Single molecule detection system and single molecule detection method

Abstract

A single molecule detection system and a single molecule detection method. The single molecule detection system comprises: a surface plasmon resonance (SPR) sensor (100), comprising a first surface (111) and a second surface (112), wherein the first surface (111) is configured to bind to a first molecule (610), and the first molecule (610) is configured to bind to a second molecule (620) marked with a fluorescent substance (621); a light source (200), configured to provide incident light to the second surface (112) to excite surface plasmon polaritons (SPP), so as to generate SPR; a first detection portion (300), configured to detect optical parameters of the SPR; and a second detection portion (400), configured to detect a fluorescence signal generated by the second molecule (620). On the basis of the resonance and fluctuation characteristics of the SPPs, a marked signal feature and an unmarked signal feature are synchronously obtained on two sides of a surface of the SPR sensor (100), so that qualitative or quantitative measurement of features such as loading amount, positioning, affinity to a substrate, and a molecular docking rate of the unmarked first molecule (610) is realized, and dynamic interaction processes between molecules such as binding and dissociation are synchronously measured.

Description

Single molecule detection system and single molecule detection method Technical Field

[0001] The present invention belongs to the technical field of single molecule detection, and in particular relates to a single molecule detection system and a single molecule detection method. Background Art

[0002] Single-molecule technology, also known as single-molecule biology, primarily focuses on the study of individual biomolecules. It generally refers to techniques and methods that can observe or inverse biomolecular behavior, obtain characteristic molecular information, and measure the laws governing molecular interactions. Over the past two decades, this field has seen almost exponential growth in its applications in biology, physics, and chemistry, while also opening up numerous new directions in interdisciplinary fields such as life sciences, medicine, new materials science, and computer science.

[0003] Based on the characteristics of currently available single-molecule technologies, they can be roughly divided into two categories: direct methods and indirect methods. Direct methods refer to the use of physical and chemical means to locally immobilize single molecules in an observable area and combine them with optical, electrical and other means for measurement. Representative technologies of this type of method include: single-molecule Raman technology, single-molecule scanning tunneling current technology, single-molecule atomic force microscopy technology, single-molecule optical tweezers, magnetic tweezers, etc. Indirect methods do not directly detect target single molecules, but instead obtain molecular information patterns through ensemble measurements of multiple molecules, or by utilizing interactions with labeled target single molecules to evolve. Therefore, indirect methods can also be divided into: labeled methods and label-free methods.

[0004] Fluorescence labeling is the most representative labeling-based single-molecule detection technology. Due to the unique luminescence properties of fluorescent dye molecules, intrinsic properties such as fluorescence intensity, fluorescence lifetime, and fluorescence spectrum can be used to label biomolecules and measure the fluorescence patterns of the molecules, thereby inverting the target molecule information. Among them, fluorescence resonance energy transfer (FRET), which has developed rapidly in recent years, has become one of the most representative technologies of single-molecule fluorescence and has been successfully applied in membrane protein dynamics, molecular colocalization, transcription mechanisms, protein folding, protein sequencing and other fields.

[0005] However, single-molecule fluorescence detection is usually aimed at two or more biological molecules, and single-molecule information is measured by characterizing the interaction rules between them. Generally speaking, one or more molecules need to be fluorescently labeled, while the other molecule does not use fluorescent labeling. Molecules without fluorescent labels need to be immobilized on the surface of the chip, but there is a problem with this process, that is, the loading amount, immobilization position, area and affinity between the molecules without fluorescent labels and the chip are usually difficult to measure and obtain quantitative results, resulting in unclear expression of the interaction between them and subsequent fluorescent-labeled molecules, and insufficient repeatability and uniformity of test data.

[0006] Summary of the Invention

[0007] The present invention provides a single molecule detection system and a single molecule detection method, which can realize qualitative or quantitative detection of the interaction between unlabeled and fluorescently labeled molecules.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] A first aspect of the present invention provides a single molecule detection system comprising:

[0010] An SPR sensor, the SPR sensor comprising a first surface and a second surface opposite to each other, the first surface being used to bind a first molecule, and the first molecule being used to bind a second molecule labeled with a fluorescent substance;

[0011] a light source configured to provide incident light to the second surface, thereby exciting surface plasmons to generate surface plasmon resonance;

[0012] a first detection unit, configured to detect an optical parameter of the surface plasmon resonance;

[0013] The second detection unit is used to detect the fluorescence signal generated by the second molecule under the excitation of the surface plasmon.

[0014] In some embodiments of the present invention, the optical parameter includes at least one of a resonance angle, a resonance wavelength, a phase difference between p-light and s-light, a Goos-Hansen shift difference between p-light and s-light, and an intensity of reflected light.

[0015] In some embodiments of the present invention, the fluorescence signal includes at least one of fluorescence intensity, fluorescence lifetime, fluorescence spectrum, and fluorescence correlation spectrum.

[0016] In some embodiments of the present invention, the single molecule detection system further comprises a fluorescence collecting element, which collects the fluorescence signal and outputs it to the second detection unit for detection, and the fluorescence collecting element comprises at least one of an objective lens and a lens.

[0017] In some embodiments of the present invention, the SPR sensor includes a substrate and a membrane layer, the side of the membrane layer away from the substrate is the first side, the side of the membrane layer in contact with the substrate is the second side, and the membrane layer is metal or graphene.

[0018] In some embodiments of the present invention, the film layer is a film layer formed by at least one of a single substance or an alloy of gold, silver, platinum, copper, aluminum, titanium, nickel, and chromium.

[0019] In some embodiments of the present invention, the first detection portion includes a first photodetection unit, and the second detection portion includes a second photodetection unit.

[0020] In some embodiments of the present invention, the first photoelectric detection unit and the second photoelectric detection unit are independently selected from any one of a point detector, a line detector, and a surface detector.

[0021] In some embodiments of the present invention, the first photodetection unit and the second photodetection unit are independently selected from any one of APD, SPAD, PIN, CCD, PMT, and CMOS.

[0022] In some embodiments of the present invention, the light source is a pulsed light source or a continuous light source.

[0023] In some embodiments of the present invention, the light source generates the incident light at more than one wavelength.

[0024] In some embodiments of the present invention, a plurality of channels are formed on the first surface, and each channel is used to bind the same or different first molecules.

[0025] In some embodiments of the present invention, at least one of the first detection unit and the second detection unit includes an area detector.

[0026] A second aspect of the present invention provides a single molecule detection method using the aforementioned single molecule detection system, comprising the following steps:

[0027] providing incident light to the second surface using a light source;

[0028] bringing the first molecule into contact with the first surface and detecting an optical parameter using a first detection portion;

[0029] A second molecule is brought into contact with the first surface, and the fluorescent signal is detected using a second detection unit.

[0030] In some embodiments of the present invention, when the surface plasmon resonance intensity detected by the first detection portion is saturated, the second molecule is brought into contact with the first surface.

[0031] In some embodiments of the present invention, at least one of the loading amount and the binding position of the first molecule is analyzed according to the optical parameter.

[0032] In some embodiments of the present invention, at least one of affinity and kinetics between the first molecule and the second molecule is analyzed based on the fluorescent signal.

[0033] In some embodiments of the present invention, the first detection unit detects the optical parameter by any one of light intensity modulation, angle modulation, wavelength modulation, phase modulation, and Goos-Hansen shift modulation.

[0034] In some embodiments of the present invention, the optical parameter detected by the light intensity modulation is the intensity of the light reflected on the second surface; the optical parameter detected by the angle modulation is the resonance angle; the optical parameter detected by the wavelength modulation is the resonance wavelength; the optical parameter detected by the phase modulation is the phase difference between p-light and s-light; and the optical parameter detected by the Goos-Hansen shift modulation is the Goos-Hansen shift difference between p-light and s-light.

[0035] The beneficial effects of the present invention are:

[0036] The present invention mainly utilizes the resonance characteristics and fluctuation characteristics of surface plasmon (SPP) at the same time, and synchronously obtains two types of signal characteristics, labeled and unlabeled, on both sides of the SPR sensor surface, thereby achieving qualitative or quantitative measurement of characteristics such as the loading amount, positioning, affinity with the substrate, and molecular chimerism rate of the unlabeled first molecule, while at the same time synchronously measuring the dynamic interaction processes such as the binding-dissociation of the fluorescently labeled second molecule. Thus, it helps to achieve stable, accurate, high-resolution, and highly sensitive in-situ detection of the interaction between two or more molecules, and the resolution is single molecule. Based on the above conception, the single molecule detection system or single molecule detection method of the present invention is suitable for a variety of biological applications, such as enzyme kinetics, DNA transcription, translation process, protein sequencing, targeted drug efficacy analysis, etc., becoming a paradigm measurement model for exploring the interaction between molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIG1 is a schematic diagram of a single molecule detection system in one embodiment of the present invention.

[0038] Figure 2 illustrates the principle of co-excitation surface plasmon sensing and single-molecule fluorescence detection, as well as the multimodal data collected simultaneously in situ, in Example 1 of the present invention. (a) shows the fluorescence acquisition system on the upper side of the SPR sensor, (b) shows the fluorescence intensity signal detected from the interaction between the fluorescently labeled second molecule and the first molecule, (c) shows the surface plasmon sensing system on the lower side of the SPR chip, and (d) shows the SPR data detected for the unlabeled molecule. The horizontal axis is in seconds (s).

[0039] Figure 3 is a schematic diagram of multi-channel co-excitation surface plasmon sensing and single-molecule fluorescence detection in Example 2 of the present invention. (a) is a schematic diagram of a 2×2-channel SPR sensor; (b) is the fluorescence signal detection result of the 2×2 channels, with the horizontal axis being seconds (s); and (c) is the SPR sensing signal detection result of the 2×2 channels, with the horizontal axis being seconds (s).

[0040] Reference numerals: SPR sensor 100 , first surface 111 , second surface 112 , light source 200 , first detection unit 300 , second detection unit 400 , fluorescence collecting element 500 , first molecule 610 , second molecule 620 , fluorescent substance 621 . DETAILED DESCRIPTION

[0041] In the description of the present invention, the terms "first," "second," and "third" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, a feature specified as "first," "second," or "third" may explicitly or implicitly include at least one of such features. In the description of the present invention, "plurality" means at least two, for example, two, three, etc., unless otherwise specifically defined.

[0042] In a first aspect of the present invention, a single molecule detection system is provided. Referring to FIG. 1 , the system includes an SPR sensor 100 , a light source 200 , a first detection unit 300 , and a second detection unit 400 .

[0043] Among them, surface plasmon resonance (SPR) refers to a physical optical phenomenon. Specifically, when light propagates from a denser medium to a less dense medium at a certain angle, total internal reflection occurs at the interface between the two. When total internal reflection occurs, the incident light first passes through the less dense medium to a depth of about one wavelength, and then returns to the denser medium. The wave that passes through the less dense medium is called an evanescent wave. At the same time, under the action of the electromagnetic field of the incident light, the electrons in the less dense medium oscillate and propagate on the surface at a certain frequency to form a plasma wave (surface plasmon, SPP). When the frequency and wave vector of the evanescent wave are the same as those of the plasma wave, resonance occurs, which is called surface plasmon resonance. The energy of the incident light is absorbed, causing the intensity of the reflected light to decrease.

[0044] Based on the above principles, the SPR sensor 100 includes a first surface 111 and a second surface 112 facing each other. Light source 200 provides incident light, which undergoes total internal reflection on second surface 112 and forms an evanescent wave in the direction from second surface 112 toward first surface 111, thereby generating surface plasmon resonance. Because the resonance peak is sensitive to subtle changes in the refractive index of the surrounding environment, a molecular capture mechanism is designed on first surface 111, and detection is performed using the difference in the resonance peak of the surface plasmon resonance or other relevant parameters caused by the change in refractive index before and after capture. Therefore, first surface 111 is used to bind first molecule 610, while first molecule 610 is used to bind second molecule 620. The optical parameters of the surface plasmon resonance are then detected by the first detection unit 300.

[0045] In the above scheme, after first surface 111 loaded with first molecule 610 further binds to second molecule 620, its surface plasmon resonance peak changes. Detection of optical parameters, including this change, enables in situ measurement of the location, loading amount, molecular chimerism rate, and other parameters of second molecule 620. However, it is often difficult to quantitatively measure the loading amount, loading position, and region of first molecule 610, as well as its affinity for the chip. This results in unclear representation of the interaction between first molecule 610 and second molecule 620, and insufficient repeatability and uniformity in test data.

[0046] Therefore, in the single molecule detection system of some embodiments of the present application, in addition to utilizing the resonance characteristics of surface plasmons (SPPs), the wave characteristics of SPPs are also utilized. The relationship between surface plasmons and the incident excitation wavelength is: where k in is the incident light wave vector, ε m is the dielectric constant of the film, ε d is the dielectric constant of the environment outside the film layer. SPP has a slight frequency shift compared to the incident light, but can still serve as an excitation source for fluorescence excitation. Based on this principle, a fluorescent substance 621 is marked on the second molecule 620, and a second detection unit 400 is introduced to detect the fluorescence signal generated by the second molecule 620 under surface plasmon excitation. In this way, the detection of SPR in the final single-molecule detection system is reflective, and the relevant optical component system is only built on one side of the SPR sensor; on the other side, SPP is used as a fluorescence excitation source to achieve synchronous multimodal detection with and without fluorescent labels.

[0047] In some embodiments, the first molecule is selected from an antigen, an antibody, a ligand, a receptor, biotin, avidin, a substrate, an enzyme, a coenzyme, a nucleic acid molecule, etc., and correspondingly, the second molecule is selected from an antibody, an antigen, a receptor, a ligand, avidin, biotin, an enzyme, a coenzyme, a substrate, a nucleic acid molecule, a polypeptide, a cell, an extracellular vesicle, etc. In some embodiments, the first molecule can be bound to the first surface by a covalent bond, such as an amino-carboxyl group, an amino-aldehyde group, or an amino-isothiocyanate group; in other embodiments, the first molecule can also be bound to the first surface by a gold-sulfhydryl bond or other means known in the art.

[0048] Among them, a fluorescent substance refers to a substance that can be excited into an excited state under specific light irradiation conditions and then emit fluorescence when it returns to a ground state from the excited state. In some embodiments, the fluorescent substance is a fluorophore, including fluoresceins, isothiocyanates, anthocyanins, coumarins, rhodamines, BODIPYs, and the like, specifically including but not limited to FAM, VIC, Cy2, Cy3, Cy5, Cy7, NED, AMCA, Texas Red, sulforhodamine B, FITC, DEAC, TRITC, 5-TAMRA, 6-TAMRA, 7-hydroxycoumarin-3-carboxylic acid, 7-methoxycoumarin-3-carboxylic acid, eosin-5-isothiocyanate, erythrophycocyanate, AMCA-X SE, DTAF, 6-FAM, dansyl-X, 6-JOE, HEX, PyMPO, BODIPY-TMR-X, BODIPY FL, BODIPY 576 / 589, BODIPY 581 / 591, ROX, carboxy naphthofluorescein, and the like. In some other embodiments, the fluorescent substance may also be a fluorescent protein, such as phycoerythrin (PE), allophycocyanin (APC), green fluorescent protein (GFP), mGFP5, EGFP, D2EGFP, yellow fluorescent protein (YFP), EYFP, blue fluorescent protein (BFP), cyan fluorescent protein (CFP), red fluorescent protein (DsRed), DsRed2, mRFP1, mCherry, etc., and also includes quantum dots, nanocrystals, semiconductor fluorophores, etc. It will be understood that in the present application, the fluorescent substance is selected from substances that can be excited by surface plasmons and transition back to the ground state to emit fluorescence.

[0049] The second detection unit detects the fluorescence signal generated by the fluorescent substance labeled on the second molecule under the excitation of the surface plasmon. Due to the luminescence characteristics of the fluorescent substance, in some embodiments, the second detection unit inverts the information of the target molecule by measuring intrinsic properties such as fluorescence intensity, fluorescence lifetime, fluorescence spectrum, fluorescence correlation spectrum, etc. Since the signal of single-molecule fluorescence is weak, in some embodiments, the single-molecule detection system also includes a fluorescence collection element, which collects the fluorescence signal of the fluorescent substance and outputs it to the second detection unit for detection, so as to improve the fluorescence signal collection effect of the second molecule. In some embodiments, the fluorescence collection element includes at least one of an objective lens and a lens, for example, it can be a single lens, a single objective lens, a lens group, a combination of one or more objective lenses and one or more lenses, etc.

[0050] In some embodiments, the SPR sensor includes a membrane layer, and the first surface and the second surface are two surfaces arranged opposite to each other on the membrane layer. The material of this surface plasmon resonance membrane layer is generally a metal, such as a membrane layer formed by at least one of a single substance or alloy of metal elements such as gold, silver, platinum, copper, aluminum, titanium, nickel, chromium, etc. In some embodiments, the membrane layer material can also be other materials with matching dielectric constants, such as semiconductors (such as graphene). In some embodiments, the surface of the membrane layer can be further modified to improve the detection sensitivity, for example, at least one of nanoparticles or two-dimensional nanomaterials is modified on the surface of the membrane layer. In some embodiments, the nanoparticles include at least one of noble metal nanoparticles (such as gold nanoparticles, silver nanoparticles, etc.) and magnetic nanoparticles (such as Fe3O4 nanoparticles). In some embodiments, the two-dimensional nanomaterial includes at least one of an inorganic two-dimensional nanomaterial, an organic two-dimensional nanomaterial, and an inorganic-organic two-dimensional nanomaterial. Taking inorganic two-dimensional nanomaterials as an example, it includes at least one of graphene, graphene oxide, two-dimensional transition metal chalcogenides (such as MX2, M is at least one of W and Mo, and X is at least one of S and Se), two-dimensional black phosphorus, etc.

[0051] In some embodiments, the thickness of the film layer is 1 to 500 nm, specifically 1 nm, 2 nm, 3 nm, 5 nm, 10 nm, 15 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 120 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, for example, the thickness of the film layer is 2 to 400 nm, 5 to 200 nm, or 10 to 100 nm. SPP At the same frequency, it is usually larger than the wave vector k of the incident light. in , so incident light cannot directly excite SPPs. A coupling mechanism is required to achieve wave vector matching between the two. Therefore, in some embodiments, the SPR sensor further includes a substrate on which the film layer is formed. The substrate can be, for example, at least one of a prism, a waveguide, an optical fiber, a grating, and the like.

[0052] According to the principles of SPR sensors, surface plasmon resonance is determined by parameters such as the wavelength, angle, film layer, substrate, and refractive index of the incident light. Changes in the refractive index caused by the first molecule or the first and second molecules detected by the SPR sensor can be expressed through changes in these parameters. Therefore, in some embodiments, SPR sensing employs five detection methods: light intensity modulation, angle modulation, wavelength modulation, phase modulation, and Goos-Hansen shift modulation. Depending on these different detection methods, the first detection section detects different optical parameters during the surface plasmon resonance process. For example, when light intensity modulation is used, the incident light provided by the light source is irradiated at a fixed incident angle. As the first molecule and the second molecule are bound to the first surface, the change in surface plasmon resonance causes the intensity of the reflected light to change, so the first detection unit detects the intensity of the reflected light; when angle modulation is used, the angle of the incident light provided by the light source is changed directly or indirectly, and the change in the intensity of the reflected light with the incident angle is recorded to obtain a reflected light intensity curve based on the angle change, where the angle corresponding to the position with the lowest intensity is the resonance angle, and the resonance angle reflects the change in the surface refractive index, so the first detection unit detects the resonance angle, for example, via the reflected light intensity and the incident angle; when wavelength modulation is used, the light source provides incident light with a fixed incident angle, and the reflectivity changes accordingly with the different wavelengths of the incident light, and the lowest reflectivity corresponds to The wavelength is the resonant wavelength at the corresponding incident angle, and the resonant wavelength changes when the surface refractive index changes. Therefore, the first detection unit detects the resonant wavelength, for example, through the incident light wavelength and the reflectivity; during phase modulation, the light source provides p-polarized light (p-light). When the surface refractive index changes, the phase of the p-light changes, while the s-polarized light (s-light) used as a reference does not participate in the SPR effect and therefore the phase remains basically unchanged. Therefore, the first detection unit detects the phase difference between the p-light and the s-light; during Goos-Hansen shift modulation, similar to phase modulation, since the Goos-Hansen shift is related to the phase, the phase of the reflected light of the p-light changes significantly due to the influence of SPR, so the Goos-Hansen shift changes greatly, while the s-light remains almost unchanged. Therefore, the first detection unit detects the Goos-Hansen shift difference between the p-light and the s-light.

[0053] In some embodiments, the first detection unit and the second detection unit detect the optical parameters of the surface plasmon resonance and the fluorescence signal generated by the second molecule under the surface plasmon excitation through a photoelectric detection unit. In some embodiments, the first detection unit includes a first photoelectric detection unit, and the second detection unit includes a second photoelectric detection unit. The first photoelectric detection unit and the second photoelectric detection unit can be any one of a point detector, a line detector, and a surface detector. For example, the first photoelectric detection unit and the second photoelectric detection unit are independently selected from any one of a photomultiplier tube (PMT), an avalanche photodiode (APD), a single photon avalanche diode (SPAD), a PIN photodiode (PIN), a charge coupled device (CCD), and a complementary metal oxide semiconductor sensor (CMOS). In some embodiments, the first photoelectric detection unit and the second photoelectric detection unit are detectors of weak light signals, independently selected from devices such as APD, SPAD, and PMT.

[0054] In some embodiments, the second surface is located at the bottom side of the SPR sensor, and the first surface is located at the top side of the SPR sensor, and the first detection unit, the second detection unit, and the light source are arranged accordingly. In some embodiments, the first surface is located at the bottom side of the SPR sensor, and the second surface is located at the top side of the SPR sensor, and the first detection unit, the second detection unit, and the light source are arranged accordingly.

[0055] In some embodiments, the light source can be a pulsed light source or a continuous light source, and can generate incident light of more than one wavelength, for example, single wavelength or multi-wavelength excitation.

[0056] In some embodiments, the first surface of the single-molecule detection system is formed with a channel, and the first molecule and the second molecule are bound to the first surface in the channel, for example, by a liquid phase containing the first molecule and a liquid phase containing the second molecule, respectively, flowing through the channel, thereby reacting and binding to the first surface. In some embodiments, the liquid phase containing the first molecule and the liquid phase containing the second molecule can each independently be an aqueous phase or an oil phase. In some embodiments, the liquid phase containing the first molecule and the liquid phase containing the second molecule are solutions.

[0057] In some embodiments, the single-molecule detection system can be a single-point detection system or a high-throughput multi-point detection system. For the latter, the first surface of the single-molecule detection system is formed with multiple channels, and each channel is used to bind the same or different first molecules. In some embodiments, multiple channels are arranged to form an array, for example, it can be a rectangular array of N1×N2, N1 and N2 are independently positive integers and are not 1 at the same time. It can be understood that the array formed by the arrangement of multiple channels can also be other regular or irregular arrays. In some embodiments, the channel can be rectangular, circular or other regular or irregular shapes. In some embodiments, for a high-throughput multi-channel single-molecule detection system, the first detection unit and the second detection unit can select a surface detector for signal acquisition and detection, and in other embodiments, the first detection unit and the second detection unit can also select one or more point detectors or line detectors to match the relative positions of multiple channels for detection.

[0058] In some embodiments, the molecular detection limit of the single molecule detection system can cover different orders of magnitude such as molar, millimolar, micromole, nanomole, picomolar, femtomolar, atmolar, and zetmolar, that is, the lowest can reach the zetmolar level (~10 -15 mol / L) or lower.

[0059] A second aspect of the present invention provides a single molecule detection method comprising the following steps:

[0060] providing incident light to the second surface using a light source;

[0061] bringing the first molecule into contact with the first surface and detecting an optical parameter using a first detection portion;

[0062] The second molecule is brought into contact with the first surface, and the fluorescent signal is detected using the second detection unit.

[0063] Since fluorescent labeling and non-fluorescent labeling techniques each have their own advantages and disadvantages, there is currently a lack of integrated technical solutions that complement the advantages of the two on the market. From the perspective of technical effects, the shortcomings of the existing technology are mainly reflected in the following aspects: label-free technology (such as SPR technology) lacks specific molecular binding signals and can only obtain average measurements of multiple molecules; fluorescent labeling technology is highly targeted and suitable for analyzing specific target single molecules, but when studying intermolecular interactions, it is often necessary to fix unlabeled molecules on the surface of a biochip, and there are few reports on the precise measurement of the positioning, immobilization amount, etc. of unlabeled molecules, and the analysis of the system or work that interacts with the fluorescent labeled molecules on the basis of quantitative measurement. The above-mentioned detection method provided in the embodiment of the present application mainly utilizes the resonance characteristics and fluctuation characteristics of surface plasmon (SPP) at the same time, and obtains two types of signal characteristics of labeled (second molecule) and unlabeled (first molecule) on both sides of the sensor, thereby realizing the qualitative and quantitative measurement of the loading amount, positioning, and affinity of the unlabeled molecules to the substrate, and at the same time, the dynamic processes such as binding-dissociation of the fluorescent labeled molecules are synchronously measured. By comparing the signals of the two parts, it is helpful to analyze the interaction process of unlabeled and labeled molecular information.

[0064] Therefore, the methods provided in the embodiments of this application can address the problems of existing technical solutions, such as the inability to simultaneously detect labeled and unlabeled molecules, the high randomness of single-molecule measurements, and the lack of stable and reliable technical solutions for precise in situ measurement of single molecules. They also help to achieve stable, accurate, high-resolution, and highly sensitive detection of interactions between two or more molecules. Biological applications suitable for the above methods include, but are not limited to, enzyme kinetics, DNA transcription, translation processes, protein sequencing, and targeted drug efficacy analysis.

[0065] In some embodiments, when the surface plasmon resonance intensity detected by the first detection unit reaches saturation, the second molecule is brought into contact with the first surface. Surface plasmon resonance intensity saturation refers to a state in which the SPR signal intensity reaches relative equilibrium in the time domain, indicating that the binding of the first molecule to the first surface is near saturation, i.e., the first molecule has bound to the first surface, thereby preparing for the next step of single-molecule fluorescence detection between the second molecule and the first molecule.

[0066] In some embodiments, at least one of the loading amount and the binding location of the first molecule is analyzed based on optical parameters.

[0067] In some embodiments, at least one of affinity and kinetics between the first molecule and the second molecule is analyzed based on the fluorescent signal.

[0068] In some embodiments, the first detection unit detects optical parameters using any of the following methods: light intensity modulation, angle modulation, wavelength modulation, phase modulation, or Goos-Hansen shift modulation. Specifically, the optical parameter detected by light intensity modulation is the intensity of light reflected from the second surface; the optical parameter detected by angle modulation is the resonance angle; the optical parameter detected by wavelength modulation is the resonance wavelength; the optical parameter detected by phase modulation is the phase difference between p-light and s-light; and the optical parameter detected by Goos-Hansen shift modulation is the Goos-Hansen shift difference between p-light and s-light.

[0069] The present invention is further described in detail below through specific examples.

[0070] It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0071] The experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or the conditions recommended by the manufacturers. The materials and reagents used in these examples were commercially available unless otherwise specified.

[0072] Example 1

[0073] This example is used to illustrate the working principle and results of a single-molecule detection system of co-excited surface plasmon resonance sensing and single-molecule fluorescence detection.

[0074] The single molecule detection system in this embodiment refers to Figure 1 and includes an SPR sensor 100, a light source 200, a first detection unit 300, a second detection unit 400, and a fluorescence collection element 500. The first molecule 610 is an SPR sensor 100, which is an SPR biochip, including a prism located below, a 100nm thick gold film coated on the prism, and a channel formed on the gold film. The first molecule used in the detection process is a polypeptide, and the second molecule is a fluorescently labeled first molecule that specifically recognizes the protein ClpS. The detector 2 of the first detection unit 300 and the detector 1 of the second detection unit 400 are SPAD devices, and the beam collection system of the fluorescence collection element 500 is a lens group.

[0075] The single-molecule detection system in this embodiment uses bottom-side SPR excitation and top-side single-molecule fluorescence collection. A light source 200 is positioned below the SPR sensor 100. This light source 200 emits incident light onto the second surface 112 of the gold film, and the intensity of the reflected light is detected by a first detection unit 300.

[0076] First, referring to FIG2 c and in combination with FIG1, a surface plasmon sensing system on the bottom side of a biochip is shown, wherein a solution containing a first molecule 610 flows through a channel on the first surface 111, wherein the unlabeled first molecule 610 (non-fluorescently labeled molecule) gradually binds to the first surface 111 of the gold film of the biochip by free diffusion or chemical bond modification. At this time, the incident light below excites an SPR signal, and the amount of the unlabeled first molecule loaded, the immobilized position / area, and other measurements are obtained by detecting the resonance peak shift (scanning angle) of the reflectance spectrum or converting it into detecting the energy difference (light intensity difference) brought about by the peak shift. Moreover, by judging the strength of the SPR signal, the affinity of the unlabeled molecule to the substrate can be inferred. As shown in FIG2 d, an SPR signal curve can represent the binding strength of a molecule to the gold film, while different SPR signal curves can represent the binding strength of different molecules. When the SPR signal reaches a relative equilibrium in the time domain (i.e., the signal intensity gradually stops changing over time), it indicates that the immobilization amount of the first molecule is close to saturation, that is, the unlabeled first molecule has been bound to the first side of the biochip, preparing for the next step of single-molecule fluorescence detection.

[0077] Referring to FIG2a, the single-molecule detection system also includes a fluorescence collection system on the upper side of the biochip. When a solution of a second molecule 620 (with a fluorescently labeled molecule) labeled with a fluorescent substance 621 flows through the channel of the first surface 111 and interacts with the first molecule 610 immobilized on the first surface, the fluorescently labeled molecule enters the evanescent field and scatters fluorescence. At this time, the affinity between the first molecule and the second molecule, as well as the kinetic process, can be inverted through the fluorescence signal. The weak signal is collected by the fluorescence collection element 500 and a fluorescence signal in the time domain is formed on the detector 1. The type of fluorescence signal detected is fluorescence intensity, see FIG2b.

[0078] Because the fluorescence signal is derived solely from the fluorescent substance on the fluorescently labeled molecules, it is difficult to directly measure the distribution of molecules on the chip surface through fluorescence measurement, making it impossible to estimate parameters such as the location and loading amount of unlabeled molecules. This embodiment of the present application leverages the dual nature of SPP, enabling simultaneous observation of the interaction between labeled and unlabeled molecules through the simultaneous detection and comparison of SPR and fluorescence signals.

[0079] Example 2

[0080] This embodiment provides a single molecule detection system, which differs from Example 1 in that a multi-pixel array detector is used for parallel spatial array measurement to achieve high-throughput single molecule detection. Referring to a in Figure 3, a 2×2 channel array is formed on the biochip including four channels C_1, C_2, C_3, and C_4. The first detection unit and the second detection unit use an imaging system of a multi-pixel array detector to obtain signal differences at different spatial positions in the plane, and perform parallel acquisition of multi-region labeled and unlabeled molecular signals, which can realize the measurement of in-situ multi-channel co-excitation signals, thereby achieving the purpose of high-throughput measurement. Among them, the unlabeled molecules are different polypeptides, and the labeled molecules are fluorescently labeled specific recognition proteins of the corresponding polypeptides. Refer to Figure 2 b and c, which show the changes in fluorescence intensity and SPR intensity in channels C_1, C_2, C_3, and C_4 over time (seconds). By comparing the fluorescence intensity and SPR intensity in channels C_1, C_2, C_3, and C_4, respectively, the interaction process between the labeled and unlabeled molecules in channels C_1, C_2, C_3, and C_4 can be analyzed.

[0081] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A single molecule detection system, characterized in that, Comprising: An SPR sensor, the SPR sensor comprising opposite first and second surfaces, the first surface being for binding a first molecule, the first molecule being for binding a second molecule labeled with a fluorescent substance; A light source for providing incident light to the second surface, thereby exciting surface plasmons to generate surface plasmon resonance; A first detection unit for detecting optical parameters of the surface plasmon resonance; A second detection unit for detecting a fluorescence signal generated by the second molecule under excitation of the surface plasmons.

2. The single molecule detection system according to claim 1, characterized in that: The optical parameters include at least one of a resonance angle, a resonance wavelength, a phase difference between p-light and s-light, a Goos-Hänchen shift difference between p-light and s-light, and an optical intensity of reflected light; and / or, the fluorescence signal includes at least one of a fluorescence intensity, a fluorescence lifetime, a fluorescence spectrum, and a fluorescence correlation spectrum.

3. The single molecule detection system according to claim 1, characterized in that: The single molecule detection system further includes a fluorescence collection element that collects the fluorescence signal and outputs it to the second detection unit for detection, the fluorescence collection element including at least one of an objective lens and a lens.

4. The single molecule detection system according to claim 1, characterized in that: The SPR sensor includes a substrate and a film layer, a surface of the film layer away from the substrate being the first surface, a surface of the film layer in contact with the substrate being the second surface, the film layer being metal or graphene.

5. The single molecule detection system according to claim 4, characterized in that: The film layer is a film layer formed of at least one of a single substance or an alloy of gold, silver, platinum, copper, aluminum, titanium, nickel, and chromium.

6. The single molecule detection system according to claim 1, characterized in that: The first detection unit includes a first photoelectric detection unit, and the second detection unit includes a second photoelectric detection unit.

7. The single molecule detection system according to claim 6, characterized in that: The first photoelectric detection unit and the second photoelectric detection unit are each independently selected from any one of a point detector, a line detector, and a surface detector.

8. The single molecule detection system according to claim 6, characterized in that: The first photoelectric detection unit and the second photoelectric detection unit are each independently selected from any one of an APD, an SPAD, a PIN, a CCD, a PMT, and a CMOS.

9. The single molecule detection system according to claim 1, characterized in that: The light source is a pulsed light source or a continuous light source; and / or, the light source generates the incident light of more than one wavelength.

10. The single molecule detection system according to claim 1, characterized in that: The first surface is formed with a plurality of channels, each channel being respectively for binding the same or different first molecules.

11. The single molecule detection system according to claim 10, characterized in that: At least one of the first detection unit and the second detection unit includes a surface detector.

12. A single molecule detection method using the single molecule detection system according to any one of claims 1 to 11, characterized in that, Comprising the following steps: Using a light source to provide incident light to the second surface; Bringing the first molecule into contact with the first surface and detecting optical parameters using the first detection unit; Bringing the second molecule into contact with the first surface and detecting the fluorescence signal using the second detection unit.

13. The single molecule detection method according to claim 12, characterized in that: When the surface plasmon resonance intensity detected by the first detection unit is saturated, bring the second molecule into contact with the first surface.

14. The single-molecule detection method according to claim 12, wherein: Analyzing at least one of the loading amount and the binding position of the first molecule according to the optical parameters; and / or, analyzing at least one of the affinity and the kinetic process between the first molecule and the second molecule according to the fluorescence signal.

15. The single-molecule detection method according to claim 12, wherein: Using the first detection unit to detect the optical parameters by any one of methods of intensity modulation, angle modulation, wavelength modulation, phase modulation, and Goos-Hänchen shift modulation.

16. The single-molecule detection method according to claim 15, wherein: The optical parameter detected by the intensity modulation is the optical intensity of the reflected light on the second surface; The optical parameter detected by the angle modulation is the resonance angle; The optical parameter detected by the wavelength modulation is the resonance wavelength; The optical parameter detected by the phase modulation is the phase difference between the p-wave and the s-wave; The optical parameter detected by the Goos-Hänchen shift modulation is the Goos-Hänchen shift difference between the p-wave and the s-wave.

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