CN109207555A - A kind of DNase I activity colorimetric detection method - Google Patents
A kind of DNase I activity colorimetric detection method Download PDFInfo
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- 102000007260 Deoxyribonuclease I Human genes 0.000 title abstract description 65
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract
The invention discloses a kind of active colorimetric detection methods of the deoxyribonuclease I (Deoxyribonuclease I, DNase I) for adjusting MIL-53 (Fe) peroxidase activity based on DNA.The present invention uses cheap 3,3', 5,5'- tetramethyl benzidine (TMB) is colorimetric substrates, the characteristic of MIL-53 (Fe) peroxidase activity can be effectively adjusted based on ssDNA, using DNase I to the hydrolysis of ssDNA, the achievable naked eye observation active biosensor of DNase I is constructed.Developing a kind of has many advantages, such as at low cost, simple, convenient quick colorimetric analysis.This method high sensitivity, stability are good, are suitable for promoting and applying.
Description
Technical field
The invention belongs to analytical chemistry fields, and in particular to one kind adjusts MIL-53 (Fe) peroxidase activity based on DNA
The DNase I activity colorimetric detection method of property.
Background technique
DNase I is that one kind can generate monodeoxyribonucleotide with hydrolysing single or double-stranded DNA or single-stranded or double-stranded widow is de-
The endonuclease of oxygen nucleotide.It is distributed widely in mammalian body fluid and tissue, especially hypophysis and pancreas.In addition to crowd
Outside well known pancreas digestion, DNase I plays a significant role in DNA reparation, genetic recombination and Apoptosis.
The shortage of DNase I or reduction can cause human system's property lupus erythematosus, gastric cancer and colon cancer, and breast cancer and patient of diabetes
DNase I activity in person's serum is higher than normal level.In addition, DNase I has been used as acute myocardial infarction and the transience heart
The diagnostic marker of the cardiovascular diseases such as myocardial ischemia.Therefore, a kind of high sensitivity and selective good DNase I activity point are developed
Analysis method is most important for bioanalysis, clinical diagnosis and drug screening.
Traditional detection DNase I activity methods have an enzyme linked immunosorbent assay (ELISA), minor effect genes method, electrochemical determination,
Fluorescence method, colorimetric method etc..Wherein, the colorimetric method for the advantages that there is easy to operate, cost performance height, as a result can visually observe, at
For the main stream approach of DNase I detection.
Metal organic framework (MOFs) material is a kind of relatively new porous crystalline material, by transition metal ions or cluster
It is connected with multi-functional organic ligand.It is reported that such as MIL-53 of the MOF based on iron, MIL-88, MIL-101 etc. are shown
The catalytic activity of peroxidase, they are successfully applied to H2O2, ascorbic acid, the colorimetric bio biography of the substances such as glucose
In the design of sensor.Since Mirkin and Alivisatos group foundes and starts sex work, the cross materials of DNA have been related to it
Various applications, such as the directional assembly of nanostructure, bio-sensing and drug conveying are used for by wide coverage.Benefit from its height
Programmable feature, desired specificities combine or cracking, unique interaction on structure transfer capability and bioelectric interface,
DNA is very outstanding biopolymer, and is included in bio-nanotechnology extensively.The study found that nucleic acid and MOFs are passed through
Covalent bond or intermolecular force combine the additional molecules recognition capability and stability that can promote MOFs.Oligonucleotides-
MOFs compound realizes H5N1 antibody, DNA and Hg as fluorescence probe2+Detection have been reported.Chen reports a seed nucleus
The Nanosized metal organic frame (NMOFs) of acid functionalization, for loading stimulate the reaction medium (fluorescence probe or anticarcinogen
Object).The ferriporphyrin MOFs and AuNPs and Streptavidin (SA) that Ju develops DNA functionalization are realized real as electrochemical probe
When Pb2+It is detected with DNA.However, the research for being adjusted the intrinsic peroxidase property of MIL-53 (Fe) using DNA is still rarely had
Report.
Research reports that the oligonucleotides on nano-interface is by promoting enzyme-substrate affinity to significantly increase consolidating for nano enzyme
There is an enzymatic activity, also some researches show that DNA there are inhibitory enzyme activity since physics hinders or electrostatic repulsion.It is opened by these reports
Hair and our lasting research interests, present invention discover that the single stranded DNA (ssDNA) of different length can effectively adjust MIL-53
(Fe) peroxidase activity, wherein the long-chain ssDNA for possessing longer base number possesses stronger facilitation, and base number
Short chain ssDNA on the low side but can not effectively influence the peroxidase activity of MIL-53 (Fe).Different ssDNA length provide not
Same enzyme-substrate affinity.Based on this characteristic, the property of short chain ssDNA is effectively hydrolyzed into long-chain ssDNA in conjunction with DNase I
Matter detects the activity of DNase I we have developed a kind of simple and reliable colorimetric sensor.As far as we know, this is studied
It is the rare example that the Peroxidase Activity Determination DNase I of MIL-53 (Fe) is adjusted using ssDNA.Its performance is full
Foot is convenient, quickly, sensitive, the requirement of specific detection DNase I, therefore may have very big potential clinic in the near future
Application value.
Summary of the invention
The object of the present invention is to provide a kind of deoxyribose cores that MIL-53 (Fe) peroxidase activity is adjusted based on DNA
The active colorimetric detection method of sour enzyme I (Deoxyribonuclease I, DNase I), one kind adjusting MIL-53 (Fe) mistake based on DNA
Peroxidase activity is for simple, cheap, effective, the quick detection active colorimetric method of DNase I.The method is based on DNase
I is effectively hydrolyzed into the property of short chain ssDNA to long-chain ssDNA, while using different length ssDNA to the peroxide of MIL-53 (Fe)
The difference that compound analogue enztme activity influences, the colorimetric substrates TMB of blue is shown using cheap and oxidation product, and building is practical
Feasible effective colorimetric detection scheme.
For achieving the above object, the present invention adopts the following technical scheme:
One kind adjusting the DNase I activity colorimetric detection method of MIL-53 (Fe) peroxidase activity, the side based on DNA
Method specifically comprises the following steps:
(1) with low-cost terephthalic acid (TPA) and FeCl3·6H2O synthesizes metal-organic framework materials as reaction raw materials
MIL-53 (Fe): firstly, by the FeCl of the terephthalic acid (TPA) of 0.334g and 0.545g3·6H2O is dissolved in the DMF of 10mL
In, and the mixture is stirred at room temperature 10 minutes.Acquired solution is transferred to the stainless steel of 50mL teflon lined
In autoclave, then it is sealed and placed in 150 DEG C of baking oven 17 hours.
(2) after being cooled to room temperature the product in step (1), by heavy with 3 minutes collection yellow of 6000rpm centrifugation
It forms sediment, and removes extra reactant with deionized water cleaning down.Product is dried in vacuo 24 hours at 60 DEG C.Finally, obtaining
It obtains yellow product MIL-53 (Fe).
(3) that MIL-53 (Fe) obtained in step (2) is configured to aqueous solution is stand-by, mixes every time using on preceding earthquake device
It is even.
(4) ssDNA is placed in 1 × DNase I reaction buffer (10mM Tris-HCl, 2.5mM MgCl2, 0.5mM
CaCl2, pH 7.6) in, it is slowly cooled to room temperature after five minutes in 95 DEG C of heating.Then, various concentration is added into above-mentioned solution
DNaseI and reacted 2 hours at 37 DEG C.
(5) next, the MIL-53 (Fe) prepared in step (3) is added in the system of step (4).Then, it is added dense
The TMB being dissolved in ethyl alcohol is added as reaction substrate, then as buffer solution in the NaOAc-HAc that degree is 10mM, pH 4.0
H is added2O2Initiation reaction.
(6) system of step (5) is reacted after forty minutes at 40 DEG C, Centrifuge A sample from mixture to remove MIL-53
(Fe), the active analysis of DNase I is completed by detecting the UV-Vis absorption signal of supernatant.
The size of MOF material MIL-53 (Fe) is about 1-3 μm in the step (1).
The ssDNA concentration being added in the step (4) is 200nM.
The final concentration of 5 μ g mL of MIL-53 (Fe) being added in the step (5)-1。
The preparation method of NaOAc-HAc solution in the step (5) is: NaOAc and HAc, which is hybridly prepared into pH value, is
The NaOAc-HAc solution of 4-9, the pH are preferably 4.0.
The TMB concentration for being dissolved in ethyl alcohol being added in the step (5) is 0.2mM.
The H being added in the step (5)2O2Concentration is 2.5mM.
The detectable concentration range of DNase I to be measured is 0.2~7U mL in the step (6)-1(R2=0.997), detection limit
For 0.09U mL-1。
More specifically, a kind of DNase I for adjusting MIL-53 (Fe) peroxidase activity based on DNA of the present invention
Active colorimetric detection method, includes the following steps:
(1) with the FeCl of the terephthalic acid (TPA) of 0.334g and 0.545g3·6H2O is dissolved in the DMF of 10mL as reaction raw materials
In, and the mixture is stirred at room temperature 10 minutes.Acquired solution is transferred to the height of the stainless steel in 50mL polytetrafluoroethylene (PTFE)
It presses in kettle, is then sealed and placed in 150 DEG C of baking oven 17 hours.
(2) after being cooled to room temperature the product in step (1), by heavy with 3 minutes collection yellow of 6000rpm centrifugation
It forms sediment, and removes extra reactant with deionized water cleaning down.Product is dried in vacuo 24 hours at 60 DEG C.Finally, obtaining
It obtains yellow product MIL-53 (Fe).(3) that MIL-53 (Fe) obtained in step (2) is configured to aqueous solution is stand-by, uses every time
It is mixed on preceding earthquake device.
(4) PCR pipe of 200 μ L is added in LifeECO PCR instrument (BIOER, Hangzhou) in the ssDNA of 200nM
95 DEG C are heated 5 minutes and are slowly cooled to room temperature.Then, in 1 × DNase I reaction buffer (10mM Tris-HCl, 2.5mM
MgCl2, 0.5mM CaCl2, pH 7.6) in be added various concentration DNase I and reacted 2 hours at 37 DEG C.
(5) next, the MIL-53 (Fe) configured in step (3) is added and the 5 minutes systems that step (4) are added that are vortexed.
It is the NaOAc-HAc of 10mM, pH 4.0 as buffer solution that concentration, which is added, and 0.2mM is added and is dissolved in the TMB in ethyl alcohol as anti-
Substrate is answered, 2.5mM H is then added2O2Initiation reaction.
(6) system of step (5) is reacted after forty minutes at 40 DEG C, Centrifuge A sample from mixture to remove MIL-53
(Fe), and using blue supernatant peroxidase activity is monitored.Absorption signal passes through UV-Vis spectral measurement.
The present invention adjusts MIL-53 (Fe) peroxidase activity colorimetric detection DNase I activity using DNA, uses price
Cheap and oxidation product, which shows blue, may be implemented the substrate TMB of naked eye observation.Macroscopic blue is presented in TMB after being oxidized
And it is shown when not oxidized colourless.The present invention is effectively hydrolyzed into the property of short chain DNA, while base using DNase I to long chain DNA
In different length ssDNA to the great disparity of the Mimetic Peroxidase activity influence of MIL-53 (Fe), construct a kind of feasible effective
Detect the colorimetric analysis of DNase I.The detectable concentration range of DNase I to be measured is 0.2-7.0U mL-1(R2=0.997) it, examines
Rising limit is 0.09U mL-1。
Fig. 1 is DNase I Activity determination feasibility analysis figure.It is (bent compared with the weak absorbance intensity of the sample of not DNA
Line a), its strong 2.4 times of (curve b) of the dulling luminosity ratio of the sample containing long ssDNA.However, into the sample with long ssDNA
Only add 10U mL-1After DNase I, absorbance intensity reduces immediately (curve d).In order to confirm merely due to DNaseI activity is added
And absorbance intensity is reduced, control experiment (curve e) is carried out using the DNase I of heat inactivation.As expected, it inactivates
DNase I fail to prevent long ssDNA enhancing absorbance intensity, this shows that the reduction of absorbance intensity in DNase I system can
To be truly reflected the activity of this enzyme.(the curve c) in addition, DNase I itself does not play a role in all samples.Cause
This adjusts MIL-53 (Fe) by using DNA and is used as sensing platform, and the mixing and detection process can be used for quantifying DNase I points
Analysis.Testing conditions are as follows: [M24]=200nM, [DNase I]=10U mL-1, [TMB]=0.2mM, [H2O2]=2.5mM,
[MIL-53 (Fe)]=5 μ g mL-1。
Fig. 2 is the working curve diagram of DNase I.The DNase I of various concentration absorbance at 652nm for reaction system
The variation of intensity influences.Insert pictures show that the DNase I range of linearity is 0.2-7U mL-1, detect and be limited to 0.09U mL-1.Inspection
Survey condition are as follows: [M24]=200nM, [TMB]=0.2mM, [H2O2]=2.5mM, [MIL-53 (Fe)]=5 μ g mL-1。A0With A points
It is not absorbance of the DNase I the absence and presence of lower reaction system.
The peroxidase activity for the MIL-53 (Fe) that the present invention uses is powerful reliable, and in identical pH various concentration
It is able to maintain in NaOAc 24 hours or more and is stabilized the structure of matter.MIL-53 can be effectively adjusted in view of different length ssDNA
(Fe) property of peroxidase simply, conveniently, visually, can be realized rapidly by colorimetric method and be quantified to DNase I
It is detected with bioactivity.
Detailed description of the invention
Fig. 1 DNase I Activity determination mechanism figure.
The working curve diagram of Fig. 2 DNase I.
Specific embodiment
Embodiment 1
(1) with low-cost terephthalic acid (TPA) and FeCl3·6H2O synthesizes metal-organic framework materials as reaction raw materials
MIL-53 (Fe): firstly, by the FeCl of the terephthalic acid (TPA) of 0.334g and 0.545g3·6H2O is dissolved in the DMF of 10mL, and will
The mixture is stirred at room temperature 10 minutes.Acquired solution is transferred to the stainless steel autoclave of 50mL teflon lined
In, it is then sealed and placed in 150 DEG C of baking oven 17 hours.
(2) after being cooled to room temperature the product in step (1), by heavy with 3 minutes collection yellow of 6000rpm centrifugation
It forms sediment, and removes extra reactant with deionized water cleaning down.Product is dried in vacuo 24 hours at 60 DEG C.Finally, obtaining
It obtains yellow product MIL-53 (Fe).(3) aqueous solution that MIL-53 (Fe) is configured to 0.1mg/mL obtained in step (2) is waited for
With every time using being mixed on preceding earthquake device.
(4) 2.5 μM of ssDNA (M24) of 8 μ L are added in the PCR pipe of 200 μ L, make it in LifeECO PCR instrument
It heats 5 minutes and is slowly cooled to room temperature for 95 DEG C in (BIOER, Hangzhou).Then, in 1 × DNase I reaction buffer
(10mM Tris-HCl, 2.5mM MgCl2, 0.5mM CaCl2, pH 7.6) in be added various concentration (0.2-20U mL-1)
DNase I simultaneously reacts 2 hours at 37 DEG C.
(5) next, the 0.1mg/mL MIL-53 (Fe) configured in 5 μ L steps (3) is added and shakes the step of addition in 5 minutes
Suddenly the system of (4).It is the NaOAc-HAc of 50mM, pH 4.0 as buffer solution that 20 μ L concentration are added into system, and 4 μ L are added
The TMB of 5mM in ethyl alcohol is dissolved in as reaction substrate, the 10mM H of 25 μ L is then added2O2Initiation reaction.System total volume is
100μL。
(6) system of step (5) is reacted after forty minutes at 40 DEG C, Centrifuge A sample from mixture to remove MIL-53
(Fe), and using blue supernatant peroxidase activity is monitored.Absorption signal passes through UV-Vis spectral measurement.
The final concentration of 200nM for the ssDNA being added in the step (4).
The final concentration of 5 μ g mL of MIL-53 (Fe) being added in the step (5)-1。
The preparation method of NaOAc-HAc solution in the step (5) is: NaOAc and HAc, which is hybridly prepared into pH value, is
The NaOAc-HAc solution of 4-9, the pH are preferably 4.0.NaOAc-HAc solution ultimate density in system is 10mM.
The TMB concentration for being dissolved in ethyl alcohol being added in the step (5) is 0.2mM.
The H being added in the step (5)2O2Concentration is 2.5mM.
The detectable concentration range of DNase I to be measured is 0.2-7.0U mL in the step (6)-1(R2=0.997) it, detects
It is limited to 0.09U mL-1。
Fig. 1 is DNase I Activity determination feasibility analysis figure.It is (bent compared with the weak absorbance intensity of the sample of not DNA
Line a), its strong 2.4 times of (curve b) of the dulling luminosity ratio of the sample containing long ssDNA.However, into the sample with long ssDNA
Only add 10U mL-1After DNase I, absorbance intensity reduces immediately (curve d).In order to confirm merely due to DNaseI activity is added
And absorbance intensity is reduced, control experiment (curve e) is carried out using the DNase I of heat inactivation.As expected, it inactivates
DNase I fail to prevent long ssDNA enhancing absorbance intensity, this shows that the reduction of absorbance intensity in DNase I system can
To be truly reflected the activity of this enzyme.(the curve c) in addition, DNase I itself does not play a role in all samples.Cause
This adjusts MIL-53 (Fe) by using DNA and is used as sensing platform, and the mixing and detection process can be used for quantifying DNase I points
Analysis.Testing conditions are as follows: [M24]=200nM, [DNase I]=10U mL-1, [TMB]=0.2mM, [H2O2]=2.5mM,
[MIL-53 (Fe)]=5 μ g mL-1。
Fig. 2 is the working curve diagram of DNase I.The DNase I of various concentration absorbance at 652nm for reaction system
The variation of intensity influences.Insert pictures show that the DNase I range of linearity is 0.2-7U mL-1, detect and be limited to 0.09U mL-1.Inspection
Survey condition are as follows: [M24]=200nM, [TMB]=0.2mM, [H2O2]=2.5mM, [MIL-53 (Fe)]=5 μ g mL-1。A0With A points
It is not absorbance of the DNase I the absence and presence of lower reaction system.
Although above having made detailed description to invention with a general description of the specific embodiments, at this
On the basis of invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Therefore,
These modifications or improvements without departing from theon the basis of the spirit of the present invention are fallen within the scope of the claimed invention.
A specific embodiment of the invention, but scope of protection of the present invention is not limited thereto, it is any to be familiar with this field
Within the technical scope disclosed by the invention, changes or replacements that can be thought of without creative work should all be contained technical staff
Lid is within protection scope of the present invention.Therefore, protection scope of the present invention should be to protect model defined by claims
Subject to enclosing.
Claims (8)
1. a kind of DNaseI activity colorimetric detection method, which is characterized in that specifically comprise the following steps:
(1) with terephthalic acid (TPA) and FeCl3·6H2O synthesizes metal-organic framework materials MIL-53 (Fe) as reaction raw materials: first
First, by the FeCl of the terephthalic acid (TPA) of 0.334g and 0.545g3·6H2O is dissolved in the DMF of 10mL, and by the mixture in room temperature
Acquired solution is transferred in the stainless steel autoclave in 50mL polytetrafluoroethylene (PTFE) lining z, then seals juxtaposition by lower stirring 10 minutes
17 hours in 150 DEG C of baking oven;
(2) after being cooled to room temperature the product in step (1), by being centrifuged 3 minutes collection yellow mercury oxides with 6000rpm, and
Extra reactant is removed with deionized water cleaning down;Product is dried in vacuo 24 hours at 60 DEG C, finally, obtaining yellow
Product MIL-53 (Fe);
(3) by MIL-53 (Fe) obtained in step (2) be configured to concentration be 0.1mg/mL aqueous solution it is stand-by, before each use
It is mixed on earthquake device;
(4) ssDNA is placed in 1 × DNaseI reaction buffer (10mMTris-HCl, 2.5mMMgCl2, 0.5mMCaCl2,
PH7.6 it in), is slowly cooled to room temperature after five minutes in 95 DEG C of heating, then, various concentration is added into above-mentioned solution
DNaseI simultaneously reacts 2 hours at 37 DEG C;
(5) next, the MIL-53 (Fe) prepared in step (3) is added in the system of step (4), then, concentration, which is added, is
The NaOAc-HAc of 10mM, pH=4.0 are added the TMB being dissolved in ethyl alcohol as reaction substrate, then add as buffer solution
Enter H2O2Initiation reaction;
(6) by the system of step (5) 40 DEG C reaction after forty minutes, Centrifuge A sample with from mixture remove MIL-53 (Fe),
UV-Vis absorption signal by detecting supernatant completes the active analysis of DNaseI.
2. a kind of DNaseI activity colorimetric detection method according to claim 1, which is characterized in that MOF in the step (1)
The size of material MIL-53 (Fe) is about 1-3 μm.
3. a kind of DNaseI activity colorimetric detection method according to claim 1, which is characterized in that add in the step (4)
The final concentration of 200nM of the ssDNA entered.
4. a kind of DNaseI activity colorimetric detection method according to claim 1, which is characterized in that add in the step (5)
The final concentration of 5 μ gmL of MIL-53 (Fe) entered-1。
5. a kind of DNaseI activity colorimetric detection method according to claim 1, which is characterized in that in the step (5)
The preparation method of NaOAc-HAc solution is: NaOAc and HAc are hybridly prepared into the NaOAc-HAc solution that pH value is 4-9, it is described
PH is preferably 4.0.
6. a kind of DNaseI activity colorimetric detection method according to claim 1, which is characterized in that add in the step (5)
The TMB concentration for being dissolved in ethyl alcohol entered is 0.2mM.
7. a kind of DNaseI activity colorimetric detection method according to claim 1, which is characterized in that add in the step (5)
The H entered2O2Concentration is 2.5mM.
8. a kind of DNaseI activity colorimetric detection method according to claim 1, which is characterized in that in the step (6) to
The detectable concentration range for surveying DNaseI is 0.2-7.0UmL-1, detect and be limited to 0.09UmL-1。
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