CN113008973A - Protein chip suitable for detecting low-abundance protein and preparation method and application thereof - Google Patents
Protein chip suitable for detecting low-abundance protein and preparation method and application thereof Download PDFInfo
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- CN113008973A CN113008973A CN202110134324.4A CN202110134324A CN113008973A CN 113008973 A CN113008973 A CN 113008973A CN 202110134324 A CN202110134324 A CN 202110134324A CN 113008973 A CN113008973 A CN 113008973A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
- G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry
Abstract
The invention discloses a protein chip suitable for detecting low-abundance protein, which comprises sample hole points with the diameter of 1-3mm and the depth of less than 400 mu m; wherein, the surface of the sample hole has hydrophilicity, and the other surfaces except the sample hole on the protein chip have hydrophobicity. The surface of the sample hole point on the protein chip has completely different hydrophilic and hydrophobic properties from other surfaces, and a sample always stays in the sample hole point without flowing out in the processing and detecting processes, so that the condition of cross contamination is effectively avoided; meanwhile, different silanization reagents can be bonded on the surfaces of the sample holes, so that different samples can be detected, and the multifunction of one chip is realized; and the specific surface area of the sample pore site position is large, so that the sensitivity of sample detection is effectively improved, and the chip is suitable for detecting low-abundance proteins. Therefore, the chip can be used as a sample pretreatment carrier and a sample detection platform, and the operation steps are simplified.
Description
Technical Field
The present invention relates to the field of biological detection. More particularly, relates to a protein chip suitable for detecting low-abundance protein, a preparation method and application thereof.
Background
Proteomics plays a crucial role in the field of biomedicine, and many protein molecules in human blood and body fluid have been clinically recognized as important biomarkers for disease diagnosis, drug and other therapeutic methods for effect evaluation, and the like. Even so, the proportion of the number of protein molecules in human blood, which are currently used in routine clinical tests, to the total protein molecular species is relatively small, and finding and verifying those protein molecules that are related to diseases and can be used for evaluating the effects of various therapeutic methods is still a hot spot in the current biological field. With the development of modern biotechnology, many technologies are available for detecting protein molecules in human blood, and most detection methods are limited by technical principles, and indirect detection methods are adopted, for example, by specifically binding the detected protein molecules with biomolecules with certain labels, such as luminescent labels, and then detecting the signal intensity of the labels to judge the content of the detected protein molecules, and the indirect detection methods usually suffer from non-specificity due to the similar biochemical properties of many biomolecules. The advent of matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) makes it possible to directly detect macromolecular substances such as protein molecules, and because mass spectrometry detects the mass-to-charge ratio of molecules, the mass-to-charge ratio belongs to the properties of substances and is not influenced by other substances, the detection of protein molecules by MALDI-TOF MS can avoid the problem of non-specificity in the indirect detection method.
The protein molecules in human blood, which can be used as clinical detection indexes, are generally low-abundance proteins, while MALDI-TOF MS tests samples, which are not selective for substances in the samples, all ionized substances in the samples can be detected. For the reasons, when detecting low-abundance proteins in a sample by mass spectrometry, the sample usually needs to be pretreated, for example, a protein concentration kit, a low-abundance protein enrichment kit, a surface-modified nano material or a porous material and the like are used for selectively enriching the low-abundance proteins in the sample; or an immunocapture method, such as ELISA or magnetic bead method, is adopted to capture the protein molecules to be detected by antigen/antibody specificity, and then the captured target protein is transferred to a MALDI-TOF MS target plate for detection, thereby improving the detection sensitivity of the target protein.
The main disadvantages of these current methods for the enrichment of commonly used lower abundance proteins and many newly developed materials for the enrichment of low abundance proteins are: the processing flow is complex, the cost is high, the required sample amount is large, and some methods involve sample transfer in the sample processing process to cause sample loss and the like. Therefore, it is desirable to provide a protein chip that can be directly used for detecting low-abundance proteins.
Disclosure of Invention
The invention aims to provide a protein chip suitable for detecting low-abundance protein, wherein the surface of the chip can be used for selectively enriching a sample and can also be used as a MALDI-TOF MS detection target plate, so that the sample pretreatment process is simplified, the pollution and loss of the sample in the treatment and transfer processes are avoided, and a rapid and high-flux sample pretreatment and detection method is provided for detecting low-abundance protein by MALDI-TOF MS. And the preparation method is simple, the cost is low, and the potential of large-scale production is realized.
The second purpose of the invention is to provide a preparation method of a protein chip suitable for detecting low-abundance proteins.
The third purpose of the invention is to provide the application of the protein chip which is suitable for detecting the low-abundance protein.
In order to achieve the purpose, the invention adopts the following technical scheme:
a protein chip suitable for detecting low-abundance protein comprises sample hole points with the diameter of 1-3mm and the depth of less than 400 mu m; wherein, the surface of the sample hole has hydrophilicity, and the other surfaces except the sample hole on the protein chip have hydrophobicity.
The surface of the protein chip is provided with the sample hole points, and the sample hole points have certain size and surface microstructure, so that the specific surface area of the sample point positions of the chip can be effectively improved, the sample detection sensitivity of the chip is increased, and the protein chip is further suitable for detecting low-abundance proteins; meanwhile, the sample hole points have hydrophilicity, but the surfaces of the non-sample hole points have hydrophobicity, so that the sample is always remained in the sample hole points and cannot flow out in the processing and detecting processes, the cross contamination condition is effectively avoided, and the processing and the detecting processing of multiple samples on the same chip become possible. Therefore, compared with the traditional method for enriching the sample by adopting the magnetic beads and the porous microspheres, the chip prepared by the invention can be used as a carrier for sample pretreatment and a detection platform for the sample in the using process, does not need sample transfer, has simple operation flow, and provides a rapid and high-flux sample pretreatment and detection method for MALDI-TOF MS when detecting low-abundance protein.
Preferably, the surface of the sample hole is modified with one or more silanization reagents;
preferably, the silylating agent is selected from epoxy silylating agents, amino silylating agents, mercapto silylating agents.
The surface of the hydrophilic sample pore point on the same protein chip surface can be modified with different hydrophilic silanization reagents, and the tail ends of the silanization reagents contain different functional groups, such as epoxy groups, amino groups, sulfydryl and the like. Different hydrophilic silanization reagents have different chemical properties, can be used for detecting different samples, can prevent the performance of sample cross contamination by matching sample holes, and realize the function that one chip can be used for processing various samples.
A preparation method of the protein chip comprises the following steps: cleaning a silicon wafer, carrying out oxidation treatment and hydrophobic modification on the surface of the silicon wafer, etching sample hole points on the surface of the silicon wafer by using laser, and carrying out hydrophilic modification on the surfaces of the sample hole points.
Preferably, the silicon wafer is an N-type silicon wafer doped with phosphorus, arsenic or antimony, the resistivity of the silicon wafer is less than 5 omega-m, and the thickness of the silicon wafer is 400-1000 mu m.
Cleaning is to remove particulate matter remaining on the surface as well as lipids and other organic compound molecules. The cleaning method comprises the following steps: immersing the silicon wafer in hydrofluoric acid for etching for 5-10 minutes, then cleaning the surface of the silicon wafer by flowing deionized water, and then drying the surface of the silicon wafer by nitrogen; or immersing the silicon wafer in ethanol or methanol for 5-10 minutes by ultrasonic treatment, then flushing the surface with flowing ethanol or methanol, and then blowing the surface of the silicon wafer by nitrogen.
And carrying out oxidation treatment on the surface of the cleaned silicon wafer to enable the surface to grow a thin compact oxide layer. The oxidation method comprises the following steps: putting the silicon chip into a UVO reactor to be oxidized for 10-20 minutes; or immersing the silicon wafer in hydrogen peroxide for about 30 minutes, taking out, washing with deionized water and drying with nitrogen.
Preferably, the silanization reagent used in the hydrophobic modification process is selected from long-chain alkane silane compounds or fluorine-containing silane compounds.
For example, the long chain alkane silane compounds include, but are not limited to, dodecylchlorosilane, dodecylmethoxysilane, octadecylchlorosilane, octadecylmethoxysilane, and the like; the fluorine-containing silane compounds include, but are not limited to, perfluorooctyltrichlorosilane, perfluorooctylmethoxysilane, perfluorodecyltrichlorosilane, perfluorodecylmethoxysilane, trichloro (1H, 2H-tridecafluoro-n-octyl) silane, and the like.
The silanization reagent with stronger hydrophobicity is modified to the surface of the oxidized silicon wafer in a monomolecular layer self-assembly chemical bonding mode, and the specific realization method comprises the following steps: preparing a toluene solution of a silylation reagent with a certain concentration, soaking the silicon wafer in the toluene solution of the silylation reagent, reacting for several hours at room temperature or under heating, cleaning the silicon wafer with toluene and ethanol in sequence, finally ultrasonically cleaning the silicon wafer with ethanol for 5 to 10 minutes, removing non-chemical bonding reagents on the surface, such as the silylation reagent which is physically adsorbed, and drying the silicon wafer with nitrogen.
The concentration range of the silylation reagent toluene solution is 1-10 mM;
the reaction condition of the hydrophobic modification can be adjusted according to the type of the selected silanization reagent, and if a chlorosilane reagent is adopted, the chip can be modified for 2-10 hours at room temperature due to the higher reaction activity of the reagent; if methoxy or ethoxy silanization reagents are used, the reaction temperature needs to be increased to obtain the same reaction efficiency as chlorosilane reagents, for example, the reaction is carried out for 4 to 10 hours at 50 to 80 ℃;
preferably, in the process of etching the sample hole point on the surface of the silicon wafer by using the laser, the precision of the laser is +/-3 μm, the speed is 500-1000m/s, the etching distance is 10-50 μm, and the laser energy is 90-100% of the maximum energy of the laser.
The possible implementation mode is that the laser etching template in fig. 1 is adopted, at least 16 sample hole points can be etched on the silicon wafer at the same time, laser etching parameters are set, laser etching is carried out on the surface of the silicon wafer after hydrophobic modification by using a laser marking machine, the hydrophobic layer on the selected surface is burned off, and the process mainly has the following functions: re-oxidizing the exposed silicon surface by using the energy released by the laser during firing to form a compact oxide layer; in addition, the optimized etching conditions are adopted, a micron-level uniform rough surface can be etched on the original smooth silicon wafer surface, the specific surface area of a sample processing position is effectively improved, and therefore the detection sensitivity of the chip is improved.
The etched silicon wafer is placed in a hydrophilic silanization reagent with a specific functional group at the tail end to carry out a monomolecular layer self-assembly reaction, the selected silanization reagent can be specifically bonded on sample holes etched by laser in the reaction, and active silicon hydroxyl on the surface of the silicon wafer is covered because the hydrophobic silanization reagent is bonded at other places on the surface of the silicon wafer, so that the surfaces can not react with a new silanization reagent any more, and the good hydrophobic performance is still kept.
Preferably, the silylation agent used in the hydrophilic modification of the surface of the spots is selected from the group consisting of epoxysilane compounds, aminosilane compounds and mercaptosilane compounds.
One possible embodiment is that the hydrophilic modification process for the surface of the well spot comprises the following steps: preparing a toluene solution of a silylation reagent with a certain concentration, soaking the silicon wafer in the toluene solution of the silylation reagent, reacting for several hours at room temperature or under heating, cleaning the silicon wafer with toluene and ethanol in sequence, finally ultrasonically cleaning the silicon wafer with ethanol to remove non-chemical bonding on the surface, such as the silylation reagent adsorbed physically, and drying the silicon wafer with nitrogen;
preferably, the concentration of the silylation reagent in the toluene solution is in the range of 10mM-100 mM;
preferably, a small amount of organic base such as pyridine, triethylamine and the like can be added into the silanized toluene solution reaction system as a catalyst, and the volume percentage of the organic base catalyst is 0.1-1%.
Preferably, during the preparation process, the steps of: etching sample hole points on the surface of the silicon wafer by using laser and carrying out hydrophilic modification on the surfaces of the sample hole points; the silylating agent used to hydrophilically modify the spots can be different for each cycle. The preparation process is repeated, so that different sample holes on the same chip are modified with different silanization reagents, and the method can be used for detecting different protein molecules.
The application of the protein chip as a MALD-TOF MS detection target plate.
Preferably, the application comprises the following processes:
(1) antigen or antibody molecule solution spotting;
(2) sealing treatment;
(3) enriching target molecules to be detected;
(4) and (4) dropwise adding a chemical matrix, and placing the chemical matrix into a QuandTOF detection system for testing.
In a specific implementation process, the antigen or antibody molecule solution spotting process of step (1) is as follows: spotting a trace volume of antigen or antibody solution which can be specifically combined with target protein to be detected on the sample well point, incubating for 0.5-4 hours at normal temperature to ensure that amino in the biological molecule is chemically bonded with a terminal chemical group modified by a silanization reagent on the sample well point, then sucking away the antigen or antibody solution by a pipette, and washing the sample well point with water for 4-5 times in a blowing mode.
The step (2) of sealing treatment comprises the following steps: spotting a trace volume of sealing solution, incubating for 0.5 hour at normal temperature, sealing off unreacted silanization reagent terminal groups on the surface of the 'sample well spots', and avoiding non-specific combination of biomolecules except target proteins in the subsequent sample and the chip; preferably, the blocking solution comprises ethanolamine (at a concentration of 0.1-0.5M, pH 8.0), Tris-HCl or an amino acid solution.
The enrichment process of the target molecules to be detected in the step (3) comprises the following steps: and (2) placing a sample point of diluted or undiluted blood or serum with a micro volume on a sample hole point modified by antigen or antibody molecules, incubating for 0.5-4 hours at normal temperature to ensure that target molecules to be detected in the sample with low abundance are specifically enriched on the sample hole point, sucking away the sample solution by using a pipette after enrichment, and washing the sample hole point for 4-5 times by using water in a blowing mode.
The protein chip can be used as a carrier for sample pretreatment and a detection platform of a sample, so that the sample transfer step is not involved in the process from treatment to detection, the loss of a target substance in the sample is avoided, and the operation steps are simplified.
The invention has the following beneficial effects:
the surface of the sample hole point on the protein chip has completely different hydrophilic and hydrophobic properties from other surfaces, and a sample always stays in the sample hole point without flowing out in the processing and detecting processes, so that the condition of cross contamination is effectively avoided; meanwhile, different silanization reagents can be bonded on the surfaces of the sample holes, so that different target molecules can be detected, and the multifunction of one chip is realized; and the specific surface area of the sample pore site position is large, so that the sensitivity of sample detection is effectively improved, and the chip is suitable for detecting low-abundance proteins. Therefore, the chip can be used as a sample pretreatment carrier and a sample detection platform, so that a sample transfer step is not involved in the process from treatment to detection of the sample, the loss of a target substance in the sample is avoided, and the operation steps are simplified.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
Fig. 1 shows a laser marking machine etching template in the present invention.
FIG. 2 shows the results of pretreatment and detection of a sample of β 2-microglobulin in example 1 on a chip with well spots modified with 3-glycidoxypropyltrimethoxysilylation reagent.
FIG. 3 shows the results of pretreatment and detection of a sample of aqueous beta 2-microglobulin solution of comparative example 1 on untreated and modified silicon wafers.
FIG. 4 shows the results of pretreatment and detection of beta 2-microglobulin in a human serum sample on a chip with 3-glycidoxypropyltrimethoxysilylation reagent modified at the well site in example 2.
FIG. 5 shows the results of pretreatment and detection of apolipoprotein C III in human serum samples of example 2 on a chip with well spots modified with 3-glycidoxypropyltrimethoxy silylation reagent.
FIG. 6 shows the results of the test on the chip modified with 3-glycidoxypropyltrimethoxysilylation reagent directly on the well site without pretreatment after mixing human serum samples with the chemistry matrix in comparative example 2.
Fig. 7 shows the roughness of the surface of the well spots in example 3.
Fig. 8 shows the roughness of the surface of the spot of the wells in comparative example 3.
Detailed Description
In order to more clearly illustrate the invention, the invention is further described below with reference to preferred embodiments and the accompanying drawings. Similar parts in the figures are denoted by the same reference numerals. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
Example 1
Treatment and detection of beta 2-microglobulin samples
In the embodiment, a No. 1 chip with sample holes modified by a 3-glycidyloxypropyltrimethoxysilylation reagent is used as a sample enrichment and detection platform to selectively enrich and test a low-concentration beta 2-microglobulin sample, and the specific steps are as follows:
1, sample enrichment:
loading 2 mu L of beta 2-microglobulin antibody aqueous solution (1mg/mL) on a sample hole point of a No. 1 chip (modified by a 3-glycidyloxypropyltrimethoxysilanization reagent), incubating at room temperature for 2 hours, sucking the antibody solution by using a pipette after incubation, sucking 2uL of deionized water by using the pipette, repeatedly blowing the sample hole point, and repeating the step for 4-5 times;
loading 2 mu L of 0.5M glycine aqueous solution on the sample hole point, sealing for half an hour at room temperature, and then repeating the cleaning step;
loading (1mg/mL) beta 2-microglobulin sample solution on the sample hole, incubating for 2 hours at room temperature, sucking the sample solution by using a pipette after incubation, repeating the cleaning step, and finally drying the surface by using nitrogen;
2, sample detection:
dripping 1-2 mu L of chemical matrix solution (ethanol water solution of 4-hydroxy-3, 5-dimethoxy cinnamic acid, the volume ratio of ethanol to the water solution is 1:3, the concentration of the chemical matrix is 10mg/mL) on a sample hole point of the No. 1 chip for completing sample pretreatment, drying, and putting into a QuandTOF detection system for testing, wherein the mass spectrum detection conditions are as follows: the laser wavelength is 349nm, the laser pulse frequency is 5kHz, the accelerating voltage is-20 kV, the detection range is 5k-50kDa, the delay time is 2000ns, and linear positive ion mode detection is carried out.
FIG. 2 shows the results of the treatment and detection of a β 2-microglobulin sample on a chip modified with 3-glycidoxypropyltrimethoxysilylation reagent at the well site. In the figure, the abscissa represents the mass-to-nucleus ratio of a mass peak, and the ordinate represents the peak intensity of the peak.
Comparative example 1
The chip No. 2 used in comparative example 1 was an untreated and modified silicon wafer, the other steps were exactly the same as in example 1, and fig. 3 is a result of processing and detecting a β 2-microglobulin sample on the chip No. 2. The comparison shows that the chip No. 1 has obvious enrichment effect on the beta 2-microglobulin sample in the sample.
Example 2
Simultaneous processing and detection of two low abundance proteins in serum sample
In this embodiment, a No. 3 chip with sample wells modified by 3-glycidyloxypropyltrimethoxysilylation reagent is used as a sample enrichment and detection platform, and selective enrichment and test are performed on two proteins with lower abundance, beta 2-microglobulin and apolipoprotein C iii in a human serum sample at different sample wells of the same chip, specifically, the following steps are performed:
1, sample enrichment:
loading 2 mu L of beta 2-microglobulin antibody aqueous solution (1mg/mL) and apolipoprotein CIII antibody aqueous solution (1mg/mL) on sample well points 1 and 2 of a No. 3 chip (modified by a 3-glycidyloxypropyltrimethoxysilanization reagent) respectively, incubating for 2 hours at room temperature, sucking the antibody solution away by using a pipette after incubation, sucking 2 mu L of deionized water by using the pipette, repeatedly blowing and punching the sample well points, and repeating the step for 4-5 times;
loading 2 mu L of 0.5M glycine aqueous solution on the sample hole points 1 and 2 respectively, sealing at room temperature for half an hour, and then repeating the cleaning step;
loading 2 μ L of human serum diluted with deionized water (x8) on wells 1 and 2, incubating at room temperature for 2 hours, sucking off the sample solution with a pipette after incubation, repeating the above washing steps, and finally drying the surface with nitrogen.
2, sample detection:
dripping 1-2 mu L of chemical matrix solution (ethanol water solution of 4-hydroxy-3, 5-dimethoxy cinnamic acid, the volume ratio of ethanol to the water solution is 1:3, the concentration of the chemical matrix is 10mg/mL) on a sample hole point of the No. 3 chip for completing sample pretreatment, drying, and putting into a QuandTOF detection system for testing, wherein the mass spectrum detection conditions are as follows: the laser wavelength is 349nm, the laser pulse frequency is 5kHz, the accelerating voltage is-20 kV, the detection range is 5k-50kDa, the delay time is 2000ns, and linear positive ion mode detection is carried out.
The results of the tests are shown in FIGS. 4 and 5. In the figure, the abscissa represents the mass-to-nucleus ratio of a mass peak, and the ordinate represents the peak intensity of the peak. FIG. 4 shows the results of treatment and detection of beta 2-microglobulin in human serum samples on chip well No. 3, spot 1, and FIG. 5 shows the results of treatment and detection of apolipoprotein C III in human serum samples on chip well No. 3, spot 2.
Comparative example 2
The mixed solution of human serum and chemical matrix (volume ratio 1: 100) was directly dropped on well spot 3 of chip No. 3, well spot 3 was unmodified, and fig. 6 shows the test results. The results of fig. 4, 5 and 6 illustrate in comparison: after the serum sample is pretreated on the modified chip, compared with the sample without pretreatment, the beta 2-microglobulin and the apolipoprotein CIII with lower abundance in the sample are obviously enriched and purified.
Example 3
Taking an N-type (single-side polished and non-oxide layer) silicon wafer with the size of 40mmx27mmx0.5mm, ultrasonically cleaning the silicon wafer with ethanol for 5-10 minutes, blow-drying the silicon wafer with nitrogen, and oxidizing the silicon wafer in an UVO reactor for 10 minutes;
preparing 2 mu M trichloro (1H,1H,2H, 2H-tridecafluoro n-octyl) silane toluene solution, soaking the silicon wafer in the newly prepared solution, carrying out closed reaction for 2-10 hours at room temperature, sequentially washing the surface of the silicon wafer by toluene, ethanol and the like after the reaction, carrying out ultrasonic cleaning on the silicon wafer for 5-10 minutes by using ethanol if residual silica gel polymer exists on the surface, and finally blowing the silicon wafer dry by using nitrogen;
according to the designed laser etching template, etching the sample hole points selected on the surface of the processed silicon wafer by using a laser marking machine, and optimizing specific parameters of laser etching, wherein the parameter optimization range is as follows: the laser etching speed is 500-1000m/s, and the etching interval is 10-50 μm. With fixed laser energy: 90% of the maximum energy of the laser.
The roughness of the surface of the spot after etching under each condition is shown in fig. 7. In FIG. 7, (a), (b), and (c) are roughness maps of the surface of the selected spots on the silicon wafer at etching pitches of 10 μm, 25 μm, and 50 μm at a laser etching speed of 500m/s, respectively; in FIG. 7, (d) and (e) are graphs of roughness of the surface of selected sampling holes on a silicon wafer at an etching pitch of 10 μm and 25 μm, respectively, at a laser etching speed of 1000 m/s.
Preparing 20 mu M of 3-glycidyloxy propyl trimethoxy silane toluene solution, adding 0.5 volume percent pyridine solution, soaking the silicon wafer subjected to laser etching under the conditions in the newly prepared solution, reacting for 4-36 hours at 50-80 ℃, washing the surface of the silicon wafer with toluene, ethanol and the like in sequence after reaction, and if residual silica gel polymer exists on the surface, ultrasonically cleaning the silicon wafer with ethanol for 5-10 minutes, and finally drying the silicon wafer with nitrogen.
The chips prepared under various conditions are used as a sample enrichment and detection platform to selectively enrich and test beta 2-microglobulin in a human serum sample, and the operation steps are the same as the specific steps in the embodiment 2. The results show that when the laser etching speed is 500When the etching spacing is 10 μm, 25 μm and 50 μm at m/s, the absolute intensity of the mass spectrum peak of the beta 2-microglobulin is 1.4 multiplied by 10-2volts，1.2×10-2volts，1.5×10-2volts; when the laser etching speed is 1000m/s and the etching distance is 10 mu m and 25 mu m, the absolute intensity of the mass spectrum peak of the beta 2-microglobulin is 1.2 multiplied by 10-2volts and 1.1X 10- 2volts。
Comparative example 3
The other conditions in comparative example 3 were exactly the same as those in example 2 except that the laser etching speed was 2000m/s, the etching pitches were 10 μm, 25 μm and 50 μm, and the surface roughness of the etched well spots was as shown in (a), (b) and (c) of FIG. 8. The selective enrichment and testing of beta 2-microglobulin in human serum samples were carried out using the same procedure as described in example 2. The results showed that the absolute intensities of the mass spectrum peaks of beta 2-microglobulin were 6.1X 10 at the etching pitches of 10 μm, 25 μm and 50 μm, respectively-3volts，5.5×10-3volts，2.2×10-3volts。
Comparison of the results of example 3 and example 4 shows that selection of an appropriate laser speed and an appropriate etching distance greatly affects the roughness of the surface of the sample hole point, and further affects the detection sensitivity.
Example 4
Preparation of chip for selectively enriching lower abundance protein
Taking an N-type (single-side polished and non-oxide layer) silicon wafer with the size of 40mmx27mmx0.5mm, ultrasonically cleaning the silicon wafer with ethanol for 5-10 minutes, blow-drying the silicon wafer with nitrogen, and oxidizing the silicon wafer in an UVO reactor for 10 minutes;
preparing 2 mu M trichloro (1H,1H,2H, 2H-tridecafluoro n-octyl) silane toluene solution, soaking the silicon wafer in the newly prepared solution, carrying out closed reaction for 2-10 hours at room temperature, sequentially washing the surface of the silicon wafer by toluene, ethanol and the like after the reaction, carrying out ultrasonic cleaning on the silicon wafer for 5-10 minutes by using ethanol if residual silica gel polymer exists on the surface, and finally blowing the silicon wafer dry by using nitrogen;
according to a designed laser etching template, etching the selected sample hole points on the surface of the processed silicon wafer by using a laser marking machine, wherein the laser etching speed is 500m/s, the etching interval is 25 mu m, and the laser energy is 90% of the maximum energy of a laser;
preparing 20 mu M gamma-aminopropyl trimethoxy silane toluene solution, adding 0.5 percent pyridine solution by volume ratio, soaking the silicon wafer after laser etching in newly prepared solution, reacting for 4-36 hours at 50-80 ℃, washing the surface of the silicon wafer by toluene, ethanol and the like in sequence after reaction, if residual silica gel polymer exists on the surface, carrying out ultrasonic cleaning on the silicon wafer by ethanol for 5-10 minutes, and finally blowing the silicon wafer by nitrogen.
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all embodiments may not be exhaustive, and all obvious variations or modifications may be included within the scope of the present invention.
Claims (10)
1. A protein chip suitable for detecting low-abundance protein is characterized in that the protein chip comprises sample hole points with the diameter of 1-3mm and the depth of less than 400 mu m; wherein, the surface of the sample hole has hydrophilicity, and the other surfaces except the sample hole on the protein chip have hydrophobicity.
2. The protein chip according to claim 1, wherein the surface of the well spot is modified with one or more silylation reagents; preferably, the silylating agent is selected from epoxy silylating agents, amino silylating agents, mercapto silylating agents.
3. A method for preparing a protein chip according to any one of claims 1-2, comprising the steps of: cleaning a silicon wafer, carrying out oxidation treatment and hydrophobic modification on the surface of the silicon wafer, etching sample hole points on the surface of the silicon wafer by using laser, and carrying out hydrophilic modification on the surfaces of the sample hole points.
4. The method as claimed in claim 3, wherein the silicon wafer is an N-type silicon wafer doped with P, As or Sb, and has a resistivity of less than 5 Ω -m and a thickness of 400-1000 μm.
5. The method according to claim 3, wherein the silylation agent used in the hydrophobic modification process is selected from long-chain alkane silanes or fluorine-containing silanes.
6. The method as claimed in claim 3, wherein the precision of the laser is + -3 μm, the speed is 500-1000m/s, the etching distance is 10 μm-50 μm, and the laser energy is 90% -100% of the maximum energy of the laser in the process of etching the sample hole point on the surface of the silicon wafer with the laser.
7. The method according to claim 3, wherein the silylation agent used in the hydrophilic modification of the surface of the well is selected from the group consisting of an epoxysilane compound, an aminosilane compound and a mercaptosilane compound.
8. The method of claim 3, wherein the steps of: etching sample hole points on the surface of the silicon wafer by using laser and carrying out hydrophilic modification on the surfaces of the sample hole points; the silylating agent used to hydrophilically modify the spots can be different for each cycle.
9. Use of a protein chip according to any one of claims 1-2 as a target plate for MALDI-TOF MS detection.
10. The application according to claim 9, characterized in that it comprises the following processes:
(1) antigen or antibody molecule solution spotting;
(2) sealing treatment;
(3) enriching target molecules to be detected;
(4) and (4) dropwise adding a chemical matrix, and placing the chemical matrix into a QuandTOF detection system for testing.
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