Nucleic acid aptamer functionalized ultra-small luminescent gold nanoparticles, preparation method and application thereof

Abstract

This invention discloses nucleic acid aptamer-functionalized ultrasmall luminescent gold nanoparticles, their preparation method, and applications. The method involves: reacting an aqueous solution of chloroauric acid with an aqueous solution of glutathione ligand at room temperature until the reaction color becomes colorless; then adding a thioylated nucleic acid aptamer; followed by the addition of an aqueous solution of sodium borohydride; reacting until the solution turns brown; stopping the reaction; ultrafiltration; and finally, precipitating the nucleic acid aptamer-functionalized gold nanoparticles using an ethanol solution containing magnesium chloride and sodium chloride to remove free thioylated nucleic acid aptamers from the supernatant; redissolving the precipitate with buffer and water; and storing the concentrated precipitate at 4°C to obtain the nucleic acid aptamer-functionalized gold nanoparticle material. The product of this invention exhibits good biocompatibility, low toxicity, low fluorescence background interference, high detection sensitivity, and good selectivity. It is simple to operate for cell and tumor imaging research and can achieve non-invasive, high-resolution, and low-signal-to-background early diagnosis of in vivo tumors.

Description

Technical Field

[0001] This invention discloses a method for preparing nucleic acid aptamer-functionalized near-infrared II luminescent gold nanomaterials and their application in highly selective and sensitive diagnosis of tumor models. Background Technology

[0002] Currently, the incidence of malignant tumors is rising year by year, and the mortality rate remains high, seriously threatening human health and life. Highly sensitive and selective tumor diagnostic technologies have significant clinical implications, helping doctors to develop treatment plans early, evaluate treatment effectiveness, monitor tumor recurrence and metastasis, and assess prognosis. Therefore, there is an urgent need to develop highly sensitive and selective nanomaterials for cancer diagnostic technologies to meet clinical needs.

[0003] Nucleic acid aptamers, also known as chemical antibodies, are specialized oligonucleotide sequences characterized by their small size, low cost, low immunogenicity, ease of synthesis and modification, and ability to bind with high specificity and affinity to various targets. An increasing number of nucleic acid aptamer-functionalized nanomaterials are being used as nanoprobes in fluorescence imaging technology to enhance nanomaterial recognition, signal amplification, and cellular uptake, broadening their applications in biosensing, bioimaging, and targeted drug delivery. However, these nanoprobes face several challenges. Currently, contrast agents for bioimaging include traditional small molecule probes and conventional nanoprobes. Traditional small molecule probes are small in size and have the advantage of renal clearance, but suffer from low targeting efficiency; conventional nanoprobes are large in size and have the advantage of high targeting efficiency, but are difficult to excrete. Therefore, there is a need to develop novel probes with high efficiency targeting and renal clearance for highly sensitive and selective bioimaging of diseases. Luminescent metal nanoimaging probes are a type of probe that lies between metal atoms and metal nanoparticles. Luminescent metal nanoimaging probes possess unique optical properties, tunable surface chemistry, and renal clearance. They combine the advantages of high targeting efficiency and renal clearance. Therefore, modifying nucleic acid aptamers onto ultra-small luminescent gold nanoparticles will have a wider range of clinical application prospects. Summary of the Invention

[0004] To overcome the shortcomings and deficiencies of existing technologies, this invention provides a method for preparing nucleic acid aptamer-functionalized ultrasmall gold nanoparticles and their application in in vitro and in vivo tumor diagnosis. The nucleic acid aptamer-functionalized gold nanoparticles provided by this invention can specifically bind to protein receptors overexpressed on cancer cells, thereby improving tumor targeting efficiency. Therefore, these nucleic acid aptamer-functionalized ultrasmall luminescent gold nanoparticles can be used for highly sensitive and selective in vitro diagnosis of tumor cells. Due to their near-infrared luminescence, renal clearance, high targeting efficiency, and good selectivity, they can also be used for highly sensitive and selective in vivo diagnosis of tumor tissues. Furthermore, the fluorescence changes of the material in in vitro tumor cells and in vivo tumor tissues can be observed using laser confocal microscopy and near-infrared fluorescence imaging cameras.

[0005] The present invention can be achieved through the following technical solutions.

[0006] A method for preparing nucleic acid aptamer-functionalized gold nanoparticles includes the following steps:

[0007] Aqueous solutions of chloroauric acid and glutathione ligand were stirred at room temperature for half an hour. When the reaction color changed from yellow to colorless, thioylated nucleic acid aptamers were added and reacted for 10 minutes. Then, aqueous solutions of sodium borohydride were added and the mixture was stirred for 24 hours. When the solution color turned brown, the reaction was stopped. Unreacted substrate and excess glutathione were removed by ultrafiltration. Finally, the nucleic acid aptamer-functionalized gold nanoparticles were precipitated using an ethanol solution containing magnesium chloride and sodium chloride. Free thioylated nucleic acid aptamers in the supernatant were removed. The precipitate was redissolved in buffer and water, concentrated by ultrafiltration, and stored at 4°C to obtain nucleic acid aptamer-functionalized gold nanoparticle materials.

[0008] Furthermore: the thioylated nucleic acid aptamer is thioylated AS1411, which consists of two regions: the thiophosphate backbone region can serve as a precursor binding region, and the phosphate backbone region serves as a molecular recognition region. The nucleic acid aptamer is not limited to AS1411.

[0009] Furthermore, the reducing agent is sodium borohydride, but it is not limited to sodium borohydride.

[0010] Furthermore: in the reaction solution obtained by mixing chloroauric acid and thiol ligand, the initial molar ratio of chloroauric acid to glutathione is 1:0.5 to 1:1; while the molar ratio of thioacylated nucleic acid aptamer to glutathione is 1:30 to 1:5; when sodium borohydride is added, the initial molar ratio of chloroauric acid to sodium borohydride is 1:8 to 1:20.

[0011] Furthermore, the reaction solution pH is 8-12, the reaction temperature is 4-40℃, the reaction time is 6-40h, and the nucleic acid aptamer-functionalized gold nanoparticle material is monodisperse nanoparticles with a single gold nanoparticle size of 1.0-3.0nm.

[0012] Furthermore: the ultrafiltration and concentration processes utilize ultrafiltration tubes with membrane pore sizes of 3-50 kDa; the ultrafiltration and concentration centrifugation temperatures are 4-25°C; the centrifugation speeds are 2000-5000 rpm; and the centrifugation time is 10-30 min. The precipitation method uses ethanol concentrations of 35%-60%, sodium chloride concentrations of 100-1000 mM, and magnesium chloride concentrations of 10-50 mM.

[0013] Furthermore, the synthesized product can be monovalent nucleic acid aptamer-functionalized gold nanoparticles or multivalent nucleic acid aptamer-functionalized gold nanoparticles.

[0014] The method for preparing nucleic acid aptamer-functionalized gold nanoparticles for detecting tumor cells by the above preparation method includes the following steps: seeding cells at a density of 2000 per dish in a 35 mm imaging dish, culturing them in an incubator for 24 h, adding nucleic acid aptamer-functionalized gold nanoparticles to the cells after they have adhered to the wall, incubating at 37 °C for a period of time, and measuring the fluorescence intensity of the cells in the culture dish.

[0015] This invention applies the near-infrared luminescent nucleic acid aptamer-functionalized ultrasmall gold nanoparticle material to tumor cell imaging and in vivo tumor imaging. Compared with the unfunctionalized nucleic acid aptamer ultrasmall gold nanoparticle material, it has higher sensitivity and selectivity.

[0016] Furthermore: the nucleic acid aptamer-functionalized gold nanoparticle material emits light in the near-infrared region, the detection concentration is 10-200 nM, the detection time is 1-12 h, and the cell imaging environment is phenol red-free DMEM medium.

[0017] Furthermore, the cells being tested are 4T1 cells, but not limited to 4T1 cells.

[0018] Furthermore, the excitation wavelength range during detection is 250-550nm, and the emission wavelength range is 600-1200nm.

[0019] The process of studying the cell entry pathway of nucleic acid aptamer-functionalized gold nanoparticles prepared by the above method includes the following steps: After culturing cells with a cell density of 20%-30% for 24 hours, the cells are washed twice with DPBS. An inhibitory titer is added to one dish, and after incubation for a period of time, nucleic acid aptamer-functionalized gold nanoparticles are added. Nucleic acid aptamer-functionalized gold nanoparticles are added directly to another dish. After culturing in the culture medium for different times, the imaging of the material in the cells is observed using a confocal microscope, and the cells are collected and the uptake of the material by the cells at different time points is detected by inductively coupled plasma mass spectrometry.

[0020] Furthermore: the nucleic acid aptamer-functionalized gold nanoparticle material emits light in the near-infrared region, the detection concentration is 10-200 nM, the detection time is 1-12 h, and the cell imaging environment is phenol red-free DMEM medium.

[0021] Furthermore: the excitation wavelength range during detection is 250-550nm, the emission wavelength range is 600-1200nm, the cell imaging process is observed using a confocal imaging system, and the content of material taken up by cells is detected using an inductively coupled plasma mass spectrometer.

[0022] The method for preparing nucleic acid aptamer-functionalized gold nanoparticles by the above-described preparation method for detecting tumor tissue in vivo includes the following steps: intravenously injecting the nucleic acid aptamer-functionalized gold nanoparticles into female Balb / c mice inoculated with a skin tumor model, circulating the material in the blood for a period of time, and detecting the fluorescence changes in the tumor portion.

[0023] Furthermore, the nucleic acid aptamer-functionalized gold nanoparticle material emits light in the near-infrared region, and the detection concentration is 3.5-15 μM, with a detection time of 3-24 h.

[0024] Furthermore: the tumor being detected is a 4T1-inoculated breast tumor, but not limited to 4T1-inoculated breast tumors.

[0025] Furthermore: the excitation wavelength range during detection is 600-900 nm, the emission wavelength range is 900-1200 nm, and the power density range is 30-70 mW / cm². 2 The exposure time range is 0.1-5s.

[0026] The nucleic acid aptamer-functionalized ultrasmall gold nanoparticles synthesized in this invention have advantages such as good biocompatibility, low toxicity, near-infrared luminescence, renal clearance, high targeting efficiency, and good selectivity. They can be applied to tumor detection. At the same time, confocal microscopy and deep-cooled near-infrared cameras can clearly observe the distribution of tumor cells and tumor tissues. Therefore, the nucleic acid aptamer-functionalized ultrasmall gold nanoparticles have great application prospects in the fields of biosensing, bioimaging, and targeted drug delivery.

[0027] Compared with the prior art, the present invention has the following advantages and technical effects:

[0028] (1) The nucleic acid aptamer-functionalized ultrasmall gold nanoparticle material synthesized in this invention has a simple and convenient synthesis process and is easy to industrialize.

[0029] (2) The nucleic acid aptamer-functionalized ultrasmall gold nanoparticles synthesized in this invention have good biocompatibility, low toxicity, low fluorescence background interference, high detection sensitivity and good selectivity.

[0030] (3) The nucleic acid aptamer-functionalized ultra-small gold nanoparticle material synthesized in this invention emits light in the near-infrared region. It is simple to operate for cell and tumor imaging and can achieve non-invasive, high-resolution and low signal-to-back-ratio early diagnosis of tumors in vivo. Attached Figure Description

[0031] Figure 1 The spectrum of AS1411 functionalized gold nanoparticles synthesized in Example 1 is shown.

[0032] Figure 2 and Figure 3 This is a diagram illustrating the purification method of AS1411 functionalized gold nanoparticles in Example 1.

[0033] Figure 4 This is a stability diagram of the purified AS1411 functionalized gold nanoparticles in DPBS from Example 1.

[0034] Figure 5 This is a transmission electron microscope image of the purified AS1411 functionalized gold nanoparticles from Example 1.

[0035] Figure 6 This is a particle size distribution chart of the purified AS1411 functionalized gold nanoparticles from Example 1.

[0036] Figure 7 The image shows the hydrated particle size of the purified AS1411 functionalized gold nanoparticles from Example 1.

[0037] Figure 8 This is a 5% agarose gel electrophoresis image of the purified AS1411 functionalized gold nanoparticles from Example 2.

[0038] Figure 9 This is a diagram showing the specific hybridization and complementary pairing of purified AS1411 functionalized gold nanoparticles with complementary DNA in Example 3.

[0039] Figure 10 This is a cytotoxicity graph showing the toxicity of purified AS1411 functionalized gold nanoparticles at different concentrations in Example 4.

[0040] Figure 11 This is a graph showing the uptake of purified ASl411 functionalized gold nanoparticles by 4Tl cells in Example 4.

[0041] Figure 12 This is an analytical graph comparing the uptake of purified AS1411 functionalized gold nanoparticles by 4T1 cells and 293T cells in Example 4.

[0042] Figure 13 This is an image of 4T1 tumor cells detected by purified AS1411 functionalized gold nanoparticles in Example 5.

[0043] Figure 14 The image shows the detection of 4T1 tumor cells and 293T normal cells by purified AS1411 functionalized gold nanoparticles in Example 5.

[0044] Figure 15 The effect of the AS1411 inhibitor on the uptake of purified AS1411-functionalized gold nanoparticles by cells is shown in Example 5.

[0045] Figure 16 To implement in vivo 4T1 tumor imaging using six purified AS1411-functionalized gold nanoparticles.

[0046] Figure 17 To implement the synthesis method of 7 thioacylated AS1411 functionalized gold nanoparticles.

[0047] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0048] In the following specific embodiments, the chloroauric acid involved was purchased from Shanghai Myrele Chemical Technology Co., Ltd., and the glutathione was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; the DNA sequence was purchased from Shanghai Sangon Biotech Co., Ltd. 4T1 cells were purchased from ATCC, DMEM medium (gibico, catalog number: LOT8118192), and the DNA sequence is listed in Table 1. The instruments used for observing the properties of AS1411 functionalized gold nanoparticles, cell imaging, and in vivo tumor imaging analysis mainly included a Thermo Fisher Scientific Talos F200X transmission electron microscope, a PerkinElmer fluorescence / phosphorescence / luminescence spectrophotometer (LS-55), an Olympus confocal fluorescence imaging system, a DYY-8C gel electrophoresis system, a Thermo Scientific inductively coupled plasma mass spectrometer (iCAP RQ), and a photon ZephIR 1.7 deep-cooled infrared camera.

[0049] Table 1 Nucleic acid probe sequences

[0050]

[0051] Note: A* indicates that the phosphodiester backbone of deoxynucleotide A (adenine) is modified with thiophosphate.

[0052] Example 1

[0053] The synthesis of nucleic acid aptamer-functionalized gold nanoparticles according to this invention comprises the following steps: First, 900 μL of water, 21 μL of glutathione (24 mM), and 1 μL of chloroauric acid (500.8 mM) were added to a 2.0 mL centrifuge tube. After vigorous stirring at room temperature for 30 minutes, the solution became colorless, indicating the formation of the glutathione-Au(I) complex. Then, 2.7 μL of sodium hydroxide (1 M) was added to adjust the pH of the solution to 4-5. Next, AS411 was thioacylated in the solution, and the mixture was stirred for 10 minutes. Finally, 50 μL of sodium borohydride (200 mM) was added to the mixture under vigorous stirring, and the mixture was stirred continuously for 24 hours. The resulting solutions were transferred to ultrafiltration centrifuge tubes (Amicon, 10 kDa MWCO) and centrifuged at 3750 rpm for 6 cycles, 12 minutes each time, to remove excess GSH. Finally, the thioyl AS1411 functionalized gold nanoparticles were precipitated with 40% ethanol, 500 mM NaCl and 20 mM MgCl2 solution to remove free thioyl AS1411 from the supernatant. The precipitate was redissolved with buffer and water. The above steps were repeated 3 times, followed by ultrafiltration concentration and storage at 4°C to obtain purified nucleic acid aptamer functionalized gold nanoparticle materials.

[0054] The spectrum of the synthesized thioyl AS1411 functionalized gold nanoparticles is shown in the figure. Figure 1 As shown, by Figure 1 It can be seen that the synthesized thioyl AS1411 functionalized gold nanoparticles have strong fluorescence properties, with a maximum emission peak at 1030 nm.

[0055] The purification method of the purified thioyl AS1411 functionalized gold nanoparticles is as follows: Figure 2 and Figure 3 As shown, by Figure 2 and Figure 3 It is known that 40% ethanol, centrifuged and precipitated three times, can completely remove free thioyl AS1411 in thioyl AS1411 functionalized gold nanoparticles.

[0056] The stability of purified thioyl AS1411 functionalized gold nanoparticles in buffer solution is as follows: Figure 4 As shown, by Figure 4 As shown, the thioyl AS1411 functionalized gold nanoparticles maintain good stability in buffer solution.

[0057] Transmission electron microscopy image of purified thioyl ASl411 functionalized gold nanoparticles, as shown below. Figure 5 As shown, the synthesized thioylated AS1411 functionalized gold nanoparticles were statistically analyzed using particle size analysis software (such as Nano Measurer 1.2.5), and the results are as follows. Figure 6 As shown, its particle size is 1.0–2.5 nm.

[0058] Figure 7 The purified thioylated Asl411 functionalized gold nanoparticles have a hydrated particle size of 4.2 nm to 5.3 nm.

[0059] Example 2

[0060] The synthesis of nucleic acid aptamer-functionalized gold nanoparticles according to this invention comprises the following steps: First, 900 μL of water, 21 μL of glutathione (24 mM), and 1 μL of chloroauric acid (500.8 mM) were added to a 2.0 mL centrifuge tube. After vigorous stirring at room temperature for 30 minutes, the solution became colorless, indicating the formation of the glutathione-Au(I) complex. Then, 2.7 μL of sodium hydroxide (1 M) was added to adjust the pH of the solution to 4-5. Next, AS411 was thioacylated in the solution, and the mixture was stirred for 10 minutes. Finally, 50 μL of sodium borohydride (200 mM) was added to the mixture under vigorous stirring, and the mixture was stirred continuously for 24 hours. The resulting solutions were transferred to ultrafiltration centrifuge tubes (Amicon, 10 kDa MWCO) and centrifuged at 3750 rpm for 6 cycles, 12 minutes each time, to remove excess glutathione. Finally, the thioyl AS1411-functionalized gold nanoparticles were precipitated using 40% ethanol, 500 mM NaCl, and 20 mM MgCl2 solution to remove free thioyl AS1411 from the supernatant. The precipitate was redissolved in buffer and water, and the above steps were repeated three times. After ultrafiltration and concentration, the nanoparticles were stored at 4°C to obtain nucleic acid aptamer-functionalized gold nanoparticles. A set of 12 μL of purified thioyl AS1411-functionalized gold nanoparticles was taken, and 2 μL of 5 M NaCl, 2 μL of 200 mM MgCl2, and 4 μL of 1×TE buffer solution were added. Electrophoresis was performed on a 5% agarose gel with 0.25×TBE as the buffer system at 6 V / cm and 20 mA for 60–100 min. After electrophoresis, the gel was transferred to a live fluorescence imaging system. Fluorescence signals were collected using 365 nm excitation light and a filter of 800 nm or higher for an exposure time of 1 min to obtain a fluorescence image. Fluorescence signals were also collected using 365 nm excitation light and a non-emission filter for an exposure time of 1 s to obtain a bright-field image.

[0061] Figure 8 This image shows a 5% agarose gel electrophoresis image of the purified thioacylated AS1411 functionalized gold nanoparticles. Figure 8 In the image, 'a' represents a fluorescence imaging pattern. Figure 8 In the figure, b represents a bright-field imaging pattern. As can be seen from the figure, the thioylated AS1411 has been successfully functionalized on gold nanoparticles.

[0062] Example 3

[0063] The synthesis of nucleic acid aptamer-functionalized gold nanoparticles according to this invention comprises the following steps: First, 900 μL of water, 21 μL of glutathione (24 mM), and 1 μL of chloroauric acid (500.8 mM) were added to a 2.0 mL centrifuge tube. After vigorous stirring at room temperature for 15 minutes, the solution became colorless, indicating the formation of the glutathione-Au(I) complex. Then, 2.7 μL of sodium hydroxide (1 M) was added to adjust the pH of the solution to 4-5. Next, AS411 was thioacylated in the solution, and the mixture was stirred for 10 minutes. Finally, 50 μL of sodium borohydride (200 mM) was added to the mixture under vigorous stirring, and the mixture was stirred continuously for 24 hours. The resulting solutions were transferred to ultrafiltration centrifuge tubes (Amicon, 10 kDa MWCO) and centrifuged at 3750 rpm for 6 cycles, 12 minutes each time, to remove excess GSH. Finally, the thioyl AS1411-functionalized gold nanoparticles were precipitated using 40% ethanol, 500 mM NaCl, and 20 mM MgCl2 solution. Free thioyl AS1411 in the supernatant was removed, and the precipitate was redissolved in buffer and water. This process was repeated three times, followed by ultrafiltration concentration and storage at 4°C to obtain nucleic acid aptamer-functionalized gold nanoparticles. Multiple groups of 12 μL of purified thioyl AS1411-functionalized gold nanoparticles were taken, and 2 μL of 5 M NaCl, 2 μL of 200 mM MgCl2, and 4 μL of 1×TE buffer solution were added. The first group received no material, the second group received excess complementary DNA, and the third group received an equal amount of mismatched DNA. Electrophoresis was performed on a 5% agarose gel at 6 V / cm and 20 mA for 60–100 min using 0.25×TBE as the buffer system. After electrophoresis, the gel was transferred to a live fluorescence imaging system. Fluorescence signals were collected using 365 nm excitation light and a filter of 800 nm or higher for an exposure time of 1 min to obtain a fluorescence image. Fluorescence signals were also collected using 365 nm excitation light and a non-emission filter for an exposure time of 1 s to obtain a bright-field image.

[0064] Figure 9 This image shows a 5% agarose gel electrophoresis image of purified thioyl AS1411 functionalized gold nanoparticles after the addition of complementary DNA. Figure 9 In the image, 'a' represents a fluorescence imaging pattern. Figure 9 In the figure, b represents a bright-field imaging image. As can be seen from the figure, AS1411 functionalized on gold nanoparticles with thioyl AS1411 has good bioactivity and can completely hybridize and complement complementary DNA.

[0065] Example 4

[0066] This invention prepares nucleic acid aptamer-functionalized gold nanoparticles for studying cellular uptake of the material. The specific implementation scheme is as follows: 4T1 (mouse breast cancer cells) were seeded at 5 × 10⁴ cells / well in 24-well plates and cultured at 37°C for 24 hours. The adhered cells were then washed twice with DPBS. Thionyl-S1411 functionalized gold nanoparticles (500 μL, 30 nM) were added, and the cells were cultured at 37°C for 3 hours to allow endocytosis in both 4T1 and 293T cells. After gently pipetting the cells twice, the concentration of gold in the cells was measured using inductively coupled plasma mass spectrometry.

[0067] Using 4T1 cells as test cells, the toxicity of thioacylated AS1411 functionalized gold nanoparticles at different concentrations was studied using a CCK8 assay kit.

[0068] Figure 10 The figure shows the toxicity test results of thioyl AS1411 functionalized gold nanoparticles at different concentrations. The material is essentially non-toxic.

[0069] Figure 11 The figure shows the endocytosis efficiency of thioyl AS1411 functionalized gold nanoparticles on 4T1 cancer cells and 293T normal cells under the same incubation time. As shown in the figure, the endocytosis efficiency of 4T1 cancer cells is higher than that of 293T tumor cells, indicating that the thioyl AS1411 functionalized gold nanoparticles have selectivity and specificity for the endocytosis of tumor cells.

[0070] Figure 12 The results of the analysis of the endocytosis inhibition pathway of AS1411 inhibitor on thioylated AS1411-functionalized gold nanoparticles are shown in the figure. As shown, AS1411 reduced the endocytosis efficiency of 4T1 cells after incubation for 1 h and 3 h, indicating that the endocytosis of 4T1 cells with thioylated AS1411-functionalized gold nanoparticles is AS1411-mediated active targeted endocytosis.

[0071] Example 5

[0072] The implementation scheme for preparing nucleic acid aptamer-functionalized gold nanoparticles for imaging detection of tumor cells according to this invention is as follows: Cells seeded in confocal culture dishes with a cell density of 20%-30% were cultured for 24 hours, washed twice with DPBS, and then the prepared nanomaterials (200 nM) were added. After co-incubation at 37°C for 3 hours, excess material was washed away, and phenol red-free DMEM was added. Finally, the imaging of cells on the thioacylated AS1411-functionalized gold nanoparticles at 3 hours was observed using a confocal microscope.

[0073] Figure 13This indicates that the thioyl AS1411 functionalized gold nanoparticle material can be used to detect 4T1 cells.

[0074] Figure 14 This indicates that the thioylated AS1411 functionalized gold nanoparticles exhibit selectivity and specificity for the detection of 4T1.

[0075] Figure 15 This indicates that the endocytosis of thioyl AS1411-functionalized gold nanoparticles by 4T1 cells is an active targeted endocytosis mediated by AS1411.

[0076] Example 6

[0077] The implementation scheme for preparing nucleic acid aptamer-functionalized gold nanoparticles for in vivo tumor imaging detection according to the present invention is as follows: mice subcutaneously inoculated with 4T1 tumors are intravenously injected with thioylated AS1411 functionalized gold nanoparticles (300 μL, about 3.5 μM), and near-infrared imaging is performed at different time points after injection (e.g., 10 minutes, 6, 12 and 24 hours).

[0078] Figure 16 This indicates that thioyl AS1411 functionalized gold nanoparticles can be used to detect 4T1 tumors in vivo.

[0079] Example 7

[0080] The invention is designed as follows Figure 17 The thioyl AS1411 shown was synthesized by Shanghai Sangon Biotech Co., Ltd. Thioyl AS1411 consists of two regions: a thiophosphate backbone region that serves as a precursor binding region, and a phosphate backbone region that serves as a molecular recognition region. The synthetic principle of this invention is as follows: GSH is used as a co-ligand and mixed with HAuCl4 for 30 minutes to form a glutathione-gold(I) complex. Thioyl AS1411 is added to the reaction solution as a template for gold nanoparticle growth and stirred for 10 minutes. Under the conditions of sodium borohydride reduction and a pH of 9 throughout the reaction system, thioyl AS1411-functionalized AuNPs are synthesized.

[0081] Figure 17 This describes a method for synthesizing thioyl AS1411-functionalized ultrasmall luminescent gold nanoparticles.

Claims

1. A method for preparing nucleic acid aptamer-functionalized ultrasmall luminescent gold nanoparticles, characterized in that: The process includes the following: Aqueous solutions of chloroauric acid and glutathione ligand were stirred at room temperature until the reaction color changed from yellow to colorless. Thionylated nucleic acid aptamers were then added and stirred until the solution turned brown. The reaction was then stopped. Unreacted substrate and excess glutathione were removed by ultrafiltration. Finally, the nucleic acid aptamer-functionalized gold nanoparticles were precipitated using an ethanol solution containing magnesium chloride and sodium chloride. Free thioylated nucleic acid aptamers in the supernatant were removed. The precipitate was redissolved in buffer and water, concentrated by ultrafiltration, and stored at 4°C to obtain the nucleic acid aptamer-functionalized gold nanoparticle material.

2. The method for preparing nucleic acid aptamer-functionalized ultrasmall luminescent gold nanoparticles according to claim 1, characterized in that: The thioylated nucleic acid aptamer is thioylated AS1411, which consists of two regions: a thiophosphate backbone region that can serve as a precursor binding region and a phosphate backbone region that serves as a molecular recognition region. The nucleic acid aptamer is not limited to AS1411.

3. The method for preparing nucleic acid aptamer-functionalized ultrasmall luminescent gold nanoparticles according to claim 1, characterized in that: In the reaction solution obtained by mixing chloroauric acid and thiol ligand, the initial molar ratio of chloroauric acid to glutathione is 1:0.5 to 1:1; while the molar ratio of thioyl nucleic acid aptamer to glutathione is 1:30 to 1:5; when sodium borohydride is added, the initial molar ratio of chloroauric acid to sodium borohydride is 1:8 to 1:

20.

4. The method for preparing nucleic acid aptamer-functionalized ultrasmall luminescent gold nanoparticles according to claim 1, characterized in that: After adding sodium borohydride in the final step, the pH of the entire reaction solution is 8-12, the reaction temperature is 4-40℃, and the reaction time is 6-40 h; the nucleic acid aptamer-functionalized gold nanoparticle material is a monodisperse nanoparticle with a single gold nanoparticle having a particle size of 1.0-3.0 nm.

5. The method for preparing nucleic acid aptamer-functionalized ultrasmall luminescent gold nanoparticles according to claim 1, characterized in that: The ultrafiltration and concentration processes use ultrafiltration tubes with membrane pore sizes of 3-50 kDa, centrifugation temperatures of 4-25 °C, centrifugation speeds of 2000-5000 rpm, and centrifugation times of 10-30 min. The concentrations of ethanol used are 35%-60%, sodium chloride concentration is 100-1000 mM, and magnesium chloride concentration is 10-50 mM.

6. The preparation method according to any one of claims 1 to 5 yields thioylated nucleic acid aptamer-functionalized gold nanomaterials, characterized in that: The thioylated nucleic acid aptamer-functionalized gold nanomaterials are either monovalent nucleic acid aptamer-functionalized gold nanoparticles or multivalent nucleic acid aptamer-functionalized gold nanoparticles.

7. The thioylated nucleic acid aptamer-functionalized gold nanomaterial of claim 6 is used to detect tumor cells by recognizing protein ligands on the surface of tumor cells, characterized in that: Cells were seeded in imaging dishes and cultured in an incubator. After the cells adhered to the incubator, nucleic acid aptamer-functionalized gold nanoparticles were added to the cells. After incubation at 37°C for 3-12 h, the fluorescence intensity of the cells in the culture dish was measured. The nucleic acid aptamer-functionalized gold nanoparticle material emits light in the near-infrared region. The detection concentration is 10-200 nM, the detection time is 1-12 h, and the cell imaging environment is phenol red-free DMEM medium.

8. The application according to claim 7, characterized in that: The detection cells were 4T1 cells, and the excitation wavelength range during detection was 250-550 nm, while the emission wavelength range was 600-1200 nm.

9. The application according to claim 7, characterized in that: The nucleic acid aptamer-functionalized gold nanoparticle material emits light in the near-infrared region. The detection concentration is 3.5-15 μM, and the detection time is 3-24 h.

10. The application according to claim 7, characterized in that: The detection tumor is 4T1 inoculated breast tumor, the detection is excited wavelength range of 600 - 900 nm, emission wavelength range is 900- 1200 nm, power density range is 30-70 mW / cm 2 , exposure time range is: 0.1 - 5 s.

Images