CN204405682U - The test card of use in conjunction quantum dot and rare-earth luminescent material - Google Patents
The test card of use in conjunction quantum dot and rare-earth luminescent material Download PDFInfo
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- CN204405682U CN204405682U CN201520103651.3U CN201520103651U CN204405682U CN 204405682 U CN204405682 U CN 204405682U CN 201520103651 U CN201520103651 U CN 201520103651U CN 204405682 U CN204405682 U CN 204405682U
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Abstract
The utility model provides the test card of a kind of use in conjunction quantum dot and rare-earth luminescent material, it shell comprising test strips and be coated with described test strips, wherein said test strips comprises sample application zone, be coated with the first antibody that individually marked by quantum dot and rare-earth luminescent material and the antigen-binding site by biotin labeled second antibody simultaneously, there is the detection zone of detection zone and the quality control band be coated with separately, suction zones, and base plate, and described shell comprises the Ka Gai being provided with well and detect aperture and the base be connected to each other with described Ka Gai.Test card of the present utility model has high sensitivity, the advantage such as easy to use.
Description
Technical field
The utility model relates to a kind of test card for immune detection, is specifically related to the test card of a kind of use in conjunction quantum dot and rare-earth luminescent material.
Background technology
In clinical diagnosis field, the method for testing goal mark conventional at present comprises: euzymelinked immunosorbent assay (ELISA), chemoluminescence method, latex enhancing immune turbidimetry, colloidal gold immunity chromatography, fluorescence immune chromatography method etc.Euzymelinked immunosorbent assay (ELISA) and chemoluminescence method detection sensitivity high, can accurate quantitative analysis be realized, but it is consuming time long, and this instant mensuration of single increment cannot be carried out.Though latex enhancing immune turbidimetry can realize high flux screening, due to the restriction of methodology itself, its detection sensitivity is lower, cannot carry out Accurate Measurement to low value sample.Colloidal gold immunity chromatography is simple to operate, without the need to special instrument and equipment, but can only realize sxemiquantitative.In fluorescence immune chromatography method, though organic fluorescent compounds such as fluorescein isothiocyanate (fitc), the rhodamine fluorescent dye etc. of tradition application have very high sensitivity in aqueous, but during the biological sample immune analysis determination such as serum, body fluid, cell for complexity after its labelled antibody or antigen, once the substrate concentration to be checked in sample in low concentration of water at ordinary times, the concentration of coexistent impurity will be much higher than substrate concentration to be checked.Now, the coexistent impurity reasons for its use fluorescence of high concentration will cause serious interference to the fluorometric assay of immune product, and then significantly reduces the sensitivity of fluorescence immunoassay, this greatly limits its application in clinical diagnosis field.
Comparatively speaking, quantum dot then has the optical property of some uniquenesses not available for traditional organic fluorescent dye.Quantum dot size is between several nm to tens nm.The principle of luminosity of quantum dot is, when the size of quantum dot little to certain value time, owing to being subject to the impact of quantum size effect, originally continuous print level structure becomes the discontinuous construction of similar atom.When illumination is mapped on quantum dot, quantum dot absorb photons, excited after Electron absorption energy in valence band, transit to conduction band, valence band then can produce the hole of answering with the electron pair that is excited, the electronics transitted on conduction band can come back to valence band in the mode of radiation transistion again, launches photon with the hole-recombination in valence band.
The optical property of quantum dot uniqueness is mainly reflected in the following aspects: size and composition 1, by changing quantum dot can realize the regulation and control to quantum dot emission spectrum, thus obtain the quantum dot of multiple different glow color, and the fluorescence emission spectrum of quantum dot can be made to cover whole visible region; 2, wide excitation spectrum: the light of same excitation wavelength can excite the quantum dot of different size, the fluorescence that the quantum dot emission of different size goes out is different, is conducive to the application in synchronous detection; 3, large Stokes displacement (hundreds of nm can be reached) and the narrow and emission spectrum of symmetry, this characteristic makes effectively to avoid spectra overlapping when using quantum dot; With 4, quantum dot has good light stability, and its fluorescence can keep for a long time and can not fade.
It is made to be applicable to low concentration level based on quantum dot unique optical properties, the Accurate Determining of thing content as to be checked in pg/ml-ng/ml, but its range of linearity detected is wide not, within the scope of identical a set of detection system, cannot realize the accurate quantitative analysis of high level sample.Therefore, when being used alone quantum dot, the detection sensitivity of low side and the wide range of linearity cannot be taken into account.And other to have fluorescent element or the fluorescent material of similar excitation wavelength and emission wavelength to quantum dot by use in conjunction, the wider range of linearity is obtained by being expected to, and can be implemented in the reading carrying out fluorescence signal result in same set of fluorescence detecting system, be also more conducive in clinical popularization and application.
Utility model content
technical matters
Based on lacking the method being applicable to clinical sample and fast, accurately, quantitatively detecting at present, the problem cannot taking into account low side detection sensitivity and the wide range of linearity particularly existed at present, the utility model provides the test card of a kind of use in conjunction quantum dot and rare-earth luminescent material.
technical scheme
On the one hand, the utility model provides the test card of a kind of use in conjunction quantum dot and rare-earth luminescent material, it is characterized in that, described test card comprises:
I) test strips, wherein said test strips comprises:
I-1) sample application zone,
I-2) be coated with the first antibody individually marked by quantum dot and rare-earth luminescent material and the antigen-binding site by biotin labeled second antibody simultaneously,
I-3) have the detection zone of detection zone and the quality control band be coated with separately, wherein said detection zone is fixed with Avidin, and described quality control band is fixed with the antibody of against murine IgG,
I-4) suction zones, and
I-5) base plate, the order arrangement on described base plate of wherein said sample application zone, antigen-binding site, detection zone and suction zones also overlaps in turn; And
Ii) shell of coated described test strips, wherein said shell comprises:
Ii-1) Ka Gai of well and detect aperture is provided with, wherein said well is opened on the upside of described sample application zone, and to expose the subregion of described sample application zone, described detect aperture is opened on the upside of described detection zone, to expose the Zone Full of described detection zone and described quality control band, and
Ii-2) base be connected to each other with described Ka Gai.
In one embodiment, described quantum dot is microspheres form and has the particle size range of 2nm-30nm, and described quantum dot is selected from following group: CdTe, CdSe, PbSe, InP and GaN.
In one embodiment, described antigen-binding site is coated with microballoon further, wherein said micro-ball load has described rare-earth luminescent material and described first antibody.
In one embodiment, the particle size of described microballoon is 50nm-400nm.
In one embodiment, the particle size of described microballoon is 100nm-300nm.
In one embodiment, the particle size of described microballoon is 100nm or 300nm.
In one embodiment, the different epi-positions of described first antibody and second antibody identification testing protein of the same race, described albumen is selected from following group: cardiac muscle troponin I, N terminal brain natriuretic peptide, cardic fatty acid binding protein, Procalcitonin, c reactive protein and DDi.
In one embodiment, be interposed between 0.1-0.6cm between described detection zone and described quality control band.
In one embodiment, the width of described test strips is between 0.2cm-1cm.
In one embodiment, described Ka Gai and described base are made of plastics.
beneficial effect
The major advantage of test card of the present utility model comprises: use in conjunction quantum dot and rare-earth luminescent material, the problem that the fabulous low value sensitivity overcoming application quantum dot or rare-earth luminescent material existence separately and the detection range of linearity cannot be taken into account; By introducing biotin-labeled pentylamine Cascaded amplification system, further increase detection sensitivity; Be easy to carry about with one with test card and preserve, being easy to realize in corresponding fluorescence detector the analysis of ELISA test strip result.
Accompanying drawing explanation
The vertical view of Fig. 1 test card of the present utility model.
The side view of Fig. 2 test strips of the present utility model.
The vertical view of Fig. 3 test strips of the present utility model.
Reference numeral:
1 base; 2 Ka Gai; 3 wells; 4 detect aperture; 5 detection zone exposure position; 6 quality control band exposure position; 7 sample application zone; 8 antigen-binding site; 9 detection zones; 10 detection zone; 11 quality control bands; 12 suction zones; With 13 base plates.
Embodiment
Hereinafter come with reference to the accompanying drawings to describe illustrative embodiments in more detail.Described accompanying drawing is used for illustrating the utility model, but not is limited.In the accompanying drawings, in order to clearly illustrate, be exaggerated size and the relative scale thereof of test strips subregion.
As shown in Figure 1, test card of the present utility model comprises base 1 and Ka Gai 2, card lid 2 is provided with well 3 and detect aperture 4, and wherein detect aperture 4 is to having detection zone exposure position 5 and quality control band exposure position 6.Test strips (not shown) is closely buckled in test card inside each other by base 1 and Ka Gai 2.Base 1 and Ka Gai 2 all can be made up of such as plastic or other material.Well 3 and detect aperture 4 can be the shapes such as square, rectangle, ellipse, circle.The length of well can be 0.1cm to 0.5cm, and width can be 0.2cm to 0.4cm.The length of detect aperture 4 can be 1.0cm to 2.3cm, and width can be 0.2cm to 0.4cm.Test card of the present utility model realizes quick large batch of clinical sample by automatic biochemical analyzer and detects.
Fig. 2 is the side view of reagent strip in test card of the present utility model.
As shown in Figure 2, test strips of the present utility model comprises sample application zone 7, antigen-binding site 8, detection zone 9, suction zones 12 and base plate 13, and wherein said sample application zone 7, antigen-binding site 8, detection zone 9 and suction zones 12 on base 13 order arrangement also overlap in turn.
Sample application zone 7 for receiving sample, its can by being selected from glass fibre element film or polyester film, cotton fine material makes, be preferably made up of glass fibre element film.Sample application zone 7 can play the effect of sample filtering.Such as, when detected sample is blood plasma, serum, sample application zone 7 can graininess insolubles in filtered sample.Sample application zone 7 overlaps with antigen-binding site 8 in the structure of reagent strip.The mode that sample application zone 7 and antigen-binding site 8 overlap is not by the restriction of mode shown in Fig. 1.In one embodiment, sample application zone 7 is overlapped on antigen-binding site 8.In one embodiment, antigen-binding site 8 is overlapped on sample application zone 7.
Antigen-binding site 8 between sample application zone 7 and detection zone 9, and laps one another with both.The mode that antigen-binding site 8 and sample application zone 7 and detection zone 9 overlap is not by the restriction of mode shown in Fig. 1.Antigen-binding site 8 is for arranging and the antibody that specific protein in testing sample reacts.In one embodiment, the specific protein in described testing sample is selected from the group of following albumen composition: cardiac muscle troponin I, cardic fatty acid binding protein, Procalcitonin, N terminal brain natriuretic peptide, c reactive protein and DDi.In a preferred embodiment, described specific protein is cardiac muscle troponin I.
In an embodiment of reagent strip of the present utility model, antigen-binding site 8 is coated with simultaneously the first antibody that individually marked by quantum dot and rare-earth luminescent material and and by biotin labeled second antibody.In reagent strip of the present utility model, specificity is for the different epi-positions of testing protein respectively for described first antibody and second antibody, and namely described first antibody and second antibody are respectively in conjunction with the different epi-positions of specific protein.
In the utility model, term " mark " comprises employing chemistry or physical method makes the first antibody of anti-testing protein directly be connected with quantum dot and rare-earth luminescent material.But mark also can be completed by alternate manner, the first antibody of such as rare-earth luminescent material and anti-testing protein is by such as embedding or the mode of load on microballoon reach the object of " mark " simultaneously.Therefore, in one embodiment, antigen-binding site 8 can have microballoon (not shown) further, on described microballoon, embedding or load have the first antibody of rare-earth luminescent material and anti-testing protein simultaneously.In one embodiment, embedding or can have chemistry between the rare-earth luminescent material of load on described microballoon and the first antibody of anti-testing protein and connect, such as, be present on described microballoon with the form of the first antibody of the anti-testing protein of rare-earth luminescent material mark.In another embodiment, embedding or can not have chemistry between the rare-earth luminescent material of load on described microballoon and the first antibody of anti-testing protein and connect, namely on described microballoon, embedding or load have to each other without the rare-earth luminescent material of chemistry connection and the first antibody of anti-testing protein.
In one embodiment, described microballoon can be polymer microballoon, such as polystyrene microsphere.In one embodiment, the particle size scope of microballoon, between 50nm-400nm, preferably between 100nm-300nm, is more preferably 100nm or 300nm.
The method preparing rare-earth fluorescent microballoon is that prior art is known, includes but not limited to: investment, namely in the polymerization process of polymer microballoon, adds rare earth material complex; Bonding method, even if there is coordination reaction in the functional group on rare earth element ion and polymer lateral chain obtains rare-earth fluorescent microballoon; And swelling method and cladding process.Latter two method is all on the basis of monodispersed microballoon (as: polystyrene microsphere), by the infiltration of rare earth material complex or the inside or the surface that are coated on microballoon, by controlling reaction conditions, can realize finally preparing the polymer microballoon that monodispersity is good, fluorescence intensity is high, fluorescent stability is good.
Lateral size of dots, usually between 2nm-30nm, presents microspheroidal.The electronics of quantum dot and hole are by quantum confinement, and continuous band structure becomes the discrete energy levels structure with molecular characterization, so quantum dot can emitting fluorescence after being excited.Quantum dot is adopted to carry out degree of accuracy and the sensitivity that labelled antibody can improve detection greatly.Although antibody quantum dot-labeled is separately highly sensitive, quantum dot mark is presented as that high level detection is inaccurate separately.Although the antibody of independent rare-earth luminescent material mark possesses the good range of linearity, sensitivity haves much room for improvement, and is presented as that low value detects inaccurate.
The utility model is by coupling quantum dot and rare-earth luminescent material, and quantum dot-labeled thing is conducive to the detection of low value to reach the requirement of sensitivity, and rare-earth luminescent material label makes the range of linearity detected can cover high level region.
In one embodiment, described quantum dot has the particle diameter of 2nm-30nm.In a preferred embodiment, described quantum dot has the particle diameter of 2nm-20nm.In a preferred embodiment, described quantum dot has the particle diameter of 5nm-20nm.In one embodiment, described quantum dot is II-VI group quantum dot (as CdTe, CdSe etc.).In another embodiment, described quantum dot is iii-v quantum dot (as InP, GaN etc.).In another embodiment, described quantum dot is group IV-VI quantum dot (as PbSe etc.).
In one embodiment, described rare-earth luminescent material is one or several in fluorescent material containing the various lanthanide series such as europium (Eu) ion, terbium (Tb) ion, samarium (Sm) ion, neodymium (Nd) ion, dysprosium (Dy) ion.In one embodiment, rare-earth luminescent material is that one or several in rare-earth luminescent material containing the various lanthanide series such as europium (Eu) ion, terbium (Tb) ion, samarium (Sm) ion, neodymium (Nd) ion, dysprosium (Dy) ion load to the rare-earth fluorescent microballoon that microballoon is formed.Rare-earth luminescent material is loaded to the stability that microballoon can further improve fluorescence.
Detection zone 9 between antigen-binding site 8 and suction zones 12, and laps one another with both.The mode that detection zone 9 and antigen-binding site 8 and suction zones 12 overlap is not by the restriction of mode shown in Fig. 1.Detection zone 9 is coated with detection zone 10 and quality control band 11.Detection zone 10 is fixed with Avidin, quality control band 11 is fixed with the antibody of against murine IgG.Detection zone 10 and quality control band 11 spaced apart, the distance between it can between 0.05cm to 0.8cm.In one embodiment, be interposed between between detection zone and quality control band between 0.1cm to 0.6cm, preferably between 0.3cm to 0.5cm.Detect in the process of specific protein at application reagent card of the present utility model, have employed double antibody sandwich method and biotin-avidin Cascaded amplification system simultaneously, which further improves detection sensitivity, to the requirement of sensitivity when ensure that specific protein detects.
Suction zones 12 can be made up of the material being selected from by velveteen, glass, is preferably made up of velveteen.
The entire length of test strips is between 8cm to 10cm, and overall width, between 0.2cm to 0.5cm, preferably between 0.2cm to 0.3cm, most preferably is 0.3cm.Test strips of the present utility model is being provided sensitivity and while detecting the range of linearity, can reduced the consumption of antibody, greatly saved production cost by the length and/or width reducing test strips.
Fig. 3 is the schematic side view of the vertical view of test strips of the present utility model.
As shown in Figure 3, test strips of the present utility model comprises order arrangement on base 13 and overlaps sample application zone 7, antigen-binding site 8, detection zone 9 and suction zones 12 in turn.Length after base plate 13 can overlap in turn than sample application zone 7, antigen-binding site 8, detection zone 9 and suction zones 12 and width wider and/or longer.In a preferred embodiment, base plate 13 overlap in turn with sample application zone 7, antigen-binding site 8, detection zone 9 and suction zones 12 after length and width consistent.No longer repeated description is carried out to content identical with Fig. 1 with Fig. 2 in Fig. 3.
Test card of the present utility model has at least following plurality of advantages: the use of the coupling of quantum dot and rare-earth luminescent material and double antibody sandwich method and biotin-avidin technology substantially increases and detects detection sensitivity and improve the range of linearity; Be easy to carry about with one with reagent card design, preserve and reading in fluorescence detector, and structure is simple, material therefor wide material sources, cheap, be applicable to very much large-scale promotion application clinically.
Embodiment
Following examples for illustration of the utility model, but should not be construed as and are limited to embodiment set forth herein.In addition, only to detect cardiac muscle troponin I, test card of the present utility model is described in following examples.
In following embodiment, described cardiac muscle troponin I first antibody, cardiac muscle troponin I second antibody, dynamics, Avidin, biotin, quantum dot, rare-earth europium fluorescent microsphere, europium oxide (Eu
2o
3), 4,4'-bis-(1,1', 2,2', 3, the fluoro-4' of 3'-seven, 6'-acetyl butyryl-6'-base)-chlorine sulphonyl-ortho-terphenyl (BHHCT), 2-(N-morpholine) ethyl sulfonic acid (MES), carbodiimide (EDC), N-hydroxy-succinamide (NHS), dimethyl formamide (DMF), N, N'-dicyclohexylcarbodiimide (DCC), pyridine, bovine serum albumin(BSA) (BSA), Tween-20, trehalose, Superdex200 filler, glass fibre element film, polyester film, nitrocellulose filter, high-intensity water absorbent paper is commercially available prod.
The test strips of described use in conjunction quantum dot and rare-earth luminescent material and the preparation method of test card are:
1) preparation of sample application zone
Glass fibre element film or polyester film are put into the pretreatment fluid (0.1M containing 1%BSA and 0.1%Tween-20, boric acid-the borate buffer solution of pH8.0-8.6 or the 0.05M containing 1%BSA and 0.1%Tween-20, the Tris-HCl damping fluid of pH7.4-8.2) middle after immersion treatment 3-5 hour, in 35-39 DEG C of air dry oven, dry 3-5 hour.
2) preparation of antigen-binding site
The pre-service of A, antigen-binding site
Glass fibre element film or polyester film are put into the pretreatment fluid (0.1M containing 1%BSA and 0.1%Tween-20, boric acid-the borate buffer solution of pH8.0-8.6 or the 0.05M containing 1%BSA and 0.1%Tween-20, the Tris-HCl damping fluid of pH7.4-8.2) middle after immersion treatment 3-5 hour, in 35-39 DEG C of air dry oven, dry 3-5 hour.
The preparation of B, quantum dot-labeled antibody
Use 0.05M, 2-(N-morpholine) ethyl sulfonic acid (MES) activation buffer of pH4.5-5.0 washs the quantum dot containing carboxylic group in modified surface, adding carbodiimide (EDC) and N-hydroxy-succinamide (NHS) makes the two final concentration be 15-60mmol, room temperature reaction 20-30 minute, abundant washing quantum dot, with 0.05M, the cardiac muscle troponin I first antibody that same buffer was dialysed is added after boric acid-borate buffer solution redissolution of pH8.0-8.6, the mass ratio of first antibody and quantum dot is made to be 1:5 to 1:25, room temperature reaction is after 2 hours, add the 0.02M containing 10%BSA, the phosphate buffer room temperature of pH7.2-7.6 closes 30 minutes, abundant washing quantum dot, with the 0.05M containing 1%BSA and 0.1%Tween-20, the conserving liquid of the Tris-HCl damping fluid of pH7.4-8.2 redissolves to original volume.
The preparation (1) of C, rare-earth luminescent material labelled antibody
Eu is dissolved with 6M HCl
2o
3, and be settled to final concentration 1mg/ml with ultrapure water, then use 0.05MpH8.0NH
4hCO
3solution dilution 500 times.By BHHCT anhydrous alcohol solution to final concentration 10mg/ml.Use the carbonate mark damping fluid of 0.05-0.2M, pH9.0-9.5 to cardiac muscle troponin I first antibody dialysis 4-6 time, or ultrafiltration centrifugation is washed 4-6 time, adjustment concentration is to 0.25mg/ml.Add in first antibody solution by BHHCT ethanol solution with the ratio of volume ratio 1:200, room temperature lucifuge continues mixing reaction 1-3 hour.Use 0.05M pH8.0NH
4hCO
3solution desalination.Use Superdex200 filler purifying bond in 1*30cm post, with 0.1%BSA solution equilibria 1-3 hour, with 0.05M Tris-HCl wash-out, flow velocity is 1ml/min.Protein peak is collected, with 0.22 μm of filter aseptic filtration according to 280nm absorbance.Dilute 20 times with 0.05M Tris-HCl again, add Eu afterwards
3+nH
4hCO
3solution, the ratio being 1:5 to 1:15 according to volume ratio is added, and room temperature lucifuge continues mixing reaction 1-2 hour.Clean with 0.05M Tris-HCl with ultrafiltration centrifugation and be concentrated into antibody concentration and be greater than 5mg/ml, for subsequent use.
The preparation (2) of D, rare-earth luminescent material labelled antibody
Use 0.05M, the rare-earth europium fluorescent microsphere of 2-(N-morpholine) ethyl sulfonic acid (MES) the activation buffer washing of pH4.5-5.0, adding carbodiimide (EDC) and N-hydroxy-succinamide (NHS) makes the two final concentration be 15-60mmol, room temperature reaction 20-30 minute, abundant washing rare-earth europium fluorescent microsphere, with 0.05M, the first antibody of dialysing is added after boric acid-borate buffer solution redissolution of pH8.0-8.6, the mass ratio of cardiac muscle troponin I first antibody and fluorescent microsphere is made to be 1:8 to 1:60, room temperature reaction is after 2 hours, add the 0.02M containing 10%BSA, the phosphate buffer room temperature of pH7.2-7.6 closes 30 minutes, abundant washing rare-earth europium fluorescent microsphere, with the 0.05M containing 1%BSA and 0.1%Tween-20, the conserving liquid of the Tris-HCl damping fluid of pH7.4-8.2 redissolves to original volume.
The mixing of E, quantum dot-labeled antibody and rare-earth luminescent material labelled antibody
The rare-earth luminescent material labelled antibody prepared in the quantum dot-labeled antibody prepared in the step B of this part and step C or step D is mixed in the ratio that antibody concentration is 1:5 to 5:1, namely prepares the double-tagging potpourri of the quantum dot-labeled antibody of associating and rare-earth luminescent material labelled antibody.
The preparation of F, biotinylated antibody
By cardiac muscle troponin I second antibody with phosphate buffer 4 DEG C of dialysed overnight of 0.02M, pH7.2-7.6, adjustment concentration is 5mg/ml-10mg/ml.
The pre-activate of biotin: accurately take 300mg biotin and be dissolved in the dimethyl formamide (DMF) of 8ml, the N-hydroxy-succinamide (NHS) of 183mg is added under room temperature condition, the N of 304mg, the pyridine of N'-dicyclohexylcarbodiimide (DCC) and 0.1ml, at room temperature stirs 24 hours by reaction mixture.Cross the urea filtering reaction and produce, by silica gel chromatography after filtrate is concentrated, the white solid that recrystallization obtains in isopropyl alcohol is preactivated biotin.
Preactivated biotin is dissolved with dimethyl formamide (DMF), its final concentration is made to be 0.05M, and join in the second antibody of dialysing, the mol ratio of second antibody and biotin is made to be 1:10-1:25, room temperature reaction 1 hour, with phosphate buffer 4 DEG C of dialysed overnight of 0.02M, pH7.2-7.6.
The preparation of G, antigen-binding site
The biotinylated antibody that quantum dot-labeled antibody this part step e prepared and the double-tagging potpourri of rare-earth luminescent material labelled antibody and step F prepare fully mixes, use and quantitatively spray film instrument with on the plain film of the glass fibre that 2 μ l/cm-8 μ l/cm even application are pretreated in above-mentioned steps A or polyester film, in 35-39 DEG C of air dry oven, dry 2-4 hour, add drying agent and seal up for safekeeping for subsequent use.
3) preparation of detection zone
The preparation of A, detection zone: use the coating buffer (0.01-0.02M containing 0.1-10% trehalose, the Tris-HCl damping fluid of pH7.4-8.2) Avidin is diluted to the concentration of 1mg/ml-5mg/ml, use quantitatively spray film instrument with 1.2 μ l/cm even application on nitrocellulose filter, in 35-39 DEG C of air dry oven, dry 2-4 hour.
The preparation of B, quality control band: use the coating buffer (0.01-0.02M containing 0.1-10% trehalose, the Tris-HCl damping fluid of pH7.4-8.2) anti-mouse IgG is diluted to the concentration of 0.5mg/ml-3mg/ml, use quantitatively spray film instrument with 0.8 μ l/cm even application on nitrocellulose filter, in 35-39 DEG C of air dry oven, dry 2-4 hour.
The preparation of C, detection zone: with the confining liquid (0.01-0.02M containing 1-10%BSA and 1-10% trehalose, the Tris-HCl damping fluid of pH7.4-8.2) by the cellulose nitrate membrane closure 1-2 hour containing detection zone and quality control band, take out in rearmounted 35-39 DEG C air dry oven and dry 2-4 hour, envelope is for subsequent use.
4) assembling of test strips
Be less than 30% in humidity, under temperature 20-30 DEG C of condition, carry out the assembling of test strips.Base plate selects PVC material, connects in turn and the partly overlapping sample application zone of adjacent regions, antigen-binding site, detection zone and suction zones in the direction of base plate the same face.When detection zone covers base plate, the position of detection zone is near antigen-binding site, and the position of quality control band is near suction zones.Suction zones is high-intensity water absorbent paper.Test strips is cut into after assembling.
5) assembling of test card
Be less than 30% in humidity, under temperature 20-30 DEG C of condition, carry out the assembling of test card.Above-mentioned test strips is placed in the centre position of plastic feet, the base plate of test strips is connected with plastic feet, and fastens plastic clip lid.Card covers and is provided with well and detect aperture, and well is opened on the upside of sample application zone, exposes the subregion of sample application zone, and detect aperture is opened on the upside of described detection zone, exposes the Zone Full of detection zone and quality control band.
6) mensuration of calibration object and measuring samples
The standard items of 100 μ l or measuring samples are joined the well of test card, carry out immunochromatography reaction, after 10-15 minute, test card is placed in special fluorescence detector, carry out quantitative measurement by the size reading fluorescence signal.
Above embodiment is only in order to the citing done clearly is described, the restriction not to embodiment.For those of ordinary skill in the field, other improvements and modifications can be made on the basis of the above description, exhaustive without the need to also giving all embodiments here.And apparent improvements and modifications amplified thus are still within protection domain of the present utility model.
Claims (10)
1. a test card for use in conjunction quantum dot and rare-earth luminescent material, is characterized in that, described test card comprises:
I) test strips, wherein said test strips comprises:
I-1) sample application zone,
I-2) be coated with the first antibody individually marked by quantum dot and rare-earth luminescent material and the antigen-binding site by biotin labeled second antibody simultaneously,
I-3) have the detection zone of detection zone and the quality control band be coated with separately, wherein said detection zone is fixed with Avidin, and described quality control band is fixed with the antibody of against murine IgG,
I-4) suction zones, and
I-5) base plate, the order arrangement on described base plate of wherein said sample application zone, antigen-binding site, detection zone and suction zones also overlaps in turn; And
Ii) shell of coated described test strips, wherein said shell comprises:
Ii-1) Ka Gai of well and detect aperture is provided with, wherein said well is opened on the upside of described sample application zone, and to expose the subregion of described sample application zone, described detect aperture is opened on the upside of described detection zone, to expose the Zone Full of described detection zone and described quality control band, and
Ii-2) base be connected to each other with described Ka Gai.
2. test card as claimed in claim 1, it is characterized in that, described quantum dot is microspheres form and has the particle size range of 2nm-30nm, and described quantum dot is selected from following group: CdTe, CdSe, PbSe, InP and GaN.
3. as the test card of claim 1 or 2, it is characterized in that, described antigen-binding site is coated with microballoon further, wherein said micro-ball load has described rare-earth luminescent material and described first antibody.
4. test card as claimed in claim 3, it is characterized in that, the particle size of described microballoon is 50nm-400nm.
5. test card as claimed in claim 4, it is characterized in that, the particle size of described microballoon is 100nm-300nm.
6. test card as claimed in claim 5, it is characterized in that, the particle size of described microballoon is 100nm or 300nm.
7. test card as claimed in claim 1, it is characterized in that, the different epi-positions of described first antibody and second antibody identification testing protein of the same race, described albumen is selected from following group: cardiac muscle troponin I, N terminal brain natriuretic peptide, cardic fatty acid binding protein, Procalcitonin, c reactive protein and DDi.
8. test card as claimed in claim 1, is characterized in that, be interposed between 0.1-0.6cm between described detection zone and described quality control band.
9. test card as claimed in claim 1, it is characterized in that, the width of described test strips is between 0.2cm-1cm.
10. test card as claimed in claim 1, it is characterized in that, described Ka Gai and described base are made of plastics.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107085116A (en) * | 2017-05-16 | 2017-08-22 | 张子林 | It is a kind of to detect kit of calprotectin and preparation method thereof in human faecal mass sample |
| CN107290529A (en) * | 2017-08-08 | 2017-10-24 | 广州市微米生物科技有限公司 | A kind of immune chromatography test paper for detecting heart infarction heart failure and preparation method thereof |
-
2015
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107085116A (en) * | 2017-05-16 | 2017-08-22 | 张子林 | It is a kind of to detect kit of calprotectin and preparation method thereof in human faecal mass sample |
| CN107290529A (en) * | 2017-08-08 | 2017-10-24 | 广州市微米生物科技有限公司 | A kind of immune chromatography test paper for detecting heart infarction heart failure and preparation method thereof |
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Effective date of registration: 20180613 Address after: 225300 Taizhou, Jiangsu, China, west side of Tai Chi Road, China's medicine, building G59, 63 to three, west of Lu Jia Lu. Patentee after: JIANGSU DAJUN BIOTECHNOLOGY Co.,Ltd. Address before: 100176 Beijing Daxing District Hongda North Road 12 innovation building B block 3 District 210 Patentee before: ABZYMO BIOSCIENCES Co.,Ltd. |
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