Fluorescence-based assays quantification

Fluorescence-based assays either in the measurement of enzyme activity or in the quantification of enantioselectivity all have a high degree of sensitivity, which allows the use of very dilute substrate concentrations and extremely small amounts of enzymes. Basically, there are two different approaches. One involves the use of a substrate of interest to which a fluorescent-active (or potentially active) moiety is covalently attached. The second approach makes use of a fluorescence-based sensor, which gives rise to a signal as a consequence of the enzyme-catalyzed reaction of a substrate of interest. 

Maus L, Spatz JP, Fiammengo R (2009) Quantification and reactivity of functional groups in the ligand shell of PEGylated GNPs via a fluorescence-based Assay. Langmuir 25 7910-7917... 

Total protein assays have the advantage of being relatively straightforward compared to molecular-level analyses. Methods with fluorescence-based detection are also highly sensitive, and thus amenable direcdy to DON. Quantitative interpretation for environmental mixtures such as seawater, however, may be problematic for some samples. Most methods react with specific moieties (e.g., coomassie blue binds to lysine and arginine) and thus results obtained can depend on protein composition, size distribution, and even conformation (Sapan et ai, 1999), making the careful choice of calibration standards important. In addition, common components of natural samples, such as humic materials (e.g., Mayer et ai, 1986), carbohydrates (Sapan et ai, 1999), or NH3 may interfere with quantification. Overall, colorimetric methods can be very useful as quick, Hkely semi-quantitative estimates of total protein or peptide. However, potential biases inherent in the mechanism of a specific method should be considered before one is chosen, and appHcation of newer molecular assays (e.g., CBQCA) should be carefully examined in terms of natural sample matrix (Nunn et ai, 2003). 

Fig. 3 Typical microarray-based assay. A microarray is incubated with a solution containing multiple target molecules. After removal of unbound molecules, the target-bound microarray is stained with fluorescence, followed by imaging and quantification...

Martin, H. Comeskey, D. Simpson, R. M. Laing, W. A. McGhie, T. K. Quantification of folate in fruits and vegetables A fluorescence-based homogeneous assay. Anal. Biochem. 2010,402,137-145. 

Fig. 2 Increasing content recovery by coupling luciferase-based assays with high-throughput biochemical readouts. The same cell line subjected to the luciferase-based assay protocol (HCT116 cells) is evaluated here for its reliability In reporting a biochemical readout (p53 expression) by dot blot analysis. Cells transfected with indicated expression construct and pathway reporters in a 96-well culture plates were lysed 48 h posttransfection. Following luciferase activity measurements (data not shown), protein from spent lysates was immobilized on nitrocellulose using a liquid handler and filtration manifold, (a) p53 and p-actin protein levels detected using protein-specific antibodies, infrared fluorescent dye-coupled secondary antibodies (with emissions at 680 and 800 nM), and the Li-COR imaging system. Columns of lysate corresponding to cells transfected with p53 DNA are boxed, (b) Quantification of the p53 to p-actin protein ratio...

As an example, the assay commonly used to measure the direct generation of ROS by nanoparticles is based on the conversion by ROS of the 2,7-dichlorodihydrofluorescein dye into a fluorescent product, 2,7-dichlorofluorescein. There is also a range of fluorescent probes that measure NM-induced ROS production inside the cells, in different intracellular compartments (e.g., dihydrorhoda-mine-1,2,3 in the mitochondria, 2,7-dichlorodihyydrofluorescein diacetate in the cytoplasm, dihydroethidium bromide in the nucleus) [59]. They are all relatively easy to use for quantification in a fluorimeter, multiwall plate reader, or by flow cytometry, but a potential drawback of all these assays is the background caused by particles as well as the fluorescence quenching effects that need to be controlled and taken into account to reliably measure the free radical production [59, 60]. 

As mentioned in the Section 1, physico-chemical methodology for quantitative analysis of plant hormone focuses primarily on GC-SIM, although HPLC with selective fluorescence detection continues to be used for lAA analysis in some laboratories. Procedures, such as the 2-methylindolo-a-pyrone assay for lAA analysis [82], are now rarely utilised. With the exception of ethylene quantification [2] there is little use of non-MS-based GC detection techniques, despite the fact that selective analysis at the picogram level is achieved for ABA with an electron capture detector [83], and lAA and cytokinins with a nitrogen phosphorus detector [84,85]. The reason for the demise of these GC procedures is that the detectors are destructive and this precludes the reliable recovery of labelled internal standards for radioassay and isotopic dilution analysis. The usual compromise was to take two aliquots of the purified samples, one for GC analysis and the other for the determination of radioactivity. The accuracy of this approach is dependent upon the questionable assumption that the radioactivity in the purified sample is associated exclusively with the compound under study. In an attempt to circumvent this problem, a double standard isotope dilution procedure was devised for the quantitative analysis of lAA in which one internal standard was used to correct for losses during sample preparation and a second for GC quantification [86]. This procedure was used in several... 

Various detection methods, such as fluorescence, ultraviolet absorption, and others have been combined with chromatographic methods. New methods based on the production of antibodies specific for individual mycotoxins have also beoi developed and include enzyme-linked immunosorbent assays and immunoaffinity colunms. These methods allow for specific and precise detection and quantification of specific mycotoxins. This has led to the development of test kits for mycotoxins, such as VtCAM , which are rapid and simple to use and can be used in the field and throughout the processing stages. 

Recently, a universal enzyme-coupled fluorescence assay for glycosyl transferases was developed. This method is extremely cost-effective and is based on the quantification of nucleotides produced in the glycosyl transfer reaction. The guanosine diphosphate (GDP), uridine diphosphate (UDP), and cytidine monophosphate (CMP) are phos-phorylated with nucleotide kinase in the presence of excess of ATP, generating ADP. Via coupled enzyme reactions involving ADP-hexokinase,glucose-6-phosphate dehydrogenase, and diaphorase, the ADP is utilized for the conversion of resazurin to resorufin, which is then quantified by fluorescence measurement. 

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