Quantitative evaluation of oligonucleotide surface concentrations using polymerization-based amplification

• Published in: Analytical and bioanalytical chemistry 2008-09, Vol.392 (1-2), p.167-175
• Main Authors: Hansen, Ryan R, Avens, Heather J, Shenoy, Raveesh, Bowman, Christopher N
• Format: Article
• Language: English
• Subjects: Analytical Chemistry; Binding, Competitive; Biochemistry; Biotin - chemistry; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; DNA - analysis; DNA - chemistry; DNA microarrays; Dye Dilution Technique - instrumentation; Eosine Yellowish-(YS) - analogs & derivatives; Eosine Yellowish-(YS) - chemistry; Fluorescent Dyes - chemistry; Food Science; Kinetics; Laboratory Medicine; Microscopy, Fluorescence; Molecular diagnostics; Monitoring/Environmental Analysis; Nanoparticles - chemistry; Oligonucleotide Array Sequence Analysis - methods; Oligonucleotides - analysis; Oligonucleotides - chemistry; Original Paper; Quantitative nucleic acid detection; Signal amplification; Streptavidin - chemistry; Surface Properties; Surface-initiated polymerization; Visible light photopolymerization
• ISSN: 1618-2642; 0937-0633; 1618-2650; 1432-1130
• DOI: 10.1007/s00216-008-2259-6

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/μm² (~10⁶ 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. [graphic removed]