Flavonoid antioxidant recognition colorimetric array sensing analysis method

Abstract

The invention discloses a colorimetric array sensing analysis method for identifying a flavonoid antioxidant, belongs to the technical field of detection, and particularly relates to a flavonoid antioxidant identification technology. The method disclosed by the invention is based on NiCo-LDH, H2O2 is introduced to construct a reaction system of three different chromogenic substrates, and five flavonoid antioxidants are identified by a colorimetric array sensing method. The method and colorimetric array sensing have the characteristic of high throughput, can detect a plurality of samples at the same time, greatly improve the detection efficiency, quickly screen the flavonoid derivatives with high antioxidant activity, lay a foundation for drug research and development and product development, and show better prospects in the fields of medicine, food science and the like.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, and in particular to a flavonoid antioxidant identification technology. Background Technology

[0002] In the normal physiological processes of organisms, the production and clearance of reactive oxygen species (ROS) maintain a dynamic balance, playing a crucial role in cell signal transduction and homeostasis. However, when the body is subjected to various internal and external factors such as environmental pollution, radiation, unhealthy lifestyle habits, and disease, this balance is disrupted, leading to a large accumulation of ROS. Excessive ROS triggers oxidative stress, causing severe oxidative damage to intracellular macromolecules such as proteins, lipids, and nucleic acids, thereby disrupting the normal structure and function of cells. Long-term oxidative stress is closely related to the development of various chronic diseases, including cancer, atherosclerosis, and aging.

[0003] Flavonoid derivatives are a class of promising natural bioactive ingredients, widely found in many natural plants and characterized by structural diversity and rich bioactivity. Numerous studies have demonstrated that flavonoid derivatives possess significant antioxidant capabilities, scavenging excess reactive oxygen species (ROS) in the body through multiple mechanisms. Flavonoids can protect neurons from oxidative stress more effectively than ascorbic acid, thereby protecting cells from oxidative damage. They also exhibit various bioactivities, including anti-inflammatory, antibacterial, and antitumor effects, showing great potential in the prevention and treatment of oxidative stress-related diseases.

[0004] However, while there is a wide variety of flavonoid derivatives, there is no good method for distinguishing them to provide a corresponding theoretical basis and practical guidance for their application in food, medicine, agriculture and other fields. Summary of the Invention

[0005] The purpose of this invention is to provide a colorimetric array sensing analysis method for identifying flavonoid antioxidants, which can quickly screen out flavonoid derivatives with high antioxidant activity.

[0006] A colorimetric array sensor analysis method for identifying flavonoid antioxidants is used to identify the antioxidant properties of flavonoids. The method is characterized by being based on NiCo-LDH and introducing H2O2 to construct a colorimetric array sensor for three different chromogenic substrates and five flavonoid antioxidants. The three different chromogenic substrates are: 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonicacid) diamine salt (ABTS), 3,3',5,5'-tetramethylbenzidine (TMB), and o-phenylenediamine (…). o -Phenylenediamine (OPD); The five flavonoid antioxidants mentioned are: epicatechin (EC), kaempferol (KF), hesperidin (HP), naringenin (NG), and quercetin (QC).

[0007] The use of the colorimetric array sensor specifically involves dropping NiCo-LDH, H2O2, and the target flavonoid antioxidant into three different chromogenic substrates TMB, OPD, and ABTS, respectively, and then using a UV-Vis spectrophotometer to measure and identify its oxidation performance, or comparing it with a blank array sensor to identify its oxidation performance.

[0008] The concentration of the target flavonoid antioxidant is at least 200 nM.

[0009] Colorimetric array sensors are characterized by high throughput, enabling simultaneous detection of multiple samples, which greatly improves detection efficiency and allows for rapid screening of flavonoid derivatives with high antioxidant activity. This lays the foundation for drug development and product development, and shows promising prospects in fields such as medicine and food science. Attached Figure Description

[0010] Figure 1 SEM and TEM images of ZIF-67 and NiCo-LDH, among which, Figure 1 A is a scanning electron microscope (SEM) image taken with a ZIF-67. Figure 1 B is a transmission electron microscope (TEM) image; Figure 1 C is the SEM image of NiCo-LDH. Figure 1 D is a TEM image.

[0011] Figure 2 XRD patterns of ZIF-67 and NiCo-LDH.

[0012] Figure 3 The images show the UV-Vis absorption spectra of different substrates, where... Figure 3 A represents TMB. Figure 3 B stands for OPD. Figure 3 C represents the UV-Vis absorption spectra of ABTS as substrates; where... Figure 3 Curves ae in AC represent the absorption spectra of different reaction systems, specifically: a for substrate + H2O2, b for ZIF-67+ substrate, c for ZIF-67+ substrate + H2O2, d for NiCo-LDH+ substrate, and e for NiCo-LDH+ substrate + H2O2.

[0013] Figure 4 The image shows the electromagnetic resonance spectrum of hydroxyl radicals (-OH) in the presence or absence of hydrogen peroxide, using NiCo-LDH as a peroxide mimic enzyme.

[0014] Figure 5 A graph for colorimetric array sensing to detect and identify antioxidants KF, NG, HP, EC, and QC, wherein... Figure 5 A represents the fingerprint spectrum; Figure 5 B is a heatmap; Figure 5 C is a three-dimensional spatial distribution diagram of five antioxidants in the array sensor at an antioxidant concentration of 200 nM; Figure 5 D is a three-dimensional spatial distribution diagram of five antioxidants in the array sensor at an antioxidant concentration of 50 μM.

[0015] Figure 6 A visualization of an array sensor distinguishing five antioxidants at a concentration of 50 μM.

[0016] Figure 7 Three-dimensional spatial distribution diagrams of binary antioxidant mixtures at ratios of 5:5 and 7:3.

[0017] Figure 8 Visualized images of the array-sensor colorimetric response of binary antioxidant mixtures (KF:HP, KF:NG, KF:EC, KF:QC). Detailed Implementation

[0018] Example 1: A colorimetric array sensing analysis method for identifying flavonoid antioxidants. Based on NiCo-LDH, H2O2 was introduced to construct an array sensor with 3 different chromogenic substrates and 5 flavonoid antioxidants. Five sets of data were repeated to obtain 3×5×5 array sensor data points.

[0019] The three different chromogenic substrates are: 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diamine salt (ABTS), 3,3',5,5'-tetramethylbenzidine (TMB), and o-phenylenediamine (…). o -Phenylenediamine (OPD); The five flavonoid antioxidants mentioned are: epicatechin (EC), kaempferol (KF), hesperidin (HP), naringenin (NG), and quercetin (QC).

[0020] In the presence of H₂O₂, NiCo-LDH, acting as a peroxide mimic enzyme, effectively catalyzes the generation of hydroxyl radicals (-OH) from H₂O₂. These hydroxyl radicals (-OH) oxidize ABTS, TMB, and OPD, producing green, blue, and yellow oxidation products, respectively. Flavonoid derivatives, acting as antioxidants, are then introduced into three different reaction systems to inhibit the generation of hydroxyl radicals. As the concentration of the antioxidant increases, a series of color changes occur, enabling quantitative analysis of the antioxidant capacity of the flavonoid derivatives.

[0021] The NiCo-LDH described above uses dodecahedral ZIF-67 as a precursor for metal-organic frameworks (MOFs) and introduces Ni... 2+ After ionization, a hollow NiCo-LDH-like nanostructure is formed based on the Kirkendall effect. Two metal ions, Ni... 2+ and Co 2+ Asymmetric diffusion in the solid phase leads to vacancy accumulation and structural reconstruction, as seen in Co from ZIF-67. 2+ The diffusion rate is significantly faster than that of Ni from solution. 2+ This led to Co 2+ Prioritize outward migration, Ni 2+ Inward diffusion, thus achieving ion exchange. Co 2+ The rapid loss of these vacancies leaves gaps inside ZIF-67. These gaps accumulate to form porous or hollow structures, transforming into NiCo-LDH, such as... Figure 1C and D. The crystal structures of ZIF-67 and NiCo-LDH were characterized using X-ray diffraction (XRD). After ZIF-67 was converted to NiCo-LDH, the crystal planes at 12.5°, 22.1°, 24.4°, 34.1°, 36.5°, and 59.9° corresponded to the (003), (006), (101), (009), (012), and (110) planes of NiCo-LDH, respectively. Figure 2 .

[0022] The NiCo-LDH exhibits excellent peroxidase mimicry activity, and the verification process is as follows: In the absence of NiCo-LDH nanozymes and with only H2O2 present, no significant absorption peak was observed in the TMB + H2O2 system at 652 nm. Similarly, OPD and ABTS also showed no significant absorption peaks at 450 nm and 420 nm, respectively, in the absence of NiCo-LDH nanozymes. This indicates that the substrates TMB, OPD, and ABTS are not directly oxidized in the presence of only H2O2. Figure 3 A, Figure 3 B and Figure 3 Curve a in C; When ZIF-67 is used as an oxidase mimic and no H2O2 is present, no absorption peaks were observed for TMB at 652 nm, OPD at 450 nm, and ABTS at 420 nm. Figure 3 Curve A, b Figure 3 curve B and b Figure 3 C curve b; When H2O2 is introduced, the absorbance of the three substrates TMB, OPD, and ABTS increases significantly after oxidation, such as... Figure 3 A, Figure 3 B and Figure 3 Curve c in C indicates that ZIF-67 itself does not have oxidase-mimicking activity, but it does have some weak peroxidase-mimicking activity. When NiCo-LDH is used as the oxidase mimic, in the absence of H2O2, TMB shows strong absorption peaks at 652 nm, OPD at 450 nm, and ABTS at 420 nm. Figure 3 A, Figure 3 B and Figure 3 Curve d in C; when H2O2 is introduced, TMB exhibits highly efficient peroxidase-mimicking activity at 652 nm, OPD at 450 nm, and ABTS at 420 nm, such as Figure 3 A, Figure 3 B and Figure 3 Curve e in C.

[0023] To further verify whether the reaction between NiCo-LDH and H2O2 produces reactive oxygen species (ROS), the electron paramagnetic resonance (EPR) spectra of hydroxyl radicals (-OH) were measured under conditions with and without hydrogen peroxide. Figure 4 As shown, after NiCo-LDH reacts with H2O2, a significant -OH group is generated.

[0024] The above verification process shows that the significant absorption at 652 nm, 450 nm and 420 nm in the TMB, OPD and ABTS system is because the -OH generated after NiCo-LDH reacts with H2O2 oxidizes TMB, OPD and ABTS to form oxidized oxTMB, oxOPD and oxABTS.

[0025] In this embodiment, an array sensor is used to identify five flavonoid antioxidants. Each flavonoid compound exhibits different inhibitory effects due to differences in its chemical structure, resulting in different response patterns on the array sensor. These patterns can be visually displayed through fingerprint spectra, thermograms, spatial 3D maps, and array photographs. Seventy-five data points (3 color channels × 5 flavonoid antioxidants × 5 replicates) were collected and integrated using linear discriminant analysis to identify the five flavonoid compounds.

[0026] In the detection system of this embodiment, after adding five flavonoid antioxidants (EC, KF, HP, NG, and QC), the absorbance of the sensor unit decreased, showing different absorption response modes, such as... Figure 5 A, simultaneously, heatmaps were generated for responses to five flavonoid antioxidants, such as Figure 5 B, and a three-dimensional spatial diagram, such as Figure 5 C and Figure 5 D. The results showed that even at low concentrations (200 nM), the array sensor could still distinguish different flavonoid antioxidants. Therefore, linear discriminant analysis (LDA) was used to analyze three factors: substrate type, flavonoid antioxidant type, and absorbance. These three discriminant factors were then used to transform the training data matrix into a visualized three-dimensional typical score map. From Figure 5 C and Figure 5 As can be seen from D, each flavonoid antioxidant formed an independent cluster in the three-dimensional typical scoring map at concentrations of 200 nM and 50 μM, respectively. The array image is shown below. Figure 6 The results show that each individual flavonoid antioxidant corresponds to a different colorimetric result, indicating that the array sensor has good recognition capabilities.

[0027] To further verify the array sensor's recognition capability in complex systems, a mixture of binary flavonoid antioxidants with a total concentration of 10 μM was used in mixing ratios of 5:5 and 7:3. The binary mixtures achieved complete separation in a three-dimensional spatial distribution map, as shown below. Figure 7 As shown, the array sensor can effectively distinguish the oxidative properties of different ratios of flavonoid antioxidant combinations, including but not limited to KF:HP, KF:NG, KF:EC, and KF:QC combinations. Furthermore, Figure 8 The array sensor colorimetric response map of the binary mixture is shown, and obvious color differences can be observed with the naked eye, further verifying the distinguishing ability of the array sensor.

Claims

1. A colorimetric array sensing analysis method for identifying flavonoid antioxidants, used to identify the antioxidant properties of flavonoids, characterized in that... This method is based on NiCo-LDH and introduces H2O2 to construct a colorimetric array sensor with three different chromogenic substrates and five flavonoid antioxidants; The three different chromogenic substrates are: 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) ammonium salt (ABTS), 3,3',5,5'-tetramethylbenzidine (TMB), and o-phenylenediamine (…). o -Phenylenediamine (OPD); The five flavonoid antioxidants mentioned are: epicatechin (EC), kaempferol (KF), hesperidin (HP), naringenin (NG), and quercetin (QC).

2. The colorimetric array sensing and analysis method for identifying flavonoid antioxidants as described in claim 1, characterized in that... The use of a colorimetric array sensor involves dropping NiCo-LDH, H2O2, and the target flavonoid antioxidant into three different chromogenic substrates: TMB, OPD, and ABTS. The oxidation performance is then measured using a UV-Vis spectrophotometer, or compared with a blank array sensor to identify its oxidation performance.

3. The colorimetric array sensing analysis method for identifying flavonoid antioxidants as described in claim 1, characterized in that... The concentration of the target flavonoid antioxidant is at least 200 nM.

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