CN106957892A - A kind of label-free electrochemical sensing detection method for protein tyrosine phosphatase activity - Google Patents
A kind of label-free electrochemical sensing detection method for protein tyrosine phosphatase activity Download PDFInfo
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Abstract
The invention discloses a kind of electrochemical sensing detection method of simple, quick, label-free protein tyrosine phosphatase (PTP) activity.We utilize covalent technique for fixing, PTP substrate (phosphorous phosphorylated tyrosine) peptide molecule film is constructed on semi-conducting electrode, electrochemical signals probe is used as using the tyrosine in peptide molecule, with reference to the catalysis enlarging function of the metal complex of the suitable electron mediator osmium of current potential, establish a kind of electrochemica biological sensor for quantitatively detecting PTP enzymatic activitys, lowest detection is limited to 10nM, and this method can also be applied to the rapid screening of PTP enzyme inhibitors and drug molecule.The sharpest edges of the electrochemical sensing method are not need antibody and signal mark molecule, and operating procedure is simple, is especially suitable for the quantitative detection of protein tyrosine phosphatase activity.
Description
Technical field：
The present invention relates to a kind of electrochemical biosensor method available for label-free detection protein tyrosine phosphatase activity.This method is simple to operate, spirit
Sensitivity is high, the quantitative analysis available for protein tyrosine phosphatase activity.
Technical background：
Protein-tyrosine reversible phosphorylation is the important way of eukaryotic signal path regulation and control, and it passes through protein tyrosine kinase (Protein tyrosine
Kinase, PTK) phosphorylation and protein tyrosine phosphatase (Proteintyrosine phosphatase, PTP) dephosphorylation be total to
With regulation, eukaryotic cells adjusted by this reversible balance intracellular and cell-cell communication (referring to document Lim W.A.et al.Cell, 2010,
142(5):661-667.).Compared with PTK, the research to PTP is started late, until recent two decades, and people just gradually recognize that it is strong in the mankind
Importance in health and disease.PTP huge numbers, wide expression is in various tissues and cell type, and the PTP family members having confirmed that have
More than 100, PTP has regulating and controlling effect to PTK and its downstream a series of signal approach of mediation, its signal pathway regulated and controled be related to cell propagation,
All many-sided (the den Hertog J.et al.FEBS J., 2008,275 such as differentiation, adhesion, migration, apoptosis, cell cycle:831-847.).Especially in control
On cells phosphorylation level of tyrosine processed, PTP plays the positive role of high degree of specificity, occupies leading position.PTP families be signal transduction on the way
Important regulatory factor in footpath, and there are a considerable amount of PTP to be played an important role in human diseases, PTP ectopic expressions, unconventionality expression,
Dysfunction and the generation development of a variety of diseases even cancer caused by fragment deletion and point mutation is closely related.Such as：The proprietary albumen junket of haemocyte
Propylhomoserin phosphatase (Hematopoietic protein tyrosine phosphatase, HePTP) is one of PTP related to cancer for finding earliest,
Its gene is located on chromosome LQ32.1, and research shows in preleukemia RAEB and acute myelocytic leukemia patient's body
HePTP expression excessive (Zanke B.et al.Leukemia, 1994,8 (2):236-244.)；SHP-1 is the negative regulator of hematopoietic growth signal path
The factor, its low expression can cause bone marrow cell development abnormal syndrome and lymthoma generation (Zhang Q.et al.Proc.Natl.Acad.Sci.USA,
2005,102(19):6948-6953.), SHP-1 overexpression but is found in oophoroma；The phosphatase PTEN homologous with skelemin, as
Tumor suppressor gene, the propagation of kinds of tumor cells can be significantly inhibited, adhere to, sprawl, migrating and anti-apoptotic (Suzuki A.et al.Cancer Sci., 2008,
99(2):209-213.), PTEN gene mutations or missing can induced tumor generation, PTEN missing is found in the cancer of the esophagus, in breast cancer and
Find that PTEN expression is low in prostate cancer, and PTEN point mutation can cause some genetic diseases, and such as hamartomatosis is comprehensive
Disease, Dysplastic gangliocytoma；Myotube fibroin MTMR2 missing can damage the first stage of peripheral nervous system myelin generation,
So as to cause neurotic atrophy syndrome (Chojnowski A.et al.Neurobiol.Dis., 2007,26 (2):323-331.)；HLA is related
Albumen (leukocyte antigen-related protein, LAR) belongs in receptor type PTP, breast cancer overexpression (the Yang T.et al. for finding LAR
Mol.Carcinogen.,1999,25(2):139-149.), LAR gene mutation and the generation of Human colon cancer are closely related；SHP-2 gene is dashed forward
Change can trigger the generation of a variety of diseases, such as exert southern syndrome, leopard line disease, teenager's monocytic leukemia (Muller P.J.et al.J.
Proteomics,2013,84:132-147.), and SHP-2 functional disturbances can cause the generation of certain cancers, such as stomach cancer, kidney；CD45 joins
With T, B cell system and NK cells signal transduction process and development and the function point analysis of lymphocyte, its abnormal expression can cause immune system
Lesion (Alexander D.R.et al.Semin.Immunol., 2000,12 (4):349-359.)；PTPRJ(Protein tyrosine phosphatase,
Receptor type J) (Ruivenkamp C.A.L.et al.Nat.Genet., 2002,31 (3) in colon cancer:295-300.), PTPRO (Protein
Tyrosine phosphatase, receptor type O) had been found that in liver cancer and colon cancer expression quantity it is low (Motiwala T.et al.Oncogene, 2003,
22(41):6319-6331.)；Low molecule amount PTP and mitogen-activated protein kinase phosphatase-1 (MKP-1) take part in growth factor receptors and mitogen
The signal path of former activated protein kinase (mitogen-activated protein kinase, MAPK) is related to cardiac hypertrophy；Many entities swell
PTP α unconventionality expression (Stebbing J.et al.Oncogene, 2014,33 (8) are had been found that in knurl such as Advanced Colon Cancer, lung cancer:939-953.).Base
In the important function of PTP in vivo, the medicine using PTP as therapy target is also come out one after another, and therapy mechanism is to corresponding PTP activity
(Vintonyak V.V.et al.Curr.Opin.Chem.Biol., 2009,13 (3) is adjusted:272-283.).As PTP1B makes insulin receptor dephosphorization
Acidifying, is the crucial negative growth factor of insulin signaling pathway, medicine develop by therapy target of PTP1B can be used for treating type ii diabetes with
Obesity.Although the research abnormal PTP related to disease has had made some progress, still extremely have about the research meanses of PTP activity
Limit, and then cause the research in terms of the research and development and screening for the small-molecule drug of its activity regulation, have difficulty in taking a step.PTP determinations of activity problem is urgently
To be solved, it will provide scientific basis and experimental data for the medicament research and development using PTP as therapeutic targets.
The existing detection method of research PTP activity mainly has fluorescence method (Sahoo H.et al.J.Am.Chem.Soc., 2007,129 (51) at present:
15927-15934；Freeman R.et al.Nano.lett.,2010,10(6):2192-2196；Wang F.F.et al.Chem.Commun.,2014,
50(60):8161-8163.), Capillary Electrophoresis (CE) (Phillips R.M.et al.Anal.Chem., 2014,86 (2):1291-1297.) with ELISA (Bose
A.K.et al.Mol.Cell.Proteomics,2013,12(3):797-806.) etc..Fluorescence method is utilized before substrate phosphorylation tyrosine residue dephosphorylation
The difference acted on afterwards with fluorescence marker groups causes the change of system signal to detect, but is related to more complicated chemical synthesis.Capillary electrophoresis
Mainly before and after the substrate dephosphorylation according to fluorescence labeling, the difference of electromigration speed is first separated it, but needs and laser inductive fluorescence method
Combination, operation is cumbersome, and time-consuming.ELISA is first to use antibody capture target protein based on immunoprecipitate, then adds zymolyte, profit
Detected with free phosphate radical is hydrolyzed with developer reaction.But the preparation of antibody, purifying and the factor such as whether be readily available can the side of restriction
The use of method.In summary, although the analyzing detecting method of some protein tyrosine phosphatases activity has been developed, but these methods are also deposited
In many defects, it is impossible to meet actual requirement, await the further research and development of new method and new technology.
The content of the invention：
The present invention is improved primarily directed to the deficiency of prior art the following aspects：
1) existing fluorescence method is related to the more complicated chemical synthesis of zymolyte fluorophor mark；2) Capillary Electrophoresis needs and other method combination, and behaviour
Make cumbersome time-consuming；3) ELISA such as is prepared, purified and whether is readily available at the factor restriction by antibody.
Because electrochemical method has the features such as simple to operate, response is fast, sensitivity is higher, instrument is cheap, occupy solely in analysis method research field always
Special status.To realize the improvement to the above, it can be used for simple, quick, label-free detection protein-tyrosine phosphorus the invention provides one kind
The electrochemical sensing method of sour enzyme.Particularly as being, PTP substrate (phosphorous phosphorylated tyrosine) polypeptide point on indium-tin oxide electrode surface, fixation
Son.Added into solution after PTP, phosphorylated tyrosine is by dephosphorylation.Using the suitable electron mediator of current potential, optionally catalytic tyrosine
Electrochemical oxidation reactions, produce higher electrochemical signals.And phosphorylated tyrosine can not can't detect the electro-catalysis of tyrosine by catalysis oxidation
Signal, only detects the weaker electrochemical signals of electron mediator in itself.If containing PTP inhibitor to be investigated, PTP enzymatic activitys in solution
It is suppressed, only some is smaller when detection signal is relative to no inhibitor by dephosphorylation for phosphorylated tyrosine.Pass through this small molecule of tyrosine
Electrochemical signals probe, can study the dephosphorylation reaction of protein-tyrosine, quantitatively detect PTP enzymatic activity, rapid screening compound pair
PTP inhibitory action.Compared with the conventional method, this method has that operating procedure is simple, can realize high-throughout advantage；Meanwhile, our side
Method by the use of the endogenous tyrosine of polypeptide electrochemical source of current as detection signal, remain unmarked characteristic；Introduce electron mediator catalysis amplification letter
Number, it also ensure that the high sensitivity of detection.
One aspect of the present invention provides the construction method of the sensing chip of electrochemical sensor, and it comprises the following steps：
(a) selection of matrix is semi-conducting electrode；
(b) functional modification first is carried out to electrode surface, it is necessary to consider decorative layer to electrode surface polypeptide fixed amount and the aspect of electron transport rate two because
The influence of element, carrys out density, the structure of coordination electrode finishing coat；
(c) gentle reaction condition is used, polypeptide is fixed on electrode surface in stable, orderly mode, has both ensured the identifiability of polypeptide and enzyme, again
Maintain the electrochemical response of polypeptide.
(d) suitable sealer, non-specific adsorption of the reduction enzyme in electrode surface are used.
(e) suitable reaction condition is used, the catalytic activity of enzyme is effectively kept and play.
Another aspect of the present invention provides electrochemical sensing testing mechanism, and it comprises the following steps：
(a) by the semi-conducting electrode of surface immobilized polypeptide molecule, insert complete to the reaction of polypeptide dephosphorylation in enzyme reaction solution.
(b) enzyme of electrode surface is cleaned up using suitable cleaning fluid, inserts and electrochemical measurement is carried out in the solution containing electron mediator.
(c) electrochemical signals of measurement system, according to the strong and weak to enzymatic activity progress qualitative or quantitative analysis of signal.
Its representational operating procedure is：
(a) functional modification on semi-conducting electrode surface
(b) covalent fixation of the peptide molecule in electrode surface
(c) Seal treatment of electrode surface
(d) before and after measurement enzyme reaction occurs, electrochemical signals of the system in electronic media liquid solution carry out qualitative or quantitative analysis to enzymatic activity.
In one aspect of the invention, the sensor chip provided has following feature：For semi-conducting electrode, its surface is needed by functional modification.
In another aspect of the present invention, described electrode surface peptide molecule film is covalent fixed, and reaction condition is gentle, makes polypeptide straight in electrode surface
Vertical assembling, reduces steric hindrance when enzyme is recognized with polypeptide, so as to ensure the generation of the polypeptide dephosphorylation reaction of PTP catalysis.
In another aspect of the present invention, electron mediator refers to the metal complex of terpyridyl osmium.
The present invention mainly provides a kind of electrochemical sensing method that can be used for detecting PTP enzymatic activitys, by covalently fixing PTP on semi-conducting electrode surface
Substrate polypeptide, using the tyrosine in peptide molecule as electrochemical probe, introduce electron mediator catalysis amplification tyrosine electrochemical signals, utilize
The difference of normal tyrosine and phosphorylated tyrosine electro-catalysis signal, realizes the detection to PTP enzymatic activitys.The method is simple, quick, label-free.Profit
With the method for the present invention detect PTP enzymatic activitys advantage be：(1) by the use of the tyrosine in zymolyte as signal probe, the mark without being related to complexity
Remember synthesis step, it is simple to operate, quick；(2) selection zymolyte is peptide molecule, and nonprotein, it is to avoid protein macromolecule is fixed on electricity
The deactivation phenomenom on pole surface；(3) peptide molecule be covalently fixed on electrode surface reaction condition it is gentle, fixed rate is high, it is easy to operate；(4) signal
Amplification system is the solution of electron mediator, it is easy to operated；(5) semi-conductor electricity that sensing chip is related to is extremely easy to make, with low cost.
Brief description of the drawings：
Fig. 1 is electrochemical sensor to protein tyrosine phosphatase activity and its Cleaning Principle schematic diagram of inhibitor screening.
Fig. 2 is the result that electrochemical sensor detects SHP-2 phosphatase activities.
Embodiment：
The functional modification on the semi-conducting electrode surface of embodiment 1
Because we use sensor detection mode, it is necessary to first carry out functional modification, the trim one that can be used to tin indium oxide (ITO) electrode surface
As be poorly conductive organic compound, it is possible to suppress the electrochemical response of electron mediator.We have carried out different degrees of silicon to electrode surface
Alkanisation is modified, and ITO electrode is placed in the toluene solution of the trimethoxy silane of glycydoxy containing 1%3- reacts different time respectively
(0.5,1,2,4,8,10,12,16,20,24 hour).Reaction is finished, successively each ultrasonic 5 minutes in toluene and absolute ethyl alcohol, then,
Nitrogen is dried up, and is placed in 115 DEG C of Muffle furnaces and is dried 20 hours.X-ray photoelectron power spectrum (XPS), which is characterized, finds that the surface concentrations of trim are anti-with modification
Increase between seasonable.Although decorative layer has obvious inhibitory action to the electrochemical response of the classical electrochemical probe potassium ferricyanide, to electron mediator
Os(bpy)3 2+Electrochemical reaction have little to no effect.
Covalent fixation of the peptide molecule film of embodiment 2 in indium tin oxide surfaces
First the ITO electrode of functional modification is cut into the size of 2.5cm × 0.5cm sizes, pure water and cleaned, nitrogen drying, by 10 μ L various concentrations
Polypeptide solution (2.0M phosphate buffer solutions, pH 7.4) uniform fold is laid in the ITO electrode of 0.5cm × 0.5cm sizes, be placed in one it is moist
25 DEG C are reacted 24 hours in container.After surface reaction terminates, electrode is successively in the 20mM phosphate buffers containing 0.1% Tween-20, containing 150mM
Concussion cleaning 5 minutes in the 20mM phosphate buffers and 20mM phosphate buffers of sodium chloride, nitrogen drying.Finally, ITO electrode is statically placed in
In Tris-HCl solution, the reaction site for enclosed-electrode remained on surface.Deionized water is cleaned, nitrogen drying.Using XPS and cyclic voltammetry
Determine the electro-chemical activity of polypeptide fixed amount and polypeptide.Silylation modification layer is considered to electrode surface polypeptide fixed amount and the side of electron transport rate two
The influence of face factor, gives optimal silanization organic film coverage rate.Silylation modification ITO electrode 1 hour, polypeptide optimal concentration is
0.2mg/mL。
The detection of the protein tyrosine phosphatase of embodiment 3 activity
According to the condition of optimization, the ITO electrode that surface is fixed with substrate polypeptide be placed in dephosphorylation reaction solution (NaCl containing 50mM, 1mM DTT,
0.05%Tween-20 and PTP or PTP and inhibitor) in, a period of time is reacted at 37 DEG C.Electrode is successively in the 20mM containing 0.1%Tween-20
Concussion cleaning 5 minutes in phosphate buffer, the 20mM phosphate buffers of the sodium chloride containing 150mM and 20mM phosphate buffers, nitrogen drying,
Carry out Electrochemical Detection.
The Electrochemical Detection of embodiment 4
Electrochemical appliance uses three-electrode system：ITO electrode is working electrode, and Ag/AgCl electrodes are reference electrode (3M KCl), and platinized platinum is auxiliary electricity
Pole.Electrochemical Detection is circulated voltammetric scan in the 20mM phosphate buffer solutions containing 3 μM of electron mediator terpyridyl osmiums, and it is 30 to sweep speed
mV/s.Compare the difference of electrochemical signals before and after enzymic catalytic reaction, the activity to enzyme carries out quantitative analysis and detection.
Claims (6)
1. a kind of label-free electrochemical sensing detection method for protein tyrosine phosphatase activity, core component sensing chip is related to phosphoric acid
The peptide molecule for changing tyrosine is covalently fixed on semi-conducting electrode surface, using tyrosine as electrochemical probe, introduces the metal combination of electron mediator osmium
Thing, using the difference of normal tyrosine and phosphorylated tyrosine electro-catalysis signal, realizes the detection to protein tyrosine phosphatase activity.
2. semi-conducting electrode according to claim 1, its surface needs first to carry out functional modification with silylating reagent.
3. peptide molecule according to claim 1, it, as the substrate of protein tyrosine phosphatase, is the characteristic sequence of one section of amino acid, is needed
Design, synthesize.
4. electrochemical sensing chip according to claim 1 is, it is necessary to which peptide molecule to be covalently fixed on to the semi-conductor electricity of functionalization using silane chemistries
Pole surface, forms the sensitive membrane of sensor.
5. electron mediator according to claim 1, is the metal complex of terpyridyl osmium.
6. electron mediator according to claim 1, its concentration is 3.0 μM.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111554345A (en) * | 2020-05-15 | 2020-08-18 | 吉林大学 | Method for constructing three-dimensional structure of protein based on specific cross-linked tyrosine |
| TWI706798B (en) * | 2019-01-31 | 2020-10-11 | 國立交通大學 | Electrode used for treating alzheimer's disease |
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| US20030186284A1 (en) * | 2000-07-24 | 2003-10-02 | Masaharu Noda | Method of screening ptp ç activitiy promoter or inhibitor |
| CN104849448A (en) * | 2015-05-12 | 2015-08-19 | 陕西师范大学 | Fluorescence quenching-based protein kinase activity analysis method |
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2016
- 2016-01-08 CN CN201610009584.8A patent/CN106957892A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030186284A1 (en) * | 2000-07-24 | 2003-10-02 | Masaharu Noda | Method of screening ptp ç activitiy promoter or inhibitor |
| CN104849448A (en) * | 2015-05-12 | 2015-08-19 | 陕西师范大学 | Fluorescence quenching-based protein kinase activity analysis method |
Non-Patent Citations (1)
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI706798B (en) * | 2019-01-31 | 2020-10-11 | 國立交通大學 | Electrode used for treating alzheimer's disease |
| CN111554345A (en) * | 2020-05-15 | 2020-08-18 | 吉林大学 | Method for constructing three-dimensional structure of protein based on specific cross-linked tyrosine |
| CN111554345B (en) * | 2020-05-15 | 2022-06-03 | 吉林大学 | Method for constructing protein three-dimensional structure based on specific cross-linked tyrosine |
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Application publication date: 20170718 |