CN112904013B - High-energy ultraviolet cross-linking instrument for biological sample - Google Patents
High-energy ultraviolet cross-linking instrument for biological sample Download PDFInfo
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- CN112904013B CN112904013B CN201911229070.3A CN201911229070A CN112904013B CN 112904013 B CN112904013 B CN 112904013B CN 201911229070 A CN201911229070 A CN 201911229070A CN 112904013 B CN112904013 B CN 112904013B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/76—Assays involving albumins other than in routine use for blocking surfaces or for anchoring haptens during immunisation
- G01N2333/765—Serum albumin, e.g. HSA
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/14—Measures for saving energy, e.g. in green houses
Abstract
The invention relates to a high energy ultraviolet cross-linking instrument for biological samples, comprising: (1) 365nm LED light source; (2) a biological sample cooling system; (3) a biological sample height adjustment platform. The LED light source is provided with a corresponding heat dissipation system, the ultraviolet light beams emitted by the LED light source are gathered through the plano-convex lens, and high energy is irradiated on the biological sample. The biological sample is placed on a biological sample cooling system platform so as to reduce the influence on a crosslinking system caused by heat generated by irradiation of strong ultraviolet light. The biological sample height adjustment platform can adjust the distance between the reaction system and the light source according to the energy required by the crosslinking reaction. The invention has the advantages that the high energy and low heat are realized, the LED light source provides the ultraviolet light beam with high energy density to ensure the crosslinking reaction effect, and the biological sample cooling system reduces the temperature of the crosslinking system to ensure the physiological condition of the biological sample and maintain the physiological activity of the biological sample, so that the invention provides an important instrument support for developing the photocrosslinking living experiment.
Description
Technical Field
The invention relates to a high-energy ultraviolet crosslinking instrument for biological samples, which uses a high-energy LED light source to provide high-energy density ultraviolet light beams for a crosslinking system so as to ensure the crosslinking reaction effect. Meanwhile, the biological sample cooling system is used for reducing the temperature of the crosslinking system to ensure the physiological condition of the biological sample and maintain the physiological activity of the biological sample, and the biological sample height adjusting platform can adjust the distance between the reaction system and the light source according to the energy required by the crosslinking reaction. Provides important instrument support for developing photocrosslinking in-vivo experiments.
Background
The photo-crosslinking reaction is widely applied to different research fields of chemistry, biology, medicine, materials and the like as a rapid, simple and space-time controllable crosslinking tool. In chemical biological analysis, photocrosslinking is a powerful tool in the fields of studying protein-protein interactions (PPIs), protein labeling, and the like.
Protein-protein interactions (PPIs) trigger a broad biological signaling pathway, which is critical for biomedical research and drug discovery (Jin L, wang W, fang G.targeting protein-protein interaction by small molecules Annu Rev Pharmacol Toxicol.2014; 54:435-56). Various techniques have been used to study specific proteins, including affinity chromatography, active probes, affinity probes, and photoaffinity labels (PALs). PAL has become one of the most powerful strategies to study PPIs (dorstin G, preswich GD. Perselect in biochemistry.1994; gubbens J, de Kroon AI. Proteome-wide detection of phospholipid-protein interactions in mitochondria by photocrosslinking and click chemistry.mol BioSyst.2010; 6:1751-9.). Conventional photocrosslinkers are used for benzophenones (benzophenones), aryl azides (aryl azides) and diazozines (diazoines). Under light irradiation, these photocrosslinkers produce highly reactive species that react with neighboring molecules, resulting in direct covalent modification. Because PAL is capable of sterically capturing non-covalent interaction partners, photocrosslinking has become an important tool in the study of PPIs. Photocrosslinking is widely used not only in the field of proteomics research but also in living cell imaging, drug targeting and other studies (Yao S Q, pan S, jang S Y, et al A Suite of\Mini ist\photo-Crosslinkers for Live-Cell Imaging and Chemical Proteomics: case Study with BRD Inhibitors [ J ] Angewandte Chemie International Edition, 2017.).
However, there is a significant limitation to the photocrosslinking technique: the biological (cellular) toxicity caused by illumination inevitably includes phototoxicity of ultraviolet light sources and the influence of heat generated by illumination on cell activity. The conventional photo-crosslinking apparatus has two main disadvantages: the light energy density is low and the illumination generates a lot of heat. Thereby causing the photocrosslinking reaction to be limited in the biological sample, for example, extraction of cell membrane proteins.
According to the patent, a high-energy LED ultraviolet light source, a biological sample cooling system and a biological sample height adjusting platform are utilized, and a high-energy low-heat ultraviolet cross-linking instrument is built. Due to the advantage of high energy density, the method realizes that enough photocrosslinking energy is obtained in shorter illumination time, thereby reducing the influence of phototoxicity on biological samples; meanwhile, the excellent cooling performance of the cross-linking instrument is utilized, so that the influence on biological samples caused by illumination heat generation is reduced. In a word, the invention can ensure the physiological condition of the biological sample and maintain the physiological activity of the biological sample while ensuring the crosslinking reaction effect, and provides important instrument support for developing photocrosslinking in-vivo experiments.
Disclosure of Invention
The invention relates to a high-energy ultraviolet crosslinking instrument for biological samples, which uses a high-energy LED light source to provide high-energy density ultraviolet light beams for a crosslinking system so as to ensure the crosslinking reaction effect. Meanwhile, the biological sample cooling system is used for reducing the temperature of the crosslinking system to ensure the physiological condition of the biological sample and maintain the physiological activity of the biological sample, and the biological sample height adjusting platform can adjust the distance between the reaction system and the light source according to the energy required by the crosslinking reaction. Provides important instrument support for developing photocrosslinking in-vivo experiments.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the instrument includes: (1) 365nm LED light source; (2) a biological sample cooling system; (3) a biological sample height adjustment platform. The three are indispensable. The method is characterized in that: the 365nm LED light source comprises a group of 365nm LED lamps, a corresponding heat dissipation system is configured according to the quantity of lamp strains and released energy and heat, and a plano-convex lens is arranged in front of light beams emitted by the LEDs for gathering scattered light. The biological sample liquid cooling system is a semiconductor cooling device arranged on a platform for placing an irradiated sample, so as to reduce the influence on a biological sample caused by heat generated by ultraviolet irradiation. The biological sample cell height adjustment platform can adjust the distance between the crosslinking system and the light source according to the requirement of crosslinking energy. The biological sample cooling system ensures the physiological condition of the biological sample and maintains the physiological activity of the biological sample by reducing the temperature of a crosslinking system while the LED light source provides high-energy-density ultraviolet light beams to ensure the crosslinking reaction effect.
The 365nmLED light source comprises a group of 365nmLED light strains with the width of 3-10W, and the number of the light strains can be configured according to the energy requirement of biological samples from 1 to 100. The biological sample cooling system adopts 12706 semiconductor refrigerating sheets. When the refrigerating sheet works, one end refrigerates and the other end releases heat, and the refrigerating effect of the other side can be guaranteed only by cooling the heating surface, so that a fan is arranged outside the heating surface to cool the heating surface. The temperature of the cold and hot surfaces can be within 15 ℃. The biological sample tank height adjusting platform is a spiral lifting type platform.
The 365nmLED light source has direct relation between the number of lamp strains and the released energy and heat, and a corresponding heat dissipation system can be configured according to the released heat. The heat dissipation system consists of aluminum heat dissipation fins and a heat dissipation fan. In order to enable the temperature of the semiconductor refrigerating piece to be quickly transferred to the contact surface of the reaction liquid, the upper surface of the semiconductor refrigerating piece is tightly covered and compacted by an aluminum plate, and heat-conducting silica gel is added in the middle, so that the temperature of the refrigerating piece is well transferred to the surface.
In front of the light source, a plano-convex lens is used for gathering scattered light emitted by the LED lamp strain, so that the scattered light source is as close to the parallel light source as possible. The biological sample cooling system is characterized in that a temperature sensor is arranged between the aluminum plate and the biological sample on the semiconductor refrigerating piece, so that the cooling effect of the refrigerating piece on the biological sample is conveniently monitored, and the temperature change condition is observed through liquid crystal display outside the shell.
The invention has the following advantages:
1. the advantage of high energy density is that a shorter illumination time is achieved to obtain sufficient photo-crosslinking energy, thereby reducing the effect of phototoxicity on biological samples.
2. The cross-linking instrument has excellent cooling performance, and reduces the influence of light irradiation heat generation on biological samples. The method has the advantages of simple operation steps, short time consumption and high flux, and can realize the large-scale identification of plasma membrane protein interaction.
3. The biological sample height adjustment platform can adjust the distance between the reaction system and the light source according to the energy required by the crosslinking reaction.
4. The method has the advantages of simple operation steps and short time consumption, and can realize large-scale photocrosslinking of biological samples.
Drawings
FIG. 1 is a schematic view of a high energy ultraviolet cross-linking instrument
Detailed Description
Example one photo-crosslinking experiment on bovine serum Albumin
(1) Precooling by an ultraviolet crosslinking instrument: and starting a biological sample cooling system, and cooling for 10min. The liquid crystal temperature showed that the plateau temperature was reduced from room temperature 25.6 ℃ to 14.0 ℃.
(2) Adjusting the height of the biological sample height adjustment platform: in combination with an ultraviolet energy meter, the illumination energy density is selected to be 1000mW/cm 2 Is a preferred platform height.
(3) Photo-crosslinking reaction: keeping the biological cooling system on, placing the photo-crosslinking reaction sample, namely bovine serum albumin solution, on a biological sample height adjustment platform, and precooling for 1min. And turning on a 365nmLED light source, and illuminating for 2min. The liquid crystal temperature showed that the plateau temperature was raised from 14 ℃ to 20.0 ℃.
(4) Illumination time gradient control: and turning off the light source, and taking out the reacted bovine serum albumin solution. The biological cooling system is kept on and cooled for 1-3min (temperature display: 14.0 ℃). Placing HELA cell culture solution, turning on 365nmLED light source, and illuminating for 5min. And (3) displaying the liquid crystal temperature: the plateau temperature was raised from 14 ℃ to 28.4 ℃.
(5) Identification of photocrosslinking efficiency: protein extraction, pretreatment of the obtained cross-linked protein sample, and MS identification. The obtained mass spectrum data were subjected to crosslinking site search by using pLink2 software, and the statistical photocrosslinking efficiency was 90%.
Example two in situ photo-crosslinking experiments on HELA cell proteins
(3) Precooling by an ultraviolet crosslinking instrument: and starting a biological sample cooling system, and cooling for 10min. The liquid crystal temperature showed that the plateau temperature was reduced from room temperature 25.6 ℃ to 14.0 ℃.
(4) Adjusting the height of the biological sample height adjustment platform: in combination with an ultraviolet energy meter, the illumination energy density is selected to be 1000mW/cm 2 Is a preferred platform height.
(3) Photo-crosslinking reaction: and (3) keeping the biological cooling system on, placing the photocrosslinking reaction sample HELA cell culture solution on a biological sample height adjustment platform, and precooling for 1min. And turning on a 365nmLED light source, and illuminating for 2min. The liquid crystal temperature showed that the plateau temperature was raised from 14 ℃ to 20.0 ℃.
(4) Illumination time gradient control: and (5) turning off the light source, and taking out the reacted HELA cell culture solution. The biological cooling system is kept on and cooled for 1-3min (temperature display: 14.0 ℃). Placing HELA cell culture solution, turning on 365nmLED light source, and illuminating for 5min. And (3) displaying the liquid crystal temperature: the plateau temperature was raised from 14 ℃ to 28.4 ℃.
(5) Observing the cell state: when the cell morphology is observed through a microscope, most HELA cells keep the spindle-shaped cell morphology and the adherent state, and the cell survival rate is high enough.
(6) Identification of photocrosslinking efficiency: protein extraction, pretreatment of the obtained cross-linked protein sample, and MS identification. The obtained mass spectrum data were retrieved using the pll 2 software crosslinking site and the statistical photocrosslinking efficiency was 89%.
Claims (2)
1. Use of a high energy uv cross-linker for biological samples, the apparatus comprising: (1) 365nm LED light source; (2) a biological sample cooling system; (3) Biological sample pond height adjustment platform, its characterized in that:
the 365nm LED light source comprises more than 2 365nm LED lamp beads, and scattered light emitted by the LEDs is gathered through the plano-convex lens to form light beams;
the biological sample cooling system is arranged below the light source and comprises a metal heat conduction platform for placing a biological sample tank, a semiconductor refrigerating block is arranged below the metal heat conduction platform, and the cold end of the semiconductor refrigerating block is tightly connected with the lower surface of the metal heat conduction platform so as to reduce the influence on a biological sample caused by heat generated by ultraviolet irradiation; a fan for radiating the hot end is arranged at the hot end of the semiconductor refrigerating block; the light beam irradiates the sample in the biological sample cell from the upper part of the biological sample cell;
a biological sample tank height adjusting platform is arranged below the biological sample cooling system and can drive the biological sample tank cooling system to move up and down so as to adjust the distance between the biological sample and the light source; the scattered light emitted by the LED lamp strains is gathered by a plano-convex lens, so that the scattered light is as close to a parallel light source as possible;
the biological sample cooling system is characterized in that a temperature sensor is arranged between a metal heat conduction platform and a biological sample pool on the semiconductor refrigerating block so as to conveniently monitor the cooling effect of a refrigerating sheet on the biological sample, and a liquid crystal display outside the shell is used for observing a temperature signal output by the temperature sensor and observing the temperature change condition;
the 365nm LED light source comprises a group of single LED light sources3-10WThe number of the 365nm LED lamp strains can be configured according to the energy demand of the biological sample, and the number of the lamp strains is from 1 to 100;
the biological sample cooling system adopts 12706 semiconductor refrigerating sheets; one end of the refrigerating sheet is refrigerated and the other end of the refrigerating sheet can emit heat when in operation, and the refrigerating effect of the other side can be ensured only by cooling one heat-emitting side, so that a fan is arranged outside the heat-emitting side to cool the heat-emitting side; difference in temperature of cold and hot surfaces 15 o C, within the range, the biological sample tank height adjusting platform is a spiral lifting type platform;
illumination time gradient control: turning off the light source, taking out the reacted bovine serum albumin solution, keeping the biological cooling system on, cooling for 1-3min, and displaying the temperature: 14.0 o C, placing HELA cell culture solution, starting 365nmLED light source, illuminating for 5min, and displaying liquid crystal temperature: the platform temperature is from 14 o C rises to 28.4 o C, or light time gradient control: turning off the light source, taking out the reacted HELA cell culture solution, keeping the biological cooling system on, cooling for 1-3min, and displaying the temperature: 14.0 o C, placing HELA cell culture solution, starting 365nmLED light source, illuminating for 5min, and displaying liquid crystal temperature: the platform temperature is from 14 o C rises to 28.4 o C。
2. The use according to claim 1, characterized in that: the 365nm LED light source has direct relation between the quantity of lamp plants and the released energy and heat, and a corresponding heat dissipation system can be configured according to the level of the released heat; the heat dissipation system consists of aluminum heat dissipation fins and a heat dissipation fan;
in order to enable the temperature of the semiconductor refrigeration block to be quickly transferred to the contact surface of the reaction liquid, the biological sample cooling system is formed by tightly contacting and covering the upper surface of the semiconductor refrigeration piece with an aluminum plate, compacting, and adding heat-conducting silica gel in the middle, so that the temperature of the refrigeration block is well transferred to the surface.
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| CN1412320A (en) * | 2001-10-11 | 2003-04-23 | 宋克 | Joint treatment system of gene chip and its related technique |
| CN1424405A (en) * | 2001-12-14 | 2003-06-18 | 宋克 | Gene chip molecular probe and related technology |
| CN1492051A (en) * | 2002-10-04 | 2004-04-28 | 克 宋 | Gene chip combined treating system and relative technology |
| US9702823B2 (en) * | 2011-09-30 | 2017-07-11 | Life Technologies Corporation | Optical systems and methods for biological analysis |
| TW201317561A (en) * | 2011-10-19 | 2013-05-01 | Smobio Inc | Multi-exciting light source system |
| CN202448275U (en) * | 2012-03-06 | 2012-09-26 | 哈尔滨理工大学 | Light source focusing mechanism of ultraviolet light irradiation crosslinking equipment |
| ES2943159T3 (en) * | 2015-02-06 | 2023-06-09 | Life Technologies Corp | Optical instrument for biological analysis |
| CN104726330A (en) * | 2015-03-27 | 2015-06-24 | 武汉亚心医疗科技有限公司 | Photooxidation crosslinking treatment equipment of biological tissue materials |
| CN105060739B (en) * | 2015-07-31 | 2018-06-12 | 长飞光纤光缆股份有限公司 | A kind of adjustable fibre coating ultraviolet curing equipment of light intensity |
| CN109564056B (en) * | 2016-07-29 | 2021-08-27 | 索尔库德公司 | Device for optical cooling of an object |
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| 刘银春 等编.实验半导体制冷与温度传感器.《大学物理实验学》.厦门：厦门大学出版社,2006,第118-119页. * |
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