Dual-color laser alternating excitation single molecule imaging detection device, use method and application

Abstract

The application discloses a kind of dual-color laser alternate excitation single molecule imaging detection device, use method and application, belong to biological analysis field.The device includes: dual-color laser alternate exciter;Acousto-optic tunable filter;Object platform, including microflow chamber, for placing first target sample and second target sample, first target sample is labeled by first fluorescent dye, second target sample is labeled by second fluorescent dye, during first excitation light irradiation, excite first fluorescent dye, so that first fluorescent dye emits first fluorescent signal, and when the distance between first fluorescent dye and second fluorescent dye satisfies resonance energy transfer condition, fluorescence resonance energy transfer signal is generated, during second excitation light irradiation, excite second fluorescent dye, so that second fluorescent dye emits second fluorescent signal;Fluorescence detection component is used to receive fluorescence resonance energy transfer signal, first fluorescent signal and second fluorescent signal and form target image.

Description

Technical Field

[0001] This invention relates to the field of bioanalysis, specifically to a dual-color laser alternating excitation single-molecule imaging detection device, its usage method, and its application. Background Technology

[0002] Homologous recombination is catalyzed by a series of conserved homologous recombination enzymes, including T4 UvsX in bacteriophages, RadA in archaea, RecA in bacteria, and Rad51 and DMC1 in eukaryotes. In eukaryotes, Rad51 is mainly responsible for homologous recombination repair of DNA double-strand breaks, while DMC1 specifically catalyzes homologous recombination during meiosis. Homologous recombination involves numerous steps, requiring the participation of multiple protein machines and is subject to fine regulation. Its biochemical process can be roughly divided into four stages: initiation, homologous pairing, new strand synthesis, and separation. Homologous recombination is the molecular basis of the origin of life, biodiversity, and the development of diseases, and the study of its various intricate processes is at the forefront of life sciences today. Developing new optical analysis and research methods is key to answering important scientific questions about homologous recombination.

[0003] Because traditional analytical techniques obtain information based on the average of all molecular measurements, a great deal of specific individual information is masked, making it difficult to reveal many complex biological processes such as homologous recombination. In fundamental life processes, many biomolecules perform their specific functions as single molecules or in groups of several molecules. Therefore, observing the structure and behavior of target molecules at the single-molecule level is crucial for our understanding of fundamental life processes.

[0004] Currently, various single-molecule measurement techniques have been developed, which can be broadly classified into two categories: one is imaging techniques based on fluorescence and its spectroscopy, mainly including single-molecule fluorescence imaging and single-molecule fluorescence resonance energy transfer (smFRET); the other is manipulation and detection techniques based on mechanics, mainly including optical tweezers, magnetic tweezers, and atomic force microscopy.

[0005] Optical microscopes are indispensable research tools in the biomedical field, playing a crucial role in both basic research and clinical diagnosis. Through microscopic imaging, we can obtain information not only about the presence and location of target molecules, but also, because the emission wavelength of fluorescence is longer than its absorption wavelength, background interference can be significantly reduced through proper optical path design, making it the optimal choice for single-molecule imaging. Currently, existing optical microscopes cannot accurately measure transient or unstable states, nor can they simultaneously acquire and analyze two or more fluorescence signals. Summary of the Invention

[0006] Based on this, the present invention proposes a dual-color laser alternating excitation single-molecule imaging detection device, its usage method, and its application, which can realize real-time detection and quantitative characterization of the dynamics of two interacting molecules or the pairing and separation of the two complementary strands of a double-stranded DNA molecule at the single-molecule level.

[0007] According to a first aspect of the present invention, a dual-color laser alternating excitation single-molecule imaging detection device is provided, comprising: a dual-color laser alternating exciter for exciting two beams of first and second excitation light of different wavelengths as excitation sources; an acousto-optic tunable filter adapted to control the alternating excitation of the two beams of first and second excitation light of different wavelengths to form an alternating laser and couple the alternating laser to an optical fiber; and a sample platform including a microfluidic chamber for placing a first target sample and a second target sample, wherein the first target sample is labeled with a first fluorescent dye and the second target sample is labeled with a second fluorescent dye, wherein during irradiation by the first excitation light, the first fluorescent dye is excited, such that the first target sample is labeled with a second fluorescent dye. A fluorescent dye emits a first fluorescent signal, and when the distance between the first fluorescent dye and the second fluorescent dye satisfies the resonance energy transfer condition, a fluorescence resonance energy transfer signal is generated. During the second excitation light irradiation, the second fluorescent dye is excited, causing the second fluorescent dye to emit a second fluorescent signal. A fluorescence detection component is used to receive the fluorescence resonance energy transfer signal, the first fluorescent signal, and the second fluorescent signal, and to sense and form a target image through an electron multiplication inductively coupled device. The alternating laser on the object platform utilizes total internal reflection to form an evanescent field to reduce background light interference.

[0008] According to an embodiment of the present invention, the microfluidic chamber includes: a chamber for sample retention composed of a glass slide and a PEG-modified coverslip, wherein the height of the microfluidic chamber is the height of the adhesive bonding structure; wherein the glass slide includes an inlet port and an outlet port, and the outlet port is connected to a syringe via a conduit.

[0009] According to an embodiment of the present invention, the microfluidic chamber includes: a target molecule, comprising a probe molecule labeled with a first fluorescent dye and a target molecule labeled with a second fluorescent dye; an imaging buffer for introducing the target molecule, and further comprising an oxygen removal system and a reducing agent for inhibiting triplet scintillation of fluorescent groups and photobleaching, wherein the components of the imaging buffer include D-glucose, glucose oxidase, catalase and water-soluble vitamin E.

[0010] According to an embodiment of the present invention, the device further includes an inverted fluorescence microscope disposed below the object platform, used to focus the alternating laser onto the coverslip of the object platform, or to extract the fluorescence resonance energy transfer signal, the first fluorescence signal, and the second fluorescence signal from the object platform; and an illuminator connected to the acousto-optic tunable filter via an optical fiber, used to receive the alternating laser from the acousto-optic tunable filter and to emit the alternating laser to excite the first fluorescent dye or the second fluorescent dye.

[0011] According to an embodiment of the present invention, the above-mentioned dual-color laser alternating exciter includes: an excitation light source for emitting two laser beams of different wavelengths; a reflector and a dichroic mirror for reflecting the laser emitted by the laser source into the optical path; and a lens for focusing the two laser beams onto the acousto-optic tunable filter.

[0012] According to an embodiment of the present invention, the fluorescence detection component includes: a dichroic mirror for separating the fluorescence resonance energy transfer signal to obtain two different wavelengths of fluorescence laser; and two single-band filters arranged in parallel between the electron multiplication inductively coupled device and the dichroic mirror for filtering noise from the fluorescence laser.

[0013] According to a second aspect of the present invention, an application is provided of the above-described dual-color laser alternating excitation single-molecule imaging detection device in identifying the capture and initiation of target molecules during biological chain exchange.

[0014] According to embodiments of the present invention, applications include: detection of strand separation of double-stranded DNA or RNA molecules; detection of recombination reactions between a single-stranded DNA molecule and the complementary strand of a double-stranded DNA molecule; detection of recombination reactions between a single-stranded RNA molecule and the complementary strand of a double-stranded DNA molecule; accurate identification of processes such as DNA strand capture, reaction initiation, and termination in recombination reaction detection or strand separation detection; detection of hybridization reactions between two single-stranded DNA or single-stranded RNA molecules; detection of hybridization reactions between a single-stranded RNA molecule and a single-stranded DNA molecule; real-time dynamic analysis and detection of the conformation and folding of single-stranded RNA or DNA molecules; conformation and dynamic analysis of single nucleic acid aptamer molecules; dynamic analysis of the binding of single nucleic acid aptamer molecules to target proteins; high-sensitivity detection of proteins and other target molecules using nucleic acid aptamer molecules as affinity ligands and single-molecule signal transduction; or high-sensitivity detection of other molecules, such as proteins, DNA, RNA, and various small molecule compounds, using antibodies and other affinity proteins as ligands.

[0015] According to a third aspect of the present invention, a method for using the above-described dual-color laser alternating excitation single-molecule imaging detection device is provided, comprising: exciting two laser beams of different wavelengths as excitation light sources using a dual-color laser alternating exciter; forming alternating laser beams for exciting target molecules using an acousto-optic tunable filter; receiving the alternating laser beams from the acousto-optic tunable filter via an optical fiber; emitting the alternating laser beams to excite a first fluorescent dye or a second fluorescent dye in a carrier platform using an illuminator; the alternating laser beams undergo total internal reflection on the carrier platform to form an evanescent field on the carrier platform, exciting the first fluorescent dye or the second fluorescent dye, such that a fluorescence resonance energy transfer signal is formed between the first fluorescent dye and the second fluorescent dye, or the first fluorescent dye excites a first fluorescence signal, or the second fluorescent dye excites a second fluorescence signal; receiving the fluorescence resonance energy transfer signal, the first fluorescence signal, and the second fluorescence signal via a fluorescence detection component; and sensing and forming a target image using an electron multiplication inductively coupled plasma device.

[0016] According to an embodiment of the present invention, receiving the fluorescence resonance energy transfer signal by the fluorescence detection component includes: separating the two fluorescence resonance energy transfer signals by a dichroic mirror; and filtering noise in the fluorescence resonance energy transfer signal by a parallel single-band filter.

[0017] As can be seen from the above technical solutions, the dual-color laser alternating excitation single-molecule imaging detection device, its usage method, and its application provided by the present invention have the following beneficial effects:

[0018] The dual-color laser alternating excitation single-molecule imaging detection device provided by this invention enables real-time detection and quantitative characterization of the dynamics of two interacting molecules or the pairing and separation of the two complementary strands of a double-stranded DNA molecule at the single-molecule level. It has been successfully applied to real-time analysis of strand exchange in homologous recombination processes and can eliminate false positive results in DNA strand exchange events. Furthermore, this device improves the accuracy of single-molecule fluorescence resonance energy transfer (smFRET) measurement and can identify extremely low smFRET states. It can be used for bioanalysis, detection, and imaging techniques in important biological processes such as homologous recombination, as well as for single-molecule dynamic studies. Attached Figure Description

[0019] The above and other objects, features and advantages of this application will become clearer from the following description of embodiments with reference to the accompanying drawings, in which:

[0020] Figure 1 This is a schematic diagram of the structure of the dual-color laser alternating excitation single-molecule imaging detection device according to an embodiment of the present invention;

[0021] Figure 2 This is a schematic diagram of the microfluidic chamber according to an embodiment of the present invention;

[0022] Figure 3 This is a flowchart of the dual-color laser alternating excitation single-molecule imaging detection device used in the embodiments of the present invention;

[0023] Figure 4 This is a schematic diagram of the strand exchange reaction mediated by RecA between a single-stranded DNA molecule and a homologous double-stranded DNA molecule in Example 1 of the present invention.

[0024] Figure 5 The signal curve of the DNA strand exchange reaction in Example 1 is identified using the detection device of this embodiment of the invention;

[0025] Figure 6 The signal curve diagram for identifying the release of ATP hydrolysis-promoted chain exchange reaction products in Example 1 using the detection device of this embodiment of the invention;

[0026] Figure 7 To observe in real time the graphs of strand exchange reactions mediated by ssDNA of different lengths in Example 2 using the detection device of this embodiment;

[0027] Figure 8 The detection device of this embodiment of the invention is used to characterize the strand exchange reaction efficiency of ssDNA of different lengths in Example 2.

[0028] In the picture:

[0029] Acousto-optic tunable filter-1;

[0030] Microfluidic chamber-2 on the platform;

[0031] Slide-21;

[0032] Coverslip -22;

[0033] Double-sided adhesive connection structure - 23;

[0034] Dual-color laser alternating exciter-3;

[0035] Mirror-31;

[0036] Dichroic mirror -32;

[0037] Lens-33;

[0038] Fluorescence detection component-4;

[0039] Mirror-41;

[0040] Single-band filter -42;

[0041] Electron multiplier inductively coupled plasma device-43;

[0042] illuminator-5;

[0043] Inverted fluorescence microscope-6. Detailed Implementation

[0044] The embodiments of this application will now 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 this application. In the following detailed description, numerous specific details are set forth to provide a thorough understanding of the embodiments of this application for ease of explanation. However, it will be apparent that one or more embodiments may be implemented without these specific details. Furthermore, descriptions of well-known structures and technologies are omitted in the following description to avoid unnecessarily obscuring the concepts of this application.

[0045] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. The terms “comprising,” “including,” etc., as used herein indicate the presence of the stated features, steps, operations, and / or components, but do not exclude the presence or addition of one or more other features, steps, operations, or components.

[0046] All terms used herein (including technical and scientific terms) have the meanings commonly understood by those skilled in the art, unless otherwise defined. It should be noted that the terms used herein are to be interpreted in a manner consistent with the context of this specification, and not in an idealized or overly rigid way.

[0047] When using expressions such as "at least one of A, B and C", they should generally be interpreted in accordance with the meaning that is commonly understood by those skilled in the art (e.g., "a system having at least one of A, B and C" should include, but is not limited to, a system having A alone, a system having B alone, a system having C alone, a system having A and B, a system having A and C, a system having B and C, and / or a system having A, B and C, etc.).

[0048] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0049] Homologous recombination is catalyzed by a series of conserved homologous recombination enzymes, including T4 UvsX in bacteriophages, RadA in archaea, RecA in bacteria, and Rad51 and DMC1 in eukaryotes. In eukaryotes, Rad51 is mainly responsible for homologous recombination repair of DNA double-strand breaks, while DMC1 specifically catalyzes homologous recombination during meiosis. Homologous recombination involves numerous steps, requires the participation of various protein machinery, and is subject to fine regulation. Its biochemical process can be roughly divided into the following four stages: initiation stage, homologous pairing stage, new strand synthesis stage, and separation stage. In the initiation stage, after the double-strand break occurs, various helicases and nucleases act on the ends of the double-strand break, specifically cleaving double-stranded DNA (dsDNA) to produce single-stranded DNA (ssDNA) with a 3' protrusion, which is protected by single-stranded DNA-binding proteins. In the homologous pairing stage, homologous recombination enzymes, with the support of some helper proteins, first replace the protein molecules bound to ssDNA, and then co-assemble in the ssDNA region to form nucleoprotein filaments. The resulting nucleoprotein filaments can seek out and invade homologous dsDNA regions, undergoing strand exchange reactions to form triple-stranded molecules. During the new strand synthesis stage, DNA polymerase uses the intact homologous DNA strand as a template to synthesize and extend the DNA strand at the 3' end of the broken strand, forming a new structure, the Holliday crossover. In the final separation stage, resolvases or nucleases untie the Holliday crossover, forming two independent dsDNA molecules. Thus, homologous recombination is complete. Homologous recombination is the molecular basis of the origin of life, biodiversity, and the development of diseases; the study of its intricate processes is at the forefront of life sciences today. Developing new optical analysis and research methods is key to answering important scientific questions about homologous recombination.

[0050] Because traditional analytical techniques obtain information based on the average of all molecular measurements, a large amount of specific individual information is masked, making it difficult to reveal many complex biological processes such as homologous recombination. In fundamental life processes, many biomolecules perform their specific functions as single molecules or in groups of several. Therefore, observing the structure and behavior of target molecules at the single-molecule level is crucial for our understanding of fundamental life processes. Currently, various single-molecule measurement techniques have been developed, which can be broadly classified into two categories: one is imaging techniques based on fluorescence and its spectroscopy, mainly including single-molecule fluorescence imaging (SFI) and single-molecule fluorescence resonance energy transfer (SmFRET); the other is manipulation and detection techniques based on mechanics, mainly including optical tweezers, magnetic tweezers, and atomic force microscopy.

[0051] Optical microscopes are indispensable research tools in the biomedical field, playing a crucial role in both basic research and clinical diagnosis. Through microscopic imaging, we can obtain information not only about the presence and location of target molecules, but also, because the emission wavelength of fluorescence is longer than its absorption wavelength, background interference can be significantly reduced through proper optical path design, making it the optimal choice for single-molecule imaging.

[0052] When the emission spectrum of a fluorescent molecule (donor molecule) overlaps with the excitation spectrum of another fluorescent molecule (acceptor molecule), and their spatial distance is relatively short (<10 nm), the emission energy of the donor molecule can excite the acceptor molecule to emit fluorescence (at a longer wavelength), while the fluorescence intensity emitted by the acceptor molecule itself decreases. This phenomenon is called fluorescence resonance energy transfer (FRET). The efficiency of FRET is closely related to the spatial distance between the donor and acceptor molecules, thus FRET can be used to characterize structural changes of the same molecule or interactions between different molecules. Ha et al. extended the traditional whole-molecule FRET technique to the single-molecule level, developing single-molecule FRET (smFRET) technology, which makes it possible to observe the conformational changes and motion of individual molecules in real time. Compared with whole-molecule FRET measurements, smFRET technology can not only observe the heterogeneity between molecules and determine the distribution of multiple conformations, but also observe some transient intermediate states and unstable states, and measure the kinetic parameters of biochemical reactions in real time.

[0053] Currently, optical microscopes are not specifically designed for real-time observation of conformational changes and motion of individual molecules; other specialized microscopes are typically used for observation. They cannot accurately measure transient intermediate or unstable states, nor can they simultaneously acquire and analyze two or more fluorescence signals. Therefore, an imaging and detection device specifically designed for single-molecule FRET (smFRET) technology is needed.

[0054] According to the general inventive concept of the first aspect of the present invention, a dual-color laser alternating excitation single-molecule imaging detection device is provided, comprising a dual-color laser alternating exciter, an acousto-optic tunable filter 1, a material platform 2, and a fluorescence detection component.

[0055] A dual-color laser alternating exciter excites two beams of first and second excitation light of different wavelengths as excitation sources. An acousto-optic tunable filter 1 controls the alternating excitation of the two beams to form an alternating laser, and couples the alternating laser into an optical fiber. A platform 2 holds a first target sample and a second target sample. The first target sample is marked with a first fluorescent dye, and the second target sample is marked with a second fluorescent dye. During the first excitation light irradiation, the first fluorescent dye is excited, emitting a first fluorescence signal. When the distance between the first and second fluorescent dyes satisfies the resonance energy transfer condition, a fluorescence resonance energy transfer signal is generated. During the second excitation light irradiation, the second fluorescent dye is excited, emitting a second fluorescence signal. A fluorescence detection component receives the first fluorescence signal, the fluorescence resonance energy transfer signal, and the second fluorescence signal, and senses and forms a target image through an electron-multiplying inductively coupled device 43. The alternating laser on the platform 2 forms an evanescent field using total internal reflection technology to reduce background light interference. The fluorescence detection component is used to receive the first fluorescence signal, the fluorescence resonance energy transfer signal and the second fluorescence signal generated after the target molecule is excited, and to form a target image through the electron multiplication inductive coupling device 43.

[0056] According to an embodiment of the present invention, the laser can be selected from two wavelengths, 532 nm and 647 nm, which can excite two fluorescent groups in the target molecule respectively. For example, the 532 nm laser excites the first fluorescent dye Cy3, and the 647 nm laser excites the second fluorescent dye Cy5. When the spatial distance between Cy3 and Cy5 in the target molecule is less than 10 nm, fluorescence resonance energy transfer will occur, that is, the fluorescence generated by the 532 nm laser excitation of Cy3 can be absorbed by Cy5 to generate the second fluorescence.

[0057] Taking DNA strand exchange reaction as an example. The first (donor) fluorescent dye is Cy3, and the second (acceptor) fluorescent dye is Cy5, which are labeled on the two different strands of the donor dsDNA, respectively; the two alternating laser beams are a 532 nm laser and a 647 nm laser, respectively; the 532 nm laser is used to excite Cy3, and the 647 nm laser is used to excite Cy5.

[0058] When observing the DNA strand exchange reaction in real time, the capture process of the donor dsDNA is observed first. When the donor dsDNA is captured by the invading ssDNA, and the alternating laser is at 532 nm at the current moment, the distance between Cy3 and Cy5 labeled on the donor dsDNA is close enough to satisfy the fluorescence resonance energy transfer condition. At this time, the fluorescence signal of Cy3 excited by the 532 nm laser and the fluorescence signal of Cy5 excited by fluorescence resonance energy transfer can be observed simultaneously, that is, a fluorescence resonance energy transfer signal is formed between Cy3 and Cy5.

[0059] A successful strand exchange reaction releases the Cy3-labeled strand from the donor dsDNA. The resulting dsDNA contains no Cy3 label but is labeled with Cy5. Under 532 nm laser irradiation, no Cy3 fluorescence signal is emitted, and therefore no Cy5 fluorescence signal is generated via fluorescence resonance energy transfer (FRET), resulting in the simultaneous disappearance of both Cy3 and Cy5 fluorescence signals. Therefore, the residence time of the Cy3 and Cy5 fluorescence signals can be used to characterize the time required for strand exchange to occur.

[0060] However, a successful strand exchange reaction does not cause the disappearance of the Cy5-labeled complementary strand in the resulting dsDNA. If excited only by a single-wavelength 532 nm laser, the Cy5-labeled complementary strand will not emit a fluorescent signal, making it impossible to determine whether the complementary strand disappeared along with the homologous donor dsDNA or whether strand exchange occurred. In this case, by switching the excitation wavelength to 647 nm to directly excite Cy5 and obtaining a separate second fluorescent signal, it is easy to determine whether the Cy5-labeled complementary strand has disappeared.

[0061] Although both the primary fluorescence signal and the corresponding fluorescence resonance energy transfer signal corresponding to Cy3 disappeared, two different types of molecular events could be observed using a two-color alternating laser excitation strategy. In the first type of event, the secondary fluorescence signal corresponding to Cy5 excited by a 647 nm laser and the primary fluorescence signal corresponding to Cy3 excited by a 532 nm laser both disappeared, indicating that the donor dsDNA was briefly captured by the invading ssDNA and then released, without a strand exchange reaction. In the second type of event, it could be observed that once the donor DNA was captured, Cy5 could be directly excited by a 647 nm laser to form a secondary fluorescence signal, marking the successful pairing of the complementary strand and the invading strand, i.e., the successful occurrence of the strand exchange reaction.

[0062] In summary, the observations of both the brief capture and subsequent release of donor dsDNA by invading ssDNA under 532 nm laser irradiation alone and the successful strand exchange reaction are consistent. Therefore, 532 nm laser irradiation alone cannot distinguish false positives in cases where donor dsDNA is briefly captured and then released by invading ssDNA. To address this, a dual-color alternating excitation strategy is needed. A 647 nm laser is introduced to excite Cy5, and the presence of the Cy5-labeled strand is identified by the second fluorescence signal. If the Cy5-labeled strand is present, the strand exchange reaction has occurred successfully; if the Cy5-labeled strand is absent, it is a false positive. This dual-color alternating excitation strategy can clearly distinguish between successful strand exchange reactions and false positive interference.

[0063] Furthermore, it should be noted that if a parallel excitation strategy is used instead of a dual-color alternating excitation strategy, i.e., simultaneously generating and irradiating with 532 nm and 647 nm lasers, a portion of Cy5 will be directly excited by the 647 nm laser during the chain exchange reaction, while a portion of Cy5 will be excited by the fluorescence resonance energy transfer effect of Cy3, making it impossible to clearly distinguish between them.

[0064] Therefore, the aforementioned dual-color alternating excitation strategy is a key technical means implemented to avoid false positive interference, rather than a simple timing control operation.

[0065] The dual-color laser alternating excitation single-molecule imaging detection device provided by this invention enables real-time detection and quantitative characterization of the dynamics of two interacting molecules or the pairing and separation of the two complementary strands of a double-stranded DNA molecule at the single-molecule level. It has been successfully applied to real-time analysis of strand exchange in homologous recombination processes and can eliminate false positive results in DNA strand exchange events. Furthermore, this device improves the accuracy of single-molecule fluorescence resonance energy transfer (smFRET) measurement and can identify extremely low smFRET states. It can be used for bioanalysis, detection, and imaging techniques in important biological processes such as homologous recombination, as well as for single-molecule dynamic studies.

[0066] According to an embodiment of the present invention, the operation of the dual-color laser alternating exciter, the acousto-optic tunable filter 1, the illuminator 5, and the fluorescence detection component can be controlled using Micro-Manager-1.4 software installed in a computer.

[0067] Figure 1 This is a schematic diagram of the structure of the dual-color laser alternating excitation single-molecule imaging detection device according to an embodiment of the present invention.

[0068] According to embodiments of the present invention, such as Figure 1As shown, the detection device also includes an inverted fluorescence microscope 6, which is located below the platform 2 and is used to focus alternating lasers onto the coverslip 22 of the platform 2, or to extract fluorescence resonance energy transfer signals from the platform 2.

[0069] According to an embodiment of the present invention, the detection device further includes an illuminator 5, which is connected to an acousto-optic tunable filter 1 via an optical fiber, for receiving alternating lasers from the acousto-optic tunable filter 1, and for achieving total internal reflection excitation of target molecules fixed within approximately 200 nm on the surface of the cover glass by adjusting the laser incident angle.

[0070] According to an embodiment of the present invention, a dual-color laser alternating exciter includes: a laser source, a reflector 31, a dichroic mirror 32, and a lens 33. The laser source emits two laser beams of different wavelengths. The reflector 31 and the dichroic mirror 32 reflect the laser beams emitted by the laser source into the optical path. The lens 33 focuses the two laser beams onto an acousto-optic tunable filter 1.

[0071] According to an embodiment of the present invention, an acousto-optic deflector can also be provided in the optical path, which allows two laser beams to pass through the device alternately, thereby realizing the alternating excitation of target molecules by two laser beams of different wavelengths.

[0072] According to an embodiment of the present invention, the fluorescence detection component includes a dichroic mirror 41 and two single-band filters 42. The dichroic mirror 41 is used to separate the fluorescence resonance energy transfer signal to obtain two different wavelengths of fluorescence laser. The two single-band filters 42 are arranged in parallel between the electron multiplication inductively coupled device 43 and the dichroic mirror 42 to filter fluorescence noise.

[0073] In the dual-color laser alternating excitation single-molecule imaging detection device, two laser beams of different wavelengths (532 nm and 647 nm) are coupled to an acousto-optical tunable filter (AOTF). The AOTF allows for alternating excitation of different wavelengths. The laser coupled to the AOTF only needs to be connected to the optical path of an objective-type total internal reflection fluorescent (TIRF) microscope via an optical fiber and an illuminator 5. The alternatingly excited lasers are focused onto a coverslip 22 in a microfluidic chamber by the TIRF illuminator 5 and the objective lens of the inverted fluorescence microscope. The angle of laser illumination is adjusted by the TIRF illuminator 5, and an evanescent field is generated on the other side of the medium after total internal reflection. Due to the excitation light decaying exponentially, only fluorescent molecules below 200 nm in the sample region very close to the total internal reflection surface are excited to produce fluorescence, which greatly reduces background interference and improves the resolution of single-molecule detection. The fluorescence signal generated by the sample fixed on the surface of the coverslip 22 is first separated by a dichroic mirror, and then filtered by two parallel bandpass filters. The fluorescence signal is then acquired by an electron multiplying charge coupled device (EMCCD) 43 and recorded on a computer.

[0074] Figure 2 This is a schematic diagram of the microfluidic chamber in an embodiment of the present invention.

[0075] According to embodiments of the present invention, such as Figure 2 As shown, the microfluidic chamber includes a sample retention chamber composed of a glass slide 21 and a PEG-modified coverslip 22, and the height of the microfluidic chamber is the same as the height of the adhesive bonding structure 23.

[0076] According to an embodiment of the present invention, the slide 21 includes an inlet port and an outlet port, and the outlet port is connected to a syringe via a conduit.

[0077] According to embodiments of the present invention, the adhesive bonding structure can be double-sided tape, adhesive, etc.

[0078] The microfluidic chamber allows for the introduction of target molecules through a solution, enabling real-time observation of the interaction between probe and target molecules. The double-sided adhesive tape binds the PEG-modified coverslip 22 and slide 21 together, providing a reaction chamber. PEG is neutral and biocompatible; the modification aims to passivate the slide surface, reducing the adsorption of impurities such as biomolecules, minimizing interference with single-molecule detection, and improving the detection signal.

[0079] According to an embodiment of the present invention, the microfluidic chamber includes a target molecule and an imaging buffer.

[0080] The target molecules include probe molecules and target molecules labeled with two different fluorescent dyes.

[0081] The imaging buffer is used to introduce target molecules and also consists of a deoxygenation system and a reducing agent. Its components include D-glucose, glucose oxidase, catalase, and water-soluble vitamin E.

[0082] According to embodiments of the present invention, preferably, the imaging buffer may include D-glucose (8 mg / mL), glucose oxidase (1 mg / mL), catalase (0.04 mg / mL) and water-soluble vitamin E (>3 mM).

[0083] According to the general inventive concept of the second aspect of the present invention, an application of a dual-color laser alternating excitation single-molecule imaging detection device is provided in identifying the capture and initiation of target molecules during biological chain exchange.

[0084] According to embodiments of the present invention, applications include: detection of strand separation of double-stranded DNA or RNA molecules; detection of recombination reactions between a single-stranded DNA molecule and the complementary strand of a double-stranded DNA molecule; detection of recombination reactions between a single-stranded RNA molecule and the complementary strand of a double-stranded DNA molecule; accurate identification of processes such as DNA strand capture, reaction initiation, and termination in recombination reaction detection or strand separation detection; detection of hybridization reactions between two single-stranded DNA or single-stranded RNA molecules; detection of hybridization reactions between a single-stranded RNA molecule and a single-stranded DNA molecule; real-time dynamic analysis and detection of the conformation and folding of single-stranded RNA or DNA molecules; conformation and dynamic analysis of single nucleic acid aptamer molecules; dynamic analysis of the binding of single nucleic acid aptamer molecules to target proteins; high-sensitivity detection of proteins and other target molecules using nucleic acid aptamer molecules as affinity ligands and single-molecule signal transduction; or high-sensitivity detection of other molecules, such as proteins, DNA, RNA, and various small molecule compounds, using antibodies and other affinity proteins as ligands.

[0085] The dual-color laser alternating excitation single-molecule imaging detection device provided by this invention enables real-time detection and quantitative characterization of the dynamics of two interacting molecules or the pairing and separation of the two complementary strands of a double-stranded DNA molecule at the single-molecule level. It has been successfully applied to real-time analysis of strand exchange in homologous recombination processes and can eliminate false positive results in DNA strand exchange events. Furthermore, this device improves the accuracy of single-molecule fluorescence resonance energy transfer (smFRET) measurement and can identify extremely low smFRET states. It can be used for bioanalysis, detection, and imaging techniques in important biological processes such as homologous recombination, as well as for single-molecule dynamic studies.

[0086] The dual-color laser alternating excitation single-molecule imaging detection device provided by this invention can excite two fluorescent groups in a target molecule separately, such as 532 nm laser excitation of the first (donor) fluorescent dye Cy3 and 647 nm laser excitation of the second (acceptor) fluorescent dye Cy5; it can detect the fluorescence emitted by the two excited fluorescent groups at different wavelengths; and simultaneously detect the fluorescence resonance energy transfer (FRET) signal of the two fluorescent groups under 532 nm laser excitation, i.e., the fluorescence signal of Cy5; it can identify the occurrence, development and termination of biological processes such as chain exchange, such as the capture of target molecules and the initiation of reactions.

[0087] Figure 3 This is a flowchart of the dual-color laser alternating excitation single-molecule imaging detection device used in an embodiment of the present invention.

[0088] According to the general inventive concept of the third aspect of the present invention, such as Figure 3 As shown, a method for using a dual-color laser alternating excitation single-molecule imaging detection device is provided, including operations S1 to S7.

[0089] S1: Two beams of first and second excitation light of different wavelengths are excited by a dual-color laser alternating exciter as the excitation source.

[0090] S2: Two beams of first and second excitation light of different wavelengths are controlled by an acousto-optic tunable filter to alternately excite and form an alternating laser.

[0091] S3: Receive alternating laser light from the acousto-optic tunable filter via optical fiber.

[0092] S4: The alternating laser emitted by the illuminator excites either the first or second fluorescent dye in the carrier platform.

[0093] S5: After the alternating lasers undergo total internal reflection on the platform 2, an evanescent field is formed on the platform 2. During the first excitation light irradiation, the first fluorescent dye is excited, causing the first fluorescent dye to emit a first fluorescent signal. When the distance between the first fluorescent dye and the second fluorescent dye satisfies the resonance energy transfer condition, a fluorescence resonance energy transfer signal is generated. During the second excitation light irradiation, the second fluorescent dye is excited, causing the second fluorescent dye to emit a second fluorescent signal.

[0094] S6: Receives the first fluorescence signal, the fluorescence resonance energy transfer signal, and the second fluorescence signal through the fluorescence detection component.

[0095] S7: A target image is formed by sensing fluorescence signals through an electron multiplier inductively coupled device 43.

[0096] Two-color alternating excitation (TCAE) single-molecule fluorescence imaging technology has been successfully applied to the real-time dynamic monitoring of DNA strand exchange reactions. By labeling the two strands of donor dsDNA with Cy3 and Cy5, respectively, and alternating excitation at 532 nm and 647 nm, the entire strand exchange process can be observed in real time, from the initial donor capture to the final release of the replaced strand. This alternating excitation mode allows for the identification of successful strand exchange events from false positive signals at the single-molecule level.

[0097] According to an embodiment of the present invention, receiving the fluorescence resonance energy transfer signal through the fluorescence detection component includes operations S701 to S702.

[0098] S701: Separates two fluorescence signals, donor fluorescence and acceptor fluorescence generated through fluorescence resonance energy transfer, using a dichroic mirror.

[0099] S702: Filters noise in the fluorescence resonance energy transfer signal by using parallel single-band filters.

[0100] The technical solution of the present invention will be described in detail below through preferred embodiments. It should be noted that the specific embodiments in the following text are for illustrative purposes only and are not intended to limit the present invention.

[0101] Example 1:

[0102] Figure 4 This is a schematic diagram of the strand exchange reaction between a single-stranded DNA (ssDNA) molecule and a homologous double-stranded DNA (dsDNA) molecule in Example 1 of the present invention.

[0103] The dual-color laser alternating excitation single-molecule imaging detection device provided in this embodiment of the invention is used to identify strand exchange reactions between single-stranded DNA molecules and homologous double-stranded DNA molecules.

[0104] 1. Preparation of the target sample within the microfluidic chamber

[0105] A chamber for sample retention is formed by bonding a glass slide and a PEG-modified coverslip together with double-sided adhesive.

[0106] An imaging buffer containing the target molecule is added into the chamber.

[0107] The imaging buffer consisted of D-glucose (8 mg / mL), glucose oxidase (1 mg / mL), catalase (0.04 mg / mL), and water-soluble vitamin E (>3 mM).

[0108] The target molecules include single-stranded DNA molecules of the same length (ssDNA) and homologous double-stranded DNA (dsDNA).

[0109] The two segments of the double-stranded DNA (dsDNA) were labeled with fluorescent dyes Cy3 and Cy5, respectively.

[0110] The imaging buffer also includes a homologous recombinase (RecA) for mediating strand exchange between single-stranded DNA molecules (ssDNA) and homologous double-stranded DNA (dsDNA).

[0111] The reaction process includes: the homologous recombinase RecA assembling on ssDNA to form nucleoprotein filaments;

[0112] Nucleoprotein filaments bind to homologous dsDNA to temporarily form a triple-stranded complex;

[0113] The ssDNA of nucleoprotein filaments pairs with the complementary strand on the homologous dsDNA to form a new heterologous double-stranded DNA.

[0114] Meanwhile, another strand of homologous dsDNA, identical to that of a single-stranded DNA molecule (ssDNA), is replaced and released after a strand exchange reaction with the nucleoprotein filament.

[0115] 2. The chain exchange reaction process was characterized using this dual-color laser alternating excitation single-molecule imaging detection device.

[0116] The laser can be selected from two wavelengths, 532 nm and 647 nm, which can excite two fluorescent groups in the target molecule respectively. The 532 nm laser excites the fluorescent dye Cy3, and the 647 nm laser excites the fluorescent dye Cy5.

[0117] (1) Signal recognition during DNA strand exchange reaction

[0118] Figure 5 The signal curve of the DNA strand exchange reaction in Example 1 is used to identify the detection device of this embodiment of the invention.

[0119] The detection device of this invention identifies the signal curves when nucleoprotein filaments capture homologous dsDNA and temporarily form a triple-stranded complex during the DNA strand exchange reaction, as well as the signal curves after a successful DNA strand exchange reaction. The results are as follows: Figure 5 As shown.

[0120] Figure 5 (A) shows the signal curve obtained from the transient capture of homologous dsDNA. The arrow indicates the disappearance of the Cy5 signal, i.e., the signal change caused by the release of the complementary strand as the homologous dsDNA is released. Figure 5(B) shows the signal obtained from a successful DNA strand exchange reaction.

[0121] Therefore, the detection device provided by this invention can identify signal changes in the DNA strand exchange reaction process in real time.

[0122] (2) Signal recognition during ATP hydrolysis-promoted chain exchange reaction

[0123] Figure 6 The signal curve for identifying the release of ATP hydrolysis-promoted chain exchange reaction products in Example 1 using the detection device of this embodiment of the invention.

[0124] The detection device of this invention identifies signals in ATP hydrolysis-promoted chain exchange reactions and in processes where chain exchange reactions do not occur, and the results are as follows: Figure 6 As shown.

[0125] Figure 6 (A) shows a typical signal curve obtained under ATP hydrolysis conditions. The arrows indicate the release of the replaced strand. (B) shows a typical signal curve obtained in the absence of ATP hydrolysis. (C) shows a representative photograph (24 μm × 24 μm) of the Cy5 channel taken 10 min after strand exchange.

[0126] In the image with an excitation wavelength of 532 nm, the gray arrows indicate donor ds93 molecules that are trapped but have not undergone complete chain separation, where the Cy5 signal is excited at 532 nm.

[0127] In the image with an excitation wavelength of 647 nm, the solid arrows indicate the donor ds93 molecules that have been captured and successfully chain-separated, where the Cy5 signal can only be excited at 647 nm.

[0128] Figure 6 Figure (D) shows the statistical results of the number of Cy5 signals captured in three different imaging regions (82 μm × 41 μm). It can be seen that the detection device provided by the present invention can identify the signal changes in the process of ATP hydrolysis promoting chain exchange reaction in real time.

[0129] Example 2:

[0130] 1. Preparation of the target sample within the microfluidic chamber

[0131] The preparation process is the same as in Example 1, except that single-stranded DNA molecules (ssDNA) of different lengths and homologous double-stranded DNA (dsDNA) are used.

[0132] 2. The chain exchange reaction process was characterized using this dual-color laser alternating excitation single-molecule imaging detection device.

[0133] The laser can be selected from two wavelengths, 532 nm and 647 nm, which can excite two fluorescent groups in the target molecule respectively. The 532 nm laser excites the fluorescent dye Cy3, and the 647 nm laser excites the fluorescent dye Cy5.

[0134] (1) Signal recognition in strand exchange reactions mediated by ssDNA of different lengths

[0135] Figure 7 The detection device of this embodiment of the invention is used to observe the curves of strand exchange reactions mediated by ssDNA of different lengths in Example 2 in real time.

[0136] The detection device of this invention identifies signals in strand exchange reactions mediated by ssDNA of different lengths and in processes where strand exchange reactions do not occur, and the results are as follows: Figure 7 As shown.

[0137] Figure 7 (A) shows a schematic diagram of the experimental protocol for strand exchange reactions mediated by ssDNA of different lengths. (B–D) Representative fluorescence resonance energy transfer (FRET) signals and FRET efficiency time curves when the donor ds93 undergoes strand exchange with homologous ss93 (B), ss83 (C), or ss73 (D), respectively. The solid arrow on the left indicates the capture of the donor ds93, and the dashed arrow on the right indicates the release of the homologous strand. Δt represents the lifetime of the triplet synapse (i.e., the time required for strand exchange). (E) The time distribution required for strand exchange reactions mediated by ssDNA of different lengths. Therefore, the detection device provided by this invention can identify signal changes in the strand exchange reaction process mediated by ssDNA of different lengths in real time.

[0138] (2) Signal recognition during DNA strand exchange reaction

[0139] Figure 8 The detection device of this embodiment of the invention is used to characterize the strand exchange reaction efficiency of ssDNA of different lengths in Example 2.

[0140] Figure 8 Image (A) shows the Cy5 channel fluorescence image obtained after 10 min of chain exchange reaction (left two columns) or an additional 30 min of SacI cleavage (rightmost column). In the image at excitation wavelength of 532 nm, the gray arrows indicate donor ds93 molecules that are trapped but have not undergone complete chain separation, where the Cy5 signal is excited at 532 nm.

[0141] In the image at 647 nm, the solid arrows indicate the donor ds93 molecules that have been captured and successfully chain-separated, where the Cy5 signal can only be excited at 647 nm. Figure 8(B) shows the statistical results of the number of donor ds93 molecules captured using ssDNA of different lengths. The data represent the mean and standard deviation from six different imaging regions (82 μm × 41 μm).

[0142] Therefore, the detection device provided by this invention can identify signal changes in the DNA strand exchange reaction process in real time.

[0143] Those skilled in the art will understand that the features described in the various embodiments of this application can be combined and / or combined in various ways, even if such combinations or combinations are not explicitly described in this application. In particular, the features described in the various embodiments of this application can be combined and / or combined in various ways without departing from the spirit and teachings of this application. All such combinations and / or combinations fall within the scope of this application.

[0144] The embodiments of this application have been described above. However, these embodiments are merely illustrative and not intended to limit the scope of this application. Although various embodiments have been described above, this does not mean that the measures in the various embodiments cannot be used advantageously in combination. Without departing from the scope of this application, those skilled in the art can make various substitutions and modifications, all of which should fall within the scope of this application.

Claims

1. A dual-color laser alternating excitation single-molecule imaging detection device, characterized in that, include: A dual-color laser alternating exciter is used to use two beams of first and second excitation light of different wavelengths as excitation sources; An acousto-optic tunable filter is suitable for controlling two beams of first and second excitation light of different wavelengths to alternately excite and form an alternating laser, and to couple the alternating laser into an optical fiber. The platform includes a microfluidic chamber for placing a first target sample and a second target sample. The first target sample is labeled with a first fluorescent dye, and the second target sample is labeled with a second fluorescent dye. During the first excitation light irradiation, the first fluorescent dye is excited, causing it to emit a first fluorescent signal. When the distance between the first fluorescent dye and the second fluorescent dye satisfies the resonance energy transfer condition, a fluorescence resonance energy transfer signal is generated. During the second excitation light irradiation, the second fluorescent dye is excited, causing it to emit a second fluorescent signal. A fluorescence detection component is used to receive the first fluorescence signal, the fluorescence resonance energy transfer signal and the second fluorescence signal, and to form a target image by sensing through an electron multiplication inductively coupled device. The alternating laser utilizes total internal reflection on the sample platform to form an evanescent field that excites fluorescent molecules on the sample surface, thereby reducing background fluorescence interference and improving the signal-to-noise ratio.

2. The detection device according to claim 1, wherein, The microfluidic chamber includes: A chamber for sample retention, consisting of a glass slide and a PEG-modified coverslip, wherein the height of the microfluidic chamber is the same as the height of the adhesive bonding structure; The glass slide includes an inlet port and an outlet port, and the outlet port is connected to a syringe via a conduit.

3. The detection device according to claim 2, wherein, The microfluidic chamber includes: The target molecules include probe molecules labeled with a first fluorescent dye and target molecules labeled with a second fluorescent dye; An imaging buffer, used to introduce target molecules, comprises a deoxygenation system and a reducing agent. The components of the imaging buffer include D-glucose, glucose oxidase, catalase, and water-soluble vitamin E.

4. The detection device according to claim 2, wherein, Also includes: An inverted fluorescence microscope is disposed below the object platform and is used to focus the alternating laser onto the coverslip of the object platform, and to extract the first fluorescence signal and fluorescence resonance energy transfer signal generated by the first laser excitation and the second fluorescence signal generated by the second laser excitation from the object platform. An illuminator, connected to the acousto-optic tunable filter via an optical fiber, is used to receive alternating laser light from the acousto-optic tunable filter and to emit the alternating laser light to excite the first fluorescent dye and / or the second fluorescent dye.

5. The detection device according to claim 1, wherein, The dual-color laser alternating exciter includes: An excitation light source is used to emit two laser beams of different wavelengths; The reflector and dichroic mirror are used to reflect the laser emitted by the excitation source into the optical path; A lens is used to focus the two laser beams onto the acousto-optic tunable filter.

6. The detection device according to claim 1, wherein, The fluorescence detection component includes: A dichroic mirror is used to separate the first fluorescence signal from the second fluorescence signal generated by fluorescence resonance energy transfer or by direct excitation, so as to obtain two different wavelengths of fluorescence. Two single-band filters are arranged in parallel between the electron multiplier inductively coupled device and the dichroic mirror to filter noise in the fluorescence signal.

7. The application of the dual-color laser alternating excitation single-molecule imaging detection device according to any one of claims 1-6 in identifying the capture of target molecules and the initiation and termination of reactions during biological chain exchange.

8. The application of the detection device according to claim 7, wherein, Applications include the following: Detection of strand separation in double-stranded DNA or RNA molecules; Detection of recombination reactions between a single-stranded DNA molecule and the complementary strand of a double-stranded DNA molecule; Detection of recombination reaction between a single-stranded RNA molecule and the complementary strand of a double-stranded DNA; Accurate identification of DNA strand capture, reaction initiation and termination processes in recombinant reaction detection or strand separation detection; Detection of hybridization reactions between two single-stranded DNA molecules or single-stranded RNA molecules; Detection of hybridization reaction between a single-stranded RNA molecule and a single-stranded DNA molecule; Real-time dynamic analysis and detection of conformation and folding of single-stranded RNA or DNA molecules; Conformation and dynamic analysis of single nucleic acid aptamer molecules; Dynamic analysis of the binding of a single nucleic acid aptamer molecule to a target protein; Using nucleic acid aptamer molecules as affinity ligands and single-molecule signal transduction, highly sensitive detection of proteins and other target molecules is achieved; or Using antibodies and other affinity proteins as ligands, it can detect other molecules with high sensitivity, such as proteins, DNA, RNA, and various small molecule compounds.

9. A method for using the dual-color laser alternating excitation single-molecule imaging detection device according to any one of claims 1-6, comprising: Two beams of first and second excitation light of different wavelengths are excited by a dual-color laser alternating exciter as the excitation source. Alternating laser is formed by alternating excitation with two beams of first and second excitation light of different wavelengths controlled by an acousto-optic tunable filter. Alternating laser beams are received from the acousto-optic tunable filter via optical fiber; The alternating lasers emitted by the illuminator excite either the first or the second fluorescent dye in the carrier platform; The alternating laser forms an evanescent field after total internal reflection on the platform. During the first excitation light irradiation, the first fluorescent dye is excited, causing the first fluorescent dye to emit a first fluorescent signal. When the distance between the first fluorescent dye and the second fluorescent dye satisfies the resonance energy transfer condition, a fluorescence resonance energy transfer signal is generated. During the second excitation light irradiation, the second fluorescent dye is excited, causing the second fluorescent dye to emit a second fluorescent signal. The first fluorescence signal, the fluorescence resonance energy transfer signal, and the second fluorescence signal are received by a fluorescence detection component. The target image is formed by sensing through an electron multiplier inductively coupled device.

10. The method according to claim 9, wherein, The step of receiving the fluorescence resonance energy transfer signal through the fluorescence detection component includes: The two fluorescent resonance energy transfer signals were separated by a dichroic mirror; Noise in the fluorescence resonance energy transfer signal is filtered out by parallel single-band filters.

Images