CN112904013A - High-energy ultraviolet crosslinking instrument for biological sample - Google Patents
High-energy ultraviolet crosslinking instrument for biological sample Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention relates to a high-energy ultraviolet crosslinking instrument for biological samples, which comprises: (1) a 365nm LED light source; (2) a biological specimen cooling system; (3) biological sample height adjustment platform. The LED light source is provided with a corresponding heat dissipation system, ultraviolet light beams emitted by the LED light source are gathered through the plano-convex lens, and high-energy light is irradiated on a biological sample. The biological sample is placed on a biological sample cooling system platform to reduce the influence on a crosslinking system caused by heat generated by strong ultraviolet irradiation. 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 cross-linking reaction. The invention has the advantages that the high energy and low heat are realized, the LED light source provides high-energy-density ultraviolet light beams to ensure the crosslinking reaction effect, and meanwhile, the biological sample cooling system ensures the physiological condition of a biological sample and maintains the physiological activity of the biological sample by reducing the temperature of a crosslinking system, thereby providing important instrument support for developing a photocrosslinking living experiment.
Description
Technical Field
The invention relates to a high-energy ultraviolet crosslinking instrument for a biological sample, 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 ensuring the physiological condition of the biological sample and maintaining the physiological activity of the biological sample by reducing the temperature of the crosslinking system, 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 time-space controllable crosslinking tool. In chemical biological analysis, photocrosslinking is a powerful tool for studying the fields of protein-protein interactions (PPIs), protein labeling, and the like.
Protein-protein interactions (PPIs) trigger a broad biological signaling pathway that 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 labeling (PAL). PAL has become one of the most powerful strategies for studying PPIs (Dorming G, Prestwich GD. perspectives in biochemistry.1994; Gubbens J, de Kroon AI. protome-wide detection of phospholipd-protein interactions in mitochondria by photocrosslinking and click chemistry. mol biochemistry.2010; 6: 1751-9.). Conventional photocrosslinkers are used for benzophenones (benzophenones), arylazides (aryl azides) and diazooxazines (diazorine). Under light irradiation, these photocrosslinkers generate highly reactive species that react with neighboring molecules, resulting in direct covalent modification. Due to the ability of PAL to capture non-covalent interaction partners spatially selectively, photocrosslinking has become an important tool for studying PPIs. Photocrosslinking has not only been used in Proteomics research to produce great diversity, but also has been widely used in studies such as living Cell Imaging and drug targeting (Yao S Q, Pan S, Jang S Y, et al. A Suite of "Minimalist", "Photo-Crosslinkers for Live-Cell Imaging and Chemical Proteomics: Case Study with BRD4 Inhibitors [ J ]. Angewandte Chemical Edition, 2017.).
However, photo-crosslinking techniques have a significant limitation: the biological (cellular) toxicity inevitably generated by the illumination includes the phototoxicity of an ultraviolet light source and the influence of heat generated by the illumination on the activity of cells. The conventional photo-crosslinking instrument has two main disadvantages: low light energy density and light exposure generates a lot of heat. This results in a limitation of the photocrosslinking reaction in the biological sample, for example, the extraction of cell membrane proteins.
In the patent, a high-energy LED ultraviolet light source, a biological sample cooling system and a biological sample height adjusting platform are utilized to build a high-energy low-heat ultraviolet crosslinking instrument. Due to the advantage of high energy density, the short illumination time is realized to obtain enough photo-crosslinking energy, so that the influence of phototoxicity on a biological sample is reduced; meanwhile, the excellent cooling performance of the crosslinking instrument is utilized, and the influence on the pair of biological samples caused by heat generated by illumination 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 a biological sample, 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 ensuring the physiological condition of the biological sample and maintaining the physiological activity of the biological sample by reducing the temperature of the crosslinking system, 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 purpose, the invention adopts the technical scheme that:
the instrument includes: (1) a 365nm LED light source; (2) a biological specimen cooling system; (3) biological sample height adjustment platform. None of the three should be considered. 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 number of lamp strains and the 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 that the influence of heat generated by ultraviolet irradiation on the biological sample is reduced. The biological sample pool height adjusting platform can adjust the distance between the crosslinking system and the light source according to the requirements 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 the 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 3-10W 365nmLED lamp strains, and the number of the lamp strains can be configured from 1 to 100 according to the requirement of a biological sample on energy. The biological sample cooling system adopts 12706 semiconductor refrigerating sheets. This refrigeration piece during operation one end refrigeration and the other end can be exothermic, only can guarantee the refrigeration effect of another side to the one side cooling that generates heat, consequently at the fan of the outside installation of the surface of generating heat, cools off the surface of generating heat. The temperature difference between the cold surface and the hot surface can be within 15 ℃. The biological sample pool height adjusting platform is in a spiral lifting type.
The 365nm LED light source has the advantages that the number of the lamp strains is directly related to the released energy and the heat, and a corresponding heat dissipation system can be configured according to the level of the released heat. The heat dissipation system is composed of an aluminum heat dissipation fin and a heat dissipation fan. The reaction liquid cooling system is used for enabling the temperature of the semiconductor refrigerating piece to be quickly transferred to a reaction liquid contact surface, the semiconductor refrigerating piece is covered and compacted by an aluminum plate in close contact, and the heat conducting silica gel is added in the middle of the semiconductor refrigerating piece, 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 the scattered light emitted by the LED lamp, so that the scattered light source is as close to a parallel light source as possible. Biological sample cooling system places temperature sensor with aluminum plate and biological sample centre on the semiconductor refrigeration piece to convenient monitoring refrigeration piece is to biological sample cooling effect, and observes the observation temperature change condition through the liquid crystal display outside the casing.
The invention has the following advantages:
1. the advantage of high energy density realizes shorter illumination time to obtain enough photocrosslinking energy, thereby reducing the influence of phototoxicity on the biological sample.
2. The cross-linking instrument has excellent cooling performance, and reduces the influence on the pair of biological samples caused by heat generated by illumination. The method has the advantages of simple operation steps, short time consumption and high flux, and can realize large-scale identification of the interaction of the plasma membrane protein.
3. 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 cross-linking reaction.
4. The method has simple operation steps and short time consumption, and can realize large-scale photo-crosslinking of the biological sample.
Drawings
FIG. 1 is a schematic structural diagram of a high-energy ultraviolet crosslinking instrument
Detailed Description
Example I light crosslinking experiment on bovine serum albumin
(1) Pre-cooling by an ultraviolet crosslinking instrument: and starting a biological sample cooling system, and cooling for 10 min. The liquid crystal temperature shows that the plateau temperature drops from 25.6 ℃ at room temperature to 14.0 ℃.
(2) Adjusting the height of the biological sample height adjusting platform: the ultraviolet energy measuring instrument is used in combination, and the illumination energy density is selected to be 1000mW/cm2The optimal platform height.
(3) Photo-crosslinking reaction: keeping the biological cooling system open, placing the photo-crosslinking reaction sample-bovine serum albumin solution on a biological sample height adjusting platform, and precooling for 1 min. And (4) turning on a 365nmLED light source, and illuminating for 2 min. The liquid crystal temperature shows that the plateau temperature rises from 14 ℃ to 20.0 ℃.
(4) Illumination time gradient comparison: and (5) turning off the light source, and taking out the reacted bovine serum albumin solution. The biological cooling system is kept open and cooled for 1-3min (temperature is shown: 14.0 ℃). Placing HELA cell culture solution, turning on 365nm LED light source, and illuminating for 5 min. Displaying the liquid crystal temperature: the plateau temperature rose from 14 ℃ to 28.4 ℃.
(5) And (3) identifying the photocrosslinking efficiency: and (3) extracting protein, pretreating the obtained cross-linked protein sample, and performing MS identification. The obtained mass spectrum data is subjected to cross-linking site retrieval by using pLink2 software, and the statistical photocrosslinking efficiency is 90%.
Example two in situ photocrosslinking experiments on HELA cellular proteins
(3) Pre-cooling by an ultraviolet crosslinking instrument: and starting a biological sample cooling system, and cooling for 10 min. The liquid crystal temperature shows that the plateau temperature drops from 25.6 ℃ at room temperature to 14.0 ℃.
(4) Adjusting the height of the biological sample height adjusting platform: the ultraviolet energy measuring instrument is used in combination, and the illumination energy density is selected to be 1000mW/cm2The optimal platform height.
(3) Photo-crosslinking reaction: keeping the biological cooling system on, placing the HELA cell culture solution as the photo-crosslinking reaction sample on a biological sample height adjusting platform, and precooling for 1 min. And (4) turning on a 365nmLED light source, and illuminating for 2 min. The liquid crystal temperature shows that the plateau temperature rises from 14 ℃ to 20.0 ℃.
(4) Illumination time gradient comparison: and (5) turning off the light source, and taking out the reacted HELA cell culture solution. The biological cooling system is kept open and cooled for 1-3min (temperature is shown: 14.0 ℃). Placing HELA cell culture solution, turning on 365nm LED light source, and illuminating for 5 min. Displaying the liquid crystal temperature: the plateau temperature rose from 14 ℃ to 28.4 ℃.
(5) Observation of the cell state: the cell morphology was observed by microscopy, and most of the HELA cells maintained spindle cell morphology as well as adherent state, demonstrating that the cell survival rate was sufficiently high.
(6) And (3) identifying the photocrosslinking efficiency: and (3) extracting protein, pretreating the obtained cross-linked protein sample, and performing MS identification. The obtained mass spectrum data are searched by using pLink2 software crosslinking sites, and the statistical photocrosslinking efficiency is 89%.
Claims (4)
1. A high energy uv cross-linking apparatus for biological samples, the apparatus comprising: (1) a 365nm LED light source; (2) a biological specimen cooling system; (3) biological sample cell 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 pool, a semiconductor refrigeration block is arranged below the metal heat conduction platform, and the cold end of the semiconductor refrigeration block is tightly connected with the lower surface of the metal heat conduction platform so as to reduce the influence of heat generated by ultraviolet irradiation on the biological sample; a fan for radiating heat of the hot end is arranged at the hot end of the semiconductor refrigeration block; the light beam irradiates on the sample in the biological sample pool from the upper part of the biological sample pool;
and a biological sample pool height adjusting platform is arranged below the biological sample cooling system and can drive the biological sample pool cooling system to move up and down to adjust the distance between the biological sample and the light source.
2. The ultraviolet crosslinking apparatus of claim 1, wherein: the 365nm LED light source comprises a group of 3-10W 365nm LED lamp strains, and the number of the lamp strains can be configured according to the requirement of a biological sample on energy and is from 1 to 100;
the biological sample cooling system adopts 12706 semiconductor refrigerating sheets; when the refrigerating sheet works, one end of the refrigerating sheet refrigerates and the other end of the refrigerating sheet releases heat, and the refrigerating effect of the other side can be ensured only by cooling the heating side, so that a fan is arranged outside the heat releasing side to cool the heating side; the temperature difference between the cold surface and the hot surface is less than 15 ℃.
3. The biological sample cell height adjustment platform is a spiral lifting type, and the ultraviolet crosslinking instrument according to claim 1 is characterized in that: the 365nm LED light source has the direct relation between the number of the lamp strains and the released energy and heat, and a corresponding heat dissipation system can be configured according to the height of the released heat; the heat dissipation system consists of an aluminum heat dissipation fin and a heat dissipation fan;
in order to enable the temperature of the semiconductor refrigerating block to be quickly transferred to a reaction liquid contact surface, the biological sample cooling system is covered and compacted by an aluminum plate in close contact with the semiconductor refrigerating piece, and the heat conducting silica gel is added in the middle of the semiconductor refrigerating piece, so that the temperature of the refrigerating block is well transferred to the surface.
4. The uv cross-linking apparatus of claim 1, wherein the light from the light source is collected by a plano-convex lens to diffuse the scattered light from the LED lamp, such that the scattered light source is as close as possible to a collimated light source;
in the biological sample cooling system, a temperature sensor is arranged between a metal heat conduction platform (such as an aluminum plate) and a biological sample pool on a semiconductor refrigeration block so as to conveniently monitor the cooling effect of the refrigeration piece on the biological sample, and a temperature signal output by the temperature sensor is displayed and observed through a liquid crystal outside a shell so as to observe the temperature change condition.
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