A method and device for lysing cells and application thereof

Abstract

The present invention belongs to the field of biological industry, and specifically relates to a method and device for lysing cells and their applications. The method of the present invention is based on chemical lysis and ultrasonic lysis, atomizes the lysis solution, lyses the cells with a continuous flow of atomization, and realizes the recycling of the lysis solution, so that only a small amount of lysis solution is needed, which improves the speed, yield and abundance of cell lysis; at the same time, using this method, the present invention provides a device for lysing cells. Furthermore, the present invention takes high-risk HPV home detection as an example and provides the application of the above method and device in disease detection. The speed and accuracy of detection are improved, and the convenience of detection is greatly improved, and personnel without professional knowledge and skills can also complete the operation quickly.

Description

Technical Field

[0001] The present invention belongs to the field of biological industry, and specifically relates to a method and device for lysing cells and application thereof. Background Art

[0002] Cell lysis technology, also known as cell disruption technology, refers to the use of external force to destroy the cell membrane and cell wall to release the cell contents including the target product components. It is the basis for separating and purifying non-secretory biochemical substances (products) synthesized in cells. Cell lysis is an important experimental method to change cell permeability and activity, and is widely used in many biochemical experiments and tests.

[0003] Cell lysis methods can be roughly divided into two categories: mechanical lysis and non-mechanical lysis. Mechanical lysis includes grinding, ball stirring, ultrasonic lysis, high-pressure homogenization, etc.; non-mechanical lysis includes repeated freeze-thaw lysis, high-temperature lysis, enzymatic lysis, chemical lysis, etc. Mechanical lysis is a more intense method that can lyse cells more comprehensively, but it will cause damage to the DNA in the cells; while non-mechanical lysis is usually milder and less likely to break DNA, but its efficiency is lower than mechanical lysis.

[0004] Although there are many methods for lysing cells, in general, these methods require professional equipment and certain operating techniques, and can often only be performed by professional experimenters in the laboratory. This greatly limits the use of this technology, especially in home testing. Summary of the invention

[0005] Based on the above technical problems, in order to overcome the above technical deficiencies, the present invention provides a method for room temperature atomization continuous flow cell lysis, which utilizes both chemical lysis and ultrasonic lysis, and only requires a small amount of lysis solution for cell lysis, thereby improving the speed, yield and abundance of cell lysis; at the same time, using this method, the present invention provides a cell lysis device and a sample collection device, which improve the speed and accuracy of detection, and greatly improve the convenience of detection, so that personnel without professional knowledge and skills can also quickly complete the operation.

[0006] The first aspect of the present invention aims to provide a method for cell lysis.

[0007] The second aspect of the present invention aims to provide a cell lysis device.

[0008] The third aspect of the present invention aims to provide an application.

[0009] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

[0010] The first aspect of the present invention provides a cell lysis method, comprising the following steps:

[0011] The cell sample is fixed on the surface of the carrier, the lysis solution is atomized and impacted on the cells, and the cell lysate is collected.

[0012] Preferably, the surface of the carrier is covered with a hydrophobic material.

[0013] Preferably, the hydrophobic material includes at least one of polyethylene, polypropylene, polytetrafluoroethylene, water-based acrylate, hydrophobic glass, hydrophobic ceramics, and hydrophobic metal.

[0014] Preferably, the carrier surface has a micro-nano structure.

[0015] Preferably, the micro-nano structure refers to a functional structure that is artificially designed on the surface, has a characteristic size of micrometer or nanometer scale, and is arranged in a specific manner. Increasing the specific surface area by micro-nano structure can improve the surface's adsorption capacity for sample cells, and prevent sample cells from falling off the carrier material during atomization impact, thereby affecting the lysis efficiency. Furthermore, the micro-nano structure can have a specific shape and be arranged in a directional manner, and has the function of directional liquid transportation, which can guide and accelerate the flow of lysate droplets condensed on the carrier surface, thereby preventing the lysate droplets from excessively agglomerating and affecting the lysis yield.

[0016] Preferably, the micro-nano structure is formed by fiber weaving or prepared by micro-nano processing. Currently, the manufacturing methods of micro-nano structures include micro-nano processing and fiber weaving. Micro-nano processing mainly includes photolithography, template manufacturing, etc., which can form relatively complex and rich micro-nano structures but the cost is high and not suitable for large-scale manufacturing; while the micro-nano structure formed by the fiber manufacturing method is relatively simple and has fewer types, but the cost is low and suitable for current large-scale manufacturing.

[0017] Preferably, the lysis buffer comprises at least one of RIPA lysis buffer, SDS lysis buffer, NP40 lysis buffer, Tris-Triton lysis buffer, ACK lysis buffer, Urea lysis buffer, and Tris-HCl lysis buffer.

[0018] Preferably, the cells include at least one of reproductive system exfoliated cells, respiratory system exfoliated cells, digestive system exfoliated cells, urinary system exfoliated cells, and skin exfoliated cells. In some embodiments of the present invention, the cells are reproductive system mucosal epithelial cells, including but not limited to vaginal epithelial cells and cervical epithelial cells.

[0019] Preferably, the sample is collected through natural secretions, or directly collected from epithelial tissue by brushing, dipping, etc. Preferably, the sample is tissue secretions or exfoliated material scraped by a sampling swab. In gynecological liquid-based cytology examination, a sampling brush is used to brush exfoliated cell samples and cervical secretions from the cervix.

[0020] Preferably, the method of atomizing the lysis solution includes at least one of electrofluid atomization, ultrasonic atomization, compressed air atomization, rotating disk atomization, and vortex atomization.

[0021] In some embodiments of the present invention, the method of atomizing the lysate comprises ultrasonic atomization.

[0022] Preferably, the ultrasonic atomization refers to an atomization method that generates ultrasonic waves through high-frequency vibration to break the liquid into small droplets.

[0023] Preferably, the high-frequency vibration can be generated by piezoelectric, magnetostrictive and electrostrictive methods. The piezoelectric refers to applying a voltage to an ultrasonic transducer (usually quartz or ceramic), causing the transducer to undergo mechanical deformation and generate ultrasonic high-frequency vibration. The magnetostrictive refers to applying a magnetic field to a magnetostrictive material (such as nickel or cobalt), causing it to undergo mechanical deformation and generate ultrasonic high-frequency vibration. The electrostrictive refers to applying an electric field to an electrostrictive material (such as lead zirconate titanate and other materials), causing it to undergo mechanical deformation and generate ultrasonic high-frequency vibration.

[0024] In some embodiments of the present invention, the ultrasonic atomization is performed based on piezoelectric ultrasound.

[0025] In some embodiments of the present invention, an atomizing sheet is used for ultrasonic atomization, and the oscillation frequency of the atomization is 1.7 MHz to 2.4 MHz; and the voltage is 3 to 12V.

[0026] In the first aspect of the present invention, the present invention combines chemical lysis of lysate components and physical lysis of ultrasonic atomization impact, fixes the cell sample on the carrier material, and achieves the lysis effect of the cell under the dual effects of chemical destruction and physical impact through a small amount of lysate. The method does not require a centrifuge, pipette, etc. required for conventional lysis, and can be designed in a portable device, which has the characteristics of being fast, portable, and easy to operate.

[0027] According to a second aspect of the present invention, a cell lysis device is provided, which includes a lysis chamber, an atomizer, a carrier, a substrate layer and a carrier layer. The lysis chamber is used to store a lysis solution. The atomizer includes a liquid inlet end and an atomizing end. The liquid inlet end is connected to the lysis chamber. The atomizing end is used to spray the lysis solution into a jet. A spacing space for forming a jet is provided between the carrier and the atomizing end. The carrier is used to carry a cell sample. A connecting structure connected to the carrier is provided on one side of the substrate layer. The carrier layer can be detachably provided on the other side of the substrate layer. The carrier layer is provided with pores, and the size of the pores is smaller than the size of the cell sample to be collected.

[0028] Preferably, the surface of the carrier is made of hydrophobic material.

[0029] Preferably, the cell lysis device also includes a collection chamber, the carrier includes a sample portion and a guide portion connected to each other, the lysis solution is sprayed to the sample portion through the atomization end and the spacing space, the cell sample is arranged in the sample portion, and the guide portion is used to guide the lysis solution and the lysis product to flow into the collection chamber.

[0030] Preferably, the collecting chamber comprises a collecting end, the sample portion is higher than the collecting end, and the guide portion extends obliquely downward from the sample portion toward the collecting end.

[0031] Preferably, the collecting chamber and the lysis chamber are interconnected, and a filtering structure capable of blocking the lysis products is provided between the collecting chamber and the lysis chamber.

[0032] Preferably, the collection chamber is provided with a detection port for inserting a test strip, and the cell lysis device further comprises a destructible or movable sealing structure, and the sealing structure is arranged at the detection port.

[0033] In the second aspect of the present invention, a cell lysis device that can be used to implement the cell lysis method of the first aspect of the present invention is further provided. The cell lysis device can achieve the cell lysis effect under the dual effects of chemical destruction and physical impact by using a small amount of lysis solution, and has the characteristics of rapidity, portability, and easy operation. In addition, the sample collection device can be conveniently arranged at the sampling position through the connection structure, so that the cell sample is collected into the carrier layer, and then the carrier layer is separately peeled off for cell lysis by the cell lysis device, which has the characteristics of rapidity, portability, and easy operation.

[0034] The third aspect of the present invention provides use of the cell lysis method of the first aspect of the present invention and the cell lysis device of the second aspect of the present invention in preparing a detection product.

[0035] Preferably, the product is used to detect at least one of RNA, DNA, and protein.

[0036] Preferably, the product can be used to detect the target after lysing the cells.

[0037] Preferably, the product includes at least one of a portable detector and a detection kit.

[0038] Preferably, the detection kit comprises a detection reagent, a detection device, and the cell lysis device of the second aspect of the present invention.

[0039] Preferably, the detection reagent is selected from substances for quantitative or qualitative detection of RNA, DNA, and protein, such as nucleic acid aptamers, primers, and antibodies that specifically bind to antigens.

[0040] Preferably, the detection device includes at least one of a test paper and a chip.

[0041] Preferably, the test paper comprises at least one of a colloidal gold test paper and an ELISA test paper.

[0042] In the third aspect of the present invention, the method based on the first aspect of the present invention and the application of the cell lysis device of the second aspect of the present invention are further provided. Due to the portability and ease of operation brought by the above method and device, they can be used for the development of various home detection products.

[0043] In some embodiments of the present invention, taking human papillomavirus (HPV) associated with female cervical cancer as an example, an application of the above method and device in developing a product for home detection of HPV virus is provided.

[0044] Human papillomavirus (HPV) is a spherical DNA virus that is widely present in nature. It has humans as its only host. It is very tolerant to drying and can be stored for a long time. Currently, there are only more than 150 known HPV subtypes, which can be divided into low-risk and high-risk types according to the severity of the diseases they cause. High-risk HPV can cause a variety of tumors, such as cervical cancer, anal cancer, and penile cancer. Studies have shown that E7 protein is an important cause of cervical epithelial cell disease (cervical cancer). E7 protein inhibits retinoblastoma protein (pRb) to enable cells to proliferate indefinitely. When the genetic tissue is damaged, E7 protein begins to express. Detecting E7 protein in female cervical exfoliated cells can qualitatively detect cervical cancer. The vaginal secretions of adult women are a mixture of secretions from the Bartholin's glands, cervical glands, endometrial secretions, vaginal mucosal exudates, and exfoliated vaginal epithelial cells. During the physiological period of women, there are also exfoliated cells of the endometrium. Through sanitary napkins used by women, cells shed from the female reproductive tract can be collected, and the biomass in these cells can be used for HPV home testing.

[0045] Since the abundance of E7 protein in cells is low in the early stage of HPV infection, conventional nucleic acid or PCR proliferation methods are required for accurate detection of early HPV infection; and some HPV home early screening products also have low detection accuracy due to low abundance. However, the method of the first aspect and the device of the second aspect of the present invention can achieve room-temperature efficient lysis of female reproductive tract exfoliated cells collected on female sanitary napkins at room temperature, while reducing the amount of lysis solution, greatly reducing the dilution of E7 protein, and significantly improving the detection accuracy.

[0046] Based on the same principle, the method and device of the present invention can also be applied to home detection of other diseases, including but not limited to influenza, mycoplasma, inflammation, cancer and other related diseases.

[0047] The beneficial effects of the present invention are:

[0048] The method of the present invention is based on chemical lysis and ultrasonic lysis, and the lysis solution is atomized, and the cells are lysed by a continuous flow of atomization, and the recycling of the lysis solution is realized, so that only a small amount of lysis solution is needed, and the speed, yield and abundance of cell lysis are improved; at the same time, using this method, the present invention provides a device for lysing cells. Furthermore, the present invention takes high-risk HPV home detection as an example and provides the application of the above method and device in disease detection. The speed and accuracy of detection are improved, and the convenience of detection is greatly improved, and personnel without professional knowledge and skills can also quickly complete the operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Schematic diagram of the process of cell / tissue lysis by atomization continuous flow in a cell lysis device according to an embodiment of the present invention.

[0050] Figure 2 This is a schematic diagram of a cell lysis device and a sample collection device according to an embodiment of the present invention applied to a detection product, which can be used for home detection of high-risk HPV.

[0051] Figure 3 This is a schematic diagram of a cell lysis device and a sample collection device according to an embodiment of the present invention applied to a detection product.

[0052] Figure 4 Schematic diagram of the structure of a cell lysis device according to an embodiment of the present invention, wherein a portion of the outer shell of the cell lysis device is hidden to show the internal structure.

[0053] Figure 5 Schematic diagram of the structure of the lysis chamber in the cell lysis device according to an embodiment of the present invention, wherein a portion of the outer shell of the cell lysis device is hidden to show the internal structure.

[0054] Wherein: 1-sample part, 2-guiding part, 3-cell sample, 4-atomization end, 5-lysate jet, 6-condensation droplets, 7-collection chamber, 8-liquid inlet end, 9-lysis chamber, 10-lysate, 11-filtration structure, 12-cell cluster, 13-sample collection device, 14-cell lysis device, 15-detection strip, 16-packaging shell, 17-sample chamber, 18-cover, 19-control board, 21-filter, 22-power interface, 23-detection port, 24-start switch. DETAILED DESCRIPTION

[0055] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0056] In the description of the present invention, it should be understood that descriptions involving orientations, such as up, down, front, back, left, right, etc., and orientations or positional relationships indicated are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present invention.

[0057] In the description of the present invention, "several" means one or more, "more" means more than two, "greater than", "less than", "exceed" etc. are understood as not including the number itself, and "above", "below", "within" etc. are understood as including the number itself. If there is a description of "first" or "second", it is only used for the purpose of distinguishing the technical features, and cannot be understood as indicating or implying the relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

[0058] In the description of the present invention, unless otherwise clearly defined, terms such as setting, installing, connecting, etc. should be understood in a broad sense, and technicians in the relevant technical field can reasonably determine the specific meanings of the above terms in the present invention based on the specific content of the technical solution.

[0059] The cell lysis method provided in the embodiment of the present application includes fixing cells on the surface of a carrier, atomizing a lysis solution and impacting the cells, and collecting the cell lysis solution.

[0060] On the one hand, the jet generated by atomization exerts a physical impact on the cells, and on the other hand, the microdroplets of lysate generated by atomization fully combine with the surface of the cells, causing chemical damage to the cells. The dual effects of chemical damage and physical impact can achieve the effect of cell lysis. The cell lysis method does not require the centrifuge, pipette, etc. required for conventional lysis to operate the instruments, nor does it require the use of a refrigerator for time-consuming repeated freezing and thawing. It can be designed in a portable device, which has the characteristics of being fast, portable, and easy to operate.

[0061] It is understandable that the method of atomizing the lysis solution in the cell lysis method includes at least one of electrofluid atomization, ultrasonic atomization, compressed air atomization, rotating disk atomization, and vortex atomization.

[0062] The embodiments of the present application also provide a cell lysis device, which is suitable for implementing the cell lysis method of the present application.

[0063] For example, refer to Figure 1 The cell lysis device at least includes a lysis chamber 9, an atomizer and a carrier. The lysis chamber 9 is used to store a lysis solution 10. The atomizer includes a liquid inlet end 8 and an atomization end 4. The liquid inlet end 8 is arranged in the lysis chamber 9. The atomization end 4 is used to spray a jet. The carrier is spaced apart and arranged opposite to the atomization end 4. The carrier is used to carry a sample (here, a cell sample 3).

[0064] When in use, the atomizer absorbs the lysate 10 in the lysis chamber 9 through the liquid inlet end 8, and converts the lysate 10 into a lysate jet 5, which is ejected from the atomization end 4 to impact the cell sample 3 on the stage, so that the cell sample 3 is lysed. The cell lysis device does not require a centrifuge, a pipette, etc. required for conventional lysis to operate the instrument, nor does it require a refrigerator for time-consuming repeated freezing and thawing, and has the characteristics of being fast, portable, and easy to operate.

[0065] The atomizer can realize atomization based on the principle that the control board applies a high-frequency oscillating voltage to the piezoelectric ceramic, and the atomization frequency of the piezoelectric ceramic can be 1.7 MHz to 2.4 MHz, and the voltage is 3 to 12 V. The liquid inlet end 8 can be provided with a liquid inlet, or a porous absorption material can be provided to deliver the lysate to the atomization end 4 by capillary action.

[0066] Other design details of the atomizer can be referred to the relevant prior art and will not be described in detail here.

[0067] Further, refer to Figure 2 and Figure 3 The embodiment of the present application also provides a sample collection device 13 suitable for use with a cell lysis device 14, and a detection product including the cell lysis device 14, the sample collection device 13 and a detection strip 15.

[0068] The sample collection device 13 and the detection product also have the beneficial effects produced by the cell lysis device 14, which will not be described in detail here.

[0069] Continuing to introduce the cell lysis device 14, it can be understood that, if Figure 1 As shown, the lysis liquid jet 5 will reconverge on the surface of the carrier to form the lysis liquid 10 again, and the lysis product produced by the lysis will be mixed with the lysis liquid 10. Therefore, it is necessary to collect the lysis product by collecting the lysis liquid 10 so as to further detect the lysis product later.

[0070] Optionally, considering that most of the current lysing solutions 10 are water-based, in order to prevent the lysing solution 10 and the lysed products from being adsorbed on the surface of the carrier and affecting the collection process, the surface of the carrier can be set to a hydrophobic material. The use of a hydrophobic material can significantly reduce the adsorption of the lysing solution 10 and the lysed products on the carrier, thereby improving the final lysis efficiency.

[0071] Specifically, the entire carrier may be made of a hydrophobic material, or the surface of the carrier may be covered with a hydrophobic coating made of a hydrophobic material, and the present invention is not limited to this.

[0072] Optionally, in order to collect the lysate 10, the cell lysis device further comprises a collection chamber 7, the carrier comprises a sample portion 1 and a flow guide portion 2 connected to each other, the sample portion 1 is aligned with the atomization end 4, the cell sample 3 is arranged in the sample portion 1, and the flow guide portion 2 is used to guide the lysate 10 and the lysate product (such as Figure 1 The cell mass 12 shown in FIG. 1 ) flows into the collection chamber 7 .

[0073] The flow guide 2 can guide the lysate 10 by capillary force, gravity or other forces. Figure 1 The collecting chamber 7 includes a collecting end (specifically, a collecting port formed by opening above the collecting chamber 7), the sample portion 1 is higher than the collecting end, and the flow guide portion 2 extends obliquely downward from the sample portion 1 toward the collecting end. In this way, the condensed droplets 6 generated by the lysing solution 10 can automatically flow to the collecting port with the lysed products under the action of gravity, and drip and collect in the collecting chamber 7.

[0074] The inclination angle of the air guide portion 2 may be greater than or equal to 10° and less than or equal to 90°.

[0075] It should be noted that Figure 1 The construction principle of the carrier is only shown by way of example; in an actual cell lysis device, the carrier may be constructed in different ways.

[0076] For example, refer to Figure 4 and Figure 5The cell lysis device has a packaging shell 16, an atomizer is arranged inside the packaging shell 16, the packaging shell 16 is provided with a hollow hole facing the atomization end 4, and defines a sample chamber 17 connected to the hollow hole. A cover 18 is movably mounted on the packaging shell 16, and the cover 18 can close or open the sample chamber 17.

[0077] When in use, the cell sample 3 is placed in the sample chamber 17, and the cover 18 and the packaging shell 16 clamp and position the cell sample 3, and expose a portion of the cell sample 3 through the hollow hole. At this time, the cover 18 and a portion of the packaging shell 16 corresponding to the sample chamber 17 together serve as the sample portion 1 of the carrier, and the portion of the packaging shell 16 that extends obliquely downward from the sample chamber to the collecting chamber 7 serves as the guide portion of the carrier. Condensation droplets 6 can be formed from the hollow hole and then flow along the inclined inner wall of the packaging shell 16 to the collecting chamber 7.

[0078] It is understandable that the lysis solution 10 in the collecting chamber 7 still has a certain effectiveness. In order to make more effective use of the lysis solution 10, optionally, the collecting chamber 7 and the lysis chamber 9 are interconnected, and a filtering structure 11 capable of blocking the lysis products is provided between the collecting chamber 7 and the lysis chamber 9.

[0079] At this time, the lysate 10 entering the collection chamber 7 can return to the lysis chamber 9 again, thereby reducing the total amount of lysate 10 required for lysis, which helps save reagent costs and also helps miniaturize the cell lysis device. At the same time, the lysate can be blocked by the filter structure 11 to prevent the lysate from being lost.

[0080] The filtering structure 11 can be implemented in a variety of ways. Figure 2 and Figure 3 The encapsulation shell 16 and the partition inside the encapsulation shell 16 define the collection chamber 7 and the lysis chamber 9. The filtering structure 11 can specifically adopt a filter mesh 21, which is installed on the partition between the collection chamber 7 and the lysis chamber 9. The pore size of the filter mesh 21 is smaller than the lysis product, so that the lysis product is retained while allowing the lysis solution 10 to pass through.

[0081] Alternatively, the collection chamber 7 and the lysis chamber 9 may be connected via a micro-nano scale microchannel, the diameter of the microchannel being smaller than the lysis product, thereby preventing the lysis product from passing through while allowing the lysis solution 10 to pass through.

[0082] The implementation methods of other filtering structures 11 can refer to the relevant existing technologies and will not be described in detail here.

[0083] Optionally, the collection chamber 7 may be formed with a detection port 23 communicating with the outside, and the cell lysis device may further include a destructible or movable sealing structure, which is disposed at the detection port 23. The detection port 23 may be used to conveniently detect the lysis product.

[0084] For a disposable cell lysis device, the sealing structure may be destructible, for example, a sealing film covering the detection port 23 may be used, and the lysis product may be detected by piercing the sealing film, and the use of the cell lysis device may be understood by observing the state of the sealing film.

[0085] For a reusable cell lysis device, the sealing structure may be movable, such as a sealing cap that can be twisted open, so that after the detection port 23 is opened, the sealing structure can be reset to reseal the detection port 23 for the next detection.

[0086] Optionally, a porous liquid storage material may be provided in the lysis chamber 9 , and the lysis liquid 10 is saturatedly absorbed in the porous material, so that cells can be lysed with less lysis liquid 10 .

[0087] Optionally, the power source of the cell lysis device can be a battery, or an external power source through a power interface 22. The cell lysis device provided by the present invention can be powered by a small battery or a low-voltage power interface because the amount of detection is a trace amount and the energy required is very low. The cell lysis device is also provided with a start switch 24 for the atomizer in the lysis chamber 9 to start and stop atomization lysis.

[0088] Back to Figure 2 and Figure 3 Optionally, the sample collection device 13 may include a substrate layer and a carrier layer, wherein a connection structure is provided on one side of the substrate layer, and the carrier layer is detachably provided on the other side of the substrate layer, and the carrier layer is provided with pores, the size of the pores being smaller than the size of the cell sample 3 to be collected.

[0089] The connection structure can be Velcro, double-sided tape, etc., and the substrate layer is arranged at a position suitable for collecting the cell sample 3 through the connection structure, for example, it can be attached to the surface of the sanitary napkin. The carrier layer with pores allows the aqueous substances in the female vaginal secretions to pass through and be absorbed by the sanitary napkin, and the pores of appropriate size can intercept the reproductive tract exfoliated cells flowing out with the vaginal secretions, while also ensuring a certain permeability. The carrier layer can be removed from the sanitary napkin after use, and as a carrier of the exfoliated cells, placed on the carrier of the cell lysis device 14 for lysis.

[0090] It is understandable that the vaginal secretions of adult women are a mixture of secretions from the Bartholin's glands, cervical glands, endometrium, vaginal mucosal exudates, and sloughed vaginal epithelial cells. Through sanitary napkins used by women, cells shed from the female reproductive tract can be collected, and the biomass in these cells can be used for in vitro diagnostic (IVD) home testing. For example, the test product can be used for home testing of high-risk HPV. In gynecological liquid-based cytology examinations, a sampling brush can also be used to collect sloughed cell samples and cervical secretions from the cervix.

[0091] Human papillomavirus (HPV) is a spherical DNA virus that is widely present in nature. It has humans as its only host. It is very resistant to drying and can be stored for a long time. Currently, there are only more than 150 known HPV subtypes, which can be divided into low-risk and high-risk types according to the severity of the diseases they cause. High-risk HPV can cause a variety of tumors, such as cervical cancer, anal cancer, and penile cancer. Studies have shown that E7 protein is an important cause of cervical epithelial cell disease (cervical cancer). E7 protein inhibits retinoblastoma protein (pRb) to enable cells to proliferate indefinitely. When the genetic tissue is damaged, E7 protein begins to express. Detecting E7 protein in female cervical exfoliated cells can qualitatively detect cervical cancer.

[0092] Since the abundance of E7 protein in cells is low in the early stage of HPV infection, conventional nucleic acid or PCR proliferation methods are required for accurate detection of early HPV infection; and some HPV home early screening products also have low detection accuracy due to low abundance. The method of using lysis solution atomization continuous flow can achieve room-temperature efficient lysis of female reproductive tract exfoliated cells collected on female sanitary napkins, while reducing the amount of lysis solution used, greatly reducing the dilution of E7 protein, and significantly improving the accuracy of detection.

[0093] In this scenario, the detection strip 15 is an E7 protein marker detection strip, which can be inserted into the collection chamber 7 through a pre-set detection port 23 and contacted with the lysate 10 to complete thin layer chromatography.

[0094] Furthermore, the E7 protein marker test strip can be a colloidal gold test strip or an ELISA test strip. In other words, it can be a colloidal gold test or an ELISA test, the difference being that the colloidal gold test is read by the naked eye, while the ELISA test requires a spectral detection device to read the result.

[0095] In order to further improve the collection effect, the carrier layer can be made of a hydrophobic material. In addition, the pore diameter of the carrier layer can be set to be no greater than 40 microns and no less than 15 microns.

[0096] Furthermore, the sample collection device 13 may include multiple carrier layers, each of which has a different pore diameter and is used to collect different cell samples, so that it can be used for a variety of tests.

[0097] For example, the pore diameter of the outermost carrier layer (hereinafter referred to as the first carrier layer) can be set to be no more than 40 microns and no less than 15 microns, and a second carrier layer can be further provided between the first carrier layer and the substrate layer, and the second carrier layer can be made of a hydrophilic material and have pores no less than 5 microns. These pores can separate and absorb other substances in female vaginal secretions as needed for other observations and detections. For example, the red blood cells of the menstrual blood of the female physiological period can be intercepted by the second carrier layer to observe the cell morphology, etc.

[0098] The concept and technical effects of the present invention can be further illustrated by the following specific embodiments.

[0099] Example 1: Device-based method of use

[0100] Cell lysis method using the device:

[0101] 1) Collection of reproductive exfoliated cells: Collect the reproductive exfoliated cells on the sanitary napkin or scrape the reproductive exfoliated cells after the sampler penetrates into the vagina, and contact the separation collection sheet with the sanitary napkin or brush (sampling carrier) carrying the reproductive exfoliated cells to transfer the reproductive exfoliated cells to the separation collection sheet. Install the separation collection sheet in the sample area of ​​the device.

[0102] 2) Nebulization lysis: Pour 1000ml RIPA lysis solution into the lysis solution storage chamber, cover the carrier material with the sample area, leave a distance between the sample area and the nebulizer (fixed by support rods or edge buckles, etc.), and turn on the nebulization mode with one button. The lysis solution is sprayed out and impacts the cells attached to the sample area, and then the liquid flows into the collection chamber. Part of the liquid in the collection chamber is filtered and re-enters the storage bin for recycling. The lysis product cannot be filtered and stays in the collection chamber. After 30 minutes of nebulization lysis, turn off the nebulizer and stop working.

[0103] The structure and use method of the device have been listed in detail in the above specific implementation methods, and those skilled in the art can adjust the relevant parameters according to actual needs.

[0104] Example 2 Comparison between atomization pyrolysis and conventional chemical pyrolysis

[0105] The mouse thoracic and abdominal aorta vascular tissue fragments were lysed for 30 minutes using 1000 μl RIPA chemical lysis solution in a room temperature atomization continuous flow (using the method in Example 1 of the present invention), and the lysis solution was circulated and the total protein in the final lysate was quantified by the Bradford method; the same mass of mouse thoracic and abdominal aorta vascular tissue fragments were resuspended in the same 1000 μl RIPA chemical lysis solution for lysis, first ice bathed for 30 minutes, and the cells were fully mixed by vortex oscillation every 3 minutes to fully lyse. Centrifuged at 4°C, 8000 r / min for 10 minutes, the lysate supernatant was collected, and the protein was quantified by the Bradford method.

[0106] The preparation method of mouse thoracic and abdominal aorta vascular tissue fragments is as follows: 2 ml of 10% chloral hydrate was injected intraperitoneally to anesthetize the mouse, and the mouse was immersed in 75% ethanol. The chest and abdominal cavities were opened under sterile conditions to expose the heart. The thoracic and abdominal organs were cut off in sequence until the aorta was exposed, and the aorta was completely separated and placed in a 35 mm sterile plate. 1 ml of sterile PBS (pH 7.2) was added and quickly transferred to the clean bench. The aorta was repeatedly rinsed with D-Hanks solution to remove residual blood, and the fat tissue outside the aorta was carefully peeled off with forceps, and the outer membrane was carefully peeled off until the blood vessels were smooth and transparent. The aorta was placed in DMEM solution (containing 4.5 g / L glucose, 10 mmol / L pyruvate, 2 mmol / L glutamine, 100 U / L penicillin and streptomycin, and 20% fetal bovine serum), and the aorta was cut longitudinally with ophthalmic scissors, and the intima was gently scraped 2 to 3 times with a blade to remove the intima. The stripped blood vessels were transferred to another 35 mm sterile dish containing 3 ml of DMEM / F12 culture medium containing 20% ​​FBS, and cut into pieces with ophthalmic scissors, with the size of the pieces being about 1 mm × 1 mm. RIPA lysis buffer: abs9228; active ingredients: 1 v / v% NP-40, 0.5 wt% sodium deoxycholate, 0.1 wt% SDS; protease inhibitors were added before use.

[0107] The results of protein quantification by Bradford method using the two lysis methods are shown in Table 1.

[0108] Table 1 Comparison of total protein content between atomized continuous flow lysis and conventional chemical lysis

[0109]

[0110] From the above results, it can be seen that both the room temperature atomization continuous flow method and the conventional laboratory chemical lysis method can lyse tissue cells very well, and the cell lysis rates are basically the same.

[0111] Example 3 Effect of different lysis liquid amounts on atomized continuous flow lysis

[0112] like Figure 4 and Figure 5 Different amounts of RIPA chemical lysis solution were absorbed into the porous material as an atomization source to lyse mouse thoracic and abdominal aorta vascular tissue fragments (same as in Example 2), and the lysis solution was circulated for 30 minutes. After the lysis was completed, the porous material was taken out, the lysis solution therein was squeezed out, and the total protein in the final lysate was quantified by the Bradford method.

[0113] RIPA lysis buffer: abs9228; active ingredients: 1 v / v % NP-40, 0.5 wt % sodium deoxycholate, 0.1 wt % SDS; add protease inhibitors before use

[0114] The results of using different lysis buffer dosages were compared, and the results are shown in Table 2.

[0115] Table 2 Comparison of total protein content in different lysis buffer dosages

[0116]

[0117] The above results show that atomized continuous flow pyrolysis can increase the abundance of target biomass by reducing the amount of pyrolysis solution.

[0118] Example 4 HPV simulation detection

[0119] Ordinary commercially available HELA human cervical cancer cells and normal human cervical epithelial cells were used, and cell recovery and cell passage were performed according to the product instructions. When the cells were in good growth condition, a certain amount of samples were collected and placed in the sample chamber, and lysed with 500 microliters of lysis solution for 30 minutes. Different batches of HELA human cervical cancer cells and normal human cervical epithelial cells were taken and measured 10 times, and the sample numbers were recorded as P1~P10 and N1~N10 respectively.

[0120] RIPA lysis buffer: abs9228; active ingredients: 1 v / v % NP-40, 0.5 wt % sodium deoxycholate, 0.1 wt % SDS; add protease inhibitors before use

[0121] After the lysis was completed, a conventional commercially available HPV colloidal gold test strip was inserted into the detection hole for testing, and the test results were compared with those of the conventional laboratory chemical lysis method combined with the test results of the same brand and batch of commercially available HPV colloidal gold test strips (each sample was tested once using both methods). The test results were all consistent with those using the conventional laboratory chemical lysis method (Table 3).

[0122] Table 3

[0123]

Claims

1. A cell lysis device, characterized in that: The cell lysis device comprises: A lysis chamber, the lysis chamber is used to store lysis solution; an atomizer, the atomizer comprising a liquid inlet end and an atomizing end, the liquid inlet end being in communication with the lysis chamber, the atomizing end being used to spray the lysis liquid into a jet; A carrier, wherein a space for forming a jet is provided between the carrier and the atomization end, and the carrier is used to carry a cell sample; A substrate layer, one side of which is provided with a connection structure connected to the carrier; A carrier layer, the carrier layer is detachably arranged on the other side of the substrate layer, the carrier layer is provided with pores, and the size of the pores is smaller than the size of the cell sample to be collected; The cell lysis device further comprises a collection chamber, the carrier comprises a sample portion and a flow guide portion connected to each other, the lysis solution is sprayed to the sample portion through the atomization end and the spacing space, the cell sample is arranged in the sample portion, and the flow guide portion is used to guide the lysis solution and the lysis product to flow into the collection chamber; The surface of the carrier is a hydrophobic material; The collecting chamber comprises a collecting end, the sample portion is higher than the collecting end, and the flow guide portion extends obliquely downward from the sample portion toward the collecting end; The collecting chamber and the cracking chamber are communicated with each other, and a filtering structure capable of blocking the cracking product is arranged between the collecting chamber and the cracking chamber.

2. The cell lysis device according to claim 1, characterized in that: The collection chamber is provided with a detection port for inserting a detection test paper, and the cell lysis device further comprises a destructible or movable sealing structure, and the sealing structure is arranged at the detection port.

3. A cell lysis method, characterized in that: The following steps are involved: The cell sample is fixed on the surface of the stage, the lysate is atomized and impacted on the cells, and the cell lysate is collected; Methods for atomizing the lysate include ultrasonic atomization; The cell lysis method is performed using the cell lysis device according to claim 1 or 2.

4. The cell lysis method according to claim 3, characterized in that: The lysate includes at least one of RIPA lysate, SDS lysate, NP40 lysate, Tris-Triton lysate, ACK lysate, Urea lysate, and Tris-HCl lysate.

5. Use of the cell lysis device according to any one of claims 1 to 2 in the preparation of detection products; The product is used to detect at least one of RNA, DNA, and protein.

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