Non-enzymatic electrochemical sensor and components

Abstract

The present invention relates to a thiophene based covalent organic framework (T-COF) and application of such T-COFs in electrochemical sensors for sensing glucose. In particular, the present invention relates to a T-COF comprising boronic acid-functionalized C60 (BA-C60) and the use of such T-COFs and electrochemical sensors in methods of sensing glucose such as in bioprocesses.

Description

[0001] Non-enzymatic electrochemical sensor and components

[0002] Technical field of the invention

[0003] The present invention relates to a thiophene based covalent organic framework (T-COF) and application of such T-COFs in electrochemical sensors for sensing glucose. In particular, the present invention relates to a T-COF comprising boronic acid-functionalized Ceo (BA-Ceo) and the use of such T-COFs and electrochemical sensors in methods of sensing glucose such as in bioprocesses.

[0004] Background of the invention

[0005] Glucose is a key substrate in bioprocesses, as it influences cell growth, productivity, and metabolite production. Therefore, real-time and accurate glucose measurement is vital for bioprocess control and optimization. Continuous glucose measurements can help maintain the optimal metabolic state, maximize product yields, and minimize by-product formation. Moreover, real-time data collection can improve the understanding of cellular metabolism and enable the development of predictive models for bioprocess optimization.

[0006] A multitude of glucose monitoring techniques have been developed over the years, ranging from traditional analytical methods like high-performance liquid chromatography (HPLC) and enzymatic assays to cutting-edge technologies such as biosensors, spectroscopy, and microfluidic devices. However, conventional methods for glucose monitoring, such as HPLC, are often time-consuming, labor- intensive, and prone to errors.

[0007] Hence, there is a need for alternative methods that can provide accurate, reliable, and continuous glucose monitoring in bioprocesses. Enzymatic electrochemical biosensors based on glucose oxidase (GOx) or glucose dehydrogenase have issues such as enzyme instability, short lifetime, and fouling due to biofilm formation or nonspecific protein adsorption. Enzymes can lose activity over time, leading to reduced sensor performance and necessitating frequent calibration or replacement. Although optical glucose sensors resist fouling, they have a narrow range of sensitivity and can be affected by environmental factors such as turbidity, gas bubbles, multiphase systems, and temperature. They may also need complex instrumentation to operate correctly. As an alternative, non-enzymatic electrochemical sensors have emerged for glucose monitoring. These sensors usually employ electrocatalytic materials, like noble metals, metal nanoparticles, transition-metal oxides, carbon nanomaterials or conductive polymers, to facilitate direct electron transfer between glucose and the electrode. Since they do not depend on enzymes, they are less susceptible to environmental factors and can maintain their performance for extended periods. Furthermore, most non-enzymatic sensors have the benefit of being oxygenindependent and can avoid signal fluctuations within the normal oxygen range. Non-enzymatic electrochemical sensors offer many advantages, but they still face some challenges in terms of sensitivity and selectivity. These are important parameters for accurate and reliable glucose detection in complex biological samples. To compete with the existing enzymatic and optical sensing technologies, non-enzymatic sensors need to employ novel materials and sensor designs that can enhance their performance and stability.

[0008] Hence, improved electrochemical sensors for sensing glucose are sought for, and in particular more selective and reliable electrochemical sensors with high sensitivity and stability would be advantageous.

[0009] Summary of the invention

[0010] Thus, an object of the present invention relates to providing new materials with a high specificity and sensitivity towards glucose. In particular, it is an object of the present invention to provide electrochemical sensors comprising these new materials, that solve the above-mentioned problems of the prior art.

[0011] A novel non-enzymatic glucose sensor is thus provided, based on a T-COF creating high selectivity and sensitivity. This new sensor demonstrates excellent electrochemical performance, outperforming other modified electrodes due to its large electrochemical active surface area and good conductivity. The non- enzymatic T-COF-based sensor demonstrates remarkable effectiveness in glucose detection and outstanding electrooxidation detection performance for glucose monitoring. The electrocatalytic activity can be attributed to multiple factors, such as efficient electrolyte diffusion, selective glucose interaction with BA-Ceo, rapid charge transfer, exceptional electroconductivity of the BA-Ceo@T-COF, and the efficiency of active sites due to the distinct morphology and synergistic impact between T-COF and BA-Ceo in the constructed structure.

[0012] The developed sensor exhibits a highly consistent response over consecutive measurement cycles without requiring any cleaning procedures and maintains stable sensitivity for 20 days under ambient conditions. In comparison to traditional HPLC analysis, this sensor enables dependable glucose quantification in a significantly more efficient and time-saving manner. It displays exceptional stability when in direct contact with the fermentation medium and accurately quantifies glucose even in the presence of several electroactive species. The established method can be easily regarded as a straightforward, sturdy, precise, and cost-effective solution for real-time glucose monitoring in bioprocesses.

[0013] The T-COF's- and the electrochemical sensor's functioning and production are in particular demonstrated by the examples:

[0014] Example 1-4 shows how to manufacture-, characterize- and test the performance of the T-COF and electrochemical sensor of the present invention.

[0015] Example 5 shows a structural characterization of the T-COF and the electrochemical sensor.

[0016] Example 6 shows an electrochemical evaluation.

[0017] Example 7 shows an example of the sensing performance of the electrochemical sensor, and that despite the presence of interfering sugars, the sensor is capable of measuring actual bioprocess glucose concentrations, and with a high selectivity for glucose detection. This shows that the sensor has great potential for practical applications in bioprocesses, offering good selectivity and sensitivity for glucose detection in the presence of interfering substances.

[0018] Example 8 shows the stability, repeatability, and reproducibility of the T-COF and electrochemical sensor. The sensor demonstrates a rapid recovery time (less than 3 seconds) and consistent response towards glucose detection, showing its suitability for multiple measurements without requiring cleaning after each use. After 20 days, the recorded sensitivity was 95.3% of the initial sensitivity, showing a sustained ability to measure glucose after production.

[0019] Example 9 demonstrates how the T-COF and electrochemical sensor can be applied at-line during a fermentation process.

[0020] Example 10 shows that the T-COF can be applied to different carbon based substrates, with the best results achieved on substrates with increased surface areas. Example 11 investigates the usefulness of different nanoparticles and finds the gold (Au) nanoparticles to have a size of 100 nm.

[0021] More specifically, in a first aspect, the invention relates to a thiophene based covalent organic framework (T-COF) comprising boronic acid-functionalized Ceo (BA-Ceo), the T-COF comprising or consisting of C2 and C3 arranged in a crystal lattice structure represented by formula I, wherein C3 is represented by formula II (formula II), wherein Ri is C2 and C2 is represented by formula III (formula III). As introduced earlier this T-COF can thus be used in electrochemical sensors, thus in another aspect, the invention relates to the use of the T-COF as described herein, in an electrochemical sensor for sensing glucose. Evidently, in a further aspect, the invention relates to an electrochemical sensor for sensing glucose comprising a working electrode, the working electrode comprising the T-COF as described herein.

[0022] The invention further relates to methods of sensing glucose, -manufacturing etc. In another aspect, the invention relates to the use of the electrochemical sensor as described herein, in a method for sensing glucose.

[0023] In another aspect, the invention relates to a method of sensing glucose, the method comprising:

[0024] 1. Providing an electrochemical sensor as defined herein configured to produce an electric signal when a glucose molecule is at, or near, the working electrode;

[0025] 2. Providing a sample suspected of containing glucose;

[0026] 3. Contacting the electrochemical sensor with the sample;

[0027] 4. Measuring the signal from the working electrode, thereby sensing the presence of glucose in the sample;

[0028] 5. Optionally, comparing the signal to a signal from a control electrode on the electrochemical sensor to reduce background signals; and

[0029] 6. Optionally, comparing the signal from the working electrode to a signal obtained from measuring a control sample, to obtain a level of glucose in the sample.

[0030] In another aspect, the invention relates to an apparatus for sensing metabolites, the apparatus comprising the electrochemical sensor as described herein, optionally the apparatus comprising at least one further sensor for sensing a metabolite other than glucose.

[0031] In another aspect, the invention relates to a method of producing a T-COF comprising boronic acid-functionalized Ceo (BA-Ceo), the method comprising:

[0032] 1. combining thieno[3,2-b]thiophene-2,5-diboronic acid (F TTDB) and 2,3,6,7,10,11-hexahydroxytriphenylene (HHTP) with dry dioxane and dry mesitylene;

[0033] 2. reacting the mixture in an autoclave, such as at 150 °C for 72 h;

[0034] 3. washing the product thereof, and drying the product; 4. optionally, heating the product under high vacuum, such as at app. 200 °C and IO’6torr (1.33 x 10’4Pa);

[0035] 5. obtaining a T-COF;

[0036] 6. reacting toluene, fullerene Ceo, 4-formylphenylboronic acid pinacol ester (FPBA), and sarcosine, such as at 125 °C for 12 h;

[0037] 7. washing the product thereof;

[0038] 8. removing the protective group to obtain BA-Ceo, such as by the use of methylboronic acid dissolved in dichloromethane, followed by the addition of trifluoroacetic acid (TFA); and

[0039] 9. combining the T-COF with the BA-Ceo, thereby obtaining a T-COF comprising BA-Ceo.

[0040] In another aspect, the invention relates to a method of producing an electrochemical sensor for sensing glucose, the method comprising:

[0041] 1. providing a carbon based substrate, such as a pyrolytic carbon electrode; nanoparticles comprising or consisting of a metal, such as Au, Pt, Ag, or Cu; and a T-COF comprising BA-Ceo as defined herein;

[0042] 2. depositing the nano particles on the carbon based substrate;

[0043] 3. depositing the T-COF comprising BA-Ceo on the nanoparticles;

[0044] 4. optionally, depositing a counter- and / or a reference electrode at suitable positions on the electrochemical sensor;

[0045] 5. optionally, coating with a zwitterionic polymer, such as zwitterionic polypyrrole (ZiPPy); and

[0046] 6. optionally, coating with a diffusion-limiting layer, such as a layer comprised of a trimethylolpropane triglycidyl ether

[0047] (PTGE): polyethyleneimine (PEI) layer.

[0048] In another aspect, the invention relates to a kit comprising the electrochemical sensor as described herein, and optionally means for replacing the electrochemical sensor and / or one or more further electrochemical sensors, such as one or more further electrochemical sensors for sensing one or more metabolites other than glucose, optionally the kit may also comprise components for cleaning, such as washing buffers. Brief description of the figures

[0049] Figure 1. Building blocks used for COF synthesis, classified by their connectivity leading to 2D and 3D COFs.

[0050] Figure 2. Scheme of binding equilibria of boronic acid to glucose.

[0051] Figure 3. A) 3D pyrolytic carbon microelectrode in the three-electrode-chip configuration (10 xl6 mm); B) SEM of pyrolytic carbon micropillars as WE (284 micropillars).

[0052] Figure 4. Schematic representation of the process flow for the fabrication of the 3D pyrolytic carbon microelectrodes.

[0053] Figure 5. Synthetic pathway of BA-Ceo (boronic acid-functionalized Ceo). (insets: the photographs of Ceo and BA-Ceo in toluene).

[0054] Figure 6. Schematic representation of the synthesis process for T-COF and its structure.

[0055] Figure 7. The schematic diagrams of (a) the BA-Ceo encapsulation process by T- COF. (insets: the photographs of T-COF and BA-Ceo@T-COF powders), and (b) modification of 3D WE by electrodeposition of Au NPs and then electrodeposition of the BA-Ceo@T-COF composite on the Au NPs modified electrode.

[0056] Figure 8. a) The procedure for synthesizing zwitterionic pyrrole (ZiPy) monomers, b) Schematic of the modification of the Au NPs@BA-Ceo@T-COF modified electrode surface using PTGE: PEI by spray-casting and creating an antifouling coating by electropolymerizing ZiPy into ZiPPy through cyclic voltammetry (CV), at 0.0 - +0.95 V versus Ag / AgCI, at a scan rate of 50 mV s’1.

[0057] Figure 9. (a) UV-vis spectra of pristine Ceo and BA-Ceo in in a toluene, (b) FT-IR spectra of FPBA, pristine Ceo and BA-Ceo, and (c) FT-IR spectra of F TTBD, HHTP, T-COF, BA-Ceo, and T-COF@BA-Ceo. Figure 10. (a) PXRD patterns, (b) N2 adsorption-desorption isotherms, (c) pore size distribution (inset: pore volume) and (d) isotherm of TGA under N2 of T-CO and T-COF@BA-C6O.

[0058] Figure 11. SEM images of (a) T-COF and (b) BA-Ceo@T-COF with (i) low and (ii) high magnifications.

[0059] Figure 12. SEM images of (a) bare carbon micropillars, (b-d) after electrodeposition of Au NPs with low, medium and high magnifications, (d, inset) XRD pattern of Au NPs, (e) top-view of the electrodeposited BA-Ceo@T-COF film and (f) coated with PTGE: PEI thin film and ZiPPy polymer.

[0060] Figure 13. (a) Full survey XPS and high-resolution XPS spectra of (b) C Is, (c) O Is, (d) B Is, (e) S 2p, (f) N Is, and (g) Au 4f of T-COF, BA-C6o@T-COF, and AUNPS@BA-C6O@T-COF modified 3D pyrolytic carbon WE.

[0061] Figure 14. Electrochemical behavior: (a) CVs at AuNPs@BA-Ceo@T-COF, AuNPs@BA-Ceo and AuNPs@T-COF modified 3D pyrolytic carbon WE 0.1 M PBS (pH 7.0) at 50 mV s-1scan rate without and with 10.0 mM glucose, (b) the scan rate effect (20-170 mV s-1) on the oxidation properties of 10.0 mM glucose in PBS, (c) anodic peak current (lap) vs. u1 / 2and (d) anodic peak potential vs. log(u) plots.

[0062] Figure 15. Schematic diagrams of (a) the fabricated AuNPs@BA-Ceo@T-COF based sensor and electrochemical performance towards glucose detection and (b) the glucose oxidation reaction on the surface of the AuNPs@BA-Ceo@T-COF modified electrode.

[0063] Figure 16. (a) The cyclic voltammograms of 1 mM K3Fe(CN)e for the AuNPs@BA- Ceo@T-COF electrode at various pH values, using a scan rate of 100 mV s-1. Inset: determination of pKavalue of BA-Ceo; (b) Oxidation peak current responses of glucose with increasing value of pH by AuNPs@BA-Ceo@T-COF electrode in 0.1 M PBS with a glucose concentration of 10 mM; (c) Amperometric response of AUNPS@BA-C6O@T-COF electrodes toward successive glucose addition at different applied potentials; (d) Chronoamperometric response of AuNPs@BA-Ceo@T-COF modified electrode coated with PTGA: PEI layer and without this layer towards glucose: (e) Amperometric stepwise response of AuNPs@BA-Ceo and AuNPs@BA- Ceo@T-COF electrode for successive addition of glucose to PBS buffer solution by applying +0.55 V potential and (f) change in current with glucose concentration (calibration curve).

[0064] Figure 17. Glucose detection performance of AuNPs@BA-Ceo@T-COF electrode, (a) Selective response of the sensor towards glucose in the presence of different interfering agents (inset: comparison of the current with 5 mM of glucose and 20 mM of sugars), (b) Response reproducibility of the sensor towards a single concentration of glucose, 5 mM. (c) chronoamperogram of the 1stday, 10thday and 30thday after sensor fabrication, with successive additions of 5 mM glucose in PBS solution. (Inset: Long-term reusability of the sensor for glucose detection during 20 days) and (d) top view SEM images of sensor surface after 30 days of glucose detection test in PBS.

[0065] Figure 18. Glucose concentration measured at-line with and without cells by means of the AuNPs@BA-Ceo@T-COF based sensor (coated with ZiPPy and Nation as the antifouling layers) as well as off-line (cell-free samples) by HPLC. Besides, the increase in biomass over time is demonstrated by optical density measurements (ODeoo, at-line).

[0066] Figure 19. Glucose concentration measured at-line with and without cells by means of the AuNPs@BA-Ceo@T-COF based sensor coated with ZiPPy and compared with the results from biosensors (BioProfile FLEX2)

[0067] Figure 20. Amperometric response of the AuNPs@BA-Ceo@T-COF modified graphene based Dropsens electrode (as a commercial electrode) and 3D pyrolytic electrode by successive addition of glucose solution.

[0068] Figure 21. Comparison of electrochemical responses for different metal nanoparticles; AuNPs@BA-Ceo@T-COF, Pt NPs@BA-Ceo@T-COF, Ag NPs@BA- Ceo@T-COF, and Cu NPs@BA-Ceo@T-COF for detection of 10 mM glucose in PBS buffer solution. Figure 22. Measurement of Au nanoparticle sizes.

[0069] The present invention will now be described in more detail in the following.

[0070] Detailed description of the invention

[0071] Definitions

[0072] Prior to discussing the present invention in further details, the following terms and conventions will first be defined:

[0073] In the context of the present disclosure "Ceo" or "C60" is understood as a fullerene molecule, an icosahedron composed of sp2carbons in five and six-membered rings. In furtherance, BA-Ceo is understood as the fullerene molecule comprising a boronic acid.

[0074] In the context of the present disclosure "thiophene based COF", "T-COF", or "COF" are used interchangeably to COFs of the invention, i.e. T-COFs comprising boronic acid-functionalized Ceo. As described in more detail below, covalent organic frameworks (COFs) are a unique class of porous polymers created entirely through the covalent bonding of organic building units.

[0075] In the context of the present disclosure "an electrochemical substrate" is understood as a material or surface that serves as a platform for carrying out electrochemical reactions. The substrate acts as an electrode, facilitating the transfer of electrons between the reactants and the electrode surface.

[0076] Electrochemical substrates include metals, such as Au, Pt, Ag, or Cu, or carbonbased electrodes like graphite or glassy carbon and combinations thereof.

[0077] Covalent organic framework comprising boronic acid-functionalized Ceo

[0078] Covalent organic frameworks (COFs) are a unique class of porous polymers created entirely through the covalent bonding of organic building units. These frameworks display crystalline conjugated structures with a wealth of channels, functional groups, and pores. These materials can be classified into two main categories: two-dimensional (2D) and three-dimensional (3D) COFs (Figure 1). 2D COFs are characterized by their planar, layered structures, in which monomers are covalently bonded. These layers are interconnected through n-n interactions, resulting in a conjugated system. The size and shape of 2D COFs are highly dependent on the arrangement of these layers. Conversely, 3D COFs possess larger, cage-like cavities, with monomers that can continue to react and expand outward to create complex, interwoven structures. The basic components of COFs can be described using "knots" and "links," although their connections are constrained by the inherent structure of the monomers and the reaction conditions. Common linkage structures include boroxine boronate-ester, borosilicate, triazine, imine, hydrazone, borazine, squaraine, azine, phenazine, imide, spiroborate, C=C, amide, and viologen (Figure 1). The overall structure of COFs is determined by the building blocks used and the type of connections formed. Research on COFs has produced a diverse array of 2D and 3D structures with varying shapes, geometries, and functions, expanding the potential applications of these materials.

[0079] COFs offer unique advantages that cannot be achieved by metal-organic frameworks (MOFs) and zeolitic imidazolate frameworks (ZIFs), such as biocompatibility, and higher thermal and chemical resistance due to the stronger covalent bonds. COFs can be constructed from a variety of building blocks, including boron-based, imine-based, or triazine-based molecules. Moreover, their electrochemical behavior can be adjusted by utilizing electroactive building blocks. Recently, COFs constructed from thiophene building blocks have garnered significant interest due to their promising properties, such as high conductivity, which are retained in the self-assembled COF structure. COFs can serve as a sensing element, transducer, or signal amplifier in electrochemical sensors, depending on the modification. COFs can be modified with electroactive groups or compounds to selectively interact with analytes, immobilize biomolecules on their surface, and improve the performance of electrochemical sensors.

[0080] Enhancing the selectivity of glucose detection is essential for achieving precise and dependable glucose sensing. In the present disclosure this is done by incorporating functional materials with glucose binding capabilities, such as synthetic boronic acid and its derivatives. Boronic acid exists as a trigonal planar sp2-hybridized boron in its neutral form. In aqueous media, boronic acid (and boronic acid-containing compounds) exists in an equilibrium between its neutral hydrophobic form and, after complexation with a hydroxide ion, as a hydrophilic hydroxyboronate anion (sp3-hybridized boron). When the pH is higher than the pKa, boronate esterification is favorable. Inversely, a pH lower than the pKainduces hydrolysis and reverses the reaction. Its reversible reaction with c / s-1,2- or 1,3-diols of carbohydrates to form five or six-membered cyclic boronic esters allows boronic acid to be used as a diol-responsive material, reacting with molecules such as glucose (Figure 2). These properties are also the basis for utilizing boronic acid materials for the determination of monosaccharides and other diols in multiple applications, such as fluorescence, surface attachment, and potentiometric / amperometric sensing. However, the most common pKa range of boronic acids is in the range of 8-10, and there is much ongoing research into modifying boronic acids into materials sensitive to the relevant pH range in different applications.

[0081] The grafting density of boronic acid groups can be significantly increased by functionalizing them with various materials such as redox-active macromolecules, carbon nanomaterials, and polymers. This selective modification allows for the efficient capture of glucose. This improves the sensitivity of the boronic acid based sensors and broadens their applicability to complex sample matrices. Among the carbon nanomaterials, fullerene Ceo, a zero-dimensional nanocarbon, is the most abundant member of the fullerene family (Ceo-Cso). It is a truncated icosahedron composed of sp2carbons in five and six-membered rings. Ceo exhibits an array of fascinating characteristics, including high electronic conductivity, large specific surface area, good biocompatibility, inert behavior, and thermal and chemical stable structure. Furthermore, it is free of metallic impurities and can be easily implemented to produce reproducible electrocatalytic responses.

[0082] Thus, in a first aspect, the invention relates to a thiophene based covalent organic framework (T-COF) comprising boronic acid-functionalized Ceo (BA-Ceo), the T- COF comprising or consisting of C2 and C3 arranged in a crystal lattice structure represented by formula I, wherein C3 is represented by formula II (formula II), wherein Ri is C2 and C2 is represented by formula III (formula III).

[0083] Boronic acids are considered Lewis acids. The pKavalues can vary depending on the substituents on the boronic acids derivatives. If an electron-withdrawing group (e.g. halide, carboxyl, nitro) is attached to an aromatic boronic acid, the pKa value decreases, while an electron-donating group (e.g. alkyl, alkoxy, amine) increases the pKavalue. The skilled person will find that he will be able to adjust the boronic acid, however to maintain the specificity towards glucose, the skilled person should preferably select a boronic acid with a relevant pKavalue. In some embodiments of the present disclosure, the boronic acid in the BA-Ceo is a boronic acid having a pKavalue in the range of 4'0-4'2, preferably 4'1. As shown in the examples, BA-Ceo@T-COF coated on the surface of Au NPs modified 3D pyrolytic electrode has a pKavalue of 4.1. We measured the pKaof BA-Ceo@T-COF on the electrode with the electrochemical method. Therefore, in some embodiments, the pKavalue is determined at a later stage.

[0084] In the examples of the present invention a specific example of a boronic acid is provided. However, the skilled person will also be able to select other suitable boronic acids, that can be applied in the T-COF.

[0085] In some embodiments of the present disclosure, the BA-Ceo is represented by the formula IV

[0086] (formula IV).

[0087] In some embodiments of the present disclosure, the BA-Ceo is represented by a formula selected from the following table

[0088] Table 1

[0089] In some embodiments of the present disclosure, the BA-Ceo is represented by a formula selected from the following table

[0090] The boronic acid is covalently bonded to the fullerene C60 molecule, preferably in a 1: 1 ratio. The interaction between the boronic acid-functionalized fullerene Ceo (BA-Ceo) and the T-COF is defined as host-guest interactions, involving an array of intermolecular forces (van der Waals and electrostatic interactions). The pores of the T-COF act as host sites for BA-Ceo molecules, enabling their encapsulation within the COF framework, which, in turn, grants stability to the BA-Ceo@T-COF composite.

[0091] In some embodiments of the present disclosure, the BA-Ceo is non-covalently bonded to the T-COF, such as via intermolecular forces, such as van der Waals forces, and electrostatic interactions, such as n-n stacking interaction.

[0092] As introduced, an important use of the T-COF is the application in sensing of glucose. Thus, in another aspect, the invention relates to the use of the T-COF as described herein, in an electrochemical sensor for sensing glucose.

[0093] Electrochemical sensor

[0094] As introduced above, the T-COF provided can be used in different electrochemical sensors, wherein the specificity or sensitivity can be tweaked further if need be. In another aspect, the invention relates to an electrochemical sensor for sensing glucose comprising a working electrode, the working electrode comprising the T- COF as described herein. In some embodiments of the present disclosure, the working electrode further comprising:

[0095] • an electrochemical substrate, such as a carbon-based substrate, optionally having micropillars; and

[0096] • nanoparticles comprising or consisting of a metal, such as Au, Pt, Ag, or Cu.

[0097] In some embodiments of the present disclosure, nanoparticles comprising or consisting of a metal, such as Au, Pt, Ag, or Cu deposited on the electrochemical substrate

[0098] In some embodiments of the present disclosure, the T-COF is deposited on the nanoparticles.

[0099] In some embodiments of the present disclosure, the nanoparticles have a diameter of between about 60 to about 140 nm, such as between about 80 to about 120 nm, preferably about 100 nm.

[0100] In some embodiments of the present disclosure, the sensor further comprises a counter electrode and, optionally, a reference electrode.

[0101] In some embodiments of the present disclosure, the counter electrode and / or reference electrode comprises a noble metal, such as Au, Pt, or Ag. In preferred embodiments of the present disclosure, the noble metal is Au. The purpose of counter or auxiliary electrode is to complete the circuit and allow charge to flow. Consequently, they need to be manufactured from inert materials such as carbon, gold, and platinum. Other materials that can be used for counter electrodes include titanium, brass, silver, and platinum alloys. Thus, in some embodiments, the counter electrode is made from an inert materials. In some embodiments, the counter electrode is made from a metal selected from the group consisting of gold, titanium, brass, silver, and platinum. Reference electrodes provide a stable and known potential for measuring the potential of other electrodes in electrochemical measurements. Common materials used for reference electrodes include calomel, silver / silver chloride (Ag / AgCI), and copper / copper sulfate (Cu / CuSO4), with their potentials depending on the concentration of specific ions in the solution. Thus, in some embodiments, the reference electrode is made from a material selected from calomel, (Ag / AgCI), and (Cu / CuSO4), preferably Ag / AgCI. For the easiest manufacturing the metal for the counter- and reference electrode may be the same metal, such as Au. The skilled person will know that different materials can be used, for instance to achieve even better performing chips. Using Ag / AgCI as a reference electrode may for instance provide the most optimal signal.

[0102] The COF of the present invention may be applied to both three-dimensional (3D) and 2D surfaces. A way of achieving a higher signal is to employ the COF on a 3D surface, as the examples show produce a signal can be produced with and without 3D surfaces.

[0103] 3D functional electrodes made from various materials and structures might surpass the limitations of conventional thin film electrodes. These 3D electrodes possess exceptional properties such as a large electrochemically active surface area, improved mass transport and diffusion, superior accessibility to active sites, and rapid kinetics at the electrode / electrolyte interface, potentially offering greater sensitivity compared to 2D electrodes. Using cleanroom-based microfabrication processes, 3D pyrolytic carbon electrodes can be fabricated, allowing for precise customization of electrode geometry, configuration, and properties to suit specific applications.

[0104] In some embodiments of the present disclosure, the carbon-based substrate is a pyrolytic carbon electrode, such as a 3D pyrolytic carbon microelectrode, such as comprised of glassy carbon, optionally having micropillars.

[0105] In some embodiments of the present disclosure, the pyrolytic carbon electrode is made from pyrolysing a negative photoresist, such as epoxy photoresist SU-8. In some embodiments of the present disclosure, the pyrolytic carbon electrode is exposed to UV light before pyrolysation.

[0106] In some embodiments of the present disclosure, the surface area of the carbonbased substrate is expanded by one or more 3D microstructures, such as micropillars. In some embodiments of the present disclosure, the 3D microstructures are structures of the same material as the carbon-based substrate, and extend in a perpendicular direction from the carbon-based substrate.

[0107] The specific sensor shown in the examples has a spacing between adjacent micropillars varying depending on the direction between about 100 pm and about 150 pm (125 ± 25 pm). The extra area added by the pillars is 11 mm2to 12.5 mm2(11.75 ± 0.75 mm2). However, the skilled person will be able to the design the chip with the 3D structures having different shapes, and sizes.

[0108] In some embodiments of the present disclosure, the 3-dimensional microstructures are micropillars with a height of between 100 pm to 300 pm and a diameter of 5 to 200 pm.

[0109] In some embodiments of the present disclosure, wherein the micropillars are pillars with a height of approximately 100 pm to 300 pm and a diameter of 100 pm.

[0110] In some embodiments of the present disclosure, wherein the micropillars are pillars with a height of approximately 180 pm to 200 pm and a diameter of 100 pm.

[0111] In some embodiments of the present disclosure, the sensor further comprises means for providing insulation and / or a passivation layer ensuring only active regions, such as the working-, reference, and / or counter electrode are available to a solution applied to the electrochemical sensor.

[0112] In some embodiments of the present disclosure, the means for providing insulation and / or a passivation layer is made from the same material as a pyrolytic carbon electrode, without having been pyrolysed.

[0113] In some embodiments of the present disclosure, the ability of the sensing layer to detect glucose is maintained after exposure to autoclave conditions, such as overpressure and about 120 degrees Celcius. The application of electrochemical sensors for on-line or in-line monitoring of bioprocesses is a promising technique, but it comes with certain challenges. One of the primary challenges is the susceptibility of these sensors to fouling by proteins, cells, or other biomolecules that are present in the bioprocess media. This can significantly affect the long-term accuracy and reliability of the sensors. To address this issue, sample purification and pre-treatment have been suggested as possible solutions, but this approach can be complex and impractical for in-situ measurements. Another solution is to use antifouling polymer membranes, such as poly(ethylene glycol) (PEG) derivatives, polyamides, polyurethanes , or naturally occurring polysaccharides like dextran or chitosan. These polymers can help minimize nonspecific molecular adsorption and prevent fouling. Polymers used for electrochemical sensing and biosensing applications must meet additional requirements beyond their basic functionality. Specifically, they must not interfere with electrochemical measurements, and their deposition process must be precisely controlled for reproducibility. Charged polymers can pose a challenge since they can impede the migration of ions toward electrodes, thereby hampering the measurement process.

[0114] Most polymer / biopolymer coatings exhibit hydrophilicity as the primary antifouling mechanism. This involves creating a highly hydrated environment within the coating, which establishes a steric repulsive barrier. This barrier hinders fouling molecules from forming adhesive connections with the surface. While PEGs are widely considered the leading option for antifouling coatings, their effectiveness is heavily dependent on factors such as polymerization level and surface coating density. Recently, zwitterionic polymer (ZP) coatings have emerged as a promising alternative due to their superior hydration effects and enhanced antifouling properties. Unlike PEGs, ZP coatings contain both cationic and anionic groups on the same polymer chain, leading to a powerful hydration effect. This causes water molecules to be attracted to the charged groups on the polymer surface, creating a strong hydration sphere that serves as both a physical and entropic barrier against biomolecules.

[0115] Thus, the skilled person will be able to select many different coatings to enhance the longevity of the sensor.

[0116] A zwitterionic polypyrrole (ZiPPy) polymer membrane could be employed as an anti-fouling layer. To synthesize this polymer, zwitterionic pyrrole (ZiPy) monomers were electropolymerized onto the surface of the electrode, resulting in a ZiPPy coating. The ZiPPy-coated electrodes demonstrated remarkable hydrophilicity and significantly lower protein adsorption compared to noncoated or PPy-coated electrodes. The ZiPPy approach is thus a method for producing high- performance electrochemical biosensors and sensors, as it combines the benefits of zwitterionic molecules and conducting PPy.

[0117] In some embodiments of the present disclosure, the sensor further comprises means for anti-fouling. In some embodiments of the present disclosure, the means for anti-fouling may be a coating with a zwitterionic polymer, such as zwitterionic polypyrrole (ZiPPy).

[0118] In some embodiments of the present disclosure, the sensor further comprises means for reducing diffusion, such as a diffusion-limiting layer, such as a layer comprised of a trimethylolpropane triglycidyl ether (PTGE) and polyethyleneimine (PEI).

[0119] In some embodiments of the present disclosure, the sensor comprises means for antifouling as described above and means for reducing diffusion as described above, and preferably wherein the means are combined in a ratio of about 1: 1.

[0120] Since the bioprocesses may run from 6 hours to days, and some up to several weeks, the ability to provide reliable measurements over a sustained period is well sought after. In some embodiments of the present disclosure, the electrochemical sensor's ability to sense glucose is maintained for a sustained period of time after manufacturing without affecting the sensor's ability to sense glucose, wherein the sustained period of time is at least 5 days, such as at least 10 days, such as 15 days, such as at least 20 days.

[0121] Depending on the specific composition of the sensor, the temperature at which the function of the sensor is maintained may vary. In some embodiments of the present disclosure, the electrochemical ability of the sensing layer to detect glucose is maintained after exposure to autoclave conditions, such as overpressure and about 120 degrees Celsius. When for instance the above described antifouling and / or diffusion layer is applied, it may lower the sensor's ability to maintain function, rendering these layers non-functioning under autoclave conditions. Thus, in certain embodiments of the present disclosure, the electrochemical sensor can maintain its ability to sense glucose even after being exposed to temperatures of up to 70 degrees Celsius.

[0122] In some embodiments of the present disclosure, the sensor's ability to sense glucose is defined as an ability to continuously measure the glucose content of a control sample, such as running amperometric tests at glucose concentrations ranging from 1 to 100 mM or switching between pure Phosphate-Buffered Saline (PBS) and PBS with 5 mM glucose, and maintaining sensitivity of at least about 90% such as even about 95%.

[0123] The exact way to do the measurement may vary. For instance when measuring a calibration curve, the sensor may be moved between individual solutions and rinsed in Phosphate-Buffered Saline (PBS) or the sensor can be placed in PBS where the concentration of glucose is successively increased by adding glucose directly to the solution.

[0124] In another aspect, the invention relates to the use of the electrochemical sensor as described herein, in a method for sensing glucose.

[0125] Methods for sensing glucose

[0126] Having introduced the T-COF and the application of the T-COF in electrochemical sensors, the specific methods for using the sensor, as well as manufacturing the different components are also provided. The skilled person will easily be able to understand how the embodiments disclosed above regarding the T-COF and electrochemical sensor, can be further applied in the below described aspects. Thus, in another aspect, the invention relates to a method of sensing glucose, the method comprising:

[0127] 1. Providing an electrochemical sensor as defined herein configured to produce an electric signal when a glucose molecule is at, or near, the working electrode;

[0128] 2. Providing a sample suspected of containing glucose;

[0129] 3. Contacting the electrochemical sensor with the sample;

[0130] 4. Measuring the signal from the working electrode, thereby sensing the presence of glucose in the sample; 5. Optionally, comparing the signal to a signal from a control electrode on the electrochemical sensor to reduce background signals; and

[0131] 6. Optionally, comparing the signal from the working electrode to a signal obtained from measuring a control sample, to obtain a level of glucose in the sample.

[0132] In some embodiments of the present disclosure, the method senses glucose in the range from 0.1 mM to 120 mM.

[0133] In another aspect, the invention relates to an apparatus for sensing metabolites, the apparatus comprising the electrochemical sensor as described herein, optionally the apparatus comprising at least one further sensor for sensing a metabolite other than glucose.

[0134] As described in Table 1 and Table 2 a specific set of BA-Ceo molecules are provided. These molecules may be prepared by using the Formylphenylboronic acid pinacol ester derivatives as provided in Table 3, by using suitable reaction conditions and removing the protecting group in the end of the reaction, or as described in the examples.

[0135] Thus, in an aspect is provided a method of producing a T-COF comprising boronic acid-functionalized Ceo (BA-Ceo), the method comprising:

[0136] 1. combining triformy thieno[3,2-b]thiophene-2,5-diboronic acid (F TTDB) and 2,3,6,7,10,11-hexahydroxytriphenylene (HHTP) to obtain a T-COF;

[0137] 2. performing a Prato reaction between fullerene Ceo, and a suitable pinacol ester derivative, such as a formylphenylboronic acid pinacol ester derivative, and a relevant amino acid, and removing the protective group to obtain BA-Ceo; and

[0138] 3. combining the T-COF with the BA-Ceo, thereby obtaining a T-COF comprising BA-Ceo.

[0139] In another aspect, the invention relates to a method of producing a T-COF comprising boronic acid-functionalized Ceo (BA-Ceo), the method comprising:

[0140] 1. combining thieno[3,2-b]thiophene-2,5-diboronic acid (F TTDB) and 2,3,6,7,10,11-hexahydroxytriphenylene (HHTP) with dry dioxane and dry mesitylene; 2. reacting the mixture in an autoclave, such as at 150 °C for 72 h;

[0141] 3. washing the product thereof, and drying the product;

[0142] 4. optionally, heating the product under high vacuum, such as at app. 200 °C and IO’6torr (1.33 x 10’4Pa); 5. obtaining a T-COF;

[0143] 6. performing a Prato reaction between fullerene Ceo, and a suitable pinacol ester derivative, such as a formylphenylboronic acid pinacol ester derivative, and a relevant amino acid, such as sarcosine, such as at 125°C for 12 h; 7. washing the product thereof;

[0144] 8. removing the protective group to obtain BA-Ceo, such as by the use of methylboronic acid dissolved in dichloromethane, followed by the addition of TFA; and

[0145] 9. combining the T-COF with the BA-Ceo, thereby obtaining a T-COF comprising BA-Ceo.

[0146] Table 3

[0147] In a more specific aspect, a method is provided of specifically generating the T- COF comprising the BA-Ceo of formula IV. Thus, in another aspect, the invention relates to a method of producing a T-COF comprising boronic acid-functionalized Ceo (BA-Ceo), the method comprising:

[0148] 1. combining thieno[3,2-b]thiophene-2,5-diboronic acid (F TTDB) and 2,3,6,7,10,11-hexahydroxytriphenylene (HHTP) with dry dioxane and dry mesitylene;

[0149] 2. reacting the mixture in an autoclave, such as at 150 °C for 72 h;

[0150] 3. washing the product thereof, and drying the product;

[0151] 4. optionally, heating the product under high vacuum, such as at app. 200 °C and IO’6torr (1.33 x 10’4Pa);

[0152] 5. obtaining a T-COF;

[0153] 6. reacting fullerene Ceo, 4-formylphenylboronic acid pinacol ester (FPBA), and sarcosine, in a suitable solvent such as toluene, such as at 125°C for 12 h;

[0154] 7. washing the product thereof;

[0155] 8. removing the protective group to obtain BA-Ceo, such as by the use of methylboronic acid dissolved in dichloromethane, followed by the addition of TFA; and

[0156] 9. combining the T-COF with the BA-Ceo, thereby obtaining a T-COF comprising BA-Ceo.

[0157] In another aspect, the invention relates to a method of producing an electrochemical sensor for sensing glucose, the method comprising: 1. providing a carbon based substrate, such as a pyrolytic carbon electrode; nanoparticles comprising or consisting of a noble metal, such as Au, Pt, Ag, or Cu; and a T-COF comprising BA-Ceo as defined herein;

[0158] 2. depositing the nanoparticles on the carbon based substrate;

[0159] 3. depositing the T-COF comprising BA-Ceo on the nanoparticles;

[0160] 4. optionally, depositing a counter- and / or a reference electrode at suitable positions on the electrochemical sensor;

[0161] 5. optionally, coating with a zwitterionic polymer, such as zwitterionic polypyrrole (ZiPPy); and

[0162] 6. optionally, coating with a diffusion-limiting layer, such as a layer comprised of a trimethylolpropane triglycidyl ether

[0163] (PTGE): polyethyleneimine (PEI) layer.

[0164] The skilled person will know that the sequence of the different steps may vary, depending on the specific chip and its manufacturing.

[0165] In some embodiments of the present disclosure, the counter electrode and / or reference electrode comprises a noble metal, such as Au, Pt, or Ag. In preferred embodiments of the present disclosure, the noble metal is Au. In some embodiments, the counter electrode is made from an inert material. In some embodiments, the counter electrode is made from a metal selected from the group consisting of gold, titanium, brass, silver, and platinum. In some embodiments, the reference electrode is made from a material selected from calomel, (Ag / AgCI), and (Cu / CuSO4), preferably Ag / AgCl.

[0166] In some embodiments of the present disclosure, the pyrolytic carbon electrode is made from pyrolysing a negative photoresist, such as epoxy photoresist SU-8. In some embodiments of the present disclosure, the pyrolytic carbon electrode is exposed to UV light before pyrolysation.

[0167] Kit

[0168] In another aspect, the invention relates to a kit comprising the electrochemical sensor as described herein, and optionally means for replacing the electrochemical sensor and / or one or more further electrochemical sensors, such as one or more further electrochemical sensors for sensing one or more metabolites other than glucose, optionally the kit may also comprise components for cleaning, such as washing buffers.

[0169] It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.

[0170] All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

[0171] The invention will now be described in further details in the following non-limiting examples.

[0172] Examples

[0173] Example 1 - Materials and methods

[0174] Materials

[0175] Starting materials were procured from Sigma-Aldrich, TCI or VWR and utilized without additional purification. Mesitylene, thieno[3,2-b]thiophene-2,5-diboronic acid (H4TTDB), and 1,4-dioxane were obtained from TCI. N,N-dimethylformamide, gold(III) chloride trihydrate (HAuCk-SH O), hexane, ethyl acetate, and silica-gel were sourced from VWR. Fullerene Ceo, 4-formylphenylboronic acid pinacol ester (FPBA), methylboronic acid, polyethyleneimine (PEI, 50% w / v in H2O, Mn~60000, Mw~750000), trimethylolpropane triglycidyl ether (PTGE), pyrocatechol, 1,2- octanediol, cis- 1,2-cyclohexanediol, toluene, pyrrole, acetonitrile (ACN), luteolin, hexane, 2,3,6,7,10,11-hexahydroxytriphenylene (HHTP), tetrabutylammonium bromide (TBAB, 99.0%), ethyl acetate (EtOAc), trifluoroacetic acid (TFA), and dichloromethane were obtained from Sigma-Aldrich. Sarcosine and d-xylose were procured from VWR. Silica featuring a particle size of 300-400 mesh was acquired from TCI. All stock solutions were freshly prepared prior to each experiment using double distilled water, except when noted otherwise. 6-inch single side polished silicon wafers were obtained from Siegert Wafer, while the SU-8 2000 series resist was sourced from Kayaku Advanced Materials. Surface characterization

[0176] Scanning electron microscope (SEM) Zeiss Supra VP 40 (Germany) was used to evaluate the morphology and surface topography of the as-prepared nanostructures and modified surface of electrodes. The average thickness and diameter of the nanomaterials were estimated using the Image! software, which is developed by the National Institute of Health in the USA. The PANalitycal model AERIS X-ray diffractometer (Malvern Panalytical Ltd., Almedo, The Netherlands) with Cu Ko radiation (A = 1.5406 A, voltage = 40 kV, and beam current = 30 mA) was used to obtain X-ray diffraction (XRD) spectra of the samples. Analysis of the elemental composition and chemical states of the synthesized samples before and after thermal treatment were carried out using X-ray photoelectron spectroscopy (XPS; Thermo Scientific Nexsa, USA). Attenuated total reflectance- Fourier transform infrared spectroscopy (ATR-FTIR) spectra of the aerogel and carbon aerogel electrodes were collected using a PerkinElmer Spectrum 100 FTIR spectrometer. The spectra were obtained at a temperature of 25 °C with a resolution of 4 cm-1and 16 scans in the range of 4000-500 cm-1. Data on N2 adsorption-desorption was gathered using a Quantachrome NOVA station (Boynton Beach, FL, USA) with NovaWin software (version 11.0). The specific surface area and pore volume of samples were evaluated using the multipoint BET (Brunauer, Emmett, and Teller) method in the relative pressure (p / po) range of 0.05 to 1. The samples underwent degassing at 150°C for 6 hours prior to the analysis.

[0177] Electrochemical studies Electrochemical measurements were carried out using MultiEmStat (PalmSens, Utrecht, The Netherlands) with a DRP-CAST1X8 interface (DropSens, Llanera, Spain) controlled by MultiTrace 3.4 software (PalmSens, Utrecht, The Netherlands). To conduct preliminary experiments and to compare the performance of 3D electrodes with commercial electrodes, DRP-110DGPHOX (DropSens, Llanera, Spain) screen printed electrodes (SPEs) printed on ceramic substrate were used. Each sensor consists of a carbon WE (0.4 cm diameter; 0.126 cm2apparent geometric area) modified with a graphene oxide (GO) layer, a carbon CE and a Ag / AgCI paste RE. For all electrochemical characterizations and fabrications, a silicon chip coated with 15 nm titanium (Ti) and 200 nm platinum (Pt) on both sides, having a total immersed surface area of around 200 mm2, was utilized as a CE for each WE, and an external Ag / AgCI electrode in 3 M KCI was used as a RE. To prevent the passivation or destruction of the gold CE and RE for the 3D pyrolytic carbon microelectrodes on the three-electrode-chip configuration, the surface of the electrodes was protected by using tape.

[0178] Cyclic voltammograms were obtained within a potential range of -0.8 to +0.8 V (vs. Ag / AgCI) using 10 mM PBS with a pH of 7.4 as the background electrolyte. When required, glucose solution was added to the electrolyte from a 0.1 M stock. Chronoamperometric profiles were documented at applied potentials from +0.4 to +0.6 V, with the stepwise addition of specific quantities of glucose stock solution to 10 ml_ of PBS electrolyte, while being continuously magnetically stirred under ambient conditions. The optimal potential of +0.55 V was employed for all other measurements. The recovery time, response reproducibility, and reusability of the modified AuNPs@BA-Ceo@T-COF electrodes were examined using chronoamperometric tests at +0.55 V.

[0179] Briefly, the reproducibility and recovery time were evaluated by recording the amperometric responses of the modified AuNPs@BA-Ceo@T-COF electrode through 20 consecutive cycles in both blank PBS and 50 mM glucose solution. To verify the long-term stability and reusability of the modified electrodes, their stepwise sensitivity to glucose was assessed over a 30-day period. The electrodes, after each measurement, were stored under ambient conditions without additional cleaning. To investigate the anti-interference capability of the fabricated electrochemical sensor, the chronoamperometric response of the modified AUNPS@BA-C6O@T-COF electrode was recorded for various interfering agents. The experiment involved adding glucose to the PBS background solution, followed by the stepwise addition of potential interfering substances such as ethanol, lactate, acetate, NaCI, SO42', galactose, fructose, and sucrose, all at equal concentrations of 20 mM. The study concluded with the subsequent addition of glucose and documentation of the resulting current change. For each sensor type, a linear equation was fitted to the data points within the dynamic range, with the slope representing the sensor's sensitivity. The limit of detection (LOD) for each type of sensor was calculated by: where SD is the standard deviation of the current response to the lowest analyte concentration and b is the slope of the linear regression y=a+bx. In order to examine the apparent acid dissociation constant (pKa) of the boronic acid group on the BA-Ceo@T-COF modified electrode surface, the Fe(CN)63' / 4' redox pair was employed as a probe molecule. The pH levels of aqueous solutions, ranging from 3 to 10, were adjusted using a Britton-Robinson buffer solution. This was achieved by adding appropriate amounts of 1 M KOH solution to a mixture containing 10 mM each of acetic acid, boric acid, and phosphoric acid. The ionic strength of these buffer solutions was regulated with the addition of 0.1 M KCI. The cyclic voltammetry (CV) measurements were conducted at room temperature.

[0180] Example 2 - Generation of components

[0181] Pyrolytic Carbon Electrode Fabrication

[0182] 3D pyrolytic carbon microelectrodes in a three-electrode-chip configuration were designed and fabricated. The circular 3D working electrode (WE) with a diameter of 4 mm had an additional 284 micropillars made of pyrolytic carbon (Figure 3). The fabrication of pyrolytic carbon electrodes involved using photolithography with negative epoxy photoresist SU-8, then subjecting it to pyrolysis, as shown in Figure 4. This process began by applying 6 ml_ of SU-8 2035 to a 6-inch silicon wafer with a 600 nm silicon oxide insulating layer using a spin coating technique. The coating of the substrate was done using a RCD8 spin coater (Karl-Suss, Germany) with a spreading cycle at 1000 rpm for 10 seconds at an acceleration of 200 rpm / s and a thinning cycle at 5000 rpm for 120 seconds using 1000 rpm s'1acceleration, which resulted in a film of 15 pm thickness as shown in Figure 4A. The SU-8 was baked at a low temperature of 50 °C for 15 minutes on a programmable hot plate (Harry Gestigkeit GmbH, Germany) with a temperature increase of 2 °C min-1to evaporate the solvent. The 3D WE design was then printed onto the wafer coated with photoresist by exposing it to UV light on a 365 nm MA6 mask aligner (Karl-Suss, Germany) with a total dose of 2x250 mJ cm-2as shown in Figure 4B. After exposing the areas, the material was cross-linked in a post-exposure-bake (PEB) process similar to the soft-bake, but for a longer duration of 2 hours. For the 3D WE, an extra step of SU-8 photolithography was performed to shape the micropillars. This was achieved by applying SU-8 2075 through spin coating (500 rpm for 30 seconds with 50 rpm s'1acceleration, and 1000 rpm for 60 seconds with 100 rpm s'1acceleration), followed by another soft- bake at 50 °C for 6 hours, as shown in Figure 4C. SU-8 2075 was spin-coated for a second time to achieve the desired thickness of approximately 500 pm, and then soft-baked at 50 °C for 12 hours. The design of the micropillars was transferred to the substrate by exposing it to UV light with a total dose of 4x250 mJ cm-2on the mask aligner, followed by a post-exposure- bake for 10 hours at 50°C as illustrated in Figure 4D. The non-crosslinked SU-8 photoresist was eliminated by immersing it in PGMEA for 2x40 minutes (Figure 4E), followed by washing with IPA and drying in a nitrogen gas atmosphere at room temperature. The samples were then exposed to the same dose of 4x250 mJ cm-2, after which they were hard-baked for 15 hours at 90 °C to solidify and dry out the SU-8 microstructures for 3D electrodes. After that, the SU-8 samples were heated at a high temperature of 1100 °C in a nitrogen gas atmosphere in a furnace (PEO-04, ATV Technologies, Germany) for 5 hours. The latter process is known as pyrolysis (Figure 4F). The pyrolysis causes the SU-8 microstructures to shrink, resulting in pillars with a height of approximately 180 pm to 200 pm and a diameter of 100 pm. The spacing between adjacent pillars varies depending on direction but is between 100 pm and 150 pm. The extra area added by the pillars is 11 mm2to 12.5 mm2.

[0183] The contact layer consisting of a stack of 20 nm Ti and 100 nm Au was deposited through e-beam evaporation (Alcatel SCM 600) (Figure 4G) using a shadow mask that was previously fabricated using laser machining. The on-chip reference electrode (RE) has a non-passivated area of 0.55 mm2, while the counter electrode (CE) has a non-passivated area of 49 mm2. The current collection ring around the WE base carbon layer is 250 pm wide and covers around 260 degrees of the perimeter around the WE. The entire electrode chip, with the exception of the electrodes and the metal contact pads, was covered with a 5 pm thick SU-8 layer (as shown in Figure 4H). This passivation layer limits the sensing area to the pyrolytic carbon surface, and it also protects the metal and carbon from coming apart. The passivation layer ensures only active regions are available to the solution interface, as well as providing contacts to a potentiostatic circuit. Finally, the wafer was diced into 84 electrode chips with a dimension of 10 x 16 mm2(Figure 41).

[0184] Synthesis of BA-Ceo

[0185] The synthetic pathway of BA-Ceo follows a previously reported procedure, which is illustrated in Figure 5. To prepare BA-Ceo, a mixture of toluene (200 mL), Ceo (0.6 mmol), 4-formylphenylboronic acid pinacol ester (FPBA) (3 mmol), and sarcosine (1.2 mmol) was mixed at 125°C for 12 h. Then, a rotary evaporator was used to remove the solvent before loading the product onto a silica gel chromatography column. The unreacted Ceo was eluted with a toluene: hexane mixture in varying ratios, and the brown solid product A (192 mg, 36%) was obtained by eluting with toluene:hexane (4: 1) in three-column volumes. In all eluting procedures, the aerator pump was employed to boost flow rate. The boronic ester group in product A was then deprotected by transesterification to yield product B. In brief, product A (100 mg) and methylboronic acid (30 mg) were dissolved in dichloromethane (50 mL), followed by the addition of TFA (3 mL), then the mixture was let to sit overnight with constant stirring at room temperature. After evaporating the solvent, purification of the product was carried out through a silica gel chromatography column, using a mixture of five-column volumes of toluene: EtOAc (5: 1). This process yielded the brown solid product B (BA-Ceo), with a mass of 65 mg and a yield of 72%.

[0186] Synthesis of T-COF

[0187] The procedure for synthesizing thiophene based COF (T-COF) was based on previously published studies. A solution containing 0.75 mmol (190 mg) of H4BTDB (one of the most favorable monomers for organic conducting polymers) and 0.5 mmol (165 mg) of HHTP in 15 mL of dry dioxane and 15 mL of dry mesitylene was prepared and added to a traditional stainless steel autoclave with a 100 mL Teflon liner. The solution was then purged three times with nitrogen gas and heated at 150 °C for 72 h. After the reaction, the resulting dark gray flocculent solid was subjected to a washing process using 20 mL of dry dioxane and dry dichloromethane and afterwards centrifuged. Subsequently, the remaining powder was dried for 8 hours in a vacuum oven. To achieve complete desolvation, the powder was then heated overnight at 200 °C under high vacuum (IO-6torr (1.33 x 10'4Pa)) (Figure 6).

[0188] Synthesis of BA-Ceo@T-COF

[0189] A mixture of 100 mg of BA-Ceo and 6 mL of toluene: EtOAc (5: 1) was prepared and then T-COF (50 mg) was added to it. The resulting mixture was heated in an oven preheated to 200 °C for a period of 24 hours. Subsequently, the vial was allowed to cool down to room temperature, and the supernatant was decanted. The BA- Ceo@T-COF sample obtained was then thoroughly washed with toluene: EtOAc (5: 1) (3 x 5 mL) to remove any BA-Ceo physically adsorbed on the COF surface. The solid was subsequently dried at 80 °C overnight under dynamic vacuum (the procedure is illustrated in Figure 7a).

[0190] Example 3 - Fabrication of electrochemical glucose sensor'

[0191] Modification of electrode by BA-Cso@T-COF and electrodeposition of gold nanoparticles

[0192] Initially, the pyrolytic carbon electrodes were cleansed using isopropanol and deionized water, and then dried utilizing compressed nitrogen gas. Following this, the electrodes were subjected to an oxygen plasma treatment in a Zepto plasma chamber (manufactured by Diener Electronic GmbH, Germany) for a duration of 65 seconds at an oxygen pressure of 0.6 mbar, employing 40% of the power, which is equivalent to 120 W. Next, the electrochemical behavior of the plasma- treated electrodes was evaluated via cyclic voltammetry (CV) employing a MultiEmStat potentiostat (from PalmSens, Utrecht, The Netherlands) in conjunction with a DRP-CAST1 x 8 interface (provided by DropSens, Llanera, Spain), which was managed by the MultiTrace 3.4 software. For each WE, a silicon chip coated with 15 nm titanium (Ti) and 150 nm platinum (Pt) on both sides, having a total immersed surface area of around 200 mm2, was utilized as a CE, and an external Ag / AgCI electrode in 3 M KCI was used as a RE. The cyclic voltammograms were obtained in 10 mM K3Fe(CN)e / K4Fe(CN)6 in 10 mM phosphate buffered saline (PBS) pH 7.4, with a potential range between -0.8 V and +0.8 V at a scan rate of 50 mV s’1. Electrodes exhibiting reproducible cyclic voltammograms were chosen for sensor preparation.

[0193] Au NPs were electrodeposited onto the 3D WE in 0.5 M H2SO4 solution containing 0.5 mM HAuCk through a series of five successive cyclic CV cycles, within a potential range of -0.2 to 1.5 V, while using a scan rate of 50 mV s-1. Subsequently, the prepared Au NPs modified electrode is washed with DI water and then dried. To coat the Au NPs modified electrode with the BA-Ceo@T-COF composite, the suspensions of BA-Ceo@T-COF composite were mixed with 10 mL of ethyl acetate and subjected to a sonication process (20 minutes) in an ultrasonic bath to achieve a uniform dispersion of the particles in the solvent (Figure 7b).

[0194] A potentiostatic method was applied for the electrodeposition of BA-Ceo@T-COF composite on the 3D working electrode at 0.50 V for 5 min. Following the deposition, the potential was reset to zero, and the electrodes were taken out of the solvent, thoroughly rinsed with ethanol and then dried under a flow of nitrogen gas.

[0195] Synthesis of zwiterionic pyrrole

[0196] A two-step process was used to synthesize zwitterionic pyrrole (compound 2). Firstly, a derivative of pyrrole amine (compound 1) was prepared, and subsequently, it was converted into a zwitterionic form by undergoing a ringopening reaction with g-propiolactone (Figure 8a).

[0197] The synthesis of N,N-Dimethyl-3-(lH-pyrrol-l-yl)propan-l-amine (compound 1) was conducted using a previously reported method. First, pyrrole (1 mL, 14 mmol) was dissolved in 20 mL of dimethyl sulfoxide under an argon atmosphere. Subsequently, 35 mg (0.1 mmol) of tetrabutylammonium bromide (TBAB) was added, which was then followed by the addition of 6.5 mL of 50 wt% NaOH. To facilitate mixing, 0.5 mL of water was added prior to the gradual addition of 3- dimethylaminopropylchloride hydrochloride (DMPC) (5 g, 32 mmol) dissolved in 7 mL of DMSO. After stirring the mixture for 24 hours, 20 mL of water was added to dissolve the precipitate, and the resulting mixture was subjected to extraction with diethyl ether (3 x 25 mL). The organic phase was then washed with water (2 x 25 mL) and brine (1 x 25 mL). After drying over MgSO4, the product was obtained in 70% yield as a colorless liquid after removing the solvent under vacuum. No further purification was required. The elemental analysis results for the sample are as follows: C content is 69.61%, H content is 10.94%, and N content is 19.45%. The empirical formula for the compound might be C9H17N2. The synthesis of 3-((3-(lH-Pyrrol-l-yl)propyl)dimethylammonio)-propanoate (ZiPy) (compound 2) involved drop-wise addition of p-propiolactone (0.76 mL, 12 mmol) in anhydrous THF (10 mL) to a solution of compound 1 (1.4 g, 8 mmol) in 50 mL of anhydrous THF under argon at 0 °C, followed by stirring at 15 °C for 6 h. After the addition of diethyl ether (50 mL), the product was extracted with water (2 x 25 mL) and purified via reverse-phase column chromatography (5% MeOH in water) using Thermo Scientific HyperSep C18 Cartridges (10 g), which yielded the compound as a hygroscopic white solid at 41% yield.

[0198] Coating of glucose sensor with a diffusion-limiting layer

[0199] To adjust the linear range of the glucose sensor, a diffusion-limiting layer can be incorporated. This layer restricts the diffusion of molecules to and from the active surface of the sensor, which extends the linear range to higher concentrations. To create the diffusion-limiting layer, PEI and PTGE were first prepared separately in ethanol at a concentration of 10 mg mL , then mixed at a 1: 1 weight ratio in an ultrasonic bath for 30 minutes. The resulting PTGE: PEI solution can be diluted to achieve thin films of various thicknesses. A uniform thin film of PTGE: PEI was achieved on the sensing layer by spray-casting the solution in air using an airbrush (Redox. me, Sweden) at an atomization air pressure of 1.4-1.9 bar. The spray-casting process allowed for precise application of the ink, resulting in consistent film thickness. Following spray-casting, the PTGE: PEI thin films were dried at 80°C for 3 hours in an enclosed environment (Figure 8b).

[0200] Coating of glucose sensor with zwitterionic polypyrrole (ZiPPy)

[0201] The developed glucose sensors were coated with an antifouling layer by electropolymerizing the ZiPy monomer into ZiPPy. The process was conducted in a solution containing 0.1 M of the monomer and 0.1 M NaCI, using CV to scan the potentials between 0.0 and +0.95 V versus an Ag / AgCI RE, at a scan rate of 50 mV s-1. After polymerization, the sensors were rinsed with milli-Q water and stored at room temperature until use (Figure 8b).

[0202] To compare the performance of the ZiPPy-coated glucose sensors, PPy was also electropolymerized onto the surface of the electrode using the same procedure as for ZiPPy, but with 0.1 M pyrrole instead of the ZiPy monomer.

[0203] Coating of glucose sensor with Nation

[0204] For Nation coating, the modified WE was coated with a 3 pL drop of Nafion®117, which had been diluted to 5 vol. % in water. The sensors were then left to dry overnight in a climate chamber with a humidity of 40% and a temperature of 8 °C. Example 4 - Investigation of the developed sensor for determination of glucose in different bioprocesses

[0205] Bacteria! fermentation processes

[0206] In this study, the bacterial strain Pseudomonas putida KT2440 was used and preserved at -80 °C in 50% (w / w) glycerol. LB agar medium was composed of 10 g tryptone, 5 g yeast extract, 10 g NaCI, and 20 g agar per liter. A defined basis medium was prepared by adding 3.88 g K2HPO4, 2.12 g NaH2PO4.2H2O, 2 g (NH4)2SO4, and 1 mL trace element solution (TE) per liter. The trace element solution consisted of 10g EDTA, 100 g MgCl2.6H2O, 2 g ZnSO4.7H2O, 1 g CaCl2.2H2O, 5 g FeSO4.7H2O, 200 mg Na2MoO4.2H2O, 200 mg CuSO4.5H2O, 400 mg COCI2.6H2O, and 1.22 g MnCl2.4H2O. All cultures were supplemented with 4 g L'1glucose, unless specified otherwise. Precultures were performed in three stages: firstly, glycerol stocks were activated by streaking on LB agar plates and incubating at 30 °C for 18 h. Secondly, a liquid preculture was inoculated with 3 colonies from a fresh LB agar plate in a 14 mL cultivation tube containing 5 mL basis medium and incubated at 30 °C and 180 rpm for 8 h in a rotary shaker. Thirdly, a liquid preculture was inoculated to an initial ODeoo of 0.01 in 50 mL basis medium in 500 mL baffled shake flasks and incubated at 30 °C and 180 rpm for 18 h on a rotary shaker. For all preculture incubations, an Ecotron incubation shaker (Infors HT, Switzerland) was employed.

[0207] Shake flask cultures of KT2440 were performed at 30 °C and 180 rpm in 2 L baffled shake flasks with a liquid filling volume of 1: 10 minimal medium. A 2 mL sample was withdrawn aseptically every hour, and all shake flask cultivations were performed in triplicates. Optical density of shake flask cultures was measured at a wavelength of 600 nm (ODeoo) in a UV-1800 UV-spectrophotometer (Shimadzu, Japan) and correlated to a cell dry weight concentration.

[0208] Cell cultivation

[0209] HEK293 and K562 mammalian cell lines (Thermo Fisher Scientific) were selected. HEK293 wild-type cells were expanded in 30 mL Freestyle™ 293 Expression Medium (Thermo Fischer Catalog number: 12338018), supplemented with 300 pL Penicillin-Streptomycin ((10,000 U / mL) Thermo Fischer Catalog number: 15140122), at a density of 0.35xl06cells mL1, in a 125 mL Erlenmeyer shake flask. K562 wild-type cells were expanded in 30 mL RPMI 1640 Medium (Thermo Fischer Catalog number: 11875093), at a density of 0.25x l06cells mL1, in a 125 mL Erlenmeyer shake flask. The media bottle of 500 mL was supplemented with 50 mL FBS (Fetal Bovine Serum) (Gibco™ 10500064), and 5 mL L-Glutamine- Penicillin-Streptomycin solution (Sigma-Aldrich - G6784). Both cell types were incubated in a humidified incubator at 150 rpm, 37 °C, for 7 days, in a 5% CO2 incubator. Every day, three 1 mL samples were collected from each of the cell cultures.

[0210] Yeast Fermentation Processes

[0211] The classic Saccharomyces cerevisiae Bl strain was used to carry out yeast fermentation. For both the seed and fermentation stages, the basal media consisted of the following amounts per liter: 10 g KH2PO4, 0.5 g MgSO4, 5 g yeast extract, 0.1 g CaC , and 5 g (NH4)2SO4. The glucose concentrations for the seed and batch were initially set at 20 g L'1and 100 g L’1, respectively. The seed culture was performed in a 250 mL shake flask with 100 mL of working volume at 220 rpm and 30 °C for 14 hours (optical density (ODeoo) ~ 8), then transferred into the fermentation medium at a 20% inoculum volume. A Biostat® B plus stirred tank bioreactor (Sartorius AG, Germany) was used to conduct batch cultivations. An Applikon ez controller (Applikon, Delft, the Netherlands) was used to regulate the entire system, which included dissolved oxygen (DO), pH, and temperature probes in the fermentation vessel. For the 2 L bioreactor culture, 1 L of initial volume with a 0.2 L inoculum was cultured at 30 °C and 150 rpm for 12 hours without aeration.

[0212] At-line monitoring of glucose by the developed sensors

[0213] At-line glucose measurements were conducted during bioprocessing using the developed sensors based on the calibration curve obtained in different media. For the fermentations, samples were manually withdrawn every hour, while for the cell cultures, samples were collected on a daily basis. Glucose quantification was performed using the developed sensor in both cell-free and cell-containing samples. Prior to the measurement, cell-free samples were passed through a 0.2 pm sterile filter (Sartorius, Gottingen, Germany). To complement the glucose measurements obtained from the developed sensor, glucose quantification was performed using HPLC in yeast and bacterial fermentations, and with biosensors in a BioProfile FLEX2 (NOVA Biomedical, Waltham, USA) for cell cultures. Reference analysis used for validation of the developed sensors

[0214] To prepare samples for HPLC analysis, a 1: 100 volume of 2 M H2SO4 was added to the samples immediately after withdrawal to adjust the pH and halt any reactions. The samples were then centrifuged at 10,000 x g at 4 °C for 5 minutes and filtered through a 0.2 pm cellulose acetate syringe filter. The filtered samples were stored at -18 °C before analysis. The HPLC analysis was performed using an Ultimate 3000 (Thermo Scientific, USA) equipped with an Aminex HPX-87H ion exchange column (Bio-Rad, USA) with the column temperature set to 30 °C and a mobile phase of 5 mM H2SO4 at a flow rate of 0.6 mL min-1. Detection was carried out using both refractive index and UV at 210 nm. Glucose and gluconate coelute, but their concentrations were correlated by their differential contribution in the refractive index and UV channel.

[0215] To measure total cell density, viability, cell diameter, glucose, lactate, ammonium, and pH for the cell cultures, a BioProfile FLEX2 was utilized. This device comes with the necessary solutions for the measurements, thus eliminating the need for sample preparation beforehand. The sample size for each measurement is 400 pL. BioProfile FLEX2 is an advanced analytical device that utilizes consumable microsensor cards (based on enzymatic biosensors for glucose, lactate, glutamine, and glutamate) and reagent cartridges to perform specific measurements. Once equipped, both the microsensor cards and cartridges have a limited number of measurements, and their in-use life ranges from 14 to 21 days depending on the product. The measurement of cell viability was performed using this device.

[0216] Example 5 - Structural characterization

[0217] The synthesis of BA-Ceo as a fulleropyrrolidine derivative was achieved using a one-step Prato reaction approach (1,3-dipolar cycloaddition method) between the corresponding aldehyde derivatives and sarcosine as the amino acid, as illustrated in Figure 5.

[0218] UV-vis absorption spectra (400-750 nm) of pristine Ceo and BA-Ceo were obtained in toluene solution, as shown in Figure 9a. A distinct peak appeared at 407 nm for Ceo and at 430 nm for BA-Ceo, corresponding to the 60n and 58n electron systems for Ceo and BA-Ceo, respectively. The 58n-electron system in BA-Ceo is due to the incorporation of the pyrrolidine ring, which decreases the n-electron count by two. A faint absorption at 710 nm was associated with n-n* transitions in the fullerenes. Minor absorption variances were observed between 430 and 600 nm, suggesting that the substituent groups on the fullerenes had minimal impact on the electronic structure of the fullerene n-system The synthesis of BA-Ceo is further confirmed by the FT-IR analysis (as shown in Figure 9b). Notably, the absence of the C=O stretching peak at approximately 1697 cm in the spectrum of BA-Ceo indicates the formation of the pyrrolidine ring resulting from the reaction between FPBA and sarcosine on Ceo.

[0219] A series of characteristic methods were implemented to understand the properties of as-prepared T-COF and composites. Based on the FT-IR analysis of the related materials (Figure 9c), a moderate decrease in the hydroxyl stretch was observed in T-COF compared to the initial materials. This remaining hydroxyl stretch intensity can be attributed to the hydroxyl groups present on the bis-diol units at the network termini. More significantly, the formation of boronate ester bonds (C2O2B ring) in the T-COF was verified by the appearance of vibrational C-0 bonds at 1235 cm (Figure 9c, inset). Furthermore, the prominent peaks at 1339 cm4and 1020 cm4represent the bands corresponding to the B-0 and B-C stretches within the boronate ester bonds (C2O2B ring), respectively. In T-COF, a characteristic peak at 661 cm4was observed due to the C-S bonds in the thiophene groups. Additionally, a distinct peak at 576 cm4was noticed in the spectrum of the BA-Ceo@T-COF composite compared to the spectrum of T-COF alone, which can be attributed to the encapsulated BA-Ceo molecule within the T- COF structure.

[0220] The crystal lattice structure of T-COF was determined through PXRD analysis. Figure 10a displays five distinct peaks at 3.0°, 5.3°, 6.1°, 8.2°, and 25.56° corresponding to the (100), (110), (200), (210), and (001) facets, respectively. The results verified that the T-COF structure with optimal crystallinity was synthesized. The average domain size, calculated from the XRD patterns' peak broadening, is approximately 30 nm. The PXRD profile of BA-Ceo@T-COF aligns well with the experimentally obtained pattern for pure T-COF, indicating that the encapsulation of guest molecules (BA-Ceo) does not impact its crystallinity (Figure 10a). This demonstrates the high crystallinity of both samples and the structural stability of T-COF during the BA-Ceo@T-COF synthesis process. Furthermore, the absence of characteristic diffraction peaks for BA-Ceo in the PXRD pattern of BA- C6O@T-COF suggests that BA-Ceo guest molecules are dispersed within the pores at the molecular level, without any aggregation.

[0221] The porous nature of T-COF was verified through N2 adsorption tests conducted at 77 K. The T-COF sorption isotherm curve was classified as a type IV isotherm, indicative of its microporous properties (Figure 10b). The data reveals a significant rise in adsorption due to the orderly and crystalline structure of the mesoporous pores. The Brunauer-Emmett-Teller (BET) surface area was determined to be remarkably high at 1454 m2g-1, while the pore volume measured 1.02 cm3g-1(Figure 10c, inset). As shown in Figure 10c, its pore size was found to be 3.0 nm using nonlocal density functional theory, which was in good agreement with its theoretical value of 3.0 nm. Based on the results obtained, it can be deduced that the pores of T-COF are not blocked by oligomeric fragments or solvent molecules. Figure 10b displays a noticeable decrease in the amount of N2 absorbed after incorporating BA-Ceo into T-COF. The BET surface area of BA-Ceo@T-COF composite was found to have decreased to half, measuring 703 m2g-1. Additionally, the pore volume and pore size of BA-Ceo@T-COF experienced a substantial decrease, dropping from 1.01 to 0.48 cm3g-1and from 3.0 to 2.1 nm, respectively (as shown in Figure 10c and its inset).

[0222] Thermal stability was evaluated using TGA under ambient conditions. As shown in Figure lOd, T-COF and BA-Ceo@T-COF are able to maintain their stability up to 430 °C and 410 °C, respectively. In comparison to T-COF, the additional weight loss of approximately 10% observed in BA-Ceo@T-COF can be attributed to the decomposition of BA-Ceo. To further investigate the stability of BA-Ceo@T-COF, a powder sample was dispersed in boiling water for 12 hours at room temperature, followed by collecting the precipitates through centrifugation, thorough rinsing, and drying before conducting PXRD analysis. Remarkably, the PXRD analysis showed no significant shifts in the positions and intensity of soaked BA-Ceo@T- COF, indicating its remarkable chemical robustness.

[0223] The sample morphologies were examined more closely using SEM images. SEM images for both T-COF and BA-Ceo@T-COF samples at various magnifications are displayed in Figure 11a and b, respectively. Upon comparing the BA-Ceo@T-COF SEM images to those of the pristine T-COF, it was evident that their morphologies were quite similar. This suggests that the encapsulation of BA-Ceo did not have a significant impact on the overall morphology of the T-COF structure Figures 12 presents SEM images of carbon micropillars before and after the modifications. Prior to electrodeposition, the micropillars exhibit a smooth surface and uniform structure, which is characteristic of SU-8-derived carbon materials (Figure 12a). SEM images following Au NPs electrodeposition (Figures 12b-d) verify the formation of a uniform Au NPs coating on the surface of the 3D carbon electrodes.

[0224] The XRD pattern of the deposited Au NPs displays four peaks, corresponding to standard Bragg reflections (111), (200), (220), and (311) of the face-centered cubic lattice (Figure 12d, inset), indicating the purity of the deposited Au NPs. Top-view SEM micrographs of the electrodeposited BA-Ceo@T-COF film demonstrate consistent deposits on the surface of the Au NPs-modified 3D carbon electrode (Figure 12e). These deposits comprise closely packed particles of similar size, generating interstitial voids. Notably, these voids are visible throughout the films. Lastly, Figure 12f depicts the surface of the modified electrode after being coated with PTGE: PEI thin film and ZiPPy polymer, which serve as diffusionlimited and antifouling layers, respectively.

[0225] The full-scale XPS spectra in Figure 13a revealed the surface chemical composition of the T-COF, BA-Ceo@T-COF, and AuNPs@BA-Ceo@T-COF modified 3D pyrolytic carbon WE. Four peaks corresponding to C Is, O Is, B Is and S 2p were observed in all the samples. At the same time, the BA-Ceo@T-COF and AUNPS@BA-C6O@T-COF both displayed a weak N Is signal, confirming the presence of BA-Ceo (because of the pyridine ring) in these samples. In comparison, the Au 4f peak was only present in the AuNPs@BA-Ceo@T-COF modified electrode. The deconvoluted high-resolution C Is peak of all samples exhibited four peaks centered at a binding energy of 283.7, 284.6, 285.9 and 287.6 eV, corresponding to the C-S, C=C, C-C, and C=O, respectively (Figure 13b). The high-resolution O Is spectra of the BA-Ceo@T-COF, and AuNPs@BA- Ceo@T-COF composites can be deconvoluted into three characteristic peaks at 531.8, 533.2, and 534.28 eV, corresponding to the C-O, B-O-C (C2O2B ring), and B-OH (BA-Ceo), respectively (Figure 13c). In contrast, the O Is spectrum of the T- COF structure displayed only two peaks, corresponding to C-0 and B-O-C bonds. This suggests that BA-Ceo molecules were successfully enclosed within the T-COF structure. Furthermore, the absence of the B-OH peak at 193.2 eV in the B Is spectrum of T-COF (Figure 13d), along with the presence of the N-C peak at 399.28 eV in the N Is spectra of both composites (Figure 13e), provides additional evidence that BA-Ceo molecules were encapsulated within the T-COF pores. The S 2p spectra of the AuNPs@BA-C6o@T-COF film showed a binding energy of 162.3 eV, as depicted in Figure 13f. This energy was a result of the Au- S bond, which was a result of the strong interaction between the thiophene and gold, a well-known phenomenon. In addition, the Au 4f spectra showed a peak around 84.7 eV for the Au-S bond, which overlapped with the Au 4f? / 2 peak that was associated with Au° from the nanoparticles (Figure 13g). Therefore, it is believed that the thiophene groups present in the BA-Ceo@T-COF played a role in the formation of a stable film on the surface of the 3D pyrolytic carbon WE modified by Au NPs.

[0226] Example 6 - Electrochemical evaluation

[0227] The electrochemical performance of the fabricated electrodes for glucose electrooxidation was evaluated using CV. Figure 14a displays the CV profiles for AUNPS@BA-C6O@T-COF, AuNPs@BA-Ceo, and T-COF modified 3D pyrolytic carbon WEs in 0.1 M PBS (pH 7.0) at a scan rate of 50 mV s-1, in the absence and presence of 10.0 mM glucose. As illustrated in Figure 14a, the T-COF modified electrode exhibited no catalytic activity toward glucose oxidation, as evidenced by the absence of a Faradaic peak in the CV curve. On the other hand, the AUNPS@BA-C6O@T-COF electrode generated a pronounced oxidation peak current when glucose was present in the PBS solution, while no response was detected without glucose. In contrast, the AuNPs@BA-Ceo electrode displayed a weak peak for glucose oxidation, suggesting inferior electrocatalytic activity. This can be ascribed to the reversible covalent cyclic ester formed between the boronic acids of BA-Ceo and the 1,2-cis hydroxyl groups of glucose. Furthermore, incorporating BA-Ceo into the T-COF pores in the AuNPs@BA-Ceo@T-COF modified electrode not only provides a conductive platform for the electrochemical oxidation reaction but also positions more boronic acid groups on the electrode surface, furnishing additional binding sites and rapid association kinetics.

[0228] The electron transfer kinetics of glucose oxidation were investigated by examining the impact of the scan rate (u). The results of the cyclic voltammetry (CV) experiments, conducted on 10.0 mM glucose in PBS using the AuNPs@BA-Ceo@T- COF modified electrode, are shown in Figure 14b for scan rates ranging from 20- 170 mV s-1. The data indicates that both the oxidation peak currents (Ipa) and peak potentials (Epa) exhibit a positive shift with increasing scan rates. In addition, Figure 14c shows that the Ipaof glucose oxidation is directly proportional to the scan rate, implying a surface-controlled electrode process. However, at higher scan rates (>50 mV s-1), the plot of Ipaversus u1 / 2also demonstrates a linear relationship, indicating a diffusion-controlled glucose oxidation (Figure 14c). The regression equation for the anodic region of the oxidation reaction is given by Eq. (2).

[0229] Ipa= 0.2476 u1 / 2- 0.7109 (mA, mV1 / 2s’1 / 2, R2= 0.9884) (2)

[0230] Based on the results obtained, it can be inferred that the electrochemical oxidation of glucose is a combination of both diffusion-controlled and adsorption- controlled processes, which are dependent on the scan rates used. Additionally, a linear plot was observed for the oxidation peak potentials (Epa) against the logarithm of scan rates (Figure 14d), with a corresponding correlation equation

[0231] (Eq. (3)).

[0232] Epa= 0.0451 log u + 0.5664 (V, V s’1, R2= 0.9954) (3)

[0233] As the scan rate (u) was increased, the oxidation peak potential (Epa) showed a positive shift. To estimate the on value for this irreversible glucose oxidation,

[0234] Laviron's equation (Eq. (4)) was used : which involves various parameters such as the electron transfer coefficient (a), the standard rate constant of the reaction (ks), the scan rate (u), the electron transfer number (n), and the formal potential (E°). The Epavs. In(u) plot was used to deduce an on value of 1.31, with R = 8.314 JK-1mol-1, T = 298 K, and F = 96485 Cmol-1being assumed. Since the glucose oxidation is irreversible, a was considered to be 0.5, resulting in an estimated electron transfer number (n) of 2.61. This indicates that the glucose oxidation at the AuNPs@BA-C6o@T-COF film is a two-electron transfer process. Previous studies have shown that the number of electrons and protons participating in glucose oxidation are equal, suggesting that the electrochemical glucose oxidation at the AuNPs@BA-Ceo@T-COF modified electrode involves a combination of two electrons and two protons. Figure 15a represents the schematic diagram of the fabricated AuNPs@BA-Ceo@T-COF based sensor and its electrochemical performance towards glucose detection. Figure 15b shows a schematic of the glucose oxidation reaction on the surface of the AUNPS@BA-C6O@T-COF modified electrode. The surface pKaof the BA-Ceo group in AuNPs@BA-C6o@T-COF modified electrode surface was investigated by analyzing the attenuation of redox reactions involving charged probe molecules. Figure 16a illustrates the CV response of Fe(CN)e3' / 4' on the AUNPS@BA-C6O@T-COF modified electrode under varying pH conditions. As the pH of the solution rises, the redox response of Fe(CN)e3' / 4' is noticeably diminished. It is widely recognized that the redox reaction of a charged probe molecule weakens when a charged layer on the electrode surface has the same charge as the probe molecule. Thus, the observed pH-dependent redox response can be attributed to the electrostatic repulsion between Fe(CN)e3' / 4' and the negatively charged BA-Ceo groups on the electrode surface. The decline in the current response in CV experiments was employed to approximate the pKa value of the attached BA-Ceo group. The surface pKavalue of the BA-Ceo group on the electrode was approximated to be 4.1, as determined by the optimal fit of the data in Figure 16a, inset.

[0235] The shift in the pKavalue of a functionalized molecule on a substrate surface compared to its bulk pKain solution can be attributed to several factors, such as specific interfacial effects and the structure of the surface grafted layer. Other studies have reported similar shifts in pKavalues, with some showing a positive shift for surface amino groups and a negative shift for surface carboxylic groups in polyelectrolyte layer-by-layer films. The mechanistic details of the pKashift observed in this study are not yet fully understood. However, a negative shift in pKais favorable for glucose detection using the BA-Ceo structure as a recognition site, as lower pKavalues are believed to have higher binding affinities towards glucose. Hence, there is considerable interest in developing synthetic receptors based on a boronic acid moiety having a lowered pKa.

[0236] As mentioned above, the pH value of a solution significantly influences the affinity of BA-Ceo towards glucose, affecting its overall effectiveness. In Figure 16b, the peak current responses at the AuNPs@BA-Ceo@T-COF electrode were recorded after a 10-minute incubation in a 10 mM glucose solution within a 0.1 M PBS (pH 7.0). The pH values tested ranged from 3 to 10. At lower pH values, the current responses of glucose were notably smaller due to the reduced affinity between boronic acids and diols. However, a substantial increase in current responses occurred when the pH value shifted from 5 to 6.7. This can be attributed to the significantly enhanced binding constant between boronic acids and glucose within this pH range, resulting in the oxidation of more boronate esters. These findings align with those reported by Wang's research group. Nonetheless, the current responses appeared to stabilize for pH values between 6.7 and 10. This stabilization occurs because the pH range surpasses the pKaof BA-Ceo (Figure 16a, inset), where the binding constant between BA-Ceo and glucose reaches its maximum value.

[0237] Example 7 - Sensing performance of AuNPs@BA-Ceo@T-COF modified electrode

[0238] The assessment of glucose oxidation and sensitivity in the modified 3D WEs was performed using amperometric analysis, with a gradual increases in glucose concentration in the PBS solution. For achieving the best amperometric current response, the applied potentials on the AuNPs@BA-Ceo@T-COF electrode were adjusted within a range of 0.40 to 0.60 V based on the CV results (as shown in Figure 16c). As 5.0 mM glucose was successively added to the electrolyte, the electrode demonstrated a stepwise response under varying applied potentials. As a result, 0.55 V was determined to be the optimal potential for the modified electrodes. It is important to note that the calibration range for the AuNPs@BA- Ceo@T-COF modified electrode, in the absence of a diffusion-limiting layer, is between 0.05 and 5.5 mM of glucose, as shown in Figure 16d. However, when a PTGE: PEI layer is coated on the AuNPs@BA-Ceo@T-COF modified electrode, the current response for glucose drops significantly. This decrease in current response can be attributed to the PTGE: PEI layer's ability to delay the transport of glucose molecules to the electrode surface. Although this may seem like a disadvantage, it is actually a valuable feature that expands the dynamic range of the electrode. The larger dynamic range is particularly useful for monitoring bioprocesses, where glucose concentrations can vary widely over time. Moreover, slowing down glucose transport through the PTGE: PEI layer can also prevent interference from other electroactive species that may be present in the sample matrix. This selectivity enhances the accuracy and reliability of glucose monitoring. Figure 16e illustrates the real-time amperometric response of the AuNPs@BA-C6o@T-COF and AUNPS@BA-C6O modified electrodes with PTGE: PEI layer as a diffusion-limit layer to increase the dynamic range. The current produced by AuNPs@BA-Ceo upon incremental glucose addition to the PBS solution was minimal, while the AUNPS@BA-C6O@T-COF electrode exhibited a noticeable current change even at glucose concentrations as low as 0.1 mM. This electrode displayed exceptional sensitivity across a broad range of glucose concentrations (0.1 mM to 120 mM) without necessitating any additional treatment or washing steps between tests. The impressive performance and swift response of the AuNPs@BA-C6o@T-COF modified electrode can be attributed to its large surface area and electrocatalytic activity, which are a result of BA-Ceo encapsulation within the T-COF pores. Figure 16f depicts the current response of the AuNPs@BA-C6o@T-COF modified electrode in relation to glucose concentration, demonstrating linear behavior with an R2value of 0.995. This suggests the electrode's reliability as a glucose sensor across a wide range of concentrations. The calculated slope was 0.3324 mA mM-1cm-2(equivalent to a sensitivity of 332.4 pA mM-1cm-2) for the AuNPs@BA-Ceo@T- COF modified electrode. The limit of detection (LOD) was determined to be 0.051 mM, based on an equation employing a signal-to-noise ratio of 3.

[0239] The selectivity of a glucose sensor is a critical factor in its effectiveness for real- world applications, especially in bioprocesses where interfering substances such as acetate, lactate, ethanol, NaCI, and SO42' are frequently present. Therefore, the sensor must exhibit high selectivity for glucose over these other species to be useful. The sensor exhibited consistent response to glucose in the presence of all interfering agents, with no significant change in current observed upon their addition (Figure 17a). In this study, the glucose selectivity of the AuNPs@BA- Ceo@T-COF based sensor was also investigated using sugars similar to glucose such as galactose (15.1%), fructose (8.2%), and sucrose (2.1%) (Figure 17a, inset). The results showed that the largest current response was observed for galactose, followed by fructose and sucrose. However, the peak currents of all the glucose- interfering sugars in 20 mM were lower than that obtained for the glucose (5 mM) peak current, confirming that BA-Ceo bound more strongly to glucose than to the other sugars. Moreover, this finding agreed well with the association constants reported in previous research work.

[0240] Despite the presence of interfering sugars, the sensor's response to actual bioprocess glucose concentrations was acceptable, indicating excellent selectivity for glucose detection. This suggests that the AuNPs@BA-Ceo@T-COF based sensor has great potential for practical applications in bioprocesses, offering good selectivity and sensitivity for glucose detection in the presence of interfering substances. Example 8 - Stability, repeatability, and reproducibility of sensor

[0241] Figure 17b illustrates the response reproducibility of the AuNPs@BA-C6o@T-COF modified electrode, which was subjected to 20 cycles of switching between pure PBS and PBS with 5 mM glucose. The sensor demonstrated a rapid recovery time (less than 3 seconds) and consistent response towards glucose detection, indicating its suitability for multiple measurements without requiring cleaning after each use. Figure 17c shows the chronoamperogram of the 1stday, 10thday and 30thday after sensor fabrication, with successive additions of 5 mM glucose. The electrode's long-term stability was evaluated by running amperometric tests at glucose concentrations ranging from 1 to 100 mM every week, as shown in the inset of Figure 17c. The electrodeposited structure of the AuNPs@BA-Ceo@T-COF on 3D WE and using diffusion limited and antifouling layers prevented the instability resulting from active site leaching, resulting in an outstanding reusability and a highly stable response. After 20 days, the recorded sensitivity was 95.3% of the initial sensitivity, which was attributed to the sensor's unique properties. This superiority can be attributed to the high surface area of the sensor, which provides robust electrocatalytic sites for the glucose oxidation process. Figure 17d shows that the structure of the surface of the electrode was retained at the same level as the freshly made ZiPPy coatings after the 10th, 20thand 30thday of glucose sensing. Moreover, even after 30 days of use, the ZiPPy coating still could maintain high antifouling capacity.

[0242] Example 9 - At-Line Application of the Biosensor Platform During a Fermentation Process

[0243] The AuNPs@BA-C6o@T-COF-based sensors, calibrated in different medium, were applied for at-line monitoring of glucose in bacterial, yeast fermentation and cell culture samples.

[0244] Figures 18 displays the outcomes of a Saccharomyces cerevisiae Bl yeast fermentation. The fermentation's progression is illustrated by the rising cell concentration and the diminishing glucose concentration over a 12-hour period (Figure 18). The glucose concentration was tracked using the developed sensor platforms, which measured both cell-free and cell-containing samples. These glucose profiles were then compared to HPLC results. It is important to note that HPLC analysis can only be performed on cell-free samples, as cell-containing samples would quickly clog the system's capillaries and the separation column. Figure 18 evaluates the performance of the AuNPs@BA-C6o@T-COF-based sensor relative to HPLC analysis by comparing the average standard deviation and the average time to signal stabilization (or the average time to obtain measurement results for HPLC measurements). The data depicted in the Figure 18 demonstrates the precise measurement of glucose concentrations in the cell-free supernatant during the entire fermentation process using the AuNPs@BA-Ceo@T-COF-based sensor coating, incorporating ZiPPy and Nation as antifouling layers. The results obtained from the sensor were in agreement with those from HPLC, indicating its accuracy and reliability.

[0245] The glucose concentration measurements in samples containing cells with the AuNPs@BA-C6o@T-COF-based sensor coated with Nation showed lower values compared to those obtained from cell-free samples. On the other hand, for the sensor coated with ZiPPy, the difference in glucose concentration measurements between samples with and without cells was found to be minimal. The ZiPPy- coated electrodes showed superior hydrophilicity and considerably lower protein and cell adsorption when compared to Nafion-coated electrodes. The difference in the adhesion behavior of cells and proteins on Nation compared to ZiPPy may suggest that the charge of the Nation polymer film does not significantly impact cell adhesion. Additionally, the mixed hydrophobic and hydrophilic groups in Nation, which create a poor hydration sphere, could also contribute to cell and protein adhesion. Higaki et al. showed in a recent study that the order of antifouling ability for polymers was zwitterionic polyelectrolytes > hydrophobic polymers > charged polyelectrolytes, indicating that it may be the combination of hydrophobicity and charge that results in the high cell count on Nafion-coated surfaces.

[0246] The developed ZiPPy coated AuNPs@BA-Ceo@T-COF-based sensor was also evaluated for its ability to detect glucose levels in mammalian cell cultures with HEK293 and K562 cell lines. The results obtained using the developed sensor platform were compared with those obtained using biosensors based on glucose oxidase in BioProfile FLEX2 devices, and are illustrated in Figure 19a and b. The glucose signal trends captured by both methods were in good agreement, with the developed sensor demonstrating accurate glucose signal detection. However, it should be noted that the glucose signal measured by the developed sensor was consistently slightly lower than that measured by the biosensors in the K562 cell culture (Figure 19b). This difference may be attributed to differences in sampling times, as the samples were first injected into the BioProfile FLEX2 device before being measured by the developed sensor.

[0247] Table 4 provides a comparison between glucose concentration values obtained from the developed sensor and HPLC measurements at different sampling times during the Pseudomonas putida KT2440 fermentation process. The comparison between glucose sensing values obtained from the developed sensor and HPLC measurements shows that the sensor provides reasonably accurate and reliable results. Importantly, the AuNPs@BA-Ceo@T-COF-based sensor demonstrated that the presence of various electroactive species and changes in the medium did not interfere with the glucose measurement, emphasizing the advantages of utilizing a sensor that possesses a highly specific and selective detection part. Table 4. Off-line monitoring of glucose by the developed sensor in a Pseudomonas putida KT2440 fermentation process.

[0248] Example 10 - T-COF applied on different carbon based substrates

[0249] In this study, the electrochemical performance of AuNPs@BA-Ceo@T-COF modified 3D pyrolytic carbon microelectrode and AuNPs@BA-Ceo@T-COF modified graphene based DropSens electrode were compared. Both electrodes were tested for their ability to detect glucose in different concentrations, and it was found that the DropSens electrode show no signal below 1 mM and above 6 mM glucose concentration. The superior performance of the 3D pyrolytic carbon microelectrode can be attributed to its larger surface area compared to the DropSens electrode. The larger surface area of the 3D pyrolytic carbon microelectrode provides more opportunities for the immobilization of AuNPs@BA- C6O@T-COF, which enhances its electrocatalytic activity towards the oxidation of glucose. Additionally, the 3D pyrolytic carbon microelectrode was able to attract and immobilize AuNPs@BA-C6o@T-COF more effectively than the DropSens electrode. This may be because the 3D pyrolytic carbon microelectrode has a higher density of carboxylic groups on its surface, which provides more binding sites for the AuNPs@BA-C6o@T-COF. The strong binding between the 3D pyrolytic carbon microelectrode and AuNPs@BA-Ceo@T-COF promotes efficient electron transfer, resulting in a more sensitive and stable amperometric signal for glucose detection (Figure 20).

[0250] Example 11 - nanoparticles

[0251] In the fabrication of AuNPs@BA-Ceo@T-COF, different metal nanoparticles such as Ag, Au, Pt, and Cu were electrodeposited on the surface of the electrode (Figure 21). These electrodes were then tested for their ability to detect 10 mM glucose. The results showed that gold had the highest signal for the determination of 10 mM glucose, followed by Pt. This high signal energy was a result of the Au-S bond formed due to the strong interaction between the thiophene groups present in the BA-Ceo@T-COF and the gold, which is a well-known phenomenon. The thiophene groups in the BA-Ceo@T-COF played a crucial role in the formation of a stable film on the surface of the 3D pyrolytic carbon working electrode (WE) modified by Au NPs. This stable film ensured a high electrocatalytic activity for the oxidation of glucose, resulting in a more sensitive and stable signal for glucose detection. The effectiveness of gold as the best electrode can be attributed to its high affinity towards the BA-Ceo@T-COF, resulting in an efficient immobilization of the AUNPS@BA-C6O@T-COF on the surface of the electrode.

[0252] The sizes of the nanoparticles was assessed, and the mean size of nanoparticles was found to be about 100 nm (Figure 22).

[0253] Example 12 - Conclusion

[0254] A novel non-enzymatic glucose sensor was developed, based on a fullerene AUNPS@BA-C6O@T-COF modified 3D pyrolytic carbon electrode. This new sensor demonstrated excellent electrochemical performance, outperforming other modified electrodes due to its large electrochemical active surface and good conductivity. The non-enzymatic AuNPs@BA-Ceo@T-COF-based sensor demonstrated remarkable effectiveness in glucose detection and outstanding electrooxidation detection performance for glucose monitoring. The electrocatalytic activity can be attributed to multiple factors, such as efficient electrolyte diffusion, selective glucose interaction with BA-Ceo, rapid charge transfer, exceptional electroconductivity of the BA-Ceo@T-COF, and the efficiency of active sites due to the distinct morphology and synergistic impact between T- COF and BA-Ceo in the constructed structure. To prevent cells and proteins from attaching to the surface of the electrode in real samples, the developed electrodes were coated with ZiPPy, a zwitterionic polymer. The sensor recorded a fast detection with excellent sensitivity of 332.4 pA mM-1cm-2for glucose concentrations ranging from 0.1 mM to 120 mM, with a limit of detection of 0.051 mM (S / N = 3). The developed sensor exhibited a highly consistent response over 20 consecutive measurement cycles without requiring any cleaning procedures and maintained stable sensitivity for 20 days under ambient conditions. In comparison to traditional HPLC analysis, this sensor enabled dependable glucose quantification in a significantly more efficient and time-saving manner. It displayed exceptional stability when in direct contact with the fermentation medium and accurately quantified glucose even in the presence of several electroactive species. The established method can be easily regarded as a straightforward, sturdy, precise, and cost-effective solution for real-time glucose monitoring in bioprocesses.

[0255] Items

[0256] 1. A thiophene based covalent organic framework (T-COF) comprising boronic acid-functionalized Ceo (BA-Ceo), the T-COF comprising or consisting of C2 and C3 arranged in a crystal lattice structure represented by formula I, (formula I), wherein C3 is represented by formula II (formula II), wherein Ri is C2 and C2 is represented by formula III (formula III).

[0257] 2. The T-COF according to item 1, wherein the boronic acid in the BA-Ceo is a boronic acid having a pKavalue in the range of 4.0-4.2, preferably 4.1. 3. The T-COF according to any of the preceding items, wherein the BA-Ceo is represented by the formula IV

[0258] (formula IV).

[0259] 4. The T-COF according to any of items 1-2, wherein the BA-Ceo is represented by a formula selected from the following table

[0260]

[0261] 5. The T-COF according to any of the preceding items, wherein the BA-Ceo is non- covalently bonded to the T-COF, such as via intermolecular forces, such as van der Waals forces, and electrostatic interactions, such as n-n stacking interaction. 6. Use of the T-COF according to any of the preceding items, in an electrochemical sensor for sensing glucose.

[0262] 7. An electrochemical sensor for sensing glucose comprising a working electrode, the working electrode comprising the T-COF according to any of items 1-5.

[0263] 8. The electrochemical sensor according to item 7, the working electrode further comprising:

[0264] • an electrochemical substrate, such as a carbon-based substrate, optionally having micropillars; and

[0265] • nanoparticles comprising or consisting of a metal, such as Au, Pt, Ag, or Cu.

[0266] 9. The electrochemical sensor according to any of items 7-8, wherein the sensor further comprises a counter electrode and, optionally, a reference electrode.

[0267] 10. The electrochemical sensor according to item 9, wherein the counter electrode and / or reference electrode comprises a noble metal, such as Au, Pt, or Ag.

[0268] 11. The electrochemical sensor according to any of items 9-10, wherein the noble metal is Au.

[0269] 12. The electrochemical sensor according to any of items 7-11, wherein the carbon-based substrate is a pyrolytic carbon electrode, such as a 3D pyrolytic carbon microelectrode, such as comprised of glassy carbon, optionally having micropillars.

[0270] 13. The electrochemical sensor according to item 12, wherein the pyrolytic carbon electrode is made from pyrolysing a negative photoresist, such as epoxy photoresist SU-8.

[0271] 14. The electrochemical sensor according to item 13, wherein the pyrolytic carbon electrode is exposed to UV light before pyrolysation. 15. The electrochemical sensor according to any of items 7-14, wherein the surface area of the carbon-based substrate is expanded by one or more 3- dimensional microstructures, such as micropillars.

[0272] 16. The electrochemical sensor according to item 15, wherein the 3-dimensional microstructures are structures of the same material as the carbon-based substrate, and extend in a perpendicular direction from the carbon-based substrate.

[0273] 17. The electrochemical sensor according to item 15 or item 16, wherein the 3- dimensional microstructures are micropillars with a height of between 100 pm to 300 pm and a diameter of 5 to 200 pm.

[0274] 18. The electrochemical sensor according to any of items 7-16, wherein the micropillars are pillars with a height of approximately 100 300 pm and a diameter of 100 pm.

[0275] 19. The electrochemical sensor according to any of items 7-18, wherein the sensor further comprises means for providing insulation and / or a passivation layer ensuring only active regions, such as the working-, reference, and / or counter electrode are available to a solution applied to the electrochemical sensor.

[0276] 20. The electrochemical sensor according to item 19, wherein the means for providing insulation and / or a passivation layer is made from the same material as a pyrolytic carbon electrode, without having been pyrolysed.

[0277] 21. The electrochemical sensor according to any of items 7-20, wherein the electrochemical sensor's ability to sense glucose is maintained after exposure to autoclave conditions, such as overpressure and about 120 degrees Celcius.

[0278] 22. The electrochemical sensor according to any of items 7-20, wherein the sensor further comprises means for anti-fouling, such as the electrochemical sensor being coated with a zwitterionic polymer, such as zwitterionic polypyrrole (ZiPPy). 23. The electrochemical sensor according to any of items 7-20, wherein the sensor further comprises means for reducing diffusion, such as a diffusion-limiting layer, such as a layer comprised of a trimethylolpropane triglycidyl ether (PTGE) and polyethyleneimine (PEI).

[0279] 24. The electrochemical sensor according to any of items 7-20 or any of items 22- 23, wherein the sensor comprises means for antifouling according to item 22 and means for reducing diffusion according to item 23, and preferably wherein the means are combined in a ratio of about 1: 1.

[0280] 25. The electrochemical sensor according to any of items 7-24, wherein the electrochemical sensor's ability to sense glucose is maintained for a sustained period of time after manufacturing without affecting the sensor's ability to sense glucose, wherein the sustained period of time is at least 5 days, such as at least 10 days, such as 15 days, such as at least 20 days.

[0281] 26. The electrochemical sensor according to any of items 22-25, wherein the electrochemical sensor's ability to sense glucose is maintained after exposure of up to 70 degrees Celcius.

[0282] 27. The electrochemical sensor according to any of items 7-26, wherein the sensor's ability to sense glucose is defined as an ability to continuously measure the glucose content of a control sample, such as running amperometric tests at glucose concentrations ranging from 1 to 100 mM or switching between pure PBS and PBS with 5 mM glucose, and maintaining sensitivity of at least about 90% such as even about 95%.

[0283] 28. Use of the electrochemical sensor according to any of items 7-27, in a method for sensing glucose.

[0284] 29. A method of sensing glucose, the method comprising:

[0285] 1. Providing an electrochemical sensor as defined in any of items 7-27 configured to produce an electric signal when a glucose molecule is at, or near, the working electrode;

[0286] 2. Providing a sample suspected of containing glucose; 3. Contacting the electrochemical sensor with the sample;

[0287] 4. Measuring the signal from the working electrode, thereby sensing the presence of glucose in the sample;

[0288] 5. Optionally, comparing the signal to a signal from a control electrode on the electrochemical sensor to reduce background signals; and

[0289] 6. Optionally, comparing the signal from the working electrode to a signal obtained from measuring a control sample, to obtain a level of glucose in the sample.

[0290] 30. The method according to item 29, wherein the method senses glucose in the range from 0.1 mM to 120 mM.

[0291] 31. An apparatus for sensing metabolites, the apparatus comprising the electrochemical sensor according to any of items 7-27, optionally the apparatus comprising at least one further sensor for sensing a metabolite other than glucose.

[0292] 32. A method of producing a T-COF comprising boronic acid-functionalized Ceo (BA-Ceo), the method comprising:

[0293] 1. combining thieno[3,2-b]thiophene-2,5-diboronic acid (F TTDB) and 2,3,6,7,10,11-hexahydroxytriphenylene (HHTP) with dry dioxane and dry mesitylene;

[0294] 2. reacting the mixture in an autoclave, such as at 150 °C for 72 h;

[0295] 3. washing the product thereof, and drying the product;

[0296] 4. optionally, heating the product under high vacuum, such as at app. 200 °C and IO’6torr (1.33 x 10’4Pa);

[0297] 5. obtaining a T-COF;

[0298] 6. reacting fullerene Ceo, 4-formylphenylboronic acid pinacol ester (FPBA), and sarcosine, such as at 125 °C for 12 h;

[0299] 7. washing the product thereof;

[0300] 8. removing the protective group to obtain BA-Ceo, such as by the use of methylboronic acid dissolved in dichloromethane, followed by the addition of TFA; and

[0301] 9. combining the T-COF with the BA-Ceo, thereby obtaining a T-COF comprising BA-Ceo. 33. A method of producing an electrochemical sensor for sensing glucose, the method comprising:

[0302] 1. providing a carbon based substrate, such as a pyrolytic carbon electrode; nanoparticles comprising or consisting of a metal, such as Au, Pt, Ag, or Cu; and a T-COF comprising BA-Ceo as defined in any of items 1- 5, or item 32;

[0303] 2. depositing the nanoparticles on the carbon based substrate;

[0304] 3. depositing the T-COF comprising BA-Ceo on the nanoparticles;

[0305] 4. optionally, depositing a counter- and / or a reference electrode at suitable positions on the electrochemical sensor;

[0306] 5. optionally, coating with a zwitterionic polymer, such as zwitterionic polypyrrole (ZiPPy); and

[0307] 6. optionally, coating with a diffusion-limiting layer, such as a layer comprised of a trimethylolpropane triglycidyl ether

[0308] (PTGE): polyethyleneimine (PEI) layer.

[0309] 34. The method of producing an electrochemical sensor according to item 33, wherein the counter electrode and / or reference electrode comprises a noble metal, such as Au, Pt, or Ag.

[0310] 35. The method of producing an electrochemical sensor according to any of items 33-34, wherein the noble metal is Au.

[0311] 36. The method of producing an electrochemical sensor according to any of items 33-35, wherein the pyrolytic carbon electrode is made from pyrolysing a negative photoresist, such as epoxy photoresist SU-8.

[0312] 37. The method of producing an electrochemical sensor according to item 36, wherein the pyrolytic carbon electrode is exposed to UV light before pyrolysation.

[0313] 38. A kit comprising the electrochemical sensor according to any of items 7-27, and optionally means for replacing the electrochemical sensor and / or one or more further electrochemical sensors, such as one or more further electrochemical sensors for sensing one or more metabolites other than glucose, optionally the kit may also comprises components for cleaning, such as washing buffers.

Claims

Claims1. A thiophene based covalent organic framework (T-COF) comprising boronic acid-functionalized Ceo (BA-Ceo), the T-COF comprising or consisting of C2 and C3 arranged in a crystal lattice structure represented by formula I,wherein C3 is represented by formula II(formula II), wherein Ri is C2 and C2 is represented by formula III(formula III).

2. The T-COF according to claim 1, wherein the boronic acid in the BA-Ceo is a boronic acid having a pKavalue in the range of 4.0-4.2, preferably 4.

1.

3. The T-COF according to any of the preceding claims, wherein the BA-Ceo is represented by a formula selected from the following tablepreferably formula IV.

4. An electrochemical sensor for sensing glucose comprising a working electrode, the working electrode comprising the T-COF according to any of claims 1-3.

5. The electrochemical sensor according to claim 4, the working electrode further comprising:• an electrochemical substrate, such as a carbon-based substrate, optionally having micropillars; and• nanoparticles comprising or consisting of a metal, such as Au, Pt, Ag, or Cu.

6. The electrochemical sensor according to any of claims 4-5, wherein the sensor further comprises a counter electrode and, optionally, a reference electrode such as the counter electrode and / or reference electrode comprises a noble metal, such as Au, Pt, or Ag, preferably Au.

7. The electrochemical sensor according to any of claims 4-6, wherein the surface area of the carbon-based substrate is expanded by one or more 3D microstructures, such as micropillars.

8. The electrochemical sensor according to any of claims 4-7, wherein the sensor further comprises one or more means selected from the group consisting of:• means for providing insulation and / or a passivation layer ensuring only active regions, such as the working-, reference, and / or counter electrode are available to a solution applied to the electrochemical sensor• means for providing insulation and / or a passivation layer made from the same material as a pyrolytic carbon electrode, without having been pyrolysed;• means for anti-fouling, such as the electrochemical sensor being coated with a zwitterionic polymer, such as zwitterionic polypyrrole (ZiPPy);• means for reducing diffusion, such as a diffusion-limiting layer, such as a layer comprised of a trimethylolpropane triglycidyl ether (PTGE) and polyethyleneimine (PEI); and• means for antifouling and means for reducing diffusion, combined in a ratio of about 1: 1.

9. The electrochemical sensor according to any of claims 4-8, wherein the electrochemical sensor's ability to sense glucose is maintained for a sustained period of time after manufacturing without affecting the sensor's ability to senseglucose, wherein the sustained period of time is at least 5 days, such as at least 10 days, such as 15 days, such as at least 20 days.

10. The electrochemical sensor according to any of claims 4-9, wherein the electrochemical sensor's ability to sense glucose is maintained after exposure of up to 70 degrees Celcius, such as even after exposure to autoclave conditions, such as overpressure and about 120 degrees Celcius.

11. Use of:• the T-COF according to any of claims 1-3, in an electrochemical sensor for sensing glucose; or• the electrochemical sensor according to any of claims 4-10, in a method for sensing glucose.

12. A method of sensing glucose, preferably in the range from 0.1 mM to 120 mM, the method comprising:

1. Providing an electrochemical sensor as defined in any of claims 4-10 configured to produce an electric signal when a glucose molecule is at, or near, the working electrode;2. Providing a sample suspected of containing glucose;3. Contacting the electrochemical sensor with the sample;4. Measuring the signal from the working electrode, thereby sensing the presence of glucose in the sample;5. Optionally, comparing the signal to a signal from a control electrode on the electrochemical sensor to reduce background signals; and6. Optionally, comparing the signal from the working electrode to a signal obtained from measuring a control sample, to obtain a level of glucose in the sample.

13. A method of producing a T-COF comprising boronic acid-functionalized Ceo (BA-Ceo), the method comprising:

1. combining thieno[3,2-b]thiophene-2,5-diboronic acid (F TTDB) and 2,3,6,7,10,11-hexahydroxytriphenylene (HHTP) with dry dioxane and dry mesitylene;2. reacting the mixture in an autoclave, such as at 150 °C for 72 h;3. washing the product thereof, and drying the product;4. optionally, heating the product under high vacuum, such as at app. 200 °C and IO’6torr (1.33 x 10’4Pa);5. obtaining a T-COF;6. reacting fullerene Ceo, 4-formylphenylboronic acid pinacol ester (FPBA), and sarcosine, such as at 125 °C for 12 h;7. washing the product thereof;8. removing the protective group to obtain BA-Ceo, such as by the use of methylboronic acid dissolved in dichloromethane, followed by the addition of TFA; and9. combining the T-COF with the BA-Ceo, thereby obtaining a T-COF comprising BA-Ceo.

14. A method of producing an electrochemical sensor for sensing glucose, the method comprising:

1. providing a carbon based substrate, such as a pyrolytic carbon electrode; nanoparticles comprising or consisting of a metal, such as Au, Pt, Ag, or Cu; and a T-COF comprising BA-Ceo as defined in any of claims 1- 3, or claim 13;2. depositing the nanoparticles on the carbon based substrate;3. depositing the T-COF comprising BA-Ceo on the nanoparticles;4. optionally, depositing a counter- and / or a reference electrode at suitable positions on the electrochemical sensor;5. optionally, coating with a zwitterionic polymer, such as zwitterionic polypyrrole (ZiPPy); and6. optionally, coating with a diffusion-limiting layer, such as a layer comprised of a trimethylolpropane triglycidyl ether(PTGE): polyethyleneimine (PEI) layer.

15. A kit comprising the electrochemical sensor according to any of claims 4-10, and optionally means for replacing the electrochemical sensor and / or one or more further electrochemical sensors, such as one or more further electrochemical sensors for sensing one or more metabolites other than glucose, optionally the kit may also comprises components for cleaning, such as washing buffers.

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