Near-infrared silver nanocluster, preparation method, application and experimental method

Abstract

The invention relates to the technical field of biomedicine, in particular to a near-infrared silver nanocluster, a preparation method, application and an experimental method.Reductive lipoic acid and the silver nanocluster are combined, a novel near-infrared fluorescent probe is synthesized through a microwave method, and rapid and accurate quantitative detection on hydrogen peroxide can be achieved; meanwhile, the probe has the advantages of high-sensitivity fluorescence signals and small autofluorescence interference, and can be well applied to detection of hydrogen peroxide in cells; compared with a fluorescent probe in a visible light range, the near-infrared fluorescent probe can avoid the problems of self-fluorescence interference, low tissue penetrability, low sensitivity and background fluorescence interference, and can also reduce errors caused by substrate concentration; when the near-infrared silver nanocluster is used for hydrogen peroxide detection, the technical problems that the existing detection method is weak in tissue penetrability, is easily influenced by background fluorescence and is relatively large in self-fluorescence interference are solved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and in particular to a near-infrared silver nanocluster, a preparation method, an application, and an experimental method. Background Art

[0002] With the rapid development of technology, the quality of human life has been significantly improved. However, cancer, as a major health threat globally, still has a high incidence and mortality rate. According to statistics, there were as many as 19.3 million newly diagnosed cancer cases globally in 2020, and approximately 10 million people died from it. It is predicted that by 2050, the number of cancer patients globally will exceed 28.4 million, with a rise of up to 47%. Therefore, strengthening scientific research on cancer prevention and treatment and improving early detection and diagnosis technologies for cancer are crucial for reducing the harm of cancer.

[0003] In an organism, the balance of reactive oxygen species (ROS) is crucial for the normal progress of life activities. Among them, hydrogen peroxide (H 2 O 2 ) is an important ROS substance, and its concentration change is closely related to the occurrence and development of cancer. The level of H 2 O 2 in cancer cells is usually higher than that in normal cells. Therefore, designing a method that can highly sensitively and selectively detect the content of H 2 O 2 in an organism is of great significance for the early screening of cancer.

[0004] Currently, existing methods for detecting H 2 O 2 include near-infrared analysis, chemical titration analysis, chemiluminescence, spectrophotometry, fluorescence analysis, electrochemistry, etc. Among them, fluorescence analysis is widely used in the detection of hydrogen peroxide due to its advantages such as high sensitivity, low detection limit, and high selectivity.

[0005] Traditional fluorescence analysis fixes the excitation light at a certain wavelength. In the reaction system, as different concentrations of H 2 O 2 are added, the fluorescence signal of the product changes (which can be detected by a fluorescence spectrophotometer). According to the quantitative relationship between fluorescence intensity and concentration, the concentration of H 2 O 2 is calculated. This method has high sensitivity, low detection limit, and high selectivity, and is currently a commonly used method for detecting hydrogen peroxide. Zou Yun et al.

[28] developed a new method for determining the residual amount of hydrogen peroxide in hydroponic foods based on fluorescence analysis. This method relies on the Fenton reaction, that is, in an acidic environment, specific Fe(II) ions react with hydrogen peroxide (H 2 O 2)The reaction generates hydroxyl radicals. These hydroxyl radicals then undergo a hydroxylation reaction with benzoic acid in an alkaline medium to form hydroxybenzoic acid with strong fluorescence. However, benzoic acid itself hardly produces fluorescence in fluorescence analysis. Therefore, by measuring the increase in fluorescence intensity after the reaction, the content of hydrogen peroxide can be indirectly quantified.

[0006] However, traditional fluorescence analysis methods have problems such as weak tissue penetration, susceptibility to background fluorescence, and large autofluorescence interference. Summary of the Invention

[0007] The purpose of the present invention is to provide a near-infrared silver nanocluster, a preparation method, an application, and an experimental method, aiming to solve the technical problems in the existing fluorescence analysis methods, such as weak tissue penetration, susceptibility to background fluorescence, and large autofluorescence interference.

[0008] To achieve the above purpose, a preparation method of a near-infrared silver nanocluster adopted by the present invention includes the following steps:

[0009] Step 1: Add 150 μL of a NaOH solution with a concentration of 1 M to 5 mL of a thiolic acid solution with a concentration of 30 mM, and stir well for 10 min until completely dissolved to obtain a mixed solution A for standby;

[0010] Step 2: Add 0.5 - 5 mL of an AgNO 3 solution with a concentration of 10 mM to the mixed solution A. Subsequently, under rapid stirring, slowly dropwise add 150 μL of a NaBH 4 solution with a concentration of 1 M to obtain a mixed solution B;

[0011] Step 3: Place the mixed solution B in a microwave oven, set the temperature and time of the microwave oven to 20 - 100 °C and 10 - 50 s. When the color of the mixed solution B turns black-brown and wait for the solution to cool to room temperature, and the mixed solution B turns bright brown, the near-infrared silver nanocluster can be obtained.

[0012] Among them, after the near-infrared silver nanocluster is prepared, it should be stored for standby at 4 °C.

[0013] The present invention also provides a near-infrared silver nanocluster prepared by the preparation method of the near-infrared silver nanocluster as described above.

[0014] The present invention also provides an experimental method for the near-infrared silver nanocluster, which is used to conduct experiments on the near-infrared silver nanocluster as described above.

[0015] Conducting experiments on the near-infrared silver nanocluster includes temperature condition experiments, time condition experiments, volume ratio condition experiments, morphology characterization detection, sensitivity detection to H 2 O 2 and anti-interference experiments.

[0016] Among them, the specific method of the temperature condition experiment is as follows:

[0017] By setting the temperature of the microwave oven to 20 - 100 °C, five different temperatures of 20 °C, 40 °C, 60 °C, 80 °C, and 100 °C are selected. Keeping other conditions unchanged, five different solutions are obtained through the change of temperature during the microwave method. The most suitable temperature is obtained by observing the fluorescence intensity and testing the fluorescence spectrum under natural light and ultraviolet light respectively;

[0018] The specific method of the time condition experiment is as follows:

[0019] By setting the time of the microwave oven to 10 - 50 s, five different times of 10 s, 20 s, 30 s, 40 s, and 50 s are selected. Keeping other conditions unchanged, five different solutions are obtained through the change of time during the microwave method. The most suitable time is obtained by observing the fluorescence intensity and testing the fluorescence spectrum under natural light and ultraviolet light respectively;

[0020] The specific method of the volume ratio condition experiment is as follows:

[0021] By preparing 5 mL of DHLA solution, and then adding different volumes of AgNO 3 solution to obtain DHLA - Ag NCs with different ratios. In the experiment, five different volume ratios of DHLA - AgNCs solutions of 5:0.5, 5:1, 5:2, 5:3, and 5:5 are set. Five solutions are obtained by heating through the microwave method. The most suitable volume ratio is obtained by observing the fluorescence intensity and testing the fluorescence spectrum under natural light and ultraviolet light respectively.

[0022] Among them, the specific method of the fluorescence spectrum test is as follows:

[0023] Add 2 ml of near - infrared silver nanocluster solution into a quartz cuvette, and place the cuvette in the sample chamber of a fluorescence spectrophotometer; Set the fluorescence spectrum test conditions: both the incident and exit slits are 10 nm, and the gain is medium; Select different modes to find the best excitation wavelength of the material. The best excitation wavelength range of this material is between 460 - 470 nm. Then, in the emission mode, set the best excitation, and the emission wavelength scanning range of NRI - Ag NCs is 500 - 800 nm;

[0024] At the same time, conduct the fluorescence stability test:

[0025] The test conditions are set as follows: the scanning mode is set to time scanning mode, the scanning time is 600 s, the incident slit and the exit slit are both 10 nm, and the gain is medium; the excitation and emission wavelengths of NRI-Ag NCs are set to 468 nm and 657 nm respectively; the above experimental operations are repeated 3 times, and the obtained spectra are analyzed and compared.

[0026] Among them, the method for morphological characterization detection is:

[0027] The transmission electron microscopy technology is adopted. Through the analysis of the transmission electron microscope, the morphological characteristics of these nanomaterials can be clearly depicted, and their particle size can be accurately measured.

[0028] Among them, the specific method for sensitivity detection is:

[0029] First, prepare H 2 O 2 with a concentration of 100 mM, and then dilute it to different multiples to obtain H 2 O 2 with a concentration of 1 - 50 mM, and store it at 4 °C for later use;

[0030] After that, add different concentrations of H 2 O 2 to 2 mL of NRI-Ag NCs solution, and use a pipette to blow and stir to make it react fully. Then place it under ultraviolet light to observe the fluorescence change, and then use a fluorescence spectrophotometer to record the change of the fluorescence spectrum, and statistically compare the obtained spectra and corresponding data.

[0031] Among them, the specific method for the anti-interference experiment is:

[0032] The concentration of H 2 O 2 is 100 mM, and the interfering substances are Ala, Cys, Glu, Leu, Tyr, Trp, AA, GSH, NaCl, Ser, Asp, and the concentration of all interfering substances is 100 mM;

[0033] Use a fluorescence spectrophotometer to characterize the fluorescence emission spectrum of NRI-Ag NCs with different interfering substances, and compare it with the fluorescence emission spectrum of NRI-Ag NCs after adding H 2 O 2 ;

[0034] Scan all fluorescence spectra at a slit width of 10 nm at 468 nm; the above steps are all carried out at room temperature, and all tests are repeated at least 3 times.

[0035] As described above, the near-infrared silver nanoclusters in the early detection of cancer H 2 O 2Application in Visual Detection

[0036] A near-infrared silver nanocluster, preparation method, application and experimental method of the present invention combine reducing lipoic acid with silver nanoclusters. By microwave method, a novel near-infrared fluorescent probe can be synthesized to realize rapid and accurate quantitative detection of hydrogen peroxide. At the same time, this probe has the advantages of high-sensitivity fluorescence signal and small autofluorescence interference, and can be well applied to the detection of hydrogen peroxide in cells. Compared with fluorescent probes in the visible light range (300 - 550 nm), the use of near-infrared fluorescent probes can avoid problems such as autofluorescence interference, low tissue penetration, low sensitivity, and background fluorescence interference, and can also reduce errors caused by substrate concentration. At the same time, the fluorescence color change of near-infrared fluorescent probes is more obvious than that of single-emission fluorescent probes, and visual detection can be carried out. Finally, hydrogen peroxide is quantitatively detected by fluorescence analysis method. Using the near-infrared silver nanoclusters of the present invention for hydrogen peroxide detection solves the technical problems existing in the existing detection methods, such as weak tissue penetration, being easily affected by background fluorescence, and large autofluorescence interference. Brief Description of the Drawings

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following described drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0038] Figure 1 It is the excitation (Ex) and emission (Em) spectrogram of the NRI-Ag NCs of the present invention.

[0039] Figure 2 It is the schematic diagram of spectral characterization of the present invention under different excitation lights in the range of 440 - 490 nm.

[0040] Figure 3 It is the fluorescence time-course curve of the NRI-Ag NCs of the present invention.

[0041] Figure 4 It is the solution diagram of the NRI-Ag NCs of the present invention under visible light (left) and ultraviolet light (right) and the NRI-Ag NCs added with H 2 O 2 of the NRI-Ag NCs.

[0042] Figure 5 In which A is the schematic diagram of the fluorescence spectrum of the materials obtained under different temperature conditions of the present invention, Figure 5 In which B is the schematic diagram of the materials prepared under different temperature conditions of the present invention under visible light (upper) and ultraviolet light (lower).

[0043] Figure 6 In Figure A, it is the fluorescence spectrum schematic diagram of the materials obtained under different time conditions of the present invention. Figure 6 In Figure B, it is the schematic diagram of the materials prepared under different time conditions of the present invention under visible light (upper) and ultraviolet light (lower).

[0044] Figure 7 In Figure A, it is the fluorescence spectrum schematic diagram of the materials obtained under different volume ratio conditions of the present invention. Figure 7 In Figure B, it is the schematic diagram of the materials prepared under different volume ratio conditions of the present invention under visible light (upper) and ultraviolet light (lower).

[0045] Figure 8 In Figure A, it is the particle size distribution and Gaussian fitting curve of NRI-Ag NCs of the present invention. Figure 8 In Figure B, it is the TEM image of NRI-Ag NCs of the present invention and the corresponding lattice image.

[0046] Figure 9 It is the response schematic diagram of NRI-Ag NCs of the present invention to different concentrations of H 2 O 2

[0047] Figure 10 It is the relationship diagram between the concentration of H 2 O 2 of the present invention and the fluorescence ratio (F / F 0 ).

[0048] Figure 11 It is the response schematic diagram of NRI-Ag NCs of the present invention to H 2 O 2 and different interferents.

[0049] Figure 12 It is the bar chart of the fluorescence peak ratio (F / F 2 O 2 and the response of NRI-Ag NCs to each interferent. 0 )

[0050] Figure 13 It is the fluorescence image of NRI-Ag NCs under ultraviolet light after adding H 2 O 2 and each interferent of the present invention. Detailed Description of the Specific Embodiment

[0051] The embodiments of the present invention will be described in detail below. The examples of the embodiments are shown in the drawings. The embodiments described below by referring to the drawings are exemplary and are intended to explain the present invention and should not be construed as limiting the present invention.

[0052] ​The present invention provides a near-infrared silver nanocluster, a preparation method, an application and an experimental method, including the following steps:

[0053] Step 1: Add 150 μL of a NaOH solution with a concentration of 1 M to 5 mL of a lipoic acid solution with a concentration of 30 mM, and stir well for 10 min until completely dissolved to obtain a mixed solution A for standby;

[0054] Step 2: Add 0.5 - 5 mL of an AgNO 3 solution with a concentration of 10 mM thereto. Subsequently, under rapid stirring, slowly add 150 μL of a NaBH 4 solution to obtain a mixed solution B;

[0055] Step 3: Place the mixed solution B in a microwave oven, set the temperature and time of the microwave oven to 20 - 100 °C and 10 - 50 s. The color of the mixed solution B turns black-brown. Wait for the solution to cool to room temperature, and the mixed solution B turns bright brown, then the near-infrared silver nanocluster can be obtained.

[0056] Among them, after the near-infrared silver nanocluster is prepared, it should be stored for standby at 4 °C.

[0057] Please refer to Figures 1 to 4 , Figure 1 which is the excitation (Ex) and emission (Em) spectrogram of the NRI-Ag NCs of the present invention. Figure 2 which is the schematic diagram of the spectral characterization of the present invention under different excitation lights in the range of 440 - 490 nm. Figure 3 which is the fluorescence time course curve of the NRI-Ag NCs of the present invention. Figure 4 which is the solution diagram of the NRI-Ag NCs of the present invention under visible light (left) and ultraviolet light (right) and the NRI-Ag NCs added with H 2 O 2

[0058] The present invention also provides a near-infrared silver nanocluster prepared by using the preparation method of the near-infrared silver nanocluster as described above.

[0059] This probe is prepared by a green and environmentally friendly microwave method with lipoic acid as a stabilizer and NaBH 4 as a reducing agent. At a suitable temperature and a specific pH, through the stabilizing effect of lipoic acid, electrons are efficiently transferred from NaBH 4 to Ag + in the solution, reducing it to silver nanoparticles. This method has a fast reaction rate and is easy to operate.

[0060] Spectral characterization was carried out on the NRI-Ag NCs to explore their fluorescence characteristics. From Figure 1 ​It can be obtained that the optimal excitation wavelength of NRI-Ag NCs is 468 nm, and the optimal emission wavelength is 657 nm at the optimal excitation wavelength. At the same time, the present invention explores the influence of different excitation lights on the fluorescence value of NRI-I Ag NCs, and six different excitation wavelengths from 440 to 490 nm are selected for exploration, and it is found that the fluorescence value is optimal only at 468 nm( Figure 2 ). The material is continuously scanned, and the fluorescence intensity is stable at about 2000 a.u. Within 600 s, the change in the fluorescence intensity of the probe itself can be ignored. After adding H 2 O 2 , the fluorescence intensity also remains stable within 600 s( Figure 3 ), indicating that the prepared NRI-Ag NCs have excellent fluorescence stability and are not easily affected by the external environment. Under natural light, the NRI-Ag NCs are brownish-yellow. After adding H 2 O 2 , the color of the solution changes significantly to black. Under ultraviolet light irradiation, the NRI-Ag NCs exhibit bright pink fluorescence, while the fluorescence of the solution after adding H 2 O 2 is significantly quenched, and the solution appears black( Figure 4 ).

[0061] Please refer to Figures 5 to 13 , Figure 5 where A is a schematic diagram of the fluorescence spectrum of the material obtained under different temperature conditions of the present invention, Figure 5 and B is a schematic diagram of the material prepared under different temperature conditions of the present invention under visible light (above) and ultraviolet light (below). Figure 6 where A is a schematic diagram of the fluorescence spectrum of the material obtained under different time conditions of the present invention, Figure 6 and B is a schematic diagram of the material prepared under different time conditions of the present invention under visible light (above) and ultraviolet light (below). Figure 7 where A is a schematic diagram of the fluorescence spectrum of the material obtained under different volume ratio conditions of the present invention, Figure 7 and B is a schematic diagram of the material prepared under different volume ratio conditions of the present invention under visible light (above) and ultraviolet light (below). Figure 8 where A is the particle size distribution and Gaussian fitting curve of NRI-Ag NCs of the present invention, Figure 8 and B is the TEM image of NRI-Ag NCs of the present invention and the corresponding lattice image. Figure 9 is a schematic diagram of the response of NRI-Ag NCs of the present invention to different concentrations of H 2 O 2 . Figure 10 is the concentration of H 2 O 2 of the present invention and the fluorescence ratio (F / F0 Relationship diagram of Figure 11 This is the response of the NRI-AgNCs of the present invention to H 2 O 2 And the response schematic diagram of different interferents. Figure 12 This is the response of the NRI-Ag NCs of the present invention to H 2 O 2 And the fluorescence peak ratio (F / F 0 ) bar chart of the response of each interferent. Figure 13 This is the fluorescence image of the NRI-Ag NCs under ultraviolet light after adding H 2 O 2 And each interferent.

[0062] The present invention also provides an experimental method for near-infrared silver nanoclusters, which is used to conduct experiments on the near-infrared silver nanoclusters as described above.

[0063] Conducting experiments on the near-infrared silver nanoclusters includes temperature condition experiments, time condition experiments, volume ratio condition experiments, morphology characterization detection, sensitivity detection to H 2 O 2 And anti-interference experiments.

[0064] The specific method of the temperature condition experiment is:

[0065] By setting the temperature of the microwave oven to 20 - 100 °C, five different temperatures of 20 °C, 40 °C, 60 °C, 80 °C, and 100 °C can be selected. Keeping other conditions unchanged, five different solutions are obtained by changing the temperature during the microwave method. By observing the fluorescence intensity under natural light and ultraviolet light respectively and testing the fluorescence spectrum, the most suitable temperature is obtained;

[0066] For this specific embodiment, when controlling other variables unchanged, fluorescence probes at 20 °C, 40 °C, 60 °C, 80 °C, and 100 °C were prepared successively using the microwave method. Please refer to Figure 5 Figure A, and it is concluded that the fluorescence effect of the fluorescence probe prepared at 60 °C is significantly higher than that of the materials prepared at other temperatures.

[0067] It can also be seen from under the ultraviolet light that the fluorescence effect of the material at 60 °C is better than that of other temperatures, and the color of the solution of the material prepared at 60 °C is also brighter than that of the solutions at other temperatures under visible light (refer to Figure 5 Figure B).

[0068] Therefore, the present invention selects 60 °C as the optimal temperature for preparing this probe, and the fluorescence value of the material obtained at this temperature is significantly higher than that of the materials obtained at other temperatures.

[0069] The specific method of the time condition experiment is:

[0070] By setting the time of the microwave oven to 10 - 50 s, five different times of 10 s, 20 s, 30 s, 40 s, and 50 s can be selected. Keeping other conditions unchanged, five different solutions are obtained by varying the time during the microwave method. The most suitable time is obtained by observing the fluorescence intensity and measuring the fluorescence spectrum under natural light and ultraviolet light respectively;

[0071] For this specific embodiment, when controlling other variables unchanged, fluorescent probes were prepared successively using the microwave method at times of 10 s, 20 s, 30 s, 40 s, and 50 s. It is found that the material prepared at a microwave time of 30 s has a fluorescence value significantly higher than that of the materials prepared at other times (see Figure 6 A).

[0072] It can also be seen from under the ultraviolet light that the fluorescence effect of the fluorescent probe prepared at 30 s is better than that of other times, and the color of the solution of this probe at 30 s is also lighter than that of the solutions at other times under visible light, and the solution is more transparent (see Figure 6 B).

[0073] Therefore, the present invention selects 30 s as the optimal time for preparing this probe, and the fluorescence value of the material obtained at this time is significantly higher than that of the materials obtained at other times.

[0074] The specific method for the volume ratio condition experiment is as follows:

[0075] By preparing 5 mL of DHLA solution, and then adding different volumes of AgNO 3 solution to obtain DHLA - Ag NCs with different ratios. In the experiment, five different volume ratios of DHLA - AgNCs solutions of 5:0.5, 5:1, 5:2, 5:3, and 5:5 are set. Five solutions are obtained by heating through the microwave method, and the most suitable volume ratio is obtained by observing the fluorescence intensity and measuring the fluorescence spectrum under natural light and ultraviolet light respectively.

[0076] For this specific embodiment, when controlling other variables unchanged, fluorescent probes with volume ratios of DHLA to silver nitrate of 5:0.5, 5:1, 5:2, 5:3, and 5:5 are prepared successively using the microwave method. It can be concluded that when the volume ratio is 5:2, the fluorescence value of the material is significantly higher than that of other volume ratios (see Figure 7 A).

[0077] It can also be seen from under the ultraviolet light that the fluorescence effect of the fluorescent probe prepared at a volume ratio of 5:2 is better than that of other volume ratios, and the color of the solution with a volume ratio of 5:2 is also different from that of other volume ratios under visible light (see Figure 7 B). It can also be seen that as AgNO 3The volume is increasing, the color of the solution is getting darker, and the fluorescence intensity under ultraviolet light is getting weaker. This may be due to the excessive reduction of Ag.

[0078] Therefore, the present invention selects the condition that the volume ratio of DHLA to silver nitrate is 5:2 as the optimal volume ratio for preparing the probe. The fluorescence value of the material obtained at this volume ratio is significantly higher than that of the materials obtained at other volume ratios.

[0079] The method for morphological characterization detection is as follows:

[0080] The transmission electron microscopy technique is adopted. Through transmission electron microscopy analysis, the morphological characteristics of these nanomaterials can be clearly depicted, and their particle size can be accurately measured.

[0081] For this specific embodiment, to investigate the morphology and size of NRI-Ag NCs, the high-magnification scanning images are all characterized by TEM. As Figure 8 shown in Figure B, the NRI-Ag NCs particles have good dispersibility and uniform particle size. The high-resolution image shows that the lattice of NRI-Ag NCs is obvious, and the lattice spacing is 0.22 nm. 186 NRI-Ag NCs particles are randomly selected. After statistics, it is calculated that the particle size of this probe is distributed in the range of 2.0 - 2.8 nm, and the average particle size is about 2.42 nm (see Figure 8 Figure A).

[0082] The specific method for sensitivity detection is as follows:

[0083] First, prepare H 2 O 2 with a concentration of 100 mM, and then dilute it to different multiples to obtain H 2 O 2 with different concentrations (1 mM, 5 mM, 10 mM, 50 mM), and store it at 4 °C for later use;

[0084] After that, add H 2 O 2 with different concentrations to 2 mL of NRI-Ag NCs solution, and use a pipette to blow and stir to make it react fully. Then place it under ultraviolet light to observe the fluorescence change, and use a fluorescence spectrophotometer to record the change of the fluorescence spectrum, and statistically compare the obtained spectra and corresponding data.

[0085] For this specific embodiment, add H 2 O 2 with different concentrations to the NRI-Ag NCs system and observe the change of the emission peak to explore the relationship between the concentration of H 2 O 2 and the fluorescence ratio (F / F 0 ). AsFigure 9 As shown, with the increase in the concentration of H 2 O 2 added to the NRI-Ag NCs system, the fluorescence intensity gradually decreases until the concentration of H 2 O 2 increases to 9 mM, and the fluorescence is almost completely quenched. The reason is that the reducing property of H 2 O 2 over-reduces Ag + to Ag. Due to the change in particle size, the particle size of Ag becomes larger and larger, resulting in fluorescence quenching. A large number of experimental data show that within a certain concentration range of H 2 O 2 , F / F 0 has a good linear relationship with the concentration of H 2 O 2 (see Figure 10 ): F / F0 = -0.403X + 0.478, and the linear correlation coefficient R 2 = 0.99. By fitting, the lowest detection limit of this probe is 2.5 μM, ranging from 2.5 μM to 9 mM.

[0086] The specific method of the anti-interference experiment is as follows:

[0087] The concentration of H 2 O 2 is 100 mM, and the interfering substances are Ala, Cys, Glu, Leu, Tyr, Trp, AA, GSH, NaCl, Ser, Asp. The concentration of all interfering substances is 100 mM;

[0088] Use a fluorescence spectrophotometer to characterize the fluorescence emission spectra of NRI-Ag NCs with different interfering substances, and compare them with the fluorescence emission spectra of NRI-Ag NCs after adding H 2 O 2 ;

[0089] Scan all fluorescence spectra at a slit width of 10 nm at 468 nm; The above steps are all carried out at room temperature, and all tests are repeated at least 3 times.

[0090] For this specific embodiment, different interfering substances are added to the NRI-Ag NCs system to observe the change of the emission peak to explore the anti-interference ability of this probe. There are a total of 11 interfering substances, namely Ala, Cys, Glu, Leu, Tyr, Trp, AA, GSH, NaCl, Ser, Asp. The concentrations of the interfering substances and H 2 O 2 are both 100 mM. As Figure 11 shown, the fluorescence will slightly decrease after adding the interfering substances to the NRI-Ag NCs system, but after adding H2 O 2 The fluorescence will decrease significantly later, and Figure 12 it can be seen from 0 that the fluorescence ratio F / F is around 0.8 after adding the interferent, while after adding H 2 O 2 the fluorescence ratio F / F 0 will decrease significantly to around 0.6. Figure 13 Figure shows the fluorescence images after adding H 2 O 2 and different interferents under ultraviolet light. It can be clearly seen that the fluorescence will be significantly quenched after adding H 2 O 2 in the system, which forms a distinct contrast with other interferents.

[0091] Therefore, it can be concluded that NRI-AgNCs only respond to the target analyte H 2 O 2 and can specifically recognize H 2 O 2 ,

[0092] that is, this material has good anti-interference performance.

[0093] The experimental reagents for the experimental method of near-infrared silver nanoclusters are shown in Table 1:

[0094] Table 1

[0095]

[0096] The resistivity of the ultrapure water required for the experiment is 18.2 MΩ·cm.

[0097] The experimental instruments for the experimental method of near-infrared silver nanoclusters are shown in Table 2:

[0098] Table 2

[0099]

[0100]

[0101] Using a near-infrared silver nanocluster, preparation method, application and experimental method of this embodiment, combining reducing lipoic acid with silver nanoclusters, and synthesizing a novel near-infrared fluorescent probe by microwave method can realize rapid and accurate quantitative detection of hydrogen peroxide. At the same time, this probe has the advantages of high-sensitivity fluorescence signal and small autofluorescence interference, and can be well applied to the detection of hydrogen peroxide in cells. Compared with fluorescent probes in the visible light range (300 - 550 nm), using near-infrared fluorescent probes can avoid problems such as autofluorescence interference, low tissue penetration, low sensitivity, and background fluorescence interference, and can also reduce errors caused by substrate concentration. At the same time, the near-infrared fluorescent probe has a more obvious fluorescence color change than single-emission fluorescent probes, and can be used for visual detection. Finally, hydrogen peroxide is quantitatively detected by fluorescence analysis method. Using the near-infrared silver nanocluster of the present invention for hydrogen peroxide detection solves the technical problems of weak tissue penetration, susceptibility to background fluorescence, and large autofluorescence interference existing in existing detection methods.

[0102] The present invention prepared NRI-AgNCs with pink fluorescence by microwave method, explored the optimal preparation conditions of this probe through condition optimization experiments, and obtained that the material prepared when the volume ratio is 5:2, the temperature is 60 °C, and the time is 30 s has the best fluorescence effect. Fluorescence spectrum results show that the best emission wavelength of NRI-Ag NCs at the optimal excitation wavelength of 468 nm is 657 nm, and presents pink fluorescence.

[0103] Since H 2 O 2 can quench the fluorescence of this probe, it is feasible to use NRI-Ag NCs as a near-infrared silver nanocluster fluorescent probe for the detection of H 2 O 2 . The final results show that the lowest detection limit of NRI-Ag NCs for H 2 O 2 is 2.5 μM, the linear range is 2.5 μM - 9 mM, and the fitting degree is R 2 = 0.99.

[0104] The prepared fluorescent probe is used for the detection of H 2 O 2 , which provides feasibility for the application of near-infrared fluorescent probes in the fields of biomedical detection and imaging.

[0105] In summary, the NRI-Ag NCs fluorescent probe has a good response to H 2 O 2 , and the linear range of detection can just detect the H 2 O 2 concentration produced by cancer cells, and can just quench the fluorescence of the fluorescent probe. Therefore, this near-infrared first-region fluorescent probe can detect H2 O 2 has great application prospects.

[0106] Compared with the existing preparation methods of metal nanoclusters, the near-infrared metal nanoclusters are prepared by the microwave method, which has a shorter preparation time (30 s) and simpler preparation materials.

[0107] The above-disclosed is only a preferred embodiment of the present invention. Of course, the scope of the rights of the present invention cannot be limited thereby. Those of ordinary skill in the art can understand all or part of the processes of implementing the above embodiments, and the equivalent changes made according to the claims of the present invention still fall within the scope covered by the invention.

Claims

1. A method for preparing near-infrared silver nanoclusters, characterized in that: The steps include: Step 1: Add 150 μL of 1M NaOH solution to 5mL of 30mM lipoic acid solution, stir for 10 minutes until completely dissolved, and obtain a mixed solution A for later use; Step 2: Add 0.5-5 mL of 10 mM AgNO3 solution to the mixed solution A, and then slowly add 150 μL of 1 M NaBH4 solution under rapid stirring to obtain a mixed solution B; Step 3: Place the mixed solution B in a microwave oven, set the temperature and time of the microwave oven to 20-100°C and 10-50s, the color of the mixed solution B changes to dark brown, wait for the solution to cool to room temperature, and the mixed solution B changes to bright brown, and near-infrared silver nanoclusters are obtained.

2. The method for preparing near-infrared silver nanoclusters according to claim 1, characterized in that: After the near-infrared silver nanoclusters are prepared, they should be stored at 4°C for future use.

3. A near-infrared silver nanocluster, characterized in that: The near-infrared silver nanoclusters are prepared by the preparation method of claim 2.

4. A near-infrared silver nanocluster experimental method, used for conducting an experiment on the near-infrared silver nanocluster as claimed in claim 3, characterized in that: The experiments conducted on the near-infrared silver nanoclusters include temperature condition experiments, time condition experiments, volume ratio condition experiments, morphology characterization tests, sensitivity tests to H2O2 and anti-interference experiments.

5. The experimental method of near-infrared silver nanoclusters as claimed in claim 4, characterized in that: The specific method of temperature condition experiment is: By setting the temperature of the microwave oven to 20-100°C, a total of five different temperatures of 20°C, 40°C, 60°C, 80°C, and 100°C were selected, and other conditions were kept unchanged. Five different solutions were obtained by the change of temperature during the microwave method. The most suitable temperature was obtained by observing the fluorescence intensity under natural light and ultraviolet light and testing the fluorescence spectrum. The specific method of the time condition experiment is as follows: By setting the microwave oven time to 10-50s, a total of five different times of 10s, 20s, 30s, 40s, and 50s were selected, and other conditions were kept unchanged. Five different solutions were obtained by changing the microwave method time. The most suitable time was obtained by observing the fluorescence intensity under natural light and ultraviolet light and testing the fluorescence spectrum. The specific method of the volume ratio condition experiment is: DHLA-Ag NCs with different ratios were obtained by preparing 5 mL of DHLA solution and then adding different volumes of AgNO3 solution. In the experiment, five DHLA-Ag NCs solutions with different volume ratios of 5:0.5, 5:1, 5:2, 5:3, and 5:5 were set up, and five solutions were obtained by microwave heating. The most suitable volume ratio was obtained by observing the fluorescence intensity under natural light and ultraviolet light and testing the fluorescence spectrum.

6. The experimental method of near-infrared silver nanoclusters as claimed in claim 5, characterized in that: The specific method of fluorescence spectrum testing is: Add 2 ml of near-infrared silver nanocluster solution to a quartz cuvette, and place the cuvette in the sample chamber of a fluorescence spectrophotometer; set the fluorescence spectrum test conditions: the incident and exit slits are both 10 nanometers, and the gain is medium; select different modes to find the best excitation wavelength of the material, which is between 460 and 470 nanometers, and then set the best excitation in the emission mode, and the emission wavelength scanning range of NRI-Ag NCs is 500-800 nanometers; Fluorescence stability test was also performed: The test conditions were set as follows: the scanning mode was set to time scanning mode, the scanning time was 600 s, the incident slit and the exit slit were both 10 nm, and the gain was medium; the excitation and emission wavelengths of NRI-Ag NCs were set to 468 nm and 657 nm, respectively; the above experimental operations were repeated 3 times, and the obtained spectra were analyzed and compared.

7. The experimental method of near-infrared silver nanoclusters as claimed in claim 6, characterized in that: The morphology characterization test method is: Transmission electron microscopy technology is used to clearly depict the morphological characteristics of these nanomaterials and accurately measure their particle size through transmission electron microscopy analysis.

8. The experimental method of near-infrared silver nanoclusters as claimed in claim 7, characterized in that: The specific method of sensitivity detection is: First, prepare H2O2 with a concentration of 100 mM, then dilute it to different multiples to obtain H2O2 with a concentration of 1-50 mM, and store it at 4°C until use; Then, different concentrations of H2O2 were added to 2 mL of NRI-Ag NCs solution, and the solution was blown and stirred with a pipette to allow it to react fully. It was then placed under ultraviolet light to observe the fluorescence changes, and a fluorescence spectrophotometer was used to record the changes in the fluorescence spectrum, and the obtained spectra and corresponding data were statistically compared.

9. The experimental method of near-infrared silver nanoclusters as claimed in claim 8, characterized in that: The specific method of anti-interference experiment is: The concentration of H2O2 was 100 mM, and the interfering substances included Ala, Cys, Glu, Leu, Tyr, Trp, AA, GSH, NaCl, Ser, and Asp, and the concentration of all interfering substances was 100 mM; The fluorescence emission spectra of NRI-Ag NCs in the presence of different interferents were characterized using a fluorescence spectrophotometer and compared with the fluorescence emission spectra of NRI-Ag NCs after adding H2O2; All fluorescence spectra were scanned at 468 nm with a slit width of 10 nm; the above steps were all carried out at room temperature, and all tests were repeated at least 3 times.

10. The near-infrared silver nanoclusters according to claim 3, characterized in that: Application in H2O2 visualization detection for early cancer detection.

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