This article presents a new, highly sensitive method for the identification of single nucleotide polymorphisms (SNPs) in homogeneous solutions using fluorescently labeled hairpinstructured oligonucleotides (smart probes) and fluorescence single-molecule spectroscopy. While the hairpin probe is closed, fluorescence intensity is quenched due to close contact between the chromophore and several guanosine residues. Upon hybridization to the respective target SNP sequence, contact is lost and the fluorescence intensity increases significantly. High specificity is achieved by blocking sequences containing mismatch with unlabeled oligonucleotides. Time-resolved single-molecule fluorescence spectroscopy enables the detection of individual smart probes passing a small detection volume. This method leads to a subnanomolar sensitivity for this single nucleotide specific DNA assay technique.

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Abstract:The aim of our work was to study the structure of a typical modern cosmetic oil-in-water emulsion (o/w-emulsion), based on the emulsifier polyglyceryl-3 dicitrate/stearate and glycerylstearate/stearyl alcohol as consistency enhancer. We have used a systematic approach building up the cosmetic emulsion step by step, characterizing all systems by using Small Angle Neutron Scattering (SANS), Differential Scanning Calorimetry (DSC), Freeze Fracture Transmission Electron Microscopy (FF-TEM), light microscopy and rheology. The starting point was the pure emulsifier in water, which was shown to form lamellar stacked bilayers with a spacing of 7 nm, coexisting with polydisperse unilamellar vesicles in the sub-µm range. Upon addition of consistency enhancer, also multilamellar vesicles could be obtained. Then, oil has been added stepwise, until finally a complete cosmetic o/w-emulsion was obtained. In the final emulsion, oil droplets with sizes in the µm range are surrounded by multiple, irregularly spaced bilayer structures and vesicles. Approximately 30% of the water present in the system shows a restricted mobility ("encapsulated water") according to PFG-NMR. Crucial for the viscosity build-up is the presence of the oil droplets; a cream-like consistency is obtained by steric interaction of oil droplets and crystalline bilayer structures in the aqueous phase of the emulsion.

In this article we report on two different classes of self-quenching hairpin-structured DNA probes that can be used as alternatives to Molecular Beacons. Compared to other hairpin-structured DNA probes, the so-called smart probes are labeled with only one extrinsic dye. The fluorescence of this dye is efficiently quenched by intrinsic guanine bases via a photo-induced electron transfer reaction in the closed hairpin. After hybridization to a target DNA, the distance between dye and the guanines is enlarged and the fluorescence is restored. The working mechanism of the second class of hairpin DNA probes is similar, but the probe oligonucleotide is labeled at both ends with an identical chromophore and thus the fluorescence of the closed hairpin is reduced due to formation of non-fluorescent dye dimers. Both types of probes are appropriate for the identification of single nucleotide polymorphisms and in combination with confocal single-molecule spectroscopy sensitivities in the picomolar range can be achieved.