WO2019139537A1

WO2019139537A1 - Improved biosensor and method for manufacturing such

Info

Publication number: WO2019139537A1
Application number: PCT/SE2019/050023
Authority: WO
Inventors: Tautgirdas Ruzgas; Thomas Arnebrant; Javier SOTRES; Wanida LAIWATTANAPAISAL; Juan Francisco GONZALEZ; Atefeh SHAFAAT; Anh DUC PHAM; Nutcha LARPANT
Original assignee: Tautgirdas Ruzgas; Thomas Arnebrant; Sotres Javier; Laiwattanapaisal Wanida; Gonzalez Juan Francisco; Shafaat Atefeh; Duc Pham Anh; Larpant Nutcha
Priority date: 2018-01-15
Filing date: 2019-01-15
Publication date: 2019-07-18

Abstract

An electrochemical biosensor is provided. The biosensor comprises at least one conductive nanomaterial (10) operatively connected to at least one electrode (12); and a catalyst (16) for a reduction and/or oxidation reaction of said nanomaterial (10). Further, a method for manufacturing an electrochemical biosensor (1) is provided. The method (100) comprises the steps of: depositing (110) a first conducting nanomaterial (10) onto a first electrode (12); and adding (130) a catalyst (16) for a reduction and/or oxidation reaction of said nanomaterial (10).

Description

IMPROVED BIOSENSOR AND METHOD FOR MANUFACTURING SUCH

Technical Field

The present invention relates to an electrochemical biosensor for use at or in a human body. More particularly, the present invention relates to a wireless

electrochemical biosensor for e.g. glucose monitoring and diabetes control.

Background

Research on chemical and biochemical wireless sensors and biosensors aims to introduce these devices in to the medical practice, food and environmental control, etc.

It is expected that the number of wireless sensors in use, e.g., at homes, will increase and, thus, of special interest is to develop battery -less wireless and, ideally, chip-less RFID or NFC biosensor technologies.

Most often, the development of RFID biosensors rely on capacitive or resistive mechanism by which a specific biological affinity interaction or biochemical reaction is coupled into an inductance-capacitance-based resonance circuit enabling RFID monitoring. The capacitive and resistive mechanisms of coupling proposed to date do not allow a simple and universal integration of electrochemical biosensors with battery less RFID. Specifically, the most broadly used electrochemical biosensors for glucose monitoring and diabetes control, are based on electrodes modified with redox enzymes such as glucose oxidase or glucose dehydrogenase. These biosensors usually require potentiostate for current measurements at certain applied voltage and, currently, lack a general solution enabling their easy coupling to battery -less and chip-less RFID tags.

An object of the present invention is therefore to provide such a general solution.

Summary

According to a first aspect of the invention, the above and other objects of the invention are achieved, in full or in part, by an electrochemical biosensor comprising: at least one conductive nanomaterial operatively connected to at least one electrode; and a catalyst for a reduction and/or oxidation reaction of said nanomaterial. The biosensor may further comprise an antenna operatively connected to said at least one electrode. The antenna is advantageous in that it allows for wireless monitoring.

The antenna may be an RFID antenna. The RFID antenna is advantageous in that it is efficient and standardized.

The catalyst may comprise hydrogen peroxide. This is advantageous in that it is a strong oxidizer.

The catalyst may comprise an enzyme. This is advantageous in that enzymes may already be present or easily introduced in situ.

The catalyst may comprise an enzyme being horseradish peroxidase. This is advantageous in that it is a strong oxidizer.

The catalyst may comprise an enzyme being a peroxidase and/or glucose oxidase enzyme. This is advantageous in that these are body friendly oxidizer.

The catalyst may comprise an enzyme being a reductase enzyme. This is advantageous in that these are body friendly reducing agent.

The catalyst may comprise an organelle or cells, e.g. bacteria, and/or their biofilms. These are advantageous in that they are biological, which may be easily reproduced and body friendly.

The nanomaterial may comprise silver nanoparticles. These are advantageous in that they may easily be oxidized to AgCl and the formal potential of Ag/AgCl reactions is in the middle of the formal potential range of biologically relevant reactions, and thus, may be exploited for monitoring biologically relevant oxidation and reduction reactions by choosing an appropriate biological catalyst, e.g. an enzyme.

The nanomaterial may comprise gold nanoparticles. These are advantageous in that they are easy to manufacture and highly conductive.

The nanomaterial may comprise graphene. This is advantageous in that it is easy to manufacture.

The nanomaterial may comprise zink nanoparticles. These are advantageous in that their oxidation or reduction results in a high change of the resistance or a change of the double layer capacitance. The at least one electrode may be an interdigitated electrode. This is advantageous in that it is simple to integrate into an RFID tag.

The at least one electrode may be a screen-printed electrode. This is advantageous in that it is simple to manufacture.

The biosensor may comprise at least two electrodes. This is advantageous in that it may be simpler to manufacture and may yield a stronger signal.

The at least two electrodes may be operatively connected. This is advantageous in that this may be simpler to manufacture and may yield a stronger signal.

The conductive nanomaterial deposited to each electrode may differ by at least one nanomaterial. This is advantageous in that it allows for structures that are more complex and may improve the electrical characteristics of the nanomaterial.

The reduction and/or oxidation reaction may change a double layer capacitance of said nanomaterial. This is advantageous in that it is easily measured and thereby detectable.

The reduction and/or oxidation reaction may change the resistivity of said nanomaterial. This is advantageous in that it is easily measured and thereby detectable.

The biosensor may comprise saline solution. This is advantageous in that it may guarantee a similar environment at all times or to emulate an in situ environment ex situ.

The saline solution may comprise chloride and/or phosphate. This is advantageous in that it may improve the electrical and/or chemical environment of the biosensor to enhance either the reduction and/or oxidation reaction or the signal produced.

According to a second aspect of the invention, a method for manufacturing an electrochemical biosensor is provided. The method comprises the steps of: depositing a first conducting nanomaterial onto a first electrode; and adding a catalyst for a reduction and/or oxidation reaction of said nanomaterial.

The method may comprise a step of depositing a second conducting nanomaterial onto a second electrode operatively connected to said first electrode. This step is advantageous in that it allows for more complex structures and better electrical characteristics. The method may comprise a step of registering electromagnetic reflection of said nanomaterial resulting from said reduction and/or oxidation reaction. This step is advantageous in that it allows a biological process to be detected.

Other objectives, features and advantages of the present invention will appear from the following detailed disclosure, from the attached claims, as well as from the drawings. It is noted that the invention relates to all possible combinations of features.

It should be emphasized that the term“comprises/comprising” when used in this specification is taken to specify the presence of stated features, integers, steps, or components, but does not preclude the presence or addition of one or more other features, integers, steps, components, or groups thereof. All terms used in the claims are to be interpreted according to their ordinary meaning in the technical field, unless explicitly defined otherwise herein. All references to "a/an/the [element, device, component, means, step, etc.]" are to be interpreted openly as referring to at least one instance of the element, device, component, means, step, etc., unless explicitly stated otherwise.

Brief Description of the Drawings and Tables

By way of example, embodiments of the present invention will now be described with reference to the accompanying drawings and tables, in which:

Fig. 1 shows a basic RFID biosensor design. (A) RFID tag antenna with integrated interdigitated electrodes (IDE). (B) Basic setup for RFID-based

measurements of electromagnetic reflection from the RFID biosensor.

Fig. 2 shows effect of electrochemical redox conversion between Ag and AgCl nanoparticles on electromagnetic reflection from the RFID tag comprised of IDE covered with AgNP layer. (A) Anodic part of cyclic voltammogram (solid line) recorded with IDE covered with AgNPs and immersed in PBS solution. Dashed line presents a resistance of the electrode recorded simultaneously with cyclic voltammetry measurement. (B) The attenuation dependence on frequency (calculated as a baseline corrected reflection, function Sl 1) from the measured reflection function for

AgNP/IDE-RFID tag. The Sl 1 was recorded with the tag in air before and after electrochemical oxidation of AgNP to AgCl. (C) SEM image of a AgNP covered IDE. (D) SEM image of the IDE after electrochemical oxidation of AgNP to AgCl.

Fig. 3 shows a system for enzymatically (horseradish peroxidase) driven oxidation of AgNP to AgCl. (A) Schematic representation of the IDE electrode modified with a mixture of AgNPs and HRP/AuNP nanobiomaterial. (B) Photo and schematic presentation of the IDE short-circuited by AgNPs with separate T-shaped HRP/AuNP layer, which is electrically connected on the AgNP layer. (C) Schematic presentation of AgNP modified IDE, which is electrically connected to the HRP/AuNP layer on the other electrode, i.e., biofuel cell-based biosensor design. (D) Sl l function of the IDE-RFID biosensor design where IDE with 2 mm gap comprises a layer mage from mixture of HRP/AuNPs and AgNPs.“Before reaction” shows attenuation from the RFID with the IDE immersed in PBS and“After reaction” shows the attenuation after addition of H2O2 into PBS. Resulting H2O2 concentration 25 mM. (E) The same design and experiment as in case (D), but shows the measurements of DC current that flows through the IDE; applied DC voltage between the IDE fingers equal to 5 mV.

Fig. 4 shows measurements of (A) a DC current, which flows through the biosensor electrode for glucose, which is used to construct the RFID biosensor for glucose. The biosensor consists of IDE with 2 mm gap connected by nanobiocomposite comprised of AgNPs, HRP/AuNPs, and glucose oxidase. Applied voltage between the IDE fingers is equal to 5 mV. Glucose concentration in PBS is 1 niM. (B) Attenuation (background corrected Sl l) for the RFID biosensor for glucose. The biosensor electrode in PBS (blue trace); after the addition of glucose into PBS the attenuation curve shifts indicating the shift of resonance frequency of the RFID biosensor tag due to glucose oxidation, production of H2O2 and HRP/AuNP driven oxidation of AgNPs to AgCl.

Fig. 5 shows (A) a design RFID biosensor for detection of H2Ch-based on a screen-printed electrode in a microchannel. Two electrodes on the screen-printed electrode are short-circuited by the AgNP layer. The electrode in the centre of the SPE is also connected to the layer of HRP/AuNPs. These layers are only shown on the SPE before mounting of the microchannel. PBS with different concentration of H2O2 is pipetted into a microchannel. Graph (B) shows changes in the attenuation (corrected S 11) the radio signal from the RFID biosensor with increasing H2O2 concentration. (C) and (D) show dependence of resonance frequency and attenuation on H2O2

concentration.

Fig. 6 shows a flowchart of a method for manufacturing an electrochemical biosensor according to an embodiment.

Table Sl shows resonance frequency measured with the ID E-RFID tag. IDE was covered by a layer of AgNP, immersed in PBS and dried in air. The measured frequencies are shown in columns labelled by“Before reaction”. In this case, the nanoparticles are comprised of Ag. The same measurements were repeated after the AgNPs on IDE were electrochemically oxidized in PBS by running cyclic voltammetry. The frequencies are shown in columns labelled by“After reaction”. AgNPs after the reaction constitute silver chloride deposit. The table also includes relevant figures showing dependence of the attenuation on frequency.

Table S2 shows resonance frequency measured with the AgNP/2-mm-gap- electrode-RFID tag. Gold fingers in the central part of the IDE were removed and the gap was covered by layer of AgNP, immersed in PBS and dried in air. The measured frequencies are shown in columns labelled by“Before reaction”. In this case, the nanoparticles are comprised of Ag. The same measurements were repeated after the AgNPs on the IDE were electrochemically oxidized in PBS by running cyclic voltammetry. The frequencies are shown in columns labelled by“After reaction”.

AgNPs after the reaction constitute silver chloride deposit. The same experiments were also done with a screen-printed electrode (SPE) where 2 mm gap between the electrodes have been filled with layer of AgNPs. The table also includes relevant figures showing dependence of the attenuation on frequency.

Table S3 describes the composition of the targeting sample (No. 1) and control samples (No. 2-4).“+” mean the presence of the component and“-” mean the absence of the component.

Fig. Sl shows UV-Vis absorbance of 0.01 mg/ml AuNP and 0.01 mg/ml AgNP, and the mixture - black line, 0.01 mg/ml AuNP - dot line, 0.01 mg/ml AgNP - dash line. Fig. S2 shows UV-Vis absorbance of Sample 1 - Black line, Sample 2 - Dash line, Sample 3 - Dot line, Sample 4 - Dash dotted line.

Fig. S3A shows the Thevenin equivalent circuit for the RFID reader and sensor tag; and

Fig. S3B shows a simplified circuit of the RFID sensor tag.

Detailed Description

Embodiments of the invention will now be described with reference to the accompanying drawings. The invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

The terminology used in the detailed description of the particular embodiments illustrated in the accompanying drawings is not intended to be limiting of the invention. In the drawings, like numbers refer to like elements.

Wireless sensors and biosensors are of high interest for modernisation of healthcare, food and environmental control, security, etc. Of special interest are battery less wireless and, ideally, chip-less RFID sensor and biosensor designs. However, the capacitive and resistive mechanisms of coupling proposed to date do not allow a simple and universal integration of electrochemical, or redox reaction-based, sensors and biosensors with battery-less RFID tags. In this description, we disclose a general principle for constructing RFID sensors and biosensors with a recognition mechanism- based on redox reactions. The main embodiment comprises a material, including nanomaterials and their combination, which constitutes an RFID antenna or part of the antenna. This material is made sensitive and specific to a redox reaction of interest using e.g. specific nanomaterials 10 that have a specifically favourable reduction and/or oxidation reaction or using catalysts 16 that induce a specific reduction and/or oxidation reaction. As an example in this work, we show that a coupling of a silver nanoparticle modified electrode as a part of RFID antenna allows construction of sensors and biosensors for hydrogen peroxide and glucose. Similar biosensors may be constructed with exploiting any available oxidoreductase enzyme, their combinations, bacteria or any other biological or non-biological recognition elements capable to drive reduction or oxidation of the RFID-electrode material. In this description, we demonstrate the principle using silver nanoparticles as the sensing electrode material and Ag/AgCl redox reaction as a transduction reaction coupling the specific oxidation or reduction of the compound of analytical interest to an RFID detection principle. However, any other reduction and/or oxidation reaction of said nanomaterial 10 is possible as long as it changes an electronic characteristic of the nanomaterial 10 such as capacitance or resistivity. The choice of reaction will in large part depend on the nanomaterial 10.

In this description, we propose and demonstrate that the majority of biosensors based on redox enzymes may be simply and universally coupled with battery -less and chip-less RFID tags 14. The idea of the main embodiment is based on exploitation of silver nanoparticles 10, AgNPs, as a part of RFID tag antenna 14. In brief, a AgNP layer 10 is deposited as a part of the antenna circuit 14. The AgNPs are in electronic contact with oxidoreductase enzyme 16 realising direct or mediated electron transfer contact between the enzyme and a nanoparticle. In the presence of the enzyme substrate the enzyme catalyses oxidation of AgNPs into silver chloride, AgCl. This results into a high change of RFID tag impedance, which may be measured as a change in tags resonance frequency and attenuation of the reflected electromagnetic field. We demonstrate that with an appropriate choice and combination of nanomaterials and enzymes the third generation biosensors based on direct electron transfer between the enzymes and electrodes may be easily converted into RFID-based biosensors. We demonstrate the principle for such substrates as hydrogen peroxide and glucose. The proposed RFID biosensor design using redox sensitive nanoparticles, in this particular case, based on Ag/AgCl redox conversion, is a universal design. Based on the proposed design RFID biosensors can be constructed for oxidation (transduction reaction for coupling to RFID is Ag -> AgCl) as well as for reduction reaction (transduction reaction for coupling to RFID is AgCl -> Ag). The demonstration of the proposed design is done by using redox enzymes, however, the same principle can be realised by using organelles, prokaryotic (bacteria) and eukaryotic cells or tissues. The proposed solution allows using direct and mediated electron transfer (ET) mechanisms between the redox enzymes 16 and nanoparticles 10 as well as different variety of wireless tags 14, e.g., RFID, NFC, UHF, etc.

Materials and methods

Glucose, tablets of phosphate buffer saline (PBS), 35 % hydrogen peroxide solution, AgNCh, HAuCU. tri-sodium citrate, sodium chloride, potassium chloride, glucose oxidase from Aspergillus niger, horseradish peroxidase, and L-ascorbic acid were purchased from Sigma Aldrich.

Silver and gold nanoparticles have been synthesised following protocols described in the synthesis of nanoparticles section. The nanoparticles have been characterised by dynamic light scattering. AuNPs had a mean diameter of 46 nm and zeta potential of -44 mV. AgNPs had a mean diameter of 80 nm and zeta potential of - 17 mV. NP dispersion after synthesis had NP concentration equal to 1 and 0.3 mM for AuNPs and AgNPs, respectively. Before drop casting on the electrodes, the dispersions have been concentrated 20 times.

Biosensors have been produced by drop casting an appropriate amount of dispersion of nanoparticle on gold interdigitated electrodes (IDE) with 10 pm band-gap dimensions or on screen-printed electrodes (SPE), both from Dropsens, Llanera (Asturias), Spain

All solutions have been prepared by using deionised water purified by Milli-Q system (Merck Millipore, Billerica, USA) with resistivity of 18.2 W cm.

Electrochemical verification of RFID-based registration of redox conversion Ag/AgCl

The RFID biosensor design, which enables coupling of any redox enzyme- based biosensor with the RFID principle of signal registration, is realised in two steps. First, a layer of AgNPs is deposited as a part of the RFID tag circuit, e.g., part of antenna. Secondly, the redox enzyme is electronically connected to the AgNP layer enabling the enzyme-catalysed oxidation of highly conducting AgNPs into low conductivity AgCl. This ensures that the resonance frequency as well as the attenuation of the electromagnetic signal reflected from the RFID biosensor tag change

considerably in the presence of the enzyme substrate. The first step, i.e., the deposition 110 of AgNP layer as part of RFID tag antenna and the effect of AgNP redox conversion Ag -> AgCl and AgCl -> Ag was realised and demonstrated by driving 130 Ag/AgCl redox reaction electrochemically. The tag antenna was cut and the interdigitated electrode was connected (Fig. 1 A). Next, 2 pL of concentrated dispersion of AgNPs were deposited on IDE and left to dry (the electrode is noted as AgNP/IDE). The IDE-RFID tag design and the setup for the measurements of electromagnetic reflection from the tag are shown in Fig. 1, however other designs and setups are of course possible. The setup comprises a network analyser, a reading coil, and an RFID tag 14 with a connected IDE 12; all are schematically represented in Fig. 1.

As can be seen from Fig. 2 electrochemical oxidation of AgNPs on IDE to AgCl (Eq. 1, Fig. 2A, solid curve) results in high change in the AgNP/IDE resistance (Fig. 2A, dashed line) and high shift of the resonance frequency of the RFID tag (Fig. 2B, the attenuation is calculated by monitoring electromagnetic signal reflection, i.e., function Sn).

Ag + Cl <® AgCl + e Eq. 1

The resonance frequency of the AgNP/IDE-RFID tag before and after the AgNP oxidation to AgCl strongly depends on the media in which the IDE is kept. In air, the change of frequency is equal to 4.0±0.7 MHz while in PBS it is close to zero, 0.06±0.06 MHz. This data is summarised in Table Sl.

In use, the biosensor 1 may find itself in an in situ environment that may be similar to air, PBS or any other. In order to guarantee a similar environment at all times or to emulate an in situ environment ex situ, a saline solution may be added to the biosensor 1. This saline solution may comprise chloride and/or phosphate.

It may be noted that the resonance frequency change for AgNP/IDE-RFID in air after AgNP oxidation is more than 10 time higher than for the same tag in PBS (Table Sl. In PBS, the frequency change is noticeable, equal to approx. 0.06 MHz, but due to high standard deviation is statistically not significant). High ionic conductivity of PBS limits the change of the total impedance of the IDE-RFID tag immersed in PBS when highly electrically conducting AgNP layer is converted into a low conductivity AgCl layer. At the same conditions, the resistance (impedance at zero frequency, Fig. 2A, dashed line) of the AgNP/IDE changes from approx. 0.07 to 16 kOhms (when AgNP layer is electrochemically converted into AgCl layer). These electrochemical experiments prove that conversion of AgNP into AgCl is easily detectable by measuring changes of resonance frequency of the tag. Similar experiments have been conducted also making 2 mm gap on the IDE e.g. by scratching gold fingers in the central part in the IDE and with screen-printed electrodes with a 2 mm gap between the electrodes.

The gaps were filled with the layer of AgNPs (the electrodes are shown in Table S2). Higher separation between electrodes results in much higher sensitivity of the AgNP/2- mm-gap-electrode-RFID response (resonance frequency and attenuation) upon oxidation of AgNPs to AgCl. Enclosing the electrode into a 75 pm thick microchannel further increase AgNP/2-mm-gap-electrode-RFID signal sensitivity to the conversion of AgNP to AgCl. In case of 2 mm gap electrodes the effect of ionic conductivity provided by PBS in microchannel imposes very negligible effect on the change of the resonance frequency of the RFID tag (Sl l) caused by AgNP conversion to AgCl. The resonance frequency changes from 12.19±0.05 MHz to 2l.08±0.03 MHz upon oxidation of AgNPs to AgCl in this case. This data is presented in Table S2.

Coupling of enzymatic redox reactions to RFID-based registration

Embodiment 1: RFID biosensor for peroxide-based on horseradish peroxidase driven oxidation of AgNPs to AgCl

After a high sensitivity of RFID-based detection of Ag/AgCl redox reaction has been realised and confirmed by electrochemical experiments the aim was to prove that an enzymatic reaction may convert AgNPs to AgCl on the AgNP/IDE or

AgNP/SPE containing RFID tags. For this, after the AgNPs were deposited on IDE, the horseradish peroxidase solution (1 mg/mL in water) was drop casted and let dry. It is well known that HRP adsorbs on solid surfaces and shows efficient direct electronic coupling between the active site of HRP and high variety of electrically conducting materials. Thus, it was expected that in the presence of hydrogen peroxidase (H2O2), a H2O2/HRP system will drive AgNP oxidation to AgCl in PBS, as presented by reactions 2 and 3.

HRP(Fe³⁺ ) + H₂0₂ ® CompI(Fe⁴⁺, P ^*) + H₂0 Eq. 2 HRP/AgNP

AgNP + Cl + Compl(Fe⁴⁺, P ^*) - > AgCl + HRP{Fe³⁺ ) Eq. 3

Where HRP(Fe³⁺) is a native form of horseradish peroxidase with a ferrihaeme prostetic group and CompKFe⁴⁴ , P^*) is compound I, consisting of oxyferryl iron (he⁴ ()) and a porphyrin P* cation radical. HRP/AgNP indicates that the enzyme is directly adsorbed on AgNPs, expecting that HRP is in direct electron transfer (DET) contact with AgNP surface, i.e., HRP and AgNPs are electronically connected by DET.

Unfortunately, it was not possible to confirm this reaction neither by RFID measurements, nor by amperometric or spectrophotometric monitoring (data not shown). Though, it is well known that HRP shows efficient direct electronic coupling with a number of carbon- and metal-based materials including gold nanoparticles (AuNPs). Keeping this in mind the experimental setup enabling enzymatic conversion of AgNPs to AgCl was modified. First, HRP was adsorbed on AuNPs and then this AuNP/HRP nanobiocompound was mixed with AgNPs, deposited on the IDE and was successfully used to convert AgNP to AgCl in PBS. The design, comprising AgNPs 10 and AuNPs 10 deposited on an IDE 12, is schematically shown in Fig. 3 A. This is only one example of several different nanomaterials 10 operatively connected to each other and the electrode 12, and any number and combination is possible. The reaction of enzymatically driven AgNP conversion to AgCl is presented by Eq. 4.

Where HRP/AgNP noted in Eq. 3 is now substituted to HRP/AuNP/AgNP and indicates that the enzyme is in DET contact with AuNPs and that the AuNP is electronically connected to the AgNPs. Fig. 3D presents the effect of H2O2/HRP/AUNP driven oxidation of AgNPs to AgCl on the electromagnetic reflection from the RFID tag (the tag comprised of the IDE with 2 mm gap which is covered with of AgNPs and

HRP/ AuNP mixture). The HRP/ AuNP/ AgNP/2-mm-gap-IDE was immersed in PBS and Sl l function was recorded showing resonance frequency of the IDE tag at 14 MHz. After addition of H2O2 into the PBS the resonance frequency shifted to approx. 21 MHz (Fig. 3D). The direct current measurements through IDE before and after addition of H2O2 for the similarly prepared IDE are shown in Fig. 3E. It can be seen that addition of H2O2 resulting in 25 mM concentration diminishes DC current through the IDE from 60 mA to electrical noise level (approx. 50 pA), which corresponds to the resistance change from 83 Ohms to higher than 80 MOhms. These experiments confirm enzymatically driven AgNP oxidation to AgCl on the IDE. Since HRP cannot directly oxidise AgNPs to AgCl the electron transfer pathway between the HRP and the AgNPs thus proceeds through the AuNPs, i.e., Compound I on AuNP oxidises AgNPs to AgCl. The electron transfer proceeds through the following electron transfer pathway

H₂0₂^HRP^AuNP^AgNP and results to the conversion of AgNPs to AgCl at the same time H2O2 is reduced to H2O (Eqs. 2 and 4). This reaction sequence is also confirmed by registering UV-Vis spectra during the reaction composed from different nanobiomaterial mixtures, see Figs. Sl-2.

Making HRP/ AuNP and AgNP nanobiocompound mixture on IDE

reproducibly is difficult. The dispersion of AuNPs should be quite homogeneous avoiding short-circuiting the IDE. Since, DET between HRP and the AuNP drives oxidation of the AgNPs through electronic contact between the AuNP and the AgNPs it is possible to carry out the HRP/AuNP ET process and Ag/AgCl ET processes on separate electrodes. Fig. 3B and 3C show two electrode 12 designs where the

HRP/AuNP T-shaped layer is deposited on the AgNP layer. The AgNP layer connects two sides of IDE separated by 2 mm gap. Fig. 3C shows the design, which realises the HRP/AuNP and Ag/AgCl ET reactions on totally separated electrodes. Any number of electrodes 12 may be used, and these may or may not be operatively connected. The conductive nanomaterial 10 deposited to each electrode 12 may differ by at least one nanomaterial 10. In fact, the Fig. 3C design resembles biofuel cell (BFC)-based electrode configuration and, thus, suggests that the majority of anode or cathode electrodes could be used as the biosensing electrode of the proposed RFID biosensor construction. It also suggests that instead of AgNPs, we might be able to choose other nanomaterials, e.g. graphene or zink nanoparticles, which upon oxidation or reduction experience high change of the resistance or the change of the double layer capacitance. Embodiment 2: RFID biosensor for glucose-based on glucose oxidase driven oxidation of AgNPs to AgCl Above described HRP/AuNP/AgNP nanobiomaterial is an essential element of RFID biosensor for peroxide. Since many oxidase enzyme-based biosensors produce H2O2 it is easy to make an RFID biosensor for any oxidise enzyme substrate just by adding oxidase enzyme onto HRP/AuNP nanobiomaterial. Fig. 4 presents an attenuation (Sl 1 after background subtraction) and resistance signal for glucose oxidase-based RFID biosensor for glucose. For these measurements, IDE electrode coupled to RFID tag was modifies sequentially by GOx/HRP/AuNP and AgNP nanobiomaterial starting first with deposition of the AgNP layer. The electrode immersed into the PBS has low resistance (high current). After the addition of glucose it gradually loses conductivity (current decreases, Fig. 4A) due to oxidation of glucose and generation of H2O2. H2O2 then drives the oxidation of AgNPs to AgCl as described in the previous paragraph. Attenuation registered with the RFID biosensor for glucose is shown in Fig. 4B. The glucose caused shift of the resonance frequency is obvious. This in principle discloses a general principle on how RFID biosensors based on redox enzymes may be constructed.

Dependence RFID biosensor characteristics on peroxide concentration in PBS

Trying to achieve high sensitivity of the HRP/AuNP/ AgNP -based RFID biosensor for H2O2, a number of different constructions have been tested. One of the best designs appeared to be based on screen-printed electrode, SPE-RFID tag, where the SPE is included into a microchannel. The design is shown in Fig. 5.

In this construction, AgNPs short circuit two electrodes of SPE that are coupled to an RFID tag. For the H2O2 sensing, the HRP/AuNPs layer is deposited on the SPE in a way that it contacts only part of the AgNP layer, avoiding permanent crosslinking of RFID coupled electrodes by the AuNPs. The described electrodes and sensing layers are enclosed in microchannels that have been sequentially filled with PBS containing different concentrations of H2O2. The change of attenuation (baseline corrected Sl 1) response of the RFID biosensor to H2O2 is shown in Fig. 5B. It is clearly visible that the attenuation is sensitive to H2O2 concentrations, which are in the range relevant to those, which are generated by appropriate biosensor measuring glucose and lactate in blood or sweat. An example of the dependence of resonance frequency and the attenuation of the RFID biosensor on H2O2 concentration are shown in Figs. 5C and 5D, respectively. As can be seen the resonance frequency shift strongly at lower H2O2 concentrations, while the attenuation is much more sensitive to higher H2O2 concentrations. This may be explained by the fact that the initial phase of the AgNP oxidation to AgCl strongly affects the surface properties of the AgNPs, which results in high change in the double layer capacitance of the layer and, thus, the change of the resonance frequency of the RFID biosensor. At higher concentrations of H2O2, only attenuation is changing indicating that the AgNP oxidation process involves mainly Ag, which constitutes the volume of AgNPs, i.e., Ag inside AgNPs.

The attenuation response (baseline corrected S 11) of the RFID biosensor to H2O2 can be modelled by using equivalent circuit of the RFID sensor tag. The equivalent circuit of the RFID reader and the RFID sensor tag is presented in Fig. S3A.

The RFID reader, usually, a network analyzer, measures so called Thevenin impedance, ZT¾, which is related to sensor impedance, Z_SCns, see Eqs. 5-7. Expanding Zsens to an equivalent circuit of resistor and capacitor in parallel, we arrive to impedance value sensed by the network analyzer.

Mutual inductance (M) is defined as the ratio of the total flux through both coils and the current through the coils at the reader. Then mutual inductance can be approximated by the following relationship (Eq. 8):

To calculate Sl 1 parameter the equivalent circuit of the reader and the sensor tag is simplified by the circuit of Fig. S3B. The Sl 1 parameter is calculated as follows (Eq. 9):

Synthesis of nanoparticles

Silver nanoparticles (AgNPs) were synthesized by citrate reduction method. Briefly, 95 mL of water was heated until the temperature reached 70 °C. After 1 minute 100 pL of 0.10 M ascorbic acid solution was added then the mixture solution comprising of 2 mL of the 1% citrate solution, 0.5 mL of 1% silver nitrate and 1 mL of 8 mM potassium chloride, which incubated for 5 minute was consecutively added into vigorously stirring solution. Let it stirred for 1 hr. Clear yellowish of AgNP-suspension was cooled down at room temperature. The concentration of synthesized AgNP is 0.3 mM.

Gold nanoparticles (AuNPs) were synthesized by following the reduction method of HAuCL with tri-sodium citrate (NaiG.FLO? ) in aqueous solution. Firstly, 40 ml of deionized (DI) FLO was heated to 90 °C then 5ml 0.01M HAuCh was added and stirred for 1 minute after that 5ml of NasCeFLCh (0.03M) was added under vigorously stirring. When the color change to red wine for gold, the mixture was heated and stirred for 15 min further. Let suspension of particle cooled down to room temperature and stored at 4 °C. The concentration of AuNPs is 0.20 mg/ml(l.0l54 mM).

The size and zeta potential of colloid particles was analyzed by DLS technique.

Method for manufacturing

A method 100 for manufacturing an electrochemical biosensor 1 is shown in

Fig. 6. The method 100 comprises at least two steps, and Fig. 6 shows four steps, though three, five or any other number greater than two is possible. The steps may be performed in any order and some may be skipped entirely.

A first depositing step 110 comprises depositing a first conducting

nanomaterial 10 onto a first electrode 12. The deposition may be dry or wet deposition such as electrophoresis. The deposition may also be thin film coating or casting such as drop casting or dip coating. While the previously described embodiments use drop casting, any method of depositing nanomaterial 10 is possible in any embodiment. The nanomaterial 10 may be any conducting nanomaterial as described previously.

A second depositing step 120 comprises depositing a second conducting nanomaterial 10 onto a second electrode 12 operatively connected to said first electrode 12. The deposition may be made using any of the methods described in the above paragraph, and may or may not be the same method as the first depositing step 110.

The operative connection between the first and second electrode 12 may be achieved using any connection such as being connected by an electrically conducting metal or being physically connected together.

A driving step 130 comprises a reduction and/or oxidation reaction of said nanomaterial 10. This may be achieved e.g. electrochemically by adding a catalyst 16 for said reaction. The catalyst 16 may be added e.g. directly to the nanomaterial 10 or to a solution comprising the biosensor 1. The catalyst 16 may be any catalyst as previously described. The reduction and/or oxidation reaction may be a reduction reaction, an oxidation reaction or a redox reaction as previously described.

A registering step 140 comprises registering electromagnetic reflection of said nanomaterial 10 resulting from said reduction and/or oxidation reaction. The registering may comprise any type of measurement such as voltage, resistance or radiation measurements. Using previously established measurements, a difference may then be detected and registered. This registration may be used to monitor biological functions such as glucose levels in a patient, which has many medical and diagnostic uses for e.g. diabetes control. Spectrophotometric verification of the main embodiment

To determine AgNP oxidation by H2O2 catalyzed by HRP in the presence of AuNP, the following experiment was conducted. The target sample was prepared by mixing AuNP and AgNP to similar concentration of O.Olmg/ml. HRP was injected into particle mixture to the concentration of 0.1 pg/ml. The sample was left for 10 min at room temperature for stabilization before addition of H2O2 to 0.1 mM. Further waiting time of 10 min for H2O2 action was allowed. The sample was prepared in diluted PBS. Then, the UV-Vis absorbance spectrum were examined within the wavelengths from 300 to 700 nm. The target sample contained four components, namely AgNPs, AuNPs, HRP, H2O2. To validate the reaction each of the three compounds were omitted in the control samples (Table S3). All the samples were prepared in 10 times diluted PBS. Result: The UV-Vis absorbance of gold and silver nanoparticles are given in Fig. Sl. The absorbance of AuNPs has a peak at 525 nm and AgNPs has a peak at 4l0nm. The mixture of two samples show a UV-Vis composed of two spectra with the presence of two peaks.

The absorbance graph of sample 1 to sample 4 are presented in Fig. S2.

Notably, in the sample 1 with the mixture of four substrates, the absorbance peak of AgNPs at 410 nm disappeared if compared to the other samples.

The result illustrates the disappearance of AgNPs, which is a consequence of AgNP oxidation to AgCl particles by H2O2 catalyzed by HRP. The reaction generated a potential that may convert silver into silver chloride.

Conclusions

In this description, it is described and disclosed how to make RFID biosensor based on redox enzymes. The biosensor redox reaction is coupled to the registration of electromagnetic reflection through AgNP oxidation to AgCl. The AgNP oxidation to AgCl may be driven by any appropriate redox reaction including enzymatic or non- enzymatic redox reactions. We demonstrate the principle using peroxidase and glucose oxidase enzyme; however, any other oxidoreductase can be used in the proposed RFID biosensor design. We demonstrate the RFID biosensor principle using only oxidase enzymes, which drive AgNP conversion to AgCl. However, our investigations also confirmed (data not shown) that the opposite reaction direction, i.e., AgCl -> Ag, is possible to drive electrochemically. This suggest that reductase enzyme-based biosensors should be also be possible to construct where the enzymatic reaction drives reduction of AgCl back to AgNPs. We are convinced that a similar approach could be adopted with other materials where enzymes can be used to drive nanomaterial oxidation or reduction. The highest sensitivity of the RFID biosensor may be achieved for reactions, which cause high change of the double layer of the nanomaterial or its resistivity or both. Such nanomaterials could be graphene/graphene oxide, Zn/ZnO, etc. What is probably unique with AgNPs is that the formal potential of Ag/AgCl reactions is the middle of the formal potential range of biologically relevant reactions, and thus, may be exploited for monitoring biologically relevant oxidation and reduction reactions by choosing an appropriate enzyme. Instead of the enzymes, organelles or cells, e.g., bacteria and their biofilms, can also be used in this case. We demonstrate the principle on coupling of an enzyme to nanoparticle by direct electron transfer, but it is obvious that mediated electron transfer schemes will work in similar way. In case of cell-based RFID biosensors, mediated ET might be beneficial. The proposed sensor may thus be used not only to detect enzyme substrates, but also to detect the growth of bacteria or bacterial biofilms on sensor surface or on separate surface that is electronically connected to RFID sensor comprised of nanoparticles that change their double layer capacitance or resistance as exemplified in this work for AgNPs and their redox conversion to AgCl.

In summary, this description discloses a general principle for constructing RFID sensors and biosensors with a recognition mechanisms based on redox reactions. The main embodiment comprises a material, including nanomaterials and their combination, which constitutes an RFID antenna or part of the antenna. This material is made sensitive and specific to a redox reaction of interest. As an example, in this description we show an RFID and silver nanoparticle modified electrode coupling allows construction of sensors and biosensors for hydrogen peroxide and glucose. Similar biosensors may be constructed with exploiting any available oxidoreductase enzyme, their combinations, bacteria or any other biological recognition element capable to drive reduction or oxidation of the RFID-electrode material. In this case, we demonstrate the principle using silver nanoparticles as the sensing electrode material and Ag/AgCl redox reactions as a transduction reaction coupling the compound of analytical interest and RFID detection principle.

Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Moreover, although the foregoing descriptions and the associated drawings describe exemplary

embodiments in the context of certain exemplary combinations of elements and/or functions, it should be appreciated that different combinations of elements and/or functions may be provided by alternative embodiments without departing from the scope of the appended claims. In this regard, for example, different combinations of elements and/or functions than those explicitly described above are also contemplated as may be set forth in some of the appended claims. In cases where advantages, benefits or solutions to problems are described herein, it should be appreciated that such advantages, benefits and/or solutions may be applicable to some example embodiments, but not necessarily all example embodiments. Thus, any advantages, benefits or solutions described herein should not be thought of as being critical, required or essential to all embodiments or to that which is claimed herein. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Claims

1. An electrochemical biosensor comprising:

at least one conductive nanomaterial (10) operatively connected to at least one electrode (12); and

a catalyst (16) for a reduction and/or oxidation reaction of said nanomaterial

(10).

2. The biosensor according to claim 1, further comprising an antenna (14) operatively connected to said at least one electrode (12).

3. The biosensor according to claim 2, wherein said antenna (14) is an RFID antenna.

4. The biosensor according to any one of the preceding claims, wherein said catalyst (16) comprises hydrogen peroxide.

5. The biosensor according to any one of the preceding claims, wherein said catalyst (16) comprises an enzyme.

6. The biosensor according to claim 5, wherein said enzyme (16) is a peroxidase and/or glucose oxidase enzyme.

7. The biosensor according to claim 6, wherein said enzyme (16) is horseradish peroxidase.

8. The biosensor according to claim 5, wherein said enzyme (16) is a reductase enzyme.

9. The biosensor according to any one of the claims 1-4, wherein said catalyst (16) comprises an organelle or cells, e.g. bacteria, and/or their biofilms.

10. The biosensor according to any one of the preceding claims, wherein said nanomaterial (10) comprises silver nanoparticles.

11. The biosensor according to any one of the preceding claims, wherein said nanomaterial (10) comprises gold nanoparticles.

12. The biosensor according to any one of the claims 1-9, wherein said nanomaterial (10) comprises graphene.

13. The biosensor according to any one of the claims 1-9, wherein said nanomaterial (10) comprises zink nanoparticles.

14. The biosensor according to any one of the preceding claims, wherein said at least one electrode (12) is an interdigitated electrode.

15. The biosensor according to any one of the preceding claims, wherein said at least one electrode (12) is a screen-printed electrode.

16. The biosensor according to any one of the preceding claims, comprising at least two electrodes (12).

17. The biosensor according to claim 16, wherein said at least two electrodes (12) are operatively connected.

18. The biosensor according to claim 16 or 17, wherein the conductive nanomaterial (10) deposited to each electrode (12) differs by at least one nanomaterial (10).

19. The biosensor according to any one of the preceding claims, wherein said reduction and/or oxidation reaction changes a double layer capacitance of said nanomaterial (10).

20. The biosensor according to any one of the preceding claims, wherein said reduction and/or oxidation reaction changes the resistivity of said nanomaterial (10).

21. The biosensor according to any one of the preceding claims, further comprising a saline solution.

22. The biosensor according to claim 21, wherein said saline solution comprises chloride.

23. The biosensor according to claim 21 or 22, wherein said saline solution comprises phosphate.

24. A method for manufacturing an electrochemical biosensor (1), said method (100) comprising the steps of:

depositing (110) a first conducting nanomaterial (10) onto a first electrode (12); and

driving (130) a reduction and/or oxidation reaction of said nanomaterial (10).

25. The method according to claim 24, further comprising a step of depositing (120) a second conducting nanomaterial (10) onto a second electrode (12) operatively connected to said first electrode (12).

26. The method according to claim 24 or 25, further comprising a step of registering (140) electromagnetic reflection of said nanomaterial (10) resulting from said reduction and/or oxidation reaction.