CN107976538A - A kind of immune labeled probe of silica fluorescent based on fluorescence resonance energy transfer, preparation method and application - Google Patents
A kind of immune labeled probe of silica fluorescent based on fluorescence resonance energy transfer, preparation method and application Download PDFInfo
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- CN107976538A CN107976538A CN201711150942.8A CN201711150942A CN107976538A CN 107976538 A CN107976538 A CN 107976538A CN 201711150942 A CN201711150942 A CN 201711150942A CN 107976538 A CN107976538 A CN 107976538A
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 title claims abstract description 144
- 239000000377 silicon dioxide Substances 0.000 title claims abstract description 72
- 239000000523 sample Substances 0.000 title claims abstract description 42
- 238000002866 fluorescence resonance energy transfer Methods 0.000 title claims abstract description 36
- 238000002360 preparation method Methods 0.000 title claims abstract description 33
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 claims abstract description 39
- 229940043267 rhodamine b Drugs 0.000 claims abstract description 39
- 239000002105 nanoparticle Substances 0.000 claims abstract description 35
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 claims abstract description 32
- 238000003018 immunoassay Methods 0.000 claims abstract description 7
- 239000000243 solution Substances 0.000 claims description 43
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 36
- 238000006243 chemical reaction Methods 0.000 claims description 33
- 235000012239 silicon dioxide Nutrition 0.000 claims description 25
- 238000003756 stirring Methods 0.000 claims description 21
- 238000000034 method Methods 0.000 claims description 20
- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 claims description 16
- 238000002156 mixing Methods 0.000 claims description 16
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 14
- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical class CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 claims description 12
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 12
- 239000000908 ammonium hydroxide Substances 0.000 claims description 12
- 239000007853 buffer solution Substances 0.000 claims description 11
- 238000005406 washing Methods 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 239000008351 acetate buffer Substances 0.000 claims description 6
- 230000035484 reaction time Effects 0.000 claims description 6
- 229910000033 sodium borohydride Inorganic materials 0.000 claims description 6
- 239000012279 sodium borohydride Substances 0.000 claims description 6
- 238000000108 ultra-filtration Methods 0.000 claims description 6
- 238000004140 cleaning Methods 0.000 claims description 5
- 238000013019 agitation Methods 0.000 claims description 4
- 239000011259 mixed solution Substances 0.000 claims description 4
- 239000003643 water by type Substances 0.000 claims description 4
- 239000008367 deionised water Substances 0.000 claims description 3
- 229910021641 deionized water Inorganic materials 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 2
- 230000008569 process Effects 0.000 claims description 2
- 240000007594 Oryza sativa Species 0.000 claims 1
- 235000007164 Oryza sativa Nutrition 0.000 claims 1
- 235000009566 rice Nutrition 0.000 claims 1
- 239000007850 fluorescent dye Substances 0.000 abstract description 15
- 230000008878 coupling Effects 0.000 abstract description 8
- 238000010168 coupling process Methods 0.000 abstract description 8
- 238000005859 coupling reaction Methods 0.000 abstract description 8
- 230000000694 effects Effects 0.000 abstract description 8
- 239000003431 cross linking reagent Substances 0.000 abstract description 5
- 239000003814 drug Substances 0.000 abstract description 5
- 238000001215 fluorescent labelling Methods 0.000 abstract description 4
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 238000001514 detection method Methods 0.000 abstract description 3
- 238000010791 quenching Methods 0.000 abstract description 3
- 230000000171 quenching effect Effects 0.000 abstract description 3
- 239000011159 matrix material Substances 0.000 abstract description 2
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 22
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 12
- 238000002649 immunization Methods 0.000 description 12
- 230000003053 immunization Effects 0.000 description 10
- 238000012546 transfer Methods 0.000 description 10
- 238000003760 magnetic stirring Methods 0.000 description 8
- 241000283707 Capra Species 0.000 description 7
- 239000000975 dye Substances 0.000 description 7
- 230000005284 excitation Effects 0.000 description 7
- JRNVZBWKYDBUCA-UHFFFAOYSA-N N-chlorosuccinimide Chemical compound ClN1C(=O)CCC1=O JRNVZBWKYDBUCA-UHFFFAOYSA-N 0.000 description 6
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 6
- 239000002671 adjuvant Substances 0.000 description 6
- 238000012512 characterization method Methods 0.000 description 6
- 238000002347 injection Methods 0.000 description 6
- 239000007924 injection Substances 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical class O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 4
- WTEOIRVLGSZEPR-UHFFFAOYSA-N boron trifluoride Chemical compound FB(F)F WTEOIRVLGSZEPR-UHFFFAOYSA-N 0.000 description 4
- 239000012043 crude product Substances 0.000 description 4
- 230000006837 decompression Effects 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 239000003208 petroleum Substances 0.000 description 4
- 229920000642 polymer Polymers 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 3
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical class [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- SOQBVABWOPYFQZ-UHFFFAOYSA-N oxygen(2-);titanium(4+) Chemical class [O-2].[O-2].[Ti+4] SOQBVABWOPYFQZ-UHFFFAOYSA-N 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 229910052710 silicon Inorganic materials 0.000 description 3
- 239000010703 silicon Substances 0.000 description 3
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N titanium dioxide Inorganic materials O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 description 3
- 235000010215 titanium dioxide Nutrition 0.000 description 3
- QGLWBTPVKHMVHM-KTKRTIGZSA-N (z)-octadec-9-en-1-amine Chemical compound CCCCCCCC\C=C/CCCCCCCCN QGLWBTPVKHMVHM-KTKRTIGZSA-N 0.000 description 2
- 229910015900 BF3 Inorganic materials 0.000 description 2
- 240000007711 Peperomia pellucida Species 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical class C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- 238000007792 addition Methods 0.000 description 2
- 150000001335 aliphatic alkanes Chemical class 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- HUMNYLRZRPPJDN-UHFFFAOYSA-N benzaldehyde Chemical compound O=CC1=CC=CC=C1 HUMNYLRZRPPJDN-UHFFFAOYSA-N 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 238000010241 blood sampling Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- -1 dichloromethane Alkane Chemical class 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000002114 nanocomposite Substances 0.000 description 2
- 238000011587 new zealand white rabbit Methods 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 150000003233 pyrroles Chemical class 0.000 description 2
- 238000010898 silica gel chromatography Methods 0.000 description 2
- 229910052938 sodium sulfate Inorganic materials 0.000 description 2
- 235000011152 sodium sulphate Nutrition 0.000 description 2
- 238000000967 suction filtration Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- ZHERPVLBGALJPP-UHFFFAOYSA-N trimethoxymethylsilicon Chemical compound COC([Si])(OC)OC ZHERPVLBGALJPP-UHFFFAOYSA-N 0.000 description 2
- DNXUGBMARDFRGG-UHFFFAOYSA-N 3,6-dioxocyclohexa-1,4-diene-1,2-dicarbonitrile Chemical compound O=C1C=CC(=O)C(C#N)=C1C#N DNXUGBMARDFRGG-UHFFFAOYSA-N 0.000 description 1
- AQZMINLSVARCSL-UHFFFAOYSA-N 4-chloro-3,6-dioxocyclohexa-1,4-diene-1,2-dicarbonitrile Chemical compound ClC1=CC(=O)C(C#N)=C(C#N)C1=O AQZMINLSVARCSL-UHFFFAOYSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000005864 Sulphur Substances 0.000 description 1
- LIQLLTGUOSHGKY-UHFFFAOYSA-N [B].[F] Chemical compound [B].[F] LIQLLTGUOSHGKY-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000011953 bioanalysis Methods 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 235000021393 food security Nutrition 0.000 description 1
- NXHPIYRSMIFFBV-UHFFFAOYSA-N formaldehyde;1,3,5-trimethylbenzene Chemical compound O=C.CC1=CC(C)=CC(C)=C1 NXHPIYRSMIFFBV-UHFFFAOYSA-N 0.000 description 1
- 125000003147 glycosyl group Chemical group 0.000 description 1
- 208000006278 hypochromic anemia Diseases 0.000 description 1
- 150000002460 imidazoles Chemical class 0.000 description 1
- 150000003949 imides Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- NALMPLUMOWIVJC-UHFFFAOYSA-N n,n,4-trimethylbenzeneamine oxide Chemical compound CC1=CC=C([N+](C)(C)[O-])C=C1 NALMPLUMOWIVJC-UHFFFAOYSA-N 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- QNGNSVIICDLXHT-UHFFFAOYSA-N para-ethylbenzaldehyde Natural products CCC1=CC=C(C=O)C=C1 QNGNSVIICDLXHT-UHFFFAOYSA-N 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000006862 quantum yield reaction Methods 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229940032753 sodium iodate Drugs 0.000 description 1
- 235000015281 sodium iodate Nutrition 0.000 description 1
- 239000011697 sodium iodate Substances 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- 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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- 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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
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- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- General Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Optics & Photonics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention provides a kind of immune labeled probe of silica fluorescent based on fluorescence resonance energy transfer, preparation method and application, preparation method is：Using mesoporous silica nano-particle as matrix, doping fluorescent dye, rhodamine B and BODIPY structure fluorescence resonance energy transfer system (RB BODIPY FRET).Fluorescence labeling probe is coupled using sodium periodate oxidised antibody；Reliable basis are provided for the super sensitivity detection immunoassay of nano-medicament carrier.Compared with prior art, the present invention is with fluorescent dye BODIPY and rhodamine B codope, fluorescence resonance energy transfer can occur between two kinds of fluorescent dyes, improve fluorescence intensity, weaken quenching, fluorescence labeling probe is coupled using sodium periodate oxidised antibody, coupling efficiency is improved, eliminates influence of the crosslinking agent to antibody activity.
Description
Technical field
The invention belongs to bioanalysis and immunoassay field, and in particular to it is a kind of based on fluorescence resonance energy transfer two
Silica fluorescence immunoassay label probe, preparation method and application.
Background technology
Polysuccinimide (PSI), N- (3- aminopropyls) imidazoles (NAPI) and oleyl amine (OAm) react jointly, obtain
PSI-OAm-NAPI amphiphilics polymer/nanoparticle (MFAP) is a kind of polymer nanocomposite pharmaceutical carrier.Polymer emulsion delivery system
Entrapped drug is widely used in, many polymer nanocomposite drug delivery systems are given the ratification in clinical practice, or are being used for
In the clinical experimental study for the treatment of cancer.It is excessive into the healthy inseparable of the metabolic condition after organism and organism
Drug dose may cause carrier metabolism slowly so as to produce toxic side effect to organism.Therefore, the quantitative survey of pharmaceutical carrier
Determine for it is particularly important that whether material is suitable for clinical practice investigated.
The specificity that immunofluorence technic not only takes full advantage of immunoassay is also combined with the sensitiveness of fluorescence, in disease
Sick prevention, clinical detection, drug screening, food security etc. suffer from quite varied application.The wherein specificity of antibody
Play a decisive role with activity to the accuracy of immunoassay；The fluorescence intensity and stability of label are then high sensitivities, are reappeared
The good key factor of property.Therefore, highly sensitive fluorescence immune analysis method is established in the screening of fluorescent labelling techniques and label
Play very important effect.
The content of the invention
It is an object of the invention to provide a kind of immune labeled spy of the silica fluorescent based on fluorescence resonance energy transfer
Pin and preparation method thereof, new mark spy is provided to establish good specificity, favorable reproducibility, highly sensitive immunoassay program
Pin；The method is using mesoporous silica nano-particle as matrix, doping fluorescent dye, rhodamine B and BODIPY structure fluorescence resonance energy
Measure transfering system (RB-BODIPY-FRET).
Another object of the present invention is to provide a kind of silica fluorescent based on fluorescence resonance energy transfer mark is immunized
Remember the application of probe, coupling efficiency is further increased using new labeling method coupled antibody and ensure that antibody activity, is disappeared
Except influence of the crosslinking agent to antibody activity；Reliable basis are provided for the super sensitivity detection immunoassay of nano-medicament carrier.
A kind of preparation of the immune labeled probe of silica fluorescent based on fluorescence resonance energy transfer provided by the invention
Method, comprises the following steps：
1) cetyl trimethylammonium bromide CTAB, is dissolved in deionized water；
2) ethylene glycol and ammonium hydroxide, are added；
3) ethyl orthosilicate TEOS reactions, are added afterwards；
4) the BODIPY solution and rhodamine B solution mixed, then, is added, adds trimethoxymethylsila,e
MTMS, reaction；
5), adding 3- aminopropyl triethoxysilanes APTES, the reaction was continued；Centrifuge washing, freeze-drying, obtains RB-
BODIPY-FRET Nano particles of silicon dioxide, the as immune labeled spy of the silica fluorescent based on fluorescence resonance energy transfer
Pin.
Whole reflux condensation mode in above-mentioned preparation process.Since step 1), whole process is reacted under the conditions of 60-70 DEG C.
Cetyl trimethylammonium bromide CTAB is dissolved in deionized water under 60-70 DEG C of stirring condition in step 1).
The middle interior addition ethyl orthosilicate TEOS of 3-6min after adding ethylene glycol and ammonium hydroxide of step 3)；Reaction time is 30-
60min。
Step 4) the reaction time is 2-4h.
The BODIPY solution manufacturing methods are to weigh the BODIPY that molal weight is 0.001-0.006mmol to be dissolved in
1mLDMF, the rhodamine B solution manufacturing method are that the rhodamine B of 0.001-0.006mmol is dissolved in 1mL deionized waters；Mixing
Good BODIPY solution and rhodamine B solution mixes in advance ensures that reaction process intermediary hole silica adsorbs two kinds of dyestuffs and arrives
Ability in mesoporous is identical, avoid a kind of dyestuff first add occupy it is most of mesoporous.
Further, BODIPY and rhodamine B molar ratio are 1 in step 4):1, and control mixing before BODIPY solution and
Rhodamine B solution concentration is identical, is respectively 0.001mmol/mL, 0.002mmol/mL, 0.003mmol/mL, 0.004mmol/
ML, 0.005mmol/mL, 0.006mmol/mL；A series of additions investigate optimum selecting.Preferably, it is 0.004mmol/mL,
The concentration of 0.005mmol/mL, prepared fluorescence nano label probe fluorescence intensity is high, and stability is good.
BODIPY solution concentrations are 0.001mmol/mL-0.006mmol/mL before step 4) mixing；Rhodamine B is molten before mixing
Liquid concentration is 0.001mmol/mL-0.006mmol/mL.
The BODIPY preparation methods are：
A, 1-3mL pyrroles, 1-3mL equal three are added while stirring in the round-bottomed flask containing 40-60mL water at 20-25 DEG C
Benzaldehyde, stirs；
B, the dense HCl of 0.2-1mL are added, and 12-24h is reacted in 20-25 DEG C, there are a large amount of black precipitates to generate, is used after suction filtration
Petroleum ether, obtains crude product powder 1；
C, take 200-600mg crude products powder 1 to be dissolved in 40-60mL anhydrous tetrahydro furans, add N- under magnetic stirring
Chlorosuccinimide NCS 300-700mg, 20-25 DEG C of reaction 1-3h.
D, add mixture to after completion of the reaction in 100-300mL water, with the extraction of 20-60mL dichloromethane three times, 10-
30g anhydrous sodium sulfates are dried, and decompression is spin-dried for.
E, 20-60mL dichloromethane is added in the product after being spin-dried for fully to dissolve, add 2,3- bis- under magnetic stirring
Chloro- 5,6- dicyan 1,4-benzoquinone DDQ 400-600mg react 5-20min, add 1-2mL triethylamine react 0.5-2h afterwards,
F, boron trifluoride ether 1-3mL reaction 1-3h are eventually adding, extract three with 30-60mL dichloromethane after completion of the reaction
Secondary, anhydrous sodium sulfate drying, decompression is spin-dried for, through silica gel column chromatography separating-purifying (300-400 mesh silica whites, petroleum ether/dichloromethane
Alkane=10/1, V/V) obtain pure BODIPY.
Step 5) the reaction time is 1-2h.
Further, lucifuge sealing is protected after the RB-BODIPY-FRET Nano particles of silicon dioxide that step 5) is prepared
Deposit.
Further, the preparation side of the immune labeled probe of a kind of silica fluorescent based on fluorescence resonance energy transfer
Method, specifically includes following steps：
1), at 60-70 DEG C by cetyl trimethylammonium bromide CTAB 0.03-0.08g under the conditions of magnetic agitation it is molten
In 30-70mL deionized waters；
2) 0.4-0.7mL ethylene glycol and 2-3mL ammonium hydroxide, are then added at 60-70 DEG C；
3) ethyl orthosilicate TEOS 100-200 μ L reactions 30- is added in the 3-6min, added after ethylene glycol and ammonium hydroxide
60min；
4) 1mL BODIPY solution and the mixed solution and 0.02-0.05mL trimethoxymethylsila,es of rhodamine B, are added
MTMS, reacts 2-4h；
5) 3- aminopropyl triethoxysilanes APTES the reaction was continued the 1-2h of 0.06-0.1mL, are added；
Complete to stir after reacting and be cooled to room temperature, centrifuge washing 2-4 times, is freeze-dried to obtain RB-BODIPY-FRET titanium dioxides
Silicon nano, lucifuge are sealed.
A kind of immune labeled probe of silica fluorescent based on fluorescence resonance energy transfer, is prepared into using the above method
Arrive.
Present invention also offers a kind of immune labeled probe conjugate of the silica fluorescent based on fluorescence resonance energy transfer
The application of antibody.
Concrete application method is：
A, antibody is dissolved into acetate buffer solution, adds sodium periodate solution, aoxidized in 4 DEG C of lucifuge stirrings,
Ultrafiltration removes unreacted sodium periodate, obtains oxidised antibody；
B, by after the immune labeled probe cleaning of the silica fluorescent based on fluorescence resonance energy transfer of above-mentioned preparation points
It is scattered in PBS buffer solutions；Oxidised antibody prepared by step a is added under agitation, and lucifuge stirs at 4 DEG C；
C, it is last, add sodium borohydride solution and continue 4 DEG C of lucifuge stirrings, the solution centrifuge washing after reaction is distributed to
In PBS buffer solutions, 4 DEG C store for future use.
Acetate buffer solution 0.05M, pH=4.6 described in step a；Sodium periodate the solution 2mg/mL, pH=4.6；Institute
The filter membrane for stating ultrafiltration is 0.45 μm of filter membrane；
Mixing time described in step a is 2-4h；
The preparation method of antibody is described in step a：
A-1, first immunisation：Immunogene is used with Freund's complete adjuvant in equal volume than mixing after being emulsified with syringe
Dorsal sc injection mode is immunized new zealand white rabbit, is subcutaneously injected 7-11 point, accumulated dose 1.0-2.0mg/kg/ times, three weeks
After carry out booster immunization；
A-2, booster immunization：Same dorsal sc injection mode and injection measurement is taken to carry out injecting immune, it is different
It is incomplete Freund's adjuvant for adjuvant used in booster immunization, hereafter carries out a booster immunization every two weeks, middle week is in rabbit ear edge
Potency is surveyed in blood sampling, and potency reaches 1:Final booster immunization once, is taken a blood sample in arteria carotis after a week after 64000, and purifying obtains more grams
Grand antibody.
Antibody described in step a is selected from, but not limited to, anti-MFAP high specifics polyclonal antibody or goat anti-rabbit antibody.
PBS buffer solutions 0.01M, pH=7.4 in the b.
Mixing time described in step a is 2-4h.
Mixing time described in step b is 12-36h.
Sodium borohydride solution described in step c is now with the current, concentration 2mg/mL, pH=7.1.
Mixing time described in step c is 2-4h.
Further, preferable method is：
A, antibody 1-4mg is dissolved into 100-300 μ L acetate buffer solutions, adds 300-900 μ L sodium periodate solution,
2-4h is stirred in 4 DEG C of lucifuges, ultrafiltration removes unreacted sodium periodate, obtains oxidised antibody；
B, the immune labeled probe of the silica fluorescent based on fluorescence resonance energy transfer of the above-mentioned preparations of 2-10mg is used
It is distributed to after the cleaning of PBS buffer solutions in 3-6mL PBS buffer solutions；Oxidation prepared by step a is added under magnetic stirring to resist
Body, lucifuge stirs 12-36h at 4 DEG C；
C, 100-300 μ L sodium borohydride solutions are added and continue 4 DEG C of lucifuge stirring 2-4h, by the solution centrifuge washing after reaction
It is distributed in PBS buffer solutions, 4 DEG C store for future use.
The immune labeled probe of a kind of silica fluorescent based on fluorescence resonance energy transfer provided by the invention is used to examine
Survey the application of nano-medicament carrier.
The transfer characterization of energy in RB-BODIPY-FRET silica nanometer label probe systems：
The excitation of rhodamine B is emitted as 576nm for 530nm, and the excitation of BODIPY is emitted as 530nm for 470nm, due to
The emission peak of BODIPY and the excitation overlap of peaks of rhodamine B, can occur energy transfer between two kinds of dyestuffs, with swashing for BODIPY
When sending out wavelength excitation, the fluorescence intensity of its own can weaken, and the fluorescence intensity of rhodamine B can strengthen.Fluorescence resonance does not occur
The rhodamine B of energy transfer fluorescence caused by 470nm is extremely weak, strong with the rhodamine B fluorescence that energy transfer occurs under concentration
Degree can then strengthen very much.Therefore rhodamine B Nano particles of silicon dioxide and the RB-BODIPY-FRET with concentration are excited with 470nm
Silica nanometer label probe, does fluorogram and observes the change of its fluorescence intensity then provable whether energy transfer occurs.
RB-BODIPY-FRET silica nanometers label probe success coupled antibody characterization：
Fluorogram observation RB-BODIPY-FRET Nano particles of silicon dioxide and RB-BODIPY- are done with 470nm excitations
The change of emission peak positions after FRET Nano particles of silicon dioxide coupled antibodies.
The present invention uses fluorine boron pyroles and rhodamine B, because it is with good photochemistry physical property, rubs Ru higher
Your extinction coefficient, higher fluorescence quantum yield, spectral quality are highly stable etc.；Therefore in analytical chemistry, molecular biology etc.
More favored in field.Fluorescence resonance energy transfer technology has stronger antijamming capability than conventional fluorescent technology, can
The influence of scattering light is avoided, optical efficiency greatly improves, so as to fulfill oversoul sensitive detection.But with progress of research, it is desirable to examine
Survey sensitivity further to improve, fluorescent dye is used directly to mark, the fluorescence that labeling effciency is low, photostability is poor, relatively low
The shortcomings of intensity, limits its development and application.Fluorescent nano particles can exactly overcome the shortcomings that fluorescent dye by researcher institute
Value, become new most promising label probe.The present invention is nontoxic using silica fluorescent nanoparticle, water-soluble
Good, easily modification, has good biocompatibility；And mesoporous silica nano-particle preparation method is simple, specific surface area is big,
Fluorescent dye load factor is high；The photoluminescent property of dyestuff is remained unchanged with good good light stability in nano-particle.
Compared with prior art, the present invention has the following advantages：
(1) fluorescent nano particles overcome fluorescent dye and are used directly to mark, and labeling effciency is low, photostability is poor, relatively
The shortcomings of low fluorescence intensity；
(2) Nano particles of silicon dioxide is nontoxic, good water solubility, easily modification, has good biocompatibility；And mesoporous two
Silica nano particle preparation method is simple, and specific surface area is big, and fluorescent dye load factor is high；The fluorescence of dyestuff in nano-particle
Matter is remained unchanged with good good light stability；
(3) with fluorescent dye BODIPY and rhodamine B codope, fluorescence resonance energy can occur between two kinds of fluorescent dyes
Amount transfer, improves fluorescence intensity, weakens quenching；BODIPY is the energy donor in resonance system, and rhodamine B is energy acceptor,
BODIPY is excited, energy transfer can be promoted it to shine by the BODIPY in excitation state to rhodamine B, wide to have changed rhodamine B
Excites scope, compared with directly exciting rhodamine B, this energy transfer form can effectively weaken fluorescent quenching, improve glimmering
Luminous intensity.
(4) using sodium periodate oxidised antibody coupling fluorescence labeling probe, coupling efficiency is improved, eliminates crosslinking agent pair
The influence of antibody activity.Antibody glycosyl dihydroxy is oxidized to aldehyde radical using Over-voltage protection, with the amino knot on nano-probe
Close, not using crosslinking agents such as carbodiimides, glutaraldehydes, avoid influence of the crosslinking agent to antibody activity, and Conjugate ratio is high.
Two methods add 2mg antibody, 8mg label probes, and the ultraviolet two methods that are characterized as centrifuge gained supernatant after completion of the reaction
UV absorption figure, as can be seen from Figure 5 uses sodium periodate fado using the content ratio of antibody in the supernatant of glutaraldehyde method,
The amount ratio sodium periodate fado for the antibody not combined using glutaraldehyde method with label probe, is shown even using Over-voltage protection
Connection rate is high.
Brief description of the drawings
Fig. 1 is to excite rhodamine B Nano particles of silicon dioxide and embodiment 1 of the concentration as 0.004mmol/mL using 470nm
Each dye strength is the fluorescence hair of the RB-BODIPY-FRET Nano particles of silicon dioxide of 0.004mmol/mL before the mixing of preparation
Penetrate spectrogram；
Fig. 2 is to excite RB-BODIPY-FRET Nano particles of silicon dioxide and the RB- of the preparation of embodiment 2 with 470nm
BODIPY-FRET Nano particles of silicon dioxide is coupled the fluorescent emission spectrogram after anti-MFAP antibody；
Fig. 3 is using 470nm excitation concentration as the rhodamine B Nano particles of silicon dioxide of 0.005mmol/mL and is embodiment
The fluorescence hair for the RB-BODIPY-FRET Nano particles of silicon dioxide that each dye strength is 0.005mmol/mL before 3 mixing prepared
Penetrate spectrogram；
Fig. 4 is to excite RB-BODIPY-FRET Nano particles of silicon dioxide and the RB- of the preparation of embodiment 4 with 470nm
Fluorescent emission spectrogram after BODIPY-FRET Nano particles of silicon dioxide coupling goat anti-rabbit antibody；
The comparison that Fig. 5 is sodium iodate method and glutaraldehyde method is coupled；
Fig. 6 is BODIPY preparation methods used.
Embodiment
Embodiment 1
A kind of preparation method of the immune labeled probe of silica fluorescent based on fluorescence resonance energy transfer, method include with
Lower step：
1), at 65 DEG C by cetyl trimethylammonium bromide CTAB 50mg under magnetic stirring, be dissolved in and being gone containing 50mL
In ionized water；
2) 650 μ L ethylene glycol and 2.1mL ammonium hydroxide, are then added at 65 DEG C；
3) 200 μ L reactions 30min of ethyl orthosilicate TEOS, are added in the 5min after adding ethylene glycol and ammonium hydroxide；
4), 0.5mL 0.004mmol/mL BODIPY solution and 0.5mL 0.004mmol/mL rhodamine B solution are mixed
Close, the 1mL BODIPY mixed and the mixed solution of rhodamine B are added into step 3), add trimethoxy methyl silicon afterwards
30 μ L of alkane MTMS, react 2h；
5) 3- aminopropyl triethoxysilanes APTES the reaction was continued the 1.5h of 90 μ L mL, are added；
Stirring is cooled to room temperature simultaneously centrifuge washing 3 times after completing reaction, is freeze-dried to obtain RB-BODIPY-FRET titanium dioxides
Silicon nano, the i.e. immune labeled probe of the silica fluorescent based on fluorescence resonance energy transfer, lucifuge are sealed.
The transfer characterization of energy in RB-BODIPY-FRET silica nanometer label probe systems：
Each dyestuff before exciting concentration as the rhodamine B Nano particles of silicon dioxide of 0.004mmol/mL and mixing using 470nm
Concentration is the RB-BODIPY-FRET Nano particles of silicon dioxide of 0.004mmol/mL.From figure 1 it appears that add with concentration
Enter the fluorescence that rhodamine B after BODIPY is sent to be remarkably reinforced.
BODIPY concrete structures used are Fig. 6 products, and specific preparation process is as follows：
A, 2mL pyrroles is added at 25 DEG C while stirring in the round-bottomed flask containing 50mL water, 1.5mL mesitylene formaldehyde,
Stir；
B, the dense HCl of 0.5mL are added, and 24h is reacted in 25 DEG C, there are a large amount of black precipitates to generate, is washed after suction filtration with petroleum ether
Wash, obtain crude product powder 1；
C, take 500mg crude products powder 1 to be dissolved in 50mL anhydrous tetrahydro furans, add N- chloros fourth two under magnetic stirring
Acid imide NCS 507mg, 25 DEG C of reaction 2h.
D, add mixture to after completion of the reaction in 200mL water, with the extraction of 50mL dichloromethane three times, the anhydrous sulphur of 15g
Sour sodium drying, decompression are spin-dried for.
E, 50mL dichloromethane is added in the product after being spin-dried for fully to dissolve, it is chloro- to add 2,3- bis- under magnetic stirring
5,6- dicyan 1,4-benzoquinone DDQ 517mg react 10min, add 1.5mL triethylamine react 1h afterwards,
F, boron trifluoride ether 2.5mL reaction 2h are eventually adding, after completion of the reaction with the extraction of 50mL dichloromethane three times,
15g anhydrous sodium sulfates are dried, and decompression is spin-dried for, through silica gel column chromatography separating-purifying (300-400 mesh silica whites, petroleum ether/dichloromethane
Alkane=10/1, V/V) obtain BODIPY.
Embodiment 2
A kind of application of the immune labeled probe conjugate antibody of silica fluorescent based on fluorescence resonance energy transfer, specifically
Method is：
1st, anti-MFAP high specifics polyclonal antibody is first prepared：
1-1, first immunisation：Immunogene is used with Freund's complete adjuvant in equal volume than mixing after being emulsified with syringe
New zealand white rabbit is immunized in dorsal sc injection mode.10 points, accumulated dose 1.0mg/kg/ times is subcutaneously injected.Carry out after three weeks
Booster immunization；
1-2, booster immunization：Same dorsal sc injection mode and injection measurement is taken to carry out injecting immune, it is different
It is incomplete Freund's adjuvant for adjuvant used in booster immunization.Hereafter a booster immunization is carried out every two weeks, and middle week is in rabbit ear edge
Potency is surveyed in blood sampling, and potency reaches 1:Final booster immunization once, is taken a blood sample in arteria carotis after a week after 64000, and purifying is resisted
MFAP high specific polyclonal antibodies.
2nd, RB-BODIPY-FRET silica nanometers label probe is coupled anti-MFAP high specifics using Over-voltage protection
Polyclonal antibody：
2-1, the anti-MFAP high specifics polyclonal antibody 2mg for preparing step 1-2 are dissolved into 300 μ L acetate buffer solutions
In (0.05M, pH=4.6), it is 2mg/mL to add 700 μ L concentration, and pH=4.6 sodium periodate solution, is stirred in 4 DEG C of lucifuges
2h, removes unreacted sodium periodate for 0.45 μm of ultrafiltration through membranes with filter membrane, obtains oxidised antibody；
2-2, RB-BODIPY-FRET Nano particles of silicon dioxide PBS (0.01M, the pH=for preparing 8mg embodiments 1
7.4) it is distributed to after cleaning 3 times in 3mL PBS buffer solutions in (0.01M, pH=7.4).Step 2-1 is added under magnetic stirring
The oxidised antibody of preparation, lucifuge stirs 20h at 4 DEG C；
2-3, then, it be 2mg/mL to add 100 μ L concentration, and pH=7.1 sodium borohydride solutions continue 4 DEG C of lucifuges and stir 2h,
Solution centrifuge washing after reaction is distributed in PBS buffer solutions, you can, 4 DEG C store for future use.
RB-BODIPY-FRET silica nanometers label probe success coupled antibody characterization：
RB-BODIPY-FRET Nano particles of silicon dioxide and RB-BODIPY-FRET silica nanometers are excited with 470nm
Particle is coupled the fluorescent emission spectrogram after anti-MFAP antibody.As can be seen from Figure 2 RB-BODIPY-FRET silica nanometers
Particle emission peak emission peak after 576nm, the anti-MFAP antibody of RB-BODIPY-FRET Nano particles of silicon dioxide coupling exists
582nm, the generation peak position for being coupled rhodamine B after anti-MFAP antibody move 6nm to long wave direction, show successfully to be coupled anti-
Body.
Embodiment 3
A kind of preparation method of the immune labeled probe of silica fluorescent based on fluorescence resonance energy transfer, including it is following
Step：
1), cetyl trimethylammonium bromide CTAB 50mg are dissolved in containing in 50mL deionized waters at 65 DEG C；
2) 650 μ L ethylene glycol and 2.1mL ammonium hydroxide, are then added at 65 DEG C；
3) 200 μ L reactions 30min of ethyl orthosilicate TEOS, are added in the 5min after adding ethylene glycol and ammonium hydroxide；
4), 0.5mL 0.005mmol/mL BODIPY solution and 0.5mL 0.005mmol/mL rhodamine B solution are mixed
Close, the 1mL BODIPY mixed and the mixed solution of rhodamine B are added into step 3), add trimethoxy methyl silicon afterwards
Alkane MTMS30 μ L, react 2h；
5) 3- aminopropyl triethoxysilanes APTES the reaction was continued the 1.5h of 90 μ L mL, are added；
Stirring is cooled to room temperature simultaneously centrifuge washing 3 times after completing reaction, is freeze-dried to obtain RB-BODIPY-FRET titanium dioxides
Silicon nano, the i.e. immune labeled probe of the silica fluorescent based on fluorescence resonance energy transfer, lucifuge are sealed.
The transfer characterization of energy in RB-BODIPY-FRET silica nanometer label probe systems：
Each dyestuff before exciting concentration as the rhodamine B Nano particles of silicon dioxide of 0.005mmol/mL and mixing using 470nm
Concentration is the RB-BODIPY-FRET Nano particles of silicon dioxide of 0.005mmol/mL.From figure 3, it can be seen that add with concentration
Enter the fluorescence that rhodamine B after BODIPY is sent to be remarkably reinforced.
Embodiment 4
A kind of application of the immune labeled probe conjugate antibody of silica fluorescent based on fluorescence resonance energy transfer, specifically
Method is：
Goat anti-rabbit antibody used is directly commercially available for Shanghai Sheng Gong bioengineering Co., Ltd in this example.
1st, goat anti-rabbit antibody 2mg is dissolved into 300 μ L acetate buffer solutions (0.05M, pH=4.6), it is dense adds 700 μ L
Spend for 2mg/mL, pH=4.6 sodium periodate solution, 2h are stirred in 4 DEG C of lucifuges, removed with filter membrane for 0.45 μm of ultrafiltration through membranes not anti-
The sodium periodate answered, obtains oxidised antibody；
2nd, the RB-BODIPY-FRET Nano particles of silicon dioxide PBS buffer solutions for preparing the glimmering embodiments 3 of 8mg
(0.01M, pH=7.4) is distributed in 3mL PBS buffer solutions (0.01M, pH=7.4) after cleaning 3 times；Under magnetic stirring plus
Entering the goat anti-rabbit antibody after oxidation, lucifuge stirs 20h at 4 DEG C,
3rd, it is 2mg/mL to add 100 μ L concentration, and pH=7.1 sodium borohydride solutions continue 4 DEG C of lucifuge stirring 2h, after reaction
Solution centrifuge washing be distributed in PBS, 4 DEG C store for future use.
RB-BODIPY-FRET silica nanometers label probe success coupled antibody characterization：
RB-BODIPY-FRET Nano particles of silicon dioxide and RB-BODIPY-FRET silica nanometers are excited with 470nm
Fluorescent emission spectrogram after particle coupling goat anti-rabbit antibody.As can be seen from Figure 4 RB-BODIPY-FRET silica nanometers
Particle emission peak emission peak after 576nm, the anti-MFAP antibody of RB-BODIPY-FRET Nano particles of silicon dioxide coupling exists
584nm, the generation peak position of rhodamine B moves 8nm to long wave direction after being coupled goat anti-rabbit antibody, shows successfully to be coupled anti-
Body.
Above-mentioned reference embodiment is to a kind of immune labeled probe of the silica fluorescent based on fluorescence resonance energy transfer
Prepare and using the detailed description carried out, be illustrative rather than limited, can not therefore be interpreted as to the present invention
The limitation of scope, can include several embodiments, as long as in the case where not departing from present general inventive concept according to limited scope
With replacing or equivalent transformation changes and modifications, should all be included within protection scope of the present invention.
Claims (10)
1. a kind of preparation method of the immune labeled probe of silica fluorescent based on fluorescence resonance energy transfer, its feature exist
In the preparation method comprises the following steps：
1) cetyl trimethylammonium bromide CTAB, is dissolved in deionized water；
2) ethylene glycol and ammonium hydroxide, are added；
3) ethyl orthosilicate TEOS reactions, are added afterwards；
4) the BODIPY solution and rhodamine B solution mixed, then, is added, adds trimethoxymethylsila,e MTMS,
Reaction；
5), adding 3- aminopropyl triethoxysilanes APTES, the reaction was continued；Centrifuge washing, freeze-drying, obtains RB-BODIPY-
FRET Nano particles of silicon dioxide, the as immune labeled probe of the silica fluorescent based on fluorescence resonance energy transfer.
2. preparation method according to claim 1, it is characterised in that since step 1), whole process is in 60-70 DEG C of condition
Lower reaction.
3. preparation method according to claim 1 or 2, it is characterised in that 3- after ethylene glycol and ammonium hydroxide is added in step 3)
Ethyl orthosilicate TEOS is added in 6min；Reaction time is 30-60min.
4. according to claim 1-3 any one of them preparation methods, it is characterised in that the step 4) reaction time is 2-
4h。
5. according to the preparation method described in claim 1-4, it is characterised in that the step 5) reaction time is 1-2h.
6. according to the preparation method described in claim 1-4, it is characterised in that BODIPY solution concentrations are before step 4) mixing
0.001mmol/mL-0.006mmol/mL；Rhodamine B solution concentration is 0.001mmol/mL-0.006mmol/mL before mixing；It is mixed
BODIPY and rhodamine B molar ratio are 1 after conjunction:1.
7. according to the preparation method described in claim 1-6, it is characterised in that the preparation method specifically includes following steps：
1), cetyl trimethylammonium bromide CTAB 0.03-0.08g are dissolved under the conditions of magnetic agitation at 60-70 DEG C
In 30-70mL deionized waters；
2) 0.4-0.7mL ethylene glycol and 2-3mL ammonium hydroxide, are then added at 60-70 DEG C；
3) ethyl orthosilicate TEOS 100-200 μ L reactions 30-60min is added in the 3-6min, added after ethylene glycol and ammonium hydroxide；
4) 1ml BODIPY solution and the mixed solution and 0.02-0.05mL trimethoxymethylsila,es of rhodamine B, are added
MTMS, reacts 2-4h；
5) 3- aminopropyl triethoxysilanes APTES the reaction was continued the 1-2h of 0.06-0.1mL, are added；
Stir and be cooled to room temperature after completing reaction, centrifuge washing 2-4 times, be freeze-dried RB-BODIPY-FRET silica is received
Rice corpuscles, lucifuge are sealed.
8. a kind of be prepared the silica based on fluorescence resonance energy transfer using any one of claim 1-7 the method
Fluorescence immunoassay label probe.
9. the immune labeled probe conjugate of the silica fluorescent based on fluorescence resonance energy transfer described in a kind of claim 8 resists
The application of body.
10. application according to claim 9, it is characterised in that concrete application method is：
A, antibody is dissolved into acetate buffer solution, adds sodium periodate solution, aoxidized in 4 DEG C of lucifuge stirrings, ultrafiltration
Unreacted sodium periodate is removed, obtains oxidised antibody；
B, will be distributed to after the immune labeled probe cleaning of the silica fluorescent based on fluorescence resonance energy transfer of above-mentioned preparation
In PBS buffer solutions；Oxidised antibody prepared by step a is added under agitation, and lucifuge stirs at 4 DEG C；
C, it is last, add sodium borohydride solution and continue 4 DEG C of lucifuge stirrings, by the solution centrifuge washing after reaction, be distributed to PBS and delay
Rush in solution, 4 DEG C store for future use.
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