CN109406442B - Method for rapidly measuring protein thermal denaturation temperature - Google Patents
Method for rapidly measuring protein thermal denaturation temperature Download PDFInfo
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- CN109406442B CN109406442B CN201811276965.8A CN201811276965A CN109406442B CN 109406442 B CN109406442 B CN 109406442B CN 201811276965 A CN201811276965 A CN 201811276965A CN 109406442 B CN109406442 B CN 109406442B
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 52
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 52
- 238000004925 denaturation Methods 0.000 title claims abstract description 32
- 230000036425 denaturation Effects 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims abstract description 28
- 239000012460 protein solution Substances 0.000 claims abstract description 15
- 229910052751 metal Inorganic materials 0.000 claims description 37
- 239000002184 metal Substances 0.000 claims description 37
- 238000010438 heat treatment Methods 0.000 claims description 15
- 238000010521 absorption reaction Methods 0.000 claims description 9
- 239000000758 substrate Substances 0.000 claims description 9
- 239000008367 deionised water Substances 0.000 claims description 3
- 229910021641 deionized water Inorganic materials 0.000 claims description 3
- 229910052710 silicon Inorganic materials 0.000 claims description 3
- 239000010703 silicon Substances 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 230000007423 decrease Effects 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 6
- 230000009191 jumping Effects 0.000 abstract description 3
- 230000003247 decreasing effect Effects 0.000 abstract description 2
- 230000008859 change Effects 0.000 description 7
- 238000001228 spectrum Methods 0.000 description 5
- 238000010586 diagram Methods 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 238000001035 drying Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000000691 measurement method Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000000113 differential scanning calorimetry Methods 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 238000000411 transmission spectrum Methods 0.000 description 1
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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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
- G01N21/3581—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light using far infrared light; using Terahertz radiation
-
- 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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
- G01N21/3577—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light for analysing liquids, e.g. polluted water
Abstract
The invention provides a method for rapidly measuring the thermal denaturation temperature of protein, which comprises the following steps: s1, preparing protein solutions with different concentrations from a protein sample to be detected; s2, dripping protein solution on the sensor surface of claim 2 or 3, and forming a protein film on the sensor surface; s3, the sensor is placed on a focal plane of the terahertz waves, and the terahertz waves vertically pass through the sensor; s4 the sensor heats up when the relative humidity of the air in the environment in which the sensor is located has decreased to at least below 1%. The method for measuring the protein thermal denaturation temperature by the resonance peak position jumping has the advantages of high accuracy, simple operation and high detection speed, and is particularly suitable for application occasions with high requirements on accuracy and instantaneity.
Description
Technical Field
The invention belongs to the technical field of terahertz, and particularly relates to a method for rapidly measuring protein thermal denaturation temperature.
Background
Terahertz (THz) refers to electromagnetic wave with frequency of 0.1THz-10THz, photon energy is about 1-10 mev, harmful photoionization can not be generated, and the Terahertz (THz) is particularly suitable for unmarked and harmless biological detection and identification and has important application value in the field of biomedicine.
"metamaterial" refers to some composite materials having artificially designed structures and exhibiting extraordinary physical properties not possessed by natural materials.
Proteins are the material basis of life and are the major contributors to life activities. However, proteins are denatured under certain conditions. Any process that causes a conformational change in a protein that does not involve cleavage of peptide bonds is called denaturation. Denaturation changes its physical, chemical and biological properties, and proteins exhibit various biological functions in their life activities, depending entirely on their conformation, which are lost once they change. The method has very important significance for accurately measuring the thermal denaturation rule and the thermal denaturation temperature of the protein.
At present, the measurement method of the thermal denaturation temperature of protein mainly comprises Differential Scanning Calorimetry (DSC). The method comprises the links of solution preparation, tabletting and sample preparation, temperature scanning and recording and the like, has complex test process, large calculated amount, low detection speed and low accuracy, and can not meet the requirements of many application occasions on measurement accuracy and real-time property.
Disclosure of Invention
In view of the above-mentioned shortcomings of the prior art, the present invention is directed to a method for rapidly measuring the thermal denaturation temperature of a protein, so as to solve the problems of complex testing process, large amount of calculation, slow detection speed and low accuracy in the prior art.
To achieve the above and other related objects, the present invention provides a sensor unit including a first metal bar, a second metal bar, and a third metal bar, the second metal bar and the second metal bar being connected to both ends of the first metal bar, respectively; the first metal rod, the second metal rod and the third metal rod are encircled to form an open resonator with an opening;
the sensor unit also comprises an H-shaped resonator which is arranged in a space surrounded by the split resonator;
the split resonator and the H-shaped resonator have the same resonance absorption peak in the terahertz frequency band.
In order to achieve the above objects and other related objects, the present invention further provides a sensor, including a substrate and a metal resonator array formed by a plurality of the sensor units and disposed on the substrate.
Optionally, the substrate is high-resistance silicon.
In order to achieve the above and other related objects, the present invention further provides a system for rapidly measuring the thermal denaturation temperature of protein, comprising the sensor of claim 2, a heat conducting plate 3 and a heating plate 4, wherein the sensor is disposed on the heat conducting plate, the heat conducting plate is connected to the heating plate, a circular hole is disposed on the heat conducting plate, and the sensor is disposed in the circular hole.
To achieve the above and other related objects, the present invention also provides a method for rapidly measuring a thermal denaturation temperature of a protein, the method comprising the steps of:
s1, preparing protein solutions with different concentrations from a protein sample to be detected;
s2, dripping protein solution on the sensor surface of claim 2 or 3, and forming a protein film on the sensor surface;
s3, the sensor is placed on a focal plane of the terahertz waves, and the terahertz waves vertically pass through the sensor;
s4 heating the sensor when the relative humidity of the air in the environment in which the sensor is located has decreased to at least below 1%.
1. The method for rapidly measuring the thermal denaturation temperature of the protein as claimed in claim 5, wherein the protein sample to be measured is prepared into a protein solution, and specifically comprises the following steps:
and preparing a protein solution with a certain concentration by taking a protein sample to be detected and deionized water.
2. The method for rapidly measuring the thermal denaturation temperature of the protein as claimed in claim 5, wherein the specific method for forming a protein film on the surface of the sensor is as follows:
s21, dripping protein solution with different concentrations onto the surface of the sensor;
s22, heating the sensor at the temperature of 30 ℃ to form a protein film on the surface of the sensor.
As described above, a method for rapidly measuring the thermal denaturation temperature of a protein according to the present invention has the following beneficial effects:
the method for measuring the protein thermal denaturation temperature by the resonance peak position jumping has the advantages of high accuracy, simple operation and high detection speed, and is particularly suitable for application occasions with high requirements on accuracy and instantaneity.
Drawings
To further illustrate the description of the present invention, the following detailed description of the embodiments of the present invention is provided with reference to the accompanying drawings. It is appreciated that these drawings are merely exemplary and are not to be considered limiting of the scope of the invention.
FIG. 1 is a schematic structural diagram of an apparatus for implementing a method for rapidly detecting protein thermal denaturation temperature by using a metamaterial sensor and terahertz spectroscopy according to the present invention;
FIG. 2 is a top view of a sensor and a thermally conductive sheet;
figure 3 is a diagram of an array of metamaterial sensor structures,
FIG. 4 is a diagram of a metamaterial sensor structure;
FIG. 5 is a diagram of a metamaterial sensor unit;
FIG. 6 is a terahertz transmission spectrum of a protein in a temperature rise process;
FIG. 7 is a flow chart of a method for rapidly measuring the temperature of thermal denaturation of proteins.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention. It is to be noted that the features in the following embodiments and examples may be combined with each other without conflict.
It should be noted that the drawings provided in the following embodiments are only for illustrating the basic idea of the present invention, and the components related to the present invention are only shown in the drawings rather than drawn according to the number, shape and size of the components in actual implementation, and the type, quantity and proportion of the components in actual implementation may be changed freely, and the layout of the components may be more complicated.
As shown in fig. 4, the present invention provides a sensor unit including a first metal bar, a second metal bar and a third metal bar, the second metal bar and the second metal bar being connected to both ends of the first metal bar, respectively; the first metal rod, the second metal rod and the third metal rod are encircled to form a split resonator with an opening.
In one embodiment, the second metal bar and the third metal bar are perpendicular to the first metal bar, respectively. May also be considered a U-shaped sensor.
The sensor unit also comprises an H-shaped resonator which is arranged in a space surrounded by the split resonator; the split resonator and the H-shaped resonator have the same resonance absorption peak in the terahertz frequency band.
In one embodiment, the U-shaped resonator and the H-shaped resonator are made of aluminum metal, and the thickness of the metal layer is 200 nm. The length and width of the metal rods at the two sides and the bottom edge of the U-shaped resonator are 50 mu m and 5 mu m, the length and width of the metal rods at the two sides of the H-shaped resonator are 35 mu m and 5 mu m, and the length and width of the middle transverse rod are 20 mu m and 5 mu m.
As shown in FIG. 3, the invention provides a sensor, which comprises a substrate and a metal resonator array arranged on the substrate and composed of a plurality of sensor units. In one embodiment, the substrate is high-resistance silicon.
As shown in fig. 1 and 2, the present invention provides a system for rapidly measuring the thermal denaturation temperature of protein, comprising the sensor of claim 2, a heat conducting plate 3 and a heating plate 4, wherein the sensor is arranged on the heat conducting plate, the heat conducting plate is connected to the heating plate, a circular hole is arranged on the heat conducting plate, and the sensor is arranged in the circular hole.
As shown in fig. 7, the present invention also provides a method for rapidly measuring the thermal properties of a protein, comprising the steps of:
protein solutions with different concentrations are prepared by taking a protein sample to be detected with the size of 5 mu m and 30 mu m deionized water. (to verify that the denaturation temperature of the protein is independent of concentration and only dependent on temperature.)
Using a pipette to drop 5 mul of prepared protein solution with different concentrations on the surface of the sensor, drying the sensor on a heating table at 30 ℃ until a layer of film 1 is formed, repeatedly overlapping the film on the desired concentration to be measured, and drying the film on the heating table at 30 ℃.
A metal strip with good heat conduction and a round hole is connected with the heating table, the sensor 2 is fixed on the round hole 5 and is positioned on a focal plane of the terahertz waves, so that the center of the round hole coincides with the center of the focal plane of the terahertz waves, and the terahertz waves vertically pass through the sensor.
And when the relative humidity of the air of the terahertz system is reduced to below 1%, starting a temperature control heater to heat the sample wafer. And measuring the terahertz spectrum penetrating through the sample wafer in the heating process, and mainly observing and recording the position change condition of the resonance absorption peak.
From the real-time measurement of the terahertz spectrum during the temperature rise, it was found that the position of the resonance absorption peak was almost unchanged before the thermal denaturation of the protein occurred. When thermal denaturation occurs, the position of the resonance absorption peak of the sensor jumps to high frequency, and the change value is larger than 5 GHz. Therefore, the corresponding heater temperature when the resonance absorption peak position of the sensor jumps to high frequency is the thermal denaturation temperature of the protein. The theoretical basis of this measurement method is: after the protein is denatured, the protein conformation changes, thereby causing the dielectric constant to change, and the sensor of the invention is sensitive to the change of the dielectric constant.
The invention relates to a method and a system for rapidly detecting protein thermal denaturation temperature by utilizing a metamaterial sensor and a terahertz spectrum. The sensor is formed by combining a U-shaped resonator and an H-shaped resonator surrounded by the U-shaped resonator, the sensor has a strong resonance absorption peak in a terahertz frequency band, when a layer of substance covers the surface of the sensor, the absorption peak can move to a low frequency, and the magnitude of frequency shift is closely related to the concentration of the surface substance and the dielectric property of the surface substance. When measuring the temperature of protein thermal denaturation, firstly, a trace amount of protein solution is dripped on the surface of a sensor and dried to form a layer of protein film. And then, putting the sensor into a terahertz spectrum system, heating through a heating plate, and carrying out terahertz spectrum acquisition. When the temperature does not reach the thermal denaturation temperature of the protein, the resonance peak position hardly changes. When the temperature reaches the protein thermal denaturation temperature, the resonance peak frequency jumps to high frequency, and the temperature corresponding to the resonance peak frequency jump is the protein denaturation temperature. The method for measuring the protein thermal denaturation temperature through resonance peak position jumping has the advantages of high accuracy, simple operation and high detection speed, and is particularly suitable for application occasions with high requirements on accuracy and instantaneity.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (4)
1. The system for rapidly measuring the thermal denaturation temperature of protein is characterized by comprising a sensor, a heat conducting strip (3) and a heating strip (4), wherein the sensor is arranged on the heat conducting strip, the heat conducting strip is connected to a heating platform, a round hole is formed in the heat conducting strip, the sensor is arranged in the round hole, the sensor comprises a substrate and a metal resonator array which is arranged on the substrate and consists of a plurality of sensor units, the substrate is made of high-resistance silicon, the sensor units comprise a first metal rod, a second metal rod, a third metal rod and an H-shaped resonator, and the second metal rod and the third metal rod are respectively connected to two ends of the first metal rod; the first metal rod, the second metal rod and the third metal rod enclose an open resonator with an opening, and the H-shaped resonator is arranged in a space enclosed by the open resonator;
the split resonator and the H-shaped resonator have the same resonance absorption peak in the terahertz frequency band.
2. A method for rapidly measuring the temperature of thermal denaturation of a protein using the system of claim 1, comprising the steps of:
s1, preparing protein solutions with different concentrations from a protein sample to be detected;
s2, dripping the protein solution on the surface of the sensor, and forming a layer of protein film on the surface of the sensor;
s3, the sensor is placed on a focal plane of the terahertz waves, and the terahertz waves vertically pass through the sensor;
s4 the sensor is heated when the relative humidity of the air in the environment in which the sensor is located decreases below 1%.
3. The method according to claim 2, wherein the protein sample to be tested is prepared as protein solutions of different concentrations, in particular:
and preparing protein solutions with different concentrations by taking a protein sample to be detected and deionized water.
4. The method of claim 2, wherein the specific method for forming a protein film on the surface of the sensor is as follows:
s21, dripping protein solution with different concentrations onto the surface of the sensor;
s22, heating the sensor at the temperature of 30 ℃ to form a protein film on the surface of the sensor.
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CN112098366B (en) * | 2020-07-27 | 2022-10-25 | 桂林电子科技大学 | Embedded double-U-shaped refractive index sensor for realizing three Fano resonances |
CN113030006B (en) * | 2021-03-08 | 2022-03-25 | 西南科技大学 | Reflection-type terahertz micro-flow sensor with irregular U-shaped metal microstructure |
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Application publication date: 20190301 Assignee: GUILIN GUANGLONG SCIENCE AND TECHNOLOGY GROUP Co.,Ltd. Assignor: GUILIN University OF ELECTRONIC TECHNOLOGY Contract record no.: X2023980045854 Denomination of invention: A Rapid Method for Measuring Protein Thermal Denaturation Temperature Granted publication date: 20210105 License type: Common License Record date: 20231107 |
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