Apparatus and methods for protein sample solution replacement or protein separation

Abstract

This application discloses an apparatus and method for protein sample solution replacement or protein separation. The apparatus includes a microfluidic chip, a sample cell, and an electroosmotic module. The microfluidic chip has a cavity structure with elongated ends and a central through-hole. Target background solution inlets and outlets are located at both ends of the microfluidic channel, and the central through-hole accommodates the sample cell and the electroosmotic module. The sample cell fits tightly with the top cavity of the central through-hole, and its contact portion with the target background solution is a semi-permeable membrane structure. The electroosmotic module fits tightly with the lower cavity of the central through-hole and is positioned below the sample cell. This application combines electrophoresis, a semi-permeable membrane, and electroosmosis to achieve rapid and efficient solution replacement or protein separation without introducing mechanical force, enabling the migration of non-volatile salt ions and neutral small molecules. This application utilizes a microfluidic chip for solution replacement or protein separation, facilitating automated control and exhibiting good online integration compatibility.

Description

Technical Field

[0001] This application relates to the field of protein detection technology, and in particular to an apparatus and method for replacing protein sample solutions or separating proteins. Background Technology

[0002] Proteins play vital functional roles in life activities. The biological function of proteins is closely related to their structure, and understanding protein function relies on analyzing their multi-level structures. Solution replacement of protein samples is a common step in protein analysis pretreatment, and it is essential for analytical methods such as mass spectrometry. Mass spectrometry is a tool for measuring the molecular weight of chemical substances. Due to its ultra-high sensitivity and resolution, as well as low sample consumption, it is widely used in the characterization of biological macromolecules. Expression and purification proteins are usually in non-volatile salt ion buffers. These non-volatile salt ions can severely inhibit protein ionization and detection, and can also contaminate the mass spectrometer. Therefore, in the pretreatment for protein mass spectrometry analysis, non-volatile salts and other small molecules that may cause signal interference in the protein solution must be replaced with mass spectrometry-compatible volatile salts. Besides mass spectrometry, solution replacement of protein samples is also commonly used in other analytical methods to remove interfering or contaminating small molecules, or to change solution conditions.

[0003] Ultrafiltration based on semipermeable membranes has become the most common method for ion exchange of biological macromolecules in recent years. Under the mechanical force generated by high-speed centrifugation, small molecules such as salts are largely removed along with the original protein solution through the semipermeable membrane, while larger proteins are retained within the membrane. Subsequently, a solution containing the target component is added to the sample, achieving a certain ratio of replacement between the original and target solutions. Repeating this process multiple times achieves a large-scale replacement. This method is relatively cumbersome and time-consuming, difficult to automate, and the mechanical force generated by high-speed centrifugation and the sudden changes in the solution environment caused by the introduction of the target solution can lead to protein sample loss due to degradation and aggregation. Dialysis, also based on semipermeable membranes without applying additional mechanical force, is a commonly used technique for solution replacement and protein purification and concentration. The original protein solution is placed inside a semipermeable dialysis bag, while the target solution is placed outside. The substances in both the original and target solutions diffuse due to their different concentrations inside and outside the bag until the concentrations reach equilibrium; the protein itself remains inside the dialysis bag. A large-scale solution replacement can be achieved by continuously replenishing the target solution outside the bag. Due to the lack of mechanical assistance, this method is more time-consuming. Solid-phase extraction (SPE) selectively retains proteins through hydrophobic interactions, requiring organic eluents for protein recovery. This introduces denaturation conditions, leading to changes in the higher-order structure of proteins and even aggregation, making it incompatible with non-denaturing analyses and causing sample loss. Solution replacement methods based on size exclusion chromatography (SEC), gel permeation chromatography (GPC), or ion exchange chromatography (IEX) also require mechanical assistance and often necessitate large volumes of the target mobile phase, resulting in sample dilution and hindering subsequent high-sensitivity analysis. Electrophoresis, under the influence of an electric field, utilizes the difference in electrophoretic mobility between different molecules to achieve separation, allowing proteins to leave the original solution and enter the target solution. Common electrophoresis application schemes include slab gel electrophoresis and capillary electrophoresis. The former is difficult to efficiently recover proteins under non-denaturing conditions, while the latter, due to its low flow rate, is difficult to effectively collect proteins from the target solution offline and is also difficult to directly integrate with online mass spectrometry analysis using a universal protocol.

[0004] Furthermore, whether it's biological samples extracted from bacteria or cells, or large quantities of commercially available sample solutions, proteins are preserved in complex background solutions to maintain their biological activity and physiological function. These solutions not only contain a large amount of non-volatile salt ions but also some uncharged neutral small molecules, all of which severely interfere with the mass spectrometry signal of the target sample. In particular, neutral small molecules are difficult to remove, and ion migration is restricted.

[0005] Therefore, how to quickly and effectively replace protein sample solutions and remove neutral small molecules while avoiding protein degradation and denaturation remains a key research focus in this field. Summary of the Invention

[0006] The purpose of this application is to provide a new apparatus and method for replacing protein sample solutions or separating proteins.

[0007] To achieve the above objectives, this application adopts the following technical solution:

[0008] One aspect of this application discloses a device for replacing or separating protein sample solutions, comprising a microfluidic chip, a sample cell for holding the protein sample solution to be processed, and an electroosmotic module. The microchannel of the microfluidic chip has a cavity structure with elongated ends and a through-hole in the middle. Through-holes are respectively opened at both ends of the microchannel, serving as the inlet and outlet of the target background solution. The through-hole in the middle accommodates the sample cell and the electroosmotic module. The sample cell fits tightly with the top cavity of the through-hole in the microchannel, and the part of the sample cell in contact with the target background solution in the microchannel is a semi-permeable membrane structure. The electroosmotic module fits tightly with the lower middle cavity of the through-hole in the microchannel. In use, the electroosmotic module is placed below the sample cell and directly contacts the target background solution in the microchannel. The elongated ends and through-hole structure, for example, is a spindle-shaped structure in one implementation of this application; however, other shapes with elongated ends and a through-hole in the middle are also possible. In this application, when replacing a protein sample solution, the target background solution in the microchannel is the target solution to be replaced; when separating or purifying a protein, the target background solution in the microchannel is the original sample solution of the protein sample to be processed or a similar protein dissolution solution; therefore, the target background solution in this application refers to other solution components besides protein in the solution to be replaced or the protein sample solution, such as buffer solutions, salt solutions without buffering effect, pure solvents or mixed solvents, etc.

[0009] It should be noted that the protein sample solution replacement or protein separation device of this application connects positive and negative electrodes to the inside and outside of the sample cell, respectively, combining electrophoresis, a semi-permeable membrane, and electroosmosis to achieve rapid and efficient solution replacement without introducing mechanical force. In particular, it utilizes electroosmosis to facilitate the migration of non-volatile salt ions and neutral small molecules, solving the problem of the difficulty in removing neutral small molecules. Furthermore, the microfluidic technology using microfluidic chips to construct a microscale replacement device enables automated control and has compatibility with other pretreatment modules or online integration with subsequent detection.

[0010] It should also be noted that, in addition to solution replacement of proteins, the device of this application can also separate or purify proteins of different sizes by utilizing the semi-permeable membrane structure of the sample cell; therefore, the device of this application can also be used as a protein separation or purification device.

[0011] In one implementation of this application, the electroosmotic module is a membrane or plate with microporous channels, or the electroosmotic module is an array structure composed of capillaries with an inner diameter at the micrometer or millimeter level.

[0012] It should be noted that the key to this application lies in generating electroosmosis using an electroosmotic flow module. This involves using a porous support in an electric field to adsorb positive and negative ions from the water, making the solution relatively charged. Under the influence of the electric field, the solution moves in a certain direction. Therefore, the electroosmotic flow module of this application can refer to porous supports capable of generating electroosmosis used in the prior art, including but not limited to membranes or plates with microporous channels. Alternatively, the electroosmotic flow module can be an array structure composed of capillaries with inner diameters at the micrometer or millimeter level. For example, the array structure composed of several capillaries of the same height bundled together can serve as the electroosmotic flow module.

[0013] In one implementation of this application, the electroosmotic module is made of a material containing silicon dioxide.

[0014] In one implementation of this application, the material containing silicon dioxide is silicone, quartz, or glass.

[0015] It should be noted that the key to this application is the combination of an electroosmotic flow module capable of generating electroosmosis with electrophoresis and a semi-permeable membrane. The porous support material used as the electroosmotic flow module includes, but is not limited to, silicone, quartz and glass.

[0016] In one implementation of this application, the electroosmotic flow module is at least one of the following structures.

[0017] (1) Silicone with microporous channels; for example, silicone membrane;

[0018] (2) Hollow glass tube array; for example, hollow glass tube bundle;

[0019] (3) Fused silica capillary array; for example, capillary bundle;

[0020] (4) Hollow glass tube array with glass or quartz wire inserted into the internal channel;

[0021] (5) A glass plate with a microporous channel array; for example, a microporous glass plate.

[0022] It is understood that electroosmotic flow, or electroosmotic effect, is essentially the application of voltage across a porous medium, microchannel, or other fluid conduit to induce fluid flow. Therefore, the electroosmotic flow module of this application only needs to have a porous medium, microchannel, or other fluid conduit, including but not limited to silicone membranes, glass tube bundles, glass capillary bundles, or microporous glass plates.

[0023] In one implementation of this application, the pore size of the micropore channels in the silicone or glass plate is at the micrometer or millimeter level.

[0024] Preferably, the pore size of the micropore channel in the silicone or glass plate is 0.1 mm. For example, in one implementation of this application, the pore size of both the silicone film micropore channel and the glass plate micropore channel is 0.1 mm.

[0025] Preferably, the inner diameter of the fused silica capillary is less than or equal to 1 mm.

[0026] Preferably, the inner diameter of the fused silica capillary is 0.1 mm.

[0027] Preferably, the inner diameter of the hollow glass tube is 0.6-0.7 mm.

[0028] Preferably, the diameter of the glass or quartz wire is 0.1 mm.

[0029] It should be noted that the specific dimensions of the silicone membrane, glass tube, glass fiber, quartz fiber, glass capillary, and glass plate mentioned above are only the dimensions specifically used in one implementation of this application. Under the same inventive concept, appropriate adjustments can be made based on the above dimensions. For example, the pore diameter of the micropore channel or the inner diameter of the capillary can be in the range of a few micrometers to a few millimeters, and no specific limitation is made here.

[0030] In one implementation of this application, the device for replacing or separating protein sample solutions further includes a circuit assembly, which includes a conductive electrode and a power supply. In use, the conductive electrode of the positive terminal of the power supply is fixed in the sample cell and in contact with the protein sample solution to be processed, while the conductive electrode of the negative terminal of the power supply is fixed at the target background solution inlet or target background solution outlet of the microfluidic chip.

[0031] In one implementation of this application, the apparatus for replacing or separating protein sample solutions further includes a target background solution component. The target background solution component includes a liquid propulsion device for regulating the flow rate of the target background solution in the microchannels of the microfluidic chip. In use, the liquid propulsion device is connected to the target background solution inlet of the microfluidic chip. The liquid propulsion device includes, but is not limited to, an injection pump.

[0032] In one implementation of this application, the target background solution component further includes a waste liquid tank; in use, the waste liquid tank is connected to the target background solution outlet of the microfluidic chip.

[0033] It should be noted that the circuit components of this application, such as conductive electrodes and power supplies, can all be obtained from conventional laboratory electrophoresis equipment; similarly, the target background solution components, such as syringe pumps and waste liquid tanks, can also be obtained from conventional laboratory injection equipment. It is understood that the key components of this application are the microfluidic chip, sample cell, and electroosmotic module; other components can be selectively combined into the device used for protein sample solution replacement or protein separation, depending on the requirements.

[0034] In one implementation of this application, the microfluidic chip is made of polydimethylsiloxane and carried on a glass slide.

[0035] It should be noted that dimethylsiloxane is only one specific microfluidic chip fabrication material used in this application, and it is possible that other conventional microfluidic chip fabrication materials can also be used.

[0036] In one implementation of this application, the sample cell is made of polyurethane resin, and a semi-permeable membrane is sealed at the bottom of the sample cell.

[0037] It should be noted that polyurethane resin is only one specific sample cell preparation material used in this application, and it is not excluded that other materials may also be used to prepare the sample cell.

[0038] In one implementation of this application, the bottom of the sample cell is a regenerated fiber dialysis bag.

[0039] Another aspect of this application discloses a method for replacing or separating a protein sample solution, comprising replacing or separating the original solution of the protein sample solution to be treated using the apparatus of this application for replacing or separating a protein sample solution.

[0040] In one implementation of this application, the protein sample solution replacement or protein separation method specifically includes the following steps:

[0041] The electroosmotic flow module and sample cell are placed in the central through-hole of the microfluidic chip, and the sample cell is placed above the electroosmotic flow module. The target background solution is introduced into the target background solution inlet of the microfluidic chip at a set flow rate. The target background solution passes through the central through-hole of the microchannel and is finally discharged from the target background solution outlet. Specifically, when replacing the protein sample solution, the target background solution, i.e., the target solution to be replaced, is introduced into the microchannel. When separating or purifying proteins, the target background solution, i.e., the original sample solution of the protein sample to be processed or a similar protein dissolution solution, is introduced into the microchannel.

[0042] Clean the sample cell thoroughly, and place the protein sample solution to be processed into the sample cell so that the target background solution comes into contact with the protein sample solution to be processed in the sample cell;

[0043] The conductive electrode of the positive terminal of the power supply is fixed in the sample cell and in contact with the protein sample solution to be processed. The conductive electrode of the negative terminal of the power supply is fixed at the inlet or outlet of the target background solution of the microfluidic chip to form a closed loop.

[0044] By applying voltage through a power source, ions with particle sizes smaller than the pore size of the semipermeable membrane in the protein sample solution to be processed are driven to pass through the semipermeable membrane. At the same time, under the action of the electroosmotic module, neutral small molecules with particle sizes smaller than the pore size of the semipermeable membrane are also driven through the semipermeable membrane and the electroosmotic module through the electroosmotic effect, thereby achieving the replacement of the original solution of the protein sample solution to be processed; or enabling proteins with sizes smaller than the pore size of the semipermeable membrane to pass through the semipermeable membrane, thereby achieving protein separation of the protein sample solution to be processed.

[0045] In one implementation of this application, the target background solution introduced into the microchannel is the target solution to be replaced or a protein dissolution solution.

[0046] Preferably, the target background solution introduced into the microchannel is an ammonium acetate solution, an ammonium bicarbonate solution, or an ammonium acetate or ammonium carbonate solution with a pH adjuster.

[0047] It should be noted that, in order to preserve the native conformation of proteins, the pH of the protein sample solutions before and after electrophoresis should be kept within a relatively small range. Simultaneously, mass spectrometry requires that the sample solution not contain non-volatile salts to prevent signal interference and contamination of the mass spectrometer. Therefore, this application preferably uses ammonium acetate solution as the target background solution. It is understood that using ammonium acetate solution as the target background solution is primarily due to the requirements of mass spectrometry detection; in other applications, the solution requiring replacement can also be used directly as the target background solution.

[0048] In one implementation of this application, the method further includes checking for leakage before placing the protein sample solution to be processed into the sample cell. If leakage is found, the microfluidic chip and / or the sample cell needs to be replaced. Checking for leakage includes observing whether there is any seepage in the microfluidic chip and the gap between the microfluidic chip and the sample cell.

[0049] Due to the adoption of the above technical solutions, the beneficial effects of this application are as follows:

[0050] The protein sample solution replacement or protein separation device and method of this application combine electrophoresis, semi-permeable membranes, and electroosmosis to achieve rapid and efficient solution replacement or protein separation without introducing mechanical force. Electroosmosis facilitates the migration of non-volatile salt ions and neutral small molecules, solving the problem of difficult removal of neutral small molecules. Furthermore, the use of microfluidic chips for solution replacement or protein separation not only enables automated control but also provides excellent online integration compatibility with pretreatment modules or subsequent detection. Attached Figure Description

[0051] Figure 1 This is a schematic diagram of the microfluidic chip structure in the embodiments of this application;

[0052] Figure 2 This is a schematic diagram of the sample cell structure in an embodiment of this application;

[0053] Figure 3 This is a schematic diagram of the protein sample solution replacement device and its overall process in the embodiments of this application;

[0054] Figure 4 The results are from the solution displacement test of the organic dye tartrazine in the embodiments of this application;

[0055] Figure 5 These are the test results of cyt c in 0.25mM KCl solution before and after solution displacement in the embodiments of this application;

[0056] Figure 6 These are the mass spectrometry detection results of cyt c in PBS solutions of different concentrations in the embodiments of this application;

[0057] Figure 7 These are the test results of Ub in the 1×PBS solution before and after solution replacement in the embodiments of this application;

[0058] Figure 8 These are mass spectra of avidin in 1×PBS solution after electrophoretic replacement at different times, under SF 20eV conditions, used to show the complete tetramer signal.

[0059] Figure 9 These are mass spectra of avidin in 1×PBS solution after electrophoretic displacement at different times, under SF 60eV conditions, used to show the monomer signals obtained by gas phase dissociation.

[0060] Figure 10 These are the test results of solution displacement and protein separation after mixing proteins with different charges in the embodiments of this application;

[0061] Figure 11 This is a schematic diagram of the structure of another microfluidic chip in the embodiments of this application;

[0062] Figure 12 This is a schematic diagram of the electroosmotic flow module in an embodiment of this application;

[0063] Figure 13 This is a schematic diagram of another protein sample solution replacement device and its overall process in the embodiments of this application;

[0064] Figure 14 The absorbance detection results of Rhodamine B after electrophoresis with and without the electroosmotic flow module in the embodiments of this application are shown. Detailed Implementation

[0065] This application addresses the pretreatment needs of protein analysis by introducing the principle of electrophoresis combined with the principle of semi-permeable membranes to achieve rapid and efficient solution replacement without introducing mechanical force. Furthermore, to improve the solution replacement performance, an electroosmotic flow module is introduced into the developed replacement device, utilizing the electroosmotic flow effect to drive the migration of ions and neutral small molecules.

[0066] Electroosmotic flow is the movement of liquid caused by an applied electric potential in porous materials, capillaries, membranes, microchannels, or other fluid conduits. It is an important technique in chemical separation, such as capillary electrophoresis. The quartz material used in capillaries leaves numerous exposed silanol groups on the inner wall. These silanol groups dissociate in solutions with a pH above 3, forming negatively charged groups. Cations in the solution accumulate near these negative charges, forming an electrical double layer and a diffusion layer. When a voltage for electrophoresis is applied across the capillary, a large number of positive charges in the diffusion layer move towards the negative electrode. Since these positive charges are carried by solvated cations, they can drag a large amount of solution along with them. Because the inner diameter of the capillary is small, the internal solution can move as a whole under this effect, which macroscopically manifests as electroosmotic flow. Electroosmotic flow is a holistic effect of the solution within the capillary; the velocity of the electroosmotic flow itself provides a common mobility component for all substances within the capillary. The direction of this mobility is the same as that of cation electrophoresis and opposite to that of anion electrophoresis, and its value is often greater than that of electrophoretic mobility. Therefore, the combined effect is that cations move faster, and neutral substances and anions can also move in the same direction as cations with relatively small mobility. The positive significance of this result is that substances with different charges can be separated in the same direction in a single operation. Changing the surface properties of the capillary inner wall and changing the ionic strength of the solution can both change the Zeta potential ζ of the electric double layer, and changing the solution temperature can change the solution viscosity. Therefore, these parameters can all be used to regulate the strength of electroosmotic flow.

[0067] Electrophoresis is the movement of charged particles in the direction opposite to their charge under the influence of an electric field. Electroosmosis, on the other hand, occurs when porous materials adsorb positive and negative ions from water, making the solution relatively charged. Under the influence of an electric field, the solution moves in one direction, meaning all ions in the solution can move in the same direction. This application creatively proposes that if a device capable of generating electroosmotic flow is applied to the solution replacement device of this application, the migration time of non-volatile salt ions and neutral small molecules in the solution will be shortened, thus improving performance.

[0068] The protein sample solution replacement or protein separation device of this application combines the principles of electrophoresis, the separation effect of a semi-permeable membrane, and electroosmosis, solving the problem of the difficulty in removing neutral small molecules; and uses microfluidic technology to construct a microscale replacement or protein separation device, which can be automatically controlled and has compatibility with online integration with other pretreatment modules.

[0069] The apparatus and method for protein sample solution replacement or protein separation in this application have the following advantages compared with the prior art:

[0070] 1. Utilizing the principle of electrophoresis to drive small molecule ions in the original solution through a semipermeable membrane, while retaining proteins, thereby achieving the separation of proteins from the original solution environment or the separation and purification of proteins of different sizes.

[0071] 2. Utilize the principle of electrophoresis to drive small molecular ions in the target solution through a semipermeable membrane and merge with the protein, thereby placing the protein in a new solution environment; or enable small-sized proteins to pass through a semipermeable membrane for protein separation or purification.

[0072] 3. The electroosmotic flow module is used to achieve the migration of non-volatile salt ions and neutral small molecules, which solves the problem of the difficulty in removing neutral small molecules.

[0073] 4. Continuous perfusion of the target solution improves replacement efficiency and facilitates automated control.

[0074] 5. Constructing the device in the form of a microfluidic chip reduces sample consumption and facilitates automated control and online integration with other functional modules.

[0075] 6. Solution displacement using the electrophoresis effect involves only ion replacement, without introducing solvent into the sample, and therefore does not cause dilution. For example, the electrophoresis process is accompanied by electroosmotic flow, in which case the solvent can enter or leave the semipermeable membrane under the action of electroosmotic flow.

[0076] 7. A novel semi-permeable membrane encapsulation method was used to prepare the sample cell.

[0077] The present application will now be described in further detail with reference to specific embodiments and accompanying drawings. In the following embodiments, many details are described to facilitate a better understanding of the present application. However, those skilled in the art will readily recognize that some features may be omitted in different situations, or may be replaced by other devices, materials, or methods. In some cases, certain operations related to the present application are not shown or described in the specification to avoid obscuring the core parts of the application with excessive description. For those skilled in the art, detailed description of these related operations is not necessary; a complete understanding of the related operations can be obtained from the description in the specification and general technical knowledge in the art.

[0078] Example 1

[0079] I. Microfluidic Chip Design and Fabrication

[0080] The microfluidic chip in this example has a microchannel structure with elongated ends and a through-hole in the middle. Specifically, this example uses a spindle-shaped design. Figure 1 As shown, the spindle-shaped design reduces flow resistance and dead volume during fluid flow; the microchannel has a height of 240 μm and a width of 300 μm. At both ends of the channel are 0.7 mm diameter through-holes, serving as the buffer inlet and outlet, respectively (target background solution inlet 11 and target background solution outlet 12). The central through-hole, with a diameter of 5 mm (central through-hole 13), can be embedded in the sample cell. The maximum diameter of the spindle-shaped cavity is larger than the sample cell diameter, ensuring that the dialysis membrane at the bottom of the sample cell can fully contact the continuously changing target background solution within the chip's flow channels.

[0081] A microfluidic chip structure with a spindle shape was designed and a film was fabricated. A microfluidic chip template was then created using photolithography. SU-8 photoresist was uniformly coated onto a silicon wafer and baked dry. Exposure was then performed to transfer the microfluidic pattern from the photomask to the silicon wafer. Polydimethylsiloxane (PDMS) solutions A and B were mixed at a mass ratio of 10:1 and poured into the photolithography plate. The mixture was placed at 80°C and allowed to stand for 60 minutes. Demolding yielded the corresponding PDMS chip channels. Single-sided vias with a diameter of 0.7 mm were drilled at the inlet and outlet, and a single-sided via with a diameter of 5 mm was drilled at the center of the spindle-shaped region.

[0082] II. Sample Cell Fabrication

[0083] In this example, the sample cell, such as Figure 2 As shown, the circular bowl-shaped structure 21 has a semi-permeable membrane structure 22 sealed at its bottom.

[0084] A sample cell mold was fabricated, and the regenerated fiber dialysis bag was placed at the bottom of the mold and secured. Polyurethane resin (C3H8N2O, PU resin) solutions A and B were mixed evenly at a 1:1 mass ratio and quickly poured into the mold. After allowing it to stand freely for 20 minutes, the sample cell was removed. The protein sample solution to be processed was placed in the sample cell; the target solution was continuously infused into the microchannels.

[0085] Semipermeable membranes are powerful tools for separating particles of different sizes and are widely used in the pretreatment of biological samples, such as dialysis and ultrafiltration. Electrophoresis is the process by which charged ions migrate in the opposite direction to their charge under the influence of an electric field. Applying an electric field causes proteins and coexisting non-volatile salt ions in the sample solution to migrate towards the semipermeable membrane. Due to the interception effect of the semipermeable membrane, small anions or cations pass through, while larger proteins are retained in the sample cell. To ensure a stable circuit and replenish ions to the sample solution, a stable solution needs to be provided on the other side of the semipermeable membrane, i.e., the chip channel layer, ensuring good contact with the sample solution. During electrophoresis, ion exchange occurs between the solutions on both sides of the semipermeable membrane. To preserve the native conformation of proteins, the pH of the sample solution before and after electrophoresis should be kept within a relatively small range. Simultaneously, mass spectrometry requires that the sample solution does not contain non-volatile salts to prevent signal interference and contamination of the mass spectrometer; therefore, ammonium acetate solution, which is compatible with mass spectrometry, was chosen as the target background solution in this example.

[0086] In this example, a semi-permeable membrane is sealed at the bottom of the sample cell. The target background solution flowing rapidly at the bottom has good contact with the sample in the sample cell, so the exchanged ions can be removed while maintaining the electrical pathway.

[0087] III. Circuit Components and Target Background Solution Components

[0088] In this example, high-purity platinum wire is selected as the conductive electrode, fixed inside the electrophoretic pool and in contact with the protein sample solution to be processed. The other end of the electrode is fixed at the inlet and outlet of the target background solution. A power supply device capable of switching the positive and negative terminals is connected in series in the entire circuit. In this example, a microcontroller with switchable polarity is used to periodically switch the power direction to allow both anions and cations in the original sample solution to pass through the semipermeable membrane. The direction of protein migration changes with the change of electrode direction. It is worth noting that the final electrode switch should ensure that the electrode direction causes the protein to migrate away from the semipermeable membrane, thereby reducing the adsorption of protein by the semipermeable membrane.

[0089] The protein sample solution replacement procedure in this example includes:

[0090] Connect the completed chip to the solution tubing and the syringe for injecting the target solution; clamp the bottom of the sample cell containing the ion exchange membrane at the maximum diameter of the chip cavity; connect the circuit after leak testing.

[0091] Among them, the pipe connection is as follows Figure 3As shown: First, one end of the first solution tubing 31 is connected to the first steel needle 32, and the other end is connected to the needle of the syringe 33; one end of the second solution tubing 34 is connected to the second steel needle 35; the first steel needle 32 on the first solution tubing 31 is inserted into the target background solution inlet, and the other end is connected to the syringe 33; the second steel needle 35 on the second solution tubing 34 is inserted into the target background solution outlet, and the other end is placed in the waste liquid pool 36; the syringe 33, the first solution tubing 31, the first steel needle 32, the microchannel, the second steel needle 35, the second solution tubing 34, and the waste liquid pool 36 constitute a liquid passage; prepare a power supply 37, a power supply device 38 for switching the positive and negative terminals of the power supply, a wire 39, and a 99.99% high-purity platinum wire 310; the two ends of the power supply device 38 are respectively connected to the high-purity platinum wire 310 placed in the sample pool and the second steel needle 35 at the target background solution outlet to form a closed loop, as shown. Figure 3 As shown.

[0092] The syringe draws up the target background solution, i.e., 150mM ammonium acetate solution. The liquid propulsion device is adjusted to 200μL / min in this example. The target background solution is injected into the microchannel at a stable flow rate. The sample volume of about 20μL is added to the sample cell. The power supply device's working time and power electrode switching time are set and then the power is turned on.

[0093] IV. Protein Sample Solution Displacement Test

[0094] 1. Feasibility verification

[0095] The effectiveness of the protein sample solution replacement device in this example was tested using the organic dye tartrazine. Since this dye develops normally in negative ion form, the negative terminal of the power supply was connected to the sample cell, and the positive terminal was connected to the outlet of the target solution flow channel. The target background solution was 150 mM NH4Ac solution. In this example, the absorbance values ​​of the sample solution were measured after electrophoresis at 0 min, 15 min, 30 min, 40 min, and 50 min. Three parallel measurements were performed at each time point, and the results are as follows: Figure 4 As shown in the figure. The electrophoresis voltage is 60V, and 0min indicates that electrophoresis was not performed.

[0096] like Figure 4 As shown, the color of the sample solution containing approximately 0.02 micrograms of dye initially faded with increasing electrophoresis time, and almost disappeared after 40 minutes. After 50 minutes, the absorbance of the solution decreased by approximately 500 times. This experiment demonstrates the effectiveness of this device in replacing a single ion in the original solution using a single electric field direction.

[0097] Based on this, this example tested the displacement effect on a cyt c solution containing 0.25 mM KCl. Electrophoresis was performed under a constant current of 300 μA provided by a flow-controlled power supply; the electrode orientation was switched every 20 s.

[0098] Figure 5 The images show the mass spectra of the Cyt C samples before and after electrophoresis. Image A is the global signal spectrum, and image B is a magnified spectrum of the +6 valent Cyt C ion signal. After solution replacement using this apparatus, the high-valent (+8 to +10) ion signals disappeared, indicating that the protein denaturation caused by the presence of KCl was eliminated; the adduct formed by the protein and K ions is [Cyt C + 5H + 1K]. 6+ and [cyt c+4H+2K] 6+ Such levels are significantly reduced, and protein ions are mainly [cyt c+6H]. 6+ The form exists, and the data quality has been greatly improved.

[0099] 2. Displacement effect of Cyt C solution under different concentrations of PBS

[0100] Phosphate buffered saline (PBS) is one of the most widely used buffer solutions in biochemical research. Its main components are Na₂HPO₄·7H₂O, KH₂PO₄, and NaCl. It provides suitable pH buffering and salt balance for biomolecules. In this example, cyt c in 0.2×, 0.5×, and 1× PBS buffers were subjected to solution displacement. Figure 6 As shown, the left and right sides are the mass spectra of the cyt c protein samples before and after solution replacement, respectively. The main image is the global spectrum, and the upper part is a magnified spectrum of the cyt c +7 valent ion.

[0101] The results showed that PBS components in the protein solution significantly interfered with the protein signal. At lower PBS concentrations, a large number of alkali metal cation adducts were formed, dispersing and suppressing the protein signal. At higher PBS concentrations, signal suppression became the dominant factor, potentially leading to the complete disappearance of the protein signal. In this example, a 150 mM ammonium acetate solution was used for replacement. To ensure that both anions and cations in the original PBS solution were replaced, the electric field direction was switched every 20 seconds, and the electrophoresis process lasted for 1 hour. The current was kept constant at 300 μA using a flow-controlled power supply. After the replacement operation, the number of alkali metal cation adducts in the mass spectrometry detection of each sample was significantly reduced, with protonated molecular ions becoming the main protein ion presentation. The spectral quality was significantly improved, and the original PBS concentration had little impact on the replacement results, demonstrating that this device can efficiently perform solution replacement for actual protein samples.

[0102] 3. Displacement effect of other types of protein solutions under PBS environment

[0103] (1) Solution displacement of ubiquitin

[0104] Ubiquitin (Ub) is a small protein found in eukaryotes with a molecular weight of approximately 8.6 kDa. In this example, the Ub sample in 1×PBS was subjected to solution replacement, and a constant current of 300 μA was provided by a flow-controlled power supply. The electrode orientation was switched every 20 s. The mass spectrometry results are as follows: Figure 7 The image shows the mass spectra of the samples before and after solution replacement. The main image below is the global spectrum, and the inset above is an amplified spectrum of the +6 and +5 valent protein ion signals. The signal dispersion and protein signal suppression caused by alkali metal cation adducts are significantly improved after solution replacement.

[0105] (2) Solution displacement of recombinant avidin

[0106] Recombinant avidin is a glycoprotein extracted from egg white, consisting of a complex of four identical subunits linked by non-covalent interactions, with a molecular weight of approximately 60 kDa. In mass spectrometry detection of the pure protein under non-denaturing conditions, a complete tetramer signal can be detected at low source fragmentation (SF) conditions (e.g., 20 eV). Figure 8 As shown, the avidin sample under 1×PBS conditions could not directly obtain a mass spectrometry signal due to the signal suppression effect of non-volatile ions. However, by using this device to perform solution replacement at a constant current of 300 μA for 10 min, 30 min, and 60 min, high-quality signals of intact protein complexes were obtained, with molecular weights matching theoretical values, proving that the solution replacement effect achieved the expected results.

[0107] Under high SF conditions (e.g., 60 eV), the tetrameric complex of avidin can dissociate into monomers via a gas-phase dissociation reaction. Figure 9 The images show the mass spectra of the monomers obtained from gas-phase dissociation before and after solution replacement, under the same electrophoresis conditions. With prolonged electrophoresis, the number of alkali metal cation adducts significantly decreased, the glycoform distribution of the protein monomers became clearer, and the spectral quality significantly improved.

[0108] 4. Protein separation effect in samples with multiple coexisting proteins

[0109] The semi-permeable membrane used in this device separates different proteins based on their pore size. In this example, Ub and β-lactoglobulin (β-Lg) are smaller than the semi-permeable membrane pore size, while immunoglobulin G (IgG) is larger. In this example, a sample containing a 1:3 molar mixture of Ub and IgG was used for protein separation. The results are as follows... Figure 10 As shown in Figure A; a protein separation experiment was performed on a sample in which β-Lg and IgG were mixed at a molar ratio of 1:3, and the results are as follows. Figure 10 As shown in Figure B. Protein separation experiments were performed using this device with a constant current of 300 μA for solution replacement, and the electrode orientation was switched every 20 seconds.

[0110] Figure 10 The results showed that small protein components in the solution could be removed in 40 minutes, leaving only large IgG proteins, thus effectively achieving protein separation.

[0111] Example 2

[0112] I. Design and Fabrication of Novel Microfluidic Chips

[0113] This example is an improvement on Example 1. The microfluidic chip in Example 1 contained only one layer of PDMS; this example adds another layer of PDMS, such as... Figure 11 As shown, the first PDMS layer is the same as in Example 1, the only difference being that the diameter of the central through-hole is 7mm; the second PDMS layer does not require flow channels, only a 5mm central through-hole for holding the sample cell. It is important to note that the 5mm central through-hole of the second PDMS layer completely overlaps with the 7mm central through-hole of the first PDMS layer to ensure sufficient contact between the semi-permeable membrane of the sample cell and the solution at the bottom during electrophoresis. In use, as... Figure 13 As shown, the electroosmotic flow module 131 and the sample cell 132 are both placed in the middle through hole of the microfluidic chip 133, and the sample cell 132 is placed above the electroosmotic flow module 131.

[0114] II. Electroosmotic Flow Module

[0115] The electroosmotic module 131 is a membrane or plate with microporous channels, or an array structure composed of capillaries with inner diameters in the micrometer or millimeter range. In this example, the electroosmotic module is made of a material containing silica, such as silicone, quartz, or glass. The pore size of the microporous channels or the inner diameter of the capillaries is in the micrometer or millimeter range, ranging from a few micrometers to a few millimeters, as long as it can achieve the electroosmotic effect.

[0116] This example demonstrates the design and testing of five different electroosmotic flow modules, such as... Figure 12 As shown, the specific structure is as follows:

[0117] Method 1: Place a silicone film with a diameter of 7mm, a thickness of 2mm, and microporous channels in the 7mm central through hole of the first layer of PDMS. The pore size of the microporous channels is 0.1mm, i.e., silicone with microporous channels.

[0118] Method 2: Cut a glass tube with an inner diameter of 0.68 mm and an outer diameter of 1.20 mm into glass tubes with a height of about 2 mm. Then arrange the glass tubes in close contact in an array to form a hollow glass tube array, which is placed in the 7 mm central through hole of the first layer of PDMS.

[0119] Method 3: Cut a glass capillary tube with an inner diameter of 0.10 mm and an outer diameter of 0.30 mm into a small tube with a height of about 2 mm. Then arrange the capillary tubes in close contact array to form a capillary array, and place it in the 7 mm central through hole of the first layer of PDMS.

[0120] Method 4: Cut a hollow glass tube with an inner diameter of 0.68 mm and an outer diameter of 1.20 mm into glass tubes with a height of about 2 mm. Arrange the hollow glass tubes in close contact to form a hollow glass tube array. Insert 0.10 mm diameter optical fibers into the glass tubes to increase the flow channel and place them in the 7 mm central through hole of the first layer of PDMS.

[0121] Method 5: Fabricate a microchannel glass plate with a diameter of 7mm and a height of 2mm, with a microchannel aperture of 0.10mm.

[0122] III. Electroosmotic Flow Effect Test

[0123] In this example, Rhodamine B was used as an indicator, and the absorbance of the solution to 552nm visible light was used as an evaluation index. By comparing the results before and after setting up the electroosmotic flow module, the solution replacement effect of the above five electroosmotic flow modules was tested.

[0124] During electrophoresis, cations in the solution migrate towards the negative electrode, and anions migrate towards the positive electrode; however, the direction of electroosmotic flow is always from the negative electrode to the positive electrode. Rhodamine B develops normally in its cationic form. If the positive electrode of the power supply is connected to the sample cell and the negative electrode is connected to the outlet of the target background solution channel, as shown... Figure 13 As shown, the indicator can move from the sample cell through the semi-permeable membrane to the outlet of the target background solution channel, whether under electrophoresis or electroosmosis.

[0125] This example tested the electrophoresis results at 0 min, 10 min, 20 min, 30 min, and 40 min before and after setting five different electroosmotic modules under constant electrode conditions. Three parallel measurements were performed at each time point, and the results are as follows: Figure 14 As shown in Table 1. The electrophoresis voltage is 60V, and 0min indicates that electrophoresis has not been performed. Figure 14 In the diagram, the top curve represents normal electrophoresis, which is electrophoresis performed under the same conditions without adding an electroosmotic flow module. The five curves below, from top to bottom, represent the curves using the electroosmotic flow module in mode 3, mode 2, mode 5, mode 1, and mode 4, respectively.

[0126] Table 1 compares the absorbance at 552 nm of the Rhodamine B sample solution before and after solution displacement using the electroosmotic flow module.

[0127]

[0128]

[0129] Table 1 summarizes the results of three parallel experiments. For example, "Method 1" refers to using Method 1 as the electroosmotic flow module, "0 min" refers to the absorbance value without electrophoresis measurement, and "9.25 / 9.25 / 9.25" refers to the test results of the three parallel experiments. For another example, the data "3.86 / 2.61 / 4.27" corresponding to "Method 1" and "10 min" indicate that after using Method 1 as the electroosmotic flow module for 10 min of electrophoresis, the absorbance measured in the three parallel experiments were 3.86, 2.61, and 4.27, respectively.

[0130] Figure 14 The results in Table 1 show that after setting up the electroosmotic flow module, the indicator concentration was significantly reduced under electrophoresis, and the solution replacement effect was significantly enhanced.

[0131] The above description, in conjunction with specific embodiments, provides a further detailed explanation of this application and should not be construed as limiting the specific implementation of this application to these descriptions. Those skilled in the art to which this application pertains can make several simple deductions or substitutions without departing from the concept of this application.

Claims

1. An apparatus for replacing protein sample solutions or separating proteins, characterized in that: It includes a microfluidic chip, a sample cell for holding the protein sample solution to be processed, and an electroosmotic module; The microfluidic chip has a cavity structure with elongated ends and a through hole in the middle. The two ends of the microfluidic channel are respectively provided with through holes as the target background solution inlet and the target background solution outlet, and the through hole in the middle is used to accommodate the sample cell and the electroosmotic flow module. The sample cell fits tightly with the top cavity of the central through hole of the microchannel, and the part of the sample cell that contacts the target background solution in the microchannel is a semi-permeable membrane structure. The electroosmotic module fits tightly with the lower cavity of the central through hole of the microchannel. When in use, the electroosmotic module is placed below the sample cell and comes into direct contact with the target background solution in the microchannel. The bottom of the sample cell is sealed with a semi-permeable membrane; In use, the sample cell is detachably embedded in the central through-hole of the microfluidic chip.

2. The apparatus according to claim 1, characterized in that: The electroosmotic module is a membrane or plate with microporous channels, or the electroosmotic module is an array structure composed of capillaries with an inner diameter at the micrometer or millimeter level.

3. The apparatus according to claim 2, characterized in that: The electroosmotic module is made of a material containing silicon dioxide.

4. The apparatus according to claim 3, characterized in that: The material containing silicon dioxide is silicone, quartz, or glass.

5. The apparatus according to claim 4, characterized in that: The electroosmotic flow module is at least one of the following structures. (1) Silica gel with microporous channels; (2) Hollow glass tube array; (3) Fused silica capillary array; (4) An array of hollow glass tubes with glass or quartz wires inserted into the internal channels; (5) A glass plate with a microporous channel array.

6. The apparatus according to claim 5, characterized in that: The pore size of the micropore channels in the silicone or glass plate is at the micrometer or millimeter level.

7. The apparatus according to claim 6, characterized in that: The pore size of the micropore channels in the silicone or glass plate is 0.1 mm.

8. The apparatus according to claim 5, characterized in that: The inner diameter of the fused silica capillary is less than or equal to 1 mm.

9. The apparatus according to claim 8, characterized in that: The inner diameter of the fused silica capillary is 0.1 mm.

10. The apparatus according to claim 5, characterized in that: The inner diameter of the hollow glass tube is 0.6-0.7 mm.

11. The apparatus according to claim 5, characterized in that: The diameter of the glass or quartz wire is 0.1 mm.

12. The apparatus according to any one of claims 1-11, characterized in that: It also includes circuit components, which include conductive electrodes and a power source; In use, the conductive electrode of the positive terminal of the power supply is fixed in the sample cell and in contact with the protein sample solution to be processed, while the conductive electrode of the negative terminal of the power supply is fixed at the target background solution inlet or target background solution outlet of the microfluidic chip.

13. The apparatus according to any one of claims 1-11, characterized in that: It also includes a target background solution assembly, which includes a liquid propulsion device for regulating the flow rate of the target background solution in the microchannel of the microfluidic chip; in use, the liquid propulsion device is connected to the target background solution inlet of the microfluidic chip.

14. The apparatus according to claim 13, characterized in that: The target background solution assembly also includes a waste liquid tank; in use, the waste liquid tank is connected to the target background solution outlet of the microfluidic chip.

15. The apparatus according to any one of claims 1-11, characterized in that: The microfluidic chip is made of polydimethylsiloxane and is mounted on a glass slide.

16. The apparatus according to any one of claims 1-11, characterized in that: The sample cell is made of polyurethane resin.

17. The apparatus according to claim 16, characterized in that: The bottom of the sample cell is a regenerated fiber dialysis bag.

18. A method for replacing protein sample solutions or separating proteins, characterized in that: This includes using the apparatus described in any one of claims 1-17 to replace the original solution or separate the protein sample solution to be treated.

19. The method according to claim 18, characterized in that: The method involves placing an electroosmotic flow module and a sample cell in the central through-hole of the microfluidic chip, placing the sample cell above the electroosmotic flow module, and introducing the target background solution into the target background solution inlet of the microfluidic chip at a set flow rate. The target background solution passes through the central through-hole of the microchannel and is finally discharged from the target background solution outlet. Clean the sample cell thoroughly, and place the protein sample solution to be processed into the sample cell so that the target background solution comes into contact with the protein sample solution to be processed in the sample cell; The conductive electrode of the positive terminal of the power supply is fixed in the sample cell and in contact with the protein sample solution to be processed. The conductive electrode of the negative terminal of the power supply is fixed at the inlet or outlet of the target background solution of the microfluidic chip to form a closed loop. By applying voltage through a power source, ions with particle sizes smaller than the pore size of the semipermeable membrane in the protein sample solution to be processed are driven to pass through the semipermeable membrane. At the same time, under the action of the electroosmotic module, neutral small molecules with particle sizes smaller than the pore size of the semipermeable membrane are also driven through the semipermeable membrane and the electroosmotic module through the electroosmotic effect, thereby achieving the replacement of the original solution of the protein sample solution to be processed; or enabling proteins with sizes smaller than the pore size of the semipermeable membrane to pass through the semipermeable membrane, thereby achieving protein separation of the protein sample solution to be processed.

20. The method according to claim 19, characterized in that: The target background solution introduced into the microchannel is the target solution or protein dissolution solution that needs to be replaced.

21. The method according to claim 20, characterized in that: The target background solution introduced into the microchannel is an ammonium acetate solution, an ammonium bicarbonate solution, or an ammonium acetate or ammonium carbonate solution with a pH adjuster.

22. The method according to any one of claims 18-21, characterized in that: It also includes checking for leaks before putting the protein sample solution to be processed into the sample cell. If there are leaks, the microfluidic chip and / or sample cell need to be replaced. The check for leakage includes observing whether there is any seepage in the microfluidic chip and the gap between the microfluidic chip and the sample cell.

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