CN114674807B - Up-conversion Raman sensing method and application - Google Patents
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Abstract
The embodiment of the invention discloses an up-conversion Raman sensing method and application, wherein the method comprises the following steps: selecting a Raman light source with a first wavelength and an infrared light source with a second wavelength according to the standard Raman spectrum and the infrared absorption spectrum of the analyte; and simultaneously irradiating the analyte by adopting a Raman light source with a first wavelength and an infrared light source with a second wavelength to detect an up-conversion Raman signal of the analyte. The method can not only obviously improve the specificity identification capability of the molecular fingerprint in a complex environment, but also effectively separate the background noise which often appears in the traditional Raman technology, and greatly improve the sensitivity and the signal-to-noise ratio of the molecular signal in the aspects of spectrum and imaging.
Description
Technical Field
The invention relates to the technical field of optical sensing, in particular to an up-conversion Raman sensing method and application.
Background
With the development of science and technology, the technology makes further breakthrough in the micro field, the existing test conditions in the micro field can not meet the requirements of science and technology, and the micro quality detection and measurement become more and more important particularly in the field of precision measurement.
Raman scattering spectroscopy, or simply raman spectroscopy, is a method of studying molecular structure and properties by observing the frequency, intensity, polarization, etc. of raman scattering from a sample under test, and identifying an analyte by determining the material characteristics of the analyte species from the spectral components contained in a response signal (e.g., raman scattering signal) generated by inelastic scattering.
At present, most analytes are in a complex detection environment, characteristic Raman signals of the analytes are easily interfered or masked by Raman signals of other molecules in the environment, so that signal analysis needs to be realized by means of a complex machine learning algorithm, and meanwhile, when the molecular Raman signals are excited, fluorescence signals of the analytes are also excited, so that the sensitivity of Raman sensing is greatly influenced.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides an up-conversion Raman sensing method and application thereof, and the up-conversion Raman sensing method provided by the embodiment of the invention not only can obviously improve the specific identification capability of molecular fingerprints in a complex environment, but also can effectively separate background noise frequently occurring in the traditional Raman technology, and greatly improve the sensitivity and the signal-to-noise ratio of molecular signals in the aspects of spectrum and imaging.
In a first aspect, an embodiment of the present invention provides an up-conversion raman sensing method, which includes:
selecting a Raman light source with a first wavelength and an infrared light source with a second wavelength according to the standard Raman spectrum and the infrared absorption spectrum of the analyte;
and simultaneously irradiating the analyte by adopting the Raman light source with the first wavelength and the infrared light source with the second wavelength so as to detect an up-conversion Raman signal of the analyte.
Preferably, in the method for up-conversion raman sensing, the selecting a raman light source with a first wavelength and an infrared light source with a second wavelength according to a standard raman spectrum and an infrared absorption spectrum of an analyte includes:
overlapping the standard Raman spectrum with the infrared absorption spectrum to obtain an overlapping peak;
determining the molecular vibration mode of the analyte according to the overlapping peaks;
and selecting the Raman light source with the first wavelength and the infrared light source with the second wavelength according to the molecular vibration mode.
Preferably, in the up-conversion raman sensing method, before the simultaneously irradiating the analyte with the raman light source with the first wavelength and the infrared light source with the second wavelength, the method further includes:
the analyte is placed in a nanogap region between the first metal nanoparticle and the second metal nanoparticle.
More preferably, in the up-conversion raman sensing method, the placing the analyte in a nanogap region between the first metal nanoparticle and the second metal nanoparticle includes:
depositing the first metal nanoparticles on a substrate;
adsorbing the analyte on the first metal nanoparticle;
adsorbing the second metal nano-particles on the analyte to achieve placement of the analyte in the nano-gap region.
More preferably, in the up-conversion raman sensing method, the first metal nanoparticle and the second metal nanoparticle are both modified with a ligand, and the analyte is disposed in the nanogap region through the ligand.
More preferably, in the up-conversion raman sensing method, the first metal nanoparticles are one or more of gold nanoparticles, silver nanoparticles, and aluminum nanoparticles.
More preferably, in the up-conversion raman sensing method, the second metal nanoparticles are one or more of gold nanoparticles, silver nanoparticles, and aluminum nanoparticles.
Preferably, in the up-conversion raman sensing method, the infrared light source is any one of a near-infrared light source, a mid-infrared light source, a far-infrared light source, and an ultra-far-infrared light source.
Preferably, in the up-conversion raman sensing method, the raman light source is a laser light source.
In a second aspect, the embodiments of the present invention further provide an application of the up-conversion raman sensing method, which is applied to virus detection, chemical toxicity detection, clinical diagnosis of diseases and cancers, drug discovery, food process control and environmental monitoring.
The embodiment of the invention provides an up-conversion Raman sensing method and application, wherein a Raman light source with a first wavelength and an infrared light source with a second wavelength which are required by the irradiation of an analyte are determined through a standard Raman spectrum and an infrared absorption spectrum of the analyte, and then the Raman light source with the first wavelength and the infrared light source with the second wavelength are used for simultaneously irradiating the analyte so as to detect an up-conversion Raman signal of the analyte. The method can not only obviously improve the specificity identification capability of the molecular fingerprint in a complex environment, but also effectively separate the background noise which often appears in the traditional Raman technology, and greatly improve the sensitivity and the signal-to-noise ratio of the molecular signal in the aspects of spectrum and imaging.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
Fig. 1 is a schematic flow chart of an up-conversion raman sensing method according to an embodiment of the present invention;
FIG. 2 is a Raman spectrum formed in a conventional Raman process;
FIG. 3 is a graph of an upconverted Raman spectrum according to an embodiment of the present invention;
fig. 4 is a schematic flow chart of an up-conversion raman sensing method according to an embodiment of the present invention;
FIG. 5 is a schematic diagram of a standard Raman spectrum superimposed with an infrared absorption spectrum according to an embodiment of the present invention;
fig. 6 is a schematic flow chart of an up-conversion raman sensing method according to an embodiment of the present invention;
fig. 7 is a schematic diagram illustrating a plasmon resonance phenomenon generated by a metal nanoparticle under the action of an electromagnetic field according to an embodiment of the present invention;
FIG. 8 is a schematic structural view of an analyte disposed in a nanogap region according to an embodiment of the invention;
FIG. 9 is a schematic diagram of the frequency matching of the resonant peak of the metal nanogap structure in the visible band and the molecular Raman scattering provided by an embodiment of the invention;
FIG. 10 is a schematic diagram of the frequency matching of the formants of the metal nanogap structure in the mid-infrared band with the infrared absorption of molecules according to an embodiment of the invention;
fig. 11 is a schematic diagram of a process for implementing up-conversion raman according to an embodiment of the present invention;
fig. 12 is a schematic flowchart of an up-conversion raman sensing method according to an embodiment of the present invention;
fig. 13 is a schematic structural diagram of an up-conversion raman sensor according to an embodiment of the present invention;
FIG. 14 is a schematic diagram of an analyte detection structure provided in an embodiment of the present invention;
FIG. 15 is a graph of an up-converted Raman spectrum in low resolution mode provided by an embodiment of the present invention;
FIG. 16 is a graph of an upconverted Raman spectrum in high resolution mode according to an embodiment of the present invention;
FIG. 17 is a chart of the upconversion Raman spectrum in the low resolution mode according to an embodiment of the present invention;
fig. 18 is a graph of an up-conversion raman spectrum in a high resolution mode according to an embodiment of the present invention.
Detailed Description
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 some, 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.
It will be understood that the terms "comprises" and/or "comprising," when used in this specification and the appended claims, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.
It is also to be understood that the terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification of the present invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
It should be further understood that the term "and/or" as used in this specification and the appended claims refers to and includes any and all possible combinations of one or more of the associated listed items.
Referring to fig. 1, fig. 1 is a schematic flow chart of an up-conversion raman sensing method according to an embodiment of the present invention. As shown in fig. 1, an up-conversion raman sensing method includes:
s10, selecting a Raman light source with a first wavelength and an infrared light source with a second wavelength according to the standard Raman spectrum and the infrared absorption spectrum of the analyte;
s20, simultaneously irradiating the analyte by using the Raman light source with the first wavelength and the infrared light source with the second wavelength to detect the up-conversion Raman signal of the analyte.
According to the up-conversion Raman sensing method provided by the embodiment of the invention, after the Raman light source with the first wavelength and the infrared light source with the second wavelength which are required by irradiating the analyte are determined through the standard Raman spectrum and the infrared absorption spectrum of the analyte, the Raman light source and the infrared light source are used for irradiating the analyte at the same time, and extra signals with the same frequency can be superposed on the specific vibration mode of the Raman spectrum of the analyte to form an up-conversion Raman signal, so that background noise which often occurs in the traditional Raman technology is effectively separated, and the sensitivity and the signal-to-noise ratio of a molecular signal in the aspects of spectrum and imaging are greatly improved.
The up-conversion Raman signal is an extra signal with the same frequency superimposed on a specific vibration mode of a Raman spectrum of an analyte, and the extra signal is consistent with most basic physical properties of a traditional Raman signal, but the spectrum shape and the peak intensity of the up-conversion Raman signal follow an infrared light source, so that various unique advantages of the traditional Raman technology are inherited, specific detection can be realized, and meanwhile, the signal is stable and cannot be quenched.
Specifically, the up-conversion raman signal mentioned in this embodiment is different from the conventional raman signal in that the up-conversion raman signal completely replicates the spectrum shape of the infrared light source in the spectrum, that is, if the infrared light source is a single peak narrower than the conventional raman peak, the up-conversion raman signal overlaps a narrower peak above the conventional raman peak; if the infrared source is a split double peak, the up-conversion signal will be a double peak superimposed on the traditional raman peak, so that the only variable in the whole process is that the wavelength is changed from the infrared band to be consistent with the frequency of the raman mode.
It can be understood that when implementing the up-conversion raman of the analyte, a molecular vibration mode of the analyte is selected, the molecular vibration mode can be obtained from the standard raman spectrum and the infrared absorption spectrum of the analyte, the analyte needs to have both raman scattering and infrared absorption activities in the mode, so that the visible infrared laser can be coupled with the selected molecular vibration mode by means of the infrared absorption process, and simultaneously the raman scattering process is excited by the raman light source, so as to up-convert the signal into the stokes (difference frequency process of infrared light and raman light source) and anti-stokes (sum frequency process of infrared light and raman light source) light of the visible band.
It will also be appreciated that the molecular switching raman sensing process can be viewed as a Sum Frequency Generation (SFG) and Difference Frequency Generation (DFG) process in second order nonlinear optics, i.e. a raman source of a first wavelength is combined with an infrared source of a second wavelength to form a beam of higher energy anti-stokes raman light (SFG, Sum Frequency process) or the energy of the raman source of the first wavelength is subtracted from the energy of the infrared source of the second wavelength to form a beam of slightly lower energy stokes raman light (DFG, Difference Frequency process).
It should be noted that the sum frequency process and the difference frequency process cannot occur in the free space by means of the analyte. Specifically, the energy of the infrared light source with the second wavelength is firstly absorbed by the analyte and converted into the molecular vibration energy by means of the infrared absorption process, and meanwhile, the raman light source with the first wavelength is used for irradiating the analyte to generate the raman scattering process, so that the converted molecular vibration energy and the raman light source with the first wavelength are subjected to energy combination (sum frequency) or subtraction (difference frequency), and an up-conversion raman signal is obtained.
In some embodiments, the infrared light source may be any one of a near-infrared light source, a mid-infrared light source, a far-infrared light source, and an extreme far-infrared light source; the raman light source may be any one of a near-infrared light source and a visible light source. When the infrared light source and the Raman light source are near-infrared light sources, the two near-infrared light sources irradiating on the object to be analyzed are near-infrared light sources with two different wavelengths; when the raman light source is a visible light source, the raman light source can be a laser light source, the laser light source can be a high-power laser light source, and the infrared light source can be a mid-infrared laser light source.
Referring to fig. 2 and 3, fig. 2 is a raman spectrum formed in a conventional raman process; fig. 3 is an up-conversion raman spectrum provided by an embodiment of the present invention. As shown in fig. 2 and 3, the wavelength of the raman light source is 780 nm, the wavelength of the infrared light source is 9.3 μm, and the conventional raman process is only to add or subtract the thermal vibration of the molecule and the energy generated by the raman light source to form raman light, i.e. conventional raman light = incident light ± thermal vibration; in the up-conversion raman process provided by the present application, the vibration energy level of the molecule is utilized by the infrared light source, and the up-conversion raman light is obtained by taking the vibration energy level as a springboard to directly generate a sum frequency process or a difference frequency process with the raman light source, that is, the up-conversion raman light = the raman light source ± infrared light.
In addition, the main difference between the up-conversion raman process in the present application and the conventional raman process in principle is that the vibrational energy level of the molecule in the conventional raman process is directly excited by the thermal energy of the environment (no such process at absolute zero), and thus no infrared absorption process is involved; whereas for the up-conversion raman process, the molecular vibrations are driven by the infrared light source by means of the infrared absorption process. Meanwhile, as shown in fig. 3, when the analyte is subjected to the up-conversion raman process, the conventional raman process is synchronized, i.e., conventional raman + up-conversion raman = incident light ± infrared light + incident light ± thermal vibration, where the conventional raman signal brought by the thermal vibration is inverted to the thermal noise to be eliminated. In the embodiment shown in fig. 3, both near-infrared signal lights obtained based on sum-frequency and difference-frequency processes are called up-conversion raman, i.e. a low-energy infrared light source of 9.3 microns is converted to a high-energy visible or near-infrared band, however, considering the signal-to-noise ratio in sensing applications and in order to avoid a fluorescent background below the laser energy, the application usually detects only the sum-frequency up-conversion raman signal higher than the laser energy, i.e. raman source + infrared source.
In some embodiments, as shown in fig. 4, step S10 includes sub-steps S101, S102, and S103.
S101, overlapping the standard Raman spectrum and the infrared absorption spectrum to obtain an overlapping peak;
s102, determining the molecular vibration mode of the analyte according to the overlapping peaks;
s103, selecting the Raman light source with the first wavelength and the infrared light source with the second wavelength according to the molecular vibration mode.
The standard raman spectrum and the infrared absorption spectrum are obtained by characterizing an analyte, and are mainly used for determining a molecular vibration mode with up-conversion activity in the embodiment.
It can be understood that when the raman light source with the first wavelength and the infrared light source with the second wavelength are selected, the analyte needs to be characterized to obtain the standard raman spectrum and the infrared absorption spectrum of the analyte, then the standard raman spectrum and the infrared absorption spectrum of the analyte are overlapped to determine an overlapping peak, and the molecular vibration mode of the analyte is determined through the overlapping peak, so that the wavelength of the preset infrared laser is determined.
Referring to fig. 5, fig. 5 is a schematic diagram illustrating the standard raman spectrum and the infrared absorption spectrum overlapped according to the embodiment of the present invention. In the example shown in FIG. 5, where the analyte is BPT molecule (biphenyl-4-thiol, CAS number 19813-90-2) powder, multiple candidate molecular vibrational modes of BPT molecules can be obtained by measuring the standard Raman spectrum and the infrared absorption spectrum of BPT molecules and then overlapping the standard Raman spectrum and the infrared absorption spectrum of BPT molecules. Wherein, the molecular vibration mode of the BPT molecular candidate is preferably 1080cm -1 And 1580cm -1 . In addition, when the molecular vibration mode of the BPT molecular candidate is 1580cm -1 The same may also exist for other interfering molecules in the environmentCarbon peak, therefore, the molecular vibrational mode of BPT in the examples of the present application is preferably 1080cm -1 。
In the embodiment shown in FIG. 5, the molecular vibrational mode of the BPT molecule candidate is preferably 1080cm -1 For the infrared light source with the second wavelength, the second wavelength can be directly 1080cm -1 Performing a unit conversion to obtain a second wavelength, i.e. a second wavelength equal to 9.3 microns; while for the traditional Raman scattering process of molecules, the corresponding mode R = +/-1080 cm -1 Is also dependent on the first wavelength lambda L The conversion formula of the Raman light source is R/10 7 =1/λ L -1/λ. If the first wavelength λ L 740 nm, corresponding Stokes (Stokes) and Anti-Stokes (Anti-Stokes) raman wavelengths are 804 nm and 685 nm, respectively.
In the above embodiment, the 9.3 micron infrared source and the 740 nm raman source are combined into a higher energy, 685 nm wavelength anti-stokes raman light (SFG, sum frequency process), or the 740 nm raman source is subtracted from the 9.3 micron infrared source to form a slightly lower energy, 804 nm wavelength stokes raman light (DFG, difference frequency process).
In some embodiments, as shown in fig. 6, step S20 is preceded by step S11.
S10, selecting a Raman light source with a first wavelength and an infrared light source with a second wavelength according to the standard Raman spectrum and the infrared absorption spectrum of the analyte;
s11, placing the analyte in the nanogap region between the first and second metal nanoparticles 120;
s20, simultaneously irradiating the analyte by using the Raman light source with the first wavelength and the infrared light source with the second wavelength to detect the up-conversion Raman signal of the analyte.
Specifically, theoretically, an upconversion raman signal can be obtained by simply relying on molecules, but the upconversion efficiency is extremely low, so that the actual signal is too weak, or a raman light source (such as a high-power laser) with extremely high power is needed for excitation. In order to solve the above-mentioned existing drawbacks, in the present embodiment, the analyte is placed in the nanogap region between the first metal nanoparticle and the second metal nanoparticle 120 to enable the analyte to be molecularly coupled, and the nanogap region between the first metal nanoparticle and the second metal nanoparticle 120 can provide a huge electromagnetic field enhancement, thereby realizing a large amplitude enhancement of the upconverted raman signal.
It is understood that metal nanoparticles can be physically simplified as a combination of negatively charged free electron clouds and positively charged atomic entities. As shown in fig. 7, when incident light irradiates on the particle, the electromagnetic wave periodically drives the electron cloud to generate collective oscillation, in the process, the light energy and the electron energy interact, so that the energy of the electromagnetic field is bound to a region of several nanometers on the metal surface, and the phenomenon is called a surface plasmon resonance phenomenon. By means of the surface plasmon resonance phenomenon, the energy of incident electromagnetic waves distributed throughout the free space is compressed into a high-local-area optical field with only a few nanometers, so that the intensity of the electromagnetic field is greatly enhanced relative to the intensity of the incident light, and the optical local area and field enhancement effect of a plasmon resonance structure is generated; when two metal nanoparticles are close to each other, the plasmon resonance modes of the two metal nanoparticles are hybridized and coupled, so that most of energy of an electromagnetic field is concentrated in a gap region, and the smaller the gap distance is, the more concentrated the energy of the electromagnetic field is, and the stronger the energy is.
It should be noted that, the analyte may be detected by a single metal nanoparticle to achieve detection of the up-converted raman signal, or may be detected by two or more metal nanoparticles to achieve detection of the up-converted raman signal, and in the metal nanostructure composed of a single metal nanoparticle or multiple metal nanoparticles, the electromagnetic field localization and enhancement provided by the nanogap structure formed between two metal nanoparticles are often the strongest, which is the preferred structure in the present application. Referring to FIG. 8, by placing the analyte in the region where the electromagnetic field is most enhanced (i.e., the nanogap region), various optical processes of the molecule can be greatly enhanced.
In addition, the analyte can also be placed near the nanogap region between the first metal nanoparticle and the second metal nanoparticle 120, and after the analyte is simultaneously irradiated by the raman light source with the first wavelength and the infrared light source with the second wavelength, the up-conversion raman signal of the analyte can also be detected. It should be noted that, when the analyte is disposed near the nanogap region between the first metal nanoparticle and the second metal nanoparticle 120, the distance between the analyte and the nanogap region may be selected according to practical applications, and is not specifically limited in this embodiment.
In one embodiment, as shown in FIGS. 9 and 10, if the analyte is BPT, the molecular vibrational mode is 1080cm -1 The wavelength of the infrared light source is 9.3 micrometers, the wavelength of the light source can be 740 nanometers, the wavelength of raman scattering is 685 nanometers, and a nanogap region between the first metal nanoparticle and the second metal nanoparticle 120 has surface plasmon resonance response, and the two processes are enhanced while the two wavelengths are respectively covered, so that matching on frequency is realized.
In addition, the plasmon resonance needs to be matched in a spatial vector with the optical process of the molecule. Referring to fig. 11, only specific directions of electromagnetic field can effectively excite the ir absorption and raman scattering processes of immobilized molecules, which are simplified as ir absorption dipole and raman dipole processes, respectively. Wherein the dipoles are two opposite positive and negative charges separated by a distance. Meanwhile, in order to effectively realize signal amplification of molecules, the molecules are arranged in a nanogap region with the strongest electromagnetic field, and the directions of infrared absorption and Raman dipoles of the molecules are consistent with the direction of a local electromagnetic field of the nanogap. If the electromagnetic field direction is perpendicular to the dipole, the infrared absorption and raman process of the molecule cannot be excited, or the excitation efficiency is low. Meanwhile, since the infrared absorption and raman scattering processes required for up-conversion raman occur on the same (regional) molecule, the two plasmon resonance modes of the metal nanostructure in the infrared and visible bands need to coincide with each other in space.
Therefore, it can be understood that several necessary conditions and technical points for realizing the efficient up-conversion raman process can be specifically as follows:
(1) the metal nano structure has mutually overlapped regions in the local electromagnetic field modes of infrared and visible wave bands in space, and molecules are positioned in the overlapped regions;
(2) the infrared absorption dipole of the molecule is matched with the infrared electromagnetic field mode in frequency, and the orientation of the infrared absorption dipole and the infrared electromagnetic field mode is kept approximately consistent;
(3) the raman dipole of the molecule matches the visible electromagnetic field pattern in frequency and the orientation of the two remains substantially the same.
In some embodiments, as shown in fig. 12, step S11 includes sub-steps S111, S112, and S113.
S111, depositing the first metal nanoparticles on a substrate;
s112, adsorbing an analyte on the first metal nanoparticles;
s113, adsorbing the second metal nano on the analyte to realize that the analyte is placed in the nano-gap area.
When the substrate is selected, if the Raman light source or the infrared light source needs to be projected on the substrate, the substrate needs to select a material capable of projecting the Raman light source or the infrared light source; the substrate can be any material substrate if the raman light source or infrared light source does not need to project on the substrate. For example, the substrate may be BaF 2 、CaF 2 KBr, diamond, germanium sheet, zinc sulfide, silicon wafer, and the like.
In some specific embodiments, the first metal nanoparticle and the second metal nanoparticle 120 are modified with a ligand, and the analyte is disposed in the nanogap region through the ligand. Wherein, the ligand and the analyte can be connected by hydrogen bond, van der waals force or covalent bond, etc. to realize the placing of the analyte in the nanogap region between the first metal nanoparticle and the second metal nanoparticle 120. The first metal nanoparticles and the second metal nanoparticles 120 in this embodiment are chemically synthesized, and the surface of the first metal nanoparticles and the second metal nanoparticles is provided with a ligand, and the ligand may be sodium citrate.
In some embodiments, the first metal nanoparticles are one or more of gold nanoparticles, silver nanoparticles, aluminum nanoparticles; the second metal nanoparticles 120 are one or more of gold nanoparticles, silver nanoparticles, and aluminum nanoparticles. The first metal nanoparticles and the second metal nanoparticles 120 may be the same metal nanoparticles or different metal nanoparticles.
In this embodiment, the first metal nanoparticles and the second metal nanoparticles 120 are preferably one or more of gold nanoparticles, silver nanoparticles, and aluminum nanoparticles, but not limited to gold nanoparticles, silver nanoparticles, and aluminum nanoparticles, and may also include other metal nanoparticles that can greatly reduce light loss.
It is understood that different metal nanostructures can be constructed between the first metal nanoparticles and the second metal nanoparticles 120 to realize the placement of the analyte in the metal nanogap, such as metal nanoparticle-metal nano groove, metal nanoparticle-metal nano wire, metal nano bow-micro bow, metal nanoparticle-molecule-metal micro disc, metal nanoparticle close-packed-gold film, and other configurations.
The up-conversion raman sensing method provided herein is specifically described below in a metal nanoparticle-metal nano-groove configuration.
As shown in fig. 13, an up-conversion raman sensor includes:
the substrate is polished on both sides, and a plurality of grooves 130 with metal inner walls 131 are arranged on the substrate 110; a metal connection layer 140, a first metal layer 150 and a second metal layer 160 are sequentially deposited on the substrate 110, and after the metal connection layer 140, the first metal layer 150 and the second metal layer 160 are deposited on the substrate 110, exposure and etching are performed, so that a groove 130 is formed on the substrate 110; wherein the first metal layer 150 acts as a first metal nanoparticle and can capture an analyte via ligands on its particles;
a second metal nanoparticle 120, the second metal nanoparticle 120 being movable to fill the groove 130;
wherein the analyte fixed between the second metal nanoparticles 120 and the grooves 130 is irradiated with visible laser, and the bottom of the substrate 110 is irradiated with a predetermined infrared laser based on a molecular vibration pattern of the analyte, so as to detect an up-conversion raman signal of the analyte.
Specifically, the metal connection layer 140, the first metal layer 150, and the second metal layer 160 are deposited on the substrate 110 by using an electron beam evaporation or magnetron sputtering technique. The deposition speed of the metal connecting layer 140, the first metal layer 150 and the second metal layer 160 should be in the range of 0.1-2nm/s, after deposition is completed, the root mean square of the roughness of the surface of the sample in the range of 10 square microns should be kept within 10nm, and the surface of the sample is too rough to affect the optical performance.
Meanwhile, after the metal connection layer 140, the first metal layer 150, and the second metal layer 160 are sequentially deposited on the substrate 110, the pattern of the array groove 130 is written on the substrate 110 by electron beam exposure. Specifically, before pattern writing, the sample is baked at a high temperature (e.g., 180 ℃) for 2-5min to remove moisture adsorbed on the surface, and then left to stand for 1-3min to keep the sample at room temperature, then 1-3mL of PMMA (polymethyl methacrylate) solution (molecular weight 950K, dilution a4, company MircoChem) is spin-coated on the surface of the sample at a spin-coating speed of 1500-.
Wherein the spatial resolution of pattern exposure is 5-30nm, preferably 20nm (namely, the geometric pattern is converted into a square pixel element with the size of 20 nm), the voltage of an electron beam is 50-100kv (preferably 100 kv), the current is 60-80nA (preferably 70 nA), the exposure dose is 1000-. The developing solution is prepared by mixing methyl isobutyl ketone and isopropanol, wherein the ratio of methyl isobutyl ketone to isopropanol is preferably 1: 3.
in addition, after the substrate 110 is exposed and developed, an argon ion etching technique may be used to etch the substrate 110 to form the array groove 130. Wherein, the etching voltage, current and etching time depend on the thickness of the first metal layer 150, and the etching angle is-10 ° (the sample surface is-10 ° +90 ° =80 ° with the direction of the argon ion beam). The substrate 110 is kept rotating at a speed of 5-15rpm (preferably 10 rpm) in the etching process, after the etching is completed, PMMA on the surface of the substrate 110 needs to be removed, specifically, the substrate 110 is immersed in acetone and subjected to ultrasonic treatment (for example, 30 min), after the ultrasonic treatment is completed, the substrate is placed in etching equipment again, the power is kept unchanged, the etching angle is set to-70 degrees for etching, after the etching is completed, the substrate is placed in an oxygen plasma environment for surface oxidation treatment (for example, 10 min), the maximum power does not exceed 400w, further, the surfaces of the metal connecting layer 140 and the second metal layer 160 are oxidized into metal oxide layers, only the first metal layer 150 on the inner wall of the groove 130 is exposed to the outside, and the inner wall of the groove 130 is in a state without strong adsorption molecules.
It is understood that the first metal layer 150 provided in this embodiment is formed by depositing first metal nanoparticles, preferably gold nanoparticles, on a substrate, and the thickness of the first metal layer 150 is preferably 30-200nm, and a thickness below 30nm may cause the optical performance of the device to be reduced, while a thickness above 200nm may significantly increase the cost, and may also cause side effects such as optical interference and transmittance reduction. The material of the metal connection layer 140 is preferably aluminum, which has a thickness of preferably 1-2nm, because aluminum has less influence on the optical properties of gold.
In some embodiments, the material of the second metal layer 160 may be aluminum or a metal material that can be oxidized in an oxygen plasma environment, in this embodiment, the material of the second metal layer 160 is preferably aluminum, when the substrate 110 is exposed, etched, and oxidized, the surface of the second metal layer 160 is oxidized by aluminum, and since the hydrophilicity of aluminum oxide is better than that of gold, the substrate 110 after the groove 130 is formed may be compatible with the subsequent assembly application of the second metal nanoparticles 120. In addition, if a hydrophobic surface is desired, silane molecules can be adsorbed onto the alumina surface to form a hydrophobic surface.
In the above embodiment, if the argon ion etching technique is used for etching, the thickness of the first metal layer 150 is preferably 150nm, the etching voltage is preferably 300V, the current is preferably 500 mA, the first etching time is preferably 240s, and the second etching time is preferably 30 s.
In some embodiments, the grooves 130 are rectangular grooves, the long sides of the grooves 130 are 1 to 5 micrometers, the short sides of the grooves 130 are 40 to 500 nanometers, the diameter of the second metal nanoparticles 120 is 40 to 500 nanometers, the grooves 130 are arranged in an array on the substrate 110, the distance between the adjacent grooves 130 on two long sides is 1 to 4 micrometers, preferably 2 micrometers, and the distance between the adjacent grooves 130 on two short sides is 1 to 4 micrometers, preferably 2 micrometers. The long sides of the grooves 130 are preferably 2 micrometers, the long sides of the grooves 130 determine infrared resonance wavelengths, plasmon resonance peaks corresponding to the grooves are about 9.3 micrometers and matched with a 1080cm & lt-1 & gt molecular mode, the short sides of the grooves 130 are preferably 150nm, the diameter of the second metal nanoparticles 120 is preferably 150nm, and the length of the short sides is not less than 10nm or one fifth of the thickness of the first metal layer 150.
It is understood that after the groove preparation is completed, a plurality of batches of groove 130 samples can be put into the BPT molecule solution with different concentrations for 1-3h, preferably 2h, because the thiol group and gold atom of the BPT molecule have strong binding effect, the BPT molecule can automatically assemble into a monolayer of molecules on the surface of the groove 130, and finally the samples are taken out, washed with ethanol, and dried with nitrogen for standby.
Specifically, when the BPT molecules are adsorbed on the groove 130, the second metal nanoparticles 120 may be prepared into a solution, and the solution may be dropped on samples of BPT molecules with different concentrations, and after standing for 1-30min (preferably 10 min), the samples may be cleaned, and at this time, the groove 130 structure is filled with the second metal nanoparticles 120, so that the BPT molecules are sandwiched between the first metal nanoparticles and the second metal nanoparticles 120, thereby having a stronger electromagnetic field enhancing effect. In addition, a more complicated and reliable capillary self-assembly technique may be adopted to complete the structure of the second metal nanoparticles 120 filling the grooves 130, and the implementation manner thereof may be modified according to specific situations, which is not specifically limited in this embodiment.
The process of preparing the second metal nanoparticle 120 solution in the above embodiment may be: the second metal nanoparticles 120 are prepared according to the size of the grooves 130, and the second metal nanoparticles 120 are dispersed in a solvent, wherein the concentration of the second metal nanoparticles 120 solution may be OD1 (OD, optical density, OD1 here means that the light intensity of 520nm light passing through the solution is attenuated to 10% after 1 cm), and the solvent may be a common chemical or biological sensing solvent such as deionized water, ethanol, 1 XPBS (phosphate buffered saline), 1 XPS (triethanolamine buffered saline), and the like.
In addition, if a specific chemical molecule or biological protein is detected, a specific ligand or antibody is modified on the surface of the first metal layer 150, so that the analyte can be captured on the surface of the inner wall of the groove 130.
Meanwhile, when the analyte needs to be detected by the up-conversion raman signal, referring to fig. 14, after the groove 130 is fixed in the nanogap region between the first metal nanoparticle and the second metal nanoparticle 120, the analyte is placed under a sample stage of a microscope, a laser with 785 nm wavelength is selected, aligned and converged to the position where the nanoparticle is located through an objective lens, a scattering signal is collected through the same objective lens, and an optical signal is introduced into a spectrometer through a series of optical elements to obtain a raman spectrum, and at the same time, a waveband of 1080cm can be measured -1 The infrared laser is converged to the back of the sample through another objective lens, and simultaneously, the infrared light spot is overlapped with the visible light spot in space, so that an up-conversion Raman spectrum can be generated. As shown in fig. 15-18, if the spectrometer is in low resolution mode (>1 nm), the information of the analyte can be judged by observing the intensity of the up-conversion Raman spectrum to analyze; if the spectrometer is switched to high resolution mode (<1nm, preferably<0.1 nm), the sensitivity and the specific recognition degree of the analyte can be further improved by observing the peak type information of the peak. In addition, if the analyte concentration is too low, the analyte concentration is too lowThe signal can be amplified by increasing the power of the infrared laser light if the signal is weak.
Although the physical principle of the up-conversion Raman sensing adopted by the invention is slightly different from that of the traditional Raman sensing, the up-conversion Raman sensing provided by the invention can be directly compatible with most of technical optimization based on the traditional Raman molecular sensing, and comprises the following steps: the signal amplification technology, for example, combines an analyte with a metal nano mechanism with plasmon resonance to realize surface enhanced Raman spectroscopy technology of Raman signal exponential amplification, and simultaneously, the preparation technology of the surface enhanced Raman substrate, namely the optimal design of optical resonance and antenna effect, can be optimized on the basis. In addition, the molecular diffusion and substrate adsorption techniques necessary for molecular detection can also be implemented.
In addition, compared with the traditional Raman sensing, the up-conversion Raman sensing provided by the embodiment of the invention has higher detection sensitivity. Traditional raman sensing often requires signal amplification techniques to achieve sufficient sensitivity, i.e., combining the analyte with metal nanostructures with plasmon resonance to achieve exponential amplification of the raman signal. The upconversion Raman sensing provided by the embodiment of the invention not only can completely inherit the characteristics of the surface enhanced Raman spectroscopy technology, but also generates an upconversion Raman signal in direct proportion to the power of the infrared laser, so that the intensity of the signal can be further improved only by improving the power of the infrared laser, and the detection sensitivity is further greatly improved.
The up-conversion Raman sensing combined high-resolution spectrometer provided by the embodiment of the invention has higher signal-to-noise ratio compared with the traditional Raman spectrum, the up-conversion Raman signal is stronger than the traditional Raman signal in the low-resolution spectrum environment, and the signal-to-noise ratio of the generated up-conversion Raman signal can be further amplified by a plurality of times compared with the up-conversion Raman signal in the low-resolution mode when the same measurement is carried out in the high-resolution spectrum. Because the real peak width of the up-conversion Raman peak is consistent with the infrared incident light source and is far smaller than the resolution of the conventional Raman spectrometer, the signal of the up-conversion Raman signal in a low-resolution spectrum becomes short and wide, while the signal in a high-resolution spectrum is high and narrow, and the sizes of the geometrical areas of the spectra in the two cases are not changed. Therefore, further improving the spectral resolution can further improve the intensity of the signal, and the proportion of the wide background noise below the up-converted raman signal occupying the whole spectrum is relatively reduced, so as to obtain a higher signal-to-noise ratio.
It can be understood that the raman spectrum generated by the upconversion raman sensing technology provided by the embodiment of the present invention in combination with a high resolution spectrometer has a higher signal-to-noise ratio than the conventional raman spectrum, so the technology is particularly suitable for realizing specific sensing of a molecule to be detected (only staring at an upconversion signal with a much narrower peak width) in a complex detection environment (such as an intracellular) with a high fluorescence background and multiple interfering molecules. In addition, different molecules to be detected have different characteristic up-conversion vibration modes, so that infrared light sources with various wavelengths are used in combination, and high signal-to-noise ratio, multi-molecule and multi-index molecule sensing can be realized.
In addition, the up-conversion Raman sensing provided by the embodiment of the invention has larger imaging sensing application potential than the traditional Raman sensing. The up-conversion Raman signal is a narrow-band signal, so other background signals can be effectively isolated on the spectrum, and the Raman imaging with higher signal-to-noise ratio than the traditional Raman sensing can be obtained by matching with the narrow-band optical filter.
In addition, the embodiment of the invention also provides an application of the up-conversion Raman sensing method, and the up-conversion Raman sensing method is applied to virus detection, chemical toxicity detection, disease and cancer clinical diagnosis, drug discovery, food process control and environmental monitoring.
While the invention has been described with reference to specific embodiments, the invention is not limited thereto, and various equivalent modifications and substitutions can be easily made by those skilled in the art within the technical scope of the invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (7)
1. An up-conversion raman sensing method, comprising:
selecting a Raman light source with a first wavelength and an infrared light source with a second wavelength according to the standard Raman spectrum and the infrared absorption spectrum of the analyte;
simultaneously irradiating the analyte by adopting the Raman light source with the first wavelength and the infrared light source with the second wavelength to detect an up-conversion Raman signal of the analyte, and realizing virus detection, chemical toxicity detection, drug discovery, food process control and environmental monitoring through the up-conversion Raman signal;
wherein, the Raman light source with the first wavelength and the infrared light source with the second wavelength are selected according to the standard Raman spectrum and the infrared absorption spectrum of the analyte, and the method comprises the following steps:
overlapping the standard Raman spectrum with the infrared absorption spectrum to obtain an overlapping peak;
determining the molecular vibration mode of the analyte according to the overlapping peaks;
selecting the Raman light source with the first wavelength and the infrared light source with the second wavelength according to the molecular vibration mode; specifically, unit conversion is carried out according to the molecular vibration mode to determine the infrared light source with the second wavelength, meanwhile, the Raman light source with the first wavelength in the near-infrared light source and the visible light source is selected, and the conversion formula R/10 is used 7 =1/λ L 1/λ determines the up-converted stokes and anti-stokes raman light wavelengths;
wherein R is molecular vibration mode, lambda L λ is the upconverted stokes or anti-stokes raman light wavelength, which is the first wavelength.
2. The method of claim 1, further comprising, prior to simultaneously illuminating the analyte with the raman light source of the first wavelength and the infrared light source of the second wavelength:
the analyte is placed in a nanogap region between the first metal nanoparticle and the second metal nanoparticle.
3. The up-conversion raman sensing method according to claim 2, wherein said placing the analyte in a nanogap region between a first metal nanoparticle and a second metal nanoparticle comprises:
depositing the first metal nanoparticles on a substrate;
adsorbing the analyte on the first metal nanoparticles;
adsorbing the second metal nano-particles on the analyte to achieve placement of the analyte in the nano-gap region.
4. The up-conversion raman sensing method according to claim 2, wherein the first metal nanoparticle and the second metal nanoparticle are each modified with a ligand, and the analyte is placed in the nanogap region through the ligand.
5. The upconversion raman sensing method according to claim 2, wherein the first metal nanoparticle is one or more of a gold nanoparticle, a silver nanoparticle, and an aluminum nanoparticle.
6. The upconversion raman sensing method according to claim 2, wherein the second metal nanoparticles are one or more of gold nanoparticles, silver nanoparticles, and aluminum nanoparticles.
7. The up-conversion raman sensing method according to claim 1, wherein the infrared light source is any one of a near-infrared light source, a mid-infrared light source, a far-infrared light source, and an extreme far-infrared light source.
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