WO2014043923A1 - 用多波长吸收单通道同步测量多种酶活性的方法 - Google Patents

用多波长吸收单通道同步测量多种酶活性的方法 Download PDF

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WO2014043923A1
WO2014043923A1 PCT/CN2012/081869 CN2012081869W WO2014043923A1 WO 2014043923 A1 WO2014043923 A1 WO 2014043923A1 CN 2012081869 W CN2012081869 W CN 2012081869W WO 2014043923 A1 WO2014043923 A1 WO 2014043923A1
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absorption
chromogenic
product
enzyme
wavelength
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PCT/CN2012/081869
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French (fr)
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廖飞
杨晓兰
刘红博
党济政
龙高波
李元丽
刘霖
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重庆医科大学
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Priority to US14/430,391 priority Critical patent/US9611502B2/en
Publication of WO2014043923A1 publication Critical patent/WO2014043923A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/25—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
    • G01N21/272—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration for following a reaction, e.g. for determining photometrically a reaction rate (photometric cinetic analysis)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2334/00—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N2021/3125—Measuring the absorption by excited molecules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/904—Oxidoreductases (1.) acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/91045—Acyltransferases (2.3)
    • G01N2333/91074—Aminoacyltransferases (general) (2.3.2)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
    • G01N2333/938—Hydrolases (3) acting on glycosyl compounds (3.2) acting on beta-galactose-glycoside bonds, e.g. beta-galactosidase

Definitions

  • the invention relates to a method for synchronously measuring a plurality of enzyme activities by using a multi-wavelength absorption synchronous measurement technique, and particularly relates to a method for simultaneously measuring multi-wavelength absorption by a single channel and simultaneously measuring the activity of a plurality of enzymes.
  • the sensitivity of the enzyme activity measurement is defined as the slope of the response curve of the enzyme change rate.
  • the limit of quantification (LOQ) of the enzyme activity measurement is the linear section intercept in the above response curve plus the regression estimation standard deviation of 5 times corresponding to the enzyme amount. Determination of enzyme activity requires sensitivity as high as possible, as low as possible, and as high as possible.
  • the enzyme activity can be determined by continuously and sensitively measuring the absorption change reaction curve with a highly specific substrate for a specific enzyme; the enzyme activity measurement process is routinely used for clinical biochemical tests, hygienic tests for detecting enzyme inhibitor foods or Environmental pollutants, high-throughput screening enzyme inhibitor combination libraries and enzyme-labeled immunoassays.
  • the high specificity and high sensitivity of enzyme activity assays allow the enzyme to be used as a marker signal for immunoassays, a representative technique, Enzyme-linked immunoassay (ELISA), which is a selective assay in the field of clinical testing.
  • ELISA Enzyme-linked immunoassay
  • the capture component is coated with a microplate to separate the immune response complex, and the washing process is required.
  • the single analysis often takes more than 130 min, and the analysis efficiency is low. Therefore, improving the efficiency of enzyme activity measurement is of great significance in applications.
  • the enzymatic activity was determined by continuously tracking the enzymatic reaction curve each time using an chromogenic substrate to determine an enzyme activity, i.e., only one enzyme was determined per reaction channel at a time.
  • determination of liver function status requires determination of alanine aminotransferase, aspartate aminotransferase, ⁇ -glutamyltransferase, alkaline phosphatase, etc. in the same specimen.
  • amylase and lipase in the same specimen should be determined.
  • the main disadvantage of ELISA is its low analytical efficiency and long time-consuming analysis.
  • the simplest strategy for improving the efficiency of enzymatic analysis is to continuously measure the absorption curve of multiple enzymes in a single enzyme reaction system, ie, an enzyme reaction channel, using a variety of chromogenic substrates in real-time or substantially real-time synchronization.
  • a variety of enzyme activities Simultaneous measurement of multi-wavelength absorption photometers has become popular.
  • the Biotek ELX800 microplate reader can simultaneously measure three-wavelength absorption
  • the Microscope UV1600PC general photometer can simultaneously measure seven-wavelength absorption.
  • These conventional devices simultaneously measure the absorption of different wavelengths and actually have a shorter time difference; a photometer with a dual monochromator or a diode array detector can measure multi-wavelength absorption in substantially real time.
  • the phenolphthalein monophosphate is used as the alkaline phosphatase chromogenic substrate, and the specific activity is lower than that of p-nitrophenyl phosphate.
  • the ester is a chromogenic substrate, it is lower than 7% of the activity.
  • the sensitivity is low, and the cost of phenolphthalein monophosphate is high, making this technology not practical.
  • the two enzyme reactions and the detection of the two enzyme chromogenic products were not synchronized, and the two enzyme reaction curves could not be continuously tracked (Dean et al, Clin Chem, 1983; 29(6) : 1051-1056. Ng, et al. Clin Chem, 1987; 33(12): 2286-2288.
  • the invention uses a multi-wavelength absorption single channel to simultaneously measure a plurality of enzyme activities, comprising the following steps:
  • Al selects the chromogenic substrate combination that satisfies the following conditions according to the specificity of the enzyme to be tested:
  • the selected chromophore can be used to prepare the chromogenic substrate of the enzyme to be tested, respectively;
  • the maximum wavelength of the difference absorption peak of the coloring product obtained by the coloring substrate corresponding to the enzyme to be tested is arranged from large to small, and the maximum wavelength of the difference absorption peak of the adjacent color developing products is greater than 30 nm.
  • each chromogenic substrate is obtained under the action of the corresponding enzyme.
  • the maximum wavelength of the difference absorption peak of the chromogenic product is less than 25 nm and is as short as possible to the maximum isother absorption wavelength of a chromogenic substrate in the array; a2 synthesizing the corresponding enzyme to be tested by the chromophore selected in step a Color substrates and use in combination;
  • the chromogenic substrate closest to the infrared end of the color difference product absorption peak is the chromogenic substrate A, and the color product absorption is measured at the maximum wavelength of the chromogenic product differential absorption peak of the chromogenic substrate A. ; measuring the absorption of the remaining enzyme color developing product or chromogenic substrate at the equal absorption wavelength of the chromogenic substrate A;
  • a data processing method for eliminating the overlapping interference of the chromogenic substance in the reaction channel is established, and the color developing product or the chromogenic substrate of each enzyme to be tested is obtained after eliminating the overlapping interference of the absorption spectrum. No interference absorption curve; If a certain enzyme to be tested is not interfered by the products and substrates of other enzymes in the reaction system and the substrate concentration is more than 3 times the Mie's constant of the corresponding enzyme, the color is analyzed by the classical initial velocity method.
  • the classical initial velocity method and the reaction process analysis method analyze the color-developing product or the chromogenic substrate without interference absorption curve to determine the initial velocity; when a certain enzyme to be tested is subjected to its own and/or other enzyme substrate and/or Or the interference of the product, the differential velocity equations describing the kinetics of the reaction system are numerically integrated, and the activity of the color-developing product or the chromogenic substrate without interference absorption is analyzed.
  • the maximum reaction rate is determined to be
  • the initial velocity indicates the enzyme activity when the activity, or its differential velocity equation and the maximum reaction rate obtained, are converted to a substrate concentration of 93% of the starting substrate concentration.
  • the maximum wavelength of the difference absorption peak of the chromogenic substrate relative to the chromogenic substrate is above 300 nm and the maximum wavelength of the absorption peak of the chromogenic substrate is smaller than the maximum wavelength of the absorption peak of the corresponding chromogenic product, otherwise the enzyme is determined by a reverse reaction.
  • the activity of the chromogenic substrate in the combination of the chromogenic substrate of the chromogenic product, the maximum absorption wavelength of the chromogenic product, the maximum absorption wavelength of the chromogenic product, and the maximum absorption wavelength of the chromogenic product which is closer to the infrared end.
  • the maximum absorption wavelength of the chromogenic product is closer to the ultraviolet end.
  • each chromogenic substrate in the chromogenic substrate combination used is only one of the samples.
  • the enzyme to be tested produces a color-developing product, and is not affected by any other enzyme in the sample or the effect is not obvious; the effect described herein is not affected by any other enzyme or the effect is not obvious, and refers to the color development corresponding to the enzyme I in the sample.
  • concentration of the substrate differs from the Michaelis constant by less than 50%, the specific activity of any other enzyme in the sample acting on the chromogenic substrate is lower than the specific activity of the enzyme I acting on the chromogenic substrate.
  • the buffer medium and the reaction conditions for selecting the enzyme to be tested for the simultaneous determination of activity are selected, and the specific conditions include: the pH of the buffer medium is between pH 5.0 and 9.0 and the activity is the lowest to be tested.
  • the optimum pH of the sample, the reaction temperature of the sample used is between 20 and 40 degrees Celsius.
  • the chromogenic substrate comprises a natural chromogenic substrate and a non-natural chromogenic substrate;
  • the natural chromogenic substrate comprises nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate
  • the non-natural chromogenic substrate consists of a chromophore and an enzyme-recognizing group, including but not limited to 4-nitrophenol, 4-nitrothiophenol, 4-nitroaniline, 4-nitro 1-naphthol, 4-nitro-1-naphthol, 4-nitro-1-naphthylamine, 2-naphthol, 4-chlorophenol, rose acid or a derivative thereof.
  • the method is applied to synchronously measure the activity of two enzymes.
  • the two enzymes to be tested are named as enzyme A and enzyme B, respectively, and the two chromogenic substrates are respectively named as color base.
  • a and chromogenic substrate B under the action of two enzymes to be tested, two chromogenic products were named as chromogenic product A and chromogenic product B, and the combination of these chromogenic substrates and chromogenic products was to satisfy the following Conditions: (1) The maximum wavelength XI of the color difference product A is close to the infrared end, and the maximum wavelength X2 of the color difference product is close to the ultraviolet end, and the maximum wavelength X2 of the color difference product B is poorly absorbed by the color product A.
  • the peak maximum wavelength XI is greater than 30 nm and as far as possible; (2)
  • the maximum absorption wavelength of the color-developing product A is Y1 and is less than 25 nm from the maximum wavelength X2 of the color-developing product B, and is as short as possible.
  • the method is applied to the steps b and c and d for simultaneously measuring the activity of two enzymes, and the corresponding chromogenic substrate A and the synthesis of the two enzymes for chromophore A and chromophore B are synthesized in a synchronous reaction sample in one reaction channel.
  • the wavelength XI is ⁇ ⁇ to measure the chromogenic product A absorption; by rapidly changing the measurement wavelength, the two-wavelength absorption synchronous measurement realizes the simultaneous tracking of the reaction process of two enzymes to be tested in a single reaction system.
  • the method is applied to the step e of synchronously measuring the activity of two enzymes, and the method for eliminating data interference by multi-substance absorption is established based on the absorption linear addition;
  • the wavelength of the absorption peak of the color-developing product A is not more than 25 nm before and after the maximum wavelength. Is ⁇ ⁇ and absorbs Al at this wavelength, and does not exceed 25 in the vicinity of the maximum absorption wavelength of the color developing product ⁇ and the chromogenic substrate A
  • the wavelength of nm is ⁇ 2 and the absorption at this wavelength is ⁇ 2.
  • a 2 ⁇ 20 + ⁇ 32 XPi + E35 XP 2
  • A ⁇ io+ ⁇ ia + R33 XA 2h
  • a 2 ⁇ 20 + ⁇ 2b + R31 X a
  • Pi is the instantaneous concentration of the chromogenic product A
  • P 2 is the instantaneous concentration of the chromogenic product B
  • E31 is the difference molar extinction coefficient between the chromogenic product A and the chromogenic substrate A at ⁇
  • ⁇ 32 is the chromogenic product ⁇ and the chromogenic substrate A is the molar extinction coefficient ⁇ 32 at ⁇ 2
  • ⁇ 34 is the difference molar extinction coefficient between the color-developing product ⁇ and the chromogenic substrate ⁇ at ⁇
  • ⁇ 35 is the color-developing product ⁇ and the chromogenic substrate ⁇ under the ⁇ 2 differential molar extinction coefficient
  • ⁇ is the instantaneous total absorption at ⁇ before absorbing interference
  • ⁇ is the instantaneous total absorption at ⁇ 2 before correcting the absorption interference
  • a is the instantaneous net absorption of the chromogenic product ⁇ at ⁇ after correcting the absorption interference, which is equal
  • the non-interference absorption curves A la and A 2b of the chromogenic product ⁇ and the chromogenic product B under ⁇ ⁇ and at ⁇ 2 are determined ;
  • the concentration of the chromogenic substrate A is Cl, and the concentration of the initial chromogenic substrate B is C2; the maximum reaction rate of the enzyme B is mB , and the preset chromogenic substrate concentration used for conversion to the initial velocity is equal to 93% of C 2 .
  • the Michaelis constant of enzyme B on the chromogenic substrate B is ⁇ ; if the chromogenic product A is competitively inhibited by the enzyme B, the competitive inhibition constant is a , and the kinetic equation of the enzyme B is: 2 E M K mB (1 + ⁇ 1 ⁇ / ⁇ ⁇ /£ 31 ) + C 2
  • reaction time is integrated from 0 ⁇ t on both sides of the above equation.
  • the integral term on the left is the net absorption change of the color product B between the reaction time t and the reaction time 0; the integral term on the right side of the equation is numerically integrated with the constant concentration of the color product A in dt, that is, the calculation
  • V mB C 2 x E 34 x sumx x At) 0.93 x 2 x V mB /(K mB +0.93x 2 )
  • the measurement relationship of the enzyme A converting the chromogenic substrate A into the color developing product A is 1:1, and the measurement relationship of the enzyme B converting the chromogenic substrate B into the color developing product B is also 1:1;
  • the method for simultaneously measuring the activity of two enzymes has the following characteristics:
  • the chromophore combination is selected as follows: chromophore A and chromophore B.
  • the maximum absorption wavelength of the chromogenic product A is equal to or greater than the maximum wavelength X2 of the chromogenic product B.
  • the measurement wavelength ⁇ 2 is selected as follows: The chromogenic product ⁇ and the chromogenic substrate ⁇ difference molar extinction coefficient is higher at the maximum absorption wavelength Y1 of the chromogenic product ⁇ At 30% of the maximum wavelength X2 of the difference absorption peak of the chromogenic product B, the maximum absorption wavelength Y1 of the chromogenic product A is selected to be ⁇ 2, otherwise, the color difference product B of the maximum absorption wavelength Y1 closest to the chromogenic product ⁇ is used.
  • the absorption peak wavelength is ⁇ 2, or the closest to the color-developing product ⁇ -difference absorption peak and the other color-developing products to be tested and the color-developing chromogenic substrate are different in molar extinction coefficient than the chromogenic product ⁇ and the chromogenic substrate ⁇
  • the extinction coefficient of 20% of the wavelength is ⁇ 2 to measure the color product ⁇ absorption;
  • step e it is necessary to determine the correction coefficient of R31 as the absorption interference of the color-developing product A; when determining R31, only all the chromogenic substrates required for the enzyme A are used without any other chromogenic substrate of the enzyme to be tested, so that the reaction
  • the system does not absorb from the chromogenic substrate B and the chromogenic product B at two measurement wavelengths ⁇ ⁇ and ⁇ 2 , at which time the change in absorption at the two measurement wavelengths in the reaction channel is simultaneously measured until the absorption change at ⁇ 2 is greater than 0.005 or
  • the ⁇ absorption change is greater than 0.500; the absorption is ⁇ as the abscissa, and the absorption at ⁇ 2 is the ordinate for regression analysis, and the obtained regression line slope is the correction coefficient R31;
  • step e it is necessary to determine the correction coefficient of R33 as the absorption interference of the chromogenic product B; when determining R33, only the chromogenic substrate required for the enzyme B is used without any chromogenic substrate of the enzyme to be tested, so that the reaction
  • the system does not absorb from the chromogenic substrate A and the chromogenic product A at two measurement wavelengths ⁇ ⁇ and ⁇ 2 , at which time the change in absorption at the two measurement wavelengths in the reaction channel is simultaneously measured until the absorption change at ⁇ 2 is greater than 0.500 or
  • the absorption of ⁇ is greater than 0.005; the absorption is ⁇ 2 as the abscissa, and the absorption under ⁇ is the ordinate for regression analysis.
  • the slope of the regression line obtained is the correction coefficient R33;
  • the corresponding chromogenic substrate of the enzyme ⁇ includes 4-nitrophenyl acetate having a maximum absorption wavelength between 310 and 330 nm, and the hydrolysis reaction is maximum.
  • the chromogenic substrate B corresponding to the chromogenic substrate A of such enzyme A is mainly a natural chromogenic substrate, including nicotinamide adenine dinucleotide or nicotinamide adenine dinucleoside Acid phosphoric acid;
  • the enzyme A corresponding to the above chromogenic substrate A includes, but is not limited to, an aromatic esterase, a phosphatase, a sulfatase, a ⁇ -glutamy
  • Enzyme B enzyme B corresponding to such enzymes, including but not limited to lactate dehydrogenase LDH, malate dehydrogenase MDH, LDH-coupled valley Alanine aminotransferase and aspartate aminotransferase coupled to MDH.
  • the method is used for synchronously measuring the activity of three enzymes, and the three enzymes to be tested are respectively named as enzyme A, enzyme B and enzyme C, and the desired chromogenic substrates are respectively named as chromogenic substrate A and chromogenic bottom.
  • the substance B and the chromogenic substrate C which are colored under the action of the corresponding enzymes, are named as the color developing product, the color developing product B and the color developing product C; the combination of these coloring substrate needs to satisfy the following conditions: (1)
  • the maximum absorption peak of the color product A is XI and is closest to the infrared end.
  • the maximum wavelength of the difference absorption peak of the color product C is X3 and is closest to the ultraviolet end.
  • the maximum wavelength of the difference absorption peak of the color developing product B is X2 and is located at XI and X3. Between X2 and XI and X3 are both greater than 30 nm and as far as possible; (2) The maximum absorption wavelength of color product A is Y1 and the second largest absorption wavelength is Ys; chromogenic substrate B produces chromogenic product B. The maximum equal absorption wavelength is Y2 and is separated from Y1 The distance is far from Ys as short as possible; (3) Y1 and X2 are less than 25 nm apart and the distance is as short as possible; Y2 and Ys and X3 are less than 25 nm apart and the distance is as short as possible;
  • step b the reaction of the three enzymes against the chromogenic substrate A, the chromogenic substrate B and the chromogenic substrate C is simultaneously initiated in one reaction channel, and color development is obtained.
  • step c when the method is used to simultaneously measure the activity of three enzymes, in step c, the maximum absorption wavelength Y1 of the color product A or the wavelength within 25 nm is measured as ⁇ 2
  • the product ⁇ absorption, the color product ⁇ difference absorption peak maximum wavelength XI or the distance within 25 nm is ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ,
  • step d the measurement process of the two samples in a single reaction system is synchronously tracked by rapidly changing the measurement wavelength and the simultaneous measurement of the three-wavelength absorption;
  • step e the data is resolved based on the linear addition of absorption. Eliminating the overlapping interference of multiple substances requires solving the following ternary equations:
  • a x A w +A la + R33 A 2h + R35 XA 3c
  • a 2 A 20 ⁇ 2b + R31X ⁇ ia + R36X ⁇ 3c
  • a 3 A 30 +A 3c + R32X ⁇ ia + R34X ⁇ 2b
  • ⁇ is the instantaneous absorption at ⁇ before the correction of the absorption interference
  • ⁇ is the instantaneous absorption at ⁇ 2 before the correction of the absorption interference
  • ⁇ is the instantaneous absorption at ⁇ 3 before the correction of the absorption interference
  • la is the chromogenic product at the ⁇ after the corrected absorption interference Net absorption
  • ⁇ 2b is the net absorption of chromogenic product ⁇ at ⁇ 2 after correction absorption interference
  • e is the instantaneous net absorption of chromogenic product C at ⁇ 3 after correcting absorption interference
  • no chromogenic product is formed before absorption in ⁇
  • 2Q is absorbed in ⁇ 2 before the reaction system generates any color developing product.
  • E31 is the color difference product ⁇ and chromogenic substrate A under the ⁇ difference molar extinction coefficient
  • ⁇ 32 is the color product ⁇ and the chromogenic substrate A is lower at ⁇ 2 Molar extinction coefficient
  • ⁇ 33 is the difference molar extinction coefficient between chromogenic product ⁇ and chromogenic substrate A at ⁇ 3
  • ⁇ 34 is the difference molar extinction coefficient between chromogenic product ⁇ and chromogenic substrate ⁇ at ⁇
  • ⁇ 35 is the color development product ⁇
  • the chromogenic substrate ⁇ at ⁇ 2 is the molar extinction coefficient
  • ⁇ 36 is the chromogenic product ⁇ and the chromogenic substrate ⁇ at ⁇ 3, the molar extinction coefficient
  • ⁇ 37 is the chromogenic product C and the chromogenic substrate C at the measurement wavelength ⁇
  • the lower molar extinction coefficient, ⁇ 38 is the difference molar extinction coefficient of the chromogenic product C and
  • the interference-free absorption curves A la and A 2b of chromogenic product A and chromogenic product B under ⁇ ⁇ and at ⁇ 2 are determined ;
  • the concentration of the chromogenic substrate A is Cl, and the concentration of the initial chromogenic substrate B is C2; the maximum reaction rate of the enzyme B is mB , and the preset chromogenic substrate concentration used for conversion to the initial velocity is equal to 93% of C 2 .
  • the Michaelis constant of enzyme B on the chromogenic substrate B is ⁇ ; if the chromogenic product A is competitively inhibited by the enzyme B, the competitive inhibition constant is a , and the kinetic equation of the enzyme B is:
  • the measurement relationship of the enzyme A to convert the chromogenic substrate A into the chromogenic product A is 1: 1, and the measurement relationship of the enzyme B to the chromogenic substrate B to the chromogenic product B is also 1:1;
  • the color rendering product A needs to absorb the interference correction coefficients R31 and R32 in step e; only all the chromogenic substrates of the enzyme A are used in the determination without any other enzymes to be tested.
  • the chromogenic substrate is such that the reaction system does not absorb from the chromogenic substrate B and the chromogenic product B, the chromogenic substrate C, and the chromogenic product C at three measurement wavelengths ⁇ , ⁇ 2, and ⁇ 3, and the reaction channels are simultaneously measured in three Absorption changes at the measured wavelengths until the absorption changes at ⁇ 2 and ⁇ 3 are greater than 0.005 or the absorption changes at ⁇ are greater than 0.500; the absorption changes at ⁇ are plotted on the abscissa, and the absorption changes at ⁇ 2 and at ⁇ 3 are plotted as ordinates.
  • the slope of the regression curve of the absorption change at ⁇ 2 is the correction coefficient R31
  • the slope of the regression curve of the absorption change at ⁇ 3 is the correction coefficient R32;
  • the color-developing product B needs to absorb the interference correction coefficients R33 and R34 in step e; only all the color-developing substrates of enzyme B are used in the determination without any color development of other enzymes to be tested.
  • the substrate is such that the reaction system does not absorb from the chromogenic substrate A and the chromogenic product A, the chromogenic substrate C, and the chromogenic product C at three measurement wavelengths ⁇ 1, ⁇ 2, and ⁇ 3, and simultaneously measures three wavelengths in the reaction channel.
  • the obtained regression curve of the absorption change under ⁇ is the correction coefficient R33, and the slope of the regression curve of the absorption change at ⁇ 3 is the correction coefficient R34;
  • the color-developing product C needs to absorb the interference correction coefficients R35 and R36 in step e; only all the color-developing substrates of enzyme C are used in the determination without any color development of other enzymes to be tested.
  • the substrate is such that the reaction system does not absorb from the chromogenic substrate A and the chromogenic product A, the chromogenic substrate B and the chromogenic product B at three measurement wavelengths ⁇ ⁇ , ⁇ 2 and ⁇ 3 , and simultaneously measures three wavelengths in the reaction channel.
  • the slope of the regression curve of the absorption change under ⁇ is the correction coefficient R35, and the slope of the regression curve of the absorption change at ⁇ 2 is the correction coefficient R36;
  • the chromogenic substrate combination can be selected by the following principle:
  • the chromogenic substrate A, the chromogenic substrate B and the chromogenic substrate C should be used to make the chromogenic product A
  • Maximum equal absorption wavelength Y1 and color rendering product B The absorption wavelengths of the difference absorption peaks X2 are equal or not more than 5 nm, the absorption wavelength Y2 of the chromogenic product B is large, and the absorption wavelength Ys of the chromogenic product A is large, and the maximum wavelength X3 of the chromogenic product C difference absorption peak is equal or both are separated.
  • the maximum wavelength XI of the difference absorption peak of the color-developing product A is selected as ⁇ 1
  • the other two measurement wavelengths ⁇ 2 and ⁇ 3 can be selected according to the following principle:
  • the color product ⁇ and the chromogenic substrate ⁇ difference molar extinction coefficient is higher than the chromogenic product B and the chromogenic substrate B at the maximum absorption wavelength Y1 of the chromogenic product ⁇ at the maximum wavelength X2 of the difference absorption peak of the difference absorption molar extinction coefficient At 30%, Y1 is selected as the measurement wavelength ⁇ 2.
  • the wavelength difference of the color-developing product B which is closest to the color-developing product ⁇ maximum absorption wavelength Y1, or the difference absorption peak of the color-developing product B and other color-developing products are used.
  • the molar extinction coefficient with the color developing chromogenic substrate is smaller than the coloring product B and the chromogenic substrate B.
  • the molar extinction coefficient is 20%.
  • the wavelength is ⁇ 2.
  • the color product ⁇ absorption is measured; the color developing product C and the chromogenic substrate C
  • the difference molar extinction coefficient is larger in the color rendering product A
  • the absorption wavelength Ys or the maximum absorption wavelength Y2 of the color developing product B and the chromogenic substrate B should be higher than 30% of the difference molar extinction coefficient at the maximum wavelength X3 of the difference absorption peak, then Y2 or Ys is selected.
  • the chromogenic substrate difference molar extinction coefficient is smaller than the chromogenic product C and the chromogenic substrate C difference molar extinction coefficient 20% of the wavelength is ⁇ 3 measurement color product C absorption;
  • the chromogenic substrate corresponding to the enzyme ⁇ includes 4-nitro-1-naphthyl phosphate and the two absorption wavelengths of the hydrolysis are respectively 315 to 335 nm.
  • 4-nitro-1-naphthyl sulfate and its two absorption wavelengths are between 315 and 335 nm and between 395 and 415 nm, 4-nitro- 1-naphthyl acetate and its two absorption wavelengths between 310 and 330 nm and between 375 and 1 395 nm, 4-nitro-1-naphthyl-D-galactoside and hydrolysis
  • 4-nitro-1-(N-lysyl)naphthylamine and the maximum absorption wavelength of the hydrolysis is 385 to 410 nm, respectively.
  • the chromogenic substrate A is activated by the enzyme A to form the chromogenic product A as 4-nitro-1-naphthol or 4-nitro-1-naphthylamine or a derivative thereof; and such a chromogenic substrate A
  • the corresponding chromogenic substrate B is mainly a 4-nitrophenol or 4-nitroaniline derivative, and the color developing product B is formed by the corresponding enzyme B to be 4-nitrophenol or 4-nitroaniline or 4-nitro Phenyl Alcohol; with such a color corresponding to the A and B chromogenic substrate substrate comprises a chromogenic substrate C nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate;
  • the enzyme A corresponding to the chromogenic substrate A includes, but is not limited to, an aromatic esterase, a phosphatase, a sulfatase, a ⁇ -glutamyltransferase, a peptidase, that is, a natural amino acid ⁇ .
  • the enzyme oxime corresponding to the chromogenic substrate ⁇ includes other hydrolase different from the selected oxime but belonging to the enzyme A;
  • the enzyme corresponding to the chromogenic substrate C includes, but is not limited to, lactic acid Dehydrogenase LDH, malate dehydrogenase MDH, alanine aminotransferase coupled to LDH, and aspartate aminotransferase coupled to MDH.
  • the present invention simultaneously measures multiple enzyme activities by multi-wavelength absorption single channel, and establishes enzyme activity based on absorption linearity of non-interacting substance absorption, enzyme chromogenic substrate and corresponding chromogenic product.
  • the classical initial velocity method only analyzes the average velocity of the linearly increasing portion of the color product absorption at the initial stage of the reaction, and the classical initial velocity indicates the enzyme activity.
  • the photometer has a limited absorption range.
  • the chromogenic substrate A and the color-developing product A and the absorption wavelength Y1 are ⁇ 2, and the chromogenic product ⁇ absorption can reduce the interference, but the chromogenic substrate ⁇ absorption at Y1 constitutes a background; in order to make the chromogenic product B absorb the measurement range Sufficiently wide, the background absorption from the chromogenic substrate A at Y1 or ⁇ 2 should not be too high, and the concentration range of the chromogenic substrate used is limited. At the limited chromogenic substrate A concentration, the upper limit of the classical initial rate for the determination of enzyme A is lower.
  • a new strategy for calculating the initial velocity and the classical initial velocity method can be used to determine the enzyme activity by analyzing the enzymatic reaction process; this new strategy is not the present invention.
  • the claimed content is the content that has been protected by the Chinese invention patent (ZL 200710093081.4); when it is necessary to apply this new strategy, it belongs to the intersection of ZL 200710093081.4 and the platform technology of the present invention.
  • the invention proposes a new method for synchronously tracking multiple enzyme reaction processes by single-channel multi-wavelength absorption synchronous measurement, thereby realizing simultaneous measurement of single-channel multiple enzyme activities, which relies on chromogenic substrate combination screening, measurement wavelength combination selection, and rapid Transform absorption measurement wavelength synchronous tracking of multi-wavelength absorption changes, data processing methods to eliminate absorption interference, data processing methods to eliminate mutual interference between substrates and products of different enzymes (Figure 1).
  • the two chromophores should satisfy the following requirements: (a) The color product A has the largest absorption peak The wavelength XI is close to the infrared end, and the color-developing product B has a maximum absorption wavelength of X2 near the ultraviolet end. The distance between X2 and XI is greater than 30 nm and the farther the better, (b) the maximum absorption of the color-developing product A by the chromogenic substrate A. The wavelength Y1 is different from the maximum wavelength X2 of the color-developing product B by less than 25 nm and the shorter the distance is (Fig. 1 and Table 1); (c) The ideal chromophore A and chromophore B should be Y1 and X2 Equal or apart no more than 5 nm;
  • the data processing method for eliminating the overlapping interference in the above item (4) does not depend on the measurement wavelength combination, nor on the combination of the chromogenic substrates; however, when (c) of the above (1) is not satisfied, the enzyme The limit of quantitation of the activity measurement will be inferior to the one-component assay; the combination of the ideal chromogenic substrates A and B as described in (c) above (b) is obtained and the measurement wavelength combination is selected as described in paragraph (3). , the performance of this method is comparable to the one-component determination.
  • the chromogenic products commonly used in the enzyme activity assay are mainly 4-nitrophenol, 4-nitroaniline and its derivatives, and NADH and NADPH (Fig. 2). Determination of Enzyme Activity
  • the natural chromogenic substrates NADH and NADPH are typically measured at wavelengths between 320 and 355 nm.
  • the maximum isother absorption wavelength of 4-nitrophenol and 4-nitroaniline chromogenic substrate under the action of enzyme is between 310 and 350 nm, and the maximum absorption peak of the color product is close to 400 nm; under the action of enzyme, 4 -nitrophenol or 4-nitrobenzene
  • the absorption of the amine at its maximum equal absorption wavelength did not change significantly when it absorbed more than 1.0 at around 400 nm (Fig. 3).
  • 4-nitrophenol and 4-nitroaniline derivatives are selected as the chromogenic substrate A, and NADH or NADPH is used as the color developing product B, and the maximum absorption wavelength Y1 of the coloring substrate A is selected as ⁇ 2.
  • the absorption of the color product ruthenium is substantially unaffected by the change in the concentration of the chromogenic substrate A and the chromogenic product A. Therefore, the 4-nitrophenol and 4-nitroaniline chromophore corresponding chromogenic substrate can be combined with NADH or NADPH, and the two enzyme activities can be measured simultaneously in a single channel.
  • the molar extinction coefficient of 4-nitro-1-naphthol near 450 nm at pH 7.4 is about SJxlO ⁇ mol/L ⁇ xcm- 4-nitrophenol molar extinction coefficient at 405 nm is about l.lxl0 4 (mol/L) _ ⁇ cm- immunoassay commonly used calf intestinal alkaline phosphatase (CIAP) as a tool enzyme, 4-nitro-1-naphthyl phosphate as a chromogenic substrate, its Michaelis constant 10 ⁇ /L; Determination of the specific activity of CIAP with 4-nitro-1-naphthyl phosphate can reach about 40% of the chromogenic substrate with 4-nitrophenyl phosphate; considering the difference in extinction coefficient of the color product, The two phosphates measure the quantitative limit of the alkaline phosphatase.
  • 4-nitro-1-naphthyl phosphate can be used to determine the alkaline phosphatase of bovine small intestine mucosa (Fig. 4), and to determine ⁇ -galactosidase by using 4-nitrophenyl- ⁇ -galactoside ( ⁇ -D-galacotosidase, hereinafter referred to as ⁇ -Gal) combination or other enzyme combination for producing 4-nitrophenol, is used for immunoassay for single-channel multi-wavelength absorption continuous tracking and simultaneous measurement of two component contents.
  • ⁇ -Gal 4-nitrophenyl- ⁇ -galactoside
  • glycosidase specificity, optimal pH and buffer are consistent, and are not easily interfered by mutual products and substrates; 4-nitro-1-naphthyl- ⁇ -D-galactoside and 4-nitro Phenyl- ⁇ -D-glucoside glycosides are substrates, and ⁇ -galactosidase and ⁇ -glucosidase combinations can be used for single-channel two-component ELISA; however, the optimal measurement wavelength combination is 400 nm and 460 or 450. Nm, using a common filter microplate reader to provide only 405 nm light source, is not conducive to the combination of advantages.
  • the data processing method for correcting the absorption overlap interference established by the invention and the data processing method for correcting the interference effect when the substrate and the product of the plurality of enzymes interfere with each other are common to the enzyme reaction system which is stable to the chromogenic substrate and the chromogenic product, and
  • the key to the application of the invention is to screen the chromogenic substrate combination such that the sensitivity, limit of quantitation and linear range of the simultaneous determination of the two enzyme activities in a single channel are comparable to the individual assays.
  • a representative chromogenic substrate combination of the present invention comprises: a hydrophilic derivative of 4-nitrophenol and 4-nitro-1-naphthol as a hydrolase chromogenic substrate combination, and a 4-nitroaniline chromogenic substrate The combination is combined with NADH or NADPH.
  • FIG. 1 Schematic diagram of the simultaneous measurement of two enzyme activities by single-channel multi-wavelength absorption
  • Phenylacetic acid (DTNA) + acetylcholinesterase (Sigma C2888) Absorption spectra during the reaction
  • Phenylacetic acid (DTNA) + acetylcholinesterase (Sigma C2888) Absorption spectra during the reaction
  • FIG. Schematic diagram of the operation of two-component simultaneous enzyme-linked immunosorbent assay
  • the method of the present invention for simultaneously measuring the activity of a plurality of enzymes by multi-wavelength absorption single channel comprises the following steps:
  • the selected chromophore can be used separately for preparation a chromogenic substrate of the enzyme to be tested; in the combination of the chromogenic substrates obtained, the maximum wavelength of the difference absorption peak of the chromogenic substrate relative to the chromogenic substrate obtained by the chromogenic substrate corresponding to the enzyme to be tested is from large to small Arrangement, the adjacent wavelengths of the difference between the maximum wavelengths of the color-developing products are greater than 30 nm and as far as possible; in the arrangement of the maximum wavelength of the absorption peak of the color-developing product, the closest absorption peak of the color-developing product is closest.
  • the maximum wavelength of the difference absorption peak of the chromogenic product obtained by the corresponding chromogenic substrate under the action of the corresponding enzyme is less than 25 nm from the maximum equal absorption wavelength of a chromogenic substrate in the array.
  • a2 synthesize the corresponding chromogenic substrate of the enzyme to be tested by using the chromophore selected in step a, and use in combination;
  • the chromogenic substrate closest to the infrared end of the color difference product absorption peak is the chromogenic substrate A, and the color product absorption is measured at the maximum wavelength of the chromogenic product differential absorption peak of the chromogenic substrate A. ; measuring the absorption of the remaining enzyme color developing product or chromogenic substrate at the equal absorption wavelength of the chromogenic substrate A;
  • a data processing method for eliminating the overlapping interference of the chromogenic substance in the reaction channel is established, and the color developing product or the chromogenic substrate of each enzyme to be tested is obtained after eliminating the overlapping interference of the absorption spectrum. No interference absorption curve; If a certain enzyme to be tested is not interfered by the products and substrates of other enzymes in the reaction system and the substrate concentration is more than 3 times the Mie's constant of the corresponding enzyme, the color is analyzed by the classical initial velocity method.
  • the product or chromogenic substrate has an interference-free absorption curve to determine the initial velocity; if a certain enzyme to be tested is not interfered with the products and substrates of other enzymes in the reaction system, the chromogenic substrate concentration used is 5% of the Michaelis constant of the enzyme. Above and below 3 times the Michaelis constant, the classical initial velocity method and the reaction process analysis method are combined to analyze the color change product or the chromogenic substrate without interference absorption curve to determine the initial velocity; when a certain enzyme to be tested is subjected to the reaction system In the case of interference with the substrate and/or product of itself and/or other enzymes, the differential velocity equations describing the kinetics of the reaction system are numerically integrated and the activity is interfered with the enzyme reaction to be tested.
  • the color product or the chromogenic substrate has no interference absorption change curve, and the maximum reaction rate is determined to indicate its activity, or the initial velocity is expressed according to the differential velocity equation and the maximum reaction rate obtained, and the substrate concentration is 93% of the initial substrate concentration. Enzyme activity.
  • the maximum wavelength of the difference absorption peak of the chromogenic substrate relative to the chromogenic substrate is above 300 nm and the maximum wavelength of the absorption peak of the chromogenic substrate is smaller than the maximum wavelength of the absorption peak of the corresponding chromogenic product, otherwise the reverse reaction is used.
  • the activity of the enzyme is determined; any two chromogenic substrates adjacent to the maximum wavelength of the difference in absorption peak of the chromogenic product in the enzyme chromogenic substrate combination, and the maximum wavelength of the difference absorption peak of the chromogenic product closer to the infrared end
  • the absorption wavelength is different from the maximum wavelength of the color-developing product.
  • the difference between the maximum absorption wavelength of the absorption peak and the maximum wavelength of the absorption peak is equal to or less than 5 nm.
  • the chromogenic substrate combination is used in the sample.
  • An enzyme to be tested produces a chromogenic product, which is not affected by any other enzyme in the sample or has an ineffective effect; the effect described herein is not affected by any other enzyme or the effect is not obvious, and refers to the enzyme I in the sample.
  • concentration of the corresponding chromogenic substrate differs from the Michaelis constant by less than 50%, the specific activity of any other enzyme in the sample acting on the chromogenic substrate is lower than the ratio of the enzyme I acting on the chromogenic substrate.
  • the buffer medium and the reaction conditions for selecting the enzyme to be tested for the simultaneous determination of the activity are selected, and the specific conditions include: the pH of the buffer medium is between pH 5.0 and 9.0 and the activity is close to the lowest activity.
  • the optimum pH of the sample to be tested, the reaction temperature of the sample used is between 20 and 40 degrees Celsius.
  • the chromogenic substrate comprises a natural chromogenic substrate and a non-natural chromogenic substrate when the method is applied;
  • the natural chromogenic substrate comprises nicotinamide adenine dinucleotide or nicotinamide adenine dinuclear Glycosyl phosphate; non-natural chromogenic substrate
  • the chromophore is composed of an enzyme recognition group, including but not limited to 4-nitrophenol, 4-nitrothiophenol, 4-nitroaniline, 4-nitro-1-naphthol, 4- Nitro-1-naphthylthiophenol, 4-nitro-1-naphthylamine, 2-naphthol, 4-chlorophenol, rosin or a substituted aromatic phenol/amine derivative.
  • the method is applied to synchronously measure two enzyme activities.
  • the two enzymes to be tested are named as enzyme A and enzyme B, respectively, and the two chromogenic substrates are respectively named as The chromogenic substrate A and the chromogenic substrate B, two kinds of chromogenic products formed under the action of two enzymes to be tested are respectively named as chromogenic product A and chromogenic product B, and the combination of these chromogenic substrates and chromogenic products
  • the following conditions should be met: (1) The maximum wavelength XI of the color difference product A is close to the infrared end, and the maximum wavelength X2 of the color difference product is close to the ultraviolet end, and the maximum wavelength X2 of the color difference product B is different from the color product.
  • the maximum wavelength XI of the difference absorption peak is greater than 30 nm and as far as possible; (2) The maximum absorption wavelength of the color-developing product A is Y1 and is less than 25 nm from the maximum wavelength X2 of the chromogenic product B, and is as short as possible.
  • the method is applied to the steps b and c and d for simultaneously measuring the activity of two enzymes, and the corresponding chromogenic substrate A and the synthesis of the two enzymes for chromophore A and chromophore B are synthesized in a synchronous reaction sample in one reaction channel.
  • the wavelength XI is ⁇ ⁇ to measure the chromogenic product A absorption; by rapidly changing the measurement wavelength, the two-wavelength absorption synchronous measurement realizes the simultaneous tracking of the reaction process of two enzymes to be tested in a single reaction system.
  • the method is applied to the step e of synchronously measuring the activity of two enzymes, and the method for eliminating data interference by multi-substance absorption is established based on the absorption linear addition;
  • the wavelength of the absorption peak of the color-developing product A is not more than 25 nm before and after the maximum wavelength. Is ⁇ ⁇ and absorbs Al at this wavelength, and the wavelength of the chromogenic product ⁇ and the chromogenic substrate A is not more than 25 nm near the absorption wavelength ⁇ 2 and the absorption at this wavelength is ⁇ 2, based on the absorption linear addition elimination
  • the material absorption overlap interference needs to be solved as follows:
  • ⁇ 2 ⁇ 20 + ⁇ 32 ⁇ ⁇ + E35 XP 2
  • Pi is the instantaneous concentration of the chromogenic product A
  • P 2 is the instantaneous concentration of the chromogenic product B
  • E31 is the difference molar extinction coefficient between the chromogenic product A and the chromogenic substrate A at ⁇
  • ⁇ 32 is the chromogenic product ⁇ and the chromogenic substrate A is the molar extinction coefficient ⁇ 32 at ⁇ 2
  • ⁇ 34 is the difference molar extinction coefficient between the color-developing product ⁇ and the chromogenic substrate ⁇ at ⁇
  • ⁇ 35 is the color-developing product ⁇ and the chromogenic substrate ⁇ under the ⁇ 2 differential molar extinction coefficient
  • ⁇ is the instantaneous total absorption at ⁇ before absorbing interference
  • ⁇ is the instantaneous total absorption at ⁇ 2 before correcting the absorption interference
  • a is the instantaneous net absorption of the chromogenic product ⁇ at ⁇ after correcting the absorption interference, which is equal
  • the non-interference absorption curves A la and A 2b of the chromogenic product ⁇ and the chromogenic product B under ⁇ ⁇ and at ⁇ 2 are determined ;
  • the concentration of chromogenic substrate A is Cl
  • initial display The concentration of the color substrate B is C2; the maximum reaction rate of the enzyme B is mB , the preset chromogenic substrate concentration used for conversion to the initial velocity is equal to 93% of C 2 , and the Michaelis constant of the enzyme B to the chromogenic substrate B
  • the chromogenic product A is competitively inhibited by enzyme B
  • its competitive inhibition constant is a
  • the enzyme B kinetic equation is:
  • the integral term on the left is the net absorption change of the color product B between the reaction time t and the reaction time 0; the integral term on the right side of the equation is numerically integrated with the constant concentration of the color product A in dt, that is, the calculation
  • V mB x E 34 x sumx x At) 0.93 x C 2 x V mB /(K mB + 0.93 x 2 )
  • the measurement relationship of the enzyme A to convert the chromogenic substrate A into the chromogenic product A is 1: 1, and the measurement relationship of the enzyme B to the chromogenic substrate B to the chromogenic product B is also 1:1;
  • the method for simultaneously measuring the activity of two enzymes has the following characteristics:
  • the chromophore combination is selected as follows: chromophore A and chromophore B.
  • the maximum absorption wavelength of the chromogenic product A is equal to or greater than the maximum wavelength X2 of the chromogenic product B.
  • the measurement wavelength ⁇ 2 is selected as follows: The chromogenic product ⁇ and the chromogenic substrate ⁇ difference molar extinction coefficient is higher at the maximum absorption wavelength Y1 of the chromogenic product ⁇ At 30% of the maximum wavelength X2 of the difference absorption peak of the chromogenic product B, the maximum absorption wavelength Y1 of the chromogenic product A is selected to be ⁇ 2, otherwise, the color difference product B of the maximum absorption wavelength Y1 closest to the chromogenic product ⁇ is used.
  • the absorption peak wavelength is ⁇ 2, or the closest to the color-developing product ⁇ -difference absorption peak and the other color-developing products to be tested and the color-developing chromogenic substrate are different in molar extinction coefficient than the chromogenic product ⁇ and the chromogenic substrate ⁇
  • the extinction coefficient of 20% of the wavelength is ⁇ 2 to measure the color product ⁇ absorption;
  • step e it is necessary to determine the correction coefficient of R31 as the absorption interference of the color-developing product A; when determining R31, only all the chromogenic substrates required for the enzyme A are used without any other chromogenic substrate of the enzyme to be tested, so that the reaction
  • the system does not absorb from the chromogenic substrate B and the chromogenic product B at two measurement wavelengths ⁇ ⁇ and ⁇ 2 , at which time the change in absorption at the two measurement wavelengths in the reaction channel is simultaneously measured until the absorption change at ⁇ 2 is greater than 0.005 or
  • the ⁇ absorption change is greater than 0.500; the absorption is ⁇ as the abscissa, and the absorption at ⁇ 2 is the ordinate for regression analysis, and the obtained regression line slope is the correction coefficient R31;
  • step e it is necessary to determine the correction coefficient of R33 as the absorption interference of the chromogenic product B; when measuring R33, only the enzyme B is used. All the chromogenic substrates required without any other chromogenic substrate of the enzyme to be tested, so that the reaction system does not absorb from the chromogenic substrate A and the chromogenic product A at two measurement wavelengths ⁇ ⁇ and ⁇ 2 . Simultaneously determine the change in absorption at the two measurement wavelengths in the reaction channel until the change in absorption at ⁇ 2 is greater than 0.500 or the change in absorption at ⁇ is greater than 0.005; the absorption at ⁇ 2 is plotted on the abscissa and the absorption at ⁇ is plotted on the ordinate. , the slope of the obtained regression line is the correction coefficient R33;
  • the corresponding chromogenic substrate of the enzyme ⁇ includes 4-nitrophenyl acetate having a maximum absorption wavelength between 310 and 330 nm, and hydrolysis.
  • the maximum absorption wavelength of the reduction reaction is between 350 and 370 nm
  • the chromogenic substrate is disulfide
  • the chromogenic substrate B corresponding to the chromogenic substrate A of such enzyme A is mainly a natural chromogenic substrate, including nicotinamide adenine dinucleotide or nico
  • the method is used for synchronously measuring three enzyme activities, and the three enzymes to be tested are respectively named enzyme A, enzyme B and enzyme C, and the required chromogenic substrates are respectively named as chromogenic substrate A,
  • the chromogenic substrate B and the chromogenic substrate C which are colored under the action of the corresponding enzymes, are named as the chromogenic product A, the chromogenic product B and the chromogenic product C; these chromogenic substrate combinations are required to satisfy the following conditions: (1)
  • the maximum wavelength of the difference absorption peak of the color-developing product A is XI and is closest to the infrared end.
  • the maximum wavelength of the difference absorption peak of the color-developing product C is X3 and is closest to the ultraviolet end, and the maximum wavelength of the difference absorption peak of the color-developing product B is X2 and is located at Between X and X3, X2 is more than 30 nm away from XI and X3 and as far as possible; (2) The maximum absorption wavelength of color product A is Y1 and the second absorption wavelength is Ys; the chromogenic substrate B is generated.
  • the maximum isother absorption wavelength of the color product B is Y2 and is as far as possible from Y1 and as short as possible from Ys; (3) Y1 and X2 are less than 25 nm apart and the distance is as short as possible; Y2 and Ys and X3 are less than 25 nm apart and spaced apart As short as possible;
  • step b the reaction of the three enzymes against the chromogenic substrate A, the chromogenic substrate B and the chromogenic substrate C is simultaneously initiated in one reaction channel, and correspondingly obtained.
  • step c the maximum absorption wavelength Y1 of the color product A or the wavelength within 25 nm is measured as ⁇ 2, and the color product ⁇ absorption peak is selected.
  • the wavelength XI or the wavelength within 25 nm is ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ;
  • step d the measurement process of the two samples in a single reaction system is synchronously tracked by rapidly changing the measurement wavelength and the simultaneous measurement of the three-wavelength absorption;
  • step e the data is resolved based on the linear addition of absorption. Eliminating the overlapping interference of multiple substances requires solving the following ternary equations:
  • a x A w +A la + R33 A 2h + R35 XA 3c
  • a 2 A 20 10 2b + R31 X ⁇ ia + R36 X ⁇ 3c
  • a 3 A 30 +A 3c + R32 X ⁇ ia + R34 X ⁇ 2b
  • R31 E32/E31
  • ⁇ is the instantaneous absorption at ⁇ before correcting the absorption interference
  • ⁇ is the instantaneous absorption at ⁇ 2 before correcting the absorption interference
  • ⁇ is the instantaneous absorption at ⁇ 3 before correcting the absorption interference
  • a is the chromogenic product at the ⁇ after correcting the absorption interference Net absorption
  • ⁇ 2b is the net absorption of the chromogenic product ⁇ at ⁇ 2 after correcting the absorption interference
  • no chromogenic product is formed before absorption in ⁇
  • 2Q is absorbed in ⁇ 2 before the reaction system generates any color developing product.
  • E31 is the color difference product ⁇ and chromogenic substrate A under the ⁇ difference molar extinction coefficient
  • ⁇ 32 is the color product ⁇ and the chromogenic substrate A is lower at ⁇ 2 Molar extinction coefficient
  • ⁇ 33 is the difference molar extinction coefficient between chromogenic product ⁇ and chromogenic substrate A at ⁇ 3
  • ⁇ 34 is the difference molar extinction coefficient between chromogenic product ⁇ and chromogenic substrate ⁇ at ⁇
  • ⁇ 35 is the color development product ⁇
  • the chromogenic substrate ⁇ at ⁇ 2 is the molar extinction coefficient
  • ⁇ 36 is the chromogenic product ⁇ and the chromogenic substrate ⁇ at ⁇ 3, the molar extinction coefficient
  • ⁇ 37 is the chromogenic product C and the chromogenic substrate C at the measurement wavelength ⁇
  • the lower molar extinction coefficient, ⁇ 38 is the difference molar extinction coefficient of the chromogenic product C and
  • the color rendering product A needs to absorb the interference correction coefficients R31 and R32 in step e; only all the color developing substrates of the enzyme A are used in the determination without using other tests.
  • Any chromogenic substrate of the enzyme so that the reaction system is free from the chromogenic substrate B and the chromogenic product B, the chromogenic substrate C and the chromogenic product C are absorbed at three measuring wavelengths, 1, 2 and 3, synchronized
  • the change in absorption at the three measurement wavelengths in the reaction channel is determined until the absorption change at ⁇ 2 and ⁇ 3 is greater than 0.005 or the absorption change at ⁇ is greater than 0.500; the absorption change at ⁇ is plotted on the abscissa, and at ⁇ 2 and at ⁇ 3.
  • the change is the ordinate and the regression analysis is performed.
  • the slope of the regression curve of the absorption change at ⁇ 2 is the correction coefficient R31, and the slope of the regression curve of the absorption change at
  • the color-developing product B needs to absorb the interference correction coefficients R33 and R34 in step e; only all the color-developing substrates of enzyme B are used in the determination without any color development of other enzymes to be tested.
  • the substrate is such that the reaction system does not absorb from the chromogenic substrate A and the chromogenic product A, the chromogenic substrate C, and the chromogenic product C at three measurement wavelengths ⁇ 1, ⁇ 2, and ⁇ 3, and simultaneously measures three wavelengths in the reaction channel.
  • the obtained regression curve of the absorption change under ⁇ is the correction coefficient R33, and the slope of the regression curve of the absorption change at ⁇ 3 is the correction coefficient R34;
  • the color-developing product C needs to absorb the interference correction coefficients R35 and R36 in step e; only all the color-developing substrates of enzyme C are used in the determination without any color development of other enzymes to be tested.
  • the substrate is such that the reaction system does not absorb from the chromogenic substrate A and the chromogenic product A, the chromogenic substrate B and the chromogenic product B at three measurement wavelengths ⁇ ⁇ , ⁇ 2 and ⁇ 3 , and simultaneously measures three wavelengths in the reaction channel.
  • the chromogenic substrate combination can be selected as follows: The chromogenic substrate A, the chromogenic substrate B and the chromogenic substrate C should be used to make the chromogenic product A
  • the maximum absorption wavelength Y1 is equal to or different from the maximum wavelength X2 of the color difference product B absorption peak, or the absorption wavelength Y2 of the color product B is large, and the absorption color Ys of the color product A is large and the absorption of the color product C is poor.
  • the peak maximum wavelength X3 is equal or neither of them is more than 5 nm; when the method is used to simultaneously measure the three enzyme activities, in step c, the maximum color XI of the difference absorption peak of the color-developing product A is selected as ⁇ 1, and
  • the other two measurement wavelengths ⁇ 2 and ⁇ 3 are selected as follows:
  • the chromogenic product ⁇ and the chromogenic substrate ⁇ difference molar extinction coefficient is higher than the chromogenic product B and the chromogenic substrate B at the maximum absorption wavelength Y1 of the chromogenic product ⁇ When the difference absorption peak is 30% of the molar extinction coefficient at the maximum wavelength X2, Y1 is selected as the measurement wavelength ⁇ 2, otherwise, the chromogenic product B difference absorption peak wavelength closest to the chromogenic product ⁇ maximum absorption wavelength Y1 is used, or Close to color
  • the difference between the absorption peak of the substance B and the difference between the other color-developing products and the color-developing substrate is that
  • the chromogenic substrate A corresponding to the enzyme ⁇ includes 4-nitro-1-naphthyl phosphate and the two absorption wavelengths of the hydrolysis are respectively Between 315 and 335 nm and between 395 and 415 nm, 4-nitro-1-naphthyl sulfate and its two absorption wavelengths are between 315 and 335 nm and between 395 and 415 nm, respectively.
  • two equal absorption wavelengths are between 320 and lj 345 nm and between 390 and 410 nm, 4-nitro-1-(N-lysyl)naphthylamine and the maximum absorption wavelength of the hydrolysis is 385 to 385
  • these chromogenic substrates A are activated by enzyme A to form chromogenic product A as 4-nitro-1-naphthol or 4-nitro-1-naphthylamine or a derivative thereof;
  • Substrate A corresponding to chromogenic substrate B is mainly 4-nitrophenol or 4-nitroaniline derivative, under the action of corresponding enzyme B to produce color product B is 4-nitrophenol or 4-nitroaniline or 4 -nitrobenzene a thiol;
  • the enzyme A corresponding to the chromogenic substrate A includes, but is not limited to, an aromatic esterase, a phosphatase, a sulfatase, a ⁇ -glutamyltransferase, a peptidase, that is, a natural amino acid ⁇ .
  • the enzyme oxime corresponding to the chromogenic substrate ⁇ includes other hydrolase different from the selected oxime but belonging to the enzyme A;
  • the enzyme corresponding to the chromogenic substrate C includes, but is not limited to, lactic acid Dehydrogenase LDH, malate dehydrogenase MDH, alanine aminotransferase coupled to LDH, and aspartate aminotransferase coupled to MDH.
  • novel chromogenic substrate required for the application of the examples was prepared by conventional organic synthesis, purified by silica gel column, and subjected to high resolution mass spectrometry.
  • Heli R determines the structure; the synthesis method and process of these novel chromogenic substrates are not within the protection scope of the present invention, and will be the protection content of the separately applied invention patent.
  • Common chromophores suitable for use in the present invention are shown in Figure 2; the equal absorption wavelengths of common enzyme reactions are shown in Figures 3 and 4;
  • the change in the absorption spectrum of the reaction process was measured using a wavelength uncalibrated Shimad Z u UV2550 luminometer.
  • the enzyme activity was determined by using the corresponding control software on a MapAda UV1600 PC or by using a Gene 5.0 software control instrument on a Biotek ELX800 plate reader. Unless otherwise stated, all of the following measured temperatures are room temperature, i.e., 25 degrees.
  • Example 1 Simultaneous determination of LDH and GGT in rabbit kidney homogenate by single channel two-wavelength absorption
  • the chromogenic substrate of lactate dehydrogenase (abbreviated as LDH, Shanghai Shenggong, No. NB0642) is pyruvic acid and reduced nicotinamide adenine dinucleotide, NADH, which are all domestically analyzed and pure;
  • the chromogenic substrate of glutamyl transferase (GGT) is ⁇ -glutamyl-(4-nitro)aniline (Sigma, No. 49525), and the diglyceride is domestically pure analytically; the buffer used is 0.10 mol/L.
  • the pH is 7.0 sodium phosphate
  • the GGT chromogenic substrate is dissolved in the buffer to be a chromogenic substrate solution A
  • LDH chromogenic substrate solids are weighed and dissolved in the buffer to be a chromogenic substrate solution B
  • the mixture of chromogenic substrates is the chromogenic substrate solution C
  • the chromogenic substrate of GGT ⁇ -glutamyl-(4-nitro)aniline is the chromogenic substrate A
  • the corresponding chromogenic product A is 4-nitroaniline.
  • NADH is the LDH chromogenic substrate but is used as the chromogenic product B; the solution used only contains the GGT chromogenic substrate and does not contain NADH, the system light absorption background is zero;
  • GGT is from the rabbit kidney, with 0.1% surface activity Rabbit kidney homogenate was prepared by using NP40 and 0.10 mol/L acid salt buffer of pH 7.0, and the supernatant was taken as GGT-rich after 20 min of 4 Q C centrifugation at 10,000 rpm. The sample to be tested, the homogenate without surfactant was centrifuged and the supernatant was a sample containing low activity GGT.
  • the method for determining the GGT activity is the invention patent ZL 200710093081.4 and J Zhejiang Univ Sci B, 2011, 12 3): 180-188; the GGT coloring product is set to a GGT inhibition constant of 185 ⁇ /L, GGT to ⁇ when data processing.
  • - glutamyl-C4-nitro)aniline has a Michaelis constant of 1.00 mmol/L.
  • the software used is the same as J ZA 'awg Univ Sci B, 2011, 12(3): 180-188 o
  • GGT is an enzyme.
  • LDH is enzyme B
  • chromogenic product A is 4-nitroaniline
  • chromogenic product B is NADH but it is actually a chromogenic substrate
  • ⁇ is 405 nm in the measurement wavelength
  • measurement wavelength ⁇ 2 is 344 nm (see Figure 3b).
  • the representative process for simultaneous determination of rabbit kidney GGT and rabbit muscle LDH using the present invention is as follows:
  • Preparation buffer According to the "General Manual for Biochemistry and Molecular Biology Experiments" (Science Press, 2000), prepare 0.20 mol / L and pH 7.0 sodium phosphate buffer diluted to 0.10 mol / L; 25 ° constant temperature;
  • chromogenic substrate solution Prepare three solutions of the chromogenic substrate; chromogenic substrate stock solution A contains 1.5 mm 0 l/L Y - glutamyl-(4-nitro)aniline and 0.70 mmol /L diglycine; chromogenic substrate stock solution B contains 1.50 mmol / L of NADH and 30.0 mmol / L pyruvic acid; chromogenic substrate stock solution C is chromogenic substrate stock solution A and chromogenic substrate stock solution An equal mixture of B; all assays have a total reaction volume of 1.0 ml, a final concentration of ⁇ -glutamyl-(4-nitro)aniline of 0.15 mmol/L and a diameptane of 70 mmol/L and a NADH of 0.15 mmol/1 The pyruvic acid was 3.0 mmol/L, and the rest were buffer and moderately diluted samples;
  • Multi-wavelength absorption interference correction coefficient Using chromogenic substrate stock solution A plus appropriate buffer and sample to make the final concentration of diglycopeptide 70 mmol / L and chromogenic substrate A 0.15 mmol / L, ⁇ ⁇ and ⁇ 2
  • the relationship between the lower absorption changes (Fig. 6a), the reaction time of 10.0 min increases the absorption at 405 nm by more than 0.55; the sensitized substrate stock solution B is used to supplement the buffer solution and the LDH sample solution to determine the absorption changes under ⁇ and ⁇ 2.
  • the relationship (Fig. 6b) the reaction time of 10.0 min reduces the absorption at 344 nm by 0.55.
  • the slopes of the statistically significant response curves in Figures 7a and 7b are the interference correction coefficients R31 and R33, respectively;
  • NADH has no absorption at 405 nm, so the measured 405 nm absorption comes from the background and the chromogenic product 4-nitroaniline, and does not need to correct the interference of NADH absorption on 405 nm absorption;
  • the absorption of the sample and chromogenic substrate A at 405 nm is the base, and the absorption at 405 nm minus the background is the absorption of 4-nitroaniline, which is used to correct the interference of 4-nitroaniline on LDH activity;
  • a total of 15 min is recorded for 20 s;
  • the specific processing method for the obtained two-wavelength absorption change data is as follows:
  • the interference-free data of the two color products can be obtained without interference at two wavelengths; the color product A is relatively measured and the chromogenic substrate A is in the ⁇ ⁇ difference molar small light coefficient, color product ⁇ Compared with the chromogenic substrate ⁇ 2 in the ⁇ 2 difference molar light coefficient, the reaction product chromogenic product ⁇ and the chromogenic product ⁇ instantaneous concentration can be obtained; the classical initial velocity method or the process analysis method can be used to separately treat the interference-free absorption change or the display Color product concentration data, thereby simultaneously measuring the activity of the two enzymes in a single channel;
  • LDH has a NADH Michaelis constant of 0.040 mmol/L
  • p-nitroaniline has a differential extinction coefficient of 9.8 nm at 405 nm of 9.87 L-(mmol.cm)" 1
  • NADH at 344 nm millimolar difference extinction coefficient is ⁇ ⁇ ⁇ )- 1
  • the concentration of pyruvic acid in the reaction system is twenty times higher than that of NADH
  • LDH acts as a single chromogenic substrate enzyme for NADH
  • the initial concentration of NADH is 0.14 mmol/L when designing LDH activity
  • the maximum reaction rate of LDH is VmB
  • After correcting the interference NADH absorbs b at 344 nm instantaneously.
  • V mB of LDH 1
  • the concentration of p-nitroaniline was constant during the sampling interval, and the integral was obtained as Sumx.
  • the mB of LDH was obtained and the initial LDH velocity was obtained with the initial NADH concentration of 0.14 mmol/L.
  • the obtained response curve is shown in Figure 6d. Correction of p-nitroaniline interference The effect is shown in Table 2; this calibration allows the two enzymes to be synchronized to the limit of quantitation alone.
  • the limit of GGT for LDH is ⁇ . ⁇ ⁇ ⁇ ⁇ - 1 ; When the LDH is 8.2 ⁇ ⁇ ) -1 with chromogenic substrate solution, the GGT limit of quantification is 0.14 ⁇ ⁇ ) -1 ; When the activity is 20 ⁇ ⁇ ) -1 , the GGT limit of quantification is 0.17 (Table 1). Quantification of LDH without GGT, determination of GG with chromogenic substrate solution When T is l J mol ⁇ L in) -1 , the limit of LDH is 0.6. The chromogenic substrate solution A is determined to have a limit of LDH of 0.5 when GGT is 10 mol ⁇ L in) -1 . The results are compared in Table 3.
  • Example 2 Simultaneous determination of alkaline phosphatase and natural ⁇ -galactosidase mixture by single-channel multi-wavelength absorption
  • the small intestinal mucosal alkaline phosphatase (Sigma P0114, CIAP) was used as the enzyme A, and the natural ⁇ -D-galactosidase ( ⁇ -Gal) in the E. coli cell line BL21 (DE3) was passed through Sephadex G25. After removal of the small molecule by the column, the enzyme B (Sephadex G25 column height 15 cm inner diameter 10 mm volume about 10 ml, equilibrated with 50 mmol/L and pH 7.5 tris). 4-Nitro-1-naphthyl phosphate is a chromogenic substrate A and synthesized as shown in the literature (MM Mhala, Puma Nand.
  • the content of the effective chromogenic substrate in the 4-nitro-1-naphthyl phosphate is about 68%, and the free 4-nitro-1-naphthol is less than 2%; 27%), 4-nitrophenyl- ⁇ -D-galactoside is a BBI product (BBI, No. NB2361-lg).
  • the chromogenic product A is 4-nitro-1-naphthol, and the chromogenic product B is 4-nitrophenol; the wavelength ⁇ ⁇ is 450 nm (microplate reader), and ⁇ 2 is 405 nm (Fig. 4b and Fig. 3a).
  • the microplate reader used was Biotek E1X800 and the accompanying Gene 5.0 software.
  • the buffer used was pH 7.5 and contained 5.0 ⁇ /L citric acid 50 mmol/L Tris-HCl; the chromogenic substrate A was dissolved in the buffer to be a chromogenic substrate solution A, and the chromogenic substrate B was dissolved.
  • the buffer solution is a chromogenic substrate solution B; the chromogenic substrate mixture containing enzyme A and enzyme B is a chromogenic substrate solution C.
  • chromogenic substrate solution A contains only 4-nitro-1-naphthyl phosphate at a final concentration of 0.20 mmol/L, and the chromogenic substrate solution is prepared with dimethyl sulfoxide.
  • chromogenic substrate solution B contained only 6.0 mmol/L 4-nitrophenyl- ⁇ -D-half Glycoside, dissolved directly in buffer Solution, used as the day after preparation
  • chromogenic substrate solution C contains 0.20 mmol/L 4-nitro-1-naphthyl phosphate and 6.0 mmol/L 4-nitrophenyl- ⁇ -galactoside.
  • the chromogenic substrate solution ⁇ and ⁇ are used only on the same day after the configuration;
  • Multi-wavelength absorption interference correction coefficient The same concentration of 4-nitro-1-naphthyl phosphate and CIAP were used to continuously record the absorption changes at 450 nm and 405 nm. Under the conditions used, the absorption was substantially linear before the absorption at 450 nm increased by 0.60. , the response curve of 405 nm absorption to 450 nm absorption is analyzed, and the obtained slope is the interference correction coefficient R31 (Fig. 7a); the 450 nm and 405 nm absorption are continuously measured by 4-nitrophenyl- ⁇ -D-galactoside and ⁇ -Gal. The change is linear before the absorption at 405 nm increases to 1.00. The response of the absorption at 450 nm to the absorption at 405 nm is analyzed. The slope of the obtained curve is the interference correction coefficient R33 (Fig. 7b);
  • Vl sl+ R33xs2
  • V2 s2+ R31 xsl
  • ⁇ -D-galactosidase (Sigma G4155, ⁇ -Gal) is used as enzyme A
  • ⁇ -D-glucosidase (Sigma G0660, ⁇ -Glu) as enzyme B
  • 4-nitro- 1-naphthyl- ⁇ -D-galactoside is a chromogenic substrate A, synthesized by the synthesis of 4-nitrophenyl- ⁇ -D-galactoside, and purified by gel column chromatography (using ⁇ -D) -galactosidase to completely hydrolyze the chromogenic substrate and determine the resulting 4-nitro-1-naphthol to determine the purity)
  • 4-nitrophenyl- ⁇ -D-glucoside (Sigma N1377) for color development
  • Substrate B 100 mmol/L phosphate buffer with buffer pH 7.4.
  • the chromogenic product A is 4-nitro-1-naphthol, and the chromogenic product B is 4-nitrophenol; the measurement wavelength ⁇ is 450 nm and the measurement wavelength ⁇ 2 is 405 nm (provided in the standard configuration of the Biotek ELX 800 microplate reader) Filter, Figure 4a).
  • the mixture enzyme sample in this application example is a mixture of enzyme A and enzyme B which are separated by an enzyme-linked immunosorbent assay (ELISA) but bound to a microplate.
  • Preparation buffer According to the "General Manual for Biochemistry and Molecular Biology Experiments” (Science Press, 2000), 100 mmol/L and pH 7.4 sodium phosphate buffer solution; 25 ° C water bath constant temperature standby;
  • chromogenic substrate A is prepared with dimethyl sulfoxide to prepare 20 mmol/L chromogenic substrate A stock solution.
  • the sodium phosphate buffer was diluted to 0.20 mmol/L as the chromogenic substrate solution A; the chromogenic substrate B was formulated with 20% dimethyl sulfoxide to prepare a 50 mmol/L chromogenic substrate B stock solution, buffered with sodium phosphate.
  • the solution was diluted to 2.6 mmol/L as the chromogenic substrate solution B; the stock solution of the chromogenic substrate A and the chromogenic substrate B were diluted with sodium phosphate buffer to a chromogenic substrate A concentration of 0.20 mmol/ L and chromogenic substrate B is 2.6 mmol / L, to obtain a chromogenic substrate solution C;
  • Penicillin G activation 0.73 g of penicillin G sodium salt is dissolved in 2.0 ml of hydrazine, ⁇ '-dimethylformamide, and 0.40 g of 1-ethyl-(3-dimethylaminopropyl)-carbodiimide is added. (abbreviated as EDC) hydrochloride, and 0.30 g of 1-hydroxybenzotriazole for 30 min to obtain a dark yellow reaction solution; take 4 ul of the above dark yellow solution and dilute to 54 ul with 54 ul of buffer to activate the penicillin G solution;
  • EDC 1-ethyl-(3-dimethylaminopropyl)-carbodiimide
  • Dosage When applied; After optimizing the amount of monoclonal antibody, gradually increase the dosage of penicillin-labeled ⁇ -D-galactosidase solution until the bound hapten-labeled ⁇ -D-galactosidase activity reaches saturation; in 96-well plate System, when 20 ⁇ l diluted 500 times of monoclonal antibody, add penicillin labeled ⁇ -D-galactosidase solution 20 ul, to achieve saturation;
  • Post-labeling specific activity The specific activity was determined to be 90 mol L_min_mg) with a final concentration of 2.6 mmol/L of 4-nitrophenyl-aD-glucoside pH 7.4 in 100 mmol/L phosphate buffer - retention 85 %;

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Abstract

本发明公开了一种用多波长吸收单通道同步测量多种酶活性的方法；基于光吸收线性加和性及酶显色底物与其对应显色产物的等吸收波长，建立多波长吸收同步测量单通道同步测定多种酶活性所需显色底物组合设计原理及消除吸收光谱重叠干扰的数据处理方法，并以数值积分的速度方程分析酶反应过程测定最大反应速度消除各种底物及产物对待测酶活性的干扰，使单通道同步测定多种酶活性的范围及检测限都与单独测定时相当；该方法可用于同步测定生物样品多种酶活性、酶标记免疫分析同步测定多组分含量、同步筛选不同靶酶的抑制剂，可用于临床生化分析和临床免疫检验、卫生检验、酶抑制剂筛选等常规实践，也可用于这类酶法分析技术适用的各种基础研究。

Description

说 明 书
用多波长吸收单通道同步测量多种酶活性的方法
技术领域
本发明涉及一种用多波长吸收同步测量技术实现多种酶活性同步测量方法， 具体涉及 一种单通道同步测定多波长吸收而同步测量多种酶活性的方法。 背景技术
定义酶活性测量灵敏度为吸收变化速度对酶量响应曲线斜率，酶活性测量的定量限 (limit of quantification, LOQ)为上述响应曲线中直线部分截距加上回归估计标准差 5倍对应酶量。 测定酶活性都要求灵敏度尽量高、 定量限尽量低而分析效率尽量高。用针对特定酶的高专一 性显色底物可简便高灵敏度连续测定吸收变化反应曲线而测定酶活性； 这类酶活性测定过程 常规用于临床生化检验、 卫生检验检测酶抑制剂类食品或环境污染物、 高通量筛选酶抑制剂 类组合库和酶标记免疫分析等。 另一方面， 酶活性测定的高特异性和高灵敏度使酶作为标记 信号用于免疫分析， 代表性技术即酶联免疫吸附分析 (Enzyme-linked immunoassay, ELISA), 这是临床检验领域选择性测定混合物中特定大分子或小分子的最常用方法之一。 ELISA应用 过程中需用捕获成分包被微孔板以便分离免疫反应复合物，并需洗涤过程，单次分析常需 130 min以上， 分析效率低。 所以， 提高酶活性测量效率在应用中有重要意义。
连续跟踪酶反应曲线测定酶活性都每次使用一个显色底物测定一种酶活性， 即每个反应 通道每次只测定一种酶。 在临床检验领域经常需测定相同标本中的两种甚至多种酶活性， 如 测定肝功状态需测定相同标本中的谷丙转氨酶、 谷草转氨酶、 γ-谷氨酰基转移酶、 碱性磷酸 酶等， 诊断急性胰腺炎时需测定相同标本中的淀粉酶和脂肪酶。 另一方面， ELISA技术主要 缺点是其分析效率低， 单次分析耗时长。 再有， 筛选酶抑制剂组合化学库时， 每次只测定一 种靶酶活性的筛选成本很高而效率低， 对组合库样品的消耗量大。发展新技术显著提高测定 酶活性效率， 并保障测量酶活性的灵敏度和定量限与单组分测定时相当， 无疑有重要意义。
提高酶法分析效率的最简单策略是在一个酶反应体系， 即酶反应通道， 每次使用多种显 色底物实时同步或基本实时同步地连续跟踪多种酶显色产物吸收变化曲线同步测定多种酶 活性。同步测定多波长吸收光度计已普及，如 Biotek ELX800酶标仪可同步测量三波长吸收、 美谱达 UV1600PC普通光度计可同步测定七波长吸收。这些常规设备同步测定不同波长吸收 实际上有较短时差； 用有双单色器或用二极管阵列检测器的光度计可基本实时同步测量多波 长吸收。 所以， 建立单通道同步测定多种酶的显色底物组合原理及所需数据处理方法， 就有 望实现单通道多波长吸收同步跟踪多种酶反应过程同步测量多种酶活性并保障与单组分测 定定量限和灵敏度相当， 此方法用于 ELISA及酶抑制剂高内涵筛选都可显著提高效率。
很早报道用两种酶标记单通道测量两组分的 ELISA技术， 但没有实现单通道两种酶同 步反应和吸收连续跟踪。 Blake等 1982年报道用碱性磷酸酶和 β-半乳糖苷酶标记两种抗原竞 争结合测定单通道同步测定半抗原 (Clin Chem 1982; 28(7):1469-1473) o 但这两种酶反应是在 不同反应体系进行而非同步进行， 且不能连续跟踪任一标记酶反应的过程， 其使用酚酞单磷 酸酯为碱性磷酸酶显色底物， 比活性低于对硝基苯基磷酸酯为显色底物时比活性的 7%而降 低了灵敏度， 而酚酞单磷酸酯的成本又很高， 使得此技术没有得到实际应用。 其它单通道检 测两组分的 ELISA技术中两种酶反应及两种酶显色产物检测都不同步， 也不能连续跟踪两 种酶反应曲线 (Dean et al, Clin Chem, 1983; 29(6): 1051-1056。Ng， et al. Clin Chem, 1987; 33(12): 2286-2288。 Choi, et al. Clin Chem, 1991 ; 37(5): 673-677。 Porstmann T, et al. J Immunol Methods . 1993; 158:95-106。 Krambovitis E， et al. Clin chem 1995; 41 :48-53。 Osuchowski, et al. Methods 2006; 38:304-31 1和 Sun J， et al. Anal Chim Acta 2010; 666: 76-82)。 因此， 现有 ELISA技术都 未实现单通道同步反应同步连续跟踪多种酶显色显色产物； 在筛选酶抑制剂领域， 也没有实 现单通道同步测量多种酶活性筛选酶抑制剂的报道。
显然， 需建立测定酶活性的显色底物设计组合原理及对应的特殊数据处理方法， 才可能 用常规设备多波长吸收单通道同步测量多种酶活性。在 863项目 2011AA02A108资助下， 发 明人建立了本发明权利要求书中所提出的方法。 发明内容
本发明的目的是提供一种用多波长吸收单通道同步测量多种酶活性的方法，其基于无相 互作用物质吸收的线性加和性、酶显色底物和相应显色产物的等吸收波长建立所需显色底物 设计和组合应用的新原理， 并建立多种物质吸收光谱重叠干扰的数据处理方法， 以及消除不 同酶的底物与产物之间相互干扰的数据处理方法， 单通道多波长吸收同步测定多种酶活性。
本发明用多波长吸收单通道同步测量多种酶活性的方法， 包括下列步骤：
a. 确定用多波长吸收单通道同步测定多种酶活性所需显色底物组合：
al根据待测酶的专一性筛选满足下列条件的显色底物组合： 所选显色团能分别用于制备 待测酶的显色底物； 所得这些显色底物组合中， 将显色底物在对应待测酶作用下所得显色产 物相对该显色底物的差吸收峰最大波长从大到小排列，相邻的显色产物差吸收峰最大波长之 间相距大于 30 nm且尽量远； 在按显色产物差吸收峰最大波长从大到小的排列中， 除了显色 产物差吸收峰最靠近红外端的显色底物外，每个显色底物在对应酶作用下所得显色产物差吸 收峰最大波长， 都与此排列中某个显色底物的最大等吸收波长相差小于 25 nm且尽量短； a2 用步骤 al所选显色团分别合成待测酶的对应显色底物并组合使用；
b . 在一个反应混合物或酶反应体系即单反应通道中， 同步启动多个待测酶针对组合使用 的显色底物混合物对应显色底物的专一催化反应；
c 选择测量波长组合： 以显色产物差吸收峰最靠近红外端的显色底物为显色底物 A， 在 显色底物 A的显色产物差吸收峰最大波长下测定该显色产物吸收； 在显色底物 A的等吸收 波长处测定其余待测酶显色产物或显色底物的吸收；
d. 在所选多个测量波长组合下， 通过快速变换测量波长， 多波长吸收同步测量实现同步 跟踪单通道中多个待测酶的反应过程；
e. 基于无相互作用物质吸收的线性加和性建立消除反应通道中显色物质吸收重叠干扰的 数据处理方法， 获得消除吸收光谱重叠干扰后每个待测酶的显色产物或显色底物无干扰吸收 变化曲线； 如某个待测酶不受反应体系其它酶的产物及底物的干扰且其底物浓度在对应酶米 氏常数的 3倍以上，用经典初速度法分析其显色产物或显色底物无干扰吸收变化曲线确定初 速度； 如某个待测酶不受反应体系其它酶的产物及底物的干扰但所用显色底物浓度在该酶米 氏常数的 5 %以上而低于米氏常数的 3倍， 联用经典初速度法和反应过程分析法分析其显色 产物或显色底物无干扰吸收变化曲线确定初速度；当某个待测酶受反应体系中其自身和 /或其 它酶的底物和 /或产物的干扰时，将描述反应体系动力学的微分速度方程组数值积分后分析活 性受到干扰待测酶反应的显色产物或显色底物无干扰吸收变化曲线，确定其最大反应速度表 示其活性，或据其微分速度方程和所得最大反应速度换算成底物浓度为起始底物浓度 93 %时 初速度表示酶活性。
进一步， 应用该方法时， 显色产物相对显色底物差吸收峰最大波长在 300 nm以上且其 显色底物吸收峰最大波长小于相应显色产物吸收峰最大波长， 否则用逆反应测定该酶的活 性； 对待测酶显色底物组合中显色产物差吸收峰最大波长相邻的任两个显色底物， 显色产物 差吸收峰最大波长更靠近红外端者的最大等吸收波长与显色产物差吸收峰最大波长更靠近 紫外端者的差吸收峰最大波长相等或相距不超过 5nm为优化组合;所用显色底物组合中每个 显色底物都仅被样品中的一种待测酶作用生成显色产物， 而不被样品中其它任何酶作用或作 用效果不明显； 此处所述不被其它任何酶作用或作用效果不明显， 指当样品中酶 I所对应显 色底物的浓度与其米氏常数相差小于 50%时，样品中其它任何酶作用于该显色底物的比活性 都低于酶 I作用于该显色底物比活性的 1%; 权利要求 1的步骤 b中， 选择适于同步测定活 性的待测酶发挥作用的缓冲介质和反应条件， 具体包括： 缓冲介质 pH位于 pH 5.0到 9.0之 间且接近比活性最低待测样品的最适 pH， 所用样品反应温度在摄氏 20到 40度之间。
进一步， 该方法应用时所述显色底物包括天然显色底物和非天然显色底物； 天然显色底 物包括尼克酰胺腺嘌呤二核苷酸或尼克酰胺腺嘌呤二核苷酸磷酸； 非天然显色底物由显色 团和被酶识别基团组成， 显色团包括但不限于 4-硝基苯酚、 4-硝基苯硫酚、 4-硝基苯胺、 4-硝基 -1-萘酚、 4-硝基 -1-萘硫酚、 4-硝基 -1-萘胺、 2-萘酚、 4-氯苯酚、 玫红酸或其衍生物。
进一步， 该方法应用于同步测量两种酶活性， 步骤 a中， 所述样品中含两种待测酶分别 命名为酶 A和酶 B，所需两种显色底物分别命名为显色底物 A和显色底物 B，在两种待测酶 作用下生成两种显色产物分别命名为显色产物 A和显色产物 B，这些显色底物及显色产物的 组合需满足下列条件： （1 ) 显色产物 A差吸收峰最大波长 XI靠近红外端， 显色产物 B差 吸收峰最大波长 X2靠近紫外端， 显色产物 B差吸收峰最大波长 X2与显色产物 A差吸收峰 最大波长 XI相距大于 30 nm且尽量远； （2)显色产物 A的最大等吸收波长为 Y1且与显色 产物 B差吸收峰最大波长 X2相距小于 25 nm且且尽量短。
该方法应用于同步测量两种酶活性的步骤 b和 c及 d中， 在一个反应通道同步启动样品 中两种酶针对显色团 A和显色团 B合成的对应显色底物 A和显色底物 B的反应， 并得到显 色产物 A和显色产物 B; 以显色产物 A的最大等吸收波长 Y1为 λ2处测定显色产物 Β吸收， 以显色产物 Α的差吸收峰最大波长 XI为 λΐ处测定显色产物 A吸收;通过快速变换测量波长， 两波长吸收同步测量实现同步跟踪单个反应体系中两种待测酶的反应过程。
该方法应用于同步测量两种酶活性的步骤 e中， 基于吸收线性加和性建立数据解析消 除多种物质吸收重叠干扰方法； 以显色产物 A差吸收峰最大波长前后不超过 25 nm的波长 为 λΐ且在此波长下吸收为 Al， 以显色产物 Α和显色底物 A最大等吸收波长附近不超过 25 nm的波长为 λ2且此波长下吸收为 Α2， 基于吸收线性加和性消除物质吸收重叠干扰需解如 下二元一次方程组： = Aw+E31ΧΡι + Ε34Χ P2
A2 = Α20+Έ32 XPi + E35 X P2
进一步可转变成如下二元一次方程组
A = ^io+^ia + R33 XA2h A2 = ^20+^2b + R31 X a
R31= E32/E31 R33= E34/E35
Pi为显色产物 A瞬时浓度； P2为显色产物 B瞬时浓度； E31为显色产物 A与显色底物 A在 λΐ下差摩尔消光系数； Ε32为显色产物 Α与显色底物 A在 λ2下差摩尔消光系数 Ε32; Ε34为显色产物 Β与显色底物 Β在 λΐ下差摩尔消光系数； Ε35为显色产物 Β与显色底物 Β 在 λ2下差摩尔消光系数； ^为校正吸收干扰前在 λΐ瞬时总吸收， Α为校正吸收干扰前在 λ2 瞬时总吸收； a为校正吸收干扰后显色产物 Α在 λΐ瞬时净吸收， 其等于 1>1和£31乘积， ^2b为校正吸收干扰后显色产物 Β在 λ2瞬时净吸收， 其等于 Ρ2和 Ε35乘积； 。为反应体 系未生成任何显色产物前在 λΐ的吸收， 为来自反应体系的本底， 相当于含相同浓度全部显 色底物反应体系在 λΐ吸收与含相同浓度样品反应体系在 λΐ吸收的加和； 2Q为反应体系未 生成任何显色产物前在 λ2的吸收， 为来自反应体系的本底， 相当于含浓度相同全部显色底 物的反应体系在 λ2吸收与含相同浓度样品反应体系在 λ2吸收的加和；
当仅某种酶显色产物对另一种酶有竞争抑制作用时， 确定显色产物 Α及显色产物 B在 λΐ下和在 λ2下的无干扰吸收变化曲线 Ala和 A2b;设初始显色底物 A的浓度为 Cl，初始显 色底物 B的浓度为 C2; 设酶 B最大反应速度为 mB， 由 换算成初速度所用预设显色底 物浓度等于 C2的 93 %， 酶 B对显色底物 B的米氏常数为 ^; 如显色产物 A对酶 B为竞 争性抑制， 其竞争性抑制常数为 a， 酶 B动力学方程为： 2 EM KmB (1 + Α1α /Κία /£31) + C2
对上述方程两边对反应时间从 0〜t进行积分得到
Figure imgf000006_0001
左边的积分项为反应时刻 t和反应时刻 0之间显色产物 B的净吸收变化；方程右边积分 项以 dt内显色产物 A浓度恒定进行数值积分， 即计算
1
X -■
KmB (\ + A, KiaIE^) + C2
以 dt为 Δ ， 计算每个记录点的 χ， 将所有 X求和再减去起点和终点 X得到 sumx， 用如 下公式计 并据其微分动力学方程得无干扰初速度 。
VmB C2 x E34 x sumx x At) = 0.93 x 2x VmB /(KmB +0.93x 2)
Figure imgf000006_0002
上述计算公式中， 酶 A将显色底物 A转变成显色产物 A的计量关系为 1:1， 酶 B将显 色底物 B转变成显色产物 B的计量关系也为 1:1;
当酶 A的产物对酶 B抑制作用类型不同时， 所得酶 B的米氏动力学方程不同而所需数 值积分过程相同， 并仍用 93 % C2为初始底物浓度计算 s2;
进一步， 该方法用于同步测量两种酶活性有如下特征：
步骤 a中， 如下选择显色团组合： 显色团 A和显色团 B使显色产物 A最大等吸收波长 Y1与显色产物 B差吸收峰最大波长 X2相等或相距不超过 5 nm;
步骤 c中， 选择产物 A差吸收峰最大波长 XI为 λΐ后， 如下选择测量波长 λ2: 显色产物 Β与显色底物 Β差摩尔消光系数在显色产物 Α最大等吸收波长 Y1处高于在显色产物 B差吸 收峰最大波长 X2处的 30%， 则选择显色产物 A最大等吸收波长 Y1为 λ2， 否则， 用最靠近 显色产物 Α最大等吸收波长 Y1的显色产物 B差吸收峰波长为 λ2， 或最靠近显色产物 Β差 吸收峰且其它待测显色显色产物与显色显色底物差摩尔消光系数都小于显色产物 Β 与显色 底物 Β差摩尔消光系数 20%的波长为 λ2测量显色产物 Β吸收；
步骤 e中， 需测定 R31为显色产物 A吸收干扰的校正系数； 测定 R31时， 仅用酶 A所 需的全部显色底物而不加其它待测酶的任何显色底物， 使反应体系无来自显色底物 B及显 色产物 B在两个测量波长 λΐ和 λ2的吸收， 此时同步测定反应通道中在两个测量波长下的 吸收变化， 直到在 λ2吸收变化大于 0.005或在 λΐ吸收变化大于 0.500; 以在 λΐ下吸收为横 坐标， 在 λ2下吸收为纵座标进行回归分析， 所得回归直线斜率为校正系数 R31 ;
步骤 e中， 需测定 R33为显色产物 B吸收干扰的校正系数； 测定 R33时， 仅用酶 B 所需的全部显色底物而不加其它待测酶的任何显色底物， 使反应体系无来自显色底物 A及 显色产物 A在两个测量波长 λΐ和 λ2的吸收， 此时同步测定反应通道中在两个测量波长下 的吸收变化， 直到在 λ2吸收变化大于 0.500或在 λΐ吸收变化大于 0.005 ; 以在 λ2下吸收为 横坐标， 在 λΐ下吸收为纵座标进行回归分析， 所得回归直线斜率为校正系数 R33 ;
进一步， 该方法用于同步测量两种酶活性时， 酶 Α对应显色底物包括水解反应最大等 吸收波长在 310到 330 nm之间的 4-硝基苯基乙酸酯， 水解反应最大等吸收波长在 330到 350nm之间的 4-硝基苯基磷酸酯、 4-硝基苯基硫酸酯、 4-硝基苯基 -β-D-半乳糖苷和 γ-谷氨 酰基 -4-硝基苯胺， 还原反应最大等吸收波长在 350到 370nm之间 5-巯基 -2-硝基苯甲酸及 5-巯基 -2-硝基苯乙酸， 其显色底物为二硫化物被酶作用所释放的硫醇还原； 与此类酶 A的 显色底物 A对应的显色底物 B主要是天然显色底物，包括尼克酰胺腺嘌呤二核苷酸或尼克 酰胺腺嘌呤二核苷酸磷酸； 上述显色底物 A对应的酶 A包括但不限于芳香酯酶、 磷酸酶、 硫酸酯酶、 γ_谷氨酰基转移酶、 肽酶即天然氨基酸 α-羧基对应的酰胺酶和糖苷酶； 与此类 酶 Α对应的酶 B包括但不限于乳酸脱氢酶 LDH、 苹果酸脱氢酶 MDH、 偶联 LDH的谷丙 转氨酶和偶联 MDH的谷草转氨酶。
进一步， 该方法用于同步测量三种酶活性， 三种待测酶分别命名为酶 A、 酶 B和酶 C， 所需显色底物三种分别命名为显色底物 A、显色底物 B和显色底物 C， 在对应酶作用下生成 显色产物分别命名为显色产物 、显色产物 B和显色产物 C; 这些显色底物组合需满足下列 条件： （1 ) 显色产物 A差吸收峰最大波长为 XI且最靠近红外端， 显色产物 C差吸收峰最 大波长为 X3且最靠近紫外端， 显色产物 B差吸收峰最大波长为 X2且位于 XI和 X3之间， X2与 XI及 X3相距都大于 30 nm且尽量远； （2 ) 显色产物 A的最大等吸收波长为 Y1且 次大等吸收波长为 Ys;显色底物 B生成显色产物 B的最大等吸收波长为 Y2且与 Y1相距尽 量远而和 Ys相距尽量短； （3) Y1与 X2相距小于 25 nm且相距尽量短； Y2和 Ys及 X3相 距都小于 25 nm且相距尽量短；
该方法用于同步测量三种酶活性时， 步骤 b中， 在一个反应通道同步启动三种酶针对显 色底物 A、显色底物 B和显色底物 C的反应，并得到显色产物 A、显色产物 B和显色产物 C; 该方法用于同步测量三种酶活性时， 步骤 c中， 选显色产物 A最大等吸收波长 Y1或相 距 25nm以内的波长为 λ2测显色产物 Β吸收， 选显色产物 Α差吸收峰最大波长 XI或相距 25nm以内波长为为 λΐ测显色产物 Α吸收， 选显色产物 B最大等吸收波长 Y2或相距 25nm 以内的波长为 λ3测显色产物 C吸收；
该方法用于同步测量三种酶活性时， 步骤 d中， 通过快速变换测量波长， 三波长吸收同 步测量实现同步跟踪单个反应体系中两种样品的反应过程；
该方法用于同步测量三种酶活性时， 步骤 e 中， 基于吸收的线性加和性建立数据解析 消除多种物质吸收重叠干扰需解如下三元一次方程组：
Ax = Aw+Ala + R33 A2h + R35 XA3c
A2 = A20十 2b + R31X^ia + R36X^3c
A3 = A30 +A3c + R32X^ia + R34X^2b
R31= E32/E31 R32= E33/E31 R33= E34/E35
R34= E36/E35 R35= E37/E39 R36= E38/E39
上述公式中， ^为校正吸收干扰前在 λΐ瞬时吸收， ^为校正吸收干扰前在 λ2瞬时吸 收， ^为校正吸收干扰前在 λ3瞬时吸收； la为校正吸收干扰后显色产物 Α在 λΐ瞬时净吸 收， ^2b为校正吸收干扰后显色产物 Β在 λ2瞬时净吸收， e为校正吸收干扰后显色产物 C 在 λ3瞬时净吸收； 。为反应体系未生成任何显色产物前在 λΐ吸收， 2Q为反应体系未生成 任何显色产物前在 λ2吸收， 。为反应体系未生成任何显色产物前在 λ3吸收； E31为显色 产物 Α与显色底物 A在 λΐ下差摩尔消光系数， Ε32为显色产物 Α与显色底物 A在 λ2下差 摩尔消光系数， Ε33为显色产物 Α与显色底物 A在 λ3下差摩尔消光系数； Ε34为显色产物 Β与显色底物 Β在 λΐ下差摩尔消光系数， Ε35为显色产物 Β与显色底物 Β在 λ2下差摩尔消 光系数， Ε36为显色产物 Β与显色底物 Β在 λ3下差摩尔消光系数； Ε37为显色产物 C与显 色底物 C在测量波长 λΐ下差摩尔消光系数， Ε38为显色产物 C与显色底物 C在测量波长 λ2下差摩尔消光系数， Ε39为显色产物 C与显色底物 C在测量波长 λ3下差摩尔消光系数； 上述公式中， 显色底物 Α转变成显色产物 Α计量关系为 1:1; 显色底物 B转变成显色产物 B的计量关系为 1:1; 显色底物 C转变成显色产物 C的计量关系为 1:1;
当仅某种酶显色产物对另一种酶有竞争抑制作用时， 确定显色产物 A及显色产物 B在 λΐ下和在 λ2下的无干扰吸收变化曲线 Ala和 A2b;设初始显色底物 A的浓度为 Cl，初始显 色底物 B的浓度为 C2; 设酶 B最大反应速度为 mB， 由 换算成初速度所用预设显色底 物浓度等于 C2的 93 %， 酶 B对显色底物 B的米氏常数为 ^; 如显色产物 A对酶 B为竞 争性抑制， 其竞争性抑制常数为 a， 酶 B动力学方程为：
C2 EM KmB x (l + Ala /Kia /Eu) + C
对上述方程两边对反应时间从 0〜t进行积分得到 d 2b = Vm£ x
KmB (\ + A,a I KJ E^) + C2 左边的积分项为反应时刻 t和反应时刻 0之间显色产物 B的净吸收变化；方程右边积分 项以 dt内显色产物 A浓度恒定进行数值积分， 即计算
KmB (\ + A, Kia I E^) + C2
以 dt为 Δ ， 计算每个记录点的 χ， 将所有 X求和再减去起点和终点 X得到 sumx， 用如 下公 无干扰初速度 。
Figure imgf000009_0001
上述计算公式中， 酶 A将显色底物 A转变成显色产物 A的计量关系为 1 : 1， 酶 B将显 色底物 B转变成显色产物 B的计量关系也为 1 : 1 ;
当酶 A的产物对酶 B抑制作用类型不同时， 所得酶 B的米氏动力学方程不同而所需数 值积分过程相同， 并仍用 93 %C2为初始底物浓度计算 s2;
进一步， 该方法用于同步测量三种酶活性时， 步骤 e中需显色产物 A吸收干扰校正系 数 R31和 R32; 测定时仅用酶 A的全部显色底物而不用其它待测酶的任何显色底物， 使得 反应体系无来自显色底物 B及显色产物 B、 显色底物 C及显色产物 C在三个测量波长 λΐ、 λ2和 λ3吸收， 同步测定反应通道中在三个测量波长下的吸收变化， 直到在 λ2及 λ3吸收变 化都大于 0.005或在 λΐ吸收变化大于 0.500; 以在 λΐ下吸收变化为横坐标， 在 λ2下和在 λ3 下吸收变化为纵座标进行回归分析， 所得在 λ2下吸收变化回归直线斜率为校正系数 R31， 在 λ3下吸收变化回归直线斜率为校正系数 R32;
该方法用于同步测量三种酶活性时， 步骤 e中需要显色产物 B吸收干扰校正系数 R33 和 R34; 测定时仅用酶 B的全部显色底物而不用其它待测酶的任何显色底物， 使得反应体 系无来自显色底物 A及显色产物 A、 显色底物 C及显色产物 C在三个测量波长 λ1、 λ2和 λ3吸收贡献， 同步测定反应通道中三个波长下的吸收变化， 直到在 λ2及 λ3吸收变化都大 于 0.005或在 λΐ吸收变化大于 0.500; 以在 λ2下吸收变化为横坐标， 在 λΐ下和在 λ3下吸收 变化为纵座标进行回归分析， 所得在 λΐ下吸收变化回归直线斜率为校正系数 R33， 在 λ3下 吸收变化回归直线斜率为校正系数 R34;
该方法用于同步测量三种酶活性时， 步骤 e中需要显色产物 C吸收干扰校正系数 R35 和 R36; 测定时仅用酶 C的全部显色底物而不用其它待测酶的任何显色底物， 使得反应体 系无来自显色底物 A及显色产物 A、 显色底物 B及显色产物 B在三个测量波长 λΐ、 λ2和 λ3吸收贡献， 同步测定反应通道中三个波长下的吸收变化， 直到在 λ2及 λΐ吸收变化都大 于 0.005或在 λ3吸收变化大于 0.500; 以在 λ3下吸收变化为横坐标， 在 λΐ下和在 λ2下吸收 变化为纵座标进行回归分析， 所得在 λΐ下吸收变化回归直线斜率为校正系数 R35， 在 λ2下 吸收变化回归直线斜率为校正系数 R36;
该方法用于同步测量三种酶活性时， 步骤 a中， 可用如下原则选择显色底物组合： 所用 显色底物 A、 显色底物 B及显色底物 C应使显色产物 A最大等吸收波长 Y1与显色产物 B 差吸收峰最大波长 X2相等或相距不超过 5nm、 显色产物 B最大等吸收波长 Y2和显色产物 A次大等吸收波长 Ys及显色产物 C差吸收峰最大波长 X3相等或任两者相距不超过 5nm; 该方法用于同步测量三种酶活性时， 步骤 c中， 选择显色产物 A差吸收峰最大波长 XI 为 λ1， 还可按如下原则选择另外两个测量波长 λ2和 λ3: 显色产物 Β与显色底物 Β差摩尔消 光系数在显色产物 Α最大等吸收波长 Y1处高于显色产物 B与显色底物 B在二者差吸收峰最 大波长 X2处差摩尔消光系数的 30%时， 选择 Y1为测量波长 λ2， 否则， 用最靠近显色产物 Α最大等吸收波长 Y1的显色产物 B差吸收峰波长，或靠近显色产物 B差吸收峰且其它显色 产物与显色显色底物差摩尔消光系数都小于显色产物 B与显色底物 B差摩尔消光系数 20% 的波长为 λ2测量显色产物 Β吸收； 显色产物 C与显色底物 C差摩尔消光系数在显色产物 A 次大等吸收波长 Ys处或显色产物 B与显色底物 B最大等吸收波长 Y2处应高于二者在其差 吸收峰最大波长 X3处差摩尔消光系数的 30%时， 则选择 Y2或 Ys为为 λ3测量显色产物 Β 吸收， 否则用最靠近显色产物 Β最大等吸收波长 Υ2或显色产物 Α次大等吸收波长 Ys或靠 近显色产物 C 差吸收峰最大波长且其它显色产物与显色底物差摩尔消光系数都小于显色产 物 C与显色底物 C差摩尔消光系数 20%的波长为 λ3测量显色产物 C吸收；
进一步， 该方法用于同步测量三种酶活性时， 与酶 Α对应的显色底物 Α包括 4-硝基 -1- 萘基磷酸酯且其水解的两个等吸收波长分别在 315到 335nm之间及 395到 415 nm之间、 4-硝基 -1-萘基硫酸酯且其水解的两个等吸收波长分别在 315到 335nm之间及 395到 415 nm 之间、 4-硝基 -1-萘基乙酸酯且其水解时两个等吸收波长分别在 310到 330nm之间及 375 至 lj 395 nm之间、 4-硝基 -1-萘基 -D-半乳糖苷且其水解时两个等吸收波长分别在 320 到 345nm 之间及 390到 410nm之间、 4-硝基 -1-(N-赖氨酰)萘胺且其水解最大等吸收波长分别在 385 到 410 nm之间， 这些显色底物 A被酶 A作用生成显色产物 A为 4-硝基 -1-萘酚或 4-硝基 -1- 萘胺或其衍生物；与此类显色底物 A对应显色底物 B主要是 4-硝基苯酚或 4-硝基苯胺衍生 物，在相应酶 B作用下生成显色产物 B为 4-硝基苯酚或 4-硝基苯胺或 4-硝基苯基硫醇； 与 此类显色底物 A和显色底物 B对应显色底物 C包括尼克酰胺腺嘌呤二核苷酸或尼克酰胺腺 嘌呤二核苷酸磷酸；
该方法用于同步测量三种酶活性时， 与显色底物 A对应酶 A包括但不限于芳香酯酶、 磷酸酶、 硫酸酯酶、 γ_谷氨酰基转移酶、 肽酶即天然氨基酸 α-羧基对应的酰胺酶和糖苷酶； 与显色底物 Β对应的酶 Β包括与已选酶 Α不同但属于酶 A的其它水解酶； 与显色底物 C对 应的酶包括但不限于乳酸脱氢酶 LDH、 苹果酸脱氢酶 MDH、 偶联 LDH的谷丙转氨酶和偶 联 MDH的谷草转氨酶。
本发明的有益效果： 本发明用多波长吸收单通道同步测量多种酶活性， 基于无相互作用 物质吸收线性加和性及酶显色底物和相应显色产物等吸收波长特征， 建立酶活性测定显色底 物设计和组合应用的新原理及解决多种物质吸收光谱重叠干扰的数据处理方法，及消除不同 酶的底物及产物之间相互干扰的数据处理方法， 实现基于多波长吸收同步测量单通道同步测 定多种酶活性； 可同步测定生物样品中多种酶活性、 酶标记免疫分析同步测定多种组分、 同 步筛选针对不同靶酶的抑制剂； 可用于临床生化分析和临床免疫检验、 卫生检验、 筛选酶抑 制剂等常规实践， 也可用于这类酶法分析技术适用的基础研究。 用本发明所述方法单通道同步测定多种酶活性还有特殊情况需考虑。经典初速度法仅分 析反应初始阶段显色产物吸收线性增加部分的平均速度为经典初速度表示酶活性。光度计可 测吸收范围有限。 用显色底物 A和显色产物 A等吸收波长 Y1为 λ2测定显色产物 Β吸收可 降低干扰， 但显色底物 Α吸收在 Y1处构成本底； 为使显色产物 B吸收测量范围足够宽， 来 自显色底物 A在 Y1处或 λ2的本底吸收就不能太高， 所用显色显色底物 Α浓度范围就有限。 在有限显色底物 A浓度下， 测定酶 A的经典初速度上限就较低。 为了在有限显色底物 A浓 度下拓宽测定酶 A初速度的上限，可用分析酶反应过程获得计算初速度和经典初速度法联用 的新策略测定酶活性； 这种新策略不是本发明所要求保护的内容， 而是授权中国发明专利 (ZL 200710093081.4)已保护的内容； 当需要应用这种新策略时， 属于 ZL 200710093081.4与 本发明所述平台技术的交叉。
本发明提出通过单通道多波长吸收同步测量而同步跟踪多种酶反应过程， 实现单通道多 种酶活性同步测定的新方法学， 其依赖于显色底物组合筛选、 测量波长组合选择、 快速变换 吸收测量波长同步跟踪多波长吸收变化、 消除吸收干扰的数据处理方法、 消除不同酶的底物 和产物间相互干扰的数据处理方法 (图 1)。
实现两波长吸收单通道同步测定两种酶活性的代表性过程如下：
(1) 筛选显色团 A和显色团 B用于合成两种待测酶的显色底物， 此两种显色团应同时 满足如下要求： （a) 显色产物 A差吸收峰最大波长 XI靠近红外端、 显色产物 B差吸收峰最 大波长 X2靠近紫外端， X2与 XI相距大于 30 nm且越远越好， （b) 显色底物 A生成显色产 物 A的最大等吸收波长 Y1与显色产物 B差吸收峰最大波长 X2相距小于 25 nm且相距越短 越好 (图 1和表 1); (c)理想的显色团 A和显色团 B应使 Y1与 X2相等或相距不超过 5nm;
(2) 合成对应的两种显色显色底物； 这些显色底物合成细节和合成工艺将作为其它发 明申请保护的内容而不是本发明所要求保护的内容；
(3) 在一个反应通道同步启动两种酶针对这两种显色显色底物的反应， 以 Y1为 λ2测 定显色产物 Β吸收， 以 XI处为 λΐ测定显色产物 Α吸收； 通过快速变换测量波长， 两波长 吸收同步跟踪单个反应体系中两个酶的反应过程；
(4) 建立消除多种物质吸收光谱重叠干扰的特殊数据处理方法， 获得每种待测酶显色 产物或底物的无干扰吸收变化曲线；
(5) 建立消除不同酶的底物及产物间相互干扰的数据处理方法， 分析两种酶反应的无 干扰吸收变化曲线， 数值积分拟合获得无干扰的酶最大反应速度；
上述第 (4) 条消除吸收重叠干扰的数据处理方法不依赖于测量波长组合，也不依赖于显 色底物的组合；但是，当上述第 (1)条的 (c)不满足时，酶活性测量的定量限将逊于单组分测定; 获得按上述第 (1)条的 (c)所述的理想显色底物 A和 B 组合并按第 (3)条所述选择测量波长组 合， 则此方法的性能与单组分测定相当。
在酶活性测定中常用显色产物主要是 4-硝基苯酚、 4-硝基苯胺及其衍生物和 NADH及 NADPH等 (图 2)。 测定酶活性常用天然显色底物 NADH和 NADPH的测量波长可选 320到 355 nm之间。 4-硝基苯酚和 4-硝基苯胺类显色底物在酶作用下的最大等吸收波长在 310到 350 nm之间而显色产物差吸收峰最大波长接近 400nm;在酶作用下， 4-硝基苯酚或 4-硝基苯 胺在 400 nm附近吸收超过 1.0时在其最大等吸收波长的吸收都无显著改变 (图 3)。 所以， 选 用 4-硝基苯酚、 4-硝基苯胺类衍生物为显色底物 A， 以 NADH或 NADPH为显色产物 B， 选 显色底物 A的最大等吸收波长 Y1为 λ2测定显色产物 Β的吸收，可基本不受显色显色底物 A 及显色产物 A浓度变化的干扰。 所以， 4-硝基苯酚和 4-硝基苯胺显色团对应显色底物可与 NADH或 NADPH组合， 单通道同步测量两种酶活性。
pH7.4时 4-硝基 -1-萘酚 450 nm附近摩尔消光系数约 SJxlO^mol/L^xcm- 4-硝基苯酚在 405 nm的摩尔消光系数约 l.lxl04(mol/L)_ ^cm- 免疫分析常用牛小肠粘膜碱性磷酸酶 (calf intestinal alkaline phosphatase, 简称 CIAP)为工具酶， 以 4-硝基 -1-萘基磷酸酯为显色底物， 其 米氏常数约 10 μηιοΙ/L; 用 4-硝基 -1-萘基磷酸酯测定 CIAP比活性可达到用 4-硝基苯基磷酸 酯为显色底物的 40%左右； 考虑显色产物消光系数差异， 两种磷酸酯测量该碱性磷酸酶的定 量限相当。 所以， 4-硝基 -1-萘基磷酸酯可用于测定牛小肠粘膜碱性磷酸酶 (图 4)， 与用 4-硝 基苯基 -β -半乳糖苷测定 β -半乳糖苷酶 (β-D-galacotosidase, 以下简称 β-Gal)组合或其它生成 4-硝基苯酚的酶组合，用于免疫分析单通道多波长吸收连续跟踪同步测量两组分含量。另外， 糖苷酶专一性高、 最适 ρΗ和缓冲液一致、 不易受相互产物和底物的干扰； 用 4-硝基 -1-萘基 -β -D-半乳糖苷和 4-硝基苯基 -α -D-葡萄糖苷糖苷为底物， β -半乳糖苷酶和 α -葡萄糖苷酶组 合可用于单通道两组分 ELISA; 但其最优测量波长组合为 400 nm和 460或 450 nm， 用普通 滤光片酶标仪仅提供 405 nm光源， 不利于发挥其组合优势。
本发明所建立的校正吸收重叠干扰的数据处理方法及多种酶的底物和产物相互干扰时 校正干扰作用的数据处理方法， 对显色底物和显色产物稳定的酶反应体系通用， 而本发明应 用关键是筛选显色底物组合以使得单通道同步测定两种酶活性的灵敏度、定量限和线性范围 与单独测定相当。本发明的代表性显色底物组合包括： 4-硝基苯酚和 4-硝基 -1-萘酚的亲水衍 生物为水解酶显色底物组合、 4-硝基苯胺类显色底物与 NADH或 NADPH组合。
附图说明
下面结合附图和实施例对本发明作进一步描述。
图 1单通道多波长吸收同步测量两种酶活性的原理示意图
图 2 测定酶活性常用显色团及常用显色显色底物
图 3 常见酶作用于 4-硝基苯酚和 4-硝基苯胺类显色底物的光谱特征
3-a. pH 7.4, 0.050 mol/L Tris-HCl, 0.50mmol/L 4-硝基苯基 -β-D-半乳糖苷 +大肠杆菌
β-D-半乳糖苷酶 （Sigma G4155)反应过程中的吸收光谱
3-b. pH 7.4， 0.050 mol/L Tris-HCl, 0.50mmol/L γ-谷氨酰基 -(4-硝基)苯胺 +兔肾脏匀浆
中 γ-谷氨酰基转移酶反应过程中的吸收光谱
3-c. pH 7.4， 0.050 mol/L Tris-HCl, 0.50 mmol/L 4-硝基苯基磷酸酯 + 牛小肠粘膜碱性磷
酸酶 (Sigma P0114) 反应过程中的吸收光谱
3-d. pH 8.5， 0.050 mol/L Tris-HCl, 0.50 mmol/L 4-硝基苯基磷酸酯 + 牛小肠粘膜碱性磷
酸酶 (Sigma P0114) 反应过程中的吸收光谱
3-e. pH 7.4, 0.050 mol/L Tris-HCl, 0.50 mmol/L硫代乙酰胆碱 + Ellman试剂 5，5 ' -二硫
代-双 (2-硝基苯甲酸）（DTNB) + 乙酰胆碱酯酶 （Sigma C2888) 反应过程中的吸收 光谱
-f. H 7.4, 0.050 mol/L Tris-HCl, 0.50 mmol/L硫代乙酰胆碱 + 5,5 ' -二硫代-双 (2-硝基
苯乙酸）（DTNA) +乙酰胆碱酯酶 （Sigma C2888) 反应过程中的吸收光谱
-g. pH 7.4, 0.050 mol/L Tris-HCl, 0.50 mmol/L硫代乙酰胆碱 + Ellman试剂 5，5 ' -二硫
代-双 (2-硝基苯甲酸）（DTNB) + 乙酰胆碱酯酶 （Sigma C2888) 反应过程中的吸收 光谱
- h. pH 7.4, 0.050 mol/L Tris-HCl, 0.50 mmol/L硫代乙酰胆碱 + 5,5 ' -二硫代-双 (2-硝基
苯乙酸）（DTNA) +乙酰胆碱酯酶 （Sigma C2888) 反应过程中的吸收光谱
图 4常见酶作用于 4-硝基 -1-萘酚类显色底物的光谱特征
- a. pH 7.4, 0.050 mol/L Tris-HCl, 0.25 mmol/L (4-硝基 -1-萘酚) -β-D-半乳糖苷 +大肠杆
菌 β-D-半乳糖苷酶 (Sigma G4155) 反应过程中的吸收光谱
-b. pH 7.4, 0.050 mol/L Tris-HCl, 0.50 mmol/L 4-硝基 -1-萘基磷酸酯 + 牛小肠粘膜碱
性磷酸酶 (Sigma P0114) 反应过程中的吸收光谱
-c. pH 7.4, 0.050 mol/L Tris-HCl, 0.50 mmol/L 4-硝基 -1-萘基硫酸酯 + 蜗牛芳香硫酯
酶 (Sigma S9626) 反应过程中的吸收光谱
图 5. 两组分同步酶联免疫吸附分析的操作过程示意图
图 6. 同步测量兔组织匀浆中的 γ-谷氨酰基转移酶 GGT和乳酸脱氢酶 LDH
-a. 单用 GGT显色底物测定 4-硝基苯胺对 344 nm吸收干扰校正系数 R31
回归分析得 zW344 = 0.0289x/W405+0.0167， i?2 > 0.902, 故 R31=0.0289。
-b. 单用 LDH显色底物测定 NADH对 405 nm吸收干扰校正系数 R33
回归分析得
Figure imgf000013_0001
= 0.0088x/L4344-0.0002， i?2 > 0.993 , 故 R31=0.0088。
此干扰太小， 故可不校正； 分析实验数据校正与否结果无统计差异。
-c. 同步测量 GGT和 LDH中 GGT校正效果比较
A: 校正前 （zW/min) VI对酶量响应不成线性
B: 校正后 （zW/min) si = 2.6565x -0.0061 , i?2>0.996
C: 单独测定 (zW/min) si = 2.6971 x -0.0059, i?2>0.997
- d. 同步测量 GGT和 LDH中 LDH校正效果比较
A: 校正前 （zW/min) V2对酶量响应不成线性
B: 校正后 （zW/min) s2= 1.1028x x + 0.0025， R2 >0.996
C: 单独测定 (zW/min) s2= 1.1715x x +0.0016， i?2>0.994
图 7.用 4-硝基 -1-萘基磷酸酯和 4-硝基苯基 -β-D-半乳糖苷同步测量碱性磷酸酶和 β-D-半乳糖 苷酶
- a. 单用碱性磷酸酶显色底物测定 4-硝基 -1-萘酚对测定 405 nm吸收干扰校正系数 R31 回归分析得^ 4405 = -0.015x/L4450+0.0107， i?2 > 0.88， 故 R31= -0.015。
-b. 单用 β-D-半乳糖苷酶的显色底物测定 4-硝基苯酚对测定 450 nm 吸收的干扰校正系数 R33
回归分析得^ 445。 = 0.193 l x/W4Q5-0.0817， ?2 > 0.999， 故 R33= 0.1931。 7-c. 同步测量碱性磷酸酶和 β-D-半乳糖苷酶中碱性磷酸酶校正效果比较
A: 校正前 （zW/min) VI = 0.7425x -0.0003, R2 >0.99
B: 校正后 （zW/min) si =0.6333x -0.0008, i?2>0.995
C: 单独测定 (zW/min) si =0.6200x +0.0009, i?2>0.998
7- d. 同步测量碱性磷酸酶和 β-D-半乳糖苷酶中 β-D-半乳糖苷酶校正效果比较
A: 校正前 （zW/min) V2 = 0.6267x +0.0017, i?2>0.993
B: 校正后 （zW/min) s2 = 0.6067x +0.0023, i?2>0.993
C: 单独测定 (zW/min) s2 = 0.6133x +0.0021, i?2>0.998
图 8. 用青霉素标记 β-D-半乳糖苷酶和瘦肉精标记的 α-D-葡萄糖苷酶
8- a. 单用 4-硝基 -1-萘基 -β-D-半乳糖苷单独测定 β-D-半乳糖苷酶反应响应曲线时 4-硝基 -1- 萘酚对同步测定 405 nm吸收干扰校正系数 R31
回归分析得 zW405 = 0.0665x/W450+0.0015， i?2> 0.998， 故 R31=0.0665。
8-b. 用 4-硝基苯基 -α-D-葡萄糖苷单独测定 α-D-葡萄糖苷酶反应时 4-硝基苯酚对同步测定 450 nm吸收干扰校正系数 R33
回归分析得^445 = 0.1957 5-0.0874， R2> 0.992, 故 R33=0.1957
8-c. 同步测量 β-D-半乳糖苷酶和 α-D-葡萄糖苷酶中 β-D-半乳糖苷酶校正效果
A: 校正前 （zW/min) VI =2.2871x -0.0073, R2 >0.997
B: 校正后 （zW/min) si = 1.9216x x -0.0034， i?2>0.996
C: 单独测定 (zW/min) si =2.0291x -0.0027, i?2>0.999
8-d. 同步测量 β-D-半乳糖苷酶和 α-D-葡萄糖苷酶中 α-D-葡萄糖苷酶校正效果
A: 校正前 （zW/min) VI = 1.9065x X -0.0123， R2 >0.989
B: 校正后 （zW/min) si = 1.7543x X -0.0124， i?2>0.989
C: 单独测定 (zW/min) si = 1.7945x x -0.0118， i?2>0.992
8-e. 同步测量时 β-D-半乳糖苷酶标记物结合率校正前后结果比较
A: 校正前 结合率(％) = -6.4865 1^( +87.332， i?2>0.987
B: 校正后 结合率(％) = -6.2685 1^( +86.886， i?2>0.990
C: 单独测 β-D-半乳糖苷酶标记物 （未加入 a-D-葡萄糖苷酶显色底物） 时的曲线 为： 结合率(％) = -6.8541 xLn(x )+87.689， i?2>0.977
8-f. 同步测量时 a-D-葡萄糖苷酶标记物结合率校正前后结果比较
A: 校正前 结合率(％) = -11.3572 01 +92.27， i?2>0.991
B: 校正后 结合率(％) = -12.054 1^( +93.755， i?2>0.955
C: 单独测 a-D-葡萄糖苷酶标记物 （未加入 β-D-半乳糖苷酶显色底物） 时的曲线 为： 结合率 (％) = -13.145xLn(x )+104.47， i?2>0.990
具体实施方式
本发明的用多波长吸收单通道同步测量多种酶活性的方法， 包括下列步骤：
a. 确定用多波长吸收单通道同步测定多种酶活性所需显色底物组合：
al根据待测酶的专一性筛选满足下列条件的显色底物组合： 所选显色团能分别用于制备 待测酶的显色底物； 所得这些显色底物组合中， 将显色底物在对应待测酶作用下所得显色产 物相对该显色底物的差吸收峰最大波长从大到小排列，相邻的显色产物差吸收峰最大波长之 间相距大于 30 nm且尽量远； 在按显色产物差吸收峰最大波长从大到小的排列中， 除了显色 产物差吸收峰最靠近红外端的显色底物外，每个显色底物在对应酶作用下所得显色产物差吸 收峰最大波长， 都与此排列中某个显色底物的最大等吸收波长相差小于 25 nm且尽量短； a2 用步骤 al所选显色团分别合成待测酶的对应显色底物并组合使用；
b. 在一个反应混合物或酶反应体系即单反应通道中， 同步启动多个待测酶针对组合使用 的显色底物混合物对应显色底物的专一催化反应；
c 选择测量波长组合： 以显色产物差吸收峰最靠近红外端的显色底物为显色底物 A， 在 显色底物 A的显色产物差吸收峰最大波长下测定该显色产物吸收； 在显色底物 A的等吸收 波长处测定其余待测酶显色产物或显色底物的吸收；
d. 在所选多个测量波长组合下， 通过快速变换测量波长， 多波长吸收同步测量实现同步 跟踪单通道中多个待测酶的反应过程；
e. 基于无相互作用物质吸收的线性加和性建立消除反应通道中显色物质吸收重叠干扰的 数据处理方法， 获得消除吸收光谱重叠干扰后每个待测酶的显色产物或显色底物无干扰吸收 变化曲线； 如某个待测酶不受反应体系其它酶的产物及底物的干扰且其底物浓度在对应酶米 氏常数的 3倍以上，用经典初速度法分析其显色产物或显色底物无干扰吸收变化曲线确定初 速度； 如某个待测酶不受反应体系其它酶的产物及底物的干扰但所用显色底物浓度在该酶米 氏常数的 5 %以上而低于米氏常数的 3倍， 联用经典初速度法和反应过程分析法分析其显色 产物或显色底物无干扰吸收变化曲线确定初速度；当某个待测酶受反应体系中其自身和 /或其 它酶的底物和 /或产物的干扰时，将描述反应体系动力学的微分速度方程组数值积分后分析活 性受到干扰待测酶反应的显色产物或显色底物无干扰吸收变化曲线，确定其最大反应速度表 示其活性，或据其微分速度方程和所得最大反应速度换算成底物浓度为起始底物浓度 93 %时 初速度表示酶活性。
本实施例中， 应用该方法时， 显色产物相对显色底物差吸收峰最大波长在 300 nm以上 且其显色底物吸收峰最大波长小于相应显色产物吸收峰最大波长， 否则用逆反应测定该酶的 活性； 对待测酶显色底物组合中显色产物差吸收峰最大波长相邻的任两个显色底物， 显色产 物差吸收峰最大波长更靠近红外端者的最大等吸收波长与显色产物差吸收峰最大波长更靠 近紫外端者的差吸收峰最大波长相等或相距不超过 5nm为优化组合;所用显色底物组合中每 个显色底物都仅被样品中的一种待测酶作用生成显色产物， 而不被样品中其它任何酶作用或 作用效果不明显； 此处所述不被其它任何酶作用或作用效果不明显， 指当样品中酶 I所对应 显色底物的浓度与其米氏常数相差小于 50%时，样品中其它任何酶作用于该显色底物的比活 性都低于酶 I作用于该显色底物比活性的 1%; 权利要求 1的步骤 b中， 选择适于同步测定 活性的待测酶发挥作用的缓冲介质和反应条件， 具体包括： 缓冲介质 pH位于 pH 5.0到 9.0 之间且接近比活性最低待测样品的最适 pH， 所用样品反应温度在摄氏 20到 40度之间。
本实施例中， 该方法应用时所述显色底物包括天然显色底物和非天然显色底物； 天然显 色底物包括尼克酰胺腺嘌呤二核苷酸或尼克酰胺腺嘌呤二核苷酸磷酸； 非天然显色底物由 显色团和被酶识别基团组成， 显色团包括但不限于 4-硝基苯酚、 4-硝基苯硫酚、 4-硝基苯 胺、 4-硝基 -1-萘酚、 4-硝基 -1-萘硫酚、 4-硝基 -1-萘胺、 2-萘酚、 4-氯苯酚、 玫红酸或有取代 基的这些芳香酚 /胺衍生物。
本实施例中， 该方法应用于同步测量两种酶活性， 步骤 a中， 所述样品中含两种待测酶 分别命名为酶 A和酶 B，所需两种显色底物分别命名为显色底物 A和显色底物 B，在两种待 测酶作用下生成两种显色产物分别命名为显色产物 A和显色产物 B，这些显色底物及显色产 物的组合需满足下列条件： （1 )显色产物 A差吸收峰最大波长 XI靠近红外端， 显色产物 B 差吸收峰最大波长 X2靠近紫外端， 显色产物 B差吸收峰最大波长 X2与显色产物 A差吸收 峰最大波长 XI相距大于 30 nm且尽量远； （2 )显色产物 A的最大等吸收波长为 Y1且与显 色产物 B差吸收峰最大波长 X2相距小于 25 nm且且尽量短。
该方法应用于同步测量两种酶活性的步骤 b和 c及 d中， 在一个反应通道同步启动样品 中两种酶针对显色团 A和显色团 B合成的对应显色底物 A和显色底物 B的反应， 并得到显 色产物 A和显色产物 B; 以显色产物 A的最大等吸收波长 Y1为 λ2处测定显色产物 Β吸收， 以显色产物 Α的差吸收峰最大波长 XI为 λΐ处测定显色产物 A吸收;通过快速变换测量波长， 两波长吸收同步测量实现同步跟踪单个反应体系中两种待测酶的反应过程。
该方法应用于同步测量两种酶活性的步骤 e中， 基于吸收线性加和性建立数据解析消 除多种物质吸收重叠干扰方法； 以显色产物 A差吸收峰最大波长前后不超过 25 nm的波长 为 λΐ且在此波长下吸收为 Al， 以显色产物 Α和显色底物 A最大等吸收波长附近不超过 25 nm的波长为 λ2且此波长下吸收为 Α2， 基于吸收线性加和性消除物质吸收重叠干扰需解如 下二元一次方程组：
/4ι = /4ιο+Ε31 Χ Ρι + Ε34 Χ Ρ2
Α2 = Α20+Ε32 Χ Ρι + E35 X P2
进一步可转变成如下二元一次方程组
^l = ^10+^la + R33 X^2b ^2 = ^2o+^2b + R31 X^ia
R31= E32/E31 R33= E34/E35
Pi为显色产物 A瞬时浓度； P2为显色产物 B瞬时浓度； E31为显色产物 A与显色底物 A在 λΐ下差摩尔消光系数； Ε32为显色产物 Α与显色底物 A在 λ2下差摩尔消光系数 Ε32; Ε34为显色产物 Β与显色底物 Β在 λΐ下差摩尔消光系数； Ε35为显色产物 Β与显色底物 Β 在 λ2下差摩尔消光系数； ^为校正吸收干扰前在 λΐ瞬时总吸收， Α为校正吸收干扰前在 λ2 瞬时总吸收； a为校正吸收干扰后显色产物 Α在 λΐ瞬时净吸收， 其等于 1>1和£31乘积， ^2b为校正吸收干扰后显色产物 Β在 λ2瞬时净吸收， 其等于 Ρ2和 Ε35乘积； 1Q为反应体 系未生成任何显色产物前在 λΐ的吸收， 为来自反应体系的本底， 相当于含相同浓度全部显 色底物反应体系在 λΐ吸收与含相同浓度样品反应体系在 λΐ吸收的加和； 2Q为反应体系未 生成任何显色产物前在 λ2的吸收， 为来自反应体系的本底， 相当于含浓度相同全部显色底 物的反应体系在 λ2吸收与含相同浓度样品反应体系在 λ2吸收的加和；
当仅一种酶显色产物对另一种酶有竞争抑制作用时， 确定显色产物 Α及显色产物 B在 λΐ下和在 λ2下的无干扰吸收变化曲线 Ala和 A2b ;设初始显色底物 A的浓度为 Cl，初始显 色底物 B的浓度为 C2; 设酶 B最大反应速度为 mB， 由 换算成初速度所用预设显色底 物浓度等于 C2的 93 %， 酶 B对显色底物 B的米氏常数为 ^ ; 如显色产物 A对酶 B为竞 争性抑制， 其竞争性抑制常数为 a， 酶 B动力学方程为：
dA ib VmB x dt
2 x EM KmB (\ + A ,a I KJ E^ ) + C2 对上述方程两边对反应时间从 0〜t进行积分得到
Figure imgf000017_0001
左边的积分项为反应时刻 t和反应时刻 0之间显色产物 B的净吸收变化；方程右边积分 项以 dt内显色产物 A浓度恒定进行数值积分， 即计算
1 以 dt为 Δ ， 计算每个记录点的 χ， 将所有 X求和再减去起点和终点 X得到 sumx， 用如 下公式计 并据其微分动力学方程得无干扰初速度 。
VmB x E34 x sumx x At) = 0.93 x C2 x VmB /(KmB + 0.93 x 2)
Figure imgf000017_0002
上述计算公式中， 酶 A将显色底物 A转变成显色产物 A的计量关系为 1 : 1， 酶 B将显 色底物 B转变成显色产物 B的计量关系也为 1 : 1 ;
当酶 A的产物对酶 B抑制作用类型不同时， 所得酶 B的米氏动力学方程不同而所需数 值积分过程相同， 并仍用 93 % C2为初始底物浓度计算 s2;
本实施例中， 该方法用于同步测量两种酶活性有如下特征：
步骤 a中， 如下选择显色团组合： 显色团 A和显色团 B使显色产物 A最大等吸收波长 Y1与显色产物 B差吸收峰最大波长 X2相等或相距不超过 5 nm;
步骤 c中， 选择产物 A差吸收峰最大波长 XI为 λΐ后， 如下选择测量波长 λ2: 显色产物 Β与显色底物 Β差摩尔消光系数在显色产物 Α最大等吸收波长 Y1处高于在显色产物 B差吸 收峰最大波长 X2处的 30%， 则选择显色产物 A最大等吸收波长 Y1为 λ2， 否则， 用最靠近 显色产物 Α最大等吸收波长 Y1的显色产物 B差吸收峰波长为 λ2， 或最靠近显色产物 Β差 吸收峰且其它待测显色显色产物与显色显色底物差摩尔消光系数都小于显色产物 Β 与显色 底物 Β差摩尔消光系数 20%的波长为 λ2测量显色产物 Β吸收；
步骤 e中， 需测定 R31为显色产物 A吸收干扰的校正系数； 测定 R31时， 仅用酶 A所 需的全部显色底物而不加其它待测酶的任何显色底物， 使反应体系无来自显色底物 B及显 色产物 B在两个测量波长 λΐ和 λ2的吸收， 此时同步测定反应通道中在两个测量波长下的 吸收变化， 直到在 λ2吸收变化大于 0.005或在 λΐ吸收变化大于 0.500; 以在 λΐ下吸收为横 坐标， 在 λ2下吸收为纵座标进行回归分析， 所得回归直线斜率为校正系数 R31 ;
步骤 e中， 需测定 R33为显色产物 B吸收干扰的校正系数； 测定 R33时， 仅用酶 B 所需的全部显色底物而不加其它待测酶的任何显色底物， 使反应体系无来自显色底物 A及 显色产物 A在两个测量波长 λΐ和 λ2的吸收， 此时同步测定反应通道中在两个测量波长下 的吸收变化， 直到在 λ2吸收变化大于 0.500或在 λΐ吸收变化大于 0.005 ; 以在 λ2下吸收为 横坐标， 在 λΐ下吸收为纵座标进行回归分析， 所得回归直线斜率为校正系数 R33 ;
本实施例中， 该方法用于同步测量两种酶活性时， 酶 Α对应显色底物包括水解反应最 大等吸收波长在 310到 330 nm之间的 4-硝基苯基乙酸酯， 水解反应最大等吸收波长在 330 到 350nm之间的 4-硝基苯基磷酸酯、 4-硝基苯基硫酸酯、 4-硝基苯基 -β-D-半乳糖苷和 γ-谷 氨酰基 -4-硝基苯胺， 还原反应最大等吸收波长在 350到 370nm之间 5-巯基 -2-硝基苯甲酸 及 5-巯基 -2-硝基苯乙酸， 其显色底物为二硫化物被酶作用所释放的硫醇还原； 与此类酶 A 的显色底物 A对应的显色底物 B主要是天然显色底物，包括尼克酰胺腺嘌呤二核苷酸或尼 克酰胺腺嘌呤二核苷酸磷酸；上述显色底物 A对应的酶 A包括但不限于芳香酯酶、磷酸酶、 硫酸酯酶、 γ_谷氨酰基转移酶、 肽酶即天然氨基酸 α-羧基对应的酰胺酶和糖苷酶； 与此类 酶 Α对应的酶 B包括但不限于乳酸脱氢酶 LDH、 苹果酸脱氢酶 MDH、 偶联 LDH的谷丙 转氨酶和偶联 MDH的谷草转氨酶。
本实施例中， 该方法用于同步测量三种酶活性， 三种待测酶分别命名为酶 A、 酶 B和酶 C， 所需显色底物三种分别命名为显色底物 A、 显色底物 B和显色底物 C， 在对应酶作用下 生成显色产物分别命名为显色产物 A、显色产物 B和显色产物 C; 这些显色底物组合需满足 下列条件： （1 ) 显色产物 A差吸收峰最大波长为 XI且最靠近红外端， 显色产物 C差吸收 峰最大波长为 X3且最靠近紫外端，显色产物 B差吸收峰最大波长为 X2且位于 XI和 X3之 间， X2与 XI及 X3相距都大于 30 nm且尽量远； （2 )显色产物 A的最大等吸收波长为 Y1 且次大等吸收波长为 Ys;显色底物 B生成显色产物 B的最大等吸收波长为 Y2且与 Y1相距 尽量远而和 Ys相距尽量短； （3 ) Y1与 X2相距小于 25 nm且相距尽量短； Y2和 Ys及 X3 相距都小于 25 nm且相距尽量短；
该方法用于同步测量三种酶活性时， 步骤 b中， 在一个反应通道同步启动三种酶针对显 色底物 A、 显色底物 B和显色底物 C的反应， 并得到对应的显色产物 A、 显色产物 B和显 色产物 C;
该方法用于同步测量三种酶活性时， 步骤 c中， 选显色产物 A最大等吸收波长 Y1或相 距 25nm以内的波长为 λ2测显色产物 Β吸收， 选显色产物 Α差吸收峰最大波长 XI或相距 25nm以内波长为为 λΐ测显色产物 Α吸收， 选显色产物 B最大等吸收波长 Y2或相距 25nm 以内的波长为 λ3测显色产物 C吸收；
该方法用于同步测量三种酶活性时， 步骤 d中， 通过快速变换测量波长， 三波长吸收同 步测量实现同步跟踪单个反应体系中两种样品的反应过程；
该方法用于同步测量三种酶活性时， 步骤 e 中， 基于吸收的线性加和性建立数据解析 消除多种物质吸收重叠干扰需解如下三元一次方程组：
Ax = Aw+Ala + R33 A2h + R35 XA3c
A2 = A20十 2b + R31 X^ ia + R36 X^3c
A3 = A30 +A3c + R32 X^ ia + R34 X^2b R31= E32/E31
R32= E33/E31
R33= E34/E35
R34= E36/E35
R35= E37/E39
R36= E38/E39
上述公式中， ^为校正吸收干扰前在 λΐ瞬时吸收， ^为校正吸收干扰前在 λ2瞬时吸 收， ^为校正吸收干扰前在 λ3瞬时吸收； a为校正吸收干扰后显色产物 Α在 λΐ瞬时净吸 收， ^2b为校正吸收干扰后显色产物 Β在 λ2瞬时净吸收， 为校正吸收干扰后显色产物 C 在 λ3瞬时净吸收； 。为反应体系未生成任何显色产物前在 λΐ吸收， 2Q为反应体系未生成 任何显色产物前在 λ2吸收， 。为反应体系未生成任何显色产物前在 λ3吸收； E31为显色 产物 Α与显色底物 A在 λΐ下差摩尔消光系数， Ε32为显色产物 Α与显色底物 A在 λ2下差 摩尔消光系数， Ε33为显色产物 Α与显色底物 A在 λ3下差摩尔消光系数； Ε34为显色产物 Β与显色底物 Β在 λΐ下差摩尔消光系数， Ε35为显色产物 Β与显色底物 Β在 λ2下差摩尔消 光系数， Ε36为显色产物 Β与显色底物 Β在 λ3下差摩尔消光系数； Ε37为显色产物 C与显 色底物 C在测量波长 λΐ下差摩尔消光系数， Ε38为显色产物 C与显色底物 C在测量波长 λ2下差摩尔消光系数， Ε39为显色产物 C与显色底物 C在测量波长 λ3下差摩尔消光系数； 上述公式中， 显色底物 Α转变成显色产物 Α计量关系为 1 :1 ; 显色底物 B转变成显色产物 B的计量关系为 1 :1 ; 显色底物 C转变成显色产物 C的计量关系为 1 :1。
本实施例中， 该方法用于同步测量三种酶活性时， 步骤 e中需显色产物 A吸收干扰校 正系数 R31和 R32; 测定时仅用酶 A的全部显色底物而不用其它待测酶的任何显色底物， 使得反应体系无来自显色底物 B及显色产物 B、显色底物 C及显色产物 C在三个测量波长 入1、 人2和人3吸收， 同步测定反应通道中在三个测量波长下的吸收变化， 直到在 λ2及 λ3吸 收变化都大于 0.005或在 λΐ吸收变化大于 0.500; 以在 λΐ下吸收变化为横坐标， 在 λ2下和 在 λ3下吸收变化为纵座标进行回归分析， 所得在 λ2 下吸收变化回归直线斜率为校正系数 R31 , 在 λ3下吸收变化回归直线斜率为校正系数 R32;
该方法用于同步测量三种酶活性时， 步骤 e中需要显色产物 B吸收干扰校正系数 R33 和 R34; 测定时仅用酶 B的全部显色底物而不用其它待测酶的任何显色底物， 使得反应体 系无来自显色底物 A及显色产物 A、 显色底物 C及显色产物 C在三个测量波长 λ1、 λ2和 λ3吸收贡献， 同步测定反应通道中三个波长下的吸收变化， 直到在 λ2及 λ3吸收变化都大 于 0.005或在 λΐ吸收变化大于 0.500; 以在 λ2下吸收变化为横坐标， 在 λΐ下和在 λ3下吸收 变化为纵座标进行回归分析， 所得在 λΐ下吸收变化回归直线斜率为校正系数 R33， 在 λ3下 吸收变化回归直线斜率为校正系数 R34;
该方法用于同步测量三种酶活性时， 步骤 e中需要显色产物 C吸收干扰校正系数 R35 和 R36; 测定时仅用酶 C的全部显色底物而不用其它待测酶的任何显色底物， 使得反应体 系无来自显色底物 A及显色产物 A、 显色底物 B及显色产物 B在三个测量波长 λΐ、 λ2和 λ3吸收贡献， 同步测定反应通道中三个波长下的吸收变化， 直到在 λ2及 λΐ吸收变化都大 于 0.005或在 λ3吸收变化大于 0.500; 以在 λ3下吸收变化为横坐标， 在 λΐ下和在 λ2下吸收 变化为纵座标进行回归分析， 所得在 λΐ下吸收变化回归直线斜率为校正系数 R35， 在 λ2下 吸收变化回归直线斜率为校正系数 R36;
该方法用于同步测量三种酶活性时， 步骤 a中， 可如下选择显色底物组合： 所用显色底 物 A、 显色底物 B及显色底物 C应使显色产物 A的最大等吸收波长 Y1与显色产物 B差吸 收峰最大波长 X2相等或相距不超过 5nm、 显色产物 B最大等吸收波长 Y2和显色产物 A次 大等吸收波长 Ys及显色产物 C差吸收峰最大波长 X3之间相等或任两者相距都不超过 5nm; 该方法用于同步测量三种酶活性时， 步骤 c中， 选择显色产物 A差吸收峰最大波长 XI 为 λ1， 还可按如下原则选择另外两个测量波长 λ2和 λ3: 显色产物 Β与显色底物 Β差摩尔消 光系数在显色产物 Α最大等吸收波长 Y1处高于显色产物 B与显色底物 B在二者差吸收峰最 大波长 X2处差摩尔消光系数的 30%时， 选择 Y1为测量波长 λ2， 否则， 用最靠近显色产物 Α最大等吸收波长 Y1的显色产物 B差吸收峰波长，或靠近显色产物 B差吸收峰且其它显色 产物与显色显色底物差摩尔消光系数都小于显色产物 B与显色底物 B差摩尔消光系数 20% 的波长为 λ2测量显色产物 Β吸收； 显色产物 C与显色底物 C差摩尔消光系数在显色产物 A 次大等吸收波长 Ys处或显色产物 B与显色底物 B最大等吸收波长 Y2处应高于二者在其差 吸收峰最大波长 X3处差摩尔消光系数的 30%时， 则选择 Y2或 Ys为为 λ3测量显色产物 Β 吸收， 否则用最靠近显色产物 Β最大等吸收波长 Υ2或显色产物 Α次大等吸收波长 Ys或靠 近显色产物 C 差吸收峰最大波长且其它显色产物与显色底物差摩尔消光系数都小于显色产 物 C与显色底物 C差摩尔消光系数 20%的波长为 λ3测量显色产物 C吸收；
本实施例中， 该方法用于同步测量三种酶活性时， 与酶 Α对应的显色底物 A包括 4-硝 基 -1-萘基磷酸酯且其水解的两个等吸收波长分别在 315到 335nm之间及 395到 415 nm之 间、 4-硝基 -1-萘基硫酸酯且其水解的两个等吸收波长分别在 315到 335nm之间及 395到 415 nm之间、 4-硝基 -1-萘基乙酸酯且其水解时两个等吸收波长分别在 310到 330nm之间及 375 到 395 nm 之间、 4-硝基 -1-萘基 -D-半乳糖苷且其水解时两个等吸收波长分别在 320 至 lj 345nm之间及 390到 410nm之间、 4-硝基 -1-(N-赖氨酰)萘胺且其水解最大等吸收波长分别 在 385 到 410 nm之间， 这些显色底物 A被酶 A作用生成显色产物 A为 4-硝基 -1-萘酚或 4-硝基 -1-萘胺或其衍生物；与此类显色底物 A对应显色底物 B主要是 4-硝基苯酚或 4-硝基 苯胺衍生物，在相应酶 B作用下生成显色产物 B为 4-硝基苯酚或 4-硝基苯胺或 4-硝基苯基 硫醇；与此类显色底物 A和显色底物 B对应显色底物 C包括尼克酰胺腺嘌呤二核苷酸或尼 克酰胺腺嘌呤二核苷酸磷酸；
该方法用于同步测量三种酶活性时， 与显色底物 A对应酶 A包括但不限于芳香酯酶、 磷酸酶、 硫酸酯酶、 γ_谷氨酰基转移酶、 肽酶即天然氨基酸 α-羧基对应的酰胺酶和糖苷酶； 与显色底物 Β对应的酶 Β包括与已选酶 Α不同但属于酶 A的其它水解酶； 与显色底物 C对 应的酶包括但不限于乳酸脱氢酶 LDH、 苹果酸脱氢酶 MDH、 偶联 LDH的谷丙转氨酶和偶 联 MDH的谷草转氨酶。
以下为本发明具体实验数据及实施例
应用实施例所需新型显色底物用常规有机合成方法制备、 过硅胶柱纯化， 用高分辨质谱 和丽 R确定结构； 这些新型显色底物的合成方法和工艺不在本发明专利的保护范围之内， 将 作为另行申请的发明专利的保护内容。 适用于本发明常见显色团见图 2; 常见酶反应的等吸 收波长见图 3和 4; 两种酶同步测量 ELISA操作流程见图 5。
实施例中， 测定反应过程的吸收光谱变化都用波长未校准的 ShimadZu UV2550光度计。 测定酶活性时在 MapAda UV1600 PC上用对应控制软件进行，或在 Biotek ELX800酶标仪上 用 Gene 5.0软件控制仪器测定。 如未说明， 以下所有测定温度为室温即 25度。
实施例一： 单通道两波长吸收同步测定兔肾脏匀浆中 LDH和 GGT
本应用实例中测定乳酸脱氢酶 （简称 LDH， 上海生工， 编号 NB0642)显色底物为丙酮酸 和还原型尼克酰胺腺嘌呤二核苷酸即 NADH,均为国产分析纯；测定 γ-谷氨酰基转移酶 (简称 GGT)显色底物为 γ-谷氨酰基 -(4-硝基)苯胺 (Sigma, 编号 49525)， 双甘肽为国产分析纯； 所用 缓冲液为 0.10 mol/L且 pH 为 7.0磷酸钠； 将 GGT显色底物溶解于缓冲液为显色底物溶液 A、 LDH显色底物固体称量溶解于缓冲液为显色底物溶液 B; 含有 LDH和 GGT显色底物的混合物 为显色底物溶液 C ; GGT的显色底物 γ-谷氨酰基 -(4-硝基)苯胺为显色底物 A， 对应显色产物 A 为 4-硝基苯胺； NADH为 LDH显色底物但当作显色产物 B; 所用溶液中只含有 GGT显色底物 而不含有 NADH时将系统光吸收本底调零； GGT来自兔子肾脏， 用 0.1%表面活性剂 NP40和 pH 7.0的 0.10 mol/L酸盐缓冲液制备兔肾脏匀浆， 4 QC以 10000 rpm离心后 20 min后取上清液 为富含 GGT的待测样品， 不加表面活性剂所得匀浆离心后上清为含低活性 GGT的样品。
测定 GGT活性方法为发明专利 ZL 200710093081.4和 J Zhejiang Univ Sci B, 2011 , 12 3): 180-188所述； 数据处理时设定 GGT显色产物对 GGT抑制常数为 185 μηιοΙ/L, GGT对 γ- 谷氨酰基 -C4-硝基)苯胺米氏常数为 1.00 mmol/L， 所用软件同 J ZA 'awg Univ Sci B, 2011 , 12(3): 180-188 o 本应用实例中， GGT为酶 A、 LDH为酶 B; 显色产物 A为 4-硝基苯胺、 显色产 物 B为 NADH但实际其为显色底物； 测量波长中 λΐ为 405 nm、测量波长 λ2为 344 nm (；见图 3b)。
应用本发明同步测定兔子肾脏 GGT和兔子肌肉 LDH的代表性过程如下：
1. 配制缓冲液： 按 《生物化学与分子生物学实验常用数据手册》 （科学出版社， 2000)配 制 0.20 mol/L且 pH为 7.0磷酸钠缓冲液稀释到 0.10 mol/L; 25度恒温；
2. 配制显色底物溶液： 共配制显色底物的三种溶液； 显色底物储备液 A含有 1.5 mm0l/L Y- 谷氨酰基 -(4-硝基)苯胺和 0.70 mmol/L双甘肽； 显色底物储备液 B含有 1.50 mmol/L的 NADH和 30.0 mmol/L丙酮酸；显色底物储备液 C为显色底物储备液 A和显色底物储备液 B的等比例混合 物；所有测定反应总体积 1.0 ml， γ-谷氨酰基 -(4-硝基)苯胺终浓度为 0.15 mmol/L而双甘肽为 70 mmol/L, NADH为 0.15 mmol/1而丙酮酸 3.0 mmol/L， 其余为缓冲液和适度稀释的样品；
3. 制备样品：用 0.1%的 NP40和 pH 7.0的 0.10 mol/L磷酸钠缓冲液，在冰水浴中制备兔肾脏 的匀浆， 4 QC高速离心， 10000 rmpx20 min后取上清液为富含 GGT样品； 稀释时用所配制磷 酸钠缓冲液； 用 LDH与此 GGT混合为含两种酶的样品；
4. 多波长吸收干扰校正系数： 用显色底物储备液 A加适量缓冲液和样品使双甘肽终浓度 为 70 mmol/L且显色底物A为0.15 mmol/L， 测定 λΐ和 λ2下吸收变化之间的联系 (图 6a)， 反应时 间 10.0 min使得在 405 nm吸收增加 0.55以上；用显色底物储备液 B补适量缓冲液和 LDH样品溶 液， 测定 λΐ和 λ2下吸收变化之间的联系 (图 6b)， 反应时间 10.0 min使得在 344 nm吸收降低 0.55 以上； 图 7a和图 7b中有统计意义的响应曲线斜率分别为干扰校正系数 R31和 R33 ;
5. LDH和 GGT的单通道同步测定： 用显色底物储备液 C加缓冲液和适量样品到 1.0 ml且 含有 γ-谷氨酰基 -(4-硝基)苯胺为 0.15 mmol/L而双甘肽为 70 mmol/L, NADH为 0.15 mmol/1而丙 酮酸 3.0 mmol/L; 旋涡振荡混匀后， 同步启动两种酶反应测量波长 λΐ和 λ2下吸收变化， 共测 定 15.0 min以便分析反应过程； NADH在 405 nm基本无吸收， 故所测定的 405 nm吸收来自 本底和显色产物 4-硝基苯胺， 不需要校正 NADH吸收对 405 nm吸收的干扰； 在不同加样量时 分别测定样品及显色底物 A在 405 nm吸收之和为本底， 在 405nm的吸收扣除本底后为 4-硝基 苯胺的吸收， 用于校正 4-硝基苯胺对 LDH活性的干扰； 记录间隔 20 s共记录 15 min; 所得两 波长吸收变化数据的具体处理方法如下：
(1) 获得两个波长下无干扰吸收变化数据
设未加酶样品时独立测定显色底物浓度全部相同的反应体系中来自两种显色显色底物在 λΐ吸收为 1Q、在 λ2吸收为 2Q; 校正吸收干扰前在 λΐ瞬时吸收为 A 在 λ2瞬时吸收为 Α2 ; 校正吸收干扰后显色产物 Α4-硝基苯胺在 λΐ瞬时吸收为 Ala， 显色产物 B即 NADH在 λ2瞬 时吸收为 A2b; 据无相互作用稳定显色团吸收的线性加和性有如下方程：
= 10十 la + R33 X^2b
2 = 20+ 2b + R31 X^la
解此二元一次方程组， 可获得无干扰的两波长下两种显色产物吸收无干扰数据； 独立测 量显色产物 A相对显色底物 A在 λΐ差摩尔小光系数、 显色产物 Β相对于显色底物 Β在 λ2差摩尔 小光系数， 可获得反应体系显色产物 Α和显色产物 Β瞬时浓度； 可再用经典初速度法或过程 分析法分别处理无干扰吸收变化或显色产物浓度数据， 从而单通道同步测定两种酶活性；
(2)联用法分析 GGT反应过程测定其活性
用发明专利 ZL 200710093081.4和 J ZA^/Yawg Univ Sci B, 2011, 12(3): 180-188所述方法用分 析过程和经典初速度法联用测定 GGT初速度 (这不是本发明所需保护的权利要求， 而是专利 ZL 200710093081.4在本发明中的应用)； 数据处理时设转谷氨酰基显色产物对 GGT抑制常数 为 185 μηιοΙ/L, GGT对 γ-谷氨酰基 -(4-硝基)苯胺米氏常数为 1.00 mmol/L,所用软件同 JZ/^ awg Univ Sci B, 2011, 12(3): 180-188； 将最大反应速度转变成初速度所用显色底物浓度为 0.14 mmol/L; 所得 GGT活性有很宽线性范围； 结果见图 6c;
(3)消除 4-硝基苯胺干扰测定 LDH 活性
将无干扰并扣除样品本底 405 nm吸收 Ala用 9.87 L mm0l.Cm)- 1换算成 4-硝基苯胺的浓 度； 测得对硝基苯胺对 LDH竞争性抑制常数为 0.024 mmol/L, LDH对 NADH米氏常数为 0.040 mmol/L; 对硝基苯胺在 405nm毫摩尔差消光系数为 9.87 L-(mmol.cm)"1 , NADH在 344nm毫摩 尔差消光系数为 ό^ ΜηηηοΙ ηι)-1 , 反应体系丙酮酸浓度比 NADH高二十倍， LDH为作用于 NADH单显色底物酶； 设计算 LDH活性时 NADH初始浓度为 0.14 mmol/L; LDH最大反应速 度为 VmB; 校正干扰后， NADH 在 344 nm瞬时吸收为 b， 按照如下公司分析关联的 GGT和 LDH反应过程， 如下方程用于获得 LDH的 VmB : 1
X ~ 0.040 x (1 + Aia / 0.024 / 9.87) + 0.14
sumx = f 1
画 X― 87) + 0.14
VmB x sumx]
Figure imgf000023_0001
取采样间隔内对硝基苯胺浓度恒定， 数值积分获其积分为 Sumx ; 可得 LDH的 mB并用 0.14 mmol/L初始 NADH 浓度得 LDH初速度； 所得响应曲线见图 6d; 校正对硝基苯胺干扰的 效果见表 2; 此校正使两酶同步与单独测定定量限相当。
6. 无 LDH 时 GGT定量限为 Ο. Ι Ι μηιοΚ ηώ -1 ; 在用显色底物溶液 Β测定 LDH为 8.2 μηιοΚ ηώι)-1时， GGT定量限为 0.14 μηιοΚ ηώι)-1 ;在 LDH活性为 20 μηιο ηώι)-1时， GGT 定量限为 0.17 (表 1)。无 GGT时 LDH定量 , 在用显色底物 溶液 Α测定 GG
Figure imgf000023_0002
T为 l J mol^L in)-1时 LDH定量限为 0.6 显色底物溶液 A 测定 GGT为 lO mol^L in)-1时 LDH定量限为 0.5
Figure imgf000023_0003
; 结果比较见表 3。 实施例 2: 单通道多波长吸收同步测定碱性磷酸酶和天然 β_半乳糖苷酶混合物
本应用实例中以牛小肠粘膜碱性磷酸酶 (Sigma P0114, CIAP)为酶 A、 大肠杆菌细胞株 BL21(DE3)裂解液中天然 β-D-半乳糖苷酶 (β-Gal)过 Sephadex G25柱除去小分子物质后为酶 B (Sephadex G25柱高15cm内径10mm容积约10 ml， 用 50 mmol/L且 pH 为 7.5的三羟甲基氨基 甲烷 (Tris)-HCl平衡洗脱)。 4-硝基 -1-萘基磷酸酯为显色底物 A并按文献所示合成（ MM Mhala, Puma Nand. Hydrolysis of organic phosphatases. Part IX -hydrolysis of 1 -nitro-2-naphthyl- and 4-nitro- 1 -naphthyl-phosphate moniesters [J]. Indian Journal of Chemistry, 1976, Vol 14A: 344-346)后用硅胶柱层析纯化 (用含 10%氯仿的甲醇洗脱， 薄板层析无其它成分； 用碱性磷酸 酶将其全部水解后测定所释放 4-硝基 -1-萘酚在 460 nm吸收定含量； 以 4-硝基 -1-萘酚 (Alfa-Aesar编号 L19782)为标准品， 得此 4-硝基 -1-萘基磷酸酯中有效显色底物含量约为 68%， 含游离 4-硝基 -1-萘酚小于 2% ; 按钠离子算氯化钠约 27%)， 4-硝基苯基 -β-D-半乳糖苷为 BBI 产品 (BBI， 编号 NB2361-lg)。 显色产物 A为 4-硝基 -1-萘酚， 显色产物 B为 4-硝基苯酚； 波长 λΐ为 450 nm (酶标仪)、 λ2为 405nm (图 4b和图 3a)。
所用酶标仪为 Biotek E1X800及配套 Gene 5.0软件。所用缓冲液为 pH 为 7.5并含 5.0 μηιοΙ/L 柠檬酸 50 mmol/L Tris-HCl; 将显色底物 A溶解于此缓冲液为显色底物溶液 A、 显色底物 B称 量溶解于此缓冲液为显色底物溶液 B ; 含酶 A和酶 B的显色底物混合物为显色底物溶液 C。
应用本发明所述方法同步测定 CIAP和 β-Gal的代表性过程如下：
1.配制缓冲液： 按 《生化实验方法和技术》 （高等教育出版社， 1981 ; 第一版， 第 374页) 配制 50 mmol/L且 pH为 7.5含 5umol/L柠檬酸的 Tris-HCl缓冲液； 摄氏 25度水浴恒温备用；
2.配制显色底物溶液： 显色底物溶液 A仅含终浓度为 0.20 mmol/L的 4-硝基 -1-萘基磷酸酯， 该显色底物溶液用二甲亚砜配制成 20 mmol/L储备液，在临用前用上述 Tris-HCl缓冲液稀释到 0.20 mmol/L; 显色底物溶液 B仅含 6.0 mmol/L的 4-硝基苯基 -β-D-半乳糖苷， 直接用缓冲液溶 解， 配制后当天用； 显色底物溶液 C含有 0.20 mmol/L4-硝基 -1-萘基磷酸酯和 6.0 mmol/L的 4- 硝基苯基 -β-半乳糖苷， 配制方法参照显色底物溶液 Α和 Β， 当天配置后也仅在当天使用；
3. 制备样品： 将 CIAP按商家标示活性浓度用 Tris-HCl缓冲液稀释到约 800 U/L, 将 β-Gal 用 Tris-HCl缓冲液稀释到约 600 U/L; 两种酶液等比例混合为含两种酶的混合样品；
4.多波长吸收干扰校正系数：用相同浓度的 4-硝基 -1-萘基磷酸酯和 CIAP,连续记录 450 nm 和 405 nm吸收变化， 所用条件下在 450 nm吸收增加 0.60以前基本呈线性， 分析 405nm吸收对 450nm吸收的响应曲线， 所得斜率为干扰校正系数 R31(图 7a); 用 4-硝基苯基 -β-D-半乳糖苷和 β-Gal连续测定 450 nm和 405 nm吸收变化， 在 405 nm吸收增加到 1.00以前都成线性， 分析 450nm吸收对 405nm吸收的响应， 所得曲线的斜率为干扰校正系数 R33 (；图 7b);
5.碱性磷酸酶和 β-半乳糖苷酶的单通道同步测定： 用显色底物溶液 C稀释到两种显色底物 达到测定酶活性的终浓度时， 用于将测量波长 405 nm和 450 nm下吸收同步调零； 取显色底物 C共 1.50 ml， 加混合样品 20到 400 μΐ并补充缓冲液到 2.0 ml; 旋涡振荡混匀后， 摄氏 25度同步 测量波长 405 nm和 450 nm下吸收变化， 记录间隔 20s， 共测定 3.0 min; 两波长下吸收在 90 s 以内都是线性变化， 可用如下的简便方法计算表观初速度对酶量的响应 (图 7c和图 7d);
6. 解下列方程组消除干扰后得 450 nm无干扰吸收变化初速度 si、405nm无干扰吸收变化 初速度 s2(图 7c和图 7d); 可见经干扰校正后单独测定和两种酶同步测定响应曲线斜率无差异；
Vl= sl+ R33xs2
V2= s2+ R31 xsl
7.无 β-D-半乳糖苷酶时， 碱性磷酸酶定量限约 0.06 糖苷酶活性为 0.6 μηιοΐ^ΐ^ηώι)-1时， 两种酶同测碱性磷酸酶定量限
Figure imgf000024_0001
β-D-半乳糖苷 酶活性为 2.5 μηιοΐ^ΐ^ηώι)-1时， 两种酶同测时碱性磷酸酶定量限约为 0.08
Figure imgf000024_0002
无 碱性磷酸酶时， β-D-半乳糖苷酶定量限约为 0.07
Figure imgf000024_0003
; 用显色底物溶液 Α测定碱性 磷酸酶的活性为 0.7 μηκ^Ο^ηώι)-1时， 两种酶单通道同测时 β-D-半乳糖苷酶定量限为 0.09
Figure imgf000024_0004
; 碱性磷酸酶的活性为 5.6 μηιοΐ^ΐ^ηώι)-1时， 两种酶单通道同测时 β-D-半乳糖 苷酶定量限为 0.08
Figure imgf000024_0005
结果比较见表 4。 实施例 3 : 酶联免疫吸附分析单通道同步测定牛奶中青霉素和瘦肉精
本应用实例中以 β-D-半乳糖苷酶 (Sigma G4155 , β-Gal))为酶 A、 α-D-葡萄糖苷酶 (Sigma G0660, α-Glu)为酶 B; 4-硝基 -1-萘基 -β-D-半乳糖苷为显色底物 A， 按合成 4-硝基苯基 -β-D- 半乳糖苷的路线合成、过胶柱层析纯化 (用 β-D-半乳糖苷酶将显色底物完全水解后测定所生成 的 4-硝基 -1-萘酚确定纯度)， 4-硝基苯基 -α-D-葡萄糖苷 (Sigma N1377)为显色底物 B; 所用缓 冲液 pH 为 7.4的 100 mmol/L磷酸盐缓冲液。显色产物 A为 4-硝基 -1-萘酚， 显色产物 B为 4-硝基 苯酚； 测量波长 λΐ为 450 nm、 测量波长 λ2为 405 nm (Biotek ELX 800酶标仪标准配置中提供 的滤光片， 附图 4a)。 本应用实施例中的混合物酶样品为经过酶联免疫吸附分析 (ELISA)过程 后， 分离出来但结合在微孔板上的酶 A和酶 B混合物。
本实施例中使用 Pierce的预包被羊抗小鼠 IgG的多克隆抗体的微孔板 （产品编号 15134， 结合容量每孔 7 pmole); 用 Abeam的抗青霉素鼠源 IgG单抗 ab 15070、 抗瘦肉精鼠源 IgG单抗 ab32005 ;用青霉素 G修饰 β-D-半乳糖苷酶完全同应用实施例 3 ;修饰显色产物过 Sephadex G25 柱除去游离的半抗原； 瘦肉精修饰 α-Glu见如下所述， 并过 Sephadex G25柱纯化。 用本发明 所述的方法单通道同步测定纯牛奶中外加的青霉素 G和瘦肉精过程如下：
1.配制缓冲液： 按 《生物化学与分子生物学实验常用数据手册》 （科学出版社， 2000)所述配 制 100 mmol/L且 pH为 7.4磷酸钠缓冲液； 摄氏 25度水浴恒温备用；
2.配置显色底物溶液 A、 显色底物溶液 B和显色底物溶液 C: 显色底物 A用二甲亚砜配制成 20 mmol/L的显色底物 A储备液， 用磷酸钠缓冲液稀释到 0.20 mmol/L为显色底物溶液 A; 显色底 物 B用 20%的二甲亚砜配制成 50 mmol/L的显色底物 B储备液， 用磷酸钠缓冲液稀释到 2.6 mmol/L为显色底物溶液 B;将显色底物 A的储备液和显色底物 B的储备液用磷酸钠缓冲液稀释 到显色底物 A浓度为 0.20mmol/L且显色底物 B为 2.6 mmol/L, 得到显色底物溶液 C;
3.青霉素 G标记 β-D-半乳糖苷酶：
a) 青霉素 G活化： 0.73 g青霉素 G钠盐溶于 2.0 ml Ν,Ν'-二甲基甲酰胺，加入 0.40 g 1-乙基 -(3-二甲氨基丙基) -碳酰二亚胺 (縮写为 EDC)盐酸盐，及 0.30 g 1-羟基苯并三唑反应 30 min得深 黄色反应液； 取 4 ul 上述深黄色溶液用 54 ul 缓冲液稀释到 58 ul为活化青霉素 G溶液；
b) 取上述稀释活化青霉素 G溶液 50 ul, 与用 0.10 mol/L的磷酸盐缓冲液 (pH 7.4)共 0.50 ml融解并稀释的 β-D-半乳糖苷酶 0.44 mg混匀； 摄氏 4度反应 120 min; 过 Sephadex G25柱 (；高 15 cm内径 10 mm容积约 10 ml, pH 7.5的 50 mmol/L Tris-HCl缓冲液平衡）， 用 280 nm吸收监测， 收集相同 Tris-HCl缓冲液的第一个洗脱峰为青霉素 G修饰的 β-D-半乳糖苷酶， 所得青霉素 G修 饰 β-D-半乳糖苷酶溶液共 2.0 ml含有 0.22 mg蛋白质；
c)标记后比活性： 在 pH 7.5的 50 mmol/L Tris-HCl缓冲液中， 用终浓度为 6.0 mmol/L的 4- 硝基苯基 -β-D-半乳糖苷时酶比活性为 30 保留近 100%; 用 4-硝基 -1- 萘基 -β-D-半乳糖苷为显色底物测定比活
Figure imgf000025_0001
, 保留约 70%活性；
d)用量： 应用时； 优化单抗用量后， 逐步提高青霉素标记 β-D-半乳糖苷酶溶液用量， 直 到结合的半抗原标记 β-D-半乳糖苷酶活性达到饱和； 在 96孔板体系， 用 20 ul稀释 500倍的单 抗时加入青霉素标记 β-D-半乳糖苷酶溶液 20 ul, 即可达到饱和；
4. 瘦肉精标记 α-D-葡萄糖苷酶：
a) 瘦肉精 0.8 g溶于 1.0 ml pH 1.0的盐酸水溶液中， 加 24 ul 亚硝酸钠水溶液 (1.0 mol/L)， 室温反应 30 min; 用淀粉 -碘化钾试纸检测为蓝色表明反应完全， 即为活化瘦肉精重氮盐； b) 用 pH 7.8的 200 mmol/L磷酸盐缓冲液 (；含 10%甘油)溶解 α-D-葡萄糖苷酶到 2.0 mg/ml;取 此 a-D-葡萄糖苷酶 100 ul加入 2 ul活化瘦肉精重氮盐， 摄氏 4度反应 12 h; 过 Sephadex G25柱 (高 15 cm内径 10 mm容积约 10 ml， 用 pH 7.4的 100 mmol/L 磷酸钠缓冲液平衡)， 用 280 nm吸 收监测， 收集此磷酸盐缓冲液第一个洗脱峰 2.0 ml为瘦肉精标记 α-D-葡萄糖苷酶溶液；
c) 标记后比活性： 用终浓度为 2.6 mmol/L的 4-硝基苯基 -a-D-葡萄糖苷 pH 7.4的 100 mmol/L磷酸盐缓冲液测得比活性为 90 mol L_min_mg)- 保留 85%;
d) 将抗瘦肉精单抗稀释 5000倍后取 80 ul/孔， 加入瘦肉精标记 a-D-葡萄糖苷酶溶液 10 ul, 结合的瘦肉精标记 a-D-葡萄糖苷酶活性即达到饱和；
5.优化检测单抗 abl5070和 ab32005的混合比例： 青霉素单抗稀释比例 1 :500; 瘦肉精单抗稀释 比例为 1 :5000; 上述稀释的单抗从 20 ul到 80 ul优化组合， 加入上述瘦肉精标记 α-D-葡萄糖苷 酶溶液及青霉素标记 β-半乳糖苷酶溶液各 40 ul; 当反应体系在 405 nm和 450 nm吸收变化速度 相当时， 所需抗青霉素单抗为 20 ul且抗瘦肉精单抗也为 80 ul; 以下实验中， 两种单抗即以此 比例混匀后， 在 96孔板上每孔加入 100 ul单抗混合物；
6.优化两种酶标半抗原混合比例： 取上述瘦肉精标记 a-D-葡萄糖苷酶 300 ul和青霉素标记 β- 半乳糖苷酶溶液 600 μΐ混合后，用 ρΗ 7.4的 100 mmol/L 磷酸盐缓冲液稀释到 3.0 ml; 以下实验 中每孔加入 100 ul半抗原标记酶混合物；
7.在 Pierce的预包被羊抗小鼠 IgG多克隆抗体 96孔板上如下操作：（1)每孔加入 100 ul单抗混合 物与捕获多抗在 25度低速震荡 60 min; (2) 用含 0.05%吐温 -20的 pH 7.4的 100 mmol/L 磷酸盐 缓冲液为洗涤液， 每孔每次加入洗涤液 0.20 ml, 每次洗涤 3 min后去洗涤液，共重复三次； (3) 将纯牛奶在 4度 8000rpm离心 30min去上层脂肪获得脱脂牛奶， 用 pH 7.4的 100 mmol/L 磷酸盐 缓冲液稀释 1倍加入不同比例青霉素和瘦肉精为样品； （4)用步骤 (2)洗涤液洗涤 3次；
8. 在 96孔板中每孔加入显色底物溶液 、 显色底物溶液 B或显色底物溶液 C中的一种共 200 ul; 中速震荡 10 min， 在 Biotek ELX 800酶标仪上， 用仪器配套软件同步测量 405 nm和 450 nm 吸收， 每隔 3 min读数跟踪反应过程， 共记录 30 min;
9. 干扰校正系数的测定：操作同应用实施例 2;用相同终浓度 4-硝基 -1-萘基 -β-D-半乳糖苷为 显色底物和 β-Gal, 连续记录 450 nm和 405 nm吸收变化， 间隔 20s， 共记录 3.0 min， 使得在 450 nm吸收增加到 0.80之前都成线性， 回归分析 405 nm吸收对 450 nm吸收的响应曲线， 所得斜率 为干扰校正系数 R31(图 8a); 用相同浓度的 α-D-葡萄糖苷和 a-Glu， 连续记录 450 nm和 405 nm 吸收变化， 间隔 20 s， 共记录 3.0 min， 使得在 405 nm吸收增加到 0.80之前都成线性， 回归分 析 450 nm吸收对 405 nm吸收的响应曲线， 所得斜率为干扰校正系数 R33(图 8b);
10. 两种糖苷酶同步测定与单独测定的比较： 用显色底物溶液 C稀释到两种显色底物达到测 定酶活性的终浓度时，用于将测量波长 405 nm和 450 nm下吸收同步调零；取显色底物 C共 1.50 ml, 加混合样品 20到 400 μΐ并补充缓冲液到 2.0 ml; 旋涡振荡混匀后， 摄氏 25度同步测量波长 405 nm和 450 nm下吸收变化， 记录间隔 20s， 共测定 3.0 min; 两波长下吸收在 90 s以内都是线 性变化， 可用如下的简便方法计算表观初速度对酶量的响应 (图 8c和图 8d);
11. 响应曲线制作： 按照实施例 2中所用校正系数， 将 450 nm吸收变化表观初速度 VI和 405 nm吸收变化表观初速度 V2换算成无干扰的吸收变化速度并扣除自发反应本底； 以无外加标 准品混合物时两种酶活性为 100%， 计算加入两种抗生素标准品混合物后的酶活性剩余率；
12. 抗生素标记酶结合率； 以此结合率对反应体系的抗生素标准品浓度对数作图； 结果在结 合率在 10%到 85%之间为线性响应 (图 8e和 8f)；
13. 数据处理和结果报告：两种糖苷酶标记单通道 ELISA同步测定两种半抗原的定量限比较 见表 5、 回收率比较见表 6。
表 1单通道多波长吸收同步测量三种酶活性所需物质、 参数表 多组分测定所涉及的光谱特征等参数及定量关系 成分 A B C 测定波长 1 2 3 表观初速度 Vi v2 v3 真实初速度 Sl s2 S3
A成分对各表观初速度相对贡献 1 R31 R32
B成成分对各表观初速度相对贡献 R33 1 R34
C成成分对各表观初速度相对贡献 R35 R36 1
A成分表观初速度 Vi= Si+ S2 XR33 + S3 XR35
B 成分表观初速度 V2= S2+ Si XR31 + S3 XR36
C 成分表观初速度 V3= S3+ Si XR32 + S2 XR34
Figure imgf000027_0001
Figure imgf000027_0002
表 4 同步测定 CIAP和 β-Gal的定量限 >3) 酉每量 μηιο1·(ί·ηώι)-1 LOQ
Figure imgf000028_0001
CIAP β-Gal CIAP β-Gal
0 0 0.06 0.07
0.7 0 1 0.09
5.6 0 1 0.08
0 0.6 0.06 1
0 2.5 0.08 1 表 5 用 β-Gal和 α-Glu为标记酶单通道同步测定瘦肉精和青霉素的结合率定量限
Figure imgf000028_0002
表 6 用 β-Gal和 α-Glu标记记单通道同步测定瘦肉精和青霉素的回收率 （w>5)
Figure imgf000028_0003
最后说明的是， 以上实施例仅用以说明本发明的技术方案而非限制；本领域的普通技术人 员应当理解， 可以对本发明的技术方案进行修改或者等同替换， 而不脱离本发明技术原理， 其均应涵盖在本发明的权利要求范围当中； 除了容易获得的 4-硝基 -1-萘酚、 4-硝基苯酚、 4- 硝基苯胺、 4-硝基 -1-萘胺类显色团， 其它满足光谱特征要求的显色团也可用于特定酶所需要 的显色底物， 用于实现多波长吸收单通道同步测定多种酶活性， 并使其定量限、 灵敏度和线 性测量范围与用相同浓度底物在相同波长下测定单个酶活性时相当。

Claims

权 利 要 求 书
1.一种用多波长吸收单通道同步测量多种酶活性的方法， 其特征在于： 包括下列步骤： a. 确定用多波长吸收单通道同步测定多种酶活性所需显色底物组合：
al根据待测酶的专一性筛选满足下列条件的显色底物组合： 所选显色团能分别用于制备 待测酶的显色底物； 所得这些显色底物组合中， 将显色底物在对应待测酶作用下所得显色产 物相对该显色底物的差吸收峰最大波长从大到小排列，相邻的显色产物差吸收峰最大波长之 间相距大于 30 nm且尽量远； 在按显色产物差吸收峰最大波长从大到小的排列中， 除了显色 产物差吸收峰最靠近红外端的显色底物外，每个显色底物在对应酶作用下所得显色产物差吸 收峰最大波长， 都与此排列中某个显色底物的最大等吸收波长相差小于 25 nm且尽量短； a2 用步骤 al所选显色团分别合成待测酶的对应显色底物并组合使用；
b. 在一个反应混合物或酶反应体系即单反应通道中， 同步启动多个待测酶针对组合使用 的显色底物混合物对应显色底物的专一催化反应；
c 选择测量波长组合： 以显色产物差吸收峰最靠近红外端的显色底物为显色底物 A， 在 显色底物 A的显色产物差吸收峰最大波长下测定该显色产物吸收； 在显色底物 A的等吸收 波长处测定其余待测酶显色产物或显色底物的吸收；
d. 在所选多个测量波长组合下， 通过快速变换测量波长， 多波长吸收同步测量实现同步 跟踪单通道中多个待测酶的反应过程；
e. 基于无相互作用物质吸收的线性加和性建立消除反应通道中显色物质吸收重叠干扰的 数据处理方法， 获得消除吸收光谱重叠干扰后每个待测酶的显色产物或显色底物无干扰吸收 变化曲线； 如某个待测酶不受反应体系其它酶的产物及底物的干扰且其底物浓度在对应酶米 氏常数的 3倍以上，用经典初速度法分析其显色产物或显色底物无干扰吸收变化曲线确定初 速度； 如某个待测酶不受反应体系其它酶的产物及底物的干扰但所用显色底物浓度在该酶米 氏常数的 5 %以上而低于米氏常数的 3倍， 联用经典初速度法和反应过程分析法分析其显色 产物或显色底物无干扰吸收变化曲线确定初速度；当某个待测酶受反应体系中其自身和 /或其 它酶的底物和 /或产物的干扰时，将描述反应体系动力学的微分速度方程组数值积分后分析活 性受到干扰待测酶反应的显色产物或显色底物无干扰吸收变化曲线，确定其最大反应速度表 示其活性，或据其微分速度方程和所得最大反应速度换算成底物浓度为起始底物浓度 93 %时 初速度表示该待测酶的活性。
2. 根据权利要求 1所述用多波长吸收单通道同步测量多种酶活性的方法， 其特征在于： 显色产物相对显色底物差吸收峰最大波长在 300 nm以上且其显色底物吸收峰最大波长小于 其对应显色产物吸收峰最大波长， 否则测定该酶催化逆反应的速度表示该酶的活性； 显色产 物差吸收峰最大波长相邻的任两个显色底物中， 显色产物差吸收峰最大波长更靠近红外端显 色底物的最大等吸收波长与显色产物差吸收峰最大波长更靠近紫外端显色产物的差吸收峰 最大波长相等或相距不超过 5nm为优化的显色底物组合;所用显色底物组合中每个显色底物 都仅被样品中的一种待测酶作用生成对应显色产物， 而不被样品中其它任何酶作用或作用效 果不明显； 步骤 b中， 选择适于同步测定活性的待测酶发挥作用的缓冲介质和反应条件， 即 缓冲介质 pH位于 pH 5.0到 9.0之间且接近比活性最低待测样品的最适 pH，所用反应温度在 摄氏 20到 40度之间。
3. 根据权利要求 2所述的用多波长吸收单通道同步测量多种酶活性的方法， 其特征在 于： 所述显色底物包括天然显色底物和非天然显色底物； 天然显色底物包括尼克酰胺腺嘌 呤二核苷酸或尼克酰胺腺嘌呤二核苷酸磷酸； 非天然显色底物由显色团和被酶识别基团组 成， 显色团包括但不限于 4-硝基苯酚、 4-硝基苯硫酚、 4-硝基苯胺、 4-硝基 -1-萘酚、 4-硝基 -1-萘硫酚、 4-硝基 -1-萘胺、 2-萘酚、 4-氯苯酚、 玫红酸或这些芳香酚及芳香胺的衍生物。
4. 根据权利要求 1、 2或 3所述的用多波长吸收单通道同步测量多种酶活性的方法， 其 特征在于： 采用两波长吸收单通道同步测量两种酶活性：
步骤 a中，将两种待测酶分别命名为酶 A和酶 B，所需两种显色团分别为显色团 A和显 色团 B， 对应的两种显色底物分别为显色底物 A和显色底物 B， 在两种待测酶作用下生成的 两种显色产物分别为显色产物 A和显色产物 B，这些显色底物及显色产物的组合需满足下列 条件： （1 ) 显色产物 A差吸收峰最大波长 XI靠近红外端， 显色产物 B差吸收峰最大波长 X2靠近紫外端， 显色产物 B差吸收峰最大波长 X2与显色产物 A差吸收峰最大波长 XI相 距大于 30 nm且尽量远； （2) 显色产物 A的最大等吸收波长为 Y1且与显色产物 B差吸收 峰最大波长 X2相距小于 25 nm且尽量短；
步骤 b、 c和 d中， 在一个反应通道同步启动样品中两种酶针对显色底物 A和显色底物 B的反应， 并得到显色产物 A和显色产物 B; 选择显色产物 A的差吸收峰最大波长 XI为 λΐ 测定显色产物 Α吸收， 选择显色产物 A的最大等吸收波长 Y1为 λ2处测定显色产物 Β吸收； 通过快速变换测量波长， 两波长吸收同步测量同步跟踪单通道中两种待测酶的反应过程； 步骤 e中， 基于吸收的线性加和性建立消除多种物质吸收重叠干扰的数据处理方法， 获得消除吸收光谱重叠后两个待测酶的显色产物或显色底物无干扰吸收变化曲线； 如某个 待测酶不受反应体系其它酶的产物及底物的干扰且其底物浓度在对应酶米氏常数的 3倍以 上， 则用经典初速度法分析其显色产物或显色底物无干扰吸收变化曲线确定初速度； 如某 个待测酶不受反应体系其它酶的产物及底物的干扰但所用底物浓度在该酶米氏常数的 5 % 以上而低于米氏常数的 3倍， 联用经典初速度法和反应过程分析法分析其显色产物或显色 底物无干扰吸收变化曲线确定初速度； 当某个待测酶受到反应体系中其自身或其它酶的底 物或产物的干扰时， 将描述反应体系动力学的微分速度方程组数值积分后分析活性受到干 扰待测酶反应的显色产物或显色底物无干扰吸收变化曲线， 确定其最大反应速度表示其活 性， 或据其微分速度方程和所得最大反应速度换算成底物浓度为起始底物浓度 93 %时初速 度表示酶活性；以显色产物 A差吸收峰最大波长前后不超过 25 nm的波长为 λΐ并测定在此 波长下吸收为 Al， 以显色产物 Α和显色底物 A最大等吸收波长附近不超过 25 nm的波长 为 λ2下并测定此波长下吸收为 Α2，基于吸收线性加和性消除多种物质吸收重叠干扰计算无 干扰的显色产物或底物吸收， 需解下列二元一次方程组：
/4ι = /4ιο+Ε31 Χ Ρι + Ε34 Χ Ρ2
^ ^o+ESS X Pi + Ε35 Χ Ρ2
进一步可转变成如下二元一次方程组 2 = 20+ 2b + R31 X^la R31= E32/E31
R33= E34/E35
上述公式中， Pi为显色产物 A瞬时浓度； P2为显色产物 B瞬时浓度； E31为显色产物 A与显色底物 A在 λΐ下差摩尔消光系数； Ε32为显色产物 Α与显色底物 A在 λ2下差摩尔 消光系数 Ε32; Ε34为显色产物 Β与显色底物 Β在 λΐ下差摩尔消光系数； Ε35为显色产物 Β与显色底物 Β在 λ2下差摩尔消光系数； Αχ为校正吸收干扰前在 λΐ瞬时总吸收， Α2为校正 吸收干扰前在 λ2瞬时总吸收； a为校正吸收干扰后显色产物 Α在 λΐ瞬时净吸收， 其等于 ?1和 E31乘积， ^2b为校正吸收干扰后显色产物 B在 λ2瞬时净吸收，其等于 Ρ2和 Ε35乘积； 0为反应体系未生成任何显色产物前在 λΐ 的吸收， 为来自反应体系的本底， 相当于含相 同浓度全部显色底物反应体系在 λΐ 吸收与含相同浓度样品反应体系在 λΐ 吸收的加和； ^20 为反应体系未生成任何显色产物前在 λ2的吸收， 为来自反应体系的本底， 相当于含浓度相 同全部显色底物的反应体系在 λ2吸收与含相同浓度样品反应体系在 λ2吸收的加和；
上述计算公式中， 酶 Α将显色底物 Α转变成显色产物 Α的计量关系为 1 : 1， 酶 B将显 色底物 B转变成显色产物 B的计量关系也为 1 : 1 ;
如反应通道中某种酶的底物或产物抑制或激活另一种酶， 则如下测量被干扰酶活性： 获得两种显色底物或显色产物的无干扰吸收变化曲线， 据此换算出产生干扰作用物质 的浓度变化曲线； 用含干扰作用微分动力学方程数值积分计算理论反应曲线拟合被干扰酶 显色底物或显色产物的无干扰吸收变化曲线，对应最好拟合的最大反应速度 m为无干扰时 的待测酶活性； 据该酶的微分动力学方程、 m和其初始底物浓度的 93%， 计算在刚启动反 应时干扰物质还没有明显积累的初速度表示该待测酶活性；
当仅一种酶显色产物对另一种酶有竞争抑制作用时， 先确定显色产物 A及显色产物 B 在 λΐ下和在 λ2下的无干扰吸收变化曲线 Ala和 A2b;设初始显色底物 A的浓度为 Cl，初始 显色底物 B的浓度为 C2; 设酶 B最大反应速度为 mB， 由 换算成初速度所用预设显色 底物浓度等于 C2的 93 %， 酶 B对显色底物 B的米氏常数为 ^; 如显色产物 A对酶 B为 竞争性抑制， 其竞争性抑制常数为 a， 酶 B动力学方程为：
C2 xE34 KmB x(l + Ala/Kia/E31) + C2 对上述方程两边对反应时间从 0〜t进行积分得到 X [ dA7, - VmR X ί X dt
C2xE34 i { KmBx(l + Ala/Kia/E3l) + C2 左边的积分项为反应时刻 t和反应时刻 0之间显色产物 B的净吸收变化；方程右边积分 项以 dt内显色产物 A浓度恒定进行数值积分， 即计算
1
X
KmB x(l + Ala/K E l) + C2 以 dt为 Δ ， 计算每个记录点的 χ， 将所有 X求和再减去起点和终点 X得到 sumx， 用如 下公式计算 并据其微分动力学方程得无干扰初速度 。
Figure imgf000032_0001
5. 根据权利要求 4所述的用多波长吸收单通道同步测量多种酶活性的方法，特征在于： 步骤 a中，如下选择显色团 A和显色团 B组合： 显色产物 A的最大等吸收波长 Y1与显 色产物 B差吸收峰最大波长 X2相等或相距不超过 5 nm;
步骤 c中， 选择产物 A差吸收峰最大波长 XI为 λΐ后， 如下选择测量波长 λ2: 显色产物 Β与显色底物 Β差摩尔消光系数在显色产物 Α最大等吸收波长 Y1处高于在显色产物 B差吸 收峰最大波长 X2处的 30%， 则选择显色产物 A最大等吸收波长 Y1为 λ2， 否则， 用最靠近 显色产物 Α最大等吸收波长 Y1的显色产物 B差吸收峰波长为 λ2， 或最靠近显色产物 Β差 吸收峰且其它待测显色显色产物与显色显色底物差摩尔消光系数都小于显色产物 Β 与显色 底物 Β差摩尔消光系数 20%的波长为 λ2测量显色产物 Β吸收； 步骤 e中， 需测定 R31为显色产物 A吸收干扰的校正系数； 测定 R31时， 仅用酶 A 所需的全部显色底物而不加其它待测酶的任何显色底物， 使反应体系无来自显色底物 B及 显色产物 B在两个测量波长 λΐ和 λ2的吸收， 此时同步测定反应通道中在两个测量波长下 的吸收变化， 直到在 λ2吸收变化大于 0.005或在 λΐ吸收变化大于 0.500; 以在 λΐ下吸收为 横坐标， 在 λ2下吸收为纵座标进行回归分析， 所得回归直线斜率为校正系数 R31 ;
步骤 e中， 需测定 R33为显色产物 B吸收干扰的校正系数； 测定 R33时， 仅用酶 B 所需的全部显色底物而不加其它待测酶的任何显色底物， 使反应体系无来自显色底物 A及 显色产物 A在两个测量波长 λΐ和 λ2的吸收， 此时同步测定反应通道中在两个测量波长下 的吸收变化， 直到在 λ2吸收变化大于 0.500或在 λΐ吸收变化大于 0.005; 以在 λ2下吸收为 横坐标， 在 λΐ下吸收为纵座标进行回归分析， 所得回归直线斜率为校正系数 R33;
6. 根据权利要求 4或 5所述的用多波长吸收单通道同步测量多种酶活性的方法， 其特 征在于：
与酶 Α对应的显色底物包括水解反应最大等吸收波长在 310到 330 nm间的 4-硝基苯 基乙酸酯， 水解反应最大等吸收波长在 330到 350nm间的 4-硝基苯基磷酸酯、 4-硝基苯基 硫酸酯、 4-硝基苯基 -β-D-半乳糖苷和 γ-谷氨酰基 -4-硝基苯胺， 还原反应最大等吸收波长在 350到 370nm间的 5-巯基 -2-硝基苯甲酸及 5-巯基 -2-硝基苯乙酸对应显色底物为二硫化物可 被酶作用所释放的硫醇还原； 与此类酶 A的显色底物 A对应的酶 B的显色底物 B主要是 天然显色底物， 包括尼克酰胺腺嘌呤二核苷酸或尼克酰胺腺嘌呤二核苷酸磷酸；
与显色底物 A对应的酶 A包括但不限于芳香酯酶、 磷酸酶、 硫酸酯酶、 γ-谷氨酰基转 移酶、 天然氨基酸 α-羧基对应的酰胺酶和糖苷酶； 与此类酶 Α对应的酶 B包括但不限于乳 酸脱氢酶 LDH、 苹果酸脱氢酶 MDH、 偶联 LDH的谷丙转氨酶和偶联 MDH的谷草转氨酶。
7. 根据权利要求 1、 2或 3所述的用多波长吸收单通道同步测量多种酶活性的方法， 其 特征在于： 采用三波长吸收单通道同步测量三种酶活性：
步骤 a中， 将三种待测酶分别命名为酶 A、 酶 B和酶 C， 所需显色底物分别为显色底物 A、 显色底物 B和显色底物 C， 在对应酶作用下生成显色产物分别为显色产物 A、 显色产物 B和显色产物 C; 这些显色底物和显色产物的组合需满足下列条件： （1 ) 显色产物 A差吸 收峰最大波长为 XI且最靠近红外端，显色产物 C差吸收峰最大波长为 X3且最靠近紫外端， 显色产物 B差吸收峰最大波长为 X2且位于 XI和 X3之间， X2与 XI及 X3相距都大于 30 nm 且尽量远； （2) 显色产物 A的最大等吸收波长为 Y1且次大等吸收波长为 Ys; 显色底物 B 生成显色产物 B的最大等吸收波长为 Y2且与 Y1相距尽量远而和 Ys相距尽量短； （3 ) Y1 与 X2相距小于 25 nm且相距尽量短； Y2和 Ys及 X3相距都小于 25 nm且相距尽量短； 步骤 b中， 在一个反应通道同步启动三种酶针对显色底物 A、 显色底物 B和显色底物 C 的反应， 并得到对应的显色产物 、 显色产物 B和显色产物 C;
步骤 c中， 以显色产物 A差吸收峰最大波长 XI或相距 25nm以内波长为 λΐ测定显色产 物 Α吸收， 选择显色产物 A最大等吸收波长 Y1或相距 25nm以内波长为 λ2测定显色产物 Β 吸收，选择显色产物 Β最大等吸收波长 Υ2或相距 25nm以内波长为 λ3测定显色产物 C吸收; 步骤 d中， 通过快速变换测量波长， 三波长吸收同步测量实现同步跟踪单个反应体系中 两种样品的反应过程；
步骤 e 中， 先建立消除多种物质吸收重叠干扰的数据处理方法， 获得消除吸收光谱重 叠后每个待测酶的显色产物或显色底物无干扰吸收变化曲线； 某个待测酶不受反应体系其 它酶的产物及底物的干扰且其底物浓度在对应酶米氏常数的 3倍以上， 则用经典初速度法 分析其显色产物或显色底物无干扰吸收变化曲线确定初速度； 如某个待测酶不受反应体系 其它酶的产物及底物的干扰但所用底物浓度在该酶米氏常数的 5 %以上而低于米氏常数的 3 倍， 联用经典初速度法和反应过程分析法分析其显色产物或显色底物无干扰吸收变化曲 线确定初速度； 当某个待测酶受到反应体系中其自身或其它酶的底物或产物的干扰时， 将 描述反应体系动力学的微分速度方程组数值积分后分析活性受到干扰待测酶反应的显色产 物或显色底物无干扰吸收变化曲线， 确定其最大反应速度表示其活性， 或据其微分速度方 程和所得最大反应速度换算成底物浓度为起始底物浓度 93 %时初速度表示酶活性； 基于吸 收线性加和性消除多种物质吸收重叠干扰需解如下三元一次方程组：
Ax = Aw+Ala + R33 A2h + R35 XA3c
2 = 20十 2b + R31 X a + R36 X 3c
A3 = A30 +A3c + R32 X/4ia + R34 X/42b
R31= E32/E31
R32= E33/E31
R33= E34/E35
R34= E36/E35
R35= E37/E39
R36= E38/E39
上述公式中， ^为校正吸收干扰前在 λΐ瞬时吸收， ^为校正吸收干扰前在 λ2瞬时吸 收， ^为校正吸收干扰前在 λ3瞬时吸收； la为校正吸收干扰后显色产物 Α在 λΐ瞬时净吸 收， ^2b为校正吸收干扰后显色产物 Β在 λ2瞬时净吸收， e为校正吸收干扰后显色产物 C 在 λ3瞬时净吸收； 。为反应体系未生成任何显色产物前在 λΐ吸收， 2Q为反应体系未生成 任何显色产物前在 λ2吸收， 。为反应体系未生成任何显色产物前在 λ3吸收； E31为显色 产物 Α与显色底物 A在 λΐ下差摩尔消光系数， Ε32为显色产物 Α与显色底物 A在 λ2下差 摩尔消光系数， Ε33为显色产物 Α与显色底物 A在 λ3下差摩尔消光系数； Ε34为显色产物 Β与显色底物 Β在 λΐ下差摩尔消光系数， Ε35为显色产物 Β与显色底物 Β在 λ2下差摩尔消 光系数， Ε36为显色产物 Β与显色底物 Β在 λ3下差摩尔消光系数； Ε37为显色产物 C与显 色底物 C在测量波长 λΐ下差摩尔消光系数， Ε38为显色产物 C与显色底物 C在测量波长 λ2下差摩尔消光系数， Ε39为显色产物 C与显色底物 C在测量波长 λ3下差摩尔消光系数； 上述公式中， 显色底物 Α转变成显色产物 Α计量关系为 1:1; 显色底物 B转变成显色产物 B的计量关系为 1:1; 显色底物 C转变成显色产物 C的计量关系为 1:1;
如反应通道中某种酶的底物或产物抑制或激活另一种酶， 则如下测量被干扰酶活性： 获得两种显色底物或显色产物的无干扰吸收变化曲线， 据此换算出产生干扰作用物质 的浓度变化曲线； 用含干扰作用微分动力学方程数值积分计算理论反应曲线拟合被干扰酶 显色底物或显色产物的无干扰吸收变化曲线，对应最好拟合的最大反应速度 m为无干扰时 的待测酶活性； 据该酶的微分动力学方程、 m和其初始底物浓度的 93%， 计算在刚启动反 应时干扰物质还没有明显积累的初速度表示该待测酶活性；
当仅一种酶显色产物对另一种酶有竞争抑制作用时， 先确定显色产物 A及显色产物 B 在 λΐ下和在 λ2下的无干扰吸收变化曲线 Ala和 A2b;设初始显色底物 A的浓度为 Cl，初始 显色底物 B的浓度为 C2; 设酶 B最大反应速度为 mB， 由 换算成初速度所用预设显色 底物浓度等于 C2的 93 %， 酶 B对显色底物 B的米氏常数为 ^; 如显色产物 A对酶 B为 竞争性抑制， 其竞争性抑制常数为 a， 酶 B动力学方程为：
dA2b _ VmB x dt
2 xEM KmB (\ + A,aIKJE^) + C2 对上述方程两边对反应时间从 0〜t进行积分得到
Figure imgf000035_0001
左边的积分项为反应时刻 t和反应时刻 0之间显色产物 B的净吸收变化；方程右边积分 项以 dt内显色产物 A浓度恒定进行数值积分， 即计算
1
X~ KmBx(l + Ala/Kia/E31) + C2
以 dt为 Δ ， 计算每个记录点的 χ， 将所有 X求和再减去起点和终点 X得到 sumx， 用如 下公式计算 并据其微分动力学方程得无干扰初速度 。
VmB - dA2b l(C2 x Ε , x sumx x At) s2 ^ 0.93 x C2 x VmB/(KmB + 0.93 x C2)
8. 根据权利要求 7所述的用多波长吸收单通道同步测量多种酶活性的方法， 其特征 在于： 采用三波长吸收单通道同步测量三种酶活性：
步骤 a中， 可用如下原则选择显色底物组合： 所用显色底物 A、显色底物 B及显色底 物 C使显色产物 A的最大等吸收波长 Y1与显色产物 B差吸收峰最大波长 X2相等或相距 不超过 5nm、 显色产物 B最大等吸收波长 Y2和显色产物 A次大等吸收波长 Ys及显色产 物 C差吸收峰最大波长 X3之间相等或任两者相距都不超过 5nm;
步骤 c中， 选择显色产物 A差吸收峰最大波长 XI为 λΐ后， 按如下原则选择另外两个 测量波长 λ2和 λ3 : 显色产物 Β与显色底物 Β差摩尔消光系数在显色产物 Α最大等吸收波 长 Y1处高于显色产物 B与显色底物 B在二者差吸收峰最大波长 X2处差摩尔消光系数的 30%时， 选择 Y1为测量波长 λ2， 否则， 用最靠近显色产物 Α最大等吸收波长 Y1的显色产 物 B差吸收峰波长， 或靠近显色产物 B差吸收峰且其它显色产物与显色显色底物差摩尔消 光系数都小于显色产物 B与显色底物 B差摩尔消光系数 20%的波长为 λ2测量显色产物 Β 吸收； 显色产物 C与显色底物 C差摩尔消光系数在显色产物 Α次大等吸收波长 Ys处或显 色产物 B与显色底物 B最大等吸收波长 Y2处应高于二者在其差吸收峰最大波长 X3处差 摩尔消光系数的 30%时，则选择 Y2或 Ys为为 λ3测量显色产物 Β吸收，否则用最靠近显色 产物 Β最大等吸收波长 Υ2或显色产物 Α次大等吸收波长 Ys或靠近显色产物 C差吸收峰 最大波长且其它显色产物与显色底物差摩尔消光系数都小于显色产物 C与显色底物 C差摩 尔消光系数 20%的波长为 λ3测量显色产物 C吸收；
步骤 e中需测定显色产物 A吸收干扰校正系数 R31和 R32;测定时仅用酶 A的全部显 色底物而不用其它待测酶的任何显色底物， 使反应体系无来自显色底物 B及显色产物 B、 显色底物 C及显色产物 C在 λ1、 λ2和 λ3的吸收， 同步测定反应通道中三个波长下的吸收 变化， 直到在 λ2及 λ3吸收变化都大于 0.005或在 λΐ吸收变化大于 0.500; 以在 λΐ下吸收变 化为横坐标， 在 λ2下和在 λ3下吸收变化为纵座标进行回归分析， 所得在 λ2下吸收变化回归 直线斜率为校正系数 R31 , 在 λ3下吸收变化回归直线斜率为校正系数 R32;
步骤 e中需测定显色产物 B吸收干扰校正系数 R33和 R34 ;测定时仅用酶 B的全部显 色底物而不用其它待测酶的任何显色底物， 使反应体系无来自显色底物 A及显色产物 A、 显色底物 C及显色产物 C在 λ1、 λ2和 λ3的吸收， 同步测定反应通道中三个波长下的吸收 变化， 直到在 λ2及 λ3吸收变化都大于 0.005或在 λΐ吸收变化大于 0.500; 以在 λ2下吸收变 化为横坐标， 在 λΐ下和在 λ3下吸收变化为纵座标进行回归分析， 所得在 λΐ下吸收变化回 归直线斜率为校正系数 R33, 在 λ3下吸收变化回归直线斜率为校正系数 R34;
步骤 e中需测定显色产物 C吸收干扰校正系数 R35和 R36;测定时仅用酶 C的全部显 色底物而不用其它待测酶的任何显色底物， 使反应体系无来自显色底物 A及显色产物 A、 显色底物 B及显色产物 B在 λ1、 λ2和 λ3的吸收， 同步测定反应通道中三个波长下的吸收 变化， 直到在 λ2及 λΐ吸收变化都大于 0.005或在 λ3吸收变化大于 0.500; 以在 λ3下吸收变 化为横坐标， 在 λΐ下和在 λ2下吸收变化为纵座标进行回归分析， 所得在 λΐ下吸收变化回 归直线斜率为校正系数 R35, 在 λ2下吸收变化回归直线斜率为校正系数 R36。
9. 根据权利要求 7或 8所述的用多波长吸收单通道同步测量多种酶活性的方法， 其特 征在于：
步骤 a中， 与酶 A对应显色底物 A包括 4-硝基 -1-萘基磷酸酯且其水解两个等吸收波长 分别在 315到 335nm之间及 395到 415 nm之间、 4-硝基 -1-萘基硫酸酯且其水解两个等吸 收波长分别在 315到 335nm之间及 395到 415 nm之间、 4-硝基 -1-萘基乙酸酯且其水解时 两个等吸收波长分别在 310到 330nm之间及 375 到 395 nm之间、 4-硝基 -1-萘基 -D-半乳糖 苷且其水解时两个等吸收波长分别在 320 到 345nm之间及 390到 410nm之间、 4-硝基 -1-(N- 赖氨酰)萘胺且其水解的最大等吸收波长分别在 385 到 410 nm之间， 这些显色底物 A被酶 A作用生成 4-硝基 -1-萘酚或 4-硝基 -1-萘胺或其衍生物作为显色产物 A;与此类显色底物 A 对应的显色底物 B主要是 4-硝基苯酚或 4-硝基苯胺衍生物，在相应的酶 B作用下生成显色 产物 B为 4-硝基苯酚或 4-硝基苯胺或 4-硝基苯基硫醇； 与此类显色底物 A和显色底物 B 对应的显色底物 C包括尼克酰胺腺嘌呤二核苷酸或尼克酰胺腺嘌呤二核苷酸磷酸；
与显色底物 A对应酶 A包括但不限于芳香酯酶、 磷酸酶、 硫酸酯酶、 γ_谷氨酰基转移 酶、 肽酶即天然氨基酸 α-羧基对应的酰胺酶和糖苷酶； 与显色底物 Β对应的酶 Β包括与已 选酶 Α不同但属于酶 A 的其它水解酶； 与显色底物 C对应的酶包括但不限于乳酸脱氢酶 LDH、 苹果酸脱氢酶 MDH、 偶联 LDH的谷丙转氨酶和偶联 MDH的谷草转氨酶。
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