Mxene-based electrochemical phosphate sensors

Abstract

Rapid and accurate detection of molecular species with a high degree of selectivity and sensitivity constitutes the ultimate goal of designing sensors for various applications, from studying nutrients in soil-water systems to assessing physiological conditions in human health. For the first time, MXene systems, such as atomically thin two-dimensional Ti3C2Tx layered materials, can be used for electrochemical detection of phosphates, one of the key molecules for sustainability of life on earth, and one of the major contributors to environmental pollution. The MXene sensors are highly selective towards phosphates.

Description

MXENE-BASED ELECTROCHEMICAL PHOSPHATE SENSORSSTATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0001] This invention was made with government support under Award Nos. 1935676 and CMMI-2134607 awarded by the National Science Foundation (NSF). The government has certain rights in the invention.RELATED APPLICATIONS

[0002] This application claims the priority benefit of U.S. Provisional Patent Application Serial No. 63 / 713,842 entitled “MXENE-BASED ELECTROCHEMICAL PHOSPHATE SENSORS,” filed October 30, 2024, the entire disclosure of which is incorporated herein by reference.BACKGROUND1. Field of the Invention

[0003] The present disclosure is generally related to MXene-based electrochemical phosphate sensors with high sensitivity, selectivity, and reliability.2. Description of the Related Art

[0004] Electrochemical sensors are ubiquitous due to their ability to interact and measure the presence of desired species in molecular form in diverse fields, including environmental monitoring, health diagnostics, industrial processes, and many more. The key information is derived from the fundamental interactions between the sensor surface and its immediate environment (typically, a fluid environment in close contact with the sensor). In addition to the interfacial phenomena, such as diffusion, adsorption, charge-transfer, and double layer formation, several other factors are also crucial in evaluating the success of an electrochemical sensor device, particularly in complex real-world applications. These include the degree of signal transduction (sensitivity), reliability under repeated cycling, impact of physical stimuli such as pH and temperature, and selectivity amidst other interfering species. Nanoscale materials with unique geometries, such as nanowires, nanotubes, and nanosheets with semiconducting and metallic type electronic properties, have demonstrated transformative sensing characteristics in the past forelectrochemical sensing addressing low sensitivity issues. However, these materials are often functionalized with recognition agents that can cause instabilities, such as leaching and low binding affinities. Therefore, achieving an optimal balance between sensitivity, stability, and reliability, while ensuring selective detection of molecular species in the environment through improved functionalization strategies or with materials that do not require functionalization is the primary goal in developing state-of-the art sensor materials and devices.

[0005] Phosphorus in the form of phosphates is essential for all life forms on Earth to carry out many biological processes, such as synthesis of genetic material (DNA and RNA), formation of cell membrane (phospholipids), cellular energy transfer (ATP), activation of enzymes, maintenance of blood pH, and many more. In the agricultural ecosystem, phosphate is one of the major macronutrients for plant growth and reproduction. However, the available phosphate for plants in the soil (inorganic orthophosphates) is often limited due to its tendency to bind with soil particles and metals, forming complexes that render it inaccessible to plants, thereby resorting farmers to apply phosphate fertilizers to maximize the availability of P in soil for plant uptake. Adversely, unregulated use of phosphate as fertilizer disrupts soil microbial activity and poses risks to crop health, ultimately reducing the crop yield. Furthermore, excess phosphate gets leached into nearby water bodies, causing eutrophication. Therefore, maintaining an optimal presence of phosphorus in the soil environment is necessary. This urges careful monitoring of phosphates with the development of reliable sensor technologies. While many laboratory-based techniques exist for detecting phosphate, developing an in-field electrochemical phosphate sensor is preferred for its portability, affordability, rapid response time, and sensitivity.

[0006] Several mechanisms have been proposed in the past to develop phosphate-based electrochemical sensors. A common approach involved immobilization of a synthetic anion receptor with an integrated redox active element on a working electrode in voltammetry techniques to create an electrochemical signal for detection. Some sensing of phosphate ions has been conducted in solution phase anion receptors with redox elements as opposed to invariably immobilizing the receptor on the electrode as mentioned before. On the other hand, potentiometric technique often involved the use of an ionophore in a membrane matrix or metal as ion selective electrode (“ISE”) for selective detection of phosphate ions. In other cases, the classical EPA- approved molybdenum blue method used in colorimetric technique to detect phosphates has been utilized.

[0007] In the classical method, the phosphomolybdenum complex (“PMC”) formed in the presence of phosphate and molybdate in an acidified medium (Equation (1)) is reduced to a mixed molybdenum oxidation state using an ascorbic acid as a reducing agent and potassium antimony tartrate as a catalyst (Equation (2)). The reduced PMC appears blue, and the intensity of the color is quantified using spectrometric technique and is directly proportional to phosphate concentration, as measured with a spectrophotometer.

[0008] However, these conventional techniques are labor intensive, costly, timeconsuming, and in-field incompatible.

[0009] Recently, atomically thin two-dimensional (2D) nanomaterials, such as graphene, have been used as working electrodes for various electrochemical sensors, including graphenebased electrochemical phosphate sensors. However, the use of 2D materials for phosphate sensing remains in its early stages, presenting opportunities to build on previous research and explore additional 2D materials for this purpose.

[0010] Discovery and scientific research on atomically thin two-dimensional nanomaterials have been on the surge since the isolation of graphene from graphite in 2004. Research has been undertaken on the understanding of the two-dimensional nanomaterials and their properties for development of electrochemical phosphate sensors. Phosphate is a molecule of interest for sensing owing to its importance in soil and water ecosystems. Excess phosphate in water contributes to eutrophication and deficiency / excess phosphate in soil is detrimental to plant growth. Hence, strict monitoring of phosphate in these ecosystems is necessary.

[0011] Rapid and accurate detection of molecular species with a high degree of selectivity and sensitivity constitutes the ultimate goal of designing sensors for various applications, from studying nutrients in soil-water systems to assessing physiological conditions in human health.SUMMARY

[0012] One or more embodiments of the present disclosure generally concern a method of testing a sample for the presence of a target substance. Generally, the method comprises: (a) dispersing the sample within a medium comprising a recognition agent that is operable to react with the target substance, wherein the target substance comprises phosphorous, a phosphorouscompound, and / or a phosphate; (b) contacting the medium within which the sample is dispersed with a sensing device, the sensing device comprising a MXene electrode containing a MXene, wherein the MXene has a chemical formula of Mn+iXnTx, where “M” denotes a transition metal atom, “X” denotes either C or N, “T.v” denotes one or more surface termination groups, and “n” denotes an integer number ranging from 1 to 3; (c) inducing an electrochemical reaction between the target substance and the recognition agent; and (d) detecting a peak current signal at a characteristic applied voltage within the appropriate electrochemical window with the sensing device, the peak current signal being proportional to the concentration of the target substance within the sample.

[0013] One or more embodiments of the present disclosure generally concern a method of testing a sample for the presence of phosphate. Generally, the method comprises: (a) dispersing the sample within an electrolyte medium comprising a molybdenum recognition agent that is operable to react with the phosphate and form a phosphomolybdenum complex; (b) contacting the electrolyte medium within which the sample is dispersed with a sensing device, the sensing device comprising a MXene electrode containing a MXene, wherein the MXene has a chemical formula of Mn+iXn r, where “M” denotes Ti, Sc, Mo, Zr, V, or Cr, “X” denotes either C or N, “Tx” denotes one or more surface termination groups, and “n” denotes an integer number ranging from 1 to 3; (c) inducing an electrochemical reaction of the phosphomolybdenum complex on a surface of the MXene electrode to thereby form a quantifiable electrochemical signal; and (d) detecting the quantifiable electrochemical signal with the sensing device, the quantifiable electrochemical signal being proportional to the concentration of the phosphate within the sample.

[0014] One or more embodiments of the present disclosure generally concern a phosphate electrochemical sensing system. Generally, the system comprises: (a) a molybdenum recognition agent; and (b) an MXene electrode containing an MXene, wherein the MXene has a chemical formula of Mn+iXnTY, where “M” denotes a transition metal atom, “X” denotes either C or N, “Tx” denotes one or more surface termination groups, and “n” denotes an integer number ranging from 1 to 3.BRIEF DESCRIPTION OF THE FIGURES

[0015] Embodiments of the present invention are described herein with reference to the following drawing figures, wherein:

[0016] FIG. 1 is a schematic flow diagram depicting the synthesis of a MXene; and

[0017] FIG. 2 is a schematic flow diagram depicting how the MXene sensors are produced and used.DETAILED DESCRIPTION

[0018] Two-dimensional transition metal carbides / nitrides, such as MXene, have shown great potential for various applications, such as energy storage and electromagnetic interference shielding, with recent progress in sensing. However, the fundamental electrochemical studies and subsequent applications of MXene for molecular sensing are still in their infancy. Due to their nanometer-scale thickness, MXene materials exhibit unique electrical, thermal, mechanical, chemical, and biochemical properties that are unattainable in their bulk counterparts. These exceptional characteristics have the potential to significantly improve the performance and sensitivity of phosphate sensors. Such sensors could also enable fundamental insights into 2D material-environmental fluid (nutrient in solution) interactions by detecting electronic signals originating from the nanoscale solid-liquid interface.

[0019] One or more embodiments of the present disclosure generally concern a method of testing a sample for the presence of phosphate. Generally, the method comprises: (a) examining and determining the safe electrochemical window of the MXene “working electrode” to avoid electrochemical oxidation or reduction and corresponding ORR (oxygen reduction reaction) and HER (hydrogen evolution reaction), respectively; (b) dispersing the sample within an electrolyte medium comprising a molybdenum recognition agent that is operable to react with the phosphate and form a phosphomolybdenum complex; (c) contacting the electrolyte medium within which the sample is dispersed with a sensing device, the sensing device comprising a MXene electrode containing a MXene, wherein the MXene has a chemical formula of Mn+iXnT , where “M” denotes Ti, Sc, Mo, Zr, V, or Cr, “X” denotes either C or N, “Tx” denotes one or more surface termination groups, and “n” denotes an integer number ranging from 1 to 3; (d) inducing an electrochemical reaction that gives rise to the formation of phosphomolybdenum complex on the surface of the MXene electrode to thereby form a quantifiable electrochemical signal; and (e) detecting the quantifiable electrochemical signal with the sensing device, the quantifiable electrochemical signal being proportional to the concentration of the phosphate within the sample

[0020] MXene, a class of atomically thin two-dimensional materials, generally with metallicity characteristics but not limited to, is proposed for the first time for electrochemical phosphate sensors. An exemplary type of MXene is titanium carbide (Ti iC^T J is presently being studied for scalable manufacturing; however, its use in electrochemical phosphate sensing has not been developed. The present disclosure presents the first MXene-based electrochemical phosphate sensor with high sensitivity, selectivity, and reliability.

[0021] In particular, we have leveraged the metal -like electrical conductivity and surface properties of TriC^T? MXene to discover electrode processes that elucidate the electrochemical redox activities of heteropolymetalate ions, specifically PMCs, in the modified MB method. Consequently, we utilize this underlying mechanism to design a TisC2TxMXene-based electrochemical phosphate sensor. The designed phosphate sensor demonstrated a limit of detection (LOD) of 1.31 pM and a linear sensing range of 1-350 pM while being very selective to phosphate amongst other interfering ions.

[0022] MXenes generally exhibit high intrinsic hydrophilicity and high electrical conductivity, which make them desirable for the uses described herein. Generally, MXenes have a general formula of Mn+iXnTx, where “M” denotes a transition metal atom (such as Ti, Sc, Mo, Zr, V, Cr, etc.); “X” denotes either a carbon or nitrogen atom; “Tx” denotes one or more surface termination groups (such as O, -F, -Cl, and / or -OH); and “n” denotes an integer number ranging from 1 to 3. Typically, such MXenes exhibit atomically thin 2D characteristics with active inplane sites that allow for functionalization to detect environmental species.

[0023] MXenes are synthesized by selectively etching A atoms (i.e., an element from group 13 to 16 of periodic table, such as Al, Si, and / or Ga) from parent MAX crystals (general formula of Mn+iAXn) using either a fluorine-based or non-fluorine-based etchants, such as HF and / or HC1.

[0024] The ease of solution processibility and the high metallic-type electrical conductivity of MXenes has attracted much attention, with applications spanning from energy storage, electromagnetic interference shielding, electrochemical sensors, catalysis, drug-delivery, and water purification. However, MXene has shown limitations due to its rapidly oxidizing nature, thereby limiting its long-term use and environmental stability. Electrochemical oxidation of MXene has also been demonstrated, recently, within a specific potential window (vs. Ag / AgCl reference electrode). This constrains the potential range applicable for the electrochemical redox activities at the MXene surface, thereby affecting its use for various analyte sensing. Therefore,investigating MXene’s potential as an electrochemical sensor for various analytes despite its drawbacks is a valuable area of study.

[0025] Furthermore, in the electrochemical systems, TLC2T.V MXene may be used in the cathodic potential window as it undergoes an irreversible electrochemical oxidation in the anodic potential window, with the oxidation potential being primarily influenced by the electrolyte and the pH of the electrochemical system. Therefore, identifying the appropriate potential range in investigating TisC^T. MXene’s promise as an electrochemical sensor for various analytes is a valuable area of study.

[0026] We have leveraged the metal-like electrical conductivity and surface properties of TiaC2TxMXene to discover electrode processes that elucidate the electrochemical redox activities of heteropolymetalate ions, specifically PMCs, in the modified molybdenum blue method. Consequently, we utilized the underlying mechanism to design a TisC2Tv MXene based electrochemical P sensor. The designed P sensor demonstrated a limit of detection (LOD) of 1.31 pM and a linear sensing range of 1 pM to 350 pM while being very selective to P amongst other interfering ions.

[0027] Herein, we demonstrate for the first time atomically thin TLCLTv layers as a model MXene system for electrochemical detection of phosphates, one of the key molecules for sustainability of life on earth, and major contributors to environmental pollution. The MXene sensors were demonstrated to be highly selective towards phosphates, among other potential interfering molecules with a sensing range from 1 pM to 350 pM and a detection limit (LOD) of 1.31 pM. This work provides a foundational discovery in the molecular sensing of TisCTTv and other MXene materials in complex environments.

[0028] More particularly, we exploit exceptional the electrical conductivity and surface catalytic properties of Mxene, such as Ti3C2Tx, the first two-dimensional atomically thin metallic material discovered to have electrode processes that elucidate the electrochemical redox activities of heteropolymetalate ions, specifically phosphomolybdenum complexes (“PMC”). Consequently, we utilized this underlying mechanism to design the first MXene based electrochemical phosphate sensor. The designed phosphate sensor demonstrated an LOD of 1.31 pM and linear sensing range of 1-350 pM while being very selective to phosphate amongst other interfering ions.

[0029] Phosphate ions have significant impact in life and environment - both human life and plant life, as well as environmental sustainability. Therefore, monitoring phosphates in soil,water, and human health using a portable device in a cost-effective manner with high sensitivity, selectivity, and reliability has a great potential in developing a transformative technology for societal benefit. The present disclosure uses an MXene, such as TFCTTv, to function as an excellent nanomaterial platform to build phosphate sensors. Real water samples were tested using such sensors.

[0030] MXene related phosphate sensors, such as dye-sensitized TisC2 photoelectrochemical sensing of phosphate have been used; however, pure Ti3C2Tv MXene has not been used as a phosphate sensor. This is the first comprehensive work on phosphate sensors from MXenes, such as Ti3C2Tx. It was previously observed that Ti^CTTv MXene usually gets oxidized in atmospheric moisture and might not be suitable for sensing purposes. However, high response after the 135thday indicated that the material has the potential for long term use as phosphate sensors. As described herein, the MXene sensors of the present disclosure can retain at least 90% of their phosphate sensing capacity after 135 days of storage, indicating reasonable stability under our testing conditions.

[0031] The overall process for developing the MXene phosphate sensors and the operation of the sensors are described below.Production of Etched and Delaminated Few Layer Tii ICb

[0032] In one or more embodiments, the stock materials may be synthesized via wet chemical (e g., acid-based with HC1 and / or HF) etching of a TisAlC2 MAX phase material, followed by delamination of the resultant multilayer MXene, as described herein. X-ray diffraction (“XRD”), scanning electron microscopy (“SEM”), tip-enhanced Raman spectroscopy (“TERS”), (Scanning) Transmission Electron Microscopy (S / TEM), and atomic force microscopy (“AFM”), characterization were utilized to measure various properties.

[0033] All the results consistently showed the presence of a few layers of TnCTTv. FIG. 1 provides a schematic diagram representing the synthesis of TisCTTv. As shown in FIG. 1, a TiaAlC2 MAX precursor phase material 10 is subjected to etching with etchants (HF and HC1) to thereby form a multilayer Ti3C2TxMXene 12. Subsequently, as shown in FIG. 1, the multilayer Ti3C2TxMXene 12 is then subjected to delamination with a delamination intercalant (e.g., LiCl) to thereby form a suspension 16 containing MXene with reduced layers 18.

[0034] FIG. 2 depicts that the MXene suspension 16 can be drop-coated onto a bare glassy carbon electrode (GCE) 18 to thereby form a Ti3C2Tx / GCE electrode 20. Subsequently, the Ti3C2Tx / GCE electrode 20 can be used to measure phosphates via electrochemical redox activity of PMC 22, which provides a quantifiable signal 24 that can be analyzed and measured.

[0035] An exemplary method for producing a MXene-containing electrode, particularly a Ti3C2T.Y-containing electrode is provided as follows. Initially, a bare glassy carbon electrode (“GCE”) from BASi MF-2012 was polished with alumina slurry and rinsed using distilled water followed by methanol. It was then water sonicated for 10 minutes. Meanwhile, 10 mg / ml of Ti3C2TAstock solution was prepared and further diluted to 4 mg / ml. Subsequently, 5 pl of the 4 mg / ml Ti3C2T was then drop coated on the clean GCE and dried under an IR lamp for a few minutes before use in electrochemical studies. Before each experiment, the GCE was polished with an alumina-water slurry and rinsed with distilled water followed by drop coating of MXene as described herein.

[0036] Another exemplary process for producing MXene phosphate sensors is provided below.

[0037] In various embodiments, the stock material may be synthesized via wet chemical (acid-based, e.g., via HC1 and / or HF) etching of H3AIC2 MAX phase material, followed by delamination of the resultant multilayer MXene. Our observations consistently show the presence of a few layers of Ti3C2T in the resultant multilayer MXene.

[0038] All chemicals used may be of analytical grade and may be used as obtained from the suppliers without further purification. Potassium chloride, hexaammineruthenium (III) chloride (HisChNeRu), sulfuric acid (H2SO4), ammonium heptamolybdate tetrahydrate ((NH4)6Mo?O24.4H2O), potassium dihydrogen phosphate (KH2PO4), and sodium nitrate (NaNCh) were purchased from Sigma-Aldrich. Potassium sulfate (K2SO4) and sodium carbonate anhydrous (Na2CC>3) were purchased from Thermo Fisher Scientific. All solutions were prepared using ultrapure water with a resistivity of 18.2 MQ.cm at 25°C. Real sample test was performed using tap water obtained directly from the laboratory and environmental water sample collected from a nearby lake (Tuttle Creek Lake located in Manhattan, Kansas).

[0039] The exemplary process steps may include the following sequential steps.

[0040] TisCsT.v MXene may be derived from MAX phase U3AIC2 using a HF-HC1 etchant and LiCl intercalant from known protocols. In particular, the material was procured from Anasori’ s group at Purdue University.

[0041] The highly concentrated material (called “MXene clay”) may be diluted further in water to obtain a concentration of about 4 mg / ml. MXene is much more stable from water molecule-induced degradation when stored in the clay form and preserved at low temperature. Therefore, the clay was stored in air-tight Eppendorf cryo-storage vials (vol. 50 m ) in a refrigerator at 4°C (henceforward assigned as “fresh MXene” or “fresh Ti3C2Txclay”).

[0042] About 5 pl of this concentration may be drop coated on a commercially available glassy carbon electrode (“GCE”) for electrochemical sensing measurements.

[0043] After drop coating, the electrode may be dried for 2-5 minutes using an IR lamp.

[0044] Cyclic voltammetry (“CV”) was first employed in a -IV to +1V potential window in a KC1 supporting electrolyte with the modified glassy carbon electrode with Ti3C2Tx(GCE / MXene) as a working electrode, Ag / AgCl as reference electrode, and platinum wire as a counter electrode. An irreversible oxidation of MXene initiates at approximately 0.25V indicating to avoid any studies at or above 0.25 V.

[0045] Hexaammineruthenium (III) chloride may be used as a standard redox probe to identify the potential sensing capabilities of the MXene. In our studies, good cathodic and anodic peaks were obtained in all scan rates.

[0046] A comparative CV study may also be conducted between bare GCE and the GCE / MXene for the standard redox probe. Our studies indicated larger cathodic and anodic current peaks for GCE / MXene.

[0047] Phosphate sensing may be conducted using the GCE / MXene electrode in the presence of 1 mM ((NH4)6Mo?O24 4H2O), 0.1 M H2SO4, and 0.1 M KC1 as the base electrolyte, with KH2PO4 analyte as the source of phosphate molecules. This study utilized a potential window of 0 V to 0.25 V. In such embodiments, the electrolyte can comprise a recognition agent, such as the molybdenum in this case.

[0048] Phosphate sensing peaks in CV may be observed from 10 pM to 500 pM concentration.

[0049] However, for more precise measurements, the differential pulse voltammetry (“DPV”) technique may be followed in similar concentration range. Significant clarity in phosphate ion sensitivity in the concentration range of 1 pM to 350 pM was obtained.

[0050] Interfering ions, such as chlorine, nitrate, carbonate, etc., may also be added to the system to measure the selectivity of the sensor. Based on our observations, the sensor showed a prominent signal only in the presence of phosphate, which indicates its great selectivity towards this molecule.

[0051] Real sample analysis may also be conducted by spiking tap water and water from local water bodies with a known amount of phosphate. Based on our observations, the recovery percentage of phosphate ion was close to 100% for both samples indicating the reliability of the sensors for the usage in real environments.

[0052] Hysteresis (with respect to gradual increase and decrease in concentration) measurements conducted on the sensor indicated no significant hysteresis present at the sensorwater interface.

[0053] The sensor was also tested for its performance stability on days 0, 5, 10, and 135. The sensor showed nominal change in the signal although it showed an apparent near saturation in current for phosphate sensing after day 10. Given that MXene is a material prone to degradation from atmospheric moisture and other potential contaminants, this result was a significant achievement to demonstrate MXene’ s potential for phosphate sensor.

[0054] In this embodiment, the proposed sensor had a limit of detection of 1.31 pM with linear sensing range of 1 pM to 350 pM.Synthesized MXene Flake Characterization

[0055] Prior to the electrochemical characterization of the TiaC^Tv MXene electrode, the individual Ti3C2Tv flakes were characterized for their quality, as the source and purity of MAX phase significantly influence the quality of MXene material. The MXene quality and structural characteristics are also critically important for the electrochemical detection of the molecules. XRD measurements of the crystalline structure of the D3AIC2 MAX showed sharp peaks, which indicated a well-defined TisAlC2 MAX crystalline structure, with the (002) plane representing the preferential growth direction, despite the poly crystalline nature of the material. XRD measurements also showed, after etching the interlayer aluminum, the MAX phase materialtransformed into the TisC^T.v MXene crystals. In this transformation, the

[0002] direction emerged as the predominant crystal orientation, indicated by the X-ray scattering intensity from this crystal plane. Additionally, there was an increase in the c-lattice parameter, as evidenced by the peak shift towards lower diffraction angle for the (002) peak. This shift confirmed the presence of surface functional groups and micro-molecules, such as water, between the MXene sheets, which originated from the synthesis protocol. In various embodiments, the average lateral size and the representative flake thickness of the TiiC^E. MXene crystal flakes were estimated to be about 4.69 pm and 1.78 nm, respectively (corresponding to 1 to 2 MXene layers), as confirmed via SEM and the AFM testing. The flake size was estimated from the statistical analysis of- 60 TiiC^T- MXene flakes observed in the SEM images while the flake thickness was estimated from - 9 flakes observed in AFM

[0056] XRD patterns of the as-synthesized TisAlC2 MAX and TLC2TA MXene were analyzed using a Bruker D8 X-ray diffractometer with a Cu Ka (X = 1.5406 A) emitter and a VANTEC 500 two-dimensional x-ray detector (XRD2). A corundum standard was used to ensure each detector was calibrated. The precursor samples were mounted on Kapton tapes and scanned from 5° to 80° with a step size of 5 ° and a dwell time of 30 seconds per step. Traditional XRD plots were obtained by merging and integrating the XRD2data in DIFFRAC. SUITE EVA software. SEM was performed on a JEOL JSM-7800F at an acceleration voltage of 15 kV to study the flake size and surface morphology. The solution concentration was maintained at < 0.1 mg / mL and loaded on an anodic disc followed by vacuum drying for 2 h. The samples were gold sputtered to reduce the charging. FEI Tecnai Osiris S / TEM was used to obtain low and high-resolution images of the Ti3C2Txflakes using an accelerating voltage of 200 kV. AFM and TERS characterization were performed on a LabRAM-Nano system (HORIBA Scientific). 785nm excitation with the laser power on the sample of ~200pW was used for collecting TERS spectra. Both excitation and collection were done through the side 100X, 0.7 NA objective (Mitutoyo), inclined at 25 degrees to the sample plane. Access-SNC-Ag TERS probes (APPNano) were used for the TERS imaging in Spec-Top™ (HORIBA) mode. The ex-situ Raman spectro- electrochemical study was conducted using Renishaw Invia Raman equipped with an argon laser (Ao = 532 nm, source power 20 mW). 100X objective lens (NA = 0.90) was used to focus the laser beam with a power of about 1 mW at the Ti3C2TxMXene coated glassycarbon surface. The Raman spectrometer was operated in an extended mode with spectral window covering 50 to 800 cm'1.

[0057] Ti3C2 v MXene was further characterized using the High-Angle Annular Dark Field (HAADF)-STEM measurements coupled with Energy Dispersive X-ray spectroscopy (EDX) to clearly distinguish the elemental composition of the material. It was observed that the flakes consist of not just Ti and C but also the functional groups such as -O, -F, -Cl on the basal plane and edges originating from the wet-chemical synthesis protocol, useful for functionalization in multitude of sensor applications. The TERS spectrum imaging of the Ti3C2Tx MXene flakes demonstrated that Ti3C2Tx MXene flakes exhibited absolute intensity of most prominent TERS spectral features at ~ 203 cm'1, -570 cm'1and 732 cm'1, which decreased with the increase of the flake thickness, while the intensity of the Elg peak at - 125 cm'1, being practically negligible in the monolayer, became visible in the few-layer thick part of the flake.

[0058] As confirmed via transmission electron microscope (HR-TEM) imaging, the MXene flake is very transparent to electron beams, thereby indicating its atomically thin nature. Furthermore, HR-TEM imaging also confirmed the atomic arrangements and the interplanar spacing in the representative MXene flakes, which showed the hexagonal symmetry of the Ti3C2TxMXene structure.Electrochemical Characterization of MXene and Phosphate Electrochemistry

[0059] Cyclic voltammetry (“CV”) is a powerful electroanalytical method to understand the fundamental charge transfer at the electrode-electrolyte interface. Before examining the electrode processes for phosphate ions, the charge transfer via electrochemistry was studied using CV at Ti?,C2T< / GCE electrode. This study was conducted within the potential window of -1.0 V to +1.0 V using a 0.1 M KC1 supporting electrolyte. TiA^Tv has been widely recognized for its stability in cathodic potential window, while exhibiting an irreversible oxidation peak within the anodic potential window. This irreversibility arises from the voltage-induced MXene oxidation leading to an electrical conductivity degradation in turn negatively affecting the charge-transfer ability of MXene in sensing applications. A CV of TiiC+Tv / GCE confirmed the presence of an irreversible oxidation peak of MXene in the very first cycle of operation and its disappearance in the remaining cycles as reported before. Although the prominent oxidation peak occurred at 0.84 V, the oxidation was initiated at a much earlier stage (~ 0.25 V). Therefore, all electrochemicalsensing experiments were conducted below 0.25 V to minimize the possible oxidation of the MXene material.

[0060] To explore the electrochemical properties of MXene within the above potential range, a standard redox active probe, [RuCNHsf,]3 2was employed according to Equations (3) and (4), below. Testing CV between a bare GCE and TisC2TVGCE showed an increase in both anodic (Ipa) and cathodic (Ipc) peak currents for MXene following baseline correction, which confirmed the inherent higher electrical conductivity and active electrochemical sites of the material, facilitating more efficient electron transfer between the working electrode and the redox active species. The testing was conducted in a 0.1 M KC1 supporting electrolyte from -0.3 V to 0.03V at 50 mV / s. A CV of TijC Tv / GCE at different scan rates was also ran, which revealed an anodic peak potential (Epa) and a cathodic peak potential (Epc) of -0.11 V and -0.19 V, respectively. Despite the slightly larger peak -to-peak separation (AEP) of 78 mV (derived from equation 5 across all scan rates) compared to the thermodynamically derived value of 57 / n for an ideal reversible system (n is the number of electrons transferred in the redox reaction, n=l for [RulNHij.-,]3 2redox event shown in Formulas (3) and (4)), this value remained consistent across all scan rates, due to constant Epaand Epcvalues indicating the reversible nature of the reaction. Additionally, a corresponding plot of current versus square root of scan rate for both anodic (lpa) and cathodic (Ipc) currents showed linearity, confirming the diffusion-controlled process as opposed to adsorption- controlled process.Ep= Epa- Epc—— (5)

[0061] As mentioned earlier, in a typical lab oratory -based molybdenum blue method, phosphate detection is facilitated though spectrophotometric measurements involving a chelation reaction between phosphate and molybdate in an acidic medium. This reaction forms a PMC, which is then reduced to phosphomolybdenum blue (“PMB”) using ascorbic acid as a reducing agent. In contrast, in an electrochemical reaction, the application of a reduction potential (and / or oxidation potential, if the process is reversible) typically induces a corresponding redox reaction that reduces (and / or oxidizes) the PMC on the electrode surface. In other words, in the modified method, such as the electrochemical method, the PMC formed is directly reduced at the workingelectrode surface without an external reducing agent and a catalyst leading to a quantifiable electrochemical signal with respect to phosphate concentration. The nature, efficacy, and reliability of the electrode process is fundamentally dependent on the electrode material and the charge transfer process between the electrode surface and the analyte. Therefore, understanding the efficiency of the PMC’s redox activity via electrochemical reactions at the TrsC^Tv MXene electrode surface was paramount.

[0062] Given the lack of fundamental understanding for the mechanistic view of the MXene-phosphate electrode process, we conducted a CV measurement of Ti C^Tv / GCE in a base solution consisting of the KC1 supporting electrolyte, ((NH4)6Mo?O24 ' 4H2O) [abbreviated as Mo], and H2SO4 (to maintain a pH of 0.65), along with 100 pM phosphate. The measurements were performed over a potential window between -0.2 V vs. Ag / AgCl and 1.0 V vs. Ag / AgCl.The CV data was obtained after the first cycle oxidation of Ti.sC^Tv MXene, primarily to explore all the possible phosphate electrochemical reactions at the MXene surface. Subsequent observations related to phosphate sensing were conducted below the oxidation region of MXene (< 0.25 V).

[0063] A series of reversible electrochemical oxidation and reduction peak pairs were apparent from CV measurements. Appearance of multiple voltammetric waves signified more than one electroactive species involving a multiple electrode reaction. The presence of multiple voltammetric peaks indicates the involvement of more than one electroactive species, each participating in distinct redox processes at the electrode interface. Various parameters, such as the solvent ratio, and pH conditions, have been shown to influence the number and strength of voltammetric waves associated with the redox activity of PMC electroactive species.

[0064] Analogous electrode processes involving multiple voltammogram peaks in PMCs in an acidic pH range have been studied previously with the use of gold mini-grid working electrodes in water-dioxane (50% v / v) solutions and in 0.2 M H2SO4. These processes were ascribed to three two-electron reversible reactions, with two showing remarkable robustness regardless of the dioxane concentration. The redox peaks at half wave potential (E1 / 2) of - 0.36 V (BB’), - 0.18 V (CC’), and -0.02 V (DD’) in our case closely resemble those in this previous goldbased study, despite the use of Ti3C2Tv as the active material. We have identified the presence of three pairs of two electron reversible processes (BB’, CC’, and DD’). Each of these three redox active two-electron processes can be linked to the electronic structure of the PMC, which relates to the structural phase transition associated with the diverse Keggin structure of electron richpolyoxometalates. Some have explored MXene-based phosphate sensors and absorbers, but the formation of heteropoly acid and the mechanistic understanding of the electrode process related to the electronic and molecular structure at the MXene surface remains unexplored. This disclosure aims to address these gaps. The origin of the observed peaks is attributed to equations 5-7, below, where the PMC species formed in our electrochemical system (as described in equation 1 from classical molybdenum blue method), undergoes three distinct two-electron electrochemical reduction processes corresponding to peaks B, C, and D. Each step results in a progressively reduced PMC with a mixed molybdenum oxidation state, followed by successive oxidation of the species resulting in B’, C’ and D’ peaks.

[0065] The origin of the redox active PMC species in our electrochemical system is attributed to the equation described earlier in the introduction (Equation (1)), which is relevant to the classical molybdenum blue method. Without wishing to be bound by theory, it is believed that this PMC formed undergoes three distinct two-electron electrochemical reduction processes (peak B to C to D), resulting in a mixed molybdenum oxidation state shown in the successive equations below in equations (5)-(7).(Peak C) [H4PMO4(7)MO8(W)O40]3- + 2e~ + 2H+- [H6PMo6(7)Mo6(W)O40]3“- — (7) (Peak D)

[0066] In other systems (using different working electrode and different solvent), despite acidic pEI conditions, the first two two-electron processes (BB’ and CC’) are shown to be reversible, however, the third two-electron process (DD”) is irreversible possibly due to homogenous reaction in the solution forming a different unidentified PMC.

[0067] In the classical method, a stable mixed molybdenum state complex is often achieved after a four-electron reduction process of the PMC from Equation (1), leading to the intense blue color. In this context, Equations (2) and (6) are equivalent. However, due to the higher sensitivity of the electrochemical technique, less dominant reaction steps, such as Equations (6) and (7), are still detected.

[0068] Moreover, the aforementioned redox activities at the surface of MXene electrode leads to structural changes of PMC. Without wishing to be bound by theory, it is believed that thetransitions are from an a-isomer structure of PMC to a stable 0-isomer structure. During each of the reduction reactions, the PMC obtained in Equation (1) is proposed to be converted from an a isomer to a 0 isomer by rotating a single triad at 60°. The isomer regains its a form upon complete oxidation of the reduced complexes. However, this needs further validation through more extensive ex-situ and in-situ experiments in the future.Electrochemical Sensing of Phosphates with MXene

[0069] To characterize Ti CXE. MXene as P sensor, CV was initially employed in a voltage window between 0 V vs. Ag / AgCl and 0.25 V vs. Ag / AgCl. Despite observing three 2-electron processes within the potential window of -0.2 V to 1 V, further measurements focused specifically on CC’ peaks as they are more distinct compared to the other peaks in the CV data. The potential range associated with the CC’ peaks also helps to avoid any unwanted oxidation of MXene at higher voltage values. A CV using the base solution consisting of the KC1 supporting electrolyte, Mo, and H2SO4 showed no signs of electrochemical sensing. However, upon the addition of 100 pM KH2PO4 (hereby referred to as “P”), a sharp oxidation / anodic peak pair at around 0.2 V and a reduction / cathodic peak at 0.15 V against Ag / AgCl were observed.

[0070] As previously confirmed for [Ru(NHs)6]3+ / 2+active probe, the Ti3C2TA / GCE working electrode presents a larger peak current on both the anodic and cathodic sides, attributed to its high electrical conductivity and larger surface-to-volume ratio, characteristics of two- dimensional nanomaterials. Increasing the concentration of P from 10 pM to 500 pM showed a consistent increase in the anodic and cathodic peak current in CV, indicating a sensor behavior. The CV of 100 pM of P at different scan rates had a corresponding current versus square root of scan rate plot in the inset that proved that the P redox reaction as diffusion limited. The system provided a AEPof 32 mV, a value slightly larger than the thermodynamically derived value of 28.5 mV for a two-electron redox process. However, this value was constant for all the scan rates affirming the system’s reversibility.

[0071] Although CV technique detects the increase in P concentration efficiently, the prominence of the redox peaks diminishes for lower P concentrations. Hence, DPV was employed to enhance the sensitivity for P sensing by minimizing the background current and amplifying the peak associated with the P sensing. The results obtained for the oxidation peak at 0.2 V showed sensing was observed from 1 pM to 350 pM beyond which saturation prevails. Using analyticaldata, two linear ranges were observed in a calibration plot, one from 1 pM to 10 pM of P and the other from 10 pM to 350 pM of P with a sensitivity of 0.14835 pA pM"10.03393 pA pM"1, respectively. The limit of detection (LOD) was found to be 1.31 pM for Ti3C2TY / GCE sensor using the standard formula for LOD , which is 3s / m, where m is slope of the calibration curve and s is standard deviation of the blank solution. Presence of two linear regime in the calibration plot was due to (1) higher number of active sites present at lower concentration of P that results in simultaneous occurrence of redox reaction aiding better sensitivity, and (2) higher concentration region where fewer simultaneous redox reaction takes places because of excessive presence of PMCs that surpasses the number of MXene active sites and crowding caused by oxidized / reduced PMCs near the working electrode surface. To ensure subsequent addition of P did not alter the pH of the base solution, pH vs concentration measurement was performed. The results did not show any deviation in pH.

[0072] Although 2D materials have been widely explored for chemical and molecular sensing, the work presented herein represents the efforts toward developing an electrochemical phosphate sensor based on TijC2TxMXene. TABLE 1, below, presents a summary of pristine 2D materials such as Ti C^Tv MXene and graphene that have been investigated as working electrodes for phosphate sensors using the modified molybdenum blue method. While the linear detection range of graphene sensor appears broader, the Ti3C2TxMXene sensor demonstrates lower LOD, likely due to its superior electrical conductivity and higher density of active sites, making its performance comparable to that of state-of-the-art 2D sensors. Further enhancement of sensor performance could be achieved through optimization of TisC ^ MXene concentration and film thickness on GCE, tuning of surface terminations, and the introduction of structural porosity to improve redox activity. An extended list of a few other nanomaterials and their phosphate sensing metrics is also presented in TABLE 1, below.TABLE 1: Performance Comparison of Different 2D Material Based Working Electrode forPhosphate Sensing using the Modified Molybdenum Blue MethodN / A-data not available, CV-Cyclic Voltammetry, DPV - Differential Pulse Voltammetry', SWV - Square Wave Voltammetry, Mo-Molybdenum, PVC - Polyvinyl chloride, Au - gold, MWCNT - multiwall carbon nanotube, NP- nanoparticles, PVDF-poly(vinylidene fluoride-co-hexafluoropropylene), PANI-Polyaniline, AHM-Ammonium Heptamolybdate, CC - Coconut shell-derived carbonInterference and Real Sample Analysis of TisCiT / GCE for Phosphate Sensing

[0073] The reliability of environmental sensors requires analysis in the presence of interfering species. As such, the Ti3C2T, / GCE sensor was subjected to measurements in the presence of common ions found in environmental water, such as sulfate, carbonate, and nitrate. 100 pM of each interfering species was added to the base solution for current measurement followed by the addition of 100 pM of P ions. The Ti3C2Tx / GCE sensor showed a significant change in the current only in the presence of phosphate and no other ions. The individual change in current for 100 pM interfering ions was also recorded and was insignificant compared to the Ti3C2TA / GCE sensors’ response to P moleculesHysteresis and Stability

[0074] Hysteresis assessment was conducted for MXene sensors to witness the difference in change in current across different concentrations of P over two cycles of increasing and decreasing concentrations. Our studies showed no significant hysteresis in one cycle of changing the concentration from low to high and vice versa. The MXene working electrode exhibited excellent retention in the change in current for all concentrations of P during multiple cycles making it a viable option for future P sensors. The electrodes were also tested for stability over a period of 135 days. The Ti3C2T.v / GCE was used for DPV sensing of 100 pM P on day 0 and stored in ambient condition for 5 days before the next measurement. A similar protocol was carried out for the 10thday measurement and 135thday measurement. Although there was no significant difference in change in current between days 0 and 5 (0.26 pA, 7% change), a decrease in change in current was observed between day 0 and day 10 by 14% (0.55 pA). This could be ascribed to possible oxidation of MXene working electrode in the air. By day 135, the change in current between day 10 and day 135 was observed to be only 5% (0.2 pA). These studies indicate that MXene, even in its oxidized state, still shows its potential for phosphate sensing. Our observations highlight the promising sensing capabilities of MXene at its oxidized state, which can be pursued for sensing applications. Although MXene oxidizes in air, few reports show their stability over time following certain degrees of oxidation and their potential for various applications, including in electrochemical applications.

[0075] In summary, the present disclosure presents the first-time demonstration of intrinsic property of atomically thin TisC T MXene for detection of phosphate ions via electrochemicalsensing. Electrochemical response of TisC2Tywas studied with a supporting electrolyte to determine the electrochemical window for sensing of molecular phosphates. At the identified potential window, phosphate detection at the surface of T C^Tv was facilitated by PMC via an electrode-analyte charge transfer mechanism. Despite widespread belief in MXene’s (TijC^Tx) air instability, the sensor retains its phosphate sensing capacity by 90% after 135 days of storage. MXene phosphate sensors exhibited consistent and highly selective response towards phosphate molecules among other potential interfering ions with a sensing ranging from 1 pM to 350 pM with a detection limit (LOD) of 1.31 pM. This work paves the way for TisCsTv and other MXene materials for exploration of phosphate molecular sensing in complex environments, such as agricultural, ecological, and biomedical domains.

[0076] All electrochemical measurements were performed at room temperature using Interface 1010E potentiostat from Gamry Instruments along with Ag / AgCl (3 M NaCl) reference electrode, platinum wire counter electrode and TfCE v modified GCE working electrode Tr^T. / GCE. The redox reactions at the TEC^Tv / GCE working electrode were investigated by first performing the cyclic voltammetry (“CV”) measurements in a 0.1 M KC1 supporting electrolyte with a potential window between -1 V to IV. The presence of an oxidation peak within the specified potential window required setting the potential range for subsequent redox probe and phosphate sensing measurements below the oxidation potential of MXene. The redox probe sensing was carried out in 0.1 M KC1 and 5 mM HisChNeRu from -0.3 V to 0.03 V. Phosphate sensing utilized a solution consisting of 1 mM ((NH4)6Mo?O24 TBEO), 0.1 M H2SO4, 0.1 M KC1 as the base solution, with KH2PO4 analyte concentrations ranging from 1 pM to 1000 pM. The CV corresponding to the phosphate sensing measurements were taken between 0 V and 0.25 V voltage window. Each CV measurement employed a 2 mV step size over 3 cycles. The differential pulse voltammetry (“DPV”) technique was used primarily for electrochemical sensing of various phosphate concentrations, interference studies, and the real sample analysis as well as the hysteresis assessment. The DPV phosphate sensing utilized the same solution medium as the CV phosphate sensing. Interference studies were conducted within the base solution with the addition of 100 pM each of NaNCh, K2SO4, and Na2CCh. The real samples were measured using DPV within the base solution along with an addition of 1 ml of real sample. The optimized experimental parameters for DPV were as follows: potential window of 0 V to 0.24 V, step size of 2 mV, sampleperiod of 2 s, pulse time of 0.1 s and pulse size of 50 mV. All the electrochemical experiments were repeated n number of times (n > 3) to ensure reproducibility of the results.The MXene Sensors and Sensing Devices

[0077] One or more embodiments of the present disclosure concern a sensing device for a target substance, wherein the sensing device comprises, consists essentially of, or consists of an MXene sensor and a reference electrode. In such embodiments, the MXene sensor comprises, consists essentially of, or consists of a MXene, which has a general formula of Mn+iXnTx, where “M” denotes a transition metal atom (such as Ti, Sc, Mo, Zr, V, or Cr); “X” denotes either a carbon or nitrogen atom; “Tx” denotes one or more surface termination groups (such as O, -F, -Cl, and / or -OH); and “n” denotes an integer number ranging from 1 to 3. In certain embodiments, the MXene can be Ti3C2Tx.

[0078] These MXenes generally exhibit high intrinsic hydrophilicity and high electrical conductivity, which make them desirable for the uses described herein. Generally, MXenes have a general formula of Mn+iXnTx-, where “M” denotes a transition metal atom (such as Ti, Sc, Mo, Zr, V, Cr, etc.); “X” denotes either a carbon or nitrogen atom; “Tx” denotes one or more surface termination groups (such as O, -F, -Cl, and / or -OH); and “n” denotes an integer number ranging from 1 to 3.

[0079] One or more embodiments of the present disclosure generally concern a phosphate electrochemical sensor comprising, consisting essentially of, or consisting of a MXene, such as Ti3C2Tx, where “Tx” corresponds to surface termination groups, such as -O, -F, -Cl, and / or -OH.

[0080] In various embodiments, the MXene, such as Ti3C2Tx, may be free and devoid of any added dyes.

[0081] The MXene sensors described herein may be utilized to test for a target substance, such as phosphate, in various sample types. Generally, the sensors can be used in a method involving: (a) dispersing a sample within a medium comprising a recognition agent that is operable to react with a target substance (e.g., a phosphate ion); (b) contacting the medium comprising the dispersed sample with a sensing device comprising the MXene sensor along with a reference electrodeand a counter electrode; (c) inducing an electrochemical reaction between the target substance and the recognition agent; and (d) detecting a peak current signal at a characteristic applied voltage to the MXene sensor with respect to the reference electrode, the peak current signalbeing proportional to the concentration of the target substance within the sample. The target substance can be phosphorous, a phosphorous compound, and / or a phosphate ion. Generally, phosphorous is in the form of phosphate ions, which is important for all life forms.

[0082] Although the target substance is indicated above being phosphate, other target substances may be tested with the MXene sensors described herein. For example, TisC^Tv has shown compatibility with small molecules (e.g., hydrogen peroxide, dopamine, epinephrine, ascorbic acid, uric acid, acetaminophen, serotonin, and cysteine) and environmental contaminants (e g., pesticides, dye molecules, and heavy metal ions).

[0083] In one or more embodiments, the recognition agent may comprise molybdenum.

[0084] In one or more embodiments, the medium comprises an electrolyte, a chloride (e.g., potassium chloride), and / or a proton donating species (e.g. sulfuric acid).

[0085] As noted above, sensing devices may be produced that contain one or more TisCsT^ sensors described herein, along with a working commercial electrode (e.g., the GCE).

[0086] Generally, the ThCFT-, sensors may be produced by: (1) chemical (acid-based) etching of a Ti AlC2 MAX precursor phase material, usually in the presence of HC1 and / or HF, to form a Ti.sC^Tv multilayer MXene and (2) delaminating the resultant multilayer MXene with a delamination intercalant (e.g., LiCl) to form a suspension containing MXene with reduced layers. Afterwards, at least a portion of the suspension containing MXene with reduced layers may be drop-coated onto an electrode, such as a bare glassy carbon electrode (GCE). As noted above, the GCE may be polished with an alumina slurry.

[0087] Generally, the individual TisCdT, flakes forming the layered structures may be analyzed as this reflects the source and purity of MAX precursor phase, which significantly influences the quality of the resulting MXene material. The MXene quality and structural characteristics are also critically important for the electrochemical detection of the phosphate molecules. Typically, after etching the interlayer aluminum, the MAX phase material may be transformed into TisC^T. MXene crystals. This shift confirms the presence of surface functional groups and micro-molecules, such as water, between the MXene sheets, which originate from the synthesis process.

[0088] In one or more embodiments, the average lateral size of the TiaC^T, MXene crystal flakes can be at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, or 4.5 pm and / or less than 10, 9, 8, 7, 6, or 5 pm. Additionally, in such embodiments, the flake thickness of the Ti3C2T.vMXene crystal flakes canbe at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, or 4 nm and / or less than 10, 9, 8, 7, 6, or 5 nm. Furthermore, the reduced layered MXene after the delamination step may contain 1 to 10, 1 to 8, 1 to 6, 1 to 4, 2 to 4, 2 to 3, or 1 to 2 MXene layers. The flake size may be estimated from the statistical analysis of ~ 60 or ~9 TisC^Tv MXene flakes observed in the SEM images. Thus, the resulting Ti^CTU MXene crystal flakes provide a very thin profile, which can be beneficial in the detection of phosphate ions via electrochemical sensing.

[0089] Subsequently, the resulting sensing device (i.e., the coated MXene sensor) may be utilized to measure the desired target substance (e.g., phosphate ions) via electrochemical redox activity of PMC as described herein. More particularly, the modified molybdenum blue method described herein may be used as a sensing mechanism for phosphate detection.

[0090] Utilizing the sensing devices described herein, sensing the target substance (e.g., phosphate ion) was observable at concentrations from 1 to 350 pM. In addition, the sensing devices exhibited superior reliability and demonstrated very little interference or hysteresis. As a reflection of its inherent stability, the sensing devices described herein may retain at least 70, 75, 80, 85, or 90 percent of its target substance (e.g., phosphate ions) sensing after 50, 75, 100, 110, 125, or 135 days in storage at room temperature.

[0091] As demonstrated herein, the MXene sensors exhibited consistent and highly selective response towards phosphate molecules among other potential interfering ions with a sensing ranging from 1 pM to 350 pM and a detection limit (LOD) of 1.31 pM. This paves the way for Ti3C2Txand other MXene materials for exploration of phosphate molecular sensing in complex environments, such as agricultural, ecological, and biomedical domains.

[0092] Accordingly, the phosphate sensors described herein can be used for water, soil (or other agricultural uses), and / or biomedical applications.

[0093] The successful integration of MXene materials into modem electronic devices depends not only on scalable material synthesis techniques, but also on the ability to fabricate high- performance devices in a cost-effective and scalable manner. Additive manufacturing has emerged as a promising next-generation manufacturing approach for addressing this need by enabling fabrication of printed electronics using functional inks formulated from the MXene materials described herein. Unlike conventional subtractive manufacturing, which removes material through processes such as etching or machining, additive manufacturing builds up material layer by layer or deposits it directly onto substrates.

[0094] Additive manufacturing comprises a wide range of techniques, which can be broadly categorized as traditional and digital methods. Traditional techniques can include, for example, drop casting, spin coating, spray coating, screen printing, gravure printing, and flexographic printing. Techniques like drop casting and spin coating offer limited patterning capabilities, while others in this category often rely on pre-designed master templates or stencils, which can be time consuming and inflexible when modifications to the design are required. On the other hand, digital additive techniques, such as inkjet printing and extrusion -based printing offer superior design flexibility through the use of CAD models, enabling on demand customization without the need for physical masks or patterns. While all additive manufacturing techniques aim to reduce material waste and lower processing costs, their resolution, throughput, and scalability differ significantly.

[0095] The integration of these techniques with functional MXene inks holds immense promise for the development of flexible, lightweight, and low-cost electronic devices for applications ranging from sensors and wearables to energy storage and loT systems.

[0096] In one or more embodiments, an electrochemical sensor device is provided for sensing phosphate. Generally, these devices can contain the MXene electrodes described herein. In various embodiments, these electrochemical sensor devices can comprise an amperometric sensor, a potentiometric sensor, an impedimetric sensor, a conductometric sensor, or a voltametric sensor.

[0097] In one or more embodiments, a portable soil phosphate sensing system with wireless communication can be produced, which contains an electrochemical sensor comprising the MXene-coated electrode discussed above. This soil phosphate sensing system can placed in- situ and utilize the MXene-coated electrode to monitor phosphate levels in real-time within the soil and convey the real-time measurements to a device or application. The portable device can contain a photodiode to measure the intensity of blue color from the reduced PMC with respect to output voltage derived from the absorption values.

[0098] In various embodiments, the portable soil phosphate sensing system can comprise a phosphate recognition system and a physiochemical transducer, which converts the chemical interaction at the phosphate receptor site into a measurable electrical signal.

[0099] The MXene sensors described herein can be used in electrochemical applications for sensing and quantifying phosphate in various mediums, such as soil. In these electrochemicalapplications, the phosphate may be detected via the modified MB method described herein, where the PMC formed is directly reduced at the working electrode surface without an external reducing agent, such as ascorbic acid, and a potassium antimonyl tartrate catalyst. Two different approaches may use this technique; one uses molybdenum in the electrolyte solution while the other incorporates immobilized molybdenum at the working electrode surface.

[0100] In the realm of internet of things (“loT”), deployment of in-field sensors has the potential to revolutionize the way farmers manage their crops, addressing many aforementioned challenges associated with traditional methods. These sensors embedded in vast areas of the agricultural fields can provide real-time data of phosphate levels. This wirelessly transmitted data will help farmers not only make informed decisions on agricultural practices but also automate responses for irrigation, pest control schedules, and fertilizer applications when integrated with loT system.

[0101] This invention can be further illustrated by the following examples of embodiments thereof, although it will be understood that these examples are included merely for the purposes of illustration and are not intended to limit the scope of the invention unless otherwise specifically indicated.EXAMPLESExample 1

[0102] 2D Ti.iCATv MXene was synthesized from the optimized 3D T AlCA MAX phase crystals. In short, for the synthesis of ThCAT- MXene, 1 g of optimized T13AIC2 MAX was first washed using 9 M hydrochloric acid (HC1) for 18 h to remove intermetallic impurities and mixed with an etchant solution (6:3:1 mixture (by volume) of 12 M HC1, deionized water, and 50 wt % hydrofluoric acid (HF)). The etching reaction was set up for 24 h at 35 °C with continuous stirring at 400 RPM. The etched multilayered TisC2TxMXene was washed with deionized water via repeated centrifugation at 3234 RCF (4-5 cycles with -200 mb of deionized water) until the supernatant reached pH ~6. For delamination, the multilayered ThCAT? MXene was added to lithium chloride solution, typically 50 mL / per gram of starting etched powder, and stirred at 400 RPM for 1 h at 65 °C under an inert environment. The mixture was then washed with deionized water via centrifugation at 3234 RCF for 5, 10, 15, and 20 minutes. Then, the final Ti iCATv MXene mixture was vortexed, followed by centrifugation at 2380 RCF for 30 minutes to ensure theTi3C2TxMXene solutions were single-to-few-layered flakes. The synthesized U3C2TX MXene had a density (p), zeta potential ( ) and bulk electrical conductivity of 4.21 g cm3, -40 mV, and -21000 S / cm, respectively. The freshly prepared Ti3C2TxMXene was used for electrochemical studies.

[0103] The Ti3AlC2 MAX phase was transformed into the TisC^T. MXene after selectively etching the aluminum layers. The increase in the c-lattice parameter confirmed successful selective etching and the presence of surface functional groups and intercalated molecules, such as water, between the TisC2TxMXene flakes. The average lateral size and the average flake thickness of the Ti3C2TxMXene flakes were estimated to be 4.69 pm and 1.78 nm respectively (corresponding to 1-2 MXene layers).Example 2

[0104] The MXene sensors were produced as follows. Initially, a bare GCE (BASi, MF- 2012) was polished with alumina slurry and rinsed using distilled water, followed by methanol. It was then water-sonicated for 10 minutes. 10 mg / mL of Ti3C2Tv MXene stock solution was prepared from Example 1 and further diluted to 4 mg / mL. 5 pL of the 4 mg / mL Ti3C2Txwas then drop coated on the clean GCE and dried under an IR lamp for a few minutes before use in the electrochemical study. Before each experiment, the GCE was polished with an alumina-water slurry and rinsed with distilled water followed by a drop coating of Ti3C2TxMXene.Example 3

[0105] The reliability of environmental sensors required an analysis in the presence of interfering species. As such, the ' isCL'I'v / GCE sensor was subjected to measurements in the presence of common ions found in environmental water, such as sulfate, carbonate, and nitrate. Testing using 100 pM of each interfering species in the base solution was carried out for current measurement followed by addition of 100 pM of P ions.

[0106] P recovery in real samples, such as tap water, lake water and agricultural runoff, were also tested by spiking the respective samples with 100 pM of P. DPV measurements were conducted and the recovery results are depicted in TABLES 2-4. The DPV measurements of the sensor involved using the MXene / GCE sensor in 0.1 M KC1 supporting electrolyte, 1 mM Mo, and 0.1 M H2SO4 from 0 V vs. Ag / AgCl to 0.24 V vs. Ag / AgCl with the addition of 100 pM of all the interfering ions (sulfate, carbonate and nitrate) and 100 pM P. Using DPV analysis, theindividual change in current was observed for respective 100 pM of interfering ions vs. change in current for 100 pM P in the presence of interfering ions. Furthermore, DPV analysis of the MXene / GCE sensor in 0.1 M KC1 supporting electrolyte, 1 mM Mo, and 0.1 M H2SO4 from 0 V to 0.24 V with the addition of the tap water, environmental water, and agricultural runoff (collected from Kansas Agricultural Watershed Field Laboratory) was conducted at various concentrations of P. The change in current values from these DPV analyses was used to construct the recovery rates depicted in TABLES 2-4, below.

[0107] The sensor showed a change in current only in the presence of P and no other ions. The individual change in current for 100 pM interfering ions was also recorded and was insignificant compared to the sensors’ response to P ions.

[0108] The pH of the base solution was also monitored after the addition of real samples. The tap water and lake water (from Tuttle Creek Lake in Manhattan, Kansas) did not alter the pH of the base solution significantly. However, there was a small deviation in pH observed for agricultural runoff sample.TABLE 2: Determination of Phosphate in Tap Water Using the TisCiTr / GCETABLE 3: Determination of Phosphate in Tuttle Creek Using the TijCiTv / GCETABLE 4: Determination of Phosphate in Agricultural Runoff Using the TisCiTx / GCE

[0109] Overall, tap water exhibited the lowest relative standard deviation (RSD), followed by lake water and agricultural runoff. Notably, the RSD values for lake water and agricultural runoff appeared much higher at lower phosphate concentrations. This is likely due to complex and heterogenous composition of the lake water and agricultural runoff sample, which introduced a higher degree of variability and lower signal to noise ratio compared to more homogenous matrices such as tap water. These results suggest that, while the sensor is responsive to phosphate in real samples, additional validation and optimization may be required to achieve improved performance.

[0110] This showed that the sensor had superior performance in the presence of multitude of interfering agents. The real sample measurements did not alter the pH of the base solution.

[0111] The complete list of chemical and mineralogical elements present in the real samples was measured using various techniques and are shown TABLE 5, below.TABLE 5: Concentration of Ions Presented in Real SamplesICP - Inductively Coupled Plasma Spectroscopy, RFA - Rapid Flow AnalyzerBoth NH4-N and NO3-N are well known interference species to phosphates

[0112] Humic acid, a naturally occurring organic compound formed from the decomposition of plant and animal matter, is also often regarded as a potential interfering species in agricultural sensor development. Thus, organic matter (OM) absorbance fluorescence spectroscopy method was used to characterize humic acid content in these real samples, and the detailed information is present in TABLE 6, below.TABLE 6: Results from OM Absorption Fluorescence SpectroscopyFI: fluorescence Index; HI: Humification Index

[0113] An organic matter (OM) absorbance fluorescence spectroscopy method was used to characterize the presence and possible interference of the humic acid component in the real sample, tap water, lake water, and agricultural runoff. Distilled water (DI) was used as a calibration standard. In short, a HORIBA Aqualog fluorimeter was used for the optical absorbance measurement, where excitation wavelengths of 240 nm - 450 nm window was used with a 3 nm step size. Simultaneously, the fluorescence was measured for emission wavelengths of 300 nm- 600 nm (instrument default increment of 3.28 nm was used for the emission signal to collect). The Rayleigh scattering was blocked by a scatter masking and other filter corrections were used inside the fluorimeter to the raw fluorescent data and the processed data was corrected and normalized. Dissolved organic matter (DOM) was calculated by using the specific ultraviolet absorbance at 254 nm (Abs254). The Fluorescence Index (FI) was calculated from the fluorescence data as a measure of DOM. The humification index (HI) was calculated to estimate the extent of humification. The humic-like contributions were obtained from the intensity peaks of Peaks A, M and C.

[0114] Briefly, although the humification index (HI) seemed to be identical for all samples, there was more contribution coming from the standard humic like peaks (A, M, and C) in the agricultural runoff sample compared to the tap water and lake water samples. This couldalso potentially be why the RSD values shown for agricultural samples for lower concentration of phosphate were the highest among all other samples causing a lower signal to noise ratio.Example 4

[0115] To enhance further mechanistic understanding of the P redox processes at the surface of TisC2TxMXene, an ex-situ Raman spectro-electrochemical measurement was conducted. The outcome of the experiment was used in correlating the structural changes in Ti3C2U MXene to the underlying P redox process. We hypothesized that the formation of the a- isomer in the electrolyte, followed by its isomeric transition (a-to-P) near the TisCiTx MXene sensor surface during electron transfer, induced strain in the MXene crystal lattice manifesting as either compressive or tensile stress. Thus, comparing the Raman spectra of MXene before and after electrochemical cycling in the phosphate containing electrolyte medium provided further insight into the P mechanism proposed in this work. The unprocessed Raman spectra of pristine MXene, MXene after immersion in a-isomer PMC solution, and MXene after electrochemical cycling involving a-to-P isomeric transition at its surface were gathered and analyzed.

[0116] The characteristic lattice vibrational modes corresponding to various atomic bonds within lattice carbon, surface terminal groups, and the skeletal regions were identified in the ThCTTv MXene sample before and after electrochemical cycling. The comparable intensities of the deconvoluted peaks in the lattice carbon spectral region, relative to those in the skeletal and surface group regions, indicated that our ThCTU MXene primarily consists of single to few layer flakes, corroborating the AFM analysis. Upon introduction of the a-isomer and subsequent electrochemical a-to-P isomeric transition near the Ti3C2Tv MXene surface, a systematic increase in the Raman spectral intensity of the skeletal vibrational modes (within the 50 - 300 cm'1spectral region) was observed relative to the lattice carbon region (560 - 800 cm'1). This could be due to the skeletal strain imparted to the ThCTU MXene lattice by the PMC a-isomer formation and structural transition of the isomer from a to P in the vicinity of the electrode.

[0117] All electrochemical measurements were performed at room temperature using an Interface 1010E potentiostat from Gamry Instruments along with Ag / AgCl (3 M NaCl) reference electrode, platinum wire counter electrode, and TECTT. MXene modified GCE working electrode (TfCTU MXene / GCE). The redox reactions at the ThCTU MXene / GCE working electrode were investigated by first performing the cyclic voltammetry (CV) measurements in a 0.1 M KC1supporting electrolyte with a potential window between -1 V vs. Ag / AgCl to 1 V vs. Ag / AgCl. The presence of an oxidation peak within the specified potential window required setting the potential range for subsequent redox probe and P sensing measurements below the oxidation potential of TisC^Tv MXene. The redox probe sensing was carried out in 0.1 M KC1 and 5 mM HisChNeRu from -0.3 V vs. Ag / AgCl to 0.03 V vs. Ag / AgCl. P sensing utilized a solution consisting of 1 mM ((NH4)6Mo?O24 4H2O), 0.1 M H2SO4, 0.1 M KC1 as the base solution, with KH2PO4 (the source of P) analyte concentrations ranging from 1 pM to 1000 pM. The CV corresponding to the P sensing measurements was taken between 0 V vs. Ag / AgCl and 0.25 V vs. Ag / AgCl voltage window. Each CV measurement employed a 2 mV step size over 3 cycles. The differential pulse voltammetry (DPV) technique was used primarily for electrochemical sensing of various P concentrations, interference studies, the real sample analysis, and the hysteresis assessment. The DPV-based P sensing utilized the same solution medium as the CV P sensing. Interference studies were conducted in the base solution with the addition of 100 pM each of NaNOs, K2SO4, and Na2CCh. The real samples were measured using DPV within the base solution, and 1 ml of real sample was added. The optimized experimental parameters for DPV were as follows: the potential window of 0 V vs. Ag / AgCl to 0.24 V vs. Ag / AgCl, step size of 2 mV, sample period of 2 seconds, pulse time of 0.1 seconds, and pulse size of 50 mV. All the electrochemical experiments were repeated n number of times (n > 3) to ensure reproducibility of the results.

[0118] By deconvoluting the three spectral regions (skeleton, surface group, and lattice carbon), the source of the individual phonon peaks in the pristine Ti3C2TxMXene were identified. The peak shifts in each region for a-isomer and P-isomer related to Ti3C2TxMXene are reported in TABLE 7, below. Almost all the peaks demonstrated either compressive (Raman blue shift) or tensile (Raman red shift) stresses, indicating the role of electron transfer at the isomeric transition to the individual and / or collective Ti3C2TxMXene crystal lattice.TABLE 7: Raman peak origin and nature of shifts in TisCiTx MXene / a-isomer and a-to-P isomeric transitionDEFINITIONS

[0119] It should be understood that the following is not intended to be an exclusive list of defined terms. Other definitions may be provided in the foregoing description, such as, for example, when accompanying the use of a defined term in context.

[0120] As used herein, the terms “a,” “an,” and “the” mean one or more.

[0121] As used herein, the term “and / or,” when used in a list of two or more items, means that any one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed. For example, if a composition is described as containingcomponents A, B, and / or C, the composition can contain A alone; B alone; C alone; A and B in combination; A and C in combination, B and C in combination; or A, B, and C in combination.

[0122] As used herein, the terms “comprising,” “comprises,” and “comprise” are open- ended transition terms used to transition from a subject recited before the term to one or more elements recited after the term, where the element or elements listed after the transition term are not necessarily the only elements that make up the subject.

[0123] As used herein, the terms “having,” “has,” and “have” have the same open-ended meaning as “comprising,” “comprises,” and “comprise” provided above.

[0124] As used herein, the terms “including,” “include,” and “included” have the same open-ended meaning as “comprising,” “comprises,” and “comprise” provided above.NUMERICAL RANGES

[0125] The present description uses numerical ranges to quantify certain parameters relating to the invention. It should be understood that when numerical ranges are provided, such ranges are to be construed as providing literal support for claim limitations that only recite the lower value of the range as well as claim limitations that only recite the upper value of the range. For example, a disclosed numerical range of 10 to 100 provides literal support for a claim reciting “greater than 10” (with no upper bounds) and a claim reciting “less than 100” (with no lower bounds).CLAIMS NOT LIMITED TO DISCLOSED EMBODIMENTS

[0126] The preferred forms of the invention described above are to be used as illustration only, and should not be used in a limiting sense to interpret the scope of the present invention. Modifications to the exemplary embodiments, set forth above, could be readily made by those skilled in the art without departing from the spirit of the present invention.

[0127] The inventors hereby state their intent to rely on the Doctrine of Equivalents to determine and assess the reasonably fair scope of the present invention as it pertains to any apparatus not materially departing from but outside the literal scope of the invention as set forth in the following claims.

Claims

1. What is claimed is:

1. A method of testing a sample for the presence of a target substance comprising:(a) dispersing the sample within a medium comprising a recognition agent that is operable to react with the target substance, wherein the target substance comprises phosphorous, a phosphorous compound, and / or a phosphate;(b) contacting the medium within which the sample is dispersed with a sensing device, the sensing device comprising an MXene electrode containing an MXene, wherein the MXene has a chemical formula of Mn i XtlTv, where “M” denotes a transition metal atom, “X” denotes either C or N, “Tx” denotes one or more surface termination groups, and “n” denotes an integer number ranging from 1 to 3;(c) inducing an electrochemical reaction between the target substance and the recognition agent; and(d) detecting a peak current signal at a characteristic applied voltage with the sensing device, the peak current signal being proportional to the concentration of the target substance within the sample.

2. The method of claim 1, wherein the target substance comprises a phosphate.

3. The method of claim 2, wherein the recognition agent comprises molybdenum.

4. The method of claim 1, wherein “Tx” denotes O, -F, -Cl, and / or -OH.

5. The method of claim 4, wherein “M” denotes Ti.

6. The method of claim 5, wherein “X” denotes a carbon.

7. The method of claim 1, wherein the chemical formula of the MXene is ThC^Tv8. The method of claim 1, wherein the MXene is in the form of crystals having an average lateral size of 0.5 to 10 pm.

9. The method of claim 1, wherein the MXene is multilayered and comprises 1 to 10 layers.

10. The method of claim 1, wherein the medium comprises an electrolyte.

11. The method of claim 10, wherein the medium further comprises a chloride and a proton donating species.

12. The method of claim 11, wherein the proton donating species comprises sulfuric acid and the chloride comprises potassium chloride.

13. The method of claim 1, wherein the sample comprises soil or water.

14. The method of claim 1, wherein the step of detecting the peak current signal comprises performing cyclic voltammetry or differential pulse voltammetry analysis.

15. A method of testing a sample for the presence of a phosphate comprising:(a) dispersing the sample within an electrolyte medium comprising a molybdenum recognition agent that is operable to react with the phosphate and form a phosphomolybdenum complex;(b) contacting the electrolyte medium within which the sample is dispersed with a sensing device, the sensing device comprising an MXene electrode containing an MXene, wherein the MXene has a chemical formula of Mn+iXnTx, where “M” denotes Ti, Sc, Mo, Zr, V, or Cr, “X” denotes either C or N, “Tx” denotes one or more surface termination groups, and “n” denotes an integer number ranging from 1 to 3 ;(c) inducing an electrochemical reaction of the phosphomolybdenum complex on a surface of the MXene electrode to thereby form a quantifiable electrochemical signal; and(d) detecting the quantifiable electrochemical signal with the sensing device, the quantifiable electrochemical signal being proportional to the concentration of the phosphate within the sample.

16. The method of claim 15, wherein the inducing occurs in the absence of an external reducing agent and a reduction catalyst.

17. The method of claim 15, wherein “Tx” denotes O, -F, -Cl, and / or -OH.

18. The method of claim 15, wherein “X” denotes a carbon.

19. The method of claim 15, wherein the chemical formula of the MXene is Ti3C2TA20. The method of claim 15, wherein the sample comprises soil or water.

21. The method of claim 15, wherein the step of detecting the electrochemical signal comprises performing cyclic voltammetry or differential pulse voltammetry analysis.

22. A phosphate electrochemical sensing system comprising:(a) a molybdenum recognition agent; and(b) an MXene electrode containing an MXene, wherein the MXene has a chemical formula of Mn+iXnTx, where “M” denotes a transition metal atom, “X” denotes either C or N, “Tx” denotes one or more surface termination groups, and “n” denotes an integer number ranging from 1 to 3.

23. The system of claim 22, wherein “Tx” denotes O, -F, -Cl, and / or -OH.

24. The system of claim 22, wherein “M” denotes Ti.

25. The system of claim 22, wherein “X” denotes a carbon.

26. The system of claim 22, wherein the chemical formula of the MXene is TisC2Tx.

27. The system of claim 22, wherein the MXene is multilayered and comprises 1 to 10 layers.

28. The system of claim 22, further comprising an electrolyte, wherein the molybdenum recognition agent is present in the electrolyte.

29. The system of claim 28, wherein the medium further comprises a chloride and a proton donating species.

30. The system of claim 29, wherein the proton donating species comprises sulfuric acid and the chloride comprises potassium chloride.

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