Mercury ion interference resistant method for detecting fe3+ fluorescence and application thereof

Abstract

This invention discloses a Fe ion-resistant material. 3+ The fluorescence detection method and its application include the following steps: (1) adding soluble iodide to the sample solution for pretreatment; (2) mixing the pretreated sample solution, nitrogen-doped carbon dot solution, and acetate buffer solution to obtain a mixed detection system; (3) after the mixed detection system is incubated at room temperature to reach equilibrium, measuring its fluorescence intensity at an emission wavelength of 450 nm, and quantitatively analyzing the Fe in the sample solution by the degree of quenching of the fluorescence intensity. 3+ Concentration. This invention ingeniously utilizes the extremely strong coordination affinity between iodide ions and mercury ions to form a power mask, specifically eliminating the interference of mercury ions without affecting the detection of the target ion. This allows the sensor to accurately identify Fe even in complex interference systems where dozens of competing metal ions coexist. 3+ It exhibits extremely high analytical specificity and strong anti-interference performance.

Description

Technical Field

[0001] This invention belongs to the field of luminescent nanomaterials and analytical chemistry detection technology, specifically relating to a Fe nanomaterial resistant to mercury ion interference. 3+ Fluorescence detection methods and their applications. Background Technology

[0002] Iron is one of the essential trace elements for the human body, playing a crucial role in various biological processes such as oxygen transport, electron transfer, and enzymatic reactions. In the serum of healthy individuals, iron is primarily composed of Fe²⁺. 3+ It exists in the form of iron. When the human body is in a pathological state such as liver dysfunction, hereditary hemochromatosis, hemolytic disease, or excessive iron intake, serum iron may be elevated. 3+ An abnormally high level of Fe, with excessive Fe 3+ It catalyzes the generation of reactive oxygen species through Fenton-type reactions, leading to oxidative stress and cell damage. Therefore, it is necessary to construct a simple, rapid, highly sensitive, and highly selective Fe2+ oxidase inhibitor suitable for serum samples. 3+ The new detection method has significant research value and application implications for clinical iron metabolism assessment, bioanalysis, and auxiliary diagnosis of diseases.

[0003] Currently, various Fe products have been developed both domestically and internationally. 3+ Quantitative analysis techniques mainly include atomic absorption spectrometry, inductively coupled plasma mass spectrometry, electrochemical methods, and colorimetric analysis. However, while atomic absorption spectrometry and inductively coupled plasma mass spectrometry offer excellent sensitivity and accuracy, their high cost and complex operation limit their application in routine detection and on-site analysis. Electrochemical methods utilize Fe... 3+ The redox behavior provides good sensitivity, but its stability and anti-interference ability in complex matrices still need to be improved; colorimetric methods are simple and fast, but generally suffer from low sensitivity and insufficient selectivity, and are easily affected by matrix interference in complex biological samples such as whole blood and serum.

[0004] In contrast, fluorescence sensing detection has attracted increasing attention due to its high sensitivity, rapid response, and ease of operation. Among various fluorescence sensing materials, nitrogen-doped carbon dots (NCDs) have become highly promising fluorescent probes for metal ion detection due to their excellent photoluminescence properties, good water solubility, low toxicity, and abundant surface functional groups (such as hydroxyl, carboxyl, and amino groups). Previous studies have confirmed that Fe... 3+ Fluorescence of carbon dots can be effectively quenched through mechanisms such as surface complexation and electron transfer. Based on this mechanism, researchers have developed various methods for quenching the fluorescence of Fe. 3+ The detected carbon dot fluorescent probe.

[0005] However, most existing research on carbon dot sensors has been conducted in pure water systems, and exploration of practical applications in complex biological matrices such as human serum remains limited. In actual sample detection, coexisting heavy metal ions often cause severe specific interference to the detection system. For example, when mercury ions (Hg) are present in the sample... 2+ When this occurs, it also induces significant fluorescence quenching of nitrogen-doped carbon dots, thereby affecting the fluorescence of Fe. 3+ Fluorescence detection systems cause significant interference, reducing the specificity and accuracy of analytical methods. Therefore, it is crucial to find a simple and efficient way to specifically eliminate Hg while maintaining the excellent luminescence properties of carbon dots. 2+ To mitigate quenching interference, a Fe2-based novel quenching agent with both strong anti-interference capability and high selectivity is constructed. 3+ Fluorescence detection platforms are currently the most advanced technology for detecting Fe in complex biological systems. 3+ Technical bottlenecks that urgently need to be addressed in the field of quantitative analysis. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide an Fe that resists mercury ion interference. 3+ Fluorescence detection method.

[0007] Another object of the present invention is to provide the above-mentioned Fe 3+ Application of fluorescence detection methods.

[0008] The technical solution of the present invention is as follows:

[0009] A Fe resistant to mercury ion interference 3+ The fluorescence detection method includes the following steps:

[0010] (1) Add soluble iodide to the test sample solution containing or suspected of containing mercury ion interference for pretreatment;

[0011] (2) The sample solution to be tested, nitrogen-doped carbon dot solution and acetate buffer solution after pretreatment in step (1) are mixed to obtain a mixed detection system containing free iodide ions;

[0012] (3) After the mixed detection system obtained in step (2) is incubated at room temperature to reach equilibrium, its fluorescence intensity at an emission wavelength of 450 nm is measured at an excitation wavelength of 355 nm. The degree of quenching of this fluorescence intensity is used to quantitatively analyze the Fe in the sample solution to be tested. 3+ concentration;

[0013] In the reaction environment maintained by the aforementioned acetate buffer solution, iodide ions ionized from the soluble iodide bind to mercury ions in the system, and nitrogen-doped carbon dots bind to Fe in the system through their surface hydroxyl, carboxyl, or amino functional groups. 3+ It exhibits a ground-state chemical coordination complex structure.

[0014] In a preferred embodiment of the present invention, the acetate buffer solution in step (2) has a pH of 3.5 and a concentration of 2 M.

[0015] In a preferred embodiment of the present invention, the nitrogen-doped carbon dot solution in step (2) is a solution with a concentration of 0.2 mg / mL prepared by dissolving nitrogen-doped carbon dot powder in deionized water.

[0016] More preferably, the preparation of the nitrogen-doped carbon dot powder includes: mixing and stirring citric acid, formamide, and N,N-dimethylformamide, and maintaining the reaction at a constant temperature of 180°C for 4 h; after the reaction, centrifuging the resulting solution to remove insoluble particles, adding acetone as an eluent to the supernatant until there is no fluorescence in the supernatant, collecting the precipitate, and finally dissolving the precipitate in water and vacuum drying; the ratio of citric acid, formamide, and N,N-dimethylformamide is 1 g: 1.4 mL: 10 mL.

[0017] In a preferred embodiment of the present invention, the mixed detection system in step (2) is composed of a nitrogen-doped carbon dot solution with a concentration of 0.2 mg / mL, an acetate buffer solution with a concentration of 2 M and a pH of 3.5, and the sample solution to be tested after pretreatment in step (1) in a volume ratio of 50: 50: 890-900.

[0018] In a preferred embodiment of the present invention, the room temperature incubation time in step (3) is 10 min.

[0019] In a preferred embodiment of the present invention, when the sample solution to be tested is a serum sample, the following serum pretreatment step is included before step (1): adding a 20 wt% trichloroacetic acid solution to the serum, mixing, stirring and heating to 90°C for 15 min; cooling and then sonicating, then centrifuging the mixture to remove protein precipitate, collecting the supernatant and adjusting the pH of the supernatant to 7.0 as the sample solution to be tested; the volume ratio of serum to trichloroacetic acid solution is 1:1.

[0020] A Fe resistant to mercury ion interference 3+ Fluorescence detection sensor, including:

[0021] Nitrogen-doped carbon dots are prepared by reacting a precursor system including citric acid, formamide and N,N-dimethylformamide. They serve as fluorescent probes and have hydrophilic functional groups including hydroxyl, carboxyl and amino groups on their surface.

[0022] An acetate buffer solution with a concentration of 2 M and a pH of 3.5 is used to maintain Fe.3+ The mixed reaction system for the fluorescence sensor to operate is in an environment with a pH of 3.5.

[0023] Iodide ion masking agent, which is a soluble iodide that can ionize into free iodide ions;

[0024] In the reaction environment of pH 3.5 maintained by the above-mentioned acetate buffer solution, the iodide ions ionized from the iodide ion masking agent are bound to the mercury ions in the system, and the nitrogen-doped carbon dots bind to the Fe in the system through the hydroxyl, carboxyl, or amino functional groups on their surface. 3+ It exhibits a ground-state chemical coordination complex structure.

[0025] In a preferred embodiment of the present invention, the ratio of citric acid, formamide and N,N-dimethylformamide is 1g: 1.4 mL: 10 mL.

[0026] More preferably, the reaction is carried out under constant temperature at 180°C for 4 hours.

[0027] The beneficial effects of this invention are:

[0028] 1. This invention introduces a specific iodine ion masking pretreatment step. In heavy metal ion screening of complex systems, mercury ions often interact strongly with carbon dot surface groups, producing severe non-specific fluorescence quenching interference. This invention cleverly utilizes the extremely strong coordination affinity between iodine ions and mercury ions to form a power mask, specifically eliminating mercury ion interference without affecting the detection of the target ion. This allows the sensor to accurately identify Fe even in complex interference systems where dozens of competing metal ions coexist. 3+ It exhibits extremely high analytical specificity and strong anti-interference performance.

[0029] 2. The Fe based on fluorescence quenching effect constructed in this invention 3+ The sensing platform exhibits a very wide linear detection range and a relatively low detection limit. This excellent sensitivity and wide detection range not only sensitively capture trace changes in iron ion concentration, but also perfectly cover Fe in human serum. 3+ The concentration range of physiological and pathological characteristics allows for flexible application to various routine clinical monitoring, emergency physical examination screenings, and quantitative analysis in specific pathological scenarios.

[0030] 3. The nitrogen-doped carbon dots (NCDs) fluorescent probes used as the sensing core in this invention can be easily and efficiently synthesized using a one-step lyothermal method involving citric acid, formamide, and DMF. In subsequent quantitative detection, the entire reaction system only requires a brief incubation at room temperature to reach absolute equilibrium, allowing for rapid data recording and processing using general-purpose fluorescence detection instruments. The overall method is extremely simplified, effectively overcoming the high cost and complex operational limitations of traditional large-scale mass spectrometry or spectroscopy techniques, making it highly suitable for routine clinical analysis and rapid on-site screening.

[0031] 4. The nano-carbon dot probe prepared by this invention exhibits excellent environmental tolerance, showing no significant attenuation under prolonged continuous excitation light irradiation, demonstrating strong resistance to photobleaching. Simultaneously, its fluorescence intensity remains essentially constant in high-concentration salt solutions, exhibiting good high-salt tolerance. Furthermore, it maintains good luminescence efficiency over a wide pH range. Combined with excellent batch-to-batch reproducibility, this ensures that the sensor can stably provide highly reliable and reproducible detection results in complex and variable biological matrix environments.

[0032] 5. This invention not only designs a scientific and thorough pretreatment process for deproteinizing and dissociating bound iron in serum, but also demonstrates satisfactory spike recovery and accuracy in actual human serum sample testing. Through parallel comparative testing with the industry-standard thiocyanate spectrophotometric method, the results of this invention show high consistency and reliability with the industry standard method. It can accurately and sensitively detect abnormally elevated iron ion levels in the serum of clinical patients, providing a simple, feasible, and promising new analytical method for assessing iron metabolism and assisting in the diagnosis of diseases in complex biological systems. Attached Figure Description

[0033] Figure 1 The synthesis of NCDs and Fe in Example 1 of this invention 3+ A schematic diagram of fluorescence detection.

[0034] Figure 2 The results of the microscopic structural and morphological characterization of the prepared NCDs in Example 1 of this invention are shown. Among them: (A) TEM image of NCDs; (B) Normal distribution of NCD diameter and actual images of NCDs in aqueous solution under white light and 365 nm excitation light; (C) X-ray diffraction pattern of NCDs.

[0035] Figure 3 The results of characterizing the surface functional groups and basic optical behavior of the prepared NCDs in Example 1 of this invention are shown. Among them: (A) FTIR spectra of NCDs and citric acid; (B) UV-vis and FL spectra of NCDs; (C) Fluorescence emission spectra of NCDs at different excitation wavelengths.

[0036] Figure 4 The figure shows the experimental results of multi-dimensional environmental stability assessment of the prepared NCDs in Example 1 of this invention. (A) Changes in fluorescence intensity of NCDs under continuous excitation light irradiation for 30 min; (B) Fluorescence intensity of NCDs in NaCl solutions of different concentrations; (C) Relative fluorescence intensity of different batches of synthesized NCDs; (D) Fluorescence intensity of NCDs in different pH environments.

[0037] Figure 5 This invention presents a performance comparison of three materials—NCDs, F-NCDs, and D-NCDs—in Example 1. Specifically: (A) Comparative photographs of the three materials under white light and 365nm excitation light; (B) A bar chart comparing the fluorescence quantum yields of NCDs and F-NCDs.

[0038] Figure 6 This is a comparative diagram of the experimental characterizations used in Example 1 of the present invention to deduce and confirm the intrinsic mechanism of Fe3+ surface complexation fluorescence quenching. Among them: (A) NCDs and NCDs+Fe 3+ (A) FTIR spectra of NCDs and NCDs+Fe 3+ UV-vis contrast; (C) NCDs and NCDs+Fe 3+ E g Calculation; (D) NCDs and Fe at different concentrations 3+ Comparison of Zeta potentials after addition; (E) NCDs and NCDs+Fe 3+ Comparison of fluorescence lifetimes; (F) Quenching constants at different temperatures; (G) Blank, Fe 3+ NCDs and NCDs+Fe 3+ Comparison of CV curves; (H) E of NCDs red (I) NCDs + Fe 3+ E red

[0039] Figure 7 In Example 1 of this invention, NCDs affect Fe 3+ Fluorescence quenching mechanism and schematic diagram of LUMO / HOMO energy levels.

[0040] Figure 8 This is a response diagram showing the results of specificity and selectivity testing and anti-heavy metal interference masking experiments in Example 1 of the present invention. Wherein: (A) Fluorescence response of different metal ions to NCDs; (B) Fe 3+ Fluorescence response of mixed solutions with different metal ions to NCDs; (C) Comparison with pretreated Hg 2+ The effect on the fluorescence intensity of NCDs.

[0041] Figure 9 For Fe in Embodiment 1 of the present invention 3+ Parameter curves showing comprehensive optimization of the fluorescence sensor detection environment and kinetic time. Among them: (A) NCDs and NCDs+Fe 3+ Fluorescence intensity in the pH range of 3.0–6.5; (B) NCDs and NCDs+Fe 3+ Comparison of F0 / F and ΔF in the pH range of 3.0–6.5; (C) Fe 3+ Changes in NCD fluorescence intensity within 30 minutes after addition.

[0042] Figure 10 In Example 1 of this invention, under optimal conditions, the sensor detects different concentrations of Fe. 3+ Standard curves for quantitative analysis and sensitivity assessment. (A) Fe at different concentrations. 3+ FL spectra of NCDs; (B) Fe at different concentrations 3 + Linear fitting with F0 / F; (C) Fe at different concentrations 3+ Physical image of NCDs fluorescence quenching. Detailed Implementation

[0043] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0044] Example 1

[0045] I. Experimental Methods

[0046] 1. Synthesis of NCDs

[0047] 1 g of citric acid, 1.4 mL of formamide, and 10 mL of N,N-dimethylformamide were thoroughly stirred in a beaker. The mixture was then transferred to a 50 mL polytetrafluoroethylene reactor and placed in a 180°C constant temperature drying oven for 4 h (e.g., ...). Figure 1 (As shown). The reacted solution was then centrifuged at 8000 r / min for 10 min to remove insoluble particles. A large amount of acetone was added as an eluent until no fluorescence was observed in the supernatant, and the product was collected. Finally, the product was dissolved in water and vacuum dried at 60 °C to collect the NCDs.

[0048] Simultaneously, the material synthesis was compared while maintaining a constant volume. 1g of citric acid and 11.4mL of formamide were thoroughly stirred in a beaker, then the mixture was transferred to a 50mL polytetrafluoroethylene (PTFE) reactor and placed in a 180℃ constant temperature drying oven for 4 hours to synthesize F-NCDs. 1g of citric acid and 11.4mL of N,N-dimethylformamide were thoroughly stirred in a beaker, then the mixture was transferred to a 50mL PTFE reactor and placed in a 180℃ constant temperature drying oven for 4 hours to synthesize D-NCDs.

[0049] 2. Fe 3+ Fabrication of fluorescence sensors

[0050] NCDs powder was prepared into a 0.2 mg / mL solution using deionized water, and a 2 M acetate buffer solution with pH 3.5 was prepared simultaneously. (The sentence fragment about Fe is incomplete and lacks context. It's unclear what "Fe" refers to.) 3+ In the detection experiment, 50 μL of NCDs solution and 50 μL of acetate buffer were added to 900 μL of Fe at different concentrations. 3+ In the solution, the final mixed volume is 1 mL. The mixture is incubated at room temperature for 10 min, and then the fluorescence intensity at the emission wavelength of 450 nm is recorded at an excitation wavelength of 355 nm (e.g., [missing information]). Figure 1 (As shown). The obtained data is processed, with F0 / F as y (F0 is Fe). 3+ The fluorescence intensity at a concentration of 0 mg / L, where F is Fe. 3+ Fluorescence intensity at a certain concentration), Fe 3+ The concentration of Fe is x, and linear fitting is used to determine the concentration of Fe. 3+ The detection range.

[0051] 3. Fe resisting mercury ion interference 3+ Fabrication of fluorescence sensors

[0052] KI was prepared into a 1 M KI solution using deionized water, and each solution was prepared fresh for each use. Simultaneously, a 0.2 mg / mL NCDs solution and a 2 M acetate buffer solution at pH 3.5 were prepared. To achieve a masking effect, 10 μL of KI solution and 50 μL of acetate buffer were added to 890 μL of Fe at different concentrations. 3+ The sample was thoroughly mixed for 5 min, and then 50 μL of NCDs solution was added, bringing the final mixture volume to 1 mL. The mixture was incubated at room temperature for 10 min, and then the fluorescence intensity was recorded at an excitation wavelength of 355 nm and an emission wavelength of 450 nm.

[0053] 4. Pretreatment of actual serum samples

[0054] To determine the Fe in human serum3+ To determine the concentration, the serum was first treated with trichloroacetic acid (TCA) to reduce Fe... 3+ The protein was released, and the procedure was as described in the literature. Add 1 mL of 20% TCA to 1 mL of serum, then stir the mixture and heat to 90°C for 15 min. After cooling, sonicate the mixture for 2 min. Centrifuge the mixture at 10000 r / min for 10 min to remove protein precipitate, then adjust the pH to 7.0 and use the supernatant for Fe... 3+ The determination.

[0055] 5. Stern-Volmer equation

[0056] Fluorescence quenching efficiency is usually quantified by equations.

[0057]

[0058] In this equation, F0 and F represent the steady-state fluorescence intensity before and after quenching, respectively; the parameter Ksv represents the quenching constant, reflecting the quenching efficiency; and [Q] is the concentration of the quencher, i.e., Fe. 3+ The concentration.

[0059] II. Results and Discussion

[0060] 1. Material Characterization

[0061] Characterization of NCDs

[0062] The morphology and particle size distribution of NCDs synthesized by hydrothermal method were characterized by TEM. Figure 2 As shown in (A), the NCDs exhibit good dispersion and a quasi-spherical morphology. Particle size distribution analysis reveals that the NCDs are mainly distributed in the range of 1.33–2.62 nm, with an average diameter of 1.83 nm. The NCDs are uniformly distributed without significant aggregation, confirming the formation of ultra-small NCDs. High-resolution TEM images reveal clear lattice fringes with an interplanar spacing of approximately 0.30 nm, which is close to the characteristic (002) interplanar spacing of graphitic carbon. This observation indicates the presence of partially graphitized regions within the NCDs.

[0063] The crystal structure of NCDs was further investigated using X-ray diffraction (XRD). Figure 2The XRD pattern in (C) shows two broad diffraction peaks at 2θ ≈ 21° and 26°. The broad peak at approximately 26° can be attributed to the (002) crystal plane of graphitic carbon, while the peak at approximately 21° is generally associated with amorphous carbon structures or carbon frameworks containing numerous structural defects and surface functional groups. The broadening of these diffraction peaks indicates low crystallinity and small grain size, consistent with the nanoscale results observed in TEM analysis. In summary, the TEM and XRD results demonstrate that the synthesized NCDs consist of ultrasmall nanoparticles with a structure characterized by short-range ordered graphitized regions embedded in an amorphous carbon matrix. This partially graphitized structure, combined with abundant defect sites, is expected to play a crucial role in determining the physicochemical and optical properties of NCDs.

[0064] FITR spectra such as Figure 3 As shown in (A), the infrared spectrum of citric acid is in the range of 3500–3000 cm⁻¹. -1 Within the area, at 3280cm -1 The broad absorption peak at 3490 cm⁻¹ is due to the hydrogen bonds formed between the carboxyl group (-COOH) and the hydroxyl group (-OH) in the citric acid molecule; the peak at 3490 cm⁻¹ is due to the hydrogen bonds formed between these groups. -1 The absorption peaks at these locations are sharper and typically correspond to free or weakly hydrogen-bonded hydroxyl groups; both absorption peaks are attributed to the OH stretching vibration. The absorption peaks at 1750–1700 cm⁻¹ are... -1 A strong absorption peak appears at [value missing], corresponding to the C=O stretching vibration of the carboxyl group. Furthermore, [value missing] in the 1200–1000 cm⁻¹ range... -1 Multiple absorption bands were observed within the range, mainly attributed to C–O and C–O–C stretching vibrations. The infrared spectra of NCDs showed significant changes. At 3400 cm⁻¹... -1 A broad peak, corresponding to O–H and N–H stretching vibrations, is still observed nearby, indicating that hydrophilic functional groups such as –OH or –NH are retained on the NCDs surface, which contributes to their dispersion stability in aqueous solution. These groups may originate from the incomplete carbonization of citric acid during the hydrothermal reaction, as well as nitrogen-containing groups introduced by formamide and DMF, thus achieving nitrogen doping of the NCDs. Secondly, the peak located at 1700 cm⁻¹ in the NCDs... -1 The C=O absorption peak near the NCDs shows a significant decrease and broadening, indicating that some carboxyl groups participated in dehydration condensation or carbonization reactions during NCDs formation, generating new chemical bonds (such as amide bonds). The absorption peak at 1600–1500 cm⁻¹ is also significant. -1 The absorption peaks appearing in the region can be attributed to C=C or C=N stretching vibrations, indicating that a certain degree of graphite-like or conjugated sp24-like structures have formed inside the NCDs. 2 Carbon structure. NCDs are located at 1200–1000 cm⁻¹. -1The absorption characteristics of C–O are retained within the range, indicating that there are residual oxygen-containing functional groups such as hydroxyl groups, ether bonds, or carboxyl groups on its surface.

[0065] The UV-vis spectrum of NCDs is as follows Figure 3 As shown in (B), a strong absorption band is observed in the ultraviolet region below 300 nm, which can be attributed to aromatic sp. 2 π–π* transitions in the carbon domain were observed. A relatively weak but broad absorption band was also observed in the approximately 300–450 nm range, typically associated with n–π* transitions of surface functional groups such as C=O and C–N, indicating abundant surface states on the NCD surface. Excitation spectra monitored at the maximum emission wavelength showed a broad excitation band centered at approximately 350–360 nm, suggesting the existence of multiple emission centers or surface-related energy levels within the NCDs. Under optimal wavelength excitation, the NCDs exhibited strong blue emission, with the maximum emission peak located at approximately 440–450 nm. The large Stokes shift between the excitation and emission bands helped minimize self-absorption effects and indicated that emission was primarily dominated by surface defect states rather than intrinsic band-edge transitions.

[0066] To further investigate the photoluminescence behavior of NCDs, emission spectra were recorded at different excitation wavelengths in the range of 300–400 nm, such as… Figure 3 As shown in (C), NCDs exhibit wavelength-dependent emission behavior, with both emission intensity and peak position varying with the excitation wavelength. As the excitation wavelength increases, the emission peak shows a slight redshift, while the fluorescence intensity changes. This wavelength-dependent emission behavior is a common feature of NCDs and is generally attributed to the non-uniform distribution of surface states, particle size effects, and various emission traps associated with surface functional groups.

[0067] Stability of NCDs

[0068] The stability of the synthesized NCDs was systematically evaluated under continuous 30 min excitation light irradiation, different concentrations of NaCl solution, different synthesis batches, and different pH environments.

[0069] The photostability of NCDs was evaluated by continuously irradiating them with 355 nm excitation light for 30 min, with fluorescence intensity measured every 5 min during this period. The results are as follows: Figure 4 (A) The fluorescence intensity did not decrease significantly over time.

[0070] NCDs were dispersed in NaCl solutions of different concentrations (0–2.0 M), and the effect of ionic strength on fluorescence was investigated. Figure 4 As shown in (B), the fluorescence intensity remains essentially unchanged within this concentration range, indicating that it has good high salt tolerance.

[0071] To compare the differences between different batches of NCDs synthesis, five batches of NCDs were synthesized under the same synthetic conditions, such as... Figure 4 As shown in (C), there was no significant difference in fluorescence intensity, indicating that the synthesis of NCDs has good reproducibility and stable synthesis effect.

[0072] The fluorescence intensity of NCDs at different pH values ​​was tested, and the results are as follows: Figure 4 (D) NCDs exhibit high fluorescence intensity within the pH range of 4 to 10, with minimal differences in fluorescence intensity between different pH levels. However, the fluorescence intensity of NCDs decreases significantly in both acidic and alkaline environments. This may be because surface carboxyl groups are protonated in acidic environments, altering the electron distribution on the NCD surface and thus reducing fluorescence intensity. In alkaline environments, carboxyl groups on the NCD surface are deprotonated, and a large number of OH- ions in the solution bind to the NCD surface, altering the surface state of the NCDs. This may lead to the destruction of existing high-efficiency luminescent centers or the formation of new luminescent centers. Such drastic changes in surface state are often accompanied by a significant decrease in the original fluorescence intensity.

[0073] Material comparison

[0074] The synthesized NCDs, F-NCDs, and D-NCDs materials, under white light and 365nm excitation light irradiation, show the following: Figure 5 As shown in Figure A, D-NCDs did not exhibit fluorescence, and no carbon dots were synthesized. Quantum yield tests were performed on NCDs and F-NCDs; the quantum yield of NCDs was 3.78%, while that of F-NCDs was 3.69%. The quantum yield of NCDs was superior to that of F-NCDs. Figure 5 B).

[0075] 2. NCDs and Fe 3+ Study on the quenching mechanism of surface complexation fluorescence

[0076] Fe 3+ The addition of Fe caused fluorescence quenching in NCDs at the 450 nm emission wavelength. This observed quenching behavior is related to the structure of NCDs and their interaction with Fe. 3+ The interactions of ions are closely related. To elucidate its underlying mechanism, the study of Fe... 3+ The structures of NCDs before and after the introduction were characterized. Based on comparative analysis, a possible quenching mechanism was proposed.

[0077] Introducing Fe 3+ After ionization, such as Figure 6 As shown in (A), NCDs–Fe 3+ Significant changes were observed in the FTIR spectra of the complex. The intensity of the –OH / –NH stretching vibration was significantly weakened and slightly shifted, indicating that Fe… 3+There are strong coordination interactions between the oxygen and hydroxyl or amino groups on the NCD surface. Simultaneously, the absorption band associated with C=O shows significant changes in both peak shape and intensity, indicating that the oxygen atom in the carboxyl or amide group participates in the interaction with Fe. 3+ Coordination. Furthermore, the fingerprint region (1000–1500 cm²) -1 The changes in Fe, particularly those related to C–O and C–N vibrations, further confirm the presence of Fe. 3+ Coordination bonds were formed between the ions and multiple surface functional groups of NCDs. These results indicate that Fe 3+ Ions can coordinate with the hydroxyl, carboxyl, and amino groups on the surface of NCDs to form stable NCDs–Fe 3+ Complexes. This coordination interaction significantly alters the surface electronic structure of NCDs, promoting nonradiative recombination pathways and thus leading to effective fluorescence quenching.

[0078] For NCDs and the addition of Fe 3+ UV-vis spectral tests were performed on the NCDs, from Figure 6 (B) It can be seen that the addition of Fe 3+ Subsequently, the position of the ultraviolet absorption peak did not show a significant red shift or blue shift, and the absorption peak at 550 nm was significantly weakened. (Fe) 3+ Coordination with NCDs alters the original electron distribution on the NCD surface, weakening the n–π* transitions associated with surface states, and may be accompanied by electron transfer, thus leading to a reduction in characteristic absorption peaks. The optical band gap (E0) is calculated using UV-Vis spectroscopy and the following Tauc formula. g ):

[0079]

[0080] Where α is the absorption coefficient and absorbance value; h is Planck's constant; ν is the frequency; and A is a constant. Figure 6 (C) It can be seen that E is obtained through calculation. g It is 4.1 eV.

[0081] Fe 3+ The coordination interactions with the hydroxyl, carboxyl, and amino groups on the NCD surface were further confirmed by Zeta potential measurements. Figure 6 As shown in (D), individual NCDs exhibit a negative Zeta potential due to the abundance of –COOH groups on their surface. The addition of Fe... 3+ Afterwards, the Zeta potential changed from negative to positive, indicating that the positively charged Fe... 3+ Successful binding to NCDs surfaces resulted in a significant surface charge reversal. This observation relates to the electrostatic relationship between NCDs–Fe 3+ Evidence was provided for the formation of surface composite structures.

[0082] Further analysis of NCDs and the addition of Fe 3+ The resulting mixed solution (NCDs + Fe) 3+ Fluorescence lifetime was tested. The results are as follows: Figure 6 (E) shows that the fluorescence lifetime of NCDs alone is 1.28 ns, and the addition of Fe... 3+ The resulting mixed solution (NCDs + Fe) 3+ The fluorescence lifetime was 1.08 ns, indicating a decrease in fluorescence lifetime, suggesting that Fe... 3+ The interaction between ions and NCDs involves dynamic binding, leading to dynamic quenching.

[0083] In addition, NCDs affect Fe 3+ The fluorescence quenching response can also be well described by the Stern–Volmer equation, where the dynamic quenching constant increases with increasing temperature, while the static quenching constant decreases. .See Figure 6 As shown in (F), F0 / F and Fe 3+ A good linear relationship exists between concentration and Ksv, and the value of Ksv is derived from the slope of the fitted linearity. Ksv increases with increasing temperature, which is consistent with the dynamic quenching mechanism. In F0 / F and Fe... 3+ The linear relationship of concentration (y = 0.042x + 1.28) shows an intercept significantly greater than 1, deviating from the theoretical expectation of a purely dynamic quenching process. This indicates that fluorescence quenching is also related to non-fluorescent NCDs–Fe 3+ It is related to the formation of coordination compounds, which is a characteristic of the static quenching mechanism.

[0084] The effects of cyclic voltammetry (CV) on NCDs and NCDs+Fe were investigated. 3+ Electrochemical properties, such as Figure 6 As shown in (G), when only Fe is present 3+ At this point, a strong reduction current appears, with a reduction peak at 0.07 V, corresponding to irreversible Fe. 3+ Reduced to Fe 2+ The process; when only NCDs are present, there are no sharp oxidation / reduction peaks, indicating that NCDs themselves do not have strong redox capabilities; when Fe 3+ When present simultaneously with NCDs, a reduction peak appears at 0.19 V and an oxidation peak appears at 0.45 V. Fe is generated during the reduction scan. 2+ It is oxidized back to Fe during the forward scan. 3+ The highest occupied (HOMO) and lowest occupied (LUMO) energy levels of NCDs and added NCDs can be calculated using the following formula:

[0085]

[0086]

[0087] Among them, E ox Represents oxidation potential; E red This represents the reduction potential.

[0088] Through CV curves, as shown Figure 6 (H) It can be seen that the E of NCDs is determined red The value is -0.21 V, and the corresponding E LUMO The calculated value is -4.19 eV. The E of NCDs... g It is 4.1 eV, according to the formula: E HOMO = E LUMO -E g Therefore, the E of NCDs HOMO The value is -8.29 eV. The same method was used to add Fe... 3+ Calculations were performed on NCDs, NCDs + Fe 3+ like Figure 6 (I) Shown E red It is 0.16 V, E LUMO The value is -4.56 eV, and E is calculated. HOMO is -8.66 eV. E HOMO Energy level and E LUMO The energy levels all decrease, E g Without change, further proof of Fe 3+ Coordination with the NCDs surface forms a ground-state complex, altering the potential distribution around the NCDs, leading to an overall decrease in energy levels and static quenching. The schematic diagram of the overall decrease in LUMO / HOMO energy levels and the nonradiative electron transfer process before and after the interaction is shown below. Figure 7 As shown.

[0089] In summary, these results indicate that Fe 3+ Ions coordinate with the surface functional groups of NCDs to form stable NCDs–Fe 3+ The complex, through the formation of a non-fluorescent complex, dominated the static quenching. Meanwhile, Fe... 3+ Ions further enhance the nonradiative electron or energy transfer processes in the excited state, as evidenced by the shortened fluorescence lifetime. The synergistic effect of dynamic and static quenching mechanisms ultimately leads to the efficient quenching of NCD fluorescence (see the complete schematic diagram of the fluorescence quenching mechanism). Figure 7 ).

[0090] 3. Fe 3+ Fabrication of fluorescence sensors

[0091] Selectivity and anti-interference study

[0092] The selectivity of NCDs for various metal ions was examined to evaluate their effect on Fe. 3+ Specificity of the detection. For example... Figure 8 As shown in (A), the results indicate that Fe 3+ The presence of Hg can effectively quench NCDs, while when Hg 2+ The presence of these substances can also cause fluorescence quenching in NCDs, affecting Fe. 3+ The fluorescence detection system introduces interference. Therefore, by using I... - The sample solution was pretreated with Hg. 2+ The fluorescence quenching interference caused by it was effectively eliminated. Figure 8 (C)). Other tested metal ions did not induce significant fluorescence quenching in NCDs, indicating that NCDs are not effective against Fe. 3+ It exhibits excellent selectivity. Furthermore, it can detect Fe in the presence of other metal ions. 3+ The system's anti-interference capability was examined. For example... Figure 8 As shown in (B), fluorescence quenching can still be observed in the mixed ion system, which confirms the strong anti-interference performance of the proposed method.

[0093] NCDs for Fe 3+ Optimization of fluorescence detection conditions

[0094] NCDs exhibited varying fluorescence intensities under different pH conditions. Although the maximum fluorescence intensity was observed in 0.1 M PBS buffer (pH 7.4), this was not necessarily due to Fe. 3+ It is readily hydrolyzed under alkaline conditions. Therefore, acetate buffer was used in subsequent experiments. In this medium, the effects of adding Fe were evaluated within a pH range of 3.0–6.5. 3+ Changes in fluorescence intensity of NCDs before and after, such as Figure 9 As shown in (A, B), the group with the most significant change in fluorescence intensity was selected, and the acetate buffer at pH 3.5 was determined to be the optimal condition for all further Fe... 3+ Testing.

[0095] In the study of Fe 3+ In the fluorescence detection, Fe was further optimized. 3+ The reaction time for binding with NCDs. For example... Figure 9 As shown in (C), when Fe is added 3+ Then, the fluorescence intensity of the system was measured every 5 minutes. The fluorescence intensity tended to stabilize after 10 minutes. Therefore, all subsequent fluorescence measurements were performed after the addition of Fe. 3+ Perform in the last 10 minutes.

[0096] Sensitivity Research

[0097] Under optimal conditions, a fluorescence detection method for Fe was established using NCDs.3+ The standard curve. For example... Figure 10 As shown in (A, B), in a 0.1M acetate buffer system with pH=3.5, the F0 / F ratio and Fe 3+ The concentration showed a linear relationship in the range of 0.60–9.00 mg / L, and the linear regression equation was y = 0.042x + 1.28 (where x is Fe). 3+ The concentration of y is expressed in mg / L; y is F0 / F, and the correlation coefficient is R. 2 The standard deviation of the blank was σ = 0.977, and the standard deviation of the blank was σ = 0.003. The calculated LOD was 0.21 mg / L.

[0098] Compared with other methods in Table 1, this embodiment has a wider linear detection range and a relatively low detection limit, covering Fe in serum. 3+ Physiological and pathological concentration ranges, suitable for various clinical scenarios.

[0099] Table 1 Fe 3+ Comparison of detection methods

[0100]

[0101] Comparison with standard methods

[0102] To further verify the reliability of the method proposed in this embodiment, it was compared with the thiocyanate spectrophotometric method specified in industry standard DZ / T 0064.24-2021 for Fe... 3+ Comparison of measurement results. As shown in Table 2, the method of this embodiment is consistent with the colorimetric method specified in the industry standard, and has high reliability.

[0103] Table 2. Comparison of the accuracy of the detection method in this embodiment with the industry standard method (n=3)

[0104]

[0105] 4. Fe in actual serum samples 3+ Detection

[0106] The established method was used to analyze serum samples from healthy individuals and patients. The Fe content in normal human serum... 3+ The concentration was below the linear detection range of this method. Therefore, spiked recovery experiments were performed on normal serum. As shown in Table 2, the method exhibited satisfactory recovery and accuracy in the serum matrix. In the analysis of patient serum (as shown in Table 3), Fe was detected. 3+ A significantly elevated concentration, exceeding the normal range, may indicate liver disease. In conclusion, the analytical results of actual samples confirm the effectiveness of this method in determining Fe in clinical serum samples, particularly under pathological conditions.3+ Its practical application value.

[0107] Table 3. Detection of Fe in actual samples 3+ Performance study (n=3)

[0108]

[0109] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

[0110]

Claims

1. A Fe ion resistant to mercury ion interference 3+ The fluorescence detection method is characterized by: Includes the following steps: (1) Add soluble iodide to the test sample solution containing or suspected of containing mercury ion interference for pretreatment; (2) The sample solution to be tested, nitrogen-doped carbon dot solution and acetate buffer solution after pretreatment in step (1) are mixed to obtain a mixed detection system containing free iodide ions; (3) After the mixed detection system obtained in step (2) is incubated at room temperature to reach equilibrium, its fluorescence intensity at an emission wavelength of 450 nm is measured at an excitation wavelength of 355 nm. The degree of quenching of this fluorescence intensity is used to quantitatively analyze the Fe in the sample solution to be tested. 3+ concentration; In the reaction environment maintained by the aforementioned acetate buffer solution, iodide ions ionized from the soluble iodide bind to mercury ions in the system, and nitrogen-doped carbon dots bind to Fe in the system through their surface hydroxyl, carboxyl, or amino functional groups. 3+ It exhibits a ground-state chemical coordination complex structure.

2. The Fe as described in claim 1 3+ The fluorescence detection method is characterized by: The acetate buffer solution in step (2) has a pH of 3.5 and a concentration of 2 M.

3. The Fe as described in claim 1 3+ The fluorescence detection method is characterized by: The nitrogen-doped carbon dot solution in step (2) is a solution with a concentration of 0.2 mg / mL prepared by dissolving nitrogen-doped carbon dot powder in deionized water.

4. The Fe as described in claim 3 3+ The fluorescence detection method is characterized by: The preparation of the nitrogen-doped carbon dot powder includes: mixing and stirring citric acid, formamide and N,N-dimethylformamide, and maintaining the reaction at 180°C for 4 h; after the reaction, centrifuging the resulting solution to remove insoluble particles, adding acetone as an eluent to the supernatant until there is no fluorescence in the supernatant, collecting the precipitate, and finally dissolving the precipitate in water and vacuum drying; the ratio of citric acid, formamide and N,N-dimethylformamide is 1 g: 1.4 mL: 10 mL.

5. The Fe as described in claim 1 3+ The fluorescence detection method is characterized by: The mixed detection system in step (2) consists of a nitrogen-doped carbon dot solution with a concentration of 0.2 mg / mL, an acetate buffer solution with a concentration of 2 M and a pH of 3.5, and the sample solution to be tested after pretreatment in step (1) in a volume ratio of 50: 50: 890-900.

6. The Fe as described in claim 1 3+ The fluorescence detection method is characterized by: The room temperature incubation time in step (3) is 10 min.

7. The Fe as described in claim 1 3+ The fluorescence detection method is characterized by: When the sample solution to be tested is a serum sample, the following serum pretreatment steps are included before step (1): add a 20 wt% trichloroacetic acid solution to the serum, mix, stir and heat to 90°C for 15 min; after cooling, perform ultrasonic treatment, then centrifuge the mixture to remove protein precipitate, collect the supernatant and adjust the pH of the supernatant to 7.0 as the sample solution to be tested; the volume ratio of serum to trichloroacetic acid solution is 1:

1.

8. A Fe ion resistant to mercury ion interference 3+ A fluorescence detection sensor, characterized in that: include: Nitrogen-doped carbon dots are prepared by reacting a precursor system including citric acid, formamide and N,N-dimethylformamide. They serve as fluorescent probes and have hydrophilic functional groups including hydroxyl, carboxyl and amino groups on their surface. An acetate buffer solution with a concentration of 2 M and a pH of 3.5 is used to maintain Fe. 3+ The mixed reaction system for the fluorescence sensor to operate is in an environment with a pH of 3.

5. Iodide ion masking agent, which is a soluble iodide that can ionize into free iodide ions; In the reaction environment of pH 3.5 maintained by the above-mentioned acetate buffer solution, the iodide ions ionized from the iodide ion masking agent are bound to the mercury ions in the system, and the nitrogen-doped carbon dots bind to the Fe in the system through the hydroxyl, carboxyl, or amino functional groups on their surface. 3+ It exhibits a ground-state chemical coordination complex structure.

9. The Fe as described in claim 8 3+ A fluorescence detection sensor, characterized in that: The ratio of citric acid, formamide, and N,N-dimethylformamide is 1 g: 1.4 mL: 10 mL.

10. The Fe as described in claim 9 3+ A fluorescence detection sensor, characterized in that: The reaction was carried out under constant temperature at 180°C for 4 hours.

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