CN115652440A - Low-density aldehyde-based substrate and preparation method of low-density biochip - Google Patents
Low-density aldehyde-based substrate and preparation method of low-density biochip Download PDFInfo
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Abstract
The application relates to a low-density aldehyde-based substrate and a preparation method of a low-density biochip, and the low-density aldehyde-based substrate comprises the following steps: the porous membrane plate is made of a hydrophobic material and is provided with a through hole array, and the porous membrane plate is attached to the aldehyde modified surface of the aldehyde modified substrate. According to the low-density aldehyde-based substrate, the porous membrane plate made of the hydrophobic material is attached to the aldehyde-based substrate, when the low-density aldehyde-based substrate is used for preparing a biochip, the sample solution can be gathered in the hydrophobic through holes by utilizing hydrophobicity, so that the preparation of the biochip is finished, and the method is simple and easy to implement; and the chip can be prepared by using a liquid-transfering gun, so that the applicability is wide.
Description
Technical Field
The application relates to the field of biochips, in particular to a low-density aldehyde-based substrate and a preparation method of the low-density biochip.
Background
Biochips are currently widely used in the field of biological detection. At present, the biochip, especially the high-density biochip, is restricted by many aspects of manufacturing cost, sample processing time and high-resolution detecting instrument, and the market of the high-density biochip is greatly squeezed along with the rapid development of sequencing technology and the reduction of sequencing cost. However, the low-density chip has the advantages of low manufacturing cost, high sample processing speed and low detection cost, and has a good development trend in the field of POCT.
The number of probes on the low-density biochip is small, and low-cost POCT detection can be realized by properly increasing the quantity of samples and increasing the intervals among probe sites. The special substrate suitable for producing low-density biochip can raise the quality of low-density biochip and lower the cost of substrate. There is a need in the art for such a substrate for low density biochips.
Disclosure of Invention
The invention provides a low-density aldehyde substrate and a method for preparing a low-density biochip aiming at the defect that a special substrate for a low-density chip is absent in the current market, aldehyde modification on a specific position can be carried out according to point position information of a target probe, and the modified aldehyde is only arranged on a position which reacts with the probe and is combined with the probe with amino; the other sites are hydrophobic environment, which is not combined with the probe to reduce the imaging detection background, and the probe is driven to gather to the aldehyde group modification site through hydrophobic effect to improve the combination of the probe and the aldehyde group site, and simultaneously, the chip can be manufactured without depending on a professional sample applicator.
The application provides a low density aldehyde base plate, includes:
an aldehyde-based modified substrate, and
a porous membrane plate made of a hydrophobic material and having an array of through holes, the porous membrane plate attached to the aldehyde-modified surface of the aldehyde-modified substrate.
The application also provides a preparation method of the low-density biochip, which comprises the following steps:
s1, providing a substrate with aldehyde group modification;
s2, adhering a porous membrane plate with a through hole array made of a hydrophobic material to the aldehyde-group modified surface of the aldehyde-group modified substrate to obtain the low-density aldehyde-group substrate;
and S3, applying a probe with amino groups into the through hole, and fixing the probe on the substrate to obtain the low-density biochip.
According to the low-density aldehyde-based substrate, the porous membrane plate made of the hydrophobic material is attached to the aldehyde-based substrate, so that when the low-density aldehyde-based substrate is used for preparing a biochip, the sample solution can be gathered in the hydrophobic through holes by utilizing hydrophobicity, the preparation of the biochip is completed, and the method is simple and easy to implement; and can use the liquid-transfering gun to prepare the chip, the serviceability is extensive.
Drawings
FIG. 1 is a schematic diagram of a low-density aldehyde-based substrate, wherein 1 is a porous membrane plate, 2 is a through hole of the porous membrane plate, and 3 is an aldehyde-based modified substrate;
fig. 2 shows the detection results of example 1.
Detailed Description
The present application will be described in further detail below with reference to examples. The features and advantages of the present application will become more apparent from the description.
The word "exemplary" is used exclusively herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.
In addition, the technical features described below in the different embodiments of the present application may be combined with each other as long as they do not conflict with each other.
As shown in fig. 1, the present application provides a low density aldehyde-based substrate comprising:
aldehyde group-modified substrate 3, and
a porous membrane plate 1 made of a hydrophobic material and having an array of through holes, the porous membrane plate being attached to the aldehyde-modified surface of the aldehyde-modified substrate.
In the present disclosure, "low density" means less than 4000 probes per square centimeter, and particularly within 1000 probes per square centimeter, relative to a high density chip with over ten thousand probes per square centimeter.
In the present disclosure, the low-density aldehyde-based substrate includes the aldehyde-based modified substrate 3. The aldehyde-modified substrate 3 may be a glass substrate such as a low-fluorescence glass substrate, one surface of which is subjected to aldehyde modification treatment to form an aldehyde-modified surface.
Providing an aldehyde-based modified substrate comprising:
s11, activating the surface of the substrate;
s12, performing amino modification on the substrate with the activated surface;
and S13, performing aldehyde group modification on the substrate modified by the amino group to obtain the aldehyde group modified substrate.
The surface of a substrate such as a low-fluorescence white glass sheet can be cleaned to remove oil stains and dust, surface activation can be performed (for example, concentrated sulfuric acid and hydrogen peroxide (95 to 5 vol.%) can be used for treatment), and surface amino modification can be performed after water washing.
The substrate may be subjected to surface amino modification in an amino modification solution. In one embodiment, making the amino modification comprises: treating with amino modifier solution for 0.5-2 hr.
The amino modifier is a compound having the following formula I:
wherein R is 1 、R 2 、R 3 Each independently selected from C 1 -C 6 Alkyl or C 1 -C 6 Alkoxy radical, wherein R 1 、R 2 、R 3 At least one of them being C 1 -C 6 An alkoxy group;
R 4 is selected from C 1 -C 6 An alkylene group.
Preferably, the amino modifier may be 3-aminopropyltriethoxysilane or the like. A solution of the amino modifier may be used for 0.5-2 hours with the aid of gentle shaking. For example, 1mol/L acetone solution of 3-aminopropyltriethoxysilane can be allowed to act at room temperature for 0.5 to 2 hours.
And (3) modifying the amino modified substrate with a dialdehyde compound to obtain the aldehyde modified substrate. The substrate can be placed in a dialdehyde compound solution for aldehyde group modification to obtain the aldehyde group modified substrate. The aldehyde group modification conditions are as follows: after the dialdehyde compound solution is added to the substrate, it is allowed to react at room temperature for 0.5 to 2 hours. The dialdehyde compound used in general may be selected from terephthalaldehyde, glutaraldehyde and the like, and for example, 1mol/L acetone solution of the dialdehyde compound may be used as the aldehyde group modifier solution.
When the amino modified substrate is placed in a dialdehyde compound, one aldehyde group of the amino modified substrate and the amino can be dehydrated to form Schiff base, and the other aldehyde group is exposed on the surface of the substrate and can be combined with probe molecules with the amino.
The low-density aldehyde-based substrate comprises a porous membrane plate 1, wherein the porous membrane plate 1 is made of a hydrophobic material and is provided with a through hole array of through holes 2, and the porous membrane plate 1 is attached to an aldehyde-based modified surface of an aldehyde-based modified substrate 3.
In the present application, the porous membrane sheet 1 is made of a hydrophobic material. The use of a porous membrane sheet 1 made of a hydrophobic material is critical to the present application. When the biochip is prepared by the low-density aldehyde-based substrate, the probe solution is dripped onto the through hole, the liquid drops automatically gather into the through hole due to the hydrophobicity of the hydrophobic material of the porous membrane plate, and the amino group of the probe in the liquid drops and the aldehyde group on the substrate form Schiff base, so that the probe is fixed on the substrate, and the preparation of the biochip is completed. Moreover, the hydrophobic material and the through holes on the hydrophobic material provide a hydrophobic fence for the hydrophilic sites which can be combined by each probe, so that the flow of the sampling liquid to the periphery is prevented, the edge deformation of the probe sites caused by the edge evaporation of the sampling liquid is prevented, and the quality of the biochip is improved. The hydrophobic environment is arranged around the hydrophilic site where each probe can be combined, so that the displacement of probe points and the formation of noise points outside the probes caused by the complete activation of the surface are overcome in the manufacturing process of the biochip by utilizing the hydrophobic environment, and the yield and the quality of the biochip are improved. In one embodiment, materials having hydrophobic properties such as metals, wax films, oilpaper, ceramics, resins, etc. may be used to make the porous membrane sheet.
The lattice density of the low-density aldehyde-based substrate of the present disclosure depends on the density of the through holes in the through hole array in the porous membrane plate 1. In one embodiment, the through hole array comprises a plurality of through holes 2 distributed in an array, the pore diameter of the through hole 2 of the porous membrane plate is 100-300 μm, and the distance between the two through holes is 300-500 μm. The aldehyde group modified surface of the aldehyde group substrate can be exposed at the through hole 2; and in other positions, the aldehyde modified surface of the aldehyde substrate is covered. Therefore, when the biochip is prepared, the hydrophilic sites which can be combined by the probes on the low-density aldehyde-based substrate are arranged at a larger distance and are surrounded by the hydrophobic regions, the spotting fluid can be automatically gathered on the hydrophilic sites, and the preparation process of the biochip can be completed without using a special spotting instrument.
Thus, the present application provides a method for preparing a low-density biochip, comprising:
s1, providing a substrate with aldehyde group modification;
s2, adhering a porous membrane plate with a through hole array made of a hydrophobic material to the aldehyde-group modified surface of the aldehyde-group modified substrate to obtain the low-density aldehyde-group substrate;
and S3, applying a probe with amino groups into the through hole, and fixing the probe on the substrate to obtain the low-density biochip.
Wherein, providing the substrate with aldehyde group modification can be performed as above, and is not described in detail here.
In one embodiment, the porous membrane sheet may be adhered to the aldehyde-modified surface of the aldehyde-modified substrate by an adhesive. The adhesive may be selected from a uv curable adhesive or a moisture curable adhesive. The adhesive may be coated on the porous membrane plate and then adhered to the aldehyde-modified surface of the aldehyde-modified substrate, and after curing, a low-density aldehyde-based substrate may be obtained.
The amino group of the probe can only be connected with the aldehyde group exposed at the through hole through Schiff base reaction, so that the probe molecule is connected to the substrate, unreacted probes are removed through washing, and the biochip for detection is obtained after drying. The probe solution may be added to the low-density aldehyde-based substrate by a robot-operated pipette. Especially, a multi-channel liquid-transfering gun can be used, and the preparation efficiency of the low-density biochip is greatly improved. Useful biological probes include various genetic probes, protein probes, and the like in the art. Because the hydrophilic site capable of being combined with the probe is surrounded by the hydrophobic region, when the probe is combined, the sample solution can be automatically gathered on the aldehyde group modified site, so that the preparation process of the chip can be completed without using a special sample applicator.
Example 1
(1) Activating the glass surface: carrying out surface cleaning on the low-fluorescence white glass sheet to remove oil stains and dust, carrying out surface activation by using concentrated sulfuric acid and hydrogen peroxide (volume ratio of 95 to 5), and carrying out surface amino modification after washing;
(2) Amino modification: adding an amino modifier for amino modification: using 1mol/L acetone solution of 3-aminopropyltriethoxysilane, acting for 0.5-2 hours at room temperature, and slightly oscillating with assistance to obtain an amino modified substrate;
(3) Aldehyde group modification: carrying out dialdehyde compound modification on the amino modified substrate: reacting 1mol/L acetone solution of terephthalaldehyde at room temperature for 0.5-2 hours to obtain an aldehyde substrate;
(4) Designing a porous membrane plate: the aperture of the through hole of the porous membrane plate is 100-300 mu m, and the distance between the two through holes is 300-500 mu m; in the embodiment, the aluminum foil is used, the manufacturing is convenient, the porous membrane plate of the microarray through hole with the through hole aperture of 200 mu m and the distance between two through holes of 500 mu m is manufactured;
(5) Adhering a porous membrane plate made of a hydrophobic material to an aldehyde-modified glass substrate to prepare a low-density aldehyde-based substrate, wherein each through hole corresponds to a hydrophilic site to which a probe can be combined; adhering by using (Loctite) UV glue, wherein the using method comprises the following steps: coating UV glue on the porous membrane plate of the microarray through hole, scraping the UV glue into a thin layer by using a glass rod, and directly adhering the thin layer to an aldehyde-based substrate to ensure that only the position of the through hole is exposed to aldehyde groups, and the aldehyde groups at other positions are covered by the porous membrane plate; irradiating with 365nm ultraviolet lamp at a lamp distance of 3cm for 15 s to complete primary curing, and standing for 1 hr;
(6) Preparing a gene chip: dripping a sample solution of an oligonucleotide probe (purchased from Biotechnology engineering (Shanghai) Co., ltd., use concentration of 15mmol/L; sample solution is a crystal core gene chip sample solution and purchased from Beijing Boao biology Co., ltd.) with a modified amino group at the 5' end onto the through hole, automatically gathering the liquid drops into the through hole due to the hydrophobicity of a hydrophobic material, forming Schiff base by the amino group of the probe in the liquid drops and the aldehyde group on the substrate, fixing the probe on the substrate, and cleaning and removing the unreacted probe after combining the liquid drops and the gene chip.
(7) And (3) detection process: adding target DNA to be detected to the gene chip combined with the probe, completing the hybridization process of the target gene by utilizing the existing chip gene microarray chip hybridization box (purchased from Beijing Boao biology, ltd.), and reading the hybridization result by utilizing a gene chip reader. The results of the detection are shown in FIG. 2. It can be seen that each hybridization point is round and full and the boundary is clear, because when the biochip is prepared, the hydrophobic porous material of the substrate and the through holes on the hydrophobic porous material can provide a hydrophobic fence for the hydrophilic sites which can be combined by each probe, and the edge deformation of the probe sites caused by the flow of the sample solution to the periphery is prevented, so that each hybridization point is round and full and the boundary is clear when the prepared biochip is subjected to hybridization detection, and the quality of the biochip is high.
The present application has been described above with reference to preferred embodiments, but these embodiments are merely exemplary and merely illustrative. On the basis of the above, the present application can be subjected to various substitutions and improvements, and the substitutions and the improvements are all within the protection scope of the present application.
Claims (10)
1. A low density aldehyde based substrate comprising:
an aldehyde-based modified substrate, and
a porous membrane plate made of a hydrophobic material and having an array of through holes, the porous membrane plate attached to the aldehyde-modified surface of the aldehyde-modified substrate.
2. The low-density aldehyde-based substrate according to claim 1, wherein the porous membrane plate has a through-hole diameter of 100 to 300 μm and a spacing between two through-holes of 300 to 500 μm.
3. The low-density aldehyde-based substrate according to claim 1, wherein the hydrophobic material is selected from a metal material, a wax film, an oilpaper material, a ceramic material, or a resin material.
4. A method for preparing a low-density biochip, comprising:
s1, providing a substrate with aldehyde group modification;
s2, adhering a porous membrane plate with a through hole array made of a hydrophobic material to the aldehyde-group modified surface of the aldehyde-group modified substrate to obtain the low-density aldehyde-group substrate;
and S3, applying a probe with amino groups into the through hole, and fixing the probe on the substrate to obtain the low-density biochip.
5. The production method according to claim 4, wherein the porous membrane sheet is adhered to the aldehyde-modified surface of the aldehyde-modified substrate by an adhesive.
6. The production method according to claim 4, wherein the adhesive is selected from an ultraviolet curable adhesive or a moisture curable adhesive.
7. The method of claim 4, wherein providing the aldehyde-based modified substrate comprises:
s11, activating the surface of the substrate;
s12, performing amino modification on the substrate with the activated surface;
and S13, performing aldehyde group modification on the substrate modified by the amino group to obtain the aldehyde group modified substrate.
8. The method of claim 7, wherein the amino group modification comprises: treating with amino modifier solution for 0.5-2 hr.
9. The method of claim 8, wherein the amino modifier is a compound having the following formula I:
wherein R is 1 、R 2 、R 3 Each independently selected from C 1 -C 6 Alkyl or C 1 -C 6 Alkoxy radical, wherein R 1 、R 2 、R 3 At least one of them being C 1 -C 6 An alkoxy group;
R 4 is selected from C 1 -C 6 An alkylene group.
10. The method of claim 7, wherein performing aldehyde group modification comprises: treating with anhydrous solution of dialdehyde compound with two aldehyde groups for 0.5-2 hr.
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| CN116003865A (en) * | 2023-02-24 | 2023-04-25 | 北京百奥纳芯生物科技有限公司 | Flexible aldehyde base sheet, preparation method and biochip |
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