CN108008002A - Correct the method and product of the accuracy rate of Mass Spectrometer Method nucleic acid samples - Google Patents

Abstract

The present invention provides a kind of method by improving sample of nucleic acid 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 special ratios carries out point sample crystallization on chip.Present invention also offers the mass spectrum bearing calibration for the accuracy rate for improving Mass Spectrometer Method nucleic acid 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 sample of nucleic acid simultaneously crystallizes, and the sample of nucleic acid of correction hole position is bombarded by mass spectrum, it is corrected with the mass spectral results to sample to be tested.Present invention also offers the chip 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, its bearing calibration helps to obtain and more stablizes and accurate Mass Spectrometer Method result.

Description

Correct the method and product of the accuracy rate of Mass Spectrometer Method nucleic acid samples

Technical field

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

Background technology

Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF MS) technology becomes 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 is combined with suitable organic substrate, 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 sample molecule and matrix is formed uniformly altogether Crystallization.MALDI methods analysis one of macromolecular, important key is the suitable matrix of selection.The result shows that the preferable base of effect Matter only has several.Water solubility synthesis macromolecule such as polyethylene glycol (PEG), polypropylene glycol often arise in the MALDI researchs of early stage In, this is because the matrix of analysis polypeptide can also be applied to them, can be from macromolecule and base for other macromolecule The thinking of some selection matrix is found in the dissolubility of matter.In general, select that during matrix matrix and high molecular polarity should be made Than more consistent, there is compatibility each other.

Micro-array chip is characterized by high density arrays.Microarray technology is exactly to utilize 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 as developing a most rapid part in chip field.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 specimen 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 parcel 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 is high.Pass through Software analysis compares, and screens and determines 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 gun for 100~ 1000 000 biomolecule, is acted on by high-voltage electricity and short laser pulse, allows host crystal literization, makes matrix and sample molecules Gasification enters mass spectrometric gas phase, and the characteristics of it is most prominent is that quasi-molecular ionization is very strong, big to the dosis tolerata of impurity in sample, The mixture that Direct Analysis nucleic acid, protein are produced through ionization, can provide effectively for clinical examination Plays thing Quality Research Technical guarantee, is the first choice of the reference method of clinical examination.Therefore the sample crystal habit and the direct shadow of quality on chip Ring 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 setting hydrophobic and hydrophilic region to prepare detection chip on chip, Wherein using screen printing technique by hydrophobic polymer (dimethyl silicone polymer or polymethyl methacrylate) according to design For good template brush on electro-conductive glass, 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 has been opened, including by base Constructed on bottom and be used as barrier with larger-diameter polymer coating (such as polymethyl methacrylate, polystyrene or photoresist) Circle area, is then attached in whole substrate using fluorine-containing monolayer or evaporated metal layer as hydrophobic 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 so that it is (fluorine-containing 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, have impact on the application of common lab.

Chinese patent application 200610023671.5, disclose " 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 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 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, increase cost, prepares Complexity, is not appropriate for being not easy the detection and application of 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 extra device and inspection software are needed, 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.

It is immediate in the prior art, inventor formerly before submit Chinese patent (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, its 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 Studied on basis.

In view of MALDI-TOF MS can be used for analyzing polytype sample, including organic molecular solution, nucleic acid, protein And whole microorganism, wherein gene, protein and microorganism is at present in the most widely used item in mass spectrometry clinical testing laboratory Mesh, thus on existing chip basis, it is necessary to one kind by improving sample crystallization condition to improve MALDI-TOF Mass Spectrometer Methods The method of nucleic acid target 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 nucleic acid target molecule on chip crystallographic property, there is provided a kind of bio-target molecule Generic crystallization method.

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 of nucleic acid 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 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 Filter 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 wherein in step 2, and are led to 2000-3000rpm concussion 3-10min, 8000-12000rpm centrifugation 3-10min are crossed, obtain 3- hydroxyl -2- pyridine carboxylic acid solution； In a specific embodiment, the 3- hydroxyl -2- pyridine carboxylic acids of 0.05mg are added.

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, 8000-12000rpm centrifugation 3-10min, it is molten to obtain lemon acid diamine 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 are selected from 100,000 grades of cleanliness factors of operation room, 20-25 DEG C of environment temperature, 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, its 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 the titration location verification correction hole of calibration nucleic acid master sample, are used for Verify 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 its 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 correction of accuracy rate for improving Mass Spectrometer Method nucleic acid target molecule Method, step include：

(1) as described above, preparing matrix solution, and the solution of separate target nucleic acid subsample 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 90% UEP peak molecular weights deviation is less than 200PPM, 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 a specific embodiment, the bio-target molecule is nucleic acid, and the calibration samples UEP is to be used to expand target The primer of nucleic acid, i.e. deafness UEP, verify deaf 20 UEP peak molecular weights of UEP spectrograms, wherein 18 UEP peak molecular weights Deviation is less than 200PPM, and as correction is effective.

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

Turing mode:linear；

Mass Range:3000-9000；

Max Laser Rep Rate:10.0；

Power:105；

Profiles:40；

Shots:10。

In a preferred embodiment, the mass spectrum is 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.

Brief description of the drawings

Fig. 1 is biological specimen 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 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 verifies correction product spectrogram for deafness UEP；

Fig. 5 is 5 mass spectrograms of deaf sample；

Technique effect

1st, nucleic acid Mass Spectrometer Method of the invention is to be based on microarray nucleic acid detection chip, its micro-array chip surface texture increases Correction up hole, improves mass spectra peak Mass accuracy, so as to improve the accuracy of nucleic acid Mass Spectrometer Method；

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

3rd, the present invention proposes that a kind of preferable 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, 20 deaf samples are chosen, identify accuracy Reach 100%；

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 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 and deionized water volume mL ratios are 1-5：100, shake 3-10min, 8000-12000rpm centrifugations in 2000-3000rpm 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 specimen primary crystallization form

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

As 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 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

The mass spectrum chip of clean surface, and the matrix solution configured according to embodiment one are got out, respectively in chip surface 3 holes midpoint, 0.5,0.75,1 tri- kinds of volumes of μ L matrix solution, form primary crystallization after volatilizing naturally；

Then on three kinds of primary crystallization surfaces, 0.5 μ L master sample UEP is put, secondary crystallization is formed after volatilizing naturally；

The contrast of secondary crystallization is as shown in Figure 2.Wherein ,+0.5 μ L samples two of+0.5 μ L samples of 0.5 μ L matrix and 1 μ L matrix The crystallization that kind proportioning is formed is irregular, in uneven thickness, middle empty, thick around.Therefore when laser bombardment sample, sample peak is accurate Exactness is poor, and noise is high, and baseline is high.

And the crystallization rule that the proportioning of+0.5 μ L samples of 0.75 μ L matrix is formed, thickness is uniform, and crystal orientation grows careful, quality Intensive, when laser bombardment sample, the accuracy of sample peak is high, and noise is low, and baseline is low.

Thereby determine that and select 0.75 μ L matrix solutions and 0.5 μ L samples to be crystallized, be preferred plan.

Example IV, deaf gene UEP corrections

(1) deaf gene is chosen

20 are respectively with the relevant gene polymorphic site of hereditary hearing impairment：GJB2 gene 35delG, GJB2 genes 167delT, GJB2 gene 17 6_191del16, GJB2 gene 235delC, GJB2 gene 299_300delAT, GJB3 gene C538T, GJB3 gene G547A, SLC26A4 Gene C2 81T, SLC26A4 gene G589A, SLC26A4 gene IVS7-2, SLC26A4 Gene A 1174T, SLC26A4 gene G1226A, SLC26A4 gene C 1229T, SLC26A4 genes IVS15_5, SLC26A4 gene G1975A, SLC26A4 gene T2027A, SLC26A4 Gene C2 162T, SLC26A4 Gene As 2168G, 12S RRNA gene C 1494 Ts, 12S rRNA Gene A 1555G, primer is designed to the deaf gene.

Table 1 is primer information table, and each site primer sequence is SEQ ID NO respectively:1-20：

Table 1

(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 deafness UEP standard items are put, after volatilizing naturally, microarray Mass Spectrometer Method chip is loaded into adaptor chip, into Sample, is 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:3000-9000；

Max Laser Rep Rate:10.0；

Power:105；

Profiles:40；

Shots:10。

(d) vacuum threshold：

As vacuum ＜ 5E-6, start to detect；

(3) UEP is corrected

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

Deaf 20 UEP peak molecular weights of UEP spectrograms are corrected, wherein 18 UEP peak molecular weight deviations are less than 200PPM, you can.

Secondth, the deaf UEP of laser bombardment verification correction hole position

Deaf 20 UEP peak molecular weights of UEP spectrograms are verified, wherein 18 UEP peak molecular weight deviations are less than 200PPM, as correction are effective.

The Mass Spectrometer Method part spectrogram of verification correction hole position is shown in attached drawing 4.

In 3000-9000Da mass ranges, 20 UEP peaks, resolution ratio 400-600, baseline is low, and signal-to-noise ratio is high.

20 UEP peak positions deviations of verification correction hole position see the table below：

Table 2 verifies correction hole UEP peak deviations：

Deaf 20 UEP peaks of UEP standard items, remove 6782.4Da, 6789.4Da two, and remaining 18 UEP peaks are maximum inclined Difference is 147.3PPM ＜ 200PPM.

After correcting successfully, you can detection deaf gene sample.

Embodiment five, deaf gene pattern detection

(1) preparation：

Prepared matrix solution, 20, the deaf plasmid sample that pre-treatment is completed, the microarray mass spectrum inspection cleaned up Survey chip.

(2) point sample：

20 hydrophilic region location holes and cent(e)ring hole, verification correction are chosen in microarray Mass Spectrometer Method chip surface Hole, puts 0.75 μ L matrix solutions, after volatilizing naturally, 0.5 μ L deafness UEP standard items of cent(e)ring hole and verification correction hole point, and 20 The clinical deaf samples of a 0.5 μ L of hydrophilic region location hole point, after volatilizing naturally, microarray Mass Spectrometer Method chip loading chip is fitted Orchestration, 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:3000-9000；

Max Laser Rep Rate:10.0；

Power:105；

Profiles:40；

Shots:10。

Vacuum threshold：

As vacuum ＜ 5E-6, start to detect；

(4) UEP is corrected

1st, the deaf UEP of laser bombardment cent(e)ring hole position

Deaf 20 UEP peak molecular weights of UEP spectrograms are corrected, wherein 18 UEP peak molecular weight deviations are less than 200PPM, you can.

2nd, the deaf UEP of laser bombardment verification correction hole position

Deaf 20 UEP peak molecular weights of UEP spectrograms are verified, wherein 18 UEP peak molecular weight deviations are less than 200PPM, as correction are effective.

After correcting successfully, you can the deaf sample of detection.

(5) sample collection

The deaf plasmid sample of laser bombardment hydrophilic region location hole, gathers 20 deaf sample mass spectrograms；Refer to attached drawing 5。

(6) spectrum analysis

The BESNP softwares of Beijing Yixin Bochuang Biotechnology Co., Ltd. are selected to carry out deaf sample analysis, 20 sites Analysis result refer to report.

By BESNP software analysis probation reports, draw, 20 deaf plasmid samples, common 20*20=400 site, identification As a result it is all correct, i.e., deaf sample qualification result accuracy 100%.

BESNP software analysis probation reports are as follows：

Table 3

It is reported above to draw, deaf 20, sample, every 20 heavy spectral peak of example, detection range 3000-9000Da, totally 400 Site, through Beijing Yixin Bochuang Biotechnology Co., Ltd.'s BE-SNP software analysis, each site correct parting of energy, identification knot Fruit accuracy 100%.

In conclusion matrix solution formula provided by the invention and matrix and sample ratio, it is preferable can to obtain form Primary crystallization and secondary crystallization, obtained beneficial effect is that the baseline of Mass Spectrometer Method spectrogram is low, and signal-to-noise ratio is high；According to the present invention The bearing calibration of offer, obtained beneficial effect are to improve Mass Spectrometer Method Mass accuracy, improve identification accuracy rate, the present embodiment ear Deaf pattern detection, qualification result accuracy 100%.

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 mass spectrum bearing calibration for the accuracy rate for improving Mass Spectrometer Method nucleic acid target molecule, step include：

(1) matrix solution is prepared, and configures the solution of separate target nucleic acid subsample and calibration samples UEP；

(2) respectively in hydrophilic region location hole, cent(e)ring hole and verification correction hole, point 0.5-1 μ L matrix solutions, are waved naturally Primary crystallization is formed after dry；

(3) in hydrophilic region location hole, on primary crystallization surface, point 0.5-1 μ L separate target nucleic acids subsample, forms after volatilizing naturally Secondary crystallization；

(4) respectively in cent(e)ring hole, verification correction hole, on primary crystallization surface, point 0.5-1 μ L calibration samples UEP, are waved naturally Secondary crystallization is formed after dry；

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

(6) when 90% UEP peak molecular weights deviation is less than 200PPM, as correction is effective；

(7) after correcting successfully, you can carry out Mass Spectrometer Method to the determined nucleic acid target molecule sample of hydrophilic region location hole.

Wherein, in step (1) by acetonitrile solution with 1-10：The ratio of 100 (mg/ml), adds 3- hydroxyl -2- pyridine carboxylic acids Fully dissolving obtains mixed liquor, then by 3- hydroxyls -2- pyridine carboxylic acids solution and citric acid diamine solution, according to 1-3:1 volume Than being uniformly mixed, filter up to matrix solution.

2. the method for claim 1 wherein choose 0.75 μ L matrix solutions and 0.5 μ L target molecules samples or master sample solution into Row crystallization.

3. the method for claim 2, wherein the calibration samples UEP is the primer for being used to expand target nucleic acid as described in Table 1, i.e., Deaf UEP.

4. the method for any one of claim 1-3, wherein the mass spectrographic parameter setting is as follows：

Turing mode:linear；

Mass Range:3000-9000；

Max Laser Rep Rate:10.0；

Power:105；

Profiles:40；

Shots:10。

5. the method for claim 5, wherein MALDI-TOF mass spectrums are CLIN-TOF-II flight time mass spectrums.

6. the mass spectrum chip of the accuracy rate that can improve Mass Spectrometer Method nucleic acid 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, its 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 the titration location verification correction hole of calibration nucleic acid standard, for verifying correction 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 its 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 8, wherein the silane coupling agent is selected from vinyl silanes, amino silane or dimethyl dichloro Silane.