A spatiotemporal decoupling mass spectrometry imaging method based on droplet storage

Abstract

This invention discloses a spatiotemporally decoupled mass spectrometry imaging method based on droplet storage, comprising the following steps: (1) micro-area sampling; (2) liquid bridge capture; (3) droplet encapsulation; (4) generation of droplet array; and (5) droplet release and mass spectrometry analysis. This invention completely decouples imaging sampling from subsequent analysis in space and time. Through steps such as micro-area sampling and droplet encapsulation, sample information is stored in the form of droplets, and subsequent analysis is performed. This achieves independent optimization of the three major processes of sampling, storage, and analysis, which not only greatly expands the range of analyzable samples but also eliminates the negative impact of preprocessing on imaging resolution from the root, meeting the requirements of different application scenarios for analytical efficiency and accuracy.

Description

Technical Field

[0001] This invention relates to the field of mass spectrometry analysis technology, and in particular to a spatiotemporal decoupling mass spectrometry imaging method based on droplet storage. Background Technology

[0002] Mass spectrometry imaging technology can simultaneously provide spatial distribution information of multiple molecules in a sample, and has become an important tool in life sciences, medical diagnostics, materials science, and other fields. Matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MSI) and desorption / electrospray ionization imaging (DESI-MSI) are among the mainstream mass spectrometry imaging methods. However, traditional MALDI-MSI requires matrix spraying or other pretreatment of tissue sections before analysis. These processes often lead to lateral migration of analytes on the tissue surface, resulting in degraded imaging resolution. Although DESI-MSI eliminates the matrix spraying step, its solvent spraying process still inevitably leads to tissue surface wetting, causing analyte migration and thus limiting further improvement in imaging resolution. Therefore, there is an urgent need to develop a new method that can fundamentally avoid the impact of pretreatment processes on imaging resolution.

[0003] On the other hand, existing mass spectrometry imaging techniques have the following inherent limitations: First, sampling and analysis must be performed simultaneously. The sample stage must be placed near the vacuum interface of the mass spectrometer or directly inside the ion source, which greatly limits the size and shape of the sample to be analyzed (for example, large tissue sections are difficult to analyze directly). Second, because the desorbate needs to be delivered to the mass spectrometer in real time, the entire imaging process must be completed continuously on the mass spectrometer without interruption, resulting in long instrument occupancy times and making it difficult to achieve high throughput or remote sampling. Finally, real-time analysis has stringent requirements for matching the laser repetition frequency and the mass spectrometry scanning speed, limiting further improvements in imaging resolution and speed.

[0004] Therefore, there is an urgent need in this field for a new mass spectrometry imaging technique that can break the strong spatiotemporal coupling between preprocessing, sampling, and analysis. Summary of the Invention

[0005] This invention aims to solve the resolution limitation problem caused by the mutual coupling of the "preprocessing-sampling-mass spectrometry analysis" process in existing mass spectrometry imaging. It provides a spatiotemporally decoupled mass spectrometry imaging method based on droplet storage. By completely decoupling the imaging sampling from the subsequent analysis in time and space, the negative impact of the preprocessing process on the imaging resolution is eliminated from the root, and the independent optimization of the three major processes of sampling, storage and analysis is achieved.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: a spatiotemporal decoupling mass spectrometry imaging method based on droplet storage, comprising the following steps: (1) Micro-area sampling: After fixing the sample on the sample stage and setting the sampling parameters, the micro-area sampling probe is used to scan the sample surface pixel by pixel according to the preset path, so that the sample at the corresponding pixel position produces desorption material.

[0007] (2) Liquid bridge capture: The generated desorbate is captured by a stable-flowing liquid bridge located above the sample, forming a capture liquid. A suitable solvent system can be selected according to the properties of the target analyte, and the liquid bridge may contain derivatizing reagents or other reagents depending on the actual situation.

[0008] (3) Droplet encapsulation: The capture liquid is introduced into the droplet generation flow path as a dispersed phase and a continuous phase to generate droplets containing the chemical composition of the sample at the corresponding pixel position. The droplets enter the storage pipeline for storage along with the continuous phase in the droplet generation flow path. The dispersed phase and the continuous phase are immiscible.

[0009] (4) Generation of droplet array: Repeat steps (1) to (3) to complete the micro-area sampling, liquid bridge capture, and droplet encapsulation process for all pixel positions on the sample surface. The generated droplets enter the storage pipeline and are arranged sequentially at intervals to form a droplet array. Each droplet in the droplet array contains the chemical composition of the corresponding pixel position on the sample. The generated droplets enter the storage pipeline in the sampling order to achieve physical isolation of information from different pixels. Post-processing of the droplets can be performed in the storage pipeline. The flow rate of the droplets in the storage pipeline can be controlled by the positive / negative pressure in the storage pipeline; the storage pipeline can be completely removed; the storage pipeline can be surface modified according to the actual situation.

[0010] (5) Droplet release and mass spectrometry analysis: The droplets in the storage tube are released one by one in sequence and transported to the mass spectrometer for chemical composition analysis to obtain the chemical distribution image of each droplet.

[0011] Preferably, in step (1), the sample is a solid sample with a smooth surface and a surface roughness ≤100μm.

[0012] Preferably, the micro-area sampling probe is a focused laser probe or a particle beam probe, and the sampling parameters of the micro-area sampling probe are: sampling diameter of 0.1μm-500μm, pulse energy of 1nJ-10mJ, sampling spacing of 0.1μm-500μm, and scanning speed of 0.1-1000pixel / s.

[0013] Preferably, in step (3), the droplet diameter is 20 μm-200 μm, and the droplet is an oil-in-water droplet, a water-in-oil droplet, a water-in-oil-in-water droplet, or an oil-in-water-in-oil droplet.

[0014] Preferably, in step (3), when the droplet is a water-in-oil or water-in-oil droplet, the continuous phase is a mineral oil or fluorinated oil containing a surfactant; when the droplet is an oil-in-water or oil-in-water droplet, the continuous phase is water or an aqueous solution containing a surfactant.

[0015] Preferably, the droplets are generated by a flow focusing structure, a T-junction, a co-flow focusing structure, an electric field driving, a magnetic field driving, an acoustic field driving, a pneumatic driving, or a mechanical driving, and the above structures can be fabricated using microfluidic technology.

[0016] Preferably, the storage pipeline is a section of surface-modified inorganic pipeline or flexible polymer material pipeline. The inorganic pipeline is a quartz capillary or stainless steel pipe, and the flexible polymer material is polyurethane, polyether ether ketone, or polytetrafluoroethylene. Surface modification refers to the treatment of the inner wall of the pipeline with a hydrophobic or oleophobic coating.

[0017] Preferably, in step (5), the mass spectrometer is an ion trap mass spectrometer, a time-of-flight mass spectrometer, a quadrupole mass spectrometer, an orbital trap mass spectrometer, a Fourier transform ion cyclotron resonance mass spectrometer, or a combination thereof, and the ion source of the mass spectrometer is an electrospray ionization source, a nanoliter electrospray ionization source, an atmospheric pressure chemical ionization source, or a matrix-assisted laser desorption / ionization source.

[0018] Therefore, the present invention has the following beneficial effects: (1) In terms of imaging resolution, since imaging sampling and subsequent analysis are completely decoupled in time and space, the negative impact of preprocessing on imaging resolution in traditional methods is avoided, enabling more accurate imaging analysis of samples. At the same time, the lateral migration of analytes on tissue surface caused by matrix spraying, reagent incubation and other steps in traditional mass spectrometry imaging is completely avoided, so that the imaging resolution is no longer constrained by the preprocessing process and can approach the limit resolution of the sampling technology itself, which significantly improves the imaging resolution and can more clearly present the spatial distribution information of molecules in the sample. (2) In terms of sample adaptability, the present invention breaks through the strict limitations of traditional mass spectrometry imaging technology on sample size and shape. Through micro-area sampling, droplet encapsulation and other steps, the sample information is stored in the form of droplets and then analyzed. It no longer relies on real-time sampling of the sample near the mass spectrometer, which greatly expands the range of analyzable samples. Whether it is a large tissue section or an irregularly shaped sample, it can perform effective mass spectrometry imaging analysis. (3) In terms of analytical efficiency, this invention achieves independent optimization of the three major processes of sampling, storage, and analysis. Sampling and droplet storage are performed first, and then mass spectrometry analysis is performed on the stored droplets at appropriate times and locations, which improves the efficiency of instrument use and facilitates high-throughput analysis and remote sampling. In addition, this invention no longer has stringent requirements for matching laser repetition frequency and mass spectrometry scanning speed, which can improve imaging resolution and speed to a certain extent and meet the requirements of analytical efficiency and accuracy for different application scenarios; (4) It is not limited to specific pretreatment methods or target molecule types. It can be directly used for high-resolution imaging of various molecules such as lipids, drugs, and endogenous metabolites. After droplet storage, it can be flexibly pretreated offline as needed (such as enzymatic digestion, derivatization, etc.) or directly injected for analysis. This greatly expands the application scenarios of mass spectrometry imaging, bringing new breakthroughs and developments to mass spectrometry imaging technology. It is expected to be widely used in many fields such as life sciences, medical diagnosis, and materials science. (5) It can be adapted to various micro-area sampling methods such as laser desorption and desorption electrospray, as well as various ionization sources such as ESI and MALDI, and has high platform compatibility and scalability. Detailed Implementation

[0019] The present invention will be further described below through specific embodiments.

[0020] (1) Micro-area sampling: The sample (a mouse kidney tissue slice with a thickness of 10 μm) was fixed on the sample stage. After setting the sampling parameters, the micro-area sampling probe (focused laser probe) was used to scan the sample surface pixel by pixel according to the preset path, so that the sample at the corresponding pixel position would generate desorption. The sampling parameters of the micro-area sampling probe were: sampling diameter of 100 μm, pulse energy of 200 uJ, sampling interval of 100 μm, and scanning speed of 20 pixel / s. (2) Liquid bridge capture: The generated desorbate is captured by a stable liquid bridge located above the sample, forming a capture liquid. The liquid bridge is a 50% acetonitrile aqueous solution containing 0.1% (wt%) formic acid. (3) Droplet encapsulation: The capture liquid is introduced into the droplet generation flow path as the dispersed phase. A T-junction design is adopted in the microfluidic chip. Under the combined action of shear force and interfacial tension between the continuous phase (perfluoronaphthalene containing 1% EA surfactant) and the dispersed phase, droplets containing the chemical composition of the sample at the corresponding pixel position are generated. The droplet diameter is 100μm. The droplets are stored in the storage pipeline (quartz capillary with polyimide coating, inner diameter of 100μm and length of 2m) along with the continuous phase. (4) Generate droplet array: Repeat steps (1) to (3) to complete the micro-area sampling, liquid bridge capture and droplet encapsulation process for all pixel positions on the sample surface. The generated droplets enter the storage pipeline and are arranged in sequence at intervals to form a droplet array. Each droplet in the droplet array contains the chemical composition of the corresponding pixel position on the sample. (5) Droplet release and mass spectrometry analysis: The droplets in the storage tube are released one by one in sequence and transported to the mass spectrometer for chemical composition analysis. The mass spectrometry analysis uses a time-of-flight mass spectrometer and the ion source of the mass spectrometer is a nano-electrospray ionization source (nano-ESI) to obtain the chemical distribution image of the chemical components in each droplet.

[0021] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications are possible without departing from the technical solutions described in the claims.

Claims

1. A spatiotemporal decoupling mass spectrometry imaging method based on droplet storage, characterized in that, Includes the following steps: (1) Micro-area sampling: After fixing the sample on the sample stage and setting the sampling parameters, the micro-area sampling probe is used to scan the sample surface pixel by pixel according to the preset path, so that the sample at the corresponding pixel position produces desorption material. (2) Liquid bridge capture: The generated desorbate is captured by a stable liquid bridge located above the sample, forming a capture liquid; (3) Droplet encapsulation: The capture liquid is introduced into the droplet generation flow path as a dispersed phase and a continuous phase to generate droplets containing the chemical composition of the sample at the corresponding pixel position. The droplets enter the storage pipeline for storage along with the continuous phase in the droplet generation flow path. The dispersed phase and the continuous phase are immiscible. (4) Generate droplet array: Repeat steps (1) to (3) to complete the micro-area sampling, liquid bridge capture and droplet encapsulation process for all pixel positions on the sample surface. The generated droplets enter the storage pipeline and are arranged in sequence at intervals to form a droplet array. Each droplet in the droplet array contains the chemical composition of the corresponding pixel position on the sample. (5) Droplet release and mass spectrometry analysis: The droplets in the storage tube are released one by one in sequence and transported to the mass spectrometer for chemical composition analysis to obtain the chemical distribution image of each droplet.

2. The spatiotemporal decoupling mass spectrometry imaging method based on droplet storage according to claim 1, characterized in that, In step (1), the sample is a solid sample with a smooth surface and a surface roughness ≤100μm.

3. A spatiotemporal decoupling mass spectrometry imaging method based on droplet storage according to claim 1 or 2, characterized in that, The micro-area sampling probe is a focused laser probe or a particle beam probe. The sampling parameters of the micro-area sampling probe are: sampling diameter of 0.1μm-500μm, pulse energy of 1nJ-10mJ, sampling interval of 0.1μm-500μm, and scanning speed of 0.1-1000pixel / s.

4. The spatiotemporal decoupling mass spectrometry imaging method based on droplet storage according to claim 1, characterized in that, In step (3), the droplet diameter is 20 μm-200 μm, and the droplet is an oil-in-water droplet, a water-in-oil droplet, a water-in-oil-in-water droplet, or an oil-in-water-in-oil droplet.

5. The spatiotemporal decoupling mass spectrometry imaging method based on droplet storage according to claim 4, characterized in that, In step (3), when the droplet is a water-in-oil or water-in-oil droplet, the continuous phase is a mineral oil or fluorinated oil containing a surfactant; when the droplet is an oil-in-water or oil-in-water droplet, the continuous phase is water or an aqueous solution containing a surfactant.

6. A spatiotemporal decoupling mass spectrometry imaging method based on droplet storage according to claim 1 or 4, characterized in that, The droplets are generated through a flow focusing structure, a T-junction, a co-flow focusing structure, electric field driving, magnetic field driving, acoustic field driving, pneumatic driving, or mechanical driving.

7. The spatiotemporal decoupling mass spectrometry imaging method based on droplet storage according to claim 1, characterized in that, The storage pipeline is a section of surface-modified inorganic pipeline or flexible polymer material pipeline. The inorganic pipeline is a quartz capillary or stainless steel pipe, and the flexible polymer material is polyurethane, polyether ether ketone, or polytetrafluoroethylene.

8. The spatiotemporal decoupling mass spectrometry imaging method based on droplet storage according to claim 1, characterized in that, In step (5), the mass spectrometer is an ion trap mass spectrometer, a time-of-flight mass spectrometer, a quadrupole mass spectrometer, an orbital trap mass spectrometer, a Fourier transform ion cyclotron resonance mass spectrometer, or a combination thereof, and the ion source of the mass spectrometer is an electrospray ionization source, a nanoliter electrospray ionization source, an atmospheric pressure chemical ionization source, or a matrix-assisted laser desorption / ionization source.