CN106706578A - Fluorescence detection method for hydrolase activity - Google Patents
Fluorescence detection method for hydrolase activity Download PDFInfo
- Publication number
- CN106706578A CN106706578A CN201611025012.5A CN201611025012A CN106706578A CN 106706578 A CN106706578 A CN 106706578A CN 201611025012 A CN201611025012 A CN 201611025012A CN 106706578 A CN106706578 A CN 106706578A
- Authority
- CN
- China
- Prior art keywords
- substrate
- detection method
- disulfonic acid
- fluorescence
- hydrolase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
Abstract
The invention discloses a fluorescence detection method for hydrolase activity. The method comprises the following steps: hatching detection liquid containing a substrate and a Cu<2+>-bathocuproine disulfonic acid complex with a to-be-detected hydrolase solution, taking the Cu<2+>-bathocuproine disulfonic acid complex as a fluorescent probe, and reducing the fluorescent probe by a reducing product obtained by hydrolyzing the substrate by virtue of hydrolase, so as to generate the Cu<2+>-bathocuproine disulfonic acid complex and weaken a fluorescent signal of the detection liquid. The higher is the activity of the hydrolase in a sample, the lower fluorescence emission intensity of the detection liquid is. The fluorescence detection method disclosed by the invention has high detection sensitivity on hydrolase activity, good selectivity, strong interference resistance, good reproducibility, simple operation and short detection time and can be used for quick and high-sensitivity detection of the hydrolase activity in a clinical sample, and the detection cost is greatly reduced.
Description
Technical field
The invention belongs to bioassay technique field, it is related to a kind of fluorescence detection method of hydrolytic enzyme activities.
Background technology
Hydrolase is the general name of the class of enzymes of catalytic hydrolysis reaction, is widely present in various animals, plant and microorganism.
Activity that is easy, quick and delicately detecting hydrolase, early diagnosis, observation of curative effect and Prognosis scoveillance for disease have pole
Its important meaning.
At present, for hydrolase determination of activity more use XRF and colorimetric analysis.Wherein, XRF
The fluorescence intensity change based on material in solution come method that quantitative analysis is carried out to content of material, with testing cost it is low,
The good characteristic such as equipment is simple, easy to operate, the degree of accuracy is high.By taking alkaline phosphatase as an example, its determination of activity is presently available for
Fluorescence analysis method has two categories below：(1) principle lighted based on polymerisation induced, alkaline phosphatase is by water miscible phosphoric acid-four
The water insoluble tetraphenyl ethylene of styrene hydrolysis generation, can produce fluorescence, so as to realize to alkaline phosphatase after tetraphenyl ethylene polymerization
The fluorescence analysis of enzyme, but there is poor selectivity in the method, and sensitivity is low and defect (the 1.Gu X, et such as antijamming capability deficiency
al.(2013).A new fluorometric turn-on assay for alkaline phosphatase and
inhibitor screening based on aggregation and deaggregation of
tetraphenylethylene molecules[J].Analyst,138(8):2427-2431；2.Liang J,et al.
(2013).Fluorescent light-up probe with aggregation-induced emission
characteristics for alkaline phosphatase sensing and activity study[J].ACS
Applied Materials&Interfaces,5(17):8784-8789.)；(2) using carbon quantum dot as fluorescence probe, carbon amounts
The carboxylic group of son point surface enrichment can cause Quenching of fluorescence when being initiated aggregation, so as to realize the fluorescence to alkaline phosphatase
Analysis, however the method to there is testing cost high, poor anti jamming capability and may have defect (the 1.Qian Z, et such as toxicity
al.(2015).Carbon quantum dots-based recyclable real-time fluorescence assay
for alkaline phosphatase with adenosine triphosphate as substrate[J]
.Analytical Chemistry,87(5):2966-2973；2.Qian Z S,et al.(2015).A real-time
fluorescent assay for the detection of alkaline phosphatase activity based on
carbon quantum dots[J].Biosensors and Bioelectronics,68:675-680.)。
The content of the invention
In view of the shortcomings of the prior art, the invention provides a kind of fluorescence detection method of hydrolytic enzyme activities, the method base
Hydrolytic enzyme activities are determined in fluorescence analysis, sensitivity is high, selectivity is good, and strong antijamming capability, reproducibility is good, simple to operate,
Detection time is short, with low cost, can be used for the clinical detection of hydrolytic enzyme activities.
Technical scheme is as follows：
A kind of fluorescence detection method of hydrolytic enzyme activities, comprises the following steps：Substrate and Cu will be contained2+The sulphur of-bathocuproine two
The detection liquid of sour fluorescence probe is incubated with hydrolysis enzyme solutions to be measured, generates Cu+- bathocuproine disulfonic acid complex compound, determines reaction solution
Fluorescent emission intensity at 402nm, according to fluorescent emission intensity and the standard curve relation of hydrolytic enzyme activities, is calculated and treats
Survey the activity of hydrolysis enzyme solutions reclaimed water solution enzyme；Described substrate is irreducibility substrate, with hydrolysis enzyme reaction generation reduction to be measured
Property product.
Described Cu2+- bathocuproine disulfonic acid fluorescence probe is by Cu2+It is mixed with bathocuproine disulfonic acid.
Described hydrolase be selected from alpha-galactosidase, beta galactosidase, alpha-glucosidase, beta-glucosidase,
Alpha-Mannosidase, beta-Mannosidase, alkaline phosphatase, Soil neutral phosphatase or acid phosphatase.
When described hydrolase is alpha-galactosidase, substrate can be p-amino phenyl- α-D- galactopyranosides.
When described hydrolase is beta galactosidase, substrate can be p-amino phenyl- β-D- galactopyranosides.
When described hydrolase is alpha-glucosidase, substrate can be p-amino phenyl- α-D- glucopyranosides.
When described hydrolase is beta-glucosidase, substrate can be p-amino phenyl- β-D- glucopyranosides.
When described hydrolase is alpha-Mannosidase, substrate can be p-amino phenyl- α-D- mannopyranose glycosides.
When described hydrolase is beta-Mannosidase, substrate can be p-amino phenyl- β-D- mannopyranose glycosides.
When described hydrolase is alkaline phosphatase, substrate can be ascorbic acid -2- phosphoric acid.
When described hydrolase is Soil neutral phosphatase, substrate can be ascorbic acid -2- phosphoric acid.
When described hydrolase is acid phosphatase, substrate can be ascorbic acid -2- phosphoric acid.
The fluorescence detection method of hydrolytic enzyme activities of the invention, substrate does not have reproducibility, it is impossible to reduce Cu2+- bathocuproine
Disulfonic acid fluorescence probe, substrate can generate reproducibility product in the presence of hydrolase, and the reproducibility product of generation can be by Cu2 +- bathocuproine disulfonic acid fluorescence probe is reduced into Cu+- bathocuproine disulfonic acid complex compound.Containing substrate and Cu2+The sulphur of-bathocuproine two
During the detection liquid of sour fluorescence probe is incubated with hydrolysis enzyme solutions to be measured, substrate generates reproducibility product with hydrolysis enzyme reaction,
Reproducibility product is by Cu2+- bathocuproine disulfonic acid fluorescence probe is reduced into Cu+- bathocuproine disulfonic acid complex compound, causes detection liquid to exist
Fluorescent emission signals at 402nm decline.The present invention by determining fluorescence intensity of the reaction solution in 402nm, finally according to fluorescence
Intensity and the standard curve relation of hydrolytic enzyme activities, obtain the activity of hydrolysis enzyme solutions reclaimed water solution enzyme to be measured.
The present invention only needs single stepping to be capable of achieving the measure to hydrolytic enzyme activities, and fluorescence probe preparation process is extremely simple
It is single, with good characteristics such as stable chemical nature, good water solubility and good biocompatibilities.Fluorescence detection method of the invention has
Sensitivity is high, and selectivity is good, and strong antijamming capability, reproducibility is good, simple to operate, and it is excellent that detection time is short and with low cost etc.
Point, can be used for quick, the highly sensitive detection of hydrolytic enzyme activities in clinical sample, significantly reduce testing cost.
Brief description of the drawings
Fig. 1 is the fluorescence emission spectrogram of compound of the detection liquid containing different component in embodiment 1, wherein, a is to be not added with alkaline phosphorus
Sour enzyme, to be not added with ascorbic acid -2- phosphoric acid, to be not added with bathocuproine disulfonic acid, d is to be not added with Cu to c to b2+- bathocuproine disulfonic acid fluorescence is visited
Pin, e adds for all reagents.
Fig. 2 is the graph of relation between fluorescent emission intensity and alkaline phosphatase activities in embodiment 2.
Fig. 3 is the comparing of the detection liquid fluorescent emission intensity at 402nm containing different proteins sample in embodiment 3
Figure, wherein, a is alkaline phosphatase, and b is acid phosphatase, and c is human albumin's enzyme, and d is trypsase, and e is fibrin ferment, and f is
Hemoglobin, g is blank.
Fig. 4 is the graph of relation between fluorescent emission intensity and blood serum sample volume in embodiment 4.
Fig. 5 is the fluorescent emission intensity spectrogram of the detection liquid containing different component in embodiment 5, wherein, a for be not added with α-
Galactosidase, to be not added with p-amino phenyl- α-D- galactopyranosides, to be not added with bathocuproine disulfonic acid, d is to be not added with Cu to c to b2+-
Bathocuproine disulfonic acid fluorescence probe, e adds for all reagents.
Fig. 6 is the fluorescent emission intensity spectrogram of the detection liquid containing different component in embodiment 6, wherein, a for be not added with β-
Galactosidase, to be not added with p-amino phenyl- β-D- galactopyranosides, to be not added with bathocuproine disulfonic acid, d is to be not added with Cu to c to b2+-
Bathocuproine disulfonic acid fluorescence probe, e adds for all reagents.
Fig. 7 is the fluorescent emission intensity spectrogram of the detection liquid containing different component in embodiment 7, wherein, a for be not added with α-
Glucuroide, to be not added with p-amino phenyl- α-D- glucopyranosides, to be not added with bathocuproine disulfonic acid, d is to be not added with Cu to c to b2+-
Bathocuproine disulfonic acid fluorescence probe, e adds for all reagents.
Fig. 8 is the fluorescent emission intensity spectrogram of the detection liquid containing different component in embodiment 8, wherein, a for be not added with β-
Glucuroide, to be not added with p-amino phenyl- β-D- glucopyranosides, to be not added with bathocuproine disulfonic acid, d is to be not added with Cu to c to b2+-
Bathocuproine disulfonic acid fluorescence probe, e adds for all reagents.
Fig. 9 is the fluorescent emission intensity spectrogram of the detection liquid containing different component in embodiment 9, wherein, a for be not added with α-
Mannosidase, to be not added with p-amino phenyl- α-D- mannopyranose glycosides, to be not added with bathocuproine disulfonic acid, d is to be not added with Cu to c to b2+-
Bathocuproine disulfonic acid fluorescence probe, e adds for all reagents.
Figure 10 is the fluorescent emission intensity spectrogram of the detection liquid containing different component in embodiment 10, wherein, a is to be not added with
Beta-Mannosidase, to be not added with p-amino phenyl- β-D- mannopyranose glycosides, to be not added with bathocuproine disulfonic acid, d is to be not added with to c to b
Cu2+- bathocuproine disulfonic acid fluorescence probe, e adds for all reagents.
Figure 11 is the fluorescent emission intensity spectrogram of the detection liquid containing different component in embodiment 11, wherein, a is to be not added with
Soil neutral phosphatase, to be not added with ascorbic acid -2- phosphoric acid, to be not added with bathocuproine disulfonic acid, d is to be not added with Cu to c to b2+- bathocuproine disulfonic acid
Fluorescence probe, e adds for all reagents.
Figure 12 is the fluorescent emission intensity spectrogram of the detection liquid containing different component in embodiment 12, wherein, a is to be not added with
Acid phosphatase, to be not added with ascorbic acid -2- phosphoric acid, to be not added with bathocuproine disulfonic acid, d is to be not added with Cu to c to b2+- bathocuproine disulfonic acid
Fluorescence probe, e adds for all reagents.
Specific embodiment
With reference to embodiment and accompanying drawing, the invention will be further described.
Embodiment 1
The inventive method is used for the feasibility analysis experiment of detection of alkaline phosphatase activity.
By 20 μ L 0.05mg mL-1Alkaline phosphatase enzyme sample be added to 2980 μ L contain 2mM ascorbic acid -2- phosphoric acid and
0.03mM Cu2+In the detection liquid of-bathocuproine disulfonic acid fluorescence probe, after being incubated 15min at 25 DEG C, record detects the glimmering of liquid
Optical emission spectroscopy.
Feasibility analysis experiment in, institute scarce component same volume buffer solution replacement.It will be seen from figure 1 that in inspection
When being not added with alkaline phosphatase (a) or substrate (b) in survey liquid, fluorescent emission intensity of the detection liquid at 402nm is stronger；In detection liquid
In be not added with bathocuproine disulfonic acid (c), be not added with Cu2+- bathocuproine disulfonic acid fluorescence probe (d) or all components all in the presence of
E when (), the fluorescent emission intensity at~402nm is very weak.As can be seen here, fluorescent method of the invention can be used to detect alkaline phosphorus
The activity of sour enzyme.
Embodiment 2
The inventive method is used between the fluorescent emission intensity of detection of alkaline phosphatase activity and alkaline phosphatase activities
Relation.
It is 0,20,40,60,80,100,120,140,160,180 and 220mU mL by the concentration of 20 μ L-1Alkaline phosphatase
Enzyme sample is added separately to 2980 μ L and contains 2mM ascorbic acid -2- phosphoric acid and 0.03mM Cu2+- bathocuproine disulfonic acid fluorescence probe
Detection liquid in, after being incubated 15min at 25 DEG C, the fluorescence emission spectrum of record detection liquid.
Figure it is seen that with the increase of sample activity change of Alkaline phosphatase, fluorescence of the detection liquid at~402nm
Emissive porwer accordingly weakens, and fluorescent emission intensity has good quantitative relationship with alkaline phosphatase activities.As can be seen here, originally
The fluorescence analysis method of invention can be used for the quantitative determination of sample activity change of Alkaline phosphatase.
Embodiment 3
The inventive method is used for the selectivity of detection of alkaline phosphatase activity.
By 20 μ L 0.05mg mL-1Alkaline phosphatase or 20 μ L 0.5mg mL-1Other protein solutions be added to
2980 μ L contain 2mM ascorbic acid -2- phosphoric acid and 0.03mM Cu2+In the detection liquid of-bathocuproine disulfonic acid fluorescence probe, 25
After 15min being incubated at DEG C, fluorescent emission intensity of the record detection liquid at~402nm.
From figure 3, it can be seen that compared with blank, although the concentration of other protein is the 10 of alkaline phosphatase concentration
Times, but only when alkaline phosphatase is added, fluorescent emission intensity of the detection liquid at~402nm can just be remarkably decreased.By
This is visible, with selectivity very high when fluorescence analysis method of the invention is for detection of alkaline phosphatase activity.
Embodiment 4
Detection performance of the inventive method for detection of alkaline phosphatase when active in blood serum sample.
The normal human sera samples of 4,8,12,16 and 20 μ L are added separately to respectively contain 2mM ascorbic acid -2- phosphoric acid
With 0.03mM Cu2+In the detection liquid of-bathocuproine disulfonic acid fluorescence probe, after being incubated 15min at 25 DEG C, record detection liquid
Fluorescence emission spectrum.
From fig. 4, it can be seen that with the increase of blood serum sample volume, fluorescent emission intensity of the detection liquid at~402nm
It is corresponding to decline, and fluorescent emission intensity has good quantitative relationship with the volume of blood serum sample.As can be seen here, it is of the invention glimmering
Light analysis method can be used for the quantitative determination of blood serum sample activity change of Alkaline phosphatase, therefore with good practicality.
Embodiment 5
The inventive method is used to detect the feasibility analysis experiment of alpha-galactosidase activity.
By 20 μ L 0.01mg mL-1Alpha-galactoside enzyme sample enters to 2980 μ L to contain 2mM p-amino phenyl- α-D- pyrans
Galactoside and 0.03mM Cu2+In the detection liquid of-bathocuproine disulfonic acid fluorescence probe, after being incubated 15min at 25 DEG C, record
Detect the fluorescence emission spectrum of liquid.
Feasibility analysis experiment in, institute scarce component same volume buffer solution replacement.From fig. 5, it can be seen that in inspection
Survey in liquid when being not added with alpha-galactosidase (a) or substrate (b), fluorescent emission intensity of the detection liquid at~402nm is stronger；In inspection
Survey in liquid and be not added with bathocuproine disulfonic acid (c), be not added with Cu2+The feelings that-bathocuproine disulfonic acid fluorescence probe (d) or all components are all present
Under condition when (e), the fluorescent emission intensity at~402nm is very weak.As can be seen here, fluorescence analysis method of the invention can be used to examine
Survey the activity of alpha-galactosidase.
Embodiment 6
The feasibility analysis that the inventive method is used for detectionofβ-galactosidaseactivity is tested.
By 20 μ L 0.01mg mL-1Beta galactosidase sample is added to 2980 μ L and contains 2mM p-amino phenyl- β-D- pyrroles
Mutter galactoside and 0.03mM Cu2+In the detection liquid of-bathocuproine disulfonic acid fluorescence probe, after being incubated 15min at 25 DEG C, note
The fluorescence emission spectrum of record detection liquid.
Feasibility analysis experiment in, institute scarce component same volume buffer solution replacement.From fig. 6, it can be seen that in inspection
Survey in liquid when being not added with beta galactosidase (a) or substrate (b), fluorescent emission intensity of the detection liquid at~402nm is stronger；In inspection
Survey in liquid and be not added with bathocuproine disulfonic acid (c), be not added with Cu2+The feelings that-bathocuproine disulfonic acid fluorescence probe (d) or all components are all present
Under condition when (e), the fluorescent emission intensity at~402nm is very weak.As can be seen here, fluorescence analysis method of the invention can be used to examine
Survey the activity of beta galactosidase.
Embodiment 7
The inventive method is used to detect the feasibility analysis experiment of alpha-glucosidase activity.
By 20 μ L 0.05mg mL-1Alpha-glucosaccharase enzyme sample is added to 2980 μ L and contains 2mM p-amino phenyl- α-D- pyrroles
Glucopyranoside glycosides and 0.03mM Cu2+In the detection liquid of-bathocuproine disulfonic acid fluorescence probe, after being incubated 15min at 25 DEG C, note
The fluorescence emission spectrum of record detection liquid.
Feasibility analysis experiment in, institute scarce component same volume buffer solution replacement.From figure 7 it can be seen that in inspection
Survey in liquid when being not added with alpha-glucosidase (a) or substrate (b), fluorescent emission intensity of the detection liquid at~402nm is stronger；In inspection
Survey in liquid and be not added with bathocuproine disulfonic acid (c), be not added with Cu2+The feelings that-bathocuproine disulfonic acid fluorescence probe (d) or all components are all present
Under condition when (e), the fluorescent emission intensity at~402nm is very weak.As can be seen here, fluorescence analysis method of the invention can be used to examine
Survey the activity of alpha-glucosidase.
Embodiment 8
The inventive method is used to detect the feasibility analysis experiment of activity of beta-glucosidase.
By 20 μ L 0.05mg mL-1Beta-glucosidase enzyme sample is added to 2980 μ L and contains 2mM p-amino phenyl- β-D- pyrroles
Glucopyranoside glycosides and 0.03mM Cu2+In the detection liquid of-bathocuproine disulfonic acid fluorescence probe, after being incubated 15min at 25 DEG C, note
The fluorescence emission spectrum of record detection liquid.
Feasibility analysis experiment in, institute scarce component same volume buffer solution replacement.From figure 8, it is seen that in inspection
Survey in liquid when being not added with beta-glucosidase (a) or substrate (b), fluorescent emission intensity of the detection liquid at~402nm is stronger；In inspection
Survey in liquid and be not added with bathocuproine disulfonic acid (c), be not added with Cu2+The feelings that-bathocuproine disulfonic acid fluorescence probe (d) or all components are all present
Under condition when (e), the fluorescent emission intensity at~402nm is very weak.As can be seen here, fluorescence analysis method of the invention can be used to examine
Survey the activity of beta-glucosidase.
Embodiment 9
The inventive method is used to detect the feasibility analysis experiment of alpha-Mannosidase activity.
By 20 μ L 0.01mg mL-1Alpha-Mannosidase sample is added to 2980 μ L and contains 2mM p-amino phenyl- α-D- pyrroles
Mutter mannoside and 0.03mM Cu2+In the detection liquid of-bathocuproine disulfonic acid fluorescence probe, after being incubated 15min at 25 DEG C, note
The fluorescence emission spectrum of record detection liquid.
Feasibility analysis experiment in, institute scarce component same volume buffer solution replacement.From fig. 9, it can be seen that in inspection
Survey in liquid when being not added with alpha-Mannosidase (a) or substrate (b), fluorescent emission intensity of the detection liquid at~402nm is stronger；In inspection
Survey in liquid and be not added with bathocuproine disulfonic acid (c), be not added with Cu2+The feelings that-bathocuproine disulfonic acid fluorescence probe (d) or all components are all present
Under condition when (e), the fluorescent emission intensity at~402nm is very weak.As can be seen here, fluorescence analysis method of the invention can be used to examine
Survey the activity of alpha-Mannosidase.
Embodiment 10
The inventive method is used to detect the feasibility analysis experiment of beta-Mannosidase activity.
By 20 μ L 0.01mg mL-1Beta-Mannosidase sample is added to 2980 μ L and contains 2mM p-amino phenyl- β-D- pyrroles
Mutter mannoside and 0.03mM Cu2+In the detection liquid of-bathocuproine disulfonic acid fluorescence probe, after being incubated 15min at 25 DEG C, note
The fluorescence emission spectrum of record detection liquid.
Feasibility analysis experiment in, institute scarce component same volume buffer solution replacement.From fig. 10 it can be seen that in inspection
Survey in liquid when being not added with beta-Mannosidase (a) or substrate (b), fluorescent emission intensity of the detection liquid at~402nm is stronger；In inspection
Survey in liquid and be not added with bathocuproine disulfonic acid (c), be not added with Cu2+The feelings that-bathocuproine disulfonic acid fluorescence probe (d) or all components are all present
Under condition when (e), the fluorescent emission intensity at~402nm is very weak.As can be seen here, fluorescence analysis method of the invention can be used to examine
Survey the activity of beta-Mannosidase.
Embodiment 11
The inventive method is used to detect the feasibility analysis experiment of Soil neutral phosphatase activity.
By 20 μ L 0.05mg mL-1Soil neutral phosphatase sample be added to 2980 μ L contain 2mM ascorbic acid -2- phosphoric acid and
0.03mM Cu2+In the detection liquid of-bathocuproine disulfonic acid fluorescence probe, after being incubated 15min at 25 DEG C, record detects the glimmering of liquid
Optical emission spectroscopy.
Feasibility analysis experiment in, institute scarce component same volume buffer solution replacement.It can be seen from figure 11 that in inspection
Survey in liquid when being not added with Soil neutral phosphatase (a) or substrate (b), fluorescent emission intensity of the detection liquid at~402nm is stronger；In detection
Bathocuproine disulfonic acid (c) is not added with liquid, Cu is not added with2+The situation that-bathocuproine disulfonic acid fluorescence probe (d) or all components are all present
Under (e) when, the fluorescent emission intensity at~402nm is very weak.As can be seen here, fluorescence analysis method of the invention can be with detection
The activity of acid phosphatase.
Embodiment 12
The inventive method is used to detect the feasibility analysis experiment of activity of acid phosphatase.
By 20 μ L 0.05mg mL-1Acid phosphatase enzyme sample be added to 2980 μ L contain 2mM ascorbic acid -2- phosphoric acid and
0.03mM Cu2+In the detection liquid of-bathocuproine disulfonic acid fluorescence probe, after being incubated 15min at 25 DEG C, record detects the glimmering of liquid
Optical emission spectroscopy.
Feasibility analysis experiment in, institute scarce component same volume buffer solution replacement.It can be recognized from fig. 12 that in inspection
Survey in liquid when being not added with acid phosphatase (a) or substrate (b), fluorescent emission intensity of the detection liquid at~402nm is stronger；In detection
Bathocuproine disulfonic acid (c) is not added with liquid, Cu is not added with2+The situation that-bathocuproine disulfonic acid fluorescence probe (d) or all components are all present
Under (e) when, the fluorescent emission intensity at~402nm is very weak.As can be seen here, fluorescence analysis method of the invention can be used to detect
The activity of acid phosphatase.
Claims (10)
1. a kind of fluorescence detection method of hydrolytic enzyme activities, it is characterised in that comprise the following steps：Substrate and Cu will be contained2+- bath
The detection liquid of copper spirit disulfonic acid fluorescence probe is incubated with hydrolysis enzyme solutions to be measured, generates Cu+- bathocuproine disulfonic acid complex compound, surveys
Fluorescent emission intensity of the reaction solution at 402nm is determined, according to fluorescent emission intensity and the standard curve relation of hydrolytic enzyme activities, meter
Calculation obtains the activity of hydrolysis enzyme solutions reclaimed water solution enzyme to be measured；Described substrate is irreducibility substrate, with hydrolysis enzyme reaction to be measured
Generation reproducibility product.
2. the fluorescence detection method of hydrolytic enzyme activities according to claim 1, it is characterised in that described Cu2+- bathocuproine
Disulfonic acid fluorescence probe is by Cu2+It is mixed with bathocuproine disulfonic acid.
3. the fluorescence detection method of hydrolytic enzyme activities according to claim 1, it is characterised in that described hydrolase is selected from
Alpha-galactosidase, beta galactosidase, alpha-glucosidase, beta-glucosidase, alpha-Mannosidase, β-mannoside
Enzyme, alkaline phosphatase, Soil neutral phosphatase or acid phosphatase.
4. the fluorescence detection method of hydrolytic enzyme activities according to claim 1, it is characterised in that described hydrolase be α-
Galactosidase, substrate is p-amino phenyl- α-D- galactopyranosides.
5. the fluorescence detection method of hydrolytic enzyme activities according to claim 1, it is characterised in that described hydrolase be β-
Galactosidase, substrate is p-amino phenyl- β-D- galactopyranosides.
6. the fluorescence detection method of hydrolytic enzyme activities according to claim 1, it is characterised in that described hydrolase be α-
Glucuroide, substrate is p-amino phenyl- α-D- glucopyranosides.
7. the fluorescence detection method of hydrolytic enzyme activities according to claim 1, it is characterised in that described hydrolase be β-
Glucuroide, substrate is p-amino phenyl- β-D- glucopyranosides.
8. the fluorescence detection method of hydrolytic enzyme activities according to claim 1, it is characterised in that described hydrolase be α-
Mannosidase, substrate is p-amino phenyl- α-D- mannopyranose glycosides.
9. the fluorescence detection method of hydrolytic enzyme activities according to claim 1, it is characterised in that described hydrolase be β-
Mannosidase, substrate is p-amino phenyl- β-D- mannopyranose glycosides.
10. the fluorescence detection method of hydrolytic enzyme activities according to claim 1, it is characterised in that described hydrolase is
Alkaline phosphatase, Soil neutral phosphatase or acid phosphatase, substrate are ascorbic acid -2- phosphoric acid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611025012.5A CN106706578A (en) | 2016-11-22 | 2016-11-22 | Fluorescence detection method for hydrolase activity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611025012.5A CN106706578A (en) | 2016-11-22 | 2016-11-22 | Fluorescence detection method for hydrolase activity |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106706578A true CN106706578A (en) | 2017-05-24 |
Family
ID=58940115
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611025012.5A Pending CN106706578A (en) | 2016-11-22 | 2016-11-22 | Fluorescence detection method for hydrolase activity |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106706578A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108827948A (en) * | 2018-08-08 | 2018-11-16 | 福建医科大学 | Acid phosphatase electrogenerated chemiluminescence measuring method based on gold nano cluster probe |
| CN109270041A (en) * | 2018-10-29 | 2019-01-25 | 济南大学 | A kind of method of quantitative detection alkaline phosphatase activities |
| CN111879741A (en) * | 2020-07-15 | 2020-11-03 | 安徽师范大学 | Method for detecting activity of alpha-glucosidase |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1697880A (en) * | 2002-09-09 | 2005-11-16 | 阿普尔拉股份有限公司 | Fluorescent enzyme assay methods and compositions |
| CN1774267A (en) * | 2003-01-22 | 2006-05-17 | 综合医院有限公司 | Amyloid-binding, metal-chelating agents |
| CN105259168A (en) * | 2015-10-15 | 2016-01-20 | 南京理工大学 | Method for measuring alkaline phosphatase activity |
| CN105543335A (en) * | 2016-01-08 | 2016-05-04 | 南京理工大学 | Measurement method of acid phosphatase activity |
-
2016
- 2016-11-22 CN CN201611025012.5A patent/CN106706578A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1697880A (en) * | 2002-09-09 | 2005-11-16 | 阿普尔拉股份有限公司 | Fluorescent enzyme assay methods and compositions |
| CN1774267A (en) * | 2003-01-22 | 2006-05-17 | 综合医院有限公司 | Amyloid-binding, metal-chelating agents |
| CN105259168A (en) * | 2015-10-15 | 2016-01-20 | 南京理工大学 | Method for measuring alkaline phosphatase activity |
| CN105543335A (en) * | 2016-01-08 | 2016-05-04 | 南京理工大学 | Measurement method of acid phosphatase activity |
Non-Patent Citations (1)
| Title |
|---|
| 贲长恩: "《组织化学》", 30 November 2001 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108827948A (en) * | 2018-08-08 | 2018-11-16 | 福建医科大学 | Acid phosphatase electrogenerated chemiluminescence measuring method based on gold nano cluster probe |
| CN109270041A (en) * | 2018-10-29 | 2019-01-25 | 济南大学 | A kind of method of quantitative detection alkaline phosphatase activities |
| CN111879741A (en) * | 2020-07-15 | 2020-11-03 | 安徽师范大学 | Method for detecting activity of alpha-glucosidase |
| CN111879741B (en) * | 2020-07-15 | 2023-03-28 | 安徽师范大学 | Method for detecting activity of alpha-glucosidase |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wang et al. | A bioluminescent sensor for highly selective and sensitive detection of human carboxylesterase 1 in complex biological samples | |
| CN102375021B (en) | Electrochemical method employing DNA as probe to detect environmental pollutant | |
| Chen et al. | Determination of bisphenol‐A levels in human amniotic fluid samples by liquid chromatography coupled with mass spectrometry | |
| Wang et al. | Hydroxylamine amplified gold nanoparticle-based aptameric system for the highly selective and sensitive detection of platelet-derived growth factor | |
| Xia et al. | Gold nanoparticle-based colorimetric method for the detection of prostate-specific antigen | |
| CN105510420A (en) | Method for determining ATP content on basis of magnetic bead separation and DNA marker gold nanoparticle probe | |
| Pan et al. | Turn-on fluorescence measurement of acid phosphatase activity through an aggregation-induced emission of thiolate-protected gold nanoclusters | |
| CN106706578A (en) | Fluorescence detection method for hydrolase activity | |
| de Pedro et al. | Analytical validation of telomere analysis technology® for the high-throughput analysis of multiple telomere-associated variables | |
| Zhang et al. | A path-choice-based biosensor to detect the activity of the alkaline phosphatase as the switch | |
| CN113340863B (en) | Enzyme-free circulating amplification aptamer sensor and preparation method and application thereof | |
| Ye et al. | Sensitive detection of alkaline phosphatase based on terminal deoxynucleotidyl transferase and endonuclease IV-assisted exponential signal amplification | |
| CN109632757A (en) | Fluorescence analysis method based on carbon quantum dot detection activity of acid phosphatase | |
| Lv et al. | On-line galvanic cell generated electrochemiluminescence determination of acyclovir based on the flow injection sampling | |
| KR100449216B1 (en) | Measurement method of test substance by chemiluminescence amount control | |
| WO2007011778A2 (en) | Use of raman spectroscopy in enzyme activity assays | |
| Li et al. | Sensitive fluorometric detection of alkaline phosphatase using a water-soluble conjugated polymer | |
| Huang et al. | An enzyme-activatable dual-readout probe for sensitive β-galactosidase sensing and Escherichia coli analysis | |
| CN109946355A (en) | A kind of electrochemical luminescence method detecting tumor markers | |
| Zhou et al. | An enzyme-induced metallization-based electrochemical signal amplification strategy for ultrahigh sensitive alkaline phosphatase detection at attomolar concentrations | |
| Shang et al. | A turn-on fluorescent strategy for alkaline phosphatase detection based on enzyme-assisted signal amplification | |
| CN109971463B (en) | Ratiometric fluorescent probe for detecting intracellular lysosome NO and application thereof | |
| CN104360074A (en) | Time-resolved fluorescence immunoassay method of Lp-PLA2 and kit | |
| CN104359957B (en) | A kind of electrochemical sensor and preparation and application thereof | |
| CN107356577A (en) | A kind of universal sulfotransferase activity assays |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170524 |