WO2025155514A1 - System and method for sucralose measurement - Google Patents

Abstract

A sucralose electrochemical sensor includes a counter electrode and a reference electrode. The sensor also includes a working electrode having a distal electrode portion and a layer formed from a metal and metal oxide disposed on the distal electrode portion for oxidation of the sucralose contained in the fluid sample.

Description

SYSTEM AND METHOD FOR SUCRALOSE MEASUREMENT

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of, and priority to, U.S. Provisional Patent Application Serial No. 63/621,361 filed on January 16, 2024. The entire contents of the foregoing application are incorporated by reference herein.

BACKGROUND

[0002] The use of sweeteners, such as sugar to enhance the taste of food, has had an adverse impact on human health. This includes alteration to the gut microbiota, increase in obesity, and diabetes. This has led to the development and abundant use of non-nutritive sweeteners in food and beverages over the past decade. In contrast to sweeteners, a minimal amount of non- nutritive/artificial sweeteners can achieve the same amount of sweetness as sugars. With a market cap of above $500 million, artificial sweeteners have become popular and are currently present in thousands of food and beverage products worldwide.

[0003] Among several sweeteners used in food and beverage products, the use of sucralose (1,6- dichloro-l,6-dideoxy-P-D-fructofuranosyl-4-chloro-4-deoxy-agalactopyranoside) has been widely adopted since its commercial inception in 1991 due to its exceptional heat stability (melting point 130 °C), excellent solubility characteristics, and high compatibility with commonly used food ingredients. Sucralose is a white crystalline odorless powder obtained by selectively substituting three hydroxyl groups of sucrose with three chlorine atoms. Sucralose is used as a non-caloric, high intensity sweetener that is 600 times sweeter than sugar and is used in many food and beverage industries.

[0004] Stability experiments conducted on carbonated soft drinks, still drinks, dry mixes and strawberry milk sweetened with sucralose exhibited extremely high stability even during extended storage. In another experiment, a gelatin mixture showed no noticeable degradation of sucralose when stored at 30 °C for 6 months followed by two years storage at room temperature and in cola beverage stored at 20 °C.

[0005] Sucralose is not hydrolyzed in the intestinal lumen and about 92% of the ingested quantity is excreted unchanged in the feces. Even under very highly acidic conditions (pH 3.0), it was observed that after 1 year, only 1 % of sucralose decomposed to 1,6-dichlorofructose and 4- chlorogalactose at 25 °C. Most of the sucralose entering wastewater remains intact as no hydrolysis occurs at neutral pH. High aqueous solubility, excellent stability, and low affinity for solid phase partitioning together with high frequency of detection in wastewater treatment facility effluents make sucralose an ideal candidate as a wastewater tracer. Wastewater contamination of groundwater, drinking water treatment plants, surface waters, estuaries and coastal waters has been successfully established using sucralose as the tracer.

[0006] Commercial quantitative determination of sucralose has been routinely carried out using chromatography, such as high or ultra-performance liquid chromatography (HPLC or UPLC), gas chromatography-mass spectrometry (GC-MS), and combinations thereof. These quantitative methods involve complex sample processing, are labor intensive and do not provide real time results. For example, GC requires salinization to increase volatility of sucralose. Similarly, since sucralose has very low UV absorption, derivatization reagents are added prior to quantification with HPLC that utilizes UV detectors. Fourier Transform Infrared Spectroscopy (FTIR) has also been employed for quantification of sucralose. Of all the reported methods, the least time consuming is capillary electrophoresis (CE) with results being available in about sixteen minutes.

But these methods often suffer from limitations that arise due to complex and long detection processes coupled with low sensitivity. Thus, a simple, real-time sensor for quantifying sucralose is desired.

SUMMARY

[0007] Biosensors utilizing biorecognition elements (such as enzymes, antibodies or DNA/aptamers, etc.), function as an independent integrated receptor transducer devices and can provide instantaneous, selective, quantitative /semi -quantitative analytical information. Of the different transducers used to develop biosensors, electrochemical methods are better suited to develop sensors that are robust, portable, and tailored for a specific application. These sensors may use electrode material that can directly catalyze the oxidation of sugars. These electrodes may be constructed using a variety of materials including metals, alloys and bimetallic systems, carbonbased materials, metal/metal oxides, heterogeneous nanocomposites and layered double hydroxides.

[0008] Metal oxide-based sensors are relatively inexpensive to fabricate, exhibit excellent sensitivity along with the added advantage of rapid response. Transition metal oxides and alloys significantly enhance direct oxidation of sucralose compared to other metals due to the catalytic effect resulting from the multi-electron oxidation mediated by surface metal oxide layers. Transition metals such as copper (Cu) and nickel (Ni) can oxidize carbohydrate easily without surface poisoning. Unlike metallic Cu and Ni, their corresponding oxides or hydroxides are relatively stable in air and in solution.

[0009] The present disclosure provides an electrochemical sensor for quantifying sucralose. The sensor may be an electrochemical sucralose test strip which may include a Cu/CuO modified screen-printed carbon electrode (SPCE) or a platinum (Pt) wire electrode. The Cu/CuO modified SPCE exhibits a linear range of detection up to about 25 mM while the Cu/CuO modified platinum wire electrode detects sucralose up to 75 mM. The Cu/CuO composition acts as an electrocatalyst for the direct electrooxidation of sucralose. Both sucralose test strips exhibited a rapid response towards sucralose with a response time of less than 5 seconds. Nanomaterials of metals and metal oxides are excellent electrocatalysts due to their increased surface area and enhanced electronic properties while oxides of metals such as Cu, Ni, and cobalt (Co) exhibit high catalytic efficiency towards electrooxidation of carbohydrates. Thus, it is envisioned that the electrochemical sucralose sensor according to the present disclosure may include other metals (e g., Ni, Co, etc.) and their oxides.

[0010] Performance of the test strips was characterized morphologically and electrochemically and tested for its response towards sucralose in an alkaline condition. The analysis suggests that sucralose with stoichiometric amount of 1 M NaOH initially undergoes a mono SN2-cyclization at one of the secondary hydroxyl groups and primary chloride of the fructose ring giving a crystalline tricyclic compound. Reaction with three equivalents of IM NaOH undergoes a double SN2- reaction to afford a stable tetracyclic ring involving the fructose moiety of sucralose. The tricyclic product was confirmed by X-ray crystallography. Both the tricyclic and the tetracyclic products were characterized by ¹ H NMR and ¹³C NMR spectroscopy. Structure of the tricyclic product was also refined based on ID and 2D NMR techniques.

[0011] According to one embodiment of the present disclosure, a sucralose electrochemical sensor is disclosed. The sucralose electrochemical sensor includes a counter electrode and a reference electrode. The sensor also includes a working electrode having a distal electrode portion and a layer formed from a metal and a metal oxide disposed thereon for oxidation of the sucralose contained in the fluid sample. [0012] Implementations of the above embodiment may include one or more of the following features. According to one aspect of the above embodiment, the sucralose electrochemical sensor may also include a substrate formed from a dielectric material, where the counter electrode, the reference electrode, and the working electrode are disposed on the substrate. The metal may be one of copper, nickel, or cobalt. The metal oxide may be one of copper oxide, nickel oxide, or cobalt oxide. Each of the counter electrode, the reference electrode, and the working electrode may be screen-printed carbon electrodes. Each of the counter electrode, the reference electrode, and the working electrode may be formed from a metallic wire. The layer formed from the metal and the metal oxide may include a plurality of metal and metal oxide nanoclusters.

[0013] According to another embodiment of the present disclosure, a sucralose measurement system for measuring concentration of sucralose in a fluid sample is disclosed. The sucralose measurement system includes a sucralose electrochemical sensor including a counter electrode, a reference electrode, and a working electrode having a distal electrode portion and a layer of a metal and a metal oxide disposed thereon for oxidation of the sucralose contained in the fluid sample. The system also includes a sucralose meter having a port for receiving a proximal portion of the sucralose electrochemical sensor. The sucralose meter also includes a measurement circuit for applying a constant voltage potential to the reference electrode and the working electrode to oxidize the sucralose contained in the fluid sample and for measuring current due to oxidation of the sucralose. The sucralose meter further includes a controller for converting the measured current into a voltage signal and calculating the concentration of the sucralose based on the voltage signal.

[0014] Implementations of the above embodiment may include one or more of the following features. According to one aspect of the above embodiment, the sucralose electrochemical sensor may also include a substrate formed from a dielectric material, where the counter electrode, the reference electrode, and the working electrode are disposed on the substrate. The metal may be one of copper, nickel, or cobalt. The metal oxide may be one of copper oxide, nickel oxide, or cobalt oxide. Each of the counter electrode, the reference electrode, and the working electrode may be screen-printed carbon electrodes. Each of the counter electrode, the reference electrode, and the working electrode may be formed from a metallic wire. The layer formed from the metal and the metal oxide may include a plurality of metal and metal oxide nanoclusters. The sucralose meter may further include a display screen for displaying the concentration of the sucralose. The measurement circuit may include a potentiostat and an operational amplifier.

[0015] According to another embodiment of the present disclosure, a method for forming a sucralose electrochemical sensor for measuring concentration of sucralose in a fluid sample is disclosed. The method includes placing a working electrode and a reference electrode of the sucralose electrochemical sensor in a first solution including a salt of a metal dissolved therein. The method also includes applying an electrical potential at a first voltage between the reference electrode and the working electrode in the first solution to form a metal layer on the working electrode from the salt of the metal. The method further includes placing the working electrode and the reference electrode of the sucralose electrochemical sensor in a second solution and applying the electrical potential at a second voltage between the reference electrode and the working electrode in the second solution to oxidize the metal layer to form a layer having the metal and metal oxide nanoclusters.

[0016] Implementations of the above embodiment may include one or more of the following features. According to one aspect of the above embodiment, the metal may be one of copper, nickel, or cobalt. The metal oxide may be one of copper oxide, nickel oxide, or cobalt oxide. The first voltage may be negative with respect to the reference electrode and the second voltage may be positive with respect to the reference electrode.

BRIEF DESCRIPTION OF DRAWINGS

[0017] Various embodiments of the present disclosure are described below with reference to the following figures:

[0018] FIG. 1 is a top view of a sucralose test strip according to an embodiment of the present disclosure;

[0019] FIG. 2 is atop view of a sucralose test strip according to another embodiment of the present disclosure;

[0020] FIG. 3 is a perspective view of a sucralose measurement system including a sucralose meter according to an embodiment of the present disclosure;

[0021] FIG. 4 is a top view of electronic components of the sucralose meter of FIG. 3 according to an embodiment of the present disclosure;

[0022] FIG. 5 shows a method of using the sucralose measurement system of FIG. 3 according to an embodiment of the present disclosure;

[0023] FIG. 6 shows scanning electron microscopy (SEM) image of uniform Cu/CuO nanoclusters deposited on the surface of an SPCE according to an embodiment of the present disclosure;

[0024] FIG. 7 shows a plot of atomic composition of the Cu/CuO on the SPCE following electrooxidation of Cu and further oxidation for 60 seconds according to an embodiment of the present disclosure; [0025] FIGS. 8A-C show scanning electron microscopy-energy dispersive spectroscopy (SEM- EDS) maps of Cu (FIG. 8A), carbon (C) (FIG. 8B), and oxygen (0) (FIG. 8C) according to an embodiment of the present disclosure;

[0026] FIG. 9 shows linear sweep voltammetry (LSV) plots (a-f bottom to top) of Cu/CuO on SPCE in 0.1 M NaOH with increasing concentrations of sucralose from 5 mM to 25 mM at a scan rate of 100 mV/s according to an embodiment of the present disclosure;

[0027] FIG. 10 shows LSV plots (a-o bottom to top) of Cu/CuO/SPCE in 0.1 MNaOH containing 3 mM sucralose at different scan rate from 10 mV/s to 150 mV/s according to an embodiment of the present disclosure;

[0028] FIG. 11 shows a plot of current vs sucralose concentration illustrating amperometric response at +0.65 V on the Cu/CuO/SPCE with sucralose concentrations from 1 mM to 20 mM in 0.1 mol L'¹ NaOH according to an embodiment of the present disclosure;

[0029] FIG. 12 shows a plot of current vs sucralose concentration illustrating amperometric response at +0.65 V on the Cu/CuO/Pt wire electrode with sucralose concentrations from 1 mM to 75 mM in 0.1 mol L'¹ NaOH according to an embodiment of the present disclosure;

[0030] FIG. 13 shows a reaction scheme for intramolecular SN2 mono-cyclization of trichlorogalactosucrose and heteronuclear multiple bond correlation (HMBC) and correlated spectroscopy (COSY) correlations for the dicyclized fructofuranose;

[0031] FIG. 14 shows a reaction scheme for intramolecular SN2 di-cyclization of trichlorogalactosucrose and HMBC and COSY correlations for dicyclic derivative of trichlorogalactosucrose; and

[0032] FIG. 15 shows a schematic diagram of a sucralose meter according to an embodiment of the present disclosure. DETAILED DESCRIPTION

[0033] As used herein the term “distal” refers to that portion of an apparatus, or component thereof, farther from the user, while the term “proximal” refers to that portion of the surgical instrument, or component thereof, closer to the user. As used herein, the term “nanoparticle” denotes a particle having any shape and a maximum dimension in any direction of from about 1 nm to about 100 nm. As used herein the terms “about”, “approximately”, and other relative terms, denote a range of ± 5% of the stated value.

[0034] The present disclosure provides an electrochemical sucralose meter that uses a plurality of electrodes to determine concentration, i.e., quantity, of sucralose in a solution. The electrodes may be formed as screen-printed electrodes on a disposable test strip (FIG. 1) or in any other suitable arrangement, such as wire electrodes (FIG. 2) with or without use of a strip. FIG. 1 shows a sucralose electrochemical sensor 10, which may be configured as a test strip. The sucralose electrochemical sensor 10 may include a substrate 12 that may be formed from any suitable dielectric material, such as plastics (e.g., PET, PETG, polyimide, polycarbonate, polystyrene), silicon, ceramic, glass, adhesives, and combinations thereof. The substrate 12 includes a proximal end portion 12a insertable into a sucralose meter (FIGS. 3 and 4) and a distal end portion 12b for receiving a sample being measured.

[0035] The sucralose electrochemical sensor 10 also includes a plurality of electrodes that are used for the electrochemical measurement of sucralose and are disposed on the substrate 12. In particular, the electrodes include a reference electrode 14, a counter (i.e., auxiliary) electrode 16, and a working electrode 18. Each of the electrodes 14, 16, 18 includes a distal electrode portion 14a, 16a, 18a coupled to a contact 14c, 16c, 18c disposed at the proximal end portion 12a of the substrate 5. Corresponding traces 14b, 16b, 18b connect the distal electrode portions 14a, 16a, 18a to the contacts 14c, 16c, 18c, respectively.

[0036] The electrodes 14, 16, 18 may be wholly or partially made from any suitable electrically conductive material such as, for example, gold, palladium, carbon, silver, platinum, tin oxide, iridium, indium, or combinations thereof (e.g., indium doped tin oxide). In embodiments, the electrodes 14, 16, 18 may be formed using a carbon ink that is screen-printed onto substrate 12 as shown in FIG. 1. In another embodiment shown in FIG. 2, the electrodes 14, 16, 18 may be formed from a metallic wire, which may be formed from any suitable metal or alloys. The wires may be used with or without the substrate 12.

[0037] The reference electrode 14 may be a silver/silver chloride (Ag/AgCl) electrode. The reference electrode 14 may be formed by applying Ag to the distal electrode portion 14a followed by AgCl coating using screen printing, electrodeposition, and the like. The distal electrode portion 16a may be formed from the same material as the rest of the counter electrode 16, e.g., carbon.

[0038] The working electrode 18 may include a metal/metal oxide layer deposited on the distal electrode portion 18a. Metal/metal oxide composition acts as an electrocatalyst for the direct electrooxidation of sucralose. Suitable metals and metal oxides include Cu, Ni, Co, and their corresponding oxides. The metal/metal oxide layer may be formed by initially electrodepositing the metal (e.g., Cu, Ni, Co, etc.) followed by oxidation of the metal to form the metal oxide. Electrodeposition may be performed by submerging the working electrode 18 in an acidic solution including a salt of the metal (e.g., CuSC ) and applying an electric potential at a negative voltage (e.g., -0.6 V with respect to the reference electrode 14) between the reference electrode 14 and the working electrode 18. [0039] Oxidation may be achieved using electrochemical oxidation, which may be performed by submerging the electrodeposited working electrode 18 in a basic solution and applying an electrical potential at a positive voltage (e.g., 0.8 V with respect to the reference electrode 14) between the reference electrode 14 and the working electrode 18. This process forms metal/metal oxide nanoclusters as shown in FIG. 6. Electrodeposition and oxidation processes form metal nanoclusters, which are clusters of a plurality of nanoparticles of the metal/metal oxide, on the surface of the distal electrode portion 18a.

[0040] The sucralose electrochemical sensor 10 may also include a port (not shown) for introducing a quantity of a fluid sample being analyzed, where the port is fluidly coupled to the electrodes 14, 16, 18. In one aspect, the port may be configured such that capillary action causes the fluid sample to contact the distal electrode portions 14a, 16a, 18a. The electrodes 14, 16, 18 may be coated with a hydrophilic reagent to promote the capillarity of the fluid port. In further embodiments, the electrodes 14, 16, 18 may be separated by spacer layers or disposed directly on the substrate 12.

[0041] FIG. 3 shows a sucralose measurement system 1, which includes a sucralose meter 20 for receiving the sucralose electrochemical sensor 10 and determining concentration of sucralose through an electrochemical reaction at the electrochemical sensor 10. The sucralose meter 20 may include a housing 21, user interface buttons 26, 28, 30, a display 24, and a strip port 32. User interface buttons 26, 28, 30 may be configured to allow the entry of data, navigation of menus, and execution of commands though a graphical user interface (GUI) displayed on the display screen 24. Data may include values representative of sucralose concentration. The electronic components of meter 20 may be disposed on a circuit board 44 that is within housing 11. User interface button 28 may be in the form of a two-way toggle switch. The display screen 24 may be a touchscreen to allow for additional user input in addition to or in place of the buttons 26, 28, 30.

[0042] FIG. 4 shows electronic components disposed on a circuit board 44. The electronic components include the strip port 32, a measurement circuit 45, a controller 48, a display screen connector 25, a non-volatile memory 50, a clock 52, and a communication module 56. Clock 52 may be configured to keep current time related to the geographic region in which the user is located and also for measuring time. The clock 52 may also include a crystal that provides a clock signal to the controller 38. The circuit board 44 also includes a battery connector 23 for coupling to a battery (not shown), which may be a rechargeable embedded battery, a removable single use battery, and the like. Controller 48 may be electrically connected to strip port 32, measurement circuit 45, communication module 56, display screen 24, non-volatile memory 50, clock 52, battery, and user interface buttons 26, 28, 30.

[0043] The measurement circuit 45 may include two or more operational amplifiers configured to provide a portion of the potentiostat function and the current measurement function. In embodiments, a standalone potentiostat, such as LMP91000 available from Texas Instruments may be used. Operational amplifier may be LMP7721, available from Texas Instruments. The potentiostat applies a test voltage between at least two electrodes 14, 16, 18 of the electrochemical sensor 10. The current function may refer to the measurement of a test current resulting from the applied test voltage. The current measurement may be performed with a current-to-voltage converter. The controller 38 may be a signal processor, such as a mixed signal microprocessor (MSP) such as, for example, the Texas Instrument MSP 430. The TI-MSP 430 may be configured to also perform a portion of the potentiostat function and the current measurement function. In addition, the MSP 430 may also include volatile and non-volatile memory. In another embodiment, many of the electronic components may be integrated with the controller in the form of an application-specific integrated circuit (ASIC). In further embodiments, a PIC 16LF 1783 controller may be used.

[0044] Strip port 32 may be configured to form an electrical connection to the electrochemical sensor 10. In particular, the port 32 may include individual connectors for electrically coupling each of the contacts 14c, 16c, 18c of the electrodes 14, 16, 18 to the operational amplifier circuit 35. Display screen connector 25 may be configured to attach to display screen 24. The display screen 24 may be a liquid crystal display, organic light emitting diode display, etc. for reporting measured sucrose concentration. The communication module 56 may provide a wired and/or wireless access to an external device such as a personal computer. The communication module 56 may include a data port that allows for transmission of data such as, for example, a serial, USB, or a parallel port. Wireless communication may be achieved via one or more wireless configurations, e.g., radio frequency, optical, Wi-Fi, Bluetooth (an open wireless protocol for exchanging data over short distances, using short length radio waves, from fixed and mobile devices, creating personal area networks (PANs), ZigBee® (a specification for a suite of high level communication protocols using small, low-power digital radios based on the IEEE 122, 15.4-1203 standard for wireless personal area networks (WPANs)).

[0045] Referring to FIG. 5, a method 100 for determining an analyte concentration (e.g., sucralose concentration) that uses the sucralose measuring system 1, i.e., sucralose meter 20 and the electrochemical sensor 10 embodiments will now be described. In exemplary method 100, the sucralose meter 20 and the test strip(s) 10 are provided as part of the method. The sucralose meter 20 performs amperometric detection of sucralose and includes electronic circuitry described above that is used to apply a plurality of voltages to the electrochemical sensor 10 and to measure a current transient output resulting from an electrochemical reaction at the distal electrode portions 14a, 16a, 18a of the electrodes 14, 16, 18 of the electrochemical sensor 10. The controller 38 executes a set of instructions stored in memory 50 for the method of determining an analyte concentration in a fluid sample as disclosed herein.

[0046] The method can be achieved starting with step 102 with insertion of the electrochemical sensor 10 into the strip port 32 of the sucralose meter 20 to connect the electrodes 14, 16, 18 of the electrochemical sensor 10 to the measurement circuit 45. In this method, the sucralose meter 20 may apply a test voltage or a current between any two or more of the contacts 14c, 16c, 18c of the electrodes 14, 16, 18. Once the sucralose meter 20 recognizes that the electrochemical sensor 10 has been inserted from step 102, the sucralose meter 20 turns on and initiates a fluid detection mode. In the exemplary method, the fluid detection mode causes sucralose meter 20 to apply a constant test current, which may be about 1 microampere, between the counter electrode 16 and the working electrode 18. Because the electrochemical sensor 10 is initially dry, the sucralose meter 20 measures a relatively large voltage. When the fluid sample is deposited onto the test chamber in step 104, the sample bridges the gap between the distal electrode portions 14a, 16a, 18a and the sucralose meter 20 will measure a decrease in measured voltage that is below a predetermined threshold causing sucralose meter 20 to initiate the sucralose test in step 106 by application of a voltage potential between the reference electrode 14 and the working electrode 18. The application of the measurement signal, e.g., constant voltage, may be done automatically in response to detection of the fluid sample at step 104 or manually in response to use input via one of the buttons 26, 28, 30 or the display screen 24.

[0047] Constant potential voltage is applied between the reference electrode 14 and the working electrode 18. The applied voltage potential may be from about +0.5 V to about +0.7 V, and in embodiments may be about +0.65 V. The applied potential results in oxidation of sucralose in the fluid sample contacting the working electrode 18. The working electrode 18 serves as a surface on which the electrochemical reaction takes place since the metal/metal oxide layer acts as an electrocatalyst for the direct electrooxidation of sucralose. Thus, the working electrode 18 senses the current of the oxidation reaction. The reference electrode 14 is used to hold a constant voltage with respect to the working electrode 18 to aid with the chemical reaction. The constant potential is maintained by the measurement circuit 45 using the potentiostat.

[0048] At step 108, the current at the working electrode 18 is also measured by the measurement circuit 45 using its potentiostat, which converts the detected current due to oxidation of sucralose into a corresponding voltage signal. The converted voltage signal is provided to the controller 38, which calculates the concentration of the sucralose based on the converted voltage signal. At step 110, the controller 38 may use a transfer function, a lookup table, or another calibration algorithm to convert the voltage signal to corresponding sucralose concentration. At step 120, the calculated concentration is displayed on the display screen 24 as a graphical, text, and/or numerical value.

[0049] The following Examples illustrate embodiments of the present disclosure. These Examples are intended to be illustrative only and are not intended to limit the scope of the present disclosure. Also, parts and percentages are by weight unless otherwise indicated. As used herein, “room temperature” or “ambient temperature” refers to a temperature from about 20 °C to about 25 °C.

EXAMPLES

EXAMPLE 1

[0050] This example describes preparation of sucralose sensors.

[0051] Two different electrode sensors were fabricated. The first electrode was formed by electrodepositing copper onto the SPCE from a solution containing 0.1 M CuSCL in 0.1 M H2SO4 at a constant potential of -0.6 V versus Ag/AgCl reference electrode. The deposited Cu was then electrochemically oxidized to CuO by applying a constant potential of 0.8 V in a stirred solution of 0.1 M NaOH. The first fabricated electrode is denoted herein as Cu/CuO/SPCE.

[0052] The second electrode was formed in a similar manner. Cu was electrodeposited on a platinum wire electrode from a solution containing 0.1 M CuSCL in 0.1 M. H2SO4 at a constant potential of -0.6 V versus Ag/AgCl reference electrode followed by electrochemically oxidized to CuO of 0.1 M NaOH. The fabricated electrode is denoted as Cu/CuO/Pt wire.

[0053] The Cu/CuO mesoclusters were electrodeposited on the SPCE and oxidized to Cu/CuO mesoclusters. The surface morphology of the SPCE modified with Cu/CuO was studied with scanning electron microscope (SEM) while elemental analysis was carried out with energy- dispersive X-ray spectroscopy (EDS) spectrum. Both the studies were carried out using the Thermo Scientific Apreo equipment.

[0054] The SEM image of FIG. 6 illustrates that the Cu/CuO mesoclusters are evenly distributed on the surface of the SPCE. The Cu/CuO mesoclusters were formed as aggregates of smaller spherical Cu deposits that ranged in size from 50 nm to 300 nm. Element compositions of the electrode shown in a plot FIG. 7 confirmed the presence of Cu and oxygen (O) in the atomic ratio of about 85.5 % and 1.7%. Since the surface of the bare SPCE is made up of carbon, an atomic percentage of about 16.9% of carbon was observed. The distribution of Cu in FIG. 8 A further confirms the uniform deposition of Cu on the electrode surface. The distribution of oxygen is shown in FIG. 8B while that of carbon is shown in FIG. 8C. Since oxidation of the deposited Cu was carried out in sodium hydroxide, the distribution of oxygen is observed over the region of copper deposits as shown in FIG. 8C.

EXAMPLE 2

[0055] This example describes electrochemical oxidation of sucralose on Cu/CuO/SPCE of Example 1.

[0056] Electrochemical studies were carried out using CHI 1030C electrochemical workstation (CH Instruments, TX, USA). The electrochemical cell consisted of a three-electrode system with SPCE as working electrode for the first study and Pt wire for the second set of studies, a Pt wire as counter electrode and saturated Ag/AgCl reference electrode. All potentials described in these Examples are in reference to Ag/AgCl electrode.

[0057] All electrochemical reactions were performed in 0.1 M NaOH. Electrooxidation of sucralose was studied using linear sweep voltammetry (LSV) at a potential window of 0 to 0.9 V with a scan rate of 100 mV/s. Amperometric detection of sucralose was carried out at a constant potential by injecting different concentrations of sucralose into a constantly stirred solution of 0.1 MNaOH. The effect of interfering species was studied by injecting ascorbic acid (AA), uric acid (UA), dopamine (DA) and sugars (glucose, fructose, maltose, sucrose and galactose) for their possible interference under similar experimental conditions. [0058] The electrochemical oxidation of sucralose on the Cu/CuO/SPCE was studied using linear sweep voltammetry (LSV). FIG. 9 shows LSV plots a-f (bottom to top) recorded using Cu/CuO/SPCE in 0.1 M NaOH with increasing sucralose concentrations. The LSV plots illustrate that in the absence of sucralose (plot a) no characteristic peak was obtained, but a small shoulder was observed around +0.45 V which may be due to the redox wave of Cu(II)/Cu(III). But, in the presence of sucralose (5 mM), a distinct peak at +0.50 V was obtained (plot b) and this can be attributed to the oxidation of sucralose. During each addition of 5 mM of sucralose (plots b-f), the peak current increased linearly up to about 25mM.

[0059] LSV’s were also recorded on the Cu/CuO/SPCE with 3 mM sucralose in 0.1 M NaOH while increasing the scan rate (10 mV/s to 150 mV/s). As observed in FIG. 10, as the scan rate increased from 10 mV/s to 150 mV/s plots a-o (bottom to top), the Faradaic current increased linearly with square root of scan rate with a regression equation Ip(mA)=0.1025v^1/2(mV/s)+(- 0.02836), o=0.01003, n=16 and r=0.9937. This suggests that sucralose oxidation on the Cu/CuO/SPCE may involve a diffusion-controlled mechanism.

EXAMPLE 3

[0060] This example describes amperometric detection of sucralose using the electrodes of Example 1.

[0061] The steady state response current towards different concentrations of sucralose in 0.1 M NaOH on the modified electrode was studied at various potentials on Cu/CuO/SPCE. It was found that the response current for unit concentration of sucralose is maximum at +0.65 V with a rapid response of less than 2 sec. Consequently, all further experiments were conducted at +0.65 V. FIG. 11 shows plots representing the amperometric response towards five additions of 1 mM and four additions of 5 mM of sucralose. The Cu/CuO/SPCE sensor exhibited a linear response from 1 mM up to 25 mM of sucralose with a regression value close to 1 which is an important characteristic for a biosensor.

[0062] Similarly, the steady state current response of the Cu/CuO/Pt wire electrode towards different concentrations of sucralose was studied at +0.65 V. As shown in FIG. 12, the Cu/CuO/Pt wire electrode exhibited excellent electrocatalytic activity towards sucralose with detection in the concentration range of 1 mM to 75 mM. Both the Cu/CuO/SPCE and Cu/CuO/Pt wire electrodes were tested for their repeatability and reproducibility by making five electrodes under similar experimental conditions and testing them amperometrically at +0.65 V with 3 mM of sucralose in 0.1 M NaOH. A variation of less than ±3% was observed for both the electrodes. This indicates that the electrode modification is highly reliable.

EXAMPLE 4

[0063] This example describes electrooxidation of sucralose on Cu/CuO/SPCE and Cu/CuO/Pt electrodes.

[0064] To elucidate the mechanism of direct electrooxidation of sucralose on CuO modified electrodes, electrooxidation of 75 mM of sucralose in 0.1 M NaOH was carried out at +0.65V for about 90 minutes on the Cu/CuO/SPCE electrode. After the electrooxidation, the spent electrolyte was analyzed by and ¹³C NMR spectroscopy. From the NMR data, formation of formate was evident. ¹³C NMR analysis revealed the presence of small amounts of sucralose-derivatives, in addition to the formate peak. Analysis showed that these sucralose-derivatives were generated by the reaction of sucralose with deuterated 0. IM NaOH and were not the products of electrooxidation of sucralose. [0065] To elucidate the structures of the sucralose-derived products and to further understand the stability of sucralose in an alkaline medium, IM sucralose (1 mL) was mixed with IM of sodium hydroxide (ImL) in a total volume of 2 mL and heated at 40 °C for 5 minutes. Comparison of the ¹³C NMR data of the spent electrolyte with the spectrum of sucralose in D2O revealed a major product with a downfield signal (8C 70.00) and loss of an upfield sucralose signal (5C 43.45). Overnight storage of the reaction mixture at 25 °C deposited a crystalline precipitate (crystalline compound A). Melting point analysis of the crystal revealed a temperature of 100 °C. X-ray crystallographic analysis showed that the fructofuranose moiety undergoes an intermolecular SN2 cyclization in the presence of an alkaline medium and confirmed the absolute stereochemistry of this known compound.

[0066] The ¹³C NMR data of the starting reagents for the crystalline compound A matched closely with a pentacyclic sucrose derivative, which was confirmed using ID and 2D NMR analyses as shown in Table 1 below. A summary of 2D NMR heteronuclear multiple bond correlation (HMBC) and correlated spectroscopy (COSY) correlations for the dicyclized fructofuranose moiety is shown in FIG. 13.

Table 1. *H (500 MHz) and ¹³C (125 MHz) NMR Data for Compound A in DMSO-D6

[0067] The initial mono-cyclization of the fructofuranose functionality of sucralose occurs between the hydroxide at C9 with the 1° Cl-atom at C12 under an alkaline condition via an intramolecular SN2 reaction (Scheme 1). This reaction is very slow in low temp and basic conditions (0. IM NaOH at 25 °C), however, it proceeds fast enough under higher temp and basic conditions (1 ,0M NaOH at 40 °C) to isolate compound A.

[0068] NMR analysis of the residual mother liquor, obtained after isolation compound A, showed the presence of another sucralose derivative with a downfield signal shift (5C 72.51) at position C7 and minor unreacted sucralose impurities. It is believed that a second intramolecular SN2 cyclization had taken place based on the C7 shift and subsequent lack of a C-Cl bond signal. To synthesize more pure dicyclic derivative B (scheme 2 of FIG. 14), 1 ,0M sucralose was added to 3.0M sodium hydroxide at 40 °C for 5h. 'H and ¹³C NMR analyses indicated that a that a second SN2 cyclization had occurred on the fructofuranose functionality between the CIO hydroxide and the C7 1° Cl-atom. However, due to ¹³C NMR spectrum signal overlap (6C 72.78) of compound B, these signals could not be unambiguously assigned. However, 2D NMR analysis allowed elucidation of compound B (Table 2). Key HMBC and COSY correlations are shown in FIG. 14. Based on these findings, it is believed that synthesis of compound A during electrooxidation of sucralose is not possible and that this reaction would instead yield compound B.

Table 2. ' H (500 MHz) and ¹³C (125 MHz) NMR Data for Compound B in D₂O

[0069] Amperometric experiments carried out in the presence of compound A and compound B in 0.1 M NaOH at +0.65 V did not yield any increase in current. This clearly indicates that the amperometric response obtained on the Cu/CuO/Pt and Cu/CuO/SPCE electrodes is due to the direct electrooxidation of sucralose and not from the SN2 compounds. EXAMPLE 5

[0070] This example describes an electric meter module for use with the electrode sensors of Example 1.

[0071] Detection of sucralose concentration is accomplished by applying a constant potential between the working electrode with reference to the reference electrode. The constant potential, which was about +0.65 V, resulted in the oxidation of sucralose. The liberated electrons were measured as current. A potentiostat circuit was used to maintain a constant potential. The current produced during the reaction was first converted into the corresponding voltage before being fed to the microcontroller. The microcontroller used a calibration program to convert the voltage into its corresponding sucralose concentration and displayed the results on an LCD display. The various components of the sucralose meter module are shown in FIG. 15.

[0072] The sucralose meter module included a potentiostat LMP91000 and an operational amplifier LMP7721. An advanced low power microcontroller was used in conjugation with an 8MHz crystal that provided a clock signal to the microcontroller. Microchip’s advanced core microcontroller PIC16LF1783 was used as the meter controller with a 12-bit ADC resolution. Its Extreme Low Power (XLP) technology reduced the power consumption, making it well suited for battery powered applications. Its wide voltage range and in-circuit serial programming features provided an added advantage for portable device development. The meter module used a single Li-Ion battery, and two low dropout regulators were used for providing constant voltage. A push button switch was used to give user input to the meter. The result and status were provided through the graphical LCD. I2C communication was used for communicating the LMP91000 with the microcontroller. SCL and SDA pins were used for I2C communication. The output from the AFE (Vout) was fed to the controller’s ADC channel. The graphical LCD used serial SPI communication. [0073] This disclosure provided two types of enzyme-free electrodes for the quantitative determination of sucralose concentration. The first sensor type involved the use of Cu/CuO on screen-printed carbon electrodes to act as catalyst for sucralose oxidation. The sensor was able to linearly detect sucralose concentration up to 25 mM. Moreover, the mechanism of oxidation of sucralose was observed to be diffusion controlled. For the second sensor, a bare platinum wire electrode coated with Cu/CuO was utilized for sucralose oxidation up to 75 mM. The response time for both the sensor electrodes was less than 5 seconds with negligible impact to oxidation of sucralose in the presence of other sugars. The fabrication process was also found to be highly reliable. The developed sensors are highly sensitive and provide instantaneous results without sample processing and pretreatment, thus overcoming the drawbacks of the commercially available sucralose quantification systems and methods.

[0074] It will be appreciated that of the above-disclosed and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications. Various presently unforeseen or unanticipated alternatives, modifications, variations or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims. Unless specifically recited in a claim, steps or components of claims should not be implied or imported from the specification or any other claims as to any particular order, number, position, size, shape, angle, or material.

Claims

WHAT IS CLAIMED IS:

1. A sucralose electrochemical sensor for measuring concentration of sucralose in a fluid sample, the sucralose electrochemical sensor comprising: a counter electrode; a reference electrode; and a working electrode including a distal electrode portion and a layer formed from a metal and a metal oxide disposed on the distal electrode portion for oxidation of the sucralose contained in the fluid sample.

2. The sucralose electrochemical sensor according to claim 1, further comprising: a substrate formed from a dielectric material, wherein the counter electrode, the reference electrode, and the working electrode are disposed on the substrate.

3. The sucralose electrochemical sensor according to claim 1, wherein the metal is at least one of copper, nickel, or cobalt.

4. The sucralose electrochemical sensor according to claim 1, wherein the metal oxide is at least one of copper oxide, nickel oxide, or cobalt oxide.

5. The sucralose electrochemical sensor according to claim 1, wherein each of the counter electrode, the reference electrode, and the working electrode are screen-printed carbon electrodes.

6. The sucralose electrochemical sensor according to claim 1, wherein each of the counter electrode, the reference electrode, and the working electrode are formed from a metallic wire.

7. The sucralose electrochemical sensor according to claim 1, wherein the layer formed from the metal and the metal oxide includes a plurality of metal and metal oxide nanoclusters.

8. A sucralose measurement system for measuring concentration of sucralose in a fluid sample, the system comprising: a sucralose electrochemical sensor including: a counter electrode; a reference electrode; and a working electrode including a distal electrode portion and a layer including a metal and a metal oxide disposed on the distal electrode portion for oxidation of the sucralose contained in the fluid sample; and a sucralose meter including: a port for receiving a proximal portion of the sucralose electrochemical sensor; a measurement circuit for applying a constant voltage potential to the reference electrode and the working electrode to oxidize the sucralose contained in the fluid sample and for measuring current due to oxidation of the sucralose; and a controller for converting the measured current into a voltage signal and calculating a concentration of the sucralose based on the voltage signal.

9. The sucralose measurement system according to claim 8, wherein the sucralose electrochemical sensor further includes a substrate formed from a dielectric material, wherein the counter electrode, the reference electrode, and the working electrode are disposed on the substrate.

10. The sucralose measurement system according to claim 8, wherein the metal is at least one of copper, nickel, or cobalt.

11. The sucralose measurement system according to claim 8, wherein the metal oxide is at least one of copper oxide, nickel oxide, or cobalt oxide.

12. The sucralose measurement system according to claim 8, wherein each of the counter electrode, the reference electrode, and the working electrode are screen-printed carbon electrodes.

13. The sucralose measurement system according to claim 8, wherein each of the counter electrode, the reference electrode, and the working electrode are formed from a metallic wire.

14. The sucralose measurement system according to claim 8, wherein the layer formed from the metal and the metal oxide includes a plurality of metal and metal oxide nanoclusters.

15. The sucralose measurement system according to claim 8, wherein the sucralose meter further includes a display screen for displaying the concentration of the sucralose.

16. The sucralose measurement system according to claim 8, wherein the measurement circuit includes a potentiostat and an operational amplifier.

17. A method for forming a sucralose electrochemical sensor for measuring concentration of sucralose in a fluid sample, the method comprising: placing a working electrode and a reference electrode of the sucralose electrochemical sensor in a first solution including a salt of a metal dissolved therein; applying an electrical potential at a first voltage between the reference electrode and the working electrode in the first solution to form a metal layer on the working electrode from the salt of the metal; placing the working electrode and the reference electrode of the sucralose electrochemical sensor in a second solution; and applying the electrical potential at a second voltage between the reference electrode and the working electrode in the second solution to oxidize the metal layer to form a layer having the metal and metal oxide nanoclusters.

18. The method according to claim 17, wherein the metal is at least one of copper, nickel, or cobalt.

19. The method according to claim 17, wherein metal oxide of the metal and metal oxide nanoclusters is at least one of copper oxide, nickel oxide, or cobalt oxide.

20. The method according to claim 17, wherein the first voltage is negative with respect to the reference electrode and the second voltage is positive with respect to the reference electrode.