CN104459117A - Carbon nano-tube procalcitonin detection kit and preparation method thereof - Google Patents
Carbon nano-tube procalcitonin detection kit and preparation method thereof Download PDFInfo
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- CN104459117A CN104459117A CN201410740721.6A CN201410740721A CN104459117A CN 104459117 A CN104459117 A CN 104459117A CN 201410740721 A CN201410740721 A CN 201410740721A CN 104459117 A CN104459117 A CN 104459117A
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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Abstract
The invention discloses a carbon nano-tube procalcitonin detection kit and a preparation method thereof. A test paper base plate is sequentially adhered with a sample pad, a carbon nano-tube colloidal gold pad, a nitrocellulose membrane and a water absorbent pad. The carbon nano-tube procalcitonin detection kit is characterized in that the carbon nano-tube colloidal gold pad is adhered with a carbon nano-tube colloidal gold compound labeled procalcitonin monoclonal antibody 1, and the nitrocellulose membrane is coated with a detection line and a quality control line of a procalcitonin monoclonal antibody 2. According to the kit disclosed by the invention, procalcitonin in a sample is detected by adopting a carbon nano-tube colloidal gold labelling technology so as to judge whether bacterial infection occurs. The carbon nano-tube procalcitonin detection kit disclosed by the invention has the characteristics of simplicity in operation, rapid reaction, high sensitivity, strong specificity and the like, and is suitable for field detection and self-detection.
Description
Technical field
The present invention relates to a kind of external diagnosis reagent case, specifically, relate to a kind of carbon nano-tube Procalcitonin detection kit and preparation method thereof, be applicable to the Procalcitonin in clinical examination sample.
Background technology
Colloidal gold immunochromatographimethod technology is widely used in multiple fields such as disease markers detection, food inspection, scientific research, selected trace labelling material is generally collaurum, latex particle etc., chromatographic material is generally cellulose membrane, comprise nitrocellulose filter, cellulose acetate membrane, cellulose mixture film etc., wherein the most frequently used is nitrocellulose filter.Under current technical conditions, the tracer materials such as collaurum and nitrocellulose filter is used to be difficult to improve detection sensitivity again as carrier.
The not high reason of sensitivity is a lot, comprise raw material, technique etc., but the underlying cause is the limitation of collaurum method itself, improves sensitivity again by traditional collaurum method is very difficult.Now general resolving ideas increases amplification system in collaurum system, such as adopts biotin-avidin amplification system.
Summary of the invention
The invention provides a kind of carbon nano-tube Procalcitonin detection kit and preparation method thereof, its technical matters to be solved is: provide a kind of carbon nano-tube colloidal gold composite, as trace labelling material, for marking Procalcitonin antibody, improve the sensitivity detected.
the present invention adopts following technical scheme to realize:
A kind of carbon nano-tube Procalcitonin detection kit, is characterized in that: this kit is with carbon nano-tube colloidal gold composite mark Procalcitonin antibody.
Test paper base plate is pasted with successively sample pad, carbon nano-tube colloidal gold composite pad, nitrocellulose filter and adsorptive pads; Described carbon nano-tube colloidal gold composite pad is attached with the Procalcitonin monoclonal antibody 1 of carbon nano-tube colloidal gold composite mark, described nitrocellulose filter is coated with detection line and the nature controlling line of Procalcitonin monoclonal antibody 2.
Carbon nano-tube 4 used is the one in Single Walled Carbon Nanotube, multi-walled carbon nano-tubes, hydroxylation carbon nano-tube, carboxylic carbon nano-tube, water-soluble carbon nanometer tube.
Carbon nano-tube 4 and collaurum 3 form carbon nano-tube colloidal gold composite by self assembly.
Described carbon nano-tube colloidal gold composite, wherein carbon nano-tube 4 accounts for colloidal gold composite gross mass is 0.1 ~ 20.0%.
A kind of preparation method of carbon nano-tube Procalcitonin detection kit as above, it is characterized in that: the Procalcitonin monoclonal antibody 1 described carbon nano-tube colloidal gold composite pad being attached with carbon nano-tube colloidal gold composite mark, described nitrocellulose filter is coated with detection line and the nature controlling line of Procalcitonin monoclonal antibody 2; Concrete steps are as follows:
(1) preparation of carbon nano tube dispersion liquid: get the surfactant adding 0.5 ~ 5.0g in 100ml pure water, after stirring, adds 10 ~ 1000mg carbon nano-tube 4, and the ultrasonic 2 ~ 10min of ultrasonic cell disruptor, obtains carbon nanotube dispersed solution;
(2) preparation of carbon nano-tube colloidal gold composite: take the Procalcitonin antibody that collaurum 3 has marked, adds the above-mentioned carbon nano tube dispersion liquid of mass ratio 0.1 ~ 30.0%, stirs 5 ~ 30 minutes; Centrifugal 30 ~ 60min;
(3) preparation of carbon nano-tube colloidal gold composite pad: abandoning supernatant, then in centrifuge tube, add gold mark dilution, after mixing, by above-mentioned solution spraying on the gold mark pad processed, drying for standby;
(4) detection line, nature controlling line draw film: be affixed on base plate by NC film, stand-by; The Procalcitonin monoclonal antibody 2 of preparation 0.5mg/ml; Compound concentration is 1.0mg/ml sheep anti-mouse antibody; Respectively on ready NC film before, draw film with 1.0 μ l/cm by point film instrument again, be respectively T, C line, drying at room temperature 2 hours;
(5) assemble: more dried colloidal gold pad and thieving paper, sample pad, the base plate that posts NC film are assembled, then the test strips of 4.0mm is cut into by automatic cutting machine, again test strips and cartridge are carried out pressure shell, then kit and drying agent, suction pipe are assembled and sealed.
Advantage and effect: it is generally acknowledged are 10 when the colloid gold particle of every square millimeter gathers
7time individual, detection line could obviously develop the color, and just can improve detection sensitivity by the accumulated amount increasing collaurum.In line with this thinking, adopt carbon nano-tube and collaurum to combine the method forming compound and improve detection sensitivity.
Carbon nano-tube is a kind of novel nano-material, lightweight, and hexagonal structure connects perfect, has many abnormal mechanics, electricity and chemical property.Carbon nano-tube is existing many research in immunoassay, mainly concentrates on immunosensor field, can increase substantially detection sensitivity.Carbon nano-tube is divided into Single Walled Carbon Nanotube, multi-walled carbon nano-tubes, hydroxylation carbon nano-tube, carboxylic carbon nano-tube, also can obtain water-soluble carbon nanometer tube after treatment, has good adsorption separation performance, and the carbon nano-tube of functionalization more easily and protein bound.The compound labelled protein of carbon nano-tube and collaurum, can improve the sensitivity detected.
Procalcitonin (PCT) is a kind of protein, and when serious bacterial, fungi, parasitic infection and pyemia and MOFE, its level in blood plasma raises.When autoimmunity, allergy and virus infections, PCT can not raise.
In respiratory tract bacterial infection patient, the Procalcitonin Concentrations in sputum and pharyngeal juice all can raise, and its elevated levels, generally higher than blood, so detect the Procalcitonin content in sputum and throat swab, can diagnose the type of respiratory tract infection.
accompanying drawing illustrates:
Fig. 1 is carbon nano-tube colloidal gold composite micromechanism schematic diagram of the present invention.
Mark in figure: collaurum 3, carbon nano-tube 4.
embodiment:
A carbon nano-tube can adsorb multiple colloid gold particle; as long as wherein have one or several antibody and determined antigen to form sandwich complex on detection line; just can other colloid gold particles in carbon nano-tube be accumulated on detection line simultaneously, increase colored intensity, thus improve detection sensitivity.
Fig. 1 is carbon nano-tube colloidal gold composite schematic diagram, and collaurum and carbon nano-tube can form compound by self assembly, and a carbon nano-tube can adsorb multiple gold grain.In general colloidal gold method, a gold grain marked and a determined antigen combine, and on detection line, form centre-fills colour developing.After adopting carbon nano-tube colloidal gold composite, if a gold grain in carbon nano-tube and determined antigen combine, take detection line to together with the gold grain that so just other in carbon nano-tube can not combined with determined antigen and stop colour developing, increase the accumulated amount of gold grain at detection line, color developing effect is more obvious.In most cases, determined antigen will be less than the antibody of gold mark, and antibody is often excessive in other words, especially all the more so when detecting trace antigen, such as detects trace P CT.In this case, a large amount of gold grains do not combined with determined antigen does not stop at detection line, and direct chromatography is gone over, and colloidal gold method sensitivity that Here it is is difficult to the basic reason improved.By adopting carbon nano-tube colloidal gold composite technology, the colloid gold particle in conjunction with determined antigen just can be allowed to stop on detection line, thus improve detection sensitivity.
A kind of carbon nano-tube Procalcitonin detection kit and preparation method thereof, described carbon nano-tube colloidal gold composite pad is attached with the Procalcitonin monoclonal antibody 1 of carbon nano-tube colloidal gold composite mark, described nitrocellulose filter is coated with detection line and the nature controlling line of Procalcitonin monoclonal antibody 2.
The preparation of carbon nano tube dispersion liquid: get the surfactant adding 0.5 ~ 5.0g in 100ml ultrapure water, after stirring, adds 10 ~ 1000mg carbon nano-tube, and the ultrasonic 2 ~ 10min of ultrasonic cell disruptor, obtains carbon nanotube dispersed solution;
The preparation of carbon nano-tube colloidal gold composite: take the Procalcitonin antibody that colloid gold label is good, adds the above-mentioned carbon nano tube dispersion liquid of mass ratio 0.1 ~ 30.0%, stirs 5 ~ 30 minutes.Centrifugal 30 ~ 60min.
The preparation of carbon nano-tube colloidal gold composite pad: abandoning supernatant, then in centrifuge tube, add gold mark dilution, after mixing, by above-mentioned solution spraying on the gold mark pad processed, drying for standby.
Below in conjunction with embodiment, the present invention is further elaborated, but protection scope of the present invention not limit by embodiment.
Embodiment
1, the preparation of carbon nano tube dispersion liquid: get the Tween-20 adding 2.0g in 100ml pure water, after stirring, adds 20mg Single Walled Carbon Nanotube, and the ultrasonic 2min of ultrasonic cell disruptor, obtains the carbon nano-tube solution of 0.2mg/ml;
2, colloid gold label Procalcitonin antibody: get the colloidal gold solution 100ml prepared and add in beaker, measured by acidometer, with the solution of potassium carbonate adjust pH of 0.2M for 8.0, adds the Procalcitonin monoclonal antibody 1, magnetic agitation 30min of 0.5mg.
3, the preparation of carbon nano-tube colloidal gold composite: take the Procalcitonin antibody that colloid gold label is good, adds the above-mentioned carbon nano tube dispersion liquid of 0.1ml, stirs 30 minutes.Low-temperature centrifugation 30min.
4, the preparation of carbon nano-tube colloidal gold composite pad: abandoning supernatant, then in centrifuge tube, add gold mark dilution, after mixing, by above-mentioned solution spraying on gold mark pad, drying for standby.
5, detection line, nature controlling line draw film: be affixed on base plate by NC film, stand-by.The Procalcitonin monoclonal antibody 2 of preparation 0.5mg/ml; Compound concentration is 1.0mg/ml sheep anti-mouse antibody; Respectively on ready NC film before, draw film with 1.0 μ l/cm by point film instrument again, be respectively T, C line, drying at room temperature 2 hours.
6, assemble: more dried colloidal gold pad and thieving paper, sample pad, the base plate that posts NC film are assembled, then the test strips of 4.0mm is cut into by automatic cutting machine, again test strips and cartridge are carried out pressure shell, then kit and drying agent, suction pipe are assembled and sealed.
Minimum detectability
The Procalcitonin quality-control product (coming from Landau company) of preparation variable concentrations, detect by this detection kit, result shows that this detection kit minimum detectability is 0.1ng/ml.About 5 times are improved than traditional collaurum method sensitivity.
Clinical examination
The clinical testing of this kit, via the routine clinical research of two provincial Grade A hospital 240, may be used for the sxemiquantitative inspection of Procalcitonin in sputum or throat swab, and the coincidence rate of blood testing is 100%.
Claims (5)
1. a carbon nano-tube Procalcitonin detection kit, is characterized in that: this kit carbon nano-tube colloidal gold composite mark Procalcitonin antibody; Test paper base plate is pasted with sample pad, carbon nano-tube colloidal gold composite pad, nitrocellulose filter and adsorptive pads successively; Described carbon nano-tube colloidal gold composite pad is attached with the Procalcitonin monoclonal antibody 1 of carbon nano-tube colloidal gold composite mark, described nitrocellulose filter is coated with detection line and the nature controlling line of Procalcitonin monoclonal antibody 2.
2. carbon nano-tube Procalcitonin detection kit according to claim 1, is characterized in that: carbon nano-tube (4) used is Single Walled Carbon Nanotube, one in multi-walled carbon nano-tubes, hydroxylation carbon nano-tube, carboxylic carbon nano-tube, water-soluble carbon nanometer tube.
3. carbon nano-tube Procalcitonin detection kit according to claim 1, is characterized in that: carbon nano-tube (4) and collaurum (3) form carbon nano-tube colloidal gold composite by self assembly.
4. carbon nano-tube Procalcitonin detection kit according to claim 1, is characterized in that: described carbon nano-tube colloidal gold composite, and wherein carbon nano-tube (4) accounts for colloidal gold composite gross mass is 0.1 ~ 20.0%.
5. the preparation method of a carbon nano-tube Procalcitonin detection kit as claimed in claim 1, it is characterized in that: the Procalcitonin monoclonal antibody 1 described carbon nano-tube colloidal gold composite pad being attached with carbon nano-tube colloidal gold composite mark, described nitrocellulose filter is coated with detection line and the nature controlling line of Procalcitonin monoclonal antibody 2; Concrete steps are as follows:
(1) preparation of carbon nano tube dispersion liquid: get the surfactant adding 0.5 ~ 5.0g in 100ml pure water, after stirring, add 10 ~ 1000mg carbon nano-tube (4), the ultrasonic 2 ~ 10min of ultrasonic cell disruptor, obtains carbon nanotube dispersed solution;
(2) preparation of carbon nano-tube colloidal gold composite: take the Procalcitonin antibody that collaurum (3) has marked, adds the above-mentioned carbon nano tube dispersion liquid of mass ratio 0.1 ~ 30.0%, stirs 5 ~ 30 minutes; Centrifugal 30 ~ 60min;
(3) preparation of carbon nano-tube colloidal gold composite pad: abandoning supernatant, then in centrifuge tube, add gold mark dilution, after mixing, by above-mentioned solution spraying on the gold mark pad processed, drying for standby;
(4) detection line, nature controlling line draw film: be affixed on base plate by NC film, stand-by; The Procalcitonin monoclonal antibody 2 of preparation 0.5mg/ml; Compound concentration is 1.0mg/ml sheep anti-mouse antibody; Respectively on ready NC film before, draw film with 1.0 μ l/cm by point film instrument again, be respectively T, C line, drying at room temperature 2 hours;
(5) assemble: more dried colloidal gold pad and thieving paper, sample pad, the base plate that posts NC film are assembled, then the test strips of 4.0mm is cut into by automatic cutting machine, again test strips and cartridge are carried out pressure shell, then kit and drying agent, suction pipe are assembled and sealed.
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| CN201410740721.6A CN104459117A (en) | 2014-12-08 | 2014-12-08 | Carbon nano-tube procalcitonin detection kit and preparation method thereof |
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| CN201410740721.6A CN104459117A (en) | 2014-12-08 | 2014-12-08 | Carbon nano-tube procalcitonin detection kit and preparation method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105424933A (en) * | 2016-01-26 | 2016-03-23 | 姜竹泉 | Collaurum immunochromatography test strip detecting mycoplasma pneumoniae and preparing method thereof |
| CN107543933A (en) * | 2016-06-24 | 2018-01-05 | 江苏雷森生物科技有限公司 | A kind of preparation method of carbon nano-particle of antibody labeling and the early pregnancy test strips using its preparation |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN202794178U (en) * | 2012-08-07 | 2013-03-13 | 天津中新科炬生物制药有限公司 | Fast quantitative immunochromatographic assay kit for procalcitonin |
| CN103278637A (en) * | 2013-06-05 | 2013-09-04 | 姜竹泉 | Carbon nanotube test paper for detecting helicobacter pylori, and preparation method thereof |
| CN104090109A (en) * | 2014-07-25 | 2014-10-08 | 胡晓武 | Colloidal gold immunochromatography test paper and colloidal gold immunochromatography test method for quickly detecting human blood procalcitonin |
-
2014
- 2014-12-08 CN CN201410740721.6A patent/CN104459117A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN202794178U (en) * | 2012-08-07 | 2013-03-13 | 天津中新科炬生物制药有限公司 | Fast quantitative immunochromatographic assay kit for procalcitonin |
| CN103278637A (en) * | 2013-06-05 | 2013-09-04 | 姜竹泉 | Carbon nanotube test paper for detecting helicobacter pylori, and preparation method thereof |
| CN104090109A (en) * | 2014-07-25 | 2014-10-08 | 胡晓武 | Colloidal gold immunochromatography test paper and colloidal gold immunochromatography test method for quickly detecting human blood procalcitonin |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105424933A (en) * | 2016-01-26 | 2016-03-23 | 姜竹泉 | Collaurum immunochromatography test strip detecting mycoplasma pneumoniae and preparing method thereof |
| CN107543933A (en) * | 2016-06-24 | 2018-01-05 | 江苏雷森生物科技有限公司 | A kind of preparation method of carbon nano-particle of antibody labeling and the early pregnancy test strips using its preparation |
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Application publication date: 20150325 |