Fluorescence of enzymes

Galban J, Andreu Y, Sierra IF, de Marcos S, Castillo JR. Intrinsic fluorescence of enzymes and fluorescence of chemically modified enzymes for analytical purposes a review. Luminescence 2001 16 199-210. 

Ha T, Ting A Y, Liang J, Caldwell W B, Deniz A A, Chemla D S, Schultz P G and Weiss S 1999 Single-molecule fluorescence spectroscopy of enzyme conformational dynamics and cleavage mechanism Proc. Natl Acad. Sc/. USA 96 893-8... 

The dye is excited by light suppHed through the optical fiber (see Fiber optics), and its fluorescence monitored, also via the optical fiber. Because molecular oxygen, O2, quenches the fluorescence of the dyes employed, the iatensity of the fluorescence is related to the concentration of O2 at the surface of the optical fiber. Any glucose present ia the test solution reduces the local O2 concentration because of the immobilized enzyme resulting ia an iacrease ia fluorescence iatensity. This biosensor has a detection limit for glucose of approximately 100 ]lM , response times are on the order of a miaute. 

Quantitative polymerase chain reaction, also called real-time RT-PCR or QPCR, is a method which employs insertion of a signal, such as fluorescence or enzyme activity, into PCR products generated by RT-PCR to determine the amount of messenger RNA (mRNA) in a tissue accurately. 

The assay of enzymes whose reactions are not accompanied by a change in absorbance or fluorescence is generally more difficult. In some instances, the product or remaining substrate can be transformed into a more readily detected compound. In other instances, the reaction product may have to be separated from unreacted substrate prior to measurement—a process facili-... 

Several types of labels have been used in immunoassays, including radioactivity, enzymes, fluorescence, luminescence and phosphorescence. Each of these labels has advantages, but the most common label for clinical and environmental analysis is the use of enzymes and colorimetric substrates. 

Secondary antibody and determination. A secondary antibody labeled with an enzyme is added which binds to the primary antibody that is bound to the coating antigen. If the primary antibody were produced in a rabbit, an appropriate secondary antibody would be goat anti-rabbit immunoglobulin G (IgG) conjugated with horseradish peroxidase (HRP) (or another enzyme label). Excess secondary antibody is washed away. An appropriate substrate solution is added that will produce a colored or fluorescent product after enzymatic conversion. The amount of enzyme product formed is directly proportional to the amount of first antibody bound to the coating antigen on the plate and is inversely proportional to the amount of analyte in the standards. 

Figure 18 Flow chart of the automated on-line flow injection immunoassay (FllA). Six steps are involved in each cycle (1) addition of antibody and incubation (2) addition of analyte (or standard) and incubation (3) addition of enzyme-tracer and incubation (4) addition of substrate and incubation (5) downstream measurement of fluorescence (6) regeneration of affinity column...

Proteases are one of the largest families of enzymes and are involved in a multitude of vital processes. Due to their biological relevance and diversity, multiple fluorescent reporters monitoring their activity have been designed and successfully applied in vitro and in vivo [112-114]. Standard small molecule FRET probes for proteases consist of an amino acid sequence flanked by a FRET pair, consisting of two fluorophores or one fluorophore and a quencher molecule. Upon cleavage of the peptide sequence, the emission of the donor fluorophore is dequenched and the intensity increases whereas the emission of the acceptor decreases and vanishes more or less completely in those cases where the acceptor is fluorescent (see Fig. 6.11). 

Volkl K.P., Opitz N., Lubbers D.W., Continuous measurement of concentrations of alcohol using a fluorescence-photometric enzymic method, Fresenius J. Anal. Chem. 1980 301 162. 

Lanthanide chelates also can be used in FRET applications with other fluorescent probes and labels (Figure 9.51). In this application, the time-resolved (TR) nature of lanthanide luminescent measurements can be combined with the ability to tune the emission characteristics through energy transfer to an organic fluor (Comley, 2006). TR-FRET, as it is called, is a powerful method to develop rapid assays with low background fluorescence and high sensitivity, which can equal the detection capability of enzyme assays (Selvin, 2000). 

The rapid turnover rate of some enzymes allows ELISAs to be designed that surpass the sensitivity of radiolabeling techniques. In addition, substrates can be chosen to produce soluble products that can be accurately quantified by their absorbance or fluorescence. Alternatively, substrates are available which form insoluble, highly colored precipitates, excellent for localizing antigens in blots, cells, or tissue sections. The flexibility of enzyme-based assay systems makes the chemistry of enzyme conjugation one of the most important application areas in bioconjugate techniques. 

Enzymes useful for detection purposes in ELISA techniques (Chapter 26) also can be employed in the creation of highly sensitive DNA probes for hybridization assays. The attached enzyme molecule provides detectability for the oligonucleotide through turnover of substrates that can produce chromogenic or fluorescent products. Enzyme-based hybridization assays are perhaps the most common method of nonradioactive detection used in nucleic acid chemistry today. The sensitivity of enzyme-labeled probes can approach or equal that of radiolabeled nucleic acids, thus eliminating the need for radioactivity in most assay systems. 

The dianion 117 bonded to enzyme (firefly luciferase) appears to be the emitter in blue-green firefly bioluminescence as the emission spectrum exactly matches the fluorescence of 117, and in an analogous way in the case of red bioluminescence the emitter is 115. 

The performance of a biotreatment system ultimately depends on optimization of the activity of microbes and the ability to control the process parameters of the treatment system [157]. In this respect, the ability to monitor gene copy numbers and gene expression is highly useful for real time optimization of the efficiency of a biotreatment system. Advanced molecular techniques as well as low cost methods (e.g., antibody detection of enzymes based on color reaction strips fluorescence i.e., GFP marked organisms with UV light detection) can also be applied to monitor the microbial community structure, persistence of the added bacteria, and their interactions with indigenous populations. 

Measurement of blue and green fluorescence of NADH and FAD in living tissues Quantitative fluorescent cytochemistry Using permeable fluorogenic substrates of enzymes, specific inhibitors, and kinetic analysis... 

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