CN108051503A - Improve the method and product of Mass Spectrometer Method microorganism crystallization - Google Patents

Abstract

The present invention provides a kind of methods by improving micro-biological samples secondary crystallization, it is included in acetonitrile solution and is separately added into 3 hydroxyl, 2 pyridine carboxylic acid, citric acid diamine solution to prepare matrix solution, the sample solution for then adding in special ratios carries out point sample crystallization on chip.The present invention also provides the mass spectrum bearing calibrations for the accuracy rate for improving Mass Spectrometer Method bio-target molecule, it is included on the chip of the hydrophilic region location hole with multiple vertical cross arrangements, weep hole exterior domain, cent(e)ring hole, verification correction hole, spare correction hole, point sample matrix solution crystallization respectively, point sample micro-biological samples simultaneously crystallize, and the micro-biological samples of correction hole position are bombarded by mass spectrum, it is corrected with the mass spectral results to sample to be tested.The present invention also provides the chips for being applicable in above-mentioned mass spectrum correction and detection.The present invention improves matrix solution formula, can obtain the preferable primary crystallization of crystal habit and secondary crystallization effect, and bearing calibration helps to obtain and more stablize and accurate Mass Spectrometer Method result.

Description

Improve the method and product of Mass Spectrometer Method microorganism crystallization

Technical field

The present invention relates to one kind MALDI-TOF Mass Spectrometer Methods are being improved by improving biological sample crystallization and correcting mode just The method of true rate, energy Mass Spectrometer Method microorganism, belongs to mass spectrum detection field.

Background technology

Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF MS) technology has become current protein group Learn the classical technology in research.During the successful application of the technology, suitable sample-pretreating method is played primary and closed The effect of key.Only suitable sample-pretreating method with suitable organic substrate is combined, could successfully be realized to core The precise Identification of acid and protein and other.Choosing for matrix, solvent, salt (metal ion) and sample preparation methods Select be MALDI analysis high polymer success or failure key factor.The condition of optimization is that sample molecule and matrix is made to be formed uniformly altogether Crystallization.MALDI methods analysis one of macromolecular, important key is the suitable matrix of selection.The result shows that effect is preferable Matrix only has several.Water solubility synthesis macromolecule such as polyethylene glycol (PEG), polypropylene glycol often arise in the MALDI of early stage and grind In studying carefully, this is because the matrix of analysis polypeptide can also be applied to them, for other macromolecule, can from macromolecule and The thinking of some selection matrix is found in the dissolubility of matrix.In general, select that during matrix matrix and high molecular pole should be made Property than more consistent, each other have compatibility.

Micro-array chip is characterized by high density arrays.Microarray technology is exactly using molecule Hybridization principle, is made while quilt The sample and microarray hybridization compared, by detecting hybridization signal intensities and data processing, them is changed into different specimens The abundance of specific gene, so as to compare the difference of the gene expression dose of different specimens comprehensively.Micro-array chip is equal because of its height One property, structural stability, amount of samples is few and high-throughput and becomes in chip field and develops a most rapid part.Microarray is not Only there is extensive purposes in biological heredity field, also have potential use in terms of other quantitative and relative quantitative assay On the way.

For micro-array chip because its pore size is identical, micropore and surrounding hydrophilic and hydrophobic qualitative difference preferably limit sample Scope residing for product so that the region analyzed is consistent, and makes it possible quantitative analysis.Mass spectrum is into figure because it is without mark Note without separation, by carrying out the quantitative analysis of component in mixture to the analysis of image, makes multi-component while examines Survey is possibly realized, therefore carries out multi-component Simultaneous Quantitative Analysis into figure using mass spectrum in micro-array chip, is that one kind has extensively The quantitative analysis tech of general application prospect.Application of the mass spectrum into figure in quantitative analysis is largely depended in micro-array chip In the homogeneity of sample crystallization, the quality in being gathered due to mass spectrometric data is discriminated against, and the peak height or peak area of mass spectrogram can not conducts The foundation of the quantitative analysis of sample, therefore how to obtain reliably, stable quantitative data is just particularly important.

In application MALDI-TOF MS, usually by biological sample and a kind of low molecular weight inorganic compound solution of saturation (being known as matrix) carries out mixing and is added on target plate, and sample after matrix cocrystallization with foring with the sample of matrix package framework after dry This solid precipitates.For sample matrix crystalline solid through laser emission, matrix absorbs energy, energy accumulation and rapid heat production from laser, So that host crystal distils, make sample adsorption, electric charge transfer occurs between matrix and sample and causes ionized sample molecule, ion Identical kinetic energy is obtained under accelerating field, time-of-flight detector is entered after high pressure accelerates, focuses on.During according to ion flight Between difference carry out analysis and draw ion mass-to-charge ratio (m/z) and ion peak value, form quality collection of illustrative plates, detection accuracy height.Pass through Software analysis is compared, and is screened and is determined specific finger-print, so as to fulfill the differentiation to objective microbe kind or bacterial strain and Identification.MALDI-TOF MS can be used for analyzing polytype sample, including organic molecular solution, nucleic acid, protein and whole A microorganism, wherein gene, protein and microorganism are at present in the most widely used project in mass spectrometry clinical testing laboratory.

The ionizable relative molecular mass of substance assistant laser desorpted ionized mass spectrum (MALDI-TOF MS) ion source is 100 ~1,000 000 biomolecule is acted on by high-voltage electricity and short laser pulse, allows host crystal literizations, divides matrix and sample Son gasification enters mass spectrometric gas phase, it is most prominent the characteristics of be quasi-molecular ionization it is very strong, to the dosis tolerata of impurity in sample Greatly, the mixture that Direct Analysis nucleic acid, protein are generated through ionization, can provide effectively for clinical examination Plays object Quality Research Technical guarantee, be the reference method of clinical examination first choice.Therefore the sample crystal habit on chip and quality are direct Influence mass spectrographic verification result.

In the sample crystal habit and matter quantifier elimination for improving chip, existing research emphasis is how to improve chip Quality.For example, Chinese granted patent 201410090967.3, " preparing the alternate micro-array chip of hydrophobe and its for matter The method of spectrum imaging quantitative analysis " provides one kind by the way that hydrophobic and hydrophilic region is set to prepare detection chip on chip, Wherein using screen printing technique by hydrophobic polymer (dimethyl silicone polymer or polymethyl methacrylate) according to design Good template brush is on electro-conductive glass, and wherein applying area is hydrophobic region, and clear area is as reserved hydrophilic area.Then, use is hydrophilic Material (such as aqueous solution of sample system) is coated with clear area, forms hydrophilic area and the hydrophobic region at homogeneous interval.This method passes through profit With screen printing technique come design template shape, the area that leaves some space in advance in hydrophilic area (also known as " being left white processing "), it is therefore desirable to Special printing equipment and operation software, while target plate will definitely be stood when being coated with hydrophobic region.Meanwhile screen printing technique Precision is low, and the boundary of close and distant aqua region cannot reach micron accuracy.Furthermore, it is desirable to by testing mixture system aqueous solution equably Hydrophilic area is layered on, chip baking and curing is placed in 60 degrees Celsius of curing 2h in baking oven, adds production time and cost.

Chinese granted patent 201110401165.6, " be enriched with to biological sample and the method for desalination purification processing " are public A kind of method that the detection target plate with closed pattern surface is prepared using hydrophobic and water wetted material is opened, including by base It is constructed on bottom and is used as barrier with larger-diameter polymer coating (such as polymethyl methacrylate, polystyrene or photoresist) Circle area, is then attached to as hydrophobic layer in entire substrate using fluorine-containing monolayer or evaporated metal layer.Since the hydrophobic layer cannot Above-mentioned barrier circle area is adhered to, therefore target plate is immersed in organic solvent and is ultrasonically treated, barrier circle area can be removed.Most Afterwards, then by the polymer coating round area's internal modifications small diameter concentric circles, it is (fluorine-containing so as to obtain hydrophobic outer ring Monolayer or evaporated metal layer)-hydrophilic centre circle-hydrophobic inner ring (polymer coating) concentric circles mass spectrum target plate, because This this method is also known as " barrier coating ".However, this method constructs the closed pattern of hydrophobe-hydrophile-hydrophobic region in substrate Surface, it is necessary to carry out two step hydrophobic treatments, i.e., using fluorine-containing reagent at 100~250 DEG C 1~5 hour of heat growth, work Skill is complicated, and process takes.Since this method needs the spacing of accurate control polymer coating twice, spacing is too small cause outer ring and Inner ring is connected, simultaneously because being mutated different substrate surfaces respectively using two kinds of different hydrophobic materials, what is obtained is hydrophobic The contact angle of area's drop is too small, causes hydrophobic effect poor, affects the application of common lab.

Chinese patent application 200610023671.5 discloses " a kind of low-abundance protein target previous step desalination and enrichment Method ", wherein by hydrophobic polymer (polymethyl methacrylate, polyethylene, polystyrene, polyvinyl fluoride etc.) in advance in target The sample cell middle body of plate is coated, and then carries out albumen point sample so that protein sample is enriched on hydrophobic polymer layer. Then, matrix solution is added in into point sample area so that pollutant and inorganic salts in protein sample are spread out, and finally obtain sample With the crystallization of matrix uniform and delicate.Since this method is that protein sample directly is added to hydrophobic layer, by adding in excessive matrix Solution is purified, although objectively there is certain purification effect, causes the waste of protein sample.Meanwhile if manually Operation in the aperture of each Kapton films, takes 0.2 μ l hydrophobic polymers solution points, and the precision of 0.2 μ l solution is difficult With control, the hydrophobic homogeneity of orifice surface is influenced；If be automatically brought into operation, it is necessary to configure corresponding point sample equipment, prepared by increase cost Complexity is not appropriate for being not easy the detection and application of the trace protein sample prepared.

Due in the first research of above-mentioned target plate or target plate surface does not have hydrophilic-hydrophobic difference, (such as Chinese patent 200610023671.5), cause crystal habit poor or form the coating of hydrophilic-hydrophobic difference, but hydrophobe boundary cannot reach It is too small to micron accuracy or liquid-drop contact angle, but preparation process is excessively complicated, it is impossible to and save time and cost (such as Chinese patent 201410090967.3rd, patent 201110401165.6) or need additional device and inspection software or need excessive Precious albumen sample be detected, the accuracy that these result in Mass Spectrometer Method sample peak is low, and signal-to-noise ratio is low, and baseline is high.

The immediate Chinese patent that in the prior art, inventor submits before formerly (CN201710539756, for flying Row time mass spectrum detects the preparation method of the general-purpose chip of albumen and nucleic acid；CN201710539893, for flight time mass spectrum Detect the general-purpose chip of albumen and nucleic acid) in, it is proposed that improve sample crystal habit and matter by preparing new mass spectrum chip Amount, the chip mainly include the hydrophily loading wells and hydrophobic pores outskirt that body surfaces have the arrangement of micro- permutation, hydrophilic points Sample hole covers the hydrophilic film of 150-800nm, and hydrophobic pores outskirt covers 150nm-2 μm of hydrophobic film, surface water droplet 120 ° of contact angle ＞, wherein the hydrophilic film is special silica oxides film, zinc-oxide film, aluminum oxide film Film etc., hydrophobic pores outskirt carry out processing by silane coupling agent and form hydrophobic film.The invention to a certain extent can Improve the crystalline quality of sample.However, the research emphasis of the invention essentially consists in the mass spectrum chip for providing a kind of dual-purpose type, joint The adaptor chip of special designing is designed by card slot different on adapter, can place multiple chips, can realize nucleic acid and egg Bai Butong samples while sample detection save mass spectrum enabling lockup.Since the research emphasis of the invention has not focused on pin To in the Mass Spectrometer Method of single sample, how to improve the crystalline quality of single sample, thus the present inventor still need as It is studied on basis.

In view of MALDI-TOF MS can be used for analyzing polytype sample, including organic molecular solution, nucleic acid and whole In the most widely used project in mass spectrometry clinical testing laboratory, therefore a microorganism, wherein gene, protein and microorganism are at present , it is necessary to which one kind improves MALDI-TOF Mass Spectrometer Method biological targets by improving sample crystallization condition on existing chip basis The method of molecule accuracy rate.

The content of the invention

One of principle of the invention is, MALDI-TOF Mass Spectrometer Method biological targets are improved by improving sample crystallization condition The method of molecule accuracy rate, this method are not directed to how to improve existing mass spectrum chip, but by groping and optimizing mass spectrum core The crystallization condition of piece, to improve the level of crystallization to bio-target molecule on chip, so as to make full use of existing mass spectrum Chip, to reduce testing cost.

The two of the principle of the invention are, for microbial target molecule on chip crystallographic property, a kind of biological target point is provided The generic crystallization method of son.

The three of the principle of the invention are, on the basis of sample crystallization condition is improved, it is further provided a kind of sample mass spectrum The bearing calibration of detection, so as to determine precondition to improve MALDI-TOF Mass Spectrometer Method bio-target molecule accuracys rate.

Therefore, first purpose of the invention is to provide a kind of method for improving sample primary crystallization, and step includes：

(1) using the acetonitrile of deionized water and high-efficiency liquid chromatographic-grade according to 1:1-0.5:1 volume ratio mixing, is mixed Close solution；

(2) add in 3- hydroxyl -2- pyridine carboxylic acids fully to dissolve, wherein 3- hydroxyls -2- pyridine carboxylic acids：Mixed solution=1- 10： 100(mg/ml)；

(3) 3- hydroxyls -2- pyridine carboxylic acids solution and citric acid diamine solution are taken, according to 1-3:1 volume ratio is uniformly mixed, mistake It filters up to matrix solution.

(4) 0.5-1 μ L matrix solutions and 0.5-1 μ L samples are chosen, point sample crystallization is carried out on chip.

In one embodiment, the 3- hydroxyl -2- pyridine carboxylic acids of 0.01-0.2mg are added in wherein in step 2, and are led to 2000-3000rpm concussion 3-10min, 8000-12000rpm centrifugation 3-10min are crossed, it is molten to obtain 3- hydroxyl -2- pyridine carboxylic acids Liquid；In a specific embodiment, the 3- hydroxyl -2- pyridine carboxylic acids of 0.05mg are added in.

In another embodiment, citric acid diamine solution wherein in step 3 is formulated as follows：Weigh lemon acid diamine It is placed in centrifuge tube, adds deionized water and dissolved, the wherein weight mg of lemon acid diamine and deionized water volume mL ratios For 1-5：100,3-10min is shaken in 2000-3000rpm, it is molten to obtain lemon acid diamine by 8000-12000rpm centrifugation 3-10min Liquid.In a specific embodiment, filtering is to select the filtering of 0.22um pin types filter.

In other embodiments, 0.75 μ L matrix solutions are wherein chosen in step 4 and 0.5 μ L samples are crystallized.

In any of the above-described embodiment, aforesaid operations be selected from 100,000 grades of cleanliness factors of operation room, 20-25 DEG C of environment temperature, It is carried out under the conditions of ambient humidity 20-30%.

Second purpose of the invention is to provide a kind of mass spectrum core for the accuracy rate that can improve Mass Spectrometer Method bio-target molecule Piece, the chip include：The chip body being made of silicon materials or glass or titanium alloy, surface have multiple vertical cross arrangements Hydrophilic region location hole, weep hole exterior domain, cent(e)ring hole, verification correction hole, spare correction hole；

Wherein, location hole has water-wet behavior, for titrating matrix solution or sample；Weep hole exterior domain, surface have Hydrophobic property；The structure for the close and distant water spacer in micro-array chip surface that location hole and hole exterior domain are formed, can be with auxiliary liquid sample This contraction is condensed upon in hydrophilic region, concentrates sample crystallization, and standard circular is presented, and improves Ionization Efficiency；

Cent(e)ring hole and verification correction hole, for correcting the titration location verification correction hole of microbial standard, for testing Demonstrate,prove calibration result；

Spare correction hole, as spare, cent(e)ring hole or verification correction hole are if there is exception, available backup correction Hole.

In one embodiment, location hole is the vertical crisscross arrangement of (4-8) × (4-8), center on chip Correction hole, verification correction hole and spare correction hole in the chips between position vertical array, quantity is 1 or 2.In a tool In body embodiment, the quantity in cent(e)ring hole, verification correction hole and spare correction hole is 1.

In another embodiment, the hydrophilic film of the chip hydrophily location hole covering 150-800nm, hydrophobicity Hole outskirt covers 150nm-2 μm of hydrophobic film, and 120 ° of surface water droplet contact angle ＞ is in a specific embodiment, described Hydrophilic film is silica oxides film, zinc-oxide film, aluminum oxide film etc., and hydrophobic pores outskirt is even by silane Connection agent carries out processing and forms hydrophobic film.In another embodiment, the silane coupling agent be selected from vinyl silanes, Amino silane or dimethyldichlorosilane.

3rd purpose of the invention is to provide a kind of mass spectrum school for the accuracy rate for improving Mass Spectrometer Method microbial target molecule Correction method, step include：

(1) as described above, preparing matrix solution, and the solution of target molecule sample and calibration samples UEP are configured；

(2) respectively hydrophilic region location hole, cent(e)ring hole and verification correction hole, point 0.5-1 μ L matrix solutions, oneself Primary crystallization is formed after so volatilizing；

(3) in hydrophilic region location hole, on primary crystallization surface, point 0.5-1 μ L bio-target molecule samples, after volatilizing naturally Form secondary crystallization；

(4) respectively cent(e)ring hole, verification correction hole, on primary crystallization surface, point 0.5-1 μ L calibration samples UEP, oneself Secondary crystallization is formed after so volatilizing；

(5) cent(e)ring hole position, the calibration samples UEP of verification correction hole are bombarded respectively by Laser time-flight MS, Obtain the spectrogram of multiple UEP peak molecular weights；

(6) when at least 60% UEP peak molecular weights deviation is less than 700PPM, as correction is effective

(7) after correcting successfully, you can carry out Mass Spectrometer Method to the bio-target molecule sample to be measured of hydrophilic region location hole.

In one embodiment, 0.75 μ L matrix solutions are chosen and 0.5 μ L samples is crystallized.

In another embodiment, the bio-target molecule is micro-biological samples, and the calibration samples UEP is 8739 micro- lifes Object standard solution.

In any of the above-described embodiment, the mass spectrum, parameter setting is as follows：

Turing mode:linear；

Mass Range:2000-20000；

Max Laser Rep Rate:20.0；

Power:65；

Profiles:100；

Shots:5。

In a preferred embodiment, the MALDI-TOF mass spectrums are CLIN-TOF-II flight time mass spectrums.

4th purpose of the invention is to protect the standard that can improve Mass Spectrometer Method bio-target molecule used in the above method The mass spectrum chip of true rate.

Description of the drawings

Fig. 1 is biological sample primary crystallization figure；

Fig. 2 is matrix solution and sample secondary crystallization comparison diagram, wherein+0.5 μ L samples of (a) 0.5 μ L matrix solutions, (b) 1 + 0.5 μ L samples of μ L matrix solutions ,+0.5 μ L samples of (c) 0.75 μ L matrix solutions.

Fig. 3 is improved microarray Mass Spectrometer Method chip surface structure diagram, including：(1) hydrophilic region location hole； (2) weep hole exterior domain；(3) cent(e)ring hole；(4) correction hole is verified；(5) spare correction hole；

Fig. 4 detects mass spectrogram for microorganism UEP standard items；

Fig. 5 is 5 mass spectrograms of micro-biological samples；

Technique effect

1st, nucleic acid Mass Spectrometer Method of the invention is based on microarray nucleic acid detection chip, and micro-array chip surface texture increases Mass spectra peak Mass accuracy is improved, so as to improve the accuracy of nucleic acid Mass Spectrometer Method in correction up hole；

2nd, the present invention proposes a kind of preferred matrix solution formula and cleanliness factor ambient temperature and humidity condition, makes biological sample Primary crystallization form is preferable；

3rd, the present invention proposes that a kind of preferred matrix solution and sample volume match, and makes matrix solution with sample in chip list Evenly, testing result is more preferably for face cocrystallization.

4th, it is proposed by the present invention by improving crystal habit and bearing calibration, choose 20 Clinical microorganism samples, identification Accuracy reaches 100%, particularly with staphylococcus aureus, Klebsiella Pneumoniae, Bacillus cereus, salmonella, excrement Enterococcus, can quickly, it is clear, accurately tell unknown microorganism.

Specific embodiment

The present invention will be further described with reference to the accompanying drawings and embodiments.

The preparation of embodiment one, matrix solution

The matrix solution main component is 3- hydroxyl -2- pyridine carboxylic acids, adds in a certain proportion of acetonitrile, accelerates matrix one Subcrystalline volatilization quickly forms homogeneous intact primary crystallization.

The preparation steps of matrix solution are as follows：

A, using the acetonitrile of deionized water and high-efficiency liquid chromatographic-grade according to V deionized waters：V acetonitrile=1:1-0.5:1 Volume ratio mixes, and obtains mixed solution I；

B, 3- hydroxyl -2- pyridine carboxylic acids are weighed to be placed in centrifuge tube, adds mixed solution I and dissolved, wherein 3- hydroxyls Weight mg and mixed liquor volume the mL ratio of base -2- pyridine carboxylic acids are 1-10：100；

C, 2000-3000rpm shakes 3-10min, and 8000-12000rpm centrifugation 3-10min obtain 3- hydroxyl -2- pyridines Formic acid solution；

D, weigh lemon acid diamine to be placed in centrifuge tube, add deionized water and dissolved, wherein lemon acid diamine Weight mg is 1-5 with deionized water volume mL ratios：100,2000-3000rpm shake 3-10min, 8000-12000rpm from Heart 3-10min obtains citric acid diamine solution；

E, 3- hydroxyls -2- pyridine carboxylic acids solution and citric acid diamine solution are taken, according to V3- hydroxyl -2- pyridine carboxylic acids：V lemons Lemon acid diamine=1-3:1 volume ratio mixes；

F, 3-10min being shaken in 2000-3000rpm, 8000-12000rpm centrifugation 3-10min obtain mixed solution II, Filtering, obtains matrix solution.

Above-mentioned 3- hydroxyls -2- pyridine carboxylic acids specification will can choose SIGMA brands in content more than 99%.

Above-mentioned 3- hydroxyls -2- pyridine carboxylic acid powder 0.01-0.2mg are claimed with electronic balance of the precision more than 0.001g Amount, it is preferable that the present embodiment weighs 3- hydroxyl -2- pyridine carboxylic acids 0.05mg.

Above-mentioned lemon acid diamine powder 0.010-0.020mg is weighed with electronic balance of the precision more than 0.001g, can To choose Chinese medicines group brand, analyze pure (AR), concentration ＞ 99%.

Above-mentioned deionized water can choose thermo Fisher brands, the deionized water of specification 100ml；Acetonitrile can select The silent winged generation that brand of purchase match, high-efficiency liquid chromatographic-grade (HPLC).

Step (F) filtering is to select the filtering of 0.22um pin types filter, can choose Millipore brands.

Embodiment two, biological sample primary crystallization form

In (1) hydrophilic region location hole, point 0.5-1 μ L matrix solutions form primary crystallization after volatilizing naturally；

(2) cent(e)ring hole, point 0.5-1 μ L matrix solutions, primary crystallization is formed after volatilizing naturally；

(3) correction hole is verified, point 0.5-1 μ L matrix solutions form primary crystallization after volatilizing naturally；

If Fig. 1 is shown, left figure is the crystallization figure of hydrophilic region location hole, the crystallization figure of correction hole centered on right figure.Two Regular circle is presented in crystal shape, and the mellow and full such as jade in surface, quality rule is homogeneous, and crystal is respectively careful to growing, for ideal Protein crystal form.

It should be pointed out that embodiment one and two should be in 100,000 grades of cleanliness factors of operation room, 20-25 DEG C of environment temperature, ring It is carried out under the conditions of the humidity 20-30% of border.

Embodiment three, by secondary crystallization, determine the optimal proportion of matrix and sample solution

According to substance assistant laser desorpted ionized principle, the biological sample (Escherichia coli 8739) of selection standard, respectively 3 A 0.5,0.75,1 μ L matrix solutions of hole midpoint, primary crystallization is formed after volatilizing naturally；

Then on primary crystallization surface, 0.5 μ L master samples is put, secondary crystallization is formed after volatilizing naturally；

The comparison of secondary crystallization is as shown in Figure 2.Wherein, the crystallization of 0.5 μ L and 1 μ L matrix solutions is irregular, uneven thickness Even, intermediate empty, surrounding is thick.Therefore when laser bombardment sample, sample peak poor accuracy, noise is high, and baseline is high.

And 0.75 μ L matrix solutions are regular, thickness is uniform, and crystal orientation growth is careful, and quality is intensive, it is contemplated that when laser bangs When hitting sample, the accuracy of sample peak is high, and noise is low, and baseline is smooth.

It thereby determines that and 0.75 μ L matrix solutions and 0.5 μ L samples is selected to be crystallized, be preferred plan.

The correction of example IV, microorganism clinical sample standard

(1) 8739 solution of microorganism clinical sample reference culture is chosen (in the management of Chinese industrial Microbiological Culture Collection The heart, gram-Negative bacillus)

(2) pre-process

In the cent(e)ring hole of microarray Mass Spectrometer Method chip and verification correction hole, 0.75 μ L matrix solutions, natural wind are put After dry, then 0.5 μ L UEP standard items (8739 solution of bacterial strain) are put, after volatilizing naturally, microarray Mass Spectrometer Method chip is loaded into core Piece adapter, sample introduction are put into CLIN-TOF-II flight time mass spectrums sample room.

CLIN-TOF-II flight time mass spectrum parameters are set：

Turing mode:linear；

Mass Range:2000-20000；

Max Laser Rep Rate:20.0；

Power:65；

Profiles:100；

Shots:5。

(d) vacuum threshold：

As vacuum degree ＜ 5E-6, start to detect；

(3) UEP (8739 solution) is corrected

Firstth, the UEP of laser bombardment cent(e)ring hole position

8739 spectrogram, 13 peak molecular weights are corrected, wherein 8 8739 peak molecular weight deviations are less than 700PPM, i.e., It can.

Secondth, the UEP of laser bombardment verification correction hole position

8739 spectrogram, 13 peak molecular weights are verified, wherein 8 8739 peak molecular weight deviations are less than 700PPM, are Correction is effective, attached drawing 4.Such as Fig. 4, wherein 4365.4,5096.8,5381.4,6255.4,6316.2,6411.6,7158.8, 7274.5,7872.1,9742.0Da 10 peaks are satisfied by molecular weight deviation less than 700PPM.

After correcting successfully, you can detection micro-biological samples.

Embodiment five, micro-biological samples detection

(1) preparation：

Prepared matrix solution；20, Clinical microorganism sample (the firm new rich wound biotechnology in Beijing that pre-treatment prepares Microbiological Lab of Co., Ltd preserves), each centrifuge tube (200 μ l) is middle to add in 20 μ l-30 μ l component I, with 1 μ l aseptic inoculations Ring or pipette tips picking single bacterium are fallen in upper tube, mixing, concussion mixing 5min；The microarray Mass Spectrometer Method chip cleaned up.

(2) point sample：

20 hydrophilic region location holes and cent(e)ring hole, verification correction are chosen in microarray Mass Spectrometer Method chip surface 0.75 μ L matrix solutions are put in hole, after volatilizing naturally, 0.5 μ L Clinical microorganism samples of cent(e)ring hole and verification correction hole point UEP standard items, 20 0.5 μ L Clinical microorganism samples of hydrophilic region location hole point, after volatilizing naturally, by microarray Mass Spectrometer Method Chip is loaded into adaptor chip, and sample introduction is put into CLIN-TOF-II flight time mass spectrums sample room.

(3) CLIN-TOF-II flight time mass spectrum parameters are set：

Turing mode:linear；

Mass Range:2000-20000；

Max Laser Rep Rate:20.0；

Power:65；

Profiles:100；

Shots:5。

(d) vacuum threshold：

As vacuum degree ＜ 5E-6, start to detect；

(4) sample collection

The sample of laser bombardment hydrophilic region location hole gathers 20 micro-biological samples mass spectrograms；Partial detail view is shown in attached drawing 5。

(5) spectrum analysis

The microbial identification platform BE3.1.6 of Beijing Yixin Bochuang Biotechnology Co., Ltd. is selected to carry out micro-biological samples Analysis, the analysis result in 20 sites refer to report；

It is analyzed and identified and is reported as follows by microbial identification platform BE3.1.6：

The probation report in 3 20 sites of table

It is reported above to draw, 20 micro-biological samples, every 20 heavy spectral peak of example, detection range 2000-20000, totally 400 Site, each site correct parting of energy, qualification result accuracy 100%.

Wherein, as shown in figure 5, sequentially consisting of staphylococcus aureus, Klebsiella Pneumoniae, waxy brood cell's bar Bacterium, salmonella, enterococcus faecalis, can quickly, it is clear, accurately tell unknown microorganism.

In summary, the present invention improves matrix solution formula, can obtain the preferable primary crystallization of crystal habit and secondary Crystallization effect, and its bearing calibration can aid in acquisition and more stablize and accurate Mass Spectrometer Method result.

Claims (10)

1. a kind of method for the crystallization for improving Mass Spectrometer Method micro-biological samples, step include：

(1) using the acetonitrile of deionized water and high-efficiency liquid chromatographic-grade according to 1:1-0.5:1 volume ratio mixing, obtains mixing molten Liquid；

(2) add in 3- hydroxyl -2- pyridine carboxylic acids fully to dissolve, wherein 3- hydroxyls -2- pyridine carboxylic acids：Mixed solution=1-10：100 (mg/ml)；

(3) 3- hydroxyls -2- pyridine carboxylic acids solution and citric acid diamine solution are taken, according to 1-3:1 volume ratio is uniformly mixed, and filtering is Obtain matrix solution.

(4) 0.5-1 μ L matrix solutions and 0.5-1 μ L samples are chosen, point sample crystallization is carried out on chip.

2. the method for claim 1 wherein the 3- hydroxyl -2- pyridine carboxylic acids that 0.01-0.2mg is added in step 2, and pass through 2000-3000rpm shakes 3-10min, and 8000-12000rpm centrifugation 3-10min obtain 3- hydroxyl -2- pyridine carboxylic acid solution.

3. the method for claim 1 wherein the 3- hydroxyl -2- pyridine carboxylic acids of 0.05mg in step 3.

4. the method for claim 1 wherein being formulated as follows for the citric acid diamine solution in step 3：Lemon acid diamine is weighed to put It in centrifuge tube, adds deionized water and is dissolved, the wherein weight mg of lemon acid diamine is with deionized water volume mL ratios 1-5：100,3-10min is shaken in 2000-3000rpm, it is molten to obtain lemon acid diamine by 8000-12000rpm centrifugation 3-10min Liquid.

5. the method for claim 1 wherein choose 0.75 μ L matrix solutions and 0.5 μ L samples are crystallized in step 4.

6. the mass spectrum chip of the accuracy rate that can improve Mass Spectrometer Method bio-target molecule used in the method for claim 1-5, The chip includes：The chip body being made of silicon materials or glass or titanium alloy, surface have multiple vertical cross arrangements Hydrophilic region location hole, weep hole exterior domain, cent(e)ring hole, verification correction hole, spare correction hole；

Wherein, location hole has water-wet behavior, for titrating matrix solution or sample；Weep hole exterior domain, surface have hydrophobic Characteristic；The structure for the close and distant water spacer in micro-array chip surface that location hole and hole exterior domain are formed, can be received with auxiliary liquid sample Contracting is condensed upon in hydrophilic region, concentrates sample crystallization, and standard circular is presented, and improves Ionization Efficiency；

Cent(e)ring hole and verification correction hole, for correcting the titration location verification correction hole of microbial standard, for verifying school Plus effect；

Spare correction hole, as spare, cent(e)ring hole or verification correction hole are if there is exception, available backup correction hole.

7. the method for claim 6, wherein location hole are the vertical crisscross arrangement of (4-8) × (4-8) on chip, center Correction hole, verification correction hole and spare correction hole in the chips between position vertical array, quantity is 1 or 2.

8. the method for claim 7, wherein the hydrophilic film of chip hydrophily location hole covering 150-800nm, hydrophobic Property hole outskirt cover 150nm-2 μm of hydrophobic film, 120 ° of surface water droplet contact angle ＞.

9. the method for claim 8, wherein the hydrophilic film is silica oxides film, zinc-oxide film, oxidation Aluminium film etc., hydrophobic pores outskirt carry out processing by silane coupling agent and form hydrophobic film.

10. the method for claim 9, wherein the silane coupling agent is selected from vinyl silanes, amino silane or dimethyl dichloro Silane.