Method for simultaneously measuring activity of various enzymes by using multi-wavelength absorption single channel

摘要

A method for simultaneously measuring the activities of multiple kinds of enzymes in single channel through concimitantly monitoring multiple wavelength absorbances is claimed; based on linear additivity of absorbances and the isoabsorbance wavelengths of chromogenic substrates and their chromogenic products, both the principles for the combination of chromogenic substrates required for simultaneously measuring the activities of multiple kinds of enzymes in single channel through concimitantly monitoring multiple wavelength absorbances and an approach for data processing to eliminate the interference of the overlapped absorbances are developed, kinetic analysis of reaction curves via numerical integration eliminates the interference of all substrates and products and estimates the maximal reaction rates of the enzymes to be measured, giving the linear ranges and the limits of quantification for simultaneously measuring the activities of multiple kinds of enzymes in single channel comparable to those by separate assays; the present invention is applicable for simultaneously measuring the activities of multiple kinds of enzymes in biological samples, simultaneously measuring multiple components by enzyme-labeled immunoassays, screening simultaneously of inhibitors against multiple targets, for the practice in clinical biochemical analyses, clinical immunoassays, health laboratory analyses, the discovery of inhibitors, and the basical researches which need the present invention.

主权项

1. A method for simultaneously measuring the activities of multiple kinds of enzymes in single channel through concomitantly monitoring multiple wavelength absorbances the method comprising:
a. determining the combination of chromogenic substrates required for simultaneously measuring the activities of multiple kinds of enzymes: a1. selecting the combination of chromogenic substrates that meets the following criteria according to the specificity of the enzymes to be measured: the selected chromophores can be respectively used to prepare the chromogenic substrates of the enzymes to be measured; in the obtained combination of chromogenic substrates, the difference absorbance peak maximum wavelengths of the chromogenic products formed under the actions of the respective enzymes to be measured with respect to their chromogenic substrates are arranged from large to small to give a one-dimension array, the distance between any two adjacent difference absorbance peak maximum wavelengths of the chromogenic products in the aforementioned array is greater than 30 nm and preferably as far as possible; in the array of the difference absorbance peak maximum wavelengths of the chromogenic products from large to small, except for the chromogenic substrate whose chromogenic product has the difference absorbance peak maximum wavelength nearest to the infrared end, the difference absorbance peak maximum wavelength of each chromogenic product obtained from its chromogenic substrate under the action of the corresponding enzyme is less than 25 nm and preferably as shorter as possible away from the maximum isoabsorbance wavelength of one of the chromogenic substrates in the aforementioned array; a2. synthesizing corresponding chromogenic substrates of the enzymes to be measured respectively by the chromophores selected in step a1, and using them in combination; b. in one reaction mixture or enzyme reaction system, that is a single reaction channel, synchronously initiating the specifically-catalyzed reactions of multiple kinds of the enzymes to be measured with their corresponding chromogenic substrates combined for use as a mixture; c. selecting a combination of wavelengths for measuring absorbance: taking the chromogenic substrate whose chromogenic product thereof has the difference absorbance peak maximum wavelength nearest to the infrared end as chromogenic substrate A, determining the absorbance of the chromogenic product at the difference absorbance peak maximum wavelength of the chromogenic product of chromogenic substrate A; determining the absorbance of the chromogenic products or chromogenic substrates of the other enzymes whose activities are to be measured at the isoabsorbance wavelengths of chromogenic substrate A; d. realizing simultaneous measurement of multiple wavelength absorbances for concomitantly monitoring the reaction processes of multiple kinds of the enzymes to be measured in a single channel through swift alteration of the wavelengths selected in the combination; e. developing an approach of data processing for eliminating the interference of the overlapped absorbance of chromogens involved in enzyme reactions based on linear additivity of absorbances of non-interacting compounds, to obtain an absorbance change curve of the chromogenic product or substrate of each enzyme to be measured after the elimination of the overlapping interference in absorbance spectra; if the action of an enzyme to be measured is not subject to the interference by any enzyme products and substrates in the reaction system, and the substrate concentration is over three times of a Michaelis constant of the corresponding enzyme, the classical initial rate method is used to analyze the absorbance change curve of the chromogenic product or substrate with no disturbing absorbance to determine the initial rate; if the action of an enzyme to be measured is not disturbed by any enzyme products and substrates in the reaction system, however, the concentration of the used chromogenic substrate is over 5% of a Michaelis constant of the corresponding enzyme but less than three times of the Michaelis constant, the classical initial rate method is integrated with kinetic analysis of reaction process to analyze the reaction curve as the absorbance change of the chromogenic product or substrate thereof with no disturbing absorbance to determine the initial rate; when the action of an enzyme to be measured is disturbed by substrates and/or products of itself and/or other enzymes in the reaction system, a group of the differential rate equations that describe the kinetics of the reaction system is numerically integrated for fitting to the reaction curve as the absorbance change of the chromogenic product or substrate of the enzyme to be measured with no disturbing absorbance of chromogenic products and chromogenic substrates in the reaction system, to estimate the maximum reaction rate thereof, which is used directly as an index of the activity of the enzyme to be measured, or is further converted according to the differential rate equations into an initial rate at the substrate concentration of 93% of the initial one for reflecting the activity of the enzyme to be measured.