Fluorescent conjugates, detection

Kumaraswamy S, Bergstedt T, Sh X, Rininsland F, Kushon S, Xia W, Ley K, Achyuthan K, McBranch D, Whitten D (2004) Fluorescent-conjugated polymer superquenching facilitates highly sensitive detection of proteases. Proc Natl Acad Sci 101 7511-7515... 

Thomas SW, Swager TM (2006) Trace hydrazine detection with fluorescent conjugated polymers a tum-on sensory mechanism. Adv Mater 18 1047-1050... 

Fig. 1. (opposite page) Distribution of FITC-conjugated BSA in various fibroblast cell lines under different fixation/permeabilization regimes. (A-D) Protein distribution in living cells (A) PtKj, (B) CHO, (C) 3T3, and (D) HeLa cells. The protein is excluded from the nuclei of all cells. (E-H) Protein distribution in cells extracted for 10 min with 0.1% Triton X-100 before fixation for 30 min with 3.7% formaldehyde (E) PtKi, (F) CHO, (G) 3T3, and (H) HeLa cells. Nuclear fluorescence is seen in (E) PtKj and (G) 3T3 cells. (I-L) Protein distribution in cells extracted for 10 min with 1% Triton X-100 before fixation for 30 min with 3.7% formaldehyde (I) PtKj, (J) CHO, (K) 3T3, and (L) HeLa cells. No fluorescence is detected in the cells with the exception of some nuclear fluorescence seen in (L) HeLa cells. (M-P) Protein distribution in cells fixed for 30 min with 3.7% paraformaldehyde before permeabilization for 10 min with 0.1% Triton X-100. Fluorescence is seen primarily in the cytoplasm with the exception that nuclear fluorescence is seen in (M) PtKi and (N) CHO cells. (Q-T) Protein distributions in cells fixed for 5 min with 90% methanol, 50 vaM EGTA at -20°C (Q) PtKj, (R) CHO, (S) 3T3, and (T) HeLa cells. All cells show an overall low fluorescence, fibrous-textured cytoplasmic fluorescence, and bright staining at the periphery of the nucleus. 10 mm per scale division (black bar). (Reproduced with permission from ref. 6.)... 

Lucifer Yellow probes are water-soluble to at least 1.5%. The absorbance maximum of the derivatives occurs at about 426—428 nm with an emission peak at about 530—535 nm, in the yellow region of the spectrum. The quantum yield of Lucifer dyes is about 0.25. The good intensity of luminosity from these dyes makes possible detection of small quantities of labeled molecules intracellularly. The fluorescent conjugates are readily visible in living cells at concentrations that are nontoxic to cell viability. The low molecular weight and water solubility of these dyes allow passage of labeled compounds from one cell to another, potentially revealing molecular relationships... 

In another approach, a fluorescent conjugated polymer was used as the material for the preparation of a chemosensor to detect 2,4,6-trinitrotoluene (TNT) and its related nitroaromatic compounds. To this end, microparticles, made of three-dimensionally cross-linked poly(l,4-phenylene vinylene) (PPV) via emulsion polymerization, were synthesized [61]. This material was chosen due to its high fluorescence intensity and sensitivity to changes in its microenvironment. The chemosensor was exposed to vapour containing different amounts of TNT and quenching of the polymer luminescence at 560 nm was observed after excitation at 430 nm. The dependence of the fluorescence signal in response to the analyte was described by a modified Stem-Volmer equation that assumes the existence of two different cavity types. The authors proposed the modified Stem-Volmer equation as follows ... 

The effect of temperature on fluorescence has been studied, as has the effect of salt concentration and water-soluble conjugated polymers. A method for the quantification of ssDNA dsDNA is described, as well as kinetics of mismatch hybridization and the kinetics of collision in short ss-nucleic acids. Fluorescence quenching of Cy-5 labelled oligonucleotides by poly(phenylene ethynylene) particles has been shown to be a more sensitive method than excitation of the Cy-5 fluorophore. An ultrasensitive method for the detection of DNA uses highly fluorescent conjugated nanoparticles, and detection limits below IfM were achieved. DNA transport through a carbon nanotube has also been observed using fluorescence microscopy. " ... 

Detection methods are critical for future automation, since detection can be a rate-limiting step in an automated process. In addition to common detection methods used in manual IA techniques, other detection methods have been applied to automation. For example, ECL technology has been used for nucleic acids in the IGEN system. Cell surface markers are detected by the Copalis system using a fluorescent conjugate and flow cytometry. New detection methods for drug IAs as well as PD measurements for efficacy and safety should prove useful in the future. 

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