CN215963614U - Immunodetection chip - Google Patents
Immunodetection chip Download PDFInfo
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- CN215963614U CN215963614U CN202023078697.4U CN202023078697U CN215963614U CN 215963614 U CN215963614 U CN 215963614U CN 202023078697 U CN202023078697 U CN 202023078697U CN 215963614 U CN215963614 U CN 215963614U
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Abstract
The utility model discloses an immunodetection chip which comprises a substrate, wherein the substrate is made of an ultraviolet curing material, a periodic nano groove array is arranged on the surface of the substrate, a metal layer is arranged on the upper surface of the substrate, an oxide layer is arranged on the upper surface of the metal layer, and the groove is sealed by sealing liquid after the upper surface of the oxide layer is modified by a polymer layer. The utility model also provides a preparation method of the immunodetection chip, which is used for producing the detection chip with the organic material as the substrate in batch through the silicon-based template. The chip has high sensitivity, high flux and short production period, can realize batch production, and has wide application range when being used for immunodetection.
Description
Technical Field
The utility model belongs to the field of biochip manufacturing, and particularly relates to an immunodetection chip.
Background
As an important modern detection technology, fluorescence detection has the advantages of high sensitivity, various methods and the like compared with other detection technologies, and is one of the most common analysis and test technologies in the fields of diagnostics, bioscience, biotechnology and the like. However, due to the diversity and specificity of the samples, the sensitivity of fluorescence detection in practical processes still cannot meet the requirements of all assays, and especially has great limitation on the detection of weak fluorescence molecular systems and trace target molecules.
The traditional fluorescence detection technology is simple and crude in means and limited in technology, manual operation is mostly needed, so that the error is large, and the detection result of a sample is greatly related to the detection level of detection personnel. The biochip technology can integrate massive information on a tiny size, can realize rapid and efficient measurement and analysis, is orderly fixed on the surface of a carrier according to the preset arrangement, measures and analyzes biomolecules by utilizing the specific affinity reaction between the biomolecules, avoids multiple manual operations, reduces time consumption, reduces the system deviation caused by a detection result, improves the detection efficiency, and has great value in the aspect of high-throughput application.
The common preparation materials of the biochip at present are monocrystalline silicon wafers, glass and quartz. Glass and quartz have good surface properties and optical properties, and the manufacturing method mainly comprises a standard photoetching technology and wet etching, but the manufacturing process of the chip is complicated and high in cost, and the processing process of the micro-nano structure is not mature. The monocrystalline silicon is used as the primary material of the biochip, and the processing technology of the monocrystalline silicon is mature, and has the advantages of high strength, high purity, high cleanliness, good scattering property, corrosion resistance and the like. However, the silicon material has poor insulation and light transmission, great difficulty in deep etching, poor adhesion of the silicon substrate, and relatively high cost of the silicon chip compared with other materials.
The development of biochip technology is still in its early stage, and its complex theory and technology need further research, so its accuracy and stability are very affected. The biochip is usually made of silicon substrate, and is usually applied to photoetching film and ion etching technology, and has high preparation cost, long period and difficult popularization, and the chip design and the development and application of new products are seriously influenced, which is the limitation of the biochip technology. For example, the preparation cost of the photoetching mask is high and the period is long in the preparation process of the gene chip. Biochips are started late in China, large-scale production is not realized until now, and more biochips depend on foreign import. The chinese biochip research started in the 90 s of the 20 th century, and increased at a rate of over 20% each year from 2008 to 2019, the market size of which in 2019 has exceeded us $ 9 billion, 2020 is expected to exceed 20 billion in chinese market size in 2021 because of the outbreak of new coronavirus worldwide, and the late growth rate will exceed 40%. The biochip market reaches $ 39 billion worldwide in 2014, develops at a growth rate of over 31.6% in 2015-2020, has reached $ 184 billion in 2020, and the north american region dominates the global biochip market. The investment of China in the basic research field and the new drug research and development field is relatively small, international chip products compete over domestic products, and the international chip is basically used in international high-level research.
The prepared fluorescence detection biochip has high flux and sensitivity and is suitable for batch industrial production, and has great value.
SUMMERY OF THE UTILITY MODEL
Aiming at least one technical problem in the prior art, the utility model provides the immunoassay chip which has the advantages of high chip sensitivity, high flux, short production period, realization of batch production and wide application range for immunoassay.
The technical scheme for solving the technical problems is as follows: the utility model provides an immunoassay chip, includes the base, the base adopts ultraviolet curing material, the base surface is provided with periodic nanometer groove array, the base upper surface is provided with the metal level, the metal level upper surface is provided with the oxide layer, the oxide layer upper surface is through the modification of macromolecular layer after, will the recess is sealed with the confining liquid.
On the basis of the technical scheme, the utility model can be further improved as follows.
Further, the thickness of the metal layer is 100nm to 500 nm.
Further, the metal layer includes a metal adhesion layer of a lower layer and a noble metal layer of an upper layer.
Preferably, the thickness of the metal adhesion layer is 4 nm-50 nm.
Preferably, the thickness of the noble metal layer is 100nm to 400 nm.
Preferably, the metal adhesion layer is made of Cr or Ti or an alloy of Cr and Ti.
Preferably, the material of the noble metal layer is Au, Ag, Cu or Pt, or an alloy of Au, Ag, Cu and Pt.
Further, the period of the periodic nano groove array is 100 nm-3000 nm.
Further, the diameter of a single groove of the periodic nano groove array is 50 nm-2000 nm.
Further, the depth of a single groove of the periodic nano groove array is 10 nm-500 nm.
Further, the thickness of the oxide layer is 100 nm-600 nm.
Further, the oxide layer is SiO2、ZnO、Al2O3、TiO2Or MgF2Material layer, or SiO2、ZnO、Al2O3、TiO2And MgF2A layer of material prepared from the mixture of (a).
The utility model has the beneficial effects that: the silicon substrate is not adopted, the production period is short compared with the traditional production method, the cost is low, and the mass and rapid production can be realized; the chip of the utility model enhances fluorescence by plasmon resonance of micro/nano periodic structure, and can significantly improve the flux and sensitivity of immunoassay by an organic material micro-substrate; the utility model is used for immunodetection, has extremely wide application range, and can detect biomolecules such as nucleic acid, protein, polypeptide and the like; the utility model has very important significance for the development of the biological detection microchip market and has very great economic value.
Drawings
FIG. 1 is a schematic process flow diagram of the present invention;
FIG. 2 is a schematic structural view of the present invention;
FIG. 3 is a graph showing the effects of tests on examples of the present invention and comparative examples;
FIG. 4 is a graph showing fluorescence detection intensity of examples of the present invention and comparative examples;
in the drawings, the components represented by the respective reference numerals are listed below:
1. the device comprises a substrate, 2, a metal layer, 21, a metal adhesion layer, 22, a noble metal layer, 3, an oxidation layer, 4 and sealing liquid.
Detailed Description
The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the utility model.
As shown in fig. 2, the immunoassay chip designed by the present invention includes a substrate 1, wherein the substrate 1 is made of an ultraviolet light curing material, a periodic nano groove array is disposed on the surface of the substrate 1, a metal layer 2 is disposed on the upper surface of the substrate 1, an oxide layer 3 is disposed on the upper surface of the metal layer 2, and the groove is sealed by a sealing liquid 4 after the upper surface of the oxide layer 3 is modified by a polymer layer.
The core of the utility model is that the substrate 1 made of non-silicon-based material is adopted, and the substrate 1 made of ultraviolet curing material is adopted.
At present, in the field of chips, particularly in the field of precise chip manufacturing, a silicon-based material is used as a substrate, a very precise chip structure can be prepared by utilizing a photoetching mode, and the performance of the chip can be greatly improved by depositing a corresponding metal or non-metal material in a matching manner.
However, the inventor finds that in the field of biochips, the silicon-based substrate has high preparation cost and long cycle, and the development of the biochips is directly restricted by the high cost of the silicon-based substrate. The utility model has the advantages of low flux and low detection sensitivity of a chip adopting the silicon material during fluorescence detection, along with poor insulation and light transmission of the silicon material, high deep etching difficulty and poor adhesion of the silicon substrate. The inventor researches and discovers that the special characteristics of fluorescence detection cause different requirements on materials, and simultaneously discovers that the ultraviolet curing material has good insulating property and light transmittance and is very suitable for preparing biochips, so that the substrate 1 is prepared from an organic polymer material.
The ultraviolet curing material has strong machinability and can be processed by an ultraviolet hardening imprint lithography technology. The surface processing precision of the biochip is extremely high, and is in a nanometer level. The inventor finally determines that the silicon material is used as the template, the high-precision template can be prepared by utilizing the advantage of high photoetching precision of the silicon material through the ultraviolet hardening imprint lithography process, and the high-precision micropore array can be imprinted on the surface of the ultraviolet curing material by utilizing the template. Through repeated verification experiments of the inventor, the precision of the processed micropore array can completely meet the precision requirement of a biochip.
The utility model also provides an improved technical scheme, wherein in the improved technical scheme, the thickness of the metal layer 2 is 100 nm-500 nm.
The utility model also provides an improved technical scheme, in the improved technical scheme, the metal layer 2 comprises a metal adhesion layer 21 at the lower layer and a noble metal layer 22 at the upper layer.
Preferably, the thickness of the metal adhesion layer 21 is 4nm to 50 nm.
Preferably, the thickness of the noble metal layer 22 is 100nm to 400 nm.
Preferably, the metal of the metal adhesion layer 21 is one of Cr and Ti or an alloy thereof.
Preferably, the metal of the noble metal layer 22 is one of Au, Ag, Cu and Pt or an alloy thereof.
The improved scheme can enable the chip to have stronger sensitivity and photoelectric performance.
The utility model also provides an improved technical scheme, wherein in the improved technical scheme, the period of the periodic nano groove array is 100 nm-3000 nm.
The utility model also provides an improved technical scheme, wherein in the improved technical scheme, the diameter of each groove of the periodic nano groove array is 50 nm-2000 nm.
The utility model also provides an improved technical scheme, wherein in the improved technical scheme, the depth of a single groove of the periodic nano groove array is 10 nm-500 nm.
The improved technical scheme can enable the surface of the chip to have a micro/nano periodic structure, and plasmon resonance enhanced fluorescence can be obtained, so that the sensitivity of the chip is further improved.
The utility model also provides an improved technical scheme, wherein in the improved technical scheme, the thickness of the oxide layer is 100 nm-600 nm.
The utility model also provides an improved technical scheme, wherein in the improved technical scheme, the oxide of the oxide layer is SiO2、ZnO、Al2O3、TiO2、MgF2One or a combination of more than one of them.
The utility model also provides an improved technical scheme, and in the improved technical scheme, the upper surface of the oxide layer is modified by a silane coupling agent.
Preferably, the silane coupling agent is one of (3-glycidoxypropyl) trimethoxysilane, 1H,2H, 2H-perfluorooctyltriethoxysilane, KH-1332 tridecafluorooctyltriethoxysilane and 3-mercaptopropyl-trimethoxysilane.
The improved technical scheme can make the chip surface easier to combine with the sealing liquid 4.
The preparation method comprises the following steps:
s1, preparing a template: taking a monocrystalline silicon/quartz substrate with a silicon oxide layer, and processing a nanopore array with the shape opposite to that of a periodic nanometer groove array arranged on the surface of a substrate 1 on the surface in a photoetching mode to prepare a template;
s2, preparing a substrate 1: preparing a substrate 1 of the ultraviolet curing material with the periodic nano groove array by using the template prepared in the step S1 through an ultraviolet hardening imprint lithography mode;
s3, deposition: sequentially depositing a metal layer 2 and an oxide layer 3 on the substrate 1 prepared in the step S2 to prepare a chip substrate;
s4, coating an antibody: and (4) modifying the surface of the chip substrate prepared in the step (S3) by using a polymer layer, and then sealing by using sealing liquid 4 to prepare the final biochip.
On the basis of the technical scheme, the utility model can be further improved as follows.
The step S1 includes the steps of:
1) taking a monocrystalline silicon/quartz substrate with a silicon oxide layer, and coating an electron beam photoresist layer on the silicon oxide layer;
2) etching a processing surface of a nanopore array with the shape opposite to that of the periodic nanometer groove array arranged on the surface of the substrate 1 on the electron beam photoresist layer by using an electron beam exposure method to expose the silicon oxide layer of the photoetching part;
3) and chemically etching the processing surface, processing a nanopore array with the shape opposite to that of the periodic nanometer groove array arranged on the surface of the substrate 1 on the monocrystalline silicon/quartz substrate, cleaning the residual glue after etching, and blow-drying to obtain the template.
Preferably, the chemical etching is performed by introducing trifluoromethane gas and oxygen gas in sequence.
In the step S2, the ultraviolet hardening imprint lithography specifically includes the steps of spreading a layer of ultraviolet curing nanoimprint lithography glue on the surface of the template, imprinting the nanoimprint lithography glue with the template, and etching the residual layer after ultraviolet exposure and demolding to obtain the substrate 1.
In step S4, the polymer layer modification specifically includes placing the chip substrate in a polymer modification reagent solution for reaction, and after the reaction is finished, cleaning and drying the chip substrate.
The polymer modification reagent is a silane coupling agent, and the reaction is carried out for 2 hours in a constant temperature shaking table under the condition of 30 ℃.
Example 1
Taking a quartz substrate (purchased commercially), coating an electron beam photoresist layer (adopting PMMA photoresist), processing a cylindrical matrix formed by periodically arranging convex surfaces on the electron beam photoresist layer by using an electron beam exposure method, wherein the period of the matrix is 2200nm, the height of the cylinder is 260nm, and the diameter is 1350nm, then obtaining a substrate A by using a developing technology, then placing the substrate A in a reactive ion etching device, introducing trifluoromethane gas into the reactive ion etching device, wherein the flow rate of the trifluoromethane gas is 310sccm, the gas pressure is 1.65Pa, the radio frequency power is 150W, and the etching time is 280 s; introducing oxygen after the etching of the trifluoromethane is finished, wherein the oxygen flow is 108sccm, the gas pressure is 1.14Pa, the radio frequency power is 110W, and the etching time is 63 s; and taking out the substrate A after etching, dropwise adding 0.5mL of trimethylchlorosilane into a vessel, placing the etched substrate A into the vessel without contacting with the trimethylchlorosilane, sealing the vessel and standing for 30min, taking out the substrate A subjected to standing treatment, cleaning the substrate A with ethanol, and drying the substrate A by blowing to obtain the nano-imprint template.
Taking a silicon substrate (purchased commercially), spin-coating ultraviolet curing nanoimprint lithography glue with the thickness of 600-700 nm on the substrate, then pressing the silicon substrate with the nanoimprint lithography template in an opposite manner, curing the ultraviolet curing glue through template ultraviolet irradiation, and etching the substrate by using ion etching equipment after demolding to obtain the chip silicon substrate B with the periodic nano matrix. Depositing a 24nm Cr layer on a chip substrate B, then laminating a 2400nm Au layer, and finally depositing a 25nm SiO layer2And oxidizing the layer to obtain a chip substrate C.
And (2) placing the chip substrate C in an ethanol solution containing 0.1 percent (3-glycidoxypropyl) trimethoxysilane and 0.1 percent of 1H,1H,2H, 2H-perfluorooctyltriethoxysilane, shaking for 2 hours at 30 ℃, washing for 3 times respectively by using ethanol and distilled water, drying the chip substrate D, sealing the biochip substrate D by using a sealing solution for 2 hours, removing the redundant sealing solution, and drying the substrate to obtain the final fluorescence pre-amplification immunoassay chip.
Example 2
Taking a quartz glass substrate (purchased commercially), coating an electron beam photoresist layer (adopting PMMA photoresist) on the substrate, processing a cuboid matrix with convex surfaces arranged periodically on the electron beam photoresist layer by using an electron beam exposure method, wherein the period of the matrix is 2200nm, the height of the cuboid is 260nm, and the length of the cuboid is 1300nm, then obtaining a substrate A by using a developing technology, then placing the substrate A in a reactive ion etching device, introducing trifluoromethane gas into the reactive ion etching device, wherein the flow rate of the trifluoromethane gas is 310sccm, the gas pressure is 1.65Pa, the radio frequency power is 150W, and the etching time is 280 s; introducing oxygen after the etching of the trifluoromethane is finished, wherein the oxygen flow is 108sccm, the gas pressure is 1.14Pa, the radio frequency power is 110W, and the etching time is 63 s; and taking out the substrate A after etching, dropwise adding 0.5mL of trimethylchlorosilane into a vessel, placing the etched substrate A into the vessel without contacting with the trimethylchlorosilane, sealing the vessel and standing for 30min, taking out the substrate A subjected to standing treatment, cleaning the substrate A with ethanol, and drying the substrate A by blowing to obtain the nano-imprint template.
Taking a silicon substrate (purchased commercially), spin-coating ultraviolet curing nanoimprint lithography glue with the thickness of 600-700 nm on the substrate, then pressing the silicon substrate with the nanoimprint lithography template in an opposite manner, curing the ultraviolet curing glue through template ultraviolet irradiation, and etching the substrate by using ion etching equipment after demolding to obtain the chip silicon substrate B with the periodic nano matrix. Depositing a 24nm Cr layer on a chip substrate B, then laminating a 2400nm Au layer, and finally depositing a 25nm SiO layer2And oxidizing the layer to obtain a chip substrate C.
And (2) placing the chip substrate C in an ethanol solution containing 0.1 percent (3-glycidoxypropyl) trimethoxysilane and 0.1 percent of 1H,1H,2H, 2H-perfluorooctyltriethoxysilane, shaking for 2 hours at 30 ℃, washing for 3 times respectively by using ethanol and distilled water, drying the chip substrate D, sealing the biochip substrate D by using a sealing solution for 2 hours, removing the redundant sealing solution, and drying the substrate to obtain the final fluorescence pre-amplification immunoassay chip.
And (3) testing and analyzing:
the fluorescence immunoassay biochip of example 1 and example 2 and a commonly used glass substrate chip were used to detect VEGF in ocular tissue fluid of a patient with ocular fundus disease.
First, since the sealing liquid sealing is performed in examples 1 and 2, the sealing liquid treatment is also required in the conventional glass substrate detection, and the detection time is shortened by not less than 2h by using the chips in examples 1 and 2. Finally, immunofluorescence detection is performed on the biochip substrate using a microarray scanner.
The experimental results are as follows:
the results of the tests of examples 1 and 2, and the conventional glass substrate chip are shown in FIG. 3. As can be seen from fig. 3, the fluorescence effect was clearly observed in both example 1 and example 2, whereas the fluorescence detection effect of the conventional glass substrate chip was not significant.
Next, the fluorescence detection intensity was measured for example 1 and example 2, and a conventional glass substrate chip, and the results are shown in FIG. 4. As can be seen from fig. 4, the fluorescence detection intensities of the three samples are 18600, 19200 and 105, respectively, and the detection intensities of example 1 and example 2 are increased 177 and 182 times, respectively, compared with those of the conventional glass substrate chip. Meanwhile, the detection limits of the three samples are respectively 38pg/ml, 42pg/ml and 1.2pg/ml through the test, and the detection intensity of the example 1 and the example 2 is respectively increased by 31 times and 35 times compared with the detection intensity of the conventional glass substrate chip.
As can be seen from the above experiments, the biochip of the present invention has greatly improved sensitivity and flux compared to the conventional silicon substrate chip.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the utility model, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (9)
1. An immunoassay chip comprising a substrate (1), characterized in that: the substrate (1) is made of an ultraviolet curing material, the surface of the substrate (1) is provided with a periodic nano groove array, the upper surface of the substrate (1) is provided with a metal layer (2), the upper surface of the metal layer (2) is provided with an oxide layer (3), and after the upper surface of the oxide layer (3) is modified by a polymer layer, the groove is sealed by sealing liquid (4).
2. The immunoassay chip of claim 1, wherein: the thickness of the metal layer (2) is 100 nm-500 nm.
3. The immunoassay chip of claim 1, wherein: the metal layer (2) comprises a metal adhesion layer (21) at the lower layer and a noble metal layer (22) at the upper layer.
4. The immunoassay chip of claim 3, wherein: the thickness of the metal adhesion layer (21) is 4 nm-50 nm.
5. The immunoassay chip of claim 3, wherein: the thickness of the noble metal layer (22) is 100 nm-400 nm.
6. The immunoassay chip of claim 1, wherein: the period of the periodic nano groove array is 100 nm-3000 nm.
7. The immunoassay chip of claim 1, wherein: the diameter of a single groove of the periodic nano groove array is 50 nm-2000 nm.
8. The immunoassay chip of claim 1, wherein: the depth of a single groove of the periodic nano groove array is 10 nm-500 nm.
9. The immunoassay chip of claim 1, wherein: the thickness of the oxide layer (3) is 100 nm-600 nm.
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| CN202023078697.4U CN215963614U (en) | 2020-12-20 | 2020-12-20 | Immunodetection chip |
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| CN202023078697.4U CN215963614U (en) | 2020-12-20 | 2020-12-20 | Immunodetection chip |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112611861A (en) * | 2020-11-23 | 2021-04-06 | 武汉世纪康敏生物科技有限公司 | Fluorescence immunoassay chip and preparation method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112611861A (en) * | 2020-11-23 | 2021-04-06 | 武汉世纪康敏生物科技有限公司 | Fluorescence immunoassay chip and preparation method thereof |
| CN112611861B (en) * | 2020-11-23 | 2024-03-29 | 武汉世纪康敏生物科技有限公司 | Fluorescent immunodetection chip and preparation method thereof |
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