Flow type single cell scale adjustable device and adjusting method for electrospray ionization mass spectrometry

Abstract

The invention relates to the technical field of single cell analysis of cell biology and analytical chemistry, in particular to a flow type single cell scale adjustable device for electrospray ionization mass spectrometry and an adjusting method. According to the present invention, the detection requirements of multi-size cells can be met, the target cells with different sizes in the range of 10-100 [mu] m can be screened through the arched channel switching of the arched shunting capture module, and the comprehensiveness of the detection sample can be ensured. According to the device, the arched channels can be rapidly switched to adapt to cells of different sizes without disassembling the device, continuous operation of sample introduction, screening, extraction, ionization and detection is realized through integrated flow design, high throughput and convenience are taken into account, and the device is adaptive to diversified environmental pollutant exposure metabolism research scenes. The cell suspension and the diluent are mixed on line to reduce non-volatile salt interference, the high-voltage electric field and the sheath gas cooperate to improve ionization efficiency, qualitative and quantitative analysis is achieved through orbitrap mass spectrometry, reliable data support is provided for accumulation, distribution and metabolic transformation characteristics of pollutants in cells, and detection accuracy is guaranteed.

Description

Technical Field

[0001] This invention relates to the field of single-cell analysis technology in cell biology and analytical chemistry, specifically to a single-cell scale adjustable device and adjustment method for electrospray mass cytometry. Background Technology

[0002] The intracellular accumulation and transformation processes of emerging pollutants that are of widespread concern in the environment (including persistent organic pollutants, endocrine disruptors, antibiotics, microplastics, etc.) are a core element in assessing ecotoxicity and health risks.

[0003] Traditional population cell analysis can mask differences in pollutant levels and metabolic transformations between individual cells, while single-cell mass spectrometry, with its high sensitivity, strong specificity, and ability to provide molecular information, has become a core technological support in this field. Among them, electrospray ionization mass cytometry (ESI-MS) is increasingly being applied to the detection of unknown environmental pollutants in single cells due to its ability to achieve label-free detection and adaptability to simultaneous multi-component analysis.

[0004] Existing mass spectrometry flow cytometry single-cell capture technology mainly focuses on the precise separation of individual cells, and is geared towards biomarker detection or routine cell sorting, taking into account the characteristics of environmental pollutants.

[0005] For example, Union Biometrica's Copas large particle sorter is based on pneumatic drive and mechanical screening principles and can process cells / biological particles of 10-1500μm. However, traditional mechanical screening can easily cause physical disturbance to cells, leading to cell rupture and thus affecting the accuracy of detection.

[0006] For example, On-chip Biotechnologies' On-chip SPiS microfluidic chip single-cell separation system achieves non-destructive separation of 10-200μm particles through real-time imaging recognition and piezoelectric ceramic driving principle. Although it solves the problem of easy damage, the imaging-guided capture system has a long processing time and can only analyze 384 wells per hour, which is difficult to meet the needs of large-scale cell screening in environmental samples.

[0007] For example, although the Fluigent inertial flow cytometry sorting system has a throughput of up to 3 ml / min, its scale resolution is limited and it is easy for non-target cells to be mixed in, leading to metabolite sample contamination.

[0008] For example, the invention patent application with patent number 202310989300.5 discloses a mass spectrometry flow cytometry single-cell analysis system that uses a capillary or arc-shaped structure with a fixed inner diameter, which can only be adapted to single cells of size 30-50μm.

[0009] In other words, the existing technology has the following problems:

[0010] 1. In current research on exposure to environmental pollutants, the single cell size of tissues such as heart, liver, brain, lungs and kidneys in model organisms such as zebrafish and mice varies greatly (10-100μm). Among them, small cells such as zebrafish glial cells (10-25μm) and mouse alveolar type I cells (25-40μm) are prone to transport deviation and have low capture efficiency, while larger cells are prone to congestion or structural damage in the channels.

[0011] The existing devices lack fine-grained control over channel scale adjustment, resulting in insufficient size adaptability and inability to meet the needs of multi-size analysis. This leads to signal loss due to cell retention and channel adsorption issues when low-content metabolites are present. Furthermore, the coarse nature of scale adjustment makes it difficult to guarantee the uniqueness of single-cell capture, which can easily lead to multi-cell co-capture and interfere with the qualitative and quantitative analysis of metabolites.

[0012] 2. Existing devices often use unmodified materials for their channel surfaces (such as quartz and polydimethylsiloxane), failing to consider the small molecule characteristics and adsorption properties of single-cell metabolites. After exposure to environmental pollutants, differentially expressed metabolites with extremely low background levels within cells are easily adsorbed onto the channel walls, leading to signal loss and problems such as false negatives in metabolite identification, misjudgment of fold differences, and inability to screen for characteristic metabolic markers. Simultaneously, cells tend to adhere at channel shunt nodes, resulting in low screening throughput and making it difficult to meet the needs of large-scale metabolomics analysis. Summary of the Invention

[0013] The purpose of this invention is to address the problems existing in the prior art by providing a single-cell scale adjustable device and adjustment method for electrospray mass cytometry.

[0014] To address the problems existing in the prior art, the present invention adopts the following technical solution:

[0015] A single-cell scale tunable device for electrospray mass cytometry includes:

[0016] The sample injection control module includes a sample and a sample delivery device to provide sample fluid to downstream modules;

[0017] The bow-shaped shunt capture module has its input end connected to the output end of the sample injection control module. The bow-shaped shunt capture module includes a bow-shaped shunt cavity, a waste liquid port, and a cell inlet port. The waste liquid port and the cell inlet port are respectively connected to the bow-shaped shunt cavity.

[0018] The arc-shaped shunt cavity includes at least three arc-shaped channels with different curvatures for cell shunt, and the at least three arc-shaped channels are switched by a drive mechanism to screen target cells of different sizes;

[0019] An online extraction electrospray ionization module is provided, the input of which is connected to the output of the bow-shaped shunt capture module. The online extraction electrospray ionization module includes an atmospheric pressure flow chamber and a hollow sleeve disposed in the atmospheric pressure flow chamber. At least three capillaries are coaxially disposed in the hollow sleeve, and it is equipped with an external high-voltage power supply system to provide charged ions for mass spectrometry detection.

[0020] The orbital trap mass spectrometer receives charged ions from the online extraction electrospray ionization module and detects cells.

[0021] The sample injection control module provides sample fluid to the bow-shaped shunt capture module. Cells of different sizes are shunted through bow-shaped channels with different curvatures in the bow-shaped shunt capture module. The switching output of different bow-shaped channels is realized by the drive mechanism. In the online extraction electrospray ionization module, the high-voltage electric field formed by the external high-voltage power supply system and the atmospheric pressure flow cytometry chamber are combined to construct a mass spectrometry flow cytometry environment.

[0022] As an improvement to the technical solution of the single-cell scale adjustable device for electrospray mass cytometry of the present invention, the bow-shaped shunt capture module is molded from transparent quartz glass, and the inner wall of the bow-shaped shunt capture module is coated with a membrane containing biocompatible protein (BSA) molecules and glutaraldehyde crosslinking.

[0023] As an improvement to the technical solution of the single-cell scale adjustable device for electrospray mass cytometry of the present invention, the curvatures of at least three of the said arc-shaped channels are 150μm, 100μm and 50μm respectively, which can correspond to the output cells with sizes of 60-100μm, 30-60μm and 10-30μm respectively.

[0024] As an improvement to the technical solution of the single-cell scale adjustable device for electrospray mass cytometry of the present invention, the bow-shaped shunt capture module has a conical structure or a tree-like structure.

[0025] As an improvement to the technical solution of the single-cell scale adjustable device for electrospray mass cytometry of the present invention, the sample injection control module includes sheath fluid, cell suspension sample and cell diluent and corresponding delivery pipes. A peristaltic pump is correspondingly provided on the delivery pipe, and the three delivery pipes are connected to the input end of the bow-shaped shunt capture module to provide sample fluid to the downstream module.

[0026] As an improvement to the technical solution of the single-cell scale adjustable device for electrospray mass cytometry of the present invention, the online extraction electrospray ionization module includes an atmospheric pressure flow chamber, the end of the hollow sleeve is an outwardly expanding funnel shape, at least three capillaries are arranged in the atmospheric pressure flow chamber, and three cell sample introduction channels are formed in the at least three capillaries.

[0027] The at least triple capillary includes an inner capillary that serves as a cell sampling channel, an intermediate capillary for delivering sheath fluid, and an outer capillary that serves as a sheath gas conduit.

[0028] As an improvement to the technical solution of the single-cell scale adjustable device for electrospray mass cytometry of the present invention, the outer diameter and inner diameter of the inner capillary are 200 μm and 100 μm, respectively; the outer diameter and inner diameter of the middle capillary are 350 μm and 250 μm, respectively; and the outer diameter and inner diameter of the outer capillary are 700 μm and 500 μm, respectively.

[0029] As an improvement to the technical solution of the single-cell scale adjustable device for electrospray mass cytometry of the present invention, the voltage of the external high-voltage power supply system is 1-3kV.

[0030] As an improvement to the technical solution of the single-cell scale tunable device for electrospray mass cytometry of the present invention, the orbital trap mass spectrometer is an Orbitrap MS.

[0031] A method for single-cell scale modulation in electrospray mass cytometry, using the single-cell scale modulation device for electrospray mass cytometry as described above, includes the following steps:

[0032] S1. Start the sample injection control module to extract the sample into the bow-shaped shunt capture module and the online extraction electrospray ionization device;

[0033] S2. When the sample passes through the bow-shaped shunt capture module, it is sorted and shunt according to the different sizes of the cells, and the target size cells are output.

[0034] S3. The cells output from the bow-shaped diversion and capture module come into contact with the sheath fluid inside the hollow sleeve and flow to the outlet of the atmospheric pressure flow chamber under the action of the sheath fluid. At the same time, the intracellular substances are extracted online by the sheath fluid.

[0035] S4. Start the external high-voltage power supply system. The intracellular substances extracted by the sheath fluid form charged droplets in the high-voltage electric field, and the charged droplets are evaporated in at least three capillaries to obtain charged ions.

[0036] S5. After evaporation, the charged ions enter the orbital trap mass spectrometer to complete the detection and analysis of substances within a single cell.

[0037] The beneficial effects of this invention are:

[0038] 1. In this invention, at least three arc-shaped channels with different curvatures of the arc-shaped shunt capture module are used, and these three arc-shaped channels can be switched by a drive mechanism. This enables the screening of cells ranging from 10 to 100 μm, covering metabolic research cells sensitive to pollutant exposure, such as zebrafish glial cells (10-25 μm), mouse alveolar type I cells (25-40 μm), and mouse cardiomyocytes (60-100 μm). This fully reflects the heterogeneity of metabolic responses of different cell types to pollutants and provides comprehensive sample support for metabolite differential analysis.

[0039] 2. In this invention, by using the sheath flow and hollow sleeve as fluid focusing and guiding modules, single cells are transferred to the arc-shaped channel of the central axis of the online extraction electrospray ionization module. Combined with the electrospray capillary, the cell rupture rate is reduced, and metabolite leakage is minimized. At the same time, the ionization efficiency of the focused cells during electrospraying is improved, which helps in the screening of characteristic metabolic markers.

[0040] 3. Because at least three arc-shaped channels with different curvatures are formed in the arc-shaped shunt capture module, and the switching output of different arc-shaped channels is realized through the drive mechanism, the screening and adaptation of target cells of different sizes can be completed without disassembling the device. For example, switching from detecting zebrafish glial cells (10-25μm, high curvature R=50μm) to mouse cardiomyocytes (60-100μm, low curvature R=150μm) takes only a few seconds. It can flexibly adapt to metabolic research scenarios of different model organisms, different tissues, and different pollutant exposure types, greatly improving the applicability and experimental flexibility of the device. Attached Figure Description

[0041] Figure 1 This is a schematic diagram of the structure of the present invention;

[0042] Figure 2 This is a schematic diagram of the bow-shaped shunt capture module in this invention;

[0043] Figure 3 This is a schematic diagram of the hollow sleeve in the online extraction electrospray ionization module of the present invention;

[0044] Figure 4 This is a static contact angle image of water after surface modification in Example 2 of the present invention.

[0045] Figure labeling: 1 - Sample injection control module; 11 - Peristaltic pump; 12 - Sheath fluid; 13 - Cell suspension sample; 14 - Cell diluent; 2 - Bow-shaped shunt capture module; 21 - Waste liquid port; 22 - Cell inlet port; 3 - Online extraction electrospray ionization module; 31 - External high-voltage power supply system; 32 - Sheath gas port; 33 - Atmospheric pressure flow chamber; 34 - Capillary; 35 - Sheath fluid port; 4 - Orbital trap mass spectrometer; 41 - Mass spectrometer inlet. Detailed Implementation

[0046] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments.

[0047] like Figures 1 to 4 As shown, one aspect of the present invention provides a single-cell scale adjustable device for electrospray mass cytometry, comprising:

[0048] The sample injection control module 1 includes a sample and a sample delivery device to provide sample fluid to downstream modules;

[0049] The bow-shaped shunt capture module 2 has its input end connected to the output end of the sample injection control module 1. The bow-shaped shunt capture module 2 includes a bow-shaped shunt cavity, a waste liquid port 21 and a cell inlet port 22, and the waste liquid port 21 and the cell inlet port 22 are respectively connected to the bow-shaped shunt cavity.

[0050] The arc-shaped shunt cavity includes at least three arc-shaped channels with different curvatures for cell shunt, and the at least three arc-shaped channels are switched by a drive mechanism to screen target cells of different sizes;

[0051] The online extraction electrospray ionization module 3 has its input end connected to the output end of the bow-shaped shunt capture module 2. The online extraction electrospray ionization module 3 includes a spray flow module and an atmospheric pressure flow chamber 33 connected to each other, and a hollow sleeve disposed in the atmospheric pressure flow chamber 33. At least three capillaries 34 are coaxially disposed in the hollow sleeve, and it is equipped with an external high-voltage power supply system 31 to provide charged ions for mass spectrometry detection.

[0052] The orbital trap mass spectrometer 4 receives charged ions from the online extraction electrospray ionization module 3 and detects cells;

[0053] The sample injection control module 1 provides sample fluid to the bow-shaped shunt capture module 2. The bow-shaped channels with different curvatures in the bow-shaped shunt capture module 2 are used to shunt cells of different sizes. The switching output of different bow-shaped channels is realized by the drive mechanism. In the online extraction electrospray ionization module 3, the high-voltage electric field formed by the external high-voltage power supply system 31 and the atmospheric pressure flow chamber 33 are combined to construct the mass spectrometry flow environment.

[0054] In use, the output of sample injection control module 1 is connected to the input of bow-shaped shunt capture module 2. Cell screening utilizes the principle of curvature and contact angle difference to shunt cells of different sizes. Finally, the valve core is switched via an internal drive mechanism to achieve different channel outputs. The output of bow-shaped shunt capture module 2 is connected to the inlet of the hollow sleeve in the spray flow module of the online extraction electrospray ionization module 3. The spray flow module is fixed to the tabletop by an iron frame, and the outlet of the capillary 34 in the atmospheric pressure flow chamber 33 is aligned with the mass spectrometry inlet 41 of the track trap mass spectrometer 4. Nitrogen gas is used as the sheath gas and methanol as the sheath liquid, which are assembled on the outside of the atmospheric pressure flow chamber 33 through a connecting nut. The mass spectrometry flow environment is then constructed by an external high-voltage power supply system 31 in conjunction with the atmospheric pressure flow chamber 33. Preferably, the drive mechanism is a motor, and the structure of the motor is as described in the prior art, which will not be repeated here.

[0055] In this invention, the bow-shaped diversion chamber in the bow-shaped diversion capture module 2 is equipped with at least three bow-shaped channels with different curvatures. These three channels can be flexibly switched via a drive mechanism. Utilizing the principle that the contact angle between the fluid and the edge of the bow-shaped channel differs due to cell size differences, cells of different sizes are diverted to the bow-shaped channel with the corresponding curvature. As an embodiment of this invention, the bow-shaped diversion chamber is equipped with three bow-shaped channels with curvatures of 150μm, 100μm, and 50μm, respectively, corresponding to the output of cells with sizes of 60-100μm, 30-60μm, and 10-30μm. By utilizing the principle that the contact angle between the fluid and the edge of the bow-shaped channel differs due to cell size differences, cells of different sizes are diverted to the bow-shaped channel with the corresponding curvature, thereby achieving precise screening and adaptation of cells within the 10-100μm range. The capillary 34 is preferably a fused silica capillary 34.

[0056] In addition, the drive mechanism is located at the outlet confluence node of the bow-shaped channel. An external controller controls the drive mechanism to switch between different bow-shaped channels to ensure that the bow-shaped channel corresponding to the target size cell enters the downstream module, avoids interference from non-target bow-shaped channels, improves the accuracy and efficiency of the diversion, and makes up for the uncertainty of pure physical diversion.

[0057] Waste liquid outlet 21 and cell inlet 22 are located at the rear end of the arc-shaped diversion cavity. Waste liquid outlet 21 is used to discharge non-target cells and waste liquid, while cell inlet 22 is used to lead target cells out of the arc-shaped channel. The end of the hollow sleeve is shaped like an outwardly expanding trumpet to facilitate cell focusing.

[0058] In detail, firstly, in this invention, at least three arc-shaped channels with different curvatures of the arc-shaped shunt capture module 2 are used, and these at least three arc-shaped channels can be switched by a drive mechanism, enabling the screening of 10-100μm cells. This covers pollutant-sensitive metabolic research cells such as zebrafish glial cells (10-25μm), mouse alveolar type I cells (25-40μm), and mouse cardiomyocytes (60-100μm), fully reflecting the heterogeneity of metabolic responses of different cell types to pollutants and providing comprehensive sample support for metabolite differential analysis.

[0059] Secondly, in this invention, by using the sheath flow and hollow sleeve as fluid focusing and guiding modules, single cells are transferred to the central axis channel of the online extraction electrospray ionization module 3 after screening, combined with the electrospray capillary 34, so as to reduce the cell rupture rate and minimize metabolite leakage; at the same time, the ionization efficiency of the focused cells during electrospraying is improved, which helps to screen characteristic metabolic markers.

[0060] In some embodiments of the present invention, the bow-shaped shunt capture module 2 is molded from transparent quartz glass, and the inner wall of the bow-shaped shunt capture module 2 is coated with a membrane containing biocompatible protein (BSA) molecules and glutaraldehyde crosslinking.

[0061] In detail, in this invention, membrane modification is performed inside the bow-shaped shunt capture module 2. BSA, as a biocompatible protein, contains a large number of hydrophilic groups (such as carboxyl and amino groups) in its molecules. It can cover the active sites on the surface of quartz glass through physical adsorption, blocking the van der Waals forces and hydrogen bonding between cells and glass, and reducing non-specific adhesion. Preferably, the concentration is 0.1%, as described in the literature "Microfluidics and Nanofluidics" 2021, 25 (8): 92. This concentration is a commonly used anti-adhesion concentration for microfluidic devices.

[0062] In some embodiments of the present invention, the curvatures of at least three arc-shaped channels are 150 μm, 100 μm, and 50 μm, respectively, which can correspond to the output cells with sizes of 60-100 μm, 30-60 μm, and 10-30 μm, respectively. Furthermore, the arc-shaped shunt capture module 2 has a conical structure or a tree-like structure.

[0063] In detail, because at least three arc-shaped channels are formed in the arc-shaped shunt capture module 2, and the switching output of different arc-shaped channels is realized through the drive mechanism, the screening and adaptation of target cells of different sizes can be completed without disassembling the device. For example, switching from detecting zebrafish glial cells (10-25μm, high curvature R=50μm) to mouse cardiomyocytes (60-100μm, low curvature R=150μm) takes only a few seconds. It can flexibly adapt to metabolic research scenarios of different model organisms, different tissues, and different pollutant exposure types, greatly improving the applicability and experimental flexibility of the device.

[0064] More specifically, in this invention, an adjustable capture device with a single-cell scale of 10-100μm is constructed through the bow-shaped shunt capture module 2. This device is suitable for the analysis of single-cell metabolites exposed to environmental pollutants, which will greatly simplify the connection process between single-cell separation and metabolite detection, and achieve low-interference and high-precision detection and analysis of single-cell metabolites under environmental pollutant exposure compared with existing technologies.

[0065] The bow-shaped flow diversion and capture module 2 has a conical or tree-like structure. Internally, it utilizes the curvature of the bow-shaped edge structure and the principle of contact angle difference to divert flow between cells of different sizes. When fluid containing single cells passes through the bow-shaped edge, the cell size changes the contact angle (θ) between the fluid and the bow-shaped edge. Only when the contact angle exceeds a certain threshold (θ0) will the fluid deviate towards the corresponding bow-shaped channel. The change in contact angle is determined by the spatial volume occupied by the cell. When large cells occupy a large space, the fluid is compressed, and the contact angle increases significantly (θ > θ0); small cells occupy a small space, resulting in a small change in contact angle (θ < θ0), and the fluid is diverted along its original path. The conical or tree-like structure here is as follows: Figure 2 As shown, the overall width gradually increases along the direction of cell flow.

[0066] That is, when cells flow through the arc-shaped channel in a fluid, their different sizes occupy different volumes of space, causing differences in the contact angle (θ) between the fluid and the edge of the arc-shaped channel. For example, large cells (such as 60-100 μm mouse cardiomyocytes) occupy a large space, resulting in significant fluid compression and a significantly increased contact angle (θ > threshold θ0); small cells (such as 10-25 μm zebrafish glial cells) occupy a small space, resulting in a smaller change in contact angle (θ < threshold θ0). The three sets of arc-shaped channels with different curvatures (R=50 / 100 / 150 μm) correspond to different ranges of contact angle variation. The high curvature (R=50 μm) arc-shaped channel is adapted to the small-amplitude contact angle variation of small cells, ensuring that they are accurately diverted along the original path; the low curvature (R=150 μm) arc-shaped channel is adapted to the large-amplitude contact angle variation of large cells, causing them to deviate towards the corresponding arc-shaped channel output; and the medium curvature (R=100 μm) arc-shaped channel is adapted to the contact angle variation of medium-sized cells.

[0067] In some embodiments of the present invention, the sample injection control module 1 includes a sheath fluid 12, a cell suspension sample 13, and a cell diluent 14, as well as corresponding delivery pipes. A peristaltic pump 11 is correspondingly provided on the delivery pipes, and the three delivery pipes converge and are connected to the input end of the bow-shaped diversion and capture module 2 to provide sample fluid to the downstream module.

[0068] In detail, the sample injection control module 1 is used to adjust the constant speed and deliver the corresponding liquids in each arc-shaped channel. It mainly includes sheath fluid 12, cell suspension sample 13 and cell diluent 14 and corresponding delivery pipes, forming a sheath fluid 12 input channel for extracting intracellular substances, a cell suspension sample 13 channel for maintaining uniform dispersion and suspension of cells in liquid culture medium, and a cell diluent 14 channel for diluting high concentrations of non-volatile salts in the cell suspension.

[0069] In one embodiment of this method, the sheath fluid 12 in the sheath fluid inlet channel uses chromatographic grade methanol, and its rate is controlled at 1-10 μL / min by a peristaltic pump 11 installed on the sheath fluid 12 delivery pipeline. A cell suspension apparatus is installed on the cell suspension sample 13 delivery pipeline to maintain the uniform dispersion and suspension of cells in the liquid culture medium. In the cell diluent 14 channel, ammonium methyl methacrylate aqueous solution is used as the buffer solution, with a concentration of 140 mM, and its rate is controlled at 1-10 μL / min by a peristaltic pump 11 installed on the cell diluent 14 delivery pipeline.

[0070] In some embodiments of the present invention, the online extraction electrospray ionization module 3 includes an atmospheric pressure flow chamber 33, and at least three capillaries 34 are disposed in the atmospheric pressure flow chamber 33, forming three cell sample introduction channels in the at least three capillaries 34.

[0071] The at least triple capillary 34 includes an inner capillary 34 serving as a cell sample introduction channel, a middle capillary 34 for transporting sheath fluid 12, and an outer capillary 34 serving as a sheath gas conduit. The atmospheric pressure flow chamber 33 is also equipped with a sheath gas port 32 and a sheath fluid port 35 to correspondingly introduce sheath gas and sheath fluid into the atmospheric pressure flow chamber 33. The sheath fluid containing intracellular substances is extracted and forms charged droplets under the action of a high-voltage electric field.

[0072] Furthermore, the outer diameter and inner diameter of the inner capillary 34 are 200 μm and 100 μm, respectively; the outer diameter and inner diameter of the middle capillary 34 are 350 μm and 250 μm, respectively; and the outer diameter and inner diameter of the outer capillary 34 are 700 μm and 500 μm, respectively.

[0073] In detail, such as Figure 3As shown, the online extraction electrospray ionization module 3 includes an atmospheric pressure flow chamber 33, which contains at least three capillary tubes 34. The at least three capillary tubes 34 are fixedly connected by a metal tee and a graphite gasket to form a coaxial nested structure.

[0074] The inner capillary 34 has an outer diameter of 200 μm and an inner diameter of 100 μm, serving as a cell sample introduction channel and connecting to the inlet of the arc-shaped shunt capture module 2. The middle capillary 34 has an outer diameter of 350 μm and an inner diameter of 250 μm, serving as a sheath fluid 12 channel to transport the sheath fluid 12 from the sample introduction control module 1 to the arc-shaped shunt capture module 2. Furthermore, the middle capillary 34 is longer than the inner capillary 34, and its outlet end is 5 mm longer than that of the inner capillary 34. This system enables the focusing, separation, and online extraction of intracellular substances from cells, and forms charged droplets from the extracted intracellular substances under a high-voltage electric field. The outer capillary 34 has an outer diameter of 700 μm and an inner diameter of 500 μm, serving as a sheath gas conduit. It coaxially wraps around the middle capillary 34, with the outer capillary 34 being slightly shorter than the middle capillary 34. The outlet end of the outer capillary 34 is 1 mm shorter than the outlet end of the middle capillary 34, used to load sheath gas. In conjunction with an external high-voltage power supply system 31, it accelerates the evaporation of the low-charge solution, improving ionization efficiency. Furthermore, the voltage of the external high-voltage power supply system 31 is 1-3 kV.

[0075] In this invention, the sheath gas is mainly nitrogen, and the sheath gas pipeline is used to promote the ionization process. The input pressure of nitrogen is 0.2-0.5 MPa, preferably 0.3 MPa.

[0076] In some embodiments of the present invention, the orbital trap mass spectrometer 4 is an Orbitrap MS. Based on the characteristics of Orbitrap MS, it has high sensitivity and high specificity, can accurately receive charged ions and complete the qualitative and quantitative detection of substances in single cells, and is suitable for the analysis needs of single-cell metabolites.

[0077] The present invention will be described below through several embodiments.

[0078] Example 1

[0079] In Example 1, which is a specific embodiment of the bow-shaped shunt module, details are as follows:

[0080] Cell suspension sample 13 and cell diluent 14 are drawn into bow-shaped shunt capture module 2 by peristaltic pump 11 at a constant flow rate. They are then contacted with sheath fluid 12 in online extraction electrospray ionization module 3, where intracellular substances are extracted online. The extracted substances are then ionized with the assistance of sheath gas and high voltage, and finally enter orbital trap mass spectrometer 4 for detection.

[0081] Furthermore, such as Figure 2 As shown, the uppermost arc-shaped channel (with a curvature of 150 μm) is the widest (W=150 μm, θ=60°) and has the largest arc curvature (R=150 μm), exhibiting the highest "tolerance" for contact angles. Cells must overcome the critical interfacial tension value to compress the fluid, resulting in a contact angle θ>120°. Larger cells (60-100 μm) (such as mouse cardiomyocytes and zebrafish gill chlorinating cells) can generate sufficient spatial compression to achieve a contact angle exceeding 120°, thus allowing them to enter the arc-shaped channel.

[0082] Secondly, the middle arc-shaped channel has a moderate width (an arc-shaped channel with a curvature of 100 μm), a transitional arc-shaped structure (W=150 μm-100 μm, θ=60°), and a contact angle in the range of 90° to 120°. For example, the compression generated by medium-sized cells (30-60 μm) such as zebrafish hepatocytes and mouse glomerular podocytes causes the contact angle to fall within this range and enter this arc-shaped channel.

[0083] Third, the lowest arc-shaped channel (with a curvature of 50 μm) is the narrowest (W=100 μm, θ=30°) and has the smallest arc curvature, resulting in the lowest "tolerance" for contact angles; only a contact angle θ < 90° is required. The compression generated by small cells of 10-30 μm (such as zebrafish liver macrophages and mouse alveolar type II cells) is insufficient to cause a contact angle exceeding 90°, therefore they flow out along this arc-shaped channel. Detailed data are shown in Table 1 below.

[0084] Table 1

[0085] Each arc-shaped channel has an independent diversion chamber downstream of its arc edge (200 μm long and the width of the arc-shaped channel). The diversion chamber is divided into two paths: an outlet (outputting cells of the corresponding size) and a bypass (expelling cells of non-target size). The isolation wall between the arc-shaped channels is ≥500 μm thick, and the one-piece molded structure avoids crossflow, ensuring that cells of different sizes only enter the corresponding arc-shaped channel.

[0086] Example 2

[0087] In Example 2, which is an embodiment of the modification of the bow-shaped channel in the bow-shaped diversion and capture module 2, details are as follows:

[0088] Example 2-1 Uniform BSA film formation on the inner wall of the buffer cavity

[0089] A 0.1% BSA solution was prepared using 0.01 mol / L PBS (pH=7). The solution was preheated to 30°C to reduce viscosity and improve fluidity. At the same time, 0.05% Tween-20 was added to reduce the surface tension of the solution, making it easier to spread the solution on the inner wall of the arc-shaped channel, especially improving the wetting effect in the cone angle and narrow arc-shaped channel area.

[0090] Then, the prepared BSA solution was slowly injected into the narrow end of the multi-arched channel on the right side using a syringe. After injection, the solution was allowed to stand at room temperature for 30 minutes, and then purged with a dry nitrogen gradient (first purging at 0.02 MPa for 10 minutes, then purging at 0.05 MPa for 20 minutes) to avoid local BSA membrane detachment or uneven distribution caused by direct high-pressure purging.

[0091] Then, slowly inject the BSA solution again from the wide end in the forward direction. After inverting the buffer chamber, lay the other side flat at room temperature for 30 minutes to achieve secondary immersion and ensure that the BSA is uniformly adsorbed on the inner wall of the conical triple-arched channel buffer chamber. Then, use a dry nitrogen gradient purging (purge at 0.02 MPa for 10 minutes first, then at 0.05 MPa for 20 minutes).

[0092] Finally, rinse the arcuate channel 2-3 times with PBS buffer to remove unadsorbed BSA and impurities.

[0093] Example 2-2 Glutaraldehyde Crosslinking

[0094] Prepare a 0.5% glutaraldehyde solution using 0.01 mol / L PBS (pH=7), ensuring only a uniform BSA adsorption layer remains on the inner wall of the arcuate channels. Slowly inject the prepared glutaraldehyde solution from the narrow end, ensuring the solution completely fills all arcuate channels, and let stand at room temperature for 3 hours. Then, slowly flush the arcuate channels forward with PBS buffer to thoroughly remove unreacted glutaraldehyde and prevent local residues from affecting subsequent performance.

[0095] In Examples 2-1 and 2-2, as Figure 4 As shown, the contact angle of water on the surface of Implementation Case 2 was detected using a contact angle measuring instrument. The contact angle on the left side of the figure is 50.4° and the contact angle on the right side is 46.3°, both of which are less than 60°. This indicates that the material surface is hydrophilic and can easily form a dense water film with water, which physically blocks the direct contact between cells and the material surface, weakening the van der Waals forces and hydrophobic interactions required for cell adhesion.

[0096] Another aspect of the present invention provides a method for single-cell scale adjustment in electrospray mass cytometry, using the single-cell scale adjustment device for electrospray mass cytometry as described above, comprising the following steps:

[0097] S1. Start the sample injection control module 1 to extract the sample into the bow-shaped diversion capture module 2 and the online extraction electrospray ionization device;

[0098] S2. When the sample passes through the bow-shaped shunt capture module 2, it is sorted and shunt according to the different sizes of the cells, and the target size cells are output.

[0099] S3. The cells output from the bow-shaped diversion capture module 2 come into contact with the sheath fluid 12 in the hollow sleeve and flow to the outlet of the atmospheric pressure flow chamber 33 under the action of the sheath fluid 12. At the same time, the intracellular substances are extracted online by the sheath fluid 12.

[0100] S4. Start the external high-voltage power supply system 31. The intracellular substances extracted by the sheath fluid 12 form charged droplets in the high-voltage electric field, and the charged droplets are evaporated in at least three capillaries 34 to obtain charged ions.

[0101] S5. After evaporation, the charged ions enter the orbital trap mass spectrometer 4 to complete the detection and analysis of substances within a single cell.

[0102] This invention is applicable to the detection needs of cells of various sizes. Through the switching of the arc-shaped channel in the arc-shaped shunt capture module 2, it accurately screens target cells of different sizes within the 10-100μm range, covering single cells from multiple tissues of mainstream model organisms in environmental pollutant research. This avoids the problems of small cell transport deviation and large cell congestion and damage, ensuring the comprehensiveness of the detected samples. The BSA-glutaraldehyde cross-linked membrane modification design of the arc-shaped shunt capture module 2 reduces cell adhesion and adsorption of trace metabolites. Online extraction of sheath fluid 12, combined with the focusing effect of the coaxial capillary 34, reduces cell rupture and metabolite leakage, improving detection sensitivity and avoiding false negatives or misjudgments of metabolite identification. The arc-shaped channel can be quickly switched to adapt to different cell sizes without disassembling the device. The integrated workflow design enables continuous operation of sample introduction, screening, extraction, ionization, and detection, balancing high throughput and convenience, and adapting to diverse environmental pollutant exposure metabolism research scenarios. Online mixing of cell suspension and diluent reduces interference from non-volatile salts, while high-voltage electric field and sheath gas work together to improve ionization efficiency. Orbital trap mass spectrometry enables qualitative and quantitative analysis, providing reliable data support for the accumulation, distribution, and metabolic transformation characteristics of pollutants within cells, and ensuring the accuracy of detection.

[0103] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

Claims

1. A single-cell scale tunable device for electrospray mass spectrometry flow, characterized in that, The application relates to a sample injection control module, an arcuate split capture module, an online extraction electrospray ionization module and an orbitrap mass spectrometer. The sample injection control module comprises a sample and a sample conveying device to provide sample fluid for the downstream module. The arcuate split capture module is connected with the output end of the sample injection control module, and comprises an arcuate split cavity, a waste liquid port and a cell introduction port. The arcuate split cavity comprises at least three arcuate channels with different curvatures for cell split, and the at least three arcuate channels are switched by a driving mechanism to screen target cells with different sizes. The online extraction electrospray ionization module is connected with the output end of the arcuate split capture module, and comprises an atmospheric flow cavity and a hollow sleeve arranged in the atmospheric flow cavity. The hollow sleeve is coaxially provided with at least three capillaries, and is matched with an external high-voltage power supply system to provide charged ions for mass spectrometric detection. The orbitrap mass spectrometer receives the charged ions of the online extraction electrospray ionization module and detects cells.

2. The single-cell scale tunable device for electrospray mass spectrometry flow of claim 1, wherein, The sample injection control module provides sample fluid for the arcuate split capture module, the arcuate split capture module splits cells with different sizes through arcuate channels with different curvatures, and the driving mechanism realizes switching output of different arcuate channels.

3. The single-cell scale tunable device for electrospray mass spectrometry flow of claim 1, wherein, The online extraction electrospray ionization module cooperates with the high-voltage electric field formed by the external high-voltage power supply system and the atmospheric flow cavity to construct a mass spectrometric flow environment.

4. The worry packet size adjustment apparatus for electrospray mass spectrometry flow according to claim 1, wherein, The arcuate split capture module is molded by transparent quartz glass, and the inner wall of the arcuate split capture module is coated with a film containing biocompatible protein (BSA) molecules and glutaraldehyde cross-linking.

5. The single-cell scale tunable device for electrospray mass spectrometry flow of claim 1, wherein, The curvatures of the at least three arcuate channels are 150 mu m, 100 mu m and 50 mu m, and the corresponding output sizes of the cells are 60-100 mu m, 30-60 mu m and 10-30 mu m.

6. The single-cell scale tunable device for electrospray mass spectrometry flow of claim 1, wherein, The arcuate split capture module is a conical structure or a tree structure. The sample injection control module comprises sheath liquid, cell suspension sample and cell diluent, and corresponding conveying pipelines are provided with peristaltic pumps.

7. The single-cell scale tunable device for electrospray mass spectrometry flow of claim 6, wherein, The online extraction electrospray ionization module comprises an atmospheric flow cavity, the end of the hollow sleeve is a flared horn-shaped port, and at least three capillaries are arranged in the atmospheric flow cavity.

8. The single-cell scale tunable device for electrospray mass spectrometry flow of claim 1, wherein, The at least three capillaries comprise an inner capillary as a cell sample introduction channel, a middle capillary for conveying sheath liquid and an outer capillary as a sheath gas pipeline. The outer diameter and the inner diameter of the inner capillary are 200 mu m and 100 mu m, the outer diameter and the inner diameter of the middle capillary are 350 mu m and 250 mu m, and the outer diameter and the inner diameter of the outer capillary are 700 mu m and 500 mu m. The voltage of the external high-voltage power supply system is 1-3 kV.

9. The single-cell scale tunable device for electrospray mass spectrometry flow of claim 1, wherein, The orbitrap mass spectrometer is an Orbitrap MS.

10. A method for single-cell scale modulation for electrospray mass spectrometry flow, comprising: The single-cell scale regulating device for electrospray mass spectrometry flow as claimed in any one of claims 1-9 comprises the following steps: S1, starting the sample injection control module to draw the sample to the arcuate split capture module and the online extraction electrospray ionization device; S2, when the sample passes through the arcuate split capture module, the cells are screened and split according to different sizes, and the target size cells are outputted; S3, the cells outputted in the arcuate split capture module are in contact with the sheath liquid in the hollow sleeve, and flow to the outlet of the atmospheric flow chamber under the action of the sheath liquid, while the intracellular substances are extracted online by the sheath liquid; S4, starting the external high-voltage power supply system, the intracellular substances extracted by the sheath liquid form charged droplets in the high-voltage electric field, and the charged droplets are subjected to evaporation treatment in at least three capillaries to obtain charged ions; S5, the charged ions after evaporation treatment enter the orbitrap mass spectrometer to complete the detection and analysis of the intracellular substances of the single cell.

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