Chemiluminescence coupled assay

Determination of pyrogallol. Pyrogallol was assayed by HRP catalyzed imidazole chemiluminescence coupled to the micro-flow injection system at room temperature. Pyrogallol specimens (50 pL) were injected using an autosampler (AS-950, JASCO, Tokyo, Japan) every five min into a stream of water (100 uL/min) using a HPLC pump (PU-980, JASCO), and the other mobile phase (imidazole 100 mmol/L in the Tricine buffer 50 mmol/L, pH 9.3) was delivered at 100 pL/min. The light emitted from the reactor tube was detected with a... 

Clinical Analysis. A wide range of clinically important substances can be detected and quantitated using chemiluminescence or bioluminescence methods. Coupled enzyme assay protocols permit the measurement of kinase, dehydrogenase, and oxidases or the substrates of these enzymes as exemplified by reactions of glucose, creatine phosphate, and bile acid in the following ... 

Several other chromophores have been used in the development of sensors based upon ECL. For example, the luminol reaction is a conventional chemi-luminence reaction that has been studied in detail and it is believed that the mechanism of the ECL reaction is similar, if not identical, to that of the chemiluminescence. As shown in Fig. 2, the luminol ion undergoes a one-electron oxidation to yield a diazaquinone, which then reacts with peroxide or superoxide ( OOH) to give the excited 3-aminophthalate which has an emission maximum of 425 nm. This reaction is particularly versatile and has been utilized in a variety of ECL assays, many of which have been previously summarized by Knight [1], The luminol ECL reaction can be used for the determination of any species labeled with luminol derivatives, hydrogen peroxide, and other peroxides or enzymatic reactions that produce peroxides. A couple of examples are described later. 

Chemiluminescent assays are conventionally monitored using photomultiplier-based instruments. However, portable instruments are becoming available that use as photodetectors silicon photodiodes, charge-coupled devices, and instant photographic or x-ray film (58, 63-68). Spacially resolved, quantitative light measurements are pcirticularly advantageous for assays based on membranes or microtiter plates. 

An indirect chemiluminescence immunoassay is an assay, with another component than the primary chemiluminescent emitter coupled to the antigen or antibody. This can be a cofactor or a catalyst or even a molecule capable of converting a non-chemiluminescent precursor to a chemiluminescent or potentially chemiluminescent species. Most indirect assays are enzyme mediated. 

Chemiluminescent labels may be employed in sandwich or competitive antigen assays. In sandwich assays, a solid support holds a primary antibody, and incubation with ligand yields a species that is detectable following a second incubation step with a labeled second antibody. Luminol has been tested as an immunoassay label it may be coupled to proteins through its primary amino group. Luminol reacts with hydrogen peroxide and hydroxide in a microperoxidase-catalyzed reaction, which yields light at 430 nm (Eq. 6.8) ... 

Two-site immunometric or sandwich assays that made use of two or more antibodies directed at different parts of the PRL molecule were next to be developed. As with other two-site IRMA assays, the capture antibody is attached to a solid phase separation system and the second or signal antibody is labeled with a detection molecule (e.g., radio-isotope, enzyme,fluorophor, or chemiluminescence tag ). In some assays, the capture antibody is attached to the wall of test tubes, plastic beads, microtiter plates, ferromagnetic particles, or glass-fiber paper. Other assays have used the strep-avidin approach that couples biotin to the signal antibody with avidin linked to a solid phase. Most of the current immunometric assays for PRL have been adapted to fully automated immunoassay systems. Compared with the older traditional RIA methods, these automated immunometric assays for PRL generally achieve lower detection limits (0.2 to 1.0 ig/L) and improved precision (interlaboratory coefficients of variation of <8% at all concentrations), and have superior specificity (<0.05% crossreactivity with GH). 

Areas in which technological innovations have been accomplished include detection instruments [ranging from small portable luminometers (M3) to camera-type detectors (B32, K30) and charge-coupled devices (L3)], data analysis (E3, SI2), and assay formatting, e.g., immunofiltration (HI6). The so-called camera luminometers (B32) offer the joint advantages of ease of use, portability, and detectability without an electrical power supply. With fast film such as Polaroid 612 (20,000 ASA), exposure times can be as short as 5 sec. Examples of assays using enhanced chemiluminescence and Polaroid 612 film include a-fetoprotein (L9), albumin (L8), immunoglobulins and transferrin (J3), and ferritin (07). 

The first type of enzyme application in microfluidics is chemical sensing. Sensors can be constmeted in cases where an enzyme turns over a particular smaU-molecule substrate to produce a product quantifiable by fluorescence, chemiluminescence, absorbance spectroscopy, or electrochemical detectors. In cases where the substrate is not detectable itself, an enzymatic product can often be coupled to another enzyme that produces a detectable product. For example, there are a wide variety of small molecules (such as nutrients, amino acids, and sugars) that can be coupled to the chemiluminescent reaction of luminol and peroxide in the presence of horseradish peroxidase. These enzyme-substrate assays were the first to be adapted to microfluidic devices — a great number of small-molecule sensors have been developed based on microfluidic channels with electroosmotic or hydrodynamic flow, and pre-loaded microfluidic cartridges containing nanoliter volumes of reagents have... 

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