Preparation method of quantum dot fluorescent probe for online monitoring of comprehensive toxicity of water quality

Abstract

This invention relates to the field of water pollution detection technology, specifically to a method for preparing quantum dot fluorescent probes for online monitoring of comprehensive water toxicity. The method uses L-histidine and levofloxacin as raw materials, and through hydrothermal reaction, centrifugation, dialysis, pH adjustment, filtration, and freeze-drying, prepares histidine-modified carbon quantum dot fluorescent probes (His-CQDs), which are further formulated into a working solution. The histidine is used to simulate key toxicity recognition sites in the luciferase system of luminescent bacteria, enabling the His-CQDs to produce fluorescence quenching responses to heavy metal ions and fluorescence enhancement responses to organic pollutants, thereby achieving online monitoring of the comprehensive toxicity of the water sample. During detection, the working solution is mixed with the water sample, and the fluorescence intensity is measured. The water toxicity evaluation result is obtained by calculating the fluorescence change rate. This invention achieves a materialized simulation of the toxicity recognition mechanism of luminescent bacteria, and has the advantages of fast response, good stability, and suitability for online monitoring of comprehensive water toxicity.

Description

Technical Field

[0001] This invention relates to the field of water pollution detection technology, specifically to a method for preparing quantum dot fluorescent probes for online monitoring of comprehensive water toxicity. Background Technology

[0002] Comprehensive water toxicity monitoring is an important component of environmental safety monitoring and aquatic ecological risk assessment. Among existing comprehensive toxicity detection methods, the luminescent bacteria method is a widely used approach. Its principle is based on the luciferase bioluminescence system within luminescent bacteria, specifically the reaction: FMNH2 + R-CHO + O2 → FMN + R-COOH + H2O + hν. When toxic substances are present in the water, they act on key functional components of the luminescent bacteria, causing enzyme denaturation and inactivation or impaired electron transport, thus reducing the luminescence intensity and achieving toxicity detection. Histidine (His) is a key amino acid residue in the luciferase system of luminescent bacteria. Its imidazole ring can coordinate with heavy metal ions, and its amino and carboxyl groups can form hydrogen bonds or electrostatic interactions with organic pollutants, making it an important functional group for luminescent bacteria to recognize toxicity.

[0003] However, existing technologies still have the following problems: 1) Traditional luminescent bacteria method relies on intact live bacteria. The bacteria themselves are sensitive to the environment, require strict temperature control, have a short shelf life, and have limited stability time after reconstitution, making it difficult to meet the needs of rapid on-site detection and long-term online monitoring. 2) Although unmodified carbon quantum dots have good fluorescence properties, they lack the ability to specifically recognize toxic substances, making it difficult to distinguish different toxic mechanisms, especially the comprehensive toxic response of heavy metal ions and organic pollutants. 3) The toxic response of luminescent bacteria depends on their luciferase system and key amino acid components. However, there is no effective solution in the current technology for how to extract and stably immobilize such bioactive recognition elements on the surface of materials while retaining their toxic response characteristics.

[0004] Therefore, it is necessary to provide a method for preparing a quantum dot fluorescent probe that can reflect the toxic response mechanism of luminescent bacteria, possess the advantages of carbon quantum dots in terms of stability and rapid detection, and can be used for online monitoring of comprehensive water toxicity. Summary of the Invention

[0005] To address the above problems, this invention provides a method for preparing a quantum dot fluorescent probe for online monitoring of comprehensive water toxicity, comprising the following steps: SA1. Preparation of histidine-functionalized carbon quantum dots: SA1-1. Weigh L-histidine and levofloxacin and add them to DMF. Stir ultrasonically for 10-15 min until completely dissolved to form a homogeneous solution. Transfer the resulting solution to a stainless steel reactor lined with polytetrafluoroethylene and hydrothermally react at 200℃ for 12 h. After the reaction is complete, allow it to cool naturally to room temperature to obtain the reaction solution. Let n be the multiplier and n∈R + The addition range of each component in SA1-1 is as follows: L-histidine (carbon source) is [1.2n, 1.5n] g, levofloxacin is [0.5n, 1n] g, and DMF is [20n, 25n] mL. SA1-2. Centrifuge the reaction solution obtained in SA1-1 at 10000~12000 rpm for 15~20 min, collect the supernatant and place it in a dialysis bag with a molecular weight cutoff of 500~1000 Da, and dialyze in deionized water for 24 h, changing the dialysis solution every 4 h during the period. SA1-3. After dialysis, the pH of the solution was adjusted to 7.3-7.5 with 1 mol / L NaOH solution, then filtered through a 0.22 μm microporous membrane, and then freeze-dried for 48 h to obtain histidine-modified carbon quantum dot fluorescent probes, denoted as His-CQDs. SA2, Preparation of working solution: The His-CQDs prepared in SA1 were dispersed in pure water and ultrasonically dispersed evenly to prepare a His-CQDs working solution with a concentration of 0.01-0.1 mg / mL, which was then stored at 4°C in the dark.

[0006] Explanation: The toxic response of luminescent bacteria originates from key functional components in their luciferase system. When toxic substances are present in the environment, the active sites in this system interact with these substances, thereby affecting electron transfer processes and altering luminescence intensity. Histidine residues are important functional amino acids in the luciferase system; their imidazole ring structure can coordinate with heavy metal ions, while their amino and carboxyl groups can form hydrogen bonds, electrostatic interactions, or hydrophobic interactions with various organic pollutants. This invention introduces L-histidine into a carbon quantum dot structure through a one-step hydrothermal reaction, enriching the surface of the resulting carbon quantum dots with histidine functional groups, thereby obtaining a histidine-modified carbon quantum dot fluorescent probe with toxicity recognition capabilities.

[0007] In this process, levofloxacin acts as an auxiliary carbon source in the hydrothermal reaction, participating in the formation of carbon quantum dot structures. DMF serves as the reaction solvent and medium, facilitating the uniform dispersion of the precursor and the generation of carbon quantum dots. Through centrifugation, dialysis, pH adjustment, filtration, and freeze-drying, unreacted small molecule impurities are effectively removed, yielding stable His-CQDs quantum dot fluorescent probe materials. This invention achieves a materialized simulation of the toxicity recognition mechanism of luminescent bacteria by introducing the key functional group histidine from the luminescent bacterial toxicity recognition mechanism into the carbon quantum dot structure.

[0008] Furthermore, the His-CQDs prepared in SA1 were further coated with polydopamine to construct quantum dot fluorescent probes with selective mass transfer function. The steps are as follows: SB1. Preparation of polydopamine shell: The His-CQDs prepared in SA1 were dispersed in Tris buffer solution, and the pH of the system was adjusted to 8.0-9.0 with 1 mol / L NaOH solution. Then, dopamine monomer was added and stirred at room temperature for 2-3 h to allow dopamine to undergo oxidative self-polymerization reaction and form a polydopamine shell on the surface of His-CQDs, thus obtaining solution A. Let n be the multiplier and n∈R+, then the addition range of each component in SB1 is: His-CQDs is [100n, 110n] mg, Tris buffer solution is [100n, 120n] mL, and dopamine monomer is [50n, 60n] mg; SB2, Purification Process: The solution A prepared in SB1 was centrifuged at 10,000 to 12,000 rpm for 15 to 20 min, the supernatant was discarded, the precipitate was resuspended in deionized water and washed repeatedly 3 to 5 times, filtered and freeze-dried for 48 h to obtain a quantum dot fluorescent probe with a gate-type shell, denoted as His-CQDs@PDA. SB3. Preservation of quantum dot fluorescent probes: Redisperse His-CQDs@PDA in pure water to prepare a His-CQDs@PDA working solution with a concentration of 0.01~0.1 mg / mL, and store at 4°C protected from light.

[0009] Description: By introducing dopamine monomers under alkaline conditions, an oxidative self-polymerization reaction is induced on the surface of carbon quantum dots, forming a stable polydopamine shell. This shell exhibits good biocompatibility and chemical stability, while also possessing abundant aromatic structures and hydrogen bonding sites, which are beneficial for the enrichment and adsorption of organic pollutants. The thickness of the polydopamine shell can be adjusted by controlling the dopamine concentration and reaction time, thereby regulating the mass transfer behavior of pollutants. This preparation method is simple to operate, has good reproducibility, and is easy to scale up, providing a stable and reliable probe material for the time-resolved fluorescence detection method proposed in this invention.

[0010] Furthermore, in SB2, the gate shell thickness of His-CQDs@PDA has the following relationship with the pollutant mass transfer behavior: When the shell thickness is 3–8 nm, the diffusion time of organic pollutants is less than that of metal ions. When the shell thickness is 8–15 nm, the diffusion time of metal ions is significantly delayed and a fluorescence quenching hysteresis effect is produced. When the shell thickness is 15–20 nm, the enrichment rate of organic pollutants remains at a high level, while the diffusion rate of metal ions decreases to 0.3–0.6 times that of organic pollutants.

[0011] Explanation: By adjusting the thickness of the gate-type shell, the diffusion rates of organic pollutants and metal ions in the probe structure can be effectively modulated, thereby altering the temporal order of the two types of pollutants in the fluorescence response. When the shell thickness is small, organic pollutants can rapidly diffuse into the probe structure and produce an enhanced fluorescence response, while metal ions, due to the presence of a hydrated shell structure, diffuse at a relatively slower rate, resulting in a delayed fluorescence quenching effect. As the shell thickness further increases, the diffusion resistance of metal ions further increases, thus further delaying their influence on the fluorescence signal in the time dimension, while organic pollutants can still achieve rapid enrichment through hydrophobic interactions and π-π interactions.

[0012] Through this thickness control mechanism, the present invention can form a time-resolved response with distinct characteristics in the fluorescence trajectory, thereby enabling the detection method to more accurately distinguish the action mechanisms of different types of pollutants and improve the reliability and discrimination ability of pollutant detection in complex aquatic environments.

[0013] As another aspect of the present invention, a method for online monitoring of comprehensive water toxicity based on the above-mentioned His-CQDs is also provided: The His-CQDs were formulated into a working solution (i.e., His-CQDs working solution) and brought into contact with the water sample to be tested. The overall toxicity of the water sample was monitored online by detecting changes in its fluorescence signal. Histidine was used to simulate the key toxicity recognition functional site in the luciferase system of luminescent bacteria, enabling His-CQDs to produce a fluorescence quenching response to heavy metal ions and a fluorescence enhancement response to organic pollutants, thereby characterizing the overall toxicity of the water sample to be tested.

[0014] Note: The bioluminescent reaction of luminescent bacteria depends on the luciferase system, in which histidine residues participate in electron transfer and substrate binding in the enzyme's active site, serving as crucial functional groups for the luminescent bacteria's response to external toxic substances. This invention functionalizes histidine into the surface of carbon quantum dots, endowing them with a chemical environment similar to the key recognition sites of luminescent bacteria. Specifically, the imidazole ring of histidine can react with Hg... 2+ Cu 2+ Cd 2+ The coordination of heavy metal ions alters the electronic structure of quantum dots, leading to fluorescence quenching. Meanwhile, the amino and carboxyl groups of histidine can bind to organic pollutants such as polycyclic aromatic hydrocarbons, phenols, and pesticides through hydrogen bonds, electrostatic interactions, or π-π interactions, inhibiting non-radiative transitions and thus producing a fluorescence enhancement effect. Based on this dual-mode fluorescence response mechanism, rapid characterization of the overall toxicity of water samples can be achieved.

[0015] Furthermore, the steps for online monitoring of the comprehensive water toxicity based on the above His-CQDs are as follows: 1) Mix an equal volume of His-CQDs working solution with the water sample to be tested; 2) React at room temperature for 30~120 s; 3) Measure the fluorescence intensity using a fluorescence spectrometer or portable fluorescence detection device; 4) Calculate ΔF / F0 and obtain the TU value by referring to the standard curve; Wherein, F0 is the initial fluorescence intensity of His-CQDs working solution without water sample added, F is the fluorescence intensity after adding water sample to be tested, ΔF is the difference between F0 and F, and TU is the comprehensive toxicity intensity of water quality. When ΔF / F0 < -20%, it is determined to be a quenching toxicity response; When ΔF / F0 > +20%, it is determined to be an enhanced toxicity response.

[0016] Explanation: When His-CQDs come into contact with the water sample, the toxic substances in the sample rapidly interact with the histidine functional groups, thereby altering the electronic structure and energy transition processes on the carbon quantum dot surface, leading to changes in the fluorescence signal. The degree of influence of the toxic substances on the fluorescence system can be reflected by measuring the fluorescence change rate ΔF / F0. Here, F0 is the initial fluorescence intensity without the water sample, and F is the fluorescence intensity after the water sample is added. By establishing a pre-defined correlation between the fluorescence change rate and the toxicity unit TU using standard toxic substances, the fluorescence change signal can be converted into a comprehensive quantitative toxicity index.

[0017] The above detection process is simple to operate, requiring only mixing the quantum dot fluorescent probe with the water sample and measuring the fluorescence signal to complete the detection. The detection time is 30-120 seconds, with a fast response speed, making it suitable for online monitoring. Furthermore, by establishing a quantitative relationship between the fluorescence change rate and the toxicity unit TU, rapid quantitative evaluation of the overall toxicity of the water sample can be achieved, improving detection efficiency and on-site applicability.

[0018] As another aspect of the present invention, a specific structure of the His-CQDs@PDA is also provided, wherein the quantum dot fluorescent probe includes a carbon quantum dot core, a histidine functional layer covering the outside of the carbon quantum dot core, and a gate-type shell covering the outside of the histidine functional layer. Among them, the carbon quantum dot core is a nano-carbon source with fluorescence emission properties; The histidine functional layer is a surface functional layer containing imidazole ring coordination sites, which can undergo coordination reactions with heavy metal ions; The gate-type shell is a coating layer with selective mass transfer properties, formed by polydopamine. Its surface contains aromatic structures and hydrogen bonding sites, which can promote the enrichment of hydrophobic or aromatic organic pollutants into the probe and delay the diffusion of hydrated metal ions to histidine coordination sites, thereby producing differentiated fluorescence responses of organic pollutants and metal ions in the time dimension.

[0019] Note: The aforementioned quantum dot fluorescent probe employs a core-shell structure consisting of a carbon quantum dot core, a histidine functional layer, and a gated shell. The carbon quantum dot core provides a stable fluorescence emission signal, while the histidine functional layer provides metal ion coordination sites through an imidazole ring structure, enabling heavy metal ions to specifically interact with the probe. Simultaneously, the outer gated shell, formed by a polydopamine structure, acts as a coating layer with selective mass transfer properties. This shell contains aromatic structures and hydrogen bonding sites, which can promote the enrichment of hydrophobic or aromatic organic pollutants into the probe interior through hydrophobic interactions and π-π interactions.

[0020] Because the polydopamine shell impedes the diffusion of hydrated metal ions, a mass transfer difference is created within the probe structure, resulting in different diffusion rates for organic pollutants and metal ions as they enter the probe structure. This structural design causes organic pollutants to preferentially generate a fluorescence-enhancing response, while metal ions exhibit fluorescence quenching at a later stage, thus forming distinguishable fluorescence response trajectories over time. Through the synergistic effect of this core-shell structure, the quantum dot fluorescent probe of this invention can effectively amplify the response differences between different pollutants, thereby providing a reliable structural basis for time-resolved fluorescence detection methods.

[0021] As another aspect of the present invention, a method for online monitoring of comprehensive water toxicity based on the above-mentioned His-CQDs@PDA is also provided: 1) Establish reference parameters: Prepare standard reference samples and obtain probe baseline parameters. The standard reference samples include: standard organic contamination samples and standard metal contamination samples. His-CQDs@PDA were added to a standard sample, and the fluorescence trajectory curve F(t) of fluorescence intensity changing over time was recorded in a fluorescence spectrometer. Reference parameters were obtained, including the time t for the reference peak. ref and reference peak fluorescence intensity F ref ; Reference peak time t ref This represents the time when the fluorescence intensity in the fluorescence trajectory curve increases from the initial fluorescence intensity F0 and reaches its maximum value; F ref The fluorescence trajectory corresponding to the reference peak time t ref fluorescence intensity; 2) Testing the sample to be tested: Add His-CQDs@PDA to the water sample to be tested, and record the fluorescence trajectory curve F(t) of fluorescence intensity change over time under the same experimental conditions; 3) Extract fluorescence kinetic parameters: The following parameters are extracted from the fluorescence trajectory curve F(t): Initial fluorescence intensity F0, peak fluorescence intensity F peak Peak time t peak ; Where F peak t represents the maximum fluorescence intensity within the fluorescence trajectory. peak For corresponding to F peak The moment; 4) Construct the discriminant index: Based on the above parameters, the following discriminant index is constructed: Dynamic delay index K d : Fluorescence response index I r : 5) Pollution type determination: Determining the type of water pollution based on the discriminant index: When K is satisfied: d >1 and I r When the value is less than 1, it is determined that there are metallic pollutants in the water sample; When K is satisfied: d <1 and I r When the value is greater than 1, the concentration of organic pollutants in the water sample is determined to be higher than that in the standard sample. When the fluorescence trajectory exhibits a two-stage change of first increasing and then decreasing, it is determined that both metallic and organic pollutants are present in the water sample.

[0022] Description: This invention proposes a water pollutant detection method based on time-resolved fluorescence trajectories. By introducing time-resolved fluorescence kinetic parameters, it transforms the traditional single fluorescence intensity endpoint detection into fluorescence kinetic trajectory detection. This is achieved by establishing a standard reference sample and obtaining the reference peak time t. ref and reference peak fluorescence intensity F ref This provides a unified benchmark for the detection system, enabling standardized processing of fluorescence responses in complex aquatic environments. During the detection process, the initial fluorescence intensity F0 and peak fluorescence intensity F0 are extracted. peak and peak time t peak And construct the dynamic delay exponent K d and fluorescence response index I r This enables quantitative characterization of the mechanisms of action of different pollutants.

[0023] Metal contaminants coordinate with the imidazole ring coordination sites in the histidine functional layer, resulting in electron or energy transfer effects, which quench fluorescence and delay the appearance of the fluorescence peak. Hydrophobic or aromatic organic contaminants, on the other hand, accumulate in the probe structure through hydrophobic interactions or π-π interactions, thereby enhancing fluorescence and accelerating the fluorescence response process. This is determined by the kinetic delay index K. d With fluorescence response index I r The combined analysis can effectively distinguish the different mechanisms by which metallic and organic pollutants affect fluorescence signals.

[0024] Compared with existing technologies, this invention avoids the misjudgment problem caused by signal superposition or cancellation in complex systems by using the joint discrimination of time parameters and intensity parameters, thereby significantly improving the accuracy and reliability of water pollutant detection.

[0025] Compared with existing methods for detecting the toxicity of water pollutants, the advantages of this invention are: (1) This invention uses His, a key toxicity recognition functional group in the luciferase system of luminescent bacteria, as a biomimetic source. By modifying His onto the surface of carbon quantum dots, the material simulation of the toxicity recognition mechanism of luminescent bacteria is realized.

[0026] (2) This invention does not require maintaining the complete activity of luminescent bacteria, thus avoiding the problem that traditional luminescent bacteria methods are highly sensitive to temperature, osmotic pressure and storage conditions, making it convenient for long-term storage, transportation and on-site use; the working solution has good stability and can be stored for a long time (>6 months).

[0027] (3) This invention can respond to both heavy metal ions and organic pollutants, and can be used to characterize the comprehensive toxicity of the water sample to be tested, rather than just for the detection of single pollutant concentration.

[0028] (4) The present invention has a fast response speed and is suitable for online monitoring. His-CQDs can complete the fluorescence signal change instantly when they interact with toxic substances, and the detection step can be completed within 30~120 s. Attached Figure Description

[0029] Figure 1 The following are fluorescence emission spectra of His-CQDs working solution under the action of water samples with different total toxicity intensities (TU) in Experiment Example A1 (characteristic band 410–470 nm); each curve represents the fluorescence spectrum obtained after the reaction of water samples with different total toxicity intensities (TU) and quantum dot fluorescent probes. Detailed Implementation

[0030] To further illustrate the methods and effects of this invention, the technical solution of this invention will be clearly and completely described below in conjunction with experiments.

[0031] For ease of explanation and to avoid confusion, in the following examples and experimental cases, examples based on His-CQDs are designated as Example A and Experiment A, and examples based on His-CQDs@PDA are designated as Example B and Experiment B. Example A illustrates the preparation and application of the His-CQDs of the present invention in online monitoring of comprehensive water toxicity, while Example B illustrates the further preparation of His-CQDs@PDA and its time-resolved fluorescence trajectory analysis method.

[0032] Example A1: This example provides a method for preparing a quantum dot fluorescent probe for online monitoring of comprehensive water toxicity, the steps of which are as follows.

[0033] SA1. Preparation of histidine-functionalized carbon quantum dots: SA1-1: Weigh 1.35 g of L-histidine and 0.75 g of levofloxacin, add them to 22 mL of DMF, and stir under ultrasonic conditions for 12 min until completely dissolved to form a homogeneous solution; transfer the resulting solution to a polytetrafluoroethylene-lined stainless steel reactor, and hydrothermally react at 200℃ for 12 h. After the reaction is complete, allow it to cool naturally to room temperature to obtain a brownish-yellow reaction solution; SA1-2. Centrifuge the reaction solution at 11,000 rpm for 18 min, collect the supernatant, and place it in a dialysis bag with a molecular weight cutoff of 1000 Da. Dialyze in deionized water for 24 h, changing the dialysis solution every 4 h during the process. SA1-3. After dialysis, the pH of the solution was adjusted to 7.4 with 1 mol / L NaOH solution, then filtered through a 0.22 μm microporous membrane and freeze-dried for 48 h to obtain histidine-modified carbon quantum dot fluorescent probes, denoted as His-CQDs. SA2, Preparation of working solution: The His-CQDs were dispersed in pure water and ultrasonically dispersed to prepare a His-CQDs working solution of 0.05 mg / mL, which was then stored at 4°C in the dark. The resulting His-CQDs working solution was uniformly dispersed and had stable fluorescence emission characteristics, and could be used for subsequent comprehensive water toxicity testing.

[0034] Example A2: The preparation method of this example is basically the same as that of Example A1. The difference is that all reaction parameters are taken as the lower limit value. The rest are the same as those of Example A1 and are not included in the experimental comparison.

[0035] SA1-1: L-histidine 1.2 g, levofloxacin 0.5 g, DMF 20 mL, sonication time 10 min; In SA1-2: Centrifugation conditions: 10000 rpm, 15 min, molecular weight cutoff of dialysis bag 500 Da; In SA1-3: Adjust the pH to 7.3; In SA2, the concentration of His-CQDs working solution is 0.01 mg / mL.

[0036] Example A3: The preparation method of this example is basically the same as that of Example A1. The difference is that all reaction parameters are taken at the upper limit value. The rest are the same as those of Example A1 and are not included in the experimental comparison.

[0037] SA1. Preparation of histidine-functionalized carbon quantum dots: SA1-1: L-histidine 1.5 g, levofloxacin 1.0 g, DMF 25 mL, sonication time 15 min; In SA1-2: centrifugation conditions: 12000 rpm, 20 min; molecular weight cutoff of dialysis bag: 1000 Da. In SA1-3: Adjust the pH to 7.5; In SA2: His-CQDs working solution concentration is 0.1 mg / mL.

[0038] Experimental Example A1: To verify the application effect of the quantum dot fluorescent probe of the present invention in online monitoring of comprehensive water toxicity, the His-CQDs prepared in Example A1 were used to conduct a comprehensive toxicity detection experiment.

[0039] Experimental conditions: The experimental water samples were simulated water bodies with different integrated toxicity intensities (TU), the experimental temperature was 25℃, the pH of the water samples was 7, and the concentration of His-CQDs working solution was 0.05 mg / mL.

[0040] The overall toxicity intensity (TU) is set in six gradients: 0 TU, 0.2 TU, 0.4 TU, 0.8 TU, 1.2 TU, and 2.0 TU.

[0041] Fluorescence detection was performed using a fluorescence spectrometer with an excitation wavelength of 240 nm and an emission spectrum scanning range of 240–840 nm.

[0042] Experimental procedure: The His-CQDs working solution (10 mg / L) prepared in Example A1 was used as the quantum dot fluorescent probe system. The His-CQDs working solution was mixed with the water samples of different toxicity intensities (TU) in equal volumes. After reacting at room temperature for 60 s, the fluorescence emission spectrum of the system was measured.

[0043] For ease of analysis, the characteristic wavelength range of fluorescence emission (410~470 nm) was selected for comparison, and the results are as follows: Figure 1 As shown.

[0044] Experimental results: Under the influence of water samples with different overall toxicity intensities, the fluorescence emission spectrum shapes of His-CQDs were basically consistent, but the peak intensity showed significant differences with the toxicity intensity. When the overall toxicity of the water sample increased, the fluorescence intensity generally showed a decreasing trend, exhibiting a fluorescence quenching effect; local enhancement phenomena were also observed in some water samples, indicating that the system has a differentiated response capability to different types of toxic substances.

[0045] Toxicity evaluation methods: To further quantitatively evaluate the toxicity of water samples, the fluorescence change rate can be calculated: Where F0 is the initial fluorescence intensity of the His-CQDs working solution without water sample, and F is the fluorescence intensity after the water sample to be tested is added.

[0046] When ΔF / F0 < -20%, it is determined to be a quenching toxicity response; When ΔF / F0 > +20%, it is determined to be an enhanced toxicity response.

[0047] Experimental results show that the His-CQDs quantum dot fluorescent probe of the present invention can produce a significant fluorescent response to toxic substances in water samples within 30~120 s, enabling rapid monitoring of the comprehensive toxicity of water bodies.

[0048] Experimental Example A2: To verify the detection capability of the quantum dot fluorescent probe of this invention for heavy metal ions, Hg was selected. 2+ Gradient experiments were conducted using representative heavy metal pollutants.

[0049] Experimental conditions: Fluorescence detection was performed using a fluorescence spectrometer with an excitation wavelength of 240 nm and an emission spectrum scanning range of 240–840 nm.

[0050] Experimental procedure: The His-CQDs working solution (0.05 mg / mL) prepared in Example A1 was used as the quantum dot fluorescent probe system. The fluorescence intensity of the His-CQDs working solution without the added analyte was taken as the initial fluorescence intensity F0, which was denoted as 1000 in this experiment.

[0051] Preparation of Hg at different concentrations 2+ Standard solutions, grouped as follows: Experimental Example A2-1: 0 μM; Experimental Example A2-2: 5 μM; Experimental Example A2-3: 10 μM; Experimental Example A2-4: 20 μM; Experimental Example A2-5: 40 μM; Experimental Example A2-6: 80 μM.

[0052] The above Hg 2+ The solutions were mixed with equal volumes of His-CQDs working solution and reacted at room temperature for 60 s. The fluorescence intensity of the system was then measured using a fluorescence spectrometer. The data are shown in the table below:

[0053] Experimental results show that as Hg 2+ With increasing concentration, the fluorescence intensity of His-CQDs gradually decreased, exhibiting a significant fluorescence quenching phenomenon.

[0054] This is due to Hg 2+ It can coordinate with the imidazole ring coordination site in the histidine functional group on the surface of His-CQDs, thereby changing the electronic structure of the quantum dot surface and enhancing the nonradiative energy transfer process, resulting in a decrease in fluorescence emission intensity.

[0055] When Hg 2+When the concentration reaches a certain level, ΔF / F0 is less than -20%, which shows a clear quenching-type toxicity response.

[0056] Experimental results show that the quantum dot fluorescent probe of this invention can target Hg. 2+ Heavy metal ions produce a rapid and stable fluorescence quenching response.

[0057] Experimental Example A3: To verify the detection capability of the quantum dot fluorescent probe of the present invention for organic pollutants, phenol was selected as a representative organic pollutant for gradient experiments.

[0058] Fluorescence detection was performed using a fluorescence spectrometer with an excitation wavelength of 240 nm and an emission spectrum scanning range of 240–840 nm.

[0059] Experimental procedure: The His-CQDs working solution (0.05 mg / mL) prepared in Example A1 was used as the quantum dot fluorescent probe system. The fluorescence intensity of the His-CQDs working solution without the added analyte was taken as the initial fluorescence intensity F0, which was denoted as 1000 in this experiment.

[0060] Prepare phenol standard solutions of different concentrations, grouped as follows: Experimental Example A3-1: 0 μM; Experimental Example A3-2: 10 μM; Experimental Example A3-3: 20 μM; Experimental Example A3-4: 40 μM; Experimental Example A3-5: 80 μM; Experimental Example A3-6: 160 μM.

[0061] The above phenol solution was mixed with an equal volume of His-CQDs working solution and reacted at room temperature for 60 s. The fluorescence intensity of the system was then measured using a fluorescence spectrometer. The data are shown in the table below:

[0062] Experimental results show that the fluorescence intensity of His-CQDs gradually increases with the increase of phenol concentration.

[0063] This is because phenol molecules can interact with functional groups on the surface of carbon quantum dots through hydrogen bonding and hydrophobic interactions, thereby suppressing nonradiative transition processes and enhancing fluorescence emission intensity.

[0064] As the concentration of phenol increases, ΔF / F0 gradually increases and shows a good correlation with the concentration of phenol within a certain concentration range.

[0065] When ΔF / F0 > +20%, the system exhibits a significant enhanced toxicity response.

[0066] Experimental results show that the quantum dot fluorescent probe of this invention can produce a stable fluorescence enhancement response to organic pollutants.

[0067] Example B1: A method for preparing and detecting His-CQDs@PDA under preferred parameters.

[0068] Weigh 1.30 g of L-histidine and 0.70 g of levofloxacin, add them to 22 mL of DMF, and stir under ultrasonic conditions for 12 min until completely dissolved to form a homogeneous solution; transfer the resulting solution to a stainless steel reactor lined with polytetrafluoroethylene, and perform a hydrothermal reaction at 200℃ for 12 h; after the reaction is completed, allow it to cool naturally to room temperature to obtain the reaction solution; The reaction solution was centrifuged at 11,000 rpm for 18 min, the supernatant was collected and placed in a dialysis bag with a molecular weight cutoff of 800 Da, and dialyzed in deionized water for 24 h, with the dialysate being changed every 4 h. After dialysis, the pH was adjusted to 7.4 with 1 mol / L NaOH solution, filtered through a 0.22 μm microporous membrane and freeze-dried for 48 h to obtain histidine-modified carbon quantum dot fluorescent probes, denoted as His-CQDs. Weigh 105 mg of His-CQDs and add 110 mL of Tris buffer solution (10 mmol / L). Adjust the pH of the system to 8.5 using 1 mol / L NaOH solution. Add 55 mg of dopamine hydrochloride and stir magnetically at room temperature for 2.5 h to allow dopamine to undergo an oxidative self-polymerization reaction, forming a polydopamine shell on the surface of His-CQDs. The resulting shell is approximately 10 nm thick, yielding solution A. Solution A was then centrifuged at 11,000 rpm for 18 min, washed four times with deionized water, and freeze-dried for 48 h to obtain a quantum dot fluorescent probe with a gated shell, denoted as His-CQDs@PDA. Dissolve His-CQDs@PDA in pure water to prepare a His-CQDs@PDA working solution with a concentration of 0.10 mg / mL, and store at 4°C protected from light. Establish reference parameters: The standard organic contaminant sample is a phenol solution (10 μM), and the standard metal contaminant sample is Cu. 2+ Solution (10 μM); After adding His-CQDs@PDA, the fluorescence trajectory curve F(t) was recorded, and the reference parameter t was obtained from the standard sample. ref = 41 s, F ref = 1320; The test sample yielded: F0 = 520, F peak = 1280, t peak = 78 s; Calculate: K d = 1.90, Ir = 0.95 It was identified as a metallic contaminant.

[0069] Examples B2 to B4: Shell thickness variable group.

[0070] Example B2: Compared with Example B1, only the amount of dopamine added was changed to 30 mg, the reaction time was 1 h, and the resulting shell thickness was about 4 nm, while the other conditions were the same.

[0071] Example B3: Dopamine addition amount 40 mg, reaction time 2 h, shell thickness approximately 7 nm.

[0072] Example B4: Dopamine addition amount 70 mg, reaction time 4 h, shell thickness approximately 17 nm.

[0073] Examples B5 to B7: Structural control group.

[0074] Example B5: Steps SB1 and SB2 are not performed, that is, His-CQDs are not coated with polydopamine, and His-CQDs are used only as quantum dot fluorescent probes.

[0075] Example B6: Carbon quantum dots were prepared using levofloxacin as the carbon source without adding L-histidine during the preparation process.

[0076] Example B7: Using commercially available carbon quantum dots (Sigma-Aldrich) as quantum dot fluorescent probes.

[0077] Examples B8 to B11: Histidine ratio variable group, with other conditions the same as in Example B1.

[0078] Example B8: L-histidine 1.0 g.

[0079] Example B9: L-histidine 1.6 g.

[0080] Example B10: L-histidine 2.0 g.

[0081] Example B11: L-histidine 2.5 g.

[0082] Examples B12 to B15: Gate-type shell variable group.

[0083] Example B12: Except for the following conditions, it is the same as Example B1.

[0084] The amount of His-CQDs added was 105 mg, the amount of Tris buffer solution was 110 mL, the pH of the system was 8.5, the amount of dopamine monomer added was 20 mg, and the reaction was carried out at room temperature with stirring for 2.5 h. The thickness of the polydopamine shell was measured to be approximately 3.4 nm by transmission electron microscopy.

[0085] Example B13: Except for the following conditions, it is the same as Example B1.

[0086] The amount of His-CQDs added was 105 mg, the amount of Tris buffer solution was 110 mL, the pH of the system was 8.5, the amount of dopamine monomer added was 35 mg, and the reaction was carried out at room temperature with stirring for 2.5 h. The thickness of the polydopamine shell was measured to be approximately 7.8 nm by transmission electron microscopy.

[0087] Example B14: Except for the following conditions, it is the same as Example B1.

[0088] The amount of His-CQDs added was 105 mg, the amount of Tris buffer solution was 110 mL, the pH of the system was 8.5, the amount of dopamine monomer added was 55 mg, and the reaction was carried out at room temperature with stirring for 3.5 h. The thickness of the polydopamine shell was measured to be approximately 12.6 nm by transmission electron microscopy.

[0089] Example B15: Except for the following conditions, it is the same as Example B1.

[0090] The amount of His-CQDs added was 105 mg, the amount of Tris buffer solution was 110 mL, the pH of the system was 8.5, the amount of dopamine monomer added was 80 mg, and the reaction was carried out at room temperature with stirring for 4.5 h. The thickness of the polydopamine shell was measured to be approximately 18.4 nm by transmission electron microscopy.

[0091] Example B16: Except for the following conditions, it is the same as Example B1.

[0092] The amount of His-CQDs added was 105 mg, the amount of Tris buffer solution was 110 mL, and the amount of dopamine monomer added was 55 mg. The reaction was carried out at room temperature with stirring for 2.5 h, but the pH of the system was adjusted to 7.0 using 1 mol / L NaOH solution. Transmission electron microscopy showed that the resulting polydopamine shell was not uniformly formed, with an average thickness of approximately 5.1 nm.

[0093] Example B17: Except for the following conditions, it is the same as Example B1.

[0094] The amount of His-CQDs added was 105 mg, the pH of the system was 8.5, the amount of dopamine monomer added was 55 mg, and the reaction was carried out at room temperature with stirring for 2.5 h, but the volume of Tris buffer solution was adjusted to 80 mL. The average thickness of the polydopamine shell was measured to be approximately 11.8 nm by transmission electron microscopy.

[0095] Example B18: Except for the following conditions, it is the same as Example B1.

[0096] The system pH was 8.5, the Tris buffer solution volume was 110 mL, the dopamine monomer dosage was 55 mg, and the reaction was carried out at room temperature with stirring for 2.5 h, but the His-CQDs dosage was adjusted to 70 mg. The average thickness of the polydopamine shell was measured to be approximately 13.2 nm by transmission electron microscopy.

[0097] Examples B19 to B20 only provide support for the range of essential parameters and do not participate in experimental test cases.

[0098] Example B19: The His-CQDs@PDA preparation method of the present invention was followed, but all parameters were taken at the lower limit value.

[0099] Weigh 1.20 g of L-histidine and 0.50 g of levofloxacin, add them to 20 mL of DMF, and sonicate for 10 min until completely dissolved to form a homogeneous solution. Transfer the resulting solution to a stainless steel reactor lined with polytetrafluoroethylene, and hydrothermally react at 200℃ for 12 h. After the reaction is complete, allow it to cool naturally to room temperature to obtain the reaction solution. The resulting reaction solution was centrifuged at 10,000 rpm for 15 min, the supernatant was collected and placed in a dialysis bag with a molecular weight cutoff of 500 Da, and dialyzed in deionized water for 24 h, with the dialysate being changed every 4 h during the process. After dialysis, the pH of the solution was adjusted to 7.3 with 1 mol / L NaOH solution, then filtered through a 0.22 μm microporous membrane, and freeze-dried for 48 h to obtain histidine-functionalized carbon quantum dot powder His-CQDs; Weigh 100 mg of His-CQDs and disperse them in 100 mL of Tris buffer solution. Adjust the pH of the system to 8.0 with 1 mol / L NaOH solution. Add 50 mg of dopamine monomer and stir at room temperature for 2 h to allow dopamine to undergo an oxidative self-polymerization reaction, forming a polydopamine shell on the surface of His-CQDs, thus obtaining solution A of the quantum dot fluorescent probe. Solution A was centrifuged at 10,000 rpm for 15 min, the supernatant was discarded, the precipitate was resuspended in deionized water and washed three times, filtered and freeze-dried for 48 h to obtain quantum dot fluorescent probe powder with a gate-type shell structure, denoted as His-CQDs@PDA; the thickness of the polydopamine shell was measured to be approximately 3.0 nm.

[0100] Example B20: The quantum dot fluorescent probe preparation method of the present invention was followed, but all parameters were taken at their upper limits.

[0101] Weigh 1.50 g of L-histidine and 1.00 g of levofloxacin, add them to 25 mL of DMF, and sonicate for 15 min until completely dissolved to form a homogeneous solution. Transfer the resulting solution to a stainless steel reactor lined with polytetrafluoroethylene, and hydrothermally react at 200℃ for 12 h. After the reaction is completed, allow it to cool naturally to room temperature to obtain the reaction solution. The resulting reaction solution was centrifuged at 12,000 rpm for 20 min, the supernatant was collected and placed in a dialysis bag with a molecular weight cutoff of 1,000 Da, and dialyzed in deionized water for 24 h, with the dialysate being changed every 4 h during the process. After dialysis, the pH of the solution was adjusted to 7.5 with 1 mol / L NaOH solution, then filtered through a 0.22 μm microporous membrane, and freeze-dried for 48 h to obtain histidine-functionalized carbon quantum dot powder His-CQDs; Weigh 110 mg of His-CQDs and disperse them in 120 mL of Tris buffer solution. Adjust the pH of the system to 9.0 with 1 mol / L NaOH solution. Add 60 mg of dopamine monomer and stir at room temperature for 3 h to allow dopamine to undergo an oxidative self-polymerization reaction, forming a polydopamine shell on the surface of His-CQDs, thus obtaining solution A of the quantum dot fluorescent probe. Solution A was centrifuged at 12,000 rpm for 20 min, the supernatant was discarded, the precipitate was resuspended in deionized water and washed 5 times, filtered and freeze-dried for 48 h to obtain quantum dot fluorescent probe powder with gate shell structure, denoted as His-CQDs@PDA; the thickness of the polydopamine shell was measured to be approximately 20.0 nm.

[0102] Experimental Example B1: Pollutant Identification Ability Test.

[0103] Experimental conditions: The experimental water sample was an artificially prepared simulated water body: pH = 7.2, temperature = 25℃. Initial pollutant concentration: Cu 2+ = 10 μM, phenol = 10 μM Detection method: fluorescence excitation wavelength 365 nm.

[0104] Comparison objects: Example B1, Example B2, Example B3, Example B4, Example B5, Example B6, Example B7.

[0105] As can be seen from the table above, in the water pollutant detection method based on time-resolved fluorescence trajectories proposed in this invention, the gate-type shell structure and histidine functional layer have a significant impact on the pollutant recognition ability. Example B1 shows that, under the synergistic effect of the carbon quantum dot core, histidine functional layer, and gate-type shell, the kinetic delay index K... d The fluorescence response index I reached 1.90. r A value of 0.95 indicates that the presence of metallic pollutants in the water sample can be stably determined. In Examples B2-B4, by changing the thickness of the gate-type shell, the system could still identify metallic pollutants, but the index value changed, indicating that the shell thickness significantly affects the pollutant diffusion kinetics. In Example B5, after removing the gate-type shell, K... d with I r The values ​​are all close to 1, indicating that the system cannot effectively distinguish between pollutant types. In Example B6, after removing the histidine functional layer, the system mainly exhibits an enhanced effect on organic pollutants. In Example B7, when commercially available carbon quantum dots are used, the lack of a specific functional structure also prevents stable pollutant differentiation.

[0106] Experimental Example B2: Detection Sensitivity Test.

[0107] Experimental conditions: Testing Cu 2+ Concentration gradient: 0–50 μM.

[0108] Experimental subjects: Examples B1, B8, B9, B10, and B11.

[0109] As can be seen from the table above, in the preferred embodiment B1 of the present invention, Cu 2+ The detection showed a minimum detection limit of 0.45 μM and a wide linear detection range. In Example B8, the reduced histidine content weakened the metal ion coordination ability, resulting in a significant decrease in detection sensitivity. In Example B9, increasing the histidine ratio improved detection sensitivity, but an excessively high nitrogen source ratio affected the fluorescence efficiency of the carbon quantum dots. Examples B10 and B11, by changing the carbon quantum dot particle size, revealed that carbon quantum dots with a suitable particle size exhibited better fluorescence response performance. Therefore, the histidine ratio and carbon quantum dot structure used in Example B1 can enhance the recognition ability of metal ions while maintaining fluorescence intensity, thereby achieving optimal detection sensitivity.

[0110] Experimental Example B3: Experiment on the effect of gate-type shell formation process on time-resolved dynamic performance.

[0111] Experimental conditions: The water samples used in the experiment were from the same batch of artificially simulated water, with a pH of 7.2 and a temperature of 25℃. The organic pollutant was phenol, with an initial concentration of 10 μM; the metallic pollutant was Cu. 2+ The initial concentration was 10 μM. Unless otherwise specified, the preparation conditions, reference parameter establishment conditions, quantum dot fluorescent probe addition concentration, and fluorescence detection conditions for histidine-functionalized carbon quantum dots were performed as described in each example. The fluorescence detection excitation wavelength was 365 nm, the fluorescence trajectory curve F(t) was recorded, and the peak time t was calculated. peak The time separation value Δt was determined, and the thickness of the polydopamine shell was measured using transmission electron microscopy.

[0112] As can be seen from the table above, in the present invention, the amount of dopamine monomer added, the oxidative self-polymerization reaction time, the system pH, the volume of the Tris buffer solution, and the amount of His-CQDs added all affect the polydopamine shell formation process and further affect the time-resolved kinetic performance of His-CQDs@PDA. Examples B12 and B13, by reducing the amount of dopamine monomer added, form thinner shells. Although the overall response speed is faster, the diffusion retardation effect on metal ions is limited, resulting in a smaller Δt. Examples B14 and B15, by extending the reaction time and increasing the amount of dopamine monomer added, obtain thicker shells. The metal ion diffusion delay effect is enhanced, and Δt increases, but at the same time, t... peak The significant increase in time resolution indicates that an excessively thick shell layer increases overall mass transfer resistance. In Example B16, lowering the pH of the shell reaction resulted in insufficient polydopamine formation, leading to an uneven shell layer and decreased time resolution. Examples B17 and B18, by varying the volume of the Tris buffer solution and the amount of His-CQDs added, also caused changes in the shell structure and kinetic response. A comprehensive comparison shows that the shell formation process corresponding to Example B1 achieves a better balance between response speed and time resolution, thus exhibiting the best overall kinetic performance.

Claims

1. A method for preparing quantum dot fluorescent probe for online monitoring of comprehensive toxicity of water quality, characterized in that, Includes the following steps: SA1. Preparation of histidine-functionalized carbon quantum dots: SA1-1. Weigh L-histidine and levofloxacin and add them to DMF. Stir ultrasonically for 10-15 min until completely dissolved to form a homogeneous solution. Transfer the resulting solution to a stainless steel reactor lined with polytetrafluoroethylene and hydrothermally react at 200℃ for 12 h. After the reaction is complete, allow it to cool naturally to room temperature to obtain the reaction solution. Let n be the scale factor and n∈R + Then the addition amount interval of each component in SA1-1 is: L-histidine is [1.2n, 1.5n] g, levofloxacin is [0.5n, 1n] g, and DMF is [20n, 25n] mL. SA1-2. Centrifuge the reaction solution obtained in SA1-1 at 10000~12000 rpm for 15~20 min, collect the supernatant and place it in a dialysis bag with a molecular weight cutoff of 500~1000 Da, and dialyze in deionized water for 24 h, changing the dialysis solution every 4 h during the period. SA1-3. After dialysis, the pH of the solution was adjusted to 7.3-7.5 with 1 mol / L NaOH solution, then filtered through a 0.22 μm microporous membrane, and then freeze-dried for 48 h to obtain histidine-modified carbon quantum dot fluorescent probes, denoted as His-CQDs. SA2, Preservation of quantum dot fluorescent probes: The His-CQDs prepared in SA1 were dispersed in pure water and ultrasonically dispersed evenly to prepare a His-CQDs working solution with a concentration of 0.01~0.1 mg / mL, which was then stored at 4℃ in the dark.

2. The production method according to claim 1, wherein The His-CQDs prepared in SA1 were further coated with polydopamine to construct quantum dot fluorescent probes with selective mass transfer function. The steps are as follows: SB1. Preparation of polydopamine shell: The His-CQDs prepared in SA1 were dispersed in Tris buffer solution, and the pH of the system was adjusted to 8.0-9.0 with 1 mol / L NaOH solution. Then, dopamine monomer was added and stirred at room temperature for 2-3 h to allow dopamine to undergo oxidative self-polymerization reaction and form a polydopamine shell on the surface of His-CQDs, thus obtaining solution A. Let n be the multiplier and n∈R+, then the addition range of each component in SB1 is: His-CQDs is [100n, 110n] mg, Tris buffer solution is [100n, 120n] mL, and dopamine monomer is [50n, 60n] mg; SB2, Purification Process: The solution A prepared in SB1 was centrifuged at 10,000 to 12,000 rpm for 15 to 20 min, the supernatant was discarded, the precipitate was resuspended in deionized water and washed repeatedly 3 to 5 times, filtered and freeze-dried for 48 h to obtain a quantum dot fluorescent probe with a gate-type shell, denoted as His-CQDs@PDA. SB3. Preservation of quantum dot fluorescent probes: Redisperse His-CQDs@PDA in pure water to prepare a His-CQDs@PDA working solution with a concentration of 0.01~0.1 mg / mL, and store at 4°C protected from light.

3. A method for online monitoring of comprehensive toxicity of water quality, characterized in that, The quantum dot fluorescent probe prepared by the method of claim 1 comprises: The His-CQDs working solution described in SA2 is brought into contact with the water sample to be tested, and the overall toxicity of the water sample is monitored online by detecting changes in its fluorescence signal. The histidine is used to simulate the key toxicity recognition functional site in the luciferase system of luminescent bacteria, so that the His-CQDs produce a fluorescence quenching response to heavy metal ions and a fluorescence enhancement response to organic pollutants, thereby characterizing the overall toxicity of the water sample to be tested.

4. The method of claim 3, wherein, The steps are as follows: 1) Mix an equal volume of His-CQDs working solution with the water sample to be tested; 2) React at room temperature for 30~120 s; 3) Measure the fluorescence intensity using a fluorescence spectrometer or portable fluorescence detection device; 4) Calculate ΔF / F0 and obtain the TU value by referring to the standard curve; Wherein, F0 is the initial fluorescence intensity of His-CQDs working solution without water sample added, F is the fluorescence intensity after adding water sample to be tested, ΔF is the difference between F0 and F, and TU is the comprehensive toxicity intensity of water quality. When ΔF / F0 < -20%, it is determined to be a quenching toxicity response; When ΔF / F0 > +20%, it is determined to be an enhanced toxicity response.

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