CN106404719A - Method for detecting protein kinase based on surface plasmon resonance - Google Patents
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Abstract
A method for detecting protein kinase based on surface plasmon resonance comprises the following steps: A, a gold film is used as a sensing layer, the sensing layer, first, is soaked with ethanol, then ultrasonically cleaned, H2 flame is used for annealing on the surface of the gold film; B, the gold film processed by the step A is soaked in a polypeptide P1 solution with an amino acid sequence shown as SEQIDNO:1, then soaked with the gold film in a polypeptide P2 solution with an amino acid sequence shown as SEQIDNO:2, and blown to dry with N2, and sealed storage of the prepared polypeptide P1 and P2-modified sensing gold film is performed at-18 DEG C for standby use; and C, the polypeptide P1 and P2-modified sensing gold film prepared by the step B is put on a SPR (surface plasmon resonance) apparatus, protein kinase flow injection is performed on the sensing gold film, when the sensing gold film is bonded with polypeptides on the surface of the gold film, a new resonance angle is produced, and the size of the resonance angle is a SPR response signal.
Description
Technical field
The present invention relates to a kind of sufacing for protein kinase detection, particularly to based on surface plasma excimer
The method of resonance detection protein kinase, belongs to chemical field.
Background technology
The phosphorylation of protein has certainly at cell proliferation and the aspect such as differentiation, intercellular information transmission, gene expression
Qualitative effect.Protein kinase is a kind of enzyme being capable of catalytic proteins generation phosphorylation reaction.Research shows, some major diseases
（As diabetes, Alzheimer, cancer, metabolism syndrome etc.）Generation close with the content of protein kinase and activity change
Related.The detection of therefore protein kinase is significant at the aspect such as the diagnosis of these major diseases and Therapy study.At present
Method for detecting protein kinase mainly has radioisotope method, fluorescence method, electrochemistry and mass spectrography etc., but these methods
Take time and effort, to analyze testing cost higher, and need to enzyme catalysiss substrate（Typically adenosine triphosphate ATP）It is marked.It is situated between
In significant role in research vital movement and diseases prevention and treatment for the phosphorylation reaction, set up a kind of inexpensive, simple and quick, be suitable to
The method that analysis measures protein kinase content in common laboratory or clinic is one of focus of scientific research.Surface plasma
Body plasmon resonance（Surface Plasmon Resonance, SPR）Technology be by measure super thin metal adsorption layer change Lai
A kind of sufacing of the interphase interaction of research biomolecule.It the aspects such as dynamics research, quantitative analyses have sensitive,
Quickly, without the advantage that labelling, real-time monitoring etc. are unique, have wide in terms of the biomolecule detection such as protein and nucleic acid
Application prospect.Most-often used detection system is to capture target protein using antibody molecule, is acted on by Ag-Ab and realizing
The detection by quantitative of protein.However, the preparation cost of antibody is higher, less stable, it is in the fixed form of SPR chip surface
And space orientation orientation can have a strong impact on the interaction of Ag-Ab.During protein phosphorylation, protein kinase is the bottom of to
The identification of thing（Interaction typically with polypeptide fragment a certain in substrate protein）It is the committed step of phosphorylation reaction.It is based on
There is phosphorylation reaction in the characteristic of this catalytic reaction and the feature of protein kinase and substrate interaction, with substrate protein
The peptide molecule that polypeptide fragment structure is similar to is often the effective inhibitor suppressing protein kinase activity, and it can be with protein kinase
There is stronger effect, but phosphorylation reaction will not occur.For example, the peptide molecule containing RRNAI structure fragment is suppression egg
Effective inhibitor of white kinase A activity, it can occur stronger effect with the catalysed partial of protein kinase A.With respect to antibody,
Peptide molecule has the advantages that preparation cost is low, stability is preferable, sequence is controlled.So, the peptide inhibitor of protein kinase
It is expected to replace antibody, become a kind of new target molecule being capable of identify that protein kinase.Based on this identification ability of polypeptide
There is not been reported for sufacing, therefore, using polypeptide as protein kinase receptor, develops one kind and is used for protein kinase Gao Ling
Quick and selective enumeration method surface sensing technology is very necessary.In addition, biomolecule in suprabasil fixed form and is covered
Lid density, protein are surface sensing technology key issues to be solved in the non-specific adsorption of sensor surface.Early stage
Biomolecule fixation mode is directly by biomolecule fixation to golden film or silverskin surface by physisorption, however, this
Kind of adsorption is not only easily caused biomolecule degeneration or loss of activity, is also easy to make to analyze sample very strong with substrate generation
Non-specific adsorption.Self-assembled film containing specific groups or biological function film substrate enable to biomolecule and keep activity,
Non-specific adsorption can also be reduced simultaneously.Most successful self-assembled film be modify in golden film containing sulfydryl or disulfide bond point
Son, this self-assembled film is to be formed according to strong golden sulfide linkage between gold and sulfydryl.According to thiol molecule other end group（Logical
It is often carboxyl or amido）Reactivity, suitable cross-linking agent can be passed through, by biomolecule fixation in substrate surface.SPR
In technology, conventional self-assembled film mainly includes mercaptan carboxylic acid, Sensor Chip CM 5, polyvinyl alcohol, polylysine, strepto- is affine
Element, triacetic acid ammonia etc..However, in place of these self-assembled films there is also some shortcomings, for example：Preparation process is complicated, relatively costly,
Prevent the nonspecific ability of protein poor etc..
Content of the invention
It is an object of the invention to provide a kind of method that protein kinase is detected based on surface plasmon resonance.
The method detecting protein kinase based on surface plasmon resonance provided by the present invention, walks including following
Suddenly：A：Using golden film as inductive layer, described golden film is first used soaked in absolute ethyl alcohol 10 minutes before the use, is cleaned by ultrasonic 5
Minute, then rinsed well with secondary water, and use N2Dry up, after cleaning up, use H2Flame, in gold plaque flash annealing, first preheats,
By golden film and H2Flame is in 45 degree of angle homo genizing annelaings 30 seconds, in order to clean surface, removes the material of surface non-specific adsorption；
B：By the golden film after processing of step A in aminoacid sequence such as SEQ ID NO：Soak 12 hours in 1 polypeptide P1 solution,
The aminoacid sequence of polypeptide P1 is CPPPPTTYADFIASGRTGRRNAIHD, then rinses golden film secondary water well, N2 blows
Dry, then by above-mentioned golden film in aminoacid sequence such as SEQ ID NO：Closing in 2 hours is soaked unreacted in 2 polypeptide P2 solution
Gold surface, the aminoacid sequence of polypeptide P2 is CPPPP, then is rinsed well with DMSO, secondary water successively and blown with N2 behind golden film surface
Dry, prepared P1Polypeptide, P2The sensing golden film sealed storage modified is standby at -18 DEG C；
C：The polypeptide P that step C is obtained1, polypeptide P2Modify sensing golden film be placed on SPR instrument, when incident illumination from prism to
When metallic film is propagated, when angle of incidence is more than critical angle, then can total internal reflection, produce evanescent waves, evanescent waves can induce gold
Metal surface produces plasma, and when evanescent waves are identical with the wave vector of plasma, the two resonates, and total reflection condition breaks
Bad, by metal surface Electron absorption, light-wave energy becomes the motion of metal surface electronics to incident illumination energy, leads to the energy of reflected light
Amount drastically declines, and therefore formant in reflectance spectrum, formant is reflex strength minimum, now corresponding angle of incidence is
For resonance angle, because the change of the structure on golden film surface or thickness shows as the change of resonance angle, after baseline stability, correspond to
One resonance angle, i.e. SPR response signal, flow injection protein kinase in above-mentioned sensing golden film, when protein kinase and gold
After the polypeptide on film surface combines, a new resonance angle can be produced, the size of resonance angle is SPR response signal, further
, after protein kinase acts on golden film surface, then flow injection NaOH solution, can destroy between polypeptide and protein kinase
Interact, SPR response value is reduced to initial size, so that chip regeneration.
The present invention introduces cysteine residues in the nitrogen end of protein kinase identification polypeptide（C）The dried meat stronger with four rigidity
Histidine residue（PPPP）, polypeptide pass through cysteine residues in sulfydryl and golden film between Au-S effect be fixed in golden film table
Face, four stronger proline residues of rigidity enable polypeptide to stand on golden film surface, thus being conducive to polypeptide to swash with albumen
The interaction of enzyme.Meanwhile, in order to prevent other oroteins in serum, in the non-specific adsorption of chip surface, the present invention adopts five
PEPC PPPP closes unreacted golden film surface, desirably prevents the non-specific adsorption of protein, does not affect protein kinase simultaneously
Interaction with identification polypeptide.Polypeptide fixing meanss according to the present invention have the advantages that step is simple, low cost, effect are good.
It is simple, without labelling, the renewable, real-time monitoring of sensing chip etc. that SPR sensorgram technology in the present invention has sensitive quick, step
Advantage；The receptor of the protein kinase adopting in the present invention is peptide molecule, with respect to traditional receptor（Antibody）, polypeptide has system
The advantages of standby simple, low cost, good stability；In the present invention, the fixing meanss of polypeptide are simple to operate, low cost, and can have
Effect prevents the non-specific adsorption of protein.
Brief description
Fig. 1. it is the experimental principle figure that SPR technique detects protein kinase A.
Fig. 2 is the SPR response curve to different sensing golden film surfaces for the flow injection protein kinase A.
Fig. 3 is the regeneration Journal of Sex Research of SPR sensorgram golden film.
Fig. 4 is the linear relationship chart between SPR response value and protein kinase A concentration.
Fig. 5 is SPR kinetic curve.
Fig. 6 is the SPR response signal in sensing golden film surface injection different proteins.
Specific embodiment
More fully explain the enforcement of the present invention, the embodiment of the present invention is provided.These embodiments are only right
The elaboration of this technique, does not limit the scope of the invention, and is illustrated with following examples in the present invention, but is not limited to following embodiments,
Any change is included in the technical scope of the present invention.In the present invention, the representative material of various abbreviations is respectively：
CPPPPTTYADFIASGRTGRRNAIHD：P1The aminoacid sequence of polypeptide, CPPPP：P2The aminoacid sequence of polypeptide, TCEP：Three
(2- carboxyethyl) phosphine, PBS：Phosphate buffer, SPR：Surface plasmon resonance.MM, M, nM are concentration unit,
Represent 10 respectively-3mol/L、10-6mol/L、10-9mol/L.Following examples are entered using protein kinase A as testing protein kinases
Row is specifically described.To accompanying drawing further explanation, in Fig. 1, represent polypeptide P1/P2The golden film modified, represents protein kinase A, in order to
Clear expression, in figure molecule is not all given by actual ratio.In Fig. 2, curve a is in polypeptide P1/P2The golden film Surface runoff modified
The SPR curve of injection protein kinase A, curve b is in polypeptide P2The SPR that the golden film Surface runoff modified injects protein kinase A is bent
Line.In Fig. 2, the flow velocity of carrier fluid is 10 L/min.Fig. 3 is polypeptide P1/P2The golden film modified regenerates after different number of times through NaOH solution
The SPR response signal of flow injection protein kinase A again.Fig. 4 is the linear relationship chart of SPR response signal and protein kinase A concentration,
The concentration of protein kinase A is followed successively by 0.1,1,5,10 and 20 nM.Solid line in Fig. 5 is in polypeptide P1/P2The golden film table modified
The SPR response curve of the protein kinase A of three variable concentrations of face flow injection, dotted line is to simulate out according to test result
SPR kinetic curve, the concentration of protein kinase A is followed successively by 12.5,25 and 50 nM, and the flow velocity of carrier fluid is 30 L/min.Fig. 6 is
In polypeptide P1/P2The golden film Surface runoff injection bovine serum albumin modified（1）, immunoglobulin G（2）, thrombin（3）, lysozyme
（4）, protein kinase A（5）SPR response signal size.The concentration of protein kinase A is 20 nM, and 1 ~ 4 concentration is 0.1 mg/
mL.
Embodiment 1
The preparation of SPR sensorgram chip, step is as follows：
（1）The golden film of 50 nm thickness is purchased from Biosensing Instrument company of the U.S..The basic structure of the golden film of the said firm
One-tenth is on the microscope slide of 18 mm × 18 mm, first plates the thick Cr metal film of 2 nm, in order to strengthen between gold and glass
Adhesion, is then plated with the thick gold of 50 nm as inductive layer；
（2）Golden film first uses soaked in absolute ethyl alcohol 10 minutes before the use, ultrasonic 5 minutes, then is rinsed well with secondary water, is used in combination
N2Dry up；
（3）After cleaning up, use H2Flame, in gold plaque flash annealing, first preheats, then with 45 degree of angles H2Even flame annealing 30
Second, in order to clean surface, eliminate non-specific adsorption；
（4）Polypeptide P1It is dissolved to 1 mM with secondary water, be then diluted to 10 M, polypeptide with the PBS solution of 10 mM pH 7.2
P2It is dissolved to 5 mM with DMSO, be then diluted to 0.5 mM, TCEP water dissolution to 10 with the PBS solution of 10 mM pH 7.2
mM；
（5）Clean golden film is comprised 50 M TCEP and 10 M polypeptide P in 5 mL 10 mM1PBS solution in soak 12 little
When, then golden film secondary water is rinsed well, N2Dry up.The polypeptide P being 10 M by above-mentioned golden film in concentration again2Soak in solution
Bubble 4 hours, closes unreacted gold surface.Golden film surface is rinsed well with 1% DMSO, secondary water successively, then uses N2Dry up.System
The polypeptide P obtaining1/P2The golden film sealed storage modified is standby in -18 DEG C of refrigerators；
（6）Polypeptide P2The preparation process of golden film modified is：The polypeptide P that clean golden film is 0.5 mM in concentration2In solution
Soak 4 hours, then rinsed well with 1% DMSO, secondary water successively, finally use N2Dry up.Prepared polypeptide P2The golden film modified is close
Envelope is stored in stand-by in -18 DEG C of refrigerators.
Embodiment 2：
The protein kinase A specificity on golden film surface in embodiment 1 and nonspecific action, step is as follows：
（1）Protein kinase A is diluted with 10 mM pH 7.2 PBS；
（2）By the P preparing1/P2The golden film modified is placed on SPR instrument, after baseline stability, the egg of flow injection 25 nM
White kinases solution A, records SPR response signal, until steady.Carrier fluid is the PBS of 10 mM pH 7.2, and flow velocity is 10
L/min；
（3）By the P preparing2The golden film modified is placed on SPR instrument, and after baseline stability, the albumen of flow injection 25 nM swashs
Enzyme solution A, records SPR response signal, until steady.Carrier fluid is the PBS of 10 mM pH 7.2, and flow velocity is 10 L/
min；
（4）Relatively inject the SPR response signal of protein kinase A solution in different golden film Surface runoff, as shown in Figure 2：Albumen swashs
Enzyme A is in polypeptide P1/P2The golden film surface modified creates stronger SPR response signal, and this is by protein kinase A and polypeptide P1It
Between specificity interact caused by.Protein kinase A is in polypeptide P2The golden film surface modified almost does not have SPR response signal, table
Bright protein kinase A is in P2The golden film surface modified does not have non-specific adsorption to act on.This illustrates polypeptide P2Can close unreacted
Gold surface, thus effectively prevent non-specific adsorption.
Embodiment 3：The regeneration of sensing chip, step is as follows：
（1）Protein kinase A 10 mM, pH 7.2 PBS dilutes；
（2）By the P preparing1/P2The golden film modified is placed on SPR instrument, after baseline stability, the egg of flow injection 25 nM
White kinases solution A, records SPR response signal, until steady.Carrier fluid is the PBS of 10 mM pH 7.2, and flow velocity is 10
L/min；
（3）In step（2）Sense channel in inject 50 mM NaOH solution, flow velocity is 10 L/min, be incorporated into sensing gold
The protein kinase A eluting on film surface so that sensing golden film regeneration, until spr signal reach big before injection protein kinase A
Little, the detection for next sample is prepared；
（4）To step（3）Injection and step on the surface obtaining（2）The protein kinase A solution of middle same concentration, record SPR letter
Number, until baseline is steady, with step（2）Middle signal intensity is compared；
（5）Repeat step 2-4, compares spr signal change.From figure 3, it can be seen that polypeptide P1/P2The golden film modified is through 3 times again
After life, SPR response signal does not almost change, and illustrates that this sensing golden film can regenerate recycling, thus reduce analysis cost,
Save detection time.After 8 regeneration cycle, SPR response signal reduces 10.5%.
Embodiment 4：SPR response value and the relation of protein kinase A concentration, step is as follows：
（1）Protein kinase A is diluted to variable concentrations with 10 mM pH 7.2 PBS；
（2）By polypeptide P1/P2The golden film modified is placed on SPR instrument, flow injection certain density protein kinase A solution, note
Record SPR response signal, until steady.Carrier fluid is the PBS of 10 mM pH 7.2, and flow velocity is 10 L/min；
（3）In step（2）Sense channel in inject 50 mM NaOH solution, be incorporated into sense golden film surface protein kinase
A eluting so that sensing golden film regenerates, prepare by the detection for next sample；
（4）In step（3）Sense channel in inject the protein kinase A solution of another concentration, record SPR response signal, until
Steadily；
（5）Repeat step 2-4, records SPR response signal.After the protein kinase A of variable concentrations is contacted with sensing golden film, it is attached to
The protein kinase A quantity on surface is different, therefore causes SPR response signal also different.With protein kinase A concentration as abscissa, SPR
Response value is vertical coordinate, obtains canonical plotting.Figure 4, it is seen that in the concentration range of 0.1 ~ 20 nM, SPR rings
Induction signal is linear with protein kinase A concentration.After protein kinase A concentration is higher than 20 nM, SPR response signal is with egg
The amplitude that white kinases A concentration increases and increases reduces, and not in this range of linearity, this is because protein kinase A is in sensing golden film
The adsorbance on surface has reached certain saturation degree, is unfavorable for many peptide interactions of protein kinase A and golden film surface.
Embodiment 5：SPR dynamic analyses, step is as follows：
（1）Protein kinase A is diluted with 10 mM pH 7.2 PBS；
（2）By the P preparing1/P2The golden film modified is respectively placed on SPR instrument, after baseline stability, flow injection respectively
12.5th, the protein kinase A solution of 25 and 50 nM, records SPR response signal, until steady.Carrier fluid is 10 mM pH 7.2
PBS, flow velocity is 30 L/min；
（3）Srubber program using SPR system carries out dynamic analyses to above-mentioned test result, as shown in figure 5, kinetics
Simulation curve（Dotted line）Curve with practical measurement（Solid line）Identical property preferably, calculates polypeptide P1Work with protein kinase A
It is 13.8 nM with coefficient.
Embodiment 6：The selectivity analysis of sensing golden film, step is as follows：
（1）All of protein is all with 10 mM pH 7.2 PBS prepared and diluted；
（2）By polypeptide P1/P2The golden film modified is placed on SPR instrument, respectively the different protein solution of flow injection, Zhi Daoji
Line steadily, records SPR response value.As shown in fig. 6, other oroteins are in polypeptide P1/P2The SPR that the golden film surface modified causes rings
The signal that induction signal produces compared to protein kinase A is negligible, and the selectivity to protein kinase A for this sensing technology is described
Preferably, can be used for the detection of protein kinase A.In figure protein is：1- bovine serum albumin, 2- immunoglobulin G, 3- blood coagulation
Enzyme, 4- lysozyme, 5- protein kinase A.The concentration of protein kinase A is 20 nM, and 1 ~ 4 concentration is 0.1 mg/mL.
After describing embodiments of the present invention in detail, one of ordinary skilled in the art is clearly understood that, is not taking off
Various change and modification can be carried out under above-mentioned claim with spirit, all technical spirit according to the present invention are to above reality
Apply any simple modification, equivalent variations and the modification that example is made, belong to the scope of technical solution of the present invention, and the present invention is also not
It is limited to the embodiment of example in description.
SEQUENCE LISTING
<110>Anyang Teachers College
<120>The method that protein kinase is detected based on surface plasmon resonance
<130> 2010
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 25
<212> PRT
<213>Artificial sequence
<400> 1
Cys Pro Pro Pro Pro Thr Thr Tyr Ala Asp Phe Ile Ala Ser Gly Arg
1 5 10 15
Thr Gly Arg Arg Asn Ala Ile His Asp
20 25
<210> 2
<211> 5
<212> PRT
<213>Artificial sequence
<400> 2
Cys Pro Pro Pro Pro
1 5
Claims (2)
1. detect the method for protein kinase it is characterised in that comprising the following steps based on surface plasmon resonance：
A：Using golden film as inductive layer, described golden film is first used soaked in absolute ethyl alcohol 10 minutes before the use, is cleaned by ultrasonic 5
Minute, then rinsed well with secondary water, and use N2Dry up, after cleaning up, use H2Flame, in gold plaque flash annealing, first preheats,
Golden film and H2 flame are in 45 degree of angle homo genizing annelaings 30 seconds, in order to clean surface, remove the material of surface non-specific adsorption；
B：By the golden film after processing of step A in aminoacid sequence such as SEQ ID NO：Soak 12 hours in 1 polypeptide P1 solution,
The aminoacid sequence of polypeptide P1 is CPPPPTTYADFIASGRTGRRNAIHD, then rinses golden film secondary water well, N2 blows
Dry, then by above-mentioned golden film in aminoacid sequence such as SEQ ID NO：Closing in 2 hours is soaked unreacted in 2 polypeptide P2 solution
Gold surface, the aminoacid sequence of polypeptide P2 is CPPPP, then is rinsed well with DMSO, secondary water successively and blown with N2 behind golden film surface
Dry, prepared P1Polypeptide, P2The sensing golden film sealed storage modified is standby at -18 DEG C；
C：The polypeptide P that step C is obtained1, polypeptide P2The sensing golden film modified is placed on SPR instrument, when incident illumination from prism to gold
When belonging to thin film and propagating, when angle of incidence is more than critical angle, then can total internal reflection, produce evanescent waves, evanescent waves can induce metal
Surface produces plasma, and when evanescent waves are identical with the wave vector of plasma, the two resonates, and total reflection condition breaks
Bad, by metal surface Electron absorption, light-wave energy becomes the motion of metal surface electronics to incident illumination energy, leads to the energy of reflected light
Amount drastically declines, and therefore formant in reflectance spectrum, formant is reflex strength minimum, now corresponding angle of incidence is
For resonance angle, because the change of the structure on golden film surface or thickness shows as the change of resonance angle, after baseline stability, correspond to
One resonance angle, i.e. SPR response signal, flow injection protein kinase in above-mentioned sensing golden film, when protein kinase and gold
After the polypeptide on film surface combines, a new resonance angle can be produced, the size of resonance angle is SPR response signal.
2. the method detecting protein kinase based on surface plasmon resonance according to claim 1, its feature exists
In：After protein kinase acts on golden film surface, then flow injection NaOH solution, can destroy between polypeptide and protein kinase
Interact, SPR response value is reduced to initial size, so that chip regeneration.
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| CN108287148A (en) * | 2017-12-11 | 2018-07-17 | 深圳大学 | A kind of mercury ion sensor, preparation method and application |
| CN108226098A (en) * | 2017-12-20 | 2018-06-29 | 中国科学院微电子研究所 | Fano resonance optical hydrogen sensor and preparation method and application system thereof |
| CN108593601A (en) * | 2018-04-11 | 2018-09-28 | 珠海国际旅行卫生保健中心 | The SPRi biochip preparing methods of quick detection HIV-1 a kind of and its application |
| CN110579612A (en) * | 2019-10-17 | 2019-12-17 | 桂林理工大学 | Surface plasma resonance immunoassay method for embedded Au nano-film electrode |
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