GB2588780A - Biocompatible electrodes for electro-chemical biosensors - Google Patents

Abstract

An electrode suitable for biosensing comprises a conductive substrate and a porous biocompatible surface. The surface material comprises nanoparticles of titanium dioxide manganese (TiO2 Mn) forming a nanocrystalline layer, applied as a paste. The electrodes can readily adsorb on their surface biomolecules that act as recognition agents for a variety of analytes within an electrochemical three-electrode system. The biomolecules are not denatured and remain active, while the working electrode can operate in the absence of additional electron mediators in the analyte solution.

Description

DESCRIPTION

FIELD OF TIIE INVENTION

The present invention relates to electrodes for use in electrochemical biosensors and methods of making the electrodes. They are particularly useful as working electrodes when the recognition biomolecules need to be adsorbed directly on the electrode surface without being denatured, remaining active. The electrodes of the invention can operate in the absence of additional mediators in the analyte solution.

BACKGROUND OF THE INVENTION

Electrochemical biosensors provide an attractive means to detect specific analytes in solution by providing an electronic signal that is proportional to the concentration of the analyte. The most common electrochemical set up used in biosensing is the three-electrode set-up comprising of a working electrode (WE), a counter electrode (CE) and a reference electrode (RE). In particular, the reaction of interest takes place on the working electrode (WE), while the counter electrode (CE) is used to close the current circuit. The reference electrode (RE) is an electrode with a stable and well-known potential and is used as a point of reference in the electrochemical cell for potential control during measurements. The operating principle of such a three-electrode system can be seen in Figure 1.

oj The signal transduction and the general performance of an electrochemical biosensor is often determined by the surface architecture and properties of the working electrode Enzymatic sensors have been around the longest in the field of hiosensing. Until recently commercial biosensors placed the enzyme in the analyte solution along with electron mediators to facilitate electron transport LCD between the enzymatic active site and the working electrode to enhance the electronic signal. For example waier-soluble poly(o-)ylylviologen dihronnde) and polyip-xylylviologen dibromicle) can efficiently mediate electron transfer from reduced glucose oxidase (GOD) to the working electrode within a three electrode glucose bioseuE;ing system (Heller etal., 2008). Nevertheless, during recent years, it is desirable that the recognition biomolecules are in direct contact with the working electrode, rather than in the analyte solution, in order to further enhance the electrical signal and reduce noise, while working in the absence of expensive electron mediators. In particular, as stated by the Marcus theory, electron transfer can decay exponentially with increasing distance (Bard et al, 2001).

In such applications where biomolecules are immobilized on an electrode surface, bioactivity, stability and quantity of biological recognition elements on the working electrode plays an important role. In general, the direct adsorption of biomolecules on naked surfaces of materials may frequently result in the denaturation and loss of bioactivity. Thus, it is required that electrodes are surface modified to avoid biomolecules denaturing as well as providing anchoring sites for biomolecule attachment (Karunakaran et al, 2015). Various methods have been employed for biomolecule immobilization on electrode surfaces and can be classified into two broad categories: irreversible (covalent, cross-linking, entrapement-beads or fibers) and reversible methods (adsorption, bioaffinity, chelation) (Liebana et al, 2016).

Usually, metallic electrodes and in particular Au or carbon materials have been used as electrode materials. In the case of Au self-assembled monolayers are employed on the electrode surface based on the attachment of thiol (SH) or disulfide (-S-S-) functional groups before hiomolecules can he covalently attached. This is both to protect biomolecules from denaturing, while also providing anchoring sites. In other applications, carbon-based working electrodes have been used, such as graphite, graphene or reduced graphene oxide (rGO) to provide surfaces for the direct adsorption of biomolecules on a biocompatible surface. However, the hydrophobicity of graphite/graphene makes it incompatible with aqueous electrolyte solutions, leading to significant impediment to the effective adsorption of biomolecules and ultimately to the electron transfer process. For this purpose, it often needs to be modified for application in electrochemical sensors (Akkarachanchainon et al, 2017).

DETAILED DESCRIPTION OF THE INVENTION

The electrodes of the present invention are comprised of a conductive substrate and a biocompatible surface based on a nanocrystalline Ti02:Mn paste that is screen printed and immobilized via thermal annealing. This results in electrodes with a porous surface where biomolecules can be directly adsorbed without being denatured and thus retaining their activity. The substrate can be any conductive substrate for example FTO or ITO coated glass, conductive paper or a conductive polymer. The nanocrystalline Ti02:Mn porous paste used comprises from about 1-10% by weight of Ti02:Mn nanoparticles, with an average primary particle size not exceeding 100nm and where the titania is primarily in the rutile phase and the manganese ions are in the 3' state. The amount of Mn doping ranges between 0.1 to 1 %. The Ti02:Mn particles may have an organic coating, without restricted to a complete covering. For example, they may be coated with one or more organic materials such as polyols, amines, alkanolamines, polymeric organic silicon compounds, hydrophilic polymers or surfactants.

In order to prepare the paste. Ti02:Mn nanoparticles are placed in a mortar, where AcOH is added as well as organic solvents that maybe selected from lower alcohols and polyols such as ethanol, C\I isopropanol, propylene glygol, glycerine and sorbitol, before the solution is sonicated and surfactants such as Terpineol, along with ethylcellulose are added under magnetic stirring. The final mixture o consists of 1-10% by weight of Ti02:Mn nanoparticles as described above and AcOH at less than 0.5% by weight, surfactants at 5-30% by weight and 1-20% by weight ethylcellulose. The organic 1.0 solvents make up to 50-70% by weight of the mixture. The mixture is then placed in a rotary evaporator and heated until a viscous paste is formed.

The electrodes according to the present invention may find application in a variety of electrochemical biosensors, such as enzymatic, immunosensors or DNA sensors as well as any application that would require such a conductive biosensing platform that preserves biomolecule activity.

DESCRIPTION OF A PREFERRED EMBODIMENT:

The example which follows further illustrates the present invention:

EXAMPLE

The nanocrystalline Ti:M n paste was prepared using Manganese doped titani a nanoparticles of the type described by Knowland et al in US 6869596 and US 8642019. It was then doctor-bladed on 6cm2 commercial fluorine tin oxide (FTO) conductive glass with thickness 3ium and surface resistivity of -8 Q/sq purchased from Sigma Aldrich Immobilization is achieved via annealing at a temperature of 500°C for 15 minutes to prepare the working electrode (WE). In Figure 2, scanning electron microscopy shows the mesoporous surface of the electrode, while Figure 3 shows the X-ray diffraction pattern of the nanocrystalline Ti02:Mn preparation revealing that it is predominantly in the rutile phase. The thickness of the TiO2 paste as immobilized on the FTO glass was lOttm.

Subsequently 5mg/mL of the glucose oxidase enzyme (GOD) was prepared in a sodium acetate buffer with pH=5.1 at 25°C and 300411 were pipetted on the samples. The samples were then left overnight to adsorb the enzyme at 4°C. GOD was from Aspergillus Niger Type XS Lyophilized Powder by Sigma Alchich noting a 149,500 Unit s/2 solid activity for the batch. The enzymatic activity of glucose oxidase (GOD) was studied prior to the enzyme adsorbed on the working electrode, using the Enzymatic Assay of GOD by Sigma Aldrich noted as (EC 1.1.3.4), which is based on the spectrophotometric determination of 11202, as a product of enzymatic activity. In particular o-Di an i sidine changes distinctively colour in the presence of 11202. The detailed method and procedures of the assay can be found at [Sigma, 1996]. The conditions of the assay in our case were T = 25°C, pH = 5.1, A500nm, Light path = 1 cm. POD stands for the peroxidase enzyme. The principle can be seen below: -D-Glucose + 02 + H20 GOD > D-Glucono-1,5-Lactone + 11202 11202 + o-Diunisidine (reduced) POD > o-Dianisidine (oxidized) Consequently, according to the EC 1.1.3.4 GOD assay the activity of the commercial GOD used was determined to be approximately 18% less than the indicated units, which could be as a result of storage or environmental conditions. Thus the 5mg/mL of GOD used to prepare the enzyme solution before pipetting it onto the electrode corresponded to 614 Units/ml, versus a 748 Units/ml as indicated by the commercial vial. In this regard, the Units on our electrode are calculated to be 184 Units of GOD enzyme. Subsequently, the adsorption of GOD on the surface of the electrode was established as described in the method below: Method to establish GOD adsorption on the electrode surface "1-In order to check for the adsorption of the enzyme on the electrode surface, each sample was washed 04,1 out (4) times with 3.5ml of 50mM sodium acetate buffer with P11=5.1 at 25°C until no enzymatic activity was detected. In particular, the resulting wash-out was pipetted into suitable cuvettes along with the reagents as described in the EC1.1.3.4 assay to measure the amount of enzyme lost in Units. CD each time. After all (4) washes, a total of approximately 32 Units were lost from the initial 184 Units, If) corresponding to more than 80% of the enzyme being adsorbed and retained.

Determination of glucose in human serum samples using a GOD ninctionalized working electrode based on Ti02:Mn Using a Pt counter electrode, a GOD functionalized working electrode based on Ti02:Mn and an Ag/AgC1 reference electrode, cyclic voltammetry measurements were performed in human scrum with different glucose concentrations (Figure 4). The serum was buffered each time using 0.1M Sodium acetate at pH=5.1 at room temperature. The working electrode had a surface area of 6cm2. Figure 5, shows that the greater the glucose concentration in the scrum sample, the greater the resulting current was observed, demonstrating a liner response. In the absence of the enzyme on the electrode surface, there was no current detected even in the presence of glucose.

References Cited

1. U.S. Patent Documents 8642019 February 4.2014 Knowland.IS et al. 6869596 March 22, 2005 Knowland JS et al. 2. Other references Aklutrachanchainon N. et al, 'Hydrophilic graphene surface prepared by electrochemically reduced inicellar graphene oxide as a platform for electrochemical sensor', Talanta (2017), 165 (12), pp. 692-701.

Bard A..I. et al, 'Electrochemical Methods Fundamentals and Applications' 2nd ed. John Wiley & Sons; Hoboken, Ni, USA', (2001) Kinetics of Electrode Reactions; pp. 117-132.

Heller A. et of, 'Electrochemical glucose sensors and their applications in diabetes management', Chem. Rev. (2008) 108 (7), pp. 2482-2505.

Karunakaran C. et al, 'Biosensors and Biocicclronics' Elsevier Inc (2015) Liebana S. et al, 'Bioconjugation and stabilisation of biomolecules in biosensors Essays Biochem. (2016) 60(U pp 5968.

Sigma. SIGMA QUALITY CONTROL TEST PROCEDURE, Enzymatic Assay of GLUCOSE OXIDASE (EC 1.1.3.4). Last revised 08/30/96

Claims (1)

1. Claims (7) What is claimed is: I. An electrode comprising of a conductive substrate and a porous biocompatible surface for applications in biosensing 2. A method to prepare the electrode of claim I, where a nanocrystalline paste based on the Ti02:Mn nanoparticles is deposited and immobilized via annealing on the conductive substrate to provide the porous biocompatible surface of the electrode 3. The nanocrystalline paste of claim 2 has a composition based on Ti02:Mn nanoparticles where: a) their average primary particle size does not exceed 100nm b) the titania is primarily in the rutile phase c) the manganese ions are in the 3 state.d) the amount of Mn doping is from about 0.1 to 1 atom %.e) the Ti02:Mn particles may have an organic coating 4. A method to prepare the nanocrystalline paste of claim 2 such that the final mixture consists of: 1-10% Ti02:Mn nanoparticles, Ac01-1 at less than 0.5% by weight, surfactants at 5-30% by weight and 1-20% by weight ethylcel I ulose. The organic solvents make up to 50-70% by weight of the mixture. The mixture is then converted into a viscous paste.5. The electrodes of claim I, can be used for the direct adsorption of biomolecules on their porous surface, without denaturing them 6. The electrodes of claim 1 are stable in a range of electrolyte environments and buffers ranging from 0-14 pH 7. The biocompatible nanocrystalline paste based on Ti02:Mn nanoparticles of claim I can be easily screen printed on any conductive surface, leading to a biocompatible electrode in a quick, cost-effective way.