Ascorbic acid high-sensitivity colorimetric sensor as well as preparation method and application thereof

Abstract

The invention provides an ascorbic acid high-sensitivity colorimetric sensor as well as a preparation method and application thereof, and aims to solve the problem of low sensitivity of an MOFs (Metal-Organic Frameworks) nano material in ascorbic acid detection. Ce-UiO-66-NH2 nano enzyme particles can catalyze O2 to oxidize TMB (Tetramethylbenzidine) to generate a blue product (TMBOX) under an acidic condition; based on the principle that the absorbance of a mixed solution is reduced due to the fact that the ascorbic acid can react with TMBOX, the high-sensitivity ascorbic acid colorimetric sensor composed of Ce-UiO-66-NH2 nano-enzyme particles with the diameter of 10-50 nm and a chromogenic substrate 3, 3 ', 5, 5'-tetramethyl benzidine (TMB) is constructed. Compared with MnO2, Pt-BGP NCs, CeO2 (at) PTA HNS and Co-PB NCs, the prepared ascorbic acid high-sensitivity colorimetric sensor has the advantages that the reaction time is shorter, and the content of ascorbic acid can be rapidly measured within 15 seconds. Based on the principle, the invention further provides a portable colorimetric test paper for visually detecting the ascorbic acid, and the ascorbic acid in the liquid lemon water can be semi-quantitatively detected by naked eyes within 30 seconds.

Description

Technical Field

[0001] The invention belongs to the field of analysis and detection, and particularly relates to a highly sensitive ascorbic acid colorimetric sensor and a preparation method thereof. Background Art

[0002] Ascorbic acid (AA) is considered a novel dietary protective agent that can effectively prevent and alleviate the inflammatory cascade associated with Parkinson's disease. However, excessive ascorbic acid intake can lead to elevated levels in the human body, which can have harmful effects and even contribute to a variety of diseases. Various methods have been developed for the detection of ascorbic acid, including chromatography, fluorescence spectroscopy, and electrochemistry. Chromatographic methods require complex sample pretreatment and long measurement times, making them unsuitable for portable, on-site determination of ascorbic acid in beverages. Electrochemical analysis requires constant electrode cleaning and is susceptible to external interference. Currently available spectrophotometric methods are complex, subject to numerous interference factors, exhibit a single color change, and require specialized personnel for analysis, making them incapable of on-site visual detection of ascorbic acid in food samples. Colorimetric methods have garnered significant attention in recent years due to their simplicity, convenience, and low cost. In the past, natural enzymes were often used to develop colorimetric sensors. However, natural enzymes suffer from numerous limitations, such as low stability and storage difficulties, which significantly restrict their application.

[0003] Nanozymes are nanomaterials that possess the catalytic activity of natural enzymes and can overcome the inherent limitations of natural enzymes. Metal-organic frameworks (MOFs), with their advantages such as high stability, large surface area, and unsaturated metal sites, have attracted significant interest in exploring nanozyme activity, offering the potential for developing simple, low-cost, and visually detectable technologies. Although MOFs have been demonstrated to possess peroxidase, oxidase, and haloperoxidase activities, and extensive research has been conducted on ascorbic acid detection, achieving high-sensitivity detection remains a significant challenge for researchers in this field. Summary of the Invention

[0004] The present invention addresses the problem of low sensitivity of MOFs nanomaterials in ascorbic acid detection and provides a highly sensitive ascorbic acid colorimetric sensor and a preparation method thereof. The technical solutions adopted by the present invention are as follows: The present invention provides a highly sensitive colorimetric sensor for ascorbic acid, which is composed of Ce-UiO-66-NH2 nanozyme particles with a diameter of 10-50 nm and a chromogenic substrate 3, 3', 5, 5'-tetramethylbenzidine (TMB); Under acidic conditions, Ce-UiO-66-NH2 nanozyme particles can catalyze O2 to oxidize TMB to produce a blue product (TMBOX); ascorbic acid can react with TMBOX, resulting in a decrease in the absorbance of the mixed solution, thereby constructing a highly sensitive colorimetric sensor for ascorbic acid.

[0005] Preferably, the acidic condition is pH = 2.0~5.0.

[0006] The steps for preparing the highly sensitive ascorbic acid colorimetric sensor of the present invention are as follows: 600 μL of 0.1 mol / L Na2HPO4-NaH2PO4 buffer solution (pH=2.0~5.0) and 150 μL of 30~600 μg / mL Ce-UiO-66-NH2 dispersion were added with 150 μL of 0.2~2.0 mM TMB solution, and incubated at 20~60℃ for 5-30 min to prepare the ascorbic acid highly sensitive colorimetric sensor.

[0007] Preferably, the pH of the Na2HPO4-NaH2PO4 buffer solution is 5.0; Preferably, the concentration of Ce-UiO-66-NH2 dispersion is 500 μg / mL; Preferably, the concentration of the TMB solution is 1.2 mM; Preferably, the incubation temperature is 30°C; Preferably, the incubation time is 5 min.

[0008] The application of the ascorbic acid highly sensitive colorimetric sensor described in the present invention is: Add 300 μL of ascorbic acid solution and incubate at room temperature for 1-180 s. Then use a UV spectrophotometer to measure the spectrum of the mixed solution within 500-800 nm or the absorbance at the maximum absorption wavelength (652 nm).

[0009] Preferably, the incubation time is 15 seconds.

[0010] The test paper is immersed in the ascorbic acid highly sensitive colorimetric sensor solution, and after drying, a portable visual colorimetric test paper for detecting ascorbic acid is prepared, and the ascorbic acid concentration is detected by a colorimetric card.

[0011] Compared with the prior art, the advantages of the present invention are: (1) The Ce-UiO-66-NH2 nanozyme prepared in the present invention has better catalytic performance than Cr-N / C, Ce–N / C, TTPA-COF, and MnO2@PLNPs.

[0012] (2) The high-sensitive colorimetric sensor for ascorbic acid prepared in the present invention has a faster response time than MnO2, Pt-BGP NCs, CeO2@PTAHNS, and Co-PB NCs, and can quickly determine the ascorbic acid content within 15 seconds.

[0013] (3) Based on the above principle, the present invention also proposes a portable colorimetric test strip for visual detection of ascorbic acid, which can semi-quantitatively detect ascorbic acid in liquid lemonade with the naked eye within 30 seconds. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the preparation process roadmap of Ce-UiO-66-NH2; Figure 2 This is a diagram exploring the catalytic activity of Ce-UiO-66-NH2 nanozyme; Figure 3 This is the absorbance image of Ce-UiO-66-NH2 in different systems of a single variable; Figure 4 This is a diagram showing the catalytic mechanism of Ce-UiO-66-NH2 nanozyme; Figure 5 Figure 1 is the absorption spectrum (A) and standard curve (B) of the ascorbic acid detection reaction system; Figure 6 This is an experimental diagram of the anti-interference performance of ascorbic acid detection; Figure 7 The following are pictures of the detection results of the paper-based sensor on AA solutions of different concentrations. DETAILED DESCRIPTION

[0015] The present invention will be described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the scope of protection of the present invention. Any improvements and changes made on the basis of the present invention are within the scope of protection of the present invention.

[0016] Example 1: Preparation of Ce-UiO-66-NH2 nanozyme, the synthetic route is shown in Figure 1 (1) Ce(NH3)2(NO3)6 (4.110 g) was dissolved in ultrapure water (20 mL) and formic acid (6.5 mL) at room temperature to obtain solution I. (2) Disperse 2-aminoterephthalic acid (1.358 g) in anhydrous ethanol (50 mL) to obtain solution II; (3) The two solutions were mixed and stirred at room temperature for 8 h; (4) After the reaction was completed, the brown precipitate was collected by centrifugation (10,000 r / min, 10 min) and washed with water and ethanol three times. The sample was dried at 60 °C for 8 h to obtain a brown solid Ce-UiO-66-NH2. (5) Finally, 50 mg of Ce-UiO-66-NH2 solid powder was mixed with 100 mL of water, and the mixture was sonicated in an ultrasonic machine (200 W) for 24 h. The dispersion was centrifuged at 3000 rpm for 3 min to collect the supernatant, and then centrifuged at 10000 rpm for 5 min to collect the precipitate. The collected precipitate was further redispersed in water and sonicated for 2 h to obtain a Ce-UiO-66-NH2 dispersion. The concentration of the dispersion was adjusted to 1 mg / mL.

[0017] Example 2: Preparation of a highly sensitive colorimetric sensor for ascorbic acid 150 μL of Ce-UiO-66-NH2 dispersion (500 μg / mL) and 150 μL of TMB (1.0 mmol / L) were added to 600 μL of Na2HPO4-NaH2PO4 buffer solution (pH 4.0, 0.1 mol / L), mixed well, and incubated in a metal bath at 35 °C for 5 min; 150 μL of 31.25-500 μg / mL Ce-UiO-66-NH2 dispersion and 150 μL of 0-2.0 mM TMB solution were added to 600 μL of 0.1 mol / L Na2HPO4-NaH2PO4 buffer solution (pH = 2.2-9.0), mixed evenly, and incubated in a metal bath at 20-70°C for 1-30 min to obtain the highly sensitive colorimetric sensor for ascorbic acid. The corresponding absorption intensity (A) was detected by UV-visible spectroscopy.

[0018] Figure 2 The results showed that both the TMB-added Ce-UiO-66-NH2 group and the Ce-UiO-66-NH2 and H2O2 group changed from colorless to blue within 5 minutes. The UV absorbance at 652 nm after the color change was essentially identical for both groups, a characteristic peak for charge-transfer complexes derived from the single-electron oxidation of TMB. The TMB-alone group showed no color change. Therefore, Ce-UiO-66-NH2 possesses excellent oxidase activity, rapidly oxidizing TMB to produce blue TMBOX.

[0019] Figure 3The effects of reaction pH, TMB concentration, temperature, Ce-UiO-66-NH2 concentration and reaction time on the absorbance of Ce-UiO-66-NH2-TMB system are shown. This example uses a single variable to optimize different experimental conditions, such as Figure 3 a and Figure 3 The pH value of the buffer solution shown in b has a certain influence on the absorbance of the solution. It has good catalytic activity under weak acidic conditions. The absorbance reaches the maximum when the pH is 5.0, and Ce-UiO-66-NH2 has the highest catalytic activity. Figure 3 c and Figure 3 As shown in d, when the TMB concentration is in the range of 0-1.2 mmol / L, the absorbance of the mixed solution increases rapidly with the increase of TMB concentration; when the TMB concentration exceeds 1.2 mmol L -1 After that, the absorbance of the mixed solution also tended to be flat. Therefore, 1.2 mmol L -1 As the optimal concentration of TMB. Figure 3 e and Figure 3 As shown in Figure f, as the reaction temperature increases, the absorbance value continues to increase. When the temperature is 30°C, the absorbance reaches the highest. If the temperature continues to rise, the absorbance will decrease. Figure 3 g and Figure 3 As shown in Figure 3, with the increase of Ce-UiO-66-NH2 concentration, the absorbance of the mixed solution also gradually increased. Considering various factors, the concentration of Ce-UiO-66-NH2 was selected as 500 μg / mL. Figure 3 i and Figure 3 Figure j shows that the absorbance of the mixed solution gradually increases with the increase of reaction time. When the reaction time reaches 30 minutes, the absorbance reaches its maximum value. After that, the absorbance of the mixed solution hardly changes with the extension of reaction time. Therefore, 30 minutes is the optimal reaction time.

[0020] Example 3: Catalytic mechanism of Ce-UiO-66-NH2 nanozyme In order to explore the types of reactive oxygen species generated, 150 μL of Ce-UiO-66-NH2 (500 μg / mL) dispersion and 150 μL of TMB (1.2 mmol L -1 ) was added into 600 μL of Na2HPO4-NaH2PO4 buffer solution (pH 5.0, 0.1 mol / L) and mixed evenly to form the Ce-UiO-66-NH2-TMB system; Sodium oxalate, urea, 2,2,6,6-tetramethylpiperidinyl oxide (TEMPO), and L-histidine at three concentrations (5 μmol / L, 50 μmol / L, and 100 μmol / L) were added to the Ce-UiO-66-NH2-TMB system as holes (h + ), hydroxyl radicals (•OH), superoxide radicals (•O2 − ) and singlet oxygen (1O2) scavengers, and incubated in a metal bath for 5 min, and the corresponding absorption intensity was detected by UV-visible spectroscopy; In order to explore the effect of O2 production on the Ce-UiO-66-NH2-TMB system, 150 μL Ce-UiO-66-NH2 (500 μg / mL) dispersion and 150 μL TMB (1.2 mmol L -1 ) were added to 600 μL of Na2HPO4-NaH2PO4 buffer solution (pH 5.0, 0.1 mol / L), mixed evenly, and incubated in a metal bath for 5 min. The corresponding absorption spectra were detected by UV-visible spectroscopy; from Figure 4 As can be seen in a, after adding TEMPO, the absorbance of the system decreases with the increase of TEMPO concentration. The absorbance does not decrease significantly when other scavengers are added. This result indicates that the main catalytic role in the TMB oxidation reaction is played by •O2 − In addition, the effect of oxygen on the Ce-UiO-66-NH2-TMB system was investigated. Oxygen and nitrogen were respectively introduced into the reaction system. Figure 4 As shown in Figure b, when oxygen was introduced, the absorbance of the system increased, while when nitrogen was introduced, the absorbance of the system decreased significantly, indicating that the oxidation of TMB requires the participation of oxygen.

[0021] Example 4: Application of ascorbic acid colorimetric sensor 150 μL of Ce-UiO-66-NH2 (500 μg / mL) dispersion and 150 μL of TMB (1.2 mmol L -1 ) was added to 600 μL of Na₂HPO₄-NaH₂PO₄ buffer solution (pH 5.0, 0.1 mol / L), mixed thoroughly, and incubated for 5 minutes to prepare the substrate. After the reaction was complete, 300 μL of ascorbic acid solution of varying concentrations was added. After incubation at room temperature for 15 seconds, the mixed solution was measured using a UV spectrophotometer to measure the absorbance at the wavelength of maximum absorption (652 nm) within the 500-800 nm range. Figure 5 As shown in a, as the concentration of AA increases, the color of the reaction system becomes lighter. Figure 5 b It can be observed that the absorbance at 652 nm showed a good linear relationship with the AA concentration in the range of 1-50 μmol / L (R 2 =0.99749). The detection limit was 0.43 μmol / L, calculated using the formula 3σ / k. Compared with other AA colorimetric sensors, the AA colorimetric sensor based on Ce-UiO-66-NH2 exhibits a wider detection range and lower detection limit. This may be attributed to the nanozyme's large surface area, which enhances its oxidase-like activity and detection sensitivity. Compared with other methods reported in the literature, this method has a shorter response time for AA detection, enabling rapid detection.

[0022] Example 5: Interference Experiment of Ascorbic Acid Colorimetric Sensor 150 μL Ce-UiO-66-NH2 (500 μg / mL) dispersion and 150 μL TMB (1.2 mmol L -1 ) was added to 600 μL of Na2HPO4-NaH2PO4 buffer solution (pH 5.0, 0.1 mol / L), mixed evenly, and incubated for 5 min to prepare the substrate; After the reaction was completed, 300 μL of common metal ions (Na + , K + , Ca 2+ Mg 2+ 、Zn 2+ 、Cl - 、SO4 2- ), citric acid and glucose solution 500 μmol L -1 After incubation at room temperature for 15 seconds, the spectrum of the mixed solution within 500-800 nm and the absorbance at the maximum absorption wavelength (652 nm) were detected using an ultraviolet spectrophotometer; like Figure 6 It was observed that the absorbance of the system remained essentially unchanged after the introduction of various interfering substances, while the absorbance decreased significantly after the addition of AA. Therefore, the Ce-UiO-66-NH2-TMB developed in this work has strong specificity and anti-interference properties for the quantitative detection of AA.

[0023] Example 6: Preparation of colorimetric test paper for ascorbic acid 150 μL Ce-UiO-66-NH2 (500 μg / mL) dispersion and 150 μL TMB (1.2 mmol L -1) was added to 600 μL of Na2HPO4-NaH2PO4 buffer solution (pH 5.0, 0.1 mol / L), mixed evenly, and incubated for 5 min to form a Ce-UiO-66-NH2-TMBOX substrate solution; Several circular test paper sheets with a diameter of 1 cm were made using a hole punch, and then immersed in the Ce-UiO-66-NH2-TMB substrate solution for 5 min. After vacuum drying at room temperature, the ascorbic acid paper-based sensor was formed. like Figure 7 As shown in a, the color of the paper-based sensor of ascorbic acid is uniform and no longer changes. AA solutions of different concentrations were added to the portable paper-based sensor and reacted for 30 seconds. Figure 7 As shown in b, the results show that as the concentration of AA gradually increases, the color of the paper base gradually becomes lighter and visible to the naked eye. After the paper-based sensor is placed for 2 hours, the color still does not change ( Figure 7 c). This result shows that the paper-based sensor can quantitatively and instantly detect the AA content in beverages. According to the colorimetric card ( Figure 7 d). The color ranges of samples 6 to 9 ranged from 0-5 μM, 5-10 μM, 10-20 μM, and 20-50 μM. HPLC analysis revealed that the ascorbic acid concentrations in samples 6 to 9 were 4.95 μM, 9.88 μM, 23.97 μM, and 32.45 μM, respectively. These results demonstrate that the portable paper-based sensor can be used for the semi-quantitative detection of ascorbic acid in liquid lemonade by visual inspection.

Claims

1. A highly sensitive colorimetric sensor for ascorbic acid, characterized in that: The sensor consists of Ce-UiO-66-NH2 nanozyme particles with a diameter of 10-50 nm and a chromogenic substrate 3, 3', 5, 5'-tetramethylbenzidine. Under acidic conditions, the Ce-UiO-66-NH2 nanozyme particles can catalyze the oxidation of 3, 3', 5, 5'-tetramethylbenzidine by O2 to generate a blue product TMBOX. Ascorbic acid can react with TMBOX, resulting in a decrease in the absorbance of the mixed solution, thereby constructing a highly sensitive colorimetric sensor for ascorbic acid.

2. The highly sensitive colorimetric sensor for ascorbic acid according to claim 1, wherein: The acidic condition is pH=2.0~5.

0.

3. The method for preparing the highly sensitive colorimetric sensor of ascorbic acid according to claim 1, wherein: The steps of this method are: 600 μL of 0.1 mol / L Na2HPO4-NaH2PO4 buffer solution (pH = 2.0~5.0) and 150 μL of 30~600 μg / mL Ce-UiO-66-NH2 dispersion were added with 150 μL of 0.2~2.0 mM 3,3', 5, 5'-tetramethylbenzidine solution, and incubated at 20~60°C for 5-30 min to prepare the ascorbic acid highly sensitive colorimetric sensor.

4. The method for preparing the highly sensitive ascorbic acid colorimetric sensor according to claim 3, wherein: Buffer solution pH=5.

0.

5. The method for preparing the highly sensitive colorimetric sensor for ascorbic acid according to claim 3, wherein: The concentration of Ce-UiO-66-NH2 dispersion is 500 μg / mL.

6. The method for preparing the highly sensitive colorimetric sensor for ascorbic acid according to claim 3, wherein: The concentration of 3,3', 5, 5'-tetramethylbenzidine solution was 1.2 mM.

7. The method for preparing the highly sensitive colorimetric sensor for ascorbic acid according to claim 3, wherein: The incubation temperature was 30°C.

8. The method for preparing the highly sensitive ascorbic acid colorimetric sensor according to claim 3, wherein: The incubation time is 5 min.

9. Use of the highly sensitive colorimetric sensor for ascorbic acid according to claim 1, characterized in that: The method is as follows: add 300 μL of ascorbic acid solution, incubate at room temperature for 1-180 s, and then use an ultraviolet spectrophotometer to detect the spectrum of the mixed solution within 500-800 nm or the absorbance at the maximum absorption wavelength.

10. Use of the highly sensitive colorimetric sensor for ascorbic acid according to claim 1, characterized in that: The test paper is immersed in the ascorbic acid highly sensitive colorimetric sensor solution, and after drying, a portable visual colorimetric test paper for detecting ascorbic acid is prepared, and the ascorbic acid concentration is detected by a colorimetric card.

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