CN106589321B - Preparation method and applications based on cationic conjugated polymer Yu the fluorescent optical sensor system of complex of iridium FRET effect - Google Patents
Preparation method and applications based on cationic conjugated polymer Yu the fluorescent optical sensor system of complex of iridium FRET effect Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G61/00—Macromolecular compounds obtained by reactions forming a carbon-to-carbon link in the main chain of the macromolecule
- C08G61/02—Macromolecular compounds containing only carbon atoms in the main chain of the macromolecule, e.g. polyxylylenes
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic System
- C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic System compounds of the platinum group
- C07F15/0033—Iridium compounds
- C07F15/004—Iridium compounds without a metal-carbon linkage
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G2261/00—Macromolecular compounds obtained by reactions forming a carbon-to-carbon link in the main chain of the macromolecule
- C08G2261/10—Definition of the polymer structure
- C08G2261/18—Definition of the polymer structure conjugated
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G2261/00—Macromolecular compounds obtained by reactions forming a carbon-to-carbon link in the main chain of the macromolecule
- C08G2261/30—Monomer units or repeat units incorporating structural elements in the main chain
- C08G2261/31—Monomer units or repeat units incorporating structural elements in the main chain incorporating aromatic structural elements in the main chain
- C08G2261/314—Condensed aromatic systems, e.g. perylene, anthracene or pyrene
- C08G2261/3142—Condensed aromatic systems, e.g. perylene, anthracene or pyrene fluorene-based, e.g. fluorene, indenofluorene, or spirobifluorene
Abstract
The invention discloses the preparation method and applications based on cationic conjugated polymer Yu the fluorescent optical sensor system of complex of iridium FRET effect, feature is the synthesis step for including complex of iridium；The synthesis step of cationic conjugated polymer；The step of cationic conjugated polymer and complex of iridium occur FRET, obtain the fluorescent optical sensor system based on cationic conjugated polymer and complex of iridium FRET effect；The fluorescent optical sensor system can be applied to detection fibrin ferment, atriphos, platelet derived growth factor, cocaine and lysozyme, and advantage is that good specificity, high sensitivity, result are accurate and reliable, at low cost, quick, and process is extremely simple.
Description
Technical field
The present invention relates to a kind of fluorescent optical sensor systems, are matched based on cationic conjugated polymer with iridium more particularly, to one kind
Close the preparation method and applications of the fluorescent optical sensor system of object FRET effect.
Background technique
Fluorescence resonance energy transfer (FRET) is that current design fluorescent optical sensor uses relatively broad principle.Fluorescence resonance
Energy transfer refers to that two fluorescence chromophoric groups when sufficiently closing to, are excited after the photon that donor molecule absorbs certain frequency
To higher electron energy state, before the electronics returns to ground state, by dipole-dipole interaction, energy is realized to neighbouring receptor
Resonance energy transfer occurs for molecule transfer.It is harsh that the condition of effective energy transfer occurs between energy supply body-acceptor
, specifically include that the emission spectrum of (1) energy donor must be overlapped with the absorption spectrum of energy acceptor；(2) energy donor and energy
The fluorescence chromophore of amount receptor must arrange in the right way；(3) must be close enough between energy donor, energy acceptor,
The probability that energy transfer occurs so just can be high.Fluorescence sense method based on fluorescence resonance energy transfer has operating process letter
The features such as list, high sensitivity, good, the response time is short selectivity.
The sensibility that FRET adjusts the distance by it is widely used to structure of biological macromolecule, property, reaction mechanism and determines
The research of amount analysis etc..Therefore, energy transfer fluorescent analysis is very suitable for environment, biomedical science and clinicization
One of learn etc. complexity, the analysis of low content component and genetic engineering new tool.
It is well known that be equally the metal iridium complex of octahedral coordination, with strong visible absorbance and good photic
Luminescent properties, compared with bipyridyl ruthenium class complex, complex of iridium has high quantum efficiency, emission lifetime length, emission wavelength can
Adjust, big Stokes displacement and excited by visible light and the features such as photostability, make its chemistry, bio-sensing and biology at
As field attracts wide public concern.
Cationic conjugated polymer (CCP) the water soluble fluorescence molecule novel as one kind, big with molar absorption coefficient,
The advantages that fluorescence quantum yield is high.Meanwhile conjugated polymer has the function of molecular wire, can be realized putting for fluorescence signal
Greatly.Conjugation can make conjugated polymer light energy collecting effect with higher and good fluorescent characteristic, and hydrophilic
Group can assign their some other outstanding characteristics, such as: can with the matter interaction of oppositely charged, to extinguish base
The highly sensitive response of group, and have more it is attractive that it can be dissolved in water, and based on this, may be implemented to DNA, egg
The highly sensitive detection of the biomolecule such as white matter.Based on these unique photoluminescent properties, cationic conjugated polymer is in biology
There is boundless application prospect in terms of chemical analysis.It has no and is cooperated based on cationic conjugated polymer and iridium both at home and abroad at present
The fluorescent optical sensor system of object FRET effect and its relevant report of application.
Summary of the invention
Good, high sensitivity that technical problem to be solved by the invention is to provide a species specificity, result be accurate and reliable, cost
It is low, quick, and the extremely simple fluorescence sense body based on cationic conjugated polymer Yu complex of iridium FRET effect of process
The preparation method and applications of system.
The technical scheme of the invention to solve the technical problem is: a kind of based on cationic conjugated polymer and iridium
The preparation method of the fluorescent optical sensor system of complex FRET effect, the specific steps are as follows:
(1) synthesis of complex of iridium
A. by 10~15mmol 2- chlorine pyrimidine, 10~20mmol 2,4- difluorophenyl boronic acid, 1.25~1.5mmol triphen
Base phosphine, 10~15mL glycol dimethyl ether and 15~20mL solution of potassium carbonate are added in three-neck flask, lead to nitrogen 30 at room temperature
~60min, then in three-neck flask be added 0.3~0.5mmol palladium acetate, magnetic agitation, and in 110~150 DEG C flow back 15~
Stop reaction after 20h, is cooled to room temperature, separates organic phase and water phase；Water phase 30~60mL ethyl acetate is extracted, then by water
Mutually extraction is repeated three times with 30~60mL ethyl acetate；After organic phase is merged successively with 40~80mL water and 40~80mL salt water
Washing, then it is dry with anhydrous magnesium sulfate, revolving removes acetate extractant, and obtained solid is dried in vacuo 12 in 60~80 DEG C
~for 24 hours；By dried object, with methylene chloride/acetone soln (volume ratio 10:1), column is chromatographed on a silica gel column, passes through chromatographic silica gel plate
Analysis ingredient merges the eluent at second peak, removes methylene chloride and acetone, vacuum at 65 DEG C by vacuum rotary steam
Dry 12~for 24 hours, obtain light yellow solid 4- (2,4- difluorophenyl) pyrimidine (DFPPM)；
B. by 2~4mmol IrCl3·3H2O, 4~5mmol 4- (2,4 difluorobenzene base) pyrimidine (DFPPM), 30~50mL
Ethylene glycol monoethyl ether and 10~30mL water are added in three-neck flask, after leading to 30~60min of nitrogen at room temperature, in 120~150 DEG C
Flow back 24~36h, and cooled and filtered obtains solid and filtrate；Solid 2~5mL ethylene glycol monoethyl ether is extracted, merges and extracts
Liquid and filtrate are added the aquatic yellowly precipitating of 20~30mL, yellow mercury oxide are successively washed with water, acetone, dehydrated alcohol, is obtained
Chlorine bridging；
C. it is added in reaction flask after 0.07~0.10mmol chlorine bridging being dissolved in 10~20mL methylene chloride, by 0.10
4,4 '-dicarboxylic acids -2 of~0.20mmol assistant ligand, 2 '-bipyridyls are added in reaction flask after being dissolved in 10~20mL methanol,
Reaction flask is flowed back 2~5h at 60~100 DEG C；0.2~0.4g sodium acetate is dissolved in after 2~5mL methanol again and is added to reaction
In bottle, flow back 1~5h, is cooled to room temperature；Reaction is added to after ammonium hexafluorophosphate is added to 2~5mL methanol solution to saturation
In bottle, 30~60min of stirring at normal temperature, revolving removes solvent methanol, then the salt of 1~3M, 10~20mL is added in obtained solid
Acid solution, suspension stir 10~30min, filtering；By solid with after water washing 2 times of 10~20mL, by solid 2mL methanol
It extracts, the ammonium hexafluorophosphate methanol solution of 2~5mL saturation is added in extracting solution, stir 30~60min, revolving removes solvent
Methanol, obtained solid is placed in a vacuum drying oven dry 12~for 24 hours；By dried object methylene chloride/methanol solution (volume ratio
5:1) column chromatographs on a silica gel column, and by chromatographic silica gel plate analysis ingredient, the eluent at second peak is merged, is revolved by decompression
It is evaporated off methylene chloride and methanol, vacuum drying 12 at 65 DEG C~for 24 hours, obtain yellow complex of iridium；
(2) synthesis of cationic conjugated polymer
A. the potassium hydroxide solution of 30~60wt% is poured into round-bottomed flask, be cooled to room temperature, 0.1~0.5mmol is added
Tetrabutylammonium bromide, be placed in oil bath pan, open magnetic agitation, oil bath heating to after 75~100 DEG C, sequentially add 0.9~
1, the 6- dibromo-hexane of 1.2mmol and 2, the 7- dibromo fluorenes of 0.1~0.5mmol, are vigorously stirred, and react 30~60min, stop stirring
It mixes, standing is cooled to room temperature；Then 20mL methylene chloride is added to extract 3~5 times, with successively water and 0.1 after organic phase is merged
The dilute hydrochloric acid of~0.5M washs, then with after anhydrous magnesium sulfate drying, vacuum rotary steam removes methylene chloride, obtains clear yellow viscous liquid
Body；It by yellow viscous liquid under the conditions of 120 DEG C, is evaporated under reduced pressure with oil pump, removes most of excessive 1,6- dibromo-hexane, then
Silicagel column is rinsed with petroleum ether, by chromatographic silica gel plate analysis ingredient, the eluent at third peak is merged, vacuum rotary steam removes
Solvent petroleum ether obtains white crystal conjugated polymer 1；1HNMR (400MHz, CDCl3, ppm): δ 7.43-7.54 (m, 6H),
3.28-3.31 (t, 4H), 1.90-1.95 (m, 4H), 1.65-1.69 (m, 4H), 1.08-1.20 (m, 8H), 0.48-0.61 (m,
4H)；
B. first by bromo- 9,9- bis- (the 6 '-bromine hexyl) fluorenes of 2, the 7- of 0.5~1mmol bis-, Isosorbide-5-Nitrae-Asia of 0.05~0.1mmol
The ferrocene palladium chloride of phenyl hypoboric acid, 0.01~0.05mmol is added in round-bottomed flask, and repetition is filled after being vacuumized with oil pump
Enter argon gas to operate 2~3 times；Then 3mL water and 6mL tetrahydrofuran are added in round-bottomed flask, be placed in 10 in ice-water bath~
30min is evacuated behind solution temperature to -7~-10 DEG C and is re-filled with argon gas operation 2~3 times；Restore to solution temperature to room
Round-bottomed flask is placed in oil bath pan by Wen Hou, opens magnetic agitation, cooling after reacting 24~36h under the conditions of 85~100 DEG C
It to room temperature, 10mL methanol is then added in round-bottomed flask, stands and precipitating is precipitated, filtering takes precipitating successively with pre-cooling
After methanol and acetone washing, vacuum rotary steam removes solvent methanol, obtains grey black solid；In grey black solid be added 100~
150mL tetrahydrofuran, ultrasonic dissolution after standing 1~2h, are filtered with 0.45 μm of nylon membrane, remove solid palladium black, filtrate is revolved
It is dry, obtain yellow solid conjugated polymer 2；1H NMR(400MHz,CDCl3):δ7.84(m,5H),7.70-7.60(m,4H),
7.5(m,1H),3.31(t,4H),2.11(m,4H),1.70(m,4H),1.26-1.15(m,8H),0.80(m,4H)；
C. the conjugated polymer of 60~80mg 2 and 10~30mL tetrahydrofuran are added in round-bottomed flask, in -78~-
15~30min is pre-chilled at 100 DEG C, after solution cools to -78~-100 DEG C, is evacuated and is re-filled with argon gas operation 2~3 times；
After 2~5mL trimethylamine is slowly added dropwise in round-bottomed flask with syringe, refrigeration is closed, is waited round-bottomed flask after 1~2h
It is placed in ice-water bath, after being gradually brought to room temperature, magnetic agitation reacts 24~36h, then 10 are added dropwise by syringe~
20mL water after stirring and evenly mixing, is cooled to -78~-100 DEG C, is evacuated after being filled with argon gas, 2~5mL trimethylamine is added dropwise,
Refrigeration is closed, round-bottomed flask is placed in ice-water bath after 1~2h, after being gradually brought to room temperature, magnetic agitation, reaction 24~
Then most tetrahydrofuran solvent is removed under reduced pressure in 36h, add the 10mL acetone of pre-cooling, and precipitating is precipitated, careful with dropper
Supernatant liquor is drawn on ground, by precipitating with acetone washing 2~3 times of pre-cooling, after sinking completely per inferior precipitating, carefully with dropper
Supernatant liquor is drawn, supernatant is merged, vacuum rotary steam removes solvent acetone, is dried to obtain cationic conjugated polymer；1H
NMR(400MHz,CD3OD):δ8.07-7.51(m,10H),3.31-3.22(t,4H),3.04(s,18H),2.25(br,4H),
1.57(br,4H),1.18(br,8H),0.77(br,4H)；
(3) FRET occurs for cationic conjugated polymer and complex of iridium
By cationic conjugated polymer, (final concentration is 2.95 × 10-6M), complex of iridium (final concentration be 1 ×
10-5M) 90:1:9 mixes to get to glimmering based on cationic conjugated polymer and complex of iridium FRET effect by volume with water
Optical sensor system.
The above-mentioned application based on cationic conjugated polymer Yu the fluorescent optical sensor system of complex of iridium FRET effect, this is glimmering
Optical sensor system is for detecting fibrin ferment, and steps are as follows for specific method: 10 μM of 2 μ L of thrombin aptamer solution is added
Into the above-mentioned fluorescent optical sensor system based on cationic conjugated polymer and complex of iridium FRET effect of 100 μ L, then distinguish
The thrombin solution of various concentration is added in fluorescent optical sensor system, buffer solution is then added, incubates 40 at 37 DEG C
~60min, the variation of fluorescence intensity calculate the fluorescence intensity and complex of iridium of cationic conjugated polymer (415nm)
The fluorescence intensity ratio of (530nm) obtains a series of corresponding fluorescence intensity ratio of fibrin ferment of various concentrations, it is strong to establish fluorescence
Spend the quantitative relationship between ratio and concentration of thrombin；According to the two quantitative relationship, the concentration of fibrin ferment in sample to be tested is detected；
The thrombin aptamer is a bit of high-affinity and strong specificity knot to be carried out with fibrin ferment through what in-vitro screening obtained
The oligonucleotide sequence of conjunction, nucleotides sequence are classified as 5'-AGT CCG TGG TAG GGC AGG TTG GGG TGACT-3'.
The buffer solution is the phosphate buffer solution of pH=7.0~8.0, and concentration is 0.1~0.2M.
The fluoremetry condition is as follows: excitation wavelength is 380nm, and slit width is 10nm, and voltage is 700V, response
Time is 0.01s, and scanning speed is 1200nm/min.
The above-mentioned application based on cationic conjugated polymer Yu the fluorescent optical sensor system of complex of iridium FRET effect, this is glimmering
For optical sensor system for detecting atriphos (ATP), steps are as follows for specific method: 2 μ L, 10 μM of atriphos is fitted
Ligand solution is added to the above-mentioned fluorescent optical sensor system based on cationic conjugated polymer Yu complex of iridium FRET effect of 100 μ L
In, then the atriphos thrombin solution of various concentration is added in fluorescent optical sensor system respectively, is then added slow
Solution is rushed, 40~60min is incubated at 37 DEG C, the variation of fluorescence intensity calculates cationic conjugated polymer (415nm)
The fluorescence intensity ratio of fluorescence intensity and complex of iridium (530nm), a series of atriphos for obtaining various concentrations are corresponding
Fluorescence intensity ratio establishes the quantitative relationship between fluorescence intensity ratio and atriphos concentration；According to the two quantitative relationship,
Detect the concentration of atriphos in sample to be tested.
The above-mentioned application based on cationic conjugated polymer Yu the fluorescent optical sensor system of complex of iridium FRET effect, this is glimmering
Optical sensor system is for detecting platelet derived growth factor (PDGF), and steps are as follows for specific method: by 2 μ L, 10 μM of blood
It is above-mentioned based on cationic conjugated polymer and complex of iridium FRET effect that platelet derivative growth factor adaptation liquid solution is added to 100 μ L
In the fluorescent optical sensor system answered, the platelet derived growth factor solution of various concentration is then added to fluorescence sense respectively
In body system, buffer solution is then added, 40~60min is incubated at 37 DEG C, the variation of fluorescence intensity calculates cation
The fluorescence intensity of conjugated polymer (415nm) and the fluorescence intensity ratio of complex of iridium (530nm), obtain a series of various concentrations
The corresponding fluorescence intensity ratio of platelet derived growth factor, it is dense with platelet derived growth factor to establish fluorescence intensity ratio
Quantitative relationship between degree；According to the two quantitative relationship, the concentration of platelet derived growth factor in sample to be tested is detected.
The above-mentioned application based on cationic conjugated polymer Yu the fluorescent optical sensor system of complex of iridium FRET effect, this is glimmering
Optical sensor system is for detecting cocaine (Cocaine), and steps are as follows for specific method: by 2 μ L, 10 μM of cocaine aptamers
Solution is added in the above-mentioned fluorescent optical sensor system based on cationic conjugated polymer and complex of iridium FRET effect of 100 μ L,
Then the cocaine solution of various concentration is added in fluorescent optical sensor system respectively, buffer solution is then added, in 37 DEG C
40~60min of lower incubation, the variation of fluorescence intensity calculate the fluorescence intensity and iridium of cationic conjugated polymer (415nm)
The fluorescence intensity ratio of complex (530nm) obtains a series of corresponding fluorescence intensity ratio of cocaine of various concentrations, establishes
Quantitative relationship between fluorescence intensity ratio and cocaine concentration；According to the two quantitative relationship, cocaine in sample to be tested is detected
Concentration.
The above-mentioned application based on cationic conjugated polymer Yu the fluorescent optical sensor system of complex of iridium FRET effect, this is glimmering
Optical sensor system is for detecting lysozyme (Lysozyme), and steps are as follows for specific method: 10 μM of 2 μ L of lysozyme is adapted to
Liquid solution is added to the above-mentioned fluorescent optical sensor system based on cationic conjugated polymer Yu complex of iridium FRET effect of 100 μ L
In, then the lysozyme soln of various concentration is added in fluorescent optical sensor system respectively, buffer solution is then added, in 37
Incubate 40~60min at DEG C, the variation of fluorescence intensity, calculate the fluorescence intensity of cationic conjugated polymer (415nm) with
The fluorescence intensity ratio of complex of iridium (530nm) obtains a series of corresponding fluorescence intensity ratio of lysozyme of various concentrations, builds
Quantitative relationship between vertical fluorescence intensity ratio and lysozyme concentration；According to the two quantitative relationship, bacteriolyze in sample to be tested is detected
The concentration of enzyme.
Inventive principle: single sensitive molecule is become a phase by chemical conjugation key connection by cationic conjugated polymer
The entirety of mutual correlation, the electron cloud in pi bond can be spread along the entire conjugated backbone of conjugated polymer.Long-range electron is conjugated not only
It is substantially reduced the energy gap of bonding and antibonding energy interband, and keeps two energy bands broadening, energy band inner orbit number increases, interorbital energy gap
Reduce.The electron transition from bonding energy band to antibonding energy band can be easily achieved by absorbing photon or chemical doping, electronics.
Multiple sensitive monomers are connected with each other by pi-electron conjugated system, form the conjugated polymer with specific structure, thus sun from
Sub- conjugated polymer is a kind of very efficient energy/electron transfer mediator, is had the function of " molecular wire ", i.e., is swashed by photon
Hair generate exciton (exciton) can excited energy can be along conjugated polymer in other words along conjugated main chain free migration
Main chain is transmitted.Further, since the presence of conjugation, cationic conjugated polymer is in addition to having the characteristics that signal amplifies
Outside, it also has biggish molar extinction coefficient, and value is up to 106M-1cm-1The order of magnitude, absorbing ability is strong, fluorescence quantum yield
It is high.Complex of iridium has the Stokes displacement that quantum efficiency is high, emission lifetime is long, emission wavelength is adjustable, big, so that it is obtained extensively
General application.FRET has high-resolution, high sensitivity, simple as the optical molecular ruler in 1.0~10.0nm distance range
The advantages that facilitating.
Principle of the present invention according to fluorescence resonance energy transfer constructs a kind of simple, highly sensitive, highly selective " turn-
On " analyzes method for sensing.Since cationic conjugated polymer is positively charged, made between the complex of iridium with negative electricity by electrostatic
With FRET occurs, when the two exists simultaneously, the fluorescence intensity ratio of the two changes.There are the object aptamers of proper proportion
When, the fluorescence intensity ratio of the two is reduced；In the presence of having object, since the specific binding of object and its aptamers is made
With the fluorescence intensity ratio increase for making the two.Target concentration is bigger, and the fluorescence intensity ratio of the two is bigger, fluorescence intensity ratio
It is in a linear relationship between value and the logarithm of target concentration.The detection of the concentration of object in sample to be tested may be implemented accordingly.
The present invention is applicable in other determinand acting bodies and determinand and detects testing concentration by fluorescence intensity change.
Compared with the prior art, the advantages of the present invention are as follows:
(1) highly sensitive, detection sensitivity of the invention is about 10 times of existing method or more, and sensitivity of the present invention is
2.3fM, reason are: firstly, the sensitivity of fluorescent optical sensor is significantly larger than other sensors；Secondly, cation conjugation is poly-
Closing object has the function of molecular wire, can be realized the amplification of fluorescence signal, itself has stronger fluorescence.
(2) high specific, common others enzyme is noiseless to this detection architecture, and reason is: the present invention is based on mesh
It marks the specific recognition between object aptamers and object and combines and the fluorescent optical sensor of building, interfering substance is not object
The object of aptamers, therefore the interfering substance in prepare liquid can not be in conjunction with object aptamers, therefore to this detection architecture
It is noiseless.
(3) detection process is simple, quick, and cationic conjugated polymer is mixed and can be occurred with complex of iridium
FRET is added after the completion of object incubation, can be detected to obtain fluorescence radiation signal, realizes quantitative detection.
(4) result is accurate, and the rate of recovery is between 90%~110%.
In conclusion the present invention is glimmering based on above-mentioned water-soluble cationic conjugated polymer and complex of iridium FRET effect
Optical sensor system has many advantages, such as high sensitivity, high specific, simple, quick, easily operated.
Detailed description of the invention
Fig. 1 is that (A and B are cationic conjugation respectively for the excitation and emission spectra of cationic conjugated polymer and complex of iridium
The excitation and emission spectra of polymer；C and D is the excitation and emission spectra of complex of iridium respectively)；
Fig. 2 is under the conditions of the fibrin ferment of various concentration, based on cationic conjugated polymer and complex of iridium detection method
Normalization fluorescence emission spectrum；
Fig. 3 is to normalize the linear relationship chart of fluorescence intensity and log concentration value under the conditions of the fibrin ferment of various concentration；
Fig. 4 is selective lab diagram of the fluorescent optical sensor system of the present invention to fibrin ferment；
Fig. 5 is interference--free experiments figure of the fluorescent optical sensor system of the present invention to fibrin ferment；
Fig. 6 is to normalize the line of fluorescence intensity and log concentration value under the conditions of atriphos (ATP) of various concentration
Sexual intercourse figure；
Fig. 7 is to normalize fluorescence intensity and concentration under the conditions of platelet derived growth factor (PDGF) of various concentration
The linear relationship chart of logarithm；
Fig. 8 is to normalize the line of fluorescence intensity and log concentration value under the conditions of cocaine (Cocaine) of various concentration
Sexual intercourse figure；
Fig. 9 is normalization fluorescence intensity and log concentration value under the conditions of lysozyme (Lysozyme) of various concentration
Linear relationship chart.
Specific embodiment
The present invention will be described in further detail below with reference to the embodiments of the drawings.
One, specific embodiment
Embodiment 1
A kind of preparation method based on cationic conjugated polymer Yu the fluorescent optical sensor system of complex of iridium FRET effect,
Specific step is as follows:
(1) synthesis of complex of iridium
A. by 10~15mmol 2- chlorine pyrimidine, 10~20mmol 2,4- difluorophenyl boronic acid, 1.25~1.5mmol triphen
Base phosphine, 10~15mL glycol dimethyl ether and 15~20mL solution of potassium carbonate are added in three-neck flask, lead to nitrogen 30 at room temperature
~60min, then in three-neck flask be added 0.3~0.5mmol palladium acetate, magnetic agitation, and in 110~150 DEG C flow back 15~
Stop reaction after 20h, is cooled to room temperature, separates organic phase and water phase；Water phase 30~60mL ethyl acetate is extracted, then by water
Mutually extraction is repeated three times with 30~60mL ethyl acetate；After organic phase is merged successively with 40~80mL water and 40~80mL salt water
Washing, then it is dry with anhydrous magnesium sulfate, revolving removes acetate extractant, and obtained solid is dried in vacuo 12 in 60~80 DEG C
~for 24 hours；By dried object, with methylene chloride/acetone soln (volume ratio 10:1), column is chromatographed on a silica gel column, passes through chromatographic silica gel plate
Analysis ingredient merges the eluent at second peak, removes methylene chloride and acetone, vacuum at 65 DEG C by vacuum rotary steam
Dry 12~for 24 hours, obtain light yellow solid 4- (2,4- difluorophenyl) pyrimidine (DFPPM)；
B. by 2~4mmol IrCl3·3H2O, 4~5mmol 4- (2,4 difluorobenzene base) pyrimidine (DFPPM), 30~50mL
Ethylene glycol monoethyl ether and 10~30mL water are added in three-neck flask, after leading to 30~60min of nitrogen at room temperature, in 120~150 DEG C
Flow back 24~36h, and cooled and filtered obtains solid and filtrate；Solid 2~5mL ethylene glycol monoethyl ether is extracted, merges and extracts
Liquid and filtrate are added the aquatic yellowly precipitating of 20~30mL, yellow mercury oxide are successively washed with water, acetone, dehydrated alcohol, is obtained
Chlorine bridging；
C. it is added in reaction flask after 0.07~0.10mmol chlorine bridging being dissolved in 10~20mL methylene chloride, by 0.10
4,4 '-dicarboxylic acids -2 of~0.20mmol assistant ligand, 2 '-bipyridyls are added in reaction flask after being dissolved in 10~20mL methanol,
Reaction flask is flowed back 2~5h at 60~100 DEG C；0.2~0.4g sodium acetate is dissolved in after 2~5mL methanol again and is added to reaction
In bottle, flow back 1~5h, is cooled to room temperature；Reaction is added to after ammonium hexafluorophosphate is added to 2~5mL methanol solution to saturation
In bottle, 30~60min of stirring at normal temperature, revolving removes solvent methanol, then the salt of 1~3M, 10~20mL is added in obtained solid
Acid solution, suspension stir 10~30min, filtering；By solid with after water washing 2 times of 10~20mL, by solid 2mL methanol
It extracts, the ammonium hexafluorophosphate methanol solution of 2~5mL saturation is added in extracting solution, stir 30~60min, revolving removes solvent
Methanol, obtained solid is placed in a vacuum drying oven dry 12~for 24 hours；By dried object methylene chloride/methanol solution (volume ratio
5:1) column chromatographs on a silica gel column, and by chromatographic silica gel plate analysis ingredient, the eluent at second peak is merged, is revolved by decompression
It is evaporated off methylene chloride and methanol, vacuum drying 12 at 65 DEG C~for 24 hours, obtain yellow complex of iridium；
(3) synthesis of cationic conjugated polymer
A. the potassium hydroxide solution of 30~60wt% is poured into round-bottomed flask, be cooled to room temperature, 0.1~0.5mmol is added
Tetrabutylammonium bromide, be placed in oil bath pan, open magnetic agitation, oil bath heating to after 75~100 DEG C, sequentially add 0.9~
1, the 6- dibromo-hexane of 1.2mmol and 2, the 7- dibromo fluorenes of 0.1~0.5mmol, are vigorously stirred, and react 30~60min, stop stirring
It mixes, standing is cooled to room temperature；Then 20mL methylene chloride is added to extract 3~5 times, with successively water and 0.1 after organic phase is merged
The dilute hydrochloric acid of~0.5M washs, then with after anhydrous magnesium sulfate drying, vacuum rotary steam removes methylene chloride, obtains clear yellow viscous liquid
Body；It by yellow viscous liquid under the conditions of 120 DEG C, is evaporated under reduced pressure with oil pump, removes most of excessive 1,6- dibromo-hexane, then
Silicagel column is rinsed with petroleum ether, by chromatographic silica gel plate analysis ingredient, the eluent at third peak is merged, vacuum rotary steam removes
Solvent petroleum ether obtains white crystal conjugated polymer 1；1HNMR (400MHz, CDCl3, ppm): δ 7.43-7.54 (m, 6H),
3.28-3.31 (t, 4H), 1.90-1.95 (m, 4H), 1.65-1.69 (m, 4H), 1.08-1.20 (m, 8H), 0.48-0.61 (m,
4H)；
B. first by bromo- 9,9- bis- (the 6 '-bromine hexyl) fluorenes of 2, the 7- of 0.5~1mmol bis-, Isosorbide-5-Nitrae-Asia of 0.05~0.1mmol
The ferrocene palladium chloride of phenyl hypoboric acid, 0.01~0.05mmol is added in round-bottomed flask, and repetition is filled after being vacuumized with oil pump
Enter argon gas to operate 2~3 times；Then 3mL water and 6mL tetrahydrofuran are added in round-bottomed flask, be placed in 10 in ice-water bath~
30min is evacuated behind solution temperature to -7~-10 DEG C and is re-filled with argon gas operation 2~3 times；Restore to solution temperature to room
Round-bottomed flask is placed in oil bath pan by Wen Hou, opens magnetic agitation, cooling after reacting 24~36h under the conditions of 85~100 DEG C
It to room temperature, 10mL methanol is then added in round-bottomed flask, stands and precipitating is precipitated, filtering takes precipitating successively with pre-cooling
After methanol and acetone washing, vacuum rotary steam removes solvent methanol, obtains grey black solid；In grey black solid be added 100~
150mL tetrahydrofuran, ultrasonic dissolution after standing 1~2h, are filtered with 0.45 μm of nylon membrane, remove solid palladium black, filtrate is revolved
It is dry, obtain yellow solid conjugated polymer 2；1H NMR(400MHz,CDCl3):δ7.84(m,5H),7.70-7.60(m,4H),
7.5(m,1H),3.31(t,4H),2.11(m,4H),1.70(m,4H),1.26-1.15(m,8H),0.80(m,4H)；
C. the conjugated polymer of 60~80mg 2 and 10~30mL tetrahydrofuran are added in round-bottomed flask, in -78~-
15~30min is pre-chilled at 100 DEG C, after solution cools to -78~-100 DEG C, is evacuated and is re-filled with argon gas operation 2~3 times；
After 2~5mL trimethylamine is slowly added dropwise in round-bottomed flask with syringe, refrigeration is closed, is waited round-bottomed flask after 1~2h
It is placed in ice-water bath, after being gradually brought to room temperature, magnetic agitation reacts 24~36h, then 10 are added dropwise by syringe~
20mL water after stirring and evenly mixing, is cooled to -78~-100 DEG C, is evacuated after being filled with argon gas, 2~5mL trimethylamine is added dropwise,
Refrigeration is closed, round-bottomed flask is placed in ice-water bath after 1~2h, after being gradually brought to room temperature, magnetic agitation, reaction 24~
Then most tetrahydrofuran solvent is removed under reduced pressure in 36h, add the 10mL acetone of pre-cooling, and precipitating is precipitated, careful with dropper
Supernatant liquor is drawn on ground, by precipitating with acetone washing 2~3 times of pre-cooling, after sinking completely per inferior precipitating, carefully with dropper
Supernatant liquor is drawn, supernatant is merged, vacuum rotary steam removes solvent acetone, is dried to obtain cationic conjugated polymer；1H
NMR(400MHz,CD3OD):δ8.07-7.51(m,10H),3.31-3.22(t,4H),3.04(s,18H),2.25(br,4H),
1.57(br,4H),1.18(br,8H),0.77(br,4H)；
(3) FRET occurs for cationic conjugated polymer and complex of iridium
By cationic conjugated polymer, (final concentration is 2.95 × 10-6M), complex of iridium (final concentration be 1 ×
10-5M) 90:1:9 mixes to get to glimmering based on cationic conjugated polymer and complex of iridium FRET effect by volume with water
Optical sensor system.
The excitation spectrum and emission spectrum for measuring cationic conjugated polymer and complex of iridium respectively determine that the two can be sent out
Raw fluorescence resonance energy transfer, as shown in Figure 1, it can be seen that the emission spectrum (B) and complex of iridium of cationic conjugated polymer
(C) excitation spectrum has overlapping, so this system is likely to occur FRET, cationic conjugated polymer does donor, complex of iridium
Do receptor.
The most important condition that FRET occurs is that the emission spectrum of energy donor and the absorption spectrum of energy acceptor must be overlapped,
It is secondary be between energy donor, energy acceptor must it is close enough, in this way generation energy transfer probability just understand height.Measurement sun respectively
The excitation spectrum and emission spectrum of ion conjugated polymer and complex of iridium determine that the two can occur fluorescence resonance energy and turn
It moves.Measure cationic conjugated polymer, complex of iridium Zeta potential.Experimental data shows pure cationic conjugated polymer
Zeta potential is 103mV, and after mixing with complex of iridium, Zeta potential drops to 13.1mV, this shows because of electrostatic interaction
So that complex of iridium is gathered in around cationic conjugated polymer, and the positive charge on complex of iridium surface reduces cationic conjugation
The Zeta potential of polymer forms compound by conjugation.
Two, Application Example
Embodiment 1
Fluorescent optical sensor system based on cationic conjugated polymer and complex of iridium FRET effect detects fibrin ferment
1, detection method
2 μ L, 10 μM of thrombin aptamer solution is added to prepared in the above-mentioned specific embodiment one of 100 μ L based on sun
In the fluorescent optical sensor system of ion conjugated polymer and complex of iridium FRET effect, then respectively by the fibrin ferment of various concentration
Solution is added in fluorescent optical sensor system, and buffer solution is then added, and 40~60min is incubated at 37 DEG C, and detection fluorescence is strong
The variation of degree calculates the fluorescence intensity of cationic conjugated polymer (415nm) and the fluorescence intensity ratio of complex of iridium (530nm)
Value, obtains a series of corresponding fluorescence intensity ratio of fibrin ferment of various concentrations, establishes fluorescence intensity ratio and concentration of thrombin
Between quantitative relationship；According to the two quantitative relationship, the concentration of fibrin ferment in sample to be tested is detected；
Wherein thrombin aptamer is a bit of to carry out high-affinity and Qiang Te with fibrin ferment through what in-vitro screening obtained
The oligonucleotide sequence that the opposite sex combines, nucleotides sequence are classified as 5'-AGT CCG TGG TAG GGC AGG TTG GGG TGA
CT-3'。
Above-mentioned buffer solution is the phosphate buffer solution of pH=7.0~8.0, and concentration is 0.1~0.2M；Fluoremetry item
Part is as follows: excitation wavelength is 380nm, and slit width is 10nm, and voltage is 700V, and the response time is 0.01s, and scanning speed is
1200nm/min。
Experimental result as shown in Fig. 2, with thrombin solution addition, destroy DNA, cationic conjugated polymer and iridium
Complex has occurred FRET again, the fluorescence enhancement at 530nm, the fluorescent weakening at 415nm.According to concentration of thrombin and fluorescence
The relation curve of intensity rate is as shown in figure 3, illustrate the sensor to the linear sound of the thrombin solution of 0.01pM~200pM
It answers, linear equation are as follows: I=0.20logc+0.83, coefficient R2=0.9971, detection is limited to 2.3fM.
2, specificity experiments
Selectivity experiment is as follows with the abbreviation of other enzymes used in interference--free experiments: horseradish peroxidase (HRP), rotten
Protease (CHY), carboxypeptidase (CPY), lysozyme (Lysozyme), acetylcholinesterase (AchE), papain
(Papain), bovine serum albumin (BSA).
Selective experimental design explanation, as a result as shown in Figure 4.As can be seen from Fig., when other enzymes (concentration: 10nM) of addition
When, FRET efficiency does not change compared with blank control, and the ratio of the fluorescence intensity of 100pM fibrin ferment has been added to reach maximum,
Illustrate that this system is selectively good.
Interference--free experiments specification, as a result as shown in figure 5, respectively by horseradish peroxidase, chymotrypsin, carboxylic peptide
Enzyme, lysozyme, acetylcholinesterase, papain, bovine serum albumin enzyme (each enzyme concentration is 10nM) and 100pM fibrin ferment
When mixing, the fluorescence intensity ratio at 415nm and 530nm has almost no change compared with single fibrin ferment, in addition, this eight kinds
Substance will not significantly affect sensor to the response signal of fibrin ferment when coexisting, illustrate that this system anti-interference is good.
Embodiment 2
Fluorescent optical sensor system based on cationic conjugated polymer and complex of iridium FRET effect detects atriphos,
Steps are as follows for specific method: 10 μM of 2 μ L of atriphos adaptation liquid solution is added to the above-mentioned specific embodiment one of 100 μ L
In the fluorescent optical sensor system based on cationic conjugated polymer and complex of iridium FRET effect of middle preparation, then respectively will not
Atriphos solution with concentration is added in fluorescent optical sensor system, and buffer solution is then added, and incubates 40 at 37 DEG C
~60min, the variation of fluorescence intensity calculate the fluorescence intensity and complex of iridium of cationic conjugated polymer (415nm)
The fluorescence intensity ratio of (530nm) obtains a series of corresponding fluorescence intensity ratio of atriphos of various concentrations, establishes glimmering
Quantitative relationship between intensity ratio and atriphos concentration；According to the two quantitative relationship, three phosphorus in sample to be tested are detected
The concentration of adenosine monophosphate.Above-mentioned buffer solution is the phosphate buffer solution of pH=7.0~8.0, and concentration is 0.1~0.2M；Fluorescence
Determination condition is as follows: excitation wavelength is 380nm, and slit width is 10nm, and voltage is 700V, and the response time is 0.01s, scanning speed
Degree is 1200nm/min.
Experimental result is as shown in fig. 6, be presented linear close between fluorescence intensity ratio and the logarithm of atriphos concentration
System, linear equation are as follows: I=0.19logc+0.24, coefficient R2=0.9947, the range of linearity is 10pM~20nM, detection limit
For 2pM.It is linear good, it can be used for unknown sample detection.
Embodiment 3
It is platelet-derived based on cationic conjugated polymer and the fluorescent optical sensor system detection of complex of iridium FRET effect
Growth factor, steps are as follows for specific method: 10 μM of 2 μ L of platelet derived growth factor adaptation liquid solution is added to 100 μ L
The fluorescence sense body based on cationic conjugated polymer Yu complex of iridium FRET effect prepared in above-mentioned specific embodiment one
In system, then the platelet derived growth factor solution of various concentration is added in fluorescent optical sensor system respectively, then plus
Enter buffer solution, 40~60min is incubated at 37 DEG C, the variation of fluorescence intensity calculates cationic conjugated polymer
The fluorescence intensity of (415nm) and the fluorescence intensity ratio of complex of iridium (530nm), a series of blood platelet for obtaining various concentrations spread out
The corresponding fluorescence intensity ratio of life growth factor, establishes between fluorescence intensity ratio and platelet derived growth factor concentration and determines
Magnitude relation；According to the two quantitative relationship, the concentration of platelet derived growth factor in sample to be tested is detected.Above-mentioned buffer solution is
The phosphate buffer solution of pH=7.0~8.0, concentration are 0.1~0.2M；Fluoremetry condition is as follows: excitation wavelength is
380nm, slit width are 10nm, and voltage is 700V, and the response time is 0.01s, and scanning speed is 1200nm/min.
Experimental result as shown in fig. 7, be between fluorescence intensity ratio and the logarithm of platelet derived growth factor concentration
Existing linear relationship, linear equation are as follows: I=0.58+0.22logc, coefficient R2=0.9855, the range of linearity be 0.1pM~
1000pM, detection are limited to 0.01pM.It is linear good, it can be used for unknown sample detection.
Embodiment 4
Fluorescent optical sensor system based on cationic conjugated polymer and complex of iridium FRET effect detects cocaine, specifically
Method and step is as follows: 10 μM of 2 μ L of cocaine adaptation liquid solution being added to and is prepared in the above-mentioned specific embodiment one of 100 μ L
In fluorescent optical sensor system based on cationic conjugated polymer and complex of iridium FRET effect, then respectively by various concentration
Cocaine solution is added in fluorescent optical sensor system, and buffer solution is then added, and 40~60min, detection are incubated at 37 DEG C
The variation of fluorescence intensity, the fluorescence intensity for calculating cationic conjugated polymer (415nm) and the fluorescence of complex of iridium (530nm) are strong
Ratio is spent, a series of corresponding fluorescence intensity ratio of cocaine of various concentrations is obtained, establishes fluorescence intensity ratio and cocaine
Quantitative relationship between concentration；According to the two quantitative relationship, the concentration of cocaine in sample to be tested is detected.Above-mentioned buffer solution is
The phosphate buffer solution of pH=7.0~8.0, concentration are 0.1~0.2M；Fluoremetry condition is as follows: excitation wavelength is
380nm, slit width are 10nm, and voltage is 700V, and the response time is 0.01s, and scanning speed is 1200nm/min.
Experimental results are shown in figure 8, fluorescence intensity ratio and logarithm between linear relationship, linear equation is presented are as follows: I=
0.78+0.20logc, coefficient R2=0.9822, the range of linearity is 0.01nM~150nM, and detection is limited to 3pM.It is linear good
It is good, it can be used for unknown sample detection.
Embodiment 5
Fluorescent optical sensor system based on cationic conjugated polymer and complex of iridium FRET effect detects lysozyme, specifically
Method and step is as follows: 10 μM of 2 μ L of lysozyme adaptation liquid solution being added to and is prepared in the above-mentioned specific embodiment one of 100 μ L
In fluorescent optical sensor system based on cationic conjugated polymer and complex of iridium FRET effect, then respectively by various concentration
Lysozyme soln is added in fluorescent optical sensor system, and buffer solution is then added, and 40~60min, detection are incubated at 37 DEG C
The variation of fluorescence intensity, the fluorescence intensity for calculating cationic conjugated polymer (415nm) and the fluorescence of complex of iridium (530nm) are strong
Ratio is spent, a series of corresponding fluorescence intensity ratio of lysozyme of various concentrations is obtained, establishes fluorescence intensity ratio and lysozyme
Quantitative relationship between concentration；According to the two quantitative relationship, the concentration of lysozyme in sample to be tested is detected.Above-mentioned buffer solution is
The phosphate buffer solution of pH=7.0~8.0, concentration are 0.1~0.2M；Fluoremetry condition is as follows: excitation wavelength is
380nm, slit width are 10nm, and voltage is 700V, and the response time is 0.01s, and scanning speed is 1200nm/min.
Experimental result is as shown in figure 9, be presented linear relationship, linear equation between fluorescence intensity ratio and the logarithm of lysozyme
Are as follows: I=1.16+0.32logc, coefficient R2=0.9907, the range of linearity is 0.005nM~2nM, and detection is limited to 1pM.Line
Property it is good, can be used for unknown sample detection.
Certainly, above description is not limitation of the present invention, and the present invention is also not limited to the example above.The art
The variations, modifications, additions or substitutions that those of ordinary skill makes within the essential scope of the present invention also should belong to protection of the present invention
Range.
Sequence table
<110>University Of Ningbo
<120>preparation based on cationic conjugated polymer Yu the fluorescent optical sensor system of complex of iridium FRET effect
Method and its application
<130>
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 29
<212> DNA
<213>artificial sequence
<220>
<223>thrombin aptamer
<400> 1
5'-AGT CCG TGG TAG GGC AGG TTG GGG TGA CT-3' 29
Claims (6)
1. a kind of preparation method based on cationic conjugated polymer Yu the fluorescent optical sensor system of complex of iridium FRET effect,
It is characterized in that specific step is as follows:
(1) synthesis of complex of iridium
A. by 10~15mmol 2- chlorine pyrimidine, 10~20mmol 2,4- difluorophenyl boronic acid, 1.25~1.5mmol triphenyl
Phosphine, 10~15mL glycol dimethyl ether and 15~20mL solution of potassium carbonate are added in three-neck flask, lead at room temperature nitrogen 30~
60min, then 0.3~0.5mmol palladium acetate is added in three-neck flask, magnetic agitation, and in 110~150 DEG C of 15~20h of reflux
Stop reaction afterwards, is cooled to room temperature, separates organic phase and water phase；Water phase 30~60mL ethyl acetate is extracted, then by water phase
Extraction is repeated three times with 30~60mL ethyl acetate；It is successively washed after organic phase is merged with 40~80mL water and 40~80mL salt water
It washs, then dry with anhydrous magnesium sulfate, revolving removes acetate extractant, obtained solid is dried in vacuo in 60~80 DEG C to 12~
24h；By dried object methylene chloride/acetone soln in a volume ratio of 10:1, column is chromatographed on a silica gel column, passes through chromatographic silica gel plate
Analysis ingredient merges the eluent at second peak, removes methylene chloride and acetone, vacuum at 65 DEG C by vacuum rotary steam
Dry 12~for 24 hours, obtain light yellow solid 4- (2,4- difluorophenyl) pyrimidine；
B. by 2~4mmol IrCl3·3H2O, 4~5mmol4- (2,4 difluorobenzene base) pyrimidine, 30~50mL ethylene glycol monoethyl ether
It is added in three-neck flask with 10~30mL water, after leading to 30~60min of nitrogen at room temperature, in 120~150 DEG C of 24~36h of reflux,
Cooled and filtered obtains solid and filtrate；Solid 2~5mL ethylene glycol monoethyl ether is extracted, combined extract and filtrate are added
The aquatic yellowly precipitating of 20~30mL, yellow mercury oxide is successively washed with water, acetone, dehydrated alcohol, chlorine bridging is obtained；
C. it is added in reaction flask after 0.07~0.10mmol chlorine bridging being dissolved in 10~20mL methylene chloride, by 0.10~
4,4 '-dicarboxylic acids -2 of 0.20mmol assistant ligand, 2 '-bipyridyls are added in reaction flask after being dissolved in 10~20mL methanol, will
Reaction flask flows back 2~5h at 60~100 DEG C；Reaction flask is added to after 0.2~0.4g sodium acetate is dissolved in 2~5mL methanol again
In, flow back 1~5h, is cooled to room temperature；Reaction flask is added to after ammonium hexafluorophosphate is added to 2~5mL methanol solution to saturation
In, 30~60min of stirring at normal temperature, revolving removes solvent methanol, then the hydrochloric acid of 1~3M, 10~20mL is added in obtained solid
Solution, suspension stir 10~30min, filtering；By solid with after water washing 2 times of 10~20mL, solid is mentioned with 2mL methanol
It takes, the ammonium hexafluorophosphate methanol solution of 2~5mL saturation is added in extracting solution, stir 30~60min, revolving removes solvent first
Alcohol, obtained solid is placed in a vacuum drying oven dry 12~for 24 hours；The methylene chloride/methanol for being 5:1 with volume ratio by dried object
Column chromatographs solution on a silica gel column, and by chromatographic silica gel plate analysis ingredient, the eluent at second peak is merged, is revolved by decompression
It is evaporated off methylene chloride and methanol, vacuum drying 12 at 65 DEG C~for 24 hours, obtain yellow complex of iridium；
(2) synthesis of cationic conjugated polymer
A. the potassium hydroxide solution of 30~60wt% is poured into round-bottomed flask, be cooled to room temperature, the four of 0.1~0.5mmol are added
Butylammonium bromide is placed in oil bath pan, opens magnetic agitation, oil bath heating to after 75~100 DEG C, sequentially add 0.9~
1, the 6- dibromo-hexane of 1.2mmol and 2, the 7- dibromo fluorenes of 0.1~0.5mmol, are vigorously stirred, and react 30~60min, stop stirring
It mixes, standing is cooled to room temperature；Then 20mL methylene chloride is added to extract 3~5 times, with successively water and 0.1 after organic phase is merged
The dilute hydrochloric acid of~0.5M washs, then with after anhydrous magnesium sulfate drying, vacuum rotary steam removes methylene chloride, obtains clear yellow viscous liquid
Body；It by yellow viscous liquid under the conditions of 120 DEG C, is evaporated under reduced pressure with oil pump, removes most of excessive 1,6- dibromo-hexane, then
Silicagel column is rinsed with petroleum ether, by chromatographic silica gel plate analysis ingredient, the eluent at third peak is merged, vacuum rotary steam removes
Solvent petroleum ether obtains white crystal conjugated polymer 1, i.e. bromo- 9,9- bis- (the 6 '-bromine hexyl) fluorenes of 2,7- bis-；
B. first by bromo- 9,9- bis- (the 6 '-bromine hexyl) fluorenes of 2, the 7- of 0.5~1mmol bis-, Isosorbide-5-Nitrae-phenylene of 0.05~0.1mmol
The ferrocene palladium chloride of hypoboric acid, 0.01~0.05mmol is added in round-bottomed flask, and repetition is filled with argon after being vacuumized with oil pump
Gas operates 2~3 times；Then 3mL water and 6mL tetrahydrofuran are added in round-bottomed flask, is placed in 10~30min in ice-water bath, to
Solution temperature is re-filled with argon gas and operates 2~3 times to being evacuated after -7~-10 DEG C；Restore after solution temperature to room temperature, it will
Round-bottomed flask is placed in oil bath pan, opens magnetic agitation, under the conditions of 85~100 DEG C, after reacting 24~36h, is cooled to room temperature,
Then 10mL methanol is added in round-bottomed flask, stands and precipitating is precipitated, filtering takes precipitating successively with the methanol and third pre-cooled
After ketone washing, vacuum rotary steam removes solvent methanol, obtains grey black solid；100~150mL tetrahydro furan is added in grey black solid
It mutters, ultrasonic dissolution after standing 1~2h, is filtered with 0.45 μm of nylon membrane, removes solid palladium black, filtrate is spin-dried for, yellow is obtained
Solid conjugated polymer 2；
C. the conjugated polymer of 60~80mg 2 and 10~30mL tetrahydrofuran are added in round-bottomed flask, in -78~-100 DEG C
15~30min of lower pre-cooling is evacuated after solution cools to -78~-100 DEG C and is re-filled with argon gas operation 2~3 times；With injection
After 2~5mL trimethylamine is slowly added dropwise in round-bottomed flask in device, refrigeration is closed, waits and round-bottomed flask is placed in ice after 1~2h
In water-bath, after being gradually brought to room temperature, magnetic agitation reacts 24~36h, then 10~20mL water is added dropwise by syringe,
After stirring and evenly mixing, -78~-100 DEG C are cooled to, is evacuated after being filled with argon gas, 2~5mL trimethylamine is added dropwise, closes system
It is cold, round-bottomed flask is placed in ice-water bath after 1~2h, after being gradually brought to room temperature, magnetic agitation reacts 24~36h, then
Most solvents tetrahydrofurane is removed under reduced pressure, adds the 10mL acetone of pre-cooling, precipitating is precipitated, is carefully drawn with dropper
Layer clear liquid after sinking completely per inferior precipitating, carefully draws upper layer with dropper by precipitating with acetone washing 2~3 times of pre-cooling
Clear liquid, merges supernatant, and vacuum rotary steam removes solvent acetone, is dried to obtain cationic conjugated polymer；
(3) FRET occurs for cationic conjugated polymer and complex of iridium
By cationic conjugated polymer, complex of iridium and water, 90:1:9 is mixed to get poly- to being conjugated based on cation by volume
Close the fluorescent optical sensor system of object and complex of iridium FRET effect.
2. the fluorescent optical sensor system based on cationic conjugated polymer Yu complex of iridium FRET effect prepared in claim 1
Application in terms of preparing fibrin ferment detection architecture, it is characterised in that steps are as follows for specific method: the fibrin ferment of 2 μ L10 μM is fitted
Ligand solution is added to described in 100 μ L claims 1 based on cationic conjugated polymer and complex of iridium FRET effect
In fluorescent optical sensor system, then the thrombin solution of various concentration is added in fluorescent optical sensor system respectively, then plus
Enter buffer solution, 40~60min is incubated at 37 DEG C, the variation of fluorescence intensity calculates the glimmering of cationic conjugated polymer
The fluorescence intensity ratio of luminous intensity and complex of iridium obtains a series of corresponding fluorescence intensity ratio of fibrin ferment of various concentrations,
Establish the quantitative relationship between fluorescence intensity ratio and concentration of thrombin；According to the two quantitative relationship, detects in sample to be tested and coagulate
The concentration of hemase；The thrombin aptamer be it is a bit of through in-vitro screening obtain can with fibrin ferment carry out high-affinity and
The oligonucleotide sequence specifically bound by force, nucleotides sequence are classified as 5'-AGT CCG TGG TAG GGC AGG TTG GGG
TGA CT-3'。
3. the fluorescent optical sensor system based on cationic conjugated polymer Yu complex of iridium FRET effect prepared in claim 1
Application in terms of preparing atriphos detection architecture, it is characterised in that steps are as follows for specific method: by three phosphorus of 2 μ L10 μM
Adenosine monophosphate adaptation liquid solution is added to described in 100 μ L claims 1 based on cationic conjugated polymer and complex of iridium
In the fluorescent optical sensor system of FRET effect, the atriphos thrombin solution of various concentration is then added to fluorescence respectively
In sensor systems, buffer solution is then added, 40~60min is incubated at 37 DEG C, the variation of fluorescence intensity calculates sun
The fluorescence intensity of ion conjugated polymer and the fluorescence intensity ratio of complex of iridium, obtain a series of Adenosine triphosphate of various concentrations
The corresponding fluorescence intensity ratio of glycosides, establishes the quantitative relationship between fluorescence intensity ratio and atriphos concentration；According to the two
Quantitative relationship detects the concentration of atriphos in sample to be tested.
4. the fluorescent optical sensor system based on cationic conjugated polymer Yu complex of iridium FRET effect prepared in claim 1
Application in terms of preparing platelet derived growth factor detection architecture, it is characterised in that steps are as follows for specific method: by 2 μ L
10 μM of platelet derived growth factor adaptation liquid solution is added to described in 100 μ L claims 1 to be conjugated based on cation
In the fluorescent optical sensor system of polymer and complex of iridium FRET effect, then respectively by the platelet derived growth of various concentration
Factor solutions are added in fluorescent optical sensor system, and buffer solution is then added, and 40~60min is incubated at 37 DEG C, are detected glimmering
The variation of luminous intensity calculates the cationic fluorescence intensity of conjugated polymer and the fluorescence intensity ratio of complex of iridium, obtains a system
The corresponding fluorescence intensity ratio of the platelet derived growth factor of column various concentration, establish fluorescence intensity ratio with it is platelet-derived
Quantitative relationship between growth factor concentration；According to the two quantitative relationship, platelet derived growth factor in sample to be tested is detected
Concentration.
5. the fluorescent optical sensor system based on cationic conjugated polymer Yu complex of iridium FRET effect prepared in claim 1
Application in terms of preparing cocaine detection architecture, it is characterised in that steps are as follows for specific method: by 2 μ L, 10 μM of cocaine
Adaptation liquid solution is added to described in 100 μ L claims 1 based on cationic conjugated polymer and complex of iridium FRET effect
Fluorescent optical sensor system in, then the cocaine solution of various concentration is added in fluorescent optical sensor system respectively, then
Buffer solution is added, 40~60min is incubated at 37 DEG C, the variation of fluorescence intensity calculates cationic conjugated polymer
The fluorescence intensity ratio of fluorescence intensity and complex of iridium obtains a series of corresponding fluorescence intensity ratio of cocaine of various concentrations
Value, establishes the quantitative relationship between fluorescence intensity ratio and cocaine concentration；According to the two quantitative relationship, detect in sample to be tested
The concentration of cocaine.
6. the fluorescent optical sensor system based on cationic conjugated polymer Yu complex of iridium FRET effect prepared in claim 1
Application in terms of preparing lysozyme detection architecture, it is characterised in that steps are as follows for specific method: by 2 μ L, 10 μM of lysozyme
Adaptation liquid solution is added to described in 100 μ L claims 1 based on cationic conjugated polymer and complex of iridium FRET effect
Fluorescent optical sensor system in, then the lysozyme soln of various concentration is added in fluorescent optical sensor system respectively, then
Buffer solution is added, 40~60min is incubated at 37 DEG C, the variation of fluorescence intensity calculates cationic conjugated polymer
The fluorescence intensity ratio of fluorescence intensity and complex of iridium obtains a series of corresponding fluorescence intensity ratio of lysozyme of various concentrations
Value, establishes the quantitative relationship between fluorescence intensity ratio and lysozyme concentration；According to the two quantitative relationship, detect in sample to be tested
The concentration of lysozyme.
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