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

Abstract

The invention provides a dual-laser excitation capillary electrophoresis imaging detection device and a use method. The detection device comprises a dual-laser excitation assembly, a control unit, a separation assembly and a fluorescence detection assembly. The double-laser excitation assembly is suitable for exciting two laser beams with different wavelengths; the control unit is suitable for controlling the double-laser excitation assembly to alternately excite laser according to a preset time sequence to form detection laser composed of two beams of laser with different wavelengths alternately; the separation assembly is suitable for applying an electric field to the sample solution, so that target molecules marked by the two fluorescent substances in the sample solution are migrated and separated, and the laser with different wavelengths in the detection laser is suitable for exciting the two fluorescent substances respectively, so that the two fluorescent substances emit fluorescence signals; the fluorescence detection assembly is adapted to receive and form a separation process image of the target molecule according to the fluorescence signal.

Description

Technical Field

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

[0002] Capillary electrophoresis (CE) is a separation analysis technique based on the difference in migration speed of charged particles in an electric field. In this technique, the sample solution is injected into a thin capillary filled with an electrolyte solution, and after a high voltage is applied, the charged particles migrate under the action of the electric field force. Due to the different charges, sizes and shapes of different particles, their migration speeds in the electric field are different, thus achieving separation.

[0003] Although traditional capillary electrophoresis detection methods, such as ultraviolet or fluorescence detection, can achieve high separation efficiency, they have limitations in the visualization of analyte distribution and the intuitive observation of the separation process. In order to improve the intuitiveness and resolution of the analysis, researchers have developed capillary electrophoresis imaging technology, which combines the high separation efficiency of capillary electrophoresis with the direct visualization of imaging technology, allowing real-time monitoring of the distribution of analytes during the separation process, thereby providing more intuitive separation efficiency and characteristic information.

[0004] However, existing imaging technologies still have difficulty in achieving high-resolution visual detection when dealing with complex samples, especially in distinguishing more similar analytes. Therefore, it is particularly important to develop an electrophoresis imaging detection device that can perform high-resolution imaging of complex samples. Summary of the invention

[0005] In view of this, the present disclosure provides a dual 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 two fluorescent substances under electric field manipulation, thereby distinguishing two different target objects in a sample.

[0006] One aspect of the present disclosure provides a dual-laser excitation capillary electrophoresis imaging detection device, including a dual-laser excitation component, suitable for exciting two beams of laser light with different wavelengths; a control unit, suitable for controlling the dual-laser excitation component to alternately excite the laser light according to a preset timing to form a detection laser light composed of two beams of laser light with different wavelengths; a separation component, suitable for applying an electric field to a sample solution to cause target molecules in the sample solution that are labeled with two fluorescent substances to migrate and separate, the laser light of different wavelengths in the detection laser light being suitable for respectively exciting the two fluorescent substances to cause the two fluorescent substances to emit fluorescent signals; and a fluorescence detection component, suitable for receiving and forming an image of the separation process of the target molecules based on the fluorescent signals.

[0007] According to an embodiment of the present disclosure, the above-mentioned dual laser excitation component includes: two lasers, each suitable for exciting a laser of a specific wavelength to respectively excite the two fluorescent substances marked with the above-mentioned target molecules; and a first optical component, suitable for merging the lasers excited by the two lasers into the same optical path for propagation, and coupling them into the optical fiber.

[0008] According to an embodiment of the present disclosure, the separation component includes: a support frame; a capillary, which is arranged on the support frame, and a separation channel for accommodating a sample solution is formed in the capillary; two buffer tanks, which are symmetrically arranged at both ends of the capillary, and both ends of the capillary are immersed in the buffer tanks; and a power supply, wherein two platinum electrodes of the power supply are respectively arranged in the two buffer tanks, and are electrically connected to the sample solution in the separation channel through the buffer solution in the buffer tanks; wherein, when the power supply is activated, an electric field is formed in the sample solution, driving the target molecules in the sample solution to be electrophoretically separated along the separation channel.

[0009] According to an embodiment of the present disclosure, it also includes an observation unit, which is arranged below the above-mentioned support frame and is suitable for focusing the above-mentioned detection laser on the above-mentioned separation channel in the above-mentioned capillary, or extracting the above-mentioned fluorescence signal from the above-mentioned separation channel.

[0010] 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 provided to face the observation unit.

[0011] According to an embodiment of the present disclosure, it also includes a lighting unit, which is connected to the above-mentioned dual laser excitation component through an optical fiber and emits the above-mentioned detection laser to the above-mentioned observation unit; wherein the above-mentioned lighting unit is suitable for making the above-mentioned detection laser reach the critical angle of the evanescent wave so as to cause total reflection in the above-mentioned observation unit, form an evanescent field on the surface of the above-mentioned sample solution, and reduce background signal interference.

[0012] According to an embodiment of the present disclosure, the fluorescence detection component includes: a second optical component, suitable for separating the fluorescence signals emitted by the two fluorescent substances according to wavelength; and a detector, configured to respectively sense the separated fluorescence signals and form the separation process image.

[0013] According to an embodiment of the present disclosure, the second optical component includes a spectroscope, which is suitable for separating the two fluorescence signals of different wavelengths; two filters are respectively arranged on the light output side of the spectroscope to filter the two separated fluorescence signals.

[0014] One aspect of the present disclosure provides a method for using a dual-laser excitation capillary electrophoresis imaging detection device, comprising: controlling a dual-laser excitation component to produce a detection laser consisting of two laser beams of different wavelengths alternating with each other through a control unit; exciting two fluorescent substances on target molecules separated in the separation component through the detection laser to obtain a fluorescence signal; receiving the fluorescence signal through a fluorescence detection component and forming an image of the separation process of the target molecules.

[0015] According to an embodiment of the present disclosure, it also includes using the fluorescent substances corresponding to the two laser beams of different wavelengths to respectively label the target molecules in the sample solution; applying an electric field or applying pressure to migrate the target molecules in the sample solution to a separation channel in the capillary; forming an electric field in the separation channel through a power supply, and electrophoretically separating the target molecules labeled with different fluorescent substances.

[0016] According to the embodiments of the present disclosure, by precisely controlling the alternating excitation of two laser beams of different wavelengths, selective excitation and imaging of target molecules labeled with two fluorescent substances is achieved, thereby achieving the technical effect of efficiently distinguishing and visualizing different targets in complex samples. Not only does it improve the specificity and sensitivity of the detection, but it also enhances the accuracy and intuitiveness of the analysis by forming an image of the separation process of the target molecules, providing a powerful tool for the field of biological analysis and molecular detection. 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 dual laser excitation capillary electrophoresis imaging detection device according to an embodiment of the present disclosure is schematically shown.

[0019] Figure 2 The structure diagram of the separation component according to an embodiment of the present disclosure is schematically shown.

[0020] Figure 3 The flowchart schematically shows a method for using the dual-laser excitation capillary electrophoresis imaging detection device according to an embodiment of the present disclosure.

[0021] Figure 4 The timing curve diagram schematically shows the separation of target molecules labeled with two fluorescent substances according to an embodiment of the present disclosure.

[0022] Figure 5 The first-time imaging diagram of target molecules labeled with two fluorescent substances according to an embodiment of the present disclosure is schematically shown.

[0023] Figure 6The imaging diagram of the target molecules labeled with two fluorescent substances at the second time according to the embodiment of the present disclosure is schematically shown.

[0024] In the drawings, the meanings of the reference numerals are as follows:

[0025] 1. Dual laser excitation components;

[0026] 11. Laser;

[0027] 12. a first optical component;

[0028] 121. Reflector;

[0029] 122. Dichroic mirror;

[0030] 123. Lens;

[0031] 2. Control unit;

[0032] 3. Separate components;

[0033] 31. Support frame;

[0034] 32. Capillary;

[0035] 321, separation channel;

[0036] 33. Buffer pool;

[0037] 34. Power supply;

[0038] 341, platinum electrode;

[0039] 35. Fixing parts;

[0040] 36. Sample tray;

[0041] 361, accommodating chamber;

[0042] 362, shaft;

[0043] 4. Fluorescence detection component;

[0044] 41. A second optical component;

[0045] 411, spectroscope;

[0046] 412, filter;

[0047] 42. Detector;

[0048] 5. Observation unit;

[0049] 51. Objective lens;

[0050] 6. Lighting unit;

[0051] 7. Optical fiber; DETAILED DESCRIPTION

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.).

[0056] As an advanced separation and analysis method, capillary electrophoresis relies on the different migration rates of charged particles in an electric field to achieve separation. In short, the sample solution is injected into a capillary filled with electrolytes, and after a high voltage is applied, the particles begin to move under the action of the electric field force. The differences in charge, size and shape of these particles cause them to move at different speeds in the capillary, thereby achieving the purpose of separation.

[0057] Capillary electrophoresis has high separation ability, high sensitivity, extremely low sample consumption, fast analysis process and low-cost operation. These characteristics make it play an important role in many fields such as analytical chemistry, biochemistry and biomedicine. The diversified forms of capillary electrophoresis, such as zone electrophoresis, isoelectric focusing electrophoresis, micellar electrokinetic chromatography, etc., continue to broaden its application areas.

[0058] Furthermore, the development of capillary electrophoresis imaging technology combines the high efficiency of separation with the intuitive visualization of imaging technology, allowing the distribution of analytes during the separation process to be directly observed. This technology provides intuitive information about separation efficiency and analyte characteristics, which has significant advantages over traditional ultraviolet or fluorescence detection methods. Through imaging technology, researchers can track the migration and distribution of analytes in real time and gain a deep understanding of the separation process and the factors behind it.

[0059] This disclosure proposes a dual laser excitation capillary electrophoresis imaging detection device for the field of biological analysis, which achieves high-resolution separation effects and obtains high-resolution images by combining advanced imaging technology, effectively distinguishing similar analytes. This technological advancement not only improves the accuracy and sensitivity of analysis, but also provides more powerful technical support for the field of biological analysis and promotes the further development of related research.

[0060] Figure 1 The structure diagram of the dual laser excitation capillary electrophoresis imaging detection device according to an embodiment of the present disclosure is schematically shown.

[0061] The embodiment of the present disclosure provides a dual laser excitation capillary electrophoresis imaging detection device, such as Figure 1 As shown, it includes: a dual laser excitation component 1, which is suitable for exciting two laser beams with different wavelengths; a control unit 2, which is suitable for controlling the dual laser excitation component 1 to alternately excite the laser according to a preset timing to form a detection laser composed of two laser beams with different wavelengths alternately; a separation component 3, which is suitable for applying an electric field to a sample solution to make the target molecules marked by two fluorescent substances in the sample solution migrate and separate, and the lasers of different wavelengths in the detection laser are suitable for respectively exciting the two fluorescent substances to make the two fluorescent substances emit fluorescent signals; and a fluorescence detection component 4, which is suitable for receiving and forming a separation process image of the target molecules according to the fluorescent signals.

[0062] According to the above-mentioned setting mode, through the coordinated work of the dual laser excitation component 1 and the control unit 2, lasers of different wavelengths are alternately excited to effectively excite the two fluorescent substances respectively, so that the target molecules migrate and separate under the action of the electric field applied by the separation component 3. The fluorescence detection component 4 further receives the fluorescence signal to form a clear image of the separation process. The resolution and sensitivity of electrophoresis imaging are significantly improved, making it possible to distinguish similar analytes in complex samples, greatly enhancing the research capabilities and application scope of biological analysis.

[0063] The dual laser excitation capillary electrophoresis imaging detection device disclosed in the present invention includes the following application fields: capillary electrophoresis separation and imaging technology of single or two fluorescent small molecules; capillary electrophoresis separation and imaging of nucleic acid molecules using one or two fluorescent labels; capillary electrophoresis separation and imaging of single-stranded DNA and its hybridized double-stranded DNA with two fluorescent labels; capillary electrophoresis separation and imaging of single-stranded RNA, single-stranded DNA and its hybrid chains using two fluorescent labels; capillary electrophoresis separation and imaging of one or two fluorescent molecular labels or fluorescent protein fusion proteins; and capillary electrophoresis separation and imaging of nucleic acid molecules with dual fluorescent labels, fluorescent molecular labels, fluorescent protein fusion proteins and their protein-nucleic acid complexes.

[0064] In an illustrative embodiment, Figure 1 As shown, the dual laser excitation component 1 includes: two lasers 11, each suitable for exciting lasers of specific wavelengths to respectively excite two fluorescent substances that mark the target molecules; and a first optical component 12, suitable for merging the lasers excited by the two lasers into the same optical path for propagation, and coupling them into the optical fiber 7.

[0065] According to the above-mentioned setting mode, the precise merging and transmission of the laser beams of specific wavelengths emitted by the two lasers 11 are realized, and the first optical component 12 effectively merges the two laser beams into the same optical path and couples them into the optical fiber 7. It ensures that the two fluorescent substances can be excited synchronously and independently, thereby improving the efficiency and selectivity of the detection, simplifying the optical path design, reducing the complexity and cost of the system, and providing a stable and reliable light source for capillary electrophoresis imaging detection.

[0066] According to an embodiment of the present disclosure, the control unit 2 is connected to the dual laser excitation assembly 1. The control unit 2 includes a high-precision timing synchronization controller, and through its driver, it realizes fast and accurate synchronization control of the two lasers 11 of the dual laser excitation assembly 1. This ensures the coordinated operation of the entire detection device and improves the synchronization of the experiment and the accuracy of data collection.

[0067] In an illustrative embodiment, Figure 1 As shown, the first optical component 12 is composed of a reflector 121, a dichroic mirror 122 and a lens 123 arranged in sequence. Specifically, the function of the reflector 121 is to reflect the first laser beam to ensure that the laser beam propagates along a predetermined path. The dichroic mirror 122 has a special property that allows the first reflected laser beam to pass through while reflecting the second laser beam, thereby achieving separation and guidance of the two laser beams. The lens 123 is responsible for focusing the two reflected laser beams and accurately directing them into the optical fiber 7 for subsequent optical processing and transmission.

[0068] Figure 2The structure diagram of the separation component according to an embodiment of the present disclosure is schematically shown.

[0069] In an illustrative embodiment, Figure 1 and Figure 2 As shown, the separation component 3 includes: a support frame 31; a capillary 32, which is arranged on the support frame 31, and a separation channel 321 for accommodating a sample solution is formed in the capillary 32; two buffer tanks 33, which are symmetrically arranged at both ends of the capillary 32, and both ends of the capillary 32 are immersed in the buffer tanks 33; and a power supply 34, wherein two platinum electrodes 341 of the power supply 34 are respectively arranged in the two buffer tanks 33, and are electrically connected to the sample solution in the separation channel 321 through the buffer solution in the buffer tanks 33; wherein, when the power supply 34 is activated, an electric field is formed in the sample solution, driving the target molecules in the sample solution to be electrophoretically separated along the separation channel 321.

[0070] According to the above-mentioned setting method, the structural design of the support frame 31 and the capillary 32 ensures the stable containment of the sample solution and the effective formation of the separation channel 321; secondly, the symmetrical setting of the two buffer pools 33 and the immersion of the two ends of the capillary 32, as well as the electric field formed after the power supply 34 is activated, work together to achieve efficient electrophoretic migration and separation of the target molecules in the separation channel 321; improve the controllability and repeatability of the separation process, provide a guarantee for obtaining high-resolution and accurate electrophoretic separation results, thereby enhancing the performance of the overall detection device.

[0071] In an alternative embodiment, Figure 2 As shown, it includes two sample trays 36, which are symmetrically arranged on the support frame 11. The end surface of the sample tray 36 is provided with a plurality of accommodating cavities 361 spaced apart in the circumferential direction, which are suitable for accommodating NaOH solution, water, sample solution and buffer solution respectively. The accommodating cavity 361 for accommodating the buffer solution is used as the buffer pool 33; further, the other end of the sample tray 36 is also concentrically provided with a rotating shaft 362, so that the sample tray 36 is rotatably mounted on the support frame 31, so as to quickly switch the connection between the solution in each accommodating cavity 3612 and the capillary 32 through rotation.

[0072] According to an embodiment of the present disclosure, the power supply 34 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.

[0073] According to the embodiments of the present disclosure, Figure 2 As shown, it also includes a fixing member 35 suitable for fixing the capillary 32 and two platinum electrodes 341 to the support frame 31.

[0074] In an illustrative embodiment, Figure 1As shown, it also includes an observation unit 5, which is arranged below the support frame 31 and is suitable for focusing the detection laser on the separation channel 321 in the capillary 32, or extracting the fluorescence signal from the separation channel 321.

[0075] According to the above-mentioned configuration, accurate focusing of the sample solution and efficient extraction of the fluorescence signal are achieved through the observation unit 5. The optimized configuration of the observation unit 5 significantly improves the focusing accuracy of the detection laser, ensures the effective excitation of the target molecules in the sample, and enhances the collection efficiency of the fluorescence signal, thereby improving the clarity of the imaging and the sensitivity of the detection.

[0076] In detail, the observation unit 5 includes an inverted fluorescence microscope, which is a widely used tool in cell and molecular biology research. Its unique inverted design allows the sample to be located below the objective lens, which is convenient for illumination and signal collection from below. The microscope is equipped with a variety of fluorescent light sources and filters, which are specially used to excite fluorescent markers in the sample and collect corresponding signals, allowing researchers to observe cell structures, proteins or molecules in detail.

[0077] According to the embodiments of the present disclosure, Figure 1 As shown, the observation unit includes an objective lens 51, which adopts a high-resolution total internal reflection oil immersion objective lens. This objective lens supports the laser to be incident at the critical angle of the evanescent wave, thereby realizing efficient total internal reflection excitation. The total internal reflection oil immersion objective lens has a high numerical aperture, can provide excellent spatial resolution, and excite fluorescent molecules near the sample surface through the total internal reflection phenomenon. When the laser is incident on the interface between the sample and the objective lens at an angle exceeding the critical angle, the generated evanescent wave only exists near the interface, effectively exciting the fluorescent markers close to the interface. By using a specific immersion oil to match the refractive index between the sample and the objective lens, the refraction and reflection losses of light at the interface are reduced, thereby significantly improving the imaging quality and detection efficiency.

[0078] In an illustrative embodiment, the cross section of the capillary 32 is configured to be square, and a detection window is disposed on one side of the square capillary 32 , and the detection window is disposed facing the observation unit 5 .

[0079] According to the above-mentioned configuration, by adopting the design of the square capillary 32, this embodiment not only increases the cross-sectional surface area of ​​the capillary, thereby improving the efficiency of electrophoretic separation, but also enables the objective lens 51 of the observation unit 5 to directly image the sample inside the capillary through a specially set detection window. This directly opposite configuration greatly improves the collection efficiency of the fluorescent signal and significantly improves the brightness and contrast of the imaging. In addition, the precise alignment of the square capillary with the detection window effectively optimizes the optical path, reduces the interference of stray light and background noise, and thus improves the overall quality of the imaging.

[0080] In an illustrative embodiment, it also includes an illumination unit 6, which is connected to the dual laser excitation assembly 1 through an optical fiber 7 and emits a detection laser to the observation unit 5; wherein the illumination unit 6 is suitable for making the detection laser reach the critical angle of the evanescent wave so as to cause total reflection in the observation unit 5, form an evanescent field on the surface of the sample solution, and reduce background signal interference.

[0081] According to an embodiment of the present disclosure, the lighting unit 6 is a total internal reflection laser illuminator.

[0082] According to the above-mentioned setting mode, the integration of the illumination unit 6 is connected to the dual laser excitation component 1 through the optical fiber 7, and the detection laser is emitted to the observation unit 5, so that the detection laser reaches the critical angle of the evanescent wave, realizes total reflection inside the observation unit 5, and then forms an evanescent field on the surface of the sample solution. The background signal interference is effectively reduced, the signal specificity and the clarity of the imaging are improved, thereby significantly improving the sensitivity of the detection and the accuracy of the data.

[0083] When a laser beam is incident on a medium interface (such as the interface between glass and water) at an angle exceeding the critical angle, total internal reflection will occur. At this time, the laser will not penetrate into the light-scarce medium, but will be completely reflected at the interface. Under this condition, if the target molecules in the sample solution are labeled with fluorescent dyes or fluorescent proteins and are located in the penetration area of ​​the evanescent wave (usually tens to hundreds of nanometers below the interface), the electric field of the evanescent wave can effectively excite these fluorescent markers. Due to the limited penetration depth of the evanescent wave, the nonspecific fluorescence background signal of deep samples is significantly suppressed, thereby significantly improving the signal-to-noise ratio. In this way, high-resolution, low-background interference surface layer imaging is achieved.

[0084] According to an embodiment of the present disclosure, the objective lens 31 used is a high-resolution total internal reflection oil immersion objective lens, which is specially designed for laser incidence that reaches the critical angle of the evanescent wave. This objective lens has a high numerical aperture (NA), which ensures extremely high spatial resolution, and uses the principle of total internal reflection to excite fluorescent molecules close to the sample surface. When the laser irradiates the interface between the sample and the objective lens at an angle exceeding the critical angle, an evanescent wave is generated near the interface, which can efficiently excite fluorescent markers near the interface. By using a specific immersion oil to match the refractive index of the sample and the objective lens, the refraction and reflection losses of light during interface transmission are reduced, thereby significantly improving the clarity of imaging and the efficiency of detection.

[0085] In an illustrative embodiment, the fluorescence detection component 4 includes a second optical component 41, which is suitable for separating the fluorescence signals emitted by two fluorescent substances according to wavelengths; and a detector 42, which is configured to respectively sense the separated fluorescence signals and form a separation process image.

[0086] According to the above-mentioned configuration, the separation and detection of the fluorescence signals emitted by two different fluorescent substances are achieved through the second optical component 41 and the detector 42. The second optical component 41 is specially designed to distinguish the two fluorescence signals according to the wavelength, ensuring the minimum cross-interference between the signals, thereby improving the specificity of the detection. The detector 42 is configured to sense these separated fluorescence signals separately and convert them into a separation process image. Such a configuration not only improves the signal-to-noise ratio of the image, but also allows researchers to obtain more accurate molecular separation data, thereby conducting more in-depth analysis and research.

[0087] In an illustrative embodiment, the second optical component 41 includes: a spectroscope 411, which is used to separate two fluorescent signals of different wavelengths; and two filters 412, which are respectively arranged on the light-emitting sides of the spectroscope 411 to filter the two separated fluorescent signals.

[0088] According to the above-mentioned setting mode, the function of the spectroscope 411 is to separate the incident composite fluorescent signal according to the wavelength, ensuring that the fluorescent signals of different wavelengths can be effectively distinguished. Subsequently, two filters 412 are respectively arranged on the light-emitting side of the spectroscope 411, and they each filter the separated fluorescent signals at a specific wavelength, thereby further purifying the signal and reducing background noise and cross-interference. Such a configuration not only improves the detection sensitivity and selectivity of the fluorescent signal, but also provides a guarantee for the formation of high-quality, high-contrast separation process images, and enhances the performance and reliability of the entire detection system.

[0089] Figure 3 The flowchart schematically shows a method for using the dual-laser excitation capillary electrophoresis imaging detection device according to an embodiment of the present disclosure.

[0090] Another aspect of the embodiment of the present disclosure provides a method for using a dual laser excitation capillary electrophoresis imaging detection device, including: operations SS110 to S130:

[0091] Operation S110: Control the dual laser excitation assembly 1 to produce detection lasers composed of two laser beams with different wavelengths alternately through the control unit 2;

[0092] Operation S120: obtaining a fluorescence signal by detecting two fluorescent substances on the target molecules separated in the laser excitation separation component 3;

[0093] Operation S130: receiving the fluorescence signal through the fluorescence detection component 4 and forming a separation process image of the target molecule.

[0094] In an exemplary embodiment, operations S210 to S230 are also included.

[0095] Operation S210: using fluorescent substances corresponding to two laser beams with different wavelengths to respectively label target molecules in the sample solution;

[0096] Operation S220: applying an electric field or applying pressure to migrate the target molecules in the sample solution to the separation channel 321 in the capillary 32;

[0097] Operation S230: An electric field is formed in the separation channel 321 by the power supply 34 to electrophoretically separate the target molecules labeled with different fluorescent substances.

[0098] The present technical solution is described in detail below through a preferred embodiment. It should be noted that the following specific embodiments are only examples and do not limit the scope of the present disclosure.

[0099] First, two lasers 11 were selected according to the fluorescence characteristics of the fluorescent substance that marked the target molecule. Two lasers 11 were used to emit two laser beams of different wavelengths as excitation light sources. In the specific implementation, we used two fluorescent dyes, Rhodamine B and Alexa Fluor 488, so 488 nm and 532 nm lasers 11 were selected. Among them, the 532 nm laser was used to excite Rhodamine B and receive its fluorescence signal at 580 nm; the 488 nm laser was used to excite Alexa Fluor488 and receive its fluorescence signal at 520 nm.

[0100] Using a high-precision timing synchronization controller, we can achieve alternating excitation of the target molecule by two laser beams. Through the control program, we can flexibly adjust the laser exposure interval in the range of 10-500 milliseconds. The detection laser is accurately guided into the optical fiber through a carefully configured reflector, dichroic mirror and lens system. In order to further accurately define the laser band, we use a suitable narrow-band filter to screen the laser.

[0101] We adjusted the angle of the detection laser to achieve total internal reflection illumination using a total internal reflection laser illuminator. At the same time, we used a high-resolution total internal reflection oil immersion objective lens coated with immersion oil with a refractive index of about 1.5 to fix a 25 to 100 micron square capillary and ensure that one side of the capillary was parallel to the stage.

[0102] In this embodiment, we prepared a square capillary with a length of 23 cm, an inner diameter of 50 μm, and an outer diameter of 360 μm. A detection window was opened by firing technology at the effective length of 14 cm of the capillary. The capillary was placed in the receiving chamber 431, activated with a 100 mM NaOH solution for 2 hours, and then rinsed with deionized water for 10 minutes to remove residual NaOH. Finally, the capillary was equilibrated for 1 hour using a 1x TG buffer (containing 25 mM Tris and 192 mM glycine) to create suitable conditions for electrophoretic separation and fluorescence detection.

[0103] At 5 kV, nM samples (containing rhodamine B and Alexa Fluor 488) were injected. Buffer containing 1xTG, deoxygenation system and reducing agent was loaded at both ends of the square capillary as separation buffer, and capillary zone electrophoresis separation was performed at the same voltage.

[0104] The fluorescence detection component is responsible for receiving the time series fluorescence signals of the entire separation process. When the target molecule moves to the detection window with the electroosmotic flow, the laser excites the labeled fluorescent dye to generate a fluorescence signal. The detector captures these signals, forms a target image, and records the complete time series image from the beginning to the end of the separation through the software.

[0105] Figure 4 The timing curve diagram schematically shows the separation of target molecules labeled with two fluorescent substances according to an embodiment of the present disclosure, including rhodamine B dye and Alexa flour 488 dye.

[0106] The laser can be selected with two wavelengths of 532 nm and 488 nm, which can simultaneously excite two fluorescent groups in the target molecule. The 532 nm laser excites the fluorescent dye Rhodamine B, and receives the fluorescence signal of the fluorescent dye Rhodamine B at 580 nm. The 488 nm laser excites the fluorescent dye Alexa flour 488, and receives the fluorescence signal of the fluorescent dye Alexa flour 488 at 520 nm.

[0107] When the detection device of the above embodiment is operated under the above method, rhodamine B is separated in about 2 min and Alexaflour 488 is separated in about 5 min.

[0108] Figure 5 The first-time imaging diagram of target molecules labeled with two fluorescent substances according to an embodiment of the present disclosure is schematically shown. Figure 6 The imaging diagram of the target molecules labeled with two fluorescent substances at the second time according to the embodiment of the present disclosure is schematically shown.

[0109] like Figure 5 and Figure 6As shown, the fluorescence imaging images at 2.11 min and 4.97 min were selected. The fluorescence imaging of the fluorescent dye Rhodamine B was received at 2.11 min, while the fluorescence imaging of Alexa flour 488 was not detected. The fluorescence imaging of the fluorescent dye Alexa flour 488 was received at 4.97 min, while the fluorescence imaging of Rhodamine B was not detected. The two dyes were separated by capillary electrophoresis.

[0110] 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.

[0111] 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 dual laser excitation capillary electrophoresis imaging detection device, characterized in that: include: A dual laser excitation component (1) is suitable for exciting two laser beams with different wavelengths; A control unit (2) adapted to control the dual laser excitation component (1) to alternately excite lasers according to a preset time sequence, thereby forming a detection laser composed of two laser beams of different wavelengths alternately; The separation component (3) is adapted to apply an electric field to the sample solution to cause the target molecules in the sample solution labeled with two fluorescent substances to migrate and separate, and the lasers of different wavelengths in the detection laser are adapted to respectively excite the two fluorescent substances to cause the two fluorescent substances to emit fluorescent signals; as well as The fluorescence detection component (4) is adapted to receive and form an image of the separation process of the target molecule based on the fluorescence signal.

2. The dual laser excitation capillary electrophoresis imaging detection device according to claim 1, characterized in that: The dual laser excitation component (1) comprises: two lasers (11), each adapted to excite lasers of a specific wavelength to respectively excite the two fluorescent substances marking the target molecules; and The first optical component (12) is suitable for combining the lasers excited by the two lasers (11) to propagate in the same optical path and coupling them into the optical fiber (7).

3. The dual laser excitation capillary electrophoresis imaging detection device according to claim 1, characterized in that: The separation component (3) comprises: Support frame (31); A capillary tube (32) is arranged on the support frame (31), wherein a separation channel (321) for accommodating a sample solution is formed in the capillary tube (32); Two buffer pools (33) are symmetrically arranged at two ends of the capillary tube (32), and both ends of the capillary tube (32) are immersed in the buffer pools (33); and A power supply (34), wherein two platinum electrodes (341) of the power supply (34) are respectively disposed in the two buffer pools (33) and are electrically connected to the sample solution in the separation channel (321) via the buffer solution in the buffer pool (33); When the power source (34) is activated, an electric field is formed in the sample solution, driving the target molecules in the sample solution to be electrophoretically separated along the separation channel (321).

4. The dual laser excitation capillary electrophoresis imaging detection device according to claim 3, characterized in that: Also includes: The observation unit (5) is arranged below the support frame (31) and is suitable for focusing the detection laser on the separation channel (321) in the capillary (32) or extracting the fluorescence signal from the separation channel (321).

5. The dual laser excitation capillary electrophoresis imaging detection device according to claim 4, characterized in that: The cross section of the capillary tube (32) is configured to be square, and a detection window is provided on one side of the square capillary tube (32), wherein the detection window is arranged facing the observation unit (5).

6. The dual laser excitation capillary electrophoresis imaging detection device according to claim 4, characterized in that: Also includes: An illumination unit (6) is connected to the dual laser excitation assembly (1) via an optical fiber (7) and emits the detection laser to the observation unit (5); The illumination unit (6) is adapted to make the detection laser reach the critical angle of the evanescent wave, so as to cause total reflection in the observation unit (5), form an evanescent field on the surface of the sample solution, and reduce background signal interference.

7. The dual laser excitation capillary electrophoresis imaging detection device according to any one of claims 1 to 6, characterized in that: The fluorescence detection component (4) comprises: A second optical component (41) is adapted to separate the fluorescent signals emitted by the two fluorescent substances according to wavelength; and The detector (42) is configured to respectively sense the separated fluorescence signals and form an image of the separation process.

8. The dual laser excitation capillary electrophoresis imaging detection device according to claim 7, characterized in that: The second optical component (41) comprises: A spectroscope (411), adapted to separate the fluorescent signals of two different wavelengths; Two filters (412) are respectively arranged on the light-emitting sides of the beam splitter (411) to filter the two separated fluorescence signals.

9. A method for using the dual laser excitation capillary electrophoresis imaging detection device according to any one of claims 1 to 8, characterized in that: include: The control unit (2) controls the dual laser excitation component (1) to excite a detection laser composed of two laser beams with different wavelengths alternately; Exciting two fluorescent substances on the target molecules separated in the separation component (3) by the detection laser to obtain a fluorescent signal; The fluorescence signal is received by a fluorescence detection component (4) and an image of the separation process of the target molecule is formed.

10. The method of use according to claim 9, characterized in that: Also includes: Using the fluorescent substances corresponding to the two laser beams with different wavelengths to respectively mark the target molecules in the sample solution; Applying an electric field or applying pressure causes target molecules in the sample solution to migrate to a separation channel (321) in the capillary (32); An electric field is formed in the separation channel (321) by a power source (34), and target molecules labeled with different fluorescent substances are electrophoretically separated.

Images