CN113884479B - Active intracellular Raman spectrum detection method - Google Patents
Active intracellular Raman spectrum detection method Download PDFInfo
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- CN113884479B CN113884479B CN202111487496.6A CN202111487496A CN113884479B CN 113884479 B CN113884479 B CN 113884479B CN 202111487496 A CN202111487496 A CN 202111487496A CN 113884479 B CN113884479 B CN 113884479B
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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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- 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
Abstract
The invention provides an active intracellular Raman spectrum detection method. The active intracellular Raman spectrum detection method comprises the following steps: providing a thermal gradient generation layer on which a cell fluid is disposed; injecting particles capable of exciting surface plasmons into the cell sap; injecting a surfactant into the cell sap; after the surfactant is injected into the cell sap, introducing the particles into the interior of the cells in the cell sap by using optical tweezers; after the particles are introduced into the interior of the cell, controlling the particles to move to a designated area in the cell through optical tweezers; after the particles move to the designated area, the cells are subjected to raman spectroscopy. The Raman spectrum detection method induces particles capable of exciting surface plasmons to enter cells, the surface plasmons generated by the excited particles enhance Raman signals of molecules in the cells, the enhancement of Raman scattering of the particles in the cells is realized, and the detection intensity is improved.
Description
Technical Field
The invention relates to the technical field of Raman signal sample detection, in particular to an active intracellular Raman spectrum detection method.
Background
Cells are the basic structural and functional units of an organism, consisting of complex biomolecules encapsulated within membranes, which are distributed at different locations within the cell to perform their respective functions. The accurate detection and identification of these elements is an important problem in life science research, and the current methods for studying cell phenotype at home and abroad comprise surface enhanced Raman scattering, but the traditional Raman signal detection strength is too weak, so that the signal acquisition time is too long, and the practical application of the method is greatly limited.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to overcome the defect that the detection intensity is too weak when the Raman spectrum detection is carried out on molecules in the cell, thereby providing an active intracellular Raman spectrum detection method.
The invention provides an active intracellular Raman spectrum detection method, which comprises the following steps: providing a thermal gradient generation layer on which a cell fluid is disposed; injecting particles capable of exciting surface plasmons into the cell sap; injecting a surfactant into the cell sap; after the surfactant is injected into the cell sap, introducing the particles into the interior of the cells in the cell sap by using optical tweezers; after the particles are introduced into the interior of the cell, controlling the particles to move to a designated area in the cell through optical tweezers; after the particles move to the designated area, raman spectroscopy is performed on the interior of the cell.
Optionally, before injecting the surfactant into the cell sap, capturing the cells by using optical tweezers; injecting a surfactant into the cell sap so that cells in the cell sap are bound to the surface of the thermal gradient generation layer; injecting a surfactant into the cell sap, wherein the surfactant comprises cations and anions, and part of the cations are wrapped on the surfaces of the particles to form a cationic membrane; the step of introducing the particles into the interior of the cells in the cell sap using optical tweezers comprises: capturing the particles with the surface wrapped by the cationic membrane by using optical tweezers, wherein in the process of capturing the particles with the surface wrapped by the cationic membrane by using the optical tweezers, captured light emitted by the optical tweezers irradiates the surface of the thermal gradient generation layer to form a thermal gradient field, and the thermal gradient field drives the free cations and anions in the cell sap to displace so as to form a local electric field; the capture light moves, so that the particles coated with the cationic membrane move to the periphery of the cells on the surface of the thermal gradient generation layer, and the particles coated with the cationic membrane are introduced into the interior of the cells under the action of the capture light and the electric field.
Optionally, the surfactant comprises a cetyltrimethyl ammonium chloride solution, a cetyltrimethyl ammonium bromide solution, a sodium dodecyl sulfate solution or a polydimethyldiallylammonium chloride solution.
Optionally, the concentration of the surfactant in the cell sap is 1-20 mmol/L.
Optionally, the particles capable of generating the surface enhanced raman scattering effect comprise metal nanoparticles.
Optionally, the metal nanoparticles comprise gold nanoparticles, silver nanoparticles, or copper nanoparticles.
Optionally, the particle diameter is 100 nm-800 nm.
Optionally, the thermal gradient generation layer includes a graphene thermal gradient generation layer or a metal thermal gradient generation layer.
Optionally, the material of the metal thermal gradient generation layer comprises gold or silver.
Optionally, the step of performing raman spectroscopy detection on the interior of the cell comprises: exciting the cells with a raman generating module to generate an initial raman signal; and collecting the Raman signal generated by the particles after the surface plasmon enhancement by adopting a Raman collection module.
Optionally, the step of performing raman spectroscopic detection on the cell comprises: performing in situ Raman spectroscopy on the designated area inside the cell.
Optionally, the step of performing raman spectroscopic detection on the cell comprises: and controlling the particles to move in the designated area through the optical tweezers, and performing surface scanning Raman imaging on the designated area.
The technical scheme of the invention has the following advantages:
according to the active intracellular Raman spectrum detection method provided by the technical scheme of the invention, a three-dimensional optical trap is formed in cell sap through an optical tweezers, after a surfactant is added, particles capable of exciting surface plasmons are induced to enter cells, free electrons on the surfaces of the particles interact with photons of the optical tweezers to form collective oscillation, the collective electromagnetic oscillation is called surface plasmons, a strong electromagnetic field is generated through the surface plasmons, and the Raman scattering intensity is in direct proportion to the fourth power of the electromagnetic field intensity, so that the intracellular particle enhanced Raman scattering signal is realized, and the detection intensity is effectively improved.
Further, a surfactant is injected into the cell fluid, so that the cells in the cell fluid are bound to the surface of the thermal gradient generation layer. Because the surfactant changes the water solubility of the cell membrane, the cell bottom is fixed on the surface of the thermal gradient generation layer, and the cell becomes permeable, thereby being beneficial to capture and conveying the particles to the inside of the cell under the action of the thermal electric field. Meanwhile, the surface active agent can be coated on the surface of the particles to form a cationic membrane, which is beneficial to being captured by the optical tweezers. And in the process of capturing the particles with the cationic membranes on the surface by using the optical tweezers, the captured light emitted by the optical tweezers irradiates the surface of the thermal gradient generation layer to form a thermal gradient field, and the thermal gradient field drives the free cations and anions in the irradiated area in the cell sap to displace to form a local electric field. The capture light moves so that the particles coated with the cationic membrane move to the periphery of the cells on the surface of the thermal gradient generation layer, and the particles coated with the cationic membrane are introduced into the interior of the cells under the action of the capture light and the electric field. Combining the optical force of the trapped light, the photo-induced thermal gradient field and the surfactant, the particles are easily introduced into the interior of the cell.
Furthermore, the graphene thermal gradient generation layer is used as the thermal gradient generation layer, so that the graphene thermal gradient generation layer has excellent thermal conductivity, can generate strong thermoelectric force, and can play a good role in inhibiting fluorescence generated by a detection sample during Raman detection. In addition, the graphene thermal gradient generation layer also has certain biocompatibility.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic flow chart of the active intracellular Raman spectroscopy method of the present invention;
FIG. 2 is a bright field image of the cell captured by the optical tweezers in this example;
FIG. 3 is a bright field image of the particles captured by the optical tweezers in this embodiment;
FIG. 4 is a bright field image of the particles moved by the optical tweezers close to the cell in this embodiment;
FIG. 5 is a bright field image of the present example in which particles are introduced into the interior of a cell;
FIG. 6 is a comparison graph of Raman spectra obtained by detecting a particle-introduced cell and a particle-free cell in this example.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. 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.
In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, and are only for convenience of description and simplicity of description, but do not indicate or imply that the device or element being referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus, should not be construed as limiting the present invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.
In the description of the present invention, it should be noted that, unless otherwise explicitly specified or limited, the terms "mounted," "connected," and "connected" are to be construed broadly, e.g., as meaning either a fixed connection, a removable connection, or an integral connection; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.
In addition, the technical features involved in the different embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The embodiment of the invention provides a Raman spectrum detection method, the flow of which is shown in figure 1, and the method comprises the following steps:
s1: providing a thermal gradient generation layer on which a cell fluid is disposed;
s2: injecting particles capable of exciting surface plasmons into the cell sap;
s3: injecting a surfactant into the cell sap;
s4: after the surfactant is injected into the cell sap, introducing the particles into the interior of the cells in the cell sap by using optical tweezers;
s5: after the particles are introduced into the interior of the cell, controlling the particles to move to a designated area in the cell through optical tweezers;
s6: after the particles move to the designated area, raman spectroscopy is performed on the interior of the cell.
In one embodiment, the thermal gradient generating layer comprises a graphene thermal gradient generating layer or a metal thermal gradient generating layer. The material of the metal thermal gradient generation layer comprises gold or silver. In this embodiment, the graphene thermal gradient generation layer is used as the thermal gradient generation layer, has excellent thermal conductivity, can generate strong thermoelectric force, and can play a good role in inhibiting fluorescence generated by a detection sample during raman detection. In addition, the graphene thermal gradient generation layer also has certain biocompatibility. The substrate, which in one particular embodiment is a glass substrate, serves as a support structure for the thermal gradient generating layer.
In this embodiment, a surfactant is injected into the cell fluid, so that the cells in the cell fluid are bound to the surface of the thermal gradient generation layer; and in the step of injecting a surfactant into the cell sap, the surfactant comprises cations and anions, and part of the cations are wrapped on the surfaces of the particles to form a cation membrane.
The step of introducing the particles into the interior of the cells in the cell sap using optical tweezers comprises: capturing the particles with the surface wrapped by the cationic membrane by using optical tweezers, wherein in the process of capturing the particles with the surface wrapped by the cationic membrane by using the optical tweezers, captured light emitted by the optical tweezers irradiates the surface of the thermal gradient generation layer to form a thermal gradient field, and the thermal gradient field drives the free cations and anions in the cell sap to displace so as to form a local electric field; the capture light moves, so that the particles coated with the cationic membrane move to the periphery of the cells on the surface of the thermal gradient generation layer, and the particles coated with the cationic membrane are introduced into the interior of the cells under the action of the capture light and the electric field.
In this embodiment, the surfactant includes a cetyltrimethylammonium chloride solution, a cetyltrimethylammonium bromide solution, a sodium dodecyl sulfate solution, or a poly dimethyl diallyl ammonium chloride solution.
The concentration of the surfactant in the cell sap is 1 mmol/L-20 mmol/L, such as 1mmol/L, 10mmol/L or 20 mmol/L. Too low a concentration of surfactant does not form enough anions and cations in the cell sap, resulting in difficulties in forming local electric fields driven by thermal gradient fields, rendering the cells and the particles uncontrollable. In addition, the permeability of the cell membrane of the cell to the particles can be adjusted by controlling the concentration of the surfactant, and the surfactant with proper concentration is beneficial to controlling the introduction of the particles into the cell by the optical tweezers.
In one embodiment, the cell sap is in direct contact with the graphene thermal gradient generating layer. The cells were captured with optical tweezers and are shown in FIG. 2. After the surfactant is added, the process of inducing the particles to enter the cells by the optical tweezers is taken by an electron microscope, fig. 3 shows that the particles are captured by the optical tweezers, the particles are in black circles in fig. 3, and the arrow direction is the moving direction of the optical tweezers. Fig. 4 shows the process of moving the particles close to the cell by the optical tweezers, and the particles are in the black circles in fig. 4. FIG. 5 shows the introduction of particles into the interior of a cell, the particles being introduced within the black circles in FIG. 5. The introduction of the particles into the interior of the cell is achieved by means of optical tweezers through modification with a surfactant.
In this embodiment, the wavelength of the trapping light is 500nm to 1064nm, such as 532nm or 660 nm. The wavelength of the light is selected according to the size and the material of the particles.
In the embodiment, the intensity of the capture light is 1.0 mW-5.0 mW, such as 2.4mW, 3.6mW or 4.8 mW. The intensity of the capture light meets the premise that the optical tweezers can capture cells or particles, the cells are prevented from being damaged by illumination, and simultaneously, Raman scattering signals with enough intensity can be generated by excitation.
In one embodiment, the particles comprise metal nanoparticles. The metal nanoparticles include gold nanoparticles, silver nanoparticles, or copper nanoparticles.
In one embodiment, the particles are 100nm to 800nm in diameter. The smaller the particle diameter is, the more accurate the Raman spectrum detection positioning is, but if the particle diameter is less than 100nm, the Brownian motion of the particles is enhanced, and the operation difficulty of capturing the particles by the optical tweezers is obviously improved; if the diameter of the particle is larger than 800nm, the detection and positioning precision of Raman spectrum is reduced, the difficulty of the particle penetrating through a cell membrane is increased, and even the acting force of the optical tweezers is insufficient to control the particle. Therefore, the diameter of the particles is within the range of 100 nm-800 nm, and good Raman detection precision can be obtained within the range allowed by guaranteed conditions.
In one embodiment, the step of performing raman spectroscopic detection of the cell comprises: performing in situ Raman spectroscopy on the designated area inside the cell. In other embodiments, the step of performing raman spectroscopic detection of the cell comprises: and controlling the particles to move in the designated area through the optical tweezers, and performing surface scanning Raman imaging on the designated area. The step of performing raman spectroscopic detection of the cells may further comprise performing in situ raman spectroscopic detection and surface scanning raman imaging sequentially.
In this embodiment, the step of performing raman spectroscopy detection on the inside of the cell includes: exciting the cells with a raman generating module to generate an initial raman signal; and collecting the Raman signal generated by the particles after the surface plasmon enhancement by adopting a Raman collection module.
FIG. 6 is a Raman spectrum contrast chart of a particle-introduced cell and a particle-free cell, in which the horizontal axis represents Raman shift in cm-1The vertical axis represents diffraction intensity. In FIG. 6, the upper solid line shows a diffraction peak generated by Raman spectroscopy on a cell into which a particle is introduced, and the lower dotted line shows a diffraction peak generated by Raman spectroscopy on a cell without a particle. As can be seen from the figure, the raman signal significantly increases the diffraction peak intensity under the surface plasmon enhancement by the particle.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (11)
1. An active intracellular raman spectroscopy detection method, comprising:
providing a thermal gradient generation layer on which a cell fluid is disposed;
injecting particles capable of exciting surface plasmons into the cell sap;
injecting a surfactant into the cell sap to enable cells in the cell sap to be bound on the surface of the thermal gradient generation layer, wherein the surfactant comprises cations and anions, a part of the cations are coated on the surfaces of the particles to form a cation membrane, and the concentration of the surfactant in the cell sap is 10-20 mmol/L;
after the surfactant is injected into the cell sap, introducing the particles into the interior of the cells in the cell sap by using optical tweezers; the step of introducing the particles into the interior of the cells in the cell sap using optical tweezers comprises: capturing the particles with the surface wrapped by the cationic membrane by using optical tweezers, wherein in the process of capturing the particles with the surface wrapped by the cationic membrane by using the optical tweezers, captured light emitted by the optical tweezers irradiates the surface of the thermal gradient generation layer to form a thermal gradient field, and the thermal gradient field drives the free cations and anions in the cell sap to displace so as to form a local electric field; the capture light moves, so that the particles coated with the cationic membrane move to the periphery of the cells on the surface of the thermal gradient generation layer, and the particles coated with the cationic membrane are introduced into the interior of the cells under the action of the capture light and the electric field;
after the particles are introduced into the interior of the cell, controlling the particles to move to a designated area in the cell through optical tweezers;
after the particles move to the designated area, raman spectroscopy is performed on the interior of the cell.
2. The active intracellular raman spectroscopy method of claim 1, further comprising: before the surfactant is injected into the cell sap, the cells are captured by using optical tweezers.
3. The active intracellular raman spectroscopy method of claim 1, wherein the surfactant comprises a cetyltrimethylammonium chloride solution, a cetyltrimethylammonium bromide solution, a sodium dodecyl sulfate solution, or a polydimethyldiallylammonium chloride solution.
4. The active intracellular raman spectroscopy method of claim 1, wherein the particles comprise metal nanoparticles.
5. The active intracellular raman spectroscopy detection method of claim 4, wherein the metal nanoparticles comprise gold nanoparticles, silver nanoparticles, or copper nanoparticles.
6. The active intracellular Raman spectroscopy method of claim 1, 4, or 5, wherein the particle has a diameter of 100nm to 800 nm.
7. The active intracellular raman spectroscopy method of claim 1, wherein the thermal gradient generation layer comprises a graphene thermal gradient generation layer or a metal thermal gradient generation layer.
8. The active intracellular raman spectroscopy method of claim 7, wherein the material of the metallic thermal gradient generation layer comprises gold or silver.
9. The active intracellular raman spectroscopy method of claim 1, wherein the step of performing raman spectroscopy on the interior of the cell comprises:
exciting the cells with a raman generating module to generate an initial raman signal;
and collecting the Raman signal generated by the particles after the surface plasmon enhancement by adopting a Raman collection module.
10. The active intracellular raman spectroscopy method of claim 1, wherein the step of performing raman spectroscopy on the cell comprises:
performing in situ Raman spectroscopy on the designated area inside the cell.
11. The active intracellular raman spectroscopy method of claim 1, wherein the step of performing raman spectroscopy on the cell comprises:
and controlling the particles to move in the designated area through the optical tweezers, and performing surface scanning Raman imaging on the designated area.
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| CN111487757A (en) * | 2020-05-21 | 2020-08-04 | 深圳大学 | Thermoelectric optical tweezers device and particle capture method based on thermoelectric optical tweezers device |
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| CN111487757A (en) * | 2020-05-21 | 2020-08-04 | 深圳大学 | Thermoelectric optical tweezers device and particle capture method based on thermoelectric optical tweezers device |
Non-Patent Citations (2)
| Title |
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| Plasmonic tweezers: for nanoscale optical trapping and beyond;Yuquan Zhang et al.;《Light: Science & Applications》;20210317;第10卷(第1期);第59-1至59-41页 * |
| Thermophoretic Tweezers for Low-Power and Versatile Manipulation of Biological Cells;Linhan Lin et al.;《ACS Nano》;20170223;第11卷;第3147-3154页 * |
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