CN106872435B - Protein bio luminescence imaging sensor based on acid imide multiarm polymers - Google Patents
Protein bio luminescence imaging sensor based on acid imide multiarm polymers Download PDFInfo
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Abstract
The present invention relates to a kind of protein bio luminescence imaging sensor based on acid imide multiarm polymers, use Fluc for donor, 6 kinds of acid imide multiarm polymers are receptor, testing protein competitively with acceptor polymer by noncovalent interaction in conjunction with, donor luciferase is replaced, so as to cause the variation of bioluminescence resonance energy transfer signal, signal is acquired by toy biodiversity resources instrument, every kind of albumen has collectively constituted its unique response modes to the response signal of 6 sensing units, similar to finger-print, to be identified.Identify 10 kinds of protein using linear discriminant analysis, accuracy is up to 100%.In conjunction with ultraviolet spectral technique, 76 agnoprotein samples are successfully authenticated, accuracy is up to 95%.This method does not need light source, it is only necessary to can acquire signal by being simply loaded to take pictures, have many advantages, such as that quick and convenient, stability is good, high sensitivity, high specificity, low in cost.
Description
Technical field
The invention belongs to technical field of analytical chemistry, it is related to the protein bio based on acid imide multiarm polymers and shines
Imaging sensor.
Background technique
Protein detection is an important topic in analytical chemistry, and the application in medicine and pharmacology has also been weighed extensively
Depending on, especially the diagnosis of disease marker, drug screening, disease in terms of be of great significance.However, by
Become an extremely challenging problem in the detection of the diversity and complexity of target analytes structure, protein.Array passes
Sensor, also referred to as " chemical nose/tongue ", the fingerprint image formed using the cross response between multiple sensing units and analyte
Spectrum realizes the identification to many kinds of substance and complex mixture, good etc. with fast and convenient, selectivity height, high sensitivity, stability
Advantage provides an ideal strategy for the detection of protein.In the past few years, several albumen based on different optics strategies
Quality detection sensor array has been developed that, such as fluorescence and fluorescence resonance energy transfer (FRET).However, these systems
Excitation light source is generally needed, the interference of background matrix is inevitably received and does not have the possibility for developing into portable device
Property, it is unfavorable for realizing the device of sensor.Therefore, it develops a kind of with high accuracy, high sensitivity and simple portable new
Type Protein Detection sensor array is still extremely challenging.
In recent years, bioluminescence resonance energy transfer technology, abbreviation BRET technology, is concerned, is widely used in albumen
The research of matter interaction.BRET refers to when two protein moleculars interact, the donor fluorescent of a protein fusion expression
The excitation spectrum phase of luminous signal spectrum and the acceptor fluorophore of another protein labeling that plain zymoprotein zymetology reaction generates
Overlapping induces acceptor molecule and issues fluorescence.BRET technology can overcome the problems, such as that fluorescence and FRET technology exist, and have without excitation
Light, background is lower, is easy to implement the device of sensor；True reflection testing protein surface nature is small to sample broke；It keeps away
Exempt from the interference of autofluorescence；The advantages that data window is wider has very big application prospect in the detection of protein.Common BRET is supplied
Body mainly has Fluc, bacteriofluorescein enzyme and renilla luciferase etc., and receptor is generally selected to be sent out with donor organism
Fluorescin, quantum dot or the fluorophor that light spectrum matches.
Acid imide is a kind of polycyclic aromatic hydrocarbon compound with intense fluorescence, has good light, heat, chemical stabilization
Property and higher fluorescence quantum yield.In recent years, imide derivative, which has begun, applies in bio-imaging, biomarker
And the fields such as genophore.So need a kind of acid imide multiarm polymers using amino acid modification make BRET receptor with
Protein is in conjunction with come the method that identifies albumen.
Summary of the invention
In view of this, one of the objects of the present invention is to provide a kind of, the protein based on acid imide multiarm polymers is raw
Object luminescence imaging sensor, the second object of the present invention, which is provided, detects albumen using protein bio luminescence imaging sensor
Method.
In order to achieve the above objectives, the invention provides the following technical scheme:
1. a kind of protein bio luminescence imaging sensor based on acid imide multiarm polymers, which is characterized in that institute
Stating protein bio luminescence imaging sensor is BRET receptor, BRET donor luciferase and luciferase substrate, the BRET
Receptor is acid imide multiarm polymers.
Further, the BRET donor luciferase is Fluc, and luciferase substrate is D- fluorescein.
Further, for the acid imide multiarm polymers structure as shown in general formula I, n is the integer of 10-20；R is stood alone asWherein when
R isWhen acid imide multiarm polymers are named as P1, wherein when R isBy acid imide multi-arm
Polymer is named as P2, wherein when R isWhen acid imide multiarm polymers are named as P3, wherein when R isWhen acid imide multiarm polymers are named as P4, wherein when R isWhen by acid imide multi-arm
Polymer is named as P5, wherein when R isWhen acid imide multiarm polymers are named as P6；
Further, the preparation method of the acid imide multiarm polymers P1 to P6 is following steps: 1 equivalent inducing agent N,
N'- bis- (2,6- diisopropyl phenyl) -1,6,7,12- four [4- (2- isobutyl ethyl bromide) phenoxy group] -3,4,9,10- tetracarboxylic acid
The monomer of 400 equivalent orresponding amino acids modification, 2000 equivalent butanone are added in pyromellitic imide, 1000 equivalents of methanol and 1000 are worked as
Measure water；Reaction vessel is deaerated 3 times with freeze-thaw pump circulation method, and 80 equivalent pentamethyldivinyltriamine and 28 equivalent bromines are added
Change cuprous；Nitrogen protection stirs after ten minutes at 18-25 DEG C, and 60 DEG C of polymerization reactions are reacted after overnight with liquid nitrogen quenching, and solution falls
Enter precipitation polymers in excess diethyl ether, remove ether solution, will precipitate soluble in water and be freeze-dried after dialysis 3 days, obtains acyl Asia
Amine multiarm polymers P1 to P6, the monomer of the amino acid modification are respectively phenylalanine monomer, serine monomer, aspartic acid
Monomer, leucine monomer, histidine monomer or histidine methylester monomer.
Further, the final concentration of 500nM of luciferase, acid imide multiarm polymers P1's, P2, P3, P4, P5 and P6
Final concentration is respectively 500nM, 50nM, 50nM, 250nM, 167nM and 100nM, and luciferase substrate concentration is 0.04M.
2. using the method for the protein bio luminescence imaging sensor detection albumen based on acid imide multiarm polymers,
Described method includes following steps:
(1) sensing unit is constructed, the sensing unit is BRET receptor acid imide multiarm polymers and BRET donor firefly
Light element enzyme is determined the stoichiometric ratio of BRET receptor Yu BRET donor luciferase using titration, makes luciferase concentration
Centainly, it is gradually increased polymer concentration, records BRET efficiency change situation, when curve, which tends to be steady, no longer to be risen, takes BRET
The stoichiometric ratio of BRET receptor and BRET donor luciferase constructs sensing unit when maximum efficiency, by structure needed for detecting
Build sensing unit quantity；Different acid imide multiarm polymers constitute different types of sensing unit；
(2) known albumen and agnoprotein are all diluted to same level, are respectively added in different sensing units incubate respectively
20~40min is educated, known albumen is added is configured to training set, and agnoprotein is added is configured to test set；
(3) luciferase substrate of final concentration of 0.04M is added in training set and test set respectively, it is raw by toy
Object luminescence imaging instrument detects BRET signal intensity, and Detection wavelength is 555nm and 610nm；
(4) BRET signal intensity data are obtained to step (3) using statistical software selection analysis method to analyze, is calculated
The square value of the mahalanobis distance of albumen, agnoprotein are classified as and known albumen geneva known to test set agnoprotein and training set
That the smallest albumen of the square value of distance.
It further, further include the initial concentration that detecting step (5) is obtained agnoprotein by dilution level calculation.
Further, BRET donor luciferase described in step (1) is Fluc, the BRET receptor acyl
Imines multiarm polymers are any one in P1-P6, and the final concentration of 500nM of luciferase, described in the sensing unit
The corresponding final concentration of acid imide multiarm polymers P1, P2, P3, P4, P5 and P6 is respectively 500nM, 50nM, 50nM, 250nM,
167nM and 100nM.
Further, the BRET signal intensity, abbreviation BR, calculation are as follows:
Wherein, R is the ratio of acceptor luminescence intensity and donor luminescence intensity when protein is added, R0For protein is not added
When acceptor luminescence intensity and donor luminescence intensity ratio.
Further, the sensing unit system is the 0.01M sodium phosphate buffer of pH 7.8, and the same level is albumen
Ultraviolet absorption value at 280nm is 0.005.
The beneficial effects of the present invention are: using the acid imide multiarm polymers simulated albumin matter of amino acid modification, with
Firefly luciferase interaction, constructs the sensor based on BRET technology, has successfully distinguished not homologous protein, accuracy
Reach 100%, specifically being identical property, accuracy are up to 95% in the detection of protein example.The technical program method tool
Have the advantage that 1, light source from bioluminescence, is not necessarily to exciting light, it is simple portable；2, with the acid imide of amino acid modification
Multiarm polymers simulated albumin matter, not only can be with the size of simulated albumin matter but also can be with the interaction of simulated albumin matter, to albumen
Sample more really reflects the characteristic of every kind of albumen without destructiveness；3, Detection wavelength can avoid bio-matrix interference in 610nm；4,
It only needs simply to be loaded, take pictures, then can be differentiated through mathematical statistics method analysis as a result, quick and convenient, it is low in cost；5, exist
It detects in the application of agnoprotein sample, minimal detectable concentration reaches 110nM (cromoci), and accuracy is up to 95%, explanation
This method high sensitivity, high specificity, stability are good.
Detailed description of the invention
In order to keep the purpose of the present invention, technical scheme and beneficial effects clearer, the present invention provides following attached drawing and carries out
Illustrate:
Fig. 1 is the protein bio luminescence imaging sensor detection provided by the invention based on acid imide multiarm polymers
Albumen schematic diagram；
Fig. 2 is acid imide multiarm polymers P1 synthetic route chart；
Fig. 3 is that acid imide multiarm polymers P1 nuclear-magnetism characterizes spectrogram；
Fig. 4 is that acid imide multiarm polymers P2 nuclear-magnetism characterizes spectrogram；
Fig. 5 is that acid imide multiarm polymers P3 nuclear-magnetism characterizes spectrogram；
Fig. 6 is that acid imide multiarm polymers P4 nuclear-magnetism characterizes spectrogram；
Fig. 7 is that acid imide multiarm polymers P5 nuclear-magnetism characterizes spectrogram；
Fig. 8 is that acid imide multiarm polymers P6 nuclear-magnetism characterizes spectrogram；
The A of Fig. 9 is luciferase emission spectrum provided in an embodiment of the present invention feelings Chong Die with the sharp P1-P6 luminous spectrum of polymer
Condition spectrogram, the B of Fig. 9 are the result figure that Detection wavelength is 500-700nm wave band detection BRET signal；
Figure 10 is the titration curve figure for 6 kinds of polymer that the embodiment of the present invention 3 provides；
Figure 11 is the 96 orifice plate biodiversity resources result figures that the embodiment of the present invention 3 provides；
The A of Figure 12 is that the use that the embodiment of the present invention 4 provides is sent out based on the protein bio of acid imide multiarm polymers
Light imaging sensor detects the BRET response modes figure of protein, and the B of Figure 12 is that the use that the embodiment of the present invention 4 provides is based on
The LDA shot chart of the protein bio luminescence imaging sensor detection protein of acid imide multiarm polymers；
The A of Figure 13 is that the use that the embodiment of the present invention 5 provides is sent out based on the protein bio of acid imide multiarm polymers
Light imaging sensor detects the BRET response modes figure of agnoprotein sample, and the B of Figure 13 is the use that the embodiment of the present invention 5 provides
The LDA shot chart of protein bio luminescence imaging sensor detection agnoprotein sample based on acid imide multiarm polymers；
Figure 14 is the sample-adding design table of embodiment 4.
Specific embodiment
Below in conjunction with attached drawing, a preferred embodiment of the present invention will be described in detail.It is not specified in embodiment specific
The experimental method of condition, usually according to conventional conditions or according to the manufacturer's recommendations.
Protein bio luminescence imaging sensor provided by the invention based on acid imide multiarm polymers detects albumen
Schematic diagram is as shown in Figure 1.
Embodiment 1: the preparation of acid imide polymerized monomer.
(1) N- acryloyl-L-phenylalanine (NALP) preparation: by 3.3038g phenylalanine (0.02mol), 0.01g
2,6-di-tert-butyl p-cresol, 0.8g NaOH (0.02mol) and 20mL distilled water are placed in 100ml round-bottomed flask, are clarified
1.63ml acryloyl chloride (0.02mol) is added dropwise into the solution in solution (pH12), ice bath stirring.After being added dropwise, make mixed liquor
It is warming up to room temperature, and is stirred at room temperature 1 hour.Then clear aqueous solution is acidified to pH 1-2 with concentrated hydrochloric acid.It will precipitating
Object is filtered and is recrystallized, yield: 62%.
(2) N- acryloyl-Serine (NALS), N- acryloyl-L-Aspartic acid (NALA), the bright ammonia of N- acryloyl-L-
It is raw material that sour (NALL) and the preparation of N- acryloyl-L-Histidine (NALH), which use the corresponding amino acid of 0.02mol, it then follows with
The identical preparation step of NALP.It should be strongly noted that the preparation of NALA adds a little more NaOH additionally to neutralize aspartic acid
β carboxyl, make initial pH 12, yield is respectively as follows: 28%, 37%, 38% and 54%.
(3) N- acryloyl-L-Histidine methyl esters (NALHH) preparation: nitrogen protection, to 3.3836g group ammonia under ice bath
50mL is added in acid methyl ester hydrochloride salt (0.02mol) solution newly to steam methylene chloride and 12.46mL triethylamine (90mmol, 4.5 work as
Amount).1.63ml acryloyl chloride (0.02mol) is added dropwise in 1 hour.After being added dropwise, mixture is warmed to room temperature, is stirred for 4
Hour.Reaction solution is acidified with hydrochloric acid, and with 3.5% salt acid elution.Organic layer is separated, washs three with saturation Na2CO3 solution
It is secondary.With anhydrous MgSO4 drying and solvent is removed under reduced pressure in isolated organic phase, yield: 37%.
Embodiment 2: the preparation of acid imide multiarm polymers P1-P6.
6 kinds of acid imide multiarm polymers, i.e. P1, P2, P3, P4, P5 and P6, preparation method are as follows:
The preparation of P1: polymerization reaction carries out in strictly dry Schlenk pipe, and initiator 4Br-PDI is added thereto
(10mg, 5.4 × 10-3mmol, 1 equivalent), NALP (equivalent of 508mg, 2.16mmol, 4 × 100) and butanone/methanol/water (2:1:
1, amount to 2.0mL).Then pentamethyldivinyltriamine is added by freezing-pump three times-thaw cycles degassing in reaction tube
(equivalent of 77.8mg, 0.45mmol, 4 × 21) and CuBr (equivalent of 21.4mg, 0.15mmol, 4 × 7).It is stirred at room temperature 10 points
After clock is to ensure that catalyst forms complex compound completely, nitrogen protection is polymerize at 60 DEG C.It is sudden with liquid nitrogen after reaction 5 hours
Reaction is put out, reaction mixture is poured into excess diethyl ether and is precipitated, precipitate soluble in water and is dialysed 3 days.Solvent, product is removed under reduced pressure
Vacuum drying, obtains P1 acid imide multiarm polymers, yield: 63%；Fig. 2 is acid imide multiarm polymers P1 synthetic route
Figure.
P2 is using 2.16mmolNALS as raw material, other reagents and the same P1 of step.
P3 is using 2.16mmolNALA as raw material, other reagents and the same P1 of step.
P4 is using 2.16mmolNALL as raw material, other reagents and the same P1 of step.
P5 is using 2.16mmolNALH as raw material, other reagents and the same P1 of step.
P6 is using 2.16mmolNALHH as raw material, other reagents and the same P1 of step.
P1-P6 structure respectively corresponds the acid imide multiarm polymers that R is different aminoacids substituent group as shown in general formula I,
N is the integer of 10-20, and the basic structure and physical property of each polymer are shown in Table shown in one.
One polymer basic structure of table and physical property
The nuclear-magnetism characterization spectrogram of polymer P 1 provided in an embodiment of the present invention is shown in that attached drawing 3, the nuclear-magnetism of polymer P 2 characterize spectrum
Figure is shown in that attached drawing 4, the nuclear-magnetism characterization spectrogram of polymer P 3 are shown in that attached drawing 5, the nuclear-magnetism characterization spectrogram of polymer P 4 are shown in attached drawing 6, polymer
The nuclear-magnetism characterization spectrogram of P5 is shown in that attached drawing 7, the nuclear-magnetism characterization spectrogram of polymer P 6 are shown in attached drawing 8.
Embodiment 3: the protein bio luminescence imaging sensor based on acid imide multiarm polymers
Luciferase emission spectrum and polymer P 1-P6 excitation spectrum overlapping cases spectrogram are shown in the A of attached drawing 9, Detection wavelength
The result figure of BRET signal is detected as shown in the B of Fig. 9 for 500-700nm wave band.
Sensing unit is constructed, the sensing unit is BRET receptor acid imide multiarm polymers and BRET donor luciferin
Enzyme is determined the stoichiometric ratio of BRET receptor acid imide multiarm polymers and donor luciferase using titration, makes firefly
Light element enzyme concentration is certain, is gradually increased polymer concentration, records BRET efficiency change situation, no longer rises when curve tends to be steady
When, the BRET efficiency of the point is maximum BRET efficiency, BRET receptor and BRET donor luciferin when taking BRET maximum efficiency
The stoichiometric ratio of enzyme constructs sensing unit, by building sensing unit quantity needed for detection.
In 96 orifice plates, every hole adds 50 μ L of 2mM Fluc, then is separately added into the acid imide of various concentration gradient
Multiarm polymers P1-P6 is incubated for 15min, and 50 μ L of 0.2M luciferase substrate D- fluorescein is added and is examined after 1 minute with microplate reader
The bioluminescence signal at 555nm is surveyed, the BRET efficiency of polymer and Fluc various concentration than under, knot are recorded
Fruit is as shown in Table 2.
Table imidodicarbonic diamide multiarm polymers P1-P6 and Fluc titration data
The titration curve of 6 kinds of polymer is shown in attached drawing 10, according to two result of table obtain P1-P6 polymer maximum BRET efficiency and
Stoichiometric ratio, as shown in Table 3:
Table trimerization object maximum BRET efficiency and stoichiometric ratio
Every final concentration of 500nM of hole luciferase is finally determined according to the sensitivity for testing sepectrophotofluorometer used,
It is separately added into one in acid imide multiarm polymers P1-P6 again as a kind of sensing unit, takes the corresponding biography of 1-6 kind polymer
Feeling unit is one group, and according to above-mentioned stoichiometric ratio, P1, P2, P3, P4, the final concentration of P5 and P6 are respectively 500nM, 50nM,
50nM, 250nM, 167nM and 100nM, buffer used are the 0.01M sodium phosphate buffer that pH is 7.8 (luciferase optimal pHs)
Liquid, is incubated for 15min jointly, and every group of sensing unit is added same testing protein and is incubated for 30min, after add final concentration of 0.04M
Luciferase substrate D- fluorescein.Every kind of albumen has collectively constituted its uniqueness to the response signal of different 1-6 kind sensing units
Response modes, be similar to finger-print, to be identified.
Embodiment 4: the foundation of training set
Testing protein stacked by hydrogen bond, hydrophobic, π-π and the noncovalent interactions such as electrostatic interaction competitively with receptor knot
It closes, donor luciferase is replaced, is changed so as to cause BRET signal.Pass through toy biodiversity resources instrument again
It detects BRET signal intensity (BR), selects suitable analysis method to analyze data using statistical software.
Protein bio luminescence imaging sensor based on acid imide multiarm polymers detects known protein sample, this reality
It applies 10 kinds of albumen shown in example selection table four to be tested, experimental protein final concentration is 500nM, but this should not be interpreted as to this
Invention application range is only limitted to example below:
Four the present embodiment of table detects protein varieties and albumen fundamental property
Protein bio luminescence imaging sensor based on acid imide multiarm polymers detects known protein sample, step
It is as follows:
(1) the 50 μ L of luciferase of 2000nM is added into 96 orifice plates, then respectively by each hole of table of sample-adding design shown in Figure 14
The polymer P 1 of addition 2000nM, the P6 of the P5 or 400nM of P4,668nM of P3,1000nM of P2,200nM of 200nM, each 50
μ L is incubated for 15min；Practical sample-adding can according to experiment designed, designed Loading sequence, take 1-6 kind polymer can group be combined into one
Group sensing unit carries out linear discriminant analysis further according to the data obtained；
(2) various corresponding 100 μ L of 1000nM testing protein solution are added in the hole Xiang Shangshu, are incubated for 30min jointly, at this time
The final concentration of the final concentration of 500nM, P1, P2, P3, P4, P5 and P6 of luciferase are respectively 500nM, 50nM, 50nM in hole,
250nM, 167nM and 100nM, the final concentration of 500nM of testing protein；
(3) be added 50 μ L of 0.2M luciferase substrate D- fluorescein, after 1 minute, with small animal living body optical imaging instrument into
The imaging of 96 orifice plate of row, shooting wavelength are 555nm and 610nm, and acquired results are shown in attached drawing 11；
(4) image is analyzed with Living Image software, obtains the mean radiation intensity in each hole based on
Calculate BR, BR calculation method are as follows:
Wherein, receptor is in 610nm average radiation luminous intensity and donor in 555nm average radiation when R is addition protein
The ratio of luminous intensity, R0Receptor is average in 555nm in 610nm average radiation luminous intensity and donor when for protein not being added
The ratio of radioluminescence intensity.6 parallel laboratory tests are carried out, the response modes of 10 kinds of albumen are obtained, as shown in the A of attached drawing 12；
(5) table five is the BRET response modes of 10 kinds of protein and LDA discriminant scores in the present embodiment, to above-mentioned data into
Row linear discriminant analysis obtains discriminant scores figure shown in the B of attached drawing 12, is successfully authenticated 10 kinds of protein.
The BRET response modes of 10 kinds of protein and LDA discriminant scores in five the present embodiment of table
Embodiment 5: the foundation and calculating of test set
Protein bio luminescence imaging sensor based on acid imide multiarm polymers detects agnoprotein sample, this reality
It applies 10 kinds of albumen shown in example selection table four to be tested, but should not be construed as application range of the present invention and be only limitted to this.
Protein bio luminescence imaging sensor based on acid imide multiarm polymers detects agnoprotein sample, step
It is as follows:
(1) known protein standard substance is diluted to same level, making its ultraviolet absorption value at 280nm is 0.005,
Known albumen is detected using the protein bio luminescence imaging sensor based on acid imide multiarm polymers by described in embodiment 4
The method of matter constructs training set；
(2) random 80 protein samples with 10 kinds of albumen various concentrations shown in tabulation four, as agnoprotein sample
Carry out blind experiment；
(3) above-mentioned 80 agnoprotein samples are diluted to same level, make its ultraviolet absorption value at 280nm be
0.005, it is detected by described in embodiment 4 using the protein bio luminescence imaging sensor based on acid imide multiarm polymers
Know that the method for protein obtains test set, calculates the mahalanobis distance of albumen known to every kind in test set and the present embodiment training set
Square, sample to be tested is by that the smallest albuminoid of the square value for being classified as mahalanobis distance；
(4) by dilution horizontal integration Beer-Lambert Law, by the anti-initial concentration for releasing agnoprotein matter of simple computation,
There are 76 correctly to be identified in 80 unknown samples, accuracy is up to 95%, and concentration deviation is within ± 10%, testing number
According to as shown in Table 6.
Calculation method: C=nA/ ε l
N is protein solution extension rate, and ε is the molar absorption coefficient of the albumen, and A is that ultraviolet 280 absorbance (takes herein
0.005), l is absorber thickness (usually 1cm).
The A of Figure 13 is protein bio luminescence imaging of the use provided in this embodiment based on acid imide multiarm polymers
Sensor detects the BRET response modes figure of agnoprotein sample, and the B of Figure 13 is provided in this embodiment using sub- based on acyl
The LDA shot chart of the protein bio luminescence imaging sensor detection agnoprotein sample of amine multiarm polymers；
6 80 sample parameters of table and test data
Finally, it is stated that preferred embodiment above is only used to illustrate the technical scheme of the present invention and not to limit it, although logical
It crosses above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be
Various changes are made to it in form and in details, without departing from claims of the present invention limited range.
Claims (9)
1. a kind of protein bio luminescence imaging sensor based on acid imide multiarm polymers, which is characterized in that the egg
White matter biodiversity resources sensor is BRET receptor, BRET donor luciferase and luciferase substrate, the BRET receptor
For acid imide multiarm polymers；The acid imide multiarm polymers structure such as general formulaShown, n is the integer of 10-20；R is only
It stands and is、、、Or；Wherein when R is
When acid imide multiarm polymers are named as P1, wherein when R is, acid imide multiarm polymers are named as
P2, wherein when R isWhen acid imide multiarm polymers are named as P3, wherein when R isShi Jiang
Acid imide multiarm polymers are named as P4, wherein when R isWhen acid imide multiarm polymers are named as P5,
Wherein when R isWhen acid imide multiarm polymers are named as P6；
。
2. sensor as described in claim 1, which is characterized in that the BRET donor luciferase is light of firefly luciferin
Enzyme, the luciferase substrate are D- fluoresceins.
3. sensor as described in claim 1, which is characterized in that the preparation side of the acid imide multiarm polymers P1-P6
Method is following steps: -1,6,7,12- tetra- [4-(2- isobutyl bromide second of 1 equivalent inducing agent N, N'- bis- (2,6- diisopropyl phenyl)
Ester) phenoxy group] -3, the monomer of 400 equivalent orresponding amino acids modification is added in 4,9,10- tetracarboxylic acid diimides, 2000 work as
Measure butanone, 1000 equivalents of methanol and 1000 equivalent water；Reaction vessel is deaerated 3 times with freeze-thaw pump circulation method, and 80 equivalents are added
Pentamethyldivinyltriamine and 28 equivalent cuprous bromides；Nitrogen protection stirs after ten minutes at 18-25 DEG C, and 60 DEG C of polymerizations are anti-
It is reacted after should staying overnight with liquid nitrogen quenching, solution pours into precipitation polymers in excess diethyl ether, removes ether solution, will precipitate soluble in water
And be freeze-dried after dialysing 3 days, acid imide multiarm polymers P1 to P6 is obtained, the monomer of the amino acid modification is respectively phenylpropyl alcohol
Propylhomoserin monomer, serine monomer, aspartic acid monomer, leucine monomer, histidine monomer or histidine methylester monomer.
4. sensor as described in claim 1, which is characterized in that the final concentration of 500nM of luciferase, acid imide multi-arm
The final concentration of polymer P 1, P2, P3, P4, P5 and P6 are respectively 500nM, 50nM, 50nM, 250nM, 167nM and 100nM, fluorescent
Plain zymolyte concentration is 0.04M.
5. utilizing the method for protein bio luminescence imaging sensor detection albumen described in claim 1, which is characterized in that institute
The method of stating includes the following steps:
(1) sensing unit is constructed, the sensing unit is BRET receptor acid imide multiarm polymers and BRET donor luciferin
Enzyme is determined the stoichiometric ratio of BRET receptor Yu BRET donor luciferase using titration, keeps luciferase concentration certain,
It is gradually increased polymer concentration, records BRET efficiency change situation, when curve, which tends to be steady, no longer to be risen, takes BRET efficiency most
The stoichiometric ratio of BRET receptor and BRET donor luciferase constructs sensing unit when big value, by building sensing needed for detection
Element number；Different acid imide multiarm polymers constitute different types of sensing unit；
(2) known albumen and agnoprotein are all diluted to same level, be respectively added to respectively in different sensing units be incubated for 20 ~
40min, known albumen is added is configured to training set, and agnoprotein is added is configured to test set；
(3) luciferase substrate of final concentration of 0.04M is added in training set and test set respectively, is sent out by toy biology
Light imager detects BRET signal intensity, and Detection wavelength is 555nm and 610nm；
(4) BRET signal intensity data are obtained to step (3) using statistical software selection analysis method to analyze, calculates test
Collect the square value of the mahalanobis distance of albumen known to agnoprotein and training set, agnoprotein is classified as and known albumen mahalanobis distance
That the smallest albumen of square value.
6. according to the method described in claim 5, it is characterized in that, further including that detecting step (5) is obtained not by dilution level calculation
Know the initial concentration of albumen.
7. according to the method described in claim 5, it is characterized in that, BRET donor luciferase described in step (1) is the light of firefly
Firefly luciferase, the BRET receptor acid imide multiarm polymers are any one of P1 into P6, in the sensing unit
The corresponding final concentration of the final concentration of 500nM of luciferase, acid imide the multiarm polymers P1, P2, P3, P4, P5 and P6
Respectively 500nM, 50nM, 50nM, 250nM, 167nM and 100nM.
8. according to the method described in claim 5, it is characterized in that, the BRET signal intensity, abbreviation BR, calculation is such as
Under:
Wherein, R is the ratio of acceptor luminescence intensity and donor luminescence intensity when protein is added, R0When for protein not being added by
The ratio of body luminous intensity and donor luminescence intensity.
9. according to the method described in claim 5, it is characterized in that, the sensing unit system is the 0.01M sodium phosphate of pH7.8
Buffer, the same level are that ultraviolet absorption value of the albumen at 280nm is 0.005.
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