CN114942240A - Up-conversion Raman sensor and application - Google Patents
Up-conversion Raman sensor and application Download PDFInfo
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- CN114942240A CN114942240A CN202210597176.4A CN202210597176A CN114942240A CN 114942240 A CN114942240 A CN 114942240A CN 202210597176 A CN202210597176 A CN 202210597176A CN 114942240 A CN114942240 A CN 114942240A
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Abstract
The embodiment of the invention discloses an up-conversion Raman sensor and application thereof. The sensor includes: a substrate; a first metal nanoparticle deposited on a substrate; a second metal nanoparticle movable to be close to the first metal nanoparticle; the method comprises the steps of fixing an analyte in or near a nanogap region between first metal nanoparticles and second metal nanoparticles, and simultaneously irradiating the analyte by adopting a Raman light source and an infrared light source based on a molecular vibration mode of the analyte so as to detect an up-conversion Raman signal of the analyte. The up-conversion Raman sensor provided by the invention not only can realize molecule specificity detection and stable signal, but also can be directly compatible with most of technical optimization based on the traditional Raman molecule sensing, and compared with the traditional Raman technology, the up-conversion Raman sensor can effectively separate background noise which often appears in the traditional Raman technology, and greatly improve the sensitivity and signal-to-noise ratio of molecular signals 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 sensor and application thereof.
Background
Currently, microscopic detection usually employs optical detection and sensing of raman scattering to obtain 'fingerprint' spectrum of molecular vibration, thereby realizing label-free high-specificity detection. Based on this unique advantage, raman detection and sensing play an important role in a variety of biochemical related applications, including viral detection, chemical toxicity detection, clinical diagnosis of disease and cancer, drug discovery, food process control, and environmental monitoring, among others.
However, many difficulties are still not solved in practical applications of raman sensing, for example, when the raman sensing is applied to a multi-molecule or complex detection environment (such as in vivo of a cell), the characteristic raman signal of a true molecule to be detected is easily interfered or masked by the raman signals of other molecules in the environment, so that it is difficult to perform signal analysis simply and reliably, and in this case, a complex machine learning algorithm is required to implement, which results in greatly reduced specificity of raman sensing and also affects the quantitative property of molecule detection.
In addition, when a molecular raman signal is excited, a fluorescence signal of the molecule is often excited, and the fluorescence signal is shown as a broadband strong background signal (a large packet below the raman spectrum) below a raman peak on a spectrum, and even the raman signal is directly covered, so that the sensitivity of raman sensing is greatly influenced.
The defects cause that the Raman sensing is only limited to spectral detection and analysis, compared with other optical processes such as fluorescence and the like, the imaging application of the Raman sensing is severely limited by the fluorescence background, and meanwhile, the specific identification advantage of the Raman imaging can be obviously reduced due to multi-molecule interference.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides the up-conversion Raman sensor and the application thereof, and the up-conversion Raman sensor 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, the present invention provides an up-conversion raman sensor comprising:
a substrate;
a first metal nanoparticle deposited on the substrate;
a second metal nanoparticle movable to be close to the first metal nanoparticle;
wherein an analyte is immobilized at or near a nanogap region between the first metal nanoparticle and the second metal nanoparticle, and the analyte is simultaneously irradiated with a raman light source and an infrared light source based on a molecular vibration mode of the analyte to detect an up-conversion raman signal of the analyte.
Preferably, in the up-conversion raman sensor, the first metal nanoparticles are deposited on the substrate to form a plurality of grooves on the substrate.
More preferably, in the up-conversion raman sensor, a metal connection layer, the first metal nanoparticles, and the second metal layer are sequentially deposited on the substrate, and are exposed and etched to form the grooves on the substrate.
More preferably, in the up-conversion raman sensor, the groove is a rectangular groove, and the long side of the groove is 1 to 5 micrometers and the short side is 10 to 1000 nanometers.
More preferably, in the up-conversion raman sensor, the grooves are arranged in an array on the substrate, a distance between two adjacent grooves with long sides is greater than 0.5 micrometers, and a distance between two adjacent grooves with short sides is greater than 0.5 micrometers.
Preferably, in the up-conversion raman sensor, the diameters of the first metal nanoparticle and the second metal nanoparticle are both 10 to 1000 nm.
Preferably, in the up-conversion raman sensor, the first metal nanoparticles and the second metal nanoparticles are one or more of gold nanoparticles, silver nanoparticles and aluminum nanoparticles.
Preferably, in the up-conversion raman sensor, the first metal nanoparticle and the second metal nanoparticle are both modified with a ligand, and the analyte is immobilized in the nanogap region through the ligand.
Preferably, in the up-conversion raman sensor, the infrared light source is any one of a near-infrared light source, a mid-infrared light source, a far-infrared light source, and a very far-infrared light source, and the raman light source is a visible laser.
In a second aspect, the present invention also provides the use of an up-conversion raman sensor according to the first aspect in viral 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 sensor and application thereof. The up-conversion Raman sensor provided by the embodiment of the invention not only can realize molecule specificity detection and stable signal, but also can be directly compatible with most of technical optimization based on the traditional Raman molecule sensing, and compared with the traditional Raman technology, the up-conversion Raman sensor can effectively separate background noise which often appears in the traditional Raman technology, and greatly improve the sensitivity and signal-to-noise ratio of the molecule 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 required to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the description below are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.
Fig. 1 is a schematic structural diagram of an up-conversion raman sensor according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of a standard Raman spectrum and an infrared absorption spectrum overlapped according to an embodiment of the present invention
FIG. 3 is a Raman spectrum formed in a conventional Raman process;
FIG. 4 is a graph of an upconverted Raman spectrum according to an embodiment of the present invention;
fig. 5 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. 6 is a schematic structural view of an analyte disposed in a nanogap region according to an embodiment of the invention;
FIG. 7 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 the embodiment of the invention;
FIG. 8 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. 9 is a schematic diagram of a process for implementing up-conversion raman according to an embodiment of the present invention;
fig. 10 is a schematic structural diagram of an up-conversion raman sensor according to an embodiment of the present invention;
fig. 11 is another schematic structural diagram of an up-conversion raman sensor according to an embodiment of the present invention;
FIG. 12 is an optical diagram of an upconverted Raman signal for detecting an analyte according to an embodiment of the present invention;
FIG. 13 is a graph of an upconverted Raman spectrum in low resolution mode according to an embodiment of the present invention;
FIG. 14 is a graph of an upconverted Raman spectrum in high resolution mode according to 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 the upconversion Raman spectrum in high resolution mode according to an embodiment of the present invention;
fig. 17 is a schematic flow chart of an up-conversion raman sensing method according to an embodiment of the present invention;
FIG. 18 is another optical diagram of an upconverted Raman signal for detecting an analyte according to embodiments 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 of the invention. 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 structural diagram of an up-conversion raman sensor according to an embodiment of the present invention. As shown in fig. 1, an up-conversion raman sensor includes:
a substrate 110;
a first metal nanoparticle 111, the first metal nanoparticle 111 being deposited on the substrate 110;
a second metal nanoparticle 120 movable to be close to the first metal nanoparticle 111;
wherein an analyte is immobilized at or near a nanogap region between the first metal nanoparticle 111 and the second metal nanoparticle 120, and the analyte is simultaneously irradiated with a raman light source or an infrared light source based on a molecular vibration mode of the analyte to detect an up-conversion raman signal of the analyte.
According to the up-conversion raman sensor provided by the embodiment of the present invention, by depositing the first metal nanoparticles on the substrate 110, when an analyte needs to be detected, the analyte only needs to be adsorbed on the first metal nanoparticles 111, and the second metal nanoparticles 120 are adsorbed on the analyte, so that the analyte is in a nanogap between the first metal nanoparticles 111 and the second metal nanoparticles 120, and an up-conversion raman signal of the analyte can be detected by simultaneously irradiating the analyte with a raman light source and an infrared light source based on a molecular vibration mode of the analyte.
According to the up-conversion Raman sensor provided by the embodiment of the invention, on the basis of the traditional Raman detection technology, the infrared light source is added to superpose the extra signals with the same frequency on the specific vibration mode of the Raman spectrum of the analyte to form the up-conversion Raman signal, the up-conversion Raman signal is consistent with most basic physical properties of the traditional Raman signal, but the spectrum shape and the peak intensity of the signal follow the infrared light source, so that various unique advantages of the traditional Raman technology are inherited, the specific detection can be realized, and meanwhile, the signal is stable and cannot be quenched.
Specifically, when the up-conversion raman of the analyte is realized, a molecular vibration mode of the analyte needs to be selected, and the analyte in the mode needs to have both raman scattering activity and infrared absorption activity, so that the infrared light source can be coupled with the selected molecular vibration mode by means of an infrared absorption process, and simultaneously, a raman scattering process excited by visible pump light is used for up-converting a signal into stokes (a difference frequency process between infrared light and a raman light source) and anti-stokes (a sum frequency process between infrared light and a raman light source) light in a visible waveband.
It is also understood that the conversion raman sensing process of molecules can be viewed as a Sum Frequency Generation (SFG) and Difference Frequency Generation (DFG) process in second order nonlinear optics, i.e., a raman light source and an infrared light source are combined into a higher energy anti-stokes raman light (SFG, Sum Frequency process) or the energies of the raman light source and the infrared light source are subtracted to form a 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 is firstly absorbed by the analyte and converted into molecular vibration energy by means of an infrared absorption process, and meanwhile, the analyte is irradiated by the raman light source to generate a raman scattering process, so that the converted molecular vibration energy and the raman light source 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.
It is understood that when the infrared light source is selected, the analyte is characterized to obtain a standard raman spectrum and an infrared absorption spectrum of the analyte, then the standard raman spectrum and the infrared absorption spectrum of the analyte are overlapped to determine overlapped peaks, and a molecular vibration mode of the analyte is determined through the overlapped peaks, so that the wavelength of the preset infrared laser is determined.
Referring to fig. 2, fig. 2 is a schematic diagram illustrating a standard raman spectrum and an infrared absorption spectrum overlapped according to an embodiment of the present invention. In the example shown in FIG. 2, if the analyte is BPT molecule (biphenyl-4-thiol, CAS number 19813-90-2) powder, the standard Raman spectrum and the infrared absorption spectrum of the BPT molecule are measured, and then the standard Raman spectrum and the infrared absorption spectrum of the BPT molecule are overlapped, and the candidate molecular pattern of the BPT molecule is 1080cm -1 And 1580cm -1 Wherein 1580cm is adopted -1 In the mode, other interfering molecules in the environment may have the same carbon peak, so that the molecular mode of the BPT molecule is determined to be 1080cm -1 So that the adopted wave band is 1080cm -1 The bottom of the substrate 110 is illuminated to detect the up-converted raman signal of the BPT molecules.
In the embodiment shown in FIG. 2, the molecular vibration 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; and for the traditional Raman scattering process of molecules, the corresponding mode R = +/-1080 cm -1 The wavelength lambda of the Raman light 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 and 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).
Referring to fig. 3 and 4, fig. 3 is a raman spectrum formed in a conventional raman process; fig. 4 is an up-conversion raman spectrum provided by an embodiment of the present invention. As shown in fig. 3 and 4, 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 sum frequency process or the difference frequency process is directly generated between the jumpers and the raman light source, so as to obtain the up-conversion raman light, i.e., the up-conversion raman light = 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. 4, when the analyte is subjected to the upconversion raman process, the conventional raman process is synchronized, i.e. conventional raman + upconversion raman = incident light ± infrared light + incident light ± thermal vibration, where the conventional raman signal brought by the thermal vibration is inverted to thermal noise to be suppressed. In the embodiment shown in fig. 4, 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.
Although the upconversion raman signal can be theoretically obtained by simply relying on molecules, 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 required 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 111 and the second metal nanoparticle 120 to enable molecular coupling, and the nanogap region between the first metal nanoparticle 111 and the second metal nanoparticle 120 can provide a huge electromagnetic field enhancement, thereby realizing a large-scale 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. 5, 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 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 a plurality of metal nanoparticles, the electromagnetic field localization and enhancement provided by the nanogap structure formed between two metal nanoparticles is often the strongest, which is the preferred structure of the present application. Referring to FIG. 6, 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. 7 and 8, 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 raman light source is 740 nanometers, the wavelength of the up-conversion raman scattering is 685 nanometers, and the nanogap region between the first metal nanoparticle 111 and the second metal nanoparticle 120 has surface plasmon resonance response, and the two wavelengths are respectively covered while the two processes are enhanced, so that matching in 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. 9, for an immobilized molecule, only a specific direction of the electromagnetic field can effectively excite the ir absorption and raman scattering processes, which are simplified as ir absorption dipole and raman dipole processes, respectively. Wherein the dipoles are two opposite charges, positive and negative, 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, the substrate 110 is BaF 2 、CaF 2 One or more of KBr, diamond, germanium sheet, zinc sulfide and silicon wafer; the thickness of the substrate 110 is 100-400 microns, and the substrate 110 is polished on both sides. When the substrate 110 is selected, both sides of the substrate 110 need to be polished, and the substrate 110 is primarily polished on both sides to ensure that the infrared light can penetrate through the back side of the substrate 110 to irradiate the structure on the front side of the substrate 110. The substrate 110 in this embodiment is preferably a silicon wafer, typically about 350 microns thick, having a 30% transmission of infrared light at a wavelength of 9.3 microns.
It should be noted that the size of the silicon wafer selected in the above embodiments is not particularly limited, and may be selected according to practical applications. In order to be compatible with the existing micromachining technology, the silicon wafer provided by the embodiment is generally a 4-inch silicon wafer by default.
In some embodiments, the first metal nanoparticles 111 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 111 and the second metal nanoparticles 120 may be the same metal nanoparticles or different metal nanoparticles.
It should be noted that, in this embodiment, the diameters of the first metal nanoparticles and the second metal nanoparticles are both 10 to 1000 nanometers; the first and second metal nanoparticles 111 and 120 are not limited to gold nanoparticles, silver nanoparticles, and aluminum nanoparticles, but include other materials that can greatly reduce light loss.
It is understood that different metal nanostructures can be constructed between the first metal nanoparticle 111 and the second metal nanoparticle 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.
In some embodiments, please refer to fig. 10, fig. 10 is a schematic structural diagram of an up-conversion raman sensor according to an embodiment of the present invention.
As shown in fig. 10, an up-conversion raman sensor includes: the substrate 110 is provided with a plurality of grooves 130 with metal inner walls 131; a second metal nanoparticle 120, the second metal nanoparticle 120 being movable to fill the recess 130. Wherein the first metal nanoparticles 111 are deposited on the substrate to form several grooves on the substrate 110. Meanwhile, the inner wall of the groove exposes the first metal nanoparticles 111, and the surface of the substrate 110 does not expose the first metal nanoparticles 111.
In the embodiment shown in fig. 10, by providing a plurality of grooves 130 with metal inner walls 131 on a substrate 110, when an analyte needs to be detected, the analyte is adsorbed on the metal inner walls 131, and the grooves 130 are filled with second metal nanoparticles 120, so that the analyte is fixed between the second metal nanoparticles 120 and the grooves 130, and simultaneously, the analyte is irradiated with visible laser, and the bottom of the substrate 110 is irradiated with a predetermined infrared laser based on the molecular vibration mode of the analyte, so that an up-conversion raman signal of the analyte can be detected.
In some embodiments, please refer to fig. 11, and fig. 11 is another schematic structural diagram of an up-conversion raman sensor according to an embodiment of the present invention.
As shown in fig. 11, a metal connection layer 140, a first metal layer 150, and a second metal layer 160 are sequentially deposited on a 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. 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 first metal layer is formed by depositing the first metal nanoparticles 111 on the substrate 110, the deposition speed of the metal connecting layer 140, the first metal layer 150 and the second metal layer 160 is within the range of 0.1-2nm/s, after the deposition is completed, the root mean square of the roughness of the sample surface within the range of 10 square microns is kept within 10nm, and the optical performance is affected due to the fact that the sample surface is too rough.
In some embodiments, the material of the metal connection layer 140 may be chromium or aluminum, chromium may be used as a standard connection material, and the thickness deposited on the substrate 110 is between 0.5 and 2 nanometers, and excessively thick chromium may significantly reduce the plasmon resonance performance of the first metal layer 150.
In some embodiments, 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-.
In this embodiment, the spatial resolution of the pattern exposure is 5-30nm, preferably 20nm (i.e. transforming the geometric pattern into square pixel elements of 20 nm), and the voltage of the electron beam is 50-100kv (preferably100 kv) is selected, the current is 60-80nA (preferably 70 nA), the exposure dose is 1000-2000 mu C/cm 2 (preferably 1500. mu.C/cm) 2 ) After the electron beam exposure is completed, the sample is immersed in a developing solution for 1-3min (preferably 2 min), and the developing solution is cleaned and blown dry by nitrogen. 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 some embodiments, 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 ° (sample surface is-10 ° +90 ° =80 ° with the direction of 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 side of the groove 130 is preferably 2 microns, the long side of the groove 130 determines the infrared resonance wavelength, and the corresponding plasmon resonance peak is about 9.3 microns and 1080cm -1 The molecular pattern is matched, the short side of the groove 130 is preferably 150nm, the diameter of the second metal nanoparticle 120 is preferably 150nm, wherein the length of the short side should not be less than 10nm or one fifth of the thickness of the first metal layer 150.
In some embodiments, the inner walls of the groove 130 are exposed with first metal nanoparticles. Specifically, when the analyte is immobilized between the second metal nanoparticles 120 and the inner walls of the grooves 130, the gap regions between the second metal nanoparticles 120 and the inner walls of the grooves 130 generate a huge electromagnetic field enhancement, and the right sign of the electromagnetic field enhancement coincides with the molecules of the analyte in space, so that both processes of infrared absorption and raman scattering of the analyte are resonance-enhanced. Meanwhile, when the second metal nanoparticles 120 are filled in the grooves 130, resonance modes having sufficient electromagnetic field enhancement can be formed in both the infrared and visible wavelength bands.
It can be understood that the two electromagnetic field resonance modes need to overlap in space and simultaneously overlap with the molecule in space, i.e. the space of the molecule-the infrared resonance mode-the visible resonance mode coincides with the molecule in space. The plurality of grooves 130 on the substrate 110 may be in an array configuration, or may be in other configurations, and only the above conditions are required to be satisfied.
In some embodiments, the first metal nanoparticle 111 and the second metal nanoparticle 120 are modified with a ligand, and the second metal nanoparticle 120 fixes the analyte between the metal nanoparticle 120 and the groove 130 through the ligand, i.e., the analyte is located in the nanogap region between the first metal nanoparticle 111 and the second metal nanoparticle 120. Wherein, the first metal nanoparticle 111 and the second metal nanoparticle 120 have a ligand on their surfaces when chemically synthesized, and the ligand may be sodium citrate.
In some embodiments, the analyte may be BPT molecules, the material of the second metal layer 160 may be gold, multiple batches of the sample of the groove 130 may be put into a solution of BPT molecules with different concentrations for 1-3h, preferably 2h, since the thiol group of the BPT molecules and the gold atom have strong binding effect, the BPT molecules will automatically assemble a monolayer of molecules on the surface of the nano-groove 130, and finally the sample is taken out, washed with ethanol, and dried with nitrogen for use.
Specifically, when the BPT molecules in the above embodiment are adsorbed on the groove 130, the second metal nanoparticles 120 may be prepared into a solution, and the solution is dropped on samples of BPT molecules with different concentrations, and after standing for 1-30min (preferably 10 min), the samples are 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 particles and the groove 130, thereby having a strong 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 some embodiments, when specific chemical molecule or biological protein detection is involved, a specific ligand or antibody is modified on the surface of the first metal layer 150, so that the surface of the inner wall of the groove 130 can capture the analyte.
In some embodiments, as shown in fig. 12, after the analyte is fixed between the second metal nanoparticles 120 and the grooves 130, the sample is placed under a sample stage of a microscope, a visible laser with a wavelength of 785 nm is selected, reflected by the reflection sheet 220 into the objective lens 210, and focused by the objective lens 210 to the position where the nanoparticles are located, and meanwhile, a scattering signal is collected by the objective lens 210, and the light signal is guided to a spectrometer through a series of optical elements to obtain a raman spectrum, and meanwhile, the wavelength band can be 1080cm -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. 13-16, 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 concentration of the analyte is too low, resulting in a weak signal, the signal can be amplified by increasing the power of the infrared laser.
An embodiment of the present invention further provides an up-conversion raman sensing method, as shown in fig. 17, the method includes:
s101, 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;
s102, placing the analyte in a nanogap region between the first metal nanoparticle 111 and the second metal nanoparticle 120;
s103, 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.
It can be understood that the up-conversion raman sensing method provided by the embodiment of the present invention is not limited to only injecting the raman light source into the top of the substrate 110 and injecting the infrared light source into the bottom of the substrate 110 to achieve the detection of the up-conversion raman signal of the analyte, and the method shown in fig. 18 can also be adopted to inject the infrared light source and the raman light source into the upper surface of the substrate 110 to achieve the detection of the up-conversion raman signal of the analyte.
In the optical path diagram shown in fig. 18, the raman light source employs a visible laser, the infrared light source employs a mid-infrared laser, the visible laser is incident into the objective lens above the substrate 110 through the notch filter 230, and the mid-infrared laser is incident into the objective lens 210 above the substrate 110 through the mid-infrared dichroic mirror 240, so that the analyte in the nanogap is simultaneously irradiated above the substrate 110, at this time, the infrared light spot is overlapped with the visible light spot in space, and the detection of the up-conversion raman signal of the analyte is realized. However, although the notch filter 230 reflects only laser light and transmits other wavelengths, the intermediate infrared dichroic mirror 240 reflects infrared laser light, but is transparent to visible light, and the objective lens 210 may be a reflective objective lens or an intermediate infrared transmissive objective lens.
Although the physical principle of the up-conversion Raman sensing technology adopted by the invention is slightly different from that of the traditional Raman sensing technology, the up-conversion Raman sensing technology 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 the analyte with the 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 110, that is, the optimal design of optical resonance and antenna effect, can be optimized on the basis. In addition, the molecular diffusion and substrate 110 adsorption techniques necessary for molecular detection can also be implemented.
In addition, compared with the traditional Raman sensing technology, the up-conversion Raman sensing technology provided by the embodiment of the invention has higher detection sensitivity. The traditional raman sensing technology often needs to adopt a signal amplification technology to obtain enough sensitivity, namely, an analyte is combined with a metal nano structure with plasmon resonance to realize raman signal exponential amplification. The upconversion Raman sensing technology provided by the embodiment of the invention not only can completely inherit the characteristics of the surface enhanced Raman spectroscopy technology, but also can generate 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 technology provided by the embodiment of the invention is combined with a high-resolution spectrometer, the generated Raman spectrum has higher signal-to-noise ratio than the traditional Raman spectrum, the up-conversion Raman signal is stronger than the traditional Raman signal in a 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 a 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 improvement of spectral resolution can further improve the intensity of the signal, and at the same time, the proportion of the wide background noise below the up-converted raman signal occupying the whole spectrum is relatively reduced, so that a higher signal-to-noise ratio is obtained.
It can be understood that the up-conversion raman sensing technology provided by the embodiment of the present invention, in combination with the raman spectrum generated by the 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 aiming at up-conversion signals 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 technology provided by the embodiment of the invention has larger imaging sensing application potential than that of the traditional Raman sensing technology. 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.
The embodiment of the invention also provides application of the up-conversion Raman sensor, which is applied to virus detection, chemical toxicity detection, clinical diagnosis of diseases and cancers, 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 (10)
1. An up-conversion raman sensor, comprising:
a substrate;
a first metal nanoparticle deposited on the substrate;
a second metal nanoparticle movable to be close to the first metal nanoparticle;
wherein an analyte is immobilized at or near a nanogap region between the first metal nanoparticle and the second metal nanoparticle, and the analyte is simultaneously irradiated with a raman light source and an infrared light source based on a molecular vibration mode of the analyte to detect an up-conversion raman signal of the analyte.
2. The up-conversion raman sensor according to claim 1, wherein the first metal nanoparticles are deposited on the substrate to form a plurality of grooves on the substrate.
3. The up-conversion raman sensor according to claim 2, wherein a metal connection layer, the first metal nanoparticles, and a second metal layer are sequentially deposited on the substrate, and exposed and etched to form the groove on the substrate.
4. An up-conversion raman sensor according to claim 2, wherein said grooves are rectangular grooves having a long side of 1-5 μm and a short side of 10-1000 nm.
5. The up-conversion Raman sensor of claim 4, wherein the grooves are arranged in an array on the substrate, a spacing between adjacent grooves on two long sides is greater than 0.5 microns, and a spacing between adjacent grooves on two short sides is greater than 0.5 microns.
6. The up-conversion raman sensor according to claim 1, wherein the first metal nanoparticle and the second metal nanoparticle each have a diameter of 10 to 1000 nm.
7. The up-conversion raman sensor according to claim 1, wherein the first metal nanoparticles and the second metal nanoparticles are one or more of gold nanoparticles, silver nanoparticles, and aluminum nanoparticles.
8. The up-conversion raman sensor according to claim 1, wherein the first metal nanoparticle and the second metal nanoparticle are each modified with a ligand, and the analyte is immobilized in the nanogap region by the ligand.
9. The up-conversion raman sensor 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 ultra-far-infrared light source, and the raman light source is any one of a near-infrared light source and a visible light source.
10. Use of an up-converting raman sensor according to any one of claims 1 to 9 for virus detection, chemical toxicity detection, clinical diagnosis of diseases and cancers, drug discovery, food process control, and environmental monitoring.
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Address after: Room 606-05, Building 2, No. 333, Xingpu Road, Suzhou Industrial Park, Suzhou Pilot Free Trade Zone, China (Jiangsu) 215000 Patentee after: Suzhou Taihe Optics Technology Co.,Ltd. Address before: 430000 plant 401, 4 / F, building B9, phase I, block B, Wuhan hi tech medical equipment Park, 818 Gaoxin Avenue, Donghu New Technology Development Zone, Wuhan City, Hubei Province (Wuhan area of free trade zone) Patentee before: Wuhan Taihe Optical Technology Co.,Ltd. |