Patent Application
20210010050
The present invention relates to a liquid composition comprising a protein, a polyphenol and a morphogen; to a solid composition comprising the cross-linked product of a protein and a polyphenol; to a biosensor and a biofuel cell comprising said solid composition bound to a surface of an electrochemical probe; to a process for the detection of an analyte with said biosensor; and to the use of a ferrocene as a morphogen in an electrodeposition process.
The integration of biomolecules, such as proteins and antibodies, onto electronic platforms is used to obtain functional bioelectronic devices for the detection of biomarkers. In biomedical applications, proteins are commonly used as recognition elements due to their high specificity, selectivity and catalyst efficiency in physiological conditions. Protein-based biosensors are based on the use of proteins to transduce biorecognition as electric output signals. In medical diagnosis and environmental-monitoring, protein-based biosensors are widely applied due to their high sensitivity, potential selectivity, and their possibility of miniaturization/automation.
Protein-based biosensors are a combination of an electrochemical probe, such as an electrode, having an active protein immobilized on a surface thereof. An extensive variety of redox enzymes, such as glucose oxidase (GOX), horseradish peroxidase, lactate oxidase, alcohol dehydrogenase, aldehyde dehydrogenase or urease, have been used for the elaboration of glucose, H2O2, lactate, ethanol, aldehyde or urea biosensors, respectively. Enzymatic micro-biosensors have also been developed for short-term brain applications in animal models to monitor neurotransmitters, such as glutamate or choline, using immobilized glutamate or choline oxidase, respectively. GOX biosensors have been extensively developed due to their ability to detect blood glucose and their effectiveness in the diagnostic analysis of diabetes. A representative case of success is the glucometer with test strips.
Protein immobilization is a critical process in the development of biosensors, as is the necessity to avoid their denaturation and ensure their accessibility towards the analyte.
Different techniques have been implemented for the immobilization of proteins onto solid surfaces, including adsorption, covalent binding, entrapment, cross-linking or affinity. However, protein adsorption and entrapment exhibit undesirable protein leaking issues. Covalent binding and cross-linking are able to increase enzyme stability but often exhibit reduced activity. The lack of accuracy of the systems obtained with these known techniques is a major concern. Low accuracy of the fabrication protocols, high sensitivity of proteins to the immobilization protocol or environmental factors can affect the reliability and reproducibility of glucose measurements.
Electrodeposition of macromolecules is increasingly considered to be the most suitable method for the design of biosensors. Indeed, said method is a simple and attractive bottom-up approach and can be used to finely control the immobilization of a protein on a surface of an electrode with an electrical stimulus. Conventional electrodeposition processes used to develop protein-based biosensors can be divided into three main categories:
(i) precipitation of polyelectrolytes or proteins through change of solubility,
(ii) self-assemblies of polyelectrolytes through electrostatic/ionic interactions; and
(iii) formation of covalent bonds between monomers (electropolymerization).
Another recent electrodeposition method is the electro-cross-linking process. Protein immobilization by electro-cross-linking is more efficient than conventional manual cross-linking by drop- or dip-coating of liquid protein preparations containing suitable cross-linkers, such as glutaraldehyde. Indeed, electro-cross-linking offers better reproducibility and better control of the immobilization process and can be used for the development of miniaturized biosensors through the functionalization of specific electrodes out of an assorted microelectrode array.
The Applicants have developed a concept named morphogenic electrotriggered self-construction of films, based on electro-cross-linking between two polymers in one pot using a morphogen. A morphogen is a molecule or an ion that is produced at an interface and diffuses into the solution, thus creating a concentration gradient and locally inducing a chemical reaction or interaction between two non-interacting species.
Rydzek, G. et al. “Electrochemically Triggered Assembly of Films: A One-Pot Morphogen-Driven Buildup”, Angew. Chemie—Int. Ed., 2011, 50, 4374-4377 discloses the use of Cu(I) as a morphogen to obtain polymeric films using a Cu(I)-catalyzed click reaction between azides and alkynes. However, said method is not adapted to the electro-cross-linking of proteins since proteins do not comprise alkyne or azide functional groups.
Maerten, C. et al. “Electrochemically Triggered Self-Construction of Polymeric Films Based on Mussel-Inspired Chemistry”, Langmuir, 2015, 31, 13385-13393 discloses the electro-cross-linking of polyamines based on mussel-inspired chemistry using a homobifunctional catechol ethylene oxide molecule, named bis-catechol, as a cross-linker and a morphogen. Indeed, the exceptional ability of mussels to adhere on almost any type of surfaces is based on catechol biochemistry (i.e. hydrogen bonds, metal-ligand complexes and covalent bond formation). In particular, when oxidized into the corresponding quinone, a catechol is able to react with a nucleophilic group, such as an amine or thiol, through Michael addition and Schiff's base formation. However, when said method was applied to the electro-cross-linking of enzymes, the resulting film did not exhibit any enzymatic activity and could therefore not be used in the preparation of a biosensor for the detection of an analyte.
After extensive research, Applicants have developed a one-pot electrotriggered self-construction of protein-based films for second-generation biosensors by contacting an electrochemical probe with a liquid composition comprising a protein, a polyphenol and a morphogen; and applying an electric stimulus to the electrochemical probe so as to form a solid composition comprising the cross-linked product of an protein and a polyphenol on a surface of the electrode. The protein is covalently bound to the surface of the electrochemical probe and is not prone to leaking. The biosensor obtained with said solid composition allows the electrochemical detection of an analyte, such as glucose, with excellent sensitivity and selectivity. Further, the electro-cross-linking process of the invention is regioselective and can be used to develop miniaturized biosensors through functionalization of specific electrodes out of a microelectrode array.
A first object of the invention is a liquid composition comprising a protein, a polyphenol, and a morphogen.
Another object of the invention is a solid composition comprising a cross-linked product of a protein and a polyphenol.
Yet another object of the invention is a process for the preparation of the solid composition of the invention, wherein the process comprises the steps of:
The present invention is also directed to a biosensor comprising an electrochemical probe, wherein a solid composition comprising a cross-linked product of a protein and a polyphenol is bound to a surface thereof.
Further the present invention also aims at providing a process for the detection of an analyte in a sample, wherein the process comprises the steps of:
Another object of the invention is a biofuel cell comprising:
wherein a solid composition comprising a cross-linked product of a protein and a polyphenol is bound to a surface of the positive electrode and/or the negative electrode.
A final object of the invention is the use of a ferrocene as a morphogen in the formation of a solid composition on the surface of an electrode by electrodeposition.
GOX/polyphenol film of example 4 upon addition of 600 μL of different concentrations of glucose in the presence of 0.5 mmol/L ferrocene methanol during the application of +0.25 V.
As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.
Additionally, the use of “or” is intended to include “and/or”, unless the context clearly indicates otherwise.
As used herein, the term “(C1-C6)alkyl” refers to a straight or branched saturated hydrocarbon having 1 to 6 carbon atoms. Examples of such groups include, but are not limited to, methyl, 2-propyl, 1-butyl, 2-butyl, 2-methyl-2-propyl, 2-methyl-1-butyl and 1-hexyl.
As used herein, the term “(C2-C6)alkene” refers to a refers to a straight or branched unsaturated hydrocarbon having 1 to 6 carbon atoms.
As used herein, the term “(C1-C6)hydroxyalkyl” refers to a (C1-C6)alkyl that is substituted with at least one hydroxy (—OH) group.
As used herein, the term “(C1-C6)haloalkyl” refers to a″(C1-C6)alkyl that is substituted with at least one halogen atom, such as Cl, Br, I or F.
As used herein, the term “azide” refers to a —N3 group.
As used herein, the term “formyl” refers to a —CHO group.
As used herein, the term “carboxyl” refers to a —COOH group.
As used herein, the term “(C1-C6)alkanoic acid” refers to a (C1-C6)alkyl that is substituted with at least one carboxyl (—COOH) group.
As used herein, the term “(C1-C6)aminoalkyl” refers to a (C1-C6)alkyl that is substituted with at least one amino (—NH2) group.
As used herein the term “(C1-C6)alkylamino” refers to a —NHRa group wherein Ra is a (C1-C6)alkyl group.
As used herein the term “di((C1-C6)alkyl)amino” refers to a —NRaRb group wherein Ra and Rb are a (C1-C6)alkyl.
As used herein, the term “(C1-C6)alkyl-(C1-C6)alkylamino” refers to a (C1-C6)alkylamino group that is connected by a (C1-C6)alkyl radical.
As used herein, the term “(C1-C6)alkyl-di((C1-C6)alkyl)amino” refers to a di((C1-C6)alkyl)amino group that is connected by a (C1-C6)alkyl radical.
As used herein, the term “(C1-C6)carbonylalkyl” refers to a (C1-C6)alkyl group that is connected by a carbonyl (C═O) group.
As used herein, the term “(C1-C6)carboxyalkyl” refers to a (C1-C6)carbonylalkyl wherein the carbonyl group is connected by an oxygen bridge.
As used herein, the term “(C1-C6)alkyl(C1-C6)carboxyalkyl” refers to a “(C1-C6)carboxyalkyl that is connected by a (C1-C6)alkyl radical.
As used herein, the term “(C1-C6)carbonylhaloalkyl” refers to a (C1-C6)haloalkyl group that is connected by a carbonyl (C═O) group.
Liquid Composition
The liquid composition of the invention comprises a protein, a polyphenol and a morphogen.
As used herein, the term “liquid composition” refers to a composition that flows under its own weight within a temperature range of 5° C. to 50° C.
In particular, the liquid composition may be an aqueous composition. The term “aqueous composition” refers to a composition that comprises water. The aqueous composition may further optionally comprise a non-aqueous solvent.
More particularly, the liquid composition of the invention may be an aqueous solution. The term “solution” refers to homogenous liquid composition in which the different constituents, namely the protein, the polyphenol and the morphogen, are dissolved.
The liquid composition of the invention may be a buffered solution. As used herein, the term “buffered solution” relates to a solution comprising a buffering agent. Buffering agents may be weak acids or bases used to maintain the acidity (pH) of a solution near a chosen value even when further acids or bases are added. In the present invention, the oxidation of the polyphenol during the electro-cross-linking process generates H+ ions. The presence of a buffering agent in the liquid composition of the invention advantageously prevents the pH from becoming too acidic which is detrimental for the reactivity of the amine functions of the protein.
Preferably, the liquid composition of the invention may be a phosphate buffered solution. The liquid composition may, for example, comprise 50 to 250 mmol, in particular 100 to 200 mmol, more particularly 120 to 170 mmol, of phosphate per liter of solution. The liquid composition may further comprise NaCl and/or KCl. For example, the liquid composition may comprise 1 to 3 mol, in particular, 1.5 to 2.5 mol, more particularly 1.8 to 2.2 mol of NaC1 per liter of solution. The liquid composition may also comprise 0.01 to 0.1 mol, in particular, 0.02 to 0.6 mol, more particularly 0.3 to 0.5 mol, of KCl per liter of solution.
The liquid composition of the invention may exhibit a pH of 5 to 9, in particular 6 to 8, more particularly 7 to 7.8.
In the liquid composition of the invention, the protein and the polyphenol are in their free form, i.e. they are not covalently linked one with another.
The liquid composition may be flushed with nitrogen so as remove dissolved oxygen in order to prevent oxidation of the polyphenol.
Protein
The liquid composition of the invention comprises a protein.
As used herein, the term “protein” refers to a polymer of amino acids joined together by peptide bonds. Proteins perform a vast array of functions within organisms, including catalyzing metabolic reactions, DNA replication, responding to stimuli, and transporting molecules from one location to another. Proteins differ from one another primarily in their sequence of amino acids, which is dictated by the nucleotide sequence of their genes. Proteins usually exhibit a three-dimensional structure that determines its activity.
The protein may be an enzyme or an antibody.
In one embodiment, the protein may be an enzyme.
As used herein, the term “enzyme” refers to a biological macromolecule that is capable of catalyzing biochemical reactions. Generally, enzymes are proteins, such as globular proteins. The enzyme may optionally be associated with a cofactor. As used herein, the term “cofactor” refers to a non-protein compound, such as an organic molecule or a metallic ion, required for an enzyme's activity.
The enzyme may be a redox enzyme. As used herein, the term “redox enzyme” refers to an enzyme that catalyzes the transfer of electrons from one molecule, the reductant, also called the electron donor, to another, the oxidant, also called the electron acceptor.
In particular, the enzyme may be selected from a dehydrogenase, a reductase, an oxidase, an oxygenase, a peroxidase, a catalase, a transhydrogenase, a transferase, a hydrolase, a lyase, an isomerase, a ligase and a urease.
More particularly, the enzyme may be selected from glucose oxidase (GOX), horseradish peroxidase (HRP), lactate oxidase, alcohol dehydrogenase, aldehyde dehydrogenase, urease, glutamate oxidase, choline oxidase, glucose dehydrogenase, laccase, bilirubin oxidase, ascorbate oxidase, formate dehydrogenase, lactate dehydrogenase, pyruvate dehydrogenase, malate dehydrogenase, p-cresolmethylhydroxylase, methylamine dehydrogenase, succinate dehydrogenase, fumarate reductase, D-fructose dehydrogenase, D-gluconate dehydrogenase, cytochrome c, peroxidase, ferredoxin, plastocyanin, azurin, and azotoflavin.
Even more particularly, the enzyme may be GOX.
In another embodiment, the protein may be an antibody.
As used herein, the term “antibody” refers to a protein that is produced by specialized B cells after stimulation by an antigen, such as a pathogenic bacteria or a virus, and acts specifically against the antigen in an immune response.
In particular, the antibody may be an immunoglobulin, for example immunoglobulin G (IgG).
The protein may optionally be labeled with a fluorescent tag, such as rhodamine, so as to monitor the localization of the protein by fluorescence.
The liquid composition may comprise from 0.5 to 100 mmol, in particular 1 to 50 mmol, more particularly 5 to 10 mmol, of protein per liter of liquid composition.
Polyphenol
The liquid composition of the invention comprises a polyphenol. As used herein, the term “polyphenol” refers to a molecule comprising more than one phenyl group, preferably 2 to 10 phenyl groups, wherein each phenyl group is substituted by more than one hydroxy group, preferably by two or three hydroxy groups. The polyphenol is capable of being oxidized into the corresponding quinone by application of a potential with an electrode. In particular, the oxidized polyphenol is able to establish cross-links between proteins and cross-links between proteins and the surface of the electrode.
In particular, the polyphenol may correspond to the following formula (I):
wherein R1-R10 are each independently selected from H and OH provided that at least two of R1-R5 are OH at least two of R6-R10 are OH;
the linker is a hydrocarbon chain optionally interrupted by one or more heteroatoms selected from N, O and S, wherein the hydrocarbon chain is optionally substituted by one or more functional groups selected from carbonyl, thiocarbonyl, C1-C8 alkyl, halogen, —COOH; or the linker is a heteroaryl, in particular a triazole.
More particularly, the polyphenol may correspond to the following formula (II):
wherein R1, R2, R5, R6, R9, and R10 are each independently selected from H and OH;
X1, X3, X4 and X6 are each independently a bond, O, N or S;
X2 and X5 are each independently O or S;
n, m, p, q are r are integers that are independently equal to 0, 1, 2, 3, 4, 5, 6, 7 or 8, provided that n, m, p, q are r are not all equal to 0.
Even more particularly, the polyphenol may correspond to the following formula (III):
In another embodiment, the polyphenol may be a tannic acid, for example a tannic acid that corresponds to one of the following formulae:
The liquid composition may comprise from 0.5 to 100 mmol, in particular 1 to 50 mmol, more particularly 5 to 10 mmol, of polyphenol per liter of liquid composition.
In one embodiment, the amount of polyphenol in the composition is determined as a function of the number of accessible amine groups of the protein, i.e. the number of amine groups on the protein that are able to react with a quinone through Michael addition and Schiff's base condensation reaction. In particular, the liquid composition may exhibit a [phenol]/[amine] molar ratio of 0.05 to 0.5, in particular 0.08 to 0.4, more particularly 0.1 to 0.3, wherein [phenol] corresponds to the number of phenyl groups bearing OH substituents of the polyphenol, and [amine] corresponds to the number of accessible amine groups of the protein.
Morphogen
The liquid composition of the invention comprises a morphogen. As used herein, the term “morphogen” refers to a molecule or ion that is capable of being oxidized by application of a potential with an electrode. In particular, the oxidized morphogen is able to induce cross-linking reactions between the protein and the polyphenol and between the protein, the polyphenol, and the surface of the electrode. The morphogen is thus distinct from the polyphenol and the protein.
The morphogen is selected from a ferrocene, a source of protons, a ruthenium complex and a ferricyanide complex, a source of hydroxide, a nickelocene, an osmium complex, an iron complex, a cobalt complex, methylene blue, dihydroxybenzoquinone, manganese cyclopentadienyl and an oxidized viologen.
In one embodiment, the morphogen is a ferrocene. As used herein, the term “ferrocene” refers to an organometallic chemical compound consisting of two five-membered rings bound on opposite sides of a central iron atom. The five-membered rings of the ferrocene comprise 5 ring atoms selected from C, N, P, preferably 5 carbon ring atoms. Further, each five-membered ring may be independently substituted by 1 to 5 substituents and/or may be fused with another 6-membered ring. The substituents of the five-membered ring may each be independently selected from (C1-C6)alkyl, (C2-C6)alkene, (C1-C6)hydroxyalkyl, (C1-C6)haloalkyl, azide, formyl, carboxyl, (C1-C6)alkanoic acid, (C1-C6)aminoalkyl, (C1-C6)alkyl-(C1-C6)alkylamino, (C1-C6)alkyl-di((C1-C6)alkyl)amino, (C1-C6)carbonylalkyl, (C1-C6)carboxyalkyl, (C1-C6)alkyl(C1-C6)carboxyalkyl and (C1-C6)carbonylhaloalkyl.
In particular, the ferrocene may comprise two cyclopentadienyl rings and may be a 1-substituted ferrocene, i.e. a ferrocene comprising one substituent on one ring; or a 1,1′-disubstituted ferrocene i.e. a ferrocene comprising one substituent on each ring, wherein the one or two substituents are independently selected from (C1-C6)alkyl, (C2-C6)alkene, (C1-C6)hydroxyalkyl, (C1-C6)haloalkyl, azide, formyl, carboxyl, (Ci-C6)alkanoic acid, (C1-C6)aminoalkyl, (C1-C6)alkyl-(C1-C6)alkylamino, (C1-C6)alkyl-di((C1-C6)alkyl)amino, (C1-C6)carbonylalkyl, (C1-C6)carboxyalkyl, (C1-C6)alkyl(C1-C6)carboxyalkyl and (C1-C6)carbonylhaloalkyl.
More particularly, the ferrocene may be selected from di(cyclopentadienyl)iron, methanol ferrocene, acetylferrocene, 1,1′-diacetylferrocene, (dimethylaminomethyl)ferrocene, ferrocenecarboxaldehyde, (1-acetoxyethyl)ferrocene, ferrocenoyl azide, α-methylferrocenemethanol, 1-(dimethylamino)ethyl]ferrocene, aminomethylferrocene, 1,1′-di(aminomethyl)ferrocene, aminoethylferrocene, 1,1′-di(aminoethyl)ferrocene, ferrocenecarboxylic acid, 1,1′-ferrocenedicarboxylic acid, and (6-Bromo-1-oxohexyl)ferrocene.
Even more particularly, the morphogen may be methanol ferrocene.
In yet another embodiment, the morphogen is a source of protons.
In particular, the source of protons may be generated electrochemically using the electrolysis of water above 1.5 V (vs Ag/AgCl) or oxidation of hydroquinone above 30 μA/cm2.
In yet another embodiment, the morphogen is a ruthenium complex.
In particular, the ruthenium complex may be selected from Ru(NH3)6Cl3, [Ru(2,2′,2″-terpyridine)(1,10-phenanthroline)(OH2)]2+, trans-[Ru(2,2′-bipyridine)2(OH2)(OH)]2+, [(2,2′-bipyridine)2(OH)RuORu(OH)(2,2′bpy)2]4+ and [Ru(4,4′-bipyridine)(NH3)5]2+.
In yet another embodiment, the morphogen is a ferricyanide complex.
In particular, the ferricyanide complex may be potassium ferricyanide.
The liquid composition may comprise from 0.01 to 10 mmol, in particular 0.05 to 5 mmol, more particularly 0.1 to 1 mmol, of morphogen per liter of liquid composition.
Solid Composition
The solid composition of the invention comprises the cross-linked product of a protein and a polyphenol, in particular the cross-linked product of an enzyme and a polyphenol.
As used herein, the term “solid composition” refers to a composition that does not flow under its own weight within a temperature range of 5° C. to 50° C.
As used herein, the term “cross-linked product of a protein and a polyphenol” refers to a copolymer comprising two different monomeric units derived from a protein and a polyphenol. The proteins are covalently cross-linked with one another by the polyphenols. Indeed, in its oxidized state, the polyphenol exhibits an affinity for the amino groups of the protein. The polyphenol thus acts as a cross-linking agent between the proteins, in particular by binding with some of the amino groups of the proteins.
The protein and the polyphenol in the cross-linked product of the solid composition may be as defined hereinabove. In one embodiment, the solid composition of the invention does not comprise ferrocene. More particularly, the solid composition consists essentially of the cross-linked product of a protein and a polyphenol.
In one embodiment, the solid composition of the invention may be in the form of a film. As used herein, the term “film” refers to tridimensional material in the form of a thin layer.
More particularly, the solid composition may be in the form of a film bound to the surface of an electrochemical probe. As used herein, the term “film bound to the surface of” refers to a layer of solid composition covalently bound to the surface of the electrochemical probe. As such, the present invention is not directed to the physical deposition of the protein on the surface of the electrochemical probe or to the physical immobilization or the protein by embedding the protein in a matrix, such as a paint.
Even more particularly, the solid composition may be bound to the surface of the electrochemical probe by means of the polyphenol. Indeed, in its oxidized state, the polyphenol exhibits an affinity for the surface of the electrode and the amino groups of the protein. The polyphenol thus acts as a cross-linking agent between the protein and the surface of the electrode, in particular by binding with some of the amino groups of the protein and the surface of the electrode.
In one embodiment, the solid composition of the invention is in the form of a film that exhibits a thickness of 40 to 150 nm, in particular 45 to 120 nm, more particularly 50 to 100 nm. The thickness of the film may be measured according to the Thickness Test Method described herein.
Further, the solid composition of the invention may be in the form of a film comprising the cross-linked product of an enzyme and a polyphenol that exhibits an enzymatic activity (Kmapp) that is lower than 100 mmol/L, in particular lower than 20 mmol/L, more particularly lower than 10 mmol/L, even more particularly lower than 7 mmol/L. The enzymatic activity (Kmapp) may be determined according to the Electrochemical Enzymatic Activity Test Method disclosed herein.
The solid composition of the composition may exhibit a percentage of enzyme leaking of less than 5%, in particular less than 1%, more particularly 0%, wherein the percentage of enzyme leaking corresponds to the molar percentage of immobilized enzyme that is removed from the solid composition by washing with a detergent according to the Enzyme Leaking Test Method disclosed herein.
The solid composition of the invention may be obtained by applying an electric stimulus to an electrochemical probe in contact with the liquid composition of the invention. The process for the preparation of said solid composition is described hereinafter.
Process for the Preparation of a Solid Composition
The process for the preparation of the solid composition of the invention comprises the step of contacting an electrochemical probe with the liquid composition of the invention. The liquid composition is as defined herein above.
The electrochemical probe may an amperometric, voltammetric or conductimetric electrochemical probe. In particular, the electrochemical probe may be an electrode, more particularly a working electrode.
The electrochemical probe preferably exhibits an affinity for the polyphenol. Examples of suitable materials for the electrochemical probe in the context of the invention include gold, platinum, silver, copper, palladium, iridium, ruthenium, aluminum, nickel, titanium, indium tin oxide (ITO), zinc oxide (ZnO), iron and carbon (graphite, graphene, carbon nanotubes); preferably gold. In another embodiment, the electrochemical probe may be a screen printed electrode.
The process for the preparation of the solid composition of the invention further comprises the step of applying an electric stimulus to the electrochemical probe so as to form a solid composition comprising the cross-linked product of a protein and a polyphenol on a surface of the electrochemical probe.
The electric stimulus should be selected so as to induce electro-oxidation of the morphogen and create a gradient of oxidized morphogen from the surface of the electrochemical probe. The oxidized morphogen will then oxidize the phenol groups of the polyphenol into quinone groups which will in turn enable the chemical reaction of the quinone groups with free amino moieties of the protein through Michael addition and Schiff's base condensation reaction.
In one embodiment, the application of an electric stimulus may be the application of cyclic voltammetry, for example between −2 and 1.5 V, in particular between 0 and 0.7 V, with a scan rate of 0.01 to 0.2 V/s, in particular 0.05 V/s.
The process of the invention may be conducted in a 3-electrode cell comprising a working electrode, a counter-electrode and a reference electrode. The working electrode is immersed in the liquid composition of the invention and corresponds to the electrochemical probe on which the film of solid composition is formed. The counter-electrode may be a platinum electrode. The reference electrode may be a Ag/AgCl electrode.
The electric stimulus may be applied for a period of time sufficient to form a film of solid composition having the desired thickness. In particular, the period of time may be 1 to 120 minutes, in particular 15 to 90 minutes, more particularly 30 to 60 minutes, even more particularly 45 minutes.
After application of the electric stimulus, the process of the present invention may comprise a rinsing step by contacting the electrochemical probe with an aqueous solution, in particular a buffered solution, more particularly a phosphate buffered solution.
Use of Ferrocene as a Morphogen
The present invention is also directed to the use of a ferrocene as a morphogen in the formation of a solid composition on the surface of an electrode by electrodeposition.
The ferrocene for use as a morphogen may be as described above. In particular, the ferrocene may be methanol ferrocene.
In particular, the ferrocene is used to form of a solid composition on the surface of an electrode by electrodeposition by applying an electric stimulus to an electrochemical probe so as to form a solid composition on a surface of the electrochemical probe. The solid composition obtained by electrodeposition may result from one of the following processes:
(i) precipitation of polyelectrolytes or proteins through change of solubility,
(ii) self-assemblies of polyelectrolytes through electrostatic/ionic interactions;
(iii) formation of covalent bonds between monomers (electropolymerization); or
(iv) formation of cross-links between polymer chains (electro-cross-linking).
Biosensor and Process for the Detection of an Analyte
The biosensor of the invention comprises an electrochemical probe.
As used herein, the term “biosensor” refers to a self-contained analytical device that combines a biological component with a physicochemical device for the detection of an analyte of biological importance within a sample, such as a biological sample.
The electrochemical probe of the biosensor comprises the solid composition comprising a cross-linked product of a protein and a polyphenol according to the invention bound to a surface thereof.
The electrochemical probe may an amperometric, voltammetric or conductimetric electrochemical probe. In particular, the electrochemical probe may be an electrode, more particularly a working electrode.
The electrochemical probe preferably exhibits an affinity for the polyphenol. Examples of suitable materials for the electrochemical probe in the context of the invention include gold, platinum, silver, copper, palladium, iridium, ruthenium, aluminum, nickel, titanium, indium tin oxide (ITO), zinc oxide (ZnO) iron and carbon (graphite, graphene, carbon nanotubes); preferably gold. In another embodiment, the electrochemical probe may be a screen printed electrode.
In one embodiment, the biosensor comprises a housing. The housing may comprise a solution containing a mediator.
As used herein, the term “mediator” refers to a molecule or ion that is capable of being oxidized by application of a potential with an electrode. The mediator advantageously exhibits an adequate solubility in both oxidized and reduced states for a rapid diffusion between the redox center of the protein and the electrode surface as well as a fast reaction with the reduced form of the protein. In particular, the mediator may be selected from a ferrocene, a source of protons, a ruthenium complex, a ferricyanide complex, a source of hydroxide, a nickelocene, an osmium complex, an iron complex, a cobalt complex, methylene blue, dihydroxybenzoquinone, manganese cyclopentadienyl, an oxidized viologen; more particularly the mediator may be a ferrocene.
The solution comprising the mediator is preferably a buffered solution, more preferably a phosphate buffered solution. The solution comprising the mediator may, for example, comprise 1 to 50 mmol, in particular 2 to 50 mmol, more particularly 5 to 20 mmol, of phosphate per liter of solution. The solution comprising the mediator may exhibit a pH of 5 to 9, in particular 6 to 8, more particularly 7 to 7.8.
The electrochemical probe comprising the solid composition of the invention may be immersed in the solution comprising the mediator. The sample containing the analyte to be detected may be injected in said housing.
The biosensor may further comprise a counter-electrode and optionally a reference electrode.
In one embodiment, the biosensor comprises a means to measure the electric signal variation in terms of current, electric potential or inductance in case of amperometric, voltammetric or conductimetric biosensors. Further, the biosensor may comprise a means to correlate the electric signal variation to the concentration of the analyte within the sample.
The present invention also relates to a process for the detection of an analyte in a sample with the biosensor of the invention.
The process of the invention comprises the steps of:
The mediator used in the process of the invention is as defined above. In one embodiment, the mediator is reduced by application of an adequate potential. The potential may be determined by cyclic voltammetry to identify the oxidation and reduction peaks. When the mediator is methanol ferrocene, the potential that is applied may be 0.25 V.
The variation of electric signal generated by the oxidation of the mediator may be the variation of intensity generated by the oxidation of the mediator.
The analyte detected by the biosensor may be a substance which is derived from a living body and which may serve as an index for a disease or health condition. The substance to be measured may be glucose (e.g., blood sugar), cholesterol, alcohol, sarcosine, fructosyl amine, pyruvic acid, lactic acid, and hydroxybutyric acid, for example. The sample that contains the analyte may be a biological sample. Examples of the biological sample include blood and urine.
Biofuel Cell
The biofuel cell of the invention comprises:
a positive electrode;
a negative electrode;
an electrolyte; and
an external circuit electrically connecting the positive electrode and the negative electrode;
wherein a solid composition comprising a cross-linked product of a protein and a polyphenol is bound to a surface of the positive electrode and/or the negative electrode.
The present invention is further detailed in the non-limiting examples below.
Materials and Test Methods
Chemicals Alkaline Phosphatase (AP from bovine intestinal mucosa, CAS 9001-78-9), Glucose oxidase (GOX from Aspergillus niger, CAS 9001-37-0), paranitrophenyl phosphate liquid substrate (pNPP, P7998, CAS 4264-83-9), salicylic acid (SA, M=138.12 g/mol, CAS 69-72-7), ascorbic acid (AA, M=176.12 g/mol, CAS 50-81-7) sodium nitrate (NaNO3, M=84.99 g/mol, CAS 7631-99-4), potassium hexacyanoferrate(II) (M=422.41 g/mol, CAS 14459-95-1), o-dianisidine peroxidase (M=244.29 g/mol, CAS 119-90-4), glucose (M=180.16 g/mol, CAS 50-99-7), phosphate buffered saline tablet (PBS, P4417), tris(hydroxymethyl)aminomethane (Tris, M=121.14 g/mol, CAS 77-86-1) and ferrocene methanol (FC, M=216.06 g/mol, CAS 1273-86-5) were purchased from Sigma-Aldrich. Dopamine hydrochloride was purchased from Aldrich. TUDA was purchased from Iris Biotech. All dried solvents were purchased from Acros Organics. Acetaminophen (AP, M=151.17 g/mol, CAS 103-90-2) and uric acid (UA, M=168.11 g/mol, CAS 69-93-2) were purchased from Alfa Aesar. All chemicals were used as received and dissolved in aqueous solution using MilliQ water (resistivity of 18.2 MΩ·cm at 25° C.). Phosphate buffer solution was prepared at 150 mmol/L phosphate, 2.06 mol/L NaCl and 0.041 mol/L KCl and adjusted at pH 7.4.
Electrochemical Cell
The electrochemical cell used to prepare the solid composition by electrodeposition and measure the enzymatic activity is an Electrochemical Quartz Crystal Microbalance (EC-QCM) with Dissipation Monitoring. Q-Sense El apparatus from Q-Sense AB (Gothenburg, Sweden) was used to perform the electrochemical quartz microbalance (EC-QCM) experiments by monitoring the changes in the resonance frequency fv and the dissipation factor Dv of an oscillating quartz crystal upon adsorption of a viscoelastic layer (v represents the overtone number, equal to 1, 3, 5 and 7). The measurements were executed at the first, third, fifth, and seventh overtones, corresponding to 5, 15, 25, and 35 MHz after the excitation of the quartz crystal at its fundamental frequency (5 MHz). The QCM measurement is sensitive to the amount of water associated with the adsorbed molecules and senses the viscoelastic changes in the interfacial material. Only the third overtone at 15 MHz is presented. Electrochemical measurements were performed on a CHI660E apparatus from CH instrument (Austin, Tex.) coupled on the QCM-D (named EC-QCM) apparatus with a three electrode system: The gold-coated QCM sensor acted as working electrode. A platinum electrode (counter electrode) on the top wall of the chamber and a no-leak Ag/AgCl reference electrode fixed in the outlet flow channel were used respectively as counter and reference electrodes. Before the electrodeposition of the solid composition, in order to test the quality of the EC-QCM cell, a capacitive current in the presence of phosphate buffer and a faradic current of 1 mmol/L of potassium hexacyanoferrate (II) aqueous solution (prepared in phosphate buffer) was recorded by application of cyclic voltammetry (5 cycles at 50 mV/s between 0 and 0.7 V vs Ag/AgCl).
NMR
1H NMR spectra were recorded on Bruker Advance DPX400 (400 MHz) spectrometers.
Film Thickness and Roughness
The thickness and roughness of the film is measured by Atomic Force Microscopy (AFM) on a film bound to the surface of an electrochemical probe. The film is obtained with the process of the invention as detailed in the examples below. AFM images were obtained in contact mode in liquid conditions with the Nanoscope IV from Veeco (Santa Barbara, CA). Cantilevers with a spring constant of 0.03 N/m and silicon nitride tips (model MSCTAUHW, Veeco) were used. Several scans were performed over a given surface area. These scans had to produce comparable images to ascertain that there is no sample damage induced by the tip. Deflection and height images were scanned at a fixed scan rate (1 Hz) with a resolution of 512×512 pixels. The film thickness was measured by using the “scratch” method. The scratches were achieved with a plastic cone tip and were always imaged perpendicular to the fast scan axis. Profilometric section analysis of a scratched film was used to determine the thickness of the film over the scanned area. The film thickness is the minimal z distance between the bare substrate and the surface of the film which covers the whole substrate. The mean thickness of the scratched film was determined by measuring the thickness on at least six areas. The film roughness is the RMS given by the AFM software on 3×3 μm2 images. Data evaluations were performed with the NanoScope software version 5.31r1 (Digital Instruments, Veeco).
Colorimetric Enzymatic Activity of Cross-linked Enzyme/Polyphenol Film
A multidetector spectroscope UV (Xenius XC, SAFAS, Monaco) equipped with a microplate reader was used to monitor the catalytic activity of the enzyme within the film using o-dianisidine assay. The films were disposed in a 24 wells plate with 1 mL of a solution containing glucose (1 mg/mL), horseradish peroxidase (HRP) (1 mg/mL) and o-dianisidine (10−3 M) prepared in 150 mmol/L NaNO3-10 mmol/L Tris buffer at pH=8.0. The enzyme's activity was monitored by using a second enzyme, HRP, which will use the H2O2 produced during the reaction of the immobilized enzyme with glucose and will react with o-dianisidine (colourless) to obtain oxidized o-dianisidine, which is brown. The reaction was followed at 440 nm.
Electrochemical Enzymatic Activity of Cross-Linked Enzyme/Polyphenol Film
All measurements were carried out on a CHI 660B electrochemical workstation (CH Instruments, USA). The same electrode set-up used during the electrodeposition, was used for the electrochemical performance study. The gold electrode was used as the working electrode. Cyclic Voltammetry and Chronoamperometric measurements were carried out after injecting 600 μL of solutions of different concentration of glucose at 1 mL/min in the absence and in the presence of a mediator (0.5 mmol/L of ferrocene methanol, FC). The chronoamperometric measurements of glucose were performed in Ar-saturated solution at constant potential of 0.25 V (vs Ag/AgCl). A surface area of 0.8 cm2, corresponding to the exposed area of the gold QCM sensor, was used for current density calculations as explained in Singh, K.; McArdle, T.; Sullivan, P. R.; Blanford, C. F. “Sources of Activity Loss in the Fuel Cell Enzyme Bilirubin Oxidase”, Energy Environ. Sci., 2013, 6, 2460.
The Michaelis-Menten constant (Kmapp) was determined to evaluate the biological activity of the immobilized enzyme and is estimated using the following derived equation from Lineweaver-Burk equation as disclosed in Kamin, R. A.; Wilson, G. S. “Rotating Ring-Disk Enzyme Electrode for Biocatalysis Kinetic Studies and Characterization of the Immobilized Enzyme Layer”, Anal. Chem., 1980, 52, 1198-1205:
where iss is the steady-state current after the addition of substrate, imax is the maximum current measured under saturated substrate condition, and C is the bulk concentration of the substrate. A low Kmapp indicates a high enzymatic activity of the immobilized enzyme.
Enzyme Leaking
Chronoamperometric measurements of glucose were performed in Ar-saturated solution at constant potential of 0.25 V (vs Ag/AgCl) after injection of 600 μL of different concentration of glucose in the presence of 0.5 mmol/L of methanol ferrocene as disclosed in the electrochemical enzymatic activity test method. A surface area of 0.8 cm2, corresponding to the exposed area of the gold QCM sensor, was used for current density calculations. To prove the covalent immobilization of GOX, three sequential washes (for 5 min) by injection of 0.01% of detergent Tween® 20, prepared in phosphate buffer, were performed during the chronoamperometric test. These washes are expected to remove any physically bound GOX.
Polyphenol of formula (III) was prepared in two steps from dopamine (CAS 62-31-7, Sigma) and 3,6,9-trioxaundecandioic acid (TUDA, CAS 13887-98-4, Iris Biotech) according to the following Scheme 1:
Preparation of N,N-succinimide trioxaundecanediamide
3,6,9-trioxaundecandioic acid (TUDA, 0.99 g, 4.46 mmol, 1.0 eq.) was mixed with 5.31 g of molecular sieves in CH2Cl2 (20 mL). DCC (3.27 g, 16.19 mmol, 3.6 eq.) and NHS (1.83 g, 15.91 mmol, 3.6 eq.) were added and the mixture was stirred overnight. The solution was filtered over celite. The volume of the solvent was reduced under vacuum. Cold Et2O was then added to induce the precipitation of the product and the mixture was stored in a cold medium overnight. After filtration, the precipitate was purified with flash chromatography (eluent DCM) affording of solid white product (0.28 g, 15%).
1H NMR (MeOD-d4, 400 MHz) δ 2.83 (s, 8H) 3.68 (m, 4H) 3.79 (m, 4H) 3.78 (m, 4H) 4.52 (s, 4H).
Preparation of Polyphenol of Formula (III)
N,N-succinimide trioxaundecanediamide (0.28 g, 0.67 mmol, 1 eq.) was dissolved in 5 ml of chloroform. Then dopamine hydrochloride (0.25 g, 1.34 mmol, 2 eq.), dissolved in 5 ml of EtOH, was added followed by N-methylmorpholine (0.40 ml, 3.35 mmol, 5 eq.) and the solution was stirred for 48 h at RT. The solvent was evaporated, and the residual solid was purified by flash chromatography eluting with CH2Cl2/MeOH (1:0 to 97:3) affording 0.25 g of the desired biscatechol of formula (III) (492.52 g/mol, 70%).
1H NMR (D2O, 400 MHz) δ 2.7 (t, J=7.2 Hz, 4H), 3.4 (t, J=7.1 Hz, 4H), 3.52 (s, 8H), 3.96 (s, 4H), 6.68 (dd, J=2 Hz, 8Hz, 2H), 6.77 (d, J=1.9 Hz, 2H), 6.82 (d, J=7.9 Hz, 2H).
Electrochemical Characterization Polyphenol of Formula (III)
The electrochemical response of a solution of polyphenol of formula (III) and a mixture of polyphenol of formula (III)/methanol ferrocene was determined by cyclic voltammetry (CV) in phosphate buffer solution (
In the presence of methanol ferrocene, the redox peaks are slightly shifted at 0.35 and 0.02 V with higher values of intensity measured, in particular, for the oxidation peak (
100 mg of GOX was dissolved in 80 ml of a solution of Na2CO3 (0.1 M, pH 8.5) and stirred at 4° C. for 1 h. 340 μl of rhodamine B isocyanate solution (1.5 mg in 0.7 ml of DMSO) was added to the GOX solution and remained stirred at 4° C. for 4 h. Rhodamine labeled GOX (GOXRho) was purified by first dialyzing it overnight in a solution of 0.25 M of NaCl and then in pure water for several days.
A liquid composition comprising GOX as the protein, the polyphenol of formula (III) and ferrocene methanol as the morphogen was prepared. Knowing that GOX have 15 accessible lysine residues for chemical modification, the [phenol]/[amine] ratio was set to 0.13 to favor the cross-linking of GOX with polyphenol compared to polyphenol self-cross-linking. The liquid composition comprised 1 mg/mL of GOX (6.25 mmol/L), 3 mg/mL polyphenol of formula (III) (6.1 mmol/L) and 0.5 mmol/L of methanol ferrocene in phosphate buffer at pH 7.4. Nitrogen was flushed in the solution to prevent oxidation of the polyphenol due to dissolved oxygen.
A solid composition comprising the cross-linked product of GOX and the polyphenol of formula (III) was obtained by applying an electric stimulus to the liquid composition of example 3 in the electrochemical cell described above (EC-QCM). Once the QCM signal in contact with the buffer solution was stable, a mixture of the liquid composition of Example 3 was injected in the electrochemical cell (600 μL) at a flow rate of 600 μL/min with a peristaltic pump. After stabilization of the signal, a cyclic voltammetry between 0 and 0.7 V (vs Ag/AgCl, scan rate 0.05 V/s) was applied to trigger the polyphenol oxidation into the corresponding bis-quinone and start the self-construction of the film. A rinsing step was performed by injection in the flux of an aqueous solution of phosphate buffer at the end of the self-construction. The gold working electrode was then un-mounted from the EC-QCM cell and stored into the buffer solution for further characterizations or kept in the cell for electrochemical characterization of the enzymatic activity. The solid composition obtained is referred to GOX/polyphenol film.
AFM measurements were performed in contact mode and liquid state to characterize the topography and the thickness of the self-constructed films.
The enzymatic activity of GOX/polyphenol film of example 4 was first investigated using the colorimetric enzymatic activity test method described above for different electrodeposition times going from 15 to 60 min. The enzymatic analysis of the different films showed the best response for the self-constructed film at 30 min (
To demonstrate the effective biofunctionalization of the electrode by GOX/polyphenol film of example 4, the electrochemical biosensing capabilities of the immobilized enzyme were determined using standard enzyme-catalyzed glucose oxidation in the presence of ferrocene methanol (FC) as described in the electrochemical enzymatic activity test method above. FC mediator was used to enhance the electron transfer rate between GOX and the electrode and no enzymatic activity was detected in the absence of FC, presumably because the enzymatic active site was inaccessible to direct electron transfer from the electrode.
FC→FC++e− (at electrode)
GOXox+β-D-Glucose→GOXred+D-glucono-δ-lactone
GOXred+2 FC+→GOXox+2 H++2 FC
In typical electrochemistry test, the reduced form of the mediator, FC, is oxidized into FC+ by the application of an appropriate potential. The introduction of glucose triggers an increase of the anodic current caused by the regeneration of FC through the catalytic cycle depicted in
The Michaelis-Menten constant (Kmapp) was determined according to the Electrochemical enzymatic activity test method described above. The Kmapp of self-constructed GOX/polyphenol film of example 4 is about 6.3 mM which is lower than the reported 10.36 mM obtained for GOX/polyaniline,50 19 mM for GOX/ZnO nanotubes51 and 21.4 mM GOX/CaCO352 biosensors. The above result further indicates that the electrodeposited films exhibit a high affinity to glucose with Imax=25 μA/mM.
The selectivity of GOX/polyphenol functionalized biosensor was evaluated using common blood interfering substances, such as salicylic acid (SA, 0.75 mM), acetaminophen (AP, 0.35 mM), uric acid (UA, 0.5 mM)) and ascorbic acid (AA, 0.15 mM), which could have a contribution on the amperometric signal because of their low redox potentials. Thus, the maximum common concentration of these molecules in blood (Medscape) was added to the buffer solution both in the absence and the presence of 5 mM glucose to measure the current response at 0.25 V. There are not significant differences in the biosensor response due to the presence of these interfering species, suggesting an excellent anti-interference ability of the biosensor (
The enzymatic activity of GOX/polyphenol film of example 4 was first investigated using the colorimetric enzymatic activity test for different electrodeposition times going from 15 to 60 min. The enzymatic analysis of the different films showed the best response for the self-constructed film at 30 min with no leaking of the GOX from the matrix due to the covalent cross-linking with the polyphenol in the film (
Enzyme leaking was first assessed by conducting the colorimetric enzymatic activity test (absorbance of the supernatant as a function of time at 440 nm) on the GOX/polyphenol film of example 4 obtained after 30 min of self-construction. The film was removed from the medium after 30 min of enzymatic reaction. The absorbance remained constant after removal of the film from the medium (
This conclusion is further confirmed by the enzyme leaking test defined above, since the current densities measured before and after 3 washes with 0.01% of detergent Tween® 20 are similar (
Since the electro-cross-linking of protein using polyphenol is localized near the electrode, it can be used to functionalize microelectrodes. A solid composition comprising the cross-linked product of rhodamine-labeled GOX (GOXRho) and the polyphenol of formula (III) was obtained by applying an electric stimulus to an interdigitated array electrode (IDA ref: A-012125, Biologic) (
| Number | Date | Country | Kind |
|---|---|---|---|
| 18305325.5 | Mar 2018 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2019/057246 | 3/22/2019 | WO | 00 |
