Mass spectrum detection chip preparation system and method

Abstract

The invention belongs to the field of mass spectrometric detection, and provides a mass spectrometric detection chip preparation system and method. The system comprises an ion generation module, a solvent removal transmission module and an ion landing module, and sample transmission lines among the three modules are on the same straight line; the ion generation module is used for generating charged sample liquid drops, and the charged sample liquid drops enter the solvent removal and transmission module under the combined action of a first electric field and a first airflow channel; the solvent removal and transmission module is used for carrying out desolvation operation on the charged sample liquid drops to obtain charged sample molecules; and the charged sample molecules land on a target plate of the ion landing module under the combined action of the second electric field and the second airflow channel. The system has the advantages of being convenient to operate, simple in working mode switching, high in ion transmission efficiency, low in sample consumption and the like, and various types of mass spectrum detection chips can be prepared.

Description

Technical Field

[0001] The present invention belongs to the field of mass spectrometry detection, and in particular relates to a mass spectrometry detection chip preparation system and method. Background Art

[0002] The statements in this section merely provide background information related to the present invention and do not necessarily constitute prior art.

[0003] Detection chips usually require specific capture reagents or materials to be fixed on the surface, such as antibodies, lectins or other biomolecules with sufficient affinity for the target molecule and selective solid phase materials. After specific binding to specific target analytes in the experimental sample, after a washing step to remove non-specific binding medium components, the specifically bound target is analyzed using appropriate technology. The combination of mass spectrometry and detection chips provides a label-free and multi-channel detection platform. As long as there are differences in the molecular weight, fragment ions or gas phase ion conformations of the analytes to be analyzed, this technology can separate and distinguish different analytes bound to the same fixed phase from the background signal from the chip, achieving rapid analysis.

[0004] Currently, there is an urgent need to develop a mass spectrometry-specific detection chip that can be coupled with various types of ionization methods to further improve the throughput and speed of mass spectrometry analysis and analyze trace levels of targets in complex samples. Summary of the invention

[0005] In order to solve the technical problems existing in the above-mentioned background technology, the present invention provides a mass spectrometry detection chip preparation system and method, which can be applied to prepare various types of mass spectrometry detection chips, can reduce the demand for expensive biological samples, and has simple operation for switching between multiple working modes.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A first aspect of the present invention provides a mass spectrometry detection chip preparation system.

[0008] A mass spectrometry detection chip preparation system comprises: an ion generation module, a solvent removal transmission module and an ion landing module, wherein the sample transmission routes between the three modules are on the same straight line; a first electric field and a first air flow channel are arranged between the ion generation module and the solvent removal transmission module; a second electric field and a second air flow channel are arranged between the solvent removal transmission module and the ion landing module;

[0009] The ion generation module is used to generate charged sample droplets, and the charged sample droplets enter the solvent removal and transmission module under the combined action of the first electric field and the first air flow channel;

[0010] The solvent removal and transmission module is used to perform a desolvation operation on the charged sample droplets to obtain charged sample molecules; the charged sample molecules land on the target plate of the ion landing module under the combined action of the second electric field and the second air flow channel.

[0011] As an embodiment, the displacements of the ion generation module, the solvent removal transmission module and the ion landing module are independently controlled.

[0012] As an embodiment, the ion generating module includes a power supply, a gas circuit, a peristaltic pump, an injection needle, a spray needle and a spray head;

[0013] The injection needle is loaded on the peristaltic pump; the positive electrode of the power supply is connected to the needle of the injection needle, so that the sample forms a charged sample droplet; the injection needle is connected to the front end of the spray needle through a quartz capillary, and the rear end of the spray needle extends into the nozzle and extends out a set distance;

[0014] The gas path is connected to the rear end of the spray needle, and the gas flow enters the spray head from the gas path, moves around the injection capillary inserted into the spray head, and sprays out at the spray head, forming a first gas flow channel;

[0015] When preparing the mass spectrometry detection chip, the charged sample droplets are ejected from the nozzle under the joint promotion of the first electric field and the first air flow channel and enter the solvent removal and transmission module.

[0016] As an embodiment, the spray needle adopts a fused silica capillary with a stretched tip. After the inner diameter is stretched, the length of the spray needle is controlled within the range of 10-50um by adjusting the stretching temperature and distance to ensure the occurrence of non-denatured protein molecular ions.

[0017] As an embodiment, the speed of the peristaltic pump is used to adjust the flow rate of the sample.

[0018] As an implementation method, the rear end of the spray needle extends out of the spray head by 1-2 mm.

[0019] As an embodiment, the solvent removal transmission module includes a transmission cavity and a first temperature controller; the transmission cavity is an inner and outer double-layer structure, the outer layer is sleeved on the outside of the inner layer, the inner layer is a desolventizing tube, and the outer layer is directly assembled with the first temperature controller.

[0020] As an embodiment, the ion landing module includes a target plate, a target plate holder, a second temperature controller and a two-dimensional automated platform; the target plate is fixed on the target plate holder, and the second temperature controller is connected to the target plate to control the temperature of the target plate so that the charged sample molecules on the target plate can be quickly cooled; the two-dimensional automated platform is connected to the target plate holder to control the target plate holder to perform two-dimensional movement.

[0021] As an implementation manner, the temperature control range of the second temperature controller is -25°C to 120°C.

[0022] The second aspect of the present invention provides a method for preparing a mass spectrometry detection chip.

[0023] A method for preparing a mass spectrometry detection chip, based on the mass spectrometry detection chip preparation system as described above, comprises:

[0024] The ion generation module generates charged sample droplets, so that the charged sample droplets enter the solvent removal and transmission module under the combined action of the first electric field and the first air flow channel;

[0025] The solvent removal and transmission module performs a desolvation operation on the charged sample droplets to obtain charged sample molecules, so that the charged sample molecules land on the target plate of the ion landing module under the combined action of the second electric field and the second air flow channel.

[0026] The beneficial effects of the present invention are:

[0027] The present invention provides a system for preparing a mass spectrometry detection chip, which is used to prepare a mass spectrometry detection chip system compatible with matrix-assisted laser desorption and micro-droplet surface sampling probe ionization. The system can achieve soft landing of protein biomolecules on the chip, and can also be prepared by realizing in-situ gas phase reaction of the stationary phase material on the chip surface.

[0028] The system of the present invention has the advantages of simple operation of switching working modes, convenient temperature control of the transmission process and landing area, high ion transmission efficiency, accurate positioning, high degree of automation and low sample consumption, and can realize the preparation of various types of mass spectrometry detection chips.

[0029] Advantages of additional aspects of the present invention will be given in part in the following description, and in part will become obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0031] Figure 1 is a schematic diagram of the structure of a mass spectrometry detection chip preparation system according to an embodiment of the present invention;

[0032] Figure 2 This is a fluorescence microscope image of fluorescein-labeled lysozyme on the chip surface;

[0033] Figure 3 This is the mass spectrum of in situ enzymatic protein digestion on the trypsin chip surface.

[0034] Among them, 1, peristaltic pump; 2, injection needle; 3, spray needle; 4, desolvation tube; 5, mass spectrometry detection chip; 6, target plate bracket; 7, second temperature controller; 8, two-position automated displacement platform; 9, second three-dimensional displacement platform; 10, first three-dimensional displacement platform; 11, gas path inlet; 12, high voltage power supply; 13, first module temperature controller. DETAILED DESCRIPTION

[0035] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0036] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0037] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0038] The surface and substrate of the mass spectrometry detection chip must be compatible with the selected desorption and ionization mass spectrometry method, such as matrix-assisted laser desorption / ionization, which requires the sample substrate to be conductive and compatible with the matrix. Protein detection chips must ensure that proteins can be fixed and maintained without destroying the secondary or tertiary structure to avoid losing their biological activity. Commonly used protein chips for fluorescence detection are made of glass slide substrates, which are coated with substances that passively absorb array proteins or allow them to crosslink to reactive surfaces. Many other matrices are developed based on adsorption, crosslinking, affinity or hybridization. Self-assembled monolayers using gold or gold deposition are also used to couple protein chips. The gold surface can crosslink proteins through covalent bonds; connecting between surfaces and proteins on porous silicon or special polymers is also a way to prepare chips. The main limitation of this type of method is that gold, silicon or polymer surfaces can non-specifically bind different substances present in the same sample. Due to non-specific interactions, protein chips are interfered by other molecules from the sample being studied, which will hinder the analysis of ultra-low concentration targets.

[0039] In addition, for mass spectrometry detection chips based on stationary phase materials, the loading methods of the stationary phase include physical adhesion, surface chemical reaction or direct printing to fix the material on the chip. The physical adhesion method is easy to fall off during use; the chemical reaction method usually requires the use of multiple reagents for inert metal surfaces, and requires multiple operations on the chip target plate; the direct printing method consumes stationary phase materials, especially adsorption materials, and the cost is relatively high. At present, there are no reports on the preparation of multifunctional mass spectrometry detection chips. It is of great application value to develop a device that can directly fix proteins and / or materials on inert metal / metal oxide surfaces and other related conductive substrates (without any soft intermediate layer) without losing their activity or adsorption properties.

[0040] The technical solution of the present invention is as follows:

[0041] Microflow and nanoflow electrospray are used in combination with transmission temperature-controlled solvent removal technology. By controlling parameters such as the temperature and scale of the chip landing point, the in-situ reaction and ion internal energy are regulated to achieve non-denaturing landing of biomacromolecules on the chip surface and automated in-situ preparation of stationary phase materials.

[0042] according to Figure 1 A mass spectrometry detection chip preparation system according to an embodiment of the present invention comprises: an ion generation module, a solvent removal transmission module and an ion landing module, wherein the sample transmission routes between the three modules are on the same straight line; a first electric field and a first air flow channel are arranged between the ion generation module and the solvent removal transmission module; a second electric field and a second air flow channel are arranged between the solvent removal transmission module and the ion landing module;

[0043] The ion generation module is used to generate charged sample droplets, and the charged sample droplets enter the solvent removal and transmission module under the combined action of the first electric field and the first air flow channel;

[0044] The solvent removal and transmission module is used to perform a desolvation operation on the charged sample droplets to obtain charged sample molecules; the charged sample molecules land on the target plate of the ion landing module under the combined action of the second electric field and the second air flow channel.

[0045] In the embodiment of the present invention, the displacements of the ion generation module, the solvent removal transmission module and the ion landing module are independently controlled. The solvent removal transmission module is connected to the first three-dimensional displacement platform 10. The first three-dimensional displacement platform 10 can be a manual three-dimensional displacement platform.

[0046] In the specific real-time process, the ion generating module includes a power supply, a gas circuit, a peristaltic pump 1, an injection needle 2, a spray needle 3 and a spray head;

[0047] The injection needle 2 is loaded on the peristaltic pump 1; the positive electrode of the power supply is connected to the needle of the injection needle 2, so that the sample forms a charged sample droplet; the injection needle is connected to the front end of the spray needle through a quartz capillary, and the rear end of the spray needle extends into the nozzle and extends a set distance (such as 1-2mm); wherein the power supply is a high voltage power supply 12;

[0048] The gas path is connected to the rear end of the spray needle, and the gas flow enters the spray head from the gas path inlet 11, moves around the injection capillary inserted into the spray head and sprays out at the spray head, forming a first gas flow channel;

[0049] When preparing the mass spectrometry detection chip 5, the charged sample droplets are ejected from the nozzle under the joint promotion of the first electric field and the first air flow channel and enter the solvent removal and transmission module.

[0050] The spray needle 3 is a fused silica capillary with a stretched tip. After the inner diameter is stretched, the length of the spray needle is controlled within the range of 10-50 um by adjusting the stretching temperature and distance to ensure the generation of non-denatured protein molecular ions.

[0051] In this embodiment, the speed of the peristaltic pump is used to adjust the flow rate of the sample.

[0052] Specifically, the solvent removal transmission module includes a transmission cavity and a first temperature controller 13 ; the transmission cavity is an inner and outer double-layer structure, the outer layer is sleeved on the outer side of the inner layer, the inner layer is a desolventizing tube 4 , and the outer layer is directly assembled with the first temperature controller 13 .

[0053] For example, the inner layer of the desolventizing tube 4 is a stainless steel tube with an inner diameter of 3-5mm and a length of 10cm; the outer layer is made of PEEK material. The first temperature controller is arranged in the sleeve, and its length is 9cm, to ensure that the desolventizing tube is evenly heated as a whole, and the temperature of the whole desolventizing process is controllable. It is combined with the PEEK tube through a hoop, and the assembled joint is grounded. The rear end of the desolventizing tube is docked with the landing module, and the outlet size is controlled by an external porous metal cover. The diameter of the holes on the surface of the outer cover can be adjusted according to the needs of the target plate. The overall setting is simple, physical adhesion, and single target plate is convenient for disposable use.

[0054] In this embodiment, the ion landing module includes a target plate, a target plate bracket, a second temperature controller 7 and a two-dimensional automated platform 8; the target plate is fixed on the target plate bracket, and the second temperature controller is connected to the target plate to control the temperature of the target plate so that the charged sample molecules on the target plate can be quickly cooled; the temperature control range of the second temperature controller is -25°C to 120°C.

[0055] The target plate support is also connected to the second three-dimensional displacement platform 9. Wherein, the second three-dimensional displacement platform 9 can be a manual three-dimensional displacement platform.

[0056] The two-dimensional automated platform is connected to the target plate support and is used to control the target plate support to perform two-dimensional movement. For example, the two-dimensional automated platform can move in the xy axis direction through a programming control module, with a range of ±100 mm and a step length of 0.001 mm.

[0057] Each module of this embodiment can be positioned individually by a three-dimensional manual platform, and the two-dimensional automated platform of the ion landing module is used to control the movement of the target plate and the preparation of the detection chip array. The ion generating module is a nanoliter spray emitter obtained by a fused silica capillary. The ion desolvation chamber consists of a stainless steel tube and a temperature-controlled sleeve. In order to adapt to the geometric shape of the bracket, different types of target plates are designed for scanning mode mass spectrometry analysis. The size of the landing area is controlled by the preset program of the automatic platform. The sample injection speed is 120μL / h, the current is generally controlled at about 100nA, the temperature range of the chip landing point is -55℃~120℃, the size range of the landing point, the metal cover can be customized as needed to control the shape and size of the landing point, and the prepared mass spectrometry chip can be made of stainless steel or conductive glass with a size of 75.0mm×25.0mm×2.0mm.

[0058] A specific embodiment is given below.

[0059] Specific parameter settings and models of each module in this embodiment:

[0060] Power supply, voltage range: Tesman TRC2020 series high voltage power supply.

[0061] Input: AC220V±10%, 50 / 60Hz. Output: 1kV to 100kV and other maximum output voltages are optional, the maximum output power is 300W. 0~ to the maximum voltage is continuously adjustable, the output is positive and negative single polarity.

[0062] Gas path, gas flow rate range is: 0~2L / min.

[0063] Inner and outer diameters of the nozzle / needle: The outer diameter of the metal needle is 1.0mm, and the inner diameter is 0.5mm. The specific parameters can be adjusted according to the chip customization needs.

[0064] The ion landing module includes the target plate bracket dimensions: target plate bracket 100.0mm×100.0mm×6.2mm; target plate slot 75.2mm×25.2mm×2.5mm; target plate 75.0mm×25.0mm×2.0mm.

[0065] The temperature control range of the thermostat: 0 ~ 600 degrees Celsius.

[0066] Displacement and step length of the two-dimensional automated platform: the movable range of the xy axis is ±100mm; the step length is 0.001mm.

[0067] During the preparation of the lysozyme chip:

[0068] A 1.0 mg / mL lysozyme solution was delivered to the nano-electrospray emitter at a flow rate of 120 μL / h. A positive voltage of +1.5 kV was applied to the injection needle from an external high-voltage power supply. A nitrogen flow rate of 1.6 L / min was used for gas atomization. The charged aerosol was dried through a 10 cm long desolvation capillary, which was kept at ground potential and externally heated to 45 °C. The dried ions were deposited on a custom steel plate and the lysozyme solution was sprayed for 30 minutes. After ion landing deposition, the functionalized surface was washed in water for 5 minutes and dried at room temperature. The prepared lysozyme protein chip was placed in a solution of Micrococcus luteus, and the activity of the lysozyme landed on the chip was calculated based on the absorbance change. Figure 2 This is a fluorescence microscope image of fluorescein-labeled lysozyme on the chip surface.

[0069] During the preparation of the trypsin chip:

[0070] The prepared trypsin solution was delivered to the nano-electrospray emitter at a flow rate of 120 μL / h, and a positive voltage (+1.2 kV) provided by an external high-voltage power supply was applied to the liquid joint (metal joint). 1.6 L / min of nitrogen was applied to the atomizing gas to further improve atomization and evaporation. The charged aerosol was dried through a 10 cm long desolvation tube, which was maintained at ground potential and externally heated to 40 ° C. The dried ions were deposited on a custom steel plate and landed for 30 minutes. After the landing time, the protein chip was removed from the target holder, the functionalized surface was treated as needed, the HCCA matrix was added, and it was placed in the MALDI mass spectrometer for detection. Figure 3 This is the mass spectrum of in situ enzymatic protein digestion on the trypsin chip surface.

[0071] In one or more embodiments, a method for preparing a mass spectrometry detection chip, based on the mass spectrometry detection chip preparation system as described above, comprises:

[0072] The ion generation module generates charged sample droplets, so that the charged sample droplets enter the solvent removal and transmission module under the combined action of the first electric field and the first air flow channel;

[0073] The solvent removal and transmission module performs a desolvation operation on the charged sample droplets to obtain charged sample molecules, so that the charged sample molecules land on the target plate of the ion landing module under the combined action of the second electric field and the second air flow channel.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A mass spectrometry detection chip preparation system, characterized in that: include: The sample transmission routes among the ion generation module, the solvent removal transmission module and the ion landing module are on the same straight line; a first electric field and a first air flow channel are arranged between the ion generation module and the solvent removal transmission module; a second electric field and a second air flow channel are arranged between the solvent removal transmission module and the ion landing module; The ion generation module is used to generate charged sample droplets, and the charged sample droplets enter the solvent removal and transmission module under the combined action of the first electric field and the first air flow channel; The solvent removal and transmission module is used to perform a desolvation operation on the charged sample droplets to obtain charged sample molecules; the charged sample molecules land on the target plate of the ion landing module under the combined action of the second electric field and the second air flow channel.

2. The mass spectrometry detection chip preparation system according to claim 1, characterized in that: The displacements of the ion generation module, the solvent removal transmission module and the ion landing module are independently controlled.

3. The mass spectrometry detection chip preparation system according to claim 1, characterized in that: The ion generating module comprises a power supply, an air circuit, a peristaltic pump, an injection needle, a spray needle and a spray head; The injection needle is loaded on the peristaltic pump; the positive electrode of the power supply is connected to the needle of the injection needle, so that the sample forms a charged sample droplet; the injection needle is connected to the front end of the spray needle through a quartz capillary, and the rear end of the spray needle extends into the nozzle and extends out a set distance; The gas path is connected to the rear end of the spray needle, and the gas flow enters the spray head from the gas path, moves around the injection capillary inserted into the spray head, and sprays out at the spray head, forming a first gas flow channel; When preparing the mass spectrometry detection chip, the charged sample droplets are ejected from the nozzle under the joint promotion of the first electric field and the first air flow channel and enter the solvent removal and transmission module.

4. The mass spectrometry detection chip preparation system according to claim 3, characterized in that: The spray needle adopts a fused silica capillary with a stretched tip. After the inner diameter is stretched, the length of the spray needle is controlled within the range of 10-50um by adjusting the stretching temperature and distance to ensure the generation of non-denatured protein molecular ions.

5. The mass spectrometry detection chip preparation system according to claim 3, characterized in that: The speed of the peristaltic pump is used to adjust the flow rate of the sample.

6. The mass spectrometry detection chip preparation system according to claim 3, characterized in that: The rear end of the spray needle extends out of the spray head by 1-2 mm.

7. The mass spectrometry detection chip preparation system according to claim 1, characterized in that: The solvent removal transmission module includes a transmission cavity and a first temperature controller; the transmission cavity is an inner and outer double-layer structure, the outer layer is sleeved on the outer side of the inner layer, the inner layer is a desolventizing tube, and the outer layer is directly assembled with the first temperature controller.

8. The mass spectrometry detection chip preparation system according to claim 1, characterized in that: The ion landing module includes a target plate, a target plate bracket, a second temperature controller and a two-dimensional automated platform; the target plate is fixed on the target plate bracket, and the second temperature controller is connected to the target plate to control the temperature of the target plate so that the charged sample molecules on the target plate can be quickly cooled; the two-dimensional automated platform is connected to the target plate bracket to control the target plate bracket to perform two-dimensional movement.

9. The mass spectrometry detection chip preparation system according to claim 8, characterized in that: The temperature control range of the second temperature controller is -25°C to 120°C.

10. A method for preparing a mass spectrometry detection chip, based on the mass spectrometry detection chip preparation system according to any one of claims 1 to 9, characterized in that: include: The ion generation module generates charged sample droplets, so that the charged sample droplets enter the solvent removal and transmission module under the combined action of the first electric field and the first air flow channel; The solvent removal and transmission module performs a desolvation operation on the charged sample droplets to obtain charged sample molecules, so that the charged sample molecules land on the target plate of the ion landing module under the combined action of the second electric field and the second air flow channel.

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