CN109444251B

CN109444251B - Application of nano matrix in nucleic acid detection

Info

Publication number: CN109444251B
Application number: CN201811404804.2A
Authority: CN
Inventors: 万馨泽; 吴舒; 刘彬; 陈伟
Original assignee: Yinapu Zhejiang Biotechnology Co ltd
Current assignee: Yinapu Zhejiang Biotechnology Co ltd
Priority date: 2018-11-23
Filing date: 2018-11-23
Publication date: 2021-12-21
Anticipated expiration: 2038-11-23
Also published as: CN109444251A

Abstract

The invention belongs to the technical field of mass spectrometry detection, and relates to application of a nano matrix in nucleic acid detection. The application of the nano-matrix in nucleic acid detection is characterized in that the structure of the nano-matrix is a nuclear layer/an intermediate shell layer/an outer shell layer; the core layer is made of magnetic oxide, the middle shell layer is made of silicon oxide, and the outer shell layer is made of platinum. The core-shell structure nano matrix is easy to prepare, separate and purify, the condition of nonuniform crystallization of the organic matrix can be improved, the desorption ionization effect of the oligonucleotide is increased through the plasmon effect of the noble metal, and the sensitivity of nucleic acid mass spectrum detection is increased. As a means for simply, quickly and high-flux detecting an oligonucleotide sample, the invention has good application prospect and is worthy of popularization and application.

Description

Application of nano matrix in nucleic acid detection

Technical Field

The invention belongs to the technical field of mass spectrometry detection, and particularly relates to application of a nano matrix in nucleic acid detection.

Background

Matrix-assisted laser desorption ionization (MALDI), which is a "soft ionization" mode, can generate stable gaseous ions with less energy, and is one of the most important ionization modes for high molecular weight compounds that are less volatile. The ionization technology is combined with time of flight mass spectrometry (TOF-MS), and the biomacromolecule can be detected quickly, efficiently and reliably. Research shows that matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) can be effectively applied to the detection of oligonucleotides, such as the analysis of Single Nucleotide Polymorphisms (SNPs) and cytosine methylation markers; especially, the method plays an important role in the detection of SNP in a plurality of fields such as biology, medicine, agriculture and the like. Compared with other research methods, MALDI-TOF-MS is an analysis mode which has high flux and high precision and is relatively simple and convenient, so that MALDI-TOF-MS is widely applied.

Fragmentation of DNA fragments during laser desorption ionization is a common phenomenon, which can be induced by protonation of bases, resulting in loss of bases and ultimately in loss of signal strength of intact DNA fragments. The fragmentation of DNA is mainly determined by the nature of the matrix used, and in the process, the matrix plays a role in absorbing, transmitting laser energy to the sample to be tested and ionizing the sample to be tested. MALDI-TOF-MS conventional matrices are typically organic matrices. Ferulic acid, 2, 5-dihydroxybenzoic acid (2, 5-DHB), was used as a substrate for mass spectrometric analysis of nucleic acids, but it has been rarely used in practice due to its low sensitivity. 2,4, 6-trihydroxyacetophenone (2, 4, 6-THAP) and 3-hydroxypicolinic acid (3-HPA) are commonly used as matrixes for nucleic acid mass spectrometry, but because of nonuniform crystallization, the matrixes can only play a role at high concentration, and because the excessive quantity of the matrixes can cause excessive energy after laser energy is absorbed, nucleic acid fragments are fragmented. At present, the common problem of all the traditional organic matrixes is the problem of 'effective points', so that the repeatability of experimental results is poor; meanwhile, because the effect of desorption ionization on nucleic acid samples is not ideal enough, only nucleic acid molecules within a specific molecular weight range can be detected, and the nucleic acid molecules are generally below 10000 Da.

In addition, with the research and development of matrix of MALDI-TOF-MS, some inorganic nano materials are beginning to be explored as matrix of MALDI-TOF-MS or as reinforcing material of organic matrix, such as carbon nano tube, graphene, porous silicon, silicon nano particle, gold nano particle, etc., and have shown a certain application prospect. However, in nucleic acid samples, due to the complexity of the samples, the ionization efficiency of the samples to be detected can be seriously disturbed, and the matrix is still difficult to satisfy the application in the detection of nucleic acid mass spectrometry. Therefore, for nucleic acid analysis by MALDI-TOF-MS, it is desirable to provide a matrix material with higher efficiency, sensitivity, and reproducibility, which can solve at least one of the above problems.

In view of this, the invention is particularly proposed.

Disclosure of Invention

An object of the present invention is to provide a method for applying nanomatrix in nucleic acid detection, which has the characteristics of high sensitivity, good repeatability, simple operation, rapidness, high efficiency, etc., and can overcome the above problems or at least partially solve the above technical problems.

In order to achieve the purpose, the invention adopts the technical scheme that:

according to one aspect of the present invention, the present invention provides a use of a nanomatrix in nucleic acid detection, wherein the nanomatrix has a structure of a core layer/an intermediate shell layer/an outer shell layer;

the core layer is made of magnetic oxide, the middle shell layer is made of silicon oxide, and the outer shell layer is made of platinum.

As a further preferable technical solution, the nanomatrix has a rough surface;

and/or the particle size of the nano matrix is less than 1000nm, preferably less than or equal to 900nm, more preferably less than or equal to 800nm, and more preferably 200-700 nm.

According to a further preferable technical scheme, the core layer is spherical or spheroidal particles, and the particle size of the particles is 100-400 nm, preferably 120-380 nm, and further preferably 150-350 nm;

and/or the thickness of the intermediate shell layer is 20-200 nm, preferably 50-180 nm, and further preferably 80-160 nm;

and/or the thickness of the shell layer is 5-80 nm, preferably 10-50 nm, and further preferably 20-30 nm.

As a further preferable technical solution, the magnetic oxide includes one or more of ferroferric oxide, iron sesquioxide, or ferrite, and is preferably ferroferric oxide;

and/or the silicon oxide comprises silicon monoxide and/or silicon dioxide, preferably silicon dioxide.

As a further preferred technical solution, the method for preparing the nanomatrix comprises:

adding magnetic oxide particles into a solution system for generating silicon oxide to react to obtain silicon oxide coated magnetic oxide particles;

and adding the silicon oxide coated magnetic oxide particles into a solution system for generating platinum through reaction to react to obtain the core-shell structure nano matrix with the magnetic oxide/silicon oxide/platinum structure.

In a further preferred embodiment, the method for detecting nucleic acid comprises:

carrying out composite sample application on a nucleic acid sample and the co-matrix on a target plate of a matrix-assisted laser desorption ionization mass spectrometer, drying and carrying out mass spectrometry detection;

wherein the co-matrix comprises an organic matrix and the nanomatrix;

preferably, the organic matrix comprises 2,4,6-THAP and/or 3-HPA;

preferably, the target plate is pre-treated prior to complex spotting in a manner comprising: and (3) carrying out ultrasonic cleaning on the target plate by using a low-carbon alcohol solution and water in sequence, wherein the cleaning time is 30-90 min, and preferably 50-60 min.

As a further preferred technical solution, the complex spotting method comprises:

firstly spotting the nano matrix aqueous solution on a mass spectrum target plate, then spotting the nucleic acid sample on the surface of the dried nano matrix, finally spotting the organic matrix solution on the surface of the dried nucleic acid sample, and drying.

firstly spotting a nano matrix aqueous solution on a mass spectrum target plate, then uniformly mixing a nucleic acid sample and an organic matrix solution to obtain a mixture, spotting the mixture on the surface of a dried nano matrix, and drying.

As a further preferred embodiment, the nucleic acid sample comprises at least one of a biologically extracted DNA fragment, a PCR product, and an oligonucleotide fragment.

As a further preferred embodiment, the nucleic acid detection method has a detection molecular weight of 30000Da or less;

and/or the detection conditions of the matrix-assisted laser desorption ionization mass spectrometer comprise: and detecting positive ions, wherein the detected mass spectrum signal with the signal-to-noise ratio of more than 6 is an effective signal which can be analyzed.

Compared with the prior art, the invention has the beneficial effects that:

(1) the nanometer matrix with a core-shell structure is applied to the field of nucleic acid detection, the nanometer matrix takes a magnetic oxide as a core layer, a silicon oxide is coated to form an intermediate shell layer, platinum nanoparticles are further coated to form an outer shell layer structure, the nanometer matrix is convenient to be separated and purified by equipment due to the magnetic property of the magnetic oxide, and the nanometer matrix has a stable structure, a large surface area and unique surface plasma resonance and hot carriers provided by the platinum nanoparticles, so that the problem of uneven crystallization of the existing organic matrix is relieved on one hand, and the desorption ionization effect of a nucleic acid sample is promoted on the other hand.

(2) The detection sensitivity is improved, and the repeatability is good: the nano matrix with the core-shell structure provided by the invention can obviously improve the desorption ionization effect of a nucleic acid sample, realize accurate identification of the sample and greatly improve the sensitivity and the repeatability of detection.

(3) The consumption of the detection sample is low: the nano matrix of the invention greatly improves the desorption ionization of nucleic acid samples, combines the characteristics of mass spectrum detection, can obviously reduce the consumption of the samples and realize the detection of low-concentration samples.

(4) The detection method is simple and efficient: the detection process is simple and convenient to operate and very quick, and the final detection result can be obtained only in a few minutes in the whole detection process.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 shows Fe according to an embodiment of the present invention₃O₄/SiO₂Pt core-shell junctionA characterization map of the structured nanoparticles; FIG. 1(a) is an SEM image, and FIG. 1(b) is a TEM image;

FIG. 2 shows Fe according to an embodiment of the present invention₃O₄/SiO₂A characterization diagram of the Pt core-shell structure nano matrix and the organic matrix during co-crystallization;

FIG. 3 shows Fe according to an embodiment of the present invention₃O₄/SiO₂The Pt core-shell structure nano-particles are mass spectrograms obtained by detecting a section of oligonucleotide standard (with the molecular weight of 5605.7) with the sequence of GCAGACGATCCTGGGGGG by using a co-matrix;

FIG. 4 shows Fe provided in an embodiment of the present invention₃O₄/SiO₂The Pt core-shell structure nano-particles are mass spectrograms obtained by detecting multiple PCR product oligonucleotide samples of 34 SNP sites related to the neonatal deafness gene by using a co-matrix.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to embodiments and examples, but those skilled in the art will understand that the following embodiments and examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. Those who do not specify the conditions are performed according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

In a first aspect, there is provided in at least one embodiment a use of a nanomatrix in nucleic acid testing, the nanomatrix having the structure of a core layer/intermediate shell layer/outer shell layer;

the core layer is made of magnetic oxide, the middle shell layer is made of silicon oxide, and the outer shell layer is made of platinum (Pt).

In view of the defects of the prior MALDI-TOF-MS matrix in nucleic acid detection, which limits the application of MALDI-TOF-MS in nucleic acid sample analysis and detection, a new matrix is needed and applied in nucleic acid sample detection to improve the defects of the prior art. Based on this, the invention proposes a new matrix, and the related acquisition method and application.

The nano matrix with the core-shell structure provided by the invention is mainly applied to matrix-assisted laser desorption ionization time-of-flight mass spectrometry detection of nucleic acid samples, and can overcome the defects of the traditional matrix. Furthermore, the invention changes the material structure of the nanometer matrix, uses the magnetic oxide as a core layer, uses the silicon oxide to continuously coat and form a middle shell layer, and uses the platinum nanometer particle to continuously coat and form an outer shell layer structure.

Therefore, the core-shell structure nano matrix is easy to prepare, separate and purify, the condition of nonuniform crystallization of the organic matrix can be improved, the desorption ionization effect of the oligonucleotide is increased through the plasmon effect of the noble metal, and the sensitivity of nucleic acid mass spectrum detection is increased. The invention is a simple, rapid, high-sensitivity, good-repeatability and high-throughput means for detecting nucleic acid samples, has good application prospect and is worth popularizing and applying.

The core-shell structure nanomatrix of the core layer/intermediate layer/outer shell layer of the present invention may also be expressed as a core-shell structure nanomatrix of the core layer @ intermediate layer @ outer shell layer.

The following is a detailed description of the nanomatrix material and the application method thereof in nucleic acid detection according to the embodiments of the present invention:

the nano matrix contains particles with a core-shell structure, and the particles are of a multilayer core-shell structure; further, the particulate matter generally has a three-layer structure including a core layer, an intermediate shell layer, and an outer shell layer, and the intermediate shell layer covers the core layer and the outer shell layer covers the intermediate shell layer. It is noted that in some examples, the intermediate shell layer is wrapped outside the core layer, and is not intended to limit the intermediate shell layer to have to be completely wrapped outside the core layer. For example, the silicon oxide particles constituting the intermediate shell layer may be mixed in an appropriate amount in the magnetic oxide of the core layer; the same is true for the outer shell layer that is wrapped around the intermediate shell layer.

The nano matrix is generally spherical (or sphere-like) nano particles, and in general, the nano matrix material provided by the embodiment of the invention is nano-scale particles with uniform granularity; the core layer, the intermediate shell layer and the outer shell layer are all particles, and further, the core layer, the intermediate shell layer and the outer layer are all in a nanometer scale.

In some examples of the invention, the nanomatrix has a rough surface;

According to the invention, the nanomatrix has a rough surface, in other words the outer shell layer has a rough surface. The term "rough surface" is understood to mean a surface which is not fine, smooth or delicate. In some examples of the invention, the platinum nanoparticles comprising the outer shell are loosely packed outside the intermediate shell, with appropriate nanoscale gaps formed between the platinum nanoparticles. The nano matrix adopts metal platinum nano particles to form a shell layer and has a rough surface, and when the nano matrix is applied to nucleic acid mass spectrometry detection, the desorption ionization process can be promoted to the maximum extent in the contact process of the nano particles and oligonucleotide molecules.

The particle size of the nano matrix is less than 1 mu m, the particle size is uniform, the particle size of the nano matrix (nano spherical particles) is preferably less than or equal to 900nm, more preferably less than or equal to 800nm, and more preferably 200-700 nm; typical but non-limiting examples are 200nm, 30nm, 400nm, 500nm, 600nm, 700nm or 800 nm.

In some examples of the present invention, the core layer is spherical or spheroidal particle, and the particle size of the particle is 100 to 400nm, preferably 120 to 380nm, and more preferably 150 to 350 nm; typical but non-limiting examples are 100nm, 120nm, 150nm, 180nm, 200nm, 250nm, 300nm, 350nm, 380nm or 400 nm.

And/or the thickness of the intermediate shell layer is 20-200 nm, preferably 50-180 nm, and further preferably 80-160 nm; typical but non-limiting examples are 20nm, 50nm, 80nm, 100nm, 120nm, 150nm, 160nm, 180nm or 200 nm.

And/or the thickness of the shell layer is 5-80 nm, preferably 10-50 nm, and further preferably 20-30 nm; typical but non-limiting examples are 5nm, 10nm, 20nm, 30nm, 40nm, 50nm, 60nm, 70nm or 80 nm.

The particle size of the core layer particles and the thicknesses of the intermediate shell layer and the outer layer are suitable, so that the spherical particle core can be well protected, the hot electron generation rate can be higher, and the hot carrier effect is promoted.

In some examples of the invention, the magnetic oxide comprises one or more of ferroferric oxide, ferric oxide, or ferrite;

and/or, the silicon oxide comprises silicon monoxide and/or silicon dioxide;

preferably, the magnetic oxide is ferroferric oxide;

preferably, the silicon oxide is silicon dioxide.

It is understood that there are many alternative materials for the magnetic oxide constituting the core layer of the present invention, such as one or a mixture of several of ferroferric oxide, ferric oxide, or ferrite, but not limited thereto, and in other embodiments of the present invention, there may be other various choices for the magnetic oxide. Preferably, the magnetic oxide forming the core layer of the invention is selected from ferroferric oxide (Fe) which has wide source, is easy to obtain and has better matching application effect with the intermediate shell layer and the outer shell layer₃O₄)。

It is understood that there are many alternative materials for the silicon oxide constituting the intermediate shell layer of the present invention, such as one or a mixture of two of silicon monoxide and silicon dioxide, but not limited thereto, and in other embodiments of the present invention, there may be other various choices for the silicon oxide. Preferably, constitute the present inventionThe silicon oxide of the bright intermediate shell layer is selected from silicon dioxide (SiO) which has wide source, low cost and better matching application effect with the nuclear layer and the outer shell layer₂)。

In a preferred embodiment of the present invention, the structure of the nanomatrix is ferroferric oxide/silica/platinum, i.e. Fe₃O₄/SiO₂/Pt。

According to the invention, the core layer, the intermediate shell layer and the outer shell layer of the core-shell structure nano matrix particles are obtained by layer-by-layer (successive) reaction. For example, a magnetic oxide constituting a core layer is first produced by a reaction, an intermediate shell layer is grown on the surface of the core layer based on the core layer, and an outer shell layer is further grown on the surface of the intermediate shell layer based on the intermediate shell layer.

In some examples of the invention, the method of preparing the nanomatrix comprises:

adding the prepared magnetic oxide particles into a solution system for generating silicon oxide to react to obtain silicon oxide coated magnetic oxide particles;

and adding the silicon oxide coated magnetic oxide particles into a solution system for generating platinum through reaction to react to prepare the magnetic oxide/silicon oxide/platinum structure core-shell structure nano matrix.

It should be noted that the preparation method of the nanomatrix of the present invention can adopt a method commonly used in the art, and the present invention is not limited to the specific preparation process. The following description will be made in more detail mainly by taking the example of the nano-matrix with a ferroferric oxide/silica/platinum structure, however, it should be understood that the manufacturing method is also applicable to the preparation of the nano-matrix with a similar structure such as a ferric oxide/silica/platinum structure.

In a preferred embodiment, the method for preparing a nanomatrix comprises the steps of:

s1 using FeCl₃·6H₂Taking O as an iron source, simultaneously adding trisodium citrate and sodium acetate for modification, and taking ethylene glycol as a solvent for thermal reaction to prepare the monodisperse Fe₃O₄A nanoparticle; reaction stripThe piece of equipment includes: the temperature is 200 ℃, and the time is 8-72 h.

Wherein, the glycol is used as a solvent and a reducing agent; the trisodium citrate and the sodium acetate can improve the stability and the dispersibility of the system. Trisodium citrate can be used as a stabilizer and complexed with iron ions, so that the agglomeration of the iron ions is prevented; sodium acetate can increase the electrostatic stability of the magnetic oxide particles, preventing their agglomeration.

Fe obtained₃O₄The particle size of the nanoparticles can be adjusted by adjusting the amount of the iron source and/or trisodium citrate to meet the actual demand.

S2 production of Fe from S1₃O₄Dispersing the nano particles in a mixed solvent of water and ethanol, catalyzing tetraethyl orthosilicate with ammonia water for hydrolytic polycondensation nucleation, and reacting for 2-8 h at normal temperature to obtain Fe₃O₄Forming silicon dioxide layers with different thicknesses on the surfaces of the nano particles to obtain Fe₃O₄/SiO₂Core-shell structured nanoparticles.

Wherein, water and ethanol promote tetraethyl orthosilicate to hydrolyze to form gel, and alkaline ammonia water is used as a catalyst to promote the formation of silicon dioxide microspheres. In general, in Fe₃O₄The thickness of the silica layer formed on the surface of the nanoparticles is relatively uniform, but in some examples, the thickness of the silica layer may be slightly different, and the thickness of the layer may be controlled within a preferred range by controlling the reaction conditions.

S3 production of Fe from S2₃O₄/SiO₂The core-shell structure nano particles are evenly dispersed in absolute ethyl alcohol by ultrasonic, 3-aminopropyl triethoxysilane is added to be stirred for 6 to 12 hours to form Fe₃O₄/SiO₂－NH₂A nanoparticle;

s4, dissolving polyvinylpyrrolidone in a chloroplatinic acid solution to obtain a platinum source solution;

the polyvinylpyrrolidone therein can stabilize the system against agglomeration.

S5, Fe formed from S3₃O₄/SiO₂－NH₂Dispersing the nano particles in a platinum source solution of S4, and stirring for 2h to ensure that the platinum is dispersedIons are uniformly distributed on the surface of the nano-particles to obtain Fe₃O₄/SiO₂－NH₂A nanoparticle dispersion solution;

s6 Fe obtained at S5₃O₄/SiO₂－NH₂Adding reducing agent sodium borohydride into the nanoparticle dispersion solution, stirring for 8-12 h at normal temperature to obtain Fe₃O₄/SiO₂Pt core-shell structure nanoparticles;

s7, washing Fe obtained in S6 repeatedly with ethanol and deionized water₃O₄/SiO₂And drying the Pt core-shell structure nano particles into powder in a drying oven at the temperature of 60 ℃.

The method is simple, easy to operate and easy to control, the prepared product has good performance stability, the regulation and control of the structure, the thickness and the like of the nuclear layer, the intermediate shell layer and the outer shell layer are realized, the yield is high, and the large-scale production is easy to realize.

The invention provides Fe₃O₄/SiO₂The Pt core-shell structure nano particles can be used as a matrix to be applied to mass spectrometry. For example in matrix assisted laser desorption ionization mass spectrometry detection analyzers. In application, the nanomatrix is dispersed (resuspended) in a dispersion liquid as a mass spectrometry matrix; among them, the dispersion liquid is preferably water.

Therefore, the invention also provides a detection method, which adopts the nano matrix and utilizes the matrix-assisted laser desorption ionization mass spectrometer for detection. In view of the specificity of the nanomatrix, the target detection object in the detection method is mainly a nucleic acid sample, and therefore, the invention provides the Fe₃O₄/SiO₂The Pt core-shell structure nano-particles are applied to nucleic acid mass spectrometry detection.

In some examples of the invention, the nucleic acid sample comprises biologically extracted DNA fragments, PCR products, and oligonucleotide fragments derived from other pathways.

Furthermore, in the mass spectrometric detection of the nucleic acid sample, the molecular weight range is less than or equal to 30000 Da.

Further, the laser desorption ionization mass spectrometer is bruker autoflex^TMspeed MALDI-TOF (TOF) Mass Spectrometry using either a smartpeak-II laser at 355nm or bruker microflex-TOF (TOF) Mass Spectrometry using a nitrogen laser at 337 nm.

Further, analysis processing of data was performed using flexAnalysis.

Therefore, the nano matrix disclosed by the invention is applied to the mass spectrometry of the nucleic acid sample, the molecular weight range of detection is widened, the existing nucleic acid molecules which can only detect a specific molecular weight (generally below 10000 Da) range are relieved, and the application range is wider.

In some examples of the present invention, the core-shell structure nanomatrix is used as a matrix for nucleic acid mass spectrometry detection, and a specific analysis detection method thereof comprises the following steps:

(a) the method comprises the following steps Preparation of instruments and reagents: the laser desorption ionization mass spectrometer adopts a pulse electric field delay extraction signal and a positive ion linear mode, and only adopts a signal with a signal-to-noise ratio larger than 6 for analysis;

(b) the method comprises the following steps Resuspending the core-shell structure nano matrix in deionized water to serve as a co-matrix;

(c) the method comprises the following steps Preparing a nucleic acid sample, wherein the oligonucleotide sample is subjected to desalting treatment to be used as an analyte;

(d) the method comprises the following steps Preparing organic matrix (such as 2,4,6-THAP or 3-HPA) into solution according to different modes to serve as co-matrix;

it should be noted here that the method for preparing the organic matrix is prior art, and the present invention is not described in detail herein.

(e) The method comprises the following steps Carrying out composite sample application on the core-shell structure nano matrix, the oligonucleotide sample and the organic matrix solution on a target plate;

(f) the method comprises the following steps And analyzing the mass spectrum detection result to obtain a conclusion.

Furthermore, the mass spectrometry target plate needs to be a clean target plate, and the target plate needs to be pretreated before use, for example, the target plate can be sequentially cleaned by absolute ethyl alcohol and deionized water for about 60min, so as to remove the interference of impurities on the target plate.

Further, the composite sample application comprises two optional sample application modes, namely:

the first spotting method: firstly spotting the nano matrix aqueous solution on a mass spectrum target plate by using a pipette, drying at room temperature, then spotting the nucleic acid sample on the surface of the nano matrix by using the pipette, drying at room temperature, finally spotting the organic matrix solution on the surface of the dried nucleic acid sample, and drying at room temperature.

The second spotting method: firstly, spotting the nano matrix aqueous solution on a mass spectrum target plate by using a pipette, drying at room temperature, then uniformly mixing the nucleic acid sample and the organic matrix solution, spotting on the dried nano matrix surface by using the pipette, and drying at room temperature.

The present invention will be further described with reference to specific examples, comparative examples and the accompanying drawings.

Example 1

Use of a nanomatrix in nucleic acid detection comprising:

1. preparing a nano matrix:

s1 using FeCl₃·6H₂Taking O as an iron source, simultaneously adding trisodium citrate and sodium acetate for modification, and taking ethylene glycol as a solvent for thermal reaction to prepare the monodisperse Fe₃O₄A nanoparticle; the reaction conditions include: the temperature is 200 ℃, and the time is 8-72 h;

s2 production of Fe from S1₃O₄Dispersing the nano particles in a mixed solvent of water and ethanol, catalyzing tetraethyl orthosilicate with ammonia water for hydrolytic polycondensation nucleation, and reacting for 2-8 h at normal temperature to obtain Fe₃O₄Forming silicon dioxide layers with different thicknesses on the surfaces of the nano particles to obtain Fe₃O₄/SiO₂Core-shell structured nanoparticles;

s5, Fe formed from S3₃O₄/SiO₂－NH₂Dispersing the nano particles in a platinum source solution of S4, stirring for 2h to ensure that platinum ions are uniformly distributed on the surfaces of the nano particles to obtain Fe₃O₄/SiO₂－NH₂A nanoparticle dispersion solution;

s7, washing Fe obtained in S6 repeatedly with ethanol and deionized water₃O₄/SiO₂Drying Pt core-shell structure nano particles into powder in a drying oven at 60 ℃;

s8, resuspending the nanoparticles obtained in S8 in deionized water, and using them as a mass spectrometry matrix.

Characterization of the matrix:

the instrumentation used for characterization included: the size and morphology characterization of the product is completed on a JEOL JEM-2100F Transmission Electron Microscope (TEM), a JEOL JEM-2100F high-resolution transmission electron microscope (HRTEM) and a Hitachi S-4800 Scanning Electron Microscope (SEM).

The characterization result is as follows:

as shown in fig. 1, fig. 1(a) is an SEM characterization of the nanoparticles, and fig. 1(b) is a TEM characterization. Fe₃O₄/SiO₂The Pt core-shell structure nano particles are nano spherical particles, the particle size is uniform, and the average particle size is about 400 nm. The results of the high-resolution scanning electron microscope show that the particle surface is rough and not smooth. The transmission electron microscope result shows that the roughness of the surface is composed of nano-spheres with the diameter of about 5-10 nm.

2. Detection of nucleic acid samples

A standard oligonucleotide whose detection sequence is GCAGACGATCCTGGGGGG

Utilizing the above-mentioned Fe₃O₄/SiO₂Matrix-assisted laser desorption ionization flight time of oligonucleotide standard substance by taking Pt core-shell structure nano particles as co-matrixMass spectrum detection, the detection steps are as follows:

(a) preparation of instruments and reagents: laser desorption ionization mass spectrometers, only mass spectra signals with a signal-to-noise ratio of greater than 6 are used for analysis. The Bruker microflex MALDI-TOF (TOF) mass spectrum was used with a nitrogen laser at 337 nm. And a pulse electric field delay extraction and linear working mode and a positive ion mode are adopted for detection. Data were observed, processed, and analyzed using flexAnalysis.

(b)Fe₃O₄/SiO₂Preparing Pt core-shell structure nano particles.

(c) Preparation of oligonucleotide standard samples: synthesized by Competition Biotechnology engineering (Shanghai) Ltd, and purified by HPLC CE method.

(d) And ultrasonically cleaning the mass spectrum target plate for 1h by using absolute ethyl alcohol and deionized water in sequence.

(e) Mixing Fe₃O₄/SiO₂The Pt core-shell structure nano particles are ultrasonically dispersed in deionized water, are spotted on a mass spectrum target plate by a pipette, and are dried at room temperature.

(f) And (3) spotting the oligonucleotide standard sample on the dried surface of the nano matrix, and drying at room temperature.

(g) Dissolving the organic matrix in a solvent in a ratio, and spotting on the surface of a dried oligonucleotide standard sample to form recrystallization for laser desorption ionization mass spectrometry.

The mass spectrum detection result is shown in fig. 3, and it can be seen from fig. 3 that the invention can obtain a mass spectrum signal with high quality.

In addition, FIG. 2 shows Fe₃O₄/SiO₂The characterization chart of the Pt core-shell structure nano matrix and the organic matrix during cocrystallization shows that the core-shell structure nano matrix can enable the organic matrix to be crystallized more uniformly as shown in figure 2.

Example 2

Use of a nanomatrix in nucleic acid detection comprising:

1. the nanomatrix was prepared as in example 1.

2. Detection of nucleic acid samples

Multiplex PCR product sample oligonucleotide fragment for detecting 34 SNP sites related to neonatal deafness gene

Utilizing the above-mentioned Fe₃O₄/SiO₂Performing matrix-assisted laser desorption ionization time-of-flight mass spectrometry detection on a PCR product sample by taking Pt core-shell structure nanoparticles as a co-matrix, wherein the detection steps are as follows:

(b)Fe₃O₄/SiO₂Preparing Pt core-shell structure nano particles.

(c) Preparing an oligonucleotide biological sample: comprises extracting DNA, carrying out PCR on a target fragment and carrying out single base extension on an SNP locus.

(f) And (3) spotting the oligonucleotide biological sample on the dried surface of the nano matrix, and drying at room temperature.

(g) Dissolving the organic matrix in a solvent in a ratio, and spotting on the surface of the dried oligonucleotide biological sample to form recrystallization for laser desorption ionization mass spectrometry.

The mass spectrum detection result is shown in FIG. 4, and it can be seen from FIG. 4 that the invention can obtain a high-quality spectrogram for a multiplex PCR product, and can detect all 34 SNP sites.

Comparative example 1

The application of the nano matrix in nucleic acid detection is different from the application of example 1 in that:

of the nanomatrixThe structure is ferroferric oxide/silicon dioxide/silver, namely Fe₃O₄/SiO₂/Ag。

The detection method was the same as in example 1.

Comparative example 2

the structure of the nano matrix is ferroferric oxide/silicon dioxide/silver platinum alloy, namely Fe₃O₄/SiO₂/Ag－Pt。

The detection method was the same as in example 1.

Comparing the comparative example 1 and the comparative example 2 with the above examples, or comparing other nanomatrix with the above examples, a large number of experimental verification results show that the nanomatrix of the invention has the best effect in the detection of nucleic acid mass spectrometry. Other noble metals besides Pt may have influence on the nucleic acid itself, and desorption ionization is difficult, so that the quality of the obtained mass spectrum signal is poor.

From the above, the nano-matrix of the invention is applied to nucleic acid mass spectrometry detection, and mainly has the following characteristics:

the sensitivity is improved: the synergistic effect of the oligonucleotide and the organic matrix improves the desorption ionization effect of the oligonucleotide and realizes the detection of low-concentration samples.

The repeatability is good: the organic matrix is more uniformly crystallized, so that the repeatability of the detection result is better.

The whole process of sample detection is very quick: the whole detection process only needs a few minutes to obtain the final detection result.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of the nano matrix in nucleic acid detection is characterized in that the structure of the nano matrix is a nuclear layer/an intermediate shell layer/an outer shell layer;

the core layer is made of magnetic oxide, the middle shell layer is made of silicon oxide, and the outer shell layer is made of platinum;

the detection molecular weight of the nucleic acid detection method is below 30000 Da;

the nucleic acid sample comprises at least one of biologically extracted DNA fragments, PCR products, and oligonucleotide fragments.

2. Use of a nanomatrix according to claim 1 in nucleic acid detection, wherein the nanomatrix has a rough surface;

and/or the particle size of the nano matrix is less than 1000 nm.

3. The use of nanomatrix of claim 2 in nucleic acid testing, wherein the nanomatrix has a particle size of 900nm or less.

4. The use of nanomatrix of claim 3 in nucleic acid testing, wherein the nanomatrix has a particle size of 800nm or less.

5. The use of the nanomatrix of claim 4 in nucleic acid detection, wherein the nanomatrix has a particle size of 200 to 700 nm.

6. The application of the nanomatrix of claim 1 in nucleic acid detection, wherein the core layer is spherical or spheroidal particles, and the particle size of the particles is 100-400 nm;

and/or the thickness of the intermediate shell layer is 20-200 nm;

and/or the thickness of the shell layer is 5-80 nm.

7. The use of the nanomatrix of claim 6 in nucleic acid detection, wherein the core layer is spherical or spheroidal particles, and the particle size of the particles is 120-380 nm.

8. The use of the nanomatrix of claim 6 in nucleic acid detection, wherein the core layer is spherical or spheroidal particles, and the particle size of the particles is 150 to 350 nm.

9. The use of the nanomatrix of claim 6 in nucleic acid testing, wherein the thickness of the intermediate shell layer is 50 to 180 nm.

10. The use of the nanomatrix of claim 6 in nucleic acid detection, wherein the thickness of the intermediate shell layer is 80 to 160 nm.

11. The use of the nanomatrix of claim 6 in nucleic acid testing, wherein the shell layer has a thickness of 10 to 50 nm.

12. The use of a nanomatrix of claim 11 in nucleic acid testing, wherein the shell layer has a thickness of 20 to 30 nm.

13. The use of the nanomatrix of any one of claims 1 to 12 in nucleic acid detection, wherein the magnetic oxide comprises one or more of ferroferric oxide, ferric oxide, or ferrite;

and/or the silicon oxide comprises silicon monoxide and/or silicon dioxide.

14. The use of a nanomatrix of claim 13 in nucleic acid testing, wherein the magnetic oxide is ferroferric oxide.

15. Use of a nanomatrix according to claim 13 in nucleic acid testing, wherein the silicon oxide is silica.

16. The use of the nanomatrix of any one of claims 1 to 12 in nucleic acid testing, wherein the method of making the nanomatrix comprises:

17. Use of a nanomatrix of any one of claims 1 to 12 in nucleic acid testing, wherein the method of nucleic acid testing comprises:

wherein the co-matrix comprises an organic matrix and the nanomatrix.

18. Use of a nanomatrix of claim 17 in nucleic acid detection, wherein the organic matrix comprises 2,4,6-THAP and/or 3-HPA.

19. Use of a nanomatrix of claim 17 in nucleic acid testing, wherein the target plate is pre-treated prior to complex spotting by means of: and (3) carrying out ultrasonic cleaning on the target plate by using a low-carbon alcohol solution and water in sequence, wherein the cleaning time is 30-90 min.

20. The use of the nanomatrix of claim 19 in nucleic acid testing, wherein the washing time is 50 to 60 min.

21. Use of a nanomatrix according to claim 17 in nucleic acid testing, wherein the complex spotting means comprises:

22. The use of nanomatrix of claim 21 in nucleic acid testing, wherein the complex spotting format comprises:

23. The use of a nanomatrix of claim 17 in nucleic acid detection, wherein the detection conditions of a matrix assisted laser desorption ionization mass spectrometer comprise: and detecting positive ions, wherein the detected mass spectrum signal with the signal-to-noise ratio of more than 6 is an effective signal which can be analyzed.