A method for surface enhanced fluorescence and its application

Abstract

The present invention provides a method and application for surface enhanced fluorescence, wherein a nano material is used as an enhanced substrate, a fluorescent substance to be tested is placed on the surface of the enhanced substrate, and then the fluorescent substance to be tested is stimulated by excitation light, and the multiple light scattering effect of the nano material on the excitation light and the radiated fluorescence is utilized to achieve enhancement of the fluorescence signal. According to the present invention, the problems existing in the existing surface enhanced fluorescence technology, such as large light loss, difficulty in control, complex process, high cost, etc., are solved, especially the problem of the current high dependence of the fluorescence enhancement substrate on metals and high refractive index materials is solved, and a lossless, non-metal-dependent surface enhanced fluorescence method is developed, which is not only suitable for the research of basic scientific problems, but also suitable for a wide range of practical applications.

Description

Technical Field

[0001] The present invention relates to the field of surface enhanced fluorescence, and more specifically to a method and application for surface enhanced fluorescence. Background Art

[0002] Fluorescence has a wide range of applications in optical devices, microscopic imaging, biology, medical research and diagnosis. With the increasing requirements for fluorescence sensitivity and detection limit, surface enhanced fluorescence (SEF) technology has become an important branch of modern fluorescence spectroscopy technology. Especially in the field of fluorescence detection with high sensitivity requirements, surface enhanced fluorescence technology can greatly reduce the concentration of the detected object, even to the level of single molecule detection, which is of great significance for early screening of disease markers when the concentration is very low and trace detection of harmful substances. In addition, in single-molecule fluorescence imaging, due to the weak fluorescence of single molecules, it is usually necessary to configure a high numerical aperture objective lens and a highly sensitive and efficient detector. Surface enhanced fluorescence technology can increase the luminescence intensity of fluorescent labeled molecules, reduce the requirements for objective lenses and detectors, improve imaging quality and reduce costs.

[0003] The working principle of SEF is to enhance fluorescence emission and improve fluorescence emission and detection efficiency by regulating the local electromagnetic environment of fluorescent materials. The key point is to design a "surface structure that provides electromagnetic environment" to maximize the number of photons emitted by fluorescent materials located on or near its surface. At present, there are two main types of "surface structures" used for SEF: metal structures and dielectric structures.

[0004] Metal structure: mainly precious metal structures such as gold and silver. By designing metal nanostructures or nanogaps formed by nanostructures, strong surface plasmon resonance or localized plasmon resonance is generated under the excitation of incident light to enhance the fluorescence emission of nearby fluorescent substances. The enhanced electric field intensity generated by the metal structure is high, and the fluorescence enhancement factor is usually high. However, the problem with the metal structure is that the cost is high, and the light loss itself is large, which is prone to local overheating, resulting in inactivation of fluorescent detection substances. In addition, there is a competitive relationship between surface enhanced fluorescence and surface enhanced Raman in the test process. The distance between the test substance and the metal structure needs to be controlled at about 10 nanometers to achieve a good enhancement effect. If it is less than 10 nanometers, Raman enhancement will occur, and if it is greater than 10 nanometers, the enhancement effect will be poor. However, this will be difficult to control in actual operation.

[0005] Dielectric structure: mainly high-refractive index material structures such as silicon and germanium. By designing dielectric nanostructures of specific shapes or nanogaps / arrays formed by nanostructures, the electric and magnetic fields based on Mie theory are resonated under the excitation of incident light to form an enhancement field to enhance fluorescence, which can achieve the enhancement effect of metal structures. However, there are few types of high-refractive index materials that can be used, mainly silicon in the visible light region, and the preparation process mainly relies on micro-nano processing technology (electron beam etching, ion beam etching, etc.). The equipment required is expensive, the process is complex, and the cost is high. These structures are suitable for basic scientific research, but not suitable for widespread practical applications.

[0006] In order to solve these problems of metal structures and high refractive index dielectric structures, some low refractive index metal oxides (such as titanium dioxide, tungsten oxide, etc.) are used for surface enhancement. However, because the enhancement process of metal oxides involves charge / energy transfer processes, they are mainly used for surface enhanced Raman, which has a quenching effect on fluorescence, and the fluorescence enhancement effect is not ideal. In addition, among low refractive index materials, polymer materials are of many types, easy to process, and have high transparency, and are often used in optical structures. However, because polymer materials have a lower refractive index than metal oxides, it is usually necessary to form a composite system of polymer nanocolumn arrays and gold films to enhance fluorescence by forming an optical mode through metal-dielectric resonance hybridization (ACS Photonics 2019, 6, 2612-2617). Although good fluorescence enhancement effects can be achieved, metal-polymer hybrid structures still rely mainly on metal properties to achieve fluorescence enhancement. Therefore, it is urgent to develop a lossless, non-metal-dependent low refractive index polymer structure for surface enhanced fluorescence. Summary of the invention

[0007] The purpose of the present invention is to provide a method and application for surface enhanced fluorescence, so as to solve the problems existing in the existing surface enhanced fluorescence technology, such as large light loss, difficult control, complex process, high cost, etc., especially to solve the problem that the current fluorescence enhancement substrate is highly dependent on metals and high refractive index materials.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0009] According to the first aspect of the present invention, a method for surface enhanced fluorescence is provided, wherein a nanomaterial is used as an enhanced substrate, a fluorescent substance to be tested is placed on the surface of the enhanced substrate, and then the fluorescent substance to be tested is stimulated by excitation light. The fluorescence signal can be enhanced by utilizing the multiple light scattering effect of the nanomaterial on the excitation light and the radiated fluorescence.

[0010] The nanomaterials include: dielectric nanomaterials, semiconductor nanomaterials and metal nanomaterials. Among them, the dielectric nanomaterials may optionally include polymer nanomaterials, glass, ceramics, quartz, silicon, germanium and other nanomaterials.

[0011] According to a preferred embodiment of the present invention, the method uses polymer nanomaterials as a reinforcing substrate, and the polymer nanomaterials are films, gels, colloids, or powders formed by nanostructures made of polymer materials, such as semi-solid gels, liquid colloids, powder agglomerates, etc.; the nanostructures can be in any suitable shape, such as nanofibers, nanowires, nanospheres, nanocolumns, nanodisks, nanopolyhedrons, nanostars, etc.

[0012] According to another preferred embodiment of the present invention, the polymer nanomaterial is a polymer nanofilm formed by stacking any one or more nanostructures such as nanofibers, nanowires, nanospheres, nanocolumns, nanodisks, nanopolyhedrons, nanostars, etc. made of polymer materials.

[0013] According to another preferred embodiment of the present invention, the polymer nanomaterial is a polymer nanofiber membrane formed by stacking nanofibers made of polymer material.

[0014] It should be known that nanofiber membranes can be prepared by electrospinning, wet spinning, suction deposition, template pressing, etc., and can even be obtained by manual arrangement. These preparation methods are conventional technical means in the field.

[0015] The stacking method includes: random disordered stacking or regular ordered stacking. If a special arrangement is required, some electric field, magnetic field, force field induction or special substrate needs to be added.

[0016] Preferably, the thickness of the polymer nanofiber membrane is not less than 1 micron, preferably 1-20 microns.

[0017] According to the present invention, the polymer nanofibers constituting the polymer nanofiber membrane may be polymer nanofibers of a single size or polymer nanofibers of multiple sizes.

[0018] According to the present invention, the fluorescent substance to be detected is a fluorescent substance or a mixture of two or more fluorescent substances with distinguishable fluorescence wavelengths.

[0019] The fluorescent substance to be tested is a substance with fluorescence emission characteristics, including but not limited to organic fluorescent molecules, inorganic quantum dots, rare earth elements, carbon-based quantum dots and other luminescent materials, biological and immune-related substances with fluorescence emission characteristics or proteins, RNA, DNA, antibodies and other biological molecules marked with fluorescent substances, mitochondria, exosomes, cell membranes and other organelles, tissues and cells.

[0020] According to the present invention, the method of placing the fluorescent substance to be tested on the surface of the enhanced substrate includes placing the dispersion or powder of the fluorescent substance to be tested on the surface of the enhanced substrate by amplification, cultivation, drip coating, spin coating, immersion, pulling, spraying, etc.

[0021] According to a second aspect of the present invention, there is provided an application of the method for surface enhanced fluorescence as described above in fluorescence correlation detection.

[0022] The nanomaterial can be used as an enhanced substrate for fluorescence-related detection, including but not limited to the inner wall of the well plate reaction tank of fluorescent PCR, immunofluorescence detection chip, fluorescent imaging substrate, food safety detection chip, etc. By enhancing the original fluorescence signal intensity, the detection concentration can be reduced, the detection sensitivity can be improved, the detection credibility can be increased, and the requirements for detection equipment can be reduced.

[0023] According to a method for surface enhanced fluorescence provided by the present invention, the working principle is:

[0024] The fluorescent substance to be tested is placed on the surface of an enhanced substrate made of nanomaterials, and then the fluorescent substance to be tested is irradiated with excitation light. The excitation light excites the radiant fluorescence of the fluorescent substance. The excitation light and the radiant fluorescence are multiply scattered in the nanomaterial structure. The excitation light forms an enhanced field after multiple scattering. At the same time, the fluorescence is diffused and amplified after multiple scattering. These two processes work together to enhance the fluorescence of the fluorescent substance, thereby realizing surface enhanced fluorescence signals.

[0025] Compared with the prior art, the method for surface enhanced fluorescence provided by the present invention has the following beneficial effects:

[0026] 1) The present invention provides a fully dielectric low refractive index enhancement structure, whose enhancement mechanism is multiple light scattering, which does not rely on metal, especially polymer materials with high transparency, weak self-absorption, no local heat generation, and no damage to the fluorescent substance to be tested;

[0027] 2) The present invention does not need to control the distance between the substance to be detected and the enhanced surface, has no surface enhanced Raman scattering competition, has a high fluorescence enhancement factor, can significantly reduce the concentration of the detected substance, and improves the analytical sensitivity;

[0028] 3) Polymer materials are low-cost and can be prepared using common methods such as electrospinning, making them easy to mass-produce. Flexible polymer nanofiber membranes can be reprocessed and conveniently used as fluorescence enhancement surfaces in various types of fluorescence detection. In addition, they can be expanded to other low-refractive index materials to construct similar structures.

[0029] In summary, different from the existing surface enhanced structures, the present invention proposes a surface enhanced fluorescence method for all-dielectric low-refractive index nanomaterials based on the multiple scattering enhancement mechanism, which has good fluorescence enhancement effect, can greatly reduce the concentration of the detected object, and improve the analysis sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a schematic diagram of a process of enhancing fluorescence on the surface of a polymer nanofiber membrane according to a preferred embodiment of the present invention;

[0031] Figure 2 is a scanning electron microscope image of a typical microstructure of a polymer nanofiber membrane according to a preferred embodiment of the present invention;

[0032] Figure 3 The concentration is 1×10 -10 Fluorescence spectrum test results of Rhodamine 6G on the surface of silica (solid line) and polymer nanofiber membrane (dashed line) of M;

[0033] Figure 4 It is a schematic diagram of applying a polymer nanofiber membrane to a PCR well plate reaction tank according to a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0034] The following describes the embodiments of the present invention by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the following embodiments and features in the embodiments can be combined with each other without conflict.

[0035] It should be noted that the illustrations provided in the following embodiments are only schematic illustrations of the basic concept of the present invention, and thus the drawings only show components related to the present invention rather than being drawn according to the number, shape and size of components in actual implementation. In actual implementation, the type, quantity and proportion of each component may be changed arbitrarily, and the component layout may also be more complicated.

[0036] First embodiment of the polymer nanofiber membrane surface enhanced fluorescence method

[0037] According to the preferred embodiment, a method for enhancing fluorescence on the surface of a polymer nanofiber membrane is provided, wherein a fluorescent substance to be tested is placed on a polymer nanofiber membrane substrate, and then the fluorescent substance to be tested is excited by irradiation with excitation light. When the excitation light excites the fluorescent substance, both the excitation light and the emitted fluorescence are multiply scattered in the nanofiber membrane structure. After the multiple scattering, the excitation light forms an enhanced field, and at the same time, the fluorescence is diffused and amplified after the multiple scattering. These two processes enhance the fluorescence of the fluorescent substance, thereby achieving enhancement of the surface fluorescence signal.

[0038] Its working principle is as follows Figure 1As shown, multiple scattering mainly includes two types of scattering. One is shown in light color, which is the same color as the excitation light, representing the scattering of the excitation light; the other is shown in dark color, which is the same color as the enhanced fluorescence signal, and is the scattering of the fluorescence signal. Through these two processes, the enhancement of the surface fluorescence signal can be achieved.

[0039] It should be understood that the method for surface enhanced fluorescence provided according to the present invention is not limited to polymer nanofiber membranes. The applicable nanomaterials can be membranes formed by nanostructures made of polymer materials, or gels, colloids, or powders assembled from these nanostructures, such as semi-solid gels, liquid colloids, powder agglomerates, etc.

[0040] The method of placing the fluorescent substance to be tested on the surface of the polymer nanofiber membrane includes but is not limited to placing the dispersion or powder of the fluorescent substance to be tested on the substrate surface by amplification, cultivation, drip coating, spin coating, immersion, pulling, spraying and the like.

[0041] The fluorescent substance to be tested refers to a substance with fluorescence emission characteristics, including but not limited to organic fluorescent molecules, inorganic quantum dots, rare earth elements, carbon-based quantum dots and other luminescent materials, biological and immune-related substances with fluorescence emission characteristics or proteins, RNA, DNA, antibodies and other biological molecules marked with fluorescent substances, mitochondria, exosomes, cell membranes and other organelles, tissues and cells.

[0042] The fluorescent substance to be detected can be a single fluorescent substance or a mixture of two or more fluorescent substances with distinguishable fluorescence wavelengths.

[0043] The excitation light needs to be selected according to the self-luminescence characteristics of the fluorescent substance, and the maximum absorption wavelength of each fluorescent substance is preferably the excitation light; according to the currently commonly used fluorescent substances and detection instruments, the excitation light wavelength range is from ultraviolet to near-infrared, and can also be in the deep ultraviolet region, or mid-infrared region, or far-infrared region according to special fluorescent substances.

[0044] To enhance the fluorescence signal, a fluorescence spectrometer, a photodetector, a photon counter or other devices that can receive photons can be used to obtain the corresponding fluorescence spectrum, fluorescence image, or fluorescence photon count, etc.

[0045] Second Example: Randomly stacked polymer nanofiber membrane

[0046] like Figure 2 The figure shows a scanning electron microscope image of the microstructure of a polymer nanofiber disordered stacked fiber membrane prepared according to the preferred embodiment. The fiber diameter distribution is a multi-level structure of 200nm-1100nm, which is randomly crossed and stacked in multiple layers, and can effectively perform multiple scattering of light irradiated to the surface.

[0047] According to this preferred embodiment, the polymer nanofiber diameter is distributed in multiple scales. The preparation method can also be controlled to control the nanofiber diameter distribution of 20nm-2μm, or a single size distribution, or a mixture of one single size or several single size diameter fibers.

[0048] According to this preferred embodiment, the polymer nanofibers are randomly crossed and stacked in multiple layers in a disordered manner, and the nanofibers can also be arranged in a regular array and stacked in multiple layers in an orderly manner. The polymer nanofibers in this embodiment are prepared by electrostatic spinning. It should be understood that during the preparation process of the nanofibers formed by electrostatic spinning, as time goes by, the nanofibers will randomly stack in multiple layers to form a polymer nanofiber membrane.

[0049] According to this preferred embodiment, polyvinyl acetate is selected as the polymer. It should be understood that other polymers may also be selected, including but not limited to polymethyl methacrylate, polycarbonate, polyvinyl chloride, polyvinylidene fluoride, polyimide, polyvinyl alcohol, cellulose, silk protein and the like to prepare polymers for nanofibers.

[0050] According to the preferred embodiment, the preparation method of polymer nanofibers adopts electrostatic spinning technology, and other spinning methods may also be adopted, including but not limited to melt spinning, solution spinning and other preparation methods.

[0051] According to the preferred embodiment, a polymer nanofiber membrane is selected. It should be understood that nanostructures such as nanowires, nanospheres, columns, triangles, polygons, etc. made of polymer materials can also be used, and then multi-layer stacking can be used to form a polymer nanomaterial to form multiple scattering.

[0052] Third Example Polymer Nanofiber Membrane Surface Enhanced Low Concentration Rhodamine 6G Fluorescence Emission

[0053] In this example, the concentration is 1×10 -10 Rhodamine 6G with a wavelength of 532 nm was used as the test sample, and 5 μL of the solution was dropped on the surface of the silica and the surface of the polymer nanofiber membrane prepared in the second embodiment, and then dried naturally. The fluorescence spectrum was tested with an excitation light with a wavelength of 532 nm. The fluorescence spectrum of Rhodamine 6G on the silica surface is shown in FIG. Figure 3 The middle black solid line, the maximum fluorescence peak intensity is 76, and the fluorescence integrated area is 3628; the fluorescence spectrum of rhodamine 6G on the surface of polymer nanofiber membrane is shown in Figure 3 The middle black dashed line, the maximum fluorescence peak intensity is 6728, and the fluorescence integrated area is 315523. Compared with the fluorescence of rhodamine 6G on the silica surface, the surface of the polymer nanofiber membrane enhances the fluorescence emission of rhodamine 6G, and the fluorescence enhancement factor is 87 under the same detection concentration and excitation power conditions.

[0054] Fourth embodiment: polymer nanofiber membrane is applied to PCR well plate reaction tank

[0055] like Figure 4 As shown, in this embodiment, the polymer nanofiber membrane is used for the bottom of the sample slot of the PCR detection reaction well plate, and the PCR well plate structure with the polymer nanofiber membrane is added as shown in FIG. Figure 4 As shown, the purpose of this embodiment is to utilize the enhancement of fluorescence by polymer nanofibers to improve the detection sensitivity and detection limit of fluorescence signals during PCR detection.

[0056] In this embodiment, the PCR well plate reaction tank is cylindrical, and can also be used for a square reaction tank. The shape of the polymer nanofiber membrane at the bottom of the reaction tank is determined according to the different shapes of the reaction tank.

[0057] In this embodiment, the polymer nanofiber is placed at the bottom of the sample slot of the PCR detection reaction well plate. Specifically, according to the shape of the sample slot, the polymer nanofiber membrane can be processed into different shapes (cut into a circle or a square). The simplest is to adhere to the bottom surface, or spray the nanofiber on the bottom of the sample slot of the PCR detection reaction well plate. Other nanostructured membranes that can achieve multiple scattering effects can also be used, such as titanium dioxide, zinc dioxide and other oxide nanowires or particles. If it is other nanowires or nanoparticles, spraying, evaporation or immersion adsorption can also be used.

[0058] In this embodiment, the polymer nanofiber membrane is used in the reaction groove of the PCR well plate, but it should be understood that the polymer nanofiber membrane can also be used on the inner wall of a similar centrifuge tube or the inner wall of other biochip channels.

[0059] The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. The above embodiments of the present invention can also be modified in various ways. All simple, equivalent changes and modifications made according to the claims and the description of the present invention fall within the scope of protection of the claims of the present invention. The contents not described in detail in the present invention are all conventional technical contents.

Claims

1. A method for surface enhanced fluorescence, characterized in that: A polymer nanomaterial is used as an enhancement substrate, a fluorescent substance to be tested is placed on the surface of the enhancement substrate, and then the fluorescent substance to be tested is stimulated by excitation light, and the fluorescence signal can be enhanced by utilizing the multiple light scattering effect of the polymer nanomaterial on the excitation light and the radiated fluorescence; wherein the polymer nanomaterial is a film, gel, colloid, or powder formed by a nanostructure made of polymer material, which is then stacked in multiple layers to form multiple scattering. The method of placing the fluorescent substance to be tested on the surface of the enhancement substrate includes placing the dispersion or powder of the fluorescent substance to be tested on the surface of the enhancement substrate in any one of the following ways: amplification, cultivation, drip coating, spin coating, immersion, pulling, and spraying.

2. The method according to claim 1, characterized in that The nanostructures include nanofibers, nanowires, nanospheres, nanocolumns, nanodisks, nanopolyhedrons, or nanostars.

3. The method according to claim 2, characterized in that The polymer nanomaterial is a polymer nanofiber membrane formed by stacking nanofibers made of polymer material, and the stacking method includes: free random disordered stacking, or regularly arranged ordered stacking.

4. The method according to claim 3, characterized in that The polymer nanofibers constituting the polymer nanofiber membrane are polymer nanofibers of a single size or polymer nanofibers of multiple sizes.

5. The method according to claim 1, characterized in that The fluorescent substance to be detected is a fluorescent substance or a mixture of two or more fluorescent substances with distinguishable fluorescence wavelengths.

6. The method according to claim 5, characterized in that The fluorescent substance to be detected is a substance with fluorescence emission characteristics, including: organic fluorescent molecules.

7. The method according to claim 5, characterized in that The fluorescent substance to be detected is a substance with fluorescence emission characteristics, including: inorganic quantum dots.

8. The method according to claim 5, characterized in that The fluorescent substance to be detected is a substance having fluorescent emission characteristics, including rare earth elements.

9. The method according to claim 5, characterized in that The fluorescent substance to be tested is a substance with fluorescence emission characteristics, including: carbon-based quantum dots.

10. The method according to claim 5, characterized in that The fluorescent substance to be detected is a biological or immune-related substance with fluorescent emission characteristics or a substance marked with a fluorescent substance.

11. Use of the method for surface enhanced fluorescence according to any one of claims 1 to 10 in fluorescence correlation detection.

12. The use according to claim 11, characterized in that: The method for surface enhanced fluorescence is applied to a well plate reaction tank of fluorescent PCR, an immunofluorescence detection chip, a fluorescent imaging substrate, a fluorescent spectrum trace detection substrate or a food safety detection chip to enhance the original fluorescent signal intensity.

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