High-flux optical mass spectrometer and single-molecule small protein detection system
By introducing a reference optical module and an interference module into an optical mass spectrometer, combined with a sheath flow device, high-throughput detection of small protein molecules is achieved, the problem of insufficient detection throughput and speed in the prior art is solved, and rapid and dynamic proteomic analysis is achieved.
Patent Information
- Application Number
- CN202411837659.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-13
- Publication Date
- 2025-03-28
AI Technical Summary
The prior art is difficult to achieve rapid, high-throughput and dynamic detection in proteomic analysis, especially in the detection of small protein molecules, with insufficient detection throughput and speed.
A high-throughput optical mass spectrometer was designed. By introducing a reference optical module, a scattered optical module and an interference module, the scattered light is coherently amplified by the principle of interference scattering, which reduces the lower limit of detection and realizes the detection of small single molecule proteins through the sheath flow device.
High-throughput detection of small protein molecules is achieved, lower detection limits, improve detection throughput and speed, and can perform proteomic analysis quickly and dynamically.
Smart Images
Figure CN119715328A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to biological detection technology, and in particular to a high-throughput optical mass spectrometer and a single-molecule small protein detection system. Background Art
[0002] With the growing demand for proteomics analysis in biomedicine, how to achieve short-term and rapid proteomics analysis while maintaining a relatively ideal coverage depth has become one of the key issues that need to be solved urgently. The technologies currently used in proteomics analysis include biological mass spectrometry and mass photometry. Biological mass spectrometry technology is developed from mass spectrometers. The current technical level can draw 100 cross-species proteome maps, including 2 million peptides and 340,000 protein identification information. It is the most widely used biomolecular analysis technology. Mass photometry technology is a protein molecule detection technology developed from interference scattering microscopy in the past decade. It does not require complex pre-processing and directly images biological molecules. The measurement range of molecules can cover 30kDa to 5MDa. It is a dynamic and intuitive measurement solution.
[0003] Currently, the relevant technologies include (1) biomass spectrometry technology; (2) mass spectrometry technology and (3) nanoflow technology. The relevant technologies are described in detail below.
[0004] (1) Biomass spectrometry: Biomass spectrometry analyzes the composition and structure by distinguishing and analyzing the mass-to-charge ratio (m / z) of different biomolecules. The corresponding instrument is a biomass spectrometer. Early chemical mass spectrometers were based on hard ionization technology, which transferred high energy to molecules, resulting in a large number of fragmented ions. Due to the serious damage to biomolecules, they could not be used for biomass spectrometry analysis. It was not until the 1980s that soft ionization technologies represented by ESI and MALDI technologies emerged, allowing biomolecules to retain their complete characteristics, and biomass spectrometers came into being. After the biomolecules are softly ionized, they are filtered by quadrupoles, time-of-flight and other principles, and finally the detector is used to distinguish the protein components with different charge-to-mass ratios, and finally the biomass spectrum of the sample can be obtained. Biomass spectrometry is a static biomolecule analysis technology with high precision and strong protein detection specificity, but low throughput. Although biomass spectrometry has the ability to detect with high precision and high specificity, the system has a low detection throughput and is static, which cannot meet the needs of high-speed and dynamic detection.
[0005] (2) Mass photometry: Mass photometry is based on the principles of interference reflection microscopy and interference scattering microscopy. In the early 21st century, Professor Kukura's team at Oxford University proved that the tiny amount of light scattered by a single molecule on the observation interface can be reliably detected and can be used to calculate the number of molecules in the solution. The intensity of the scattered light is directly related to the molecular mass and can be used to measure the mass of the molecule. The specific implementation method is: when a beam of light is irradiated onto the molecules on the interface (such as a glass slide), two signals, the scattered light of the molecules and the reflected light of the interface, will be generated. These two signals interfere, causing the scattering signal of the protein to be amplified. When received by the detector, more photons can be obtained than by directly measuring the scattered light signal, that is, smaller protein molecules can be measured. However, due to the integration time of the imaging system, it often takes several seconds to image a single small protein, and the measurement throughput of the system is low.
[0006] (3) Nanoflow technology: Nanoflow technology uses fluid mechanics to arrange the molecules to be detected in a row in the sample flow and pass through the focused light spot one by one. Proteins of different sizes scatter light with different intensities, so the size of the protein can be detected; after pretreatment, different types of proteins emit different fluorescent signals, so different types of proteins can be sorted in conjunction with the sorting device. Since the molecules continue to flow in the solution, the detection speed of this method is very fast. However, due to the influence of the scattered light intensity, when the protein size is small to a certain extent, the scattered light intensity is less than the detection limit of the detector and cannot be detected. Summary of the invention
[0007] In view of this, it is necessary to provide a high-throughput optical mass spectrometer and single-molecule small protein detection system that can both reduce the size detection limit and increase the detection throughput to address the current technical defects of high-throughput biomolecule detection.
[0008] To solve the above problems, this application adopts the following technical solutions:
[0009] One of the purposes of the present application is to provide a high-throughput optical mass spectrometer, comprising: a reference light module, a scattered light module and an interference module;
[0010] The reference light module includes a light source, a beam splitter, a half glass slide, a first plane mirror, a first beam expander lens, a scanning lens, a second plane mirror, an adjustable attenuator and a first focusing lens;
[0011] The scattered light module includes the light source, the beam splitter, a plane mirror group and a second focusing lens;
[0012] The interference module includes a focusing lens group and a single photon counting module;
[0013] The laser beam emitted by the light source is split into reference light and scattered light by the beam splitter;
[0014] The reference light sequentially passes through the half glass slide and the first plane mirror and enters the first beam expanding lens, the first beam expanding lens controls the spot size of the incident reference light, and then enters the second plane mirror through the second beam expanding lens, and enters the adjustable attenuator after being reflected by the second plane mirror, and then enters the first focusing lens after the adjustable attenuator controls the optical power of the incident reference light;
[0015] The scattered light enters the second focusing lens after passing through the plane mirror group;
[0016] The focusing lens group performs superposition interference on the reference light passing through the first focusing lens and the scattered light passing through the second focusing lens, and then the light is received by the single photon counting module.
[0017] In some embodiments, the plane mirror group includes a third plane mirror, a fourth plane mirror, a fifth plane mirror and a sixth plane mirror, and the scattered light enters the second focusing lens after passing through the third plane mirror, the fourth plane mirror, the fifth plane mirror and the sixth plane mirror in sequence.
[0018] In some of the embodiments, the fifth plane mirror and the sixth plane mirror are disposed on a translation stage, and the fifth plane mirror and the sixth plane mirror can be moved on the translation stage. The optical path of the scattered light path can be adjusted by adjusting the positions of the fifth plane mirror and the sixth plane mirror to make it consistent with the optical path of the reference light path.
[0019] In some embodiments, the focusing lens group includes a third focusing lens, a fourth focusing lens, a fifth focusing lens and a sixth focusing lens. The reference light passing through the first focusing lens and the scattered light passing through the second focusing lens enter the third focusing lens and form an interference light signal through the third focusing lens. The interference light signal is then acquired by the single photon counting module through the fourth focusing lens, the fifth focusing lens and the sixth focusing lens.
[0020] In some of the embodiments, the focusing lens group further includes a pinhole, and the third focusing lens, the fourth focusing lens and the pinhole constitute a confocal system.
[0021] In some embodiments, the reference light has the same numerical aperture as the scattered light when it is received by the third focusing lens after passing through the first beam expanding lens.
[0022] In some embodiments, the single photon counting module is a photon detection module using a silicon-based avalanche diode as a detector.
[0023] The second purpose of the present application is to provide a single-molecule small protein detection system, including the high-throughput optical mass spectrometer and the sheath flow device, wherein the molecular sample liquid to be tested and the sheath liquid pass through the sheath flow device, wherein the molecular sample liquid to be tested flows in the center of the sheath flow device, and the sheath liquid flows around the sheath flow device and squeezes the molecular sample liquid to be tested, so that the molecular sample liquid to be tested forms a single arranged molecular flow in the middle of the sheath flow device, and the molecular flow is sequentially irradiated by the light source of the high-throughput optical mass spectrometer, thereby realizing the detection of single molecules.
[0024] In some embodiments, the size of the molecular sample solution to be detected is smaller than the spot size of the reference light.
[0025] In some embodiments, the sheath flow device includes a sheath flow cell or a capillary or other forms of micro-flow channels.
[0026] This application adopts the above technical solution, and its beneficial effects are as follows:
[0027] The high-throughput optical mass spectrometer and single-molecule small protein detection system provided by the present application, the laser beam emitted by the light source is divided into reference light and scattered light through the beam splitter; the reference light sequentially passes through the half glass slide and the first plane mirror into the first beam expanding lens, the first beam expanding lens controls the spot size of the incident reference light, and then enters the second plane mirror through the second beam expanding lens, and enters the adjustable attenuator after being reflected by the second plane mirror, and then enters the first focusing lens after controlling the optical power of the incident reference light; the scattered light enters the second focusing lens after passing through the plane mirror group; The focusing lens group superimposes and interferes the reference light passing through the first focusing lens and the scattered light passing through the second focusing lens, and then the light is received by the single-photon counting module. Compared with flow cytometry for high-throughput biological molecule detection, the high-throughput optical mass spectrometer and single-molecule small protein detection system provided by the present application no longer rely solely on detecting scattered light signals, but introduces reference light to coherently amplify the scattered light, thereby reducing the size detection limit of the system; in addition, compared with the mass photometry technology for detecting small-sized biological molecules by imaging, the present application abandons the detection principle of imaging, thereby obtaining a shorter detection time and improving the detection throughput of the system. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments of the present application or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
[0029] Figure 1 A schematic diagram of the structure of a high-throughput optical mass spectrometer provided in an embodiment of the present application. DETAILED DESCRIPTION
[0030] Embodiments of the present application are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present application, and should not be construed as limiting the present application.
[0031] In the description of the present application, it should be understood that the terms "upper", "lower", "horizontal", "inside", "outside", etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present application.
[0032] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of this application, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.
[0033] In order to make the objectives, technical solutions and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments.
[0034] See also Figure 1 , is a schematic diagram of the structure of a high-throughput optical mass spectrometer provided in an embodiment of the present application, comprising: a reference light module 100, a scattered light module 200 and an interference module 300. The technical solution for its implementation is described in detail below.
[0035] The reference light module 100 includes a light source 110 , a beam splitter 120 , a half glass 130 , a first plane mirror 140 , a first beam expander lens 150 , a second beam expander lens 160 , a second plane mirror 170 , an adjustable attenuator 180 and a first focusing lens 190 .
[0036] The scattered light module 200 includes the light source 110 , the beam splitter 120 , a plane lens assembly 210 , and a second focusing lens 220 .
[0037] The interference module 300 includes a focusing lens group 310 and a single photon counting module 320 .
[0038] The high-throughput optical mass spectrometer provided in the above embodiment of the present application works as follows:
[0039] The laser beam emitted by the light source 110 is split into reference light and scattered light by the beam splitter 120 .
[0040] It should be noted that: after the laser beam emitted by the light source 110 passes through the beam splitter 120, a part of the beam is reflected by the beam splitter 120 to form a reference light; the other part of the beam is transmitted by the beam splitter 120 to form a scattered light. The function of the reference light part is to generate a path with attenuated intensity and higher intensity than the scattered light. The reference light signal does not carry the size information of the particles, and is only used to provide a stable light signal that interferes with the scattered light of the particles.
[0041] The reference light enters the first beam expanding lens 150 in sequence through the half glass slide 130 and the first plane mirror 140. The first beam expanding lens 150 controls the spot size of the incident reference light. The reference light enters the second plane mirror 170 through the second beam expanding lens 160 and enters the adjustable attenuator 180 after being reflected by the second plane mirror 170. The adjustable attenuator 180 controls the optical power of the incident reference light before entering the first focusing lens 190.
[0042] It can be understood that the purpose of introducing the adjustable attenuator 180 in the optical path system of this embodiment is to control the optical power of the reference light so that it has sufficient amplification effect after interference and superposition with the scattered light, and the final detectable photon number of the system does not exceed the saturation photon number of the single photon counting module.
[0043] It can be understood that the present embodiment introduces the first beam expanding lens 150 and the second beam expanding lens 160 into the optical path system. The first beam expanding lens 150 and the second beam expanding lens 160 form a beam expander to control the spot size of the reference light so that the reference light has the same numerical aperture as the scattered light when it passes through the first focusing lens 190 and is received by the focusing lens group 310, so that the two beams of light interfere better.
[0044] The scattered light enters the second focusing lens 220 after passing through the plane mirror assembly 210 .
[0045] In this embodiment, the plane mirror group 210 includes a third plane mirror 211, a fourth plane mirror 212, a fifth plane mirror 213 and a sixth plane mirror 214. The scattered light enters the second focusing lens 220 after passing through the third plane mirror 211, the fourth plane mirror 212, the fifth plane mirror 213 and the sixth plane mirror 214 in sequence.
[0046] In this embodiment, the fifth plane mirror 213 and the sixth plane mirror 214 are arranged on a displacement stage (not shown), and the fifth plane mirror 213 and the sixth plane mirror 214 can be moved on the displacement stage. The positions of the fifth plane mirror 213 and the sixth plane mirror 214 are adjusted to adjust the optical path of the scattered light so that it is consistent with the optical path of the reference light.
[0047] The focusing lens group 310 performs superposition interference on the reference light passing through the first focusing lens 190 and the scattered light passing through the second focusing lens 220 , and the light is then received by the single photon counting module 320 .
[0048] It can be understood that the optical power P received by the system det It can be expressed by the following formula
[0049] P det =P ref +P int +P scat
[0050] Among them, P ref is the optical power of the reference light, P int is the interference light power generated by the interference of the reference light and the scattered light, P scat is the light power scattered by the biomolecules, without considering the phase,
[0051] In this embodiment, the focusing lens group 310 includes a third focusing lens 311, a fourth focusing lens 312, a fifth focusing lens 313 and a sixth focusing lens 314. The reference light through the first focusing lens 190 and the scattered light through the second focusing lens 220 enter the third focusing lens 311, and an interference light signal is formed through the third focusing lens 311. The interference light signal is then acquired by the single photon counting module 320 through the fourth focusing lens 312, the fifth focusing lens 313 and the sixth focusing lens 314.
[0052] It can be understood that the interference module 300 provided in this embodiment is mainly composed of a third focusing lens 311, a fourth focusing lens 312, a fifth focusing lens 313 and a sixth focusing lens 314 and a single photon counting module 320, wherein the third focusing lens 311 is an objective lens responsible for collecting the light signal scattered by the particles and the reference light signal emitted through the first focusing lens 190.
[0053] Furthermore, the focusing lens group 310 further includes a pinhole 315 , and the third focusing lens 311 , the fourth focusing lens 312 and the pinhole 315 constitute a confocal system.
[0054] It can be understood that since the third focusing lens 311, the fourth focusing lens 312 and the pinhole constitute a confocal system, the stray ambient light around the focal point of the light spot can be filtered out. hole =f2 / f1*r paticle , f1 and f2 are respectively the focal lengths of the third focusing lens 311 and the fourth focusing lens 312. After the interference light signal passes through the fourth focusing lens 312, it passes through the fifth focusing lens 313 and the sixth focusing lens 314 and is received by the single photon counting module 320, and then uniformly processed.
[0055] In this embodiment, the single photon counting module 320 is a photon detection module using a silicon-based avalanche diode as a detector, and has an ultra-high photon detection sensitivity.
[0056] In some embodiments, a group of specific embodiments of the key components of the above-mentioned optical system are given: 1) Laser: 405nm long coherence laser (Changchun New Industries Laser: MDL-C-405); 2) Detector (Excelitas: SPCM-AQRH-14-TR); 3) Spectrum BS: intensity spectrometry T:R=99 / 1; 4) The first focusing lens / the third focusing lens is Nikon CFI60; 5) The focal length of the second focusing lens: 12.5mm; 6) The focal length of the third focusing lens: 250mm; 7) Pinhole diameter: 50μm.
[0057] It should be noted that: the high-throughput optical mass spectrometer provided in the present application, based on the principle of interference scattering, can be regarded as consisting of three parts: a reference light module, a scattered light module and an interference module. The function of the reference light part is to generate a reference light with attenuated intensity and an intensity higher than the intensity of the scattered light. This part of the light signal does not carry the size information of the particles and is only used to provide a stable light signal that interferes with the scattered light of the particles; the function of the scattered light module is to focus the laser emitted by the light source to the particles to be measured, so that the light is Rayleigh scattered in all directions under the action of the particles, and this scattered light intensity carries the size signal of the particles; the core function of the interference module is to superimpose and interfere the reference light with the scattered light, so as to achieve the amplification effect of the scattered light signal.
[0058] It should be pointed out that the high-throughput optical mass spectrometer provided in this application only provides a system implementation embodiment by constructing a reference light and a light scattered by a biomolecule to interfere with each other. There are other light path implementation methods for constructing a reference light, and the spatial structure of the light path is not limited to the structure shown in the figure. Since the implementation methods of the light path vary greatly, it is impossible for this material to traverse all possibilities, but the core idea is to achieve the amplification effect of the scattered light signal by artificially constructing a reference light beam for interfering with the scattered light of the biomolecule.
[0059] The present application also provides a single-molecule small protein detection system, including the high-throughput optical mass spectrometer and the sheath flow device, wherein the molecular sample liquid to be detected and the sheath liquid pass through the sheath flow device, the molecular sample liquid to be detected flows in the center of the sheath flow device, the sheath liquid flows around the sheath flow device and squeezes the molecular sample liquid to be detected, so that the molecular sample liquid to be detected forms a single arranged molecular flow in the middle of the sheath flow device, and the molecular flow is sequentially irradiated by the light source of the high-throughput optical mass spectrometer, thereby realizing the detection of single molecules.
[0060] It can be understood that sheath flow is a technology that controls the flow of biomolecules in liquid. A capillary or pinhole device is used to align the small hole tube, and the cell suspension is ejected from the capillary. At the same time, it flows through the sensitive area together with the sheath liquid flowing out from all sides, which can ensure that the sample flow where the biomolecule is located is surrounded by sheath liquid, forming a single arranged molecular flow in the middle, thereby realizing the detection of single molecules.
[0061] It should be noted that the device through which the biomolecules flow in the above embodiment is a sheath flow cell, but it is also feasible to use other devices that can generate sheath flow, such as capillaries, or other forms of micro-channels. The core function of the device is to restrict the biomolecules from passing through one by one under the light spot by means of fluid dynamics.
[0062] In this embodiment, the size of the molecular sample liquid to be tested is smaller than the spot size of the reference light, and under the action of a high-throughput optical mass spectrometer, imaging of a single arranged molecular flow of the molecular sample liquid to be tested can be achieved.
[0063] Compared with flow cytometry for high-throughput biomolecule detection, the high-throughput optical mass spectrometer and single-molecule small protein detection system provided by the present application no longer rely solely on detecting scattered light signals, but instead introduce reference light to coherently amplify the scattered light, thereby reducing the size detection limit of the system; in addition, compared with mass photometry technology for detecting small-sized biomolecules by imaging, the present application abandons the detection principle of imaging, thereby obtaining a shorter detection time and improving the detection throughput of the system.
[0064] The above are only preferred embodiments of the present application, and only specifically describe the technical principles of the present application. These descriptions are only for explaining the principles of the present application and cannot be interpreted as limiting the scope of protection of the present application in any way. Based on the explanation here, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application, and other specific implementation methods of the present application that can be associated with the technicians in this field without creative work, should be included in the scope of protection of the present application.
Claims
1. A high-throughput optical mass spectrometer, characterized in that: include: Reference light module, scattered light module and interference module; The reference light module includes a light source, a beam splitter, a half glass slide, a first plane mirror, a first beam expander lens, a second beam expander lens, a second plane mirror, an adjustable attenuator and a first focusing lens; The scattered light module includes the light source, the beam splitter, a plane mirror group and a second focusing lens; The interference module includes a focusing lens group and a single photon counting module; The laser beam emitted by the light source is split into reference light and scattered light by the beam splitter; The reference light sequentially passes through the half glass slide and the first plane mirror and enters the first beam expanding lens, the first beam expanding lens controls the spot size of the incident reference light, and then enters the second plane mirror through the second beam expanding lens, and enters the adjustable attenuator after being reflected by the second plane mirror, and then enters the first focusing lens after the adjustable attenuator controls the optical power of the incident reference light; The scattered light enters the second focusing lens after the optical path is controlled by the plane mirror group; The focusing lens group performs superposition interference on the reference light passing through the first focusing lens and the scattered light passing through the second focusing lens, and then the light is received by the single photon counting module.
2. The high-throughput optical mass spectrometer according to claim 1, characterized in that: The plane mirror group includes a third plane mirror, a fourth plane mirror, a fifth plane mirror and a sixth plane mirror. The scattered light enters the second focusing lens after passing through the third plane mirror, the fourth plane mirror, the fifth plane mirror and the sixth plane mirror in sequence.
3. The high-throughput optical mass spectrometer according to claim 2, characterized in that: The fifth plane mirror and the sixth plane mirror are arranged on a displacement platform, and the fifth plane mirror and the sixth plane mirror can be moved on the displacement platform. The optical path of the scattered light path can be adjusted by adjusting the positions of the fifth plane mirror and the sixth plane mirror to make it consistent with the optical path of the reference light path.
4. The high-throughput optical mass spectrometer according to claim 1, characterized in that: The focusing lens group includes a third focusing lens, a fourth focusing lens, a fifth focusing lens and a sixth focusing lens. The reference light passing through the first focusing lens and the scattered light passing through the second focusing lens enter the third focusing lens and form an interference light signal through the third focusing lens. The interference light signal is then acquired by the single-photon counting module through the fourth focusing lens, the fifth focusing lens and the sixth focusing lens.
5. The high-throughput optical mass spectrometer according to claim 4, characterized in that: The focusing lens group also includes a pinhole, and the third focusing lens, the fourth focusing lens and the pinhole constitute a confocal system.
6. The high-throughput optical mass spectrometer according to claim 4, characterized in that: After passing through the first beam expanding lens, the reference light has the same numerical aperture as the scattered light when received by the third focusing lens.
7. The high-throughput optical mass spectrometer according to claim 1, characterized in that: The single photon counting module is a photon detection module using a silicon-based avalanche diode as a detector.
8. A single molecule small protein detection system, characterized in that: It comprises the high-throughput optical mass spectrometer and sheath flow device as described in claim 1, wherein the molecular sample liquid to be measured and the sheath liquid pass through the sheath flow device, wherein the molecular sample liquid to be measured flows in the center of the sheath flow device, the sheath liquid flows around the sheath flow device and squeezes the molecular sample liquid to be measured, and makes the molecular sample liquid to be measured form a single arranged molecular flow in the middle of the sheath flow device, and the molecular flow is sequentially irradiated by the light source of the high-throughput optical mass spectrometer, thereby realizing the detection of single molecules.
9. The single-molecule small protein detection system according to claim 8, characterized in that: The size of the molecular sample liquid to be detected is smaller than the spot size of the reference light.
10. The single-molecule small protein detection system according to claim 8, characterized in that: The sheath flow device includes a sheath flow pool or a capillary or other forms of micro flow channels.