A microfluidic chip detection system and method for flow cytometry detection

Abstract

This invention discloses a microfluidic chip detection system and method for flow cytometry, relating to the field of microfluidic particle metrology. The method includes: calculating the optical path disturbance depth and performing calibration based on the optical baseline light intensity, transmitted light intensity time series, and lowest transmitted light intensity to obtain a calibrated particle event set; matching the calibrated particle event set with the transmitted light intensity time series to calculate the optical path disturbance integral; constructing an optical path disturbance stability threshold using the median and discrete values ​​of the optical path disturbance integral, and forming an optically stable event set; and estimating the optical equivalent diameter using the optically stable event set and the calibrated particle event set to form an equivalent optical size sequence and construct a size distribution histogram. This invention achieves continuous metrological expression of the particle size of the sample by establishing a linear mapping relationship between the calibrated optical path disturbance depth and the particle diameter.

Description

Technical Field

[0001] This invention relates to the field of microfluidic particle metering technology, and in particular to a microfluidic chip detection system and method for flow cytometry detection. Background Technology

[0002] Microfluidic chips, as an important carrier for flow cytometry, are widely used in microscale detection scenarios such as particle size characterization, cell analysis, and functional material evaluation. Conventional methods typically utilize a stable laminar flow within the chip to deliver particles one by one to an optical detection window. Under fixed light source conditions, the change in transmitted light over time is recorded, and particle size parameters or structural properties are inferred by analyzing features such as the magnitude of light intensity decrease, the duration of the decrease, or the overall optical path perturbation. With the continuous improvement of detection requirements, flow cytometry optical measurement has gradually formed a basic workflow based on reference particle calibration, event fragment analysis, and signal statistical feature description. This allows for a more systematic analytical framework for the structured representation of particle events, the quantification of optical path perturbation, and size estimation models.

[0003] However, existing methods have two shortcomings in terms of metrological consistency and reliability: First, optical metrology often relies on a single reference particle or a fixed optical response ratio, which makes it difficult to fully reflect the differences in optical path perturbation under different refractive indices and particle sizes, making the linear mapping relationship of particle size metrology susceptible to the influence of medium conditions and optical deviations; Second, the quality judgment of particle events is mainly based on local light intensity thresholds or characteristics of falling segments, making it difficult to use the complete temporal perturbation level for robust evaluation, thus affecting the stability and repeatability of particle metrology data. Summary of the Invention

[0004] In view of the aforementioned existing problems, the present invention is proposed.

[0005] Therefore, this invention provides a microfluidic chip detection method for flow cytometry, which solves the problems of optical metrology being affected by medium conditions and the difficulty in uniformly assessing the stability of particle events.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides a microfluidic chip detection method for flow cytometry detection, comprising: acquiring transmitted light intensity under constant volume flow rate and constant light source brightness conditions, obtaining optical baseline light intensity by moving average, and obtaining a time series of transmitted light intensity and the lowest transmitted light intensity during the flow of a mixed suspension.

[0008] Based on the optical baseline light intensity, the time series of transmitted light intensity and the minimum transmitted light intensity, the optical path perturbation depth is calculated and calibration is completed to obtain the calibrated set of particle events.

[0009] The calibrated set of particle events is matched with the transmitted light intensity time series to calculate the optical path disturbance integral. The optical path disturbance stability threshold is constructed by the median and discrete values ​​of the optical path disturbance integral, and an optically stable event set is formed.

[0010] The optical equivalent diameter is estimated by calculating the optically stable event set and the calibrated particle event set, forming an equivalent optical size sequence and constructing a size distribution histogram.

[0011] As a preferred embodiment of the microfluidic chip detection method for flow cytometry described in this invention, the method for acquiring transmitted light intensity and obtaining optical baseline light intensity by moving average includes: under constant volume flow rate and constant light source brightness, a suspension medium without particles is flowed through an optical detection window, and the transmitted light intensity is continuously acquired by a photodetector, and multiple moving average operations are performed on the continuously acquired transmitted light intensity.

[0012] The stable average value of the transmitted light intensity obtained through multiple moving average calculations is used as the optical baseline light intensity.

[0013] As a preferred embodiment of the microfluidic chip detection method for flow cytometry described in this invention, wherein: obtaining the transmission light intensity time series and the lowest transmission light intensity during the flow of the mixed suspension includes: injecting a mixed suspension composed of sample particles to be tested, first type reference particles, and second type reference particles under constant volume flow conditions; flowing the mixed suspension through an optical detection window; and recording the transmission light intensity at a fixed sampling frequency using a photodetector to form a transmission light intensity time series.

[0014] By scanning the time series of transmitted light intensity point by point to identify the decreasing interval, the minimum transmitted light intensity within the decreasing interval is found and recorded as the lowest transmitted light intensity of the particle event.

[0015] As a preferred embodiment of the microfluidic chip detection method for flow cytometry described in this invention, the calculation of the optical path perturbation depth includes: performing a ratio calculation between the difference between the optical baseline light intensity and the lowest transmitted light intensity relative to the optical baseline light intensity to obtain the optical path perturbation depth of each particle event.

[0016] As a preferred embodiment of the microfluidic chip detection method for flow cytometry described in this invention, wherein: the calibration is completed and the calibrated particle event set is obtained by constructing a first type of reference particle event set, a second type of reference particle event set, and a sample particle event set based on the joint distribution of optical path disturbance depth and transmission light intensity decrease duration;

[0017] Based on the refractive difference relationship between the diameter of the first type of reference particle, the diameter of the second type of reference particle, the refractive index of the first type of reference particle, the refractive index of the second type of reference particle, and the refractive index of the suspension medium, a theoretical optical path perturbation ratio is constructed.

[0018] The actual optical path perturbation ratio is formed by calculating the average optical path perturbation depth of the second type of reference particle and the average optical path perturbation depth of the first type of reference particle and performing a ratio calculation.

[0019] The ratio of the theoretical optical path perturbation ratio to the actual optical path perturbation ratio is calculated to form the optical path perturbation gain calibration coefficient.

[0020] The optical path perturbation depth of each particle event is linearly scaled by the optical path perturbation gain calibration coefficient to obtain the calibrated optical path perturbation depth and form a calibrated set of particle events.

[0021] As a preferred embodiment of the microfluidic chip detection method for flow cytometry described in this invention, wherein: the step of performing corresponding matching and calculating the optical path disturbance integral includes matching the start time point and end time point of the transmitted light intensity decrease interval in the calibrated particle event set with the same time position in the transmitted light intensity time series to obtain a unique transmitted light intensity decrease interval for each particle event in the transmitted light intensity time series.

[0022] The optical path perturbation integral for each particle event is calculated using the start time point of the transmitted light intensity decrease interval, the end time point of the transmitted light intensity decrease interval, the transmitted light intensity time series, and the optical baseline light intensity.

[0023] As a preferred embodiment of the microfluidic chip detection method for flow cytometry described in this invention, the step of constructing an optical path disturbance stability threshold and forming an optical stability event set includes: sorting all optical path disturbance integrals according to their numerical values, selecting the optical path disturbance integral at the median position of the sorting position as the median optical path disturbance integral, calculating the absolute difference sequence between all optical path disturbance integrals and the median optical path disturbance integral, and taking the value corresponding to the median position of the absolute difference sequence as the optical path disturbance integral discrete quantity;

[0024] The optical path perturbation stability threshold is obtained by summing the median of the optical path perturbation integral and the discrete value of the optical path perturbation integral.

[0025] When the integral of the optical path disturbance is less than or equal to the optical path disturbance stability threshold, the particle event is added to the optically stable event set.

[0026] As a preferred embodiment of the microfluidic chip detection method for flow cytometry described in this invention, the step of calculating and estimating the optical equivalent diameter to form an equivalent optical size sequence includes: matching the optically stable event set with the calibrated particle event set according to the start time point and end time point of the transmitted light intensity decrease interval; and including particle events with completely consistent start and end times of the transmitted light intensity decrease interval into the first type of optically stable reference particle event set, the second type of optically stable reference particle event set, and the optically stable particle event set of the sample to be tested, respectively.

[0027] The average calibrated optical path perturbation depth of all particle events in the first type of optically stable reference particle event set is taken as the average calibrated optical path perturbation depth of the first type of reference particle.

[0028] The average calibrated optical path perturbation depth of all particle events in the second type of optically stable reference particle event set is taken as the average calibrated optical path perturbation depth of the second type of reference particle.

[0029] Based on the average calibrated optical path perturbation depth of the first and second type reference particles, and combined with the calibrated optical path perturbation depth in the optically stable particle event set of the sample under test, a linear mapping relationship between the calibrated optical path perturbation depth and the particle diameter is constructed to obtain the estimated optical equivalent diameter of the particles in the sample under test.

[0030] As a preferred embodiment of the microfluidic chip detection method for flow cytometry described in this invention, the construction of the size distribution histogram includes dividing the equivalent optical size sequence into several equal-width size intervals according to the minimum and maximum values ​​of the estimated optical equivalent diameter, counting the number of particles in each size interval, and constructing a size distribution histogram based on the estimated optical equivalent diameter.

[0031] Secondly, the present invention provides a microfluidic chip detection system for flow cytometry detection, comprising,

[0032] The optical baseline construction module collects transmitted light intensity under constant volume flow rate and constant light source brightness conditions, obtains the optical baseline light intensity through moving average, and obtains the time series of transmitted light intensity and the lowest transmitted light intensity when the mixed suspension is flowing.

[0033] The optical path disturbance calibration module calculates the optical path disturbance depth and completes calibration based on the optical baseline light intensity, the time series of transmitted light intensity and the minimum transmitted light intensity, and obtains the calibrated set of particle events.

[0034] The optical path stability screening module matches the calibrated set of particle events with the transmitted light intensity time series, calculates the optical path disturbance integral, constructs the optical path disturbance stability threshold by using the median and discrete values ​​of the optical path disturbance integral, and forms an optically stable event set.

[0035] The particle size measurement module estimates the optical equivalent diameter by calculating the optical stable event set and the calibrated particle event set, forming an equivalent optical size sequence and constructing a size distribution histogram.

[0036] The beneficial effects of this invention are as follows: by constructing an optical path perturbation stability threshold, stable screening of particle events on a uniform perturbation scale is achieved; by establishing a linear mapping relationship between the calibrated optical path perturbation depth and the particle diameter, continuous quantitative expression of the particle size of the sample to be tested is achieved. Attached Figure Description

[0037] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0038] Figure 1 This is a flowchart of a microfluidic chip detection method for flow cytometry.

[0039] Figure 2 This is a schematic diagram of a microfluidic chip detection system used for flow cytometry.

[0040] Figure 3 This is a flowchart for optical path perturbation depth calibration.

[0041] Figure 4 A flowchart for screening optically stable events. Detailed Implementation

[0042] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0043] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0044] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0045] Reference Figures 1-4 This is one embodiment of the present invention, which provides a microfluidic chip detection method for flow cytometry detection, comprising the following steps:

[0046] S1. Under constant volumetric flow rate and constant light source brightness, the transmitted light intensity is collected, the optical baseline light intensity is obtained by moving average, and the time series of transmitted light intensity and the lowest transmitted light intensity are obtained when the mixed suspension is flowing.

[0047] Furthermore, a particle-free suspension medium is injected at the inlet of the microfluidic chip, allowing the suspension medium to pass through the optical detection window of the microfluidic chip at a constant volumetric flow rate. The constant volumetric flow rate is maintained at a constant value by controlling the pressure difference between the inlet and outlet sides of the suspension medium, so that the suspension medium exhibits a stable, uniform flow state in the flow channel of the microfluidic chip without abrupt changes.

[0048] To ensure that the transmitted light intensity acquisition process is unaffected by light source fluctuations, the illumination beam maintains a constant brightness during acquisition and continuously illuminates the optical detection window throughout the entire particle-free flow process.

[0049] The transmitted light intensity is continuously recorded by a photodetector under stable illumination conditions and during periods of no particle flow. The continuously collected transmitted light intensity is then subjected to multiple moving average calculations over time. The moving average window is set to a value between 5 and 20 milliseconds. The time step of the moving average should be smaller than the duration of the moving average window to smooth out short-term fluctuations in the transmitted light intensity.

[0050] The stable average value of the transmitted light intensity obtained by multiple moving average calculations is used as the optical baseline light intensity; the optical baseline light intensity reflects the actual transmission capability of the optical detection window under conditions without particle interference.

[0051] Furthermore, after obtaining the optical baseline light intensity, the sample particles to be tested, the first type of reference particles, and the second type of reference particles are mixed to form a mixed suspension.

[0052] The geometric size of the first type of reference particle is smaller than that of the second type of reference particle. This results in a significantly smaller decrease in transmitted light intensity when the first type of reference particle passes through the optical detection window compared to the second type of reference particle. Consequently, the first type of reference particle exhibits two different intensity reduction characteristics in optical observation.

[0053] The mixed suspension is injected into the microfluidic chip at the same constant volumetric flow rate as the suspension medium, so that the mixed suspension forms a continuous and stable laminar flow in the flow channel of the microfluidic chip.

[0054] The photodetector records the transmitted light intensity of the mixed suspension flowing through the optical detection window at a fixed sampling frequency. An example of a fixed sampling frequency is a range of 5 kHz to 50 kHz. The range of 5 kHz to 50 kHz ensures that the changes in light intensity of particles passing through the optical detection window are completely recorded on a time scale. The light intensity decrease process will not be truncated due to excessively long sampling intervals, nor will redundant data occupy too much processing time due to excessively high sampling frequencies.

[0055] The transmitted light intensity of the continuously collected mixed suspension is used to construct a time series of transmitted light intensity.

[0056] It should be noted that after obtaining the transmitted light intensity time series, the transmitted light intensity time series is scanned point by point. When the transmitted light intensity is continuously lower than the optical baseline light intensity, and the duration of the transmitted light intensity being lower than the optical baseline light intensity is longer than the duration of the moving average window, the time range in which the transmitted light intensity is continuously lower than the optical baseline light intensity is determined as the decreasing interval.

[0057] Each descending interval corresponds to the passage of a particle event through the optical detection window.

[0058] Perform a local search on the descent interval corresponding to each particle event to find the minimum transmitted light intensity within the descent interval, and record the minimum transmitted light intensity as the lowest transmitted light intensity of the particle event.

[0059] The minimum transmitted light intensity of a particle event reflects the maximum transmitted optical path impact caused by the particle as it passes through the optical detection window, and can be used as an optical feature to distinguish the degree of optical path disturbance of different particle events.

[0060] S2. Based on the optical baseline light intensity, the time series of transmitted light intensity and the minimum transmitted light intensity, calculate the optical path perturbation depth and complete the calibration to obtain the calibrated set of particle events.

[0061] Furthermore, the optical path perturbation depth is calculated for each particle event. Specifically, the difference between the optical baseline light intensity and the lowest transmitted light intensity is calculated as a ratio with the optical baseline light intensity to obtain the optical path perturbation depth corresponding to each particle event.

[0062] The optical path perturbation depth characterizes the relative perturbation intensity caused by particle events to the transmitted optical path, so that each particle event in the mixed suspension can obtain a comparable amount of optical perturbation.

[0063] Furthermore, by analyzing the joint distribution of optical path perturbation depth and transmission light intensity decrease duration, the first type of reference particle event set, the second type of reference particle event set, and the test sample particle event set are identified respectively.

[0064] When the optical path disturbance depth of a particle event falls within the range of optical path disturbance depth values ​​formed by the first type of reference particle under the condition of single injection, and the duration of the decrease in transmitted light intensity falls within the range of the decrease duration values ​​formed by the first type of reference particle under the condition of single injection, the particle event is classified into the first type of reference particle event set.

[0065] The range of optical path disturbance depth values ​​formed by the first type of reference particle under individual injection conditions is obtained by statistically analyzing all optical path disturbance depth values ​​formed by the first type of reference particle during individual injection, and constructing a closed interval using the minimum and maximum values; the range of descent duration values ​​formed by the first type of reference particle under individual injection conditions is obtained by statistically analyzing all descent duration values ​​formed by the first type of reference particle during individual injection, and constructing a closed interval using the minimum and maximum values.

[0066] When the optical path disturbance depth of a particle event falls within the range of optical path disturbance depth values ​​formed by the second type of reference particle under the condition of single injection, and the duration of the decrease in transmitted light intensity falls within the range of the decrease duration values ​​formed by the second type of reference particle under the condition of single injection, the particle event is classified into the set of second type of reference particle events.

[0067] The range of optical path disturbance depths formed by the second type of reference particles under individual injection conditions is obtained by statistically analyzing all optical path disturbance depths formed by the second type of reference particles during individual injection, and using the minimum and maximum values ​​to form a closed interval; the range of descent durations formed by the second type of reference particles under individual injection conditions is obtained by statistically analyzing all descent durations formed by the second type of reference particles during individual injection, and using the minimum and maximum values ​​to form a closed interval.

[0068] The single injection condition refers to the condition where only the first type of reference particles or only the second type of reference particles are injected without the sample particles to be tested.

[0069] All particle events that do not belong to the first type of reference particle event set or the second type of reference particle event set are included in the particle event set of the sample to be tested.

[0070] Furthermore, after obtaining the first set of reference particle events, the second set of reference particle events, and the set of particle events of the sample to be tested, the optical path perturbation depth of each particle event is calibrated.

[0071] The calibration process is based on the refractive difference relationships among the diameters of the first type of reference particles, the diameters of the second type of reference particles, the refractive indices of the first type of reference particles, the refractive indices of the second type of reference particles, and the refractive index of the suspension medium. A theoretical optical path perturbation ratio is constructed, expressed as:

[0072] ;

[0073] in, This represents the theoretical optical path perturbation ratio. The diameter of the first type of reference particle. The diameter of the second type of reference particle. The refractive index of the first type of reference particle, The refractive index of the second type of reference particle. is the refractive index of the suspension medium.

[0074] The average optical path perturbation depth of the first type of reference particle event set is obtained by averaging the optical path perturbation depths; the average optical path perturbation depth of the second type of reference particle event set is obtained by averaging the optical path perturbation depths.

[0075] The average optical path perturbation depth of the second type of reference particles is calculated as a ratio to the average optical path perturbation depth of the first type of reference particles to form the actual optical path perturbation ratio under actual measurement conditions.

[0076] The ratio of the theoretical optical path perturbation ratio to the actual optical path perturbation ratio is calculated to form the optical path perturbation gain calibration coefficient.

[0077] The optical path perturbation gain calibration factor is used to reflect the overall proportional deviation between ideal optical conditions and actual measurement conditions.

[0078] It should be noted that after the optical path perturbation gain calibration coefficient is formed, the optical path perturbation depth corresponding to each particle event is linearly scaled according to the optical path perturbation gain calibration coefficient to obtain the calibrated optical path perturbation depth of each particle event, expressed as:

[0079] ;

[0080] in, For the first The calibrated optical path perturbation depth for each particle event. This is the optical path perturbation gain calibration coefficient. For the first The optical path perturbation depth of each particle event.

[0081] After calibration, the optical path perturbation depth expresses the optical path perturbation intensity of all particle events under the same metrological scale, enabling the first type of reference particles, the second type of reference particles, and the sample particles to be tested to be distinguished and analyzed under a unified metrological framework.

[0082] It should also be noted that after completing the optical path perturbation depth calibration of all particle events, a calibrated particle event set is formed, including the calibrated first-class reference particle event set, the calibrated second-class reference particle event set, and the calibrated sample particle event set.

[0083] The calibrated set of first-type reference particle events consists of the calibrated optical path perturbation depth of all first-type reference particle events and the transmission intensity decrease interval corresponding to each particle event in the transmission intensity time series; the calibrated set of second-type reference particle events consists of the calibrated optical path perturbation depth of all second-type reference particle events and the transmission intensity decrease interval corresponding to each particle event in the transmission intensity time series; the calibrated set of particle events of the test sample consists of the calibrated optical path perturbation depth of all test sample particle events and the transmission intensity decrease interval corresponding to each particle event in the transmission intensity time series.

[0084] S3. Match the calibrated set of particle events with the transmitted light intensity time series, calculate the optical path disturbance integral, construct the optical path disturbance stability threshold by the median and discrete values ​​of the optical path disturbance integral, and form an optically stable event set.

[0085] Furthermore, the calibrated set of particle events is matched with the transmitted light intensity time series.

[0086] The matching was completed by the start and end times of the transmission intensity decrease interval, both of which were derived from the optical path perturbation depth calculation process. The start time of the transmission intensity decrease interval was matched with the same time position in the transmission intensity time series, and the end time of the transmission intensity decrease interval was matched with the same time position in the transmission intensity time series. Through two independent matchings, a unique transmission intensity decrease interval in the transmission intensity time series for each particle event was determined.

[0087] The transmitted light intensity decrease range includes the time point when the transmitted light intensity begins to decrease and the time point when the transmitted light intensity recovers to near the optical baseline light intensity. It is used to represent the complete transmitted light intensity change segment formed by each particle event before entering the optical detection window, within the optical detection window, and when leaving the optical detection window.

[0088] For each particle event, the transmitted light intensity change process is constructed, and the optical path perturbation integral is expressed as:

[0089] ;

[0090] in, For the first The integral of optical path perturbation for a single particle event For the first The starting time point of the transmission light intensity decrease range for each particle event. For the first The end time of the transmission light intensity decrease interval for each particle event. For the time series of transmitted light intensity in time The value of , The optical baseline light intensity.

[0091] The optical path perturbation integral is used to characterize the combined optical path perturbation level, which is the depth of the optical path perturbation and the duration of the perturbation.

[0092] After obtaining the optical path disturbance integrals for all particle events, all optical path disturbance integrals are sorted according to their numerical values. The optical path disturbance integral at the median position in the sorting is selected as the median optical path disturbance integral. The absolute difference sequence between all optical path disturbance integrals and the median optical path disturbance integral is calculated. The value corresponding to the median position of the absolute difference sequence is taken as the discrete optical path disturbance integral. The median optical path disturbance integral and the discrete optical path disturbance integral are summed to form the optical path disturbance stability threshold, expressed as:

[0093] ;

[0094] in, This is the optical path perturbation stability threshold. This is the median calculation function.

[0095] It should be noted that the median of the optical path perturbation integral reflects the overall stability level of all particle events, while the discrete quantity of the optical path perturbation integral reflects the degree of fluctuation of the optical path perturbation integral around the overall stability level.

[0096] The optical path disturbance stability threshold reflects the reasonable fluctuation range of particle events flowing through the optical detection window near the overall disturbance level, and is used to distinguish between stable optical path disturbance processes and abnormal optical path disturbance processes.

[0097] Furthermore, the integral of the optical path disturbance for each particle event is compared with the optical path disturbance stability threshold. When the integral of the optical path disturbance is less than or equal to the optical path disturbance stability threshold, the particle event is determined to be an optically stable event; when the integral of the optical path disturbance is greater than the optical path disturbance stability threshold, the particle event is excluded from the category of optically stable events.

[0098] All particle events that are determined to be optically stable events are included in the optically stable event set.

[0099] The optically stable event set is used to represent the set of particle events in which the transmitted light intensity change process is not affected by non-ideal factors such as optical noise disturbance, flow field pulsation, local deflection, and vibration under constant fluid driving conditions and constant light source brightness conditions.

[0100] S4. Estimate the optical equivalent diameter by calculating the optically stable event set and the calibrated particle event set, form the equivalent optical size sequence, and construct the size distribution histogram.

[0101] Furthermore, the optically stable event set and the calibrated particle event set are matched one by one according to the start time point and end time point of the transmitted light intensity decrease interval. Particle events with completely consistent start and end times of the transmitted light intensity decrease interval are respectively included in the first type of optically stable reference particle event set, the second type of optically stable reference particle event set, and the optically stable particle event set of the sample to be tested.

[0102] The average calibrated optical path perturbation depth of the first type of optically stable reference particle event set is obtained by taking the average value of all particle events in the set.

[0103] The average calibrated optical path perturbation depth of the second type of optically stable reference particle is obtained by averaging the calibrated optical path perturbation depth of all particle events in the second type of optically stable reference particle event set.

[0104] Based on the average calibrated optical path perturbation depth of the first and second type reference particles, and combined with the calibrated optical path perturbation depth in the optically stable particle event set of the sample under test, a linear mapping relationship between the calibrated optical path perturbation depth and the particle diameter is constructed, yielding the estimated optical equivalent diameter of the sample particles, expressed as:

[0105] ;

[0106] in, For the sample particles to be tested The estimated optical equivalent diameter, This represents the calibrated optical path perturbation depth during optically stable particle events in the sample under test. The average calibrated optical path perturbation depth of the first type of reference particle. This represents the average calibrated optical path perturbation depth of the second-class reference particles.

[0107] Substituting all optically stable particle events of the sample under test into the linear mapping relationship between the optical path perturbation depth and the particle diameter after calibration, the estimated optical equivalent diameter of all particles of the sample under test is obtained, forming an equivalent optical size sequence of the particles of the sample under test.

[0108] Furthermore, the equivalent optical size sequence of the particles in the test sample is divided into several equal-width size intervals according to the minimum and maximum values ​​of the estimated optical equivalent diameter. The number of particles in each size interval is counted, and a size distribution histogram based on the estimated optical equivalent diameter is constructed.

[0109] The arithmetic mean of all estimated optical equivalent diameters is used to characterize the center position of the size distribution, the standard deviation of all estimated optical equivalent diameters relative to the center position of the size distribution is used to characterize the width of the size distribution, and the shape of the size distribution is characterized by comparing the trend of particle number variation in each size interval.

[0110] By jointly analyzing the center position, width, and shape of the size distribution, a unified quantitative expression is obtained for the average estimated optical equivalent diameter, the dispersion of the estimated optical equivalent diameter, and the particle aggregation in abnormal estimated optical equivalent diameter regions of the sample particles under flow cytometry detection conditions. This enables accurate identification of the overall size structure, size uniformity, and abnormal size distribution characteristics of the sample particle population in the flow cytometry detection environment, thereby improving the stability, repeatability, and consistency of the particle size measurement results across detection batches.

[0111] This embodiment also provides a microfluidic chip detection system for flow cytometry, including:

[0112] The optical baseline construction module collects transmitted light intensity under constant volume flow rate and constant light source brightness conditions, obtains the optical baseline light intensity through moving average, and obtains the time series of transmitted light intensity and the lowest transmitted light intensity when the mixed suspension is flowing.

[0113] The optical path disturbance calibration module calculates the optical path disturbance depth and completes calibration based on the optical baseline light intensity, the time series of transmitted light intensity and the minimum transmitted light intensity, and obtains the calibrated set of particle events.

[0114] The optical path stability screening module matches the calibrated set of particle events with the transmitted light intensity time series, calculates the optical path disturbance integral, constructs the optical path disturbance stability threshold by using the median and discrete values ​​of the optical path disturbance integral, and forms an optically stable event set.

[0115] The particle size measurement module estimates the optical equivalent diameter by calculating the optical stable event set and the calibrated particle event set, forming an equivalent optical size sequence and constructing a size distribution histogram.

[0116] In summary, this invention achieves stable screening of particle events at a uniform perturbation scale by constructing an optical path perturbation stability threshold; and achieves continuous metrological expression of particle size of the sample under test by establishing a linear mapping relationship between the calibrated optical path perturbation depth and particle diameter.

[0117] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention 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 the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A microfluidic chip detection method for flow cytometry, characterized in that: include, Under constant volumetric flow rate and constant light source brightness, a particle-free suspension medium is flowed through an optical detection window, and the transmitted light intensity is continuously collected by a photodetector. Multiple moving average calculations are performed on the continuously collected transmitted light intensity, and the stable average value of the transmitted light intensity obtained after multiple moving average calculations is used as the optical baseline light intensity. Under constant volumetric flow rate, a mixed suspension composed of the sample particles to be tested, first-type reference particles, and second-type reference particles is injected. The geometric size of the first-type reference particles is smaller than that of the second-type reference particles. The time series of transmitted light intensity and the lowest transmitted light intensity are obtained when the mixed suspension flows. Based on the optical baseline light intensity, the time series of transmitted light intensity and the minimum transmitted light intensity, the optical path perturbation depth is calculated and calibration is completed to obtain the calibrated set of particle events. The calculation of the optical path perturbation depth includes performing a ratio calculation between the difference between the optical baseline light intensity and the lowest transmitted light intensity relative to the optical baseline light intensity to obtain the optical path perturbation depth for each particle event. The calibration process is completed, and the resulting set of particle events includes the construction of a first set of reference particle events, a second set of reference particle events, and a set of particle events of the sample to be tested, based on the joint distribution of optical path perturbation depth and duration of transmission light intensity decrease. When the optical path disturbance depth of a particle event falls within the range of optical path disturbance depth values ​​formed by the first type of reference particle under the condition of individual injection, and the duration of the decrease in transmitted light intensity falls within the range of the decrease duration values ​​formed by the first type of reference particle under the condition of individual injection, the particle event is classified into the first type of reference particle event set; the range of optical path disturbance depth values ​​formed by the first type of reference particle under the condition of individual injection is obtained by statistically analyzing all optical path disturbance depths formed by the first type of reference particle during the individual injection process, and constructing a closed interval using the minimum and maximum values; The range of descent duration values ​​formed by the first type of reference particle under the condition of individual injection is obtained by statistically analyzing all descent durations formed by the first type of reference particle during individual injection and constructing a closed interval using the minimum and maximum values. When the optical path disturbance depth of a particle event falls within the range of optical path disturbance depth values ​​formed by the second type of reference particle under the condition of individual injection, and the duration of the decrease in transmitted light intensity falls within the range of the decrease duration values ​​formed by the second type of reference particle under the condition of individual injection, the particle event is classified into the second type of reference particle event set; the range of optical path disturbance depth values ​​formed by the second type of reference particle under the condition of individual injection is obtained by statistically analyzing all optical path disturbance depths formed by the second type of reference particle during the individual injection process, and using the minimum and maximum values ​​to form a closed interval; The range of descent duration values ​​formed by the second type of reference particles under individual injection conditions is obtained by statistically analyzing all descent durations formed by the second type of reference particles during individual injection, and using the minimum and maximum values ​​to form a closed interval. The single injection condition refers to the condition of injecting only the first type of reference particles or only the second type of reference particles without the sample particles to be tested; all particle events that do not belong to the first type of reference particle event set or the second type of reference particle event set are included in the sample particle event set to be tested. Based on the refractive difference relationship between the diameters of the first type of reference particles, the diameters of the second type of reference particles, the refractive indices of the first type of reference particles and the second type of reference particles, and the refractive index of the suspension medium, a theoretical optical path perturbation ratio is constructed, expressed as: ； in, This represents the theoretical optical path perturbation ratio. The diameter of the first type of reference particle. The diameter of the second type of reference particle. The refractive index of the first type of reference particle, The refractive index of the second type of reference particle. The refractive index of the suspension medium; The actual optical path perturbation ratio is formed by calculating the average optical path perturbation depth of the second type of reference particle and the average optical path perturbation depth of the first type of reference particle; the optical path perturbation gain calibration coefficient is formed by calculating the ratio of the theoretical optical path perturbation ratio and the actual optical path perturbation ratio. The optical path perturbation depth of each particle event is linearly scaled using the optical path perturbation gain calibration coefficient to obtain the calibrated optical path perturbation depth, forming a calibrated set of particle events. The calibrated optical path perturbation depth is expressed as: ； in, For the first The calibrated optical path perturbation depth for each particle event. This is the optical path perturbation gain calibration coefficient. For the first The optical path perturbation depth of each particle event; The calibrated set of particle events is matched with the transmitted light intensity time series to calculate the optical path disturbance integral. The optical path disturbance stability threshold is constructed by the median and discrete values ​​of the optical path disturbance integral, and an optically stable event set is formed. The process of matching and calculating the integral of optical path disturbance includes matching the start time and end time of the transmission light intensity decrease interval in the calibrated particle event set with the same time position in the transmission light intensity time series to obtain a unique transmission light intensity decrease interval for each particle event in the transmission light intensity time series. By using the start time point of the transmitted light intensity decrease interval, the end time point of the transmitted light intensity decrease interval, the transmitted light intensity time series, and the optical baseline light intensity, the optical path perturbation integral for each particle event is calculated. The optical path perturbation integral is expressed as: ； in, For the first The integral of optical path perturbation for a single particle event For the first The starting time point of the transmission light intensity decrease range for each particle event. For the first The end time of the transmission light intensity decrease interval for each particle event. For the time series of transmitted light intensity in time The value of , The optical baseline light intensity; The process of constructing the optical path disturbance stability threshold and forming an optical stability event set includes: sorting all optical path disturbance integrals according to their numerical values; selecting the optical path disturbance integral at the median position in the sorting as the median optical path disturbance integral; calculating the absolute difference sequence between all optical path disturbance integrals and the median optical path disturbance integral; taking the value corresponding to the median position of the absolute difference sequence as the optical path disturbance integral discrete quantity; and summing the median optical path disturbance integral and the optical path disturbance integral discrete quantity to obtain the optical path disturbance stability threshold, which is expressed as: ； in, This is the optical path perturbation stability threshold. This is the median calculation function; When the integral of the optical path disturbance is less than or equal to the optical path disturbance stability threshold, the particle event is added to the optically stable event set. The optical equivalent diameter is estimated by calculating the optically stable event set and the calibrated particle event set, forming an equivalent optical size sequence and constructing a size distribution histogram. The calculation and estimation of the optical equivalent diameter to form an equivalent optical size sequence includes matching the optically stable event set with the calibrated particle event set one by one according to the start time point and end time point of the transmitted light intensity decrease interval. Particle events with completely consistent start and end times of the transmitted light intensity decrease interval are respectively included in the first type of optically stable reference particle event set, the second type of optically stable reference particle event set, and the optically stable particle event set of the sample to be tested. The average calibrated optical path perturbation depth of the first type of optically stable reference particle event set is obtained by averaging the calibrated optical path perturbation depth of all particle events in the first type of optically stable reference particle event set; the average calibrated optical path perturbation depth of the second type of optically stable reference particle event set is obtained by averaging the calibrated optical path perturbation depth of all particle events in the second type of optically stable reference particle event set. Based on the average calibrated optical path perturbation depth of the first and second type reference particles, and combined with the calibrated optical path perturbation depth in the optically stable particle event set of the sample under test, a linear mapping relationship between the calibrated optical path perturbation depth and the particle diameter is constructed to obtain the estimated optical equivalent diameter of the sample particles. The estimated optical equivalent diameter is expressed as: ； in, For the sample particles to be tested The estimated optical equivalent diameter, This represents the calibrated optical path perturbation depth during optically stable particle events in the sample under test. The average calibrated optical path perturbation depth of the first type of reference particle. The average calibrated optical path perturbation depth of the second type of reference particles; Substituting all optically stable particle events of the sample under test into the linear mapping relationship between the optical path perturbation depth and the particle diameter after calibration, the estimated optical equivalent diameter of all particles of the sample under test is obtained, forming an equivalent optical size sequence of the particles of the sample under test.

2. The microfluidic chip detection method for flow cytometry as described in claim 1, characterized in that: The step of obtaining the time series of transmitted light intensity and the lowest transmitted light intensity when the mixed suspension is flowing includes passing the mixed suspension through an optical detection window and recording the transmitted light intensity at a fixed sampling frequency through a photodetector to form a time series of transmitted light intensity. By scanning the time series of transmitted light intensity point by point to identify the decreasing interval, the minimum transmitted light intensity within the decreasing interval is found and recorded as the lowest transmitted light intensity of the particle event.

3. The microfluidic chip detection method for flow cytometry as described in claim 1, characterized in that: The construction of the size distribution histogram includes dividing the equivalent optical size sequence into several equal-width size intervals according to the minimum and maximum values ​​of the estimated optical equivalent diameter, counting the number of particles in each size interval, and constructing a size distribution histogram based on the estimated optical equivalent diameter.

4. A microfluidic chip detection system for flow cytometry, based on the microfluidic chip detection method for flow cytometry according to any one of claims 1 to 3, characterized in that: include, The optical baseline construction module collects transmitted light intensity under constant volume flow rate and constant light source brightness conditions, obtains the optical baseline light intensity through moving average, and obtains the time series of transmitted light intensity and the lowest transmitted light intensity when the mixed suspension is flowing. The optical path disturbance calibration module calculates the optical path disturbance depth and completes calibration based on the optical baseline light intensity, the time series of transmitted light intensity and the minimum transmitted light intensity, and obtains the calibrated set of particle events. The optical path stability screening module matches the calibrated set of particle events with the transmitted light intensity time series, calculates the optical path disturbance integral, constructs the optical path disturbance stability threshold by using the median and discrete values ​​of the optical path disturbance integral, and forms an optically stable event set. The particle size measurement module estimates the optical equivalent diameter by calculating the optical stable event set and the calibrated particle event set, forming an equivalent optical size sequence and constructing a size distribution histogram.

Images