阅读说明：本技术 一种碱性磷酸酶的检测方法 (Detection method of alkaline phosphatase ) 是由 杨华林 周玉 马丽圆 张兴平 于 2021-09-27 设计创作，主要内容包括：本发明公开一种碱性磷酸酶的检测方法,属于检测碱性磷酸酶的技术领域。该碱性磷酸酶的检测方法,包括以下步骤：S1、将N-甲基卟啉二丙酸IX、G-四聚体和4-硝基苯基磷酸酯六水合物添加至缓冲液中,之后继续加入待测液得到待测体系；S2、检测所述待测体系的荧光发射光谱并获取在610nm处的荧光发射峰；S3、根据所述荧光发射峰结合碱性磷酸酶的浓度与荧光发射峰的标准方程获取所述待测液中碱性磷酸酶的浓度。本发明提出的检测方法灵敏度高且检测速度快。(The invention discloses a detection method of alkaline phosphatase, belonging to the technical field of detection of alkaline phosphatase. The detection method of alkaline phosphatase comprises the following steps: s1, adding N-methyl porphyrin dipropionic acid IX, a G-tetramer and 4-nitrophenyl phosphate hexahydrate into a buffer solution, and then continuously adding a solution to be detected to obtain a system to be detected; s2, detecting the fluorescence emission spectrum of the system to be detected and acquiring a fluorescence emission peak at 610 nm; and S3, obtaining the concentration of the alkaline phosphatase in the liquid to be detected according to the standard equation of the concentration of the fluorescence emission peak combined with the alkaline phosphatase and the fluorescence emission peak. The detection method provided by the invention has high sensitivity and high detection speed.)

1. A method for detecting alkaline phosphatase, comprising the steps of:

s1, adding N-methyl porphyrin dipropionic acid IX, a G-tetramer and 4-nitrophenyl phosphate hexahydrate into a buffer solution, and then continuously adding a solution to be detected to obtain a system to be detected;

s2, detecting the fluorescence emission spectrum of the system to be detected and acquiring a fluorescence emission peak at 610 nm;

and S3, obtaining the concentration of the alkaline phosphatase in the liquid to be detected according to the standard equation of the concentration of the fluorescence emission peak combined with the alkaline phosphatase and the fluorescence emission peak.

2. The method for detecting alkaline phosphatase according to claim 1, wherein in step S3, the standard equation is Y-62375.4834X +2028450, where X represents the concentration of alkaline phosphatase and Y represents a fluorescence emission peak.

3. The method for detecting alkaline phosphatase according to claim 1, wherein the standard equation is obtained by the following steps in step S3:

adding alkaline phosphatase solutions with different known concentrations into a buffer solution containing N-methylporphyrin dipropionate IX, G-tetramer and 4-nitrophenyl phosphate hexahydrate disodium salt to react to obtain a mixture; detecting the fluorescence emission spectrum of the mixture and recording the fluorescence emission peak at 610nm, and establishing the standard equation according to the relation between the concentration of the alkaline phosphatase solution and the corresponding fluorescence emission peak.

4. The method for detecting alkaline phosphatase according to claim 1, wherein the sequence of the G-tetramer is 5'-GTGGGTCATTGTGGGTGGGTGTGG-3' in step S1.

5. The method for detecting alkaline phosphatase according to claim 1, wherein the concentration of the G-tetramer in the buffer solution is 500nM or more in step S1.

6. The method for detecting alkaline phosphatase according to claim 1, wherein the concentration of 4-nitrophenylphosphate hexahydrate in the buffer is 500 μ M or more in step S1.

7. The method for detecting alkaline phosphatase according to claim 1, wherein the pH of the buffer solution is 8 to 8.5 in step S1.

8. The method for detecting alkaline phosphatase according to claim 3, wherein the concentrations of the alkaline phosphatase solutions having different concentrations are 0U/L, 2.5U/L, 5U/L, 10U/L, 15U/L, 20U/L, 25U/L, 30U/L, 40U/L, and 60U/L, respectively.

9. The method for detecting alkaline phosphatase according to claim 1, wherein the solution to be detected is added and mixed for 30 to 40 minutes in step S1, and then the reaction is continued at 36 to 37 ℃ to obtain the solution to be detected.

10. The method for detecting alkaline phosphatase according to claim 9, wherein the reaction is continued at 36 to 37 ℃ for 30 to 40 minutes to obtain the test solution.

Technical Field

The invention relates to the technical field of alkaline phosphatase detection, in particular to a detection method of alkaline phosphatase.

Background

Alkaline phosphatase (ALP) is widely found in human liver, bone, intestine, kidney, placenta and other tissues. Since it plays an important role in signal transduction, cell growth and apoptosis, it is often used as a marker for specific diseases such as biliary obstruction, liver dysfunction and renal osteodystrophy. Therefore, detection of alkaline phosphatase levels is clinically significant.

Many methods for determining alkaline phosphatase have been developed, including electrochemical, colorimetric and fluorescent methods. Among them, the fluorescence method is attracting attention because of its high sensitivity. Some fluorescent materials, such as nanoclusters, organic fluorophores, quantum dots, carbon dots, and the like, have been successfully used to monitor alkaline phosphatase. However, most of these methods have the limitations of expensive reagents, long time, complicated synthetic route, etc. Therefore, it is necessary to develop a method for detecting alkaline phosphatase with high sensitivity and high detection speed.

Disclosure of Invention

The invention aims to overcome the technical defects and provide a method for detecting alkaline phosphatase, which solves the technical problems of low sensitivity and low detection speed of detecting alkaline phosphatase in the prior art.

In order to achieve the above technical object, the present invention provides a method for detecting alkaline phosphatase, comprising the steps of:

s1, adding N-methyl porphyrin dipropionic acid IX, a G-tetramer and 4-nitrophenyl phosphate hexahydrate into a buffer solution, and then continuously adding a solution to be detected to obtain a system to be detected;

s2, detecting the fluorescence emission spectrum of the system to be detected and acquiring a fluorescence emission peak at 610 nm;

and S3, obtaining the concentration of the alkaline phosphatase in the liquid to be detected according to the standard equation of the concentration of the fluorescence emission peak combined with the alkaline phosphatase and the fluorescence emission peak.

Further, in step S3, the standard equation is Y ═ 62375.4834X +2028450, where X denotes the concentration of alkaline phosphatase and Y denotes the fluorescence emission peak.

Further, in step S3, the standard equation is obtained by:

adding alkaline phosphatase solutions with different known concentrations into a buffer solution containing N-methylporphyrin dipropionate IX, G-tetramer and 4-nitrophenyl phosphate hexahydrate disodium salt to react to obtain a mixture; detecting the fluorescence emission spectrum of the mixture and recording the fluorescence emission peak at 610nm, and establishing the standard equation according to the relation between the concentration of the alkaline phosphatase solution and the corresponding fluorescence emission peak.

Further, in step S1, the sequence of the G-tetramer is 5'-GTGGGTCATTGTGGGTGGGTGTGG-3'.

Further, in step S1, the concentration of the G-tetramer in the buffer is 500nM or more.

Further, in step S1, the concentration of the 4-nitrophenyl phosphate hexahydrate in the buffer is 500 μ M or more.

Further, in step S1, the pH of the buffer is 8-8.5.

Further, the concentration of the alkaline phosphatase solution with different concentration is 0U/L, 2.5U/L, 5U/L, 10U/L, 15U/L, 20U/L, 25U/L, 30U/L, 40U/L and 60U/L respectively.

Further, in step S1, the solution to be tested is added and mixed for 30-40 minutes, and then the reaction is continued at 36-37 ℃ to obtain the solution to be tested.

Further, the reaction is continued for 30 to 40 minutes at the temperature of 36 to 37 ℃ to obtain the solution to be detected.

Compared with the prior art, the invention has the beneficial effects that: in the absence of alkaline phosphatase, the complex formed between the G-tetramer and N-methylporphyrindipropionic acid IX in the buffer fluoresced strongly (400nm excitation, 610nm emission). When alkaline phosphatase exists, 4-nitrophenyl phosphate hexahydrate in buffer solution is hydrolyzed into p-nitrophenol, then the p-nitrophenol is excited by 400nm fluorescence, and the energy of the p-nitrophenol is absorbed, so that a G-tetramer/N-methylporphyrin dipropionate IX complex cannot be excited, the fluorescence is reduced, a fluorescence emission spectrum of a system to be detected is detected, a fluorescence emission peak at 610nm is obtained, the concentration of the alkaline phosphatase in the liquid to be detected can be obtained by combining a standard equation of the concentration of the alkaline phosphatase and the fluorescence emission peak, and the method is high in sensitivity and high in detection speed.

Drawings

FIG. 1A is a graph of the UV-VIS absorption spectra of PNPP and PNP of the present invention.

FIG. 1B shows fluorescence excitation spectra of G4/NMM complex of the present invention.

FIG. 2 is a schematic diagram of the detection of alkaline phosphatase according to the present invention based on G4/NMM and the effect on PNP internal filtration.

FIG. 3 is a fluorescence spectrum under different conditions in example 1 of the present invention.

FIG. 4A is a graph showing the results of fluorescence intensity at G4/NMM at 1610 nm in example of the present invention.

FIG. 4B is a graph showing the effect of different amounts of PNPP on the fluorescence intensity of G4/NMM complex in example 1.

FIG. 4C is a graph showing the effect of pH on the fluorescence intensity of the G4/NMM complex in the buffer of example 1 of the present invention.

FIG. 5A is a graph of the fluorescence spectrum of the response of ALP at different concentrations in example 1 of the present invention.

FIG. 5B is a graph showing the results of fluorescence signals at 610nm for different concentrations of ALP in example 1 of the present invention, wherein the inset is a linear relationship of the fluorescence signals in the range of 2.5-25U/L.

FIG. 6 is a graph showing the results of selective analysis according to the method of example 1 of the present invention.

Detailed Description

The present embodiment provides a method for detecting alkaline phosphatase (i.e., ALP), comprising the steps of:

s1, adding N-methyl porphyrin dipropionic acid IX (namely NMM), G-tetramer (namely G4) and 4-nitrophenyl phosphate hexahydrate (namely PNPP) into a buffer solution, then continuously adding the solution to be detected, mixing for 30-40 minutes, and then continuously reacting for 30-40 minutes at 36-37 ℃ to obtain a system to be detected; the sequence of the G-tetramer is 5'-GTGGGTCATTGTGGGTGGGTGTGG-3'; the concentration of the G-tetramer in the buffer solution is more than 500 nM; the concentration of the 4-nitrophenyl phosphate hexahydrate in the buffer is above 500 [ mu ] M; the pH of the buffer solution is 8-8.5.

S2, detecting the fluorescence emission spectrum of the system to be detected and acquiring a fluorescence emission peak at 610 nm;

s3, obtaining the concentration of alkaline phosphatase in the liquid to be detected according to the fluorescence emission peak and the standard equation of the concentration of the alkaline phosphatase and the fluorescence emission peak; the standard equation is Y-62375.4834X +2028450, wherein X represents the concentration of alkaline phosphatase and Y represents the fluorescence emission peak; the standard equation is obtained by the following steps:

adding alkaline phosphatase solutions with different known concentrations into a buffer solution containing N-methylporphyrin dipropionate IX, G-tetramer and 4-nitrophenyl phosphate hexahydrate disodium salt to react to obtain a mixture; detecting the fluorescence emission spectrum of the mixture and recording the fluorescence emission peak at 610nm, and establishing the standard equation according to the relation between the concentration of the alkaline phosphatase solution and the corresponding fluorescence emission peak; the concentrations of the alkaline phosphatase solutions with different concentrations are 0U/L, 2.5U/L, 5U/L, 10U/L, 15U/L, 20U/L, 25U/L, 30U/L, 40U/L and 60U/L respectively.

The invention principle is as follows:

referring to FIG. 2, wherein G4/NMM represents the G-tetramer/N-methylporphyrin dipropionate IX complex, Alkaline phosphatase is Alkaline phosphatase, PNPP is 4-nitrophenylphosphate hexahydrate disodium salt, and PNP is p-nitrophenol. PNPP is a substrate of ALP, and by hydrolysis of ALP, p-nitrophenol (i.e., PNP) can be generated as a product. As shown in FIG. 1A, PNPP has an absorption peak at 310nm, and the maximum absorption peak shifts to 400nm when PNP is generated. As shown in FIG. 1B, the maximum excitation wavelength of G4/NMM is exactly 400 nm. Thus, the PNP produced can quench the fluorescence of G4/NMM by the internal filtering effect.

The maximum absorption peak of PNP and the maximum excitation peak of G4/NMM were found to be close to each other by the absorption spectrum of PNP and the excitation spectrum of G4/NMM. By this phenomenon, we have devised a new fluorescent method for detecting ALP.

The detection principle is as follows: in the absence of ALP, G4/NMM fluoresced strongly (400nm excitation, 610nm emission). When ALP is added, PNPP is hydrolyzed to PNP, which is then excited by 400nm fluorescence, and the energy is absorbed by PNP, so that G4/NMM is not excited, resulting in fluorescence decrease. In this process, since the role of ALP is a catalyst, an ultra-sensitive ALP detection method can be established.

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Example 1

The embodiment comprises the following steps: scanning ultraviolet visible absorption spectra of PNPP and PNP and fluorescence excitation spectra of G4/NMM by using a microplate reader, and researching the feasibility of the method; the feasibility of the method is researched by utilizing a microplate reader; optimizing the G4 concentration, the PNPP concentration and the pH value of the buffer solution; the sensitivity and linear detection range of the method; selectivity of the process; and (5) detecting a real sample.

(1) Feasibility of the research method

To evaluate the feasibility of our design, first fluorescence spectrum scans were performed on G4/NMM (sequence G4: 5'-GTGGGTCATTGTGGGTGGGTGTGG-3'), G4+ NMM + PNPP, G4+ NMM + PNP, G4+ NMM + PNPP + ALP, respectively, and we prepared four groups of samples, 500nM G4+500nM NMM, respectively; 500nM G4+500nM NMM + 500. mu.M PNPP; 500nM G4+500nM NMM +500 μ M PNP; 500nM G4+500nM NMM +500 μ M PNPP +100U/L ALP; the above samples were reacted in a buffer solution (50mM HEPES, 0.5M NaCl) of pH 8 for 30min, and 200. mu.L of each sample was added to a 96-well microplate, and the change in the fluorescence signal was recorded by a microplate reader.

The results are shown in FIG. 3: the G4/NMM complex showed strong fluorescence at 610 nm. When PNPP is added, fluorescence is not affected. However, the addition of PNP can significantly suppress fluorescence. When ALP was added to the PNPP and G4/NMM mixture, fluorescence quenching was also clearly observed due to the hydrolysis of PNPP to PNP. Thus, these results indicate that the proposed method can be used to detect alkaline phosphatase.

(2) Optimization of reaction conditions

In order to achieve the best analytical performance of the alkaline phosphatase detection method, several experimental parameters involved in this experiment were optimized:

since the fluorescence intensity of NMM will increase with increasing G-tetramer concentration, the concentration of NMM was fixed at 500nM, optimizing the G-tetramer concentration. 100nM, 200nM, 300nM, 400nM, 500nM, 600nM, 700nM, 800nM G-tetramer and 500nM NMM were added to pH 8 buffer (50mM HEPES, 0.5M NaCl) for 30min, 200. mu.L of each sample was added to a 96-well microplate, and the change in fluorescence signal was recorded with a microplate reader. The results are shown in FIG. 4A, where the G-quadruplex concentration increased in the range of 100-800nM and the fluorescence intensity plateaued at 500nM, indicating that NMM is completely bound by G4. Therefore, the 500nM G-quadruplex concentration was chosen as the optimal concentration.

Since in the experiment, enough reaction of the substrate PNPP and ALP is ensured, and the principle of saving experimental materials is followed, the influence of the concentration of PNPP on the fluorescence quenching effect is researched. When the concentration of ALP was fixed at 100U/L, PNPP at a concentration of 100-800. mu.M was added to a buffer solution (50mM HEPES, 0.5M NaCl) containing 500nM G4 and 500nM NMM, respectively, for reaction for 30 minutes, 200. mu.L of each sample was added to a 96-well microplate, and the change in fluorescence signal was recorded with a microplate reader. As shown in FIG. 4B, when the concentration reached 500. mu.M, the fluorescence did not decrease further. Therefore, an excessive amount of PNPP cannot lower the fluorescence intensity any more. Therefore, PNPP was selected as the optimum concentration at a concentration of 500. mu.M.

Finally, the buffer pH was optimized in consideration of its influence on the fluorescence quenching effect, and as shown in fig. 4C, when the pH was 8.0, the fluorescence quenching effect was the best, and therefore, pH 8.0 was selected for subsequent experiments.

(3) Sensitivity and Linear detection Range of the method

First, alkaline phosphatase was added to a buffer solution (50mM HEPES, 0.5M NaCl, pH 8) containing 500nM NMM, 500nM G4, 500. mu.M PNPP at various concentrations for 30 minutes, mixed well, and reacted at 37 ℃ for 30 minutes. Finally, the spectral change was recorded with a microplate reader. As shown in FIG. 5A, in the concentration range of 0-30U/L, the fluorescence intensity decreased with increasing ALP concentration, and then reached a plateau, and the fluorescence change was not significant with increasing ALP concentration. Subsequently, we analyzed the peak at 610nm, and as a result, as shown in FIG. 5B, the fluorescence intensity has a good linear correlation with the ALP concentration in the range of 2.5-25U/L. The calibration equation is-62375.4834X +2028450 (R)²0.995), X represents the concentration of alkaline phosphatase, and Y represents the fluorescence emission peak. The detection limit was 0.81U/L from the calibration curve based on 3 times blank error/slope.

(4) Selectivity of the process

By comparing the response of ALP with interfering substances (BSA, glycine, glucose, Na) possibly present in the serum⁺、K⁺、Ca²⁺、Fe³⁺、Mg²⁺) The selectivity of the biosensor we designed was investigated. Protein interference was studied with BAS (bovine serum albumin). As a result, as shown in FIG. 6, only ALP produced a large change in fluorescence, while other interfering substances were present at the background level, wherein F0 indicates the fluorescence intensity without ALP, F indicates the fluorescence intensity with ALP, and F0-F indicates the difference therebetween. These results indicate that the method has high selectivity for the detection of ALP in serum.

(5) Detection of alkaline phosphatase in bovine serum samples

To evaluate the applicability and reliability of the proposed detection method in real biological samples, the analytical performance of the ALP assay was performed in bovine serum. Prior to measurement, serum samples were treated with 10-fold dilutions to reduce matrix interference. Then, different concentrations of ALP were added to the serum samples using standard addition methods. Finally, the content of alkaline phosphatase was determined by the detection method, and the recovery rate was calculated. As shown in table 1, the recovery rate was between 90.46% and 98.90%, which demonstrates the reliability of the proposed detection method to detect ALP in biological samples.

TABLE 1 analysis of recovery of alkaline phosphatase

The above-described embodiments of the present invention should not be construed as limiting the scope of the present invention. Any other corresponding changes and modifications made according to the technical idea of the present invention should be included in the protection scope of the claims of the present invention.