CN103197055B - Method for reducing background of vector in biological reaction - Google Patents
Method for reducing background of vector in biological reaction Download PDFInfo
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- CN103197055B CN103197055B CN201310078156.7A CN201310078156A CN103197055B CN 103197055 B CN103197055 B CN 103197055B CN 201310078156 A CN201310078156 A CN 201310078156A CN 103197055 B CN103197055 B CN 103197055B
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- carrier
- dye
- deionized water
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- dye liquor
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Abstract
The present invention relates to a method for reducing background of a vector in a biological reaction, and belongs to the technical field of biological detection. According to the method for reducing the background of the vector in the biological reaction, the used biological reaction vector is a colored vector formed by coloring process. The biological reaction vector is a vector which can be combined with coated biological materials. The method of the present invention can significantly reduce the background during biological reaction, and improve the detection sensitivity, and has the advantages of low cost and easiness to operate.
Description
Technical field
The present invention relates to a kind of method reducing carrier background when biological respinse, belong to technical field of biological.
Background technology
Current, bio-reaction system is widely used in each research field of the life sciences such as genome research, medical research and study of pharmacy, and progressively to each large field infiltration such as preferred good child-rearing of new drug development, clinical diagnosis, health forecast, disease treatment, judicial expertise, military protection, Food Hygiene Surveillance, commerce and trade inspection and quarantine, environmental monitoring control and farming, forestry, husbandary and fishing crop.Particularly in disease detection diagnosis, bio-reaction system has unique advantage.A carrier can carry out multiple patient the detection of various diseases or index simultaneously, thus at short notice for medical worker provides a large amount of medical diagnosis on disease information.Such as to the clinical examination of the common diseases such as genetic disease, tumour and communicable disease and frequently-occurring disease, all can applying biological reaction technology.People even can have in " individual laboratory " from now on, can monitor whenever and wherever possible to the health status of oneself.
Bio-reaction system is according to interactional principle special between biomolecule, by biochemical analysis process integration in carrier surface, thus realizes detecting fast the high flux of DNA, RNA, polypeptide, protein and other biological composition.Preparing bio-reaction system at present mainly adopts the method for surface chemistry or the method for combinatorial chemistry to process solid-phase matrix as polystyrene board, silicon chip, cellulose membrane or nylon membrane etc., then makes DNA, RNA, polypeptide, protein and other biological composition be arranged on carrier by particular requirement.But usually run into that background values is high, the problem of difference in signal strength in detecting.
Application number is that the Chinese invention patent of 200710132686.X discloses a kind of biological chip glass carrier, under the making of this biological chip glass carrier comprises the steps: (1) room temperature condition, first silica sand and sodium carbonate is mixed; (2) MnO is added
2, borax, NaNO
3, carbon dust, titanium dioxide two arsenic, K
2cO
3, stir and evenly mix; (3) above-mentioned compound is dropped into smelting furnace, heating is melt into pulpous state, and stirs 1 hour continuously; (4) 25 ~ 40 DEG C are cooled to rapidly; (5) cut into slices; (6) polishing, shaping.The background values of this invention sheet base is better than common microslide sheet base, signal intensity aspect, and this invention microslide average signal strength is better than common microslide.But these advantages all need to be further improved.Due to glass carrier shortcoming inherently, make it that there is limitation, the carrier of what current most biological detection reagent kit adopted is polystyrene board carrier, filter membrane class.
Summary of the invention
The object of the invention is for solving the problems of the technologies described above, providing a kind of method reducing carrier background when biological respinse.
Above-mentioned purpose of the present invention is achieved by the following technical programs:
Reduce a method for carrier background when biological respinse, before biological respinse, colouring process is carried out to biological respinse carrier.
Preferred as technique scheme, described biological respinse carrier be can with wrap the carrier that be combined by biological substance.
Described colouring process Toner used is the Toner that significantly can reduce the background of carrier when biological respinse after carrier colouring, improve detection sensitivity.
Preferred as technique scheme, described carrier is the supporting body that miillpore filter or tygon, polystyrene, Polyvinylchloride, polypropylene or silastic material are made; Described colouring is treated to carrier in the fabrication process, adopts coloring matter to be scattered in the raw material manufacturing described carrier, through moulding process, described coloring matter is evenly blended in carrier.
Preferred as technique scheme, any one in the supporting body that described carrier is miillpore filter or tygon, polystyrene, Polyvinylchloride, polypropylene, silastic material are made; Described colouring process be making biological detection reagent before, adopt contaminate or be coated with dye technique, by described carrier impregnation in dye liquor or be coated be covered with dye liquor, make described carrier become coloured carrier.
Preferred as technique scheme, any one in the supporting body that described carrier is miillpore filter or tygon, polystyrene, Polyvinylchloride, polypropylene, silastic material are made; Described colouring process be make biological detection reagent process in, adopt contaminate or be coated with dye technique, by described carrier impregnation in dye liquor or be coated be covered with dye liquor, make described carrier become coloured carrier.
Preferred as technique scheme, described carrier is miillpore filter.
Described cellulose family miillpore filter refers to film, cellulose membrane, glass fibre element film etc. that the potpourri of nitrocellulose filter, cellulose acetate membrane, cellulose acetate and cellulose nitrate is made.
Described polyvinyl-fluoride miillpore filter refers to polyvinylidene fluoride film or poly tetrafluoroethylene.
Preferred as technique scheme, described Toner is direct dyes, azoic dyes insoluble azo dyes, reactive dye, reducing dye, soluble vat dye, sulfur dye, sulphur vat dye, phthalocyanine dye, oxidation dye, the condensation dye, disperse dyes, acid dyes, acidic intermedium and acid containing mordant dye, alkalescence and the dye of positive ion, coating.
Preferred as technique scheme, described painting methods is for contaminating.
Preferred as technique scheme, described colouring process is: in the Tween-20 solution be first soaked in by described carrier, take out after soaking at room temperature; Described carrier deionized water rinsing; Immerse in dye liquor and start dyeing, raised temperature is also incubated; Take out carrier, in dye liquor, add HAC, put into carrier, soak; Remove dye liquor, with deionized water rinsing; Be soaked in a period of time in deionized water; Remove deionized water, wash with cleansing solution; Remove cleansing solution, with deionized water rinsing; Take out washed carrier to dry.
As technique scheme further preferably, described colouring process is: be first soaked in the Tween-20 solution of 0.1 ~ 0.5% by described carrier, and soaking at room temperature was taken out after 3 hours; Described carrier deionized water rinsing; Immerse in dye liquor and start dyeing, by 2 ~ 4 DEG C/min raised temperature, rise to 90 ~ 95 DEG C of insulation 20min, period, every 8 ~ 10min jiggled once; Take out carrier, in dye liquor, add HAC, put into carrier, 90 ~ 95 DEG C of insulation 15 ~ 20min; Remove dye liquor, with deionized water rinsing; Be soaked in deionized water, room temperature more than 12 hours; Remove deionized water, wash 3 ~ 5 hours with cleansing solution; Remove cleansing solution, with deionized water rinsing; Take out washed 35 ~ 37 DEG C, carrier oven dry.
Preferred as technique scheme, described colouring process is when making described carrier, is mixed in raw material by the dyestuff of 0.1 ~ 5% and paints.
As technique scheme further preferably, described colouring process is when making described carrier, is mixed in raw material by the dyestuff of 0.1 ~ 0.5% and paints.
Preferred as technique scheme, described colouring process: be add in confining liquid massfraction be 0.1 ~ 5% dyestuff carrier surface is closed; Described biological detection reagent is the carrier that biological substance not of the same race is coated in carrier surface, through the biological detection reagent be assembled into.
As technique scheme further preferably, described colouring process: be add in confining liquid massfraction be 0.1 ~ 0.5% dyestuff carrier surface sheet is closed; Described biological detection reagent is the carrier that biological substance not of the same race is coated in carrier surface, through the biological detection reagent be assembled into.
The present invention has following beneficial effect:
The present invention significantly can reduce the background of carrier when biological respinse, improve detection sensitivity, in addition the present invention also has that cost is low, simple operation and other advantages.
Accompanying drawing explanation
Fig. 1 does not paint and the bio-reaction system experimental image of painting;
Fig. 2 is the analysis of results table of experimental image.
Embodiment
below in conjunction with accompanying drawing, the present invention is further explained.
In Fig. 1, I1 and I 2 holes are negative sample; III 1 and III 2 holes are positive sample.Test figure is concrete as shown in Figure 2, and as can be seen from Figure 1, in III 1, background colour is dusky comparatively fuzzy, and in III 2, background colour is totally limpid; As can be seen from the data in the table of Fig. 2 also, the membrane carrier through colouring process has and improves signal to noise ratio (S/N ratio) and reduce the advantage of background signal value, and with the contrast of not painting, difference is clearly.
Embodiment one
The preparation of genetic chip
(1) NC film dyeing:
1. be soaked in by NC film in 0.2% Tween-20 solution, soaking at room temperature was taken out after 3 hours; Film after soaking is immersed in dyestuff, and 85 DEG C contaminate 1 hour, and period, every 5min jiggled once; 2. dye liquor is removed, with deionized water rinsing 3 times;
3. the film rinsed is soaked in deionized water, room temperature more than 12 hours;
4. remove deionized water, and with after deionized water rinsing twice, add cleansing solution (0.2M PBS-T), wash 5 hours for 50 DEG C;
5. cleansing solution is removed, with deionized water rinsing 3 times;
6. add deionized water, 50 DEG C, soak 1 hour, and repeat again once;
7. washed film is taken out, after deionized water rinsing one time, 37 DEG C of oven dry, for subsequent use.
(2) bag quilt: different nucleic acid probes is coated in nitrocellulose filter surface with certain requirement.
(3) assemble: by the nitrocellulose filter containing biological substance after bag is done, be assembled into genetic chip.
(4) close: with the confining liquid of every hole 100ul, biochip is closed.
(5) dry: biochip carries out drying after having closed in drying box.
(6) pack: pack after biochip drying.
Embodiment two
The preparation of immuno-chip (fluorescence method)
(1) bag quilt: biological substance not of the same race is coated in cellulose acetate membrane surface with certain requirement.
(2) assembling bag be done after by the cellulose acetate membrane containing biological substance, be assembled into immuno-chip.
(3) liquid of painting is modulated: aniline blue (aniline blue) 0.5 gram is dissolved in the distilled water of 100ml, boils rear cooling, then adds orange G 1 gram, acid fuchsin 1.5 grams, corrects pH to 1.9, can use with HCC.
(4) close: add 1.0% colouring liquid in confining liquid, with the confining liquid of every hole 100ul, immuno-chip is closed.
(5) dry: immuno-chip carries out drying after having closed in drying box.
(6) pack: pack after immuno-chip drying.
Embodiment three
The preparation of biochip
(1) bag quilt: biological substance not of the same race is coated in nitrocellulose filter surface with certain requirement.
(2) assembling bag be done after by the nitrocellulose filter containing biological substance, be assembled into biochip.
(3) close: add 0.4% methyl violet in confining liquid, with the confining liquid of every hole 100ul, biochip is closed.
(4) dry: biochip carries out drying after having closed in drying box.
(5) pack: pack after biochip drying.
Embodiment four
The preparation of biochip
(1) making of carrier:
1. be soaked in by nylon membrane in 0.2% Tween-20 solution, soaking at room temperature was taken out after 3 hours; 2. the nylon membrane leaching deionized water rinsing 3 times after soaking;
3. immerse in dyestuff, 40 DEG C of dyeing of beginning, by 3 DEG C/min raised temperature, rise to 95 DEG C of insulation 20min; Period, every 10min jiggled once;
4. take out nylon membrane, in dye liquor, add HAC, put into nylon membrane, 95 DEG C of insulation 20min;
5. dye liquor is removed, with deionized water rinsing 3 times;
6. the film rinsed is soaked in deionized water, room temperature more than 12 hours;
7. remove deionized water, and with after deionized water rinsing twice, add cleansing solution (0.2M PBS-T), wash 5 hours for 40 DEG C;
8. cleansing solution is removed, with deionized water rinsing 3 times;
9. add deionized water, 40 DEG C, soak 1 hour, and repeat again once;
10. washed film is taken out, after deionized water rinsing one time, 37 DEG C of oven dry, for subsequent use.
(2) bag quilt: biological substance not of the same race is coated in the nylon membrane after dyeing with certain requirement.
(3) assembling bag be done after by the nylon membrane containing biological substance, be assembled into biochip.
(4) close: with the confining liquid of every hole 100ul, biochip is closed.
(5) dry: biochip carries out drying after having closed in drying box.
(6) pack: pack after biochip drying.
Embodiment five
The preparation of biological detection reagent
(1) making of carrier: under the prerequisite that the original technique of polystyrene batten is constant, 0.2% black dyes is mixed in polystyrene raw material and paints, carry out lath production by original technique.
(2) bag quilt: envelope antigen bag is buffered liquid preparation coating buffer, every hole is got 100 mL and added ELISA Plate, builds cover plate film, and 4 DEG C of bags are by 16-20h.
(3) wash plate: wrap by after to carry out twice washing, 250 microlitres/hole, after washing, should at towel arsis trepang and liquid, and carry out next step in time and close.
(4) close: confining liquid should shift to an earlier date taking-up in a hour and tentatively rise again, and with front shaking up, every hole is got 180 mL and added ELISA Plate, builds cover plate film, and 37 DEG C of closed 1.5h, the spacing between ELISA Plate should be consistent.
(5) dry: after having closed, fall down liquid in hole, and pat dry on towel, next take 37 DEG C to be inverted the mode finish-drying ELISA Plate of drying or drying.
(6) pack: after plank drying, valve bag should be loaded in time, load according to the amount of every block plate 1 bag of drying agent, finish writing plank outside sack and prepare the date, put into this warehouse for finished product cold storage environment and save backup.
Claims (2)
1. reduce a method for carrier background when biological respinse, it is characterized in that: before biological respinse, colouring process is carried out to biological respinse carrier;
Described biological respinse carrier is the miillpore filter that can be combined by biological substance with bag;
Described colouring process is making before biological detection reagent or in the process making biological detection reagent, adopting the technique contaminated or be coated with dye, by described carrier impregnation in dye liquor or be coated with and be covered with dye liquor, makes described carrier become coloured carrier;
Described colouring process is specially: be first soaked in the Tween-20 solution of 0.1 ~ 0.5% by described carrier, and soaking at room temperature was taken out after 3 hours; Described carrier deionized water rinsing; Immerse in dye liquor and start dyeing, by 2 ~ 4 DEG C/min raised temperature, rise to 90 ~ 95 DEG C of insulation 20min, period, every 8 ~ 10min jiggled once; Take out carrier, in dye liquor, add HAC, put into carrier, 90 ~ 95 DEG C of insulation 15 ~ 20min; Remove dye liquor, with deionized water rinsing; Be soaked in deionized water, room temperature more than 12 hours; Remove deionized water, wash 3 ~ 5 hours with cleansing solution; Remove cleansing solution, with deionized water rinsing; Take out washed 35 ~ 37 DEG C, carrier oven dry.
2. a kind of method reducing carrier background when biological respinse according to claim 1, is characterized in that: described dye liquor is that direct dyes, azoic dyes insoluble azo dyes, reactive dye, reducing dye, soluble vat dye, sulfur dye, sulphur vat dye, phthalocyanine dye, oxidation dye, the condensation dye, disperse dyes, acid dyes, acidic intermedium and acidity are containing any one in mordant dye, alkalescence and the dye of positive ion, coating.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310078156.7A CN103197055B (en) | 2013-03-12 | 2013-03-12 | Method for reducing background of vector in biological reaction |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310078156.7A CN103197055B (en) | 2013-03-12 | 2013-03-12 | Method for reducing background of vector in biological reaction |
Publications (2)
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| CN103197055A CN103197055A (en) | 2013-07-10 |
| CN103197055B true CN103197055B (en) | 2015-04-15 |
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| CN201310078156.7A Expired - Fee Related CN103197055B (en) | 2013-03-12 | 2013-03-12 | Method for reducing background of vector in biological reaction |
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Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB9215003D0 (en) * | 1992-07-15 | 1992-08-26 | Courtaulds Plc | Coloured film |
| US20030219816A1 (en) * | 2001-07-02 | 2003-11-27 | Keith Solomon | Composite microarray slides |
| CN100347545C (en) * | 2003-04-08 | 2007-11-07 | 成都夸常科技有限公司 | Method of preoceeding qualitative and/or quantitative analysis against target substance in sample and its detecting device |
| CN201096776Y (en) * | 2007-10-30 | 2008-08-06 | 江苏三联生物工程有限公司 | Biologic chip |
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2013
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