Multi-laser excitation capillary electrophoresis imaging detection device and use method

Abstract

The invention provides a multi-laser excitation capillary electrophoresis imaging detection device and a use method. The detection device comprises a laser generation assembly, a control unit, an illumination assembly, an electrophoresis assembly and a fluorescence detection assembly. The laser generating assembly is configured to excite multiple beams of laser with different wavelengths; the control unit is configured to control the laser generation assembly to excite laser in turn according to a preset time sequence; the illumination assembly is configured to receive multiple beams of laser and combine the multiple beams of laser into the same light path to form detection laser formed by alternating laser with different wavelengths; the electrophoresis assembly is configured to apply an electric field to the sample solution so as to migrate and separate target molecules respectively marked by multiple fluorescent substances in the sample solution, and the detection laser is used for exciting the multiple fluorescent substances to generate fluorescence signals; the fluorescence detection assembly is configured to form an image of the target molecule according to the received fluorescence signal.

Description

Technical Field

[0001] The present disclosure relates to the field of bioanalysis technology, and more specifically, to a multi-laser excitation capillary electrophoresis imaging detection device and a use method thereof. Background Art

[0002] Capillary electrophoresis (CE) is a separation and analysis technique based on the difference in migration speed of charged particles under the action of an electric field. It uses the difference in mobility of charged particles in an electric field to drive the charged particles in the sample solution to migrate in a thin capillary filled with an electrolyte solution through high voltage. Due to the different charges, sizes and shapes of different particles, their migration speeds vary, thus achieving efficient separation.

[0003] Although traditional capillary electrophoresis detection methods (such as ultraviolet or fluorescence detection) can provide high separation efficiency, they have certain limitations in the visualization of analyte distribution and the intuitive observation of the separation process. In order to overcome this shortcoming, researchers have begun to explore the combination of capillary electrophoresis and imaging technology in recent years and developed capillary electrophoresis imaging technology. By combining the high separation efficiency of capillary electrophoresis with the direct visualization of imaging technology, the distribution of analytes during the separation process can be observed in real time, providing intuitive information on separation efficiency and analyte characteristics, thereby significantly improving the accuracy and resolution of the analysis.

[0004] However, current imaging technology still has difficulty in performing high-resolution visual detection of complex samples, and in distinguishing more similar analytes with high-resolution imaging technology. Therefore, an electrophoresis imaging detection device for complex samples is urgently needed. Summary of the invention

[0005] In view of this, the present disclosure provides a multi-laser excitation capillary electrophoresis imaging detection device and a method of using the same, which can realize capillary electrophoresis imaging of target molecules labeled with different fluorescent substances under electric field manipulation, thereby distinguishing similar analytes in complex samples.

[0006] One aspect of an embodiment of the present disclosure provides a multi-laser excitation capillary electrophoresis imaging detection device, characterized in that it includes: a laser generating component, configured to excite multiple beams of lasers with different wavelengths; a control unit, configured to control the above-mentioned laser generating component to excite the above-mentioned lasers in turn according to a preset time sequence; an illumination component, configured to receive the multiple beams of the above-mentioned lasers, and merge the multiple beams of the above-mentioned lasers into the same optical path to form a detection laser composed of the above-mentioned lasers of different wavelengths alternatingly; an electrophoresis component, configured to apply an electric field to a sample solution to migrate and separate target molecules in the sample solution that are respectively labeled with multiple fluorescent substances, wherein the above-mentioned detection laser is used to excite the multiple fluorescent substances to generate fluorescent signals; and a fluorescence detection component, configured to form an imaging map of the above-mentioned target molecules according to the received above-mentioned fluorescent signals.

[0007] According to an embodiment of the present disclosure, the multi-laser excitation capillary electrophoresis imaging detection device also includes: an inverted fluorescence microscope, which is arranged directly below the sample solution in the electrophoresis component and is configured to focus the detection laser from the illumination component on the sample solution, or to lead the fluorescence signal out of the electrophoresis component.

[0008] According to an embodiment of the present disclosure, the above-mentioned lighting component includes: a lighting unit, configured to make multiple beams of the above-mentioned lasers reach the critical angle of the evanescent wave at the same time; and a first optical component, configured to merge the multiple beams of the above-mentioned lasers from the above-mentioned lighting unit into the same optical path to obtain the above-mentioned detection laser.

[0009] According to an embodiment of the present disclosure, the laser generating assembly includes a plurality of exciters, each of which is configured to excite the lasers of different wavelengths, and the lighting unit includes three illuminators, which are respectively connected to the exciters via optical fibers; wherein at least one of the illuminators is configured to be connected to at least two of the exciters.

[0010] According to an embodiment of the present disclosure, the electrophoresis component includes: a carrier, which is arranged above the inverted fluorescence microscope; a capillary, which is arranged on the carrier, and a microfluidic channel for accommodating the sample solution is formed in the capillary; two sample trays, which are symmetrically arranged on the carrier and used to accommodate a buffer solution, and both ends of the capillary are respectively immersed in the buffer solution; and a power supply, wherein two platinum electrodes of the power supply are respectively arranged in the two sample trays and electrically connected to the capillary through the buffer solution, so as to form an electric field in the microfluidic channel when power is turned on.

[0011] According to an embodiment of the present disclosure, the cross section of the capillary is configured to be square, a detection window is provided on one side of the square capillary, and the detection window is arranged to face the objective lens of the inverted fluorescence microscope.

[0012] According to an embodiment of the present disclosure, the fluorescence detection component includes: a second optical component configured to separate the fluorescence signals emitted by the different fluorescent substances according to wavelengths; and a detector configured to sense the separated fluorescence signals and form the target image.

[0013] On the other hand, an embodiment of the present disclosure provides a method for using a multi-laser excited capillary electrophoresis imaging detection device, comprising: controlling a laser generating component by a control unit to alternately excite multiple beams of lasers with different wavelengths according to a preset timing; merging the multiple beams of the above lasers into the same optical path by an illumination component to form a detection laser composed of the above lasers of different wavelengths alternatingly, and emitting it to an electrophoresis component; exciting different fluorescent substances separated from multiple target molecules in the above electrophoresis component by the above detection laser to obtain a fluorescence signal; receiving the above fluorescence signal by a fluorescence detection component to form an imaging map of the above target molecules.

[0014] According to an embodiment of the present disclosure, the method of use also includes: receiving the above-mentioned detection laser from the above-mentioned illumination component through an inverted fluorescence microscope, and the above-mentioned detection laser is totally reflected at the objective lens of the above-mentioned inverted fluorescence microscope to form an evanescent wave on the surface of the sample solution of the above-mentioned electrophoresis component, thereby exciting the above-mentioned multiple different fluorescent substances marking the target molecules and obtaining the above-mentioned fluorescence signal.

[0015] According to an embodiment of the present disclosure, the method of use also includes: using the fluorescent substances corresponding to the multiple beams of laser light with different wavelengths to respectively label the target molecules in the sample solution; migrating the multiple target molecules in the sample solution to the microfluidic channel of the capillary by applying an electric field or applying pressure; and forming an electric field in the microfluidic channel by a power supply to separate the target molecules labeled with different fluorescent substances.

[0016] According to the embodiments of the present disclosure, efficient and highly sensitive target molecule detection is achieved by precisely controlling the excitation and electrophoresis separation process of multiple laser beams of different wavelengths. This solution can simultaneously excite multiple fluorescently labeled target molecules, and through time series control, it can achieve the alternating excitation of lasers of different wavelengths, and through the effective merging of multiple laser beams by the lighting component, a detection laser is formed by lasers of different wavelengths continuously alternating in time sequence. The detection laser can simultaneously detect target molecules labeled with different fluorescent substances in the same detection area, providing effective distinction and high-resolution imaging of similar analytes in complex samples; at the same time, the efficient separation of the electrophoresis component, combined with the imaging technology of the fluorescence detection component, provides intuitive and accurate imaging of target molecules, greatly improving the readability and analysis quality of experimental data. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The above and other objects, features and advantages of the present disclosure will become more apparent through the following description of the embodiments of the present disclosure with reference to the accompanying drawings, in which:

[0018] Figure 1 The structure diagram of the multi-laser excitation capillary electrophoresis imaging detection device according to an embodiment of the present disclosure is schematically shown;

[0019] Figure 2 Schematically shows a structural diagram of an electrophoresis assembly according to an embodiment of the present disclosure;

[0020] Figure 3 A flow chart schematically shows a method for using the multi-laser excitation capillary electrophoresis imaging detection device according to an embodiment of the present disclosure;

[0021] Figure 4 Schematic diagram showing the timing curve of separation of target molecules labeled with different fluorescent substances according to an embodiment of the present disclosure

[0022] Figure 5 The imaging diagram of target molecules labeled with different fluorescent substances according to the embodiments of the present disclosure is schematically shown.

[0023] In the drawings, the meanings of the reference numerals are as follows:

[0024] 1. Laser generating components;

[0025] 11. Exciter;

[0026] 2. Lighting components;

[0027] 21. Lighting unit;

[0028] 211. Illuminator;

[0029] 22. a first optical component;

[0030] 221. Reflector;

[0031] 222, dichroic mirror;

[0032] 23. Optical fiber;

[0033] 231, coupling unit;

[0034] 3. Inverted fluorescence microscope;

[0035] 31. Objective lens;

[0036] 32. Aperture;

[0037] 33. Lens;

[0038] 4. Capillary electrophoresis components;

[0039] 41. Carrying rack;

[0040] 42. Capillary;

[0041] 421. Microfluidic channel;

[0042] 43. Sample tray;

[0043] 431, accommodating chamber;

[0044] 432, shaft;

[0045] 44. Power supply;

[0046] 441, platinum electrode;

[0047] 45. Fixing parts;

[0048] 5. Target molecules;

[0049] 6. Fluorescence detection component;

[0050] 61. A second optical component;

[0051] 62. Detector;

[0052] 7. Control unit;

[0053] 8. Data processing equipment. DETAILED DESCRIPTION

[0054] Hereinafter, embodiments of the present disclosure will be described with reference to the accompanying drawings. However, it should be understood that these descriptions are exemplary only and are not intended to limit the scope of the present disclosure. In the following detailed description, for ease of explanation, many specific details are set forth to provide a comprehensive understanding of the embodiments of the present disclosure. However, it is apparent that one or more embodiments may also be implemented without these specific details. In addition, in the following description, descriptions of known structures and technologies are omitted to avoid unnecessary confusion of the concepts of the present disclosure.

[0055] The terms used herein are only for describing specific embodiments and are not intended to limit the present disclosure. The terms "include", "comprising", etc. used herein indicate the existence of the features, steps, operations and / or mechanisms, but do not exclude the existence or addition of one or more other features, steps, operations or mechanisms.

[0056] All terms (including technical and scientific terms) used herein have the meanings commonly understood by those skilled in the art unless otherwise defined. It should be noted that the terms used herein should be interpreted as having a meaning consistent with the context of this specification and should not be interpreted in an idealized or overly rigid manner.

[0057] When using expressions such as "at least one of A, B, and C, etc.", they should generally be interpreted according to the meaning of the expression commonly understood by those skilled in the art (for example, "a system having at least one of A, B, and C" should include but is not limited to a system having A alone, B alone, C alone, A and B, A and C, B and C, and / or A, B, C, etc.).

[0058] Capillary electrophoresis is an efficient separation and analysis technology that achieves separation based on the difference in migration speed of charged particles in an electric field. In operation, the sample solution is injected into a capillary filled with electrolyte. After high voltage is applied, the particles migrate at different speeds due to differences in charge, size and shape, achieving the purpose of separation. Capillary electrophoresis technology plays an important role in many fields such as analytical chemistry, biochemistry and biomedicine with its advantages such as excellent separation efficiency, high sensitivity, low sample consumption, rapid analysis and low cost.

[0059] In order to further enhance the analytical capabilities of capillary electrophoresis, capillary electrophoresis imaging technology has been developed: it combines the efficient separation capabilities of capillary electrophoresis with the intuitive visualization advantages of imaging technology, allowing researchers to directly observe the distribution of analytes during the separation process. Compared with traditional ultraviolet or fluorescence detection methods, imaging technology provides more intuitive separation efficiency and characteristic information, making real-time monitoring and in-depth understanding of the separation process possible.

[0060] The present disclosure relates to the field of bioanalysis technology, and proposes a multi-laser excited capillary electrophoresis imaging detection device and its operation method. The device achieves efficient separation performance, and by combining advanced imaging technology, it achieves the acquisition of high-definition images, so that even very similar analytes can be effectively distinguished. This technological innovation not only significantly improves the accuracy and sensitivity of analysis, but also brings more powerful technical means to the field of bioanalysis, and promotes the development of related scientific research to a deeper level.

[0061] Figure 1 The structure diagram of the multi-laser excitation capillary electrophoresis imaging detection device according to an embodiment of the present disclosure is schematically shown;

[0062] The embodiment of the present disclosure provides a multi-laser excitation capillary electrophoresis imaging detection device, such as Figure 1As shown, it includes a laser generating component 1, a control unit 7, an illumination component 2, an electrophoresis component 4 and a fluorescence detection component 6. The laser generating component 1 is configured to excite multiple laser beams with different wavelengths; the control unit 7 is configured to control the laser generating component 1 to excite the lasers in turn according to a preset time sequence; the illumination component 2 is configured to receive multiple laser beams and merge the multiple laser beams into the same optical path to form a detection laser composed of laser beams of different wavelengths alternately; the electrophoresis component 4 is configured to apply an electric field to the sample solution to migrate and separate the target molecules 5 in the sample solution that are respectively labeled by multiple fluorescent substances, wherein the detection laser is used to excite multiple fluorescent substances to generate fluorescence signals; the fluorescence detection component 6 is configured to form an imaging map of the target molecule 5 according to the received fluorescence signals.

[0063] According to the above-mentioned setting mode, efficient and high-resolution separation imaging is achieved through the laser generating component 1, the control unit 7, the lighting component 2, the electrophoresis component 4 and the fluorescence detection component 6. The device can alternately excite multiple beams of laser light of different wavelengths, and accurately control the excitation sequence through the control unit, so that the lighting component 2 can be combined to form a multi-wavelength detection laser, thereby achieving effective excitation of multiple fluorescently labeled target molecules. The electrophoresis component 4 can accurately separate the target molecules during the application of the electric field, and the fluorescence detection component 6 generates a clear imaging image based on the generated fluorescence signal. Not only the sensitivity and resolution of the analysis are improved, but also the effective distinction of similar analytes in complex samples is achieved through imaging technology, which has brought significant technological progress to the field of biological analysis.

[0064] In an illustrative embodiment, the control unit 7 is communicatively connected with the laser excitation component 1 and the fluorescence detection component 6, and the control unit 7 includes a high-precision timing synchronization controller, which can quickly and accurately synchronize and control the laser excitation component 1 and the fluorescence detection component 6 through a real-time synchronization controller driver.

[0065] In an illustrative embodiment, Figure 1 As shown, the multi-laser excitation capillary electrophoresis imaging detection device also includes an inverted fluorescence microscope 3, which is arranged directly below the sample solution in the electrophoresis component 4 and is configured to focus the detection laser from the illumination component 2 on the sample solution, or to lead the fluorescence signal out of the electrophoresis component 4.

[0066] According to the above-mentioned configuration, accurate focusing of the sample solution and effective extraction of the fluorescence signal are achieved through the inverted fluorescence microscope 3. The configuration of the inverted fluorescence microscope 3 not only improves the focusing efficiency of the detection laser, ensuring that the target molecules in the sample solution can be fully excited, but also enhances the collection capability of the fluorescence signal, thereby improving the clarity of the imaging and the sensitivity of the detection.

[0067] In detail, the inverted fluorescence microscope 3 is a microscope commonly used in cell and molecular biology research. The design of the inverted fluorescence microscope 3 allows the sample to be placed under the microscope, and the objective lens is located below the sample. The inverted fluorescence microscope 3 can be equipped with a variety of fluorescent light sources and filters to excite fluorescent markers in the sample and collect fluorescent signals. This allows researchers to observe specific cell structures, proteins or molecules.

[0068] According to an embodiment of the present disclosure, the objective lens 31 includes a high-resolution total internal reflection oil immersion objective lens, which supports laser incidence to reach the evanescent wave critical angle.

[0069] Detailed, high-resolution total internal reflection oil immersion objectives have a high numerical aperture (NA) that provides extremely high spatial resolution and can use the phenomenon of total internal reflection to excite fluorescent molecules near the sample surface. When the laser is incident on the interface between the sample and the objective at an angle greater than the critical angle, an evanescent wave is generated. This wave only exists near the interface and can effectively excite fluorescent markers close to the interface. Special immersion oil is used to match the refractive index between the sample and the objective, thereby reducing the refraction and reflection losses of light when passing through the interface, improving imaging quality and efficiency.

[0070] In an illustrative embodiment, Figure 1 As shown, the lighting assembly 2 includes a lighting unit 21 and a first optical assembly 22. The lighting unit 21 is configured to make multiple laser beams reach the evanescent wave critical angle simultaneously; the first optical assembly 22 is configured to merge the multiple laser beams from the lighting unit 21 into the same optical path to obtain the detection laser.

[0071] According to the above-mentioned configuration, the lighting assembly 2 realizes efficient merging and optimized use of multiple laser beams by including the lighting unit 21 and the first optical assembly 22. The lighting unit 21 enables multiple laser beams to reach the critical angle of the evanescent wave at the same time, while the first optical assembly 22 merges these laser beams into the same optical path to form a detection laser. The effect of this technical configuration is to improve the utilization efficiency of the laser, ensure the concentration and uniform distribution of the laser energy, and thus enhance the excitation effect of the fluorescence signal.

[0072] In detail, when a laser beam is incident on a medium interface (e.g., the interface between glass and water) at an angle greater than the critical angle, total internal reflection occurs. In this case, the laser does not pass through the interface into the light-scarce medium, but is completely reflected at the interface. If the target molecules in the sample solution are labeled with fluorescent dyes or fluorescent proteins, and these fluorescent substances are within the penetration range of the evanescent wave (usually within a few tens to hundreds of nanometers from the interface), the electric field of the evanescent wave can effectively excite these fluorescent molecules. Due to the limited penetration depth of the evanescent wave, the background signal (non-specific fluorescence from deep in the sample) is greatly reduced, thereby improving the signal-to-noise ratio. Therefore, high-resolution, low-background surface imaging can be achieved.

[0073] According to the embodiment of the present disclosure, the inverted fluorescence microscope is a total internal reflection fluorescence microscope. The detection laser is irradiated to the objective lens 31 through the aperture 32 and lens 33 of the microscope, and total reflection occurs. When the detection laser is incident on the bottom of the lens of the objective lens 31 (the surface in contact with the sample) at an angle greater than the critical angle, total internal reflection occurs at the interface between the lens and the sample. In this process, an evanescent wave is generated along the sample surface and extends into the interior of the sample.

[0074] In an illustrative embodiment, Figure 1 As shown, the first optical component 22 includes a reflector 221 and two dichroic mirrors 222. In detail, the reflector 221 and the two dichroic mirrors 222 are arranged at intervals, the reflector 221 is suitable for reflecting the first laser beam, the first dichroic mirror 222 is suitable for transmitting the reflected first laser beam and reflecting the second laser beam, and the second dichroic mirror 222 is suitable for transmitting the reflected first laser beam and the second laser beam and reflecting the third laser beam. In this way, through the combination of the reflector 221 and the two dichroic mirrors 222, the three laser beams can be merged into the same optical path.

[0075] In an illustrative embodiment, Figure 1 As shown, the laser generating component 1 includes multiple exciters 11, each exciter 11 is configured to excite lasers of different wavelengths, and the lighting unit 21 includes three illuminators 211, which are respectively connected to the exciters 11 through optical fibers 23; wherein, at least one illuminator 211 is configured to be connected to at least two exciters 11.

[0076] In detail, the control unit 7 is configured to control any three exciters 11 to excite lasers of different wavelengths in turn according to a preset time sequence, and the lasers are transmitted to the connected illuminator 211 through the optical fiber.

[0077] According to the above-mentioned setting mode, the laser generating component 1 realizes efficient excitation and transmission of multi-wavelength lasers through the precise configuration of multiple exciters 11 and lighting units 21. Each exciter 11 is dedicated to excitation of lasers of a specific wavelength, and three illuminators 211 are connected to the exciter 11 through optical fibers 23, and at least one illuminator 211 can simultaneously receive lasers from at least two exciters 11. The control unit 7 is responsible for controlling any three exciters 11 to excite in turn according to a preset time sequence to ensure that the laser is transmitted to the illuminator 211 through the optical fiber 23. The types of lasers that can be excited by the device are increased to accommodate more fluorescent substances, the types of detectable target molecules are increased, and the effective use and precise control of laser energy are ensured, thereby realizing multi-channel, multi-wavelength fluorescence excitation, and greatly improving the sensitivity and multi-parameter analysis capabilities of capillary electrophoresis imaging detection.

[0078] According to an embodiment of the present disclosure, the illuminator 211 is a total internal reflection laser illuminator.

[0079] According to an embodiment of the present disclosure, a coupling unit 231 is further provided at the end of the optical fiber 23, which is suitable for introducing the laser excited by the exciter 11 into the optical fiber 23. In detail, the coupling unit 231 includes: a fiber coupler, which is used to couple the output of the laser into the optical fiber to ensure efficient energy transmission; or, a fiber end face (Fiber Endpoint): refers to the cut and polished end of the optical fiber 23, which can be directly connected to the laser 11 or connected through a fiber connector; or, a fiber lens, which is used to focus or collimate the laser beam so as to efficiently introduce it into the optical fiber.

[0080] Figure 2 The structure of an electrophoresis assembly according to an embodiment of the present disclosure is schematically shown.

[0081] In an illustrative embodiment, Figure 2 As shown, the electrophoresis component 4 includes: a carrier 41, which is arranged above the inverted fluorescence microscope 3; a capillary 42, which is arranged on the carrier 41, and a microfluidic channel 421 for accommodating a sample solution is formed in the capillary 42; two sample trays 43, which are symmetrically arranged on the carrier 41 and are used to accommodate a buffer solution, and both ends of the capillary 42 are respectively immersed in the buffer solution; and a power supply 44, two platinum electrodes 441 of the power supply 44 are respectively arranged in the two sample trays 43, and are electrically connected to the capillary 42 through the buffer solution, so as to form an electric field in the microfluidic channel 421 when power is turned on.

[0082] According to the above-mentioned configuration, a complete capillary electrophoresis analysis system can be formed above the inverted fluorescence microscope 3 by configuring the sample rack 41, the capillary 42, the sample tray 43 and the power supply 44. The microfluidic channel 421 in the capillary 42 is used to accommodate the sample solution, and the buffer solution in the two sample trays 43 is connected to the sample solution through the two ends of the capillary 42. The power supply 44 forms an electric field in the sample solution between the buffer solutions through the platinum electrode 441, thereby realizing electrophoretic separation in the microfluidic channel 421. The efficiency and reproducibility of sample separation are improved, and the high-resolution separation of capillary electrophoresis provides clear and orderly sample zones for subsequent fluorescence imaging detection, thereby improving the accuracy and reliability of the overall detection.

[0083] In an illustrative embodiment, Figure 2 As shown, the cross section of the capillary 42 is configured to be square, and a detection window is provided on one side of the square capillary 42 . The detection window is arranged to face the objective lens 31 of the inverted fluorescence microscope 3 .

[0084] According to the above-mentioned setting mode, the design of the square capillary 42 increases the cross-sectional surface area, effectively improving the electrophoretic separation efficiency, and at the same time, its specially set detection window allows the objective lens 31 of the inverted fluorescence microscope 3 to directly image the sample inside the capillary. This face-to-face configuration ensures the efficient collection of fluorescence signals and significantly enhances the brightness and contrast of the imaging. In addition, the precise alignment of the square capillary and the detection window optimizes the optical path, reduces stray light and background noise, thereby improving the imaging quality, ensuring the accurate capture of high-resolution imaging, and providing researchers with clear and accurate sample analysis results.

[0085] According to the embodiments of the present disclosure, Figure 2 As shown, a plurality of accommodating cavities 431 are arranged on the end surface of the sample tray 43 at intervals along the circumferential direction, which are suitable for accommodating NaOH solution, water, sample solution and buffer solution respectively. Furthermore, a rotating shaft 432 is also concentrically arranged at the other end of the sample tray 43, so that the sample tray 43 is rotatably mounted on the carrier 41, so as to quickly switch the connection between the solution in each accommodating cavity 431 and the capillary 42 through rotation.

[0086] According to an embodiment of the present disclosure, the power supply 44 is a high voltage DC power supply, which is suitable for providing a 0-30 kV positive voltage or a negative voltage to the platinum electrodes at both ends.

[0087] According to the embodiments of the present disclosure, Figure 2 As shown, it also includes a fixing member 45 suitable for fixing the capillary 42 and two platinum electrodes 441 to the carrier 41.

[0088] In an illustrative embodiment, Figure 1As shown, the fluorescence detection component 6 includes: a second optical component 61, configured to separate the fluorescence signals emitted by different fluorescent substances according to wavelengths; and a detector 62, configured to sense the separated fluorescence signals and form a target image.

[0089] According to the above-mentioned configuration, the fluorescence detection component 6 realizes separation and sensing of fluorescence signals of different wavelengths through the integrated second optical component 61 and the detector 62, thereby effectively converting the fluorescence signal of the target molecule into a clear image. The sensitivity and specificity of the detection are significantly improved, so that a variety of fluorescently labeled substances can be detected and distinguished at the same time in the same experiment, providing imaging results with high signal-to-noise ratio and resolution for the analysis of complex samples.

[0090] According to an embodiment of the present disclosure, the second optical component 61 is a three-screen device.

[0091] In an illustrative embodiment, Figure 1 As shown, it also includes a data processing device 8, which is communicatively connected to the control unit 7 and the detector 62, and is suitable for configuring the control unit 7 and recording and saving the imaging images from the detector 62.

[0092] Figure 3 The flowchart schematically shows a method for using the multi-laser excitation capillary electrophoresis imaging detection device according to an embodiment of the present disclosure.

[0093] Another aspect of the present disclosure provides a method for using a multi-laser excitation capillary electrophoresis imaging detection device, such as Figure 3 As shown, including operations S110~S140:

[0094] Operation S110: controlling the laser generating assembly 1 to alternately excite multiple laser beams with different wavelengths according to a preset timing sequence through the control unit 7;

[0095] Operation S120: merging multiple laser beams into the same optical path through the illumination component 2 to form a detection laser composed of laser beams of different wavelengths alternately, and emitting the detection laser beam to the electrophoresis component 4;

[0096] Operation S130: obtaining a fluorescence signal by detecting different fluorescent substances separated from the multiple target molecules 5 in the laser-excited electrophoresis component 4;

[0097] Operation S140 : receiving the fluorescence signal through the fluorescence detection component 6 to form an imaging image of the target molecule 5 .

[0098] In an illustrative embodiment, operation S330 includes receiving a detection laser from the illumination component 2 through an inverted fluorescence microscope 3, and the detection laser is totally reflected at the objective lens 31 of the inverted fluorescence microscope 3 to form an evanescent wave on the surface of the sample solution of the electrophoresis component 4, thereby exciting a plurality of different fluorescent substances marking the target molecules 5 and obtaining a fluorescence signal.

[0099] In an illustrative embodiment, before the target molecules 5 in the sample solution are electrophoretically separated, operations S210 to S230 are also included:

[0100] Operation S210: using fluorescent substances corresponding to multiple laser beams with different wavelengths to respectively label target molecules 5 in the sample solution;

[0101] Operation S220: applying an electric field or applying pressure to allow the plurality of target molecules 5 in the sample solution to migrate to the microfluidic channel 421 of the capillary 42;

[0102] Operation S230 : An electric field is formed in the microfluidic channel 421 by the power supply 44 to separate the target molecules 5 labeled with different fluorescent substances.

[0103] The technical solution of the present disclosure is described in detail below through preferred embodiments. It should be noted that the specific embodiments below are only used for illustration and are not used to limit the present invention.

[0104] First, three laser exciters 11 with different wavelengths are selected according to the target molecule fluorescence of the sample. Three laser beams with different wavelengths are excited by three exciters 11 as excitation light sources. In a specific embodiment, four fluorescent substances, namely, rhodamine B dye, Alexa fluor 488-ds20, TMR-ds20 and Cy5-ds20, are used to label the target molecule 5, and corresponding lasers 11 with excitation lasers of 488 nm, 532 nm and 640 nm are selected as laser light sources. Among them, the 488 nm laser excites the fluorescent dye Alexa flour 488-ds20, and receives the fluorescence signal of the fluorescent dye Alexa flour 488 at 520 nm. The 532 nm laser excites the fluorescent dyes rhodamine B and TMR-ds20, and receives the fluorescence signals of the fluorescent dyes rhodamine B and TMR-ds20 at 580 nm. The 640 nm laser excites Cy5-ds20, and receives the fluorescence signal of Cy5-ds20 at 690 nm.

[0105] Through a high-precision timing synchronization controller, the three exciters 11 are used to alternately excite lasers of different wavelengths according to a preset timing, and the incident angles of the three laser beams are adjusted by a total internal reflection laser illuminator, and the three laser beams are merged into the same optical path by a reflector 221 and a dichroic mirror 222, to form a detection laser composed of laser beams of different wavelengths that are continuously and alternately distributed according to a preset timing.

[0106] Furthermore, by fine-tuning the high-precision timing synchronization controller through a fine control program, the laser exposure interval can be precisely adjusted, ranging from 10 to 500 milliseconds. In order to improve the accuracy of the laser band, a suitable narrow-band filter can also be selected to finely filter the laser.

[0107] Using a high-resolution total internal reflection oil immersion objective lens, the detection laser is fully reflected inside it, thereby generating an evanescent wave on the sample surface to efficiently excite the fluorescent substance on the target molecule 5. In this process, the total internal reflection oil immersion objective lens is coated with a mirror oil with a refractive index of about 1.5 to fix a square capillary of 25 to 100 microns. By accurately adjusting the position of the square capillary to ensure that one side of it is parallel to the carrier 41, the efficiency of fluorescence excitation and signal collection are optimized.

[0108] Further, according to this embodiment, a square capillary with a total length of 23 cm, an inner diameter of 50 microns, and an outer diameter of 360 microns is cut out. Then, a detection window is created by firing technology at the effective length of 14 cm of the capillary. Subsequently, the capillary is placed in the accommodating chamber 431 and activated with a 100 mM NaOH solution for 2 hours. After activation, the capillary is rinsed with deionized water (ddH2O) for 10 minutes to remove residual NaOH. Finally, the capillary is balanced with 1x TG buffer (containing 25 mM Tris and 192 mM glycine) for 1 hour to ensure that the conditions of the inner wall of the capillary are suitable for subsequent electrophoretic separation and fluorescence detection.

[0109] Next, a voltage of 5 kV was applied, and a sample solution containing nM concentrations (including Rhodamine B dye, AlexaFluor 488-ds20, TMR-ds20, and Cy5-ds20) was injected into the square capillary within 5 seconds. The two ends of the capillary were loaded with buffers containing 1xTG buffer, deoxidizer, and reductant, respectively, to perform capillary zone electrophoresis at a voltage of 5 kV to separate the target molecules in the sample solution. The composition of the deoxidizer and reductant included D-glucose (8 mg / mL), glucose oxidase (1 mg / mL), catalase (0.04 mg / mL), and water-soluble vitamin E (concentration greater than 3 mM). This configuration is designed to provide a low oxygen environment and reducing conditions to protect the fluorescent markers in the sample from oxidation, ensuring the stability of electrophoretic separation and the efficient detection of fluorescent signals.

[0110] During the experiment, the fluorescence detection component is responsible for capturing the time series fluorescence signals during the entire electrophoretic separation process. When the electroosmotic flow drives the target molecule to move to the detection window position, the laser excites the fluorescent groups of the three fluorescent dyes (Rhodamine B, Alexa Fluor 488-ds20, TMR-ds20 and Cy5-ds20) fixed on the target molecule, thereby generating fluorescence signals. These fluorescence signals are received by the detector 62 and converted into target images. At the same time, the supporting software system can record the complete time series images from the beginning to the end of the electrophoretic separation for subsequent analysis and processing. In this way, researchers can obtain detailed information on the separation process and the dynamic behavior of the target molecules, thereby performing accurate quantitative and qualitative analysis.

[0111] Figure 4 The timing curve diagram of the separation of target molecules labeled with different fluorescent substances according to the embodiment of the present disclosure is schematically shown. In detail, the fluorescent substances include rhodamine B dye, Alexa fluor 488-ds20, TMR-ds20 and Cy5-ds20.

[0112] Three different wavelengths of lasers, 640 nm, 532 nm, and 488 nm, were used to simultaneously excite three different fluorescent groups in the target molecule. Specifically, the 488 nm laser was used to excite the fluorescent dye Alexa Fluor 488-ds20, and its fluorescence signal was received at 520 nm. The 532 nm laser was used to excite the fluorescent dyes Rhodamine B and TMR-ds20, and the corresponding fluorescence signals were detected at 580 nm. The 640 nm laser was used to excite Cy5-ds20, and its fluorescence signal was received at 690 nm. Through this multi-wavelength laser excitation and signal reception in a specific band, the synchronous detection of multiple fluorescent dyes was achieved.

[0113] With the application of the above detection device and method, it was observed that rhodamine B was separated at about 1.75 minutes, while Alexa Fluor 488-ds20, TMR-ds20 and Cy5-ds20 were separated at about 2.95 minutes. This time-resolved separation data provides researchers with precise molecular dynamics information, which helps to carry out efficient biological analysis and molecular recognition.

[0114] Figure 5 The imaging diagram of target molecules labeled with different fluorescent substances according to the embodiments of the present disclosure is schematically shown.

[0115] In the experiment, the imaging at two time points, 1.75 minutes and 2.95 minutes, was specially selected for analysis. At 1.75 minutes, the fluorescence imaging of the fluorescent dye Rhodamine B was successfully captured, which reflects the specific separation state of Rhodamine B during electrophoresis. Then, at 2.95 minutes, the fluorescence imaging of the three fluorescent dyes Alexa Fluor 488-ds20, TMR-ds20 and Cy5-ds20 were received respectively, which revealed the separation of these three fluorescent labeled molecules. Through the imaging data at these two time points, the researchers were able to clearly observe the separation dynamics of different fluorescent dyes, providing strong visual evidence for analyzing the properties and interactions of the target molecules.

[0116] According to the embodiments of the present disclosure, the multi-laser excitation capillary electrophoresis imaging detection device and the use method provided have a wide range of applications, including but not limited to the following fields:

[0117] 1. Capillary electrophoresis separation and imaging of three fluorescent small molecules: can be used to analyze and image small molecule compounds with different fluorescent labels.

[0118] 2. Capillary electrophoresis separation and imaging of nucleic acid molecules labeled with three fluorescent molecules: suitable for the analysis of nucleic acid fragments, such as the separation and imaging of DNA or RNA.

[0119] 3. Capillary electrophoresis separation and imaging of single-stranded DNA and its hybridized double-stranded DNA with three kinds of fluorescence: can be used to study the hybridization dynamics and structural analysis of DNA.

[0120] 4. Capillary electrophoresis separation and imaging of single-stranded RNA, single-stranded DNA and their hybrid chains with three kinds of fluorescence: suitable for analyzing the interaction between RNA and DNA and their structural characteristics.

[0121] 5. Capillary electrophoresis separation and imaging of three fluorescent molecular markers or fluorescent protein fusion proteins: can be used for protein analysis, including the separation and imaging of fluorescently labeled proteins or fluorescent protein fusions.

[0122] 6. Capillary electrophoresis separation and imaging of nucleic acid molecules with three fluorescent molecules, fluorescent molecular markers or fluorescent protein fusion proteins and their protein-nucleic acid complexes: suitable for studying the interaction between proteins and nucleic acids and the formation of complexes.

[0123] It will be appreciated by those skilled in the art that the features described in the various embodiments of the present disclosure may be combined and / or combined in a variety of ways, even if such combinations or combinations are not explicitly described in the present disclosure. In particular, without departing from the spirit and teachings of the present disclosure, the features described in the various embodiments of the present disclosure may be combined and / or combined in a variety of ways. All of these combinations and / or combinations fall within the scope of the present disclosure.

[0124] The embodiments of the present disclosure are described above. However, these embodiments are only for illustrative purposes and are not intended to limit the scope of the present disclosure. Although the embodiments are described above, this does not mean that the measures in the various embodiments cannot be used in combination to advantage. Without departing from the scope of the present disclosure, those skilled in the art may make a variety of substitutions and modifications, which should all fall within the scope of the present disclosure.

Claims

1. A multi-laser excitation capillary electrophoresis imaging detection device, characterized in that: include: A laser generating component (1) is configured to excite a plurality of laser beams having different wavelengths; A control unit (7) configured to control the laser generating components (1) to stimulate the lasers in turn according to a preset time sequence; An illumination component (2) is configured to receive multiple beams of the laser light and combine the multiple beams of the laser light into the same optical path to form a detection laser light composed of laser light of different wavelengths alternating with each other; The electrophoresis component (4) is configured to apply an electric field to the sample solution to migrate and separate target molecules (5) in the sample solution that are respectively labeled with a plurality of fluorescent substances, wherein the detection laser is used to excite the plurality of fluorescent substances to generate fluorescent signals; as well as The fluorescence detection component (6) is configured to form an imaging image of the target molecule (5) based on the received fluorescence signal.

2. The multi-laser excitation capillary electrophoresis imaging detection device according to claim 1, characterized in that: Also includes: An inverted fluorescence microscope (3) is disposed directly below the sample solution in the electrophoresis component (4) and is configured to focus the detection laser from the illumination component (2) on the sample solution or to lead the fluorescence signal out of the electrophoresis component (4).

3. The multi-laser excitation capillary electrophoresis imaging detection device according to claim 1, characterized in that: The lighting assembly (2) comprises: An illumination unit (21) configured to make the plurality of laser beams reach the evanescent wave critical angle simultaneously; and The first optical component (22) is configured to combine the multiple laser beams from the lighting unit (21) into the same optical path to obtain the detection laser.

4. The multi-laser excitation capillary electrophoresis imaging detection device according to claim 3, characterized in that: The laser generating assembly (1) comprises a plurality of exciters (11), each of the exciters (11) being configured to excite the laser light of a different wavelength, and the lighting unit (21) comprises three illuminators (211), each of which is connected to the exciters (11) via an optical fiber (23); Wherein, at least one of the illuminators (211) is configured to be connected to at least two of the exciters (11).

5. The multi-laser excitation capillary electrophoresis imaging detection device according to claim 2, characterized in that: The electrophoresis assembly (4) comprises: A specimen carrier (41) disposed above the inverted fluorescence microscope (3); A capillary tube (42) is arranged on the carrier (41), and a microfluidic channel (421) for accommodating the sample solution is formed in the capillary tube (42); Two sample trays (43) are symmetrically arranged on the carrier (41) and are used to contain a buffer solution, and both ends of the capillary tube (42) are respectively immersed in the buffer solution; and A power source (44), wherein two platinum electrodes (441) of the power source (44) are respectively disposed in the two sample disks (43) and are electrically connected to the capillary (42) via the buffer solution so as to form an electric field in the microfluidic channel (421) when power is supplied.

6. The multi-laser excitation capillary electrophoresis imaging detection device according to claim 5, characterized in that: The cross section of the capillary (42) is configured to be square, and a detection window is provided on one side of the square capillary (42), wherein the detection window is arranged to face the objective lens (31) of the inverted fluorescence microscope (3).

7. The multi-laser excitation capillary electrophoresis imaging detection device according to claim 1, characterized in that: The fluorescence detection component (6) comprises: A second optical component (61) configured to separate the fluorescent signals emitted by different fluorescent substances according to wavelengths; and The detector (62) is configured to sense the separated fluorescence signals and form the target image.

8. A method for using the multi-laser excitation capillary electrophoresis imaging detection device according to any one of claims 1 to 7, characterized in that: include: The control unit (7) controls the laser generating component (1) to alternately excite multiple laser beams with different wavelengths according to a preset time sequence; The plurality of laser beams are combined into the same optical path through the illumination component (2) to form a detection laser beam composed of laser beams of different wavelengths alternately, and emitted to the electrophoresis component (4); Exciting different fluorescent substances separated with the multiple target molecules (5) in the electrophoresis component (4) by the detection laser to obtain fluorescent signals; The fluorescence signal is received by a fluorescence detection component (6) to form an imaging image of the target molecule (5).

9. The method of use according to claim 8, characterized in that: Also includes: The detection laser light from the illumination component (2) is received by an inverted fluorescence microscope (3), and the detection laser light is totally reflected by the objective lens (31) of the inverted fluorescence microscope (3) to form an evanescent wave on the surface of the sample solution of the electrophoresis component (4), thereby exciting the multiple different fluorescent substances marking the target molecules (5) and obtaining the fluorescence signal.

10. The method of use according to claim 8, characterized in that: Also includes: Using the fluorescent substances corresponding to the laser beams of different wavelengths to respectively label the target molecules in the sample solution (5); Applying an electric field or applying pressure causes a plurality of target molecules (5) in a sample solution to migrate to a microfluidic channel (421) of a capillary (42); An electric field is formed in the microfluidic channel (421) by a power source (44) to separate target molecules (5) labeled with different fluorescent substances.

Images