CN111175280A - Cytochrome detection method based on surface enhanced Raman spectroscopy - Google Patents
Cytochrome detection method based on surface enhanced Raman spectroscopy Download PDFInfo
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- CN111175280A CN111175280A CN202010112056.1A CN202010112056A CN111175280A CN 111175280 A CN111175280 A CN 111175280A CN 202010112056 A CN202010112056 A CN 202010112056A CN 111175280 A CN111175280 A CN 111175280A
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- 238000001514 detection method Methods 0.000 title claims abstract description 41
- 102000018832 Cytochromes Human genes 0.000 title claims abstract description 29
- 108010052832 Cytochromes Proteins 0.000 title claims abstract description 29
- 238000004416 surface enhanced Raman spectroscopy Methods 0.000 title claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 18
- 239000000758 substrate Substances 0.000 claims description 14
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 claims description 13
- 238000001069 Raman spectroscopy Methods 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 9
- 239000002073 nanorod Substances 0.000 claims description 9
- 229910052709 silver Inorganic materials 0.000 claims description 9
- 239000004332 silver Substances 0.000 claims description 9
- 238000000479 surface-enhanced Raman spectrum Methods 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 230000010354 integration Effects 0.000 claims description 5
- 102000004169 proteins and genes Human genes 0.000 abstract description 7
- 108090000623 proteins and genes Proteins 0.000 abstract description 7
- 230000008859 change Effects 0.000 abstract description 4
- 238000004458 analytical method Methods 0.000 abstract description 2
- 239000003814 drug Substances 0.000 abstract description 2
- 238000012544 monitoring process Methods 0.000 abstract description 2
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- 238000012512 characterization method Methods 0.000 abstract 1
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- 241001148470 aerobic bacillus Species 0.000 description 1
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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
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
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- Health & Medical Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Physics & Mathematics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention discloses a cytochrome detection and characterization method based on Surface Enhanced Raman Spectroscopy (SERS). the method directly collects SERS signals of cytochrome, and is simple to operate and free of marks. Information on the change of the secondary structure of the cytochrome with concentration can be obtained through further analysis. Can provide a new method for the real-time monitoring of the secondary structure change of the biological medicine and the protein.
Description
Technical Field
The invention relates to a cytochrome detection method based on surface enhanced Raman, in particular to a method for measuring a secondary structure of cytochrome.
Background
Cytochrome is an electron transfer protein which takes ferriporphyrin (or heme) as a prosthetic group and widely participates in the redox reaction of animals, plants, yeast, aerobic bacteria, anaerobic photosynthetic bacteria and the like. Cytochrome is a good bioelectronic carrier, which passes through the reduced state of the iron atom (Fe) in the heme prosthetic group2+) And oxidation state (Fe)3+) The reversible change between the two realizes the transport of electrons. The porphyrin ring is attached to the iron atom with four coordinate bonds, forming a tetradentate chelated complex, commonly referred to as heme. Depending on the structure of the heme prosthetic group, cytochromes can be classified into A, B, C and D groups. Taking the common cytochrome C as an example, the reduced structure is a dispersed needle crystal, and the oxidized structure is a petal-like crystal. Cytochrome C can be used in various groups clinicallyThe auxiliary treatment of tissue hypoxia emergency treatment, such as treatment of myocardial hypoxia caused by dyspnea caused by carbon monoxide poisoning, cyanide poisoning, neonatal asphyxia and the like and various heart diseases. Has rapid enzymatic action on the oxidation and reduction processes of cells in tissues.
At present, the detection methods for proteins such as cytochrome are mainly X-ray crystal diffraction, nuclear magnetic resonance, fluorescence detection, attenuated total reflection Fourier infrared, circular dichroism, electron spin resonance, surface plasmon resonance, atomic force microscope, neutron reflection, ellipsometry and the like. By using these techniques, information can be measured at some molecular level of the protein, but these techniques have certain limitations. For example, X-ray crystallography is dependent on the crystalline state of the protein, and the crystallization process is likely to result in denaturation of the protein; although widely used in protein research, nuclear magnetic resonance has relatively low sensitivity and large sample usage in experiments. In addition, the method is complex to operate, has high requirements on the quality of personnel operating the instrument and the performance of the instrument and has high test cost.
Surface-enhanced Raman scattering (SERS) spectroscopy is a surface-interface sensitive detection technique. The enhancement factor can be as high as 10 by enhancing Raman scattering through molecular or plasma magnetic silicon nanotube and other nano structures adsorbed on the rough metal surface10To 1011This means that the technique can detect a single molecular level. SERS has the advantages of high sensitivity, short detection time, direct in-situ analysis and the like, and draws wide attention of scholars in the field of biological analysis. Based on the high-sensitivity detection capability and good signal light stability of an SERS technology to a sample with extremely low concentration, the SERS technology is gradually developed into an effective means for researching biophysics.
Disclosure of Invention
In order to solve the above problems, the present invention provides a method for rapidly and sensitively detecting and analyzing a cytochrome, which can rapidly detect and analyze the cytochrome in a very simple and accurate manner. The method can be used for direct detection, and the silver nanorod array substrate which is obliquely arranged is used during detection, so that the detection sensitivity is higher and more stable, the influence of the external temperature and the sample on the detection qualitative and quantitative properties is greatly reduced, the detection is more convenient, and the operation process is simple.
In order to achieve the purpose, the invention adopts the following technical means:
the invention provides a cytochrome detection method based on surface enhanced Raman spectroscopy, which comprises the following steps:
1. a cytochrome detection method based on Surface Enhanced Raman Scattering (SERS) is characterized by comprising the following steps:
a. dissolving the cytochrome to be detected in water, preparing water solutions with different concentration gradients and uniformly mixing;
b. dropwise adding 2 mu L of the solution obtained in the step a onto a silver nanorod array substrate, and concentrating the solution to obtain a detection solution;
c. performing Raman scattering detection on the detection liquid obtained in the step b, setting the laser power to be 30 mW and the integration time to be 10s, and measuring the SERS spectrum of the detection liquid;
d. and (c) according to the SERS spectrum of the detection liquid obtained in the step (c), utilizing Lorentz linear fitting to obtain structural information.
Further, the silver nanorod array substrate used in the step b is a silver nanorod array substrate arranged obliquely. The silver nanorods have the length of 900 +/-100 nm and the diameter of 150 +/-40 nm, the interval between two adjacent silver nanorods is 110 +/-10 nm, and the inclination angle is 73 +/-5 degrees.
Further, in the step c: laser wavelength, power and integration time are set, a measured object on the substrate is scanned, and the obtained characteristic peak in the surface enhanced Raman spectrum can be used as a reference peak for detection.
Has the advantages that:
the invention realizes the detection and the structural analysis of the cytochrome by utilizing the Surface Enhanced Raman Scattering (SERS) technology. The method adopts direct detection, has simple operation method, and is not easily interfered by the surrounding environment. The invention overcomes the defect of complex sample pretreatment in the prior detection and can carry out real-time detection. The invention has simple operation and conforms to the rapid inspection standard. The silver nanorod array substrate based on the inclined arrangement can enable the detection sensitivity to be higher and more stable, greatly reduces the influence of the external temperature and the sample on the detection, enables the detection to be more convenient and fast, is simple in operation process, and can provide a new method for the real-time monitoring of clinical medicine.
Drawings
FIG. 1 is a scanning electron microscope picture of a substrate with surface enhanced Raman scattering as used in example 1;
FIG. 2 is a graph of the spectra of cytochrome C of different concentrations based on surface enhanced Raman in example 1;
FIG. 3 is a graph showing the change in secondary structure of cytochrome C according to concentration in example 1.
Detailed Description
The invention is further described with reference to the following drawings and specific embodiments.
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1: the embodiment provides a cytochrome detection method based on surface enhanced Raman, which comprises the following steps:
dissolving cytochrome to be detected in water, preparing water solutions with different concentration gradients and uniformly mixing;
step two, dripping 2 mu L of the solution obtained in the step b onto a silver nanorod array substrate, and concentrating the solution to obtain a detection solution;
step three, performing SERS detection on the detection liquid obtained in the step two, setting the laser power to be 30 mW and the integration time to be 10s, and measuring an SERS spectrum of the detection liquid;
and step four, according to the SERS spectrum of the detection liquid obtained in the step three, utilizing Lorentz linear fitting to obtain structural information.
FIG. 1 is a scanning electron microscope image of a surface enhanced Raman substrate used in example 1, which shows that the substrate has good uniformity.
FIG. 2 is a surface enhanced Raman based spectrum of cytochrome C with different concentrations, and it can be seen that the invention has good sensitivity and can be used for FMN sample detection.
The obtained sample signals were further analyzed by origin analysis software to obtain the variation of different secondary structures of cytochrome C with concentration, as shown in fig. 3.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned. The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and any minor modifications, equivalent replacements and improvements made to the above embodiment according to the technical spirit of the present invention should be included in the protection scope of the technical solution of the present invention.
Claims (3)
1. A cytochrome detection method based on Surface Enhanced Raman Scattering (SERS) is characterized by comprising the following steps:
a. dissolving the cytochrome to be detected in water, preparing water solutions with different concentration gradients and uniformly mixing;
b. dropwise adding 2 mu L of the solution obtained in the step a onto a silver nanorod array substrate, and concentrating the solution to obtain a detection solution;
c. performing Raman scattering detection on the detection liquid obtained in the step b, setting the laser power to be 30 mW and the integration time to be 10s, and measuring the SERS spectrum of the detection liquid;
d. and (c) according to the SERS spectrum of the detection liquid obtained in the step (c), utilizing Lorentz linear fitting to obtain structural information.
2. The method for detecting surface-enhanced Raman based cytochrome according to claim 1, wherein the silver nanorod array substrate used in step b is an obliquely arranged silver nanorod array substrate, the silver nanorods are 900 ± 100nm in length and 150 ± 40nm in diameter, the space between two adjacent silver nanorods is 110 ± 10nm, and the inclination angle is 73 ± 5 °.
3. The method for rapid detection of surface-enhanced Raman based cytochrome as claimed in claim 1, wherein in the step c: laser wavelength, power and integration time are set, a measured object on the substrate is scanned, and the obtained characteristic peak in the surface enhanced Raman spectrum can be used as a reference peak for detection.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112279909A (en) * | 2020-10-21 | 2021-01-29 | 吉林师范大学 | Method for reducing oxidized cytochrome c by laser induction |
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2020
- 2020-02-24 CN CN202010112056.1A patent/CN111175280A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| KANG-ZHEN TIA ET AL.: "Sensitive and Label-Free Detection of Protein Secondary Structure by Amide III Spectral Signals using Surface-Enhanced Raman Spectroscopyby Surface Enhanced Raman Spectroscopy", 《CHINESE JOURNAL OF CHEMICAL PHYSICS》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112279909A (en) * | 2020-10-21 | 2021-01-29 | 吉林师范大学 | Method for reducing oxidized cytochrome c by laser induction |
| CN112279909B (en) * | 2020-10-21 | 2022-08-30 | 吉林师范大学 | Method for reducing oxidized cytochrome c by laser induction |
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Application publication date: 20200519 |