Quantitative evaluation of oligonucleotide surface concentrations using polymerization-based amplification

Quantitative evaluation of minimal polynucleotide concentrations has become a critical analysis among a myriad of applications found in molecular diagnostic technology. Development of high-throughput, nonenzymatic assays that are sensitive, quantitative and yet feasible for point-of-care testing are thus beneficial for routine implementation. Here, we develop a nonenzymatic method for quantifying surface concentrations of labeled DNA targets by coupling regulated amounts of polymer growth to complementary biomolecular binding on array-based biochips. Polymer film thickness measurements in the 20–220 nm range vary logarithmically with labeled DNA surface concentrations over two orders of magnitude with a lower limit of quantitation at 60 molecules/μm(2) (∼10(6) target molecules). In an effort to develop this amplification method towards compatibility with fluorescence-based methods of characterization, incorporation of fluorescent nanoparticles into the polymer films is also evaluated. The resulting gains in fluorescent signal enable quantification using detection instrumentation amenable to point-of-care settings. [Figure: see text]