Cell detection device and method based on paper-based discharge technology

Abstract

The application provides a cell detection device and method based on paper discharge technology, which comprises a mass spectrometer; a paper with a tip, a conductor connected to the paper and adapted to connect a power supply; a carrier with a plurality of carrier positions for carrying cells; an end of a fluid conduit cooperates with the carrier positions to form a space isolated from the outside between the carrier positions and the fluid conduit, and the cells in the carrier positions are in the space; the fluid conduit has an inlet and an outlet respectively communicating with the space; a gas source is used to supply gas into the space to drive the substances in the space into the fluid conduit, and the substances discharged from the outlet of the fluid conduit are sprayed onto the paper between the tip and the conductor. The application has the advantages of accurate detection, automation and the like.

Description

[0001] The present application is a divisional application of Chinese Patent Application No. 2021101342985, the original application of which has an application date of February 1, 2021, and is entitled "Cell detection device and method based on paper-based discharge technology". TECHNICAL FIELD

[0002] The present application relates to cell analysis, in particular to a cell detection device and method based on paper-based discharge technology. BACKGROUND

[0003] Single-cell technology has developed rapidly in recent years. With the continuous development of single-cell sequencing and single-cell transcriptome technology, humans have a deep understanding of cell population heterogeneity from the DNA and RNA levels. Single-cell analysis is of great significance for studying cell heterogeneity. Mass spectrometers are widely used in single-cell analysis due to their high sensitivity, accurate structure identification, and quantitative analysis. However, single cells have a small volume of fL~pL. If the extraction liquid volume is too large, the metabolites in the cell will be diluted too much, resulting in a concentration that is too low to be detected by the mass spectrometer. If the dilution is too small, the amount of sample available for analysis is too small, and the difficulty of sample manipulation is greatly increased.

[0004] Researchers have used paper spray mass spectrometry technology for cell analysis. This method uses a triangular paper base as a carrier, adds samples to the surface of the paper base, applies a high voltage, and uses the voltage to drive the solvent to dissolve and extract target substances from the sample, allowing them to migrate and ionize. The paper base tip is vaporized to form an electrospray that enters the mass spectrometer for analysis. This method is easy to operate and does not cause excessive dilution. However, paper spray mass spectrometry technology has the following problems:

[0005] 1. When used with a mass spectrometer with a gas curtain, the solvent quickly dries on the paper, resulting in a short and unstable spray, which leads to unstable mass spectrometry signals and low sensitivity.

[0006] 2. This method generally uses a pipette to spot samples, which cannot guarantee analysis of samples at the single-cell level.

[0007] 3. Due to matrix interference, this method has limitations when analyzing complex biological and environmental complex matrix samples. SUMMARY

[0008] To solve the above problems in the prior art, the present application provides a cell detection device based on paper-based discharge technology with good accuracy and stability.

[0009] The object of the present application is achieved by the following technical solution:

[0010] A cell detection device based on paper-based discharge technology, comprising a mass spectrometer; the cell detection device based on paper-based discharge technology further comprises:

[0011] A paper having a tip and a conductive body connected to the paper and adapted to be connected to a power source;

[0012] A carrier having a plurality of carrier sites for carrying cells;

[0013] An end of a fluid conduit is fitted to the carrier site, forming a space isolated from the outside between the carrier site and the fluid conduit, and the cells of the carrier site are in the space; the fluid conduit has an inlet and an outlet respectively communicating with the space;

[0014] A gas source for supplying gas into the space, driving substances in the space into the fluid conduit, and ejecting substances discharged from the outlet of the fluid conduit onto the paper between the tip and the conductive body;

[0015] The fluid conduit comprises:

[0016] A capillary tube and a transmission tube arranged outside the capillary tube and forming a sandwich extending vertically between part of the outer wall of the capillary tube; when the first open end of the capillary tube and the open end of the transmission tube are in contact with the carrier site, the space is formed between the capillary tube, the transmission tube and the carrier site; substances in the space enter from the first open end of the capillary tube and are discharged from the second open end of the capillary tube; the gas source communicates with the inside of the transmission tube.

[0017] Another object of the present application is to provide a cell detection method based on paper-based discharge technology, which is achieved by the following technical solutions:

[0018] A cell detection method based on paper-based discharge technology, comprising the following steps:

[0019] (A1) One end of a fluid conduit is fitted to a carrier site on a carrier, forming a space isolated from the outside, and cells of the carrier site are in the space; the carrier has a plurality of carrier sites;

[0020] (A2) Gas enters the space, driving extracted substances of the cells to be ejected from the open end of the fluid conduit;

[0021] (A3) The conductive body connected to the paper discharges, and the extracted substances ejected onto the paper are ionized, and ions are ejected from the tip of the paper into the mass spectrometer;

[0022] (A4) The mass spectrometer analyzes the ions to obtain information of the cells.

[0023] Compared with the prior art, the present application has the beneficial effects of:

[0024] 1. The detection result is accurate;

[0025] The extraction liquid is of fixed volume, and the single cell is extracted by the extraction liquid without mixing other substances, so that the dilution concentration is better controlled, and the comparability between groups is improved;

[0026] The single cell is in sufficient contact with the extraction liquid, so that the matrix interference is reduced, and the accuracy of cell detection is improved;

[0027] 2. The detection stability is good;

[0028] The fluid pipeline and the bearing position form a space isolated from the outside, and then the extraction material is continuously supplied to the paper base by the thrust of the gas entering the space through the inlet, so that the problem of single-point sample injection of the paper base electrospray cannot form stable and continuous electrospray is overcome, so that the signal stability of the paper spray mass spectrum is improved, and additional continuous solvent supplement to the paper base is not required. BRIEF DESCRIPTION OF DRAWINGS

[0029] The disclosure of the present application will become more apparent with reference to the drawings. It is easy for those skilled in the art to understand that these drawings are only used to illustrate the technical solutions of the present application, and are not intended to limit the protection scope of the present application. In the drawings:

[0030] Figure 1 is a flow chart of a cell detection method based on paper base discharge technology according to an embodiment of the present application;

[0031] Figure 2 is a longitudinal sectional structure diagram of a fluid pipeline according to embodiment 2 of the present application;

[0032] Figure 3 is a longitudinal sectional structure diagram of a fluid pipeline according to embodiment 3 of the present application;

[0033] Figure 4 is a longitudinal sectional structure diagram of a fluid pipeline according to embodiment 4 of the present application. DETAILED DESCRIPTION

[0034] Figures 1-4 The optional embodiments of the present application described in the following description and the accompanying drawings are presented to teach those skilled in the art how to make and use the present application. Some conventional aspects have been simplified or omitted in order to explain the technical solutions of the present application. Those skilled in the art should understand that variations or substitutions derived from these embodiments will be within the scope of the present application. Those skilled in the art should understand that the following features can be combined in various ways to form multiple variations of the present application. Therefore, the present application is not limited to the following optional embodiments, but is only limited by the claims and their equivalents.

[0035] Example 1:

[0036] A cell detection device based on paper-based electro-discharge technology, comprising:

[0037] A mass spectrometer having a sample inlet;

[0038] Paper having a tip, such as triangular paper, and an electrically conductive body connecting the paper and adapted to connect to a power source;

[0039] A carrier having a plurality of carrier sites, such as a matrix of carrier sites, for holding cells, such as single cells;

[0040] A fluid conduit having an end portion mating with the carrier site and forming a space between the carrier site and the fluid conduit, the space being isolated from the outside, the cell of the carrier site being in the space, the fluid conduit having an inlet and an outlet respectively communicating with the space;

[0041] A gas source for supplying gas into the space through the inlet, driving substances in the space into the fluid conduit, and ejecting substances discharged from the outlet of the fluid conduit onto the paper between the tip and the electrically conductive body.

[0042] To form the space, further, the fluid conduit comprises:

[0043] A capillary tube and a transfer tube, the transfer tube being disposed outside the capillary tube, the capillary tube having a first open end and a second open end, and the space being formed inside the transfer tube between the first open end of the capillary tube and the carrier site, the substances in the space entering the capillary tube from the first open end and being discharged from the second open end of the capillary tube, the gas source communicating with the inside of the transfer tube.

[0044] To form the space, further, the fluid conduit comprises:

[0045] A capillary tube and a transfer tube, the transfer tube being disposed outside the capillary tube and forming a sandwich with part of the outer wall of the capillary tube extending upward and downward, the space being formed between the capillary tube, the transfer tube and the carrier site when the first open end of the capillary tube and the open end of the transfer tube are in contact with the carrier site, the substances in the space entering the capillary tube from the first open end and being discharged from the second open end of the capillary tube, the gas source communicating with the inside of the transfer tube.

[0046] To form the space, further, the fluid conduit comprises:

[0047] A capillary and a transmission pipe, when a first open end of the capillary is in contact with the bearing position, the space is formed between the first open end of the capillary and the bearing position; the transmission pipe is connected with the capillary and communicates with the space; the substance in the space is discharged from a second open end of the capillary; and the gas source communicates with the inside of the transmission pipe.

[0048] To detect the cells in each bearing position, further, the cell detection device further comprises:

[0049] A driving unit, which drives the fluid pipe or the bearing position, so that the fluid pipe is in contact with any bearing position, thereby forming the space.

[0050] To apply the quantitative extraction liquid into the capillary, further, the cell detection device further comprises:

[0051] A container, which is open at the upper end and is adapted to contain the cell extraction liquid;

[0052] The driving unit drives the capillary to move up and down and to turn upside down.

[0053] To form the space and to transport the cells, further, the inner diameter of the first open end is 0.5mm-1.2mm, the inner diameter of the second open end is 10μm-100μm; the first open end is in the transmission pipe, and the distance between the first open end and the end of the transmission pipe which is in contact with the bearing position is 0.5mm-2mm.

[0054] To send the quantitative extraction liquid into the space to complete the extraction of the cells, further, the cell detection device further comprises:

[0055] A liquid source and a quantitative unit, the extraction liquid provided by the liquid source enters the transmission pipe after being quantified by the quantitative unit.

[0056] Figure 1 A flow chart of the cell detection method based on the paper-based discharge technology according to the embodiment of the application is given, that is, the working method of the cell detection device according to the embodiment, as shown in Figure 1 The cell detection method comprises the following steps:

[0057] (A1) One end of the fluid pipe is matched with the bearing position on the bearing member, thereby forming a space isolated from the outside, and the cells in the bearing position are in the space; the bearing member has a plurality of bearing positions;

[0058] (A2) The gas enters the space, and drives the extraction substance of the cells to be ejected from the open end of the fluid pipe;

[0059] (A3) the discharge of the conductive body connecting the paper, the extractant sprayed on the paper is ionized, and the ions are ejected from the tip of the paper into the mass spectrometer;

[0060] (A4) the mass spectrometer analyzes the ions to obtain information of the cells.

[0061] To extract the cells, further, the extraction method is:

[0062] The cells are extracted by:

[0063] The cells are extracted by the extractant first entering the space, and the extractant is driven by the gas entering the space into the fluid conduit; or,

[0064] The cells are driven by the gas entering the space into the fluid conduit, and are extracted by the extractant in the fluid conduit.

[0065] Embodiment 2:

[0066] Application example of the cell detection device and method based on the paper-based discharge technology according to Embodiment 1 of the present application.

[0067] In this application example, as shown in Figure 2 the fluid conduit includes a fixedly connected capillary tube 11 and a transmission tube 21, the first open end 12 of the capillary tube 11 has an inner diameter of 0.5mm-1.2mm, which is larger than the diameter of the cells, and the second open end 13 (the outlet of the fluid conduit) has an inner diameter of 10μm-100μm; the transmission tube 21 is wrapped around the outside of the capillary tube 11, has an inner diameter of 2mm-10mm, and the first open end 12 is located in the transmission tube 21 and is 0.5mm-2mm away from one end 22 of the contact bearing position of the transmission tube 21; the upper end inlet 23 of the transmission tube 21 is the inlet of the fluid conduit;

[0068] The paper is triangular, and the conductive clip (the conductive body) clamps the bottom side, and the angle (acute angle) opposite to the bottom side is the tip for ion ejection;

[0069] The gas source is connected to the inlet 23, i.e. the interlayer between the transmission tube 21 and the capillary tube 11;

[0070] The driving unit is used to translate the transmission tube 21 (capillary tube 11) in the vertical direction and the horizontal direction, and to overturn the transmission tube 21 (capillary tube 11), so that the transmission tube 21 contacts any bearing position to form a space isolated from the outside world;

[0071] The bearing member is plate-shaped and horizontally placed; a plurality of bearing positions are distributed in a matrix manner on the upper side of the bearing member, and each bearing position has a ring-shaped groove made of flexible material on the outer side, and the contact bearing position one end 22 of the transmission tube 21 is adapted to be inserted into the ring-shaped groove and kept sealed.

[0072] The cell detection method based on the paper-based discharge technology comprises the following steps:

[0073] (A1) The second opening end 13 of the capillary tube 11 is turned downward and enters the extraction liquid in the container by driving the driving unit, and part of the extraction liquid enters the inside of the second opening end 13; the volume of the extraction liquid entering the second opening end 13 is fixed due to the capillary effect;

[0074] The capillary tube 11 moves upward and turns over, and the second opening end 13 faces upward;

[0075] Under the action of the driving unit, the capillary tube 11 moves horizontally to the upper side of the selected cell carrying position, and then moves downward;

[0076] The bottom end of the transmission tube 21 is inserted into the annular groove of the selected carrying position to form a space isolated from the outside, and the dehydrated cells in the carrying position are located in the space at the bottom of the carrying position; the dehydrated cells are obtained in the following manner:

[0077] The cells to be detected are obtained from blood, ascites or amniotic fluid and the like, and are centrifuged at a low speed of 1000 r / min for 10 min, and are washed with phosphate buffered saline (PBS) for 2-3 times, and are washed with a culture medium once, and are then divided into test tubes for culture after adjusting the cell concentration;

[0078] The culture solution in the test tube is mixed by gently blowing with a pipette, 1 ml of the proliferated culture solution is sucked into 5 ml of washing liquid (PBS or normal saline), and is centrifuged at a low speed of 1000 r / min for 5 min, and the supernatant is discarded, and the precipitated cells are collected;

[0079] The precipitated cells are sequentially soaked and dried with 70%, 80% and 90% alcohol, and finally 1 ml of anhydrous ethanol is added, and the mixture is poured into the carrying member, and the cells enter the carrying positions, and the ethanol solution is then volatilized; only single cells are left in the carrying positions;

[0080] (A2) The gas provided by the gas source enters the transmission tube 21 and the space in sequence through the inlet 23, and the single cells in the carrying position enter the capillary tube 11 from the first opening end 12;

[0081] The cells moving upward are extracted by the extraction liquid in the second opening end 13;

[0082] Under the driving of the gas, the extracted substances of the cells are sprayed from the second opening end 13 of the capillary tube 11; the distance between the second opening end 13 and the paper is 10-20 mm, such as 10 mm, 12 mm, 17 mm or 20 mm;

[0083] (A3) the conductive clip discharges electricity to the paper, the extractant sprayed on the lower side of the paper between the conductive clip and the tip is ionized, and the ions are ejected from the tip into the mass spectrometer inlet; the distance between the tip and the inlet is 3-10 mm, such as 3 mm, 5 mm, 8 mm, or 10 mm;

[0084] (A4) the mass spectrometer analyzes the ions to obtain information of the single cell in the loading position.

[0085] Embodiment 3:

[0086] Application of the cell detection device and method based on the paper-based discharge technology according to Embodiment 1 of the present application.

[0087] In this application example, as shown in FIG. 1, the inner diameter of the first open end 12 of the capillary tube 11 is 0.5-1.2 mm, which is larger than the diameter of the cell, and the inner diameter of the second open end 13 (i.e. the outlet of the fluid conduit) is 10-100 μm; the delivery tube 21 is connected to the bottom end of the capillary tube 11, and the inside of the delivery tube 21 is in communication with the space; when the fluid enters the capillary tube 11 from the delivery tube 21, it is inclined downward. Figure 3

[0088] The driving unit is used to translate the capillary tube 11 in the vertical and horizontal directions, so that the capillary tube 11 is in contact with any loading position to form a space isolated from the outside world.

[0089] The carrier is in the form of a plate and is placed horizontally; the plurality of loading positions are distributed in a matrix on the upper side of the carrier.

[0090] The gas source and the liquid source are selectively connected to the inlet of the delivery tube 21, and a liquid metering unit is arranged between the inlet and the liquid source.

[0091] The cell detection method based on the paper-based discharge technology according to the embodiment of the present application comprises the following steps:

[0092] (A1) the first open end 12 of the capillary tube 11 is lowered by driving the driving unit;

[0093] The bottom end of the capillary tube 11 is in contact with the selected loading position to form a space isolated from the outside world, and the dehydrated cells in the loading position are in the space; the dehydrated cells are obtained in the following manner:

[0094] The cells to be detected are from solid tissue material, and the cells in the tissue are dispersed by mechanical dispersion or digestion separation method to prepare a cell suspension material, which is then cultured in a test tube after adjusting the cell concentration.

[0095] ​Gently pipette the culture medium in the test tube to mix it. Take 1 ml of the proliferated culture medium into 5 ml of washing solution (PBS or physiological saline), centrifuge at 1000 r / min for 5 min, discard the supernatant and collect the precipitated cells.

[0096] The precipitated cells are soaked in a sodium chloride solution with a concentration exceeding 10%, and the solution is removed by centrifugation or cell filter. After repeating this process multiple times, the cells are transferred to the carrier position of the carrier by means of purging or other methods.

[0097] (A2) A quantitatively measured volume of extract first enters the space through the transfer tube 21, and the cells at the carrier site are extracted.

[0098] The gas then passes through the transmission tube 21 and enters the space, carrying the extracted substance from the first opening end 12 into the capillary 11;

[0099] Driven by gas, the cell extract is ejected from the second opening end 13 of the capillary 11; the distance between the second opening end 13 and the paper is 10-20 mm, such as 10 mm, 12 mm, 17 mm, or 20 mm.

[0100] (A3) The conductive clip (conductor) connecting the paper discharges, and the extractant sprayed onto the paper (the lower side of the paper between the conductive clip and the tip) is ionized. Ions are ejected from the tip of the paper and enter the mass spectrometer inlet; the distance between the tip and the inlet is 3-10 mm, such as 3 mm, 5 mm, 8 mm, 10 mm.

[0101] (A4) Mass spectrometry analyzes ions to obtain information about cells.

[0102] Example 4:

[0103] The application example of the cell detection device and method based on paper-based discharge technology according to Embodiment 1 of the present invention differs from Embodiment 2 in that:

[0104] like Figure 4 As shown, the transfer tube 21 is disposed outside the capillary tube 11, and a vertically extending interlayer 24 is formed between a portion of the outer wall of the capillary tube 11 and the transfer tube 21. The bottom of the interlayer 24 is connected to the bottom inside the capillary tube 11. The first open end 12 of the transfer tube 21 and the capillary tube 11 contact the bearing position 22, thereby forming a space isolated from the outside. As needed, the extractant and gas are successively introduced into the space through the inlet 23 of the transfer tube 21 (i.e., the inlet of the fluid pipe), and the extracted substances of the cells are discharged from the second open end 13 of the capillary tube 11 (i.e., the outlet of the fluid pipe).

[0105] The above embodiments exemplarily show the case of using the driving unit to move the fluid pipe, of course, the manual insertion of the bearing position can also be used to form the space isolated from the outside.

Claims

1. A cell detection method based on paper-based discharge technology implemented using a cell detection device based on paper-based discharge technology, wherein the cell detection device includes a mass spectrometer, and further includes: Paper and a conductor, the paper having a tip, the conductor being connected to the paper and adapted to be connected to a power source; A carrier having multiple carrier positions for carrying cells; A fluid conduit, the end of which mates with the bearing position, forming a space isolated from the outside between the bearing position and the fluid conduit, the cells of the bearing position being located within the space; the fluid conduit having an inlet and an outlet respectively connected to the space; A gas source is used to supply gas into the space, drive the substance in the space into the fluid pipe, and spray the substance discharged from the outlet of the fluid pipe onto the paper between the tip and the conductor. The fluid conduit includes a capillary tube and a transmission tube. The transmission tube is disposed outside the capillary tube and forms a vertically extending interlayer between it and a portion of the outer wall of the capillary tube. When the first open end of the capillary tube and the open end of the transmission tube contact the bearing position, a space is formed between the capillary tube, the transmission tube, and the bearing position. The substance in the space enters from the first open end of the capillary tube and exits from the second open end of the capillary tube. The gas source is connected to the interior of the transmission tube. Each bearing position has an annular groove made of flexible material on its outer side, and one end of the transmission tube that contacts the bearing position is adapted to be inserted into the annular groove and remain sealed. The cell detection method includes the following steps: (A1) One end of the fluid pipe and the bearing position on the bearing member cooperate to form a space isolated from the outside world, and the cells of the bearing position are located in the space; the bearing member has multiple bearing positions; (A2) Gas enters the space, driving the extracted substances of the cells to be ejected from the opening end of the fluid pipe; (A3) The conductor connected to the paper discharges, the extractant sprayed onto the paper is ionized, and the ions are ejected from the tip of the paper and enter the mass spectrometer; (A4) Mass spectrometry analyzes ions to obtain information about cells.

2. The cell detection method based on paper-based discharge technology according to claim 1, characterized in that, The cells are extracted in the following manner: The cells are extracted by an extractant that first enters the space, and the extracted substance is then driven into the fluid channel by a gas that subsequently enters the space; or... The cells are driven into the fluid channel by the gas entering the space, where they are extracted by the extractant inside the fluid channel.

3. The cell detection method based on paper-based discharge technology according to claim 1, characterized in that, The cell detection device also includes: A driving unit drives the fluid pipe or bearing position, causing the fluid pipe to contact any bearing position, thereby forming the space.

4. The cell detection method based on paper-based discharge technology according to claim 3, characterized in that, The cell detection device also includes: A container, the container having an opening at the top, suitable for containing cell extract; The driving unit drives the capillary to move up and down and flip up and down.

5. The cell detection method based on paper-based discharge technology according to claim 3, characterized in that, The cell detection device also includes: The liquid source and the metering unit, wherein the extract provided by the liquid source is metered by the metering unit and then enters the transfer tube.

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