CN109557060B - Based on NH2Method for visually detecting alkaline phosphatase activity in serum by using (E) -Cu-MOF - Google Patents
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Abstract
The invention discloses a method based on NH2Method for visually detecting alkaline phosphatase activity in serum by using-Cu-MOF, comprising the steps of firstly synthesizing NH2-Cu-MOF nanoenzyme material; reuse of NH2-preparing a gel detection tube for detecting alkaline phosphatase activity from Cu-MOF nanoenzyme and agarose; finally, after the alkaline phosphatase is added into the gel detection tube, placing the gel detection tube under an ultraviolet lamp, and realizing semi-quantitative detection by observing color change; then, a mobile phone or a digital camera is used for photographing, RGB values in the measured photograph are read through RGB color extractor software, and the oxidation degree of OPD is indicated by analyzing the RGB values, so that the field visual multicolor fluorescence quantitative detection of the activity of alkaline phosphatase in serum is realized; during detection, a working curve of the change of the gel color along with the activity of the alkaline phosphatase is drawn, and then the actual serum sample is detected. The method can realize the on-site visual multicolor fluorescence determination of the alkaline phosphatase in the serum without expensive instruments and equipment, participation of biological enzymes and operation of professionals.
Description
Technical Field
The invention relates to a method for detecting alkaline phosphatase activity, in particular to a method based on NH2-visual detection of alkaline phosphatase activity in serum by Cu-MOF.
Background
Alkaline phosphatase (ALP) is a hydrolase enzyme commonly found in prokaryotic and eukaryotic cells that catalyzes the removal of phosphate groups from phosphate-containing polyphosphate substrates. In the human body, alkaline phosphatase is widely present in tissues (e.g., bone, kidney, liver, etc.) and plays an important role in cell signaling, cell division, differentiation, bone calcification, and many other normal cellular functions. On the other hand, down-regulation of alkaline phosphatase activity is closely related to various human diseases, including hypophosphatasia, primary biliary cirrhosis, diabetes, various cancers, and the like. Therefore, the development of a method for reliably and easily detecting alkaline phosphatase activity is of great research value for the diagnosis of clinical diseases and the study of the role of alkaline phosphatase in physiological and pathological processes.
In related patents that have disclosed detecting alkaline phosphatase at present, CN108375616A is a liquid crystal biosensor for detecting alkaline phosphatase and a method for preparing the same, wherein the liquid crystal biosensor requires the use of a polarizing microscope to obtain an optical response image; CN106248644A A method for detecting alkaline phosphatase based on carbon point fluorescence quenching-recovery requires a fluorescence spectrometer to obtain a fluorescence spectrum; CN107422014A modified electrode for detecting alkaline phosphatase, and its preparation method and detection method, need to utilize electrochemical workstation to obtain electrochemical signal to draw working curve. Therefore, the existing alkaline phosphatase activity detection method mainly realizes quantitative detection by means of analytical instruments such as a fluorescence spectrometer, an electrochemical workstation and the like. However, these methods require a high degree of expertise on the operator, and in particular, the instruments used are expensive and bulky, which is not suitable for widespread application and popularization.
In addition, the existing visual detection method of alkaline phosphatase activity, CN106066325A, is a method for detecting alkaline phosphatase, which utilizes the gradient color change of the reaction solution color changing from blue to colorless with the increase of the alkaline phosphatase concentration to realize the quantitative determination of alkaline phosphatase activity, however, the disadvantages of this method are single color change of concentration gradient color development and low resolution; also, CN106596532A, a simple and low-cost method for detecting alkaline phosphatase activity, has the same problems. The prior patent CN106596532A discloses a simple and low-cost method for detecting alkaline phosphatase activity, which uses hydrophilic paper to design a paper device, and the flow length of a test agent solution in the paper device is inversely proportional to the activity of alkaline phosphatase in a sample. However, the device needs biological enzymes such as saccharifying enzymes, the activity of the biological enzymes is influenced by a plurality of factors in the processes of production, transportation and use, and the accuracy and the precision of measuring and manually reading the flowing length of the solution by only using a ruler are still to be improved; CN107677804A A kit for detecting bone-derived alkaline phosphatase and its application method, the detection time of the kit is short, but the detection is still realized by a full-automatic biochemical analyzer. Therefore, the development of a visual, portable, color-change multiplex and high-resolution detection method for the alkaline phosphatase in the serum has important practical significance.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for preparing a catalyst based on NH2The method for visually detecting the activity of the alkaline phosphatase in the serum by using the Cu-MOF does not need expensive instruments and equipment, does not need the participation of biological enzymes, can judge the activity of the alkaline phosphatase by the fluorescence color change of a solution or a gel tube without the operation of a professional, and can realize the on-site visual multicolor fluorescence measurement of the alkaline phosphatase in the serum.
The technical scheme for realizing the purpose of the invention is as follows:
based on NH2-method for visual detection of alkaline phosphatase activity in serum of Cu-MOF comprising the steps of:
(1) synthesizing NH with certain morphological characteristics2-Cu-MOF nanoenzyme material;
(2) by means of NH2-preparing a gel detection tube for detecting alkaline phosphatase activity from Cu-MOF nanoenzyme and agarose;
(3) after the alkaline phosphatase is added into the gel detection tube, the strength of the yellow fluorescence intensity of the gel is in positive correlation with the concentration of the oxidation product 2, 3-Diaminophenazine (DAP) of o-phenylenediamine (OPD), and the NH is used2The Cu-MOF nanoenzyme has blue fluorescence, so that the gel detection tube is placed under an ultraviolet lamp to form ratio fluorescence with DAP, high-resolution color change is generated, and the color change is observed through observationThe color change is observed to realize semi-quantitative detection; then, a mobile phone or a digital camera is used for photographing, RGB values in the measured photograph are read through RGB color extractor software, and the oxidation degree of OPD is indicated by analyzing the RGB values, so that the field visual multicolor fluorescence quantitative detection of the activity of alkaline phosphatase in serum is realized; during detection, a working curve of the change of the gel color along with the activity of the alkaline phosphatase is drawn, and then the actual serum sample is detected.
Synthesis of NH in step (1)2-a Cu-MOF nanoenzyme material comprising the steps of:
(1.1) adding Cu (NO)3)2·3H2O dissolved in deionized water, Cu (NO)3)2·3H2The mass ratio of O to deionized water is 1: 10;
(1.2) mixing the DMF solution dissolved with the 2-amino terephthalic acid and ethanol, adding the mixture into the solution obtained in the step (1.1) after mixing, and then uniformly mixing;
the volume ratio of DMF to ethanol solvent is 1:1, Cu (NO)3)2·3H2The mass concentration ratio of the O to the 2-amino terephthalic acid is 2:1-3: 1;
(1.3) after fully and uniformly mixing, putting the solution into a Teflon-lined high-pressure kettle, and reacting at 80-90 ℃;
(1.4) finally, NH was collected by centrifugation2Washing the Cu-MOF with DMF, ethanol and deionized water in sequence, and naturally drying to synthesize NH2-Cu-MOF nanoenzyme material.
The preparation method of the gel detection tube in the step (2) comprises the following steps:
(2.1) firstly, dissolving agarose in boiling water to prepare an agarose gel solution, wherein the mass concentration of the agarose is 5g/L-15 g/L;
(2.2) reacting NH2Adding the solution of the-Cu-MOF nanoenzyme into the agarose gel solution, and uniformly mixing; NH (NH)2-the concentration of Cu-MOF nanoenzyme is 0.3-0.6 g/L;
(2.3) after mixing, taking the mixed solution with the volume of 0.050-0.250mL into a cover of a test tube, standing for 5 min, solidifying the gel, covering the cover, and storing at 4 ℃.
The detection in the step (3) is specifically as follows: during detection, firstly, drawing a working curve of the change of the gel color along with the change of the activity of alkaline phosphatase, adding alkaline phosphatase standard solutions with different activity sizes and 67 mu L of ppi solution into a gel test tube, vertically placing the gel test tube in a constant-temperature culture oscillator at 37 ℃ for incubation for 30 min, then adding 80 mu L of OPD solution, placing the gel test tube in an inverted state in the constant-temperature culture oscillator at 37 ℃ for incubation for 40 min, taking a picture under an ultraviolet lamp, reading RGB values of the solutions in the picture by using ColorSchemer Studio software, calculating R/(R + G + B) of a blank solution and a liquid to be detected, and taking a delta R/(R + G + B) value as a longitudinal coordinate to obtain a change curve of the delta R/(R + G + B) value of the gel color along with the change of the activity of the alkaline phosphatase, namely the working curve for detecting the activity of the alkaline phosphatase in serum;
secondly, detecting the actual sample: sucking 60 mu L of serum and 67 mu L of ppi solution, adding into a gel test tube, vertically placing in a constant temperature culture oscillator at 37 ℃ for incubation for 30 min, then adding 80 mu L of OPD solution, inversely placing in a constant temperature culture oscillator at 37 ℃ for incubation for 40 min, and taking a picture under an ultraviolet lamp to obtain color change; and reading the RGB value of the solution in the picture by using ColorSchemer Studio software, and substituting the RGB value into the working curve to obtain the activity value of the alkaline phosphatase in the serum.
The method of the invention comprises the steps of firstly synthesizing NH with certain morphological characteristics2The catalytic activity of the nano enzyme is inhibited by PPi, and then the inhibition of PPi is released by the hydrolysis of PPi by alkaline phosphatase, so that NH is recovered2Catalytic oxidation of OPD by Cu-MOF, binding of NH2The self fluorescent signal of the Cu-MOF ensures that the mixed solution presents different degrees of ratio fluorescence under the irradiation of ultraviolet light after alkaline phosphatase solutions with different activity sizes are added, thereby realizing the quantitative detection of the alkaline phosphatase activity.
In addition, agarose gel binding, using NH2Cu-MOF material prepared a portable gel detection tube for detecting alkaline phosphatase in serum. Because the intensity of yellow fluorescence of the alkaline phosphatase solution is in positive correlation with the concentration of the oxidation product DAP of OPD, professional color matching can be combined by a mobile phone or a digital cameraSoftware or RGB color extractor software analyzes the RGB values in the measured photograph to indicate the degree of oxidation of o-phenylenediamine, thereby enabling quantitative multicolor fluorescence detection of alkaline phosphatase activity.
The invention is based on NH2Method for multicolor visual detection of alkaline phosphatase in serum by using-Cu-MOF nanoenzyme2-Cu-MOF material, which is an oxidase-like mimic enzyme, a fluorescence donor and a function of a cascade reaction participant, respectively. The principle of multicolor fluorescence detection is based on that hydrolysis of PPi by alkaline phosphatase can remove NH by PPi2Coordination of Cu in Cu-MOF to recover NH2The catalytic oxidation effect of the Cu-MOF on the OPD is combined with the autofluorescence of the material, so that alkaline phosphatase solutions with different activities show different-degree ratio fluorescence signals under the irradiation of ultraviolet light, the color change of the system is richer, and the system is easy to distinguish by naked eyes. The method does not need expensive instruments and equipment, does not need biological enzyme to participate, and can realize semi-quantitative analysis by naked eyes; quantitative analysis can be realized by using a portable mobile phone with a photographing function as an analysis tool. According to the invention, the activity of the alkaline phosphatase in the serum can be analyzed through the color change of the gel tube under the irradiation of the ultraviolet lamp without the operation of a professional, and the field visual multicolor fluorescence quantitative detection of the alkaline phosphatase in the serum can be realized by reading RGB values through mobile phone software.
Detailed Description
The present invention will be further described with reference to the following examples, but the present invention is not limited thereto.
Examples
Based on NH2-a method for multicolor visualization of Cu-MOF nanoenzymes for detection of alkaline phosphatase activity in serum comprising the steps of:
(1) synthesizing NH with certain morphological characteristics2-Cu-MOF nanoenzyme material;
1g of Cu (NO)3)2·3H2Dissolving O in 10mL of deionized water; mixing 10mL of DMF (dimethyl formamide) and 10mL of ethanol in which 0.4 g of 2-amino terephthalic acid is dissolved, adding the mixture into the solution, and uniformly mixing; is sufficientAfter uniform mixing, putting the solution into a Teflon-lined high-pressure kettle, and reacting for 24 hours at 80 ℃; finally, the NH was collected by centrifugation2Cu-MOF, and washing with DMF, ethanol and deionized water in sequence, and naturally drying.
(2) By means of NH2-preparing a gel detection tube for detecting alkaline phosphatase activity from Cu-MOF nanoenzyme and agarose;
adding 300 μ L of Tris-HCl buffer solution with pH 7.2 and 100 μ L of NH2-an ethanolic solution of Cu-MOF (2.5 g/L) was added to 1 mL of agarose gel, the temperature of the gel water bath was controlled at 40 ℃; after mixing, 200 muL of the mixed solution is taken out and placed in a cover of a test tube, after 5 min, the gel is solidified, the cover is covered, and the gel is stored at 4 ℃ and is colorless and transparent.
(3) Placing the gel detection tube under an ultraviolet lamp, taking a picture by a mobile phone, reading RGB values in the measured picture through RGB color extractor software, and analyzing the RGB values to indicate the oxidation degree of OPD, thereby realizing the field visual multicolor fluorescence quantitative detection of the activity of alkaline phosphatase in serum.
During detection, firstly, a working curve of the change of the gel color along with the activity of the alkaline phosphatase is drawn:
different activity levels of alkaline phosphatase standard solutions (0U/L, 5U/L, 10U/L, 30U/L, 60U/L, 90U/L, 120U/L), 67. mu.L ppi (30 mmol L)-1) The solution was added to a gel test tube, incubated immediately in a 37 ℃ incubator shaker for 30 min, and then 80. mu.LOPD (0.4mmol L) was added-1) The solution is placed in a 37 ℃ Zhicheng constant temperature culture oscillator in an inverted mode for incubation for 40 min, a picture is taken under an ultraviolet lamp, RGB values of the solution in the picture are read by ColorSchemer Studio software, R/(R + G + B) of a blank solution and a solution to be detected are calculated, and a delta R/(R + G + B) value is taken as a vertical coordinate, so that a change curve of the delta R/(R + G + B) value of the gel color along with the change of the activity of alkaline phosphatase, namely a working curve for detecting the activity of the alkaline phosphatase in serum can be obtained, wherein the color change is shown as the following table I:
TABLE 1 is based on NH2-data of multicolor visual detection of alkaline phosphatase activity in standard solution of Cu-MOF nanoenzyme
Secondly, detecting the actual sample: aspirate 60. mu.L of serum, 67. mu.L of ppi (30 mmol L)-1) Adding the solution into a gel test tube, immediately incubating in 37 deg.C Zhicheng constant temperature culture shaker for 30 min, and adding 80 μ L OPD (0.4mmol L)-1) Standing upside down, incubating in a 37 deg.C Zhicheng constant temperature culture oscillator for 40 min, and taking a picture under an ultraviolet lamp to obtain color change; the RGB value of the solution in the picture is read by using ColorSchemer Studio software and is substituted into the working curve to obtain the activity value of the alkaline phosphatase in the serum, and the activity value is shown in the table II:
TABLE II is based on NH2-multicolor visualization data of alkaline phosphatase activity in serum for Cu-MOF nanoenzyme
Claims (1)
1. NH-based non-disease diagnosis2-visual detection of alkaline phosphatase activity in serum by Cu-MOF, characterized in that it comprises the following steps:
(1) synthesis of NH2-a Cu-MOF nanoenzyme material comprising the steps of:
(1.1) first, Cu (NO)3)2·3H2O dissolved in deionized water, Cu (NO)3)2·3H2The mass ratio of O to deionized water is 1: 10;
(1.2) mixing the DMF solution dissolved with the 2-amino terephthalic acid and ethanol, adding the mixture into the solution obtained in the step (1.1), and uniformly mixing;
the volume ratio of DMF to ethanol solvent is 1:1, Cu (NO)3)2·3H2The mass concentration ratio of the O to the 2-amino terephthalic acid is 2:1-3: 1;
(1.3) after fully and uniformly mixing, putting the solution into a Teflon-lined high-pressure kettle, and reacting at 80-90 ℃;
(1.4) finally, NH was collected by centrifugation2Washing the Cu-MOF with DMF, ethanol and deionized water in sequence, and naturally drying to synthesize NH2-Cu-MOF nanoenzyme material;
(2) by means of NH2-preparing a gel detection tube for detecting alkaline phosphatase activity from Cu-MOF nanoenzyme and agarose, comprising the steps of:
(2.1) firstly, dissolving agarose in boiling water to prepare an agarose gel solution, wherein the mass concentration of the agarose is 5g/L-15 g/L;
(2.2) reacting NH2Adding the solution of the-Cu-MOF nanoenzyme into the agarose gel solution, and uniformly mixing, wherein the NH is2-the concentration of Cu-MOF nanoenzyme is 0.3-0.6 g/L;
(2.3) after uniformly mixing, putting the mixed solution with the volume of 0.050-0.250mL into a cover of a test tube, standing for 5 min, solidifying gel, covering the cover, and storing at 4 ℃;
(3) during detection, firstly, drawing a working curve of the change of the gel color along with the change of the activity of alkaline phosphatase, adding alkaline phosphatase standard solutions with different activity sizes and 67 mu L of ppi solution into a gel test tube, vertically placing the gel test tube in a constant-temperature culture oscillator at 37 ℃ for incubation for 30 min, then adding 80 mu L of OPD solution, placing the gel test tube in an inverted state in the constant-temperature culture oscillator at 37 ℃ for incubation for 40 min, taking a picture under an ultraviolet lamp, reading RGB values of the solutions in the picture by using ColorSchemer Studio software, calculating R/(R + G + B) of a blank solution and a liquid to be detected, and taking a delta R/(R + G + B) value as a longitudinal coordinate to obtain a change curve of the delta R/(R + G + B) value of the gel color along with the change of the activity of the alkaline phosphatase, namely the working curve for detecting the activity of the alkaline phosphatase in serum;
secondly, detecting the actual sample: sucking 60 mu L of serum and 67 mu L of ppi solution, adding into a gel test tube, vertically placing in a constant temperature culture oscillator at 37 ℃ for incubation for 30 min, then adding 80 mu L of OPD solution, inversely placing in a constant temperature culture oscillator at 37 ℃ for incubation for 40 min, and taking a picture under an ultraviolet lamp to obtain color change; and reading the RGB value of the solution in the picture by using ColorSchemer Studio software, and substituting the RGB value into the working curve to obtain the activity value of the alkaline phosphatase in the serum.
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