CN105445449A - Apparatus for rapidly and semi-quantitatively detecting uric acid in saliva, and making method thereof - Google Patents

Apparatus for rapidly and semi-quantitatively detecting uric acid in saliva, and making method thereof Download PDF

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CN105445449A
CN105445449A CN201510938602.6A CN201510938602A CN105445449A CN 105445449 A CN105445449 A CN 105445449A CN 201510938602 A CN201510938602 A CN 201510938602A CN 105445449 A CN105445449 A CN 105445449A
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uric acid
concentration
lysine
poly
saliva
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CN105445449B (en
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杨小军
李欣
赵旻
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Jilin Shuan Zheng Medical Science And Technology Co Ltd
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Jilin Shuan Zheng Medical Science And Technology Co Ltd
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Abstract

The invention relates to an apparatus for rapidly and semi-quantitatively detecting uric acid in saliva, and a making method thereof, and belongs to the field of medical detection apparatuses. The apparatus is obtained through pasting a nitrocellulose membrane of solid phase uric acid-carrier protein and goat anti-rabbit IgG polyclonal antibody, glass fibers adsorbed with colloidal gold labeled anti-uric acid antibody, a sample pad, absorbent paper and other auxiliary materials. The apparatus has the advantages of novel structure, strong specificity, rapid and semi-quantitative detection of the content of uric acid in saliva, and no increase of the production operation complexity. The nitrocellulose membrane is preprocessed with a polylysine treatment liquid, the uric acid-carrier protein and silica nanoparticles are combined, the obtained combination material is adsorbed to the nitrocellulose membrane, and a proper gold spray buffer solution and a proper sample pad treatment liquid are matched, so the reaction sensitivity is effectively improved on the basis of guaranteeing of complete release of immune colloid gold, the use amount of the immune colloid gold is reduced under same threshold, and the cost is saved. The detection apparatus has the advantages of high sensitivity, strong specificity, simple operation, time saving and strong practicality.

Description

Saliva uric acid rapid semi-quantitative pick-up unit and preparation method thereof
Technical field
The present invention relates to medical treatment detection device field, in particular to saliva uric acid rapid semi-quantitative pick-up unit and preparation method thereof, utilize pick-up unit of uric acid content and preparation method thereof in colloidal gold immunochromatographimethod technology and double antibody sandwich method principle half-quantitative detection saliva sample, can realize saliva uric acid sensitive, special, detect fast.
Background technology
Uric acid is the dead end product of mankind's purine metabolism, is formed by yellow fat cry of certain animals dehydrogenasa or yellow fat cry of certain animals oxidase degraded purine.Uric acid depends on uric acid formation speed and the equilibrium relation between kidney is discharged in the concentration of extracellular fluid, uric acid source is too much or cause serum uric acid level to raise through renal excretion deficiency, it is the Etiological of hyperuricemia, the urate of high concentration can be deposited on the positions such as articular cartilage, tendon, kidney by crystallization, thus causes disease.Hyperuricemia and obesity, blood fat disorder, hypertension, abnormal carbohydrate metabolism etc. are also deposited, and hyperuricemia is the independent hazard factor of coronary heart disease.
Saliva is the physiological functions such as the mixing material secreted by the parotid gland, sublingual gland, salivary gland, helpful digestion, protection mucous membrane.Saliva is the same with other samples such as body fluids such as blood, urine, and inclusions is very abundant, in close relations to the corresponding composition in blood.Taken the lead in using salivary analysis as since the method for diagnosing the illness from a Michaets and 1903 year Kirk in 1901, the importance of salivary analysis has obtained increasing clinical accreditation.Compared with blood preparation, saliva is owing to drawing materials conveniently, and to health without wound, patient is more acceptant.In saliva, in uric acid concentration and blood, uric acid concentration has good correlativity, and the concentration therefore measuring uric acid in saliva can reflect the concentration of uric acid in blood, and in saliva, uric acid is the potential source biomolecule mark of diagnosis hyperuricemia and gout.Detecting common method for uric acid is at present that automatic clinical chemistry analyzer detects, the method needs specific apparatus, can only detect in hospital, be not suitable for the daily uric acid monitoring of the patient such as hyperuricemia or gout, therefore finding sensitive, the special fast simple device development that can be used for uric acid content mensuration is also research in recent years focus.
Colloidal gold immunochromatographimethod technology (goldimmunochromatographyassay, GICA) is that a kind of what colloidal gold-labeled method and protein chromatography technology combined take miillpore filter as the immobilon-p immuno analytical method of carrier.Colloidal gold immunochromatographimethod technology is a kind of conventional immunochromatography detection method, because it is simple to operate, save time, the feature such as manufacturing cost is lower, the easy interpretation of result, be very suitable for Site Detection, be widely used in the fields such as biology, medicine, food.Because colloidal gold immunochromatographimethod technology is that a step completes detection, therefore the disturbing factor of testing process is more, its sensitivity is low is the principal element limiting colloidal gold immunochromatographimethod range of application, and the detectability of traditional colloidal gold immunochromatographimethod technology is higher than methods such as ELISA.
In colloidal gold immunochromatographimethod detects, protein is bonded to the capture agent of nitrocellulose filter (NC film) as sample to be tested.Because testing result depends on the adsorption effect that capture agent reaches good on film completely, the absorption that therefore protein is homogeneous, good on film is extremely important to collaurum testing result.If the protein content that NC film combines is not enough or protein combination power is strong not, just there will be considerable problem, on the detection line of testing result clearly.If the protein content that film combines is too low, the more weak and detection sensitivity of detection line colour developing reduces so in the result.If albumen can not firmly be adsorbed in NC film, so spread before NC film in protein adsorption, thus cause detection line wider, colour developing more weak instead of bright-coloured and clear, make testing result be difficult to explain.Under extreme conditions, if the physisorption of albumen and NC film is too weak, the albumen of set may be washed off from NC film by the Protein Detection thing flow through and surfactant solution, thus display is wider or detection line not clearly, is difficult to explain testing result.
Summary of the invention
The invention provides a kind of saliva uric acid rapid semi-quantitative pick-up unit and preparation method thereof, solve the uric acid concentration not using large-scale instrument and detect, and solve the NC film adhesion protein quantity not sufficient that prior art exists, the problem that adhesion is not strong.Saliva uric acid rapid semi-quantitative pick-up unit prepared by the present invention, can realize saliva uric acid sensitive, special, detect fast, improve right combination judgement Patients with Hyperuricemia being carried out to uric acid metabolism, fast, accurately can carry out hyperuricemia early warning and state of an illness risk judgment.Have the nitrocellulose filter of purifying uric acid-carrier protein and goat anti-rabbit igg polyclonal antibody by solid phase, be adsorbed with other auxiliary material such as glass fibre, sample pad, thieving paper of the anti-uric acid antibody of colloid gold label rabbit paste make.Can realize saliva uric acid sensitive, special, detect fast.
The technical scheme that the present invention takes is:
Saliva uric acid rapid semi-quantitative pick-up unit: sample pad (1), immune colloid gold glass fibre membrane (2), nitrocellulose filter (3), absorption pad (4) are pasted onto plastic plate (5) respectively, the two ends of described nitrocellulose filter (3) overlap with absorption pad (4), immune colloid gold glass fibre membrane (2) respectively, and the other end and the sample pad (1) of described immune colloid gold glass fibre membrane (2) overlap; Described nitrocellulose filter (3) is arranged detection line (T) and nature controlling line (C), or the first detection line (T1), the second detection line (T2), the 3rd detection line (T3) and nature controlling line (C) are set, the upper solid phase of described detection line (T) has uric acid-carrier protein; Or the first described detection line T1, the second detection line T2, solid phase has concentration uric acid-carrier protein from small to large respectively on the 3rd detection line T3, the upper specking goat anti-rabbit igg polyclonal antibody of described nature controlling line (C).
The upper solid phase of described detection line (T) has uric acid-carrier protein, and this uric acid-carrier protein comprises: ovalbumin (OVA), bovine serum albumin(BSA) (BSA), hemocyanin (KLH).
The preparation method of saliva uric acid rapid semi-quantitative pick-up unit of the present invention, is characterized in that: comprise the following steps:
A () adopts trisodium citrate reduction method to prepare collaurum;
B () adopts the anti-uric acid antibody of colloid gold label rabbit obtained in step (a), adaptive immune collaurum;
C () adopts the immune colloid gold adaptive immune colloidal gold solution of metal spraying damping fluid dilution step (b), with immune colloid gold solution spraying in fiberglass packing, and obtained immune colloid gold glass fibre membrane;
(d) by nitrocellulose filter with after the pre-service of poly-D-lysine treating fluid, specking uric acid-carrier protein is detection line T or variable concentrations uric acid-carrier protein is the first detection line (T1), the second detection line (T2), the 3rd detection line (T3), specking goat anti-rabbit igg antibody, as nature controlling line, obtains immune nitrocellulose filter;
Immune nitrocellulose filter, thieving paper prepared by e immune colloid gold glass fibre membrane, step (d) that pretreated sample pad, step (c) are prepared by () are pasted onto on offset plate successively, cutting is obtained detects reagent strip, finally will detect reagent strip and load plastic casing.
Colloid gold particle particle diameter prepared by the employing trisodium citrate reduction method described in step (a) of the present invention is 20 ~ 60nm.
Metal spraying damping fluid described in step (c) of the present invention is made up of Tris-HCL liquid, sucrose, trehalose, bovine serum albumin(BSA) BSA, pH value 8.5, wherein Tris-Hcl concentration is 0.02mol/L, sucrose concentration is 5 ~ 20%, trehalose concentration is 1 ~ 5%, and bovine serum albumin(BSA) BSA concentration is 0.5 ~ 1%.
It is 0.5% to form that poly-D-lysine treating fluid described in step (d) of the present invention is diluted to concentration by poly-D-lysine, through 0.22 μm of membrane filtration, wherein poly-D-lysine, and SIGMA, 150KD ~ 300KD, for subsequent use.
Poly-D-lysine treating fluid described in step (d) of the present invention to be mixed with methyl alcohol by poly-D-lysine and forms, and wherein poly-D-lysine concentration is 0.5% composition, and methanol concentration is 10%, through 0.22 μm of membrane filtration, wherein poly-D-lysine, SIGMA, 150KD ~ 300KD, for subsequent use.
Poly-D-lysine treating fluid described in step (d) of the present invention is mixed formed by poly-D-lysine, methyl alcohol, PEG20000, wherein poly-D-lysine concentration is 0.5% composition, methanol concentration is 10%, PEG20000 concentration is 0.2%, through 0.22 μm of membrane filtration, wherein poly-D-lysine, SIGMA, 150KD ~ 300KD, for subsequent use.
Step (d) uric acid-carrier protein of the present invention is combined with nano SiO 2 particle, method is: using silicon dioxide as carrier, gets 1mL uric acid-carrier protein solution, mixes with ethanol and deionized water and stirring, add quantitative ammoniacal liquor and the positive silicic acid of 0.3mL ester again, stir after 1 hour, add the positive silicic acid of residue ester, Keep agitation, whole reaction lucifuge is carried out, within centrifugal 10 minutes, collect with 12000rpm, 8500rpm and 7000rpm respectively, absolute ethyl alcohol, deionized water respectively wash 2 times.
The preparation of the described nano SiO 2 particle of step (d) of the present invention is: be mixed in conical flask by the ammoniacal liquor of 2.48mL volume fraction 25 and 43.2mL absolute ethyl alcohol, under about 35 DEG C and ultrasound condition, with the speed of 0.4mL/min instillation 3.5mL ethyl orthosilicate, after dripping off ultrasonic 5 minutes again, the particle that particle diameter 120nm is homogeneous can be obtained, respectively with 12000rpm, 8500rpm and 7000rpm collects for centrifugal 10 minutes, outwell supernatant, add water ultrasonic disperse again. and repeated multiple times washing is to weakly acidic pH, finally be settled to volume with water identical with volume when just preparing, deposit stand-by.
In step (d) of the present invention, uric acid-carrier protein comprises ovalbumin (OVA), bovine serum albumin(BSA) (BSA), hemocyanin (KLH).
The sample pad treating fluid that pretreated sample pad described in step (e) of the present invention adopts is made up of Tris-HCL liquid, bovine serum albumin(BSA) BSA, casein, surfactant AEO, wherein the concentration of Tris-HCL liquid is 0.1mol/L, bovine serum albumin(BSA) BSA concentration is 0.5 ~ 1%, and casein concentration is 0.1 ~ 0.2%, surfactant AEO concentration is 0.5 ~ 1%.
Saliva uric acid rapid semi-quantitative pick-up unit prepared by the present invention can detect patient's saliva uric acid content, in the body of clinical hyperuricemia and patient with gout, uric acid level monitoring is significant.
The present invention has the nitrocellulose filter of the uric acid-carrier protein of purifying (detection line T) and goat anti-rabbit igg antibody (nature controlling line) by solid phase, be adsorbed with other auxiliary material such as glass fibre, sample pad, thieving paper of the anti-uric acid antibody of colloid gold label rabbit pastes and makes, and has special, quick, sensitive feature.Poly-D-lysine is adopted to carry out pre-service to nitrocellulose filter, and prepare suitable metal spraying damping fluid and sample pad treating fluid, ensureing that immune colloid gold discharges completely on basis, effectively raise the sensitivity of reaction, under same threshold value, also can reduce the consumption of immune colloid gold, cost-saving.
Can supply coupling containing multiple amino in the structure of poly-D-lysine (Poly-L-Lysine, PLL), reactivation process is simple, facilitates NC film surface ankyrin.Poly-D-lysine is with a large amount of positive charges, significantly can increase the positive charge quantity of avtive spot on NC film, and antibody is electronegative under condition at routine bag, can in conjunction with the antibody of greater number on the NC film of such unit area, this significantly can increase the bag of antibody by efficiency.Under normal condition, on the NC film of unit area, the quantity of binding antibody is limited, after adopting poly-D-lysine process, binding antibody quantity on the NC film of unit area can be allowed to increase, and then can realize higher detection sensitivity.Research finds that methyl alcohol has good heavy wetting state, can ensure not produce bubble when pre-service NC film in order to avoid the existence of bubble causes NC film process local insufficient, also can activate NC film simultaneously, allow NC film strips positive charge, this is similar to polylysine, both synergies, better effects if.Can there is suction-operated with the site of NC film in PEG20000, rear at albumen bag, can absorb the Bound moisture of albumen, causes albumen more hydrophobic, and the combination of such albumen and NC film is more firm, significantly improves the bag of albumen by efficiency by hydrophobic effect.To sum up, poly-D-lysine, methyl alcohol and PEG20000 combination formula, can improve the bag of NC memebrane protein by efficiency from charge effect and hydrophobic effect two aspect.
In order to improve the sensitivity of colloidal gold immunochromatographimethod technology, we are by having carried out pre-service to nitrocellulose filter poly-D-lysine treating fluid, reach the object improving test paper sensitivity.
Beneficial effect of the present invention is:
1, structure of the detecting device of the present invention is novel, uric acid antibody is coated on nitrocellulose membrane film, high specificity, uric acid content can be detected by rapid semi-quantitative, namely read value instrument by colorimetric card or collaurum and judge uric acid content, also the colour developing situation by three detection lines judges body uric acid content, does not also increase the complexity of production operation.
2, in immune colloid gold preparation process, by coordinating suitable metal spraying damping fluid and sample pad treating fluid, can ensure that immune colloid gold discharges completely on basis, effectively raise the sensitivity of reaction, under same threshold value, also can reduce the consumption of immune colloid gold, cost-saving.
3, the present invention carries out pre-service to nitrocellulose filter, improves Test paper sensitivity, specificity.
4, pick-up unit of the present invention is without any need for special instruments and equipment, and testing cost is low.
5, pick-up unit of the present invention is easy and simple to handle, does not need professional to operate.Practical.
Accompanying drawing explanation
Fig. 1 is structural representation of the present invention, is a detection line in figure;
Fig. 2 is structural representation of the present invention, is three detection lines in figure.
Embodiment
Shown in Figure 1, saliva uric acid rapid semi-quantitative pick-up unit of the present invention, sample pad (1), immune colloid gold glass fibre membrane (2), nitrocellulose filter (3), absorption pad (4) are pasted onto plastic plate (5) respectively, the two ends of described nitrocellulose filter (3) overlap with absorption pad (4), immune colloid gold glass fibre membrane (2) respectively, and the other end and the sample pad (1) of described immune colloid gold glass fibre membrane (2) overlap; Described nitrocellulose filter (3) is arranged detection line (T) and nature controlling line (C), the upper solid phase of described detection line (T) has uric acid-carrier protein, the upper specking goat anti-rabbit igg polyclonal antibody of described nature controlling line (C).
Shown in Figure 2, saliva uric acid rapid semi-quantitative pick-up unit of the present invention, sample pad (1), immune colloid gold glass fibre membrane (2), nitrocellulose filter (3), absorption pad (4) are pasted onto plastic plate (5) respectively, the two ends of described nitrocellulose filter (3) overlap with absorption pad (4), immune colloid gold glass fibre membrane (2) respectively, and the other end and the sample pad (1) of described immune colloid gold glass fibre membrane (2) overlap; Described nitrocellulose filter (3) is arranged the first detection line (T1), the second detection line (T2), the 3rd detection line (T3) and nature controlling line (C), on the first described detection line T1, the second detection line T2, the 3rd detection line T3, solid phase has concentration uric acid-carrier protein from small to large respectively, the upper specking goat anti-rabbit igg polyclonal antibody of described nature controlling line (C); Wherein on the first detection line T1, the second detection line T2, the 3rd detection line T3, solid phase has the uric acid-carrier protein of 1mg/ml, 1.5mg/ml, 2mg/ml concentration respectively;
The preparation method of saliva uric acid rapid semi-quantitative pick-up unit of the present invention, comprises the following steps:
A () adopts trisodium citrate reduction method to prepare collaurum;
B () adopts the anti-uric acid antibody of colloid gold label rabbit obtained in step (a), adaptive immune collaurum;
C () adopts the immune colloid gold adaptive immune colloidal gold solution of metal spraying damping fluid dilution step (b), with immune colloid gold solution spraying in fiberglass packing, and obtained immune colloid gold glass fibre membrane;
(d) by nitrocellulose filter with after the pre-service of poly-D-lysine treating fluid, specking uric acid-carrier protein is detection line T or variable concentrations uric acid-carrier protein is the first detection line (T1), the second detection line (T2), the 3rd detection line (T3), specking goat anti-rabbit igg antibody, as nature controlling line, obtains immune nitrocellulose filter; Wherein T1 detection line can detect 250umol/L uric acid, and T2 detection line can detect 250-350umol/L uric acid, and T3 detection line can detect 350-500umol/L uric acid;
Immune nitrocellulose filter, thieving paper prepared by e immune colloid gold glass fibre membrane, step (d) that pretreated sample pad, step (c) are prepared by () are pasted onto on offset plate successively, cutting is obtained detects reagent strip, finally will detect reagent strip and load plastic casing;
Colloid gold particle particle diameter prepared by the employing trisodium citrate reduction method described in step (a) is 20 ~ 60nm.
Metal spraying damping fluid described in step (c) is made up of Tris-HCL liquid, sucrose, trehalose, bovine serum albumin(BSA) BSA, pH value 8.5, wherein Tris-Hcl concentration is 0.02mol/L, sucrose concentration is 5 ~ 20%, trehalose concentration is 1 ~ 5%, and bovine serum albumin(BSA) BSA concentration is 0.5 ~ 1%.
It is 0.5% to form that poly-D-lysine treating fluid described in step (d) is diluted to concentration by poly-D-lysine, through 0.22 μm of membrane filtration, wherein poly-D-lysine, and SIGMA, 150KD ~ 300KD, for subsequent use.
Poly-D-lysine treating fluid described in step (d) to be mixed with methyl alcohol by poly-D-lysine and forms, and wherein poly-D-lysine concentration is 0.5% composition, and methanol concentration is 10%, through 0.22 μm of membrane filtration, wherein poly-D-lysine, SIGMA, 150KD ~ 300KD, for subsequent use.
Poly-D-lysine treating fluid described in step (d) is mixed formed by poly-D-lysine, methyl alcohol, PEG20000, wherein poly-D-lysine concentration is 0.5% composition, methanol concentration is 10%, PEG20000 concentration is 0.2%, through 0.22 μm of membrane filtration, wherein poly-D-lysine, SIGMA, 150KD ~ 300KD, for subsequent use.
Step (d) uric acid-carrier protein is combined with nano SiO 2 particle, method is: using silicon dioxide as carrier, gets 1mL uric acid-carrier protein solution, mixes with ethanol and deionized water and stirring, add quantitative ammoniacal liquor and the positive silicic acid of 0.3mL ester again, stir after 1 hour, add the positive silicic acid of residue ester, Keep agitation, whole reaction lucifuge is carried out, within centrifugal 10 minutes, collect with 12000rpm, 8500rpm and 7000rpm respectively, absolute ethyl alcohol, deionized water respectively wash 2 times.(this section of emphasis is looked at)
The preparation of the described nano SiO 2 particle of step (d) is: be mixed in conical flask by the ammoniacal liquor of 2.48mL volume fraction 25 and 43.2mL absolute ethyl alcohol, under about 35 DEG C and ultrasound condition, with the speed of 0.4mL/min instillation 3.5mL ethyl orthosilicate, after dripping off ultrasonic 5 minutes again, the particle that particle diameter 120nm is homogeneous can be obtained, respectively with 12000rpm, 8500rpm and 7000rpm collects for centrifugal 10 minutes, outwell supernatant, add water ultrasonic disperse again. and repeated multiple times washing is to weakly acidic pH, finally be settled to volume with water identical with volume when just preparing, deposit stand-by.
In step (d), uric acid-carrier protein comprises ovalbumin (OVA), bovine serum albumin(BSA) (BSA), hemocyanin (KLH).
The sample pad treating fluid that pretreated sample pad described in step (e) adopts is made up of Tris-HCL liquid, bovine serum albumin(BSA) BSA, casein, surfactant AEO, wherein the concentration of Tris-HCL liquid is 0.1mol/L, bovine serum albumin(BSA) BSA concentration is 0.5 ~ 1%, and casein concentration is 0.1 ~ 0.2%, surfactant AEO concentration is 0.5 ~ 1%.
Embodiment 1
(1) trisodium citrate reduction method is adopted to prepare collaurum
Chlorauride is added fast in the 100ml purified water of heating, after solution seethes with excitement again, add trisodium citrate rapidly, chlorauride: trisodium citrate=1:2, continue to boil, observe solution colour by yellow blackening purpling again, after finally becoming stable claret, timing continues heating 10 minutes, and colloid gold particle is 20nm;
(2) immune colloid gold preparation
1) get 100 milliliters of 20nm colloidal gold solutions respectively, add pH adjusting agent 140 μ l, mixing; Leave standstill 5min;
2) in 20nm colloidal gold solution, the anti-uric acid antibody of rabbit is added according to the ratio of every milliliter of colloidal gold solution 10 μ g, altogether 1mg, mixing; Leave standstill 5min;
3) add collaurum stabilizing agent 0.4 milliliter according to the ratio of 0.4% respectively, mixing, leave standstill 5 minutes;
4) 10000,12000, the centrifugal 10min of 14000rpm, respectively collecting precipitation, merges the precipitation of three collections;
(3) the metal spraying damping fluid dilution immune colloid gold adaptive immune colloidal gold solution optimized is adopted, with immune colloid gold solution spraying in fiberglass packing, obtained immune colloid glass fibre membrane; Described metal spraying damping fluid comprises: concentration is 20mMTris-HCL liquid, sucrose concentration is 5%, trehalose concentration is 1%, BSA concentration be 1%, pH is 8.5;
(4) solid phase nitrocellulose filter
1) poly-D-lysine treating fluid pre-service nitrocellulose filter
Preparation poly-D-lysine treating fluid: poly-D-lysine (SIGMA, 150KD), concentration are 0.5%, through 0.22 μm of membrane filtration, for subsequent use;
Poly-D-lysine treating fluid pre-service nitrocellulose filter: nitrocellulose filter is put into poly-D-lysine treating fluid and soak 1h, and vibration is rocked at a slow speed, cleans 3 times after taking-up with distilled water, finally dry in vacuum drying chamber;
2) nano SiO 2 particle modifies uric acid-OVA antigen
The preparation of monox nanometer particle is: be mixed in conical flask by the ammoniacal liquor of 2.48mL volume fraction 25 and 43.2mL absolute ethyl alcohol, under 35 DEG C and ultrasound condition, with the speed of 0.4mL/min instillation 3.5mL ethyl orthosilicate, after dripping off ultrasonic 5 minutes again, the particle that particle diameter 120nm is homogeneous can be obtained, respectively with 12000rpm, 8500rpm and 7000rpm collects for centrifugal 10 minutes, outwell supernatant, add water ultrasonic disperse again. and repeated multiple times washing is to weakly acidic pH, finally be settled to volume with water identical with volume when just preparing, deposit stand-by.
Uric acid-OVA is combined with nano SiO 2 particle, method is: using silicon dioxide as carrier, gets 1mL uric acid-OVA antigenic solution, mixes with ethanol and deionized water and stirring, add quantitative ammoniacal liquor and the positive silicic acid of 0.3mL ester again, stir after 1 hour, add the positive silicic acid of residue ester, Keep agitation, whole reaction lucifuge is carried out, within centrifugal 10 minutes, collect with 12000rpm, 8500rpm and 7000rpm respectively, absolute ethyl alcohol, deionized water respectively wash 2 times.
3) the bag quilt of nitrocellulose filter detection line and nature controlling line antibody
When spray film amount is 5 μ l/cm, by uric acid-ovalbumin (OVA) antigen diluent to 1,1.5,2mg/ml, control line goat anti-rabbit igg antibody is diluted to 1mg/ml, wrap by the detection line of nitrocellulose filter and control line respectively, on the first described detection line T1, the second detection line T2, the 3rd detection line T3 solid phase have 1,1.5, the uric acid-OVA antigen of 2mg/ml concentration; The upper specking goat anti-rabbit igg polyclonal antibody of described nature controlling line (C).T1 detection line can detect 250umol/L uric acid, and T2 detection line can detect 250-350umol/L uric acid, and T3 detection line can detect 350-500umol/L uric acid.Dry overnight at room temperature, stores for future use.
4) sample pad pre-service
Glass fibre sample pad treating fluid is soaked 10min, its kind of sample pad treating fluid comprises: Tris-HCL liquid concentration is 0.1M, bovine serum albumin(BSA) BSA concentration is 0.5%, casein concentration is 0.1%, surfactant concentration is 0.5%, 37 DEG C of dry for standby, sample pad can improve reaction sensitivity after treatment;
(5) assemble
Pretreated sample pad, immune colloid gold glass fibre membrane, immune nitrocellulose filter, thieving paper are pasted onto successively on offset plate, cutting is obtained detects reagent strip, finally will detect reagent strip and load plastic casing.
(6) half-quantitative detection: point out uric acid content to be less than 250umol/L when T1, T2 and T3 detection line all develops the color, when the colour developing of T2, T3 detection line, prompting saliva uric acid content 250-350umol/L, T3 detection line develops the color, prompting saliva uric acid content 2350-500umol/L; Uric acid content is pointed out to be greater than 500umol/L when T1, T2 and T3 detection line does not all develop the color.
Embodiment 2
(1) trisodium citrate reduction method is adopted to prepare collaurum
Chlorauride is added fast in the 100ml purified water of heating, after solution seethes with excitement again, add trisodium citrate rapidly, chlorauride: trisodium citrate=1:1, continue to boil, observe solution colour by yellow blackening purpling again, after finally becoming stable claret, timing continues heating 10 minutes, and colloid gold particle is 40nm;
(2) immune colloid gold preparation
1) get 100 milliliters of 20nm colloidal gold solutions respectively, add pH adjusting agent 140 μ l, mixing; Leave standstill 5min;
2) in 20nm colloidal gold solution, the anti-uric acid antibody of rabbit is added according to the ratio of every milliliter of colloidal gold solution 10 μ g, altogether 1mg, mixing; Leave standstill 5min;
3) add collaurum stabilizing agent 0.4 milliliter according to the ratio of 0.4% respectively, mixing, leave standstill 5 minutes;
4) 10000,12000, the centrifugal 10min of 14000rpm, respectively collecting precipitation, merges the precipitation of three collections;
(3) the metal spraying damping fluid dilution immune colloid gold adaptive immune colloidal gold solution optimized is adopted, with immune colloid gold solution spraying in fiberglass packing, obtained immune colloid glass fibre membrane; Described metal spraying damping fluid comprises: concentration is 20mMTris-HCL liquid, sucrose concentration is 5%, trehalose concentration is 1%, BSA concentration be 1%, pH is 8.5;
(4) solid phase nitrocellulose filter
1) poly-D-lysine treating fluid pre-service nitrocellulose filter
Preparation poly-D-lysine treating fluid: poly-D-lysine (SIGMA, 150KD), concentration are 0.5%, through 0.22 μm of membrane filtration, for subsequent use;
Poly-D-lysine treating fluid pre-service nitrocellulose filter: nitrocellulose filter is put into poly-D-lysine treating fluid and soak 1h, and vibration is rocked at a slow speed, cleans 3 times after taking-up with distilled water, finally dry in vacuum drying chamber;
2) nano SiO 2 particle modifies uric acid-OVA antigen
The preparation of monox nanometer particle is: be mixed in conical flask by the ammoniacal liquor of 2.48mL volume fraction 25 and 43.2mL absolute ethyl alcohol, under 35 DEG C and ultrasound condition, with the speed of 0.4mL/min instillation 3.5mL ethyl orthosilicate, after dripping off ultrasonic 5 minutes again, the particle that particle diameter 120nm is homogeneous can be obtained, respectively with 12000rpm, 8500rpm and 7000rpm collects for centrifugal 10 minutes, outwell supernatant, add water ultrasonic disperse again. and repeated multiple times washing is to weakly acidic pH, finally be settled to volume with water identical with volume when just preparing, deposit stand-by.
Uric acid-OVA is combined with nano SiO 2 particle, method is: using silicon dioxide as carrier, gets 1mL uric acid-OVA antigenic solution, mixes with ethanol and deionized water and stirring, add quantitative ammoniacal liquor and the positive silicic acid of 0.3mL ester again, stir after 1 hour, add the positive silicic acid of residue ester, Keep agitation, whole reaction lucifuge is carried out, within centrifugal 10 minutes, collect with 12000rpm, 8500rpm and 7000rpm respectively, absolute ethyl alcohol, deionized water respectively wash 2 times.
3) the bag quilt of nitrocellulose filter detection line and nature controlling line antibody
When spray film amount is 5 μ l/cm, by uric acid-OVA antigen diluent to 1,1.5,2mg/ml, control line goat anti-rabbit igg antibody is diluted to 1mg/ml, wraps respectively by the detection line of nitrocellulose filter and control line.The upper solid phase of described first, second and third detection line (T1, T2, T3) has 1,1.5, the uric acid-OVA antigen of 2mg/ml concentration; The upper specking goat anti-rabbit igg polyclonal antibody of described nature controlling line (C).T1 detection line can detect 250umol/L uric acid, and T2 detection line can detect 250-350umol/L uric acid, and T3 detection line can detect 350-500umol/L uric acid.Dry overnight at room temperature, stores for future use.
4) sample pad pre-service
Glass fibre sample pad treating fluid is soaked 10min, its kind of sample pad treating fluid comprises: Tris-HCL liquid concentration is 0.1M, bovine serum albumin(BSA) BSA concentration is 0.5%, casein concentration is 0.1%, surfactant concentration is 0.5%, 37 DEG C of dry for standby, sample pad can improve reaction sensitivity after treatment;
(5) assemble
Pretreated sample pad, immune colloid gold glass fibre membrane, immune nitrocellulose filter, thieving paper are pasted onto successively on offset plate, cutting is obtained detects reagent strip, finally will detect reagent strip and load plastic casing.
(6) half-quantitative detection: point out uric acid content to be less than 250umol/L when T1, T2 and T3 detection line all develops the color, when the colour developing of T2, T3 detection line, prompting saliva uric acid content 250-350umol/L, T3 detection line develops the color, prompting saliva uric acid content 2350-500umol/L; Uric acid content is pointed out to be greater than 500umol/L when T1, T2 and T3 detection line does not all develop the color.
Embodiment 3
(1) trisodium citrate reduction method is adopted to prepare collaurum
Chlorauride is added fast in the 100ml purified water of heating, after solution seethes with excitement again, add trisodium citrate rapidly, chlorauride: trisodium citrate=1:0.5, continue to boil, observe solution colour by yellow blackening purpling again, after finally becoming stable claret, timing continues heating 10 minutes, and colloid gold particle is 60nm;
(2) immune colloid gold preparation
1) get 100 milliliters of 20nm colloidal gold solutions respectively, add pH adjusting agent 140 μ l, mixing; Leave standstill 5min;
2) in 20nm colloidal gold solution, the anti-uric acid antibody of rabbit is added according to the ratio of every milliliter of colloidal gold solution 10 μ g, altogether 1mg, mixing; Leave standstill 5min;
3) add collaurum stabilizing agent 0.4 milliliter according to the ratio of 0.4% respectively, mixing, leave standstill 5 minutes;
4) 10000,12000, the centrifugal 10min of 14000rpm, respectively collecting precipitation, merges the precipitation of three collections;
(3) the metal spraying damping fluid dilution immune colloid gold adaptive immune colloidal gold solution optimized is adopted, with immune colloid gold solution spraying in fiberglass packing, obtained immune colloid glass fibre membrane; Described metal spraying damping fluid comprises: concentration is 20mMTris-HCL liquid, sucrose concentration is 5%, trehalose concentration is 1%, BSA concentration be 1%, pH is 8.5;
(4) solid phase nitrocellulose filter
1) poly-D-lysine treating fluid pre-service nitrocellulose filter
Preparation poly-D-lysine treating fluid: poly-D-lysine (SIGMA, 150KD), concentration are 0.5%, through 0.22 μm of membrane filtration, for subsequent use;
Poly-D-lysine treating fluid pre-service nitrocellulose filter: nitrocellulose filter is put into poly-D-lysine treating fluid and soak 1h, and vibration is rocked at a slow speed, cleans 3 times after taking-up with distilled water, finally dry in vacuum drying chamber;
2) nano SiO 2 particle modifies uric acid-OVA antigen
The preparation of monox nanometer particle is: be mixed in conical flask by the ammoniacal liquor of 2.48mL volume fraction 25 and 43.2mL absolute ethyl alcohol, under 35 DEG C and ultrasound condition, with the speed of 0.4mL/min instillation 3.5mL ethyl orthosilicate, after dripping off ultrasonic 5 minutes again, the particle that particle diameter 120nm is homogeneous can be obtained, respectively with 12000rpm, 8500rpm and 7000rpm collects for centrifugal 10 minutes, outwell supernatant, add water ultrasonic disperse again. and repeated multiple times washing is to weakly acidic pH, finally be settled to volume with water identical with volume when just preparing, deposit stand-by.
Uric acid-OVA is combined with nano SiO 2 particle, method is: using silicon dioxide as carrier, gets 1mL uric acid-OVA antigenic solution, mixes with ethanol and deionized water and stirring, add quantitative ammoniacal liquor and the positive silicic acid of 0.3mL ester again, stir after 1 hour, add the positive silicic acid of residue ester, Keep agitation, whole reaction lucifuge is carried out, within centrifugal 10 minutes, collect with 12000rpm, 8500rpm and 7000rpm respectively, absolute ethyl alcohol, deionized water respectively wash 2 times.
3) the bag quilt of nitrocellulose filter detection line and nature controlling line antibody
When spray film amount is 5 μ l/cm, by uric acid-OVA antigen diluent to 1,1.5,2mg/ml, control line goat anti-rabbit igg antibody is diluted to 1mg/ml, wraps respectively by the detection line of nitrocellulose filter and control line.The upper solid phase of described first, second and third detection line (T1, T2, T3) has 1,1.5, the uric acid-OVA antigen of 2mg/ml concentration; The upper specking goat anti-rabbit igg polyclonal antibody of described nature controlling line (C), T1 detection line can detect 250umol/L uric acid, and T2 detection line can detect 250-350umol/L uric acid, and T3 detection line can detect 350-500umol/L uric acid, dry overnight at room temperature, stores for future use;
4) sample pad pre-service
Glass fibre sample pad treating fluid is soaked 10min, its kind of sample pad treating fluid comprises: Tris-HCL liquid concentration is 0.1M, bovine serum albumin(BSA) BSA concentration is 0.5%, casein concentration is 0.1%, surfactant concentration is 0.5%, 37 DEG C of dry for standby, sample pad can improve reaction sensitivity after treatment;
(5) assemble
Pretreated sample pad, immune colloid gold glass fibre membrane, immune nitrocellulose filter, thieving paper are pasted onto successively on offset plate, cutting is obtained detects reagent strip, finally will detect reagent strip and load plastic casing.
(6) half-quantitative detection: point out uric acid content to be less than 250umol/L when T1, T2 and T3 detection line all develops the color, when the colour developing of T2, T3 detection line, prompting saliva uric acid content 250-350umol/L, T3 detection line develops the color, prompting saliva uric acid content 2350-500umol/L; Uric acid content is pointed out to be greater than 500umol/L when T1, T2 and T3 detection line does not all develop the color.
Embodiment 4
(1) trisodium citrate reduction method is adopted to prepare collaurum
Chlorauride is added fast in the 100ml purified water of heating, after solution seethes with excitement again, add trisodium citrate rapidly, chlorauride: trisodium citrate=1:2, continue to boil, observe solution colour by yellow blackening purpling again, after finally becoming stable claret, timing continues heating 10 minutes, and colloid gold particle is 20nm;
(2) immune colloid gold preparation
1) get 100 milliliters of 20nm colloidal gold solutions respectively, add pH adjusting agent 140 μ l, mixing; Leave standstill 5min;
2) in 20nm colloidal gold solution, the anti-uric acid antibody of rabbit is added according to the ratio of every milliliter of colloidal gold solution 10 μ g, altogether 1mg, mixing; Leave standstill 5min;
3) add collaurum stabilizing agent 0.4 milliliter according to the ratio of 0.4% respectively, mixing, leave standstill 5 minutes;
4) 10000,12000, the centrifugal 10min of 14000rpm, respectively collecting precipitation, merges the precipitation of three collections;
(3) the metal spraying damping fluid dilution immune colloid gold adaptive immune colloidal gold solution optimized is adopted, with immune colloid gold solution spraying in fiberglass packing, obtained immune colloid glass fibre membrane; Described metal spraying damping fluid comprises: concentration is 20mMTris-HCL liquid, sucrose concentration is 5%, trehalose concentration is 1%, BSA concentration be 1%, pH is 8.5;
(4) solid phase nitrocellulose filter
1) poly-D-lysine treating fluid pre-service nitrocellulose filter
Preparation poly-D-lysine treating fluid: poly-D-lysine (SIGMA, 150KD), concentration are 0.5%, through 0.22 μm of membrane filtration, for subsequent use;
Poly-D-lysine treating fluid pre-service nitrocellulose filter: nitrocellulose filter is put into poly-D-lysine treating fluid and soak 1h, and vibration is rocked at a slow speed, cleans 3 times after taking-up with distilled water, finally dry in vacuum drying chamber;
2) nano SiO 2 particle modifies uric acid-OVA antigen
The preparation of monox nanometer particle is: be mixed in conical flask by the ammoniacal liquor of 2.48mL volume fraction 25 and 43.2mL absolute ethyl alcohol, under 35 DEG C and ultrasound condition, with the speed of 0.4mL/min instillation 3.5mL ethyl orthosilicate, after dripping off ultrasonic 5 minutes again, the particle that particle diameter 120nm is homogeneous can be obtained, respectively with 12000rpm, 8500rpm and 7000rpm collects for centrifugal 10 minutes, outwell supernatant, add water ultrasonic disperse again. and repeated multiple times washing is to weakly acidic pH, finally be settled to volume with water identical with volume when just preparing, deposit stand-by.
Uric acid-OVA is combined with nano SiO 2 particle, method is: using silicon dioxide as carrier, gets 1mL uric acid-OVA antigenic solution, mixes with ethanol and deionized water and stirring, add quantitative ammoniacal liquor and the positive silicic acid of 0.3mL ester again, stir after 1 hour, add the positive silicic acid of residue ester, Keep agitation, whole reaction lucifuge is carried out, within centrifugal 10 minutes, collect with 12000rpm, 8500rpm and 7000rpm respectively, absolute ethyl alcohol, deionized water respectively wash 2 times.
3) the bag quilt of nitrocellulose filter detection line and nature controlling line antibody
When spray film amount is 5 μ l/cm, by uric acid-OVA antigen diluent to 3mg/ml, control line goat anti-rabbit igg antibody is diluted to 1mg/ml, wraps by the detection line of nitrocellulose filter and control line respectively, and the upper solid phase of described detection line (T) has the uric acid-OVA antigen of 3mg/ml concentration; The upper specking goat anti-rabbit igg polyclonal antibody of described nature controlling line (C).
4) sample pad pre-service
Glass fibre sample pad treating fluid is soaked 10min, its kind of sample pad treating fluid comprises: Tris-HCL liquid concentration is 0.1M, bovine serum albumin(BSA) BSA concentration is 0.5%, casein concentration is 0.1%, surfactant concentration is 0.5%, 37 DEG C of dry for standby, sample pad can improve reaction sensitivity after treatment;
(5) assemble
Pretreated sample pad, immune colloid gold glass fibre membrane, immune nitrocellulose filter, thieving paper are pasted onto successively on offset plate, cutting is obtained detects reagent strip, finally will detect reagent strip and load plastic casing.
(6) half-quantitative detection: when the colour developing of T detection line, contrast uric acid content colorimetric card, developing concentration is respectively 200umol/L, 250umol/L, 300umol/L, 400umol/L, 500umol/L, 600umol/L, 700umol/L, 800umol/L, 900umol/L, 1000umol/L, judges saliva sample uric acid content.
Embodiment 5
(1) trisodium citrate reduction method is adopted to prepare collaurum
Chlorauride is added fast in the 100ml purified water of heating, after solution seethes with excitement again, add trisodium citrate rapidly, chlorauride: trisodium citrate=1:1, continue to boil, observe solution colour by yellow blackening purpling again, after finally becoming stable claret, timing continues heating 10 minutes, and colloid gold particle is 40nm;
(2) immune colloid gold preparation
1) get 100 milliliters of 20nm colloidal gold solutions respectively, add pH adjusting agent 140 μ l, mixing; Leave standstill 5min;
2) in 20nm colloidal gold solution, the anti-uric acid antibody of rabbit is added according to the ratio of every milliliter of colloidal gold solution 10 μ g, altogether 1mg, mixing; Leave standstill 5min;
3) add collaurum stabilizing agent 0.4 milliliter according to the ratio of 0.4% respectively, mixing, leave standstill 5 minutes;
4) 10000,12000, the centrifugal 10min of 14000rpm, respectively collecting precipitation, merges the precipitation of three collections;
(3) the metal spraying damping fluid dilution immune colloid gold adaptive immune colloidal gold solution optimized is adopted, with immune colloid gold solution spraying in fiberglass packing, obtained immune colloid glass fibre membrane; Described metal spraying damping fluid comprises: concentration is 20mMTris-HCL liquid, sucrose concentration is 5%, trehalose concentration is 1%, BSA concentration be 1%, pH is 8.5;
(4) solid phase nitrocellulose filter
1) poly-D-lysine treating fluid pre-service nitrocellulose filter
Preparation poly-D-lysine treating fluid: poly-D-lysine (SIGMA, 150KD), concentration are 0.5%, through 0.22 μm of membrane filtration, for subsequent use;
Poly-D-lysine treating fluid pre-service nitrocellulose filter: nitrocellulose filter is put into poly-D-lysine treating fluid and soak 1h, and vibration is rocked at a slow speed, cleans 3 times after taking-up with distilled water, finally dry in vacuum drying chamber;
2) nano SiO 2 particle modifies uric acid-OVA antigen
The preparation of monox nanometer particle is: be mixed in conical flask by the ammoniacal liquor of 2.48mL volume fraction 25 and 43.2mL absolute ethyl alcohol, under 35 DEG C and ultrasound condition, with the speed of 0.4mL/min instillation 3.5mL ethyl orthosilicate, after dripping off ultrasonic 5 minutes again, the particle that particle diameter 120nm is homogeneous can be obtained, respectively with 12000rpm, 8500rpm and 7000rpm collects for centrifugal 10 minutes, outwell supernatant, add water ultrasonic disperse again. and repeated multiple times washing is to weakly acidic pH, finally be settled to volume with water identical with volume when just preparing, deposit stand-by.
Uric acid-OVA is combined with nano SiO 2 particle, method is: using silicon dioxide as carrier, gets 1mL uric acid-OVA antigenic solution, mixes with ethanol and deionized water and stirring, add quantitative ammoniacal liquor and the positive silicic acid of 0.3mL ester again, stir after 1 hour, add the positive silicic acid of residue ester, Keep agitation, whole reaction lucifuge is carried out, within centrifugal 10 minutes, collect with 12000rpm, 8500rpm and 7000rpm respectively, absolute ethyl alcohol, deionized water respectively wash 2 times.
3) the bag quilt of nitrocellulose filter detection line and nature controlling line antibody
When spray film amount is 5 μ l/cm, by uric acid-OVA antigen diluent to 3mg/ml, control line goat anti-rabbit igg antibody is diluted to 1mg/ml, wraps respectively by the detection line of nitrocellulose filter and control line.The upper solid phase of described detection line (T) has the uric acid-OVA antigen of 3mg/ml concentration; The upper specking goat anti-rabbit igg polyclonal antibody of described nature controlling line (C).
4) sample pad pre-service
Glass fibre sample pad treating fluid is soaked 10min, its kind of sample pad treating fluid comprises: Tris-HCL liquid concentration is 0.1M, bovine serum albumin(BSA) BSA concentration is 0.5%, casein concentration is 0.1%, surfactant concentration is 0.5%, 37 DEG C of dry for standby, sample pad can improve reaction sensitivity after treatment;
(5) assemble
Pretreated sample pad, immune colloid gold glass fibre membrane, immune nitrocellulose filter, thieving paper are pasted onto successively on offset plate, cutting is obtained detects reagent strip, finally will detect reagent strip and load plastic casing.
(6) half-quantitative detection: when the colour developing of T detection line, contrast uric acid content colorimetric card, developing concentration is respectively 200umol/L, 250umol/L, 300umol/L, 400umol/L, 500umol/L, 600umol/L, 700umol/L, 800umol/L, 900umol/L, 1000umol/L, judges saliva sample uric acid content.
Embodiment 6
(1) trisodium citrate reduction method is adopted to prepare collaurum
Chlorauride is added fast in the 100ml purified water of heating, after solution seethes with excitement again, add trisodium citrate rapidly, chlorauride: trisodium citrate=1:0.5, continue to boil, observe solution colour by yellow blackening purpling again, after finally becoming stable claret, timing continues heating 10 minutes, and colloid gold particle is 60nm;
(2) immune colloid gold preparation
1) get 100 milliliters of 20nm colloidal gold solutions respectively, add pH adjusting agent 140 μ l, mixing; Leave standstill 5min;
2) in 20nm colloidal gold solution, the anti-uric acid antibody of rabbit is added according to the ratio of every milliliter of colloidal gold solution 10 μ g, altogether 1mg, mixing; Leave standstill 5min;
3) add collaurum stabilizing agent 0.4 milliliter according to the ratio of 0.4% respectively, mixing, leave standstill 5 minutes;
4) 10000,12000, the centrifugal 10min of 14000rpm, respectively collecting precipitation, merges the precipitation of three collections;
(3) the metal spraying damping fluid dilution immune colloid gold adaptive immune colloidal gold solution optimized is adopted, with immune colloid gold solution spraying in fiberglass packing, obtained immune colloid glass fibre membrane; Described metal spraying damping fluid comprises: concentration is 20mMTris-HCL liquid, sucrose concentration is 5%, trehalose concentration is 1%, BSA concentration be 1%, pH is 8.5;
(4) solid phase nitrocellulose filter
1) poly-D-lysine treating fluid pre-service nitrocellulose filter
Preparation poly-D-lysine treating fluid: poly-D-lysine (SIGMA, 150KD), concentration are 0.5%, through 0.22 μm of membrane filtration, for subsequent use;
Poly-D-lysine treating fluid pre-service nitrocellulose filter: nitrocellulose filter is put into poly-D-lysine treating fluid and soak 1h, and vibration is rocked at a slow speed, cleans 3 times after taking-up with distilled water, finally dry in vacuum drying chamber;
2) nano SiO 2 particle modifies uric acid-OVA antigen
The preparation of monox nanometer particle is: be mixed in conical flask by the ammoniacal liquor of 2.48mL volume fraction 25 and 43.2mL absolute ethyl alcohol, under 35 DEG C and ultrasound condition, with the speed of 0.4mL/min instillation 3.5mL ethyl orthosilicate, after dripping off ultrasonic 5 minutes again, the particle that particle diameter 120nm is homogeneous can be obtained, respectively with 12000rpm, 8500rpm and 7000rpm collects for centrifugal 10 minutes, outwell supernatant, add water ultrasonic disperse again. and repeated multiple times washing is to weakly acidic pH, finally be settled to volume with water identical with volume when just preparing, deposit stand-by.
Uric acid-OVA is combined with nano SiO 2 particle, method is: using silicon dioxide as carrier, gets 1mL uric acid-OVA antigenic solution, mixes with ethanol and deionized water and stirring, add quantitative ammoniacal liquor and the positive silicic acid of 0.3mL ester again, stir after 1 hour, add the positive silicic acid of residue ester, Keep agitation, whole reaction lucifuge is carried out, within centrifugal 10 minutes, collect with 12000rpm, 8500rpm and 7000rpm respectively, absolute ethyl alcohol, deionized water respectively wash 2 times.
3) the bag quilt of nitrocellulose filter detection line and nature controlling line antibody
When spray film amount is 5 μ l/cm, by uric acid-OVA antigen diluent to 3mg/ml, control line goat anti-rabbit igg antibody is diluted to 1mg/ml, wraps respectively by the detection line of nitrocellulose filter and control line.The upper solid phase of described detection line (T) has the uric acid-OVA antigen of 3mg/ml concentration; The upper specking goat anti-rabbit igg polyclonal antibody of described nature controlling line (C).
4) sample pad pre-service
Glass fibre sample pad treating fluid is soaked 10min, its kind of sample pad treating fluid comprises: Tris-HCL liquid concentration is 0.1M, bovine serum albumin(BSA) BSA concentration is 0.5%, casein concentration is 0.1%, surfactant concentration is 0.5%, 37 DEG C of dry for standby, sample pad can improve reaction sensitivity after treatment;
(5) assemble
Pretreated sample pad, immune colloid gold glass fibre membrane, immune nitrocellulose filter, thieving paper are pasted onto successively on offset plate, cutting is obtained detects reagent strip, finally will detect reagent strip and load plastic casing.
(6) half-quantitative detection: when the colour developing of T detection line, contrast uric acid content colorimetric card, developing concentration is respectively 200umol/L, 250umol/L, 300umol/L, 400umol/L, 500umol/L, 600umol/L, 700umol/L, 800umol/L, 900umol/L, 1000umol/L, judges saliva sample uric acid content.
Embodiment 7
Nano SiO 2 particle modifies uric acid-BSA
The preparation of nano SiO 2 particle is: be mixed in conical flask by the ammoniacal liquor of 2.48mL volume fraction 25 and 43.2mL absolute ethyl alcohol, under 35 DEG C and ultrasound condition, with the speed of 0.4mL/min instillation 3.5mL ethyl orthosilicate, after dripping off ultrasonic 5 minutes again, the particle that particle diameter 120nm is homogeneous can be obtained, respectively with 12000rpm, 8500rpm and 7000rpm collects for centrifugal 10 minutes, outwell supernatant, add water ultrasonic disperse again. and repeated multiple times washing is to weakly acidic pH, finally be settled to volume with water identical with volume when just preparing, deposit stand-by;
Uric acid-BSA antigen is combined with nano SiO 2 particle, method is: using silicon dioxide as carrier, gets 1mL uric acid-BSA solution, mixes with ethanol and deionized water and stirring, add quantitative ammoniacal liquor and the positive silicic acid of 0.3mL ester again, stir after 1 hour, add the positive silicic acid of residue ester, Keep agitation, whole reaction lucifuge is carried out, within centrifugal 10 minutes, collect with 12000rpm, 8500rpm and 7000rpm respectively, absolute ethyl alcohol, deionized water respectively wash 2 times.
All the other are with embodiment 2
Embodiment 8
Nano SiO 2 particle modifies uric acid-KLH
The preparation of nano SiO 2 particle is: be mixed in conical flask by the ammoniacal liquor of 2.48mL volume fraction 25 and 43.2mL absolute ethyl alcohol, under 35 DEG C and ultrasound condition, with the speed of 0.4mL/min instillation 3.5mL ethyl orthosilicate, after dripping off ultrasonic 5 minutes again, the particle that particle diameter 120nm is homogeneous can be obtained, respectively with 12000rpm, 8500rpm and 7000rpm collects for centrifugal 10 minutes, outwell supernatant, add water ultrasonic disperse again. and repeated multiple times washing is to weakly acidic pH, finally be settled to volume with water identical with volume when just preparing, deposit stand-by;
Uric acid-KLH antigen is combined with nano SiO 2 particle, method is: using silicon dioxide as carrier, gets 1mL uric acid-KLH solution, mixes with ethanol and deionized water and stirring, add quantitative ammoniacal liquor and the positive silicic acid of 0.3mL ester again, stir after 1 hour, add the positive silicic acid of residue ester, Keep agitation, whole reaction lucifuge is carried out, within centrifugal 10 minutes, collect with 12000rpm, 8500rpm and 7000rpm respectively, absolute ethyl alcohol, deionized water respectively wash 2 times.
All the other are with embodiment 2
Embodiment 9
Preparation poly-D-lysine treating fluid: to be mixed with methyl alcohol by poly-D-lysine (SIGMA, 150KD) and form, wherein poly-D-lysine concentration is 0.5% composition, and methanol concentration is 10%, through 0.22 μm of membrane filtration, for subsequent use.
All the other are with embodiment 2.
Embodiment 10
Preparation poly-D-lysine treating fluid: to be mixed with methyl alcohol by poly-D-lysine (SIGMA, 200KD) and form, wherein poly-D-lysine concentration is 0.5% composition, and methanol concentration is 10%, through 0.22 μm of membrane filtration, for subsequent use.
All the other are with embodiment 2.
Embodiment 11
Preparation poly-D-lysine treating fluid: to be mixed with methyl alcohol by poly-D-lysine (SIGMA, 300KD) and form, wherein poly-D-lysine concentration is 0.5% composition, and methanol concentration is 10%, through 0.22 μm of membrane filtration, for subsequent use.
All the other are with embodiment 2.
Embodiment 12
Preparation poly-D-lysine treating fluid: mixed formed by poly-D-lysine (SIGMA, 150KD), methyl alcohol, PEG20000, wherein poly-D-lysine concentration is 0.5% composition, methanol concentration is 10%, PEG20000 concentration is 0.2%, through 0.22 μm of membrane filtration, for subsequent use;
All the other are with embodiment 2.
Embodiment 13
Preparation poly-D-lysine treating fluid: mixed formed by poly-D-lysine (SIGMA, 200KD), methyl alcohol, PEG20000, wherein poly-D-lysine concentration is 0.5% composition, methanol concentration is 10%, PEG20000 concentration is 0.2%, through 0.22 μm of membrane filtration, for subsequent use;
All the other are with embodiment 2.
Embodiment 14
Preparation poly-D-lysine treating fluid: mixed formed by poly-D-lysine (SIGMA, 300KD), methyl alcohol, PEG20000, wherein poly-D-lysine concentration is 0.5% composition, methanol concentration is 10%, PEG20000 concentration is 0.2%, through 0.22 μm of membrane filtration, for subsequent use;
All the other are with embodiment 2.
Embodiment 15
Preparation poly-D-lysine treating fluid: to be mixed with methyl alcohol by poly-D-lysine (SIGMA, 150KD) and form, wherein poly-D-lysine concentration is 0.5% composition, and methanol concentration is 10%, through 0.22 μm of membrane filtration, for subsequent use.
All the other are with embodiment 5.
Embodiment 16
Preparation poly-D-lysine treating fluid: to be mixed with methyl alcohol by poly-D-lysine (SIGMA, 200KD) and form, wherein poly-D-lysine concentration is 0.5% composition, and methanol concentration is 10%, through 0.22 μm of membrane filtration, for subsequent use.
All the other are with embodiment 5.
Embodiment 17
Preparation poly-D-lysine treating fluid: to be mixed with methyl alcohol by poly-D-lysine (SIGMA, 300KD) and form, wherein poly-D-lysine concentration is 0.5% composition, and methanol concentration is 10%, through 0.22 μm of membrane filtration, for subsequent use.
All the other are with embodiment 5.
Embodiment 18
Preparation poly-D-lysine treating fluid: mixed formed by poly-D-lysine (SIGMA, 150KD), methyl alcohol, PEG20000, wherein poly-D-lysine concentration is 0.5% composition, methanol concentration is 10%, PEG20000 concentration is 0.2%, through 0.22 μm of membrane filtration, for subsequent use;
All the other are with embodiment 5.
Embodiment 19
Preparation poly-D-lysine treating fluid: mixed formed by poly-D-lysine (SIGMA, 200KD), methyl alcohol, PEG20000, wherein poly-D-lysine concentration is 0.5% composition, methanol concentration is 10%, PEG20000 concentration is 0.2%, through 0.22 μm of membrane filtration, for subsequent use;
All the other are with embodiment 5.
Embodiment 20
Preparation poly-D-lysine treating fluid: mixed formed by poly-D-lysine (SIGMA, 300KD), methyl alcohol, PEG20000, wherein poly-D-lysine concentration is 0.5% composition, and methanol concentration is 10%, PEG20000 concentration is 0.2%,
Through 0.22 μm of membrane filtration, for subsequent use;
All the other are with embodiment 5.
Further illustrate effect of the present invention by experiment below.
Experimental example 1:
1, poly-D-lysine treating fluid is to the comparison of nitrocellulose filter protein adsorption ability
1.1 materials and methods
1.1 materials: nitrocellulose filter, aperture 4.5um, purchased from American General Electric Co. Limited, poly-D-lysine (SIGMA, 150KD) available from Sigma
1.2 cellulose nitrate membrane processing methods
1.2.1 preparation poly-D-lysine treating fluid
Prepare three kinds of poly-D-lysine treating fluids: poly-D-lysine group, poly-D-lysine concentration is 0.5% composition; Poly-D-lysine treating fluid methyl alcohol group, poly-D-lysine concentration is 0.5% composition, and methanol concentration is 10%; Poly-D-lysine, methyl alcohol and PEG20000 group, wherein poly-D-lysine concentration is 0.5% composition, and methanol concentration to be 10%, PEG20000 concentration be 0.2%, three groups for the treatment of fluids are all through 0.22 μm of membrane filtration, for subsequent use.
1.2.2 cellulose nitrate membrane processing method
Nitrocellulose filter is put into poly-D-lysine treating fluid and soak 1h, and vibration is rocked at a slow speed, cleans 3 times after taking-up with distilled water, finally dry in vacuum drying chamber.
1.3 experimental technique
Respectively untreated and processed nitrocellulose filter is prepared the sxemiquantitative of saliva uric acid according to above-described embodiment technological process and detect Test paper fast, testing process, with reference to test paper instructions, compares absorption affinity and stability indicator difference after the untreated and process of nitrocellulose filter.
1.4 result
1.4.1 protein adsorption force rate comparatively
Getting 3 groups of processed group and untreated fish group test paper, add measuring samples respectively, judging that process caudacoria is to protein adsorption ability, the results are shown in Table 1 by observing colour developing situation.Result shows, and the nitrocellulose filter after process obviously will be better than untreated film in solution impregnation, and process caudacoria positive band color is slightly dark, especially when concentration is lower, improve the sensitivity of reaction, show that protein adsorption ability obviously strengthens, improve reaction sensitivity.Poly-D-lysine, methyl alcohol and PEG20000 processed group adsorption effect are obviously better than poly-D-lysine group and poly-D-lysine methyl alcohol group.
Table 1 nitrocellulose filter adsorptive power compares
1.4.2 cellulose nitrate membrane stability compares
Get 3 groups of processed group and untreated fish group test paper, observe colour developing situation by 37 DEG C of Acceleration study and judge the stability processing adhesion protein on rear nitrocellulose filter, the results are shown in Table 2.Table 2 result and table 1 results contrast find, after process, the change of nitrocellulose filter color is with basically identical before 10 days, good stability.
Table 2 nitrocellulose filter accelerated stability compares (placing 10 days for 37 DEG C)
Experimental example 2:
2, nano SiO 2 particle modifies uric acid-OVA
2.1 materials and methods
1.1 materials: nitrocellulose filter, aperture 4.5um, purchased from American General Electric Co. Limited, ethyl orthosilicate is purchased from chemical plant, the west of Gansu Province, Shantou
2.2.1 nano SiO 2 particle preparation
2.48mL ammoniacal liquor (volume fraction 25) and 43.2mL absolute ethyl alcohol are mixed in conical flask, under about 35 DEG C and ultrasound condition, people 3.5mL ethyl orthosilicate is dripped with the speed of 0.4mL/min, after dripping off ultrasonic 5 minutes again, the particle (120nm) of uniform particle diameter can be obtained.Within centrifugal 10 minutes, collect with 12000rpm, 8500rpm and 7000rpm respectively, outwell supernatant, then the ultrasonic disperse that adds water. repeated multiple times washing is to weakly acidic pH.Finally be settled to volume with water identical with volume when just preparing, deposit stand-by.
2.2.2 nano SiO 2 particle modifies uric acid-OVA
Using silicon dioxide as carrier, get 1mL uric acid-OVA solution, mix with ethanol and deionized water and stirring, then add quantitative ammoniacal liquor and the positive silicic acid of 0.3mL ester, stir after 1 hour, add the positive silicic acid of residue ester, Keep agitation, whole reaction lucifuge is carried out, respectively with 12000rpm, 8500rpm and 7000rpm collects for centrifugal 10 minutes, and absolute ethyl alcohol, deionized water respectively wash 2 times.
2.3 experimental technique
Respectively uric acid-the OVA that nano SiO 2 particle is modified is prepared saliva uric acid half-quantitative detection test paper with the uric acid-OVA of unmodified according to above-described embodiment technological process, testing process, with reference to test paper instructions, compares nano SiO 2 particle process and untreated to protein adsorption power and stability indicator difference.
2.4 result
2.4.1 protein adsorption force rate comparatively
Getting nano SiO 2 particle processed group and untreated fish group test paper, add measuring samples respectively, judging that process caudacoria is to protein adsorption ability, the results are shown in Table 3 by observing colour developing situation.Result shows, and nano SiO 2 particle modification group film positive band color is slightly dark, especially when concentration is lower, improves the sensitivity of reaction, shows that protein adsorption ability obviously strengthens, improve reaction sensitivity.
Table 3 nano SiO 2 particle is modified and is compared protein adsorption ability
2.4.2 stability compares
Get nano SiO 2 particle processed group and untreated fish group test paper, observe by 37 DEG C of Acceleration study the stability that colour developing situation judges adhesion protein on silicon dioxide modified rear nitrocellulose filter, the results are shown in Table 4.Table 4 result and table 3 results contrast find, after process, the change of nitrocellulose filter color is with basically identical before 10 days, good stability.
Table 4 accelerated stability compares (placing 10 days for 37 DEG C)
Experimental example 3
3, nano SiO 2 particle modifies uric acid-BSA
3.1 materials and methods
1.1 materials: nitrocellulose filter, aperture 4.5um, purchased from American General Electric Co. Limited, ethyl orthosilicate is purchased from chemical plant, the west of Gansu Province, Shantou
3.2.1 nano SiO 2 particle preparation
2.48mL ammoniacal liquor (volume fraction 25) and 43.2mL absolute ethyl alcohol are mixed in conical flask, under about 35 DEG C and ultrasound condition, people 3.5mL ethyl orthosilicate is dripped with the speed of 0.4mL/min, after dripping off ultrasonic 5 minutes again, the particle (120nm) of uniform particle diameter can be obtained.Within centrifugal 10 minutes, collect with 12000rpm, 8500rpm and 7000rpm respectively, outwell supernatant, then the ultrasonic disperse that adds water. repeated multiple times washing is to weakly acidic pH.Finally be settled to volume with water identical with volume when just preparing, deposit stand-by.
3.2.2 nano SiO 2 particle modifies uric acid-BSA
Using silicon dioxide as carrier, get 1mL uric acid-BSA solution, mix with ethanol and deionized water and stirring, then add quantitative ammoniacal liquor and the positive silicic acid of 0.3mL ester, stir after 1 hour, add the positive silicic acid of residue ester, Keep agitation, whole reaction lucifuge is carried out, respectively with 12000rpm, 8500rpm and 7000rpm collects for centrifugal 10 minutes, and absolute ethyl alcohol, deionized water respectively wash 2 times.
3.3 experimental technique
Respectively uric acid-the BSA that nano SiO 2 particle is modified is prepared saliva uric acid half-quantitative detection test paper with the uric acid-BSA of unmodified according to above-described embodiment technological process, testing process, with reference to test paper instructions, compares nano SiO 2 particle process and untreated to protein adsorption power and stability indicator difference.
3.4 result
3.4.1 protein adsorption force rate comparatively
Getting nano SiO 2 particle processed group and untreated fish group test paper, add measuring samples respectively, judging that process caudacoria is to protein adsorption ability, the results are shown in Table 5 by observing colour developing situation.Result shows, and nano SiO 2 particle modification group film positive band color is slightly dark, especially when concentration is lower, improves the sensitivity of reaction, shows that protein adsorption ability obviously strengthens, improve reaction sensitivity.
Table 5 nano SiO 2 particle is modified and is compared protein adsorption ability
2.4.2 stability compares
Get nano SiO 2 particle processed group and untreated fish group test paper, observe by 37 DEG C of Acceleration study the stability that colour developing situation judges adhesion protein on silicon dioxide modified rear nitrocellulose filter, the results are shown in Table 6.Table 6 result and table 5 results contrast find, after process, the change of nitrocellulose filter color is with basically identical before 10 days, good stability.
Table 6 accelerated stability compares (placing 10 days for 37 DEG C)
Experimental example 4
4, nano SiO 2 particle modifies uric acid-KLH
4.1 materials and methods
1.1 materials: nitrocellulose filter, aperture 4.5um, purchased from American General Electric Co. Limited, ethyl orthosilicate is purchased from chemical plant, the west of Gansu Province, Shantou
4.2.1 nano SiO 2 particle preparation
2.48mL ammoniacal liquor (volume fraction 25) and 43.2mL absolute ethyl alcohol are mixed in conical flask, under about 35 DEG C and ultrasound condition, people 3.5mL ethyl orthosilicate is dripped with the speed of 0.4mL/min, after dripping off ultrasonic 5 minutes again, the particle (120nm) of uniform particle diameter can be obtained.Within centrifugal 10 minutes, collect with 12000rpm, 8500rpm and 7000rpm respectively, outwell supernatant, then the ultrasonic disperse that adds water. repeated multiple times washing is to weakly acidic pH.Finally be settled to volume with water identical with volume when just preparing, deposit stand-by.
4.2.2 nano SiO 2 particle modifies uric acid-BSA
Using silicon dioxide as carrier, get 1mL uric acid-BSA solution, mix with ethanol and deionized water and stirring, then add quantitative ammoniacal liquor and the positive silicic acid of 0.3mL ester, stir after 1 hour, add the positive silicic acid of residue ester, Keep agitation, whole reaction lucifuge is carried out, respectively with 12000rpm, 8500rpm and 7000rpm collects for centrifugal 10 minutes, and absolute ethyl alcohol, deionized water respectively wash 2 times.
4.3 experimental technique
Respectively uric acid-the BSA that nano SiO 2 particle is modified is prepared saliva uric acid half-quantitative detection test paper with the uric acid-BSA of unmodified according to above-described embodiment technological process, testing process, with reference to test paper instructions, compares nano SiO 2 particle process and untreated to protein adsorption power and stability indicator difference.
4.4 result
4.4.1 protein adsorption force rate comparatively
Getting nano SiO 2 particle processed group and untreated fish group test paper, add measuring samples respectively, judging that process caudacoria is to protein adsorption ability, the results are shown in Table 7 by observing colour developing situation.Result shows, and nano SiO 2 particle modification group film positive band color is slightly dark, especially when concentration is lower, improves the sensitivity of reaction, shows that protein adsorption ability obviously strengthens, improve reaction sensitivity.
Table 7 nano SiO 2 particle is modified and is compared protein adsorption ability
2.4.2 stability compares
Get nano SiO 2 particle processed group and untreated fish group test paper, observe by 37 DEG C of Acceleration study the stability that colour developing situation judges adhesion protein on silicon dioxide modified rear nitrocellulose filter, the results are shown in Table 8.Table 8 result and table 7 results contrast find, after process, the change of nitrocellulose filter color is with basically identical before 10 days, good stability.
Table 8 accelerated stability compares (placing 10 days for 37 DEG C)
The foregoing is only preferred embodiment of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.All any amendments made for the present invention, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.

Claims (12)

1. a saliva uric acid rapid semi-quantitative pick-up unit, it is characterized in that: sample pad (1), immune colloid gold glass fibre membrane (2), nitrocellulose filter (3), absorption pad (4) are pasted onto plastic plate (5) respectively, the two ends of described nitrocellulose filter (3) overlap with absorption pad (4), immune colloid gold glass fibre membrane (2) respectively, and the other end and the sample pad (1) of described immune colloid gold glass fibre membrane (2) overlap; Described nitrocellulose filter (3) is arranged detection line (T) and nature controlling line (C), or the first detection line (T1), the second detection line (T2), the 3rd detection line (T3) and nature controlling line (C) are set, the upper solid phase of described detection line (T) has uric acid-carrier protein; Or the first described detection line T1, the second detection line T2, solid phase has concentration uric acid-carrier protein from small to large respectively on the 3rd detection line T3, the upper specking goat anti-rabbit igg polyclonal antibody of described nature controlling line (C).
2. saliva uric acid rapid semi-quantitative pick-up unit according to claim 1, it is characterized in that: the upper solid phase of described detection line (T) has uric acid-carrier protein, and this uric acid-carrier protein comprises: ovalbumin (OVA), bovine serum albumin(BSA) (BSA), hemocyanin (KLH).
3. the preparation method of saliva uric acid rapid semi-quantitative pick-up unit as claimed in claim 1 or 2, is characterized in that: comprise the following steps:
A () adopts trisodium citrate reduction method to prepare collaurum;
B () adopts the anti-uric acid antibody of colloid gold label rabbit obtained in step (a), adaptive immune collaurum;
C () adopts the immune colloid gold adaptive immune colloidal gold solution of metal spraying damping fluid dilution step (b), with immune colloid gold solution spraying in fiberglass packing, and obtained immune colloid gold glass fibre membrane;
(d) by nitrocellulose filter with after the pre-service of poly-D-lysine treating fluid, specking uric acid-carrier protein is detection line T or variable concentrations uric acid-carrier protein is the first detection line (T1), the second detection line (T2), the 3rd detection line (T3), specking goat anti-rabbit igg antibody, as nature controlling line, obtains immune nitrocellulose filter;
Immune nitrocellulose filter, thieving paper prepared by e immune colloid gold glass fibre membrane, step (d) that pretreated sample pad, step (c) are prepared by () are pasted onto on offset plate successively, cutting is obtained detects reagent strip, finally will detect reagent strip and load plastic casing.
4. the preparation method of saliva uric acid rapid semi-quantitative pick-up unit according to claim 3, is characterized in that: colloid gold particle particle diameter prepared by the employing trisodium citrate reduction method described in step (a) is 20 ~ 60nm.
5. the preparation method of saliva uric acid rapid semi-quantitative pick-up unit according to claim 3, it is characterized in that: the metal spraying damping fluid described in step (c) is made up of Tris-HCL liquid, sucrose, trehalose, bovine serum albumin(BSA) BSA, pH value 8.5, wherein Tris-Hcl concentration is 0.02mol/L, sucrose concentration is 5 ~ 20%, trehalose concentration is 1 ~ 5%, and bovine serum albumin(BSA) BSA concentration is 0.5 ~ 1%.
6. the preparation method of saliva uric acid rapid semi-quantitative pick-up unit according to claim 3, it is characterized in that: it is 0.5% to form that the poly-D-lysine treating fluid described in step (d) is diluted to concentration by poly-D-lysine, through 0.22 μm of membrane filtration, wherein poly-D-lysine, SIGMA, 150KD ~ 300KD, for subsequent use.
7. the preparation method of saliva uric acid rapid semi-quantitative pick-up unit according to claim 3, it is characterized in that: the poly-D-lysine treating fluid described in step (d) to be mixed with methyl alcohol by poly-D-lysine and forms, wherein poly-D-lysine concentration is 0.5% composition, methanol concentration is 10%, through 0.22 μm of membrane filtration, wherein poly-D-lysine, SIGMA, 150KD ~ 300KD, for subsequent use.
8. the preparation method of saliva uric acid rapid semi-quantitative pick-up unit according to claim 3, it is characterized in that: the poly-D-lysine treating fluid described in step (d) is mixed formed by poly-D-lysine, methyl alcohol, PEG20000, wherein poly-D-lysine concentration is 0.5% composition, methanol concentration is 10%, PEG20000 concentration is 0.2%, through 0.22 μm of membrane filtration, wherein poly-D-lysine, SIGMA, 150KD ~ 300KD, for subsequent use.
9. the preparation method of saliva uric acid rapid semi-quantitative pick-up unit according to claim 3, it is characterized in that: step (d) uric acid-carrier protein is combined with nano SiO 2 particle, method is: using silicon dioxide as carrier, get 1mL uric acid-carrier protein solution, mix with ethanol and deionized water and stirring, add quantitative ammoniacal liquor and the positive silicic acid of 0.3mL ester again, stir after 1 hour, add the positive silicic acid of residue ester, Keep agitation, whole reaction lucifuge is carried out, respectively with 12000rpm, 8500rpm and 7000rpm collects for centrifugal 10 minutes, absolute ethyl alcohol, deionized water respectively washes 2 times.
10. the preparation method of saliva uric acid rapid semi-quantitative pick-up unit according to claim 9, it is characterized in that: the preparation of the described nano SiO 2 particle of step (d) is: the ammoniacal liquor of 2.48mL volume fraction 25 and 43.2mL absolute ethyl alcohol are mixed in conical flask, under about 35 DEG C and ultrasound condition, with the speed of 0.4mL/min instillation 3.5mL ethyl orthosilicate, after dripping off ultrasonic 5 minutes again, the particle that particle diameter 120nm is homogeneous can be obtained, respectively with 12000rpm, 8500rpm and 7000rpm collects for centrifugal 10 minutes, outwell supernatant, add water ultrasonic disperse again. and repeated multiple times washing is to weakly acidic pH, finally be settled to volume with water identical with volume when just preparing, deposit stand-by.
The preparation method of 11. saliva uric acid rapid semi-quantitative pick-up units according to claim 3, is characterized in that: in step (d), uric acid-carrier protein comprises ovalbumin (OVA), bovine serum albumin(BSA) (BSA), hemocyanin (KLH).
The preparation method of 12. saliva uric acid rapid semi-quantitative pick-up units according to claim 3, it is characterized in that: the sample pad treating fluid that the pretreated sample pad described in step (e) adopts is made up of Tris-HCL liquid, bovine serum albumin(BSA) BSA, casein, surfactant AEO, wherein the concentration of Tris-HCL liquid is 0.1mol/L, bovine serum albumin(BSA) BSA concentration is 0.5 ~ 1%, and casein concentration is 0.1 ~ 0.2%, surfactant AEO concentration is 0.5 ~ 1%.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106370861A (en) * 2016-08-25 2017-02-01 北京金华科生物技术有限公司 C reactive protein saliva test paper strip and preparation method thereof
CN110297082A (en) * 2019-06-12 2019-10-01 杭州启创生物技术有限公司 The method of the markers such as saliva uric acid specific detection and diagnosis gout
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Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001083163A (en) * 1999-09-17 2001-03-30 Fuji Photo Film Co Ltd High density macro-array
CN101957366A (en) * 2009-07-15 2011-01-26 英科新创(厦门)科技有限公司 Human erythrocyte membrane antigen coated microsphere and application thereof
CN102135535A (en) * 2010-01-25 2011-07-27 刘凤鸣 Immune colloidal metal detection technology capable of directly performing semi-quantitative analysis, preparation method and application
CN102135498A (en) * 2010-01-25 2011-07-27 刘凤鸣 Semi-quantitative colloidal metal detection technology taking multi-capture property as characteristic and preparation method and use thereof
CN102735835A (en) * 2011-04-15 2012-10-17 佳木斯大学 Disposable whole-blood uric acid detection test strip and manufacturing method thereof
WO2013013214A1 (en) * 2011-07-20 2013-01-24 Access Bio, Inc. Method for detecting microorganisms
CN102998443A (en) * 2011-09-14 2013-03-27 佳木斯大学 Immune colloidal gold test strip for detecting uric acid in urine, and its manufacturing method
CN103954753A (en) * 2014-05-12 2014-07-30 中国科学院苏州生物医学工程技术研究所 Quantitative determination method of immune chromatography test strip
CN104749363A (en) * 2015-04-04 2015-07-01 吉林双正医疗科技有限公司 Joint detection device and preparation method of helicobacter pylori urease antibodies IgM and IgG

Family Cites Families (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6306642B1 (en) * 1997-11-24 2001-10-23 Quidel Corporation Enzyme substrate delivery and product registration in one step enzyme immunoassays
FI20012608A0 (en) * 2001-12-31 2001-12-31 Biofons Oy Diagnostic methods
US20050175980A1 (en) * 2002-04-09 2005-08-11 Government Of The United States Of America, Repres By The Secretary, Dept. Of Health And Human Serv. Quantitative assay of the angiogenic and antiangiogenic activity of a test molecule
CN201096785Y (en) * 2007-03-30 2008-08-06 万华普曼生物工程有限公司 Nitrogen-terminal brain natriuretic peptide/C-reaction albumen/troponin I diagnosis testing paper
CN102062735B (en) * 2009-11-18 2013-09-18 江苏迈迪基因生物科技有限公司 Biomarker diagnostic kit for acute coronary syndrome
PL390673A1 (en) * 2010-03-10 2011-09-12 Stanisław Kafel Urease test for detection of Helicobacter pylori
CN102323422B (en) * 2011-05-30 2014-03-12 中国科学院上海微系统与信息技术研究所 Immunochromatographic test strip for semi-quantitatively and simultaneously detecting cTnI and Myo and preparation method thereof
US20140127791A1 (en) * 2011-07-28 2014-05-08 Siemens Aktiengesellschaft Diagnostic device
CN102944677A (en) * 2012-11-26 2013-02-27 深圳市伯劳特生物制品有限公司 Type detection kit and preparation method thereof for hand-foot -mouth diseases (HFMD)
CN103267855A (en) * 2013-05-06 2013-08-28 南京凯基生物科技发展有限公司 CTnI, Myo and CK-MB three-in-one colloidal gold test strip, kit thereof, and making methods of test strip and kit
CN203241420U (en) * 2013-05-17 2013-10-16 杭州赛凯生物技术有限公司 Novel joint detection reagent
CN103954778A (en) * 2014-05-22 2014-07-30 江苏金标世纪生物科技有限公司 Myocardial infarction triple rapid detection kit and preparation method for same
CN104090098A (en) * 2014-06-13 2014-10-08 南京东检生物科技有限公司 Ultrasensitive immune detection technology based on silica nanoparticle enhancement
CN204479590U (en) * 2015-04-04 2015-07-15 吉林双正医疗科技有限公司 Helicobacter Pylori urease antibody IgM, IgG combine device for fast detecting

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001083163A (en) * 1999-09-17 2001-03-30 Fuji Photo Film Co Ltd High density macro-array
CN101957366A (en) * 2009-07-15 2011-01-26 英科新创(厦门)科技有限公司 Human erythrocyte membrane antigen coated microsphere and application thereof
CN102135535A (en) * 2010-01-25 2011-07-27 刘凤鸣 Immune colloidal metal detection technology capable of directly performing semi-quantitative analysis, preparation method and application
CN102135498A (en) * 2010-01-25 2011-07-27 刘凤鸣 Semi-quantitative colloidal metal detection technology taking multi-capture property as characteristic and preparation method and use thereof
CN102735835A (en) * 2011-04-15 2012-10-17 佳木斯大学 Disposable whole-blood uric acid detection test strip and manufacturing method thereof
WO2013013214A1 (en) * 2011-07-20 2013-01-24 Access Bio, Inc. Method for detecting microorganisms
CN102998443A (en) * 2011-09-14 2013-03-27 佳木斯大学 Immune colloidal gold test strip for detecting uric acid in urine, and its manufacturing method
CN103954753A (en) * 2014-05-12 2014-07-30 中国科学院苏州生物医学工程技术研究所 Quantitative determination method of immune chromatography test strip
CN104749363A (en) * 2015-04-04 2015-07-01 吉林双正医疗科技有限公司 Joint detection device and preparation method of helicobacter pylori urease antibodies IgM and IgG

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106370861B (en) * 2016-08-25 2018-11-20 北京金华科生物技术有限公司 A kind of c reactive protein saliva test strip and preparation method thereof
CN106370861A (en) * 2016-08-25 2017-02-01 北京金华科生物技术有限公司 C reactive protein saliva test paper strip and preparation method thereof
US11828768B2 (en) 2017-11-20 2023-11-28 Lg Chem, Ltd. Device and method for qualitative and quantitative analysis of heavy metals utilizing rotary disc system
US20200254449A1 (en) * 2017-11-20 2020-08-13 Lg Chem, Ltd. Device and Method for Qualitative and Quantitative Analysis of Heavy Metals Utilizing Rotary Disc System
US11835536B2 (en) * 2017-11-20 2023-12-05 Lg Chem, Ltd. Device and method for qualitative and quantitative analysis of heavy metals utilizing rotary disc system
US11835535B2 (en) 2017-11-20 2023-12-05 Lg Chem, Ltd. Device and method for qualitative and quantitative analysis of heavy metals utilizing rotary disc system
CN110297082A (en) * 2019-06-12 2019-10-01 杭州启创生物技术有限公司 The method of the markers such as saliva uric acid specific detection and diagnosis gout
CN111638364A (en) * 2020-04-30 2020-09-08 吉林省格瑞斯特生物技术有限公司 GDF15 rapid quantitative fluorescent microsphere detection device and preparation method thereof
CN111638366B (en) * 2020-04-30 2021-02-19 吉林省格瑞斯特生物技术有限公司 G-17, PGI and PGII combined detection device and preparation method thereof
CN111638365A (en) * 2020-04-30 2020-09-08 吉林省格瑞斯特生物技术有限公司 GDF15 quick quantitative detection device and preparation method thereof
CN111638366A (en) * 2020-04-30 2020-09-08 吉林省格瑞斯特生物技术有限公司 G-17, PGI and PGII combined detection device and preparation method thereof
CN112754535A (en) * 2021-01-20 2021-05-07 东北农业大学 Pig saliva collection system
CN114354951A (en) * 2021-12-20 2022-04-15 安徽惠邦生物工程有限公司 Folate and vitamin B12 combined detection kit and preparation method thereof

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