阅读说明：本技术 一种碱性磷酸酶活性的荧光检测方法 (A kind of fluorescence detection method of alkaline phosphatase activities ) 是由 孙健 杨秀荣 刘国永 赵佳会 卢沙沙 于 2019-08-27 设计创作，主要内容包括：本发明涉及一种碱性磷酸酶活性的荧光检测方法,将4-羟基苯基磷酸钠(4-HPP)作为底物,在水溶液中,该底物分子经碱性磷酸酶的催化水解生成1,4-苯二酚(HQ),1,4-苯二酚可以与后续加入的3-(2-氨基乙基氨基)丙基三甲氧基硅烷(DAMO)发生反应,生成绿色荧光发射的硅纳米粒子(SiNPs)。体系中的碱性磷酸酶活性越高,生成的荧光硅纳米粒子就越多,荧光强度就越大,根据检测溶液的荧光强度变化,进行碱性磷酸酶活性的定量分析检测。与现有技术相比,本发明所需试剂药品简单易得,操作方便,快速准确,为临床样品中碱性磷酸酶活性定量检测提供了一种灵敏可靠的方法。本发明的荧光检测方法,检出限为0.1mU/mL,具有高灵敏度,高选择性,宽线性范围等优点。(The present invention relates to a kind of fluorescence detection methods of alkaline phosphatase activities, it regard 4- hydroxy phenyl sodium phosphate (4-HPP) as substrate, in aqueous solution, the substrate molecule generates 1 through the catalyzing hydrolysis of alkaline phosphatase, 4- benzenediol (HQ), 1, 4-benzenediol can react with 3- (2- aminoethylamino) propyl trimethoxy silicane (DAMO) of subsequent addition, generate the silicon nano (SiNPs) of green-fluorescent emission.Alkaline phosphatase activities in system are higher, and the Fluorescent silicon nanoparticle of generation is more, and fluorescence intensity is bigger, according to the fluorescence intensity change of detection solution, carry out the quantitative analysis detection of alkaline phosphatase activities.Compared with prior art, reagent chemicals needed for the present invention are simple and easy to get, easy to operate, quick and precisely, provide a kind of sensitive reliable method for clinical sample activity change of Alkaline phosphatase quantitative detection.The advantages that fluorescence detection method of the invention, detection are limited to 0.1mU/mL, have high sensitivity, highly selective, the wide range of linearity.)

1. a kind of fluorescence detection method of alkaline phosphatase activities, which comprises the following steps:

Step 1: by 4- hydroxy phenyl sodium phosphate aqueous solution respectively from the alkaline phosphatase standard solution of different known activities slow It rushes to mix in solution and be incubated for, obtain a series of mixed solutions；

Step 2: 3- (2- aminoethylamino) propyl trimethoxy is separately added into a series of mixed solutions of step 1 preparation Base silane aqueous solution, further mixing is incubated for, and carries out fluorescence spectrum test；

Step 3: using the linear relationship of alkaline phosphatase standard solution activity and photoluminescence spectrum intensity, standard curve is drawn；

Step 4: mixing incubation with alkaline phosphatase enzyme solutions to be measured for 4- hydroxy phenyl sodium phosphate aqueous solution in buffer solution, Obtain alkaline phosphatase mixed solution to be measured；

Step 5: 3- (2- aminoethylamino) propyl three is added in the alkaline phosphatase mixed solution to be measured of step 4 preparation Methoxy silane aqueous solution, further mixing is incubated for, and is carried out fluorescence spectrum test, is obtained the fluorescence spectrum of alkaline phosphatase to be measured Intensity；

Step 6: alkaline phosphorus to be measured is calculated in the photoluminescence spectrum intensity measured using the standard curve and step 5 of step 3 The activity of sour enzyme.

2. the fluorescence detection method of alkaline phosphatase activities according to claim 1, which is characterized in that alkaline phosphatase mark The field of activity of quasi- solution are as follows: 0~50mU/mL.

3. the fluorescence detection method of alkaline phosphatase activities according to claim 1, which is characterized in that in step 1 and four The concentration of 4- hydroxy phenyl sodium phosphate aqueous solution is 0.1~1.5mM.

4. the fluorescence detection method of alkaline phosphatase activities according to claim 1, which is characterized in that in step 1 and four Buffer solution is 10~100mM Tris-HCl solution of pH=7.5~10.5, and contains MgCl₂Concentration is 20~500 μM.

5. the fluorescence detection method of alkaline phosphatase activities according to claim 1, which is characterized in that in step 1 and four Incubation temperature is 20~37 DEG C, and the time is 10~90min.

6. the fluorescence detection method of alkaline phosphatase activities according to claim 1, which is characterized in that in step 2 and five The concentration of 3- (2- aminoethylamino) propyl trimethoxy silicane aqueous solution is mass fraction 1%~5%.

7. the fluorescence detection method of alkaline phosphatase activities according to claim 1, which is characterized in that in step 2 and five Incubation temperature is 20~37 DEG C, and the time is 20~120min.

Technical field

The invention belongs to technical field of analysis and detection, and in particular to a kind of fluorescence detection method of alkaline phosphatase activities.

Background technique

Alkaline phosphatase (alkaline phosphatase, ALP, EC 3.1.3.1) be distributed widely in human liver, Bone, intestines, kidney and placenta etc. organize a kind of enzyme being discharged through liver to outside gallbladder, can be catalyzed going for a variety of phosphate ester-containing substrates Phosphorylation events have extremely important effect for the signal transduction in cell growth, Apoptosis process and into the cell adjusting.Alkalinity Phosphatase is the important indicator for reflecting the systemic diseases such as liver and gallbladder, bone, when human body suffers from yellow subcutaneous ulcer, liver cancer, Cholestatic hepatitis Etc. diseases when, the content of alkaline phosphatase in serum will be significantly raised；When occur fracture or with malacosteon, rickets, When the skeletal diseases such as bone cell cancer, osteoporosis, the content of alkaline phosphatase in serum can also be increased.Therefore, sensitive height is established It imitates and the determination of alkaline phosphatase activity method that can be detected in clinical blood is particularly important.

All the time, the analysis method that the researcher of many analytical chemistry and detection technique is dedicated to developing function admirable is used It is detected in alkaline phosphatase activities.The method of traditional detection alkaline phosphatase activities includes isotope-labelling method, complicated for operation, consumption When it is inefficient, and need to endanger biggish radioisotopic tracer.Later, researcher is based on chromatography, colorimetric method, chemistry again Luminescence method, electrochemical process, fluorescence method, the different analytical technology such as serrs create a series of alkaline phosphorus The new detecting method of phytase activity.Wherein, fluorescence sense technology due to its simple and sensitive, real-time and physical examination survey etc. advantages, The always research emphasis of people.Current alkaline phosphatase fluorescence detection method, it is most of to pass through cumbersome organic synthesis, system Standby substrate molecule more complicated out, is examined by the difference in fluorescence between substrate molecule and alkaline phosphatase enzymolysis product It surveys.However, being based on commercialized simple small molecule, reaction generates the process of fluorescent material, and the fluorescence of building " lights " type biology Analytical technology, usually the operation is more convenient, and background interference is lower, and detection sensitivity is higher, therefore based on the development of this principle Alkaline phosphatase enzymatic detection techniques have better application prospect.

Summary of the invention

The object of the present invention is to provide a kind of fluorescence detection method of alkaline phosphatase activities, the detection method agents useful for same It is simple and easy to get, it is easy to operate, quick and precisely, high sensitivity.

To achieve the goals above, technical solution of the present invention is specific as follows:

The present invention provides a kind of fluorescence detection method of alkaline phosphatase activities, comprising the following steps:

Step 1: by 4- hydroxy phenyl sodium phosphate (4-HPP) aqueous solution respectively from the alkaline phosphatase of different known activities (ALP) standard solution mixes incubation in buffer solution, obtains a series of mixed solutions；

Step 2: 3- (2- aminoethylamino) propyl three is separately added into a series of mixed solutions of step 1 preparation Methoxy silane (DAMO) aqueous solution, further mixing is incubated for, and carries out fluorescence spectrum test；

Step 3: using the linear relationship of alkaline phosphatase standard solution activity and photoluminescence spectrum intensity, it is bent to draw standard Line；

Step 4: by 4- hydroxy phenyl sodium phosphate (4-HPP) aqueous solution and alkaline phosphatase enzyme solutions to be measured in buffer solution Middle mixing is incubated for, and obtains alkaline phosphatase mixed solution to be measured；

Step 5: 3- (2- aminoethylamino) third is added in the alkaline phosphatase mixed solution to be measured of step 4 preparation Base trimethoxy silane (DAMO) aqueous solution, further mixing is incubated for, and is carried out fluorescence spectrum test, is obtained alkaline phosphatase to be measured Photoluminescence spectrum intensity；

Step 6: alkali to be measured is calculated in the photoluminescence spectrum intensity measured using the standard curve and step 5 of step 3 The activity of acid phosphatase.

In the above-mentioned technical solutions, the preferred field of activity of alkaline phosphatase standard solution are as follows: 0~50mU/mL.

In the above-mentioned technical solutions, in preferred steps one and four 4- hydroxy phenyl sodium phosphate aqueous solution concentration be 0.1~ 1.5mM。

In the above-mentioned technical solutions, in preferred steps one and four buffer solution be pH=7.5~10.5 10~100mM Tris-HCl solution, and contain MgCl₂Concentration is 20~500 μM.

In the above-mentioned technical solutions, incubation temperature is 20~37 DEG C in preferred steps one and four, and the time is 10~90min.

In the above-mentioned technical solutions, 3- (2- aminoethylamino) propyl trimethoxy silicane water in preferred steps two and five The concentration of solution is mass fraction 1%~5% (g/g).

In the above-mentioned technical solutions, incubation temperature is 20~37 DEG C in preferred steps two and five, and the time is 20~120min.

The beneficial effects of the present invention are:

The fluorescence detection method of alkaline phosphatase activities of the invention regard 4- hydroxy phenyl sodium phosphate (4-HPP) as bottom Object, in aqueous solution, the substrate molecule generate 1, 4-benzenediol (HQ) through the catalyzing hydrolysis of alkaline phosphatase, and 1, 4-benzenediol can It is reacted with 3- (2- aminoethylamino) propyl trimethoxy silicane (DAMO) with subsequent addition, generates green fluorescence hair The silicon nano (SiNPs) penetrated.Alkaline phosphatase activities in system are higher, and the Fluorescent silicon nanoparticle of generation is more, Fluorescence intensity is bigger, according to the fluorescence intensity change of detection solution, carries out the quantitative analysis detection of alkaline phosphatase activities.With The prior art is compared, and reagent chemicals needed for the present invention are simple and easy to get, easy to operate, is clinical sample neutral and alkali phosphorus quick and precisely Phytase activity quantitative detection provides a kind of sensitive reliable method.

Fluorescence detection method of the invention can carry out fluorogenic quantitative detection to alkaline phosphatase activities, and detection is limited to The advantages that 0.1mU/mL has high sensitivity, highly selective, the wide range of linearity.

Fluorescence detection method of the invention can dilution human serum sample in detect, therefore it is potential be applied to face The accurate quick detection of bed sample activity change of Alkaline phosphatase.

For the present invention to operator without special technical requirement, required instrument popularity is higher, and required reagent is business Change product, it is required easy to operate, reproducible.

Detailed description of the invention

Invention is further described in detail with reference to the accompanying drawings and detailed description.

Fig. 1 is the fluorescence detection method schematic diagram of alkaline phosphatase activities of the present invention.

Fig. 2 is that the present invention utilizes fluorescence spectrum detection of alkaline phosphatase feasibility analysis figure.

The Fluorescent silicon nanoparticle spectrogram (A) that Fig. 3 synthesizes for 1, 4-benzenediol (HQ) with DAMO, transmission electron microscope picture (B), (wherein a, b, c are respectively that HQ, DAMO and silicon nano are inhaled for Fourier transform infrared spectrogram (C) and x-ray photoelectron spectroscopy figure (D) Receive spectrum；D, e are respectively the fluorescence excitation and emission spectra of silicon nano).

Fig. 4 is solution fluorescence spectrum in the present invention with the change curve (A) and standard working curve of alkaline phosphatase activities (B)；Control alkaline phosphatase activities are 0,0.1mU/mL, 0.2mU/mL, 0.3mU/mL, 0.5mU/mL, 0.7mU/mL, 0.9mU/ ML, 1mU/mL, 2mU/mL, 3mU/mL, 5mU/mL, 10mU/mL, 15mU/mL, 20mU/mL, 30mU/mL, 40mU/mL, 50mU/ mL。

Fig. 5 is that the present invention is used for the active specific outcome figure of detection of alkaline phosphatase；The potential interference enzyme or egg of selection White matter includes lysozyme LZM, human serum albumin HSA, glucose oxidase GOx, endonuclease EcoR I, bovine serum albumin White BSA and trypsase Trypsin.

Fig. 6 is alkaline phosphatase activities testing result figure of the present invention in dilution human serum sample (1%).

Specific embodiment

Below in conjunction with embodiment, the present invention is further elaborated with attached drawing, and following embodiment is only of the invention preferred Embodiment in order to more fully understand the present invention, thus should not be taken as limiting the scope of the invention.The method is conventional side Method, the raw material can be obtained from public commercial source.