An F-LSPR sensor and its preparation method and application
By combining metal nanoparticles with nanoscale fluorescent materials, F-LSPR sensors were prepared. The principle of fluorescence resonance energy transfer was used to solve the problem of limited sensitivity of LSPR sensors, and high sensitivity and high reliability detection was achieved, which was suitable for portable devices.
Patent Information
- Application Number
- CN201910815515.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2019-08-30
- Publication Date
- 2025-08-29
- Estimated Expiration
- 2039-08-30
AI Technical Summary
The sensitivity of existing LSPR sensors is limited by the smallest spectral displacement and a single signal, which hinders its development and application.
Combining metal nanoparticles and nanoscale fluorescent materials, an F-LSPR sensor is prepared through the principle of fluorescence resonance energy transfer, and the fluorescence intensity changes are used to reflect the LSPR energy changes to achieve high sensitivity detection.
It improves the sensitivity and detection reliability of the sensor, reduces interference from other wavelength light sources, and realizes one-step detection, which is suitable for portable devices.
Smart Images
Figure CN110470641B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of biosensors, and more particularly to an F-LSPR sensor and a preparation method and application thereof. Background Art
[0002] In recent years, with the development of advanced nanotechnology, optical sensing has made explosive progress and has been widely used in important fields such as food safety, environmental monitoring, and biodiagnosis. The core of optical sensing is the change in optical signals caused by the interaction between analytes and electromagnetic fields. Therefore, common optical sensing strategies usually rely on optical phenomena such as extinction, fluorescence, electrochemiluminescence, and Raman scattering.
[0003] Among various optical sensing technologies, localized surface plasmon resonance (LSPR) sensing technology based on extinction has attracted much attention due to its advantages of simple preparation, low cost, fast response, high signal-to-noise ratio, and no need for labeling. LSPR is a strong plasma extinction phenomenon caused by the resonance of the free electron cloud on the metal surface when the frequency of the collective oscillation is the same as the frequency of the incident light. The most common LSPR substrates are nanoparticles and nanoarrays composed of precious metals (such as gold, silver, platinum, etc.), which have the following advantages: (1) High molar extinction coefficient, such as the extinction coefficient of gold nanorods (Au NRs) can reach 6 × 10 9 M −1 •cm −1 ; (2) The spectrum can be adjusted according to the shape, size, composition and mutual coupling of the nanostructures; (3) It has good chemical and optical stability; (4) It is easy to carry out surface modification. The energy of the LSPR substrate can be reflected as peaks and troughs in the extinction spectrum, which is greatly affected by the dielectric properties of the surrounding environment. Studies have shown that the peak / valley shift of the LSPR is linearly related to the refractive index (RI) of the surrounding environment. Therefore, this property has become the most concerned detection parameter in traditional LSPR sensing systems and is widely used in chemical and biological molecular analysis based on affinity recognition. However, LSPR sensing still faces some problems. For example, the peak / valley shift of the LSPR can only provide a single signal, and its sensitivity is still limited by the minimum spectral shift that can be detected. These problems have hindered the development and application of LSPR sensors. Summary of the Invention
[0004] The main purpose of the present invention is to overcome the above-mentioned shortcomings and provide an F-LSPR (fluorescence-localized surface plasmon resonance) sensor and its preparation method and application.
[0005] The technical solution of the present invention is:
[0006] The present invention provides an F-LSPR sensor comprising metal nanoparticles having an ultraviolet-visible-near-infrared light absorption peak between 650 and 950 nm, wherein the outer layer of the metal nanoparticles is coated with a nano-scale fluorescent material.
[0007] Furthermore, the metal nanoparticles include gold nanoparticles, silver nanoparticles and aluminum nanoparticles.
[0008] Furthermore, the shapes of the metal nanoparticles include sphere, rod, triangle or cube.
[0009] Furthermore, the nano-scale fluorescent material is selected from fluorescent quantum dots, polymer fluorescent nanospheres or composite fluorescent silica nanoparticles.
[0010] The present invention also provides a method for preparing the F-LSPR sensor, comprising the following steps:
[0011] Adding a nanoscale fluorescent material solution to the purified metal nanoparticle solution to obtain a metal nanoparticle-nanoscale fluorescent material composite, namely the F-LSPR sensor;
[0012] The connection method between the metal nanoparticles and the nanoscale fluorescent material includes electrostatic self-assembly or chemical bond connection;
[0013] The concentration ratio of the nanoscale fluorescent material solution to the metal nanoparticle solution is 1:100-1:1000.
[0014] The absorption peak of the metal nanoparticle solution is between 650 nm and 950 nm.
[0015] Specifically, when the metal nanoparticles and the nanoscale fluorescent material are connected by chemical bonds, a metal nanoparticle solution modified with amino groups and a nanoscale fluorescent material solution activated with carboxyl groups can be mixed to obtain a metal nanoparticle-nanoscale fluorescent material complex.
[0016] Furthermore, the preparation method needs to be completed under the conditions of avoiding light and isolating oxides.
[0017] The technical concept of this invention is to utilize the advantages of fluorescence sensing, such as simple preparation, rapid response, high sensitivity, and high temporal and spatial resolution, to combine the LSPR properties of metal nanoparticles with the highly sensitive fluorescence properties of nanofluorescent materials to produce a highly sensitive sensor. When metal nanoparticles are combined with nanoscale fluorescent materials, the sensitivity of the system and the reliability of detection can be effectively improved by combining the advantages of metal nanoparticles and nanoscale fluorescent materials. When other molecules are attached to or adsorbed on the surface of the metal nanoparticle-nanoscale fluorescent material complex, the refractive index (RI) of the metal nanoparticle-nanoscale fluorescent material complex increases due to the other molecules, causing a change in the LSPR energy. Under the action of energy transfer, this can affect the fluorescence intensity of the system. In other words, changes in fluorescence intensity can also reflect changes in LSPR energy.
[0018] Therefore, the present invention uses nanoscale fluorescent materials to coat metal nanoparticles. Through surface charge adsorption or chemical bonding, the fluorescent properties of the nanoscale fluorescent materials and the refractive index changes of the metal nanoparticles with their surroundings are combined to create a metal nanoparticle-nanoscale fluorescent material composite, the F-LSPR sensor. This strategy of using fluorescence detection to achieve refractive index sensing differs from the currently commonly used UV-visible mode.
[0019] The present invention utilizes the principles of localized surface plasmon resonance and fluorescence resonance energy transfer (FRET) to combine metal nanoparticles with nanoscale fluorescent materials to create a new biosensor, namely, a metal nanoparticle-nanoscale fluorescent material composite. The metal nanoparticle-nanoscale fluorescent material composite utilizes the overlapping absorption wavelengths of the nanoscale fluorescent material and the metal nanoparticles. Because the nanoscale fluorescent material is modified on the metal nanoparticles, FRET reduces the fluorescence energy of the nanoscale fluorescent material, causing fluorescence quenching of the nanoscale fluorescent material. When the protein concentration on the surface of the metal nanoparticles changes, the surface refractive index of the metal nanoparticle-nanoscale fluorescent material composite changes, which in turn causes a shift in the absorption wavelength of the nanoscale fluorescent material. FRET sensitizes the nanoscale fluorescent material, thereby measuring changes in fluorescence energy. Because fluorescence signals are more sensitive to changes in the nanoparticle's own optical signal, the metal nanoparticle-nanoscale fluorescent material composite can reduce interference from light sources of other wavelengths and amplify the detection signal. Consequently, the composite exhibits higher sensitivity, enabling one-step detection and meeting the needs of developing portable devices.
[0020] The present invention also provides application of the F-LSPR sensor in monitoring antigens in biological samples.
[0021] Furthermore, the biological sample is whole blood, serum or plasma.
[0022] Furthermore, the purified metal nanoparticle solution is added to a nanoscale fluorescent material solution and shaken in the dark to obtain a metal nanoparticle-nanoscale fluorescent complex base solution. This solution is then added to a PBS solution containing the antibody protein, allowed to stand overnight for 8-12 hours, centrifuged, and the supernatant discarded. The solution is then added to a PBS solution containing BSA, allowed to stand for 2 hours, and washed with a PBS solution by centrifugation to prepare a metal nanoparticle-nanoscale fluorescent material-antibody complex solution. The solution is then added to the biological sample, incubated for 2 hours, and the fluorescence intensity is measured to determine the antigen concentration.
[0023] The base solution of the metal nanoparticle-nanoscale fluorescent material complex is added to a PBS buffer solution containing an antibody or antigen protein; the antibody or antigen protein is adsorbed on the metal nanoparticle-nanoscale fluorescent material complex under the action of the charge, and the complex is blocked with a PBS solution containing BSA. After centrifugation to remove the blocking solution, the metal nanoparticle-nanoscale fluorescent material complex with the antibody or antigen protein surface modified is obtained.
[0024] The biological sample to be tested is added to a metal nanoparticle-nanoscale fluorescent material complex coated with different antibodies or antigen proteins. The adsorption of the antigen to be tested to the surface of the complex will cause the refractive index of the complex surface to increase, thereby affecting the LSPR effect of the metal nanoparticles; the influence of this LSPR effect is manifested in the spectrum as a right shift of the LSPR peak and the realization of fluorescence energy resonance transfer, accompanied by fluorescence quenching or enhancement, thereby realizing fluorescent immunoassay.
[0025] Compared with the prior art, the present invention has the following advantages:
[0026] The F-LSPR sensor provided by the present invention has the characteristics of easy detection, automation and high sensitivity, laying the foundation for the realization of convenient, rapid and automated detection equipment. The preparation method is simple, and it can realize convenient and rapid monitoring of antigen levels in the blood. It can be applied to portable mobile medical devices. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 Schematic diagram of the structure of the nanogold triangle-fluorescent quantum dot-antibody complex;
[0028] Figure 2 This is a diagram showing the binding principle of the gold nanotriangle-fluorescent quantum dot-antibody complex;
[0029] Figure 3 This is a diagram showing the effect of the gold triangle nanoparticles-fluorescent quantum dots-antibody complex binding to the antigen of the sample to be tested;
[0030] Figure 4This is the process in which the fluorescence intensity of quantum dots gradually decreases before and after the gold nanotriangle-fluorescent quantum dot-antibody complex binds to the antigen;
[0031] Figure 5 This is the process in which the fluorescence intensity of quantum dots gradually increases before and after the gold nanorod-fluorescent quantum dot-antibody complex binds to the antigen;
[0032] Figure 6 This is a transmission electron microscopy (TEM) image of the gold nanotriangle-fluorescent quantum dot-antibody complex;
[0033] Figure 7 Figure 3 is the fluorescence intensity curve of the gold triangle nanoparticles-fluorescent quantum dots-antibody complex after binding to antigens of different concentrations. The peak positions in each curve from top to bottom correspond to antigen concentrations of 0, 2, 4, 6, 8, and 10 ng / mL, respectively.
[0034] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings and in conjunction with the embodiments. DETAILED DESCRIPTION
[0035] The preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby more clearly defining the protection scope of the present invention.
[0036] Example 1
[0037] This embodiment provides an F-LSPR sensor, comprising gold triangle nanoparticles and fluorescent quantum dots, wherein the outer layer of the gold triangle nanoparticles is coated with fluorescent quantum dots.
[0038] This embodiment also provides a method for preparing the above-mentioned F-LSPR sensor, comprising the following steps:
[0039] S1. Preparation of nano-gold triangle particles (characteristics: UV-visible-near-infrared light absorption peak between 650-800 nm);
[0040] Solutions A, B, C, D, and E were prepared separately. Solutions A and B had the same formula, and NaOH (0.1 M, 0.05 mL), citric acid (0.1 M, 0.05 mL), KI (0.1 M, 4.5 µL), CTAB (0.05 M, 9.0 mL), and HAuCl4 (10 mM, 0.25 mL) were added in the above order, respectively. Solutions C, D, and E had the same formula, namely NaOH (0.1 M, 0.25 mL), citric acid (0.1 M, 0.25 mL), KI (0.1 M, 23 µL), CTAB (0.05 M, 45 mL), and HAuCl4 (10 mM, 1.25 mL), respectively. They were added in the above order, mixed, and set aside.
[0041] To synthesize the gold nanoparticle seed solution: Transfer 36 mL of ultrapure water to a reaction vessel (a clean 150 mL beaker). Refrigerate a sufficient volume of ultrapure water (greater than 10 mL). Add 1 mL of 0.01 M sodium citrate and stir (shake thoroughly). Add 1 mL of 0.01 M HAuCl₄ and stir thoroughly. Prepare a 0.1 M NaBH₄ solution in ice water and quickly add 1 mL of the mixture to the mixture. Stir rapidly for 2 minutes. After stirring for 2 minutes, store in the dark overnight (to allow any remaining NaBH₄ to decompose).
[0042] Synthesize a gold nanoparticle solution: Add 1 ml of the gold nanoparticle seed solution to solution A and shake well. Add 5 mL of the prepared solution A to solution B and shake well. Add 2, 4, and 6 mL to solutions C, D, and E, respectively, and shake well. Incubate in a 30°C water bath overnight to prepare a gold nanoparticle solution. Purify the prepared gold nanoparticle solution to obtain a gold nanoparticle solution: Add 0.2 mL of 4 M NaCl solution to the gold nanoparticle solution, shake well, and let stand for 4 hours. Pour the supernatant into another conical flask and repeat the above steps until gold nanoparticles of the appropriate size are purified.
[0043] S2, preparing nano-gold triangle-fluorescent quantum dot complex;
[0044] According to the needs, fluorescent quantum dots with appropriate wavelengths (maximum fluorescence absorption peak between 700-900 nm) were selected; nano-gold triangles and nano-fluorescent quantum dots were electrostatically self-assembled to prepare nano-gold triangle-fluorescent quantum dot complexes; the purified nano-gold triangle particle solution was washed with purified water and centrifuged for purification, and 4 ml was taken to prepare 3.0×10 －4 M solution, add 2 mL (1 nM) fluorescent quantum dot solution of specific wavelength, and shake in the dark for 1 h to obtain the nanogold triangle-fluorescent quantum dot complex base solution.
[0045] This embodiment also provides an application of an F-LSPR sensor in monitoring antigens in a biological sample. Specifically, the biological sample is whole blood, serum, or plasma.
[0046] The prepared nano-gold triangle-fluorescent quantum dot complex base solution was added to a PBS solution (pH = 6.0) containing antibody protein, with an antibody protein concentration of 15 μg / ml, and allowed to stand overnight for 8-12 hours. The solution was centrifuged and the supernatant was discarded. The nano-gold triangle-fluorescent quantum dot-antibody complex was added to a 0.1% BSA PBS solution, allowed to stand for 2 hours, and washed twice by centrifugation with a PBS solution (pH = 6.0) to prepare a nano-gold triangle-fluorescent quantum dot-antibody complex. Figures 1-3 As shown, Figure 6 is a transmission electron microscope (TEM) image of the nano-gold triangle-fluorescent quantum dot-antibody complex; the biological sample to be tested was added to the nano-gold triangle-fluorescent quantum dot-antibody complex solution, incubated for 2 hours, and the fluorescence intensity was measured; in this embodiment, Figure 4 The nano-gold triangle-fluorescent quantum dot complex (wherein the fluorescence emission wavelength and the LSPR peak do not overlap or overlap). When the nano-gold triangle-fluorescent quantum dot-antibody complex binds to the target antigen in the biological sample, thereby achieving detection, the LSPR peak shifts to a longer wavelength (i.e., the peak shifts to the right), and the fluorescence resonance energy transfer (FRET) changes from low to high, causing the quantum dot fluorescence intensity to decrease from strong to weak. Therefore, the concentration of the target antigen in the biological sample can be determined based on the change in fluorescence intensity.
[0047] Example 2
[0048] This embodiment provides an F-LSPR sensor, including gold nanorod particles and fluorescent quantum dots, wherein the outer layer of the gold nanorod particles is coated with fluorescent quantum dots.
[0049] This embodiment also provides a method for preparing the above-mentioned F-LSPR sensor, comprising the following steps:
[0050] S1. Preparation of gold nanorod particles (characteristics: UV-visible-near-infrared absorption peaks between 800-950 nm);
[0051] Synthesis of gold nanoparticle seed solution: First, prepare a 0.01 mol / L sodium borohydride solution with ice water. Then, add 25 µL of 0.1 mol / L chloroauric acid solution and 600 µL of sodium borohydride solution to 10 mL of 0.1 mol / L hexadecyltrimethylammonium bromide solution. The resulting mixed solution is stirred at 800 rpm for 5 minutes and then allowed to stand in a 30°C water bath for 4 hours to obtain the gold nanoparticle seed solution.
[0052] Synthesis of gold nanorod solution: To 100 mL of 0.1 mol / L hexadecyltrimethylammonium bromide solution, add 0.5 mL of 0.1 mol / L chloroauric acid solution, 80 µL of 0.1 mol / L silver nitrate solution, and 0.55 mL of 0.1 mol / L ascorbic acid solution in sequence. After mixing, add 100 µL of gold seed solution. Stir at 800 rpm for 5 min, then place in a 30°C water bath for 15 h. Centrifuge and reconstitute to an equal volume to obtain a gold nanorod solution.
[0053] S2, preparation of gold nanorod-fluorescent quantum dot complexes;
[0054] Amino-modification of gold nanorods: 100 µL of 50 mM mercaptoethylamine solution was added dropwise to 5 mL of the purified gold nanorod solution while ultrasonicating in a 50°C water bath. The solution was ultrasonicated at 50°C for 30 minutes and then washed three times by centrifugation.
[0055] Activation of fluorescent quantum dots: Add 100 µL of EDC / NHS mixture (40 mM) to 5 mL of fluorescent quantum dot solution (30 nM) and shake at room temperature for 2 h to activate the surface carboxyl groups of the fluorescent quantum dots.
[0056] Gold nanorod-fluorescent quantum dot composite: amino-modified gold nanorod solution was prepared into 2×10 －10 The M solution was then mixed with a carboxyl-activated fluorescent quantum dot solution in a 1:1 volume ratio. The mixed solution was shaken at room temperature for 1 hour and then allowed to stand overnight at room temperature. After the reaction, the product was centrifuged and washed three times to obtain a purified gold nanorod-fluorescent quantum dot complex substrate solution.
[0057] This embodiment also provides an application of an F-LSPR sensor in monitoring antigens in a biological sample. Specifically, the biological sample is whole blood, serum, or plasma.
[0058] The prepared gold nanorod-fluorescent quantum dot complex substrate solution was added to a PBS solution (pH = 6.0) containing antibody protein, the antibody protein concentration was 15 μg / ml, and the mixture was allowed to stand overnight for 8-12 hours, centrifuged, and the supernatant was discarded; the gold nanorod-fluorescent quantum dot-antibody complex was added to a 0.1% BSA PBS solution, allowed to stand for 2 hours, and washed twice by centrifugation with a PBS solution (pH = 6.0) to prepare a gold nanorod-fluorescent quantum dot-antibody complex; the biological sample to be tested was added to the gold nanorod-fluorescent quantum dot-antibody complex solution, allowed to stand and incubate for 2 hours, and the fluorescence intensity was measured; in this embodiment, Figure 5This is a gold nanorod-fluorescent quantum dot complex (where the fluorescence emission wavelength and LSPR peak overlap or do not overlap). When the gold nanorod-fluorescent quantum dot-antibody complex binds to the target antigen in a biological sample, enabling detection, the LSPR peak shifts to a longer wavelength (i.e., the peak shifts to the right), and the fluorescence resonance energy transfer (FRET) decreases from high to low, causing the quantum dot fluorescence intensity to increase from weak to strong. Therefore, the concentration of the target antigen in the biological sample can be determined based on the change in fluorescence intensity.
[0059] Example 3
[0060] According to Example 1, Figure 7 The curve graph shows that the fluorescence intensity of the nanogold triangle-fluorescent quantum dot-antibody complex gradually decreases as the antigen concentration increases.
[0061] The above are only preferred embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention description and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.
Claims
1. An F-LSPR sensor for antigen detection, characterized in that: The invention relates to metal nanoparticles having an ultraviolet-visible-near-infrared absorption peak between 650 nm and 950 nm, wherein the metal nanoparticles are coated with a nanoscale fluorescent material, and the absorption peak of the metal nanoparticles overlaps with the fluorescence emission peak of the nanoscale fluorescent material; the metal nanoparticles and the nanoscale fluorescent material are connected by electrostatic self-assembly or chemical bonding; and the metal nanoparticles have a rod-like, triangular, or cubic shape. When the antigen concentration on the surface of the metal nanoparticles changes, the surface refractive index of the metal nanoparticle-nanoscale fluorescent material complex changes, which in turn causes the LSPR peak to shift. Based on the fluorescence resonance energy transfer between the metal nanoparticles and the nanoscale fluorescent material, the nanoscale fluorescent material produces fluorescence sensitization, thereby measuring the change in the fluorescence energy value.
2. The F-LSPR sensor according to claim 1, wherein The metal nanoparticles include gold nanoparticles, silver nanoparticles and aluminum nanoparticles.
3. The F-LSPR sensor according to claim 1, wherein The nano-scale fluorescent material includes fluorescent quantum dots, polymer fluorescent nano-microspheres or composite fluorescent silicon dioxide nano-particles.
4. A method for preparing the F-LSPR sensor according to claim 1, characterized in that: The following steps are included: Adding a nanoscale fluorescent material solution to the purified metal nanoparticle solution to obtain a metal nanoparticle-nanoscale fluorescent material composite, namely the F-LSPR sensor; The concentration ratio of the nanoscale fluorescent material solution to the metal nanoparticle solution is 1:100-1:1000; The absorption peak of the metal nanoparticle solution is between 650 nm and 950 nm.
5. The method for preparing the F-LSPR sensor according to claim 4, wherein The preparation method needs to be completed under the conditions of avoiding light and isolating oxides.
6. Use of the F-LSPR sensor according to any one of claims 1 to 3 in monitoring antigens in biological samples.
7. Use of the F-LSPR sensor according to claim 6 in monitoring antigens in biological samples, characterized in that: The biological sample is whole blood, serum or plasma.
8. Use of the F-LSPR sensor according to claim 6 in monitoring antigens in biological samples, characterized in that: The purified metal nanoparticle solution is added to a nanoscale fluorescent material solution, shaken in the dark, and a metal nanoparticle-nanoscale fluorescent material complex base solution is obtained; the solution is added to a PBS solution containing antibody protein, allowed to stand overnight for 8-12 hours, centrifuged, and the supernatant is discarded; the solution is then added to a PBS solution containing BSA, allowed to stand for 2 hours, and washed by centrifugation with a PBS solution to prepare a metal nanoparticle-nanoscale fluorescent material-antibody complex solution; the biological sample is added, allowed to stand and incubate for 2 hours, and the fluorescence intensity is measured to measure the antigen concentration.