CN116858812A - Dual-color laser alternate excitation single-molecule imaging detection device, use method and application - Google Patents
Dual-color laser alternate excitation single-molecule imaging detection device, use method and application Download PDFInfo
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Abstract
The invention discloses a double-color laser alternate excitation single-molecule imaging detection device, a use method and application thereof, and belongs to the field of biological analysis. The device comprises: the double-color light alternate exciter is used for exciting two laser beams with different wavelengths as excitation light sources; the acousto-optic tunable filter is suitable for controlling two beams of laser with different wavelengths to be alternately excited to form alternate laser and coupling the alternate laser into one optical fiber; the carrier platform is used for placing a sample containing target molecules marked by two different fluorescent dyes, and exciting the two different fluorescent dyes of the target molecules through the alternative laser to enable the fluorescent dyes to respond to emitting fluorescent groups, and the fluorescent groups are subjected to resonance transfer to form a fluorescence resonance energy transfer signal; the fluorescence detection component is used for receiving a fluorescence resonance energy transfer signal generated after the target molecule is excited, and forming a target image through induction of the electron multiplication inductive coupling device.
Description
Technical Field
The invention relates to the field of biological analysis, in particular to a double-color laser alternate excitation single-molecule imaging detection device, a use method and application.
Background
Homologous recombination is catalyzed by a series of conserved homologous recombinases, including phage T4UvsX, archaebacterial RadA, bacterial RecA, and eukaryotic Rad51 and DMC1, among others. In eukaryotes, rad51 is mainly responsible for the homologous recombination repair process of DNA double strand breaks, while DMC1 specifically catalyzes homologous recombination during meiosis. The homologous recombination step is numerous, needs participation of various protein machines and is finely regulated, and the biochemical process can be roughly divided into the following four stages: an initiation phase, a homologous pairing phase, a new strand synthesis phase and an isolation phase. Homologous recombination is the molecular basis for the development of life origin, biodiversity and disease occurrence, and research of various fine processes is the leading field of life sciences today. Development of new optical analysis and research means is key to answer important scientific questions in homologous recombination.
Since the information obtained by conventional analytical techniques is based on the average of all molecular measurements, a large amount of specific individual information is masked, which makes it difficult to reveal many complex biological processes such as homologous recombination. In the fundamental life process, many biological macromolecules perform their specific functions through single or several molecules. Thus, observing the structure and behavior of target molecules at the single molecule level is critical to our understanding of basic life processes.
Currently, a variety of single molecule measurement techniques have been developed, which can be broadly divided into two categories: imaging technology based on fluorescence and spectroscopy mainly comprises single-molecule fluorescence imaging technology (single-molecule fluorescence imaging) and single-molecule fluorescence resonance energy transfer technology (single-molecule fluorescence resonance energy transfer, smFRET); and secondly, the mechanical-based control and detection technology mainly comprises optical tweezers, magnetic tweezers, an atomic force microscope and the like.
Optical microscopy is an indispensable research tool in the biomedical field, and plays an important role in basic research and clinical diagnosis. Through microscopic imaging, not only can the existence and position information of the target molecule be obtained, but also the rotation and orientation of the target molecule can be deduced by utilizing the characteristics of polarization response and the like. Because the emission wavelength of fluorescence is longer than the absorption wavelength, the interference of the background can be greatly reduced through reasonable light path design, thereby becoming the optimal choice of single-molecule imaging. Currently, existing optical microscopes cannot accurately measure for observing transient intermediate or unstable states, and it is also difficult to obtain and analyze two or more fluorescent signals simultaneously.
Disclosure of Invention
Based on the method, the invention provides a double-color laser alternate excitation single-molecule imaging detection device, a use method and application, and real-time detection and quantitative characterization of the dynamic state of two interaction molecules or the pairing and separation of two complementary strands of a double-stranded DNA molecule can be realized at a single molecule level.
According to a first aspect of the present invention, there is provided a dual-colour laser alternately excited single-molecule imaging detection apparatus comprising:
the double-color light alternate exciter is used for exciting two laser beams with different wavelengths as excitation light sources;
the acousto-optic tunable filter is suitable for controlling two beams of laser with different wavelengths to be alternately excited to form alternate laser and coupling the alternate laser into one optical fiber;
an object carrying platform for placing a sample comprising target molecules labeled with two different fluorescent dyes, exciting the two different fluorescent dyes of the target molecules by the alternating laser light, causing the fluorescent dyes to respond with a fluorescent group, and forming a fluorescence resonance energy transfer signal by resonance transfer of the fluorescent group;
the fluorescence detection component is used for receiving fluorescence resonance energy transfer signals generated after the target molecules are excited and inducing the fluorescence resonance energy transfer signals to form a target image through the electron multiplication inductive coupling device;
wherein the alternating laser forms a recessed evanescent field on the loading platform to reduce background light noise interference.
According to an embodiment of the present invention, the microfluidic chamber includes:
a chamber for sample retention, which is formed by combining a glass slide and a PEG modified cover glass, wherein the height of the microfluidic chamber is the height of the connecting structure;
the slide glass comprises a sample inlet and an output hole, and the output hole is connected with a syringe through a catheter.
According to an embodiment of the present invention, the microfluidic chamber includes:
target molecules, including probe molecules and target molecules labeled with two different fluorescent dyes;
imaging buffer for introducing target molecule comprising deoxidizing system and reducer, wherein the imaging buffer comprises D-glucose, glucose oxidase, catalase and water-soluble vitamin E.
According to an embodiment of the present invention, the fluorescent microscope further comprises an inverted fluorescent microscope, wherein the inverted fluorescent microscope is disposed at the lower side of the carrying platform, and is used for focusing the alternative laser on a cover glass of the carrying platform, or extracting the fluorescent resonance energy transfer signal from the carrying platform;
and the illuminator is connected with the acousto-optic tunable filter through an optical fiber and is used for receiving the alternate laser from the acousto-optic tunable filter and emitting the alternate laser to excite the target molecules.
According to an embodiment of the present invention, the dual-color light alternate activator includes:
the laser source is used for emitting two beams of laser with different wavelengths;
a reflecting mirror and a dichroic mirror for reflecting the laser light emitted from the laser source into the optical path;
and a lens for focusing the two laser beams to the acousto-optic tunable filter.
According to an embodiment of the present invention, the fluorescence detection assembly includes:
a dichroic mirror for separating the fluorescence resonance energy transfer signals to obtain two fluorescence lasers with different wavelengths;
and the two single-band filters are arranged in parallel between the electron multiplication inductive coupling device and the dichroic mirror and are used for filtering noise of the fluorescent laser.
According to a second aspect of the present invention, there is provided the use of a dual-colour laser alternating excitation single-molecule imaging detection device as described above for the identification of the capture and initiation of a reaction of a target molecule during a biological chain exchange process.
According to an embodiment of the invention, applications include the following:
strand separation detection of double-stranded DNA molecules or RNA molecules;
detecting the recombination reaction of a single-stranded DNA molecule and a complementary strand in a double-stranded DNA;
detecting the recombination reaction of a single-stranded RNA molecule and a complementary strand in a double-stranded DNA;
the accurate identification of processes such as capturing of DNA chains, starting and ending of reactions and the like in recombination reaction detection or chain separation detection;
hybridization reaction detection between two single-stranded DNA molecules or single-stranded RNA molecules;
hybridization reaction detection between a single-stranded RNA molecule and a single-stranded DNA molecule;
the conformation and folding of single-stranded RNA molecules or DNA molecules are dynamically analyzed and detected in real time;
conformation and dynamic analysis of individual nucleic acid aptamer molecules;
dynamic analysis of binding of individual aptamer molecules to target proteins;
the nucleic acid aptamer molecules are used as affinity ligands and single molecule signal transduction, so that proteins and other target molecules are detected with high sensitivity; or (b)
Antibodies and other affinity proteins are used as ligands, and other molecules such as proteins, DNA, RNA and various small molecule compounds are detected with high sensitivity.
According to a third aspect of the present invention, there is provided a method for alternately exciting a single-molecule imaging detection apparatus using the above-described two-color laser, comprising:
exciting two laser beams with different wavelengths by a double-color light alternate exciter to serve as an excitation light source;
forming two beams of laser into alternate laser for exciting target molecules through an acousto-optic tunable filter;
receiving alternating laser light from the acousto-optic tunable filter through an optical fiber;
emitting the alternating laser to excite target molecules in the carrying platform through an illuminator;
after the alternating laser is totally reflected on the carrying platform, a hidden evanescent field is formed on the carrying platform, and two fluorescent dyes marked on the target molecule are excited to generate fluorescent groups;
fluorescent resonance energy transfer signals formed by resonance transfer of fluorescent groups emitted by the two fluorescent dyes;
receiving the fluorescence resonance energy transfer signal through a fluorescence detection assembly;
the target image is inductively formed by the electron multiplying inductive coupling device.
According to an embodiment of the present invention, the receiving the fluorescence resonance energy transfer signal by the fluorescence detection assembly includes:
separating the two fluorescence resonance energy transfer signals by a dichroic mirror;
noise in the fluorescence resonance energy transfer signal is filtered through a single-band filter arranged in parallel.
According to the technical scheme, the double-color laser alternately excited single-molecule imaging detection device, the use method and the application have the following beneficial effects:
the double-color laser alternate excitation single-molecule imaging detection device provided by the invention can realize real-time detection and quantitative characterization of the dynamic state of two interaction molecules or the pairing and separation of two complementary strands of a double-stranded DNA molecule on a single-molecule level. Strand-exchange real-time analysis, which has been successfully applied to homologous recombination processes, can exclude false positive results in DNA strand-exchange events. In addition, the device improves the accurate measurement of single molecule fluorescence resonance energy transfer (smFRET), and can identify extremely low smFRET states. Can be used for biological analysis, detection and imaging technology and single molecule dynamic research in important biological processes such as homologous recombination and the like.
Drawings
FIG. 1 is a schematic diagram of a dual-color laser alternate excitation single-molecule imaging detection device according to an embodiment of the present invention;
FIG. 2 is a schematic view of a microfluidic chamber according to an embodiment of the present invention;
FIG. 3 is a flow chart of a dual-color laser alternately excited single-molecule imaging detection apparatus used in an embodiment of the present invention;
FIG. 4 is a schematic diagram showing the strand-exchange reaction between a single-stranded DNA molecule and a homologous double-stranded DNA molecule according to example 1 of the present invention;
FIG. 5 is a graph showing signals of DNA strand exchange reaction in example 1 identified by the detection device according to the embodiment of the present invention;
FIG. 6 is a graph showing the signals of the detection device according to the embodiment of the present invention for identifying the release of ATP hydrolysis promoting strand exchange reaction products in example 1;
FIG. 7 is a graph of real-time observation of different length ssDNA mediated strand exchange reactions of example 2 using a detection apparatus according to an embodiment of the present invention;
FIG. 8 is a graph depicting the efficiency of various lengths of ssDNA mediated strand exchange reactions in example 2 using the detection apparatus of an embodiment of the present invention.
In the figure:
an acousto-optic tunable filter-1;
a carrying platform-2;
slide-21;
cover glass-22;
-a connection structure-23;
a bi-color light alternate exciter-3;
a mirror-31;
dichroic mirror-32;
a lens-33;
a fluorescence detection assembly-4;
dichroic mirror-41;
a single band filter-42;
electron multiplying inductive coupling device-43;
a luminaire-5;
inverted fluorescence microscope-6.
Detailed Description
The present invention will be further described in detail below with reference to specific embodiments and with reference to the accompanying drawings, in order to make the objects, technical solutions and advantages of the present invention more apparent.
Homologous recombination is catalyzed by a series of conserved homologous recombinases, including phage T4UvsX, archaebacterial RadA, bacterial RecA, and eukaryotic Rad51 and DMC1, among others. In eukaryotes, rad51 is mainly responsible for the homologous recombination repair process of DNA double strand breaks, while DMC1 specifically catalyzes homologous recombination during meiosis. The homologous recombination step is numerous, needs participation of various protein machines and is finely regulated, and the biochemical process can be roughly divided into the following four stages: an initiation phase, a homologous pairing phase, a new strand synthesis phase and an isolation phase. At the initial stage, after the double-strand break is generated, various helicases and nucleases act on the ends of the double-strand break, specifically cleave double-strand DNA (dsDNA), generate single-strand DNA (ssDNA) with 3' -end protruding, and are protected by single-strand DNA binding proteins. In the homologous pairing phase, homologous recombinases first replace protein molecules bound to ssDNA with the support of some auxiliary protein, and cooperatively assemble in the ssDNA region to form nucleoprotein filaments. The formed nucleoprotein filament is able to find and invade the homologous dsDNA region, and undergo a strand exchange reaction with it to form a triplex-linked molecule. In the new strand synthesis stage, the DNA polymerase synthesizes and extends the DNA strand at the 3' -end of the broken strand using the entire homologous DNA strand as a template, and forms a new structure, i.e., a holliday cross structure. In the final separation stage, a resolvase (resolvase) or nuclease breaks down the hollidae cross-structure to form two separate dsDNA molecules. Thus, homologous recombination is completed. Homologous recombination is the molecular basis for the development of life origin, biodiversity and disease occurrence, and research of various fine processes is the leading field of life sciences today. Development of new optical analysis and research means is key to answer important scientific questions in homologous recombination.
Since the information obtained by conventional analytical techniques is based on the average of all molecular measurements, a large amount of specific individual information is masked, which makes it difficult to reveal many complex biological processes such as homologous recombination. In the fundamental life process, many biological macromolecules perform their specific functions through single or several molecules. Thus, observing the structure and behavior of target molecules at the single molecule level is critical to our understanding of basic life processes. Currently, a variety of single molecule measurement techniques have been developed, which can be broadly divided into two categories: imaging technology based on fluorescence and spectroscopy mainly comprises single-molecule fluorescence imaging technology (single-molecule fluorescence imaging) and single-molecule fluorescence resonance energy transfer technology (single-molecule fluorescence resonance energy transfer, smFRET); and secondly, the mechanical-based control and detection technology mainly comprises optical tweezers, magnetic tweezers, an atomic force microscope and the like.
Optical microscopy is an indispensable research tool in the biomedical field, and plays an important role in basic research and clinical diagnosis. Through microscopic imaging, not only can the existence and position information of the target molecule be obtained, but also the rotation and orientation of the target molecule can be deduced by utilizing the characteristics of polarization response and the like. Because the emission wavelength of fluorescence is longer than the absorption wavelength, the interference of the background can be greatly reduced through reasonable light path design, thereby becoming the optimal choice of single-molecule imaging.
A "DNA curtains" technique has been developed that can simultaneously fix thousands of long fragments of DNA molecules on a phospholipid-modified slide surface, spread the DNA molecules with flowing liquid to form a DNA array, and then observe the effect of a target protein on a single DNA molecule in real time by a fluorescence microscope. To reduce the interference of background noise, only the fluorescence resonance energy transfer signal on the DNA molecule is observed, and the "DNA curtain" experiment is typically performed on a total internal reflection fluorescence microscope. When excitation light is totally reflected on the surface of the slide, an evanescent field is generated on the other surface, and the energy of the excitation light decays exponentially with the space distance, so that the excitation light can only excite fluorescence resonance energy transfer signals of DNA molecules in a very thin layer (< 200 nm) on the surface of the slide.
When the emission spectrum of one fluorescent molecule (donor molecule) overlaps with the excitation spectrum of another fluorescent molecule (acceptor molecule) and their spatial distance is close (< 10 nm), the emission energy of the donor molecule can excite the acceptor molecule to emit fluorescence (wavelength longer) while the fluorescence intensity of its own emission is reduced, a phenomenon called fluorescence resonance energy transfer (fluorescence resonance energy transfer, FRET). The efficiency of FRET is closely related to the spatial distance of the donor-acceptor molecule, and thus structural changes of the same molecule or interactions between different molecules can be characterized by FRET. Ha et al extended the traditional whole FRET technique to a single molecule level, developed a single molecule FRET (smFRET) technique, making it possible to observe conformational changes and movement of single molecules in real time. Compared with the whole FRET measurement, the smFRET technology can observe the heterogeneity among molecules, determine the distribution of various conformations, observe some transient intermediate states and unstable states and measure the kinetic parameters of biochemical reactions in real time.
Currently, the optical microscope is not specially used for observing the conformational change and the motion condition of a single molecule in real time, and other professional microscopes are generally used for observation in the observation process. It is also difficult to obtain and analyze two or more fluorescent signals simultaneously, and to make accurate measurements for the observation of transient intermediate or unstable conditions. There is therefore a need for an imaging detection device that is specific for single molecule FRET (smFRET) technology.
According to the first aspect of the present invention, there is provided a dual-color laser alternate excitation single-molecule imaging detection apparatus, comprising a dual-color optical alternate exciter, an acousto-optic tunable filter 1, a carrying platform 2 and a fluorescence detection assembly.
The double-color light alternate exciter is used for exciting two laser beams with different wavelengths as excitation light sources. The acousto-optic tunable filter 1 is suitable for controlling two laser beams with different wavelengths to be alternately excited to form alternate laser beams and coupling the alternate laser beams into one optical fiber. The carrying platform 2 is used for placing a sample containing target molecules marked by two different fluorescent dyes, and exciting the two different fluorescent dyes of the target molecules through alternative lasers, so that fluorescent dyes respond to emit fluorescent groups, and the fluorescent groups are subjected to resonance transfer to form a fluorescence resonance energy transfer signal. The fluorescence detection component is used for receiving a fluorescence resonance energy transfer signal generated after the target molecule is excited, and the fluorescence resonance energy transfer signal is induced to form a target image through the electron multiplication inductive coupling device 43. Wherein the alternating lasers form an evanescent field on the object carrying platform 2 to reduce background light noise interference. The fluorescence detection component is used for receiving a fluorescence resonance energy transfer signal generated after the target molecule is excited, and the fluorescence resonance energy transfer signal is induced to form a target image through the electron multiplication inductive coupling device 43.
According to the embodiment of the invention, the laser can select laser with two wavelengths of 532nm and 647nm, and can excite two fluorescent groups in target molecules simultaneously, such as 532nm laser excited fluorescent dye Cy3 and 647nm laser excited fluorescent dye Cy5.
The double-color laser alternate excitation single-molecule imaging detection device provided by the invention can realize real-time detection and quantitative characterization of the dynamic state of two interaction molecules or the pairing and separation of two complementary strands of a double-stranded DNA molecule on a single-molecule level. Strand-exchange real-time analysis, which has been successfully applied to homologous recombination processes, can exclude false positive results in DNA strand-exchange events. In addition, the device improves the accurate measurement of single molecule fluorescence resonance energy transfer (smFRET), and can identify extremely low smFRET states. Can be used for biological analysis, detection and imaging technology and single molecule dynamic research in important biological processes such as homologous recombination and the like.
According to an embodiment of the present invention, micro-Manager-1.4 software installed in a computer may be used to control the operation of the bi-color light alternate exciter, acousto-optic tunable filter 1, illuminator 5 and fluorescence detection assembly.
Fig. 1 is a schematic structural diagram of a dual-color laser alternating excitation single-molecule imaging detection device according to an embodiment of the invention.
According to an embodiment of the invention, as shown in fig. 1, the detection device further comprises an inverted fluorescence microscope 6, the inverted fluorescence microscope 6 being arranged at the underside of the carrier platform 2 for focusing the alternating laser light on the cover glass 22 of the carrier platform 2 or for extracting the fluorescence resonance energy transfer signal from the carrier platform 2.
According to an embodiment of the invention, the detection device further comprises an illuminator 5, the illuminator 5 being connected to the acousto-optic tunable filter 1 by an optical fiber for receiving the alternating laser light from the acousto-optic tunable filter 1 and emitting said alternating laser light for exciting the target molecule.
According to an embodiment of the present invention, a dual-color light alternate activator includes: a laser source, a mirror 31, a dichroic mirror 32 and a lens 33. And the laser source is used for emitting two laser beams with different wavelengths. A mirror 31 and a dichroic mirror 32 for reflecting laser light emitted from the laser light source into the optical path. And a lens 33 for focusing the two laser beams to the acousto-optic tunable filter 1.
According to the embodiment of the invention, the acousto-optic deflector can be arranged in the light path, so that two laser beams alternately pass through the device, and the two laser beams with different wavelengths alternately excite target molecules.
According to an embodiment of the present invention, a fluorescence detection assembly includes: a dichroic mirror 41 and two single band filters 42. A dichroic mirror 41 for separating the fluorescence resonance energy transfer signals to obtain two kinds of fluorescence laser light of different wavelengths. Two single-band filters 42 are disposed in parallel between the electron multiplying inductive coupling device 4343 and the dichroic mirror 32, for filtering noise of the fluorescent laser light.
In the double-color laser alternate excitation single-molecule imaging detection device, two beams of lasers (532 nm and 647 nm) with different wavelengths are coupled with an acousto-optic tunable filter 1 (AOTF), the lasers with different wavelengths can be controlled to be alternately excited through the AOTF, and the lasers after being coupled with the AOTF are only connected with an optical path of an objective type total internal reflection fluorescence (total internal reflection fluorescent, TIRF) microscope through an optical fiber and an illuminator 5. The alternately excited laser is focused to a cover glass 22 in a microfluidic chamber through a TIRF illuminator 5 and an objective lens of an inverted fluorescence microscope, the laser irradiation angle is regulated through the TIRF illuminator 5, an evanescent field is generated on the other side of the medium after the laser is totally reflected, and because the excitation light decays exponentially, only fluorescent molecules in a sample area very close to a total internal reflection surface are excited to generate fluorescence below 200nm, the background noise interference is greatly reduced, and the resolution of single molecule detection is improved. The fluorescent signals generated by excitation of the sample fixed on the surface of the cover glass 22 are first separated by a dichroic mirror 32, and the fluorescent signals filtered by two parallel bandpass filters are collected by an electron multiplying inductive coupling device 43 (electron multiplying charge coupled device, EMCCD) and recorded on a computer.
FIG. 2 is a schematic structural view of a microfluidic chamber according to an embodiment of the present invention.
According to an embodiment of the present invention, as shown in fig. 2, the microfluidic chamber includes: the chamber for sample retention, combined by slide 21 and PEG-modified cover slip 22, has a height of the microfluidic chamber that is the height of the connecting structure 23.
According to an embodiment of the present invention, the slide 21 includes a sample inlet and an outlet, the outlet being connected to a syringe via a conduit.
According to an embodiment of the present invention, the link structure may be a double sided tape, an adhesive, or the like.
The microfluidic chamber functions to introduce target molecules through a solution so as to observe information of interactions between probe molecules and target molecules in real time. The double sided tape serves to adhere the treated cover glass 22 and the slide glass 21 together to provide a reaction chamber. PEG is neutral and has good biocompatibility, the purpose of modification is to passivate the surface of a slide, reduce the adsorption of impurities such as biomolecules on the surface, reduce the interference on single molecule detection and improve detection signals.
According to an embodiment of the invention, the microfluidic chamber includes a target molecule and an imaging buffer.
The target molecules include probe molecules and target molecules labeled with two different fluorescent dyes.
The imaging buffer is used for introducing target molecules and is mainly composed of an oxygen removal system and a reducing agent, wherein the components comprise D-glucose, glucose oxidase, catalase and water-soluble vitamin E.
According to an embodiment of the 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).
According to a second aspect of the present general inventive concept, there is provided a dual-color laser alternating excitation single-molecule imaging detection apparatus for use in identifying capture of target molecules and initiation of reactions during a biological chain exchange process.
According to an embodiment of the invention, applications include, among others: strand separation detection of double-stranded DNA molecules or RNA molecules; detecting the recombination reaction of a single-stranded DNA molecule and a complementary strand in a double-stranded DNA; detecting the recombination reaction of a single-stranded RNA molecule and a complementary strand in a double-stranded DNA; the accurate identification of processes such as capturing of DNA chains, starting and ending of reactions and the like in recombination reaction detection or chain separation detection; hybridization reaction detection between two single-stranded DNA molecules or single-stranded RNA molecules; hybridization reaction detection between a single-stranded RNA molecule and a single-stranded DNA molecule; the conformation and folding of single-stranded RNA molecules or DNA molecules are dynamically analyzed and detected in real time; conformation and dynamic analysis of individual nucleic acid aptamer molecules; dynamic analysis of binding of individual aptamer molecules to target proteins; the nucleic acid aptamer molecules are used as affinity ligands and single molecule signal transduction, so that proteins and other target molecules are detected with high sensitivity; or antibodies and other affinity proteins are used as ligands, and other molecules such as proteins, DNA, RNA and various small molecule compounds are detected with high sensitivity.
The double-color laser alternate excitation single-molecule imaging detection device provided by the invention can realize real-time detection and quantitative characterization of the dynamic state of two interaction molecules or the pairing and separation of two complementary strands of a double-stranded DNA molecule on a single-molecule level. Strand-exchange real-time analysis, which has been successfully applied to homologous recombination processes, can exclude false positive results in DNA strand-exchange events. In addition, the device improves the accurate measurement of single molecule fluorescence resonance energy transfer (smFRET), and can identify extremely low smFRET states. Can be used for biological analysis, detection and imaging technology and single molecule dynamic research in important biological processes such as homologous recombination and the like.
The double-color laser alternate excitation single-molecule imaging detection device provided by the invention can simultaneously excite two fluorescent groups in target molecules, such as 532nm laser excitation fluorescent dye Cy3 and 647nm laser excitation fluorescent dye Cy5; the fluorescence emitted by the two excited fluorophores with different wavelengths can be detected simultaneously; fluorescence Resonance Energy Transfer (FRET) signals generated by two fluorophores can be detected simultaneously; the mutual interference between fluorescence resonance energy transfer signals generated by Fluorescence Resonance Energy Transfer (FRET) and other fluorescence resonance energy transfer signals is eliminated; the occurrence, development and end of biological processes such as strand exchange, e.g., capture of target molecules and initiation of reactions, can be identified.
FIG. 3 is a flow chart of a dual-color laser alternately excited single-molecule imaging detection apparatus used in an embodiment of the present invention.
According to a third aspect of the present general inventive concept, as shown in fig. 3, there is provided a method of alternately exciting a single-molecule imaging detection apparatus using a dual-color laser, including operations S1 to S8.
S1: two laser beams with different wavelengths are excited by a double-color light alternate exciter to serve as an excitation light source.
S2: the two lasers are formed into alternate lasers for exciting the target molecules by means of an acousto-optic tunable filter 1.
S3: the alternate laser light from the acousto-optic tunable filter 1 is received through an optical fiber.
S4: the target molecules in the load platform 2 are excited by the illumination 5 emitting alternating laser light.
S5: after the alternate laser is totally reflected on the carrying platform 2, a hidden evanescent field is formed on the carrying platform 2, and two fluorescent dyes marked on target molecules are excited to generate fluorescent groups.
S6: fluorescent groups emitted by the two fluorescent dyes are subjected to resonance transfer to form fluorescent resonance energy transfer signals.
S7: the fluorescence resonance energy transfer signal is received by a fluorescence detection assembly.
S8: the target image is inductively formed by the electron multiplying inductive coupling device 43.
The double-color alternate excitation (two-color alternating excitation, TCAE) single-molecule fluorescence imaging technology is successfully applied to the real-time dynamic monitoring of DNA strand exchange reaction. Cy3 and Cy5 were labeled on both strands of donor dsDNA, respectively, and the entire process of strand exchange could be observed in real time by alternating excitation at 532nm and 647nm, from initial donor capture until release of the last replaced strand. By this alternating excitation pattern, it is possible to identify successful strand exchange events from false positive signals at the single molecule level.
According to an embodiment of the present invention, wherein receiving the fluorescence resonance energy transfer signal by the fluorescence detection assembly includes operations S701 through S702.
S701: the two fluorescence resonance energy transfer signals are separated by a dichroic mirror 32.
S702: noise in the fluorescence resonance energy transfer signal is filtered through a single-band filter arranged in parallel.
The following detailed description of the present invention is given by way of example only, and not by way of limitation.
Example 1:
FIG. 4 is a schematic diagram showing a strand-exchange reaction between a single-stranded DNA molecule and a homologous double-stranded DNA molecule according to an embodiment of the present invention.
The double-color laser alternate excitation single-molecule imaging detection device provided by the embodiment of the invention is adopted to identify the chain exchange reaction between the single-chain DNA molecules and homologous double-chain DNA molecules.
1. Preparation of target samples in microfluidic chambers
The slide glass and the PEG modified cover glass are combined into a cavity for retaining the sample through double-sided adhesive bonding.
An imaging buffer comprising a target molecule is added to the chamber.
The imaging buffer was D-glucose (8 mg/mL), glucose oxidase (1 mg/mL), catalase (0.04 mg/mL), and water-soluble vitamin E (> 3 mM).
The target molecule includes single-stranded DNA molecules (ssDNA) of the same length and homologous double-stranded DNA (dsDNA).
Wherein the two segments of double stranded DNA (dsDNA) are labeled with fluorescent dyes Cy3 and Cy5, respectively.
Also included in the imaging buffer is a homologous recombinase (RecA) for assembly of single-stranded DNA molecules (ssDNA) with homologous double-stranded DNA (dsDNA).
The reaction process comprises the following steps: the homologous recombinase RecA assembles on ssDNA to form a nucleoprotein filament;
the nucleoprotein filaments bind to the cognate dsDNA temporarily forming a triplex complex;
the ssDNA of the nucleoprotein filament pairs with complementary strands on the homologous dsDNA to form new heterologous double-stranded DNA;
and another replaced strand of homologous dsDNA identical to the single stranded DNA molecule (ssDNA) is released after the strand exchange reaction with the nucleoprotein filament.
2. The double-color laser alternately excited single-molecule imaging detection device is adopted to characterize the reaction process of the chain exchange reaction
The laser with two wavelengths of 532nm and 647nm can be adopted, so that two fluorescent groups in target molecules can be excited simultaneously, the 532nm laser excites fluorescent dye Cy3, and the 647nm laser excites fluorescent dye Cy5.
(1) Signal recognition during DNA strand exchange reactions
FIG. 5 is a graph showing signals of DNA strand exchange reaction in example 1 identified by the detection device according to the embodiment of the present invention.
The detection device of the embodiment of the invention is used for identifying a signal curve when the nuclear protein wire captures homologous dsDNA binding to temporarily form a triplex complex in the DNA strand exchange reaction process and a signal curve after the successful DNA strand exchange reaction, and the result is shown in figure 5.
The signal profile obtained by transient capture of homologous dsDNA is shown in fig. 5 (a). The arrow indicates the disappearance of the Cy5 signal, i.e., the change in signal that the complementary strand is released by another strand of the homologous dsDNA that is identical to the single-stranded DNA molecule (ssDNA) after the strand exchange reaction with the nucleoprotein filament. The signals obtained by a successful DNA strand exchange reaction are shown in FIG. 5 (B).
Therefore, the detection device provided by the invention can identify the signal change in the DNA strand exchange reaction process in real time.
(2) ATP hydrolysis facilitates signal recognition during chain exchange reactions
FIG. 6 is a graph showing the signals for identifying the release of ATP hydrolysis-promoting strand-exchange reaction products in example 1 using the detection device according to the embodiment of the present invention.
The detection device according to the embodiment of the present invention recognizes that ATP hydrolysis promotes the strand-exchange reaction and that signals during the strand-exchange reaction do not occur, and the results are shown in FIG. 6.
A typical signal profile obtained under ATP hydrolysis conditions is shown in FIG. 6 (A). The arrow indicates the release of the replaced chain. (B) Typical signal curves obtained in the absence of ATP hydrolysis are shown. (C) Representative photographs (24 μm x 24 μm) of Cy5 channels from 10min strand exchange are shown.
In the 532nm wavelength plot, the arrow indicates the captured donor ds93 molecule, where Cy5 signal is excited at 532nm, but no complete chain separation occurs;
in the picture at wavelength 647nm, the Cy5 signal can only excite at 647nm the arrow indicates the donor ds93 molecule that was captured and that successfully underwent chain separation, where the Cy5 signal can only excite at 647 nm.
Fig. 6 (D) shows statistics of the number of Cy5 signals captured in three different imaging regions (82 μm×41 μm), and thus it can be seen that signal changes during ATP hydrolysis promoting strand exchange reaction can be recognized in real time by the detection device provided by the present invention.
Example 2:
1. preparation of target samples in microfluidic chambers
The procedure was the same as in example 1, except that single-stranded DNA molecules (ssDNA) of different lengths and homologous double-stranded DNA (dsDNA) were used.
2. The double-color laser alternately excited single-molecule imaging detection device is adopted to characterize the reaction process of the chain exchange reaction
The laser with two wavelengths of 532nm and 647nm can be adopted, so that two fluorescent groups in target molecules can be excited simultaneously, the 532nm laser excites fluorescent dye Cy3, and the 647nm laser excites fluorescent dye Cy5.
(1) Signal recognition during different length ssDNA mediated strand exchange reactions
FIG. 7 is a graph showing real-time observation of ssDNA-mediated strand exchange reactions of example 2 of different lengths using the detection apparatus of the present invention.
The detection device of the embodiment of the invention is used for identifying ssDNA mediated chain exchange reactions with different lengths and signals in the process of not carrying out the chain exchange reactions, and the results are shown in FIG. 7.
A schematic of a short-chain single-molecule DNA strand exchange protocol in a different length ssDNA-mediated strand exchange reaction is shown in fig. 7 (a). Representative fluorescence resonance energy transfer signal and FRET efficiency time curves for (B-D) donor ds93 in strand exchange with homologous ss93 (B), ss83 (C) or ss73 (D), respectively. The solid arrow on the left indicates capture of donor ds93 and the dashed arrow on the right indicates release of the cognate chain. Δt represents the lifetime of the triplex (i.e., the time it takes for chain exchange to occur). (E) time distribution required for chain exchange reaction. Therefore, the detection device provided by the invention can identify the signal change in the process of the ssDNA mediated chain exchange reaction with different lengths in real time.
(2) Signal recognition during DNA strand exchange reactions
FIG. 8 is a graph depicting the efficiency of various lengths of ssDNA mediated strand exchange reactions in example 2 using the detection apparatus of an embodiment of the present invention.
The Cy5 channel fluorescence image obtained after 10min of chain exchange reaction (left two columns) or another 30min SacI cleavage (right most column) is shown in FIG. 8 (A). In the 532nm wavelength plot, the arrow indicates the captured donor ds93 molecule, where the Cy5 signal is excited at 532nm, but no complete chain separation occurs.
In the picture at wavelength 647nm, the Cy5 signal can only excite at 647nm the arrow indicates the donor ds93 molecule that was captured and that successfully underwent chain separation, where the Cy5 signal can only excite at 647 nm. Fig. 8 (B) shows statistics of the number of capture donor ds93 molecules using ssDNA of different lengths. Data represent mean and standard deviation counted from six different imaging areas (82 μm×41 μm).
Therefore, the detection device provided by the invention can identify the signal change in the DNA strand exchange reaction process in real time.
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.
Claims (10)
1. A dual-color laser alternating excitation single-molecule imaging detection device, comprising:
the double-color light alternate exciter is used for exciting two laser beams with different wavelengths as excitation light sources;
the acousto-optic tunable filter is suitable for controlling two beams of laser with different wavelengths to be alternately excited to form alternate laser and coupling the alternate laser into one optical fiber;
an cargo platform for placing a sample comprising target molecules labeled with two different fluorescent dyes, exciting the two different fluorescent dyes of the target molecules by the alternating laser, causing the fluorescent dyes to respond by emitting a fluorescent group that resonantly transfers to form a fluorescent resonance energy transfer signal;
the fluorescence detection component is used for receiving a fluorescence resonance energy transfer signal generated after the target molecule is excited, and forming a target image through induction of the electron multiplication inductive coupling device;
wherein the alternating lasers form a recessed evanescent field on the cargo platform to reduce background light noise interference.
2. The detection apparatus according to claim 1, wherein the microfluidic chamber comprises:
a chamber for sample retention combined by a slide glass and a PEG modified cover glass, wherein the height of the microfluidic chamber is the height of the connecting structure;
the glass slide comprises a sample inlet and an output hole, and the output hole is connected with a syringe through a catheter.
3. The detection apparatus according to claim 2, wherein the microfluidic chamber comprises:
target molecules, including probe molecules and target molecules labeled with two different fluorescent dyes;
an imaging buffer for introducing a target molecule comprising an oxygen scavenging system and a reducing agent, the imaging buffer comprising D-glucose, glucose oxidase, catalase, and water-soluble vitamin E.
4. The detection apparatus according to claim 2, further comprising:
the inverted fluorescence microscope is arranged on the lower side of the carrying platform and is used for focusing the alternate laser on a cover glass of the carrying platform or leading out the fluorescence resonance energy transfer signal from the carrying platform;
and the illuminator is connected with the acousto-optic tunable filter through an optical fiber and is used for receiving the alternate laser from the acousto-optic tunable filter and emitting the alternate laser to excite the target molecules.
5. The detecting device according to claim 1, wherein,
the dual-color light alternate activator includes:
a laser source for emitting two laser beams of different wavelengths;
a reflecting mirror and a dichroic mirror for reflecting the laser light emitted from the laser source into the optical path;
and the lens is used for focusing the two laser beams to the acousto-optic tunable filter.
6. The detection apparatus of claim 1, wherein the fluorescence detection assembly comprises:
a dichroic mirror for separating the fluorescence resonance energy transfer signals to obtain two fluorescence lasers with different wavelengths;
and the two single-band filters are arranged in parallel between the electron multiplication inductive coupling device and the dichroic mirror and are used for filtering noise of the fluorescent laser.
7. Use of a dual-colour laser alternating excitation single-molecule imaging detection device according to any one of claims 1-6 for the identification of capture of target molecules and initiation of reactions during a biological chain exchange process.
8. Use of the detection device according to claim 7, comprising the following:
strand separation detection of double-stranded DNA molecules or RNA molecules;
detecting the recombination reaction of a single-stranded DNA molecule and a complementary strand in a double-stranded DNA;
detecting the recombination reaction of a single-stranded RNA molecule and a complementary strand in a double-stranded DNA;
the accurate identification of processes such as capturing of DNA chains, starting and ending of reactions and the like in recombination reaction detection or chain separation detection;
hybridization reaction detection between two single-stranded DNA molecules or single-stranded RNA molecules;
hybridization reaction detection between a single-stranded RNA molecule and a single-stranded DNA molecule;
the conformation and folding of single-stranded RNA molecules or DNA molecules are dynamically analyzed and detected in real time;
conformation and dynamic analysis of individual nucleic acid aptamer molecules;
dynamic analysis of binding of individual aptamer molecules to target proteins;
the nucleic acid aptamer molecules are used as affinity ligands and single molecule signal transduction, so that proteins and other target molecules are detected with high sensitivity; or (b)
Antibodies and other affinity proteins are used as ligands, and other molecules such as proteins, DNA, RNA and various small molecule compounds are detected with high sensitivity.
9. A method of alternately exciting a single molecule imaging detection apparatus using the dual color laser of any one of claims 1-6, comprising:
exciting two laser beams with different wavelengths by a double-color light alternate exciter to serve as an excitation light source;
forming two beams of laser into alternate laser used for exciting target molecules through an acousto-optic tunable filter;
receiving alternating laser light from the acousto-optic tunable filter through an optical fiber;
emitting the alternating laser light through an illuminator to excite target molecules in the cargo platform;
after the alternating laser is subjected to total reflection on the object carrying platform, a hidden evanescent field is formed on the object carrying platform, and two fluorescent dyes marked on the target molecule are excited to generate fluorescent groups;
fluorescent groups emitted by the two fluorescent dyes are subjected to resonance transfer to form fluorescent resonance energy transfer signals;
receiving the fluorescence resonance energy transfer signal by a fluorescence detection assembly;
the target image is inductively formed by the electron multiplying inductive coupling device.
10. The method of claim 8, wherein the receiving the fluorescence resonance energy transfer signal by a fluorescence detection assembly comprises:
separating the two said fluorescence resonance energy transfer signals by a dichroic mirror;
and filtering noise in the fluorescence resonance energy transfer signal through a single-band filter arranged in parallel.
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