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Title:
SENSOR FOR MEASURING AMOUNTS OF VITAMIN C AND D
Document Type and Number:
WIPO Patent Application WO/2022/207693
Kind Code:
A1
Abstract:
The present invention relates to the measurement of vitamin C and/or D in an aqueous medium, particularly saliva using a sensor. This allows a quick, easy and cost-efficient possibility to determine the amount of vitamin C and/or D in an aqueous medium, such as human saliva, by a non-invasive way method. A fast measurement of vitamin C and/or vitamin D allows an easy detection of a need of supply in vitamin C and/or vitamin D of an individuum, and, hence, a personalized dietary supplementation of vitamin C and/or D is possible. The new sensor is produced in an easy, fast and cost-efficient way, allowing that the sensor can be broadly used for single-use allowing fast and broad testing of the vitamin C and/or D status in a large population and allowing a personalized supplementation of vitamin C and/or D of individuals in need. As vitamin C and D have a big positive impact in the body's immunity, this is particularly importance in situations of presence of pathogens or pandemia like the current COVID-19 pandemia.

Inventors:
BAILEY EILEEN MARIE (CH)
DUESTERLOH ANDRE (CH)
MAY JENNIFER YVONNE (CH)
RUIZ VALDEPEÑAS MONTIEL VICTOR (US)
SEMPIONATTO MORETO JULIANE R (US)
VARGAS ORGAZ EVA (US)
WANG JOSEPH (US)
Application Number:
PCT/EP2022/058390
Publication Date:
October 06, 2022
Filing Date:
March 30, 2022
Export Citation:
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Assignee:
DSM IP ASSETS BV (NL)
International Classes:
G01N33/82; C12Q1/00
Domestic Patent References:
WO2001038873A22001-05-31
WO2020186118A12020-09-17
WO2014025430A22014-02-13
WO2022170361A12022-08-11
Other References:
CHAUHAN DEEPIKA ET AL: "An efficient electrochemical biosensor for Vitamin-D3 detection based on aspartic acid functionalized gadolinium oxide nanorods", JOURNAL OF MATERIALS RESEARCH AND TECHNOLOGY, vol. 8, no. 6, 1 November 2019 (2019-11-01), BR, pages 5490 - 5503, XP055840436, ISSN: 2238-7854, DOI: 10.1016/j.jmrt.2019.09.017
CARLUCCI LUCIANO ET AL: "Several approaches for vitamin D determination by surface plasmon resonance and electrochemical affinity biosensors", BIOSENSORS AND BIOELECTRONICS, vol. 40, no. 1, 1 February 2013 (2013-02-01), Amsterdam , NL, pages 350 - 355, XP055840469, ISSN: 0956-5663, DOI: 10.1016/j.bios.2012.07.077
TAMAL SARKAR ET AL: "Studies on carbon-quantum-dot-embedded iron oxide nanoparticles and their electrochemical response", NANOTECHNOLOGY, INSTITUTE OF PHYSICS PUBLISHING, GB, vol. 31, no. 35, 15 June 2020 (2020-06-15), pages 355502, XP020355915, ISSN: 0957-4484, [retrieved on 20200615], DOI: 10.1088/1361-6528/AB925E
SARKAR TAMAL ET AL: "Carbon dots-modified chitosan based electrochemical biosensing platform for detection of vitamin D", INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES, ELSEVIER BV, NL, vol. 109, 21 December 2017 (2017-12-21), pages 687 - 697, XP085346929, ISSN: 0141-8130, DOI: 10.1016/J.IJBIOMAC.2017.12.122
KULYS JUOZAS ET AL: "Electrocatalytic Oxidation of Ascorbic Acid at Chemically Modified Electrodes", 1 January 1991 (1991-01-01), pages 209 - 214, XP055840546, Retrieved from the Internet [retrieved on 20210913]
ENSAFI A A ET AL: "A differential pulse voltammetric method for simultaneous determination of ascorbic acid, dopamine, and uric acid using poly (3-(5-chloro-2-hydroxyphenylazo)-4,5-dihydroxynaphthalene-2,7-disulfonic acid) film modified glassy carbon electrode", JOURNAL OF ELECTROANALYTICAL CHEMISTRY AND INTERFACIAL ELECTROCHEMISTRY, ELSEVIER, AMSTERDAM, NL, vol. 633, no. 1, 1 August 2009 (2009-08-01), pages 212 - 220, XP026349163, ISSN: 0022-0728, [retrieved on 20090606]
PADAYATTY, S.J. ET AL., J AM COLL NUTR, vol. 22, no. 1, 2003, pages 18 - 35
FEYAERTS, A.F.LUYTEN, W., NUTRITION, vol. 79, no. 80, 2020, pages 110948
BAE, M.KIM, H., MOLECULES, vol. 25, 2020, pages 5346
SEMPIONATTO, J.R. ET AL., ACS SENS., vol. 5, no. 6, 2020, pages 1804 - 1813
KAUR, A. ET AL., J ELECTROANAL CHEM, vol. 873, 2020, pages 114400
Attorney, Agent or Firm:
DUX, Roland (CH)
Download PDF:
Claims:
Claims

1. A electrode (1 ) for measuring vitamin D in an aqueous medium wherein said electrode comprises an electroconductive electrode (2) and a layer of vitamin D specific antibody (3) on the electroconductive electrode.

2. A electrode (1') for measuring vitamin C in an aqueous medium wherein said electrode comprises an electroconductive electrode (2') and a layer (4) comprising a catalyst for the oxidation of vitamin C, particularly tetrathiafulvalene.

3. The electrode according to claim 2, characterized in that the layer (4) comprising a catalyst for the oxidation of vitamin C, further comprises a polymer, preferably a polymer which is a reaction product of a polyamine and a polyaldehyde, particularly of chitosan and glutaraldehyde.

4. A sensor (10) for determining the amount of vitamin C and vitamin D in an aqueous medium, wherein said sensor comprises

- a first working electrode (WE1 ) which is an electrode (1 ) for measuring vitamin D according to claim 1 ;

- a second working electrode (WE2) which is an electrode (T) for measuring vitamin C according to claim 2 or 3; and

- a counter electrode (11 ).

5. The sensor according to claim 4, characterized in that the first electrode (1 ) and the second electrode (T) and the counter electrode (11) are localized on a non-conductive support (12).

6. The sensor according to claim 5, characterized in that the counter electrode is an Ag/AgCI electrode.

7. The electrode for measuring vitamin C or the electrode for measuring vitamin D or the sensor according to any of the preceding claims characterized in that the aqueous medium is saliva. 8. A method of determining the concentration of vitamin C and vitamin D in an aqueous medium (20) comprising the following steps: a) providing a sensor (10) according to any of the preceding claims 4-7 ; b) admixing horseradish peroxidase-conjugated vitamin D (19) to the aqueous medium (20) to be tested to yield a contact solution (21); c) contacting at least the sensing area (5,5') of the working electrodes (1,1') and the counter electrode (11 ) of the sensor with the contact solution (21); d) measuring the cathodic current at the electrode for measuring vitamin C (1 ') by an amperometer (22) and counter electrode (11 ) to determine the concentration of vitamin C; e) removing the contact solution (21 ); f) providing a redox solution (23) of H2O2 and a mediator, preferably 3,3',5,5'-tetramethylbenzidine; g) contacting at least the sensing area (5) of the working electrode for vitamin D (1 ) and the counter electrode (11 ) of the sensor with the redox solution (23); h) measuring the cathodic current at the electrode for measuring vitamin D (1 ) by an amperometer and counter electrode (11 ) to determine the concentration of vitamin D.

9. A non-invasive method of determining the amount of vitamin C and vitamin D to be provided to an animal, particularly a human, comprising the steps in an aqueous medium comprising the following steps: a) providing a sensor (10) according to any of the preceding claims 4-7; b) admixing horseradish peroxidase-conjugated vitamin D to the aqueous medium (20) to be tested to yield a contact solution (21); c) contacting at least the sensing area (5,5') of the working electrodes (1,1') and the counter electrode (11 ) of the sensor (10) with the contact solution (21); d) measuring the cathodic current at the electrode for measuring vitamin C (T) by an amperometer (22) and electrode (11) to determine the concentration of vitamin C; e) removing the contact solution (21 ); f) providing a redox solution (23) of FI2O2 and a mediator, preferably 3,3',5,5'-tetramethylbenzidine; g) contacting at least the sensing area (5) of the working electrode for vitamin D (1 ) and the counter electrode (11 ) of the sensor with the redox solution (23); h) measuring the cathodic current at the electrode for measuring vitamin D (1 ) by an amperometer (22) and electrode (11 ) to determine the concentration of vitamin D; i) comparing the measured values (45) of vitamin C and vitamin D with the daily recommend dosage of vitamin C and D obtainable from a data source (46) to determine a need in additional supply of vitamin C and/or D; j) calculating the needed amount of additional supply of vitamin C, respectively vitamin D; k) suggesting an suggested amount (48) of additional supply of vitamin C respectively vitamin D, to be administrated to said animal, particularly human, in case that a need is calculated in step g).

10. The non-invasive method according to claim 9 characterized in that characterized in that that the recommended dosage of vitamin C and/or D for humans are based on the recommendation daily dosage listed particularly as nutrient reference values (NRV) in annex XIII of the EU regulation no. 1169/2011 or as daily values by the FDA (U.S. Food and Drug Administration) as of 05/05/2020.

11. The non-invasive method according to any of the preceding claims 9 or 10, characterized in that the aqueous medium (7) originating from said animal, particularly human, is salvia.

12. Use of a sensor (10) according to any of the preceding claims 4-7 for determining the composition of a personalized dietary supplement, wherein said personalized dietary supplement comprises vitamin C and/or vitamin D. 13. Kit (40) comprising a sensor (10) according to any of the preceding claims 4-7 ; and a container (41 ) comprising at least horseradish peroxidase-conjugated vitamin D (HRP-VitD); and a container (42) comprising at least H2O2.

14. Process of manufacturing of a personalized dietary supplement (44) comprising the following subsequent steps a) providing a sensor (10) according to any of the preceding claims 4-7 to a consumer; b) Receiving data (45) from said consumer, wherein said data has been provided by the sensor of step a); g) Determining the need of vitamin C and D of the consumer of step a) using the data of step b); d) providing a personalized dietary supplement (44) comprising vitamin C and/or vitamin D in an amount based on the need as determined in step g).

15. Process of manufacturing a sensor (10) according to any of the preceding claims 4-7 comprising the following subsequent steps i) forming a layer of an electroconductive material on a non-conductive support (12) to produce a first electroconductive electrode (2) and a second electroconductive electrode (2'); ii) forming a counter electrode (11 ); iii) formation of a layer (3) of vitamin D specific antibody on an area of the first electroconductive electrode (2); iv) formation of a layer (4) comprising a catalyst for the oxidation of vitamin C, preferably tetrathiafulvalene, on the area of the second electroconductive electrode (2').

16. Process according to claim 16, characterized in that the electroconductive material of the first electroconductive electrode (2) and a second electroconductive electrode (2') is gold and that the process comprises furthermore the step ia) applying at least one mercaptan on the surface onto said gold layer, preferably forming a self-assembly monolayer of mercaptan on gold; wherein step ia) is performed after step i) or ii), preferably after step ii), and before step iii).

Description:
SENSOR FOR MEASURING AMOUNTS OF VITAMIN C AND D

Technical Field

The present invention relates to the fields of biosensors, particularly sensors for determining the concentrations of vitamin C and/or D in an aqueous medium.

Background of the invention

Vitamin C and D are extremely important vitamins for the health of humans and animals. Particular recent attention has been to the major benefits of vitamins C and D towards improving the immune system response toward preventing COVID-19 or slowing its progression, since these vitamins are involved in regulating the immune system function and in reducing viral replication.

Vitamin C is the most commonly vitamin associated with prevention and alleviation of viral infections as disclosed by Padayatty, S.J. et al. , J Am Coll Nutr 22:1, 18-35 (2003).

Feyaerts, A.F., Luyten, W., Nutrition 79, 80, 110948 (2020) discloses that vitamin C can reduce the inflammatory mediator cascade manifested during COVID-19 infection. Furthermore, vitamin D has gained a considerable recent attention, e.g.

Bae, M., Kim, H., Molecules 25, 5346 (2020), becoming a well-established key player in the function of the immune system by improving the physical barrier against viruses and stimulating the production of antimicrobial peptides.

Accordingly, there are urgent demands for effective decentralized self- testing multi-vitamin sensing platform that monitors the levels of these important immune-supporting micronutrients.

Personalized nutrition aims at the assessment of individual nutritional status regarding intake or metabolism of dietary constituents and reclaims tight nutrition-related biomarker monitoring in body fluids as essential to get direct feedback and guidance for supporting dietary behavior change. New approaches of portable, low-cost and easy-to-use bioelectronic sensors enabling on-the-spot multiplexed measurements of fluctuating biochemical markers in bodily fluids would provide comprehensive molecular information supporting personalized nutrition and care. Sempionatto, J.R. et al. ACS Sens. 5, 6, 1804-1813 (2020) discloses an epidermal biosensor by which vitamin C can be detected by immobilization of the enzyme ascorbate oxidase onto said electrode.

Kaur, A. et al. J Electroanal Chem 873, 114400 (2020) discloses a gold-platinum bimetallic nanoparticles coated 3-aminopropyltriethoxysilane based electrochemical immunosensor for vitamin D estimation.

These sensors, respectively their manufacturing process, however, are rather complex.

Therefore, there still is a great need for easy produced and cost-efficient electrodes and sensors to measure reliably and selectively the amounts of vitamin C and/or D in aqueous media.

Summary of the invention

Therefore, the problem to be solved by the present invention is to offer a sensor for measuring vitamin C and/or D concentrations in an aqueous medium, particularly saliva, which can be produced in a cost-efficient and easy way and offer an easy and reproducible measurement of vitamin C and D.

Surprisingly, it has been found that the electrodes of claim 1 or 2, respect ively the sensor of claim 3 , are able to solve said problem.

Particularly, it has been found that this allows electrocatalytic ampero- metric detection of vitamin C along with competitive enzyme-based immunoassay of detection of vitamin D, to perform simultaneous electrochemical detection of both vitamins in the same microliter saliva sample within a time frame of about 25 min. This sensor for vitamin C and D offers a highly selective measurements of vitamin C and D without cross-talks between the adjacent electrocatalytic and bioaffinity sensors.

It has been found that these vitamins can be measured in saliva of multiple subjects after ingestion of commercial vitamins supplements and fortified milk. The distinct profiles of vitamin C and D of the different individuals indicate the potential of the bioelectronic sensor strategy for enhancing personalized nutrition and guiding dietary changes towards fortifying the immune system.

It has been further found that particularly the electrocatalytic vitamin C detection relies on the direct oxidation of vitamin C using catalyst for the oxidation of vitamin C, particularly tetrathiafulvalene (TTF), with the chronoamperometric response is directly proportional to the vitamin C concentration, while vitamin D detection is based on a horseradish peroxidase (HRP)-tagged competitive format immunoassay employing a redox solution of H2O2 and a mediator, particularly 3,3',5,5'-tetramethylbenzidine (TMB), where the cathodic current corresponds to the HRP-catalyzed reduction of H2O2 and it is inversely proportional to the Vitamin D concentration. Systematic optimization and rational design of the sensor and its operational conditions resulted in a reliable simultaneous detection of vitamin C and D in a single aqueous medium, preferably saliva, sample within typically 25 minutes, with no apparent cross-talks between the neighboring electrocatalytic and bioaffinity sensors. Particularly, the resulting dual bioelectronic sensor have been successfully applied for the monitoring of physiological temporal profile of vitamin C and D after administration of dietary supplements or fortified foods directly in undiluted saliva. The developed decentralized dual sensor paves the way towards an effective personalized nutrition enabling enhanced prevention and recovery of infectious diseases.

Further aspects of the invention are subject of further independent claims. Particularly preferred embodiments are subject of dependent claims.

Detailed description of the invention

Electrode for measuring vitamin D

In a first aspect, the present invention relates to an electrode 1 for measuring vitamin D in an aqueous medium wherein said electrode comprises an electroconductive electrode 2 and a layer of vitamin D specific antibody 3 on the electroconductive electrode.

The electrode for measuring vitamin D comprises an electroconductive electrode. Said electroconductive electrode is made of an electroconductive material. Particularly suitable electroconductive materials are chromium, silver and gold. It is also possible, and preferred, that the electroconductive electrode has several layers of different electroconductive materials. The electroconductive material is preferably applied on a non-conductive, i.e. electro-non-conductive, support material, particularly glass or a polymer, particularly a polymer selected from the group consisting of polystyrene (PS), polymethyl(meth)acrylate (PMMA), polycarbonate (PC), polyethylenterephthalate (PET), glycol modified polyethylene- terephthalate (PETG), acrylonitrile/butadiene/styrene copolymer (ABS), polyvinyl chloride (PVC), polylactic acid (PLA), polyethylene (PE) and polypropylene (PP), preferably selected from the group consisting of polystyrene (PS), polymethyl- (meth)acrylate (PMMA), polycarbonate (PC), polyethylenterephthalate (PET), glycol modified polyethylenterephthalate (PETG), acrylonitrile/butadiene/styrene copolymer (ABS), polyvinylchloride (PVC) and polylactic acid (PLA), more prefe rably selected from the group consisting of polystyrene (PS), polymethyl- (meth)acrylate (PMMA), polycarbonate (PC), polyethylenterephthalate (PET) and glycol modified polyethylenterephthalate (PETG).

The non-conductive support material can be in different shapes, particu larly in the form of a foil, sheet, plate, strip or block. For certain applications it is advantageous if said support material has at least a certain flexibility, particularly flexibility in regard to bending.

The electroconductive material is preferably applied by metal vacuum deposition, particularly by sputtering, on the surface of the non-conductive support material. The electrode for measuring vitamin D comprises a layer of vitamin D specific antibody. Such capture antibody (CAb) is used to probe vitamin D. Said vitamin D specific antibody is particularly a vitamin D monoclonal antibody, more particularly a 25-OH Vitamin D2 and 25-OH Vitamin D3 monoclonal antibody. Particularly preferred is a 25-OH Vitamin D2 and 25-OH Vitamin D3 monoclonal antibody as available from GenScript USA Inc. under the number V00201 or V00202.

The antibody is able to bind vitamin D from the aqueous medium. It also binds horseradish peroxidase-conjugated vitamin D.

It is preferred that an adhesion promoter, particularly a mercaptan, is present between the electroconductive material, particularly in case of gold, and the vitamin D specific antibody. Said adhesion promoter ensures the adhesion, respectively the binding, of the vitamin D specific antibody onto the electrocon ductive electrode. It is preferred that the mercaptan comprises a hydroxyl or carboxyl group in its chemical structure. Particularly, the mercaptan is a Ce-ie alkane carrying a COOH group and a SH group or a Ce-ie alkane carrying a OH group and a SH group. Preferably, a self-assembly monolayer (SAM) is formed by the mercaptan on the electroconductive, particularly gold, layer.

This electrode can be used for determining the amounts of vitamin D in an aqueous medium as shown later in this document.

Electrode for measuring vitamin C

In a further aspect, the present invention relates to an electrode T for measuring vitamin C in an aqueous medium wherein said electrode comprises an electroconductive electrode 2' and a layer 4 comprising a catalyst for the oxidation of vitamin C, particularly tetrathiafulvalene.

Details of the electroconductive electrode 2' and its preparation and preferred embodiments are as described above for the electroconductive electrode 2 of the electrode for measuring vitamin D.

The electrode for measuring vitamin C comprises a layer 4 of a catalyst for the oxidation of vitamin C. The catalyst for the oxidation of vitamin C is particularly selected from the group consisting of tetrathiafulvalene (TTF), ferrocene derivates such as ferrocenecarboxylic acid, ferroceneacetic acid or ferrocenemethanol, conducting electroactive polymers such as polyaniline, polypyrrole and poly 3,4- ethylenedioxythiophene, norepinephrine, epinephrine, cyclized epinephrine quinone and 3,4-dihydroxycinnamic acid. Most preferably, the catalyst for the oxidation of vitamin C is tetrathiafulvalene (TTF).

It is preferred that between the electroconductive material, particularly in case of gold, and the layer comprising the catalyst for the oxidation of vitamin C an adhesion promoter, particularly a mercaptan, is present. Said adhesion promoter ensures the adhesion, respectively the binding, of the layer comprising the catalyst for the oxidation of vitamin C is particularly onto the electroconductive electrode. It is preferred that the mercaptan comprises a hydroxyl or carboxyl group in its chemical structure. Particularly, the mercaptan is a Ce-ie alkane carrying a COOFI group and a SH group or a Ce-ie alkane carrying a OH group and a SH group. Preferably, a self-assembly monolayer (SAM) is formed by the mercaptan on the electroconductive, particularly gold, layer.

It is preferred that the layer 4 comprising a catalyst for the oxidation of vitamin C, further comprises a polymer. It is preferred in one embodiment that said polymer is a reaction polymer, more preferably a polymer which is a reaction product of a polyamine and a polyaldehyde, particularly of chitosan and glutaraldehyde. It is preferred that the reactive monomers are selected so that at least one of the reactive polymers has more than 2 reactive sites, allowing the formation of a crosslinked polymer. It is obvious that the ratio of the reactive monomers forming the reaction polymer is selected such as to provide optimal polymerisation and crosslinking.

In the present document, any substance starting with "poly" such as polyamine or polyaldehyde refers to substances formally containing two or more of the corresponding functional groups per molecule.

In another embodiment the polymer is a sulfonated tetrafluoroethylene based fluoropolymer-copolymer, such as commercialized under the brand name Nafion by Dupont, or an electropolymerized o-phenylenediamine. In one embodiment, the catalyst for the oxidation of vitamin C is applied directly onto the electroconductive material or the adhesion promoter, respecti vely. It is preferred that said catalyst for the oxidation of vitamin C is applied as a solution, particularly a solution of tetrathiafulvalene (TTF) in a solvent selected from the group consisting of dichloromethane, acetone, ethanol, acetonitrile and toluene as well as mixtures thereof. It is preferred that the polymer, particularly the reaction polymer, as just described above is applied onto said catalyst for the oxidation of vitamin, particularly tetrathiafulvalene (TTF) after flash-off of the solvent. It is advantageous that the monomers are mixed and applied right away to obtain an in-situ curing of the polymer.

In another embodiment, the catalyst for the oxidation of vitamin C, particularly tetrathiafulvalene (TTF), is admixed to the polymer or the monomers and applied on the onto the electroconductive material or the adhesion promoter, respectively. This electrode can be used for determining the amounts of vitamin C in an aqueous medium as shown later in this document.

Sensor for determining the amount of vitamin C and vitamin D

In a further aspect, the present invention relates to a sensor 10 for determining the amount of vitamin C and vitamin D in an aqueous medium wherein said sensor comprises

- a first working electrode (WE1 ) which is an electrode 1 for measuring vitamin D as discussed above;

- a second working electrode (WE2) which is an electrode 1' for measuring vitamin C as discussed above; and

- a counter electrode 11.

In other words, the sensor 10 combines the electrodes for measuring vitamin C and D, as described above, and a counter electrode. The counter electrode 11 comprises also electroconductive material. It is also possible, and preferred, that the counter electrode has several layers of different electroconductive materials, preferably chromium, silver and gold.

Preferably, the counter electrode is an Ag/AgCI electrode. It comprises preferably a layer of Ag and a layer of AgCI. The latter is typically produced by chloridation at the surface of the silver layer.

The electroconductive material is preferably applied by metal vacuum deposition, particularly by sputtering, on the surface of the non-conductive support material

The first electrode 1 and the second electrode 1 ' and the counter electrode 11 are preferably localized on a non-conductive support 12. As the sensor 10 for determining the amount of vitamin C and vitamin D in an aqueous medium can be regarded as a combination of a sensor 10' for determining the amount of vitamin D in an aqueous medium and a sensor 10" for determining the amount of vitamin C in an aqueous medium, particularly using the same counter electrode 11.

Therefore, it is obvious that, similarly to the sensor 10 for determining the amount of vitamin C and vitamin D in an aqueous medium, also a sensor 10' for determining the amount of vitamin D in an aqueous medium can be manufactured. Such a sensor 10' comprises - a working electrode (WE) which is an electrode 1 for measuring vitamin D as discussed above; and

- a counter electrode 11. It is also further obvious that similarly to the sensor 10 for determining the amount of vitamin C and vitamin D in an aqueous medium, also a sensor 10" for determining the amount of vitamin C in an aqueous medium can be manufactured. Such a sensor 10" comprises

- a working electrode (WE) which is an electrode 1' for measuring vitamin C as discussed above; and

- a counter electrode 11.

Principally the size of the electrodes 1', 1' as well of the sensor 10, 10',

10" can be varied in a very broad range, depending on the requirement of a specific measurement and concentrations of vitamin D and C and volume of the aqueous medium 20 to be measured. Its size can range from macro scale to micro scale, particularly 20 cm - 100 pm, preferably 3 - 1 cm, in length and/or 5 cm - 50 pm, preferably 3 - 1 cm, in width, and/or 10 mm - 10 pm, preferably 2 - 0.5 mm, in thickness.

Manufacturing of electrodes and sensor

In the following, more details for the manufacturing of the electrodes 1,1',

2, 2', 11 as well as of the sensors 10, 10', 10" are discussed.

In one aspect, the present invention relates to a process of manufacturing a sensor 10' for determining the amount of vitamin D in an aqueous medium as discussed above in great detail comprising the following subsequent steps i') forming a layer of an electroconductive material on a non-conductive support 12 to produce an electroconductive electrode 2 ii') forming a counter electrode 11 iii') formation of a layer 3 of vitamin D specific antibody on an area of the electroconductive electrode 2.

In another aspect, the present invention relates to a process of manufacturing a sensor 10" for determining the amount of vitamin C in an aqueous medium as discussed above in great detail comprising the following subsequent steps i") forming a layer of an electroconductive material on a non-conductive support 12 to produce a electroconductive electrode 2' ii") forming a counter electrode 11 iii") formation of a layer 4 comprising a catalyst for the oxidation of vitamin C, preferably tetrathiafulvalene, on the area of the electroconductive electrode 2'.

In a further aspect, the present invention relates to a process of manufacturing a sensor 10 as discussed above in great detail comprising the following subsequent steps i) forming a layer of an electroconductive material on a non-conductive support 12 to produce a first electroconductive electrode 2 and a second electroconductive electrode 2'; ii) forming a counter electrode 11 ; iii) formation of a layer 3 of vitamin D specific antibody on an area of the first electroconductive electrode 2; iv) formation of a layer 4 comprising a catalyst for the oxidation of vitamin C, preferably tetrathiafulvalene, on the area of the second electroconductive electrode 2'.

In step i) a layer of an electroconductive material on a non-conductive support 12 is applied to produce a first electroconductive electrode 2 and a second electroconductive electrode 2'. The conductive material as well as the non- conductive support have been already discussed above in great detail. As also mentioned, it is preferably that several layers of electroconductive materials, particularly chromium, gold and silver, are applied. It is also preferred that the electroconductive material is applied by metal vacuum deposition, particularly by sputtering. It is preferred that that for all electrodes 1, 1' and 11 , first a layer of chromium, followed by a layer of gold followed by a layer of silver are subsequent ly formed and that the silver layers are later on removed by chemical etching, such as by nitric acid, at the areas of sensing at the first and second working electrodes 1,1' whereas the silver layer remains untouched for the counter electrode.

To obtain on the same non-conductive support 12 separate electrodes 2,

2', the conductive material needs to be applied at those area where the electroconductive electrode are to be produced and these areas need to be separated from each other. The electroconductive electrodes 2,2' assure the electric connections between the area of measuring, respectively sensing, 5, 5' of the sensor, and the connectors to the measuring device on the sensor 10, preferably an amperometer. The conductive material is preferably applied on specific areas of the non- conductive support 12 by means of respective masks. The location of the conductive material can be chosen so to fit the connectors of a respective amperometer.

The dimension of the non-conductive support 12, respectively sensor 10, 10' or 10" can be broadly varied in dimension. Typically, said dimensions are in the range of 20 cm - 100 pm, preferably 3 - 1 cm, in length and/or 5 cm - 50 pm, preferably 3 - 1 cm, in width, and/or 10 mm - 10 pm, preferably 2 - 0.5 mm, in thickness.

Analogously to the sensor 10 for determining the amount of vitamin C and vitamin D in an aqueous medium, also a sensor 10' for determining the amount of vitamin D in an aqueous medium and a sensor 10" for determining the amount of vitamin C in an aqueous medium can be manufactured.

In step ii) a counter electrode 11 is formed. The preferred counter electrode is an Ag/AgCI counter electrode. It is preferred that said counter electrode is formed from a electroconductive electrode, the surface of which is at least partially covered by a layer of AgCI, which is preferably produced by chloridation of the Ag surface by a respective chloridation agent, such as preferably ferric chloride.

Preferably, in the above process, the electroconductive material of the first electroconductive electrode 2 and a second electroconductive electrode 2' is gold and comprises furthermore the step ia) ia) applying at least one mercaptan on the surface onto said gold layer, preferably forming a self-assembly monolayer of mercaptan on gold; wherein step ia) is performed after step i) or ii), preferably after step ii), and before step iii). It is preferred that the mercaptan comprises a hydroxyl or carboxyl group in its chemical structure. Particularly, the mercaptan is a Ce-ie alkane carrying a COOH group and a SH group or a Ce-ie alkane carrying a OH group and a SH group. Preferably, a self-assembly monolayer (SAM) is formed by the mercaptan on the electroconductive, particularly gold, layer. The mercaptan has the function of an adhesion promoter ensuring a better adhesion, respectively the binding, of the vitamin D specific antibody or the catalyst for oxidation of vitamin C, particularly tetrathiafulvalene, respectively, onto the electroconductive electrode

In case of the binding to the vitamin D specific antibody, it is preferred that the mercaptan is activated. Said activation of mercaptans, particularly of mercaptans carrying COOH groups, is particularly provoked by treatment with an activating agent, which is particularly a carbodiimide, preferably 1 -ethyl-3-(3- (dimethylamino)propyl) carbodiimide (EDC), more preferably a mixture of 1-ethyl- 3-(3-(dimethylamino)propyl) carbodiimide and N-hydroxysuccinimide.

As shown, the sensors 10, 10' and 10" for determining the amount of vitamin C and/or vitamin D in an aqueous medium can be produced in an easy and efficient way. The preferred embodiments can be produced in a cheap way which offer the possibility of offering affordable single use sensors allowing a fast, easy and cost-efficient manner for the measurement of the amount of vitamin C and/or D in an aqueous medium. In the most preferred embodiment, the concent ration of vitamin D and/or C can be measured in saliva. This ease of measuring the concentration without any invasive techniques, increases the acceptance of the public, respectively the customers, to determine their individual concentration of vitamin C and/or D and, therefore, increases the willingness to check if there is a need for additional supply of vitamin C and/or D. As vitamin C and/or D are known to have a big positive impact on the immunity system of animals, assuring a sufficient supply of vitamin C and/or vitamin D is essential for maintaining the individual and the whole population healthy and for preventing the spread or figh ting against the spread of infections, particularly infections like COVID-19 or flu.

Therefore, this invention offers an important positive contribution to increase and maintain global health. Measurement of vitamin C and/or D in an aqueous medium

The electrode 1 for measuring vitamin D, particularly integrated in a sensor 10' for determining the amount of vitamin D or a sensor 10 for determining the amount of vitamin C and vitamin D, can be used for the measuring, respectively determining, of the amount of vitamin D in an aqueous medium.

For such a measurement, either the electrode 1 in combination with a counter electrode, particularly an external Ag/AgCI counter electrode, or a sensor 10' for determining the amount of vitamin D or a sensor 10 for determining the amount of vitamin C and vitamin D, is brought in contact with a defined amount an aqueous medium 20, so that at least the area 5 of sensing and the counter electrode are in contact with the aqueous medium.

Horseradish peroxidase-conjugated vitamin D (HRP-VitD) is added to the aqueous medium to be tested to yield a contact solution 21.

The contacting can be performed in one embodiment by dipping the sensor 10 into the contact solution 21 , respectively the aqueous medium 20. In another embodiment, a specific volume of the contact solution 21 is given by a respective mean, such as a pipette, onto the electrodes 1, T at the sensing area 5,5' as well as onto the counter electrode 11.

The horseradish peroxidase-conjugated vitamin D and the vitamin D in the contact solution compete with each other for binding to the vitamin D specific antibody. After an incubation time of typically about 20 minutes all sites of the vitamin D specific antibody are either occupied by vitamin D or horseradish peroxidase-conjugated vitamin D. After removal of the contact solution 21 , preferably followed by rinsing of the electrode or the sensor 10' or the sensor 10, the electrode 1 or sensor 10' or sensor 10 is brought in contact with a redox solution 23 of H2O2 and a mediator, so that at least the area 5 of sensing of the electrode 1 and the counter electrode 11 are in contact with the said redox solution 23. The mediator is preferably selected from the group consisting of 3,3’,5,5’-tetramethylbenzidine (TMB), 2,2'-azino-bis(3-ethylbenzothiazoline-6- sulphonic acid), o-phenylenediamine dihydrochloride, hydroquinone, hydroxymethyl ferrocene, ferrocene, and tetrathiafulvalene.

The preferred mediator is 3,3',5,5'-tetramethylbenzidine.

As the horseradish peroxidase-conjugated vitamin D reacts with the redox solution whereas vitamin D does not react, an electrochemical signal can be measured by means of the electrode 1 / counter electrode and an amperometer being connected to them. This signal corresponds to the amount of the horseradish peroxidase-conjugated vitamin D bound to the vitamin D specific antibody, respectively corresponds inversely to the amount of vitamin D bound to the vitamin D specific antibody. Hence, based on the known concentration of the horseradish peroxidase-conjugated vitamin D in the contact solution 21, the measurement can deliver the concentration of vitamin D in the contact solution 21 or the aqueous medium 20 to be tested.

The electrochemical signal is preferably measured as a function of time Hence, the amount of vitamin D in the aqueous medium is preferably determined by chronoamperometry. The measurement of the electrochemical signal is typically finished within 5 minutes, particularly within 1.5 minutes. The electrode T for measuring vitamin C particularly integrated in a sensor 10" for determining the amount of vitamin C or a sensor 10 for determining the amount of vitamin C and vitamin D can be used for the measuring, respectively determining, the amount of vitamin C in an aqueous medium .

For such a measurement, either the electrode T in combination with a counter electrode, particularly an external Ag/AgCI counter electrode, or a sensor 10" for determining the amount of vitamin C or a sensor 10 for determining the amount of vitamin C and vitamin D is brought in contact with a defined amount an aqueous medium, so that at least the area 5' of sensing and the counter electrode are in contact with the aqueous medium. By the catalyst for the oxidation of vitamin C, being particularly tetrathiafulvalene, vitamin C is oxidized. Said oxidation produces an electrochemical signal which can be measured by means of the electrode T / counter electrode and an amperometer being connected to them. This signal corresponds to the amount of vitamin C in the aqueous medium to be tested 20. The electrochemical signal is preferably measured as a function of time.

Hence, the amount of vitamin C in the aqueous medium is preferably determined by chronoamperometry. The measurement of the electrochemical signal is typically finished within 5 minutes, particularly within 1 minute. With a sensor 10 as described above in great details, the amount of both vitamin C and vitamin D can be measured in an aqueous medium. Hence, the present invention relates, in a further aspect, to a method of determining the concentration of vitamin C and vitamin D in an aqueous medium 20 comprising the following steps: a) providing a sensor 10 as described above in great detail; b) admixing horseradish peroxidase-conjugated vitamin D to the aqueous medium 20 to be tested to yield a contact solution 21 ; c) contacting at least the sensing area 5,5' of the working electrodes 1,1' and the counter electrode 11 of the sensor 10 with the contact solution

21 ; d) measuring the cathodic current at the electrode 1' for measuring vitamin C by an amperometer 22 and counter electrode 11 to determine the concentration of vitamin C; e) removing the contact solution 21 ; f) providing a redox solution 23 of H2O2 and a mediator, preferably 3,3',5,5'-tetramethylbenzidine; g) contacting at least the sensing area 5 of the working electrode 1 for vitamin D and the counter electrode 11 of the sensor with the redox solution 23; h) measuring the cathodic current at the electrode for measuring vitamin D 1 by an amperometer and counter electrode 11 to determine the concentration of vitamin D. It is of course also possible that, in an alternative way, steps b) and c) in the above process are replaced by steps b') and c') b') contacting at least the sensing area 5,5' of the working electrodes 1,1' and the counter electrode 11 of the sensor 10 with the aqueous medium 20 to be tested; c') admixing horseradish peroxidase-conjugated vitamin D to the aqueous medium 20 to yield a contact solution 21. It is obvious, that the details are the same as those which have been just discussed above for the electrode 1 , electrode T, sensor 10, sensor 10' and sensor 10".

In the present document the term "aqueous medium" means a solution, dispersion or an emulsion comprising water. Said aqueous medium is preferably an aqueous medium originating from an animal, particularly a human, most preferred from human. Accordingly, the term "animal" embraces in the present document also humans. Preferred animals are mammals, most preferred animals are humans.

Such an aqueous medium originating from animals is particularly blood, urine, sweat, tears or saliva, preferably salvia, most preferred human salvia.

This method is particularly advantageous because it is preferably non- invasive and offers an easy, cheap and fast possibility of determining the amount of vitamin C and D in an aqueous medium.

As the measurement of vitamin C and/or D is very sensitive, it is possible to have measured vitamin C and/or D in very low amounts in the aqueous medium. Typically the measurement can take place already in a few microliters, e.g. 10 pi, of an aqueous medium.

Based on the measuring of the amount of vitamin C and D in aqueous medium originating from an animal, the amount of vitamin C and D can be determined which is to be provided to said animal.

Hence, in a further aspect, the present invention relates to a non-invasive method of determining the amount of vitamin C and vitamin D to be provided to an animal, particularly a human, comprising the steps a) providing a sensor 10 as discussed above in great detail; b) admixing horseradish peroxidase-conjugated vitamin D to the aqueous medium 20 to be tested to yield a contact solution 21 ; c) contacting at least the sensing area 5,5' of the working electrodes 1,1' and the counter electrode 11 of the sensor 10 with the contact solution 21 ; d) measuring the cathodic current at the electrode 1 ' for measuring vitamin C by an amperometer 22 and counter electrode 11 to determine the concentration of vitamin C; e) removing the contact solution 21 ; f) providing a redox solution 23 of H2O2 and a mediator, preferably 3,3',5,5'-tetramethylbenzidine; g) contacting at least the sensing area 5 of the working electrode 1 for vitamin D and the counter electrode 11 of the sensor with the redox solution 23; h) measuring the cathodic current at the electrode for measuring vitamin D 1 by an amperometer 22 and counter electrode 11 to determine the concentration of vitamin D; i) comparing the measured values 45 of vitamin C and vitamin D with the daily recommend dosage of vitamin C and D obtainable from a data source 46 to determine a need in additional supply of vitamin C and/or D; j) calculating the needed amount of additional supply of vitamin C respectively vitamin D; k) suggesting an suggested amount 48 of additional supply of vitamin C respectively vitamin D, to be administrated to said animal, particularly human, in case that a need is calculated in step g).

The steps a) to h), including the alternative using steps b') and c'), have been already discussed above.

In step i) the measured values of vitamin C and vitamin D are compared with the daily recommend dosage of vitamin C and D to determine a need in additional supply of vitamin C and/or D. The calculation in step j) is preferably performed by a computer 47 using a respective computer programme taking into account of specific parameters such as kind of animal, sex of the animal, age, weight or kind of aqueous medium originating from animal. The computer 47 can be a personal computer, a server or a mobile computer such particularly a laptop, a tablet, an iPad or a mobile phone.

The computer programme can be a computer software or an app (application) for a mobile computer device, such as a mobile phone.

The recommended dosage of vitamin C and/or D for humans are based on the recommendation daily dosage listed particularly as nutrient reference values (NRV) in annex XIII of the EU regulation no. 1169/2011 (https://eur- lex.europa.eu/leaal-content/EN/TXT/PDF/?uri=CELEX:02011 R1169-20180101 &from=FRl or as daily values by the FDA (U.S. Food and Drug Administration) as of 05/05/2020 (https://www.fda.qov/food/new-nutrition-facts-label/dailv-va lue-new-nutrition-and-supplement-facts- labels).

Preferred are the dosage as recommended nutrient reference values (NRV) listed in annex XIII of the EU regulation no. 1169/2011.

The sensor 10, 10' or 10" as discussed above in great detail can be used for determining the amounts of vitamin C and D in an aqueous medium. It can be particularly used for determining the composition of a personalized dietary supplement, wherein said personalized dietary supplement comprises vitamin C and/or vitamin D.

If said aqueous medium is originating from an animal, and is particularly saliva, it can be determined if said animal received its daily recommended dosage of vitamin C and/or D, and finally a personalized dietary supplement can be produced which supplies the need of said animal for vitamin C and/or D.

The sensor 10, 10' or 10" can be can be particularly used for determining the composition of a personalized dietary supplement, wherein said personalized dietary supplement comprises vitamin C and/or vitamin D. Hence, in a further aspect, the present invention relates to a process of manufacturing of a personalized dietary supplement 44 comprising the following subsequent steps a) providing a sensor 10 as discussed above in great detail to a consumer; b) Receiving data 34 from said consumer, wherein said data has been provided by the sensor of step a); g) Determining the need of vitamin C and D of the consumer of step a) using the data of step b); d) providing a personalized dietary supplement 44 comprising vitamin C and/or vitamin D in an amount based on the need as determined in step g).

The data 34 can be sent from the sensor to a computer, which is either part of the measing device including at least the sensor 10, 10', 10" and amperometer 22, or attached to it, or located at another site. In the later case, the data 34 is preferably sent to said computer 47 by internet. Step g) is preferably performed by a computer programme running on said computer 47 accessing data for recommended daily dosages for the animal at issue and taking into account specific parameters such as kind, sex, age or weight of the animal as well as the kind of aqueous medium originating from animal. The personalized dietary supplement 44 is preferably provided to the customer.

The sensor 10', respectively sensor 10, can be part of a kit for measuring the amount of vitamin D and/or vitamin C.

Hence, the present invention relates in a further aspect to a kit 40 which comprises a sensor 10 or 10' as discussed above in great detail; and - a container 41 comprising at least horseradish peroxidase-conjugated vitamin D (HRP-VitD); and a container 42 comprising at least H2O2 Furthermore, it is preferred that said kit comprises also a mediator. The mediator is preferably selected from the group consisting of 3, 3’, 5,5’- tetramethylbenzidine (TMB), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), o-phenylenediamine dihydrochloride, hydroquinone, hydroxymethyl ferrocene, ferrocene, and tetrathiafulvalene. The preferred mediator is 3, 3', 5,5'- tetramethylbenzidine.

Said mediator may be provided in the container 42 or in a separate container 43.

It is preferred that the kit 40 comprises a vessel for taking up the aqueous medium in a pre-defined amount for the measurement. Said predefined amount may be preferably showed by indicators on said vessel. In another embodiment the container 41 has empty volume space, which can serve as vessel for taking up the pre-defined amount of aqueous medium to form the contact solution 21.

A syringe may be a further item which is part of such a kit 40.

Such a kit is very interesting for any testing purposes as all necessary sensors and ingredients are readily available for use in form of kit.

This is very useful for mass testing and screening.

Figures

Figure 1a shows a schematic cross-sectional view of an electrode 1 for measuring vitamin D in an aqueous medium.

Figure 1b shows a schematic cross-sectional view of an electrode T for measuring vitamin C in an aqueous medium.

Figure 1a' shows a schematic cross-sectional view of a preferred embodiment of an electrode 1 for measuring vitamin D in an aqueous medium or a sensor 10 or 10' (cutting line AA) of figure 2a or 2b.

Figure 1b' shows a schematic cross-sectional view of a preferred embodiment of an electrode 1 ' for measuring vitamin C in an aqueous medium or a sensor 10 or 10" (cutting line BB) of figure 2a or 2c. Figure 1c' shows a schematic cross-sectional view of a preferred embodiment of a counter electrode 11 or a sensor 10 or 10' or 10" (cutting line CC) of figure 2a or 2b or 2c. Figure 2a shows a schematic view of a preferred sensor 10 for determining the amount of vitamin C and vitamin D in an aqueous medium.

Figure 2b shows a schematic view of a preferred sensor 10' for determining the amount of vitamin D in an aqueous medium.

Figure 2c shows a schematic view of a preferred sensor 10" for determining the amount of vitamin C in an aqueous medium.

Figures 3a-j show schematic views of different stages in a preferred process of the manufacturing of a preferred sensor 10 for determining the amount of vitamin C and vitamin D in an aqueous medium.

Figures 4a-i show schematic views of different stages in a preferred process of the manufacturing of a preferred sensor 10' for determining the amount of vitamin D in an aqueous medium.

Figures 5a-g show schematic views of different stages in a preferred process of manufacturing of a preferred sensor 10" for determining the amount of vitamin C in an aqueous medium.

Figure 6a shows a schematic representation of a process of determining the concentration of vitamin C and vitamin D in an aqueous medium 20.

Figure 6b shows a schematic representation of a process of determining the amount of vitamin C and vitamin D to be provided to an animal, particularly a human.

Figure 7 shows a schematic representation of a kit 40. Figure 8a shows a photograph of the sensor (SD) for measuring vitamin D which is used in the experimental part.

Figure 8b shows a photograph of the sensor (SCD) for measuring vitamin C and D which is used in the experimental part.

Figure 9 shows a graph of the measurements of current versus time for different concentrations of vitamin D in saliva and the correlation between hso s and the concentration of vitamin D (see example 1).

Figure 10 shows the graph of the measurements of current versus time in saliva measured after an intake of specific amounts of vitamin D (see example 2) (using sensor SD) and the insert graph of Ai versus the intake of specific amounts of vitamin D (see example 2) (using sensor SD).

Figure 11 shows the graph of measurement of current versus time in saliva of two individuals after different waiting time after ingestion time of a pill of 10Ό00 IU (IU=lnternational Units) of vitamin D (see example 3) (using sensor SD) and the insert graph of Ai os versus the time after intake (tint).

Figure 12 shows the graph of Ai os in saliva versus time of an individual with regular intake of a pill of 2Ό00 IU of vitamin D (see example 4) (using sensor SD). Figure 13 shows a graphic representation of the measurements undertaken in example 5 using the sensor SC for measuring different amount of vitamin C added to saliva and the correlation between o s and the concentration of vitamin C.

Figure 14 shows a graphic representation of the measurements undertaken in example 6 using the sensor SC for measuring different ingredients added to saliva. Figure 15 shows a graph of the measurements of current versus time for different concentrations of vitamin C in saliva and the correlation between o s and the concentration of vitamin C (see example 7) (using sensor SCD). Figure 16 shows a graph of the measurements of current versus time for different concentrations of vitamin D in saliva and the correlation between hso s and the concentration of vitamin D (see example 8) (using sensor SCD).

Figure 17 shows a series of graphs of the measurements of current versus time for different concentrations of vitamin C and D in saliva for the evaluation of cross talk of the sensor SCD (see example 9).

Figure 18a shows in the upper part graphs of the measurements of current versus time for different concentrations of vitamin C at a fixed concentration of vitamin D (see example 10) by the sensor SCD. The insert graph in figure 18a shows a representation of o s for vitamin C and hso for vitamin D for these solutions.

Figure 18b shows graphs of the measurements of current versus time for different concentrations of vitamin D at a fixed concentration of vitamin C (see example 10) by the sensor SCD. The insert graph in figure 18b shows a representation of o s for vitamin C and hso for vitamin D for these solutions.

Figure 1a shows schematically an electrode 1 for measuring vitamin D in an aqueous medium. It comprises an electroconductive electrode 2 and a layer of vitamin D specific antibody 3 on the electroconductive electrode. This representation shows that the layer of vitamin D specific antibody 3 is preferably located at the area of sensing 5 of vitamin D. Figure 1 a' shows a schematic cross-sectional view of a preferred embodiment of an electrode 1 for measuring vitamin D in an aqueous medium. It comprises a non-conductive support material 12 on which the electroconductive electrode 2 is applied. More precisely, in the embodiment as shown, said electro conductive electrode 2 is composed of two layers of electroconductive materials, a layer of chromium 32a and a layer of gold 31 b. A layer of activated mercaptan 34 localized on the gold layer 31 b at the sensing area 5 of the working electrode 1 is acting as bonding bridge for the layer of vitamin D specific antibody 3.

An electrode 1 for measuring vitamin D in an aqueous medium can be also part of a sensor 10' for determining the amount of vitamin D in an aqueous medium or a sensor 10 for determining the amount of vitamin C and vitamin D in an aqueous medium. Hence, figure 1a' represents also schematic cross-sectional view of a preferred sensor 10 or 10' (cutting line AA) of figure 2a or 2b.

Figure 1b shows schematically an electrode 1' for measuring vitamin C in an aqueous medium. It comprises an electroconductive electrode 2' and a layer (4) comprising a catalyst for the oxidation of vitamin C, particularly tetrathiafulvalene.

Figure 1b' shows a schematic cross-sectional view of a preferred embodiment of an electrode 1' for measuring vitamin C in an aqueous medium. It comprises a non-conductive support material 12 on which the electroconductive electrode 2' is applied. More precisely, in the embodiment as shown, said electroconductive electrode 2' is composed of two layers of electroconductive materials, a layer of chromium 32'a and a layer of gold 31 b. A layer of mercaptan 33' localized on the gold layer 31 b at the sensing area 5' of the working electrode 1', preferably as a self-assembly monolayer (SAM), is acting as bonding bridge for the layer comprising the catalyst for oxidation of vitamin C, particularly tetrathiafulvalene.

An electrode 1' for measuring vitamin C in an aqueous medium can be also part of a sensor 10" for determining the amount of vitamin C in an aqueous medium or a sensor 10 for determining the amount of vitamin C and vitamin D in an aqueous medium. Hence, figure 1b' represents also schematic cross-sectional view of a preferred sensor 10 or 10" (cutting line BB) of figure 2a or 2c.

Figure 1c' shows a schematic cross-sectional view of a preferred embody- ment of a counter electrode 11. It comprises a non-conductive support material 12 on which the electroconductive electrode is applied. More precisely, in the embodiment as shown, said electroconductive electrode is composed of three layers of electroconductive materials, a layer of chromium 31a and a layer of gold 31 b and a layer of silver 31 c. The surface of the silver layer is chloridated to form a layer of AgCI 31'c. In the present embodiment, as shown here, the whole electrode is covered by AgCI. An counter electrode 11 can be also part of a sensor 10' for determining the amount of vitamin D in an aqueous medium or a sensor 10" for determining the amount of vitamin C in an aqueous medium or a sensor 10 for determining the amount of vitamin C and vitamin D in an aqueous medium. Hence, figure 1c' represents also schematic cross-sectional view of a preferred sensor 10 or 10' or 10" (cutting line CC) of figure 2a or 2b or 2c.

Figure 2a shows a schematic view of a preferred sensor 10 for determining the amount of vitamin C and vitamin D in an aqueous medium. The sensor comprises a first working electrode (WE1 ) which is an electrode 1 for measuring vitamin D and separated thereof a second working electrode (WE2) which is an electrode T for measuring vitamin C and a counter electrode 11. In the here showed embodiment the electrodes 1 , T and the counter electrode 11 are localized on a non-conductive support 12. The electrode 1 for measuring vitamin D comprises an electroconductive electrode 2, which consists of a layer of chromium and gold. In the sensing area 5, a layer of vitamin D specific antibody 5 is attached to the layer of gold surface. An adhesion promoter, particularly a mercaptan, respectively activated mercaptan, can be used to ensure good bonding of a layer 5 of vitamin D specific antibody 5 to the layer of gold. The electrode T for measuring vitamin C, which is localized separated from the first working electrode 1 and the counter electrode 11 , comprises an electroconductive electrode 2', which consists of a layer of chromium and gold. In the sensing area 5', a layer 4 comprising a catalyst for the oxidation of vitamin C, particularly tetrathiafulvalene, is attached to layer of gold. An adhesion promoter, particularly a mercaptan, respectively activated mercaptan, can be used to ensure good bonding of a layer 4 of the catalyst for oxidation of the vitamin C to the layer of gold. The catalyst for the oxidation of vitamin C, particularly tetrathiafulvalene, is preferably covered by a polymer, particularly a reaction polymer obtained preferably by the reaction of chitosan and glutaraldehyde. The counter electrode 11 is localized on the non-conductive support 12 in a distance suitable for measuring from the first working electrode 1 and the second working electrode T. The shape of the first working electrode 1 and the second working electrode T in the area of sensing 5, 5' are preferrable circular and the shape of the counter electrode 11 is preferably such that the distance between counter electrode 11 and first working electrode 1 respectively second working electrode T is constant over a large portion of the sensing area 5,5'.

Therefore, an arrangement of a counter electrode in a T-type shape and a circular sensing area 5 for the first working electrode 1 , respectively 5' of the second working electrode T, localized in the proximity of the horizontal part of the T, as schematically shown in figure 2a, is preferred.

Figure 2a also shows the line AA, BB resp. CC, for which a cross- sectional view is shown in figures 1a', 1b' resp. 1c'.

Figure 2b shows a schematic view of a preferred sensor 10' for determining the amount of vitamin D in an aqueous medium. Details for this figure are as shown for the figure 2a.

Figure 2b shows a schematic view of a preferred sensor 10" for determining the amount of vitamin C in an aqueous medium. Details for this figure are as shown for the figure 2a.

Figures 3a-j show schematic views of different stages in a preferred process of the process of manufacturing a preferred sensor 10 for determining the amount of vitamin C and vitamin D in an aqueous medium.

On a non-conductive support 12, preferably in form of a polymer foil or plate, an electroconductive material is applied. As schematically shown in figure 3a a layer of metallic chromium 31a, 32a, 32'a is applied on the area where the first working electrode 1 , second working electrode 1 ' and counter electrode 11 are to be formed. The selection of these areas are preferably realized by the use of masks. The chromium is preferably applied by sputter deposition in vacuum. Subsequently, a layer of metallic gold 31 b is applied on top of the layer of chromium 31a, 32a, 32'a as schematically shown in figure 3b in the areas of first working electrode 1 , second working electrode 1 ' and counter electrode 11 , so that in the area of first working electrode 1 and second working electrode 1', the electroconductive electrode 2, 2' of the electrodes 1,1' are formed.

The gold is preferably applied by sputter deposition in vacuum.

Subsequently, a layer of metallic silver 31c, 32c, 32'c is applied on top of the layer of gold layer 31 b as schematically shown in figure 3c in the areas of first working electrode 1 , second working electrode 1 ' and counter electrode 11.

The silver is preferably applied by sputter deposition in vacuum.

As schematically shown in figure 3d, in the areas of first working electrode 1 and second working electrode T the silver layer 31c, 32c, 32'c formed in pre vious step is now removed by chemical etching, particularly by HNO3. Therefore, in the areas of first working electrode 1 and second working electrode T, the gold layers as electroconductive electrode 2, 2' are again present on top of the electrodes, whereas in the area of the counter electrode 11 the silver layer 31c remains unchanged.

As schematically shown in figure 3e, the silver layer 31c of the counter electrode 11 is treated in a subsequent step with a respective chloridation agent, such as preferably ferric chloride, to yield at the surface AgCI 31'c, hence, yielding the counter electrode 11 of the sensor 10. The counter electrode 11 of the sensor 10 is finalized by this step.

In figure 3f it is schematically shown that in a further step, mercaptan is applied on the surface of said gold layer of the electroconductive electrode 2, 2', so that a mercaptan layer 33, 33' is formed. It is particularly applied to the area 5,5' of sensing of vitamin C, respectively D, of the final sensor 10. The mercaptan is preferably a mercaptan comprising a hydroxyl or carboxyl group. Particularly, the mercaptan is a Ce-18 alkane carrying a COOH group and a SH group or a Ce-18 alkane carrying a OH group and a SH group. Preferably, a self-assembly monolayer (SAM) 33,33' is formed by the mercaptan on the gold layer. As shown schematically in figure 3g, in a following step, which is dealing with the vitamin C part of the sensor only, a catalyst 4 for oxidation of vitamin C, preferably tetrathiafulvalene (TTF) is applied, preferably together with a polymer, to the electroconductive electrode 2', respectively to the layer of mercaptan 33' layer, in the area of the vitamin C sensing area 5' of the final sensor 10. It is preferred that in a first step, the catalyst for the oxidation of vitamin C, preferably tetrathiafulvalene, is applied as a solution, followed by covering said catalyst by a polymer coating. The preferred polymer covering said catalyst, preferably tetrathiafulvalene, is a reaction polymer of chitosan and glutaraldehyde, which is formed in situ, i.e. that chitosan and glutaraldehyde are applied particularly in their respective stoichiometric amounts onto said catalyst, preferably tetrathiafulvalene, to result in said reaction polymer. The electrode T for measuring vitamin C of the sensor 10 is finalized by this step.

In the area of the vitamin D sensing area of the final sensor 10, in a subsequent step, as shown schematically in figure 3h, the mercaptan layer 33 is activated to form an activated mercaptan layer 34. The activation is provoked by an activating agent, which is particularly a carbodiimide, preferably 1-ethyl-3-(3- (dimethylamino)propyl) carbodiimide (EDC), more preferably a mixture of 1-ethyl- 3-(3-(dimethylamino)propyl) carbodiimide and N-hydroxysuccinimide.

In a following step, the vitamin D specific antibody 35 is applied to the activated mercaptan layer, as shown schematically in figure 3i. The vitamin D specific antibody binds covalently to the activated mercaptan. However, not all activated mercaptans are reacted and some residual activated mercaptan remain accessible at the surface.

In the final step, as schematically shown in figure 3j, these residual activated sites are blocked by a suitable blocking agent, such as ethanolamine to form the final layer of vitamin D specific antibody 3.

After rinsing, the electrode 1 for measuring vitamin D of the sensor 10 is finalized by this step, so that the complete sensor 10 for vitamin C and D is finished after performing this step. Accordingly, figures 4 a-i show schematically the individual steps of a very preferred embodiment of the process of manufacturing a sensor 10' for determining the amount of vitamin D in an aqueous medium. These step shown in figures 4 a-i correspond to the respective steps relating to the vitamin D part of the sensor 10 as described above for figures 3a-f and 3h-j.

Accordingly, figures 5 a-g show schematically the individual steps of a very preferred embodiment of the process of manufacturing a sensor 10" for determining the amount of vitamin C in an aqueous medium. These step shown in figures 5 a-g correspond to the respective steps relating to the vitamin C part of the sensor 10 as described above for figures 3a-g.

Figure 6a shows a schematic representation of a process of determining the concentration of vitamin C and vitamin D in an aqueous medium 20.

A sensor 10 is provided in a first step, i.e. step a). In step b) horseradish peroxidase-conjugated vitamin D 19 is admixed to the aqueous medium 20, which is to be tested, to yield a contact solution 21. In step c) at least the sensing area 5,5' of the working electrodes 1,1' and the counter electrode 11 of the sensor 10 is contacted with the contact solution 21. In step d) the cathodic current at the electrode 1' for measuring vitamin C is measured amperometer 22 and the counter electrode 11 to determine the concentration of vitamin C. In step e) the contact solution 21 is removed and the sensor 10 is contacted in step g) with the redox solution 23 of H2O2 and a mediator, preferably 3,3',5,5'-tetramethyl- benzidine, which is provided by step f), so that at least the sensing area 5 of the working electrode 1 for vitamin D and the counter electrode 11 of the sensor are in contact with the redox solution 23. In step h) the cathodic current at the electrode 1 for measuring vitamin D is measured by an amperometer 22 and a counter electrode 11 to determine the concentration of vitamin D.

Figure 6b shows a schematic representation of a process of determining the amount of vitamin C and vitamin D to be provided to an animal, particularly a human. The steps a) to h) are identical to the steps a) to h) as described for figure 6a above. In step i) the measured values 45 of vitamin C and vitamin D are compared with the daily recommend dosage of vitamin C and D from a data source 46 to determine a need in additional supply of vitamin C and/or D. In step j) the needed amount of dosage of amount of additional supply of vitamin C respectively vitamin D, are calculated. This calculation preferably is performed on a computer 47 using a respective software on the basis of which the an amount 48 of additional supply of vitamin C, respectively vitamin D, is suggested to be administrated in step k) to said animal, particularly human, in case that a need is calculated in step g). Figure 7 shows a schematic representation of a kit 40. Said kit comprises a sensor 10 for determining the amount of vitamin C and vitamin D in an aqueous medium and a container 41 comprising at least horseradish peroxidase- conjugated vitamin D (HRP-VitD) and a container 42 comprising at least H2O2. Furthermore, it is preferred that said kit 40 comprises also a mediator, which is preferably 3,3',5,5'-tetramethylbenzidine. Said mediator may be provided in the container 42 or in a separate container 43. In the present representation the mediator is comprised in a separate container 43.

A similar kit 40 comprises a sensor 10' instead of the sensor 10. Figure 8a shows a photograph of the sensor 10' (SD) for measuring vitamin D which is used in the experimental part. Said sensor has a working electrode 1 for measuring vitamin D and the counter electrode 11. The photograph also shows how the sensor with the dimensions of 13 mm x 20 mm x 0.8 mm, is hold by a human hand in a plastic glove. Finally, the photograph shows the sensor being in contact with the contact solution 21.

Figure 8b shows a photograph of the sensor (SCD) for measuring vitamin C and D which is used in the experimental part.

On the left side of figure 8b a photograph of the sensor 10 (SCD) for measuring vitamin C and D, which is used in the experimental part, is shown on a fingertip . Said sensor has a working electrode 1 for measuring vitamin D, a working electrode T for measuring vitamin C, and a counter electrode 11. The sensor has the dimension 13 mm x 15mm x 0.8 mm. Furthermore, the photograph of figure 8b also shows on the right side that the sensor is connected to the amperometer 22 which is connected to a smartphone 47, on which a programme is running. In the photograph, on the display a graph (in red) of the measured electric current as function of time is displayed.

Figure 9 shows a graphic representation of the measurements undertaken in example 1 using the sensor SD for measuring different amounts of vitamin D added to saliva. The insert shows that there exists an inverse linear correlation between the measured current hso s and the concentration of vitamin D.

Figure 10 shows a graphic representation of the measurements under taken in example 2 using the sensor SD for measuring the amount of vitamin D in saliva after the intake of specific amounts of vitamin D. The insert graph shows a graphic representation of the difference (Ai) of measurements between the blank (no intake) and after intake of vitamin D against the amounts of vitamin D.

Figure 11 shows the graph of measurement of current versus time in saliva of two individuals after different of ingestion time of a pill of 10Ό00 IU (IU=lnternational Units) of vitamin D (see example 3) (using sensor SD) and the insert graph of Ai os versus the time after intake (tint).

Figure 12 shows the graph of Ai in saliva versus time of regular intake of a pill of 2Ό00 IU of vitamin D (see example 4) (using sensor SD). It shows how fast an increase of vitamin D can be observed and how fast the increased level in vitamin D is lost after stopping of the regular supplementation of vitamin D.

Figure 13 shows a graphic representation of the measurements undertaken in example 5 using the sensor SC for measuring different amount of vitamin C added to saliva. The insert shows that there exists a linear correlation between the measured current hso s and the concentration of vitamin D. Figure 14 shows a graphic representation of the measurements undertaken in example 6 using the sensor SC for measuring different ingredients added to saliva. Figure 15 shows a graphic representation of the measurements undertaken in example 7 using the sensor SCD for measuring different amount of vitamin C added to saliva. The insert shows that there exists linear correlation between the measured current o s and the concentration of vitamin C. Figure 16 shows a graphic representation of the measurements undertaken in example 8 using the sensor SCD for measuring different amount of vitamin D added to saliva. The insert shows that there exists an inverse linear correlation between the measured current hso s and the concentration of vitamin D. Figure 17 shows graphs of the measurements of current versus time for different concentrations of vitamin C and D in saliva for the evaluation of cross-talk of the sensor SCD as undertaken in example 9.

Figure 18a shows graphs of the measurements of current versus time for different concentrations of vitamin C at a fixed concentration of vitamin D as measured in example 10 by the sensor SCD. The insert graph in figure 18a shows a representation of o s for vitamin C and -hso for vitamin D for these solutions.

Figure 18b shows graphs of the measurements of current versus time for different concentrations of vitamin D at a fixed concentration of vitamin C as measured in example 10 by the sensor SCD. The insert graph in figure 18b shows a representation of o s for vitamin C and -hso for vitamin D for these solutions. List of reference signs

1 Electrode for measuring vitamin D, working electrode for vitamin D 1 ' Electrode for measuring vitamin C, working electrode for vitamin C

2 Electroconductive electrode, first electroconductive electrode 2' Electroconductive electrode, second electroconductive electrode

3 Layer of vitamin D specific antibody

4 Layer comprising catalyst for oxidation of vitamin C, particularly tetrathiafulvalene

5,5' Sensing area of working electrode 1,1' 10 Sensor for vitamin C and D 10' Sensor for vitamin D 10" Sensor for vitamin C

11 Counter electrode

12 Non-conductive support 20 Aqueous medium to be tested

21 Contact solution

22 Amperometer

23 Redox solution of H2O2 and mediator, preferably 3,3',5,5'-tetramethyl- benzidine

31 a, 32a, 32'a Layer of chromium

31b Layer of gold

32c,32'c,31c Layer of silver

31'c Layer of AgCI

33,33' Layer of mercaptan 34 Layer of activated mercaptan

35 Layer of vitamin D specific antibody and unreacted sites

40 Kit

41 Container comprising horseradish peroxidase-conjugated vitamin D

42 Container comprising at least H2O2 43 Container comprising a mediator

44 Personalized dietary supplement

45 Data

46 Source of data of recommended dosage of vitamin C and/or D

47 Computer 48 Suggested amount of additional supply of vitamin C respectively vitamin D Examples

The present invention is further illustrated by the following experiments.

Preparation of sensor for measuring vitamin D A sensor for measuring vitamin D was prepared in the following way:

At the area of the working and counter electrode, a layer of chromium was applied by Cr-sputtering under argon on the surface of a PETG plate (13mm x 20 mm x 0.8 mm), followed by a layer of gold followed by silver by sputter deposition under argon.

At the working electrode, the layer of Ag was removed by etching with nitric acid. At the counter electrode the silver was treated by FeCta to form a Ag/AgCI counter electrode.

The working electrode was dipped into a solution of 11-mercaptoundeca- noic acid / 6-mercapto-1-hexanol (I.OmM / 0.1 mM) in ethanol to form a SAM on the gold surface. After washing and drying, the SAM layer was treated with a solution of 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide / N-hydroxysuccin- imide (0.4/.01 M). After washing and drying, the electrode was incubated with 10 pL of 50 pg mL 1 Vit D-specific antibody (CAb) (V00202, Genscript) solution (pH 6.5) during 60 minutes for the covalent attachment of the CAb on the activated carboxylic groups of the SAM. Then the free unreacted carboxylic groups were deactivated by incubating 10 pL of 2 M ethanolamine solution to yield the working electrode for measuring vitamin D, respectively the sensor for measuring vitamin D ("SD") used in the following. Figure 8a shows a photograph of this sensor.

Preparation of sensor for measuring vitamin C A sensor for measuring vitamin C was prepared in the following way:

At the area of the working and counter electrode, a layer of chromium was applied by Cr-sputtering under argon on the surface of a PETG plate (13 mm x 20 mm x 0.8 mm), followed by a layer of gold followed by silver by sputter deposition under argon.

At the working electrode, the layer of Ag was removed by etching with nitric acid. At the counter electrode the silver was treated by FeCta to form a Ag/AgCI counter electrode. The working electrode was dipped into a solution of 11-mercaptoundeca- noic acid / 6-mercapto-1-hexanol (lOmM / 0.1 mM) in ethanol to form a SAM on the gold surface. After washing and drying, the SAM layer was treated with a solution of TTF solution (5 mM in 1:1 acetone/ethanol) was drop cast on vitamin C sensor. Next, 2 pL of chitosan (1.0%) and 0.5 pl_ of glutaraldehyde (0.5%) were sequentially cast on the TTF-modified electrode to yield the working electrode for measuring vitamin C ("SC") used in the following.

Preparation of sensor for measuring vitamin C and D A sensor for measuring vitamin C and D was prepared in the following way:

At the area of the working and counter electrode, a layer of chromium was applied by Cr-sputtering under argon on the surface of a PETG plate (13 mm x 15 mm x 0.8 mm), followed by a layer of gold followed by silver by sputter deposition under argon.

At the working electrode, the layer of Ag was removed by etching with nitric acid. At the counter electrode the silver was treated by FeCta to form a Ag/AgCI counter electrode.

The working electrode was dipped into a solution of 11-mercaptoundeca- noic acid / 6-mercapto-1-hexanol (lOmM / 0.1 mM) in ethanol to form a SAM on the gold surface. After washing and drying, the SAM layer was treated with a solution of TTF solution (5 mM in 1 : 1 acetone/ethanol) was drop cast on vitamin C sensor. Next, 2 mI_ of chitosan (1.0%) and 0.5 mI_ of glutaraldehyde (0.5%) were sequentially cast on the TTF-modified electrode to yield the working electrode for measuring vitamin C.

After washing and drying, the SAM layer at the vitamin D electrode was treated with a solution of 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide / N- hydroxysuccinimide (0.4/.01 M). After washing and drying, the electrode was incubated with 10 pL of 50 pg mL-1 Vit D-specific antibody (CAb) (V00202, Genscript) solution (pH 6.5) during 60 minutes for the covalent attachment of the CAb on the activated carboxylic groups of the SAM. Then the free unreacted carboxylic groups were deactivated by incubating 10 pL of 2 M ethanolamine solution to yield the working electrode for measuring vitamin D, respectively the sensor for measuring vitamin C and D, ("SCD") used in the following. General procedure for measuring vitamin D

10 mI_ of the aqueous medium to be measured a solution is supplemented with 200 ng/ml_ of horseradish peroxidase-conjugated vitamin D forming the contact solution. This solution was deposited on the area of sensing of the vitamin D working electrode during 20 minutes. After washing of the sensor with water and drying, 20 mI_ of a redox solution of H2O2 and 3,3',5,5'-tetramethylbenzidine as mediator were applied on the area of sensing of the vitamin D working electrode. After said application, the vitamin D related electrochemical signal (current I) is detected and measured during the time t by chronoamperometry by applying -0.1 V for 2.5 minutes, so that the cathodic current obtained corresponding to the HRP- catalyzed reduction of H2O2 is inversely proportional to the Vit D concentration.

I iso s represents the current measured after 2.5 minutes (150 seconds) of contact.

General procedure for measuring vitamin C and D 10 mI_ of the aqueous medium to be measured a solution is supplemented with 200 ng/mL of horseradish peroxidase-conjugated vitamin D forming the contact solution. This solution was deposited on the area of sensing of both the vitamin C and D working electrodes as well as the counter electrode. The amperometric response is immediately recorded on working electrode for vitamin C at +0.1 V for 60 seconds. o s represents the current measured after 60 seconds of contact. The electrodes are allowed to incubate for 20 min with the contact solution. After washing the sensor with water and drying, 20 mI_ of a redox solution of H2O2 and 3,3',5,5'-tetramethylbenzidine as mediator were applied on the area of sensing of the vitamin D working electrode. After said application the vitamin D related electrochemical signal is detected and measured by chronoamperometry by applying -0.1 V for 2.5 minutes, so that the cathodic current obtained corresponding to the HRP-catalyzed reduction of H2O2 is inversely proportional to the Vit D concentration l o s represents the current measured after 2.5 minutes (150 seconds) of contact. Example 1: Measuring different concentration of vitamin D using Sensor SD

In a first example, different concentrations in human saliva of vitamin D ranging from 0 to 200 ng/mL in 50 ng/mL increments, have been prepared as contact solution by adding the respective amounts of vitamin D to saliva and measured using the sensor SD according to the method described above.

In figure 9 the chronoamperometric responses of the sensor SD measured for the different solutions of saliva are represented. The analysis further show an in verse linear correlation between hso s and the concentration of vitamin D in the con- tact solution, respectively saliva, as can be seen from the insert graph in figure 9.

These findings of example 1 can also be found when using a sensor SCD instead of the sensor SD (see example 8).

Example 2: Measuring concentration of vitamin D in saliva after intake of vitamin D using Sensor SD

Different amounts of vitamin D (0, 1000 IU, 2000 IU, 5000 IU, 10000 IU) have been administered to a human. After 30 minutes, a sample of saliva taken from said human has been measured using the sensor SD according to the method described above. Figure 10 show the chronoamperometric response of the different saliva samples. The saliva measured with no administered vitamin D serves as reference (blank) for the further analysis.

In the insert graph in figure 10, the differences Ahsos of the values hsos between the measured saliva and the blank are plotted against the specific intake of amount of vitamin D.

These findings of example 2 can also be found when using a sensor SCD instead of the sensor SD (see example 8) Example 3: Measuring concentration of vitamin D in saliva after intake of vitamin D by different individuals using Sensor SD

A pill of 10000 IU of vitamin D has been administered to two different humans. A sample of saliva has been taken from both individuals after 20 minutes, 40 minutes and 60 minutes and measured using the sensor SD according to the method described above.

Figure 11 shows the chronoamperometric response for the first individuum ( Indivl ) and the second individuum ( Indiv2 ) after different time of ingestion (ti nt ). The respective insert graph shows the difference of hsos as compared Ahsos after different time of ingestion (ti nt ).

The analysis shows that the first individuum has a higher level of vitamin D as detected in the saliva of reference (i.e. without any intake of vitamin D pills) (represented by dotted line) as compared to the second individuum. The analysis further shows that it takes longer to show an increase in vitamin D level in the saliva of the person having an low initial level of vitamin D ( Indiv2 ) as compared to the person having a higher initial level in vitamin D ( Indiv2 ).

These findings of example 3 can also be found when using a sensor SCD instead of the sensor SD (see example 8) Example 4: Measuring concentration of vitamin D in saliva after regular intake of vitamin D using Sensor SD

To two individuals a pill of 2000 IU vitamin D has been daily administered over a longer period of 4 days, respectively 4 weeks (marked by hatching in figure 12) and tested each 2 days, respectively each week. Before the first intake and after the last intake of vitamin D further measurements of saliva have been additionally measured. The chronoamperometric response has been measured using the sensor SD according to the method described above.

The graph for the first individuum in figure 12 shows that the concentration of vitamin D increases gradually over the 4 days of intake of vitamin D. It also shows that after stopping the supplementation, the concentration drops again to roughly the initial level of vitamin D at a same rate as the increase has occurred.

The graph for the second individuum shows in the lower part of figure 12 that the concentration of vitamin D increases significantly in the first week and less in the following 2 weeks to reach a peak of about 3 weeks. After stopping the supplementation, the concentration of vitamin D in the saliva gradually decreases, however, still remains after additional 2 weeks at a significantly higher level as compared to the initial level of vitamin D.

These findings of example 4 can also be found when using a sensor SCD instead of the sensor SD (see example 8)

Example 5: Measuring different concentration of vitamin C using Sensor SC

Different concentrations of vitamin C ranging from 0 to 550 mM in 50 pM increments, in human saliva as contact solution have been prepared by adding the respective amounts of vitamin C to saliva and measured using the sensor SC according to the method described above.

In figure 13 the chronoamperometric responses of sensor SC measured for the different solutions of saliva are represented. The analysis further show a linear correlation between o s and the concentration of vitamin C in the contact solution, respectively saliva, as can be seen from the insert graph in figure 13.

These findings of example 5 can also be found when using a sensor SCD instead of the sensor SC (see example 7)

Example 6: Measuring different ingredients using Sensor SC Untreated saliva and saliva with a specific added concentrations of 500 pM of the ingredients as listed in table 2

Table 2. Different ingredients added to saliva. Their chronoamperometric responses has been measured using the sensor SC to check the selectivity of the sensor.

In figure 14 the chronoamperometric responses of sensor SC measured for these different solutions of saliva are represented. It shows that the sensor responses selectively to vitamin C.

These findings of example 6 can also be found when using a sensor SCD instead of the sensor SC (see example 7)

Example 7: Measuring different concentration of vitamin C using Sensor SCD

Different concentrations of vitamin C ranging from 0 to 800 mM in 100 pM increments, in human saliva as contact solution have been prepared by adding the respective amounts of vitamin C to saliva and measured using the sensor SCD for measuring vitamin C and D according to the method described above.

In figure 15 the chronoamperometric responses of sensor SCD measured for the different solutions of saliva are represented. The analysis further shows a linear correlation between o s and the concentration of vitamin C in the contact solution, respectively saliva, as can be seen from the insert graph in figure 15.

Both sensors SC and SCD measure adequately the concentration of vitamin C in a very high reproducibility and reliability as can be derived from the comparison of figure 15 and 13 (example 5).

Example 8: Measuring different concentration of vitamin D using Sensor SCD

Different concentrations of vitamin D ranging from 0 to 200 ng/mL in 50 ng/mL increments, in human saliva as contact solution have been prepared by adding respective amounts of vitamin D to saliva and measured using the sensor SCD for measuring vitamin C and D according to the method described above.

In figure 16 the chronoamperometric responses of sensor SCD measured for the different solutions of saliva are represented. The analysis further show an inverse linear correlation between hso s and the concentration of vitamin D in the contact solution, respectively saliva, as can be seen from the insert graph in figure 16.

Both sensors SD and SCD measure adequately the concentration of vitamin D in a very high reproducibility and reliability as can be derived from the comparison of figure 16 and 9 (example 1). Example 9: Measuring different concentration of vitamin C and D using Sensor SCD: Crosstalk-Evaluation

Different combinations of concentrations of vitamin C and vitamin D have been prepared (table 3) and the chronoamperometric responses measured using the sensor SCD for measuring vitamin C and D according to the method described above to check if there is crosstalk between the electrodes, respectively the vitamins.

Table 3. Concentration of different combinations of vitamin C and D for evaluation of cross-talk.

In figure 17 the chronoamperometric responses of sensor SCD measured for the different solutions of saliva of table 3 are represented.

The comparison of the chronoamperometric responses show that there exists no cross-talk in sensor SDC.

The results of example 9 shows that aqueous media comprising vitamin C or D can be excellently measured with the same sensor SCD.

Example 10: Measuring different concentration of vitamin C and D using Sensor SCD Different combinations of concentrations of vitamin C and vitamin D have been prepared (table 4) and the chronoamperometric responses measured using the sensor SCD for measuring vitamin C and D according to the method described above to check the response of the sensor.

Table 4. Concentration of different combinations of vitamin C and D. In figure 18a the chronoamperometric responses of sensor SCD measured for the different solutions A',B' and C of saliva of table 4, which have a variable concentrations of vitamin C at a fixed concentration of vitamin D, are represented. The insert graph in figure 18a shows a representation of the values of le os for vitamin C and -hso for vitamin D for these solutions.

In figure 18b the chronoamperometric responses of sensor SCD measured for the different solutions D',E' and F' of saliva of table 4, which have a variable concentrations of vitamin D at a fixed concentration of vitamin C, are represented. The insert graph in figure 18b shows a representation of the values of /60 s for vitamin C and -hso for vitamin D for these solutions.

The results of example 10 conclude that the sensor SCD shows an inverse linear response for concentrations in vitamin D and a linear response for concentrations of vitamin C also in the presence of the other vitamin.

This shows that the sensor SCD is excellently suited for the measurement of solutions comprising both vitamin C and D.