Mass spectrum sampling method and device for trace cell lysis, atomization and ionization

Abstract

The invention provides a mass spectrum sampling method and a mass spectrum sampling device for trace cell lysis, atomization and ionization. Comprising a surface acoustic wave device which comprises a surface acoustic wave chip, a chip clamp, a power amplifier and a signal generator; the surface acoustic wave chip comprises a piezoelectric substrate and an interdigital electrode; the surface acoustic wave chip is clamped on the chip clamp; the signal generator is used for generating a sinusoidal signal of the center frequency of the surface acoustic wave device, the sinusoidal signal is amplified by the power amplifier, and an electric signal amplified by the power amplifier is applied to the interdigital electrode to drive the surface acoustic wave chip to vibrate so as to realize cracking and atomization of trace cells; the nanoliter electric spraying device comprises an electric spraying head, a two-way connecting piece, an adjustable direct-current power supply and a solvent conveying device, the adjustable direct-current power supply applies high voltage to the electric spray head through the two-way connecting piece to generate electric spray; the front of the mass spectrometer and the mass spectrum sample inlet corresponds to the tip of the electric spray head, and the working area of the surface acoustic wave chip is located under the tip of the electric spray head.

Description

Technical Field

[0001] The invention belongs to the technical field of mass spectrometry detection, and in particular relates to a mass spectrometry sampling method and a device thereof for micro-cell lysis, atomization and ionization. Background Art

[0002] Mass spectrometry (MS) is an analytical technique that determines the composition and structure of a substance by measuring the mass-to-charge ratio (m / z) of ions. Its basic process includes sample ionization and ion detection. Mass spectrometry is used to identify the structure and composition of compounds in chemical analysis. It is also widely used in proteomics and metabolomics in the biomedical field to analyze biological samples. In addition, it is also used for environmental monitoring, detection of pollutants and chemical components in environmental samples, and even detection of drugs and explosives. The diversity of mass spectrometry technology enables it to play an important role in various fields.

[0003] Mass spectrometry (MS) can quickly and extremely accurately determine the molecular weight of biological macromolecules. Mass spectrometry analysis has the advantages of high sensitivity, low sample usage, fast analysis speed, and simultaneous separation and identification. Applying mass spectrometry technology to cells can analyze biological macromolecules in cells with high precision and high throughput, including proteins and metabolites, and can deeply reveal the biochemical characteristics and functional status of cells. This is of great significance for understanding the complexity of cell populations, analyzing cell functions, and deconstructing disease mechanisms. However, cell analysis research was once hindered due to the extremely small size of cells, the large number of substance types, the extremely low compound content, and the significant concentration differences between different compounds. Therefore, in-depth research on cell analysis technology has become extremely necessary.

[0004] The key to the application of mass spectrometry technology in the analysis of intracellular chemical substances lies in the study of the injection system. The micro-cell mass spectrometry injection system requires the study of several key steps: cell lysis, chemical ionization, and injection mass spectrometry. Among them, the quality of cell lysis determines the quality of subsequent analysis. In the past, surface acoustic wave cell lysis often adopted the method of adding microparticles, magnetic beads, and micro-nanowires to the droplets containing cells. Under the drive of surface acoustic waves, due to the different accelerations of cells and doped substances, the collision between cells and doped substances led to cell lysis. Due to the introduction of doping substances, the cell solution after lysis by the surface acoustic wave device is often unable to be directly injected into mass spectrometry analysis; the electrospray ionization source is the most common and widely used ionization source. The sample solution containing the substance to be tested flows through the capillary, and a high DC voltage is applied at the outlet of the capillary. At the same time, the capillary coaxially carries a heated atomizing gas. Under the dual effects of electric field force and nebulizing gas, the solvent molecules will be sprayed at the capillary outlet and atomized into small droplets with electric charge at the micron level. After solvent evaporation and Coulomb explosion, the substance to be tested will eventually become gaseous ions and enter the mass spectrometer for analysis.

[0005] For example, CN108414303A discloses a particle collision cell lyser based on surface acoustic wave, which utilizes the acoustic microfluidic effect of surface acoustic wave to drive droplets and cells and particles in the droplets to move at high speed, thereby achieving the effect of cell lysing. However, this method is not designed to be stable. Under the drive of unilateral surface acoustic wave, the droplets will be adsorbed to 3M glue on the other side, causing the loss of cell samples, and introducing particulate impurities. There are particulate impurities in the cell droplets after lysis, which affects the subsequent biological experiments, resulting in the invention being unable to be subsequently combined with mass spectrometry detection. CN114724920A discloses a microporous oscillating atomization electrospray extraction ionization device for mass spectrometry analysis, which utilizes a microporous atomization sheet to atomize the sample solution into micron-sized droplets. In the extraction ionization chamber, the sample droplets collide with the solvent droplets for extraction ionization. The sampling device of this method is a microporous atomization sheet. The presence of the microporous structure is prone to blockage when facing complex cell samples, and the sample accumulates for a long time at the microporous structure. In addition, the microporous structure makes it difficult to clean the injection device, resulting in cross-contamination between samples, and the data obtained after injection into the mass spectrometer has greater noise.

[0006] Therefore, how to achieve excellent cell lysis effect while avoiding infection of the sample to be tested and possible clogging problems when electrospray is directly used for cell mass spectrometry is a hot topic that urgently needs to be studied. Summary of the invention

[0007] In view of the deficiencies of the prior art, the object of the present invention is to provide a mass spectrometry sampling method and device for micro-cell lysis, atomization and ionization. The present invention uses the same surface acoustic wave device to achieve high-efficiency cell lysis and atomization mass spectrometry sampling under different conditions, which can not only effectively avoid the problem of blockage in electrospray injection, but also is easy to clean and can effectively avoid cross contamination between samples. In addition, the extraction ionization solvent droplets generated by the nanoliter electrospray device and the atomized sample droplets to be tested are extracted and charge transferred before entering the mass spectrometer, thereby achieving the ionization of the substance to be tested. This selective extraction not only increases the concentration of the substance to be tested, but also reduces the concentration of interfering components, thereby reducing the influence of the matrix on the ionization process, and is suitable for use with subsequent mass spectrometer analysis.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a device for micro-cell lysis, atomization and ionization, comprising:

[0010] A surface acoustic wave device comprises a surface acoustic wave chip, a chip clamp, a power amplifier and a signal generator; the surface acoustic wave chip comprises a piezoelectric substrate and interdigital electrodes processed on the surface of the piezoelectric substrate; the surface acoustic wave chip is clamped on the chip clamp; the signal generator is used to generate a sinusoidal signal of the center frequency of the surface acoustic wave chip, and the sinusoidal signal is amplified by the power amplifier; the chip clamp contains four conductive spring legs, and the electrical signal amplified by the power amplifier is applied to the four pins of the interdigital electrodes through the conductive spring legs, so as to drive the surface acoustic wave chip to vibrate, thereby realizing the lysis and atomization of trace cells.

[0011] A nanoliter electrospray device comprises an electrospray head, a two-way connector, an adjustable direct current power supply and a solvent delivery device; one end of the two-way connector is connected to the electrospray head, and the other end of the two-way connector is connected to the solvent delivery device through an infusion capillary for delivering an extraction ionization solvent to the electrospray head; the adjustable direct current power supply applies high voltage to the electrospray head through the two-way connector to generate electrospray.

[0012] A mass spectrometer comprises a mass spectrometer injection port, the front of the mass spectrometer injection port corresponds to the tip of the electrospray head of the nanoliter electrospray device, and the working area of ​​the surface acoustic wave chip is located directly below the tip of the electrospray head.

[0013] The present invention uses the same surface acoustic wave device to achieve high-efficiency cell lysis and atomization mass spectrometry sampling under different input conditions, which can not only effectively avoid the problem of blockage in electrospray injection, but also is easy to clean and can effectively avoid cross contamination between samples. In addition, the extraction ionization solvent droplets generated by the nanoliter electrospray device and the atomized sample droplets to be tested are extracted and charge transferred before entering the mass spectrometer, thereby achieving ionization of the substance to be tested. This selective extraction not only increases the concentration of the substance to be tested, but also reduces the concentration of interfering components, thereby reducing the influence of the matrix on the ionization process, and is suitable for use with subsequent mass spectrometer analysis.

[0014] It should be noted that the working principle of the surface acoustic wave device is: when high-frequency alternating current is applied to the interdigitated electrodes, the piezoelectric substrate undergoes mechanical deformation due to the inverse piezoelectric effect, converting the input electrical signal into an acoustic signal, which propagates along the surface of the substrate.

[0015] It should be noted that cell lysis refers to breaking the cell barrier by physical or chemical means to release the internal substances of the cell, including chemical lysis, mechanical lysis, extraction, cell electrolysis and acoustic lysis.

[0016] It should be noted that atomization means dispersing liquid into droplets with smaller diameters; ionization means that ionization refers to the process in which atoms or molecules in a substance lose or gain electrons to form charged ions. When an atom or molecule loses one or more electrons, it becomes a positive ion. Conversely, when it gains electrons, it becomes a negative ion. Ionization can be achieved in a variety of ways, including high temperature, radiation, chemical reactions, or electric fields. In fields such as mass spectrometry and gas discharge, ionization is a key step that can convert samples into a measurable ionic state.

[0017] Extraction electrospray ionization is a technique used in the present invention. It is an analytical method that combines extraction and electrospray ionization techniques. Its basic process is to first separate the target compound from the sample by liquid-liquid extraction or solid-liquid extraction, and then use electrospray technology to convert the extracted sample into ions in the gas phase for mass spectrometry analysis. The advantage of this method is that it can improve analytical sensitivity and reduce matrix interference, and is particularly suitable for the analysis of complex samples, such as biological samples or environmental samples.

[0018] Preferably, the material of the piezoelectric substrate includes lithium niobate.

[0019] Preferably, the interdigital electrodes include a stacked adhesion layer and a conductive electrode layer along a direction away from the piezoelectric substrate.

[0020] In the present invention, the function of the adhesive layer is to connect the conductive electrode layer and the piezoelectric substrate and enhance the adhesion between the conductive electrode layer and the piezoelectric substrate.

[0021] Preferably, the material of the adhesion layer includes chromium.

[0022] Preferably, the thickness of the adhesion layer is 10-30 nm, for example, 10 nm, 15 nm, 20 nm, 25 nm or 30 nm.

[0023] Preferably, the material of the conductive electrode layer includes gold.

[0024] Preferably, the thickness of the conductive electrode layer is 50-100 nm, for example, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm or 100 nm.

[0025] Preferably, the interdigitated electrodes include:

[0026] The first interdigitated electrode comprises a reflective electrode and an excitation electrode, wherein the reflective electrode comprises a plurality of electrode strips arranged in a grid shape, and the excitation electrode comprises a plurality of cross-arranged finger strips.

[0027] The second interdigital electrode comprises a reflection electrode and an excitation electrode. The reflection electrode comprises a plurality of electrode strips arranged in a grid shape, and the excitation electrode comprises a plurality of cross-arranged finger strips.

[0028] The interval area between the first interdigital electrodes and the second interdigital electrodes is a working area of ​​the sample to be tested.

[0029] The present invention optimizes each interdigitated electrode. In addition to the excitation electrode, a reflection electrode is also designed so that the energy of the surface acoustic wave is reflected and focused on the working area in the middle of the surface acoustic wave chip through the reflection electrode. The present invention does not need to introduce particle collision to lyse cells, but can directly use the shear stress caused by the focused surface acoustic wave to lyse cells, and can be more conveniently used in combination with mass spectrometry.

[0030] In the present invention, the working area helps to superimpose the surface acoustic waves generated by the interdigitated electrodes, so that the droplets absorb more energy, thereby completing the lysis of trace cells in the droplets in a short time, and the lysis efficiency is higher.

[0031] Preferably, the length of the working area is 6-6.5 mm, for example, it may be 6 mm, 6.1 mm, 6.2 mm, 6.3 mm, 6.4 mm or 6.5 mm.

[0032] In the present invention, the working area of ​​suitable length is helpful for the superposition of surface acoustic waves generated by the interdigitated electrodes, so that the droplets absorb more energy, thereby completing the lysis of trace cells in the droplets in a short time, and the lysis efficiency is higher.

[0033] Preferably, in the first interdigitated electrode, the overlapping length between adjacent fingers is the electrode aperture, the electrode aperture is the width of the working area, the width of the working area is 4-6mm, for example, it can be 4mm, 4.5mm, 5mm, 5.5mm or 6mm.

[0034] Preferably, in the first interdigitated electrodes, the reflective electrode is located at a side away from the working area, and the shortest distance between the reflective electrode and the exciting electrode is 40-60 μm, for example, 40 μm, 50 μm or 60 μm.

[0035] Preferably, in the second interdigitated electrodes, the reflective electrode is located at a side away from the working area, and the shortest distance between the reflective electrode and the exciting electrode is 40-60 μm, for example, 40 μm, 50 μm or 60 μm.

[0036] In the present invention, the reflection electrodes and the excitation electrodes with appropriate spacing are helpful to enhance the focusing effect of the surface acoustic wave in the working area.

[0037] Preferably, in the first interdigitated electrode and the second interdigitated electrode, the intervals between adjacent fingers of the excitation electrode are 30-100μm, for example, 30μm, 40μm, 50μm, 60μm, 70μm, 80μm, 90μm or 100μm, etc., and the widths of the fingers are 30-100μm, for example, 30μm, 40μm, 50μm, 60μm, 70μm, 80μm, 90μm or 100μm, etc.

[0038] In the present invention, the center frequency of the surface acoustic wave chip is determined by the excitation electrodes with appropriate finger width and inter-finger spacing.

[0039] Preferably, in the first interdigitated electrodes and the second interdigitated electrodes, the number of pairs of fingers of the excitation electrodes is 10-30 pairs, for example, 10 pairs, 20 pairs or 30 pairs.

[0040] Preferably, in the first interdigital electrodes and the second interdigital electrodes, the number of electrode strips of the reflective electrode is 10-30, for example, 10, 15, 20, 25 or 30.

[0041] Preferably, the output frequency of the signal generator is 0-40 MHz, for example, 5 MHz, 10 MHz, 15 MHz, 20 MHz, 25 MHz, 30 MHz, 35 MHz or 40 MHz.

[0042] Preferably, the output voltage of the signal generator is 0-10V, for example, 2V, 4V, 6V, 8V or 10V.

[0043] In the present invention, the output frequency of the signal generator is 0-40MHz, and the output voltage is 0-10V, which is used to generate a sinusoidal signal of the center frequency of the surface acoustic wave chip.

[0044] Preferably, the operating frequency of the power amplifier is 1-130 MHz, for example, it may be 1 MHz, 10 MHz, 30 MHz, 50 MHz, 70 MHz, 90 MHz, 100 MHz, 120 MHz or 130 MHz.

[0045] It should be noted that the power amplifier is powered by a 12V adjustable DC power supply, and the gain of the RF power amplifier is 37dB.

[0046] Preferably, the electrospray head is a spray needle.

[0047] Preferably, the spray needle comprises a quartz capillary needle.

[0048] In the present invention, the spray needle may be a quartz capillary needle or other spray needles capable of producing an electrospray effect, such as a steel needle.

[0049] Preferably, the quartz capillary needle has a caliber of 10-50 μm, for example, 10 μm, 20 μm, 30 μm, 40 μm or 50 μm.

[0050] In the present invention, a quartz capillary needle of appropriate caliber helps to reduce the diameter of solvent droplets, helps to facilitate subsequent desolvation sampling and mass spectrometry detection, and helps to enhance the intensity of mass spectrometry signals.

[0051] Preferably, the two-way connector is made of metal, for example, stainless steel, aluminum or copper.

[0052] Preferably, the adjustable DC power supply has an output voltage range of -8 to 8 kV, for example, -8 kV, -6 kV, -4 kV, -2 kV, 2 kV, 4 kV, 6 kV or 8 kV.

[0053] Preferably, the solvent delivery device comprises a syringe pump and a syringe.

[0054] Preferably, the distance between the tip of the electrospray head and the mass spectrometer injection port is 1-2 cm, for example, 1 cm, 1.2 cm, 1.4 cm, 1.6 cm, 1.8 cm or 2 cm.

[0055] In the present invention, the appropriate distance between the tip of the electrospray head and the mass spectrometer injection port can enhance the spray effect and enhance the intensity of the mass spectrometer signal.

[0056] Preferably, the distance between the working area of ​​the surface acoustic wave chip and the tip of the electrospray head is 1-2 cm, for example, 1 cm, 1.2 cm, 1.4 cm, 1.6 cm, 1.8 cm or 2 cm.

[0057] In the present invention, the working area of ​​the surface acoustic wave chip and the tip of the electrospray head at an appropriate distance can improve the collision extraction efficiency between the sample droplets and the electrospray solvent droplets and enhance the mass spectrometry signal intensity.

[0058] In a second aspect, the present invention provides a mass spectrometry sampling method for micro-cell lysis, atomization and ionization, wherein the mass spectrometry sampling method uses the device as described in the first aspect, and the mass spectrometry sampling method comprises the following steps:

[0059] The adjustable DC power supply and the solvent delivery device are started, and high voltage is applied to the electrospray head through a two-way connector. Then, the extraction ionization solvent is introduced into the electrospray head through the solvent delivery device, so that electrospray is generated at the tip of the electrospray head and atomized into charged solvent droplets.

[0060] A sample solution containing trace cells is placed on a surface acoustic wave chip, and then the surface acoustic wave device is driven under a first condition to lyse the cells, and then the surface acoustic wave device is driven under a second condition to atomize the sample solution containing the lysed cells.

[0061] The sample droplets atomized on the surface acoustic wave device and the charged solvent droplets atomized on the nano-electrospray device cross-collide to extract and ionize the atomized sample droplets, thereby ionizing the substance to be tested, and then entering the mass spectrometer through the mass spectrometer inlet.

[0062] It should be noted that when preparing the sample solution, the type of cells may be, for example, Hela cells.

[0063] Preferably, the voltage setting interval of the signal generator that meets the first condition is 0.15-0.25V, for example, it can be 0.15V, 0.2V or 0.25V.

[0064] In the present invention, under the first condition, the cells in the sample solution can be fully lysed, and the cell lysis rate can be accelerated.

[0065] Preferably, the voltage setting interval of the signal generator that meets the second condition is 0.3-0.5V, for example, it can be 0.3V, 0.4V or 0.5V.

[0066] In the present invention, under the second condition, the sample atomization rate can be controlled, the sample injection rate can be increased, the sample droplets and the electrospray solvent droplets can be fully collided to extract the injection mass spectrum, and the mass spectrometry signal intensity can be enhanced.

[0067] It should be noted that the sample droplet size of the sample solution containing lysed cells after atomization is between submicron and micron levels.

[0068] In the present invention, droplets of suitable size cross-collide with atomized solvent droplets with charges, which helps to fully extract and ionize the substances to be tested in the sample droplets and enhance the intensity of mass spectrometry signals.

[0069] Preferably, the extractive ionization solvent comprises a methanol aqueous solution.

[0070] Preferably, the flow rate of the extraction and ionization solvent is 0.2-6 μL / min, for example, it can be 0.2 μL / min, 0.6 μL / min, 1 μL / min, 3 μL / min, 5 μL / min or 6 μL / min.

[0071] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0072] Compared with the prior art, the present invention has the following beneficial effects:

[0073] (1) The present invention uses the same surface acoustic wave device to achieve high-efficiency cell lysis and atomization mass spectrometry injection under different input conditions, which can not only effectively avoid the problem of blockage in electrospray injection, but also is easy to clean and can effectively avoid cross contamination between samples. In addition, the extraction ionization solvent droplets generated by the nanoliter electrospray device and the atomized sample droplets to be tested are extracted and charge transferred before entering the mass spectrometer, thereby achieving ionization of the substance to be tested. This selective extraction not only increases the concentration of the substance to be tested, but also reduces the concentration of interfering components, thereby reducing the influence of the matrix on the ionization process, and is suitable for use with subsequent mass spectrometer analysis.

[0074] (2) The surface acoustic wave device designed in the present invention can lyse cells within 5 seconds, with a lysis efficiency of more than 95% and a good lysis effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 This is a schematic diagram of the device structure provided in Example 1 of the present invention.

[0076] Figure 2 This is a schematic diagram of the interdigital electrode structure provided in Example 1 of the present invention.

[0077] Figure 3 This is a comparison diagram of the fluorescence signals of Hela cells before and after lysis in Example 1 of the present invention.

[0078] Figure 4 Schematic diagram of charged solvent droplets in Example 1 of the present invention.

[0079] Figure 5 Ion flow diagram for performance testing of the devices provided in Example 1 and Comparative Example 1 of the present invention.

[0080] Figure 6 The mass spectra are obtained by performing performance tests on the devices provided in Example 1 and Comparative Example 1 of the present invention.

[0081] Figure 7 This is a mass spectrometry analysis diagram after mass spectrometry injection based on the devices provided in Example 1 and Comparative Example 1.

[0082] Among them, 1-surface acoustic wave chip; 2-chip clamp; 3-power amplifier; 4-signal generator; 5-electrospray head; 6-two-way connector; 7-adjustable DC power supply; 8-injection pump; 9-mass spectrometer; 10-first interdigitated electrode; 11-second interdigitated electrode; 12-working area. DETAILED DESCRIPTION

[0083] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only used to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0084] Example 1

[0085] This embodiment provides a device for micro-cell lysis, atomization and ionization, such as Figure 1 As shown, including:

[0086] A surface acoustic wave device comprises a surface acoustic wave chip 1, a chip clip 2, a power amplifier 3 and a signal generator 4; the surface acoustic wave chip 1 comprises a piezoelectric substrate and interdigital electrodes processed on the surface of the piezoelectric substrate; the piezoelectric substrate is a 4-inch, 0.5 mm thick double-sided polished 128° YX lithium niobate wafer; the interdigital electrodes comprise a stacked adhesion layer with a thickness of 20 nm and a conductive electrode layer with a thickness of 80 nm along a direction away from the piezoelectric substrate, the adhesion layer is made of chromium, and the conductive electrode layer is made of gold; the interdigital electrodes are as follows Figure 2 As shown, it includes: a first interdigitated electrode 10, the first interdigitated electrode 10 includes a reflection electrode and an excitation electrode, the reflection electrode includes a plurality of electrode strips arranged in a grid shape, the number of electrode strips is 25, the excitation electrode includes a plurality of cross-arranged fingers, the interval between adjacent fingers is 100 μm, the width of the finger is 100 μm, and the number of finger pairs is 10; a second interdigitated electrode 11, the second interdigitated electrode 11 includes a reflection electrode and an excitation electrode, the reflection electrode includes a plurality of electrode strips arranged in a grid shape, the number of electrode strips is 25, the excitation electrode includes a plurality of cross-arranged fingers, the interval between adjacent fingers is 100 μm, the width of the finger is 100 μm, and the number of finger pairs is 10; The interval area between the first interdigitated electrode 10 and the second interdigitated electrode 11 is the working area 12 of the sample to be tested, the length of the working area 12 is 6.2 mm, the overlapping length between adjacent fingers is the electrode aperture, the electrode aperture is the width of the working area 12, and the width of the working area 12 is 5 mm; in the first interdigitated electrode 10, the reflection electrode is located on the side away from the working area 12, and the shortest distance between the reflection electrode and the excitation electrode is 50 μm; in the second interdigitated electrode 11, the reflection electrode is located on the side away from the working area 12, and the shortest distance between the reflection electrode and the excitation electrode is 50 μm; the size of the surface acoustic wave chip 1 is 1.3 cm×3.2 cm;

[0087] The surface acoustic wave chip 1 is clamped on the chip clamp 2; the signal generator 4 is used to generate a sinusoidal signal of the center frequency of the surface acoustic wave chip, and the sinusoidal signal is amplified by the power amplifier 3; the output frequency of the signal generator 4 is 10MHz, and the output voltage includes low voltage and high voltage, the low voltage is 0.2V, and the high voltage is 0.4V; the operating frequency of the power amplifier 3 is 10MHz, and the gain of the power amplifier 3 is 37dB; the chip clamp 2 contains four conductive spring legs, and the electrical signal amplified by the power amplifier 3 is applied to the four pins of the interdigital electrode through the conductive spring legs, which is used to drive the surface acoustic wave chip 1 to vibrate, so as to realize the lysis and atomization of trace cells.

[0088] The nanoliter electrospray device comprises an electrospray head 5, a two-way connector 6, an adjustable DC power supply 7 and a solvent delivery device; one end of the two-way connector is connected to the electrospray head 5, and the other end of the two-way connector 6 is connected to the injection pump 8 through an infusion capillary for delivering the extraction ionization solvent to the electrospray head 5; the adjustable DC power supply 7 applies high voltage to the electrospray head 5 through the two-way connector to generate electrospray. The electrospray head 5 is a quartz capillary needle with a diameter of 20 μm; the material of the two-way connector is stainless steel; the output voltage of the adjustable DC power supply 7 is 3500V, the positive and negative of the DC power supply output voltage is controlled by a pin, and the output size is adjusted by a 20kΩ potentiometer. The infusion capillary is a liquid phase capillary with an inner diameter of 0.5 mm and an outer diameter of 1.59 mm; the solvent delivery device comprises an injection pump 8 and an injection tube.

[0089] A mass spectrometer 9, wherein the mass spectrometer 9 comprises a mass spectrometer injection port, the front of the mass spectrometer injection port corresponds to the tip of the electrospray head 5 of the nanoliter electrospray device, the working area of ​​the surface acoustic wave chip 1 is located directly below the tip of the electrospray head 5; the distance between the tip of the electrospray head 5 and the mass spectrometer injection port is 1.5 cm; the distance between the working area of ​​the surface acoustic wave chip 1 and the tip of the electrospray head 5 is 1.5 cm.

[0090] This embodiment also provides a mass spectrometry sampling method for micro-cell lysis, atomization and ionization, wherein the mass spectrometry sampling method uses the device as described above, and the mass spectrometry sampling method comprises the following steps:

[0091] (1) The adjustable DC power supply and the solvent delivery device are started, and a high voltage of 3500 V is applied to the electrospray head through a two-way connector. Then, a methanol aqueous solution (the volume ratio of methanol to water is 1:1) is sucked into a 1000 μL syringe by a syringe pump and passed into the electrospray head at a flow rate of 3 μL / min, so that an electrospray is generated at the tip of the electrospray head and atomized into charged solvent droplets.

[0092] (2) Using Hela cells as experimental materials, the cultured Hela cells were stained with fluorescein diacetate, resuspended with 150 mmol / L ammonium bicarbonate aqueous solution, and diluted to a concentration of 10^5 cells / mL; then, a pipette was used to drop 2 μL of the prepared fluorescent-stained cell suspension onto the working area of ​​the surface acoustic wave chip, the output frequency of the signal generator was set to 10 MHz, the output voltage was adjusted to 0.2 V, the switch was turned on, the surface acoustic wave device was driven to work for 5 seconds, and then the signal generator and the power switch were turned off to cause the cells to lyse.

[0093] The output frequency of the signal generator was set to 10 MHz, the output voltage was adjusted to 0.4 V, and the switch was turned on. The sample solution containing Hela cells was vibrated by the surface acoustic wave chip to generate atomized droplets.

[0094] (3) The sample droplets atomized on the surface acoustic wave device and the charged solvent droplets atomized on the nano-electrospray device cross-collide to extract and ionize the atomized sample droplets, thereby achieving ionization of the substance to be tested. The ionized sample enters the vacuum interface area of ​​the mass spectrometer through the mass spectrometer inlet for desolvation and Coulomb explosion, followed by mass spectrometry analysis.

[0095] Figure 3 The comparison diagram of the fluorescence signals of Hela cells before and after lysis in this embodiment is shown. It can be seen from the figure that after the Hela cells are lysed, the fluorescence signal disappears, indicating that the cells are effectively lysed.

[0096] Figure 4 A schematic diagram of the charged solvent droplets in this embodiment is shown. As can be seen from the figure, effective and stable electrospray can be observed from the tip of the electrospray head in the figure, and the shape of the spray Taylor cone is full and stable.

[0097] Comparative Example 1

[0098] The difference between this comparative example and Example 1 is that the silent surface wave device is only composed of a nanoliter electrospray device and a mass spectrometer, and the nanoliter electrospray device is used for electrospray injection mass spectrometry of the sample to be tested.

[0099] The remaining parameters remain the same as those in Example 1.

[0100] The present invention uses reserpine as the detection object, and performs performance tests on the devices provided in Example 1 and Comparative Example 1, that is, 1 ppm of reserpine is injected into the device provided in Comparative Example 1, and 1 ppm of reserpine is injected into the sample area of ​​the surface acoustic wave chip. The test results are as follows: Figure 5 and Figure 6 As shown in the figure, it can be seen that the device of Example 1 provided by the present invention has a good sampling effect.

[0101] Figure 7The mass spectrometry analysis diagrams after mass spectrometry injection using the devices provided in Example 1 and Comparative Example 1 are shown. It can be seen from the diagrams that both devices can obtain obvious lipid information by performing mass spectrometry analysis.

[0102] The applicant declares that the present invention illustrates the process method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned process steps, that is, it does not mean that the present invention must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A device for micro-cell lysis, atomization and ionization, characterized in that: include: A surface acoustic wave device comprises a surface acoustic wave chip, a chip clamp, a power amplifier and a signal generator; the surface acoustic wave chip comprises a piezoelectric substrate and interdigital electrodes processed on the surface of the piezoelectric substrate; the surface acoustic wave chip is clamped on the chip clamp; the signal generator is used to generate a sinusoidal signal of the center frequency of the surface acoustic wave chip, and the sinusoidal signal is amplified by the power amplifier; the chip clamp contains four conductive spring legs, and the electrical signal amplified by the power amplifier is applied to the four pins of the interdigital electrodes through the conductive spring legs, so as to drive the surface acoustic wave chip to vibrate, thereby realizing the lysis and atomization of trace cells; A nanoliter electrospray device comprises an electrospray head, a two-way connector, an adjustable DC power supply and a solvent delivery device; one end of the two-way connector is connected to the electrospray head, and the other end of the two-way connector is connected to the solvent delivery device through an infusion capillary for delivering an extraction ionization solvent to the electrospray head; the adjustable DC power supply applies high voltage to the electrospray head through the two-way connector to generate electrospray; A mass spectrometer comprises a mass spectrometer injection port, the front of the mass spectrometer injection port corresponds to the tip of the electrospray head of the nanoliter electrospray device, and the working area of ​​the surface acoustic wave chip is located directly below the tip of the electrospray head.

2. The device according to claim 1, characterized in that The material of the piezoelectric substrate includes lithium niobate; Preferably, the interdigital electrodes include a stacked adhesion layer and a conductive electrode layer along a direction away from the piezoelectric substrate; Preferably, the material of the adhesion layer includes chromium; Preferably, the thickness of the adhesion layer is 10-30 nm; Preferably, the material of the conductive electrode layer includes gold; Preferably, the thickness of the conductive electrode layer is 50-100 nm.

3. The device according to claim 1 or 2, characterized in that: The interdigitated electrodes include: a first interdigitated electrode, wherein the first interdigitated electrode comprises a reflection electrode and an excitation electrode, wherein the reflection electrode comprises a plurality of electrode strips arranged in a grid shape, and the excitation electrode comprises a plurality of cross-arranged finger strips; a second interdigital electrode, wherein the second interdigital electrode comprises a reflection electrode and an excitation electrode, the reflection electrode comprises a plurality of electrode strips arranged in a grid shape, and the excitation electrode comprises a plurality of cross-arranged finger strips; The interval area between the first interdigital electrode and the second interdigital electrode is a working area of ​​the sample to be tested; Preferably, the length of the working area is 6-6.5 mm; Preferably, in the first interdigitated electrode, the overlapping length between adjacent fingers is the electrode aperture, the electrode aperture is the width of the working area, and the width of the working area is 4-6 mm.

4. The device according to claim 3, characterized in that In the first interdigitated electrodes, the reflective electrode is located at a side away from the working area, and the shortest distance between the reflective electrode and the exciting electrode is 40-60 μm; Preferably, in the second interdigitated electrodes, the reflective electrode is located at a side away from the working area, and the shortest distance between the reflective electrode and the exciting electrode is 40-60 μm; Preferably, in the first interdigitated electrode and the second interdigitated electrode, the intervals between adjacent fingers of the excitation electrode are 30-100 μm, and the widths of the fingers are 30-100 μm; Preferably, in the first interdigitated electrodes and the second interdigitated electrodes, the number of pairs of fingers of the excitation electrodes is 10-30 pairs.

5. The device according to any one of claims 1 to 4, characterized in that: The output frequency of the signal generator is 0-40MHz; Preferably, the output voltage of the signal generator is 0-10V; Preferably, the operating frequency of the power amplifier is 1-130 MHz.

6. The device according to any one of claims 1 to 5, characterized in that: The electrospray head is a spray needle; Preferably, the spray needle comprises a quartz capillary needle; Preferably, the quartz capillary needle has a caliber of 10-50 μm; Preferably, the material of the two-way connector is metal; Preferably, the adjustable DC power supply has an output voltage range of -8 to 8 kV; Preferably, the solvent delivery device comprises a syringe pump and a syringe.

7. The device according to any one of claims 1 to 6, characterized in that: The distance between the tip of the electrospray head and the mass spectrometer injection port is 1-2 cm; Preferably, the distance between the working area of ​​the surface acoustic wave chip and the tip of the electrospray head is 1-2 cm.

8. A mass spectrometry sampling method for micro-cell lysis, atomization and ionization, characterized in that: The mass spectrometry injection method adopts the device according to any one of claims 1 to 7, and the mass spectrometry injection method comprises the following steps: Starting the adjustable DC power supply and the solvent delivery device, applying high voltage to the electrospray head through the two-way connector, and then passing the extraction ionization solvent from the solvent delivery device into the electrospray head, so that the tip of the electrospray head generates electrospray and atomizes into charged solvent droplets; Placing a sample solution containing a trace amount of cells on a surface acoustic wave chip, then driving the surface acoustic wave device under a first condition to lyse the cells, and then driving the surface acoustic wave device under a second condition to atomize the sample solution containing the lysed cells; The sample droplets atomized on the surface acoustic wave device and the charged solvent droplets atomized on the nano-electrospray device cross-collide to extract and ionize the atomized sample droplets, thereby ionizing the substance to be tested, and then entering the mass spectrometer through the mass spectrometer inlet.

9. The mass spectrometry sampling method according to claim 8, characterized in that: The voltage setting interval of the signal generator that meets the first condition is 0.15-0.25V; Preferably, the voltage setting interval of the signal generator that meets the second condition is 0.3-0.5V.

10. The mass spectrometry sampling method according to claim 8 or 9, characterized in that: The extractive ionization solvent includes methanol-water solution; Preferably, the flow rate of the extraction and ionization solvent is 0.2-6 μL / min.

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