A copper ion ratio fluorescent probe based on carbon dots and its preparation method and application

Abstract

The present invention discloses a copper ion ratio fluorescent probe based on carbon dots, its preparation method and application. The ratio fluorescent probe is prepared as follows: polyethyleneimine and cysteine ​​are dissolved in deionized water or deionized water containing formamide, and the reaction is refluxed at 60-140°C for 0.5-24h. The ratio fluorescent probe is obtained by filtration, column chromatography separation, concentration and drying. The obtained ratio fluorescent probe is used to detect the copper ion ratio in different aqueous environments. 2+ The results showed that the different Cu 2+ The ratiometric fluorescent probe has a wide concentration range, with emission wavelengths of 489 and 680 nm serving as references. It exhibits high copper ion targeting specificity, rapid sensing, high sensitivity, and high accuracy, achieving a detection limit of as low as 3.13 nM for copper ions in complex physiological matrices. The ratiometric fluorescent probe exhibits high photostability, good water solubility, and high biosafety, making it suitable for the specific and precise detection of copper ions in biological samples and environmental water.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanometer ratio fluorescent probes, and in particular relates to a copper ion ratio fluorescent probe based on carbon dots, and a preparation method and application thereof. Background Art

[0002] Since their initial discovery by Sun et al. in 2004, carbon dots have been widely used in optoelectronic devices, photocatalysis, drug delivery, bioimaging, and fluorescence sensing due to their nanoscale size, unique physicochemical properties, high photostability, high fluorescence quantum yield, tunable emission spectra, and excellent biosafety. However, current carbon dots used for fluorescence sensing are primarily based on single-emission fluorescence intensity, which is susceptible to interference from factors unrelated to copper ions, such as light source fluctuations, uneven probe distribution, and microenvironmental changes, leading to false-positive results. In contrast, carbon dot-based ratiometric fluorescence probes use the fluorescence intensity ratio of two (or more) well-resolved emission peaks at the same excitation wavelength as the response signal. They have a built-in calibration function that can effectively avoid interference from factors unrelated to the analyte, thereby effectively improving sensing sensitivity, reliability, and accuracy, providing strong support for the accurate monitoring of target analytes.

[0003] Copper ions, essential trace elements for life, play a crucial role in human physiological and pathological processes. Copper ions possess strong redox properties, and imbalances in their concentration can contribute to cancer, Parkinson's disease, and other biological disorders. my country's drinking water hygiene standard, GB 5749-2006, stipulates that the copper ion content should not exceed 15.7 μM / mL. However, excessive copper ion concentrations in environmental water resources can easily accumulate in organisms and cause toxicity. Therefore, the development of highly sensitive and accurate carbon-dot-based copper ion ratiometric fluorescent probes has significant scientific significance and application value. Summary of the Invention

[0004] This invention addresses the current bottlenecks in the construction of ratiometric fluorescent probes based on carbon dots and the serious diseases caused by copper ion concentration imbalance. It provides a copper ion ratiometric fluorescent probe based on the inherent dual-emission properties of carbon dots, as well as its preparation method and application. This invention utilizes dual-emission carbon dots to construct a novel copper ion ratiometric fluorescent probe, enabling ratiometric fluorescence sensing of copper ions. The probe features a simple preparation process, high detection selectivity and accuracy, and an extremely low detection limit.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] A method for preparing a carbon dot-based copper ion ratio fluorescent probe (RCDs) comprises the following steps: dissolving polyethyleneimine and cysteine ​​in deionized water or deionized water containing formamide, reacting the resulting mixed solution at 60-140°C for 0.5-24 hours using a reflux reaction method, removing raw materials and intermediates that do not participate in the reaction by filtration and column chromatography, and finally concentrating and drying to obtain a carbon dot-based copper ion ratio fluorescent probe that emits at 489 and 680 nm.

[0007] Preferably, the mass feed ratio of polyethyleneimine to cysteine ​​is 1:100-50:1, the molecular weight of polyethyleneimine is 600-10000, and the mass volume ratio of polyethyleneimine to deionized water or deionized water containing formamide is 1g:50-100mL.

[0008] Preferably, the eluent used in the column chromatography is a mixture of methanol: water: n-butanol: acetic acid in a volume ratio of 4:1:2:0.5-4:0.5:1:0.1.

[0009] Preferably, the volume ratio of formamide to deionized water in the formamide-containing deionized water is 1:10-20:1.

[0010] The present invention also provides a carbon dot-based copper ion ratio fluorescent probe prepared according to the above preparation method.

[0011] Furthermore, the present invention provides application of the ratiometric fluorescent probe in copper ion detection.

[0012] Preferably, the application comprises the following steps: adding the copper ion solution to be tested and the copper ion standard solution to the ratio fluorescence probe aqueous solution, respectively, measuring the fluorescence intensity F at wavelengths of 489 and 680 nm after the reaction is balanced, and 680 / F 489 or F 489 / F 680 The ratio is linearly related to the copper ion concentration of the standard solution, and the concentration of copper ions in the copper ion solution to be detected is calculated.

[0013] Preferably, the concentration of the ratiometric fluorescent probe aqueous solution is 10-200 μg / mL.

[0014] Preferably, the fluorescence response range of the ratiometric fluorescent probe to copper ions is 0-1000 μM, and the reaction equilibrium time is 1.5 min.

[0015] The present invention has the following advantages and effects compared to the prior art:

[0016] (1) The copper ion ratio fluorescent probe based on carbon dots of the present invention has the advantages of simple preparation, long wavelength emission, high fluorescence quantum yield and high biosafety.

[0017] (2) It has the characteristics of specificity, high sensitivity and accuracy in copper ion sensing applications, and can be used for detection in environmental water and biomass samples.

[0018] (3) The present invention expands the types of copper ion ratio fluorescent probes based on carbon dots, and provides technical support for the preparation of new ratio fluorescent probes with high photostability and low toxicity and their sensing applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Transmission electron microscopy image (left) and particle size distribution diagram (right) of the ratiometric fluorescent probe.

[0020] Figure 2 The UV-visible absorption spectrum (left) and fluorescence spectrum (right) of the ratiometric fluorescent probe.

[0021] Figure 3 This is the ratio fluorescence response diagram of the ratio fluorescence probe to copper ions and other ions.

[0022] Figure 4 The left figure shows the time response of the ratiometric fluorescent probe to copper ions and the right figure shows the response to different concentrations of copper ions.

[0023] Figure 5 This is a ratiometric fluorescence response curve of the ratiometric fluorescent probe to copper ions in the copper ion concentration range of 0-5 μM and 5-1000 μM.

[0024] Figure 6 This is the ratio fluorescence response curve of the ratio fluorescence probe to copper ions in the cell matrix. DETAILED DESCRIPTION

[0025] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.

[0026] Example 1

[0027] A method for preparing a carbon dot-based copper ion ratiometric fluorescent probe comprises the following steps: 100 mg of polyethyleneimine (molecular weight 10,000) and 1 g of cysteine ​​are weighed and dissolved in a reaction flask containing 10 mL of deionized water. The resulting mixed solution is stirred on a magnetic stirrer and refluxed in a 60°C oil bath for 24 hours. The reaction solution is then filtered and separated by column chromatography using an alumina column and an eluent (a mixture of methanol, water, n-butanol, and acetic acid in a volume ratio of 4:0.5:1:0.1). The eluate is collected, concentrated, and dried to obtain the carbon dot-based copper ion ratiometric fluorescent probe.

[0028] Example 2

[0029] A method for preparing a carbon dot-based copper ion ratiometric fluorescent probe comprises the following steps: 350 mg of polyethyleneimine (molecular weight 1800) and 650 mg of cysteine ​​are weighed and dissolved in a reaction flask containing 20 mL of a formamide / deionized water (20:1, volume ratio) mixed solution. The resulting mixed solution is stirred on a magnetic stirrer and refluxed in a 140°C oil bath for 0.5 h. The reaction solution is then filtered and separated by column chromatography using an alumina column and an eluent (a mixture of methanol: water: n-butanol: acetic acid in a volume ratio of 4:1:2:0.5). The eluate is collected, concentrated, and dried to obtain the carbon dot-based copper ion ratiometric fluorescent probe.

[0030] Example 3

[0031] A method for preparing a carbon dot-based copper ion ratiometric fluorescent probe comprises the following steps: 1g of polyethyleneimine (molecular weight 600) and 20mg of cysteine ​​are weighed and dissolved in a reaction flask containing 50mL of a formamide / deionized water (1:10, volume ratio) mixture. The resulting mixture is stirred on a magnetic stirrer and refluxed in a 100°C oil bath for 12 hours. The reaction solution is then filtered and separated by column chromatography using an alumina column and an eluent (a mixture of methanol, water, n-butanol, and acetic acid in a volume ratio of 4:0.8:1.5:0.3). The eluate is collected, concentrated, and dried to obtain the carbon dot-based copper ion ratiometric fluorescent probe.

[0032] Example 4

[0033] The morphology and optical properties of the ratiometric fluorescent probe prepared in Example 2 of the present invention were characterized. Figure 1 and 2 shown. Figure 1 Transmission electron microscopy (LEFT) and particle size distribution (RIGHT) show that the prepared ratiometric fluorescent probe has nanometer size, with an average particle size of approximately 2.25 nm; Figure 2 The UV-visible absorption spectrum (left) shows that the ratiometric fluorescent probe has broad absorption peaks at 268nm, 400nm, 635nm and 679nm; it exhibits dual emission characteristics in the excitation wavelength range of 350-450nm ( Figure 2 : right figure), where the emission peak at 400-600nm is excitation-dependent, while the emission peak at 680nm is independent of wavelength.

[0034] Example 5

[0035] (1) This example uses the ratiometric fluorescent probe prepared in Example 2 to investigate its specific response to copper ions: 0.3 mL of different types of cations including NH4 + 、Na + Mg 2+ 、Al3+ , K + , Ca 2+ Cr 3+ 、Mn 2+ 、Fe 2+ 、Fe 3 + 、Co 2+ 、Ni 2+ 、Cu 2+ 、Zn 2+ 、Cd 2+ , Pb 2+ and Ag + (The final concentration of each ion is 1 mM) was added to 2.7 mL of a 60 μg / mL ratiometric fluorescent probe aqueous solution (phosphate buffer, pH = 7.4), mixed and allowed to stand for a certain period of time, and the fluorescence intensity changes at 489 and 680 nm emission wavelengths of the ratiometric fluorescent probe under the action of a 425 nm excitation wavelength were measured using a fluorescence spectrometer, and the fluorescence intensity ratio F was calculated. 680 / F 489 , the results are as follows Figure 3 As shown. Figure 3 It can be seen that the prepared ratiometric fluorescent probe has a highly specific ratiometric response ability to copper ions.

[0036] (2) Evaluation of the time and concentration response performance of the ratiometric fluorescent probe prepared in Example 2 to copper ions: 0.3 mL of copper ion aqueous solution (final concentration of 10 μM) was added to 2.7 mL of 60 μg / mL ratiometric fluorescent probe aqueous solution (phosphate buffer, pH = 7.4), and the fluorescence spectrum changes at different time points of the reaction between the ratiometric fluorescent probe and copper ions were measured using a fluorescence spectrometer. Figure 4 (Left) Figure 4 (Left) It can be seen that the ratiometric fluorescent probe has a fast response speed to copper ions. Subsequently, different concentrations of copper ions were added to the ratiometric fluorescent probe aqueous solution and reacted for 1.5 minutes, and the emission spectrum changes of the ratiometric fluorescent probe were measured ( Figure 4 : right). Figure 4 (Right) As can be seen, with the increase of copper ion concentration, the emission peak intensity at 680nm gradually weakened, while the emission peak intensity at 489nm changed slightly, and the probe fluorescence changed from blue-white to bright blue. Fluorescence intensity ratio F 680 / F 489 It showed good linearity and high sensitivity in the copper ion concentration range of 0-5μM, with a detection limit of 10.86nM ( Figure 5 : Left Figure); and F 489 / F 680 The log10 logarithm showed a good linear relationship in the copper ion concentration range of 5-1000μM ( Figure 5These results indicate that the prepared ratiometric fluorescent probe has high specificity, high sensitivity, and fast response to copper ions.

[0037] Example 6

[0038] The copper ion ratio fluorescence probe based on carbon dots prepared in Example 2 was dissolved in cell matrix (DMEM cell culture medium, pH = 7.2-7.4), and the ratio fluorescence response of the probe to different copper ion concentrations was measured ( Figure 6 ).Depend on Figure 6 It can be seen that the prepared ratiometric fluorescent probe also has ratiometric response capability at copper ion concentrations of 0-1000 μM, and has higher sensitivity with a detection limit of 3.13 nM, and has the potential to detect cellular copper ions.

[0039] Example 7

[0040] The ratio fluorescent probe prepared in Examples 1, 2 and 3 of the present invention is applied to the detection of copper ions in laboratory tap water. The detection method is as described in Example 5 (2), and the calculation formula is as described in Example 5 (2). Figure 5 The linear regression curve (F 680 / F 489 =-1.80967C(Cu 2+ )+10.18885, R 2 =0.991), and the relevant results are shown in Table 1. Table 1 shows that the recovery rate of the prepared ratio fluorescent probe for detecting copper ions in tap water is 97.2%-104.2%, which meets the detection standard, indicating that the prepared ratio fluorescent probe can be used for copper ion detection in the environment.

[0041] Table 1. Results of copper ion concentration detection in tap water using ratiometric fluorescence probe

[0042]

[0043]

[0044] In summary, the copper ion ratio fluorescent probe based on carbon dots of the present invention can be applied to the detection of copper ions in biological samples and environmental water.

[0045] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. Persons skilled in the art will appreciate that improvements and modifications may be made without departing from the spirit and scope of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a copper ion ratio fluorescent probe based on carbon dots, characterized in that: The following steps are involved: Polyethyleneimine and cysteine ​​were dissolved in deionized water containing formamide. The resulting mixed solution was reacted at 60-140°C for 0.5-24 hours using a reflux reaction method. The unreacted raw materials and intermediates were removed by filtration and column chromatography. Finally, the mixture was concentrated and dried to obtain a carbon-dot-based copper ion ratiometric fluorescent probe emitting at 489 and 680 nm. The mass feed ratio of polyethyleneimine to cysteine ​​is 1:100-50:1, the molecular weight of polyethyleneimine is 600-10000, and the mass volume ratio of polyethyleneimine to deionized water containing formamide is 1 g:50-100 mL; The eluent used in the column chromatography is a mixture of methanol: water: n-butanol: acetic acid in a volume ratio of 4:1:2:0.5-4:0.5:1:0.1; The volume ratio of formamide to deionized water in the formamide-containing deionized water is 1:10-20:

1.

2. The ratiometric fluorescent probe prepared by the preparation method according to claim 1.

3. The use of the ratiometric fluorescent probe according to claim 2 in copper ion detection, characterized in that: The following steps are involved: The copper ion solution to be tested and the copper ion standard solution were added to the ratio fluorescence probe aqueous solution respectively. After the reaction was balanced, the fluorescence intensity F at wavelengths of 489 and 680 nm was measured, and the fluorescence intensity F was calculated based on the F 680 / F 489 or F 489 / F 680 The ratio is linearly related to the copper ion concentration of the standard solution, and the concentration of copper ions in the copper ion solution to be detected is calculated.

4. The use according to claim 3, characterized in that The concentration of the ratiometric fluorescent probe aqueous solution is 10-200 μg / mL.

5. The use according to claim 3, characterized in that The fluorescence response range of the ratiometric fluorescent probe to copper ions is 0-1000 μM, and the reaction equilibrium time is 1.5 min.

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