Molecular fishing system and elution process thereof

Abstract

This invention discloses a molecular elution system and its elution process, belonging to the field of molecular elution technology. The system includes a chip, a flow cell, a light source, a camera device, a sampling needle, a reservoir ring, a first pumping mechanism, a second pumping mechanism, and multiple valve groups. Through the coordination of these valve groups, a complete flow path is formed, encompassing quantitative sample aspiration, injection into the flow cell, selectable elution modes, and automatic recovery. Based on this system, this invention provides three elution processes: direct elution, eluent elution, and washing elution, which can be flexibly selected according to the dissociation characteristics of the target molecule (fast or slow dissociation) and the complexity of the sample. This invention avoids the loss of rapidly dissociated samples caused by traditional rinsing steps, significantly improving the recovery rate. Simultaneously, the optional washing step effectively removes impurities, improving sample purity, and is applicable to fields such as antibody drug screening, proteomics, and biomarker discovery.

Description

Technical Field

[0001] This invention belongs to the field of molecular fishing technology, and particularly relates to a molecular fishing system and its elution process. Background Technology

[0002] Molecular fishing is a separation and purification technique based on the specific interactions of biomolecules. Its basic principle involves immobilizing known "bait" molecules (such as antigens, antibodies, receptors, and nucleic acid aptamers) on a solid matrix surface (such as sensor chips or gel matrices) via covalent coupling or other methods. When a complex sample containing the target molecule flows through the solid surface, the target molecule specifically binds to the bait molecule and is captured, while non-specific components are eluted. Finally, the enriched target molecule is eluted and recovered by changing the buffer conditions (such as pH, ionic strength, or adding a competing agent). This technique, due to its high specificity, high sensitivity, and ability to capture target molecules in their native conformation, is widely used in proteomics research, drug target screening, biomarker discovery, and antibody drug development.

[0003] Currently, commercially available mainstream molecular fishing systems, such as the Biacore series platform based on surface plasmon resonance (SPR) technology, have become common tools in this field due to their ability to monitor intermolecular interactions in real time. These systems typically integrate sophisticated microfluidic systems, optical detection units, and pre-set control software. In a standard operating procedure, the typical "bind-wash-elute" process is as follows: First, the sample is injected into the flow cell, allowing the target molecule to bind to the decoy molecules immobilized on the chip surface; then, a running buffer is injected for a brief wash to remove unbound impurities and non-specific adsorption; finally, an eluent is injected to dissociate and recover the bound target molecule.

[0004] However, the existing fixed procedure has significant limitations. Experimental studies show that the dissociation kinetics between different target molecules and decoys vary significantly, and can be divided into fast dissociation and slow dissociation types. For fast dissociation target molecules, the binding complex with the decoy has a short half-life. In the standard "rinse" step of the existing technology, the continuously flowing running buffer causes a large number of bound fast dissociation target molecules to dissociate prematurely and be flushed into the waste liquid, resulting in a significant reduction in the final recovery rate, and even making it difficult to obtain sufficient sample for subsequent analysis. In addition, the flow path design of the existing system is relatively fixed, and the operation mode is singular, making it difficult to flexibly adjust experimental parameters and procedures according to the specific characteristics of the sample. For example, it is not possible to selectively add a washing step before elution to remove specific impurities, nor can it be eluted immediately after binding to avoid sample loss. This lack of flexibility limits its applicability and scientific validity when handling complex and diverse samples.

[0005] Therefore, how to provide a molecular fishing system and its elution process that can flexibly adjust the elution strategy according to the dissociation characteristics of different samples, thereby improving the recovery efficiency and purity of target molecules, is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] This invention aims to address the problems of low recovery rates and poor experimental flexibility in existing molecular elution systems due to their fixed programs, especially when eluting samples with different dissociation characteristics (particularly fast dissociation). This invention provides a molecular elution system with a rational structural design and precise control, along with multiple selectable elution processes to meet the experimental needs of different samples and improve the recovery efficiency and purity of target molecules.

[0007] To solve the above-mentioned technical problems, the specific technical solution of the present invention is as follows:

[0008] In some embodiments of this application, a molecular fishing system is provided, comprising: The chip has a flow pool on it; A light source and a camera device are disposed on one or both sides of the chip; Sampling needle; A liquid storage ring is used to quantitatively store fluid. The first pumping mechanism and the second pumping mechanism are used to drive fluid flow; The first valve assembly connects the sampling needle, the reservoir ring, the flow cell, and the buffer source, and can switch between a first state and a second state. In the first state, the sampling needle is connected to the reservoir ring to draw up the sample. In the second state, the reservoir ring is connected to the flow cell to inject the sample into the flow cell. The second valve assembly is connected between the flow cell and the sample collector and the waste bottle, and is used to selectively direct the flow cell effluent to the sample collector or the waste bottle; The third valve group is connected to the pipeline between the first pumping mechanism, the second pumping mechanism, and the flow pool and the first valve group, and is used to control the connection or disconnection between the second pumping mechanism and the flow pool.

[0009] In some embodiments of this application, the first valve group is a six-way valve, the second valve group includes a combination of a six-way valve and a three-way valve, and the third valve group is a three-way valve.

[0010] In some embodiments of this application, the sample collector includes: A sample tray with multiple sample collection positions; A rotary drive mechanism is used to drive the sample disk to rotate so as to align different sample collection positions with the recovery pipeline; A lifting drive mechanism is used to drive the recovery pipeline to move up or down to insert or withdraw from the sample collection position.

[0011] In some embodiments of this application, the rotary drive mechanism includes a rotary motor and a synchronous belt drive mechanism, and the lifting drive mechanism includes a lifting motor and a lead screw drive mechanism.

[0012] In some embodiments of this application, a direct elution process using any of the molecular fishing systems described above is disclosed, including a sample binding step and an elution step; the elution step is one of a direct elution mode, an eluent elution mode, or a washing elution mode.

[0013] In some embodiments of this application, the direct elution mode includes: After the sample binding step, without a rinsing step, the second pumping mechanism is immediately started to introduce the running buffer into the flow cell through the third valve group for elution, and the eluent is guided to the sample collector through the second valve group for recovery.

[0014] In some embodiments of this application, the elution mode of the eluent includes: After the sample binding step, the eluent is quantitatively drawn into the reservoir ring through the first valve group and the sampling needle; The eluent in the storage ring is injected into the flow cell for elution through the first valve group; The eluent is guided through the second valve assembly to the sample collector for recovery.

[0015] In some embodiments of this application, the washing and elution mode includes: After the sample binding step, the eluent is quantitatively drawn into the reservoir ring through the first valve group and the sampling needle; The process is paused after the eluent is pushed to a predetermined position before the inlet of the flow tank. The second pumping mechanism is activated to introduce the running buffer into the flow pool through the third valve group for cleaning; Stop the second pumping mechanism, while continuing to push the eluent into the flow tank for elution; The eluent is guided through the second valve assembly to the sample collector for recovery.

[0016] In some embodiments of this application, the sample binding step includes: The sample is quantitatively drawn into the reservoir ring through the sampling needle and the first valve group in an air-sample-air intermittent manner. The sample in the storage ring is injected into the flow cell through the first valve group, so that the target molecules combine with the decoy molecules fixed on the chip surface, and the outflow liquid is guided to the waste liquid bottle through the second valve group.

[0017] Compared with existing technologies, the advantages of this invention are as follows: the system, through a unique flow path design, realizes three different elution processes, which can be flexibly selected according to the dissociation kinetics of the target molecules. Among them, the direct elution process can elute immediately after sample binding, effectively avoiding the loss of rapidly dissociated samples caused by traditional rinsing steps and significantly improving the recovery rate. Secondly, the washing-elution process, by adding an optional washing step before elution, can effectively remove non-specific adsorbed impurities in complex samples, which helps to improve the purity of the recovered samples. In addition, the system integrates an automated sample collector, which can realize continuous collection from multiple tubes, improving the convenience and throughput of experimental operations. Attached Figure Description

[0018] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings: Figure 1 This is a schematic diagram of the molecular fishing system provided in an embodiment of the present invention; Figure 2 A schematic diagram of the collector provided in an embodiment of the present invention; Figure 3 This is a schematic diagram of a molecular fishing system provided in an embodiment of the present invention; Figure 4 This is a graph showing the verification and comparison of fishing performance. In the graph, a: M: pre-stained protein molecular weight standard; 1: total protein recovered from 6 BLI tests; 2: total protein recovered from 6 T200 tests; 3: total protein recovered from 6 S200F tests; C: 0.9 μg mepolizumab; b: Western Blot results; c: protein quantification standard curve. Figure 5This is a comparison chart of the anti-interference capability verification of the S200F platform. In a, M: pre-stained protein molecular weight standard; 1: 293F cell lysis buffer (containing mepolizumab) sample; 2: sample recovered under "elution and dissociation" mode; 3: sample recovered under "washing & elution and dissociation" mode. b: M: pre-stained protein molecular weight standard; 1-1: BLI SSA blank sensor recovered sample; 1-2: BLI SSA-Protein A recovered sample; 2-1: T200 CM5 blank chip recovered sample; 2-2: T200 CM5-Protein A recovered sample; 3-1: S200F CM5 blank chip recovered sample; 2-2: S200F CM5-Protein A recovered sample; C: mepolizumab. c M: Prestained protein molecular weight standard; 1: BLI SSA-Bat ACE2 recovered sample; 2: T200 CM5-Bat ACE2 recovered sample; 3: S200F CM5-Bat ACE2 recovered sample; C: Mepocillinumab.

[0019] In the diagram, 401 is the waste liquid bottle; 402 is the light source; 403 is the chip; 404 is the camera device; 405 is the first six-way valve; 406 is the sampling needle; 407 is the liquid storage ring; 408 is the first plunger pump; 409 is the second plunger pump; 410 is the first three-way valve; 411 is the buffer bottle; 412 is the second six-way valve; 413 is the second three-way valve; 414 is the collector; 415 is the flow cell; 51 is the lifting motor; 52 is the outer casing; 53 is the centrifuge tube trough; 54 is the centrifuge tube position; 55 is the rotary motor; 56 is the synchronous belt; 57 is the sample tray; 58 is the waste liquid level; and 59 is the recovery pipeline. Detailed Implementation

[0020] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0021] To better understand the purpose, structure, and function of this invention, the invention will be described in further detail below with reference to the accompanying drawings.

[0022] like Figures 1-3 As shown, a molecular fishing system includes a chip 403, a light source 402 (for providing a continuous and stable light signal), a camera device 404 (preferably a CCD camera for real-time data acquisition), a first six-way valve 405, a second six-way valve 412, a first three-way valve 410, a second three-way valve 413, a sampling needle 406, a liquid storage ring 407, a first plunger pump 408, a second plunger pump 409, a collector 414, a buffer bottle 411, and a waste bottle 401. The chip 403 has a flow cell 415 (preferably a flow cell 415 is attached to it), and a light source 402 and a camera device 404 are arranged nearby. A first six-way valve 405 is connected to the flow cell 415, a sampling needle 406 is connected to the first six-way valve 405, one end of a liquid storage ring 407 is connected to the first six-way valve 405, and the other end is connected to a buffer bottle 411. A first plunger pump 408 and a second plunger pump 408 are respectively connected to the pipeline connecting the liquid storage ring 407 and the buffer bottle 411. The plunger pump 409 has one end of the first three-way valve 410 connected to the pipeline between the first six-way valve 405 and the flow pool 415, and the other end connected to the second plunger pump 409. The first plunger pump 408 and the second plunger pump 409 are connected. One end of the second six-way valve 412 is connected to the pipeline between the first six-way valve 405 and the flow pool 415, and the other end is connected to one end of the second three-way valve 413. The other end of the second three-way valve 413 is connected to the collector 414 and the waste liquid bottle 401 respectively.

[0023] In this invention, collector 414 includes: The outer casing 52 contains a sample tray 57, which has a waste liquid level 58, centrifuge tube positions 54, and a recovery pipeline 59. Preferably, there is one waste liquid level 58 and five centrifuge tube positions 54, with a centrifuge tube groove 53 for fixing the centrifuge tubes.

[0024] A rotating device, located at the bottom of the housing 52, is used to drive the waste liquid level 58 and the centrifuge tube level 54 to rotate. Preferably, the rotating device includes a rotary motor 55 and a synchronous belt 56, with the rotary motor 55 driving the sample tray 57 to rotate via the synchronous belt 56.

[0025] A lifting device is installed on the top of the outer casing 52 to drive the recovery pipeline 59 to rise and fall. Preferably, the lifting device includes a lifting motor 51 and a lead screw. The lifting motor 51 drives the recovery pipeline 59 to rise and fall via the lead screw, thereby achieving automated sample collection and automated pipeline cleaning.

[0026] In this invention, the imaging device 404 is a CCD camera used for real-time data acquisition.

[0027] The molecular elution system provided by this invention is designed based on in-depth research and understanding of dissociation models for different samples (including both fast and slow dissociation types). This system not only supports multiple elution processes but can also be flexibly adjusted according to specific experimental needs, making the entire experimental process more scientific and efficient. The connectivity between the components will be described in detail in the following three elution processes: Secondly, the present invention provides a direct elution process for the above-mentioned molecular fishing system, comprising the following steps: Step (1) System preparation: The first plunger pump 408 and the second plunger pump 409 draw running buffer from the buffer bottle 411 and inject it into all the pipelines and valves of the molecular fishing system. At the same time, the light source 402 illuminates the chip 403, and the camera device 404 receives the signal from the chip 403 and begins real-time data acquisition. Step (2): Flushing the pipeline, the second plunger pump 409 draws the running buffer to the maximum volume, the first three-way valve 410 is switched to OFF, the second six-way valve 412 is switched to position 2, the second three-way valve 413 is switched to OFF, and the second plunger pump 409 injects the running buffer to clean the pipeline. Step (3): Sample sampling. The first six-way valve 405 is switched to position 1, the sampling needle 406 is moved to the sample location, and the first plunger pump 408 sequentially draws air, sample, and air through the sampling needle 406, and then continues to draw air and sample into the liquid storage ring 407. Step (4): Sample combination. The first six-way valve 405 is switched to position 2, the second six-way valve 412 is switched to position 3, and the second three-way valve 413 is switched to ON. The first plunger pump 408 pushes air and sample from the liquid storage ring 407 through the first six-way valve 405 to the position next to the chip 403. The first section of air enters the pipeline connected to position 3 of the second six-way valve 412. The second six-way valve 412 is switched to position 2, and the first plunger pump 408 continues to inject. The sample passes through the flow cell 415, through the pipeline at position 2 of the second six-way valve 412, through the ON end of the second three-way valve 413, and is discharged into the waste liquid bottle 401 until the second section of air is injected into the flow cell 415. Step (5), elution: the first three-way valve 410 is switched to ON, and the second plunger pump 409 injects running buffer, which enters the flow cell 415 through the ON end of the first three-way valve 410 for elution; Step (6) Sample recovery: The second plunger pump 409 continues to inject running buffer, pushing the running buffer from step (5) to the C end of the second three-way valve 413. The first three-way valve 410 is switched to the OFF end, the second three-way valve 413 is switched to the OFF end, the second plunger pump 409 continues to inject running buffer, and the running buffer from step (5) is pushed to the collector 414 for collection.

[0028] The direct elution process provided by this invention exhibits significant advantages in terms of production efficiency, cost control, product quality, and environmental benefits. These are specifically described in the section on beneficial effects.

[0029] Thirdly, the present invention provides an elution process for the above-mentioned molecular fishing system eluent, comprising the following steps: Step (1) System preparation: The first plunger pump 408 and the second plunger pump 409 draw running buffer from the buffer bottle 411 and inject it into all the pipelines and valves of the molecular fishing system. At the same time, the light source 402 illuminates the chip 403, and the camera device 404 receives the signal from the chip 403 and begins real-time data acquisition. Step (2): Flushing the pipeline, the second plunger pump 409 draws the running buffer to the maximum volume, the first three-way valve 410 is switched to OFF, the second six-way valve 412 is switched to position 2, the second three-way valve 413 is switched to OFF, and the second plunger pump 409 injects the running buffer to clean the pipeline. Step (3): Sample sampling. The first six-way valve 405 is switched to position 1, the sampling needle 406 is moved to the sample location, and the first plunger pump 408 sequentially draws air, sample, and air through the sampling needle 406, and then continues to draw air and sample into the liquid storage ring 407. Step (4): Sample combination. The first six-way valve 405 is switched to position 2, the second six-way valve 412 is switched to position 3, and the second three-way valve 413 is switched to ON. The first plunger pump 408 pushes air and sample from the liquid storage ring 407 through the first six-way valve 405 to the position next to the chip 403. The first section of air enters the pipeline connected to position 3 of the second six-way valve 412. The second six-way valve 412 is switched to position 2, and the first plunger pump 408 continues to inject. The sample passes through the flow cell 415, through the pipeline at position 2 of the second six-way valve 412, through the ON end of the second three-way valve 413, and is discharged into the waste liquid bottle 401 until the second section of air is injected into the flow cell 415. Step (5): Eluent sampling. The first six-way valve 405 is switched to position 1, the sampling needle 406 is moved to the blank position, the first plunger pump 408 draws running buffer from the buffer bottle 411 and injects it into the reservoir ring 407. It is discharged through the first six-way valve 405 and the sampling needle 406 to complete the cleaning of the reservoir ring 407 and the sampling needle 406. The sampling needle 406 is moved to the position of the eluent. The first plunger pump 408 draws air, eluent and air in sequence through the sampling needle 406, and then continues to draw air and eluent into the reservoir ring 407. Step (6), elution: the first six-way valve 405 is switched to position 2, the second six-way valve 412 is switched to position 3, the second three-way valve 413 is switched to ON, the first plunger pump 408 pushes air and eluent from the reservoir ring 407 through the first six-way valve 405 to the position next to the chip 403, the first section of air enters the pipeline connected to position 3 of the second six-way valve 412, the second six-way valve 412 is switched to position 2, the first plunger pump 408 continues to inject, and the eluent passes through the flow cell 415; Step (7) Sample recovery: The first plunger pump 408 continues to inject the eluent, pushing the eluent from step (6) to the C end of the second three-way valve 413. The first three-way valve 410 switches to the OFF end, the second three-way valve 413 switches to the OFF end, and the second plunger pump 409 injects the running buffer, pushing the eluent from step (6) to the collector 414 for collection.

[0030] The elution process provided by this invention has advantages such as high efficiency, flexibility, stability, ease of operation and environmental friendliness, and is suitable for the separation and purification of a variety of substances.

[0031] Fourthly, the present invention provides a cleaning and elution process for the above-mentioned molecular fishing system, comprising the following steps: Step (1) System preparation: The first plunger pump 408 and the second plunger pump 409 draw running buffer from the buffer bottle 411 and inject it into all the pipelines and valves of the molecular fishing system. At the same time, the light source 402 illuminates the chip 403, and the camera device 404 receives the signal from the chip 403 and begins real-time data acquisition. Step (2): Flushing the pipeline, the second plunger pump 409 draws the running buffer to the maximum volume, the first three-way valve 410 is switched to OFF, the second six-way valve 412 is switched to position 2, the second three-way valve 413 is switched to OFF, and the second plunger pump 409 injects the running buffer to clean the pipeline. Step (3): Sample sampling. The first six-way valve 405 is switched to position 1, the sampling needle 406 is moved to the sample location, and the first plunger pump 408 sequentially draws air, sample, and air through the sampling needle 406, and then continues to draw air and sample into the liquid storage ring 407. Step (4): Sample combination. The first six-way valve 405 is switched to position 2, the second six-way valve 412 is switched to position 3, and the second three-way valve 413 is switched to ON. The first plunger pump 408 pushes air and sample from the liquid storage ring 407 through the first six-way valve 405 to the position next to the chip 403. The first section of air enters the pipeline connected to position 3 of the second six-way valve 412. The second six-way valve 412 is switched to position 2, and the first plunger pump 408 continues to inject. The sample passes through the flow cell 415, through the pipeline at position 2 of the second six-way valve 412, through the ON end of the second three-way valve 413, and is discharged into the waste liquid bottle 401 until the second section of air is injected into the flow cell 415. Step (5): Eluent sampling. The first six-way valve 405 is switched to position 1, the sampling needle 406 is moved to the blank position, the first plunger pump 408 draws running buffer from the buffer bottle 411 and injects it into the reservoir ring 407. It is discharged through the first six-way valve 405 and the sampling needle 406 to complete the cleaning of the reservoir ring 407 and the sampling needle 406. The sampling needle 406 is moved to the position of the eluent. The first plunger pump 408 draws air, eluent and air in sequence through the sampling needle 406, and then continues to draw air and eluent into the reservoir ring 407. Step (6), cleaning: the first six-way valve 405 is switched to position 2, the second six-way valve 412 is switched to position 3, the second three-way valve 413 is switched to ON, the first plunger pump 408 pushes air and eluent from the reservoir ring 407 through the first six-way valve 405 to the position next to the chip 403, the first section of air enters the pipeline connected to position 3 of the second six-way valve 412, the second six-way valve 412 is switched to position 2, the first three-way valve 410 is switched to ON, the second plunger pump 409 injects running buffer for cleaning; Step (7), elution, the second plunger pump 409 stops, while the first plunger pump 408 continues to inject, and the eluent passes through the flow cell 415; Step (8) Sample recovery: The first plunger pump 408 continues to inject the eluent, pushing the eluent from step (6) to the C end of the second three-way valve 413. The first three-way valve 410 switches to the OFF end, the second three-way valve 413 switches to the OFF end, and the second plunger pump 409 injects the running buffer, pushing the eluent from step (6) to the collector 414 for collection.

[0032] The cleaning and elution process provided by this invention can significantly improve cleaning efficiency. By optimizing the fluid dynamics design, the cleaning solution can more evenly cover the surface to be cleaned, thereby achieving a deeper cleaning effect.

[0033] The technical solution of the present invention will be clearly and thoroughly described below with reference to specific embodiments.

[0034] Example 1 1. Materials and Methods 1.1 Experimental Platform The S200F SPR molecular interaction analyzer was used as the core hardware platform. Its main functional modules include: a fluid control system (flow rate 0.005-1 mL / min), an optical detection system (light source wavelength 624 nm, refractive index range 1.30-1.39), and a sensor chip. The S200F molecular interaction platform, along with its control and data analysis software, enables automation of the fishing process and data analysis. The Biacore T200 and BLI R8 were used as auxiliary equipment for fishing tests and affinity detection. In addition, this study also utilized a low-temperature high-speed centrifuge, an AKTA protein purification system, a mass spectrometer, a fully automated digital Western blotting system, and a protein electrophoresis apparatus during sample preparation and identification.

[0035] 1.2 Main Reagents The supernatants from Bat ACE2 protein and SARS-CoV-2 RBD secretion expression were cryopreserved in our laboratory. Protein A was purchased from Yisheng Biotechnology Co., Ltd.; mepolizumab was purchased from GlaxoSmithKline. Pre-stained protein molecular weight standards were purchased from Thermo Fisher Scientific; SDA-PAGE precast gels were purchased from Genscript Biotech Co., Ltd.; 293T I serum-free medium was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.; anti-rabbit secondary antibody detection kit was purchased from ProteinSimple, USA; amino-coupled reagent kit, CM5 chip and Glycine 2.0 were purchased from Cytiva; SSA sensor was purchased from Sartorius; PBS and Tween-20 were purchased from Shanghai Beyotime Biotechnology Co., Ltd.; other reagents were all domestically produced analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd.

[0036] 1.3 Construction of Molecular Fishing Platform The CM5 carboxymethyl dextran fishing chip was selected and fabricated by Beijing Yingbo Biotechnology. The chip consists of seven channels connected end-to-end to form a serpentine flow cell 50 μm high, with a flow cell surface area of ​​approximately 43 mm². The target protein was immobilized using an amino covalent coupling method: first, equal volumes of 0.4 M EDC and 0.1 M NHS were mixed and flowed over the chip surface at a rate of 10 μL / min for 10 minutes; then, Protein A (20-100 μg / ml, dissolved in sodium acetate at pH 4.0-5.5) was injected at a rate of 10 μL / min for 10 minutes, with a coupling amount between 600-3000 milligrams; finally, unreacted sites were blocked with 1 M ethanolamine (pH 8.5).

[0037] After immobilizing Protein A onto the chip surface, the third fishing process (molecular fishing system cleaning and elution process) described above was used for fishing mepolizumab: PBST (containing 0.05% Tween-20) was selected as the buffer, mepolizumab or a mixed solution containing mepolizumab was used as the sample to flow through the chip, and 10mM glycine (pH 2.0) was used as the elution buffer for elution. During the process, running buffer PBST (containing 0.05% Tween-20) was injected for cleaning.

[0038] 1.4 Performance Evaluation 1.4.1 Fishing ability test To quantitatively evaluate the fishing capabilities of the S200F platform, an antigen-antibody system was used in the experiment, with Protein A and mepolizumab selected as test samples. Control experiments were also conducted on two imported platforms, the T200 and BLI R8.

[0039] For the S200F platform fishing test, the CM5 fishing chip from Beijing Yingbo Biotechnology was used. First, equal volumes of 0.4M EDC and 0.1M NHS were mixed and flowed through the chip surface at a flow rate of 10 μL / min for 10 minutes. Then, Protein A was diluted to 200 μg / ml with sodium acetate (pH 4.0) and injected at a flow rate of 10 μL / min for 10 minutes, with a coupling volume of 2220.08 milligrams. Next, a third fishing process was selected, in which mepolizumab (100 nM) was injected at a flow rate of 10 μL / min for 5 minutes, followed by elution with 10 mM glycine (pH 2.0) for 30 μL, neutralization with 1M Tris-HCl (pH 9.0), and sample recovery. This injection-elution process was repeated 12 times, and the eluent was collected and concentrated.

[0040] For fishing tests on the T200 platform, the Cytiva S-series CM5 chip was used. Protein A was coupled to channels 1-4 via amino covalent coupling, with a coupling amount of 6000 RU. Then, the T200 control software's preset method "Inject and recover" was selected, with an injection flow rate of 10 μL / min, a time of 5 minutes, an eluent of 10 mM glycine (pH 2.0), and a neutralization solution of 1 M Tris-HCl (pH 9.0). The injection-elution cycle was repeated 12 times, and the eluent was collected and concentrated.

[0041] For the fishing test on the BLI platform, Sartorius' SSA sensor was used. Biotin-linked Protein A was immobilized onto the SSA sensor at a thickness of 3.4 nm. Then, the sensor was immersed in a mepolizumab (100 nM) solution for 15 minutes to bind, followed by elution in a 10 mM glycine (pH 2.0) solution for 30 seconds. This binding-elution cycle was repeated 12 times, and the eluent was collected and concentrated.

[0042] Finally, the concentrated sample was run on an SDS-PAGE gel and quantified using a fully automated Western blotting analyzer from ProteinSimple, USA.

[0043] 1.4.2 Fishing capabilities of complex systems To verify the capture specificity and anti-interference capability of the S200F platform, the second and third fishing processes of the S200F platform were first tested, in which Protein A captured the purity of the target antibody from 293F cell lysate (containing mepolizumab). Next, on the S200F, T200, and BLI fishing platforms, two sets of samples were tested: Protein A & 293F cell lysate (containing mepolizumab) and Bat ACE2 & 293F cell supernatant (containing SARS-CoV-2 RBD). The operational details are the same as in 1.4.1.

[0044] 2 Results 2.1 Fishing Capabilities of the S200F Platform and Comparison In the S200F and T200 platform tests, Protein A was conjugated to the chip, combined with mepolizumab (100 nM) for 300 seconds, eluted with 10 mM glycine (pH 2.0), neutralized with 1 M Tris-HCl (pH 9.0), and the eluent was recovered. In the BLI platform test, biotin-Protein A was immobilized on the SSA sensor, combined with mepolizumab (100 nM) for 300 seconds, eluted with 10 mM glycine (pH 2.0), the eluent was recovered, and neutralized with 1 M Tris-HCl (pH 9.0). The eluent was concentrated and analyzed by SDS-PAGE and a fully automated Western blotting analyzer. The S200F platform accurately captured and enriched the target molecule, and the mass recovery ratio of the S200F, T200, and BLI platforms was 25:3:1. Figure 4 As shown.

[0045] 2.2 Fishing specificity and anti-interference ability The second and third leaching processes on the S200F platform involve Protein A capturing the target antibody from 293F cell lysates (containing mepolizumab). The third leaching process yields samples with a purification rate exceeding 60%, significantly higher than the purity obtained using the second process. Figure 4 In the test of Protein A&293F cell lysate (containing mepolizumab) samples, the S200F platform showed significantly better anti-interference ability than BLI, but slightly lower than T200. Figure 4In the testing of Bat ACE2 & 293F cell supernatant (containing SARS-CoV-2 RBD), the S200F platform and T200 showed comparable performance with no significant difference, both being stronger than BLI. Figure 5 As shown.

[0046] In the description of this application, it should be understood that the terms "center", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this application.

[0047] The terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, unless otherwise stated, "a plurality of" means two or more.

[0048] In the description of this application, it should be noted that, unless otherwise expressly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection between two components. Those skilled in the art can understand the specific meaning of the above terms in this application based on the specific circumstances.

[0049] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. For the apparatus disclosed in the embodiments, since they correspond to the methods disclosed in the embodiments, the description is relatively simple; relevant parts can be referred to the method section.

[0050] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A molecular fishing system, characterized in that, include: The chip has a flow pool on it; A light source and a camera device are disposed on one or both sides of the chip; Sampling needle; A liquid storage ring is used to quantitatively store fluid. The first pumping mechanism and the second pumping mechanism are used to drive fluid flow; The first valve assembly connects the sampling needle, the reservoir ring, the flow cell, and the buffer source, and can switch between a first state and a second state. In the first state, the sampling needle is connected to the reservoir ring to draw up the sample. In the second state, the reservoir ring is connected to the flow cell to inject the sample into the flow cell. The second valve assembly is connected between the flow cell and the sample collector and the waste bottle, and is used to selectively direct the flow cell effluent to the sample collector or the waste bottle; The third valve group is connected to the pipeline between the first pumping mechanism, the second pumping mechanism, and the flow pool and the first valve group, and is used to control the connection or disconnection between the second pumping mechanism and the flow pool.

2. The molecular fishing system according to claim 1, characterized in that, The first valve group is a six-way valve, the second valve group includes a combination of a six-way valve and a three-way valve, and the third valve group is a three-way valve.

3. The molecular fishing system according to claim 1, characterized in that, The sample collector includes: A sample tray with multiple sample collection positions; A rotary drive mechanism is used to drive the sample disk to rotate so as to align different sample collection positions with the recovery pipeline; A lifting drive mechanism is used to drive the recovery pipeline to move up or down to insert or withdraw from the sample collection position.

4. The molecular fishing system according to claim 3, characterized in that, The rotary drive mechanism includes a rotary motor and a synchronous belt drive mechanism, and the lifting drive mechanism includes a lifting motor and a lead screw drive mechanism.

5. A process for elution using the molecular fishing system according to any one of claims 1-4, characterized in that, It includes a sample binding step and an elution step; the elution step is one of a direct elution mode, an eluent elution mode, or a washing elution mode.

6. The elution process according to claim 5, characterized in that, The direct elution mode includes: After the sample binding step, without a rinsing step, the second pumping mechanism is immediately started to introduce the running buffer into the flow cell through the third valve group for elution, and the eluent is guided to the sample collector through the second valve group for recovery.

7. The elution process according to claim 5, characterized in that, The elution mode of the eluent includes: After the sample binding step, the eluent is quantitatively drawn into the reservoir ring through the first valve group and the sampling needle; The eluent in the storage ring is injected into the flow cell for elution through the first valve group; The eluent is guided through the second valve assembly to the sample collector for recovery.

8. The elution process according to claim 5, characterized in that, The washing and elution modes include: After the sample binding step, the eluent is quantitatively drawn into the reservoir ring through the first valve group and the sampling needle; The process is paused after the eluent is pushed to a predetermined position before the inlet of the flow tank. The second pumping mechanism is activated to introduce the running buffer into the flow pool through the third valve group for cleaning; Stop the second pumping mechanism, while continuing to push the eluent into the flow tank for elution; The eluent is guided through the second valve assembly to the sample collector for recovery.

9. The elution process according to any one of claims 6-8, characterized in that, The sample binding step includes: The sample is quantitatively drawn into the reservoir ring through the sampling needle and the first valve group in an air-sample-air intermittent manner. The sample in the storage ring is injected into the flow cell through the first valve group, so that the target molecules combine with the decoy molecules fixed on the chip surface, and the outflow liquid is guided to the waste liquid bottle through the second valve group.

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