A method for detecting hydrogen peroxide and glucose

Abstract

The application discloses a method for detecting H2O2 and glucose, and constructs an AMP-Tb / CPBA probe by using adenosine monophosphate (AMP), terbium (Tb) and 3-carboxyphenylboronic acid (CPBA). 3+ CPBA can open the fluorescence of Tb 3+ at 488 and 544 nm as a sensitizing agent. The probe can be used for quantitative detection of H2O2 and glucose, and the sensitivity and selectivity of the detection are good, and the detection limits of H2O2 and glucose can be as low as 0.23 μM and 0.49 μM respectively.

Description

TECHNICAL FIELD

[0001] The present application relates to a fluorescent method for detecting hydrogen peroxide and glucose, and in particular to the construction of an AMP-Tb / CPBA probe system and the quantitative detection of hydrogen peroxide and glucose concentrations. BACKGROUND

[0002] Hydrogen peroxide (H2O2) is a strong oxidant and is often used as a bleaching agent in industry (e.g. bleaching cotton cloth for printing and dyeing). H2O2 is also an effective bactericide and is therefore often used for disinfection and sterilization of foodstuffs such as dairy products (including milk), chicken claws and soy products. However, residual H2O2 in foodstuffs can cause serious harm to human health. Excessive intake of H2O2 can accelerate aging and cause various diseases such as cardiovascular disease, diabetes and cancer. Therefore, it is very important to remove H2O2 from foodstuffs. In addition, H2O2 residue and illegal addition of H2O2 in foodstuffs are also one of the serious food safety problems.

[0003] Glucose, as a monosaccharide, is widely present in living organisms and is a component of certain disaccharides (such as sucrose, maltose, etc.) and polysaccharides (such as starch, cellulose, etc.), and is the main energy substance in living organisms. The glucose molecule is the main component of blood sugar and urine sugar, and the detection of the glucose molecule has important significance in the early detection, diagnosis and treatment of diabetes. Glucose, as a major physiological analyte, affects all aspects of people's daily life, and the detection of glucose also has important significance for quality control in the food industry.

[0004] To date, most fluorescent probes for detecting H2O2 and glucose only involve changes in fluorescence intensity at a single wavelength, and the results are extremely susceptible to changes in probe concentration, excitation intensity and external environment. However, ratiometric fluorescent probes can avoid such influences. Dual-emission ratiometric fluorescent probes measure the ratio of the fluorescence intensity of the reference fluorophore and the response fluorophore at different emission wavelengths, which avoids errors caused by single signal detection systems. As a new type of hybrid material, lanthanide coordination polymers (CPs) have unique fluorescence properties such as large stokes shift, long fluorescence lifetime and high photochemical stability, and have been used for the development and synthesis of fluorescent probes and have important applications in the field of fluorescent detection. However, due to the prohibition of f-f electronic transitions, the excitation efficiency of Ln 3+ is very low. When Ln 3+ is coordinated with a certain high molar absorbance organic ligand (referred to as an "antenna ligand"), the ligand can transfer the absorbed energy to the Ln 3+ to which it is coordinated, so that the Ln 3+The luminescence is more sensitive. Lanthanide CPs have been used to synthesize fluorescent probes for detecting anions, biomolecules, metal ions and small organic molecules. However, in most of such probes the antenna ligand only acts as a sensitizing ligand, providing a change in the intensity of monochromatic fluorescent signal, and the selectivity of these probes is poor, and the accuracy is low. Therefore, it is still challenging to develop a fluorescent probe with simplicity, high sensitivity and selectivity for determining H2O2 and glucose. SUMMARY

[0005] One of the purposes of the present application is to construct a new type of lanthanide coordination polymer fluorescent probe as a probe for detecting H2O2. The lanthanide coordination polymer is spontaneously assembled from one adenosine monophosphate (AMP), Tb 3+ and 3-carboxyphenylboronic acid (CPBA) in aqueous solution, and CPBA acts as a sensitizer (through antenna effect) to turn on the Tb 3+ fluorescence at 488 and 544 nm, and the probe is labeled as AMP-Tb / CPBA.

[0006] The second purpose of the present application is to provide the application of the AMP-Tb / CPBA probe for detecting H2O2 and glucose.

[0007] The third purpose of the present application is to provide a method for detecting H2O2. When H2O2 is added to the AMP-Tb / CPBA probe system, the nucleophilic H2O2 reacts with the electrophilic boronic acid in CPBA and is hydrolyzed to produce m-hydroxybenzoic acid (HBA), and the product HBA emits strong blue fluorescence at 401 nm. At the same time, the intramolecular energy transfer efficiency of CPBA to Tb 3+ is reduced due to the oxidation of CPBA by H2O2, so that the fluorescence of AMP-Tb / CPBA at 544 nm is quenched. Therefore, the ratio of the fluorescence intensities at 401 nm and 544 nm (I 401 / I 544 ) can be used as the signal output of the ratio fluorescent probe for sensitively detecting the concentration of H2O2, and the linear range of the detection system for detecting H2O2 is 0.1-60 μM, and the detection limit is 0.23 μM.

[0008] The fourth purpose of the present application is to provide a method for detecting glucose. Glucose can be oxidized by glucose oxidase to H2O2 and gluconic acid, and the product H2O2 produces a fluorescent response to the probe AMP-Tb / CPBA. Accordingly, the AMP-Tb / CPBA probe can also be used to determine the hydroxyl radical hydrogen peroxide produced by glucose, so as to quantitatively detect glucose. The linear range of the detection system for detecting glucose is 1-60 μM, and the detection limit is 0.49 μM.

[0009] To achieve the above purposes, the technical solutions adopted by the present application are as follows:

[0010] A preparation method of a novel AMP-Tb / CPBA coordination polymer fluorescent probe, comprising the following steps:

[0011] (1) slowly adding a Tb(NO3)3 aqueous solution (2.0 mM) into a HEPES buffer solution (100 mM; pH 7.4) in which AMP disodium salt (2.0 mM) is dissolved, a white precipitate is formed immediately after the Tb(NO3)3 solution is added, then 100 μL of a CPBA aqueous solution (0.1 M) is added, and stirring is conducted at room temperature.

[0012] (2) centrifuging the above solution, collecting the white precipitate, dispersing the white precipitate in 10 mL of ultrapure water after washing and drying to obtain an AMP-Tb / CPBA suspension.

[0013] In step (1), the volume of the Tb(NO3)3 aqueous solution and the AMP disodium salt solution is 4.0 mL.

[0014] In step (1), the stirring time is 2.5-3.5 h, and preferably 3 h.

[0015] In step (2), the centrifugal speed is 8000 rpm, and the centrifugal time is 10 min.

[0016] In step (2), the washing solution is ultrapure water.

[0017] The AMP-Tb / CPBA prepared according to the above method is composed of a nanoscale fiber network structure and can emit green fluorescence, the emission peak of the coordination polymer is located at 488 and 544 nm under excitation of 290 nm excitation light, the fluorescence intensity of the emission peaks at 488 and 544 nm can be quenched by H2O2, and the non-fluorescent CPBA can be oxidized to HBA by H2O2, and HBA has a new fluorescence emission peak at 410 nm.

[0018] The application further provides application of the AMP-Tb / CPBA in detection of H2O2, which can realize quantitative detection of H2O2.

[0019] The application further provides a detection method of H2O2, which comprises the following steps: mixing 100 μL of the AMP-Tb / CPBA suspension and a HEPES buffer solution, adding H2O2 with different concentrations into the mixture, and finally adding ultrapure water to make the volume 1.0 mL. After a period of reaction, the fluorescence emission spectrum in the wavelength range of 310-560 nm under excitation of 290 nm is tested, the ratio I401 / I544 of the fluorescence intensity at 401 nm and 544 nm is taken as the ordinate, the concentration of the H2O2 solution is taken as the abscissa to draw a standard curve, and then the concentration of H2O2 in the to-be-tested solution is detected. 401 / I 544 is taken as the ordinate, the concentration of the H2O2 solution is taken as the abscissa to draw a standard curve, and then the concentration of H2O2 in the to-be-tested solution is detected.

[0020] The final concentration of the HEPES buffer solution is 100 μM, and the pH is 7.4.

[0021] The reaction condition is standing at room temperature for 1 h.

[0022] The final concentration of the H2O2 is 0, 0.1, 1, 2, 5, 10, 15, 20, 30, 40, 50, 60 μM, respectively.

[0023] The linear equation of the standard curve is: I 401 / I 544 = 0.2287 + 0.0530C, the correlation coefficient R 2 is 0.998, and C is the concentration of H2O2, in units of μM.

[0024] The application also provides a detection method of glucose, which comprises the following steps: mixing a small amount of glucose oxidase and glucose solution, and placing in a constant temperature water bath at 37℃ for 40 min, and cooling to room temperature. Different volumes of glucose reaction solution and HEPES buffer solution are added into 100 μL of AMP-Tb / CPBA suspension, and the volume is made up to 1.0 mL. After a period of reaction, the fluorescence emission spectrum of each system in the wavelength range of 310-560 nm under the excitation wavelength of 290 nm is tested, and the ratio I 401 / I 544 of the fluorescence intensity at 401 nm and 544 nm is taken as the ordinate, and the concentration of the glucose solution is taken as the abscissa to draw a standard curve, so as to detect the glucose concentration in the to-be-tested solution.

[0025] The final concentration of the glucose oxidase is 0-0.01 mg / mL.

[0026] The final concentration of the HEPES buffer solution is 100 μM, and the pH is 7.4.

[0027] The final concentration of the glucose is 0, 1, 5, 10, 15, 20, 25, 30, 40, 50, 60 μM, respectively.

[0028] The reaction condition is that the glucose oxidase and the glucose solution are mixed and placed in a constant temperature water bath at 37℃ for 40 min, and the glucose hydrolysis product is mixed with the probe and stands at room temperature for 1 h.

[0029] The linear equation of the standard curve is: I 401 / I 544 = 0.2223 + 0.0254C0, the correlation coefficient R 2 is 0.994, and C0 is the concentration of glucose, in units of μM.

[0030] The application provides a technical scheme, wherein a novel lanthanide coordination polymer AMP-Tb / CPBA is synthesized, the coordination polymer has two characteristic emission peaks of Tb 3+ at 488 and 544 nm under excitation of a 290 nm wavelength. H2O2 can oxidize the non-fluorescent substance CPBA in the AMP-Tb / CPBA into HBA, and the product HBA emits strong blue fluorescence at 401 nm. Meanwhile, the oxidation of CPBA by H2O2 reduces the energy transfer efficiency of CPBA to Tb 3+ , so that the fluorescence of AMP-Tb / CPBA at 544 nm is quenched. Based on the above principle, a novel ratio-type fluorescent probe is designed for quantitative detection of H2O2. Since glucose oxidase catalyzes the reaction of glucose and oxygen to generate gluconic acid and hydrogen peroxide, the product H2O2 produces a fluorescent response to the probe AMP-Tb / CPBA. Therefore, the AMP-Tb / CPBA probe can also be used for determination of the hydrolysis product hydrogen peroxide of glucose, so as to realize quantitative detection of glucose.

[0031] Compared with single-emission fluorescent probes, the ratio-type fluorescent probe designed by the application for detection of H2O2 and glucose has good selectivity in the detection process and is not interfered by other related substances. The method can be used for detection of the content of hydrogen peroxide and glucose in tap water, and has good sensitivity and selectivity, and the detection limits of H2O2 and glucose can be as low as 0.23 μM and 0.49 μM, respectively. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 A transmission electron microscope image of AMP-Tb prepared in Example 1;

[0033] Figure 2 A transmission electron microscope image of AMP-Tb / CPBA prepared in Example 1;

[0034] Figure 3 Infrared spectra of AMP, AMP-Tb, CPBA, AMP-Tb / CPBA and AMP-Tb / CPBA+H2O2 prepared in Example 1;

[0035] Figure 4 Fluorescence emission spectra of AMP-Tb, AMP-Tb / CPBA and AMP-Tb / CPBA+H2O2 prepared in Example 1, wherein the final concentration of H2O2 is 110 μM;

[0036] Figure 5 A mechanism diagram of detection of H2O2 by the AMP-Tb / CPBA probe in Example 2;

[0037] Figure 6Fluorescence lifetime map of AMP-Tb / CPBA, AMP-Tb / CPBA+H2O2 in Example 2;

[0038] Figure 7 Fluorescence intensity ratio I 401 / I 544 vs. H2O2 concentration between 0 and 110 μM, where the final concentration of H2O2 was 0, 0.1, 1, 2, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 μM, respectively;

[0039] Figure 8 Standard curve of AMP-Tb / CPBA system for detecting H2O2 in Example 3, where the final concentration of H2O2 was 0, 0.1, 1, 2, 5, 10, 15, 20, 30, 40, 50, 60 μM, respectively;

[0040] Figure 9 Fluorescence intensity ratio I 401 / I 544 vs. glucose concentration between 0 and 100 μM, where the final concentration of glucose was 0, 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 μM, respectively;

[0041] Figure 10 Standard curve of AMP-Tb / CPBA system for detecting glucose in Example 4 in the concentration range of 1 to 60 μM, where the final concentration of glucose was 0, 1, 5, 10, 15, 20, 25, 30, 40, 50, 60 μM, respectively;

[0042] Figure 11 Effect of pH on the fluorescence intensity ratio (I 401 / I 544 ) of AMP-Tb / CPBA probe for detecting H2O2 system in Example 5, where the final concentration of H2O2 was 30 μM;

[0043] Figure 12 Effect of reaction time on the fluorescence intensity ratio (I 401 / I 544 ) of AMP-Tb / CPBA probe for detecting H2O2 system in Example 6, where the final concentration of H2O2 was 30 μM;

[0044] Figure 13 Effect of reaction time on the fluorescence intensity ratio (I 401 / I 544 ) of AMP-Tb / CPBA probe for detecting glucose system in Example 7, where the final concentration of H2O2 was 30 μM;

[0045] Figure 14 The figure shows the selective detection of H2O2 by the AMP-Tb / CPBA system in Example 8, where the final concentration of H2O2 was 30 μM and the concentrations of other ions were 10 times the final concentration of H2O2. Detailed Implementation

[0046] The present invention will now be described in detail with reference to the embodiments.

[0047] Unless otherwise specified, all solutions in this invention are aqueous solutions of the respective substances.

[0048] Example 1

[0049] The preparation method of AMP-Tb and AMP-Tb / CPBA CPs includes the following steps:

[0050] (1) 4.0 mL of Tb(NO3)3 solution (2.0 mM) was slowly added to 4.0 mL of HEPES buffer (100 mM; pH 7.4) containing AMP (2.0 mM). A white precipitate formed immediately upon addition of the Tb(NO3)3 solution. The mixture was stirred at room temperature for 3 h. The solution was centrifuged at 8000 rpm for 10 min, and the white precipitate was collected and washed three times with ultrapure water to remove unreacted reagents. The precipitate was dried and dispersed in 10 mL of ultrapure water to obtain an AMP-Tb suspension, which was stored at 4 °C for subsequent experiments.

[0051] (2) 4.0 mL of Tb(NO3)3 solution (2.0 mM) was slowly added to 4.0 mL of HEPES buffer (100 mM; pH 7.4) containing AMP (2.0 mM). A white precipitate formed immediately upon the addition of the Tb(NO3)3 solution. Then, 100 μL of CPBA solution (100 mM) was added, and the mixture was stirred at room temperature for 3 h. Subsequent steps were the same as in (1) to obtain the AMP-Tb / CPBA suspension, which was stored at 4 °C for subsequent experiments.

[0052] Its transmission electron microscope image is as follows Figure 1 As shown in the transmission electron microscope (TEM) image, the prepared AMP-Tb is a nanoscale fiber network structure. Figure 1 Meanwhile, the TEM images of AMP-Tb / CPBA show that its fiber network structure is similar to that of AMP-Tb. Figure 2 ).

[0053] Its infrared spectrum is as follows Figure 3 As shown, the infrared spectra of AMP at 1105 and 978 cm⁻¹ -1The absorption peak at that location belongs to the stretching vibration of the phosphate group. Comparing the infrared spectra of AMP and AMP-Tb, the adenine C2-N1 stretching vibration peak (1484-1476 cm⁻¹) is observed. -1 ) and phosphate antisymmetric peaks (1105-1099 cm⁻¹) -1 The shifts in these peaks indicate that AMP interacts with Tb via two phosphate groups and the N1 and NH2 sites. 3+ Binding. In the AMP-Tb / CPBA infrared spectrum, 1688-1648 cm⁻¹ -1 The shift of the OC=O tensile vibration peak at 1648 cm⁻¹ -1 The absorption peak intensity at this point is lower than that in the CPBA infrared spectrum, indicating that the carboxyl group of CPBA reacts with Tb. 3+ Successful coordination was achieved. These results suggest that both AMP and CPBA may be involved in the formation of the AMP-Tb / CPBA coordination polymer network structure.

[0054] Example 2

[0055] Mechanism of H2O2 detection by AMP-Tb / CPBA prepared in Example 1

[0056] The fluorescence emission spectrum of AMP-Tb / CPBA prepared in Example 1 at an excitation wavelength of 290 nm is as follows. Figure 4 As shown, without the addition of H2O2, due to the interaction between CPBA and Tb 3+ Intramolecular energy transfer causes AMP-Tb / CPBA to emit strong green fluorescence. When H₂O₂ solution is added, the nucleophilic H₂O₂ reacts with the electrophilic borate group in CPBA and hydrolyzes to generate m-hydroxybenzoic acid (HBA). The product HBA emits strong blue fluorescence at 401 nm (e.g., ...). Figure 4 , Figure 5 Simultaneously, the CPBA in AMP-Tb / CPBA is oxidized by H2O2, causing CPBA to react with Tb. 3+ The reduced energy transfer efficiency between them leads to fluorescence quenching at 544 nm. The maximum emission peak of HBA (401 nm) and Tb... 3+ The maximum emission peak (544 nm) differs by 143 nm, providing extremely favorable conditions for using AMP-Tb / CPBA as a ratiometric fluorescent probe for detecting H2O2. Therefore, the ratio of fluorescence intensity I can be used... 401 / I 544 It is used as an output signal to detect the concentration of H2O2.

[0057] H₂O₂ exhibits chemoselectivity towards borate groups, capable of hydrolyzing the CB bond in CPBA into a CO bond (e.g., Figure 5The infrared spectra of AMP-Tb / CPBA in both the absence and presence of H2O2 reveal the mechanism of interaction between AMP-Tb / CPBA and H2O2. Figure 3 As shown, the addition of H2O2 resulted in a higher 1381 cm⁻¹ in the AMP Tb / CPBA infrared spectrum. -1 The disappearance of the BO stretching vibration peak at 1648 cm⁻¹ indicates that the boronic acid group of CPBA in AMP-Tb / CPBA is hydrolyzed by H₂O₂. In the AMP-Tb / CPBA infrared spectrum, the peak at 1648 cm⁻¹ is... -1 The position of the OC=O stretching vibration peak at CPBA is almost unaffected by H2O2. These results indicate that although H2O2 hydrolyzes the boronic acid group of CPBA, it does not affect the interaction between CPBA and Tb in AMP-Tb. 3+ Coordination.

[0058] To further investigate the luminescence mechanism, we measured the fluorescence lifetime of the system, and the results are as follows: Figure 6 As shown, the fluorescence lifetime of AMP-Tb / CPBA in the presence of H2O2 is 0.984 ms, slightly shorter than that of AMP-Tb / CPBA without H2O2 (1.066 ms). This slight change in fluorescence lifetime further indicates that H2O2 does not affect the interaction between CPBA or HBA and Tb in AMP-Tb. 3+ Coordination.

[0059] Furthermore, since glucose oxidase catalyzes the reaction of glucose and oxygen to produce gluconic acid and H2O2, AMP-Tb / CPBA exhibits a fluorescent response to the glucose hydrolysis product H2O2, enabling quantitative detection of glucose. When glucose reaction solution is added to the AMP-Tb / CPBA probe system, the fluorescence intensity at 544 nm (Ig) is [not specified]. 544 The fluorescence intensity at 401 nm decreased, while the fluorescence intensity at 401 nm (I) decreased. 401 Enhancement can also be achieved by the ratio of fluorescence intensity I. 401 / I 544 It is used as an output signal to detect glucose concentration.

[0060] Example 3

[0061] Quantitative Detection of H2O2

[0062] The quantitative detection method of H2O2 includes the following steps: 100 μL of AMP-Tb / CPBA suspension is added to 10 μL of HEPES buffer solution (100 mM; pH 7.4), then different volumes of H2O2 are added to the mixture to make the final concentration of H2O2 in the range of 0-110 μM, and finally ultrapure water is added to make the volume to 1.0 mL. After being placed at room temperature for 1 h, the emission spectrum in the wavelength range of 310-560 nm is determined under the excitation wavelength of 290 nm. The ratio of fluorescence intensities I 401 / I 544 is taken as the ordinate, and the concentration of H2O2 solution is taken as the abscissa. The final concentrations of H2O2 are 0, 0.1, 1, 2, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 μM, respectively.

[0063] As can be seen from Figure 7 , with the increase of the concentration of H2O2, the fluorescence intensity of the AMP-Tb / CPBA probe system at 544 nm gradually increases, and the fluorescence at 401 nm gradually decreases.

[0064] As can be seen from Figure 8 , the ratio of fluorescence intensities I 401 / I 544 shows a good linear relationship with the concentration of H2O2, and the linear equation is I 401 / I 544 = 0.2287 + 0.0530C, and the correlation coefficient R 2 is 0.998, where C is the concentration of H2O2 in μM. The linear concentration range of H2O2 in this method is 0.1-60 μM. The detection line of H2O2 is 0.23 μM calculated by the formula 3σ / k (where σ is the standard deviation of 5 blank measurements, and k is the slope of the standard curve).

[0065] Example 4

[0066] Quantitative detection of glucose

[0067] The quantitative detection method of glucose includes the following steps: 10 μL of glucose oxidase (10 mg / mL) and 1 mL of glucose solution (1 mM) are mixed and placed in a constant temperature water bath at 37°C for 40 minutes, and then cooled to room temperature. Then, different volumes of glucose reaction solution and 10 μL of HEPES buffer (100 mM, pH 7.4) are added to 100 μL of AMP-Tb / CPBA probe, and reacted at room temperature for 1 h. The ratio of fluorescence intensities I 401 / I 544Plot a graph with the glucose solution concentration on the x-axis and the glucose oxidase concentration on the y-axis. The final concentration of glucose oxidase in the system was 0-0.01 mg / mL, and the final glucose concentrations were 0, 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, and 100 μM.

[0068] from Figure 9 As can be seen, when glucose reaction solution is added to the AMP-Tb / CPBA system, the fluorescence emission peak at 544 nm weakens with increasing glucose concentration, while the fluorescence emission at 401 nm gradually strengthens. This is because glucose oxidase reacts with glucose, and the H2O2 generated by hydrolysis oxidizes CPBA in the probe to HAB. HBA has a strong fluorescence emission peak at 401 nm, and the change in CPBA caused by the oxidation process causes CPBA to convert to Tb. 3+ Intramolecular energy transfer efficiency decreases, and fluorescence quenching occurs at 544 nm.

[0069] from Figure 10 As can be seen from this, with fluorescence intensity higher than I... 401 / I 544 Plotting glucose concentrations revealed that the fluorescence intensity was higher than that of I. 401 / I 544 The fluorescence intensity gradually increased with increasing glucose concentration, and within the range of 1.0-60 μM, the fluorescence intensity was higher than that of I. 401 / I 544 It is positively correlated with glucose concentration, with a correlation coefficient R. 2 The value is 0.994, and the linear equation is I. 401 / I 544 =0.2223 + 0.0254C0, where C0 is the concentration of glucose in μM. The limit of detection for glucose is calculated to be 0.49 μM using the formula 3σ / k, where σ is the standard deviation of five blank experiments and k is the slope of the standard curve.

[0070] Example 5

[0071] Effect of pH of HEPES buffer solution on the detection system

[0072] HEPES buffer solutions with different pH values ​​were added to the AMP-Tb / CPBA probe. Then, equal concentrations of H₂O₂ were added to each solution, and the mixture was thoroughly mixed and brought to a final volume of 1.0 mL. The mixtures were allowed to react at room temperature for 1 h. The fluorescence emission spectra of each system were measured at an excitation wavelength of 290 nm. The difference in fluorescence intensity ratio ΔI was calculated by plotting the pH of the HEPES buffer solution on the x-axis. 401 / I 544 Plot the ordinate (ΔI) 401 / I 544 =(I 401 / I 544 )-(I 401 / I 544 )0, wherein I 401 / I 544 ) and I 401 / I 544 )0 are the fluorescence intensity ratios of AMP-Tb / CPBA system with and without H2O2, respectively, at the same pH, as shown in Fig. 1. The final concentration of HEPES buffer solution is 100 μM, and the final concentration of H2O2 is 30 mM. The pH values of the system are 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, and 8.2, respectively. Figure 11

[0073] As shown in Fig. 2, the pH of the HEPES buffer solution used in the detection system affects the sensitivity of the ratiometric fluorescent probe. When the pH of the buffer solution is 7.4, the difference ΔI 401 / I 544 between the fluorescence intensity ratios reaches a maximum. Therefore, when detecting H2O2 and glucose, the HEPES buffer solution with pH = 7.4 is selected as the optimal reaction value. Figure 11

[0074] Example 6

[0075] Effect of reaction time of H2O2 with the probe on the sensitivity of the detection system

[0076] The HEPES buffer solution and the AMP-Tb / CPBA suspension are mixed, and the same concentration of H2O2 is added to the system, which is then mixed thoroughly and diluted to 1.0 mL. The emission spectrum is detected every 10 min under excitation at 290 nm, and the reaction time is plotted as the abscissa, and I 401 / I 544 is plotted as the ordinate, as shown in Fig. 3. The final concentration of HEPES buffer solution in the detection system is 100 μM, and the final concentration of H2O2 is 30 mM. Figure 12 As shown in Fig. 4, the fluorescence intensity ratio I 401 / I 544 increases with the increase of the reaction time, and gradually stabilizes when the reaction time reaches 1 h. In order to make the reaction more thorough, the optimal reaction time is selected as 1 h.

[0077] Figure 12 Example 7

[0078] Effect of incubation temperature of glucose and glucose oxidase on the sensitivity of the detection system

[0079]

[0080] ​​​​A small amount of glucose oxidase was added to the glucose solution and mixed thoroughly. The mixture was then incubated at different water bath temperatures for 40 minutes each, followed by cooling to room temperature. Next, the glucose reaction solution and HEPES buffer were added to 100 μL of AMP-Tb / CPBA suspension, and the volume was adjusted to 1.0 mL using ultrapure water. Plotting the reaction temperature on the x-axis, I... 401 / I 544 Plot a bar chart with the vertical axis as shown below. Figure 13 As shown in the figure. The water bath temperatures were 27℃, 37℃, 47℃, and 57℃. The final concentration of glucose oxidase in the detection system was 3 μg / mL, and the final concentration of glucose was 30 μM.

[0081] Depend on Figure 13 It can be seen that when the water bath temperature is 37℃, the fluorescence intensity ratio I 401 / I 544 The maximum. Therefore, 37°C is selected as the optimal water bath temperature for incubating glucose and glucose oxidase.

[0082] Example 8

[0083] Selective experiment

[0084] A high-performance fluorescent probe must possess good selectivity and interference resistance. The selectivity of the AMP-Tb / CPBA ratiometric fluorescent probe for H2O2 and glucose was investigated through comparative experiments. Under the same experimental conditions, 30 μM H2O2 and 10-fold concentrations of other ions (L-cysteine, fructose, glutathione, glycine, histidine, sucrose, Ca2+) were selected. 2+ ,K + Mg 2+ Na + ,S 2+ ,Cl - ,ClO - NO2 - NO3 2- and SO4 2- After reacting with the AMP-Tb / CPBA probe system at room temperature for 1 hour, the fluorescence intensity was measured at an excitation wavelength of 290 nm to calculate I. 401 / I 544 .

[0085] like Figure 14 Experimental results show that, except for H2O2, which can increase the fluorescence intensity compared to I... 401 / I 544 Aside from a significant increase in ion concentration, the influence of other ions on this probe is almost negligible. Therefore, the ratiometric fluorescent probe designed in this invention is considered to have good selectivity and anti-interference capabilities.

[0086] Example 9

[0087] Determination of H2O2 and glucose in real samples

[0088] To verify the practicability of the detection method provided by the present application, the concentrations of H2O2 and glucose in laboratory tap water were detected by the standard addition method, and the results are listed in Table 1 and Table 2.

[0089] In the laboratory tap water, the presence of H2O2 and glucose was not detected, and two different concentrations were added for recovery experiments, and it was found that the recovery rate of this method was between 97.5% and 103.7%, and the average relative standard deviation was controlled within 3.6%. This data shows that this method is practical and reliable.

[0090] Table 1 Determination of H2O2 concentration in tap water

[0091]

[0092] Table 2 Determination of glucose concentration in tap water

[0093]

[0094]

[0095] The above reference examples for a new type of fluorescent probe AMP-Tb / CPBA preparation method and H2O2 and glucose detection method are illustrative rather than limiting, and several examples can be listed according to the limited range, therefore the changes and modifications without departing from the overall concept of the present application shall be within the protection scope of the present application.

Claims

1. A method for preparing a novel AMP-Tb / CPBA fluorescent probe, characterized in that, Includes the following steps: (1) Slowly add 4.0 mL of 2.0 mM Tb(NO3)3 solution to 4.0 mL of 100 mM pH 7.4 HEPES buffer solution containing 2.0 mM AMP. A white precipitate forms immediately after the Tb(NO3)3 solution is added. Then add 100 μL of 0.1 M 3-carboxyphenylboronic acid aqueous solution, mix well, and stir at room temperature. The final concentrations of the added 3-carboxyphenylboronic acid and the buffer solution are 1.0-1.5 mM and 45-55 mM, respectively. (2) Centrifuge the above solution, collect the white precipitate, wash and dry it, and disperse it in 10 mL of ultrapure water to obtain the AMP-Tb / CPBA fluorescent probe. The probe is fluorescent at 544 nm. AMP refers to adenosine monophosphate.

2. A method for detecting H2O2 using the AMP-Tb / CPBA fluorescent probe as described in claim 1, characterized in that, Includes the following steps: Mix 100 μL of the AMP-Tb / CPBA fluorescent probe according to claim 1 with HEPES buffer solution, then add different concentrations of H2O2, and finally add ultrapure water to make up to 1.0 mL; after reacting for a period of time, test the fluorescence emission spectrum in the wavelength range of 310-560 nm at an excitation wavelength of 290 nm, and use the ratio of fluorescence intensity at wavelengths of 401 nm and 544 nm as the ratio. I 401 / I 544 A standard curve was plotted with the H2O2 solution concentration on the x-axis and the H2O2 concentration on the y-axis. The final H2O2 concentrations were 0, 0.1, 1, 2, 5, 10, 15, 20, 30, 40, 50, and 60 μM. The H2O2 concentration in the test solution was detected by the standard curve. In the detection system, the final concentration of the HEPES buffer solution was 100 μM and the pH was 7.

4. The reaction time was 1 h at room temperature.

3. A method for detecting glucose using the AMP-Tb / CPBA fluorescent probe according to claim 1, characterized in that, Includes the following steps: Glucose oxidase and glucose solution were mixed and placed in a constant temperature water bath at 37 ℃ for 40 min. After cooling to room temperature, different volumes of glucose reaction solution and HEPES buffer were added to 100 μL of AMP-Tb / CPBA fluorescent probe, and the volume was adjusted. After reacting for a period of time, the fluorescence emission spectra of each system in the wavelength range of 310-560 nm at an excitation wavelength of 290 nm were measured. The ratio of fluorescence intensity at 401 nm and 544 nm was used as the analytical term. I 401 / I 544 A standard curve was plotted with the glucose solution concentration on the x-axis and the glucose concentration on the y-axis. The final glucose concentrations were 0, 1, 5, 10, 15, 20, 25, 30, 40, 50, and 60 μM. The glucose concentration in the test solution was detected by the standard curve. In the detection system, the final concentration of HEPES buffer solution was 100 μM and the pH was 7.

4. The final concentration of glucose oxidase was 0-0.01 mg / mL. The reaction time was 1 h at room temperature.

Images