CN107177689B - Universal chip for detecting protein and nucleic acid by time-of-flight mass spectrometry - Google Patents
Universal chip for detecting protein and nucleic acid by time-of-flight mass spectrometry Download PDFInfo
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- CN107177689B CN107177689B CN201710539893.0A CN201710539893A CN107177689B CN 107177689 B CN107177689 B CN 107177689B CN 201710539893 A CN201710539893 A CN 201710539893A CN 107177689 B CN107177689 B CN 107177689B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
- C12Q1/6872—Methods for sequencing involving mass spectrometry
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- 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
Abstract
The invention provides a universal chip for detecting protein and nucleic acid by using time-of-flight mass spectrometry, wherein the chip comprises: (1) a chip main body: the surface of the main body is provided with hydrophilic spot-like holes and hydrophobic hole outer zones which are arranged in micro-arrays; (2) the chip adapter is composed of a metal bottom plate, wherein a plurality of chip clamping grooves matched with a chip main body are distributed on the surface of the adapter, the chip main body is selected from a chip which is tough in texture and good in flatness and is made of single-side polished diamond, monocrystalline silicon and quartz crystals, the hydrophilic sample application holes are covered with a hydrophilic thin film of 150 nm-800 nm, the hydrophobic hole outer region is covered with a hydrophobic thin film of 150nm-2 mu m, and the contact angle of water drops on the surface of the hydrophobic thin film is more than 120 degrees; wherein the wells are pretreated beforehand with a coating matrix solution selected from a matrix solution for protein mass spectrometry or a matrix solution selected from a matrix solution for nucleic acid mass spectrometry.
Description
Technical Field
The invention relates to a dual-purpose chip for detecting protein and nucleic acid by flight time mass spectrometry, which can detect protein and nucleic acid molecules by mass spectrometry and belongs to the field of mass spectrometry.
Background
Proteomics is a new science in the age of functional genomics, which studies the composition and activity of proteins in cells and the interaction between proteins on an overall level. Including the identification of protein expression, modifications, structures, functions, interactions, and the like. Proteomics can be classified into expression proteomics, structural proteomics, and functional proteomics according to the purpose of research. Expression proteomics is used for quantitative studies of intracellular protein sample expression. Proteomics, which is aimed at mapping the structure of protein complexes or proteins present in a particular organelle, is called structural proteomics, and is used to map intracellular signal transduction networks and explain the effect of expression of certain specific proteins on cells [2 ]. Functional proteomics aims to study the functions of intracellular proteins and interactions between proteins, and by studying and analyzing a selected proteome, important information on phosphorylation, glycosylation, and the like of proteins can be provided.
At the heart of proteomics research is the ability to systematically identify each protein and the properties of proteins expressed in a cell or tissue. The main related technologies of proteomics include two-dimensional gel electrophoresis, two-dimensional fluorescence difference gel electrophoresis, mass spectrometry, etc. [2 ]. Due to the high complexity of proteins and the presence of large amounts of low abundance proteins, the analysis technique presents a huge challenge, the biological mass spectrometry technique is a necessary choice for adapting to the challenge, and the recent rapid development is MALDI-TOF mass spectrometry.
Compared with the traditional method and the automatic instrument mainly used at present, the proteomics technology based on the MALDI-TOF mass spectrum for identifying and classifying the microorganisms has the following characteristics:
the operation is simple and quick. Single colonies of microorganisms or other biological material can be added directly to a MALDI sample target and analyzed using a MALDI-TOF mass spectrometer, the spectral identification can be done within minutes, and the data evaluation is directly linked to the assay. This simple and unique workflow is sufficient for the identification of most microorganisms and does not require gram staining, oxidase testing or PCR primer and condition selection.
② the repeatability is good. MALDI methods have proven to be very stable over a wide range of conditions. The different compositions of the growth medium have very little effect on the peak pattern distribution, as in the range from 4000 to 12000Da, little effect of the medium is observed. Also, the growth state of the cells had no effect on the peak pattern, and the cells in the slow growth phase had a pattern similar to those in the logarithmic, plateau, or death phase. After sample preparation and measurement under standard conditions, the mass spectra obtained on different MALDI-TOF instruments are highly comparable, e.g. the spectra measured on the same sample target are practically identical on 3 different instruments. Therefore, spectra from different MALDI-TOF mass spectrometers can be used to build a truly reliable database. This high reproducibility is based on the measurement of highly abundant proteins for stable expression, e.g. ribosomal proteins. The spectrum can be observed in the mass range of 2000 to 20000Da where few metabolites are present. Bacterial spores can produce significantly different peak patterns compared to live cells, and these "spore patterns" are also reproducible. Currently, the high sensitivity of the instrument can detect as low as 100ng or 10 ng5And (4) cells. For the use of the CLIN-TOF instrument, 25ng of biomaterial was sufficient.
And high accuracy. The protein fingerprint obtained by MALDI-TOFMS is used as pattern matching, and the matching score is used for grading and classifying the identification result. MALDI software analyzes and unifies the obtained spectrum, and the correction and statistical operation ensure the accuracy of identification, so that the mass spectrum deviating from 5000ppm can be identified at present. The protein fingerprint is mainly focused on the continuous high expression protein with 2-20kDa which is slightly influenced by the growth environment and the state of the microorganism.
The biological mass spectrometry has incomparable superiority and becomes an indispensable technical platform in proteomics research. With the continuous development of sensitivity, accuracy and high throughput of mass spectrometry, mass spectrometry plays an increasingly important role in proteomics research. It has been widely used in protein identification, sequence analysis, quantification, post-translational processing, protein interaction, etc. Similarly, the high sensitivity, high accuracy and high throughput of MALDI-TOF mass spectrometry are also particularly advantageous in the field of genomics detection.
Genomic research should include two aspects: structural genomics (structural genomics) targeting whole genome sequencing and functional genomics (functional genomics) targeting gene function identification, also known as post-genome (postgenome) research, are important methods in system biology.
Sequencing of genomic DNA is the first step in human understanding of the self genome. With the completion of sequencing, functional genomics research becomes the mainstream of research, and elucidation of the functions of genomes is performed from the high degree of interaction between genome information and the external environment. The research content of functional genomics is as follows: human genome DNA sequence variability research, genome expression regulation research, model organism research, bioinformatics research and the like.
(1) And (3) researching genome expression and regulation. At the level of whole cells, all genome expression products mRNA and protein are identified, and the interaction of the two is identified, and a temporal and spatial overall regulatory network of genome expression in the development process and under different environmental stresses is elucidated.
(2) Identification and characterization of human genetic information. The basic work necessary for extracting genome function information, and identifying gene sequences is essential. The gene identification needs to adopt bioinformatics, computational biology techniques and biological experimental means, and combines theoretical methods and experiments. Theoretical-based methods have been developed primarily from a large number of nucleic acid sequence data already known, and theoretical methods of sequence comparison, genome comparison, and gene prediction have been developed. The biological means for identifying genes are mainly based on the following principles and ideas: based on the expressible Sequence Tags (STS); carrying out direct cDNA selection on chromosome specific cosmids; according to CpG island; differential display and related principles; exon capture and related principles; gene chip technology; genome scanning; mutation detection systems, and the like.
(3) And extracting and identifying gene function information. The method comprises the following steps: systematic identification of human gene mutants; drawing a gene expression profile; identification of "gene alteration-functional alteration"; detection of protein levels, modification status and interactions.
(4) In sequencing and gene diversity analysis. Although the genome sequence obtained by the human genome project is representative, the genome of each person is not completely the same, and the genome sequence differs. Differences in the genome reflect differences in phenotype that form individuals, such as differences between black and white, between tall and short, between healthy and genetic patients, and so forth. The most frequently occurring gene polymorphisms are Single Nucleotide Polymorphisms (SNPs).
(5) And (5) comparing genomics. Comparing the human genome with the genome of a model organism helps, on the one hand, to analyze the function of the human genes according to the homology method and, on the other hand, to discover the essential differences between human and other organisms, exploring the mysteries of the genetic language.
The accuracy rate of SNP detection finished by MALDI-TOF flight time mass spectrum can reach 99.9%, and the method has the advantages of high accuracy, strong flexibility, large flux, short detection period and the like, and is most attractive and should also have the cost performance. The MALDI-TOF time-of-flight mass spectrometry platform is a research platform for international universal gene Single Nucleotide Polymorphism (SNP), and the method has become a new standard in the field by virtue of the scientificity and accuracy.
Although MALDI-TOF time-of-flight mass spectrometry can be used for detecting protein or nucleic acid molecules, from the application point of view, the traditional MALDI chip has a single application field, such as a DE1580TA steel sheet target plate produced by SHIMADZU, is only used for protein microorganism identification, and is suitable for MALDI-TOF AXIMA of SHIMADZU; the L24 SpectroCHIP chip produced by AGENA is only used for gene detection, is suitable for MALDI-TOF MASSARRY of the AGENA, and does not have a chip which can cover two detection fields of genomics detection or proteomics identification at the same time at present.
From the perspective of mass spectrum chip material and surface characteristics, the traditional metal chip is repeatedly used and has scratches, the surface is not chemically modified, the crystallization form is poor, the accuracy of a mass spectrum detection sample peak is low, the signal-to-noise ratio is low, and the baseline is high. And for nucleic acid samples, micro-nucleic acid residues can affect mass spectrometry results. Therefore, the metal chip is mainly used for protein mass spectrometry detection and cannot detect nucleic acid samples in a cross-border manner. The nucleic acid chip mainly uses silicon chip materials, but is expensive, the formula of the matrix is monopolized abroad, and the surface of the purchased imported chip is directly covered with the matrix, so that the nucleic acid chip cannot be used for protein mass spectrometry.
Therefore, there is a need for a universal chip for protein and nucleic acid detection by time-of-flight mass spectrometry, and a substrate formula for covering the surface of the chip.
Disclosure of Invention
In order to solve the above technical problems, a first object of the present invention is to provide a universal chip for protein and nucleic acid detection by time-of-flight mass spectrometry, comprising:
(1) a chip main body: the surface of the main body is provided with hydrophilic spot-like holes and hydrophobic hole outer zones which are arranged in micro-arrays;
(2) the chip adapter is composed of a metal bottom plate, wherein a plurality of chip clamping grooves matched with a chip main body are distributed on the surface of the adapter;
wherein, the chip main body is selected from the chips of diamond, monocrystalline silicon and quartz crystal which have tough texture and good planeness;
the hydrophilic spotting hole covers a hydrophilic film with the thickness of 150-800nm, the outer region of the hydrophobic hole covers a hydrophobic film with the thickness of 150-2 μm, and the contact angle of a water drop on the surface is more than 120 degrees.
In one embodiment, the hydrophilic film is a silica oxide film, a zinc oxide film, an alumina film, or the like, and the hydrophobic extracellular region is treated with a silane coupling agent to form a hydrophobic film. In a particular embodiment, the silane coupling agent is selected from the group consisting of vinyl silane, amino silane, or dimethyldichlorosilane.
In another embodiment, the chip is a single side polishing silicon chip or quartz chip, the hydrophilic sample porous layer film thickness is 500nm, the hole region contact angle is > 120 °, preferably about 135 °, 155 °.
In one embodiment, the number of the hydrophilic spotting wells on the chip is 24, 40, 70, 384, etc. wells, or the shape of the hydrophilic spotting wells can be selected from a circle, a square, a triangle, a polygon, etc. according to the detection amount of the biological sample in the actual clinical trial or scientific research process. In a specific embodiment, the pore size of the hydrophilic spotting wells ranges from 0.5 to 2.8mm, and the chip size ranges from 20X 30 to 83X 125 mm. In another embodiment, the hydrophilic spot area is circular and has a diameter of 0.7mm, and the appropriate amount of matrix is selected according to the diameter of the circular hole, such as 0.5 μ l, and the biomarker sample is titrated, and the crystal morphology is regular and the crystal orientation is uniform, which is the best.
In other embodiments, the chip adapter can be designed with different card slots according to the size of the chip, and the size of the card slot is 20 × 30-83 × 125mm, so that one adapter can ensure that chips with different specifications enter the detection chamber.
In another embodiment, the microarray chip card slots on the adapter can be designed in different shapes, such as square, rectangle, etc., and each slot has a size of 20 × 20mm to 25 × 60 mm. In one embodiment, the number of card slots of the chip adapter is 2, 4, 6, 8, 12, 16 or 20.
In further embodiments, the chip surface flatness is less than 10 μm. In one embodiment, the chip is previously processed by Chemical Mechanical Polishing (CMP) to have a single-side polished mirror effect. And (3) polishing the chip twice by a CMP method, namely rough polishing and fine polishing. The rough polishing is intended to remove residual mechanical damage from the chip surface, typically to a thickness in the range of 30um from the surface. The fine polishing aims to remove slight damage and haze defects left on the surface of the chip by the first polishing, and generally removes 2-3 um from the surface. The surface of the chip polished twice has a mirror effect, and in the process of mass spectrometry detection, the bombarded condition of the sample on the surface of the chip can be directly observed through illumination, light path reflection and real-time display of a camera, so that the position of laser bombardment is changed, and the optimal map is obtained. In a more specific embodiment, the chip is a single-side polished silicon chip or a quartz chip.
The second object of the present invention is to provide a dual-purpose chip for pre-treating the surface of hydrophilic wells with a coating substrate, wherein the wells are pre-treated in advance with a coating substrate solution on the basis of the above-mentioned embodiment.
In one embodiment, the chip may be used for mass spectrometric detection of proteins when the matrix solution is selected from matrix solutions used for protein mass spectrometry. In another embodiment, the chip may be used for mass spectrometric detection of nucleic acids when the matrix solution is selected from matrix solutions used for nucleic acid mass spectrometry.
In one embodiment, the matrix for mass spectrometric detection of proteins is selected from the group consisting of alpha-cyano-4-hydroxycinnamic acid (CHCA), 3, 5-dimethoxy-4-hydroxycinnamic acid (SA), and the matrix for mass spectrometric detection of nucleic acids is 3-hydroxy-2-picolinic acid.
The third purpose of the invention is to provide a dual-purpose chip for preparing protein and nucleic acid for flight time mass spectrum detection, which comprises:
(1) selecting a chip with tough texture and good planeness as a substrate or a chip main body;
(2) carrying out hydrophilic treatment on the surface of the chip;
(3) sealing the hydrophilic spot-like holes by using photoresist;
(4) treating the chip with a silane coupling agent;
(5) restoring the spot sample area sealed by the photoresist;
(6) and matching and mounting the chip and the metal chip adapter.
In one embodiment, in the step (2), the oxygen flow rate is 60-140 ml/min, the power is 100-300W, and the treatment time is 1-10 min; and ultrasonically cleaning the surface of the substrate for 2-3 times by using high-purity water, wherein the time for each time is 2-5 min, so that the surface is thoroughly cleaned and hydroxylated.
In another embodiment, in the step (3), the photoresist is spin-coated on the treated substrate by a spin coating method, the spin coating speed is 1000-8000 rpm, then the substrate is placed under a photoetching mask plate, and after exposure is carried out for 20 s-5 min under 390nm ultraviolet light, the substrate is eluted by corresponding developing solution, so that a sample application hole region with an ordered structure is constructed. The photoresist is made of acid-resistant polymethyl methacrylate (PMMA) or polystyrene.
In other embodiments, step (4) comprises:
(i) cleaning the chip: sequentially immersing into acetone, methanol water solution and chloroform, respectively cleaning, taking out and drying;
(ii) under the heating condition, slowly adding concentrated acid and hydrogen peroxide into an aqueous solution containing the chip, carrying out full oxidation reaction, and then respectively cleaning in chloroform and ultrapure water, wherein the step can be repeated for multiple times;
(iii) putting the chip into a clean container, adding a silane coupling agent solution hydrolyzed in advance, and adding ammonia water for catalysis until the silanization reaction is fully completed;
(iv) taking out the chip, respectively placing in ethanol, ultrapure water and chloroform, and ultrasonically cleaning;
(v) measuring and selecting the chip with a contact angle larger than 120 degrees as a qualified chip;
in one embodiment, step (5) comprises:
(i) placing the silanized chip into the degumming solution, and soaking for 3 hours;
(ii) taking out the chip, putting the chip into acetone solution, carrying out ultrasonic treatment for 2 minutes,
the photoresist can be removed, and the hydrophilic surface of the point sample area is restored, so that the surface of the microarray chip with the hydrophilic-hydrophobic difference is formed.
In one embodiment, the degumming solution is polyethylene glycol monobutyl ether or a polydimethylsiloxane solution.
In another embodiment, the chip of step (1) is a tough and well-planed diamond, single crystal silicon, or quartz crystal chip. In one embodiment, the chip is previously processed by Chemical Mechanical Polishing (CMP) to have a single-side polished mirror effect. And (3) polishing the chip twice by a CMP method, namely rough polishing and fine polishing. The rough polishing is intended to remove residual mechanical damage from the chip surface, typically to a thickness in the range of 30um from the surface. The fine polishing aims to remove slight damage and haze defects left on the surface of the chip by the first polishing, and generally removes 2-3 um from the surface. The surface of the chip polished twice has a mirror effect, and in the process of mass spectrometry detection, the bombarded condition of the sample on the surface of the chip can be directly observed through illumination, light path reflection and real-time display of a camera, so that the position of laser bombardment is changed, and the optimal map is obtained. In a more specific embodiment, the chip is a single-side polished silicon chip or a quartz chip.
In another embodiment, the silane coupling agent of step (4) is selected from the group consisting of vinyl silane, amino silane, dimethyl dichlorosilane. The operation of the dimethyldichlorosilane for growing the hydrophobic membrane is simple, the contact is large, the hydrophobicity is good, and the thickness of the hydrophobic layer can reach the magnitude of nanometer to micrometer, so that the silane coupling agent is preferably dimethyldichlorosilane. In a specific embodiment, the hydrophobic surface, which is bulk silanized by the coupling agent, has a contact angle > 120 °, in a preferred embodiment the contact angle > 150 °, to form a superhydrophobic surface. In other preferred embodiments, the thickness of the hydrophobic surface is on the order of nanometers to micrometers.
In one embodiment, the number of the hydrophilic spotting wells on the chip is 24, 40, 70, 384, etc. wells, or the shape of the hydrophilic spotting wells may be selected from a circle, a square, a triangle, a polygon, etc. according to the detection amount of the biological sample in the actual clinical trial or scientific research process. In a specific embodiment, the pore size of the hydrophilic spotting wells ranges from 0.5 to 2.8mm, and the chip size ranges from 20X 30 to 83X 125 mm. In another embodiment, the hydrophilic wells are circular and have a diameter of 0.7mm, and the appropriate amount of matrix, e.g., 0.5 μ l, is selected based on the diameter of the circular wells, and the biomarker samples are titrated to have a regular crystal morphology with a uniform crystal orientation being the best.
In other embodiments, the microarray chip card slot may be square, rectangular, etc. as desired, each slot ranging from 20X 20mm to 25X 60 mm. In a specific embodiment, the number of card slots of the chip adapter is 2, 4, 6, 8, 12, 16 or 20, or even more.
The fourth purpose of the invention is to provide a chip for preparing a protein or nucleic acid for detecting flight time mass spectrum, which comprises: the steps of any of the above protocols, and pre-treating the spotting sites with a cover matrix solution. In one embodiment, the chip may be used for mass spectrometric detection of proteins when the matrix solution is selected from the group consisting of protein mass spectrometric matrix solutions. In another embodiment, the chip may be used for mass spectrometric detection of nucleic acids when the matrix solution is selected from the group consisting of matrix solutions for nucleic acid mass spectrometry.
The fifth purpose of the invention is to provide a mass spectrum dual-purpose detection kit comprising the chip, which comprises:
a plurality of vacuum-packed dual-purpose chips and metal chip adapters;
nucleic acid and/or protein mass spectrometry matrix solutions;
and instructions for use.
In one embodiment, the dual-purpose chip has been pre-inserted into a metal chip adapter.
In another embodiment, the chip has been pre-coated with a coating matrix solution. In a particular embodiment, the kit is useful for mass spectrometric detection of proteins when the matrix solution is selected from matrix solutions for protein mass spectrometry. In another embodiment, the kit can be used for mass spectrometric detection of nucleic acids when the matrix solution is selected from the group consisting of matrix solutions for nucleic acid mass spectrometry.
In one embodiment, the number of the hydrophilic spotting wells on the chip is 24, 40, 70, 384, etc. wells, or the hydrophilic spotting wells may be set according to the detection amount of the biological sample in the actual clinical trial or scientific research process, and the hydrophilic spotting wells may be selected from a circle, a square, a triangle, a polygon, etc. In a specific embodiment, the pore size of the hydrophilic spotting wells ranges from 0.5 to 2.8mm, and the chip size ranges from 20X 30 to 83X 125 mm. In another embodiment, the hydrophilic spot area is circular and has a diameter of 0.7mm, and the appropriate amount of matrix is selected according to the diameter of the circular hole, such as 0.5 μ l, and the biomarker sample is titrated, and the crystal morphology is regular and the crystal orientation is uniform, which is the best.
In other embodiments, the microarray chip card slot may be square, rectangular, etc. as desired, each slot ranging from 20X 20mm to 25X 60 mm. In one embodiment, the chip adapter has 2, 4, 6, 8, 12, 16, or 20 card slots, or more.
The sixth purpose of the invention is to provide the use of the chip or the kit for detecting the biomolecule by mass spectrometry.
In one embodiment, the biomolecule is a nucleic acid and/or protein molecule.
The effects of the invention include:
1. the application field of the invention covers genomics detection and proteomics identification, and the same chip can be used for gene detection and protein or polypeptide identification and covers two detection fields.
2. The chip surface has the hydrophilic hydrophobic difference design, can effectively enrich the sample, the crystal orientation grows uniformly, the sample peak accuracy of mass spectrometric detection is high, the signal-to-noise ratio is high, the base line is low.
3. The substrate is covered on the surface of the chip sample application area through automatic sample application, and the substrate and the sample are crystallized on the surface of the chip more uniformly through a special substrate formula, so that the detection result is better.
4. The invention also provides a mass spectrum detection kit containing the chip, which is convenient for a client to detect a nucleic acid or protein sample at any time according to the requirement.
5. The invention can also provide a kit comprising the chip with the surface covering matrix according to the needs of users so as to meet the personalized needs.
6. According to the kit, the main body of the chip is made of disposable materials, and a new chip can be replaced to be matched with the adapter after the kit is used, so that the cleaning of the existing metal target plate and the sample pollution are avoided.
7. The chip adapter disclosed by the invention can be used for placing a plurality of chips due to different card slot designs, can realize simultaneous sample injection detection of different samples of nucleic acid and protein, and saves the time for opening and closing the door of a mass spectrum.
Drawings
FIG. 1 is a schematic diagram of a microarray chip, in which 1 is a hydrophilic spotting well and 2 is a hydrophobic well outer region.
Fig. 2a and 2b are schematic diagrams of a microarray chip adapter, in which a bottom plate is a metal plate, a square groove is a microarray chip slot, and microarray chips of different specifications are embedded in the square groove.
FIG. 3 is a schematic view of a thermal oxidizer.
FIG. 4 is a schematic illustration of cleaning a substrate in a concentrated acid solution.
FIG. 5 is a diagram showing a crystal comparison between a microarray dual-purpose chip and a conventional chip.
FIG. 6 is a graph showing the effect of spotting a microarray chip by coating the surface of a substrate with spotting wells.
FIG. 7 shows the result of mass spectrometric detection of gene samples on microarray chips.
FIG. 8 is the result of mass spectrum detection of protein sample by microarray chip.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that the upper and lower values of the range, and each intervening value therebetween, is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.
Hydrophobicity (hydrophobicity): in the chemical discipline, hydrophobicity refers to the physical property of a molecule (hydrophobe) to repel water. Hydrophobic molecules tend to be non-polar and therefore more soluble in neutral and non-polar solutions (e.g., organic solvents). Hydrophobic molecules generally gather in a cluster in water, and water forms a large contact angle on the surface of a hydrophobic solution to form a drop shape. For example, hydrophobic molecules include alkanes, oils, fats and most lipid-containing materials.
Hydrophilic property (hydrophilic property): the molecule has polar groups, has high affinity to water, and can attract water molecules or dissolve in water. The surface of the solid material formed by the molecules is easy to be wetted by water. This property is the hydrophilicity of the material.
Contact angle (contact angle): it is the angle theta where the tangent to the gas-liquid interface at the intersection of the gas, liquid and solid phases passes through the boundary between liquid and solid-liquid, which is a measure of the degree of wetting. If θ <90 °, the solid surface is hydrophilic, i.e. the liquid wets the solid more easily, with smaller angles indicating better wetting; if θ >90 °, the solid surface is hydrophobic, i.e. the liquid does not easily wet the solid and easily moves over the surface. There are many methods for measuring the contact angle, angle measurement (droplet angle measurement), length/height measurement, force measurement, and the like. The most common method for measuring the angle of a droplet is to drop a small droplet on a flat surface and measure the angle by using a protractor of a low power microscope.
The matrix is a special solution which has strong absorption to specific wavelength and is easy to be excited, and transfers energy and charges to molecules to be detected to be gasified and ionized, so that the contained molecules to be detected enter a mass spectrum detection channel together.
The good matrix has the following characteristics: a. the most important function of the matrix is to absorb the energy of the pulsed laser, either by ionizing and absorbing the energy of the laser in the ultraviolet region or by vibrating the chemical bonds of the molecule, depending on the wavelength of the laser used. b. The matrix should be able to disperse the sample molecules well, prevent their aggregation, and increase ionization efficiency. c. When the solid-liquid phase matrix and the sample mixture are desorbed, matrix molecules do not generate extra thermal effect any more and molecules to be detected cannot be damaged. d. The matrix must be able to help the sample molecules ionize well. In the case of a liquid matrix, it must also have good vacuum stability.
The compounds used as matrix can be alpha-cyano-4-hydroxycinnamic acid, 3, 5-dimethoxy-4-hydroxycinnamic acid, 2, 5-dihydroxybenzoic acid. Depending on the sample, different matrices may be used. In general, α -cyano-4-hydroxycinnamic acid (CHCA) is suitable for peptide samples, 3, 5-dimethoxy-4-hydroxycinnamic acid (SA) is suitable for protein samples, and 2, 5-dihydroxybenzoic acid (DHB) is suitable for peptide, carbohydrate and glycolipid samples.
In genomics detection, the MALDI method is suitable for 3-hydroxy-2-picolinic acid matrix, the prepared matrix is automatically spotted by BIODOT and covered in sample holes on the surface of a microarray chip, and the matrix crystal grows finely and has uniform shape due to the hydrophilic and hydrophobic microarray structure on the surface. The accuracy of a sample peak detected by mass spectrometry is high, the signal-to-noise ratio is high, and the base line is low.
"surface-coated substrate" means a protein or nucleic acid substrate which is coated in advance with a spot region depending on the kind of a molecule to be detected and dried to form crystals. The surface of the chip is covered with a substrate in advance, so that a product or a kit for sample application and detection of a sample at one time or directly can be prepared.
EXAMPLE I hydrophilic treatment of microarray chip surface
The chip main body is characterized by tough texture and good planeness; diamond, monocrystalline silicon, quartz crystal, etc.; the chip is divided into a sheet structure suitable for the size of the surface unit of the chip adapter, the thickness of the chip is 0.5-1.8mm, and the flatness of the surface of the chip is less than 10 mu m for ensuring the quality detection precision. For example, the microarray chip substrate is a monocrystalline silicon wafer, a silicon dioxide oxide film, a zinc oxide film and an aluminum oxide film can be fired and grown on the surface of the silicon wafer structure, the thickness is 150-800nm, preferably 500nm, the film is characterized by hydrophilicity, and a thermal oxidation process is adopted.
As shown in fig. 3, the step of firing the grown silica oxide thin film using the thermal oxidizer is as follows:
putting a silicon chip into a reaction tube made of quartz glass;
heating the reaction tube by a resistance wire heating furnace at a certain temperature, wherein the common temperature is 900-1200 ℃;
thirdly, oxygen or water vapor passes through the reaction tube at the air flow speed of 1 cm/s to carry out chemical reaction on the surface of the silicon wafer:
si (solid) + O2(gaseous) → SiO2(solid state) or Si (solid state) +2H2O (vapor state) → SiO2(solid) +2H2(gas phase) formation of SiO2And (3) a layer.
The silicon thermal oxidation process can be divided into the following steps according to the used oxidation atmosphere: dry oxygen oxidation, water vapor oxidation and wet oxygen oxidation. The dry oxidation is to take dry pure oxygen as an oxidation atmosphere, and the oxygen directly reacts with silicon at high temperature to generate silicon dioxide.
The steam oxidation is to take high-purity steam as an oxidation atmosphere and react silicon atoms on the surface of a silicon wafer with water molecules to generate silicon dioxide. The oxidation rate of the water vapor oxidation is greater than that of the dry oxygen oxidation.
Wet oxygen oxidation is essentially a mixture of dry oxygen oxidation and water vapor oxidation, with oxidation rates in between.
When the water vapor oxidation is carried out by a method of directly synthesizing water vapor by high-purity hydrogen and oxygen at the inlet of a quartz reaction tube, the water vapor pressure can be adjusted by changing the proportion of the hydrogen and the oxygen, the contamination is reduced, and the quality of the thermally grown silicon dioxide is improved.
EXAMPLE two hydrophobic treatment of microarray chip surface
1. Sealing treatment of hydrophilic sample application hole area
Cleaning organic matters remained on the surface of the chip: firstly, passing through an oxygen flow rate of 60-140 ml/min, a power of 100-300W and a treatment time of 1-10 min; and ultrasonically cleaning the surface of the substrate for 2-3 times by using high-purity water, wherein the time for each time is 2-5 min, so that the surface is thoroughly cleaned and hydroxylated.
Sealing the sample application hole area by using a photoresist technology: and spin-coating the photoresist on the treated substrate by adopting a spin-coating method, wherein the spin-coating speed is 1000-8000 rpm, then placing the substrate under a photoetching mask plate, exposing the substrate for 20 s-5 min under 390nm ultraviolet light, and eluting the substrate by using corresponding developing solution, thereby constructing a sample application hole region with an ordered structure. Wherein the photoresist uses polymethyl methacrylate (PMMA) and/or polystyrene that is resistant to acids.
2. Surface hydrophobic treatment of micro-array chip hole outer zone
The process of surface treatment of metal or non-metal material with silane coupling agent as main material. Is characterized in that a hydrophobic area is manufactured by a chemical surface modification mode, the thickness range of the hydrophobic film layer is 150nm-2 mu m, and the contact angle of a water drop on the surface of the hydrophobic area is more than 120 degrees.
The silane coupling agent is an organic compound with a special structure, contains organic functional groups and inorganic functional groups, and simultaneously generates binding force with a cathode and a nonpolar substance. The silane coupling agent has a chemical general formula of Y-R-SiX3, wherein Y is a non-hydrolytic organic functional group connected with silicon through a carbon atom and can react with resin in a binder body to improve compatibility, such as amino, vinyl, epoxy, sulfydryl, acryloxypropyl and the like; r is a carbon chain with saturated and unsaturated bonds connecting Y and Si atoms; x is a hydrolyzable group such as a halogen group, an alkoxy group, an isopropenyloxy group or the like. The silanol formed by hydrolyzing the groups can react with the oxide or the alkyl on the surface of the metal, so that a silane film with a Si-O-Si three-dimensional network structure is formed on the surface of the metal, the corrosion of the metal is prevented, and the surface is hydrophobic.
The method comprises the following specific steps:
(1) cleaning the chip with a concentrated acid solution:
put the chip in a clean beaker, and the beaker is fixed on a support.
Secondly, preparing a water tank, filling the water tank with water, placing the water tank below a beaker, and heating the water tank; (see FIG. 4)
Thirdly, slowly adding concentrated acid and hydrogen peroxide into the beaker with the chip according to a certain ratio (1: 5-1: 20), and starting an oxidation reaction for 40min when small bubbles continuously appear.
Note that: concentrated acids are dangerous and require careful handling under fume hood conditions. Attention is paid to personal protection.
Fourthly, taking out the chip, putting the chip into a beaker filled with chloroform, and carrying out ultrasonic treatment for 2 min;
taking out the chip, placing the chip in a beaker filled with ultrapure water, and carrying out ultrasonic treatment for 2 min.
Sixthly, repeating the step IV;
and (c) waste liquid treatment: a large amount of water was added to the concentrated acid solution, slowly diluted, and neutralized with NaOH.
(2) Silanization treatment
The chip was placed in a clean beaker.
Hydrolyzing a silane coupling agent, wherein the silane coupling agent is prepared by the following steps: water: mixing ethanol in a ratio of 1:1:8, wherein the silane coupling agent is a dimethyldichlorosilane solution with the concentration of 5-10%, the water adopts deionized water, and the concentration of the ethanol is 99%.
Adding a proper amount of the hydrolyzed silanization solution into a beaker containing the chip;
adding a certain amount of ammonia water (13-30%) for catalysis, and accelerating the reaction according to the ratio of ammonia water to silanization which is 1: 5;
when the solution was left to stand, white smoke was generated on the surface of the solution, and the reaction was ongoing for 30 minutes (in a fume hood).
(3) Post-silylation cleaning
Taking out the chip, placing into a new beaker containing ethanol, and performing ultrasonic treatment for 10 min.
Taking out the chip, placing in a new beaker containing ultrapure water, and performing ultrasonic treatment for 10 min.
Taking out the chip, placing in a new beaker containing chloroform, and performing ultrasonic treatment for 10 min.
After drying the chip, 1 μ l of water drop was dropped on the surface of the chip, and the contact angle of the water drop was measured using a protractor of a low power microscope, to obtain two kinds of initial chips having contact angles of about 135 ° and 155 °, respectively.
(4) Degumming the sealed sample application holes
And (3) putting the silanized chip into a degumming solution to be soaked for 3 hours, taking out the chip and putting the chip into an acetone solution to be subjected to ultrasonic treatment for 2 minutes, so that the photoresist can be removed, and the hydrophilic surface of the sample application area is restored. Forming a micro array chip surface with hydrophilic-hydrophobic difference. The main component of the degumming solution is polyethylene glycol monobutyl ether or polydimethylsiloxane, etc.
EXAMPLE III crystallization comparison of microarray Dual-purpose chip with conventional chip
The same protein or nucleic acid sample is selected, and appropriate amount of corresponding matrix solution is dropped on the dual-purpose chip and the traditional chip, and after the matrix is naturally dried, the crystal form is observed under a microscope.
As shown in FIG. 5, the conventional bioassay chip has an irregular crystal form (left picture), a non-uniform thickness, a coffee ring shape, a hollow center and a thick periphery. The microarray chip provided by the invention has regular crystal form (right picture), uniform thickness and fine crystal growth.
EXAMPLE four surface of microarray chip well was pretreated with a covering substrate
Selecting a 3-hydroxy-2-picolinic acid matrix solution, automatically spotting the hydrophilic spotting holes by a BIODOT instrument to perform surface covering matrix pretreatment, and observing after the matrix is naturally dried.
As shown in FIG. 6, after the hydrophilic pores are covered on the substrate, the target spot is crystallized uniformly, the profile is uniform and smooth, no solution overflows, and the consistency is good.
EXAMPLE V preparation of Dual-purpose Mass Spectrometry kit for microarray chip
As shown in FIG. 2, the prepared microarray dual-purpose chips with different quantities or specifications are subjected to vacuum sealing packaging under a sterile environment according to needs.
Meanwhile, the kit is respectively attached with a matrix solution and an instruction for use.
In addition, according to the fourth embodiment, the surface of the chip is pretreated by the surface covering matrix by the automatic sampling method, and then the chip is vacuumized, sealed and packaged, and the molecular type of the sample to be detected is marked.
And packaging the kit, and storing at room temperature in a dark place.
When purchased by a user, the kit for surface covering substrate pretreatment or the ordinary kit can be selected as required.
EXAMPLE VI microarray Dual-purpose chip kit for Gene detection
Using a micropipette to absorb 1ul of purified deafness gene standard plasmid sample, directly spotting the sample on a chip with a surface covering matrix for pretreatment, and using a Clin-TOF type time-of-flight mass spectrometer produced by Beijing Yixinbo Biotech limited to detect and judge the result of the spotted chip.
The standard plasmid has 20 mass spectrum peaks, the mass range is 3000-9000Da,5 times of the same plasmid sample are repeated, and the typing accuracy of the detection result is analyzed.
Setting parameters:
Turing mode:linear
Mass Range:3000-9000
Max Laser Rep Rate:10.0
Power:105
Profiles:40
Shots:10
and (3) typing results:
the result is shown in FIG. 7, the detected nucleic acid sample has 20 heavy peaks, the baseline is low, the signal-to-noise ratio is high, the detection range is 3000-9000Da, the consistency of the peak position and the peak intensity of the mass spectrum curve with 5 times of repetition is good, the repeatability is good, and the accuracy of the identification result is 100 percent by the BE-SNP software analysis of Beijing Yixinbo Biotech Co. The kit is proved to have excellent resolution for mass spectrometric detection of nucleic acid samples.
EXAMPLE seventhly, microarray Dual-purpose chip kit for protein detection
Adding matrix CHCA into sample application hole of the chip by using micropipette, then adding 1ul of purified protein standard substance BPB (molecular weight 3000-.
The standard protein sample has 6 mass spectrum peaks in total, 20 repeats in total, and the CV value of a spectrogram of a detection result is analyzed.
Setting parameters:
Turing mode:linear
Mass Range:3000-40000
Max Laser Rep Rate:30.0
Power:65
Profiles:100
Shots:5
the result is shown in FIG. 8, 20 mass spectrum peaks only appear in 20 repetitions, the discrimination of peak lines is good, no obvious interference exists, the distribution range is 3014-38384, the peak lines of all the repeated tests have consistent correspondence, the repeatability CV of the spectrogram is less than 2.6%, and the accuracy of typing of the preset detection result is completely consistent, which indicates that the kit has excellent resolution for the mass spectrum detection of the protein sample.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the foregoing description only for the purpose of illustrating the principles of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims, specification, and equivalents thereof.
Claims (5)
1. A dual-purpose chip for time-of-flight mass spectrometry detection of proteins and nucleic acids, wherein the chip comprises:
(1) a chip main body: the surface of the main body is provided with hydrophilic spot-like holes and hydrophobic hole outer regions which are arrayed in a micro-array manner;
(2) the chip adapter is composed of a metal bottom plate, wherein a plurality of chip clamping grooves matched with a chip main body are distributed on the surface of the adapter, the chip main body is selected from a single-side polished monocrystalline silicon or quartz crystal chip which is tough in texture and has the surface flatness less than 10 mu m, the hydrophilic spotting holes cover a hydrophilic thin film with the thickness of 150-800nm, the hydrophobic outer region of the hydrophobic holes cover a hydrophobic thin film with the thickness of 150-2 mu m, and the contact angle of water drops on the surface of the hydrophobic thin film is about 135 degrees or 155 degrees;
wherein the spotting wells are pretreated in advance with an alpha-cyano-4-hydroxycinnamic acid or 3, 5-dimethoxy-4-hydroxycinnamic acid solution, or with a 3-hydroxy-2-picolinic acid solution, and,
the hydrophilic film is a silicon dioxide oxide film, and the hydrophobic hole outer region is processed by a silane coupling agent to form a hydrophobic film.
2. The dual-purpose chip of claim 1, wherein the silane coupling agent is selected from the group consisting of vinyl silane, amino silane, or dimethyldichlorosilane.
3. The dual-purpose chip of claim 1 or 2, wherein the thickness of the film of the hydrophilic spot-like pore layer is 500nm, the number of the hydrophilic spot-like pores on the chip main body is 24, 40, 70 or 384 pores, the pore diameter of the hydrophilic spot-like pores ranges from 0.5 to 2.8mm, the chip size is (20 x 30) - (83 x 125) mm, and the hydrophilic spot-like pores are circular.
4. The dual-purpose chip of claim 1 or 2, wherein the chip adapter is designed with different card slots according to the size of the chip body, and the size of the card slot is (20 x 30) - (83 x 125) mm, so that one adapter can ensure that the chip bodies with different specifications enter the detection chamber.
5. The dual-purpose chip of claim 4, wherein the number of card slots on the adapter is 2, 4, 6, 8, 12, 16, or 20.
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| CN107192757B (en) * | 2017-07-05 | 2018-10-19 | 北京毅新博创生物科技有限公司 | A kind of dual-purpose detection kit of mass spectrum |
| CN108008003A (en) * | 2017-10-31 | 2018-05-08 | 北京毅新博创生物科技有限公司 | Improve the method and product of Mass Spectrometer Method nucleic acid crystallization |
| CN108051503A (en) * | 2017-10-31 | 2018-05-18 | 北京毅新博创生物科技有限公司 | Improve the method and product of Mass Spectrometer Method microorganism crystallization |
| CN108051504A (en) * | 2017-10-31 | 2018-05-18 | 北京毅新博创生物科技有限公司 | Correct the method and product of Mass Spectrometer Method glycosyl accuracy |
| CN107991491A (en) * | 2017-10-31 | 2018-05-04 | 北京毅新博创生物科技有限公司 | Correct the method and product of the accuracy rate of Mass Spectrometer Method protein sample |
| CN107941894A (en) * | 2017-10-31 | 2018-04-20 | 北京毅新博创生物科技有限公司 | Improve the method and product of Mass Spectrometer Method protein crystal |
| CN108008002A (en) * | 2017-10-31 | 2018-05-08 | 北京毅新博创生物科技有限公司 | Correct the method and product of the accuracy rate of Mass Spectrometer Method nucleic acid samples |
| CN107884466A (en) * | 2017-10-31 | 2018-04-06 | 北京毅新博创生物科技有限公司 | Correct the method and product of the accuracy rate of Mass Spectrometer Method microbiological specimens |
| CN107884467A (en) * | 2017-10-31 | 2018-04-06 | 北京毅新博创生物科技有限公司 | Improve the method and product of Mass Spectrometer Method glycosyl crystallization |
| CN107983601A (en) * | 2017-11-17 | 2018-05-04 | 华南理工大学 | A kind of micro-patterning wettable surfaces and its preparation method and application |
| CN110767544A (en) * | 2019-10-31 | 2020-02-07 | 华南理工大学 | Thin film electrode and liquid phase self-climbing preparation method and application thereof |
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