CN102192986A - Cardiac troponin I detection reagent and preparation method thereof and application thereof - Google Patents
Cardiac troponin I detection reagent and preparation method thereof and application thereof Download PDFInfo
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- CN102192986A CN102192986A CN2010101278834A CN201010127883A CN102192986A CN 102192986 A CN102192986 A CN 102192986A CN 2010101278834 A CN2010101278834 A CN 2010101278834A CN 201010127883 A CN201010127883 A CN 201010127883A CN 102192986 A CN102192986 A CN 102192986A
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- 238000001514 detection method Methods 0.000 title claims abstract description 30
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- 102100036859 Troponin I, cardiac muscle Human genes 0.000 title abstract 4
- 101710128251 Troponin I, cardiac muscle Proteins 0.000 title abstract 4
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Abstract
The invention discloses a cardiac troponin I detection reagent and a preparation method thereof and an application thereof. The cardiac troponin I detection reagent is characterized in that: the detection reagent contains a nano-gold particle wrapped by organosilicon, wherein a particle size of the nano-gold particle is 10 to 150 nm and the thickness of the nano-silica coating is 5 to 100 nm; functional groups of amidine, carboxyl, hydroxy, hydrosulfuryl, cyclopropyl, aldehyde group are introduced into the surface of the organosilicon coating; and a biomolecule such as protein or antibody is marked by the nano gold with the functionalized organosilicon surface by chemical reaction so as to form a stable covalent bond connection and to maintain the biological activity of the marked biomolecule. The modified nano-gold particle, which can substitute for a traditional colloidal gold particle, can be used in the immunology field to prepare immunization reagent; and the modified nanometer colloid can be used to prepare the cardiac troponin I detection reagent.
Description
Technical field
The present invention relates to detectable of a kind of cardiac muscle troponin I (TROPONIN I) and its production and use, belong to biological medicine detectable field.
Background technology
Angiocardiopathy is first major disease that threatens human life's health.According to World Health Organization's recent statistics, the whole world has 1,670 ten thousand people to die from all kinds of cardiovascular and cerebrovascular diseases every year, and this numeral accounts for 29.2% of all disease deaths, and the acute myocardial infarction AMI of dying from over half is wherein arranged.In China, over nearly 10 years, the incidence of disease of China's acute myocardial infarction AMI obviously rises, near the international average level.Incidence of CHD accounts for about 6 ‰ of total population, and actual patients with coronary heart disease number reaches 7,800,000 people.And owing to lack the general knowledge of preventing and treating of coronary heart disease, a lot of patients reach an advanced stage when going to a doctor, and are first symptom with acute myocardial infarction AMI (AMI).
Be clinical symptoms such as pectoralgia to the AMI diagnostic criteria both at home and abroad at present, electrocardiographic abnormality and serum cardiac muscle biochemical marker are unusual.But at the AMI initial stage, about 25% patient does not have typical clinical symptoms, the patient's cardiogram of half does not have typical ANOMALOUS VARIATIONS approximately, and the blood sample mesophytization mark when therefore detecting the damage of AMI initial stage heart of patient has the important references meaning unusually in the AMI clinical diagnosis.
In China hospitals at different levels, the main biochemical marker that detects in the laboratory comprises: TnT roponin-I (TNI), Troponin-T (TNT), myoglobins Myoglobin and cretinephosphokinase CK-MB.Wherein CK-MB is present in the cardiac muscle in a large number, generally raises in 4-8 hour in the AMI morbidity, peaks in 12-20 hour, has been acknowledged as " goldstandard " that detect AMI since the early seventies.But the CK-MB specificity is relatively poor, and marathon race, Skeletal muscle injury, renal failure person CK-MB content all can increase unusually.The time that CK-MB exists in blood is also lacked (1-4 days).Along with the development of detection technique, it is the most special that cardiac muscle troponin I (CTnI) has been proved to be myocardial damage, one of the most responsive blood serum designated object.The content of CTnI generally is lower than 0.4ng/ml in the normal human blood, and after AMI took place, CTnI can raise in 6-8 hour fast, reached peak value in 18-24 hour, and all kept higher concentration in 10-15 days.Clinical fast detecting to CTnI has important society and economic benefit.
Existing C TnI detection method mainly contains euzymelinked immunosorbent assay (ELISA) (ELISA, chemoluminescence method and immunofluorescence technique.The lowest detection sensitivity of ELISA can reach 0.2ng/mL, and the mensuration cycle is long, reaches 5-6 hour.Chemoluminescence method is luminesceence analysis and immune response to be combined and the micro-antigen set up or the labelled immune analytical technology of antibody, its sensitivity is up to 0.03ng/mL, and detection time is also shorter, about 1-2 hour, but need to adopt special-purpose main equipment, complicated operation.Immunofluorescence technique adopts method sandwich and that enzyme connection fluorescence combines, and detection time is very fast, and maximum sensitivity can reach 0.1ng/mL, also needs specialized equipment.These detection methods all need expensive instrument and professional's operation, only be applicable in centralab and use, the time that testing result arrives in doctor's hand is longer, can not satisfy clinical fast detecting needs, also is not suitable for the township hospital that middle and small hospital does not especially have centralab.
The kit of domestic existing fast detecting CTnI mainly adopts traditional colloidal gold immunity chromatography preparation.Its ultimate principle is to adopt the monoclonal antibody of colloid gold label individual plant or the anti-CTnI of many strains, adopts the sandwich CTnI that detects of two anti-immunity, Chinese patent 200710022815.x, 200810036418.2 existing reports.On the market all based on CTnI quick detection kit detection sensitivity generally in the 2-10ng/ml scope.
Collaurum promptly refers to the suspending liquid of golden particulate, and its diameter has high electron density, dielectric property and catalytic action at 1～100nm, can combine with multiple biomacromolecule, and not influence its biologically active.Its color takes on a red color to purple according to diameter.The building-up process of tradition collaurum, it is to the mark of monoclonal antibody, and the preparation of solid-phase immunity chromatograph test strip all has report.In the 1980s, collaurum at first is applied to the mark and the vitro detection field of protein molecular.
Collaurum is electronegative under the weak base environment, can form firm combining with the positive charge group of protein molecule, this combination is the static combination, though do not influence the biological nature of protein, also more stable, but this combination is subjected to solution ion strength, the pH influence is very big, especially in high inonic strength solution, collaurum condenses, biomolecule with nano gold mark also becomes unstable in time, has influenced collaurum to the mark of a lot of biomolecule and the application in biological sample.
Summary of the invention
The objective of the invention is provides detectable of a kind of cardiac muscle troponin I and its production and use at the deficiencies in the prior art, is characterized in the synthesizing new nanoparticle, nano-particle surface functionalization, and the labeling method of nanoparticle antagonist.It also comprises reagent strip, contains the two anti-of anti-cardiac muscle troponin I monoclonal antibody and modified Nano gold anti-cardiac muscle troponin I monoclonal antibody of mark and anti-cardiac muscle troponin I monoclonal antibody on the reagent strip.
Purpose of the present invention is realized by following technical measures: wherein said raw material umber is parts by weight except that specified otherwise.
Detectable of cardiac muscle troponin I and preparation method thereof:
The detectable of cardiac muscle troponin I
The particle diameter of organosilicon parcel nm of gold particulate is 10-150nm, be preferably 50～100nm, most preferably be 55～75nm, nanometer silicon coating is thick to be 5-100nm, amido, carboxyl, hydroxyl, sulfhydryl, trimethylene base and aldehyde radical functional group are introduced in described organic silicon coating surface, mark is carried out by chemistry and biomolecule such as protein, antibody in the organosilicon surface of functionalization, forms stable covalent bond and connect, and maintenance is labeled the biologically active of molecule.
The preparation method of cardiac muscle troponin I detectable may further comprise the steps:
1. the preparation of traditional collaurum
With concentration is 100 parts of heating of 0.01～4% chlorauric acid solution, under stirring, boiled 10～120 minutes 100～400rpm, add fast concentration and be 0.1～5 part of 2N ammonia spirit and concentration and be 2～10 parts of 1% sodium citrates, high-speed stirred to solution presents orange red, cooling back high speed centrifugation purifying, obtain the collaurum particulate of particulate footpath, and the collaurum packing that makes is preserved at 10～200nm;
2. the preparation of the nm of gold particulate of organosilicon parcel
With 100 parts of the above-mentioned colloidal gold solutions that makes, add 0.1～2 part of three amido propyl-triethoxysilicane, stirred 15～120 minutes at 100～400rpm, add 200～500 parts of solvents, to alkalescence, add 0.1～2 part of silicon ethyl alkylating mixture with ammoniacal liquor regulator solution pH value, at room temperature continue to stir 18-24 hour, product at 2000～10000rpm high speed centrifugation purifying, is obtained the nm of gold particulate of organosilicon parcel;
3. organosilicon parcel nm of gold is surface-functionalized
The above-mentioned collaurum that makes is added 200～500 parts of solvents for 100 parts, stirred 15～120 minutes at 100～400rpm, regulate the pH value to alkalescence with ammoniacal liquor, add 0.1～2 part of 1: 1 tetraethyl silane and the mixed solution that contains organic functional group, at room temperature continue to stir 18～24 hours, product obtains the nanoparticle of the surface-functionalized modification of organic parcel nm of gold at 2000～10000rpm high speed centrifugation purifying;
4. the nm of gold of organosilicon parcel is to the mark of myocardium calcium protein I monoclonal antibody
Adopt surperficial carboxylated organosilicon parcel nm of gold to activate its surperficial carboxyl and react with the amido of cardiac muscle troponin I monoclonal antibody Fc by ethyl-3-(3-dimethyl aminopropyl)-carbodiimides (EDC), form the amide structure of stable chemical performance, this stable chemical bond is not subjected to the ionic strength of solution, Acidity of Aikalinity influences, thereby increases the chemistry and the storage stability of tagged cardiac muscle troponin I monoclonal antibody.The cardiac muscle troponin I monoclonal antibody that mark is good is sprayed on the glass fibre pad, forms gold mark pad.
5. the preparation of kit
Is the antibody phosphate buffer solution of 1.5mg/ml with the monoclonal antibody of a strain CTnI as detecting the antibody compound concentration, adds 1～20 part of 2% trehalose, and spray printing forms detection line to the nitrocellulose filter of activation; With two anti-as sheep anti-mouse igg spray printings of the monoclonal antibody of CTnI formation quality control line to the nitrocellulose filter; With dry 2-10 hour of nitrocellulose filter, preparation detected the nitrocellulose filter of CTnI.With gold mark pad, sample pad, nitrocellulose filter and adsorptive pads stick on the plastic plate, and plastic plate is cut into 5mm width detection CTnI test strips, and the plastic casing of again the CTnI test strip being packed into is made the kit that detects CTnI.
6. the method for quick of cardiac muscle troponin I
With the sample well on whole blood/blood serum sample adding kit, be penetrated on the sample pad, CTnI meets to combine with the monoclonal detection antibody of the CTnI of organosilicon parcel nano gold mark and forms compound in the sample, CTnI monoclonal capture antibodies on the compound continuation is penetrated into detection line and is fixed on detection line along nitrocellulose filter forms the compound of sandwich double antibody and combines, show the redness of nm of gold on detection line, testing result is positive; The CTnI monoclonal of unnecessary nano gold mark detects antibody when the quality control line is arrived in dialysis, with be fixed on the quality control line on two anti-form sandwich the combination, make nature controlling line also show redness, show that the dialysis of sample on nitrocellulose filter is complete, this testing result is effective.If CTnI content is lower than detection sensitivity in the sample, the CTnI monoclonal of nano gold mark detects antibody can not form effectively sandwich compound the combination with fixing capture antibodies on the detection line, detection line can not show redness, testing result is negative, but detecting antibody, the CTnI monoclonal of nano gold mark still can resist sandwich combination of formation with the monoclonal two on being fixed on nature controlling line, on nature controlling line, show redness, show that testing result is effective.This method is a technology well known to those skilled in the art.
The organic functions group is any in amido, carboxyl, sulfhydryl, hydroxyl, trimethylene base or the aldehyde radical functional group.
Solvent is any in ethanol, isopropyl alcohol, methyl alcohol, the normal butyl alcohol.
The nm of gold particulate of the surface-functionalized modification of organic parcel nm of gold carries out mark by chemistry and biomolecule and forms stable covalence key and be connected.
Detectable is used for the content of the cardiac muscle troponin I of clinical fast detecting infraction blood.
The present invention has following advantage:
1, the present invention adopts organosilicon that traditional nm of gold particulate is wrapped up, and forms the silicon coating of nano thickness on its surface, and chemical modification is carried out on the silicon coating surface, introduces carboxyl, amido, hydroxyl, sulfhydryl, trimethylene base and aldehyde radical functional group.The collaurum that organic-silicon-modified nm of gold is more traditional has better stability, is not easy to take place the cohesion of gold grain.Chemical functional group and biomolecule such as albumen that silicon face is introduced, antibody, antigen form stablizes chemical covalent bonds, prepares stabilized nano gold label more.The nm of gold of this modification can replace traditional collaurum and be used for field of immunology, the preparation immunoreagent.
2, utilize this nm of gold to bioprotein, antibody carries out mark, forms between biomolecule and nm of gold particulate and stablizes covalent bond, has improved the chemistry and the storage stability that are labeled molecule.
3, the surface silicon coating can improve the surface reflection light intensity of nano material, thereby improves the naked eyes resolution of this material, and the sensitivity that has improved immunoreagent can reach 0.4ng/mL.
4, utilize the nm of gold of this organosilicon parcel to prepare the detection kit that detects cardiac muscle troponin I, improved the detection speed of cardiac muscle troponin I greatly to the labeling of monoclonal antibody of cardiac muscle troponin I and by immunochromatographic method.
Embodiment
Below by embodiment the present invention is carried out concrete description; be necessary to be pointed out that at this present embodiment only is used for the present invention is further specified; can not be interpreted as limiting the scope of the invention, the person skilled in the art in this field can make some nonessential improvement and adjustment according to the content of the invention described above.
Embodiment 1
1: the preparation and the surface of organosilicon parcel nanogold particle are aminated
The preparation of nanogold particle solution: adopt the sodium citrate reducing process to prepare collaurum particulate solution, the particle size of collaurum is controlled at 10-150nm.With concentration is 100 parts of 1% gold chloride deionized water solutions, and high-speed stirred was also boiled 10 minutes.Add fast concentration and be 0.1 part of the hydrogen amine aqueous solution of 2N and concentration and be 2 parts of 1% sodium citrates, high-speed stirred, solution presents orange red, the particle diameter that cooling back high speed centrifugation purifying obtains particulate is the collaurum particulate of 55-75nm, the collaurum that obtains is preserved a period of time and then organosilicon parcel and aminated modification is carried out in the collaurum surface, 100 parts of the colloidal gold solutions of obtaining, add 300 parts of ethanol, stirred 15 minutes at 100～400rpm, extremely alkaline with ammoniacal liquor regulator solution pH, the tetraethyl silane and the 3-aminopropyltriethoxywerene werene mixed solution that add 1 part of mixing in 1: 1, at room temperature continue to stir 18-24 hour, product obtains the aminated novel nano gold particulate in organosilicon parcel surface at 2000～10000rpm high speed centrifugation purifying.
The anti-cardiac muscle troponin I monoclonal antibody of 2 surperficial aminated organosilicon parcel nano gold marks
In the aminated nano-Au solution in the organosilicon parcel surface of the method for 100 parts of foregoing descriptions preparation, add the anti-cardiac muscle troponin I monoclonal antibody of 10 parts of crosslinking chemicals and 100 micrograms and continue reaction 2 hours, add concentration then and be 0.1 part of the phosphate buffer of 10% bovine serum albumin(BSA), continue reaction 1 hour.Obtain the anti-cardiac muscle troponin I monoclonal antibody of surperficial aminated organosilicon parcel nano gold mark behind the high speed centrifugation purifying.
The preparation of 3 cardiac muscle troponin I kits
The monoclonal antibody of the anti-cardiac muscle troponin I of the modified Nano of above-mentioned preparation gold mark is sprayed to the immune modified Nano gold of the preparation scraps of paper on the glass fibre scraps of paper.With the specking instrument with the monoclonal capture antibodies of cardiac muscle troponin I and two anti-IgG (1.5mg/mL) speckings on detection line and nature controlling line on the nitrocellulose filter, 37 ℃ of dryings of temperature 2 hours, acquire immune cellulose nitrate film strip, thieving paper is pasted on the immune cellulose nitrate film strip, and cut into the wide test strips of 5mm, with the test strips of cutting, golden millimeter paper and sample pad, filter pad is assembled in the sample box, makes the kit of cardiac muscle troponin I.
Embodiment 2
The preparation and the surface of 1 organosilicon parcel nanogold particle are carboxylated
Prepare the collaurum particulate that particle diameter is 55-75nm by embodiment 1 described method, get above-mentioned 100 parts of the colloidal gold solutions that make, add 250 parts of ethanol, stirred 65 minutes at 100～400rpm, extremely alkaline with ammoniacal liquor regulator solution pH, add 1 part of tetraethyl silane, reacted 2 hours, add 1 part of maleamic acid propyl-triethoxysilicane again, at room temperature continue to stir 18-24 hour, product obtains the carboxylated novel nano gold particulate in organosilicon parcel surface at 2000～10000rpm high speed centrifugation purifying.
The anti-cardiac muscle troponin I monoclonal antibody of 2 surperficial carboxylated organosilicon parcel nano gold marks
In the carboxylated nano-Au solution in the organosilicon parcel surface of the method for 100 parts of foregoing descriptions preparation, the concentration that adds 5 parts of mixing in 1: 1 is that 20mmol 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides (EDC) and concentration are 20mmol N-hydroxy-succinamide (NHS) mixed solution, at room temperature reacted 45 minutes, add the anti-cardiac muscle troponin I monoclonal antibody of 50 micrograms and continue reaction 2 hours, add concentration then and be 0.1 part of the phosphate buffer of 10% bovine serum albumin(BSA), continue reaction 1 hour.Obtain the anti-cardiac muscle troponin I monoclonal antibody of surperficial carboxylated organosilicon parcel nano gold mark behind the high speed centrifugation purifying.
The preparation of 3 cardiac muscle troponin I kits
The monoclonal antibody of the anti-cardiac muscle troponin I of the modified Nano of above-mentioned preparation gold mark is sprayed to the immune modified Nano gold of the preparation scraps of paper on the glass fibre scraps of paper.With the specking instrument with the monoclonal capture antibodies of cardiac muscle troponin I and two anti-IgG (1.5mg/mL) speckings on detection line and nature controlling line on the nitrocellulose filter, 37 ℃ of dryings of temperature 2 hours, acquire immune cellulose nitrate film strip, thieving paper is pasted on the immune cellulose nitrate film strip, and cut into the wide test strips of 5mm, with the test strips of cutting, golden millimeter paper and sample pad, filter pad is assembled in the sample box, makes the kit of cardiac muscle troponin I.
Embodiment 3
1: the preparation and the surperficial sulfhydrylization of organosilicon parcel nanogold particle
Prepare the collaurum particulate that particle diameter is 55-75nm by embodiment 1 described method, get 100 parts of the above-mentioned colloidal gold solutions that makes, add 200 parts of isopropyl alcohols, stirred 120 minutes at 100～400rpm, extremely alkaline with ammoniacal liquor regulator solution pH, add part tetraethyl silane and the 3-sulfenyl propyl-triethoxysilicane mixed solution of 0.1 part of mixing in 1: 1, at room temperature continue to stir 18-24 hour.Product obtains the novel nano gold particulate that organosilicon wraps up surperficial sulfhydrylization at 2000～10000rpm high speed centrifugation purifying.
The anti-cardiac muscle troponin I monoclonal antibody of 2 surperficial sulfhydryl organosilicon parcel nano gold marks
To add 5 microgram activators in the phosphate buffer of 100 microgram cardiac muscle troponin I monoclonal antibodies, reaction is 20 minutes under the room temperature, with gel chromatographic columns purification reaction thing, the cardiac muscle troponin I monoclonal anti liquid solution that obtains activating.The nano-Au solution of the organosilicon parcel of describing among 1 part of embodiment 3 will be added in the monoclonal antibody of activation, reaction is 1 hour under the room temperature, add concentration and be 0.1 part of the phosphate buffer of 5% bovine serum albumin(BSA), continue reaction 1 hour, obtain the anti-cardiac muscle troponin I monoclonal antibody of surperficial sulfhydryl organosilicon parcel nano gold mark behind the high speed centrifugation purifying, form stable chemical covalent bond between the antibody of mark and the silicon parcel nm of gold and be connected, increased the chemical stability and the storage stability of labelled antibody.
The preparation of 3 cardiac muscle troponin I kits
The monoclonal antibody of the anti-cardiac muscle troponin I of the modified Nano of above-mentioned preparation gold mark is sprayed to the immune modified Nano gold of the preparation scraps of paper on the glass fibre scraps of paper.With the specking instrument with the monoclonal capture antibodies of cardiac muscle troponin I and two anti-IgG (1.5mg/mL) speckings on detection line and nature controlling line on the nitrocellulose filter, 37 ℃ of dryings of temperature 2 hours, acquire immune cellulose nitrate film strip, thieving paper is pasted on the immune cellulose nitrate film strip, and cut into the wide test strips of 5mm, with the test strips of cutting, golden millimeter paper and sample pad, filter pad is assembled in the sample box, makes the kit of cardiac muscle troponin I.
Embodiment 4
1: the preparation and the surperficial trimethylene baseization of organosilicon parcel nanogold particle
Prepare the collaurum particulate that particle diameter is 55-75nm by embodiment 1 described method, get above-mentioned 100 parts of the colloidal gold solutions that make, add 250 parts of ethanol, stirred 75 minutes at 100～400rpm, extremely alkaline with ammoniacal liquor regulator solution pH, add 1 part of tetraethyl silane, reacted 2 hours, add 1 part of (3-glycidoxy propyl group) dimethylethoxysilane again, at room temperature continue to stir 18-24 hour, product obtains the novel nano gold particulate that organosilicon wraps up surperficial trimethylene baseization at 2000～10000rpm high speed centrifugation purifying.
The anti-cardiac muscle troponin I monoclonal antibody of 2 surperficial trimethylene base organosilicon parcel nano gold marks
Organosilicon in the preparation of the method for 100 parts of foregoing descriptions wraps up in the nano-Au solution of surperficial propyl, add the anti-cardiac muscle troponin I monoclonal antibody of 50 micrograms and continue reaction 2 hours at 38 ℃, add concentration then and be 0.1 part of the phosphate buffer of 10% bovine serum albumin(BSA), continue reaction 1 hour.Obtain the anti-cardiac muscle troponin I monoclonal antibody of surperficial trimethylene base organosilicon parcel nano gold mark behind the high speed centrifugation purifying.
The preparation of 3 cardiac muscle troponin I kits
The monoclonal antibody of the anti-cardiac muscle troponin I of the modified Nano of above-mentioned preparation gold mark is sprayed to the immune modified Nano gold of the preparation scraps of paper on the glass fibre scraps of paper.With the specking instrument with the monoclonal capture antibodies of cardiac muscle troponin I and two anti-IgG (1.5mg/mL) speckings on detection line and nature controlling line on the nitrocellulose filter, 37 ℃ of dryings of temperature 2 hours, acquire immune cellulose nitrate film strip, thieving paper is pasted on the immune cellulose nitrate film strip, and cut into the wide test strips of 5mm, with the test strips of cutting, golden millimeter paper and sample pad, filter pad is assembled in the sample box, makes the kit of cardiac muscle troponin I.
Claims (8)
1. the detectable of a cardiac muscle troponin I, it is characterized in that the particle diameter that this detectable contains the nm of gold particulate of organosilicon parcel is 10～150nm, nanometer silicon coating is 5～100nm, amido, carboxyl, hydroxyl, sulfhydryl, trimethylene base and aldehyde radical functional group are introduced in described organic silicon coating surface, mark is carried out by chemistry and biomolecule such as protein, antibody in the organosilicon surface of functionalization, form stable covalent bond and connect, and keep being labeled the biologically active of molecule.
2. the detectable of cardiac muscle troponin I according to claim 1 is characterized in that the particle diameter of the nm of gold particulate of organosilicon parcel is 50～100nm.
3. the detectable of cardiac muscle troponin I according to claim 1 is characterized in that the particle diameter of the nm of gold particulate of organosilicon parcel is 55-75nm.
4. as the preparation method of the detectable of cardiac muscle troponin I as described in one of claim 1～3, it is characterized in that this method may further comprise the steps:
(1) preparation of traditional collaurum
With concentration is the heating of 0.01～4% chlorauric acid solution, 100 weight portions, under stirring, boiled 10～120 minutes 100～400rpm, adding concentration fast is that 2N ammonia spirit 0.1～5 weight portion and concentration are 1% sodium citrate 2～10 weight portions, high-speed stirred to solution presents orange red, cooling back high speed centrifugation purifying, obtain the collaurum particulate of particulate footpath, and the collaurum packing that makes is preserved at 10～150nm;
(2) preparation of the nm of gold particulate of organosilicon parcel
With the above-mentioned colloidal gold solution that makes 100 weight portions, add 0.1～2 weight portion, three amido propyl-triethoxysilicanes, stirred 15～120 minutes at 100～400rpm, add 200～500 parts by weight solvent, to alkalescence, add 0.1～2 weight portion silicon ethyl alkylating mixture with ammoniacal liquor regulator solution pH value, at room temperature continue to stir 18-24 hour, product at 2000～10000rpm high speed centrifugation purifying, is obtained the nm of gold particulate of organosilicon parcel;
(3) organosilicon parcel nm of gold is surface-functionalized
The above-mentioned collaurum that makes 100 weight portions are added 200～500 parts by weight solvent, stirred 15～120 minutes at 100～400rpm, regulate the pH value to alkalescence with ammoniacal liquor, add 1: 1 tetraethyl silane of 0.1～2 weight portion and the mixed solution that contains functional group, at room temperature continue to stir 18～24 hours, product at 2000～10000rpm high speed centrifugation purifying, is obtained the nanoparticle of the surface-functionalized modification of organosilicon parcel nm of gold;
(4) preparation of the detectable of cardiac muscle troponin I
Adopt two anti-immune sandwich methods that the cardiac muscle troponin I monoclonal of the above-mentioned nanoparticle mark that makes is detected the antibody specking on sample pad, two anti-speckings of another cardiac muscle troponin I capture antibodies and monoclonal capture antibodies form the quick detection reagent paper slip for preparing anti-cardiac muscle troponin I in nitrocellulose filter, again with this detectable paper slip generate a reagent box.
5. as the preparation method of the detectable of cardiac muscle troponin I as described in the claim 4, it is characterized in that containing organic functional group and be in amido, carboxyl, sulfhydryl, hydroxyl, trimethylene base or the aldehyde radical functional group any.
6. as the preparation method of the detectable of cardiac muscle troponin I as described in the claim 4, it is characterized in that solvent is any of ethanol, isopropyl alcohol, methyl alcohol, normal butyl alcohol.
7. as the preparation method of cardiac muscle troponin I detectable as described in the claim 4, the nm of gold particulate that it is characterized in that the surface-functionalized modification of organic polymer parcel nm of gold carries out mark by chemistry and biomolecule and forms stable covalence key and be connected.
8. the purposes of cardiac muscle troponin I detectable according to claim 1 is characterized in that this detectable is used for the content of the cardiac muscle troponin I of clinical fast detecting infraction blood.
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