Patent Application
20240420940
The present disclosure belongs to the technical field of cell biology, and specifically relates to an artificial cell for single-cell mass spectrometry (SCMS) measurement and a preparation method thereof.
In recent years, as a label-free detection technology with high sensitivity and specificity, single-cell mass spectrometry (SCMS) has been increasingly used in the measurement of single-cell metabolomics and proteomics. Although there are currently some other commonly used single-cell measurement methods (such as electrochemical methods and fluorescence methods), these methods generally require molecules to be measured to have specific physical and chemical properties (redox properties) or need to modify molecular structures (fluorescent labeling). This greatly limits the application scope of these methods. Compared with these methods, the SCMS can achieve highly-sensitive simultaneous detection of multiple biomolecules in cells without the need for amplification or labeling. Secondly, mass spectrometry (MS) can also achieve structural analysis of unknown compounds in a single cell by using the characteristic fragment information of molecules through multi-level fragmentation. In addition, through isotope labeling, SCMS can track the metabolic process of specific compounds without affecting the metabolic behavior of cells. At the same time, isotope dilution is currently a quantitative analysis method with the highest stoichiometry level and can directly trace the Système International d'Unités (International System of Units, SI). These excellent characteristics make the SCMS have desirable application prospects in the precise measurement of chemical components in single cells.
At present, SCMS analysis technology has achieved qualitative and quantitative analysis of some compounds within single cells. However, due to the significant individual differences and instability of physiological states in natural single cells, SCMS measurement studies still suffer from great inaccuracy and unreliability. First of all, there are an unusually wide variety of cells in nature. These cells can be divided into plant cells, animal cells, nerve cells, white blood cells, red blood cells, platelets, phagocytes, epithelial cells, cardiomyocytes, stem cells, and cancer cells and the like. Secondly, cells of a same type may also show significant differences in size, appearance, and internal omics due to their different growth stages in the organisms. These factors lead to significant sampling and measurement differences when conducting SCMS methodology research or analytical measurements, leading to problems such as poor reproducibility, low stability, and untraceable measurement results in the final measurement results.
In order to better study the physical and chemical properties and biological functions of cells, such as microscopic anatomy, signaling networks, omics libraries, and gene regulation, the concept of “artificial cell” has been widely proposed by scientists. Currently, various types of ideal artificial cell models have been developed in the field, including: liposomes, polymersomes, and vesosomes. These artificial cells can simulate the structure of cell compartments, internal compound types, and biochemical functions on the cell membrane surface through structural regulation of vesicle materials, encapsulation of target molecules, and modification of functional molecules based on the structure and physiological characteristics of the cells. Ultimately, the artificial cells are successfully used to study the physical and chemical properties and biological functions of various types of cells. Among these artificial cell models, liposomes have received the most widespread attention and reports. Liposomes are generally composed of naturally-synthesized phospholipid bilayers or vesicles prepared from artificial self-assembled materials, and have attracted widespread interest in the fields of targeted drug delivery, membrane protein research, bioreactors, and biosensors. The liposomes can simulate the size of a single living cell through the regulation of amphipathic molecular segments, and characteristics of encapsulating biomolecules and carrying specific molecular physiological functions make the liposome a potential substitute for SCMS research.
After recent years of development, research on the preparation methods of artificial cells has achieved certain results, achieving preliminary simulation and construction of the basic structure and biochemical properties of natural cells. However, the current development of liposome-based artificial cells for SCMS measurement is still in a blank stage. The main reasons are as follows:
In view of the above-mentioned deficiencies in the prior art, an objective of the present disclosure is to provide an artificial cell for single-cell mass spectrometry (SCMS) measurement and a preparation method thereof. In the present disclosure, the batched, uniform, and stable preparation is achieved for an artificial cell with different physiological characteristics. Moreover, the unification and mutual comparability of the SCMS measurement are realized through the application of artificial single-cell materials in SCMS measurement research.
To achieve the above objective, the present disclosure adopts the following technical solutions:
Further, the internal aqueous phase channel is an intermediate channel; the oil phase channel is divided into two sub-channels on two sides of the internal aqueous phase channel; and the external aqueous phase channel is divided into two sub-channels outside the internal aqueous phase channel.
Further, in step S2, the PEG and the PVA aqueous solution are added into the internal aqueous phase channel; the chloroform-hexane mixture of the L-α-phosphatidylcholine is added into the intermediate oil phase channel; and the PVA and the F-68 aqueous solution are added into the external aqueous phase channel.
Further, an organic dye or a fluorescent dye is further added into the internal aqueous phase channel in step S2 in order to clearly observe and collect a target artificial cell.
Further, in step S2, target compounds of different contents are added into the internal aqueous phase channel in order to accurately simulate an omics environment of an internal single cell; and
The present disclosure has the following beneficial effects:
The specific embodiment of the present disclosure will be described below so that those skilled in the art can understand the present disclosure, but it should be clear that the present disclosure is not limited to the scope of the specific embodiment. For those of ordinary skill in the art, as long as various changes fall within the spirit and scope of the present disclosure defined and determined by the appended claims, these changes are apparent, and all inventions and creations using the concept of the present disclosure are protected.
Referring to
In order to realize the homogenized, stabilized, and batched production of artificial cells, it was first planned to design and build a set of the microfluidic platform for the preparation of artificial cells. The microfluidic platform was composed of a three-phase channel, including: an internal aqueous phase channel (W1), an oil phase channel (O), and an external aqueous phase channel (W2). A three-phase liquid was introduced through five microfluidic channels, of which a W1 channel was an intermediate channel; there was one O channel on each side of the intermediate channel; and there was one W2 channel on an outside of each of the two O channels. A size and a structure of the artificial cell, a speed of preparation, and a microstructure of the artificial cell were regulated by controlling a width, a flow rate, and a compound composition of the channels.
In order to further achieve homogeneous, batch-based, and parameter-controllable preparation of an artificial single cell, it was planned to develop a preparation method of a water-in-oil-in-water (W/O/W) double-emulsion liposome-derived artificial cell based on a microfluidic device. The double-emulsion liposome-derived artificial cell was mainly composed of a three-phase liquid, including: an internal aqueous phase (W1), an intermediate oil phase (O), and an external aqueous phase (W2). The W1 phase was mainly composed of PEG and a PVA aqueous solution; the O phase was mainly composed of a chloroform-hexane mixture of L-α-phosphatidylcholine (PC); and the W2 phase was mainly composed of PVA and an F-68 aqueous solution.
The preparation principle was shown in
This method pre-selected PC, a common phospholipid molecule, for the construction of artificial cell lipid membranes, which could spontaneously form a double-emulsion vesicle structures in microfluidics, simulating the topography of cell membranes. In addition, this method adopted amphipathic macromolecular polymers PEG and PVA to improve the preparation success rate and stability of the emulsion, and was also planned to achieve high stability and uniformity of emulsion vesicles through optimization for a proportion of different polymers.
In order to clearly observe and collect a target artificial cell, an organic dye or fluorescent dye could be added to the internal aqueous phase to achieve better positioning of the artificial cell under the microscope. Moreover, in order to accurately simulate the omics environment of an internal single cell, target compounds (such as ATP, a common compound in cells) of different contents could be added to the internal aqueous phase for mass spectrometry research on compounds within the single cells. Through the control of different compound parameters, precisely quantitative optimization of size, appearance, and internal compound content of the artificial cell could be achieved, thereby enabling methodological research on SCMS metrology and evaluation of measurement accuracy.
In order to prove the superiority of the prepared target artificial cell material, a series of parameters of the target material were characterized in this example. First, the uniformity and stability of the artificial cell materials were evaluated.
W/O/W artificial cell vesicles could maintain a relatively uniform morphological structure (
In order to better realize the application of SCMS metrology, the uniformity, stability, and accuracy of artificial cell measurement were investigated. A representative compound ATP in cells was selected to simulate the mass spectrometry detection of specific compounds in single cells; by preparing artificial cells wrapped with the specific metabolite ATP, the measurement evaluation of artificial cells was achieved for SCMS measurement. Single cells were sampled through droplet microextraction, and a microextraction droplet had a volume of about 1 nL. The sampling was shown in
First, the measurement uniformity of artificial cell was investigated: the measurement of artificial single-cell compounds was compared in different batches, where each batch was measured 5 times, and an ATP concentration was 1 μg/mL. The measurement results were shown in
Compared with natural cell samples, new artificial single cells based on microfluidic self-assembly developed by the present disclosure have better uniformity, stability, and controllability. The artificial cell can effectively avoid significant measurement differences between individual single-cell samples, and effectively solve the problem of difficulty in stably preserving biological samples. The proposal of the preparation method of the artificial cell can largely avoid the lack of standard reference materials in the field of SCMS measurement, thereby making SCMS methodology research results more accurate, reliable, and mutually comparable.
For those skilled in the art, it is obvious that the present disclosure is not limited to the details of the above embodiments, and the present disclosure can be implemented in other specific forms without departing from the spirit or basic features of the present disclosure. Therefore, the embodiments should be regarded as exemplary and non-limiting in every respect, and the scope of the present disclosure is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of equivalent elements of the claims should be included in the present disclosure.
In addition, it should be understood that although this specification is described in accordance with the implementations, not each implementation only contains an independent technical solution, and this description in the specification is only for clarity. Those skilled in the art should take the specification as a whole. The technical solutions in the embodiments can also be properly combined to form other implementations that can be understood by those skilled in the art.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202310272610.6 | Mar 2023 | CN | national |
The present application is a national stage application of International Patent Application No. PCT/CN2023/116383, filed on Sep. 1, 2023, which claims priority to the Chinese Patent Application No. 202310272610.6, filed with the China National Intellectual Property Administration (CNIPA) on Mar. 20, 2023, and entitled “ARTIFICIAL CELL FOR SINGLE-CELL MASS SPECTROMETRY (SCMS) MEASUREMENT AND PREPARATION METHOD THEREOF”, which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2023/116383 | 9/1/2023 | WO |
