CN101857900B - Method for preparing double-epoxy modified biochip substrate - Google Patents
Method for preparing double-epoxy modified biochip substrate Download PDFInfo
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- CN101857900B CN101857900B CN 201010167831 CN201010167831A CN101857900B CN 101857900 B CN101857900 B CN 101857900B CN 201010167831 CN201010167831 CN 201010167831 CN 201010167831 A CN201010167831 A CN 201010167831A CN 101857900 B CN101857900 B CN 101857900B
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Abstract
The invention relates to a method for preparing a double-epoxy modified biochip substrate, which comprises processes of surface treatment, preparation of silane coupling agent solution, amino modification, preparation of double-epoxy reagent solution and double-epoxy modification. One of two epoxy groups in a double-epoxy reagent is reacted with an amino on the substrate and opened, and the other epoxy group is exposed on the surface of the substrate. The double-epoxy modified biochip substrate well solves the problems of small acting force of amino substrate contacts and non-uniform point samples, promotes the signal intensity of a chip to a great extent compared with an aldehyde group modified biochip substrate, and can be widely applied to various biochips.
Description
Technical field
The invention belongs to the preparation field of bio-chip substrate, particularly a kind of preparation method of bio-chip substrate of bis-epoxy base modification.
Background technology
Biochip technology is to utilize fine process to process the microstructure of preparation, reaction and detection for biological sample at glass, plastics or other substrate materials, under the form of highly in parallel, lower volume, carry out simultaneously thousands of tests, can carry out accurately biologically active substances such as gene, part, antigens, fast, the determination and analysis of large information capacity, greatly saved time and cost.
Oligonucleotide, gene fragment or proteinaceous antigen-antibody fixedly are the prerequisites that biochip is made substrate surface, therefore improving the crystallized ability of oligonucleotide, gene fragment or proteinaceous antigen-antibody, is one of gordian technique of preparation high quality biochip.Will there be the active function groups that can carry out chemical reaction on the surface of bio-chip substrate, and biomolecules carries out coupling, and substrate itself should have enough stability and bio-compatibility.Sheet glass is abundant because originating, and price is low, has good physics and chemistry inertia, and is transparent, is convenient to detect, and ripe surface treatment method is arranged, and forms one of main carriers of making into bio-chip substrate.
Biochip take sheet glass as substrate, mainly be by use after the surface treatment silane coupling agent modify in case with the aglucon covalent attachment, in early days the modification of sheet glass mostly is that simple individual layer is modified as: amido modified, aldehyde group modified, sulfydryl modification and polylysine modification etc.Amido modified bio-chip substrate is one of bio-chip substrate that uses at present more monomolecular modification.But because the link of monomolecular, so that sterically hindered larger, non-specific adsorption is higher, and point sample is all once low, and bonding force is relatively poor, and strength of signal is not high, and stability is not good during long-time some superchip processed.After occurring subsequently sheet glass modified with silane coupling agent carry out small numerator modified.Wherein with aldehyde radical amido modified bio-chip substrate is carried out secondary modification, optimization is to a certain degree arranged, but well do not improve bonding force and detection signal strength.This just needs a kind of linkage force strong, and non-specific adsorption is little, the bio-chip substrate that strength of signal is high.
Summary of the invention
Technical problem to be solved by this invention provides a kind of preparation method of bio-chip substrate of bis-epoxy base modification, the bio-chip substrate that the prepared bis-epoxy base that gets is modified, solved well amino sheet contact reactive force little, point sample is irregular, with the aldehyde group modified strength of signal that has promoted to a great extent chip of comparing, can be widely used on the different kind organism chip.
The preparation method of the bio-chip substrate that a kind of bis-epoxy base of the present invention is modified comprises:
(1) process of surface treatment: it is in 4: 1: 20 the vitriol oil, hydrogen peroxide and the ultrapure water mixed solution that the sheet glass of at first supersound process being crossed is immersed in volume ratio, 80～130 ℃ of lower placements 30～60 minutes, cooling, ultrapure water flushing 3～5 times, then be soaked in volume ratio and be in 1: 1 concentrated hydrochloric acid and the alcohol mixed solution 3～24 hours, ultrapure water cleans 3～5 times, dries 15～30 minutes for 110～140 ℃;
(2) preparation of silane coupler solution: preparation contains the ethanol solution of silane coupling agent 1～6vol%, Glacial acetic acid 0.01～1.5vol%, pH value of solution=5～6, magnetic agitation 60～90 minutes;
(3) amido modified technique: surface treated sheet glass is put into rapidly the silane coupler solution for preparing, at 25～30 ℃, relative humidity is to place 7～12 hours in 30～50% the climatic chamber, cleans 110～140 ℃ of oven dry 15～30 minutes after taking out with dehydrated alcohol;
(4) preparation of bis-epoxy base reagent solution: preparation contains the ethanol solution of bis-epoxy base reagent 1～6vol%, magnetic agitation 3～5 minutes, whole process lucifuge;
(5) bis-epoxy base modification process: the sheet glass that surface amino groups was processed is put into bis-epoxy base reagent solution, at 25～30 ℃, relative humidity is to place 10～24 hours in 30～50% the climatic chamber, after taking out with the dehydrated alcohol flushing, 110～140 ℃ of oven dry 15～30 minutes.
Silane coupling agent in the described step (2) is 3-trimethoxysilyl-1-propylamine.
Bis-epoxy base reagent in the described step (4) is BDDE.
Reagent used in the present invention (BDDE) cannot twist arbitrarily for stiff molecule, and two epoxy group(ing) in its molecular formula only have one and on-chip amino reaction and open loop, and another then is exposed on the substrate surface.Because in the simple modification of having carried out again the bis-epoxy base after amido modified, increased the substrate surface space, reduced sterically hindered, reduced to a great extent non-specific adsorption, linkage force is strong, can finely fix the materials such as amido modified nucleic acid probe and proteinaceous antigen-antibody, some shape is full, regular, and strength of signal has largely lifting.
Beneficial effect
The preparation method of biological substrate provided by the present invention is simple, the bio-chip substrate background of preparing is low, uniform surface, point of sample is more regular, bonding force is strong, non-specific adsorption is less, there is not conditions of streaking, not only can well be combined with amido modified nucleic acid, can also react with amino, carboxyl, hydroxyl, the sulfydryl of protein surface.It is a kind of desirable bio-chip substrate.
Description of drawings
Fig. 1 is epoxy substrate synthesis mechanism figure;
Fig. 2 is point sample matrix schematic diagram in the test of epoxy substrate point sample;
Fig. 3 is the fluorescent scanning picture behind the epoxy substrate point sample;
Fig. 4 is the atomic force microscopy of epoxy substrate;
Fig. 5 is that the bis-epoxy base is modified sheet and aldehyde group modified, common amino sheet fluorescence signal intensity comparison diagram, the wherein common amino sheet of a-; Aldehyde group modified of b-; C-bis-epoxy base is modified sheet.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims limited range equally.
Embodiment 1:
(1) measure respectively 99.8% dehydrated alcohol 115.7ml, silane coupling agent 7.5ml, Glacial acetic acid 1.8ml joins in the there-necked flask, and the room temperature lower magnetic force stirred 60 minutes.
(2) sheet glass was put into the staining jar that ultrapure water is housed ultrasonic 10 minutes, twice of ultrapure water flushing, then be soaked in the mixed solution of the vitriol oil, hydrogen peroxide and ultrapure water (volume ratio is 4: 1: 20), in 130 ℃ air dry oven, placed 30 minutes, cooling is with ultrapure water flushing 3 times; Then soaked 24 hours in concentrated hydrochloric acid and ethanol (volume ratio is 1: 1) mixing solutions, ultrapure water cleans 5 times, dries 30 minutes, and is cooled to room temperature for 110 ℃.
(3) surface treated sheet glass is put into amino silicane coupling agent solution, then put into climatic chamber, relative humidity is controlled at 50%, Temperature Setting is 30 ℃, place respectively 7,12 hours, with dehydrated alcohol flushing 3 times, 110 ℃ of oven dry namely obtained amido modified bio-chip substrate in 30 minutes after taking out.
(4) measure respectively 99.8% dehydrated alcohol 123.1ml, bis-epoxy base reagent 1.9ml joins in the beaker, magnetic agitation 3 minutes, lucifuge; The sheet glass that surface amino groups was processed is put into wherein, at 25 ℃, relative humidity is to place 10,15,20,24 hours in 30% the climatic chamber, washes with dehydrated alcohol after taking out, dried 30 minutes for 110 ℃, namely obtain the bio-chip substrate that the bis-epoxy base is modified.
Embodiment 2
(1) measure respectively 99.8% dehydrated alcohol 122.5ml, silane coupling agent 1.9ml, Glacial acetic acid 0.5ml joins in the there-necked flask, and at room temperature magnetic agitation is 90 minutes.
(2) sheet glass was put into the staining jar that ultrapure water is housed ultrasonic 5 minutes, twice of ultrapure water flushing, then be soaked in the mixed solution of the vitriol oil, hydrogen peroxide and ultrapure water (volume ratio is 4: 1: 20), in 80 ℃ air dry oven, placed 60 minutes, cooling is with ultrapure water flushing 3 times; Then soaked 3 hours in concentrated hydrochloric acid and ethanol (volume ratio is 1: 1) mixing solutions, ultrapure water cleans 3 times, dries 15 minutes, and is cooled to room temperature for 140 ℃.
(3) surface treated sheet glass is put into amino silicane coupling agent solution, then put into climatic chamber, relative humidity is controlled at 30%, Temperature Setting is 25 ℃, place respectively 7,12 hours, with dehydrated alcohol flushing 3 times, 140 ℃ of oven dry namely obtained amido modified bio-chip substrate in 15 minutes after taking out.
(4) measure respectively 99.8% dehydrated alcohol 117.5ml, bis-epoxy base reagent 7.5ml joins in the beaker, magnetic agitation 5 minutes, lucifuge; The sheet glass that surface amino groups was processed is put into wherein, and at 30 ℃, relative humidity is to place 10,12,24 hours in 50% the climatic chamber, and with the dehydrated alcohol flushing, 140 ℃ of oven dry 15 minutes namely obtain the bio-chip substrate that the bis-epoxy base is modified after taking out.
Embodiment 3
The detection of the bio-chip substrate crystallized ability that the bis-epoxy base is modified
The bio-chip substrate that the used bis-epoxy base of the present embodiment is modified is by embodiment 1 described method preparation, and the present embodiment is to pass through Arrayit
2 point sample instruments carry out point sample test to the epoxy substrate, and point sample matrix design schematic diagram and then passes through Axon as shown in Figure 2
4000B:Cy3 PMT600, Power100 scanner detect the crystallized ability that its fluorescence signal intensity reflects the bio-chip substrate that the bis-epoxy base is modified, and concrete operations are as follows:
(1) prepares the point sample sample
A. under the room temperature, dna probe divided with the concentration of 0.05 μ M, 0.1 μ M, 0.5 μ M, 1 μ M be clipped to 50%DMSO/50%H
2In the damping fluid of O.Antibody probe divided with the concentration of 0.5 μ g/ μ l, 1 μ g/ μ l, 2.5 μ g/ μ l, 5 μ g/ μ l be clipped to 20% glycerine/80%H
2In the damping fluid of O;
B. sample evidence matrix design schematic diagram is transferred in 96 orifice plates or 384 orifice plates, rapped microwell plate liquid is flow at the bottom of the hole, slide neatly is placed on the dot matrix instrument chassis;
C. start dot matrix instrument, from top to bottom at the array of 5 No. 1 damping fluids 5 * 4 of the right of substrate dot matrix to the substrate of selecting, at the array of 5 No. 2 damping fluids 5 * 4 of the left side of substrate dot matrix to the substrate that is well placed.Temperature is controlled at 25 ℃ of room temperatures, and humidity is controlled at about 70%.
(2) combination of probe and epoxy group(ing): behind the point sample, chip is put into chip cartridges, in 37 ℃ of climatic chambers static 24 hours.
(3) develop a film
A. preparing washing liquid 1 (2x SSC/0.2%SDS), washings 2 (1x SSC);
B. chip is placed the chip staining jar, be immersed in the washings 1, put down lightly, mention chip under the room temperature 15 times; Chip is transferred in the staining plate that fills washings 2, put into lightly, mention chip under the room temperature 15 times;
C. shift out staining jar.Use the deionized water cleaning down, washing time is no less than 2 times; With the dry chip of centrifuging (200 * g for 10s).
(4) scanning
A. a slice sample is put into scanner, by dedicated scan instrument software photomultiplier intensity is adjusted to Cy3/PMT600, Power100 clicks start button, and beginning coarse scan sample obtains scanned picture as shown in Figure 4;
B. carefully sweep 25 * 4 protein arrays of signal the best, 25 * 4DNA arrays.Read Cy3 background signal intermediate value, some signal intermediate value and SD value reading, and calculate its discrimination factor CV value and signal to noise ratio S/N value, as shown in table 1;
C. finish scanning, take out sample.
Table 1 bis-epoxy substrate scan-data
Dna probe
The albumen probe
Claims (1)
1. the preparation method of the bio-chip substrate modified of a bis-epoxy base comprises:
(1) process of surface treatment: it is in 4: 1: 20 the vitriol oil, hydrogen peroxide and the ultrapure water mixed solution that the sheet glass of at first supersound process being crossed is immersed in volume ratio, 80～130 ℃ of lower placements 30～60 minutes, cooling, ultrapure water flushing 3～5 times, then be soaked in volume ratio and be in 1: 1 concentrated hydrochloric acid and the alcohol mixed solution 3～24 hours, ultrapure water cleans 3～5 times, dries 15～30 minutes for 110～140 ℃;
(2) preparation of silane coupler solution: preparation contains the ethanol solution of silane coupling agent 1～6vol%, Glacial acetic acid 0.01～1.5vol%, pH value of solution=5～6, magnetic agitation 60～90 minutes;
(3) amido modified technique: surface treated sheet glass is put into rapidly the silane coupler solution for preparing, at 25～30 ℃, relative humidity is to place 7～12 hours in 30～50% the climatic chamber, cleans 110～140 ℃ of oven dry 15～30 minutes after taking out with dehydrated alcohol;
(4) preparation of bis-epoxy base reagent solution: preparation contains the ethanol solution of bis-epoxy base reagent 1～6vol%, magnetic agitation 3～5 minutes, whole process lucifuge;
(5) bis-epoxy base modification process: the sheet glass that surface amino groups was processed is put into above-mentioned bis-epoxy base reagent solution, at 25～30 ℃, relative humidity is to place 10～24 hours in 30～50% the climatic chamber, with the dehydrated alcohol flushing, dried 15～30 minutes for 110～140 ℃ after taking out;
Silane coupling agent in the described step (2) is 3-trimethoxysilyl-1-propylamine;
Bis-epoxy base reagent in the described step (4) is BDDE.
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| CN111266142A (en) * | 2020-03-19 | 2020-06-12 | 京东方科技集团股份有限公司 | Detection chip, modification method thereof and reaction system |
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| CN101241124A (en) * | 2008-02-18 | 2008-08-13 | 博奥生物有限公司 | Biological chip substrate and method for making same |
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| CN101241124A (en) * | 2008-02-18 | 2008-08-13 | 博奥生物有限公司 | Biological chip substrate and method for making same |
Non-Patent Citations (2)
| Title |
|---|
| 曲瑞芳等.超大孔聚苯乙烯微球固定蛋白质.《过程工程学报》.2009,第9卷(第6期),1164-1168. * |
| 蒋犁等.不同方法修饰玻璃表面制备蛋白质芯片的对比.《东南大学学报(自然科学版)》.2007,第37卷(第1期),136-140. * |
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