A mass spectrometry sample introduction method and apparatus for micro-cell lysis, nebulization and ionization

Abstract

This invention provides a mass spectrometry sample introduction method and apparatus for micro-cell lysis, nebulization, and ionization. It includes: a surface acoustic wave (SAW) device, comprising a SAW chip, a chip holder, a power amplifier, and a signal generator; the SAW chip includes a piezoelectric substrate and interdigitated electrodes; the SAW chip is held in the chip holder; the signal generator generates a sinusoidal signal at the center frequency of the SAW device, which is amplified by the power amplifier, and the amplified electrical signal is applied to the interdigitated electrodes to drive the SAW chip to vibrate, thereby achieving micro-cell lysis and nebulization; a nanoliter electrospray device, comprising an electrospray head, a two-way connector, an adjustable DC power supply, and a solvent delivery device; the adjustable DC power supply applies high voltage to the electrospray head through the two-way connector to generate electrospray; and a mass spectrometer, with the mass spectrometer inlet directly in front of the tip of the electrospray head, and the working area of ​​the SAW chip located directly below the tip of the electrospray head.

Description

Technical Field

[0001] This invention belongs to the field of mass spectrometry detection technology, specifically relating to a mass spectrometry sample introduction method and apparatus for micro-cell lysis, nebulization and ionization. Background Technology

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

[0003] Mass spectrometry (MS) can rapidly and extremely accurately determine the molecular weight of biological macromolecules. MS analysis offers advantages such as high sensitivity, small sample volume, fast analysis speed, and simultaneous separation and identification. Applying MS technology to cells allows for high-precision, high-throughput analysis of biological macromolecules within cells, including proteins and metabolites, revealing in-depth insights into the biochemical characteristics and functional states of cells. This is crucial for understanding the complexity of cell populations, elucidating cell function, and deconstructing disease mechanisms. However, cell analysis research has been hampered by the extremely small size of cells, the vast variety of substances, the very low concentrations of compounds, and the significant concentration differences between different compounds. Therefore, in-depth research into cell analysis techniques has become exceptionally necessary.

[0004] The key to applying mass spectrometry to the analysis of intracellular chemicals lies in the research of the sample introduction system. Micro-cell mass spectrometry sample introduction systems require research into several key steps: cell lysis, chemical ionization, and sample introduction mass spectrometry. Among these, the quality of cell lysis determines the quality of subsequent analysis. Previous surface acoustic wave (SAW) cell lysis methods often involved adding microparticles, magnetic beads, or micro / nanowires to cell-containing droplets. Driven by SAW, the difference in acceleration between the cells and dopants led to collisions and cell lysis. Due to the introduction of dopants, the cell solution after SAW lysis often could not be directly introduced for mass spectrometry analysis. Electrospray ionization (ESI) sources are the most common and widely used ionization sources. The sample solution containing the analyte flows through a capillary, and a high DC voltage is applied at the capillary outlet, while a heated nebulizer gas is coaxially mounted on the capillary. Under the combined influence of electric field and atomizing gas, solvent molecules are sprayed at the capillary outlet, atomizing into micron-sized charged droplets. After solvent evaporation and coulomb explosion, the analyte is finally converted into gaseous ions and enters the mass spectrometer for analysis.

[0005] For example, CN108414303A discloses a particle-collision cell lysator based on surface acoustic waves (SAWs). It utilizes the acoustic microfluidic effect of SAWs to drive droplets and the cells and particles within them at high speeds, thereby achieving cell lysis. However, this method is not stable. Driven by SAWs on one side, droplets can adhere to 3M adhesive on the other side, resulting in cell sample loss and introducing particulate impurities. The presence of particulate impurities in the lysed cell droplets affects subsequent biological experiments, making it impossible to use this invention for subsequent mass spectrometry detection. CN114724920A discloses a microporous oscillating atomization electrospray extraction ionization device for mass spectrometry analysis. It uses a microporous atomizing plate to atomize the sample solution into micron-sized droplets. Within the extraction ionization chamber, the sample droplets collide with solvent droplets for extraction ionization. The sample introduction device in this method is a microporous atomizing plate. The presence of microporous structures can lead to blockage of complex cell samples due to prolonged accumulation of sample within the microporous structure. In addition, the microporous structure makes the sample introduction device difficult to clean, cross-contamination between samples occurs, and the data obtained after mass spectrometry is noisier.

[0006] Therefore, how to achieve excellent cell lysis while avoiding infection of the test sample and potential clogging problems when electrospray is used directly for cell mass spectrometry is a hot research topic that urgently needs to be addressed. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a mass spectrometry sample introduction method and apparatus for micro-cell lysis, nebulization, and ionization. This invention utilizes the same surface acoustic wave (SAW) device to achieve highly efficient cell lysis and nebulized mass spectrometry sample introduction under different conditions. This not only effectively avoids the clogging problem associated with electrospray injection but also facilitates cleaning and effectively prevents cross-contamination between samples. Furthermore, the extraction and ionization solvent droplets generated by the nanoliter electrospray device undergo extraction and charge transfer with the nebulized sample droplets before entering the mass spectrometer, thereby achieving the ionization of the analyte. This selective extraction not only increases the concentration of the analyte but also reduces the concentration of interfering components, thus minimizing the influence of the matrix on the ionization process. This method is suitable for coupling with subsequent mass spectrometry analysis.

[0008] To achieve this objective, the present invention employs the following technical solution:

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

[0010] A surface acoustic wave (SAW) device includes a SAW chip, a chip holder, a power amplifier, and a signal generator. The SAW chip includes a piezoelectric substrate and interdigitated electrodes processed on the surface of the piezoelectric substrate. The SAW chip is held in the chip holder. The signal generator generates a sinusoidal signal at the center frequency of the SAW chip, which is amplified by the power amplifier. The chip holder contains four conductive spring legs. The amplified electrical signal is applied to the four pins of the interdigitated electrodes through the conductive spring legs to drive the SAW chip to vibrate, thereby achieving the lysis and atomization of micro-cells.

[0011] A nanoliter electrospray device includes 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 a liquid delivery capillary tube for delivering 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.

[0012] The mass spectrometer includes a mass spectrometer inlet, with the tip of the electrospray head of the nano-electrospray device directly in front of the mass spectrometer inlet, and the working area of ​​the surface acoustic wave chip located directly below the tip of the electrospray head.

[0013] This invention utilizes the same surface acoustic wave (SAW) device to achieve highly efficient cell lysis and nebulized mass spectrometry (MS / MS) sample introduction under different input conditions. This not only effectively avoids the clogging problems associated with electrospray injection but also facilitates cleaning and prevents cross-contamination between samples. Furthermore, the extraction and ionization solvent droplets generated by the nanoliter electrospray device undergo extraction and charge transfer with the nebulized sample droplets before entering the mass spectrometer, thereby achieving ionization of the analyte. This selective extraction not only increases the concentration of the analyte but also reduces the concentration of interfering components, thus minimizing the influence of the matrix on the ionization process. This makes it suitable for coupling with subsequent mass spectrometry analysis.

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

[0015] It should be noted that cell lysis refers to breaking the cell barrier through 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's important to clarify that atomization means dispersing a liquid into droplets with even smaller diameters; ionization refers to the process by which atoms or molecules in a substance lose or gain electrons, forming 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 various ways, including through high temperatures, radiation, chemical reactions, or the action of electric fields. In fields such as mass spectrometry and gas discharge, ionization is a crucial step, converting samples into measurable ionic states.

[0017] Extractive electrospray ionization (ESI), the technique employed in this invention, is an analytical method combining extraction and ESI. The basic process involves first separating the target compound from the sample using liquid-liquid or solid-liquid extraction, and then using ESI to convert the extracted sample into ions in the gas phase for mass spectrometry analysis. The advantages of this method include improved analytical sensitivity and reduced matrix interference, making it particularly suitable for the analysis of complex samples, such as biological or environmental samples.

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

[0019] Preferably, the interdigitated electrode comprises a stacked adhesive layer and a conductive electrode layer along a direction away from the piezoelectric substrate.

[0020] In this invention, the adhesive layer serves to connect the conductive electrode layer and the piezoelectric substrate, thereby enhancing the adhesion between the conductive electrode layer and the piezoelectric substrate.

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

[0022] Preferably, the thickness of the adhesive layer is 10-30 nm, for example, it can be 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 50-100nm, for example, it can be 50nm, 60nm, 70nm, 80nm, 90nm or 100nm.

[0025] Preferably, the interdigitated electrode comprises:

[0026] The first interdigital electrode includes a reflective electrode and an excitation electrode. The reflective electrode includes a plurality of grid-arranged electrode strips, and the excitation electrode includes a plurality of intersecting finger strips.

[0027] The second interdigital electrode includes a reflective electrode and an excitation electrode. The reflective electrode includes a plurality of grid-arranged electrode strips, and the excitation electrode includes a plurality of intersecting finger strips.

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

[0029] This invention optimizes each interdigital 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 onto the working area in the middle of the surface acoustic wave chip. This allows the invention to lyse cells directly by utilizing the shear stress caused by the focused surface acoustic wave without introducing particle collisions to lyse the cells. It can also be more conveniently coupled with mass spectrometry.

[0030] In this invention, the working area helps the surface acoustic waves generated by the interdigitated electrodes to superimpose, allowing the droplet to absorb more energy, thereby completing the lysis of micro-cells within the droplet in a short time and achieving higher lysis efficiency.

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

[0032] In this invention, a working area of ​​suitable length helps the surface acoustic waves generated by the interdigitated electrodes to superimpose, allowing the droplet to absorb more energy and thus complete the lysis of micro-cells within the droplet in a short time, resulting in higher lysis efficiency.

[0033] Preferably, in the first interdigital electrode, the overlap 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 4-6mm, for example, it can be 4mm, 4.5mm, 5mm, 5.5mm or 6mm, etc.

[0034] Preferably, in the first interdigitated electrode, the reflective electrode is located on the side away from the working area, and the shortest distance between the reflective electrode and the excitation electrode is 40-60 μm, for example, it can be 40 μm, 50 μm or 60 μm.

[0035] Preferably, in the second interdigitated electrode, the reflective electrode is located on the side away from the working area, and the shortest distance between the reflective electrode and the excitation electrode is 40-60 μm, for example, it can be 40 μm, 50 μm or 60 μm.

[0036] In this invention, the appropriately spaced reflective and excitation electrodes help to enhance the focusing effect of surface acoustic waves in the working area.

[0037] Preferably, in both the first and second interdigital electrodes, the spacing between adjacent fingers of the excitation electrode is 30-100 μm, for example, it can be 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm or 100 μm, and the width of the fingers is 30-100 μm, for example, it can be 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm or 100 μm.

[0038] In this invention, the excitation electrode with appropriate finger width and finger spacing determines the center frequency of the surface acoustic wave chip.

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

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

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

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

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

[0044] Preferably, the power amplifier operates at a frequency of 1-130MHz, such as 1MHz, 10MHz, 30MHz, 50MHz, 70MHz, 90MHz, 100MHz, 120MHz, or 130MHz.

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

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

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

[0048] In this invention, the spray needle can be a quartz capillary needle, or other spray needles capable of producing an electro-spray effect, such as steel needles.

[0049] Preferably, the diameter of the quartz capillary needle is 10-50 μm, for example, it can be 10 μm, 20 μm, 30 μm, 40 μm or 50 μm.

[0050] In this invention, a quartz capillary needle of suitable diameter helps to reduce the diameter of the solvent droplets, which is beneficial for subsequent desolventizing and mass spectrometry detection, and helps to enhance the mass spectrometry signal intensity.

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

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

[0053] Preferably, the solvent delivery device includes an injection pump and a syringe.

[0054] Preferably, the distance between the tip of the electrospray nozzle and the mass spectrometer inlet is 1-2 cm, for example, it can be 1 cm, 1.2 cm, 1.4 cm, 1.6 cm, 1.8 cm or 2 cm.

[0055] In this invention, the appropriate distance between the tip of the electrospray head and the mass spectrometer inlet can enhance the spraying effect and 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, it can be 1 cm, 1.2 cm, 1.4 cm, 1.6 cm, 1.8 cm or 2 cm, etc.

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

[0058] In a second aspect, the present invention provides a mass spectrometry injection method for micro-cell lysis, nebulization, and ionization, wherein the mass spectrometry injection method employs the apparatus described in the first aspect, and the mass spectrometry injection method includes the following steps:

[0059] Start the adjustable DC power supply and solvent delivery device, apply high voltage to the electrospray head through the two-way connector, and then introduce the extraction ionization solvent into the electrospray head through the solvent delivery device, so that the tip of the electrospray head generates an electrospray, which is atomized into charged solvent droplets.

[0060] A sample solution containing trace amounts of cells is placed on a surface acoustic wave (SAW) chip. The SAW device is then driven under a first condition to cause cell lysis. The SAW device is then 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 collide with each other to extract and ionize the atomized sample droplets, thereby ionizing the analyte, which is then introduced into the mass spectrometer through the mass spectrometer inlet.

[0062] It should be noted that the cell types used in preparing the sample solution can be, for example, HeLa cells.

[0063] Preferably, the voltage setting range 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 this invention, under the first condition, cells in the sample solution can be fully lysed, thereby accelerating the cell lysis rate.

[0065] Preferably, the voltage setting range 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, etc.

[0066] In this invention, the sample atomization rate can be controlled under the second condition to increase the sample injection rate, so that the sample droplets can fully collide with the electrospray solvent droplets to extract the mass spectrometer and enhance the mass spectrometry signal intensity.

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

[0068] In this invention, the collision between appropriately sized droplets and atomized charged solvent droplets facilitates the full extraction and ionization of the analyte within the sample droplets, thereby enhancing the mass spectrometry signal intensity.

[0069] Preferably, the extraction ionization solvent comprises an aqueous methanol solution.

[0070] Preferably, the flow rate of the extraction 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, etc.

[0071] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

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

[0073] (1) This invention uses the same surface acoustic wave device to achieve high-efficiency cell lysis and nebulized mass spectrometry injection under different input conditions. This not only effectively avoids the clogging problem of electrospray injection, but is also easy to clean and can effectively avoid cross-contamination between samples. In addition, the extraction ionization solvent droplets generated by the nano-electrospray device and the nebulized sample droplets undergo extraction and charge transfer before entering the mass spectrometer, thereby realizing the ionization of the analyte. This selective extraction not only increases the concentration of the analyte, but also reduces the concentration of interfering components, thereby reducing the influence of the matrix on the ionization process, making it suitable for coupling with subsequent mass spectrometry analysis.

[0074] (2) The surface acoustic wave device designed in this invention can lyse cells within 5 seconds with a lysis efficiency of over 95% and a good lysis effect. Attached Figure Description

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

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

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

[0078] Figure 4 This is a schematic diagram of a charged solvent droplet in Embodiment 1 of the present invention.

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

[0080] Figure 6 Mass spectra of the devices provided in Embodiment 1 and Comparative Example 1 of the present invention for performance testing.

[0081] Figure 7 The mass spectrometry results are shown in the mass spectrometry analysis diagrams after mass spectrometry injection based on the apparatus provided in Example 1 and Comparative Example 1.

[0082] Among them, 1-Surface acoustic wave chip; 2-Chip clip; 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 interdigital electrode; 11-Second interdigital electrode; 12-Working area. Detailed Implementation

[0083] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0084] Example 1

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

[0086] A surface acoustic wave (SAW) device includes a SAW chip 1, a chip clip 2, a power amplifier 3, and a signal generator 4. The SAW chip 1 includes a piezoelectric substrate and interdigitated electrodes processed on the surface of the piezoelectric substrate. The piezoelectric substrate is a 4-inch, 0.5mm thick, double-sided polished 128°YX lithium niobate wafer. The interdigitated electrodes, along a direction away from the piezoelectric substrate, include a 20nm thick adhesive layer and an 80nm thick conductive electrode layer. The adhesive layer is made of chromium, and the conductive electrode layer is made of gold. The interdigitated electrodes are as follows: Figure 2 As shown, it includes: a first interdigital electrode 10, which includes a reflective electrode and an excitation electrode. The reflective electrode includes a plurality of grid-arranged electrode strips, with a total of 25 electrode strips. The excitation electrode includes a plurality of interdigitated finger strips arranged in a cross pattern, with a spacing of 100 μm between adjacent finger strips, a width of 100 μm for each finger strip, and 10 pairs of finger strips. A second interdigital electrode 11, which includes a reflective electrode and an excitation electrode. The reflective electrode includes a plurality of grid-arranged electrode strips, with a total of 25 electrode strips. The excitation electrode includes a plurality of interdigitated finger strips arranged in a cross pattern, with a spacing of 100 μm between adjacent finger strips, a width of 100 μm for each finger strip, and 10 pairs of finger strips. The space between the first interdigital electrode 10 and the second interdigital electrode 11 is the working area 12 of the sample to be tested. The length of the working area 12 is 6.2 mm, and the overlap length between adjacent interdigital electrodes is the electrode aperture. The electrode aperture is the width of the working area 12, which is 5 mm. In the first interdigital electrode 10, the reflecting electrode is located on the side away from the working area 12, and the shortest distance between the reflecting electrode and the excitation electrode is 50 μm. In the second interdigital electrode 11, the reflecting electrode is located on the side away from the working area 12, and the shortest distance between the reflecting 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 (SAW) chip 1 is clamped on the chip holder 2; the signal generator 4 is used to generate a sinusoidal signal at the center frequency of the SAW 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, with low voltage being 0.2V and high voltage being 0.4V; the operating frequency of the power amplifier 3 is 10MHz, and the gain of the power amplifier 3 is 37dB; the chip holder 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 to drive the SAW chip 1 to vibrate, thereby realizing the lysis and atomization of micro-cells.

[0088] A nanoliter electrospray device includes 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 6 is connected to the electrospray head 5, and the other end is connected to the injection pump 8 via a liquid infusion capillary tube, for delivering 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 two-way connector is made of stainless steel. The adjustable DC power supply 7 has an output voltage of 3500V, the positive and negative values ​​of which are controlled by pins, and the output magnitude is adjusted by a 20kΩ potentiometer. The liquid infusion capillary tube is a liquid phase capillary tube with an inner diameter of 0.5 mm and an outer diameter of 1.59 mm. The solvent delivery device includes the injection pump 8 and an injection tube.

[0089] The mass spectrometer 9 includes a mass spectrometer inlet, with the tip of the electrospray head 5 of the nano-electrospray device directly in front of the mass spectrometer inlet. 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 inlet is 1.5 cm.

[0090] This embodiment also provides a mass spectrometry injection method for micro-cell lysis, nebulization, and ionization. The mass spectrometry injection method uses the apparatus described above and includes the following steps:

[0091] (1) Start the adjustable DC power supply and solvent delivery device, apply a high voltage of 3500V to the electrospray head through the two-way connector, and then draw a 1000μL syringe of methanol aqueous solution (methanol and water volume ratio of 1:1) into the electrospray head at a flow rate of 3μL / min, so that the tip of the electrospray head generates an electrospray and atomizes into charged solvent droplets.

[0092] (2) Using HeLa cells as experimental material, the cultured HeLa cells were stained with fluorescein diacetate, resuspended in 150 mmol / L ammonium bicarbonate aqueous solution, and diluted to a concentration of 10^5 cells / mL. Then, 2 μL of the prepared fluorescently stained cell suspension was added to the working area of ​​the surface acoustic wave chip using a pipette. The signal generator output frequency was set to 10 MHz and the output voltage was adjusted to 0.2 V. The switch was turned on, and the surface acoustic wave device was driven to work for 5 seconds. Then the signal generator and the power switch were turned off to allow the cells to lyse.

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

[0094] (3) The atomized sample droplets on the surface acoustic wave device and the charged solvent droplets atomized on the nano-electrospray device collide with each other to extract and ionize the atomized sample droplets, thereby achieving the ionization of the analyte. The ionized sample enters the vacuum interface region of the mass spectrometer through the mass spectrometer inlet for desolvation and coulomb explosion, followed by mass spectrometry analysis.

[0095] Figure 3 The figure shows a comparison of the fluorescence signals of HeLa cells before and after lysis in this embodiment. As can be seen from the figure, the fluorescence signal disappeared after the HeLa cells were lysed, indicating that the cells were effectively lysed.

[0096] Figure 4 A schematic diagram of charged solvent droplets in this embodiment is shown. As can be seen from the figure, an effective and stable electrospray can be observed from the tip of the electrospray head, 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 it does not have a surface acoustic wave device, but consists only of a nanoliter electrospray device and a mass spectrometer. The nanoliter electrospray device is used for electrospray mass spectrometry of the sample to be tested.

[0099] The remaining parameters are consistent with the structure and Example 1.

[0100] This invention uses reserpine as the detection target and performs performance tests on the devices provided in Example 1 and Comparative Example 1. Specifically, 1 ppm of reserpine was injected into the device provided in Comparative Example 1, and 1 ppm of reserpine was 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, the device of Embodiment 1 provided by the present invention has a good sample introduction effect.

[0101] Figure 7The mass spectrometry analysis results after mass spectrometry injection based on the devices provided in Example 1 and Comparative Example 1 are shown. As can be seen from the figure, both devices can obtain obvious lipid information through mass spectrometry analysis.

[0102] The applicant declares that the present invention is illustrated by the above embodiments, but the present invention is not limited to the above process steps, that is, it does not mean that the present invention must rely on the above process steps to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials used in 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 (SAW) device includes a SAW chip, a chip holder, a power amplifier, and a signal generator. The SAW chip includes a piezoelectric substrate and interdigitated electrodes processed on the surface of the piezoelectric substrate. The SAW chip is held in the chip holder. The signal generator generates a sinusoidal signal at the center frequency of the SAW chip, which is amplified by the power amplifier. The chip holder contains four conductive spring legs. The amplified electrical signal is applied to the four pins of the interdigitated electrodes through the conductive spring legs to drive the SAW chip to vibrate, thereby achieving the lysis and atomization of micro-cells. A nanoliter electrospray device includes 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 a liquid delivery capillary tube for delivering 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, wherein the mass spectrometer has a mass spectrometer inlet, the front of which corresponds to the tip of the electrospray head of the nano-electrospray device, and the working area of ​​the surface acoustic wave chip is located directly below the tip of the electrospray head; The interdigitated electrodes include: The first interdigital electrode includes a reflective electrode and an excitation electrode. The reflective electrode includes a plurality of grid-arranged electrode strips, and the excitation electrode includes a plurality of intersecting finger strips. In the first interdigital electrode, the overlap length between adjacent finger strips is the electrode aperture, and the electrode aperture is the width of the working area, which is 4-6 mm. The second interdigital electrode includes a reflective electrode and an excitation electrode. The reflective electrode includes a plurality of grid-arranged electrode strips, and the excitation electrode includes a plurality of intersecting finger strips. The interval between the first interdigital electrode and the second interdigital electrode is the working area of ​​the sample to be tested; the length of the working area is 6-6.5 mm. In the first interdigital electrode, the reflective electrode is located on the side away from the working area, and the shortest distance between the reflective electrode and the excitation electrode is 40-60 μm; in the second interdigital electrode, the reflective electrode is located on the side away from the working area, and the shortest distance between the reflective electrode and the excitation electrode is 40-60 μm; in both the first and second interdigital electrodes, the spacing between adjacent fingers of the excitation electrode is 30-100 μm, and the width of the fingers is 30-100 μm; in both the first and second interdigital electrodes, the number of finger pairs of the excitation electrode is 10-30 pairs.

2. The apparatus according to claim 1, characterized in that, The piezoelectric substrate is made of lithium niobate.

3. The apparatus according to claim 1, characterized in that, The interdigitated electrodes comprise stacked adhesive layers and conductive electrode layers along a direction away from the piezoelectric substrate.

4. The apparatus according to claim 3, characterized in that, The material of the adhesive layer includes chromium.

5. The apparatus according to claim 3, characterized in that, The thickness of the adhesive layer is 10-30 nm.

6. The apparatus according to claim 3, characterized in that, The material of the conductive electrode layer includes gold.

7. The apparatus according to claim 3, characterized in that, The thickness of the conductive electrode layer is 50-100 nm.

8. The apparatus according to claim 1, characterized in that, The output frequency of the signal generator is 0-40MHz.

9. The apparatus according to claim 1, characterized in that, The output voltage of the signal generator is 0-10V.

10. The apparatus according to claim 1, characterized in that, The power amplifier operates at a frequency of 1-130MHz.

11. The apparatus according to claim 1, characterized in that, The electro-spray head is a spray needle.

12. The apparatus according to claim 11, characterized in that, The nozzle comprises a quartz capillary needle.

13. The apparatus according to claim 12, characterized in that, The diameter of the quartz capillary needle is 10-50 μm.

14. The apparatus according to claim 1, characterized in that, The material of the two-way connector is metal.

15. The apparatus according to claim 1, characterized in that, The adjustable DC power supply has an output voltage range of -8 to 8kV.

16. The apparatus according to claim 1, characterized in that, The solvent delivery device includes an injection pump and a syringe.

17. The apparatus according to claim 1, characterized in that, The distance between the tip of the electrospray nozzle and the mass spectrometer inlet is 1-2 cm.

18. The apparatus according to claim 1, characterized in that, The distance between the working area of ​​the surface acoustic wave chip and the tip of the electrospray nozzle is 1-2 cm.

19. A mass spectrometry sample introduction method for micro-cell lysis, nebulization, and ionization, characterized in that, The mass spectrometry injection method employs the apparatus described in any one of claims 1-18, and the mass spectrometry injection method includes the following steps: Start the adjustable DC power supply and solvent delivery device, apply high voltage to the electrospray head through the two-way connector, and then introduce the extraction ionization solvent into the electrospray head through the solvent delivery device, so that the tip of the electrospray head generates an electrospray, which is atomized into charged solvent droplets; A sample solution containing trace amounts of cells is placed on a surface acoustic wave (SAW) chip. The SAW device is then driven under a first condition to cause cell lysis. The SAW device is then driven 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 collide with each other to extract and ionize the atomized sample droplets, thereby ionizing the analyte, which is then introduced into the mass spectrometer through the mass spectrometer inlet.

20. The mass spectrometry injection method according to claim 19, characterized in that, The voltage setting range of the signal generator that meets the first condition is 0.15-0.25V.

21. The mass spectrometry injection method according to claim 19, characterized in that, The voltage setting range for the signal generator that meets the second condition is 0.3-0.5V.

22. The mass spectrometry injection method according to claim 19, characterized in that, The extraction ionization solvent includes an aqueous methanol solution.

23. The mass spectrometry injection method according to claim 19, characterized in that, The flow rate of the extraction ionization solvent is 0.2-6 μL / min.

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