CN108802026B - Method and device for automatically measuring peroxidase activity and ascorbic acid simultaneously - Google Patents
Method and device for automatically measuring peroxidase activity and ascorbic acid simultaneously Download PDFInfo
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- 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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/00584—Control arrangements for automatic analysers
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- Chemical Kinetics & Catalysis (AREA)
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- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a method and a device for simultaneously and automatically and rapidly measuring the activity concentration of peroxidase and the content of ascorbic acid in a biological sample, which belong to the technical field of biochemical analysis and quantification and are used for physiological and biochemical research and enzyme method application. The enzyme reagent and the ascorbic acid sample in the quantitative ring are respectively pushed by two current carriers to enter a reaction coil, the enzyme catalysis color development reaction and the ascorbic acid color fading reaction are sequentially carried out, finally, the ascorbic acid is quantified through the absorbance reduction value of the colored product by a flow-through photometric detector, after the quantitative ring is filled with the enzyme sample, a valve rotates to the original position, the current carriers push the enzyme sample to generate the enzyme catalysis color development reaction in the reaction coil, and the enzyme activity is quantified by utilizing the absorbance increment of the product; the measuring process and the result are processed by a computer, so that the automatic and synchronous sample collection, quantification and measurement are realized.
Description
Technical Field
The invention relates to a method for simultaneously and automatically measuring peroxidase activity and ascorbic acid and a measuring device for realizing the method, belonging to the technical field of biochemical analysis and quantification. The method is suitable for quickly determining the peroxidase activity and the ascorbic acid in the biological sample.
Background
Peroxidase (POD, EC 1.11.1.7) is a biological enzyme widely existing in plants and animals, can participate in a plurality of cell metabolic cycles, and is a key enzyme for eliminating toxicity of intracellular hydrogen peroxide. Currently, a representative Horseradish peroxidase (HRP) in POD is commercialized, widely used in the fields of enzyme-linked immunoassay, clinical examination, biological investigation, sewage treatment, organic synthesis, food analysis, and the like, and has become one of the commonly used biological reagents in green chemical reactions. In the above-mentioned applications of cell physiology, biochemistry and bio-enzyme, the most important index of POD is its enzyme activity, so accurate quantification of POD activity is particularly important. However, Ascorbic Acid (AsA) coexisting in the biological sample has a strong negative interference with all POD catalytic reaction systems, thereby causing a great error in the POD activity or the quantification of POD catalytic substrates. Then, in order to solve this problem, if other chemical reagents are not added, the method directly utilizes the interference mechanism of AsA on the reaction system for measuring POD activity in biological samples to quantify AsA, and in addition, the interference is quantified, so as to obtain an accurate POD activity value, which is a green, economic and innovative idea, and no relevant report is found at home and abroad at present.
The national standard gives methods for the determination of peroxidase activity (GB/T32131-2015). The method has the determination principle that horseradish peroxidase can rapidly catalyze hydrogen peroxide and guaiacol to generate reddish brown tetramethyl catechol, and the enzyme activity of the horseradish peroxidase is calculated through the change of absorbance values at 436 nm. The disadvantages of this method are: 1) in the measurement principle, recent research at present proves that the chromogenic product of the hydrogen peroxide/peroxidase/guaiacol reaction system is guaiacol dimer instead of tetramethyl catechol, and the product has characteristic absorption at 420nm and 470nm, and the detection at 436nm inevitably reduces the quantitative sensitivity of peroxidase; 2) in the detection process, the interference of reducing substances such as ascorbic acid and the like in the sample on the enzyme activity quantification is not considered; 3) all detection processes are conventional manual operations, so that the time consumption is long, the operation is complicated, and the samples are wasted. The methods for determination of ascorbic acid in food (GB 5009.86-2016) given in the national standard are mainly high performance liquid chromatography, fluorescence method, 2, 6-dichloroindophenol titration method. Among them, the high performance liquid chromatography is a method for quantifying ascorbic acid by using the characteristic absorption of the ascorbic acid at 245nm after separating a sample, and the method does not need an additional reagent, but has the following disadvantages: expensive instruments and slow analysis speed; the fluorescence method needs to oxidize ascorbic acid into dehydroascorbic acid by activated carbon and then react with o-phenylenediamine to generate fluorescent quine oh, and has the following disadvantages: the manual operation is complicated and the instrument is expensive; the titration method is based on the principle that 2, 6-dichloroindophenol, a blue basic dye, is used for carrying out redox titration on an ascorbic acid sample acid leachate, 2, 6-dichloroindophenol is reduced to be colorless, when the titration end point is reached, redundant 2, 6-dichloroindophenol shows light red in an acid medium, and the content of ascorbic acid in a sample is calculated according to the consumption of 2, 6-dichloroindophenol.
The Flow-injection analysis (FIA) technology is an important hinge for connecting a manual analysis instrument and an analysis instrument, all manual analyses can be rapidly and automatically quantified by using the FIA, and the FIA technology is the technology which has the advantages of minimum artificial error, best reproducibility (RSD < 0.5%), obvious sample and reagent (mL grade) saving, and environmental friendliness and environmental protection. At present, the technology is being applied to the fields of biochemical analysis, chemical production, environmental monitoring, food safety and the like. A POD catalytic reaction system and an AsA interference mechanism are introduced into an FIA system, so that the POD activity and the AsA content in a biological sample can be analyzed automatically, quickly and accurately at the same time.
Disclosure of Invention
The invention aims to develop a method for simultaneously and automatically and rapidly determining the peroxidase activity concentration and the ascorbic acid content in a biological sample and a device for realizing the method, so as to solve the main problems in the prior art, simplify the operation process, save the reagent consumption, improve the economy, facilitate the environmental protection, accelerate the analysis speed and the automation level, and improve the precision and the accuracy.
The technical scheme of the invention is composed of a measuring method and a measuring device.
The determination principle of the invention is as follows: the sample containing peroxidase catalyzes guaiacol and hydrogen peroxide to carry out redox reaction to generate an amber product (namely guaiacol dimer) with characteristic absorption at 420nm and 470nm, and the activity of the peroxidase can be indirectly quantified by utilizing the absorbance increase value of the colored product; the ascorbic acid coexisting in the biological sample can generate a fading reaction with the colored product, and the ascorbic acid can be quantified by utilizing the absorbance reduction value of the colored product.
The determination process of the invention comprises the following steps: when the multifunctional valve is in an ascorbic acid sample 'filling' state, the three-way valve is communicated with the enzyme reagent, under the power action of the pump, the enzyme reagent passes through the enzyme reagent/enzyme sample pump pipe, the enzyme reagent/enzyme sample flow path and the enzyme reagent quantitative ring to finish the quantitative filling of the enzyme reagent, and redundant reagent flows out from the first waste discharge port; the ascorbic acid sample passes through the ascorbic acid sample pump tube, the ascorbic acid sample flow path and the ascorbic acid sample quantitative ring to finish the quantitative filling of the ascorbic acid sample, and the redundant sample flows out from the second waste discharge port; simultaneously, one path of carrier flow passes through a first carrier fluid pump pipe, a first carrier fluid flow path, an enzyme sample quantitative ring, an enzyme reagent/enzyme sample fluid outflow pipe, is converged with a mixed color developing agent passing through a mixed color developing agent pump pipe and a mixed color developing agent flow path in a first tee joint, then jointly passes through a first reaction coil pipe and a first connecting pipe of a constant temperature system, is converged with the other path of carrier flow passing through a second carrier fluid pump pipe, a second carrier fluid flow path, a delay coil pipe in a multifunctional combined block and a second connecting pipe in a second tee joint, finally passes through a second reaction coil pipe and a flow type photometric detector together, and flows out from a fifth waste discharge port, and the baseline absorbance value given out by the computer is recorded; then, the multifunctional valve rotates clockwise to an ascorbic acid sample injection state/enzyme sample filling state, and one path of carrier fluid passes through the first carrier fluid pump pipe and the first carrier fluid flow path, the enzyme reagent in the enzyme reagent quantitative ring is pushed into the first tee joint through the enzyme reagent/enzyme sample fluid flow pipe; the mixed color developing agent enters a first tee joint through a mixed color developing agent pump pipe and a mixed color developing agent flow path, an enzyme reagent plug is converged with the mixed color developing agent and then enters a first reaction coil pipe to generate color developing reaction, and a generated colored product belt enters a second tee joint through a first connecting pipe; meanwhile, the other path of carrier fluid pushes the ascorbic acid sample in the ascorbic acid sample quantitative ring through a second carrier fluid pump pipe and a second carrier fluid flow path, passes through a delay coil pipe and a second connecting pipe, enters a second tee joint, and after a colored product belt is converged with an ascorbic acid sample plug, a fading reaction is carried out in a second reaction coil pipe, and finally, the colored product belt enters a flow type photometric detector to give a product absorbance measured value, an actual measurement curve is recorded by a computer, a measured result is output according to a stored linear equation, and finally the colored product belt flows out from a third waste discharge port; meanwhile, the three-way valve is communicated with an enzyme sample, the enzyme sample passes through the enzyme reagent/enzyme sample pump pipe, the enzyme reagent/enzyme sample flow path and the enzyme sample quantifying ring to finish the quantitative filling of the enzyme sample, and redundant samples flow out from the fourth waste discharge port; when the multifunctional valve rotates anticlockwise to an ascorbic acid sample filling state/an enzyme sample injecting state, carrier fluid is pushed into a first tee joint through an enzyme reagent/enzyme sample fluid outflow pipe through a first carrier fluid pump pipe and a first carrier fluid flow path, an enzyme sample in an enzyme sample quantitative ring is converged with a mixed color developing agent through a mixed color developing agent pump pipe and the mixed color developing agent flow path, the converged mixture sequentially enters a first reaction coil pipe and a second reaction coil pipe to generate color development reaction, a generated colored product belt enters a flow type photometric detector to measure the absorbance value of a product, an actually measured curve is recorded and output by a computer to measure the result, and finally the result flows out from a third waste discharge port; in the measuring process, the computer automatically controls the state switching of the multifunctional valve, the rotation of the pump and the operation of the constant temperature system.
The measuring device comprises a pump, a mixed color reagent pump pipe, a mixed color reagent flow path, an enzyme reagent/enzyme sample pump pipe, an enzyme reagent/sample flow path, a first carrier fluid pump pipe, a first carrier fluid flow path, a second carrier fluid pump pipe, a second carrier fluid flow path, an ascorbic acid sample pump pipe, an ascorbic acid sample flow path, a mixed color reagent, an enzyme sample, an enzyme reagent, a carrier fluid, an ascorbic acid sample, a three-way valve, an enzyme reagent quantitative ring, an enzyme sample quantitative ring, a first waste discharge port, an ascorbic acid sample quantitative ring, a second waste discharge port, a third waste discharge port, a fourth waste discharge port, a multifunctional valve, an enzyme reagent/enzyme sample flow outlet pipe, a first three-way valve, a first reaction coil pipe, a constant temperature system, a delay coil pipe, a first connecting pipe, a second three-way valve, a second reaction coil pipe, a multifunctional combination block, a flow-through type photometric detector, a first three-way valve, a second, A fifth waste discharge port and a computer.
In the device, a mixed color reagent pump pipe is connected with a tee joint in a constant temperature system through a mixed color reagent flow path; the enzyme reagent/enzyme sample pump pipe is connected with the enzyme sample and the enzyme reagent through a three-way valve and is connected with an enzyme reagent quantitative ring of a multifunctional valve through an enzyme reagent/sample flow path; the first carrier fluid pump pipe is connected with the enzyme sample quantitative ring of the multifunctional valve through a first carrier fluid flow path, and the outlet of the first carrier fluid pump pipe is connected with a tee joint in the constant temperature system through an enzyme reagent/enzyme sample fluid outflow pipe; the second carrier fluid pump pipe is connected with the multifunctional valve through a second carrier fluid flow path, and a corresponding outlet of the second carrier fluid pump pipe is connected with the delay coil pipe; the ascorbic acid sample pump tube is connected with the ascorbic acid sample quantitative ring of the multifunctional valve through the ascorbic acid sample flow path; the outlets of the reaction coil and the delay coil in the constant temperature system are respectively connected with the tee joint of the second reaction coil in the multifunctional combined block through a first connecting pipe and a second connecting pipe; the outlet of the second reaction coil is connected with a flow-through photometric detector; the measured result is processed by a computer, and the rotation of the valve and the pump and the operation of the thermostat are controlled.
In the measuring apparatus of the present invention, the structural features of the respective components are as follows: the enzymatic chromogenic product hyperchromic reaction and the colored product extinction reaction in the same reaction system are utilized to simultaneously quantify the activity concentration of the peroxidase and the content of the ascorbic acid.
When the content of the ascorbic acid is measured, a sample is combined in a second tee joint (29) in a 'sample plug' form with a 'colored product band' generated by the reaction of the 'enzyme reagent plug' in the first reaction coil (24), and then enters a second reaction coil (30) for further extinction reaction; meanwhile, the enzyme sample quantitative ring (15) is in a sample filling state and is prepared for the next enzyme activity measurement, and the simultaneous measurement of two indexes is realized.
Ascorbic acid was added to the enzyme reagent in a series of different concentrations, and the mixed sample was introduced into an enzyme reagent/enzyme sample quantitative loop to measure the enzyme activity therein, whereby the influence coefficient of ascorbic acid on the measurement of the enzyme activity was obtained.
In the extracted enzyme extract, an ascorbic acid sample is obtained in one step by adding a protein denaturant so as to eliminate interference of enzyme coexisting in the sample on ascorbic acid quantification.
The volume of the ascorbic acid sample quantifying ring (12) is larger than that of the enzyme reagent quantifying ring (14), and the length of the first connecting pipe (27) is larger than that of the second connecting pipe (28), so that a 'sample plug' of the injection system reaches the second tee joint (29) first, and a 'ascorbic acid sample plug' completely wraps a 'colored product strip' mode is formed.
The invention has the advantages and positive effects that: according to the method, an additional reaction reagent is not needed, and only a single reaction system is utilized, so that the automatic simultaneous determination of the activity concentration of the peroxidase and the content of the ascorbic acid in the biological sample is realized; meanwhile, the influence of the ascorbic acid on the catalytic reaction of the peroxidase can be quantified, and the influence coefficient is corrected, so that the accurate value of the activity of the peroxidase can be obtained. The quantification, injection, enzyme catalysis reaction, ascorbic acid extinction reaction, temperature control, product determination and flow path cleaning of the reagent and the sample are automatically carried out; the method designed by the invention is simple and convenient to operate, low in reagent consumption, high in analysis speed and high in precision. Therefore, the invention is a new method for simultaneously measuring two indexes in a biological sample and a full-automatic rapid analysis device. In addition, the invention combines various subjects such as biochemical analysis, instrument analysis automation and the like, integrates chemical, optical, mechanical, electrical and computer, and has great practical innovation and social value.
Drawings
FIG. 1 is a schematic view of an apparatus for simultaneously and automatically measuring peroxidase activity and ascorbic acid
In the figure, (1) peristaltic pump, (2) mixed developer pump tube, (2a) mixed developer flow path, (3) enzyme reagent/enzyme sample pump tube, (3a) enzyme reagent/sample flow path, (4) first carrier liquid pump tube, (4a) first carrier liquid flow path, (5) second carrier liquid pump tube, (5a) second carrier liquid flow path, (6) ascorbic acid sample pump tube, (6a) ascorbic acid sample flow path, (7) mixed developer, (8) enzyme sample, (9) enzyme reagent, (10) carrier liquid, (11) carrier liquid, (12) ascorbic acid sample, (13) three-way valve, (14) enzyme reagent quantitative loop, (15) enzyme sample quantitative loop, (16) first waste discharge port, (17) ascorbic acid sample quantitative loop, (18) second waste discharge port, (19) third waste discharge port, (20) fourth waste discharge port, (21) multi-function valve, (22) An enzyme reagent/enzyme sample liquid outflow pipe, (23) a first tee joint, (24) a first reaction coil pipe, (25) a constant temperature system, (26) a delay coil pipe, (27) a first connecting pipe, (28) a second connecting pipe, (29) a second tee joint, (30) a second reaction coil pipe, (31) a multifunctional combined block, (32) a flow-type photometric detector, (33) a fifth waste discharge port, and (34) a computer.
FIG. 2 standard graphs of peroxidase and ascorbic acid in example 1.
FIG. 3 actual recorded graphs of peroxidase and ascorbic acid in example 1.
FIG. 4 is a graph of the sensitive effect of total reaction coil length on peroxidase activity assay in example 2.
FIG. 5 graph of the effect of ring volume on ascorbic acid assay sensitivity in ascorbic acid samples of example 3.
FIG. 6 is a graph of the effect of the second reaction coil length on ascorbic acid assay sensitivity.
FIG. 7 is a graph of the sensitive effect of guaiacol concentration in mixed color developers on peroxidase activity assays.
FIG. 8 is a graph of the sensitive effect of hydrogen peroxide concentration in mixed colour reagent on peroxidase activity assay in example 6.
FIG. 9 is a graph showing the effect of ascorbic acid concentration on peroxidase activity assay in the enzyme samples of example 7.
Detailed Description
Embodiments of the invention are further described with reference to the accompanying drawings:
example 1
The peroxidase activity and the ascorbic acid content in the plant samples were determined simultaneously using the method and apparatus of the invention (see FIG. 1). The experimental conditions are as follows: mixing 25mmol/L guaiacol and 5mmol/L hydrogen peroxide as color developing agent (7), 1000U/L horseradish peroxidase as enzyme reagent (9), and 0.05mol/L phosphate buffer (pH6.0) as carrier fluid (10) (11); the volumes of the enzyme reagent quantitative ring (14) and the enzyme sample quantitative ring (15) are both 20 mu L; the volume of the ascorbic acid sample quantification ring (17) is 150 mu L; the first reaction coil pipe (24) is a polytetrafluoroethylene pipe with the length of 400cm and the inner diameter of 0.8 mm; the delay coil pipe (26) is a polytetrafluoroethylene pipe with the length of 190cm and the inner diameter of 0.8 mm; the second reaction coil (30) is a polytetrafluoroethylene tube with the length of 25cm and the inner diameter of 0.8 mm; the multifunctional combined block (31) is made of organic glass material; the flow-through photometric detector (32) is a visible spectrophotometer.
Under the above conditions, the obtained standard curves (linear range: 20-1100U/L peroxidase, 5-50 mg/L ascorbic acid) of peroxidase and ascorbic acid are shown in FIG. 2, and the test curves and the system reproducibility test thereof are shown in FIG. 3. The peroxidase activity concentration and the ascorbic acid content in the actual plant samples of 6 were measured, and the results are shown in table 1. The result of the comparison experiment shows that the measuring result of the method is consistent with that of the conventional method, but the operation is simpler, more convenient and quicker.
Example 2
In this example, the effect of total reaction coil length in peroxidase activity measurement was examined using horseradish peroxidase as a sample. The results in FIG. 4 show that when the total reaction coil (first reaction coil + second reaction coil) length is less than 400cm, the response value obtained for the injected enzyme sample increases linearly with increasing reaction coil length; when the length thereof is more than 400cm, the response value of the enzyme sample deviates from the linearly increasing tendency.
Example 3
This example examines the effect of its quantitative loop volume on sensitivity with different concentrations of ascorbic acid standard solutions, respectively. As can be seen from fig. 5, when the ascorbic acid quantification ring volume is less than 150 μ L, the ascorbic acid response value gradually increases as the quantification ring volume increases, and then when the quantification ring volume is more than 150 μ L, the ascorbic acid response value does not change any more.
Example 4
This example examines the sensitive effect of the second reaction coil length on ascorbic acid determination. As can be seen from FIG. 6, when the ascorbic acid concentration is in the range of 20-80 mg/L and the second reaction coil is changed in the range of 5-25 cm, the ascorbic acid response value increases with the increase of the second reaction coil, and when the length of the second reaction coil is greater than 25cm, the ascorbic acid response value begins to gradually decrease.
Example 5
This example examined the effect of guaiacol concentration in the mixed color reagent on the sensitivity of the peroxidase activity assay. As can be seen from FIG. 7, there is a linear correlation between the guaiacol concentration and the peroxidase response value when the guaiacol concentration is 0.5 to 10 mmol/L; when the guaiacol concentration is respectively more than 10mmol/L, the increasing trend of the response value of the peroxidase becomes slow until the response value is stable.
Example 6
This example examined the effect of hydrogen peroxide concentration in the mixed developer on the sensitivity of peroxidase activity assays. As can be seen from FIG. 8, H2O2When the concentration is in the range of 0.5-1.5 mmol/L, the response value of the peroxidase linearly increases along with the increase of the hydrogen peroxide concentration, and then the response value is increasedThe trend becomes gentle until stable.
Example 7
This example examined the effect of ascorbic acid concentration in an enzyme sample on peroxidase activity measurement by adding a series of ascorbic acid standard solutions of different concentrations to a horseradish peroxidase solution and injecting it as a mixed enzyme sample into the system. As can be seen from FIG. 9, as the ascorbic acid concentration in the mixed enzyme sample increases, the absorbance of the chromogenic product for quantifying the peroxidase activity concentration gradually decreases, and under strictly controlled conditions in a flow injection system, the response value shows a negative good linear correlation with the ascorbic acid concentration, and the slope thereof is not affected by the peroxidase activity concentration, and the influence coefficient of the ascorbic acid concentration on the determination of the peroxidase activity (i.e., the slope of the influence curve) is calculated to be 0.0024L/mg.
Claims (7)
1. A method for simultaneously measuring the activity concentration of peroxidase and the content of ascorbic acid is characterized in that: when the multifunctional valve (21) is in an ascorbic acid sample filling state, the three-way valve (13) is communicated with the enzyme reagent (9), under the power action of the pump (1), the enzyme reagent (9) passes through the enzyme reagent/enzyme sample pump tube (3), the enzyme reagent/enzyme sample flow path (3a) and the enzyme reagent quantitative ring (14) to complete the quantitative filling of the enzyme reagent, and the redundant enzyme reagent flows out from the first waste discharge port (16); the ascorbic acid sample (12) passes through the ascorbic acid sample pump tube (6), the ascorbic acid sample flow path (6a) and the ascorbic acid sample quantitative ring (17) to complete the quantitative filling of the ascorbic acid sample, and the redundant ascorbic acid sample flows out from the second waste discharge port (18); meanwhile, a first carrier fluid (10) flows through a first carrier fluid pump tube (4), a first carrier fluid flow path (4a), an enzyme sample quantitative ring (15) and an enzyme reagent/enzyme sample liquid outflow tube (22), after being merged with the mixed developer (7) passing through the mixed developer pump tube (2) and the mixed developer flow path (2a) by a first tee (23), the first reaction coil (24) and the first connecting pipe (27) of the constant temperature system (25) are jointly passed through, then are merged with a second carrier fluid (11) which passes through a second carrier fluid pump pipe (5), a second carrier fluid flow path (5a), a delay coil (26) in a multifunctional combined block (31) and a second connecting pipe (28) in a second tee joint (29), finally pass through a second reaction coil (30) and a flow-type photometric detector (32) together and flow out from a fifth waste discharge port (33), and a baseline absorbance value given by the detector is recorded by a computer (34); subsequently, the multifunctional valve (21) is rotated clockwise to the ascorbic acid sample injection/enzyme sample filling state, and the first carrier fluid (10) is pushed into the first tee (23) through the first carrier fluid pump tube (4), the first carrier fluid flow path (4a), the enzyme reagent in the enzyme reagent quantifying ring (14), and through the enzyme reagent/enzyme sample flow-out tube (22); the mixed color developing agent (7) enters a first tee joint (23) through a mixed color developing agent pump pipe (2) and a mixed color developing agent flow path (2a), an enzyme reagent plug is converged with the mixed color developing agent (7) and then enters a first reaction coil pipe (24) for color developing reaction, and a generated colored product belt enters a second tee joint (29) through a first connecting pipe (27); meanwhile, a second carrier fluid (11) passes through a second carrier fluid pump pipe (5) and a second carrier fluid flow path (5a), an ascorbic acid sample in an ascorbic acid sample quantitative ring (17) is pushed, passes through a delay coil pipe (26) and a second connecting pipe (28) and enters a second tee joint (29), after a colored product tape is converged with an ascorbic acid sample plug, a fading reaction occurs in a second reaction coil pipe (30), and finally, the colored product tape enters a flow-type photometric detector (32) to give a product absorbance measured value, a computer (34) records an actually measured curve, a measured result is output according to a stored linear equation, and finally, the measured result flows out from a fifth waste discharge port (33); meanwhile, the three-way valve (13) is communicated with an enzyme sample (8), the enzyme sample (8) passes through the enzyme reagent/enzyme sample pump pipe (3), the enzyme reagent/enzyme sample flow path (3a) and the enzyme sample quantitative ring (15) to finish the quantitative filling of the enzyme sample, and redundant samples flow out from the fourth waste discharge port (20); when the multifunctional valve (21) rotates anticlockwise to an ascorbic acid sample filling/enzyme sample injection state, a carrier fluid (10) is pushed into a first tee joint (23) through a first carrier fluid pump pipe (4) and a first carrier fluid flow path (4a) and an enzyme sample in an enzyme sample quantification ring (15) through an enzyme reagent/enzyme sample liquid outflow pipe (22), an enzyme sample plug is converged with a mixed color developing agent passing through a mixed color developing agent pump pipe (2) and a mixed color developing agent flow path (2a), then sequentially enters a first reaction coil pipe (24) and a second reaction coil pipe (30) to generate a color development reaction, a generated colored product is brought into an inflow general photometric detector (32) to measure the absorbance value of the product, an actual measurement curve is recorded and a measurement result is output by a computer (34), and finally, the colored product is discharged from a fifth waste discharge port (33); during the measurement, the computer (34) automatically controls the state switching of the multifunctional valve (21), the rotation of the pump (1) and the operation of the thermostatic system (25).
2. An automatic analyzer for simultaneously measuring the activity concentration of peroxidase and the content of ascorbic acid, characterized in that: comprises a pump (1), a mixed color reagent pump tube (2), a mixed color reagent flow path (2a), an enzyme reagent/enzyme sample pump tube (3), an enzyme reagent/enzyme sample flow path (3a), a first carrier fluid pump tube (4), a first carrier fluid flow path (4a), a second carrier fluid pump tube (5), a second carrier fluid flow path (5a), an ascorbic acid sample pump tube (6), an ascorbic acid sample flow path (6a), a mixed color reagent (7), an enzyme sample (8), an enzyme reagent (9), a first carrier fluid (10), a second carrier fluid (11), an ascorbic acid sample (12), a three-way valve (13), an enzyme reagent quantitative ring (14), an enzyme sample quantitative ring (15), a first waste discharge port (16), an ascorbic acid sample quantitative ring (17), a second waste discharge port (18), a third waste discharge port (19), a fourth waste discharge port (20), The device comprises a multifunctional valve (21), an enzyme reagent/enzyme sample liquid outflow pipe (22), a first tee joint (23), a first reaction coil pipe (24), a constant temperature system (25), a delay coil pipe (26), a first connecting pipe (27), a second connecting pipe (28), a second tee joint (29), a second reaction coil pipe (30), a multifunctional combination block (31), a flow-through photometric detector (32), a fifth waste discharge port (33) and a computer (34); the mixed color developing agent pump pipe (2) is connected with a first tee joint (23) in a constant temperature system (25) through a mixed color developing agent flow path (2 a); the enzyme reagent/enzyme sample pump tube (3) is connected with an enzyme sample (8) and an enzyme reagent (9) through a three-way valve (13) and is connected with an enzyme reagent quantitative ring (14) of a multifunctional valve (21) through an enzyme reagent/enzyme sample flow path (3 a); the first carrier fluid pump pipe (4) is connected with an enzyme sample quantitative ring (15) of the multifunctional valve (21) through a first carrier fluid flow path (4a), and the outlet of the enzyme sample quantitative ring (15) is connected with an enzyme reagent/enzyme sample fluid outflow pipe (22) and a first tee joint (23) in a constant temperature system (25); the second carrier fluid pump pipe (5) is connected with the multifunctional valve (21) through a second carrier fluid flow path (5a), and the outlet of the second carrier fluid flow path (5a) is connected with the delay coil pipe (26) through the multifunctional valve (21); the ascorbic acid sample pump tube (6) is connected with the ascorbic acid sample quantitative ring (17) of the multifunctional valve (21) through an ascorbic acid sample flow path (6 a); the outlets of a first reaction coil (24) and a delay coil (26) in the constant temperature system (25) are respectively connected with a second tee joint (29) of a second reaction coil (30) in a multifunctional combination block (31) through a first connecting pipe (27) and a second connecting pipe (28); the outlet of the second reaction coil (30) is connected with a flow-through photometric detector (32); the computer (34) processes the measured result and controls the state switching of the multifunctional valve (21), the rotation of the pump (1) and the operation of the constant temperature system (25).
3. The method for simultaneously measuring the activity concentration of peroxidase and the content of ascorbic acid according to claim 1, wherein: the enzymatic chromogenic product hyperchromic reaction and the colored product extinction reaction in the same reaction system are utilized to simultaneously quantify the activity concentration of the peroxidase and the content of the ascorbic acid.
4. The method for simultaneously measuring the activity concentration of peroxidase and the content of ascorbic acid according to claim 1, wherein: when the content of the ascorbic acid is measured, the ascorbic acid sample is combined in a second tee joint (29) in the form of an ascorbic acid sample plug and a colored product band generated by the reaction of an enzyme reagent plug in the first reaction coil (24), and then enters a second reaction coil (30) for further extinction reaction; meanwhile, the enzyme sample quantitative ring (15) is in a sample filling state and is prepared for the next enzyme activity measurement, and the simultaneous measurement of two indexes is realized.
5. The method for simultaneously measuring the activity concentration of peroxidase and the content of ascorbic acid according to claim 1, wherein: a series of ascorbic acid with different concentrations are added into the enzyme reagent, the mixed solution of the enzyme reagent and the ascorbic acid is introduced into an enzyme reagent/enzyme sample quantitative ring, and the enzyme activity is measured, so that the influence coefficient of the ascorbic acid on the enzyme activity measurement can be obtained.
6. The method for simultaneously measuring the activity concentration of peroxidase and the content of ascorbic acid according to claim 1, wherein: adding a protein denaturant into the extracted enzyme sample solution to obtain an ascorbic acid sample so as to eliminate interference of enzyme coexisting in the enzyme sample on ascorbic acid quantification.
7. The automatic analyzer for simultaneously measuring the activity concentration of peroxidase and the content of ascorbic acid according to claim 2, wherein: the volume of the ascorbic acid sample quantifying ring (17) is larger than that of the enzyme reagent quantifying ring (14), and the length of the first connecting pipe (27) is larger than that of the second connecting pipe (28), so that the sample plug injected into the system reaches the second tee joint (29) first, and a mode that the ascorbic acid sample plug completely wraps the colored product strip is formed.
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| JPH04204054A (en) * | 1990-11-30 | 1992-07-24 | Hitachi Ltd | Analysis device of catechol amine metabolic object and creatinine |
| EP0860695B1 (en) * | 1995-10-30 | 2006-01-04 | Arkray, Inc. | Method for measuring an analyte and corresponding device |
| CA2249227A1 (en) * | 1997-01-20 | 1998-07-23 | Kyowa Medex Co., Ltd. | Methods and reagents for quantitatively determining ascorbic acid |
| AU2008362976B2 (en) * | 2008-10-17 | 2013-03-28 | Actherm Inc. | Liquid test strip and the method |
| CN201518030U (en) * | 2009-10-14 | 2010-06-30 | 北京吉天仪器有限公司 | Full automatic multi-channel multi-parameter simultaneous measuring flow injection analyzer |
| CN101813704A (en) * | 2010-04-08 | 2010-08-25 | 四川大学 | Automatic and quick measurement method and device of high-concentration silicate |
| CN102072930A (en) * | 2010-11-11 | 2011-05-25 | 四川大学 | Flow injection serially connected microelectrode electrochemical automatic method and device for simultaneous measurement of various electrolytes in blood sample |
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