Fluorescent probes passive

Fluorescent probes. Fluorescence probes are materials which indicate, by fluorescence, the presence of some specific chemical species or environment. There are two types passive and active. Passive probes indicate the presence of the material of interest simply by physically or chemically binding to it. They thus indicate the presence of an analyte substrate by their own fluorescence (although this may also be altered by the presence of the analyte), the intensity of which can be used to determine the presence of the analyte either qualitatively or in some cases quantitatively. A typical use of such probes is in fluorescence microscopy, and there are a wide range of commercially available probes for this purpose (e.g. 4.3). Active probes undergo a photochemical change in the presence of the chemical or environmental feature to which they are sensitive. This results in a change in some emission property such as wavelength, intensity, polarisation, or lifetime (e.g. 1.4, 4.8). Optical probes are discussed in detail in Chap. 12. 

It is common to use the term "probe to describe a small dye molecule added passively to a system for study. By contrast, a label is attached covalently to some component of the system. Fluorescent probe experiments are easy to carry out, but the onus rests with the experimenter to prove where in the system the probe is located. Labelling experiments are more demanding because of the need to synthesize the labelled component. Data interpretation is often easier because the dilemma of dye location is less severe. We like the phrase luminescent sensor to describe the general case where a dye is used either as a label or as a probe. 

Accumulation/efflux studies can be performed on different cell systems or membrane vesicle preparations. In the accumulation assays, uptake of a probe over time, typically either fluorescent (e.g. calcein-AM (CAM) [25-27]) or radiolabeled, into the cell or membrane vesicles is measured in the presence or absence of a known P-gp inhibitor. As P-gp transports substrates out of the cells, the inhibition of the protein would result in an increase in the amount of the probe in the cell. Accumulation studies in cells that overexpress P-gp can be compared to those obtained in the parental cell line that does not have as high a level of P-gp expression. The probe in the absence of inhibitors shows lower accumulation in P-gp expressing cells than in P-gp deficient cells. Similarly, probe accumulation is increased under conditions where P-gp is inhibited such that the difference in accumulation in P-gp deficient and overexpressing cells, respectively, becomes smaller. Accumulation assays poorly distinguish substrates and inhibitors of P-gp and, as far as transport assays are concerned, are also influenced by a passive diffusion property of molecules [20]. In contrast to transport assays, both accumulation (i.e. calcein-AM assay) and ATPase assays tend to fail in the identification ofrelatively low permeable compounds as P-gp active compounds [20]. 

QDs may act as an active sensory element (for chemical analysis applications) when the intrinsic fluorescence properties of the QDs are changed upon reaction with the analyte (e.g., by surface modification). In probes with QDs as passive labels, however, selective receptor molecules (such as antibodies) are conjugated to the surface of QDs. In this latter approach, QD-labeled antibodies are used to recognize the analyte and, in this way, visualize the eventually bound antigen. 

Probes—Natural Fluorophores and Fluorescent Indicators 2.2.1. PASSIVE REPORTER GROUPS... 

This definition also clarifies what is not a chemical sensor or biosensor for purposes of market projections and commercialization. Passive assays such as, for example, colorimetric chemical reactions, immunoassays, and nucleic acid probes are not sensors. While such assays do result in a quantifiable entity (such as color production, fluorescence, etc), the assay itself does not provide the means to quantify the response. Rather, a separate quantification system is required. 

A particularly useful technique for studying such systems involves labelling one individual component with a dye. Depending upon the dye one can study the quenching of its fluorescence or phosphorescence by a mobile probe added passively to the system. From the rates and efficiency of the quenchin, one can learn about local morphology (with resolution of 10 A to 100 A) one can measure the extent of interphase formation and one can study molecular relaxation processes in individual phases of the material. 

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