The synthesis of a polyphenylene dendrimer carrying three perylenemonoimide dyes as well as one biotin group is presented. Due to the hydrophobic polyphenylene scaffold, this dendrimer is insoluble in water thus preventing investigations in aqueous media. However, the use of an appropriate detergent results in the formation of well-defined supramolecular dendrimer-detergent complexes being soluble in aqueous media. The dendrimer-detergent complexes have a constant hydrodynamic radius of 7.1 nm measured by light scattering and fluorescence correlation spectroscopy and exhibit a high stability in the presence of blood serum proteins. The specific binding of the dendrimer-detergent complexes carrying a single biotin group to the protein streptavidin is demonstrated using a magnetic bead assay.

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.

ABSTRACT:A water-soluble statistical poly(N-acryloylmorpholine-co-N-acryloxysuccinimide) [poly(NAM/NAS)] copolymer was studied for polymer-oligonucleotide (ODN) conjugate elaboration and for further use in diagnostic applications. Three different copolymers were first prepared by free-radical solution polymerization with different Nacryloylmorpholine (NAM) and N-acryloxysuccinimide (NAS) molar ratios (80/20, 70/30, and 60/40). Their number-average molecular weights ranged from 98,000 to 120,000 g/mol, as determined by aqueous size exclusion chromatography with an online light-scattering detector. Then, polymer-ODN conjugates were obtained via a strategy consisting of the direct synthesis of ODNs onto polymer chains previously grafted onto a controlled pore glass support. Before the grafting of the polymer onto the solid support, a preliminary step was performed to bind a nucleotide starter along the polymer chain (via the reactive NAS units) to initiate automated DNA synthesis. To multiply the number of ODNs growing from starters, a branched phosphoramidite synthon [bearing two O-dimethoxytrityl groups] was introduced at the first step of ODN elongation as a short sequence of four branched synthons alternated with three thymidine residues. Conjugates were assessed in a DNA sandwich hybridization test developed for hepatitis B virus detection. Sensitivity limits were evaluated and compared to those obtained with an other polymer, poly(maleic anhydride-alt-methyl vinyl ether) [poly(MA/MVE)]. A sensitivity limit of 2.6 ϫ 10 7 DNA copies/mL was reached with the poly(MA/MVE)-ODN conjugate at the capture phase and with the poly(NAM/NAS)-branched ODN conjugate at the detection phase of the test.