Biosensor for continuous monitoring of metabolites and proteins and methods of manufacture thereof

• US 8,608,922 B2
• Filed: 11/09/2009
• Issued: 12/17/2013
• Est. Priority Date: 11/07/2008
• Status: Active Grant

• Current Assignee: University of Connecticut (State of Connecticut)
• Original Assignee: University of Connecticut (State of Connecticut)
• Inventors: Papadimitrakopoulos, Fotios, Vaddiraju, Santhisagar, Jain, Faquir Chand, Tomazos, Ioannis C.
• Primary Examiner(s): Ball, J. Christopher
• Application Number:
  US12/614,893
• Publication Number:
  US 20100116691A1
• Time in Patent Office: 1,499 Days
• Field of Search: 204/400, 2044031-40315, 205/775, 205/777.5, 205/778, 205/792, 600345-348
• US Class Current: 204/403.12
• CPC Class Codes: C12Q 1/002 Electrode membranes
• CPC Class Codes: G01N 33/6803 General methods of protein analysis not limited to specific proteins or families of proteins

Abstract

A biosensor comprises a substrate; a reference electrode; a working electrode; a counter electrode; and a plurality of permeability adjusting spacers. The reference electrode, the working electrode and the plurality of permeability adjusting spacers are all being disposed to be substantially parallel to each other to create a plurality of enzyme containing porous sections. The enzyme containing porous sections contain an enzyme; where the enzyme is operative to react with a metabolite to determine the concentration of the metabolite. By combining a number of the aforementioned biosensors, the differential concentration of a target enzyme or protein is determined by monitoring the changes on its metabolite substrates.

Claims

1. A measuring device for determining the concentration of metabolite in a sample media, comprising:

a reference electrode;

a counter electrode;

a working electrode;

a first permeability-adjusting spacer;

the first permeability adjusting spacer being opposedly disposed to the working electrode and forming a first enzyme-containing porous section between the working electrode and the first permeability-adjusting spacer, the first enzyme-containing porous section being in direct contact with the working electrode;

a first set of enzymes the first set of enzymes being disposed on a first porous matrix that is disposed in the first enzyme-containing porous section;

a second permeability-adjusting spacer;

the second separating spacer being opposedly disposed to the first permeability-adjusting spacer and forming a second enzyme-containing porous section between the first permeability-adjusting spacer and the second permeability adjusting spacer;

a second set of enzymes;

the second set of enzymes being disposed on a second porous matrix that is disposed in the second enzyme-containing porous section;

a third permeability-adjusting spacer;

the third permeability-adjusting spacer being opposedly disposed to the second permeability-adjusting spacer and forming a third enzyme-containing porous section between the second permeability-adjusting spacer and the third permeability-adjusting spacer;

a third set of enzymes;

the third set of enzymes being disposed on a third porous matrix that is disposed in the third enzyme-containing porous section;

wherein the first set of enzymes initiates a sequence of reactions that transform the metabolite into an electrochemically active species whose concentration can be sensed by the working electrode;

wherein the second set of enzymes has the ability to recycle the electrochemically species generated by the first set of enzymes and transform it to a co-substrate for at least one enzyme of the first set of enzymes; and

wherein the second set of enzymes has an ability to store excess of the co-substrate and provide it to the first set of enzymes as well as the third set of enzymes;

wherein the third set of enzymes converts interfering electrochemically active species into electrochemically inactive byproducts in conjunction with the co-substrate stored in the second set of enzymes;

wherein the third set of enzymes contains a portion of enzymes from the first set of enzymes to prevent by-products of the enzymatic sequence of reactions established by first set of enzymes that are present in the sample media to diffuse inwards and contribute to a production of the electrochemically active species that is sensed by the working electrode; and

wherein the permeability-adjusting spacers assist in controlling diffusion of the metabolite, its enzymatic byproducts, co-substrates and interfering electrochemically active species through the first enzyme containing porous section, the second enzyme containing porous section and the third enzyme containing porous section in order to establish operative detection of metabolite over a range of concentration and minimum signal from other metabolites and interfering electrochemically active species.

2. The measuring device of claim 1, further comprising a fourth set of enzymes;

the fourth set of enzymes being added to assist with the removal of enzymatic byproducts from said first, second and third enzyme-containing porous sections.

3. The measuring device of claim 1, further comprising the formation of a plurality of vertical cavities throughout the first enzyme containing porous section, the second enzyme containing porous section and the third enzyme containing porous section.

4. The measuring device of claim 1, where the first permeability adjusting spacer, the second permeability adjusting spacer and the third permeability adjusting spacer assist with the removal of enzymatic byproducts from said first enzyme containing porous section, the second enzyme-containing porous section and/or the third enzyme-containing porous section.

5. The measuring device of claim 1, where the electrochemically active species is H₂O₂.

6. The measuring device of claim 1, where the co-substrate is oxygen.

7. The measuring device of claim 1, where the sample media is a sample fluid, a body fluid, a tissue fluid or serum.

8. The measuring device of claim 1, where the sample media is the body of a living being.

9. The measuring device of claim 1, where the metabolite is glucose, lactate, oxygen, glutamate, choline, phosphate, acetylcholine, dioxybutyrate, homocysteine, cysteine, creatine, creatinine, sucrose, fructose, nitric oxide, galactose, arsenite, cholesterol, fructosamine, bilirubin, glycine, methionine, L-citrulline, phosphatidic acid, lysophospatidic acid, arachidonic acid, asymmetric dimethylarginine, 1,3-diaminopropane, 21-deoxycortisol, aminoadipic acid, D-2-hydroxyglutaric acid, L-2-hydroxyglutaric acid, aminoadipic acid, 2-hydroxyadipic acid, oxoadipic acid, oxoglutaric acid, 7-hydroxyprogesterone, 3-hydroxyisovaleric acid, 3-hydroxymethylglutaric acid, 3-methylcrotonylglycine, 3-methylglutaconic acid, adipic acid, ammonia, methylglutaric acid, (S)-3dydroxyisobutyric acid, 3-hydroxyisovaleric acid, 3-methylcrotonylglycine, 3-hydroxyisovaleric acid, pyruvic acid, (S)-3,4-dihydroxybutyric acid, pyroglutamic acid, ganglioside GM3, glucosylceramide, lactosylceramide, tetrahexosylceramide, trihexosylceramide, 2-hydroxyestradiol, 2-hydroxyestrone, 20-hydroxyeicosatetraenoic acid, 5-acetylamino-6-amino-3-methyluracil, alpha-N-phenylacetyl-L-glutamine, androstenedione, benzoic acid, bromide, cadaverine, cholic acid, coproporphyrin I, coproporphyrin III, deoxycholic acid, deoxycytidine, DHEA sulfate, DL-homocystine, estradiol, estriol, estrone, estrone sulfate, fluorine, glycocholic acid, guanine, hexanal hydroxyphenyllactic acid, iodide, L-aspartic acid, L-cystine, L-glutamine, L-lactic acid, L-malic acid, L-mefhionine, malondialdehyde, myo-inositol hexakisphosphate, N-acetylaspartylglutamic acid, orotidine, progesterone, salicyluric acid, selenomethionine, thymine, uric acid, vanilpyruvic acid, Cortisol, anabasine, cotinine, cydroxycotinine, L(−

)-nicotine pestanal nornicotine, L-lactic acid, heptacarboxylporphyrin I, enkephalin L, 24-hydroxycholesterol, 27-hydroxycholesterol, epinephrine, deoxyadenosine, 1-methyladenine, succinyladenosine, hexacosanoic acid, phytanic acid, pristanic acid, L-pipecolic acid, erucic acid, 7C-aglycone, 5C-aglycone, (R)-salsolinol, alpha-carotene, 5-meflyvltetrahydrofolic acid, butyric acid, mannitol meopterin, quinolinic acid, 2-butanol, acetone, butanone, ethanol, isopropyl alcohol, methanol, acetaldehyde, nicotinic acid, pantothenic acid, riboflavin, scyllitol, thiamine, homnogentisic acid, aminoadipic acid, L-histidine, 1,5-anhydrosorbitol, 1-methylhistidine, 3,4-dihydroxybenzeneacetic acid, 3-methylhistidine, 4-hydroxy-L-proline, 4-hydroxynonenal, 5-hydroxylysine, 8-hydroxyguanine, 8-hydroxyguanosine, anscrine, carnosine, citrulline, dopamine, epsilon-(gamma-glutamyl)-lysine, folic acid, fumaric acid, galactitol, gamma-aminobutyric acid, glycerophosphocho line, glycylproline, hydroxyproline, L-2,4-diaminobutyric acid, L-alpha-aminobutyric acid, L-arabitol, L-arginine, L-asparagine, L-cystathionine, L-DOPA, L-glutamic acid, L-isoleucine, L-leucine, L-lysine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, methylmalonic acid, myoinositol, ornithine, pentosidine, phosphorylcholine, prolylhydroxyproline, ribitol, sorbitol, succinic acid, thiamine monophosphate, thiamine pyrophosphate, estrioB-sulfate 16-glucuronide, estriol-3-glucuronide, acetylglycine, N-acetylscrine, L-thyronine, prostaglandin E2, kynurenic acid, 24,25-dihydroxyvitamin D, 25,26-dihydroxyvitamin D, 25-hydroxyvitamin D2, calcidiol, ergocalciferol, vitamin D3, 11-dehydro-thromboxane B2, 5a-tetrahydrocortisol, ethylmalonic acid, FAD, flavin mononucleotide, glutaric acid, isovalerylglycine, liothyronine, suberic acid, tetrahydrocortisone, thyroxine, 3-hydroxybutyric acid, acetoacetic acid, isocitric acid, L-glutamic acid, L-malic acid, oxalacetic acid, indolcacetic acid, argininosuccinic acid, uracil, 3-methoxytyrosine, 5-mydroxyindoleacetic acid, homovanillic acid, N-acetyl-L-tyrosine, N-acetylvanilalanine, vanillylmandelic acid, vanylglycol, taurocyamine, aspartylglycosamine, 1,3,7-trimethyluric acid, 1,3-dimethyluric acid, 1,7-dimethyluric acid, 1-methylxanthine, 1 lb-PGF2a, 3-chlorotyrosine, 3-methylxanfhine, 5-HETE, 7-methylxanthine, caffeine, paraxanthine, theobromine, theophylline, iodotyrosine, dimethyl-L-arginine, 13S hydroxyoctadecadienoic acid, symmetric dimethylarginine, androstanediol, trans-trans-muconic acid, 2-methyl-3-hydroxybutyric acid, 2-methylacetoacetic acid, tiglylglycine, acetaminophen glucuronide, ubiquinol, dihydrothymine, urcidoisobutyric acid, chenodeoxycholic acid, chenodeoxycholic acid glycine conjugate, hyaluronic acid, taurochenodesoxycholic acid, taurocholic acid, lb,3a,12a-trihydroxy-5b-cholanoic acid, hyocholic acid, hyodeoxycholic acid, ursodeoxycholic acid, isoursodeoxycholic acid, lithocholic acid, ursocholic acid, 2-methylcitric acid, 3-methylcrotonylglycine, hydroxypropionic acid, 2-pyrrolidinone, dimethylamine, 8-isoprostane, ascorbic acid, glutathione, o-phosphoethanolamine, 3,5-diiodothyronine, 1,3-diaminopropane, 1-methylguanosine, 16α

-hydroxy estrone, enterodiol, enterolactone, Nl-acetylspermidine, N8-acetylspermidine, perillic acid, perillyl alcohol, ribothymidine, xanthosine, testosterone, 1-methyluric acid, 3-methyladenine, citric acid, cytidine, hypoxanthine, inosine, N-acetylL-aspartic acid, orotic acid, oxidized glutathione, pseudouridine, thymidine, uridine, xanthine, 1-methylinosine, 16ahydroxydehydroisoandrosterone, 5a-tetrahydrocorticosterone, alpha-linolenic acid, alpha-tocopherol, B-carotene, beta-cortol, docosahexaenoic acid, docosapentaenoic acid, gama-tocopherol, linoleic acid, lycopene, putrescine, tetrahydrodeoxycorticosterone, tetrahydrodeoxycortisol, vitamin A, L-fucose, prostaglandin F2a, leukotriene B4, 6-keto-prostaglandin Fla, sebacic acid, butyrylcarnitine, decanoylcarnitine, dodecanoylcarnitine, isovalerylcarnitine, L-hexanoylcarnitine, L-octanoylcarnitine, L-palmitoylcarnitine, lactulose, propionylcarnitine, stearoylcarnitine, tiglylcarnitine, dihydrouracil, 5alphacholestanol, lathosterol, 1-methyladenosine, 3,5-diiodo-Ltyrosine, betaine, cyclic AMP, guanidine, guanidinosuccinic acid, guanidoacetic acid, methylguanidine, picolinic acid, 2,3-butanediol, 2-hydroxyphenethylamine, 2-oxoarginine, 4-guanidinobutanoic acid, 7a-hydroxycholesterol, argininic acid, cholesterol sulfate, homo-L-arginine, methanethiol, p-octopamine, propylene glycol, sulfolithocholylglycine, tyramine, urea, L-kynurenine, beta-leucine, cob(I)alamin, inosinic acid, 16-a-hydroxypregnenolone, pyridinoline, histamine, lipoxin A4, hydrogen peroxide, thromboxane A2, D-xylose, 19-hydroxyandrost-4-ene-3,17-dione, glyceric acid, L-a-glutamyl-L-lysine, corticosterone, cortisone, 1-methylhistamine, (R)-3-hydroxybutyric acid, (R)-3-hydroxyisobutyric acid, (S)-3-hydroxyisobutyric acid, 1-butanol, 4-heptanone, D-lactic acid, glycerol, hyaluronan, L-carnitine, pyruvaldehyde, S-adenosylmefhionine, hydrogen carbonate, ureidopropionic acid, beta-alanine, cortol, cortolone, leukotriene C4, leukotriene E4, adenosine triphosphate, ADP, guanosine diphosphate, guanosine triphosphate, p-hydroxyphenylacetic acid, taurine, 2-methylbutyrylglycine, isobutyrylglycine, methylsuccinic acid, N-butyrylglycine, epitestosterone, thyroxine sulfate, norepinephrine, etiocholanolone, diphenhydramine, 3-hydroxydodecanoic acid, diadenosine hexaphosphate, diadenosine pentaphosphate, diadenosine tetraphosphate, diadenosine triphosphate, xanthurenic acid, cyanocobalamin, pyridoxine, hydrogen sulfide, thiosulfate, aldosterone 18-glucuronide, p-synephrine, m-tyramine, serotonin, 1-naphthol, 2-naphthol, retinyl ester, 2-pyrocatechuic acid, gentisic acid, dopamine glucuronide, isomaltose, melanin, N2,N2-dimethylguanosine, phenylacetic acid, trimethylamine N-oxide and a combination comprising at least one of the foregoing metabolites.

10. The measuring device of claim 1, where the metabolite comprises an ion.

11. The measuring device of claim 10, where the wherein the ion is a hydrogen ion, a chlorine ion, a potassium ion, a calcium ion, an irons ion, a phosphate ion, an aluminum ion, an barium ion, a beryllium ion, a bismuth ion, a cadmium ion, a cobalt ion, a copper ion, a palladium ion, a lithium ion, a magnesium ion, a manganese ion, a mercury ion, a molybdenum ion, a nickel ion, a silicon ion, a strontium ion, a tin ion, a titanium ion, a tungsten ion, a vanadium ion, a zinc ion, a nitrate ion, a chromium ion, or a combination comprising at least one of the foregoing ions.

12. The measuring device of claim 1, where the working electrode comprises a metal or a carbonaceous material;

where the metal is platinum, gold, silver, rhodium, iridium or a combination comprising at least one of the foregoing metals.

13. The measuring device of claim 12, where the carbonaceous material comprises carbon nanotubes, carbon black, graphite, graphene sheets, or a combination comprising at least one of the foregoing carbonaceous materials.

14. The measuring device of claim 1, where the working electrode comprises metals that are in nanosized forms:

the nanosized forms being nanotubes, nanorods, nanowhiskers, nanoonions, nanohorns, nanoparticles, nanoplatelets or a combination comprising at least one of the foregoing nanosized forms.

15. The measuring device of claim 1, where the working electrode is covered with a permeability adjusting membrane to limit diffusion of molecules larger than H₂O₂ towards the said working electrode.

16. The measuring device of claim 15, where the permeability adjusting membrane is an electropolymerized film.

17. The measuring device of claim 16, where the electropolymerized film is composed by a conducting polymer and its composites with biological mediators, nanotubes, graphene, nanoparticles, nanorods, nanowhiskers, nanoonions, nanohorns, nanoplatelets and mixtures thereof.

18. The measuring device of claim 17, where the conducting polymer and its composites is composed by poly(o-phenylene diamine) and its composites with flavin mononucleotide wrapped single walled carbon nanotubes.

19. The measuring device of claim 1, where the first set of enzymes, the second set of enzymes and the third set of enzymes are hydrolases, transferases, reductases, oxidases, polymerases, kinases, transferases, peroxidases, kinases, superoxidases, phosphatases, pyrophosphatases, oxygenases, nucleases, lipases, peptidases, transacetylases, hydroxylases, dioxygenases, dehydrogenases, carboxylases, aminases, catalases, phosphohydrolases, diaminases, reductases, synthases, kinases, caspases, methionine synthase, cystathionases, or a combination comprising at least one of the foregoing enzymes.

20. The measuring device of claim 1, where the first set of enzymes are transferases, hydrolases, oxidases, peroxidases, kinases, superoxidases, phosphatases, or a combination comprising at least one of the foregoing enzymes.

21. The measuring device of claim 1, where the first set of enzymes initiates a reaction sequence with the metabolite of choice to produce hydrogen peroxide.

22. The measuring device of claim 1, where the second set of enzymes are transferases, hydrolases, oxidases, peroxidases, kinases, superoxidases and phosphatases or a combination comprising at least one of the foregoing enzymes.

23. The measuring device of claim 1, where the second set of enzymes comprise two or more enzymes, one of which is operative to store oxygen and release it in oxygen deficient conditions, while the other is operative to generate oxygen.

24. The measuring device of claim 23, where the second set of enzymes comprise myoglobin and catalase.

25. The measuring device of claim 1, where the third set of enzymes are transferases, hydrolases, oxidases, peroxidases, kinases, superoxidases, phosphatases, pyrophosphatases, oxygenases, nucleases, lipases, peptidases, transacetylases, hydroxylases, dioxygenases, dehydrogenases, carboxylases, aminases, catalases, phosphohydrolases, diaminases, reductases, synthases, kinases, caspases, methionine synthase, cystathionases, or a combination comprising at least one of the foregoing enzymes.

26. The measuring device of claim 1, where the first enzyme containing porous section, second enzyme containing porous section, and third enzyme containing porous section each comprise a network of polymer brushes.

27. The measuring device of claim 26, where the network of polymer brushes comprises water soluble polymers;

the water soluble polymers being polyethylene oxide, polyvinyl acetate, hydroxypropylcellulose, polyvinyl alcohol, polyhexaethyl methacrylate, polyallyl amine, poly(hyaluronic acid), chitosan, polysugars, polyitaconic acid, or a combination comprising at least one of the foregoing water soluble polymers.

28. The measuring device of claim 26, where the network of polymer brushes comprises of a network of porous conductive materials.

29. The measuring device of claim 26, where the network of polymer brushes comprises water soluble forms of intrinsically conducting polymers;

the intrinsically conducting polymers being polyaniline, substituted polyanilines, polypyrroles, substituted polypyrroles, polythiophenes, substituted polythiophenes, polyacetylenes, polyethylene dioxythiophenes, polyethylenedioxypyrroles, polyp-phenylene vinylenes, polycarbazoles, substituted polycarbazoles, polyindoles, poly(o-phenylene diamine)s or a combination comprising at least one of the foregoing intrinsically conducting polymers.

30. The measuring device of claim 26, where the network of polymer brushes is a network of porous conductive materials;

the network of porous conductive materials being realized by electropolymerization ofo-phenylene diamine, pyrrole, aniline, aniline, sulfonated aniline, sulfonated thiophenes, flavin mononucleotide, substituted anilines, substituted pyrroles, substituted thiophenes, acetylenes, polyethylene dioxythiophenes, ethylenedioxypyrroles, phenylene vinylenes, carbazoles, substituted carbazoles, indoles, carboxy-functionalized aqueously dispersed carbon nanotubes, flavin mononucleotide coated single wall carbon nanotubes, aqueous dispersed nanoparticles with aniline functionalities, and zirconium phosphate nanoplatelets with aniline functionalities.

31. The measuring device of claim 26, where the electropolymerized network of porous conductive materials is sequentially conducted in the presence of the desired enzyme or mixture of enzymes to lead in stratified layers of enzyme contained porous conductive materials.

32. The measuring device of claim 26, where the network of polymer brushes is a network of porous conductive materials;

the network of porous conductive materials being realized by electropolymerization of monomers;

where the electropolymerized monomers are further grafted with a polyethylene oxide oligomer.

33. The measuring device of claim 26, where the network of polymer brushes comprises a vertical forest of nanorods attached at one of their two ends on the working electrode.

34. The measuring device of claim 33, where the nanorod forest is made of carbon nanotubes.

35. The measuring device of claim 1, wherein the first permeability-adjusting spacer, the second permeability-adjusting spacer and the third permeability-adjusting spacer comprise an organic polymer.

36. The measuring device of claim 35, where the organic polymer is a polyacetal, a polyolefin, a polyacrylic, a polycarbonate, a polystyrene, a polyester, a polyamide, polyamideimides, a polyarylate, a polyarylsulfone, a polyethersulfone, a polyphenylene sulfide, a polyvinyl chloride, a polyethylene oxide, a polysulfone, a polyimide, a polyetherimide, a polytetrafluoroethylene, a polyetherketone, a polyether etherketone, a polyether ketone ketone, a polybenzoxazole, a polyphthalide, a polyacetal, a polyanhydride, a polyvinyl ether, a polyvinyl thioether, a polyvinyl alcohol, a polyvinyl ketone, a polyvinyl halide, a polyvinyl nitrile, a polyvinyl ester, a polysulfonate, a polysulfide, a poly(allyl amine), a polythioester, a polysulfone, a polysulfonamide, a polyurea, a polyphosphazene, a polysilazane, a polyvinylchloride, a polyvinyl acetate, a humic acid, a cellulose acetate, a polythiophene, a polyphenylene diamine, a polypyrrole, a polynaphthalene a polyurethane, an ethylene propylene diene rubber, a polytetrafluoroethylene, a fluorinated ethylene propylene, a perfluoroalkoxyethylene, a polychlorotrifluoroethylene, a polyvinylidene fluoride, a polysiloxane, or a combination comprising at least one of the foregoing organic polymers.

37. The measuring device of claim 35, where the organic polymer is poly(o-phenylene diamine).

38. The measuring device of claim 35, where the organic polymer is realized by electropolymerization from a water solution containing one or more monomers selected from o-phenylene diamine, pyrrole, aniline, aniline, sulfonated aniline, sulfonated thiophenes, flavin mononucleotide, substituted anilines, substituted pyrroles, substituted thiophenes, acetylenes, polyethylene dioxythiophenes, ethylenedioxypyrroles, phenylene vinylenes, carbazoles, substituted carbazoles, indoles, carboxy-functionalized aqueously dispersed carbon nanotubes, flavin mononucleotide coated single wall carbon nanotubes, aqueous dispersed nanoparticles with aniline functionalities, and a combination comprising at least one of the foregoing monomers.

39. The measuring device of claim 2, where the fourth set of enzymes are transferases, hydrolases, oxidases, peroxidases, kinases, superoxidases, phosphatases, pyrophosphatases, oxygenases, nucleases, lipases, peptidases, transacetylases, hydroxylases, dioxygenases, dehydrogenases, carboxylases, aminases, catalases, phosphohydrolases, diaminases, reductases, synthases, kinases, caspases, methionine synthase, cystathionases, or a combination comprising at least one of the foregoing enzymes.

40. The measuring device of claim 39, where the fourth set of enzymes is distributed within the first enzyme containing porous section, second enzyme containing porous section, and third enzyme containing porous section, or in a combination comprising at least one of the first enzyme containing porous section, the second enzyme containing porous section, and the third enzyme containing porous section.

41. The measuring device of claim 2, where the said fourth set of enzymes is distributed within hydrogels that impregnate and fills a plurality of vertical cavities throughout the first enzyme containing porous section, the second enzyme containing porous section and the third enzyme containing porous section and/or the first permeability adjusting spacer, the second permeability adjusting spacer and the third permeability adjusting spacer.

42. The measuring device of claim 1, where the at least one of the first enzyme containing porous section, the second enzyme containing porous section and the third enzyme containing porous section and/or the first permeability adjusting spacer, the second permeability adjusting spacer and the third permeability adjusting spacers can be deposited by spin coating, drop casting, dip coating, knife coating, spray coating, inkjet printing, or electropolymerization.

43. The measuring device of claim 38, where electropolymerization of o-phenylene diamine is performed at different pHs.