A biological detection method and system based on fluorescence and microfluidics

Abstract

The present application provides a biological detection method based on fluorescence and microfluidics, the method comprising: continuously injecting a first solution comprising at least one fluorescent molecular probe into a fluorescent chamber and a microfluidic chamber of a microfluidic chip, wherein a biological body is placed in the microfluidic chamber; obtaining first imaging information corresponding to the fluorescent chamber and second imaging information corresponding to the microfluidic chamber; and determining the kinetic information of the biological body with respect to the fluorescent molecular probe based on the first imaging information and the second imaging information.

Description

Technical Field

[0001] The present application relates to biological detection technology, and in particular to a biological detection method and system based on fluorescence and microfluidics. Background Art

[0002] By studying the interaction between molecules and organisms or biological samples, we can evaluate the biological properties of molecules and obtain physiological and pathological information of organisms, which is of great significance for disease research and drug screening. Therefore, how to determine the interaction between molecules and organisms in real time and efficiently is a goal that has been pursued in the field of biological detection. Summary of the invention

[0003] The embodiments of the present application provide a fluorescence and microfluidics-based biological detection method and system, which can determine the interaction between molecules and organisms in real time and efficiently.

[0004] The technical solution of the embodiment of the present application is implemented as follows:

[0005] In a first aspect, the present application provides a biological detection method based on fluorescence and microfluidics, comprising:

[0006] Continuously injecting a first solution including at least one fluorescent molecular probe into the fluorescent chamber and the microfluidic chamber of the microfluidic chip, wherein a biological body is placed in the microfluidic chamber;

[0007] Acquire first imaging information corresponding to the fluorescence chamber and second imaging information corresponding to the microfluidic chamber;

[0008] Based on the first imaging information and the second imaging information, kinetic information of the organism with respect to the fluorescent molecular probe is determined.

[0009] In some optional embodiments, different fluorescent molecular probes correspond to different wavelengths.

[0010] In some optional embodiments, after the first solution is continuously injected into the fluorescence chamber and the microfluidic chamber for a first period of time, the method further comprises:

[0011] continuously injecting a basic culture solution into the microfluidic chamber, wherein the basic culture solution does not include the fluorescent molecular probe;

[0012] detecting third imaging information corresponding to the microfluidic chamber;

[0013] The kinetic information of the organism with respect to the fluorescent molecular probe is determined based on the second imaging information and the third imaging information.

[0014] In some optional embodiments, the continuously injecting the first solution including the fluorescent molecular probe into the fluorescent chamber and the microfluidic chamber of the microfluidic chip comprises:

[0015] continuously injecting the first solution into the fluorescence chamber;

[0016] The first solution flows into the microfluidic chamber via a pipe connected to the fluorescence chamber and the microfluidic chamber.

[0017] In some optional embodiments, the continuously injecting the first solution including the fluorescent molecular probe into the fluorescent chamber and the microfluidic chamber of the microfluidic chip comprises:

[0018] continuously injecting the first solution into the fluorescence chamber and the microfluidic chamber respectively;

[0019] Wherein, the fluorescence chamber and the microfluidic chamber are independent chambers.

[0020] In some optional embodiments, determining the kinetic information of the organism with respect to the fluorescent molecular probe based on the first imaging information and the second imaging information includes:

[0021] Acquire second concentration information of the fluorescent molecular probe in the organism based on the second imaging information;

[0022] Acquire first concentration information of the fluorescent molecular probe in the fluorescent chamber based on the first imaging information;

[0023] Based on the first concentration information and the second concentration information, kinetic information of the organism with respect to the fluorescent molecular probe is determined.

[0024] In some optional embodiments, determining the kinetic information of the organism with respect to the fluorescent molecular probe based on the first concentration information and the second concentration information includes:

[0025] Determining a concentration difference of the fluorescent molecular probe in the fluorescent chamber and the microfluidic chamber based on the second concentration information and the first concentration information;

[0026] The absorption of the fluorescent molecular probe by the organism is determined based on the concentration difference.

[0027] In some optional embodiments, determining the kinetic information of the organism with respect to the fluorescent molecular probe based on the second imaging information and the third imaging information includes:

[0028] Acquire second concentration information of the fluorescent molecular probe in the organism based on the second imaging information;

[0029] Acquire third concentration information of the fluorescent molecular probe in the organism based on the third imaging information;

[0030] Based on the third concentration information and the second concentration information, determining a concentration difference of the fluorescent molecules before and after the basic culture solution is injected into the microfluidic chamber;

[0031] The release of the fluorescent molecular probe by the organism is determined based on the concentration difference.

[0032] In a second aspect, an embodiment of the present application provides a biological detection system based on fluorescence and microfluidics, the system comprising:

[0033] A fluid control unit, used for controlling the continuous injection of a first solution including at least one fluorescent molecular probe into the fluorescent chamber and the microfluidic chamber of the microfluidic chip, wherein the microfluidic chamber contains a biological body;

[0034] An imaging detector, used to obtain first imaging information corresponding to the fluorescence chamber and second imaging information corresponding to the microfluidic chamber;

[0035] The microfluidic chip is used to determine the kinetic information of the organism with respect to the fluorescent molecular probe based on the first imaging information and the second imaging information.

[0036] In some optional embodiments, different fluorescent molecular probes correspond to different wavelengths.

[0037] In some optional embodiments, the fluid control unit is further used to continuously inject a basic culture solution into the microfluidic chamber, wherein the basic culture solution does not include the fluorescent molecular probe;

[0038] detecting third imaging information corresponding to the microfluidic chamber;

[0039] The kinetic information of the organism with respect to the fluorescent molecular probe is determined based on the second imaging information and the third imaging information.

[0040] In some optional embodiments, the fluid control unit is used to continuously inject the first solution into the fluorescence chamber;

[0041] The first solution flows into the microfluidic chamber via a pipe connected to the fluorescence chamber and the microfluidic chamber.

[0042] In some optional embodiments, the fluid control unit is used to continuously inject the first solution into the fluorescence chamber and the microfluidic chamber respectively;

[0043] Wherein, the fluorescence chamber and the microfluidic chamber are independent chambers.

[0044] In some optional embodiments, the microfluidic chip is used to obtain second concentration information of the fluorescent molecular probe in the organism based on the second imaging information;

[0045] Acquire first concentration information of the fluorescent molecular probe in the fluorescent chamber based on the first imaging information;

[0046] Based on the first concentration information and the second concentration information, kinetic information of the organism with respect to the fluorescent molecular probe is determined.

[0047] In some optional embodiments, the microfluidic chip is used to determine the concentration difference of the fluorescent molecules in the fluorescence chamber and the microfluidic chamber based on the second concentration information and the first concentration information;

[0048] The absorption of the fluorescent molecular probe by the organism is determined based on the concentration difference.

[0049] In some optional embodiments, the microfluidic chip is used to obtain second concentration information of the fluorescent molecular probe in the organism based on the second imaging information;

[0050] Acquire third concentration information of the fluorescent molecular probe in the organism based on the third imaging information;

[0051] Based on the third concentration information and the second concentration information, determining the concentration difference of the fluorescent molecular probe before and after the basic culture solution is injected into the microfluidic chamber;

[0052] The release of the fluorescent molecular probe by the organism is determined based on the concentration difference.

[0053] The fluorescence and microfluidics-based biological detection method provided in the embodiment of the present application includes: continuously injecting a first solution including at least one fluorescent molecular probe into a fluorescent chamber and a microfluidic chamber of a microfluidic chip, wherein a biological body is placed in the microfluidic chamber; obtaining first imaging information corresponding to the fluorescent chamber and second imaging information corresponding to the microfluidic chamber; based on the first imaging information and the second imaging information, determining the kinetic information of the biological body with respect to the fluorescent molecular probe. In this way, by continuously injecting the first solution into the fluorescent chamber and the microfluidic chamber, the interaction between the fluorescent molecules and the biological body can be detected in real time. Since the types of fluorescent molecular probes are one or more, the interaction between multiple fluorescent molecules and the biological body can be detected simultaneously, thereby improving the efficiency of detecting the interaction between fluorescent molecules and the biological body. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1This is a schematic diagram of an optional processing flow of a biological detection based on fluorescence and microfluidics provided in an embodiment of the present application;

[0055] Figure 2-1 This is a schematic diagram of a connection between a fluorescence chamber and a microfluidic chamber provided in an embodiment of the present application;

[0056] Figure 2-2 This is another schematic diagram of connecting the fluorescence chamber and the microfluidic chamber provided in an embodiment of the present application;

[0057] Figure 2-3 This is another schematic diagram of the fluorescence chamber and the microfluidic chamber provided in the embodiment of the present application;

[0058] Figure 2-4 This is another schematic diagram of connecting the fluorescence chamber and the microfluidic chamber provided in the embodiment of the present application;

[0059] Figure 2-5 This is another connection schematic diagram of the fluorescence chamber and the microfluidic chamber provided in the embodiment of the present application;

[0060] Figure 3 It is a schematic diagram of a specific implementation process of determining the kinetic information of an organism with respect to a fluorescent molecular probe based on the first imaging information and the second imaging information provided in an embodiment of the present application;

[0061] Figure 4 It is another optional processing flow diagram of the fluorescence and microfluidics-based biological detection method provided in the embodiment of the present application;

[0062] Figure 5-1 The embodiment of the present application provides Figure 2-1 Schematic diagram of the fluorescence channels acquired for the structure shown;

[0063] Figure 5-2 is a frame of image in the fluorescence channel provided in an embodiment of the present application;

[0064] Figure 5-3 The embodiment of the present application provides Figure 2-2 Schematic diagram of imaging of the chip obtained by the structure shown under a fluorescence microscope;

[0065] Figure 5-4 yes Figure 5-3 An enlarged schematic diagram of the portion framed by black lines;

[0066] Figure 5-5 A schematic diagram of imaging of three fluorescent channels over a period of time provided in an embodiment of the present application;

[0067] Figure 5-6A schematic diagram of a dynamic curve of the interaction between three fluorescent molecular probes and an organism in the same sample obtained simultaneously in an embodiment of the present application;

[0068] Figure 6 Schematic diagram of the composition structure of the fluorescence and microfluidics-based biological detection system provided in the embodiment of the present application;

[0069] Figure 7 This is a schematic diagram of a practical application of a fluorescence and microfluidics-based biological detection system provided in an embodiment of the present application. DETAILED DESCRIPTION

[0070] In order to make the purpose, technical solutions and advantages of the present application clearer, the present application will be further described in detail below in conjunction with the accompanying drawings. The described embodiments should not be regarded as limiting the present application. All other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of this application.

[0071] In the following description, reference is made to “some embodiments”, which describe a subset of all possible embodiments, but it can be understood that “some embodiments” may be the same subset or different subsets of all possible embodiments and may be combined with each other without conflict.

[0072] In the following description, the terms "first\second" involved are merely used to distinguish similar objects and do not represent a specific ordering of the objects. It can be understood that "first\second" can be interchanged with a specific order or sequence where permitted, so that the embodiments of the present application described herein can be implemented in an order other than that illustrated or described herein.

[0073] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein are only for the purpose of describing the embodiments of this application and are not intended to limit this application.

[0074] It should be understood that in the various embodiments of the present application, the size of the serial number of each implementation process does not mean the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0075] Before further describing the embodiments of the present application in detail, the nouns and terms involved in the embodiments of the present application are explained. The nouns and terms involved in the embodiments of the present application are subject to the following interpretations.

[0076] 1) Fluorescent molecular probe: refers to a class of molecules that use fluorescent signals to identify the analyte, thereby achieving qualitative or quantitative analysis of the analyte.

[0077] 2) Microfluidic chips: They are the main platform and technical device for the implementation of microfluidic technology.

[0078] 3) Microfluidic chip technology: It integrates the basic operation units of biological, chemical and medical analysis processes such as sample preparation, reaction, separation and detection into a micrometer-scale chip to automatically complete the entire analysis process.

[0079] 4) Organism: refers to a living individual or object. Organisms include cells and have characteristics such as metabolism, growth, and stress response.

[0080] 5) Basic solution: including biological culture fluid or buffer.

[0081] In the related art, two fixed indicators, extracellular acidity rate and oxygen consumption rate, can be obtained simultaneously by using the Seahorse XF analyzer by using film fixation and well plate detection; however, this scheme can only obtain two fixed indicators, and cannot obtain three or more parameters of the interaction between molecules and organisms. In the related art, fluorescent proteins can also be transferred into cells, and pharmacokinetic information can be obtained by using the method of collecting microscopic images at different times; however, the operation of this scheme is complicated, and not only pre-treatment operations such as transfection of cells and introduction of fluorescent proteins into cells are required, but also image segmentation, quantitative detection and other operations are required. In the related art, it is also possible to use radionuclide labeled molecules for drug metabolism kinetic detection based on microfluidic radiobiological analysis technology (CIMR), and obtain drug metabolism kinetic information of molecules and biological samples, but for the same test sample, only one type of drug metabolism kinetic information of molecules and biological samples can be obtained in one test.

[0082] When the applicant detected the interaction between molecules and organisms based on relevant technologies, it was found that there was a lack of a solution for real-time and simultaneous detection of the interaction between the same organism and multiple different molecules.

[0083] Based on this, the embodiments of the present application provide a fluorescence and microfluidics-based biological detection method and system, which can detect the interaction between molecules and organisms in real time and efficiently.

[0084] An optional processing flow of the fluorescence and microfluidics-based biological detection method provided in the embodiment of the present application is as follows: Figure 1 As shown, at least the following steps are included:

[0085] Step S101 : continuously injecting a first solution including at least one fluorescent molecular probe into a fluorescent chamber and a microfluidic chamber of a microfluidic chip, wherein a biological body is placed in the microfluidic chamber.

[0086] In some optional embodiments, a schematic diagram of a connection between a fluorescence chamber and a microfluidic chamber is shown as follows: Figure 2-1 As shown, the fluorescence chamber and the microfluidic chamber are connected via a chamber partition, the first solution is injected into the fluorescence chamber through the entrance of the fluorescence chamber, and then enters the microfluidic chamber through the chamber partition. The microfluidic chamber and the fluorescence chamber can belong to one channel, and the chamber partition ensures the physical isolation between the fluorescence chamber and the microfluidic chamber, and ensures that the liquid can flow between the microfluidic chamber and the fluorescence chamber.

[0087] In some optional embodiments, another schematic diagram of connecting the fluorescence chamber and the microfluidic chamber is as follows: Figure 2-2 As shown, the fluorescence chamber and the microfluidic chamber can be two independent channels, and the fluorescence chamber and the microfluidic chamber are connected by a chamber partition, and the first solution is injected into the fluorescence chamber through the entrance of the fluorescence chamber, and then enters the microfluidic chamber through the chamber partition. Among them, the microfluidic chamber and the fluorescence chamber can belong to one channel, and the chamber partition ensures the physical isolation between the fluorescence chamber and the microfluidic chamber, and ensures that the liquid can flow between the microfluidic chamber and the fluorescence chamber.

[0088] In some optional embodiments, another schematic diagram of the fluorescence chamber and the microfluidic chamber is as follows: Figure 2-3 As shown, the fluorescence chamber and the microfluidic chamber can be two parallel channels, and the fluorescence chamber and the microfluidic chamber can be connected by a pipeline. The fluorescence chamber and the microfluidic chamber may not be connected by a pipeline; in the process of injecting the first solution into the fluorescence chamber and the microfluidic chamber, the fluid control unit can control the continuous injection of the first solution into the fluorescence chamber and the microfluidic chamber respectively; the fluid control unit can control the continuous injection of the first solution into the fluorescence chamber and the microfluidic chamber at the same time; the fluid control unit can also control the continuous injection of the first solution into the fluorescence chamber first, and then into the microfluidic chamber; the fluid control unit can also control the continuous injection of the first solution into the microfluidic chamber first, and then into the fluorescence chamber.

[0089] for Figure 2-3 The schematic diagram of the fluorescence chamber and the microfluidic chamber shown in the embodiment of the present application may also include multiple fluorescence chambers and multiple microfluidic chambers, and the fluorescence chamber and the multiple microfluidic chambers constitute multiple parallel channels.

[0090] In some optional embodiments, another connection diagram of the fluorescence chamber and the microfluidic chamber is as follows: Figure 2-4 As shown, a groove is provided at the connection between the fluorescence chamber and the microfluidic chamber, and the groove is used to place the microorganisms to be detected.

[0091] In some optional embodiments, there is another connection diagram between the fluorescence chamber and the microfluidic chamber, such as Figure 2-5 As shown, the connection between the fluorescent chamber and the microfluidic chamber is provided with a groove and a boss; wherein the groove is used to place the microorganisms to be detected. The boss is used to prevent the suspension of microorganisms from overflowing into the fluorescent chamber when the microorganisms are inoculated; wherein, the inoculation of cells is to first allow the suspension containing microorganisms to enter the microfluidic chamber, but the liquid cannot overflow into the microfluidic chamber; after the microorganisms sink and stick to the microfluidic chamber, the microorganisms are fixed on the bottom surface of the microfluidic chamber; after that, fresh culture fluid is added, and the fresh culture fluid will run through the entire channel, so that the culture fluid is connected between the microfluidic chamber and the fluorescent chamber.

[0092] This application Figure 2-1 to Figure 2-5 In the structure shown, when biological detection is not performed, dust covers can be placed at the entrance and exit of the microfluidic chamber and the fluorescence chamber to prevent dust from falling into the microfluidic chamber and the fluorescence chamber.

[0093] Among them, the fluorescent chamber is a chamber that only injects the first solution including one or more fluorescent molecular probes, and there is no organism in the fluorescent chamber. The microfluidic chamber can be a channel in a microfluidic chip, and the organism is placed in the microfluidic chamber. The number of microfluidic chambers can be one or more. Different organisms can be placed in different microfluidic chambers, and the organisms can include cells (such as tumor cells, nerve cells or endothelial cells), organoids, microorganisms, nematodes, zebrafish eggs, 3D cell balls and biopsy tissues; as an example, microorganisms are placed in the first microfluidic chamber, and nematodes are placed in the second microfluidic chamber.

[0094] In some optional embodiments, a first solution can be continuously injected into a microfluidic chamber of a microfluidic chip; the first solution can include a plurality of fluorescent molecular probes, and different fluorescent molecular probes correspond to different wavelengths. As an example, the fluorescent molecular probes are glucose labeled with red fluorescent molecules, nucleic acids labeled with blue fluorescent molecules, and amino acids labeled with green fluorescent molecules.

[0095] In other optional embodiments, the first solution can be continuously injected into multiple microfluidic chambers of the microfluidic chip, different organisms can be placed in each microfluidic chamber, and the first solution can include multiple fluorescent molecular probes. Therefore, the interaction between different organisms and multiple fluorescent molecular probes can be monitored simultaneously. For example, the first solution includes glucose labeled with a red fluorescent molecular probe and nucleic acids labeled with a blue fluorescent molecular probe, and the three microfluidic chambers included in the microfluidic chip are respectively equipped with three different organisms, namely, microorganisms, nematodes, and zebrafish eggs; by injecting the first solution into the three microfluidic chambers, the interaction between microorganisms and glucose labeled with a red fluorescent molecular probe, the interaction between nematodes and glucose labeled with a red fluorescent molecular probe, the interaction between zebrafish eggs and glucose labeled with a red fluorescent molecular probe, the interaction between microorganisms and nucleic acids labeled with a blue fluorescent molecular probe, the interaction between nematodes and nucleic acids labeled with a blue fluorescent molecular probe, and the interaction between zebrafish eggs and nucleic acids labeled with a blue fluorescent molecular probe can be detected.

[0096] Step S102, obtaining first imaging information corresponding to the fluorescence chamber and second imaging information corresponding to the microfluidic chamber.

[0097] In some embodiments, first imaging information corresponding to the fluorescence chamber and second imaging information corresponding to the microfluidic chamber may be collected by an imaging detector.

[0098] In a specific implementation, the imaging detector may be disposed above the fluorescence chamber and the microfluidic chamber, so that the imaging detector can be directly aligned with the internal area of ​​the fluorescence chamber and the microfluidic chamber.

[0099] In some embodiments, the imaging detector may be a positron emission tomography (PET) detector or a positron camera. The embodiments of the present application do not limit the type or form of the imaging detector.

[0100] Step S103: determining the kinetic information of the organism with respect to the fluorescent molecular probe based on the first imaging information and the second imaging information.

[0101] In some embodiments, based on the first imaging information and the second imaging information, a specific implementation process of determining the kinetic information of the organism with respect to the fluorescent molecular probe is as follows: Figure 3 As shown, it can at least include the following steps:

[0102] Step S301: acquiring second concentration information of the fluorescent molecular probe in the organism based on second imaging information.

[0103] In some embodiments, the microfluidic chip analyzes the second imaging information to obtain second concentration information of the fluorescent molecular probe in the fluorescent chamber.

[0104] Step S302: acquiring first concentration information of the fluorescent molecular probe in the fluorescent chamber based on the first imaging information.

[0105] In some embodiments, the microfluidic chip analyzes the first imaging information to obtain first concentration information of the fluorescent molecular probe in the fluorescent chamber.

[0106] Step S303: determining the kinetic information of the organism to the fluorescent molecular probe based on the first concentration information and the second concentration information.

[0107] In some embodiments, since the organism in the microfluidic chamber absorbs the fluorescent molecular probe in the first solution, the concentration of the fluorescent molecular probe in the microfluidic chamber is less than the concentration of the fluorescent molecular probe in the fluorescence chamber; by calculating the difference between the first concentration information and the second concentration information, the concentration difference of the fluorescent molecular probe in the fluorescence chamber and in the microfluidic chamber can be obtained. According to the concentration difference of the fluorescent molecular probe in the fluorescence chamber and in the microfluidic chamber, the amount of fluorescent molecular probe absorbed by the organism in the microfluidic chamber can be calculated, and then the kinetic information of the organism for the fluorescent molecular probe can be determined. Among them, the volume of the microfluidic chamber can be the same as or different from the volume of the fluorescence chamber; if the shape and size of the microfluidic chamber are the same as the shape and size of the fluorescence chamber, the volume of the microfluidic chamber is the same as the volume of the fluorescence chamber.

[0108] The embodiment of the present application breaks through by combining microfluidic technology with fluorescent molecular probe technology to detect the interaction between organisms and fluorescent molecules; by continuously injecting a first solution including fluorescent molecules into a microfluidic chamber and a fluorescent chamber, the interaction between the organism and the fluorescent molecules in the microfluidic chamber can be detected in real time, such as detecting the absorption of fluorescent molecules by the organism. When the first solution includes multiple fluorescent molecular probes, the interaction between an organism in a microfluidic chamber and multiple fluorescent molecular probes can be determined simultaneously; when the microfluidic chip includes multiple microfluidic chambers, and different organisms are placed in the multiple microfluidic chambers, the interaction between multiple organisms and multiple fluorescent molecular probes can be determined simultaneously, such as the absorption of fluorescent molecular probes by the organism.

[0109] Another optional processing flow of the fluorescence and microfluidics-based biological detection method provided in the embodiment of the present application is as follows: Figure 4 As shown, at least the following steps are included:

[0110] Step S401 : continuously injecting a first solution including at least one fluorescent molecular probe into a fluorescent chamber and a microfluidic chamber of a microfluidic chip, wherein a biological body is placed in the microfluidic chamber.

[0111] Step S402, obtaining first imaging information corresponding to the fluorescence chamber and second imaging information corresponding to the microfluidic chamber.

[0112] Step S403: determining the kinetic information of the organism with respect to the fluorescent molecular probe based on the first imaging information and the second imaging information.

[0113] In some embodiments, the specific implementation process of step S401 to step S403 is the same as step S101 to step S103, and will not be repeated here.

[0114] Step S404: continuously injecting a basic culture solution into the microfluidic chamber, wherein the basic culture solution does not include the fluorescent molecular probe.

[0115] In some embodiments, the basic culture medium may be injected into the microfluidic chamber after the first solution is continuously injected for a first time. The first time may be flexibly set according to the actual application scenario, such as after the first solution is injected within the first time, the organism no longer absorbs the fluorescent molecular probe.

[0116] In some embodiments, the basic culture fluid may also be referred to as a basal solution, and the basal solution may include a biological culture fluid or a buffer. Among them, the cell culture medium in the biological culture fluid includes various types, such as DM EM culture medium, RPMI1640 culture medium, endothelial cell culture medium, etc.

[0117] The function of the basic solution is to maintain the normal growth state or basic survival state of the biological extract, and the biological culture fluid is used to promote the growth and metabolism of biological samples; the biological culture fluid includes a variety of nutrients, and the different components of the biological culture fluid can be used to study the effects of different metabolites.

[0118] Step S405 , detecting third imaging information corresponding to the microfluidic chamber.

[0119] In some embodiments, an imaging detector may be used to detect third imaging information corresponding to the microfluidic chamber.

[0120] Step S406: determining the kinetic information of the organism with respect to the fluorescent molecular probe based on the second imaging information and the third imaging information.

[0121] In a specific implementation, the microfluidic chip can obtain the second concentration information of the fluorescent molecular probe in the organism based on the second imaging information; the microfluidic chip can obtain the third concentration information of the fluorescent molecular probe in the organism based on the third imaging information; based on the third concentration information and the second concentration information, determine the concentration difference of the fluorescent molecules before and after the basic culture solution is injected into the microfluidic chamber; based on the concentration difference, determine the release of the fluorescent molecular probe by the organism.

[0122] Specifically, the second concentration information in the microfluidic chamber at the first moment can be recorded, and then the basic culture fluid is continuously injected into the microfluidic chamber; as the basic culture fluid in the microfluidic chamber flows out, the fluorescent molecular probe in the organism will flow out of the organism, and the concentration of the fluorescent molecular probe in the organism will decrease. By calculating the difference between the second concentration information and the third concentration information, the release of the fluorescent molecular probe by the organism can be determined.

[0123] The embodiment of the present application uses a breakthrough method of combining microfluidic technology with fluorescent molecular probe technology to detect the interaction between organisms and fluorescent molecules. By continuously injecting basic culture fluid into the fluorescent chamber, it is possible to detect the interaction between organisms and fluorescent molecules in the microfluidic chamber in real time, such as detecting the release of fluorescent molecules by the organism. When the first solution includes multiple fluorescent molecular probes, the interaction between an organism in a microfluidic chamber and multiple fluorescent molecular probes can be determined simultaneously, such as the release of fluorescent molecular probes by the organism; when the microfluidic chip includes multiple microfluidic chambers, and different organisms are placed in the multiple microfluidic chambers, the interaction between multiple organisms and multiple fluorescent molecular probes can be determined simultaneously, such as the release of fluorescent molecular probes by the organism.

[0124] According to the fluorescence and microfluidics-based biological detection method provided in the embodiment of the present application, based on Figure 2-1 Schematic diagram of the fluorescence channels obtained by the structure shown, Figure 5-1 As shown; among them, Figure 5-1 From top to bottom are the imaging of DAPI, FITC, and TexRed fluorescence channels over a period of time. Figure 5-2 is a frame of image in the fluorescence channel; Figure 5-2 From top to bottom are images of DAPI, FITC, and TexRed fluorescence channels; the image in the dotted box on the left represents the imaging of the fluorescence chamber; the image in the dotted box on the right represents the imaging of the microfluidic chamber.

[0125] based on Figure 2-2 Schematic diagram of the imaging of the chip under a fluorescence microscope, as shown in Figure 5-3 As shown; among them, Figure 5-3The enlarged schematic diagram of the part framed by black lines is shown in Figure 5-4 As shown, Figure 5-4 From left to right, the images are three different fluorescence channels and bright field imaging. Bright field imaging can only reflect the structure of the chip and cannot provide corresponding data for biological detection. Figure 5-4 In the figure, the image corresponding to the upper dotted box represents the imaging of the fluorescence chamber, and the image corresponding to the lower dotted box represents the imaging of the microfluidic chamber. Figure 5-5 From top to bottom, it shows the imaging of the three fluorescent channels DAPI, FITC, and TexRed over a period of time.

[0126] In the embodiment of the present application, a schematic diagram of a dynamic curve of the interaction between three fluorescent molecular probes and an organism in the same sample is obtained simultaneously. Figure 5-6 As shown, Figure 5-6 Figures A, B, and C in the figure are the fluorescence kinetic curves obtained under three fluorescence channels. Figure 5-6 Figure D in the figure is a fusion of the fluorescence kinetic curves shown in Figures A, B, and C. Figure A is a kinetic curve obtained through the blue fluorescence channel (DAPI), and uses the live cell dye Hoechst 33342 to indicate the number and metabolic intensity of live cells. Figure B is a kinetic curve obtained through the green fluorescence channel (FITC), and uses the calcium ion fluorescent probe Calcein AM to indicate the calcium ion metabolism of cells. Figure C is a kinetic curve obtained through the red fluorescence channel (TexRed), and uses the BCECF AM fluorescent dye to indicate the pH changes of cells and surrounding solutions; among them, the BCECFAM fluorescent dye is a pH fluorescent probe.

[0127] Based on the fluorescence and microfluidics-based biological detection method provided in the embodiment of the present application, the embodiment of the present application also provides a fluorescence and microfluidics-based biological detection system, the composition structure diagram of the system is as follows: Figure 6 As shown, including at least:

[0128] A fluid control unit 601, used for controlling the continuous injection of a first solution including at least one fluorescent molecular probe into a fluorescent chamber 604 and a microfluidic chamber 605 of a microfluidic chip, wherein a biological body is placed in the microfluidic chamber;

[0129] An imaging detector 602, used to obtain first imaging information corresponding to the fluorescence chamber 604 and second imaging information corresponding to the microfluidic chamber 605;

[0130] The microfluidic chip 603 is used to determine the kinetic information of the organism with respect to the fluorescent molecular probe based on the first imaging information and the second imaging information.

[0131] In some embodiments, different fluorescent molecular probes correspond to different wavelengths.

[0132] In some embodiments, the fluid control unit 601 is further used to continuously inject a basic culture solution into the microfluidic chamber 605, wherein the basic culture solution does not include the fluorescent molecular probe;

[0133] Detecting third imaging information corresponding to the microfluidic chamber 605;

[0134] The kinetic information of the organism with respect to the fluorescent molecular probe is determined based on the second imaging information and the third imaging information.

[0135] In some embodiments, the fluid control unit 601 is used to continuously inject the first solution into the fluorescence chamber 604;

[0136] The first solution flows into the microfluidic chamber 605 via a pipe connecting the fluorescence chamber 604 and the microfluidic chamber 605 .

[0137] In some embodiments, the fluid control unit 601 is used to continuously inject the first solution into the fluorescence chamber 604 and the microfluidic chamber 605 respectively;

[0138] The fluorescence chamber 604 and the microfluidic chamber 605 are independent chambers.

[0139] In some embodiments, the microfluidic chip 603 is used to obtain second concentration information of the fluorescent molecular probe in the organism based on the second imaging information;

[0140] Acquire first concentration information of the fluorescent molecular probe in the fluorescent chamber 604 based on the first imaging information;

[0141] Based on the first concentration information and the second concentration information, kinetic information of the organism with respect to the fluorescent molecular probe is determined.

[0142] In some embodiments, the microfluidic chip 603 is used to determine the concentration difference of the fluorescent molecules in the fluorescent chamber 604 and the microfluidic chamber 605 based on the second concentration information and the first concentration information;

[0143] The absorption of the fluorescent molecular probe by the organism is determined based on the concentration difference.

[0144] In some embodiments, the microfluidic chip 603 is used to obtain second concentration information of the fluorescent molecular probe in the organism based on the second imaging information;

[0145] Acquire third concentration information of the fluorescent molecular probe in the organism based on the third imaging information;

[0146] Based on the third concentration information and the second concentration information, determining the concentration difference of the fluorescent molecular probe before and after the basic culture solution is injected into the microfluidic chamber 605;

[0147] The release of the fluorescent molecule by the organism is determined based on the concentration difference.

[0148] In some embodiments, the fluid control unit 601 is used to continuously inject drugs into the fluorescence chamber 604 and the microfluidic chamber 605 respectively;

[0149] The microfluidic chip is used to determine the effect of the drug on the metabolism of the fluorescent molecular probe by the organism based on the fourth imaging information corresponding to the fluorescent chamber 604 after the drug is injected and the fifth imaging information corresponding to the microfluidic chamber 605.

[0150] A schematic diagram of a practical application of a biological detection system based on fluorescence and microfluidics provided in an embodiment of the present application is as follows: Figure 7 As shown, the fluid control unit controls the first solution to be injected into the microfluidic chamber and the fluorescence chamber in real time, and the imaging detector detects the image information corresponding to the microfluidic chamber and the imaging information corresponding to the fluorescence chamber in real time. The microfluidic chip is placed on the sample platform, and the fluorescence chamber is placed on the sample support to make the microfluidic chip and the fluorescence chamber stable.

[0151] The above are only embodiments of the present application and are not intended to limit the protection scope of the present application. Any modifications, equivalent substitutions and improvements made within the spirit and scope of the present application are included in the protection scope of the present application.

Claims

1. A biological detection method based on fluorescence and microfluidics, characterized in that: The method comprises: Continuously injecting a first solution including at least one fluorescent molecular probe into the fluorescent chamber and the microfluidic chamber of the microfluidic chip, wherein a biological body is placed in the microfluidic chamber; Acquire first imaging information corresponding to the fluorescence chamber and second imaging information corresponding to the microfluidic chamber; Determining kinetic information of the organism with respect to the fluorescent molecular probe based on the first imaging information and the second imaging information; Wherein, determining the kinetic information of the organism with respect to the fluorescent molecular probe based on the first imaging information and the second imaging information includes: acquiring second concentration information of the fluorescent molecular probe in the organism based on the second imaging information; acquiring first concentration information of the fluorescent molecular probe in the fluorescent chamber based on the first imaging information; determining the kinetic information of the organism with respect to the fluorescent molecular probe based on the first concentration information and the second concentration information; Among them, determining the kinetic information of the organism to the fluorescent molecular probe based on the first concentration information and the second concentration information includes: determining the concentration difference of the fluorescent molecular probe in the fluorescent chamber and the microfluidic chamber based on the second concentration information and the first concentration information; and determining the absorption of the fluorescent molecular probe by the organism based on the concentration difference.

2. The method according to claim 1, characterized in that: Different fluorescent molecular probes correspond to different wavelengths.

3. The method according to claim 1 or 2, characterized in that: After continuously injecting the first solution into the fluorescence chamber and the microfluidic chamber for a first period of time, the method further includes: continuously injecting a basic culture solution into the microfluidic chamber, wherein the basic culture solution does not include the fluorescent molecular probe; detecting third imaging information corresponding to the microfluidic chamber; The kinetic information of the organism with respect to the fluorescent molecular probe is determined based on the second imaging information and the third imaging information.

4. The method according to claim 1 or 2, characterized in that: The step of continuously injecting a first solution including a fluorescent molecular probe into the fluorescent chamber and the microfluidic chamber of the microfluidic chip comprises: continuously injecting the first solution into the fluorescence chamber; The first solution flows into the microfluidic chamber via a pipe connected to the fluorescence chamber and the microfluidic chamber.

5. The method according to claim 1 or 2, characterized in that: The step of continuously injecting a first solution including a fluorescent molecular probe into the fluorescent chamber and the microfluidic chamber of the microfluidic chip comprises: continuously injecting the first solution into the fluorescence chamber and the microfluidic chamber respectively; Wherein, the fluorescence chamber and the microfluidic chamber are independent chambers.

6. The method according to claim 3, characterized in that The determining of the kinetic information of the organism with respect to the fluorescent molecular probe based on the second imaging information and the third imaging information comprises: Acquire second concentration information of the fluorescent molecular probe in the organism based on the second imaging information; Acquire third concentration information of the fluorescent molecular probe in the organism based on the third imaging information; Based on the third concentration information and the second concentration information, determining a concentration difference of the fluorescent molecules before and after the basic culture solution is injected into the microfluidic chamber; The release of the fluorescent molecular probe by the organism is determined based on the concentration difference.

7. A biological detection system based on fluorescence and microfluidics, characterized in that: The system comprises: A fluid control unit, used for controlling the continuous injection of a first solution including at least one fluorescent molecular probe into the fluorescent chamber and the microfluidic chamber of the microfluidic chip, wherein the microfluidic chamber contains a biological body; An imaging detector, used to obtain first imaging information corresponding to the fluorescence chamber and second imaging information corresponding to the microfluidic chamber; A microfluidic chip, used for determining the kinetic information of the organism with respect to the fluorescent molecular probe based on the first imaging information and the second imaging information; Wherein, the microfluidic chip is specifically used to obtain the second concentration information of the fluorescent molecular probe in the organism based on the second imaging information; obtain the first concentration information of the fluorescent molecular probe in the fluorescent chamber based on the first imaging information; and determine the kinetic information of the organism with respect to the fluorescent molecular probe based on the first concentration information and the second concentration information; The microfluidic chip is specifically used to determine the concentration difference of the fluorescent molecular probe in the fluorescent chamber and the microfluidic chamber based on the second concentration information and the first concentration information; and determine the absorption of the fluorescent molecular probe by the organism based on the concentration difference.

8. The system according to claim 7, characterized in that Different fluorescent molecular probes correspond to different wavelengths.

9. The system according to claim 7 or 8, characterized in that: The fluid control unit is further used to continuously inject a basic culture solution into the microfluidic chamber, wherein the basic culture solution does not include the fluorescent molecular probe; detecting third imaging information corresponding to the microfluidic chamber; The kinetic information of the organism with respect to the fluorescent molecular probe is determined based on the second imaging information and the third imaging information.

10. The system according to claim 7 or 8, characterized in that: The fluid control unit is used to continuously inject the first solution into the fluorescence chamber; The first solution flows into the microfluidic chamber via a pipe connected to the fluorescence chamber and the microfluidic chamber.

11. The system according to claim 7 or 8, characterized in that: The fluid control unit is used to continuously inject the first solution into the fluorescence chamber and the microfluidic chamber respectively; Wherein, the fluorescence chamber and the microfluidic chamber are independent chambers.

12. The system according to claim 9, characterized in that The microfluidic chip is used to obtain second concentration information of the fluorescent molecular probe in the organism based on the second imaging information; Acquire third concentration information of the fluorescent molecular probe in the organism based on the third imaging information; Based on the third concentration information and the second concentration information, determining the concentration difference of the fluorescent molecular probe before and after the basic culture solution is injected into the microfluidic chamber; The release of the fluorescent molecule by the organism is determined based on the concentration difference.

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