Quenching | EPFL Graph Search

In chemistry, quenching refers to any process which decreases the fluorescent intensity of a given substance. A variety of processes can result in quenching, such as excited state reactions, energy transfer, complex-formation and collisions. As a consequence, quenching is often heavily dependent on pressure and temperature. Molecular oxygen, iodine ions and acrylamide are common chemical quenchers. The chloride ion is a well known quencher for quinine fluorescence. Quenching poses a problem for non-instant spectroscopic methods, such as laser-induced fluorescence. Quenching is made use of in optode sensors; for instance the quenching effect of oxygen on certain ruthenium complexes allows the measurement of oxygen saturation in solution. Quenching is the basis for Förster resonance energy transfer (FRET) assays. Quenching and dequenching upon interaction with a specific molecular biological target is the basis for activatable optical contrast agents for molecular imaging. Many dyes undergo self-quenching, which can decrease the brightness of protein-dye conjugates for fluorescence microscopy, or can be harnessed in sensors of proteolysis. Förster resonance energy transfer There are a few distinct mechanisms by which energy can be transferred non-radiatively (without absorption or emission of photons) between two dyes, a donor and an acceptor. Förster resonance energy transfer (FRET or FET) is a dynamic quenching mechanism because energy transfer occurs while the donor is in the excited state. FRET is based on classical dipole-dipole interactions between the transition dipoles of the donor and acceptor and is extremely dependent on the donor-acceptor distance, R, falling off at a rate of 1/R6. FRET also depends on the donor-acceptor spectral overlap (see figure) and the relative orientation of the donor and acceptor transition dipole moments. FRET can typically occur over distances up to 100 Å. Dexter electron transfer Dexter (also known as Dexter exchange or collisional energy transfer, colloquially known as Dexter Energy Transfer) is another dynamic quenching mechanism.

Novel Chemosensors for Biologically Important Analytes

Ziya Köstereli

In this work, fluorescent-based chemosensors for the detection of important analytes in aqueous solution are described. The sensors are based on different detection concepts, including analyte-induced aggregation/de-aggregation, pattern-based sensing and micelle-based sensing. In chapter 2, we present an analyte-induced aggregation-type chemosensor for the sensing of spermine. A charge-frustrated amphiphile composed of a highly fluorescent pyrene-1,3,6-trisulfonate head group and an eicosane side chain was used as a fluorescence chemosensor. Analyte-induced aggregation of the dye upon addition of spermine results in pronounced fluorescence quenching. The sensor enables the detection of spermine down to the nanomolar concentration range with good selectivity over closely related biogenic amines such as spermidine. In chapter 3, we describe a conceptually new âone-cuvetteâ sensing system for the pattern-based analysis of seven aminoglycosides antibiotics. The antibiotics include amikacin, apramycin, gentamicin, kanamycin A and B, neomycin, and paromomycin. A mixture of two amphiphiles with fluorescent head groups was used as a sensing ensemble. In buffered aqueous solution, the amphiphiles form a dynamic mixture of micellar aggregates. In the presence of aminoglycosides, the relative amount and the composition of the micelles is modified. Accurate differentiation in the low micromolar concentration range is achieved by a principal component analysis of the spectral data. Also, the sensing system allows the differentiation of pure aminoglycosides from their mixtures. In chapter 4, a fluorescent chemosensor based on analyte-induced aggregation/de-aggregation is described for the sensing of Al3+ and citric acid. In buffered aqueous solution, the amphiphilic dye can form aggregates and the aggregation of the dye is associated with a strong quenching of its fluorescence. The Al3+-induced aggregation is used to sense Al3+ in the low micromolar concentration range with high selectivity. Furthermore, we demonstrate that the non-fluorescent dye-Al3+ complex can be used as a sensing ensemble for the detection of citric acid. The assay is able to quantify the citric acid content of commercial beverages such as energy drinks. A simple micelle-based assay for the fluorescence sensing of vitamin K1 is described. In the following chapter 5, the assay enables the detection of vitamin K1 in the low micromolar concentration range. As a sensing ensemble, a mixture of the surfactant triton X-100 and 1 aminopyrene in buffered aqueous solution is employed. Vitamin K1 co-localizes with the fluorescent pyrene dye in the micelle, resulting in fluorescence attenuation by dynamic quenching. The assay displays good selectivity and can be used to determine the concentration of vitamin K1 in a commercial preparation. In chapter 6, a fluorescent-based sensor array for the optical analysis of purine derivatives is described. The array is composed of four polysufonated fluorescent dyes. The complexation of the analytes results in partial quenching of the fluorescence. The Sensor array enables the identification of ten purine derivatives including caffeine, theophylline, theobromine, purine, hypoxanthine, paraxanthine, 8-chlorotheophylline, 6-mercaptopurine, cladribine and penciclovir at low millimolar concentration. Furthermore, it is possible to use the array for obtaining information about the quantity and the purity of samples.

EPFL2015

Optimalisation de la photothérapie dynamique et de la photodétection de cancers précoces par spectroscopie résolue en temps de luminophores endogènes et exogènes

Pascal Uehlinger

The aim of this thesis is to optimize and obtain fundamental information on i) the photodetection (PD) of early stage cancers in the tracheo-bronchial tree by spectroscopic imaging of the tissue autofluorescence, ii) photodynamic therapy (PDT) applied in dermatology based on the use of protoporphyrin IX (PPIX) precursors. These two areas of photomedecine have been the subject of investigation at the air and soil pollution laboratory (LPAS) for the last 10 years. PD and PDT are areas of research that evolve in parallel, both having similar objectives: i.e. improving the patients' chances of survival by the early detection and treatment of cancer. Three main studies are presented, each in relation to a particular type of chromophore : Endogenous fluorophores generate the autofluorescence of biological tissue; some of these fluorophores can be synthesized endogenously in excess of their normal concentration following the administration of a biochemical precursor; finally, exogenous luminophores are artificially synthesized and administered to the organism where they can be used for detection. The introduction of this thesis presents several aspects of cancer as well as the theoretical background necessary for this study. At first we address time-resolved autofluorescence spectroscopy of bronchial tissue and the mechanisms at the origin of the contrasts existing between normal tissue and early stage tumours. Several hypotheses explain the reduction in intensity and spectral changes of the autofluorescence observed between early epidermal carcinoma and normal bronchial mucosa : 1) the reduction in the concentration of fluorophores in the malignant tissue, 2) the quenching of the fluorescence due to a change in the physico-chemical environment of the tumour, 3) an architectural effect such as a thickening of the malignant epithelium due to an abnormal cellular proliferation and 4) an increase in the concentration of light absorbers such as haemoglobin. In the course of our measurements no statistically significant difference in the fluorescence lifetime was observed between carcinoma in situ, moderate dysplasia and normal tissue, which leads us to conclude that hypotheses 1, 3 and/or 4 may well be at the origin of the differences in fluorescence intensity. The fluorescence lifetime decays are consistent, identifiable by a triple-exponential decay in the green and red part of the spectrum. This seems to indicate that one or two dominant fluorophores are involved which possibly, given the measured lifetimes, could be collagen and/or elastine. The second part of this thesis concerns the modulation of the production of PPIX (an endogenously induced fluorophore) in the skin, following the administration of 5-aminolevulinic acid or one of its esterified derivatives. These investigations have been carried out on the epidermis of human volunteers as well as on the epidermal equivalent (Epidex™). The final goal of these studies was on the one hand to increase the production of PPIX well below the skin surface to improve certain dermatological treatments such as the PDT of nodular basal cell carcinomas (BCC), and, on the other hand, the ability to use the results obtained with the Epidex™ model to produce PPIX specifically in hair follicles, which permits the development of a new method of hair removal by PDT. The tests using the Epidex™ model demonstrated that it is possible, by adding Desferal® (an iron chelator) or (L+) ascorbic acid iron (II) salt, to increase respectively decrease the production of PPIX as well as to modulate its elimination. These results open new perspectives such as the specific use of zinc-protoporphyrin (produced by skin cells following the dermatological use of Desferal®), and the identification of a specific precursor of PPIX for hair follicles. This precursor is an excellent "candidate" in the field of research for the development of a new method of hair removal by PDT. In the third part of this thesis we describe a preliminary study of optical sensors for the intravascular partial pressure of oxygen via the measurement of phosphorescence lifetime quenching. The phosphorescence lifetime of these species is directly related to the partial pressure of the oxygen dissolved (pO2). These oxygen sensors allow us, for instance, to measure the reduction of the pO2 during PDT, without phototoxicity. Palladium and ruthenium complexes were among the most promising compounds, and certain types of nanoparticules were used as vectors. Another interesting lead consists in the covalent modification of these "luminophores-molecules": in this context, the experiments conducted in collaboration with the "Laboratoire de photonique et interfaces" of the EPFL, demonstrate that we can drastically reduce the speed of leakage outside the vascularisation of these molecules and/or improve their retention by the nanoparticules.

EPFL2004