Sensor for ion detection, methods of construction, and uses thereof

Abstract

Various examples are provided related to a sensor (e.g., biosensor) for selective ion-detection in a sample. In one example, a potentiometric sensor includes a base; a working electrode (WE) set into the base; and a reference electrode (RE) set into the base adjacent to the working electrode. The working electrode includes a WE well filled with a WE solid-state material and the reference electrode includes a RE well, adjacent to the WE well, filled with a RE solid-state material. An example of a method for measuring a concentration of ions in a sample using the potentiometric sensor includes obtaining a biological sample; bringing the biological sample into contact with both the WE well and the RE well; and measuring the open circuit potential between the working electrode and the reference electrode. Also disclosed is a system comprising the disclosed sensor and a microfluidic device in fluid communication with the sensor.

Description

SENSOR FOR ION DETECTION, METHODS OF CONSTRUCTION, ANDUSES THEREOFCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of, U.S. provisional application entitled “Sensor for Ion Detection, Methods of Construction and of Use” having serial no. 63 / 579,189, filed August 28, 2023, which is hereby incorporated by reference in its entirety.BACKGROUND

[0002] Hypocalcemia, colloquially known as Milk Fever, is a common metabolic disorder caused by severe calcium (Ca2+) deficiency most prevalent in dairy cows. This disorder has clinical and subclinical levels of severity, the latter often going undetected due to lack of symptoms. In clinical cases, the cow becomes susceptible to other infections, causing up to 5% of cows to be unable to recover from the fever. Additionally, cows can become partially to fully paralyzed. Without treatment, up to 60-80% of cases result in death. Further, milk fever significantly reduces milk yield and productivity. As a result, hypocalcemia can not only have serious effects on the long-term health of the cow but is costly to farmers financially.SUMMARY

[0003] Aspects of the present disclosure are related to a sensor (e.g. biosensor) for selective ion-detection in a sample. In one aspect, among others, a potentiometric sensor comprises a base; a working electrode (WE) set into the base, the working electrode comprising a WE well at a first distal end, the WE well filled with a WE solid-state material; and a reference electrode (RE) set into the base adjacent to the working electrode, the reference electrode comprising a RE well at the first distal end adjacent to the WE well, the RE well filled with a RE solid-state material. In one or more aspects, the potentiometric sensor can comprise a base well extending from an outer surface of the base to an inner surface of the base well at a depth of about 0.2 pm to about 0.4 pm, the base well surrounding the WE well and RE well, wherein the inner surface of base well can be flush with a surface of the WE well solid-state material and the RE well solid-state material. The WE well can be connected via a first channel to a first pad at a second distal end. The RE well can be connected via a second channel to a second pad at the second distal end adjacent to the first pad. The WE well can be circular and can have a diameter of about 5 mm to about 11 mm. The RE wellcan be circular and can have a diameter of about 1 mm to about 5 mm. The WE solid-state material and RE solid-state material can each comprise a plurality of layers of material.

[0004] In another aspect, a method for measuring a concentration of ions in a sample using the potentiometric sensor comprises obtaining a biological sample; bringing the biological sample into contact with both the WE well and the RE well; and measuring the open circuit potential between the working electrode and the reference electrode. In another aspect, a system is disclosed comprising: the potentiometric sensor of claim 2; and a microfluidic device in fluid communication with the potentiometric sensor, comprising: a reservoir configured to be in contact with the base well of the potentiometric sensor; an inlet, configured to receive a fluid sample and be in fluid communication with the reservoir; and a pump, configured to pump the fluid sample from the inlet to the reservoir.

[0005] Other systems, methods, features, and advantages of the present disclosure will be or become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be protected by the accompanying claims. In addition, all optional and preferred features and modifications of the described embodiments are usable in all aspects of the disclosure taught herein. Furthermore, the individual features of the dependent claims, as well as all optional and preferred features and modifications of the described embodiments are combinable and interchangeable with one another.BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Many aspects of the present disclosure can be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present disclosure. Moreover, in the drawings, like reference numerals designate corresponding parts throughout the several views.

[0007] FIG. 1A-1C illustrates examples of a 3D-printed sensor and an electrode layout, in accordance with various embodiments of the present disclosure.

[0008] FIG. 1 D illustrates an example of a cross-sectional view of a sensor, in accordance with various embodiments of the present disclosure.

[0009] FIGS. 2A-2D illustrates examples of results from a sensor calibration and validation study, in accordance with various embodiments of the present disclosure.

[0010] FIG. 3 illustrates an example of results from a sensor selectivity study, in accordance with various embodiments of the present disclosure.

[0011] FIGS. 4A-4F illustrates examples of structural components of the constructed sensor and electrochemical characterization of the sensor, in accordance with various embodiments of the present disclosure.

[0012] FIGS. 5A-5D illustrates examples of results from a study of a sensor’s sensing performance of ionized calcium ions, in accordance with various embodiments of the present disclosure.

[0013] FIGS. 6A-6E illustrates examples of results from testing a sensor’s sensing performance at low concentrations of ionized calcium ions, in accordance with various embodiments of the present disclosure.

[0014] FIGS. 7A-7D illustrates examples of results from selectivity and validation studies of a sensor, in accordance with various embodiments of the present disclosure.

[0015] FIGS. 8A-8D illustrates examples of results from testing a variety of sensors with milk samples containing different Ca2+concentrations, in accordance with various embodiments of the present disclosure.

[0016] FIGS. 9A-9D illustrates examples of sensors and results from phosphate sensing calibration testing, in accordance with various embodiments of the present disclosure.

[0017] FIGS. 10A-10C illustrates examples of scanning electron microscopy images of surface morphologies of sensors, in accordance with various embodiments of the present disclosure.

[0018] FIGS. 11A-11D illustrates examples of results from elemental (energy-dispersive X-ray spectroscopy) studies of various layers of a sensor, in accordance with various embodiments of the present disclosure.

[0019] FIG. 12A illustrates an example of a sensor manufacturing process, in accordance with various embodiments of the present disclosure.

[0020] FIG. 12B illustrates the computer-assisted design (CAD) of the sensor, electrodes, and the sample holder, including the WE and RE, in accordance with various embodiments of the present disclosure.

[0021] FIG. 12C depicts a photograph of a 3D printed calcium sensor displaying the WE, RE, dielectric-coated wiring, and connectors, in accordance with various embodiments of the present disclosure.

[0022] FIG. 12D illustrates a schematic showing the cross-sectional view of an iron sensor, in accordance with various embodiments of the present disclosure.

[0023] FIG. 12E shows OOP and membrane potential developed at each interface of a 3D printed Fe sensor, in accordance with various embodiments of the present disclosure.

[0024] FIG. 13A depicts a photograph of a 3D printed sensor after Au coating (top view) and an SEM image of the sensor surface, in accordance with various embodiments of the present disclosure. The sensors feature a periodic pattern with each lateral layer width measuring 80 pm.

[0025] FIGS. 13B-13C show carbon (FIG. 13B) and sulfur (FIG. 13C) elemental mapping of a POT coated Au surface, in accordance with various embodiments of the present disclosure.

[0026] FIG. 13D depicts a photograph of a sensor with POT coating on Au-coated 3D electrodes and an Sem image showing the 3D Au layer with POT layer on a printed Ca-sensor, in accordance with various embodiments of the present disclosure.

[0027] FIG. 13E shows an energy-dispersive X-ray spectrum of the POT-coated Au layer, in accordance with various embodiments of the present disclosure.

[0028] FIG. 13F depicts a photograph of a calcium sensor composed of a Ca-ISM on a POT layer, along with SEM images of the ISM surface, in accordance with various embodiments of the present disclosure.

[0029] FIG. 14A shows measurements of OOP response of an Fe-sensor with dosedependent solutions (56 ppb -5580 ppm) of Fe2+, in accordance with various embodiments of the present disclosure.

[0030] FIG. 14B shows a calibration plot of an Fe-Sensor (Pearsons, r= 0.96), in accordance with various embodiments of the present disclosure.

[0031] FIG. 14C shows a selectivity study of the Fe-sensor in the presence of K+(39 ppm) and Mg2+(23.4 ppm), in accordance with various embodiments of the present disclosure.

[0032] FIG. 14D shows measurements of OCP response of a Ca-Sensor with dosedependent solutions (20 - 400 ppm) of Ca2+, in accordance with various embodiments of the present disclosure.

[0033] FIG. 14E shows a calibration plot of a Ca-Sensor (Pearson’s r = 0.99), in accordance with various embodiments of the present disclosure.

[0034] FIG. 14F shows a selectivity study involved testing the Ca-Sensor's discrimination capability against potassium ions, chloride ions, bicarbonate ions, and sodium ions, in accordance with various embodiments of the present disclosure.

[0035] FIG. 14G shows measurements of OCP response of an N-sensor with dosedependent solutions (1-1200 ppm) of NO3-, in accordance with various embodiments of the present disclosure.

[0036] FIG. 14H shows a calibration plot of an N-Sensor (Pearson’s r = -0.99) , in accordance with various embodiments of the present disclosure.

[0037] FIG. 141 shows a selectivity study involving testing the N-Sensor against potassium ions, chloride ions, and sodium ions, in accordance with various embodiments of the present disclosure.

[0038] FIG. 14J shows measurements of OCP response of a P-Sensor with dosedependent solutions (960 ppb - 240 ppm) of HPO42", in accordance with various embodiments of the present disclosure.

[0039] FIG. 14K shows a calibration plot of a P-Sensor (Pearson’s r = -0.98), in accordance with various embodiments of the present disclosure.

[0040] FIG. 14L shows a selectivity study including testing a P-sensor against potassium ions, chloride ions, and sodium ions, in accordance with various embodiments of the present disclosure.

[0041] FIGS. 15A-15B show results from CV tests of a POT layer, depicting the potential waveform (FIG. 15A) and the resulting CV output waveform (FIG. 15B), in accordance with various embodiments of the present disclosure.

[0042] FIG. 15C shows the OCP of a Fe-sensor in a standard solution, in accordance with various embodiments of the present disclosure.

[0043] FIG. 15D shows the OCP of a Ca-sensor in a standard solution, in accordance with various embodiments of the present disclosure.

[0044] FIG. 15E shows the OCP of an N-sensor in a standard solution, in accordance with various embodiments of the present disclosure.

[0045] FIG. 15F shows the OCP of a P-sensor in a standard solution con, in accordance with various embodiments of the present disclosure.

[0046] FIGS. 15G-15H show the OCP of a fabricated RE of Ag / AgCI with and without Nation™, in accordance with various embodiments of the present disclosure.

[0047] FIG. 16A illustrates a schematic of a microfluidic device integrated with a 3D printed sensor, in accordance with various embodiments of the present disclosure.

[0048] FIG. 16B depicts an image of a PDMS microfluidic system fabricated as a negative replica mold, in accordance with various embodiments of the present disclosure.

[0049] FIG. 16C depicts an image of a calcium sensor integrated with a PDMS microfluidic system, in accordance with various embodiments of the present disclosure.

[0050] FIG. 16D illustrates a schematic of a microfluidic device integrated with a 3D printed sensor and featuring a top and bottom 3D printed body made of clear resin, encasing the 3D sensor and the PDMS microfluidic systems, in accordance with various embodiments of the present disclosure.

[0051] FIG. 16E-16F depict a top view (FIG. 16D) photo and a side view photo (FIG. 16F) of the device of FIG. 16D, in accordance with various embodiments of the present disclosure.

[0052] FIG. 16G shows the OCP of whole milk spiked with a standard concentration of calcium, in accordance with various embodiments of the present disclosure.

[0053] FIG. 16H shows a calibration graph obtained from whole milk samples using a 3D- printed sensor, in accordance with various embodiments of the present disclosure.

[0054] FIG. 17A illustrates a schematic of a microfluidic device integrated with a Ca- sensor interfaced into a milking parlor, in accordance with various embodiments of the present disclosure.

[0055] FIG. 17B shows real-time snapshot images of a microfluidic system with a 3D printed calcium sensor in action as it pumps milk samples, in accordance with various embodiments of the present disclosure.

[0056] FIG. 18 shows a schematic for integration of 3D printed sensor with milking machine for cow side and continuous measurement of Ca2+and P in milk samples, in accordance with various embodiments of the present disclosure.

[0057] FIG. 19A illustrates a calcium ion sensing mechanism (left) and phosphate ion sensing mechanism (right) on each WE, in accordance with various embodiments of the present disclosure.

[0058] FIGS. 19B-19E show SEM images of Au deposited on a wrinkled 3D printed sensor in different magnifications with primary and secondary pattern indication, in accordance with various embodiments of the present disclosure.

[0059] FIG. 19F shows CV results performed in 0.1 M ferro / ferricyanide solution at a fixed scan rate of 1 V / s, in accordance with various embodiments of the present disclosure.

[0060]

[0061] FIG. 19G shows electrochemical impedance spectroscopy studies of a sensor, in accordance with various embodiments of the present disclosure.

[0062] FIG. 20A shows an OOP responses of a Ca2+-sensor, in accordance with various embodiments of the present disclosure.

[0063] FIG. 20B shows resulting calibration graphs of the two successive measurements of FIG. 20A, in accordance with various embodiments of the present disclosure. Error bars are the standard deviation (SD) of three repeated measurements (n = 3 biologically independent experiments). Error bars, mean ± SD.

[0064] FIG. 20C shows calibration curves and slopes of the two successive sensing measurements of calcium ions (FIG. 20A), in accordance with various embodiments of the present disclosure.

[0065] FIG. 20D shows an OOP responses of a P-sensor, in accordance with various embodiments of the present disclosure.

[0066] FIGS. 20E-20F show resulting calibration graphs, slope, and calibration equation of the two successive measurements of FIG. 20D, in accordance with various embodiments of the present disclosure. Error bars are the SD of three repeated measurements (n = 3 biologically independent experiments). Error bars, mean ± SD.

[0067] FIGS. 21A-21 B shows Ca-sensor performance at a low concentration of calcium ions, in accordance with various embodiments of the present disclosure. FIG. 21 B provides a close-up of a selected portion of FIG. 21A, showing the potential response at low concentrations.

[0068] FIG. 21 C shows a bar graph of the sensor calibration at low concentrations of Ca2+, in accordance with various embodiments of the present disclosure. Error bars are the SD of three repeated measurements (n = 3 biologically independent experiments). Error bars, mean ± SD.

[0069] FIG. 21 D shows correlation in responses of a commercial Ca-meter and 3D printed Ca-sensor with theoretical values of calcium ions, in accordance with various embodiments of the present disclosure.

[0070] FIG. 21 E shows a Ca-sensor responses with repeated high (2.0 mM) and low (0 mM) concentrations of calcium ions, in accordance with various embodiments of the present disclosure.

[0071] FIG. 21 F shows an OCP vs concentration plot obtained from graph along with RSD, in accordance with various embodiments of the present disclosure. Error bars are the SD of three repeated measurements (n = 3 biologically independent experiments). Error bars, mean ± SD.

[0072] FIG. 22A shows OCP measurements of a Ca-sensor in the presence of interfering ions, in accordance with various embodiments of the present disclosure.

[0073] FIG. 22B shows OCP measurements of a Ca-sensor in fetal bovine serum samples, in accordance with various embodiments of the present disclosure.

[0074] FIG. 22C shows OCP measurements of a Ca-sensor in whole milk samples, in accordance with various embodiments of the present disclosure.

[0075] FIG. 22D depicts a visual comparison of Ca-sensor responses to each interference, in accordance with various embodiments of the present disclosure. Error bars are the SD of three repeated measurements (n = 3 biologically independent experiments). Error bars, mean ± SD.

[0076] FIG. 22E depicts a visual comparison of Ca-sensor responses in fetal bovine serum samples, in accordance with various embodiments of the present disclosure. Error barsare the SD of three repeated measurements (n = 3 biologically independent experiments). Error bars, mean ± SD.

[0077] FIG. 22F depicts a visual comparison of Ca-sensor responses in whole milk samples, in accordance with various embodiments of the present disclosure. Error bars are the SD of three repeated measurements (n = 3 biologically independent experiments). Error bars, mean ± SD.

[0078] FIGS. 22G and 22H show reproducibility tests of Ca-sensor, in accordance with various embodiments of the present disclosure. Error bars are the SD of three repeated measurements (n = 3 biologically independent experiments). Error bars, mean ± SD.

[0079] FIG. 221 shows detection time for both a Ca-sensor and a P-sensor, in accordance with various embodiments of the present disclosure.

[0080] FIGS. 23A-23B show Ca-sensor (FIG. 23A) and P-sensor (FIG. 23B) OCP results in milk samples obtained from dairy herds, in accordance with various embodiments of the present disclosure.

[0081] FIGS. 23C-23D show estimated co-relation coefficients for a fabricated and commercial Ca-sensor (FIG. 23C) and P-sensor (FIG. 23D), in accordance with various embodiments of the present disclosure.

[0082] FIG. 23E shows levels of Ca2+and P in ppm for real milk samples, in accordance with various embodiments of the present disclosure. Error bars are the SD of three repeated measurements (n = 3 biologically independent experiments). Error bars, mean ± SD.

[0083] FIG. 23F shows the ratio of P to Ca2+levels for each sample, in accordance with various embodiments of the present disclosure.

[0084] FIG. 24A illustrates a schematic of the 3D printed sensor interfacing with a potentiostat to detect hypocalcemia in different mediums of biofluids, in accordance with various embodiments of the present disclosure.

[0085] FIG. 24B illustrates a schematic of a 3D printing process for sensor manufacturing, in accordance with various embodiments of the present disclosure.

[0086] FIG. 24C illustrates a schematic of wrinkled surfaces coated with a thin layer of Au using an e-beam evaporation technique, in accordance with various embodiments of the present disclosure.

[0087] FIG. 24D illustrates a schematic for a printed sensor containing three electrodes, in accordance with various embodiments of the present disclosure.

[0088] FIG. 24E depicts an image of a sensing set-up for on-site testing of hypocalcemia in dairy cows, in accordance with various embodiments of the present disclosure.

[0089] FIGS. 25A-25B show an illustration of a front view (FIG. 25A) and a side view (FIG. 25B) of a sensor with indicated dimensions, in accordance with various embodiments of the present disclosure. The diameter of each electrode is 3 mm and depth are 2 mm. An ellipticalshaped sample holder was created, and all three electrodes are placed onto the sample holder.

[0090] FIGS. 25C-25E shows rendered designs of the sensor of FIGS. 25A-25B from different angles, in accordance with various embodiments of the present disclosure.

[0091] FIG. 25F shows a photograph of the device depicting overall size of a sensor, in accordance with various embodiments of the present disclosure.

[0092] FIG. 25G shows a printed sensor and its dimensions, shown after gold coating onto the electrodes, in accordance with various embodiments of the present disclosure.

[0093] FIGS. 26A-26D show from sensor morphological studies including SEM images of a POT-Au electrode (FIGS. 26A and 26B) and a ISM-coated POT-Au electrode (FIGS. 26C and 26D), in accordance with various embodiments of the present disclosure.

[0094] FIGS. 27A-27B show SEM images of a wrinkled structure of a 3D printed sensor coated with 100 nm Au at different magnifications, in accordance with various embodiments of the present disclosure.

[0095] FIG. 27C shows an SEM images of a 3D printed sensor and ten randomly selected points with ruler scale to calculate 3D printer’s accuracy in printing sensor with micro-structure patterns, in accordance with various embodiments of the present disclosure.

[0096] FIG. 27D shows an SEM image indicating primary (lateral patterns - longer lines) and secondary (wrinkled structures - shorter, closer packed lines) patterns.

[0097] FIGS. 28A-28C show EDS analyses of different sensor surfaces, in particular a Au@3D sensor (FIG. 28A), POT-Au@3D sensor (FIG. 28B), and ISM-POT-Au@3D sensor (FIG. 28C), in accordance with various embodiments of the present disclosure.

[0098] FIG. 28D shows a table detailed the weight % of gold and carbon for different layers of the sensors, in accordance with various embodiments of the present disclosure.

[0099] FIGS. 29A-29B show elemental mapping analysis of a POT-Au@3D sensor (FIG. 29A) and an ISM-POT-Au@3D sensor (FIG. 29B) to confirm the before and after coating ISM on POT-Au electrode, in accordance with various embodiments of the present disclosure.

[0100] FIGS. 30A-30B show X-ray Photoelectron Spectroscopy analysis survey scans on a 3D printed sensor base made of UV cured epoxy resin (FIG. 30A), and a gold coated 3D printed sensor (FIG. 30B), in accordance with various embodiments of the present disclosure.

[0101] FIGS. 30C-30E show deconvoluted high resolution XPS scans of carbon (C1s, FIG. 30C), oxygen (O1s, FIG. 30D), and gold (Au4f, FIG. 30D), in accordance with various embodiments of the present disclosure.

[0102] FIG. 31A shows a calibration curve of a commercially available calcium ion meter and the associated calibration equation, in accordance with various embodiments of the present disclosure.

[0103] FIG. 31 B shows a calibration graph of the commercial sensor, in accordance with various embodiments of the present disclosure, in accordance with various embodiments of the present disclosure.

[0104] FIG. 31 C shows a bar graph highlighting the difference in a commercial sensor's and theoretical sensor’s response to standard calcium ion solutions, in accordance with various embodiments of the present disclosure.

[0105] FIG. 32A-32C shows calibration graphs of identical Ca-sensors fabricated with similar methods but different concentrations of POT solution used to produce the POT layer: 1.1 pm / mm2(FIG. 32A), 2.2 pm / mm2(FIG. 32B), and 3.6 pm / mm2(FIG. 32C), in accordance with various embodiments of the present disclosure.

[0106] FIG. 32D shows a bar graph comparing the sensitivity of the different sensors of FIGS. 32A-32C with different concentrations of POT casted on the surface.

[0107] Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to beunderstood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.DETAILED DESCRIPTION

[0108] Disclosed herein are various examples related to a sensor (e.g. biosensor) used for selective ion-detection in a given sample. Reference will now be made in detail to the description of the embodiments as illustrated in the drawings, wherein like reference numbers indicate like parts throughout the several views.

[0109] Many modifications and other embodiments disclosed herein will come to mind to one skilled in the art to which the disclosed compositions and methods pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the disclosures are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. The skilled artisan will recognize many variants and adaptations of the aspects described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein.

[0110] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.

[0111] It should be noted that ratios, concentrations, amounts, and other numerical data may be expressed herein in a range format. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a concentration range of “about 0.1% to about 5%” should be interpreted to include not only the explicitly recited concentration of about 0.1 wt% to about 5 wt%, but also include individual concentrations (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5%, 1.1 %, 2.2%, 3.3%, and 4.4%) withinthe indicated range. The term “about” can include traditional rounding according to significant figures of numerical values. In addition, the phrase “about ‘x’ to ‘y’” includes “about ‘x’ to about .y„.

[0112] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0113] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methods belong. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.A. Abbreviations

[0114] Ca-ISM calcium-ion selective membrane

[0115] CV cyclic voltammetry

[0116] EDX energy-dispersive X-ray spectroscopy

[0117] FBS fetal bovine serum

[0118] ISM ion selective membrane

[0119] LOD limit of detection

[0120] OCP open-circuit potential

[0121] P phosphate (HPO42")

[0122] P-ISM phosphate-ion selective membrane

[0123] PDMS polydimethylsiloxane

[0124] POT poly (3-octyl-thiophene)

[0125] PVC polyvinyl chloride

[0126] RE reference electrode

[0127] RSD relative standard deviation

[0128] SEM scanning electron microscope

[0129] SLA stereolithography

[0130] THF tetra hydrofuran

[0131] WE working electrode

[0132] XPS X-ray photoelectron spectroscopyB. Discussion

[0133] Design Criteria and Standards. The criteria that guided the design and material considerations for this system were portability, selectivity, and affordability. Ideally, the sensor is designed for use on a farm, not in a laboratory setting, and therefore the sensor device is pocket sized, in one embodiment 4 inches by 6 inches. In this aspect, the sensor is designed to be selective to specific ions, such as calcium ions (Ca2+), phosphate ions (HPO42"), nitrate ions (NO3"), or iron ions (Fe2+). This sensor can be utilized in areas such as plant science (e.g., detecting Fe2+in plants), soil and water science (e.g., monitoring NO3" and HPO42" levels), and animal science (e.g., measuring Ca2+and HPO42" in lactating cows) for diagnostic and health evaluation purposes. The sensor can be configured to be sensitive to only a single ion or multiple different ions. For example, the ratio of HPO42" to Ca2+levels in ppm concentrations can serve as an indicator of clinical hypocalcemia (or milk fever), and subclinical hypocalcemia in mammals such as lactating cows. The sensor can provide an affordable option for farms as well as less expensive than a commercially available calcium sensor.

[0134] Similarly in this aspect, design constraints related to accessibility and accuracy were identified. Ideally, the sensor is easy to use for a farmer in the field, optionally able to pair to a smartphone device via Bluetooth. This would allow for more streamlined integration of the device into farms. It is also important that the sensor maintains accuracy in the field. With possible interference occurring from outside sources, it is important that the design still gives an accurate reading to ensure that the sensor is useful.

[0135] The following non-exhaustive list of standards was used during the development of the sensor:• CLSI AUTO1-A: Laboratory Automation: Specimen Container / Specimen Carrier;Approved Standard• ISO / TR 19693:2018 - Surface chemical analysis• ISO / IEC 23510:2021 - Information technology• ISO 10993-2:2022 - Biological evaluation of medical devices

[0136] At least these standards are taken into consideration during the design process because, during the testing of the sensor, proper precautions must be taken during contact with biological specimens. ISO / TR 19693:2018 - Surface chemical analysis is included to provide guidance concerning the cleaning and storage of the sensor. ISO / IEC 23510:2021 - Information technology provides recommendations for the 3D printing design of the sensor base in conjunction with additive manufacturing to improve the sensor-building workflow. Finally, ISO 10993-2:2022 - Biological evaluation of medical devices was considered due to the use of the device around animals.

[0137] Biosensor Design. Referring now to FIGS. 1A-1 B, in an aspect, the sensor can have two electrodes: a WE 106 and a RE 109 set into a base 103. In one embodiment, the base 103 is 33 mm in length, 25 mm in width, and 2 mm in height. In another embodiment, the WE 106 is 4 mm in diameter and the RE 109 is 3 mm in diameter. Each electrode and channel can be about 0.3mm in depth. An approximately 0.2 mm outer circle, referred to herein as a base well 112, can be cut out of the base 103 around both electrodes to hold liquid samples.

[0138] In another aspect, WE 106 can comprise a WE well at a first distal end, the WE well filled with a WE solid-state material. RE 109 can be set into base 103 adjacent to WE 106, RE 109 comprising a RE well at the first distal end adjacent to the WE well, the RE well filled with a RE solid-state material. Base well 112 can extend from an outer surface of base 103 to an inner surface of base well 112 at a depth of about 0.2 pm to about 0.4 pm, where base well 112 surrounds the WE well and the RE well, wherein the inner surface of the base well is flush with a surface of the WE well solid-state material and the RE well solid-state material. The WE well can be connected via a first channel 115 to a first pad 118 at a second distal end of the sensor. The RE well can be connected via a second channel 121 to a second pad 124 at the second distal end adjacent to the first pad 118. In one aspect, the WE well is circular and has a diameter of about 5mm to about 11 mm, about 6 mm to about 10 mm, or about 7 mm to about 9 mm. In another aspect, the RE well is circular and has a diameter of about 1 mm to about 5 mm or about 2 mm to about 4 mm. In another aspect, the WE solid- state material and RE solid-state material each comprise a plurality of layers of material.

[0139] As another example, illustrated in FIGS. 25A-25E, the sensor can have three electrodes. The three electrodes can be a first WE 206, a second WE 209, and a shared RE 212 set into a base 203. The second WE 209 can be adjacent to at least one of the first WE 206 or the RE 212. In one embodiment, the base 203 is 28 mm in length, 24 mm in width, and 2 mm in height. In another embodiment, the first WE 206 and second WE 209 are 3 mm in diameter and the RE 212 is 3 mm in diameter. Each electrode and channel can be about 0.3mm in depth. An approximately 0.2 mm outer circle, referred to herein as a base well 215, can be cut out of the base 203 around the electrodes to hold liquid samples.

[0140] In another aspect, first WE 206 can comprise a first WE well at a first distal end, where the first WE well can be filled with a first WE solid-state material. RE 212 can be set into base 203 adjacent to first WE 206, RE 212 comprising an RE well at the first distal end adjacent to the first WE well, where the RE well can be filled with a RE solid-state material. Second WE 209 can comprise a second WE well at a first distal end adjacent to either at least one of the first WE 206 or the RE 212, where the second WE well can be filled with a second WE solid-state material. The first WE well can be connected via a first channel 218 to a first pad 221 at a second distal end of the sensor. The second WE well can be connected via a second channel 224 to a second pad 227 at a second distal end of the sensor. The RE well can be connected via a third channel 230 to a third pad 233 at the second distal end. The third pad 233 can be adjacent to the first pad 221 . The second pad 227 can be adjacent to at least one of the first pad 221 or the third pad 233. In one aspect, both the first WE and second WE well can individually be circular and have a diameter of about 1 mm to about 5 mm or about 2 mm to about 4 mm. In another aspect, the RE well can be circular and have a diameter of about 1 mm to about 5 mm or about 2 mm to about 4 mm. In another aspect, the first WE solid-state material, second WE solid-state material, and RE solid-state material each comprise a plurality of layers of material.

[0141] Base well 215 can extend from an outer surface of base 203 to an inner surface of base well 215 at a depth of about 0.2 pm to about 0.4 pm, where base well 215 surrounds the first WE well, the second WE well, and the RE well, wherein the inner surface of the base well can be flush with a surface of the first WE well solid-state material, the second WE well solid- state material, and the RE well solid-state material.

[0142] Biosensor Construction. A base 103 and / or base 203 (base 103 / 203) of a sensor can be 3D printed using a high-temperature resistant resin that can be cured by UV light. In one aspect, the high-temperature resistant resin can be Grey Pro Resin, Clear Resin, or Rigid10K Resin from Formlabs. In one embodiment, the resin used has a heat deflection temperature of at least 230 °C at about 0.45 MPa. Following printing, the base 103 / 203 can then be washed to remove any liquid resin, e.g., using isopropyl alcohol, and cured in UV light. In one embodiment, the printing and curing process produces microstructures on the surface of the base 103 / 203 in the form of a periodic pattern of wrinkles. This can increase the surface area of the base 103 / 203, in comparison to a base 103 / 203 with a relatively smooth or flat surface. The microstructures or wrinkles can be parallel-patterned or zig-zag-patterned, with a periodicity or wavelength of about 20 pm to about 200 pm, about 20 pm to about 150 pm, about 40 pm to about 200 pm, about 40 pm to about 150 pm, about 40 pm to about 100 pm, or about 60 pm to about 100 pm. Further details and specific examples related to the production of a 3D-printed base 103 / 203 can be found in the Examples.

[0143] A layer of gold can be added to the base 103 / 203 as a layer on each electrode (e.g., WE 106, first WE 206, second WE 209, and RE 109 and / or RE 212 (RE 109 / 212)), depicted, for example, in FIG. 1C. This can be done using techniques such as electron-beam evaporation. The layer of gold can have a thickness of from about 50 nm to about 300 nm, about 50 nm to about 200 nm, or about 75 nm to about 125 nm. A layer of chromium or titanium can be deposited onto each electrode prior to adding the gold later. This layer can have a thickness of from about 5 nm to about 50 nm, from about 5 nm to about 25 nm, from about 5 nm to about 20 nm, or from about 5 nm to about 15 nm. In one embodiment, the gold layer maintains the wrinkles surface structure of the base 103, with a similar or the same periodicity and / or pattern (parallel or zig-zag). Further details and specific examples related to the deposition and surface structure of a chromium layer and a gold layer on a base 103 can be found in the Examples.

[0144] In FIGS. 1 D and 12D, a cross-section view of layers of electrodes can be seen. Following the gold layer deposition, for RE 109 / 212, an Ag / AgCI layer can be deposited onto the gold layer and dried. In one embodiment, the Ag / AgCI layer can be formed by applying an Ag / AgCI paste. The Ag / AgCI layer can have a thickness ranging from about 500 nm to about 10 pm, about 300 nm to about 5 pm, or about 100 nm to about 1 pm. An anti-leaching layer comprising a tetrafluoroethylene and fluorocarbon copolymer can be applied onto the Ag / AgCI layer. In one embodiment, the anti-leaching layer comprises Nation™, a copolymer of perfluoro-3,6-dioxa-4-methyl-7octene-sulfonic acid and tetrafluoroethylene. In one embodiment, the anti-leaching layer can increase the longevity of the electrode. The antileaching layer can have a thickness ranging from about 5 nm to about 200 nm, about 5 nm toabout 100 nm, about 5 nm to about 50 nm, or about 5 nm to about 10 nm. Further details and specific examples related to the deposition and surface structure of an Ag / AgCI layer and an anti-leaching layer can be found in the Examples.

[0145] Following the gold layer deposition, for WE 106, first WE 206, and / or second WE 209 a POT layer can be deposited. A POT transducing layer solution can be made through mixing POT with THF. The POT solution can be drop-casted on the gold layer and left to dry overnight. The POT layer can have a thickness of from about 500 nm to about 5000 nm or about 300 nm to about 1000 nm. In one embodiment, as with the base 103 / 203 and the gold layer, the POT layer can maintain a wrinkled surface structure. An ISM layer can be applied onto the POT layer. The composition of the ISM layer can vary depending on the ion to be detected by the sensor, and a variety of ISM layer compositions are discussed in detail below. The ISM layer can be applied by forming an ISM solution and depositing it onto the POT layer, for example, by drop-casting the solution. The thickness of the ISM layer can range from about 1 pm to about 10 pm, about 500 nm to about 5 pm, or about 100 nm to about 3 pm.

[0146] In one embodiment, the ISM solution or ISM layer can comprise a mixture by weight of about 33% of PVC, about 0.05% of an electrolyte (e.g., sodium tetrakis[3,5- bis(trifluoromethyl)phenyl]borate), about 65% of a plasticizer (e.g., dioctyl sebacate), and about 1 % of Calcium Ionophore II. In a further embodiment, the ISM solution can further comprise an organic solvent such as THF (e.g., 660 pl of tetrahydrofuran per 100 mg of the weight mixture). In one embodiment, this ISM layer composition is sensitive to Ca2+.

[0147] In another embodiment, the ISM solution or ISM layer can comprise a mixture by weight of about 1% tributyltin chloride, about 66% 2-nitrophenyl octyl ether, and about 33% polyvinyl chloride. In a further embodiment, the ISM solution can further comprise an organic solvent such as THF and / or further comprise bis(trifluoromethyl)phenyl]borate (e.g., about 1 mg to about 5 mg or about 1 mg to about 3 mg of bis(trifluoromethyl)phenyl]borate). In one embodiment, this ISM layer composition is sensitive to HPO42".

[0148] In another embodiment, the ISM solution or ISM layer can comprise a mixture by weight of about 6.7% 1 ,10- phenanthroline, about 3.3% potassium tetrakis chlorophenyl borate, about 60% dibutylphthalate, and about 30% polyvinyl chloride. In a further embodiment, the ISM solution can further comprise an organic solvent such as THF. In one embodiment, this ISM layer composition is sensitive to Fe2+.

[0149] In another embodiment, the ISM solution or ISM layer can comprise a mixture by weight of about 0.25% methyltriphenylphosphonium bromide, about 1.95% nitrocellulose (optionally moistened with 2-propanol), about 16.25% 2-nitrophenyl octyl ether, about 5.75% polyvinyl chloride, about 74.30% THF, and about 1.50% tridodecylmethylammonium nitrate. In one embodiment, this ISM layer composition is sensitive to NO3".

[0150] In another embodiment, the ISM solutions or ISM layers of the first WE 206 and second WE 209 can differ from one another. For example, a sensor can comprise a first WE 206 with an ISM layer sensitive to Ca2+and a second WE 209 with an ISM layer sensitive to HPO42" (or vice versa).

[0151] System Comprising the Biosensor. Also disclosed herein is a system comprising a biosensor as disclosed herein and a microfluidic device in fluid communication with the potentiometric sensor. The microfluidic device can be comprised of polydimethylsiloxane. The microfluidic device can comprise: a reservoir configured to be in contact with the base well of the potentiometric sensor; an inlet, configured to receive a fluid sample and be in fluid communication with the reservoir; and a pump, configured to pump the fluid sample from the inlet to the reservoir. In one embodiment, the reservoir can be in fluid communication with the inlet via a single or multiple microchannels. The system can further comprise an outlet configured to be in fluid communication with the reservoir and allow the fluid to exit the reservoir. In a further embodiment, the pump can also be configured to pump the fluid sample from the inlet to the outlet through the reservoir. The microchannels can have a cylindrical shape. The longest diameter of the microchannels can be from about 200 pm to about 800 pm, about 200 pm to about 600 pm, about 400 pm to about 800 pm, about 400 pm to about 600 pm or about 450 pm to about 550 pm. In one embodiment, the pump is a peristaltic pump. In one embodiment, the system can allow for reduction in temperature and volume fluctuations from one use or sample testing to another.C. Aspects

[0152] The following listing of exemplary aspects supports and is supported by the disclosure provided herein.

[0153] Aspect 1. A potentiometric sensor comprising a base; a working electrode (WE) set into the base, the working electrode comprising a WE well at a first distal end, the WE well filled with a WE solid-state material; and a reference electrode (RE) set into the base adjacentto the working electrode, the reference electrode comprising a RE well at the first distal end adjacent to the WE well, the RE well filled with a RE solid-state material.

[0154] Aspect 2. The potentiometric sensor of aspect 1 , further comprising a base well extending from an outer surface of the base to an inner surface of the base well at a depth of about 0.2 pm to about 0.4 pm, the base well surrounding the WE well and the RE well, wherein the inner surface of the base well is flush with a surface of the WE well solid-state material and the RE well solid-state material.

[0155] Aspect 3. The potentiometric sensor of aspect 1 or aspect 2, wherein the WE well is connected via a first channel to a first pad at a second distal end.

[0156] Aspect 4. The potentiometric sensor of any one of aspects 1-3, wherein the RE well is connected via a second channel to a second pad at the second distal end adjacent to the first pad.

[0157] Aspect 5. The potentiometric sensor of any one of aspects 1-4, wherein the WE well has a depth of about 0.2mm to about 0.4 mm.

[0158] Aspect 6. The potentiometric sensor of any one of aspects 1-5, wherein the WE well is circular and has a diameter of about 5mm to about 11 mm.

[0159] Aspect 7. The potentiometric sensor of any one of aspects 1-6, wherein the RE well has a depth of about 0.2 to about 0.4 mm.

[0160] Aspect 8. The potentiometric sensor any one of aspects 1-7, wherein the RE well is circular and has a diameter of about 1mm to about 5 mm.

[0161] Aspect 9. The potentiometric sensor of any one of aspects 1-8, wherein the WE solid-state material and RE solid-state material each comprise a plurality of layers of material.

[0162] Aspect 10. The potentiometric sensor of aspect 9, wherein the WE plurality of layers of material comprise a first layer material, wherein the first layer material comprises chromium or titanium; a first patterned gold layer superimposed on the first layer material; a poly(3-octylthiophene-2,5-diyl) (POT) layer superimposed on the patterned gold layer; and an ion-selective membrane superimposed on the POT layer.

[0163] Aspect 11. The potentiometric sensor of aspect 10, wherein the ion-selective membrane comprises about 33 wt% polyvinyl chloride, about 0.05 wt% sodium tetrakis [3,5-bis(trifluoromethyl)phenyl]borate, about 65 wt% plasticizer dioctyl sebacate, and about 1 wt% of a calcium ionophore II.

[0164] Aspect 12. The potentiometric sensor of aspect 11 , wherein the ion-selective membrane blocks all ions except for Ca2+.

[0165] Aspect 13. The potentiometric sensor of aspect 10, wherein the ion-selective membrane comprises about 1 wt% tributyltin chloride, about 66 wt% 2-nitrophenyl octyl ether, and about 33 wt% polyvinyl chloride.

[0166] Aspect 14. The potentiometric sensor of aspect 13, wherein the ion-selective membrane blocks all ions except for HPO42".

[0167] Aspect 15. The potentiometric sensor of aspect 10, wherein the ion-selective membrane comprises about 6.7 wt% 1 ,10- phenanthroline, about 3.3 wt% potassium tetrakis chlorophenyl borate, about 60 wt% dibutylphthalate, and about 30 wt% polyvinyl chloride.

[0168] Aspect 16. The potentiometric sensor of aspect 15, wherein the ion-selective membrane blocks all ions except for Fe2+.

[0169] Aspect 17. The potentiometric sensor of aspect 10, wherein the ion-selective membrane comprises about 0.3 wt% methyltriphenylphosphonium bromide, about 1.9 wt% nitrocellulose, about 16.3% 2-nitrophenyl octyl ether, about 5.8 wt% polyvinyl chloride, about 74.3 wt% THF, and about 1.5% tridodecylmethylammonium nitrate.

[0170] Aspect 18. The potentiometric sensor of aspect 17, wherein the ion-selective membrane blocks all ions except for NO3".

[0171] Aspect 19. The potentiometric sensor of any one of aspects 9-18, wherein the RE layers of material comprise a second layer material, wherein the second layer material comprises chromium or titanium; a second patterned gold layer superimposed on the second layer material; a silver / silver-chloride layer superimposed on the second patterned gold layer; and an anti-leaching membrane layer superimposed on the silver / silver-chloride layer.

[0172] Aspect 20. The potentiometric sensor of aspect 19, wherein the anti-leaching membrane comprises a polytetrafluoroethylene copolymer.

[0173] Aspect 21. The potentiometric sensor of any one of aspects 1-20, wherein the base has dimensions of about 20mm to about 50 mm, about 15mm to about 35 mm, or about 1mm to about 4 mm.

[0174] Aspect 22. The potentiometric sensor of any one of aspects 1-21, further comprising a second WE set into the base adjacent to at least one of the WE or the RE, the second working electrode comprising a second WE well at the first distal end adjacent to at least one of the WE well or the RE well, the second WE well filled with a second WE solid- state material.

[0175] Aspect 23. The potentiometric sensor of any one of aspects 3-21, further comprising a second WE set into the base adjacent to at least one of the WE or the RE, the second working electrode comprising a second WE well at the first distal end adjacent to at least one of the WE well or the RE well, the second WE well filled with a second WE solid- state material, wherein the second WE well is connected via a third channel to a third pad at the second distal end adjacent to at least one of the first pad or the second pad.

[0176] Aspect 24. The potentiometric sensor of aspect 22 or aspect 23, wherein the second WE well has a depth of about 0.2mm to about 0.4 mm.

[0177] Aspect 25. The potentiometric sensor of any one of aspects 22-24, wherein the second WE well is circular and has a diameter of about 5mm to about 11mm.

[0178] Aspect 26. The potentiometric sensor of any one of aspects 22-25, wherein the second WE solid-state material comprises a plurality of layers of material.

[0179] Aspect 27. The potentiometric sensor of any one of aspects 22-25, wherein the second WE plurality of layers of material comprise a third layer material, wherein the third layer material comprises chromium or titanium; a third patterned gold layer superimposed on third first layer material; a second poly(3-octylthiophene-2,5-diyl) (POT) layer superimposed on the third patterned gold layer; and a second ion-selective membrane superimposed on the second POT layer.

[0180] Aspect 28. The potentiometric sensor of aspect 27, wherein the second ion- selective membrane comprises about 33 wt% polyvinyl chloride, about 0.05 wt% sodium tetrakis [3,5-bis(trifluoromethyl)phenyl]borate, about 65 wt% plasticizer dioctyl sebacate, and about 1 wt% of a calcium ionophore II.

[0181] Aspect 29. The potentiometric sensor of any one of aspects 22-28, wherein the ion-selective membrane blocks all ions except for Ca2+.

[0182] Aspect 30. The potentiometric sensor of aspect 27, wherein the second ion- selective membrane comprises about 1 wt% tributyltin chloride, about 66 wt% 2-nitrophenyl octyl ether, and about 33 wt% polyvinyl chloride.

[0183] Aspect 31. The potentiometric sensor of any one of aspects 22-27 and 30, wherein the ion-selective membrane blocks all ions except for HPO42".

[0184] Aspect 32. The potentiometric sensor of aspect 27, wherein the second ion- selective membrane comprises about 6.7 wt% 1 ,10- phenanthroline, about 3.3 wt%, potassium tetrakis chlorophenyl borate, about 60 wt% dibutylphthalate, and about 30 wt% polyvinyl chloride.

[0185] Aspect 33. The potentiometric sensor of any one of aspects 22-27 and 32, wherein the ion-selective membrane blocks all ions except for Fe2+.

[0186] Aspect 34. The potentiometric sensor of aspect 27, wherein the second ion- selective membrane comprises about 0.3 wt% methyltriphenylphosphonium bromide, about 1.9 wt% nitrocellulose, about 16.3 wt% 2-nitrophenyl octyl ether, about 5.8 wt% polyvinyl chloride, about 74.3 wt% THF, and about 1.5% tridodecylmethylammonium nitrate.

[0187] Aspect 35. The potentiometric sensor of any one of aspect 22-27 and 34, wherein the ion-selective membrane blocks all ions except for NO3".

[0188] Aspect 36. A system comprising the potentiometric sensor of any one of aspects 2-35; and a microfluidic device in fluid communication with the potentiometric sensor, comprising a reservoir configured to be in contact with the base well of the potentiometric sensor; an inlet, configured to receive a fluid sample and be in fluid communication with the reservoir; and a pump, configured to pump the fluid sample from the inlet to the reservoir.

[0189] Aspect 37. A method for measuring a concentration of ions in a sample using the potentiometric sensor of any one of aspects 1-35, comprising obtaining a fluid sample; bringing the fluid sample into contact with both the WE well and the RE well; and measuring the open circuit potential between the working electrode and the reference electrode.

[0190] From the foregoing, it will be seen that aspects herein are well adapted to attain all the ends and objects hereinabove set forth together with other advantages which are obvious and which are inherent to the structure.

[0191] While specific elements and steps are discussed in connection to one another, it is understood that any element and / or steps provided herein is contemplated as beingcombinable with any other elements and / or steps regardless of explicit provision of the same while still being within the scope provided herein.

[0192] It will be understood that certain features and subcombinations are of utility and may be employed without reference to other features and subcombinations. This is contemplated by and is within the scope of the claims.

[0193] Since many possible aspects may be made without departing from the scope thereof, it is to be understood that all matter herein set forth or shown in the accompanying drawings and detailed description is to be interpreted as illustrative and not in a limiting sense.

[0194] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting. The skilled artisan will recognize many variants and adaptations of the aspects described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein.

[0195] Now having described the aspects of the present disclosure, in general, the following Examples describe some additional aspects of the present disclosure. While aspects of the present disclosure are described in connection with the following examples and the corresponding text and figures, there is no intent to limit aspects of the present disclosure to this description. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of the present disclosure.D. Examples1. Example 1 - Biosensor for Ca2+Detection

[0196] Rationale and Final Design Considerations. There are several different types of electrochemical biosensors that could be used to measure calcium concentrations in cows’ milk and blood plasma samples. However, the development of a potentiometric biosensor was determined to be the optimal type of sensor. A potentiometric biosensor benefits in that it has a relatively short development time, it is easily reproducible, and it can provide farmers with highly sensitive and accurate measurements in the field.

[0197] An additive manufacturing, i.e. , 3D printing approach was used for construction to try to cut down on costs of each sensor. In an attempt to keep costs down and make the sensor easier, an all-solid-state sensor was constructed using a transducing polymer layer instead of a calibrated liquid chamber (or inner filling solution) used in other ion-selective membrane-based sensors.

[0198] The final design uses two electrodes: a larger working electrode (WE) and a smaller reference electrode (RE). Both electrodes have a gold layer for conductivity and collecting sensing data. The reference electrode includes a Silver / Silver-Chloride (Ag / AgCI) layer and a Nation™ anti-leaching layer. The working electrode includes a Poly(3- octylthiophene-2,5-diyl) (POT) transducing layer with a Ca2+ion-selective membrane (ISM). The sensor is designed such that samples can be loaded into a well over the two electrodes or the sensor can be dipped into the sample. A voltmeter can then be used to measure open circuit potential (OOP) between the two electrodes. Due to the ion exchange mechanism, the ISM will only allow Ca2+ions to enter the working electrode and reject all other inferring ions. This will lead to the development of a potential corresponding to the concentration of Ca2+ions which will be measured with respect to the reference electrode at a constant potential.

[0199] Construction. A 3D printed solid-state potentiometric biosensor was constructed to measure the amount of free Ca2+ions in a liquid sample. First, a 3D model or base was constructed in Solidworks as seen in FIGS. 1A and 1 B. The sensor has two electrodes: the working electrode (WE) and the reference electrode (RE). In one embodiment, the base is 33 mm in length, 25 mm in width, and 2 mm in height. In a another embodiment, the working electrode is 4 mm in diameter and the reference electrode is 3 mm in diameter. Each electrode and channel are 0.3 mm in depth. A 0.2 mm outer circle, referred to herein as a base well, was cut out around both electrodes to hold the liquid samples.

[0200] The sensor was constructed of a 3D printed base out of a high-temperature resistant resin V2 from Form Labs. The base was printed on the Form Labs (Form 3L) printer. The base 103 was then washed in isopropyl alcohol to remove any liquid resin and cured in UV light. A 100 nm layer of gold was then added to the base 103 through electron beam evaporation for each electrode which can be seen in FIG. 1C.

[0201] In FIG. 1 D, a cross-section view of the layers of each electrode can be seen. For the WE, the POT transducing layer solution was made through mixing POT with Tetra hydrofuran (THF). The POT solution was drop-casted on the WE (gold) and left to dry overnight. The ISM was constructed by a mixture by weight of 33% of polyvinyl chloride (PVC), 0.05% of an electrolyte sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate, 65% of plasticizer dioctyl sebacate and 1% of selective material Calcium Ionophore II mixed with 660ul of tetrahydrofuran per 100mg of the weight mixture, (Nyein et al., 2016). 10ul of the ISM was then added on top of the POT transducing layer. For the RE 109, a Ag / AgCI ink was layered onto the gold layer and dried before adding a Nation™ anti-leaching layer to try to increasethe longevity of the electrode. The layer below the gold layer of both the WE 106 and the RE 109 can comprise materials such as chromium (illustrated in FIG. 1 D), titanium, and the like.

[0202] After the final layer for the electrode was added, copper conductive tape was attached to the ends of the gold layer below the electrodes. A silver conductive paint was used to keep the tape from moving or falling off.

[0203] Economic Analysis. The economic cost of hypocalcemia can be large. Per cow, milk fever can cost a farmer around $650 dollars per year, with an incidence rate of 6%. (Horst et al., 1997 & Oetzel et al., 2012). The average US dairy farm has a herd size of 320 cows (NASS USDA, 2022). Clinical hypocalcemia will therefore cost a farmer over $12,000 dollars per year. Additionally, subclinical hypocalcemia is a larger burden on a farmer. Subclinical hypocalcemia will affect the amount of and the quality of the milk produced, however not to the degree that hypocalcemia does. Subclinical hypocalcemia results in an average loss of $240 per cow per year (Horst et al., 1997). However, subclinical hypocalcemia has an incidence rate close to 60% (Oetzel et al., 2012). On the average dairy farm, this can cost the farmer $46,080 dollars per year. Since subclinical hypocalcemia presents no symptoms, a mechanism such as this sensor could help identify cows with subclinical hypocalcemia.

[0204] The lowest market price for a calcium ion sensor is around $420. Table 1 denotes the total construction cost of one sensor base 103 to be $4.60. However, this sensor cost can be reduced when scaling up the manufacturing of sensor. While this does not include electronics associated with the sensor and labor costs, keeping the product well under $10 is possible.

[0205] Method. The sensor was tested with an open circuit potential test using a VersaSTAT 4 Potentiostat Galvanostat. Calcium ion samples were prepared using CaCh dilutions of 0.25mM, 0.5mM, 1.OmM, 2.5mM, 3.0mM. Whole milk samples were prepared from store-bought milk and measured Ca2+concentration using a commercial sensor (HORIBA Ca meter). The whole milk sample was then diluted to different concentrations of Ca2+ions by adding deionized water to create samples of 0.77mM, 1.1mM, 1.42mM, 1.97mM, 3.2mM of Ca2+ions. Before testing a 3M potassium chloride (KCI) solution was added to the reference electrode 109 to saturate it with ions and washed off with deionized water. Three open circuit potential measurements were performed per dilution of the sample each lasting 3 mins. After each test, the sensor was washed with deionized water.Table 1. Cost of materials for construction of one sensor basePVC 2 mg $0.41 / g $0,00NaTFPB 0.003mg $0.69 / mg $0.00DOS 3.93 mL $0.69, 'mL $2.73ETH129 0.06 mg $6.08 / mg $0,37THF 70 uL $0.75 / mL $0.05Nation 30 uL $9.79 / mL $0.29High Temp Resin 3.3 mL S199.99T $0.66POT 0.078 mg $588 / g $0,46

[0206] The sensor was tested for selectivity with sodium (Na+) and potassium (K+) ions. First, the sensor was tested with 2 mM of Ca2+to establish a baseline. Then samples of 2 mM Na++ 2mM Ca2+, and 2 mM K++ 2 mM Ca2+were tested. Then samples of only 2 mM Na+and 2 mM K+were tested on the sensor.

[0207] Results and Discussions. Two calibration tests were performed on the sensor. The first with dilutions of Ca2+ions an increasing potential difference is seen between the two electrodes as the amount of calcium ions increases. This is due to the ISM creating the difference by only allowing Ca2+ions to affect the working electrode while all ions in the solution affect the potential in the reference electrode. FIG. 2A shows the response time of the sensor while testing with calcium titrations. A stepwise pattern is shown as each increment of calcium titration is put on the sample. FIG. 2B shows the calibration curve of the sensor with a slope value of 0.089 giving good sensitivity in the 0.5mM to the 3.0mM range.

[0208] The second test was using whole milk samples (FIGS. 2C-2D). The response time starts to show drifting between amounts of calcium, especially in the lower ranges. This maybe due to some interference from other ions or molecules in the whole milk samples or an ISM that is not selective enough for just calcium ions. The calibration curve has a slope of 0.053 which indicates a decrease in sensitivity compared to the 0.089 slope of calcium titration samples. The sensor does show changes to the amount of calcium in whole milk but iterative changes to the ISM would be needed to bring the sensitivity in line with the calcium titration tests.

[0209] A selectivity test was performed with Na+and K+ions. From FIG. 3, in the sample of both 2mM Ca2+and 2 mM K+, little difference is seen between the potential showing good selectivity for Ca2+. In the sample of both 2 mM Ca2+and 2 mM Na+there is an upwards shift. This could be due to some interference from the sodium ions for the sensor. Changes to the ISM may need to be tested to try to reduce this shift upwards. In the non-calcium testing, 2 mM K+has very little effect on the potential of the sensor whereas the 2 mM Na+leads to a potential shift. Overall, the selectivity of the ISM is working for potassium ions but net working as well for sodium ions.

[0210] FIG. 4A depicts a sensor comprising a WE and a RE, and FIGS. 4B-4C depict SEM images of various surface layers on the WE. FIGS. 4E and 4F show electrochemical characterization of the sensor of FIG. 4A. FIGS. 5A-5C show OCP results for a Ca-sensor at various concentrations of calcium ions. FIG. 5D shows correlation in responses of a commercial Ca-sensor and fabricate Ca-sensor with theoretical values of calcium ions. FIGS. 6A-6C show OCP results from tests of the Ca-sensors performance with low concentrations of calcium ions. FIGS. 7A and 7B show OCP measurements of a Ca-sensor in the presence of interfering ions and in FBS samples, respectively. FIGS. 7C and 7D depict visual comparisons of Ca-sensor responses in the presence of interfering ions and in FBS samples, respectively. FIG. 8A shows OCP measurements of a Ca-sensor in whole milk. FIGS. 8B depicts visual comparisons of Ca-sensor responses in the presence of whole milk. FIG. 8C shows OCP measurements comparing the various sensor responses, with a visual comparison of the various sensor responses shown in FIG. 8D. FIG. 9A shows a photograph of four sensors tested. FIGS. 9B-9D show electrochemical results from testing the sensors. FIGS. 10A-10B show SEM images of the electrode surface, depicting the various patterned microstructures that appear. FIGS. 11A-11C show EDS analyses of different sensor surfaces, in particular an Au@3D sensor (FIG. 11 A), POT-Au@3D sensor (FIG. 11 B), and Ca2+-ISM- POT-Au@3D sensor (FIG. 11C). The weight percentages of gold, carbon, and oxygen present are listed in FIGS. 11A-11C as well as in FIG. 11 D.

[0211] Conclusion. Based on the current results, the sensor shows a promising outlook for the detection of subclinical hypocalcemia, since the average range of calcium in cows blood is between 8.5 and 10.5 milligrams per deciliter (or approximately 2.15 - 2.55 mM), with less than 8.5 mg / dL categorized as subclinically hypocalcemic, the detection levels needed for practical application are well within the sensitivity range for the sensor. From this, one can conclude that the prototype will be not only able to subclinical hypocalcemia but also can quantify the level of calcium when tested on live animals. Results also suggested that the sensor has very good selectivity in the presence of the other interfering ions.

[0212] From the economic analysis, this manufacturing and testing method might be employed to produce cheaper sensors for the agricultural industry. This would greatly help commercial use by the target audience, farmers, who tend to have a low-profit threshold, and would only invest if there was a significant price difference between the sensors and traditional methods. Ideally, the upfront cost is low enough to encourage the adoption of this new device without significant financial risk, especially if farmers need to purchase multiple sensors per year. In another embodiment, when the cost of electronics associated with the sensor necessary for commercial use is anticipated to be much higher, the sensor includes an outer covering where replacement bits with the coating could be interchanged. This would present a higher upfront cost, but lower subsequent costs, making it a more appealing long-term investment.

[0213] Other Embodiments. In another embodiment, a small, contained circuit and chassis system are constructed for the sensor. The circuit includes a voltmeter from the sensor, a microcontroller, and a Bluetooth component to receive the signal from the sensor, translate it based on calibration data and connect it to a smartphone for data visualization. This allows for mobile testing setup that a farmer can use by hand. A continuous monitoring system built into farm equipment can also be constructed.In another embodiment, the sensor can be constructed to detect other common diseases and metabolite deficiencies, making the sensor useful in early disease prevention. For example, selenium deficiencies in goats are equivalently problematic as hypocalcemia is in livestock.

[0214] In another embodiment, the sensor has applications in preventative medicine. Being able to connect to a smartphone makes the device more appealing to mass consumer markets. Additional examples of other applications include testing for selenium and irondeficiencies, reading hormone and fertility levels on breeding animals, or even identifying antibiotic uptake and immunity levels to decrease the need for mass antibiotic dosing in food.

[0215] In another embodiment, the sensor can easily be modified for human use, monitoring glucose levels in diabetics and preventing iron deficiencies in those with anemia. It is also possible that biosensors could potentially be used to detect proteins and viral infection with minor modification in sensor surfaces. This could allow users to self-diagnose a disease even before symptoms begin to appear. This could have enormous implications for preventing the spread of diseases, where infected individuals would quarantine even if ‘asymptomatic’. Further, multiplexity of the sensor can be possible by adding more sensor units into the same sensor chip.

[0216] To make the biosensor more profitable, the cost per unit of the biosensor can be reduced by increasing the production volume. To scale up sensor manufacturing, adding more nozzles into the same printing machine can reduce sensor cost per test. If demand for the biosensor is high enough, larger production runs can be scheduled to take advantage of economies of scale. This will make the cost lower than $4.60 for each, enabling it to be more cost effective for use on farms. Additive manufacturing for fabricating all-solid-state sensors is a promising manufacturing modality for next-generation agriculture sensors with high performance.2. Example 2 - Biosensors for Multi-Ion Detection

[0217] Herein is demonstrated high-resolution and continuous sensing of multiple ions using a universal 3D-printed sensor featuring periodic patterns of POT layers with surface wrinkles. This sensor is characterized by its low cost, miniaturization, customizability, selectivity, and capability for long-term ion measurements. The sensor comprises a simple geometry with two electrodes — an extrusion 3D-printed working electrode and reference electrode. The periodic layers (width ~80pm) and surface wrinkles are achieved through lateral layer-by-layer 3D printing, enabling sensitivity down to 1 ppm of target ions. Specific ISMs on these 3D-printed sensors selectively measure ions such as iron (Fe2+), nitrate (NO3"), calcium (Ca2+), and phosphate (PO42"). The periodic POT layer acts as a redox capacitive solid-contact layer, facilitating ion-to-electron transduction through an ion-exchange mechanism. This versatile 3D sensing platform finds practical applications in plant science (e.g., detecting Fe2+in plants), soil and water science (monitoring NO3" and PO42" levels), and animal science (measuring Ca2+in lactating cows) for diagnostic and health evaluation purposes. For instance, in livestock monitoring, the sensor integrates with a PDMS-based long-channelmicrofluidic system, a peristaltic pump, and a compact 3D printed holder with a universal design. This configuration minimizes temperature fluctuations, thereby enhancing ion sensing accuracy. Its durability and portability enable seamless integration into milking parlors for direct monitoring of hypocalcemia (calcium ion tracking) in dairy cows.

[0218] Sensor Manufacturing. This 3D-printed sensor was manufactured using a stereolithography (SLA) printer, detailed in prior work and outlined in the Material and Methods section. FIGS. 12A-12E illustrates the manufacturing process, involving 3D printing with a photopolymer resin, UV curing of the printed sensor, gold and chromium deposition on the uncovered areas using a shadow mask, and application of a dielectric coating to the sensor’s wiring, sensor’s structure, and sensing principles. In FIG. 12A, the manufacturing process for the sensor base starts with 3D printing the photopolymer resin using an SLA printer. This is followed by curing the resin with UV light at a wavelength of 405 nm at 80 °C for 2 hours, forming a microscale periodic polymer pattern with a lateral layer width of approximately 80 pm and surface wrinkles. It is noted that the high temp resin used in this study offers a heat deflection temperature of 238 °C at 0.45 MPa, according to the manufacturer. This chosen resin facilitates post-processing techniques like e-beam evaporation and heat treatment for RE fabrication. The resulting periodic patterns and wrinkles on the sensor's surface create micro- geometries that enhance the surface area. Next, the sensor was covered with a shadow mask patterned with electrode geometry, and electron-beam evaporation was used to deposit a 10 nm chromium layer as an adhesion material, followed by a 100 nm gold layer as the electrode.

[0219] The connections and wiring were coated with a thin layer of dielectric material to prevent access to these areas, ensuring that modifications were restricted to the circular electrodes only. During the electrode modification steps, such as drop-casting of POT and ISM, capillary forces caused the liquid solutions to be drawn into the wiring and connection areas. The dielectric coating blocked these regions, limiting the electrode modification to the circular electrode area and improving sensor-to-sensor reproducibility. In FIG. 12C, the sensor base is designed to have two solid-state electrodes: the 8 mm diameter working electrode (WE) and the 3 mm diameter reference electrode (RE), each 1 mm deep. The sample holder, 0.3 mm deep, accommodates approximately 200 pL of testing sample. The SLA printer enabled the simultaneous production of 20 sensors within 2 hours at room temperature. Both electrodes, WE and RE consist of stacked multiple layers, as depicted in FIGS. 12B and 12D. A calcium sensor (Ca-sensor) fabricated is shown in FIG. 12C. It depicts the WE, RE,dielectric-coated wiring, and the connectors. The wires can be connected to the sensor connectors using wire soldering or conductive silver paint. For the RE, a thick layer (5 pm) of silver / silver chloride (Ag / AgCI) was applied over the Au layer, followed by a protonated Nation™ layer. The Nation™ layer effectively prevents chloride ions from leaching out of the Ag / AgCI layer. FIG. 12E illustrates the interfacial potentials of a potentiometric Fe-sensor. The total sensor potential, known as the OCP, denoted as E, combines the constant potential of the RE (Eo) with the concentration-dependent potential of the WE (Ei). The specific ionophore within each ISM generates a membrane potential (Ei) when exposed to target ions, facilitated by an ion exchange mechanism. These sensors measure the OCP difference generated between an ion-selective electrode-based WE and an RE due to the selective interaction of analyte ions with the ISM. The voltage at the RE remains constant and is independent of the concentration of the target analyte ions. According to the Nernst equation, Ei at the WE of the sensor directly correlates with the logarithmic activity of the target ions.

[0220] Sensor Geometry with Morphology. The morphology of sensors along with their elements is investigated using a scanning electron microscope (SEM) and energy-dispersive X-ray spectroscopy (EDX). The SEM images reveal the wrinkled gold surfaces on the periodic polymer patterns (FIG. 13A), alongside small, non-patterned microscopic holes within the gold layer formed during the curing process of the 3D-printed polymer, which were not filled due to the 100 nm thickness of the gold deposition. The small holes aid in adhering the POT layer. Separately, a POT solution at a concentration of 1 mg / mL (see the Methods sections) was prepared and applied onto the gold layer. The thin POT coating maintained the wrinkled structure of the gold surface (FIG. 13D), as observed in SEM images where all holes in the gold film were filled with POT polymer. Note that the wrinkled patterns of the POT layer enhance the sensor's sensitivity and improve the signal-to-noise ratio. This is because the POT functions as the ion-to-electrode transducing interface in this sensor geometry, resulting in a higher signal generation.

[0221] The EDX spectrum of the deposited Au layer confirms the presence of various elements following POT coating, including carbon and sulfur (FIG. 13E). The molecular structure of POT is composed of these two elements (see inset of FIG. 13E), and the estimated weight percentages of carbon (70.8%) and sulfur (6.9%) in the EDX spectrum. Furthermore, the EDX spectrum reveals substantial amounts of Au within the POT layer (12.4%), indicating a thin POT layer coating on the electrode surface. The elemental mapping analysis of the gold electrode coated with POT in FIGS. 13B and 13C demonstrates a uniform coating of the POTlayer. The thin, POT coating maintained the wrinkled structure of the gold surface. The capillary force of tetra hydrofuran, used as the solvent for the POT solution, contributes to the uniform distribution of POT on the gold electrode. In this study, four different ion selective membrane (ISM) cocktail solutions were separately prepared for Fe2+(Fe-Sensor), NOa- (N- Sensor), Ca2+(Ca-Sensor), and PO43" (P-Sensor). The primary component of all these membranes is polyvinyl chloride (PVC). This polymer matrix encases the ionophore. FIG. 13F shows the electrode coated with the membrane (Ca-ISM). The microporous membrane forms as the cocktail solvent (THF) evaporates, which further enhances the sensor’s sensitivity by expanding the membrane's surface area (FIG. 13F). Detailed preparation methods for these ISMs are provided in the Methods sections. Each sensor's WE were prepared by applying 30 pL of the respective cocktail solution onto the surface of the POT film. Detailed principles of operation for all sensors (Fe-Sensor, Ca-Sensor, N-Sensor, and P-Sensor) are also outlined in the Methods sections.

[0222] Sensing of Multiple Ions using 3D Printed Sensors. The sensing performance, selectivity capabilities, and calibration equation for each sensor targeting specific ions have been evaluated using OCP measurements between the WE and the RE. FIGS. 14A-14L demonstrate the potentiometric sensing results, calibration graphs, and the selectivity performance for multiple ions, including Fe2+, NO3", Ca2+, and HPO42". The Fe-sensor monitored the OCP for over 60 seconds between electrodes, with Fe2+titrate concentrations ranging from 56 ppb (low) to 5580 ppm (high). In DI water, the Fe-sensor baseline was obtained at 82 mV without any target Fe2+. Upon the introduction of 56 ppb Fe2+, the sensors signal increased to 104 mV, indicating interaction between the Fe ions and the Fe-ISM, leading to the observed potential development associated with Fe2+ions. As Fe2+concentrations further increased from 55.8 ppm to 5580 ppm, the sensor's OCP demonstrated a direct proportional increase. The sensor was rinsed with DI water before being exposed to a new concentration. Calibration of the Fe-sensor involved plotting OCP values (FIG. 14B) against Fe2+concentrations, employing logarithmic scales. This analysis effectively characterized the relationship between the sensor's OCP and varying Fe2+concentrations. For most target concentrations, the Fe-Sensor reaches saturation within 18 seconds, indicating rapid detection of Fe2+(FIGS. 14A and 14C). In regards to FIG. 14C, the ions were combined with a fixed concentration of iron (II) ion (56 ppb) in a 1 :1 ratio to evaluate the sensor's discrimination capability against interferents. Each of the four sensors has been examined in terms of response time and will be discussed in the following section. The shaded error region(SER), representing the standard deviation of three independent measurements (n=3), was calculated to assess the precision of the sensors.

[0223] Next, the performance of the Ca-Sensor for calcium ion, the N-Sensor for nitrate ion detection, and the P-Sensor for the detection of hydrogen phosphate ion was evaluated. Each sensor utilized a POT layer as a transduction material along with a specific ISM layer tailored to its target ion. Calibration of these sensors was carried out using dose-dependent titrate solutions to establish their quantitative analytical capabilities. Dose-dependent solutions of nitrate, calcium, and hydrogen phosphate ions were prepared by diluting stock solutions in deionized water to concentrations ranging from 1 ppm to 1200 ppm for nitrate, 20 ppm to 400 ppm for calcium, and 94 ppb to 94.7 ppm for hydrogen phosphate, respectively. The N-Sensor, Ca-Sensor, and P-Sensor were employed to measure the OCP responses in the presence of these solutions. Each measurement was independently repeated three times (n=3) to ensure reproducibility and evaluate the error bars. Sensing measurements were conducted using the Ca-Sensor in DI water, spanning concentrations of Ca2+ions from 20 ppm to 400 ppm. The baseline signal of the Ca-Sensor was found at -217 mV, and upon exposure to 20 ppm of calcium concentration, this value increased to 133 mV. Across the range up to 400 ppm of calcium concentration, the Ca-Sensor consistently exhibited a proportional response to the Ca levels (FIG. 14D). Like the Fe-Sensor, the Ca-Sensor incorporates a specific Ca-ISM with an ionophore designed to selectively capture Ca2+ions from the analyte solution. Calibration plots, both with and without logarithmic transformation, demonstrated high linearity, yielding a Pearson’s r value of 0.99 (FIG. 14E). The analytical sensitivity of the Ca-Sensor was found to be below 20 ppm concentration.

[0224] The OCP responses of the N-Sensor in the presence of various nitrate ion concentrations exhibited a clear dose-dependent relationship (FIG. 14G). The baseline signal of the N-Sensor initially found at 421 mV in DI water. Upon testing with a nitrate ion concentration of 1 ppm, the OCP decreased to 281 mV, indicating a detectable response to the presence of nitrate ions. (FIG. 14G). Further, as the nitrate concentration is increased from 1 ppm to 1200 ppm, the N-sensor OCP decreased from 281 mV to 160 mV. This change is attributed to the nitrate ionophore present in the nitrate-ISM on the sensor surface, which selectively binds nitrate ions from the samples, thereby generating a corresponding potential response. Calibration plots of N-Sensor with logarithmic transformation showed high linearity (Pearson’s r = -0.99), indicating robust performance across the tested concentration range (FIG. 14H). The analytical sensitivity of the N-sensor is 1 ppm concentration. In addition to thesensors designed for iron, calcium and nitrate, a hydrogen phosphate ion-selective membrane (P-ISM) integrated onto the POT surface of the P-Sensor for phosphate detection. The baseline signal of the P-Sensor exhibited an initial OOP response of 112 mV. Upon introduction of a phosphate concentration of 94 ppb, the OOP of the P-Sensor decreased to 21 mV (FIG. 14J). Subsequently, testing with a higher concentration of 96 ppm phosphate resulted in a corresponding OOP variation to -111 mV. The P-Sensor demonstrated robust responsiveness to phosphate ions, as evidenced by a calibration plot with a high coefficient of determination (Pearson’s r = -0.96) (FIG. 14K). The shaded error region, representing the standard deviation from three independent measurements, was included to illustrate the precision of the sensors for N, Ca, and P ions. The analytical sensitivity of the P-Sensor was determined to be 94 ppb concentration.

[0225] The selectivity of each sensor was evaluated in the presence of the most likely interfering ions that could affect the sensor’s performance in real-world environments. Ion concentrations were selected that were equal to or higher than those found in various real environmental, clinical, or biological samples, such as milk, lake water, or industrial wastewater. FIGS. 14C, 14F, 141, and 14L present the results of selectivity studies conducted for the Fe-Sensor, Ca-Sensor, N- Sensor, and P-Sensor, each evaluated under controlled conditions to assess their specificity. The Fe-sensor demonstrated exceptional selectivity towards non-specific target ions, as evidenced in FIG. 14C. To evaluate its selectivity, interference studies were conducted using potassium (39 ppm) and magnesium (23.4 ppm) as common interfering ions, deliberately chosen at elevated concentrations. These interferents were mixed in a 1 : 1 ratio with a fixed Fe2+concentration of 56 ppb, which is chosen to be approximately 1000 times lower in concentration compared to interfering ions The chosen concentrations are selected to encompass the ion levels found in river water, taking into account their practical applications. The typical ionic concentrations in ppm for Fe2+, Mg2+, and K+are 0.01-1.0, 1-100, and 0.5-10 in river water, respectively, and less than 0.3, 30, and 10 in high-quality drinking water, respectively. The resulting relative standard deviation (RSD) of approximately 5.78% highlights the sensor's robust capability to accurately detect Fe2+in the presence of interferents. This high selectivity is primarily attributed to the incorporation of a specific Fe2+ionophore within the Fe-ISM. This specialized ionophore facilitates the preferential exchange of Fe2+ions while effectively inhibiting interactions with non-specific targets. As a result, the Fe-sensor ensures precise and reliable detection of Fe2+, underscoring its suitability for sensitive analytical applications. FIG. 14F depicts the Ca-Sensor's responseto a target concentration of 20 ppm calcium, where potassium, sodium, and chloride ions were present as non-target interferents at 200 ppm each, in a 1 :1 ratio. In cow's milk, the concentration of calcium ions is roughly equal to that of other ions. In contrast, in bovine serum, calcium ion concentration is about one-tenth that of sodium, potassium, and chloride ions. Therefore, the concentration of interfering ions was selected to be ten times higher than that of calcium. The RSD of the Ca-Sensor in the presence of these interferents was found to be 6.04%, highlighting its high selectivity. In FIG. 141, the N-Sensor was tested with a target concentration of 50 ppm nitrate, where potassium, sodium, and chloride ions were present as non-target interferents at 100 ppm each, in a 1 :1 ratio. In agriculture runoff, typical ionic concentrations in ppm are approximately 5-100 for nitrate ion, 1- 50 for sodium, potassium, and chloride ions that are well covered by the proposed N-sensor. The RSD for the N-Sensor under these conditions was determined to be 4.5%, indicating its excellent selectivity. Similarly, Fig. 14L demonstrates the performance of the P-Sensor with a target concentration of 94 ppm phosphate, under the same mixing conditions of potassium, sodium, and chloride ions at 50 ppm each, in a 1 :1 ratio. The P-Sensor exhibited an RSD of 9.1 %, confirming its good selectivity.

[0226] Based on the detailed evaluation of Fe, Ca, N, and P sensors based on wrinkled POT layer for detecting respective ions, it is evident that each sensor demonstrates robust performance in terms of sensing accuracy, calibration reliability, and selectivity against interfering ions. The sensors exhibit high sensitivity across a range of concentrations, as indicated by their calibration plots and Pearson’s correlation coefficients. Furthermore, their ability to maintain low RSDs in the presence of significant interferents indicates their suitability for practical applications. The observed high selectivity can be attributed to the specific ISM and the hydrophobic POT layer that effectively repels other ions from the sensor interface. The hydrophobic property of polymer POT helps to improve sensor selectivity by repelling interfering ions, stabilizing the sensor interface, selectively permitting target ions, and reducing background noise. Specifically, the hydrophobic nature of solid-contact POT creates a barrier that prevents non-target ions from samples from approaching or accumulating at the interface of POT / ISM or POT / Au. Such characteristics improve the sensors' reliability in various environmental, clinical, agricultural, and livestock applications where precise ion detection is required.

[0227] Electrochemical studies. T o investigate the specific redox properties imparted by a POT layer in four different sensors, cyclic voltammetry (CV) tests lasting 30 seconds wereconducted. These CV tests were performed in phosphate-buffered saline (pH 7.4) containing a mediator, 5 mM Fe(CN)63- / 4-, at an equimolar concentration of 5 mM. This test involves applying a potential waveform (FIG. 15A) between the WE and RE, sweeping linearly from -0.7 V to +0.7 V at a constant scan rate of 50 mV / s, with a subsequent reverse sweep to the initial potential. The resulting CV output waveform (FIG. 15B) depicts the current plotted against applied potential. During the potential sweep, electrochemical reactions occur at the surfaces of both POT-modified and bare Au electrodes, reflecting electro-catalytic activity toward oxidation and reduction processes of mediator at the electrode interface. Notably, the sensor with a bare Au electrode exhibited higher oxidation or reduction peaks owing to the excellent conductivity of the gold metallic layer. Precious metals like platinum and gold exhibit excellent electrocatalytic activity towards the ferro / ferricyanide (Fe(CN)63" / 4") redox reaction due to their favorable electronic properties. These metals provide efficient electron transfer kinetics and stable surface adsorption sites, which facilitate the redox processes. Additionally, their resistance to corrosion and surface oxidation ensures long-term catalytic performance and reliability. Introducing a POT layer onto the gold surface suppressed the electrochemical response approximately threefold (FIG. 15B). Despite POT's favorable redox properties, its negatively charged layer repels ferro / ferricyanide molecules, leading to decreased current output. Despite the decrease in electrochemical current observed with the POT layer, these redox properties are valuable for sensors because the electrochemical reaction in potentiometric sensors is minimal, it generates almost no current during the measurement process. This study indicates the capability of POT as a modifier of electrode characteristics in potentiometric sensor applications, providing valuable insights into enhancing sensor sensitivity and selectivity through surface modifications with ion-selective membranes, as illustrated in FIG. 12D.

[0228] Detection time and Long Term Stability. Experiments were designed and conducted to measure the response time of each of the four sensors. During the experiments, each sensor measured a standard solution of the target ion for 60 seconds without any incubation. The OOP signal at the 60thsecond was taken as the sensor's response signal and 99% of this signal was calculated to determine the response time. The moment each sensor first reached this 99% signal, the time was recorded as the sensor's response time. In this context, the Fe-sensor was tested using a 55.8 ppm Fe2+standard solution, with the resulting OOP response shown in FIG. 15C. The OOP signal of the Fe-sensor at 60 seconds into the experiment measured 115.93 mV, and the sensor reached 99% of this signal after 18 seconds.Thus, the response time of the Fe-sensor is 18 seconds. The same experiment was conducted for the Ca-sensor (FIG. 15D), N-sensor (FIG. 15E), and P-sensor (FIG. 15F) using standard solutions of 20 ppm Ca2+, 100 ppm NO3" , and 96 ppm HPO42", respectively. These sensors achieved 99% of their final signal immediately at the beginning of the experiment for Ca-sensor and N-sensor and 24 seconds from the start of the experiment for the P-sensor. This demonstrates the rapid response nature of all four sensors, attributed to the ISM that quickly reaches equilibrium with the targeted ions. The thin ISM layer also reduces the path length required for an ion to reach the ionophore and reduces the time needed for the sensor to reach equilibrium, enhancing its response speed. Additionally, the flexible polymer chains in POT quickly adapt to changes in the ionic environment in the adjacent ISM layer, allowing the sensor to achieve equilibrium faster after exposure to the target ions. Furthermore, the oxidation of POT is a surface-confined process, which means that the redox reactions are restricted to the surface layer of the POT, thereby accelerating the overall response time.

[0229] FIG. 15G presents stability and long-term measurement data for the Nation™ on Ag / AgCI (Nafion™@Ag / AgCI) reference electrode (RE), focusing on its performance under varying chloride ion concentrations. The sensor stability was verified by investing the sensor’s response with chloride ion activity as the chloride ion leaching from RE is a major concern. A commercial RE was taken to measure the OOP of fabricated RE. In FIG. 15G, the fabricated RE (Ag / AgCI) with and without Nation™ coating was utilized to assess chloride ion activity. Chloride ion concentrations ranging from 0.01 M to 3 M (0.01 M, 0.1 M, 1 M, 2 M, and 3 M KCI) were prepared for this study. The Ag / AgCI electrode with protonated Nation™ coating was specifically chosen to mitigate chloride ion leaching, thereby enhancing stability in sensor’s response. Nation™ coating effectively maintains chloride ion activity and minimizes potential interference, crucial for accurate electrochemical measurements. The protonated Nation™ on the Ag / AgCI layer does not allow Cl- to leach and provides more stability. The fabricated RE exhibited a stable OCP when coated with Nation™ across a range of chloride ion concentrations from 0.01 M to 3 M. In contrast, the RE sensor without Nation™ showed varying OCP values in response to different chloride ion concentrations. FIG. 15H illustrates the results of long-term continuous measurements over 45 hours using the Nafion™-coated RE immersed in a 1 M chloride ion solution. This extended-duration study provides insights into the electrode's performance stability under continuous exposure to a high chloride ion concentration. Initially, within approximately 15 hours of sensor pre-conditioning, the OCP changed from -40.5 mV to -93.4 mV. Subsequently, over the next 30 hours, the OCPremained nearly unchanged at around -93 mV, indicating a stabilization rate of approximately ±1 to 2 mV per hour. The long-term measurement data in FIG. 15H confirm the electrode's robustness in maintaining stable OCP readings over an extended period in high chloride ion environments, highlighting its suitability for applications requiring continuous monitoring.

[0230] Sensor Integration with Microfluidic Devices in a Milking Parlor. In one embodiment, microfluidic devices are integrating with the biosensor to enable real-time analysis of milk samples, providing precise control and manipulation of fluid volumes. The seamless interfacing with milking machines enhances the efficiency and reliability of milk quality assessment. By incorporating electrochemical sensors, this integrated approach enhances sensitivity, specificity, and reproducibility, enabling detection of low-abundance analytes in complex matrices. These advancements support real-time monitoring for applications in point-of-care diagnostics, personalized medicine, and environmental assessments, contributing to significant advancements in dairy production and quality control practices. Furthermore, temperature (T) plays a role in the potentiometric sensors with ISMs, as governed by the Nernst equation (eq. 1). This equation describes the potential difference across the membrane as a function of ion concentration, with temperature being one factor. As temperature increases, the mobility of ions and the membrane's response typically become more pronounced, affecting the accuracy and precision of the measurements. Proper temperature control and calibration are essential to ensure reliable and reproducible results, as deviations can lead to significant errors in detecting ions. It is therefore helpful to understanding and compensating for temperature effects in order to improve accuracy of biometric analyses of all of the four proposed sensors. For example, milk samples from a milking machine or a portable milker are warmer than room temperature, and using a sensor calibrated with standard solutions at room temperature might lead to errors in ion detection. This issue also arises in other applications, such as wastewater measurement.

[0231] To equalize the temperature of milk samples between lab and farm measurements and reduce sample-to-sample temperature and volume fluctuations, a polydimethylsiloxane (PDMS) microfluidic system was designed with a serpentine microchannel of 500 pm diameter and 171 mm length (FIG. 16A). The microchannel extends to the 3D sensor's sample holder, which shares the same oval shape. This serpentine channel allows the milk sample to cool down while the increased surface-to-volume ratio of the microchannel enhances exposure tothe cool channel walls. Additionally, it allows for a controlled volume of the sample to be pumped onto the sensor's surface to minimize sample-to-sample fluctuations, improving repeatability. The PDMS channel was fabricated using a 3D-printed master mold, and the PDMS microfluidic system is shown in FIG. 16B. The CAD design of the sensor and microfluidic system are complementary, with the microfluidic system featuring a sample reservoir precisely aligned with the oval sample holder of the sensor. FIG. 16C shows the integration of the 3D-printed sensor with the microfluidic system. For farm deployability and to ensure mechanical stability for unsupervised end-users, a robust, pocket-size device was designed that integrates and embeds both the sensor and the PDMS microfluidic system. FIG. 16D illustrates this design, which includes 3 mm diameter holes for screws and 1.5 mm diameter holes for the microtube inlet and outlet. Top and side views are shown in FIGS. 16E and 16F, respectively The device body was printed using photopolymer resin, with the detailed manufacturing procedure provided in the Methods sections. FIGS. 16E and 16F display the top and side views of the assembled device, showcasing the integrated Ca-sensor and microfluidic system. The two wires protruding from the device connect to the RE (green) and WE (yellow). This durable sensor is paired with a miniaturized peristaltic micropump, powered by a 3V battery, capable of pumping 300 pL / min.

[0232] The Ca-sensor was evaluated using whole milk samples to detect calcium in this complex matrix. The samples were spiked with calcium ions, and their calcium levels were measured in ppm using a commercial calcium meter. The calcium levels in the five milk samples were 30.8, 44.0, 56.9, 78.9, and 128.2 ppm. The Ca-sensor successfully detected the differences in calcium levels, as shown by the OCP in FIG. 16G. The calibration graph for these milk samples, displayed in FIG. 16H, has a Pearson’s r of 0.96, demonstrating the sensor's ability to produce a linear response according to the Nernstian equation, even in the complex matrix of milk.

[0233] FIG. 17A shows the microfluidic integrated Ca-sensor interfaced into a milking parlor. An instance of using the sensor device in a milking parlor involves its Ca-sensor for real-time monitoring of milk calcium levels (FIG. 17A). This application serves to enhance milk quality control and detect postpartum hypocalcemia efficiently. According to FIG. 17B, the milk sample spends approximately ten seconds in the microchannels before reaching the reservoir region for sensing. The sensing region is also filled with milk samples after 35 seconds, and the entire process of cooling down and pumping takes enabled by using a peristaltic micropump less than 50 seconds. Each image of FIG. 17B is accompanied by a schematicthat indicates the volume of milk being pumped (via a peristaltic micropump) into the system. Additionally, an inset in each image displays the elapsed time during the pumping process. As previously mentioned, full sensing cycle with OCP technique takes 60 seconds, although sensors exhibit faster detection times. Therefore, the complete sensing procedure takes less than 2 minutes. Moreover, continuous pumping of the sample enables online monitoring. As previously discussed, the N-sensor and Ca-sensor can achieve 99% of their signal immediately, while the Fe-sensor and P-sensor generate their responses 18 and 24 seconds, respectively, after coming into full contact with the sample. This rapid reaction, coupled with the capabilities of the microfluidic system, allows the sensing device to be suitable for continuous and real-time monitoring across diverse applications.

[0234] Discussion. POT's oxidation process is surface-confined and rapid, ensuring the membrane-sample interface remains near thermodynamic equilibrium across a broad concentration range. This equilibrium allows for simplified theoretical predictions of sensor behavior without extensive diffusion coefficient considerations, enhancing sensor robustness and design simplicity. POT-based membranes also achieve low detection limits, similar to traditional stripping voltammetry, making them effective at low sample concentrations. These features make POT-based sensors highly effective for precise and reliable ion detection, with applications in clinical analysis and multianalyte detection in complex samples like human serum.

[0235] Additionally, the sensors show very fast response which is due to both the POT layer and the thin ISM layer atop it. The thin membrane, often in the micrometer to submicrometer range, significantly reduces the diffusion distance for ions. This allows for rapid ion transfer across the membrane-sample interface. Additionally, the oxidation of POT is a surface-confined process, meaning that the redox reactions are limited to the surface layer of the POT, further speeding up the overall response time. This makes the sensor favorable for the applications in continuous measurements and online monitoring.

[0236] Extrusion printing via SLA is used for lateral layer-by-layer deposition of high- temperature resin, creating periodic wrinkled structures on the electrode surface during curing. This 3D printing method allows for the fabrication of sensor bases, the proposed device, and the master mold for microfluidic system materials, enabling complex geometries, controlled structures, and a variety of material combinations. Importantly, it circumvents the costly clean room microfabrication processes (such as photolithography or e-beam lithography) typically used for sensor prototyping. Unlike other 3D printing methods, including direct nanoparticleprinting, this approach is cost-effective and maintains excellent sensor reproducibility while offering superior sensing properties. The wrinkled surface topology in the printed sensors provides a high surface area conducive to incorporating conducting polymers like POT, thereby enhancing sensing performance including limit-of-detection and response time compared to traditional 2D sensing geometries. 3D electrodes enhance analyte diffusion compared to 2D electrodes due to their increased surface area, which provides more active sites for reactions, and their structure, which facilitates better mass transport. The periodic wrinkled structures of 3D electrode allows ions to diffuse more efficiently, while the geometry promotes convective flows that improve transport to the electrode surface, reducing diffusion layer thickness. Additionally, the shorter diffusion paths in 3D electrodes speed up the analyte's access to reactive sites, and the full utilization of the electrode material increases overall performance, making diffusion faster and more efficient than in 2D electrodes.

[0237] The all-solid-state 3D printed sensors based on a solid-contact POT layer underwent comprehensive characterization, including electrochemical testing, to evaluate their multi-ion sensing and selectivity performances. FIGS. 14A-14L demonstrates potentiometric sensing results for multi-ions such as Fe2+, NO3", Ca2+, and HPO42" wherein the solid-contact POT layer acted as a universal ion-to-electron transducing layer for all sensors. The precision of each sensor in FIGs. 14A-14L in sensing, calibration, and selectivity studies was assessed by calculating the standard deviation based on three independent measurements (n=3). The results are represented visually using error bars and shaded error regions. The Fe-sensor (FIG. 14B) exhibited a clear relationship between OOP and Fe2+concentrations, reaching saturation within 18 seconds for most concentrations, indicating the sensor detection time is less than 20 s without any incubation (FIG. 15C). Interference studies (FIG. 14C) demonstrated the Fe-sensor's exceptional selectivity, showing an RSD of approximately 5.78% when tested against common interferents. Similarly, the N-Sensor, Ca- Sensor, and P-Sensor displayed dose-dependent responses to nitrate, calcium, and hydrogen phosphate ions, respectively. The N-Sensor, for instance, showed a linear relationship (Pearson’s r = -0.99) between OOP and nitrate concentrations, with a sensitivity down to 1 ppm. Further, the Ca-Sensor displayed a linear response to calcium concentrations ranging from 20 ppm to 400 ppm, achieving a Pearson’s r value of 0.99. Similarly, the P-Sensor demonstrated strong responsiveness to hydrogen phosphate ions with a high coefficient of determination (Pearson’s r = -0.98). Selectivity studies confirmed the sensors' ability to distinguish target ions from interferents such as potassium, sodium, and chloride. The Ca-Sensor exhibited an RSD of 6.04%, the N-Sensor 4.50%, and the P-Sensor 9.10% under challenging conditions, indicating their good selectivity. These results indicate the sensors' reliability and suitability for precise ion detection, facilitated by their specific ion-selective membranes and hydrophobic POT polymer layers that minimize interference from non-target ions. The solid-contact POT layer, recognized for its outstanding redox and hydrophobic properties, serves as a highly effective ion-to-electron transducer.

[0238] The stability of the sensor was significantly enhanced by incorporating a thin protonated Nation™ layer onto the reference electrode, as illustrated in FIG. 12D. This addition effectively prevented chloride ion leaching from the reference electrode throughout the measurement period. Remarkably, this enhanced stability enabled the sensor to maintain consistent long-term sensing performance, with minimal deviation in the output signal observed after 45 hours of continuous measurements. These findings underscore the electrode's robust resilience even under prolonged exposure to high concentrations of chloride ions, confirming its reliability for continuous sensing applications.

[0239] Integrating microfluidic systems with sensors is useful because it enables precise, real-time monitoring and analysis of small volumes of fluids. A microfluidic system was successfully integrated featuring a serpentine microchannel with dimensions of 500 pm in diameter and 171 mm in length. This setup effectively facilitates the rapid equilibration of milk samples, ensuring they reach ambient temperature efficiently. During testing, a peristaltic pump was utilized (300 pL / min) to drive the sample through the microchannel, which was integrated with a Ca-sensor. This approach not only demonstrates the system's capability to handle complex fluid dynamics but also validates its potential for precise, real-time monitoring of calcium levels in milk samples. This integration marks a significant step forward in developing a robust platform for sensitive and portable dairy quality assessment applications. Finally, a robust, pocket-size, field-deployable system was designed for unsupervised use in advancing the sensor’s technology for real-world applications. The developed device integrates any of the four sensors and the microfluidic channel within a 3D-printed enclosure, ensuring mechanical stability and ease of handling. This design not only protects delicate components from environmental factors but also facilitates seamless integration with a peristaltic pump for precise fluid manipulation. FIG. 16D illustrates the CAD rendered schematic, showcasing how the sensor interfaces with the microchannel, optimizing detection efficiency. Such advancements are poised to revolutionize on-site analysis, particularly in scenarios like milk sample analysis where reliability and portability are paramount.

[0240] Summary. This example explores the application of stereolithography-based extrusion printing to fabricate sensors device using photopolymer resin, resulting in structures with unique wrinkled patterns. This additive manufacturing method enables the creation of sensors with intricate geometries and diverse material combinations, avoiding the need for costly cleanroom processes. Incorporating a universal solid-contact POT layer, these sensors exhibit exceptional capabilities in multi-ion sensing and selectivity. They offer rapid response times and enhanced stability, supported by a protonated Nation™ layer that extends their performance longevity in multi-ion detection. The hydrophobic nature of the solid-contact POT enhances selectivity by repelling interfering ions, stabilizing sensor interfaces, and facilitating precise ion detection while minimizing background noise. The microfluidic system aims to counteract the negative effects of temperature fluctuations on accurate measurements, while the compact 3D printed device enables convenient on-site application of the sensors. This is an advancement in microelectronics fabrication for multi-ion sensing, enabling detection even at low concentration ranges across various media.

[0241] Methods - Chemicals. Deionized water with a resistivity of 18.2 MQ cm was obtained using a Millipore purification system from the US. Chemicals including calcium chloride (CaCh), sodium chloride (NaCI), sodium phosphate dibasic (Na2HPC>4), potassium chloride (KCI), and Ag / AgCI ink (composed of finely dispersed chloridized silver flakes) were obtained from Fisher Scientific, Waltham, MA, USA. Polyvinyl chloride (PVC), POT, tetra hydrofuran (THF), 1 ,10- phenanthroline, potassium tetrakis chlorophenyl borate (KTCpB), dibutylphthalate (DBP), sodium tetrakis[3,5-bis(trifluoromethyl)phenyl] borate, dioctyl sebacate (DOS), tributyltin chloride, 2-Nitrophenyl octyl ether (NPOE), methyltriphenylphosphonium bromide, tridodecylmethylammonium nitrate, tributyltin chloride, Sodium tetrakis[3,5- bis(trifluoromethyl)phenyl]borate (NaTFPB), calcium ionophore II, and Nation™ were purchased from Sigma-Aldrich (St. Louis, MO, USA). SYLGARD 184 PDMS Elastomer and curing agent purchased from Sigma Aldrich. The high-temperature resin cartridge (V2) used for manufacturing the sensor base was purchased from Dynamism, Inc., Chicago, IL.

[0242] Methods - Sensor Manufacturing. FIG. 12C displays photographs of the actual sensors alongside surface geometry views of the printed sensor. The entire sensor geometry was manufactured using an extrusion-based stereolithography printing tool. Prior to printing, a detailed 3D model was designed using Autodesk Fusion software. Each sensor included one working electrode and a reference electrode, situated within a base measuring 33 mm inlength, 25 mm in width, and 2 mm in height. The WE had a diameter of 4 mm, while the RE was 3 mm in diameter. Both electrodes and channels were 0.3 mm deep, with a 0.2 mm outer circle around each electrode designed to contain liquid samples. The sensor base was fabricated using a high-temperature resistant resin from Formlabs, Inc., utilizing an extrusionbased 3D printer. 10 sensors were successfully printed simultaneously. Post-printing, each sensor underwent cleaning with isopropyl alcohol using a FormWash L unit. Subsequently, the sensors were cured for 2 hours in a FormCure L system at 80 °C under 405 nm UV light (FIG. 12A). This curing process enabled the cross-linking of epoxy resin molecules, forming a cohesive polymer network. Subsequently, a layer of gold was deposited on each electrode. This coating was accomplished by placing a shadow mask. The mask, made from Kapton tape cut to the 2D sensor design using an automated silhouette cutter, was applied. A 10 nm thick chromium layer served as an adhesive, followed by deposition of a 100 nm thick gold layer using an e-beam evaporator. This gold layer provided conductivity on the wrinkled sensor surface.

[0243] For the modification of WE, a POT transducing layer solution was prepared by mixing POT with THF. This POT solution was drop-casted (20 pL) onto the gold-coated WE and allowed to dry overnight. For the reference electrode, a silver / silver chloride solution was applied over the gold layer and dried. Subsequently, a Nation™ anti-leaching layer was added to enhance the electrode's longevity. To complete the electrode assembly, copper conductive tape was affixed to the ends of the gold layer beneath the electrodes. Silver conductive paint was then used to secure the tape in place, preventing movement or detachment.

[0244] For the Fe-Sensor, the Fe-ISM cocktail solution was prepared using a mixture of 1 ,10- phenanthroline, KTCpB, DBP, and PVC in proportions of 6.73%, 3.33%, 60%, and 30%, respectively, along with THF. A 20 pL of this solution of the Fe-ISM was then added on top of the POT layer and dried overnight. For the Ca-Sensor, a Ca-ISM cocktail solution was prepared with 1% Ca2+selective ligand, 65% plasticizer, 1% additive, and 33% polyvinyl chloride powder. A 20 pL solution of this cocktail was drop-casted onto the POT layer. For the N-Sensor, an N-ISM cocktail contained methyltriphenylphosphonium bromide (0.25%), nitrocellulose (moistened with 2-propanol, 1.93%), 2-nitrophenyl octyl ether (16.25%), polyvinyl chloride (5.75%), THF (74.3%), and tridodecylmethylammonium nitrate (1.50%). It was sealed and stored at -20 °C before use. For the P-sensor, the P-ISM cocktail was prepared using 3.0 mg (1 wt%) of tributyltin chloride, 198 mg (66 wt%) of NPOE, 99 mg (33 wt%) of PVC, and 2.0 mg of NaTFPB in 3 mL of tetrahydrofuran. A 20 pL of this cocktail willbe coated on the POT surface to create the P-Sensor. All four sensors were conditioned by immersing them in highly concentrated (3 M) solutions of Fe2+, NO3", Ca2+, and HPO42" for over 12 hours. This step aimed to replace interfering ions in the membrane with desired ions, ensuring reliable sensor performance. Conditioning was performed before calibration and selectivity tests, to achieve stable and accurate responses.

[0245] The PDMS elastomer was mixed with its curing agent in a ratio of 10 parts elastomer to 1 part curing agent by weight. After degassing in a vacuum desiccator, the mixture was poured onto the master mold placed in a petri dish and allowed to sit at ambient temperature for 24 hours. Subsequently, the master mold was removed from the PDMS negative replica, which was then washed several times with isopropyl alcohol and DI water.

[0246] Software. The following software tools were utilized for various aspects of Example 2: OriginPro 2023b and GraphPad Prism 6.02 for statistical analysis, Autodesk Fusion 2024 and AutoCAD 2024 for designing the 3D models used in 3D printing, and Biorender 2023 for creating schematics. Additionally, software associated with the extrusion SLA printer, equipped with different control managers, was employed to manage the photopolymer resin 3D printing process of sensor base, device, and master mold.3. Example 3 - Biosensor for Dual-Ion Detection

[0247] In this study, a lithography-free and low-cost 3D printed sensor was developed to simultaneously detect Ca2+and P at the same time in milk and serum samples to identify an important metabolic disease, i.e., Hypocalcemia in transitional dairy cows. The innovative design of the sensor was manufactured using an extrusion-based 3D printer that allowed instant milk-fever diagnosis without the need for invasive blood sampling. In this device, due to the 3D geometry, which is a periodic structure of polymer (~78pm) along with secondary hierarchical wrinkled surfaces, a detection time of 10 seconds and an attomole sensitivity has been achieved. Furthermore, it can be integrated with an electrical milking machine, dairy industry piping, and milk parlors to facilitate in-field milk testing by untrained farmers (FIG. 18). The unique microscopic structures of the 3D sensor surface, along with wrinkled surfaces of POT, which served as a solid-contact ion-to-electron transducing layer, were created using extrusion 3D printing. This wrinkled structure significantly enhances the sensitivity of the sensor by increasing the surface area of the transducer. This enlarged surface area enhanced the capability of the sensor to detect and respond to even slight variations in the calcium and phosphate ion concentrations in milk and serum samples. This technology may be useful for detecting other biomarkers in milk samples.

[0248] Sensor Manufacturing. The sensor is manufactured using multiple electrodes for the detection of milk Ca2+and P ions. The low-cost 3D printed sensors in this Example were manufactured using an extrusion-based additive manufacturing technique, SLA, to detect the concentrations of ionized Ca2+and P ions in dairy cows. This fabrication method does not require sophisticated tools or cleanroom facilities, such as lithography. The details of sensor manufacturing using a 3D printer are explained in the Methods sections, FIGS. 24A-24E and FIGS. 25A-25E. For manufacturing, a 3D design was first created using “SolidWorks” software (FIGS. 25C-25E). The sensor has three functional electrodes with unique wrinkled geometries. One electrode served as a shared reference electrode (RE) and the other two served as indicators or working electrodes (WEs).

[0249] WEs were used to detect the Ca2+and P concentrations in the samples. All the electrodes were made of commercially available UV-curable high-temperature resins. The printed electrodes were placed in a small elliptical circle or sample holder, where the test samples were placed for detection (FIGS. 25A-25E). A photograph of the sensor is presented in FIGS. 25F and 25G. The sensor is connected with a Blue-tooth enabled portable potentiostat circuit to collect the data. An elliptical sample holder was created and all three electrodes were placed on it. The sensor was 33 mm in length, 25 mm in width, and 2 mm in height. The sensing geometry of each sensing unit was built via the layer-by-layer lateral printing of the polymer and cured using ultraviolet light. The width of the lateral layer of the polymer is about 78 ±2.5 pm, and the geometry of the sensor is composed of several periodic patterns of polymer containing many secondary wrinkled surfaces. All three electrodes have the same diameters of 3 mm and were placed at a 300 pm depth. This design was chosen to use a minimal amount of milk (-200 pL). In one fabrication batch, 20 sensors were printed at room temperature within 2 hours. Thus, it is reasonable to expect the sensors can be manufactured at a low cost. Once printed, a post-curing step was performed by applying UV light for 120 minutes at 80 °C to cure the device. As expected, the curing temperature used during the manufacturing process created secondary wrinkles on the sensor surface. The wrinkled surfaces of the sensor acted as a microelectrode and provided larger surface areas to develop an interfacial potential, even at low concentrations of target ions, particularly Ca2+.

[0250] After printing the sensor base, a 100 nm-thick layer of gold (Au) was deposited on the surface of the electrode by placing a polyimide tape-based shadow mask using an e-beam evaporator. The Au and chromium (Cr) layers were selected because of their high stability over time. A conducting polymer layer, i.e., a POT layer, was then introduced onto the two Auelectrodes, except for the shared RE, to serve as an ion-to-electron transducing layer in the sensor. The POT concentration was then optimized (FIGS. 32A-32D). In addition to excellent redox properties, the POT layer shows a hydrophobic surface that reduces water accumulation at the interface and high stability by reducing signal drifting over the testing time, resulting in high selectivity. Furthermore, two cocktail solutions of Ca-ISM and P-ISM were prepared separately and placed on top of the two POT / Au electrodes. Detailed explanations of these membranes are provided in the Methods section.

[0251] This sensor was based on the potentiometric principle described in FIG. 19A, wherein the OOP between the two electrodes was measured. The ion-exchange mechanisms of Ca2+(left) and P (right) through specific ion-selective membranes and POTs are described. In this sensor, the presence of specific ionophores in the respective ISMs was responsible for the selectivity. These Ca- or P-ionophores embedded in PVC membranes on sensor surfaces are responsible for changing the potential while detecting Ca2+and P ions selectively. This is achieved by the accumulation of target ions at the interfaces between the POT and ISM, which generate a potential in the presence of target ions concerning the constant potential of the RE. The details of the sensing principle are described in the Methods sections.

[0252] The field-emission SEM images in FIGS. 19B-19E depicts the surface morphology of the sensor after Au coating, modification with the POT layer, and ISM, respectively. The Au coating applied to the epoxy resin of the sensor base exhibits two distinct patterns. The first is the primary periodic pattern of the lateral polymer layer (FIG. 19B). The micro- structure (primary pattern) is achievable through 3D printing, offering precise control over the structure at a resolution of 25 pm, specifically tailored for the extrusion printer. Each printed layer maintains a consistent lateral distance (~78 pm), with the underlying polymer layers interconnected through the UV curing step. The secondary pattern revealed a wrinkled and non-uniform texture attributed to the uneven underlying layer of the 3D-printed base. Both the primary and secondary patterns are marked with lines (FIGS. 19B-19C). Such randomness in the pattern developed due to both the printing and curing steps during sensor manufacturing (FIG. 19D). FIG. 19D shows an SEM image of Au-coated 3D printed sensor showing the interconnection of a layer after the curing step using a UV light. After curing, a 100 nm-thick gold layer was selectively deposited on the patterns using an e-beam evaporator by placing a shadow mask. The coating was stabilized by adding an adhesive layer of chromium with a thickness of 10 nm. Finally, the shared RE was modified with a commercially available thick (5 pm) layer of silver / silver-chloride (Ag / AgCI) ink. A thick layer enhances device stability. Oneof the major issues of potentiometric sensors is signal drifting due to chloride (Cl") leaching over time. Thus, the RE was modified with a thin protonated membrane, / . e., Nation™, to block Cl" leaching. Note that all sensor elements in the sensor are solid-state elements; thus, the sensor does not require any internal filling reference solution, resulting in miniaturized sensors for farm measurements.

[0253] The smooth surface of the ion-selective membrane covering the wrinkled POT is revealed in FIG. 19E. FIG. 19E shows an SEM image of ISM-coated POT@Au film that shows a uniformly smooth surface of the membrane. The ion-selective membrane atop the POT-Au layer exhibited uniformity because the main constituent of the membrane cocktail, PVC, filled both the secondary and primary patterns, forming both smooth and even layers. In FIGS. 26A- 26D, an SEM analysis was conducted to investigate the reproducibility of the printed geometry of the sensor. As evidenced by a low standard deviation (SD) of ±2.518 pm for printing microstructures (Table 2), the sensor provides a high reproducibility. This confirms the random nature of the secondary pattern, as its size falls below the precision of the printer and contributes to the significantly increased surface area of the sensor. The POT layer is partially covering the wrinkled Au surface. The ISM coating on POT-Au showed full coverage with a thick film formation. The full coverage of ISM is to prevent water entering into the POT layer during measurement, hence increasing the selectivity of the sensor.Table 2. Lateral distances (pm) of periodic printed layers of the 3D printed sensor.

[0254] Surface Wrinkles Formation. Photosensitive polymer surfaces tend to form wrinkled surfaces owing to mechanical instabilities. In the device disclosed herein, the formation of wrinkles on the polymer surfaces were observed to be enabled by the combinedeffect of simultaneous thermal and UV light treatments during the curing step. The surface wrinkles on the patterned polymer are shown in FIGS. 27A-27D. These wrinkles are complex and hierarchical. Although it was a single layer of polymer (2 mm thick), the top surface of the printed structure formed wrinkles. The heights of the wrinkles were a few microns, and they were confined within periodic patterns on the surface. Unlike temperature treatment, UV light treatment allows only the top surface of the device to shine. As a result, the volume contraction and capillary forces acted simultaneously, and thus, the surface volume of the device shrank to form hierarchical wrinkles. In this study, a temperature of 80°C was maintained along with and an ultraviolet irrigation having a wavelength of 405 nm to produce reproducible geometry of the sensor. In this work, the wrinkled feature of the sensor provides a higher signal-to-noise ratio, larger surface area, and ultra-high sensitivity compared to traditional sensors with planar surfaces. Such features of the device are expected to provide ultrasensitive detection of calcium and phosphate ions, even in milk samples.

[0255] Surface Analysis of the Sensor. Because the sensor has multiple layers, the chemical composition of each layer was evaluated using Energy-dispersive X-ray spectroscopy (EDX), mapping, and X-ray photoelectron spectroscopy (see FIGS. 28A-28D, 29A-29B, and 30A-30E). The layers were composed of bare Au, POT-Au, and ISM-POT-Au films. In EDX analysis, only Au was observed on the Au film. With the POT layer, carbon traces notably increased to 55.6%, further increasing to 76.3% upon the incorporation of the ISM into the POT layer. The uniform coverage of the surface by the POT and ISM layers is evidenced by a notable reduction in the Au percentage in the film (FIG. 28D). SEM images revealed the thorough coverage of both the lateral patterns (primary pattern) and the wrinkled surface (secondary pattern) by a smooth ISM-POT layer (FIG. 19E). In addition, the surface mapping analysis of the POT layer shown in FIG. 29A reveals the presence of sulfur and carbon atoms, as POT is comprised of sulfur and carbon. The calcium and phosphate ion-selective membranes predominantly consisted of PVC, with carbon and chloride as the primary atoms in the ISM coating mapping analysis (see FIG. 29B).

[0256] X-ray photoelectron spectroscopy provides a deeper analysis of each layer of the sensor. The X-ray photoelectron spectroscopy spectra of the resin polymer and Au films are shown in FIGS. 30A-30E. The gold coating on the polymer showed multiple Au peaks owing to the disappearance of the C1 s and O1s peaks (FIGS. 30A-B) of only the polymer. As shown in FIG. 30C, there are three major peaks found at the binding energies of 284.8 eV, 286.2 eV, and 289.0 eV for printed sensors without any coating corresponding to the functional groupsof C-C, C-O, and 0-C=0, respectively. In the O1s peak (FIG. 30D) of polymer, three distinct peaks appeared at binding energies of 530.9 eV, 532 eV, and 529.1 eV, indicating oxygencarbon (O-C), oxygen-hydrogen (O-H) and oxygen- carbon (O=C) bonds, respectively. These peaks disappeared because of the gold coating (FIG. 30E). The gold layer revealed distinct peaks corresponding to the Au4f? / 2 and Au4fs / 2 binding energies, confirming the gold coating.

[0257] Electrochemical Characterization of the Sensor. POT is known as a hydrophobic conductive polymer and has potential to serve as an ion-to-electron transducer layer to develop a solid contact-based potentiometric sensor. In addition, the POT layer is highly stable and reproduces the interfacial potential between the gold and ISM layers, resulting in the high potential stability of the sensor.

[0258] To investigate the redox properties of the POT layer, the CV technique was performed, wherein the current was recorded over a range of potentials. CV experiments were performed in the presence of a 50 mM phosphate-buffered saline (PBS, pH 7.4) solution containing an equimolar (5 mM) concentration of a ferro / ferricyanide mediator. The current was recorded from -0.5 V to +0.5 V to show clear oxidation and reduction of ferro / ferricyanide. The CV study was conducted for Au, POT@Au, and ISM-POT@Au electrodes. The electrode with the POT layer shows excellent oxidation and reduction properties. With POT, the current of the Au electrode decreases, and the further current of the electrode decreases while the ISM layer is coated on POT-Au. As indicated in FIG. 19F, the CV of the Au electrode shows clear oxidation and reduction peaks due to the redox reaction of [Fe(CN)6]3- / 4-probe. Further, the peak-to-peak potential difference (AE) for the Au electrode was estimated to be 0.1 V. Such a small AE of the Au electrode indicates an excellent redox property and higher electron transfer kinetics. This is due to the wrinkled surface of the three-dimensional gold sensor. Introducing a POT layer on Au, the sensor generates a lesser electrochemical current (1.7 pA) due to the inherent insulating nature of POT compared to a bare Au electrode (3.4 pA). Peak current is one parameter that provides information about electrode kinetics, surface area, species concentration, and reaction reversibility. The AE remains almost unchanged, indicating that the POT layer does not influence the kinetics of the mediator redox reaction on its surface. The POT-Au electrode ISM coating significantly reduced the electrochemical current (0.7 pA). This is due to the ISM layer, which repels [Fe(CN)6]3" / 4" ions to reach the ion- to-electron transducing layer, and the sluggish ion exchanges on the ISM electrode surface, which acts as an isolator. Consequently, both the oxidation and reduction peaks disappeared from the recorded signals.

[0259] The wrinkled surface on the sensor acted as a microelectrode; thus, a higher diffusion coefficient of the redox species is expected due to the spherical diffusion of ions. The diffusion coefficient (Do) for each mediator using the Randles-Sevcik equation applied to individual layers of the electrode. It shows diffusion coefficients of 3.038 x 10"1°, 8.013 x 10-11, and 1.401 x 10"11mm2s-1, respectively, for [Fe(CN)e]4" diffusion on Au, POT-Au, and ISM- POT-Au sensors. In the reverse scan, the calculated diffusion coefficients of [Fe(CN)6]3" on Au, POT-Au, and ISM-POT-Au sensors are 4.752 x 10"1°, 1.087 x 10"1°, and 1.207 x 10"11mm2s-1, respectively. Both the POT and ISM layers on the wrinkled Au surfaces cause sluggish mediator diffusion towards the electrodes, resulting in lower oxidation and reduction currents in their CVs.

[0260] Electrochemical impedance spectroscopy was used to investigate the surface electrochemical properties of the sensors. This study was conducted on sensors with and without ISM and POT layers to investigate the interfacial properties between the electrode and electrolyte. An AC signal having an amplitude of 10 mV and a range of frequency of 1-10,000 Hz was applied to the sensor, and electrode impedance was measured in the presence of PBS solution containing [Fe(CN)6]3- / 4-. As shown in FIG. 19G, the Nyquist plots corresponding to each electrode indicate a significant change in the charge-transfer resistance (Rct) of the electrode. For this study, the amplitude and detection frequency were set to 10 mV and 10,000-1 Hz, respectively. These Rct values were estimated by fitting the raw data to an equivalent Randles circuit. The wrinkled Au electrode without ISM and POT layers provided an Rct value of 4.9 kQ. ISM and POT coating enhances the electrode’s impedance to a value of 69.3 kQ. This enhancement in Rct was mainly due to the resistive properties of both the ISM and POT layers, which hindered electron transfer.

[0261] Sensor Calibration. The sensor was calibrated before testing the spiked solutions and real samples. This was done by recording the OOP, which is also known as the electromotive force (emf) and is measured in millivolts (mV) between two electrodes (WE and RE) when no current flows between them. The change in the sensor output (mV) was measured using several standard solutions of calcium and phosphate ions. Note that the working electrode develops a specific potential due to ion exchange through the ISM, which varies with concentration, while the RE develops a fixed potential. The sensing principles and mechanisms are discussed in detail in the Methods sections. Considering the application of the sensor in farms, OCP measurements may be the best choice for the sensing modality.OCP sensors can provide stable measurements over extended periods. This stability is important for applications that require long-term monitoring.

[0262] The calibration of the calcium sensor (Ca-sensor) in FIG. 20A was established using standard solutions of Ca2+with concentrations ranging from 0.5 mM to 10 mM in deionized (DI) water (0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 5 mM, and 10 mM). To ensure reliability and accuracy, three consecutive readings were recorded for each measurement. The OCP of the sensor was measured for one minute. Error bars corresponding to the standard deviation of measurements derived from a minimum of n three biologically independent experiments were incorporated into each calibration plot. For the Ca- sensor, the baseline of the sensor was set to -0.190 V with DI water without any Ca2+ions. A drastic change in the potential of the sensor (0.130 V) was observed when a 0.5 mM concentration of Ca2+ions was introduced into the sensor. A specific calcium ionophore embedded in the PVC membrane was responsible for changing the sensor potential. Then, serial titration concentrations of Ca2+ranging from 1 to 10 mM were introduced and their OCP were measured. The OCP increased with elevated concentrations of Ca2+ions. The sensor was then washed with DI water to reset its baseline value. A minute change in the potential (0.004 mV) was observed during resetting of the sensor. The sensor was re-calibrated with titration concentrations of Ca2+ions (0.5-10 mM) and showed a baseline signal when tested with DI water. A similar result was observed for the sensor OCP when the sensor was tested for a second calibration. A plot of the concentration versus OCP of the sensor is shown in FIG. 20B, and the resulting calibration curves, along with the regression coefficients, are depicted in FIG. 20C. These results indicate the ability of the sensor to detect multiple measurements of Ca2+without recalibration. The sensor's sensitivity, determined by analyzing the slope of the associated calibration curve, is calculated to be 24.3 mV / dec (r2 = 0.97). A minor decrease in the sensitivity of the sensor was observed during the second measurement across the entire Ca2+concentration range, demonstrating the repeatability of successive measurements.

[0263] For the P-sensor, the sensor was calibrated with titration concentrations of HPO42" ranging from 10 pM to 30 pM (FIG. 20D). The baseline of the P-sensor was set to 358 mV using DI water without any HPO42" ions. When a concentration (10 pM) of HPO42" ions was introduced to the P-sensor, an OCP of 10 mV was decreased compared to the baseline signal. This change in the OCP is due to the presence of a phosphate ionophore embedded in the PVC layer on the sensor surface, which traps HPO42-ions from the samples and develops a potential at the membrane interface. Further, as se the concentration of HPO42-ions isincreased from 15 pM to 30 pM, the sensor potential found to be decline. Unlike the Ca-sensor, the P-sensor showed the opposite signal trend with increasing concentrations. As expected, an increase in the HPO42" concentration of the negatively charged anions led to a reduction in the OCP response. Further, the P-sensor turned into the baseline when the sensor was tested with DI water, and the measurements were repeated with 10 pM to 30 pM of concentrations of HPC>42“ ions to set the second baseline of the P-sensor. These repeated measurements indicated that the sensor did not require recalibration. The calibration plot of the P sensor is shown in FIG. 20E. The sensitivity of the P-sensor was estimated by evaluating the slope of the calibration curve, resulting in a calculated value of -152.09 mV / dec (r2 = 0.99, FIG. 20F). The analytical sensitivity of the P-sensor was 10pM.

[0264] Sensing Performance at Low Concentration and Repeatability. The baseline signal of the Ca sensor had a large gap with the response signal of the Ca2+ions (0. 5mM). Therefore, several low concentrations of Ca2+ions were introduced to evaluate the limit of detection of the sensor. Furthermore, during the transition period of dairy cows, blood Ca2+ions become too low, and it is expected that this requirement will be met using the Ca sensor. For this, a series of standard titration concentrations of Ca2+ions was prepared ranging from 1 aM, 1 fM, and 1 pM to 1 pM in DI water. The results are shown in FIG. 21A. For the baseline measurement, the sensor was tested in the absence of Ca2+ions and showed a negative OCP. Subsequently, standard solutions of Ca2+ions were introduced onto the surface of the sensor and recorded the corresponding OCP graphs over 100 seconds to ensure that the sensor delivered a stable response. As shown in FIG. 21 B, discernible details can be observed of the sensing responses at low concentrations of Ca2+ions. This demonstrates the capacity of the sensor to distinguish the attomole (1 aM) concentration of Ca2+ions from the baseline signal. Finally, the OCP responses of the sensor were plotted against each concentration of Ca2+, as depicted in FIG. 21 C. The 3D-printed sensor responses were compared with the theoretical values of Ca2+ions and those of a commercially available Ca-meter. Both sensors (printed and commercial) provided similar results, as evidenced by the minimal difference in their regression coefficients (FIG. 21 D). However, a major limitation of commercial Ca-meters is that they cannot detect Ca2+at concentrations as low as 1 mM. The analytical sensitivities for Ca-sensor and P-sensor are 1 fM and 10 pM, respectively. Such sensitivities were mainly due to the 3D geometry of the electrode and wrinkled surfaces.

[0265] The LOD is an important parameter for an ISM-based potentiometric sensor. The LOD was calculated for both Ca-sensors using previously reported literature; LOD calculationsare described in the Additional Data - Calculation of LOD section. The LOD of the Ca-sensor and P-sensor were calculated as 138 aM and 2.6 pM. The LOD in this study was useful for detecting low concentrations of Ca2+in milk samples during the transition period in dairy cows. Unlike traditional 2D surface-based sensors, the larger surface area of the wrinkled surfaces of the sensors was the main reason for the low LOD. In contrast to the findings outlined in Table 3 from various research endeavors, the sensor demonstrated a detection limit of approximately ten orders of magnitude lower, highlighting the impact of the 3D-printed sensor structure on its performance. This represents a significant milestone as it is the inaugural instance of a compact and affordable sensor that can successfully measure Ca2+ions at the attomolar level. Additionally, the linear ranges for the calcium and phosphate sensors were 10"18~ 10"2M and 1.0 x 10"5~ 3.0 x 10"5M, respectively, effectively encompassing the target physiological concentrations in cow's milk and serum samples. The broader range of detection of the Ca-sensor can maximize the efficacy beyond the physiological range of dairy cows and can also promote its use in other applications.Table 3. Comparison of the analytical properties of the Ca-sensor and P-sensor with previous literature.

[0266] For reproducibility, an experiment was conducted involving consecutive introductions of identical Ca2+concentrations of 0 mM (low) and 2 mM (high), followed by a control sample without any Ca2+ions (FIGS. 21 E-21 F). For this study, a specific high-low concentration of Ca2+that aligns with the physiological range of Ca2+in milk was chosen to investigate the sensing performance. The baseline exhibited a minimum deviation of 6.7%, whereas the sensor response showed a deviation of 1.1 % after testing the sensor sequentially (low-high-low-high) at least seven times. This confirmed the minimal potential drift of the sensor, even with a drastic change in the Ca2+level, owing to the hydrophobic surface and high selectivity of the sensor. These excellent features of the 3D printed sensor could also be of interest to the food sector, where compact sensors with high sensitivity, minimum potential drift, signal calibration, and cost-effectiveness are required.

[0267] Selectivity, Reproducibility, and Detection Time. Given the significant presence of coexisting and isostructural ionic species within clinical and biological samples, which could potentially lead to interference with the target analyte, the ability of the developed sensor to accurately measure Ca2+selectively was assessed via an interference test (FIGS. 22A, 22D, and Additional Data - Ion Selectivity Evaluation section). Notably, two major ions, sodium (Na+) and potassium (K+), were chosen because they could potentially influence target test samples such as milk and bovine serum. A constant concentration of Ca2+(2 mM) was used in the absence and presence of varying concentrations of Na+and K+. As illustrated inFIG. 22A, the sensor OCP was measured when exposed to a mixture of 2 mM Ca2+along with 2 mM and 28 mM Na+ions, as well as 2 mM and 36 mM K+ion solutions. At low concentrations of Na+and K+(2 mM), the sensor demonstrates high selectivity for the detection of Ca2+ions, as indicated by its minimal relative standard deviation (RSD ~ ±1.1%). Even with a high concentration of Na+and K+(28 and 36 mM, respectively) ions, the sensor showed a minute enhancement of RSD (~ ±6.8%). FIG. 22D depicts a visual comparison of the sensor responses to each interference, along with their corresponding error bars. These results demonstrate the exceptional ability of the sensor to selectively detect calcium ions even when solutions containing extremely high concentrations of other ions are present. The high selectivity of the sensor is due to the lipophilic ionophore embedded in the membrane along with the hydrophobic POT layer.

[0268] Furthermore, to validate the sensor specificity in complex real samples, an experiment has been designed to measure spiked Ca2+in the fetal bovine serum (FBS) samples. To accomplish this, the sensor was tested in an undiluted FBS solution, followed by dilution with DI water at ratios of 1 :10, 1 :100, and 1 :200, to measure Ca2+. These diluted concentrations of FBS were spiked with 2 mM of Ca2+concentration. As illustrated in FIGS. 22B and 22E and 5e, the sensor effectively identified the presence of calcium ions within the FBS matrix. Upon dilution with DI water, the OCP signal of the sensor decreased because of the reduction in the Ca2+concentration. In subsequent experiments, the concentration of Ca2+was maintained at 2 mM, and the matrix solution was changed from pure FBS (1 :0) to ratios of 1 :10 and 1 :200 of FBS to DI water. The sensor response remained unchanged owing to the constant Ca2+concentration. With the spiking analysis, these results suggested that the sensor exhibited excellent performance in serum solutions.

[0269] Furthermore, to assess the inter-sensor variability, four identical sensors were chosen to evaluate the reproducibility. In this study, the sensor test was conducted in the presence of 0.5 mM Ca2+ions. The sensor’s reproducibility was calculated by evaluating sensor-to-sensor variation. Each sensor was calibrated and subjected to three repeated measurements in independent biological experiments. The resulting standard deviation (SD) for sensors responding to the same Ca2+concentration was determined to be ±4.69 mV, with an RSD of 2.95% (FIG. 22G and 22H). This result indicates that the sensor provides high reproducibility owing to the precise additive manufacturing of sensors with high-resolution features.

[0270] Next, the detection time for both Ca and P sensors (FIG. 221) is discussed by calculating the OCP response percentage every tenth second to reach one minute. This result indicates their ability to measure target ions in less than 10 seconds. It is noted that the sensors reach their outputs of 100% (Ca-sensor) and 99% (P-sensor) signals within 10 s, compared to the sensor’s saturation signals (see FIG. 22I). The signals from the sensors were stable within the first 10 s of the measurement. Fast detection by both sensors was achieved owing to the potentiometric detection modality along with the wrinkled surfaces of the sensor geometry. This swift sensing facilitates the real-time identification of calcium and phosphorus levels on dairy farms and enables the continuous monitoring of milk composition in food industry pipelines. The swift response times of both sensors open up possibilities for various applications, including hypocalcemia treatment and best-practice management of dairy herds.

[0271] In vitro Tests with Biological Fluids. To ensure the reliability and effectiveness of the developed sensors for calcium and phosphate ion measurements in biological samples and consider their ultimate application for the analysis of milk samples. Before testing the milk samples, the performance of the sensor was tested on whole milk samples obtained from Walmart. Inc. In this regard, a whole milk sample was chosen as the stock solution for the milk sample studies. Three different dilutions (30%, 60%, and 80%) were prepared from whole milk in DI water. Next, the baseline of the sensor was set, and all milk samples were tested using a Ca-sensor. The maximum sensor output was achieved with whole milk because the concentration of Ca was high. As expected, the sensor response (OCP) decreased with the milk dilution from 30% to 80% (FIGS. 22C and 22F). The results were validated using a commercial calcium meter to measure the Ca2+content in all diluted milk samples.

[0272] The sensors were tested using fresh milk samples obtained from 15 lactating Holstein multiparous cows from the local dairy farm. As a proof-of-concept, these samples were obtained during a regular milking period rather than their transition period. The samples were stored at 4 °C. The Institutional Animal Care and Use Committee (IACUC) protocol was approved before the milk samples were obtained. Next, immediate sensing analyses of Ca2+and P ions was conducted using the developed sensors and commercial meters. For Ca2+measurement, the samples were utilized without any modification or dilution, whereas for P measurement, the samples were diluted with DI water. Detailed information on parameters such as days in milk (DIM), milk yield, and parity are described in Tables 4 and 5. The sensing results for the detection of Ca2+and P ions are shown in FIGS. 23A-23B. The recorded signals from these real samples were extracted to evaluate the unknown Ca2+and P levels using theircalibrations. Simultaneously, Ca2+and P levels were evaluated using commercial meters. Tables 4 and 5 show the sensing results for Ca2+and P levels in fresh milk samples. The converted Ca2+and P levels (ppm) from the developed sensors and commercial meters were compared as shown in FIGS. 23C and 23D, to estimate regression coefficients (r2). The correlation studies revealed r2 values of 0.90 for Ca2+and 0.95 for P ions. These rQ. values confirm the capability of the sensors to analyze both ions in milk samples, indicating the reliability of the sensors for on-site tests at the farm. Furthermore, the precision of the sensors, assessed by RSD values for the measurement of Ca2+and P ions, was found to be 8.97% and 5.53%, respectively. Furthermore, compared with the Ca2+concentration (FIG. 23E), the level of P in ppm varied significantly. To assess milk fever in dairy cows, the ratio of P to Ca2+ions was evaluated in milk samples and presented the results in parts per million (ppm), as shown in FIG. 23F. The ratio of P to Ca2+levels in ppm concentrations serves as an indicator of healthy milk fever, hypocalcemia, and hypercalcemia. Thus, in addition to serum (Fig. 23E), the ratio of P to Ca2+in milk samples was a more reliable indicator of hypocalcemia.Table 4. Milk sample analysis data for calcium ion measurements with both commercial and 3D printed sensors. Precision of the sensor (RSD) and accuracy of the sensor with average percentage of % 8.97.Table 5. Milk sample analysis data for phosphate ion measurements with both commercial and 3D printed sensors. Precision of the sensor (RSD) and accuracy of the sensor with average percentage of % 5.53. All milk samples have been diluted for phosphate analysis.

[0273] Discussion. In the US, approximately 30-50% of dairy cows are affected by metabolic disorders due to both nutritional deficiencies and inaccurate management. Hypocalcemia in dairy cows especially causes huge economic losses due to decreased milk yield. The Nutritional management of dairy cows is thus important to improve health and milk productivity. Ca plays a vital role in muscle and nerve function, supporting skeletal muscle strength, and immune function, laboratory screening cost is $60 per sample, and the availablecommercial on-site testing cost is about $1000. As the prevalence of subclinical and clinical hypocalcemia is approximately 33% and 5%, respectively, in the US dairy herds, there is an unmet requirement to develop diagnostic tools, such as smart and low-cost, and user-friendly sensors, to produce large-scale data to support the best management practices. Early diagnosis may prevent or treat diseases; however, it relies on rapid, economically efficient, farm-side, 550 and accurate diagnostic assessment of calcium and phosphate levels.

[0274] Unlike clinical hypocalcemia, subclinical hypocalcemia in dairy cows after calving does not cause any symptoms. Hence, it is difficult to measure at the early stage. Though several sensors and spectroscopic tests have been explored to monitor Ca2+and P, such tests are not feasible or widely adopted by dairy farmers due to the labor-intensive sample processing and long testing time. It is important to note that not all commercial meters are useful for identifying hypocalcemia in dairy farms using milk samples. One simplest way is to estimate the Ca2+ / P ratio, which is challenging because they are bound together while temperature and pH vary in the laboratory environment. In addition, unlike the blood Ca2+, detection of subclinical hypocalcemia by measuring milk Ca2+in dairy cows is challenging as milk contains huge amounts of Ca2+that make the negligible Ca2+decline, resulting in an inaccurate diagnosis of hypocalcemia. Thus, measuring the ratio of milk Ca2+to P would solve this issue.

[0275] In this study, lithography-free and low-cost 3D printed sensors were developed by additive manufacturing to analyze instant samples without any sample preprocessing and treatment. From a manufacturing perspective, the extrusion printing of sensors allows easy manufacturing, miniaturization, customization, rapid prototyping, multiplexity, and complex geometry. The additive manufacturing technique used in this study does not require cleanroom facilities, such as lithography. In addition to these features of the printed sensor, it can also provide excellent geometry with wrinkled surfaces, which improves the sensor performance. Owing to these features, the printed sensor can be used to manage dairy farms, particularly for the detection and treatment of metabolic diseases during the transition period of dairy cows. The multiplexed sensor developed in this study could simultaneously detect calcium and phosphate in milk samples at the same time. In particular, the developed sensors not only provide a low cost per test but can also detect ultralow concentrations of Ca2+ions. Furthermore, the sensor can be integrated with a milking machine to collect data in real-time to identify healthy or hypocalcemic cows. One innovation of this device is to provide economic benefits for dairy farmers by improving milk production, health management, and decision-making through the early assessment of the prevalence of clinical and subclinical hypocalcemia. This device has the potential to provide a sustainable solution to dairy farmers for the best management practices for dairy cows.

[0276] This study demonstrates the successful detection of ionic analytes at the attomolar level within seconds by enabling additive manufacturing. The 3D printed sensor base (FIG. 25A-25E) possessed a lateral pattern and wrinkled microstructure, as shown in FIGS. 27A- 27D. The unique geometry of the wrinkled surface of the sensor enhanced the sensitivity of the calcium and phosphate ion measurements. It offers a substantial surface area for the ion- to-electron (POT) solid-state layer, facilitating the detection of calcium and phosphate ions with LODs of 1.38 x 10"16M and 2.6 x 10"6M, respectively. Another important aspect of this study is the ability of the sensor to be utilized repeatedly without compromising its functionality. By employing OOP as the sensing method, electrochemical reactions were inhibited on the electrode surface, thereby preventing corrosion or leaching. This safeguard ensures that each sensor can be employed multiple times without diminishing selectivity or sensitivity. Additionally, the use of the OOP as a sensing modality contributes to the repeatability of the sensor, allowing for consistent performance across multiple applications. Additionally, the sensor’s 3D design necessitates less than 200 pL of sample for calcium and phosphate ions measurements, rendering it suitable for analyzing minute sample quantities. Finally, a compact design of the sensor, measuring 2 mm in thickness and 2 cm in width (FIG. 25B) enables seamless integration into milking machines or food industry piping through a straightforward rectangular slot with similar dimensions.

[0277] The 3D printed sensor offers several advantages over alternative approaches for measuring calcium and phosphate levels. Unlike other methods, this sensor was designed for use by unsupervised farm personnel, whereas existing techniques are primarily laboratorybased and require users to possess specific expertise. Additionally, the limited time available for diagnosing hypocalcemia necessitates prompt and regular milk analysis in transition cows. This is impractical when conventional methods are used. The cost-effective multiuse sensor, coupled with its ease of use, presents a compelling alternative, especially for remote farms where farmers may be hesitant to invest substantial amounts in acquiring existing field- deployable calcium / phosphate sensors. Moreover, the sensor design requires only a small sample size for analysis, offering convenience in situations where invasive blood sampling methods are inevitable. A mere blood droplet is ample for measurement, facilitating the early detection of diseases that require prompt investigation.

[0278] In summary, on-site monitoring of asymptomatic subclinical hypocalcemia is of great importance in the dairy industry, as it can improve the health, productivity, economy, and well-being of dairy cows. In this study, an attomole-sensitive sensor was developed that utilizes extrusion-based 3D printed sensing structures to detect hypocalcemia in milk samples, which represents a significant advancement in addressing this issue. The wrinkled structures of the 3D printed sensors enable a remarkable limit of detection for the attomole concentration of ionized calcium in milk. Unlike traditional sensors, the innovative design of the sensor featuring wrinkled POT, acts as an efficient ion-to-electron transducer for rapid calcium and phosphate detection. The ability of a multiplex sensor to provide disease indications based on the calcium-to-phosphate ratio in milk samples is a valuable diagnostic dimension. With its low cost, this sensor has emerged as a promising tool for on-site diagnostics of dairy cows, contributing to enhanced farm management. The integration of this technology has the potential to improve the overall health, productivity, and well-being of dairy cows, thereby addressing critical needs in the dairy farming industry.

[0279] Methods - Details of Sensor Manufacturing using 3D Printing. Additional Data - Chemicals section describes all the chemicals used in this study. FIG. 24A shows the overall sensor interfacing with a measurement unit i.e., a portable potentiostat, to test the milk sample. The design of the sensor used in this study is illustrated in FIGS. 25A-25E. This design was realized using the 3D CAD SolidWorks software. The sensor was 33 mm long, 25 mm wide, and 2 mm high. The device had two WEs and an RE. All electrodes are circular having diameters of 3 mm and a depth of 300 pm. The device consists of an elliptical sample holder in which all three electrodes are placed. This design allowed for a minimal amount of milk sample (ranging from 50 pL to 200 pL) to be used.

[0280] An extrusion-based high-resolution 3D printing technique called SLA was used to build the base of the sensor. A 3D sensor base was built by layer-by-layer lateral printing (layer thickness can be maintained within 25 - 300 microns) of a high-temperature polymeric resin using a 3D printer obtained from Form Labs (Form 3 L). Form 3 L is an ease-of-use SLA 3D printer with a large build volume of 11.8 x 13.2 x 7.9. It features an XY resolution of just 25 microns. 3 L is one of the only SLA machines that offers durable, flexible, and general-use resins on one platform. The 3L's wide range of material options allows it to excel when printing for various applications, from medical to mechanical.

[0281] The printing process using epoxy resin in this study is shown in FIG. 24B. 10 sensors were printed simultaneously. In one embodiment, an extrusion 3D printer was usedto build sensor structures using a high-temperature polymeric resin. A lateral layer-by-layer printing results in a periodic structure (~80 pm) of polymer. The printed structure of high- temperature resin was cured by exposing a UV light resulting in wrinkled surfaces along with a constant temperature of 80°C. These lateral layers are interconnected with each other. A photograph of the printed device is presented in FIGS. 25F and 25G. After printing, the sensor was removed from the printer tray and washed with isopropyl alcohol washing using a FormWash L unit. Subsequently, the sensor part was cured for 2 hours by exposing it to a UV light with a wavelength of 405 nm in a FormCure L system at 80°C. This curing step allowed the cross-linking of the epoxy resin molecules and formed an interconnected polymer (FIG. 24B). Herein is discussed the formation of wrinkled surfaces on the top layer of the polymer during this curing step.

[0282] After printing, a thin layer of gold was deposited onto the sensor using an electronbeam evaporator, following a specific pattern created by a shadow mask (FIG. 24C). A 2D design of a shadow mask was created using Auto CAD software. The mask was built using Kapton tape, which was cut according to the 2D sensor design using an automated silhouette cutter. Once the shadow mask was placed, a 10 nm thick chromium layer was applied as an adhesive layer, and then a 100 nm thick gold layer was deposited onto the sensor using an e- beam evaporator. This gold layer functioned as a conductive layer on the wrinkled surface of the sensor.

[0283] Next, a solid-contact layer of POT was applied by drop-casting onto the gold surface of the WE (FIG. 24D). In 24D, two of the three electrodes serve as WEs for calcium and phosphate detectors and one electrode is shared RE made of Ag / AgCI and Nation™ layers. Because of its high redox and hydrophobic properties, the POT layer serves as an ion- to-electron transducing layer. After coating the WE with POT layers, they are then coating with either a Ca-ISM solution or a P-ISM solution. The solid-state RE was modified by applying a silver-silver chloride (Ag / AgCI) paste (~5pm) along with Nation™ on the middle gold electrode to serve as an anti-leaching layer (FIG. 24D). FIGS. 32A-32D show the optimization of the POT layer. Three different amounts of POT, such as 1.1 pg / mm2, 2.2 pg / mm2, and 3.6 pg / mm2were chosen for this study and calibrations were established. The sensitivity of each sensor to these POT polymer concentrations was identical. Based on these results, a thin POT layer was selected for the development of the 3D sensor.

[0284] For the Ca sensor, a Ca2+-ISM was prepared. The Ca2+-ISM consisted of 1 wt% of the Ca2+selective ligand, 65 wt% of the plasticizer, 1 wt% of the additive, and 33 wt% of poly(vinyl chloride) powder.66This cocktail solution of 40 pL was drop-casted onto the POT layer. The ionophores in the Ca2+-ISM trap Ca2+from the samples via an ion exchange mechanism and develop a potential difference at the membrane-solid interface. For the P-sensor, the P- ISM was prepared with a mixture of 3.0 mg (1 wt%) of tri butyltin chloride, 198 mg (66 wt%) of NPOE, 99 mg (33 wt %) of PVC, and 2.0 mg of NaTFPB in 3 mL of tetrahydrofuran. This cocktail will be coated on the POT surface to develop the P-sensor. The printed sensor, shown in FIG. 24E was connected to a portable potentiostat to collect the readout signal via a Bluetooth network. Before testing the sensor with real samples, a commercial Ca meter was tested, its performance was evaluated with Ca standard solutions, and the results were compared with theoretical Ca2+concentrations (FIGS. 31A-31C). The calibration process involved using standard calcium ion solutions ranging from 10 mM to 1M in concentration. The commercial sensor exhibits two recommended product solutions for calibration at 150 ppm and 2000 ppm concentrations (depicted in the longer dotted line of FIG. 31 B). Experimentation involved testing the sensor with calcium ion standard solutions at concentrations of 80, 120, 160, 190, and 230 ppm (illustrated in the shorter dotted line of FIG. 31 B). These concentrations were strategically chosen to encompass the calcium concentration range found in cow's milk or blood samples. The meter in FIG. 31 C tends to indicate higher calcium ion concentrations than the actual values, particularly in the low concentration range using for milk sample testing.

[0285] Methods - Electrode Conditioning. The conditioning of the electrodes is a crucial initial stage to ensure reliable outcomes. Thus, the developed sensors were conditioned by treating them in a concentrated solution of CaCh and K2HPO4 for several hours (more than 12 hours). This process aims to replace all the interfering ions in the membrane with the desired ions. Prior to the calibration, selectivity tests, and analysis of real samples, both sensors were conditioned to achieve a stable and reliable response.

[0286] Methods - Sensing Mechanism. The POT layer, which serves as an ion-to- electron transduction layer, undergoes redox reactions during sensing. The cation exchange process through the POT layer was elucidated through cyclic voltammetry (CV) experiments conducted on an ion-selective membrane with a POT electrode. The process for extracting cations (specifically Ca2+) into the Ca-ISM through electrochemical mediation consists of three sequential steps. These steps include (1) Reduction of POT+to POT: In this initial step, the cation exchange layer, often referred to as PO (also known as P+), is reduced to its neutral form, known as POT (or P). (2) Extraction of hydrophilic ions (Ca2+): Following the reduction of POP, this step involves the facilitation of the extraction of the hydrophilic ion, in this caseCa2+, from the test sample. (3) Redistribution of lipophilic anion (R-): The final step involves the redistribution of the lipophilic anion (represented as R") from the POT layer to the ISM layer. These three consecutive stages collectively describe the mechanism of electrochemically mediated cation (Ca2+) extraction into the ISM. The redox reactions that occur concurrently with the transfer of Ca2+in the ISM are as follows:P(p) + e“ + R(p)+ Ca2a+q)P + R(m)+ Ca^ (eq. 2)

[0287] where m, p, and aq represent the membrane (ISM), ion-to-electron transducer (POT), and aqueous phases (cow milk), respectively. In addition, P<P) and P(P)+correspond to a small number of individual building blocks within the POT chain in two states: an electrically neutral insulating state and an oxidized state with polaronic sites.

[0288] Due to the reduction process, POT removes cations in the form of Ca2+(aq) from the sample and causes the movement of lipophilic anions (R") from POT into the ISM layer. Consequently, when the redox and ion-exchange processes at the WE are combined, an equilibrium is reached at the interfaces between the aqueous solution, transducer, and ISM. This equilibrium results in the separation of charges at each interface, leading to the creation of a phase boundary potential.70 The phase-boundary potential Ei is determined using the following equation:Inaj (eq. 3)

[0289] where R, T, z, F, and ai represent the gas constant, temperature, charge of the target ion, Faraday constant, and primary ion activity without interfering ions, respectively. In addition, the RE of the sensor underwent a redox reaction while maintaining a constant potential (Eo). A Nation™ layer is applied to the surface of Ag / AgCI, serving the dual purpose of preventing the leaching of chloride ions from Ag / AgCI and blocking other external anions from entering the RE. When anions or cations move from high to low concentrations, the ion exchange generates a potential difference. Consequently, the potential I is dependent on the logarithm of ion activity, as described by the Nernst equation:E = Eo+ E-L = Eo+ — In aj (eq. 4)

[0290] The anion exchange process in the POT layer follows a mechanism similar to that of cation exchange but in the opposite direction. The exchange of PO42" ions also involves a similar three-step mechanism, but it is based on the oxidation of POT to PO as described in eq. 5.

[0291] where m, p, and aq represent the membrane (ISM), ion-to-electron transducer (POT) phase, and aqueous phases (milk samples), respectively.

[0292] Methods - Software. OriginPro 2023b and GraphPad Prism 6.02 were used for statistical analysis. The 3D CAD SolidWorks 2020 and AutoCAD software were used to create the 3D design for 3D printing. Software associated with the extrusion SLA printer with different control managers was used to control the 3D printing of the polymer. Biorender 2023 software was used to generate the 3D schematics.

[0293] Additional Data - Chemicals. Poly(3-octylthiophene-2,5-diyl) (POT), tetra hydrofuran (THF), polyvinyl chloride (PVC), sodium tetrakis[3,5- bis(trifluoromethyl)phenyl] borate, dioctyl sebacate (DOS), tributyltin chloride, 2-Nitrophenyl octyl ether (NPOE), calcium ionophore II, and Nation™ were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium phosphate dibasic (Na2HPO4), calcium chloride (CaCI2), sodium chloride (NaCI), potassium chloride (KCI), Ag / AgCI ink (composed of finely dispersed chloritized silver flakes) were obtained from Fisher Scientific, Waltham, MA, USA. Deionized (DI) water having a resistivity of 18.2 MQ.cm was obtained using a purification system from Millipore, US. The cocktail of the calcium-ion selective membrane (Ca-ISM) is composed of 33% polyvinyl chloride (PVC), 0.05% sodium tetrakis[3,5- bis(trifluoromethyl)phenyl] borate, 65% dioctyl sebacate (DOS) and 1% calcium ionophore II mixed with 660 pl of tetrahydrofuran per 100 mg of the weighted mixture. This solution was sealed and stored at -4 °C. The cocktail of the phosphate-ISM is composed of 1% tributyltin chloride, 66% NPOE (2-nitrophenyl octyl ether), 33% polyvinyl chloride (PVC), 1% Sodium tetrakis[3,5 bis(trifluoromethyl)phenyl] borate 660 pl of tetrahydrofuran per 100 mg of the weighted mixture. This solution was sealed and stored at -4 °C.

[0294] The high-temperature resin cartridge (V2) was bought from Dynamism, Inc., Chicago, IL, and is used to manufacture the sensor base.

[0295] Additional Data - Ion Selectivity Evaluation. To evaluate the selectivity using the Nikolskii-Eisenman formalism, the logarithmic component in the following equation can be replaced with a sum of activities adjusted by selectivity factors, as outlined in the following equation.

[0296] whererepresents the selectivity coefficient, ai and aj stand for the activities of ions I and J in the test solution, and Zi and Zj denote the charges of the primary and interfering ions, respectively.

[0297] Additional Data - Calculation of LOD. The limit-of-detection (LoD) is calculated in three steps.

[0298] where ob, Oi, x, xb, c, m, LoB, and YLOD are standard deviation of blank sample, standard deviation at low concentration, sample mean, blank mean, intercept of the calibration equation, slope of the sensor calibration, Limit of Blank, and LoD of the signal, respectively.

[0299] For Ca-sensor, the calculation based on the three step LoD equations are: xb= -0.1937 V, Gb= 0.0015 [n, replicate = 3]LoB = -0.1937 + 1.645 x 0.0015 = - 0.1912 V

[0300] Mean signal at the lowest concentration (1 aM of calcium ion) is 0.0895 V, with a standard deviation of 3.87 x 10-4V for [n, replicate = 3],YLOD -0.1912 + 1.645 x 3.87 10‘4= - 0.1905 V

[0301] The Ca-sensor calibration equation is YLOD (V) = 0.0243 x log [X (mM)] + 0.122. Hence, the LoD of the Ca-sensor in calculated to be 10'12 86mM or 138 aM of Ca2+ions.

[0302] For P-sensor, the calculation based on the three-step LoD equations are: xb= 0.358 V, ob= 0.00116 [zz, replicate = 3]LoB = 0.358 + 1.645 x 0.00116 = 0.3599 V

[0303] Mean signal at the lowest concentration (10 pM of phosphate ion) is 0.335 V, with a standard deviation of 5.1 x 10-3V for [n, replicate = 3],YLoD■■■■■■ 0.3599 1.645 x 5.1 x It)- - 0.3582 V

[0304] For P-sensor, the calibration equation is YLOD (V) = 0.152 x log [X (mM)] + 0.341. Hence, the LoD of the calcium sensor in calculated to be 2.6 pM of P or PC>42“ ions.

[0305] Additional Data - Instruments. The morphology of sensor microstructures was scanned with a scanning electron microscopy (SEM, JEOL IT500 SEM). The elemental analysis (i.e. EDS) and mapping analysis of the sensor’ materials were conducted using the Oxford Aztec EDS detector integrated into the SEM instrument. A 3D Solid work CAD design software was employed to generate a 3D design and schematic of the device depicted in FIGS. 25A-25E. The VersaSTAT 4 Potentiostat / Galvanostat from Princeton Applied Research-Ameteck, with VersaStudio software, was utilized to record and analyze Cyclic Voltammetry (CV), Electrochemical Impedance Spectroscopy (EIS), and Open Circuit Potentiometry (OCP) signals. For applications beyond the laboratory, the OCP was recorded using the portable EmStat3 Blue (PalmSens) Potentiostat / Galvanostat. During EIS measurements, the AC signal had a 1 mV amplitude and a frequency range of 10,000-1 Hz. All electrochemical analyses were conducted at room temperature.

[0306] Chromium and gold layers were deposited on the high-temperature resin sensor base substrates using an e-beam evaporator (PVD 250).

[0307] Lithography-free, 3D sensor was printed using an extrusion-based Form 3L 3D printer (FormLabs), followed by IPA washing with Form Wash L (FormLabs) and curing with UV light using Form Cure L (FormLabs). The AutoCAD software (AutoCAD 2022; Autodesk Inc.) was employed to design the 2D sensor and create CAD programming file to shadow mask. A Kapton tape-based shadow mask for the device was created using an automated cutter from Silhouette Curio™, Silhouette America®, Inc. Graphs and statistical analysis were generated using OriginPro 2023b and Graphpad prism software. All the graphical illustrations were generated using Biorender 2023 software.

[0308] Additional Analyses. The morphological investigation of the sensor geometry was conducted by SEM studies. The SEM images depicting POT-Au@3D Sensor (FIGS. 26A-26B) reveal the presence of a POT layer covering the gold-coated sensor base. This is a partial coverage to demonstrate the surface morphology. This coverage elucidates the observed decline in electrical conductivity as indicated in the cyclic voltammogram (FIG. 19F). The POT layer is responsible for this decrease the sensor conductivity due to the insulating nature of it. However, the POT layer is ideal to build a solid-state type of potentiometric sensor as thesensor doesn’t flow current through it except develops the membrane potential. The coverage of POT was enhanced by increasing its concentration to develop sensors.

[0309] FIGS. 26C-26D show the SEM images after coating ion-selective membrane on POT-Au layer. SEM images a seamlessly uniform surface of ISM, indicating that the membrane envelops the entire sensor surface along with the underlying micro-structures.

[0310] SEM analysis was conducted to assess the variation of sensor geometry featuring lateral patterns. Ten random points were chosen for measuring the thickness of the lateral layers, selected from various locations on both distinct and identical lateral structures. The width of average lateral layer was -78.065 pm, accompanied by a standard deviation (SD) of ± 2.518 pm (Table 2). This low deviation serves as a measure of the printer's accuracy in replicating micro-structures during the printing process. It is important to note that the combination of the sensor base's wrinkled structure and the lateral pattern micro-structure serves to amplify the sensor's surface area, thereby enhancing the analytical sensitivity of the sensor.

[0311] EDS technique was used to analyze the elemental composition of materials on each layer of the sensor. In EDS analysis, a sample is bombarded with electrons, causing it to emit X- rays characteristic of its elemental composition. These X-rays are then detected and used to identify the elements present in the sample and quantify their concentrations. We conducted EDS analysis on every layer of the sensor, namely Au, POT-Au, and ISM-POT-Au. The EDS graphs depicting the results can be found in FIGS. 28A, 28B, and 28C. The observed reduction in gold's surface weight percentage and the corresponding increase in carbon's surface weight percentage provide confirmation of the effective coverage of POT and ISM layers over the gold layer. Considering the reported electron beam penetration depth of approximately 10 pm at a potential of 20.0 kV on the sample surface, and it can be inferred that the thickness of each POT and membrane layer exceeds 10 pm.

[0312] The mapping results of the POT-Au@3D sensor (FIG. 29A) validate the existence of sulfur and carbon atop the gold layer, corresponding to poly(3-octyl-thiophene) (POT), which solely comprises carbon and sulfur. Additionally, the predominant constituent of the membrane is poly-vinyl chloride (PVC), identified through the mapping analysis of ISM-POT- Au@3D in FIG. 29B, revealing the presence of carbon and chloride.

[0313] XPS was used to investigate the elemental composition and chemical state of materials. XPS provides detailed information about the outermost atomic layers (up to 1-10nanometers) of each layer of the sensor. By analyzing the binding energies, XPS can identify the elements present on the surface of a material and provide information about their chemical bonding environments. In this regard, an XPS survey analysis was performed on 3D printed sensor base made of epoxy resin and the gold coated layer of the sensor. As indicated FIG. 30A, the XPS survey shows peaks characteristics of O1s, C1s, Si2s, Si2p, and 02s demonstrating the presence of oxygen, carbon, and silicon on the surface of freshly printed and cured epoxy resin 3D printed base. Carbon and oxygen are expected as these are the main constituent of epoxy resin, however, the presence of silicon is the minimal contamination of the sensor surface during the curing and washing process. FIG. 30B also indicates the XPS survey results on gold coated 3D sensor base. As indicated in this figure, the main element identified on this surface is gold along with carbon and oxygen coming from negligible organic contamination on the surface.

[0314] The deconvoluted XPS result for the C1s peak in FIG. 30C reveals distinct contributions from different carbon chemical states on the surface of 3D printed base. Three major peaks are identified at specific binding energies: the peak centered at 284.8 eV corresponds to C-C bonds, indicative of carbon-carbon interactions. The peak at 286.2 eV is attributed to carbon-oxygen (C-0) bonds, suggesting the presence of functional groups in epoxy resin. Furthermore, the peak at 289.0 eV is associated with the 0-0=0 configuration, characteristic of carbonyl groups.

[0315] Additionally in FIG. 30D, slow XPS scan result for the 01s peak is shown. Three distinct peaks are observed at binding energies of 530.9 eV, 532 eV, and 529.1 eV. The peak at 530.9 eV is attributed to oxygen-carbon (0-C) bonds, indicating the presence of carbonoxygen functionalities in the material. The peak at 532 eV signifies oxygen-hydrogen (0-H) bonds, indicative of hydroxyl groups on the surface. Lastly, the peak at 529.1 eV is associated with oxygen double-bonded to carbon (0=0), suggesting the presence of carbonyl groups.

[0316] Finally, high-resolution XPS scan conducted on a gold-coated 3D printed sensor base has revealed distinct peaks corresponding to the Au4f7 / 2 and Au4f5 / 2 binding energies, demonstrating the gold coating in its atomic state (FIG. 30E). The appearance of two satellite peaks at higher binding energies for each of these main peaks further enriches the spectral information obtained. In XPS analysis, the satellite effect is a phenomenon observed as additional peaks adjacent to the main photoelectron peaks, resulting from the relaxation process of the core hole created during the photoemission.

[0317] To enhance comprehension and comparison of the proposed sensor with a commercial calcium ion sensor, a calibration of the commercial calcium meter was performed using standard solutions ranging from 10 mM to 1 M. The calibration equation revealed that the commercial meter adheres to the Nernst equation, as indicated by a slope of +29.78 mV / dec matching the 0.0592 / ZA (V / dec) term in the Nernst equation, where ZA represents the number of electrons in the electrochemical half reaction. Subsequently, the meter's response to standard calcium solutions was examined, following the company's recommendation to calibrate the meter using commercial solutions with concentrations of 150 ppm and 2000 ppm. In FIG. 31 B, the sensor calibration is presented using provided standard solutions and showcases the sensor's response to standard solutions with concentrations of 80, 120, 160, 190, and 230 ppm. This concentration range was strategically chosen to encompass the calcium concentration found in cow’s milk samples. The calibration graphs highlight a disparity in the response of the commercial sensors, particularly in the low concentration region where the calcium ion concentration in milk samples exists. The commercial meter was also subjected to testing with standard solutions, and its response in calcium ion detection is illustrated in FIG. 31C.

[0318] In this Example, the POT or poly(3-octyl-thiophene) was utilized as a sensor material. The amount of POT concentration on the sensor surface was optimized. It is a conducting polymer acted as an ion-to-electron transducer layer and this layer converts signals between ions and electrons in this device. To investigate the impact of POT concentration on the proposed sensor’s sensing capabilities, three identical Ca-sensors were constructed with varying amounts of POT layer and established calibration curves for each sensor (FIGS. 32A-32C). Three different amounts of POT such as 1.1 pg / mm2, 2.2 pg / mm2 and 3.6 pg / mm2 were chosen for this study. This exploration aimed to understand the influence of POT on sensor performance and potentially optimize the POT concentration. Illustrated in FIGS. 32A, 32B, and 32C, the calibration graphs exhibit nearly identical slopes (sensitivity), indicating that the transducer amount does not affect sensing performance. Interestingly, the superior sensing performance and lower detection limit of the sensor are attributed to the increased surface area of the wrinkled 3D printed sensor base, as evidenced. Based on these results, a thin layer of POT layer covering the sensor surface proves sufficient for providing high sensor performance.

[0319] It will be apparent to those skilled in the art that various modifications and variations can be made in the present disclosure without departing from the scope or spirit of thedisclosure. Other aspects of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the disclosure being indicated by the following claims.

Claims

ClaimsTherefore, at least the following is claimed:

1. A potentiometric sensor comprising: a base; a working electrode (WE) set into the base, the working electrode comprising a WE well at a first distal end, the WE well filled with a WE solid-state material; and a reference electrode (RE) set into the base adjacent to the working electrode, the reference electrode comprising a RE well at the first distal end adjacent to the WE well, the RE well filled with a RE solid-state material.

2. The potentiometric sensor of claim 1, further comprising a base well extending from an outer surface of the base to an inner surface of the base well at a depth of about 0.2 pm to about 0.4 pm, the base well surrounding the WE well and the RE well, wherein the inner surface of the base well is flush with a surface of the WE well solid- state material and the RE well solid-state material.

3. The potentiometric sensor of claim 1 , wherein the WE well is connected via a first channel to a first pad at a second distal end and wherein the RE well is connected via a second channel to a second pad at the second distal end adjacent to the first pad.

4. The potentiometric sensor of claim 1 , wherein the WE well is circular and has a diameter of about 5mm to about 11 mm.

5. The potentiometric sensor of claim 1, wherein the RE well is circular and has a diameter of about 1 mm to about 5 mm.

6. The potentiometric sensor of claim 1, wherein the WE solid-state material and RE solid-state material each comprise a plurality of layers of material.

7. The potentiometric sensor of claim 6, wherein the WE plurality of layers of material comprise: a first layer material, wherein the first layer material comprises chromium or titanium; a first patterned gold layer superimposed on the first layer material; a poly(3-octylthiophene-2,5-diyl) (POT) layer superimposed on the patterned gold layer; and an ion-selective membrane superimposed on the POT layer.

8. The potentiometric sensor of claim 7, wherein the ion-selective membrane comprises about 33 wt% polyvinyl chloride, about 0.05 wt% sodium tetrakis [3,5- bis(trifluoromethyl)phenyl]borate, about 65 wt% plasticizer dioctyl sebacate, and about 1 wt% of a calcium ionophore II.

9. The potentiometric sensor of claim 7, wherein the ion-selective membrane comprises about 1 wt% tributyltin chloride, about 66 wt% 2-nitrophenyl octyl ether, and about 33 wt% polyvinyl chloride.

10. The potentiometric sensor of claim 7, wherein the ion-selective membrane comprises about 6.7 wt% 1,10- phenanthroline, about 3.3 wt% potassium tetrakis chlorophenyl borate, about 60 wt% dibutylphthalate, and about 30 wt% polyvinyl chloride.

11. The potentiometric sensor of claim 7, wherein the ion-selective membrane comprises about 0.3 wt% methyltriphenylphosphonium bromide, about 1.9 wt% nitrocellulose, about 16.3% 2-nitrophenyl octyl ether, about 5.8 wt% polyvinyl chloride, about 74.3 wt% THF, and about 1.5% tridodecylmethylammonium nitrate.

12. The potentiometric sensor of claim 6, wherein the RE layers of material comprise: a second layer material, wherein the second layer material comprises chromium or titanium; a second patterned gold layer superimposed on the second layer material;a silver / silver-chloride layer superimposed on the second patterned gold layer; and an anti-leaching membrane layer superimposed on the silver / silver-chloride layer.

13. The potentiometric sensor of claim 12, wherein the anti-leaching membrane comprises a polytetrafluoroethylene copolymer.

14. The potentiometric sensor of claim 3, further comprising a second WE set into the base adjacent to at least one of the WE or the RE, wherein the second working electrode comprises a second WE well at the first distal end adjacent to at least one of the WE well or the RE well, the second WE well filled with a second WE solid-state material, and wherein the second WE well is connected via a third channel to a third pad at the second distal end adjacent to at least one of the first pad or the second pad.

15. The potentiometric sensor of claim 14, wherein the second WE well is connected via a third channel to a third pad at the second distal end adjacent to at least one of the first pad or the second pad.

16. The potentiometric sensor of claim 14, wherein the second WE well is circular and has a diameter of about 5mm to about 11mm.

17. The potentiometric sensor of claim 14, wherein the second WE solid-state material comprises a plurality of layers of material.

18. The potentiometric sensor of claim 17, wherein the second WE plurality of layers of material comprise: a third layer material, wherein the third layer material comprises chromium or titanium; a third patterned gold layer superimposed on third first layer material; a second poly(3-octylthiophene-2,5-diyl) (POT) layer superimposed on the third patterned gold layer; and a second ion-selective membrane superimposed on the second POT layer.

19. A system comprising: the potentiometric sensor of claim 2; and a microfluidic device in fluid communication with the potentiometric sensor, comprising: a reservoir configured to be in contact with the base well of the potentiometric sensor; an inlet, configured to receive a fluid sample and be in fluid communication with the reservoir; and a peristaltic pump, configured to pump the fluid sample from the inlet to the reservoir.

20. A method for measuring a concentration of ions in a sample using the potentiometric sensor of claim 1, comprising: obtaining a fluid sample; bringing the fluid sample into contact with both the WE well and the RE well; and measuring the open circuit potential between the working electrode and the reference electrode.

Images