CN109444413B - A kind of detection device - Google Patents
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- CN109444413B CN109444413B CN201811123774.8A CN201811123774A CN109444413B CN 109444413 B CN109444413 B CN 109444413B CN 201811123774 A CN201811123774 A CN 201811123774A CN 109444413 B CN109444413 B CN 109444413B
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- 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 kind of detection devices, the content for the target substance in quantitative detection sample.Quantitative detection can be carried out to the target molecule in sample by comparing detection zone signal strength and reference region signal strength.The detection device production cost is low, and testing result is accurate and reliable.
Description
Technical field
The present invention relates to field of biological detection, and in particular to a kind of for detecting the detection device of ingredient in sample.
Background technique
Current Fast Detection Technique development is swift and violent, has been widely used for clinic, food safety, legal medical expert, environment, military affairs etc.
Numerous areas.But these technology major applications are difficult to carry out quantitative detection in qualitative detection field.Marking signal it is uniform
Property, reaction time, reaction temperature and humidity, reaction speed etc. factors can all influence reaction result, and then increase variation lines
Number, constrains the development of Fast Detection Technique precise quantitative, so that the target of quantitative detection be not achieved.With presently most common
For immuno-chromatographic test paper strip, low temperature can significantly reduce test strips detection signal strength；Reaction time it is too long and it is too short all will be straight
Connect the signal ratio influenced between detection zone and reference region；Tomography Velocity of the different samples in chromatographic film can difference,
It equally will affect quantitative result；The homogeneity of chromatography membrane material also will affect the signal ratio between detection zone and reference region.Inspection
The inconsistent reaction time of survey area and reference region is also a major reason for causing immunochromatography quantitative result unstable.
Summary of the invention
The present invention provides a kind of detection device, make detection zone and reference region the same time, it is same under the conditions of react,
To improve the accuracy of quantitative detection.
The technical problems to be solved by the invention are achieved by the following technical programs:
A kind of detection device, the detection device are equipped with mutually independent detection zone and reference region, and detection zone and reference
Area is located at the same cross-section of sample flow direction.
The detection device can be immuno-chromatographic test paper strip, can also be micro-fluidic chip or other pass through sample flow
Detection zone and reference region are crossed to realize the device of detection, the detection zone and reference that the detection device passes through measurement adjacent position
The signal ratio in area determines the content of target molecule in sample.
The detection device sample flow direction same cross-section there are two or more than two detection zones, one or
More than one reference region.
In the present invention, the detection zone and reference region are according to " detection zone, reference region, detection zone, reference region ... "
Sequence is arranged in the same cross-section of sample flow direction.
In the present invention, the shape of the detection zone and/or reference region be regular pattern, line it is any, be also possible to
Other shapes；Wherein, the regular pattern includes round, rectangular, rectangle, ellipse, polygon, star-shaped at least one.
In the present invention, same cross-section difference detection zone the concentration of coated capture molecule can be different.
The invention has the following beneficial effects:
Detection device provided by the invention makes detection zone and reference region simultaneously participate in reaction, the various items in reaction process
Part infinite approach, quantitative result are more accurate.In order to further increase quantitative accuracy, the present invention also provides multiple detections
The detection device in area and reference region.Detection scheme provided by the present invention in addition to testing result accurately other than, be also easy to scale metaplasia
It produces, not will increase production cost.
Detailed description of the invention
Fig. 1 is that the structure of the chromatography membrane element of 6 immuno-chromatographic test paper strip of option A and embodiment in the embodiment of the present invention 1 is shown
It is intended to (detection zone, a reference region)；
Note: 11. detection zones；12. reference region；13. chromatographic film；Arrow: liquid flow direction.
Fig. 2 is structural schematic diagram (one of the chromatography membrane element of option b immuno-chromatographic test paper strip in the embodiment of the present invention 1
Detection zone, a reference reference region)；
Note: 14. detection zones；15. reference reference region；13. chromatographic film；Arrow: liquid flow direction.
Fig. 3 is that the structural schematic diagram of the chromatography membrane element of option A immuno-chromatographic test paper strip in the embodiment of the present invention 2 is (multiple
Detection zone, multiple reference regions)；
Note: 21. detection zones；22. reference region；23. chromatographic film；Arrow: liquid flow direction.
Fig. 4 is the structural schematic diagram of the chromatography membrane element of option b immuno-chromatographic test paper strip in the embodiment of the present invention 2；
Note: 24. detection zones；25. reference region reference region；23. chromatographic film；Arrow: liquid flow direction.
Fig. 5 is the relevant piping schematic of option A micro-fluidic chip detection zone and reference region in the embodiment of the present invention 3；
Note: 31. detection zones；32. reference region；Arrow: liquid flow direction.
Specific embodiment
The present invention is described in detail with reference to the accompanying drawings and examples.
1 immuno-chromatographic test paper strip quantitative detection Serum Alpha Fetoprotein (AFP) of embodiment
A. this embodiment scheme:
1. nanoparticle label detection antibody (nano particle-detection antibody coupling matter): taking 100 μ L concentration is 10mg/mL
Rare-earth fluorescent nano particle (partial size: 300nm) in 900 μ L MES(2- (N- morpholine) ethanesulfonic acids, 50mM, PH6.1) in, be vortexed
It mixes；20mg NHS(N- HOSu NHS is added), 20mg EDC(1- (3- dimethylamino-propyl) -3- ethyl carbon two it is sub-
Amine), it is vortexed and mixes；React at room temperature 30min；10000rpm is centrifuged 10min, abandons supernatant；Vortex resuspended particle；AFP detection is added
20 μ g of antibody reacts at room temperature 2~4h；Casein is added, make its final concentration of 0.1%, the reaction was continued 2~4h；10000rpm centrifugation
10min abandons supernatant；With PBST(phosphate Tween buffer) wash particle three times；2mL particle re-suspension liquid (10mM is added
Tris-HCl buffer, 1%BSA(bovine serum albumin(BSA)), 5% trehalose) resuspended particle, it is spare.
2. nanoparticle label reference antibody (nano particle-reference antibody conjugate): taking 100 μ L concentration is 10mg/mL
Rare-earth fluorescent nano particle (partial size: 300nm) in 900 μ L MES(50mM, PH6.1) in, be vortexed mix；20mg is added
NHS, 20mg EDC are vortexed and mix；React at room temperature 30min；10000rpm is centrifuged 10min, abandons supernatant；Vortex resuspended particle；Add
Enter 40 μ g of melamine monoclonal antibody, reacts at room temperature 2~4h；Casein is added, make its final concentration of 0.1%, the reaction was continued 2
~4h；10000rpm is centrifuged 10min, abandons supernatant；Particle is washed three times with PBST；2mL particle re-suspension liquid (10mM is added
Tris-HCl, 1%BSA, 5% trehalose) resuspended particle, it is spare.
3. the preparation of bonding pad: the nano particle prepared-detection antibody coupling matter and nano particle-reference antibody is even
Join object to mix according to isometric, then be sprayed on glass fibre element film according to 3 μ L/cm, is placed in 37 DEG C of oven drying, it is spare.
4. the assembling of test strips: nitrocellulose filter (chromatographic film 13) is pasted to the middle position of PVC bottom plate.Then,
Bonding pad, sample pad and blotting paper are pasted in corresponding position, takes sample pad, bonding pad, chromatographic film 13, blotting paper successively
It connects and is pasted on PVC bottom plate, there is the overlap joint of about 1mm or so between any two；Finally it is cut into the immuno-chromatographic test paper strip of 5.5mm wide.
5. the processing of test strips: using 2mg/ml respectively in the middle position (at distance sample pad end 30mm) of chromatographic film 13
AFP captures antibody and 2mg/ml melamine-BSA(bovine serum albumin(BSA)) conjugate specking, respectively as detection zone 11 and ginseng
Than area 12, the detection zone 11 and reference region 12 are disposed side by side on the x wire perpendicular to the test strips long side, convenient for detection
Area 11 and reference region 12 contact and react with sample to be tested simultaneously.37 DEG C of drying are placed in, 2-8 DEG C is subsequently placed in, drying, is protected from light place
It saves.
6. AFP in quantitative detection serum: test strips are lain against on level table, 180 μ L serum are added dropwise in sample pad,
15min is stood, quantitative detection is carried out using quantitative detection equipment.
B. scheme is compared: existing immuno-chromatographic test paper strip
1. the preparation that nanoparticle label detects antibody (nano particle-detection antibody coupling matter): referring to the present embodiment " A.
This embodiment scheme ".
2. the preparation of nanoparticle label reference antibody (nano particle-reference antibody conjugate): referring to the present embodiment
" A. this embodiment scheme ".
3. the preparation of bonding pad: referring to the present embodiment " A. this embodiment scheme ".
4. the preparation of chromatographic film 13: according to 0.8 μ L/cm amount toward in chromatographic film 13 along chromatography direction successively spraying concentration
Antibody (detection zone 14), 2mg/mL melamine-BSA(bovine serum albumin(BSA) are captured for the AFP of 2mg/mL) conjugate (reference region
15) 37 DEG C of dry 2h, are placed in, it is spare.
5. the assembling of test strips: nitrocellulose filter (chromatographic film 13) is pasted to the middle position (detection zone of PVC bottom plate
14 close to sample pad end, reference region 15 close to blotting paper end).Then, bonding pad, sample pad and suction are pasted in corresponding position
Water paper makes sample pad, bonding pad chromatographic film 13, blotting paper successively overlap and be pasted on PVC bottom plate, has about 1mm left between any two
Right overlap joint.It is finally cut into the immuno-chromatographic test paper strip of 5.5mm wide, is saved in 2-8 DEG C, the dry, place of being protected from light.
6. AFP in quantitative detection serum: test strips are lain against on level table, 180 μ L serum are added dropwise in sample pad,
15min is stood, quantitative detection is carried out using quantitative detection equipment.
Evaluation of result: compared with the prior art (option b), detection stability obviously mentions immuno-chromatographic test paper strip (option A)
It rises, experimental data is for statistical analysis with two schemes replication 10 times referring to table 1(same concentration).
1 this embodiment scheme of table (option A) and existing immuno-chromatographic test paper strip (option b) detection AFP comparison
Note: the coefficient of variation (coefficient of variation), also known as coefficient of dispersion (coefficient of
Dispersion) or relative deviation (rsd), it is the ratio between standard deviation sd and average value mean, is indicated with percentage, calculation formula
Are as follows: cv=sd/mean × 100%.The coefficient of variation is bigger, illustrates more unstable.
2 immuno-chromatographic test paper strip quantitative detection galectin-3 of embodiment
A. this embodiment scheme:
1. nanoparticle label detection antibody (nano particle-detection antibody coupling matter): taking 100 μ L concentration is 10mg/mL
Rare-earth fluorescent nano particle (partial size: 300nm) in 900 μ L MES(50mM, PH6.1) in, be vortexed mix；20mg is added
NHS, 20mg EDC are vortexed and mix；React at room temperature 30min；10000rpm is centrifuged 10min, abandons supernatant；Vortex resuspended particle；Add
Enter galectin-3 detection 50 μ g of antibody, reacts at room temperature 2~4h；Casein is added, make its final concentration of 0.1%, continue anti-
Answer 2~4h；10000rpm is centrifuged 10min, abandons supernatant；Particle is washed three times with PBST；2mL particle re-suspension liquid (10mM is added
Tris-HCl, 1%BSA, 5% trehalose) resuspended particle, it is spare.
2. nanoparticle label reference antibody (nano particle-reference antibody conjugate): taking 100 μ L concentration is 10mg/mL
Rare-earth fluorescent nano particle (partial size: 300nm) in 900 μ L MES(50mM, PH6.1) in, be vortexed mix；20mg is added
NHS, 20mg EDC are vortexed and mix；React at room temperature 30min；10000rpm is centrifuged 10min, abandons supernatant；Vortex resuspended particle；Add
Enter 40 μ g of melamine monoclonal antibody, reacts at room temperature 2~4h；Casein is added, make its final concentration of 0.1%, the reaction was continued 2
~4h；10000rpm is centrifuged 10min, abandons supernatant；Particle is washed three times with PBST；2mL particle re-suspension liquid (10mM is added
Tris-HCl, 1%BSA, 5% trehalose) resuspended particle, it is spare.
3. the preparation of bonding pad: the nano particle prepared-detection antibody coupling matter and nano particle-reference antibody is even
Join object to mix according to isometric, then be sprayed on glass fibre element film according to 3 μ L/cm, is placed in 37 DEG C of oven drying, it is spare.
4. the assembling of test strips: nitrocellulose filter (chromatographic film 23) is pasted to the middle position of PVC bottom plate.Then,
Bonding pad, sample pad and blotting paper are pasted in corresponding position, takes sample pad, bonding pad chromatographic film 23, blotting paper successively
It connects and is pasted on PVC bottom plate, there is the overlap joint of about 1mm or so between any two.Finally it is cut into the immuno-chromatographic test paper strip of 6.0mm.
5. the processing of test strips: as shown in figure 3, in the middle position (at distance sample pad end 30mm) of chromatographic film 23 difference
Antibody and 2mg/ml melamine-BSA conjugate specking are captured with 2mg/ml galectin-3, as detection zone 21 and ginseng
Than area 22(according to " detection zone 21, reference region 22, detection zone 21, reference region 22, detection zone 21, reference region 22 " sequence side by side
It is placed perpendicular to an x wire of the test strips long side, is contacted simultaneously with sample to be tested convenient for detection zone 21 and reference region 22
And reaction).37 DEG C of drying are placed in, is subsequently placed at dry and saves.
6. galectin-3 in quantitative detection serum: test strips being lain against on level table, 180 μ L serum are added dropwise
In in sample pad, 15min is stood, quantitative detection is carried out using quantitative detection equipment.
B. scheme is compared:
1. the preparation that nanoparticle label detects antibody (nano particle-detection antibody coupling matter): referring to the present embodiment " A.
This embodiment scheme ".
2. the preparation of nanoparticle label reference antibody (nano particle-reference antibody conjugate): referring to the present embodiment
" A. this embodiment scheme ".
3. the preparation of bonding pad: referring to the present embodiment " A. this embodiment scheme ".
4. the preparation of chromatographic film 23: as shown in figure 4, according to 0.8 μ L/cm amount toward in chromatographic film 23 along chromatography direction according to
The galectin-3 that secondary spraying concentration is 2mg/mL captures antibody (detection zone 24), 2mg/mL melamine-BSA(ox blood
Pure albumen) conjugate (reference region 25), 37 DEG C of dry 2h are placed in, it is spare.
5. the assembling of test strips: nitrocellulose filter (chromatographic film 23) is pasted to the middle position (detection zone of PVC bottom plate
24 close to sample pad end, reference region 25 close to blotting paper end).Then, bonding pad, sample pad and suction are pasted in corresponding position
Water paper makes sample pad, bonding pad chromatographic film 23, blotting paper successively overlap and be pasted on PVC bottom plate, has about 1mm left between any two
Right overlap joint.It is finally cut into the immuno-chromatographic test paper strip of 5.5mm wide, is saved in 2-8 DEG C, the dry, place of being protected from light.
6. galectin-3 in quantitative detection serum: test strips being lain against on level table, 180 μ L serum are added dropwise
In in sample pad, 15min is stood, quantitative detection is carried out using quantitative detection equipment.
Evaluation of result: compared with the prior art (option b), detection stability obviously mentions immuno-chromatographic test paper strip (option A)
It rises, experimental data is for statistical analysis with two schemes replication 10 times referring to table 2(same concentration).
2 this embodiment scheme of table (option A) and existing immuno-chromatographic test paper strip (option b) detect galectin-3 pair
Than
3 micro-fluidic chip quantitative detection insulin-like growth factor binding protein (IGFBP-7) of embodiment
Reference region 32 and detection zone 31 on the micro-fluidic chip are located at the same cross-section of liquid flowing, or are located at
The differential responses region that liquid flows through simultaneously.Although detection zone 31 and reaction zone are located at the differential responses region of chip, to be checked
Liquid flows through two regions simultaneously, to guarantee that the synchronous of reaction carries out.
Evaluation of result: being to be located at same liquid flow direction cross-section using reference region 32 and detection zone 31 in table 3
(reference region and detection zone 31 are located at different liquids flow direction cross to micro-fluidic chip (option A, as shown in Figure 5) with the prior art
At section, option b) experimental result of IGFBP-7 in detection serum.It can be seen that option A detection stability is obviously improved (same concentration
It is for statistical analysis with two schemes replication 10 times).
3 this embodiment scheme of table (A) and existing scheme (B) detect IGFBP-7
4 immunochromatography of embodiment detects quantitative detection Serum Alpha Fetoprotein (AFP)
Concrete scheme is referring to A and B in embodiment 1.
This embodiment differs from embodiment 1 in that: the processing of step 5 test strips is changed in option A: in chromatographic film
Middle position (at distance sample pad end 30mm) is respectively with 2mg/ml, 1.2mg/ml, 0.6mg/ml AFP capture antibody and 2mg/
Ml melamine-BSA(bovine serum albumin(BSA)) conjugate specking, as detection zone and reference region (according to " detection zone (2mg/ml
AFP captures antibody), reference region (2mg/ml melamine-BSA), detection zone (1.2mg/ml AFP capture antibody), reference region
The sequence of (2mg/ml melamine-BSA), detection zone (0.6mg/ml AFP captures antibody) " is disposed side by side on perpendicular to described
One x wire of test strips long side is contacted and is reacted with sample to be tested simultaneously convenient for detection zone and reference region).It is placed in 37 DEG C of bakings
It is dry, it is subsequently placed in 2-8 DEG C, the dry, place's of being protected from light preservation.
Evaluation of result: compared with the prior art (option b), detection stability obviously mentions immuno-chromatographic test paper strip (option A)
It rises, and compared with option A in embodiment 1, the coefficient of variation is significantly reduced.Experimental data is referring to table 4(same concentration with two kinds
Scheme replication 10 times for statistical analysis).
4 this embodiment scheme of table (option A) and existing immuno-chromatographic test paper strip (option b) detection AFP comparison
5 immuno-chromatographic test paper strip quantitative detection chloramphenicol of embodiment
A. this embodiment scheme:
1. nanoparticle label detection antibody (nano particle-detection antibody coupling matter): taking 100 μ L concentration is 10mg/mL
Rare-earth fluorescent nano particle (partial size: 300nm) in 900 μ L MES(50mM, PH6.1) in, be vortexed mix；20mg is added
NHS, 20mg EDC are vortexed and mix；React at room temperature 30min；10000rpm is centrifuged 10min, abandons supernatant；Vortex resuspended particle；Add
Enter chloramphenicol detection 15 μ g of antibody, reacts at room temperature 2~4h；Casein is added, make its final concentration of 0.1%, the reaction was continued 2~4h；
10000rpm is centrifuged 10min, abandons supernatant；Particle is washed three times with PBST；Addition 2mL particle re-suspension liquid (10mM Tris-HCl,
1%BSA, 5% trehalose) resuspended particle, it is spare.
2. nanoparticle label reference antibody (nano particle-reference antibody conjugate): taking 100 μ L concentration is 10mg/mL
Rare-earth fluorescent nano particle (partial size: 300nm) in 900 μ L MES(50mM, PH6.1) in, be vortexed mix；20mg is added
NHS, 20mg EDC are vortexed and mix；React at room temperature 30min；10000rpm is centrifuged 10min, abandons supernatant；Vortex resuspended particle；Add
Enter 15 μ g of melamine monoclonal antibody, reacts at room temperature 2~4h；Casein is added, make its final concentration of 0.1%, the reaction was continued 2
~4h；10000rpm is centrifuged 10min, abandons supernatant；Particle is washed three times with PBST；2mL particle re-suspension liquid (10mM is added
Tris-HCl, 1%BSA, 5% trehalose) resuspended particle, it is spare.
3. the preparation of bonding pad: the nano particle prepared-detection antibody coupling matter and nano particle-reference antibody is even
Join object to mix according to isometric, is then sprayed on glass fibre element film (width: 6mm) according to 2 μ L/cm, be placed in 37 DEG C of ovens
Drying, it is spare.
4. the assembling of test strips: nitrocellulose filter (chromatographic film) is pasted to the middle position of PVC bottom plate.Then, exist
Corresponding position pastes bonding pad, sample pad and blotting paper, overlaps sample pad, bonding pad chromatographic film, blotting paper successively viscous
It is attached on PVC bottom plate, there is the overlap joint of about 1mm or so between any two.Finally it is cut into the immuno-chromatographic test paper strip of 6.0mm.
5. the processing of test strips: as shown in Figure 1, the middle position (at distance sample pad end 30mm) in chromatographic film is used respectively
2mg/ml chloramphenicol-BSA conjugate and 2mg/ml melamine-BSA conjugate spray line, as detection zone and reference region.Two
Line is independent of one another, does not overlap, and the detection zone and reference region are disposed side by side on the transverse direction perpendicular to the test strips long side
Line is contacted and is reacted with sample to be tested simultaneously convenient for detection zone and reference region.37 DEG C of drying are placed in, is subsequently placed at dry and protects
It deposits.
6. the chloramphenicol in quantitative detection milk: test strips being lain against on level table, 180 μ L milk are added dropwise in sample
On pad, 5min is stood, quantitative detection is carried out using quantitative detection equipment.
B. scheme is compared:
1. the preparation that nanoparticle label detects antibody (nano particle-detection antibody coupling matter): referring to the present embodiment " A.
This embodiment scheme ".
2. the preparation of nanoparticle label reference antibody (nano particle-reference antibody conjugate): referring to the present embodiment
" A. this embodiment scheme ".
3. the preparation of bonding pad: referring to the present embodiment " A. this embodiment scheme ".
4. the preparation of chromatographic film: as shown in Fig. 2, the amount according to 0.8 μ L/cm is successively sprayed toward in chromatographic film along chromatography direction
Applying concentration is chloramphenicol-BSA conjugate (detection zone) of 2mg/mL, 2mg/mL melamine-BSA(bovine serum albumin(BSA)) coupling
Object (reference region) is placed in 37 DEG C of dry 2h, spare.
5. the assembling of test strips: nitrocellulose filter (chromatographic film) is pasted the middle position of PVC bottom plate, and (detection zone leans on
Nearly sample pad end, reference region are close to blotting paper end).Then, bonding pad, sample pad and blotting paper are pasted in corresponding position,
So that sample pad, bonding pad chromatographic film, blotting paper is successively overlapped and is pasted on PVC bottom plate, there is taking for about 1mm or so between any two
It connects.It is finally cut into the immuno-chromatographic test paper strip of 5.5mm wide, is saved in 2-8 DEG C, the dry, place of being protected from light.
6. the residual chloromycetin in quantitative detection milk: test strips are lain against on level table, be added dropwise 180 μ L milk in
In sample pad, 5min is stood, quantitative detection is carried out using quantitative detection equipment.
Evaluation of result: compared with the prior art (option b), detection stability obviously mentions immuno-chromatographic test paper strip (option A)
It rises, experimental data is for statistical analysis with two schemes replication 10 times referring to table 5(same concentration).
5 this embodiment scheme of table (option A) and existing immuno-chromatographic test paper strip (option b) detection chloramphenicol comparison
6 Acritamer 940 of embodiment detects the influence of stability for improving
1. nanoparticle label detection antibody (nano particle-detection antibody coupling matter): taking 100 μ L concentration is 10mg/mL
Rare-earth fluorescent nano particle (partial size: 300nm) in 900 μ L MES(2- (N- morpholine) ethanesulfonic acids, 50mM, PH6.1) in, be vortexed
It mixes；20mg NHS(N- HOSu NHS is added), 20mg EDC(1- (3- dimethylamino-propyl) -3- ethyl carbon two it is sub-
Amine), it is vortexed and mixes；React at room temperature 30min；10000rpm is centrifuged 10min, abandons supernatant；Vortex resuspended particle；C is added and reacts egg
White (CRP) detects 20 μ g of antibody, reacts at room temperature 2~4h；Casein is added, make its final concentration of 0.1%, the reaction was continued 2~4h；
10000rpm is centrifuged 10min, abandons supernatant；With PBST(phosphate Tween buffer) wash particle three times；2mL particle weight is added
Suspension (10mM Tris-HCl buffer, 1%BSA(bovine serum albumin(BSA)), 5% trehalose) resuspended particle, it is spare.
2. nanoparticle label reference antibody (nano particle-reference antibody conjugate): taking 100 μ L concentration is 10mg/mL
Rare-earth fluorescent nano particle (partial size: 300nm) in 900 μ L MES(50mM, PH6.1) in, be vortexed mix；20mg is added
NHS, 20mg EDC are vortexed and mix；React at room temperature 30min；10000rpm is centrifuged 10min, abandons supernatant；Vortex resuspended particle；Add
Enter 40 μ g of melamine monoclonal antibody, reacts at room temperature 2~4h；Casein is added, make its final concentration of 0.1%, the reaction was continued 2
~4h；10000rpm is centrifuged 10min, abandons supernatant；Particle is washed three times with PBST；2mL particle re-suspension liquid (10mM is added
Tris-HCl, 1%BSA, 5% trehalose) resuspended particle, it is spare.
3. the preparation of bonding pad: bonding pad (glass fibre) being previously placed in the Acritamer 940 gel of various concentration and is soaked
5min is steeped, taking-up is placed in 65 DEG C of drying, by the nano particle prepared-detection antibody coupling matter and nano particle-reference antibody
Conjugate is mixed according to isometric, is then sprayed on bonding pad according to 3 μ L/cm, and 37 DEG C of oven drying are placed in, spare.
4. the assembling of test strips: nitrocellulose filter (chromatographic film) is pasted to the middle position of PVC bottom plate.Then, exist
Corresponding position pastes bonding pad, sample pad and blotting paper, overlaps sample pad, bonding pad, chromatographic film, blotting paper successively viscous
It is attached on PVC bottom plate, there is the overlap joint of about 1mm or so between any two；Finally it is cut into the immuno-chromatographic test paper strip of 5.5mm wide.
5. the processing of test strips: using 2mg/ml CRP respectively in the middle position (at distance sample pad end 30mm) of chromatographic film
Capture antibody and 2mg/ml melamine-BSA(bovine serum albumin(BSA)) conjugate specking, respectively as detection zone and reference region,
The detection zone and reference region are disposed side by side on the x wire perpendicular to the test strips long side, same convenient for detection zone and reference region
When contact and react (reference can be made to Fig. 1) with sample to be tested.37 DEG C of drying are placed in, 2-8 DEG C, the dry, place's of being protected from light preservation are subsequently placed in.
6. CRP in quantitative detection serum: test strips are lain against on level table, 180 μ L serum are added dropwise in sample pad,
15min is stood, quantitative detection is carried out using quantitative detection equipment.
7. the test strips prepared are placed in 37 DEG C of different times, the CRP sample of measurement same concentrations, replication are taken out
It five times, is averaged.The results are shown in Table 6.As it can be seen that adding appropriate (0.1%-0.2%) Acritamer 940 can be improved test strips inspection
The stability of survey.
Influence of the 6 various concentration Acritamer 940 of table for detection stability
| Acritamer 940 concentration (%, w/v) | 0 | 0.1 | 0.2 | 0.3 |
| It measures average value (mg/L) | 12.95 | 13.42 | 14.88 | 10.27 |
| Actual value (mg/L) | 15 | 15 | 15 | 15 |
| Deviation value (%) | -13.67 | -10.53 | -0.80 | -31.53 |
Note: deviation value=(measurement average value-actual value)/actual value × 100%).
Embodiments of the present invention above described embodiment only expresses, the description thereof is more specific and detailed, but can not
Therefore limitations on the scope of the patent of the present invention are interpreted as, as long as skill obtained in the form of equivalent substitutions or equivalent transformations
Art scheme should all be fallen within the scope and spirit of the invention.
Claims (1)
1. a kind of detection device, which is characterized in that the detection device is immuno-chromatographic test paper strip, preparation method are as follows:
(1) nano particle-detection antibody coupling matter preparation: take 100 μ L concentration be 10mg/mL rare-earth fluorescent nano particle in
900 μ L, 50mM, PH6.1 2- (N- morpholine) ethanesulfonic acid in, be vortexed mix；Wherein, the grain of the rare-earth fluorescent nano particle
Diameter is 300nm；20mg n-hydroxysuccinimide, 20mg 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide, whirlpool is added
Rotation mixes；React at room temperature 30min；10000rpm is centrifuged 10min, abandons supernatant；Vortex resuspended particle；C reactive protein detection is added
20 μ g of antibody reacts at room temperature 2~4h；Casein is added, make its final concentration of 0.1%, the reaction was continued 2~4h；10000rpm centrifugation
10min abandons supernatant；Particle is washed three times with phosphate Tween buffer；2mL particle re-suspension liquid resuspended particle is added, it is spare；
Wherein, the particle re-suspension liquid includes 10mM Tris-HCl buffer, 1% bovine serum albumin(BSA) and 5% trehalose；
(2) nano particle-reference antibody conjugate preparation: take 100 μ L concentration be 10mg/mL rare-earth fluorescent nano particle in
900 μ L, 50mM, PH6.1 2- (N- morpholine) ethanesulfonic acid in, be vortexed mix；Wherein, the grain of the rare-earth fluorescent nano particle
Diameter is 300nm；20mg n-hydroxysuccinimide, 20mg 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide, whirlpool is added
Rotation mixes；React at room temperature 30min；10000rpm is centrifuged 10min, abandons supernatant；Vortex resuspended particle；Melamine monoclonal is added
40 μ g of antibody reacts at room temperature 2~4h；Casein is added, make its final concentration of 0.1%, the reaction was continued 2~4h；10000rpm centrifugation
10min abandons supernatant；Particle is washed three times with PBST；2mL particle re-suspension liquid resuspended particle is added, it is spare；Wherein, the particle
Re-suspension liquid includes 10mM Tris-HCl buffer, 1% bovine serum albumin(BSA) and 5% trehalose；
(3) preparation of bonding pad: bonding pad is previously placed in the Acritamer 940 gel that bulking value specific concentration is 0.2% and is impregnated
5min, taking-up are placed in 65 DEG C of drying, and the nano particle prepared-detection antibody coupling matter and nano particle-reference antibody is even
Join object to mix according to isometric, then be sprayed on bonding pad according to 3 μ L/cm, is placed in 37 DEG C of oven drying, it is spare；
(4) chromatographic film the assembling of test strips: is pasted to the middle position of PVC bottom plate；Then, knot is pasted in corresponding position
Pad, sample pad and blotting paper are closed, sample pad, bonding pad, chromatographic film, blotting paper is overlapped successively and is pasted on PVC bottom plate, two-by-two
Between have the overlap joint of 1mm；Finally it is cut into the immuno-chromatographic test paper strip of 5.5mm wide；
(5) processing of immuno-chromatographic test paper strip: 2mg/ml CRP capture antibody and 2mg/ are used respectively in the middle position of chromatographic film
Ml melamine-bovine serum albumin(BSA) conjugate specking, respectively as detection zone and reference region, the detection zone and reference region are side by side
Be placed perpendicular to an x wire of the immuno-chromatographic test paper strip long side, be placed in 37 DEG C of drying, be subsequently placed in 2-8 DEG C, it is dry,
Place is protected from light to save.
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| CN1892223A (en) * | 2005-04-11 | 2007-01-10 | 兰州大学 | Colloidal gold semi-quantitative quick immunity diagnosis test-paper stripe |
| CN102105795A (en) * | 2008-06-04 | 2011-06-22 | 阿莱瑞士股份有限公司 | Assay reader, device and method for measuring hCG |
| CN102305854A (en) * | 2011-07-20 | 2012-01-04 | 汤凌霄 | Ultrasensitive and quantitative immunochromatographic device and detection method using same |
| CN102520194A (en) * | 2011-12-29 | 2012-06-27 | 深圳康美生物科技股份有限公司 | Fluorescence immunochromatographic assay method for quantitatively detecting heart fatty acid binding protein and kit for quantitatively detecting same |
| CN104820094A (en) * | 2014-05-14 | 2015-08-05 | 陈岩松 | Immune-chromatographic detection test paper and detection method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1892223A (en) * | 2005-04-11 | 2007-01-10 | 兰州大学 | Colloidal gold semi-quantitative quick immunity diagnosis test-paper stripe |
| CN102105795A (en) * | 2008-06-04 | 2011-06-22 | 阿莱瑞士股份有限公司 | Assay reader, device and method for measuring hCG |
| CN102305854A (en) * | 2011-07-20 | 2012-01-04 | 汤凌霄 | Ultrasensitive and quantitative immunochromatographic device and detection method using same |
| CN102520194A (en) * | 2011-12-29 | 2012-06-27 | 深圳康美生物科技股份有限公司 | Fluorescence immunochromatographic assay method for quantitatively detecting heart fatty acid binding protein and kit for quantitatively detecting same |
| CN104820094A (en) * | 2014-05-14 | 2015-08-05 | 陈岩松 | Immune-chromatographic detection test paper and detection method |
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