Quantitative evaluation of oligonucleotide surface concentrations using polymerization-based amplification

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/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.

Photopolymerization as a promising method to sense biorecognition events

“…The spectrometer is set in the transmission mode and the detection wavelength is fixed at a value where the monomer double bond exhibits a discrete and intense adsorption: some authors measure the twisting of -CH=CH2 at 810 cm -1 187, 239, 247-248 . Others track the peak centered approximately in the close IR at 6175 cm -1 to monitor the polymerization in real time [77][78]249 . The evolution of the peak assigned to NVP reactant, centered at 1334 cm -1 may also be followed 172 .…”

“…Studies realized on oligonucleotide dilution chips show a critical dependence on the amount of polymer growth with the superficial biomolecules density achieved during the photoinitiator coating step. Manipulation of this parameter by changing the binding conditions brings the chance to tune the amount of amplification generated product 77 . For example, the concentration of a photoinitiator streptavidin-eosin conjugate solution must be optimized as high concentration values might lead to nonspecific adsorption of the initiator species on the support surface, raising the background signal, thus reducing sensitivity 76 .…”

“…The thickness of fully dehydrated polymer films grown from microarray spots may be measured by a profilometer 72,77,161,245,255 . Measurements are made before and after polymer growth and the thickness differences reflect the amount of polymer formed.…”

“…In order to get quantitative results some approaches can be considered:  Incorporation of fluorescent molecules to the monomer mixture produces a fluorescent signal that can be related with the polymer growth by the simple assessment: the most polymer formed onto a surface spot, the most fluorescent moieties will be captured inside the polymer formed 77 . With this in mind, a recognition assay of a biotin labelled antibody with eosin labelled streptavidin was developed in our lab with a reactive mixture containing Cy5 fluorescent moieties.…”

“…The main procedures are based on amine 77,161,[242][243] and aldehyde 74,76,244 pending groups introduced by chemical reaction with appropriate precursors. Moreover, epoxy-functionalized glass slides may be used in array design for protein detection 78 .…”