Nano-enzyme-based ratiometric fluorescence sensing platform for determining total antioxidant capacity and application thereof

Abstract

The invention relates to a nano-enzyme-based ratiometric fluorescence sensing platform for determining total antioxidant capacity and application thereof, and belongs to the technical field of preparation of fluorescence sensors. The ratio fluorescence sensing platform provided by the invention comprises MnSiO3 with oxidase-like activity, MnSiO3 with oxidase-like activity and MnSiO3 with oxidase-like activity, the invention relates to two fluorescent substrates with opposite responses. According to the ratio fluorescence sensing platform disclosed by the invention, the ratio fluorescence sensor is constructed by utilizing two fluorescent substrates, namely nano-enzyme MnSiO3 with oxidase-like activity and SC / OPD with opposite response, and is used for detecting TAC, so that the detection of three different antioxidants is realized. The ratio fluorescence sensing platform provided by the invention has the advantages of strong anti-interference capability and high sensitivity. The ratio fluorescence sensing platform has oxidase-like properties, does not introduce hydrogen peroxide, and has small interference on experiments.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent sensor preparation, and in particular relates to a nanoenzyme-based ratio fluorescent sensor platform for total antioxidant capacity determination and an application thereof. Background Art

[0002] Total antioxidant capacity (TAC) refers to the sum of the antioxidant capacities of all antioxidants in the sample to be tested. It is a clinical biochemical test indicator that can be evaluated by detecting the antioxidant content in blood, urine or other body fluids. Total antioxidant capacity can reflect the body's ability to fight free radicals and oxidative stress, and is an important tool for assessing health status and disease risk.

[0003] In order to accurately detect the total antioxidant capacity, people have constructed a fluorescence and colorimetric sensing platform based on nanozymes. For example, Tao et al. used the synthesized Cu-N / C nanozyme to oxidize ascorbic acid (AA) to produce hydrogen peroxide (H2O2), thereby designing a fluorescence and colorimetric sensing platform for TAC detection. The detection limits of this technology are 0.7μΜ and 0.3μΜ, respectively. However, the synthesis of the materials of this technology requires high temperature treatment, high cost, and a single detection target cannot meet TAC detection. (C.Tao, Y.Jiang, S.Chu, Y.Miao, J.Zhang, Y.Lu, et al., Anal.Chem.2024, 96, 3107-3115.). Yan et al. designed a fluorescence and colorimetric sensing platform based on the persistent luminescent nanozyme ZGO-Pt with peroxidase-like activity (POD). By adding H2O2, free radicals oxidize TMB, and Ox-TMB quenches the fluorescence of ZGO-Pt itself, thus enabling the detection of TAC. However, the H2O2 introduced by this technology will interfere with the determination of TAC (L.-X.Yan, Z.-Y.Yan, X.Zhao, L.-J.Chen, X.-P.Yan, Sensor.Actuat.B-Chem., 2024, 405, 135333.). Based on Co-Ir / C nanozymes with peroxidase-like activity, Chen's team used TMB as a probe to construct a colorimetric sensing platform for the detection of TAC. However, this technology uses a single signal output, which is easily affected by the environment / background, resulting in false positive / false negative results, which is not conducive to the detection of TAC (S.Li, P.Keoingthong, J.Xu, Y.Yang, J.Shen, Y.Xu, et al., Biosens.Bioelectron., 2023, 236, 115416.).

[0004] In summary, the defects of existing nanozyme-based fluorescence and colorimetric sensing platforms for detecting TAC are mainly:

[0005] (1) The single signal output is easily affected by background / environmental interference, resulting in false positive / false negative results, which is not conducive to the detection of TAC.

[0006] (2) Most materials are peroxidase-like nanozymes, which will introduce hydrogen peroxide molecules during work, causing certain background interference to TAC determination.

[0007] (3) In terms of synthetic materials, most materials require certain high temperature or ultra-high temperature treatment, which makes the synthesis complex and costly. Summary of the invention

[0008] The present invention aims to solve the technical problems in the prior art and provides a nanoenzyme-based ratio fluorescence sensing platform for determining total antioxidant capacity and its application.

[0009] In order to solve the above technical problems, the technical solutions of the present invention are as follows:

[0010] A nanozyme-based ratiometric fluorescence sensing platform for total antioxidant capacity determination, comprising:

[0011] MnSiO3 with oxidase-like activity;

[0012] Two fluorescent substrates with opposite responses.

[0013] In the above technical solution, preferably, among the fluorescent substrates, one fluorescent substrate is Scopoletin (SC) and the other fluorescent substrate is o-phenylenediamine (OPD).

[0014] In the above technical solution, preferably, in the ratiometric fluorescence sensing platform system:

[0015] The mass concentration of MnSiO3 with oxidase-like activity is 6.4 mg·mL -1 , volume 10 μL;

[0016] The molar concentration of the fluorescent substrate SC was 50 μM, and the volume was 5 μL;

[0017] The molar concentration of the fluorescent substrate OPD was 300 mM and the volume was 50 μL.

[0018] In the above technical solution, preferably, the MnSiO3 having oxidase-like activity is synthesized by a one-pot method.

[0019] In the above technical solution, it is further preferred that the method for synthesizing MnSiO3 having oxidase-like activity comprises the following steps:

[0020] MnCl2·4H2O is added to an aqueous solution of NaSiO3·9H2O and stirred; then, the resulting solution is centrifuged and washed with deionized water; finally, a brown precipitate is collected by centrifugation to obtain the MnSiO3 having oxidase-like activity.

[0021] In the above technical solution, it is further preferred that the molar concentration of MnCl2·4H2O is 0.5 mM and the volume is 10 mL; the molar concentration of NaSiO3·9H2O is 0.5 mM and the volume is 90 mL.

[0022] In the above technical solution, it is further preferred that the stirring time is 20 minutes.

[0023] In the above technical solution, it is further preferred that the centrifugal condition is: centrifugation at 11000 rpm for 12 minutes.

[0024] A nanozyme-based ratiometric fluorescence sensing platform for total antioxidant capacity determination was developed for the determination of total antioxidant capacity (TAC) in food and serum.

[0025] The beneficial effects of the present invention are:

[0026] The nanozyme-based ratiometric fluorescence sensing platform for total antioxidant capacity determination of the present invention utilizes nanozyme MnSiO3 with oxidase-like activity and fluorescent substrates with opposite responses such as SC / OPD to construct a ratiometric fluorescence sensor for the detection of TAC, thereby realizing the detection of three different antioxidants.

[0027] The nanozyme-based ratiometric fluorescence sensing platform for total antioxidant capacity determination of the present invention has similarities when the TAC value calculated from the dual output signals of the platform system is compared with that of a commercial kit, which proves the accuracy and practicality of the system.

[0028] The nanoenzyme-based ratiometric fluorescence sensing platform for total antioxidant capacity determination of the present invention also exhibits satisfactory applicability in human serum samples.

[0029] The nanoenzyme-based ratio fluorescence sensing platform for total antioxidant capacity determination of the present invention has the advantages of strong anti-interference ability and high sensitivity.

[0030] The nanozyme-based ratio fluorescence sensing platform for total antioxidant capacity determination of the present invention uses the oxide-like nanozyme MnSiO3 synthesized by a one-pot method at room temperature, and has the characteristics of simple operation and low cost.

[0031] The nanoenzyme-based ratio fluorescence sensing platform for total antioxidant capacity determination of the present invention has oxidase-like properties, does not introduce hydrogen peroxide, and has little interference with the experiment. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0033] Figure 1 Schematic diagram of the operating mechanism of the MnSiO3-based ratiometric fluorescence sensing platform for TAC determination.

[0034] Figure 2 This is a transmission electron microscope image of MnSiO3 at 50nm size.

[0035] Figure 3 Schematic diagram of the MnSiO3-based ratiometric fluorescence sensing platform of the present invention for determining TAC in real samples.

[0036] Figure 4 Figure 3 is a linear detection limit diagram of the ratio fluorescence sensing platform based on MnSiO3 of the present invention for actual sample detection, wherein A is the SC fluorescence spectrum of AA with different concentrations (from a to m, respectively, 0.1, 1, 5, 20, 40, 50, 70, 90, 120, 150, 200, 300 and 500 μM) in the presence of MnSiO3 nanozymes and different concentrations of AA; D is the SC fluorescence spectrum of AA with different concentrations (from a to k, respectively, 0.1, 10, 30 (A) SC fluorescence spectra of (B) AA, (E) Cys and (H) GSH at different concentrations (a, j, 0.1, 10, 40, 70, 100, 120, 150, 200, 300 and 400 μM); (B) SC fluorescence spectra of (E) AA, (E) Cys and (H) GSH at different concentrations; (C) SC fluorescence spectra of (D) AA, (E) Cys and (H) GSH at different concentrations; (D ... 465 / FI 562 Calibration plots and linear relationships of the values ​​with different concentrations of (C)AA, (F)Cys, and (I)GSH (error bars were obtained from three independent measurements).

[0037] Figure 5 Schematic diagram of the selectivity and anti-interference of the MnSiO3-based ratiometric fluorescence sensing platform of the present invention, wherein A is AA, B is Cys, and C is GSH (the concentrations of the three antioxidants are 200 μM, 400 μM, and 350 μM, respectively, and the concentration of other interferents is 500 μM).

[0038] Figure 6 This is a comparison chart of the results of the MnSiO3-based ratio fluorescence sensing platform of the present invention and the commercial kit for actual sample detection. DETAILED DESCRIPTION

[0039] The inventive concept of the present invention is: the present invention adopts a one-pot method at room temperature to synthesize MnSiO3 with oxidase-like activity (OXD), and combines it with fluorescent substrates SC and OPD with opposite responses to construct a new ratiometric fluorescence sensing platform with high sensitivity and strong anti-interference ability. Thanks to the dual signal output of opposite effects, the accuracy of experimental detection is improved, and the low cost of material synthesis and the lack of introduction of hydrogen peroxide provide advantages for the determination of TAC. Therefore, the present invention very cleverly solves the bottleneck problem existing in previous work.

[0040] The present invention is described in detail below with reference to the accompanying drawings.

[0041] A nanozyme-based ratiometric fluorescence sensing platform for total antioxidant capacity determination, comprising:

[0042] MnSiO3 with oxidase-like activity;

[0043] The two fluorescent substrates have opposite responses, preferably one of the fluorescent substrates is Scopoletin (SC) and the other fluorescent substrate is o-phenylenediamine (OPD).

[0044] Preferably, in the ratiometric fluorescence sensing platform system:

[0045] The mass concentration of MnSiO3 with oxidase-like activity is 6.4 mg·mL -1 , volume is 10μL; the molar concentration of the fluorescent substrate SC is 50μM, volume is 5μL; the molar concentration of the fluorescent substrate OPD is 300mM, volume is 50μL.

[0046] The MnSiO3 with oxidase-like activity of the present invention is synthesized by a one-pot method, and the synthesis steps are as follows:

[0047] MnCl2·4H2O (0.5 mM, 10 mL) was added dropwise to an aqueous solution of NaSiO3·9H2O (0.5 mM, 90 mL) and stirred for 20 min. The resulting solution was then centrifuged at 11000 rpm for 12 min and carefully washed with deionized water three times. Finally, the brown precipitate was collected by centrifugation and then dispersed with ultrapure water. The final light brown stock solution was stored at 4 °C for further use.

[0048] The nanozyme-based ratio fluorescence sensing platform for total antioxidant capacity determination of the present invention has the following mechanism of action:

[0049] MnSiO3 has OXD-like activity. In the absence of antioxidants, MnSiO3 oxidizes the fluorescent substrates SC / OPD, and the two show low and high fluorescence signals, respectively. In the presence of antioxidants, MnSiO3 is etched by the antioxidants and cannot oxidize the fluorescent substrates SC / OPD, producing high and low signals, respectively (the emission wavelengths of SC and OPD are 465nm and 562nm, respectively).

[0050] A nanozyme-based ratiometric fluorescence sensing platform for total antioxidant capacity determination was developed for the determination of total antioxidant capacity (TAC) in food and serum.

[0051] The nanoenzyme-based ratiometric fluorescence sensing platform system of the present invention is used for the detection of antioxidants in the following steps:

[0052] Different concentrations of AA and MnSiO3 (6.4 mg mL -1 , 10 μL) was incubated in 385 μL Tris-HCl buffer at room temperature for 40 minutes. Then, OPD (300 mM, 50 μL) was added to the system and incubated for 40 min, and the fluorescence intensity (FI) of ox-OPD at 562 nm after excitation at 370 nm was monitored. 562 Subsequently, SC (50 μM, 5 μL) was introduced into the above mixture, and ox-SC (FI 465 ) is the fluorescence intensity. Finally, the FI can be correctly calculated 465 / FI 562 The ratio was used as the final response signal. In addition, the analysis steps for Cys and GSH were the same as those for AA determination (see Figure 1 ).

[0053] See also Figure 3 The nanozyme-based ratio fluorescence sensing platform system of the present invention is used for the determination of TAC as follows:

[0054] Two brands of vitamin C tablets and two types of beverages were measured according to the detection method of antioxidants. Vitamin C tablets were subjected to the pretreatment steps of grinding, dissolution and centrifugation. The collected supernatant and two types of beverages were diluted 50 times, 18 times and 150 times respectively to make their different concentrations within the linear range of AA detection. Fluorescence measurement and calculation of FI 465 / FI 562 After the ratio was calculated, the expression of TAC content and concentration equivalent AA was obtained.

[0055] For TAC determination of human serum samples, the standard addition method was used. Serum samples were diluted with Tris-HCl buffer solution (5‰), and the TAC value of blank serum samples was measured. At the same time, different concentrations of AA standard solution were added to the diluted serum, and the FI was obtained using the above process.465 / FI 562 value.

[0056] Instrument parameters: excitation wavelength: OPD 370nm, SC 380nm; excitation / emission slit width: OPD 5 / 10nm, SC 5 / 5nm.

[0057] The technical scheme of the present invention is clearly and completely described below through examples, but it should be understood that the following examples do not limit the protection scope of the present invention.

[0058] The reagents used in the following examples are all commercially available products, among which SC (Scopoletin) was purchased from Beijing Bailingwei Technology Co., Ltd.

[0059] Example 1 Synthesis of MnSiO3 with oxidase-like activity:

[0060] MnCl2·4H2O (0.5 mM, 10 mL) was added dropwise to an aqueous solution of NaSiO3·9H2O (0.5 mM, 90 mL) and stirred for 20 min. The resulting solution was then centrifuged at 11000 rpm for 12 min and carefully washed with deionized water three times. Finally, the brown precipitate was collected by centrifugation and then dispersed with ultrapure water. The final light brown stock solution was stored at 4 °C for further use.

[0061] Figure 2 The transmission electron microscopy image of the prepared MnSiO3 with oxidase-like activity at a size of 50 nm proves the successful synthesis of MnSiO3.

[0062] Example 2 Feasibility of constructing a ratiometric fluorescence system based on MnSiO3

[0063] Add 440 μL Tris-HCl buffer to the centrifuge tube, and then add 10 μL MnSiO3 (6.4 mg mL -1 ), 50 μL OPD (500 mM), shake, place in a dark environment to react for 30 min, and record the fluorescence intensity of OPD on a Hongdong fluorescence instrument; add 435 μL Tris-HCl buffer to another centrifuge tube, and then add 10 μL MnSiO3 (6.4 mg mL -1 ), 50μL OPD (500mM), shake, place in the dark environment to react for 30min, then add 5μL SC (50μM), shake, react again for 40min, and record the fluorescence intensity of SC on the Gangdong fluorescence instrument. The fluorescence intensity value of OPD increased from 3.056 to 859.992, and the fluorescence intensity value of SC decreased from 529.369 to 174.979.

[0064] Example 3 Confirmatory experiment based on ratiometric fluorescence platform for detecting antioxidants (taking AA as an example):

[0065] In 395 μL Tris-HCl buffer, AA with a final concentration of 500 μΜ was reacted with MnSiO3 in the dark for 40 min, and then 10 μL MnSiO3 (6.4 mg mL -1 ), 50 μL OPD (500 mM), shaken, placed in a dark environment for 30 min, and the fluorescence intensity of OPD was recorded on a Hongdong fluorescence instrument; in 390 μL Tris-HCl buffer, AA with a final concentration of 500 μΜ was reacted with MnSiO3 in the dark for 40 min, and then 10 μL MnSiO3 (6.4 mg·mL -1 ), 50 μL of OPD (500 mM), shake, and react in a dark environment for 30 min, then add 5 μL of SC (50 μM), shake, and react again for 40 min, and record the fluorescence intensity of SC on a Gangdong fluorescence instrument.

[0066] The fluorescence intensity value of OPD decreased from 859.992 to 15.027, and the fluorescence value of SC increased from 174.979 to 379.937.

[0067] Results Feasibility and verification experiments of Examples 2 and 3. After optimization: In the nanozyme-based ratio fluorescence sensing platform system: the mass concentration of MnSiO3 with oxidase-like activity was 6.4 mg·mL -1 , volume is 10μL; the molar concentration of the fluorescent substrate SC is 50μM, volume is 5μL; the molar concentration of the fluorescent substrate OPD is 300mM, volume is 50μL.

[0068] Example 4

[0069] Using the method of Example 3 and the optimized ratio fluorescence sensing platform system based on MnSiO3, AA, Cys and GSH were measured respectively, as well as two brands of vitamin C tablets (Shenyang No. 1 Pharmaceutical Co., Ltd. of Northeast Pharmaceutical Group and Shaanxi Yishengtang Pharmaceutical Co., Ltd.) and two types of beverages (water-soluble C and ripe fruit). Vitamin C tablets were subjected to the pretreatment steps of grinding, dissolving and centrifugation in sequence. The collected supernatant and the two types of beverages were diluted 50 times, 18 times and 150 times respectively, so that their different concentrations were within the linear range of AA detection. Fluorescence measurement and calculation of FI 465 / FI 562 After the ratio was calculated, the expression of TAC content and concentration equivalent AA was obtained.

[0070] For TAC determination of human serum samples, the standard addition method was used. Serum samples were diluted with Tris-HCl buffer solution (5‰), and the TAC value of blank serum samples was measured. At the same time, different concentrations of AA standard solution were added to the diluted serum, and the FI was obtained using the above process. 465 / FI 562 value.

[0071] Instrument parameters: excitation wavelength: OPD 370nm, SC 380nm; excitation / emission slit width: OPD 5 / 10nm, SC 5 / 5nm.

[0072] Figure 4 This is a linear detection limit diagram of the MnSiO3-based ratiometric fluorescence sensing platform of the present invention for actual sample detection. It can be seen from the figure that the AA detection limit is: 0.057μΜ, the Cys detection limit is: 1.78μΜ, and the GSH detection limit is: 0.18μΜ.

[0073] The present invention also studies the selectivity and anti-interference ability of the ratio fluorescence sensing platform based on MnSiO3, see Figure 5 Here, the same concentration (500 μM) of metal ions and amino acids (such as Na + , Glu, Fru, Suc, Thr, Gly, Arg, His, Lys) were used as alternatives to evaluate selectivity. Figure 5 As shown, only AA, Cys, or GSH revealed significant FI465 / FI562 ratios, while the other counterparts produced negligible ratio values. This result can be attributed to the specific interaction between the antioxidants and the MnSiO3 nanozymes and their strong reducing ability.

[0074] The present invention also used a commercial kit to test two brands of vitamin C tablets (Northeast Pharmaceutical Group Shenyang No. 1 Pharmaceutical Co., Ltd., Shaanxi Yishengtang Pharmaceutical Co., Ltd.) and two types of beverages (water-soluble C, ripe fruit) in Example 4. The test results were compared with those of Example 4 of the present invention as shown in the following table and Figure 6 .

[0075] Commercial kit: Manufacturer: Suzhou Gres Biotechnology Co., Ltd.; Name: ABTS Clearance Ability Kit; Item No.: G0127F; Detection method: Visible spectrophotometry.

[0076] Comparison of TAC value and kit value:

[0077]

[0078] As can be seen from the above table, the TAC value calculated from the dual output signals of the ratio fluorescence sensing platform based on MnSiO3 of the present invention is compared with that of the commercial test kit, which shows similarity, proving the accuracy and practicality of the system.

[0079] like Figure 6 As shown, by comparing the calculated values ​​with the labeled contents indicated in the specifications or the results from the commercially available 2,2'-diazobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) TAC assay kit, it is almost impossible to monitor the difference. All these facts confirm the accuracy and reliability of the MnSiO3-based ratiometric fluorescence sensing platform of the present invention and highlight its great potential in food and drug quality assessment.

[0080] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A nanozyme-based ratiometric fluorescence sensing platform for total antioxidant capacity determination, characterized in that: include: MnSiO3 with oxidase-like activity; Two fluorescent substrates with opposite responses.

2. The nanozyme-based ratiometric fluorescence sensing platform according to claim 1, characterized in that: Among the fluorescent substrates, one fluorescent substrate is Scopoletin (SC), and the other fluorescent substrate is o-phenylenediamine (OPD).

3. The nanozyme-based ratiometric fluorescence sensing platform according to claim 1, characterized in that: In the ratiometric fluorescence sensing platform system: The mass concentration of MnSiO3 with oxidase-like activity is 6.4 mg.mL -1 , volume 10 μL; The molar concentration of the fluorescent substrate SC was 50 μM, and the volume was 5 μL; The molar concentration of the fluorescent substrate OPD was 300 mM and the volume was 50 μL.

4. The nanozyme-based ratiometric fluorescence sensing platform according to claim 1, characterized in that: The MnSiO3 with oxidase-like activity is synthesized by a one-pot method.

5. The nanozyme-based ratiometric fluorescence sensing platform according to claim 4, characterized in that: The method for synthesizing MnSiO3 having oxidase-like activity comprises the following steps: MnCl2·4H2O is added to an aqueous solution of NaSiO3·9H2O and stirred; then, the resulting solution is centrifuged and washed with deionized water; finally, a brown precipitate is collected by centrifugation to obtain the MnSiO3 having oxidase-like activity.

6. The nanozyme-based ratiometric fluorescence sensing platform according to claim 5, characterized in that: The molar concentration of MnCl2·4H2O is 0.5mM and the volume is 10mL; the molar concentration of NaSiO3·9H2O is 0.5mM and the volume is 90mL.

7. The nanozyme-based ratiometric fluorescence sensing platform according to claim 5, characterized in that: The stirring time is 20 minutes.

8. The nanozyme-based ratiometric fluorescence sensing platform according to claim 5, characterized in that: The centrifugal conditions were: 11000 rpm for 12 minutes.

9. A nanozyme-based ratiometric fluorescence sensing platform as described in any one of claims 1-8 for the determination of total antioxidant capacity (TAC) in food and serum.

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