CN117783262A - Chip matrix composition for time-of-flight mass spectrometry detection and application thereof - Google Patents

Abstract

The application belongs to the technical field of mass spectrometry, and particularly relates to a chip matrix composition for time-of-flight mass spectrometry detection and a method for detecting nucleic acid by using the time-of-flight mass spectrometry. The present application provides a chip matrix composition for time-of-flight mass spectrometry detection, comprising 3-hydroxy-2-picolinic acid, tri-ammonium citrate, ascorbic acid and ammonium oxalate. The solution components are prepared to ensure that the matrix solution can be evenly and densely separated out at the whole target position, and after the nucleic acid sample is added, the sample can be adsorbed on the matrix layer to form complete co-crystals without damaging the rigid structure of the co-crystals. The dried crystal can realize conventional automatic targeting on a mass spectrometer, the acquisition success rate reaches 100% by a uniform and complete recrystallization layer, the quality deviation of a detection result is less than 300ppm, the quality resolution can reach more than 1000, and the bottom of a spectrogram is clean.

Description

Chip matrix composition for time-of-flight mass spectrometry detection and application thereof

Technical Field

The application belongs to the technical field of mass spectrometry, and particularly relates to a chip matrix composition for time-of-flight mass spectrometry detection and application thereof.

Background

The advent of matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) in the 80 s of the 20 th century has brought a revolutionary breakthrough to the biological and medical fields. The analysis and research of biomacromolecules such as nucleic acid, protein and the like can be carried out by a mass spectrometer, so that the development of genomics and proteomics is greatly promoted. In 2014, the United states Food and Drug Administration (FDA) approved MALDI-TOF MS for clinical nucleic acid detection.

The nucleic acid mass spectrum combines biotechnology and mass spectrum technology, after the sample to be detected is subjected to multiplex PCR specific amplification designed by polymorphic sites and single base extension of SNP sites, the reacted product and a specific matrix form a co-crystal, then the co-crystal absorbs incident laser and provides energy for desorption and ionization of the nucleic acid sample in a mass spectrometer to generate ions with different mass-to-charge ratios, and a mass analyzer is used for measuring the molecular weight of different types of ions in the sample, wherein the specific matrix is the key of the nucleic acid mass spectrum analysis in the process.

In the analysis of biological macromolecules by MALDI, it is important to select an appropriate matrix, which is generally considered to have three functions: 1. absorbing energy; 2. so that the biological macromolecules are separated from each other; 3. the analyzed biomacromolecule is ionized. The substrates commonly used in the mass spectrometry of nucleic acids at present are organic substances such as 3-hydroxy-2-picolinic acid (3-HPA), 2,4, 6-trihydroxyacetophenone (2, 4, 6-THAP), alpha-cyano-4-hydroxycinnamic acid, 2, 5-dihydroxybenzoic acid, sinapic acid and the like, but as the relative molecular mass of DNA increases, the tendency of cleavage of DNA and formation of adducts increases, resulting in a decrease in the molecular ion yield of DNA. This undoubtedly increases the difficulty in identifying trace analytes from complex matrices by the instrument, and also causes problems of large mass deviation, low resolution and the like of detection results, thereby affecting the site weight of nucleic acid mass spectrometry detection.

In summary, the time-of-flight mass spectrometry detection in the traditional technology has the problems of low success rate of mass spectrometry signal acquisition and poor mass resolution.

Disclosure of Invention

Based on this, an embodiment of the present application provides a chip matrix composition for time-of-flight mass spectrometry detection and an application thereof, so as to improve the success rate of signal acquisition and mass resolution of nucleic acid mass spectrometry.

A chip matrix composition for time-of-flight mass spectrometry detection comprises a solvent, and 0.01-1 g/mL of 3-hydroxy-2-picolinic acid, 0.01-1 mol/L of 2-picolinic acid, 0.01-2 mol/L of tri-ammonium citrate, 0.01-2 mol/L of ascorbic acid and 0.01-2 mol/L of ammonium oxalate added in the solvent.

In one embodiment, the concentration of 3-hydroxy-2-pyridinecarboxylic acid is 0.6 g/mL-0.8 g/mL, the concentration of 2-pyridinecarboxylic acid is 0.65 mol/L-0.85 mol/L, the concentration of tri-ammonium citrate is 1 mol/L-1.5 mol/L, the concentration of ascorbic acid is 0.6 mol/L-1.4 mol/L, and the concentration of ammonium oxalate is 0.8 mol/L-1.2 mol/L.

In one embodiment, the solvent is selected from the group consisting of a mixed solvent of acetonitrile and water.

In one embodiment, the volume ratio of acetonitrile to water is (1:10): 20.

A method of time-of-flight mass spectrometry for detecting a nucleic acid comprising the steps of:

and co-crystallizing the sample to be detected and the chip matrix composition for the time-of-flight mass spectrometry detection on a chip substrate, and carrying out mass spectrometry detection.

In one embodiment, the method comprises the following steps:

and carrying out sample application, drying and crystallization on the chip matrix composition, prefabricating the chip matrix composition on the chip substrate to obtain a prefabricated chip substrate, taking a sample to be tested, and adding the sample to the prefabricated chip substrate.

In one embodiment, the sample to be tested is added in a spotted manner.

In one embodiment, the sample volume added at each spot is 0.1 μl to 0.2 μl.

In one embodiment, the co-crystallization is followed by a drying step.

In one embodiment, the sample to be tested comprises one or both of an oligonucleotide and a polynucleotide.

The application also provides application of the chip matrix composition for time-of-flight mass spectrometry detection in MALDI-TOFMS detection.

The application provides a chip matrix composition for time-of-flight mass spectrometry detection, which comprises a solvent, and 0.01-1 g/mL of 3-hydroxy-2-picolinic acid, 0.01-1 mol/L of 2-picolinic acid, 0.01-2 mol/L of tri-ammonium citrate, 0.01-2 mol/L of ascorbic acid and 0.01-2 mol/L of ammonium oxalate which are added in the solvent. The matrix solution can be ensured to be evenly and densely separated out at the whole target position through specific allocation of solution components. After addition of the nucleic acid sample, the sample can also adsorb to the matrix layer to form complete co-crystals without breaking its rigid structure. The dried crystal can realize conventional automatic targeting on a mass spectrometer, the acquisition success rate reaches 100% by a uniform and complete recrystallization layer, the quality deviation of a detection result is less than 300ppm, the quality resolution can reach more than 1000, and the bottom of a spectrogram is clean.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more fully understand the present application and its advantageous effects, the following brief description will be given with reference to the accompanying drawings, which are required to be used in the description of the embodiments. It is obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings may be obtained from these drawings without inventive effort to a person skilled in the art.

FIG. 1 is a diagram of a chip matrix composition and sample recrystallization according to the present application;

FIG. 2 is a schematic diagram of the success rate of mass spectrometry acquisition after use of the matrices of the present application;

FIG. 3 is a schematic diagram of mass deviation of an acquired mass spectrum;

FIG. 4 is a diagram of the bottom noise of the acquired mass spectrum;

FIG. 5 is a case of comparative example 1 recrystallization;

FIG. 6 is a diagram showing the case of comparative example 1 in which a single base extension product of a human ALDH2 genotyping detection reagent is tested;

FIG. 7 is a single base extension product of a genotyping test reagent for hereditary hearing loss of comparative example 1;

FIG. 8 is a recrystallization case of comparative example 2;

FIG. 9 is a single base extension product of a test reagent for genotyping human ALDH2 of comparative example 2;

FIG. 10 is a single base extension product of a genotyping test reagent for hereditary hearing loss of comparative example 2;

wherein intensity is intensity.

Detailed Description

The present application will be described in further detail with reference to embodiments and examples. It should be understood that these embodiments and examples are provided solely for the purpose of illustrating the application and are not intended to limit the scope of the application in order to provide a more thorough understanding of the present disclosure. It is also to be understood that this application may be embodied in many different forms and is not limited to the embodiments and examples described herein, but is capable of numerous changes or modifications without departing from the spirit of the application, as equivalent forms are intended to be within the scope of this application. Furthermore, in the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present application, it being understood that the present application may be practiced without one or more of these details.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

Terminology

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Unless otherwise conflict with the purpose and/or technical solution of the present application, the present application relates to the cited documents which are incorporated by reference in their entirety for all purposes. When reference is made to a cited document in this application, the definitions of the relevant technical features, terms, nouns, phrases, etc. in the cited document are also incorporated by reference. Examples of the relevant technical features and preferred modes to be cited in the present application when the cited documents are referred to in the present application are incorporated by reference in the present application, but are not limited to being able to implement the present application. It should be understood that when a reference is made to the description herein, it is intended to control or adapt the present application in light of the description herein.

Unless otherwise indicated or contradicted, terms or phrases used herein have the following meanings:

the term "and/or," "and/or," as used herein, includes any one of two or more of the listed items in relation to each other, as well as any and all combinations of the listed items in relation to each other, including any two of the listed items in relation to each other, any more of the listed items in relation to each other, or all combinations of the listed items in relation to each other. It should be noted that, when at least three items are connected by a combination of at least two conjunctions selected from "and/or", "or/and", "and/or", it should be understood that, in this application, the technical solutions certainly include technical solutions that all use "logical and" connection, and also certainly include technical solutions that all use "logical or" connection. For example, "a and/or B" includes three parallel schemes A, B and a+b. For another example, the technical schemes of "a, and/or B, and/or C, and/or D" include any one of A, B, C, D (i.e., the technical scheme of "logical or" connection), and also include any and all combinations of A, B, C, D, i.e., any two or three of A, B, C, D, and also include four combinations of A, B, C, D (i.e., the technical scheme of "logical and" connection).

In this application, reference is made to a numerical interval (i.e., a numerical range), where the optional numerical distribution is considered continuous, and includes two numerical endpoints (i.e., a minimum value and a maximum value) of the numerical range, and each numerical value between the two numerical endpoints, unless otherwise indicated. Unless otherwise indicated, when a numerical range merely refers to integers within the numerical range, both end integers of the numerical range are included, as well as each integer between the two ends, herein, each integer is recited directly, such as t is an integer selected from 1-10, and t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Further, when a plurality of range description features or characteristics are provided, these ranges may be combined. In other words, unless otherwise indicated, the ranges disclosed herein are to be understood to include any and all subranges subsumed therein.

The term "mass spectrometry is an analytical technique that ionizes a sample into charged molecules and can measure their mass-to-charge ratio (m/z). In Matrix assisted laser desorption ionization time of flight mass spectrometry MALDI-TOF mass spectrometry, the ion source is Matrix-assisted laser desorption ionization (MALDI), and the mass analyzer is a time of flight (TOF) analyzer.

The term "MALDI-TOF MS detection" MALDI is a soft ionization technique that uses a laser to impinge on a small molecule matrix to bring analyte molecules into the gas phase without being fragmented or decomposed. Some biomolecules are too large and decompose when heated, and conventional techniques can fracture or destroy the macromolecules. MALDI is suitable for analysis of biomolecules such as peptides, lipids, carbohydrates or other organic macromolecules. The analyte is embedded in a large excess of a matrix compound that is deposited on a solid surface called a target, which is typically made of a conductive metal and has spots for a variety of different samples. After a very short laser pulse, the irradiated spot is rapidly heated and excited by vibration. The matrix molecules are largely ablated from the sample surface, absorbing the laser energy and bringing the analyte molecules into the gas phase. During ablation, analyte molecules are typically ionized by being protonated or deprotonated by nearby matrix molecules. The most common form of MALDI ionization is to carry a single positive charge on the analyte molecules.

The term "mass resolution" resolution is defined as IUPAC, resolution r=m/Δm. In particular, resolution refers to the ability of a mass spectrometer to distinguish between two ions of similar mass. Approximately, a resolution of 500 refers to an ion that can distinguish between 500 and 1 (i.e., 501/499) from its mass number.

The method aims at the problems of low ionization efficiency and difficult analysis of DNA molecules with large relative molecular mass in the nucleic acid mass spectrometry of a conventional organic matrix, so that the problems of large mass deviation, low resolution and the like of detection results are caused, the site weight of the nucleic acid mass spectrometry detection is further influenced, matrix components are improved, the rigid structure of crystallization is remodeled, a uniform and compact crystallization layer is obtained, the adsorption and detection of a nucleic acid sample are facilitated, the acquisition success rate of mass spectrometry signals is greatly improved, the mass deviation of detection results is less than 300ppm, and the mass resolution is more than 1000.

The specific matrix formula is developed based on nano-liter-level sample preparation and disposable chip target plates commonly used by MALDI-TOF MS at present, and the ionization efficiency of biological macromolecules such as DNA is improved and the acquisition success rate is improved while the overall rigid structure of recrystallization is improved.

Meanwhile, the reinforcing of the matrix rigid structure ensures that nano-liter level sample preparation is not limited by a sample application instrument, accidental errors of manual sample application do not influence the recrystallization effect and subsequent mass spectrum acquisition, and a customer can select a sample preparation mode according to respective application requirements, so that the application scene of MALDI-TOF MS is expanded in a large scale, and a powerful technical support is provided for the application of MALDI-TOF MS in clinical medical diagnosis.

The crystallization of the matrix layer is critical in this application. The solution components are prepared to ensure that the matrix solution can be evenly and densely separated out at the whole target position, and then the nucleic acid sample is adsorbed on the matrix layer again to form complete co-crystals without damaging the rigid structure of the co-crystals.

In one aspect, the present application provides a chip matrix composition for time-of-flight mass spectrometry detection, comprising the following components: 0.01 g/mL-1 g/mL of 3-hydroxy-2-picolinic acid, 0.01 mol/L-1 mol/L of 2-picolinic acid, 0.01 mol/L-2 mol/L of tri-ammonium citrate, 0.01 mol/L-2 mol/L of ascorbic acid and 0.01 mol/L-2 mol/L of ammonium oxalate.

For example, 0.01g/mL, 0.1 g/mL, 0.2 g/mL, 0.3 g/mL, 0.4 g/mL, 0.5 g/mL, 0.6g/mL, 0.7 g/mL, 0.8g/mL, 0.9 g/mL, 1.0 g/mL of 3-hydroxy-2-pyridinecarboxylic acid.

0.01g/mL, 0.1 g/mL, 0.2 g/mL, 0.3 g/mL, 0.4 g/mL, 0.5 g/mL, 0.6g/mL, 0.7 g/mL, 0.8g/mL, 0.9 g/mL, 1.0 g/mL of 2-picolinic acid.

0.01g/mL, 0.1 g/mL, 0.2 g/mL, 0.3 g/mL, 0.4 g/mL, 0.5 g/mL, 0.6g/mL, 0.7 g/mL, 0.8g/mL, 0.9 g/mL, 1.0 g/mL, 1.1 g/mL, 1.2 g/mL, 1.3 g/mL, 1.4 g/mL, 1.5 g/mL, 1.6 g/mL, 1.7 g/mL, 1.8 g/mL, 1.9 g/mL, 2.0 g/mL of tri-ammonium citrate.

0.01g/mL, 0.1 g/mL, 0.2 g/mL, 0.3 g/mL, 0.4 g/mL, 0.5 g/mL, 0.6g/mL, 0.7 g/mL, 0.8g/mL, 0.9 g/mL, 1.0 g/mL, 1.1 g/mL, 1.2 g/mL, 1.3 g/mL, 1.4 g/mL, 1.5 g/mL, 1.6 g/mL, 1.7 g/mL, 1.8 g/mL, 1.9 g/mL, 2.0 g/mL of ascorbic acid.

0.01g/mL, 0.1 g/mL, 0.2 g/mL, 0.3 g/mL, 0.4 g/mL, 0.5 g/mL, 0.6g/mL, 0.7 g/mL, 0.8g/mL, 0.9 g/mL, 1.0 g/mL, 1.1 g/mL, 1.2 g/mL, 1.3 g/mL, 1.4 g/mL, 1.5 g/mL, 1.6 g/mL, 1.7 g/mL, 1.8 g/mL, 1.9 g/mL, 2.0 g/mL of ammonium oxalate.

In one specific example, the following components are included: 0.6-0.8 g/mL of 3-hydroxy-2-picolinic acid, 0.65-0.85 mol/L of 2-picolinic acid, 1-1.5 mol/L of tri-ammonium citrate, 0.6-1.4 mol/L of ascorbic acid and 0.8-1.2 mol/L of ammonium oxalate.

Alternatively, the solvent is selected from the group consisting of a mixed solvent of acetonitrile and water.

Further alternatively, the volume ratio of acetonitrile to water is (1-10): 20. For example (1, 2, 3, 4, 5, 6, 7, 8, 9, 10): 20.

In another aspect, the present application provides a method for detecting nucleic acid by time-of-flight mass spectrometry, comprising the steps of:

and co-crystallizing the sample to be detected and the chip matrix composition for the time-of-flight mass spectrometry detection on a chip substrate, and carrying out mass spectrometry detection.

In one specific example, the method comprises the steps of: and carrying out sample application, drying and crystallization on the chip matrix composition, prefabricating the chip matrix composition on the chip substrate to obtain a prefabricated chip substrate, taking a sample to be tested, and adding the sample to the prefabricated chip substrate.

Alternatively, the sample to be measured is added in a spotting manner.

Further alternatively, the sample volume added at each spot at the time of spotting is 0.1. Mu.L to 0.2. Mu.L.

It will be appreciated that the co-crystallization may further comprise a drying step.

In another aspect, the present application provides a method for detecting nucleic acid by time-of-flight mass spectrometry, comprising the steps of: and co-crystallizing the sample to be detected and the chip matrix composition for time-of-flight mass spectrometry detection on the chip by adopting the chip for time-of-flight mass spectrometry detection.

The reinforcing of matrix rigid structure makes the sample preparation of nanoliter level not need not be limited by the sample application instrument, the accidental error of manual sample application can not influence the recrystallization effect and the subsequent mass spectrum acquisition, and the sample preparation mode can be selected according to the respective application requirements, thereby expanding the application scene of MALDI-TOF MS in a large scale and providing powerful technical support for the application of MALDI-TOF MS in clinical medical diagnosis.

Alternatively, the sample to be tested includes, but is not limited to, oligonucleotides and polynucleotides. Also included are peptides, proteins, carbohydrates, lectins, lipids, glycoproteins, lipoproteins, and combinations thereof.

In general, oligonucleotides (oligonucleotides) are linear polynucleotide fragments in which 2 to 10 nucleotide residues are linked by phosphodiester bonds, but the number of nucleotide residues is not strictly defined when this term is used, and polynucleotide molecules containing 30 or more nucleotide residues are also called oligonucleotides in many documents.

The english name of a polynucleotide is generally a macromolecule formed by connecting more than 20 nucleotides through 3'-5' phosphodiester bonds.

The application also provides a chip matrix composition for time-of-flight mass spectrometry detection and application of the chip for time-of-flight mass spectrometry detection in MALDI-TOF MS detection.

Embodiments of the present application will be described in detail below with reference to examples. It should be understood that these examples are illustrative only of the present application and are not intended to limit the scope of the present application. The experimental methods, in which specific conditions are not noted in the following examples, are preferably referred to in the guidelines given in the present application, may be according to the experimental manual or conventional conditions in the art, may be according to the conditions suggested by the manufacturer, or may be referred to experimental methods known in the art.

In the specific examples described below, the measurement parameters relating to the raw material components, unless otherwise specified, may have fine deviations within the accuracy of weighing. Temperature and time parameters are involved, allowing acceptable deviations from instrument testing accuracy or operational accuracy.

The "matrix" referred to in the examples below is a chip matrix composition for time-of-flight mass spectrometry detection.

Example 1

1. The components of the chip matrix composition for time-of-flight mass spectrometry detection include:

1. 3-hydroxy-2-picolinic acid (3-HPA): 0.01 g/mL-0.33 g/mL;

2. 2-picolinic acid (2-PA): 0.01mol/L to 0.25mol/L;

3. tri-ammonium citrate: 0.01mol/L to 0.20mol/L;

4. acetonitrile: 5% -35%;

5. ascorbic acid: 0.01mol/L to 0.20mol/L;

6. ammonium oxalate: 0.01mol/L to 0.20 mol/L.

2. Solution formulation of a chip matrix composition for time-of-flight mass spectrometry detection comprises the steps of:

1. respectively preparing 10mL of 2 mol/L2-picolinic acid solution, citric acid triammonium solution, ascorbic acid solution and ammonium oxalate solution for later use.

2. Weighing 0.01 g-0.63 g of 3-hydroxy-2-picolinic acid (3-HPA), and dissolving with acetonitrile containing 5% -35% and water until the mixture is clear for later use.

3.1 mL of matrix liquid is prepared according to the proportion, wherein the matrix liquid contains 3-hydroxy-2-picolinic acid with the final concentration of 0.01 g-0.33 g/mL, 2-picolinic acid with the final concentration of 0.01-0.25 mol/L, tri-ammonium citrate with the final concentration of 0.01-0.20 mol/L, ascorbic acid with the final concentration of 0.01-0.20 mol/L and ammonium oxalate with the final concentration of 0.01-0.20 mol/L, and water is added to fix the volume to 1mL for standby.

Among them, each experiment is recommended to be made up at present due to the storage specificity of the matrix solution.

3. Specific process of matrix and sample recrystallization:

the prepared matrix liquid is prefabricated on a chip by a matrix prefabricated sample application instrument, and then naturally dried to separate out crystals. The crystals are observed to be white, round and full round small dots with naked eyes. And sucking and directly spotting the sample to be detected on the substrate by using a pipette, wherein the sucked sample quantity is approximately in the range of 0.1 mu L-0.2 mu L. The manual sample application process can not drag the liquid drop, so that cross contamination between two adjacent substrates is prevented. After sample application, the sample can be placed in a dryer for natural air drying, and mass spectrum detection can be carried out when no obvious drop trace exists on the surface of the chip. The recrystallized morphology is still complete and flat, the recrystallization conditions of the matrix and the sample are shown in fig. 1, it is obvious that the matrix crystallization of the application cannot collapse an original rigid structure due to the adhesion of the sample to be measured, the adduct of the sample to be measured is prevented from being introduced due to recrystallization after the matrix is mixed with the sample to be measured, and the transmission of laser energy is also prevented from being weakened after the matrix is mixed in the sample to be measured, so that signals are greatly reduced.

4. And (3) verifying results:

fig. 2 is a schematic diagram showing the mass spectrum acquisition success rate of 100% after using the matrix of the present invention, using an enterprise self-made calibrator directly spotted on the matrix of the present application as an effect verification test. The self-made calibrator for enterprises is formed by mixing 8 oligonucleotide single chains, and the mass-to-charge ratios of the self-made calibrator are 3052.10Da, 3655.50Da, 5523.70Da, 6511.30Da, 8100.30Da, 9006.90Da, 9022.90Da and 9938.50Da respectively. The verification collects 192 flux altogether, contains 8×192=1536 mass spectrum peak data, and calculates mass deviation (mass accuracy) average value and mass resolution average value according to each oligonucleotide single chain. The data are shown in table 1 below:

table 1: quality accuracy and resolution data summary obtained using the applied matrix acquisition

Two experiments were performed using single base extension products directly spotted on the above substrates. In a first experiment, the single base extension product of a human ALDH2 genotyping assay reagent was tested on the substrate of the invention. The mass deviation of the collected mass spectrum is shown in FIG. 3, and the mass deviation of the double peaks (6702.4 Da and 6718.4 Da) of the detection site rs671 is 153ppm and 153.4ppm respectively.

In a second experiment, the products of single base extension of the genotyping test agent for hereditary hearing loss were tested. As shown in FIG. 4, the single hole detects 20 mutation sites, the collected mass spectrogram is smooth and clean in background noise, and the interference to the site judgment result is avoided.

Comparative example 1

Compared with the examples, the composition does not contain 2-picolinic acid, and the concentration range of the rest components is the same

1. The components of the chip matrix composition for time-of-flight mass spectrometry detection are specifically:

1. 3-hydroxy-2-picolinic acid (3-HPA): 0.01 g/mL-0.33 g/mL;

2. tri-ammonium citrate: 0.01mol/L to 0.20mol/L;

3. acetonitrile: 5% -35%;

4. ascorbic acid: 0.01mol/L to 0.20mol/L;

5. ammonium oxalate: 0.01mol/L to 0.20 mol/L.

The specific process of recrystallization of the matrix and the sample is the same as in example 1.

2. Result verification

The situation of hollowing and incomplete recrystallization appears in the appearance of the matrix, and the situation of recrystallization of the matrix and the sample is shown in fig. 5, and the matrix crystal in comparative example 1 collapses the original rigid structure due to the adhesion of the sample to be measured, and the phenomenon of hollowing and incomplete recrystallization appears in the crystals to be separated out again. This may greatly reduce the success rate of signal acquisition and the intensity of the mass spectrum signal, increasing the mass deviation of the mass spectrum signal.

Two experiments were performed using single base extension products directly spotted on the substrate of comparative example 1 described above. In a first experiment, the single base extension product of a human ALDH2 genotyping assay reagent was tested on the matrix of comparative example 1. The mass deviation of the collected mass spectrum is schematically shown in FIG. 6, and in FIG. 6, the mass deviation of the double peaks (6702.4 Da and 6718.4 Da) of the detection site rs671 is 357.2ppm and 343.1ppm respectively.

In a second experiment, the products of single base extension of the genotyping test agent for hereditary hearing loss were tested. As shown in FIG. 7, 20 mutation sites are detected in a single hole, the acquired mass spectrogram has serious target peak deletion and more bottom noise peaks, and the typing result is greatly interfered.

Comparative example 2

Compared with the example, the addition amount of the 2-picolinic acid (2-PA) is 0.35 mol/L, and the concentration range of the other components is the same

The components of the chip matrix composition for time-of-flight mass spectrometry detection are specifically:

1. 3-hydroxy-2-picolinic acid (3-HPA): 0.01 g/mL-0.33 g/mL;

2. 2-picolinic acid (2-PA): 0.35 mol/L;

3. tri-ammonium citrate: 0.01mol/L to 0.20mol/L;

4. acetonitrile: 5% -35%;

5. ascorbic acid: 0.01mol/L to 0.20mol/L;

6. ammonium oxalate: 0.01mol/L to 0.20 mol/L.

The specific process of recrystallization of the matrix and the sample is the same as in example 1.

2. Result verification

The recrystallized morphology is uneven, the rod-shaped crystals are arranged irregularly, the recrystallization conditions of the matrix and the sample are shown in fig. 8, the matrix crystal of comparative example 2 has a originally flat rigid structure reconstructed due to the adhesion of the sample to be measured, and the re-precipitated crystals are irregular rod-shaped crystals, so that the crystals are uneven. This may greatly increase the mass bias of the mass spectrum signal, affecting the success rate of signal acquisition and the outcome of the typing analysis.

Two experiments were performed using single base extension products directly spotted on the substrate of comparative example 2 described above. In the first experiment, the matrix of comparative example 1The single base extension products of the human ALDH2 genotyping assay reagents were tested above. As shown in FIG. 9, the mass deviation of the acquired mass spectrogram is shown in the form of a bimodal (6702.4 Da and 6718.4 Da) mass deviation of the detection site rs671 far exceeding the maximum allowable mass-to-charge ratio error specified in the industry standard by less than or equal to 5×10 ^-4 (i.e., 500 ppm) and cannot be properly analyzed for typing.

In a second experiment, the products of single base extension of the genotyping test agent for hereditary hearing loss were tested. As shown in FIG. 10, the single hole is used for detecting 20 mutation sites, and the collected mass spectrogram is also serious in target peak deletion and more in bottom noise peak, so that the typing result is greatly interfered.

The above examples merely represent a few embodiments of the present application, which facilitate a specific and detailed understanding of the technical solutions of the present application, but are not to be construed as limiting the scope of the claims. It should be noted that it would be apparent to those skilled in the art that various modifications and improvements could be made without departing from the spirit of the present application, which would be within the scope of the present application. Further, it will be understood that various changes or modifications may be made to the present application by those skilled in the art after reading the foregoing teachings, and equivalents thereof will be within the scope of the present application. It should also be understood that those skilled in the art, based on the technical solutions provided in the present application, can obtain technical solutions through logical analysis, reasoning or limited experiments, all fall within the protection scope of the claims attached to the present application. The scope of the patent application is therefore intended to be limited by the content of the appended claims, which description and drawings may be interpreted accordingly.

Claims (10)

1. A chip matrix composition for time-of-flight mass spectrometry detection is characterized by comprising a solvent, and 0.01-1 g/mL of 3-hydroxy-2-picolinic acid, 0.01-1 mol/L of 2-picolinic acid, 0.01-2 mol/L of tri-ammonium citrate, 0.01-2 mol/L of ascorbic acid and 0.01-2 mol/L of ammonium oxalate added in the solvent.

2. The chip matrix composition for time-of-flight mass spectrometry according to claim 1, wherein the concentration of 3-hydroxy-2-picolinic acid is 0.6g/mL to 0.8g/mL, the concentration of 2-picolinic acid is 0.65mol/L to 0.85mol/L, the concentration of tri-ammonium citrate is 1mol/L to 1.5mol/L, the concentration of ascorbic acid is 0.6mol/L to 1.4mol/L, and the concentration of ammonium oxalate is 0.8mol/L to 1.2mol/L.

3. The chip matrix composition for time-of-flight mass spectrometry according to any one of claims 1 to 2, wherein the solvent is selected from the group consisting of a mixed solvent of acetonitrile and water.

4. A chip matrix composition for time-of-flight mass spectrometry according to claim 3, characterized in that the volume ratio of acetonitrile to water is (1:10): 20.

5. A method for detecting nucleic acid by time-of-flight mass spectrometry, comprising the steps of:

and co-crystallizing the sample to be detected and the chip matrix composition for the time-of-flight mass spectrometry detection on a chip substrate, and carrying out mass spectrometry detection.

6. The method for detecting nucleic acid by time-of-flight mass spectrometry according to claim 5, comprising the steps of:

and carrying out sample application, drying and crystallization on the chip matrix composition, prefabricating the chip matrix composition on the chip substrate to obtain a prefabricated chip substrate, taking a sample to be tested, and adding the sample to the prefabricated chip substrate.

7. The method for detecting nucleic acid by time-of-flight mass spectrometry according to claim 6, wherein the sample to be detected is added by spotting;

alternatively, the volume of the sample amount added at each spot at the time of spotting is 0.1. Mu.L to 0.2. Mu.L.

8. The method for detecting nucleic acid according to claim 5, further comprising a step of drying after the co-crystallization.

9. The method for detecting nucleic acid according to any one of claims 5 to 8, wherein the sample to be detected comprises one or both of an oligonucleotide and a polynucleotide.

10. Use of the chip matrix composition for time-of-flight mass spectrometry according to any one of claims 1 to 4 in MALDI-TOF MS detection.