CN107723339A - Detect the nano-sensor and its detection method of O acetylglucosamine transferases - Google Patents
Detect the nano-sensor and its detection method of O acetylglucosamine transferases Download PDFInfo
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Abstract
The invention belongs to bioanalysis detection technique field, discloses a kind of nano-sensor and its detection method based on single quantum dots characterization O acetylglucosamine transferases.Nano-sensor of the present invention includes：The peptide chain of the flower biotin modification of cyanines 5/；On-radiation uridine acetylglucosamine and by the coated quantum dot of Streptavidin；The peptide chain of the biotin modification of colored cyanines 5/ is：Biotin‑Ser‑Thr‑Pro‑Val‑Ser‑Arg‑Ala‑Asn‑‑Met‑Lys‑Cy5；Nano-sensor detection method of the present invention is very simple, it is not necessary to carries out enzyme purification, it is not necessary to the synthesis of the saccharide donor of labelled with radioisotope, specific antibody and fluorescence UDP GlcNAc analogs, and test limit can be reduced to 3.47 × 10‑13Mole every liter, there is very high sensitivity.Nano-sensor of the present invention applies also for the quantitative context of detection of the measurement of enzyme kinetics parameter, the screening of OGT inhibitor and cell OGT activity.
Description
Technical field
The invention belongs to bioanalysis detection technique field, and in particular to one kind is based on single quantum dots characterization O- acetyl Portugal
The nano-sensor and its detection method of grapes glucosamine transferase.
Background technology
Protein glycosylation is a kind of posttranslational modification of generally existing, and wherein albumen is by the N- acetyl Portugal of an oxygen connection
Osamine (O-GlcNAc) monomer is modified in serine or threonine residues, is referred to as the glycosylation of oxygen connecting-type acetylglucosamine.
O-GlcNAc plays an important role during various kinds of cell, including signal transduction, gene expression, protein degradation, new old generation
Thank, circadian rhythms, and nerve degenerative diseases.Mankind's oxygen connection acetylglucosamine transferase (OGT) is a kind of cell
Endogenous enzymes, the GlcNAc monomers being catalyzed from uridine-acetylglucosamine (UDP-GlcNAc) be transferred to protein serine or
GlcNAc- β-serine/threonine that threonine residues are formed, and the imbalance of OGT activity is related to a variety of diseases, such as cancer,
Diabetes, alzheimer's disease.Therefore, OGT is likely to become the potential therapeutic targets of disease treatment.
At present, detect OGT activity generally by monitor radiolabeled glycosyl from saccharide donor (for example, UDP- [3H]-
GlcNAc protein substrate (for example, Nup62)) is transferred to measure.But radiolabeled method is related to the labelled reagent of costliness
With cumbersome separable programming.In order to overcome these limitations, a variety of Non-radioactive methods have been exploited for detecting OGT, including
Adsorb enzyme-linked immunoassay (ELISA), Azide-ELISA, nickel-nitrilotriacetic acid plate immunoassay, antiprotease cracking fluorescence
Resonance energy transfer (FRET) method etc..However, these methods are since it is desired that shift solution enzyme reaction product to microwell plate, spy
Heterogenetic antibody (for example, specific glycopeptide monoclonal antibody, anti-tetramethylrhodamine antibody), fluorescence UDP-GlcNAc analog
Complicated chemical synthesis, complex designing FRET dyestuffs equity, so efficiency is low, sensitivity is relatively poor.In addition, OGT is pair
Temperature sensitive enzyme, and the loss for the enzymatic activity that may occur during enzyme purification, therefore quantitatively detect in actual sample
Intracellular OGT activity is a huge challenge.
Therefore, there is an urgent need to develop it is a kind of do not need enzyme purification process can simply, quickly quantitatively detect OGT activity
With the method for screening OGT inhibitor.
The content of the invention
In order to solve the above problems, shifted the invention provides one kind based on single quantum dots characterization O- acetylglucosamines
The nano-sensor and its detection method of enzyme, nano-sensor detection method of the present invention are very simple, it is not necessary to enzyme purification is carried out,
The synthesis of the saccharide donor of labelled with radioisotope, specific antibody and fluorescence UDP-GlcNAc analogs is not needed, and is examined
3.47 × 10 can be reduced to by surveying limit-13Mole every liter, there is very high sensitivity.
The present invention is achieved through the following technical solutions：
One of the object of the invention is：A kind of nanometer based on single quantum dots characterization O- acetylglucosamine transferases is provided
Sensor, it includes：The peptide chain of the flower biotin modification of cyanines 5/；On-radiation uridine-acetylglucosamine, Proteinase K and by chain
The mould coated quantum dot of Avidin.
The two of the object of the invention are：A kind of nanometer based on single quantum dots characterization O- acetylglucosamine transferases is provided
The detection method of sensor, comprises the following steps：
S1. the peptide of the flower biotin modification of cyanines 5/ is glycosylated by the glycosyl of saccharide donor uridine-acetylglucosamine；
S2. Proteinase K distinguishes glycosylation and non-glycosylated peptide；
S3. glycosylated peptide self-assembles to quantum dot surface forming amount by the interaction of Streptavidin and biotin
The measurement of sub- point-peptide-Hua Jing 5 nanostructured and follow-up FRET signal.
The three of the object of the invention are：Above-mentioned nano-sensor is provided in the measurement of enzyme kinetics parameter, the sieve of OGT inhibitor
The application of the quantitative context of detection of choosing and cell OGT activity.
Nano-sensor of the present invention is to spend the peptide chain of cyanines 5 (Cy5)/biotin (biotin) modification to be used as substrate, this peptide chain
With the site that can be identified by the glycosylated serines of OGT and protease adjacent thereto, with common on-radiation UDP-
GlcNAc's is used as glycosyl donor.In the presence of OGT, it is catalyzed glycosylation to produce glycosylated peptide, the sugar of peptide chain
Base can be protected it from by protease cracking.Add by the coated quantum dot of Streptavidin (QD), the glycosylated Cy5/
The peptide of biotin modification may be connected to QD surfaces by the interaction of biotin-Streptavidin, to form QD- peptides-Cy5's
Nanostructured, so as to which FRET can occur between QD and Cy5.
Compared with prior art, the present invention has following technological merit：
(1) nano-sensor of the present invention has high sensitivity：Given birth in the technical program using the flower cyanines 5/ of OGT inductions
The glycosylation and the cracking of subsequent Proteinase K of the peptide of thing element modification can distinguish glycosylated peptide and nonglycosylated peptide, and this
Multiple glycosylated peptide substrates is assembled into single QD surfaces for invention so that FRET efficiency highs, in addition, the present invention uses
Single Molecule Detection, it has the advantages of high s/n ratio, and therefore, nano-sensor of the present invention has fabulous selectivity and very
High sensitivity；
(2) nano-sensor principle of the present invention is simple, and cost is low：Whole process does not need the purifying of enzyme, mustn't need yet
The saccharide donor and specific antibody of synthesizing radioactive mark and the UDP-GlcNAc homologues with fluorophor；
(3) OGT activity intracellular in actual sample can be detected.
Brief description of the drawings
Fig. 1 is the mechanism figure of nano-sensor of the present invention.
Fig. 2 is the measurement in QD the and Cy5 fluorescence in the absence of OGT (control) and when OGT be present.
Fig. 3 is by the single molecular imaging based on utilizing total internal reflection fluorescence microscope while detects a variety of DNA glycosylases.
In the single molecular fluorescence image in the absence of (A-C) and when OGT (D-F) be present.A, D are QD fluoroscopic examination result；B, E Cy5
Fluoroscopic examination result, C, F are that QD and Cy5 overlap testing result.The OGT concentration is that every liter of 0.5 nanomole is with QD concentration
0.02 every liter of nanomole.Engineer's scale is 8 microns.
Fig. 4：The counting of flower cyanines 5 corresponding to the OGT of various concentrations.Illustration display flower cyanines 5 are counted with OGT concentration to number form
Linear relationship between formula.What error bar represented is the standard deviation for repeating experiment three times.
Fig. 5：DNA methylation transferase (Dam MTase) (40 units per mls), phosphokinase (PNK) (40 units
Every milliliter), the count measurement of the flower cyanines 5 in the presence of protein kinase A (PKA) (40 units per mls), without any ferment treatment
Sample as control.What error bar represented is the standard deviation for repeating experiment three times.
Fig. 6：OSMI-1 suppresses the analysis of OGT activity.What error bar represented is the standard deviation for repeating experiment three times.
Embodiment
It is noted that described further below is all exemplary, it is intended to provides further instruction to the present invention.It is unless another
Indicate, all technologies used herein and scientific terminology are with usual with general technical staff of the technical field of the invention
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe embodiment, and be not intended to restricted root
According to the illustrative embodiments of the present invention.As used herein, unless the context clearly indicates otherwise, otherwise singulative
It is also intended to include plural form, additionally, it should be understood that, when in this manual using term "comprising" and/or " bag
Include " when, it indicates existing characteristics, step, operation, part and/or combinations thereof.
As background technology is introduced, the method for detecting OGT activity in the prior art has that efficiency is low, sensitivity is relative
The problem of poor.In addition, OGT is thermally sensitive enzyme, and the loss for the enzymatic activity that may occur during enzyme purification,
Therefore it is difficult quantitatively to detect intracellular OGT activity in actual sample.In order to solve the above problems, an object of the present invention is to carry
For a kind of nano-sensor based on single quantum dots characterization O- acetylglucosamine transferases, it includes：The flower biotin of cyanines 5/
The peptide chain of modification；On-radiation uridine-acetylglucosamine, Proteinase K and by the coated quantum dot of Streptavidin.
In currently preferred technical scheme, the peptide chain of the colored biotin modification of cyanines 5/ is：Biotin-Ser-Thr-
Pro-Val-Ser-Arg-Ala-Asn-Met-Lys-Cy5。
The two of the object of the invention are：A kind of nanometer based on single quantum dots characterization O- acetylglucosamine transferases is provided
The detection method of sensor, comprises the following steps：
S1. the peptide of the flower biotin modification of cyanines 5/ is glycosylated by the glycosyl of saccharide donor uridine-acetylglucosamine；
S2. Proteinase K distinguishes glycosylation and non-glycosylated peptide；
S3. glycosylated peptide self-assembles to quantum dot surface forming amount by the interaction of Streptavidin and biotin
The measurement of sub- point-peptide-Hua Jing 5 nanostructured and follow-up FRET signal.
In currently preferred technical scheme, step S1 operating procedure is：Contain every liter of flower cyanines 5/ of 2-3 micromoles
The peptide of biotin modification, uridine-acetylglucosamine of every liter of 90-100 micromoles, the alkaline phosphatase of 0.01-0.1 units,
The OGT and 10 × reaction buffer of various concentrations, composition cumulative volume are 1 small to be incubated in 20 microlitres of reaction solution at 37 DEG C
When；The reaction buffer is by 500 mMs of every liter of trishydroxymethylaminomethanes (Tris), 200 mMs every liter of calcium chloride,
0.5% Tween-20 and 0.5%NP-40 compositions, pH value 7.8.
In further optimal technical scheme of the invention, step S1 concrete operation step is：It is every containing 2.4 micromoles
The peptide of the biotin modification of flower cyanines 5/ risen, uridine-acetylglucosamine of 96 every liter of micromoles, the alkaline phosphatase of 0.05 unit
Enzyme, the OGT and 10 × reaction buffer of various concentrations, composition cumulative volume are to incubate 1 at 37 DEG C in 20 microlitres of reaction solution
Individual hour.
In currently preferred technical scheme, step S2 operating procedure is：1-2 nanogram Proteinase Ks are added to sugar
In glycosylation reaction product, reaction cumulative volume is 30 microlitres of system, and 1-3 hours are reacted at 50-60 DEG C.
In further preferable technical scheme of the invention, the concrete operation step of the step S2 is：By 1.5 nanograms
Proteinase K is added in glycosylation product, the system that reaction cumulative volume is 30 microlitres, is reacted 2 hours at 55 DEG C.
In currently preferred technical scheme, step S3 operating procedure is：Take 20-40 microlitres of step S2 reaction production
Thing is reacted 10-30 minutes at room temperature with every liter of quantum dot of 5-15 nanomoles in 100 microlitres of culture buffer solution, forms quantum
Point-peptide-Hua Jing 5 nanostructured；Wherein, it is described culture buffer solution by：3 mMs every liter of magnesium chloride, 100 mMs every liter
PH 8.0 trishydroxymethylaminomethane-hydrochloric acid and 10 mMs every liter ammonium sulfate composition, pH value 8.0.
In the further preferred technical scheme of the present invention, step S3 concrete operation step is：Take 30 microlitres of step S2
Reaction product reacted at room temperature 20 minutes in 100 microlitres of culture buffer solution with every liter of quantum dot of 10 nanomole, formed
Quantum dot-peptide-Hua Jing 5 nanostructured.
The third object of the present invention is to provide a kind of measurement for providing above-mentioned nano-sensor in enzyme kinetics parameter, OGT
The application of the quantitative context of detection of the screening of inhibitor and cell OGT activity.
In order that technical scheme can clearly be understood by obtaining those skilled in the art, below with reference to tool
The embodiment of body describes technical scheme in detail.
A kind of nano-sensor based on single quantum dots characterization O- acetylglucosamine transferases of embodiment 1
The nano-sensor based on single quantum dots characterization O- acetylglucosamine transferases includes：The biology of flower cyanines 5/
The peptide chain of element modification；On-radiation uridine-acetylglucosamine, Proteinase K and by the coated quantum dot of Streptavidin.Its
In, the peptide chain of the colored biotin modification of cyanines 5/ is：Biotin-Ser-Thr-Pro-Val-Ser-Arg-Ala-Asn-Met-
Lys-Cy5。
A kind of detection of the nano-sensor based on single quantum dots characterization O- acetylglucosamine transferases of embodiment 2
Method
The detection method of the O- acetylglucosamine transferases, comprises the following steps：
S1. the peptide of the biotin modification of flower cyanines 5/ containing every liter of 2-3 micromoles, uridine-acetyl Portugal of 90 every liter of micromoles
Grapes glucosamine, the alkaline phosphatase of 0.01 unit, OGT and 10 × reaction buffer to be measured, the reaction that composition cumulative volume is 20 microlitres
1 hour is incubated in solution at 37 DEG C；The reaction buffer is by 500 mMs of every liter of trishydroxymethylaminomethanes
(Tris), 200 mMs every liter of calcium chloride, 0.5% Tween-20 and 0.5%NP-40 compositions, pH value 7.8；
S2. 1 nanogram Proteinase K is added in glycosylation product, the system that reaction cumulative volume is 30 microlitres, 50
Reacted 1 hour at DEG C；
S3. take 20 microlitres of step S2 reaction product with every liter of quantum dot of 5 nanomole in 100 microlitres of culture buffer solution
React 10 minutes at room temperature, form quantum dot-peptide-Hua Jing 5 nanostructured；Wherein, it is described culture buffer solution by：3 mmoles
The magnesium chloride of every liter of that, 100 mMs every liter of pH 8.0 trishydroxymethylaminomethane-hydrochloric acid and 10 mMs every liter of sulphur
Sour ammonium composition, pH value 8.0.
Single Molecule Detection and data analysis：By reaction product, with imaging cushioning liquid, (1 milligram every milliliter of grape is glycoxidative
Enzyme, 0.4% (w/v) D-Glucose, 0.04% milligram every milliliter of catalase, the bovine serum albumin of 50 micrograms per millilitres
In vain, 67 mMs every liter of glycine-potassium hydroxide, 1 milligram every milliliter of watermiscible vitamin E, 2.5 mMs every liter of chlorine
Change magnesium, pH 9.4) 500 times of dilution.It is imaged for total internal reflection fluorescent, 10 microlitres of samples is directly drawn on cover glass.Make
With 488 nanometers of laser excitation quantum dot (605QD).Quantum dot and Hua Jing 5 photon are collected by 100 × object lens, and is passed through
EMCCD detectors are with 500 milliseconds of exposure time imaging.Using image J (image J) software selection image-region (500 ×
500 pixels) it is used to spend the counting of cyanines 5 (Cy5) molecule.
A kind of detection of the nano-sensor based on single quantum dots characterization O- acetylglucosamine transferases of embodiment 3
Method
The detection method of the O- acetylglucosamine transferases, comprises the following steps：
S1. the peptide of the biotin modification of flower cyanines 5/ containing every liter of 2-3 micromoles, uridine-acetyl Portugal of 100 every liter of micromoles
Grapes glucosamine, the alkaline phosphatase of 0.1 unit, OGT and 10 × reaction buffer to be measured, the reaction that composition cumulative volume is 20 microlitres are molten
1 hour is incubated in liquid at 37 DEG C；The reaction buffer by 500 mMs of every liter of trishydroxymethylaminomethanes (Tris),
200 mMs every liter of calcium chloride, 0.5% Tween-20 and 0.5%NP-40 compositions, pH value 7.8；
S2. 2 nanogram Proteinase Ks are added in glycosylation product, the system that reaction cumulative volume is 30 microlitres, 60
Reacted 3 hours at DEG C；
S3. 40 microlitres of step S2 reaction product culture buffer solution of the every liter of quantum dot of 15 nanomole at 100 microlitres is taken
In react at room temperature 30 minutes, formed quantum dot-peptide-Hua Jing 5 nanostructured；Wherein, it is described culture buffer solution by：3 millis
Mole every liter of magnesium chloride, 100 mMs every liter of pH 8.0 trishydroxymethylaminomethane-hydrochloric acid and 10 mMs every liter
Ammonium sulfate forms, pH value 8.0.
Single Molecule Detection and data analysis are as described in Example 2.
A kind of detection of the nano-sensor based on single quantum dots characterization O- acetylglucosamine transferases of embodiment 4
Method
The detection method of the O- acetylglucosamine transferases, comprises the following steps：
S1. the peptide of the biotin modification of flower cyanines 5/ containing 2.4 every liter of micromoles, uridine-acetyl Portugal of 96 every liter of micromoles
Grapes glucosamine, the alkaline phosphatase of 0.05 unit, OGT and 10 × reaction buffer to be measured, the reaction that composition cumulative volume is 20 microlitres
1 hour is incubated in solution at 37 DEG C；
S2. 1.5 nanogram Proteinase Ks are added in glycosylation product, the system that reaction cumulative volume is 30 microlitres,
Reacted 2 hours at 55 DEG C；
S3. 30 microlitres of step S2 reaction product culture buffer solution of the every liter of quantum dot of 10 nanomole at 100 microlitres is taken
In react at room temperature 20 minutes, formed quantum dot-peptide-Hua Jing 5 nanostructured.
Single Molecule Detection and data analysis are as described in Example 2.
The OGT of embodiment 5 suppresses experiment
Various concentrations (α R)-α-(((1,2- dihydro-2-oxo -6- quinolyls) sulfonyl) amino)-N- (2- furans first
Base) -2- methoxyl groups-N- (2- thenyls) phenyl acetamides (OSMI-1) add and the reaction solution of OGT fixed concentrations (250nM)
In, and incubated 60 minutes at 37 DEG C.Then 1.5 milligrams of Proteinase Ks are added to glycosylation product, and reaction cumulative volume is 30 μ L
System, reacted 2 hours at 55 DEG C.Then the detection of O- acetylglucosamine transferases is carried out by the detection method of embodiment 4.
O- acetylglucosamine transferases detection in the cells in sample of embodiment 6
O- acetylglucosamine transferases detection in the cells in sample, is divided into following steps：
(1) cell culture and the preparation of cell extract：HEK-293 cells are in 10% hyclone (FBS) and 1% mould
Plain streptomysin, culture medium are placed in 37 DEG C, are cultivated in the incubator containing 5% carbon dioxide to cell maturation.By under cell dissociation
After coming and collecting, washed with ice-cold 1 × phosphate buffer (PBS), then in 1% lauryl sodium sulfate (SDS)/20 mmoles
Cracked in the 4- hydroxyethyl piperazineethanesulfonic acids (HEPES) (pH value 7.9) of every liter of that.Then, 1 mM every liter of benzyl sulphonyl
Fluorine (PMSF), 10 every liter of micromole O- (2- acetamido -2- deoxidation-D- glucopyranosyls amino) N- carbanilates
(PUGNAC), 10 every liter of micromoles (3AR, 5R, 6S, 7R, 7AR) -2- (ethylamino) -3a, 6,7,7a tetrahydrochysene -5- (hydroxyl first
Base) simultaneously to be added to this molten for [3,2-d] thiazole -6,7- glycol (Thiamet G) and 1 × protease inhibitor cocktail for -5H- pyrans
In liquid, it is ultrasonically treated.Finally, sample is centrifuged 15 minutes under 12000rpm, collects supernatant；
(2) supernatant for obtaining above-mentioned steps (1), O- acetylglucosamines are carried out by the detection method of embodiment 4 immediately
Transferase detects.
Test example 1 detects the feasibility Experiment of O- acetylglucosamine transferases
The FRET occurred in embodiment 4 between QD and Cy5 is verified using fluorescence measurement.As shown in Fig. 2 in control experiment
In there is no OGT (Fig. 2), be detected without significant Cy5 signals, illustrate from QD to Cy5 there is no FRET generation because not
When OGT be present, QD surfaces that the peptides of no Cy5/ biotin modifications is assembled into.In contrast, when adding OGT, can be catalyzed
The glycosylation of the oxygen connection of the peptide of Cy5/ biotin modifications, the peptide for producing antiprotease cracking can be assembled on QD surfaces, with shape
Into QD- PEPCs y5 nanostructure, cause the FRET from QD to Cy5, therefore be observed that QD fluorescence is reduced and Cy5 fluorescence
Increase (Fig. 2).
For further confirmatory experiment feasibility, in single molecules level detection OGT activity.As shown in figure 3, in the absence of
Observable only QD fluorescence signals (Fig. 3 A), but no Cy5 fluorescence signal is detected (Fig. 3 B) during OGT.On the contrary,
In the presence of OGT, QD (Fig. 3 D) and Cy5 (Fig. 3 E) fluorescence signal can be detected simultaneously by, and QD and Cy5 position can be very
Good coincidence (Fig. 3 F), this illustrates the generation of FRET between QD and Cy5.In addition, compared with no OGT (Fig. 3 A), in OGT (figures
In the presence of 3D), it was observed that, because results of the FRET from QD to Cy5 so that QD fluorescence substantially weakens, this and fluorescence measurement
Result be consistent (Fig. 2).Therefore, Cy5 signal may indicate that OGT presence, and can be with Cy5 countings come quantitative survey
Determine OGT activity.
The above results clearly demonstrate that the method for embodiment 4 can be used to detect OGT.
The sensitivity technique of test example 2
In order to determine the sensitivity that embodiment 4 detects OGT, the counting of flower cyanines 5 is measured under the OGT of various concentrations.Such as Fig. 4
It is shown, as OGT concentration is from 4.0 × 10-13Mole every liter increases to 2.5 × 10-7Mole every liter, Hua Jing 5 is counted to be increased successively
Add.It is worth noting that, on logarithmic scale, Hua Jing 5 counting and OGT concentration are from 4.0 × 10-13Mole often it is raised to 2.5 ×
10-10Presented in the range of mole every liter linearly related (Fig. 4 insertions figure).Regression equation is N=2251.1+172.8log10C(R2
=0.986) detection, is calculated and is limited to 3.47 × 10-13Mole every liter, the test limit of technology is low than ever, or even than electrification
The test limit for learning analysis method is low 4 times.
The specific detection of test example 3
In order to verify the specificity of the detection method of embodiment 4, with DNA methylation enzyme (Dam), phosphokinase (PNK), albumen
Kinases A (PKA) is used as negative control group, carries out specific detection.As shown in figure 5, add Dam, PNK and PKA three groups and blank
Group is similar, does not observe Cy5 signals.On the contrary, the experimental group for adding OGT observes the Cy5 signals significantly improved.This result
Show the method for embodiment 4 when detecting OGT with good specificity.
The inhibitor of test example 4 is analyzed
In order to verify feasibility of the method for embodiment 4 in Inhibition test, tested using OSMI-1 as model inhibitor
Card.As shown in fig. 6, with the increase of OSMI-1 concentration, OGT activity significantly reduces.Use half-inhibition concentration (IC50, OGT is lived
Property inhibitor concentration required when being suppressed to original 50%) verify inhibitory action of the OSMI-1 to OGT.OSMI-1 is calculated
503nhibiting concentration be 3.05 every liter of micromoles, this and the 503nhibiting concentration (IC reported50) it is that every liter of 2.7 micromole is consistent
's.It follows that the method for embodiment 4 can be used for Inhibition test.
SEQUENCE LISTING
<110>Shandong Normal University
<120>Detect the nano-sensor and its detection method of O- acetylglucosamine transferases
<130>
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 10
<212> PRT
<213>Manually
<400> 1
Ser Thr Pro Val Ser Arg Ala Asn Met Lys
1 5 10
Claims (10)
1. a kind of nano-sensor of detection O- acetylglucosamine transferases, it is characterised in that the nano-sensor includes：
The peptide chain of the flower biotin modification of cyanines 5/；On-radiation uridine-acetylglucosamine, Proteinase K and coated by Streptavidin
Quantum dot.
2. the nano-sensor of O- acetylglucosamine transferases is detected according to claim 1, it is characterised in that the flower
The peptide chain of the biotin modification of cyanines 5/ is：
Biotin-Ser-Thr-Pro-Val-Ser-Arg-Ala-Asn-Met-Lys-Cy5。
3. according to the detection method of the nano-sensor of claims 1 or 2, it is characterised in that comprise the following steps：
S1. the peptide of the flower biotin modification of cyanines 5/ is glycosylated by the glycosyl of saccharide donor uridine-acetylglucosamine；
S2. Proteinase K distinguishes glycosylation and non-glycosylated peptide；
S3. glycosylated peptide self-assembles to quantum dot surface by the interaction of Streptavidin and biotin and forms quantum
The measurement of point-peptide-Hua Jing 5 nanostructured and follow-up FRET signal.
4. detection method according to claim 3, it is characterised in that the operating procedure of the step S1 is：It is micro- containing 2-3
The peptide of mole every liter of the biotin modification of flower cyanines 5/, uridine-acetylglucosamine of every liter of 90-100 micromoles, 0.01-0.1 are mono-
The alkaline phosphatase of position, OGT and 10 × reaction buffer to be measured, form in the reaction solution that cumulative volume is 20 microlitres at 37 DEG C
1 hour of lower incubation；The reaction buffer is by 500 mMs of every liter of trishydroxymethylaminomethanes, 200 mMs every liter of chlorine
Change calcium, 0.5% Tween-20 and 0.5%NP-40 compositions, pH value 7.8.
5. detection method according to claim 4, it is characterised in that the concrete operation step of the step S1 is：Contain
The peptide of the biotin modification of flower cyanines 5/ of 2.4 every liter of micromoles, uridine-acetylglucosamine of 96 every liter of micromoles, 0.05 unit
Alkaline phosphatase, OGT and 10 × reaction buffer to be measured, composition cumulative volume be in 20 microlitres of reaction solution at 37 DEG C
Incubate 1 hour.
6. detection method according to claim 3, it is characterised in that the operating procedure of the step S2 is：By 1-2 nanograms
Proteinase K is added in glycosylation product, the system that reaction cumulative volume is 30 microlitres, it is small that 1-3 is reacted at 50-60 DEG C
When.
7. detection method according to claim 6, it is characterised in that the concrete operation step of the step S2 is：By 1.5
Nanogram Proteinase K is added in glycosylation product, the system that reaction cumulative volume is 30 microlitres, is reacted 2 hours at 55 DEG C.
8. detection method according to claim 3, it is characterised in that the operating procedure of the step S3 is：Take 20-40 micro-
The reaction product for rising step S2 is reacted at room temperature with every liter of quantum dot of 5-15 nanomoles in 100 microlitres of culture buffer solution
10-30 minutes, form quantum dot-peptide-Hua Jing 5 nanostructured；Wherein, it is described culture buffer solution by：3 mMs every liter of chlorine
Change magnesium, 100 mMs every liter of pH 8.0 trishydroxymethylaminomethane-hydrochloric acid and 10 mMs every liter of ammonium sulfate composition,
PH value is 8.0.
9. detection method according to claim 8, it is characterised in that the concrete operation step of the step S3 is：Take 30
Microlitre step S2 reaction product reacts 20 at room temperature with every liter of quantum dot of 10 nanomole in 100 microlitres of culture buffer solution
Minute, formation quantum dot-peptide-Hua Jing 5 nanostructured.
10. nano-sensor according to claim 1 or claim 2 the measurement of enzyme kinetics parameter, the screening of OGT inhibitor and
The application of the quantitative context of detection of cell OGT activity.
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Cited By (5)
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| CN109100340A (en) * | 2018-08-23 | 2018-12-28 | 浙江理工大学 | A kind of preparation method of the implantable sensor of cadmiumsulfide quantum dot modification |
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| CN112485233A (en) * | 2020-11-03 | 2021-03-12 | 山东师范大学 | Nano sensor for detecting deacetylase and detection method and application thereof |
| CN112485233B (en) * | 2020-11-03 | 2022-08-26 | 山东师范大学 | Nano sensor for detecting deacetylase and detection method and application thereof |
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