CN109557060A - One kind being based on NH2The method of the Visual retrieval serum activity change of Alkaline phosphatase of-Cu-MOF - Google Patents
One kind being based on NH2The method of the Visual retrieval serum activity change of Alkaline phosphatase of-Cu-MOF Download PDFInfo
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Abstract
The invention discloses one kind to be based on NH2The method of the Visual retrieval serum activity change of Alkaline phosphatase of-Cu-MOF, first synthesizes NH2- Cu-MOF nano enzyme material；Recycle NH2- Cu-MOF nano enzyme and agarose prepare the active gel detection pipe of detection of alkaline phosphatase；After alkaline phosphatase is finally added to gel detection pipe, gel detection pipe is placed under ultraviolet lamp, half-quantitative detection is realized by observation color change；Then it is taken pictures with mobile phone or digital camera, by RGB color extractor software, reads rgb value in measurement photo, analyze rgb value to indicate the degree of oxidation of OPD, to realize the visualization of presence multicolor fluorescence quantitative detection to serum activity change of Alkaline phosphatase；When detection, gel color is first drawn with the working curve of alkaline phosphatase activity size variation, then detect to the practical sample of serum.This method does not need expensive instrument and equipment, does not need the participation of biological enzyme, and the visualization of presence multicolor fluorescence measurement to serum alkaline phosphatase can be realized without professional's operation.
Description
Technical field
It is specifically a kind of to be based on NH the present invention relates to the active method of detection of alkaline phosphatase2The visualization of-Cu-MOF is examined
The method for surveying serum activity change of Alkaline phosphatase.
Background technique
Alkaline phosphatase (ALP) is a kind of hydrolase generally existing in prokaryotes and eukaryotic cells, is catalyzed phosphorus
Acid groups are removed from the polyphosphoric acid substrate containing phosphate.In the body of the mankind, alkaline phosphatase is widely present in tissue
In (such as bone, kidney, liver etc.), in a variety of normal cell functions such as cell signalling, cell division, differentiation, bone calcification
It plays an important role.On the other hand, the downward of alkaline phosphatase activities and a variety of human diseases are closely related, including low-phosphorous acid
Esterase disease, primary biliary cirrhosis, diabetes and various cancers etc..Therefore, exploitation is reliable easily detects alkaline phosphorus
The research that the method for phytase activity acts on the diagnosis of clinical disease and alkaline phosphatase in physiology and pathologic process has
Important researching value.
In the related patents for having disclosed detection of alkaline phosphatase at present, a kind of detection alkaline phosphatase of CN108375616A
Liquid crystal biosensor of enzyme and preparation method thereof, liquid crystal biosensor need to obtain optic response using petrographic microscope
Image；CN106248644A one kind is based on the detection method of the alkaline phosphatase of carbon dots fluorescence " quenching-recovery ", needs using glimmering
Photothermal spectroscopic analyzer obtains fluorescence spectra；CN107422014A is for the modified electrode and preparation method of detection of alkaline phosphatase and inspection
Survey method needs to obtain electrochemical signals using electrochemical workstation to draw working curve.Therefore existing alkaline phosphatase
Activity test method mainly realizes quantitative detection by analysis instruments such as Fluorescence Spectrometer, electrochemical workstations.However, these sides
The method professional skill more demanding to operator, especially used instrument price is costly and bulky, unfavorable
In being widely applied and promote.
In addition, the method for existing Visual retrieval alkaline phosphatase activities, a kind of alkaline phosphorus of detection of CN106066325A
The method of sour enzyme, using reaction solution color with alkaline phosphatase concentration increase and from blue become colourless gradient color variation come
Alkaline phosphatase activities quantitative determination is realized, however, being that concentration gradient colour developing changes single, resolution ratio the shortcomings that this method
It is low；There are also a kind of detection method of the alkaline phosphatase activities of simple low cost of CN106596532A, there is also same problems.?
There is a kind of detection method of the alkaline phosphatase activities of simple low cost of patent CN106596532A using hydrophily paper design
Paper device is inversely proportional to the active size of sample alkaline phosphatase using the length of flow of detection reagent solution in paper device.So
And the device needs to use biological enzyme such as carbohydrase, and biological enzyme is during production, transport and use, active shadow
The factor of sound is more, and only need to be mentioned in accuracy and accuracy by ruler measurement, the artificial length of flow for reading solution
It rises；A kind of bone-derived alkaline phosphatase detection kit of CN107677804A and its application method, bone-derived alkaline phosphatase inspection
Though test agent box detection time is short, automatic clinical chemistry analyzer is still needed to realize detection.Therefore, it develops and is used for serum neutral and alkali
The visualization of phosphatase, portable, color change is polynary, high-resolution detection method has important practical significance.
Summary of the invention
The purpose of the present invention is in view of the deficiencies of the prior art, and provide a kind of based on NH2The visualization of-Cu-MOF is examined
The method for surveying serum activity change of Alkaline phosphatase, this method do not need expensive instrument and equipment, do not need the participation of biological enzyme,
The vigor size for judging alkaline phosphatase can be changed by the fluorescence color of solution or gel tube without professional's operation, it can
To realize that the visualization of presence multicolor fluorescence to serum alkaline phosphatase measures.
Realizing the technical solution of the object of the invention is:
One kind being based on NH2The method of the Visual retrieval serum activity change of Alkaline phosphatase of-Cu-MOF, includes the following steps:
(1) synthesis has the NH of certain shape characteristic2- Cu-MOF nano enzyme material；
(2) NH is utilized2- Cu-MOF nano enzyme and agarose prepare the active gel detection pipe of detection of alkaline phosphatase；
(3) after alkaline phosphatase is added to gel detection pipe, the power and the oxygen of o-phenylenediamine (OPD) of the yellow fluorescence intensity of gel
Change product 2, the concentration of 3- diaminophenazine (DAP) is positively correlated, and due to NH2- Cu-MOF nano enzyme itself has indigo plant
Color fluorescence, therefore gel detection pipe is placed under ultraviolet lamp, ratio fluorescent is formed with DAP, generates high-resolution color change, is led to
It crosses observation color change and realizes half-quantitative detection；Then it is taken pictures with mobile phone or digital camera, by RGB color extractor software,
Rgb value in measurement photo is read, analyzes rgb value to indicate the degree of oxidation of OPD, to realize living to serum alkaline phosphatase
The visualization of presence multicolor fluorescence quantitative detection of property；When detection, gel color is first drawn with alkaline phosphatase activity size variation
Working curve, then the practical sample of serum is detected.
Step (1) the synthesis NH2- Cu-MOF nano enzyme material, includes the following steps:
(1.1) by Cu (NO3)·3H2O dissolves in deionized water, Cu (NO3)·3H2O and the mass ratio of deionized water are 1:
10；
(1.2) DMF solution for being dissolved with 2- amino terephthalic acid (TPA) and ethyl alcohol are mixed, the molten of step (1.1) is added after mixing
In liquid, remix uniformly；
The volume ratio of DMF and alcohol solvent is 1:1, Cu (NO3)·3H2The mass concentration ratio of O and 2- amino terephthalic acid (TPA) is
2:1-3:1；
(1.3) after mixing well, solution is placed in the autoclave of teflon lining, is reacted under 80 °C -90 °C；
(1.4) finally, by the way that NH is collected by centrifugation2- Cu-MOF, and rear natural drying is successively washed with DMF, ethyl alcohol and deionized water,
Synthesize NH2- Cu-MOF nano enzyme material.
The preparation of step (2) the gel detection pipe, includes the following steps:
(2.1) first agarose is dissolved in boiling water and Ago-Gel solution is made, agar Sugar concentration is 5g/L-15g/L；
(2.2) by NH2The solution of-Cu-MOF nano enzyme is added in Ago-Gel solution, is mixed； NH2- Cu-MOF nanometers
Enzyme concentration is 0.3-0.6g/L；
(2.3) after mixing, taking volume is to cool down one section at room temperature in 0.050-0.250mL mixed liquor to the lid of testing tube
After time molding, encapsulation storage.
The NH that the method for the present invention, first synthesis have certain shape characteristic2- Cu-MOF nano enzyme material, is inhibited using PPi
The catalytic activity of nano enzyme releases the inhibition of PPi then again by alkaline phosphatase to the hydrolysis of PPi, restores
NH2- Cu-MOF is to the catalysed oxidn of OPD, in conjunction with NH2The fluorescence signal of-Cu-MOF itself, so that it is big that different vigor are added
After small alkaline phosphatase enzyme solutions, mixed liquor shows different degrees of ratio fluorescent under ultraviolet light, determines to realize
Measure detection of alkaline phosphatase activity.
In addition, utilizing NH in conjunction with Ago-Gel2- Cu-MOF material is prepared for portable inspectiont serum neutral and alkali phosphoric acid
The gel detection pipe of enzyme.Since the concentration of the oxidation product DAP of the yellow fluorescence intensity power and OPD of alkaline phosphatase enzyme solutions is big
It is small to be positively correlated, therefore can be by mobile phone or digital camera, in conjunction with profession color matching software or the analysis of RGB color extractor software
The method of rgb value in photo is measured to indicate the degree of oxidation of o-phenylenediamine, to realize the polychrome to alkaline phosphatase activities
Fluorogenic quantitative detection.
The present invention is based on NH2The method of the polychrome Visual retrieval serum alkaline phosphatase of-Cu-MOF nano enzyme utilizes
There are three types of the NH of function for tool2- Cu-MOF material is class oxidase mimetic enzyme, fluorogenic donor and cascade reaction participant respectively
Function.Multicolor fluorescence testing principle is that PPi can be released based on hydrolysis of the alkaline phosphatase to PPi to NH2-Cu-MOF
In Cu coordination, restored NH2Catalysed oxidn of-the Cu-MOF to OPD, bond material autofluorescence, so that plus
The alkaline phosphatase enzyme solutions for entering different vigor show different degrees of ratio fluorescent signal, system color under ultraviolet light
Variation is more abundant, is easy to naked eyes and distinguishes.This method does not need expensive instrument and equipment, does not need biological enzyme participation, utilizes
Semi-quantitative analysis can be achieved in naked eyes；Using the Portable mobile phone with camera function as analysis tool, it can be achieved that quantitative analysis.
The present invention without professional operation can by gel tube ultraviolet light irradiation hypostome color mutation analysis serum neutral and alkali phosphorus
The vigor size of sour enzyme, reading RGB numerical value by cell phone software may be implemented the visualization of presence to serum alkaline phosphatase
Multicolor fluorescence quantitative detection.
Specific embodiment
The content of present invention is further described below with reference to embodiment, but is not limitation of the invention.
Embodiment
One kind being based on NH2The method of the polychrome Visual retrieval serum activity change of Alkaline phosphatase of-Cu-MOF nano enzyme, packet
Include following steps:
(1) synthesis has the NH of certain shape characteristic2- Cu-MOF nano enzyme material；
By 1g Cu (NO3)·3H2O is dissolved in the deionized water of 10 mL；0.4 g 2- amino terephthalic acid (TPA) will be dissolved with
10mL DMF and 10 mL ethyl alcohol mixing, be added in above-mentioned solution be uniformly mixed；After mixing well, solution is placed in teflon
In the autoclave of lining, 24 h are reacted under 80 °C；Finally, by the way that NH is collected by centrifugation2- Cu-MOF, and with DMF, ethyl alcohol and go
Ionized water successively washes rear natural drying.
(2) NH is utilized2- Cu-MOF nano enzyme and agarose prepare the active gel detection pipe of detection of alkaline phosphatase；
By 300 μ L, the NH of Tris-HCl buffer solution, 100 μ L that pH is 7.22- Cu-MOF(2.5 g/L) ethanol solution
It is added in 1 mL Ago-Gel, gel temperature controlled water baths are at 40 DEG C；After mixing, take 200 μ L mixed liquors to testing tube
In lid, gel sets after 5 min are placed, closes the lid, is stored at 4 DEG C, gel shows colorless and transparent.
(3) gel detection pipe is placed under ultraviolet lamp, measurement is read by RGB color extractor software with mobile phone photograph
RGB numerical value in photo is analyzed rgb value to indicate the degree of oxidation of OPD, is showed to realize to serum activity change of Alkaline phosphatase
Field visualized multicolor fluorescence quantitative detection.
When specific detection, gel color is drawn first with the working curve of alkaline phosphatase activity size variation:
By alkaline phosphatase standard solution (0 U/L, 5 U/L, 10 U/L, 30 U/L, 60 U/L, 90 U/ of different vigor sizes
L, 120 U/L), 67 μ L ppi(30 mmol L-1) solution is added in gel testing tube, it is upright to be put in 37 °C of constant temperature incubations vibrations
It swings and is incubated for 30 min in device, 80 μ LOPD (0.4mmol L are then added-1) solution, it stands upside down and is placed in 37 °C of intelligence city constant temperature incubations vibrations
It swings and is incubated for 40 min in device, take pictures in the UV lamp, read solution in photo using ColorSchemer Studio software
Rgb value, calculates R/(R+G+B) of blank solution and prepare liquid, and makees ordinate with △ R/(R+G+B) value, can be obtained solidifying
The △ R of glue color/(R+G+B) value is with the change curve of alkaline phosphatase activity size variation, i.e. detection serum neutral and alkali phosphoric acid
The working curve of enzyme activity, color change are as shown in Table 1:
Table one is based on NH2The data of the polychrome Visual retrieval standard solution phosphatase activity of-Cu-MOF nano enzyme
；
Secondly, being detected to practical sample: drawing serum, the 67 μ L ppi(30 mmol L of 60 μ L-1) solution is added to gel
It in testing tube, is just being placed in 37 °C of intelligence city constant temperature culture oscillators and is being incubated for 30 min, 80 μ L OPD (0.4 are then added
mmol L-1) solution, handstand, which is placed in 37 °C of intelligence city constant temperature culture oscillators, is incubated for 40 min, takes pictures in the UV lamp, can obtain face
Color change；The rgb value of solution in photo is read using ColorSchemer Studio software, substituting into working curve can obtain
To the energy value of serum alkaline phosphatase, as shown in Table 2:
Table two is based on NH2The data of the polychrome Visual retrieval serum phosphatase activity of-Cu-MOF nano enzyme
。
Claims (3)
1. one kind is based on NH2The method of the Visual retrieval serum activity change of Alkaline phosphatase of-Cu-MOF, which is characterized in that including
Following steps:
(1) NH is synthesized2- Cu-MOF nano enzyme material；
(2) NH is utilized2- Cu-MOF nano enzyme and agarose prepare the active gel detection pipe of detection of alkaline phosphatase；
(3) gel detection pipe is placed under ultraviolet lamp, is taken pictures with mobile phone or digital camera, by RGB color extractor software, read
Rgb value in measurement photo is taken, analyzes rgb value to indicate the degree of oxidation of o-phenylenediamine, to realize to serum neutral and alkali phosphoric acid
The visualization of presence multicolor fluorescence quantitative detection of enzymatic activity；When detection, gel color is first drawn with alkaline phosphatase activity size
The working curve of variation, then the practical sample of serum is detected.
2. according to claim 1 be based on NH2The method of the Visual retrieval serum activity change of Alkaline phosphatase of-Cu-MOF,
It is characterized by: step (1) the synthesis NH2- Cu-MOF nano enzyme material, includes the following steps:
(1.1) first by Cu (NO3)·3H2O dissolves in deionized water, Cu (NO3)·3H2O and the mass ratio of deionized water are 1:
10；
(1.2) it after mixing the DMF solution for being dissolved with 2- amino terephthalic acid (TPA) and ethyl alcohol, is added in the solution of step (1.1),
It is uniformly mixed；
The volume ratio of DMF and alcohol solvent is 1:1, Cu (NO3)·3H2The mass concentration ratio of O and 2- amino terephthalic acid (TPA) is 2:
1-3:1；
(1.3) after mixing well, solution is placed in the autoclave of teflon lining, is reacted under 80 °C -90 °C；
(1.4) finally, by the way that NH is collected by centrifugation2- Cu-MOF, and rear natural drying is successively washed with DMF, ethyl alcohol and deionized water, it closes
At NH2- Cu-MOF nano enzyme material.
3. according to claim 1 be based on NH2The method of the Visual retrieval serum activity change of Alkaline phosphatase of-Cu-MOF,
It is characterized by: step (2) the gel detection control is standby, include the following steps:
(2.1) first agarose is dissolved in boiling water and Ago-Gel solution is made, agar Sugar concentration is 5g/L-15g/L；
(2.2) by NH2The solution of-Cu-MOF nano enzyme is added in Ago-Gel solution, is mixed, wherein NH2- Cu-MOF receives
Rice enzyme concentration is 0.3-0.6g/L；
(2.3) after mixing, taking volume is to be cooled and shaped at room temperature in 0.050-0.250mL mixed liquor to the lid of testing tube
Afterwards, encapsulation storage.
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| CN109557060B (en) | 2020-10-20 |
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