CN106589321A - Preparation method of fluorescent sensor system based on cationic conjugated polymer and iridium complex FRET effect and application thereof - Google Patents
Preparation method of fluorescent sensor system based on cationic conjugated polymer and iridium complex FRET effect and application thereof Download PDFInfo
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- CN106589321A CN106589321A CN201610941728.3A CN201610941728A CN106589321A CN 106589321 A CN106589321 A CN 106589321A CN 201610941728 A CN201610941728 A CN 201610941728A CN 106589321 A CN106589321 A CN 106589321A
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Classifications
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- 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 Table
- C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table compounds of the platinum group
- C07F15/0033—Iridium compounds
- C07F15/004—Iridium compounds without a metal-carbon linkage
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- G—PHYSICS
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- 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
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Abstract
The invention discloses a preparation method of fluorescent sensor system based on cationic conjugated polymer and iridium complex FRET effect and application thereof and is characterized in that it comprises the synthesis step of the iridium complex; the synthesis step of the cationic conjugated polymer;the cationic conjugated polymer and iridium complexes give rise to FRET and obtain the fluorescence sensor system steps based on the FRET effect of the cationic conjugated polymer and iridium complex; the fluorescence sensor system can be used to detect thrombin, adenosine triphosphate, platelet derived growth factor, cocaine and lysozyme . The invention has the advantages of good specificity, high sensitivity, accurate and reliable results, low cost, high speed and simple process.
Description
Technical field
The present invention relates to a kind of fluorescent optical sensor system, is matched somebody with somebody based on cation conjugated polymer more particularly, to one kind with iridium
The preparation method and applications of the fluorescent optical sensor system of compound FRET effects.
Background technology
FRET (fluorescence resonance energy transfer) (FRET) is to design fluorescent optical sensor at present to use relatively broad principle.Fluorescence resonance
Energy transfer refers to two fluorescence chromophoric groups when sufficiently closing to, and is excited after donor molecule absorbs the photon of certain frequency
To higher electron energy state, before the electronics returns to ground state, by dipole-dipole interaction, energy is realized to neighbouring receptor
There is resonance energy transfer in molecule transfer.The condition that effective energy transfer occurs between energy donor-acceptor is harsh
, mainly include：(1) emission spectrum of energy donor must be overlap with the absorption spectrum of energy acceptor；(2) energy donor and energy
The fluorescence chromophore of amount receptor must be arranged by rights；(3) must be close enough between energy donor, energy acceptor,
The probability that energy transfer so occurs just can be high.Operating process letter is had based on the fluorescence sense method of FRET (fluorescence resonance energy transfer)
The features such as single, sensitivity is high, selectivity is good, response time is short.
The sensitivity that FRET adjusts the distance because of it, is widely used to structure of biological macromolecule, property, reaction mechanism and determines
The research of the aspects such as amount analysis.Accordingly, it is capable to measure transfer fluorescence analysiss be very suitable for environment, Biomedical Science and clinicization
Etc. aspect is complicated, the analysis of low content component, is also a kind of new tool in genetic engineering.
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 coordination compound, complex of iridium has quantum efficiency height, emission lifetime length, emission wavelength can
Adjust, big Stokes displacements, and the features such as excited by visible light and light stability so as to chemistry, bio-sensing and biology into
As field causes extensive concern.
Cation conjugated polymer (CCP) as a kind of new water soluble fluorescence molecule, with molar absorption coefficient it is big,
The advantages of fluorescence quantum yield is high.Meanwhile, conjugated polymer has the function of molecular wire, can realize putting for fluorescence signal
Greatly.Conjugation can make conjugated polymer have higher light energy collecting effect and good fluorescent characteristic, and hydrophilic
Group can give 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 more it is attractive that it can be dissolved in water, and based on this, it is possible to achieve to DNA, egg
The highly sensitive detection of the biomolecule such as white matter.Based on these unique photoluminescent properties, cation conjugated polymer is in biology
Chemical analyses aspect has boundless application prospect.Have no both at home and abroad at present and coordinated based on cation conjugated polymer and iridium
The fluorescent optical sensor system of thing FRET effects and its relevant report of application.
The content of the invention
The technical problem to be solved is to provide that a species specificity is good, sensitivity is high, result accurately and reliably, cost
It is low, quick, and the extremely simple fluorescence sense body based on cation conjugated polymer and complex of iridium FRET effects of process
The preparation method and applications of system.
The present invention solve the technical scheme that adopted of above-mentioned technical problem for：One kind is based on cation conjugated polymer and iridium
The preparation method of the fluorescent optical sensor system of coordination compound FRET effects, comprises the following steps that：
(1) synthesis of complex of iridium
A. by 10～15mmol 2- chloropyrimide, 10～20mmol 2,4 difluorobenzene ylboronic acids, 1.25～1.5mmol triphenyls
Phosphine, 10～15mL glycol dimethyl ethers and 15～20mL solution of potassium carbonate are added in three-neck flask, under room temperature lead to nitrogen 30～
60min, then 0.3～0.5mmol palladium is added in three-neck flask, magnetic agitation, and the 15～20h that flows back in 110～150 DEG C
Afterwards stopped reaction, is cooled to room temperature, separates organic faciess and water phase；Water is mutually extracted with 30～60mL ethyl acetate, then by water phase
Repeat extraction three times with 30～60mL ethyl acetate；Washed successively with 40～80mL water and 40～80mL saline after organic faciess are merged
Wash, then with anhydrous magnesium sulfate be dried, revolving remove acetate extractant, by gained solid in 60～80 DEG C be vacuum dried 12～
24h；By dried object dichloromethane/acetone soln (volume ratio 10:1) column chromatography on a silica gel column, by chromatographic silica gel plate point
Analysis composition, the eluent at second peak is merged, and by vacuum rotary steam methylene chloride and acetone are removed, and vacuum is done at 65 DEG C
Dry 12～24h, obtains light yellow solid 4- (2,4 difluorobenzene base) pyrimidine (DFPPM)；
B. by 2～4mmol IrCl3·3H2O, 4～5mmol 4- (2,4 difluorobenzene base) pyrimidine (DFPPM), 30～50mL second
2-ethoxyethanol and 10～30mL water are added in three-neck flask, are led under room temperature after 30～60min of nitrogen, are returned in 120～150 DEG C
24～36h of stream, cooled and filtered obtains solid and filtrate；Solid is extracted with 2～5mL ethylene glycol monoethyl ethers, united extraction liquid
And filtrate, 20～30mL water generating yellow mercury oxides are added, yellow mercury oxide is used successively water, acetone, absolute ethanol washing, obtain chlorine
Bridging；
C. it is added in reaction bulb after 0.07～0.10mmol chlorine bridgings is dissolved in 10～20mL dichloromethane, by 0.10～
0.20mmol assistant ligands 4,4 '-dicarboxylic acids -2,2 '-bipyridyl is added in reaction bulb after being dissolved in 10～20mL methanol, will
Reaction bulb flows back 2～5h at 60～100 DEG C；0.2～0.4g sodium acetates are dissolved in after 2～5mL methanol are again added to reaction bulb
In, flow back 1～5h, is cooled to room temperature；Ammonium hexafluorophosphate is added to into 2～5mL methanol solutions and is added to reaction bulb 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 gained solid
Solution, suspension stirs 10～30min, filters；After by water washing 2 times of the solid with 10～20mL, solid is carried with 2mL methanol
Take, the ammonium hexafluorophosphate methanol solution of 2～5mL saturations is added in extracting solution, stir 30～60min, revolving removes solvent first
Alcohol, gained solid is placed in vacuum drying oven and is dried 12～24h；By dried object methylene chloride/methanol solution (volume ratio 5:
1) column chromatography on a silica gel column, by chromatographic silica gel plate analysis composition, the eluent at second peak is merged, by vacuum rotary steam
Methylene chloride and methanol are removed, 12～24h is vacuum dried at 65 DEG C, obtain yellow complex of iridium；
(2) synthesis of cation conjugated polymer
A. pour the potassium hydroxide solution of 30～60wt% into round-bottomed flask, be cooled to room temperature, add the four of 0.1～0.5mmol
Butylammonium bromide, in being 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-hexanes of 1.2mmol and 2, the 7- dibromo fluorenes of 0.1～0.5mmol, are stirred vigorously, and react 30～60min, stop stirring
Mix, standing is cooled to room temperature；It is subsequently adding 20mL dichloromethane to extract 3～5 times, with water and 0.1 successively after organic faciess are merged
The dilute hydrochloric acid washing of～0.5M, then after being dried with anhydrous magnesium sulfate, vacuum rotary steam removes methylene chloride, obtains clear yellow viscous liquid
Body；By yellow viscous liquid under the conditions of 120 DEG C, oil pump vacuum distillation is used, remove most of excessive 1,6- dibromo-hexanes, then
Silicagel column is rinsed with petroleum ether, by chromatographic silica gel plate analysis composition, the eluent at the 3rd peak is merged, vacuum rotary steam is removed
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 2, the 7- bis- of 0.5～1mmol bromo- 9,9- bis- (6 '-bromine hexyl) fluorenes, the Isosorbide-5-Nitrae-phenylene of 0.05～0.1mmol
Hypoboric acid, the ferrocene palladium chloride of 0.01～0.05mmol is added in round-bottomed flask, and after repetition oil pump evacuation argon is filled with
Gas is operated 2～3 times；Then 3mL water and 6mL tetrahydrofurans are added in round-bottomed flask, 10～30min in ice-water bath is placed in, is treated
Solution temperature is evacuated and is re-filled with argon operation 2～3 times to after -7～-10 DEG C；Recover to room temperature after solution temperature, will
Round-bottomed flask is placed in oil bath pan, opens magnetic agitation, under the conditions of 85～100 DEG C, after 24～36h of reaction, is cooled to room temperature,
Then 10mL methanol is added in round-bottomed flask, is stood and is separated out precipitation, filtered, take precipitation successively with the methanol and third for pre-cooling
After ketone washing, vacuum rotary steam removes solvent methanol, obtains grey black solid；100～150mL tetrahydrochysene furans are added in grey black solid
Mutter, ultrasonic dissolution after standing 1～2h, with 0.45 μm of nylon membrane sucking filtration, removes solid palladium black, and filtrate is spin-dried for, and obtains 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 2 and 10～30mL tetrahydrofurans of 60～80mg are added in round-bottomed flask, in -78～-100 DEG C
15～30min of lower pre-cooling, after solution cools to -78～-100 DEG C, is evacuated and is re-filled with argon operation 2～3 times；With injection
Device is lentamente added dropwise over after 2～5mL trimethylamines in round-bottomed flask, closes refrigeration, waits and round-bottomed flask is placed in into ice after 1～2h
In water-bath, after being gradually brought to room temperature, magnetic agitation reacts 24～36h, then is added dropwise over 10～20mL water by syringe,
After stirring and evenly mixing, -78～-100 DEG C are cooled to, are evacuated and are filled with after argon, be added dropwise over 2～5mL trimethylamines, close 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 tetrahydrofuran solvent is removed under reduced pressure, the 10mL acetone of pre-cooling is added, precipitation is separated out, is carefully drawn with dropper
Layer clear liquid, by the washing with acetone 2～3 times of precipitation pre-cooling, after sinking completely per inferior precipitation, with dropper upper strata is carefully drawn
Clear liquid, merges supernatant, and vacuum rotary steam removes solvent acetone, is dried to obtain cation 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) with complex of iridium there is FRET in cation conjugated polymer
By cation conjugated polymer, (final concentration is 2.95 × 10-6M), (final concentration is 1 × 10 to complex of iridium-5M)
With water by volume 90：1：9 mixing, that is, obtain the fluorescence sense with complex of iridium FRET effects based on cation conjugated polymer
Body system.
The above-mentioned application based on cation conjugated polymer and the fluorescent optical sensor system of complex of iridium FRET effects, this is glimmering
Optical sensor system is used to detect thrombin that concrete grammar step to be as follows：2 μ L, 10 μM of thrombin aptamer solution is added
To in the above-mentioned fluorescent optical sensor systems based on cation conjugated polymer and complex of iridium FRET effects of 100 μ L, then distinguish
The thrombin solution of variable concentrations is added in fluorescent optical sensor system, buffer solution is subsequently adding, at 37 DEG C 40 are incubated
～60min, the change of fluorescence intensity calculates the fluorescence intensity and complex of iridium of cation conjugated polymer (415nm)
(530nm) fluorescence intensity ratio, obtains a series of corresponding fluorescence intensity ratio of thrombin of variable concentrations, sets up fluorescence strong
Quantitative relationship between degree ratio and concentration of thrombin；According to both quantitative relationships, the concentration of thrombin in testing sample is detected；
Described thrombin aptamer be a bit of Jing in-vitro screenings obtain can with thrombin carry out high-affinity and strong specificity knot
The oligonucleotide sequence of conjunction, its nucleotides sequence is classified as 5'-AGT CCG TGG TAG GGC AGG TTG GGG TGACT-3'.
Described buffer solution is the phosphate buffered solution of pH=7.0～8.0, and concentration is 0.1～0.2M.
Described 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 cation conjugated polymer and the fluorescent optical sensor system of complex of iridium FRET effects, this is glimmering
Optical sensor system is used to detect adenosine triphosphate (ATP) that concrete grammar step to be as follows：2 μ L, 10 μM of adenosine triphosphate is fitted
Ligand solution is added to the above-mentioned fluorescent optical sensor systems based on cation conjugated polymer and complex of iridium FRET effects of 100 μ L
In, then the adenosine triphosphate thrombin solution of variable concentrations is added in fluorescent optical sensor system respectively, it is subsequently adding slow
Solution is rushed, 40～60min is incubated at 37 DEG C, the change of fluorescence intensity calculates cation conjugated polymer (415nm)
A series of fluorescence intensity ratio of fluorescence intensity and complex of iridium (530nm), the adenosine triphosphate for obtaining variable concentrations is corresponding
Fluorescence intensity ratio, the quantitative relationship set up between fluorescence intensity ratio and adenosine triphosphate concentration；According to both quantitative relationships,
The concentration of adenosine triphosphate in detection testing sample.
The above-mentioned application based on cation conjugated polymer and the fluorescent optical sensor system of complex of iridium FRET effects, this is glimmering
Optical sensor system is used to detect platelet derived growth factor (PDGF) that concrete grammar step to be as follows：By 2 μ L, 10 μM of blood
Platelet derivative growth factor adaptation liquid solution is added to that 100 μ L are above-mentioned to be imitated based on cation conjugated polymer and complex of iridium FRET
In the fluorescent optical sensor system answered, then respectively the platelet derived growth factor solution of variable concentrations is added to into fluorescence sense
In body system, buffer solution is subsequently adding, 40～60min is incubated at 37 DEG C, the change 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 variable concentrations
The corresponding fluorescence intensity ratio of platelet derived growth factor, set up fluorescence intensity ratio dense with platelet derived growth factor
Quantitative relationship between degree；According to both quantitative relationships, the concentration of platelet derived growth factor in testing sample is detected.
The above-mentioned application based on cation conjugated polymer and the fluorescent optical sensor system of complex of iridium FRET effects, this is glimmering
Optical sensor system is used to detect cocaine (Cocaine) that concrete grammar step to be as follows：By 2 μ L, 10 μM of cocaine aptamers
Solution is added in the above-mentioned fluorescent optical sensor systems based on cation conjugated polymer and complex of iridium FRET effects of 100 μ L,
Then the cocaine solution of variable concentrations is added in fluorescent optical sensor system respectively, buffer solution is subsequently adding, in 37 DEG C
40～60min of lower incubation, the change of fluorescence intensity calculates the fluorescence intensity and iridium of cation conjugated polymer (415nm)
The fluorescence intensity ratio of coordination compound (530nm), obtains a series of corresponding fluorescence intensity ratio of cocaine of variable concentrations, sets up
Quantitative relationship between fluorescence intensity ratio and cocaine concentration；According to both quantitative relationships, cocaine in detection testing sample
Concentration.
The above-mentioned application based on cation conjugated polymer and the fluorescent optical sensor system of complex of iridium FRET effects, this is glimmering
Optical sensor system is used to detect lysozyme (Lysozyme) that concrete grammar step to be as follows：By 2 μ L, 10 μM of lysozyme adaptations
Liquid solution is added to the above-mentioned fluorescent optical sensor systems based on cation conjugated polymer and complex of iridium FRET effects of 100 μ L
In, then the lysozyme soln of variable concentrations is added in fluorescent optical sensor system respectively, buffer solution is subsequently adding, in 37
At DEG C incubate 40～60min, the change of fluorescence intensity, calculate cation conjugated polymer (415nm) fluorescence intensity with
The fluorescence intensity ratio of complex of iridium (530nm), obtains a series of corresponding fluorescence intensity ratio of lysozyme of variable concentrations, builds
Quantitative relationship between vertical fluorescence intensity ratio and lysozyme concentration；According to both quantitative relationships, bacteriolyze in detection testing sample
The concentration of enzyme.
Inventive principle：Single sensitive molecule is become a phase by cation conjugated polymer by the way that chemical conjugation is bonded
The entirety of mutual correlation, the electron cloud in π keys can spread along the whole conjugated backbone of conjugated polymer.Long-range electron is conjugated not only
The energy gap of bonding and antibonding energy interband is substantially reduced, and makes two can increase with inner orbit number, interorbital energy gap with broadening
Reduce.By absorbing photon or chemical doping, electronics can be easily achieved from the electron transition for taking antibonding energy band to into bond energy.
Multiple sensitive monomers are connected with each other by pi-electron conjugated system, define the conjugated polymer with ad hoc structure, thus sun from
Sub- conjugated polymer is a kind of very efficient energy/electron transfer mediator, and the function with " molecular wire " is swashed by photon
Send out produce exciton (exciton) can in other words excited energy can be along conjugated polymer along conjugated main chain free migration
Main chain is transmitted.Further, since the presence of conjugation, the characteristics of cation conjugated polymer with signal except amplifying
Outward, it also has larger molar extinction coefficient, and its value is up to 106M-1cm-1The order of magnitude, absorbing ability is strong, fluorescence quantum yield
It is high.Complex of iridium has quantum efficiency height, emission lifetime length, emission wavelength is adjustable, big Stokes displacements so that it is obtained extensively
General application.FRET as the optical molecular chi in 1.0～10.0nm distance ranges, with high-resolution, high sensitivity, simple
The advantages of facilitating.
The present invention builds a kind of simple, highly sensitive, high selectivity " turn- according to the principle of FRET (fluorescence resonance energy transfer)
On " analyzes method for sensing.Due to being made by electrostatic between cation conjugated polymer positively charged, with the complex of iridium with negative electricity
With there is FRET, when both simultaneously in the presence of, 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, because the specific binding of object and its aptamers is made
Increased with the fluorescence intensity ratio of the two is caused.Target concentration is bigger, and the fluorescence intensity ratio of the two is bigger, fluorescence intensity ratio
It is linear between value and the logarithm of target concentration.The detection of the concentration of object in testing sample can be realized accordingly.
The present invention is suitable for other determinand acting bodies and detects testing concentration by fluorescence intensity change with determinand.
Compared with prior art, it is an advantage of the current invention that：
(1) high sensitivity, the detection sensitivity of the present invention is about more than 10 times of existing method, and sensitivity of the present invention is
2.3fM, reason is：First, the sensitivity of fluorescent optical sensor is significantly larger than other sensors；Secondly, cation conjugation is poly-
Compound has the function of molecular wire, can realize the amplification of fluorescence signal, and itself has stronger fluorescence.
(2) high specific, common other enzymes are noiseless to this detection system, and reason is：The present invention is based on mesh
Specific recognition between mark thing aptamers and object and the fluorescent optical sensor for combining and building, interfering material is not object
The object of aptamers, therefore the interfering material in prepare liquid can not be combined with object aptamers, therefore to this detection system
It is noiseless.
(3) detection process is simple, quick, and cation conjugated polymer and complex of iridium are mixed just can be occurred
FRET, after the completion of adding object incubation, you can detection obtains fluorescence radiation signal, realizes detection by quantitative.
(4) accurately, the response rate is between 90%～110% for result.
In sum, the present invention is glimmering with complex of iridium FRET effects based on above-mentioned water-soluble cationic conjugated polymer
Optical sensor system, has the advantages that high sensitivity, high specific, simple, quick, easily operated.
Description of the drawings
Fig. 1 is that (A and B are respectively cation conjugation for the excitation and emission spectra of cation conjugated polymer and complex of iridium
The excitation and emission spectra of polymer；C and D are respectively the excitation and emission spectras of complex of iridium)；
Fig. 2 is under the conditions of the thrombin of variable concentrations, based on returning for cation conjugated polymer and complex of iridium detection method
One changes fluorescence emission spectrum；
Fig. 3 is the linear relationship chart of normalization fluorescence intensity and log concentration value under the conditions of the thrombin of variable concentrations；
Fig. 4 is selectivity lab diagram of the fluorescent optical sensor system of the present invention to thrombin；
Fig. 5 is interference--free experiments figure of the fluorescent optical sensor system of the present invention to thrombin；
Fig. 6 is the linear pass of normalization fluorescence intensity and log concentration value under the conditions of the adenosine triphosphate (ATP) of variable concentrations
System's figure；
Fig. 7 is normalization fluorescence intensity and log concentration under the conditions of the platelet derived growth factor (PDGF) of variable concentrations
The linear relationship chart of value；
Fig. 8 is the linear pass of normalization fluorescence intensity and log concentration value under the conditions of the cocaine (Cocaine) of variable concentrations
System's figure；
Fig. 9 is that normalization fluorescence intensity is linear with log concentration value under the conditions of the lysozyme (Lysozyme) of variable concentrations
Graph of a relation.
Specific embodiment
The present invention is described in further detail below in conjunction with accompanying drawing embodiment.
First, specific embodiment
Embodiment 1
A kind of preparation method based on cation conjugated polymer and the fluorescent optical sensor system of complex of iridium FRET effects, specifically
Step is as follows：
(1) synthesis of complex of iridium
A. by 10～15mmol 2- chloropyrimide, 10～20mmol 2,4 difluorobenzene ylboronic acids, 1.25～1.5mmol triphenyls
Phosphine, 10～15mL glycol dimethyl ethers and 15～20mL solution of potassium carbonate are added in three-neck flask, under room temperature lead to nitrogen 30～
60min, then 0.3～0.5mmol palladium is added in three-neck flask, magnetic agitation, and the 15～20h that flows back in 110～150 DEG C
Afterwards stopped reaction, is cooled to room temperature, separates organic faciess and water phase；Water is mutually extracted with 30～60mL ethyl acetate, then by water phase
Repeat extraction three times with 30～60mL ethyl acetate；Washed successively with 40～80mL water and 40～80mL saline after organic faciess are merged
Wash, then with anhydrous magnesium sulfate be dried, revolving remove acetate extractant, by gained solid in 60～80 DEG C be vacuum dried 12～
24h；By dried object dichloromethane/acetone soln (volume ratio 10:1) column chromatography on a silica gel column, by chromatographic silica gel plate point
Analysis composition, the eluent at second peak is merged, and by vacuum rotary steam methylene chloride and acetone are removed, and vacuum is done at 65 DEG C
Dry 12～24h, obtains light yellow solid 4- (2,4 difluorobenzene base) pyrimidine (DFPPM)；
B. by 2～4mmol IrCl3·3H2O, 4～5mmol 4- (2,4 difluorobenzene base) pyrimidine (DFPPM), 30～50mL second
2-ethoxyethanol and 10～30mL water are added in three-neck flask, are led under room temperature after 30～60min of nitrogen, are returned in 120～150 DEG C
24～36h of stream, cooled and filtered obtains solid and filtrate；Solid is extracted with 2～5mL ethylene glycol monoethyl ethers, united extraction liquid
And filtrate, 20～30mL water generating yellow mercury oxides are added, yellow mercury oxide is used successively water, acetone, absolute ethanol washing, obtain chlorine
Bridging；
C. it is added in reaction bulb after 0.07～0.10mmol chlorine bridgings is dissolved in 10～20mL dichloromethane, by 0.10～
0.20mmol assistant ligands 4,4 '-dicarboxylic acids -2,2 '-bipyridyl is added in reaction bulb after being dissolved in 10～20mL methanol, will
Reaction bulb flows back 2～5h at 60～100 DEG C；0.2～0.4g sodium acetates are dissolved in after 2～5mL methanol are again added to reaction bulb
In, flow back 1～5h, is cooled to room temperature；Ammonium hexafluorophosphate is added to into 2～5mL methanol solutions and is added to reaction bulb 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 gained solid
Solution, suspension stirs 10～30min, filters；After by water washing 2 times of the solid with 10～20mL, solid is carried with 2mL methanol
Take, the ammonium hexafluorophosphate methanol solution of 2～5mL saturations is added in extracting solution, stir 30～60min, revolving removes solvent first
Alcohol, gained solid is placed in vacuum drying oven and is dried 12～24h；By dried object methylene chloride/methanol solution (volume ratio 5:
1) column chromatography on a silica gel column, by chromatographic silica gel plate analysis composition, the eluent at second peak is merged, by vacuum rotary steam
Methylene chloride and methanol are removed, 12～24h is vacuum dried at 65 DEG C, obtain yellow complex of iridium；
(3) synthesis of cation conjugated polymer
A. pour the potassium hydroxide solution of 30～60wt% into round-bottomed flask, be cooled to room temperature, add the four of 0.1～0.5mmol
Butylammonium bromide, in being 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-hexanes of 1.2mmol and 2, the 7- dibromo fluorenes of 0.1～0.5mmol, are stirred vigorously, and react 30～60min, stop stirring
Mix, standing is cooled to room temperature；It is subsequently adding 20mL dichloromethane to extract 3～5 times, with water and 0.1 successively after organic faciess are merged
The dilute hydrochloric acid washing of～0.5M, then after being dried with anhydrous magnesium sulfate, vacuum rotary steam removes methylene chloride, obtains clear yellow viscous liquid
Body；By yellow viscous liquid under the conditions of 120 DEG C, oil pump vacuum distillation is used, remove most of excessive 1,6- dibromo-hexanes, then
Silicagel column is rinsed with petroleum ether, by chromatographic silica gel plate analysis composition, the eluent at the 3rd peak is merged, vacuum rotary steam is removed
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 2, the 7- bis- of 0.5～1mmol bromo- 9,9- bis- (6 '-bromine hexyl) fluorenes, the Isosorbide-5-Nitrae-phenylene of 0.05～0.1mmol
Hypoboric acid, the ferrocene palladium chloride of 0.01～0.05mmol is added in round-bottomed flask, and after repetition oil pump evacuation argon is filled with
Gas is operated 2～3 times；Then 3mL water and 6mL tetrahydrofurans are added in round-bottomed flask, 10～30min in ice-water bath is placed in, is treated
Solution temperature is evacuated and is re-filled with argon operation 2～3 times to after -7～-10 DEG C；Recover to room temperature after solution temperature, will
Round-bottomed flask is placed in oil bath pan, opens magnetic agitation, under the conditions of 85～100 DEG C, after 24～36h of reaction, is cooled to room temperature,
Then 10mL methanol is added in round-bottomed flask, is stood and is separated out precipitation, filtered, take precipitation successively with the methanol and third for pre-cooling
After ketone washing, vacuum rotary steam removes solvent methanol, obtains grey black solid；100～150mL tetrahydrochysene furans are added in grey black solid
Mutter, ultrasonic dissolution after standing 1～2h, with 0.45 μm of nylon membrane sucking filtration, removes solid palladium black, and filtrate is spin-dried for, and obtains 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 2 and 10～30mL tetrahydrofurans of 60～80mg are added in round-bottomed flask, in -78～-100 DEG C
15～30min of lower pre-cooling, after solution cools to -78～-100 DEG C, is evacuated and is re-filled with argon operation 2～3 times；With injection
Device is lentamente added dropwise over after 2～5mL trimethylamines in round-bottomed flask, closes refrigeration, waits and round-bottomed flask is placed in into ice after 1～2h
In water-bath, after being gradually brought to room temperature, magnetic agitation reacts 24～36h, then is added dropwise over 10～20mL water by syringe,
After stirring and evenly mixing, -78～-100 DEG C are cooled to, are evacuated and are filled with after argon, be added dropwise over 2～5mL trimethylamines, close 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 tetrahydrofuran solvent is removed under reduced pressure, the 10mL acetone of pre-cooling is added, precipitation is separated out, is carefully drawn with dropper
Layer clear liquid, by the washing with acetone 2～3 times of precipitation pre-cooling, after sinking completely per inferior precipitation, with dropper upper strata is carefully drawn
Clear liquid, merges supernatant, and vacuum rotary steam removes solvent acetone, is dried to obtain cation 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) with complex of iridium there is FRET in cation conjugated polymer
By cation conjugated polymer, (final concentration is 2.95 × 10-6M), (final concentration is 1 × 10 to complex of iridium-5M)
With water by volume 90：1：9 mixing, that is, obtain the fluorescence sense with complex of iridium FRET effects based on cation conjugated polymer
Body system.
The excitation spectrum and emission spectrum of cation conjugated polymer and complex of iridium are determined respectively, it is determined that both can send out
Raw FRET (fluorescence resonance energy transfer), as shown in Figure 1, it can be seen that the emission spectrum (B) of cation conjugated polymer and complex of iridium
(C) excitation spectrum has overlap, so this system is likely to occur FRET, cation 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 overlap, its
It is secondary be between energy donor, energy acceptor must it is close enough, so occur energy transfer probability just understand height.Sun is determined respectively
The excitation spectrum and emission spectrum of ion conjugated polymer and complex of iridium, it is determined that both can occur fluorescence resonance energy turning
Move.Determine cation conjugated polymer, complex of iridium Zeta potential.Experimental data shows, pure cation conjugated polymer
Zeta potential is 103mV, and after mixing with complex of iridium, Zeta potential drops to 13.1mV, and this shows because electrostatic interaction
So that complex of iridium is gathered in around cation conjugated polymer, and the positive charge on complex of iridium surface reduces cation conjugation
The Zeta potential of polymer, by conjugation complex is formed.
2nd, Application Example
Embodiment 1
Fluorescent optical sensor system detection thrombin based on cation conjugated polymer and complex of iridium FRET effects
1st, detection method
By 2 μ L, 10 μM of thrombin aptamer solution be added in the above-mentioned specific embodiments one of 100 μ L prepare based on cation
In the fluorescent optical sensor system of conjugated polymer and complex of iridium FRET effects, then respectively by the thrombin solution of variable concentrations
In being added to fluorescent optical sensor system, buffer solution is subsequently adding, 40～60min is incubated at 37 DEG C, fluorescence intensity
Change, calculates the fluorescence intensity of cation conjugated polymer (415nm) and the fluorescence intensity ratio of complex of iridium (530nm), obtains
A series of corresponding fluorescence intensity ratio of thrombin of variable concentrations is obtained, is set up between fluorescence intensity ratio and concentration of thrombin
Quantitative relationship；According to both quantitative relationships, the concentration of thrombin in testing sample is detected；
Wherein thrombin aptamer be a bit of Jing in-vitro screenings obtain can carry out high-affinity and strong specificity with thrombin
With reference to oligonucleotide sequence, its nucleotides sequence is classified as 5'-AGT CCG TGG TAG GGC AGG TTG GGG TGA CT-
3'。
Above-mentioned buffer solution is the phosphate buffered solution of pH=7.0～8.0, and concentration is 0.1～0.2M；Fluoremetry bar
Part is as follows：Excitation wavelength is 380nm, and slit width is 10nm, and voltage is 700V, and response time is 0.01s, and scanning speed is
1200nm/min。
Experimental result is as shown in Fig. 2 with the addition of thrombin solution, destroy DNA, cation conjugated polymer and iridium
Coordination compound there occurs FRET again, the Fluorescence Increasing 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 thrombin solution linear sound of the sensor to 0.01pM～200pM
Should, linear equation is：I=0.20logc+0.83, coefficient R2=0.9971, detection is limited to 2.3fM.
2nd, specificity experiments
Selectivity experiment is as follows with the abbreviation of other enzymes used in interference--free experiments：Horseradish peroxidase (HRP), rotten albumen
Enzyme (CHY), carboxypeptidase (CPY), lysozyme (Lysozyme), acetylcholinesterase (AchE), papain (Papain), cattle
Serum albumin (BSA).
The explanation of selectivity experimental design, as a result as shown in Figure 4.As can be seen from Fig., when other enzyme (concentration of addition：10nM)
When, FRET efficiency is not changed in compared with blank, and has added the ratio of the fluorescence intensity of 100pM thrombins to reach maximum,
Illustrate that body series selectivity is 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 thrombins
During mixing, the fluorescence intensity ratio at 415nm and 530nm has almost no change compared with single thrombin, additionally, this eight kinds
Also response signal of the sensor to thrombin will not be significantly affected when material coexists, illustrates that body series anti-interference is good.
Embodiment 2
Fluorescent optical sensor system detection adenosine triphosphate based on cation conjugated polymer and complex of iridium FRET effects, specifically
Method and step is as follows：2 μ L, 10 μM of adenosine triphosphate adaptation liquid solutions are added in the above-mentioned specific embodiments one of 100 μ L and are made
It is then respectively that difference is dense in the standby fluorescent optical sensor system based on cation conjugated polymer and complex of iridium FRET effects
The adenosine triphosphate solution of degree is added in fluorescent optical sensor system, is subsequently adding buffer solution, at 37 DEG C incubate 40～
60min, the change of fluorescence intensity calculates the fluorescence intensity and complex of iridium of cation conjugated polymer (415nm)
(530nm) fluorescence intensity ratio, obtains a series of corresponding fluorescence intensity ratio of adenosine triphosphate of variable concentrations, sets up glimmering
Quantitative relationship between intensity ratio and adenosine triphosphate concentration；According to both quantitative relationships, three phosphorus in detection testing sample
The concentration of adenosine monophosphate.Above-mentioned buffer solution is the phosphate buffered solution of pH=7.0～8.0, and concentration is 0.1～0.2M；Fluorescence
Condition determination is as follows：Excitation wavelength is 380nm, and slit width is 10nm, and voltage is 700V, and response time is 0.01s, scanning speed
Degree is 1200nm/min.
Experimental result is as shown in fig. 6, be presented linear closing between the logarithm value of fluorescence intensity ratio and adenosine triphosphate concentration
It is that linear equation is：I=0.19logc+0.24, coefficient R2=0.9947, the range of linearity be 10pM～20nM, test limit
For 2pM.It is linear good, can be used for unknown sample detection.
Embodiment 3
Fluorescent optical sensor system detection platelet derived growth based on cation conjugated polymer and complex of iridium FRET effects
The factor, concrete grammar step is as follows：2 μ L, 10 μM of platelet derived growth factor adaptation liquid solutions are added to into 100 μ L above-mentioned
In the fluorescent optical sensor system based on cation conjugated polymer and complex of iridium FRET effects prepared in specific embodiment one,
Then the platelet derived growth factor solution of variable concentrations is added in fluorescent optical sensor system respectively, is subsequently adding buffering
Solution, incubates 40～60min at 37 DEG C, and the change of fluorescence intensity calculates the glimmering of cation conjugated polymer (415nm)
Light intensity and the fluorescence intensity ratio of complex of iridium (530nm), obtain a series of platelet derived growth factor of variable concentrations
Corresponding fluorescence intensity ratio, the quantitative relationship set up between fluorescence intensity ratio and platelet derived growth factor concentration；Root
According to both quantitative relationships, the concentration of platelet derived growth factor in testing sample is detected.Above-mentioned buffer solution be pH=7.0～
8.0 phosphate buffered solution, concentration is 0.1～0.2M；Fluoremetry condition is as follows：Excitation wavelength is 380nm, slit width
It is 10nm, voltage is 700V, and response time is 0.01s, and scanning speed is 1200nm/min.
Experimental result as shown in fig. 7, be between the logarithm value of fluorescence intensity ratio and platelet derived growth factor concentration
Show linear relationship, linear equation is：I=0.58+0.22logc, coefficient R2=0.9855, the range of linearity be 0.1pM～
1000pM, detection is limited to 0.01pM.It is linear good, can be used for unknown sample detection.
Embodiment 4
Fluorescent optical sensor system detection cocaine based on cation conjugated polymer and complex of iridium FRET effects, concrete grammar
Step is as follows：The cocaine adaptation liquid solution of 10 μM of 2 μ L is added to prepare in the above-mentioned specific embodiments one of 100 μ L based on
In the fluorescent optical sensor system of cation conjugated polymer and complex of iridium FRET effects, then respectively by the cocker of variable concentrations
Because solution is added in fluorescent optical sensor system, buffer solution is subsequently adding, 40～60min is incubated at 37 DEG C, detect fluorescence
The change of intensity, calculates the fluorescence intensity of cation conjugated polymer (415nm) and the fluorescence intensity ratio of complex of iridium (530nm)
Value, obtains a series of corresponding fluorescence intensity ratio of cocaine of variable concentrations, sets up fluorescence intensity ratio and cocaine concentration
Between quantitative relationship；According to both quantitative relationships, the concentration of cocaine in testing sample is detected.Above-mentioned buffer solution is pH=
7.0～8.0 phosphate buffered solution, concentration is 0.1～0.2M；Fluoremetry condition is as follows：Excitation wavelength is 380nm, narrow
Seam width is 10nm, and voltage is 700V, and response time is 0.01s, and scanning speed is 1200nm/min.
Experimental result as shown in figure 8, fluorescence intensity ratio and logarithm between linear relationship is presented, linear equation is：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, can be used for unknown sample detection.
Embodiment 5
Fluorescent optical sensor system detection lysozyme based on cation conjugated polymer and complex of iridium FRET effects, concrete grammar
Step is as follows：The lysozyme adaptation liquid solution of 10 μM of 2 μ L is added to prepare in the above-mentioned specific embodiments one of 100 μ L based on
In the fluorescent optical sensor system of cation conjugated polymer and complex of iridium FRET effects, then respectively by the bacteriolyze of variable concentrations
Enzymatic solution is added in fluorescent optical sensor system, is subsequently adding buffer solution, and 40～60min is incubated at 37 DEG C, detects fluorescence
The change of intensity, calculates the fluorescence intensity of cation conjugated polymer (415nm) and the fluorescence intensity ratio of complex of iridium (530nm)
Value, obtains a series of corresponding fluorescence intensity ratio of lysozyme of variable concentrations, sets up fluorescence intensity ratio and lysozyme concentration
Between quantitative relationship；According to both quantitative relationships, the concentration of lysozyme in testing sample is detected.Above-mentioned buffer solution is pH=
7.0～8.0 phosphate buffered solution, concentration is 0.1～0.2M；Fluoremetry condition is as follows：Excitation wavelength is 380nm, narrow
Seam width is 10nm, and voltage is 700V, and 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
For：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, described above not limitation of the present invention, the present invention is also not limited to the example above.The art
Change, remodeling, addition or replacement that those of ordinary skill makes in the essential scope of the present invention, should also belong to protection of the present invention
Scope.
Sequence table
<110>University Of Ningbo
<120>Preparation based on cation conjugated polymer and the fluorescent optical sensor system of complex of iridium FRET effects
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 (8)
1. a kind of preparation method based on cation conjugated polymer and the fluorescent optical sensor system of complex of iridium FRET effects, its
It is characterised by comprising the following steps that：
（1）The synthesis of complex of iridium
A. by 10～15 mmol 2- chloropyrimide, 10～20 mmol 2,4 difluorobenzene ylboronic acids, 1.25～1.5 mmol triphens
Base phosphine, 10～15 mL glycol dimethyl ethers and 15～20 mL solution of potassium carbonate are added in three-neck flask, and nitrogen is led under room temperature
30～60 min, then 0.3～0.5 mmol palladium is added in three-neck flask, magnetic agitation, and in 110～150 DEG C of backflows
Stopped reaction after 15～20 h, is cooled to room temperature, separates organic faciess and water phase；Water is mutually extracted with 30～60 mL ethyl acetate,
Again water is mutually repeated into extraction three times with 30～60 mL ethyl acetate；With 40～80 mL water and 40～80 after organic faciess are merged
ML saline is washed successively, then is dried with anhydrous magnesium sulfate, and revolving removes acetate extractant, by gained solid in 60～80
DEG C vacuum drying 12～24 h；It is 10 by dried object volume ratio:1 dichloromethane/acetone soln column chromatography on a silica gel column,
By chromatographic silica gel plate analysis composition, the eluent at second peak is merged, by vacuum rotary steam remove methylene chloride and
Acetone, is vacuum dried 12～24 h at 65 DEG C, obtain light yellow solid 4- (2,4 difluorobenzene base) pyrimidine；
B. by 2～4 mmol IrCl3·3H2O, 4～5 mmol 4- (2,4 difluorobenzene base) pyrimidine, 30～50 mL ethylene glycol lists
Ether and 10～30 mL water are added in three-neck flask, are led under room temperature after the min of nitrogen 30～60, in 120～150 DEG C of backflows
24～36 h, cooled and filtered obtains solid and filtrate；Solid is extracted with 2～5 mL ethylene glycol monoethyl ethers, united extraction liquid
And filtrate, 20～30 mL water generating yellow mercury oxides are added, yellow mercury oxide is used successively water, acetone, absolute ethanol washing, obtain
Chlorine bridging；
C. it is added in reaction bulb after 0.07～0.10 mmol chlorine bridgings is dissolved in 10～20 mL dichloromethane, by 0.10～
0.20 mmol assistant ligands 4,4 '-dicarboxylic acids -2,2 '-bipyridyl is added in reaction bulb after being dissolved in 10～20 mL methanol,
Reaction bulb is flowed back at 60～100 DEG C 2～5 h；0.2～0.4 g sodium acetates are dissolved in after 2～5 mL methanol being again added to
In reaction bulb, flow back 1～5 h, is cooled to room temperature；Ammonium hexafluorophosphate is added to into 2～5 mL methanol solutions to add to saturation
To in reaction bulb, the min of stirring at normal temperature 30～60, revolving removes solvent methanol, then add in gained solid 1～3 M 10～
The hydrochloric acid solution of 20 mL, suspension stirs 10～30 min, filters；After by water washing 2 times of the solid with 10～20 mL, will be solid
Body adds the ammonium hexafluorophosphate methanol solution of 2～5 mL saturations, stirring 30～60 with 2 mL methanol extraction in extracting solution
Min, revolving removes solvent methanol, gained solid is placed in vacuum drying oven and is dried 12～24 h；By dried object volume ratio
For 5:1 methylene chloride/methanol solution column chromatography on a silica gel column, by chromatographic silica gel plate analysis composition, by second peak
Eluent merges, and by vacuum rotary steam methylene chloride and methanol are removed, and 12～24 h are vacuum dried at 65 DEG C, obtains yellow
Complex of iridium；
（2）The synthesis of cation conjugated polymer
A. pour the potassium hydroxide solution of 30～60 wt% into round-bottomed flask, be cooled to room temperature, add the four of 0.1～0.5 mmol
Butylammonium bromide, in being placed in oil bath pan, opens magnetic agitation, and oil bath heating sequentially adds 0.9～1.2 to after 75～100 DEG C
1, the 6- dibromo-hexanes of mmol and 2, the 7- dibromo fluorenes of 0.1～0.5 mmol, are stirred vigorously, and react 30～60 min, stop stirring
Mix, standing is cooled to room temperature；It is subsequently adding 20 mL dichloromethane to extract 3～5 times, with water and 0.1 successively after organic faciess are merged
The dilute hydrochloric acid washing of～0.5 M, then after being dried with anhydrous magnesium sulfate, vacuum rotary steam removes methylene chloride, obtains clear yellow viscous
Liquid；By yellow viscous liquid under the conditions of 120 DEG C, use oil pump vacuum distillation, remove most of excessive 1,6- dibromos oneself
Alkane, then silicagel column is rinsed with petroleum ether, by chromatographic silica gel plate analysis composition, the eluent at the 3rd peak is merged, decompression rotation
Solvent petroleum ether is evaporated off, white crystal conjugated polymer 1 is obtained；
B. first by 2, the 7- bis- of 0.5～1 mmol bromo- 9,9- bis- (6 '-bromine hexyl) fluorenes, the Isosorbide-5-Nitrae-Asia benzene of 0.05～0.1 mmol
Base hypoboric acid, the ferrocene palladium chloride of 0.01～0.05 mmol is added in round-bottomed flask, is filled with after repetition oil pump evacuation
Argon is operated 2～3 times；Then 3 mL water and 6 mL tetrahydrofurans are added in round-bottomed flask, is placed in 10～30 in ice-water bath
Min, behind solution temperature to -7～-10 DEG C, is evacuated and is re-filled with argon operation 2～3 times；Treat that solution temperature recovers to room
Wen Hou, round-bottomed flask is placed in oil bath pan, opens magnetic agitation, cold after 24～36 h of reaction under the conditions of 85～100 DEG C
But 10 mL methanol are then added to room temperature, in round-bottomed flask, is stood and is separated out precipitation, filtered, take precipitation successively with pre-cooling
Methanol and washing with acetone after, vacuum rotary steam remove solvent methanol, obtain grey black solid；In grey black solid add 100～
150 mL tetrahydrofurans, ultrasonic dissolution after standing 1～2 h, with 0.45 μm of nylon membrane sucking filtration, removes solid palladium black, will filter
Liquid is spin-dried for, and obtains yellow solid conjugated polymer 2；
C. the mL tetrahydrofurans of conjugated polymer 2 and 10～30 of 60～80 mg are added in round-bottomed flask, in -78～-100
The min of pre-cooling 15～30 at DEG C, after solution cools to -78～-100 DEG C, is evacuated and is re-filled with argon operation 2～3 times；With
Syringe is lentamente added dropwise over after 2～5 mL trimethylamines in round-bottomed flask, closes refrigeration, waits round-bottomed flask after 1～2 h
In being placed in ice-water bath, after being gradually brought to room temperature, magnetic agitation reacts 24～36 h, then 10 are added dropwise over by syringe～
20 mL water, after stirring and evenly mixing, are cooled to -78～-100 DEG C, are evacuated and are filled with after argon, are added dropwise over 2～5 mL front threes
Amine, closes refrigeration, and round-bottomed flask is placed in ice-water bath after 1～2 h, after being gradually brought to room temperature, magnetic agitation, and reaction 24
～36 h, are then removed under reduced pressure most solvents tetrahydrofurane, add 10 mL acetone of pre-cooling, separate out precipitation, use dropper
The supernatant is carefully drawn, it is little with dropper after sinking completely per inferior precipitation by the washing with acetone 2～3 times of precipitation pre-cooling
Heart draws the supernatant, merges supernatant, and vacuum rotary steam removes solvent acetone, is dried to obtain cation conjugated polymer；
（3）There is FRET with complex of iridium in cation conjugated polymer
By cation conjugated polymer, complex of iridium and water by volume 90：1：9 mixing, that is, obtain poly- based on cation conjugation
The fluorescent optical sensor system of compound and complex of iridium FRET effects.
2. the fluorescent optical sensor system based on cation conjugated polymer and complex of iridium FRET effects described in claim 1
Using, it is characterised in that the fluorescent optical sensor system is used to detect thrombin that concrete grammar step to be as follows：By 10 μM of 2 μ L's
Thrombin aptamer solution be added to described in 100 μ L claim 1 based on cation conjugated polymer and complex of iridium FRET
In the fluorescent optical sensor system of effect, then the thrombin solution of variable concentrations is added in fluorescent optical sensor system respectively,
Buffer solution is subsequently adding, 40～60 min are incubated at 37 DEG C, the change of fluorescence intensity calculates cation conjugation poly-
A series of fluorescence intensity of compound and the fluorescence intensity ratio of complex of iridium, the corresponding fluorescence of thrombin for obtaining variable concentrations is strong
Degree ratio, the quantitative relationship set up between fluorescence intensity ratio and concentration of thrombin；According to both quantitative relationships, test sample is treated in detection
The concentration of thrombin in product；Described thrombin aptamer be a bit of Jing in-vitro screenings obtain can carry out high parent with thrombin
With power and the oligonucleotide sequence of strong specific binding, its nucleotides sequence is classified as 5'-AGT CCG TGG TAG GGC AGG TTG
GGG TGA CT-3'。
3. the fluorescence sense body based on cation conjugated polymer and complex of iridium FRET effects according to claim 2
The method of system's detection thrombin, it is characterised in that：Described buffer solution is the phosphate buffered solution of pH=7.0～8.0, dense
Spend for 0.1～0.2 M.
4. the fluorescence sense body based on cation conjugated polymer and complex of iridium FRET effects according to claim 2
The method of system's detection thrombin, it is characterised in that：Described fluoremetry condition is as follows：Excitation wavelength is 380 nm, slit width
Degree is 10 nm, and voltage is 700 V, and response time is 0.01 s, and scanning speed is 1200 nm/min.
5. the fluorescent optical sensor system based on cation conjugated polymer and complex of iridium FRET effects described in claim 1
Using, it is characterised in that the fluorescent optical sensor system is used to detect adenosine triphosphate that concrete grammar step to be as follows：By 2 μ L 10
μM adenosine triphosphate adaptation liquid solution be added to matching somebody with somebody based on cation conjugated polymer and iridium described in 100 μ L claim 1
In the fluorescent optical sensor system of compound FRET effects, then the adenosine triphosphate thrombin solution of variable concentrations is added to respectively
In fluorescent optical sensor system, buffer solution is subsequently adding, 40～60 min of incubation at 37 DEG C, the change of fluorescence intensity,
The fluorescence intensity of cation conjugated polymer and the fluorescence intensity ratio of complex of iridium are calculated, the three of a series of variable concentrations are obtained
The corresponding fluorescence intensity ratio of adenosine phosphate, the quantitative relationship set up between fluorescence intensity ratio and adenosine triphosphate concentration；Root
According to both quantitative relationships, the concentration of adenosine triphosphate in testing sample is detected.
6. the fluorescent optical sensor system based on cation conjugated polymer and complex of iridium FRET effects described in claim 1
Using, it is characterised in that the fluorescent optical sensor system is used to detect platelet derived growth factor that concrete grammar step to be as follows：Will
2 10 μM of μ L platelet derived growth factor adaptation liquid solution be added to described in 100 μ L claim 1 based on cation
In the fluorescent optical sensor system of conjugated polymer and complex of iridium FRET effects, then respectively by the platelet-derived of variable concentrations
Growth factor solution is added in fluorescent optical sensor system, is subsequently adding buffer solution, and 40～60 min are incubated at 37 DEG C,
The change of fluorescence intensity, calculates the fluorescence intensity of cation conjugated polymer and the fluorescence intensity ratio of complex of iridium, obtains
A series of corresponding fluorescence intensity ratio of platelet derived growth factor of variable concentrations is obtained, fluorescence intensity ratio is set up little with blood
Quantitative relationship between plate derivative growth factor concentration；According to both quantitative relationships, platelet-derived life in detection testing sample
The concentration of the long factor.
7. the fluorescent optical sensor system based on cation conjugated polymer and complex of iridium FRET effects described in claim 1
Using, it is characterised in that the fluorescent optical sensor system is used to detect cocaine that concrete grammar step to be as follows：By 10 μM of 2 μ L's
Cocaine adaptation liquid solution be added to described in 100 μ L claim 1 based on cation conjugated polymer and complex of iridium FRET
In the fluorescent optical sensor system of effect, then the cocaine solution of variable concentrations is added in fluorescent optical sensor system respectively,
Buffer solution is subsequently adding, 40～60 min are incubated at 37 DEG C, the change of fluorescence intensity calculates cation conjugation poly-
A series of fluorescence intensity of compound and the fluorescence intensity ratio of complex of iridium, the corresponding fluorescence of cocaine for obtaining variable concentrations is strong
Degree ratio, the quantitative relationship set up between fluorescence intensity ratio and cocaine concentration；According to both quantitative relationships, test sample is treated in detection
The concentration of cocaine in product.
8. the fluorescent optical sensor system based on cation conjugated polymer and complex of iridium FRET effects described in claim 1
Using, it is characterised in that the fluorescent optical sensor system is used to detect lysozyme that concrete grammar step to be as follows：By 10 μM of 2 μ L's
Lysozyme adaptation liquid solution be added to described in 100 μ L claim 1 based on cation conjugated polymer and complex of iridium FRET
In the fluorescent optical sensor system of effect, then the lysozyme soln of variable concentrations is added in fluorescent optical sensor system respectively,
Buffer solution is subsequently adding, 40～60 min are incubated at 37 DEG C, the change of fluorescence intensity calculates cation conjugation poly-
A series of fluorescence intensity of compound and the fluorescence intensity ratio of complex of iridium, the corresponding fluorescence of lysozyme for obtaining variable concentrations is strong
Degree ratio, the quantitative relationship set up between fluorescence intensity ratio and lysozyme concentration；According to both quantitative relationships, test sample is treated in detection
The concentration of lysozyme in product.
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| CN110438202A (en) * | 2019-08-15 | 2019-11-12 | 青岛科技大学 | The preparation method of fibrin ferment optical electro-chemistry sensor of the one kind based on ring metal Ir (III) complex |
| CN110438202B (en) * | 2019-08-15 | 2022-09-20 | 青岛科技大学 | Preparation method of thrombin photoelectrochemical sensor based on cyclometall Ir (III) complex |
| CN110514632A (en) * | 2019-08-19 | 2019-11-29 | 齐鲁工业大学 | A kind of conjugated polymer nanoparticle fluorescence probe based on fluorescence resonance energy transfer and the application in detection glutathione |
| CN112945923A (en) * | 2021-02-03 | 2021-06-11 | 长沙理工大学 | Interface sensitization type detection reagent and preparation method and application thereof |
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