Chemiluminescent substrates properties

The amino group can be diazotized for coupling to various substrates, as is done in CL immunoassay, without loss of the chemiluminescent properties of the cyclic hydrazide. 

Methods in which some property related to substrate concentration (such as absorbance, fluorescence, chemiluminescence, etc.) is measured at two fixed times during the course of the reaction are known as two-point kinetic methods. They are theoreticahy the most accurate for the enzymatic determination of substrates. However, these methods are technically more demanding than equifibrium methods and all the factors that affect reaction rate, such as pH, temperature, and amount of enzyme, must be kept constant from one assay to the next, as must the timing of the two measurements. These conditions can readily be achieved in automatic analyzers. A reference solution of the analyte (substrate) must be used for calibration. To ensure first-order reaction conditions, the substrate concentration must be low compared to the K, (i.e., in the order of less than 0.2 X K, . Enzymes with high K , values are therefore preferred for kinetic analysis to give a wider usable range of substrate concentration. 

The main applications of oxalyl chloride, as described in Chapter 4, are the formation of aryl isocyanates and chloroformates (by reactions with amines and hydroxylic substrates, respectively), and the formation of acyl chlorides from carboxylic acids under very mild conditions. Oxalyl chloride reacts with amides to give acyl isocyanates, and it is used with dimethyl sulfoxide as a mild reagent for the oxidation of alcohols (Swern-type oxidation). It is also used with N,N-dimethylformamide as a mild reagent for chlorination and formylation. Oxalyl chloride is widely used in commercial formulations of speciality polymers, antioxidants, photographic chemicals, X-ray contrasting agents, and chemiluminescent materials. Other physical properties are presented in Chapter 3. 

One motivation for coupling SECM with ECL is to be able to use the SECM tip as a controllable light source that locally illuminates the sample surface. To achieve this, the tip is biased so as to trigger an electrochemical reaction that produces light by chemiluminescence. The sample surface has to be at least partly transparent and the emitted light is collected by a PMT tube located below the substrate. The ECL intensity measured by the PMT can then be plotted as a function of the tip position to form an image that reflects the local optical properties of the substrate, for example, its absorbance. The setup is similar to the one used for SECM-OM described earlier in Section 17.5.2. 

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