CN103033618A - Novel synthesis method of polyfluorene derivative and application in immunosensor for detecting tumor marker - Google Patents

Novel synthesis method of polyfluorene derivative and application in immunosensor for detecting tumor marker Download PDF

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CN103033618A
CN103033618A CN2012105771552A CN201210577155A CN103033618A CN 103033618 A CN103033618 A CN 103033618A CN 2012105771552 A CN2012105771552 A CN 2012105771552A CN 201210577155 A CN201210577155 A CN 201210577155A CN 103033618 A CN103033618 A CN 103033618A
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dibromo
reaction
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颜梅
楚成超
葛慎光
葛磊
于京华
黄家栋
李蒙
李龙
刘芳
刘伟艳
王衍虎
张彦
王盼盼
李伟平
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University of Jinan
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Abstract

The invention discloses a novel synthesis method of a polyfluorene derivative and an application in a flow injection fluorescent sensor for a tumor marker. The synthesis method of the polyfluorene derivative comprises the following steps that: 1,4-dibromo p-phenylenediamine (a compound 1) is synthesized; 2,7-dibromo-9,9-bi(3'-(N,N'-dimethylamino) propyl)-fluorene (a compound 2) is synthesized; p-vinyltoluene (a compound 3) is synthesized; the three monomers are polymerized into the polyfluorene derivative by a Heck reaction. A fabrication method of the sensor comprises the following steps that polymethyl methacrylate is adopted to fabricate a flow injection fluorescent flow cell; ferroferric oxide magnetic particles and gold nanoparticles are prepared and assembled into gold covered ferroferric oxide; a primary antibody is modified on gold cladded ferroferric oxide; the polyfluorene derivative is subjected to silicon cladding treatment; a secondary antibody is modified on the surface of the silicon cladded polyfluorene derivative; an immune reaction is conducted; and flow injection fluoroscopic detection is conducted on the tumor marker. The sensor is strong in specificity, high in sensitivity, simple to operate, and low in detecting line.

Description

A kind of applied technical field of synthetic and detection tumor markers immunosensor of novel poly-fluorene derivative
The present invention relates to the synthetic and technical field of immunoassay of immune marker, say more specifically a kind of synthetic basis of novel poly-fluorene derivative and utilize this novel poly-fluorene derivative to be the method for the flow injection fluorescent optical sensor of immune marker.
Background technology
Conjugated polymer is the polymkeric substance that has long-range π key conjugated system on the class formation.Grip altogether the polymkeric substance development so far, its kind is more, mainly contains polyhenylene (PPP), polyphenylene vinylene (PPV), poly-second fast (PA), poly-saliva fen (PT), polyaniline (PANI), polyphenylene sulfide (PPS), gathers to adjoin and cough up (PPY), polydiacetylene (PDA) equiconjugate polymkeric substance.Conjugated polymer generally is easy to and has an effect with group or the material of receptor property, thereby so that physics, the chemical property of these polymkeric substance itself change, generates the material that some have specific function.The conjugated polymer covalent bond consists of chain structure, and the HOMO energy is higher, and the electronics that is stimulated can be along whole strand delocalization on the pi-conjugated passage that produces, and the molecular chain structure of conjugated polymer has had the feature of " molecular wire ".Since the self-conjugate polymkeric substance has the Fluorescence amplification effect and is found, the fast development of conjugated polymer Fluorescence sensitized material, concrete application mainly concentrates on ion detection, and organic molecule detection and biological systems detection etc. have deep development potentiality and wide application prospect.
A kind of flat crystal with fluorescence is arranged in coal tar, and Berthelot is with its called after fluorenes (fluorene).Because 9,2 of fluorene structural units and 7 carbon are easy decorating sites, not only can be modified into the derivant of fluorenes by these easy decorating sites, and can synthesize poly-fluorenes.Poly-fluorenes (PF) is a kind of compound of rigid plane biphenyl structural, itself has higher light and thermally stable.Because these characteristics that poly-fluorenes has, scientist begins character and the function of poly-fluorenes are carried out deep research.Through people's unremitting effort, poly-fluorenes and derivant thereof synthetic had obvious progress.The solid film of poly-fluorenes and derivant thereof has higher fluorescence quantum yield about 60%～80%, and band gap is greater than 2.90 eV, and therefore its fluorescence emission peak is considered to the most promising blue emitting material about 450 nm.And it also has good chemical stability, thermal stability and dissolubility, easily adjustable colors and good cavity transmission ability.Poly-fluorenes can also obtain lighting function material adjustable in whole visible-range by methods such as copolymerization, blend and modifications.Fluorenes class chromophore is widely applied at aspects such as the adjustment of the blue light electroluminescence material of efficient stable, white light material, multifunction, organic laser/material of main part, phase structure and organic nano luminescent materials, becomes gradually the star molecule in organic electronics field.
In recent years, malignant tumour namely cancer become a serious threat of human health and life security.Detection to tumor markers is called immunoassay.Immunosensor is the specific immune response that utilizes between antigen and the antibody, and antibody is that up to a hundred amino acid molecular high-sequentials are arranged the biomacromolecule that forms.When immune system is exposed to antigenic substance, have in the antibody that pair antigenic structure is identified, the position of combination, according to " spoon-lock " model, antibody can its corresponding antigen height single-minded ground combination.The mensuration of the early diagnosis of cancer and low content antigen had great importance also become numerous analysts' important pursuit.And along with the development of modern biosensor technology, immunoassay is widely used in fields such as Micro biological Tests, Pharmaceutical Analysis, clinical diagnosis, food security and environmental monitorings.The complex operations such as traditional immuno analytical method enzyme linked immunosorbent assay, radiommunoassay, the general length consuming time of colloid gold immune technology and application of sample, incubation, washing, and the general instruments that need to be valuable such as new-type immune analysis method high performance liquid chromatography chemiluminescence immune assay, fluidic chip chemiluminescence immunoassay, capillary electrophoresis chemiluminescence immunoassay are unfavorable for popularizing.In the nineties, a kind of novel immunoassay---flow-injection immunoassay has appearred both at home and abroad.Flow-injection immunoassay combines the characteristics of flow injection and immunoassay, can utilize flow velocity, the auto injection of computer control damping fluid, detection and the data of detecting device to process, and has advantages of very outstanding at immunoassay.Fluorescence analysis is a kind of nondestructive, highly sensitive analytical approach.Just combine both advantages by the fluoroimmunoassay of flow injection, necessarily have preferably application prospect.
Summary of the invention
The technical problem to be solved in the present invention is to have synthesized a kind of poly-fluorene derivative of blue light-emitting, and utilize its as signaling molecule provide a kind of highly sensitive, detection speed is fast, reagent dosage is few, detects the flow injection fluorescent optical sensor of tumor markers.
In order to solve the problems of the technologies described above, the present invention realizes by following measures: a kind of preparation method who synthesizes and be used for detecting the flow injection fluorescent optical sensor of tumor markers of poly-fluorene derivative, and it may further comprise the steps:
(1) synthetic (PF-NH of poly-fluorene derivative 2);
(2) the poly-fluorene derivative (PF-NH of synthetic silica coating 2@SiO 2);
(3) with the two anti-PF-NH that are modified at 2@SiO 2
(4) prepare golden nanometer particle (AuNPs) according to existing method, Fe 3 O 4 magnetic particle (Fe 3O 4) tri-iron tetroxide (Fe that coats of golden nanometer particle 3O 4@Au);
(5) primary antibodie is modified at Fe 3O 4@Au surface;
(6) utilize polymethylmethacrylate (PMMA) preparation flow injection fluorescence flow cell;
(7) chemiluminescence sensor of making is detected immune marker in conjunction with the portable injection chemiluminescence instrument.
The preparation of the poly-fluorene derivative of coated with silica of the present invention and the tri-iron tetroxide of coated with silica may further comprise the steps:
(1) Isosorbide-5-Nitrae-dibromo p-phenylenediamine (PPD) (compound 1)
(a) in nitrogen environment, add 250 mL anhydrous tetrahydro furans (THF), and then add 4.2 g sodium hydrides (NaH), add 5.17 g and add hot reflux 1 h after to diphenylamine.Be cooled to and add 25 g dimethyl dicarbonate butyl esters after the room temperature, add 4.2 g NaH after stirring 30 min, 12 h reflux.Add large water gaging and make too much reactant hydrolysis, then use a large amount of ethyl acetate extractions, organic layer anhydrous magnesium sulfate (MgSO 4) drying, filter, decompression distillation, crystallization obtains the N of white, N '-2(tertbutyloxycarbonyl in ethyl acetate)-Isosorbide-5-Nitrae-diaminobenzene.
(b) in nitrogen environment, add 9 mL absolute ethers and the anhydrous THF of 9 mL, add (1)-(a) gained N, N '-2(tertbutyloxycarbonyl)-and Isosorbide-5-Nitrae-diaminobenzene, be cooled to-45 ℃, the pentane solution that dropwise adds the tert-butyl lithium of 25 mL, 1.3 M, stir 3 h at-45 ℃, then dropwise add 2 mL, one chlorine trimethyl silane at-55 ℃, reaction room temperature stirring reaction 12 h.Add the hydrochloric acid solution of 10 mL 10% in the reactant liquor, use ethyl acetate extraction, organic layer MgSO 4Drying is filtered, and decompression distillation is crossed post and can be got white N, N '-2(tertbutyloxycarbonyl)-Isosorbide-5-Nitrae-diamido-2, the 5-2(TMS) benzene.
(c) in nitrogen environment, add anhydrous methylene chloride, add (1)-(b) gained N, N '-2(tertbutyloxycarbonyl)-Isosorbide-5-Nitrae-diamido-2, the 5-2(TMS) benzene, add 1.53 g NBS, reaction adds 5 mL water at stirring at room 3 h again, use dichloromethane extraction, organic layer MgSO 4Drying is filtered, decompression distillation, recrystallization can get white N, N '-2(tertbutyloxycarbonyl)-Isosorbide-5-Nitrae-diamido-2, the 5-2 bromobenzene.
(d) in nitrogen environment, add 10 mL trifluoroacetic acids and 10 mL anhydrous methylene chlorides in device, add (1)-(c) gained N, N '-2(tertbutyloxycarbonyl)-Isosorbide-5-Nitrae-diamido-2,5-2 bromobenzene, stirring at room 12 h, then decompression distillation, evaporate to dryness.To wherein adding 5 mL anhydrous methylene chlorides, add again without saturation of the air sodium bicarbonate solution, control pH 8～9 stirs 3 h, with the dichloromethane extraction of deacration, merges organic layer, uses MgSO 4Drying, decompression distillation, recrystallization obtains Isosorbide-5-Nitrae-dibromo p-phenylenediamine (PPD).
2) 2,7-two bromo-9,9-two (3 '-(N, N '-dimethylamino) propyl group)-fluorenes (compound 2) synthetic
In nitrogen environment, add 4 g 2,7-dibromo fluorenes and 80 mg tetrabutyl ammonium bromide add 60 mL dimethylene sulfoxides (DMSO), directly add 8 ml, 50% NaOH (NaOH) solution.The hydrochloride ((CH of 5 g 3-dimethylaminopropyl chlorine will be dissolved with 3) 2N (CH 2) 3ClHCl) solution joins 20 mL(DMSO) and be added drop-wise in the potpourri, 12 h stirred.The water that adds 50 mL in the reactant liquor, dissolving residue salt.With product extraction, the washing that obtains.The anhydrous MgSO of solution after the washing 4Drying, filtration under diminished pressure revolves steaming and desolventizes, and obtains yellow 2,7-, two bromo-9,9-two (3 '-(N, N '-dimethylamino) propyl group)-fluorenes.
(3) p-divinyl benzene (compound 3) is synthetic
(a) in nitrogen environment, in 100 mL three-neck flasks, add 5.8 g N-bromosuccinimides (NBS), 0.10 g benzoyl peroxide adds 2.0 mL P-xylene and 60 mL phenixin (CCl again 4).Magnetic stirrer to all solids dissolves.Add hot reflux 12 h.After stopping reaction, filter while hot, washing precipitation is carried out recrystallization after the filtrate cooling, obtains white, needle-shaped crystals, namely to dibromo xylene.
B) in nitrogen environment, in 100 mL three-neck flasks, add 4.94 g(3)-(a) make dibromo xylene and 9.0 g triphenyl phosphorus, add again 30 mL anhydrous methylene chlorides and 30 mL formaldehyde.Filtration, washing precipitation obtain white powder to the quaternary alkylphosphonium salt of dibromo xylene.
(c) in nitrogen environment, (3)-(b) gained is transferred to the quaternary alkylphosphonium salt of dibromo xylene in the three-neck flask of 250 mL, add again 30 mL anhydrous methylene chlorides and 30 mL formaldehyde.Dropwise drip 20 mL, 5% NaOH solution in reaction solution, reaction stirred 12 h stop reaction.The extracting and washing precipitation, anhydrous MgSO 4Dried overnight.Remove solvent under reduced pressure, obtain the white needles p-divinyl benzene.
(4) poly-fluorene derivative (PF-NH 2) synthetic
In nitrogen environment, in 100 mL three-neck flasks, add successively 0.19g(1) make 1,4-dibromo p-phenylenediamine (PPD), 0.10 g (2) makes 2,7-two bromo-9,9-two (3 '-(N, N '-dimethylamino) propyl group)-fluorenes, 0.10 g(3) makes the palladium (Pd (OAc) of divinylbenzene, 25 mg 2), the triphenyl phosphorus (PPh of 0.3 g 3), 60 mL DMF, 50mL triethylamine, add hot reflux 48 h at 150 ℃.After reaction stops, using chloroform extraction, organic layer MgSO 4Drying is filtered, and decompression distillation obtains the novel poly-fluorene derivative (PF-NH of brown color 2).
(5) PF-NH that (4) is prepared 2Obtain solution (1 nM is dissolved among the DMF).In the single port bottle of 100 mL, add 1 mL PF-NH 2Solution, 8 mL ethanol, dispersed with stirring is even.Then add 0.1 mL ammonia spirit (25%), the tetraethyl orthosilicate solution of 10 μ L (TEOS, the TEOS ethanolic solution of volumetric concentration 1/20) stirs 30 min.Continue to add 10 μ L TEOS solution and stir 30 min until the TEOS solution total amount that adds is 50 μ L.After reaction finishes, at 10000 rmin -1The ethanol washing precipitation is used in centrifuging under the rotating speed, three times repeatedly.In vacuum drying chamber, obtain the poly-fluorene derivative (PF-NH of coated with silica with 50 ℃ of drying 5 h 2@SiO 2).
(6) in the small beaker of 10 mL, add the PF-NH that (5) prepare 2@SiO 20.02 g, with the freshly prepd N of 1 mL, N '-carbonyl dimidazoles solution (CDI, 100 mgmL -1, be dissolved in DMSO) add and stir 4 h.After reacting completely, adding excessive intermediate water is that excessive CDI decomposes, at 10000 rmin -1The intermediate water washing precipitation is used in centrifuging under the rotating speed, three times repeatedly.With the PF-NH that modifies 2@SiO 2Be dissolved in (PBS, pH 7.4) in the phosphate buffer solution, add a certain amount of two anti-(Ab 2) original solution and activation 2 h under 4 ℃.At 10000 rmin -1Centrifuging under the rotating speed adds 1 mL bovine serum albumin(BSA) (BSA, 1%) at 4 ℃ of lower activation 2 h.At 10000 rmin -1The PBS washing precipitation is used in centrifuging under the rotating speed, three times repeatedly.In precipitation, add at last PBS to Ab 2Concentration is 20 μ gmL -1
(7) utilize classic method to prepare golden nanometer particle (AuNPs) and Fe 3 O 4 magnetic particle (Fe 3O 4) iron.In the round-bottomed flask of 100 mL, add 0.1 g Fe 3O 4Nano particle adds 10 mL ethanol, then adds 0.5 mL APTES and magnetic agitation 1 h, separates with magnet, washes with intermediate water.Add 1 mL AuNPs(1%), add 10 mL and magnetic agitation 2 h.Separate with magnet, wash with intermediate water.In vacuum drying chamber, namely get the tri-iron tetroxide (Fe that golden nanometer particle coats with 50 ℃ of drying 10 h 3O 4@Au).
(8) with (7) Fe 3O 4@Au prepares particle 0.01 g and is dissolved among the PBS, adds a certain amount of primary antibodie (Ab 1) original solution and activation 2 h under 4 ℃.Separate with magnet, add 1 mL BSA solution, at 4 ℃ of lower activation 2 h.Separate with magnet, use the PBS washing precipitation, three times repeatedly.In precipitation, add at last PBS to Ab 1Concentration is 20 μ gmL -1
(9) adding 10 μ L(7 in the centrifuge tube of the 1 mL) Ab of preparation 1Then solution add the certain density antigens of 10 μ L (Ag) solution, and at 4 ℃ of lower activation 2 h.Add 10 μ L(6) preparation Ab 2Solution, and at 4 ℃ of lower activation 2 h.Separate with magnet, use the careful absorption supernatant of imbibition rifle, and wash three times repeatedly with PBS.
(10) utilize the fluorescence signal of gained material in the Flow Injection/Chemiluminescene Determination (9).
Tumor markers of the present invention is human chorionic gonadotrophin (HCG) CA19-9, alpha-fetoprotein (AFP), carcinomebryonic antigen.
Beneficial effect of the present invention:
(1) the present invention utilizes and has synthesized a kind of novel poly-fluorene derivative, and has showed preferably blue light, and fluorescence efficiency is just high, has preferably using value.Process by this poly-fluorene derivative being wrapped silicon, improved biocompatibility, reduced the impact of the toxicity of organism own.
(2) tri-iron tetroxide that utilizes the gold coating can well be tested and control as base material.
(3) to carry out measurement operation simple fast for the method for portable injection chemiluminescence, avoided the impact of subjective factor, and ensured good repeatability, is convenient to Site Detection.
Description of drawings
Fig. 1 is the synthetic schemes of compound 1.
Fig. 2 is compound 2,3 synthetic schemes.
Fig. 3 is poly-fluorene derivative PF-NH 2Synthetic schemes.
Fig. 4 A is the modification flow process of primary antibodie; B is two anti-modification flow processs; C is the overhaul flow chart of sensor.
Embodiment
A kind of preparation method of flow injection fluorescent optical sensor of detection immune marker, it may further comprise the steps:
(1) synthetic (PF-NH of poly-fluorene derivative 2);
(2) the poly-fluorene derivative (PF-NH of synthetic silica coating 2@SiO 2);
(3) with the two anti-PF-NH that are modified at 2@SiO 2
(4) prepare golden nanometer particle (AuNPs) according to existing method, Fe 3 O 4 magnetic particle (Fe 3O 4) tri-iron tetroxide (Fe that coats of golden nanometer particle 3O 4@Au);
(5) primary antibodie is modified at Fe 3O 4@Au surface;
(6) utilize polymethylmethacrylate (PMMA) preparation flow injection fluorescence flow cell;
(7) chemiluminescence sensor of making is detected immune marker in conjunction with the portable injection chemiluminescence instrument.
The preparation of the poly-fluorene derivative of coated with silica of the present invention and the tri-iron tetroxide of coated with silica may further comprise the steps:
(1) Isosorbide-5-Nitrae-dibromo p-phenylenediamine (PPD) (compound 1)
(a) in nitrogen environment, add 250 mL anhydrous tetrahydro furans (THF), and then add 4.2 g sodium hydrides (NaH), add 5.17 g and add hot reflux 1 h after to diphenylamine.Be cooled to and add 25 g dimethyl dicarbonate butyl esters after the room temperature, add 4.2 g NaH after stirring 30 min, 12 h reflux.Add large water gaging and make too much reactant hydrolysis, then use a large amount of ethyl acetate extractions, organic layer anhydrous magnesium sulfate (MgSO 4) drying, filter, decompression distillation, crystallization obtains the N of white, N '-2(tertbutyloxycarbonyl in ethyl acetate)-Isosorbide-5-Nitrae-diaminobenzene.
(b) in nitrogen environment, add 9 mL absolute ethers and the anhydrous THF of 9 mL, add (1)-(b) gained N, N '-2(tertbutyloxycarbonyl)-and Isosorbide-5-Nitrae-diaminobenzene, be cooled to-45 ℃, the pentane solution that dropwise adds the tert-butyl lithium of 25 mL, 1.3 M, stir 3 h at-45 ℃, then dropwise add 2 mL, one chlorine trimethyl silane at-55 ℃, reaction room temperature stirring reaction 12 h.Add the hydrochloric acid solution of 10 mL 10% in the reactant liquor, use ethyl acetate extraction, organic layer MgSO 4Drying is filtered, and decompression distillation is crossed post and can be got white N, N '-2(tertbutyloxycarbonyl)-Isosorbide-5-Nitrae-diamido-2, the 5-2(TMS) benzene.
(c) in nitrogen environment, add anhydrous methylene chloride, add (1)-(b) gained N, N '-2(tertbutyloxycarbonyl)-Isosorbide-5-Nitrae-diamido-2, the 5-2(TMS) benzene, add 1.53 g NBS, reaction adds 5 mL water at stirring at room 3 h again, use dichloromethane extraction, organic layer MgSO 4Drying is filtered, decompression distillation, recrystallization can get white N, N '-2(tertbutyloxycarbonyl)-Isosorbide-5-Nitrae-diamido-2, the 5-2 bromobenzene.
(d) in nitrogen environment, add 10 mL trifluoroacetic acids and 10 mL anhydrous methylene chlorides in device, add (1)-(c) gained N, N '-2(tertbutyloxycarbonyl)-Isosorbide-5-Nitrae-diamido-2,5-2 bromobenzene, stirring at room 12 h, then decompression distillation, evaporate to dryness.To wherein adding 5 mL anhydrous methylene chlorides, add again without saturation of the air sodium bicarbonate solution, control pH 8～9 stirs 3 h, with the dichloromethane extraction of deacration, merges organic layer, uses MgSO 4Drying, decompression distillation, recrystallization obtains Isosorbide-5-Nitrae-dibromo p-phenylenediamine (PPD).
(2) 2,7-two bromo-9,9-two (3 '-(N, N '-dimethylamino) propyl group)-fluorenes (compound 2) synthetic
In nitrogen environment, add 4 g 2,7-dibromo fluorenes and 80 mg tetrabutyl ammonium bromide add 60 mL dimethylene sulfoxides (DMSO), directly add 8 ml, 50% NaOH (NaOH) solution.The hydrochloride ((CH of 5 g 3-dimethylaminopropyl chlorine will be dissolved with 3) 2N (CH 2) 3ClHCl) solution joins 20 mL(DMSO) and be added drop-wise in the potpourri, 12 h stirred.The water that adds 50 mL in the reactant liquor, dissolving residue salt.With product extraction, the washing that obtains.The anhydrous MgSO of solution after the washing 4Drying, filtration under diminished pressure revolves steaming and desolventizes, and obtains yellow 2,7-, two bromo-9,9-two (3 '-(N, N '-dimethylamino) propyl group)-fluorenes.
(3) p-divinyl benzene (compound 3) is synthetic
(a) in nitrogen environment, in 100 mL three-neck flasks, add 5.8 g N-bromosuccinimides (NBS), 0.10 g benzoyl peroxide adds 2.0 mL P-xylene and 60 mL phenixin (CCl again 4).Magnetic stirrer to all solids dissolves.Add hot reflux 12 h.After stopping reaction, filter while hot, washing precipitation is carried out recrystallization after the filtrate cooling, obtains white, needle-shaped crystals, namely to dibromo xylene.
(b) in nitrogen environment, in 100 mL three-neck flasks, add 4.94 g(3)-(a) make dibromo xylene and 9.0 g triphenyl phosphorus, add again 30 mL anhydrous methylene chlorides and 30 mL formaldehyde.Filtration, washing precipitation obtain white powder to the quaternary alkylphosphonium salt of dibromo xylene.
(c) in nitrogen environment, (3)-(b) gained is transferred to the quaternary alkylphosphonium salt of dibromo xylene in the three-neck flask of 250 mL, add again 30 mL anhydrous methylene chlorides and 30 mL formaldehyde.Dropwise drip 20 mL, 5% NaOH solution in reaction solution, reaction stirred 12 h stop reaction.The extracting and washing precipitation, anhydrous MgSO 4Dried overnight.Remove solvent under reduced pressure, obtain the white needles p-divinyl benzene.
(4) poly-fluorene derivative (PF-NH 2) synthetic
In nitrogen environment, in 100 mL three-neck flasks, add successively 0.19g(1) make 1,4-dibromo p-phenylenediamine (PPD), 0.10 g (2) makes 2,7-two bromo-9,9-two (3 '-(N, N '-dimethylamino) propyl group)-fluorenes, 0.10 g(3) makes the palladium (Pd (OAc) of divinylbenzene, 25 mg 2), the triphenyl phosphorus (PPh of 0.3 g 3), 60 mL DMF, 50mL triethylamine, add hot reflux 48 h at 150 ℃.After reaction stops, using chloroform extraction, organic layer MgSO 4Drying is filtered, and decompression distillation obtains the novel poly-fluorene derivative (PF-NH of brown color 2).
(5) PF-NH that (4) is prepared 2Obtain solution (1 nM is dissolved among the DMF).In the single port bottle of 100 mL, add 1 mL PF-NH 2Solution, 8 mL ethanol, dispersed with stirring is even.Then add 0.1 mL ammonia spirit (25%), the tetraethyl orthosilicate solution of 10 μ L (TEOS, the TEOS ethanolic solution of volumetric concentration 1/20) stirs 30 min.Continue to add 10 μ L TEOS solution and stir 30 min until the TEOS solution total amount that adds is 50 μ L.After reaction finishes, at 10000 rmin -1The ethanol washing precipitation is used in centrifuging under the rotating speed, three times repeatedly.In vacuum drying chamber, obtain the poly-fluorene derivative (PF-NH of coated with silica with 50 ℃ of drying 5 h 2@SiO 2).
(6) in the small beaker of 10 mL, add the PF-NH that (5) prepare 2@SiO 20.02 g, with the freshly prepd N of 1 mL, N '-carbonyl dimidazoles solution (CDI, 100 mgmL -1, be dissolved in DMSO) add and stir 4 h.After reacting completely, adding excessive intermediate water is that excessive CDI decomposes, at 10000 rmin -1The intermediate water washing precipitation is used in centrifuging under the rotating speed, three times repeatedly.With the PF-NH that modifies 2@SiO 2Be dissolved in (PBS, pH 7.4) in the phosphate buffer solution, add a certain amount of two anti-(Ab 2) original solution and activation 2 h under 4 ℃.At 10000 rmin -1Centrifuging under the rotating speed adds 1 mL bovine serum albumin(BSA) (BSA, 1%) at 4 ℃ of lower activation 2 h.At 10000 rmin -1The PBS washing precipitation is used in centrifuging under the rotating speed, three times repeatedly.In precipitation, add at last PBS to Ab 2Concentration is 20 μ gmL -1
(7) utilize classic method to prepare golden nanometer particle (AuNPs) and Fe 3 O 4 magnetic particle (Fe 3O 4).In the round-bottomed flask of 100 mL, add 0.1 g Fe 3O 4Nano particle adds 10 mL ethanol, then adds 0.5 mL APTES and magnetic agitation 1 h, separates with magnet, washes with intermediate water.Add 1 mL AuNPs(1%), add 10 mL and magnetic agitation 2 h.Separate with magnet, wash with intermediate water.In vacuum drying chamber, namely get the tri-iron tetroxide (Fe that golden nanometer particle coats with 50 ℃ of drying 10 h 3O 4@Au).
(8) with (7) Fe 3O 4@Au prepares particle 0.01 g and is dissolved among the PBS, adds a certain amount of primary antibodie (Ab 1) original solution and activation 2 h under 4 ℃.Separate with magnet, add 1 mL BSA solution, at 4 ℃ of lower activation 2 h.Separate with magnet, use the PBS washing precipitation, three times repeatedly.In precipitation, add at last PBS to Ab 1Concentration is 20 μ gmL -1
(9) adding 10 μ L(7 in the centrifuge tube of the 1 mL) Ab of preparation 1Then solution add the certain density antigens of 10 μ L (Ag) solution, and at 4 ℃ of lower activation 2 h.Add 10 μ L(6) preparation Ab 2Solution, and at 4 ℃ of lower activation 2 h.Separate with magnet, use the careful absorption supernatant of imbibition rifle, and wash three times repeatedly with PBS.
(10) utilize the fluorescence signal of gained material in the Flow Injection/Chemiluminescene Determination (9).
Embodiment 1(the carbohydrate mark is such as CA19-9)
(1) at first synthetic three kinds of monomers: Isosorbide-5-Nitrae-dibromo p-phenylenediamine (PPD) (compound 1), 2,7-, two bromo-9,9-two (3 '-(N, N '-dimethylamino) propyl group)-fluorenes (compound 2) synthetic, p-divinyl benzene (compound 3) synthetic.Aggregate into PF-NH by Heck 2Synthetic.
(2) PF-NH that (1) is prepared 2Obtain solution (1 nM is dissolved among the DMF).In the single port bottle of 100 mL, add 1 mL PF-NH 2Solution, 8 mL ethanol, dispersed with stirring is even.Then add 0.1 mL ammonia spirit (25%), the TEOS solution of 10 μ L stirs 30 min.Continue to add 10 μ L TEOS solution and stir 30 min until the TEOS solution total amount that adds is 50 μ L.After reaction finishes, at 10000 rmin -1The ethanol washing precipitation is used in centrifuging under the rotating speed, three times repeatedly.In vacuum drying chamber, obtain PF-NH with 50 ℃ of drying 5 h 2@SiO 2
(3) in the small beaker of 10 mL, add the PF-NH that (2) prepare 2@SiO 20.02 g adds 1 mL CDI solution and stirs 4 h.After reacting completely, adding excessive intermediate water is that excessive CDI decomposes, at 10000 rmin -1The intermediate water washing precipitation is used in centrifuging under the rotating speed, three times repeatedly.With the PF-NH that modifies 2@SiO 2Be dissolved in PBS, add a certain amount of CA19-9 two anti-(Ab 2) original solution and activation 2 h under 4 ℃.At 10000 rmin -1Centrifuging under the rotating speed adds 1 mL BSA solution, at 4 ℃ of lower activation 2 h.At 10000 rmin -1The PBS washing precipitation is used in centrifuging under the rotating speed, three times repeatedly.In precipitation, add at last PBS to Ab 2Concentration is 20 μ gmL -1
(4) utilize classic method to prepare AuNPs and Fe 3O 4In the round-bottomed flask of 100 mL, add 0.1 g Fe 3O 4Nano particle adds 10 mL ethanol, then adds 0.5 mL APTES and magnetic agitation 1 h, separates with magnet, washes with intermediate water.Add 1 mL AuNPs(1%), add 10 mL and magnetic agitation 2 h.Separate with magnet, wash with intermediate water.In vacuum drying chamber, namely get Fe with 50 ℃ of drying 10 h 3O 4@Au.
(5) with (4) Fe 3O 4@Au prepares particle 0.01 g and is dissolved among the PBS, adds a certain amount of CA19-9 primary antibodie (Ab 1) original solution and activation 2 h under 4 ℃.Separate with magnet, add 1 mL BSA solution, at 4 ℃ of lower activation 2 h.Separate with magnet, use the PBS washing precipitation, three times repeatedly.In precipitation, add at last PBS to Ab 1Concentration is 20 μ gmL -1
(6) adding 10 μ L(5 in the centrifuge tube of the 1 mL) Ab of preparation 1Then solution add the certain density CA19-9 antigens of 10 μ L (Ag) solution, and at 4 ℃ of lower activation 2 h.Add 10 μ L(4) preparation Ab 2Solution, and at 4 ℃ of lower activation 2 h.Separate with magnet, use the careful absorption supernatant of imbibition rifle, and wash three times repeatedly with PBS.
(7) utilize the fluorescence signal of gained material in the Flow Injection/Chemiluminescene Determination (6).
CA19-9 flow injection fluorescent optical sensor detects the CA19-9 in human body, the animal blood serum sample, the results are shown in Table 1.Utilize existing method, preparation CA19-9 electrochemical immunosensor connects electrochemical workstation, and the CA19-9 in human body, the animal blood serum sample extracting solution is carried out actual detection, the results are shown in Table 1.
Table 1 CA19-9 flow injection of the present invention Fluoro-Immnnosensor and CA19-9 electrochemical immunosensor detect Contrast on effect
Figure 2012105771552100002DEST_PATH_IMAGE002
The result can find out that CA19-9 flow injection Fluoro-Immnnosensor has the wider range of linearity, higher sensitivity and lower detectability than CA19-9 electrochemical immunosensor from table 1.
Embodiment 2(steroids is such as HCG)
(1) at first synthetic three kinds of monomers: Isosorbide-5-Nitrae-dibromo p-phenylenediamine (PPD) (compound 1), 2,7-, two bromo-9,9-two (3 '-(N, N '-dimethylamino) propyl group)-fluorenes (compound 2) synthetic, p-divinyl benzene (compound 3) synthetic.Aggregate into PF-NH by Heck 2Synthetic.
(2) PF-NH that (1) is prepared 2Obtain solution (1 nM is dissolved among the DMF).In the single port bottle of 100 mL, add 1 mL PF-NH 2Solution, 8 mL ethanol, dispersed with stirring is even.Then add 0.1 mL ammonia spirit (25%), the TEOS solution of 10 μ L stirs 30 min.Continue to add 10 μ L TEOS solution and stir 30 min until the TEOS solution total amount that adds is 50 μ L.After reaction finishes, at 10000 rmin -1The ethanol washing precipitation is used in centrifuging under the rotating speed, three times repeatedly.In vacuum drying chamber, obtain PF-NH with 50 ℃ of drying 5 h 2@SiO 2
(3) in the small beaker of 10 mL, add the PF-NH that (2) prepare 2@SiO 20.02 g adds 1 mL CDI solution and stirs 4 h.After reacting completely, adding excessive intermediate water is that excessive CDI decomposes, at 10000 rmin -1The intermediate water washing precipitation is used in centrifuging under the rotating speed, three times repeatedly.With the PF-NH that modifies 2@SiO 2Be dissolved in PBS, add a certain amount of HCG two anti-(Ab 2) original solution and activation 2 h under 4 ℃.At 10000 rmin -1Centrifuging under the rotating speed adds 1 mL BSA solution, at 4 ℃ of lower activation 2 h.At 10000 rmin -1The PBS washing precipitation is used in centrifuging under the rotating speed, three times repeatedly.In precipitation, add at last PBS to Ab 2Concentration is 20 μ gmL -1
(4) utilize classic method to prepare AuNPs and Fe 3O 4In the round-bottomed flask of 100 mL, add 0.1 g Fe 3O 4Nano particle adds 10 mL ethanol, then adds 0.5 mL APTES and magnetic agitation 1 h, separates with magnet, washes with intermediate water.Add 1 mL AuNPs(1%), add 10 mL and magnetic agitation 2 h.Separate with magnet, wash with intermediate water.In vacuum drying chamber, namely get Fe with 50 ℃ of drying 10 h 3O 4@Au.
(5) with (4) Fe 3O 4@Au prepares particle 0.01 g and is dissolved among the PBS, adds a certain amount of HCG primary antibodie (Ab 1) original solution and activation 2 h under 4 ℃.Separate with magnet, add 1 mL BSA solution, at 4 ℃ of lower activation 2 h.Separate with magnet, use the PBS washing precipitation, three times repeatedly.In precipitation, add at last PBS to Ab 1Concentration is 20 μ gmL -1
(6) adding 10 μ L(5 in the centrifuge tube of the 1 mL) Ab of preparation 1Then solution add the certain density HCG antigens of 10 μ L (Ag) solution, and at 4 ℃ of lower activation 2 h.Add 10 μ L(4) preparation Ab 2Solution, and at 4 ℃ of lower activation 2 h.Separate with magnet, use the careful absorption supernatant of imbibition rifle, and wash three times repeatedly with PBS.
(7) utilize the fluorescence signal of gained material in the Flow Injection/Chemiluminescene Determination (6).
The HCG Flow-injection/Chemiluminescence Sensor detects the HCG in human body, the animal blood serum sample, the results are shown in Table 1.Utilize existing method, preparation HCG electrochemical immunosensor connects electrochemical workstation, and the HCG in human body, the animal blood serum sample extracting solution is carried out actual detection, the results are shown in Table 2.
Table 2 HCG portable injection chemiluminescence of the present invention immunosensor and HCG electrochemical immunosensor detect Contrast on effect
Figure 2012105771552100002DEST_PATH_IMAGE004
The result can find out that HCG portable injection chemiluminescence immunosensor has the wider range of linearity, higher sensitivity and lower detectability than HCG electrochemical immunosensor from table 2.
Embodiment 3(the embryonal antigen class is such as AFP)
(1) at first synthetic three kinds of monomers: Isosorbide-5-Nitrae-dibromo p-phenylenediamine (PPD) (compound 1), 2,7-, two bromo-9,9-two (3 '-(N, N '-dimethylamino) propyl group)-fluorenes (compound 2) synthetic, p-divinyl benzene (compound 3) synthetic.Aggregate into PF-NH by Heck 2Synthetic.
(2) PF-NH that (1) is prepared 2Obtain solution (1 nM is dissolved among the DMF).In the single port bottle of 100 mL, add 1 mL PF-NH 2Solution, 8 mL ethanol, dispersed with stirring is even.Then add 0.1 mL ammonia spirit (25%), the TEOS solution of 10 μ L stirs 30 min.Continue to add 10 μ L TEOS solution and stir 30 min until the TEOS solution total amount that adds is 50 μ L.After reaction finishes, at 10000 rmin -1The ethanol washing precipitation is used in centrifuging under the rotating speed, three times repeatedly.In vacuum drying chamber, obtain PF-NH with 50 ℃ of drying 5 h 2@SiO 2
(3) in the small beaker of 10 mL, add the PF-NH that (2) prepare 2@SiO 20.02 g adds 1 mL CDI solution and stirs 4 h.After reacting completely, adding excessive intermediate water is that excessive CDI decomposes, at 10000 rmin -1The intermediate water washing precipitation is used in centrifuging under the rotating speed, three times repeatedly.With the PF-NH that modifies 2@SiO 2Be dissolved in PBS, add a certain amount of AFP two anti-(Ab 2) original solution and activation 2 h under 4 ℃.At 10000 rmin -1Centrifuging under the rotating speed adds 1 mL BSA solution, at 4 ℃ of lower activation 2 h.At 10000 rmin -1The PBS washing precipitation is used in centrifuging under the rotating speed, three times repeatedly.In precipitation, add at last PBS to Ab 2Concentration is 20 μ gmL -1
(4) utilize classic method to prepare AuNPs and Fe 3O 4In the round-bottomed flask of 100 mL, add 0.1 g Fe 3O 4Nano particle adds 10 mL ethanol, then adds 0.5 mL APTES and magnetic agitation 1 h, separates with magnet, washes with intermediate water.Add 1 mL AuNPs(1%), add 10 mL and magnetic agitation 2 h.Separate with magnet, wash with intermediate water.In vacuum drying chamber, namely get Fe with 50 ℃ of drying 10 h 3O 4@Au.
(5) with (4) Fe 3O 4@Au prepares particle 0.01 g and is dissolved among the PBS, adds a certain amount of AFP primary antibodie (Ab 1) original solution and activation 2 h under 4 ℃.Separate with magnet, add 1 mL BSA solution, at 4 ℃ of lower activation 2 h.Separate with magnet, use the PBS washing precipitation, three times repeatedly.In precipitation, add at last PBS to Ab 1Concentration is 20 μ gmL -1
(6) adding 10 μ L(5 in the centrifuge tube of the 1 mL) Ab of preparation 1Then solution add the certain density AFP antigens of 10 μ L (Ag) solution, and at 4 ℃ of lower activation 2 h.Add 10 μ L(4) preparation Ab 2Solution, and at 4 ℃ of lower activation 2 h.Separate with magnet, use the careful absorption supernatant of imbibition rifle, and wash three times repeatedly with PBS.
(7) utilize the fluorescence signal of gained material in the Flow Injection/Chemiluminescene Determination (6).
The AFP Flow-injection/Chemiluminescence Sensor detects the AFP in human body, the animal blood serum sample, the results are shown in Table 3.Utilize existing method, preparation AFP electrochemical immunosensor connects electrochemical workstation, and the AFP in human body, the animal blood serum sample extracting solution is carried out actual detection, the results are shown in Table 3.
Table 3 AFP portable injection chemiluminescence of the present invention immunosensor and AFP electrochemical immunosensor detect Contrast on effect
Figure 2012105771552100002DEST_PATH_IMAGE006
The result can find out from table 3: AFP portable injection chemiluminescence immunosensor has the wider range of linearity, higher sensitivity and lower detectability than AFP electrochemical immunosensor.
Figure 2012105771552100002DEST_PATH_IMAGE008

Claims (1)

1. preparation method who detects the flow injection fluorescent optical sensor of immune marker, it may further comprise the steps:
(1) synthetic (PF-NH of poly-fluorene derivative 2);
(2) the poly-fluorene derivative (PF-NH of synthetic silica coating 2@SiO 2);
(3) with the two anti-PF-NH that are modified at 2@SiO 2
(4) prepare golden nanometer particle (AuNPs) according to existing method, Fe 3 O 4 magnetic particle (Fe 3O 4) tri-iron tetroxide (Fe that coats of golden nanometer particle 3O 4@Au);
(5) primary antibodie is modified at Fe 3O 4@Au surface;
(6) utilize polymethylmethacrylate (PMMA) preparation flow injection fluorescence flow cell;
(7) chemiluminescence sensor of making is detected immune marker in conjunction with the portable injection chemiluminescence instrument;
The preparation of the poly-fluorene derivative of coated with silica of the present invention and the tri-iron tetroxide of coated with silica may further comprise the steps:
(1) Isosorbide-5-Nitrae-dibromo p-phenylenediamine (PPD) (compound 1)
(a) in nitrogen environment, add 250 mL anhydrous tetrahydro furans (THF), and then add 4.2 g sodium hydrides (NaH), add 5.17 g and add hot reflux 1 h after to diphenylamine; Be cooled to and add 25 g dimethyl dicarbonate butyl esters after the room temperature, add 4.2 g NaH after stirring 30 min, 12 h reflux; Add large water gaging and make too much reactant hydrolysis, then use a large amount of ethyl acetate extractions, organic layer anhydrous magnesium sulfate (MgSO 4) drying, filter, decompression distillation, crystallization obtains the N of white, N '-2(tertbutyloxycarbonyl in ethyl acetate)-Isosorbide-5-Nitrae-diaminobenzene;
(b) in nitrogen environment, add 9 mL absolute ethers and the anhydrous THF of 9 mL, add (1)-(a) gained N, N '-2(tertbutyloxycarbonyl)-and Isosorbide-5-Nitrae-diaminobenzene, be cooled to-45 ℃, the pentane solution that dropwise adds the tert-butyl lithium of 25 mL, 1.3 M, stir 3 h at-45 ℃, then dropwise add 2 mL, one chlorine trimethyl silane at-55 ℃, reaction room temperature stirring reaction 12 h; Add the hydrochloric acid solution of 10 mL 10% in the reactant liquor, use ethyl acetate extraction, organic layer MgSO 4Drying is filtered, and decompression distillation is crossed post and can be got white N, N '-2(tertbutyloxycarbonyl)-Isosorbide-5-Nitrae-diamido-2, the 5-2(TMS) benzene;
(c) in nitrogen environment, add anhydrous methylene chloride, add (1)-(b) gained N, N '-2(tertbutyloxycarbonyl)-Isosorbide-5-Nitrae-diamido-2, the 5-2(TMS) benzene, add 1.53 g NBS, reaction adds 5 mL water at stirring at room 3 h again, use dichloromethane extraction, organic layer MgSO 4Drying is filtered, decompression distillation, recrystallization can get white N, N '-2(tertbutyloxycarbonyl)-Isosorbide-5-Nitrae-diamido-2, the 5-2 bromobenzene;
(d) in nitrogen environment, add 10 mL trifluoroacetic acids and 10 mL anhydrous methylene chlorides in device, add (1)-(c) gained N, N '-2(tertbutyloxycarbonyl)-Isosorbide-5-Nitrae-diamido-2,5-2 bromobenzene, stirring at room 12 h, then decompression distillation, evaporate to dryness; To wherein adding 5 mL anhydrous methylene chlorides, add again without saturation of the air sodium bicarbonate solution, control pH 8～9 stirs 3 h, with the dichloromethane extraction of deacration, merges organic layer, uses MgSO 4Drying, decompression distillation, recrystallization obtains Isosorbide-5-Nitrae-dibromo p-phenylenediamine (PPD);
(2) 2,7-two bromo-9,9-two (3 '-(N, N '-dimethylamino) propyl group)-fluorenes (compound 2) synthetic
In nitrogen environment, add 4 g 2,7-dibromo fluorenes and 80 mg tetrabutyl ammonium bromide add 60 mL dimethylene sulfoxides (DMSO), directly add 8 ml, 50% NaOH (NaOH) solution; The hydrochloride ((CH of 5 g 3-dimethylaminopropyl chlorine will be dissolved with 3) 2N (CH 2) 3ClHCl) solution joins 20 mL(DMSO) and be added drop-wise in the potpourri, 12 h stirred; The water that adds 50 mL in the reactant liquor, dissolving residue salt; With product extraction, the washing that obtains; The anhydrous MgSO of solution after the washing 4Drying, filtration under diminished pressure revolves steaming and desolventizes, and obtains yellow 2,7-, two bromo-9,9-two (3 '-(N, N '-dimethylamino) propyl group)-fluorenes;
(3) p-divinyl benzene (compound 3) is synthetic
(a) in nitrogen environment, in 100 mL three-neck flasks, add 5.8 g N-bromosuccinimides (NBS), 0.10 g benzoyl peroxide adds 2.0 mL P-xylene and 60 mL phenixin (CCl again 4); Magnetic stirrer to all solids dissolves; Add hot reflux 12 h; After stopping reaction, filter while hot, washing precipitation is carried out recrystallization after the filtrate cooling, obtains white, needle-shaped crystals, namely to dibromo xylene;
(b) in nitrogen environment, in 100 mL three-neck flasks, add 4.94 g(3)-(a) make dibromo xylene and 9.0 g triphenyl phosphorus, add again 30 mL anhydrous methylene chlorides and 30 mL formaldehyde; Filtration, washing precipitation obtain white powder to the quaternary alkylphosphonium salt of dibromo xylene;
(c) in nitrogen environment, (3)-(b) gained is transferred to the quaternary alkylphosphonium salt of dibromo xylene in the three-neck flask of 250 mL, add again 30 mL anhydrous methylene chlorides and 30 mL formaldehyde; Dropwise drip 20 mL, 5% NaOH solution in reaction solution, reaction stirred 12 h stop reaction; The extracting and washing precipitation, anhydrous MgSO 4Dried overnight; Remove solvent under reduced pressure, obtain the white needles p-divinyl benzene;
(4) poly-fluorene derivative (PF-NH 2) synthetic
In nitrogen environment, in 100 mL three-neck flasks, add successively 0.19g(1) make 1,4-dibromo p-phenylenediamine (PPD), 0.10 g (2) makes 2,7-two bromo-9,9-two (3 '-(N, N '-dimethylamino) propyl group)-fluorenes, 0.10 g(3) makes the palladium (Pd (OAc) of divinylbenzene, 25 mg 2), the triphenyl phosphorus (PPh of 0.3 g 3), 60 mL DMF, 50mL triethylamine, add hot reflux 48 h at 150 ℃; After reaction stops, using chloroform extraction, organic layer MgSO 4Drying is filtered, and decompression distillation obtains the novel poly-fluorene derivative (PF-NH of brown color 2);
(5) PF-NH that (4) is prepared 2Obtain solution (1 nM is dissolved among the DMF); In the single port bottle of 100 mL, add 1 mL PF-NH 2Solution, 8 mL ethanol, dispersed with stirring is even; Then add 0.1 mL ammonia spirit (25%), the tetraethyl orthosilicate solution of 10 μ L (TEOS, the TEOS ethanolic solution of volumetric concentration 1/20) stirs 30 min; Continue to add 10 μ L TEOS solution and stir 30 min until the TEOS solution total amount that adds is 50 μ L; After reaction finishes, at 10000 rmin -1The ethanol washing precipitation is used in centrifuging under the rotating speed, three times repeatedly; In vacuum drying chamber, obtain the poly-fluorene derivative (PF-NH of coated with silica with 50 ℃ of drying 5 h 2@SiO 2);
(6) in the small beaker of 10 mL, add the PF-NH that (5) prepare 2@SiO 20.02 g, with the freshly prepd N of 1 mL, N '-carbonyl dimidazoles solution (CDI, 100 mgmL -1, be dissolved in DMSO) add and stir 4 h; After reacting completely, adding excessive intermediate water is that excessive CDI decomposes, at 10000 rmin -1The intermediate water washing precipitation is used in centrifuging under the rotating speed, three times repeatedly; With the PF-NH that modifies 2@SiO 2Be dissolved in (PBS, pH 7.4) in the phosphate buffer solution, add a certain amount of two anti-(Ab 2) original solution and activation 2 h under 4 ℃; At 10000 rmin -1Centrifuging under the rotating speed adds 1 mL bovine serum albumin(BSA) (BSA, 1%) at 4 ℃ of lower activation 2 h; At 10000 rmin -1The PBS washing precipitation is used in centrifuging under the rotating speed, three times repeatedly; In precipitation, add at last PBS to Ab 2Concentration is 20 μ gmL -1
(7) utilize classic method to prepare golden nanometer particle (AuNPs) and Fe 3 O 4 magnetic particle (Fe 3O 4); In the round-bottomed flask of 100 mL, add 0.1 g Fe 3O 4Nano particle adds 10 mL ethanol, then adds 0.5 mL APTES and magnetic agitation 1 h, separates with magnet, washes with intermediate water; Add 1 mL AuNPs(1%), add 10 mL and magnetic agitation 2 h; Separate with magnet, wash with intermediate water; In vacuum drying chamber, namely get the tri-iron tetroxide (Fe that golden nanometer particle coats with 50 ℃ of drying 10 h 3O 4@Au);
(8) with (7) Fe 3O 4@Au prepares particle 0.01 g and is dissolved among the PBS, adds a certain amount of primary antibodie (Ab 1) original solution and activation 2 h under 4 ℃; Separate with magnet, add 1 mL BSA solution, at 4 ℃ of lower activation 2 h; Separate with magnet, use the PBS washing precipitation, three times repeatedly; In precipitation, add at last PBS to Ab 1Concentration is 20 μ gmL -1
(9) adding 10 μ L(7 in the centrifuge tube of the 1 mL) Ab of preparation 1Then solution add the certain density antigens of 10 μ L (Ag) solution, and at 4 ℃ of lower activation 2 h; Add 10 μ L(6) preparation Ab 2Solution, and at 4 ℃ of lower activation 2 h; Separate with magnet, use the careful absorption supernatant of imbibition rifle, and wash three times repeatedly with PBS;
(10) utilize the fluorescence signal of gained material in the Flow Injection/Chemiluminescene Determination (9).
Figure 2012105771552100001DEST_PATH_IMAGE002
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