Plasma exosome purification system

Abstract

The application provides a plasma exosome purification system, and belongs to the field of biomedical engineering. The system comprises: an affinity chromatography pretreatment module, which is used for specifically adsorbing exosomes from a plasma sample, and obtaining an exosome eluate through washing and elution; an ultrasonic nanofiltration module, which is used for filtering the exosome eluate under the action of ultrasonic waves to retain the exosomes and remove small molecular impurities, and obtaining an exosome concentrate; a pipetting module, which is used for performing liquid transfer operations of the plasma sample, the washing liquid, the eluate and the exosome eluate among the modules; and a main control module, which is in communication connection with the pipetting module and is used for controlling the pipetting module to perform the liquid transfer operations. The application combines the affinity chromatography and ultrasonic nanofiltration purification technologies, and realizes automatic pipetting control with the help of the main control module and the pipetting module, thereby significantly improving the purity of separation and purification while ensuring the integrity of the exosomes.

Description

Technical Field

[0001] This application relates to the field of biomedical engineering, specifically to a plasma exosome purification system. Background Technology

[0002] Exosomes are extracellular vesicles with a diameter of 30-200 nanometers, possessing a lipid bilayer membrane structure. They carry various biological information related to cell origin, such as proteins, lipids, and nucleic acids, and have important applications in disease diagnosis, treatment, and drug delivery. However, efficiently isolating exosomes from complex biological samples (such as plasma) with high purity remains a significant challenge. Currently, commonly used exosome extraction methods include ultracentrifugation, precipitation, size exclusion chromatography, and affinity chromatography. Ultracentrifugation is time-consuming, expensive, and may damage the integrity of exosomes; precipitation is simple to operate but yields low purity; size exclusion chromatography can maintain exosome activity but has limited separation efficiency; affinity chromatography, based on the principle of antigen-antibody specific binding, has high specificity but is still insufficient to obtain high-purity exosomes. Summary of the Invention

[0003] In view of the above problems, this application provides a plasma exosome purification system that can effectively solve the technical problem that it is difficult to obtain high-purity exosomes in the prior art.

[0004] This application provides a plasma exosome purification system, comprising: an affinity chromatography pretreatment module for adsorbing exosomes from a plasma sample, removing unadsorbed impurities with a washing solution, and detaching the adsorbed exosomes with an elution solution to obtain an exosome eluent; an ultrasonic nanofiltration module for filtering the exosome eluent under ultrasonic waves to retain the exosomes and remove small molecule impurities to obtain an exosome concentrate; a pipetting module for performing a liquid transfer operation, comprising: adding the plasma sample, the washing solution, and the elution solution to the affinity chromatography pretreatment module, and transferring the exosome eluent from the affinity chromatography pretreatment module to the ultrasonic nanofiltration module; and a main control module communicatively connected to the pipetting module for controlling the pipetting module to perform the liquid transfer operation.

[0005] The plasma exosome purification system of this application first utilizes the high specificity of the affinity chromatography pretreatment module to adsorb exosomes from complex plasma samples, removing a large amount of unadsorbed impurities and obtaining exosome eluent. Subsequently, the exosome eluent is filtered under ultrasonic waves by an ultrasonic nanofiltration module to further retain exosomes and remove small molecule impurities. This combined purification method ensures high-purity exosomes while maintaining their integrity. Furthermore, during the exosome purification process, the main control module can control the pipetting module to control liquid transfer, reducing reliance on manual operation during the extraction and storage process, and significantly improving exosome separation efficiency and the reproducibility of purification results.

[0006] In one possible implementation, the affinity chromatography pretreatment module includes: a first protein concentration detection unit, which is communicatively connected to the main control module. The first protein concentration detection unit is used to detect the protein concentration of the washing solution and send the detected protein concentration of the washing solution to the main control module. The main control module is further used to control the pipetting module to add the elution solution to the affinity chromatography pretreatment module when the protein concentration of the washing solution is lower than a preset first protein concentration threshold.

[0007] The first protein concentration detection unit can detect the protein concentration in the washing solution during the impurity removal process in the affinity chromatography pretreatment module, thus characterizing the amount of impurities washed out. When the main control module detects that the protein concentration is consistently below a preset threshold, it indicates that the impurities have been sufficiently removed. Based on this, the main control module can automatically stop washing and switch to the elution step. This ensures the washing effect of the affinity chromatography pretreatment module while avoiding impurity residue caused by insufficient washing or waste caused by excessive washing, thereby improving the controllability and consistency of results in the exosome purification process.

[0008] In one possible implementation, the first protein concentration detection unit is further configured to detect the protein concentration of the exosome eluent and send the detected protein concentration of the exosome eluent to the main control module; the main control module is further configured to control the pipetting module to transfer the exosome eluent to the ultrasonic nanofiltration module when the protein concentration of the exosome eluent is lower than a preset second protein concentration threshold.

[0009] The first protein concentration detection unit monitors the protein concentration of the effluent during exosome elution in real time, providing the main control module with a basis for determining the end of elution. When the main control module detects that the protein concentration is consistently below a preset threshold, it indicates that the exosomes have been basically eluted and the content of the target product in the effluent has been significantly reduced. At this point, the main control module can automatically stop the collection of elution buffer and start the liquid transfer to the next module. This method can ensure the exosome recovery rate while avoiding exosome loss due to premature termination of collection, or buffer waste, impurity introduction, and prolonged processing time due to premature termination of collection, further improving the purification effect of automated control and ensuring the consistency of purification effect.

[0010] In one possible implementation, the ultrasonic nanofiltration module includes: a nanofiltration device for containing the exosome eluent and filtering small molecule impurities in the exosome eluent; an ultrasonic generator for applying ultrasonic waves to the nanofiltration device; and a liquid level detection unit communicatively connected to the main control module, which monitors the liquid level of the exosome eluent in the nanofiltration device in real time and sends the monitored liquid level data to the main control module. The main control module is also communicatively connected to the ultrasonic generator and adjusts the infusion rate of the exosome eluent added to the ultrasonic nanofiltration module by the pipetting module according to the liquid level data of the exosome eluent to maintain a stable liquid level in the ultrasonic nanofiltration module.

[0011] Real-time monitoring of the liquid level within the nanofiltration unit by the liquid level detection unit provides the main control module with real-time data for stable control of the filtration process. When the liquid level fluctuates, the main control module automatically adjusts the rate at which the pipetting module adds liquid to the nanofiltration unit, thereby maintaining the liquid level within a preset range. This method replaces traditional experience-based operation relying on fixed flow rates or times with real-time liquid level feedback control, ensuring a continuous and stable liquid supply while avoiding the risk of overflow due to excessively high liquid levels or the decrease in filtration efficiency due to excessively low liquid levels. By maintaining a stable liquid level, the driving force of the filtration process remains balanced, which helps maintain a constant membrane flux and improve filtration efficiency.

[0012] In one possible implementation, the ultrasonic nanofiltration module further includes: a negative pressure application unit, which applies negative pressure to the nanofiltration device to provide the transmembrane pressure difference required for filtration; wherein the transmembrane pressure difference characterizes the pressure difference between the inlet and outlet sides of the nanofiltration membrane in the nanofiltration device; a pressure detection unit, which is communicatively connected to the main control module, and is used to monitor the transmembrane pressure difference of the nanofiltration device in real time and send the monitored transmembrane pressure difference to the main control module; the main control module is communicatively connected to the negative pressure application unit, and is further used to adjust the output negative pressure of the negative pressure application unit according to the transmembrane pressure difference, so that the transmembrane pressure difference of the nanofiltration device is maintained within a preset pressure difference range.

[0013] The pressure detection unit monitors the transmembrane pressure difference of the nanofiltration unit in real time, providing the main control module with a real-time basis for stable control of the filtration driving force. When the transmembrane pressure difference data deviates from the preset range, the main control module can dynamically adjust the output pressure of the negative pressure application unit to maintain the filtration pressure difference at the predetermined level. By feeding real-time pressure feedback to the main control module and then responding with control, the system ensures stable driving force during the filtration process while avoiding the risk of accelerated membrane fouling or damage to the membrane structure due to excessive pressure difference, and also prevents the risk of inconsistent filtration effects or decreased efficiency due to insufficient pressure difference.

[0014] In one possible implementation, the pressure detection unit is further configured to monitor the membrane flux of the nanofiltration device in real time and send the monitored membrane flux to the main control module; the main control module is further configured to adjust the intensity of the ultrasonic waves emitted by the ultrasonic generator according to the membrane flux, so that the membrane flux of the nanofiltration device is maintained within a preset membrane flux range; wherein, the membrane flux represents the volume of liquid passing through the nanofiltration membrane in the nanofiltration device per preset unit time.

[0015] The pressure detection unit monitors the membrane flux of the nanofiltration unit in real time, providing the main control module with an indicator reflecting changes in filtration efficiency. When the membrane flux decreases due to membrane fouling or other reasons and deviates from the preset range, the main control module can correspondingly increase the working intensity of the ultrasonic generator to maintain stable filtration efficiency. Based on the real-time feedback of membrane flux from the pressure detection unit, the main control module can dynamically control the working intensity of the high-intensity ultrasonic generator during the filtration process to suppress poor membrane flux caused by membrane fouling. This not only ensures the high efficiency and continuity of the filtration process but also reduces the extended processing time and increased energy consumption caused by flux decay. Furthermore, it helps extend the service life of the filter membrane and improves the purification efficiency and effect of exosomes.

[0016] In one possible implementation, the main control module is further configured to control the ultrasonic generator and the negative pressure application unit to stop working when the liquid level data of the exosome eluent is less than or equal to a preset liquid level threshold.

[0017] The liquid level detection unit monitors the liquid level within the nanofiltration unit in real time, providing the main control module with real-time data to determine whether the filtration process has ended. When the liquid level reaches or falls below a preset threshold, it indicates that the exosome eluent has been largely depleted, and purification is complete. At this point, the main control module can control the ultrasonic generator and negative pressure application unit to stop operating. This method allows the exosome purification system to stop operating promptly near the end of the filtration process, avoiding equipment damage and energy waste caused by the ultrasonic generator continuing to run unloaded after the liquid has been drained, thus greatly improving the safety and controllability of the exosome purification system.

[0018] In one possible implementation, the pipetting module includes: a pipette for performing displacement, aspiration, and dispensing operations under the control of the main control module; and a capacitive liquid detection sensor for detecting whether the pipette is in contact with the liquid surface, and sending a liquid surface contact signal to the main control module when the pipette is in contact with the liquid surface, so that the main control module controls the pipette to immerse itself in the liquid surface to perform aspiration operations.

[0019] A capacitive liquid detection sensor monitors the contact between the pipette and the liquid surface in real time, providing the main control module with a precise signal to determine the liquid surface position. Upon detecting a contact signal, the main control module immediately controls the pipette to immerse itself below the liquid surface to a preset depth before aspirating the liquid. This allows the exosome purification system to achieve high-precision, automated liquid transfer while effectively preventing aspiration problems caused by inaccurate liquid surface positioning, significantly improving the accuracy and reliability of liquid transfer.

[0020] In one possible implementation, the pipetting module further includes: a second protein concentration detection unit, which is communicatively connected to the main control module, for real-time monitoring of the protein concentration of the exosome eluent in the ultrasonic nanofiltration module, and sending the monitored protein concentration in the ultrasonic nanofiltration module to the main control module; the main control module is further configured to adjust the ultrasonic intensity according to the rate of change of the protein concentration in the ultrasonic nanofiltration module per unit time when the protein concentration abnormally increases.

[0021] The protein concentration in the ultrasonic nanofiltration module is monitored in real time by a second protein concentration detection unit, providing the main control module with a real-time indicator of the filtration efficiency. When the second protein concentration detection unit detects an abnormally high rate of change in protein concentration per unit time in the ultrasonic nanofiltration module, it indicates that the filtration process may be experiencing a decrease in separation efficiency. Based on this, the main control module can dynamically increase the working intensity of the ultrasonic generator to enhance its anti-fouling effect, promptly suppress the adsorption and accumulation of impurities on the membrane surface, help restore or improve the filtration effect of the membrane, and improve the stability of the ultrasonic nanofiltration process.

[0022] In one possible implementation, the affinity chromatography pretreatment module further includes an RFID unit for recording the number of times the affinity chromatography pretreatment module is used.

[0023] The use of the affinity chromatography pretreatment module is recorded by RFID units, providing the main control module with an objective basis for determining its usability. Before initialization or each use, the main control module reads the number of uses of the affinity chromatography pretreatment module and compares it with the preset safe usage limit. When the usage reaches or exceeds the limit, a corresponding warning is automatically triggered, thus avoiding the problem of poor exosome purification caused by excessive use of the affinity chromatography pretreatment module. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the composition and structure of the plasma exosome purification system provided in the embodiments of this application.

[0025] Figure 2 This is a schematic diagram of liquid transfer provided in an embodiment of this application.

[0026] Figure 3 This is a schematic diagram of the composition of the affinity chromatography pretreatment module provided in an embodiment of this application.

[0027] Figure 4 This is a schematic diagram of the composition structure of the ultrasonic nanofiltration module provided in the embodiments of this application.

[0028] Figure 5 This is a schematic diagram of the pipetting module structure provided in an embodiment of this application.

[0029] Explanation of main component symbols

[0030] 12. Affinity chromatography pretreatment module; 13. Ultrasonic nanofiltration module; 14. Main control module; 15. Pipetting module; 21. RFID unit; 22. Affinity chromatography column; 23. First protein concentration detection unit; 24. Product cell; 31. Nanofiltration device; 32. Pressure detection unit; 33. Liquid level detection unit; 34. Negative pressure application unit; 35. Ultrasonic generator; 41. Pipette; 42. Second protein concentration detection unit; 43. Capacitive liquid probe sensor; 44. Pipette drive device.

[0031] The following detailed description, in conjunction with the accompanying drawings, will further illustrate this application. Detailed Implementation

[0032] In the description of the embodiments in this application, the words "exemplary," "or," and "for example" are used to indicate examples, illustrations, or descriptions. Any embodiment or design scheme described as "exemplary" or "for example" in the embodiments of this application should not be construed as being more preferred or advantageous than other embodiments or design schemes. Specifically, the use of the words "exemplary," "or," and "for example" is intended to present the relevant concepts in a specific manner.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The terminology used in this application's specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. It should be understood that, unless otherwise stated, " / " in this application means "or". For example, A / B can mean A or B. "And / or" in this application is merely a description of the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, and B alone. "At least one" refers to one or more. "More than one" refers to two or more. For example, at least one of a, b, or c can represent: a, b, c, a and b, a and c, b and c, and a, b, and c (seven cases).

[0034] It should also be noted that the terms "first" and "second" in the specification, claims, and drawings of this application are used to distinguish similar objects, not to describe a specific order or sequence. The methods disclosed in the embodiments of this application or the methods shown in the flowcharts include one or more steps for implementing the method. Without departing from the scope of the claims, the execution order of multiple steps can be interchanged, and some steps can also be deleted.

[0035] In the field of plasma exosome isolation and purification technology, obtaining structurally intact and highly bioactive exosomes efficiently and with high purity is of paramount importance for drug delivery vehicle development and basic biological research. However, achieving this goal from plasma samples with complex compositions still faces significant challenges. Currently, mainstream exosome isolation methods all have their limitations: ultracentrifugation, as the "gold standard," is widely used, but the process is time-consuming and the equipment is expensive. Furthermore, the strong shear force generated by high-speed centrifugation may damage the vesicle integrity and biological activity of exosomes; precipitation methods (such as PEG precipitation) are simple to operate, but they co-precipitate a large number of impurity proteins and lipoproteins, resulting in low product purity, and the high concentration of polymers introduced is difficult to remove completely; size exclusion chromatography can better maintain exosome activity, but the separation efficiency is limited, the sample dilution is high, and the ability to distinguish impurities of similar particle size (such as lipoprotein particles and protein aggregates) is insufficient; affinity chromatography is based on antigen-antibody specific binding and has high selectivity, but it is usually used as a capture step. Its eluent still contains small molecule impurities such as soluble impurity proteins, free antibodies, and buffer salts, which is not enough to directly obtain high-purity exosomes suitable for high-sensitivity downstream analysis (such as proteomics and RNA sequencing). These methods generally rely on a lot of manual operation, and the steps are complicated, resulting in low separation efficiency, poor reproducibility, and difficulty in achieving standardization and high throughput, which has become a bottleneck restricting exosome research.

[0036] To address this issue, this application proposes a fully automated, integrated plasma exosome purification system. This system integrates two core purification units: highly specific affinity chromatography capture and ultrasound-enhanced nanofiltration purification. The entire liquid transfer process is automated through a main control module coordinating with the pipetting module. Specifically, the plasma exosome purification system first utilizes an affinity chromatography pretreatment module, which uses specific antibodies immobilized in the module to efficiently capture target exosomes from plasma. After washing and elution steps, a preliminarily purified exosome eluent is obtained. Subsequently, this eluent is automatically transferred to the ultrasound nanofiltration module. With the assistance of ultrasound, the exosomes are precisely retained through the nanofiltration membrane, and residual impurities such as salt ions and small protein molecules are thoroughly removed, ultimately yielding a high-concentration, high-purity exosome concentrate. By combining the methods of "affinity chromatography specific capture and ultrasound nanofiltration purification and concentration" with fully automated control, many shortcomings of existing technologies, such as low product purity, low purification efficiency, and poor result reproducibility, can be overcome.

[0037] The plasma exosome purification system and its working method of this application will be described in further detail below with reference to the accompanying drawings.

[0038] Please see Figure 1 and Figure 2 , Figure 1 and Figure 2The diagrams show the structural components and liquid transfer of a plasma exosome purification system. The system includes: an affinity chromatography pretreatment module 12, an ultrasonic nanofiltration module 13, a pipetting module 15, and a main control module 14. Under the coordinated control of the main control module 14, each module purifies a high-purity exosome concentrate from a plasma sample.

[0039] The affinity chromatography pretreatment module 12 is used to pretreat the collected plasma samples and perform affinity chromatography. Pretreatment removes large particulate impurities (such as cell debris and platelet aggregates) that may interfere with subsequent affinity chromatography. The pretreatment process includes: first, diluting a certain volume of plasma sample (e.g., 1 mL) with an appropriate amount of physiological saline at a ratio between 1:1 and 1:5 to reduce sample viscosity and adjust ionic strength. Then, the diluted sample is centrifuged in a low-temperature centrifuge to precipitate residual cells and large particles, and the supernatant is collected. Finally, the supernatant is mixed with an appropriate amount of binding buffer to obtain the pretreated plasma sample.

[0040] The affinity chromatography pretreatment module 12 includes an affinity chromatography column. This column performs affinity chromatography on the pretreated plasma sample, selectively adsorbing exosomes from complex plasma samples based on the principle of antigen-antibody specific binding. The affinity chromatography column mainly consists of a column tube and specific antibodies filling the inner surface of the tube. When the pretreated plasma sample flows through the column, the specific antigens on the exosome surface bind with the specific antibodies with high affinity, selectively adsorbing the exosomes within the column. Unadsorbed components in the plasma sample, such as proteins and lipids, flow out with the sample, thus achieving preliminary separation of exosomes from impurities. Subsequently, a washing buffer removes residual non-specific adsorbed impurities from the column, and an elution buffer, such as a low-pH buffer, dissociates and elutes the bound exosomes, yielding an exosome eluent.

[0041] The ultrasonic nanofiltration module 13 is used to filter the exosome eluent from the affinity chromatography pretreatment module 12 under the action of ultrasound. The main principle is to use a nanofiltration membrane to filter the exosome eluent. During the filtration process, the sieving effect of the nanofiltration membrane pore size retains larger exosomes while allowing impurities such as salt ions and residual small molecules of protein to pass through, thereby further removing small molecule impurities. During this process, the ultrasound applied to the nanofiltration membrane effectively agitates the liquid on the membrane surface, preventing impurities from accumulating on the membrane surface and avoiding membrane clogging, thus maintaining stable filtration efficiency. Finally, by retaining exosomes and removing small molecule impurities, a high-purity exosome concentrate is obtained.

[0042] The pipetting module 15 is used to perform liquid transfer operations during exosome extraction and storage. These operations include: aspirating a pre-treated plasma sample and adding it to the affinity chromatography column; subsequently, sequentially aspirating washing and elution buffers from their respective reagent reservoirs and adding them to the affinity chromatography column; and finally, aspirating the exosome eluent from the affinity chromatography pretreatment module 12 and adding it to the ultrasonic nanofiltration module 13. When the washing buffer is added to the affinity chromatography column, it washes away impurities that are not specifically bound to the column, i.e., washes away unadsorbed impurities. When the elution buffer is added to the affinity chromatography column, it dissociates the exosomes specifically adsorbed on the column and obtains the exosome eluent.

[0043] The main control module 14 serves as the central processing module of the plasma exosome purification system. The main control module 14 is communicatively connected to the pipetting module 15, the affinity chromatography pretreatment module 12, and the ultrasonic nanofiltration module 13. The main control module 14 is used to control the pipetting module 15 to perform liquid transfer operations based on the purification status of the affinity chromatography pretreatment module 12 and the ultrasonic nanofiltration module 13.

[0044] In one embodiment, the affinity chromatography pretreatment module is as follows: Figure 3 As shown, it includes an RFID unit 21, an affinity chromatography column 22, a first protein concentration detection unit 23, and a product cell 24;

[0045] An RFID unit 21 is disposed on the outer surface of the affinity chromatography column 22. The RFID unit includes an RFID chip and an RFID reader (not shown in the figure). The RFID chip stores key information of the affinity chromatography pretreatment module, including: column model, production batch, first use time, cumulative number of uses, cumulative sample throughput, regeneration times, and performance evaluation parameters. The RFID reader is communicatively connected to the main control module and is used to read the information in the RFID chip to confirm that the column type and number of uses have not exceeded the limits.

[0046] The affinity chromatography column 22 is filled with a specific chromatography medium. These media are immobilized with antibodies that specifically bind to specific antigens (such as CD9, CD63, etc.) on the surface of exosomes in the plasma sample. When the plasma sample flows through, exosomes are precisely adsorbed onto the chromatography medium, while other proteins and impurities that are not adsorbed are removed by a washing buffer, thus achieving the adsorption of exosomes from the plasma sample. The adsorbed exosomes are eluted with an elution buffer to obtain the exosome eluent. The affinity chromatography column of this application includes an albumin-removing column, an IgG-removing column, or a combination of both; the chromatography medium is Cibacon Blue 3GA ligand or Protein A / G ligand.

[0047] The first protein concentration detection unit 23 is located in the pipeline between the affinity chromatography column 22 and the product cell 24. It is used to detect the protein concentration of the washing solution flowing out of the affinity chromatography column 22 and send the detected protein concentration to the main control module in real time. When the detected protein concentration is lower than the preset first protein concentration threshold, it indicates that washing is complete. Specifically, the first protein concentration detection unit 23 is a linear ultraviolet (UV) absorption detector. The detection principle is based on the ultraviolet absorption characteristics of proteins. It uses dual wavelength detection at 280nm and 320nm: 280nm is the main detection wavelength, used to capture the characteristic absorption of aromatic amino acids in proteins; 320nm is the reference wavelength, used to correct for optical interferences such as turbidity and background absorption of the liquid itself. A more accurate protein concentration value is obtained by calculating the difference in absorbance at the two wavelengths.

[0048] Product pool 24 includes a container for holding washing solution and a container for holding exosome eluent, for collecting washing solution and exosome eluent flowing out from affinity chromatography pretreatment module, respectively.

[0049] During the initialization phase of the plasma exosome purification system, the main control module identifies the RFID chip on the affinity chromatography column 22 using an RFID reader. It reads key information from the RFID chip regarding the affinity chromatography pretreatment module to confirm the availability of the affinity chromatography column 22. If unavailable, it triggers relevant alarms and pauses the process. For example, when efficiency falls below a preset threshold, the main control module automatically triggers a warning and pauses the process; or, it checks whether the column type and usage limits have been exceeded. If limits are exceeded, the main control module triggers an alarm, prompting the replacement of the chromatography column and pausing the process.

[0050] In the plasma exosome purification stage, after the main control module controls the pipetting module to add the plasma sample to the affinity chromatography pretreatment module, it allows the sample to stand for a preset time to allow the exosomes to fully bind with the affinity medium. This preset time can be set according to the actual purification effect, and this application does not impose a specific limitation. Next, the main control module controls the pipetting module to obtain washing buffer from a designated location and adds the washing buffer to the affinity chromatography pretreatment module. Subsequently, based on the real-time concentration data provided by the first protein concentration detection unit 23, when the detected protein concentration is lower than a preset first protein concentration threshold, the main control module determines that the impurities have been washed away and triggers the elution procedure. For example, when the main control module determines that the 280nm wavelength absorption signal detected by the first protein concentration detection unit 23 drops to below 10% of the preset baseline and remains stable for 5-10 seconds, or when the corresponding UV absorption curve is obtained through real-time data and the signal peak generated by impurity washing completely falls back to the baseline and no longer shows a new upward trend, the main control module determines that the impurities have been fully washed away.

[0051] After washing, the main control module immediately triggers the elution program. The main control module controls the pipetting module to acquire the eluent from a designated location and add it to the affinity chromatography pretreatment module. Simultaneously, it switches the flow path valve (not shown in the diagram) to direct the effluent to the product tank 24, beginning the collection of the exosome-containing eluent. The main control module continues to monitor protein concentration data in real-time using the first protein concentration detection unit 23 to determine the elution completion point: for example, when a clear elution peak appears on the UV absorption curve of the exosome eluent, and the signal value drops back and stabilizes below the preset second protein concentration threshold, such as below the baseline level of 5% of the elution peak value, the main control module determines that the exosomes have been basically eluted. At this point, the main control module controls the pipetting module to transfer the collected exosome eluent to the ultrasonic nanofiltration module.

[0052] In one embodiment, the ultrasonic nanofiltration module is as follows: Figure 4 As shown, the ultrasonic nanofiltration module includes: a nanofiltration device 31, a pressure detection unit 32, a liquid level detection unit 33, a negative pressure application unit 34, and an ultrasonic generator 35; the pressure detection unit 32, the liquid level detection unit 33, the negative pressure application unit 34, and the ultrasonic generator 35 are all communicatively connected to the main control module.

[0053] The nanofiltration device 31 contains a nanofiltration membrane and an inlet. The inlet receives the exosome eluent from the pipetting module. The nanofiltration membrane filters out small molecule impurities, such as salt ions and small proteins, from the exosome eluent and retains the exosomes. The nanofiltration membrane comprises a ceramic or polymer membrane with a pore size of 30-100 nm. The membrane material includes alumina ceramic, zirconia ceramic, or polyethersulfone polymer. The membrane structure is asymmetric, with a surface layer thickness of 1-2 μm and a support layer thickness of 100-200 μm.

[0054] The ultrasonic generator 35 is tightly fitted to a specific location on the outer wall of the nanofiltration device 31 via a coupling agent or mechanical clamp, thereby effectively transmitting ultrasonic energy to the surface of the nanofiltration membrane inside the nanofiltration device 31 to prevent membrane fouling. The ultrasonic generator operates in pulse mode, with a working cycle of 1-2 seconds on, 2-4 seconds pause, and repeating cyclically; the ultrasonic power density is 0.1-0.5 W / cm², and the frequency is between 20-40 kHz, preferably 30 kHz.

[0055] The liquid level detection unit 33 is a machine vision-based liquid level sensing system, comprising an industrial camera (not shown in the figure), an LED backlight (not shown in the figure), and a corresponding image processing algorithm, which is executed by the main control module. The industrial camera and LED backlight are installed around the nanofiltration device 31. The LED backlight provides stable and uniform background illumination, enabling the industrial camera to clearly capture the liquid level height within the nanofiltration device 31. The image processing algorithm in the main control module analyzes the acquired images in real time, extracts the accurate contour of the liquid surface through an edge detection algorithm, and establishes a mapping relationship between pixel coordinates and the actual liquid level height based on a pre-calibrated scale in the observation area, thereby calculating the real-time liquid level. Based on the liquid level, the main control module also sets corresponding high, medium, and low liquid level thresholds to clearly distinguish the current liquid level height. The liquid level detection unit 33 has the advantages of non-contact and anti-interference, and its detection accuracy can reach ±0.5mm.

[0056] The liquid level detection unit 33 also has an anomaly recognition function. Through continuous analysis of the liquid surface status, it can automatically identify abnormal situations such as liquid splashing and bubble accumulation. Once such an anomaly is identified, the main control module can trigger corresponding processing procedures, such as pausing the current process, issuing an alert, or initiating a cleaning mechanism.

[0057] The negative pressure application unit 34 includes a precision vacuum pump. The power end of the negative pressure application unit 34 is connected to the filtrate outlet pipeline of the nanofiltration device 31 via a pipeline, and is used to apply a controllable negative pressure on the outlet side of the nanofiltration membrane to provide driving force for the filtration of the nanofiltration membrane. The output negative pressure of the negative pressure application unit 34 is typically set to -5 to -30 kPa.

[0058] The pressure detection unit 32 includes a pressure sensor installed on the pipeline where the negative pressure application unit 34 applies negative pressure. The pressure detection unit 32 is used to monitor the pressure at the nanofiltration membrane outlet in real time. Since the pressure difference at the nanofiltration membrane inlet is atmospheric, the transmembrane pressure difference and membrane flux of the nanofiltration device 31 are calculated based on the membrane outlet pressure and atmospheric pressure. The transmembrane pressure difference provided across the nanofiltration membrane is a stable sealing pressure provided by a sealed tank. The liquid passing through the nanofiltration membrane is collected in this sealed negative pressure tank. Therefore, the volume of liquid passing through the nanofiltration membrane per unit time can be determined by the rate of pressure drop in this sealed tank, thereby calculating the membrane flux. Optionally, the membrane flux can also be calculated directly based on the liquid level height detected by the liquid level detection unit within the nanofiltration device 31, determining the corresponding liquid volume change and time.

[0059] The main control module is also used to dynamically adjust the output negative pressure of the negative pressure application unit according to the transmembrane pressure difference, so as to maintain the transmembrane pressure difference of the nanofiltration device within a preset pressure difference range. This ensures that the nanofiltration process is carried out under a stable and controllable driving force, guarantees the consistency of filtration efficiency and product recovery rate, and effectively prevents accelerated membrane fouling or membrane structure damage caused by excessive pressure or drastic fluctuations. The initial control strategy of the main control module for the pressure detection unit 32 includes: applying a negative pressure of -5 to -10 kPa in the early stage of concentration; adjusting to -10 to -20 kPa in the middle stage of concentration according to the membrane flux; and reducing to -5 kPa near the end of concentration.

[0060] When the transmembrane pressure difference changes abruptly, the main control module triggers a relevant alarm. For example, if the transmembrane pressure difference drops by more than 50% in a short period of time, it is determined to be a risk of membrane rupture, and the system is immediately shut down and an alarm is issued.

[0061] The preset pressure difference range is the working pressure range determined based on the nanofiltration membrane's tolerance pressure and optimal filtration efficiency, for example, 0.1 to 0.3 MPa. The preset pressure difference range can also be set according to actual needs, and this application does not impose any specific limitations.

[0062] The main control module is also used to dynamically adjust the infusion rate of the exosome eluent added by the pipetting module to the ultrasonic nanofiltration module based on the liquid level data detected by the liquid level detection unit 33, thereby maintaining the liquid level in the ultrasonic nanofiltration module within a preset liquid level range to ensure the stable operation of the filtration process. When the liquid level data of the exosome eluent in the nanofiltration device is less than or equal to the preset liquid level threshold, the main control module determines that the ultrasonic nanofiltration is completed and controls the ultrasonic generator and the negative pressure application unit to stop working.

[0063] The main control module is also used to dynamically adjust the intensity of the ultrasonic waves emitted by the ultrasonic generator based on the membrane flux, so as to maintain the membrane flux of the nanofiltration unit within a preset membrane flux range, thereby maintaining stable filtration efficiency, preventing membrane flux from decreasing due to fouling, and realizing intelligent dynamic suppression and cleaning of membrane fouling, ensuring continuous high efficiency and stability of the separation process. For example, when the flux drops by more than 30% / 10 minutes, the main control module determines that it is membrane fouling and triggers the ultrasonic cleaning program; or, when a drop in membrane flux of more than 30% is detected, it automatically increases the ultrasonic power or extends the ultrasonic on-time to clean contaminants on the membrane surface.

[0064] Wherein, membrane flux represents the volume of liquid passing through the nanofiltration membrane in the nanofiltration device per unit time; the preset membrane flux range is a percentage range set based on the standard initial flux value of the filter membrane in the nanofiltration device, such as 60% to 90% of the standard initial flux, and the specific preset membrane flux range is not specifically limited in this application.

[0065] In one embodiment, the pipetting module is as follows: Figure 5 As shown, the pipetting module includes a pipetting needle 41, a second protein concentration detection unit 42, a capacitive liquid detection sensor 43, and a pipetting needle drive device 44.

[0066] The second protein concentration detection unit 42, the capacitive liquid sensor 43, and the pipette drive device 44 are connected to the main control module.

[0067] One end of the pipette 41 is located at the mechanical movement end of the pipette drive device 44; the pipette drive device 44, under the control of the main control module, is used to control the pipette 41 to perform up-down and left-right displacement, as well as liquid aspiration and dispensing operations.

[0068] A capacitive liquid level sensor 43 is mounted on the pipette 41. The sensor detects whether the pipette is in contact with the liquid surface and sends a liquid surface contact signal to the main control module when the pipette contacts the surface. This allows the main control module to control the pipette to immerse itself in the liquid for aspiration. For example, the capacitive liquid level sensor 43 has a resolution of 0.1 mm. The pipette descends at an adjustable speed, and upon contact with the liquid surface, the capacitance changes abruptly, thus determining the liquid surface position and controlling the pipette to immerse itself 2-3 mm below the surface for aspiration. The capacitive liquid level sensor 43 can be a three-dimensional liquid level detection sensor. The sensor also has a bubble detection function, identifying bubbles by monitoring the continuity of the capacitance signal. When a bubble is detected, the main control module immediately controls the pipette 41 to stop aspiration and reposition itself to the liquid surface.

[0069] The second protein concentration detection unit 42 is mounted on the pipette 41. It is used to monitor the protein concentration changes in the exosome eluent in the nanofiltration device in real time after adding exosome eluent to the ultrasonic nanofiltration module via the pipette 41, and to send the monitored protein concentration in the nanofiltration device to the main control module. The second protein concentration detection unit 42 is also a linear ultraviolet (UV) absorption detector, and its testing principle is consistent with that of the first protein concentration detection unit.

[0070] The main control module is also used to adjust the intensity of the ultrasonic waves emitted by the ultrasonic generator to enhance the anti-fouling effect of the membrane surface, and / or adjust the intensity of the negative pressure applied to the nanofiltration device by the negative pressure application unit to optimize the filtration kinetics, thereby dynamically responding to the changing trend of product purity during the filtration process, so as to enhance or restore the nanofiltration performance of the nanofiltration membrane and ensure the purification efficiency and high purity of the exosome concentrate.

[0071] The pipetting module also includes a syringe pump (not shown in the figure), which is connected to the pipetting needle 41 via tubing. Under the control of the main control module, the volume and flow rate of liquid aspiration and dispensing are precisely controlled. The main control module receives real-time liquid level data from the liquid level detection unit 33 and compares this data with a preset liquid level target value. This target threshold can be set according to actual needs, and this application does not impose specific limitations. When the liquid level is detected to be lower than the target value, the main control module sends a command to the syringe pump to increase its output flow rate to replenish the liquid more quickly. Conversely, when the liquid level is higher than the target value or close to the overflow warning line, the syringe pump is commanded to reduce its flow rate or even stop, thereby dynamically stabilizing the liquid level in the nanofiltration device of the ultrasonic nanofiltration module within a preset range, providing stable fluid conditions for constant pressure or constant flow filtration.

[0072] In one embodiment, the plasma exosome purification system further includes a human-machine interface. The human-machine interface is preferably a touchscreen, with a size of not less than 5 inches and a resolution of not less than 800×480 pixels, and is communicatively connected to the main control module, serving as the core terminal for user interaction with the plasma exosome purification system.

[0073] The human-machine interface is primarily used for setting parameters and monitoring the operational status of the plasma exosome purification system. Regarding parameter settings, users can conveniently input or select various key parameters of the purification process through this interface, such as: the plasma sample loading volume, the type and amount of washing and elution buffers in the affinity chromatography pretreatment module, the ultrasonic intensity, cycle time, target transmembrane pressure difference, or concentration endpoint volume in the ultrasonic nanofiltration module. Simultaneously, users can also perform tasks, select and start programs, and pause or stop them.

[0074] Regarding status display, the human-machine interface provides a real-time and intuitive overview of the system's operation, including but not limited to: the current execution step, the real-time working status of each module (such as "sample loading," "washing," and "ultrasonic nanofiltration"), key process parameters (such as real-time liquid level, transmembrane pressure difference, membrane flux, and UV absorbance curve), estimated completion time, and system alarms and prompts. All historical operating data and results can be queried, reviewed, and exported in a standardized report format through this interface.

[0075] This integrated human-machine interface, through its graphical and wizard-driven interactive design, transforms complex multi-module collaborative operation processes into clear and simple visual control, significantly reducing the professional threshold and training costs for operators. Its close collaboration with the main control module allows the entire process, from method establishment and process monitoring to result analysis, to be completed on a single terminal, greatly improving the automation, ease of operation, and traceability of the entire exosome purification process.

[0076] The plasma exosome purification system also has a cloud data synchronization function, which can upload the operating parameters, quality control data and chromatography column usage records of each batch to the cloud server to achieve batch traceability and remote diagnosis of related equipment.

[0077] In one embodiment, the plasma exosome purification system also has the function of multi-channel parallel configuration. The system can be configured with 2 to 8 independent sample processing channels with the same structure. Each channel includes its own dedicated affinity chromatography column, nanofiltration device, pipette, pressure sensor, capacitive liquid probe sensor and tubing system, so that multiple plasma samples can be purified independently at the same time, which significantly improves the equipment throughput and processing efficiency.

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit it. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the spirit and scope of the technical solutions of this application.

Claims

1. A plasma exosome purification system, characterized in that, The system includes: An affinity chromatography pretreatment module is used to adsorb exosomes in plasma samples, remove remaining unadsorbed impurities with washing solution, and detach the adsorbed exosomes with elution solution to obtain exosome eluent. An ultrasonic nanofiltration module is used to filter exosome eluent under the action of ultrasound to retain exosomes and remove small molecule impurities, obtaining an exosome concentrate. The ultrasonic nanofiltration module includes: a nanofiltration device, an ultrasonic generator, a liquid level detection unit, a negative pressure application unit, and a pressure detection unit. The nanofiltration device contains the exosome eluent and filters out small molecule impurities. The ultrasonic generator applies ultrasound to the nanofiltration device. The liquid level detection unit monitors the liquid level of the exosome eluent in the nanofiltration device in real time. The pressure detection unit monitors the transmembrane pressure difference and membrane flux of the nanofiltration device in real time. The negative pressure application unit applies negative pressure to the nanofiltration device to provide the transmembrane pressure difference required for filtration. The transmembrane pressure difference represents the pressure difference between the inlet and outlet sides of the nanofiltration membrane in the nanofiltration device, and the membrane flux represents the volume of liquid passing through the nanofiltration membrane in the nanofiltration device per preset unit time. A pipetting module is provided to perform liquid transfer operations, including: adding the plasma sample, the washing buffer, and the eluent to the affinity chromatography pretreatment module, and transferring the exosome eluent from the affinity chromatography pretreatment module to the ultrasonic nanofiltration module; the pipetting module includes a second protein concentration detection unit, which is communicatively connected to the main control module, for real-time monitoring of the protein concentration of the exosome eluent in the ultrasonic nanofiltration module, and sending the monitored protein concentration in the ultrasonic nanofiltration module to the main control module; the main control module is also used to adjust the ultrasonic intensity based on the rate of change of the protein concentration in the ultrasonic nanofiltration module per unit time when the protein concentration abnormally increases; The main control module is communicatively connected to the pipetting module, the ultrasonic generator, the liquid level detection unit, the pressure detection unit, and the negative pressure application unit. The main control module is used to: control the pipetting module to perform liquid transfer operations; adjust the infusion rate of the exosome eluent from the pipetting module to the ultrasonic nanofiltration module based on the liquid level data of the exosome eluent to maintain a stable liquid level in the ultrasonic nanofiltration module; adjust the output negative pressure of the negative pressure application unit based on the transmembrane pressure difference to maintain the transmembrane pressure difference of the nanofiltration device within a preset pressure difference range; and adjust the intensity of the ultrasonic waves emitted by the ultrasonic generator based on the membrane flux to maintain the membrane flux of the nanofiltration device within a preset membrane flux range.

2. The plasma exosome purification system as described in claim 1, characterized in that, The affinity chromatography pretreatment module includes: A first protein concentration detection unit is communicatively connected to the main control module. The first protein concentration detection unit is used to detect the protein concentration of the washing liquid and send the detected protein concentration of the washing liquid to the main control module. The main control module is also used to control the pipetting module to add the elution solution to the affinity chromatography pretreatment module when the protein concentration of the washing solution is lower than a preset first protein concentration threshold.

3. The plasma exosome purification system as described in claim 2, characterized in that, The first protein concentration detection unit is also used to detect the protein concentration of the exosome eluent and send the detected protein concentration of the exosome eluent to the main control module; The main control module is also used to control the pipetting module to transfer the exosome eluent to the ultrasonic nanofiltration module when the protein concentration of the exosome eluent is lower than a preset second protein concentration threshold.

4. The plasma exosome purification system as described in claim 1, characterized in that, The main control module is also used to control the ultrasonic generator and the negative pressure application unit to stop working when the liquid level data of the exosome eluent is less than or equal to a preset liquid level threshold.

5. The plasma exosome purification system as described in claim 1, characterized in that, The pipetting module includes: A pipette, which is used to perform displacement, aspiration and dispensing operations under the control of the main control module; A capacitive liquid detection sensor is used to detect whether the pipette is in contact with the liquid surface, and when the pipette is in contact with the liquid surface, it sends a liquid surface contact signal to the main control module so that the main control module controls the pipette to immerse itself in the liquid surface to perform a liquid aspiration operation.

6. The plasma exosome purification system as described in claim 2, characterized in that, The affinity chromatography pretreatment module further includes an RFID unit, which is used to record the number of times the affinity chromatography pretreatment module is used.

Images