Real-time biochemical monitoring method and system based on microfluidic optical tweezers

Abstract

The invention relates to the field of biochemical monitoring, and provides a real-time biochemical monitoring method and system based on micro-fluidic optical tweezers, and the real-time biochemical monitoring method based on micro-fluidic optical tweezers comprises the following steps: obtaining the type of particles of a to-be-tested reagent applied to a first chamber of a micro-fluidic chip; dispersing the particles of the to-be-tested reagent into a plurality of sub-chambers in a first chamber of the micro-fluidic chip according to the types of the particles; based on the dispersion results of the particles of the to-be-tested agent in the plurality of sub-chambers, performing optical tweezers treatment on the particles of the to-be-tested agent in different channels, and performing real-time monitoring on the channels corresponding to the optical tweezers treatment to determine suspicious target particles, the particles including the suspicious target particles, the suspicious target particles are associated with target particles. According to the technical scheme provided by the embodiment of the invention, effective separation and property determination can be carried out on the particles.

Description

Technical Field

[0001] The invention belongs to the field of biochemical monitoring, and in particular relates to a real-time biochemical monitoring method and system based on microfluidic optical tweezers. Background Art

[0002] Microfluidics is a systematic science and technology that uses pipes with a scale of tens to hundreds of microns to process or manipulate very small amounts of fluids. Laser tweezers, also known as optical tweezers or optical traps, are a technology that uses a three-dimensional potential well formed by a highly focused laser beam to capture, manipulate and control tiny particles.

[0003] In related technologies, microfluidics or optical tweezers technology is used to manipulate and monitor particles in fluids, and this is often based on the premise that the properties of the particles are already fully understood. However, for unknown particles, it is often impossible to reliably analyze their properties and / or to separate such particles. Summary of the invention

[0004] The purpose of the embodiments of the present invention is to provide a real-time biochemical monitoring method and system based on microfluidic optical tweezers, aiming to solve the problem that the properties of unknown particles cannot be reliably analyzed or separated.

[0005] The embodiment of the present invention is implemented as follows: on the one hand, a real-time biochemical monitoring method based on microfluidic optical tweezers comprises:

[0006] Acquiring the type of particles of the test agent applied to the first chamber of the microfluidic chip;

[0007] Dispersing the particles of the test agent into a plurality of sub-chambers in the first chamber of the microfluidic chip according to the types of the particles;

[0008] Based on the dispersion results of the particles of the test agent in the multiple sub-chambers, the particles of the test agent are subjected to optical tweezers processing under different channels, and the channels corresponding to the optical tweezers processing are monitored in real time to determine suspicious target particles, the particles include the suspicious target particles, and the suspicious target particles are associated with the target particles;

[0009] Based on the distribution of the suspicious target particles in the different channels, the properties of the suspicious target particles and / or the determined positions of the target particles in the different channels are determined.

[0010] As a further embodiment of the present invention, dispersing the particles of the test agent into a plurality of sub-chambers in the first chamber of the microfluidic chip according to the types of the particles comprises:

[0011] Determining variable centrifugation parameter data according to the type of the particles;

[0012] According to the variable centrifugal parameter data, the microfluidic chip is controlled to rotate around the rotation center until it is detected that the particles in the subchambers meet the preset dispersion conditions, and the preset dispersion conditions include that at least the target particles exist in a subchamber, wherein the first chamber includes a plurality of subchambers whose distances from the rotation center are gradually changed.

[0013] As a further solution of the present invention, based on the dispersion results of the particles of the test agent in multiple sub-chambers, the particles of the test agent are subjected to optical tweezers processing under different channels, and the channels corresponding to the optical tweezers processing are monitored in real time to determine suspicious target particles, the particles include the suspicious target particles, and the suspicious target particles are associated with the target particles, including:

[0014] determining a second dispersion parameter according to at least two of the plurality of sub-chambers having adjacent dispersion characteristics different from each other;

[0015] Attempting to disperse the particles of the test agent in at least two of the sub-chambers having adjacent dispersion characteristics different to different channels according to a second dispersion parameter, and performing optical tweezers processing based on the channels, wherein the parameters of the optical tweezers processing are associated with the target particles;

[0016] Among the particles captured by the optical tweezers process, the suspected target particles and the target particles are determined according to the relevant properties of the target particles.

[0017] As a further solution of the present invention, the optical tweezers processing based on the channel includes:

[0018] Based on the set position of the channel in the vertical direction, the light beam is turned on for irradiation.

[0019] As a further solution of the present invention, the method of obtaining the type of particles of the test agent applied to the first chamber of the microfluidic chip includes:

[0020] Acquire the formation period of the test agent, and determine the reduction data of all the reagents in the first area where the test agent is located within the formation period;

[0021] Determining maximum possible type data of particles of the test agent based on a combination of at least two of all the agents in the reduced data;

[0022] Determine abnormal category data based on abnormal combination of reagents among all reagents;

[0023] Based on the most likely species data and the abnormal species data, the species of the particles of the test agent are determined.

[0024] As a further solution of the present invention, the step of obtaining the formation period of the test reagent and determining the reduction data of all reagents in the first region where the test reagent is located during the formation period includes:

[0025] Determine the initial injection position corresponding to the test agent according to the injection image of the test agent in the microfluidic chip, wherein the injection image is the image when the test agent starts to be injected from the microfluidic chip;

[0026] Determining a time variation image of the test agent according to an initial injection position of the test agent;

[0027] Determining a formation period of the test agent according to the time variation image and a formation time node of a stable state of the test agent;

[0028] The reagent consumption data in the first area is acquired, and the formation period is used as a screening period to screen the consumption data to obtain the reduction data.

[0029] As a further solution of the present invention, determining the properties of the suspicious target particles and / or the determined positions of the target particles in different channels based on the distribution of the suspicious target particles in different channels includes:

[0030] Locating a first channel where a suspected target particle is captured by optical tweezers, the channel including the first channel;

[0031] According to the number of types of the suspected target particles captured by the optical tweezers in the first channel and the particles remaining at the end of the first channel, the properties of the suspected target particles and / or the determined positions of the target particles in different channels are determined.

[0032] As a further solution of the present invention, the method of determining the properties of the suspicious target particles and / or the determined positions of the target particles in different channels according to the number of types of the suspicious target particles captured by the optical tweezers in the first channel and the particles remaining at the end of the first channel includes:

[0033] If it is monitored that the number of types of the suspicious target particles captured by the optical tweezers in the first channel is at least one, based on the structure of the target particle, the determined position of the target particle or the determined position of the suspicious target particle is determined;

[0034] According to the process of the optical tweezers processing and the dispersion process of the particles, the correlation properties between the captured particles and the target particles are determined.

[0035] As a further solution of the present invention, the method of determining the properties of the suspicious target particles and / or the determined positions of the target particles in different channels according to the number of types of the suspicious target particles captured by the optical tweezers in the first channel and the particles remaining at the end of the first channel includes:

[0036] The characteristics of the remaining particles are determined based on the dispersion relationship between the particles remaining at the end of the first channel and the target particles, and / or the process of the optical tweezers treatment.

[0037] In another aspect, a real-time biochemical monitoring system based on microfluidic optical tweezers comprises:

[0038] A type determination module, used to obtain the type of particles of the test agent applied to the first chamber of the microfluidic chip;

[0039] A dispersion control module, used for dispersing the particles of the test agent into a plurality of sub-chambers in the first chamber of the microfluidic chip according to the types of the particles;

[0040] An optical tweezers and monitoring module, for performing optical tweezers processing on the particles of the test agent under different channels based on the dispersion results of the particles of the test agent in the multiple sub-chambers, and performing real-time monitoring on the channels corresponding to the optical tweezers processing to determine suspicious target particles, wherein the particles include the suspicious target particles, and the suspicious target particles are associated with the target particles;

[0041] The particle analysis module is used to determine the properties of the suspicious target particles and / or the determined positions of the target particles in the different channels based on the distribution of the suspicious target particles in the different channels.

[0042] The embodiment of the present invention provides a real-time biochemical monitoring method and system based on microfluidic optical tweezers, which obtains the type of particles of a test agent in a first chamber of a microfluidic chip; disperses the particles of the test agent into a plurality of sub-chambers in the first chamber of the microfluidic chip according to the type of the particles; performs optical tweezers treatment on the particles of the test agent under different first channels based on the dispersion results of the particles of the test agent in the plurality of sub-chambers, and monitors the first channel corresponding to the optical tweezers treatment in real time to determine suspicious target particles, wherein the particles include the suspicious target particles, and the suspicious target particles are associated with the target particles; based on the suspicious target particles, The distribution of the target particles in different first channels is determined, the properties of the suspected target particles and / or the determined positions of the target particles in different first channels are determined, and the suspected target particles are associated with the target particles, which not only realizes the separation of the target particles, but also determines the particles in the test agent that are likely to be close to the target particles in terms of mass, size, density and light scattering characteristics, as well as particles that may be close to the target particles in properties during the dispersion process but have large differences in mass, refractive index, etc. with the target particles, thereby avoiding tedious separation steps and providing a more reliable and effective reference for subsequent analysis based on the physical properties and optical properties of the particles. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 It is a main flow chart of a real-time biochemical monitoring method based on microfluidic optical tweezers.

[0044] Figure 2 The invention discloses a flow chart for determining the suspected target particle and the target particle according to the relevant properties of the target particle in a real-time biochemical monitoring method based on microfluidic optical tweezers.

[0045] Figure 3 The present invention is a flow chart for obtaining the types of particles of a test agent in a first chamber of a microfluidic chip in a real-time biochemical monitoring method based on microfluidic optical tweezers.

[0046] Figure 4 The invention is a flow chart for determining the correlation property between the captured particle and the target particle in a real-time biochemical monitoring method based on microfluidic optical tweezers.

[0047] Figure 5 This is one of the example diagrams of the main structure distribution of a microfluidic chip.

[0048] Figure 6 This is the main structural diagram of a real-time biochemical monitoring system based on microfluidic optical tweezers. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0050] In the related art, microfluidics technology or optical tweezers technology is used to manipulate and monitor particles in the fluid, and it is often based on the premise that the properties of the particles are all clear. However, for unknown particles, it is often impossible to effectively separate such particles and / or it is impossible to reliably analyze their properties. In order to solve this technical problem, the following solution is proposed.

[0051] First, let's introduce microfluidics and optical tweezers. Microfluidics is a scientific technology that precisely controls and manipulates microscale fluids, with the main feature of manipulating fluids in micro-nanoscale space. It has the ability to miniaturize the basic functions of biological and chemical laboratories, such as sample preparation, reaction, separation and detection, onto a chip of a few square centimeters. Its basic feature and greatest advantage is the flexible combination and large-scale integration of multiple unit technologies on an overall controllable micro-platform. The principle of optical tweezers is based on the radiation force of light. Quantum theory believes that light is a group of photons moving at the speed of light. The interaction between the light radiation field and the object will cause the object to be affected by the light radiation force. The light radiation force is divided into two types: the scattering force that pushes the object along the direction of light propagation and the gradient force that pulls the object in the direction of high light intensity density. When the gradient force exceeds the scattering force, the tiny particles are attracted to the point with the highest light intensity density and are stably captured at this point.

[0052] like Figure 1 As shown, it is a main flow chart of a real-time biochemical monitoring method based on microfluidic optical tweezers provided by an embodiment of the present invention, and the real-time biochemical monitoring method based on microfluidic optical tweezers includes:

[0053] Step S10: obtaining the type of particles of the test agent applied to the first chamber of the microfluidic chip 1;

[0054] Specifically, the test agent contains multiple particles, and the particles include target particles, suspected target particles and other particles. The properties of the target particles are at least partially known, and can also be determined based on non-target particles. The test agent is generally formed by erroneous operations (such as incorrect injection) or general operations (such as measuring relevant data of products of the same type of reactions or similar reactions). The types of particles in the test agent can be divided through artificial experience or obtained by tracing the formation process of the test agent. This type of type generally only requires a rough type.

[0055] Step S11 : Dispersing the particles of the test agent into a plurality of sub-chambers 101 in the first chamber of the microfluidic chip 1 according to the types of the particles.

[0056] Specifically, the geometric shape of the channel of the microfluidic chip 1 can be used (the geometric shape is set differently, not Figure 5 The first channel in the microfluidic chip 1 guides different particles into each sub-chamber 101, or the particles of the test agent are centrifuged to disperse the particles into multiple sub-chambers 101 in the first chamber of the microfluidic chip 1. The purpose of dispersion is to determine whether there are particles with at least one property that is close to or the same as the target particles.

[0057] Step S12: Based on the dispersion results of the particles of the test agent in multiple sub-chambers 101, the particles of the test agent are subjected to optical tweezers processing under different first channels, and the first channels corresponding to the optical tweezers processing are monitored in real time to determine suspicious target particles, wherein the particles include the suspicious target particles, and the suspicious target particles are associated with the target particles.

[0058] Specifically, when the particles of the test agent are subjected to optical tweezers treatment under different first channels, the particles preliminarily determined in the sub-chamber to have at least one characteristic close to or identical to the target particles can be taken out for operation (such as pumping out by a micropump), and then dispersed in the channel of the microfluidic chip 1. Optical tweezers treatment is performed during dispersion, and comparative analysis of the particles captured by the optical tweezers can help further determine whether the properties of these particles are close to the target particles. If the size (before specific monitoring is performed, it is determined that a particle may be a suspected target particle) and shape, light scattering characteristics, motion state, etc. (at least two) of the particles captured by the optical tweezers are similar to those of the target particles, then it is determined to be a suspected target particle.

[0059] Step S13: Based on the distribution of the suspicious target particles in different first channels, determine the properties of the suspicious target particles and / or the determined positions of the target particles in different first channels, and the suspicious target particles are associated with the target particles.

[0060] Specifically, the target particle will be captured by the optical tweezers with a high probability, and the exact position of the particle in the first channel can be accurately located by electron microscopy and high-energy electron beam imaging, and / or, for suspected target particles, one or more of the mass, size, density and light scattering characteristics are likely to be close to those of the target particles, or in other words, the properties are similar during the dispersion process. For particles that are not captured, the properties may be close to those of the target particles during the dispersion process (which may only be manifested as similar density), but in fact it may be that the excessively high particle mass significantly increases the inertia of the particle before the laser, and its mass is generally greater than that of the target particle. If it is clear that the mass difference is not large, it means that the particle is not sensitive to the response in the light beam, its refractive index is generally poor, the particle scatters light inefficiently, and may not be effectively captured.

[0061] When this embodiment is applied, the type of particles of the test agent applied to the first chamber of the microfluidic chip 1 is obtained; according to the type of the particles, the particles of the test agent are dispersed into a plurality of sub-chambers 101 in the first chamber of the microfluidic chip 1; based on the dispersion results of the particles of the test agent in the plurality of sub-chambers 101, the particles of the test agent are subjected to optical tweezers treatment under different first channels, and the first channel corresponding to the optical tweezers treatment is monitored in real time to determine suspicious target particles, the particles including the suspicious target particles, and the suspicious target particles are associated with the target particles ... dispersion results of the particles of the test agent in the plurality of sub-chambers 101, the first channel corresponding to the dispersion results of the particles of the test agent is subjected to optical tweezers treatment, and the first channel corresponding to the dispersion results of the particles of the test agent is subjected to optical tweezers treatment, and the first channel corresponding to the dispersion results of the particles of the test agent is subjected to optical tweezers treatment, and the first channel corresponding to the dispersion results of the particles of the test agent is subjected to optical tweezers treatment, and the first channel corresponding to the dispersion results of the particles of the test agent is subjected to optical tweezers treatment, and the first channel corresponding to the dispersion results of the particles of the test agent is subjected to optical tweezers treatment, and the first channel corresponding to the dispersion results of the particles of the The distribution of the suspected target particles in the first channels is determined, the properties of the suspected target particles and / or the determined positions of the target particles in different first channels are determined, and the suspected target particles are associated with the target particles, which not only realizes the separation of the target particles, but also determines the particles in the test agent that are likely to be close to the target particles in terms of mass, size, density and light scattering characteristics, as well as particles that may be close to the target particles in properties during the dispersion process but have large differences in mass, refractive index, etc. with the target particles, thereby avoiding cumbersome separation steps and providing a more reliable and effective reference for subsequent analysis based on the physical properties and optical properties of the particles.

[0062] As a preferred embodiment of the present invention, the particles of the test agent are dispersed into the multiple sub-chambers 101 in the first chamber of the microfluidic chip 1 according to the type of the particles, including: the microfluidic chip 1 slowly injects the particle suspension to be tested into the front storage chamber of the first chamber through a micropump, adjusts the injection flow rate according to the size and properties of the particles (such as target particles) to avoid particle aggregation or excessive disturbance to the flow, and guides different particles into each sub-chamber 101 (different from each other) according to the fluid dynamics characteristics by using the geometric shape of the channel. Figure 5In a preferred embodiment, the particles of the test agent are dispersed into the first chamber of the microfluidic chip 1 according to the type of the particles, and the plurality of sub-chambers 101 include:

[0063] Step S111: determining variable centrifugal parameter data according to the type of the particles.

[0064] Specifically, the theoretical centrifugal parameter data of the target particles are known. Under the conditions of the test agent, the range in which the target particles can actually be centrifuged is determined based on the theoretical centrifugal parameter data, and this range is used as the actual centrifugal parameter data of the target particles. In practice, there is a corresponding range of centrifugation for most types of other particles. Since the types of particles are roughly determined possible types, it is possible to query in the centrifugal correspondence table (type and centrifugal parameter correspondence table) based on the type to determine the centrifugal parameters, such as speed and time, that is, the variable centrifugal parameter data, because the particles in the test agent are products of the same reaction, similar reactions, or directly injected Therefore, it is determined that the variable centrifugal parameter data and the actual centrifugal parameter data of the target particles have an intersection, or the variable centrifugal parameter data is the actual centrifugal parameter data of the target particles multiplied by a finite coefficient, such as 0.8-1.3, (if the centrifugation time is long, the gradient centrifugation parameter data can be set, a rapid separation example), for example, the separation of red blood cells and plasma is achieved under the cooperation of high-speed centrifugation (including but not limited to 1000-6000rpm), and for particles of non-similar products, other centrifugal parameters can be determined; there are also examples such as red blood cells and white blood cells, microglia, polymer microspheres, etc., which are not described in detail here.

[0065] Step S112: According to the variable centrifugal parameter data, the microfluidic chip 1 is controlled to rotate around the rotation center 2 until it is detected that the particles in the sub-chamber 101 meet the preset dispersion conditions, and the preset dispersion conditions include that at least the target particles exist in a sub-chamber 101, wherein the first chamber includes a plurality of sub-chambers 101 with a gradually changing distance from the rotation center 2, so that the distances between the plurality of sub-chambers 101 and the rotation center 2 are different, such as setting Figure 5 As shown in FIG. 1 , there is a liquid storage chamber 102 , and the sub-chamber 101 and the liquid storage chamber 102 can be connected.

[0066] Specifically, the preset dispersion condition includes the presence of a single target particle in the subchamber 101 or a mixture of the target particle and a limited number of particles, such as one or two.

[0067] It should be understood that, through the setting of variable centrifugal parameter data and the provision of multiple sub-chambers 101 with a gradually varying distance from the rotation center 2, particles such as suspicious target particles can enter sub-chambers 101 that are close to or not far from the position of the target particles (such as adjacent sub-chambers 101 in the radial direction), indicating that these particles may be close to or not much different from the properties such as density and mass of the target particles.

[0068] like Figure 2 and Figure 5 ( Figure 5 As shown in one of the main structure distribution example diagrams of a microfluidic chip, other structures mentioned in the embodiments may also exist, as a preferred embodiment of the present invention, based on the dispersion results of the particles of the test agent in multiple sub-chambers 101, the particles of the test agent are subjected to optical tweezers treatment under different first channels, and the first channels corresponding to the optical tweezers treatment are monitored in real time to determine suspicious target particles, the particles include the suspicious target particles, and the suspicious target particles are associated with the target particles, including:

[0069] Step S20: Determine a second dispersion parameter according to at least two of the plurality of sub-chambers 101 having adjacent dispersion characteristic differences. The adjacent dispersion characteristic differences include adjacent time periods and / or adjacent centrifugal depths (the closer to the outside, the deeper).

[0070] Specifically, due to the difference in properties between particles (such as density and viscosity), the filling time of particles in the subchamber 101 may be sequential, and it is very likely that at least two particles with similar properties enter the same subchamber 101. At this time, the filling time of all particles is sorted according to the time sequence to obtain the sorting time, and then the sorting time is evenly divided into multiple groups of sorting time periods. At this time, it is considered that each sorting time period corresponds to at least two adjacent subchambers 101 with different dispersion characteristics. The second dispersion method includes at least one of a fluid flow pattern (the microfluidic chip 1 can be designed as a specific first flow channel, such as staggered, bifurcated, etc.), an electric field or a magnetic field, etc., and two adjacent groups in the multiple groups of sorting time periods are taken for trial dispersion of the second dispersion method, thereby obtaining a basic The second dispersion parameter can be calculated based on the dispersion parameter ratio per unit time. The corresponding second dispersion parameters of other groups are determined according to the time between them and the two adjacent groups and the dispersion parameter ratio. For example, after trial dispersion, the basic second dispersion parameters of A and B are determined to be A and B (for example, the size of the bifurcation of the first channel, where the size mainly refers to the width, in μm). The time between the two groups is C, and the dispersion parameter ratio a=C / (AB). The time between any group and A is D (longer than A), and the corresponding second dispersion parameter of any group is: {A-(D / C)*a)}*β (generally speaking, if the time is less, it means that the particle density is greater, or the mass is greater, then the minus sign should be replaced by a plus sign), and β is the error correction coefficient, which can be determined based on actual experience.

[0071] A method for determining the second dispersion parameter more quickly is given above. Another method is given below. The second dispersion parameter can also be determined based on attempts to determine each of at least two of the sub-chambers 101 one by one. After a group is determined, the shorter the time (or the deeper the sedimentation range), the greater the particle density or the greater the mass. In this case, the size of the first channel should be increased or a stronger electric field should be applied or the flow rate should be increased. Otherwise, the size of the first channel should be reduced or a weaker electric field should be applied. In theory, it is possible to disperse the particles in each of at least two of the sub-chambers 101.

[0072] Step S21: Attempt to disperse the particles of the test agent in at least two of the sub-chambers 101 to different first channels according to second dispersion parameters, and perform optical tweezers processing at set positions of the first channels, wherein the parameters of the optical tweezers processing are associated with the target particles.

[0073] The parameters of the optical tweezers processing are determined based on the target particles, and try to cover particles with the same or similar properties as the target particles. Therefore, the parameters of the optical tweezers processing need to be set to a wider range and be able to capture the target particles, thereby achieving the association of the parameters of the optical tweezers processing with the target particles.

[0074] After determining the second dispersion parameter, based on the aforementioned second dispersion method, each time the particles of the test agent in at least two of the sub-chambers 101 are dispersed into different first channels of the (fluidic chip), and the at least two adjacent sub-chambers 101 with different dispersion characteristics are taken as a group, and the second dispersion coefficient is the same. If it is monitored that the particles of the test agent in at least two of the sub-chambers 101 are dispersed into different first channels, it means that under the same second dispersion coefficient, the particles in at least two adjacent sub-chambers 101 with different dispersion characteristics are indeed separated, and the at least two adjacent sub-chambers 101 with different dispersion characteristics are separated. The property that some properties of particles in 01 are likely to be similar has been verified (such as properties such as density and mass are close). If not, or the dispersion is not obvious, for example, in at least two adjacent sub-chambers 101 with different dispersion characteristics, the particles in one sub-chamber 101 enter the first channel more obviously, while the other one does not, indicating that some properties of particles in at least two adjacent sub-chambers 101 with different dispersion characteristics may be similar, but factors such as the viscosity of the particles also need to be considered, that is, factors such as viscosity have a greater impact, especially in the second dispersion mode. In some cases, the shape and surface characteristics of the particles themselves also need to be considered.

[0075] Step S22: among the particles captured by the optical tweezers, determining the suspicious target particles and the target particles according to the relevant properties of the target particles.

[0076] Specifically, by comparing and analyzing the particles captured by the optical tweezers based on the target particles' particle size and shape, light scattering characteristics, motion state (such as recording the particle's motion trajectory and speed, and analyzing its behavior in the flow field) and other characteristics, it can help determine whether the properties of these particles are close to the target particles. If the size (before specific monitoring, it may be determined that a particle may be a suspected target particle) and shape, light scattering characteristics, motion state, etc. of a particle captured by the optical tweezers are similar to those of the target particle, it is determined to be a suspected target particle; or if only at least one characteristic of the target particle is known, it is determined whether a particle is the target particle based on at least one of the above similar characteristics. For example, by monitoring the shape and size of the suspected target particles, if only one particle meets the requirements, then this particle is most likely the target particle.

[0077] The above steps can identify suspicious target particles or even target particles, thereby analyzing and comparing some properties or characteristics of the particles to obtain useful monitoring information.

[0078] As a preferred embodiment of the present invention, performing optical tweezers processing at a set position of the first channel includes:

[0079] Based on the set position on the first channel, the light beam is turned on for irradiation.

[0080] Specifically, the beam parameters are first determined and modulated: the power, focal length, wavelength and other parameters of the beam can be adjusted. The modulation requirements are based on the target particles, that is, it is necessary to ensure that the target particles can be captured, and set the parameter range as wide as possible to ensure that particles with similar properties to the target particles can also be captured; the setting requirements for the position setting include: at least two groups of beams are set in parallel along the length of the first channel, and each group of beams has at least two beams evenly arranged to ensure equal opportunities for capturing particles. In some cases (if it is determined that there is a certain difference in the mobility of the particles, additional inspection can be performed ), then at least two groups of light beams are staggered, the width of the first channel is generally sufficient (can be set to be greater than 2 times and less than 3 times the size of the target particle). In the application of optical tweezers, the focus of the light beam should be as close as possible to the flow position of the target particle in the flow channel to increase the probability of the particle being captured; it should be noted that the components providing optical tweezers include a laser source, a diffraction optical element (a diffraction light field is generated after the laser penetrates) and a focusing mirror (receives the laser after being penetrated by the diffraction optical element and focuses it on the plane of the test sample), etc. The components providing optical tweezers themselves belong to the prior art and are not described here.

[0081] As a preferred embodiment of the present invention, the types of particles of the test agent applied to the first chamber of the microfluidic chip 1 are directly provided by the experimenter through actual deduction and experience.

[0082] like Figure 3 As shown, in another embodiment, the method of obtaining the type of particles of the test agent applied to the first chamber of the microfluidic chip 1 includes:

[0083] Step S30: Acquire the formation period of the test reagent, and determine the reduction data of all reagents in the first area where the test reagent is located within the formation period.

[0084] Specifically, the time when the test agent is formed can be traced. As a reagent of the test cost, its formation is related to the reduction of other reagents, and may be formed by erroneous operation (such as wrong injection) or general operation (such as just to measure relevant data of the product, etc.).

[0085] Step S31: Determine the maximum possible type data of particles of the test agent according to the combination of at least two of all the test agents in the reduced data.

[0086] Suppose we have 4 reagents: A, B, C, and D. If we want to choose 2 reagents for testing, we can calculate all possible combinations, choose 2: C(4,2) = 6; choose 3: C(4,3) = 4; choose 4 (that is, all reagents): C(4,4) = 1, the total number of types is 11.

[0087] Step S32: Determine abnormal category data based on abnormal combination reagents among all reagents.

[0088] For example, there may be proteins in the serum that compete with or interact with a compound, affecting the activity and effect of a compound. Antigens in the serum may react with specific antibodies. Similarly, these abnormal data types need to be eliminated based on the formation of precipitation, explosion, gas generation, large-scale consumption of reagents, etc., and then the objects to be eliminated (specific to the type of particles) can be manually confirmed. For example, there are three abnormal combinations of reagents. If it is caused by inorganic reagents, the inorganic reagents should be removed directly. If it is caused by two biological reagents that interact with each other, it is preferred to remove one that affects multiple. If there is a one-to-one interaction, they should be removed separately and the other particle type to be retained as the test agent should be retained accordingly.

[0089] Step S33: Determine the type of particles of the test agent based on the most likely type data and the abnormal type data.

[0090] Generally speaking, the difference between the maximum possible type data and the abnormal type data is generally the type of particles of the test agent, which is a possible type. The purpose of removing the abnormal type data is to ensure the application of the test agent as a flowable particle in the microfluidic chip 1.

[0091] It can be understood that through the above steps, it can be understood that the above steps, to a certain extent, ensure the comprehensiveness and accuracy of the types of particles to be tested.

[0092] As a preferred embodiment of the present invention, the step of obtaining the formation period of the test reagent and determining the reduction data of all reagents in the first region where the test reagent is located during the formation period includes:

[0093] Step: Determine the initial injection position corresponding to the test agent according to the injection image of the test agent in the microfluidic chip 1 , wherein the injection image is the image when the test agent starts to be injected from the microfluidic chip 1 .

[0094] Specifically, the test reagent is generally placed in an instrument such as a reagent bottle before injection, and the reagent bottle is placed at the reagent storage location, such as a reagent rack. When the test reagent is injected into the injection port (or reagent storage cavity) of the microfluidic chip 1, an image will be recorded, namely the injection image. The injection image includes the injection port (or reagent storage cavity) and meets the coverage of a preset range size. When the injection image identifies the position where the instrument first appears in the microfluidic chip 1, it is the initial injection position.

[0095] Step: Determine the time variation image of the test agent according to the initial injection position of the test agent.

[0096] Specifically, based on the coexisting image of the instrument and the instrument identification at the initial injection position, multiple frames of images that meet the time sequence are identified in the image of the first area, and the multiple frames of images all meet the third similarity threshold with the coexisting image to obtain the time change image of the test agent.

[0097] Step: determining a formation period of the test agent according to the time variation image and a formation time node of a stable state of the test agent.

[0098] Specifically, the formation time node of the stable state of the test agent indicates that the test agent has been determined and will no longer change. Generally, the test agent has been formed and stabilized before being moved to the microfluidic chip 1. According to at least one frame of the image at the front time node in the time change image, the position where the reagent is stored is located (each frame of the image carries the coordinates in the first area, and the position change can be determined according to the coordinates, and then the coordinates that meet the position where the reagent is stored are found), such as a reagent rack. There is a relatively strict correspondence between the positions where the reagents are stored, such as cells, microorganisms, etc., which strictly correspond to each experiment, or correspond to the same link of a type of experiment, such as the product link. The position where the reagent is stored is provided with a volume metering or weight metering device, and the metering parameters of the reagent storage position are read and analyzed. When the volume or weight begins to be measured to a constant time period, it is the formation period of the test agent.

[0099] Step: acquiring reagent consumption data in the first area, taking the formation period as a screening period, screening the consumption data, and obtaining the reduction data.

[0100] Specifically, the reagent consumption data is strictly related to the location where the reagents are stored. The reagent consumption data is the data consumed by each experiment or a type of experiment corresponding to the location where the reagents are stored. This can be counted (each relevant consumption is determined by manually uploading records or reading the experimental consumption sampling table for each experiment or a type of experiment corresponding to the location where the reagents are stored). After obtaining the reagent consumption data, the formation time period is screened. The reagents consumed in the formation time period are strictly related to the reagents to be tested. The former are mixed to form the latter, or the former reacts to form the latter.

[0101] This embodiment can trace the source of the test agent used in the microfluidic chip 1 and accurately locate its source and formation time, thereby determining the consumption data closely related to its components, thereby ensuring the accuracy of the type coverage of the test agent particles to a certain extent.

[0102] As a preferred embodiment of the present invention, the determining of the properties of the suspicious target particles and / or the determined positions of the target particles in different first channels based on the distribution of the suspicious target particles in different first channels, wherein the suspicious target particles are associated with the target particles, comprises:

[0103] Step: locating a first channel where a suspected target particle is captured by optical tweezers, wherein the channel includes the first channel.

[0104] Specifically, the target particles are likely to be captured by the optical tweezers, and the probability of suspicious target particles being captured by the optical tweezers is also relatively high. When locating the first channel, the image within the preset range corresponding to the focus of the detected light beam is compared with the standard image in the captured state. When the two meet the first preset threshold, they are considered to be captured. At this time, the first channel can be located by the channel identifier corresponding to the image within the preset range.

[0105] Step: Determine the properties of the suspicious target particles and / or the determined positions of the target particles in different first channels based on the number of types of the suspicious target particles captured by the optical tweezers in the first channel and the particles remaining at the end of the first channel.

[0106] Specifically, if the number of suspected target particles in different first channels is significantly greater than the number of remaining particles at the end, it means that the particles in this area have a higher capture efficiency, which may mean that they are likely to be similar to the target particles in certain key properties (such as at least one of the refractive index and size); if the number of remaining particles is large and exhibits significantly different physical properties, it may mean that there are significant differences between those uncaptured particles and the target particles, and the specific position of the target particles in the first channel can be determined based on optical tweezers behavior or supplementary microscope imaging comparison.

[0107] like Figure 4 As shown, as a preferred embodiment of the present invention, the method of determining the properties of the suspicious target particles and / or the determined positions of the target particles in different first channels according to the number of types of the suspicious target particles captured by the optical tweezers in the first channel and the particles remaining at the end of the first channel includes:

[0108] Step S40: If it is monitored that the number of types of the suspicious target particles captured by the optical tweezers in the first channel is at least one, the determined position of the target particle or the determined position of the suspicious target particle is determined based on the structure of the target particle.

[0109] Specifically, the structure of the particle may include its physical shape (such as spherical, rod-shaped, etc.), size, density, surface charge, material composition, etc. These characteristic information are very important for the identification and classification of particles; after being captured by optical tweezers, the precise position of the particle in the first channel can be located by using microscopy technology or other imaging methods (such as fluorescence imaging, scattering imaging). For example, electron microscopy uses high-energy electron beam imaging, which can be accurate to the nanometer level, and provides detailed information on the shape and structure of particles (including target particles). When the monitored high-resolution imaging and the preset imaging of the (target) particle meet the first preset threshold, it is considered that the particle in the high-resolution imaging is included in the optional range of the target particle (it is very likely to be the target particle). Further, mass spectrometry and chromatography technology (such as gas chromatography and liquid chromatography) can be combined to separate and identify the particles according to their behavior in different chemical media. Due to the position of the optical tweezers, the position of the suspicious target particle can be determined (of course, non-suspicious target particles may also be captured). The optical tweezers capture particles by generating light pressure, and the particles are captured at the focus of the beam, and the position of the focus of the beam is determined.

[0110] Step S41: determining the correlation properties between the captured particles and the target particles according to the optical tweezers processing process and the particle dispersion process.

[0111] Specifically, the suspected target particle may have some relationship with the target particle, such as homologous particles, byproducts formed in the reaction, or aggregation phenomena. In many biological and chemical experiments, the behavior or characteristics of the target particle will affect the surrounding particles (i.e., the suspected target particles). Different technical means (such as particle tracking technology, dynamic light scattering, etc.) can be used to analyze and determine the characteristics of these suspected target particles to further understand the role or influence of the target particles; when both the target particle and the suspected target particle are captured by light, it can be considered that the two captured particles may be similar in physical properties (such as one or more of the size and refractive index), resulting in similar responses to the same laser beam, which indicates that optical tweezers can effectively capture particles with certain common characteristics, combined with the dispersion process (the dispersion process in the first chamber); record the corresponding sub-chamber. 101, and then trace the dispersion process of the target particles through experimental data, and determine the suspicious target particles that are close to the target particles in dispersion time (within the first deviation time) and far from the target particles in dispersion time (outside the first deviation time). The properties of these particles in certain dispersion processes indicate that they are connected or different from the target particles. If they are within the first deviation time and are all captured, it means that the size, refractive index, density, etc. are similar (suspected target particles), and if they are outside the first deviation time and are captured, it means that although there may be some differences in mass and density (non-suspected target particles, and most likely not target particles), they can show similar characteristics in the optical tweezers environment and can all be captured. In fact, it is also achieved to separate non-suspected target particles and determine their properties.

[0112] By systematically recording and analyzing the dispersion process of the target particles and their associated particles, the behaviors of different particles in the optical tweezers environment and the performance of different particles in the dispersion process can be effectively identified and compared, which not only helps to understand the interactions between particles, but also provides important basic data for subsequent research.

[0113] As a preferred embodiment of the present invention, the method of determining the properties of the suspicious target particles and / or the determined positions of the target particles in different first channels according to the number of types of the suspicious target particles captured by the optical tweezers in the first channel and the particles remaining at the end of the first channel comprises:

[0114] Step: Determine the characteristics of the remaining particles based on the dispersion relationship between the particles remaining at the end of the first channel and the target particles, and / or the process of the optical tweezers treatment.

[0115] Specifically, the volume at the end of the first channel is relatively large, that is, larger than the size of the front end of the first channel (the entry end of the particle). The end of the first channel can also be set as a second chamber. By performing high-resolution monitoring on the particles remaining at the end of the first channel, it is determined that several types of particles are contained and / or at least one particle is directly measured by detecting the reagent (especially when only one type of particle is contained). The dispersion relationship between the remaining particles and the target particles includes that the two have similar properties under the first dispersion, such as the density difference is not large (entering a certain sub-chamber 101 together), or similar dissolution, and combining with each other through hydrogen bonds (the two are found to be separated after heating, etc.). The above can determine or roughly determine the physical properties and chemical properties. For the optical tweezers processing process, the remaining particles (generally excluding suspicious target particles) indicate that they are not captured by the optical tweezers. If the density is similar When , it means that the excessively high particle mass will significantly increase the inertia of the particle before the laser, and its mass is generally greater than that of the target particle. If it is clear that the mass difference is not much, it means that the particle is insensitive to the response in the light beam, its refractive index is generally poor, the particle scatters light inefficiently, and may not be effectively captured; if the spacing is significantly smaller than the set spacing, it means that the viscosity of the liquid medium affects the effectiveness of the optical tweezers operation, because in a high viscosity environment, the particle movement encounters greater resistance and manipulation may be more difficult. Combined with the above dispersion relationship between the particles and the target particles, and / or the process of the optical tweezers processing, the relevant properties of the remaining particles can be analyzed, mainly including physical properties and optical properties, for reference in subsequent processes of biochemical monitoring, and actual process parameters can be attached, such as the size of the first channel, the target particles, and the beam parameters of the optical tweezers processing.

[0116] like Figure 6 As shown, as another preferred embodiment of the present invention, on the other hand, a real-time biochemical monitoring system 200 based on microfluidic optical tweezers comprises:

[0117] The type determination module 210 is used to obtain the type of particles of the test agent applied to the first chamber of the microfluidic chip 1;

[0118] A dispersion control module 220, for dispersing the particles of the test agent into a plurality of sub-chambers 101 in the first chamber of the microfluidic chip 1 according to the types of the particles;

[0119] An optical tweezers and monitoring module 230, for performing optical tweezers processing on the particles of the test agent under different first channels based on the dispersion results of the particles of the test agent in the multiple sub-chambers 101, and performing real-time monitoring on the first channels corresponding to the optical tweezers processing to determine suspicious target particles, the particles including the suspicious target particles, and the suspicious target particles are associated with the target particles;

[0120] The particle analysis module 240 is used to determine the properties of the suspicious target particles and / or the determined positions of the target particles in different first channels based on the distribution of the suspicious target particles in different first channels, and the suspicious target particles are associated with the target particles.

[0121] It should be noted that, referring to the specific implementation description of a real-time biochemical monitoring method based on microfluidic optical tweezers in the aforementioned embodiment, the present system completely corresponds to the implementation method of the method and will not be described again here.

[0122] The above-mentioned embodiment of the present invention provides a real-time biochemical monitoring method based on microfluidic optical tweezers, and based on the real-time biochemical monitoring method based on microfluidic optical tweezers, provides a real-time biochemical monitoring system based on microfluidic optical tweezers, by obtaining the type of particles of the test agent in the first chamber of the microfluidic chip 1; according to the type of the particles, the particles of the test agent are dispersed into multiple sub-chambers 101 in the first chamber of the microfluidic chip 1; based on the dispersion results of the particles of the test agent in the multiple sub-chambers 101, the particles of the test agent are subjected to optical tweezers treatment under different first channels, and the first channel corresponding to the optical tweezers treatment is monitored in real time to determine suspicious target particles, the particles including the Suspected target particles, the suspected target particles are associated with target particles; based on the distribution of the suspected target particles in different first channels, the properties of the suspected target particles and / or the determined positions of the target particles in different first channels are determined, the suspected target particles are associated with the target particles, not only the separation of the target particles is achieved, but also the particles in the test agent whose mass, size and density are likely to be close to the target particles; in the dispersion process, the particles whose properties may be close to the target particles but whose mass, refractive index, etc. are greatly different from the target particles are determined, avoiding tedious separation steps, and can provide a more reliable and effective reference for the subsequent analysis of the physical properties, chemical properties and optical properties of the particles.

[0123] In order to load the above-mentioned method and system and enable it to run smoothly, the system, in addition to the various modules mentioned above, may also include more or fewer components than described above, or a combination of certain components, or different components, for example, it may include input and output devices, network access devices, buses, processors and memories, etc.

[0124] The processor may be a central processing unit (CPU), other general-purpose processors, digital signal processors (DSP), application-specific integrated circuits (ASIC), field-programmable gate arrays (FPGA) or other programmable logic devices, discrete gate or transistor logic devices, discrete hardware components, etc. The general-purpose processor may be a microprocessor or any conventional processor, etc. The processor is the control center of the system, and various interfaces and lines are used to connect various parts.

[0125] It should be understood that, although each step in the flow chart of each embodiment of the present invention is shown in sequence according to the indication of the arrow, these steps are not necessarily performed in sequence according to the order indicated by the arrow. Unless there is a clear explanation in this article, the execution of these steps does not have a strict order restriction, and these steps can be performed in other orders. Moreover, at least a portion of the steps in each embodiment may include a plurality of sub-steps or a plurality of stages, and these sub-steps or stages are not necessarily performed at the same time, but can be performed at different times, and the execution order of these sub-steps or stages is not necessarily performed in sequence, but can be performed in turn or alternately with at least a portion of other steps or sub-steps or stages of other steps.

[0126] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0127] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

[0128] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A real-time biochemical monitoring method based on microfluidic optical tweezers, characterized in that: include: Acquiring the type of particles of the test agent applied to the first chamber of the microfluidic chip; Dispersing the particles of the test agent into a plurality of sub-chambers in the first chamber of the microfluidic chip according to the types of the particles; Based on the dispersion results of the particles of the test agent in the multiple sub-chambers, the particles of the test agent are subjected to optical tweezers processing under different channels, and the channels corresponding to the optical tweezers processing are monitored in real time to determine suspicious target particles, the particles include the suspicious target particles, and the suspicious target particles are associated with the target particles; Based on the distribution of the suspicious target particles in the different channels, the properties of the suspicious target particles and / or the determined positions of the target particles in the different channels are determined.

2. The real-time biochemical monitoring method based on microfluidic optical tweezers according to claim 1, characterized in that: The step of dispersing the particles of the test agent into a plurality of sub-chambers in the first chamber of the microfluidic chip according to the types of the particles comprises: Determining variable centrifugation parameter data according to the type of the particles; According to the variable centrifugal parameter data, the microfluidic chip is controlled to rotate around the rotation center until it is monitored that the particles in the sub-chamber meet the preset dispersion conditions, and the preset dispersion conditions include that at least the target particles exist in a sub-chamber, wherein the first chamber includes a plurality of sub-chambers whose distances from the rotation center are gradually changed.

3. The real-time biochemical monitoring method based on microfluidic optical tweezers according to claim 2, characterized in that: Based on the dispersion results of the particles of the test agent in the multiple sub-chambers, the particles of the test agent are subjected to optical tweezers processing under different channels, and the channels corresponding to the optical tweezers processing are monitored in real time to determine suspicious target particles, the particles include the suspicious target particles, and the suspicious target particles are associated with the target particles, including: determining a second dispersion parameter according to at least two of the plurality of sub-chambers having adjacent dispersion characteristics different from each other; Attempting to disperse the particles of the test agent in at least two of the sub-chambers having adjacent dispersion characteristics different to different channels according to the second dispersion parameter, and performing optical tweezers processing based on the channels, wherein the parameters of the optical tweezers processing are associated with the target particles; Among the particles captured by the optical tweezers process, the suspected target particles and the target particles are determined according to the relevant properties of the target particles.

4. The real-time biochemical monitoring method based on microfluidic optical tweezers according to claim 3 is characterized in that: The optical tweezers processing based on the channel comprises: Based on the set position of the channel in the vertical direction, the light beam is turned on for irradiation.

5. The real-time biochemical monitoring method based on microfluidic optical tweezers according to claim 3, characterized in that: The method of obtaining the type of particles of the test agent applied to the first chamber of the microfluidic chip includes: Acquire the formation period of the test agent, and determine the reduction data of all the reagents in the first area where the test agent is located within the formation period; Determining maximum possible type data of particles of the test agent based on a combination of at least two of all the agents in the reduced data; Determine abnormal category data based on abnormal combination of reagents among all reagents; Based on the most likely species data and the abnormal species data, the species of the particles of the test agent are determined.

6. The real-time biochemical monitoring method based on microfluidic optical tweezers according to claim 5, characterized in that: The step of obtaining the formation period of the test reagent and determining the reduction data of all reagents in the first area where the test reagent is located within the formation period includes: Determine the initial injection position corresponding to the test agent according to the injection image of the test agent in the microfluidic chip, wherein the injection image is the image when the test agent starts to be injected from the microfluidic chip; Determining a time variation image of the test agent according to an initial injection position of the test agent; Determining a formation period of the test agent according to the time variation image and a formation time node of a stable state of the test agent; The reagent consumption data in the first area is acquired, and the formation period is used as a screening period to screen the consumption data to obtain the reduction data.

7. The real-time biochemical monitoring method based on microfluidic optical tweezers according to any one of claims 1 to 6, characterized in that: Determining the properties of the suspicious target particles and / or the determined positions of the target particles in the different channels based on the distribution of the suspicious target particles in the different channels comprises: Locating a first channel where a suspected target particle is captured by optical tweezers, the channel including the first channel; According to the number of types of the suspected target particles captured by the optical tweezers in the first channel and the particles remaining at the end of the first channel, the properties of the suspected target particles and / or the determined positions of the target particles in different channels are determined.

8. The real-time biochemical monitoring method based on microfluidic optical tweezers according to claim 7, characterized in that: Determining the properties of the suspicious target particles and / or the determined positions of the target particles in different channels according to the number of types of the suspicious target particles captured by the optical tweezers in the first channel and the particles remaining at the end of the first channel includes: If it is monitored that the number of types of the suspicious target particles captured by the optical tweezers in the first channel is at least one, based on the structure of the target particle, the determined position of the target particle or the determined position of the suspicious target particle is determined; According to the optical tweezers processing process and the particle dispersion process, the correlation properties between the captured particles and the target particles are determined, and the captured particles include suspicious target particles.

9. The real-time biochemical monitoring method based on microfluidic optical tweezers according to claim 8, characterized in that: Determining the properties of the suspicious target particles and / or the determined positions of the target particles in different channels according to the number of types of the suspicious target particles captured by the optical tweezers in the first channel and the particles remaining at the end of the first channel includes: The characteristics of the remaining particles are determined based on the dispersion relationship between the particles remaining at the end of the first channel and the target particles, and / or the process of the optical tweezers treatment.

10. A real-time biochemical monitoring system based on microfluidic optical tweezers, characterized in that: The real-time biochemical monitoring method based on microfluidic optical tweezers as claimed in any one of claims 1 to 9 comprises: A type determination module, used to obtain the type of particles of the test agent applied to the first chamber of the microfluidic chip; A dispersion control module, used for dispersing the particles of the test agent into a plurality of sub-chambers in the first chamber of the microfluidic chip according to the types of the particles; An optical tweezers and monitoring module, for performing optical tweezers processing on the particles of the test agent under different channels based on the dispersion results of the particles of the test agent in the multiple sub-chambers, and performing real-time monitoring on the channels corresponding to the optical tweezers processing to determine suspicious target particles, wherein the particles include the suspicious target particles, and the suspicious target particles are associated with the target particles; The particle analysis module is used to determine the properties of the suspicious target particles and / or the determined positions of the target particles in the different channels based on the distribution of the suspicious target particles in the different channels.

Images