Quantitative evaluation of oligonucleotide surface concentrations using polymerization-based amplification

Anal Bioanal Chem. 2008 Sep;392(1-2):167-75. doi: 10.1007/s00216-008-2259-6. Epub 2008 Jul 26.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Binding, Competitive
  • Biotin / chemistry
  • DNA / analysis*
  • DNA / chemistry
  • Dye Dilution Technique / instrumentation
  • Eosine Yellowish-(YS) / analogs & derivatives*
  • Eosine Yellowish-(YS) / chemistry
  • Fluorescent Dyes / chemistry
  • Kinetics
  • Microscopy, Fluorescence
  • Nanoparticles / chemistry
  • Oligonucleotide Array Sequence Analysis / methods*
  • Oligonucleotides / analysis*
  • Oligonucleotides / chemistry
  • Streptavidin / chemistry*
  • Surface Properties

Substances

  • Fluorescent Dyes
  • Oligonucleotides
  • eosine-5-isothiocyanate
  • Biotin
  • DNA
  • Streptavidin
  • Eosine Yellowish-(YS)