Chemiluminescence-based detection

In glycoprotein detection systems the carbohydrate portions of proteins are oxidized with sodium metaperiodate to generate aldehydes that can react with hydrazides. A biotin hydrazide is used to attach biotin onto the oxidized carbohydrates and horseradish peroxidase-conjugated streptavidin is used for chemiluminescence-based detection (Glycoprotein Detection Module, Amersham Biosciences, Uppsala, Sweden). 

Baeynes, W.R. Schulman, S.G. Calokerinos, A.C. Zhao, Y. Garcia-Campana, A.M. Nakashima, K. De Keukeleire, D. Chemiluminescence-based detection principles and analyti- 50. cal applications in flowing streams and in immunoassays. J. 

In the chemiluminescence-based HPLC detection system, illustrated schematically in Figure 6, the oxalate ester and hydrogen peroxide are introduced to the eluent stream at postcolumn mixer Mj, which then flows through a conventional fluorescence detector with the exciting lamp turned off or a specially built chemiluminescence detector. The two reagents are combined at mixer Mj, rather than being premixed, to prevent the slow hydrolytic reactions of the oxalate ester. 

It is appropriate at this juncture to illustrate the power of chemiluminescence in an analytical assay by comparing the limits of sensitivity of the fluorescence-based and the chemllumlnescence-based detection for analytes in a biological matrix. The quantitation of norepinephrine and dopamine in urine samples will serve as an illustrative example. Dopamine, norepinephrine, and 3,4-dihydroxybenzy-lamine (an internal standard) were derivatized with NDA/CN, and chemiluminescence was used to monitor the chromatography and determine a calibration curve (Figure 15). The limits of detection were determined to be less than 1 fmol injected. A typical chromatogram is shown in Figure 16. 

AMPPD is the best chemiluminescent substrate for detecting an ALP-labeled probe [2, 3], The enhanced sensitivity of the chemiluminescence based on the reaction of AMPPD with ALP depends on the enzymatic reaction time (Fig. 2), because the slow kinetics of the signal decay result in the accumulation... 

Relative purity measurement and the relative purity-based reaction optimization have long been used in combinatorial synthesis. In order to make high-through-put purification a success, the yield-based optimization is essential. Chemiluminescent nitrogen detection (CLND) [4] with HPLC determines the quantitative yield after each reaction step during the library feasibility and rehearsal stages. The yield of each synthetic step provides guidance for the final library synthesis. 

Other detection modes employed in capillary electromigration techniques include chemiluminescence [69-71], Raman spectroscopy [72,73], refractive index [74,75], photothermal absorbance [76,77], and radioisotope detection [78]. Some of these detection modes have found limited use due to their high specificity, which restricts the area of application and the analytes that can be detected, such as radioisotope and Raman-based detection that are specific for radionuclides and polarizable molecules, respectively. On the other hand, the limited use of more universal detection modes, such as refractive index, is either due to the complexity of coupling them to capillary electromigration techniques or to the possibility of detecting the analytes of interest with comparable sensitivity by one of the less problematic detection modes described above. 

Dioxetanes, labeled with triggers sensitive to the alkaline-phosphatase enzyme, serve as highly sensitive chemiluminescent probes in numerous bioassays. Current applications include immunoassays, membrane-based detection of proteins and nucleic acids, and microplate-based and array-based nucleic-acid detection. ... 

In LC/MS analysis of combinatorial libraries, the MS determines the product identity and its purity is determined by other on-line detection techniques such as UV, evaporative light scattering detection (ELSD), and chemiluminescent nitrogen detection (CLND).17-20 UV detection is used here to assess product purity based on the assumption of similar absorption coefficients at 214 nm for the desired product and the side-products. 

Third generation immunometric assays have been developed for TSH measurements that have enhanced precision at even lower detection limits. Most of these assays use chemiluminescence-based technologies and have a functional detection limit of less than 0.02 mlU/L. Fourth generation assays having a functional detection limit of 0.001 to 0.002 mlU/L range also have been developed. ... 

Tg antibodies are directed against the Tg protein, a major constituent of thyroid colloid. Several different techniques have been used to detect and quantify TgAb in peripheral blood. These include passive hemagglutination, the agar gel diffusion precipitin technique, immunofluorescence of tissue sections, enzyme-linked immunosorbent assay (ELISA), radioassay techniques, and chemiluminescence-based immunometric assays. 

G-rich sequence-functionalized polystyrene microsphere-based instananeous derivatization for the chemiluminescence-amplified detection of DNA. 

Although it is not the purpose of this article to champion one technology over another, it is fair to say that chemiluminescence offers the sensitivity of fluorescence detection without some of the attendant problems of luminescent emission from analytical samples. Thus, chemiluminescence detection can be comparable to and, with the aid of enzyme amplification, even superior to that of I. This is not to say that there are no problems associated with chemiluminescence-based analytical techniques. As we shall see later (Section 2.5), the actual signal from chemiluminescent molecules is, in most cases, a transient flash, lasting... 

The driving force for the development of chemiluminescence-based assays (as well as any other optical or electrical detection methodology) is the replacement of radiolabels both for safety reasons and because of their intrinsic instability. Because the earliest high sensitivity immunoassays utilized antibodies with covalently attached as the label, this has served as a yardstick against which all subsequent assay technologies are measured. For this reason, it is important to understand the detection limits for I. Radioactive iodine is a y-emitter that eventually decays to a stable isotope of lead. The decay process exhibits first-order kinetics so that we can write... 

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