I. Enzymatic Analysis
This is a type of analysis, depending on the highly efficient and specific catalysis of enzymes and using an enzyme as an analytical reagent or tool for detecting the content of a substance in a sample (such as food, medicine, body fluid, etc.). It has a wide range of applications, any substances related to enzyme reactions, such as enzyme substrates, cofactors, and even enzyme inhibitors, can be analyzed by this method. In the specific implementation, it needs to select a suitable enzyme according to the object. Performing this reaction under the condition of object, and then detecting by physical or chemical methods to track the test, and finally according to the relationship between the test object and the enzyme reaction. With kinetic analysis, the content of substance can be detected. As for the determination of a certain amino acids content in the fermentation broth reaction, it is necessary to select a decarboxylase specific for this amino acid as a tool enzyme for catalysis. In this case, the test object is the substrate of the enzyme, so the amino acid content can be calculated based on the CO2 released during the enzymatic reaction.
Compared with the general chemical analysis, enzymatic analysis is characterized by its high selectivity. In the complex system of the object to be analyzed and other similar substances, the object can be catalyzed directly by the specificity of the enzyme. And then according to the measured reaction speed or enzyme activity and the corresponding correlation between the components to be tested, the content of the target component can be determined. This eliminates the need for a series of extractions and pretreatments that are required in a general chemical analysis in advance. At the same time, it can easily obtain reliable results without the interference of analogs. In addition, certain substances such as coenzyme A, organophosphorus are sometimes difficult to find, or even can not be directly gained through simple analytical chemistry methods. In this case, enzymatic analysis can be applied.
The main problem with enzymatic analysis is the need for appropriate, high-purity, high-activity tool enzymes. So the cost is high. Second, as mentioned above, the tool enzyme acts only as a mild and specific pretreatment in the enzymatic assay, ultimately chemical or physico-chemical testing is still required. Therefore, enzymatic analysis itself needs to constantly absorb analytical chemistry, especially the advanced achievements of modern instrumental analysis to strengthen and develop itself.
The trend of enzymatic analysis development: A. Simpleness–test strips and other application forms. For example, combining glucose oxidase, peroxidase, and linfenidine color source substrate together and fixing on the filter paper made of urine test strips to be soaked in patient’s urine. After a certain period of time, the glucose content can be determined according to the color depth. B. Micro-quantization, continuous and automated. Generally, the tool enzyme is made into an enzyme electrode, or the enzyme is immobilized into enzyme tube or enzyme column and coupled with a detection device. For instance, a glucose oxidase enzyme electrode developed in clinical can be used to detect the trace amounts of glucose in the body fluid by observing oxygen content changes on the cathode.
II. Enzyme Immunoassay (EIA)
In the early 1970s, an analytical method called “enzyme-labeled immunization” was developed from immunochemistry field. It is a highly specific and sensitive analytical method that combines the specificity of immunology and the efficient catalytic binding of enzymes organically. Enzyme immunoassay is based on the enzyme as a marker substance to label antigen (or antibody), to form enzyme antigen (or antibody). And then based on the specific, quantitative binding relationship between antibodies to be tested (or antigen) and ELISA antigen (or antibody), the amount of antibody (or antigen) can be calculated by measuring the activity of labeled enzyme after binding.
Enzyme immunoassay compared with immunofluorescence or radioimmunoassay has following advantages: no need of special and complicated equipment and instruments; high sensitivity; good reproducibility and harmless to health. Therefore, in recent years, enzyme immunoassay has attracted more and more attentions. This approach has now been extended to many subject areas, not only for antigens or antibodies but also for many drugs, hormones and antibiotics, and even the enzyme itself.
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