CN119290833A - A nanozyme-based ratiometric fluorescence sensing platform for total antioxidant capacity determination and its application - Google Patents
A nanozyme-based ratiometric fluorescence sensing platform for total antioxidant capacity determination and its application Download PDFInfo
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- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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Abstract
本发明涉及一种用于总抗氧化能力测定的纳米酶基比率荧光传感平台及其应用，属于荧光传感器制备技术领域。本发明的比率荧光传感平台，包括：具有类氧化酶活性的MnSiO3；两种具有相反响应的荧光底物。本发明的比率荧光传感平台，利用具有类氧化酶活性的纳米酶MnSiO3，及SC/OPD两个具有相反响应的荧光底物，构建比率荧光传感器，用于TAC的检测，实现了三种不同抗氧化剂的检测。本发明的比率荧光传感平台，具有抗干扰能力强、灵敏度高的优点。本发明的比率荧光传感平台，具有类氧化酶性质，不引入过氧化氢，对实验干扰小。
The present invention relates to a nanozyme-based ratio fluorescence sensing platform for total antioxidant capacity determination and its application, and belongs to the technical field of fluorescence sensor preparation. The ratio fluorescence sensing platform of the present invention comprises: MnSiO 3 with oxidase-like activity; two fluorescent substrates with opposite responses. The ratio fluorescence sensing platform of the present invention utilizes the nanozyme MnSiO 3 with oxidase-like activity and two fluorescent substrates SC/OPD with opposite responses to construct a ratio fluorescence sensor for the detection of TAC, thereby realizing the detection of three different antioxidants. The ratio fluorescence sensing platform of the present invention has the advantages of strong anti-interference ability and high sensitivity. The ratio fluorescence sensing platform of the present invention has oxidase-like properties, does not introduce hydrogen peroxide, and has little interference with the experiment.
Description
Technical Field
The invention belongs to the technical field of fluorescent sensor preparation, and particularly relates to a nano enzyme-based ratio fluorescent sensing platform for measuring total antioxidant capacity and application thereof.
Background
The total antioxidant capacity (total antioxidant capacity, TAC) refers to the sum of the antioxidant capacities of all antioxidants in the sample to be tested. It is an index of clinical biochemical tests that can be assessed by measuring the antioxidant content in blood, urine or other body fluids. The total antioxidant capacity can reflect the body's ability to fight free radicals and oxidative stress, an important tool for assessing health and disease risk.
In order to accurately detect the total antioxidant capacity, a fluorescence and colorimetric sensing platform based on nano-enzyme is constructed. TAC detection was performed by oxidizing Ascorbic Acid (AA) with a synthetic Cu-N/C nanoenzyme to produce hydrogen peroxide (H 2O2) designed for fluorescence and colorimetric sensing platforms as described in Tao et al. The detection limits of this technique were 0.7 μm and 0.3 μm, respectively. However, the synthesis of the material in the technology requires high-temperature treatment, the cost is high, and the TAC detection cannot be satisfied due to single detection target. (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 fluorescent and colorimetric sensing platform based on a persistent luminescence nanoenzyme ZGO-Pt with peroxidase-like activity (POD). By adding H 2O2, TMB is oxidized by free radicals, and the fluorescence of ZGO-Pt is quenched by Ox-TMB, so that detection of TAC is performed. However, H 2O2 introduced by this technique may interfere with the measurement of TAC (L.—X.yan, Z.—Y.yan, X.zhao, L.—J.Chen, X.— P.Yan, sensor.Actuat.B-chem.,2024,405,135333.). The Chen subject group constructs a colorimetric sensing platform based on Co-Ir/C nano-enzyme with peroxidase-like activity by using TMB as a probe, and is used for detecting TAC. However, this technique uses a single signal output, is subject to environmental/background interference, and presents false positive/false negative results that are detrimental to TAC detection (S.Li, P.Keoingthong, J.Xu, Y.Yang, J.Shen, Y.Xu, et al, biosens. Bioelectron.,2023,236,115416).
In summary, the existing nanoenzyme-based fluorescence and colorimetric sensing platforms for detecting TAC have the following defects:
(1) The single signal output is adopted, so that the signal is easy to be interfered by the background/environment, and the false positive/false negative result is caused, so that the detection of TAC is not facilitated.
(2) Most materials are nano enzymes with peroxidase-like activity, hydrogen peroxide molecules are introduced during operation, and a certain background interference is caused to TAC determination.
(3) In the aspect of synthesizing materials, most materials need a certain high-temperature or ultra-high-temperature treatment, so that the synthesis is complex and the cost is high.
Disclosure of Invention
The invention aims to solve the technical problems in the prior art and provides a nano enzyme-based ratio fluorescent sensing platform for measuring total antioxidant capacity and application thereof.
In order to solve the technical problems, the technical scheme of the invention is as follows:
A nanoenzyme-based ratiometric fluorescence sensing platform for total antioxidant capacity determination, comprising:
MnSiO 3 with oxidase-like activity;
two fluorogenic substrates with opposite responses.
In the above technical solution, it is preferable that one of the fluorogenic substrates is Scopoletin (SC) and the other fluorogenic substrate is o-phenylenediamine (OPD).
In the above technical solution, preferably, in the ratio fluorescent sensing platform system:
the mass concentration of MnSiO 3 with oxidase-like activity is 6.4 mg.mL -1, and the volume is 10 mu L;
The molar concentration of fluorogenic substrate SC was 50. Mu.M, and the volume was 5. Mu.L;
the molar concentration of fluorogenic substrate OPD was 300mM and the volume was 50. Mu.L.
In the above technical scheme, preferably, the MnSiO 3 with oxidase-like activity is synthesized by a one-pot method.
In the above technical solution, it is further preferable that the synthesis method of MnSiO 3 having oxidase-like activity includes the steps of:
Adding MnCl 2·4H2 O into an aqueous solution of NaSiO 3·9H2 O, stirring, centrifuging the obtained solution, washing with deionized water, and finally collecting brown precipitate by centrifugation to obtain the MnSiO 3 with oxidase-like activity.
In the above embodiment, it is still further preferable that the molar concentration of MnCl 2·4H2 O is 0.5mM, the volume is 10mL, and the molar concentration of NaSiO 3·9H2 O is 0.5mM, the volume is 90mL.
In the above technical scheme, still more preferably, the stirring time is 20min.
In the above-mentioned technical scheme, it is still further preferable that the condition of centrifugation is 11000rpm for 12 minutes.
A nano enzyme-based ratio fluorescent sensing platform for measuring total antioxidant capacity is used for measuring Total Antioxidant Capacity (TAC) in food and serum.
The beneficial effects of the invention are as follows:
The nano enzyme-based ratio fluorescence sensing platform for measuring the total antioxidant capacity, disclosed by the invention, utilizes nano enzyme MnSiO 3 with oxidase-like activity and a fluorescence substrate with opposite response such as SC/OPD to construct a ratio fluorescence sensor for detecting TAC, so that the detection of three different antioxidants is realized.
The nano enzyme-based ratio fluorescence sensing platform for measuring the total antioxidant capacity has the advantages that the TAC value calculated by the double-output signals of the platform system is compared with a commercial kit, the similarity is realized, and the accuracy and the practicability of the system are proved.
The nano enzyme-based ratio fluorescence sensing platform for measuring the total antioxidant capacity also shows satisfactory applicability in human serum samples.
The nano enzyme-based ratio fluorescent sensing platform for measuring the total antioxidant capacity has the advantages of strong anti-interference capacity and high sensitivity.
The nano enzyme-based ratio fluorescent sensing platform for measuring the total antioxidant capacity, disclosed by the invention, has the characteristics of simplicity in operation and low cost due to the adoption of the oxide-like nano enzyme MnSiO 3 synthesized by a one-pot method at normal temperature.
The nano enzyme-based ratio fluorescence sensing platform for measuring the total antioxidant capacity has the oxidase-like property, does not introduce hydrogen peroxide, and has small interference to experiments.
Drawings
The invention is described in further detail below with reference to the drawings and the detailed description.
FIG. 1 is a schematic diagram of the operating mechanism of a MnSiO 3 -based ratiometric fluorescence sensing platform for TAC determination.
FIG. 2 is a transmission electron microscope image of MnSiO 3 at a 50nm size.
FIG. 3 is a schematic diagram of the MnSiO 3 -based ratiometric fluorescence sensing platform of the present invention for determining TAC in a real sample.
FIG. 4 is a graph showing the linear limit of detection of a ratio fluorescence sensing platform based on MnSiO 3 according to the present invention for practical sample detection, wherein A is the SC fluorescence spectrum using different concentrations (0.1, 1,5, 20, 40, 50, 70, 90, 120, 150, 200, 300 and 500. Mu.M) of AA in the presence of MnSiO 3 nano-enzyme and different concentrations of AA, D is the SC fluorescence spectrum using different concentrations (0.1, 10, 30, 50, 100, 150, 200, 250, 300, 350 and 400. Mu.M) of Cys in the presence of a to k in the presence of a concentration of 0.1, 10, 40, 70, 100, 120, 150, 200, 300 and 400. Mu.M) of GSH in the presence of MnSiO 3 nano-enzyme and different concentrations of (B), and (E) and (H) of ASH, and G is the linear error plot of FI 465/FI562 values versus different concentrations (C), cys in the presence of (I) and (H) of Cys in the presence of a to j in the presence of 0.1, 10, 40, 70, 100, 120, 150, 200, 300 and 400. Mu.M in the linear error plot.
FIG. 5 is a schematic diagram showing the selectivity and anti-interference properties of a MnSiO 3 -based ratiometric fluorescence sensing platform of the present invention, wherein A is AA, B is Cys, C is GSH (the concentrations of the three antioxidants are 200 μΜ, 400 μΜ, and 350 μΜ, respectively, and the concentrations of the other interferents are 500 μΜ).
FIG. 6 is a graph of the comparative results of MnSiO 3 -based ratiometric fluorescence sensing platform and commercial kits of the present invention for actual sample detection.
Detailed Description
The invention adopts a one-pot method to synthesize MnSiO 3 with similar oxidase activity (OXD) at normal temperature, and combines with fluorogenic substrates SC and OPD with opposite responses to construct a novel ratio fluorescence sensing platform with sensitivity and strong anti-interference capability. The accuracy of experimental detection is improved due to the double signal output of opposite effects, and the low cost of material synthesis and the non-introduction of hydrogen peroxide provide advantages for the determination of TAC. Therefore, the invention solves the bottleneck problem existing in the previous work very skillfully.
The present invention will be described in detail with reference to the accompanying drawings.
A nanoenzyme-based ratiometric fluorescence sensing platform for total antioxidant capacity determination, comprising:
MnSiO 3 with oxidase-like activity;
Two fluorogenic substrates having opposite responses, preferably one of which is Scopoletin (SC) and the other of which is o-phenylenediamine (OPD).
Preferably, in the ratiometric fluorescence sensing platform system:
The mass concentration of MnSiO 3 having oxidase-like activity was 6.4 mg.mL -1 in volume of 10. Mu.L, the molar concentration of fluorogenic substrate SC was 50. Mu.M in volume of 5. Mu.L, the molar concentration of fluorogenic substrate OPD was 300mM in volume of 50. Mu.L.
The MnSiO 3 with the oxidase-like activity is synthesized by adopting a one-pot method, and the synthesis steps are as follows:
MnCl 2·4H2 O (0.5 mM,10 mL) was added dropwise to an aqueous solution of NaSiO 3·9H2 O (0.5 mM,90 mL) and stirred for 20min. The resulting solution was then centrifuged at 11000rpm for 12 minutes and carefully washed 3 times with deionized water. Finally, the brown precipitate was collected by centrifugation and then dispersed with ultrapure water. The final light brown stock was kept at 4 ℃ for further use.
The invention relates to a nano enzyme-based ratio fluorescence sensing platform for measuring total antioxidant capacity, which has the following action mechanism:
MnSiO 3 has activity similar to that of OXD, mnSiO 3 oxidizes fluorescent substrate SC/OPD in the absence of antioxidant, and both of the substrates respectively show low and high fluorescence signals, and MnSiO 3 is etched by the antioxidant in the presence of the antioxidant and cannot oxidize fluorescent substrate SC/OPD to respectively generate high and low signals (the emission wavelengths of SC and OPD are 465nm and 562nm respectively).
A nano enzyme-based ratio fluorescent sensing platform for measuring total antioxidant capacity is used for measuring Total Antioxidant Capacity (TAC) in food and serum.
The detection steps of the nano enzyme-based ratio fluorescent sensing platform system for the antioxidant are as follows:
AA at various concentrations was incubated with MnSiO 3(6.4mg·mL-1, 10. Mu.L) in 385. Mu.L Tris-HCl buffer for 40min at room temperature. OPD (300 mM, 50. Mu.L) was then added to the system and incubated for 40min, and the fluorescence intensity (FI 562) of ox-OPD at 562nm after excitation at 370nm was monitored. Subsequently, SC (50. Mu.M, 5. Mu.L) was introduced into the above mixture, and the fluorescence intensity of ox-SC (FI 465) was examined at an excitation wavelength of 380 nm. Finally, the FI 465/FI562 ratio can be correctly calculated as the final response signal. In addition, the analytical steps of Cys and GSH are identical to the AA assay (see fig. 1).
Referring to fig. 3, the nano-enzyme-based ratiometric fluorescence sensing platform system of the present invention is used for TAC determination steps as follows:
Two brands of vitamin C tablets and two types of beverages were measured separately with reference to the antioxidant detection method. The vitamin C tablets are subjected to the pretreatment steps of grinding, dissolving and centrifuging in sequence. And the collected supernatant and the two types of beverages were diluted 50-fold, 18-fold and 150-fold, respectively, so that their different concentrations were within the linear range of AA detection. After fluorescence measurement and calculation of the FI 465/FI562 ratio, the expression of TAC content and concentration equivalent AA was obtained.
For TAC determination of human serum samples, standard addition methods were used. Serum samples (5% in weight) were diluted with Tris-HCl buffer and TAC values were measured for blank serum samples. Meanwhile, AA standard solutions of different concentrations were added to the diluted serum, and FI 465/FI562 values were obtained using the procedure described above.
The instrument parameters are the excitation wavelength of OPD 370nm, SC 380nm, and the excitation/emission slit width of OPD 5/10nm, SC 5/5nm.
The following examples are given to clearly and completely describe the technical solution of the present invention, but it should be understood that the following examples do not limit the scope of protection of the present invention.
The reagents used in the examples below are all commercially available. SC (Scopoletin) of which was purchased from the scientific and technological company of belvedere.
Example 1 synthesis of MnSiO 3 with oxidase-like activity:
MnCl 2·4H2 O (0.5 mM,10 mL) was added dropwise to an aqueous solution of NaSiO 3·9H2 O (0.5 mM,90 mL) and stirred for 20min. The resulting solution was then centrifuged at 11000rpm for 12 minutes and carefully washed 3 times with deionized water. Finally, the brown precipitate was collected by centrifugation and then dispersed with ultrapure water. The final light brown stock was kept at 4 ℃ for further use.
FIG. 2 is a transmission electron micrograph of prepared MnSiO 3 with oxidase-like activity at 50nm size, demonstrating successful synthesis of MnSiO 3.
Example 2 feasibility of constructing a ratiometric fluorescent System based on MnSiO 3
Adding 440 mu L of Tris-HCl buffer solution, then sequentially adding 10 mu L of MnSiO 3(6.4mg·mL-1 and 50 mu L of OPD (500 mM), vibrating, standing in a dark environment for reacting for 30min, recording the fluorescence intensity of the OPD on a Gangdong fluorometer, adding 435 mu L of Tris-HCl buffer solution, then sequentially adding 10 mu L of MnSiO 3(6.4mg·mL-1 and 50 mu L of OPD (500 mM) into another centrifuge tube, vibrating, standing in the dark environment for reacting for 30min, then adding 5 mu L of SC (50 mu M), vibrating, reacting again for 40min, and recording the fluorescence intensity of the SC on a Gangdong fluorometer. The fluorescence intensity value of OPD increased from 3.056 to 859.992 and that of SC decreased from 529.369 to 174.979.
Example 3 validation experiment for detecting antioxidants based on a ratio fluorescence plateau (exemplified by AA):
After reacting 500. Mu.m AA and MnSiO 3 in 395. Mu.L Tris-HCl buffer for 40min in dark, 10. Mu.L MnSiO 3(6.4mg·mL-1) and 50. Mu.L OPD (500 mM) are added in sequence, shaking, standing in dark for 30min, recording the fluorescence intensity of OPD on a Gangdong fluorometer, after reacting 500. Mu.L Tris-HCl buffer for 40min in 390. Mu.L Tris-HCl buffer with 500. Mu.m AA and MnSiO 3, 10. Mu.L MnSiO 3(6.4mg·mL-1) and 50. Mu.L OPD (500 mM) are added in sequence, shaking, standing in dark for 30min, adding 5. Mu.L SC (50. Mu.M), shaking, reacting again for 40min, recording the fluorescence intensity of SC on Gangdong fluorometer.
The fluorescence intensity value of OPD was decreased from 859.992 to 15.027, and the fluorescence value of SC was increased from 174.979 to 379.937.
Results the feasibility and validation experiments of examples 2 and 3 were optimized for a nano-enzyme based ratio fluorescence sensing platform system with a mass concentration of MnSiO 3 having oxidase-like activity of 6.4 mg.mL -1 and a volume of 10. Mu.L, a molar concentration of fluorogenic substrate SC of 50. Mu.M and a volume of 5. Mu.L, and a molar concentration of fluorogenic substrate OPD of 300mM and a volume of 50. Mu.L.
Example 4
AA, cys and GSH were measured using the method of example 3, and the optimized MnSiO 3 -based ratiometric fluorescence sensor platform system, respectively, with two brands of vitamin C tablets (shenyang first pharmaceutical company of northeast pharmaceutical group, the pharmaceutical company of the Yisheng, shanxi) and two types of beverages (water soluble C, ripe fruits). The vitamin C tablets are subjected to the pretreatment steps of grinding, dissolving and centrifuging in sequence. And the collected supernatant and the two types of beverages were diluted 50-fold, 18-fold and 150-fold, respectively, so that their different concentrations were within the linear range of AA detection. After fluorescence measurement and calculation of the FI 465/FI562 ratio, the expression of TAC content and concentration equivalent AA was obtained.
For TAC determination of human serum samples, standard addition methods were used. Serum samples (5% in weight) were diluted with Tris-HCl buffer and TAC values were measured for blank serum samples. Meanwhile, AA standard solutions of different concentrations were added to the diluted serum, and FI 465/FI562 values were obtained using the procedure described above.
The instrument parameters are the excitation wavelength of OPD 370nm, SC 380nm, and the excitation/emission slit width of OPD 5/10nm, SC 5/5nm.
FIG. 4 is a graph showing the linear detection limit of the MnSiO 3 -based ratio fluorescence sensing platform for detecting an actual sample, wherein the AA detection limit is 0.057 mu m, the Cys detection limit is 1.78 mu m, and the GSH detection limit is 0.18 mu m.
The invention also investigates the selectivity and anti-interference capability of the MnSiO 3 -based ratiometric fluorescence sensing platform, see fig. 5. Here, some of the same concentrations (500. Mu.M) of metal ions and amino acids (e.g., na +, glu, fru, suc, thr, gly, arg, his, lys) were used as substitutes to evaluate selectivity. As shown in FIG. 5, only AA, cys, or GSH revealed a significant FI465/FI562 ratio, while the other counterparts produced negligible ratio values. This result can be attributed to the specific interaction between the antioxidant and MnSiO 3 nanoenzyme and its strong reducing power.
The present invention was also tested using commercial kits for two brands of vitamin C tablets (Shenyang first pharmaceutical Co., north-east pharmaceutical group, shanxi Yishengtang pharmaceutical Co., ltd.) and two types of beverages (Water soluble C, ripe fruit) in example 4, as compared to the test results of example 4 of the present invention as shown in Table 6 below.
Commercial kits were manufactured by Suzhou Grace biotechnology Co., ltd, the name of the kit for removing ABTS, the product number of the kit G0127F, and the detection method of the kit were visible spectrophotometry.
TAC value and kit comparison value:
As shown in the table, the TAC value calculated by the double-output signal of the platform system of the MnSiO 3 -based ratio fluorescence sensing platform is compared with a commercial kit, and the similarity is achieved, so that the accuracy and the practicability of the system are proved.
As shown in fig. 6, by comparing the calculated value with the label content indicated in the specification or the result from the commercially available 2,2' -diazobis (3-ethylbenzothiazoline-6-sulfonic Acid) (ABTS) TAC assay kit, the difference was hardly monitored. All these facts confirm the accuracy and reliability of the MnSiO 3 -based ratiometric fluorescence sensing platform of the present invention and highlight its great potential in food and drug quality assessment.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (9)
1. A nanoenzyme-based ratiometric fluorescence sensing platform for total antioxidant capacity determination, comprising:
MnSiO 3 with oxidase-like activity;
two fluorogenic substrates with opposite responses.
2. The nano-enzyme based ratiometric fluorescence sensing platform of claim 1, wherein one of the fluorogenic substrates is Scopoletin (SC) and the other fluorogenic substrate is o-phenylenediamine (OPD).
3. The nano-enzyme based ratiometric fluorescent sensing platform of claim 1, wherein in the ratiometric fluorescent sensing platform system:
The mass concentration of MnSiO 3 with oxidase-like activity is 6.4mg.mL -1, and the volume is 10 mu L;
The molar concentration of fluorogenic substrate SC was 50. Mu.M, and the volume was 5. Mu.L;
the molar concentration of fluorogenic substrate OPD was 300mM and the volume was 50. Mu.L.
4. The nano-enzyme-based ratiometric fluorescent sensing platform of claim 1, wherein the MnSiO 3 having oxidase-like activity is synthesized using a one-pot method.
5. The nano-enzyme based ratiometric fluorescence sensing platform of claim 4, wherein the method for synthesizing MnSiO 3 with oxidase-like activity comprises the steps of:
Adding MnCl 2·4H2 O into an aqueous solution of NaSiO 3·9H2 O, stirring, centrifuging the obtained solution, washing with deionized water, and finally collecting brown precipitate by centrifugation to obtain the MnSiO 3 with oxidase-like activity.
6. The nano-enzyme based ratio fluorescence sensing platform of claim 5, wherein the molar concentration of MnCl 2·4H2 O is 0.5mM, the volume is 10mL, the molar concentration of nasio 3·9H2 O is 0.5mM, the volume is 90mL.
7. The nano-enzyme based ratiometric fluorescence sensing platform of claim 5, wherein the agitation time is 20 minutes.
8. The nano-enzyme based ratiometric fluorescence sensing platform of claim 5, wherein the centrifugation conditions are 11000rpm for 12 minutes.
9. A nanoenzyme-based ratiometric fluorescent sensing platform according to any one of claims 1-8 for use in the determination of Total Antioxidant Capacity (TAC) in food and serum.
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