Flowing type full-automatic biological particle quantitative detection treatment method and device

Abstract

The invention discloses a flowing type full-automatic biological particle quantitative detection processing method and device, which are characterized in that detection signals are collected, processed and analyzed in real time, the concentration of a detected substance in a sample is calculated, and the dilution process is controlled, so that the accuracy and reliability of a detection result are ensured; through automatic concentration adjustment, real-time monitoring, calculation and automatic dilution of the sample concentration are realized, and the sample concentration is ensured to be adjusted to a reasonable range required by detection; the flowing of a sample and a diluent among a sample storage area, a sample dilution area, a diluent storage area, a sample detection area and a subsequent processing module is realized through a sample feeding module, and the high efficiency and continuity of a detection process are ensured through an integrated design; after the concentration of the sample reaches the standard, the sample can be subjected to follow-up treatment, such as sorting and separation, so that fine treatment of the sample is realized; the detection can cover a variety of biological particles, including nanoparticles, cells, extracellular vesicles, bacteria, viruses and the like, and has a wide application range.

Description

Technical Field

[0001] The present invention relates to, in particular, a flow-type fully-automatic biological particle quantitative detection and processing method and device. Background Art

[0002] With the development of biotechnology, the detection of nano-bioparticles is increasingly being used in medicine, pharmaceuticals, environmental science and other fields. At present, the mainstream methods of bioparticle detection technology include nano flow cytometry (Nano FCM), dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), etc. These technical means have made significant progress in detection accuracy and sensitivity. However, these methods still face some technical bottlenecks and challenges in practical applications.

[0003] Nano flow cytometer (Nano FCM) is mainly used for the detection of single nanoparticles and can simultaneously measure the size and fluorescence intensity of particles. However, nano flow cytometer has limitations on injection concentration and requires that the injection concentration of the sample be within a reasonable range to obtain accurate measurement results. However, it is often difficult to determine the exact concentration of the sample before detection, so multiple pre-dilutions and detections are required, resulting in complex operations and low efficiency.

[0004] Dynamic light scattering (DLS) is used to measure the average size of nanoparticles. The particle size is determined by analyzing the intensity changes of scattered light. However, dynamic light scattering (DLS) is sensitive to sample concentration. Under high concentration conditions, light will be scattered multiple times (multiple scattering) after entering the sample, which will cause the light signal received by the detector to become complex and difficult to resolve the light intensity fluctuations of single scattering, thus affecting the accurate measurement of particle size. Low-concentration samples can reduce the multiple scattering effect, so that the scattered light received by the detector mainly comes from single scattering, and the data is easier to parse and analyze. However, the probability of the sample entering the detection area becomes relatively small and the measurement time is longer, so the sample concentration needs to be precisely controlled, which is difficult in actual operation.

[0005] Nanoparticle tracking analysis (NTA) measures particle size and concentration by tracking the Brownian motion of particles in solution. However, NTA is sensitive to sample concentration. Under high concentration conditions, there are too many particles in the sample, which can easily lead to particle overlap and occlusion effects. This makes it difficult for the camera to accurately track the motion trajectory of a single particle, thus affecting the measurement accuracy of particle size and concentration. In low-concentration samples, particles are sparsely distributed, so the camera can more easily distinguish and track the motion trajectory of each particle, and the measurement results are more accurate. However, the sample concentration needs to be adjusted and measured multiple times to obtain satisfactory results. Therefore, NTA requires operators to have a high level of technical skills, the operation is complex, and the measurement process is time-consuming.

[0006] The above shortcomings also exist in the detection process of other biological particles (such as blood cells, extracellular vesicles, bacteria, viruses, extracellular vesicles EV, etc.). It is precisely because of these shortcomings that the existing technology has problems such as low efficiency, complex operation, and unstable measurement results in practical applications. Summary of the invention

[0007] In order to solve the shortcomings of the prior art, the concentration of the biological particle solution that is not convenient to measure the concentration in advance is adjusted to an appropriate range through efficient and automated dilution, thereby improving the efficiency and accuracy of biological particle detection, simplifying the operation process, and covering a wider range of detection needs. The present invention adopts the following technical solutions:

[0008] A flow-type fully automatic biological particle quantitative detection and processing method comprises the following steps:

[0009] Step 1: Sample loading: Place the sample to be tested into the test tube in the sample storage area and insert it into the test tube holder. After the device is started, the sample enters the sample detection area (flow cell) through the sample delivery module and is ready for detection.

[0010] Step 2: Sample detection: When the sample passes through the detection area, the detection module collects the signal of the object to be measured in the sample (such as a photoelectric signal), and the signal processing module processes and analyzes the collected signal to calculate the current concentration of the object to be measured in the sample;

[0011] Step 3: Concentration calculation and dilution formula estimation: Based on the test results, the signal processing module calculates the volume of diluent and stock solution required to dilute the sample to the target concentration, and controls the mixing ratio of the diluent and the sample based on the calculation results;

[0012] Step 4: Sample dilution; the control module controls the mixing of the diluent and the sample, and achieves rapid mixing through fluid disturbance (such as magnetic rotor, vibration, etc.) to complete the dilution of the sample;

[0013] Step 5: Retest the diluted sample; the diluted sample enters the sample detection area (flow cell) through the sample delivery module for detection again. The detection result is processed by the signal processing module to detect whether the concentration after dilution meets the standard. If not, it prompts to replace the sample or return to step 3 for repeated dilution and detection. If it meets the standard, the detection is completed;

[0014] Step 6: Send the samples that have completed the test to the subsequent processing module for subsequent testing, sorting, placement and other refined processing of the samples, and record the corresponding data.

[0015] Furthermore, in step 2, the detection module collects the signal (such as photoelectric signal) of the object to be measured in the sample, analyzes and records the unit time The amount of analyte flowing through the sample detection area With injection volume ;

[0016] In step 3, based on the detection results in step 2, the signal processing module calculates the current sample concentration. , compare the current sample concentration The concentration range of the target substance ~ Compare, among them To allow the lowest concentration, The maximum concentration allowed. If the current sample concentration is Within the allowable concentration range of the measured substance ~ If the concentration of the substance in the sample reaches the target concentration range, the sample with the concentration reaching the target concentration range will be continuously delivered to complete the subsequent processing process. Lower than When the current sample concentration is too dilute, the user is reminded that it cannot meet the subsequent minimum detection requirements; if the current sample concentration is Higher than When the signal processing module determines the concentration of the target object The required sample stock volume , and calculate the diluted sample volume according to the following formula The required dilution volume :

[0017]

[0018]

[0019] Among them, determine the concentration of the target analyte The required sample stock volume hour, Minimum number of analytes required to meet minimum statistical significance , and the sample volume after dilution Cannot be higher than the sample dilution zone volume .

[0020] Furthermore, in step 3, when the sample injection flow rate is fixed at Sample injection time The injection volume can be determined by Quantitative calculation is performed, at which point the sample concentration Time concentration .

[0021] Furthermore, in step 1, the sample delivery module introduces the sample and the sheath fluid into the sample detection area together; in step 5, the sample delivery module again introduces the diluted sample and the sheath fluid into the flow cell for detection.

[0022] Furthermore, in step 4, the control module, according to the above calculation results, respectively transfers the corresponding sample stock solution and diluent into the sample dilution zone, and completes the mixing of the sample stock solution and the diluent, wherein the volume of the sample stock solution and the diluent is By traffic With time ,through After calculation, precise control is performed, including flow Can be further increased by air pressure and flow resistance ,through Control and regulation can be performed after calculation, or by the cross-sectional area of ​​the syringe loaded with the sample And the sample feeding line speed ,through Control and adjustment are carried out after calculation.

[0023] Furthermore, in step 5, when the diluted sample passes through the detection zone, the detection module collects the signal (such as photoelectric signal) of the analyte in the sample, analyzes and records the unit time. The amount of analyte flowing through the sample detection area With injection volume , according to the formula Calculate the concentration of diluted samples ; Compare the concentration of diluted samples according to step 3 The concentration range of the target substance ~ According to the relationship between the concentration and the measured substance, adjust the concentration according to the method of steps 3 to 5 until the concentration of the measured substance falls into the allowable range. ~ , To allow the lowest concentration, The highest concentration allowed.

[0024] A mobile fully automatic biological particle quantitative detection and processing device, comprising a sample storage area, a diluent storage area, a sample detection area, a sample dilution area, a sample delivery module, a detection module, a signal processing and control module, and a subsequent processing module;

[0025] The sample storage area is used to store the sample to be tested containing the object to be tested, and the sample can be a biological particle suspension in liquid form; the specific form of the storage area is a storage container such as a test tube, a quartz tube, or a separately designed container, etc.;

[0026] The diluent storage area is used to store diluent for diluting samples. The diluent itself can be flexibly set according to user needs, such as buffer, deionized water, cell culture fluid, etc.; the specific form of the storage area is a storage container such as a test tube, a quartz tube, or a separately designed container;

[0027] The sample dilution zone is used for sample dilution and can achieve rapid mixing through fluid disturbance (such as magnetic rotor, vibration, suction and injection, etc.);

[0028] The sample detection area collects electrical signals (such as photoelectric signals) related to the quantity of the object being measured in the sample through the detection module;

[0029] The sample delivery module is used for the flow delivery of samples and diluents between the sample storage area, the sample dilution area, the diluent storage area, the sample detection area and the subsequent processing module; the power mode can be a pneumatic pump, a syringe, a peristaltic pump, a centrifuge, etc., and the liquid delivery is achieved through mechanical principles such as pressing, suction, dead weight, centrifugal throwing or pumping;

[0030] The signal processing and control module is used to collect, process and analyze the signals related to the number of analytes collected from the sample detection area, calculate the concentration of the analyte in the current sample, and calculate the dilution formula according to the detection result, control the sample and the diluent to enter the sample dilution area according to the calculated required amount, and complete the dilution of the sample stock solution; the signal processing and control module realizes real-time monitoring and automatic dilution of the sample concentration, significantly improves the detection efficiency, reduces manual operations, increases the detection speed, reduces human errors, and increases the automation level of sample processing; through accurate dilution formula calculation and control, ensure that the sample concentration is adjusted to a reasonable range, ensure the accuracy and reliability of the detection results, reduce sample waste, and improve detection efficiency;

[0031] The subsequent processing module performs subsequent processing on the sample, such as continuing detection, sorting or separation, when the concentration of the analyte in the sample is within the target concentration range, to achieve refined processing of the sample.

[0032] Furthermore, the sample dilution area and the sample storage area are the same area, and the same area is connected to the waste liquid bottle. When the volume of the area is small, a portion of the diluted sample is drawn into the waste liquid bottle.

[0033] Furthermore, the detection of the sample detection area adopts a flow-based single particle analysis (FCM) or nanoparticle tracking analysis (NTA) or dynamic light scattering analysis (DLS) method based on a flow cell or a microfluidic channel flow cell, and the detection principle of the sample includes photoelectric detection (such as scattered light, fluorescence signal) or electrical detection (such as impedance measurement), which is finally converted into an electrical signal for data information processing.

[0034] Furthermore, the device also includes a sheath liquid storage area for storing sheath liquid to form a sheath flow direction so that the sample flows and is counted in the direction of the sheath flow; the sample delivery module includes an automatic air pressure regulating air pump and a multi-channel automatic air pressure regulating valve, wherein the multi-channel automatic air pressure regulating valve of each channel includes an automatic air pressure regulating valve, a switch valve, and an air leakage valve of a single channel that work independently, the automatic air pressure regulating air pump is connected to the automatic air pressure regulating valve and the switch valve of the single channel in sequence, the switch valve and the air leakage valve are cooperatively arranged as the output of the automatic air pressure regulating valve of the single channel, and the automatic air pressure regulating valves of each channel are respectively connected to the sample storage area, the sample dilution area, the diluent storage area and the sheath liquid storage area.

[0035] The advantages and beneficial effects of the present invention are:

[0036] The present invention provides a flow-type fully automatic biological particle quantitative detection and processing method and device, which realizes efficient and rapid automated sample detection, concentration calculation and dilution adjustment by integrating sample loading, detection and dilution, reduces the need for manual operation and multiple detections, improves detection efficiency, reduces the technical requirements and operation difficulty of operators, and simplifies the operation process; at the same time, through precise signal processing and control modules, the sample concentration is monitored and adjusted in real time to ensure the accuracy and reliability of each detection result; the present invention is not only suitable for the detection of nanoparticles, but also suitable for the detection of biological particles such as cells, extracellular vesicles, bacteria, viruses, etc., covering a wide range of biological particle detection application fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of the structure of a flow-type nanoparticle detection device in an embodiment of the present invention.

[0038] Figure 2 It is a schematic diagram of the device structure in which the sample dilution area and the sample storage area are unified in an embodiment of the present invention.

[0039] Figure 3 It is a schematic diagram of the structure of the device in which the dilution area and the storage area are unified and a waste liquid bottle is added in an embodiment of the present invention.

[0040] Figure 4 It is a schematic diagram of a flow detector in which a normal flow cell is used in the sample detection area in an embodiment of the present invention.

[0041] Figure 5 Schematic diagram of a flow detector in which a microfluidic flow cell is used in the sample detection area in an embodiment of the present invention.

[0042] Figure 6 It is a schematic diagram of the structure of using nanoparticle tracking analysis NTA in the sample detection area in an embodiment of the present invention.

[0043] Figure 7It is a schematic diagram of the structure in which the sample detection area adopts dynamic light scattering DLS in an embodiment of the present invention.

[0044] Figure 8 Schematic diagram of the structure of a multi-channel air pressure regulating valve in an embodiment of the present invention.

[0045] Fig. 9 It is a schematic diagram of post-processing using a microfluidic channel-based dispensing instrument in an embodiment of the present invention.

[0046] Fig.10 Schematic diagram of the chip flow channel structure of the separation instrument in the embodiment of the present invention.

[0047] Fig.11 4 is a flow chart of a flow-type fully automatic nanoparticle detection method in an embodiment of the present invention.

[0048] Fig.12 Schematic diagram of the flow-type fully automatic nanoparticle detection method in an embodiment of the present invention.

[0049] Fig.13 It is a flow chart of signal acquisition, processing and control in an embodiment of the present invention. DETAILED DESCRIPTION

[0050] The specific implementation of the present invention is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described here is only used to illustrate and explain the present invention, and is not used to limit the present invention.

[0051] like Figure 1 As shown, a flow-type fully automatic biological particle quantitative detection and processing device is suitable for the detection of biological particles such as nanoparticles, cells, extracellular vesicles, bacteria, viruses, etc. The device integrates sample loading, detection and dilution functions, and can automatically adjust the sample concentration to ensure the accuracy and efficiency of the detection, including a sample storage area, a diluent storage area, a sample detection area, a sample dilution area, a sample delivery module, a detection module, a signal processing and control module, and a subsequent processing module. The target objects that the device can detect and process have the following key characteristics: the diameter or long axis size of the target object is preferably <100 µm (applicable to biological particles such as plant cells, bacterial aggregates, cell aggregates, etc.), <30 µm is better (applicable to most human cells, pollen and other medium-sized biological particles), and <1 µm is the best (used with small biological particles such as viruses, nucleic acid drugs, nano drug carriers, small bacteria, and extracellular vesicles).

[0052] The sample storage area is used to store samples to be tested containing the objects to be tested. The samples can be biological particle suspensions in liquid form. The specific form of the storage area is storage containers such as test tubes, quartz tubes, or separately designed containers.

[0053] The diluent storage area is used to store diluents for diluting samples. The diluent itself can be flexibly set according to user needs, such as buffer solution, deionized water, etc.; the specific form of the storage area is storage containers such as test tubes, quartz tubes, or separately designed containers, etc.

[0054] The sample dilution area is used for sample dilution and can be the same area as the sample storage area, such as Figure 2 As shown, rapid mixing can be achieved by fluid disturbance (such as magnetic rotor, vibration, suction and injection, etc.). In another embodiment, as Figure 3 As shown, if the sample area is too small, a portion of it can be extracted to waste.

[0055] The sample detection area is used to collect electrical signals related to the amount of the measured object in the sample, such as Figure 4 As shown; the sample detection area can be shared with the sample storage area, and the detection area can be solid or suspended in the air; when the detection area is solid, its material can be transparent or opaque, with transparency being preferred, and the detection area can be designed to have a cross-sectional area of ​​less than 1mm 2 Microfluidic channels such as Figure 5 As shown, high-precision detection can be achieved; the detection principle can be optical detection or electrical detection, and the optical detection method includes single particle detection (such as scattering / fluorescence) or overall detection (such as transmittance). Specific methods include flow cytometry (FCM), camera detection, nanoparticle tracking analysis (NTA, such as Figure 6 As shown, ), dynamic light scattering (DLS, as Figure 7 The electrical detection methods include impedance measurement, single particle impedance measurement or overall particle impedance measurement, etc., which will eventually be converted into electrical signals for data information processing.

[0056] The sample delivery module is used to realize the flow delivery of samples and diluents between the sample storage area, sample dilution area, diluent storage area, sample detection area and subsequent processing modules; the power mode can be a pneumatic pump, a syringe, a peristaltic pump, a centrifuge, etc., through the mechanical principles of pressing, suction, dead weight, centrifugal throwing or pumping to realize liquid delivery. In the embodiment of the present invention, if Figure 8 As shown, the sample delivery module includes an automatic air pressure regulating air pump and a multi-channel automatic air pressure regulating valve, wherein the multi-channel automatic air pressure regulating valve includes a plurality of independently working single-channel automatic air pressure regulating valves, a switch valve, and an air leakage valve.

[0057] The detection module is used to collect the signal (such as photoelectric signal) of the object being tested in the sample.

[0058] The signal processing and control module is used to collect, process and analyze the signals related to the number of analytes collected from the sample detection area, deduce the concentration of the analyte in the current sample, and calculate the dilution formula based on the detection results. By controlling the sample delivery module, the control sample and diluent are adjusted to enter the sample dilution area according to the required amount estimated to complete the dilution of the sample stock solution. The signal processing and control module's calculation result of concentration and volume is the number of particles per unit volume, which can be calculated by dividing the number by volume or the number by time (volume concentration, time concentration, weight concentration, etc.); when the target concentration is lower than the current concentration, dilution is performed, and the calculation of the dilution volume can be carried out by one-step dilution method, two-step experimental method or PID multi-step adjustment method. The original sample can be directly diluted or part of the original sample can be taken out to the sample dilution area for dilution; the diluted sample can be re-tested through the sample storage area or directly delivered to the detection area. The re-testing method and the detection area can be the same or different from the previous detection method, preferably the same;

[0059] The signal processing and control module realizes real-time monitoring and automatic dilution of sample concentration, significantly improving detection efficiency, reducing manual operations, increasing detection speed, reducing human errors, and increasing the automation level of sample processing; through precise dilution formula calculation and control, it ensures that the sample concentration is adjusted to a reasonable range, ensures the accuracy and reliability of the test results, improves the accuracy of the test results, reduces repeatability, and reduces sample waste.

[0060] The subsequent processing module can perform subsequent processing on the sample when the concentration of the analyte in the sample is within the target concentration range, such as injecting a spacer solution after dilution or detection, sorting or splitting, to achieve fine processing of the sample. The subsequent processing process includes the sample delivery, observation, flow stop, flushing and re-delivery sample injection process, and information backup can be performed before and after injection, including photos or waveforms, fluorescent signals, etc. In one embodiment of the present invention, Fig. 9 , Fig.10 As shown, post-processing was performed by a splitter.

[0061] The specific working principle of the post-processing using the microfluidic channel-based dispensing instrument in the embodiment of the present invention is as follows:

[0062] 1. Sample injection: The automatic air pressure regulating valve and the automatic air pressure regulating pump work together to control the sample flow. The sample enters the system through the sample injection port of the microfluidic chip. The width of the injection port is 100 µm, which is suitable for the entry of targets <100 µm.

[0063] 2. After the sample enters the microfluidic channel, the flow direction and speed of the sample are controlled by a multi-channel automatic air pressure regulating valve, so that the sample enters the observation field of view. The size of the field of view is 300 µm x 600 µm, and the thickness of the chip flow channel is 40 µm. The flow rate and stability of the sample flow are controlled by the injection system to meet the data acquisition requirements of the industrial camera of the detection module. The detection module collects sample signals through optical or electrical methods such as photography and scattering, and the detection module performs real-time detection and analysis of the sample.

[0064] 3. The signal processing and control module calculates the concentration and the dilution formula according to the test results, and then dilutes the sample through the sample dilution area. The diluted sample is tested again to see if the concentration meets the standard. If not, it returns to repeat the dilution and testing until the concentration meets the standard.

[0065] 4. When the concentration of the analyte in the sample reaches the standard, the signal processing and control module controls the electric orifice plate moving stage to move to the sample receiving position, and the industrial camera takes pictures of the sample in the observation area. The sheath fluid enters the fluid channel upstream of the target in the sample in the microfluidic chip through the isolation liquid inlet, and the target is moved out of the microfluidic chip in a flowing manner, so that it passes through the outlet of the microfluidic chip and enters the collection container placed on the electric orifice plate moving stage, completing the entire separation process.

[0066] The overall design of the device is compact and highly integrated, suitable for laboratory and on-site testing. The equipment occupies a small space, is easy to operate and maintain, and has a wide range of applications.

[0067] like Figures 11 to 13 As shown, a flow-type fully automatic quantitative detection and processing method for biological particles is first loaded into the sample storage area, and the sample storage area is used to store the sample to be tested. Then, the sample is tested for the first time in the sample detection area, and the sample signal is collected in the sample detection area. The concentration is calculated and the dilution formula is estimated according to the test results. Then, the sample is diluted in the sample dilution area, and the diluted sample is tested again to check whether the concentration meets the standard. If not, it returns to repeat the dilution and detection until the concentration meets the standard and enters the subsequent processing stage. The signal processing and control module performs signal processing and control of the entire device, wherein the signal sensor collects the sample signal, the signal processing unit processes the data, and the control unit controls the dilution liquid volume and sample flow according to the processing results to realize automatic detection and dilution. The detection method specifically includes the following steps:

[0068] Step 1: Sample loading: Place the sample to be tested into the test tube in the sample storage area and insert it into the test tube holder. After the device is started, the sample is sucked into the sample detection area (flow cell) through the sample delivery module and is ready for detection.

[0069] In an embodiment of the present invention, while the sample is being delivered from the storage area to the detection area, sheath fluid is added along the way to achieve concentration of the sample flow (completed in the flow cell). The power method can be pressing, suction, dead weight, centrifugal throwing or pumping. The sample and sheath fluid are introduced into the flow cell together by the sample delivery module.

[0070] Step 2: Sample detection: When the sample passes through the detection area, the detection module collects the signal of the object to be measured in the sample (such as a photoelectric signal), and the signal processing module processes and analyzes the collected signal to calculate the current concentration of the object to be measured in the sample;

[0071] In the embodiment of the present invention, when the sample and the sheath fluid flow through the detection area, the detection module collects the signal (such as the photoelectric signal) of the object to be measured in the sample, analyzes and records the unit time. The amount of analyte flowing through the sample detection area With injection volume .

[0072] Step 3: Concentration calculation and dilution formula estimation: Based on the test results, the signal processing module calculates the volume of diluent and stock solution required to dilute the sample to the target concentration, and the control module controls the mixing ratio of the diluent and the sample based on the calculation results;

[0073] In the embodiment of the present invention, according to the detection result in step 2, the signal processing and control module calculates the current sample concentration (When the sample injection flow rate is fixed at When the sample injection time and injection volume are For quantitative calculation, the sample concentration can be calculated using the time concentration ). Compare the current sample concentration The target analyte concentration range ( ~ ,in To allow the lowest concentration, is the maximum concentration allowed) Within the permissible concentration range of the substance being measured ( ~ ), it indicates that the concentration of the substance in the sample has reached the target concentration range; the sample with a concentration that has reached the target concentration range is continuously delivered to complete the subsequent processing process. Lower than When the user is reminded that the current sample concentration is too dilute and cannot meet the subsequent minimum detection requirements. Higher than When the signal processing and control module determines the concentration of the target object The required sample stock volume , and calculate the diluted sample volume according to the following formula The required dilution volume

[0074]

[0075]

[0076] The target analyte concentration is determined The required sample stock volume When At least the minimum number of samples should meet the minimum statistical significance requirement of the experiment. ,and Cannot be higher than the sample dilution zone volume .

[0077] Step 4: Sample dilution; the control module controls the mixing of the diluent and the sample, and achieves rapid mixing through fluid disturbance (such as magnetic rotor, vibration, etc.) to complete the dilution of the sample;

[0078] In the embodiment of the present invention, the control module transfers the corresponding sample stock solution and diluent into the sample dilution zone according to the above calculation results, and completes the mixing of the sample stock solution and the diluent. By flow With time ,through After calculation, precise control is performed. Can be further increased by air pressure and flow resistance ,through Control and adjustment are carried out after calculation.

[0079] Step 5: Re-test the diluted sample; the diluted sample is sucked into the flow cell for testing again through the sample delivery module. The test result is processed by the signal processing module to detect whether the concentration after dilution meets the standard. If not, it prompts to replace the sample or return to step 3 for repeated dilution and testing. If it meets the standard, subsequent processing is carried out and the sample will be sent to the subsequent processing module for refined processing such as detection, sorting, and placement.

[0080] In the embodiment of the present invention, the diluted sample is again introduced into the flow cell by the sample delivery module together with the sheath liquid for detection. When the diluted sample and the sheath liquid flow through the detection area, the detection module collects the signal (such as the photoelectric signal) of the object to be detected in the sample, analyzes and records the unit time. The amount of analyte flowing through the sample detection area With injection volume , according to the formula Calculate the concentration of diluted samples Follow step 3 to compare the concentration of the diluted samples. The target analyte concentration range ( ~ ), adjust the concentration according to the method of steps 3 to 5 until the concentration of the analyte falls within the allowable range ( ~ ).

[0081] Step 6: After the concentration reaches the standard, proceed with subsequent treatment.

[0082] In the embodiment of the present invention, when the concentration of the measured object is adjusted to the allowable range ( ~ ), the samples will be sent to the subsequent processing module to start the subsequent detection, sorting, placement and other refined processing of the samples, and record the corresponding data.

[0083] Verification test:

[0084] In order to verify the above experimental steps, we conducted a verification experiment to prove the accuracy and practicality of the flow-type fully automatic biological particle quantitative processing device of the present invention.

[0085] 1. One of the specific implementation forms of the present invention: Flow-type nanoparticle detection:

[0086] Subjects:

[0087] 1) Sample: Nanoparticle suspension.

[0088] 2) Diluent: purified water.

[0089] 3) Detection equipment: The device of the present invention is a nano-flow single particle detector.

[0090] Follow the steps shown in Table 1, step 6 is nanoparticle detection:

[0091] Table 1 Flow type nanoparticle detection table of the present invention

[0092]

[0093] 2. One of the specific implementation forms of the present invention: Single particle separation instrument based on microfluidic chip

[0094] Subjects:

[0095] 1) Sample: B cell suspension.

[0096] 2) Dilution medium: cell culture medium or physiological saline solution.

[0097] 3) Testing equipment: Traditional single particle separation instrument.

[0098] Follow the steps in Table 2. Step 6 is for separating biological single particles:

[0099] Table 2 Traditional single cell dissociation instrument detection table

[0100]

[0101] Conclusion: The above experiments have verified that the mobile fully automatic biological particle quantitative processing device of the present invention is superior to the existing technology in terms of automation. The device can significantly improve the efficiency and accuracy of biological particle detection, simplify the operation process, and has broad application prospects and significant technical advantages.

[0102] In another embodiment, in the sample dilution method, in addition to using a magnetic rotor to mix the sample and the diluent, rapid mixing can also be achieved by using ultrasonic vibration, vortex mixing, mechanical stirring, etc.; it can also be achieved by pulse flow in a microfluidic channel, jet mixing, etc.

[0103] In addition to photoelectric detection (such as scattered light and fluorescent signals), the detection module can also use other optical detection methods such as laser scattering, UV-visible spectroscopy, infrared spectroscopy, etc.; in addition to impedance measurement, other electrical detection methods such as conductivity measurement and potential measurement can also be used.

[0104] In addition to using specific concentration calculation algorithms, the signal processing and control module can also use other data processing and control methods such as neural network algorithms, machine learning algorithms, fuzzy logic control, etc.; in addition to existing control logic, distributed control systems, fuzzy control systems, expert systems, etc. can also be used to achieve sample dilution and concentration adjustment.

[0105] In addition to using pressing, suction, deadweight, spiral throwing or pumping to achieve the flow of samples and diluents, the sample delivery module can also use other power systems such as pneumatic systems, hydraulic systems, electromagnetic drives, etc.

[0106] The storage containers in the sample storage and dilution areas can be made of different materials and forms, such as glass bottles, plastic containers, stainless steel containers, etc. In addition to traditional test tubes and quartz tubes, microfluidic devices can also use microfluidic chips to achieve sample storage and dilution.

[0107] Other uses:

[0108] The technical solution of the present invention is not only applicable to the detection and processing of biological particles, but can also be extended to the following fields and uses:

[0109] 1. Environmental monitoring: Detect tiny particles in water and air (such as pollutants, heavy metal particles, microplastics, etc.) to achieve environmental quality monitoring and assessment.

[0110] 2. Food safety testing: Detect microorganisms, bacteria, viruses and other biological particles in food to ensure food safety and quality.

[0111] 3. Medical diagnosis: used for the detection of biological particles in samples such as blood, urine, and tissue fluid, to assist in disease diagnosis and evaluation of treatment effects.

[0112] 4. Pharmaceutical industry: used for particle detection and quality control in drug research and development and production to ensure the purity and effectiveness of drugs.

[0113] 5. Biological research: Applied to particle analysis and processing in basic research such as cell biology and molecular biology to support scientific research.

[0114] 6. Industrial production: used for particle detection and quality control in the industrial production process of nanomaterials, particulate products, etc. to ensure product consistency and performance.

[0115] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some or all of the technical features thereof may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A flow-type fully automatic biological particle quantitative detection and processing method, characterized in that The steps include: Step 1: Sample loading: Place the sample to be tested in the sample storage area, and the sample enters the sample testing area through the sample delivery module; Step 2: Sample detection: When the sample passes through the detection area, the detection module collects the signal of the object to be measured in the sample, and the signal processing module processes and analyzes the collected signal to calculate the current concentration of the object to be measured in the sample; Step 3: Concentration calculation and dilution formula estimation; The signal processing module determines whether the concentration of the analyte in the current sample meets the standard by comparing the current concentration of the analyte in the sample with the required target concentration range. If not, the signal processing module prompts to replace the sample or calculates the volume of diluent and the volume of stock solution required to dilute the sample to the target concentration according to the test results; Step 4: Sample dilution; the control module controls the mixing of the diluent and the sample in the sample dilution area to complete the dilution of the sample; Step 5: Retest the diluted sample; the diluted sample enters the sample testing area through the sample delivery module for testing again. The test results are processed by the signal processing module to detect whether the concentration after dilution meets the standard. If not, replace the sample or return to step 3 for repeated dilution and testing. If it meets the standard, the test is completed; Step 6: Send the tested samples to the subsequent processing module for subsequent processing of the samples.

2. A flow-type fully automatic biological particle quantitative detection and processing method according to claim 1, characterized in that: In step 2, the detection module collects the signal of the object being measured in the sample, analyzes and records the unit time. The amount of analyte flowing through the sample detection area With injection volume ; In step 3, based on the detection results in step 2, the signal processing module calculates the current sample concentration. , compare the current sample concentration The concentration range of the target substance ~ Compare, among them To allow the lowest concentration, The maximum concentration allowed. If the current sample concentration is Within the allowable concentration range of the measured substance ~ If the concentration of the substance in the sample reaches the target concentration range, the sample with a concentration that reaches the target concentration range is continuously delivered to complete the subsequent processing process. Lower than If the current sample concentration is too dilute to meet the subsequent minimum detection requirements, the user is prompted to change the sample; if the current sample concentration is Higher than When the signal processing module determines the concentration of the target object The required sample stock volume , and calculate the diluted sample volume according to the following formula The required dilution volume : ， ， Among them, determine the concentration of the target analyte The required sample stock volume hour, Minimum number of analytes required to meet minimum statistical significance , and the sample volume after dilution Cannot be higher than the sample dilution zone volume .

3. A flow-type fully automatic biological particle quantitative detection and processing method according to claim 2, characterized in that: In step 3, when the sample injection flow rate is fixed at Sample injection time The injection volume can be determined by Quantitative calculation is performed, at which point the sample concentration Time concentration .

4. A flow-type fully automatic biological particle quantitative detection and processing method according to claim 1, characterized in that: In step 1, the sample delivery module introduces the sample and the sheath fluid into the flow cell sample detection area; in step 5, the diluted sample is again introduced into the flow cell by the sample delivery module together with the sheath fluid for detection.

5. The method for the flow-type fully automatic quantitative detection and processing of biological particles according to claim 1, characterized in that: In step 4, the control module respectively transfers the corresponding sample stock solution and diluent into the sample dilution zone, and completes the mixing of the sample stock solution and the diluent, wherein the volume of the sample stock solution and the diluent is By traffic With time ,through After calculation, control is performed, where the flow The gas pressure on the sample and flow resistance ,through Calculated and controlled, or by the cross-sectional area of ​​the syringe loaded with the sample And the sample feeding line speed ,through Control and adjustment are carried out after calculation.

6. A flow-type fully automatic biological particle quantitative detection and processing method according to claim 1, characterized in that: In step 5, when the diluted sample passes through the detection zone, the detection module collects the signal of the analyte in the sample, analyzes and records the unit time. The amount of analyte flowing through the sample detection area With injection volume , according to the formula Calculate the concentration of diluted samples ; Follow step 3 to compare the concentration of diluted samples The concentration range of the target substance ~ According to the relationship between the concentration and the measured substance, adjust the concentration according to the method of steps 3 to 5 until the concentration of the measured substance falls into the allowable range. ~ , To allow the lowest concentration, The highest concentration allowed.

7. A device for a mobile fully automatic biological particle quantitative detection and processing method according to claim 1, comprising a sample storage area, a diluent storage area, a sample detection area, a sample dilution area, a sample delivery module, a detection module, a signal processing and control module, and a subsequent processing module, characterized in that: The sample storage area is used to store samples to be tested containing the object to be tested; The diluent storage area is used to store the diluent for diluting the sample; The sample dilution area is used for sample dilution; The sample detection area collects electrical signals related to the quantity of the object being tested in the sample through the detection module; The sample delivery module is used for the flow delivery of samples and diluents between the sample storage area, the sample dilution area, the diluent storage area, the sample detection area and the subsequent processing module; The signal processing and control module is used to collect, process and analyze the signals related to the amount of the analyte collected from the sample detection area, calculate the concentration of the analyte in the current sample, and calculate the dilution formula according to the detection result, and control the sample and the diluent to enter the sample dilution area according to the calculated required amount; The subsequent processing module performs subsequent processing on the sample when the concentration of the analyte in the sample is within the target concentration range.

8. The detection device according to claim 7, characterized in that: The sample dilution area and the sample storage area are the same area, which is connected to the liquid preparation bottle. When the volume of the area is small, a part of the sample is extracted or the diluted sample is put into the waste liquid bottle.

9. The detection device according to claim 7, characterized in that: The detection of the sample detection area adopts a flow single particle analysis FCM or nanoparticle tracking analysis NTA or dynamic light scattering analysis DLS method based on a flow cell or a microfluidic channel flow cell. The detection principle of the sample includes photoelectric detection or electrical detection, which is finally converted into an electrical signal for data information processing.

10. The detection device according to claim 7, characterized in that: The device also includes a sheath liquid storage area for storing sheath liquid to form a sheath flow direction so that the sample can flow and be counted in the sheath flow direction; the sample delivery module includes an automatic air pressure regulating air pump and a multi-channel automatic air pressure regulating valve, wherein the multi-channel automatic air pressure regulating valve includes an automatic air pressure regulating valve, a switch valve, and an air leakage valve of a single channel that work independently, the automatic air pressure regulating air pump is connected to the automatic air pressure regulating valve and the switch valve of the single channel in sequence, the switch valve and the air leakage valve are cooperatively arranged as the output of the automatic air pressure regulating valve of the single channel, and the automatic air pressure regulating valves of each channel are respectively connected to the sample storage area, the sample dilution area, the diluent storage area, and the sheath liquid storage area.

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