Printed electrodes modified with zinc oxide nanoparticles for quantifying biomarkers and production method

Abstract

The present invention relates to the modification of the surface of a silk-screen-printed carbon electrode by means of zinc oxide nanoparticles in the presence of carboxymethyl cellulose (ZnO-2), obtained using a new synthesis method. The ZnO-2 compound is selective and sensitive for the detection of hydrogen peroxide, and allows the adsorption of biomolecules, as well as electrochemical measurement in biosensors for the quantification of different analytes of diagnostic interest.

Description

[0001] Title: Printed electrodes modified with zinc oxide nanoparticles for the quantification of biomarkers and their production procedure

[0002] Description:

[0003] The present invention relates to the field of modified electrodes for the quantification of analytes in biological fluids, and in particular to the preparation of silk-screen printed electrodes modified with zinc oxide nanoparticles (ZnONPs). The ZnONPs were obtained, by a novel method, in the presence of carboxymethylcellulose (CMC) (ZnO-2) and used in the modification of printed electrodes. The electrodes modified with the obtained nanoparticles are used in biosensors comprising said electrodes, and to the method for the quantification of analytes in biological fluids using said electrodes.

[0004] Among the compounds used for electrode surface modification are conductive polymers, nanomaterials such as carbon nanotubes, and metal and metal oxide nanoparticles. (ZnO Nanoparticles: Recent Biomedical Applications and Interaction with Proteins, 2017, 555676).

[0005] In electrochemistry, the use of these nanomaterials has allowed the modification of the electrode surface, which has demonstrated important advantages over unmodified electrodes such as: their presence facilitates charge transfer reactions, electrocatalytic effects are observed that in many cases make it possible to decrease the value of the measurement potential, as well as the efficient adsorption of biomolecules on their surface (Electrochemical stability of screen-printed electrodes modified with Au nanoparticles for detection of methicillin-resistant Staphylococcus aureus, 2020, 123562).

[0006] The development of nanosciences has allowed the revaluation of zinc oxide (ZnO), making it a leading material in biomedicine, the manufacture of antibacterial composites, biosensors, and environmental remediation, among others. (The synthesis of novel cafestol loaded zinc oxide nanoparticles and their characterization, 2020, 4263-4272); (A Review of the Construction of Nano-Hybrids for Electrochemical Biosensing of Glucose, 2019, 46); (Efficient removal of a glyphosate-based herbicide from water using ZnO nanoparticles (ZnO-NPs), 2019, 101434); (Structural, Thermal, and Antibacterial Properties of Chitosan / ZnO Composites, 2013, 79-85).The advantages of its use are associated with its low cost, synthesis versatility, high surface area, biocompatibility, optical transparency, low toxicity, high chemical and photochemical stability (Highly Sensitive Amperometric Cholesterol Biosensor Based on Pt-Incorporated Fullerene-like ZnO Nanospheres, 2010, 243-250); (Sol-Gel Synthesis of Carbon Xerogel-ZnO Composite for Detection of Catechol, 2016, 282); (Electrochemical-Based Biosensors on Different Zinc Oxide Nanostructures: A Review, 2019, 2985).

[0007] ZnO is a II-VI type semiconductor material, which has a band gap energy of 3.37 eV (Rare earth co-doped ZnO photocatalysts: Solution combustion synthesis and environmental applications, 2020, 116328). The majority charge carriers are electrons present in the crystal structure due to oxygen vacancies. Doping with metallic elements has been studied, enhancing the formation of defects and its conductive properties (Doped Zinc Oxide Nanoparticles: Synthesis, Characterization and Potential Use in Nanomedicine, 2020, 5194).

[0008] Se informa en la literatura la obtención de ZnONPs con diversas morfologías entre las que se encuentran: nanotubos (ZnO nanotubes: preparation and photocatalytic performance evaluation, 2017, 61 -99), nanoagujas (Elongated hexagonal ZnO microfence optical resonator, 2019, 984-986), nanoalambres (Enhanced filuorescence detection of proteins using ZnO nanowiresintegrated inside microfluidic chips, 2018, 368-374), tetrápodos(D¡rect growth of freestanding ZnO tetrapod networks for multifunctional applications in photocatalysis UV photodetection, and gas sensing, 2015, 14303-14316), nanovarillas (Synthesis of ZnO nanorods and observation of resistance switching memory in ZnO based polymer nanocomposites, 2018, 030171), nanoflores (Zinc oxide nanoflower prepared by wet colloid chemical method , 2014, 453- 455), nanoesferas (Synthesis of ZnO nanospheres for water treatment through adsorption and photocatalytic degradation: Modelling and process optimization, 2019, 110584) por citar algunas.Current efforts are focused on extending synthetic protocols and relating morphology to application.

[0009] The growth of nanoparticles on a given substrate is a standardized procedure. Vapor deposition, for example, requires high temperatures, an inert atmosphere, and pressure control (Direct growth of freestanding ZnO tetrapod networks for multifunctional applications in photocatalysis, LIV photodetection, and gas sensing, 2015, 14303–14316). Electrodeposition has allowed the simultaneous synthesis and modification of electrodes (Zinc oxide-gold nanocomposite as a suitable platform for label-free DNA biosensor, 2020, 107458); (Highly sensitive and selective enzymatic biosensor based on direct electrochemistry of hemoglobin at Zinc Oxide nanoparticles modified activated Screen Printed Carbon Electrode, 2014, 1984–1993) by applying high potentials to the electrodes.The systems obtained are highly ordered (Physical vapor deposited Zinc Oxide nanoparticles for direct electron transfer of superoxide dismutase, 2008, 818-820) achieving control over the shape and thickness of the modified layer.

[0010] The potential of these systems for sensor development is undeniable (ZnO nanostructures in enzyme biosensors, 2015, 60-76); unfortunately, experimental requirements and low synthetic yields limit their scaling up (Recent Advances in ZnO nanostructures and thin films for biosensor applications: Review, 2012, 1-21); (Synthesis of Zinc Oxide nanoparticles by homogeneous precipitation method and its application in antifungal activity against Candida Albicans, 2015, 967-975). In the case of using ZnONPs in the modification of printed electrodes, scaling up in synthesis is not a barrier. In electrode modification, the volumes of nanoparticle dispersions (NPs) used are less than 5 pL, where quantities of NPs less than 0.5 mg are used.

[0011] Dissolution methodologies are less expensive, allowing for greater accessibility (Recent Advances in ZnO nanostructures and thin films for biosensor applications: Review, 2012, 1-21). Hydrothermal, solvothermal, sol-gel, and precipitation techniques have been reported, obtaining a variety of morphologies. Synthetic procedures commonly employ zinc salts (acetates, chlorides, nitrates, or sulfates), basic polar media, and surfactants.

[0012] Studies of the reaction conditions: reactant concentration, time, temperature, pH and surfactants, conclude the determining role of these in the formation of NPs (Investigation of the growth parameters hydrothermal ZnO nanowires for scale up applications, 2017, 538-545); (Ultrathin Zinc Oxide Nanofilm on zinc Substrate for High Performance Electrochemical Sensors, 2014, 186-193). Likewise, the formation begins at pH above eight, with hydroxides and carbonates commonly used for adjustment (Miniemulsions as chemical nanoreactors for the room temperature synthesis of inorganic crystalline nanostructures: ZnO colloids, 2012, 1620-1626); (Ultra-sensitive cholesterol biosensor based on low-temperature grown ZnO nanoparticles, 2009, 118-121).

[0013] The main disadvantage of solution-based procedures is the non-obtention of ZnONPs directly. The formation is mediated by synthetic intermediates (Zn(OH)2, Zns(OH)6(CO3)2) that are sintered to obtain the desired NPs (The synthesis of novel cafestol loaded zinc oxide nanoparticles and their characterization, 2020, 4263-4272); (Facile synthesis of zinc carbonate and zinc oxide nanoparticles via direct carbonation and thermal decomposition, 2013,819-827) ; (Synthesis of zinc oxide nanoparticles by homogeneous precipitation method and its application in antifungal activity against Candida albicans, 2015, 967-975) ; (Ultrathin Zinc Oxide nanofilm on Zinc Substrate for High Performance Electrochemical Sensors, 2014, 186-193). The finishing methods are diverse, varying in the presence or absence of pressure and the temperature in the range 60-700 °C.In general, at higher sintering temperatures, more crystalline and larger materials are obtained due to a rearrangement in the structure (The influence of Calcination Temperature on Structural and Optical Properties of ZnO Nanoparticles via Simple Polymer Synthesis Route, 2017, 263-275).

[0014] ZnONPs have a high isoelectric point (9.5) (Particle size and Zeta Potential of ZnO, 2014, 13-17), being a suitable matrix for immobilization by electrostatic adsorption (A Highly Sensitive and Selective Enzymatic Biosensor Based on Direct Electrochemistry of Hemoglobin at Zinc Oxide. Nanoparticles Modified Activated Screen Printed Carbon Electrode, 2014, 1984-1993) especially in the case of low isoelectric point molecules such as the redox enzymes glucose oxidase and cholesterol oxidase (Effects of morphology of nanostructured ZnO on direct electrochemistry and biosensing properties of glucose oxidase, 2011, 198-205); laccase (Electrochemical Degardation of the Recalcitrant Compound 4-Nitrophenol, Using Laccase Enzyme, 2018, 29-34); superoxide dismutase (Physical vapor deposited zinc oxide nanoparticles for direct electron transfer of superoxide dismutase, 2008, 818-820), among others.In addition, it is a good conductor of electrons, acting as a mediator between the electrode surface and the recognition element.

[0015] Its use has been reported in the detection and quantification of dopamine (Dopamine Determination with a Biosensor Based on Catalase and Modified Carbon Paste Electrode with Zinc Oxide Nanoparticles, 2012, 9892-9908) , glucose (Non-Enzymatic Glucose Sensor Composed of Carbon-Coated Nano-Zinc Oxide, 2017, 36) ;(Enzyme-conjugated ZnO nanocrystals for collisioanl quenching-based glucose sensing, 2012a, 2859-2865), cholesterol (Dependence of seed layer thickness on sensitivity of nano-ZnO cholesterol biosensor, 2017, 012021 ) 2012a, 2859-2865), hydrazine (Ultrathin Zinc Oxide nanofilm on Zinc Substratefor High Performance Electrochemical Sensors, 2014, 186-193), cancer cells (Carbon-dot wrapped ZnO nanoparticle-based photoelectrochemical sensor for selective monitoring of H2O2 released from cancer cells, 2019, 127), among other biomarkers, demonstrating the potentialities of This material has been used in the development of electrochemical sensors. Glucose quantification has been achieved with non-enzymatic sensors based on the electrooxidation-reduction of the biomarker, catalyzed on the surface of the nanomaterial (Highly Sensitive Nonenzymatic Cholesterol Sensor Based on Zinc Oxide Nanorods, 2018, 47);Non- Enzymatic glucose Sensor Composed of Carbon-Coated Nano-Zinc Oxide, 2017,36).;

[0016] Recent studies reflect the dependence of biosensor performance parameters (sensitivity, detection limit, among others) on the morphology of ZnONPs. The current challenge is to develop interconnected porous and high-surface-area structures, with nanoflower-like and nanotetrapod-like structures, among others, being leaders (Electrochemical-Based Biosensors on Different Zinc Oxide Nanostructures: A Review, 2019, 2985); (ZnO Nanostructures Application in Electrochemistry: Influence of Morphology, 2021, 1472–1482).

[0017] Various electrode modifications have been achieved, both by the deposition of previously synthesized nanoparticles and by growth on their surfaces. For example, Gózde et al. (A novel amperometric biosensor based on ZnO nanoparticles-modified carbon paste electrode for determination of glucose in human serum, 2013, 332-338) described the integration of ZnONPs with graphite for the fabrication of carbon paste electrodes. Meanwhile, Ahmad et al. (Wide linear-range detecting high sensitivity cholesterol biosensors based on aspectratio controlled ZnO nanorods grown on silver electrodes, 2012, 382-386) studied the controlled growth of nanotubes on Ag electrodes.

[0018] Giri et al. (Porous ZnO microtubes with excellent cholesterol sensing and catalytic properties, 2013, 814-822) addressed the synthesis of porous microtubes from macrostructured ZnO and evaluated the electrochemical response of glassy carbon electrodes modified with the nanomaterials cholesterol oxidase and chitosan to the presence of cholesterol. The bioelectrode obtained had a sensitivity of 54.5 mAmM'. 1 cirr 2 with a detection limit of 0.2 mM. In a later study, they modified the tubular structure with Na?S, decreasing the detection limit to 0.02 mM by improving electron transfer (An amperometric cholesterol biosensor with excellent sensitivity and limit of detection based on an enzyme-immobilized microtubular ZnO@ZnS heterostructure, 2014, 16997-17004).

[0019] The response mechanism of electrodes modified with ZnONPs is still under study. There is a general consensus that defects present in the crystalline structure are decisive in electrocatalysis. Photocurrent measurements carried out in optical devices reaffirm these ideas: an increase in the number of electron-hole pairs, a product of UV light excitation, increases the performance of the devices (Carbon-dot wrapped ZnO nanoparticle-based photoelectrochemical sensor for selective monitoring of H2O2 released from cancer cells, 2019, 127); (Enzyme-conjugated ZnO nanocrystals for collisional quenching-based glucose sensing, 2012, 2859-2865); (ZnO Nanorod-Based Non-Enzymatic Optical Glucose Biosensor, 2015, 988-996).

[0020] Some authors perform electrochemical measurements in the presence of ferrocyanide, improving electron transfer and using ZnO as an electronic conductor (Physical vapor deposited zinc oxide nanoparticles for direct electron transfer of superoxide dismutase, 2008, 818-820); (An amperometric cholesterol biosensor with excellent sensitivity and limit of detection based on an enzyme-immobilized microtubular ZnO@ZnS heterostructure, 2014, 16997-17004). Cellulose and its derivatives have gained prominence in recent years in the synthesis of NPs. Their abundance, diversity of shapes, low cost and properties make them attractive for the synthesis of composites. Their use as a reducing, stabilizing and coating agent, controlling the morphology of nanomaterials, has been reported.The groups present in its structure (hydroxyls, carboxylates) easily interact with positive ions (Ag(l), Cu(ll), Zn(ll), among others) achieving the growth of the NPs in the cellulosic material (The role of cellulosic chains of cotton in biosynthesis of ZnO nanorods producing multifunctional properties: Mechanism, characterizations and features. 2015, 122-129); (Synthesis and characterization of antibacterial carboxymethyl cellulose / ZnO nanocomposite hydrogels, 2015, 136-141).

[0021] For the synthesis of ZnONPs, different works have been carried out using these materials, for example: zinc acetate precipitation with keliab solution (a precipitating agent of natural origin that guarantees the alkaline medium) at 90 °C for 60 minutes (The role of cellulosic chains of cotton in biosynthesis of ZnO nanorods producing multifunctional properties: Mechanism, characterizations and features. 2015, 122-129); hydrogel method starting from zinc nitrate and carboxymethylcellulose (CMC) at 350 °C for 30 minutes (Synthesis of ZnO Nanoparticles using Carboxymethyl Cellulose Hydrogel, 2014, 798-803); by constant agitation to bind previously synthesized ZnONPs with bacterial cellulose (Morphological, physical, antimicrobial and release properties of ZnO nanoparticles-loaded bacterial cellulose films, 2016, 8-19). The reported strategies achieve a diversity of morphologies and sizes, demonstrating their versatility.

[0022] Mehdi Yadollahia et al. (Synthesis and characterization of antibacterial carboxymethyl cellulose / ZnO nanocomposite hydrogels, 2015, 136–141) prepared antibacterial nanocomposite hydrogels based on the combination of ZnONPs and pH-responsive biopolymer, CMC. The CMC hydrogels were successfully synthesized using epichlorohydrin in an alkaline medium. ZnONPs were formed by the interaction of zinc nitrate in CMC hydrogels with NaOH at room temperature. Structural details were provided by X-ray diffraction (XRD), ultraviolet and visible light (UV-vis) spectroscopy, and scanning electron microscopy (SEM) analysis. The swelling capacity of ZnO nanocomposite hydrogels depended on the abundance of ZnONPs in the CMC hydrogels. The antimicrobial activity of the hydrogels was examined on Escherichia coli (gram negative) and Staphylococcus aureus (gram positive) according to the agar diffusion test.CMC / ZnO nanocomposite hydrogels have demonstrated antibacterial activity against both gram-positive and gram-negative bactericides. Based on these findings, the prepared CMC / ZnO nanocomposite hydrogels can be used in various medical fields, such as drug delivery, wound healing, and tissue engineering. Further studies are needed on the biodegradability, biocompatibility, cytotoxicity, and drug release kinetics of the hydrogels.

[0023] On the other hand, Zhila et al. (pH-sensitive bionanocomposite hydrogel beads based on carboxymethyl cellulose / ZnO nanoparticle as drug carrier, 2016, 1317–1327) explain in their work the preparation of novel pH-sensitive bionanocomposite beads based on ZnONPs in CMC for use as controlled drug release systems. Fe(lll) ion as a physical crosslinking agent was used to prepare ionic crosslinked bionanocomposite hydrogel beads. Propranolol hydrochloride (PPN) has been chosen as a model drug. The characterization of pH-responsive bionanocomposite beads resulting from the incorporation of different contents of ZnONPs into CMC in the matrix was carried out using different experimental techniques: X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Thermogravimetric analysis (TGA), Scanning electron microscopy (SEM) and Energy dispersive X-ray spectroscopy (EDX).The incorporation efficiency of propranolol into beads was determined by UV-vis spectroscopy and found to be high. Furthermore, the swelling and drug release properties of the bionanocomposite hydrogels were investigated. The prepared bionanocomposite beads displayed pH-responsive swelling behavior with maximum water uptake at pH 7.4. Furthermore, the swelling ratio of ZnO / CMC hydrogels in different aqueous solutions was found to be higher compared to their neat hydrogel. In vitro drug release test was carried out to demonstrate the effectiveness of this novel type of bionanocomposite hydrogel beads as a controlled drug delivery system. More sustained and controlled drug releases were observed for ZnONPs containing sodium carboxymethylcellulose (NaCMC).

[0024] In another work, Tianyi L¡ et al. (Luminescent and UV-Shielding ZnO Quantum Dots / Carboxymethylcellulose Sodium Nanocomposite Polymer Films, 2016, 1112) propose that luminescent and UV-shielding biopolymers have rarely been reported. Zinc oxide quantum dots (ZnOQDs) (~3.2 nm) were synthesized by a short-time sol–gel method. The ZnOQD possessed a crystal lattice spacing of 0.28 nm and a hexagonal wurtzite structure. Luminescent and UV-shielding ZnOQD / sodium carboxymethylcellulose (NaCMC) nanocomposite polymer films were successfully fabricated by incorporating ZnOQDs into a CMC matrix via a solution casting method. Thermal analysis showed that ZnOQD reduces the thermal decomposition rate of CMC, and a large number of ZnOQD can promote the catalytic degradation of ZnOQD / CMC nanocomposites.Furthermore, the ZnOQD / CMC hybrid polymer films exhibited photoluminescence with a peak emission wavelength at 525 nm. More significantly, ZnOQD / CMC displayed prominent UV absorption capabilities. Such ZnOQD / CMC nanocomposite polymer films are promising for UV protection and optical applications.

[0025] Authors Si-Wei Zhao et al. (The preparation and antibacterial activity of cellulose / ZnO composite: a review, 2018, 9-20) analyzed the preparation methods of cellulose / ZnO nanocomposites from different cellulose sources (cotton fiber, cellulose acetate, among others) and their effect on the composite morphology. In addition, they referred to that, among antibacterial agents, ZnO is one of the most promising metal oxides due to its non-toxic nature and excellent mechanical properties. To expand its application, many ZnO composites have been studied. Cellulose, as one of the most abundant biopolymers, is biodegradable, biocompatible, and inexpensive. Therefore, different researches focus on various nanocomposite synthesis methods to evaluate their antibacterial activity.It was reported that the higher the percentage of ZnO incorporation in the composites, the higher the antibacterial activity. Ebrahim i et al. (Development of Antibacterial Carboxymethyl Cellulose-Based Nanobiocomposite Films Containing Various Metallic Nanoparticles for Food Packaging Applications, 2019, 2537-2548), used metallic nanoparticles of silver (Ag), ZnO and copper oxide (CuO) in the preparation of CMC nanobiocomposite films. SEM, EDXA, water vapor permeability (WVP), ultraviolet and visible light (UV-Vis) spectroscopy, and mechanical and microbial testing were used to determine the characteristics of the obtained active films. The SEM results showed that the CMC nanobiocomposite films had deviating levels of roughness and EDXA test confirmed the presence of Ag, ZnO and CuO nanoparticles in the biopolymer fabric.UV-vis spectroscopy confirmed that with the addition of metal nanoparticles to the pure CMC film, the absorption rate increased and the water vapor permeability (WVP) decreased. In mechanical tests, the addition of nanoparticles also increased the tensile strength of the films, and the nanobiocomposite films exhibited greater strength compared to the pure CMC film. Films incorporating metal nanoparticles showed antibacterial properties against the growth of Escherichia coli and Staphylococcus aureus. Therefore, nanobiocomposite films can be used as active packaging films and could extend the shelf life of food.

[0026] Authors Shivakumara et al. (Miscibility Studies of ZnO Nanoparticles Incorporated CMC / PVA Nanocomposite Films, 2017, 49-53), presented the miscibility studies of CMC / Polyvinylalcohol (PVA) blends using viscosity, refractive index, density, and ultrasonic interferometer techniques. The values ​​of molecular interaction parameters such as AB, pyo, revealed that the CMC / PVA blend is miscible when the PVA content is above 50%. The CMC / PVA blend films were characterized by Fourier transform infrared techniques. ZnO nanoparticles were prepared by precipitation method and incorporated into the CMC / PVA blend films. The microstructural properties of ZnO nanoparticles incorporated into the CMC / PVA blend films were characterized using X-ray diffraction techniques.Immiscible CMC / PVA blends were found to be miscible in all the compositions of CMC / PVA blends when ZnO nanoparticles are incorporated into polymer composite films. Moreover, Jabín et al. (Carboxymethyl Cellulose Based Zinc Oxide Nanocomposites: Synthesis, Characterization and Dye Removal Applications, 2019, 1069–1077), reported that CMC / ZnO has been used for composite hydrogel design to obtain effective 3D hydrogel network structures for industrial wastewater treatment applications. The prepared composites were characterized by FT-IR, XRD, SEM, and Transmission Electron Microscopy (TEM). Swelling studies have also been evaluated at different pH solutions. The results demonstrated that the composite hydrogel possesses excellent dye removal as compared to the raw material.The highest dye adsorption rate was found for the CMC / ZnO hydrogel. Therefore, CMC / ZnO composite hydrogels could be effectively applied to remove cationic dyes from wastewater. This type of material has been used for various industrial wastewater treatments.

[0027] Nabil A et al. (Effect of different capping agents on physicochemical and antimicrobial properties of ZnO nanoparticles, 2017, 1365–1375) investigated the influence of soluble starch; lactose; CMC; urea; and polyvinylpyrrolidone on the synthesis of zinc oxide nanoparticles (ZnONPs). Zinc acetate was used as a precursor under alkaline conditions to produce ZnONPs as a low-cost and efficient antimicrobial and UV-blocking agent. The characterization and antimicrobial functional properties of the prepared nanoparticles were investigated and reported using FTIR, TGA, XRD, TEM, and antimicrobial analyses, respectively. The results revealed that the thermal decomposition profile, size of ZnONPs, FT-IR spectra, as well as the antimicrobial activity of the prepared ZnONPs are governed by the type of capping agents.Crystallinity analysis showed identical peak intensity and width patterns, regardless of the coatings used on the NP surfaces. Furthermore, the results obtained revealed that the use of soluble starch as a coating agent results in a particle size in the 3-5 nm range and greater antimicrobial efficacy compared to other coating agents.

[0028] Issa M. El-Nahhal et al. (Stabilization of nano-structured ZnO particles onto the surface of cotton fibers using different surfactants and their antimicrobial activity, 2017, 478-487), propose that ZnONPs were prepared and deposited on cotton fibers by ultrasound irradiation. Different surfactants (sodium dodecyl sulfate (SDS or NaDS), alkylhydroxyethyldimethylammonium chloride (Praepagen HY), cetyltrimethylammonium bromide (CTAB), t-Octylphenoxypolyethoxyethanol (Triton X-100) were used to stabilize and homogenize the ZnONPs. In this way, the shape and size as encapsulated species were controlled. The use of surfactants has improved the stability of ZnONPs and decreased their leaching, particularly in the presence of SDS. The improved average crystal size for ZnO particles due to the use of surfactants is the main reason for decreasing the leaching of ZnO particles from the cotton substrate.SEM and XRD analysis revealed information on the shape and size of the ZnO-coated nanoparticles. The use of surfactants such as SDS and Praepagen HY in the synthesis of ZnONPs led to an increase in their antibacterial and antifungal activity against different pathogenic bacterial and fungal species, with a reduction of over 90%.

[0029] The authors Behzad Darbasizadeh (Crosslinked-polyvinyl alcohol-carboxymethyl cellulose / ZnO nanocomposite fibrous mats containing erythromycin (PVA-CMC / ZnO- EM): Fabrication, characterization and in-vitro release and anti-bacterial properties, 2019, 1137–1146), reported that nanofibers have been widely used for biomedical applications. Simultaneous incorporation of antibiotics and ZnO nanoparticles into nanofibers is expected to result in synergistic antibacterial effect. The main objective of the present study was to fabricate polyvinyl alcohol (PVA) / carboxymethyl cellulose (CMC)-ZnO nanocomposite fibrous mats containing the drug erythromycin (EM) and crosslink them using 2% glutaraldehyde vapor and 3% AlCh alcohol solution.The fabricated nanofibers were characterized by TGA, FTIR, TEM, and SEM, which indicated that the addition of ZnO nanoparticles and EM molecules into the fabricated nanofibers resulted in a change in their average diameter. Their antibacterial activity against Staphylococcus aureus and Escherichia coli was studied, and PVA-CMC / ZnO-EM nanofibers were found to exhibit excellent antimicrobial activity. The in vitro release profile showed that the release of EM from PVA-CMC / ZnO-EM nanofibers increased slowly. The sustained drug release profile and excellent antibacterial activity of PVA-CMC / ZnO-EM nanofiber indicated that it was an ideal biomaterial for wound dressings.Also Ahmed M Youssef (Development and Characterization of CMC / PVA Films Loaded with Zno Nanoparticles for Antimicrobial Packaging Application, 2017, 157-163) describes in his work a new nanocomposite based on polyvinyl alcohol (PVA), carboxymethylcellulose (CMC) and zinc oxide nanoparticles (ZnO NPs). The CMC / PVA / ZnO nanocomposites were fabricated by solution casting methodology. ZnONPs were first prepared by hydrothermal method, then different ratios of CMC and PVA (50:50 and 60:40 v / v) were prepared to create a new mixture, after that ZnONPs were loaded with different concentrations to the prepared mixture. The prepared ZnONPs were tested using X-ray diffraction (XRD) pattern and transmission electron microscope (TEM). Furthermore, CMC / PVA / ZnO nanocomposites were studied using Fourier Transform Infrared (FTIR), TEM, XRD, UV / Vis spectroscopy and TGA.

[0030] Films containing CMC / PVA in a ratio of (50:50) v / v) showed the best morphological, thermal, mechanical and antibacterial properties compared to films prepared using (60:40 v / v). The prepared nanocomposites showed visible antibacterial activity against gram positive (Staphylococcus aureus), gram negative (Pseudomonas aeruginosa, Escherichia coli) (Candida albicans) bacteria and fungi. Furthermore, the fabricated nanocomposites can be used as good materials applied in packaging.

[0031] As previously shown, obtaining ZnONPs from cellulose (and its derivatives) is a simple and low-cost procedure, which increases their appeal. Furthermore, cellulose has been shown to be crucial for the development of porosity in NPs (Cellulose from sources to nanocellulose and an overview of synthesis and properties of nanocellulose / zinc oxide nanocomposite materials, 2020, 1050–1073). The synthesized nanocomposites have demonstrated antibacterial activity, UV-Vis protection, and high porosity; however, their use in biosensor development has been poorly reported.

[0032] Printed electrodes

[0033] Screen-printed electrodes (SPEs) are devices that have seen increasing use in recent years as transducers in electrochemical biosensors due to their small size, low cost, ease of operation, large-scale production, and the possibility of use in portable devices. SPEs typically consist of two or three electrodes (the working electrode, the counter or auxiliary electrode, and the reference electrode). (Screen-printed electrodes: Transitioning the laboratory in-to-the-field, 2021, 1-10).

[0034] Unmodified printed electrodes exhibit a low electrochemical response and fail to detect low concentrations of electroactive species. However, one of their most important advantages lies in the possibility of modifying their surfaces with metal and metal oxide nanoparticles, carbon nanotubes, conductive polymers, and others. This ensures high sensitivity, selectivity, stability, and excellent electron transfer, making them useful in the development of electrochemical biosensors.(Gold Nanoparticle Deposited on Screen-Printed Carbon Electrode for Electrochemical Detection of Nicotine in E-cigarette, 2023, 1-12), (Cyclic Voltammetry of Screen-Printed Carbon Electrode Coated with Ag-ZnO Nanoparticles in Chitosan Matrix 2023, 3266), (Fabrication of biosensor based on Chitosan-ZnO / Polypyrrole nanocomposite modified carbón paste electrode for Modified electroanalytical application, 2017, S0928493117302850), (AuNPs-modified screen-printed electrodes (SPCE and SPPtE for enhanced direct detection of Chloramphenicol, Screen-Printed Electrodes Modified with Metal Nanoparticles for Small Molecule Sensing, 2020, 9), (AuNPs-modified screen-printed electrodes (SPCE and SPPtE) for enhanced direct detection of Chloramphenicol, 2022, 1669-1680), (A disposable colesterol enzyme biosensor based on Ferrocene-capped gold nanoparticle modified screen-printed carbón electrode, 2015, 4770-4778).

[0035] In the literature consulted to date, the synthesis method and the use of the compound obtained by the reaction of ZnONPs and CMC (ZnO-2) in the modification of printed electrodes with a high level of stability and reproducibility, with a cheap process and its application in the production of biosensors with screen-printed electrodes for the quantification of different analytes in biological fluids have not been reported.

[0036] The present invention relates to the modification of the surface of a silk-screen printed carbon electrode with zinc oxide nanoparticles in the presence of carboxymethylcellulose (ZnO-2), to the production of these ZnO-2 nanoparticles by a new process and to the manufacture of enzymatic biosensors containing these modified electrodes.

[0037] A detailed description of the features and advantages of the invention is presented below, based on 7 figures:

[0038] Description of the content of the figures

[0039] Figure No. 1. The FTIR spectra of the synthetic intermediates and the ZnONPs synthesized in KBr are presented.

[0040] Figure No. 2 Sedimentation curve for ZnONPsETG (ZnO-1) and ZnONPsETGCMC (ZnO-2) dispersed in water following the corresponding adsorption maximum in each case

[0041] Figure No. 3. UV-Vis spectra of ZnONPs in aqueous suspension.

[0042] Figure No. 4. X-ray diffraction patterns of ZnONPs.

[0043] Figure No. 5. TEM images of the synthesized ZnONPs. A, B-ZnONPsETG (ZnO-1 );

[0044] C, D-ZnONPsETGCMC (ZnO-2). C, D correspond to a lower concentration of deposited NPs.

[0045] Figure No. 6. Electrochemical response of (A) SPE / ZnO-1 / COx / CEst and (B) SPE / ZnO-2 / COx / CEst towards the presence of cholesterol ester by Cyclic Voltammetry using an electroactive mediator.

[0046] Figure No. 7. Electrochemical response of (A) SPE / ZnO-1 / GDH-FAD and (B) SPE / ZnO-2 / GDH-FAD towards the presence of glucose by Chronoamperometry using an electroactive mediator.

[0047] ZnONPsETG (ZnO-1 ) nanoparticles were first synthesized in the presence of ethylene glycol. For this, 0.3 g to 0.5 g of macrostructured zinc oxide in 10 ml to 30 mL of water were dispersed in a 250 mL flask by continuous stirring (300-500 rpm). To this mixture were added 0.5 g to 2.5 g of ammonium bicarbonate and 10 mL to 15 mL of ETG. The resulting dispersion was kept under stirring for 15 to 24 h at a temperature of 23 ° C to 25 ° C. Once the stirring was removed, the white precipitate was collected by centrifugation (2000-4000 rpm for 3 min to 5 min) and washed with water twice. The mixture was then dried in a vacuum oven at 40°C to 60°C for 15 to 24 h, and finally sintered at 300–500°C for 2 to 4 h. Both the intermediate product, hydrozincite, and the sintered ZnONPs were characterized. IR, cm' 1: hydrozincite: 3300 (vOH), 3000 (vCH), 1500 and 1377 (vCO3), 833 (5CO3), 500 (vZnO); ZnO-1 500 (vZnO). These nanoparticles were obtained using a synthesis procedure previously reported in the literature. Figure 1.

[0048] Zinc oxide nanoparticles were subsequently synthesized by a new method starting from ethylene glycol and carboxymethylcellulose with a volume ratio of 1:1 (ZnO-2). For this, 0.3 g to 0.5 g of macrostructured zinc oxide in 10 ml to 30 mL of water were dispersed in a 250 mL flask by continuous stirring (300 - 500 rpm). To this mixture were added 0.5 g to 2.5 g of ammonium bicarbonate, 10 mL to 15 mL of ETG and 10 mL to 15 mL of CMC of 0.5% to 2% by mass. The resulting dispersion was kept under stirring for 15 to 24 h at a temperature of 23 °C to 25 °C. Once the stirring was discontinued, the white precipitate was collected by centrifugation (2000–4000 rpm for 3–5 min) and washed with water two to four times. The precipitate was then dried in a vacuum oven at 40–60 °C for 15–24 h; finally, it was sintered at 300–600 °C for 2–4 h.The resulting solid (ZnO-2) is nanostructured with a quasi-spherical morphology and a size of 54 ± 11 nm. Both the intermediate product, hydrozincite, and the sintered ZnONPs were characterized. IR, cm'. 1 : hydrozincite: 3300 (vOH), 3000 (vCH), 1643 (vCO), 1500 and 1377 (vCO3), 833 (5CO3), 500 (vZnO); ZnO-1 500 (vZnO).

[0049] The white solid ZnO-2 was heated in the temperature range of 300 °C to 600 °C, which led to an increase in its adsorption capacity of biomolecules produced by the decomposition of carboxymethylcellulose.

[0050] ZnO-1 and ZnO-2 nanoparticles are poorly soluble, so a study of their dispersion stability was carried out. For this purpose, a sedimentation study in aqueous solution was performed. It turned out that the prepared dispersion of the ZnO-2 compound presented greater colloidal stability compared to the ZnO-1 compound, since in a time of 20 to 30 minutes, a decrease of 30% to 50% of the initial concentration of ZnO-2 was observed, while for ZnO-1, after three minutes, the initial concentration decreased by 50%. Figure 2. The obtained ZnO-1 and ZnO-2 nanoparticles were characterized. UV-Vis spectra were recorded for suspensions of ZnONPs with a concentration of 20 pg / mL. Absorption maxima corresponding to 371 nm (ZnO-1) and 376 nm (ZnO-2) were detected, associated in the literature (Jacobsson & Edvinsson, 2012) with the electronic transition from the valence band to the conduction band (O 2p Zn 3d ). Figure 3

[0051] The powder diffractograms obtained for the ZnONPs showed ten sharp and narrow signals that corresponded to the standard pattern of the wurtzite-type hexagonal structure of ZnO (JCPDS 36-1451). These signals were related to the Miller indices (100), (002), (101 ), (102), (110), (103), (200), (112), (201 ), (004). Figure 4

[0052] The morphology of the ZnONPs was determined by TEM, showing their spherical shape at low concentrations and the formation of interconnected structures at higher concentrations. The microscopies performed allowed determining the average diameter of the quasispheres, being 65 ± 7 nm for ZnO-1, while for ZnO-2 it was 54 ± 11 nm. Statistical analysis by comparing sample means showed that the synthetic procedures performed yielded NPs of equal size with 95% confidence. Figure 5

[0053] Given the advantages of using printed or screen-printed electrodes, a reproducible and scalable process is needed, by which the surface of screen-printed electrodes can be modified with zinc oxide nanoparticles (ZnO-1) compared to zinc oxide nanoparticles in the presence of carboxymethylcellulose (ZnO-2).

[0054] The screen-printed electrode of the present invention is composed of inert material screen-printed with carbon conductive ink for the working and auxiliary electrodes and the reference electrode with Silver / Silver Chloride conductive ink.

[0055] The screen-printed electrode modified with zinc oxide nanoparticles obtained in the presence of carboxymethylcellulose (CMC) (ZnO-2) and ZnO-1, comprises dispensing onto the surface of said printed electrode a dispersion containing the compound ZnO-2 and ZnO-1 at concentrations between 0.1 and 2 mg / mL, an amphoteric surfactant compound, with quantities between 0.01 and 1% by weight with respect to the total volume of the dispersion, in ultrapure water, MiliQ quality. A volume of between 0.1 and 2 pL is deposited on the screen-printed carbon electrode and the electrode is subsequently allowed to dry at a temperature between 20 and 25 ° C for a time between 5 to 10 minutes.

[0056] The electrode modified with zinc oxide nanoparticles in the presence of CMC (ZnO-2) prepared in this invention can be used for the preparation of enzymatic biosensors with either two or three electrodes and a receptor area for the biological fluid to be analyzed, so that the latter can come into contact with the enzyme immobilized on said modified electrode. Among the enzymes immobilized on the electrode are: FAD-dependent glucose dehydrogenase, cholesterol oxidase, cholesterol esterase, lactate oxidase, lactate dehydrogenase, glucose oxidase.

[0057] The modified electrode is selective and sensitive for the detection of hydrogen peroxide, and also allows for the adsorption of biomolecules, as well as electrochemical measurement in biosensors for the quantification of different analytes of diagnostic interest.

[0058] The present biosensor comprises the modified electrode of the invention with the GDH-FAD enzyme immobilized by physical adsorption, a procedure used in these cases, so that the glucose, which is the analyte to be detected, in biological fluids acts as a substrate for the enzyme, the latter oxidizes the glucose, producing hydrogen peroxide that is oxidized on the modified electrode due to the applied potential, which was between 0.1 V and 0.3 V, applied between the working electrode and the reference electrode, producing a maximum current signal between 30 and 45 pA, directly proportional to the amount of hydrogen peroxide and therefore to the amount of glucose in the sample analyzed.In addition, the enzymes cholesterol oxidase and cholesterol esterase were immobilized for the determination of total cholesterol concentration, where in this case first the enzyme cholesterol esterase catalyzes the hydrolysis of cholesterol esters and some other sterol esters, to release cholesterol plus a fatty acid anion. Subsequently, the enzyme cholesterol oxidase oxidizes cholesterol to 4-Cholesterol-3-one and hydrogen peroxide is produced which is oxidized on the modified electrode due to the applied potential which was between 0.1 V to 0.3 V, applied between the working electrode and the reference electrode, producing a maximum current signal between 5 and 15 pA, directly proportional to the amount of hydrogen peroxide and therefore to the amount of total cholesterol in the sample analyzed.

[0059] The present process for modifying printed electrodes with zinc oxide nanoparticles in the presence of carboxymethylcellulose (ZnO-2), as described above, exhibits high reproducibility at the dispensing stage, ensuring high reproducibility of current measurements with the modified electrodes compared to electrodes modified with zinc oxide nanoparticles using the method reported in the literature (ZnO-1 ). Furthermore, they exhibit greater stability over time during storage.

[0060] The obtaining of the nanoparticles of this invention and their use for the modification of the surface of the printed electrodes, to be applied in the obtaining of electrochemical sensors and biosensors, is illustrated by the following examples, which should not be interpreted in any way as limiting.

[0061] EXAMPLES OF IMPLEMENTATION

[0062] Example 1 Preparation of the modified printed electrode

[0063] The electrodes were printed in the three-electrode format on polyethylene terephthalate (PET) sheets using a printing machine using Silk Screen technology.

[0064] Printed electrodes comprise a working electrode, a reference electrode, and an auxiliary electrode. In the glucose biosensor, the working, auxiliary, and pseudo-reference electrodes are formed by printing conductive carbon paste. For the total cholesterol biosensor, the working and auxiliary electrodes are formed by printing carbon paste, and the reference electrode is formed by printing conductive silver-silver chloride paste.

[0065] The biosensor manufacturing procedure began with the modification of the printed electrodes following a layer-by-layer methodology. First, a 0.5 pL volume of the modification solution (ZnO-1, ZnO-2) was dispensed onto the electrode surface, allowing the PETs to dry at 23°C for 7 minutes. Then, the enzyme solution was dispensed with a volume of 0.5 pL, prepared in potassium phosphate buffer solution (K2HPO4 0.05 mol / L, KH2PO4 0.05 mol / L pH 7), Milli-Q quality ultrapure water, KsFe(CN)6 (100 mM), and FAD-dependent glucose dehydrogenase (FAD-dependent GDH) enzyme (2 mg / mL), for the glucose biosensor. For the cholesterol biosensor, the same potassium phosphate buffer solution (K2HPO4 0.05 mol / L, KH2PO4 0.05 mol / L pH 7), K3Fe(CN)e / K4Fe(CN)e (1 - 20 mM), and the enzymes cholesterol oxidase (2 mg / mL) and cholesterol esterase (3 mg / mL) were used.The enzyme solution dispensed onto the electrode surface was subsequently dried at 30°C in a drying tunnel for 15 minutes. The biosensors were then packaged in an aluminum foil sleeve containing desiccant and stored at 23°C until ready for use.

[0066] Example 2 Preparation of the total cholesterol biosensor

[0067] For the total cholesterol biosensor, the obtained bioelectrodes were named SPE / ZnO-1 / COx / CEst and SPE / ZnO-2 / COx / CEst, referring to the reported ZnO-1 nanoparticles and the obtained ZnO-2 nanoparticles and the immobilized COx and CEst enzymes.

[0068] The behavior of the bioelectrodes (A) SPE / ZnO-1 / COx / CEst and (B) SPE / ZnO-2 / COx / CEst obtained layer by layer in the presence of cholesterol ester was studied by cyclic voltammetry (CV). (Figure 6)

[0069] The SPE / ZnO-1 / COx / CEst cholesterol biosensor was evaluated by VC in a concentration range of 1.3 mmol / L to 10.5 mmol / L, at E = 0.30 V. Where it was observed that the linear working range of this biosensor was 2.6 mmol / L to 10.5 mmol / L of cholesterol ester.

[0070] Once the working range was obtained, a calibration curve was constructed (Figure 6 A inserted) in the linear response range to obtain the following analytical parameters: a sensitivity of 0.78 pA / mmol / L, a coefficient of determination of 0.937 with a detection limit of 0.42 mM. Similarly, the electrochemical response of the SPE / ZnO-2 / COx / CEst cholesterol biosensor was analyzed and evaluated by VC in a concentration range of 1.3 mmol / L to 10.5 mmol / L, at E = 0.30 V. Where it was observed that the linear working range of this biosensor is 2.6 mmol / L to 10.5 mmol / L of cholesterol ester. This result suggests a rapid electron transfer between the active site of the enzyme and the electrode surface.

[0071] Once the working range was obtained, a calibration curve (Figure 6 B inserted) was constructed in the linear response range to obtain the following analytical parameters: a sensitivity of 1.37 pA / mmol / L, a coefficient of determination of 0.9999 with a detection limit of 0.18 mM.

[0072] The differences found in the electrochemical response between the presented electrodes could be associated with the grain sizes, morphology and oxygen vacancies of the synthesized NPs. The connectivity between the grains and their defects should also be considered (Prospects of pharmaceuticals and biopharmaceuticals loaded microparticles prepared by double emulsion technique for controlled delivery, 2013, 125-141), (Sub-nanometer resolution of an organic semi-conductive crystal surface using friction force microscopy in water, 2016, 1340029).

[0073] Although there is no consensus in the scientific community on the in-depth response mechanism, the following is generally proposed:

[0074] ChOx

[0075] Cholesterol + O2- > Cholesterol — 4 — in — 3 — ona + H2O2(1 )

[0076] GCE / ZnONPs

[0077] H2O2- > O2+ 2H + + 2e~ (2)

[0078] The modified electrodes varied their response in the presence of [Fe(CN)6] 3 ' / 4 ' associated with a change in the detection mechanism. Ferrocyanides catalyze the electrooxidation of peroxide, in this case the ZnONPs being the support where the reaction occurs and the electron-transducer mediator using their semiconductor properties (An amperometric cholesterol biosensor with excellent sensitivity and limit of detection based on an enzyme-immobilized microtubular ZnO@ZnS heterostructure, 2014, 16997-17004)

[0079] ChOx

[0080] Cholesterol + O2- > Cholesterol — 4 — in — 3 — ona + H2O2(1 )

[0081] Example 3 Preparation of the glucose biosensor

[0082] Operating principle of the amperometric glucose biosensor with the enzyme FAD-dependent glucose dehydrogenase

[0083] In the reaction zone, the enzyme FAD-dependent glucose dehydrogenase (GDH-FAD) and the mediator K3Fe(CN)6 are immobilized on the transducer surface (carbon electrodes). When a drop of blood is applied to the area indicated on the biosensor, the glucose mixes with the reagents immobilized on the electrode and the glucose is oxidized to gluconolactone and the potassium hexacyanoferrate(III) is reduced to potassium hexacyanoferrate(II). Subsequently, a voltage of 0.3 V is applied, which produces the oxidation of potassium hexacyanoferrate(II) to potassium hexacyanoferrate(III). The potentiostat measures the current intensity.

[0084] Reaction of glucose with GDH-FAD dependent

[0085] Glucose + K3Fe(CN)e Gluconolactone + K4Fe(CN)6

[0086] 0.3 V

[0087] K4[Fe(CN)6] - K3[Fe(CN)6]+ separates the glucose biosensor, the obtained bioelectrodes were named SPE / ZnO-1 / GDH-FAD and SPE / ZnO-2 / GDH-FAD, alluding to the reported ZnO-1 nanoparticles, the obtained ZnO-2 nanoparticles and the immobilized GDH-FAD enzyme. The behavior of the obtained bioelectrodes (A) SPE / ZnO-1 / GDH-FAD and (B) SPE / ZnO-2 / GDH-FAD layer by layer towards the presence of glucose was studied by chronoamperometry. (Figure 7)

[0088] The SPE / ZnO-1 / GDH-FAD glucose biosensor was evaluated by Chronoamperometry in a concentration range of 0.5 mmol / L to 33.3 mmol / L, at E = 0.30 V. Where it was observed that the linear working range of this biosensor was 0.5 mmol / L to 33.3 mmol / L of glucose.

[0089] Once the working range was obtained, a calibration curve (Figure 7 A inserted) was constructed in the linear response range to obtain the following analytical parameters: a sensitivity of 1.76 pA / mmol / L, a coefficient of determination of 0.9994 with a detection limit of 0.09 mmol / L.

[0090] Similarly, the electrochemical response of the SPE / ZnO-2 / GDH-FAD glucose biosensor was analyzed and evaluated by chronoamperometry over a concentration range of 0.5 mmol / L to 33.3 mmol / L, at E = 0.30 V. Where it was observed that the linear working range of this biosensor is 0.5 mmol / L to 33.3 mmol / L of glucose. This result suggests a rapid electron transfer between the active site of the enzyme and the electrode surface.

[0091] Once the working range was obtained, a calibration curve (Figure 7 B inserted) was constructed in the linear response range to obtain the following analytical parameters: a sensitivity of 1.84 pA / mmol / L, a coefficient of determination of 0.9998 with a detection limit of 0.08 mmol / L.

Claims

CLAIMS Title: Printed electrodes modified with zinc oxide nanoparticles for biomarker quantification and their production procedure 1.- Modified printed electrode characterized in that it comprises on its surface a dispersion of zinc oxide nanoparticles, a surfactant compound and an immobilized enzyme. 2.- Printed electrode of claim 1 characterized in that the surfactant compound is selected from amphoteric surfactants in quantities between 0.01 and 1% by weight with respect to the total volume of the dispersion. 3.- Modified printed electrode according to claim 1, characterized in that said dispersion of zinc oxide nanoparticles has concentrations between 0.1 and 2 mg / mL and a volume of between 0.1 and 2 pL is deposited on the screen-printed electrode. 4.- Modified printed electrode according to claim 1, characterized in that said enzyme is FAD-dependent Glucose Dehydrogenase, Cholesterol oxidase, Cholesterol esterase, Lactate oxidase, Lactate dehydrogenase, Glucose Oxidase. 5.- A new method to obtain zinc oxide nanoparticles in the presence of carboxymethylcellulose (CMC) and ethylene glycol in a volume ratio of 1:1, ammonium bicarbonate and macrostructured ZnO. 6.- The method according to claim 5, wherein the temperature at which the solid initially obtained is heated is between 300-600 °C. 7.- In the method described in claim 5, heating the solid, initially obtained, in the temperature range described according to claim 6, leads to an increase in the adsorption capacity of the ZnO-2 compound produced by the decomposition of the CMC. 8.- The prepared dispersion of the ZnO-2 compound, obtained according to the method of claim 5, has greater colloidal stability. 9.- A biosensor characterized in that it comprises the electrode claimed from 1 to 4. 10.- The biosensor according to claim 9 with which a maximum current of between 15 and 45 pA is obtained. 11.- A biosensor according to claim 9, characterized in that the analyte is glucose and said applied potential is between 0.1 V and 0.3 V. 12.- A biosensor according to claim 9, characterized in that the analyte is cholesterol and said applied potential is between 0.1 V and 0.3 V.

Images