CN2833594Y - Solid phase white nontransparent reflection type sample cell - Google Patents
Solid phase white nontransparent reflection type sample cell Download PDFInfo
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- CN2833594Y CN2833594Y CN 200520122784 CN200520122784U CN2833594Y CN 2833594 Y CN2833594 Y CN 2833594Y CN 200520122784 CN200520122784 CN 200520122784 CN 200520122784 U CN200520122784 U CN 200520122784U CN 2833594 Y CN2833594 Y CN 2833594Y
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Abstract
The utility model belongs to the technical field of solid phase time distinguish fluorescence immunity analysis, and more specifically, the utility model relates to a solid phase white nontransparent reflection type sample pond. The utility model is closed-ended circular platform or cylinder cup-shaped body; the utility model is composed of a basal body (1), a middle layer (2) and an inner surface layer (3), wherein the basal body (1) is polystyrene (PS), and the middle layer (2) is a nylon-6 film layer which is fixed on the inner surface of the polystyrene (PS) of the basal body (1); besides, the inner surface layer (3) is a polyamine basal layer which is in covalence combination with the nylon-6 film layer of the middle layer (2). The utility model utilizes covalence chemistry combination to substitute for physical adsorption; thus the combination quantity of antigens or antibodies is enlarged, the activity of combined immune petunidin is obviously increased and immunity detection sensitivity is enhanced. The utility model can not be only used for immunity analysis but also can be used as an affinity chromatography solid phase combination agent, and the utility model can be repeatedly used; besides, the function and the cost of the utility model are better than the commonly used polyacrylamide gel.
Description
Technical field
The utility model belongs to the immuno analytical method field, is specifically related to be used for the opaque reflective sample cell of solid phase white of solid-phase time-resolved fluorescence immunoassay technology.
The opaque reflective sample cell of a kind of novel solid phase white of the utility model development can be applicable to bioanalysis fields such as fluoroimmunoassay, affinity chromatography, immobilized enzyme technology and DNA chip.
Propose time resolved fluoro-immunoassay (TRFIA) theory from Soini in 1979 and Hemmila, nineteen eighty-two has founded since first manual measurement instrument, has developed into multiple TRFIA system so far.Enhancing (DELFIA) system of wherein dissociating has realized commercialization, but because this system exists defective: can not produce special spacing wave behind the ionic dissociation, be not suitable for all application, as fluorescence imaging, SABC, location hybridization, DNA chip or online detection etc.
In order to overcome the deficiency of DELFIA system, people such as Ether had set up the novel europium sequestrant 4 that strengthens fluorescence to have in 1988,7-dichloro sulfo group benzene-1,10 ferrosins 2, the serve as a mark new system of laser excitation solid-phase time-resolved fluorescence immunoassay (DSLFIA) of thing of 9-dicarboxylic acid (BCPDA), this detection system has LASER Light Source and the reflective sample cell of White-opalescent.(with novel europium sequestrant 4, the serve as a mark laser excitation solid-phase time-resolved fluorescence immunoassay of thing of 7-dichloro sulfo group benzene-1,10 ferrosin 2,9-dicarboxylic acid (BCPDA), analytical chemistry, 1988,60,1069-1074.Laser
Excitted?Time-Resolved?Solid
Phase?Fluoroimmunoassays?with?the?New?Europium?Chelate4，7--Bis(chlorosulfophenyl)-1，10-phenanthroline-2，9-dicarboxy?Acid?as
Label.Anal.Chem.1988,60,1069-1074) [1] is for carrying out the beginning that the DSLFIA system has been started detection system in a deep going way.But owing among reagent and detection system improving, do not reach the commercialization level as yet.The reflective sample cell technology of relevant White-opalescent is not appeared in the newspapers.
Nylon (polycaprolactam) comes from the many ammonia by amino as dissociating, cause people's attention very early.But most researchs all concentrate on and biomolecule are coupled to (thermolysin is fixed on the polyamines film, biotechnology and bioengineering, 2003,82 (2) 190-199 on the nylon membrane.Immobilization of Thermolysin to polyamide Nonwoven Materials.Biotechnol and Bioeng.2003,82 (2) 190-199.) [2] can be used for spot immune analysis and immune marking technology, are not suitable for doing enzyme-linked immuno assay (ELSIA) solid phase.About nylon being incorporated into the method for polystyrene solid phase, someone report in advance with biomolecule in conjunction with nylon membrane on after be covered in again that (nylon that haptens combines is coated polystyrene board as the enzyme-linked immuno assay solid phase in the PS hole, immunization method is learned magazine .1990,127:43-49.Haptenated nyIon--coated polystyreneplates as a solid phase for ELSIA.J.Immunol Methods.1990,127:43-49) [3]. this method and the PS microwell plate that obtains with this method have been proved to be in conjunction with inhomogeneous, can not tolerate the nylon hydrolysis and discharge amino strong acid treatment and the organic solvent in the chemical coupling, cause polystyrene surface to be etched, destroy.Analyze its reason and be owing to use acetate/metacresol/methylene chloride dissolving nylon to cause two subject matters: one, methylene chloride volatilizees fast, stays micropore at the nylon film, has reduced the physical strength of film and to the adhesion of PS; Its two, hydrochloric acid directly contacts in PS by micropore with organic solvent, the former ruptures the nylon film easily, the latter can be dissolved PS, causes PS to mix with the nylon film and covers the reactive group of nylon.So this method is the binding immunoassay aglucon in advance, is covered in the PS surface again, does not possess practical value.
The utility model content
In order to solve the technical matters of above-mentioned existence, the utility model provides the fixedly nylon layer of one deck White-opalescent of a kind of surface in the polystyrene sample pond, obtains the opaque reflective sample cell of solid phase white after chemical modification.The opaque reflective sample cell of this solid phase white is used for the DSLFIA technology.
The structure of the opaque reflective sample cell of solid phase white that the utility model provides as shown in Figure 1.It is one the circular platform type at the end or the goblet of column type; Be to constitute by matrix 1, middle level 2, interior surface layers 3; Described matrix 1 is polystyrene (PS), and middle level 2 is the nylon-6 retes that are linked in the inside surface of matrix 1 polystyrene (PS), interior surface layers 3 be with the nylon-6 rete in middle level 2 with covalently bound polyamines basic unit;
The connection of the nylon-6 rete in described matrix 1 polystyrene (PS) and middle level 2 is to use pyridine/acetone inside surface of activation processing matrix 1 in advance, with acetate/metacresol/pyridine dissolving, activation nylon-6, make hydrophobic interaction takes place between the two, simultaneously pyridine has activation PS concurrently and gentle " erosions " acts on, and can make nylon-6 be linked in the PS inside surface securely fast and forms interior surface layers 2; Nylon layer to interior surface layers 2 carries out the alkylation processing, alkyl-the OR that can make the generation of nylon-6 surface is at an easy rate with 2, an amino covalence combination of 6-diaminocaproic acid, form the arm of other end free amine group, it is polyamines basic units that the free amine group of this " arm " can form more firm covalently bound interior surface layers 3 with the free amine group of protein, so obtain the opaque reflective sample cell of solid phase white.
Introduce the concrete steps and the condition of the opaque reflective sample cell method of manufacturing solid phase white of the present utility model below more in detail, so that the utility model is further described.
The utility model agents useful for same:
A. matrix 1 activator: the acetone soln of 6.0-8.0% pyridine (V/V).
B. the activator of the nylon-6 in middle level 2: the acetate/m-cresol solution (V/V) of (0.5-0.9% pyridine).
C. the diethyl ether solution of epichlorokydrin: epichlorokydrin and diethyl ether solution volume ratio are 1: 9.
D. triethyl oxygen drone tetraboric acid fat (TOTFB) reagent preparation: 40ml 5% (v/v) boron trifluoride diethyl etherate (v/v) solution is continuing under the stirring, the diethyl ether solution that adds the epichlorokydrin of 12.5ml, in 30 minutes, add, reflux is one hour afterwards, room temperature stirred 3 hours again, leach TOTFB, again with absolute ether flushing 3 times, the TOTFB that obtains.Its productive rate is more than the 90%--95%.
E.TOTFB dichloromethane solution: contain 0.01-0.05g/ml TOTFB triethyl oxygen father-in-law's tetrafluoro boric acid ester (TOTFB) dichloromethane solution.
F. pentanedial liquid: the 0.5M pentanedial liquid is dissolved in 0.2M borate buffer solution (pH=7.6) mutually with 5% (v/v).
G.1,6 hexane diamine solution: concentration is the carbonate buffer solution of (pH=9.5) of 0.5M 1,6 hexane diamine.
The utility model preparation method's step and condition:
1.nylon-6 connection with PS matrix 1
In by PS matrix 1 pond, add matrix 1 activator, room temperature was placed 1-3 minute, discard the activator that adds the middle level 2nylon-6 that contains 12-18% polyamide-6 powder behind the solution immediately, the rotary substrate 1 at a slow speed along the axis, make nylon-6 quick, firm, be incorporated into the inside surface in matrix 1 pond equably, form nylon-6 thickness and be the rete in reflection sample cell middle level 2 of the White-opalescent of 0.1-0.2mm;
2. nylon membrane surface alkylation
Add in the sample cell of TOTFB dichloromethane solution above-mentioned (1), room temperature was placed 4-10 minute, discarded, and used dichloromethane rinse 2 times, with dioxane flushing 1 time;
3. modify the nylon membrane surface
Glutaraldehyde solution is joined in the sample cell of above-mentioned (2), 37 ℃ of incubations 15 minutes, discard, add 0.2M pH=7.2 phosphate (PBS) buffer solution flushing three times, add 1,6 hexane diamine (pH=9.5) solution again, room temperature was placed 3 hours, outwell, with PBS solution flushing 3 times, promptly get the opaque reflective sample cell of solid phase white then.
The technical characteristics of the opaque reflective sample cell of solid phase white of the present utility model and useful effect are as follows:
1. utilize nylon-6 to be incorporated into the reflection sample cell of White-opalescent of the inside surface of PS, adopt new many ammonia of dissolving technology and specific PS activator, many ammonia coagulating agent, make polyamines evenly, firmly transplant surface, form white catoptrical polyamines base internal layer in nylon-6.
This polyamines inside surface solid phase room temperature expose in the air can long preservation and can tolerate strong acid, alkali, organic solvent indeformable, do not come off.For making the nylon-6 polycaprolactam ammonia structure reactive group that dissociates, used hydrochloric acid partial hydrolysis method in the past, because excessively the hydrolysis meeting makes the nylon structural break, it is insufficient that the control hydrolysis time causes free amine group to expose again, so adopt TOTFB alkanisation processing nylon layer.This method can not influence the nylon-6 structure, and can fully expose reactive group
Utilize the effect of glutaraldehyde double-functional group, the free amine group in the end combination " arm ", the free amine group of the aldehyde radical conjugated protein that the other end is free, the activity of raising binding immunoassay aglucon.
3. adopt pyridine to replace methylene chloride.Because pyridine not only volatilizees slow and can promote the nylon of dissolved state to assemble and with the combining of PS, form on the PS surface even, tough and tensile, can not be through the film of second machine solvent; And when it combines with PS, to active amino content, physical strength and desirable effect is received in aspects such as organic solvent tolerance.
4. handle the nylon surface with TOTFB, existing report in the document, but all be to use the nylon film reaction, wash-out need pass through a large amount of soaked with liquid, and the utility model adopts in the pond and washes three times, and non-specific bond is 3.41%, the experiment/non-special=12.9, be significantly higher than 2.1 times of diagnostic criteria of ELISA.
5. the opaque reflective sample cell of solid phase white is used for the radioactive label immunoassay, the aglucon combination rate reaches 43.98%, for the 2.5-13.1 of the PS sample cell that is untreated doubly.Prove that this sample cell has greater activity and sensitivity.
6. batch interior error statistics of the opaque reflective sample cell of the utility model solid phase white is tested and the average CV=5.0% of control group, and each organizes 8 parallel samples CV＜10%, is significantly higher than untreated PS sample cell.
7. the destructive test result of the opaque reflective sample cell of the utility model solid phase white proves: the temperature of the complete sex change of protein is 65 ℃, experiment adopts 50 ℃ of maintenances to heat 48 hours, the combination rate of sample cell coupling antibody still keeps 41%, than having reduced by 4.6% before the experiment.And the PS microwell plate combination rate 5% that is untreated, complete failure.
8. the performance of the opaque reflective sample cell of solid phase white of the present utility model can with compare with the Nunc Covalink-NH microwell plate that Denmark produces, practicality is then than its superior (polystyrene micropore plate amination: be used for the hybrid experiment covalency and transplant dna probe.Analytical biochemistry 1996,236:85-94.Amination?of?polystyrene?Microwell：Application?tothe?covalent?Grafting?of?DNA?probe?for?Hybridization?assays.Anal.Biochem.1996，236：85-94)[5]。For example the reactive group of Nunc plate is-NH, and the utility model is-NH
2, the amino of this group linking protein matter is easier, more stable; Expensive 2 times of Nunc plate prices than PS microwell plate.The utility model is because of easy and simple to handle, draws materials easily, and cost is only expensive about 20% than PS microwell plate, and competitiveness is arranged on market.
The utility model both can be used for immunoassay, can also be used as affinity chromatography solid phase bond, being about to diameter is that 5-8 μ m PS microballon is transplanted active amino by this method, can be used as high-effect affinity chromatographic material, and can use repeatedly, its function and cost are excellent than polyacrylamide gel commonly used all, are with a wide range of applications.
Description of drawings
Fig. 1 is the structural representation of the opaque reflective sample cell of solid phase white of the present utility model.This figure also is the specification digest accompanying drawing.
Embodiment
Reagent that the utility model embodiment is used and preparation thereof:
A. matrix 1 activator: the acetone soln of 6.0-8.0% pyridine (V/V).
B. the activator of the nylon-6 in middle level 2: the acetate/m-cresol solution (V/V) of (0.5-0.9% pyridine).
C. the diethyl ether solution of epichlorokydrin: epichlorokydrin and diethyl ether solution volume ratio are 1: 9.
D. triethyl oxygen drone tetraboric acid fat (TOTFB) reagent preparation: 40ml 5% (v/v) boron trifluoride diethyl etherate (v/v) solution is continuing under the stirring, the diethyl ether solution that adds the epichlorokydrin of 12.5ml, in 30 minutes, add, reflux is one hour afterwards, room temperature stirred 3 hours again, leach TOTFB, with absolute ether flushing 3 times, obtain TOTFB again.Its productive rate is more than the 90%--95%.
E.TOTFB dichloromethane solution: contain 0.01-0.05g/ml TOTFB triethyl oxygen father-in-law's tetrafluoro boric acid ester (TOTFB) dichloromethane solution.
F. pentanedial liquid: the 0.5M pentanedial liquid is dissolved in 0.2M borate buffer solution (pH=7.6) mutually with 5% (v/v).
G.1,6 hexane diamine solution: 1, the 6 hexane diamine solution (v/v) of the 0.5M that incites somebody to action is dissolved in the carbonate buffer solution of 0.1M (pH=9.5).
Preparation method's of the present utility model step and condition:
1.nylon-6 connection with PS matrix 1
In by PS matrix 1 pond, add matrix 1 activator A, room temperature was placed 1-3 minute, discard the activator B that adds middle level 2 nylon-6 that contain 12-18% polyamide-6 powder behind the solution immediately, the rotary substrate 1 at a slow speed along the axis, make nylon-6 quick, firm, be incorporated into the inside surface in matrix 1 pond equably, form nylon-6 thickness and be the rete in reflection sample cell middle level 2 of the White-opalescent of 0.1-0.2mm;
2. nylon membrane surface alkylation
Add the TOTFB dichloromethane solution in the sample cell of above-mentioned (1), room temperature was placed 4-10 minute, discarded, and used dichloromethane rinse 2 times, with dioxane flushing 1 time;
3. modify the nylon membrane surface
Glutaraldehyde solution is joined in the sample cell of above-mentioned (2), 37 ℃ of incubations 15 minutes, discard, add 0.2M pH=7.2 phosphate (PBS) buffer solution flushing three times, add 1,6 hexane diamine (pH=9.5) solution again, room temperature was placed 3 hours, outwell, with PBS solution flushing 3 times, promptly get the opaque reflective sample cell of solid phase white then.
Embodiment 1:
(1). in the matrix 1 that constitutes by PS, add 400 μ l matrixes, 1 activator, room temperature was placed 1 minute, add the 12%nylon-6 solution of 200 μ l immediately after discarding solution by the dissolving of middle level 2 nylon-6 activators, rotate (40 times/minute) matrix 1 at a slow speed along the axis, make nylon-6 quick, firm, be incorporated into the inside surface of matrix 1 equably, forming the nylon-6 thicknesses of layers is the reflection sample cell middle level 2 nylon-6 retes of the White-opalescent of 0.1-0.2mm;
(2). the dichloromethane solution that adds 400 μ l, 5% triethyl oxygen father-in-law tetrafluoro boric acid ester (TOTFB) is in the sample cell of above-mentioned (1), and room temperature was placed 4-10 minute, discards, and uses dichloromethane rinse 2 times, dioxane flushing 1 time;
(3). 200 μ l 0.5M glutaraldehydes are added in the sample cell of above-mentioned (2), 37 ℃ of incubations 15 minutes discard.Add 0.2M pH=7.2 phosphate (PBS) buffer solution flushing three times; 1, the 6 hexane diamine solution that adds 200 μ l 0.058g/ml again, room temperature was placed 3 hours, outwelled.With PBS buffer solution flushing 3 times, promptly get the opaque reflective sample cell of solid phase white of the present utility model then.
Embodiment 2
(1). add 400 μ l matrixes, 1 activator in the matrix 1 that is made of PS, room temperature was placed 1 minute, added the 13%nylon-6 solution of 200 μ l with the dissolving of middle level 2 nylon-6 activators immediately after discarding solution.Rotate (40 times/minute) matrix 1 at a slow speed along the axis, make nylon-6 quick, firm, be incorporated into the inside surface of matrix 1 equably, forming nylon-6 thickness is the reflection sample cell middle level 2 nylon-6 retes of the White-opalescent of 0.1-0.2mm;
(2). the dichloromethane solution that adds 400 μ l 4%TOTFB is in the sample cell of above-mentioned (1), and room temperature was placed 4-10 minute, discarded. use dichloromethane rinse 2 times, dioxane flushing 1 time;
(3). add 200 μ l 0.5M glutaraldehyde borate buffer solutions in the sample cell of above-mentioned (2), 37 ℃ of incubations 15 minutes discard.Add 0.2M pH=7.2 phosphate (PBS) buffer solution flushing three times; 1, the 6 hexane diamine solution that adds 200 μ l 0.5M again, room temperature was placed 3 hours, outwelled, and with PBS buffer solution flushing 3 times, promptly obtained the opaque reflective sample cell of solid phase white of the present utility model then.
Embodiment 3
(1). in the matrix 1 that constitutes by PS, add 400 μ l matrixes, 1 activator.Room temperature was placed 1 minute, added the 14%nylon-6 solution of 200 μ l by the dissolving of middle level 2 nylon-6 activators immediately after discarding solution.Rotate (40 times/minute) matrix 1 at a slow speed along the axis, make nylon-6 quick, firm, be incorporated into the inside surface of matrix 1 equably, forming the nylon-6 thicknesses of layers is the reflection sample cell middle level 2 nylon-6 retes of the White-opalescent of 0.1-0.2mm;
(2). the dichloromethane solution that adds 400 μ l 3%TOTFB is in the sample cell of above-mentioned (1), and room temperature was placed 4-10 minute, discarded.With dichloromethane rinse 2 times, dioxane flushing 1 time; Described TOTFB synthetic method is by document: (enzyme is fixed in the chemistry of nylon, biological chemistry .J.1975,147:593-603 A chemistry for the Immobilization of Enzymeson nylon.Biochem.J.1975,147:593-603) synthetic method is synthetic;
(3). 200 μ l 0.5M glutaraldehyde borate solutions are joined in the sample cell of above-mentioned (2), and 37 ℃ of incubations 15 minutes add 0.2M pH=7.2 phosphate (PBS) buffer solution flushing three times; 1, the 6 hexane diamine solution that adds 200 μ l0.5M again, room temperature was placed 3 hours, outwelled, and with PBS solution flushing 3 times, promptly got the opaque reflective sample cell of solid phase white of the present utility model then.
Embodiment 4
(1). add 400 μ l matrix (1) activators in the matrix 1 that is made of PS, room temperature was placed 1 minute, added the 15%nylon-6 solution of 200 μ l with the dissolving of middle level 2 nylon-6 activators immediately after discarding solution.Rotate (40 times/minute) matrix 1 at a slow speed along the axis, make nylon-6 quick, firm, be incorporated into the inside surface of matrix 1 equably, forming nylon-6 thickness is the reflection sample cell middle level 2 nylon-6 retes of the White-opalescent of 0.1-0.2mm;
(2). the dichloromethane solution that adds 400 μ l 2%TOTFB is in the sample cell of above-mentioned (1), and room temperature was placed 4-10 minute, discards, and uses dichloromethane rinse 2 times, dioxane flushing 1 time;
(3). in the sample cell that joins above-mentioned (2) with 200 μ l glutaraldehyde borate buffer solutions (pH=7.6), 37 ℃ of incubations add 0.2M pH=7.2 phosphate (PBS) buffer solution flushing three times after 15 minutes; Add 200 μ again and be dissolved in 1, the 6 hexane diamine solution of 0.1M carbonate buffer solution (pH=9.5) 0.5M, room temperature was placed 3 hours, outwelled, and with PBS solution flushing 3 times, promptly got the opaque reflective sample cell of solid phase white of the present utility model then.
Embodiment 5
(1). in the matrix 1 that constitutes by PS, add 400 μ l matrixes, 1 activator.Room temperature was placed 1 minute, add the 16%nylon-6 solution of 200 μ l immediately after discarding solution by the dissolving of middle level 2 nylon-6 activators, rotate (40 times/minute) matrix 1 at a slow speed along the axis, make nylon-6 quick, firm, be incorporated into the inside surface of matrix 1 equably, forming the nylon-6 thicknesses of layers is the reflection sample cell middle level 2 nylon-6 retes of the White-opalescent of 0.1-0.2mm;
(2). the dichloromethane solution that adds 400 μ l 1%TOTFB is in the sample cell of above-mentioned (1), and room temperature was placed 4-10 minute, discarded.With dichloromethane rinse 2 times, dioxane flushing 1 time;
(3). add 200 μ l 0.5M glutaraldehyde borate buffer solutions in the sample cell of above-mentioned (2), 37 ℃ of incubations 15 minutes add 0.2M pH=7.2 phosphate (PBS) buffer solution flushing three times; Add 200 μ l0.5M1 again, 6 hexane diamine carbonate buffer solutions (pH=9.50), room temperature was placed 3 hours, outwelled.With PBS solution flushing 3 times, promptly get the opaque reflective sample cell of solid phase white of the present utility model then.
Claims (1)
1. opaque reflective sample cell of solid phase white, being one has the circular platform type at the end or the goblet of column type, it is characterized in that, is to be made of matrix (1), middle level (2), interior surface layers (3); Described matrix (1) is polystyrene (PS), and middle level (2) are the nylon-6 retes that is linked in the inside surface of matrix (1) polystyrene (PS), interior surface layers (3) be with the nylon-6 rete of middle level (2) with covalently bound polyamines basic unit.
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| CN 200520122784 CN2833594Y (en) | 2005-09-30 | 2005-09-30 | Solid phase white nontransparent reflection type sample cell |
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| CN 200520122784 CN2833594Y (en) | 2005-09-30 | 2005-09-30 | Solid phase white nontransparent reflection type sample cell |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113167790A (en) * | 2018-10-24 | 2021-07-23 | 凸版印刷株式会社 | Cup for immunoassay, method for manufacturing same, and immunoassay method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113167790A (en) * | 2018-10-24 | 2021-07-23 | 凸版印刷株式会社 | Cup for immunoassay, method for manufacturing same, and immunoassay method |
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| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20061101 Termination date: 20100930 |