Non-enzymatic electrochemical sensors for lactate for bioprocess monitoring

Abstract

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

Description

[0001] Non-enzymatic electrochemical sensors for lactate for bioprocess monitoring

[0002] Technical field of the invention

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

[0004] Background of the invention

[0005] The accurate monitoring of lactate levels is crucial in various industries, including bioprocessing and food manufacturing. Nevertheless, traditional lactate sensing methodologies, including enzymatic and optical techniques, grapple with issues related to stability, sensitivity, and selectivity.

[0006] It is important to keep track of key metabolites during the production of bioproducts and biopharmaceuticals to improve the design of the medium and feed and to optimize cell culture conditions for increased productivity. Consistent evaluation and management of culture trajectories during manufacturing processes can lead to prolonged cell life and augmented product output. The monitoring of lactate, also known as lactic acid, is crucial in evaluating the health of cells, enhancing the growth conditions, and comprehending changes in metabolism. Tracking lactate levels allows operators to identify metabolic limitations, substrate restrictions, and other factors that may impact the quality and yield of the final product.

[0007] L-lactate has quickly become a critical component in the food industry, especially in relation to fermentation and dairy processes. It serves as a determinant of a number of characteristics in the fermentation process, including quality, freshness, flavor, stability, storage lifespan, and the level of bacterial contamination. Additionally, L-lactate is used as a pickling and preserving agent. In the context of lactic acid fermentation, it helps inhibit the growth of unwanted bacteria and mould. The addition of lactic acid to the packaging of poultry and fish products can extend their shelf life. In certain cases, the fermentation of lactic acid or L-lactate can indicate the spoilage of food items. High levels of lactic acid can negatively affect the quality of wine as it can cause spoilage due to the generation of various compounds such as diacetyl and acetate. A similar pattern is noticed in juice and eggs, where an increased concentration of L-lactate can indicate spoilage, leading to changes in the taste, smell, and other sensory properties.

[0008] Lactate is a metabolic by-product of mammalian cell culture that can affect the growth and productivity of cells. Lactate accumulation can result from high glucose consumption, low oxygen availability, or inefficient mitochondrial function. High levels of lactate indicate that cells are under stress or hypoxic conditions, which can affect their viability and productivity. Therefore, measuring lactate concentration in cell culture media can provide valuable information for optimizing cell culture conditions and improving bioprocess performance.

[0009] The methods currently employed for the determination of lactate, such as high- performance liquid chromatography (HPLC), colorimetry, spectrophotometry, gas chromatography, fluorometry, chemiluminescence, and nuclear magnetic resonance (NMR), although precise and reliable, are marred by several drawbacks. These techniques tend to be laborious, requiring a significant investment of time and effort. Moreover, they are quite expensive, necessitating substantial financial resources. Further, the need for specialized laboratory equipment and trained personnel often makes it impossible to conduct these tests on-site, in real-time. This situation presents a challenge, especially for the rapidly advancing field of bioprocessing where prompt, efficient, and cost-effective testing mechanisms are crucial. The introduction of a point-of-need electrochemical measurement method could offer a significant advantage in this regard. Replacing the traditional techniques with this innovative approach might streamline lactate determination, making it quicker, less costly, and more accessible, enabling onsite analysis without the need for extensive resources or specialized facilities. Enzyme-based biosensors, particularly those that employ lactate dehydrogenase or lactate oxidase, have seen extensive development in recent years. These enzymes specifically recognize L-lactate molecules, providing the basis for the selectivity of the biosensors. The variety of output signals that these biosensors generate spans a wide range, encompassing spectroscopic, fluorescent, chemiluminescent, and amperometric detection methods. Of these, amperometric detection has been the most widely adopted and used in Bioprofile analyzers. Enzyme-based biosensors, despite their potential, carry certain drawbacks. Their operational efficiency can be influenced by environmental elements such as variations in temperature and pH. Furthermore, the inherent tendency of enzymes to degrade over time can curtail the longevity of these sensors. An additional limitation is their selectivity for L-lactate, which could lead to an underestimation of total lactate content when the sample also contains D-lactate. This selectivity diminishes their effectiveness in accurately measuring lactate, a requirement crucial in industries like food manufacturing.

[0010] In light of these challenges, there is an increasing focus on the research and development of non-enzymatic-based sensors. These advanced sensors mitigate the challenges encountered with traditional enzyme-based biosensors and provide a more accurate estimation of lactate content in various samples.

[0011] Triformylphloroglucinol-functionalized bipyridine-based covalent organic frameworks also known as TpBpy-COF have previously been produced and disclosed in the literature. "TpBpy-COF" was first introduced by Shinde and colleagues (J. Mater. Chem. A, 2016,4, 2682-2690) and has been reviewed later on, see for instance Liang et al. 2022 (Chem Catalysis, Volume 2, Issue 9, 2022, Pages 2157-2228). None of the prior art disclosures, however, disclose such a COF comprising boronic acid-functionalized Ceo nor a use of such molecules in sensing lactic acid.

[0012] Hence, improved electrochemical sensors for sensing lactate are sought for, and in particular more selective and reliable electrochemical sensors with high sensitivity and stability would be advantageous.

[0013] Summary of the invention

[0014] Thus, an object of the present invention relates to providing new materials with a high specificity and sensitivity towards lactate. 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. A novel lactate sensor is thus provided, which is non-enzymatic in nature, utilizing a TpBpy-COF comprising BA-Ceo. This avant-garde sensor has displayed superior electrochemical performance, eclipsing other modified electrodes due to its expansive electrochemical active surface and impressive conductivity. The effectiveness of this non-enzymatic sensor, built on the TpBpy-COF, is particularly noticeable in lactate detection. It exhibits exceptional electrooxidation detection capabilities, essential for effective lactate monitoring. The improved electrocatalytic activity is ascribed to multiple contributing factors. These include efficient electrolyte diffusion, selective lactate interaction with BA-Ceo, accelerated charge transfer, the remarkable electroconductivity, and the efficient employment of active sites engendered by the unique morphology and synergistic relationship between the COF and BA-Ceo in the structured assembly.

[0015] The sensor maintains consistent responses across eight consecutive measurement cycles, requiring no cleaning interventions. It sustains stable sensitivity for 25 days under normal conditions. As a comparison to the conventional HPLC analysis, our sensor offers reliable lactate quantification in a significantly more productive and time-efficient manner. It boasts remarkable stability when interacting directly with the fermentation medium, and it accurately quantifies lactate amidst several electroactive species.

[0016] The TpBpy-COF's and the electrochemical sensor's functioning and production are in particular demonstrated by the examples:

[0017] Examples 1-4 show how to manufacture, characterize and test the performance of the TpBpy-COF and electrochemical sensor of the present invention.

[0018] Example 5 shows a structural characterization of the TpBpy-COF and the electrochemical sensor.

[0019] Example 6 shows an electrochemical evaluation, and application of different nanoparticles.

[0020] Example 7 shows an example of the sensing performance of the electrochemical sensor, and that despite the presence of interfering compounds such as glucose, ethanol, pyruvate and NaCI, as well as common organic acids, the sensor is capable of measuring actual bioprocess lactate concentrations, measuring both D- lactate and L-lactate, and with a high selectivity for lactate detection. This shows that the PtNi@BA-C6o@TpBpy-COF based sensor exhibits a remarkable selectivity for lactate detection due to the selective oxidation of lactate, even in the presence of interfering substances that are typically present in bioprocesses. Example 8 shows the stability, repeatability, and reproducibility of the TpBpy-COF and electrochemical sensor. The sensor demonstrates consistent response towards lactate detection. After 25 days, the recorded sensitivity was 95.3% of the initial sensitivity, showing a sustained ability to measure lactate after production.

[0021] Example 9 demonstrates how the TpBpy-COF and electrochemical sensor can be applied at-line during a fermentation process.

[0022] Example 10 shows electrodeposition of metal and metal oxide nanoparticles on the 3D pyrolytic carbon electrode.

[0023] Example 11 investigates the usefulness of different nanoparticles and finds the PtNi nanoparticles to have a size of about 138 nm.

[0024] Thus, an aspect of the present disclosure relates to a triphenylamine- functionalized bipyridine covalent organic framework (TpBpy-COF) comprising boronic acid-functionalized Ceo (BA-Ceo), the TpBpy-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).

[0025] An even more preferred aspect of the present disclosure relates to a triformylphloroglucinol-functionalized bipyridine covalent organic framework (TpBpy-COF), comprising boronic acid-functionalized Ceo (BA-Ceo), the TpBpy-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).

[0026] Another aspect of the present disclosure relates to a use of the TpBpy-COF as described herein, in an electrochemical sensor for sensing lactate.

[0027] Another aspect of the present disclosure relates to an electrochemical sensor for sensing lactate comprising a working electrode, the working electrode comprising the TpBpy-COF as described herein.

[0028] Another aspect of the present disclosure relates to a use of the electrochemical sensor as described herein, in a method for sensing lactate.

[0029] Another aspect of the present disclosure relates to a method of sensing lactate, the method comprising:

[0030] 1. Providing an electrochemical sensor as described herein configured to produce an electric signal when a lactate molecule is at, or near, the working electrode;

[0031] 2. Providing a sample suspected of containing lactate;

[0032] 3. Contacting the electrochemical sensor with the sample;

[0033] 4. Measuring the signal from the working electrode, thereby sensing the presence of lactate in the sample;

[0034] 5. Optionally, comparing the signal to a signal from a control electrode on the electrochemical sensor to reduce background signals; and 6. Optionally, comparing the signal from the working electrode to a signal obtained from measuring a control sample, to obtain a level of lactate in the sample.

[0035] Another aspect of the present disclosure 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 lactate.

[0036] Another aspect of the present disclosure relates to a method of producing a TpBpy-COF comprising boronic acid-functionalized Ceo (BA-Ceo), the method comprising:

[0037] 1. combining triformylphloroglucinol (Tp), and 5,5'-diamino-2,2'-bipyridine (Bpy) such as with dry dioxane, dry mesitylene and aqueous acetic acid, such as in a Pyrex tube;

[0038] 2. optionally, sonicating the mixture and subjecting the pyrex tube to a step of lowering the internal pressure before sealing;

[0039] 3. reacting the mixture, such as at 120 °C for 5 days;

[0040] 4. washing the product thereof, and drying the product;

[0041] 6. optionally, soaking the product in anhydrous tetra hydrofuran (THF);

[0042] 7. optionally, heating the product under vacuum, such as at 60 °C;

[0043] 8. obtaining a TpBpy-COF;

[0044] 9. reacting paraformaldehyde, and (4-aminobutan-2-yl)boronic acid pinacol ester, such as reacting in dichlorobenzene by sonication at 180 °C under ambient air for 5 hours;

[0045] 10. washing the product thereof;

[0046] 11. removing the protective group to obtain BA-Ceo, such as by the use of methylboronic acid dissolved in CH2CI2, followed by the addition of trifluoroacetic acid (TFA); and

[0047] 12. combining the TpBpy-COF with the BA-Ceo, thereby obtaining a TpBpy- COF comprising BA-Ceo.

[0048] Another aspect of the present disclosure relates to a method of producing an electrochemical sensor for sensing lactate, the method comprising: 1. providing a carbon based substrate, such as a pyrolytic carbon electrode; nanoparticles comprising or consisting of a metal or a metal oxide, such as PtNi, Pt, Ni, CuO, or NiO; and a TpBpy-COF comprising BA- Ceo as as described herein;

[0049] 2. depositing the nanoparticles on the carbon based substrate;

[0050] 3. depositing the TpBpy-COF comprising BA-Ceo on the nanoparticles;

[0051] 4. optionally, depositing a counter- and / or a reference electrode at suitable positions on the electrochemical sensor;

[0052] 5. optionally, coating with a zwitterionic polymer, such as zwitterionic polypyrrole (ZiPPy); and

[0053] 6. optionally, coating with a diffusion-limiting layer, such as a layer comprised of a trimethylolpropane triglycidyl ether

[0054] (PTGE): polyethyleneimine (PEI) layer.

[0055] Another aspect of the present disclosure 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 lactate, optionally the kit may also comprise components for cleaning, such as washing buffers.

[0056] Another aspect of the present disclosure relates to a method of producing a TpBpy-COF comprising boronic acid-functionalized Ceo (BA-Ceo), the method comprising:

[0057] 1. combining triformylphloroglucinol (Tp), and 5,5'-diamino-2,2'-bipyridine (Bpy) to obtain a TpBpy-COF;

[0058] 2. combining Ceo, paraformaldehyde, and (4-aminobutan-2-yl)boronic acid pinacol ester, and removing the protective group to obtain BA-Ceo; and

[0059] 3. combining the TpBpy-COF with the BA-Ceo, thereby obtaining a TpBpy- COF comprising BA-Ceo.

[0060] The present invention relates to covalent organic frameworks (COF) and application of such COFs in electrochemical sensors for sensing lactate. In particular, the present invention relates to a triformylphloroglucinol-functionalized bipyridine-based covalent organic framework (TpBpy-COF) comprising boronic acid-functionalized Ceo (BA-Ceo) and the use of such TpBpy-COFs and electrochemical sensors in methods of sensing lactate such as in bioprocesses.

[0061] Likewise, the invention can be described as the present invention relates to triphenylamine-functionalized bipyridine-based covalent organic frameworks (TpBpy-COF) and application of such TpBpy-COFs in electrochemical sensors for sensing lactate. In particular, the present invention relates to a TpBpy-COF comprising boronic acid-functionalized Ceo (BA-Ceo) and the use of such TpBpy- COFs and electrochemical sensors in methods of sensing lactate such as in bioprocesses. The present invention relates to covalent organic frameworks (COF) and application of such COFs in electrochemical sensors for sensing lactate. In particular, the present invention relates to a triphenylamine-functionalized bipyridine covalent organic framework (TpBpy-COF) comprising boronic acid- functionalized Ceo (BA-Ceo) and the use of such TpBpy-COFs and electrochemical sensors in methods of sensing lactate such as in bioprocesses.

[0062] Brief description of the figures

[0063] Figure 1. Building blocks used for COF synthesis, classified by their connectivity leading to 2D and 3D COFs.

[0064] Figure 2. Schematic representation of the process flow for the fabrication of the 3D pyrolytic carbon microelectrodes

[0065] Figure 3. Synthetic pathway of BA-Ceo (boronic acid-functionalized Ceo). (insets: the photographs of Ceo and BA-Ceo in toluene).

[0066] Figure 4. Schematic representation of the synthesis process for TpBpy-COF and its structure.

[0067] Figure 5. The schematic diagrams of (a) the BA-Ceo encapsulation process by TpBpy-COF. (insets: the photographs of TpBpy-COF and BA-Ceo@TpBpy-COF powders), and (b) modification of 3D WE by electrodeposition of PtNi NPs and then electrodeposition of the BA-Ceo@TpBpy-COF nanostructure on the PtNi NPs modified electrode. Figure 6. CV profile for electrodeposition of PtNi alloy NPs on 3D pyrolytic electrode

[0068] Figure 7. (a) UV-vis spectra of pristine Ceo and BA-Ceo in toluene; (b) FT-IR spectra of (4-aminobutan-2-yl)boronic acid, pristine Ceo and BA-Ceo; (c) FT-IR spectra of Tp, Bpy, and TpBpy-COF; (d) PXRD patterns of TpBpy-COF and BA- Ceo@TpBpy-COF.

[0069] Figure 8. (a) N2 adsorption-desorption isotherms, (b) pore size distribution, (c) isotherm of TGA under air of TpBpy-COF and BA-Ceo@TpBpy-COF and (d) PXRD patterns of BA-Ceo@TpBpy-COF sample dispersed in boiling water for 0 and 12 hours.

[0070] Figure 9. SEM images of (a) TpBpy-COF and (b) BA-Ceo@TpBpy-COF with (i) low and (ii) high magnifications

[0071] Figure 10. SEM images of (a) bare carbon micropillars, (b and c) after electrodeposition of PtNi NPs with low and high magnifications, (c, inset: Pt, Ni element mapping), (d) XRD pattern of PtNi NPs modified electrode, (e) top-view of the electrodeposited BA-Ceo@TpBpy-COF film and (f) coated with PTGE: PEI thin film and ZiPPy polymer.

[0072] Figure 11. (a) Full survey XPS and high-resolution XPS spectra of (b) C Is, (c) O Is, (d) N Is, (e) B Is, (f) Pt 4f, and (g) Ni 2p of TpBpy-COF, BA-Ceo@TpBpy-COF, and PtNi@BA-Ceo@TpBpy-COF modified 3D pyrolytic carbon WE. (h) high- resolution XPS spectra of Pt 4f of Pt NPs and PtNi alloy NPs modified 3D pyrolytic carbon electrode.

[0073] Figure 12. Electrochemical behavior: (a) Cyclic voltammetry (CV) experiments at PtNi@BA-Ceo@TpBpy-COF, PtNi@BA-Ceo, PtNi@TpBpy-COF modified 3D pyrolytic carbon electrode, and bare electrode in 0.1 M PBS (pH 7.0) at 50 mV s-1scan rate without lactate; (b) the CV profile of PtNi NPs modified 3D pyrolytic carbon electrode without and with 50 mM lactate; (c) the CV profiles of PtNi@BA- Ceo@TpBpy-COF, PtNi@BA-Ceo, and PtNi@TpBpy-COF modified 3D pyrolytic carbon electrode in 0.1 M PBS at 50 mV s-1scan rate in the presence of 5 mM lactate; (d) the scan rate effect (20-120 mV s-1) on the oxidation properties of 5.0 mM lactate in PBS; (e) anodic peak current (lap) vs. u and u1 / 2(inset) and (f) anodic peak potential vs. In(u) plots.

[0074] Figure 13. CV profile for PtNi@BA-Ceo@TpBpy-COF in different lactate concentrations.

[0075] Figure 14. Schematic diagrams of (a) the fabricated of PtNi@BA-Ceo@TpBpy-COF based sensor and electrochemical performance towards lactate detection and (b) the lactate interaction and oxidation reaction on the surface of the PtNi@BA- Ceo@TpBpy-COF modified electrode.

[0076] Figure 15. (a) The cyclic voltammograms of 1 mM K3Fe(CN)e for the PtNi@BA- Ceo@TpBpy-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 lactate with increasing value of pH by PtNi@BA-Ceo@TpBpy-COF electrode in 0.1 M PBS with a lactate concentration of 5 mM; (c) Amperometric response of PtNi@BA-C6o@ TpBpy-COF electrodes toward successive lactate addition for different applied potentials; (d) Chronoamperometric response of PtNi@BA- Ceo@TpBpy-COF modified electrode coated with PTGE: PEI layer and without this layer towards lactate; (e) Amperometric stepwise response of PtNi@BA- Ceo@TpBpy-COF electrode for successive addition of lactate to PBS buffer solution by applying +0.8 V potential and (f) change in current with lactate concentration (calibration curve).

[0077] Figure 16. Comparison of electrochemical responses for PtNi@BA-Ceo@TpBpy- COF, PtNi@BA-Ceo, and PtNi@TpBpy-COF.

[0078] Figure 17. Selectivity comparison of the PtNi@BA-Ceo@TpBpy-COF based sensor for the measurement of L-lactate, D-lactate., and a mixture of both isomers.

[0079] Figure 18. Lactate detection performance of PtNi@BA-Ceo@TpBpy-COF electrode.

[0080] (a) Selective response of the sensor towards lactate in the presence of different interfering agents (inset: comparison of the current with 5 mM of lactate and 40 mM of interferences; (b) Effect of interferences on the current response of the proposed electrochemical sensor; lactate: interference at 1:0, 1: 10 and 1: 100 (n = 3); (c) Response reproducibility of the sensor towards a single concentration of lactate, 5 mM; (d) chronoamperogram of the sensor for eight runs.

[0081] Figure 19. Monitoring of lactate at-line for seven hours of C. glutamicum 2262 cultivation in CGXII medium using the PtNi@BA-C6o@TpBpy-COF-based sensor and its comparison with off-line HPLC lactate measurements. The cultivation chart also shows the growth of the bacteria as dry cell weight concentrations and glucose concentration.

[0082] Figure 20. Lactate concentration measured at-line with and without cells by means of the PtNi@BA-Ceo@TpBpy-COF-based sensor coated with ZiPPy and compared with the results from biosensors (BioProfile FLEX2)

[0083] Figure 21. (a) Effect of different metal and metal oxide nanoparticles on electrochemical signal and (b) response reproducibility of modified electrodes (PtNi@BA-Ceo@T pBpy-COF, Pt@BA-Ceo@T pBpy-COF, Ni@BA-Ceo@T pBpy-COF, NiO@BA-Ceo@TpBpy-COF, and CuO@BA-Ceo@TpBpy-COF) for detection of 10 mM lactate in 0.1 M PBS solution.

[0084] Figure 22. SEM micrograph and size distribution histograms of PtNi nanoparticles obtained from the SEM image.

[0085] The present invention will now be described in more detail in the following.

[0086] Detailed description of the invention

[0087] Definitions

[0088] Prior to discussing the present invention in further details, the following terms and conventions will first be defined:

[0089] 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. In the context of the present disclosure "triphenylamine-functionalized bipyridine based COF", or more preferably "triformylphloroglucinol-functionalized bipyridine- based COF", "TpBpy-COF", or "COF" are used interchangeably to describe COFs of the invention. In preferred embodiments "Triformylphloroglucinol-functionalized bipyridine-based COF", "TpBpy-COF", or "COF" are used interchangeably to describe COFs of the invention, the skilled person will know that such abbreviations have previously been used to describe such building blocks used in the invention. As obvious from example 2, and formula II-III, the COF is made with the starting materials triformylphloroglucinol (Tp), and 5,5'-diamino-2,2'- bipyridine (Bpy) and thus the abbreviation TpBpy. 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.

[0090] 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. Electrochemical substrates include metals or metal oxides, such as PtNi, Pt, Ni, CuO, or NiO, or carbon-based electrodes like graphite or glassy carbon and combinations thereof.

[0091] Covalent organic framework comprising boronic acid-functionalized Ceo

[0092] 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.

[0093] 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.

[0094] Enhancing the selectivity of lactate detection is essential for achieving precise and dependable lactate sensing. In the present disclosure this is done by incorporating functional materials with lactate binding capabilities, such as synthetic boronic acid and its derivatives. Boronic acid groups can serve as effective tools in lactate detection due to their unique interaction with lactate's hydroxyl compounds. This interaction forms a boron-diol bond that alters the electronic properties of the boronic acid unit, influencing its fluorescence or electrochemical characteristics. It is noteworthy that the term "boronic acid" refers to a broad class of organic compounds, rather than referring to a singular material. Boronic acids generally have the formula RB(OH)2, where R is an organic group (such as an alkyl or aryl group). This means there is an incredibly wide variety of possible boronic acids, all with different properties based on the specific R group present in their structure. For example, certain boronic acid-based fluorescent sensors, such as BODIPY boronic acid sensors, can detect lactate by significantly increasing their emission and absorption upon binding with lactate. Concurrently, electrochemical polymeric sensors can assess lactate concentrations by monitoring changes in the conductivity or impedance of the boronic acid-modified polymer. Additionally, integrating boronic acid groups into peptides or polymers can enhance their selectivity and sensitivity for lactate detection.

[0095] 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 lactate. 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. With its high electronic conductivity, biocompatibility, and stable structure, Fullerene Ceo is a suitable candidate for increasing the capture efficiency of lactate, resulting in more reliable and reproducible electrocatalytic responses.

[0096] Furthermore, the electrochemical properties for lactate determination can be amplified by encapsulating boronic acid functionalized Ceo (BA-Ceo) within a COF. The encapsulation within the COF has the potential not only to bolster the stability of the BA-Ceo complex but also to amplify its interaction with lactate. This could lead to a more reliable and reproducible electrocatalytic response, thus improving the precision of lactate detection.

[0097] Thus, in a first aspect, the invention relates to a triphenylamine-functionalized bipyridine covalent organic framework (TpBpy-COF) comprising boronic acid- functionalized Ceo (BA-Ceo), the TpBpy-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).

[0098] As obvious from example 2, the COF is made with the starting materials triformylphloroglucinol (Tp), and 5,5'-diamino-2,2'-bipyridine (Bpy) and thus the abbreviation TpBpy. Thus, in a preferred aspect, the invention relates to a triformylphloroglucinol-functionalized bipyridine covalent organic framework (TpBpy-COF) comprising boronic acid-functionalized Ceo (BA-Ceo), the TpBpy-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

[0099] (formula III). In a similar preferred aspect, the invention relates to a TpBpy-COF comprising boronic acid-functionalized Ceo (BA-Ceo), the TpBpy-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

[0100] (formula III). 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 lactate, 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 7.9-8.1, preferably 8.0. As shown in the examples, BA- Ceo@TpBpy-COF coated on the surface of PtNi NPs modified 3D pyrolytic electrode has a pKavalue of 8.0. The pKaof BA-Ceo@TpBpy-COF on the electrode was measured with the electrochemical method. Therefore, in some embodiments, the pKavalue is determined at a later stage.

[0101] In the examples of the present invention is provided a specific example of a boronic acid. However, the skilled person will also be able to select other suitable boronic acids, that can be applied in the TpBpy-COF.

[0102] In some embodiments of the present disclosure, the BA-Ceo is represented by the formula IV (formula IV).

[0103] In some embodiments of the present disclosure, the BA-Ceo is represented by a formula selected from the following table

[0104]

[0105]

[0106]

[0107] In some embodiments of the present disclosure, the BA-Ceo is represented by a formula selected from the following table

[0108]

[0109]

[0110] Table 2

[0111] The boronic acid is covalently bonded to the fullerene Ceo molecule, preferably in a 1: 1 ratio. The interaction between the boronic acid-functionalized fullerene Ceo (BA-Ceo) and the TpBpy-COF is defined as host-guest interactions, involving an array of intermolecular forces (van der Waals and electrostatic interactions). The pores of the TpBpy-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@TpBpy-COF composite. In some embodiments of the present disclosure, the BA-Ceo is non-covalently bonded to the TpBpy-COF, such as via intermolecular forces, such as van der Waals forces, and electrostatic interactions, such as n-n stacking interaction.

[0112] As introduced, an important use of the TpBpy-COF is the application in sensing of lactate. Thus, in another aspect of the present disclosure relates to a use of the TpBpy-COF as described herein, in an electrochemical sensor for sensing lactate.

[0113] Electrochemical sensor

[0114] As introduced above, the TpBpy-COF provided can be used in different electrochemical sensors, wherein the specificity or sensitivity can be tweaked further if need be.

[0115] In another aspect of the present disclosure relates to an electrochemical sensor for sensing lactate comprising a working electrode, the working electrode comprising the TpBpy-COF as described herein. The lactate may be both D-lactate and L-lactate, since the sensor is capable of detecting both types of molecules.

[0116] In some embodiments of the present disclosure, the working electrode further comprising:

[0117] • an electrochemical substrate, such as a carbon-based substrate, optionally having micropillars; and

[0118] • nanoparticles comprising or consisting of a metal or a metal oxide, such as PtNi, Pt, Ni, CuO, or NiO.

[0119] In some embodiments of the present disclosure, nanoparticles comprising or consisting of a metal or a metal oxide, such as PtNi, Pt, Ni, CuO, or NiO are deposited on the electrochemical substrate.

[0120] In some embodiments of the present disclosure, the TpBpy-COF is deposited on the nanoparticles.

[0121] In some embodiments of the present disclosure, the nanoparticles have a diameter of between about 100 to about 180 nm, such as between about 120 to about 160 nm, preferably about 140 nm, such as about 138 nm. In some embodiments of the present disclosure, the sensor further comprises a counter electrode and, optionally, a reference electrode.

[0122] 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 metal is Au.

[0123] The purpose of counter or auxiliary electrodes 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 metal. 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.

[0124] 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, however data not presented herein shows that similar COFs can be produce signal with and without 3D surfaces.

[0125] 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.

[0126] 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.

[0127] In some embodiments of the present disclosure, the pyrolytic carbon electrode is made from pyrolysing a negative photoresist, such as epoxy photoresist SU-8.

[0128] In some embodiments of the present disclosure, the pyrolytic carbon electrode is exposed to UV light before pyrolysation.

[0129] 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.

[0130] 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.

[0131] 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 design the chip with the 3D structures having different shapes, and sizes.

[0132] In some embodiments of the present disclosure, the 3D microstructures are micropillars with a height of between 100 pm to 300 pm and a diameter of 5 to 200 pm.

[0133] 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. In some embodiments of the present disclosure, the micropillars are pillars with a height of approximately 180 pm to 200 pm and a diameter of 100 pm.

[0134] 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.

[0135] 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.

[0136] In some embodiments of the present disclosure, the ability of the sensing layer to detect lactate is maintained after exposure to autoclave conditions, such as overpressure and about 120 degrees Celsius.

[0137] 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 accuracy and reliability of the sensors over the long-term. 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 polymers, 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.

[0138] 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.

[0139] Thus, the skilled person will be able to select many different coatings to enhance the longevity of the sensor.

[0140] 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.

[0141] In some embodiments of the present disclosure, the sensor further comprises means for anti-fouling, such as the electrochemical sensor being coated with a zwitterionic polymer, such as zwitterionic polypyrrole (ZiPPy).

[0142] 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).

[0143] 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. 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 lactate is maintained for a sustained period of time after manufacturing without affecting the sensor's ability to sense lactate, 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.

[0144] 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 lactate 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 lactate even after being exposed to temperatures of up to 70 degrees Celsius.

[0145] In some embodiments of the present disclosure, the sensor's ability to sense lactate is defined as an ability to continuously measure the lactate content of a control sample, such as running amperometric tests at lactate concentrations ranging from 0.01-90 mM, preferably 5 mM, and maintaining sensitivity of at least about 90% such as even about 95%.

[0146] 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 lactate is successively increased by adding lactate directly to the solution.

[0147] In another aspect the disclosure relates to a use of the electrochemical sensor as described herein, in a method for sensing lactate. Methods for sensing lactate

[0148] Having introduced the TpBpy-COF and the application of the TpBpy-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 TpBpy-COF and electrochemical sensor, can be further applied in the below described aspects. Thus, in another aspect of the present disclosure relates to a method of sensing lactate, the method comprising:

[0149] 1. Providing an electrochemical sensor as described herein configured to produce an electric signal when a lactate molecule is at, or near, the working electrode;

[0150] 2. Providing a sample suspected of containing lactate;

[0151] 3. Contacting the electrochemical sensor with the sample;

[0152] 4. Measuring the signal from the working electrode, thereby sensing the presence of lactate in the sample;

[0153] 5. Optionally, comparing the signal to a signal from a control electrode on the electrochemical sensor to reduce background signals; and

[0154] 6. Optionally, comparing the signal from the working electrode to a signal obtained from measuring a control sample, to obtain a level of lactate in the sample.

[0155] In some embodiments of the present disclosure, the method senses lactate in the range from 0.01-90 mM lactate.

[0156] In another aspect, the disclosure 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 lactate.

[0157] 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 boronic 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. Thus, in an aspect is provided a method of producing a TpBpy-COF comprising boronic acid-functionalized Ceo (BA-Ceo), the method comprising:

[0158] 1. combining triformylphloroglucinol (Tp), and 5,5'-diamino-2,2'-bipyridine (Bpy) to obtain a TpBpy-COF;

[0159] 2. combining Ceo with paraformaldehyde, and a suitable pinacol ester derivative, such as a (aminoalkyl) boronic acid pinacol ester, and removing the protective group to obtain BA-Ceo; and

[0160] 3. combining the TpBpy-COF with the BA-Ceo, thereby obtaining a TpBpy- COF comprising BA-Ceo.

[0161] An aspect of the present disclosure relates to a method of producing a TpBpy-COF comprising boronic acid-functionalized C60 (BA-Ceo), the method comprising:

[0162] 1. combining triformylphloroglucinol (Tp), and 5,5'-diamino-2,2'-bipyridine (Bpy) to obtain a TpBpy-COF;

[0163] 2. combining Ceo, paraformaldehyde, and (4-aminobutan-2-yl)boronic acid pinacol ester, and removing the protective group to obtain BA-Ceo; and

[0164] 3. combining the TpBpy-COF with the BA-Ceo, thereby obtaining a TpBpy- COF comprising BA-Ceo.

[0165] In another aspect of the present disclosure relates to a method of producing a TpBpy-COF comprising boronic acid-functionalized Ceo (BA-Ceo), the method comprising:

[0166] 1. combining triformylphloroglucinol (Tp), and 5,5'-diamino-2,2'-bipyridine (Bpy) such as with dry dioxane, dry mesitylene and aqueous acetic acid, such as in a Pyrex tube;

[0167] 2. optionally, sonicating the mixture and subjecting the pyrex tube to a step of lowering the internal pressure before sealing;

[0168] 3. reacting the mixture, such as at 120 °C for 5 days;

[0169] 4. washing the product thereof, and drying the product;

[0170] 6. optionally, soaking the product in anhydrous THF;

[0171] 7. optionally, heating the product under vacuum, such as at 60 °C;

[0172] 8. obtaining a TpBpy-COF;

[0173] 9. reacting paraformaldehyde, and suitable pinacol ester derivative, such as a (aminoalkyl) boronic acid pinacol ester, such as (4-aminobutan-2- yl)boronic acid pinacol ester, such as reacting in dichlorobenzene by sonication at 180 °C under ambient air for 5 hours;

[0174] 10. washing the product thereof;

[0175] 11. removing the protective group to obtain BA-Ceo, such as by the use of methylboronic acid dissolved in CH2CI2, followed by the addition of TFA; and

[0176] 12. combining the TpBpy-COF with the BA-Ceo, thereby obtaining a TpBpy-

[0177] COF comprising BA-Ceo.

[0178]

[0179]

[0180] Table 3

[0181] In a more specific aspect, a method is provided of specifically generating the TpBpy-COF comprising the BA-Ceo of formula IV. Thus, in another aspect, the present disclosure relates to a method of producing a TpBpy-COF comprising boronic acid-functionalized Ceo (BA-Ceo), the method comprising: 1. combining triformylphloroglucinol (Tp), and 5,5'-diamino-2,2'-bipyridine (Bpy) such as with dry dioxane, dry mesitylene and aqueous acetic acid, such as in a Pyrex tube;

[0182] 2. optionally, sonicating the mixture and subjecting the pyrex tube to a step of lowering the internal pressure before sealing;

[0183] 3. reacting the mixture, such as at 120 °C for 5 days;

[0184] 4. washing the product thereof, and drying the product;

[0185] 6. optionally, soaking the product in anhydrous THF;

[0186] 7. optionally, heating the product under vacuum, such as at 60 °C;

[0187] 8. obtaining a TpBpy-COF;

[0188] 9. reacting paraformaldehyde, and (4-aminobutan-2-yl)boronic acid pinacol ester, such as reacting in dichlorobenzene by sonication at 180 °C under ambient air for 5 hours;

[0189] 10. washing the product thereof;

[0190] 11. removing the protective group to obtain BA-Ceo, such as by the use of methylboronic acid dissolved in CH2CI2, followed by the addition of TFA; and

[0191] 12. combining the TpBpy-COF with the BA-Ceo, thereby obtaining a TpBpy- COF comprising BA-Ceo.

[0192] An aspect of the present disclosure relates to a method of producing an electrochemical sensor for sensing lactate, the method comprising:

[0193] 1. providing a carbon based substrate, such as a pyrolytic carbon electrode; nanoparticles comprising or consisting of a metal or a metal oxide, such as PtNi, Pt, Ni, CuO, or NiO; and a TpBpy-COF comprising BA- Ceo as described herein;

[0194] 2. depositing the nanoparticles on the carbon based substrate;

[0195] 3. depositing the TpBpy-COF comprising BA-Ceo on the nanoparticles;

[0196] 4. optionally, depositing a counter- and / or a reference electrode at suitable positions on the electrochemical sensor;

[0197] 5. optionally, coating with a zwitterionic polymer, such as zwitterionic polypyrrole (ZiPPy); and

[0198] 6. optionally, coating with a diffusion-limiting layer, such as a layer comprised of a trimethylolpropane triglycidyl ether

[0199] (PTGE): polyethyleneimine (PEI) layer. The skilled person will know that the sequence of the different steps may vary, depending on the specific chip and its manufacturing.

[0200] In some embodiments of the present disclosure, the counter electrode and / or reference electrode comprises a noble metal, such as Au, Pt, or Ag.

[0201] In preferred embodiments of the present disclosure, the metal is Au. In some embodiments, the counter electrode is made from an inert metal. 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.

[0202] 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.

[0203] Kit

[0204] An aspect of the present disclosure 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 lactate, optionally the kit may also comprise components for cleaning, such as washing buffers.

[0205] 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.

[0206] All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety. The invention will now be described in further details in the following non-limiting examples.

[0207] References

[0208] Digambar Balaji Shinde, Harshitha Barike Aiyappa, Mohitosh Bhadra, Bishnu P. Biswal, Pritish Wadge, Sharath Kandambeth, Bikash Garai, Tanay Kundu, Sreekumar Kurungot and Rahul Banerjee, J. Mater. Chem. A, 2016,4, 2682-2690

[0209] Zizhan Liang, Rongchen Shen, Yun Hau Ng, Yang Fu, Tianyi Ma, Peng Zhang, Youji Li, Xin Li, Chem Catalysis, Volume 2, Issue 9, 2022, Pages 2157-2228.

[0210] Examples

[0211] Example 1 - Materials and methods

[0212] Materials

[0213] Starting materials were procured from Sigma-Aldrich, TCI or VWR and utilized without additional purification. Mesitylene, triformylphloroglucinol (Tp), and 1,4- dioxane were obtained from TCI. N,N-dimethylformamide, hexane, ethyl acetate, and silica-gel were sourced from VWR. Fullerene Ceo, NiC , K2[PtCk], 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, o-dichlorobenzene (ODCB), pyrrole, acetonitrile (ACN), luteolin, hexane, 5,5'-diamino-2,2'-bipyridine (Bpy), ethyl acetate (EtOAc), trifluoroacetic acid (TFA), pinacol, and dichloromethane were obtained from Sigma-Aldrich. Sarcosine and d-xylose were procured from VWR. (4-aminobutan-2-yl)boronic acid was obtained from Chemspace. 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.

[0214] Surface characterization

[0215] 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.

[0216] Electrochemical studies

[0217] 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 150 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. Cyclic voltammograms were obtained within a potential range of -0.2 to +1.0 V (vs. Ag / AgCI) using 10 mM PBS with a pH of 7.4 as the background electrolyte. When required, lactate solution was added to the electrolyte from a 0.1 M stock. Chronoamperometric profiles were documented at applied potentials from +0.4 to +0.85 V, with the stepwise addition of specific quantities of lactate stock solution to 10 ml_ of PBS electrolyte, while being continuously magnetically stirred under ambient conditions. The optimal potential of +0.8 V was employed for all other measurements. The recovery time, response reproducibility, and reusability of the modified PtNi@BA-Ceo@TpBpy-COF electrodes were examined using chronoamperometric tests at +0.8 V.

[0218] Briefly, The reproducibility test, conducted by running the PtNi@BA-Ceo@TpBpy- COF based sensor eight times individually. To verify the long-term stability and reusability of the modified electrodes, their stepwise sensitivity to lactate was assessed over a 25-day period and also continuously monitored lactate concentration at 5 mM for 130 minutes. 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 PtNi@BA-Ceo@ TpBpy-COF electrode was recorded for various interfering agents. The experiment involved adding lactate to the PBS background solution, followed by the stepwise addition of potential interfering substances such as glucose, acetate, ethanol, pyruvate, and NaCI, all at equal concentrations of 40 mM. The study concluded with the subsequent addition of lactate 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.

[0219] In order to examine the apparent acid dissociation constant (pKa) of the boronic acid group on the BA-Ceo@TpBpy-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.

[0220] Example 2 - Generation of components

[0221] Pyrolytic Carbon Electrode Fabrication 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.

[0222] The fabrication of pyrolytic carbon electrodes involved using photolithography with negative epoxy photoresist SU-8, then subjecting it to pyrolysis, as shown in Figure 2. 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 2A. 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 2B. 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 2C. 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 2D. The non-crosslinked SU-8 photoresist was eliminated by immersing it in PGMEA for 2x40 minutes (Figure 2E), 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 2F). 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.

[0223] 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 2G) 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 2H). 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 21).

[0224] Synthesis of BA-Ceo

[0225] The synthetic pathway of BA-Ceo follows a procedure, which is illustrated in Figure 3. A 100 mL round-bottomed flask, equipped with a reflux condenser and a magnetic stirrer, was charged with Ceo (180.0 mg, 0.25 mmol), paraformaldehyde (112.5 mg, 3.75 mmol), and (4-aminobutan-2-yl)boronic acid pinacol ester (125.82.0 mg, 6.25 mmol). These components were dissolved in 30 mL of o- dichlorobenzene (ODCB) by sonication until a clear solution was obtained. The solution was then placed in an oil bath preheated to 180 °C and stirred under ambient air for 5 hours. Following the reaction, the mixture was filtered through a silica gel plug to remove any insoluble material. The solvent was evaporated under vacuum, and the residue was purified using silica gel column chromatography with CS2 / CH2CI2 as the eluent. This process yielded unreacted Ceo and product A (105 mg, 58%) as an amorphous brown solid. The boronic ester group in product A was subsequently deprotected through transesterification to obtain product B. In this step, product A (100 mg) and methylboronic acid (30 mg) were dissolved in CH2CI2 (50 mL). After adding trifluoroacetic acid (TFA) (3 mL), the mixture was stirred at room temperature overnight. The solvent was then evaporated, and the product was purified using silica gel column chromatography with a toluene:ethyl acetate (EtOAc) (5: 1) mixture as the eluent. The resulting brown solid, product B (BA-Ceo), had a mass of 96 mg and a yield of 96%.

[0226] Synthesis of TpBpy-COF

[0227] The TpBpy-COF synthesis was performed as shown in Figure 4. A Pyrex tube (with a 10 mm outer diameter, 8 mm inner diameter, and 25 cm length) was used. The tube was loaded with triformylphloroglucinol (Tp) (21 mg, 0.10 mmol), 5,5'- diamino-2,2'-bipyridine (Bpy) (27.9 mg, 0.15 mmol), 0.5 mL of 1,4-dioxane, 0.5 mL of mesitylene, and 0.1 mL of 6 M aqueous acetic acid. To achieve a uniform dispersion, the mixture was sonicated for 20 minutes. The tube was then rapidly immersed in a liquid nitrogen bath and evacuated to an internal pressure of around 0.15 mmHg before flame-sealing. The sealed tube was heated in an oven at 120 °C for 5 days, after which it was allowed to cool to room temperature. An orange-red precipitate was formed, which was collected by filtering it over a medium glass frit and washing it with anhydrous tetra hydrofuran (THF) (20.0 mL). The product was then soaked in anhydrous THF (20.0 mL) for 8 hours, and the activation solvent was drained and replaced four times during this period. Following this, the solvent was eliminated via filtration, and the remaining precipitate was dried under vacuum at 60 °C overnight to yield TpBpy-COF.

[0228] Synthesis of BA-Ceo@TpBpy-COF

[0229] To prepare the BA-Ceo@TpBpy-COF sample, 100 mg of BA-Ceo was mixed with 6 mL of toluene: EtOAc (5: 1) solution. Then, 50 mg of TpBpy-COF was added to the mixture and heated in a preheated oven at 200 °C for 24 hours. After cooling to room temperature, the supernatant was decanted, and the BA-Ceo@TpBpy-COF sample was obtained. To remove any physically adsorbed BA-Ceo on the TpBpy- COF surface, the sample was washed thoroughly with toluene: EtOAc (5: 1). This was repeated three times with 5 mL of solvent each time. The solid was finally dried under dynamic vacuum at 80 °C overnight. Figure 5a illustrates the entire procedure.

[0230] Example 3 - Fabrication of electrochemical lactate sensor

[0231] Modification of electrode by BA-Ceo@TpBpy-COF and electrodeposition of PtNi nanoparticles

[0232] The 3D pyrolytic carbon electrodes, which were fabricated as described above, were first cleaned with isopropanol and deionized water. The electrodes were then dried using compressed nitrogen gas. Following this, the electrodes underwent a 65-second oxygen plasma treatment at 0.6 mbar and 40% power, and their electrochemical behavior was evaluated by CV. A silicon chip with Ti / Pt coating and an external Ag / AgCI electrode were used as the CE and RE, respectively. The cyclic voltammograms were obtained in 10 mM K3Fe(CN)e / K4Fe(CN)6 in 10 mM PBS at pH 7.4, and electrodes with reproducible results were selected for use in sensor preparation.

[0233] The electrode was immersed in the electrolyte with a plating bath solution consisting of 50 mM NiC , 1.5 mM KzPtCk, and 500 mM NaCI, adjusted to pH 2.0 with a 0.5 M H2SO4 solution, and bubbled with N2 for 10 minutes. The PtNi alloy electrodeposition was achieved at ambient temperature using the CV technique, scanning the potential range from -0.8 to 1.25 V vs. Ag / AgCI for 30 cycles at a scan rate of 50 mV s-1(Figure 6). The prepared PtNi nanoparticles modified electrode was then washed with DI water and dried.

[0234] The process of coating the PtNi NPs modified electrode with BA-Ceo@TpBpy-COF began by mixing the BA-Ceo@TpBpy-COF sample with 10 mL of ethyl acetate and subjecting them to a 20-minute sonication process in an ultrasonic bath. This ensured that the particles were uniformly dispersed in the solvent. A potentiostatic method was then employed to electrodeposit BA-Ceo@TpBpy-COF on the 3D WE at 0.50 V for 5 minutes. Following deposition, the potential was reset to zero, and the electrodes were removed from the solvent, thoroughly rinsed with ethanol, and dried under a flow of nitrogen gas. Figure 5b illustrates the entire process.

[0235] Fabrication of electrochemical lactate sensor

[0236] Coating of lactate sensor with a diffusion-limiting layer

[0237] To adjust the linear range of the lactate 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

[0238] Coating of lactate sensor with zwitterionic polypyrrole (ZiPPy)

[0239] The developed lactate 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.

[0240] To compare the performance of the ZiPPy-coated lactate 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.

[0241] Coating of lactate sensor with Nation

[0242] 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 lactate in different bioprocesses

[0243] Bacteria! fermentation processes

[0244] To evaluate the performance of the sensors, a Corynebacterium glutamicum 2262 strain was used, which was first pre-cultivated in two consecutive steps before conducting the batch culture. In the first step (Preculture I), a cryo- preserved C. glutamicum 2262 was added to 50 mL Luria-Bertani (LB) medium supplemented with 22 g L'1glucose. The culture was grown for 4 hours at 30 °C in an orbital shaker with an amplitude of 70 mm at 200 rpm. For the second step (Preculture II), 15 mL of Preculture I was added to 85 mL of CGXII medium. This medium had a composition (per liter) of 20 g (NH4)2SO4, 1 g KH2PO4, 1 g K2HPO4, 0.25 g MgSO4.7H2O, 42 g 3-morpholinopropanesulfonic acid (MOPS), 10 mg CaC , 10 mg FeSO4.7H2O, 10 mg MnSO4-H2O, 1 mg ZnSO4.7H2O, 0.2 mg CuSO4, 0.02 mg NiCl2.6H2O and 0.2 mg biotin. Glucose was added at a concentration of 24 g L’1, and the mixture was then cultured for 20 hours at 30 °C in a rotary incubator at 200 rpm. For the batch culture, 10 mL of Preculture II was transferred to a shakeflask containing 50 mL of CGXII medium supplemented with 24 g L'1glucose. The batch culture was then grown for 7 hours at 30 °C in a rotary incubator with intermittent shaking, using 15-minute off / on intervals. Samples were collected hourly, and after centrifugation, the supernatant was frozen at -20 °C for lactate analysis. The dry cell mass was determined by duplicate gravimetric measurements.

[0245] Cell cultivation

[0246] 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.

[0247] At-line monitoring of lactate by the developed sensors

[0248] The developed sensors were used for at-line and off-line lactate measurements during bioprocessing. The calibration curve for the sensors was obtained in different media. For the fermentation, samples were manually withdrawn hourly, while for cell cultures, samples were collected daily. The developed sensor was also used for lactate quantification in both cell-free and cell-containing samples. Prior to measurement, cell-free samples were filtered using a 0.2 pm sterile filter (Sartorius, Gottingen, Germany).

[0249] Reference analysis used for validation of the developed sensors Immediately after sampling, the culture samples were filtered through a 0.2 pm cellulose acetate syringe filter. The resulting supernatant was collected in 1.5 mL tubes and stored at -80 °C until thawed for measurement. The concentration of L-lactate in the supernatant of the culture samples was determined using an Agilent 1200 system (Agilent Technologies, Santa Clara, USA) equipped with a refractive index detector and a HyperRezTM XP Carbohydrate H+column (300 x 7.7 mm, 8 pm) from Fisher Scientific. A carrier solution of 0.1 M H2SO4 was used at a flow rate of 0.5 mL min-1and a temperature of 15 °C. The resulting measurement data were analyzed with Agilent Chemstation for LC 3D systems software Rev. B.04.01.

[0250] The BioProfile FLEX2 (NOVA Biomedical, Waltham, USA) was used to measure total cell density, viability, cell diameter, glucose, lactate, ammonium, and pH in cell cultures. This device does not require sample preparation beforehand and has a sample size of 400 pL.

[0251] Example 5 - Structural characterization

[0252] A one-step thermal reaction method was used to synthesize BA-Ceo as a fulleropyrrolidine derivative. The method involves the reaction of Ceo with paraformaldehyde and primary amine in the absence of metal salts, leading to the formation of N-alkyl-2,5-unsubstituted fulleropyrrolidine (Figure 3)

[0253] The UV absorption spectra revealed distinct absorption bands at 285 nm and 325 nm (Figure 7a), indicating the presence of fullerene Ceo mono adduct.

[0254] Additionally, a new shoulder at 432 nm was observed, which corresponds to the BA-Ceo compound (Figure 7a, inset). A weak absorption band was also observed at 703 nm, indicating the Si -> So transition of the Ceo moiety. Comparison of the BA-Ceo spectrum with the parent Ceo spectrum showed that the peaks at 284, 336, and 405 nm of Ceo were significantly different, except for the characteristic peak at 284 nm, which is unique to Ceo.

[0255] The FT-IR analysis (as depicted in Figure 7b) provides additional confirmation of the synthesis of BA-Ceo. The absence of the N-H peak and the appearance of the C-N stretching peaks at approximately 1540 and 1398 cm , respectively, in the BA-Ceo spectrum suggest the formation of the pyrrolidine ring. This indicates that the reaction between (4-aminobutan-2-yl)boronic acid and paraformaldehyde on Ceo was successful in producing BA-Ceo.

[0256] Several distinct methods were employed to analyze the properties of as-prepared TpBpy-COF and its composites. Through FT-IR analysis of the related materials (as depicted in Figure 7c), strong IR peaks corresponding to the stretching vibrations of the keto-form were observed at 1606 cm-1(C=O) in the TpBpy-COF spectrum. The peak at 1450 cm-1is attributed to the C=N stretching vibration in the pyridine structure. The stretching frequency of C=C and C-N was observed at 1580 cm-1and 1260 cm-1, respectively, indicating the successful synthesis of TpBpy-COF.

[0257] The crystal lattice structure of TpBpy-COF was determined through PXRD analysis. The PXRD patterns of TpBpy-COF revealed a highly intense peak at 20 = 3.6° that corresponds to the (100) plane reflections (Figure 7d). Additionally, minor peaks at 6.0, 6.9, 9.2, and 25.1-28.6° were observed, which are in good agreement with the simulated AA stacking structure in the hexagonal space group. By using the d spacing between the (001) planes at 25.1-28.6°, the n-n stacking distance between the COF layers was determined to be 3.5 A. The results verified that the TpBpy-COF structure with optimal crystallinity was synthesized. The PXRD pattern of BA-C6o@TpBpy-COF matches well with the pure TpBpy-COF pattern, indicating that the inclusion of BA-Ceo guest molecules did not affect its crystallinity (as seen in Figure 7d). This indicates that both TpBpy-COF and BA-Ceo@TpBpy-COF have high crystallinity and structural stability during the synthesis process. Moreover, the absence of characteristic diffraction peaks for BA-Ceo in the PXRD pattern of BA-Ceo@TpBpy-COF suggests that the guest molecules are dispersed at the molecular level within the pores and not aggregated. N2 adsorption isotherms at 77 K were conducted to verify the porous nature of TpBpy-COF. The sorption isotherm curve of TpBpy-COF was classified as a type I isotherm, indicating its microporous nature (Figure 8a). The data revealed a significant rise in adsorption, indicating its microporous properties. The BET surface area was found to be remarkably high at 924 m2g-1, with a pore size of 2.19 nm, in good agreement with its theoretical value (2.1 nm) (Figure 8b). These results suggest that the pores of TpBpy-COF are not blocked by oligomeric fragments or solvent molecules. However, after incorporating BA-Ceo into TpBpy- COF, there was a noticeable decrease in the amount of N2 absorbed. The BET surface area of the composite was reduced to half, measuring 496 m2g-1, and the pore size experienced a substantial decrease from 2.19 to 1.45 nm (as shown in Figure 8b). This suggests that BA-Ceo molecules are occupying the pores, leading to the decrease in surface area and pore size.

[0258] The thermal stability of TpBpy-COF and BA-Ceo@TpBpy-COF was evaluated through TGA under ambient conditions. Results displayed in Figure 8c indicated that both materials exhibited high thermal stability up to 427 °C and 353 °C, respectively. However, a weight loss of approximately 14% was observed in BA- C6o@TpBpy-COF compared to TpBpy-COF, which could be attributed to the decomposition of BA-Ceo. The observed behaviors indicate that BA-Ceo was uniformly accommodated within the TpBpy-COF host.

[0259] In order to investigate the stability of BA-Ceo@TpBpy-COF further, a powder sample was dispersed in boiling water for 12 hours at room temperature. The precipitates were then collected through centrifugation, rinsed thoroughly, and dried before conducting PXRD analysis (Figure 8d). The remarkable result of this experiment was that the PXRD analysis showed no significant shifts in the positions and intensity of soaked BA-Ceo@TpBpy-COF, which further confirmed its exceptional chemical robustness. These findings suggest that BA-Ceo@TpBpy-COF has the potential for applications that require high chemical stability.

[0260] A closer examination of the sample morphologies was conducted using SEM images. SEM images of both TpBpy-COF and BA-Ceo@TpBpy-COF samples at different magnifications are presented in Figure 9a and b, respectively. As illustrated in Figure 9a (ii), TpBpy-COF exhibits a unique 3D flower-like structure, which is composed of intricately arranged petal-like sheets at the nanoscale. Furthermore, TpBpy-COF exhibits a nearly identical PXRD pattern, indicating that the flower-like morphology does not influence its internal crystalline structure. Upon dispersing TpBpy-COF with BA-Ceo and loading BA-Ceo onto the TpBpy-COF structure, the SEM images (Figure 9b (i) and (ii)) reveal petal-like sheet structures for BA-Ceo@TpBpy-COF, highlighting the similarity in their morphologies. This observation suggests that the encapsulation of BA-Ceo does not significantly alter the overall morphology of the TpBpy-COF.

[0261] Figure 10 displays the SEM images of carbon micropillars before and after the modification process. Initially, the micropillars exhibit a smooth surface and uniform structure, which are common attributes of SU-8-derived carbon materials (Figure 10a). Upon completing the electrodeposition of PtNi NPs, the SEM images (Figures 10b and c) confirm the successful formation of a consistent PtNi NPs layer on the surface of the 3D carbon electrodes. The inset of Figure 10c provides an elemental mapping analysis of the PtNi alloy NPs, demonstrating that the Pt and Ni components are evenly distributed across the electrode surface. Figure lOd presents the XRD pattern of the PtNi alloy NPs. The diffraction pattern of the PtNi NPs modified on a 3D pyrolytic carbon electrode displays peaks at 20 values of approximately 40.13°, 46.05°, 67.60°, and 81.42°, which correspond to the crystal planes of PtNi (111), (200), (220), and (311). Additionally, the average crystal size of the PtNi NPs was determined to be 3.36 ± 0.32 nm by employing the Scherrer equation. Moreover, the lattice parameter of the prepared materials was calculated as 3.88 A, using the PtNi (220) diffraction peak as a reference.

[0262] Top-view SEM images display the electrodeposited BA-Ceo@TpBpy-COF film, which is consistently and securely attached to the surface of the PtNi NPs-modified 3D carbon electrode (Figure lOe). The BA-Ceo@TpBpy-COF layer exhibits a 2D petallike nanosheet morphology characterized by nanoscale dimensions and a smooth surface. These BA-Ceo@TpBpy-COF nanosheets interconnect with adjacent nanosheets and are anchored to the PtNi NPs. The interconnected configuration not only provides a generous exposed surface area for ion movement within the electrolyte but also promotes effective electron transfer. Based on these observations, it can be deduced that this nanosheet structure may demonstrate outstanding sensing performance. Lastly, Figure lOf displays the modified electrode surface after being coated with PTGE: PEI thin film and ZiPPy polymer, serving as diffusion-limiting and antifouling layers, respectively. The full-scale XPS spectra presented in Figure 11a depict the surface chemical composition of TpBpy-COF, BA-Ceo@TpBpy-COF, and PtNi@BA-C6o@TpBpy-COF modified 3D pyrolytic carbon WEs. Three peaks related to C Is, O Is, and N Is were observed in all samples. Simultaneously, both BA-Ceo@TpBpy-COF and PtNi@BA-Ceo@TpBpy-COF exhibited a faint B Is signal, confirming BA-Ceo's presence (due to the boronic acid group) in these samples. In contrast, the Pt 4f and Ni 2p peaks were only found in the PtNi@BA-Ceo@TpBpy-COF modified electrode. The deconvoluted high-resolution C Is peak of all samples revealed four peaks centered at binding energies of 284.1, 284.6, 285.3, and 287.5 eV, corresponding to C-C, C=C, C-N, and C=O, respectively (Figure lib). The high- resolution O Is spectra of BA-Ceo@TpBpy-COF and PtNi@BA-Ceo@TpBpy-COF composites were deconvoluted into three characteristic peaks at 531.8, 533.2, and 534.28 eV, corresponding to C-O, C=O, and B-OH (BA-Ceo) bonds, respectively (Figure 11c). In contrast, the O Is spectrum of the TpBpy-COF structure displayed only two peaks, corresponding to C-0 and C=O bonds. This observation implies the successful incorporation of BA-Ceo molecules within the TpBpy-COF structure. The N Is spectra (Figure lid) display a profile comprising contributions from amine and imine groups at 400.2 eV and the pyridyl group at 398.8 eV. In PtNi@BA-Ceo@TpBpy-COF, the pyridinic nitrogen peak shifts to a higher binding energy (from 398.8 eV to 399.5 eV), while the secondary nitrogen peak position remains unaltered. This suggests an interaction between PtNi and the nitrogen atoms through the bipyridine units of the TpBpy-COF backbone. Hence, it is hypothesized that this interaction contributes to the formation of a stable film on the surface of the 3D pyrolytic carbon working electrode modified with PtNi nanoparticles. Additionally, Figure lie displays the XPS spectrum of the B Is region, providing further evidence of the presence of boron species on the modified electrode. The spectrum confirms the existence of both B-C and B-OH bonds. These findings substantiate the successful incorporation of BA-Ceo molecules within the TpBpy-COF structure, as well as their interaction with the modified electrode.

[0263] The Pt 4f? / 2 and Pt 4fs / 2 peaks in the PtNi@BA-Ceo@TpBpy-COF sample, which appear at binding energies of 71.2 eV and 74.5 eV, respectively, are associated with metallic Pt (Figure Ilf). The presence of Pt (II) species, such as PtO and Pt(OH)2, is indicated by peaks at 71.8 eV and 75.6 eV. Based on the relative peak intensities, Pt mainly exists in its metallic form (Pt). The metallic Ni is characterized by the Ni 2p3 / 2 and Ni 2pi / 2 peaks with binding energies of 852.8 eV and 870.2 eV, respectively (Figure 11g). The Ni 2p spectrum also includes peaks at 855.8 eV and 874.3 eV, which correspond to the oxidized state (Ni (II)) on the surface. The distinct peaks observed in the XPS spectra verify the existence of Pt and Ni within the samples. When compared to pure Pt, the Pt 4f XPS spectrum of the PtNi alloy displays a positive shift in peaks (Figure llh. This shift could likely be due to the movement of electrons from Ni to Pt, indicating a decrease in the position of the d-band center because of a change in Pt's electronic status when alloyed with Ni.

[0264] Example 6 - Electrochemical evaluation

[0265] The electrochemical characteristics of the fabricated sensors are evaluated using CV under ambient conditions, using 0.1 M PBS (pH 7.0) as the electrolyte, within a potential range of -0.2 to 1.0 V. Figure 12a presents the CV profiles for a series of modified 3D pyrolytic carbon electrodes: PtNi@BA-Ceo@TpBpy-COF, PtNi@BA- Ceo, PtNi@TpBpy-COF, as well as the bare 3D pyrolytic carbon electrode, measured in the absence of lactate. Notably, the charge under the peak for the PtNi@BA-Ceo@TpBpy-COF modified electrode is higher compared to the other electrodes. This suggests that the modification of the 3D electrode with PtNi NPs, BA-Ceo, and TpBpy-COF leads to a synergistic effect that enhances the electrochemical properties of the electrode. The improvement is likely due to the increased electron transfer kinetics and better electrical conductivity conferred by the PtNi@BA-Ceo@TpBpy-COF modification. The electrooxidation of lactate on PtNi NPs modified electrode was also investigated (Figure 12b). It is worth mentioning that this process is a non-selective reaction. While lactate is the target analyte, other compounds present in the sample may also undergo oxidation.

[0266] In order to confirm the application of the developed electrodes for lactate sensing, the current responses of the modified electrodes in the presence of 5 mM lactate were analyzed, and the results are shown in Figure 12c. As illustrated in Figure 12c, the PtNi@TpBpy-COF modified electrode exhibited no catalytic activity toward lactate, as evidenced by the absence of a Faradaic peak in the CV curve. On the other hand, the PtNi@BA-Ceo@TpBpy-COF electrode generated a pronounced oxidation peak current when lactate was present in the PBS solution, while no response was detected without lactate (Figure 12a). In contrast, the PtNi@BA-Ceo electrode displayed a weak peak for lactate, suggesting inferior electrocatalytic activity. This can be ascribed to the interaction between lactate and boronic acid leading to the formation of a cyclic intermediate. This binding is anticipated to be reinforced by the association of a hydroxide anion, thereby enhancing the stability of the complex and resulting in the formation of a tetrahedral boron species. The formation of this cyclic intermediate not only provides a stable structure, but also facilitates a pathway for the ensuing oxidation process to occur.

[0267] Moreover, the introduction of BA-Ceo in TpBpy-COF pores on the PtNi@BA- Ceo@TpBpy-COF electrode improves its function in two ways. Firstly, it increases the density of boronic acid groups on the electrode surface, providing more binding sites for analyte molecules and enhancing association kinetics. Secondly, the integration of BA-Ceo and TpBpy-COF creates a conductive framework, combining the electron affinity of BA-Ceo with the large surface area of COF. This synergistic effect significantly enhances the conductivity of the modified electrode, leading to more efficient electron transfer during electrochemical reactions.

[0268] To examine the electron-transfer kinetics, CV curves were recorded for the PtNi@BA-C6o@TpBpy-COF electrode at various scan rates (u) ranging from 20 to 120 mV s-1. The experiments were conducted in the presence of 5 mM lactate in PBS. The resulting voltammograms can be seen in Figure 12d. The data indicates that both the oxidation peak currents (Ipa) and peak potentials (Epa) exhibit a positive shift with increasing scan rates

[0269] Figure 12e provides further insights, demonstrating a direct proportionality between the Ipaof lactate oxidation and the scan rate. This observation suggests 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 that lactate oxidation becomes diffusion-controlled (Figure 12e, inset). The obtained results suggest that the electrochemical oxidation of lactate involves a combination of diffusion-controlled and adsorption-controlled processes, with the dominance of each process dependent on the chosen scan rates.

[0270] Furthermore, a linear correlation was observed between the Epaand the logarithm of scan rates, as depicted in Figure 12f. This relationship is described by a correlation equation (Eq. (1)).

[0271] Epa=0.0274 In u + 0.8395 (V, V s'1, R2= 0.9954) (1) With an increase in the scan rate (u), a positive shift was observed in the Epa. To estimate the charge transfer coefficient (an) value for the irreversible lactate oxidation, Laviron's equation (Eq. (2)) 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 an value of 0.937, with R = 8.314 JK-1mol-1, T = 298 K, and F = 96485 Cmol-1being assumed. The electron transfer coefficient (a) for the anode peak is observed to be around 0.5, indicating that the process is in the typical range for irreversible reactions. Based on this coefficient, the estimated electron transfer number (n) is calculated to be approximately 1.87. This indicates that the lactate oxidation at the PtNi@BA-Ceo@TpBpy-COF modified electrode can be described as a two-electron transfer process. Previous studies have shown that the number of electrons participating in lactate oxidation are two, indicating that the electrochemical oxidation of lactate at the PtNi@BA-Ceo@TpBpy-COF modified electrode involves the transfer of two electrons.

[0272] Furthermore, the cyclic voltammograms of the PtNi@BA-C6o@TpBpy-COF modified electrode were subsequently recorded in a 0.1 M PBS solution, with varying concentrations of lactate (0.1, 1.0, and 3.0 mM), using a scan rate of 50 mV s-1(Figure 13). It was observed that as the concentration of lactate increased, there was a significant enhancement in the anodic peak current.

[0273] Figure 14a illustrates the schematic diagram that showcases the structure of the fabricated PtNi@BA-Ceo@TpBpy-COF-based sensor and highlights its electrochemical performance in lactate detection. The diagram provides a visual representation of the sensor's components, including the sensing, diffusionlimited, and antifouling layers, emphasizing their arrangement and functionality in facilitating efficient lactate detection. Figure 14b presents a schematic representation illustrating the lactate oxidation reaction occurring on the surface of the PtNi@BA-C6o@TpBpy-COF modified electrode. Based on the obtained data, it can be concluded that the oxidation of the boronate ester complex with lactate leads to its cleavage, resulting in the formation of pyruvate and, presumably, the regeneration of the boronic acid group. Boronic acids exhibit characteristics of both Bronsted acids and weak Lewis acids. They possess the ability to donate a proton to an incoming ligand as a Bronsted acid, while also being capable of accepting a lone pair of electrons as a Lewis acid. Despite being relatively weak Lewis acids, their chemistry is notably influenced by this aspect, as evident from pKatitration curves. Their pKavalues can vary depending on the specific substituents attached to the boronic acid derivatives. In general, aryl boronic acids exhibit higher acidity compared to alkyl boronic acids. Understanding the effect of pH and pKais important in optimizing the binding affinity and selectivity of boronic acid-based sensors for lactate detection.

[0274] The determination of the surface pKaprovides important information for understanding and optimizing the performance of the PtNi@BA-C6o@TpBpy-COF modified electrode. The investigation of the surface pKaof the BA-Ceo group on the PtNi@BA-C6o@TpBpy-COF modified electrode surface involved analyzing the attenuation of redox reactions with charged probe molecules. Figure 15a demonstrates the CV response of Fe(CN)e3' / 4' on the PtNi@BA-Ceo@TpBpy-COF modified electrode under different pH conditions. As the pH of the solution increases, the redox response of Fe(CN)e3' / 4' noticeably decreases. It is well- established that the redox reaction of a charged probe molecule weakens when the charged layer on the electrode surface shares the same charge as the probe molecule. Therefore, 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 reduction in current response in the CV measurements was utilized to estimate the pKavalue of the attached boronic acid group. Through optimal fitting of the data in Figure 15a (inset), the surface pKavalue of the boronic acid group on the electrode was approximated to be 8.

[0275] The binding affinity of BA-Ceo towards lactate is greatly influenced by the pH of the solution, which has a substantial impact on its overall performance. In Figure 15b, the peak current responses were measured at the PtNi@BA-Ceo@TpBpy-COF electrode following a 10-minute incubation in 10 mM lactate solution within 0.1 M PBS (pH 7.0). The pH values examined ranged from 3 to 10, encompassing a wide pH range to assess the pH-dependence of the lactate binding response. The pH of the solution directly affects the ionization state of both the boronic acid and lactate molecules. Boronic acids exist as a dynamic equilibrium between the neutral form and the negatively charged form as the pH changes. At pH < pKa, the boronic acid predominantly exists in its neutral form, while at pH > pKa, it becomes deprotonated and becomes negatively charged as the boronate ion. The figure clearly demonstrates a notable increase in current responses of lactate at pH values lower than the pKa of the boronic acid at the electrode surface (pKa=8), while a decrease in current responses is observed at pH values higher than this pKa. This observation suggests that boronic acid (neutral form) exhibits higher reactivity compared to the boronate ion for binding with lactate. The reduction in affinity of the boronate ion for lactate binding could be attributed to electrostatic repulsions arising from the negatively polarized boron atom in the boronate ion interacting with the lactate molecule. These electrostatic interactions play a significant role in influencing the overall affinity and reactivity of the boronic acidbased sensor.

[0276] Example 7 - Sensing performance of PtNi@BA-C6o@TpBpy-COF modified electrode

[0277] The assessment of lactate oxidation and sensitivity of the modified electrodes involved the use of amperometric analysis. In this experiment, gradual increases in lactate concentration were introduced to the PBS solution. The objective was to achieve the most favorable amperometric current response. To accomplish this, the applied potentials on the PtNi@BA-Ceo@TpBpy-COF electrode were carefully adjusted within a range of 0.40 to 0.85 V, guided by the CV results depicted in Figure 15c. Throughout the experiment, 5.0 mM lactate was added successively to the electrolyte, and the modified electrode exhibited a stepwise response under varying applied potentials. After careful evaluation, it was determined that the optimal potential for the modified electrodes was 0.8 V.

[0278] It is important to underline that when the PtNi@BA-Ceo@TpBpy-COF modified electrode is coated with a PTGE: PEI layer, a noticeable decrease in the current response to lactate is observed, as demonstrated in Figure 15d. This diminished current response is due to the PTGE: PEI layer's capacity to hinder the movement of lactate molecules towards the electrode surface. However, in the absence of such a diffusion-limiting layer, the PtNi@BA-Ceo@TpBpy-COF modified ectrode possesses a calibration range spanning from 0.001 to 20 mM of lactate. This range can be subdivided into three different zones of linearity (0.001-0.2, 0.2-5, and 5 -20 mM), as depicted in Figure 15d, inset. While the decreased current response may seem disadvantageous at first glance, it actually conveys a beneficial attribute, enhancing the dynamic range of the electrode. This extended dynamic range is notably advantageous when monitoring bioprocesses, considering the significant fluctuations in lactate concentrations over time. In addition, the slowed transportation of lactate facilitated by the PTGE: PEI layer provides an added benefit. It helps to mitigate potential interference from other electroactive species that might be present in the sample matrix. This ensures that the electrode's response remains largely specific to lactate, thereby maintaining the accuracy of the lactate measurement.

[0279] Figure 15e presents the real-time amperometric response of the PtNi@BA- Ceo@TpBpy-COF modified electrode with PTGE: PEI layer coating as the diffusionlimiting layer to expand the dynamic range. Notably, when incremental doses of lactate were added to the PBS solution, the current generated by the PtNi@BA-Ceo electrode was minimal (Figure 16). In contrast, the PtNi@BA-Ceo@TpBpy-COF modified electrode demonstrated a pronounced current change, even at minimal lactate concentrations of 0.01 mM. This electrode exhibited remarkable sensitivity across a broad lactate concentration spectrum, spanning from 0.01 mM to 90 mM. This performance was achieved without the need for any additional treatment or washing steps between tests, underscoring the practical utility of this sensor. Besides, a stable current was achieved in just 20 seconds, indicating the rapid response of the PtNi@BA-Ceo@TpBpy-COF based sensor (Figure 15e, inset). Previously highlighted, the remarkable performance and prompt response of the PtNi@BA-C6o@TpBpy-COF modified electrode can be traced back to its considerable surface area and electrocatalytic activity, which are due to the encapsulation of BA-Ceo within the pores of the TpBpy-COF.

[0280] Subsequently, Figure 15f, further demonstrating the electrode's exceptional qualities, displays the current response of the PtNi@BA-Ceo@TpBpy-COF modified electrode in relation to varying lactate concentrations. It presents linear behavior with an impressively high R2value of 0.996, implying the electrode's consistent and reliable performance as a lactate sensor across a wide concentration spectrum (0.01 to 90 mM). The calculated slope for this electrode was 0.053 mA mM-1cm-2, (equivalent to a sensitivity of 53 pA mM-1cm-2), thereby emphasizing the high degree of precision of the PtNi@BA-Ceo@TpBpy-COF based sensor. Further, the LOD was determined to be 0.0082 mM, based on an equation employing a signal-to-noise ratio of 3. This reinforces the capability of the modified electrode to detect extremely low concentrations of lactate making it an effective and reliable tool for monitoring lactate levels in various contexts.

[0281] The developed PtNi@BA-C6o@TpBpy-COF lactate sensor was tested for selectivity against L-lactate, D-lactate, and a mixture of both isomers (Figure 17). The calibration curves for each isomer were in good agreement, suggesting the sensor's ability to detect both enantiomers. However, due to the structural similarity of L and D lactate, the sensor, as a chemo-sensor, did not exhibit significant selectivity for individual enantiomers.

[0282] An interference test was conducted using various common chemical substances found in bioprocesses, such as glucose, acetate, ethanol, pyruvate and NaCI, to evaluate the selectivity of PtNi@BA-C6o@TpBpy-COF based sensor. The results obtained in Figure 18a demonstrated a distinct response from the PtNi@BA- Ceo@TpBpy-COF based sensor after the addition of 10 mM lactate, while only minor fluctuations were observed following subsequent additions of 40 mM of the other species. Furthermore, the interference effects from some common organic acids were evaluated in a solution containing 0.5 mM lactate, along with a 5 mM and a 50 mM excess of the interfering substances (Figure 18b). To establish an acceptable tolerance interval, a change of 5% in the signal was considered. Notably, compounds such as formate, acetate, succinate, citric acid, ammonium, and sulfate do not interfere with the lactate measurements. This indicates that the PtNi@BA-Ceo@TpBpy-COF based sensor exhibits a remarkable selectivity for lactate detection due to the selective oxidation of lactate, even in the presence of interfering substances that are typically present in bioprocesses.

[0283] One important distinction between saccharides and hydroxy acids (like lactate acid) is the nature of their functional groups. Saccharides possess diol functional groups, whereas hydroxy acids have acid anions at a pH of 5-8. This difference in functional groups contributes to the variation in binding affinities and selectivity. Additionally, glucose is a more rigid molecule and considerably less acidic compared to lactate. These characteristics are believed to have mechanistic significance in the interaction with boronic acid. Therefore, glucose and lactate cannot be treated as comparable ligands, further supporting the notion that there is no interference from saccharides like glucose in this context.

[0284] From the provided information, we can deduce that saccharides such as glucose do not interfere with the as-prepared boronic acid intended for lactate sensing due to its high pKavalue (pKa=8). The selectivity of the boronic acid group towards saccharides and hydroxy acids is strongly influenced by the pH of the solution. Within the pH range of 5-8, which is typically associated with bioprocesses, the binding constants for saccharides with this boronic acid are significantly lower than those for lactate. This observation can be clearly inferred from the corresponding data (Figures 15a and b). This implies that the as-prepared boronic acid preferentially binds to lactate rather than saccharides such as glucose in the given pH range.

[0285] Example 8 - Stability, repeatability, and reproducibility of sensor

[0286] To assess the stability and reproducibility of the PtNi@BA-C6o@TpBpy-COF based sensor for broader applications, Figure 18c demonstrates negligible current decline while continuously monitoring lactate concentrations at 5 mM. The corresponding response remained stable and reliable, with a retention of 95.7% after 130 minutes of continuous measurement. Additionally, this sensor exhibited extraordinary stability, with 91.5 % retention after 25 days (Figure 18c, inset). The reproducibility test, conducted by running the PtNi@BA-C6o@TpBpy-COF based sensor eight times individually, is shown in Figure 18d. The sensitivity remained consistent at 48.2 pA mM-1cm-2(R2= 0.972), which was identical to the initial individual sample 53.1 pA mM-1cm-2(R2= 0.997). This demonstrates outstanding reproducibility with consistent accuracy over multiple runs. This suggests the potential for utilizing the PtNi@BA-Ceo@TpBpy-COF modified electrode for lactate sensing, offering excellent sensitivity and durability. Compared to other non-enzymatic lactate sensors (referenced in Table 4), the PtNi@BA-Ceo@TpBpy-COF based sensor exhibited outstanding sensitivity, LOD, and durability for lactate detection.

[0287] Sensor LOD ( M) Sensitivity Linear range

[0288] (mM)

[0289] GCE-Nafion / NiO 27 g235 A mM- 1 0.01-7.75 cm-2

[0290] Ti-PTFE / PPy-MWCNTs 512.9 pA mM- 1cm-2 1-15 30.59 ± 0.34 pA

[0291] SPCE-NiCo LDH 533 5-25

[0292] BDD-NiNPs 0.72 ± 0.09 (24.70 ± 0.36) pA L 6-120

[0293] C-1mol-1

[0294] NiF-NiS-NC@NiS-MS 0.5 0.39 pA pM"10.0005-0.085

[0295] GCE-Nafion / CosCU 6 - 0.5-3.0

[0296] GCE-CuO / MWCNTs / Nafion 0.088 633.0 pA mlT1cm-20.0001-10

[0297] SPCE-ZIF-67 / NiCo LDH 400 83.98 pA mlT1crrr22-26

[0298] NH2-GP-Cu3(btc)2 5 0.029 mA mM-1cm-20.05-22.6

[0299] CE-AgNWs-MIP 0.22 4.5 x IO-5A M-10.001-100

[0300] SPCE / 3-ABPA 1.5 mM - 3 -100

[0301] GCE-rGO-AuNPs-MIP 9 x 10“51.9 x 105pA L mol”11 x 10“7-l 10“5

[0302] SPCE / PdCu 0.7 / / M 396.7 / / A mM-1cm-20.5 -11

[0303] GCE-Nafion / CuO - 80.33 pA mM”10.01-27.76

[0304] 31,600 nA mM-1cm-

[0305] CO3O4 / CUO@MWCNTS NCS / N F 55 x 105 210 00

[0306] PtNi@BA-C50@TpBpy-COF 8.2 53 fifi. mM”1cm-20.01-90

[0307] Table 4 Comparisons of the analytical parameters for determination of lactate with non-enzymatic electrochemical sensors

[0308] Example 9 - At-Line application of the sensor platform during bioprocesses

[0309] The PtNi@BA-C6o@TpBpy-COF-based sensor, calibrated in different medium, was applied for at-line monitoring of lactate in C. glutamicum 2262 fermentation and cell culture samples.

[0310] Figures 19 displays the outcomes of the C. glutamicum 2262 fermentation. The fermentation's progression is illustrated by the rising cell concentration and the diminishing lactate concentration over a 7-hour period (Figure 19). The glucose consumption was tracked using the HPLC method. The lactate production profiles were measured with the developed sensor and 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. The data depicted in the Figure 19 demonstrates the precise measurement of lactate concentrations in the cell-free supernatant during the entire fermentation process using the PtNi@BA-Ceo@TpBpy-COF-based sensor coating, incorporating ZiPPy as antifouling layer. The results obtained from the sensor were in agreement with those from HPLC, indicating its accuracy and reliability. The developed ZiPPy coated PtNi@BA-C6o@TpBpy-COF-based sensor was also evaluated for its ability to detect lactate 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 lactate oxidase in BioProfile FLEX2 devices, and are illustrated in Figure 20a and b. The lactate signal trends captured by both methods were in good agreement, with the developed sensor demonstrating accurate lactate signal detection.

[0311] Example 10 - Electrodeposition of metal and metal oxide nanoparticles on the 3D pyrolytic carbon electrode

[0312] The 3D pyrolytic carbon electrode surface after cleaning with isopropanol and deionized water was coated with a CuO film through an electrodeposition process. This was achieved by submerging the electrode in a 0.1 M KCI solution with 1 mM CuCI2and maintaining it at -0.4 V for a period of 10 seconds. The resultant electrode, now coated with a film of copper, was immersed in a 0.1 M NaOH solution. It was then subjected to CV (20 cycles) within a potential range from - 0.5 to 0.3 V at a sweep rate of 50 mV s’1. This was done to transform the copper film into CuO that would attach to the surface of the 3D pyrolytic carbon electrode. The CuO modified electrode was then removed from the solution and thoroughly washed with DI water. Prior to depositing of BA-Ceo@TpBpy-COF, the modified electrode underwent three repetitive cycles between 0.2 and 1.0 V at a scanning rate of 50 mV s'1in a BPS solution with a pH of 7.0. This procedure was carried out to achieve a consistent voltammetric response.

[0313] The electrodeposition of metallic nickel was performed at a constant potential of - 0.8 V (against a reference electrode) for a duration of 5 minutes, in a pH 4 acetate buffer solution that contained 50 mM of NiC . Following this, the electrode was placed in a fresh 20 mM PBS solution, maintaining the potential at 0.8 V for 15 minutes. This allowed for the electrodissolution and passivation of NiO NPs. Finally, the modified electrode was taken out from solution, and rinsed thoroughly with DI water.

[0314] The electrode was immersed in a solution that included 1.5 mM KzPtCk and 500 mM NaCI, with its pH level adjusted to 2.0 using a 0.5 M H2SO4 solution. This solution was then purged with N2 for a period of 10 minutes. The deposition of Pt NPs was performed at room temperature utilizing the CV technique. The potential was scanned within a range from -0.8 to 1.25 V against Ag / AgCI over 30 cycles, at a scanning rate of 50 mV s’1. Following this, the modified electrode was rinsed using DI water and allowed to dry naturally under ambient conditions.

[0315] Example 11 - nanoparticles

[0316] In addition to the aforementioned analysis, the influence of different metal and metal oxide nanoparticles on the electrochemical signal of various modified electrodes was further investigated. This investigation involved the modification of electrodes with PtNi@BA-C6o@TpBpy-COF, Pt@BA-C6o@TpBpy-COF, Ni@BA- Ceo@TpBpy-COF, CuO@BA-Ceo@TpBpy-COF, and NiO@BA-Ceo@TpBpy-COF, without diffusion limited and antifouling layers, and their corresponding responses to 10 mM lactate detection were observed.

[0317] The PtNi@BA-Ceo@TpBpy-COF electrode showed a superior electrochemical response to lactate (Figure 21a) with stable signals recorded over 60 minutes (Figure 21b). This can likely be attributed to a strong synergistic effect from the combined presence of Pt and Ni, enhancing the electron transfer properties of the electrode. Additionally, this combination appears to have fostered an efficient deposition of the BA-Ceo@TpBpy-COF layer, lending to a more robust interface through a strong bonding between the PtNi alloy nanoparticles and the BA- Ceo@TpBpy-COF, thereby preventing detachment over time.

[0318] On the other hand, the Pt@BA-Ceo@TpBpy-COF electrode, despite Pt being known for its excellent catalytic properties, did not exhibit the same performance level as the PtNi combination. The singular presence of Pt might not provide an optimal environment for BA-Ceo@TpBpy-COF deposition, leading to a less stable interface and subsequently a lower lactate detection performance (Figure 21a and b). Similarly, the Ni@BA-Ceo@TpBpy-COF electrode faced issues paralleling those of the Pt@BA-Ceo@TpBpy-COF. Ni, even though acknowledged for its good electrocatalytic properties, was not as successful in facilitating BA-Ceo@TpBpy- COF deposition as the PtNi combination, which likely contributed to an unstable interface and a decrease in lactate detection performance over time (Figure 21a and b).

[0319] The NiO@BA-C6o@TpBpy-COF and CuO@BA-Ceo@TpBpy-COF electrodes may suffer from instability issues due to weaker interactions between the respective NPs and the COF. This can result in the gradual loss of BA-Ceo@TpBpy-COF from the electrode surface, limiting its effectiveness as a sensing layer for lactate detection (Figure 21a and b). Additionally, the detachment of BA-Ceo@TpBpy-COF from the metal and metal oxide nanoparticles modified electrode contributes to a shift in the lactate detection mechanism. Instead of the intended interaction between lactate and BA-Ceo, an undesired reaction occurs with the exposed nanoparticles. However, it is important to highlight that the interaction with metal and metal oxides is not a selective mechanism.

[0320] The sizes of the PtNi-nanoparticles was assessed by SEM, and the mean size of the nanoparticles was found to be about 138 nm (Figure 22).

[0321] Example 12 - Conclusion

[0322] A novel lactate sensor has been developed, which is non-enzymatic in nature, utilizing a fullerene PtNi@BA-C6o@TpBpy-COF modified 3D pyrolytic carbon electrode. This avant-garde sensor has displayed superior electrochemical performance, eclipsing other modified electrodes due to its expansive electrochemical active surface and impressive conductivity. The effectiveness of this non-enzymatic sensor, built on PtNi@BA-Ceo@TpBpy-COF, is particularly noticeable in lactate detection. It exhibits exceptional electrooxidation detection capabilities, improving effective lactate monitoring. The heightened electrocatalytic activity is ascribed to multiple contributing factors. These include efficient electrolyte diffusion, selective lactate interaction with BA-Ceo, accelerated charge transfer, the remarkable electroconductivity of the BA-Ceo@TpBpy-COF, and the efficient employment of active sites engendered by the unique morphology and synergistic relationship between TpBpy-COF and BA-Ceo in the structured assembly.

[0323] To deter cells and proteins from adhering to the electrode surface in real samples, we utilized ZiPPy, a zwitterionic polymer, to coat the developed electrodes. This sensor swiftly detects lactate with a superior sensitivity of 53 pA mM-1cm-2for lactate concentrations spanning from 0.01 mM to 90 mM. It also features an impressive detection limit of 0.0082 mM (S / N = 3).

[0324] Additionally, this sensor maintains consistent responses across eight consecutive measurement cycles, requiring no cleaning interventions. It sustains stable sensitivity for 25 days under normal conditions. As a comparison to the conventional HPLC analysis, our sensor offers reliable lactate quantification in a significantly more productive and time-efficient manner. It boasts remarkable stability when interacting directly with the fermentation medium, and it accurately quantifies lactate amidst several electroactive species.

[0325] Items

[0326] 1. A triphenylamine-functionalized bipyridine covalent organic framework (TpBpy- COF) comprising boronic acid-functionalized Ceo (BA-Ceo), the TpBpy-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

[0327] (formula III).

[0328] 2. The TpBpy-COF according to item 1, wherein the boronic acid in the BA-Ceo is a boronic acid having a pKavalue in the range of 7.9-8.1, preferably 8.0.

[0329] 3. The TpBpy-COF according to any of the preceding items, wherein the BA-Ceo is represented by the formula IV (formula IV). 4. The TpBpy-COF according to any of items 1-2, wherein the BA-Ceo is represented by a formula selected from the following table

[0330]

[0331]

[0332] 5. The TpBpy-COF according to any of the preceding items, wherein the BA-Ceo is non-covalently bonded to the TpBpy-COF, such as via intermolecular forces, such as van der Waals forces, and electrostatic interactions, such as n-n stacking interaction.

[0333] 6. Use of the TpBpy-COF according to any of the preceding items, in an electrochemical sensor for sensing lactate. 7. An electrochemical sensor for sensing lactate comprising a working electrode, the working electrode comprising the TpBpy-COF according to any of items 1-5.

[0334] 8. The electrochemical sensor according to item 7, the working electrode further comprising:

[0335] • an electrochemical substrate, such as a carbon-based substrate, optionally having micropillars; and

[0336] • nanoparticles comprising or consisting of a metal or a metaloxide, such as PtNi, Pt, Ni, CuO, or NiO.

[0337] 9. The electrochemical sensor according to any of items 7-8, wherein the sensor further comprises a counter electrode and, optionally, a reference electrode.

[0338] 10. The electrochemical sensor according to item 9, wherein the counter electrode and / or reference electrode comprises an inert metal, such as Au, Pt, or Ag.

[0339] 11. The electrochemical sensor according to any of items 9-10, wherein the metal is Au.

[0340] 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.

[0341] 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.

[0342] 14. The electrochemical sensor according to item 13, wherein the pyrolytic carbon electrode is exposed to UV light before pyrolysation.

[0343] 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 33D microstructures, such as micropillars. 16. The electrochemical sensor according to item 15, wherein 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.

[0344] 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.

[0345] 18. The electrochemical sensor according to any of items 7-16, wherein the micropillars are pillars with a height of approximately 180 pm to 200 pm and a diameter of 100 pm.

[0346] 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.

[0347] 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.

[0348] 21. The electrochemical sensor according to any of items 7-20, wherein the sensing layer's ability to detect lactate is maintained after exposure to autoclave conditions, such as overpressure and about 120 degrees Celsius.

[0349] 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).

[0350] 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). 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.

[0351] 25. The electrochemical sensor according to any of items 7-24, wherein the electrochemical sensor's ability to sense lactate is maintained for a sustained period of time after manufacturing without affecting the sensor's ability to sense lactate, 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.

[0352] 26. The electrochemical sensor according to any of items 22-25, wherein the electrochemical sensor's ability to sense lactate is maintained after exposure of up to 70 degrees Celsius.

[0353] 27. The electrochemical sensor according to any of items 7-26, wherein the sensor's ability to sense lactate is defined as an ability to continuously measure the lactate content of a control sample, such as running amperometric tests at lactate concentrations ranging from 0.01-90 mM, preferably 5 mM, and maintaining sensitivity of at least about 90% such as even about 95%.

[0354] 28. Use of the electrochemical sensor according to any of items 7-27, in a method for sensing lactate.

[0355] 29. A method of sensing lactate, the method comprising:

[0356] 1. Providing an electrochemical sensor as defined in any of items 7-27 configured to produce an electric signal when a lactate molecule is at, or near, the working electrode;

[0357] 2. Providing a sample suspected of containing lactate;

[0358] 3. Contacting the electrochemical sensor with the sample;

[0359] 4. Measuring the signal from the working electrode, thereby sensing the presence of lactate in the sample;

[0360] 5. Optionally, comparing the signal to a signal from a control electrode on the electrochemical sensor to reduce background signals; and 6. Optionally, comparing the signal from the working electrode to a signal obtained from measuring a control sample, to obtain a level of lactate in the sample.

[0361] 30. The method according to item 29, wherein the method senses lactate in the range from 0.01-90 mM lactate.

[0362] 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 lactate.

[0363] 32. A method of producing a TpBpy-COF comprising boronic acid-functionalized C60 (BA-Ceo), the method comprising:

[0364] 1. combining triformylphloroglucinol (Tp), and 5,5'-diamino-2,2'-bipyridine (Bpy) such as with dry dioxane, dry mesitylene and aqueous acetic acid, such as in a Pyrex tube;

[0365] 2. optionally, sonicating the mixture and subjecting the pyrex tube to a step of lowering the internal pressure before sealing;

[0366] 3. reacting the mixture, such as at 120 °C for 5 days;

[0367] 4. washing the product thereof, and drying the product;

[0368] 6. optionally, soaking the product in anhydrous THF;

[0369] 7. optionally, heating the product under vacuum, such as at 60 °C;

[0370] 8. obtaining a TpBpy-COF;

[0371] 9. reacting paraformaldehyde, and (4-aminobutan-2-yl)boronic acid pinacol ester, such as reacting in dichlorobenzene by sonication at 180 °C under ambient air for 5 hours;

[0372] 10. washing the product thereof;

[0373] 11. removing the protective group to obtain BA-Ceo, such as by the use of methylboronic acid dissolved in CH2CI2, followed by the addition of TFA; and

[0374] 12. combining the TpBpy-COF with the BA-Ceo, thereby obtaining a TpBpy- COF comprising BA-Ceo.

[0375] 33. A method of producing an electrochemical sensor for sensing lactate, the method comprising: 1. providing a carbon based substrate, such as a pyrolytic carbon electrode; nanoparticles comprising or consisting of a metal or a metaloxide, such as PtNi, Pt, Ni, CuO, or NiO; and a TpBpy-COF comprising BA-Ceo as defined in any of items 1-5, or item 32;

[0376] 2. depositing the nanoparticles on the carbon based substrate;

[0377] 3. depositing the TpBpy-COF comprising BA-Ceo on the nanoparticles;

[0378] 4. optionally, depositing a counter- and / or a reference electrode at suitable positions on the electrochemical sensor;

[0379] 5. optionally, coating with a zwitterionic polymer, such as zwitterionic polypyrrole (ZiPPy); and

[0380] 6. optionally, coating with a diffusion-limiting layer, such as a layer comprised of a trimethylolpropane triglycidyl ether

[0381] (PTGE): polyethyleneimine (PEI) layer.

[0382] 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.

[0383] 35. The method of producing an electrochemical sensor according to any of items 33-34, wherein the metal is Au.

[0384] 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.

[0385] 37. The method of producing an electrochemical sensor according to item 36, wherein the pyrolytic carbon electrode is exposed to UV light before pyrolysation.

[0386] 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 lactate, optionally the kit may also comprises components for cleaning, such as washing buffers. 39. A method of producing a TpBpy-COF comprising boronic acid-functionalized C60 (BA-Ceo), the method comprising:

[0387] 1. combining triformylphloroglucinol (Tp), and 5,5'-diamino-2,2'-bipyridine (Bpy) to obtain a TpBpy-COF; 2. combining Ceo, paraformaldehyde, and (4-aminobutan-2-yl)boronic acid pinacol ester, and removing the protective group to obtain BA-Ceo; and 3. combining the TpBpy-COF with the BA-Ceo, thereby obtaining a TpBpy- COF comprising BA-Ceo.

Claims

Claims1. A triformylphloroglucinol-functionalized bipyridine covalent organic framework (TpBpy-COF) comprising boronic acid-functionalized Ceo (BA-Ceo), the TpBpy-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).

2. The TpBpy-COF according to claim 1, wherein the boronic acid in the BA-Ceo is a boronic acid having a pKavalue in the range of 7.9-8.1, preferably 8.0.

3. The TpBpy-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 lactate comprising a working electrode, the working electrode comprising the TpBpy-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; andnanoparticles comprising or consisting of a metal or a metal oxide, such as PtNi, Pt, Ni, CuO, or NiO.

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 3-dimensional 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 lactate is maintained for a sustained period of time after manufacturing without affecting the sensor's ability to sense lactate, 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 lactate is maintained after exposure of up to 70 degrees Celsius, such as even after exposure to autoclave conditions, such as overpressure and about 120 degrees Celsius.

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

12. A method of sensing lactate, preferably in the range 0.01 to 90 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 lactate molecule is at, or near, the working electrode;2. Providing a sample suspected of containing lactate;3. Contacting the electrochemical sensor with the sample;4. Measuring the signal from the working electrode, thereby sensing the presence of lactate 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 lactate in the sample.

13. A method of producing a TpBpy-COF comprising boronic acid-functionalized C60 (BA-Ceo), the method comprising:

1. combining triformylphloroglucinol (Tp), and 5,5'-diamino-2,2'-bipyridine (Bpy) to obtain a TpBpy-COF;2. combining Ceo, paraformaldehyde, and (4-aminobutan-2-yl)boronic acid pinacol ester, and removing the protective group to obtain BA-Ceo; and3. combining the TpBpy-COF with the BA-Ceo, thereby obtaining a TpBpy- COF comprising BA-Ceo.

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

1. providing a carbon based substrate, such as a pyrolytic carbon electrode; nanoparticles comprising or consisting of a metal or a metal oxide, such as PtNi, Pt, Ni, CuO, or NiO; and a TpBpy-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 TpBpy-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 lactate, optionally the kit may also comprises components for cleaning, such as washing buffers.

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