CN109444251A - Application of the nanomatrix in detection of nucleic acids - Google Patents
Application of the nanomatrix in detection of nucleic acids Download PDFInfo
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- CN109444251A CN109444251A CN201811404804.2A CN201811404804A CN109444251A CN 109444251 A CN109444251 A CN 109444251A CN 201811404804 A CN201811404804 A CN 201811404804A CN 109444251 A CN109444251 A CN 109444251A
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Abstract
The invention belongs to mass spectrum detection field, it is related to a kind of application of nanomatrix in detection of nucleic acids.The application of nanomatrix of the invention in detection of nucleic acids, the structure of the nanomatrix are stratum nucleare/subshell/outer shell；Wherein, the stratum nucleare is magnetic oxide, and the subshell is Si oxide, and the outer shell is platinum.Nuclear shell structure nano matrix of the invention, which is easy to be produced, to be isolated and purified, and can be improved organic substrate and be crystallized non-uniform situation, while increase the desorption ionization effect of oligonucleotides by the phasmon effect of noble metal, increase the sensitivity of nucleic acid Mass Spectrometer Method.A kind of means of the present invention as simple, quick, high-throughput detection oligonucleotides sample, have a good application prospect, are worthy of popularization.
Description
Technical field
The invention belongs to a kind of application of mass spectrum detection field more particularly to nanomatrix in detection of nucleic acids.
Background technique
Substance assistant laser desorpted ionized (MALDI) can be generated steady as a kind of " soft ionization " mode using less energy
Fixed gas ion is one of most important ionization mode of difficult volatility high-molecular weight compounds.This ionization technique and flight
Time mass spectrum combines (TOF-MS), and the rapidly and efficiently reliable detection of large biological molecule may be implemented.Studies have shown that matrix
Assisted laser desorption ionisation flight time mass spectrum (MALDI-TOF-MS) can be effectively applied in the detection of oligonucleotides, example
Such as the analysis of single nucleotide polymorphism (SNP) and the marker of cytosine methylation；Particularly with SNP detection biology, doctor
The various fields such as, agronomy play an important role.Compared to other research methods, MALDI-TOF-MS is that one kind has height
Flux, high-precision, and a relatively easy convenient and fast analysis mode, thus be used widely.
During laser desorption ionisation, the fragmentation of DNA fragmentation is a kind of common phenomenon, the fragmentation energy quilt of DNA
The protonation of base is induced, and the loss of base is caused, and eventually leads to the loss of the signal strength of intact DNA fragments.And DNA
Fragmentation depends primarily on the property of used matrix, and in this process, matrix plays absorption, transfer laser energy to be measured
Sample and the effect for ionizing sample to be tested.MALDI-TOF-MS traditional matrix is generally organic substrate.Ferulic acid, 2,
5- dihydroxy-benzoic acid (2,5-DHB) is the matrix for being once used for nucleic acid mass spectral analysis, but since sensitivity is low, seldom by reality
Using.2,4,6- trihydroxy-acetophenones (2,4,6-THAP) and 3- hydroxyl arsenic pyridine formic acid (3-HPA) are commonly used for nucleic acid matter
The matrix of spectrum analysis, but it is uneven due to crystallizing, and only competence exertion acts at high concentrations, and because of the excess of matrix
It will lead to energy surplus after absorbing laser energy, cause the fragmentation of nucleic acid fragment.Currently, all tradition organic substrates are asked jointly
Topic is the presence of " available point ", causes experimental result repeatability very poor；Simultaneously as acting on nucleic acid samples desorption ionization
Effect is not ideal enough, the nucleic acid molecules within the scope of specified molecular weight can only be detected, generally in 10000Da or less.
In addition, the research and development of the matrix with MALDI-TOF-MS, some inorganic nano materials start to be explored work
Matrix for MALDI-TOF-MS or the reinforcing material as organic substrate, such as carbon nanotube, graphene, more empty silicon, silicon
Nano particle, gold nano grain etc. have shown certain application prospect.But in nucleic acid samples, due to the complexity of sample
Property, the Ionization Efficiency of sample to be tested can be by severe jamming, and above-mentioned matrix is still difficult to meet the application in nucleic acid Mass Spectrometer Method.Institute
With for the foranalysis of nucleic acids of MALDI-TOF-MS, it is desirable to provide it is a kind of have more efficient, sensitiveer, repeatability is more
Good host material, is able to solve at least one of above problem.
In consideration of it, the present invention is specifically proposed.
Summary of the invention
It is an object of the present invention to provide a kind of application of nanomatrix in detection of nucleic acids, have high sensitivity,
It is reproducible, it is easy to operate, quick, efficient the features such as, the above problem can be overcome or at least be partially solved above-mentioned technology
Problem.
To achieve the above object, the technical solution adopted by the present invention are as follows:
According to an aspect of the present invention, the present invention provides a kind of application of nanomatrix in detection of nucleic acids, described to receive
The structure of meter Ji Zhi is stratum nucleare/subshell/outer shell；
Wherein, the stratum nucleare is magnetic oxide, and the subshell is Si oxide, and the outer shell is platinum.
As further preferred technical solution, the nanomatrix has coarse surface；
And/or partial size < 1000nm, preferably≤900nm, the further preferably≤800nm of the nanomatrix, into
One step is preferably 200~700nm.
As further preferred technical solution, the stratum nucleare is spherical or spherical particle, the partial size of particle is 100~
400nm, preferably 120~380nm, further preferably 150~350nm；
And/or the subshell with a thickness of 20~200nm, preferably 50~180nm, further preferably 80~
160nm；
And/or the outer shell with a thickness of 5~80nm, preferably 10~50nm, further preferably 20~30nm.
As further preferred technical solution, the magnetic oxide includes ferroso-ferric oxide, di-iron trioxide or iron oxygen
One of body is a variety of, preferably ferroso-ferric oxide；
And/or the Si oxide includes silicon monoxide and/or silica, preferably silica.
As further preferred technical solution, the preparation method of the nanomatrix includes:
Magnetic oxide particle is added and is reacted into the solution system for be used to generate Si oxide, silicon oxidation is obtained
The particle of object coated magnetic oxide；
By the particle of the Si oxide coated magnetic oxide be added to be used to react generate platinum solution system in into
Row reaction, obtains magnetic oxide/Si oxide/platinum structure nuclear shell structure nano matrix.
As further preferred technical solution, the method for the detection of nucleic acids includes:
By nucleic acid samples and co-substrate, spot is carried out on substance assistant laser desorpted ionized mass spectrometric target plate
Sample is analyzed by mass spectrometry detection after dry；
Wherein, the co-substrate includes organic substrate and the nanomatrix；
Preferably, the organic substrate includes 2,4,6-THAP and/or 3-HPA；
Preferably, target plate is pre-processed before Multiple loading, pretreated mode includes: successively molten with low-carbon alcohols
Liquid and water are cleaned by ultrasonic target plate, and the time of cleaning is 30~90min, preferably 50~60min.
As further preferred technical solution, the mode of the Multiple loading includes:
First by the water-soluble liquid spotting of nanomatrix on mass spectrum target plate, then by nucleic acid samples point sample in the nanometer base dried
Matter surface, finally by organic substrate solution point sample in the nucleic acid samples surface dried, drying.
As further preferred technical solution, the mode of the Multiple loading includes:
First by the water-soluble liquid spotting of nanomatrix on mass spectrum target plate, then nucleic acid samples are mixed with organic substrate solution
It is even, mixture is obtained, by the mixture point sample in the nanomatrix surface dried, drying.
As further preferred technical solution, nucleic acid samples include that biology extracts DNA fragmentation, PCR product and oligonucleotides
At least one of segment.
As further preferred technical solution, the detection molecules amount of nucleic acid detection method is in 30000Da or less；
And/or substance assistant laser desorpted ionized mass spectrometric testing conditions include: positive ion detection, the letter detected
Making an uproar than the mass signal greater than 6 is the useful signal that can be analyzed.
Compared with prior art, the beneficial effects of the present invention are:
(1) present invention applies the nanomatrix of core-shell structure in field of nucleic acid detection, and the nanomatrix is with magnetic oxygen
Compound is stratum nucleare, coats to form subshell with Si oxide, is further coated with nano platinum particle and forms outer shell structure,
Since the magnetic characteristic of magnetic oxide is isolated and purified convenient for it by equipment, but due to the nanomatrix have stable structure, compared with
The unique surface plasma resonance and hot carrier that big surface area and Pt nanoparticle provide, on the one hand alleviate existing
Organic substrate crystallize non-uniform problem, on the other hand promote the desorption ionization effect of nucleic acid samples.
(2) sensitivity detected improves, is reproducible: the nanomatrix of core-shell structure proposed by the present invention, can be significant
The desorption ionization effect of nucleic acid samples is improved, realizes accurate identification for sample, the sensitivity and again of detection greatly improved
Renaturation.
(3) consumption of test sample is few: due to using nanomatrix of the invention the solution of nucleic acid samples being sucked away from
Sonization greatly improves, and in conjunction with the characteristics of Mass Spectrometer Method, can significantly lower the consumption of sample, and realize the inspection of low concentration sample
It surveys.
(4) detection method is simple, efficient: the detection process is easy to operate, very quickly, entire detection process, it is only necessary to
A few minutes can be obtained by last testing result.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is the Fe that an embodiment of the present invention provides3O4/SiO2The phenogram of/Pt Core-shell Structure Nanoparticles；Fig. 1
(a) scheme for SEM, Fig. 1 (b) is TEM figure；
Fig. 2 is the Fe that an embodiment of the present invention provides3O4/SiO2/ Pt nuclear shell structure nano matrix is tied altogether with organic substrate
Phenogram when brilliant；
Fig. 3 is the Fe that an embodiment of the present invention provides3O4/SiO2/ Pt Core-shell Structure Nanoparticles are cobasis quality detection sequence
It is classified as the mass spectrogram that one section of oligonucleotides standard items (molecular weight 5605.7) of GCAGACGATCCTGGGGGG obtains；
Fig. 4 is the Fe that an embodiment of the present invention provides3O4/SiO2/ Pt Core-shell Structure Nanoparticles are that cobasis quality detection is new
The mass spectrogram that the multiple PCR products oligonucleotides sample of relevant 34 SNP sites of life youngster's deaf gene obtains.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiments and examples, but this field skill
Art personnel will be understood that following embodiments and embodiment are merely to illustrate the present invention, and are not construed as limiting model of the invention
It encloses.Based on the embodiments of the present invention, obtained by those of ordinary skill in the art without making creative efforts
Every other embodiment, shall fall within the protection scope of the present invention.The person that is not specified actual conditions, according to normal conditions or manufacturer builds
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
In a first aspect, a kind of application of nanomatrix in detection of nucleic acids is provided at least one embodiment, it is described to receive
The structure of meter Ji Zhi is stratum nucleare/subshell/outer shell；
Wherein, the stratum nucleare is magnetic oxide, and the subshell is Si oxide, and the outer shell is platinum (Pt).
In view of matrix defect present in detection of nucleic acids of existing MALDI-TOF-MS, MALDI- is limited
Application of the TOF-MS in nucleic acid samples analysis detection, it is necessary to propose a kind of novel matrix, and be applied in nucleic acid sample
In the detection of product, to improve the defect of the prior art.Based on this, the invention proposes a kind of new matrix and relevant obtain
Take method, application.
The nanomatrix of core-shell structure provided by the invention is mainly used in carrying out ground substance assistant laser solution to nucleic acid samples
It inhales in ionization time of flight mass spectrometry detection, can overcome the shortcomings of traditional matrices.Further speaking, the present invention is by changing nanometer
The material structure of matrix is continuously coated with Si oxide using magnetic oxide as stratum nucleare and forms subshell, with Pt nanoparticle
Continuous cladding forms outer shell structure, since the magnetic characteristic of magnetic oxide is isolated and purified convenient for it by equipment, and due to stabilization
Structure, the unique surface plasma resonance that provides of biggish surface area and Pt nanoparticle and hot carrier, on the one hand
It alleviates existing organic substrate and crystallizes non-uniform problem, on the other hand promote the desorption ionization effect of nucleic acid samples.
Therefore, which, which is easy to be produced, isolates and purifies, and it is uneven can to improve organic substrate crystallization
Situation, while increasing by the phasmon effect of noble metal the desorption ionization effect of oligonucleotides, increase the inspection of nucleic acid mass spectrum
The sensitivity of survey.A kind of hand of the present invention as simple, quick, highly sensitive, reproducible, high-throughput detection nucleic acid samples
Section, has a good application prospect, is worthy of popularization.
It should be noted that stratum nucleare of the invention/subshell/outer shell nuclear shell structure nano matrix, it can also be with table
It is shown as the nuclear shell structure nano matrix of stratum nucleare@subshell@outer shell.
Nanomatrix material below for the embodiment of the present invention and its application method in detection of nucleic acids carry out specific
Illustrate:
Nanomatrix of the invention, the particulate matter containing core-shell structure, the particulate matter are the core-shell structure of multilayer；Into one
Step, particulate matter generally has three-decker, that is, includes stratum nucleare, subshell and outer shell, and subshell coats core
Layer, outer shell coat subshell.It should be noted that in some instances, subshell is coated on outside stratum nucleare, it is not intended to
Limiting subshell must fully be wrapped in outside stratum nucleare.For example, the silicon oxide particles of the subshell constituted can also fit
Amount is contaminated in the magnetic oxide of stratum nucleare；It is same for the outer shell being coated on outside subshell.
Nanomatrix of the invention is generally the nano particle of spherical (or spherical), on the whole for, the present invention is implemented
The nanomatrix material that example provides is the particulate matter of nanoscale, and granularity is uniform；Stratum nucleare, subshell, outer shell are
Grain, further, stratum nucleare, subshell, outer layer are also nanoscale.
In some examples of the invention, the nanomatrix has coarse surface；
And/or partial size < 1000nm, preferably≤900nm, the further preferably≤800nm of the nanomatrix, into
One step is preferably 200~700nm.
According to the present invention, nanomatrix has coarse surface, and in other words, outer shell has coarse surface.It is described " thick
Rough surface " can be understood as not fine, rough or incompact surface.In some examples of the invention, shell is constituted
The Pt nanoparticle of layer is to push away product in loose form to form nanoscale appropriate outside subshell, between Pt nanoparticle
Gap.The nanomatrix constitutes outer shell using metal Pt nanoparticle, and has coarse surface, is applied in nucleic acid matter
It in spectrum detection, can make in nano particle and oligonucleotide molecules contact process, promote desorption ionization process to greatest extent.
The partial size of the nanomatrix is less than 1 μm, and grain graininess is uniform, and the partial size of nanomatrix (nanometer spherical particle) is excellent
It is selected as≤900nm, further preferably≤800nm, further preferably 200~700nm；It is typical but non-limiting for example
Think 200nm, 30nm, 400nm, 500nm, 600nm, 700nm or 800nm.
In some examples of the invention, the stratum nucleare is spherical or spherical particle, the partial size of particle is 100~
400nm, preferably 120~380nm, further preferably 150~350nm；It is typical but non-limiting to be for example
100nm, 120nm, 150nm, 180nm, 200nm, 250nm, 300nm, 350nm, 380nm or 400nm.
And/or the subshell with a thickness of 20~200nm, preferably 50~180nm, further preferably 80~
160nm；Typical but non-limiting for example can be 20nm, 50nm, 80nm, 100nm, 120nm, 150nm, 160nm, 180nm
Or 200nm.
And/or the outer shell with a thickness of 5~80nm, preferably 10~50nm, further preferably 20~30nm；
Typical but non-limiting for example can be 5nm, 10nm, 20nm, 30nm, 40nm, 50nm, 60nm, 70nm or 80nm.
The partial size of suitable stratum nucleare particle, and the thickness of suitable subshell and outer layer, can be to spheric granules
Core plays preferable protective effect, moreover it is possible to have higher thermoelectron generation rate, while promote hot carrier's effect.
In some examples of the invention, the magnetic oxide includes ferroso-ferric oxide, di-iron trioxide or ferrite
One of or it is a variety of；
And/or the Si oxide includes silicon monoxide and/or silica；
Preferably, the magnetic oxide is ferroso-ferric oxide；
Preferably, the Si oxide is silica.
It is understood that constituting the magnetic oxide of stratum nucleare of the invention, there are the materials of plurality of optional, such as four oxygen
Change the mixture of one or more of three-iron, di-iron trioxide or ferrite, but it is not limited to this, in other realities of the invention
It applies in example, there may also be other various selections for magnetic oxide.Preferably, the magnetic oxide of stratum nucleare of the invention is constituted
Select be it is from a wealth of sources, be easily obtained, with above-mentioned subshell, the better ferroso-ferric oxide of outer shell fit applications effect
(Fe3O4)。
It is understood that constituting the Si oxide of subshell of the invention, there are the materials of plurality of optional, such as one
The mixture of one or both of silica or silica, but it is not limited to this, in other embodiments of the invention, silicon
There may also be other various selections for oxide.Preferably, constitute subshell of the invention Si oxide select be
Source is wide, it is at low cost, with above-mentioned stratum nucleare, the better silica (SiO of outer shell fit applications effect2)。
In one preferred embodiment of the invention, the structure of the nanomatrix is ferroso-ferric oxide/silica/platinum,
That is Fe3O4/SiO2/Pt。
According to the present invention, stratum nucleare, subshell and the outer shell of the nuclear shell structure nano matrix granule object be by by
Layer (gradually) reaction obtains.As first by reaction generate constitute stratum nucleare magnetic oxide, then based on stratum nucleare stratum nucleare table
Subshell is grown on face, and outer shell is further grown on the surface of subshell based on subshell.
In some examples of the invention, the preparation method of the nanomatrix includes:
Magnetic oxide particle obtained is added and is reacted into the solution system for be used to generate Si oxide, is obtained
The particle of Si oxide coated magnetic oxide；
By the particle of the Si oxide coated magnetic oxide be added to be used to react generate platinum solution system in into
Row reaction, is made the nuclear shell structure nano matrix of magnetic oxide/Si oxide/platinum structure.
It should be noted that method commonly used in the art can be used in the preparation method of nanomatrix of the invention, for it
The specific preparation process present invention does not do excessive limitation.Below mainly with ferroso-ferric oxide/silica/platinum structure nanometer base
More full and accurate elaboration is carried out for matter to its production method, it should be understood that, which is equally applicable to similar
The preparation of structure such as di-iron trioxide/silica/platinum structure nanomatrix.
In a preferred embodiment, the preparation method of nanomatrix, comprising the following steps:
S1, with FeCl3·6H2Trisodium citrate and sodium acetate modification is added as source of iron in O, using ethylene glycol as solvent
Thermal response is carried out, monodispersity Fe is made3O4Nano particle；Reaction condition includes: that temperature is 200 DEG C, and the time is 8h~72h.
Wherein, ethylene glycol is as solvent and reducing agent；Trisodium citrate and sodium acetate can increase the stability of system, divide
Dissipate property.Trisodium citrate can be used as stabilizer, be complexed with iron ion, so that iron ion be prevented to reunite；Sodium acetate can increase magnetic
The electrostatic stability of property oxide particle, prevents its reunion.
The Fe of acquisition3O4The granularity of nano particle can be adjusted by adjusting the dosage of source of iron and/or trisodium citrate,
To meet actual demand.
S2, the Fe for obtaining S13O4Nano particle is dispersed in the in the mixed solvent of water and ethyl alcohol, with the positive silicic acid of ammonia-catalyzed
Tetra-ethyl ester hydrolytie polycondensation is nucleated, and 2h~8h is reacted under room temperature, in Fe3O4The titanium dioxide of nano grain surface formation different-thickness
Silicon layer obtains Fe3O4/SiO2Core-shell Structure Nanoparticles.
Wherein, water and ethyl alcohol promote tetraethyl orthosilicate to hydrolyze to form gel, and alkaline ammonium hydroxide can promote two as catalyst
The formation of silicon oxide microsphere.In general, in Fe3O4Nano grain surface formed silicon dioxide layer thickness than more consistent, but
In some instances, the thickness of silicon dioxide layer can slightly have difference, can be by regulating and controlling reaction condition for this layer of thickness control excellent
It selects in range.
S3, the Fe for obtaining S23O4/SiO2Core-shell Structure Nanoparticles ultrasound is dispersed in dehydrated alcohol, is added
3- aminopropyl triethoxysilane stirs 6h~12h, to form Fe3O4/SiO2- NH2Nano particle；
S4, it polyethylene is given a tongue-lashing into pyrrolidone is dissolved in platinum acid chloride solution, obtain platinum source solution；
Polyethylene therein, which gives a tongue-lashing pyrrolidone, can make stable system, not reunite.
S5, the Fe for forming S33O4/SiO2- NH2Nano particle is dispersed in the platinum source solution of S4, stir 2h, make platinum from
Son is evenly distributed on nano grain surface, obtains Fe3O4/SiO2- NH2Nano particle disperses solution；
S6, the Fe obtained in S53O4/SiO2- NH2Nano particle disperses that reducing agent sodium borohydride, room temperature are added in solution
8h~12h is stirred, Fe is obtained3O4/SiO2/ Pt Core-shell Structure Nanoparticles；
S7, the Fe that S6 is obtained is washed repeatedly with ethyl alcohol and deionized water3O4/SiO2/ Pt Core-shell Structure Nanoparticles, in 60
It is dried in DEG C baking oven powdered.
The above method is simple, easy to operate, easily controllable, and properties of product stability obtained is good, still realizes to core
The regulation of structure, the thickness of layer, subshell and outer shell etc., yield are high, it is easy to accomplish large-scale production.
Fe provided by the invention3O4/SiO2/ Pt Core-shell Structure Nanoparticles can be used as matrix and be answered in mass spectral analysis
With.Such as it is applied in substance assistant laser desorpted ionized Mass Spectrometer Method analyzer.In the application, above-mentioned nanomatrix is divided
It is dispersed in and (is resuspended in) in dispersion liquid, as mass spectrometer matrix；Wherein, the dispersion liquid is preferably water.
Therefore, the present invention also provides a kind of detection methods is swashed by using above-mentioned nanomatrix using Matrix-assisted
Photodesorption ionization mass spectrometer is detected.Target detection owner in view of the particularity of above-mentioned nanomatrix, in the detection method
It to be nucleic acid samples, therefore, the present invention provides above-mentioned Fe3O4/SiO2/ Pt Core-shell Structure Nanoparticles are in nucleic acid Mass Spectrometer Method
In application.
In some examples of the invention, the nucleic acid samples include biology extract DNA fragmentation, PCR product, and and its
The oligonucleotide fragment in his approach source.
Further, in nucleic acid samples Mass Spectrometer Method, molecular weight ranges are less than or equal to 30000Da.
Further, laser desorption ionisation mass spectrograph is bruker autoflexTMSpeed MALDI-TOF (TOF) matter
Spectrum, using smartbeam-II laser, wavelength 355nm or bruker microflex MALDI-TOF (TOF) matter
Spectrum, using nitrogen laser, wavelength 337nm.
Further, it is handled using the analysis that flexAnalysis carries out data.
It can be seen from the above carrying out nucleic acid samples mass spectral analysis using nanomatrix of the invention, detection has been widened
Molecular weight ranges, alleviating existing can only detect specified molecular weight (nucleic acid molecules generally in 10000Da or less) range, are answered
It is wider with range.
In some examples of the invention, it applies using above-mentioned nuclear shell structure nano matrix as matrix in nucleic acid mass spectral analysis
In detection, specific analyzing detecting method the following steps are included:
(a): the preparation of instrument and reagent: laser desorption ionisation mass spectrograph, using impulse electric field be delayed extract signal, just from
Sub-line sexual norm, and the signal only with signal-to-noise ratio greater than 6 is for analyzing；
(b): the nuclear shell structure nano matrix being resuspended in deionized water, as co-substrate；
(c): preparing nucleic acid samples, oligonucleotides sample is used as analyte after except salt treatment；
(d): by organic substrate (such as 2,4,6-THAP or 3-HPA) differently wiring solution-forming, as co-substrate；
What needs to be explained here is that the preparation method of organic substrate is the prior art, the present invention no longer elaborates herein.
(e): by the nuclear shell structure nano matrix, oligonucleotides sample and organic substrate solution carry out on target plate
Multiple loading；
(f): analysis Mass Spectrometer Method result, it was therefore concluded that.
Further, above-mentioned mass spectrum target plate must use clean target plate, which is pre-processed before use,
Such as dehydrated alcohol, deionized water can be successively used to be cleaned by ultrasonic total 60min or so, and then the impurity on removal target plate is dry
It disturbs.
Further, the Multiple loading includes optional two kinds of spot methods, is respectively as follows:
The first spot method: first by nanomatrix aqueous solution with liquid-transfering gun point sample on mass spectrum target plate, dry at room temperature,
Then nucleic acid samples are dried at room temperature with liquid-transfering gun point sample in nanomatrix surface, finally by organic substrate solution point sample in dry
Dry nucleic acid samples surface, is dried at room temperature.
Second of spot method: first by nanomatrix aqueous solution with liquid-transfering gun point sample on mass spectrum target plate, dry at room temperature,
Then nucleic acid samples are uniformly mixed with organic substrate solution, with liquid-transfering gun point sample in the nanomatrix surface dried, room temperature
Lower drying.
Combined with specific embodiments below, comparative example and attached drawing, the invention will be further described.
Embodiment 1
A kind of application of nanomatrix in detection of nucleic acids, comprising:
1, the preparation of nanomatrix:
S1, with FeCl3·6H2Trisodium citrate and sodium acetate modification is added as source of iron in O, using ethylene glycol as solvent
Thermal response is carried out, monodispersity Fe is made3O4Nano particle；Reaction condition includes: that temperature is 200 DEG C, and the time is 8h~72h；
S2, the Fe for obtaining S13O4Nano particle is dispersed in the in the mixed solvent of water and ethyl alcohol, with the positive silicic acid of ammonia-catalyzed
Tetra-ethyl ester hydrolytie polycondensation is nucleated, and 2h~8h is reacted under room temperature, in Fe3O4The titanium dioxide of nano grain surface formation different-thickness
Silicon layer obtains Fe3O4/SiO2Core-shell Structure Nanoparticles；
S3, the Fe for obtaining S23O4/SiO2Core-shell Structure Nanoparticles ultrasound is dispersed in dehydrated alcohol, is added
3- aminopropyl triethoxysilane stirs 6h~12h, to form Fe3O4/SiO2- NH2Nano particle；
S4, it polyethylene is given a tongue-lashing into pyrrolidone is dissolved in platinum acid chloride solution, obtain platinum source solution；
S5, the Fe for forming S33O4/SiO2- NH2Nano particle is dispersed in the platinum source solution of S4, stir 2h, make platinum from
Son is evenly distributed on nano grain surface, obtains Fe3O4/SiO2- NH2Nano particle disperses solution；
S6, the Fe obtained in S53O4/SiO2- NH2Nano particle disperses that reducing agent sodium borohydride, room temperature are added in solution
8h~12h is stirred, Fe is obtained3O4/SiO2/ Pt Core-shell Structure Nanoparticles；
S7, the Fe that S6 is obtained is washed repeatedly with ethyl alcohol and deionized water3O4/SiO2/ Pt Core-shell Structure Nanoparticles, in 60
It is dried in DEG C baking oven powdered；
S8, the nano particle for obtaining S8 are resuspended in deionized water, are used as mass spectrometer matrix.
The characterization of matrix:
Characterization instrument used includes: that the size and shape of product is characterized in JEOL JEM-2100F transmission electron microscope
(TEM), in JEOL JEM-2100F high-resolution-ration transmission electric-lens (HRTEM) and Hitachi S-4800 scanning electron microscope (SEM)
It completes.
Characterization result are as follows:
As shown in Figure 1, Fig. 1 (a) is the SEM phenogram of nano particle, Fig. 1 (b) is TEM phenogram.Fe3O4/SiO2/Pt
Core-shell Structure Nanoparticles are nanometer spherical particle, and grain graininess is uniform, and average grain diameter is 400nm or so.High resolution scanning electricity
Rough is rough as the result is shown for mirror.Transmission electron microscope results show that the coarse of surface by diameter is about receiving for 5~10nm
Nano-sphere composition.
2, the detection of nucleic acid samples
Detection sequence is one section of oligonucleotides standard items of GCAGACGATCCTGGGGGG
Utilize above-mentioned Fe3O4/SiO2/ Pt Core-shell Structure Nanoparticles carry out base to oligonucleotides standard items as co-substrate
The step of matter assisted laser desorption ionisation flight time mass spectrum detection, detection, is as follows:
(a) preparation of instrument and reagent: the mass signal of laser desorption ionisation mass spectrograph, only signal-to-noise ratio greater than 6 is used for
Analysis.Using bruker microflex MALDI-TOF (TOF) mass spectrum, using nitrogen laser, wavelength 337nm.Using
Impulse electric field delay is extracted and linear working method, positive ion mode are detected.Using flexAnalysis observation, place
Reason, analysis data.
(b)Fe3O4/SiO2The preparation of/Pt Core-shell Structure Nanoparticles.
(c) preparation of oligonucleotides standard sample: commission Sangon Biotech (Shanghai) Co., Ltd.) limited liability company's synthesis, HPLC
The purifying of CE method.
(d) successively it is cleaned by ultrasonic the total 1h of mass spectrum target plate with dehydrated alcohol, deionized water.
(e) by Fe3O4/SiO2/ Pt Core-shell Structure Nanoparticles ultrasonic disperse is in deionized water, by it with liquid-transfering gun point
Sample is dried at room temperature on mass spectrum target plate.
(f) oligonucleotides standard sample point sample is dried at room temperature in the nanomatrix surface dried.
(g) organic substrate is dissolved in the solvent matched in proportion, point sample is in the oligonucleotides standard sample table dried
Face makes three form recrystallization, is used for laser desorption ionisation mass spectral analysis.
Mass Spectrometer Method result is as shown in figure 3, from figure 3, it can be seen that the present invention can obtain the mass signals of high quality.
In addition, Fig. 2 shows Fe3O4/SiO2Phenogram when/Pt nuclear shell structure nano matrix and organic substrate cocrystallization,
Figure it is seen that nuclear shell structure nano matrix of the invention can make organic substrate crystallization more evenly.
Embodiment 2
A kind of application of nanomatrix in detection of nucleic acids, comprising:
1, the preparation of nanomatrix, it is same as Example 1.
2, the detection of nucleic acid samples
Detect the multiple PCR products sample oligonucleotide segment of relevant 34 SNP sites of newborn's deaf gene
Utilize above-mentioned Fe3O4/SiO2/ Pt Core-shell Structure Nanoparticles are auxiliary to PCR product sample progress matrix as co-substrate
Laser desorption ionisation flight time mass spectrum is helped to detect, the step of detection is as follows:
(a) preparation of instrument and reagent: the mass signal of laser desorption ionisation mass spectrograph, only signal-to-noise ratio greater than 6 is used for
Analysis.Using bruker microflex MALDI-TOF (TOF) mass spectrum, using nitrogen laser, wavelength 337nm.Using
Impulse electric field delay is extracted and linear working method, positive ion mode are detected.Using flexAnalysis observation, place
Reason, analysis data.
(b)Fe3O4/SiO2The preparation of/Pt Core-shell Structure Nanoparticles.
(c) it prepares oligonucleotides biological sample: target fragment PCR prolonging SNP site as single base including extracting DNA
It stretches.
(d) successively it is cleaned by ultrasonic the total 1h of mass spectrum target plate with dehydrated alcohol, deionized water.
(e) by Fe3O4/SiO2/ Pt Core-shell Structure Nanoparticles ultrasonic disperse is in deionized water, by it with liquid-transfering gun point
Sample is dried at room temperature on mass spectrum target plate.
(f) oligonucleotides biological sample point sample is dried at room temperature in the nanomatrix surface dried.
(g) organic substrate is dissolved in the solvent matched in proportion, point sample is in the oligonucleotides biological sample table dried
Face makes three form recrystallization, is used for laser desorption ionisation mass spectral analysis.
Mass Spectrometer Method result as shown in figure 4, figure 4, it is seen that the present invention multiple PCR products can be obtained it is high-quality
Spectrogram is measured, all 34 SNP sites can be measured.
Comparative example 1
A kind of application of nanomatrix in detection of nucleic acids, the difference from embodiment 1 is that:
The structure of the nanomatrix is ferroso-ferric oxide/silica/silver, i.e. Fe3O4/SiO2/Ag。
The method of detection is the same as embodiment 1.
Comparative example 2
A kind of application of nanomatrix in detection of nucleic acids, the difference from embodiment 1 is that:
The structure of the nanomatrix is ferroso-ferric oxide/silica/silver-platinum alloy, i.e. Fe3O4/SiO2/ Ag-Pt.
The method of detection is the same as embodiment 1.
Comparative example 1, comparative example 2 are compared with above-described embodiment, or by other nanomatrixes and above-mentioned implementation
Example compares, and a large amount of experiment results show nanomatrix of the invention, and effect is most in nucleic acid Mass Spectrometer Method
It is good.Other noble metals in addition to Pt may will cause influence to nucleic acid itself, it is difficult to desorption ionization, so that obtained mass spectrum
Signal quality is bad.
It can be seen from the above nanomatrix of the invention is applied in nucleic acid Mass Spectrometer Method, mainly have the following characteristics that
Sensitivity improves: the synergistic effect with organic substrate improves the desorption ionization effect of oligonucleotides, realizes low dense
Spend the detection of sample.
It is reproducible: to help organic substrate crystallization more evenly, keep testing result repeatability more preferable.
Sample detection overall process is very quick: entire detection process, it is only necessary to which a few minutes can be obtained by last detection
As a result.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: its according to
So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into
Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme.
Claims (10)
1. a kind of application of nanomatrix in detection of nucleic acids, which is characterized in that the structure of the nanomatrix is stratum nucleare/centre
Shell/outer shell；
Wherein, the stratum nucleare is magnetic oxide, and the subshell is Si oxide, and the outer shell is platinum.
2. application of the nanomatrix according to claim 1 in detection of nucleic acids, which is characterized in that the nanomatrix tool
There is coarse surface；
And/or partial size < 1000nm, preferably≤900nm, the further preferably≤800nm of the nanomatrix, further
Preferably 200~700nm.
3. application of the nanomatrix according to claim 1 in detection of nucleic acids, which is characterized in that the stratum nucleare is spherical shape
Or spherical particle, the partial size of particle are 100~400nm, preferably 120~380nm, further preferably 150~350nm；
And/or the subshell with a thickness of 20~200nm, preferably 50~180nm, further preferably 80~
160nm；
And/or the outer shell with a thickness of 5~80nm, preferably 10~50nm, further preferably 20~30nm.
4. application of described in any item nanomatrixes in detection of nucleic acids according to claim 1~3, which is characterized in that described
Magnetic oxide includes one of ferroso-ferric oxide, di-iron trioxide or ferrite or a variety of, preferably ferroso-ferric oxide；
And/or the Si oxide includes silicon monoxide and/or silica, preferably silica.
5. application of described in any item nanomatrixes in detection of nucleic acids according to claim 1~3, which is characterized in that described
The preparation method of nanomatrix includes:
Magnetic oxide particle is added and is reacted into the solution system for be used to generate Si oxide, Si oxide packet is obtained
Cover the particle of magnetic oxide；
The particle of the Si oxide coated magnetic oxide is added anti-to being used to react progress in the solution system for generating platinum
It answers, obtains magnetic oxide/Si oxide/platinum structure nuclear shell structure nano matrix.
6. application of described in any item nanomatrixes in detection of nucleic acids according to claim 1~3, which is characterized in that described
The method of detection of nucleic acids includes:
By nucleic acid samples and co-substrate, Multiple loading is carried out on substance assistant laser desorpted ionized mass spectrometric target plate, is done
Detection is analyzed by mass spectrometry after dry；
Wherein, the co-substrate includes organic substrate and the nanomatrix；
Preferably, the organic substrate includes 2,4,6-THAP and/or 3-HPA；
Preferably, target plate is pre-processed before Multiple loading, pretreated mode include: successively with low-carbon alcohol solution and
Water is cleaned by ultrasonic target plate, and the time of cleaning is 30~90min, preferably 50~60min.
7. application of the nanomatrix according to claim 6 in detection of nucleic acids, which is characterized in that the Multiple loading
Mode includes:
First by the water-soluble liquid spotting of nanomatrix on mass spectrum target plate, then by nucleic acid samples point sample in the nanomatrix table dried
Face, finally by organic substrate solution point sample in the nucleic acid samples surface dried, drying.
8. application of the nanomatrix according to claim 6 in detection of nucleic acids, which is characterized in that the Multiple loading
Mode includes:
First by the water-soluble liquid spotting of nanomatrix on mass spectrum target plate, then nucleic acid samples are uniformly mixed with organic substrate solution,
Mixture is obtained, by the mixture point sample in the nanomatrix surface dried, drying.
9. application of described in any item nanomatrixes in detection of nucleic acids according to claim 1~3, which is characterized in that nucleic acid
Sample includes that biology extracts at least one of DNA fragmentation, PCR product and oligonucleotide fragment.
10. application of described in any item nanomatrixes in detection of nucleic acids according to claim 1~3, which is characterized in that nucleic acid
The detection molecules amount of detection method is in 30000Da or less；
And/or substance assistant laser desorpted ionized mass spectrometric testing conditions include: positive ion detection, the signal-to-noise ratio detected
Mass signal greater than 6 is the useful signal that can be analyzed.
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