ABSTRACT
Colloidal gold nanoparticles are investigated as a potential scaffold for the assisted immobilisation of probe oligonucleotides on silicon surfaces. A preliminary study is devoted to the examination of the immobilisation of DNA-modified gold nanoparticles as a function of time, concentration, salt and pH. The DNA-modified nanoparticles self-assembled onto solid surfaces in a three-dimensional self-assembled architecture. The functionalised surfaces are evaluated in diagnostic assays, where their potential to improve the efficiency of the hybridisation reaction is tested. The system utilising DNA-modified nanoparticles produced an enhancement in the hybridisation efficiency and the sensitivity limit by a factor 10 to 100 as compared to a conventional DNA immobilisation system on a planar surface.
Subject(s)
DNA/analysis , Gold/chemistry , In Situ Hybridization, Fluorescence/methods , Nanostructures/chemistry , Nanotechnology/methods , Oligonucleotide Array Sequence Analysis/methods , Adsorption , Coated Materials, Biocompatible/chemistry , In Situ Hybridization, Fluorescence/instrumentation , Nanotechnology/instrumentation , Oligonucleotide Array Sequence Analysis/instrumentation , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
(Polymer-oligonucleotide) conjugates were obtained via direct ON synthesis from the poly(ethylene-alt-maleic anhydride: PEMA) grafted onto glass bead surfaces. The effect of the ON number per polymer chain on binding was evaluated through Tm experiments. Results were correlated with ELOSA (Enzyme Linked Oligosorbent Assay) tests run with conjugates in the capture step.
Subject(s)
DNA, Viral/analysis , Maleates/chemical synthesis , Oligonucleotides/chemistry , Polyethylenes/chemical synthesis , Hepatitis B virus/genetics , Maleates/chemistry , Oligonucleotides/chemical synthesis , Polyethylenes/chemistryABSTRACT
Oligonucleotide-polymer conjugates have been described to improve the sensitivity of an enzyme-linked oligosorbent assay diagnostic test. To understand the influence of their structure and conformation in solution on the efficiency of the test during the capture step, two different ways of synthesizing these conjugates were compared. The first consisted of coupling 5' amino modified oligonucleotides to poly(maleic anhydride-alt-methylvinyl ether) and poly(maleic anhydride-alt-ethylene). The second resulted from direct synthesis of oligonucleotides from poly(maleic anhydride-alt-ethylene) previously grafted onto a controlled pore glass support. The different conjugates were analyzed by size-exclusion chromatography and viscometry. The former method for conjugate synthesis produced aggregates, which was not the case for the latter. These conjugates were then used in the capture phase of a hybridization assay using a HBV DNA target, on a bioMérieux VIDAS instrument. Different parameters were studied, such as the purity of the conjugate solution and the number of oligonucleotides per polymer chain. The amount of conjugate coated on the solid-phase receptacle surface at the time of the capture phase was evaluated by radioactive labeling. Finally, it was demonstrated that conjugates produced an amplification factor of 50 versus the capture oligonucleotide probe used as the reference. The detection limit reached 10(8) copies/mL.