Description:FIELD OF THE INVENTION
[0001] The invention relates to the field of electrochemical biosensors and medical diagnostics, and more particularly to a nanozyme-based sensor that employs copper selenite nanoparticles anchored on functionalized carbon nanofibers to enhance the accuracy of glutathione detection in biological samples, enabling improved cardiovascular disease monitoring and diagnosis through superoxide dismutase-mimicking catalytic activity and optimized electrochemical analysis.

DESCRIPTION OF THE RELATED ART
[0002] The following description of the related art is intended to provide background information pertaining to the field of disclosure. This section may include certain aspects of the art that may be related to various features of the present disclosure. However, it should be appreciated that this section is used only to enhance the understanding of the reader with respect to the present disclosure, and not as admissions of the prior art.
[0003] Glutathione (GSH) is a vital biological macromolecule and the predominant non-protein thiol in living cells that serves as a crucial antioxidant to preserve cellular redox equilibrium and safeguard against oxidative damage. GSH concentrations in cells typically range from 0.5 to 10 mM, while in biological fluids such as plasma, GSH levels are diminished, usually around 2-12 μM in healthy individuals. The equilibrium between reduced glutathione (GSH) and oxidized glutathione (GSSG) serves as a crucial indicator of cellular oxidative stress, with an elevated GSH/GSSG ratio indicating a more favorable physiological condition.
[0004] Cardiovascular diseases (CVD) including heart failure, atherosclerosis, hypertension, and coronary artery disease are often associated with oxidative stress, which plays a pivotal role in their initiation and advancement. A breakdown in the GSH/GSSG equilibrium, marked by excessive reactive oxygen species (ROS) generation and inadequate antioxidant defenses, leads to oxidative stress, thereby intensifying inflammatory responses, endothelial dysfunction, and plaque formation. Accurate monitoring of GSH levels in physiological fluids therefore offers essential insights into the body's oxidative condition, making it a crucial biomarker for evaluating cardiovascular health.
[0005] Traditional detection methods for GSH have relied primarily on natural enzyme-based sensors. However, these natural enzyme-based sensors encounter significant limitations including poor stability under various environmental conditions, rapid denaturation and activity loss under standard operating conditions, high production costs due to complex and expensive purification processes, and restricted operating parameters. Furthermore, enzyme-based biosensors frequently fail to achieve the combination of wide linear range, low detection limit, and high selectivity necessary for reliable GSH monitoring in clinical applications.
[0006] Previous approaches for GSH detection have utilized various materials including metal nanoparticles, carbon-based materials, and enzyme mimics. However, these existing methods typically suffer from narrow detection ranges, inadequate sensitivity, or insufficient selectivity when interfering substances are present. For instance, Pt-based electrodes exhibit limited linear ranges (5.0-20.0 μM, LOD 5.0 μM), while AuNPs/TiO₂-modified electrodes demonstrate elevated LODs (1300.0 μM). Additionally, many current sensors exhibit diminished performance in real physiological samples due to matrix effects and interference from other biological molecules.
[0007] Nanozymes, which are nanomaterials with enzyme-like activities, have gained attention as alternatives to natural enzymes due to their enhanced stability under severe pH, temperature, and oxidative conditions, reduced manufacturing costs, and simplified large-scale synthesis. Among these, superoxide dismutase (SOD) mimicking nanozymes are particularly important in biomedical science for regulating the amounts of reactive oxygen species in cells. However, achieving the specific combination of wide linear range, low detection limit, high selectivity, and long-term stability for GSH detection remains a substantial challenge.
[0008] Therefore, there exists a requirement for improved sensing materials that can overcome these limitations while providing efficient, stable, and selective detection of glutathione for applications in cardiovascular health monitoring and disease diagnosis, particularly sensors that can function across a wide concentration range encompassing both baseline GSH levels in healthy individuals and altered levels in disease states.

OBJECTS OF THE PRESENT DISCLOSURE
[0009] Some of the objects of the present disclosure, which at least one embodiment herein satisfies are as listed herein below.
[0010] An object of the present disclosure is to provide a nanozyme-based electrochemical sensor that eliminates the stability and denaturation issues of natural enzyme-based sensors for accurate glutathione quantification in biological samples.
[0011] An object of the present disclosure is to enable improved cardiovascular disease monitoring by utilizing copper selenite nanoparticles anchored on functionalized carbon nanofibers to achieve superoxide dismutase-mimicking catalytic activity for selective glutathione detection.
[0012] An object of the present disclosure is to enhance the electrochemical detection range of glutathione from 0.0625 μM to 7785.0 μM through the synergistic interaction between copper selenite nanoparticles and acid-functionalized carbon nanofibers.
[0013] An object of the present disclosure is to provide a sensor that achieves a detection limit of 0.0176 μM for glutathione through the formation of a nanocomposite with exposed Cu(II) coordination sites on monoclinic crystal planes.
[0014] An object of the present disclosure is to create a stable sensor that maintains at least 95% of its initial electrochemical response after 15 days of storage at room temperature without requiring specialized storage conditions.
[0015] An object of the present disclosure is to enable selective detection of glutathione in the presence of interfering substances including ascorbic acid, uric acid, dopamine, glucose, and hydrogen peroxide through specific thiol-copper interactions.
[0016] An object of the present disclosure is to provide a synthesis method that employs hydrothermal treatment without requiring additional calcination steps, utilizing a 3:1 volumetric ratio of sulfuric to nitric acid for carbon nanofiber functionalization.

SUMMARY
[0017] This section is provided to introduce certain objects and aspects of the present disclosure in a simplified form that are further described below in the detailed description. This summary is not intended to identify the key features or the scope of the claimed subject matter.
[0018] The present disclosure generally relates to electrochemical biosensors and medical diagnostics. More particularly, the present disclosure relates to a nanozyme sensor for electrochemical detection of glutathione including copper selenite nanoparticles with monoclinic crystal structure anchored to functionalized carbon nanofibers, providing enhanced cardiovascular biomarker detection through specific interaction between Cu(II) coordination sites and thiol groups of glutathione for improved sensitivity and clinical reliability.
[0019] An aspect of the present disclosure relates to a nanozyme sensor for electrochemical detection of glutathione. The sensor includes copper selenite nanoparticles with a monoclinic crystal structure and exposed crystal planes that include Cu(II) coordination sites, the copper selenite nanoparticles with a nano-spherical morphology. The sensor includes functionalized carbon nanofibers with carboxyl functional groups formed by acid treatment, wherein the copper selenite nanoparticles are anchored to the functionalized carbon nanofibers through interaction between the Cu(II) coordination sites and the carboxyl functional groups to form a nanocomposite, and wherein the copper selenite nanoparticles are distributed on the functionalized carbon nanofibers such that the Cu(II) coordination sites are accessible for binding with thiol groups of glutathione.
[0020] In another aspect, the present disclosure relates to a method for preparing a nanozyme sensor for electrochemical detection of glutathione. The method includes treating carbon nanofibers with an acid mixture to form functionalized carbon nanofibers with carboxyl functional groups. The method includes dissolving copper sulfate pentahydrate in dimethylformamide to form a copper solution. The method includes dissolving selenium dioxide in water to form a selenium solution. The method includes mixing the selenium solution with the copper solution with stirring to form a precursor mixture. The method includes subjecting the precursor mixture to hydrothermal treatment at 160°C for 3 hours to form copper selenite nanoparticles with a monoclinic crystal structure and exposed crystal planes including Cu(II) coordination sites. The method includes dispersing the copper selenite nanoparticles and the functionalized carbon nanofibers in ethanol. The method includes subjecting the dispersion to ultrasonication for 2 hours to anchor the copper selenite nanoparticles to the functionalized carbon nanofibers through interaction between the Cu(II) coordination sites and the carboxyl functional groups to form a nanocomposite, wherein the copper selenite nanoparticles are distributed on the functionalized carbon nanofibers such that the Cu(II) coordination sites are accessible for binding with thiol groups of glutathione.
[0021] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing figures in which like numerals represent like components.

BRIEF DESCRIPTION OF DRAWINGS
[0022] The accompanying drawings are included to provide a further understanding of the present disclosure and are incorporated in and constitute a part of this specification. The drawings illustrate exemplary embodiments of the present disclosure and, together with the description, serve to explain the principles of the present disclosure. The diagrams are for illustration only, which thus is not a limitation of the present disclosure.
[0023] FIG. 1 illustrates an exemplary schematic representation of the CuSeO₃@f-CNF/GCE nanozyme sensor architecture depicting the layered structure with copper selenite nanoparticles supported on functionalized carbon nanofibers deposited on a glassy carbon electrode for glutathione detection, in accordance with an embodiment of the present disclosure.
[0024] FIG. 2 illustrates an exemplary flow diagram depicting the synthesis process of CuSeO₃@f-CNF nanocomposite depicting the hydrothermal method at 160°C for 3 hours and subsequent ultrasonication step, in accordance with an embodiment of the present disclosure.
[0025] FIG. 3 illustrates exemplary electrochemical characterization graphs depicting: (A) cyclic voltammograms at different glutathione concentrations, (B) interference study bar graph, (C) amperometric response for blood sample analysis, and (D) amperometric response for urine sample analysis, in accordance with an embodiment of the present disclosure.
[0026] FIG. 4 illustrates exemplary electrochemical performance graphs depicting: (A) cyclic voltammograms of bare GCE and modified electrodes, (B) current response comparison bar graph, (C) Nyquist plots from electrochemical impedance spectroscopy, (D) charge transfer resistance values, (E) CV curves at different scan rates, and (F) peak current versus square root of scan rate calibration plot, in accordance with an embodiment of the present disclosure.
[0027] FIG. 5 illustrates exemplary amperometric detection results depicting: (A) current response at different glutathione concentrations, (B) linear calibration plot for 20-220 μM range, (C) wide range detection from 0.0625 μM to 7785.0 μM, and (D) corresponding calibration curve demonstrating the extensive linear detection range, in accordance with an embodiment of the present disclosure.

DETAILED DESCRIPTION
[0028] The ensuing description provides exemplary embodiments only, and is not intended to limit the scope, applicability, or configuration of the disclosure. Rather, the ensuing description of the exemplary embodiments will provide those skilled in the art with an enabling description for implementing an exemplary embodiment. It should be understood that various changes may be made in the function and arrangement of elements without departing from the spirit and scope of the invention as set forth.
Definitions:
Nanozyme: An inorganic nanomaterial exhibiting enzyme-like catalytic activity, specifically copper selenite nanoparticles demonstrating superoxide dismutase mimicking properties for electrochemical oxidation of glutathione through Cu²⁺/Cu⁺ redox cycling.
Functionalized Carbon Nanofibers (f-CNF): Carbon nanofibers treated with acid mixture of H₂SO₄ and HNO₃ in 3:1 volume ratio creating carboxyl functional groups on the surface, providing anchoring sites for nanoparticle attachment and enhanced electrical conductivity.
Monoclinic Crystal Structure: A crystallographic system characterized by unit cell parameters a = 7.982 Å, b = 9.364 Å, c = 6.652 Å, and β = 108.56° for copper selenite nanoparticles, featuring exposed (200) and (311) crystal planes with accessible Cu(II) coordination sites.
Electrochemical Detection: A quantitative analytical technique measuring current response generated from redox reactions between analyte and electrode surface, operating at optimized potential of 0.29 V versus Ag/AgCl reference electrode for glutathione oxidation.
Limit of Detection (LOD): The lowest concentration of glutathione reliably detectable by the nanozyme sensor, calculated as 0.0176 µM using the formula LOD = 3Sb/m where Sb represents standard deviation of blank measurements and m represents calibration curve slope.
Linear Detection Range: The concentration range over which the sensor response maintains linear relationship with glutathione concentration, spanning from 0.0625 µM to 7785.0 µM representing one of the widest ranges reported for glutathione sensors.

[0029] An aspect of the present disclosure relates to a nanozyme sensor for electrochemical detection of glutathione including copper selenite nanoparticles anchored to functionalized carbon nanofibers. The sensor includes copper selenite nanoparticles with monoclinic crystal structure and nano-spherical morphology providing Cu(II) coordination sites for specific interaction with thiol groups. The sensor includes functionalized carbon nanofibers with carboxyl groups formed by acid treatment creating a conductive matrix. The sensor includes a nanocomposite structure where copper selenite nanoparticles are distributed on functionalized carbon nanofibers through covalent bonding. The sensor includes an electrode interface modified with the nanocomposite enabling electron transfer from glutathione oxidation. The sensor includes a detection mechanism based on superoxide enzyme-mimicking activity facilitating selective glutathione quantification. The sensor includes performance characteristics demonstrating wide linear range and low detection limit suitable for clinical applications.
[0030] Various embodiments of the present disclosure are described using FIGs. 1 to 5.
[0031] FIG. 1 illustrates an exemplary schematic representation of the CuSeO₃@f-CNF/GCE nanozyme sensor architecture, in accordance with an embodiment of the present disclosure.
[0032] In an embodiment, referring to FIG. 1, the sensor (100) for electrochemical detection of glutathione can include copper selenite nanoparticles (103) exhibiting distorted nano-spherical morphology to provide high surface area, functionalized carbon nanofibers (104) forming conductive network for electron transport, a nanocomposite layer (105) created through ultrasonication ensuring uniform distribution, a glassy carbon electrode (107) serving as conductive substrate, and an electrochemical interface (108) facilitating glutathione oxidation through enzyme-mimicking mechanism.
[0033] In an embodiment, the copper selenite nanoparticles (103) can include monoclinic crystal structure with specific lattice parameters providing optimal Cu(II) site exposure for glutathione binding.
[0034] In an embodiment, the copper selenite nanoparticles (103) can be implemented in the sensor (100) to provide superoxide dismutase-mimicking catalytic activity. The nanoparticles (103) can include diameter ranging from 10-50 nm with average size of 30 nm determined through transmission electron microscopy. The nanoparticles (103) can exhibit distorted nano-spherical morphology increasing active surface area for catalytic reactions. The crystal structure can feature exposed (200) and (311) planes confirmed through X-ray diffraction peaks at 26° and 31°. The Cu(II) coordination sites can remain accessible on nanoparticle surface enabling specific thiol group interaction. The nanoparticles can maintain structural integrity during repeated electrochemical cycling demonstrating robust performance.
[0035] In an embodiment, the nanoparticles (103) can include, but are not limited to, copper selenite, copper oxide-selenite composites, doped copper selenites, and modified copper chalcogenides, to provide enzyme-mimicking activity for electrochemical sensing applications.
[0036] In an embodiment, the functionalized carbon nanofibers (104) can be implemented in the sensor (100) to provide conductive support matrix and anchoring sites for nanoparticle attachment. The nanofibers (104) can function by facilitating electron transfer between copper selenite and electrode surface. The nanofibers (104) can include diameter between 50-200 nm with surface area of 200-500 m²/g. The acid functionalization can introduce carboxyl groups with density sufficient for uniform nanoparticle distribution. The nanofibers (104) can maintain electrical conductivity of approximately 12.5 S/cm after functionalization. The three-dimensional network structure can enhance mass transport and analyte accessibility.
[0037] In an embodiment, the nanofibers (104) can include, but are not limited to, acid-treated carbon nanofibers, oxidized carbon nanotubes, functionalized graphene fibers, and modified carbon nanomaterials, to enable enhanced conductivity and nanoparticle support.
[0038] In an embodiment, the nanocomposite (105) can be implemented in the sensor (100) to act as the active sensing layer combining catalytic and conductive properties. The nanocomposite (105) can contain copper selenite and functionalized carbon nanofibers in optimized 10:90 weight ratio. The formation process can involve 2-hour ultrasonication in ethanol ensuring homogeneous dispersion. The nanocomposite (105) can exhibit synergistic effects with lower charge transfer resistance of 30 Ω compared to individual components. The uniform distribution can prevent nanoparticle aggregation maintaining consistent catalytic activity. The composite structure can provide mechanical stability during long-term operation.
[0039] In an embodiment, the nanocomposite (105) preparation methods can include, but are not limited to, ultrasonication dispersion, in-situ synthesis, chemical vapor deposition, electrochemical deposition, and solution mixing, to achieve optimal nanoparticle-nanofiber integration.
[0040] In an embodiment, the glassy carbon electrode (107) can be implemented in the sensor (100) to provide stable conductive platform for nanocomposite deposition. The electrode (107) can undergo polishing with 0.05 µm alumina powder ensuring smooth surface. The cleaned surface can enable uniform nanocomposite adhesion through drop-casting technique. The electrode (107) can maintain chemical inertness in phosphate buffer solutions. The geometric area of 0.0314 cm² can provide sufficient sensing surface. The electrode can demonstrate wide potential window suitable for glutathione oxidation.
[0041] In an embodiment, the electrode (107) can include one or more modifications to enhance performance including surface activation through electrochemical pretreatment, incorporation of conductive polymers for improved adhesion, and application of protective coatings to prevent fouling during extended use.
[0042] In an embodiment, the modifications configured on the electrode (107) can be programmed to maintain optimal surface conditions throughout sensor operation. If upon detection of decreased current response indicating surface fouling, the electrode can undergo regeneration through controlled potential cycling. The electrode (107) can ensure consistent sensitivity while extending operational lifetime.
[0043] In an embodiment, the electrochemical interface (108) can be implemented to facilitate glutathione detection through selective oxidation mechanism. The interface (108) can be configured with three-electrode system including modified working electrode, platinum counter electrode, and Ag/AgCl reference electrode. Upon detecting glutathione presence, the interface (108) can automatically generate proportional current response. The interface (108) can operate at optimized potential of 0.29 V minimizing interference from other biomolecules.
[0044] In an embodiment, the detection mechanism can involve specific interaction between glutathione thiol groups and Cu(II) sites on copper selenite surface. The mechanism can proceed through formation of Cu-S bonds facilitating electron transfer. The copper can undergo redox cycling between Cu²⁺ and Cu⁺ states during glutathione oxidation to its disulfide form. The functionalized carbon nanofibers can provide efficient electron transport pathway to electrode surface. The synergistic effect can result in enhanced sensitivity with detection limit of 0.0176 µM.
[0045] In an embodiment, glutathione oxidation can follow the reaction: Cu(Cu²⁺)SeO₃@f-CNF + 2GSH → Cu(Cu⁺)SeO₃@f-CNF + GSSG + 2H⁺ where GSH represents reduced glutathione and GSSG represents oxidized glutathione disulfide. The reaction can be pH-dependent with optimal activity at pH 3.0 due to proton involvement. The current response can show linear relationship with glutathione concentration enabling quantitative analysis. The oxidation can occur selectively without significant interference from ascorbic acid, uric acid, or dopamine.
[0046] In an embodiment, the sensor performance can be characterized through multiple electrochemical techniques. Cyclic voltammetry can reveal oxidation peak at 0.29 V with peak current proportional to glutathione concentration. Electrochemical impedance spectroscopy can demonstrate reduced charge transfer resistance of 30 Ω for the nanocomposite. Chronoamperometry can provide steady-state current measurements for quantitative analysis. The techniques can validate sensor functionality across the wide linear range.
[0047] FIG. 2 illustrates an exemplary flow diagram depicting the synthesis process of CuSeO₃@f-CNF nanocomposite, in accordance with an embodiment of the present disclosure.
[0048] In an embodiment, referring to FIG. 2, illustrating the synthesis methodology (200) for the nanozyme sensor (100). The process depicts systematic preparation starting with carbon nanofiber functionalization (201, 202) followed by copper selenite synthesis through hydrothermal treatment (203, 204). Upon completing individual component preparation, the process can automatically proceed to nanocomposite formation (205, 206). This precise synthesis protocol can remain critical for achieving optimal sensor performance. The method (200) can prevent structural defects and can maximize catalytic activity through controlled reaction conditions.
[0049] FIG. 3 illustrates exemplary electrochemical characterization graphs depicting sensor performance, in accordance with an embodiment of the present disclosure.
[0050] In an embodiment, referring to FIG. 3 illustrate comprehensive electrochemical analysis (300) for sensor (100) demonstrating detection capabilities. Panel (A) depicts cyclic voltammograms with increasing glutathione concentrations producing proportional current responses. Panel (B) displays interference study confirming selective detection with minimal response to common biomolecules. If glutathione concentration of 200 µM is present among 10-fold excess interferents, the sensor maintains over 95% selectivity. Panels (C) and (D) demonstrate real sample analysis in blood serum and urine with recovery rates of 97-99%. The enhanced selectivity translates to reliable clinical measurements without complex sample preparation.
[0051] FIG. 4 illustrates exemplary electrochemical performance evaluation, in accordance with an embodiment of the present disclosure.
[0052] In an embodiment, referring to FIG. 4 illustrate detailed performance characterization (400) for sensor (100) validating electrochemical properties. Panel (A) compares bare and modified electrodes showing enhanced current response for CuSeO₃@f-CNF. Panel (C) presents Nyquist plots revealing lowest charge transfer resistance for the nanocomposite. Panel (E) demonstrates scan rate dependence confirming diffusion-controlled process. Panel (F) depicts linear relationship between peak current and square root of scan rate with R² = 0.998. The comprehensive analysis confirms optimal electron transfer kinetics and sensor reliability.
[0053] FIG. 5 illustrates exemplary amperometric detection results demonstrating wide linear range, in accordance with an embodiment of the present disclosure.
[0054] In an embodiment, referring to FIG. 5 illustrate quantitative detection capabilities (500) for sensor (100) across extensive concentration range. Panel (A) depicts amperometric response with stepwise glutathione additions. Panel (B) presents initial linear range from 20-220 µM with excellent correlation. Panel (C) demonstrates unprecedented wide range detection from 0.0625 µM to 7785.0 µM. Panel (D) confirms linear relationship across entire range with R² = 0.997. The extraordinary linear range enables single sensor usage for both trace analysis and high concentration measurements.
[0055] In an embodiment, the sensor (100) can handle varying analytical requirements by detecting glutathione at nanomolar levels, automatically maintaining linear response across five orders of magnitude, generating accurate quantification using optimized calibration curves, activating selective detection mechanism in complex biological matrices, enabling point-of-care diagnostics via rapid response times, ensuring long-term stability through robust nanocomposite structure, and allowing clinical integration for cardiovascular biomarker monitoring.
[0056] The described disclosure presents an advanced nanozyme-based sensor (100) integrated with electrochemical detection that offers several novel features distinguishing it from existing glutathione sensors. The sensor (100) can automatically provide enzyme-like catalytic activity when glutathione is present, enabling sensitive detection without natural enzyme instability. The copper selenite nanoparticles (103) can be strategically anchored on functionalized carbon nanofibers (104) creating synergistic effects that enhance both conductivity and catalytic efficiency. During electrochemical measurement, combination of specific thiol-copper interaction and efficient electron transfer can ensure accurate glutathione quantification across unprecedented concentration range. The automated detection of sensor (100) can ensure consistent performance without requiring specialized sample preparation. The sensor parameters can effectively enable clinical diagnostics throughout varying physiological conditions.
[0057] In an exemplary embodiment, the sensor (100) can operate within validated performance metrics to ensure reliable glutathione detection across clinical populations. The sensor (100) can demonstrate sensitivity of 0.1434 µA/µM/cm² representing enhanced electrochemical response. The stability testing can show over 95% activity retention after 15 days of continuous use. The mean recovery rates in human blood serum of 98.4% and urine of 99.1% can indicate excellent accuracy in real samples. The sensor (100) can complete individual measurements within 3 minutes including sample introduction. These validated performance metrics make the sensor (100) practical, efficient, and effective for clinical cardiovascular biomarker detection applications.
[0058] While considerable emphasis has been placed herein on the preferred embodiments, it will be appreciated that many embodiments can be made and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. These and other changes in the preferred embodiments of the disclosure will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter to be implemented merely as illustrative of the disclosure and not as limitation.
[0059] If the specification states a component or feature “may”, “can”, “could”, or “might” be included or have a characteristic, that particular component or feature is not required to be included or have the characteristic.
[0060] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.
[0061] Moreover, in interpreting the specification, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms “comprises” and “comprising” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. Where the specification claims refer to at least one of something selected from the group consisting of A, B, C ….and N, the text should be interpreted as requiring only one element from the group, not A plus N, or B plus N, etc.
[0062] While the foregoing describes various embodiments of the proposed disclosure, other and further embodiments of the proposed disclosure may be devised without departing from the basic scope thereof. The scope of the proposed disclosure is determined by the claims that follow. The proposed disclosure is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.
EXAMPLES
EXAMPLE 1: Synthesis and Characterization of CuSeO₃@f-CNF Nanozyme Sensor
[0063] Copper selenite nanoparticles were synthesized using a hydrothermal method. 0.75 g of copper sulfate pentahydrate (CuSO₄·5H₂O) was dissolved in 10.0 mL of dimethylformamide (DMF) with stirring at 600 rpm for 30 minutes. Separately, 0.165 g of selenium dioxide (SeO₂) was dissolved in 10.0 mL of deionized water under identical stirring conditions. The selenium solution was gradually added to the copper solution under continuous stirring to ensure homogeneous mixing.
[0064] The mixed solution was transferred to a Teflon-lined autoclave and subjected to hydrothermal treatment at 160°C for 3 hours. After cooling to room temperature, the precipitate was washed with ethanol and deionized water, then dried in a vacuum oven at 75°C for 12 hours. XRD analysis confirmed monoclinic crystal structure with characteristic peaks at 2θ values of 15°, 16°, 18°, 23°, 26°, 29°, and 31° corresponding to (0,1,1), (1,1,0), (1,0,1), (1,2,0), (2,0,0), (2,0,1), and (0,3,1) crystal planes.
[0065] Carbon nanofibers were functionalized by treatment with H₂SO₄:HNO₃ (3:1 v/v) at 70°C for 4 hours. The functionalized CNFs were washed to neutral pH and dried. The CuSeO₃@f-CNF nanocomposite was prepared by dispersing 3.0 mg CuSeO₃ and 6.0 mg f-CNF in 3.0 mL ethanol via 2-hour ultrasonication. The resulting nanocomposite was drop-cast (8 μL) onto a polished glassy carbon electrode and dried at 40°C for 5 minutes.
EXAMPLE 2: Comparative Performance Analysis with Existing Sensors
[0066] The electrochemical performance of CuSeO₃@f-CNF/GCE was systematically compared with eight different glutathione detection systems reported in literature. Table 1 presents the comparative data:
Table 1: Comparison of GSH Detection Methods
Technique Electrode Linear Range (μM) LOD (μM) Supporting Electrolyte
CAMP Pt 5.0-20.0 5.0 0.1 M NaCl
CAMP MPT/HP-b-CD/GCE 1.0-580.0 0.287 0.1 M PB 7.0
CAMP AuNPs/TiO₂ 33.2-740.7 1300.0 PB 7.0
CAMP Cu-CoHCF/GCE 5.0-90.0 2.5 0.1 M PB 7.0
CV GCE 1000.0-12500.0 140.0 0.1 M KNO₃
CV pCAF/GCE 0.3-100.0 22.0 PB 7.0
CV NPAuNWAs 0.001-0.1 - 0.1 M PB 5.6
CV MWCNTs/SPE 0-60.0 0.11 0.15 M PB 7.0
CAMP CuSeO₃@f-CNF/GCE 0.0625-7785.0 0.0176 0.1 M PB 3.0

[0067] The CuSeO₃@f-CNF sensor demonstrated superior performance with the widest linear range (0.0625-7785.0 μM) spanning five orders of magnitude. The detection limit of 0.0176 μM represents a 284-fold improvement compared to Pt electrodes and 73,863-fold improvement compared to AuNPs/TiO₂ systems. The extraordinary range encompasses both trace analysis and high concentration detection in a single sensor platform.
[0068] Electrochemical characterization revealed synergistic effects of the nanocomposite. Charge transfer resistance decreased from 117 Ω (bare GCE) and 1026 Ω (CuSeO₃/GCE) to 30 Ω for CuSeO₃@f-CNF/GCE. The electroactive surface area increased from 0.0391 cm² (bare GCE) to 0.0789 cm² for the nanocomposite, representing 2.0-fold enhancement. The peak separation (ΔEp) improved from 0.30 V (CuSeO₃/GCE) to 0.10 V for the nanocomposite, indicating enhanced electron transfer kinetics.
EXAMPLE 3: Real Sample Analysis in Biological Fluids
[0069] The practical applicability of CuSeO₃@f-CNF/GCE was validated using human blood serum and urine samples obtained from Chang-Gung Memorial Hospital (IRB No. 201801660B), Taiwan. Samples were centrifuged at 6000 rpm for 45 minutes and filtered. For blood serum, 1.0 mL sample was mixed with 3.0 mL phosphate buffer pH 5.0 and sonicated for 15 minutes.
[0070] Recovery studies were performed by spiking known concentrations of glutathione into the biological samples. Table 2 presents the results:
Table 2: Real Sample Analysis Results
Real Sample Added (μM) Found (μM) Recovery (%) RSD (±%)
Human Serum 0 - - -
10.0 9.84 98.4 1.27
20.0 19.47 97.35 2.15
Urine 0 - - -
10.0 9.91 99.1 0.84
20.0 19.83 99.15 0.76

[0071] The sensor demonstrated excellent recovery rates ranging from 97.35% to 99.15% with relative standard deviations below 2.15%. The high accuracy in complex biological matrices confirms the sensor's reliability for clinical applications without requiring extensive sample preparation or separation techniques.
EXAMPLE 4: Interference Study and Selectivity Validation
[0072] Selectivity was evaluated by testing the sensor response to 200.0 μM glutathione in the presence of 10-fold excess (2000.0 μM) of various interfering substances. The amperometric responses were measured at 0.29 V vs. Ag/AgCl in 0.1 M phosphate buffer pH 3.0.
[0073] The interference study revealed minimal signal changes: glucose (<2%), sucrose (<2%), fructose (<2%), lactose (<2%), KCl (<1%), NaCl (<1%), H₂O₂ (<3%), dopamine (<4%), ascorbic acid (<5%), and uric acid (<4%). The exceptional selectivity results from specific interaction between glutathione thiol groups and Cu(II) coordination sites on CuSeO₃, while the f-CNF matrix minimizes non-specific adsorption.
[0074] The sensor maintained over 95% of its initial response after 15 days of continuous use when stored at room temperature in a desiccator. Repeated measurements (n=100) showed less than 3% variation in current response. The robust stability is attributed to strong covalent bonding between CuSeO₃ nanoparticles and functionalized CNFs, preventing leaching or degradation during extended operation.
[0075] The present invention enables detection of glutathione across the widest reported linear range from 0.0625 µM to 7785.0 µM through enzyme-mimicking copper selenite nanoparticles, surpassing all existing sensors including platinum (5.0-20.0 µM), gold nanoparticle (33.2-740.7 µM), and copper-cobalt (5.0-90.0 µM) systems, thereby providing single-platform analysis for both trace and high concentration measurements.
[0076] The nanozyme sensor achieves exceptional stability maintaining over 95% activity after 15 days compared to natural enzyme sensors that typically degrade within 3-7 days, while demonstrating recovery rates of 97.35-99.15% in human blood serum and urine samples with relative standard deviations below 2.15%, ensuring reliable clinical diagnostics.
[0077] The sensor provides ultra-low detection limit of 0.0176 µM representing 73,863-fold improvement over AuNPs/TiO₂ systems (1300.0 µM) and 284-fold improvement over platinum electrodes (5.0 µM), combined with selectivity showing less than 5% interference from 10-fold excess of ascorbic acid, uric acid, dopamine, and other biomolecules, enabling accurate glutathione quantification in complex biological matrices.
, Claims:1. A nanozyme sensor (100) for electrochemical detection of glutathione (109) comprising:
(a) copper selenite nanoparticles (103) with a monoclinic crystal structure and exposed crystal planes that comprise Cu(II) coordination sites, the copper selenite nanoparticles (103) with a nano-spherical morphology;
(b) functionalized carbon nanofibers (104) with carboxyl functional groups formed by acid treatment;
wherein the copper selenite nanoparticles (103) are anchored to the functionalized carbon nanofibers (104) through interaction between the Cu(II) coordination sites and the carboxyl functional groups to form a nanocomposite (105), and wherein the copper selenite nanoparticles (103) are distributed on the functionalized carbon nanofibers (104) such that the Cu(II) coordination sites are accessible for binding with thiol groups of glutathione (109).
2. The nanozyme sensor (100) as claimed in claim 1, wherein the monoclinic crystal structure of the copper selenite nanoparticles (103) comprises (200) and (311) crystal planes.
3. The nanozyme sensor (100) as claimed in claim 1, wherein the copper selenite nanoparticles (103) have a distorted nano-spherical morphology with a diameter of 10-50 nm.
4. The nanozyme sensor (100) as claimed in claim 1, wherein the functionalized carbon nanofibers (104) have a diameter of 50-200 nm.
5. The nanozyme sensor (100) as claimed in claim 1, wherein the carboxyl functional groups are formed by treating carbon nanofibers with H₂SO₄ and HNO₃ in a volume ratio of 3:1.
6. The nanozyme sensor (100) as claimed in claim 1, wherein the copper selenite nanoparticles (103) and the functionalized carbon nanofibers (104) are present in a weight ratio of 10:90 in the nanocomposite (105).
7. The nanozyme sensor (100) as claimed in claim 1, wherein the interaction between the Cu(II) coordination sites and the carboxyl functional groups comprises covalent bonding.
8. The nanozyme sensor (100) as claimed in claim 1, wherein the nanocomposite (105) is configured to be deposited on a glassy carbon electrode (107).
9. The nanozyme sensor (100) as claimed in claim 1, wherein the copper selenite nanoparticles (103) comprise a monoclinic crystal structure with unit cell parameters a = 7.982 Å, b = 9.364 Å, c = 6.652 Å, and β = 108.56°.
10. A method for preparing a nanozyme sensor (100) for electrochemical detection of glutathione (109) comprising:
(a) treating carbon nanofibers with an acid mixture (205) to form functionalized carbon nanofibers (104) with carboxyl functional groups;
(b) dissolving copper sulfate pentahydrate (101) in dimethylformamide (201) to form a copper solution;
(c) dissolving selenium dioxide (102) in water (202) to form a selenium solution;
(d) mixing the selenium solution with the copper solution with stirring (203) to form a precursor mixture;
(e) subjecting the precursor mixture to hydrothermal treatment (204) at 160°C for 3 hours to form copper selenite nanoparticles (103) with a monoclinic crystal structure and exposed crystal planes comprising Cu(II) coordination sites;
(f) dispersing the copper selenite nanoparticles (103) and the functionalized carbon nanofibers (104) in ethanol;
(g) subjecting the dispersion to ultrasonication (206) for 2 hours to anchor the copper selenite nanoparticles (103) to the functionalized carbon nanofibers (104) through interaction between the Cu(II) coordination sites and the carboxyl functional groups to form a nanocomposite (105);
wherein the copper selenite nanoparticles (103) are distributed on the functionalized carbon nanofibers (104) such that the Cu(II) coordination sites are accessible for binding with thiol groups of glutathione (109).