A dual-mode fluorescent probe based on FeCDs and a preparation method and application thereof

Abstract

This invention discloses a FeCDs-based dual-mode fluorescent probe, its preparation method, and its applications, belonging to the field of chemical synthesis and analytical technology. The probe exhibits a "fluorescence transition-new peak appearance" response to L-ascorbic acid and a "simple fluorescence quenching" response to D-ascorbic acid. It is prepared via hydrothermal synthesis of dialdehyde starch, 1,2-diaminonaphthalene, and FeCl3. During detection, the fluorescence color change is observed under a 365 nm UV lamp for visual detection, or excitation is performed at 360 nm, with L-AA exhibiting an Ig... 430 / I 610 Ratio quantification, D-AA with I 610 Intensity-based quantification enables quantitative detection. Detection limits are 0.028 μmol / L and 0.033 μmol / L, respectively, allowing for rapid qualitative differentiation and highly sensitive quantitative detection of chiral ascorbic acid.

Description

Technical Field

[0001] This invention belongs to the field of chemical synthesis and analysis technology, specifically relating to a method for preparing a dual-mode fluorescent probe based on iron-doped carbon dots (FeCDs), and its application in the highly selective identification and quantitative detection of chiral ascorbic acid (L-ascorbic acid and D-ascorbic acid). Background Technology

[0002] Chirality is a ubiquitous phenomenon in nature, and different configurations of chiral compounds often exhibit drastically different biological activities, pharmacological effects, and metabolic pathways. Ascorbic acid (AA, commonly known as vitamin C), as an essential bioactive molecule for the human body, is a key nutrient in its L-configuration (L-AA) for maintaining normal physiological metabolism, possessing important functions such as antioxidation, enhancing immunity, and promoting collagen synthesis. In contrast, the D-configuration (D-AA) has extremely low biological activity and cannot be effectively absorbed and utilized by the human body; excessive intake may also lead to adverse consequences such as gastrointestinal discomfort and metabolic disorders. Therefore, achieving efficient differentiation and accurate detection of L-AA and D-AA has significant practical implications in fields such as food nutrition assessment, pharmaceutical quality control, and biochemical metabolic analysis.

[0003] Currently, the main methods for detecting chiral ascorbic acid include high-performance liquid chromatography (HPLC), capillary electrophoresis, and electrochemical analysis. While HPLC and capillary electrophoresis offer high separation accuracy, they suffer from drawbacks such as expensive equipment, complex procedures, time-consuming sample pretreatment, and reliance on specialized technical personnel. Electrochemical analysis faces challenges such as electrode contamination, poor selectivity, and stringent environmental requirements, making it difficult to meet the practical needs of rapid on-site detection and trace analysis.

[0004] Fluorescent probe technology has become a research hotspot in the field of chiral recognition due to its advantages such as ease of operation, rapid response, high sensitivity, low cost, and no need for complex equipment. Carbon dots (CDs), as a novel class of zero-dimensional luminescent nanomaterials, have outstanding advantages such as good biocompatibility, strong photostability, low toxicity, and wide availability of raw materials, and have been widely used in the field of fluorescence sensing.

[0005] However, existing carbon dot-based ascorbic acid detection probes still have significant shortcomings: most probes can only achieve quantitative detection of total ascorbic acid, lack chiral recognition ability, and cannot distinguish the configurational differences between L-AA and D-AA; some probes with chiral recognition potential have problems such as high detection limits (usually higher than 0.1 μmol / L), narrow linear range (generally less than 100 μmol / L), and poor selectivity; at the same time, these probes are susceptible to interference from environmental factors (such as pH and temperature changes) and coexisting substances in the sample (such as anions and cations, amino acids, sugars, organic acids, etc.), resulting in insufficient detection stability and accuracy, which limits their practical application in complex matrix samples.

[0006] Although composite materials such as metal-organic frameworks (MOFs) and chiral molecularly imprinted polymers (CMIPs) have been reported in the field of chiral separation, their preparation processes are complex, their stability is insufficient, and their costs are high. Furthermore, their mechanisms of action differ fundamentally from those of fluorescent probes, making direct application to the chiral fluorescence detection of ascorbic acid difficult. While iron, as a transition metal, can theoretically enhance its interaction with target molecules by modulating the electronic structure of carbon dots, there is currently no literature reporting on whether iron-doped carbon dots can achieve chiral recognition, how to differentiate between L-AA and D-AA, and the specific dual-mode response mechanism, resulting in high uncertainty. Therefore, how to endow carbon dot materials with specific recognition capabilities for chiral ascorbic acid without increasing process complexity, while maintaining their excellent optical properties and stability, remains an unsolved technical challenge in this field. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing a dual-mode fluorescent probe based on FeCDs, and the application of this probe in the detection of chiral ascorbic acid. It aims to solve the technical problems of existing chiral ascorbic acid detection methods, such as complex operation, lack of efficient chiral recognition ability, high detection limit, and poor anti-interference ability, so as to achieve rapid qualitative differentiation and highly sensitive quantitative detection of L-AA and D-AA.

[0008] To achieve the above objectives, the specific technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a dual-mode fluorescent probe based on iron-doped carbon dots, wherein the probe is iron-doped carbon dots (FeCDs), and the FeCDs exhibit a response of fluorescence quenching at 610 nm and generating a new emission peak at 430 nm to L-ascorbic acid, and a response of fluorescence quenching only at 610 nm to D-ascorbic acid, thereby achieving dual-mode fluorescence recognition.

[0009] Furthermore, the FeCDs exhibit wavelength independence and have a characteristic emission peak at 610 nm.

[0010] Furthermore, the FeCDs exhibit a characteristic UV-Vis absorption peak at 291 nm.

[0011] Secondly, the present invention provides a method for preparing the dual-mode fluorescent probe, comprising the following steps: (1) Preparation of dialdehyde starch: Corn starch and sodium periodate solution were mixed at a liquid-solid ratio of 100 mL: 1 g and stirred at 40℃±2℃ in the dark for 4 h±0.5 h to obtain dialdehyde starch. (2) Hydrothermal synthesis of FeCDs: According to the raw material molar ratio of 1:1:1, dialdehyde starch, 1,2-diaminonaphthalene and FeCl3 were uniformly mixed, ultrapure water was added, and ultrasonic dispersion was carried out for 10 min until the mixture was uniform. The mixture was then transferred to a high-pressure reactor with a polytetrafluoroethylene liner, the pH of the system was adjusted to 4.0±0.1, and the hydrothermal reaction was carried out at 200℃±5℃ for 8 h±0.5 h. (3) Purification and drying of FeCDs: After the reaction system is cooled to room temperature, the resulting mixture is vacuum filtered through an aqueous microporous membrane. The filtrate is dialyzed in ultrapure water for 24 h using a 1000 Da dialysis bag to obtain a purified FeCDs aqueous solution. The purified FeCDs aqueous solution is freeze-dried to obtain a solid FeCDs sample.

[0012] Furthermore, in step (2), the ultrasonic power is 100-150 W.

[0013] Further, in step (3), the freeze-drying conditions are freeze-drying at -50℃±5℃ for 24 h±2 h.

[0014] Thirdly, the present invention provides an application of the dual-mode fluorescent probe described above in the detection of chiral ascorbic acid.

[0015] Furthermore, the detection method includes the following steps: (1) Preparation of FeCDs stock solution: Disperse solid FeCDs samples in deionized water to prepare a stock solution with a concentration of 200 mg / L; (2) Sample detection: Take FeCDs stock solution and mix it with 10 mmol / L PBS buffer at pH 4.0, add the sample solution to be tested, mix thoroughly and let stand at room temperature for 5 min, and observe the change in fluorescence color under a 365 nm UV lamp; (3) Result determination: If the system shows blue-green fluorescence, it indicates that the sample contains L-ascorbic acid; if the red fluorescence of the system is quenched, it indicates that the sample contains D-ascorbic acid.

[0016] Furthermore, the detection method includes the following steps: (1) Preparation of FeCDs stock solution: Disperse solid FeCDs samples in deionized water to prepare a stock solution with a concentration of 200 mg / L; (2) Sample detection: Take FeCDs stock solution and mix it with 10 mmol / L PBS buffer at pH 4.0, add the sample solution to be tested, mix thoroughly and let stand at room temperature for 5 min, measure the fluorescence emission spectrum at 360 nm as the excitation wavelength, and measure the fluorescence emission spectrum of the system in the range of 380-700 nm. (3) Take I 430 / I610 The ratio is the detection signal or I 610 To detect the signal, a standard curve is plotted, and the concentration of L-AA or D-AA in the sample is calculated based on the standard curve.

[0017] Specifically, the quantitative detection of L-ascorbic acid involves mixing FeCDs solution with the sample to be tested, measuring the fluorescence intensity ratio at 430 nm and 610 nm under an excitation wavelength of 360 nm, and calculating the L-ascorbic acid concentration based on the standard curve. The quantitative detection of D-ascorbic acid is performed by mixing FeCDs solution with the sample to be tested, measuring the fluorescence intensity at 610 nm under an excitation wavelength of 360 nm, and calculating the D-ascorbic acid concentration based on the standard curve.

[0018] Furthermore, the detection concentration range of the chiral ascorbic acid is 1.5-200 μmol / L, the detection limit of L-ascorbic acid is 0.028 μmol / L, and the detection limit of D-ascorbic acid is 0.033 μmol / L.

[0019] Fourthly, the present invention provides a method for detecting chiral ascorbic acid, using the aforementioned dual-mode fluorescent probe, comprising mixing the sample to be tested with FeCDs stock solution, and determining the presence or content of L-ascorbic acid and / or D-ascorbic acid in the sample by visual observation or fluorescence spectroscopy analysis.

[0020] The samples to be tested include food samples (such as fresh fruit and vegetable juices, vitamin C fortified foods), pharmaceutical samples (such as vitamin C tablet solutions, injection solutions), and biological samples (such as serum diluents, urine diluents); the sample pretreatment method is as follows: solid samples are ground, ultrasonically extracted with deionized water for 10 min, and the supernatant is collected by centrifugation; biological samples such as serum and urine are diluted 10 times with physiological saline, and all samples are filtered through a 0.22 μm filter membrane before use.

[0021] By adopting the above technical solution, the present invention has the following advantages and beneficial effects: (1) Outstanding chiral recognition capability: The FeCDs probe of the present invention has a unique dual-mode fluorescence response mechanism, which shows "fluorescence transition - new peak appearance" for L-AA and "fluorescence quenching" for D-AA. The two chiral configurations can be quickly distinguished by fluorescence spectrum or visual observation under ultraviolet light, which solves the technical bottleneck that existing probes cannot achieve efficient recognition of chiral ascorbic acid. Figure 6 The differences in CIE chromaticity transition paths between L-AA and D-AA recognized by FeCDs are clearly demonstrated, intuitively confirming the specificity of the dual-mode response.

[0022] (2) Excellent detection performance: The detection limit of the probe for L-AA is as low as 0.028 μmol / L and the quantitation limit is 0.084 μmol / L. The detection limit for D-AA is 0.033 μmol / L and the quantitation limit is 0.099 μmol / L. The linear range is wide (1.5-200 μmol / L), which is better than existing nanomaterial probes (most of which have detection limits higher than 0.1 μmol / L and linear ranges less than 100 μmol / L), meeting the needs of trace detection. Figure 5 Fluorescence emission spectra and linear fitting results (R) 2 (≥0.9979), further verifying its accurate quantitative detection performance.

[0023] (3) Strong anti-interference ability: Under the coexistence of 500 μmol / L common anions and cations, 200 μmol / L amino acids and structural analogs, the fluorescence response change rate of the probe is less than 3%, which can be applied to the detection of complex matrix samples; Figure 10 When various interfering substances coexist, the change rate of characteristic fluorescence signals of L-AA and D-AA by FeCDs is less than 3%, and there is no significant impact on the ultraviolet absorption stability, which fully demonstrates its anti-interference ability.

[0024] (4) Excellent stability and practicality: Figure 7 This indicates that the probe is stable in the pH range of 4.0-10.0, with fluorescence intensity fluctuations of less than 1%. Figure 8 The results showed that after storage at 298 K (25℃), 318 K (45℃), and 338 K (65℃) for 12 days, the fluorescence intensity decay rates were 0.552%, 0.585%, and 0.813%, respectively, all below 0.9%. Figure 9 The probe has been shown to have a fast response speed, reaching maximum fluorescence change within 2-2.5 s and stabilizing within 5 s. It requires no complex sample pretreatment and is highly practical.

[0025] (5) Low preparation cost and simple process: Corn starch is used as raw material, which is widely available, cheap and easy to obtain. The preparation process only involves oxidation, hydrothermal reaction and conventional purification steps. It does not require expensive equipment and complicated operation, and is easy to scale up. The probe has low toxicity and good biocompatibility, and can be used for biological sample detection. Figure 2 TEM images confirmed that FeCDs were uniformly dispersed spherical particles, indicating successful preparation with excellent morphology; Figure 4 The ultraviolet-visible absorption spectra provide mechanistic support for the different interaction modes between FeCDs and the two chiral isomers.

[0026] (6) Visual recognition capability: When irradiated with a 365 nm ultraviolet lamp, L-AA makes the system exhibit blue-green fluorescence, and D-AA quenches the red fluorescence of the system. The two chiral configurations can be quickly distinguished without any instruments or equipment, making it suitable for rapid on-site screening. Attached Figure Description

[0027] Figure 1 This is a schematic diagram illustrating the preparation process of the FeCDs dual-mode fluorescent probe of the present invention and its recognition of chiral ascorbic acid (L-AA / D-AA).

[0028] Figure 2 TEM images of FeCDs particles (Inset: Corresponding HRTEM images of FeCDs).

[0029] Figure 3 The fluorescence emission spectra of FeCDs at different excitation wavelengths are shown.

[0030] Figure 4 The UV-Vis absorption spectra of FeCDs, FeCDs+L-AA, and FeCDs+D-AA are shown.

[0031] Figure 5 (a) Fluorescence emission spectra of FeCDs after adding different concentrations of L-AA (1.5-200 μmol / L); (b) Fluorescence emission spectra of FeCDs after adding different concentrations of D-AA (1.5-200 μmol / L); (c) Linear fitting of L-AA concentration with fluorescence intensity at 430 nm; (d) Linear fitting of L-AA concentration with fluorescence intensity at 610 nm; (e) Linear fitting of D-AA concentration with fluorescence intensity at 610 nm.

[0032] Figure 6 CIE chromaticity maps for (a) L-AA and (b) D-AA to identify FeCDs.

[0033] Figure 7 The effect of pH on the fluorescence response intensity of FeCDs in recognizing different concentrations of ascorbic acid.

[0034] Figure 8 The storage stability curves of FeCDs at different temperatures are shown.

[0035] Figure 9 The reaction rates and kinetics of FeCDs reacting with 200 μmol / L L-AA and D-AA are fitted: (a, b) FeCDs + L-AA system; (c) FeCDs + D-AA system.

[0036] Figure 10Under the condition of coexistence of various interfering substances: (a) characteristic fluorescence intensity changes of the FeCDs+L-AA and (b) FeCDs+D-AA systems; (c) ultraviolet absorption stability of the FeCDs+L-AA and (d) FeCDs+D-AA systems.

[0037] Figure 11 Fluorescence emission spectrum of CDs prepared from single-component raw materials after adding L-AA / D-AA mixed solution.

[0038] Figure 12 Fluorescence emission spectra of FeCDs and undoped FeCDs after the addition of L-AA and D-AA. Detailed Implementation

[0039] To provide a clearer understanding of the present invention, the following embodiments are further illustrations of the invention. The descriptions below are illustrative and not restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0040] Example 1: Preparation of FeCDs (1) Preparation of dialdehyde starch (DAS): Accurately weigh 0.5 g of corn starch, add 50 mL of 0.5 mol / L sodium periodate solution, stir magnetically until uniform, place in a 40℃ constant temperature water bath, react for 4 h under light-protected conditions to obtain DAS solution, wash and filter through Buchner funnel and dry to obtain white DAS powder.

[0041] (2) Hydrothermal synthesis of FeCDs: Weigh 0.250 g DAS, 0.247 g 1,2-diaminonaphthalene and 0.253 g FeCl3 in a molar ratio of 1:1:1, add 30 mL of ultrapure water, and ultrasonically disperse at 120 W for 10 min until the mixture is homogeneous. Transfer to a 50 mL polytetrafluoroethylene-lined high-pressure reactor, adjust the pH of the system to 4.0 with 0.1 mol / L HCl, seal and place in a blower box, and react at 200℃ for 8 h.

[0042] (3) Purification and drying: After the reaction is completed, the reaction vessel is allowed to cool naturally to room temperature. The product is then vacuum filtered through a 0.22 μm aqueous microporous membrane and the filtrate is collected. The filtrate is placed in a 1000 Da dialysis bag and dialyzed in ultrapure water for 24 h, with the ultrapure water being replaced every 8 h to remove unreacted small molecule impurities. The dialyzed FeCDs aqueous solution is placed in a freeze dryer and freeze-dried at -50℃ for 24 h to obtain a brown-black solid FeCDs sample. The sample is then sealed and stored in a desiccator.

[0043] (4) Preparation of FeCDs stock solutions: Solid FeCDs samples were redispersed in deionized water to prepare FeCDs stock solutions of different concentrations. To ensure that the FeCDs stock solutions have optimal fluorescence performance and recognition activity, fluorescence intensity of FeCDs solutions of different concentrations (50, 100, 150, 200, 250, 300 mg / L) and their response performance to 200 μmol / L L-AA / D-AA were measured. The results showed that when the FeCDs concentration was below 200 mg / L, the initial fluorescence intensity of the system was low, and the detection signal-to-noise ratio was insufficient; when the concentration was above 200 mg / L, the fluorescence quantum yield decreased due to the enhanced internal filtration effect, and the response change rate to the target analyte decreased. At a concentration of 200 mg / L, the FeCDs stock solution could provide a sufficiently strong initial fluorescence signal to ensure detection sensitivity, while maintaining the optimal response change range to L-AA and D-AA. Therefore, 200 mg / L was selected as the optimal working concentration of the FeCDs stock solution. At this concentration, the system ensures a sufficient initial fluorescence signal-to-noise ratio while maintaining optimal fluorescence response to L-AA and D-AA. The stock solution is stored at 4°C for later use; at this concentration, the probe exhibits stable fluorescence performance and recognition activity.

[0044] like Figure 1 As shown in the figure, the preparation of dialdehyde starch (DAS) from corn starch is illustrated. Then, FeCDs are synthesized by hydrothermal method with 1,2-diaminonaphthalene and FeCl3. After purification, the FeCDs exhibit a dual-mode response of blue-green fluorescence and fluorescence quenching to L-AA and D-AA, respectively.

[0045] The morphology, structure, and optical properties of the prepared FeCDs were characterized using TEM, UV-vis, and fluorescence spectroscopy, respectively. Figure 2 It can be seen that the prepared FeCDs are uniformly dispersed spherical particles with an average particle size of 3.2 nm, clear lattice fringes, and no obvious agglomeration. Figure 3 The fluorescence emission spectra of FeCDs at different excitation wavelengths show that the position of the characteristic emission peak at 610 nm remains basically stable as the excitation wavelength increases from 320 nm to 400 nm, indicating that FeCDs are wavelength-independent. Figure 4 The UV-Vis absorption spectra showed that FeCDs had a characteristic absorption peak at 291 nm. After the addition of L-AA, the absorbance at this wavelength was significantly enhanced, while the absorbance changed slowly after the addition of D-AA, indicating that the mechanism of action of FeCDs on the two chiral isomers is different. Figure 5 The fluorescence titration results showed that L-AA induced fluorescence quenching at 610 nm and generated a new peak at 430 nm, while D-AA only caused fluorescence quenching at 610 nm, exhibiting a dual-mode response characteristic. Figure 6 The CIE chromaticity diagram confirmed that FeCDs exhibited significant differences in fluorescence color after interaction with L-AA and D-AA. These results demonstrate that FeCDs were successfully prepared and possess excellent optical properties and chiral recognition potential.

[0046] Example 2: Effect of raw materials for synthesizing FeCDs carbon dots on the detection sensitivity of analytes To investigate the effect of the raw materials used in the synthesis of FeCDs carbon dots on the detection sensitivity of the analytes, three batches of FeCDs samples were prepared in parallel using corn starch, 1,2-diaminonaphthalene, and FeCl3 from three different manufacturers and batches, following the steps described above. The results showed that there were no significant differences in particle size distribution, elemental doping ratio, fluorescence quantum yield, and detection sensitivity against ascorbic acid among the three batches of samples, and the relative standard deviation (RSD) was less than 2.0%, proving that the probe performance is only related to the properties of FeCDs themselves and is independent of the source and batch of raw materials.

[0047] Example 3: Effect of raw material ratio on the performance of FeCDs Following the preparation steps of Example 1, with the reaction conditions (pH=4.0, 200℃, 8h) and other steps unchanged, only the molar ratio of dialdehyde starch, 1,2-diaminonaphthalene, and FeCl3 was varied. When the molar ratio of dialdehyde starch, 1,2-diaminonaphthalene, and FeCl3 was 2:1:2, the yield of FeCDs was 76.2%; the highest yield (88.6%) was achieved at a molar ratio of 1:1:1; while the yield decreased to 67.8% at a molar ratio of 1:2:1. The results indicate that a molar ratio of 1:1:1 allows the precursor to participate most effectively in the reaction, forming a stable carbon dot structure; therefore, this ratio was chosen as the optimal feedstock ratio.

[0048] Example 4: Effects of reaction conditions (temperature, pH, time) on the performance of FeCDs Following the preparation steps of Example 1, with the optimal raw material molar ratio fixed (1:1:1), the effects of reaction temperature, pH value, and reaction time on the yield and fluorescence quantum yield of FeCDs were investigated sequentially. To ensure the consistency and comparability of experimental data, each optimization step was performed independently as a complete experimental series, and the results are as follows.

[0049] With fixed reaction conditions of pH=7 and reaction time of 6 h, the effect of different temperatures on the performance of FeCDs was investigated. At 160℃, the yield of FeCDs was 64.6%, and the quantum yield was 24.7%. When the temperature was increased to 200℃, the yield was 76.8%, and the quantum yield was 37.4%. Further increasing the temperature to 240℃, the yield decreased to 71.7%, and the quantum yield decreased to 28.4%. The experimental results indicate that 200℃ is the optimal reaction temperature for obtaining high yields and high quantum yields.

[0050] At the optimized temperature of 200℃ and a fixed reaction time of 6 h, the effect of different pH values ​​on the performance of FeCDs was investigated. At pH=4, the yield of FeCDs was 87.6%, and the quantum yield was 34.6%. At pH=7, the yield was 68.5%, and the quantum yield was 22.6%. At pH=11, the yield was 66.5%, and the quantum yield was 25.8%. The results indicate that the highest yield and relatively high quantum yield of FeCDs were observed at pH=4.0; therefore, pH=4.0 was selected as the optimal reaction pH.

[0051] Under the optimized conditions of 200℃ and pH 4.0, with other parameters kept constant, the effects of reaction times of 6 h, 8 h, and 10 h on the performance of FeCDs were investigated. The results showed that at 6 h, the yield of FeCDs was 87.4%, and the quantum yield was 34.3%. Extending the reaction time to 8 h increased the yield to 88.6% and the quantum yield to 38.7%. Further extending the reaction time to 10 h slightly increased the yield to 89.3%, but the quantum yield decreased to 31.5%. Considering both yield and quantum yield, 8 h was selected as the optimal reaction time.

[0052] Example 5: Effect of detection conditions on the performance of FeCDs Further investigation was conducted into the effect of detection conditions (pH value) on the ability of FeCDs to recognize ascorbic acid. For example... Figure 7 As shown, under pH=4.0 conditions, the fluorescence response intensity of FeCDs to different concentrations of ascorbic acid showed the most significant changes, and the addition of L-AA resulted in the most significant changes in I. 430 / I 610 The ratio showed the largest variation. However, at pH 10.0, the response change tended to be gradual. These results indicate that a weakly acidic environment is more conducive to the chiral recognition interaction between FeCDs and ascorbic acid, consistent with the results of the synthesis optimization.

[0053] Example 6: Storage stability and fast response detection performance of FeCDs The storage stability of FeCDs prepared under optimal conditions (200℃, pH=4.0, reaction time 8h) was investigated, such as... Figure 8 As shown, after 12 days of storage at 298 K (25℃), 318 K (45℃), and 338 K (65℃), the fluorescence intensity decay rates of FeCDs were 0.552%, 0.585%, and 0.813%, respectively, indicating that FeCDs have good storage stability.

[0054] The interaction kinetics of FeCDs prepared under optimal conditions with L-AA and D-AA were studied, such as... Figure 9As shown, when FeCDs react with L-AA, the fluorescence intensity decay at 610 nm and the fluorescence intensity increase at 430 nm both conform to the pseudo-first-order reaction kinetic equation, and the fitted R... 2 They are 0.9978 and 0.9978 respectively. Figure 9 a, 9b); When FeCDs react with D-AA, the attenuation of fluorescence intensity at 610 nm follows a pseudo-first-order reaction kinetic equation, R 2 =0.9981 ( Figure 9 c). The results show that FeCDs react rapidly with L-AA and D-AA, reaching equilibrium within seconds, and exhibiting good fit, confirming their rapid response detection performance.

[0055] Considering yield, quantum yield, pH response performance, storage stability, and reaction kinetics, the optimal synthesis conditions for FeCDs were determined to be: reaction temperature 200℃, reaction pH=4.0, and reaction time 8 h. FeCDs prepared under these conditions exhibit the best overall performance and can be used for subsequent research on the detection of chiral ascorbic acid.

[0056] Example 7: Qualitative and quantitative detection of L-AA and D-AA using FeCDs dual-mode fluorescent probes Detection of L-AA: Mix 0.5 mL of the 200 mg / L FeCDs stock solution prepared in Example 1 with 1 mL of 10 mmol / L PBS buffer (pH 4.0), add 2.5 mL of 200 μmol / L L-AA standard solution, mix well, and let stand at room temperature for 5 min. Observe under a 365 nm UV lamp; the system exhibits blue-green fluorescence, qualitatively confirming the presence of L-AA. Measure the fluorescence emission spectrum using 360 nm as the excitation wavelength, as shown below. Figure 5 As shown in (a), the results indicate that with the addition of L-AA, the fluorescence intensity at 610 nm gradually decreases, and a new emission peak appears at 430 nm. (The last sentence appears to be incomplete and possibly refers to I-AA.) 430 / I 610 The ratio was used as the detection signal to plot a standard curve. After three parallel experiments, the L-AA concentration in the sample was calculated to be 198.6 μmol / L based on the standard curve, with a relative standard deviation (RSD) of 1.2%.

[0057] Detection of D-AA: Mix 0.5 mL of the 200 mg / L FeCDs stock solution prepared in Example 1 with 1 mL of 10 mmol / L PBS buffer (pH 4.0), add 2.5 mL of 200 μmol / L D-AA standard solution, mix well, and let stand at room temperature for 5 min. Observe under a 365 nm UV lamp; the red fluorescence of the system quenches, qualitatively confirming the presence of D-AA. Measure the fluorescence emission spectrum using 360 nm as the excitation wavelength, as shown below. Figure 5 As shown in (b), the results indicate that with the addition of D-AA, the fluorescence intensity at 610 nm is significantly quenched, and no new emission peak is generated. The fluorescence intensity at 610 nm (I610) is used as the reference value. 610 Using the detection signal as a guideline, a standard curve was plotted. After three parallel experiments, the calculated concentration of D-AA in the sample based on the standard curve was 201.3 μmol / L, with a relative standard deviation (RSD) of 1.5%.

[0058] Example 8: Study on the anti-interference performance of FeCDs dual-mode fluorescent probes Experimental Methods: A series of 0.5 mL FeCDs stock solutions prepared in Example 1 were mixed with 1 mL of 10 mmol / L PBS buffer (pH 4.0). In the "FeCDs + L-AA" system, 2.4 mL of 200 μmol / L L-AA standard solution was added first, followed by 0.1 mL of various potential interfering solutions at a concentration of 10 mmol / L, to bring the final concentration of interfering substances to the corresponding levels (500 μmol / L for inorganic ions, and 200 μmol / L for organic acids, sugars, amino acids, etc.). The total volume was then increased to 4 mL with ultrapure water. After mixing, the mixture was allowed to stand at room temperature for 5 min, and the fluorescence spectrum was measured. The system without interfering substances was used as a blank control, and the fluorescence response change rate was calculated. The same method was used to test the "FeCDs + D-AA" system.

[0059] The potential interfering substances selected in this experiment include: inorganic ions (Na+). + K + Ca 2+ Mg 2+ Cu 2+ Zn 2+ HCO3 - HPO4 2 + Organic acids and sugars (L-tartaric acid, D-tartaric acid, L-malic acid, D-malic acid, uric acid, dehydroascorbic acid, glucose, maltose), amino acids and bioactive molecules (L-arginine, D-arginine, L-aspartic acid, D-aspartic acid, L-lysine, D-lysine, L-tryptophan, D-tryptophan, histidine, tyrosine, cysteine, homocysteine, glutathione, dopamine).

[0060] Experimental results are as follows Figure 10 As shown. In the presence of all listed potential interfering substances, the FeCDs system is affected by L-AA (… Figure 10 a) and D-AA ( Figure 10 (b) The rate of change of the characteristic fluorescence signal is less than 3%; at the same time... Figure 10 c and Figure 10As shown in Figure d, these interfering substances have no significant effect on the UV absorption stability of the FeCDs+L-AA and FeCDs+D-AA systems. This indicates that the FeCDs dual-mode fluorescent probe prepared in this invention has good anti-interference ability against the above substances and can achieve specific recognition and detection of L-AA and D-AA in complex sample matrices.

[0061] Example 9: Detection of L-AA in real orange juice samples using FeCDs dual-mode fluorescent probes Sample preparation: Take 5 mL of a fresh orange juice sample, filter it through a 0.22 μm filter membrane, and dilute it 10 times with deionized water to prepare the sample solution to be tested.

[0062] Detection Experiment: 0.5 mL of the 200 mg / L FeCDs stock solution prepared in Example 1 was mixed with 1 mL of 10 mmol / L PBS buffer (pH 4.0), and 2.5 mL of the sample solution was added. After mixing, the mixture was allowed to stand at room temperature for 5 min. Under UV light, the system exhibited blue-green fluorescence, indicating that the sample mainly contained L-AA. The fluorescence emission spectrum was measured at an excitation wavelength of 360 nm. Based on the L-AA standard curve in Example 7, the L-AA content in the orange juice sample was calculated to be 58.3 μmol / L. A spiked recovery experiment was conducted by adding L-AA standard to the sample (spiking concentration of 50 μmol / L). After spiking, the total L-AA content in the sample was measured to be 107.9 μmol / L, and the average recovery rate was calculated to be 99.8%. The relative standard deviation (RSD) of the three parallel determinations was 1.8%.

[0063] Example 10: Simultaneous Differentiation and Detection of L-AA and D-AA in Mixed Samples Using FeCDs Dual-Mode Fluorescent Probes Sample preparation: Prepare a mixed solution containing L-AA (final concentration 100 μmol / L) and D-AA (final concentration 100 μmol / L) using 10 mmol / L PBS buffer at pH 4.0.

[0064] Detection Experiment: 0.5 mL of the FeCDs stock solution prepared in Example 1 was mixed with 1 mL of 10 mmol / L PBS buffer (pH 4.0), and 2.5 mL of the above mixed sample solution was added. After mixing, the mixture was allowed to stand at room temperature for 5 min. First, the fluorescence color was observed under a 365 nm UV lamp. The system exhibited an intermediate color between blue-green and red, indicating the simultaneous presence of L-AA and D-AA in the system. Then, the fluorescence emission spectrum was measured using 360 nm as the excitation wavelength. The results showed a new emission peak at 430 nm, while the fluorescence intensity at 610 nm underwent partial quenching. The L-AA standard curve established in Example 6 (based on I...) was used. 430 / I 610 The concentration of L-AA in the mixed sample was calculated to be 98.5 μmol / L; using the D-AA standard curve (based on I... 610 The concentration of D-AA was calculated to be 101.2 μmol / L based on the degree of quenching. The recoveries in three parallel experiments were approximately 98.5% and 101.2%, with RSDs less than 3%. These results indicate that the probe can effectively distinguish and accurately quantify L-AA and D-AA in complex mixed systems.

[0065] Comparative Example 1: Comparison of fluorescence responses of carbon dots and FeCDs prepared from single-component raw materials to L-AA / D-AA mixed solution. Preparation of single-component carbon dots: DAS-CDs and NAP-CDs were prepared using 0.250 g DAS and 0.247 g 1,2-diaminonaphthalene as single raw materials, respectively, following the hydrothermal reaction, purification, and drying steps of Example 1, and stock solutions of 200 mg / L were prepared. The preparation of FeCDs was the same as in Example 1.

[0066] Detection experiment: 0.5 mL of each of the three carbon dot stock solutions (DAS-CDs, NAP-CDs, FeCDs) was mixed with 1 mL of 10 mmol / L PBS buffer (pH 4.0). Then, 2.5 mL of 200 μmol / L L-AA / D-AA mixed solution was added to each solution. After mixing, the mixture was allowed to stand at room temperature for 5 min. The fluorescence emission spectra were measured at an excitation wavelength of 360 nm. The results are as follows: Figure 11 As shown.

[0067] Depend on Figure 11 It was observed that only FeCDs exhibited a significant bimodal fluorescence response (fluorescence quenching at 610 nm and a new emission peak at 430 nm) after interaction with the L-AA / D-AA mixed solution, while the fluorescence spectra of the other two carbon dots showed no significant changes. This indicates that carbon dots prepared from a single component (DAS or 1,2-diaminonaphthalene) cannot achieve chiral recognition of L-AA / D-AA, and only FeCDs prepared through iron doping and the synergistic effect of bifunctional precursors possess the specific recognition ability of chiral ascorbic acid.

[0068] Comparative Example 2: Detection of chiral ascorbic acid by undoped Fe carbon dots (CDs) Preparation of CDs: Weigh 0.250 g DAS and 0.247 g 1,2-diaminonaphthalene at a molar ratio of 1:1, add 30 mL of ultrapure water, and ultrasonically disperse for 10 min until uniformly mixed. Transfer to a 50 mL polytetrafluoroethylene-lined high-pressure reactor, adjust the pH of the system to 4.0 with 0.1 mol / L HCl, seal, and place in a blower box. React at 200℃ for 8 h. Purification and drying steps are the same as in Example 1 to obtain Fe-free CDs samples, which are then prepared into a 200 mg / L stock solution.

[0069] Detection experiment: 0.5 mL of the above CDs stock solution was mixed with 1 mL of 10 mmol / L PBS buffer (pH 4.0), and 2.5 mL of 200 μmol / L L-AA and D-AA standard solutions were added respectively. After mixing, the mixture was allowed to stand at room temperature for 5 min. The fluorescence emission spectrum was measured at an excitation wavelength of 360 nm, and FeCDs were used as a comparison. The results are as follows: Figure 12 As shown.

[0070] Depend on Figure 12 It can be seen that the fluorescence emission spectra of undoped carbon dots (CDs) after the addition of L-AA or D-AA showed no significant change compared to blank CDs, and there was almost no difference between the two. However, FeCDs exhibited significant dual-mode fluorescence response characteristics after the addition of L-AA and D-AA, respectively (L-AA induced a new peak at 430 nm, and D-AA induced fluorescence quenching at 610 nm). This fully demonstrates that undoped carbon dots cannot recognize and distinguish chiral ascorbic acid, and that iron doping is the core key to endowing carbon dots with chiral recognition ability.

[0071] Comparative Example 3: Detection of L-AA / D-AA by existing nitrogen-doped carbon dot probes Preparation of nitrogen-doped carbon dots: Following existing methods, nitrogen-doped carbon dots (N-CDs) were prepared by hydrothermal method using citric acid and ethylenediamine as raw materials, and a stock solution of 200 mg / L was prepared.

[0072] Detection experiment: Mix 0.5 mL of N-CDs stock solution with 1 mL of 10 mmol / L PBS buffer at pH 4.0, add 2.5 mL of 200 μmol / L L-AA and D-AA standard solutions respectively, mix well, let stand at room temperature for 5 min, and measure the fluorescence emission spectrum.

[0073] Experimental results show that the N-CDs exhibit only a single fluorescence emission peak at 440 nm. The fluorescence intensity is quenched to the same degree upon the addition of L-AA and D-AA, indicating a lack of chiral recognition ability and an inability to distinguish between L-AA and D-AA. Its detection limit for total ascorbic acid is 0.12 μmol / L, with a linear range of 2-100 μmol / L. This detection performance is significantly inferior to the FeCDs dual-mode fluorescent probe of this invention, further demonstrating the innovation and superiority of this invention.

[0074] This invention systematically investigated the optimization of the preparation process of FeCDs dual-mode fluorescent probes and their detection performance on chiral ascorbic acid through Examples 1-10 and Comparative Examples 1-3. Figure 5 It can be seen that FeCDs exhibit distinctly different dual-mode fluorescence responses to L-AA and D-AA: L-AA induces fluorescence quenching at 610 nm and generates a new emission peak at 430 nm, while D-AA only induces fluorescence quenching at 610 nm. The two differ in their CIE chromaticity diagrams (…). Figure 6 The significant differences in fluorescent colors are beneficial for visual differentiation. Figure 10 Anti-interference experiments showed that, under the coexistence of multiple potential interfering substances, the characteristic fluorescence signal change rate of FeCDs for L-AA and D-AA was less than 3%, indicating good selectivity. Figure 7-9 Further confirmation indicates that FeCDs exhibit rapid response kinetics and excellent storage stability under optimized conditions (pH=4.0, 25℃). From Figure 11 and Figure 12 It is evident that carbon dots (DAS-CDs, NAP-CDs) prepared from single components and undoped Fe CDs cannot effectively recognize L-AA / D-AA mixed solutions. Only FeCDs prepared through iron doping and the synergistic effect of bifunctional precursors possess chiral recognition capabilities. The results of the influence of reaction conditions (temperature, pH, time) on the performance of FeCDs show that the response performance of carbon dots prepared under non-optimal synthesis conditions is significantly reduced to the target analyte, further verifying the necessity of the optimized process of this invention.

[0075] Obviously, the FeCDs dual-mode fluorescent probe prepared by this invention can be used for the detection of chiral ascorbic acid, enabling rapid qualitative differentiation and highly sensitive quantitative detection. It is beneficial for developing simple methods of visual-assisted analysis and has broad application prospects in the fields of food, medicine, and biological samples.

[0076] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A dual-mode fluorescent probe based on iron-doped carbon dots, characterized in that, The probe is an iron-doped carbon dot (FeCDs). The FeCDs exhibit a response of fluorescence quenching at 610 nm and generating a new emission peak at 430 nm to L-ascorbic acid, and a response of fluorescence quenching only at 610 nm to D-ascorbic acid, thus achieving dual-mode fluorescence recognition.

2. The dual-mode probe according to claim 1, characterized in that, The FeCDs are wavelength-independent and have a characteristic emission peak at 610 nm.

3. The dual-mode probe according to claim 1, characterized in that, The FeCDs have a characteristic UV-Vis absorption peak at 291 nm.

4. The method for preparing the dual-mode fluorescent probe according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Preparation of dialdehyde starch: Corn starch and sodium periodate solution were mixed at a liquid-solid ratio of 100 mL: 1 g and stirred at 40℃±2℃ in the dark for 4 h±0.5 h to obtain dialdehyde starch. (2) Hydrothermal synthesis of FeCDs: According to the raw material molar ratio of 1:1:1, dialdehyde starch, 1,2-diaminonaphthalene and FeCl3 were uniformly mixed, ultrapure water was added, and ultrasonic dispersion was carried out for 10 min until the mixture was uniform. The mixture was then transferred to a high-pressure reactor with a polytetrafluoroethylene liner, the pH of the system was adjusted to 4.0±0.1, and the hydrothermal reaction was carried out at 200℃±5℃ for 8 h±0.5 h. (3) Purification and drying of FeCDs: After the reaction system is cooled to room temperature, the resulting mixture is vacuum filtered through an aqueous microporous membrane. The filtrate is dialyzed in ultrapure water for 24 h using a 1000 Da dialysis bag to obtain a purified FeCDs aqueous solution. The purified FeCDs aqueous solution is freeze-dried to obtain a solid FeCDs sample.

5. The preparation method according to claim 4, characterized in that, In step (2), the ultrasonic power is 100-150 W.

6. The preparation method according to claim 4, characterized in that, In step (3), the freeze-drying conditions are freeze-drying at -50℃±5℃ for 24 h±2 h.

7. The application of a dual-mode fluorescent probe as described in any one of claims 1-3 in the detection of chiral ascorbic acid.

8. The application according to claim 7, characterized in that, The detection method includes the following steps: (1) Preparation of FeCDs stock solution: Disperse solid FeCDs samples in deionized water to prepare a stock solution with a concentration of 200 mg / L; (2) Sample detection: Take FeCDs stock solution and mix it with 10 mmol / L PBS buffer at pH 4.0, add the sample solution to be tested, mix thoroughly and let stand at room temperature for 5 min, and observe the change in fluorescence color under a 365 nm UV lamp; (3) Result determination: If the system shows blue-green fluorescence, it indicates that the sample contains L-ascorbic acid; if the red fluorescence of the system is quenched, it indicates that the sample contains D-ascorbic acid.

9. The application according to claim 7, characterized in that, The detection method includes the following steps: (1) Preparation of FeCDs stock solution: Disperse solid FeCDs samples in deionized water to prepare a stock solution with a concentration of 200 mg / L; (2) Sample detection: Take FeCDs stock solution and mix it with 10 mmol / L PBS buffer at pH 4.0, add the sample solution to be tested, mix thoroughly and let stand at room temperature for 5 min, measure the fluorescence emission spectrum at 360 nm as the excitation wavelength, and measure the fluorescence emission spectrum of the system in the range of 380-700 nm. (3) Take I 430 / I 610 The ratio is the detection signal or I 610 To detect the signal, a standard curve is plotted, and the concentration of L-AA or D-AA in the sample is calculated based on the standard curve.

10. The application according to claim 7, characterized in that, The detection concentration range of the chiral ascorbic acid is 1.5-200 μmol / L, the detection limit of L-ascorbic acid is 0.028 μmol / L, and the detection limit of D-ascorbic acid is 0.033 μmol / L.

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