SPR (Surface Plasmon Resonance) microfluidic chip and detection device
By designing the parallel runner and docking boss structure of the SPR microfluidic chip, real-time and in-situ measurement of cell membrane protein binding to antibodies is achieved, solving the complexity and non-real-time measurement of traditional methods, and improving detection efficiency and accuracy.
Patent Information
- Application Number
- CN202422226268.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Utility models(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-11
- Publication Date
- 2025-07-08
- Estimated Expiration
- 2034-09-11
AI Technical Summary
Existing SPR devices require membrane protein expression and purification when detecting the binding activity of antibodies to membrane proteins. The process is complex and time-consuming, and it is impossible to determine the binding situation in real time. The traditional ELISA method can only be used to determine the endpoint and cannot be monitored in real time.
A SPR microfluidic chip is designed, including parallel runners and docking boss structures, combined with a multi-channel microfluidic chip to achieve real-time and in-situ determination of the binding of cell membrane proteins to antibodies, and directly use cells to perform measurements to avoid allosteric and activity losses of membrane proteins.
It improves the measurement efficiency and accuracy, and can simultaneously determine the binding activity of multiple drugs or under different concentration conditions, realize high-throughput detection, ensure the sealing of the microfluidic chip and the fluid control module, and avoid the allostericity and activity loss of membrane proteins.
Smart Images
Figure CN223078186U_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of experimental detection, and more specifically, to a SPR microfluidic chip and a detection device. Background Art
[0002] According to the difference in the excitation resonance mode, the types of surface plasmon resonance sensors are mainly divided into prism type, waveguide type and grating type, that is, three coupling methods. Among them, most SPR devices use prism coupling for incident light because its production is relatively simple, but there are also limitations. For example, it is difficult to directly deposit a metal thin film on the prism, and when detecting biological samples, it is necessary to adjust the angle of the incident light, which increases the volume of the instrument and is not conducive to the development of miniaturization.
[0003] When using traditional equipment to detect the binding activity of antibodies and membrane proteins, it is necessary to express and purify the membrane protein before measuring the binding ability of the membrane protein and the antibody. This is not only complex and time-consuming, but may also cause allosteric changes and activity loss of some membrane proteins. Another traditional detection method is to use endpoint methods such as ELISA, that is, to test the endpoint result of the binding of cells and monoclonal antibodies (using the depth of color to represent different binding abilities), but it cannot measure the binding situation in real time. Summary of the Invention
[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a SPR microfluidic chip and a detection device; by improving the specific structure of the microfluidic chip, the present invention can realize real-time and in-situ determination of the binding of cell membrane proteins and antibody drugs, and there is no need to express and purify the membrane protein. Instead, cells are directly used for determination, avoiding allosteric changes and activity loss of the membrane protein.
[0005] The first aspect of the present application discloses a SPR microfluidic chip. The microfluidic chip is provided with a flow channel. The two ends of a single flow channel are respectively provided with a first microtube and a second microtube. The first microtube and the second microtube are respectively provided with an inlet end and an outlet end. The outlet end of the first microtube and the inlet end of the second microtube are connected through the flow channel; the inlet end of the first microtube and the outlet end of the second microtube are located on the first surface of the microfluidic chip, and the outlet end of the first microtube and the inlet end of the second microtube are located on the second surface of the microbody chip.
[0006] In some embodiments, the flow channel is a groove provided on the upper surface of the microfluidic chip.
[0007] In some embodiments, the flow channels are provided in several numbers, and the several flow channels are not connected to each other.
[0008] In some embodiments, the line connecting the centers of the first microtube and the second microtube in a single flow channel is used as the center line of the single flow channel, and the center lines of the several flow channels are parallel to each other.
[0009] In some embodiments, the number of the first microtubes is consistent with the number of the flow channels; the number of the second microtubes is consistent with the number of the flow channels.
[0010] In some embodiments, docking bosses are respectively arranged at the inflow end of the first microtube and the outflow end of the second microtube, and a lumen for the suspension to flow is left in the middle of the docking bosses.
[0011] The second aspect of the present application discloses an SPR microfluidic detection device, including a light source. The detection device further includes: a microfluidic chip as described in the first aspect of the present application, and a gold film layer. The flow channel is coupled with the gold film layer, and the side where the gold film layer is located faces the light source.
[0012] In some embodiments, the detection device further includes a second fluid control sub-module tightly connected to the docking boss; a first channel communicating with the first microtube and a second channel communicating with the second microtube are arranged inside the second fluid control sub-module;
[0013] Optionally, the detection device further includes a first fluid control sub-module arranged at the upper end of the second fluid control sub-module. The first fluid control sub-module includes a first connecting pipe communicating with the first microtube and a second connecting pipe communicating with the second microtube; the microfluid sequentially passes through the first connecting pipe, the first channel, the first microtube, the flow channel, the second microtube, the second channel, and returns to the second connecting pipe to enter the external structure;
[0014] Optionally, both the first connecting pipe and the second connecting pipe are communicated with a multi-channel injection pump, and the external structure is a multi-channel injection pump;
[0015] Optionally, the sizes of the first microtube, the second microtube and the flow channel are larger than the sizes of the cells in the microfluid.
[0016] In some embodiments, the detection device further includes a detection chip arranged and connected to the gold film layer.
[0017] In some embodiments, a prism is arranged on the side of the gold film layer facing the light source;
[0018] Optionally, the gold film layer is a substrate material for drug fixation and cell binding, and the gold film is coupled with an SPR detection device to excite the SPR phenomenon;
[0019] Optionally, the light source is the optical path of an SPR detection device, and the gold film layer is coupled with the optical path.
[0020] The present application has the following beneficial effects:
[0021] 1. This application innovatively discloses an SPR microfluidic chip. By setting a number of parallel flow channels and respectively arranging a first microtube and a second microtube at both ends of a single flow channel, the measurement of the specific binding activity between monoclonal antibodies and cell membrane receptor proteins effectively improves the measurement efficiency and accuracy. At the same time, through the multi-channel microfluidic chip method, the binding activities under different concentrations of multiple different drugs or the same drug can be measured simultaneously, and the detection efficiency is improved through high-throughput detection means.
[0022] 2. This application innovatively arranges docking bosses at the inflow end of the first microtube and the outflow end of the second microtube respectively, and the docking bosses are pressed tightly against the second fluid control sub-module; since the material of the microfluidic chip is PDMS (polydimethylsiloxane) polymer, the design of using bosses in the combined docking structure can increase the local pressure between the microtube and the second fluid control sub-module, thereby ensuring the tightness between the microfluidic chip and the second fluid control sub-module.
[0023] In summary, the SPR microfluidic chip and detection device disclosed in this application, by optimizing and improving the microfluidic chip, effectively solve the current situation that traditional methods and devices cannot measure the binding situation in real time when measuring the binding ability between membrane proteins and antibodies (the main feature of traditional equipment is that membrane proteins need to be expressed and purified before measuring the binding ability between membrane proteins and antibodies; another traditional method is to use endpoint methods such as ELISA, that is, to test the endpoint results of the binding of cells to monoclonal antibodies (using the depth of color to represent different binding abilities), but the binding situation cannot be measured in real time), realize real-time and in-situ measurement of the binding activity between cell membrane proteins and antibody drugs, without the need for membrane protein expression and purification, directly using cells for measurement, and avoiding conformational changes and activity loss of membrane proteins. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those skilled in the art, without creative efforts, other drawings can be obtained based on these drawings.
[0025] Figure 1 It is a schematic structural diagram of the microfluidic chip provided in the first aspect of the embodiment of the present invention;
[0026] Figure 2 It is a schematic structural diagram of the detection device provided in the second aspect of the embodiment of the present invention;
[0027] Figure 3 It is a schematic diagram of the binding between cells on the surface of the SPR detection chip and monoclonal antibodies provided in the embodiment of the present invention;
[0028] Figure 4 It is a schematic diagram of the refractive index change during the cell binding process measured at refractive indices of 52°, 54°, and 56° provided by an embodiment of the present invention;
[0029] In the figure, 30 is a detection chip; 31 is a first fluid control sub-module; 32 is a second fluid control sub-module; 33 is a gold film layer; 40 is a microfluidic chip; 41 is a docking boss; 421 is a first microtube; 422 is a second microtube; 43 is a flow channel. Detailed implementation manners
[0030] The technical solutions in the embodiments of the present invention are clearly and completely described below through specific specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation manners. Without conflict, the features in the following embodiments and the embodiments can be combined with each other. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.
[0031] The embodiments of the present disclosure and their examples are described in detail below with reference to the accompanying drawings.
[0032] Glossary:
[0033] Surface Plasmon Resonance (SPR): It is a physical optical phenomenon. When a laser with a specific wavelength is incident on a gold film at a specific angle, the coupling of the free electrons of the gold film and the evanescent wave will occur at the solid-liquid interface on the other side of the gold film, resulting in changes in the light intensity and phase of the reflected light corresponding to the incident light. The refractive index of the solid-liquid interface on the side of the gold film and the solution affects the properties of the reflected light. Therefore, the refractive index information of the solid-liquid interface can be obtained by solving the relevant information of the reflected light. The detection method based on the SPR principle can detect the dynamic binding process of antibodies near the gold film and cell membrane protein receptors without labeling and in real time, and obtain accurate and sensitive binding and dissociation curves.
[0034] Figure 1 It is a schematic diagram of the structure of the microfluidic chip provided in this embodiment. This specific implementation manner provides a SPR microfluidic chip;
[0035] For example, as Figure 1As shown, in one embodiment, the microfluidic chip 40 is provided with a flow channel 43. At both ends of a single flow channel 43, a first microtube 421 and a second microtube 422 are respectively arranged. The first microtube 421 and the second microtube 422 are respectively provided with an inlet end and an outlet end. The outlet end of the first microtube 421 and the inlet end of the second microtube 422 are connected through the flow channel 43; the inlet end of the first microtube 421 and the outlet end of the second microtube 422 are located on the first surface of the microfluidic chip 40, and the outlet end of the first microtube 421 and the inlet end of the second microtube 422 are located on the second surface of the microbody chip.
[0036] In some embodiments, the flow channel 43 is a groove provided on the upper surface of the microfluidic chip 40.
[0037] In some embodiments, a plurality of flow channels 43 are provided, and the plurality of flow channels 43 are not connected to each other.
[0038] In some embodiments, the line connecting the centers of the first microtube 421 and the second microtube 422 in a single flow channel 43 serves as the center line of the single flow channel 43, and the center lines of the plurality of flow channels 43 are parallel to each other.
[0039] In some embodiments, the number of the first microtubes 421 is the same as that of the flow channels 43; the number of the second microtubes 422 is the same as that of the flow channels 43.
[0040] In some embodiments, docking bosses 41 are respectively arranged at the inlet end of the first microtube 421 and the outlet end of the second microtube 422. A lumen for the suspension to flow is left in the middle of the docking bosses 41. Optionally, the docking bosses 41 are cylinders.
[0041] Figure 2 It is a schematic structural diagram of the detection device provided in this embodiment. The detection device includes a light source. The detection device further includes: the microfluidic chip 40 as described in the first aspect of the present application, and a gold film layer 33. The flow channel 43 is coupled with the gold film layer 33, and the side where the gold film layer 33 is located faces the light source.
[0042] In some embodiments, the detection device further includes a second fluid control sub-module 32 that is tightly connected to the docking boss 41; a first channel communicating with the first microtube 421 and a second channel communicating with the second microtube 422 are arranged inside the second fluid control sub-module 32;
[0043] Optionally, the detection device further includes a first fluid control sub-module 31 disposed at the upper end of the second fluid control sub-module 32. The first fluid control sub-module 31 includes a first connection pipe communicating with the first microtube 421 and a second connection pipe communicating with the second microtube 422. The microfluid sequentially passes through the first connection pipe, the first channel, the first microtube 421, the flow channel 43, the second microtube 422, and the second channel, and returns to the second connection pipe to enter the external structure.
[0044] Optionally, both the first connection pipe and the second connection pipe are connected to a multi-channel injection pump, and the external structure is a multi-channel injection pump.
[0045] Optionally, the sizes of the first microtube 421, the second microtube 422, and the flow channel 43 are larger than the size of the cells in the microfluid. The microtubes and the flow channel 43 are about one order of magnitude higher than the size of the cells, facilitating the effective passage of the cells and avoiding blockage. The interface of the flow channel 43 is rectangular, and the interface of the microtube is square.
[0046] In some embodiments, the detection device further includes a detection chip 30 disposed and connected to the gold film layer 33.
[0047] In some embodiments, a prism is disposed on the side of the gold film layer 33 facing the light source; the gold film and the prism are an integral structure. Refer to Figure 3 , the gold film exists in the form of a coating on the upper surface of the prism. The prism is coupled with the optical path in the SPR excitation and detection optical paths. The incident light is incident from one side of the prism, reflected by the gold film, and the SPR phenomenon occurs, and exits from the other side of the prism. In a circuit, coupling means connection, that is, sending the signal of the previous stage to the next stage. Sometimes, they cannot be directly connected together, so electronic components are needed for connection. If there is no coupling capacitor for direct connection, the DC paths of the front and rear stages will be connected to each other, affecting their respective static operating points and prone to zero drift.
[0048] Optionally, the gold film layer 33 is a substrate material for drug fixation and cell binding. The gold film is coupled with the SPR detection device to excite the SPR phenomenon.
[0049] Optionally, the light source is the optical path of the SPR detection device, and the gold film layer 33 is coupled with this optical path.
[0050] Refer to Figure 1 and Figure 2 , in the device for measuring the specific binding activity of monoclonal antibodies to cell membrane receptor proteins in this specific embodiment, the microfluidic chip 40 part is shown in the figure. Specifically, refer to Figure 2, the left side is a component diagram and the right side is an exploded view. Specifically from top to bottom in the left diagram are the first fluid control sub-module 31, the second fluid control sub-module 32, and the detection chip 30. Specifically from top to bottom in the right diagram are the first fluid control sub-module 31, the second fluid control sub-module 32, the microfluidic chip 40, the gold film, and the detection chip 30; for the specific structure of the microfluidic chip 40, refer to Figure 1 , the first microtube 421 and the second microtube 422 are respectively located at both ends of the single flow channel 43. Figure 1 Thirteen single flow channels 43 are drawn in Figure 1 The upper end of the microfluidic chip 40 in Figure 1 is the second surface of the microfluidic chip 40, and the lower end of the microfluidic chip 40 in
[0051] Figure 4 is the first surface of the microfluidic chip 40; in the embodiments of the present invention, the binding activity assay device includes three parts: an SPR detection module, a detection chip 30, and a flow path module.
[0052] SPR detection module, the SPR module implements the SPR detection method, and realizes the real-time and in-situ detection of the binding of cells and proteins on the gold film by means of phase detection and dual-CCD differential phase interference imaging, and forms a cell membrane protein-antibody binding curve; the SPR detection module realizes the SPR detection method, and realizes the real-time and in-situ detection of the binding of cells and proteins on the gold film by means of phase detection and dual-CCD differential imaging. Among them, when the detection module excites the SPR phenomenon, a 632 nanometer (nm) laser is incident on the detection chip 30, and the incident laser within a certain range can excite the SPR phenomenon. Generally, sensitive phase detection of the refractive index can be achieved within the range of 52° to 56°. Further, for the detection of the cell binding process, since the refractive index of the culture medium and extracellular fluid is about 1.35 RIU, the refractive index of intracellular fluid is about 1.37 RIU, and the refractive index of the cell membrane is about 1.46 RIU, and the thickness of the cell membrane is relatively small and can be approximately ignored. Considering comprehensively, SPR can mainly distinguish the refractive index difference between intracellular fluid and extracellular fluid. The incident angle is preferably set at 56°, and the refractive index change brought about by the cell binding process can be measured most sensitively. The specific refractive index change is as
[0053] shown.Flow path module, which includes a fluid control sub-module and a microfluidic chip 40. The fluid control sub-module is connected to a multi-channel programmable injection pump and is also connected to the microfluidic chip 40. The microfluidic chip 40 includes a docking boss 41 to ensure the tightness of the docking with the fluid control sub-module, and includes a number of parallel flow channels 43 to achieve the fixation of antibodies in a specified area and the binding of cells to antibodies. A certain concentration of antibody or cell suspension enters the microfluidic chip 40 through the fluid control sub-module, passes through a microtube and enters the flow channel 43. The flow channel 43 is tightly coupled with the gold film area of the detection chip 30. Finally, the microfluidic flows through the SPR detection chip 30 to achieve the detection functions of antibody fixation and cell binding.
[0054] Among them, the basic principle of fluid control is to utilize the dynamic characteristics of the fluid. By designing and controlling the shape, structure, position and control methods of components such as the flow channel 43, valves, and pumps, the fluid is made to move along a predetermined path and at a predetermined speed. Commonly used control parameters include flow rate, pressure, speed, state, etc.
[0055] Refer to Figure 3 , the binding of HUT-78 cells to vedolizumab is shown schematically as Figure 3 shown. The HUT-78 cells flow into the microchannel 43 with the microfluidic flow, approach the vedolizumab fixed on the gold film surface of the SPR sensing chip, and specifically bind to the α4β7 receptor protein on the cell membrane of the HUT-78 cells, causing the cells to approach the gold film surface and enter the sensitive range of the SPR method, so that they can be dynamically observed by the SPR detection method.
[0056] The usage method is as follows:
[0057] A method and device for measuring the specific binding activity of a monoclonal antibody to a cell membrane receptor protein, which can measure the specific binding activity of the HUT-78 cell membrane protein receptor to the vedolizumab monoclonal antibody under the condition of intact cells.
[0058] In the specific implementation process, the method for measuring the binding activity includes five parts: chip pretreatment, antibody fixation, cell binding, determination of the binding curve, and signal analysis and calculation of the binding activity. Chip pretreatment: The chip used for antibody fixation and cell binding is coated with carboxyl groups and activated. Antibody fixation method: Different concentrations of antibodies are fixed on the activated chip. Cell binding: The cells flow over the antibody surface and specifically bind. Signal analysis: Analyze the measured SPR curve to calculate the specific binding activity of the membrane protein receptor to the antibody.
[0059] The following points need to be explained:
[0060] (1) The drawings of the embodiments of the present disclosure only involve the structures related to the embodiments of the present disclosure, and other structures can refer to the general design.
[0061] (2) Without conflict, the embodiments of the present disclosure and the features in the embodiments may be combined with each other to obtain new embodiments.
[0062] As described above, it is only the specific implementation manner of the present disclosure, but the protection scope of the present disclosure is not limited thereto. The protection scope of the present disclosure shall be subject to the protection scope of the claimed rights.
[0063] The description of the above embodiments is only for understanding the present invention. It should be noted that for those of ordinary skill in the art, several improvements can be made to the present invention without departing from the principle of the present invention, and these improvements will also fall within the protection scope of the claims of the present invention.
Claims
1. An SPR microfluidic chip, characterized in that, The microfluidic chip is provided with flow channels. The two ends of a single flow channel are respectively provided with a first microtube and a second microtube. The first microtube and the second microtube are respectively provided with an inlet end and an outlet end. The outlet end of the first microtube and the inlet end of the second microtube are communicated through the flow channel. The inlet end of the first microtube and the outlet end of the second microtube are located on the first surface of the microfluidic chip, and the outlet end of the first microtube and the inlet end of the second microtube are located on the second surface of the microbody chip.
2. The SPR microfluidic chip according to claim 1, wherein The flow channel is a groove provided on the upper surface of the fluid pool.
3. The SPR microfluidic chip according to claim 1, characterized in that, A plurality of flow channels are provided, and the plurality of flow channels are not communicated with each other.
4. The SPR microfluidic chip according to claim 3, wherein The line connecting the centers of the first microtube and the second microtube in a single flow channel is used as the center line of the single flow channel, and the center lines of the plurality of flow channels are parallel to each other.
5. The SPR microfluidic chip according to claim 1, characterized in that, The number of the first microtubes is consistent with the number of the flow channels; the number of the second microtubes is consistent with the number of the flow channels.
6. The SPR microfluidic chip according to claim 1, characterized in that, The inlet end of the first microtube and the outlet end of the second microtube are respectively provided with docking bosses, and a lumen for the suspension or solution to flow is left in the middle of the docking bosses.
7. An SPR microfluidic detection device, comprising a light source, characterized in that, The detection device further includes: the microfluidic chip according to any one of claims 1-6, and a gold film layer. The flow channel is coupled with the gold film layer, and the side where the gold film layer is located faces the light source.
8. The SPR microfluidic detection device according to claim 7, wherein The detection device further includes a second fluid control sub-module tightly connected to the docking boss; a first channel communicating with the first microtube and a second channel communicating with the second microtube are provided inside the second fluid control sub-module. Optionally, the detection device further includes a first fluid control sub-module provided on the upper end of the second fluid control sub-module. The first fluid control sub-module includes a first connecting tube communicating with the first microtube and a second connecting tube communicating with the second microtube. The microfluid flows sequentially through the first connecting tube, the first channel, the first microtube, the flow channel, the second microtube, the second channel, and returns to the second connecting tube and enters the external structure. Optionally, both the first connecting tube and the second connecting tube are communicated with a multi-channel injection pump, and the external structure is a multi-channel injection pump. Optionally, the sizes of the first microtube, the second microtube and the flow channel are larger than the sizes of the cells in the microfluid.
9. The SPR microfluidic detection device according to claim 7, characterized in that, The detection device further includes a detection chip connected to the gold film layer.
10. The SPR microfluidic detection device according to claim 7, characterized in that, A prism is provided on the side of the gold film layer facing the light source. Optionally, the gold film layer is a base material for drug fixation and cell binding. The gold film is coupled with an SPR detection device to excite the SPR phenomenon. Optionally, the light source is the optical path of the SPR detection device, and the gold film layer is coupled with the optical path.