A dual-emission ratio fluorescent probe of carbon quantum dots and copper-silver alloy nanoclusters, its preparation method and application

Abstract

This invention discloses a carbon quantum dot (CQD) and copper-silver alloy nanocluster hybrid dual-emission ratiometric fluorescent probe, its preparation method, and its application. The invention involves mechanically mixing short-wavelength emission CQDs with long-wavelength emission CuAgNCs at room temperature to form a CQDs-CuAgNCs fluorescent probe with dual emission characteristics. The ratio of the two emission peaks is used to construct a ratiometric fluorescent probe for the quantitative detection of L-cysteine. The invented CQDs-CuAgNCs dual-emission ratiometric fluorescent probe exhibits high selectivity and sensitivity.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe preparation technology, specifically relating to a carbon quantum dot and copper-silver alloy nanocluster hybrid dual-emission ratio fluorescent probe, its preparation method and application. Background Technology

[0002] L-cysteine ​​(L-Cys), an abundant low-molecular-weight thiol in living cells, plays a crucial role in maintaining biological redox homeostasis in biological systems. Abnormal L-Cys concentrations are associated with many diseases; for example, L-Cys deficiency can lead to decreased hematopoietic function, neurotoxicity, edema, and liver damage. Conversely, L-Cys is overexpressed in patients with cardiovascular disease and Alzheimer's disease. Establishing a simple, sensitive, and low-cost method for L-Cys detection is of great significance for the diagnosis and prevention of related diseases.

[0003] Traditional methods for detecting small biological molecules, such as high-performance liquid chromatography (HPLC), electrochemistry, UV-Vis absorption spectroscopy, and capillary electrophoresis, are limited in widespread use due to high instrument costs and complex operations. In contrast, fluorescent probe detection offers advantages such as low cost, simple operation, and high sensitivity. Among fluorescent probes, dual-emission ratiometric fluorescent probes possess self-calibration capabilities, exhibiting greater stability, higher sensitivity, and more accurate detection results compared to single-emission fluorescent probes. Since individual fluorescent probe substrate materials rarely possess dual-emission characteristics, mixing two fluorescent materials with different emission wavelengths to prepare dual-emission ratiometric fluorescent probes is a promising approach. Fluorescent materials typically include organic fluorescent dyes, semiconductor quantum dots, CQDs (carbon quantum dots), and noble metal nanoclusters. Organic fluorescent dyes are unsuitable for large-scale and long-term monitoring due to complex synthesis and poor photostability, while semiconductor quantum dots, such as Cd-based and Pb-based ones, contain heavy metals, posing potential hazards. Compared to the former two, CQDs are characterized by simple preparation, strong photostability, and good biocompatibility. However, their disadvantage is the short emission wavelength, making it difficult to prepare CQDs with long-wavelength emission. Noble metal nanoclusters, such as gold nanoclusters, have long emission wavelengths and have attracted much attention due to their excellent optical properties, but their high raw material cost hinders widespread application. Compared to gold nanoclusters, copper and silver nanoclusters are much cheaper, but individual copper nanoclusters have poor stability, while individual silver nanoclusters struggle to emit long-wavelength light. Therefore, providing a noble metal nanocluster with excellent optical properties and low cost has become a challenge. Summary of the Invention

[0004] To address the problems existing in the prior art, the purpose of this invention is to provide a carbon quantum dot and copper-silver alloy nanocluster hybrid dual-emission ratio fluorescent probe, its preparation method, and its application. The carbon quantum dot and copper-silver alloy nanocluster hybrid dual-emission ratio fluorescent probe of this invention has strong stability, low cost, and long-wavelength emission characteristics.

[0005] The technical solution adopted in this invention is as follows:

[0006] A dual-emission ratio fluorescent probe composed of carbon quantum dots and copper-silver alloy nanoclusters, comprising a solvent, copper-silver alloy nanoclusters, and carbon quantum dots.

[0007] The preparation method of the carbon quantum dot and copper-silver alloy nanocluster hybrid dual-emission ratio fluorescent probe of the present invention includes the following steps:

[0008] A carbon quantum dot solution was added to a copper-silver alloy nanocluster suspension and mixed thoroughly to obtain a carbon quantum dot-copper-silver alloy nanocluster hybrid fluorescent probe with a dual emission ratio.

[0009] Preferred:

[0010] The preparation method of carbon quantum dot solution includes the following steps:

[0011] Polyethyleneimine and trisodium citrate are dissolved in water to obtain solution A;

[0012] Solution A was subjected to a hydrothermal reaction, and after the reaction was completed, it was naturally cooled to room temperature.

[0013] Impurities in the hydrothermal reaction product solution are removed to obtain the carbon quantum dot solution.

[0014] Preferred:

[0015] When preparing solution A, 45-55 mg of polyethyleneimine and 45-55 mg of trisodium citrate solution are added to every 50 mL of water.

[0016] Preferred:

[0017] During the hydrothermal reaction, the reaction temperature is 190-210 min and the reaction time is 3.5-4.5 h.

[0018] Preferably, when removing impurities from the hydrothermal reaction product solution, the hydrothermal reaction product solution is first centrifuged at 10,000 rpm for 8-12 minutes, and then the supernatant obtained by centrifugation is dialyzed using a dialysis bag with a molecular weight of 2000 Da to obtain the carbon quantum dot solution.

[0019] Preferred:

[0020] The preparation method of copper-silver alloy nanocluster suspension includes the following steps:

[0021] Dissolve Cu(NO3)2 solution and AgNO3 solution in water to obtain solution B;

[0022] DPA was added to solution B to react and a white emulsion was obtained;

[0023] The white emulsion was centrifuged to obtain a precipitate, which was then washed. The washed precipitate was then mixed with water to prepare a suspension, thus obtaining the copper-silver alloy nanocluster suspension.

[0024] Preferred:

[0025] When preparing solution B, 0.20-0.30 mL of Cu(NO3)2 solution and 0.9-1.1 mL of AgNO3 solution are added to every 10 mL of water, wherein the concentration of Cu(NO3)2 solution is 0.1 mol / L and the concentration of AgNO3 solution is 0.1 mol / L.

[0026] 0.1111-0.1121g of DPA was added to solution B.

[0027] Preferred:

[0028] Add 20-30 μL of carbon quantum dot solution to every 4 mL of copper-silver alloy nanocluster suspension.

[0029] The present invention relates to a carbon quantum dot and copper-silver alloy nanocluster hybrid dual-emission ratio fluorescent probe, as described above, for the detection of L-cysteine.

[0030] The present invention has the following beneficial effects:

[0031] The present invention comprises a carbon quantum dot and copper-silver alloy nanocluster hybrid dual-emission ratio fluorescent probe, which includes copper-silver alloy nanoclusters and carbon quantum dots. It can generate two fluorescence emission peaks at the same excitation wavelength, and when detecting L-Cys, F... 560 Changes and F 468 The accuracy remains almost unchanged, and the ratiometric fluorescent probe constructed based on this has higher accuracy. Experiments have shown that this fluorescent probe has the characteristics of high selectivity and high sensitivity.

[0032] In the preparation method of this invention, copper-silver alloy nanoclusters with high stability, low cost and long wavelength emission are prepared. Then, the copper-silver alloy nanoclusters are combined with carbon quantum dots with good biocompatibility and short emission wavelength to prepare CQDs-CuAgNCs dual emission ratio fluorescent probe for detecting L-Cys. This is of great significance for the preparation of ratio fluorescent probes and the rapid detection of L-Cys. Attached Figure Description

[0033] Figure 1The image shows a TEM image of the CQDs prepared in an embodiment of the present invention.

[0034] Figure 2 This is a particle size distribution diagram of the CQDs prepared in an embodiment of the present invention.

[0035] Figure 3 The fluorescence emission spectrum of the CQDs prepared in the embodiments of the present invention is shown.

[0036] Figure 4 This is a TEM image of CuAgNCs prepared according to an embodiment of the present invention.

[0037] Figure 5 This is a particle size distribution diagram of CuAgNCs prepared in an embodiment of the present invention.

[0038] Figure 6 The fluorescence emission spectrum of CuAgNCs prepared in the embodiments of the present invention is shown.

[0039] Figure 7 The fluorescence emission spectrum of CQDs-CuAgNCs prepared in the embodiments of the present invention is shown.

[0040] Figure 8 The selectivity diagram of L-Cys for the CQDs-CuAgNCs dual-emission ratio fluorescent probe prepared in the embodiments of the present invention is shown.

[0041] Figure 9 The fluorescence spectra of the CQDs-CuAgNCs dual-emission ratio fluorescent probe obtained in this invention, as described in the embodiments of the present invention, are obtained after adding different concentrations of L-Cys.

[0042] Figure 10 The graph shows the linear relationship after adding different concentrations of L-Cys to the CQDs-CuAgNCs dual emission ratio fluorescent probe obtained in this invention, as described in the embodiments of this invention. Detailed Implementation

[0043] The present invention will be further described in detail below with reference to embodiments and accompanying drawings. However, the present invention is not limited to the specific embodiments described below, and all equivalent changes made based on these embodiments are within the scope of protection of the present invention.

[0044] The method for preparing a dual-emission ratio fluorescent probe of carbon quantum dots and copper-silver alloy nanoclusters (CQDs-CuAgNCs) of the present invention includes the following steps:

[0045] Step 1: Preparation of CQDs

[0046] Step 1: Dissolve 50±5 mg of polyethyleneimine (PEI, molecular weight 2000) and 50±5 mg of trisodium citrate in 50 mL of water and stir for a certain period of time to dissolve the polyethyleneimine and trisodium citrate, to obtain solution A.

[0047] Step 2: Transfer the solution A obtained in Step 1 to a 100mL stainless steel hydrothermal reactor and react at 200±10℃ for 3.5-4.5h. Then wait for the reactor to cool naturally to room temperature.

[0048] Step 3: Centrifuge the hydrothermal reaction product solution obtained in Step 2 at 10,000 rpm for 8-12 min to remove large-particle impurities. Then, dialyze the supernatant using a dialysis bag with a molecular weight of 2000 Da to remove small-molecule impurities, thereby obtaining a purer CQDs solution. Store the obtained CQDs solution in a refrigerator at 4°C for further use.

[0049] Step 2: Preparation of CuAgNCs (copper-silver alloy nanoclusters) suspension:

[0050] Step 1: Add 0.25±0.05mL of Cu(NO3)2 solution (0.1mol / L) and 1±0.1mL of AgNO3 solution (0.1mol / L) to 10mL of water and stir for a certain period of time.

[0051] Step 2: Then add 0.1116±0.0005g DPA (D-penicillamine) to the solution from Step 1 and continue stirring for a certain period of time to react, resulting in a white emulsion.

[0052] Step 3: Centrifuge the white emulsion obtained in Step 2 at 10,000 rpm for 10 min, wash the precipitate three times with water, then add 100 mL of water to prepare a suspension, and store it in a refrigerator at 4°C for further use.

[0053] Step 3: Preparation of CQDs-CuAgNCs:

[0054] By adding 25±5μL of CQDs solution to 4mL of CuAgNCs solution and shaking at room temperature for 3-5min, a ratiometric fluorescent probe CQDs-CuAgNCs with dual emission characteristics was obtained.

[0055] In the above-described scheme of the present invention, the values ​​after the "±" sign represent the deviation range when weighing or measuring each parameter, which falls within the normal error range.

[0056] Example

[0057] The method for preparing a dual-emission ratio fluorescent probe composed of carbon quantum dots and copper-silver alloy nanoclusters in this embodiment includes the following steps:

[0058] Step 1: Dissolve 50 mg of polyethyleneimine (PEI, molecular weight 2000) and 50 mg of trisodium citrate in 50 mL of water and stir for a certain period of time.

[0059] Step 2: Transfer the solution from Step 1 to a 100mL stainless steel hydrothermal reactor and react at 200℃ for 4 hours. Then wait for the reactor to cool naturally to room temperature.

[0060] Step 3: Centrifuge the product solution from Step 2 at 10,000 rpm for 10 min to remove large-particle impurities. Then, dialyze the supernatant using a dialysis bag with a molecular weight of 2000 Da to remove small-molecule impurities, thereby obtaining a purer CQDs solution. Store the obtained CQDs solution in a refrigerator at 4°C for further use. Figure 1 The fluorescence and emission spectra of the obtained CQDs are shown. The emission wavelength is around 450 nm, and they exhibit bright blue fluorescence under ultraviolet light. Figure 2 The TEM image of the obtained CQDs shows an approximately spherical morphology with a lattice spacing of 0.21 nm. Figure 3 The particle size distribution of the obtained CQDs is shown in the figure. The particle size range is 1.78-4.33 nm, and the average particle size is 2.74 nm.

[0061] Step 4: Add 0.25 mL of Cu(NO3)2 solution (0.1 mol / L) and 1 mL of AgNO3 solution (0.1 mol / L) to 10 mL of water and stir for a certain period of time.

[0062] Step 5: Then add 0.1116g DPA to the solution in Step 4 and continue stirring for a certain period of time to obtain a white emulsion.

[0063] Step 6: Centrifuge the white emulsion obtained in Step 5 at 10,000 rpm for 10 min, wash the precipitate with water 3 times, and then add 100 mL of water to prepare a suspension. This solution is the CuAgNCs solution. Store it in a refrigerator at 4°C for further use. Figure 4 The image shows a TEM image of the prepared CuAgNCs, which exhibit an approximately spherical morphology. Figure 5 The particle size distribution of the prepared CuAgNCs is shown in the figure. The particle size ranges from 3.35 to 5.45 nm, with an average particle size of 4.45 nm. Figure 6 The fluorescence emission spectrum of the prepared CuAgNCs is shown. The luminescence center is located at about 560 nm, and it exhibits orange-yellow fluorescence under ultraviolet light.

[0064] Step 7: By adding 25 μL of CQDs solution (step 3) to 4 mL of CuAgNCs solution (the solution in step 6) and shaking at room temperature for 5 min, a ratiometric fluorescent probe CQDs-CuAgNCs with dual emission characteristics is obtained for the detection of L-Cys. Figure 7 The fluorescence emission spectrum of CQDs-CuAgNCs shows a dual emission characteristic, with the emission peak on the left originating from CQDs and the emission peak on the right originating from CuAgNCs.

[0065] CQDs-CuAgNCs detection of L-Cys

[0066] 4.5 mL of CQD / CuAgNC nanocomposite solution was added to a 5 mL centrifuge tube. Then, 0.5 mL of L-Cys solution containing different concentrations of L-Cys was added to the above solution. After incubation at room temperature for 5 minutes, the fluorescence emission spectrum was measured at an excitation wavelength of 365 nm. Figure 8 The diagram shows the selectivity of the CQDs-CuAgNCs dual-emission ratiometric fluorescent probe for L-Cys. It can be seen that among common biomolecular metal ions and other interfering substances, it has the best selectivity for L-Cys. Figure 9 The fluorescence emission spectra of CQDs-CuAgNCs with different concentrations of L-Cys are shown. The emission peak at 468 nm represents the emission peak of CQDs, and the emission peak at 560 nm represents the emission peak of CuAgNCs. It can be seen that as the L-Cys concentration gradually increases, the emission peak at 560 nm gradually increases, while the emission peak at 468 nm remains almost unchanged. The ratio of the emission peaks, F... 560 / F 468 The linear relationship with L-Cys in the 0-60 μM range is: F 560 / F 468 =1.180 + 0.049C [L-Cys] R 2 =0.995, LOD is 0.367μM, Figure 10 As shown.

Claims

1. A method for preparing a carbon quantum dots and copper-silver alloy nanoclusters hybrid double-emission ratio fluorescent probe, characterized in that, The method comprises the following steps: The carbon quantum dot solution is prepared by the following method: Polyethyleneimine and trisodium citrate are dissolved in water to obtain solution A; The solution A is subjected to hydrothermal reaction, and is naturally cooled to room temperature after the reaction is completed; Impurities in the hydrothermal reaction product solution are removed to obtain the carbon quantum dot solution; When preparing the solution A, 45-55 mg of polyethyleneimine and 45-55 mg of trisodium citrate are added to 50 mL of water; When the hydrothermal reaction is performed, the reaction temperature is 190-210 DEG C, and the reaction time is 3.5-4.5 h; When removing the impurities in the hydrothermal reaction product solution, the hydrothermal reaction product solution is centrifuged at 10000 rpm for 8-12 min, and then the supernatant obtained by centrifugation is dialyzed by using a dialysis bag with a molecular weight of 2000 Da to obtain the carbon quantum dot solution; The method for preparing the copper-silver alloy nanocluster suspension comprises the following steps: Cu (NO3) 2 solution and AgNO3 solution are dissolved in water to obtain solution B; DPA is added to the solution B to obtain a white emulsion; The white emulsion is centrifuged to obtain a precipitate, the precipitate is washed, and the washed precipitate is prepared into a suspension with water to obtain the copper-silver alloy nanocluster suspension; When preparing the solution B, 0.20-0.30 mL of Cu (NO3) 2 solution and 0.9-1.1 mL of AgNO3 solution are added to 10 mL of water, the concentration of the Cu (NO3) 2 solution is 0.1 mol / L, and the concentration of the AgNO3 solution is 0.1 mol / L; 0.1111-0.1121 g of DPA is added to the solution B; 20-30 μL of carbon quantum dot solution is added to 4 mL of copper-silver alloy nanocluster suspension. The carbon quantum dot and copper-silver alloy nanocluster hybrid double-emission ratio fluorescent probe is prepared by the method of claim 1.

2. A carbon quantum dots and copper-silver alloy nanoclusters hybrid double-emission ratio fluorescent probe, characterized in that, The carbon quantum dot and copper-silver alloy nanocluster hybrid double-emission ratio fluorescent probe is used for detecting L-cysteine for non-disease diagnosis and treatment purposes. 3.The application of carbon quantum dots and copper-silver alloy nanoclusters hybrid double-emission ratio fluorescent probe according to claim 2, characterized in that, ​

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