CN106872435A - The protein bio luminescence imaging sensor of Ji Yu perylene diimide multiarm polymers - Google Patents
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Abstract
The present invention relates to a kind of protein bio luminescence imaging sensor of Ji Yu perylene diimide multiarm polymers, it is donor to use Fluc, 6 Zhong perylene diimides multiarm polymers are acceptor, testing protein is competitively combined with acceptor polymer by noncovalent interaction, donor luciferase is replaced, so as to cause the change of bioluminescence resonance energy transfer signal, signal is gathered 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, it is used to be differentiated.Differentiate 10 kinds of protein using linear discriminant analysis, accuracy is up to 100%.With reference to 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 which being taken pictures by simple sample-adding just can gather signal, has the advantages that quick and convenient, good stability, sensitivity high, high specificity, with low cost.
Description
Technical field
The invention belongs to technical field of analytical chemistry, it is related to the protein bio of Ji Yu perylene diimide multiarm polymers to light
Imaging sensor.
Background technology
Protein detection is an important topic in analytical chemistry, and its application in medicine and pharmacology has also been weighed extensively
Depending on the aspect such as diagnosis, medicine examination, Prognosis scoveillance of disease particularly in disease marker is significant.However, by
In the diversity and complexity of target analytes structure, the detection of protein turns into an extremely challenging problem.Array is passed
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, with fast and convenient, selectivity height, the high, good stability of sensitivity etc.
Advantage, for the detection of protein provides an ideal strategy.In the past few years, several albumen based on different optics strategies
Quality detection sensor array has been developed that, such as fluorescence and FRET (FRET).However, these systems
Excitation source is generally needed, the interference of background matrix is inevitably received and is not possessed the possibility for developing into portable set
Property, it is unfavorable for realizing the device of sensor.Therefore, develop 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 technologies, receives much concern, and is widely used in albumen
The research that matter interacts.BRET refers to a donor fluorescent for protein fusion expression when two protein moleculars interact
The luminous signal spectrum of plain zymoprotein zymetology reaction generation and the excitation spectrum phase of the acceptor fluorophore of another protein labeling
Overlap, induce acceptor molecule and send fluorescence.BRET technologies can overcome the problem that fluorescence and FRET technologies are present, with need not excite
Light, background is lower, is easy to implement the device of sensor；True reflection testing protein surface nature is small to sample broke；Keep away
Exempt from the interference of autofluorescence；The advantages of data window is wider, there is very big application prospect in the detection of protein.Common BRET is supplied
Body mainly has Fluc, bacteriofluorescein enzyme and renilla luciferase etc., and acceptor is generally selected to be sent out with donor organism
Fluorescin, quantum dot or fluorophor that light spectrum matches.
Perylene diimide is a kind of polycyclic aromatic hydrocarbon compound with intense fluorescence, with good light, heat, chemically stable
Property and fluorescence quantum yield higher., perylene diimides derivative has begun to apply in bio-imaging, biomarker in recent years
And the field such as genophore.So need badly it is a kind of using amino acid modified perylene diimides multiarm polymers make BRET acceptors with
The method that protein combines to differentiate albumen.
The content of the invention
In view of this, an object of the present invention is to provide a kind of protein life of Ji Yu perylene diimide multiarm polymers
Thing luminescence imaging sensor, the second object of the present invention is provided and detects albumen using protein bio luminescence imaging sensor
Method.
To reach above-mentioned purpose, the present invention provides following technical scheme：
1. a kind of protein bio luminescence imaging sensor of Ji Yu perylene diimide multiarm polymers, it is characterised in that institute
Protein bio luminescence imaging sensor is stated for BRET acceptors, BRET donors luciferase and luciferase substrate, the BRET
Acceptor Wei perylene diimide multiarm polymers.
Further, the BRET donors luciferase is Fluc, and luciferase substrate is D- fluoresceins.
Further, as shown in formula I, n is the integer of 10-20 to Suo Shu perylene diimide multiarm polymers structures；R stands alone asWherein work as R
ForPerylene diimide multiarm polymers are named as P1 by Shi, wherein when R isJiang perylene diimide multi-arm
Polymer is named as P2, wherein when R isPerylene diimide multiarm polymers are named as P3 by Shi, wherein when R isPerylene diimide multiarm polymers are named as P4 by Shi, wherein when R isShi is Jiang perylene diimide multi-arm
Polymer is named as P5, wherein when R isPerylene diimide multiarm polymers are named as P6 by Shi；
Further, the preparation method of Suo Shu perylene diimide multiarm polymers P1 to P6 is following steps：1 equivalent inducing agent N,
N'- bis- (2,6- diisopropyl phenyls) -1,6,7,12- four [4- (2- isobutyl ethyl bromides) phenoxy group] perylene -3,4,9,10- tetracarboxylic acids
The monomer for adding 400 equivalent orresponding amino acids to modify in pyromellitic imide, 2000 equivalent butanone, 1000 equivalents of methanol and 1000 are worked as
Amount water；Reaction vessel is deaerated 3 times with freeze-thaw pump circulation method, adds 80 equivalent PMDETAs and 28 equivalent bromines
Change cuprous；Nitrogen is protected, and after being stirred 10 minutes at 18-25 DEG C, 60 DEG C of polymerisations are overnight reacted with liquid nitrogen quenching afterwards, and solution falls
Enter precipitation polymers in excess diethyl ether, remove ether solution, will precipitate soluble in water and freeze-drying , get perylenes acyl Asia after dialysing 3 days
Amine multiarm polymers P1 to P6, the amino acid modified monomer is respectively phenylalanine monomer, serine monomer, aspartic acid
Monomer, leucine monomer, histidine monomer or histidine methylester monomer.
Further, the final concentration of 500nM , perylene diimides multiarm polymers P1 of luciferase, P2, P3, P4, P5 and P6's
Final concentration is respectively 500nM, 50nM, 50nM, 250nM, 167nM and 100nM, and luciferase substrate concentration is 0.04M.
2. the method for detecting albumen using the protein bio luminescence imaging sensor of Ji Yu perylene diimide multiarm polymers,
Methods described comprises the following steps：
(1) sensing unit is built, the sensing unit is BRET Shou Ti perylene diimides multiarm polymers and BRET donor fireflies
Light element enzyme, the stoichiometric ratio of BRET acceptors and BRET donor luciferases is determined using titration, makes luciferase concentration
Necessarily, gradually increase polymer concentration, record BRET efficiency change situations, when curve tends to be steady no longer to be risen, take BRET
BRET acceptors build sensing unit with the stoichiometric ratio of BRET donor luciferases during maximum efficiency, by structure needed for detection
Build sensing unit quantity；Different perylene diimide multiarm polymers constitute different types of sensing unit；
(2) known albumen and agnoprotein are all diluted to same level, are respectively added to respectively in different sensing units and incubate
20~40min is educated, add known albumen is configured to training set, and add agnoprotein is configured to test set；
(3) luciferase substrate of final concentration of 0.04M is added in training set and test set respectively, is given birth to by toy
Thing luminescence imaging instrument detects BRET signal intensities, and its Detection wavelength is 555nm and 610nm；
(4) BRET signal intensity data are obtained to step (3) using statistical software selection analysis method to be analyzed, is calculated
The square value of test set agnoprotein and the mahalanobis distance of albumen known to training set, agnoprotein is classified as and known albumen geneva
That minimum albumen of the square value of distance.
Further, the initial concentration of agnoprotein is also calculated by dilution level including detecting step (5).
Further, BRET donor luciferases are Flucs described in step (1), the BRET Shou Ti perylene acyls
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 concentrations of perylene diimide multiarm polymers P1, P2, P3, P4, P5 and P6 are respectively 500nM, 50nM, 50nM, 250nM,
167nM and 100nM.
Further, the BRET signal intensities, abbreviation BR, calculation is as follows：
Wherein, R is the ratio of acceptor luminescence intensity and donor luminescence intensity when adding protein, R0Not add protein
When acceptor luminescence intensity and donor luminescence intensity ratio.
Further, the sensing unit system is the 0.01M sodium phosphate buffers of pH 7.8, and the same level is albumen
Ultraviolet absorption value under 280nm is 0.005.
The beneficial effects of the present invention are：Using amino acid modified perylene diimides multiarm polymers simulated albumin matter, with
Firefly luciferase interacts, and builds the sensor based on BRET technologies, has successfully distinguished not homologous protein, accuracy
100% is reached, specific the being identical property in the detection of protein example, accuracy is up to 95%.The technical program method has
Have the advantage that：1st, light source derives from bioluminescence, simple portable without exciting light；2nd, with amino acid modified perylene diimides
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 truly reflects the characteristic of every kind of albumen without destructiveness；3rd, Detection wavelength can avoid bio-matrix from disturbing in 610nm；4、
Only need simply to be loaded, take pictures, then can just obtain differentiating result through mathematical statistics method analysis, it is quick and convenient, it is with low cost；5th, exist
Detect in the application of agnoprotein sample, minimal detectable concentration reaches 110nM (cromoci), and accuracy is up to 95%, explanation
This method sensitivity is high, high specificity, good stability.
Brief description of the drawings
In order that the purpose of the present invention, technical scheme and beneficial effect are clearer, the present invention provides drawings described below and carries out
Explanation：
The protein bio luminescence imaging sensor detection of the Ji Yu perylene diimide multiarm polymers that Fig. 1 is provided for the present invention
Albumen schematic diagram；
The Wei perylene diimide multiarm polymers P1 synthetic route charts of figure 2；
The Wei perylene diimide multiarm polymers P1 nuclear-magnetisms of figure 3 characterize spectrogram；
The Wei perylene diimide multiarm polymers P2 nuclear-magnetisms of figure 4 characterize spectrogram；
The Wei perylene diimide multiarm polymers P3 nuclear-magnetisms of figure 5 characterize spectrogram；
The Wei perylene diimide multiarm polymers P4 nuclear-magnetisms of figure 6 characterize spectrogram；
The Wei perylene diimide multiarm polymers P5 nuclear-magnetisms of figure 7 characterize spectrogram；
The Wei perylene diimide multiarm polymers P6 nuclear-magnetisms of figure 8 characterize spectrogram；
Fig. 9 A are that luciferase emission spectrum provided in an embodiment of the present invention swashs P1-P6 luminous spectrum overlapping cases with polymer
Spectrogram, Fig. 9 B are the result figure that Detection wavelength is 500-700nm wave bands detection BRET signals；
Figure 10 is the titration curve figure of 6 kinds of polymer that the embodiment of the present invention 3 is provided；
Figure 11 is the 96 orifice plate biodiversity resources result figures that the embodiment of the present invention 3 is provided；
Figure 12 A are that the protein bio of the use Ji Yu perylene diimide multiarm polymers that the embodiment of the present invention 4 is provided lights
Imaging sensor detects the BRET response modes figures of protein, and Figure 12 B are that the use Ji Yu perylenes acyl that the embodiment of the present invention 4 is provided is sub-
The protein bio luminescence imaging sensor of amine multiarm polymers detects the LDA shot charts of protein；
Figure 13 A are that the protein bio of the use Ji Yu perylene diimide multiarm polymers that the embodiment of the present invention 5 is provided lights
Imaging sensor detects the BRET response modes figures of agnoprotein sample, and Figure 13 B are that the use that the embodiment of the present invention 5 is provided is based on
The protein bio luminescence imaging sensor of perylene diimide multiarm polymers detects the LDA shot charts of agnoprotein sample；
Figure 14 is the sample-adding design table of embodiment 4.
Specific embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.It is unreceipted specific in embodiment
The experimental technique of condition, generally according to normal condition or according to the condition proposed by manufacturer.
The protein bio luminescence imaging sensor detection albumen of the Ji Yu perylene diimide multiarm polymers that the present invention is provided
Schematic diagram is as shown in Figure 1.
Embodiment 1：The preparation of perylene diimide polymerized monomer.
(1) preparation of N- acryloyls-L-phenylalanine (NALP)：By 3.3038g phenylalanines (0.02mol), 0.01g
BHT, 0.8g NaOH (0.02mol) and 20mL distilled water are placed in 100ml round-bottomed flasks, are clarified
Solution (pH12), ice bath stirring, to dropwise addition 1.63ml acryloyl chlorides (0.02mol) in the solution.After completion of dropping, make mixed liquor
Room temperature is warming up to, and is stirred at room temperature 1 hour.Then the aqueous solution concentrated hydrochloric acid of clarification is acidified to pH 1-2.Will precipitation
Thing is filtered and recrystallized, yield：62%.
(2) N- acryloyls-Serine (NALS), N- acryloyls-L-Aspartic acid (NALA), the bright ammonia of N- acryloyls-L-
The preparation of sour (NALL) and N- acryloyls-L-Histidine (NALH) is raw material using the corresponding amino acid of 0.02mol, it then follows with
NALP identical preparation processes.It should be strongly noted that the preparation of NALA additionally adds a point NaOH to neutralize aspartic acid
β carboxyls, make initial pH for 12, yield is respectively：28%, 37%, 38% and 54%.
(3) preparation of N- acryloyls-L-Histidine methyl esters (NALHH)：Nitrogen is protected, to 3.3836g group ammonia under ice bath
(90mmol, 4.5 work as to add 50mL newly to steam dichloromethane and 12.46mL triethylamines in acid methyl ester hydrochloride salt (0.02mol) solution
Amount).1.63ml acryloyl chlorides (0.02mol) was added dropwise in 1 hour.After completion of dropping, 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, three are washed with saturation Na2CO3 solution
It is secondary.The organic phase that will be separate is dried and removal of solvent under reduced pressure, yield with anhydrous MgSO4：37%.
Embodiment 2：The preparation of perylene diimide multiarm polymers P1-P6.
6 Zhong perylene diimide multiarm polymers, i.e. P1, P2, P3, P4, P5 and P6, its preparation method are as follows：
The preparation of P1：Polymerisation is carried out in strict dry Schlenk pipes, is added thereto to initiator 4Br-PDI
(equivalent of 10mg, 5.4 × 10-3mmol, 1), NALP (508mg, 2.16mmol, 4 × 100 equivalents) and butanone/methanol/water (2:1:
1, amount to 2.0mL).Reaction tube deaerates by three freezing-pump-thaw cycles, is subsequently adding PMDETA
(77.8mg, 0.45mmol, 4 × 21 equivalents) and CuBr (21.4mg, 0.15mmol, 4 × 7 equivalents).It is stirred at room temperature 10 points
After clock is to ensure that catalyst forms complex compound completely, nitrogen protection is polymerized at 60 DEG C.It is sudden with liquid nitrogen after reaction 5 hours
Reaction is put out, reactant mixture is poured into excess diethyl ether and is precipitated, precipitated soluble in water and dialyse 3 days.Removal of solvent under reduced pressure, product
Vacuum drying, obtains P1 perylene diimide multiarm polymers, yield：63%；The Wei perylene diimide multiarm polymers P1 synthetic routes of figure 2
Figure.
P2 with 2.16mmolNALS as raw material, other reagents and the same P1 of step.
P3 with 2.16mmolNALA as raw material, other reagents and the same P1 of step.
P4 with 2.16mmolNALL as raw material, other reagents and the same P1 of step.
P5 with 2.16mmolNALH as raw material, other reagents and the same P1 of step.
P6 with 2.16mmolNALHH as raw material, other reagents and the same P1 of step.
It is that different aminoacids replace Ji perylene diimide multiarm polymers that P1-P6 structures correspond to R as shown in formula I, respectively,
N is the integer of 10-20, and the basic structure and physical property of each polymer are shown in Table shown in one.
The polymer basic structure of table one and physical property
The nuclear-magnetism of polymer P provided in an embodiment of the present invention 1 characterizes spectrogram and sees accompanying drawing 3, and the nuclear-magnetism of polymer P 2 characterizes spectrum
Figure is shown in accompanying drawing 4, and the nuclear-magnetism of polymer P 3 characterizes spectrogram and sees accompanying drawing 5, and the nuclear-magnetism of polymer P 4 characterizes spectrogram and sees accompanying drawing 6, polymer
The nuclear-magnetism of P5 characterizes spectrogram and sees accompanying drawing 7, and the nuclear-magnetism of polymer P 6 characterizes spectrogram and sees accompanying drawing 8.
Embodiment 3：The protein bio luminescence imaging sensor of Ji Yu perylene diimide multiarm polymers
Luciferase emission spectrum is shown in accompanying drawing 9A with polymer P 1-P6 excitation spectrum overlapping cases spectrograms, and Detection wavelength is
The result figure of 500-700nm wave bands detection BRET signals is as shown in Figure 9 B.
Sensing unit is built, the sensing unit is BRET Shou Ti perylene diimides multiarm polymers and BRET donor luciferins
Enzyme, the stoichiometric ratio of BRET Shou Ti perylene diimides multiarm polymers and donor luciferase is determined using titration, makes firefly
Light element enzyme concentration is certain, gradually increases polymer concentration, records BRET efficiency change situations, no longer rise when curve is tended to be steady
When, the BRET efficiency of the point is maximum BRET efficiency, BRET acceptors and BRET donor luciferins when taking BRET maximum efficiencies
The stoichiometric ratio of enzyme builds sensing unit, by structure sensing unit quantity needed for detection.
In 96 orifice plates, add the μ L of 2mM Flucs 50 per hole, then be separately added into various concentrations gradient perylene diimides
Multiarm polymers P1-P6, is incubated 15min, adds the μ L of 0.2M luciferase substrate D- fluoresceins 50, after 1 minute, is examined with ELIASA
The bioluminescence signal surveyed at 555nm, the BRET efficiency of record polymer and Fluc various concentrations than under, its knot
Fruit is as shown in Table 2.
Biao bis- perylene diimide multiarm polymers P1-P6 and Fluc titration data
The titration curve of 6 kinds of polymer is shown in accompanying drawing 10, according to the result of table two draw P1-P6 polymer maximum BRET efficiency and
Stoichiometric proportion, as shown in Table 3：
Table trimerization compound maximum BRET efficiency and stoichiometric proportion
Sensitivity finally according to experiment sepectrophotofluorometer used determines every hole final concentration of 500nM of luciferase,
One in the other Jia Ru perylene diimides multiarm polymers P1-P6 of Fen is a kind of sensing unit again, takes the corresponding biography of 1-6 kind polymer
Sense unit is one group, and according to above-mentioned stoichiometric proportion, the final concentration of P1, P2, P3, P4, P5 and P6 is respectively 500nM, 50nM,
50nM, 250nM, 167nM and 100nM, buffer solution used are that pH is the 0.01M sodium phosphate buffers of 7.8 (luciferase optimal pHs)
Liquid, is incubated 15min jointly, and every group of sensing unit adds same testing protein to be incubated 30min, after add final concentration of 0.04M
Luciferase substrate D- fluoresceins.It is unique that every kind of albumen has collectively constituted it to the response signal of different 1-6 kinds sensing units
Response modes, similar to finger-print, be used to be differentiated.
Embodiment 4：The foundation of training set
Testing protein stacked by hydrogen bond, hydrophobic, π-π and the noncovalent interaction such as electrostatic interaction competitively with acceptor knot
Close, donor luciferase is replaced, so as to cause BRET signals to change.Pass through toy biodiversity resources instrument again
Detection BRET signal intensities (BR), selects suitable analysis method to be analyzed data using statistical software.
The known protein sample of protein bio luminescence imaging sensor detection of Ji Yu perylene diimide multiarm polymers, this reality
Apply example to be tested from 10 kinds of albumen shown in table four, experimental protein final concentration is 500nM, but this should not be interpreted as into this
Invention range of application is only limitted to following example：
The present embodiment of table four detects protein varieties and albumen fundamental property
The known protein sample of protein bio luminescence imaging sensor detection of Ji Yu perylene diimide multiarm polymers, step
It is as follows：
(1) to the μ L of luciferase 50 of addition 2000nM in 96 orifice plates, then each hole of sample-adding design table shown in Figure 14 is pressed respectively
Add the polymer P 1 of 2000nM, the P6 of the P5 or 400nM of P4,668nM of P3,1000nM of P2,200nM of 200nM, each 50
μ L, are incubated 15min；Actual sample-adding can be according to experiment designed, designed Loading sequence, and taking 1-6 kinds polymer can be combined as one
Group sensing unit, linear discriminant analysis is carried out further according to the data obtained；
(2) to the various corresponding μ L of 1000nM testing proteins solution 100 are added in above-mentioned hole, 30min is incubated jointly, now
The final concentration of 500nM of luciferase in hole, the final concentration of P1, P2, P3, P4, P5 and P6 is respectively 500nM, 50nM, 50nM,
250nM, 167nM and 100nM, the final concentration of 500nM of testing protein；
(3) the μ L of 0.2M luciferase substrate D- fluoresceins 50 are added, after 1 minute, is entered with small animal living body optical imaging instrument
The orifice plate of row 96 is imaged, and shooting wavelength is 555nm and 610nm, and acquired results are shown in accompanying drawing 11；
(4) image is analyzed with Living Image softwares, obtains the mean radiation intensity in each hole based on
BR is calculated, BR computational methods are：
Wherein, when R is to add protein acceptor in 610nm average radiations luminous intensity and donor in 555nm average radiations
The ratio of luminous intensity, R0Not add acceptor during protein average in 555nm in 610nm average radiations luminous intensity and donor
The ratio of radioluminescence intensity.6 parallel laboratory tests are carried out, 10 kinds of response modes of albumen is obtained, as shown in accompanying drawing 12A；
(5) table five is BRET response modes and the LDA discriminant scores of 10 kinds of protein in the present embodiment, and above-mentioned data are entered
Row linear discriminant analysis, obtains discriminant scores figure shown in accompanying drawing 12B, is successfully authenticated 10 kinds of protein.
The BRET response modes of 10 kinds of protein and LDA discriminant scores in the present embodiment of table five
Embodiment 5：The foundation and calculating of test set
The protein bio luminescence imaging sensor detection agnoprotein sample of Ji Yu perylene diimide multiarm polymers, this reality
Apply example to be tested from 10 kinds of albumen shown in table four, but should not be construed as range of application of the present invention and be only limitted to this.
The protein bio luminescence imaging sensor detection agnoprotein sample of Ji Yu perylene diimide multiarm polymers, step
It is as follows：
(1) known protein standard substance is diluted to same level, its ultraviolet absorption value under 280nm is 0.005,
Albumen known to the protein bio luminescence imaging sensor detection of Ji Yu perylene diimide multiarm polymers is used as described in embodiment 4
The method of matter builds training set；
(2) random 10 kinds of the 80 of albumen various concentrations protein samples with shown in tabulation four, as agnoprotein sample
Carry out blind experiment；
(3) it is its ultraviolet absorption value under 280nm to same level above-mentioned 80 agnoprotein Sample Dilutions
0.005, the protein bio luminescence imaging sensor using Ji Yu perylene diimide multiarm polymers as described in embodiment 4 is detected
Know that method of protein obtains test set, calculate the mahalanobis distance of every kind of known albumen in test set and the present embodiment training set
Square, sample to be tested will classify as that albuminoid of the square value minimum of 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 differentiated in 80 unknown samples, up to 95%, within ± 10%, it detects number to concentration deviation to accuracy
According to as shown in Table 6.
Computational methods：C=nA/ ε l
N is protein solution extension rate, and ε is the molar absorption coefficient of the albumen, and A (takes herein for ultraviolet 280 absorbance
0.005), l is absorber thickness (usually 1cm).
The protein bio luminescence imaging of the use Ji Yu perylene diimide multiarm polymers that Figure 13 A are provided for the present embodiment is passed
Sensor detects the BRET response modes figures of agnoprotein sample, and Figure 13 B are more for the use Ji Yu perylene diimides that the present embodiment is provided
The protein bio luminescence imaging sensor of arm polymer detects the LDA shot charts of agnoprotein sample；
6 80 sample parameters of table and test data
Finally illustrate, preferred embodiment above is merely illustrative of the technical solution of the present invention and unrestricted, although logical
Cross above preferred embodiment to be described in detail the present invention, it is to be understood by those skilled in the art 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 (10)
1. a kind of protein bio luminescence imaging sensor of Ji Yu perylene diimide multiarm polymers, it is characterised in that the egg
White matter biodiversity resources sensor is BRET acceptors, BRET donors luciferase and luciferase substrate, the BRET acceptors
Wei perylene diimide multiarm polymers.
2. sensor as claimed in claim 1, it is characterised in that the BRET donors luciferase is light of firefly luciferin
Enzyme, luciferase substrate is D- fluoresceins.
3. sensor as claimed in claim 1, it is characterised in that Suo Shu perylene diimide multiarm polymers structure such as formula I institutes
Show, n is the integer of 10-20；R stands alone as Wherein when R isPerylene diimide multiarm polymers are named as P1 by Shi, its
In be as RPerylene diimide multiarm polymers are named as P2, wherein when R isShi is sub- Jiang perylene acyl
Amine multiarm polymers are named as P3, wherein when R isPerylene diimide multiarm polymers are named as P4 by Shi, wherein working as R
ForPerylene diimide multiarm polymers are named as P5 by Shi, wherein when R isShi is more Jiang perylene diimide
Arm polymer is named as P6；
4. sensor as claimed in claim 3, it is characterised in that the preparation side of Suo Shu perylene diimide multiarm polymers P1-P6
Method is following steps：[4- (the 2- isobutyl bromide second of 1 equivalent inducing agent N, N'- bis- (2,6- diisopropyl phenyl) -1,6,7,12- tetra-
Ester) phenoxy group] perylene -3, the monomer for adding 400 equivalent orresponding amino acids to modify in 4,9,10- tetracarboxylic acid diimides, 2000 work as
Amount butanone, 1000 equivalents of methanol and 1000 equivalent water；Reaction vessel is deaerated 3 times with freeze-thaw pump circulation method, adds 80 equivalents
PMDETA and 28 equivalent cuprous bromides；Nitrogen is protected, and after being stirred 10 minutes at 18-25 DEG C, 60 DEG C of polymerizations are anti-
Overnight should be reacted with liquid nitrogen quenching afterwards, solution pours into precipitation polymers in excess diethyl ether, removes ether solution, will precipitate soluble in water
And freeze-drying , get perylene diimide multiarm polymers P1 to P6 after dialysing 3 days, the amino acid modified monomer is respectively phenylpropyl alcohol
Propylhomoserin monomer, serine monomer, aspartic acid monomer, leucine monomer, histidine monomer or histidine methylester monomer.
5. sensor as claimed in claim 3, it is characterised in that the final concentration of 500nM , perylene diimide multi-arms of luciferase
Polymer P 1, P2, the final concentration of P3, P4, P5 and P6 is respectively 500nM, 50nM, 50nM, 250nM, 167nM and 100nM, fluorescent
Plain zymolyte concentration is 0.04M.
6. the method that albumen is detected using the protein bio luminescence imaging sensor described in claim 1, it is characterised in that institute
The method of stating comprises the following steps：
(1) sensing unit is built, the sensing unit is BRET Shou Ti perylene diimides multiarm polymers and BRET donor luciferins
Enzyme, the stoichiometric ratio of BRET acceptors and BRET donor luciferases is determined using titration, makes luciferase concentration certain,
Gradually increase polymer concentration, record BRET efficiency change situations, when curve tends to be steady no longer to be risen, take BRET efficiency most
The stoichiometric ratio of BRET acceptors and BRET donor luciferases builds sensing unit during big value, is sensed by being built needed for detection
Element number；Different perylene diimide multiarm polymers constitute different types of sensing unit；
(2) known albumen and agnoprotein are all diluted to same level, are respectively added to respectively in different sensing units and are incubated 20
~40min, add known albumen is configured to training set, and add agnoprotein 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 the way that toy is biological
Photoimaging instrument detects BRET signal intensities, and its Detection wavelength is 555nm and 610nm；
(4) BRET signal intensity data are obtained to step (3) using statistical software selection analysis method to be analyzed, calculates test
The square value of collection agnoprotein and the mahalanobis distance of albumen known to training set, agnoprotein is classified as and known albumen mahalanobis distance
Minimum that albumen of square value.
7. method according to claim 6, it is characterised in that also calculated not by dilution level including detecting step (5)
Know the initial concentration of albumen.
8. method according to claim 6, it is characterised in that BRET donor luciferases are the light of firefly described in step (1)
Firefly luciferase, the BRET Shou Ti perylene diimides multiarm polymers are any one in P1 to P6, in the sensing unit
The corresponding final concentrations of the final concentration of 500nM of luciferase, Suo Shu perylene diimide multiarm polymers P1, P2, P3, P4, P5 and P6
Respectively 500nM, 50nM, 50nM, 250nM, 167nM and 100nM.
9. method according to claim 6, it is characterised in that the BRET signal intensities, abbreviation BR, calculation is such as
Under：
Wherein, R is the ratio of acceptor luminescence intensity and donor luminescence intensity when adding protein, R0Received during protein not added
The ratio of body luminous intensity and donor luminescence intensity.
10. method according to claim 6, it is characterised in that the sensing unit system is the 0.01M phosphoric acid of pH7.8
Sodium buffer solution, the same level is that ultraviolet absorption value of the albumen under 280nm is 0.005.
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