Method for detecting Cu < 2 + > in water body based on biosynthetic cadmium sulfide quantum dot fluorescent probe
By using biosynthesized cadmium sulfide quantum dot fluorescent probes and extracellular polymers as templates, the Cu2+ detection process is simplified, solving the problems of complexity and toxicity of traditional methods, and achieving high-sensitivity and low-cost Cu2+ detection in water.
Patent Information
- Application Number
- CN202511081172.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-04
- Publication Date
- 2025-11-25
AI Technical Summary
Existing methods for detecting Cu2+ are complex to operate, require skilled personnel and expensive instruments, and traditional quantum dot synthesis is highly toxic, energy-intensive, and lacks biocompatibility.
Extracellular polymers were produced by carbon source stress/induction of *Desulfovibrio desulfurization* subsp. *desulfurization*, which were then used as biological templates to synthesize cadmium sulfide quantum dots. These dots were then used as fluorescent probes to detect Cu2+ in water, simplifying the operation and improving sensitivity.
It enables rapid, simple, sensitive and selective Cu2+ detection. The synthesis process is simple, low-cost and environmentally friendly, and suitable for actual water body detection.
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Figure CN121007874A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of environmental detection, and particularly relates to a method for detecting Cu 2+ in water bodies based on a biosynthetic cadmium sulfide quantum dot fluorescent probe. BACKGROUND
[0002] Quantum dots are widely used in the fields of biosensing, solar cells, photovoltaic devices, cell imaging, light-emitting diodes, photodetectors, etc. due to their excellent photoelectric properties. Traditional synthesis methods of quantum dots mainly include chemical synthesis and physical synthesis, but these traditional synthesis methods need to use high temperature, inert atmosphere and toxic reagents, resulting in lack of biocompatibility in application, and even a small amount of application can cause harm to the environment. In order to solve the problems of high toxicity, high energy consumption and difficult surface modification in the traditional synthesis of quantum dots, researchers have begun to turn to a green synthesis approach which is simpler, more environmentally friendly and non-toxic. Among them, EPS can be used as a biological template to synthesize cadmium sulfide quantum dots by adsorbing cadmium ions on the surface of EPS.
[0003] Cu 2+ is one of heavy metals and also an essential element for the human body, which is an important component in many physiological processes, including signal transduction, energy production and cell respiration; however, a large amount of copper intake by the human body can be harmful to the body and is related to Wilson's disease, Alzheimer's disease, etc. At present, the traditional detection methods of Cu 2+ mainly include atomic absorption spectrometry, inductively coupled plasma mass spectrometry and electrochemical analysis method, etc. However, these methods are difficult to operate, need skilled workers and expensive instruments, are not only time-consuming but also need complex pretreatment. Therefore, it is of great practical significance for environmental detection, environmental protection and human health to develop a detection technology for detecting Cu 2+ in water environment, which has fast response time, high sensitivity, good selectivity, convenient operation and low cost. SUMMARY
[0004] In order to overcome the deficiencies of the prior art, the application provides a method for detecting Cu 2+ in water bodies based on biosynthetic cadmium sulfide quantum dots fluorescent probe, which first produces extracellular polymeric substances by carbon source stress / induction of Desulfovibrio desulfuricans, biosynthesizes cadmium sulfide quantum dots with strong fluorescence outside the cells, and then uses the cadmium sulfide quantum dots as a fluorescent probe to detect Cu 2+ in water samples. The method is simple in operation, high in sensitivity and good in selectivity, and has good detection effect on Cu 2+ in actual water bodies.
[0005] In order to achieve the above purpose, the technical scheme adopted by the application is:
[0006] This invention provides a biosynthesized cadmium sulfide quantum dot fluorescent probe for detecting Cu in water. 2+ The method includes the following steps:
[0007] S1. Extracellular polymers are produced by culturing desulfurized Vibrio desulfurization subsp. desulfurization through carbon source stress / induction. The extracted extracellular polymers are then used as biological templates, cadmium salts are used as metal precursors, and sulfides are used as sulfur sources. After the extracellular polymers adsorb cadmium, they are mixed with the sulfur sources and finally dialyzed to obtain cadmium sulfide quantum dots.
[0008] S2. Using PBS buffer, cadmium sulfide quantum dots from S1 were converted into cadmium sulfide quantum dot fluorescent probe solution, and then different concentrations of Cu were added. 2+ After mixing and reacting, fluorescence emission spectroscopy was performed at an excitation wavelength of 350 nm.
[0009] S3. Based on the spectral determination in S2, calculate Cu. 2+ The fluorescence quenching ratio F0 / F before and after the addition of Cu was used to obtain the fluorescence emission intensity. 2+ Standard curve of concentration versus fluorescence intensity;
[0010] S4. Add the sample to be tested to the cadmium sulfide quantum dot fluorescent probe solution and perform fluorescence emission at an excitation wavelength of 350 nm. Observe the change in fluorescence intensity to determine whether Cu is present. 2+ Then, based on the standard curve, the Cu in the sample to be tested... 2+ Quantitative detection of concentration was performed.
[0011] This invention detects Cu 2+ The method has the advantages of simple operation, high sensitivity, low detection limit and good selectivity, and is effective for detecting Cu in actual water bodies. 2+ It has good quantitative detection capabilities, and cadmium sulfide quantum dots are simple to synthesize, low in cost, and environmentally friendly.
[0012] Preferably, the cadmium salt includes cadmium chloride, cadmium nitrate, and cadmium nitrate, and the sulfide includes sodium sulfide and potassium sulfide.
[0013] Preferably, the pH of the PBS buffer solution is 7-7.5, and the concentration of the cadmium sulfide quantum dot fluorescent probe solution is 1-4 mg / mL.
[0014] Preferably, the reaction in S2 is carried out at 20-30°C for 5-10 minutes.
[0015] Preferably, during quantitative detection, Cu 2+ The detection limit is 0.3 μM.
[0016] Preferably, the different concentrations of Cu mentioned in S2 2+Cu at 0.1, 0.3, 0.5, 0.7, 2, 4, 5, 6, 8, 10, 12, 14, 15, 16, 18, 20, 25, 30, 35, 40, 45, 50, 60 umol / L 2+ .
[0017] Preferably, in S1, the extracellular polymers produced by the Desulfovibrio desulfuricans are extracted after carbon source stress / induction culture, and then the extracellular polymers are dissolved in water, and then cadmium salt is added, and the extracellular polymers are fully adsorbed on the cadmium by oscillation, and then the pH is adjusted to 8.5-9.5, and sulfide is added and mixed, and after reaction, the cadmium sulfide quantum dots are obtained by dialysis.
[0018] More preferably, the concentration of the extracellular polymers is 1-4 mg / mL, the concentration of the cadmium salt is 1-2 mmol / L, and the concentration of the sulfide is 0.5-1 mmol / L.
[0019] More preferably, the reaction is carried out at 25-35℃ and 130-200 rpm for 10-15h.
[0020] More preferably, the dialysis is dialysis for 20-30h using a dialysis bag with a molecular weight cut-off of 4000 Da, and the water is changed every 7-9h.
[0021] More preferably, the method for producing extracellular polymers by carbon source stress / induction culture of Desulfovibrio desulfuricans is as follows: Desulfovibrio desulfuricans bacterial suspension is inoculated into Starkey culture medium, carbon source is added, and stress / induction culture is carried out for more than 3 days, the bacterial body is separated by freezing centrifugation, the bacterial body is washed with 0.6-1.2% NaCl, the extracellular polymers produced by Desulfovibrio desulfuricans are extracted by sodium hydroxide method, and then filtered and dialyzed, and freeze-dried into powder.
[0022] Compared with the prior art, the present application has the following advantages:
[0023] The present application provides a method for detecting Cu in water based on biosynthetic cadmium sulfide quantum dot fluorescent probe, which first produces extracellular polymers by carbon source stress / induction of Desulfovibrio desulfuricans, biosynthesizes cadmium sulfide quantum dots with strong fluorescence in the extracellular polymers, and then uses the cadmium sulfide quantum dots as a fluorescent probe to mix with a buffer solution to prepare a fluorescent probe solution, which is then used for detecting Cu in water. 2+ 2+ Specifically, the present application has the following advantages:
[0024] (1) The fluorescent probe of the present application is simple to prepare, has excellent recognition ability for Cu, and can quickly detect Cu without the need for skilled personnel, expensive instruments, and time-consuming and complex pretreatment. 2+ 2+ (2) The fluorescent probe of the present application can be used for the detection of Cu in water.
[0025] (2) The fluorescent probe of the present invention has good sensitivity and high selectivity, and is effective in detecting Cu in water samples. 2+ It exhibits a wide linear range during detection, from 0 μM to 60 μM, and demonstrates excellent linearity (R0) throughout the entire range. 2 =0.992), with a detection limit of 48 nM.
[0026] (3) Through the biological template effect of extracellular polymer (EPS), cadmium chloride and sodium sulfide are used as precursors. Without the need for high temperature anaerobic conditions and complex synthesis conditions such as toxic precursors, cadmium sulfide quantum dots with good fluorescence properties can be rapidly biosynthesized at room temperature and pressure. Attached Figure Description
[0027] Figure 1 Cu for CdS quantum dot fluorescence spectrum 2+ Concentration dependence;
[0028] Figure 2 The fluorescence quenching rate (F0 / F) of CdS quantum dots as a function of Cu 2+ The relationship between concentration changes;
[0029] Figure 3 Adding Cu at different pH levels 2+ Changes in fluorescence intensity of CdS quantum dots before and after;
[0030] Figure 4 For the detection of Cu by CdS quantum dot fluorescent probes for coexisting ion pairs 2+ The selective effect. Detailed Implementation
[0031] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.
[0032] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.
[0033] Example 1: Carbon source stress / induction of extracellular polymeric substances from the desulfurized subspecies of Vibrio desulfuriformis
[0034] The strain in this embodiment is Desulfovibrio desulfuricans subsp. desulfuricans, which is purchased from Beijing Bao Bo Wei Biotechnology Co., Ltd.
[0035] (1) Preparation of Starkey medium: 0.50 g of K2HPO4, 1.00 g of Na2SO4, 2.00 g of MgSO4·7H2O, 1.00 g of NH4Cl, 0.10 g of CaCl2·2H2O, 2.50 g of 60% DL-sodium lactate, and 1.00 g of yeast extract were weighed and dissolved in 1 L of pure water, and the pH was adjusted to 7.
[0036] (2) Activation culture: -80°C frozen Desulfovibrio desulfuricans subsp. desulfuricans bacterial liquid (concentration 1×10 9 CFU / mL) was thawed at room temperature, and under sterile conditions, the bacterial liquid was transferred to a sterile container containing 95 mL of Starkey medium at a 5% (v / v) inoculation amount. 1 mL of ascorbic acid solution (10.00 g / L) was added to the culture system, and high-purity nitrogen gas (≥99.99%) was continuously introduced for 1 minute to remove dissolved oxygen and establish an anaerobic environment. Then the container opening was closed with sealing film and aluminum foil, and placed in a constant temperature shaking incubator (30°C, 150 rpm) for 48 h of activation culture to obtain the target bacterial suspension.
[0037] (3) Stress / induction culture: logarithmic growth phase Desulfovibrio desulfuricans subsp. desulfuricans bacterial suspension was aseptically transferred to a sterile container containing 90 mL of Starkey medium at a 10% (v / v) inoculation amount, 1 mL of ascorbic acid solution (10.00 g / L) was injected, and 1 mL of sodium formate solution (300.00 g / L) was added to make the final concentration of sodium formate in the medium 3.0 g / L. Then high-purity nitrogen gas (≥99.99%) was continuously introduced for 1 minute to remove dissolved oxygen. Subsequently, the container opening was closed with sealing film and aluminum foil, and 72 h of sodium formate stress / induction culture was carried out in a constant temperature shaking incubator (30°C, 150 rpm).
[0038] (4) Extraction of EPS: 30 mL of bacterial solution after stress culture was taken and placed in a 50 mL centrifuge tube. The tube was centrifuged at 8000 r / min in a high-speed refrigerated centrifuge at 4°C for 10 min. The supernatant was discarded and the bacterial body was resuspended in 30 mL of 0.9% (w / v) NaCl solution. The tube was centrifuged under the same conditions and the supernatant was discarded. The washing step was repeated twice to completely remove the residual culture medium. Then, 10 mL of 0.9% (w / v) NaCl solution was used to resuspend the washed bacterial body. 0.5 mL of 1.00 mol / L NaOH solution was added, vortexed and mixed, and placed at 4°C for 3 h to lyse the cells and release EPS. Finally, the lysate was centrifuged at 10000 r / min for 20 min at 4°C. The supernatant was collected, filtered through a 0.45 μm filter membrane, and the residual bacterial body fragments were removed. The obtained filtrate was loaded into a 4000 Da dialysis bag and dialyzed in ultrapure water for 24 h (every 8 h, the water was changed). The dialyzed EPS was frozen in a -80°C refrigerator for 24 h, then transferred to a freeze dryer to freeze-dry into powder, and finally stored in a desiccator for subsequent use.
[0039] Example 2: EPS biosynthesis of cadmium sulfide quantum dots under carbon source induction
[0040] An appropriate amount of EPS was weighed and dissolved in ultrapure water to prepare an EPS solution with a concentration of 4 mg / mL. The solution was ultrasonically dispersed until it was clear. Then, 1 mM of CdCl2 solution was added to the EPS solution, and the volume ratio of the EPS solution to the CdCl2 solution was 100:1. The mixture was shaken at 30°C and 150 rpm on a constant temperature shaker for 2 h to fully adsorb Cd(II). After adjusting the pH to 9 with NaOH and HCl solutions, 50 uL of 1 mM Na2S solution was slowly added at room temperature, and the mixture was shaken well. The mixture was then reacted at 30°C and 150 rpm for 12 h. After that, the CdS QDs were dialyzed in a 4000 Da dialysis bag for 24 h (every 8 h, the water was changed), and the free ions were removed to obtain purified CdS QDs.
[0041] Example 3: A method for detecting Cu 2+ based on EPS biomimetic synthesis of cadmium sulfide quantum dot fluorescent probe
[0042] (1) 2.4 mL of phosphate buffer solution (0.1 mol / L, pH = 7) and 0.5 mL of 4 mg / mL CdS QDs solution were added to a 10 mL centrifuge tube. After mixing well, 0.1 mL of different concentrations of Cu 2+The solution (0.1, 0.3, 0.5, 0.7, 2, 4, 5, 6, 8, 10, 12, 14, 15, 16, 18, 20, 25, 30, 35, 40, 45, 50, 60 umol / L), the blank control group was added with equal amount of ultrapure water, mixed uniformly, reacted for five minutes at room temperature, then excited at 350 nm, and the change of fluorescence intensity was determined by fluorescence spectrometer, as shown in Figure 1 .
[0043] (2) The concentration of Cu 2+ was taken as the abscissa, and the ratio of the blank fluorescence intensity (F0) to the fluorescence intensity (F) of Cu 2+ added in different concentrations was taken as the ordinate, to draw a standard curve, as shown in Figure 2 ; according to the detection limit formula LOD = 3 * SD / M, the detection limit of the system was 0.3 μM, wherein SD was the standard deviation of the measured blank sample, and M represented the slope of the linear regression equation.
[0044] At the same time, the phosphate buffer solution with different pH (5, 6, 7, 8, 9, 10) was configured according to the same method for Cu 2+ detection (the concentration of Cu 2+ solution was 30 umol / L), and it was found that pH had a significant regulatory effect on the detection performance of the quantum dot fluorescence probe; as shown in Figure 3 , when the pH was from 5 to 7, the fluorescence intensity of the quantum dots gradually increased, when the pH = 7, the fluorescence intensity of the quantum dots reached the peak value, and the fluorescence quenching efficiency induced by Cu 2+ reached the maximum value, indicating that the probe was most sensitive to Cu 2+ under this pH condition; and when the pH continued to increase, the fluorescence intensity began to decrease, and when the pH continued to rise to 9-10, the quenching of Cu 2+ on the quantum dots was significantly reduced; the results showed that pH = 7 was the optimal condition for the detection of Cu 2+ by the fluorescence probe, and the probe had high fluorescence background signal and strong Cu 2+ response ability at this time.
[0045] In addition, 20 kinds of metal ions (K + , Cd 2+ , Ca 2+ , Mg 2+ , Na + , Mn 2+ , As 3+ , Ni + , Co 2+ , Li + , Zn 2+ , Sn 2+ , Fe 2+ , Cr 6+, Al 3+ , Ag + , W 6+ , Pt 2+ , Ga 3+ , La 3+ The anti-interference analysis was carried out. The concentration of all metal ions in the experiment was 30 μM. It can be seen from Figure 4 that Cu 2+ has a significant quenching effect on the fluorescence intensity of CdS QDs, while other interference ions do not show significant quenching behavior, indicating that CdS QDs have excellent selectivity.
[0046] Example 4: Detection of actual water samples
[0047] The actual water samples are tap water and Pearl River water from Guangdong University of Technology. The water samples are filtered through a 0.22 μm filter before being used for detection.
[0048] Cu 2+ was added to the tap water and river water to prepare Cu 2+ solutions of different concentrations.
[0049] The fluorescence probe of 4 mg / mL CdS QDs was used to detect Cu 2+ . 2.4 mL of phosphate buffer solution (pH = 7) and 0.5 mL of CdS QDs solution were added to the centrifuge tube, mixed thoroughly, then 0.1 mL of Cu 2+ solution of different concentrations (10, 20, 30 μM) in the actual water sample was added, mixed well, reacted for five minutes, and the change in fluorescence intensity was measured under excitation at 350 nm.
[0050] The detection results are shown in Table 1. The detection results are basically consistent with the standard addition concentration, the recovery rate is 99.52-110.11%, and the experimental results of each sample are close to 100%, indicating that the actual water sample detection has excellent accuracy and practicality.
[0051] Table 1 Recovery rate of Cu 2+ in actual water samples
[0052]
[0053] The embodiments of the present application are described in detail above, but the present application is not limited to the described embodiments. For those skilled in the art, various changes, modifications, replacements and variations of these embodiments can be made without departing from the principles and spirits of the present application, and still fall within the protection scope of the present application.
Claims
1. A biosynthesized cadmium sulfide quantum dot fluorescent probe for detecting Cu in water. 2+ The method is characterized by, Includes the following steps: S1. Extracellular polymers are produced by culturing desulfurized Vibrio desulfurization subsp. desulfurization through carbon source stress / induction. The extracted extracellular polymers are then used as biological templates, cadmium salts are used as metal precursors, and sulfides are used as sulfur sources. After the extracellular polymers adsorb cadmium, they are mixed with the sulfur sources and finally dialyzed to obtain cadmium sulfide quantum dots. S2. Using PBS buffer, cadmium sulfide quantum dots from S1 were converted into cadmium sulfide quantum dot fluorescent probe solution, and then different concentrations of Cu were added. 2+ After mixing and reacting, fluorescence emission spectroscopy was performed at an excitation wavelength of 350 nm. S3. Based on the spectral determination in S2, calculate Cu. 2+ The fluorescence quenching ratio F0 / F before and after the addition of Cu was used to obtain the fluorescence emission intensity. 2+ Standard curve of concentration versus fluorescence intensity; S4. Add the sample to be tested to the cadmium sulfide quantum dot fluorescent probe solution and perform fluorescence emission at an excitation wavelength of 350 nm. Observe the change in fluorescence intensity to determine whether Cu is present. 2+ Then, based on the standard curve, the Cu in the sample to be tested... 2+ Quantitative detection of concentration was performed.
2. The biosynthetic cadmium sulfide quantum dot fluorescent probe for detecting Cu in water as described in claim 1. 2+ The method is characterized by, The cadmium salts include cadmium chloride, cadmium nitrate, and cadmium nitrate, and the sulfides include sodium sulfide and potassium sulfide.
3. The biosynthetic cadmium sulfide quantum dot fluorescent probe for detecting Cu in water as described in claim 1. 2+ The method is characterized by, The pH of the PBS buffer solution is 7-7.5, and the concentration of the cadmium sulfide quantum dot fluorescent probe solution is 1-4 mg / mL.
4. The biosynthetic cadmium sulfide quantum dot fluorescent probe for detecting Cu in water as described in claim 1. 2+ The method is characterized by, The reaction described in S2 is carried out at 20-30°C for 5-10 minutes.
5. The biosynthetic cadmium sulfide quantum dot fluorescent probe for detecting Cu in water as described in claim 1. 2+ The method is characterized by, During quantitative detection, for Cu 2+ The detection limit is 0.3 μM.
6. The biosynthetic cadmium sulfide quantum dot fluorescent probe for detecting Cu in water as described in claim 1. 2+ The method is characterized by, S2 describes different concentrations of Cu 2+ Cu concentrations including 0.1, 0.3, 0.5, 0.7, 2, 4, 5, 6, 8, 10, 12, 14, 15, 16, 18, 20, 25, 30, 35, 40, 45, 50, and 60 μmol / L 2+ .
7. The biosynthetic cadmium sulfide quantum dot fluorescent probe for detecting Cu in water as described in claim 1. 2+ The method is characterized by, In S1, extracellular polymers are produced by carbon source stress / induced culture of *Desulfovibrio desulfurization subsp.* and the extracellular polymers are extracted. The extracellular polymers are then dissolved in water, and cadmium salts are added. The extracellular polymers are shaken to fully adsorb cadmium. The pH is then adjusted to 8.5-9.5, and sulfides are added and mixed. After the reaction, cadmium sulfide quantum dots are obtained by dialysis.
8. The biosynthetic cadmium sulfide quantum dot fluorescent probe for detecting Cu in water as described in claim 7. 2+ The method is characterized by, The concentration of the extracellular polymeric substance is 1–4 mg / mL, the concentration of the cadmium salt is 1–2 mmol / L, and the concentration of the sulfide is 0.5–1 mmol / L.
9. A biosynthesized cadmium sulfide quantum dot fluorescent probe for detecting Cu in water, as described in claim 7. 2+ The method is characterized by, The reaction is carried out at 25-35℃ and 130-200 rpm for 10-15 hours.
10. A biosynthesized cadmium sulfide quantum dot fluorescent probe for detecting Cu in water, as described in claim 7. 2+ The method is characterized by, The dialysis is performed using a 4000Da dialysis bag for 20-30 hours, with the water changed every 7-9 hours.