Description:ELECTROCHEMICAL BIOSENSING APPARATUS FOR DETECTION OF BOTH SHORT-TERM GLUCOSE LEVELS AND LONG-TERM GLYCEMIC STATUS
FIELD OF INVENTION
[0001] The present disclosure relates to electrochemical systems. More specifically, the present disclosure relates to an electrochemical biosensing apparatus for detecting both short-term glucose levels and long-term glycemic status and a method of producing the electrochemical biosensing apparatus.
BACKGROUND
[0002] Diabetes is a chronic metabolic disorder that significantly impacts millions of people worldwide. The management of diabetes requires continuous monitoring of both short-term glucose levels and long-term glycemic status, typically measured through glycated hemoglobin (HbA1c). Short-term glucose levels and HbA1c provide crucial information about a patient's immediate blood sugar levels and their average glucose control over the past two to three months, respectively. Effective diabetes management relies heavily on the accurate and timely measurement of glucose levels and HbA1c, as they guide treatment decisions, lifestyle modifications, and overall patient care strategies.
[0003] Currently, there exists a clear technological gap in the integration of glucose and HbA1c measurements into a single diagnostic platform. Most commercially available glucose meters rely solely on enzymatic sensing methods, such as glucose oxidase strips, which provide instantaneous blood glucose readings. The glucose meters are widely used for home-based monitoring and offer patients the ability to track their daily glucose fluctuations. However, they do not provide information about long-term glycemic control, which is essential for comprehensive diabetes management.
[0004] HbA1c assessment, on the other hand, is typically conducted separately using laboratory-based immunoassays. Common methods for HbA1c measurement include high-performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), and immunoturbidimetry. Such existing HbA1c testing methods, while accurate, present several limitations. The existing methods are often expensive, require specialized laboratory equipment, and necessitate sample preprocessing. Furthermore, existing methods of HbA1c measurement are not compatible with point-of-care or home-based testing, which limits their accessibility and frequency of use.
[0005] The separation of glucose and HbA1c testing into distinct processes and devices creates significant challenges for both patients and healthcare providers. Such division forces reliance on multiple devices and testing timelines, which increases the complexity of diabetes management. The use of separate testing methods also leads to higher overall costs and longer turnaround times for results. Such limiting factors can negatively impact patient adherence to regular monitoring schedules, particularly in resource-limited settings where access to healthcare facilities may be restricted.
[0006] Moreover, the lack of integration between glucose and HbA1c measurements hinders the ability to provide comprehensive, real-time assessments of a patient's glycemic status. This limitation can delay necessary adjustments to treatment plans and may result in suboptimal diabetes management. The inability to simultaneously measure both short-term and long-term glycemic markers on a single platform also presents challenges in research settings, where correlations between these parameters could provide valuable insights into diabetes progression and treatment efficacy.
[0007] Attempts have been made to develop more integrated approaches for diabetes monitoring. Some researchers have explored the use of continuous glucose monitoring (CGM) systems, which provide real-time glucose data over extended periods. While these systems offer valuable insights into daily glucose patterns, they still do not directly measure HbA1c levels. Other efforts have focused on developing non-invasive glucose monitoring techniques, such as optical or transdermal sensors, but these technologies are still in early stages of development and face challenges in terms of accuracy and reliability.
[0008] Therefore, there is a need to overcome the problems discussed above in diabetes management.
OBJECTIVES
[0009] The primary objective of the present disclosure is to provide a technical solution for detecting both short-term and long-term glycemic markers from a single sample.
[0010] Another objective of the present disclosure is to provide an integrated platform for detecting both short-term and long-term glycemic markers by combining two distinct sensing mechanisms such as enzymatic and immunosensing.
[0011] Yet another objective of the present disclosure is to enhance electron transfer kinetics, improve biomolecule immobilization, and ensure high sensitivity and stability in both sensing modes.
[0012] Yet another objective of the present disclosure is to provide a compact, real-time, and portable point-of-care diagnostics.
[0013] Yet another objective of the present disclosure is to reduce the complexity and cost associated with monitoring both short-term and long-term glycemic markers.
[0014] Yet another objective of the present disclosure is to provide a solution that improves patient compliance with regular glycemic monitoring by simplifying the testing process.
SUMMARY
[0015] The present disclosure addresses the limitations of existing approaches and provides a technical solution for simultaneous or sequential detection of both short-term glucose levels and long-term glycemic status from a single biological sample. The integrated approach overcomes the challenges associated with separate testing methods, offering a more efficient, cost-effective, and user-friendly platform for comprehensive diabetes monitoring.
[0016] According to one aspect of the present disclosure, a dual-analyte electrochemical biosensing apparatus is provided. The apparatus comprises a screen-printed electrochemical transduction platform comprising at least a first working electrode and a second working electrode spatially disposed on a substrate; a conductive nanostructured interface comprising a two-dimensional (2D) vanadium carbide-based MXene layer, operatively deposited on each of the working electrodes, the MXene layer facilitates electron transfer kinetics and provides a functional surface for biorecognition element immobilization; a glucose-sensing element formed by covalent or non-covalent immobilization of glucose oxidase (GOx) onto a MXene-modified surface of the first working electrode, for glucose sensing; and an immunoaffinity-based detection element formed by covalent or non-covalent immobilization of monoclonal anti-HbA1c antibodies onto a MXene-modified surface of the second working electrode for immunosensing of glycated hemoglobin (HbA1c). The biosensing apparatus is configured to detect both glucose and HbA1c from a unitary biological sample. The screen-printed electrochemical transduction platform is a carbon screen-printed electrochemical transduction platform.
[0017] The first working electrode is configured to detect glucose through electrochemical oxidation of hydrogen peroxide (H₂O₂) generated by enzymatic glucose oxidation. The electrochemical oxidation of H₂O₂ produces electrochemical signals that is proportional to glucose concentration in the sample.
[0018] The second working electrode is configured to detect HbA1c through measurement of interfacial electron transfer changes caused by antigen-antibody interactions. The interfacial electron transfer changes are transduced into electrochemical signals measured that is proportional to HbA1c concentration in the sample.
[0019] The screen-printed electrochemical transduction platform further comprises a shared counter electrode and a shared reference electrode for both the first working electrode and the second working electrode. The first working electrode and the second working electrode are configured to operate independently or in a sequentially triggered manner.
[0020] The 2D vanadium carbide-based MXene layer is deposited onto the first working electrode and the second working electrode via a solution-based deposition method comprising drop-casting. The MXene-modified surface of the second working electrode is activated using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in a carbodiimide-mediated coupling reaction prior to immobilization of the monoclonal anti-HbA1c antibodies.
[0021] The apparatus further comprises a portable electrochemical reader interfaced with the screen-printed electrochemical transduction platform, for applying a controlled electrochemical potential or current to the first working electrode and the second working electrode; obtaining real-time electrochemical signals generated as a result of biochemical interactions occurring at the surface of the respective electrodes; digitizing acquired analog signals via an onboard analog-to-digital converter (ADC); and determining quantitative levels of glucose and glycated hemoglobin (HbA1c) in the sample by executing embedded algorithms or firmware to analyze signal output corresponding to glucose concentration and HbA1c levels.
[0022] The electrochemical signals correspond to glucose is obtained by the portable electrochemical reader using amperometry or differential pulse voltammetry (DPV). The electrochemical signals correspond to HbA1c is obtained by the portable electrochemical reader using electrochemical impedance spectroscopy (EIS) or differential pulse voltammetry (DPV).
[0023] The apparatus is configured to be integrated into wearable or handheld personal care devices for real-time monitoring of short term and long term glucose levels.
[0024] According to another aspect of the present disclosure, a method for producing a dual-analyte electrochemical biosensing apparatus is provided. The method comprises providing a screen-printed electrochemical transduction platform comprising at least a first working electrode and a second working electrode spatially disposed on a substrate; depositing a conductive nanostructured interface comprising a two-dimensional (2D) vanadium carbide-based MXene layer on each of the working electrode; covalently or non-covalently immobilizing glucose oxidase (GOx) onto a MXene-modified surface of the first working electrode to form a glucose-sensing element; and covalently or non-covalently immobilizing monoclonal anti-HbA1c antibodies onto a MXene-modified surface of the second working electrode to form an immunoaffinity-based detection element. The biosensing apparatus is configured to detect both glucose and HbA1c from a unitary biological sample.
[0025] The 2D vanadium carbide-based MXene layer is deposited onto the first working electrode and the second working electrode via a solution-based deposition method comprising drop-casting.
[0026] The method further comprises activating the MXene-modified surface of the second working electrode using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in a carbodiimide-mediated coupling reaction prior to immobilization of the monoclonal anti-HbA1c antibodies.
[0027] The method further comprises interfacing a portable electrochemical reader screen-printed electrochemical transduction platform for: applying a controlled electrochemical potential or current to the first working electrode and the second working electrode; obtaining real-time electrochemical signals generated as a result of biochemical interactions occurring at the surface of the respective electrodes; digitizing acquired analog signals via an onboard analog-to-digital converter (ADC); and determining quantitative levels of glucose and glycated hemoglobin (HbA1c) in the sample by executing embedded algorithms or firmware to analyze signal output corresponding to glucose concentration and HbA1c levels.
[0028] The system and method of the present disclosure are advantageous in that they provide simultaneous or sequential detection of both glucose and HbA1c from a single sample, reducing testing time and sample volume requirements. By integrating enzymatic and immunosensing mechanisms on a single platform, the need for separate devices or assays is eliminated. The use of vanadium-based MXene (V2CT), enhances sensitivity and stability, while enabling real-time, label-free detection without complex sample preparation. The compact and portable design is suitable for point-of-care diagnostics and potential integration into wearable devices, offering improved cost-effectiveness and increased potential for patient compliance due to simplified testing procedures. This more comprehensive glycemic monitoring approach allows for better-informed treatment decisions and diabetes management strategies, with the potential for adaptation to detect additional biomarkers related to diabetes or other chronic conditions. Furthermore, its compatibility with existing electrochemical measurement techniques facilitates easier adoption and integration into current clinical practices.
[0029] The foregoing paragraphs have been provided by way of general introduction and are not intended to limit the scope of the following claims. The described embodiments, together with further advantages, will be best understood by reference to the following detailed description taken in conjunction with the accompanying drawings.
BRIEF DESCRIPTION OF DRAWINGS
[0030] FIG. 1 is a block diagram illustrating a dual-analyte electrochemical biosensing apparatus in accordance with the present disclosure.
[0031] FIG. 2 is a schematic illustration of a screen-printed electrochemical transduction platform in accordance with the present disclosure.
[0032] FIG. 3 is a schematic illustration of a first working electrode in accordance with the present disclosure.
[0033] FIG. 4 is a schematic illustration of a second working electrode in accordance with the present disclosure.
[0034] FIG. 5 is a flowchart illustrating a method of producing dual-analyte electrochemical biosensing apparatus in accordance with the present disclosure.
DETAILED DESCRIPTION OF THE PRESENT DISCLOSURE
[0035] Aspects of the present invention are best understood by reference to the description set forth herein. All the aspects described herein will be better appreciated and understood when considered in conjunction with the following descriptions. It should be understood, however, that the following descriptions, while indicating preferred aspects and numerous specific details thereof, are given by way of illustration only and should not be treated as limitations. Changes and modifications may be made within the scope herein without departing from the spirit and scope thereof, and the present invention herein includes all such modifications.
[0036] As mentioned above, there is a need for a technical solution to solve aforementioned technical problems in diabetes monitoring and management. The present invention provides a dual-mode electrochemical biosensing apparatus for simultaneous detection of short-term glucose levels and long-term glycemic status (HbA1c) from a unitary biological sample. The biosensing apparatus comprises an integrated sensing platform with spatially separated working electrodes on a single substrate, utilizing a conductive nanostructured interface of two-dimensional vanadium carbide-based MXene for modifying the surface of the working electrodes. The nanomaterial enhances electron transfer kinetics and biomolecule immobilization of the working electrodes. The apparatus employs dual sensing mechanisms such as enzymatic glucose detection using glucose oxidase on one working electrode, and immunosensing of HbA1c using monoclonal antibodies on the other working electrode. The apparatus offers real-time, label-free detection without complex sample preparation. Further, the compact, portable design of the apparatus suits for point-of-care diagnostics and potential wearable integration, while the Vanadium-based MXene (V2CT) enhances sensitivity and stability. Compatible with various electrochemical measurement techniques, the apparatus allows for versatile and customizable functionality. Integration of the electrochemical platform with a portable reader for enhanced usability and possesses the potential for expanding to detect additional biomarkers. The comprehensive approach significantly improves upon existing separate testing methods for glucose and HbA1c, addressing the need for efficient and user-friendly glycemic monitoring.
[0037] Now referring to FIG. 1, FIG. 1 is a block diagram illustrating a dual-analyte electrochemical biosensing apparatus 100 in accordance with the present disclosure. The biosensing apparatus 100 comprises a screen-printed electrochemical transduction platform 102 and a portable electrochemical reader 104. The electrochemical transduction platform 102 includes a first working electrode 106, a second working electrode 108, a reference electrode 110, and a counter electrode 112. The electrochemical transduction platform 102 enables simultaneous or sequential detection of glucose and glycated hemoglobin (HbA1c) from a single drop of human serum. The electrochemical transduction platform 102” refers to a sensing interface that converts a biochemical interaction (such as enzyme–substrate binding, antigen–antibody recognition, or analyte adsorption) into a measurable electrical signal, typically involving current, voltage, or impedance changes. The first working electrode 106 and second working electrode 108 refer to sensing element configured to selectively interact with target analytes and generate electrochemical signals proportional to its concentration. The reference electrode 110 refers to a stable electrode with a well-defined and constant potential that provides a fixed reference point for measuring the potential changes at the working electrodes during electrochemical measurements. The counter electrode 112 refers to electrode that completes the electrochemical circuit by allowing current to flow between itself and the working electrode(s), typically made from an inert conductive material to ensure minimal interference with the sensing process.
[0038] The screen-printed electrochemical transduction platform 102 comprises a substrate that provides mechanical support and a conductive layer which is deposited over the substrate. In some embodiments, the conductive layer is formed from materials such as carbon, gold, or platinum, selected based on the desired electrochemical performance. In one exemplary embodiment, the conductive layer is formed from carbon material. In some embodiments, the conductive layer is fabricated using screen-printing technology, which allows for cost-effective and reproducible production of electrodes on a single substrate. In some embodiments, the conductive layer is fabricated using other methods like photolithography or inkjet printing, offering different levels of precision and scalability.
[0039] The conductive layer is patterned to define two working electrodes, a first working electrode 106 and a second working electrode 108, along with corresponding separate conductive traces that terminate at individual contact pads for external connection. Patterning may be achieved during screen-printing by using discrete stencil apertures, or post-deposition via subtractive methods (e.g., laser ablation, oxygen plasma etch) or additive printing (e.g., inkjet micro-deposition of insulating barriers) to electrically isolate the two working electrodes.
[0040] An insulating overprint (dielectric layer) may be applied to cover the traces while leaving defined sensing windows (e.g., circular or rectangular apertures) over each working electrode. The inter-electrode gap can be selected to minimize cross-talk (e.g., ~200–1500 µm), and the geometric area of each electrode can be set independently (e.g., ~1–5 mm²) to tune current density and sensitivity. Registration marks may be included to ensure layer-to-layer alignment during multi-pass printing. In one embodiment, the first working electrode 106 and the second working electrode 108 are fabricated from carbon ink in the same print step.
[0041] The first working electrode 106 and the second working electrode 108 share a common reference electrode 110 and a counter electrode 112, thereby optimizing space utilization and simplifies the overall sensor architecture while maintaining accurate electrochemical measurements. The reference electrode 110 serves as a stable potential reference point for both working electrodes 106 and 108. In some embodiments, the reference electrode 110 is fabricated using silver or silver chloride (Ag/AgCl) or other materials known for their stable electrochemical potential. The reference electrode 110 ensures that the potential applied to or measured from the working electrodes 106 and 108 is accurately controlled and interpreted. The counter electrode 112, also shared by both working electrodes 106 and 108, completes the electrical circuit and balances the current generated at the working electrodes 106 and 108. In some embodiments, the counter electrode 112 is made of an inert conductive material such as platinum or carbon, chosen for its ability to support the necessary electrochemical reactions without interfering with the sensing processes. The shared electrode configuration offers several advantages. For instance, by utilizing a single reference and counter electrode for both working electrodes 106 and 108, the overall size of the sensor is reduced, making it more suitable for portable and point-of-care applications. The production process is streamlined as fewer individual components need to be manufactured and assembled. Using the same reference and counter electrodes for both sensing mechanisms ensures a consistent electrochemical environment, enhancing the comparability of measurements between the two working electrodes 106 and 108. The shared electrode configuration reduces material costs and simplifies the manufacturing process, contributing to the overall cost-effectiveness of the biosensor. The proximity of the shared electrodes to both working electrodes 106 and 108 can help minimize electrical noise and improve the quality of the electrochemical signals. The two working electrodes 106 and 108 are positioned at an appropriate distance from each other to prevent cross-talk, while remaining close enough to the shared reference and counter electrodes to maintain measurement accuracy.
[0042] The surface of the first working electrode 106 and the second working electrode 108 are further modified with vanadium-based MXene (V₂CTₓ), thereby forming a conductive nanostructured interface which will be explained in detail in FIGS. 3 and 4. The modification of the surface of the first working electrode 106 and the second working electrode 108 enhances the electrochemical performance and functionality of the working electrodes 106 and 108.
[0043] The first working electrode 106 is further functionalized with glucose oxidase (GOx), an enzyme specific for glucose detection, thereby enabling the enzymatic sensing of glucose on the MXene-modified electrode surface of the first working electrode 106. The second working electrode 108 is further functionalized with anti-HbA1c monoclonal antibodies for detection of glycated hemoglobin (HbA1c), enabling the immunosensing of HbA1c on the MXene-modified electrode surface of the second working electrode 108. It is to be noted that the functionalization of the first working electrode 106 and the second working electrode 108 will be explained in detail in FIGS 3 and 4.
[0044] The portable electrochemical reader 104 interfaces with the screen-printed electrochemical transduction platform 102 to record and analyze the electrochemical responses from the first working electrode 106 and the second working electrode 108. The portable electrochemical reader 104 includes, but not limited to, a potentiostat circuit for controlling and measuring electrode potentials and currents; an analog-to-digital converter (ADC) for signal digitization; a microcontroller for overall operation management; dedicated modules for electrochemical techniques such as amperometry, differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS); signal processing units for filtering and amplifying acquired signals; memory for data and parameter storage; a power management system for regulating power supply and battery management; a user interface with display and input options; communication modules for data transfer to external devices; and data analysis software for interpreting raw data into meaningful glucose and HbA1c readings. The portable electrochemical reader 104 also incorporates calibration and quality control features to ensure measurement accuracy. The integrated design, housed in a robust, portable casing, enables versatile measurement capabilities for both glucose and HbA1c detection, ensuring optimal signal capture, analysis, and interpretation from both the enzymatic glucose sensing and immunosensing for HbA1c. The combination of these components enhances the overall usability and effectiveness of the biosensing apparatus, making it suitable for various settings from clinical environments to point-of-care diagnostics and field testing.
[0045] In some embodiments, the portable electrochemical reader 104 is configured to apply, using the potentiostat circuit, a controlled electrochemical potential or current to the first working electrode 106 and the second working electrode 108; obtaining, using the signal processing unit, real-time electrochemical signals generated as a result of biochemical interactions occurring at the surface of the respective electrodes; digitizing, using the analog-to-digital converter (ADC), acquired analog signals; and determining, using the microcontroller, quantitative levels of glucose and glycated hemoglobin (HbA1c) in the sample by executing embedded algorithms or firmware to analyze signal output corresponding to glucose concentration and HbA1c levels.
[0046] In some embodiments, the electrochemical signals corresponding to glucose are obtained by the portable electrochemical reader 104 using amperometry or differential pulse voltammetry (DPV). The amperometry involves applying a constant potential to the working electrode and measuring the resulting steady-state current proportional to the glucose concentration. DPV involves superimposing a series of voltage pulses on a linear potential ramp to enhance sensitivity and reduce background noise for low-level glucose detection. In some embodiments, the electrochemical signals corresponding to glycated hemoglobin (HbA1c) are obtained by the portable electrochemical reader 104 using electrochemical impedance spectroscopy (EIS). The electrochemical impedance spectroscopy applies a small amplitude alternating current (AC) signal over a range of frequencies to evaluate changes in the interfacial charge transfer resistance associated with HbA1c binding events, or using differential pulse voltammetry (DPV) to resolve redox peaks corresponding to HbA1c-related electrochemical reactions with high resolution..
[0047] The dual-analyte electrochemical biosensing apparatus 100 operates by applying a single drop of human serum onto the screen-printed electrochemical transduction platform 102. The glucose present in the serum is oxidized by the GOx enzyme on the first working electrode 106, generating hydrogen peroxide (H2O2). The electrochemical oxidation of H2O2 produces a current response proportional to the glucose concentration, which is measured by the portable electrochemical reader 104. Simultaneously or sequentially, the HbA1c molecules in the sample bind to the anti-HbA1c antibodies immobilized on the second working electrode 108. The binding event alters the electrode's interfacial properties, which is detected through changes in impedance or voltammetric response.
[0048] In some embodiments, the biosensing apparatus 100 is configured to be integrated into wearable or handheld personal care devices for real-time monitoring of short term and long term glucose levels. The biosensing apparatus 100 into various form factors such as smartwatches, fitness trackers, adhesive patches, smart contact lenses, smartphone accessories, enhanced portable glucose meters, and personal digital assistants for diabetes management. Such integration offers numerous advantages including continuous monitoring, improved compliance through convenience, seamless data integration with digital health platforms, personalized alerts for glycemic events, and correlation with other health metrics for a holistic approach to diabetes management. The miniaturization and flexibility of the biosensing apparatus 100 allow for non-invasive or minimally invasive monitoring through analysis of interstitial fluid, sweat, or tears, depending on the specific device design. The approach to integrating biosensing technology into everyday wearable and handheld devices represents a significant advancement in diabetes care, potentially improving patient outcomes by providing more frequent, detailed, and easily accessible glycemic data. It enables timely interventions, personalized treatment strategies, and a more user-friendly experience in managing both short-term glucose levels and long-term glycemic status through HbA1c measurements, all within compact and convenient personal devices.
[0049] With reference to FIG. 1, FIG. 2 is a schematic illustration of the screen-printed electrochemical transduction platform 102 in accordance with the present disclosure. The screen-printed electrochemical transduction platform 102 comprises the first working electrode 106, the second working electrode 108, the reference electrode 110 and the counter electrode 112. To avoid repetition, the details of these components of the electrochemical transduction platform 102 described earlier in FIG. 1 will not be reiterated here.
[0050] With reference to FIGS 1-2, FIG. 3 is a schematic illustration of the first working electrode 106 in accordance with the present disclosure. FIG. 3 depicts a layered structure of the first working electrode 106. The first working electrode 106 comprises a carbon layer 302, a conductive nanostructured interface 304, and a glucose-sensing element 306. The first working electrode 106 enables efficient glucose detection through an enzymatic sensing mechanism, utilizing the properties of the vanadium-based MXene (V2CT) as the conductive interface. The first working electrode 106 is fabricated as part of the screen-printed carbon electrode (CSPE) substrate, providing a stable and conductive base for the subsequent layers. The carbon layer 302 on the first working electrode enhances overall conductivity and provides a suitable surface for further modifications. The carbon layer 302 may be composed of various carbon-based materials, such as graphite, carbon black, or carbon nanotubes, each offering specific advantages in terms of conductivity, surface area, and compatibility with biomolecules. In some embodiments, the carbon layer 302 is formed using a screen printed method.
[0051] The conductive nanostructured interface 304 consists of vanadium-based MXene (V2CT), which is deposited onto the carbon layer 302, thereby the modifying the electrode surface of the first working electrode 106. The MXene layer enhances the electrochemical performance of the first working electrode 106. The V2CT layer is synthesized through selective etching and delamination techniques, resulting in a two-dimensional nanostructure with high electrical conductivity and high surface area. The deposition of V2CT is typically achieved through methods such as drop-casting, spin-coating, or electrodeposition, followed by a controlled drying process to ensure uniform coverage. In some embodiments, Vanadium carbide MXene is synthesized by preparing an etching agent in deionized water through the addition of hydrofluoric acid (HF) along with hydrochloric acid (HCl). Subsequently, V2AlC MAX powder is gradually added to the aforementioned etching solution and is then stirred. Once the etching process is completed, the solution is washed multiple times with deionized water and centrifuged. After that, a greyish-black powder is formed by vacuum-drying.
[0052] . The V2CT interface plays a crucial role in enhancing electron transfer kinetics and improving the immobilization of biomolecules. Alternative MXene compositions, such as Ti3C2Tx or Nb2CTx, could potentially be used, each offering unique properties that may be advantageous for specific sensing applications.
[0053] The glucose-sensing element 306 is composed of glucose oxidase (GOx) enzymes 305 immobilized on the conductive nanostructured interface 304. The immobilization process involves depositing a GOx solution onto the conductive nanostructured interface 304, followed by a drying step to form a stable GOx/V2CT/CSPE composite. The glucose-sensing element 306 is responsible for the selective recognition and oxidation of glucose molecules. The high surface area and biocompatibility of the conductive nanostructured interface 304 contribute to efficient enzyme loading and preservation of enzymatic activity. Alternative glucose-sensing enzymes, such as glucose dehydrogenase, may be explored for specific applications requiring different catalytic properties or stability characteristics.
[0054] The glucose molecules 308 represent the target analyte in the sample. When a drop of human serum containing glucose is applied to a sensor surface of the first working electrode 106, the glucose molecules 308 molecules diffuse through the layers and interact with the immobilized GOx enzymes 305. The enzymatic reaction oxidizes glucose to gluconic acid, simultaneously producing hydrogen peroxide (H2O2) as a by-product. The reaction can be represented as:
Glucose + O2 + H2O → Gluconic acid + H2O2
[0055] The electrochemical detection of glucose is based on the subsequent oxidation of the generated H2O2 at the sensor surface of the first working electrode 106. The oxidation process produces electrons, resulting in a measurable current signal that is proportional to the glucose concentration in the sample. The reaction can be described as:
H2O2 → O2 + 2H+ + 2e-
[0056] The electron transfer process is facilitated by the conductive nanostructured interface 304, which acts as an electron mediator between the enzymatic reaction site and the electrode surface, thereby enhancing the sensitivity and response time of the glucose-sensing element 306. The generated current can be measured using various electrochemical techniques, such as amperometry or differential pulse voltammetry (DPV), providing quantitative information about glucose levels in the sample.
[0057] The glucose-sensing element 306 on the first working electrode 106 operates independently from the HbA1c detection on the second working electrode 108, allowing for simultaneous or sequential measurement of both biomarkers from a single sample. The integration of the V2CT interface and the optimized layered structure contributes to the high sensitivity, stability, and real-time detection capabilities of the dual-mode biosensor.
[0058] With reference to FIGS. 1-3, FIG. 4 is a schematic illustration of the second working electrode 108 in accordance with the present disclosure. FIG. 4 depicts the layered structure of the second working electrode 108 and the immunoaffinity-based detection process for glycated hemoglobin (HbA1c). The second working electrode 108 comprises a carbon layer 402, a conductive nanostructured interface 404, and an immunoaffinity-based detection element 406.
[0059] The carbon layer 402 forms the base of the second working electrode 108. The carbon layer 402 is typically composed of screen-printed carbon ink, which offers excellent electrical conductivity and electrochemical stability. The carbon layer 402 provides a cost-effective and versatile substrate for further modification and functionalization. Alternative materials for this layer could include graphite, carbon nanotubes, or graphene, each offering unique properties that may enhance the overall performance of the electrode.
[0060] The carbon layer 402 of the second working electrode 108 is formed simultaneously with the carbon layer 302 of the first working electrode 106 in a single screen-printing step. The simultaneous formation of the carbon layers 302 and 402 through a single printing process ensures uniformity in thickness, conductivity, and electrochemical properties across both working electrodes 106 and 108. This approach not only provides a cost-effective and versatile substrate for further modifications but also enhances the reproducibility of the sensor fabrication. The shared carbon layer serves as a consistent foundation, upon which subsequent differential functionalization will be applied to tailor each electrode for its specific sensing purpose such as glucose detection on the first working electrode 106 and HbA1c detection on the second working electrode 108.
[0061] The conductive nanostructured interface 404 is deposited on top of the carbon layer 402. The V2CT layer is synthesized via selective etching and delamination techniques, resulting in a two-dimensional nanostructure with abundant active sites for biomolecule immobilization. The conductive nanostructured interface 404 plays a crucial role in enhancing the electron transfer kinetics and improving the overall sensitivity of the HbA1c detection. The conductive nanostructured interface 404 of the second working electrode 108 is deposited simultaneously with the conductive nanostructured interface 304 of the first working electrode 106 in a single modification step. The V2CT layer is common for both working electrodes 106 and 108. The deposition of this shared V2CT layer is typically achieved through methods such as drop-casting, spin-coating, or electrodeposition, followed by a controlled drying process to ensure uniform coverage across both electrodes. This simultaneous modification ensures consistency in the enhanced electron transfer kinetics and sensitivity for both glucose and HbA1c detection. The shared V2CT layer serves as a uniform foundation for the subsequent differential functionalization of each electrode, tailoring them for their specific sensing purposes while maintaining comparable electrochemical properties.
[0062] The immunoaffinity-based detection element 406 is immobilized on the conductive nanostructured interface 404 of the second working electrode 108. The immunoaffinity-based detection element 406 consists of anti-HbA1c monoclonal antibodies 405 that are specifically configured to capture and bind HbA1c molecules from the sample. The immobilization process involves activating the V2CT surface using EDC/NHS (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-Hydroxysuccinimide) chemistry, followed by covalent or non-covalent attachment of the antibodies.
[0063] The HbA1c antigen 408 represents the target analyte in the sample. When a drop of serum is applied to the second working electrode 108, HbA1c molecules present in the sample specifically bind to the immobilized anti-HbA1c antibodies on the second working electrode 108. The antigen-antibody interaction forms a complex layer on the electrode surface of the second working electrode 108, altering the interfacial electron transfer properties. The change in electron transfer characteristics is then measured using electrochemical techniques such as electrochemical impedance spectroscopy (EIS) or differential pulse voltammetry (DPV).
[0064] This immunosensing approach allows for label-free, real-time detection of HbA1c from unprocessed human serum. The use of V2CT as the nanostructured interface enhances the sensitivity and stability of the immunosensor, while the spatial separation from the glucose-sensing electrode (W1) ensures minimal interference between the two detection modes. Alternative antibody immobilization strategies, such as biotin-streptavidin coupling or click chemistry, could be explored to further optimize the sensing performance and stability of the immunoaffinity-based detection element.
[0065] With reference to FIGS. 1-4, FIG. 5 is a flowchart illustrating a method of producing a dual-analyte electrochemical biosensing apparatus 100 and detecting both short term and long term glucose levels using the dual-analyte electrochemical biosensing apparatus 100 in accordance with the present disclosure. At step 502, the method includes providing the screen-printed electrochemical transduction platform 102 comprising at least the first working electrode 106 and the second working electrode 108 spatially disposed on a substrate. In some embodiments, the preparation of the platform 102 involves selecting and cleaning a substrate material, for instance polyethylene terephthalate (PET) or ceramic; designing a custom screen with a desired electrode pattern the layouts for the first working electrode 106, the second working electrode 108, the reference electrode 110, and the counter electrode 112.; printing carbon ink onto the substrate in a single step, forming the base layer for all working electrodes; drying and curing the printed carbon layer under controlled temperature conditions to ensure proper adhesion and conductivity; printing the reference electrode material, for instance silver/silver chloride ink, onto a designated area; if required, printing an additional layer of conductive material (e.g., platinum-based ink) over the carbon layer in a counter electrode area to improve its performance; printing an insulation layer over the electrode traces, leaving only the active areas of the electrodes and contact pads exposed. performing final curing to ensure the stability and durability of all printed layers. This multi-step screen-printing process results in the electrochemical transduction platform 102 with spatially separated working electrodes 106 and 108, ready for further modification and functionalization.
[0066] At step 504, the method includes depositing a conductive nanostructured interface 304 and 404 comprising a two-dimensional (2D) vanadium carbide-based MXene layer on each of the working electrode 106 and 108. The deposition of the 2D vanadium carbide-based MXene is performed simultaneously on both electrodes 106 and 108 to ensure uniformity and consistency. The process begins with the synthesis of V2CT MXene through selective etching of the MAX phase precursor (V2AlC) using a hydrofluoric acid solution, followed by delamination to obtain 2D nanosheets. The resulting V2CT suspension is then prepared with optimized concentration and dispersity. The deposition onto the working electrodes 106 and 108 is typically achieved through techniques such as drop-casting, spin-coating, or electrodeposition, with precise control over parameters like volume, speed, and duration to ensure uniform coverage. After deposition, the electrodes 106 and 108 undergo a controlled drying process, often involving mild heating or vacuum treatment, to remove excess solvent and promote adhesion. In some cases, a post-deposition annealing step may be employed to further enhance the electrical and mechanical properties of the MXene layer. The created a high-surface-area, conductive interface that significantly enhances electron transfer kinetics, provides abundant active sites for subsequent biomolecule immobilization, and improves the overall electrochemical performance of both working electrodes. The simultaneous modification of both electrodes with V2CT ensures that they share identical nanostructured interfaces, which is crucial for maintaining consistency in the subsequent differential functionalization for glucose and HbA1c sensing.
[0067] At step 506, the method includes covalently or non-covalently immobilizing glucose oxidase (GOx) 305 onto a MXene-modified surface 304 of the first working electrode 106 to form a glucose-sensing element 306. For covalent immobilization, the V2CT MXene surface is first activated using coupling agents like EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-Hydroxysuccinimide) to create reactive groups. The GOx solution is then applied to the electrode surface of the first working electrode 106 using techniques such as drop-casting, spin-coating, or electrochemical deposition. The immobilization occurs either through covalent bonding with the activated surface groups or via non-covalent interactions like physical adsorption or electrostatic forces. The electrode undergoes a controlled incubation period under specific temperature and humidity conditions to ensure optimal enzyme attachment and distribution. Excess or unbound enzyme is removed through gentle washing with buffer solutions. In some cases, additional cross-linking agents like glutaraldehyde may be applied to enhance the stability of the immobilized enzyme layer. Finally, the functionalized electrode is dried and stored under conditions that preserve enzyme activity.
[0068] At step 508, the method includes covalently or non-covalently immobilizing monoclonal anti-HbA1c antibodies 405 onto a MXene-modified surface 404 of the second working electrode 108 to form an immunoaffinity-based detection element 406. For covalent immobilization, the V2CT MXene surface 404 is first activated using EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-Hydroxysuccinimide) to create reactive carboxyl groups. The antibody solution is then applied to the electrode surface of the second working electrode 108 using precision techniques such as microspotting, inkjet printing, or electrochemical deposition. The immobilization occurs either through covalent bonding between the antibodies' primary amine groups and the activated surface, or via non-covalent interactions such as physical adsorption or electrostatic forces, depending on the chosen method. The second working electrode 108 undergoes a controlled incubation period under specific temperature and humidity conditions to ensure optimal antibody attachment, orientation, and distribution. Excess or unbound antibodies are removed through gentle washing with buffer solutions, and any remaining active sites on the surface are blocked with inert proteins (e.g., bovine serum albumin) to prevent non-specific binding. In some cases, additional stabilizing agents may be applied to enhance the long-term stability and activity of the immobilized antibodies. Finally, the functionalized electrode is dried and stored under conditions that preserve antibody functionality.
[0069] At step 510, the method includes applying the unitary biological sample to the electrode platform 102. For instance, a small volume (e.g., 5-50 μL) of human serum or whole blood, is applied onto the dual-functionalized electrode surface. Techniques such as micropipetting, automated dispensing systems, or specialized microfluidic channels may be employed to achieve accurate and reproducible sample delivery. The sample volume is optimized to provide sufficient analyte for both sensing mechanisms while minimizing the required blood draw from the patient. Upon application, the sample rapidly spreads across the electrode surface, facilitated by the hydrophilic nature of the MXene-modified electrodes. This spreading ensures efficient contact between the analytes (glucose and HbA1c) and their respective biorecognition elements (immobilized glucose oxidase and anti-HbA1c antibodies).
[0070] At step 512, the method includes detecting both glucose and HbA1c from the unitary biological sample. The dual detection process occurs simultaneously or in rapid succession on the functionalized electrode platform. For glucose detection, the immobilized glucose oxidase on the first working electrode 106 catalyzes the oxidation of glucose to gluconic acid and hydrogen peroxide. The electrochemical oxidation of the generated hydrogen peroxide is then measured, using amperometry or differential pulse voltammetry (DPV), producing a current signal proportional to the glucose concentration. Concurrently or sequentially, HbA1c detection takes place on the second working electrode 108, where HbA1c molecules in the sample bind specifically to the immobilized anti-HbA1c antibodies. The binding event alters the electrode's interfacial properties, which is measured using techniques such as electrochemical impedance spectroscopy (EIS) or DPV. The magnitude of signal change correlates with the HbA1c concentration.
[0071] The portable electrochemical reader 104 interfaced with the platform 102 applies the potential or current profiles to both working electrodes 106 and 108 and records the resulting electrochemical responses. Further, the signal processing algorithms analyze the responses, compensating for any background signals or interferents, and convert them into quantitative measurements of glucose and HbA1c levels. The dual-mode detection is optimized to minimize cross-talk between the two sensing mechanisms while maximizing sensitivity and accuracy for both analytes. This integrated approach allows for comprehensive glycemic assessment from a single sample application, providing immediate information on both short-term (glucose) and long-term (HbA1c) glycemic status, crucial for effective diabetes management.
[0072] Accordingly, the apparatus and method of the present disclosure offers multiple advantages over conventional approaches by integrating dual-mode sensing capabilities on the single carbon screen-printed electrode (CSPE), allowing simultaneous enzyme-based and antibody-based detection without the need for separate electrodes, thereby reducing hardware complexity, footprint, and cost. The use of vanadium-based MXene (V₂CTₓ) as a shared nanomaterial interface provides high conductivity, large surface area, and active functional groups, enabling stable immobilization of both enzymes and antibodies for enhanced sensitivity and stability. A controlled immobilization spatially separates enzyme and antibody functionalization on adjacent electrode regions, minimizing cross-contamination and signal interference. The apparatus and method supports real-time, label-free electrochemical detection from a single drop of unprocessed human serum via DPV/EIS/amperometry, eliminating complex sample preparation and enabling rapid point-of-care testing. An integrated multiplexed readout interface allows independent activation and selective or combined signal capture from the dual working electrodes 106 and 108, enhancing versatility and diagnostic capability. This combination of advanced electrode configuration, novel nanomaterial usage, and portable multiplexed measurement delivers superior detection accuracy, operational efficiency, and portability for diverse field applications..
[0073] The embodiments of the present invention disclosed herein are intended to be illustrative and not limiting. Other embodiments are possible and modifications may be made to the embodiments without departing from the spirit and scope of the invention. As such, these embodiments are only illustrative of the inventive concepts contained herein.
, Claims:1. A dual-analyte electrochemical biosensing apparatus (100), the apparatus (100) comprising:
a screen-printed electrochemical transduction platform (102) comprising at least a first working electrode (106) and a second working electrode (108) spatially disposed on a substrate;
a conductive nanostructured interface (304, 404) comprising a two-dimensional (2D) vanadium carbide-based MXene layer, operatively deposited on each of the working electrodes (106, 108), wherein the MXene layer facilitates electron transfer kinetics and provides a functional surface for biorecognition element immobilization;
a glucose-sensing element (306) formed by covalent or non-covalent immobilization of glucose oxidase (GOx) (305) onto a MXene-modified surface (304) of the first working electrode (106), for glucose sensing; and
an immunoaffinity-based detection element (406) formed by covalent or non-covalent immobilization of monoclonal anti-HbA1c antibodies (405) onto a MXene-modified surface (404) of the second working electrode (108) for immunosensing of glycated hemoglobin (HbA1c),
wherein the biosensing apparatus (100) is configured to detect both glucose and HbA1c from a unitary biological sample.
2. The apparatus (100) as claimed in claim 1, wherein the screen-printed electrochemical transduction platform (102) further comprises a shared counter electrode (112) and a shared reference electrode (110) for both the first working electrode (106) and the second working electrode (108).
3. The apparatus (100) as claimed in claim 1, wherein the screen-printed electrochemical transduction platform (102) is a carbon screen-printed electrochemical transduction platform.
4. The apparatus (100) as claimed in claim 1, wherein the 2D vanadium carbide-based MXene layer (304, 404) is deposited onto the first working electrode (106) and the second working electrode (108) via a solution-based deposition method comprising drop-casting.
5. The apparatus (100) as claimed in claim 1, wherein the MXene-modified surface (404) of the second working electrode (108) is activated using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in a carbodiimide-mediated coupling reaction prior to immobilization of the monoclonal anti-HbA1c antibodies.
6. The apparatus (100) as claimed in claim 1, wherein the first working electrode (106) is configured to detect glucose through electrochemical oxidation of hydrogen peroxide (H₂O₂) generated by enzymatic glucose oxidation, wherein the electrochemical oxidation of H₂O₂ produces electrochemical signals that is proportional to glucose concentration in the sample.
7. The apparatus (100) as claimed in claim 1, wherein the second working electrode (108) is configured to detect HbA1c through measurement of interfacial electron transfer changes caused by antigen-antibody interactions, wherein the interfacial electron transfer changes are transduced into electrochemical signals measured that is proportional to HbA1c concentration in the sample.
8. The apparatus (100) as claimed in claim 1, further comprising a portable electrochemical reader (104) interfaced with the screen-printed electrochemical transduction platform (102), for
applying a controlled electrochemical potential or current to the first working electrode (106) and the second working electrode (108);
obtaining real-time electrochemical signals generated as a result of biochemical interactions occurring at the surface of the respective electrodes (106 108);
digitizing acquired analog signals via an onboard analog-to-digital converter (ADC); and
determining quantitative levels of glucose and glycated hemoglobin (HbA1c) in the sample by executing embedded algorithms or firmware to analyze signal output corresponding to glucose concentration and HbA1c levels.
9. The apparatus (100) as claimed in claim 8, wherein the electrochemical signals correspond to glucose is obtained by the portable electrochemical reader (104) using amperometry or differential pulse voltammetry (DPV).
10. The apparatus (100) as claimed in claim 8, wherein the electrochemical signals correspond to HbA1c is obtained by the portable electrochemical reader (104) using electrochemical impedance spectroscopy (EIS) or differential pulse voltammetry (DPV).
11. The apparatus (100) as claimed in claim 1, wherein the first working electrode (106) and the second working electrode (108) are configured to operate independently or in a sequentially triggered manner.
12. The apparatus (100) as claimed in claim 1, wherein the apparatus (100) is configured to be integrated into wearable or handheld personal care devices for real-time monitoring of short term and long term glucose levels.
13. A method for producing a dual-analyte electrochemical biosensing apparatus (100), the method comprising:
providing a screen-printed electrochemical transduction platform (102) comprising at least a first working electrode (106) and a second working electrode (108) spatially disposed on a substrate;
depositing a conductive nanostructured interface (304, 404) comprising a two-dimensional (2D) vanadium carbide-based MXene layer on each of the working electrode (106, 108);
covalently or non-covalently immobilizing glucose oxidase (GOx) (305) onto a MXene-modified surface (304) of the first working electrode (106) to form a glucose-sensing element (306); and
covalently or non-covalently immobilizing monoclonal anti-HbA1c antibodies (405) onto a MXene-modified surface (404) of the second working electrode (108) to form an immunoaffinity-based detection element (406).
14. The method as claimed in claim 13, wherein the screen-printed electrochemical transduction platform (102) further comprises a shared counter electrode (112) and a shared reference electrode (110) for both the first working electrode (106) and the second working electrode (108).
15. The method as claimed in claim 13, wherein the 2D vanadium carbide-based MXene layer (304, 404) is deposited onto the first working electrode (106) and the second working electrode (108) via a solution-based deposition method comprising drop-casting.
16. The method as claimed in claim 13, further comprising activating the MXene-modified surface (404) of the second working electrode (108) using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in a carbodiimide-mediated coupling reaction prior to immobilization of the monoclonal anti-HbA1c antibodies.
17. The method as claimed in claim 13, further comprising interfacing a portable electrochemical reader (104) with the screen-printed electrochemical transduction platform (102) for:
applying a controlled electrochemical potential or current to the first working electrode (106) and the second working electrode (108);
obtaining real-time electrochemical signals generated as a result of biochemical interactions occurring at the surface of the respective electrodes (106, 108);
digitizing acquired analog signals via an onboard analog-to-digital converter (ADC); and
determining quantitative levels of glucose and glycated hemoglobin (HbA1c) in the sample by executing embedded algorithms or firmware to analyze signal output corresponding to glucose concentration and HbA1c levels.