A rapid preparation method and application of surface-enhanced Raman scattering nanoparticle clusters based on bioactive molecules

Abstract

The present invention discloses a rapid preparation method and application of surface-enhanced Raman scattering nanoparticle clusters constructed based on bioactive molecules. The method adds a precursor solution containing a metal or metal oxide to a reducing agent solution under acidic or alkaline conditions for reaction to obtain surface-enhanced Raman scattering nanoparticles without a covering molecular layer. These nanoparticles are then further mixed with Raman reporter molecules and bioactive molecules such as proteins, peptides, nucleic acids (DNA, RNA, or aptamers), cell membranes, etc. are introduced to quickly and stably fix the Raman reporter molecules to the gold nanoparticles, enhancing their sensitivity and stability to achieve better surface-enhanced Raman scattering performance.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical engineering materials and nanotechnology, and particularly relates to a rapid preparation method and application of surface-enhanced Raman scattering nanoparticle clusters constructed based on bioactive molecules. Background Art

[0002] Surface-enhanced Raman scattering (SERS) is a spectroscopic technique that uses localized plasmon resonance on metal surfaces to adsorb specific molecules or biomolecules, significantly enhancing the Raman scattering signal. Compared to conventional Raman scattering, it can provide several orders of magnitude of signal enhancement, making it possible to detect chemical and biological molecules at the single-molecule level. Currently, it has been widely used in biology, medicine, and related fields. For example, SERS can be used to monitor chemical changes in biological samples, such as the distribution and metabolism of drugs in the body, and can also be used to study the interactions between drugs and biomolecules to optimize drug design and development. In addition, it can also be used to detect changes in biomolecules and biomarkers, thereby assisting in disease diagnosis. However, nanoclusters formed by metal adsorption of specific molecules (such as Raman reporter molecules) are generally not tightly bound. In the presence of body fluids, the specific molecules easily detach from the metal surface, resulting in a significant reduction in the Raman signal. In addition, metals, especially silver, are easily oxidized when exposed to the environment and may adsorb impurities in the environment, affecting the Raman signal.

[0003] To address the above issues, some studies have covered the metal surface with an ultra-thin "protective layer" to prevent the metal from being oxidized and specific molecules from falling off the surface. However, whether it is an oxide (such as silicon oxide), carbon material, dense gold film or other shell-forming materials, it will lead to a decrease in Raman sensitivity, which is very unfavorable for detection. Therefore, we need to find a simple and efficient preparation method for surface modification and encapsulation of nanoparticle clusters based on surface-enhanced Raman scattering to enhance their sensitivity and achieve better surface-enhanced Raman scattering performance. Summary of the Invention

[0004] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a surface-enhanced Raman scattering nanoparticle without a covering molecular layer.

[0005] Another object of the present invention is to provide applications of the surface-enhanced Raman scattering nanoparticles without a covering molecular layer.

[0006] Another object of the present invention is to provide a surface-enhanced Raman scattering nanoparticle cluster.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A surface-enhanced Raman scattering nanoparticle without a covering molecular layer is prepared by any one or both of the following methods:

[0009] (1) adding a precursor solution containing a metal or metal oxide to a reducing agent solution, mixing well, adding an alkaline solution (to adjust the pH value), stirring and reacting under light-proof conditions, centrifuging and collecting the precipitate after the reaction is completed, and then resuspending it in water to obtain surface-enhanced Raman scattering nanoparticles without a covering molecular layer;

[0010] (2) adding the acidic solution to the surfactant solution, then adding the precursor solution containing the metal or metal oxide, and then adding the reducing agent solution, mixing and reacting at room temperature, and after the reaction is completed, centrifuging and washing the product to obtain surface-enhanced Raman scattering nanoparticles without a covering molecular layer.

[0011] The metal in the metal or metal oxide-containing precursor solution described in methods (1) and (2) is at least one of gold, silver, copper, palladium and aluminum (preferably at least one of gold, silver and copper; more preferably gold), and the metal oxide is at least one of gold, silver, copper, palladium and aluminum oxide (preferably at least one of gold, silver and copper oxide; more preferably gold oxide).

[0012] The metal or metal oxide-containing precursor solution in methods (1) and (2) is a gold-containing precursor solution, a silver-containing precursor solution, or a copper-containing precursor solution; wherein,

[0013] The gold-containing precursor solution is preferably at least one of chloroauric acid, sodium chloroaurate, potassium chloroaurate, sodium iodaurate and potassium iodaurate solution (for preparing gold nanoparticles or gold nanostars); more preferably chloroauric acid solution;

[0014] The silver-containing precursor solution is preferably at least one of silver nitrate, silver chloride, silver sulfate, silver acetate, silver fluoroborate, silver formate, silver citrate and silver oxalate (for preparing silver nanoparticles); more preferably, a silver nitrate solution;

[0015] The copper-containing precursor solution is preferably at least one of copper sulfate, copper chloride, copper nitrate, copper acetate, copper oxide, copper carbonate, copper oxalate, copper formate, copper bromide, copper fluoroborate and copper iodide solution (for preparing copper nanoparticles); more preferably copper sulfate solution.

[0016] The reducing agent described in method (1) is preferably at least one of hydrogen peroxide (H2O2), sodium citrate, sodium borohydride (NaBH4), ascorbic acid, hydrogen (H2), ethylene glycol, oxalic acid, trehalose, reducing sugars, ammonia, reducing amino acids, ferrocene and its derivatives, electrochemical reduction and photoreduction; more preferably, hydrogen peroxide.

[0017] The reducing sugars are preferably at least one of glucose and fructose.

[0018] The reducing amino acid is preferably arginine.

[0019] The concentration of the precursor solution containing metal or metal oxide described in method (1) is 0.1 to 25 mmol / L, preferably 10 mmol / L.

[0020] The concentration of the reducing agent solution in method (1) is 50 to 5000 mmol / L, preferably 1250 mmol / L.

[0021] The volume ratio of the metal or metal oxide-containing precursor solution to the reducing agent solution in method (1) is (1-4):(1-40), preferably 1:4.

[0022] The alkaline solution described in method (1) is at least one of NaOH solution, KOH solution and alkaline buffer solution; preferably one of NaOH solution, KOH solution, Na2HPO4 buffer solution, NaH2PO4 buffer solution, K2HPO4 buffer solution, KH2PO4 buffer solution, NaHCO3 buffer solution, Na2CO3 buffer solution, KHCO3 buffer solution and K2CO3 buffer solution; more preferably NaOH solution.

[0023] The concentration of the alkaline solution in method (1) is 1 to 100 mmol / L, preferably 12 mmol / L.

[0024] The volume of the alkaline solution in method (1) is 20-70% of the total volume of the precursor solution containing metal or metal oxide and the reducing agent solution; preferably 50% of the total volume of the precursor solution containing metal or metal oxide and the reducing agent solution.

[0025] The rotation speed of the stirring reaction in method (1) is 100 to 300 rpm, preferably 150 rpm.

[0026] The stirring reaction time in method (1) is 10 minutes to 48 hours, preferably 1 hour.

[0027] The centrifugation conditions described in method (1) are: centrifugation at 500-5000 rpm for 3-15 minutes at 4°C or room temperature; preferably: centrifugation at 3000 rpm for 5 minutes at room temperature.

[0028] The water described in method (1) is ultrapure water.

[0029] In method (1), the steps of centrifugation and resuspension are repeated three or more times.

[0030] The acidic solution described in method (2) is at least one of sulfuric acid solution, hydrochloric acid solution, nitric acid solution, acetic acid solution, citric acid solution, lactic acid solution, oxalic acid solution, phosphoric acid solution, acetic acid solution, formic acid solution and acidic buffer solution; preferably hydrochloric acid solution.

[0031] The acidic buffer solution includes at least one of acetic acid-sodium acetate buffer solution, phosphate buffer solution, citric acid-sodium citrate buffer solution, potassium tartrate-sodium tartrate buffer solution, hydrochloric acid-potassium hydrochloride buffer solution, oxalic acid-potassium oxalate buffer solution, formic acid-sodium formate buffer solution, succinic acid-sodium succinate buffer solution, lactic acid-sodium lactate buffer solution, and acetic acid-hydrogen chloride buffer solution.

[0032] The concentration of the acidic solution in method (2) is 1 to 100 mmol / L, preferably 6 mmol / L.

[0033] The surfactant described in method (2) includes at least one of sulfonates, cationic surfactants, nonionic surfactants, anionic surfactants, fluorosurfactants, fatty alcohol polyether sulfates, glucosides and fatty alcohol sulfates; preferably at least one of Tween 80, sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (cationic surfactant), dodecyl polyoxyethylene ether (nonionic surfactant), polyoxyethylene octanol ether (nonionic surfactant), sodium alkylbenzene sulfonate (anionic surfactant), sodium dodecyl fluorooctyl sulfonate (fluorosurfactant), sodium dodecyl polyoxyethylene ether sulfate (fatty alcohol polyether sulfate), octyl glucoside and sodium dodecyl sulfate (fatty alcohol sulfate); preferably Tween 80.

[0034] The concentration of the surfactant solution in method (2) is 50 to 200 mmol / L, preferably 150 mmol / L.

[0035] The concentration of the precursor solution containing metal or metal oxide described in method (2) is 0.1 to 25 mmol / L, preferably 25 mmol / L.

[0036] The reducing agent described in method (2) is preferably at least one of hydrogen peroxide (H2O2), sodium citrate, sodium borohydride (NaBH4), ascorbic acid, hydrogen (H2), ethylene glycol, oxalic acid, trehalose, reducing sugars, ammonia, reducing amino acids, ferrocene and its derivatives, electrochemical reduction and photoreduction; more preferably ascorbic acid.

[0037] The reducing sugars are preferably at least one of glucose and fructose.

[0038] The reducing amino acid is preferably arginine.

[0039] The concentration of the reducing agent solution in method (2) is 0.004 to 10 mol / L, preferably 0.2 mol / L.

[0040] The volume ratio of the precursor solution containing metal or metal oxide to the reducing agent solution in method (2) is (1-4):(1-40); preferably 1:1.

[0041] The volume ratio of the acidic solution, surfactant solution, metal or metal oxide-containing precursor solution and reducing agent solution in method (2) is 1-100:0.1-20:0.1-30:0.1-30; preferably 60:2:3:3.

[0042] The reaction time in method (2) is 10 min to 48 h, preferably 1 hour.

[0043] The centrifugal washing described in method (2) is performed by centrifugal washing with anhydrous ethanol and pure water; preferably, the centrifugal elution is performed with anhydrous ethanol and pure water for more than 3 times.

[0044] The surface-enhanced Raman scattering nanoparticles without a covering molecular layer include gold nanoparticles; preferably at least one of gold nanospheres, gold nanostars, gold nanorods, gold nanoshells, gold nanocages and gold nanopolyhedrons; more preferably gold nanostars.

[0045] Application of the surface-enhanced Raman scattering nanoparticles without a covering molecular layer in the preparation of surface-enhanced Raman scattering nanomaterials.

[0046] The surface-enhanced Raman scattering nanomaterial includes at least one of gold, silver, copper, palladium, aluminum, graphene, metal oxides, semiconductor materials and multi-element alloy particles (composite structure); preferably, the material is gold nanoparticles.

[0047] The metal in the metal oxide includes at least one of gold, silver, copper, palladium and aluminum.

[0048] The semiconductor material includes at least one of tungsten nitride, tungsten dioxide, molybdenum dioxide, titanium dioxide and vanadium dioxide.

[0049] The metal of the multi-element alloy particles includes at least one of gold, silver, copper, palladium and aluminum.

[0050] A surface-enhanced Raman scattering nanoparticle cluster is prepared by at least one of the following methods:

[0051] ① Surface-enhanced Raman scattering nanoparticle clusters modified with bioactive molecules

[0052] The surface-enhanced Raman scattering nanoparticle solution without the covering molecular layer is added to the Raman reporter molecule solution and shaken to mix evenly, and then a sodium chloride solution (for promoting the binding of nucleic acid molecules to gold nanoparticles) and a nucleic acid solution are added in sequence. The mixture is incubated at 4°C or room temperature for more than 12 hours, and resuspended by centrifugation to obtain surface-enhanced Raman scattering nanoparticle clusters. The bioactive molecule is at least one of a polypeptide, a protein, a nucleic acid, and a serum.

[0053] ②Surface-enhanced Raman scattering nanoparticle clusters for surface modification of biological cell membranes

[0054] The surface-enhanced Raman scattering nanoparticle solution without a covering molecular layer is added to a Raman reporter molecule solution and shaken to mix evenly to obtain a nanoparticle-Raman reporter molecule solution; then a biological cell membrane is added and squeezed back and forth through a porous polycarbonate membrane using a liposome extruder 20 to 30 times to obtain a surface-enhanced Raman scattering nanoparticle cluster.

[0055] The amount of the surface-enhanced Raman scattering nanoparticles without a covering molecular layer described in methods ① and ② is calculated based on a final concentration of 0.1 to 10 nmol / L in the reaction system; preferably, a final concentration of 1 nmol / L in the reaction system.

[0056] The Raman reporter molecules described in methods ① and ② include at least one of IR organic dyes, phthalocyanine dyes, cyanide dyes, fluorescent dyes (HITC), crystal violet (dye), squaraine dyes, aniline blue (dye), porphyrin dyes, quinone dyes, merocyanine dyes, p-aminobenzoic acid, pyridine, p-nitrothiophenol, p-mercaptobenzoic acid, tetraphenylporphyrin, 1,2-bis(4-pyridyl)-ethylene, naphthoquinone, fluorescein and its derivatives, and rhodamine and its derivatives; preferably at least one of IR organic dyes and fluorescent dyes (HITC); more preferably at least one of IR775, IR792, IR797, R-780, IR-783, IR-820 and fluorescent dyes (HITC); and even more preferably at least one of IR775, IR792 and IR797;

[0057] The amount of the Raman reporter molecule described in method ① is calculated based on the final concentration of the Raman reporter molecule in the reaction system being 10 to 5000 nmol / L; preferably, the final concentration of the Raman reporter molecule in the reaction system is calculated based on the final concentration of the Raman reporter molecule in the reaction system being 1250 nmol / L.

[0058] The polypeptide described in method ① includes at least one of the active antioxidant polypeptide glycomacropeptide (GMP), soybean polypeptide, casein phosphopeptide, glutathione, thymosin and thyrotropin-releasing hormone; preferably glycomacropeptide (GMP).

[0059] The protein described in method ① includes at least one of bovine serum albumin (BSA), casein, ovalbumin, collagen, insulin, growth hormone, monoclonal antibody, lectin, agglutinogen and membrane receptor; preferably at least one of bovine serum albumin (BSA) and monoclonal antibody IgG1 (immunoglobulin G1).

[0060] In method ①, when the bioactive molecule is a polypeptide or protein, the molar concentration ratio of the surface-enhanced Raman scattering nanoparticle solution without a covering molecular layer, the Raman reporter molecule and the polypeptide is (0.1-10): (10-5000): (1-1000); preferably 1:1250:100.

[0061] The nucleic acid described in method ① includes at least one of DNA, RNA and nucleic acid aptamer; and its nucleotide sequence is preferably as shown in any one of SEQ ID NOs. 1 to 3.

[0062] In method ①, when the bioactive molecule is DNA or RNA, the molar concentration ratio of the surface-enhanced Raman scattering nanoparticle solution without a covering molecular layer, the Raman reporter molecule and the nucleic acid is (0.1-10):(10-10000):(1-5000); preferably 1:1250:300.

[0063] In method ①, when the bioactive molecule is a nucleic acid aptamer, the molar concentration ratio of the surface-enhanced Raman scattering nanoparticle solution without a covering molecular layer, the Raman reporter molecule and the nucleic acid is (0.1-10): (10-100000): (1-5000); preferably 1:100000:5000.

[0064] The serum described in method ① includes mouse serum solution (Mouse serum).

[0065] In method ①, when the bioactive molecule is serum, the molar concentration ratio of the surface-enhanced Raman scattering nanoparticle solution without a covering molecular layer and the Raman reporter molecule is (0.1-10):(10-100000); preferably 1:100000; the amount of serum used accounts for 10% of the volume of the total reaction system.

[0066] The amount of sodium chloride used in method ① is calculated based on its final concentration in the reaction system being 0.3 mol / L.

[0067] The centrifugal resuspension conditions described in method ① are: centrifugation at 500-5000 rpm for 3-15 minutes at 4°C or room temperature; preferably: centrifugation at 3000 rpm for 5 minutes at 4°C.

[0068] The resuspension described in method ① is to use ultrapure water for resuspension.

[0069] The molar concentration ratio of the surface-enhanced Raman scattering nanoparticle solution without a covering molecular layer to the Raman reporter molecule described in method ② is (0.1-10):(10-5000), preferably 3:5000.

[0070] The molar concentration ratio of the nanoparticle-Raman reporter molecule solution to the biological cell membrane described in method ② is 2:1.

[0071] The biological cell membrane described in method ② includes at least one of a red blood cell membrane, a macrophage membrane, a tumor cell membrane, a normal tissue cell membrane, and an immune cell membrane; preferably, it is a cancer cell membrane vesicle (CMV), which is preferably prepared by the following method: using a liposome extruder to squeeze cancer cells back and forth through a 200nm porous polycarbonate membrane 20 to 30 times to obtain cancer cell membrane vesicles (CMV).

[0072] The cancer cells include McA-RH7777 cells (rat liver cancer cells).

[0073] The pore size of the porous polycarbonate membrane described in method ② is 200 nm.

[0074] The method further includes the step of further detecting the prepared surface-enhanced Raman scattering nanoparticle clusters using a Raman spectrometer: the test solution is dropped onto a glass slide, a 96-well plate, or a 384-well plate, and detected using a Raman spectrometer; wherein the wavelength of the Raman spectrometer is 325 nm, 488 nm, 532 nm, 633 nm, 785 nm, or 1064 nm (preferably 785 nm).

[0075] Application of the surface-enhanced Raman scattering nanoparticles without a covering molecular layer and / or the surface-enhanced Raman scattering nanoparticle clusters in the preparation of Raman imaging materials or detection reagents.

[0076] The present invention has the following advantages and effects compared to the prior art:

[0077] (1) The present invention provides a method for preparing surface-enhanced Raman scattering nanoparticles without a covering molecular layer. Compared with other current preparation methods, the method has the advantages of rapid and simple operation, no need for heating conditions, and can be prepared by reaction at room temperature. Moreover, the prepared gold nanostar particles (Gold nanostar, AuNS) can better increase the Raman signal of the Raman reporter molecules adsorbed on the surface than gold nanospheres, and are a better nanoparticle base for surface-enhanced Raman scattering. The preparation cost is low, and the prepared composite substrate has high sensitivity and strong stability in detection. Because it does not have a covering molecular layer such as adsorbent, stabilizer, optical interference substance or other pollutants and impurities that reduce the Raman signal, the Raman signal quality is higher, stronger and more stable.

[0078] (2) The present invention also provides a method for preparing surface-enhanced Raman scattering nanoparticle clusters. Taking gold nanostar (AuNS) particles as an example, gold nanoparticles are mixed with Raman reporter molecules, and bioactive molecules [such as proteins, peptides, nucleic acids (DNA, RNA or nucleic acid aptamers), cell membranes, etc.] are introduced to quickly and stably fix the Raman reporter molecules on the gold nanoparticles. Compared with other current preparation methods, this method has the advantages of being fast and simple, low cost, direct operation, and suitable for large-scale production. It also retains the function of the bioactive molecules covered by the surface modification, and stabilizes the Raman reporter molecules fixed on the surface of the gold nanoparticles, enhancing their sensitivity and stability to achieve better surface-enhanced Raman scattering performance. This method can be extended to all surface-enhanced Raman scattering nanomaterials, including gold, silver, copper, palladium, aluminum, graphene, and various metal oxides and semiconductor materials, as well as composite structures, multi-element alloy particles, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 The diagrams are the particle size distribution and absorption spectrum of the gold nanostars prepared by the present invention; wherein, a is the particle size distribution diagram of the gold nanostars; and b is the absorption spectrum diagram of the gold nanostars.

[0080] Figure 2 Schematic diagram of the preparation of gold nanoparticle clusters for surface-enhanced Raman scattering.

[0081] Figure 3 These are characterization diagrams of surface-enhanced Raman scattering gold nanoparticle clusters with five different surface modifications prepared in Examples 2-6; wherein, a is a particle size diagram; b is an absorbance spectrum diagram; and c is a Raman spectrum diagram.

[0082] Figure 4 These are the gel retardation experimental results of the five surface-modified surface-enhanced Raman scattering gold nanoparticle clusters prepared in Examples 2-6; among them, a is the gel retardation experimental result of GMP and AuNS-IR775-GMP nanoparticle clusters; b is the gel retardation experimental result of BSA and AuNS-IR775-BSA nanoparticle clusters; c is the gel retardation experimental result of IgG and AuNS-IR775-IgG nanoparticle clusters; d is the gel retardation experimental result of DNA and AuNS-IR775-DNA nanoparticle clusters; e is the gel retardation experimental result of RNA and AuNS-IR775-RNA nanoparticle clusters.

[0083] Figure 5 This is a graph showing the serum stability of five surface-enhanced Raman scattering gold nanoparticle clusters with different surface modifications prepared in Examples 2-6 (particle size changes with incubation time).

[0084] Figure 6 The serum stability diagram of AuNS-IR775-GMP gold nanoparticle clusters; a is the Raman spectra of the gold nanoparticle clusters after incubation with serum for different times; b is the Raman spectra at 1359 cm -1 intensity statistics of the gold nanoparticle clusters after incubation with serum for different times; c is the absorption spectrum of the gold nanoparticle clusters after incubation with serum for different times; d is the intensity statistics of the absorption spectrum at 600 nm.

[0085] Figure 7 The serum stability diagram of AuNS-IR775-BSA gold nanoparticle clusters; a is the Raman spectra of the gold nanoparticle clusters after incubation with serum for different times; b is the Raman spectra at 1359 cm -1 intensity statistics of the gold nanoparticle clusters after incubation with serum for different times; c is the absorption spectrum of the gold nanoparticle clusters after incubation with serum for different times; d is the intensity statistics of the absorption spectrum at 600 nm.

[0086] Figure 8 The serum stability diagram of AuNS-IR775-IgG gold nanoparticle clusters; a is the Raman spectra of the gold nanoparticle clusters after incubation with serum for different times; b is the Raman spectra at 1359 cm -1 intensity statistics of the gold nanoparticle clusters after incubation with serum for different times; c is the absorption spectrum of the gold nanoparticle clusters after incubation with serum for different times; d is the intensity statistics of the absorption spectrum at 600 nm.

[0087] Figure 9 The serum stability diagram of AuNS-IR775-DNA gold nanoparticle clusters; a is the Raman spectra of the gold nanoparticle clusters after incubation with serum for different times; b is the Raman spectra at 1359 cm -1 intensity statistics of the gold nanoparticle clusters after incubation with serum for different times; c is the absorption spectrum of the gold nanoparticle clusters after incubation with serum for different times; d is the intensity statistics of the absorption spectrum at 600 nm.

[0088] Figure 10 The cluster serum stability diagram of AuNS-IR775-RNA gold nanoparticles; a is the Raman spectrum of the gold nanoparticle cluster after incubation with serum for different times; b is the Raman spectrum at 1359 cm -1 intensity statistics of the gold nanoparticle clusters after incubation with serum for different times; c is the absorption spectrum of the gold nanoparticle clusters after incubation with serum for different times; d is the intensity statistics of the absorption spectrum at 600 nm.

[0089] Figure 11Characterization and Raman spectra of AuNP-IR775-GMP and AuNS-IR775-GMP nanoparticle clusters; among them, a is the particle size distribution diagram of spherical AuNP and star-shaped AuNS; b is the absorption spectrum of spherical AuNP and star-shaped AuNS; c is the transmission electron microscope image of spherical AuNP and star-shaped AuNS; d is the Raman imaging image of AuNP-IR775-GMP nanoparticle clusters and AuNS-IR775-GMP nanoparticle clusters formed by spherical AuNP and star-shaped AuNS; e is the Raman spectrum corresponding to d.

[0090] Figure 12 Characterization and Raman spectra of AuNS-HITC-GMP, AuNS-IR775-GMP and AuNS-IR797-GMP nanoparticle clusters; wherein, a is the particle size bar graph of the nanoparticles; b is the Zeta potential bar graph of the nanoparticles; c is the absorption spectrum of the nanoparticles; d is the Raman spectrum of the nanoparticles.

[0091] Figure 13 This is a characterization diagram of the surface-enhanced Raman scattering gold nanoparticle cluster CMV-AuNS-IR792 modified on the cell membrane surface; among them, a is the particle size diagram; b is the absorbance spectrum diagram; c is the Raman spectrum diagram; d is the transmission electron microscope diagram.

[0092] Figure 14 Figure 1 is the Raman spectrum intensity stability and absorption spectrum stability diagram of CMV-AuNS-IR792 nanoparticle clusters within 14 days; a is the Raman spectrum within 14 days; b is the Raman spectrum at 1205 cm -1 a) Statistical graph of Raman peaks; c) Absorption spectrum within 14 days; d) Statistical graph of the absorption spectrum at 650nm peak.

[0093] Figure 15 Figure 2 is the Raman spectrum intensity stability and absorption spectrum stability diagram of CMV-AuNS-IR792 nanoparticle cluster after repeated freezing and thawing; a is the Raman spectrum after 4 freeze-thaw cycles; b is the Raman spectrum at 1205 cm -1 a is the statistical graph of the Raman peak; c is the absorption spectrum after 4 freeze-thaw cycles; d is the statistical graph of the absorption spectrum at the 650nm peak.

[0094] Figure 16 Figure 2 is the Raman spectrum intensity stability and absorption spectrum stability diagram of CMV-AuNS-IR792 nanoparticle cluster after heating (50℃); a is the Raman spectrum after heating; b is the Raman spectrum at 1205cm -1 a is the statistical diagram of the Raman peak; c is the absorption spectrum after heating; d is the statistical diagram of the absorption spectrum at the 650nm peak.

[0095] Figure 17 These are characterization diagrams of the new gold nanostars AuNS, AuNS-IR797-Serum nanoparticle clusters, and AuNS-IR797-Aptamer nanoparticle clusters; among them, a is a particle size bar graph; b is a potential bar graph; c is a polymer dispersibility index PDI graph; d is a potential Zeta graph; e is a transmission electron microscope image of AuNS; and f is a Raman spectrum graph.

[0096] Figure 18 The laser exposure stability diagrams of AuNS-IR797-Serum nanoparticle clusters and AuNS-IR797-Aptamer nanoparticle clusters are shown in Figure 1. Among them, a is the Raman spectrum of the AuNS-IR797-Serum nanoparticle clusters under continuous laser exposure for 420 seconds; b is the Raman spectrum of the AuNS-IR797-Serum nanoparticle clusters at 1367 cm -1 The signal intensity statistics at the peak; c is the Raman spectrum of the AuNS-IR797-Aptamer nanoparticle cluster under continuous laser exposure for 420 seconds; d is the Raman spectrum of the AuNS-IR797-Aptamer nanoparticle cluster at 1367 cm -1 Statistical graph of signal intensity at peaks.

[0097] Figure 19 The storage stability diagram of AuNS-IR797-Mouse serum nanoparticle clusters and AuNS-IR797-Aptamer nanoparticle clusters; a is the Raman spectrum of AuNS-IR797-Mouse serum nanoparticles; b is the Raman spectrum of AuNS-IR797-Mouse serum nanoparticles at 1367 cm -1 Statistical graph of signal intensity at the peak; c is the absorbance spectrum of AuNS-IR797-Mouse serum nanoparticles; d is the statistical graph of the absorbance spectrum of AuNS-IR797-Mouse serum nanoparticles at 800nm; e is the Raman spectrum of AuNS-IR797-Aptamer nanoparticle clusters; f is the Raman spectrum of AuNS-IR797-Aptamer nanoparticle clusters at 1367cm -1 Statistical graph of signal intensity at the peak; g is the absorption spectrum of AuNS-IR797-Aptamer nanoparticle clusters; h is the statistical graph of the absorption spectrum of AuNS-IR797-Aptamer nanoparticle clusters at 800nm.

[0098] Figure 20 These are the bladder tissue pathology and Raman imaging images of cystitis rats and normal rats; among them, a is the gross pathology image and Raman imaging image of the rat bladder; b is the Raman spectrum image corresponding to the Raman imaging. DETAILED DESCRIPTION

[0099] The present invention will be described in further detail below in conjunction with the examples, but embodiments of the present invention are not limited thereto. Unless otherwise stated, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The test methods for which specific experimental conditions are not specified in the following examples are usually based on conventional experimental conditions or the experimental conditions recommended by the manufacturer. Unless otherwise stated, the reagents and raw materials used in the present invention can be obtained commercially.

[0100] Example 1 Preparation of Gold Nanostars

[0101] First, prepare a 10mM aqueous solution of chloroauric acid, along with a 1.25M solution of H₂O₂ and a 12mM solution of NaOH. Then, mix the aqueous chloroauric acid solution and the H₂O₂ solution in a ratio of 1:4 by volume. Rapidly add an equal volume of NaOH solution to the chloroauric acid and H₂O₂ mixture. Place the mixture on a homogenizer and allow it to react at 150 rpm for 1 hour at room temperature in the dark to form gold nanostars. Centrifuge the mixture at 3000 rpm at room temperature for 5 minutes and resuspend in ultrapure water. Repeat this three times to obtain gold nanostars (AuNS). Store the mixture at 4°C until ready for use.

[0102] The particle size distribution and absorption spectrum of gold nanostars are shown in Figure 2. Figure 1 As shown: Figure 1 a shows that the hydrated particle size of the prepared gold nanostars AuNS is 69 nm. Figure 1 b shows the absorption spectrum of AuNS. Transmission electron microscopy (TEM) of AuNS is shown in Figure 11 As shown in c.

[0103] Example 2 Preparation of surface-enhanced Raman scattering nanoparticle clusters modified with GMP

[0104] The AuNS solution prepared in Example 1 (final concentration of 1 nM) was added to a Raman reporter molecule IR775 (IR-775 chloride, purchased from Sigma-Aldrich, catalog number: 544914) solution (final concentration of 1250 nM), and the mixture was shaken and mixed evenly. Sodium chloride solution (final concentration of 0.3 M) was added, and then a bioactive molecule glycomacropeptide (GMP) solution (final concentration of 100 nM) was immediately added. The mixture was incubated at 4°C for more than 12 hours, and resuspended by centrifugation three times (centrifugation at 3000 rpm and 4°C for 5 minutes; resuspended in ultrapure water) to obtain surface-enhanced Raman scattering gold nanoparticle clusters (AuNS-IR775-GMP nanoparticle clusters) with GMP as the surface modification. Figure 2 Schematic diagram of the preparation process of AuNS-IR775-GMP.

[0105] Example 3 Preparation of surface-enhanced Raman scattering nanoparticle clusters modified with BSA

[0106] The AuNS solution (final concentration of 1 nM) prepared in Example 1 was added to the Raman reporter molecule IR775 solution (final concentration of 1250 nM), and the mixture was shaken and mixed evenly. Sodium chloride solution (final concentration of 0.3 M) was added, and then the bioactive molecule bovine serum albumin (BSA) solution (final concentration of 100 nM) was immediately added. The mixture was incubated at 4°C for more than 12 hours, and resuspended by centrifugation three times (centrifugation at 3000 rpm and 4°C for 5 minutes; resuspended in ultrapure water) to obtain surface-enhanced Raman scattering gold nanoparticle clusters modified with BSA (AuNS-IR775-BSA nanoparticle clusters).

[0107] Example 4 Preparation of IgG-modified surface-enhanced Raman scattering nanoparticle clusters

[0108] The AuNS solution (final concentration of 1 nM) prepared in Example 1 was added to the Raman reporter IR775 solution (final concentration of 1250 nM), and the mixture was shaken and mixed evenly. Sodium chloride solution (final concentration of 0.3 M) was added, and then the bioactive molecule IgG (Guangzhou Chuangwei Biotechnology Co., Ltd., product number: B900640) solution (final concentration of 100 nM) was immediately added. The mixture was incubated at 4°C for more than 12 hours, and the mixture was centrifuged and resuspended three times (centrifuged at 3000 rpm and 4°C for 5 minutes; resuspended in ultrapure water) to obtain surface-enhanced Raman scattering gold nanoparticle clusters (AuNS-IR775-IgG nanoparticle clusters) with IgG as the surface modification.

[0109] Example 5 Preparation of DNA-modified surface-enhanced Raman scattering nanoparticle clusters

[0110] The AuNS solution prepared in Example 1 (final concentration of 1 nM) was added to the Raman reporter IR775 solution (final concentration of 1250 nM), oscillated and mixed evenly, and sodium chloride solution (final concentration of 0.3 M) was added. Immediately, a bioactive molecule DNA solution (final concentration of 300 nM) was added, and the mixture was incubated at 4°C for more than 12 hours. The mixture was centrifuged and resuspended three times (centrifuged at 3000 rpm and 4°C for 5 minutes; resuspended in ultrapure water) to obtain surface-enhanced Raman scattering gold nanoparticle clusters modified with DNA (AuNS-IR775-DNA nanoparticle clusters). The nucleotide sequence of the DNA sample is shown in Table 1 and was synthesized by Ruibo Xingke Biotechnology Co., Ltd.

[0111] Table 1 Sequences of DNA samples used in the present invention

[0112]

[0113] Note: "SH" in Table 1 indicates a thiol functional group.

[0114] Example 6 Preparation of RNA-Modified Surface-Enhanced Raman Scattering Nanoparticle Clusters

[0115] The AuNS solution (final concentration of 1 nM) prepared in Example 1 was added to the Raman reporter IR775 solution (final concentration of 1250 nM), oscillated and mixed evenly, and sodium chloride solution (final concentration of 0.3 M) was added. Immediately, a bioactive molecule RNA solution (final concentration of 300 nM) was added, and the mixture was incubated at 4°C for more than 12 hours. The mixture was centrifuged and resuspended three times (centrifuged at 3000 rpm and 4°C for 5 minutes; resuspended in ultrapure water) to obtain surface-enhanced Raman scattering gold nanoparticle clusters modified with RNA (AuNS-IR775-RNA nanoparticle clusters). The nucleotide sequence of the RNA sample is shown in Table 2 and was synthesized by Ruibo Xingke Biotechnology Co., Ltd.

[0116] Table 2 Sequences of RNA samples used in the present invention

[0117] Sample name Sequence (from 5' to 3' end) miRNA CAG UGC AAU GAU GAA AGG GCA UAC-SH(SEQ ID NO.2)

[0118] Note: "SH" in Table 2 indicates a thiol functional group.

[0119] Example 7 Characterization of five surface-modified surface-enhanced Raman scattering nanoparticle clusters

[0120] The gold nanoparticle clusters (AuNS-IR775-GMP, AuNS-IR775-BSA, AuNS-IR775-IgG, AuNS-IR775-DNA, and AuNS-IR775-RNA) obtained in Examples 2-6 were analyzed using a Raman spectrometer. The following detection conditions were used: a laser wavelength of 785 nm, an excitation power of 100 mW, an integration time of 1 s, and a number of integrations. The Raman spectrometer was a Renishaw, Inc., Hoffman Estates, IL, facility. Three replicates were used.

[0121] The results are as follows Figure 3 As shown: Figure 3 a shows the hydrated particle size of five gold nanoparticle clusters with different surface modifications. Figure 3 b shows the absorption spectra of gold nanoparticle clusters with five different surface modifications. Figure 3 c shows the Raman spectra of gold nanoparticle clusters with five different surface modifications.

[0122] Example 8 Gel retardation experiment verifies the successful preparation of surface-enhanced Raman scattering gold nanoparticle clusters modified with polypeptides or proteins

[0123] Weigh the agarose powder and add it to the conical flask used for preparing the gel. Add 1xTBE buffer in a quantitative manner to prepare a 1.2% agarose solution. Heat it in a microwave oven to melt it. After cooling for a while, pour it into the electrophoresis tank and insert a comb. Wait until it is completely solidified. Pull out the comb, place the gel into the electrophoresis tank, and pour in TBE buffer to cover the gel surface by 1mm. Add sample peptides (GMP) or proteins (BSA, IgG) and corresponding gold nanoparticle clusters (AuNS-IR775-GMP or AuNS-IR775-BSA or AuNS-IR775-IgG) to the wells. Perform electrophoresis at a constant voltage of 100V for 30 minutes. Place the gel on a gel imager to observe the electrophoresis bands and their positions. The experiment was repeated three times.

[0124] The results are as follows Figure 4 As shown in Figures 4a, 4b, and 4c: In the gel retardation experiment, compared with the pure GMP peptide, the AuNS-IR775-GMP gold nanoparticle clusters migrated slower during gel electrophoresis due to their larger molecular weight, further proving that the AuNS-IR775-GMP gold nanoparticle clusters were successfully prepared ( Figure 4 a); Compared with pure BSA, AuNS-IR775-BSA gold nanoparticle clusters migrate slower during gel electrophoresis due to their larger molecular weight, further proving that AuNS-IR775-BSA gold nanoparticle clusters were successfully prepared ( Figure 4 b); Compared with pure IgG, AuNS-IR775-IgG gold nanoparticle clusters migrate slower during gel electrophoresis due to their larger molecular weight, further proving that AuNS-IR775-IgG gold nanoparticle clusters were successfully prepared ( Figure 4 c).

[0125] Example 9 Gel retardation experiment verifies the successful preparation of surface-enhanced Raman scattering gold nanoparticle clusters modified with nucleic acid surfaces

[0126] Weigh the agarose powder and add it to the conical flask used for preparing the gel. Add 1xTBE buffer in a quantitative manner to prepare a 2% agarose solution. Heat it in a microwave oven to melt. After cooling for a while, add EB fluorescent dye at a ratio of 1:10000. Shake it gently and pour it into the electrophoresis tank. Insert the comb and wait for it to completely solidify. Pull out the comb, place the gel into the electrophoresis tank, and pour TBE buffer to cover the gel surface by 1mm. Add DNA or RNA samples and the corresponding gold nanoparticle clusters (AuNS-IR775-DNA or AuNS-IR775-RNA) to the wells. Perform electrophoresis at a constant voltage of 100V for 30 minutes. Place the gel on a gel imager to observe the electrophoresis bands and their positions. The experiment was repeated three times.

[0127] The results are as follows Figure 4As shown in d and 4e: In the gel retardation experiment, compared with pure DNA, AuNS-IR775-DNA gold nanoparticle clusters migrated slower during gel electrophoresis due to their larger molecular weight, further proving that AuNS-IR775-DNA gold nanoparticle clusters were successfully prepared ( Figure 4 d); Compared with pure RNA, AuNS-IR775-RNA gold nanoparticle clusters migrate slower during gel electrophoresis due to their larger molecular weight, further proving that AuNS-IR775-RNA gold nanoparticle clusters were successfully prepared ( Figure 4 e).

[0128] Example 10 Surface-enhanced Raman scattering gold nanoparticle clusters in serum stability test

[0129] The gold nanoparticle clusters prepared in Examples 2-6 were mixed with equal volumes of 10% (v / v) mouse serum (purchased from Nanjing Senbega Biotechnology Co., Ltd., Catalog No. SBJ-SE-M004-50) and incubated at 37°C. After specific time periods (0, 1, 3, 6, 9, 12, and 24 hours), the nanoparticles were removed and analyzed for particle size, absorption spectra, and Raman spectroscopy to verify their stability. The experiment was repeated three times.

[0130] The particle size changes of five surface-modified gold nanoparticle clusters with surface-enhanced Raman scattering in serum with incubation time are shown in Figure 2. Figure 5 As shown in Figure 2, the Raman spectrum and the corresponding Raman intensity, the absorption spectrum and the corresponding absorption spectrum intensity change with the incubation time. Figures 6-10 As shown in Figure 4, it shows that these five gold nanoparticle clusters have good stability in serum.

[0131] Example 11 Preparation of Gold Nanoparticle Clusters for Surface Enhanced Raman Scattering Formed by Gold Nanospheres

[0132] (1) Gold nanoparticles (AuNPs) with a particle size of approximately 63 nm were prepared according to the existing seed growth method (reference: Bakshi M SA simple method of superlattice formation: step-by-step evaluation of crystal growth of gold nanoparticles through seed-growth method[J]. Langmuir, 2009, 25(21): 12697-12705.) (seed growth method).

[0133] The particle size distribution of the AuNPs prepared in this example and the AuNS prepared in Example 1 is as follows: Figure 11 The absorption spectrum is shown in a. Figure 11 As shown in b, the transmission electron microscopy image is as follows Figure 11 As shown in c.

[0134] (2) Add the AuNP solution (final concentration of 1 nM) to the Raman reporter molecule IR775 solution (final concentration of 1250 nM), shake and mix evenly, add sodium chloride solution (final concentration of 0.3 M), and immediately add the bioactive molecule GMP solution (final concentration of 100 nM). Incubate at 4°C for more than 12 hours, centrifuge and resuspend three times (centrifuge at 3000 rpm and 4°C for 5 minutes; resuspend in ultrapure water) to obtain surface-enhanced Raman scattering gold nanoparticle clusters formed by gold nanospheres (AuNP-IR775-GMP nanoparticle clusters).

[0135] The Raman imaging of the AuNP-IR775-GMP prepared in this example and the AuNS-IR775-GMP nanoparticle cluster prepared in Example 2 is shown in FIG. Figure 11 d, and its corresponding Raman spectrum is shown in Figure 11 As shown in Figure e, it can be seen that the gold spherical nanoparticles AuNP and the gold star-shaped nanoparticles AuNS in the present invention have similar particle sizes, but the Raman signal intensity of AuNS is stronger.

[0136] Example 12 Preparation of Surface Enhanced Raman Scattering Gold Nanoparticle Clusters Formed by Different Raman Reporter Molecules

[0137] The AuNS solution prepared in Example 1 (final concentration of 1 nM) was added to a Raman reporter molecule solution (HITC or IR775 or IR797) with a final concentration of 1250 nM (all purchased from Sigma-Aldrich, HITC (iodinated-1,1′,3,3,3′,3′-hexamethylindotricarbocyanine) catalog number: 252034; IR775 (IR-775 chloride) catalog number: 544914; IR797 (IR-797 chloride) catalog number: 642339). , oscillate and mix evenly, add sodium chloride solution (final concentration is 0.3M), immediately add bioactive molecule GMP solution (final concentration is 100nM), incubate at 4℃ for more than 12 hours, centrifuge and resuspend 3 times (centrifuge at 3000rpm, 4℃ for 5 minutes; resuspend in ultrapure water), and obtain surface-enhanced Raman scattering gold nanoparticle clusters (AuNS-HITC-GMP, AuNS-IR775-GMP, AuNS-IR797-GMP nanoparticle clusters) with different Raman reporter molecules as the surface modification. Characterization of surface-enhanced Raman scattering gold nanoparticle clusters formed by different Raman reporter molecules and Raman spectra are shown in Figure 2. Figure 12 shown.

[0138] Example 13 Preparation of Gold Nanoparticle Clusters CMV-AuNS-IR792 for Surface-Enhanced Raman Scattering Modified on Cell Membrane Surfaces

[0139] (1) The AuNS solution prepared in Example 1 (final concentration of 3 pM) was added to a Raman reporter molecule IR792 (IR-792 perchlorate, purchased from Sigma-Aldrich, catalog number: 425982) solution with a final concentration of 5 μM, and the mixture was shaken and mixed to obtain AuNS-IR792;

[0140] (2) Using a liposome extruder (Avanti Polar Lipids Inc), the extracted cancer cells (McA-RH7777) (purchased from Guangzhou Jibiku Biotechnology Co., Ltd., catalog number: IML-130) were squeezed back and forth through a 200 nm porous polycarbonate membrane 20-30 times to obtain cancer cell membrane vesicles (CMV);

[0141] (3) CMV and AuNS-IR792 were mixed at a molar concentration ratio of 2:1 and then squeezed back and forth through a 200 nm porous polycarbonate membrane using a liposome extruder 20-30 times. Through the extrusion force and electrostatic attraction, CMV was coated on the surface of AuNS to obtain CMV-AuNS-IR792.

[0142] Figure 13 It shows that the CMV-AuNS-IR792 nanoparticle clusters modified by cell membrane surface were successfully prepared; among them, Figure 13 a shows that the particle size of CMV-AuNS-IR792 nanoparticle clusters is approximately 120 nm; Figure 13 b shows that the CMV-AuNS-IR792 nanoparticle clusters retain the absorption peaks of each component; Figure 13 c shows that the CMV-AuNS-IR792 nanoparticle cluster has a strong Raman spectrum intensity; Figure 13 d shows that CMV-AuNS-IR792 nanoparticle clusters were successfully coated with CMV, indicating that CMV-AuNS-IR792 nanoparticle clusters were successfully prepared.

[0143] Example 14 Stability Test of Surface Enhanced Raman Scattering Gold Nanoparticle Cluster CMV-AuNS-IR792 Modified on Cell Membrane Surface within 14 Days

[0144] The CMV-AuNS-IR792 nanoparticle cluster solution prepared in Example 13 was placed in a 4°C refrigerator. After a certain period of time (0, 1, 3, 5, 7, and 14 days), 100 μL of the solution was collected for Raman and absorbance spectroscopy. The experiment was repeated three times.

[0145] Figure 14 It shows that the CMV-AuNS-IR792 nanoparticle clusters maintain good Raman spectral intensity stability and absorption spectral stability within 14 days; Figure 14 a shows Raman spectrum Figure 14 No significant changes within the day; Figure 14 b is Figure 14 a at 1205cm -1 The statistical graph of the Raman peak shows that the intensity does not change much within 14 days; Figure 14 c shows the absorption spectrum Figure 14 No significant changes within the day; Figure 14 d is Figure 14 c Statistical graph of the 650 nm peak, the intensity did not change much within 14 days. Figure 14 This shows that the CMV-AuNS-IR792 nanoparticle clusters can maintain good stability within 14 days.

[0146] Example 15 Stability Testing of Surface Enhanced Raman Scattering Gold Nanoparticle Cluster CMV-AuNS-IR792 Modified on Cell Membrane Surfaces after Repeated Freeze-Thaw

[0147] The CMV-AuNS-IR792 nanoparticle cluster solution prepared in Example 13 was frozen at -80°C for 30 minutes, then thawed at room temperature for 10 minutes. These freeze-thaw cycles were repeated four times, and 100 μL of the solution was analyzed for Raman and absorbance spectroscopy. The experiment was repeated three times.

[0148] Figure 15 The graphs show the Raman spectral intensity stability and the absorption spectral stability of CMV-AuNS-IR792 nanoparticle clusters after repeated freezing and thawing. Figure 15 a is the Raman spectrum after 4 freeze-thaw cycles, and the Raman spectrum intensity does not change much; Figure 15 b is Figure 15 a at 1205cm -1 The statistical graph of Raman peaks shows that the peak intensity does not change significantly; Figure 15 c is the absorption spectrum after 4 freeze-thaw cycles, the absorption intensity did not change significantly, and no peak shift was observed; Figure 15 d is Figure 15 c Statistical diagram of the 650 nm peak, the peak intensity does not change significantly. Figure 15 This shows that the CMV-AuNS-IR792 nanoparticle clusters can maintain good stability after four freeze-thaw cycles.

[0149] Example 16 Stability Test of Surface Enhanced Raman Scattering Gold Nanoparticle Cluster CMV-AuNS-IR792 Modified on Cell Membrane Surface after Heating

[0150] The CMV-AuNS-IR792 nanoparticle cluster solution prepared in Example 13 was heated at 50° C. for a certain period of time (0.5, 1, 2, and 24 h), and 100 μL of the solution was taken for Raman and absorbance spectroscopy. The experiment was repeated three times.

[0151] Figure 16 The graphs show the Raman spectrum intensity stability and absorption spectrum stability of CMV-AuNS-IR792 nanoparticle clusters after heating; Figure 16 a is the Raman spectrum after heating, and the Raman spectrum intensity does not change much; Figure 16 b is Figure 16 a at 1205cm -1 The statistical graph of Raman peaks shows that the peak intensity does not change significantly; Figure 16 c is the absorption spectrum after heating, the absorption intensity does not change significantly and no peak shift is observed; Figure 16 d is Figure 16 c Statistical diagram of the 650 nm peak, the peak intensity does not change significantly. Figure 16 This shows that the CMV-AuNS-IR792 nanoparticle clusters can maintain good stability after heating.

[0152] Example 17 Preparation of Novel Gold Nanostar Particles

[0153] 60 μL of 6 mol / L hydrochloric acid (HCl) solution was added to 2.0 mL of 150 mmol / L Tween 80 solution and mixed. 3.0 mL of 25 mmol / L tetrachloroauric acid (HAuCl4) solution was then added to the above solution and mixed. Finally, 3.0 mL of 0.2 mol / L antacid solution was added and rotated at room temperature for 1 hour. The final product was centrifuged and eluted three times with anhydrous ethanol and pure water to obtain the new gold nanostar AuNS.

[0154] The characterization results of the new gold nanostars AuNS are as follows Figure 17 As shown: Figure 17 a shows that the particle size of the new gold nanostar is about 80 nanometers; Figure 17 b shows that the potential of the new gold nanostar is -26 mV; Figure 17 c shows that the PDI of the new gold nanostars is about 0.20; Figure 17 d Absorption spectrum shows that the absorption peak of the new gold nanostar (compared with the gold nanostar prepared in Example 1) is broader; Figure 17 e is an electron microscope image of the new type of gold nanostar, which has more star-shaped corners. Figure 17 This indicates that the novel gold nanostars were successfully prepared in this example.

[0155] Example 18 Preparation of Novel Nanostar-Based Surface-Enhanced Raman Scattering Gold Nanoparticle Clusters AuNS-IR797-Mouse Serum

[0156] To the novel gold nanostar (AuNS) solution prepared in Example 17, a Raman reporter molecule solution (IR797) and mouse serum (available from Biosharp, Catalog No. BL1053A) were added sequentially. The mixture was mixed and rotated to react overnight. The final solution had a molar ratio of AuNS to IR797 of 1:100,000, and the volume of mouse serum accounted for 10% of the total solution volume. The mixture was eluted by centrifugation three times with pure water to obtain the AuNS-IR797-mouse serum.

[0157] The characterization results of AuNS-IR797-Mouse serum are as follows Figure 17 As shown: Figure 17 a shows that the particle size of AuNS-IR797-Mouseserum is about 110 nm; Figure 17 b shows that the potential of AuNS-IR797-Mouse serum is -21 mV; Figure 17 c shows that the PDI of AuNS-IR797-Mouse serum is approximately 0.21; Figure 17 d shows the absorbance spectrum of AuNS-IR797-Mouse serum; Figure 17 f shows the Raman spectrum of AuNS-IR797-Mouse serum, which has a strong Raman signal intensity.

[0158] Example 19 Preparation of Novel Nanostar-Based Gold Nanoparticle Clusters AuNS-IR797-Aptamer for Surface-Enhanced Raman Scattering

[0159] The novel AuNS solution prepared in Example 17 was added sequentially to the Raman reporter molecule IR797 and the nucleic acid aptamer Aptamer, resulting in a final solution with a molar ratio of AuNS, IR797, and Aptamer of 1:100,000:5000. The mixture was mixed and allowed to react overnight, and then centrifuged and eluted three times with pure water to obtain AuNS-IR797-Aptamer. The nucleic acid sequence of Aptamer is shown in Table 3 and was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0160] Table 3 Aptamer nucleic acid sequence

[0161]

[0162] Note: "SH" in Table 3 indicates a thiol functional group.

[0163] The characterization of AuNS-IR797-Aptamer was as follows Figure 17 As shown: Figure 17 a shows that the particle size of AuNS-IR797-Aptamer is about 84 nm; Figure 17 b shows that the potential of AuNS-IR797-Aptamer is −22 mV; Figure 17 c shows that the PDI of AuNS-IR797-Aptamer is approximately 0.25; Figure 17 d shows the absorption spectrum of AuNS-IR797-Aptamer; Figure 17 f shows the Raman spectrum of AuNS-IR797-Aptamer, which has a strong Raman signal intensity.

[0164] Example 20 Laser Exposure Stability Test of AuNS-IR797-Mouse Serum Nanoparticle Clusters and AuNS-IR797-Aptamer Nanoparticle Clusters

[0165] Fifty microliters of each of the AuNS-IR797-mouse serum nanoparticle clusters and AuNS-IR797-aptamer nanoparticle clusters prepared in Examples 18 and 19 were placed in a 384-well plate. The plates were continuously illuminated with a 785 nm laser (1% laser intensity) and Raman spectra were collected at fixed intervals. Three replicates were performed.

[0166] Figure 18 The laser exposure stability of AuNS-IR797-Mouse serum nanoparticle clusters and AuNS-IR797-Aptamer nanoparticle clusters was shown; Figure 18 a is the Raman spectrum of the AuNS-IR797-Serum nanoparticle cluster under continuous laser exposure for 420 seconds; Figure 18 b is Figure 18 a Raman spectrum at 1367 cm -1 The signal intensity statistical histogram at the peak shows that the signal intensity is maintained above 90%, indicating good laser exposure stability; Figure 18 c is the Raman spectrum of the AuNS-IR797-Aptamer nanoparticle cluster under continuous laser exposure for 420 seconds; Figure 18 d is Figure 18 c Raman spectrum at 1367cm -1 The signal intensity at the peak is statistically analyzed in a histogram, and the signal intensity is maintained above 80%, indicating good laser exposure stability.

[0167] Example 21 Storage Stability Test of AuNS-IR797-Mouse Serum Nanoparticle Clusters and AuNS-IR797-Aptamer Nanoparticle Clusters

[0168] The AuNS-IR797-Mouse serum nanoparticle cluster and AuNS-IR797-Aptamer nanoparticle cluster solutions prepared in Examples 18 and 19 were placed in a 4°C refrigerator. After a certain period of time (0, 1, 3, 6, 12, and 24 hours), 100 μL of the solution was collected for Raman and absorbance spectroscopy. The experiment was repeated three times.

[0169] Figure 19 The storage stability of AuNS-IR797-Mouse serum nanoparticle clusters and AuNS-IR797-Aptamer nanoparticle clusters is shown; wherein:

[0170] Figure 19 a is the Raman spectrum of AuNS-IR797-Mouse serum nanoparticle cluster; Figure 19 b is Figure 19 a at 1367cm -1 The statistical graph of signal intensity at the peak showed no obvious signal reduction; Figure 19 c is the absorption spectrum of AuNS-IR797-Mouse serum nanoparticle cluster; Figure 19 d is Figure 19 c Statistical histogram at 800 nm, the signal intensity did not change significantly.

[0171] Figure 19 e is the Raman spectrum of AuNS-IR797-Aptamer nanoparticle clusters; Figure 19 f is Figure 19 e at 1367cm -1 The statistical graph of signal intensity at the peak showed no obvious signal reduction; Figure 19 g is the absorption spectrum of AuNS-IR797-Aptamer nanoparticle clusters; Figure 19 h is Figure 19 g Statistical histogram at 800 nm, the signal intensity did not change significantly.

[0172] Example 22: Detection of Lesions in Vivo Using Raman Imaging of AuNS-IR775-GMP Nanoparticle Clusters

[0173] To establish a chronic cystitis model, SD female rats (7 weeks, 200 g, Guangdong Experimental Animal Center) were treated with cyclophosphamide CYP at 50 mg kg -1Intraperitoneal injection was performed on the 1st, 4th and 7th days of the experiment to construct a chronic cystitis model. Rats in the blank control group were intraperitoneally injected with sterile saline on the same experimental days. The AuNS-IR775-GMP nanoparticle clusters prepared in Example 2 were injected into the blank control group rats and the chronic cystitis model in a volume of 500 ml by bladder perfusion. After 12 hours of perfusion, the rats were killed, the bladders were dissected out, and the free bladder tissues were subjected to gross pathological photography and Raman imaging. Raman imaging (mapping) parameters: laser light source is 785 nm; objective lens is 50×; laser intensity is 100% (100 mW); imaging time: 0.1 s; step size is 0.1 mm; cumulative number of times is 1. The experiment was repeated three times.

[0174] Figure 20 The bladder tissue pathology and Raman imaging of cystitis rats and normal rats; Figure 20 a shows the gross pathological image and Raman imaging of rat bladder. The bladder tissue of rats with chronic cystitis has obvious bleeding and edema. The Raman imaging image shows that the Raman signal is highly matched with the lesion. Figure 20 b is the Raman spectrum corresponding to the Raman imaging image. Compared with normal bladder tissue, the Raman signal of the cystitis model is higher.

[0175] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A surface-enhanced Raman scattering nanoparticle without a covering molecular layer, characterized in that: Prepared by any of the following methods: Method (1) adding a precursor solution containing a metal or metal oxide to a reducing agent solution, mixing well, adding an alkaline solution, stirring to react under light-proof conditions, centrifuging to obtain a precipitate after the reaction is completed, and then resuspending in water to obtain surface-enhanced Raman scattering nanoparticles without a covering molecular layer; Method (2) adding an acidic solution to a surfactant solution, then adding a precursor solution containing a metal or metal oxide, and then adding a reducing agent solution, mixing and reacting at room temperature, and after the reaction is completed, centrifuging and washing the product to obtain surface-enhanced Raman scattering nanoparticles without a covering molecular layer; The concentration of the precursor solution containing metal or metal oxide described in method (1) is 0.1 to 25 mmol / L; The concentration of the reducing agent solution described in method (1) is 50 to 5000 mmol / L; The volume ratio of the metal or metal oxide precursor solution to the reducing agent solution in method (1) is 1-4:1-40; The concentration of the alkaline solution described in method (1) is 1 to 100 mmol / L; The volume of the alkaline solution in method (1) is 20 to 70% of the total volume of the precursor solution containing the metal or metal oxide and the reducing agent solution; The concentration of the acidic solution described in method (2) is 1 to 100 mmol / L; The concentration of the surfactant solution described in method (2) is 50 to 200 mmol / L; The concentration of the metal or metal oxide precursor solution described in method (2) is 0.1 to 25 mmol / L; The concentration of the reducing agent solution described in method (2) is 0.004 to 10 mol / L; The volume ratio of the metal or metal oxide-containing precursor solution to the reducing agent solution in method (2) is 1-4:1-40; The volume ratio of the acidic solution, the surfactant solution, the precursor solution containing metal or metal oxide, and the reducing agent solution described in method (2) is 1-100:0.1-20:0.1-30:0.1-30; The stirring reaction described in method (1) is performed at a speed of 100 to 300 rpm; The stirring reaction time described in method (1) is 10 minutes to 48 hours; The centrifugation conditions described in method (1) are: centrifugation at 500-5000 rpm for 3-15 minutes at 4°C or room temperature; The reaction time described in method (2) is 10 minutes to 48 hours.

2. The surface-enhanced Raman scattering nanoparticles without a covering molecular layer according to claim 1, characterized in that: The metal or metal oxide-containing precursor solution in methods (1) and (2) is a gold-containing precursor solution, a silver-containing precursor solution, or a copper-containing precursor solution; wherein, The gold-containing precursor solution is at least one of chloroauric acid, sodium chloroaurate, potassium chloroaurate, sodium iodoaurate and potassium iodoaurate solution; The silver-containing precursor solution is at least one of silver nitrate, silver chloride, silver sulfate, silver acetate, silver fluoroborate, silver formate, silver citrate and silver oxalate; The copper-containing precursor solution is at least one of copper sulfate, copper chloride, copper nitrate, copper acetate, copper oxide, copper carbonate, copper oxalate, copper formate, copper bromide, copper fluoroborate and copper iodide solution; The reducing agent in methods (1) and (2) is at least one of hydrogen peroxide, sodium citrate, sodium borohydride, ascorbic acid, hydrogen, ethylene glycol, oxalic acid, trehalose, reducing sugar, ammonia, reducing amino acids, ferrocene and its derivatives, electrochemical reduction, and photoreduction; The alkaline solution described in method (1) is at least one of a NaOH solution, a KOH solution and an alkaline buffer solution; The acidic solution described in method (2) is at least one of sulfuric acid solution, hydrochloric acid solution, nitric acid solution, acetic acid solution, citric acid solution, lactic acid solution, oxalic acid solution, phosphoric acid solution, acetic acid solution, formic acid solution and acidic buffer solution; The surfactant described in method (2) is at least one of sulfonates, cationic surfactants, nonionic surfactants, anionic surfactants, fluorine-containing surfactants, fatty alcohol polyether sulfates, glucosides and fatty alcohol sulfates.

3. The surface-enhanced Raman scattering nanoparticles without a covering molecular layer according to claim 2, characterized in that: The precursor solution containing metal or metal oxide in methods (1) and (2) is a chloroauric acid solution; The reducing agent in method (1) is hydrogen peroxide; The alkaline solution described in method (1) is a NaOH solution; The acidic solution described in method (2) is a hydrochloric acid solution; The surfactant described in method (2) is Tween 80; The reducing agent described in method (2) is ascorbic acid.

4. The surface-enhanced Raman scattering nanoparticles without a covering molecular layer according to claim 1, characterized in that: The centrifugal washing described in method (2) is centrifugal washing using anhydrous ethanol and pure water.

5. Use of the surface-enhanced Raman scattering nanoparticles without a covering molecular layer according to any one of claims 1 to 4 in the preparation of surface-enhanced Raman scattering nanomaterials.

6. A surface-enhanced Raman scattering nanoparticle cluster, characterized in that: Prepared by any of the following methods: Method ① Surface-enhanced Raman scattering nanoparticle clusters modified with bioactive molecules A solution of surface-enhanced Raman scattering nanoparticles without a covering molecular layer according to any one of claims 1 to 4 is added to a Raman reporter molecule solution and shaken to mix evenly, followed by sequential addition of a sodium chloride solution and a nucleic acid solution, incubated at 4°C or room temperature for more than 12 hours, and resuspended by centrifugation to obtain surface-enhanced Raman scattering nanoparticle clusters; wherein the bioactive molecule is at least one of a polypeptide, a protein, a nucleic acid, and a serum; Method ② Surface-enhanced Raman scattering nanoparticle clusters for surface modification of biological cell membranes A solution formed by surface-enhanced Raman scattering nanoparticles without a covering molecular layer according to any one of claims 1 to 4 is added to a Raman reporter molecule solution and shaken and mixed evenly to obtain a nanoparticle-Raman reporter molecule solution; then a biological cell membrane is added, and the solution is squeezed back and forth through a porous polycarbonate membrane using a liposome extruder 20 to 30 times to obtain surface-enhanced Raman scattering nanoparticle clusters.

7. The surface-enhanced Raman scattering nanoparticle cluster according to claim 6, characterized in that: The Raman reporter molecules described in methods ① and ② include at least one of IR organic dyes, phthalocyanine dyes, cyanide dyes, fluorescent dyes, crystal violet, chromatochrome dyes, aniline blue, porphyrin dyes, quinone dyes, melanolone dyes, p-aminobenzoic acid, pyridine, p-nitrothiophenol, p-mercaptobenzoic acid, tetraphenylporphyrin, 1,2-bis(4-pyridyl)-ethylene, naphthoquinone, fluorescein and its derivatives, and rhodamine and its derivatives; The polypeptide described in method ① includes at least one of active antioxidant polypeptide glycomacropeptide, soybean polypeptide, casein phosphopeptide, glutathione, thymosin and thyrotropin-releasing hormone; The protein in method ① comprises at least one of bovine serum albumin, casein, ovalbumin, collagen, insulin, growth hormone, monoclonal antibody, lectin, agglutinogen and membrane receptor; The nucleic acid described in method ① comprises at least one of DNA, RNA and nucleic acid aptamer; the nucleotide sequence thereof is shown in any one of SEQ ID NOs. 1 to 3; The serum described in method ① includes mouse serum solution; The biological cell membrane described in method ② includes at least one of erythrocyte membrane, macrophage membrane and tumor cell membrane.

8. The surface-enhanced Raman scattering nanoparticle cluster according to claim 6, characterized in that: The amount of surface-enhanced Raman scattering nanoparticles without a covering molecular layer described in methods ① and ② is calculated based on the final concentration of 0.1 to 10 nmol / L in the reaction system; The amount of the Raman reporter molecule described in method ① is calculated based on its final concentration in the reaction system of 10 to 5000 nmol / L; In method ①, when the bioactive molecule is a polypeptide or protein, the molar concentration ratio of the solution formed by the surface-enhanced Raman scattering nanoparticles without a covering molecular layer, the Raman reporter molecule, and the polypeptide is 0.1-10:10-5000:1-1000; In method ①, when the bioactive molecule is DNA or RNA, the molar concentration ratio of the solution formed by the surface-enhanced Raman scattering nanoparticles without a covering molecular layer, the Raman reporter molecule, and the nucleic acid is 0.1-10:10-10000:1-5000; In method ①, when the bioactive molecule is a nucleic acid aptamer, the molar concentration ratio of the solution formed by the surface-enhanced Raman scattering nanoparticles without a covering molecular layer, the Raman reporter molecule, and the nucleic acid is 0.1-10:10-100000:1-5000; In method ①, when the bioactive molecule is serum, the molar concentration ratio of the solution formed by the surface-enhanced Raman scattering nanoparticles without a covering molecular layer to the Raman reporter molecule is 0.1-10:10-100000; the amount of serum used accounts for 10% of the volume of the total reaction system; The amount of sodium chloride used in method ① is calculated based on its final concentration in the reaction system being 0.3 mol / L; The molar concentration ratio of the solution formed by the surface-enhanced Raman scattering nanoparticles without a covering molecular layer described in method ② to the Raman reporter molecule is 0.1-10:10-5000; The molar concentration ratio of the nanoparticle-Raman reporter molecule solution to the biological cell membrane described in method ② is 2:

1.

9. Use of the surface-enhanced Raman scattering nanoparticles without a covering molecular layer according to any one of claims 1 to 4 and / or the surface-enhanced Raman scattering nanoparticle clusters according to any one of claims 6 to 8 in the preparation of Raman imaging materials or detection reagents.

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