INTRODUCTION

The present invention relates a method for fabrication of an electrochemical biosensor for measurement of ketone levels.

BACKGROUND

Diabetic ketoacidosis (DKA) is a serious complication in people with diabetes when the ketones is produced at high level, resulting from insulin deficiency. This could be life-threatening if not found and treated soon. DKA mainly affects Type 1 diabetes patients, but sometimes affects patients with type 2 diabetes.

The American Diabetes Association (ADA) guidelines advises to check blood or urine ketones at sick days as often as every 4 hours, while the Australian Diabetes Educators Association (ADEA) advises that blood ketones should be checked every 2 hours at sick days for ketone levels between 0.6 - 1 mmol/L and every hour for ketone levels above 1 mmol/L.

Ketone monitoring is per March 2022 only monitored through finger-prick drawing of blood samples onto disposable sensors. This is a tedious, and physically painful procedure. Furthermore, it comes on top of monitoring the primary marker for diabetes management, glucose. In addition to being physically painful, it serves as a constant reminder to the patient that they are affected by the disease and may be disruptive to normal life. Ideally, the patient would benefit from technology making the disease manageable, minimizing the inconvenience. Lastly, and perhaps most importantly, finger pricking provides only a snapshot image of the ketone levels. It does not capture trends, peaks and valleys, and thus management interventions become less effective. The current guidelines recommend finger pricking at least once a day for Ketones.

Currently, most of the commercial ketone sensors are disposable, presented in the form of a single strip or an electrode for each single use. The drawback of these disposable sensors is the difficulty for the patients to become aware of when their ketone levels are increasing. Hence, continuous ketone monitoring is necessary for the patients to access their ketone level frequently and this is helpful for any medical intervention if necessary.

In 2019, a preliminary result from the research group of Lee et al. “The Clinical Case for the Integration of a Ketone Sensor as part of a closed loop insulin pump system”, showed that the continuous ketone sensor tested in phosphate buffer saline (PBS) solution for 7 days indicated a limit in linearity after 3 mM of hydroxybutyrate (HB). The present method retains analytical response for up too 12 days in the range 0-4 mM HB.

Commercially available point of care (POC) devices measure the ketone by detecting the concentration of sodium 3-hydroxybutyrate (3HB) by using the 3- hydroxybutyrate dehydrogenase enzyme (3HBDH). This enzyme is an oxidoreductase enzyme that catalyses the oxidation of 3HB to acetoacetate in the presence of a cofactor - nicotinamide adenine dinucleotide (NAD+). The generated NADH is oxidized at the surface of the device’s working electrode providing a current proportional to the concentration of 3HB. An in vitro and/or in- vivo ketone sensor operated continuously needs to ensure that the enzyme remains stable and sensitive under the operating conditions in the body. Challenges are, however, the low concentration of NAD+ in the human body and the electrode poisoning which reduces the potential for the subsequent NADH oxidation.

In WO2021/081456 (Teymourian et al.) proposes a microneedle sensor detecting ketone levels continuously utilizing immobilized 3HBDH. Hollow microneedles are packed with 1 ,10-Phenanthroline-5, 6-dione (PD) mediated carbon ink suspended in an ionic liquid. The HBD-NAD+ complex is then tethered in place by crosslinking. The proposed strategy opens up for the possibility of continuous ketone monitoring. Cross-linking, is however a harsh treatment to the enzyme, causing denaturation. A gentler immobilization method may limit that denaturation.

There is a need for electrochemical biosensors for measurement of ketone levels that have a simplified fabrication process, and hence caters to the need of mass distribution. The method should accommodate a conditions reminiscent of the native habitat of the enzyme, to minimize denaturation and inihibition of its function.

The aim of the present invention is to provide a method for fabrication that addresses at least one of the mentioned problems while retaining the benefits of prior art.

SUMMARY OF THE INVENTION

This is achieved according to the invention by providing a method for fabrication of an electrochemical biosensor, for measurement of ketone levels in human blood or interstitial fluid, comprising; adding at least one layer on one or more metal coated solid microneedles, from the bottom up, the at least one layer comprising an electron mediator, an enzyme and its cofactor, and an outer membrane layer, where the method comprises the steps of: a) electrodeposition of the electron mediator on the one or more metal coated microneedles by using an electrochemical waveform generator, b) coating an additional layer of electron mediator entrapped in a chitosan matrix, c) coating an enzyme-cofactor mixture on the one or more microneedles, d) coating an enzyme-cofactor mixed chitosan layer on the one or more microneedles, e) using a dense semi-permeable polymer to create an outer protective membrane on the one or more microneedles.

Further advantageous features of the present invention are defined in the dependent claims.

BRIEF DESCRIPTION OF DRAWINGS

Example embodiments will now be described with reference to the following drawings, where:

Fig. 1 shows a configuration of a ketone microneedle sensor, where the device comprises gold coated microneedles. Fig. 2 illustrates cyclic voltammogram of PD electrodeposition on the gold microneedle.

Fig. 3 illustrates first HB test of the sensor, each step is 1 -2-3-4 mM of HB.

Fig. 4 illustrates sensor fabricated with the described method working after 12 days

DETAILED DESCRIPTION

In the following, general embodiments of the invention will be described. References will be made to the accompanying drawings. It shall be noted, however, that the drawings are example embodiments only, and that other features and embodiments may well be within the scope of the invention as claimed.

The method according to the invention for fabrication of an electrochemical biosensor, for measurement of ketone levels, expressed as hydroxybutyrate levels, comprising; adding at least one layer on one or more metal coated solid microneedles, from the bottom up, comprising an electron mediator, an enzyme and its cofactor, and an outer membrane layer.

- the enzyme and cofactor is allowed freedom of movement through avoiding strong chemical bonding, but rather relies on entrapment and confinement.

The present invention relates to a method for fabrication of an electrochemical biosensor, for measurement of ketone levels, comprising the steps of: a) electrochemically depositing the electron mediator on the one or more metal coated microneedles by using an electrochemical waveform generator, b) coating an additional layer of electron mediator on the electron mediator modified one or more conductive microneedles, c) coating an enzyme-cofactor mixture on the one or more microneedles, d) coating an enzyme-cofactor mixed chitosan layer on the one or more microneedles, e) encapsulating the above layers in a protective, dense polymeric outer membrane. The layer in step a) is deposited by using an electrodeposition technique such as electro polymerization, and where the electron mediator is PD, or Meldola’s blue, or Prussian Blue mixed with ethanol or suitable solvent, and where a three- electrode system is used. The electron mediator mixture in step b) comprises PD mixed with chitosan dissolved in a suitable solvent, preferably ethanol. The enzyme-cofactor mixture of step c) comprises hydroxybutyrate dehydrogenase (3HBDH) and nicotinamide adenine dinucleotide (NAD+). The enzyme-cofactor mixed chitosan layer of step d) comprises 3HBDH enzyme and NAD+ mixed with chitosan. The outer membrane in step e) comprises dense polymeric semipermeable membrane such as polyvinylbutyral (PVB) or polyvinylchloride (PVC).

The structural material shaping the microneedles could be several types of material, but it is typically a polymer shaping the microneedles. A solid polycarbonate base carrier could be used, and where the microneedles are coated with a metal to make it an electrochemical transducer. A reaction domain is created next to the transducer by the densely packed functiounal layers, and the outer protective membrane. The outer membrane controls the influx of ketone as well as limiting the efflux of reactants. The membrane also provides a biocompatible interface. A combination of PVC and chitosan also makes the membrane respectively permselective and semipermeable (cf. Teymourian et al. 2019).

According to the invention, the cofactor and/or enzyme is not entrapped and is free to migrate through the layers. The outer membrane layer retards the migration of the NAD+Z NADH away from the formulation and into the bulk of a sample. With this formulation high analytical sensitivity, around 3 nA ml\ZT ¹ mm ^-2 is retained. This is attributed to the fact that the enzyme, and cofactor are allowed to roam freely within the reaction domain. This provides a gentler environment for the enzyme. There are accordingly less external forces contributing to changing the folding and structure of the enzyme, and consequently less denaturation. It has an in vitro operational lifetime spanning over weeks. In one embodiment the invention provides a configuration of the electrochemical biosensor for measurements of hydroxybutyrate levels as shown below. The metal coated throughout solid microneedles comprise the following layers;

PVB as the outer protective membrane, 3HBDH enzyme-NAD+ mixed chitosan, 3HBDH enzyme mixed NAD+, PD mixed chitosan,

PD electrodeposition layer on the microneedles surface, gold coated microneedle

The enzyme 3HBDH is immobilized along with its NAD+ through entrapment in a polymer layer (enzyme-NAD+ mixed chitosan) together with the addition of a protective polymer outer membrane. It is through a combination of being (i) sandwiched between and within the semipermeable chitosan layers, and (ii) “repelled” by the permselective outer membrane, that the reactans can remain within the recation domain for a time span of weeks.

A configuration of a ketone microneedle sensor, where the device comprises gold coated microneedles is illustrated in figure 1. The electrochemical biosensor according to the invention is for use in biological fluids to measure hydroxybutyrate level. The sensor can work continuously for at least 12 days (cf. figure 4).

A sensor fabricated according to the invention is used for transdermal detection of ketone level and/or glucose in blood, and also in conjunction with other sensors including lactate and glucose.

According to the present invention it is provided a versatile sensor formulation, primarily for use with electrochemical transducers, that is amenable for production, and can justify industrialization. Inspiration is drawn from the academic literature, combined, and adapted it such that is compatible with three-dimensional surfaces, targeting the application of painless, continuous monitoring on transdermal microneedles. Further, the methods described are compatible with integration with formulations targeting other diabetes markers such as glucose and ketone, on the same array of microneedles. The present invention is an improvement upon existing ketone formulations in that it concerns a very specific layering that is suited to the up-scaling of manufacturing and hence caters to the need of mass distribution. It is suited to microneedles as well as lower dimensionality structures. Additionally, the concept of allowing the reactive entities to roam free within the reaction domain allows for slower degradation of said components, and consequently improves operational lifetime and analytical sensitivity.

EXAMPLES

Fabrication of an electrochemical biosensor

Configuration of a ketone microneedle sensor, as illustraded in figure 1 , is shown below: a. Pre-treatment of sensor

The microneedle sensor consists of gold microneedles as working electrode and platinum microneedles as counter and reference electrode. The sensor was cleaned by dipping in HCI 0.1 M for three minutes to remove all contamination on the gold surface. b. Electrodeposition of electron mediator

An electrochemical waveform generator was used to electrochemically deposit the electron mediator PD on the gold microneedle. A three-electrode system was used, a standard Ag/AgCI was used as reference electrode and a Pt counter electrode. The pre-treated microneedles were dipped into the mixture containing PD 25 mM in ethanol 96% mixed with PBS 10 mM, ratio 1 :1. The electrodeposition was carried out by running cyclic voltammetry (CV) from -0.6 V to 0.6 V for 30 cycles at 100 mV/s. The sensor after electrodeposition was cleaned with DI water and let dry for 15 minutes. Figure 2 shows cyclic voltammogram of PD electrodeposition on the gold microneedle. The electrodeposition was obtained by running CV in PD solution from -0.6 V to 0.6 V at 100 mV s-1 for 30 cycles. c. Drop cast layer of electron mediator

An extra layer of of electron mediator was drop casted on the PD modified gold microneedles as follows.

A micro pipet was used to drop cast 0.3 pL of PD mixture on each microneedle. This PD mixture contained PD 25 mM in ethanol 96% mixed with chitosan (info) in 1 : 1 ratio. The sensor was then let dry for 15 m inutes. d. Drop cast layer of enzyme-cofactor

The enzyme 3-HBDH 1 mg/mL was mixed with cofactor, NAD+ 0.5 M, in a ratio 10:1 . A micro pipet was used to drop cast 0.3 pL of enzyme-cofactor mixture on every single microneedle. The microneedle sensor was then let dry for 30 minutes. e. Drop cast layers of enzyme-cofactor mixed chitosan

An enzyme-cofactor mixture of 3-HBDH enzyme 1 mg/mL and NAD+ 0.5 M, ratio 10:1 was prepared. This enzyme-cofactor mixture was then mixed with chitosan suspended in acetone (5 mg/mL) in a ratio 1 :1. A micro pipet was used to drop cast 0.3 pL of enzyme-cofactor mixed chitosan layer on every single microneedle. The microneedle sensor was then let dry for 30 minutes. This drop casting step was repeated one time to add one more layer of enzyme-cofactor mixed chitosan on the microneedles. f. Drop cast layers of polymer

A PVB solution (60 mg/mL) in cyclohexanone was prepared. A micro pipet was used to drop cast 0.3 pL of the PVB solution on every single microneedle. The microneedle sensor was then let dry for 30 minutes. This drop casting step was repeated one time to add one more layer of PVB solution.

Ketone measurement (sodium 3-hvdroxybutyrate) A potentiostat was used to perform chronoamperometric measurements, the bias voltage being maintained at +0.3 V vs. the onboard Ag/AgCI microneedle electrodes. The sensor was dipped in PBS (10 mM) solution with a magnetic stirrer inside to enhance homogenization and mass transport in the solution when sodium 3-hydroxybutyrate (HB) was added. The analyte (HB) was titrated into the test solution once the current had stabilized at a steady-state current (after 2000 seconds), with time intervals of 1000 seconds, yielding a concentration profile from 0 to 4 mM in steps of 1 mM. Sensor data:

The sensor reacts to 1 -2-3-4 mM HB presented in figure 3.