CN106483191B - A method of Mass Spectrometer Method repeatability is improved by eliminating dessert effect - Google Patents
A method of Mass Spectrometer Method repeatability is improved by eliminating dessert effect Download PDFInfo
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- CN106483191B CN106483191B CN201610974371.9A CN201610974371A CN106483191B CN 106483191 B CN106483191 B CN 106483191B CN 201610974371 A CN201610974371 A CN 201610974371A CN 106483191 B CN106483191 B CN 106483191B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
- G01N27/64—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode using wave or particle radiation to ionise a gas, e.g. in an ionisation chamber
Abstract
A method of Mass Spectrometer Method repeatability being improved by eliminating dessert effect, belongs to detection technique field.Due to weak hydrophobic effect, polymethyl methacrylate can adsorb some hydrophobic albumen and polypeptide, therefore, we provide uniform adsorption site with the hydrophobic silicon nano column array of polymethyl methacrylate point modification, it tips upside down in the solution of determinand molecule of stirring, when taking-up, due to the hydrophobic effect of the array, liquid film is not had on surface, coffee ring effect is prevented, and molecule uniform adsorption is in each nano-pillar, matrix is finally added dropwise, since the acetonitrile and trifluoroacetic acid of preparation solution are all the solvents of highly volatile, evaporation rate is greater than the speed that molecule is migrated to both ends, therefore, matrix there will not be coffee ring effect after drying.In short, our this methods ensure that being uniformly distributed for molecule and matrix, the detection repeatability of testing molecule is greatly enhanced.
Description
Technical field
The invention belongs to detection technique field, it is specially a kind of by solve due to molecule and matrix cocrystallization unevenly with
And determinand molecule caused by coffee ring effect (i.e. dessert effect) the problem of being unevenly distributed on traditional target plate, and then improve
Detect the repeatability of signal, the method to improve Mass Spectrometer Method.
Background technique
Substance assistant laser desorpted time of-flight mass spectrometer is that identification polypeptide, protein, lipid and multiple polymers are strong
Strong tool, application range are wide.Its principle is when the laser irradiation sample and substrate formed total knot with some strength
Brilliant film, matrix absorb energy from laser, and energy transfer occurs between matrix-sample so that desorption electricity occurs for sample
From, the sample of ionization accelerates to fly over dirft tube under the action of electric field, it is different according to the flight time for reaching detector and by
Detection.Actually detected process is to randomly choose the measured point of sample, it can be seen that the uniformity of detected signal is to measure
The accurate premise of detection method.The method that traditional substance assistant laser desorpted ionized mass spectrum prepares sample is (from the bottom up):
Then molecule-matrix, matrix-molecule-matrix, molecular matrix mixing again dry sample.Since matrix-molecule is total to
Coffee ring effect when crystallization is uneven and sample solution dries, molecule are distributed extremely unevenly in substrate, and due to dividing
Son aggregation causes the very strong region of signal to be referred to as " dessert ".This molecule is unevenly distributed so that detecting time point and point
Between signal difference it is especially big, there are contingency and subjectivities when eventually leading to reconnaissance, cannot carry out to experimental result accurate
Analysis.Therefore, Many researchers also did many related works, in order to weaken coffee ring effect, Electrowetting, rapid evaporation
Method increases the methods of viscosity and vacuum deposition of mixing sample and is suggested, still, due to not can control the phase between different molecular
Interaction, these work still cannot achieve the uniform cocrystallization of matrix and determinand molecule.In addition, also some studies work
Author designed prepares specific matrix to solve the problems, such as cocrystallization, but the interaction between these matrix and molecule is general
The interaction being all based between functional group, they do not have universality for the detection of determinand molecule.In addition, they
When carrying out auxiliary detection using these matrix, the effect of coffee ring present in mixed solution dry process is not accounted for nearly all
It answers, it is uneven that this also results in molecular distribution.Either from coffee ring effect is solved still from solution matrix and determinand molecule
It sets out in terms of the problem of uneven cocrystallization, although all having obtained relatively good detection repeatability, some is even lower than
10% relative standard deviation, but the defect based on technical method, the points for causing them to select are fewer, and generally less than ten
A, this does not have very big convincingness to the quality for determining solution.
Therefore, at present for the ameliorative way of dessert effect, exist solve the problems, such as it is halfway, so as to cause only selecting
The repeatability that can have just had under the premise of taking a small amount of test point has certain contingency.
Summary of the invention
The purpose of the present invention is the hydrophobic silicon nano column arrays modified using polymethyl methacrylate dots structure to solve
The bad problem of detection signal repeatability present in certainly substance assistant laser desorpted ionized mass spectral analysis.Due to weak hydrophobic work
With polymethyl methacrylate can adsorb hydrophobic albumen and polypeptide, and polymethyl methacrylate dots structure is modified
Hydrophobic silicon nano column array be capable of providing uniform determinand adsorption site, therefore we are as ground substance assistant laser solution
Inhale the substrate of ionization mass spectrometry.Substrate is placed in first on the liquid level of determinand solution, while solution is stirred, molecule is inhaled
It is attached to substrate surface.Due to the hydrophobic effect of the substrate, surface does not have solution residual when it is taken out from solution, therefore to be measured
Molecule will not generate coffee ring, to eliminate coffee ring effect.Meanwhile nano column array and the poly- first being evenly distributed on column
Base methyl acrylate is evenly distributed on testing molecule in substrate, then using the acetonitrile of highly volatile and trifluoroacetic acid as solvent
It prepares matrix solution and is added dropwise in substrate, since solvent evaporation rate is greater than the speed that molecule is migrated to edge,
Matrix will not generate coffee ring after evaporation of the solvent, and be generally evenly distributed in the substrate for having adsorbed testing molecule, ensure that point
Son is uniformly distributed with matrix.We effectively remove dessert effect at this method, improve substance assistant laser desorpted electricity
From mass spectrographic detection repeatability.
A kind of method improving Mass Spectrometer Method repeatability by eliminating dessert effect of the present invention, specific steps are such as
Under:
(1) the spin coating polymethyl methacrylate film on proposing ball silicon wafer substrate；
(2) ball silicon wafer substrate over-assemble polystyrene microsphere is mentioned in covering polymethyl methacrylate film；
(3) reactive ion etching technology is utilized, silicon is performed etching using polystyrene microsphere as exposure mask；
(4) remaining polystyrene microsphere is removed using organic solvent, to obtain orderly silicon nano column array；
(5) it is surface modified, is obtained by assembling silicon fluoride monofilm on obtained orderly silicon nano column array
To hydrophobic orderly silicon nano column array；
(6) the spin coating polymethyl methacrylate film on the hydrophobic orderly silicon nano column array of fluorine modification；
(7) the hydrophobic silicon nano column array of polymethyl methacrylate film covering is heated, obtains poly- methyl-prop
The hydrophobic silicon nano column array of e pioic acid methyl ester dots structure modification；
(8) testing molecule is adsorbed with the hydrophobic silicon nano column array that polymethyl methacrylate dots structure is modified；
(9) substrate molecule solution is added dropwise to dredge to what the polymethyl methacrylate dots structure for being adsorbed with testing molecule was modified
Water silicon nano column array；
(10) sample prepared in step (9) is dried, Mass Spectrometer Method is carried out.
In the above method, described in step (1) on proposing ball silicon wafer substrate spin coating polymethyl methacrylate film, including
The following steps:
1. cleaning silicon chip: successively using acetone, chloroform, ethyl alcohol and the deionized water ultrasound at 40~100W clear multi-disc silicon wafer
3~10min is washed, then uses NH3·H2O:H2O2: H2O volume ratio is that 1~3:1~3:5~7 mixed solution adds at 80~100 DEG C
50~70min of thermal cleaning, is finally rinsed and is used with deionized water and be dried with nitrogen, and is respectively obtained and is mentioned ball silicon wafer and vollyball silicon wafer；
2. spin coating polymethyl methacrylate: the poly- methyl-prop for being 5%~7.5% by the mass fraction prepared with dimethylbenzene
E pioic acid methyl ester (molecular weight be 97000~350000) solution is added drop-wise to that step is 1. cleaned to be mentioned on ball silicon wafer, with 2000rpm~
The speed of 7000rpm carries out spin coating, and obtain that spin coating has a polymethyl methacrylate film mentions ball silicon wafer substrate, polymethyl
The thickness range of sour methyl esters film is 100~300nm.
Described in step (2) have polymethyl methacrylate film cover mention ball silicon wafer substrate over-assemble polystyrene
Microballoon comprises the following steps:
1. preparing the ethyl alcohol of polystyrene microsphere (diameter is 100~4000nm) and the mixed solution of deionized water: will be straight
0.1~1g of Polystyrene powder of 100~4000nm of diameter is added to the mixed of 1~20mL, the deionized water of volume ratio 1:1 and ethyl alcohol
It closes in solution, 1~6h of ultrasound makes its mixing；
2. the assembling and transfer of polystyrene microsphere monofilm: deionized water being added in a clean watch glasses, and is added 20
The Surfactant SDS aqueous solution that~100 μ L mass fractions are 0.5%~2%；Then microsyringe is used
The ethyl alcohol of 1. polystyrene microsphere that step obtains and deionized water mixed solution are slowly drip onto volleyball wafers, and along
Vollyball silicon wafer slides into water, forms it into the closelypacked polystyrene microsphere monofilm of six sides, adds 5~25 μ L above-mentioned ten
Sodium dialkyl sulfate aqueous solution stablizes monofilm；It then will with the ball silicon wafer substrate that mentions with polymethyl methacrylate film
Polystyrene microsphere lifts, and slant setting is until moisture volatilizees completely.
Reactive ion etching technology is utilized described in step (3), and the etching of silicon is carried out using polystyrene microsphere as exposure mask,
Include the following steps:
1. reducing the partial size of polystyrene microsphere: by surface with polystyrene microsphere monofilm mention ball silicon wafer 5 ×
10-5~8 × 10-5Start reactive ion etching, the parameter of etching are as follows: O under Pa vacuum degree2Flow is 10~50sccm, cavity pressure
Power is 10~50mtorr, and radio-frequency power is 15~200W, and inductively coupled plasma body power is 50~150W；Etch period is 1
~15min, the partial size of polystyrene microsphere is reduced to 80~3500nm. after etching
2. the etching of silicon: mentioning ball silicon wafer 5 × 10 for reduce polystyrene microsphere partial size-5~8 × 10-5Pa vacuum
Degree is lower to start reactive ion etching, the parameter of etching are as follows: SF6Flow is 5~15sccm, CHF3Flow is 30~60sccm, cavity
Pressure is 10~50mtorr, and radio-frequency power is 15~50W, and inductively coupled plasma body power is 50~150W, and etch period is
1~9min.
Remaining polystyrene microsphere is removed using organic solvent described in step (4), to obtain orderly silicon nano-pillar
Array, the specific steps are as follows:
The ball silicon wafer that mentions that step (3) has been etched is successively placed in acetone, chloroform, ethyl alcohol and deionized water with 40~100W
Power is cleaned by ultrasonic 3~10min, until removing remaining polystyrene microsphere, to obtain orderly silicon nano-pillar battle array
The diameter of column, silicon nano-pillar is 50~3000nm, is highly 400~800nm.
Fluorine modification is carried out on the orderly silicon nano column array being prepared described in step (5), obtains hydrophobic orderly silicon
Nano column array, the specific steps are as follows:
1. hydroxylating is handled: the orderly silicon nano column array NH that will be obtained3·H2O:H2O2: H2O volume ratio is 1~3:1
The mixed solution of~3:5~7 heats 50~70min of cleaning at 80~100 DEG C, is finally rinsed with deionized water and is blown with nitrogen
It is dry；
2. fluorine modification: the orderly silicon nano column array that hydroxylating is handled is placed in glass culture dish, is added dropwise 2~10
μ L perfluor silane into glass culture dish, at 60~300 DEG C heat 0.5~6h, then successively with acetone, chloroform, ethyl alcohol, go
Ionized water is cleaned by ultrasonic 3~10min at 40~100W, to pass through self assembly perfluor silane on orderly silicon nano column array
The form of monofilm carries out fluorine modification.
Spin coating polymethyl methacrylate is thin on the hydrophobic orderly silicon nano column array of fluorine modification described in step (6)
Film, the specific steps are as follows:
By the mass fraction prepared with dimethylbenzene be 1%~5% polymethyl methacrylate (molecular weight is 97000~
350000) solution is added drop-wise on the silicon nano column array of fluorine modification, and spin speed is 1000~7000rpm, and make fluorine modification has
There is one layer of polymethyl methacrylate film on sequence silicon nano column array, the thickness range of film is 2~200nm.
The hydrophobic silicon nano column array of polymethyl methacrylate film covering is heated described in step (7), is obtained
The hydrophobic silicon nano column array modified to polymethyl methacrylate dots structure, the specific steps are as follows:
There is the silicon nano column array of the fluorine modification of polymethyl methacrylate film to be placed in thermal station spin coating, 200~
5~60min is heated under the conditions of 400 DEG C, obtains the hydrophobic silicon nano column array of polymethyl methacrylate dots structure modification.
The hydrophobic silicon nano column array absorption modified described in step (8) with polymethyl methacrylate dots structure is to be measured
Molecule, the specific steps are as follows:
It is first that the hydrophobic proteins of 1~20mL are (horse heart myoglobin, horseradish peroxidase, bovine serum albumin(BSA), thin
Cytochrome C, lysozyme) or polypeptide (angiotensinⅡ, AGT Ⅲ, bradykinin) testing molecule solution set
In small beaker, then the substrate for the hydrophobic silicon nano column array modified with polymethyl methacrylate dots structure is placed in
Testing molecule Adsorption at surface of solution testing molecule, while solution is stirred, adsorption time is 5~45min.
Substrate molecule solution is added dropwise described in step (9) to the dotted knot of polymethyl methacrylate for being adsorbed with testing molecule
The hydrophobic silicon nano column array of structure modification, the specific steps are as follows:
Draw the substrate molecule (alpha-cyano -4- hydroxycinnamic acid, sinapic acid, 2,5- dihydroxy-benzoic acid) of 0.5~10 μ L
Solution is added drop-wise on the hydrophobic silicon nano column array for the polymethyl methacrylate dots structure modification for being adsorbed with testing molecule.
The sample prepared in step (9) is dried described in step (10), carries out Mass Spectrometer Method, the specific steps are as follows:
Sample made from step (9) is dried in air, and the sample dried is pasted and is put into instrument progress matter on target plate
Spectrum test, the test be Kratos Axima CFRplus spectrometer (Shimadzu Biotech,
Manchester, UK) on carry out, laser is the laser that the wavelength that nitrogen laser issues is 355nm, acceleration voltage 20kV.
Every mass spectrogram is all to add up to obtain after 100~1000 laser irradiations.
In short, the hydrophobic silicon nano column array of this polymethyl methacrylate dots structure modification has following spy
Point: it is not had solution to remain in surface by (1) very strong hydrophobicity after taking out in solution, therefore testing molecule cannot be formed
Coffee ring.Meanwhile no solution residual can remove other chaff interferents (such as some salts), and it is sensitive to can be further improved detection
Degree reduces detection limit；(2) nano column array and polymethyl methacrylate are in being uniformly distributed on column so that testing molecule is equal
It is even to be distributed in substrate surface, and matrix solution is prepared using the acetonitrile of highly volatile and trifluoroacetic acid as solvent, when matrix is added drop-wise to
After substrate, since the evaporation rate of solvent is greater than the speed that substrate molecule is migrated to edge, after solvent volatilization, matrix will not
Coffee ring is formed, but is uniformly distributed with testing molecule.Therefore dredging with the modification of this polymethyl methacrylate dots structure
Water silicon nano column array uses as Mass Spectrometer Method substrate and first adsorbs testing molecule, then the method that substrate molecule is added dropwise is eliminated
Dessert effect obtains ideal detection repeatability, this is one in terms of substance assistant laser desorpted ionized mass spectrometric measurement
A important breakthrough.
Detailed description of the invention
Fig. 1: polymethyl methacrylate dots structure modification hydrophobic silicon nano column array construct and sample preparation is shown
It is intended to；
Fig. 2: there is the SEM picture for the silicon nano column array that the inclination angle of different surfaces modification is 45 °, inserting in all figures
Figure is the partial enlarged view of corresponding picture.Fig. 2A is silicon nano column array；Fig. 2 B silicon fluoride monofilm that has been surface modification
Silicon nano column array；Fig. 2 C is the hydrophobic silicon nano column array of polymethyl methacrylate film covering；Fig. 2 D is poly- methyl
The hydrophobic silicon nano column array of methyl acrylate dots structure modification；
Fig. 3: the water contact angle picture on following different surfaces.Fig. 3 A is silicon nano column array surface；Fig. 3 B is surface
The silicon nano column array surface for thering is silicon fluoride to modify；Fig. 3 C is the hydrophobic silicon nano-pillar battle array of polymethyl methacrylate film covering
List face；Fig. 3 D is the hydrophobic silicon nano column array surface of polymethyl methacrylate dots structure modification；
Fig. 4: using the mass spectrogram for the AGT Ⅲ that the silicon nano column array of fluorine modification is measured as substrate, angiotensins
III molecular concentration is 1pmol/ μ L, molecular weight 931.10；
Fig. 5: the hydrophobic silicon nano column array by the modification of polymethyl methacrylate dots structure and film covering is respectively
Substrate, the optics after taking out in the urea (1mol/L) containing 1pmol/ μ L AGT Ⅲ or salt (1mol/L) solution shine
Piece.1. and 2. in figure A is the substrate for the hydrophobic silicon nano column array that will have polymethyl methacrylate dots structure to modify,
Optics picture when taking out from containing 1mol/L urea and 1mol/L salting liquid respectively, scale is 5mm；4. 3. being will have
Polymethyl methacrylate film covering hydrophobic silicon nano column array substrate, respectively from contain 1mol/L urea and 1mol/L
Optics picture when being taken out in salting liquid；Figure B and figure C is 1. and 2. the sample in figure A places the light after half an hour in air
Learn picture；Figure D and figure E is that in figure A 3. and 4. sample places the optics picture after half an hour in air, and the scale for scheming B-E is
500μm；
Fig. 6: the blood vessel measured using the hydrophobic silicon nano column array that polymethyl methacrylate dots structure is modified as substrate
The mass spectrogram of Angiotensin Converting Enzyme III (1pmol/ μ L), matrix are alpha-cyano -4- hydroxycinnamic acid.Scheme A and schemes the sample of B respectively in fig. 5
1. 2. product are above obtained with sample；
Fig. 7: using the hydrophobic silicon nano column array of polymethyl methacrylate film covering as substrate, the vasotonia that measures
Plain III (1pmol/ μ L) mass spectrogram, matrix are alpha-cyano -4- hydroxycinnamic acid.Scheme A and scheme B respectively sample in fig. 5 3. and
Sample is 4. upper to be obtained；
Fig. 8: using the hydrophobic silicon nano column array of polymethyl methacrylate dots structure modification as substrate, the blood vessel that measures
Angiotensin Converting Enzyme III (10fmol/ μ L) mass spectrogram, matrix are alpha-cyano -4- hydroxycinnamic acid；
Fig. 9: is adsorbed respectively using the hydrophobic silicon nano column array of polymethyl methacrylate dots structure modification as substrate
AGT Ⅲ (1pmol/ μ L) and lysozyme (10pmol/ μ L, molecular weight are 14000 or so), and alpha-cyano -4- is added dropwise
Hydroxy cinnamic acid matrix, optical photograph (figure A and figure C) and corresponding mass spectrogram (figure B and figure D) after drying.Scheme in A and figure C
Illustration be make respectively just from two kinds of solution to be measured take out when optical photograph；Figure B and figure D is in figure A and figure C sample respectively
The mass spectrogram of 30 points of random test on product.Scale is 1mm in Fig. 9 A and Fig. 9 C, and the scale in illustration is 2mm；
Figure 10: using the hydrophobic silicon nano column array of polymethyl methacrylate film covering as substrate, it is tight blood vessel has been adsorbed
It opens III (1pmol/ μ L) of element and alpha-cyano -4- hydroxy cinnamic acid matrix is added dropwise, optical photograph (figure A) and corresponding matter after drying
Spectrogram (figure B).The illustration substrate of A is schemed just from optical photograph when solution taking-up；Scheming B is surveyed at random to the sample in Figure 10 A
The mass spectrogram of 30 points of examination.The scale for scheming A and its illustration is 1mm and 2mm respectively；
As shown in Figure 1, perfluor silane is assembled on silicon nano column array by we first, it is then spin coated onto polymethylacrylic acid
Methyl esters film, and so that polymethyl methacrylate is formed dots structure in nano-pillar top surface by heating.There to be poly- methyl-prop
The hydrophobic silicon nano column array of e pioic acid methyl ester dots structure modification is placed in testing molecule solution surface, adsorbs testing molecule, simultaneously
It is stirred continuously, sample is taken out after a period of time, matrix is added dropwise, dries in air, sample to be tested is prepared.
As shown in Fig. 2, obtained silicon nano column array is uniform Hexagonal packing arrangement, perfluor silane monolayer film is assembled
And spin coating polymethyl methacrylate film does not substantially change structure and morphology, after heating, polymethyl methacrylate exists
The top of nano-pillar forms uniform dots structure.
As shown in figure 3, the silicon nano column array of contact angle display preparation be it is hydrophilic, when assembling silicon fluoride monofilm
Afterwards, contact angle obviously increases, but after the complete polymethyl methacrylate film of spin coating, contact angle becomes smaller, and makes to gather when by heating
When methyl methacrylate film becomes dots structure, state of the contact angle almost back to only fluorine modification.
As shown in figure 4, the mass spectra peak of AGT Ⅲ does not occur, also illustrate that silicon fluoride molecule does not adsorb to be measured point
The effect of son.
As shown in figure 5, since the hydrophobic silicon nano column array of polymethyl methacrylate film covering is without strong hydrophobic work
With, therefore substrate has solution residual after taking out in the solution containing urea and the AGT Ⅲ of sodium chloride, sample dries
Afterwards, there is the crystallization of salt on surface, it will affect the detection of analyte, reduces the signal-to-noise ratio of detection signal.And poly-methyl methacrylate
Ester dots structure modification hydrophobic silicon nano column array it is very hydrophobic, from both salting liquids prepare AGT Ⅲ in it is molten
There is no solution to remain in surface when taking out in liquid, therefore the not crystallization of salt.
As shown in fig. 6, using the hydrophobic silicon nano column array of polymethyl methacrylate dots structure modification as substrate, analysis
When AGT Ⅲ molecule in urea and sodium chloride solution, signal-to-noise ratio is respectively 271.8 and 256.6, this sufficiently proves institute
Substrate has good salt tolerance in detection molecules.
As shown in fig. 7, analyzing urea using the hydrophobic silicon nano column array of polymethyl methacrylate film covering as substrate
When with AGT Ⅲ molecule in sodium chloride solution, signal-to-noise ratio is respectively 9.0 and 6.1, demonstrates poly- methyl-prop again
Superiority of the hydrophobic silicon nano column array of e pioic acid methyl ester dots structure modification as substrate.
As shown in figure 8, using the hydrophobic silicon nano column array of polymethyl methacrylate dots structure modification as substrate, blood vessel
III Molecular Detection of Angiotensin Converting Enzyme limit is very low.
As shown in figure 9, using the hydrophobic silicon nano column array of polymethyl methacrylate dots structure modification as substrate, absorption
After AGT Ⅲ molecule to be measured, when taking out from solution, surface does not have solution residual, to will not have coffee ring raw
At, then dropwise addition acetonitrile and trifluoroacetic acid be solvent prepare matrix solution, due to solvent evaporation rate be greater than molecule to
The speed of edge migration, therefore matrix will not generate coffee ring, to be evenly distributed on substrate surface with determinand molecule, detect
The relative standard deviation of signal has reached 3.8%.When the hydrophobic silicon nano-pillar modified with polymethyl methacrylate dots structure
It when array is that substrate analyzes bacteriolyze enzyme molecule (10pmol/ μ L), remains also without solution, and after dropwise addition matrix, does not also have
Coffee ring is formed, so that determinand molecule and matrix be made all to be evenly distributed, the relative standard deviation for detecting signal reaches
5.7%.
As shown in Figure 10, using the hydrophobic silicon nano column array of polymethyl methacrylate film covering as substrate, from blood vessel
When taking out in III molecular solution of Angiotensin Converting Enzyme, surface solution residual dries and generates coffee ring, although the matrix being added dropwise will not give birth to
At coffee ring.But since determinand molecular distribution is uneven, the relative standard deviation of detection signal has reached 26.0%.
Specific embodiment
Carry out the present invention is furture elucidated method and its application in Mass Spectrometer Method below by embodiment, rather than uses
These embodiments limit the present invention.The purpose of the present invention is dredging using a kind of modification of polymethyl methacrylate dots structure
Water silicon nano column array is uniformly distributed testing molecule and matrix, so that dessert effect is removed, on guaranteeing highly sensitive premise,
The reproducibility for improving Mass Spectrometer Method, has a good application prospect.
Embodiment 1
Silicon wafer is first cut into 10, wherein 9 sizes are 1.5cm × 1.5cm, a piece of size is 4cm × 6cm, so
Successively respectively it is cleaned by ultrasonic 5min at 70W with acetone, chloroform, ethyl alcohol, deionized water afterwards.Prepare NH3·H2O:H2O2: H2O=1:
The mixed solution of 1:5 (volume ratio) is heated to 90 DEG C, then cleaned two silicon wafers is immersed in the solution and boil 40min.
Finally rinsed and used with deionized water and is dried with nitrogen, respectively obtain mention ball silicon wafer (1.5cm × 1.5cm) and vollyball silicon wafer (4cm ×
6cm)。
It is with the mass fraction that dimethylbenzene is prepared with the speed spin coating of 7000rpm on mentioning ball silicon wafer with desk-top spin coating instrument
7.5% polymethyl methacrylate (relative molecular mass 97000) solution, the thickness of polymethyl methacrylate is 220nm.
The Polystyrene powder 0.1g that diameter is 600nm is added to 2mL, the deionized water of volume ratio 1:1 and ethyl alcohol
In mixed solution, ultrasonic 6h makes its mixing, obtains the ethyl alcohol and deionized water mixed solution of polystyrene microsphere.In clean table
Deionized water is added in the ware of face, and the 60 μ L of aqueous solution for the Surfactant SDS that mass fraction is 2% is added；
Then the ethyl alcohol of polystyrene microsphere and deionized water mixed solution are slowly drip onto volleyball wafers with microsyringe, and
It is slid into water along vollyball silicon wafer, forms it into the closelypacked polystyrene microsphere monofilm of six sides, add 10 μ L 12
Alkylsurfuric acid sodium water solution stablizes monofilm；Then ball silicon wafer substrate is proposed by polyphenyl second with polymethyl methacrylate
Alkene microballoon lifts, and slant setting is until moisture volatilizees completely.Surface is mentioned into ball silicon wafer with polystyrene microsphere monofilm
7.29 × 10-5Start to etch under Pa vacuum degree, the parameter of etching are as follows: O2Flow is 50sccm, chamber pressure 30mtorr,
Radio-frequency power is 30W, and inductively coupled plasma body power is 100W, etch period 2.5min.
Then by surface with partial size be reduced to 480nm polystyrene microsphere silicon wafer 7.29 × 10-5Pa vacuum degree
Under start to etch, the parameter of etching are as follows: SF6Flow is 6sccm, CHF3Flow is 45sccm, chamber pressure 8mtorr, radio frequency
Power is 25W, and inductively coupled plasma body power is 100W, etch period 8min.
The structure silicon wafer of preparation is immersed in toluene to wash away remaining polystyrene microsphere, then successively with acetone, chloroform,
Ethyl alcohol, distilled water each ultrasound 5min at 70W, it is highly the orderly silicon nano column array of 630nm that obtaining diameter, which is 380nm,.
Prepare NH3·H2O:H2O2: H2The mixed solution of O=1:1:5 (volume ratio), is heated to 90 DEG C, will clean
Orderly silicon nano column array immerse in the solution to boil 40min and finally rinsed and used with deionized water and be dried with nitrogen.By above-mentioned place
It manages obtained orderly silicon nano column array to be placed in glass culture dish, the perfluor silane of 5 μ L is added dropwise into glass culture dish,
4h is heated at 250 DEG C, is then successively cleaned by ultrasonic 5min at 70W with acetone, chloroform, ethyl alcohol, deionized water, thus by certainly
The form for assembling perfluor disilane monolayer film carries out fluorine modification to orderly silicon nano column array.
The mass fraction that the silicon nano column array of obtained fluorine modification is prepared with the speed spin coating dimethylbenzene of 7000rpm
1% polymethyl methacrylate solution (relative molecular mass 350000) obtains polymethyl methacrylate film covering
Hydrophobic silicon nano column array, wherein the polymethyl methacrylate film thickness of top end covering is 3nm, inside the groove of bottom end
The polymethyl methacrylate film thickness of covering is 45nm.The hydrophobic silicon of obtained polymethyl methacrylate film covering
Nano column array, which is placed in the thermal station for be set as 300 DEG C, heats 30min, just obtains polymethyl methacrylate dots structure and repairs
The hydrophobic silicon nano column array of decorations.
Embodiment 2
Silicon nano column array, the silicon nano column array of fluorine modification, poly- methyl-prop obtained respectively to 1 different step of embodiment
The hydrophobic silicon nano-pillar that hydrophobic silicon nano column array, the polymethyl methacrylate dots structure of e pioic acid methyl ester film covering are modified
Array is added dropwise 5 μ L water and tests contact angle.
Embodiment 3
By the silicon nano column array of fluorine modification be inverted in AGT Ⅲ (1pmol/ μ L, 3mL, solvent is deionized water,
Container is small beaker) solution surface to adsorb testing molecule adsorbs 30min under conditions of being stirred continuously, take out above structure
1 μ L alpha-cyano -4- hydroxy cinnamic acid matrix is added dropwise in substrate, and the sample dried is pasted and is put into instrument progress mass spectrum on target plate
Test, the test be Kratos Axima CFRplus spectrometer (Shimadzu Biotech, Manchester,
UK it is carried out on), laser is the laser that the wavelength that nitrogen laser issues is 355nm, acceleration voltage 20kV.Every mass spectrogram is all
It is to add up to obtain after 500 laser irradiations.
Embodiment 4
In order to detect this substrate in substance assistant laser desorpted ionized test from desalination performance, made with urea molecule
For chaff interferent, the hydrophobic silicon nano column array substrate that polymethyl methacrylate dots structure is modified is inverted in angiotensins
III (1pmol/ μ L, 3mL, solvent is the urea liquid of 1mol/L, and container is small beaker) solution surface, to adsorb testing molecule,
30min is adsorbed under conditions of being stirred continuously, and the alpha-cyano -4- hydroxy cinnamic acid matrix of 1 μ L, the sample that will be dried are added dropwise after taking-up
Product, which paste, is put into instrument progress mass spectrometric measurement on target plate, which is in Kratos Axima CFRplus
It is carried out on spectrometer (Shimadzu Biotech, Manchester, UK), laser is the wavelength that nitrogen laser issues
For the laser of 355nm, acceleration voltage 20kV.Every mass spectrogram is all to add up to obtain after 500 laser irradiations.
Embodiment 5
In order to detect this substrate in substance assistant laser desorpted ionized test from desalination performance, with chlorination sodium molecule
As chaff interferent, the hydrophobic silicon nano column array substrate that polymethyl methacrylate dots structure is modified is inverted in vasotonia
Element III (1pmol/ μ L, 3mL, solvent is 1mol/L sodium chloride solution, and container is small beaker) solution surface, to adsorb to be measured point
Son adsorbs 30min under conditions of being stirred continuously, and the alpha-cyano -4- hydroxy cinnamic acid matrix of 1 μ L is added dropwise after taking-up, will dry
Sample paste be put on target plate instrument carry out mass spectrometric measurement, which is in Kratos Axima CFRplus
It is carried out on spectrometer (Shimadzu Biotech, Manchester, UK), laser is the wavelength that nitrogen laser issues
For the laser of 355nm, acceleration voltage 20kV.Every mass spectrogram is all to add up to obtain after 500 laser irradiations.
Embodiment 6
In order to this substrate of contrast verification in substance assistant laser desorpted ionized test from the superiority of desalination, with urea
Molecule is inverted in vasotonia as chaff interferent, by the hydrophobic silicon nano column array substrate that polymethyl methacrylate film covers
Element III (1pmol/ μ L, 3mL, solvent is the urea liquid of 1mol/L, and container is small beaker) solution surface, to adsorb to be measured point
Son adsorbs 30min under conditions of being stirred continuously, and the alpha-cyano -4- hydroxy cinnamic acid matrix of 1 μ L is added dropwise after taking-up, will dry
Sample paste be put on target plate instrument carry out mass spectrometric measurement, which is in Kratos Axima CFRplus
It is carried out on spectrometer (Shimadzu Biotech, Manchester, UK), laser is the wavelength that nitrogen laser issues
For the laser of 355nm, acceleration voltage 20kV.Every mass spectrogram is all to add up to obtain after 500 laser irradiations.
Embodiment 7
In order to this substrate of contrast verification in substance assistant laser desorpted ionized test from the superiority of desalination, with chlorination
It is tight that sodium molecule as chaff interferent, by the hydrophobic silicon nano column array substrate that polymethyl methacrylate film covers is inverted in blood vessel
Element III (1pmol/ μ L, 3mL, solvent is the sodium chloride solution of 1mol/L, and container is small beaker) solution surface is opened, it is to be measured to adsorb
Molecule adsorbs 30min under conditions of being stirred continuously, and the alpha-cyano -4- hydroxy cinnamic acid matrix of 1 μ L is added dropwise after taking-up, will dry in the air
Dry sample, which pastes, is put into instrument progress mass spectrometric measurement on target plate, which is in Kratos Axima CFRplus
It is carried out on spectrometer (Shimadzu Biotech, Manchester, UK), laser is the wavelength that nitrogen laser issues
For the laser of 355nm, acceleration voltage 20kV.Every mass spectrogram is all to add up to obtain after 500 laser irradiations.
Embodiment 8
The hydrophobic silicon nano column array that polymethyl methacrylate dots structure is modified is inverted in AGT Ⅲ
(10fmol/ μ L, 3mL, solvent is deionized water, and container is small beaker) solution surface is being stirred continuously with adsorbing testing molecule
Under conditions of adsorb 30min, take out above structure substrate, 1 μ L alpha-cyano -4- hydroxy cinnamic acid matrix, the sample that will be dried be added dropwise
Product, which paste, is put into instrument progress mass spectrometric measurement on target plate, which is in Kratos Axima CFRplus
It is carried out on spectrometer (Shimadzu Biotech, Manchester, UK), laser is the wavelength that nitrogen laser issues
For the laser of 355nm, acceleration voltage 20kV.Every mass spectrogram is all to add up to obtain after 500 laser irradiations.
Embodiment 9
In order to detect by eliminating dessert effect in substance assistant laser desorpted ionized test using this substrate, to mention
The feasibility of high sample detection repeatability, using AGT Ⅲ as testing molecule, by the dotted knot of polymethyl methacrylate
The hydrophobic silicon nano column array substrate of structure modification is inverted in AGT Ⅲ solution, and (1pmol/ μ L, 3mL, solvent is deionization
Water, container are small beaker) surface to adsorb testing molecule adsorbs 30min under conditions of being stirred continuously, 1 μ L is added dropwise after taking-up
Alpha-cyano -4- hydroxy cinnamic acid matrix, the sample dried is pasted be put on target plate instrument carry out mass spectrometric measurement, the test
It is to be carried out on Kratos Axima CFRplus spectrometer (Shimadzu Biotech, Manchester, UK)
, laser is the laser that the wavelength that nitrogen laser issues is 355nm, acceleration voltage 20kV.Every mass spectrogram is all by 500
It adds up after secondary laser irradiation and obtains.
Embodiment 10
Dessert effect in substance assistant laser desorpted ionized test is eliminated in order to detect this substrate, to improve sample
The feasibility for detecting repeatability is dredged using lysozyme as determinand molecule by what polymethyl methacrylate dots structure was modified
Water silicon nano column array bottom is inverted in lysozyme soln (10pmol/ μ L, 3mL, solvent is deionized water, and container is small beaker) table
30min is adsorbed to adsorb testing molecule in face under conditions of being stirred continuously, and the alpha-cyano -4- hydroxyl meat of 1 μ L is added dropwise after taking-up
The sample dried is pasted and is put into instrument progress mass spectrometric measurement on target plate by cinnamic acid matrix, which is in Kratos Axima
It is carried out on CFRplus spectrometer (Shimadzu Biotech, Manchester, UK), laser is nitrogen laser hair
Wavelength out is the laser of 355nm, acceleration voltage 20kV.Every mass spectrogram is added up after 500 laser irradiations
It arrives.
Embodiment 11
The dessert effect in substance assistant laser desorpted ionized test is being eliminated in order to verify this substrate, to improve sample
The superiority of product examine check weighing renaturation, using AGT Ⅲ as testing molecule, by polymethyl methacrylate film covering
Hydrophobic silicon nano column array substrate is inverted in AGT Ⅲ solution, and (1pmol/ μ L, 3mL, solvent is deionized water, and container is
Small beaker) surface to adsorb testing molecule adsorbs 30min under conditions of being stirred continuously, the alpha-cyano-of 1 μ L is added dropwise after taking-up
4- hydroxy cinnamic acid matrix, the sample dried is pasted be put on target plate instrument carry out mass spectrometric measurement, the test be
It carries out, swashs on Kratos Axima CFRplus spectrometer (Shimadzu Biotech, Manchester, UK)
Light is the laser that the wavelength that nitrogen laser issues is 355nm, acceleration voltage 20kV.Every mass spectrogram is swashed by 500 times
It adds up and obtains after light irradiation.
Claims (10)
1. a kind of method for improving Mass Spectrometer Method repeatability by eliminating dessert effect, its step are as follows:
(1) the spin coating polymethyl methacrylate film on proposing ball silicon wafer substrate；
(2) ball silicon wafer substrate over-assemble polystyrene microsphere is mentioned in covering polymethyl methacrylate film；
(3) reactive ion etching technology is utilized, silicon is performed etching using polystyrene microsphere as exposure mask；
(4) remaining polystyrene microsphere is removed using organic solvent, to obtain orderly silicon nano column array；
(5) it is surface modified, is dredged by assembling silicon fluoride monofilm on obtained orderly silicon nano column array
The orderly silicon nano column array of water；
(6) the spin coating polymethyl methacrylate film on the hydrophobic orderly silicon nano column array of fluorine modification；
(7) the hydrophobic silicon nano column array of polymethyl methacrylate film covering is heated, obtains polymethylacrylic acid
The hydrophobic silicon nano column array of methyl esters dots structure modification；
(8) testing molecule is adsorbed with the hydrophobic silicon nano column array that polymethyl methacrylate dots structure is modified；
(9) hydrophobic silicon that substrate molecule solution is modified to the polymethyl methacrylate dots structure for being adsorbed with testing molecule is added dropwise
Nano column array；
(10) sample prepared in step (9) is dried, Mass Spectrometer Method is carried out.
2. a kind of method for improving Mass Spectrometer Method repeatability by eliminating dessert effect as described in claim 1, feature exist
In: described in step (1) on proposing ball silicon wafer substrate spin coating polymethyl methacrylate film, include the following steps,
1. cleaning silicon chip: successively using acetone, chloroform, ethyl alcohol and deionized water to be cleaned by ultrasonic 3 at 40~100W multi-disc silicon wafer
~10min, then use NH3·H2O:H2O2: H2O volume ratio is that 1~3:1~3:5~7 mixed solution heats at 80~100 DEG C
50~70min is cleaned, is finally rinsed and is used with deionized water and be dried with nitrogen, respectively obtains and mentions ball silicon wafer and vollyball silicon wafer；
2. spin coating polymethyl methacrylate: by the mass fraction prepared with dimethylbenzene being 5%~7.5%, molecular weight 97000
~350000 polymethyl methacrylate solution is added drop-wise to that step is 1. cleaned to be mentioned on ball silicon wafer, with 2000rpm~
The speed of 7000rpm carries out spin coating, and obtain covering polymethyl methacrylate film mentions ball silicon wafer substrate, polymethyl
The thickness range of sour methyl esters is 100~300nm.
3. a kind of method for improving Mass Spectrometer Method repeatability by eliminating dessert effect as described in claim 1, feature exist
In: ball silicon wafer substrate over-assemble polystyrene microsphere, packet are mentioned in covering polymethyl methacrylate film described in step (2)
Include following steps,
1. preparing the ethyl alcohol for the polystyrene microsphere that diameter is 100~4000nm and the mixed solution of deionized water: by diameter 100
The mixing that 0.1~1g of Polystyrene powder of~4000nm is added to 1~20mL, the deionized water of volume ratio 1:1 and ethyl alcohol is molten
In liquid, 1~6h of ultrasound makes its mixing；
2. the assembling and transfer of polystyrene microsphere monofilm: deionized water being added in a clean watch glasses, and is added 20~100
The Surfactant SDS aqueous solution that μ L mass fraction is 0.5%~2%；Then with microsyringe by step
1. the ethyl alcohol and deionized water mixed solution of obtained polystyrene microsphere are slowly drip onto volleyball wafers, and along vollyball silicon
Piece slides into water, forms it into the closelypacked polystyrene microsphere monofilm of six sides, adds 5~25 above-mentioned dodecyls of μ L
Aqueous sodium persulfate solution stablizes monofilm；Then ball silicon wafer substrate is proposed by polyphenyl second with polymethyl methacrylate film
Alkene microballoon lifts, and slant setting is until moisture volatilizees completely.
4. a kind of method for improving Mass Spectrometer Method repeatability by eliminating dessert effect as described in claim 1, feature exist
In: reactive ion etching technology is utilized described in step (3), and silicon is performed etching using polystyrene microsphere as exposure mask, including
Following steps,
1. reducing the partial size of polystyrene microsphere: surface is mentioned ball silicon wafer 5 × 10 with polystyrene microsphere monofilm-5~
8×10-5Start reactive ion etching, the parameter of etching are as follows: O under Pa vacuum degree2Flow is 10~50sccm, chamber pressure 10
~50mtorr, radio-frequency power are 15~200W, and inductively coupled plasma body power is 50~150W；Etch period be 1~
15min, the partial size of polystyrene microsphere is reduced to 80~3500nm after etching,
2. the etching of silicon: mentioning ball silicon wafer 5 × 10 for reduce polystyrene microsphere partial size-5~8 × 10-5It is opened under Pa vacuum degree
Beginning reactive ion etching, the parameter of etching are as follows: SF6Flow is 5~15sccm, CHF3Flow is 30~60sccm, and chamber pressure is
10~50mtorr, radio-frequency power be 15~50W, inductively coupled plasma body power be 50~150W, etch period be 1~
9min。
5. a kind of method for improving Mass Spectrometer Method repeatability by eliminating dessert effect as described in claim 1, feature exist
In: remaining polystyrene microsphere is removed using organic solvent described in step (4), so that orderly silicon nano column array is obtained,
Be step (3) has been etched mention ball silicon wafer be successively placed in acetone, chloroform, ethyl alcohol and deionized water it is super with 40~100W power
Sound cleans 3~10min, and until removing remaining polystyrene microsphere, to obtain orderly silicon nano column array, silicon is received
The diameter of meter Zhu is 50~3000nm, and altitude range is 400~800nm.
6. a kind of method for improving Mass Spectrometer Method repeatability by eliminating dessert effect as described in claim 1, feature exist
In: surface is carried out by assembling silicon fluoride monofilm on obtained orderly silicon nano column array described in step (5) and is repaired
Decorations, obtain hydrophobic orderly silicon nano column array, include the following steps,
1. hydroxylating is handled: the orderly silicon nano column array NH that will be obtained3·H2O:H2O2: H2O volume ratio is 1~3:1~3:5
~7 mixed solution heats 50~70min of cleaning at 80~100 DEG C, is finally rinsed and is used with deionized water and is dried with nitrogen；
2. fluorine modification: the orderly silicon nano column array that hydroxylating is handled is placed in glass culture dish, and it is complete that 2~10 μ L are added dropwise
Silicon fluoride heats 0.5~6h into glass culture dish at 60~300 DEG C, then successively uses acetone, chloroform, ethyl alcohol, deionization
Water is cleaned by ultrasonic 3~10min at 40~100W, to pass through self assembly perfluor disilane monolayer on orderly silicon nano column array
The form of film carries out fluorine modification.
7. a kind of method for improving Mass Spectrometer Method repeatability by eliminating dessert effect as described in claim 1, feature exist
In: described in step (6) on the hydrophobic orderly silicon nano column array of fluorine modification spin coating polymethyl methacrylate film, be by
The polymethyl methacrylate solution for being 97000~350000 with the molecular weight that the mass fraction that dimethylbenzene is prepared is 1%~5%
It is added drop-wise on the silicon nano column array of fluorine modification, spin speed is 1000~7000rpm, and obtaining spin coating has poly-methyl methacrylate
The orderly silicon nano column array of the fluorine modification of ester film, the thickness range of polymethyl methacrylate film is 2~200nm.
8. a kind of method for improving Mass Spectrometer Method repeatability by eliminating dessert effect as described in claim 1, feature exist
In: the hydrophobic silicon nano column array of polymethyl methacrylate film covering is heated to described in step (7), is gathered
The hydrophobic silicon nano column array of methyl methacrylate dots structure modification, is that spin coating is had polymethyl methacrylate film
The silicon nano column array of fluorine modification is placed in thermal station, and 5~60min is heated under the conditions of 200~400 DEG C, obtains polymethyl
The hydrophobic silicon nano column array of sour methyl esters dots structure modification.
9. a kind of method for improving Mass Spectrometer Method repeatability by eliminating dessert effect as described in claim 1, feature exist
In: testing molecule is adsorbed with the hydrophobic silicon nano column array that polymethyl methacrylate dots structure is modified described in step (8),
It is the small beaker that preparation fills the hydrophobic proteins of 1~20mL or the determinand molecular solution of polypeptide, by the poly- methyl of preparation
The hydrophobic silicon nano column array of methyl acrylate dots structure modification tips upside down in the small beaker for filling determinand molecular solution, stirs
5~45min is adsorbed under the conditions of mixing；The hydrophobic proteins are horse heart myoglobin, horseradish peroxidase, bovine serum albumin
White, cromoci or lysozyme, the polypeptide are angiotensinⅡ, AGT Ⅲ or bradykinin.
10. a kind of method for improving Mass Spectrometer Method repeatability by eliminating dessert effect as described in claim 1, feature exist
In: dropwise addition substrate molecule solution described in step (9) is repaired to the polymethyl methacrylate dots structure for being adsorbed with testing molecule
The hydrophobic silicon nano column array of decorations is that the substrate molecule solution of 0.5~10 μ L of absorption is added drop-wise to and is adsorbed with the poly- of determinand molecule
On the hydrophobic silicon nano column array of methyl methacrylate dots structure modification；Sample is dried described in step (10), carries out matter
Spectrum detection, is to dry sample made from step (9) in air, and the sample dried is pasted and is put into instrument progress matter on target plate
Spectrum test；The substrate molecule is alpha-cyano -4- hydroxycinnamic acid, sinapic acid or 2,5- dihydroxy-benzoic acid.
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