CN115629053B - High-flux protein thermal stability analyzer - Google Patents
High-flux protein thermal stability analyzer Download PDFInfo
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- CN115629053B CN115629053B CN202211022043.0A CN202211022043A CN115629053B CN 115629053 B CN115629053 B CN 115629053B CN 202211022043 A CN202211022043 A CN 202211022043A CN 115629053 B CN115629053 B CN 115629053B
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Classifications
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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
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
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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
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N25/00—Investigating or analyzing materials by the use of thermal means
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Abstract
The invention discloses a high-flux protein thermal stability analyzer, wherein a control system module is connected with an S-shaped motion detection system, the S-shaped motion detection system is connected with a multichannel fluorescence acquisition optical path system, the multichannel fluorescence acquisition optical path system is interacted with a sample system, and the multichannel fluorescence acquisition optical path system is connected with the control system module through a light intensity detection system. The invention adopts an independently designed multichannel fluorescence collection light path system, the range of an LED excitation light source is wide, the LED excitation light source can be rapidly switched between 340-390nm and 470-520nm, and the excitation light and the collection light path of a sample are realized through the same optical window by a coaxial light path system with the functions of dual-wavelength incidence and multi-wavelength fluorescence collection, and the accurate positioning and detection of the sample are realized by matching with a motion system. The semiconductor refrigerating/heating system is adopted, and meanwhile, the design of the heat-insulating sample cavity is adopted, so that the temperature control system can rapidly and accurately control the temperature.
Description
Technical Field
The invention relates to the technical field of detection, and aims to realize rapid protein thermal stability analysis by detecting the light intensity of emitted light generated by a sample under the irradiation of excitation light with a specific wavelength.
Background
The principle of the dye method protein thermal melting experiment is that protein and corresponding dye are mixed, the space conformation of the protein is destroyed in the continuous heating process, certain groups are exposed to combine with the dye to generate fluorescent signals, the fluorescent signals are collected in real time, and finally a specific fluorescent curve of the target protein in a certain solution is formed. Changes in the structure of the protein, the pH of the solution, the salt concentration or the buffer composition reflect the relative changes in the Tm values (melting temperatures) calculated therefrom for this fluorescent image. The difference between the fluorescence image and the Tm value may reflect the thermal stability of the protein, the higher the Tm value, the better the thermal stability of the protein. Therefore, the effect of mutation on the thermal stability of the protein can be analyzed according to the fluorescence images and the corresponding changes of Tm values of different mutant proteins under the same conditions; the effect of solution conditions or ligands on protein thermostability can be analyzed based on fluorescence images and corresponding changes in Tm of the same protein sample under different conditions.
Protein is a complex organic biological macromolecule, which accounts for about 50% of the dry weight of cells, is a controller and a direct executor of vital activities, and stabilization of protein is a basis for achieving all functions, so research on protein stability, understanding of the stabilization mechanism thereof has been one of the core contents in the field. Especially, the research on the thermal stability of the protein is not only a scientific and theoretical research, but also has an increasingly wide application value, such as in the fields of food, chemical industry, molecular design, biomedicine and the like, and the protein which can still keep the original functions at a higher temperature is often required. Therefore, improving the thermal stability of proteins by rational design modification of proteins, or changing the conditions of solutions in which the proteins are located, is becoming the focus of research by scientists. The protein thermal stability analyzer can rapidly screen stable protein samples or solution conditions in a high throughput manner, locks a plurality of target samples from a large number of samples, performs finer thermodynamic parameter analysis according to requirements, greatly saves analysis time and improves efficiency.
Disclosure of Invention
First, the technical problem to be solved
The invention develops a device capable of rapidly analyzing the thermal stability of protein with high flux, and is widely used for analyzing soluble protein and membrane protein. The equipment is widely applied, is suitable for analyzing the thermal stability of the protein in the fields of medicine and health, food, chemical industry and the like, and is used for screening in a high-flux mode: ligands that bind to the protein, small molecule and fragment library candidate drugs that bind to the protein target, antibodies that bind to the protein target, point mutations that improve the thermal stability of the protein, buffers that improve the thermal stability of the protein.
The protein thermal stability analyzer developed by the invention can carry out thermal stability analysis on protein samples with high flux (more than 30), multiple wavelengths (340-390 nm &470-510 nm) and wide temperature (10-100 ℃), adopts two excitation wavelengths of blue light and green light, rapidly increases and decreases the temperature, has a high flux design, can analyze membrane proteins detected by using a blue light channel and can analyze soluble proteins detected by using a green light channel, has simple experimental operation and rapid reaction, has small sample consumption, and can obtain batch sample results within 1-2 hours. The product is expected to make a major breakthrough in the technology of analyzing the thermal stability of the protein by using a thermal melting experiment.
(II) technical scheme
In order to solve the technical problems, the invention is realized by the following technical scheme:
The protein thermal stability analyzer mainly comprises an S-shaped motion detection system, a multichannel fluorescence acquisition light path system, a sample system, a light intensity detection system and a control system module. The control system module is connected with the S-shaped motion detection system, the S-shaped motion detection system is connected with the multichannel fluorescence acquisition optical path system, the multichannel fluorescence acquisition optical path system is interacted with the sample system, and the multichannel fluorescence acquisition optical path system is connected with the control system module through the light intensity detection system. The LED excitation light source fixing device is used for fixing the LED excitation light source on the coaxial light path mechanical device and fixing the coaxial light path mechanical device on the S-type motion detection system; the fixed multichannel fluorescence collection optical path system is positioned right above the sample system, and the condensing lens of the multichannel fluorescence collection optical path system is opposite to the sample system. The multichannel fluorescence acquisition light path system is fixed on the Z-axis direction of the S-shaped motion detection system and is positioned right above the sample system. The S-shaped motion detection system is characterized in that an S-shaped mobile detection platform is formed by matching three motors, the three motors control the multichannel fluorescence collection optical path system to move above the sample platform at a uniform speed in an S-shaped path, so that photoelectric collection is carried out on protein samples in the sample system, and meanwhile, accurate positioning and detection of the samples in the multichannel fluorescence collection optical path system are ensured.
Furthermore, the S-shaped motion detection system, the multichannel fluorescence acquisition light path system, the sample system, the light intensity detection system and the control system module are in modularized design.
Further, the S-shaped motion detection system is used for adjusting and controlling the sample tube of the multichannel fluorescence collection light path system corresponding to the specific position, so as to realize the detection of the fluorescent protein light intensity in the specific sample tube.
Furthermore, the multichannel fluorescence collection light path system has the characteristics of low temperature drift, low time drift and long service life, different excitation light sources can be adjusted at any time, multichannel fluorescence collection is carried out, and therefore thermal stability analysis of protein samples is carried out at high flux and multiple wavelengths (340-390 nm &470-510 nm).
Furthermore, the sample system is a relatively closed system with an accurate temperature control function (10-100 ℃), and consists of a sample stage and a heating/refrigerating platform, wherein the sample stage is used for storing a plurality of sample tubes to be detected, and the heating/refrigerating platform can automatically control the real-time temperature of the sample stage, so that the thermal stability analysis of the protein sample at a high flux and wide temperature (10-100 ℃) is realized.
Further, the light intensity detection system firstly collects the light signals from the multichannel fluorescence collection system through the photoelectric collection board, meanwhile converts the light signals into electric signals, sends the electric signals to the light intensity detector to perform analog-to-digital (AD) conversion to output light intensity digital signals, and finally the light intensity digital signals are subjected to data processing by the embedded system control circuit and sent to the control system module.
Further, the control system module is responsible for coordinating temperature control, motor control and optical signal consistent action and recording, finally obtaining light intensity curves of tens of protein sample liquids in the temperature rising process, and obtaining the thermal stability (melting point value Tm) of the protein under the corresponding conditions according to the temperature corresponding to the light intensity value mutation sites of the light intensity curves.
In the scheme, the multichannel fluorescence acquisition light path system adopts an LED light source, a driving circuit board is independently designed, the service life is longer than 5 ten thousand hours, and the temperature drift (10 ℃) and the time drift value (60 minutes) are both smaller than the maximum signal value of < 2%. The multichannel fluorescence acquisition light path system comprises an LED excitation light source, a dichroic filter, a lens, a coaxial light path mechanical device and an LED excitation light source fixing device.
The LED excitation light source is divided into 340-390nm and 470-520nm according to the wavelength, the dichroic filters are all dichroic mirrors with specific cut-off wavelength, and the semi-transmission and semi-reflection dichroic filters collect the light path with specific wavelength; the lens is used for focusing the light path of the collected specific wavelength, so that the light intensity loss is reduced; the coaxial light path mechanical device is a mechanical shell of the multichannel fluorescence acquisition light path system, the device is made of black PMMA, the material is light and low in cost, the device is easy to form and the like, the loss of light intensity can be greatly reduced due to the black material, and the LED excitation light source, the dichroic filter, the lens and the LED excitation light source fixing device are all arranged in the coaxial light path mechanical device; the LED excitation light source is arranged on the LED excitation light source fixing device, and a light source emitted by the LED excitation light source is emitted into the sample tube and the photodiode array after passing through the dichroic filter and the lens; preferably, the device has two LED light source incidence channels, a reflection light channel, five light intensity acquisition channels, and the distance between the five acquisition channels of design is nearer, uses the photodiode array to gather the light intensity, can change LED light source incidence channels in a flexible way according to the detection demand system, and two light source incidence channels just face two light intensity acquisition channels, and the light intensity that light intensity acquisition channel gathered can regard as the light source compensation this moment, and then improved the detection precision, from this, install two incidence lenses, a condenser lens, five dichroic filters of different wavelength and five plane mirrors in the device, five dichroic filters of different wavelength mutually support with the lens, have formed the coaxial light path system of a dual wavelength incidence and multi-wavelength fluorescence acquisition function. The lens, the dichroic filter and the plane reflecting mirror in the multichannel fluorescence collection light path system are convenient to assemble and disassemble, so that the device is easier to develop and maintain, the detection range of the instrument is increased, the service life of the instrument is prolonged, the design of the coaxial light path mechanical device enables the LED light source and the emitted light of a sample to be realized through the same optical window, and the coaxial light path mechanical device is fixed on the S-shaped motion detection system, so that the accurate positioning and detection of the sample are realized.
In the above scheme, the sample system comprises a sample stage and a heating/cooling platform, the sample system and the S-type motion detection system are in the same level, the sample stage is made of high temperature resistant materials, a plurality of sample cavities are designed on the sample stage according to the shape of the sample tube and used for placing and fixing the sample tube, the heating/cooling platform is positioned under the sample stage, the heating/cooling platform adopts a semiconductor cooling/heating system, the semiconductor temperature control has the characteristics of very low thermal inertia and rapid response, meanwhile, the sample cavities adopt the heat insulation design, the sample stage can provide a plurality of sample cavities for the sample tube to be placed, a plurality of sample measurements are realized once, and the thermal stability analysis of protein samples is realized at high flux (more than 30), multi-wavelength (340-390 nm &470-510 nm) and wide temperature (10-100 ℃).
In the above scheme, the control system can coordinate temperature control, mechanical transmission and signal detection and recording actions to be consistent, continuously control the S-shaped motion detection system in the whole heating process according to the designed functional requirements, further enable the multichannel fluorescence acquisition optical path system to move to a specific sample tube and record corresponding signal values, and then uniformly process to obtain tens of light intensity-temperature curves.
Furthermore, the instrument has the core technical points that an autonomously designed multichannel fluorescence acquisition optical path system is adopted, a modularized optical system is designed, and a semiconductor type refrigeration and heating system is adopted. The multichannel fluorescence collection optical path system is provided with two paths of LED excitation light source incidence channels and five paths of reflection fluorescence collection channels, and a coaxial optical path system is formed by two incidence lenses, a condensing lens, five dichroic filters with different wavelengths and five plane reflectors, the outlets of the five reflection fluorescence collection channels are collected by using a photodiode array, the optical system adopts a modularized design, each lens, the dichroic mirror and the plane reflectors are convenient to assemble and disassemble, the device is easier to develop and maintain, the instrument detection range is improved, the service life is prolonged, the temperature of the instrument can be accurately and rapidly controlled by the semiconductor refrigeration and heating system, and the temperature of the isolated sample cavity can be accurately controlled in a short time. When the protein thermal stability analysis is carried out, firstly, a plurality of sample tubes are placed in a sample cavity of a sample table, after the placement is finished, an LED excitation light source is turned on, a control system controls a heating/cooling platform in the sample system to heat, then, a control system controls an S-shaped motion detection system to enable a coaxial mechanical device to move above the sample table at a constant speed in an S-shaped path, when one sample tube is irradiated, the LED excitation light source with specific wavelength is reflected and conducted into the sample tubes through an incidence lens, a dichroic filter, a condensing lens and a designed coaxial mechanical device, emitted light excited by fluorescent protein in the sample tubes is sent to each collecting channel through the transmission and reflection effects of the dichroic filter with specific wavelength and the designed coaxial mechanical device, and a plane reflector in each collecting channel reflects each light path to a photodiode array to carry out photoelectric collection, so that the instrument can measure a plurality of groups of samples in a short time, and finally, high-flux (30), multi-wavelength (470-510 nm) and wide-temperature (10-100 ℃) thermal stability analysis is carried out on the protein samples.
Compared with the prior art, the invention has the following technical effects:
1) The multi-channel fluorescence collection optical path system is independently designed, the range of the LED excitation light source is wide, the LED excitation light source can be rapidly switched between 340-390nm and 470-520nm, and the excitation light and the collection optical path of the sample are realized through the same optical window by the coaxial optical path system with the dual-wavelength incidence and multi-wavelength fluorescence collection functions, and the accurate positioning and detection of the sample can be realized by matching with a motion system.
2) The optical technical scheme of the optical filter type light splitting system is utilized by a modularized optical system, and the advantages are as follows: (a) modular design is easy to technically develop and maintain; (b) No moving parts are arranged in the scheme of the optical system, so that the environmental requirements of equipment are reduced, and the service life is prolonged; (c) The optical technical scheme of the optical filter type light splitting system basically eliminates the background interference of the LED excitation light (light source) on the emitted light (signal).
3) The semiconductor refrigerating/heating system is adopted, and meanwhile, the design of the heat-insulating sample cavity is adopted, so that the temperature control system can rapidly and accurately control the temperature.
Drawings
Fig. 1 is a system block diagram of the present invention.
Fig. 2 is a schematic diagram of an S-type motion detection system. 1. The device comprises a 2-bearing support seat, a 3-bearing support seat, a 6-bearing support seat, a 7-bearing support seat, a 8-bearing support seat, a 9-bearing support seat, a 10-bearing support seat, a 11-bearing support seat, a 12-bearing support seat, a 14-bearing support seat, a 15-bearing support seat, a 16-bearing support seat, a 17-bearing support seat, a 18-stepping motor support seat, a 19-stepping motor support seat, a 20-coupling seat, a 21-right synchronous wheel, a 23-right belt, a 24-right guide rail, a 25-right slider, a 26-right belt slider fixing piece, a 27-28-left synchronous wheel, a 29-left belt, a 30-left guide rail, a 31-left slider, a 32-left belt slider fixing piece, a 33-X-axis motor, a 34-X-axis lead screw, a 35-X-axis slider, a 36-Z-axis motor, a 37-Z-axis lead screw and a 38-Z-axis slider.
Fig. 3 is an overall schematic diagram of a multichannel fluorescence collection optical path system. 39-coaxial machinery, 40-LED excitation source one, 41-LED excitation source two, 42-incidence lens one, 43-incidence lens two, 44-dichroic filter one, 45-dichroic filter two, 46-dichroic filter three, 47-dichroic filter four, 48-dichroic filter five, 49-condenser lens, 50-sample tube, 51-collector lens one, 52-collector lens two, 53-collector lens three, 54-collector lens four, 55-collector lens five, 56-plane mirror one, 57-plane mirror two, 58-plane mirror three, 59-plane mirror four, 60-plane mirror five, 61-photodiode array.
Fig. 4 is a view of an LED light source fixture. 62-LED light source fixing frame.
Fig. 5 is a top view of the coaxial mechanism. 63-roof.
Fig. 6 is a right side view of the coaxial mechanism. 64-bottom plate, 65-front side plate, 66-rear side plate, 67-reflective lens cover plate.
Fig. 7 is a front interior view of the coaxial mechanical device. 68-acquisition channel cover plate, 69-acquisition channel first inlet, 70-acquisition channel second inlet, 71-acquisition channel third inlet, 72-acquisition channel fourth inlet, 73-acquisition channel fifth inlet, 74-acquisition channel first outlet, 75-acquisition channel second outlet, 76-acquisition channel third outlet, 77-acquisition channel fourth outlet, 78-acquisition channel fifth outlet
Fig. 8 is a rear interior view of the coaxial mechanism. 79-incidence lens cover plate I, 80-incidence lens cover plate II, 81-incidence channel I and 82-incidence channel II
Fig. 9 is a top view of the sample system. 83-sample stage, 84-sample cavity
Fig. 10 is a front view of the sample system. 85-semiconductor heating/cooling stage, 86, 87-heating stage support.
Fig. 11 is a coaxial mechanical device excitation light test pattern.
Fig. 12 is a coaxial mechanical device emission light test pattern.
Detailed Description
In order to make the technical solution and advantages of the present invention more apparent, the present invention is described below with reference to the accompanying drawings. Throughout the drawings, the same or similar elements are denoted by the same or similar reference numerals. The devices in the drawings do not reflect the actual size and shape, and some structures and configurations are often omitted to avoid confusion.
As shown in fig. 1, the control system mainly comprises a signal conditioning circuit, a central processing unit and a communication interface, the semiconductor refrigeration heating stations in the light intensity detection system, the S-shaped motion detection system and the sample system are connected with the central processing unit through the signal conditioning circuit to complete signal transmission, acquisition and processing, and the central processing unit is in communication interaction with a computer through the communication interface. The control system determines the speed and direction of mechanical transmission, the temperature control of the semiconductor heating/refrigerating table and the like according to the running state of the whole system. The signal is divided into three types of light intensity, angle and temperature, when the specific sample needs to be detected, the control system regulates and controls the S-shaped motion detection system to carry out acquisition detection according to the experiment requirement, and in addition, the control system can control the temperature of the semiconductor heating/cooling table to ensure the temperature condition (such as constant temperature control or continuous temperature rising control) required by the experiment.
As shown in fig. 2, the S-type motion detection system is schematically shown in the structure, wherein four support seats (1, 2,3, 4) are respectively fixed on the left side and the right side of the instrument in a group of two support platforms (5, 6), four bearing fixing frames (7, 8, 9, 10) are respectively fixed on one side of the two support platforms (5, 6), and four bearings (12, 13, 14) are respectively fixed on the four bearing fixing frames (7, 8, 9, 10); two ends of the driving shaft (15) are fixed with the bearings (13, 14) to connect the two supporting tables (5, 6); the two straight shaft (16, 17) are respectively fixed on the other two bearings (11, 12), the stepping motor (19) is fixed in the instrument by the stepping motor supporting seat (18), the driving shaft (15) and the stepping motor (19) are respectively connected by the coupling (20), the right synchronous pulleys (21, 22) are respectively fixed on the driving shaft (15) and the straight shaft (17), the two right synchronous pulleys (21, 22) are connected by the right belt (23), the right guide rail (24) is fixed on the supporting table (5), the right slide block (25) is placed on the right guide rail (24), the right slide block (25) and the right belt (23) are connected by the right belt slide block fixing piece (26), so that the synchronous movement of the right slide block (25), the right belt (23) is realized, the left synchronous pulleys (27, 28) are respectively fixed on the driving shaft (15) and the straight shaft (16), the two left synchronous pulleys (27, 28) are connected by the left belt (29), the left guide rail (30) is fixed on the supporting table (6), the left slide block (31) is placed on the left guide rail (31) and the left slide block (31) is fixed on the left belt (31) through the left belt fixing piece (29), the left slide block (31) is fixed on the left belt fixing piece (33), the right end is fixed on the right slide block (25), the X-axis slide block (35) can slide on the X-axis screw rod (34), the Z-axis motor (36) is fixed on the X-axis slide block (35), and the Z-axis slide block (38) can slide on the Z-axis screw rod (37). When the stepping motor (19) rotates, the shaft coupler (20) drives the driving shaft (15) to rotate, so that the belts (23, 29) are driven to rotate, and finally, the left and right sliding blocks (25, 31) synchronously move on the Y axis, and the Z-axis sliding blocks (38) move in three dimensions under the cooperation of the three motors, so that accurate positioning is realized.
As shown in fig. 3, the whole schematic diagram of the multi-channel fluorescence collection optical path system is shown, the multi-channel fluorescence collection optical path system is uniformly placed (mounted) in a coaxial mechanical device (39), the first incident lens (42) and the second incident lens (43) are respectively placed in front of the first LED excitation light source (40) and the second LED excitation light source (41) so as to ensure the coaxiality of the optical paths, the first dichroic filter (44) and the third dichroic filter (46) are placed at an angle of 45 °, the second dichroic filter (45), the fourth dichroic filter (47) and the fifth dichroic filter (48) are placed at an angle of 135 °, the angles of the first dichroic filter and the fourth dichroic filter are both 90 °, the condenser lens (49) is placed between the first dichroic filter (44) and the sample tube (50), the first collecting lens (51), the second collecting lens (52), the third collecting lens (53), the fourth collecting lens (54), the fifth collecting lens (55) are respectively placed on the first dichroic filter (44), the second dichroic filter (45), the third dichroic filter (46), the fourth dichroic filter (47), the fifth dichroic filter (48), the fourth dichroic filter (48) and the fourth dichroic filter (48) are placed on a plane mirror (56), and the plane mirror (58) and the plane mirror (59) is correspondingly formed The plane reflectors (60) are respectively arranged above the first collecting lens (51), the second collecting lens (52), the third collecting lens (53), the fourth collecting lens (54) and the fifth collecting lens (55), light paths are reflected to the photodiode array (61) at the left end, and the five plane reflectors are arranged at 135 degrees, so that the distance between the light paths can be shortened, fluorescent collection can be carried out by using one photodiode array (61), and the area of a photoelectric collecting plate is reduced.
As shown in fig. 4, the structure of the LED fixing device is schematically shown, the device is composed of a first LED excitation light source (40), a second LED excitation light source (41) and a LED light source fixing frame (62), the back surfaces of the first LED excitation light source (40) and the second LED excitation light source (41) are fixed on the LED light source fixing frame (62) by using heat conducting silica gel, the LED fixing device fixes the LED light source on a coaxial mechanical device (39), the coaxial mechanical device (39) can be fixed on a Z-axis sliding block (38) of an S-type motion detection system, and a condensing lens (49) faces downwards during fixing, so that detection of a specific sample tube is realized, the LED light source fixing frame (62) is made of aluminum plates, and metal aluminum has the advantages of fast heat dissipation, good heat conductivity and the like, so that the instrument requirements are met.
As shown in fig. 5 and 6, which are top view and right view of the coaxial mechanical device, the main components of the device are a top plate (63), a bottom plate (64), a front side plate (65) and a rear side plate (66), a reflecting lens cover plate (67) is used for fixing a condensing lens (49), and the device uniformly uses black PMMA material, which has the advantages of light weight, low cost, easy molding and the like, and the black material can greatly reduce the loss of light intensity. In addition, a hole site is reserved on the front side plate (65), so that the LED fixing frame (63) is convenient for fixing the LED excitation light source I (40) and the LED excitation light source II (41) on the device.
As shown in fig. 7 and 8, which are coaxial mechanical device interior views, the first (42) and second (43) incident lenses are secured within the first (81) and second (82) incident channels, respectively, and secondarily secured within the device using the first (79) and second (80) incident lens cover plates, preventing lens fall-off, the first (51) and second (52) collecting lenses, the third (53) collecting lenses, the fourth (54) collecting lenses, the fifth (55) collecting lenses are secured within the first (69) collecting channels, the second (70) collecting channels, the third (71) collecting channels, the fourth (72) collecting channels, the fifth (73) collecting channels, and secondarily secured using the first (69) collecting channel cover plates, the first (44) and third (46) dichroic filters are disposed at an angle of 45 ° within the device, the fourth (47) dichroic filters and fifth (48) dichroic filters are disposed within the device at an angle of 135 °, the third and fourth dichroic filters are both 90 ° within the device, the bottom (64) and the top (64) and bottom (68) side plates, the top (68) and the top (65) side plates are provided with small corresponding dimensions for securing the first (79) and the respective side plates The small clamping strips on the incident lens cover plate II (80) can also assist in fixing the dichroic filter, so that the dichroic filter is prevented from loosening and falling off, the plane mirror I (56), the plane mirror II (57), the plane mirror III (58), the plane mirror IV (59) and the plane mirror V (60) are obliquely placed in the plane mirror groove of the rear side plate by 135 degrees, and the five plane mirrors are staggered and placed at a certain distance in the vertical direction, so that not only are the mutual influence of each reflected light ensured, but also the reflected light is ensured to be irradiated on different pixel points of the photodiode array, and the light intensity detection of five channels is realized.
The dichroic filters I, II, III, IV and V are semi-transparent and semi-reflective dichroic mirrors, have high reflectivity to light beams below the cut-off wavelength and high transmissivity to light beams above the cut-off wavelength, and the relation of the cut-off wavelengths of the dichroic filters is as follows: five > four > three > two > one, and the smaller the cutoff wavelength, the closer the dichroic filter spatial placement is to the sample tube.
When the LED excitation light source I (40) is turned on, the incident lens I (42) focuses and guides the excitation light to the dichroic filter I (44), the wavelength of the LED excitation light source I (40) is smaller than the cut-off wavelength of the dichroic filter I (44), so the excitation light is reflected by the dichroic filter I (44) and then passes through the condensing lens (49) and finally enters the sample tube (50), the emission wavelength excited by fluorescent protein in the sample tube is larger than the cut-off wavelength of the dichroic filter I (44) and smaller than the cut-off wavelength of the dichroic filter II (45), the emission light is transmitted through the dichroic filter I (44), then reflected on a dichroic filter II (45) and transmitted to a second acquisition channel inlet (70), and finally reflected to a second acquisition channel outlet (75) through a second plane reflector (57) for acquisition; when the LED excitation light source II (41) is turned on, the incident lens II (43) focuses and transmits the excitation light to the dichroic filter III (46), and similarly, as the LED excitation light source II (41) is smaller than the cutoff wavelength of the dichroic filter III (46) and the wavelength of the LED excitation light source II (41) is larger than the cutoff wavelengths of the dichroic filter I (44) and the dichroic filter II (45), the LED excitation light source II (41) firstly reflects on the dichroic filter III (46), then respectively transmits through the dichroic filter II (45) and the dichroic filter I (44), and finally transmits to the sample tube (50) through the condensing lens (49), at this time, the wavelength of the emitted light of the fluorescent protein in the sample tube (50) is larger than the cut-off wavelengths of the first dichroic filter (44), the second dichroic filter (45) and the third dichroic filter (46) and smaller than the cut-off wavelengths of the fourth dichroic filter (47), so that the emitted light is transmitted through the first dichroic filter (44), the second dichroic filter (45) and the third dichroic filter (46), is reflected on the fourth dichroic filter (47) and then is conducted to the fourth acquisition channel inlet (72), and is reflected to the fourth acquisition channel outlet (77) through the fourth plane mirror (59) for acquisition. If the wavelength bandwidth of the LED excitation light source I (40) is larger, the light source can reflect most of the light when passing through the dichroic filter I (44), and possibly transmit a small part of the light to the acquisition channel I inlet (69), and finally reflect the light to the acquisition channel I outlet (74) by the plane mirror I (56) for acquisition, if the wavelength bandwidth of the LED excitation light source II (41) is larger, the light source can reflect most of the light when passing through the dichroic filter III (46), and possibly transmit a small part of the light to the acquisition channel III inlet (71), and finally reflect the light to the acquisition channel III outlet (76) by the plane mirror III (58) for acquisition, therefore, the light source intensity collected at the first or third outlet of the collecting channel can compensate the fluctuation of the first or second LED excitation light source, so that the detection sensitivity of the emitted light is improved. When the emitted light excited by the LED excitation light source II (41) passes through the dichroic filter IV (47), most of the light can be reflected to the acquisition channel IV inlet (72), but a small part of the light can be transmitted to the dichroic filter V (48), then the light is reflected to the acquisition channel V inlet (73), finally the light is reflected to the acquisition channel V outlet (78) by the plane mirror V (60) for acquisition, and the light source intensity acquired at the acquisition channel V outlet (78) can be used as the reference of the emitted light intensity, so that the emitted light detection sensitivity is improved.
Referring to fig. 9 and 10, which show a top view and a front view of a sample system, a heating stage support seat (86, 88) fixes a semiconductor heating/cooling stage (85) in an instrument, a sample stage (83) is arranged above the semiconductor heating/cooling stage (85), the sample stage (83) provides more than thirty sample cavities (84) for placing sample tubes (50), the sample system adopts a semiconductor heating/cooling method, and meanwhile, a heat-insulating type sample cavity design is adopted, so that the temperature control system can rapidly and accurately control the temperature.
Example 1: the LED excitation light source with the wavelength range of 340-380nm is used for irradiating from the incident channel I of the coaxial mechanical device, the light condensing lens (49) is used for detecting by using a spectrometer, the obtained wavelength-light intensity curve is shown in figure 11, the peak wavelength is about 368.4nm, and the incident light path principle of the device is proved to work normally. And white light is used for irradiation from the reflecting lens, and a spectrometer is used for detection at the three outlets (76) of the collecting channel, so that a curve is shown in fig. 12, the peak wavelength is about 577.5nm and is smaller than the cut-off wavelength of the corresponding dichroic filter three (46) at the three inlets (71) of the collecting channel, and the detection light path of the device is verified to work normally. Thereby verifying the feasibility of the multichannel fluorescence acquisition optical path system and the coaxial mechanical device.
Example 2: in the experiment, the thermal stability of the soluble protein can be analyzed by SYPRO TM Orange dye (the excitation light wavelength is 470nm and the fluorescence wavelength is 570 nm), and the thermal stability of the membrane protein can be analyzed by CPM dye (the excitation light wavelength is 384nm and the fluorescence wavelength is 470 nm). In the embodiment, CPM dye is used for carrying out thermal stability analysis on a membrane protein sample, a plurality of sample tubes to be detected of the membrane protein sample added with the CPM dye are respectively placed in a sample cavity (84) of a sample stage (83), an LED excitation light source I (40) is switched to 384nm, the position of a coaxial mechanical device is regulated by each motor of an S-type motion detection system under the control of a control system, so that the sample tube (50) is positioned right below a condensing lens (49) in the coaxial mechanical device, an LED excitation light source I (40) is turned on to enable excitation light to be conducted into the sample tube (50) through a light path system in the coaxial mechanical device, the control system firstly controls the temperature and heating rate of a semiconductor heating/refrigerating stage (85), then controls each motor of the S-type motion detection system to regulate the position of the coaxial mechanical device, so that a plurality of sample tubes to be detected are positioned right below the condensing lens (49) in the coaxial mechanical device, the LED excitation light source I (40) is conducted into the membrane protein sample through a light path system in the coaxial mechanical device, fluorescence of 470nm is generated by excitation light, the fluorescence of the dye is conducted into a light path system of 470nm in the coaxial mechanical device, the fluorescence light path system of the fluorescent light is conducted into a fluorescent protein sample through a light path system of the coaxial mechanical device, the fluorescent light 470nm is conducted into a fluorescent light channel array (61) of a fluorescent light array, and the fluorescent signal is conducted by a fluorescent light array, and the fluorescent signal is subjected to obtain the data corresponding to the temperature data of a temperature-signal, and the temperature of a fluorescent protein array is measured in a sample, and the temperature is subjected to a temperature curve, and the temperature data of a temperature-corresponding sample is measured; and obtaining the Tm value of the corresponding film protein sample under the condition according to the temperature of the film protein sample, namely the temperature corresponding to the time when the fluorescence signal rapidly becomes strong in the fluorescence intensity curve, so that the thermal stability of the film protein can be analyzed.
Example 3: in the experiment, the thermal stability analysis can be carried out not only on a single protein, but also on two proteins at the same time, and the two proteins are not mutually influenced. In the example, CPM dye and SYPRO TM Orange dye are used for simultaneously carrying out thermal stability analysis on a membrane protein sample and a soluble protein sample, a plurality of sample tubes to be detected of the membrane protein sample added with the CPM dye and the soluble protein sample added with the SYPRO TM Orange dye are respectively put into a sample cavity (84) of a sample table (83), at the moment, the wavelength of an LED excitation light source I (40) is 384nm, the wavelength of an LED excitation light source II (41) is 470nm, the position of a coaxial mechanical device is regulated by each motor of an S-type motion detection system under the control of a control system, so that the sample tube (50) is positioned under the condensing lens (49) in the coaxial mechanical device, the first LED excitation light source (40) and the second LED excitation light source (41) are turned on, the excitation light is conducted into the sample tube (50) through a light path system in the coaxial mechanical device, the control system firstly controls the temperature and the heating rate of the semiconductor heating/refrigerating table (85), then controls each motor of the S-shaped motion detection system to adjust the position of the coaxial mechanical device, so that a plurality of sample tubes to be detected are positioned under the condensing lens (49) in the coaxial mechanical device one by one, when the film protein sample is detected, the first LED excitation light source (40) is conducted into the film protein sample through the light path system in the coaxial mechanical device, the CPM dye is excited by excitation light to generate 470nm fluorescence, the 470nm fluorescence is conducted to the photodiode array (61) through the optical path system of the coaxial mechanical device, the photodiode array (61) converts the received 470nm fluorescence signal into fluorescence intensity data and sends the fluorescence intensity data to the control system, when the soluble protein sample is detected, the second LED excitation light source (41) is conducted to the soluble protein sample through the optical path system in the coaxial mechanical device, the SYPRO TM Orange dye is excited by excitation light to generate 570nm fluorescence, the fluorescence is conducted to the photodiode array (61) through the optical path system of the coaxial mechanical device, the photodiode array (61) converts the received fluorescence signal of 570nm into fluorescence intensity data and sends the fluorescence intensity data to the control system, and the control system fits the obtained fluorescence intensity data and temperature data of the membrane protein samples and the soluble protein samples to obtain a temperature-fluorescence intensity curve corresponding to each membrane protein sample and each soluble protein sample in the heating process of the membrane protein samples and the soluble protein samples; the Tm values of the corresponding membrane protein sample and the soluble protein sample under the condition are obtained according to the temperature of the membrane protein sample and the soluble protein sample, namely the temperature corresponding to the temperature when the fluorescence signal rapidly becomes strong in the fluorescence intensity curve, so that the thermal stability of the membrane protein and the soluble protein sample can be analyzed.
In summary, the invention provides a high-flux rapid protein thermal stability analyzer based on the dye-process protein melting principle, which can rapidly analyze the thermal stability of soluble proteins and membrane proteins, and screen in a high-flux manner: ligands that bind to the protein, small molecule and fragment library candidate drugs that bind to the protein target, antibodies that bind to the protein target, point mutations that improve the thermal stability of the protein, buffers that improve the thermal stability of the protein. And because of the modularized design of the instrument, the LED excitation light source and the dichroic filter can be flexibly replaced according to the excitation wavelength and the emission wavelength of the fluorescent dye, and the application prospect is very wide.
Claims (10)
1. A high throughput protein thermal stability analyzer, characterized by: the system consists of an S-type motion detection system, a multichannel fluorescence acquisition light path system, a sample system, a light intensity detection system and a control system module; the control system module is connected with the S-shaped motion detection system, the S-shaped motion detection system is connected with the multichannel fluorescence acquisition optical path system, the multichannel fluorescence acquisition optical path system interacts with the sample system, and the multichannel fluorescence acquisition optical path system is connected with the control system module through the light intensity detection system; the LED excitation light source fixing device is used for fixing the LED excitation light source on the coaxial light path mechanical device and fixing the coaxial light path mechanical device on the S-type motion detection system; the fixed multichannel fluorescence collection optical path system is positioned right above the sample system, and a condensing lens of the multichannel fluorescence collection optical path system is opposite to the sample system; the multichannel fluorescence acquisition optical path system is fixed in the Z-axis direction of the S-shaped motion detection system and is positioned right above the sample system; the S-shaped motion detection system is characterized in that an S-shaped mobile detection platform is formed by matching three motors, the three motors control a multichannel fluorescence collection optical path system to move at a uniform speed in an S-shaped path above a sample table, so that photoelectric collection is carried out on protein samples in the sample system, and meanwhile, accurate positioning and detection of the samples in the multichannel fluorescence collection optical path system are ensured;
The multichannel fluorescence collection light path system is uniformly arranged in a coaxial mechanical device, an incidence lens I and an incidence lens II are respectively arranged in front of an LED excitation light source I and an LED excitation light source II so as to ensure the coaxial of light paths, a dichroic filter I and a dichroic filter III are arranged at an angle of 45 degrees, a dichroic filter II, a dichroic filter IV and a dichroic filter V are respectively arranged at an angle of 135 degrees, the angles of the dichroic filter I and the dichroic filter IV are 90 degrees, a condensing lens is arranged between the dichroic filter I and a sample tube, the collecting lens I, the collecting lens II, the collecting lens III, the collecting lens IV and the collecting lens V are respectively arranged above the dichroic filter I, the dichroic filter II, the dichroic filter III, the dichroic filter IV and the dichroic filter V, and are coaxial with the corresponding dichroic filters, and a plane reflector I, a plane reflector II, a plane reflector III, a plane reflector V are respectively arranged above the collecting lens I, the collecting lens II, the collecting lens III, the collecting lens IV and the collecting lens V, the collecting lens V and the collecting lens V, the light paths to the left end, and the photoelectric diode array are respectively arranged at the position of the plane reflectors 135 degrees, and the distance of each is reduced by using a fluorescent light array.
2. The high throughput protein thermal stability analyzer of claim 1, wherein: the S-type motion detection system, the multichannel fluorescence acquisition light path system, the sample system, the light intensity detection system and the control system module are in modularized design.
3. The high throughput protein thermal stability analyzer of claim 1, wherein: the S-shaped motion detection system is used for adjusting and controlling the sample tube corresponding to the multichannel fluorescence acquisition optical path system, so as to realize the detection of the fluorescent protein light intensity in the specific sample tube.
4. The high throughput protein thermal stability analyzer of claim 1, wherein: the multichannel fluorescence collection light path system can adjust different excitation light sources at any time to collect multichannel fluorescence.
5. The high throughput protein thermal stability analyzer of claim 1, wherein: the sample system consists of a sample platform and a heating/refrigerating platform, wherein the sample platform is used for storing a plurality of sample tubes to be detected, and the heating/refrigerating platform can automatically control the real-time temperature of the sample platform.
6. The high throughput protein thermal stability analyzer of claim 1, wherein: the light intensity detection system firstly collects light signals from the multichannel fluorescence collection system through the photoelectric collection plate, meanwhile converts the light signals into electric signals, sends the electric signals to the light intensity detector for analog-to-digital conversion to output light intensity digital signals, and finally the light intensity digital signals are subjected to data processing by the embedded system control circuit and sent to the control system module.
7. The high throughput protein thermal stability analyzer of claim 1, wherein: the control system module is responsible for coordinating temperature control, motor control and optical signal consistent action and recording, finally obtaining light intensity curves of tens of protein sample liquids in the temperature rising process, and obtaining the thermal stability of the protein under the corresponding conditions according to the temperature corresponding to the light intensity value mutation sites of the light intensity curves.
8. The high throughput protein thermal stability analyzer of claim 1, wherein: the multichannel fluorescence acquisition light path system comprises an LED excitation light source, a dichroic filter, a lens, a coaxial light path mechanical device and an LED excitation light source fixing device;
The LED excitation light source is divided into 340-390nm and 470-520nm according to the wavelength, the dichroic filters are all dichroic mirrors with specific cut-off wavelength, and the semi-transmission and semi-reflection dichroic filters collect the light path with specific wavelength; the LED excitation light source, the dichroic filter, the lens and the LED excitation light source fixing device are all arranged in the coaxial light path mechanical device; the LED excitation light source is arranged on the LED excitation light source fixing device, and the light source emitted by the LED excitation light source is emitted into the sample tube and the photodiode array after passing through the dichroic filter and the lens.
9. The high throughput protein thermal stability analyzer of claim 1, wherein: the sample system comprises a sample table and a heating/refrigerating platform, wherein the sample system and the S-shaped motion detection system are in the same level, the sample table is made of high-temperature resistant materials, a plurality of sample cavities are designed in the sample table according to the shape of the sample tubes and used for placing and fixing the sample tubes, the heating/refrigerating platform is located under the sample table, the heating/refrigerating platform adopts a semiconductor refrigerating/heating system, the sample cavities adopt a heat insulation design, and the sample table provides a plurality of sample cavities for placing the sample tubes.
10. The high throughput protein thermal stability analyzer of claim 1, wherein: the control system cooperatively controls temperature control, mechanical transmission and signal detection recording, controls the S-shaped motion detection system in the whole heating process according to the designed functional requirements, enables the multichannel fluorescence acquisition optical path system to move to a specific sample tube and record corresponding signal values, and then uniformly processes the obtained light intensity-temperature curve.
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