阅读说明：本技术 一种检测碱性磷酸酶活性的荧光方法 (A kind of active fluorescent method of detection of alkaline phosphatase ) 是由 孙健 杨秀荣 刘国永 邢志财 于 2019-08-27 设计创作，主要内容包括：本发明涉及一种检测碱性磷酸酶活性的荧光方法,将对氨基苯磷酸作为底物,在水溶液中,该底物分子经碱性磷酸酶的催化水解生成对氨基苯酚,对氨基苯酚可以与后续加入的乙二胺发生反应,生成强荧光发射的聚合物碳点。体系中的碱性磷酸酶活性越高,生成的荧光聚合物碳点就越多,荧光就越强,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检测。本发明的方法能够对碱性磷酸酶活性进行荧光定量检测,检出限为0.1mU/mL,具有灵敏度高,抗干扰能力强,线性关系优异等优点。本发明所用试剂简单易得,操作简便,重复性好,为临床样品中碱性磷酸酶活性的定量检测提供了一种灵敏有效且准确度高的荧光方法。(The present invention relates to a kind of active fluorescent methods of detection of alkaline phosphatase, using p-APP as substrate, in aqueous solution, the substrate molecule generates para-aminophenol through the catalyzing hydrolysis of alkaline phosphatase, para-aminophenol can react with the ethylenediamine of subsequent addition, generate the polymer carbon dots of hyperfluorescence transmitting.Alkaline phosphatase activities in system are higher, and the fluorescent polymer carbon dots of generation are more, and fluorescence is stronger, according to the fluorescence intensity change of detection solution, carry out the quantitative detection of alkaline phosphatase activities.Method of the invention can carry out fluorogenic quantitative detection to alkaline phosphatase activities, and detection is limited to 0.1mU/mL, has the advantages that high sensitivity, strong antijamming capability, linear relationship is excellent.Agents useful for same of the present invention is simple and easy to get, easy to operate, reproducible, provides a kind of sensitive effective and high accuracy fluorescent method for the quantitative detection of clinical sample activity change of Alkaline phosphatase.)

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

Step 1: p-APP aqueous solution is molten in buffering from the alkaline phosphatase standard solution of different known activities respectively It mixes and is incubated in liquid；

Step 2: being separately added into ethylenediamine solution in the mixed solution of step 1 preparation, and further mixing is incubated for, and carries out glimmering Light spectrum test；

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

Step 4: p-APP aqueous solution is mixed into incubation with alkaline phosphatase enzyme solutions to be measured in buffer solution；

Step 5: being added ethylenediamine solution in the mixed solution of step 4 preparation, and further mixing is incubated for, and carries out fluorescence light Spectrum test, obtains the photoluminescence spectrum intensity of alkaline phosphatase enzyme solutions to be measured；

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 Phytase activity.

2. the active fluorescent method of detection of alkaline phosphatase according to claim 1, which is characterized in that alkaline phosphatase mark The activity of quasi- solution are as follows: 0~70mU/mL.

3. the active fluorescent method of detection of alkaline phosphatase according to claim 1, which is characterized in that in step 1 and four The concentration of p-APP aqueous solution is 0.25~6.25mM.

4. the active fluorescent method of detection of alkaline phosphatase according to claim 1, which is characterized in that in step 1 and four Buffer solution is 10~100mM Tris-HCl solution of pH=8.0~11.0, and contains MgCl₂Concentration is 0.1~5mM.

5. the active fluorescent method of detection of alkaline phosphatase according to claim 4, which is characterized in that buffer solution is pH =10.0, the concentration of p-APP aqueous solution is 2.5mM.

6. the active fluorescent method of detection of alkaline phosphatase 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.

7. the active fluorescent method of detection of alkaline phosphatase according to claim 6, which is characterized in that in step 1 and four Incubation temperature is 37 DEG C, time 60min.

8. the active fluorescent method of detection of alkaline phosphatase according to claim 1, which is characterized in that in step 2 and five The concentration of ethylenediamine solution is 100~1000mM.

9. the active fluorescent method of detection of alkaline phosphatase 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 20~180min.

10. the active fluorescent method of detection of alkaline phosphatase according to claim 9, which is characterized in that step 2 and five Middle incubation temperature is 37 DEG C, time 120min.

Technical field

The invention belongs to chemical analysis detection technique fields, and in particular to a kind of active fluorescence side of detection of alkaline phosphatase Method.

Background technique

Alkaline phosphatase (ALP) is that one kind is widely present in human body, animal, plant and the intracorporal sugared egg of film combination of microorganism White, it directly participates in the physiology courses such as transfer and the metabolism of catalytic phosphatase group, and the signal in cell growth Apoptosis process passes It leads and plays an important role in intracellular adjustment process, the calcium-phosphorus ratio that the absorption and metabolism, maintenance to internal calcium phosphorus are suitable in vivo There is extremely important effect in the ossific process of example and animal.It is universal in the tissue such as liver, bone, intestines, kidney and placenta of human body There is alkaline phosphatases, meanwhile, the unconventionality expression of blood plasma alkaline phosphatase is related with a variety of diseases, such as skeletal diseases, sugar Urine disease, breast cancer, prostate cancer and dysfunction of liver etc..On the other hand, due to the molecular weight of alkaline phosphatase it is lower, it is active compared with The commercialization alkaline phosphatase for stablizing, being easy to purify, therefore extracting from various animals, plant, microorganism, is widely used in The research fields such as ELISA, biosensor, molecular biology.Therefore, develop easier sensitive, more preferable selecting property Alkaline phosphatase activities detection method not only can be in favor of the diagnosis and differential diagnosis of the diseases such as bone, liver and gall, can be with Binding antibody constructs novel ELISA system and detects specific disease targets.

So far, pass through chromatography, colorimetric method, chemoluminescence method, electrochemical process, fluorescence method, surface enhanced resonant drawing The detection method of a variety of alkaline phosphatase activities has been developed in the different analytical technologies such as graceful scattering.Wherein, fluorescence detection skill Art receives highest attention due to its simple and sensitive, real-time and the advantages that physical examination is surveyed.It is particularly noteworthy that being based on The fluorescence that fluorescent material generates reaction building " lights " type bioassay technique, and usually the operation is more convenient, and background interference is lower, Detection sensitivity is higher, therefore has better application prospect.

Summary of the invention

The object of the present invention is to provide a kind of active fluorescent method of detection of alkaline phosphatase, the detection method agents useful for same It is simple and easy to get, easy to operate, reproducible, high sensitivity.

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

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

Step 1: p-APP (APP) aqueous solution is molten from the alkaline phosphatase standard of different known activities respectively Liquid mixes incubation in buffer solution；

Step 2: being separately added into ethylenediamine solution in the mixed solution of step 1 preparation, and further mixing is incubated for, into The test of row fluorescence spectrum；

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: p-APP (APP) aqueous solution is mixed in buffer solution with alkaline phosphatase enzyme solutions to be measured It is incubated for；

Step 5: being added ethylenediamine solution in the mixed solution of step 4 preparation, and further mixing is incubated for, and carries out glimmering Light spectrum test obtains the photoluminescence spectrum intensity of alkaline phosphatase enzyme solutions to be measured；

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

In the above-mentioned technical solutions, the preferred activity of alkaline phosphatase standard solution are as follows: 0~70mU/mL.

In the above-mentioned technical solutions, in preferred steps one and four p-APP aqueous solution concentration be 0.25~ 6.25mM。

In the above-mentioned technical solutions, in preferred steps one and four buffer solution be pH=8.0~11.0 10~100mM Tris-HCl solution, and contain MgCl₂Concentration is 0.1~5mM.

In the above-mentioned technical solutions, further preferred buffer solution is pH=10.0, p-APP aqueous solution it is dense Degree is 2.5mM.

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, incubation temperature is 37 DEG C in further preferred step 1 and four, time 60min.

In the above-mentioned technical solutions, the concentration of ethylenediamine solution is 100~1000 in preferred steps two and five

mM。

In the above-mentioned technical solutions, incubation temperature is 20~37 DEG C in preferred steps two and five, and the time is 20~180min.

In the above-mentioned technical solutions, incubation temperature is 37 DEG C in further preferred step 2 and five, time 120min.

The beneficial effects of the present invention are:

The active fluorescent method of detection of alkaline phosphatase provided by the invention, using p-APP as substrate, in water In solution, which generates para-aminophenol through the catalyzing hydrolysis of alkaline phosphatase, and para-aminophenol can add with subsequent The ethylenediamine entered reacts, and generates the polymer carbon dots of hyperfluorescence transmitting.Alkaline phosphatase activities in system are higher, generate Fluorescent polymer carbon dots it is more, fluorescence is stronger, according to detection solution fluorescence intensity change, carry out alkaline phosphatase enzyme activity The quantitative detection of property.

Method of the invention can carry out fluorogenic quantitative detection to alkaline phosphatase activities, and detection is limited to 0.1mU/mL, has There is the advantages that high sensitivity, strong antijamming capability, linear relationship is excellent.

Method of the invention can detect in dilution human serum sample, therefore can be used for clinical sample neutral and alkali phosphorus The sensitive and accurate detection of phytase activity.

Agents useful for same of the present invention is simple and easy to get, easy to operate, reproducible, is clinical sample activity change of Alkaline phosphatase Quantitative detection provides a kind of sensitive effective and high accuracy fluorescent method.

Instrument and equipment popularity needed for the present invention is higher, and required reagent is commercially produced product, and process is simple, repeated It is good, to operator without special technical requirement.

Detailed description of the invention

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

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

Fig. 2 be the present invention using fluorescence spectrum detection of alkaline phosphatase feasibility analysis figure (wherein a is p-aminophenyl phosphorus Acid, b are p-APP+ethylenediamine, and c is alkaline phosphatase+ethylenediamine, and d is p-APP+alkaline phosphatase, and e is P-APP+alkaline phosphatase+ethylenediamine, f are para-aminophenol+ethylenediamine, are to test after solution is incubated for).

Fig. 3 is that para-aminophenol synthesizes fluorescent polymer carbon dots spectrogram (left side) and transmission electron microscope picture (right side) with ethylenediamine (wherein a, b, c are respectively ethylenediamine, para-aminophenol and carbon dots absorption spectrum；D, e are respectively the fluorescence excitation and transmitting of carbon dots Spectrum).

Fig. 4 is the optimal inspection figure of pH value of buffer solution in the present invention；Controlling pH is respectively 8,9,10,11.

Fig. 5 is the optimal inspection figure that substrate p-APP concentration is added in the present invention；It is whole to control p-APP Concentration is respectively 0,0.1mM, 0.25mM, 0.5mM, 1.0mM, 1.5mM, 2.0mM, 2.5mM.

Fig. 6 is p-APP and alkaline phosphatase enzyme solutions mixed enzymolysis incubation time in buffer solution in the present invention Optimal inspection figure；Controlled enzymatic hydrolysis incubation time is respectively 10min, 20min, 30min, 40min, 50min, 60min, 70min, 80min, 90min.

Mixing after Fig. 7 is incubated in buffer solution for p-APP in the present invention with alkaline phosphatase enzyme solutions is molten Liquid adds the optimal inspection figure of ethylenediamine mixing fluorescence reaction incubation time；Controlling fluorescence reaction incubation time is respectively 20min, 40min, 60min, 80min, 120min, 180min.

Fig. 8 is solution fluorescence spectrum in the present invention with the change curve (A) and standard working curve of alkaline phosphatase activities (B,C)；Control alkaline phosphatase activities are 0mU/mL, 0.1mU/mL, 0.5mU/mL, 1mU/mL, 2mU/mL, 3mU/mL, 4mU/ ML, 5mU/mL, 6mU/mL, 7mU/mL, 8mU/mL, 9mU/mL, 10mU/mL, 15mU/mL, 20mU/mL, 30mU/mL, 40mU/ ML, 50mU/mL, 60mU/mL, 80mU/mL.

Fig. 9 is that the present invention is used for the active specific test figure of detection of alkaline phosphatase；The potential interference enzyme or egg of selection White matter includes bovine serum albumin(BSA) BSA, endonuclease EcoR I, glucose oxidase GOx, human serum albumin HSA, bacteriolyze Enzyme Lysozyme, trypsase Trypsin.

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.