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

Abstract

The present invention provides a cell detection device and method based on paper-based discharge technology. The cell detection device includes a mass spectrometer; the paper has a tip, and a conductor is connected to the paper and is adapted to be connected to a power source; a carrier has a plurality of carrier positions for carrying cells; the end of a fluid pipeline cooperates with the carrier position to form a space isolated from the outside between the carrier position and the fluid pipeline, and the cells at the carrier position are in the space; the fluid pipeline has an inlet and an outlet that respectively communicate with the space; a gas source is used to supply gas into the space to drive substances in the space into the fluid pipeline, and the substances discharged from the outlet of the fluid pipeline are ejected onto the paper between the tip and the conductor. The present invention has the advantages of accurate detection, automation, etc.

Description

[0001] This invention patent is a divisional application of a Chinese patent with the application number 2021101342985. The filing date of the original application is February 1, 2021, and the invention title is "Cell Detection Device and Method Based on Paper-Based Discharge Technology". Technical Field

[0002] The present invention relates to cell analysis, and particularly to a cell detection device and method based on paper-based discharge technology. Background Art

[0003] Single-cell technology has developed rapidly in recent years. With the continuous development of technologies such as single-cell sequencing and single-cell transcriptomics, humans have deeply understood cell population heterogeneity at the DNA and RNA levels. Single-cell analysis is of great significance for studying intercellular heterogeneity. Mass spectrometers are widely used in single-cell analysis due to their high sensitivity, accurate structure identification, quantitative analysis, and other characteristics. However, single cells have a tiny volume of only fL~pL. If the extraction liquid volume is too large, the metabolites in the cells will be overly diluted, resulting in too low a concentration and exceeding the mass spectrometry detection range; if the dilution is too little, the amount of sample available for analysis will be too small, greatly increasing the difficulty of sample manipulation.

[0004] Some researchers have already applied paper spray mass spectrometry technology to cell analysis. This method uses a triangular paper substrate as a carrier, adds the sample on the paper substrate surface, applies a high voltage, and uses the voltage to drive the solvent to dissolve and extract the target substance in the sample, enabling it to migrate and ionize, and form an electrospray at the tip of the paper substrate to vaporize and enter the mass spectrometer for analysis. This method is convenient to operate and does not cause excessive dilution. However, paper spray mass spectrometry technology has the following problems:

[0005] 1. When combined with a mass spectrometer using curtain gas, due to the rapid drying of the solvent on the paper, a short and unstable spray is generated, resulting in unstable mass spectrometry signals and low sensitivity.

[0006] 2. This method generally spots samples by pipette, and it is impossible to ensure the analysis of samples at the single-cell level.

[0007] 3. Due to the problem of matrix interference, it has limitations in the analysis of complex biological and environmental matrix samples. Summary of the Invention

[0008] To solve the deficiencies in the above-mentioned existing technical solutions, the present invention provides a cell detection device based on paper-based discharge technology with good accuracy and stability.

[0009] The object of the present invention is achieved through the following technical solutions:

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

[0011] 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;

[0012] A carrier, the carrier having a plurality of carrier positions for carrying cells;

[0013] The end of the fluid pipeline cooperates with the carrier position to form a space isolated from the outside world between the carrier position and the fluid pipeline, and the cells at the carrier position are within the space; the fluid pipeline has an inlet and an outlet respectively communicating with the space;

[0014] A gas source for supplying gas into the space to drive the substances in the space into the fluid pipeline, and the substances discharged from the outlet of the fluid pipeline are sprayed onto the paper between the tip and the conductor;

[0015] The fluid pipeline includes:

[0016] A capillary and a transfer pipe. When the first open end of the capillary contacts the carrier position, the space is formed between the first open end of the capillary and the carrier position; the transfer pipe is connected to the capillary and communicates with the space; the substances in the space are discharged from the second open end of the capillary; the gas source communicates with the inside of the transfer pipe.

[0017] Another object of the present invention is to provide a cell detection method based on paper-based discharge technology, and this invention object is achieved through the following technical solutions:

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

[0019] (A1) One end of the fluid pipeline cooperates with the carrier position on the carrier to form a space isolated from the outside world, and the cells at the carrier position are within the space; the carrier has a plurality of carrier positions;

[0020] (A2) Gas enters the space to drive the extraction substances of the cells to spray out from the open end of the fluid pipeline;

[0021] (A3) The conductor connecting the paper discharges, and the extraction substances sprayed onto the paper are ionized, and the ions are ejected from the tip of the paper and enter the mass spectrometer;

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

[0023] Compared with the prior art, the beneficial effects of the present invention are:

[0024] 1. The detection result is accurate;

[0025] The extraction solution has a fixed volume. Single cells are extracted by the extraction solution without mixing with other substances, which can better control the dilution concentration and improve the comparability between groups;

[0026] Single cells are in full contact with the extraction solution, reducing matrix interference and improving the accuracy of cell detection;

[0027] 2. The detection stability is good;

[0028] A space isolated from the outside is formed by using a fluid pipeline and a bearing position. Then, the extraction substance is continuously supplied to the paper substrate by the thrust of the gas entering the space through the inlet, overcoming the problem that the paper-based electrospray with single-point injection cannot form a stable and continuous electrospray, thereby improving the signal stability of the paper spray mass spectrometry without the need to continuously supply additional solvent to the paper substrate. Brief Description of the Drawings

[0029] Referring to the accompanying drawings, the disclosure of the present invention will become more understandable. 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 invention and are not intended to limit the protection scope of the present invention. In the figures:

[0030] Figure 1 is a flowchart of a cell detection method based on paper-based discharge technology according to an embodiment of the present invention;

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

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

[0033] Figure 4 is a longitudinal sectional structure diagram of a fluid pipeline according to Embodiment 4 of the present invention. Detailed Embodiments

[0034] Figures 1-4 The following description and examples describe alternative embodiments of the present invention to teach those skilled in the art how to implement and reproduce the present invention. To explain the technical solutions of the present invention, some conventional aspects have been simplified or omitted. Those skilled in the art should understand that variations or substitutions derived from these embodiments will be within the scope of the present invention. Those skilled in the art should understand that the following features can be combined in various ways to form multiple variations of the present invention. Thus, the present invention is not limited to the following alternative embodiments, but is only defined by the claims and their equivalents.

[0035] Example 1:

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

[0037] A mass spectrometer having an inlet;

[0038] Paper and a conductor, the paper having a tip, such as triangular paper, the conductor being connected to the paper and adapted to be connected to a power source;

[0039] A carrier having a plurality of carrier positions, such as carrier positions distributed in a matrix, the carrier positions being for carrying cells, such as single cells;

[0040] A fluid conduit, an end of the fluid conduit cooperating with the carrier position to form a space isolated from the outside between the carrier position and the fluid conduit, the cells at the carrier position being within 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 to drive substances in the space into the fluid conduit, and the substances discharged from the outlet of the fluid conduit being ejected onto the paper between the tip and the conductor.

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

[0043] A capillary and a transfer tube, the transfer tube being sleeved outside the capillary; when an open end of the transfer tube contacts the carrier position, there is a gap between a first open end of the capillary and the carrier position; the space is formed inside the transfer tube and is between the carrier position and the first open end of the capillary; substances in the space enter the capillary from the first open end and are discharged from a second open end of the capillary; the gas source communicates with the inside of the transfer tube.

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

[0045] A capillary and a transfer tube, the transfer tube being disposed outside the capillary and forming a sandwich extending vertically between a part of the outer wall of the capillary; when a first open end of the capillary and an open end of the transfer tube contact the carrier position, the space is formed between the capillary, the transfer tube and the carrier position; substances in the space enter from the first open end of the capillary and are discharged from a second open end of the capillary; the gas source communicates with the inside of the transfer tube.

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

[0047] A capillary and a transfer tube. When a first open end of the capillary contacts a carrying position, a space is formed between the first open end of the capillary and the carrying position; the transfer tube is connected to the capillary and communicates with the space; substances in the space are discharged from a second open end of the capillary; a gas source communicates with the interior of the transfer tube.

[0048] To detect cells at each carrying position, further, the cell detection device further includes:

[0049] A driving unit that drives the fluid pipeline or the carrying position such that the fluid pipeline contacts any one of the carrying positions, thereby forming the space.

[0050] To apply a quantitative extraction solution into the capillary, further, the cell detection device further includes:

[0051] A container with an open upper end, adapted to accommodate a cell extraction solution;

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

[0053] To form the space and transfer cells, further, an inner diameter of the first open end is 0.5 mm - 1.2 mm, and an inner diameter of the second open end is 10 μm - 100 μm; the first open end is inside the transfer tube and is 0.5 mm - 2 mm away from an end of the transfer tube that contacts the carrying position.

[0054] To send a quantitative extraction solution into the space to complete cell extraction, further, the cell detection device further includes:

[0055] A liquid source and a quantification unit. The extraction solution provided by the liquid source enters the transfer tube after being quantified by the quantification unit.

[0056] Figure 1 The flowchart of the cell detection method based on the paper-based discharge technology according to an embodiment of the present invention is given, that is, the working method of the cell detection device according to this embodiment is as Figure 1 shown, and the cell detection method includes the following steps:

[0057] (A1) One end of a fluid pipeline cooperates with a carrying position on a carrier to form a space isolated from the outside, and cells at the carrying position are inside the space; the carrier has a plurality of carrying positions;

[0058] (A2) Gas enters the space, driving the extraction substances of the cells to spray out from the open end of the fluid pipeline;

[0059] The conductor of the connecting paper discharges, the extraction substance sprayed onto the paper is ionized, and ions are ejected from the tip of the paper and enter the mass spectrometer;

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

[0061] In order to extract cells, further, the extraction method is as follows:

[0062] The method for extracting the cells is as follows:

[0063] The cells are first extracted by the extraction liquid that enters the space first, and the extraction substance enters the fluid pipeline under the drive of the gas that enters the space later; or,

[0064] The cells are driven into the fluid pipeline by the gas entering the space and are extracted by the extraction liquid in the fluid pipeline.

[0065] Example 2:

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

[0067] In this application example, as Figure 2 shown, the fluid pipeline includes a capillary 11 and a transmission tube 21 fixedly connected. The inner diameter of the first opening end 12 of the capillary 11 is 0.5 mm - 1.2 mm, which is larger than the diameter of the cells, and the inner diameter of the second opening end 13 (the outlet of the fluid pipeline) is 10 μm - 100 μm; the transmission tube 21 surrounds the outside of the capillary 11, and the inner diameter is 2 mm - 10 mm. The first opening end 12 is located inside the transmission tube 21, and the distance from the contact and bearing position end 22 of the transmission tube 21 is 0.5 mm - 2 mm; the upper end inlet 23 of the transmission tube 21 is the inlet of the fluid pipeline;

[0068] The paper is triangular, and the conductive clip (conductor) clamps the bottom edge, and the angle (acute angle) opposite to the bottom edge is used as the tip for ion emission;

[0069] The gas source is connected to the inlet 23, that is, it communicates with the interlayer between the transmission tube 21 and the capillary 11;

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

[0071] The carrier is plate-shaped and placed horizontally; a plurality of bearing positions are distributed in a matrix on the upper side of the carrier, and an annular groove made of a flexible material is provided outside each bearing position, and the contact and bearing position end 22 of the transmission tube 21 is adapted to be inserted into the annular groove and maintain a seal.

[0072] The cell detection method based on paper-based discharge technology according to an embodiment of the present invention, the cell detection method comprising the following steps:

[0073] (A1)Under the drive of the drive unit, the second opening end 13 of the capillary 11 is flipped downward and enters the extraction liquid in the container, and part of the extraction liquid enters the inside of the second opening end 13; due to capillary action, the volume of the extraction liquid entering the second opening end 13 is fixed;

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

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

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

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

[0078] Gently pipette and mix the culture solution in the test tube, aspirate 1 ml of the proliferated culture liquid into 5 ml of washing solution (PBS or normal saline), centrifuge at a low speed of 1000 r / min for 5 min, discard the supernatant, and collect the precipitated cells;

[0079] The precipitated cells are successively soaked and dried with 70%, 80% and 90% alcohol, and finally 1 ml of absolute ethanol is added. After mixing, it is poured into the carrier, and the cells enter each bearing position, and then the ethanol solution volatilizes; only single cells remain in the bearing position;

[0080] (A2)The gas provided by the gas source passes through the inlet 23 and enters the transfer tube 21 and the space in turn, carrying the single cells at the bearing position from the first opening end 12 into the capillary 11;

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

[0082] Under the gas drive, the extraction substance of the cells 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, 20 mm;

[0083] The conductive clip of the connecting paper discharges, and the extraction substance 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 inlet of the mass spectrometer. The distance between the tip and the inlet is 3 - 10 mm, such as 3 mm, 5 mm, 8 mm, 10 mm.

[0084] (A4)The mass spectrometer analyzes the ions to obtain information on individual cells at the bearing positions.

[0085] Example 3:

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

[0087] In this application example, as Figure 3 shown, the inner diameter of the first opening end 12 of the capillary 11 is 0.5 mm - 1.2 mm, which is larger than the diameter of the cell, and the inner diameter of the second opening end 13 (i.e., the outlet of the fluid pipeline) is 10 μm - 100 μm. The transfer tube 21 is connected to the bottom end of the capillary 11, and the inside of the transfer tube 21 is in spatial communication. When the fluid enters the capillary 11 from the transfer tube 21, it is inclined downward.

[0088] The driving unit is used to translate the capillary 11 in the vertical and horizontal directions so that the capillary 11 contacts any bearing position to form a space isolated from the outside.

[0089] The carrier is plate-shaped and placed horizontally. A plurality of bearing 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 transfer tube 21, and a liquid metering unit is provided 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 invention, the cell detection method includes the following steps:

[0092] (A1)Using the drive of the drive unit, the first opening end 12 of the capillary 11 is moved downward.

[0093] The bottom end of the capillary 11 contacts the selected bearing position to form a space isolated from the outside, and the dehydrated cells at the bearing position are in the space. The way to obtain the dehydrated cells is:

[0094] The cells to be tested are from solid tissue materials. The cells in the tissue are fully dispersed by mechanical dispersion or digestion separation methods to make cell suspension materials. After appropriately adjusting the cell concentration, they are divided into test tubes for culture.

[0095] Gently pipette and mix the culture medium in the test tube, aspirate 1 ml of the proliferated culture liquid into 5 ml of washing solution (PBS or normal saline), centrifuge at a low speed of 1000 r / min for 5 min, discard the supernatant, and collect the precipitated cells;

[0096] Soak the precipitated cells in a sodium chloride solution with a concentration exceeding 10%, remove the solution by centrifugation or through a cell filter, repeat several times, and then transfer the cells to the bearing position of the carrier by means such as blowing;

[0097] (A2)A quantitatively determined volume of extraction liquid first enters the space through the transfer tube 21, and the cells at the bearing position are extracted;

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

[0099] Driven by the gas, the extracted substance of the cells 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, 20 mm;

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

[0101] (A4)The mass spectrometer analyzes the ions to obtain information about the cells.

[0102] Example 4:

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

[0104] As Figure 4 shown, the transfer tube 21 is arranged outside the capillary 11, and a sandwich layer 24 extending up and down is formed between a part of the outer wall of the capillary 11 and the transfer tube 21, and the bottom of the sandwich layer 24 is communicated with the bottom inside the capillary 11; the transfer tube 21 and the first opening end 12 of the capillary 11 contact the bearing position 22, thereby forming a space isolated from the outside; as needed, the extraction liquid and gas are successively input into the space through the inlet 23 of the transfer tube 21 (i.e., the inlet of the fluid pipeline), and the extracted substance of the cells is discharged from the second opening end 13 (i.e., the outlet of the fluid pipeline) of the capillary 11.

[0105] The above embodiments exemplarily show the case of using a driving unit to move a fluid pipeline. Of course, it is also possible to manually insert it into the bearing position to form a space isolated from the outside world.

Claims

1. A cell detection device based on paper-based discharge technology, the cell detection device comprising a mass spectrometer; characterized in that, The cell detection device based on the paper-based discharge technology 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, the carrier having a plurality of carrier positions for carrying cells; A fluid conduit, an end of the fluid conduit cooperating with the carrier position to form a space isolated from the outside between the carrier position and the fluid conduit, the cells at the carrier position being within the space; the fluid conduit having an inlet and an outlet respectively communicating with the space; A gas source for supplying gas into the space to drive substances in the space into the fluid conduit, and the substances discharged from the outlet of the fluid conduit being ejected onto the paper between the tip and the conductor; The fluid conduit includes: A capillary tube and a transfer tube. When a first open end of the capillary tube contacts the carrier position, the space is formed between the first open end of the capillary tube and the carrier position; the transfer tube is connected to the capillary tube and communicates with the space; the substances in the space are discharged from a second open end of the capillary tube; the gas source communicates with the interior of the transfer tube; the inner diameter of the first open end of the capillary tube is 0.5 mm - 1.2 mm, and the inner diameter of the second open end is 10 μm - 100 μm.

2. The cell detection device based on paper-based discharge technology according to claim 1, wherein, The cell detection device further includes: A driving unit for driving the fluid conduit or the carrier position so that the fluid channel contacts any one of the carrier positions, thereby forming the space.

3. The cell detection device based on paper-based discharge technology according to claim 2, characterized in that, The cell detection device further includes: A container having an open upper end and adapted to contain a cell extractant; The driving unit drives the capillary tube to move up and down and turn over up and down.

4. The cell detection device based on paper-based discharge technology according to claim 1, wherein The cell detection device further includes: A liquid source and a quantification unit, and the extractant provided by the liquid source enters the transfer tube after being quantified by the quantification unit.

5. The cell detection method based on the paper-based discharge technology of the cell detection device according to any one of claims 1 - 4, the cell detection method comprising the following steps: (A1) One end of the fluid conduit cooperates with a carrier position on the carrier to form a space isolated from the outside, and the cells at the carrier position are within the space; the carrier has a plurality of carrier positions; (A2) Gas enters the space to drive the extraction substances of the cells to be ejected from the open end of the fluid conduit; (A3) The conductor connected to the paper discharges, and the extraction substances ejected onto the paper are ionized, and the ions are ejected from the tip of the paper and enter the mass spectrometer; (A4) The mass spectrometer analyzes the ions to obtain information about the cells.

6. The cell detection method based on paper-based discharge technology according to claim 5, wherein The manner in which the cells are extracted is: The cells are first extracted by the extractant that enters the space, and the extraction substances enter the fluid conduit under the drive of the gas that enters the space later; or, The cells are driven into the fluid conduit by the gas that enters the space and are extracted by the extractant in the fluid conduit.

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