Artificial cells for single-cell mass spectrometry and methods of making the same

Abstract

The application discloses a kind of artificial cells for single-cell mass spectrometry and preparation method thereof, wherein artificial cell includes internal water phase, intermediate oil phase and external water phase;Internal water phase includes polyethylene glycol and polyvinyl alcohol aqueous solution, intermediate oil phase includes L-alpha-phosphatidylcholine chloroform and hexane mixture;External water phase includes PVA and F-68 aqueous solution.Compared with natural cell sample, the novel artificial single cell based on microfluidic self-assembly developed in the application has better uniformity, stability and controllability, effectively avoids significant measurement difference between single-cell individual samples, and effectively solves the problem of difficult stable preservation of biological samples.The proposed artificial cell preparation method will largely solve the problem of lack of standard reference material in the field of single-cell mass spectrometry, making the results of single-cell mass spectrometry method more accurate, reliable and mutually recognized.

Description

Technical Field

[0001] This invention belongs to the field of cell biology technology, specifically relating to an artificial cell for single-cell mass spectrometry measurement and its preparation method. Background Technology

[0002] In recent years, single-cell mass spectrometry (SCMS), as a label-free detection technique with high sensitivity and specificity, has been increasingly widely used in single-cell metabolomics and proteomics metrology. While other commonly used single-cell metrology methods exist (e.g., electrochemical and fluorescence methods), they often require specific physicochemical properties (redox properties) of the analyte molecules or structural modifications (fluorescent labeling), which significantly limits their application. Compared to these methods, single-cell mass spectrometry can achieve highly sensitive simultaneous detection of multiple biomolecules within cells without amplification or labeling. Secondly, mass spectrometry, through multi-stage fragmentation, can utilize characteristic fragment information to resolve the structure of unknown compounds within single cells. Furthermore, through isotope labeling, single-cell mass spectrometry can track the metabolic processes of specific compounds with minimal impact on cellular metabolic behavior. Simultaneously, isotope dilution is currently the quantitative analysis method with the highest chemometric level, allowing direct traceability to SI units. These superior characteristics make single-cell mass spectrometry a promising technology for the precise measurement of chemical components in single cells.

[0003] Although single-cell mass spectrometry has enabled qualitative and quantitative analysis of some compounds within single cells, significant individual variability and physiological instability of natural single cells still lead to substantial inaccuracies and unreliability in single-cell mass spectrometry metrology. Firstly, the variety of cell types in nature is exceptionally vast, including plant cells, animal cells, nerve cells, white blood cells, red blood cells, platelets, phagocytes, epithelial cells, cardiomyocytes, stem cells, and cancer cells. Furthermore, even cells of the same type can exhibit significant differences in size, morphology, and internal omics due to different growth stages within an organism. This results in significant sampling and measurement differences during single-cell mass spectrometry methodologies, leading to poor reproducibility, instability, and a lack of traceability in the final metrological results.

[0004] To better study the physicochemical properties and biological functions of cells, including their microscopic anatomy, signaling networks, omics libraries, and gene regulation, the concept of "artificial cells" has been widely proposed by scientists. Currently, various ideal artificial cell models have been developed, including liposomes, polymer vesicles, and multilayered lipid vesicles. These artificial cells, based on the structure and physiological characteristics of cells, simulate cell compartment structures, internal compound types, and cell membrane surface biochemical functions through structural regulation of vesicle materials, encapsulation of target molecules, and modification of functional molecules. Ultimately, they have been successfully applied to the study of various cellular physicochemical properties and biological functions. Among these artificial cell models, liposomes have received the most attention and reports. Liposomes are typically composed of vesicles made from naturally synthesized phospholipid bilayers or artificial self-assembled materials, and have attracted widespread interest in areas such as targeted drug delivery, membrane protein research, bioreactors, and biosensors. Liposomes can simulate the size of a single living cell through the regulation of amphiphilic molecular chains, and their ability to encapsulate biomolecules and carry specific molecular physiological functions makes them a potential alternative for single-cell mass spectrometry research.

[0005] In recent years, research on artificial cell preparation methods has yielded certain results, achieving preliminary simulation and construction of the basic structure and biochemical characteristics of natural cells. However, the development of liposome-based artificial cells for single-cell mass spectrometry measurement is still in its infancy, mainly due to the following reasons:

[0006] First, achieving uniform and large-scale preparation of artificial cells for single-cell mass spectrometry is challenging. Current conventional methods for synthesizing artificial cells, including porous membrane extrusion, electrodeposition, reverse-phase evaporation, droplet emulsion transfer, and freeze-drying, often result in polydisperse liposomes and inconsistent structural morphologies. A platform for the large-scale and uniform preparation of artificial cells remains undeveloped.

[0007] Second, liposome-based artificial cells suffer from poor stability and encapsulation efficiency. Currently, vesicle materials synthesized using conventional liposome methods are prone to rupture and leakage of internal compounds, which greatly limits their application in single-cell mass spectrometry, leading to unstable and inaccurate single-cell measurement results. Furthermore, current liposome encapsulation methods do not facilitate the convenient introduction of compounds, resulting in inaccuracies in the quantitative encapsulation process. Summary of the Invention

[0008] To address the aforementioned shortcomings in existing technologies, the present invention aims to provide an artificial cell for single-cell mass spectrometry metrology and its preparation method. This will enable the batch, homogenization, and stabilization of artificial cells with different physiological characteristics, and through the application of artificial single-cell materials in single-cell mass spectrometry measurement research, achieve uniformity and mutual recognition of measurement values ​​in the single-cell mass spectrometry metrology process.

[0009] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0010] In a first aspect, an artificial cell for single-cell mass spectrometry is provided, comprising an internal aqueous phase, an intermediate oil phase, and an external aqueous phase; the internal aqueous phase comprises an aqueous solution of polyethylene glycol and polyvinyl alcohol, the intermediate oil phase comprises a mixture of chloroform and hexane of L-α-phosphatidylcholine, and the external aqueous phase comprises an aqueous solution of PVA and F-68.

[0011] Secondly, a method for preparing artificial cells for single-cell mass spectrometry is provided, the method comprising the following steps:

[0012] S1. Constructing a microfluidic platform for artificial cell preparation:

[0013] The microfluidic platform is a three-phase channel, including an internal aqueous phase channel, an oil phase channel, and an external aqueous phase channel; by controlling the channel width, flow rate, and compound composition, the size and structure of artificial cells can be regulated, the preparation speed can be increased, and the microstructure of the cells can be improved.

[0014] S2. Preparation of artificial cells based on microfluidic self-assembly of dual emulsion liposomes:

[0015] Substances are added to the three-phase channel, and emulsion droplets are squeezed out through the aqueous solution to complete the initial structure of the oil-water biphase vesicle; finally, the initial structure of the vesicle automatically forms an artificial cell.

[0016] Furthermore, in step S1, the three-phase liquid is introduced through five microfluidic channels, with the inner aqueous phase channel being the middle channel, the oil phase channels being the two middle channels, and the outer aqueous phase channels being the two outer channels.

[0017] Furthermore, in step S2, an aqueous solution of polyethylene glycol and polyvinyl alcohol is added to the internal aqueous phase channel, a mixture of chloroform and hexane of L-α-phosphatidylcholine is added to the intermediate oil phase channel, and an aqueous solution of PVA and F-68 is added to the external aqueous phase channel.

[0018] Furthermore, in step S2, in order to clearly observe the collected target artificial cells, an organic dye or fluorescent staining agent is added to the internal aqueous channel.

[0019] Furthermore, in step S2, in order to accurately simulate the omics environment of the internal single cell, different amounts of the target compound are added to the internal aqueous phase channel.

[0020] The beneficial effects of this invention are as follows:

[0021] Compared to natural cell samples, the novel artificial single cells based on microfluidic self-assembly developed in this invention exhibit better uniformity, stability, and controllability. This effectively avoids significant measurement differences between individual single-cell samples and solves the problem of the difficulty in stably preserving biological samples. The proposed method for preparing artificial cells will largely address the lack of standard reference materials in the field of single-cell mass spectrometry, making the results of single-cell mass spectrometry methodologies more accurate, reliable, and mutually compatible. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the microfluidic platform for artificial cell preparation constructed in Example 1;

[0023] Figure 2 This is a CAD structural design drawing of the microfluidic platform for artificial cell preparation constructed in Example 1;

[0024] Figure 3 This is a schematic diagram illustrating the principle of the dual emulsion liposome artificial cell preparation method in Example 1;

[0025] Figure 4 This is a morphological structure diagram of the artificial cells used in Example 2 to evaluate their uniformity.

[0026] Figure 5 This is a morphological diagram of the artificial cell stability evaluation in Example 2;

[0027] Figure 6 This is a diagram of the droplet microextraction structure in Example 2;

[0028] Figure 7 This is a graph showing the uniformity evaluation of single-cell measurements from different batches in Example 2;

[0029] Figure 8 This is a graph showing the detection of ATP concentration in a single cell in Example 2;

[0030] Figure 9 The graph shows the results of ATP measurement in single cells at different times in Example 2. Detailed Implementation

[0031] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0032] Example 1

[0033] Reference Figure 1-3 This invention provides a method for preparing artificial cells for single-cell mass spectrometry measurement, the method comprising the following steps:

[0034] (1) Constructing a microfluidic platform for automated artificial cell preparation:

[0035] To achieve uniform, stable, and mass production of artificial cells, this embodiment first designs and constructs a microfluidic platform for artificial cell preparation. The microfluidic platform consists of three phase channels: an internal aqueous phase channel (W1), an oil phase channel (O), and an external aqueous phase channel (W2). These three phases of liquid are introduced through five microfluidic channels, with W1 being the central channel, O channels forming the two central side channels, and W2 channels forming the two outer side channels. By controlling the channel width, flow rate, and compound composition, the size and structure of the artificial cells, the preparation speed, and the cell microstructure can be regulated.

[0036] (2) Preparation of artificial cells based on microfluidic self-assembly of dual emulsion liposomes:

[0037] To further achieve uniform, batch, and parameter-controllable artificial single-cell preparation, this embodiment proposes to develop a method for preparing water-in-oil-in-water (W / O / W) dual-emulsion liposome artificial cells based on a microfluidic device. The dual-emulsion liposome artificial cells mainly consist of three phases of liquid: an inner aqueous phase (W1), an intermediate oil phase (O), and an outer aqueous phase (W2). Phase W1 mainly consists of aqueous solutions of polyethylene glycol (PEG) and polyvinyl alcohol (PVA); phase O mainly consists of a mixture of L-α-phosphatidylcholine (PC) in chloroform and hexane; and phase W2 mainly consists of aqueous solutions of PVA and F-68.

[0038] The preparation principle is as follows: Figure 2As shown: First, an emulsion droplet is extruded from an aqueous solution to form the initial oil-water biphase vesicle structure. Then, over time, a lipid molecular layer forms at the interface of the "vesicle," while the dopant stabilizer and excess lipids aggregate at the interface to form a small bulge. Finally, these excess "bulges" automatically separate to form a small droplet, detaching from the prepared artificial cell, thus completing the fabrication of an artificial cell encapsulated by a lipid membrane. The prepared biemulsion artificial cell can be subsequently collected in a sealed container and observed under a microscope.

[0039] This method pre-selects PC, a common phospholipid molecule, as the material for constructing artificial cell lipid membranes. PC can spontaneously form biemulsion vesicle structures in microfluidics, mimicking the morphology of cell membranes. Furthermore, this method uses amphiphilic macromolecular polymers PEG and PVA to improve the success rate and stability of emulsion preparation. It also plans to optimize the proportions of different polymers to achieve high stability and uniformity of the emulsion vesicles.

[0040] To clearly observe the collected artificial cells, organic dyes or fluorescent stains can be added to the internal aqueous phase to better locate the artificial cells under a microscope. Furthermore, to accurately simulate the omics environment of the internal single cells, different amounts of target compounds (such as adenosine triphosphate, a common compound in cells) can be added to the internal aqueous phase for mass spectrometry studies of compounds within single cells. Ultimately, by adjusting different compound parameters, precise quantitative optimization of the size, morphology, and internal compound content of the artificial cells can be achieved, enabling methodological research and evaluation of measurement accuracy in single-cell mass spectrometry.

[0041] Example 2

[0042] Evaluation of the uniformity, stability, and measurement accuracy of artificial single cells:

[0043] To demonstrate the superiority of the prepared artificial cell material, this embodiment characterizes a series of parameters of the target material. First, the uniformity and stability of the artificial cell material are evaluated.

[0044] W / O / W artificial cell vesicles can maintain a relatively uniform morphological structure. Figure 4 The particle size was approximately 28.75 ± 1.92 μm, and dynamic light scattering results showed that the particle size distribution conformed to dynamic distribution, demonstrating the good uniformity of the artificial cell material. To demonstrate the material's stability, the target artificial cell material was tested for different number of days. The stability of the artificial cells was observed under a microscope after 1, 2, 3, 4, 5, and 7 days. The results are as follows: Figure 5 As shown in the figure. The results indicate that the phospholipid W / O / W artificial cells can be preserved for approximately 7 days, and the artificial cell material exhibits good stability.

[0045] Secondly, to better realize the application of single-cell mass spectrometry, the measurement uniformity, stability, and accuracy of artificial cells were investigated. Adenosine triphosphate (ATP), a representative compound within cells, was selected. The mass spectrometry detection process of a specific compound in single cells was simulated. Artificial cells encapsulating the specific metabolite ATP were prepared to achieve the metrological evaluation of artificial cells for single-cell mass spectrometry. Single cells were sampled through a droplet microextraction process, with a droplet volume of ~1 nL. The sampling process is as follows: Figure 6 As shown. Subsequently, the extract was measured by nano-spray mass spectrometry.

[0046] First, the measurement uniformity of artificial cells was examined by comparing the measurements of compounds in artificial single cells from different batches. Each batch was measured five times, with an ATP concentration of 1 μg / mL. The measurement results are as follows: Figure 7 As shown. Furthermore, measurements were performed on different batches of artificial cells containing different concentrations of ATP, and the linear results are shown below. Figure 8 As shown, the linear correlation coefficient was 0.9998, indicating good linearity. The results demonstrate the good measurement uniformity of the artificial cells. Secondly, to evaluate the measurement stability of the artificial cells, the morphology, size, and intracellular compound content of the artificial cells were compared at different time intervals. The ATP content in the artificial cells was measured from day 1 to day 7 to assess cell stability. The measurement results are shown below. Figure 9 As shown in the figure. The results demonstrate that the artificial cells exhibit good measurement stability.

[0047] Compared to natural cell samples, the novel artificial single cells based on microfluidic self-assembly developed in this invention exhibit better uniformity, stability, and controllability. This effectively avoids significant measurement differences between individual single-cell samples and solves the problem of the difficulty in stably preserving biological samples. The proposed method for preparing artificial cells will largely address the lack of standard reference materials in the field of single-cell mass spectrometry, making the results of single-cell mass spectrometry methodologies more accurate, reliable, and mutually compatible.

[0048] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0049] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. An artificial cell for single-cell mass spectrometry measurement, characterized in that, The artificial cell comprises an internal aqueous phase, an intermediate oil phase, and an external aqueous phase; the internal aqueous phase comprises an aqueous solution of polyethylene glycol and polyvinyl alcohol, the intermediate oil phase comprises a mixture of chloroform and hexane of L-α-phosphatidylcholine, and the external aqueous phase comprises an aqueous solution of PVA and F-68.

2. A method for preparing artificial cells according to claim 1, characterized in that, Includes the following steps: S1. Constructing a microfluidic platform for artificial cell preparation: The microfluidic platform is a three-phase channel, including an internal aqueous phase channel, an oil phase channel, and an external aqueous phase channel. The three-phase liquid is introduced through five microfluidic channels, with the aqueous phase channel being the middle channel, the oil phase channels being the two middle channels, and the external aqueous phase channels being the two outer channels. By controlling the width, flow rate, and compound composition of the channels, the size and structure of artificial cells, the preparation speed, and the microstructure of the cells can be regulated. S2. Preparation of artificial cells based on microfluidic self-assembly of dual emulsion liposomes: Substances are added into the three-phase channel, and emulsion droplets are squeezed out through the aqueous solution to complete the initial structure of the oil-water biphase vesicle; finally, the initial structure of the vesicle automatically forms an artificial cell. The internal aqueous phase channel contains an aqueous solution of polyethylene glycol and polyvinyl alcohol; the oil phase channel contains a mixture of chloroform and hexane containing L-α-phosphatidylcholine; and the external aqueous phase channel contains an aqueous solution of PVA and F-68.

3. The method for preparing artificial cells according to claim 2, characterized in that, In step S2, in order to clearly observe the collected target artificial cells, organic dyes or fluorescent staining agents are added to the internal aqueous channel.

4. The method for preparing artificial cells according to claim 2, characterized in that, In step S2, in order to accurately simulate the omics environment of the internal single cell, different amounts of the target compound are added to the internal aqueous phase channel.

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