CN111220609A - Based on CeVO4Colorimetric detection method of alkaline phosphatase Activity - Google Patents
Based on CeVO4Colorimetric detection method of alkaline phosphatase Activity Download PDFInfo
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Abstract
The invention belongs to the technical field of analytical chemistry, and relates to a CeVO-based sample4Colorimetric detection of alkaline phosphatase activity of (1): alkaline phosphatase with different activities and 0.05mL of 1mM sodium hexametaphosphate (NaPO) were added to 5mL centrifuge tubes3)6(ii) a 0.1mL of 1mg/mL CeVO was added to the mixture in sequence4The suspension, 0.1mL of 5mM TMB and acetate buffer solution, wherein the final total volume of the solution is 3 mL; after the membrane is coated, an ultraviolet-visible absorption spectrophotometer is used for recording the absorbance at the wavelength of 652nm and drawing an ALP activity-absorbance standard working curve; calibrating; and (5) measuring the absorbance of the sample to be measured, and comparing to obtain the ALP activity. The invention has mild detection process conditions, low detection cost and simple operation; by TMB + CeVO4The system detects the activity of alkaline phosphatase, the detection limit is as low as 0.68U/L,the detectable range is as wide as 1-210U/L; using TMB + CeVO4The system detects the activity of alkaline phosphatase, has sensitive response to the alkaline phosphatase, has accurate and reliable measurement result of an actual sample, and realizes the detection of the activity of the alkaline phosphatase in human serum.
Description
Technical Field
The invention belongs to the technical field of analytical chemistry, relates to detection of alkaline phosphatase, and particularly relates to a method for detecting alkaline phosphatase based on CeVO4The colorimetric detection method of alkaline phosphatase activity of (1).
Background
Alkaline phosphatase (ALP) is a biological enzyme widely distributed in human liver, bone, intestine, kidney and placenta, which catalyzes dephosphorylation of a molecule of phosphate group from a multi-phosphorylated substance (e.g., nucleic acids, proteins and small molecules). The activity range of ALP is 40-190U/L. Numerous studies have shown that abnormal ALP activity is closely associated with a variety of diseases, including bone damage, liver dysfunction and prostate cancer. For example, ALP is an important indicator of osteoblast activity during early osteoblast differentiation, and its activity generally increases with increased bone activity. When its activity exceeds the upper limit, it indicates that active bone deposition may occur during this process. In addition, other bone diseases such as osteomalacia, lupus and bone cancer also cause an increase in ALP activity. In addition, patients with hepatobiliary disease often find that the ALP index is high, while severe chronic nephritis, hypothyroidism and anemia lead to low ALP activity. It is therefore widely used as a clinical biomarker for the diagnosis and treatment of these diseases. In addition, ALP is often used in various enzyme-linked immunosorbent assays (ELISA). Based on these broad demands, it is of great interest to develop an efficient, low-cost and easy-to-use ALP activity assay.
The existing alkaline phosphatase detection methods mainly comprise a fluorescence method, an electrochemical method, a colorimetric method and the like. For example:
chinese patent CN110501317A discloses a fluorescence detection method for detecting alkaline phosphatase, wherein 4-hydroxyphenyl sodium phosphate (4-HPP) is used as a substrate, in an aqueous solution, the substrate molecule is catalyzed and hydrolyzed by alkaline phosphatase to generate 1, 4-benzenediol (HQ), and the 1, 4-benzenediol can react with subsequently added 3- (2-aminoethylamino) propyl trimethoxy silane (DAMO) to generate silicon nanoparticles (SiNPs) emitting green fluorescence. The higher the alkaline phosphatase activity in the system, the more fluorescent silicon nanoparticles are produced, the higher the fluorescence intensity is, and the quantitative analysis and detection of the alkaline phosphatase activity is carried out according to the change of the fluorescence intensity of the detection solution.
Chinese patent CN108084995B discloses a CuInS/ZnS quantum dot and a method for detecting alkaline phosphatase by using the same. The CuInS/ZnS quantum dots are used as probes in a system for hydrolyzing 4-nitrophenyl disodium phosphate by alkaline phosphatase, and the content of the alkaline phosphatase is obtained through the change of fluorescence intensity and the linear relation between the alkaline phosphatase and the fluorescence intensity. The optimal excitation wavelength of the CuInS/ZnS quantum dot prepared by the method is 405nm, and the CuInS/ZnS quantum dot is overlapped with the maximum substrate absorption peak of PNP, so that an inner filter effect can be generated, and the aim of detecting ALP is fulfilled.
Chinese patent CN107422014A discloses a method for quantitatively detecting alkaline phosphatase, which comprises the steps of preparing a modified electrode, catalyzing a substrate of ascorbic acid phosphate ester by the alkaline phosphatase to remove phosphate to generate ascorbic acid with strong reducibility, reducing silver ions in a reaction solution into silver simple substances by the ascorbic acid and depositing the silver simple substances on the surface of the electrode, and finally realizing high-sensitivity detection on the alkaline phosphatase by using stripping voltammetry signals of silver deposited on the surface of the modified electrode.
Chinese patent CN107422014B discloses a modified electrode for detecting alkaline phosphatase, and a preparation method and a detection method thereof. The method comprises the steps of firstly preparing a modified electrode, then catalyzing a substrate ascorbic acid 2-phosphate ester of the modified electrode to remove phosphate groups by using alkaline phosphatase to generate ascorbic acid with strong reducibility, wherein the ascorbic acid can quickly reduce silver ions in a reaction solution into silver simple substances and deposit the silver simple substances on the surface of the electrode, and finally realizing high-sensitivity detection on the alkaline phosphatase by using stripping voltammetry signals of the silver deposited on the surface of the modified electrode. After the detection is finished, the modified electrode can be regenerated by a simple method so as to be reused.
Although the above-described disclosed alkaline phosphatase detection method has a certain detection efficiency, the following disadvantages and shortcomings still exist:
1) the detection method is relatively complex to operate;
2) some detection methods have strict requirements on environment, relatively high cost and high requirements on reagent storage conditions.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a CeVO-based food4The colorimetric detection method of alkaline phosphatase activity of (1).
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
based on CeVO4The colorimetric detection method for the activity of alkaline phosphatase, which comprises the following steps:
1) alkaline phosphatase with different activities and 0.05mL of 1mM sodium hexametaphosphate (NaPO) were added to 5mL centrifuge tubes3)6Reacting for 30-60 min, preferably 50 min; the final activity of the alkaline phosphatase in the system is 1U/L, 15U/L, 30U/L, 50U/L, 70U/L, 90U/L, 130U/L, 170U/L and 210U/L;
2) then 0.1mL of 1mg/mL CeVO is added to the mixed solution in turn4The suspension, 0.1mL of 5mM TMB and acetate buffer solution are mixed uniformly, the final total volume of the solution is determined to be 3mL, and the reaction is carried out for 1-30 min, preferably for 5 min;
3) after the mixed solution is subjected to film coating, measuring the ultraviolet absorption spectrum of the mixed solution by using an ultraviolet-visible absorption spectrophotometer, and recording the absorbance at the wavelength of 652 nm;
4) drawing a calibrated ALP activity-absorbance standard working curve by using the absorbance measurement value of the TMBox and the corresponding ALP activity;
5) and (3) repeating the steps 1) to 4) on the ALP sample to be detected, respectively measuring the absorbance at the wavelength of 652nm by using an ultraviolet-visible absorption spectrophotometer, and comparing the absorbance with a standard working curve through calculation to obtain the ALP activity.
In a preferred embodiment of the present invention, the pH of the acetate buffer solution in step 2) is 4, and the concentration is 0.2M.
In the preferred embodiment of the invention, the detectable activity range of the ALP sample to be detected in the step 5) is 1-210U/L, and the detection limit is as low as 0.68U/L.
The invention adopts CeVO4Is an oxidase-like mimic, and catalyzes the reaction between superoxide anion generated by oxygen and a color-developing agent (TMB) under acidic conditions to generate a color-developing product (TMBox), so that the whole system turns blue. Due to sodium hexametaphosphate [ (NaPO)3)6]The system has strong electronegativity, and after sodium hexametaphosphate is added into the system, positively charged TMBox and sodium hexametaphosphate can agglomerate to influence the color of the system. Alkaline phosphatase (ALP) can hydrolyze hexametaphosphateSodium produces phosphate, which has little effect on the above system. Therefore, the invention can realize the detection of the activity of the alkaline phosphatase by hydrolyzing sodium hexametaphosphate in the system with different activities in a certain time to cause the change of the color of the system. In the invention, the ALP can promote the color development of TMB, and the linear relation between the absorbance of the TMBox and the ALP activity is utilized to carry out operation processing, so that more stable and reliable detection data can be obtained, and the ALP activity in human serum can be determined.
In the present specification, the term "oxidase-like" refers to a material having an oxidase catalytic activity. Specifically, the oxidase-like enzyme of the present invention uses oxygen as an electron acceptor, and generates a colored substance by catalytic oxidation of a corresponding substrate, for colorimetric detection.
In this specification, the term "(NaPO)3)6"is the chemical formula of the compound sodium hexametaphosphate, which are used interchangeably.
In this specification, the term "TMB" is the abbreviated name of compound "3, 3 ', 5, 5' -tetramethylbenzidine", which are used interchangeably.
In this specification, the term "TMBox" is an abbreviated name for the oxidation product of the compound "3, 3 ', 5, 5' -tetramethylbenzidine", which are used interchangeably.
In the present specification, the term "ALP" refers to alkaline phosphatase, which are used interchangeably.
In this specification, the term "CeVO4"refers to the formula of cerium vanadate, which are used interchangeably.
The cerium vanadate (CeVO) provided by the invention4) Prepared according to the prior art, disclosed herein is a preparation method comprising the steps of:
1. ammonium metavanadate (NH) with the mass ratio of 1: 3-54VO3) With cerium nitrate hexahydrate [ Ce (NO)3)3·6H20]Dissolving the mixture in ultrapure water, and ultrasonically stirring for 0.5-1 h to form a yellow uniform solution, wherein the solid-to-liquid ratio of ammonium metavanadate, cerous nitrate hexahydrate and ultrapure water is 1: 3-5: 30.
2. transferring the solution into a reaction kettle, and carrying out hydrothermal reaction at 160-200 ℃ for 10-12 h;
3. naturally cooling, centrifuging at a rotating speed of 9000-11000 r/min for 5-6 min for separation, washing with ethanol, and vacuum drying at 35-45 ℃ for 12h to obtain the product.
Further, in the step 1, the solid-to-liquid ratio of the ammonium metavanadate, the cerium nitrate hexahydrate and the ultrapure water is preferably: 1: 4: 30mL, and the ultrasonic stirring time is preferably 1 h.
Further, the reaction temperature in step 2 is preferably 180 ℃, and the reaction time is preferably 12 h.
Further, the rotation speed of the centrifugal product in the step 3 is preferably 10000r/min, the centrifugal time is preferably 5min, and the vacuum drying temperature is preferably 40 ℃.
CeVO prepared by the method disclosed by the invention4The nano-particles are black solid nano-particles with the particle size of about 500nm as the quasi-oxide mimic enzyme.
The reactants and reagents used in the present invention are commercially available.
Advantageous effects
The invention utilizes TMB + CeVO4The system is used for colorimetric detection of ALP activity, the condition in the detection process is mild, other reagents are not needed, the ALP activity is conveniently and rapidly detected, the detection cost is low, and the operation is simple; by TMB + CeVO4The system detects the activity of alkaline phosphatase, the detection limit is as low as 0.68U/L, and the detection range is as wide as 1-210U/L; using TMB + CeVO4The system detects the activity of alkaline phosphatase, has sensitive response to the alkaline phosphatase, has accurate and reliable measurement result of an actual sample, and realizes the detection of the activity of the alkaline phosphatase in human serum.
Drawings
FIG. 1.TMB + CeVO4Ultraviolet-visible absorption spectrum when the system reacts for 5 min;
FIG. 2.CeVO4A peroxidase-like buffer pH optimization plot;
FIG. 3 is a schematic diagram of ALP activity detection;
FIG. 4 ALP Effect TMB + CeVO4+(NaPO3)6+(NaPO3)6Ultraviolet-visible absorption spectrum when the reaction system reacts for 5 min;
FIG. 5. Using TMB + CeVO4The detection effect of ALP activity is detected by the system (wherein, A is an ultraviolet-visible absorption spectrum when reacting for 5min, B is an absorbance fitting straight line at 652nm of an ultraviolet spectrum);
FIG. 6. Using TMB + CeVO4The system detects selectivity in ALP activity.
Detailed Description
The present invention will be described in detail below with reference to examples to enable those skilled in the art to better understand the present invention, but the present invention is not limited to the following examples.
Unless otherwise defined, terms (including technical and scientific terms) used herein should be construed to have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms used herein should be interpreted as having a meaning that is consistent with their meaning in the context of this specification and the relevant art, and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
Example 1
1. Ammonium metavanadate (NH) with the mass ratio of 1:44VO3) With cerium nitrate hexahydrate [ Ce (NO)3)3·6H20]Dissolving the mixture in ultrapure water, and ultrasonically stirring for 1h to form a yellow uniform solution A, wherein the solid-to-liquid ratio of ammonium metavanadate, cerous nitrate hexahydrate and ultrapure water is 1: 4: 30.
2. transferring the solution A into a reaction kettle, and carrying out hydrothermal reaction at 180 ℃ for 12 hours;
3. and (3) after the reaction product in the step (2) is naturally cooled, centrifuging at a rotating speed of 9000r/min for 5 minutes to separate the product, washing the product with ethanol, and drying in vacuum at 35 ℃ for 12 hours to obtain black solid nano particles with the particle size of about 500 nm.
CeVO4The application experiment of the reaction for catalyzing oxygen to oxidize TMB as the oxidase-like enzyme comprises the following steps:
1) 2.8mL of 0.2M acetate buffer (pH 4) were added to each of the buffer solutions, 0.1mL of 5mM TMB and 0.1mL of 1mg/mL CeVO4Dispersing the solution, mixing the above solutions uniformly, and controlling time to 0.5The liquid is colorless or light blue within 1 min;
2) reacting the system at room temperature for 5min, and changing the color of the liquid from colorless to blue along with the time;
3) and measuring the ultraviolet-visible absorption spectrum of the mixed solution by using an ultraviolet-visible absorption spectrophotometer.
FIG. 1 records the prepared CeVO4UV-Vis patterns of catalytic oxidation of TMB reactions in different systems. It can be found from the figure that only when TMB and CeVO are used4When the two exist together, the color reaction occurs; when TMB and CeVO4In the presence of only one of them, no color reaction occurs. The reaction is carried out by reacting TMB in CeVO4Is subjected to oxidation reduction under the catalysis of (2), and the prepared CeVO is proved4Has good oxidase activity.
Example 2
1. Mixing the components in a mass ratio of 1: 6 ammonium metavanadate (NH)4VO3) With cerium nitrate hexahydrate [ Ce (NO)3)3·6H20]Dissolving in ultrapure water, and ultrasonically stirring to form a yellow uniform solution A;
2. pouring the solution A obtained in the step 1 into a reaction kettle, and carrying out heating reaction for 6 hours at 120 ℃ in a blast type oven;
3. and (3) after the reaction product in the step (2) is naturally cooled, centrifuging at the rotating speed of 11000r/min for 10 minutes to separate the product, washing the product with ethanol, and drying in vacuum at 45 ℃ for 12 hours to obtain black solid nano particles with the particle size of about 500 nm.
CeVO4The application experiment of the reaction for catalyzing oxygen to oxidize TMB as the oxidase-like enzyme comprises the following steps:
1) respectively taking 2.8mL of acetate buffer solution or phosphate buffer solution with the pH values of 3, 4, 5, 6, 7, 8 and 9, and sequentially adding 0.1mL of 5mM TMB and 0.1mL of 1mg/mL CeVO4Dispersing the solution, and then uniformly mixing the solution, wherein the time is controlled to be 0.5-1 min, and the liquid is colorless or light blue;
2) reacting the system at room temperature for 5min, and changing the color of the liquid from colorless to blue along with the time;
3) and measuring the ultraviolet-visible absorption spectrum of the mixed solution by using an ultraviolet-visible absorption spectrophotometer.
As shown in FIG. 2, the absorbance value tended to increase and decrease with increasing pH, and was highest at pH 4, reaching a peak. When the pH is controlled to 4, the catalytic activity of the enzyme is best.
Example 3
ALP affects TMB + CeVO4+(NaPO3)6Reaction system
1) 2.8mL of 0.2M acetate buffer (pH 4) were added to each of the buffer solutions, 0.1mL of 5mM TMB and 0.1mL of 1mg/mL CeVO4Dispersing the solution, then uniformly mixing the solution for 0.5-1 min, reacting for 5min after uniformly mixing the solution and the colorless or light blue liquid, and measuring the absorbance of the mixed solution at 652nm by using an ultraviolet-visible absorption spectrophotometer;
2) 0.05mL of 1mM (NaPO)3)6Then, 2.75mL of 0.2M acetate buffer (pH 4) and 0.1mL of 1mg/mL CeVO were sequentially added4Mixing the suspension and 0.1mL of 5mM TMB, reacting for 5min, filtering, and measuring the absorbance of the mixed solution at 652nm by using an ultraviolet-visible absorption spectrophotometer;
3) 0.05mL of 1mM (NaPO)3)60.15mL of 400U/L alkaline phosphatase was added thereto, the reaction was carried out for 45min, and 2.6mL of 0.2M acetate buffer (pH 4) and 0.1mL of 1mg/mL CeVO were sequentially added to the mixture4Mixing the suspension and 0.1mL of 5mM TMB, reacting for 5min, filtering, and measuring the absorbance of the mixed solution at 652nm by using an ultraviolet-visible absorption spectrophotometer.
The results are shown in FIG. 4, where TMB + CeVO alone is shown in FIG. 44The reaction system has a larger absorption peak at 652nm, when (NaPO) is added into the mixture system3)6Then, the color changed to purple and almost colorless after filtration, and almost no absorption peak exists at 652 nm; ALP was added because of ALP hydrolysis (NaPO)3)6The degree of color change to violet decreases the bluish color after filtration, resulting in a weaker absorption peak at 652 nm.
Example 4
Using TMB + CeVO4Colorimetric detection of ALP activity in reaction system
1) 0.15mL of alkaline phosphatase with different activities and 0.05mL of 1mM (NaPO) were added to a 5mL centrifuge tube3)6Reacting for 50 min; the final activity of the alkaline phosphatase in the system is 1U/L, 15U/L, 30U/L, 50U/L, 70U/L, 90U/L, 130U/L, 170U/L and 210U/L;
2) then, 2.6mL of 0.2M acetate buffer (pH 4) and 0.1mL of 1mg/mL CeVO were sequentially added to the mixture4Suspending liquid and 0.1mL of 5mM TMB, determining the final total volume of the solution to be 3mL, and reacting for 5min after uniformly mixing the system;
3) and (3) after the mixed solution passes through a membrane, measuring the ultraviolet absorption spectrum of the mixed solution by using an ultraviolet-visible absorption spectrophotometer, recording the absorbance at the wavelength of 652nm and drawing an ALP activity-absorbance standard working curve taking TMB as a color developing agent.
The results of activity detection for ALP using the colorimetric method of the present invention are shown in FIG. 5A. Wherein, fig. 5A illustrates that the absorbance of the solution gradually increases as the activity of ALP increases; FIG. 5B is an absorbance fitting straight line at 652nm of the ultraviolet spectrum, which shows that the detectable range of ALP activity in the method is 1-210U/L, and the method has excellent detection effect.
Example 5
Using TMB + CeVO4Reaction system for detecting selectivity of ALP
0.05mL of 1mM (NaPO)3)6Adding 0.15mL of different amino acids or protease, reacting for 50min, and sequentially adding 2.6mL of 0.2M acetate buffer (pH 4) and 0.1mL of 1mg/mL CeVO4Suspending liquid and 0.1mL of 5mM TMB, then uniformly mixing the solutions, uniformly mixing the systems, reacting for 5min, filtering, and measuring the absorbance of the mixed solution at 652nm by using an ultraviolet-visible absorption spectrophotometer.
The results are shown in FIG. 7. FIG. 6 is a bar graph showing the selectivity of ALP in the TMB + CeVO4 reaction system, wherein the bar graph is blank, leucine (Leu), lysine (Lys), aspartic acid (Asp), glucose oxidase (GOx), threonine (Thr), casein (casein), alanine (Ala), urate oxidase (UOx), and Glutathione (GSH) in the order from left to right. It can be seen from the figure that only ALP can significantly promote the color reaction of TMB, and other amino acids or proteases can not influence the hydrolysis effect of ALP when coexisting, which indicates that the sensor can stably complete the colorimetric quantitative detection of ALP activity with high selectivity.
Example 6
TMB+CeVO4Reaction system for detecting ALP activity in serum
1) Respectively adding 0.15mL of alkaline phosphatase with different activities and 0.15mL of the treated serum sample into a 5mL centrifuge tube;
2) then, 2.6mL of 0.2M acetate buffer (pH 4) and 0.1L of 1mg/mL CeVO were sequentially added to the mixture4Suspending liquid and 0.1mL of 5mM TMB, determining the final total volume of the solution to be 3mL, and reacting for 5min after uniformly mixing the system;
3) and (3) measuring the ultraviolet absorption spectrum of the mixed solution by using an ultraviolet-visible absorption spectrophotometer after the mixed solution passes through a membrane, recording the absorbance at the position of 652nm of the wavelength, and substituting the absorbance at the position of 652nm of the ultraviolet spectrum to fit a straight line to convert the absorbance into ALP activity.
The measurement results are shown in the following table 1:
the method of the present invention and the comparison of the results of measured values in a certain hospital
As can be seen from the above table, TMB + CeVO4The system is sensitive to the change of ALP activity in an actual sample, and compared with an actual measurement value in a hospital, the result is similar.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present invention or directly or indirectly applied to other related technical fields are included in the scope of the present invention.
Claims (5)
1. Based onCeVO4The colorimetric detection method for the activity of alkaline phosphatase according to (1), comprising the steps of:
1) alkaline phosphatase with different activities and 0.05mL of 1mM sodium hexametaphosphate (NaPO) were added to 5mL centrifuge tubes3)6Reacting for 30-60 min, wherein the final activity of the alkaline phosphatase in the system is 1U/L, 15U/L, 30U/L, 50U/L, 70U/L, 90U/L, 130U/L, 170U/L and 210U/L;
2) 0.1mL of 1mg/mL CeVO was added to the mixture in sequence4The suspension, 0.1mL of 5mM TMB and acetate buffer solution are mixed uniformly, the final total volume of the solution is determined to be 3mL, and the reaction is carried out for 1-30 min;
3) after the mixed solution passes through a film, measuring the ultraviolet absorption spectrum of the mixed solution by using an ultraviolet-visible absorption spectrophotometer, recording the absorbance at the wavelength of 652nm and drawing an alkaline phosphatase activity-absorbance standard working curve taking TMB as a color developing agent;
4) drawing a calibrated alkaline phosphatase activity-absorbance standard working curve by using the absorbance measurement value of the TMBox and the corresponding alkaline phosphatase activity;
5) and (3) repeating the steps 1) to 4) on the alkaline phosphatase sample to be detected, respectively measuring the absorbance at the wavelength of 652nm by using an ultraviolet-visible absorption spectrophotometer, and comparing the absorbance with a standard working curve through calculation to obtain the activity of the alkaline phosphatase.
2. The CeVO-based system of claim 14The colorimetric detection method for activity of alkaline phosphatase according to (1), characterized in that: the alkaline phosphatase with different activities and 0.05mL of 1mM sodium hexametaphosphate (NaPO) were added to 5mL centrifuge tubes respectively as described in step 1)3)6And reacting for 50 min.
3. The CeVO-based system of claim 14The colorimetric detection method for activity of alkaline phosphatase according to (1), characterized in that: and (3) reacting for 5min after the uniform mixing system in the step 2).
4. According to claim 1Based on CeVO4The colorimetric detection method for activity of alkaline phosphatase according to (1), characterized in that: the pH of the acetate buffer solution in the step 2) is 4, and the concentration is 0.2M.
5. The CeVO-based system of claim 14The colorimetric detection method for activity of alkaline phosphatase according to (1), characterized in that: the detectable activity range of the alkaline phosphatase sample to be detected in the step 5) is 1-210U/L, and the detection limit is as low as 0.68U/L.
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