Integrated optical and electrochemical techniques for determining total antioxidant capacity: methodology and instrumentation design

Abstract

In this invention Total Antioxidant Capacity (TAC) is determined by a combined Spectrophotometric and Electrochemical method. A reversible spectroelectrochemical sensor consist of a covalently immobilized organic ligand to indium tin oxide (ITO) coated glass, for the binding of metal cations. A metal ion binding to an organic ligand and an auxiliary ligand gives a colored complex whose color depends on the oxidation state of the metal ion. Due to the antioxidative action of the biological sample, the metal ion is reduced in proportion to the TAC of the biological sample. The change in absorption of a thin layer on ITO glass is measured with an optical detector, based on the principle of total internal reflection. The sensor is returned to its oxidized state using electrochemical oxidation. Measuring the current required for the oxidation of reduced ions, an electrochemical signal proportional to the TAC of sample is obtained.

Description

[0001] "Integrated Optical and Electrochemical Techniques for Determining Total Antioxidant Capacity: Methodology and Instrumentation Design"

[0002] Description

[0003] Technical field to which the invention relates:

[0004] The invention relates to the field of instrumental analytical chemistry

[0005] GOIN 27 / 26 by investigating electrochemical variables; by using electrolysis or electrophoresis [2006.01]

[0006] G01N21 / 3151 Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry with comparison of measurements at specific and non-specific wavelengths using two sources of radiation of different wavelengths

[0007] GOIN 21 / 552 Attenuated total reflection [2014.01]

[0008] GOIN 21 / 47Scattering, i.e. diffuse reflection (GOIN 21 / 25, GOIN 21 / 41 take precedence) [2006.01]

[0009] Background:

[0010] The concept of total antioxidant capacity (TAC) has been used since early discovery of role of reactive oxygen species and antioxidants in living organisms.

[0011] The total antioxidant capacity (Total Antioxidant Capacity TAC) of a biological sample refers to the total capacity of that sample to neutralize or remove reactive oxygen species (ROS) and other free radicals. It is a measure of the cumulative antioxidant activity present in the sample.

[0012] TAC is determined by exposing a biological sample to a known oxidizing agent or free radical generator and then measuring the ability of the sample to neutralize or neutralize the oxidative effects of these agents. Different chemical sensors or indicators are used for its determination. TAC provides valuable information about the overall antioxidant property of a biological system, which is important for understanding its susceptibility to oxidative stress and its potential to mitigate oxidative damage. It is often determined in scientific research to evaluate the antioxidant properties of food and beverages, nutritional supplements, food products and medicines. It is also used in clinical trials, although its diagnostic role for certain diseases is limited and depends on the context. It is used as an indicator of metabolic disorders, inflammatory processes, cardiovascular diseases, and certain tumor diseases. It is also used as an indirect indicator of the effectiveness of therapy in the mentioned diseases or an indicator in determining the progress or stage of the disease.

[0013] Basic methods for determination of TAC can be divided in 4 main groups:

[0014] 1. Chemical Assays: These assays measure the ability of antioxidants to scavenge specific chemical reactive species that include: a. Ferric Reducing Ability of Plasma (FRAP): Measures the reduction of ferric ions to ferrous ions by antioxidants. b. 2,2-Diphenyl-l-picrylhydrazyl (DPPH) Assay: Measures the scavenging activity of antioxidants against the stable free radical DPPH. c. Trolox Equivalent Antioxidant Capacity (TEAC) Assay: Measures the ability of antioxidants to scavenge the radical cation ABTS+.

[0015] 2. Electrochemical Methods: Electrochemical techniques such as cyclic voltammetry and amperometry can be employed to directly measure the redox behavior of antioxidants directly. 3. Enzymatic Assays: They utilize specific enzymes to measure the total antioxidant capacity indirectly like Superoxide dismutase (SOD) or catalase.

[0016] 4. Instrumental methods: a. High-Performance Liquid Chromatography (HPLC): Coupled with various detection methods (e.g., UV-Vis, fluorescence), HPLC can separate and quantify individual antioxidants or measure the total antioxidant capacity in biological samples. b. Mass Spectrometry (MS): MS techniques can be used for the identification and quantification of antioxidants in biological samples.

[0017] The work of Helmut Sies1summarizes the advantages and limitations of TAC concept and methods for its determination. The complexity of biological systems and relations of nutritioning to the health of living organisms inspire the development of new methods to determine and evaluate TAC.

[0018] Current methods involve application of significant amounts of chemicals and solvents, which do not consent with requirements of environmental protection. This invention addresses this issue by developing a reversible sensor for multiple measurements of TAC.

[0019] In this invention TAC is determined by a combined Spectrophotometric and Electrochemical method. For this purpose, a spectroelectrochemical sensor was made consisting of a covalently immobilized organic ligand for the coordination binding of metal cations to glass coated with indium tin oxide (ITO). A metal ion binding to an organic ligand and an auxiliary ligand gives a colored complex whose color depends on the oxidation state of the metal ion. Due to the action of the antioxidative property of the biological sample, the metal ion is reduced in proportion to the TAC of the biological sample. The change in absorption of a thin layer on ITO glass is measured with an optical detector, based on the principle of total internal reflection. The sensor is returned to its original oxidized state using electrochemical oxidation. By measuring the current required for the oxidation of reduced ions, an electrochemical signal proportional to the TAC of the biological sample is obtained. In this way, a sensor with reversible operation and determination of TAC with two analytical methods and reduced measurement error was designed.

[0020] Current invention involves the development of an instrument with a reversible sensor that utilizes both electrochemical and optical methods that can offer several benefits in the measurement of TAC. Researchers can choose the most suitable detection mode based on the specific requirements of their experiments or applications, leading to more comprehensive and informative analyses. Reversible sensors enable real-time monitoring of changes in analyte concentrations, providing dynamic information about redox processes and antioxidant activity over time. This capability is particularly valuable for studying kinetic reactions or dynamic processes in biological systems. An instrument with a reversible sensor capable of both electrochemical and optical measurements can serve multiple purposes beyond TAC determination. It can be adapted for the analysis of various analytes, including different antioxidants, biomolecules, or environmental pollutants, making it a versatile tool for research and analysis in diverse fields.

[0021] Integration of electrochemical and optical detection systems into a single instrument can result in a compact and portable design, suitable for on-site or point-of-care applications. This can facilitate rapid and convenient TAC measurements in clinical settings, field research, or remote locations where access to laboratory facilities may be limited.

[0022] 1. https: / / doi.Org / 10.1093 / in / 137.6.1493 Description of images

[0023] Figure 1. Schematic presentation of chemical sensor.

[0024] Figure 2. Schematic presentation of measurement setup.

[0025] Figure 3. Technical drawing of measurement cell parts.

[0026] Figure 4. Assembly of measurement cell.

[0027] Disclosure of the invention

[0028] 1.1. Measurement methods:

[0029] To determine the total antioxidant capacity, we use methods based on one electron transfer and include modified methods for determining the reductive capacity of Fe3+, Cu2+, Ce4+and Mo6+ions in their stable complexes. The modified methods are: Ferric Reducing Antioxidant Power (FRAP), Cupric Reducing Antioxidant Power (CUPRAC), Ce(IV)-based reducing capacity (CERAC) assay, Folin— Ciocalteu test.

[0030] The modification of the method refers to the immobilization of the ligand on the surface of indium tin oxide glass (Indium Tin Oxide ITO) (Figure 1. 2.) on glass support (Figure 1. 3.) of high electrical conductivity. The ligand for the complexation of the mentioned metal ions is covalently immobilized on the ITO glass surface, followed by the complexation of the metal ions from their water-soluble salts and the addition of an auxiliary ligand.

[0031] This method prepares a two-dimensional colored sensor layer (Figure 1. 1. and Figure 2.7.). The reductive action of the antioxidants from the biological sample reduces the metal ions in the immobilized complex while changing the optical properties. The change in optical properties is proportional to the amount of antioxidants in the biological sample. The reduced metal ions in the immobilized complex are oxidized by electrochemical oxidation, and return to the original oxidized state and are ready for the measurement of the next sample. The amount of charge required for the oxidation of reduced metal ions in the complex is proportional to the amount of reduced metal ions in the immobilized complex. In this way, we get information about the amount of antioxidants in a biological sample with two methods, which reduces the measurement error of the total antioxidant capacity.

[0032] 1.2. Ligand immobilization and sensor layer synthesis:

[0033] 1.2.1. Immobilization of the sensor complex on the ITO glass surface is done in such a way that the ITO glass surface is cleaned and activated with oxygen plasma. The activated ITO glass is immediately placed from the plasma generator into a container saturated with allylamine vapors. In this way, an ITO surface covered with a covalently bound thin organic layer and primary amine functional groups is obtained .

[0034] 1.2.2. In the next step, we covalently attach a metal ion complexing ligand containing at least one carboxyl functional group to form an amide bond to the amine functional groups.

[0035] Ligand can be:

[0036] 1. 2,4,6-tri-2-pyridinyl-l,3,5-triazine-4-carboxylic acid 2. 2,2'-Bipyridine-4,4'-dicarboxylic acid

[0037] 3. 2,2'-Bipyridine-4-carboxylic acid

[0038] 4. 2,2'-Bipyridine-5-carboxylic acid

[0039] 5. 2,2'-Bi pyridine-5,5'-dica rboxylic acid

[0040] 6. l,10-Phenanthroline-2-Carboxylic Acid

[0041] 7. l,10-Phenanthroline-4,7-dicarboxylic acid

[0042] 8. l,10-phenanthroline-5,6-dicarboxylic Acid

[0043] 9. l,10-phenanthroline-2,9-dicarboxylic

[0044] 10. 4,7-diphenyl-l,10-phenanthroline-5,6-dicarboxylic acid

[0045] 11. 4,7-diphenyl-l,10-phenanthroline-2,9-dicarboxylic acid

[0046] 12. 4,7-diphenyl-l,10-phenanthroline-5,6-dicarboxaldehyde

[0047] 13. 4,7-diphenyl-l,10-phenanthroline-2,9-dicarboxaldehyde

[0048] 14. 2,2'-Biquinoline-4-carboxylic acid

[0049] 15. 2,2'-Biquinoline-6-carboxylic acid

[0050] 16. 2,2'-Biquinoline-3,3'-dicarboxylic acid

[0051] 17. 2,2 -Biquinoline-4,4 -dicarboxylic acid

[0052] 18. 2,2'-Biquinoline-5,5'-dicarboxylic acid

[0053] 19. 2,2'-Biquinoline-3,3'-dicarboxaldehyde

[0054] 20. 2,2'-Biquinoline-5,5'-dicarboxaldehyde

[0055] 1.2.3. Binding of one of the metal ions Fe3+, Cu2+, Ce4+and Mo6+to the immobilizer ligand is done from a solution of their salts in such a way that the ITO glass from step 2.2.2. is placed in the solution of their salts for a certain time at room or elevated temperature.

[0056] 1.2.4. In the last step of preparing the sensor layer, we add an auxiliary ligand. The auxiliary ligand is added in such a way that the ITO glass from step 2.2.3. is placed in the auxiliary ligand solution for a certain time at room or elevated temperature.

[0057] Auxiliary ligand can be:

[0058] 1. 2,4,6-tris-2-pyridinyl-l,3,5-triazine

[0059] 2. 2,2'-bipyridyl

[0060] 3. 1,10-phenantroline

[0061] 4. 4,7-diphenyl-l,10-phenanthroline

[0062] 5. 2,2'-biquinoline

[0063] 6. KCN 7. ortophosphate

[0064] 1.3 Spectroelectrochemical sensor

[0065] The ITO layer on the sensor has the function of a working electrode (Figure 2. 5.) with electric contact (Figure 2. 6.) and conector (Figure 2. 9.) for voltammetric, chronoamperometric, or columbmetric measurement in a three-electrode system (Figure 2.). A commercially available graphite conductive paste is used as an counter electrode (Figure 2. 12. ) and conector ((Figure 2. 14.), and a commercially available silver epoxy resin with the addition of silver chloride is used as a reference electrode (Figure 2. 13.). The electrode system is prepared using the screen printing technique on glass.

[0066] 1.4. Instrument:

[0067] 1.4.1. Measurment cell

[0068] The measuring cell consists of:

[0069] 1. Measuring head (Figure 3.1) holding two optical detectors (Figure 2. 2. and Figure 2. 3.) and a light source (Figure 2. 1.) with a special geometric arrangement.

[0070] 2. Integrated spectroelectrochemical sensor holder with silicone gasket (Figure 2.8) forming a sample space (Figure 2. 16.) with one sample inlet (Figure 2.10.) and one sample outlet (Figure 2.11.) and mechanics for easy replacement of the spectroelectrochemical sensor consisting of main body of sensor holder . (Figure 3. 2.) and flow cell (Figure 3. 3.)

[0071] 3. Meassurement cell is assambled according Figure 4.

[0072] 1.4.2. Instrument

[0073] The miniaturized device will consist of an electrochemical detection part and an optical detection part controlled by a PLC.

[0074] 1. The electrochemical detection part consists of high precision low voltage analog front ends (AFE) such as AD5940, AD5941 or equivalent AFE.

[0075] 2. For optical detection, Ampcon, Voltcon or Multiboard converters and signal amplifiers are used in combination with spectrally selective and avalanche photodiodes and optical guides. For the light source, RGB LEDs with four poles and appropriate spectral properties are used, such as (Electrospell Spectrafill LEDs )

Claims

Claims1. A method for determining the total antioxidant capacity (TAC) of a biological sample using one-electron transfer-based methods with reversible spectroelectrochemical sensor, comprising: a) Modifying the surface of indium tin oxide glass (ITO) of high electrical conductivity; with oxygenplasma activation and vapours of allyamine. b) Covalently attaching metal ion complexing ligands containing carboxyl or aldehyde functional groups to the ITO surface immobilized amine groups c) Complexing metal ions from solution of their salts with the immobilized ligands d) Adding auxiliary ligands to the sensor layer e) Measuring the change in optical properties of the sensor layer upon reduction of metal ions by antioxidants in the biological sample f) Oxidizing reduced metal ions by electrochemical oxidation to return them to the original oxidized state.

2. The method of claim 1, wherein the metal ions include Fe3+, Cu2+, Ce4+or Mo6+3. A spectroelectrochemical sensor for determining the total antioxidant capacity (TAC) of a biological sample, comprising: a) a glass substrate coated with indium tin oxide (ITO) b) ligands covalently immobilized on the ITO-coated glass substrate, wherein the ligands include metal ion complexing ligands and auxiliary ligands c) wherein the sensor layer undergoes changes in optical properties upon reduction of metal ions by antioxidants in the biological sample. d) wherein the reduced metal ions are oxidized by electrochemical oxidation providing an electrochemical analytical signal and reversible operation of sensor4. The sensor of claim 3, wherein the covalently immobilized metal ion complexing ligands include ligands selected from the group consisting of 2,2'-Bipyridine-4,4'-dicarboxylic acid; 2,2'-Bipyridine-4-carboxylic acid; 2,2'-Bipyridine-5-carboxylic acid; 2,2'-Bipyridine-5,5'- dicarboxylic acid; l,10-Phenanthroline-2-Carboxylic Acid; l,10-Phenanthroline-4,7- dicarboxylic acid; l,10-phenanthroline-5,6-dicarboxylic Acid; 10-phenanthroline-2,9- dicarboxylic; 4,7-diphenyl-l,10-phenanthroline-5,6-dicarboxylic acid; 4,7-diphenyl-l,10- phenanthroline-2,9-dicarboxylic acid; 4,7-diphenyl-l,10-phenanthroline-5,6- dicarboxaldehyde; 4,7-diphenyl-l,10-phenanthroline-2,9-dicarboxaldehyde; 2'-Biquinoline- 4-carboxylic acid; 2,2'-Biquinoline-6-carboxylic acid; 2,2'-Biquinoline-3,3'-dicarboxylic acid;2,2 -Biquinoline-4,4 -dicarboxylic acid; 2,2'-Biquinoline-5,5'-dicarboxylic acid; ,2'-Biquinoline- 3,3'-dicarboxaldehyde; 2,2'-Biquinoline-5,5'-dicarboxaldehyde5. The sensor of claim 3, wherein the auxiliary ligand or ligands is or are selected from the group consisting of 2,4,6-tris-2-pyridinyl-l,3,5-triazine; 2,2'-bi pyridyl; 1,10-phenantroline; 4,7-diphenyl-l,10-phenanthroline; 2,2'-biquinoline, CN’, ortophosphate.

6. The sensor of claim 3, further comprising an ITO layer serving as a working electrode for voltammetric, chronoamperometric, or columbmetric measurements.

7. A method for synthesizing a sensor for determining the total antioxidant capacity (TAC) of a biological sample, comprising: a) Cleaning and activating an ITO glass surface with oxygen plasma; b) Immobilizing ligands on the activated ITO surface; c) Covalently attaching metal ion complexing ligands to the immobilized ligands; d) Complexing metal ions from their water-soluble salts with the immobilized ligands; e) Adding auxiliary ligands to the sensor layer.

8. The method of claim 7, wherein the ligands are immobilized using allylamine vapors.

9. The method of claim 7, wherein the metal ion complexing ligands are attached to the immobilized ligands through amide bonds.

10. The method of claim 7, wherein the metal ions are complexed with the immobilized ligands from solutions of their salts.

11. A measuring cell for determining the total antioxidant capacity (TAC) of a biological sample, comprising: a) a measuring head with optical detectors and a light source; b) an integrated spectroelectrochemical sensor holder with a flow cell; c) wherein the measuring cell facilitates easy replacement of the spectroelectrochemical sensor.

12. The measuring cell of claim 11, wherein the optical detectors and light source have a special geometric setup.

13. An instrument for determining the total antioxidant capacity (TAC) of a biological sample, comprising: a) an electrochemical detection part comprising high precision low voltage analog front ends (AFE); b) an optical detection part comprising converters, signal amplifiers, and avalanche photodiodes;14. The instrument of claim 13, wherein the optical detection part uses RGB LEDs with appropriate spectral properties as the light source.

Images