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/microm(2) (approximately 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.
Hansen
Ryan R
RR
Department of Chemical and Biological Engineering, ECCH 111 CB 424, University of Colorado, Boulder, CO 80309, USA.
Avens
Heather J
HJ
Shenoy
Raveesh
R
Bowman
Christopher N
CN
D001667:Binding, Competitive; D001710:Biotin / Q000737:chemistry; D004247:DNA / Q000032:analysis* / Q000737:chemistry; D004395:Dye Dilution Technique / Q000295:instrumentation; D004801:Eosine Yellowish-(YS) / Q000031:analogs & derivatives* / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D007700:Kinetics; D008856:Microscopy, Fluorescence; D053758:Nanoparticles / Q000737:chemistry; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D009841:Oligonucleotides / Q000032:analysis* / Q000737:chemistry; D019809:Streptavidin / Q000737:chemistry*; D013499:Surface Properties
D005456:Fluorescent Dyes; D009841:Oligonucleotides; C012957:eosine-5-isothiocyanate; D001710:Biotin; D004247:DNA; D019809:Streptavidin; D004801:Eosine Yellowish-(YS)