CN107177689A - The general-purpose chip of albumen and nucleic acid is detected for flight time mass spectrum - Google Patents
The general-purpose chip of albumen and nucleic acid is detected for flight time mass spectrum Download PDFInfo
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- CN107177689A CN107177689A CN201710539893.0A CN201710539893A CN107177689A CN 107177689 A CN107177689 A CN 107177689A CN 201710539893 A CN201710539893 A CN 201710539893A CN 107177689 A CN107177689 A CN 107177689A
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- C—CHEMISTRY; METALLURGY
- 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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- 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
Abstract
The present invention is provided to flight time mass spectrum detection albumen and the general-purpose chip of nucleic acid, wherein the chip includes：(1) chip body：Body surfaces have the hydrophily loading wells and hydrophobic pores outskirt of micro- permutation arrangement；(2) adaptor chip being made up of metal base plate, wherein the adapter face is distributed multiple chip necks matched with chip body, wherein, chip body is selected from diamond, monocrystalline silicon, the chip of quartz crystal of the good single-sided polishing of strong but pliable in texture, flatness, the hydrophily loading wells covers 150 800nm hydrophilic film, hydrophobic pores outskirt covers 2 μm of hydrophobic films of 150nm, 120 ° of its surface water droplet contact angle ＞；Wherein covering matrix solution is used to pre-process loading wells in advance, the matrix solution is selected from the matrix solution for protein spectrum, or matrix solution is selected from for the mass spectrographic matrix solution of nucleic acid.
Description
Technical field
, can Mass Spectrometer Method albumen the present invention relates to a kind of dual-purpose chip that albumen and nucleic acid are detected for flight time mass spectrum
And nucleic acid molecules, belong to Mass Spectrometer Method field.
Background technology
Proteomics (proteomics) be studied from integral level the composition of intracellular protein, mechanics and
The interaction of protein-protein, is one new science of functional genomics epoch.Including identification protein expression,
Modified forms, structure, function and interaction etc..According to research purpose, proteomics can be divided into expression proteomics,
Structural proteomics and functional proteomicses.Expression proteomics are used for quantifying of reaching of intracellular protein sample table and ground
Study carefully.Using the albumen drawing out the structure of albumen composition or be present in a special organelle as the protein of research purpose
Group, which is learned, is referred to as structural proteomics, for setting up the network collection of illustrative plates of intracellular signal transduction and explaining the tables of some specific proteins
Up to the effect [2] to cell.Functional proteomicses using the interaction between the function and protein of intracellular protein as
Research purpose, by carrying out research and analysis to selected protein group, using the teaching of the invention it is possible to provide phosphorylation, glycosylation about protein
Deng important information.
The core of proteomics research is exactly each egg expressed in one cell or tissue of identification for be capable of system
The performance of white matter and protein.The main correlation technique of proteomics has two dimensional gel electrophore- sis, two-way fluorescent differences gel electricity
Swimming, mass spectral analysis etc. [2].High complexity and the presence of a large amount of low abundance proteinses due to protein, are carried to analytical technology
Huge challenge is gone out, tree species for bio-energy source is then the inevitable choice for adapting to this challenge, and that develop rapidly in the recent period is MALDI-
TOF mass spectrums.
Based on the mass spectrographic proteomic techniques of MALDI-TOF be used for microbial identification and classification and traditional method and
Primarily now self-reacting device compare, have the characteristics that：
1. it is simple to operate, quick.Single microbial bacterium colony or other biomaterials can be applied directly on MALDI sample targets
And analyzed using MALDI-TOF mass spectrographs, mass spectrum database can be completed within a few minutes, and data assessment is direct with determining
Connection.This simple and unique workflow is enough for the identification of most microorganisms, and need not be removed from office
Blue Albert'stain Albert, oxidizing ferment test or PCR primer and condition selection.
2. it is reproducible.In very wide condition and range, MALDI methods are all proved to be very stable.Growth medium
Different group pair peaks pattern distribution influences it is very small, such as in the range of from 4000 to 12000Da, training is almost not observed
Support the influence of base.Equally, the growth conditions of cell also do not influence on peak pattern, the cell and logarithmic growth in slow growth period
The cell of phase, plateau or death phase have similar pattern.Carry out at the standard conditions after sample preparation and measurement, not
The mass spectrogram obtained on same MALDI-TOF instruments has very high comparativity, such as on 3 different instruments, to same
The spectrogram of sample target measurement is actually consistent.Therefore, coming from the different mass spectrometric spectrograms of MALDI-TOF can be for building
Vertical true and reliable database.This high duplication is built upon in the protein Fundamentals of Measurement to the high abundance of stable expression
, such as ribosomal protein.In 2000 that metabolin seldom occur to 20000Da mass ranges, wave spectrogram can be observed.
Compared with living cells, bacterial spore can produce visibly different peak pattern, and these " gemma spectrograms " also have repeatability.
At present, the high sensitivity of instrument can detect as little as 100ng or 105Individual cell.And for using CLIN-TOF instruments, 25ng
Biomaterial with regard to demand can be met.
3. the degree of accuracy is high.The protein fingerprint spectrum that MALDI-TOFMS is obtained is used as pattern match, and matching score value is used as identification
As a result classification and classification.MALDI softwares carry out analysis to the collection of illustrative plates of gained and unitized, and this correction and statistical calculation ensure that
The accuracy of identification, can identify the mass spectrogram away from 5000ppm at present.Protein fingerprint is concentrated mainly on 2-20kDa by micro-
Biological growthing environment and state influence the lasting high expressing protein of very little.
Biological mass spectrometry turns into technology platform essential in proteomics research with its unrivaled superiority.With
Mass spectrographic sensitivity, accuracy and it is high-throughout continue to develop, mass spectrum in proteomics research performer increasingly weigh
The role wanted.It has been obtained in terms of the interaction of identification of proteins, sequence analysis, quantitative, post translational processing and protein
Wide application is arrived.Equally, the mass spectrographic high sensitivity of MALDI-TOF, pinpoint accuracy and high flux are detected in genomics
Field advantage is also especially prominent.
Genome research should include both sides content：Using genome sequencing as the structural genomics of target
(structural genomics) and functional genomics (functional by target of Functional identification of genes
Genomics), the Post genome (postgenome) that is otherwise known as is studied, the important method as systems biology.
Genomic DNA sequencing is the first step that the mankind are recognized autogene group.With the completion of sequencing, functional genome
Main flow of the research as research is learned, the height that it interacts from genomic information and external environment illustrates the function of genome.
The research contents of functional genomics：Human genome DNA's sequence variations Journal of Sex Research, the research of genomic expression regulation and control, pattern
The research of organism and the research of bioinformatics etc..
(1) research of genomic expression and regulation and control.In the level of full cell, recognize all genomic expression product mRNA and
Protein, and both interaction, illustrate genomic expression under growth course and varying environment pressure when, it is empty whole
Body regulated and control network.
(2) identification and identification of human gene information.Genome functions information is extracted, identification and identification gene order are
Essential element task.Gene identification need to use bioinformatics, calculation biology technology and biological experiment means, and
Theoretical method and experiment are combined.Mainly started with based on theoretical method from a large amount of nucleic acid sequence datas grasped,
Developmental sequence compares, genome comparison and predictive genes theoretical method.The biological means of identification gene are based primarily upon following
Principle and thinking：According to expressible nucleotide sequence label (STS)；Direct cDNA selections are carried out to chromosome specific cosmid；Root
According to CpG islands；Differential disply and relative theory；Exon trapping and relative theory；Biochip technology；Genome scanning；Mutation
Detection architecture, etc..
(3) extraction and identification of gene function information.Including：The system identification of human mutation body；Gene expression profile
Drafting；The identification of " gene alteration-function changes "；Protein level, decorating state and the detection of interaction.
(4) in sequencing and Genetical Variation.Represented although the genome sequence that the Human Genome Project is obtained has
Property, but everyone genome is not just the same, genome sequence has difference.The difference of genome is reflected in phenotype
The upper difference for just forming individual, the difference of such as Black people and white man, tall person and the difference of short, Healthy People and the difference of hereditary patient
It is different, etc..It is exactly SNP (SNPs) most gene pleiomorphisms occur.
(5) comparative genomics.Human genome is compared with Model organism genome, this aspect contributes to root
The function of human gene is analyzed according to homology method, on the other hand contributes to find the essential difference of the mankind and other biological, visits
The secret of rope genetic language.
The SNP Detection accuracies that MALDI-TOF flight time mass spectrums are completed are up to 99.9%, except accuracy is high, flexible
Property the advantage such as strong, flux is big, detection cycle is short outside, most it is attractive should or its cost performance.During MALDI-TOF flights
Between mass spectrometric platforms be international gene mononucleotide polymorphism (SNP) research platform, this method by its it is scientific and
Accuracy has become the new standard in the field.
Although MALDI-TOF flight time mass spectrums can be used in detection albumen or nucleic acid molecules, from the point of view of application angle,
Traditional MALDI chip applications field is single, the DE1580TA steel disc target plates that such as SHIMADZU is produced, and is served only for albumen microorganism
Identification, it is adaptable to SHIMADZU MALDI-TOF AXIMA；The L24 SpectroCHIP chips of AGENA productions, are served only for base
Because of detection, it is adaptable to AGENA MALDI-TOF MASSARRY, having not seen at present can covering gene group detection simultaneously or albumen
Matter group identifies the chip of two kinds of detection fields.
From mass spectrum chip material with from the point of view of surface characteristic angle, traditional metal chip, Reusability has cut, and table
Face does not have chemical modification processing, and crystal habit is poor, and the degree of accuracy at Mass Spectrometer Method sample peak is low, and signal to noise ratio is low, and baseline is high.And it is right
For nucleic acid samples, small nucleic acid residual can influence mass spectral results.Therefore, metal class chip is mainly used in protein spectrum inspection
Survey, it is impossible to transboundary detect nucleic acid samples.And nucleic acid chip mainly uses silicon chip material, but it is expensive, and matrix formulations quilt
Foreign countries' monopolization, and the chip inlet purchased, surface directly covers matrix, limits it and cannot be used for protein spectrum.
Therefore, need a kind of to be used for the general-purpose chip of flight time mass spectrum detection albumen and nucleic acid, and chip at present
The matrix formulations of surface covering.
The content of the invention
In order to solve the above-mentioned technical problem, the first object of the present invention, which is to provide one kind, can be used for flight time mass spectrum detection
The general-purpose chip of albumen and nucleic acid, including：
(1) chip body：Body surfaces have the hydrophily loading wells and hydrophobic pores outskirt of micro- permutation arrangement；
(2) distribution of the adaptor chip being made up of metal base plate, the wherein adapter face is multiple to be matched with chip body
Chip neck；
Wherein, chip body is selected from the good diamond of strong but pliable in texture, flatness, monocrystalline silicon, the chip of quartz crystal；
The hydrophily loading wells covers 150-800nm hydrophilic film, and hydrophobic pores outskirt covers 150nm-2 μm and dredged
Aqueous film, 120 ° of its surface water droplet contact angle ＞.
In one embodiment, the hydrophilic film is silica oxides film, zinc-oxide film, aluminum oxide
Film etc., hydrophobic pores outskirt carries out processing by silane coupler and forms hydrophobic film.In a specific embodiment,
The silane coupler is selected from vinyl silanes, amino silane or dimethyldichlorosilane.
In another embodiment, the chip is the silicon or quartz chip of single-sided polishing, shown hydrophily point sample
Aperture layer film thickness is 500nm, and the contact angle of the hole outskirt is 120 ° of ＞, preferably from about 135 °, 155 °.
In one embodiment, the hydrophilic point sample hole number on the chip is the holes such as 24,40,70,384, or according to
The detection limit of biological specimen is set in actual clinical experiment or process of scientific research, and the shape of hydrophily loading wells can select circle,
Square, triangle, polygon etc..In a specific embodiment, the pore diameter range of the hydrophily loading wells is 0.5-
2.8mm, the chip size is 20 × 30-83 × 125mm.In another embodiment, hydrophilic point sample area is circle, directly
Footpath is 0.7mm, and appropriate substrates quantity is selected according to Circularhole diameter, and such as 0.5 μ l titrate biomarker sample, crystal habit
Rule, homogeneous crystal orientation is optimal.
In other embodiments, the adaptor chip can design different necks, neck chi according to chip size
Very little is 20 × 30-83 × 125mm, to realize that an adapter ensures that the chip of different size enters sensing chamber.
In another embodiment, the micro-array chip neck on the adapter, can design different as needed
Shape, such as square, rectangle etc., each neck size is 20 × 20mm-25 × 60mm.In a specific embodiment
In, the neck of adaptor chip is 2,4,6,8,12,16 or 20.
In a further embodiment, the chip surface flatness is less than 10 μm.In a specific embodiment, institute
State chip has single-sided polishing mirror beforehand through chemically mechanical polishing (chemical mechanism polish, CMP) is processed
Face effect.By CMP method, chip is polished twice, rough polishing and fine polishing.Rough polishing purpose is to remove chip surface residual
Mechanical damage, typically removes the thickness in the range of 30um from surface.Fine polishing purpose is to remove polishing for the first time to stay in chip surface
Under slight damage and cloud defect, typically from surface remove 2~3um.There is minute surface by the chip surface polished twice
Effect, during Mass Spectrometer Method, is shown, can directly observe chip surface in real time by illumination, light path reflection and camera
The situation that sample is bombarded, changes the position of laser bombardment, obtains optimal collection of illustrative plates.It is described in a more specific embodiment
Chip is the silicon or quartz chip of single-sided polishing.
Second purpose of the invention is to provide a kind of surface to hydrophily loading wells and carries out the dual-purpose of covering matrix pretreatment
Chip, wherein on the basis of such scheme, being pre-processed in advance using covering matrix solution to loading wells.
In one embodiment, when matrix solution is selected from the matrix solution for protein spectrum, the chip can be used for
Mass Spectrometer Method albumen.In another embodiment, when matrix solution is selected from matrix solution mass spectrographic for nucleic acid, the chip can
For Mass Spectrometer Method nucleic acid.
In a specific embodiment, the matrix for Mass Spectrometer Method albumen is selected from α-cyanogen -4- hydroxycinnamic acids
(CHCA), 3,5- dimethoxy-4 's-hydroxycinnamic acid (SA), the matrix for Mass Spectrometer Method nucleic acid is 3- hydroxyl -2- pyridine first
Acid.
3rd purpose of the invention is to provide the dual-purpose chip for preparing flight time mass spectrum detection albumen and nucleic acid, bag
Include：
(1) substrate or chip body are used as from the good chip of strong but pliable in texture, flatness；
(2) hydrophilic treated is carried out to chip surface；
(3) Seal treatment with photoresist to hydrophily loading wells；
(4) silane coupler process chip is used；
(5) recovery processing is carried out to the point sample area that photoresist is closed；
(6) chip and metal chip adapter are subjected to Matching installation.
In one embodiment, 60~140ml/min of oxygen gas flow rate, 100~300W of power, place are passed through in step (2)
1~10min of reason time；Substrate surface is cleaned by ultrasonic 2~3 times with high purity water again, each time is 2~5min, makes surface thorough
Bottom cleaning and hydroxylating.
In another embodiment, photoresist is spun in treated substrate using the method for spin coating in step (3),
Spin speed is 1000~8000rpm, is subsequently placed under mask blank, after 390nm exposed under UV light 20s~5min
After being eluted with corresponding developer solution, so as to construct out the point sample porose area of ordered structure.Wherein photoresist uses acid proof poly- first
Base methyl acrylate (PMMA), polystyrene.
In other embodiments, step (4) includes：
(i) chip is cleaned：By priority immersion acetone, methanol aqueous solution, chloroform, after being respectively washed, drying is taken out；
(ii) in a heated condition, concentrated acid and hydrogen peroxide are slowly added in the aqueous solution containing chip, carry out abundant oxygen
Change after reaction, cleaned respectively in chloroform, ultra-pure water, the step can be repeated several times；
(iii) chip is placed in clean container, adds the silane coupler solution hydrolyzed in advance, and add ammoniacal liquor and urge
Change, until Silanization reaction is fully completed；
(iv) chip is taken out, is respectively placed in ethanol, ultra-pure water, chloroform, is cleaned by ultrasonic；
(v) by the chip after cleaning, 120 ° of contact angle ＞ chip is measured and selected, qualified chip is used as；
In one embodiment, step (5) includes：
(i) by the chip after silanization treatment, it is put into glue, soaks 3 hours；
(ii) chip takes out, and is put into acetone soln, ultrasonically treated 2 minutes,
Photoresist can be removed, recover point sample area water-wetted surface, be formed with the micro-array chip surface of hydrophobe difference.
In a specific embodiment, it is polyethylene glycol monobutyl ether or dimethyl silicone polymer solution to remove glue.
In another embodiment, step (1) described chip be diamond strong but pliable in texture, that flatness is good, monocrystalline silicon,
The chip of quartz crystal.In a specific embodiment, the chip is beforehand through chemically mechanical polishing (chemical
Mechanism polish, CMP) it is processed with single-sided polishing mirrored effect.By CMP method, chip is thrown twice
Light, rough polishing and fine polishing.Rough polishing purpose is the mechanical damage for removing chip surface residual, is typically removed from surface in the range of 30um
Thickness.Fine polishing purpose is to remove to polish the slight damage left in chip surface and cloud defect for the first time, typically from surface
Remove 2~3um.There is mirrored effect by the chip surface polished twice, during Mass Spectrometer Method, pass through illumination, light path
Reflection and camera are shown in real time, can directly observe the situation that chip surface sample is bombarded, and change the position of laser bombardment,
Obtain optimal collection of illustrative plates.In a more specific embodiment, the chip is the silicon or quartz chip of single-sided polishing.
In another embodiment, step (4) described silane coupler is selected from vinyl silanes, amino silane, diformazan
Base dichlorosilane.Wherein, dimethyldichlorosilane growth hydrophobic membrane is simple to operate, contacts larger, hydrophobicity is good, hydrophobic layer thickness
Nanometer can be reached to micron dimension, therefore silane coupler is preferably dimethyldichlorosilane.In a specific embodiment,
By the hydrophobic surface of the overall silanization of the coupling agent, 120 ° of its contact angle ＞ is in a preferred embodiment, described to connect
150 ° of feeler ＞, to form super hydrophobic surface.In other preferred embodiments, the thickness of the hydrophobic surface is arrived in nanometer
Micron dimension.
In one embodiment, the hydrophily point sample hole number on the chip is 24, the holes such as 40,70,384, or root
Factually the detection limit of biological specimen is set in border clinical test or process of scientific research, and hydrophily point sample hole shape can select circle,
Square, triangle, polygon etc..In a specific embodiment, the pore diameter range of the hydrophily loading wells is 0.5-
2.8mm, the chip size is 20 × 30-83 × 125mm.In another embodiment, hydrophily loading wells is circle,
A diameter of 0.7mm, appropriate substrates quantity is selected according to Circularhole diameter, and such as 0.5 μ l titrate biomarker sample, crystalline
State rule, homogeneous crystal orientation is optimal.
In other embodiments, the micro-array chip neck can select square, rectangle etc. as needed, often
Individual neck scope is 20 × 20mm-25 × 60mm.In a specific embodiment, the neck of adaptor chip be 2,4,6,8,
12nd, it is 16 or 20, even more many.
4th purpose of the invention is to provide the chip for preparing flight time mass spectrum detection albumen or nucleic acid, including：Above-mentioned
The step of one scheme, and, point sample area is pre-processed using covering matrix solution in advance.In one embodiment, when
When matrix solution is selected from the matrix solution of protein spectrum, the chip can be used for Mass Spectrometer Method albumen.In another embodiment, when
When matrix solution is selected from nucleic acid mass spectrographic matrix solution, the chip can be used for Mass Spectrometer Method nucleic acid.
5th purpose of the invention is to provide the dual-purpose detection kit of mass spectrum including said chip, including：
Vacuum-packed multiple dual-purpose chips and metal chip adapter；
Nucleic acid and/or protein spectrum matrix solution；
And operation instructions.
In one embodiment, the dual-purpose chip has been previously inserted in metal chip adapter.
In another embodiment, the chip is in advance using covering matrix solution.In a specific embodiment
In, when matrix solution is selected from the matrix solution of protein spectrum, the kit can be used for Mass Spectrometer Method albumen.In another embodiment party
In case, when matrix solution matrix solution mass spectrographic selected from nucleic acid, the kit can be used for Mass Spectrometer Method nucleic acid.
In one embodiment, the hydrophily point sample hole number on the chip is 24, the holes such as 40,70,384, or root
Factually the detection limit of biological specimen is set in border clinical test or process of scientific research, and hydrophily loading wells can select circle, square
Shape, triangle, polygon etc..In a specific embodiment, the pore diameter range of the hydrophily loading wells is 0.5-
2.8mm, the chip size is 20 × 30-83 × 125mm.In another embodiment, hydrophilic point sample area is circle, directly
Footpath is 0.7mm, and appropriate substrates quantity is selected according to Circularhole diameter, and such as 0.5 μ l titrate biomarker sample, crystal habit
Rule, homogeneous crystal orientation is optimal.
In other embodiments, the micro-array chip neck can select square, rectangle etc. as needed, often
Individual neck scope is 20 × 20mm-25 × 60mm.In a specific embodiment, the neck of adaptor chip be 2,4,6,8,
12nd, 16 or 20, or more.
6th purpose of the invention is to provide said chip or kit in the purposes for Mass Spectrometer Method biomolecule.
In one embodiment, the biomolecule is nucleic acid and/or protein molecular.
The effect of the present invention includes：
1st, application field covering gene group of the invention detection identifies that same chip can both be done with proteomics
Genetic test, can also do albumen or peptide identification, cover two kinds of detection fields.
2nd, chip surface has the design of hydrophilic-hydrophobic difference, can effectively be enriched with sample, the homogeneous growth of crystal orientation, Mass Spectrometer Method
The sample peak degree of accuracy it is high, signal to noise ratio is high, and baseline is low.
3rd, matrix is covered by automatic point sample on chip point sample area surface, extraordinary matrix formulations make matrix exist with sample
Evenly, testing result is more preferably for chip surface cocrystallization.
4th, present invention also offers the Mass Spectrometer Method kit comprising the chip, it is easy to client to detect at any time as needed
Nucleic acid or protein sample.
5th, the present invention can also need there is provided the kit for the chip that matrix is covered comprising surface according to user, individual to meet
Propertyization needs.
6th, kit of the invention, chip body is disposable material, i.e. replaceable new chip and adapter after use
Coordinated, it is to avoid the cleaning of existing metallic plates and sample pollution.
7th, adaptor chip of the invention, different neck designs, can place multiple chips, can realize nucleic acid with albumen not
With sample while sample detection, saves mass spectrum enabling lockup.
Brief description of the drawings
Fig. 1 is micro-array chip pictorial diagram, wherein 1 being hydrophily loading wells, 2 being weep hole outskirt.
Fig. 2 a, 2b are micro-array chip adapter schematic diagram, and wherein bottom plate is metallic plate, and square groove is micro-array chip
Neck, is embedded with the micro-array chip of different size.
Fig. 3 is thermal oxidation apparatus schematic diagram.
Fig. 4 is cleaning substrate schematic diagram in concentrated acid solution.
Fig. 5 is that the dual-purpose chip of microarray crystallizes comparison diagram with traditional die.
Fig. 6 is that micro-array chip loading wells carries out the point sample design sketch that surface covers matrix.
Fig. 7 is the spectrogram result of micro-array chip Mass Spectrometer Method gene samples.
Fig. 8 is the spectrogram result of micro-array chip Mass Spectrometer Method protein sample.
Embodiment
Now describe the various exemplary embodiment of the present invention in detail, the detailed description is not considered as the limit to the present invention
System, and it is understood as the more detailed description to certain aspects of the invention, characteristic and embodiment.
It should be understood that heretofore described term is only to be to describe special embodiment, this hair is not intended to limit
It is bright.In addition, for the number range in the present invention, it is thus understood that specifically disclose the scope upper and lower bound value and they
Between each median.Median and any other statement value in any statement value or stated ranges or in the model
The each less scope enclosed between interior median is also included in the present invention.These small range of upper and lower bounds can be only
On the spot include or exclude in the range of.
Unless otherwise stated, all technologies used herein and scientific terminology have the routine in field of the present invention
The identical meanings that technical staff is generally understood that.Although the present invention only describes preferred method and material, the present invention's
Implement or can also be used and similar or equivalent any method described herein and material in testing.
Hydrophobicity (hydrophobicity)：In chemistry subject, hydrophobicity refers to a molecule (hydrophobe) and water
Mutually exclusive physical property.Hydrophobic molecule be partial to it is nonpolar, and therefore compared with neutral and non-polar solution can be dissolved in (such as
Organic solvent).Hydrophobic molecule would generally bunch up in water, and water can then form one at the surface of hydroholic solution
Very big contact angle forms drops.For example, hydrophobic molecule includes alkane, oil, fat and majority containing grease
Material.
Hydrophily (hydrophilic property)：Refer to the molecule of molecular band polarized group, there is big affine to water
Ability, can attract hydrone, or be dissolved in water.The surface of the solid material of this quasi-molecule formation, is easily soaked by water.Have
This characteristic is all the hydrophily of material.
Contact angle (contact angle)：Refer to that the tangent line for the liquid-vapor interface made at gas, liquid, solid three-phase intersection point is worn
The angle theta crossed between liquid and solid-liquid boundary line, is measuring for wetness degree.If θ<90 °, then the surface of solids be it is hydrophilic,
I.e. liquid is easier to soak solid, and its angle is smaller, represents that wetability is better；If θ>90 °, then the surface of solids is hydrophobic, i.e. liquid
Body is not easily wetted by solid, easily moves on the surface.The assay method of contact angle is a lot, angled mensuration (drop angle
Mensuration), length/height mensuration, power mensuration etc..Wherein drop angular measurement is the most frequently used, i.e., in flat surface
A droplet is dripped, angular dimension can be measured using the protractor of low-powered microscope.
Matrix, refers to have specific wavelength and absorbs more by force and be easy to be excited, and transmits energy and electric charge to be measured point
Son occurs gasification and ionized, so that the testing molecule included enters the special solution of Mass Spectrometer Method passage together.
Good matrix will possess following several features：A. the most important function of matrix is exactly the energy for absorbing pulse laser,
Both the energy of ultra-violet (UV) band laser can be absorbed by ionizing, infrared region laser can also be absorbed by the chemical bond oscillations of molecule
Energy, this is determined by the optical maser wavelength used.B. matrix will can dispersed sample molecule well, prevent it from assembling, carry
High ionization efficiency.C. the matrix of solid, liquid phase, sample mixture are in desorption, and substrate molecule no longer produces extra fuel factor, no
Testing molecule can be destroyed.D. matrix allows for helping sample molecule to ionize well.If liquid matrix, it is necessary to have
Good vacuum stability.
Compound as matrix can be alpha-cyano -4- hydroxycinnamic acids, 3,5- dimethoxy-4 's-hydroxycinnamic acid,
2,5- dihydroxy-benzoic acids.According to the difference of sample, different substrates are can select.In general, α-cyanogen -4- hydroxycinnamic acids
(CHCA) it is applied to peptides sample, 3,5- dimethoxy-4 's-hydroxycinnamic acid (SA) are applied to protein example, and 2,5- dihydroxies
Yl benzoic acid (DHB) is then applied to peptide, carbohydrate and glycolipid class sample.
In genomics detection, what MALDI methods were applicable is 3- hydroxyl -2- pyridine carboxylic acid matrix, the matrix prepared,
By the automatic point samples of BIODOT, in the sample well for covering micro-array chip surface, due to the hydrophobe micro array structure on surface, make
Matrix crystalline growth is careful, and form is uniform.The sample peak degree of accuracy of Mass Spectrometer Method is high, and signal to noise ratio is high, and baseline is low.
" surface covering matrix ", refers to the species according to molecule to be checked, and the albumen or nucleic acid substrate in point sample area are covered in advance,
And dry formation crystallization.By the way that the surface of chip is covered into matrix in advance, it can be prepared into disposable or point directly is carried out to sample
Sample and the product or kit of detection.
Embodiment one, the processing of micro-array chip surface hydrophilic
Chip body is characterized in that strong but pliable in texture, flatness is good；Diamond, monocrystalline silicon, quartz crystal etc.；Draw and be divided into suitable core
The laminated structure of piece adapter face cell size, chip thickness 0.5-1.8mm, to ensure quality testing precision, chip surface
Flatness is less than 10 μm.For example, micro-array chip choice of the substrates monocrystalline silicon piece, growth two can be fired on silicon chip chip architecture surface
Silicon oxide film, zinc-oxide film, aluminum oxide film are aoxidized, thickness 150-800nm, preferably 500nm, film features are that have
Hydrophily, using thermal oxidation technology.
As shown in figure 3, the step of firing growth silica oxides film using thermal oxidation apparatus is as follows：
1. silicon chip is placed in the reaction tube being made of quartz glass；
2. reaction tube heats stove heat certain temperature with resistance wire, and conventional temperature is 900~1200 DEG C；
3. oxygen or steam chemically react with 1 cel air velocity by reaction tube in silicon chip surface：
Si (solid-state)+O2(gaseous state) → SiO2(solid-state) or Si (solid-state)+2H2O (steam state) → SiO2(solid-state)+2H2(gas
State) generation SiO2Layer.
Silicon thermal oxidation technique, can be divided into by oxidizing atmosphere used：Dry-oxygen oxidation, steam oxidation and wet-oxygen oxidation.Dry oxygen
Oxidation is that, using dry pure oxygen as oxidizing atmosphere, oxygen directly generates silica with pasc reaction at high temperature.
Steam oxidation is, using high-purity water vapour as oxidizing atmosphere, two to be generated by the silicon atom and water molecule reaction of silicon chip surface
Silica.The oxidation rate of steam oxidation is big than dry-oxygen oxidation.
Wet-oxygen oxidation is substantially the mixing of dry-oxygen oxidation and steam oxidation, and oxidation rate is therebetween.
When the method that water vapour is directly synthesized at crystal reaction tube import with high-purity hydrogen and oxygen carries out steam oxidation,
By changing the ratio of hydrogen and oxygen, water vapor pressure can be adjusted, reduces and stains, be favorably improved heat growth silicon dioxide
Quality.
Embodiment two, the processing of micro-array chip surface hydrophobicity
1st, the Seal treatment of hydrophily point sample porose area
Clean chip surface residual organic matter：60~140ml/min of oxygen gas flow rate, 100~300W of power are first passed through, is handled
1~10min of time；Substrate surface is cleaned by ultrasonic 2~3 times with high purity water again, each time is 2~5min, makes surface thorough
Clean and hydroxylating.
Photolithographic techniques close point sample porose area：Photoresist is spun in treated substrate, revolved using the method for spin coating
Painting speed is 1000~8000rpm, is subsequently placed under mask blank, is used after 390nm exposed under UV light 20s~5min
After corresponding developer solution elution, so as to construct out the point sample porose area of ordered structure.Wherein photoresist uses acid proof poly- methyl
Methyl acrylate (PMMA) and/or polystyrene.
2nd, the surface hydrophobicity processing of micro-array chip hole outskirt
The process that metal or nonmetallic materials are surface-treated by primary raw material of silane coupler.It is characterized in by changing
The mode for learning surface modification makes hydrophobic region, 150nm-2 μm of hydrophobic film layer thickness range, hydrophobic region surface water droplet contact angle
120 ° of ＞.
Silane coupler is the organic compound that a class has special construction, containing organo-functional group, Inorganic functional groups, together
When with negative electrode, apolar substance produce adhesion.The chemical general formula of silane coupler is Y-R-SiX3, and Y is former by carbon in formula
The non-hydrolyzable organo-functional group that son is connected with silicon, can react to improve compatibility with the resin in binding agent body,
Such as amino, vinyl, epoxy radicals, sulfydryl, acryloxypropyl；R is the carbochain with saturation and unsaturated bond, by Y and Si
Atom is connected；X is hydrolization group, such as halogen family, alkoxy, isopropyl alkenyloxy group.The silanol that these groups are hydrolyzed to form
It can be reacted with the oxide or alkyl of metal surface, so that the silane film of Si-O-Si three-dimensional net structures is formed in metal surface,
Corrosion of metal is prevented, hydrophobicity is presented in surface.
Comprise the following steps that：
(1) chip is cleaned with concentrated acid solution:
1. chip is placed in clean beaker, beaker is fixed on support.
2. prepare tank, fill water, be placed on below beaker, heating；(see Fig. 4)
3. by concentrated acid and hydrogen peroxide according to a certain percentage (1:5~1:20) it is slowly added into the beaker equipped with chip, treats not
Disconnected minute bubbles occur, oxidation reaction starts, reaction time 40min.
Note：Concentrated acid danger close, it is necessary to fume hood ventilation under conditions of handled.Note personal protection.
4. chip is taken out, be placed in the beaker for filling chloroform, ultrasonic 2min；
5. chip is taken out, be placed in the beaker for filling ultra-pure water, ultrasonic 2min.
4. 5. 6. repeat step；
7. liquid waste processing：A large amount of water are added in concentrated acid solution, slow dilution, and neutralized with NaOH.
(2) silanization treatment
In the beaker that chip is placed on to cleaning.
Silane coupler is hydrolyzed, according to proportioning silane coupler：Water：Ethanol=1:1:8 mixing, wherein silane are even
Join the dimethyldichlorosilane solution that agent is concentration 5%~10%, water uses deionized water, and concentration of alcohol is 99%.
Silanizing solution after hydrolysis is taken into appropriate add in the beaker containing chip；
A certain amount of ammoniacal liquor (13%~30%) catalysis is added, ammoniacal liquor is matched:Silanization=1:5, accelerate reaction to carry out；
Place, solution surface produces white cigarette, reaction is carried out, 30 minutes time (was carried out) in ventilating kitchen.
(3) cleaned after silanization
Chip is taken out, is placed into the new beaker for filling ethanol, ultrasound, time 10min.
Chip is taken out, is placed on into the new beaker for filling ultra-pure water water, ultrasound, time 10min.
Chip is taken out, is placed on into the new beaker for filling chloroform, ultrasound, time 10min.
Dry after chip, 1 μ l water droplets are dripped in chip surface, it is big to measure water droplet contact angle using the protractor of low-powered microscope
It is small, respectively obtain two kinds of chip first products of about 135 °, 155 ° of contact angle.
(4) loading wells of closing is removed photoresist processing
By the chip after silanization treatment, it is put into glue and soaks 3 hours, take out be put into ultrasound in acetone soln afterwards
2 minutes, you can remove photoresist, recover point sample area water-wetted surface.It is formed with the micro-array chip surface of hydrophobe difference.Remove photoresist
Liquid main component is polyethylene glycol monobutyl ether or dimethyl silicone polymer etc..
The dual-purpose chip of embodiment three, microarray and traditional die crystallization are contrasted
Identical albumen or nucleic acid samples are selected, to taking appropriate corresponding matrix solution to drop in dual-purpose chip and tradition respectively
On chip, after matrix is dried naturally, micro- Microscopic observation crystal habit.
As shown in figure 5, traditional biological detection chip crystal habit (left figure) is irregularly, and it is in uneven thickness, in coffee ring-type
State, middle empty, surrounding is thick.And micro-array chip crystal habit (right figure) rule that the present invention is provided, uniformly, crystal orientation grows thickness
It is careful.
Example IV, the surface to micro-array chip loading wells carry out covering matrix pretreatment
From 3- hydroxyl -2- pyridine carboxylic acid matrix solutions, by BIODOT instrument, hydrophily loading wells is carried out from dynamic point
Sample, to carry out surface covering matrix pretreatment, after matrix is dried naturally, is observed.
As shown in fig. 6, after hydrophilic pores covering matrix, target spot crystallization is uniform, and profile is uniformly round and smooth, and no solution overflows, tool
There is good uniformity.
Embodiment five, the dual-purpose Mass Spectrometer Method kit for preparing micro-array chip
As shown in Fig. 2 in an aseptic environment, by the dual-purpose chip of the microarray of the varying number of preparation or specification, according to need
Will, vacuum sealed package is carried out to chip.
Meanwhile, matrix solution, and operation instructions are respectively with kit.
In addition, chip surface can be carried out with automatic spotting methods by covering matrix pretreatment in surface according to example IV, then
Vacuumize and pack, and mark the molecule type of measuring samples.
After kit package, in preservation under room temperature lucifuge.
When user buys, surface can be selected to cover the kit or general reagent box of matrix pretreatment as needed.
The dual-purpose chip agent box of embodiment six, microarray is used for genetic test
Using micropipettor, the deaf gene standard plasmid sample of 1ul purifying is drawn, direct point sample covers base to surface
On the chip of matter pretreatment, and the Clin-TOF type flight time matter produced using Beijing Yixin Bochuang Biotechnology Co., Ltd.
Spectrometer is detected to the chip after point sample and result judges.
The standard plasmid totally 20 mass spectra peaks, mass range 3000-9000Da, same plasmid sample does 5 repetitions, analyzes
Testing result parting accuracy.
Parameter setting：
Turing mode:linear
Mass Range:3000-9000
Max Laser Rep Rate:10.0
Power:105
Profiles:40
Shots:10
Genotyping result：
As a result as shown in fig. 7, detecting that sample of nucleic acid has 20 heavy spectral peaks, baseline is low, signal to noise ratio is high, detection range
3000-9000Da, the mass spectrometry profile peak position of 5 repetitions and peak intensity uniformity are good, and repeatability is good, through the firm new rich creation in Beijing
Thing Science and Technology Ltd. BE-SNP software analysis, qualification result accuracy 100%.Illustrate matter of the kit for nucleic acid samples
Spectrum detection has excellent resolution ratio.
The dual-purpose chip agent box of embodiment seven, microarray is used for Protein Detection
Matrix CHCA is added to the loading wells of chip using micropipettor, then point sample adds the albumen mark of 1ul purifying
The standard plasmid sample of quasi- product BPB (molecular weight 3000-40000Da), and given birth to using YiXin Industry (Beijing) Science and Technology Ltd.
The Clin-TOF types time of-flight mass spectrometer of production is detected to the chip after point sample and result judges.
The standard protein sample totally 6 mass spectra peaks, do 20 repetitions altogether, analyze testing result spectrogram CV values.
Parameter setting：
Turing mode:linear
Mass Range:3000-40000
Max Laser Rep Rate:30.0
Power:65
Profiles:100
Shots:5
As a result as shown in figure 8,20 mass spectra peaks only occur in 20 repetitions, crest line discrimination is good, without significantly interfering with, is distributed
Scope 3014-38384, all crest lines for repeating experiment are respectively provided with consistent correspondence, spectrogram repeatability CV ＜ 2.6%, with presetting
Testing result parting accuracy it is completely the same, illustrate that the kit has excellent resolution for the Mass Spectrometer Method of protein sample
Rate.
General principle, principal character and the advantages of the present invention of the present invention has been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the simply explanation described in above-described embodiment and specification is originally
The principle of invention, without departing from the spirit and scope of the present invention, various changes and modifications of the present invention are possible, the present invention
Claimed scope is by appended claims, specification and its equivalent thereof.
Claims (6)
1. a kind of dual-purpose chip that albumen and nucleic acid are detected for flight time mass spectrum, wherein the chip includes：(1) chip master
Body：Body surfaces have the hydrophily loading wells and hydrophobic pores outskirt of micro- permutation arrangement；(2) chip being made up of metal base plate
Adapter, the wherein adapter face are distributed multiple chip necks matched with chip body, wherein, chip body is selected from quality
Diamond, monocrystalline silicon, the chip of quartz crystal of the good single-sided polishing of tough and tensile, flatness, the hydrophily loading wells covering
150-800nm hydrophilic film, hydrophobic pores outskirt covers 150nm-2 μm of hydrophobic film, its surface water droplet contact angle ＞
120°；
α-cyanogen -4- hydroxycinnamic acids (CHCA) and/or 3,5- dimethoxy-4 '-hydroxycinnamic acid (SA) solution are wherein used in advance
Loading wells is pre-processed, and/or uses 3- hydroxyl -2- pyridine carboxylic acid solution to pre-process loading wells in advance.
2. dual-purpose chip according to claim 1, wherein the hydrophilic film is silica oxides film, oxidation
Zinc film, aluminum oxide film, hydrophobic pores outskirt carry out processing by silane coupler and form hydrophobic film.
3. dual-purpose chip according to claim 2, wherein the silane coupler be selected from vinyl silanes, amino silane or
Dimethyldichlorosilane.
4. dual-purpose chip according to claim 1 or 2, wherein the chip is the silicon or quartzy core of single-sided polishing
Piece, the hydrophily point sample aperture layer film thickness is 500nm, and the contact angle of the hole outskirt is on 120 ° of ＞, the chip
Hydrophilic point sample hole number is 24,40,70 or 384 holes, and the pore diameter range of the hydrophily loading wells is 0.5-2.8mm, the core
Chip size is 20 × 30-83 × 125mm, and preferred hydrophilic loading wells is circle, a diameter of 0.7mm.
5. dual-purpose chip according to claim 1 or 2, wherein the adaptor chip can be according to chip size design not
Same neck, neck size is 20 × 30-83 × 125mm, is examined with realizing that an adapter ensures that the chip of different size enters
Survey room.
6. the micro-array chip neck size on dual-purpose chip according to claim 5, the adapter is 20 × 20mm-
25 × 60mm, neck is 2,4,6,8,12,16 or 20.
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