology. In this paper, two different silanization procedures were used to covalently bind fluorescent-labeled single-stranded DNA onto silicon dioxide or nitride chip surfaces. Effects of surface functionalization techniques for different surfaces, and immobilization conditions, including buffers and solution ionic strength, on surface probe coverage were investigated, quantifying the endpoint probe density by fluorescent measurement upon digestion with alkaline phosphatase (ALP). Digestion of surface-immobilized oligonuleotides with ALP released the fluorophore-tagged probe fragments back into the solution. The detection of DNA was accomplished by laser-induced fluorescence detection of the solution containing those cleaved fragments. The probe surface density on gold thin film, determined by ALP-digestion, was found to coincide well with that measured using the conventional alkanethiol-based fluorescence-displacement technique for the same system. The developed method has important implications for evaluating the performance of different oligonucleotide immobilization strategies. Also, it has the potential to serve as a sample-thrifty, time saving, and therefore routine tool to realize more realistic, practical quantification of the surface coverage of of oligonucleotides immobilized on any solid surfaces.