Lab-on-a-contact lens for non-invasive monitoring

Abstract

The present disclosure generally relates to smart contact lenses for non-invasive monitoring of biological markers of health and disease. In some aspects, contact lenses comprising an electrochemical sensor between two deformable polymer lenses are provided. In some embodiments, the electrochemical sensor comprises a working electrode, a counter electrode, and a reference electrode. The working electrode, in some embodiments, further comprises an electrochemically reactive compound, that when oxidized, oxidizes a target analyte in a tear fluid. Other aspects of the disclosure relate to methods of using said contact lenses. For example, in some embodiments, the methods relate to determining a concentration of the target analyte in tear fluid. In some embodiments, the target analyte is serotonin. Other embodiments are generally directed to methods of making or using the contact lenses, kits involving said contact lenses, or the like.

Description

[0001] LAB-ON-A-CONTACT LENS FOR NON-INVASIVE MONITORING

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 570,706, filed March 27, 2024, entitled “Lab-on-a-Contact Lens for Non-Invasive Monitoring,” by Zhu, et al., incorporated herein by reference in its entirety.

[0004] BACKGROUND

[0005] Tears are a complex mixture of sugars, carbohydrates, lipids, mucins, water and salts, and contain more than 1500 different proteins. Many of these compounds are known diagnostic biomarkers of disease. For example, tears are known to comprise glucose, which is known to play a key role in diabetes. Other key biomarkers include cholesterol, serotonin, and cortisol, which are implicated in heart disease, mental health disorders, and Cushing’s Syndrome, respectively. As such, tear fluid is an attractive alternative to standard biological fluids, such as blood or scrum, for monitoring specific markers commonly used to assess the risk or presence of disease. The most common strategy for tear fluid analysis consists of tear fluid collection (e.g., using either Schirmer tear strips or capillary tubes) and subsequently analysis using a combination of optical spectroscopy methods (e.g., absorption, fluorescence, scattering), separation methods (e.g., high pressure liquid chromatography (HPLC), ultra performance liquid chromatography (UPLC), SDS-PAGE), and mass spectroscopy. However, these methods do not permit real-time and on-demand monitoring, which for some diseases, is critical to the survival of the subject. Thus, improvements are needed.

[0006] SUMMARY

[0007] The subject matter of the present disclosure involves, in some cases, interrelated products, alternative solutions to a particular problem, and / or a plurality of different uses of one or more systems and / or articles.

[0008] Aspects of the present disclosure relate to a contact lens, for example, configured to measure a concentration of one or more analyte in a tear’ fluid. In some embodiments, the contact lens comprises a first polymer lens, a second polymer lens. In some embodiments, the second polymer lens defines a hole. In some embodiments, the second polymer lens is in contact with the first polymer lens, and an electrochemical sensor in fluidic communication with the hole in the second polymer lens. In some embodiments, a contact lens as disclosed herein comprises a first polymer lens, a second polymer lens defining a hole in contact with the first polymer lens, and an electrochemically reactive compound in fluidic communication with the hole.

[0009] In some embodiments, a contact lens comprises a first polymer lens, a second polymer lens in contact with the first polymer lens, and a working electrode, a reference electrode, and a counter electrode. In some embodiments, the electrodes are positioned between the first polymer lens and the second polymer lens. In certain embodiments, the working electrode comprises ferrocene.

[0010] Additionally, some embodiments, also relate to a deformable contact lens comprising an electrochemical sensor, wherein the contact lens has an elastic modulus of between 0.1 MPa and 1.5 MPa

[0011] Other aspects of the present disclosure relate to one or more methods. In some embodiments, the methods comprise exposing a contact lens to fluid produced by an eye of a subject, wherein the fluid comprises an analyte. In some embodiments, the methods comprise electrochemically reacting the analyte with an electrochemical compound (e.g., ferrocene). In some embodiments, the methods relate to determining a current produced from the electrochemical reaction.

[0012] In some embodiments, the one or more methods relate to determining a concentration of an analyte within a fluid within an eye of a subject by determining an electrical current of an electrochemical sensor within a contact lens on the eye of the subject.

[0013] In some embodiments, the one or more methods relate to determining a concentration of serotonin from a fluid within an eye of a subject, using a contact lens, by measuring a current produced from a reaction of ferrocene contained within the contact lens. Several methods are disclosed herein of administering a subject with a compound for prevention or treatment of a particular condition. It is to be understood that in each such aspect of the disclosure, the disclosure specifically includes, also, the compound for use in the treatment or prevention of that particular condition, as well as use of the compound for the manufacture of a medicament for the treatment or prevention of that particular condition.

[0014] In another aspect, the present disclosure encompasses methods of making one or more of the embodiments described herein, for example, a contact lens. In still another aspect, the present disclosure encompasses methods of using one or more of the embodiments described herein, for example, a contact lens.

[0015] Other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments of the disclosure when considered in conjunction with the accompanying figures.

[0016] BRIEF DESCRIPTION OF DRAWINGS

[0017] Non-limiting embodiments of the present disclosure will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the disclosure shown where illustration is not necessary to allow those of ordinary skill in the art to understand the disclosure. In the figures:

[0018] FIGs. 1A-1F show the structural design, device fabrication, and operation principle of the LoCL system, according to some embodiments. FIG. 1A shows a schematic representation of the LoCL system, featuring a stretchable and reversible electrochemical biosensor integrated seamlessly for the non-invasive monitoring of serotonin dynamics, according to some embodiments. FIG. IB shows a photographic depiction of the LoCL system, showcasing a closeup of the intricately printed serpentine pattern that establishes an interconnection with the serotonin biosensing interface, according to some embodiments. The scale bars are 50 mm in white and 0.25 mm in black. FIG. 1C shows a detailed schematic outlining the fabrication protocol for the LoCL system, according to some embodiments. FIG. ID shows the surface topology of the serotonin biosensing interface, showing the surface roughness (thickness) profiles of the counter electrode (CE), reference electrode (RE), and the surface-functionalized working electrode (WE) , according to some embodiments. FIG. IE shows a schematic illustration of the serotonin pathway, including its production in the brain and subsequent release through tear glands, according to some embodiments. FIG. IF shows illustrations of the capture and monitoring of serotonin released in tears via the LoCL system, according to some embodiments. FIGs. 2A-2E show surface engineering and biocompatibility evaluation, according to some embodiments. FIG. 2A shows X-ray photoclcctron spectroscopy (XPS) analysis of the LoCL system at various stages of surface engineering: (i) the original, unfunctionalized LoCL, (ii) TPSA-modified LoCL, (iii) amine / TPSA-modified LoCL, and (iv) ferrocene / amine / TPSA- modified LoCL, representing the fully functionalized system, according to some embodiments. FIG. 2B shows a top-view microscopic image of the final serotonin biosensing interface (Scale bar: 1 mm) , according to some embodiments. FIG. 2C shows fluorescence images of cell cultures after a 7-day incubation with both the unfunctionalized LoCL and functionalized LoCL, according to some embodiments. The images show live cells in green and dead cells in red. FIG. 2D shows quantification of cell viability, showing the percentage of live cells in relation to the total cell count, according to some embodiments. FIG. 2E shows the metabolic activity of the cells cultured with LoCL over 1 day and 7 days, according to some embodiments. The data are presented as mean values ± standard deviations for each group (n = 4). Statistical significance was tested using two-way ANOVA, p-values less than 0.0001 (****p<0.0001), and ns indicates no significant differences.

[0019] FIGs. 3A-3E show mechanics analysis under different loading scenarios, according to some embodiments. Finite element analysis (FEA) of the maximum principal strain distribution in the LoCL system under five typical loading conditions is shown: (FIG. 3A) flipping, (FIG. 3B) folding, (FIG. 3C) expanding, (FIG. 3D) stretching, and (FIG. 3E) twisting, according to some embodiments. For each condition, the first column shows optical images of the LoCL under the specific loading condition. The second and third columns provide the respective strain distributions in the electrodes and the contact lens. The third and fourth columns depict the maximum principal strain distribution on both the substrate and the electrode layer. Scale bar: 1 cm.

[0020] FIGs. 4A-4L show in vitro quantitative assessment, according to some embodiments. FIG. 4A illustrates the linear relationship between anodic peak currents (Ipa) and cathodic peak currents (Ipc) against the square root of the scan rate, derived from FIG. 22C, according to some embodiments. FIG. 4B presents a calibration curve showing Ipa variations in relation to serotonin concentrations up to 100 microM, according to some embodiments. The inset shows the system's linear response to nanomolar serotonin levels (1 nM-500 nM), which falls within the typical range found in human tears. FIG. 4C features bar graphs depicting relative current changes in response to serotonin concentrations ranging from 1 pM to 1 microM in both buffer and artificial tear fluids, according to some embodiments. FIG. 4D shows time-dependent responses in artificial tear solutions at escalating serotonin concentrations, according to some embodiments. FIG. 4E provides a real-time depiction of relative current changes at various serotonin concentrations (500 pM - 500 nM) in artificial tears, according to some embodiments. FIG. 4F demonstrates the LoCL system's specificity by comparing responses to serotonin with those to non-target molecules (urea, uric acid [UA], dopamine [DA], ascorbic acid [AA], and artificial tear fluids [AT]), each at a concentration of 50 microM, according to some embodiments. FIG. 4G exhibits the system's responses to various serotonin concentrations across different pH ranges (6 to 8), with each data point representing the average of three samples and error bars indicating standard deviations, according to some embodiments. FIG. 4H examines long-term stability over a 14-day period by monitoring artificial tear solutions with different serotonin concentrations (0 nM, 100 nM, 500 nM, and 1 microM), according to some embodiments. FIG. 41 analyzes mechanical durability under bending conditions (radius of curvature: 6 mm), demonstrating stable serotonin monitoring across 1,000 bending cycles, with consistent responses to 1 microM serotonin; error bars represent standard deviations, according to some embodiments. FIG. 4 J assesses the reversibility of serotonin monitoring (10 nM) over 100 washing cycles, according to some embodiments. FIG. 4K evaluates the stability of the LoCL system's serotonin monitoring across 20 washing cycles, according to some embodiments. FIG. 4L shows the repeatability of serotonin quantification through 1,000 successive measurements using the same LoCL, indicative of the long-term reversibility, according to some embodiments. All data were collected from triplicate experiments.

[0021] FIGs. 5A-5F show an ex vivo study in human tears, according to some embodiments. FIG. 5A shows an illustration of the trier social stress test (TSST) procedure, according to some embodiments. The to denotes the reference time point corresponding to the baseline stress levels. Pointed arrows indicate the moments when tear samples were collected. Both the TSST and tear sampling were performed under ambient conditions. FIG. 5B shows characterizations of the serotonin levels during the TSST. Tear samples were collected from 10 subjects (Male: Female = 1:1), according to some embodiments. The relative responses represent changes in current compared to each individual’s prestress serotonin levels, according to some embodiments. FIG. 5C shows the measured changes in serotonin levels during the TSST. The concentrations of serotonin were calculated using the linear equation from FIG. 19D, with each data point representing the average of triplicate measurements, according to some embodiments. FIG. 5D shows non-invasive monitoring of tear serotonin levels in a subject at six distinct time points (8:00 a.m., 11:00 a.m., 2:00 p.m., 4:00 p.m., 8:00 p.m., and 10:00 p.m.) during daily activities (such as waking up, exposure to sunlight, and winding down) using the LoCL system, according to some embodiments. FIGs. 5E and 5F show investigation of serotonin levels in DED. FIG. 5E shows results of the 5-item dry eye questionnaire (DEQ-5) in both healthy subjects and patients diagnosed with DED, according to some embodiments. FIG. 5F shows that human tear serotonin levels correlate with stress levels in DED patients, according to some embodiments.

[0022] FIGs. 6A-6K show in vivo performance in a porcine model, according to some embodiments. FIG. 6A shows the protocol of in vivo validation using a pig, according to some embodiments. FIG. 6B shows a photograph of the porcine model employed in the experiment, with annotations indicating the time intervals for administering eye drops containing with or without serotonin. FIG. 6C shows a magnified view of the pig eye, as shown in FIG. 6B, according to some embodiments. FIG. 6D shows a representative photograph of the LoCL worn on the eye of a live pig, according to some embodiments. FIGs. 6E and 6F show a comparison of the infrared image of the pig eye, both with (FIG. 6E) and without (FIG. 6F) the LoCL, to assess heat generation, according to some embodiments. Scale bar: 1 cm. FIG. 6G shows serotonin concentration (measured via the LoCL) in relation to the concentrations of serotonin solutions administered into the eye. Each data point represents the average result from three samples. Representative H&E staining of pig cornea showing normal intact morphology (FIG. 6H): the sclera and iris, with pupillary sphincter (FIG. 61), the sclera and ciliary muscle (FIG. 6J), retina backed by pigmented epithelium (FIG. 6K). Scale bars: 250 pm

[0023] FIGs. 7A and 7B show the pattern design of the stretchable electrodes within the LoCL system, according to some embodiments. FIG. 7A shows a schematic representation of the stretchable LoCL platform, featuring electrodes and sensor components strategically positioned around the iris perimeter, away from the pupil, ensuring no interference with vision, according to some embodiments. The combined area of the serpentine structures connecting the counter electrode (CE), working electrode (WE), and reference electrode (RE) is approximately 11.76 mm2, 11.73 mm2, and 11.85 mm2, respectively, according to some embodiments. FIG. 7B shows the horseshoe serpentine design. The horseshoe serpentine design was employed as the stretchable interconnect between the sensor and the readout, with 1 .5 mm for external diameter (D), 1 mm for internal diameter (d), and 0.25 mm for ribbon width (W), according to some embodiments. The pitch of each horseshoe is 3 mm. The total thickness of the printed stretchable electrodes is below 50 microM, according to some embodiments.

[0024] FIGs. 8A-8D show SEM analysis on biosensor working electrode (WE) modification at each step, according to some embodiments. FIG. 8A shows unfunctionalized WE, according to some embodiments; FIG. 8B shows TPSA-modified WE, according to some embodiments; FIG. 8C shows amine / TPSA-modified WE, according to some embodiments; and FIG. 8D shows FeC / Amine / TPSA-modified WE, according to some embodiments. The SEM analysis shows that surface modifications do not affect the surface morphology of the unfunctionalized WE. The SEM images show no aggregation or crack, and each component was homogeneously printed on the WE surface within the LoCL system at each modification step.

[0025] FIG. 9 shows surface engineering of the LoCL system, according to some embodiments. High-resolution energy-dispersive X-ray (EDX) analysis demonstrates the successful surface functionalization of the LoCL system (carbon (C), oxygen (O), silicon (Si), nitrogen (N), and iron (Fe)) (Scale bars: 25 microM). A bar graph shows the intensity of element components at each step.

[0026] FIGs. 10A-10C show EDX analysis on biosensor working electrode (WE) modification at each step, according to some embodiments. High-resolution EDX surface characterization of (FIG. 10A) unfunctionalized WE, (FIG. 10B) TPSA-modified WE, and (FIG. 10C) Amine / TPSA-modified WE (Scale bar: 25 microM) is shown, according to some embodiments. The O Kai, Si Kai, N Kai, and Fe Kai originate from graphene, TPSA, amine group, and FeC molecules, respectively. The EDX analysis confirms the successful modification of the biosensing WE at each step. The black image indicates that no target element was obtained from the samples.

[0027] FIGs. 11A-11D show EDS mapping to confirm the modification process of the LoCL system, according to some embodiments. From left to right, FIGs. 11A-11D show SEM images, EDS mapping overlays of C, O, Si, N, and Fe, and EDX spectra of the elemental intensity of each sample - (FIG. 11A) unfunctionalized, (FIG. 11B) TPSA- modified, (FIG. 11C) amine / TPSA-modified, and (FIG. HD) FeC / amine / TPSA-modified LoCL (final LoCL system) , according to some embodiments. Scale bars: 25 pm. FIGs. 12A-12D show an XPS survey spectrum of each step of WE surface modification, according to some embodiments. FIG. 12A shows unfunctionalized WE, FIG. 12B shows TPSA-modified WE, according to some embodiments; FIG. 12C shows Amine / TPSA-modified WE, according to some embodiments; and FIG. 12D shows FeC / amine / TPSA-modified WE, according to some embodiments. The carbon and oxygen components are attributed to the graphene layer on the WE surface. The Si component derives from the TPSA linker, and the nitrogen originates from the amine group. Compared to the previous two steps, only the final LoCL system displayed the Fe peaks, which are attributed to the modification of the ferrocene layer.

[0028] FIGs. 13A-13E show contact angle measurement of the LoCL system, according to some embodiments. FIG. 13A shows a photograph of the bare contact lens, according to some embodiments. FIG. 13B shows a photograph of the LoCL system, according to some embodiments. Scale bar: 3 mm. FIGs. 13C and 13D show zoom-in images of the LoCL system surface (FIG. 13C) without integration of the biosensing interface and (FIG. 13D) with the integration of the biosensing interface, according to some embodiments. Scale bars: 1 mm. FIG. 13E shows a bar plot of the contact angle from bare contact lens to the LoCL. The surface modification does not affect the hydrophilicity of the LoCL system compared to the pristine soft contact lens, according to some embodiments.

[0029] FIG. 14 shows optical transparency of the bare contact lens and LoCL system in the pupil zone, according to some embodiments.

[0030] FIGs. 15A-15C show mechanical analysis of the LoCL, according to some embodiments. FIG. 15A shows a load-extension graph of the LoCL platform, according to some embodiments. FIG. 15B shows a load-extension graph of the functionalized LoCL system, according to some embodiments. FIG. 15C shows a bar plot of Young's modulus of the unfunctionalized LoCL platform (0.86 ± 0.12 MPa) and the functionalized LoCL system (0.88 ± 0.06 MPa) , according to some embodiments.

[0031] FIG. 16 shows a schematic of the serotonin biosensing interface setup, according to some embodiments.

[0032] FIGs. 17A-17D show electrical characteristics of the unfunctionalized LoCL system, according to some embodiments. FIGs. 17A and 17B show CV curves of the unfunctionalized LoCL at (FIG. 17A) various scan cycles (1-5 cycles) and (FIG. 17B) wide ranges of scan rates (20-500 mV / s), according to some embodiments. The CV graphs show that the unfunctionalized LoCL system saturated after five cycles, and the oxygen and reduction peaks increased linearly with various scan ranges (20-500 mV), according to some embodiments. FIG. 17C shows a linear plot of anodic (Ipa) and cathodic peak (Ipc) currents vs. square root of scan rate, extracted from FIG. 17B, according to some embodiments. The linearity means that the unfunctionalized LoCL system followed the Randles-Sevcik equation under buffer fluids containing electrolytes (0.1 M KC1 and 5 mM [Fe(CN)6]3' / 4')- FIG. 17D shows CV curves of unfunctionalized LoCL to buffer solution with 0, 1, 10, and 100 microM, according to some embodiments. The redox interaction did not happen, and the oxidation peak was not found at 0.53 V without ferrocene molecules, according to some embodiments.

[0033] FIGs. 18A-18F show electrical characteristics of the LoCL system, according to some embodiments. FIGs. 18A and 18B show CV curves of the LoCL system to 10 nM of serotonin at (FIG. 18A) 5 scan cycles and (FIG. 18B) 10 cycles, according to some embodiments. Both CV results represent the redox interaction between ferrocene molecules and serotonin molecules saturated after five scan cycles. FIG. 18C shows CV curves of the serotonin electrochemical sensor at wide ranges of scan rates (20-500 mV), according to some embodiments. The currents of oxidation and reduction peaks increased linearly with various scan ranges (20-500 mV). FIG. 18D shows CV curves of unfunctionalized and functionalized LoCL system to 0 (buffer only) and 100 microM of serotonin, according to some embodiments. The oxidation peaks at 0.53 V display that the functionalized LoCL system can detect serotonin in the presence of serotonin (red CV). Meanwhile, no oxidation peak of the LoCL system was depicted at 0.53 V in the buffer (without serotonin, green CV). Compared to the functionalized LoCL system, the unfunctionalized LoCL system could not display the serotonin peak at 0.53 V not only in the buffer (without serotonin, black CV) but also in serotonin existence (blue CV). This means that the redox interaction does not react between graphene and serotonin molecules. FIG. 18E shows CV curves of the sensor to various serotonin concentrations of 1 pM-100 microM at applied voltage range (from -0.8 to 1.0 V). FIG. 18F shows enlarged CV curves with anodic (Ipa) and cathodic peak (Ipc), according to some embodiments. The CV graphs show that the Ipaincreased as the concentration of serotonin increased, according to some embodiments.

[0034] FIGs. 19A-19D show electrical characteristics of the FeC / amine / TPSA-modified LoCL system, according to some embodiments. FIG. 19A shows CV curves of the LoCL system to artificial tear fluids (AT) at various scan cycles (1-5 cycles), according to some embodiments. It represents the redox interaction under AT-containing electrolytes (0.1 M KC1 and 5 mM [Fe(CN)6]3 / 4’), saturated after five scan cycles. FIG. 19B shows CV graphs of the sensor to various serotonin concentrations of 1 pM-100 microM at applied voltage (from -0.8 to 1.0 V), according to some embodiments. FIG. 19C shows enlarged CV curves with anodic (Ipa) and cathodic peak (Ipc), according to some embodiments. The CV graphs show that the Ipaincreased as the concentration of serotonin increased, according to some embodiments. FIG. 19D shows the calibration curve of the Ipachanges according to the serotonin concentration (<100 microM), according to some embodiments. The inset linear graph was plotted to the serotonin concentration (0.005-10 microM). The graph shows that the Ipaof the functionalized LoCL linearly (R2=0.932) increases between 0.005-10 microM of serotonin, which includes the serotonin range in the human tears, and the detectable serotonin range of the functionalized LoCL system is 1 pM, according to some embodiments.

[0035] FIGs. 20A and 20B show response time of the LoCL system on serotonin with different concentrations, according to some embodiments. FIG. 20A shows a response time graph of the LoCL system to 1 microM serotonin at various incubation times up to 3 minutes, according to some embodiments. FIG. 20B shows a bar plot of response time (i.e.. saturated time) of the LoCL system to each concentration (0.005-1 microM) of serotonin, according to some embodiments.

[0036] FIGs. 21A-21F show CV graphs of the LoCL system to serotonin concentrations (0, 100 nM, 500 nM, and 1 microM) at different pH conditions (pH= 6.0-8.0), according to some embodiments. Each oxidation peak at 0.53 V was observed at all the ranges of buffer, suggesting that the LoCL system could stably function within the human tear’s pH range (6.5-7.4), according to some embodiments. FIG. 21F shows CV curves of the LoCL system to buffer fluids (pH 7.4) at various scan cycles (1-5 cycles), according to some embodiments. For the unfunctionalized LoCL system, the redox reaction under buffer fluids containing electrolytes (0.1 M KC1 and 5 mM [Fe(CN)6]3' / 4‘) saturated after five scan cycles, according to some embodiments.

[0037] FIG. 22A-22F shows the mechanical flexibility test of the LoCL system up to 1,000 bending cycles, according to some embodiments. Insets show the magnified view of each area highlighted in the yellow box. FIGs. 22B-22F show high-resolution SEM images of each LoCL system after (FIG. 22B) 100 cycles, (FIG. 22C) 300 cycles, (FIG. 22D) 500 cycles, (FIG. 22E) 800 cycles, and (FIG. 22F) 1,000 cycles of bending, according to some embodiments.

[0038] FIG. 23 shows reversibility of the LoCL system. The bar graph shows identical responses to 100 nM of serotonin during continuous measurements for 3 days, according to some embodiments.

[0039] FIGs. 24A-24D show human tear serotonin levels in DED patients, according to some embodiments. FIGs. 24A and 24B show serotonin responses of the LoCL system from (FIG. 24A) normal (n=9) and (FIG. 24B) dry eye (n=10) subject’s tears. FIG. 24C shows CV graphs of the LoCL system to an artificial tears fluid, normal tears, and dry eye tears, according to some embodiments. FIG. 24D shows serotonin levels of subjects’ tears (n=19), according to some embodiments.

[0040] FIGs. 25A-25C show reversibility performance in quantifying tear serotonin levels in DED patients, according to some embodiments. Cyclic voltammetry (CV) graphs of LoCL system to artificial tear- fluid from normal tear fluid (FIG. 25A) or dry eye tear fluid (FIG. 25B). FIG. 25C shows the current response to serotonin oxidation from FIG. 25A (left axis) and FIG. 25B (right axis). The magnitude of the measured current is proportional to the concentration of serotonin in the tear fluid sample, according to some embodiments.

[0041] FIG. 26 shows a bar graph of the amperometric response of the LoCL to the serotonin changes on a porcine model in real-time operation, according to some embodiments.

[0042] FIG. 27 shows an exemplary contact lens as contemplated herein, according to some embodiments. The contact lens comprises a first polymer lens and a second polymer lens, defining a hole, in contact with the first polymer lens. An electrochemical sensor is placed between the two polymer lenses. The electrochemical sensor comprises a working electrode, a counter electrode, and a reference electrode, according to some embodiments. The working electrode further comprises an electrochemically reactive compound (e.g., disposed on a surface of the working electrode), according to some embodiments. FIG. 27 also includes an inset that shows an electrochemical sensor as contemplated herein, according to some embodiments.

[0043] FIG. 28 shows the contact lens, as described in FIG. 27, connected to a potentiostat, according to some embodiments. The potentiostat completes the electrochemical circuit by placing the working electrode, counter electrode, and reference electrode into electrical contact with each other. FIG. 28 also shows provides an electrical circuit diagram of the electrochemical circuit within the contact lens, according to some embodiments.

[0044] DETAILED DESCRIPTION

[0045] The present disclosure generally relates to smart contact lenses for non-invasive monitoring of biological markers of health and disease. In some aspects, contact lenses comprising an electrochemical sensor between two deformable polymer lenses are provided. In some embodiments, the electrochemical sensor comprises a working electrode, a counter electrode, and a reference electrode. The working electrode, in some embodiments, further comprises an electrochemically reactive compound, that when oxidized, oxidizes a target analyte in a tear fluid. Other aspects of the disclosure relate to methods of using said contact lenses. For example, in some embodiments, the methods relate to determining a concentration of the target analyte in tear fluid. In some embodiments, the target analyte is serotonin. Other embodiments are generally directed to methods of making or using the contact lenses, kits involving said contact lenses, or the like.

[0046] Tear fluid is a complex mixture of sugars, carbohydrates, proteins, glycoproteins, lipids, mucins, salts, and water. Many of these compounds are known biomarkers used to assess the risk or presence of both localized ocular disease and / or systemic diseases (with or without ocular complications). For example, elevated levels of lacryglobin, Sulf-1, crystatin SA, 5-AMP- activated protein kinase subunit gamma-3, triosephosphate isomerase, microbtubule-associated tumor suppressor 1, keratin (type 1), and putative LCN-1 like protein have been identified in tears of subjects with various cancers. Other compounds in tear fluid have been associated with other diseases. For example, elevated tear levels of NGF, LCN-1, lactotransferrin, lysosome C, lacritin, lipophilin A, and Ig lambda chain is associated with DR, elevated levels of IgG is associated with multiple sclerosis (MS), and elevated levels of IL-8, IFN-gamma, MIP-lalpha, and MIP-lbeta is associated with cystic fibrosis.

[0047] As such, tear fluid has become an attractive alternative to standard biological fluids, such as blood or serum, for monitoring specific markers commonly used to assess the risk or presence of disease. The most common strategy for tear fluid analysis uses tear fluid collection (e.g., using either Schirmer tear strips or capillary tubes) and analysis using a combination of optical spectroscopy methods (e.g., absorption, fluorescence, scattering), separation methods (e.g., high pressure liquid chromatography (HPLC), ultra performance liquid chromatography (UPLC), SDS-PAGE), and mass spectroscopy. However, these methods are laborious, expensive, time consuming, and require sophisticated scientific equipment and expertise. Further, existing methods only provide an instantaneous snapshot of a biomolecules concentration at single point in time.

[0048] Accordingly, one aspect of the present disclosure is directed to the discovery that the contact lenses disclosed herein allow for the real-time monitoring of one or more biomarkers from tear fluid simultaneously over multiple time scales. As described in more detail below, some of the contact lenses disclosed herein comprise a first polymer lens and a second polymer lens, defining a hole, in contact with the first polymer lens. Further, in some embodiments, the contact lenses comprise an electrochemical sensor, located between the two polymer lenses, and in fluidic communication with the hole in the second polymer lens. The electrochemical sensor, according to some embodiments, is a three electrode electrochemical circuit. In some embodiments, electrochemical sensor comprises a working electrode, a counter electrode, and a reference electrode. In some cases, at least a portion of the working electrode comprises an electrochemically reactive compound capable of undergoing a reduction-oxidation (redox) reaction. In some embodiments, the electrochemically reactive compound is ferrocene. In some embodiments, the electrochemical circuit applies a constant electric potential across the working and counter electrodes, wherein the supplied electric potential is sufficient to oxidize the electrochemically reactive compound in the working electrode, resulting in the release of one or more electrons to the working electrode. For example, applying an electric potential of about 0.5V (e.g., via a battery) will cause an electrochemical sensor comprising ferrocene (e.g., an electrochemically reactive compound) to lose a single electron, resulting in the formation of ferrocenium. As used herein, the term “electrochemically reactive compound” and “electrochemical compound” are synonymous.

[0049] When such contact lenses are placed onto an eye of a subject, tear fluid, comprising one or more analytes, may contact the oxidized electrochemically reactive compound (e.g., ferrocenium) on the working electrode of the electrochemical sensor, e.g., via the hole in the second polymer lens. In some embodiments, the oxidized electrochemically reactive compound is configured to oxidize a target analyte (e.g., serotonin) within the pool of analytes in the tear fluid. Upon oxidizing the target analyte the oxidized electrochemically reactive compound is reduced (e.g., ferrocenium is reduced to ferrocene), thus regenerating original electrochemically reactive compound (e.g., ferrocene). For example, in some embodiments, applying an electric potential of about 0.5V to an electrochemical sensor comprising ferrocene will oxidize the ferrocene to ferrocenium. The ferrocenium may, in some embodiments, oxidize serotonin if present in tear fluid, thus regenerating the ferrocene. The skilled artisan will appreciate that the regenerated ferrocene may be repeatedly oxidized and reduced, resulting in the continued production of electrons. Because the electrochemical sensor is a complete electrochemical circuit, the electrons can flow through the circuit and can be measured as a current (e.g., via use of an ammeter). In some embodiments, the magnitude of the current produced is proportional to the concentration of the target analyte in the tear fluid.

[0050] The contact lenses disclosed herein may further comprise one or more other embodiments. For example, in some embodiments, the contact lenses comprise a first polymer lens, a second polymer lens, defining a hole, in contact with the first polymer lens, and an electrochemically reactive compound in fluidic communication with the hole. In other embodiments, the contact lenses disclosed herein comprise a first polymer lens, a second polymer lens in contact with the first polymer lens, and a working electrode, a reference electrode, and a counter electrode, each positioned between the first polymer lens and the second polymer lens. In some embodiments, the working electrode comprises ferrocene. In yet other embodiments still, the disclosure describes deformable contact lenses comprising an electrochemical sensor, wherein the deformable contact lenses have an elastic modulus of between 0.1 MPa and 1.5 MPa.

[0051] Other aspects of the disclosure relate to one or more methods. In some embodiments, the methods relate to exposing any one of the contact lenses disclosed herein to a fluid produced by an eye of a subject, wherein the fluid comprises an analyte. In some embodiments, the fluid is a tera fluid. In some embodiments, the analyte is serotonin. The methods further comprise electrochemically reacting the analyte (e.g., serotonin) with an electrochemical compound (e.g., an oxidized electrochemical compound such as ferrocenium), and determining a current produced from the electrochemical reaction.

[0052] In other embodiments, the methods relate to determining a concentration of an analyte (e.g., serotonin) within a fluid within an eye of a subject (e.g., a tear fluid). In some embodiments, the methods comprise determining the magnitude of an electrical current produced from an electrochemical sensor of any one of the contact lenses disclosed herein following placement of said contact lenses on the eye of the subject.

[0053] Additionally, in some embodiments, the methods relate to determining a concentration of serotonin for a fluid within an eye (e.g., a tear fluid) of a subject using any one of the contact lenses disclosed herein. In some cases, the methods comprise measuring a current produced from a reaction (e.g., redox reaction) of ferrocene (or the oxidized form of ferrocene, ferrocenium) contained within the contact lens.

[0054] The above discussion are non-limiting examples of certain aspects generally directed to devices or methods for sensing analytes and quantifying analyte concentration in tear fluid, e.g., for monitoring risk and / or progression of disease. However, other embodiments are also possible besides those discussed above.

[0055] For example, in some aspects, a contact lens comprising at least two polymer lenses in contact with each other is provided. FIG. 27 shows exemplary contact lens 100, comprising first polymer lens 110 and second polymer lens 150, defining hole 160, in contact with first polymer lens 110. In some embodiments, first polymer lens 110 comprises electrochemical sensor 170. Electrochemical sensor 170 comprises working electrode 120, counter electrode 130 and reference electrode 140. In some embodiments, working electrode 120, counter electrode 130, and / or reference electrode 140 are screen printed on first polymer lens 110, using for example, a suitable electrode ink (e.g., graphene ink, Ag / AgCl ink, etc.). In some embodiments, electrochemical sensor 170 is configured to measure a current produced from a reductionoxidation reaction (redox) between an electrochemically reactive compound 180 disposed on (or chemically conjugated to) working electrode 120 and a target analyte in tear fluid.

[0056] FIG. 28 shows an exemplary contact lens 200 comprising first polymer lens 205 comprising electrochemical sensor 295. Electrochemical sensor 295 comprises (1) working electrode 210 which comprises stretchable interconnect 215 and connection port 220, (2) counter electrode 230, which comprises stretchable interconnect 235 and connection port 240, and (3) reference electrode 250, which comprises stretchable interconnect 255 and connection port 250. All electrodes are contained between first polymer lens 205 and a second polymer lens (not shown). Electrochemical circuit 290 is established by connecting connection ports 220, 240, and 260 of electrochemical sensor 295 to potentiostat 270 (e.g., the potentiostat or equivalent is needed to complete the electrical circuit). Those of skill in the art will understand that the electric potential 280 of the electrochemical circuit is determined by the difference between the reference electric potential and the supplied electric potential from the potentiostat.

[0057] As stated above, electrochemical sensor 295 is a part of electrochemical circuit 290. To complete the circuit, working electrode 210, counter electrode 230, and reference electrode 250 must be placed into electrical contact with each other. In some embodiments, this is accomplished using potentiostat 270. In some embodiments, the potentiostat is further configured to apply an electric potential between the working electrode and the counter electrode. The skilled artisan will understand and appreciate that while FIG. 28 shows a potentiostat external to the contact lens, this is not a requirement, and onboard devices capable of supplying the necessary electric potential are also contemplated herein. Any suitable device capable of delivering the necessary electric potential may be used in the electrochemical circuits disclosed herein. For example, in some embodiments, a battery, e.g., a 3D silicon-based lithium- ion battery, may be used to supply the needed electric potential.

[0058] In some embodiments, an electrochemical circuit comprises one or more screen printed electrodes. In some instances, the one or more screen printed electrodes comprises a working electrode. In other cases, the one or more screen printed electrodes comprise a counter electrode. Additionally, in some embodiments, the one or more screen printed electrodes comprises a reference electrode. The electrochemical circuit may further comprise an ammeter (e.g., a device to measure electric current), in other embodiments. The ammeter, in some embodiments, is electrically connected to the working electrode and the counter electrode. In some embodiments, the ammeter is used to measure a current generated by the electrochemical circuit.

[0059] In some embodiments, a working electrode is created (e.g., screen printed) on a polymer lens using an electrode ink. Any suitable electrode ink known to the skilled artisan may be used to create (e.g., print) the working electrodes disclosed herein onto the polymer lenses. Exemplary embodiments of suitable electrode inks include, but are not limited to, graphene, gold, and platinum. Similarly, a counter electrode is created (e.g., screen printed) on the polymer lens using an electrode ink. Any suitable electrode ink known to the skilled artisan may be used to create (e.g., print) the counter electrodes disclosed herein onto the polymer lenses. Exemplary embodiments of suitable electrode inks for creating the counter electrode include, but are not limited to, graphene gold and platinum. A reference electrode may also be created (e.g., screen printed) on the polymer lens using an electrode ink. Any suitable electrode ink known to the skilled artisan may be used to create the reference electrodes disclosed herein. In some embodiments, electrode ink used to create the reference electrode comprises silver and / or silver chloride.

[0060] In some embodiments, the working electrode comprises an electrochemically reactive compound (or electrochemical compound). The electrochemically reactive compound may be introduced into the working electrode using any suitable technique. For example, in some embodiments, the electrochemically reactive compound may be mixed with an electrode ink and printed onto a polymer lens. Alternatively, in some embodiments, a surface of the working electrode is functionalized with the electrochemically reactive compound. Any suitable technique for functionalizing the working electrode with an electrochemically reactive compound may be used to create the surface functionalized working electrode. For instance, in some cases, the surface of the working electrode is modified with a reactive moiety, such as a primary amine group, carboxylic acid group, thiol, or the like, which is subsequently reacted with a electrochemically reactive compound comprising a complimentary reactive group (e.g., an amine-functionalized working electrode with a carboxylate-functionalized electrochemically reactive group). As an example, the surface of a graphene working electrode can be functionalized with primary amine groups via drop casting a 10% v / v solution of 3-triethoxy silyl propylsuccinic anhydride (TPSA) onto the working electrode of the electrochemical biosensor followed by treatment with a 3% (w / v) solution of (3-aminopropyl)triethoxysilane (APTES). This amine-functionalized graphene working electrode may then be reacted with a ferrocenecarboxaldehyde (FeC-CHO) solution (1 microL, 50 mM) to yield a working electrode comprising the electrochemically reactive compound ferrocene. Accordingly, in some embodiments, the working electrode comprises any one of the chemical reagents used to conjugate the electrochemically reactive compound to the surface of the working electrode. In some cases, the working electrode comprises a 3-triethyoxysilyl propylsuccindyl-group and / or a 3 -aminopropyl triethoxy sily 1-group .

[0061] The skilled artisan will understand and appreciate that an electrochemically reactive compound, as disclosed herein, is any compound configured to undergo a reduction-oxidation reaction. As used herein, the term “reduction-oxidation reaction” or “redox reaction” refers to a type of chemical reaction in which the oxidation states of a reactant changes. Oxidation is the loss of electrons or an increase in the oxidation state, while reduction is the gain of electrons or a decrease in the oxidation state. Again, the skilled artisan will understand that an oxidizing agent is an electron acceptor in a redox reaction that gains or “accepts” electrons from a reducing agent.

[0062] In some embodiments, an electrochemically reactive compound comprises an organometallic compound. Any suitable organometallic compound may be used. In some embodiments, the organometallic compound is ferrocene. In other embodiments, the electrochemically reactive compound comprises a multi-walled carbon nanotube (MWCNT). Other exemplary embodiments include, but are not limited to, zinc oxide (ZnO), porphyrins (e.g., 15-pcntafluorophcnyl- 10.20-p-aminophycnylprophyrin or meso-tetrakis (2-aminophenyl) porphyrin, etc.), graphene oxide, nanodiamonds, nanoparticles, poly(bromocresol green), magnetic nanoparticles, and / or polypyrrole.

[0063] The tendency of an electrochemically reactive compound to undergo oxidation or reduction may be defined by a reduction potential (e.g., measured in volts). As used herein, the term “reduction potential” refers to a measure of the tendency of a chemical species to acquire electrons from or lose electrons to an electrode (e.g., a working electrode) and thereby be reduced or oxidized, respectively. Each electrochemically reactive compound has its own intrinsic redox potential.

[0064] Based on this understanding, the skilled artisan will recognize that applying an electric potential across the working and counter electrodes greater than the reduction potential of the electrochemically reactive compound will result in the oxidation / reduction of the electrochemically reactive compound. For example, the reduction potential of ferrocene is about 0.4 V, relative to a Ag / AgCl reference electrode, such that applying an electric potential of about 0.5V across the working and counter electrodes will result in the electrochemical oxidation of ferrocene to ferrocenium on a surface of the working electrode.

[0065] Thus, in some embodiments, an electrochemically reactive compound is in a reduced state (e.g., ferrocene) at a first electric potential. In some embodiments, the first electric potential is between 0 V and 0.5 V. The first electric potential may in some cases, be greater than or equal to 0 V, greater than or equal to 0.1 V, greater than or equal to 0.2 V, greater than or equal to 0.3 V, greater than or equal to 0.4 V, or greater than or equal to 0.5V. In some embodiments, the first electric potential is less than or equal to 0.5 V, less than or equal to 0.4 V, less than or equal to 0.3 V, less than or equal to 0.2 V, less than or equal to 0.1 or less than or equal to 0 V. Combinations of the above recited ranges are also possible in some embodiments. For example, in some embodiments, the first electric potential is greater than or equal to 0 V and less than or equal to 0.5 V.

[0066] In other embodiments, an electrochemically reactive compound is electrochemically oxidized to an oxidized state (e.g., ferrocenium) at a second electric potential. In some embodiments, the second electric potential is between 0 V and 0.5 V. The second electric potential may in some cases, be greater than or equal to 0 V, greater than or equal to 0.1 V, greater than or equal to 0.2 V, greater than or equal to 0.3 V, greater than or equal to 0.4 V, or greater than or equal to 0.5V. In some embodiments, the second electric potential is less than or equal to 0.5 V, less than or equal to 0.4 V, less than or equal to 0.3 V, less than or equal to 0.2 V, less than or equal to 0.1 V, or less than or equal to 0 V. Combinations of the above recited ranges are also possible in some embodiments. For example, in some embodiments, the second electric potential is greater than or equal to 0 V and less than or equal to 0.5 V.

[0067] Thus, in some embodiments, an electrochemically reactive compound is in an oxidized state (e.g., ferrocenium) at a first electric potential. In some embodiments, the first electric potential is between 0 V and 0.5 V. The first electric potential may in some cases, be greater than or equal to 0 V, greater than or equal to 0.1 V, greater than or equal to 0.2 V, greater than or equal to 0.3 V, greater than or equal to 0.4 V, or greater than or equal to 0.5V. In some embodiments, the first electric potential is less than or equal to 0.5 V, less than or equal to 0.4 V, less than or equal to 0.3 V, less than or equal to 0.2 V, less than or equal to 0.1 or less than or equal to 0 V. Combinations of the above recited ranges are also possible in some embodiments. For example, in some embodiments, the first electric potential is greater than or equal to 0 V and less than or equal to 0.5 V.

[0068] In other embodiments, an electrochemically reactive compound is electrochemically oxidized to a reduced state (e.g., ferrocene) at a second electric potential. In some embodiments, the second electric potential is between 0 V and 0.5 V. The second electric potential may in some cases, be greater than or equal to 0 V, greater than or equal to 0.1 V, greater than or equal to 0.2 V, greater than or equal to 0.3 greater than or equal to 0.4 V, or greater than or equal to 0.5V. In some embodiments, the second electric potential is less than or equal to 0.5 V, less than or equal to 0.4 V, less than or equal to 0.3 V, less than or equal to 0.2 V, less than or equal to 0.1 V, or less than or equal to 0 V. Combinations of the above recited ranges are also possible in some embodiments. For example, in some embodiments, the second electric potential is greater than or equal to 0 V and less than or equal to 0.5 V.

[0069] As described elsewhere herein, electrochemical oxidation of the electrochemically reactive compound (e.g., ferrocene), disposed on a surface of a working electrode, produces an oxidized electrochemically reactive compound (e.g., ferrocenium). This oxidized intermediate may then oxidize a target analyte (e.g., serotonin) in a target fluid (e.g., tear fluid). This process also reduces the oxidized electrochemical reactive compound (e.g., ferrocenium) back to the starting material ( ferrocene), which may again be electrochemically oxidized. In this way, a current is generated from the continuous flow of electrons released from the electrochemical oxidation of the electrochemically reactive compound (ferrocene) and its simultaneous reduction by the target analyte (e.g., serotonin) to regenerate the starting electrochemically reactive compound (e.g., ferrocene). In some embodiments, a contact lens, as disclosed herein, is configured to measure the current generated from the continuous electrochemical oxidation of the target analyte by the electrochemically oxidized electrochemical reactive compound. In some cases, the current generated (e.g., measured) is proportional to a concentration of the target analyte.

[0070] In some embodiments, an electrochemical sensor has an average thickness of between 45 microns and 55 microns. In some embodiments, the electrochemical sensor has an average thickness of greater than or equal to 45 microns, greater than or equal to 47 microns, greater than or equal to 49 microns, greater than or equal to 50 microns, greater than or equal to 51 microns, greater than or equal to 52 microns, greater than or equal to 53 microns, greater than or equal to 54 microns, or greater than or equal to 55 microns. In some embodiments the electrochemical sensor has an average thickness of less than or equal to 55 microns, less than or equal to 54 microns, less than or equal to 53 microns, less than or equal to 52 microns, less than or equal to 51 microns, less than or equal to 50 microns, less than or equal to 49 microns, less than or equal to 47 microns, or less than or equal to 45 microns. Combinations of the above recited ranges are also possible. For example, in some embodiments the electrochemical sensor has an average thickness of greater than or equal to 45 microns and less than or equal to 55 microns.

[0071] In some embodiments, an electrochemical sensor has a total area of between 35 mm2and 40 mm2. In some embodiments the electrochemical sensor has a total area of greater than or equal to 35 mm2, greater than or equal to 37 mm2, or greater than or equal to 40 mm2. In some embodiments the electrochemical sensor as a total area of less than or equal to 40 mm2, less than or equal to 37 mm2, or less than or equal to 35 mm2. Combinations of the above recited ranges are also possible in some embodiments. For example, in some embodiments, the electrochemical sensor has a total area of greater than or equal to 35 mm2and less than or equal to 40 mm2.

[0072] In some embodiments, a working electrode has an average thickness of between 45 microns and 55 microns. In some embodiments, the working electrode has an average thickness of greater than or equal to 45 microns, greater than or equal to 47 microns, greater than or equal to 49 microns, greater than or equal to 50 microns, greater than or equal to 51 microns, greater than or equal to 52 microns, greater than or equal to 53 microns, greater than or equal to 54 microns, or greater than or equal to 55 microns. In some embodiments the working electrode has an average thickness of less than or equal to 55 microns, less than or equal to 54 microns, less than or equal to 53 microns, less than or equal to 52 microns, less than or equal to 51 microns, less than or equal to 50 microns, less than or equal to 49 microns, less than or equal to 47 microns, or less than or equal to 45 microns. Combinations of the above recited ranges are also possible. For example, in some embodiments the working electrode has an average thickness of greater than or equal to 45 microns and less than or equal to 55 microns.

[0073] In some embodiments, a working electrode has a total area of between 10 mm2and 12 mm2. In some embodiments the working electrode has a total area of greater than or equal to 10 mm2, greater than or equal to 11 mm2, or greater than or equal to 12 mm2. In some embodiments the working electrode as a total area of less than or equal tol2 mm2, less than or equal to 11 mm2, or less than or equal to 10 mm2. Combinations of the above recited ranges are also possible in some embodiments. For example, in some embodiments, the working electrode has a total area of greater than or equal to 10 mm2and less than or equal to 12 mm2.

[0074] In some embodiments, counter electrode has an average thickness of between 45 microns and 55 microns. In some embodiments, the counter electrode has an average thickness of greater than or equal to 45 microns, greater than or equal to 47 microns, greater than or equal to 49 microns, greater than or equal to 50 microns, greater than or equal to 51 microns, greater than or equal to 52 microns, greater than or equal to 53 microns, greater than or equal to 54 microns, or greater than or equal to 55 microns. In some embodiments the counter electrode has an average thickness of less than or equal to 55 microns, less than or equal to 54 microns, less than or equal to 53 microns, less than or equal to 52 microns, less than or equal to 51 microns, less than or equal to 50 microns, less than or equal to 49 microns, less than or equal to 47 microns, or less than or equal to 45 microns. Combinations of the above recited ranges arc also possible. For example in some embodiments the counter electrode has an average thickness of greater than or equal to 45 microns and less than or equal to 55 microns.

[0075] In some embodiments, a counter electrode has a total area of between 10 mm2and 12 mm2. In some embodiments the counter electrode has a total area of greater than or equal to 10 mm2, greater than or equal to 11 mm2, or greater than or equal to 12 mm2. In some embodiments the counter electrode as a total area of less than or equal to mm2, less than or equal to 11 mm2, or less than or equal to 10 mm2. Combinations of the above recited ranges are also possible in some embodiments. For example, in some embodiments, the counter electrode has a total area of greater than or equal to 10 mm2and less than or equal to 12 mm2.

[0076] In some embodiments, reference electrode has an average thickness of between 45 microns and 55 microns. In some embodiments, the reference electrode has an average thickness of greater than or equal to 45 microns, greater than or equal to 47 microns, greater than or equal to 49 microns, greater than or equal to 50 microns, greater than or equal to 51 microns, greater than or equal to 52 microns, greater than or equal to 53 microns, greater than or equal to 54 microns, or greater than or equal to 55 microns. In some embodiments the reference electrode has an average thickness of less than or equal to 55 microns, less than or equal to 54 microns, less than or equal to 53 microns, less than or equal to 52 microns, less than or equal to 51 microns, less than or equal to 50 microns, less than or equal to 49 microns, less than or equal to 47 microns, or less than or equal to 45 microns. Combinations of the above recited ranges are also possible. For example in some embodiments the reference electrode has an average thickness of greater than or equal to 45 microns and less than or equal to 55 microns.

[0077] In some embodiments, a reference electrode has a total area of between 10 mm2and 12 mm2. In some embodiments the reference electrode has a total area of greater than or equal to 10 mm2, greater than or equal to 11 mm2, or greater than or equal to 12 mm2. In some embodiments the reference electrode as a total area of less than or equal to mm2, less than or equal to 11 mm2, or less than or equal to 10 mm2. Combinations of the above recited ranges are also possible in some embodiments. For example, in some embodiments, the reference electrode has a total area of greater than or equal to 10 mm2and less than or equal to 12 mm2. In some embodiments, a working electrode, counter electrode, and / or reference electrode each comprise a stretchable interconnect. The stretchable interconnect, in some cases, is positioned away from the center of polymer lens. In some embodiments, the stretchable interconnect is positioned along a perimeter of the polymer lens. This is desirable, for example, to prevent obstructing the field of view. In some cases, at least a portion of the stretchable interconnect is serpentine. The serpentine may be defined by a ribbon width (W), external diameter (D), internal diameter (d), and pitch (L), for example, as shown in FIG. 7B.

[0078] In some embodiments, a serpentine may be defined by a ribbon width (W) of between 0.1 millimeters and 0.5 millimeters. For example, in some cases the ribbon width is greater than or equal to 0.1 millimeters, greater than or equal to 0.2 millimeters, greater than or equal to 0.3 millimeters, greater than or equal to 0.4 millimeters, or greater than or equal to 0.5 millimeters. In some cases, the ribbon width is less than or equal to 0.5 millimeters, less than or equal to 0.4 millimeters, less than or equal to 0.3 millimeters, less than or equal to 0.2 millimeters, or less than or equal to 0.1 millimeters. Combinations of the above recited ranges are also possible in other embodiments. For example, in some embodiments the ribbon width is greater than or equal to 0.1 millimeters and less than or equal to 0.5 millimeters.

[0079] In some embodiments, a serpentine may be defined by an external diameter (D) of between 1 millimeter and 2 millimeters. In some embodiments, the serpentine external diameter is greater than or equal to one millimeter, greater than or equal to 1.1 millimeter, greater than or equal to 1.2 millimeter, greater than or equal to 1.3 millimeter, greater than or equal to 1.4 millimeter, greater than or equal to 1.5 millimeters, greater than or equal to 1.6 millimeter, greater than or equal to 1.7 millimeter, greater than or equal to 1.8 millimeter, greater than or equal to 1.9 millimeter, or greater than or equal to 2 millimeters. In some embodiments, the serpentine external diameter is less than or equal to 2 millimeters, less than or equal to 1.9 millimeters, less than or equal to 1.8 millimeters, less than or equal to 1.7 millimeters, less than or equal to 1.6 millimeters, less than or equal to 1.5 millimeters, less than or equal to 1.4 millimeters, less than or equal to 1.3 millimeters, less than or equal to 1.2 millimeters, less than or equal to 1.1 millimeters, or less than or equal to 1 millimeter. Combinations of the above recited ranges are also possible in some embodiments. For example, in some embodiments the serpentine may be defined by an external diameter of greater than or equal to one millimeter and less than or equal to two millimeters. In some embodiments, a serpentine may be defined by an internal diameter of between 0.75 millimeters and 1.25 millimeters. In some embodiments, the internal diameter is greater than or equal to 0.75 millimeters, greater than or equal to 0.8 millimeter, greater than or equal to 0.85 millimeters, greater than or equal to 0.9 millimeter, greater than or equal to 0.95 millimeters, greater than or equal to 1.0 millimeter, greater than or equal to 1.05 millimeters, greater than or equal to 1.1 millimeter, greater than or equal to 1.15 millimeters, greater than or equal to 1.2 millimeter, or greater than or equal to 1.25 millimeters. In some embodiments the internal diameter is less than or equal to 1.25 millimeters, less than or equal to 1.2 millimeters, less than or equal to 1.15 millimeters, less than or equal to 1.1 millimeters, less than or equal to 1.05 millimeters, less than or equal to 1.0 millimeters, less than or equal to 0.95 millimeters, less than or equal to 0.9 millimeters, less than or equal to 0.85 millimeters, less than or equal to 0.8 millimeters, or less than or equal to 0.75 millimeters. Combinations of the above recited ranges are also possible in some embodiments. For example, in some embodiments the serpentine internal diameter is greater than or equal to 0.75 millimeters and less than or equal to 1.25 millimeters.

[0080] In some embodiments, a serpentine may be defined by a pitch (L) of between 2 millimeters and 4 millimeters. In some embodiments, the pitch is greater than or equal to 2.0 millimeters, greater than or equal to 2.2 millimeters, greater than or equal to 2.4 millimeters, greater than or equal to 2.6 millimeters, greater than or equal to 2.8 millimeters, greater than or equal to 3.0 millimeters, greater than or equal to 3.2 millimeters, greater than or equal to 3.4 millimeters, greater than or equal to 3.6 millimeters, greater than or equal to 3.8 millimeters, or greater than or equal to 4.0 millimeters. In some embodiments, the pitch is less than or equal to 4.0 millimeters, less than or equal to 3.8 millimeters, less than or equal to 3.6 millimeters, less than or equal to 3.4 millimeters, less than or equal to 3.2 millimeters, less than or equal to 3.0 millimeters, less than or equal to 2.8 millimeters, less than or equal to 2.6 millimeters, less than or equal to 2.4 millimeters, less than or equal to 2.2 millimeters, or less than or equal to 2.0 millimeters. Combinations of the above recited ranges are also possible in some embodiments. For example, in some embodiments the serpentine may be defined by a pitch that is greater than or equal to 2 millimeters and less than or equal to 4 millimeters.

[0081] A polymer lens, as disclosed herein, comprises one or more polymers. In some embodiments, the polymer lens comprises polyhdroxyethylmethyacrylate (polyHEMA). In other embodiments, the polymer lens comprises silicone. Other polymers are also possible in other embodiments. For example, in some cases, the polymer lens comprises polydimcthylsiloxanc (PDMS) or polymethyl methacrylate (PMMA). Combinations of polymer are also possible. Combinations may comprise physical mixtures of two or more polymers and / or use of copolymers. Any suitable polymer and / or copolymer known to the skilled artisan may be used in the polymer lenses disclosed herein. In some embodiments, the polymer lenses are analogous to “hard” contact lenses (e.g., PMMA-based contact lenses); in other embodiments, the polymer lenses are analogous to “soft” contact lenses (e.g., silicone- and / or polyHEMA-based contact lenses). In some embodiments, the polymer lenses are made from deformable polymers.

[0082] In some embodiments, a polymer lens is made by pouring a precursor polymer solution into a mold and polymerizing said solution. In some embodiments, the precursor solution comprises 2-hydroxy-2-methylpropiophenone, hydroxyethylmethacrylate (HEMA), and ethylene glycol dimethacrylate (EDGMA). However, the skilled artisan will understand that any known method for producing said lens may be used to produce the polymer lenses disclosed herein.

[0083] In some embodiments, a polymer lens comprises one or more electrodes. In some embodiments, the one or more electrodes is a working electrode. In some embodiments, the one or more electrodes is a counter electrode. In some embodiments, the one or more electrodes is a reference electrode. Again, while the instant disclosure generally describes electrodes defining an electrochemical circuit enclosed between two polymer lenses, the polymer lenses may comprise any suitable structure in different embodiments (e.g., a structure that defines a shape created by laser engraving at least a part of the polymer lens). Exemplary shapes that may be defined by the one or more structures, include but are not limited to, triangles, squares, rectangles, pentagons, hexagons, octagons, decagons, rhombus, parallelogram, kite, trapezium, trapezoid, or any other regular or irregular polygon known to the skilled artisan.

[0084] Further, any technique known to the skilled artisan may be used to create the one or more electrodes and / or one or more structures in the polymer lenses. For example, in some embodiments, the one or more electrode is place on at least one of the polymer lenses using screen printing. Alternatively, or additionally, in some embodiments, a space defining the one or more electrodes may be formed by a mold from which the polymer is case or engraved into the polymer lens, for example, using a laser cutter. In this way, an electrode ink, such as a polymerizable electrode ink, may be placed in the channel and cured to form the electrode. Similar processes may also be used to define one or more structures (e.g., a groove) on a first polymer lens, that when placed into contact with a second polymer lens forms a structure (e.g., a channel).

[0085] In some embodiments, a polymer lens is configured to correct a refractive error in an eye (e.g., to correct the eyesight of a subject). In other embodiments, the polymer lens is configured to dissipate eyelid pressure. Additionally, in some embodiments, the polymer lens may be used to encapsulate one or more other polymer lenses (e.g., a polymer lens comprising one or more structures).

[0086] In some embodiments, a contact lens as disclosed herein has an elastic modulus of between 0.8 MPa and 1.0 MPa. In some embodiments, the elastic modulus is greater than or equal to 0.8 MPa, greater than or equal to 0.85 MPa, greater than or equal to 0.9 MPa, greater than or equal to 0.95 MPa, or greater than or equal to 1.0 MPa. In some embodiments, the elastic modulus is less than or equal to 1.0 MPa, less than or equal to 0.95 MPa, less than or equal to 0.9 MPa, less than or equal to 0.85 MPa, or less than or equal to 0.8 MPa. Combinations of the above recited ranges are also possible according to some embodiments. For example in some embodiments, the elastic modulus is greater than or equal to 0.8 MPa in less than or equal to 1.0 MPa. In some embodiments, the contact lens has elastic modulus of 0.9 MPa.

[0087] In some embodiments, a contact lens as disclosed herein exhibits a maximal principal strain of less than 1% and no greater than 20%, during mechanical testing (e.g., flipping, folding, expanding, stretching, and twisting) as determined using finite elemental analysis (FEA). Detailed experimental parameters for exemplary tests are provided in the Examples. Accordingly, in some embodiments, the contact lens exhibits a maximal principal strain of less than 0.01%, less than 0.05%, less than 0.1%, less than 0.5%, less than 0.75% or less than 1% of the applied string. In some embodiments, the contact lens exhibits a maximal principal strain of no greater than 0.1%, no greater than 0.5%, no greater than to 0.75%, no greater than 1.0%, no greater than 5%, no greater than 7.5%, no greater than 10%, no greater than 15%, or no greater than 20% of the applied strain.

[0088] In some embodiments, the contact lenes exhibits a maximal principal strain of less than 1% following application of 600 kPa of pressure to a central circular zone (e.g., such as determined via a standard flipping test). In some embodiments, the contact lens exhibits a maximal principal strain of less than 1% when folded along a symmetrical axis (e.g., such as determined via a folding test). In some embodiments, the contact lens exhibits a maximal principal strain of less than 20% when uniaxially stretched (c.g., as determined via a standard stretching test). In some embodiments, the contact lens exhibits a maximal principal strain of less than 20% when radially expanded (e.g., as determined via a standard expansion test). In some embodiments, the contact lens exhibits a maximal principal strain of less than 20% when twisted between +180 degrees and -180 degrees along a symmetrical axis (e.g., as determined via a standard twisting test).

[0089] In some embodiments, a contact lens is configured to measure a current generated when an electrochemically oxidized electrochemically reactive compound (e.g., present on an electrochemical sensor located on the contact lens) oxidizes a target analyte. In some cases, the current generated is proportional to a concentration of the target analyte. In certain embodiments, the contact lens is configured to detect the target analyte at a concentration of between 1 picoM and 100 microM. In some embodiments the contact lens is configured to detect the target analyte at a concentration of greater than or equal to 1 picoM, greater than or equal to 10 picoM, greater than or equal to 20 picoM, greater than or equal to 30 picoM, greater than or equal to 40 picoM, greater than or equal to 50 picoM, greater than or equal to 60 picoM, greater than or equal to 70 picoM, greater than or equal to 80 picoM, greater than or equal to 90 picoM, or greater than or equal to 100 picoM. In other embodiments, the contact lens is configured to detect the target analyte at a concentration of less than or equal to 100 picoM, less than or equal to 90 picoM, less than or equal to 80 picoM, less than or equal to 70 picoM, less than or equal to 60 picoM, less than or equal to 50 picoM, less than or equal to 40 picoM, less than or equal to 30 picoM, less than or equal to 20 picoM, less than or equal to 10 picoM, or less than or equal to 1 picoM. Combinations of the above recited ranges are also possible, according to some embodiments. For example, in some embodiments, the contact lens is configured to detect the target analyte at a concentration of greater than or equal to 1 picoM and less than or equal to 100 picoM.

[0090] In some embodiments, a contact lens has a particular response time. The response time may include an inherent time delay within the electrochemical circuit. The time delay may be due reaction kinetics of the redox reaction (e.g., the redox reaction takes time to build sufficient current that can be measured) and the inherent electrical resistance of the electrodes. In some embodiments, the contact lenses disclosed herein may have a response time of between 10 seconds and 1 minute, for example, when exposed to a target analyte at a concentration of at least 1 microM. In some embodiments, the response time is greater than or equal to 10 seconds, greater than or equal to 20 seconds, greater than or equal to 30 seconds, greater than or equal to 40 seconds, greater than or equal to 50 seconds, or greater than or equal to one minute when exposed to a target analyte at a concentration of at least 1 microM. In other embodiments, the response time is less than or equal to one minute, less than or equal to 50 seconds, less than or equal to 40 seconds, less than or equal to 30 seconds, less than or equal to 20 seconds, or less than or equal to 10 seconds when exposed to the target analyte a concentration of at least 1 microM. In some embodiments, the response time is at least 40 seconds when exposed to the target analyte at a concentration of at least 1 microM. However, the skilled artisan will understand, that other response times are also possible, depending, on the setup of the electrochemical circuit.

[0091] In some embodiments, contact lens can detect an analyte concentration with a sensitivity of between 1 micro Amp s / nanoM and 5 microAmps / nanoM. In some embodiments, the contact lens can detect an analyte concentration with a sensitivity of less than or equal to 1 microAmps / nanoM, less than or equal to 2 microAmps / nanoM, less than or equal to 3 microAmps / nanoM, less than or equal to 4 microAmps / nanoM, or less than or equal to 5 microAmps / nanoM. In some embodiments, the contact lens can detect an analyte concentration of the sensitivity of greater than or equal to 5 microAmps / nanoM, greater than or equal to 4 microAmps / nanoM, greater than or equal to 3, greater than or equal to 2, or greater than or equal to 1 microAmps / nanoM. Other combinations of the above recited ranges are also possible according to other embodiments. For example, in some embodiments, the contact lens can detect analyte concentration with a sensitivity of less than or equal to 1 microAmps / nanoM and greater than or equal to 5 microAmps / nanoM. In some embodiments, the contact lens can detect an analyte concentration with a sensitivity of 3.8 microAmps / nanoM.

[0092] The analyte may be any suitable analyte within any eye fluid (e.g., tear fluid) capable of being detected using contact lenses disclosed herein. Exemplary analytes include, but are not limited to, proteins, simple sugars, carbohydrates, glycoproteins, nucleic acids, amino acids, lipids, enzymes, salts, and water. In some embodiments, the analytes are serotonin, glucose, mucin, cholesterol, triglycerides, lysozyme, lactoferrin, lipcalin, lacritin, immunoglobulin, urea, sodium, potassium, and / or cortisol. In some embodiments, the eye fluid is tear fluid. In some embodiments, a contact lens comprises a battery. Any suitable battery known to the skilled artisan may be used in the contact lenses disclosed herein. In some embodiments, the battery is a micro-battery. In some embodiments, the battery is configured for wireless monitoring.

[0093] Other aspects of the disclosure relate to methods of using one or more of the contact lenses disclosed herein. In some embodiments, the methods relate to exposing a contact lens to a fluid produced by an eye of the subject. In some embodiments, the fluid comprises an analyte (e.g., serotonin). The method, according to other embodiments, comprises electrochemically reacting the analyte with an electrochemical compound and determining a current produced from the electrochemical reaction.

[0094] The contact lenses, as described elsewhere herein, may comprise a first polymer lens and a second polymer lens, in contact with the first polymer lens. In some cases, the second polymer lens may define a hole. In some embodiments, the contact lens comprises an electrochemical circuit comprising an electrochemical sensor. In some embodiments, the electrochemical sensor is in fluidic communication with the hole in the second polymer lens. This configuration is advantageous in accordance with certain embodiments, for example, because it may allow contact between the tear fluid and electrochemical sensor without exposing other pails of the electrochemical circuit to water (e.g., it avoids short circuiting the device).

[0095] In some embodiments, contact lenses may comprise electrochemical sensors comprising a working electrode, which further comprise an electrochemical compound (or electrochemically reactive compound). In some embodiments, the electrochemical compound is any compound capable of undergoing a redox reaction (as described above). Exemplary embodiments of electrochemical compounds, as contemplated herein, include, but are not limited to ferrocene and other organometallic compound, multi- walled carbon nanotubes, porphyrins, graphene oxide, nanodiamonds, metal nanoparticles, and polypyrrole. In some embodiments, the electrochemical compound comprises ferrocene.

[0096] Additionally, contact lenses may further comprise a counter electrode, reference electrode, a potentiostat, and an ammeter. In some embodiments, the electrochemical sensor comprises the working electrode, counter electrode, and reference electrode. The potentiostat, according to some embodiments, electrically connects the working electrode to the counter electrode. In some embodiments, the potentiostat applies a voltage (electric potential) between the working electrode and the counter electrode.

[0097] In some embodiments, the methods comprise exposing the contact lens to a fluid of an eye of a subject. The fluid maybe any fluid obtained from an eye, but most commonly, will comprise tear fluid. Exposing the contact lens may comprise collecting tear fluid using, for example, a capillary tube and placing a drop of collected tear fluid into the hole of the contact lens (e.g., that is in fluidic communication with the electrochemical sensor). Alternatively, exposing the contact lens to the fluid comprising placing the contact lens onto an eye of the subject. Thus, in some embodiments, contact lenses may be configured to be worn by a subject for an extended period of time (e.g., for between 1 day and 7 days).

[0098] In some embodiments, certain methods comprise electrochemically oxidizing an electrochemical compound (e.g., ferrocene), disposed on a surface of a working electrode, to produce an oxidized electrochemical compound (e.g., ferrocenium). In some embodiments, certain methods may allow the oxidized intermediate to oxidize a target analyte (e.g., serotonin) in a target fluid (e.g., tear fluid). In some embodiments, upon oxidizing the analyte, the oxidized electrochemical reactive compound (e.g., ferrocenium) may be reduced back to the starting material (ferrocene). In certain embodiments, a current is generated from the continuous flow of electrons released from the continuous electrochemical oxidation of the electrochemically reactive compound (ferrocene) and its simultaneous reduction by the target analyte (e.g., serotonin). In some embodiments, the methods comprise measuring the current generated from the continuous electrochemical oxidation of the target analyte by the electrochemically oxidized electrochemical compound. In some cases, the current generated (e.g., measured) is proportional to a concentration of the target analyte. For example, as the concentration of target analyte is consumed, the generated current reduces in magnitude (e.g., amperes).

[0099] Accordingly, in some embodiments, some methods relate to applying a voltage to a working electrode comprising an electrochemical compound to electrochemically oxidize the electrochemical compound. As described above, applying a voltage to the working electrode, in the presence of a target analyte, causes an electrochemical compound, disposed on a surface of the working electrode, to undergo a continuous redox reaction. In some embodiments, the electrochemical compound is ferrocene and the oxidized electrochemical compound is ferrocenium. In some embodiments, the target analyte is serotonin. In some embodiments, an applied voltage is needed to trigger electrochemical oxidation of the electrochemical compound. In some embodiments, the applied voltage is between 0 V and 0.5 V. The applied voltage may in some cases, be greater than or equal to 0 V, greater than or equal to 0.1 V, greater than or equal to 0.2 V, greater than or equal to 0.3 V, greater than or equal to 0.4 V, or greater than or equal to 0.5V. In some embodiments, the applied voltage less than or equal to 0.5 V, less than or equal to 0.4 V, less than or equal to 0.3 V, less than or equal to 0.2 V, less than or equal to 0.1 V, or less than or equal to 0 V. Combinations of the above recited ranges are also possible in some embodiments. For example, in some embodiments, the applied voltage is greater than or equal to 0 V and less than or equal to 0.5 V.

[0100] The skilled artisan will understand, that while the instant disclosure demonstrates the ability of certain contact lenses to detect and quantify the concentration of serotonin from tear fluid in accordance with various embodiments, other analytes may also be quantified using the compositions and methods disclosed herein in other embodiments. For example, in some embodiments, a contact lens comprising a different electrochemical compound may be configured to detect one or more analytes, different than serotonin. Exemplary electrochemical compounds include, but are not limited to, multi-walled carbon nanotube (MWCNT), zinc oxide (ZnO), porphyrins (e.g., 15-pentafluorophenyl-10,20-p-aminophyenylprophyrin or meso-tetrakis (2- aminophenyl) porphyrin, etc.), graphene oxide, nanodiamonds, nanoparticles, poly(bromocresol green), magnetic nanoparticles, and / or polypyrrole. Exemplary analytes include, but are not limited to, proteins, simple sugars, carbohydrates, glycoproteins, nucleic acids, amino acids, lipids, enzymes, salts, and water. In some embodiments, the analytes are serotonin, glucose, mucin, cholesterol, triglycerides, lysozyme, lactoferrin, lipcalin, lacritin, immunoglobulin, urea, sodium, potassium, and / or cortisol. In some embodiments, the eye fluid is tear fluid.

[0101] Other methods contemplated herein further relate to determining a concentration of an analyte within a fluid within an eye of a subject by determining an electrical current of an electrochemical sensor within a contact lens on the eye of the subject.

[0102] Additionally, or alternatively, other methods relate determining a concentration of serotonin from a fluid within an eye of a subject, using a contact lens, by measuring a current produced from a reaction of ferrocene contained within the contact lens. U.S. Provisional Patent Application Serial No. 63 / 570,706, filed March 27, 2024, entitled “Lab-on-a-Contact Lens for Non-Invasivc Monitoring,” by Zhu, et al., is incorporated herein by reference in its entirety.

[0103] The following examples are intended to illustrate certain embodiments of the present disclosure, but do not exemplify the full scope of the disclosure.

[0104] EXAMPLES

[0105] Example 1. All-Printed Stretchable and Reversible Lab-on-a-Contact Lens Platform for Non-Invasive Monitoring of Serotonin Dynamics

[0106] This example illustrates the use of the lab-on-a-contact lens (LoCL) platform to analyze the stress-response profile of serotonin in tears, demonstrated through in vitro, ex vivo, and in vivo investigations, facilitating wearable stress analysis and enabling rapid, reliable, and decentralized monitoring of metabolic and physiological states.

[0107] Investigating stress-induced endocrine responses is useful for analyzing human performance. Currently, stress evaluation and diagnosis predominantly employ subjective questionnaires. Traditional clinical methods, such as immunoassays and chromatographic techniques, rely heavily on invasive blood sampling. In pursuit of a non-invasive, continuous approach to monitoring stress at the molecular level, this example describes a flexible lab-on-a- contact lens (LoCL) platform that offers the wearable analysis of the dynamics of the stress hormone serotonin in human tears. This example utilizes an all-printed, stretchable, and reversible electrochemical biosensing system, enhancing sensitivity, selectivity, and efficiency for wearable serotonin monitoring. The device was employed to measure serotonin levels from subjects undergoing the trier social stress test (TSST) and patients with dry eye disease and seasonal affective disorder (SAD). The present example, at least in part, aims to construct a dynamic stress-responsive profile correlating with the fluctuations of serotonin in tears. The ex vivo study in humans revealed a significant empirical correlation between stress profiles and tear serotonin dynamics. Furthermore, an in vivo study reiterated the biosafety and effectiveness of the LoCL in a live porcine model.

[0108] Introduction

[0109] Extensive research has established early life stress as a risk factor in the onset of psychiatric disorders, including major depression, schizophrenia, bipolar disorder, and attention deficit hyperactivity disorder (ADHD) Methodologies such as experience sampling, encompassing questionnaire, and diary studies, have been instrumental in identifying situational contests of stressors in relevant longitudinal stress-response research. However, the inherent variability in these methods, stemming from subjective interpretations, presents a challenge in accurately assessing stress levels. Consequently, the development of quantifiable and objective stress indicators becomes relevant, not only for screening post-traumatic stress disorder (PTSD) in the field but also for evaluating broader mental and somatic health issues.

[0110] The serotonergic system plays a role in modulating a range of behaviors Dysregulation of serotonin is linked to various mental health conditions, including depression and anxiety. Most antidepressants, particularly selective serotonin reuptake inhibitors (SSRIs), target elements of the serotonergic system, with SSRIs focusing on the serotonin transporter (SERT). Central serotonergic activity modulation is a common therapeutic approach in treating numerous psychiatric disorders, notably depression. Recent studies highlight serotonin’s dual role as a hormone and neurotransmitter, implicating its active involvement in the onset and progression of various psychiatric diseases. This positions serotonin as a potential biomarker for investigating mental disorders. However, the exact role of serotonin in the evolution of these psychiatric conditions warrants further exploration. A significant challenge in understanding serotonin dynamics stems from the limitations in non-invasively and continuously monitoring serotonin levels in a clinical setting.

[0111] Prior methods for measuring serotonin have typically relied on liquid- chromatography / mass- spectrometry (LC / MS) and enzyme-linked immunosorbent assay (ELISA) following the solid-phase extraction pre-treatment. However, these methods necessitate prolonged analysis time, incur high costs, and typically require professional expertise or invasive procedures. Smart contact lenses (SCLs) appear promising in identifying potential biomarkers related to human performance and mental health. However, the use of SCLs as sensing devices continues to face limitations due to issues associated with their mechanical softness and biocompatibility, sensitivity, and complexities related to their integrations into existing processes. An ideal SCL should 1) maintain a high degree of transparency, and 2) be comfortable to wear daily or for extended periods. This generates several challenges relating to substrate mechanical stiffness, biocompatibility, lens shape, and surface irregularities that, if not controlled, could lead to eye inflammation and irritation. This example describes an, all-printed stretchable and reversible lab-on-a-contact lens (LoCL) platform, designed for the non-invasivc monitoring of serotonin dynamics. To safeguard the ocular surface, all electronic components are encapsulated within the lens substrate. The platform incorporates serpentine graphene (silver / silver chloride, Ag / AgCl) micropattems as a flexible, biologically stable electrode pattern, ensuring seamless connectivity between the sensor and its readouts. This LoCL system is mechanically flexible, fully biocompatible, cost-effective, and easy-to-implement. The potential significance of serotonin as a biosignature indicative of stress was evaluated with the LoCL system by monitoring tear serotonin dynamics in participants undergoing the trier social stress test (TSST) and patients diagnosed with seasonal affective disorder (SAD), and dry eye disease (DED). Furthermore, the in vivo biosafety and functionality of the LoCL was confirmed in a live porcine model. Taken together, this example provides a new strategy for exploring serotonin dynamics.

[0112] Results

[0113] Example 1.1 Structural design, device fabrication, and operation principle

[0114] FIG. 1A depicts the design of the LoCL system, which features a serpentine conductive pattern and a compact serotonin biosensor. These components are strategically placed at the outer periphery of a commercial disposable contact lens (CL) made of polyhydroxyethylmethacrylate (PHEMA) or polydimethylsiloxane (PDMS). This design covers the iris region while maintaining an unobstructed visual field. The device layout is optimized for wearability, incorporating features such as oxygen permeability, flexibility, and biocompatibility. FIG. IB details the overall device architecture, highlighting the flexible, biocompatible conductive circuitry in a slender serpentine trace (width: 250 pm), which is composed of graphene and silver, and is tactically positioned along the edge of the lens, as further illustrated in FIGs. 7A and 7B.

[0115] The construction of the LoCL platform is depicted in FIG. 1C, utilizing a layer-by-layer assembly process. The lower lens serves as the base layer for optimal contact with the corneal surface. A scalable screen-printing technique facilitates the integration of a serotonin biosensor, which is realized by a three-electrode system. Such a system comprises a working electrode (WE, 2 mm diameter), a counter electrode (CE, 500 microM width), and a reference electrode (RE, 500 microM width). The serpentine conductive trace of WE and CE was printed with soft graphene ink, while the conductive trace of the RE utilizes Ag / AgCl ink. An upper lens, measuring 100 microM, was then applied to encapsulate the sensor, featuring a 5 mm diameter opening to permit tear fluid interaction with the biosensing interface. Detailed information on material selection and fabrication protocol can be found in Example 2.1. FIG. ID illustrates the surface topology of the serotonin biosensing interface, revealing peak heights of approximately 48 pm for WE, 47 pm for CE, and 28 pm for RE. This demonstrates a less than 10% increase in thickness compared to the original lens, effectively addressing potential concerns regarding discomfort from lens edge thickness and variations in lens thickness when wearing CLs.

[0116] Serotonin, a monoamine neurotransmitter in the central nervous system (CNS), is produced from L-tryptophan and stored in vesicles prior to release through serotonergic pathways. In the brain, particularly at the pituitary hypothalamus, serotonin contributes to neuronal synaptic signaling in both the brain and central nervous systems, facilitating its secretion to the lacrimal gland (FIG. IE). As a modulator of the physiological interplay among neurotransmitters such as dopamine and norepinephrine, serotonin significantly influences conditions, including anxiety, stress, depression, and chronic psychiatric disorders. It is reported that serotonin is found to exist in human tears, which is also associated with ocular discomfort and pain. This example reports an all-printed, stretchable, and reversible LoCL platform for the non-invasive monitoring of tear serotonin. It was investigated whether tear serotonin may act as a biosignature of stress, offering insights into physiological and emotional states and thereby holding potential significance in mental health evaluation.

[0117] A serotonin biosensing interface was developed within the LoCL system using ferrocene as a non-enzymatic receptor. This selection is attributed to ferrocene’s rapid electron-transfer rate and stable low-potential redox states. The process involves covalently immobilizing ferrocene onto the LoCL electrode, following activation with 3-(triethoxysilyl)propylsuccinic anhydride (TPSA) activation and subsequent amine (-NH2) modification. Such a configuration allows ferrocene to catalyze the oxidation of serotonin, a mechanism elucidated based on Nicholson and Sham's model. The ferrocene-functionalized electrode is capable of translating serotonin binding into measurable changes in surface charge, indicative of conformational alterations in ferrocene due to the binding events (FIG. IF). This label- and reagent- free approach enables the non- invasive monitoring of serotonin dynamics, allowing electronic measurement of current changes to reflect serotonin dynamics.

[0118] Example 1.2 Surface engineering and biocompatibility evaluation Soft and biocompatible graphene ink was successfully printed on the lens surface with no visible cracks or aggregations, thus ensuring robust biosensing functionality. The surface modifications within the LoCL system included sequential applications of TPSA, -NH2, and ferrocene carboxaldehyde (FeC-CHO) on the printed electrode surface (refer to FIGs. 8A-8D). TPSA, an organosiloxane compound, forms stable covalent bonds with substrate’s -OH groups, serving as an effective linker between the electrode and ferrocene to mitigate any reduction in conductivity due to ferrocene aggregation. The -NH2 groups establish covalent interactions between TPSA and FeC-CHO, facilitating the activation of ferrocene on the electrode surface. Energy-dispersive X-ray spectrometer (EDS) analysis, as illustrated in FIGs. 9-11D , verified the homogeneous and uniform distribution of graphene and ferrocene layers across the lens surface. Detailed information regarding the surface modification is presented in Example 2.2.

[0119] X-ray photoelectron spectroscopy (XPS) was performed to complement and further corroborate the findings from the EDS analysis, focusing on characterizing the surface morphology (as shown in FIG. 2A and FIGs. 12A-12D). The Fe 2p spectra distinctly revealed peaks at 708.4 eV, indicative of FeC-CHO molecules within the biosensing interface, aligning with the EDS results. Both pristine graphene and modified electrodes exhibited an absence of Fe 2p peaks. The C is and O ls spectra showcased bonds consistent with carboxyl C=O, O-C=O, hydroxyl C-O, and hydrocarbon C-C / C-H bonds at each modification stage. The Si 2p peak at 103.11 eV and N Is peak at 399.79 eV were confirmatory of TPSA and amine functionalization. Collectively, the EDS and XPS analyses corroborated the successful assembly of the LoCL platform, highlighting effective surface activation and ferrocene immobilization (FIG. 2B).

[0120] FIGs. 13A-13E, presenting contact angle analysis, confirm that the surface engineering techniques preserved the hydrophilicity of the CL. Further, as shown in FIG. 14, optical transparency measurements conducted within the pupil area (wavelength: 400-800 nm) revealed that the final LoCL system maintains over 95% transparency, ensuring their functionality across the visible light spectrum. In addition, FIGs. 15A-15C demonstrate minimal differences in mechanical properties following each modification step. The elastic modulus of the LoCL, approximately 0.9 MPa, is on par with that of the human cornea surface, which ranges from 0.1 to 1.5 MPa, thus highlighting its biomechanical compatibility.

[0121] Ensuring a direct and conformal contact between the LoCL and the eye necessitates a focus on wearability and comfort, emphasizing biocompatibility, flexibility, and stretchability. Biocompatibility, a vital factor for on-eye applications, was substantiated through in vitro tests with fibroblast cells, demonstrating low cytotoxicity. Fluorescent livc / dcad staining images, as shown in FIG. 2C, indicated that the final LoCL system had no detrimental effects on cell proliferation and spreading over a one-week period. Quantitative assessments revealed consistently high cell viability, exceeding 90% after 7 days, with no significant variances observed among the groups tested (FIG. 2D). In addition, cell metabolic activity within the LoCL system markedly increased from day 1 to day 7, showing no significant deviations from the control group(****p<0.0001), as depicted in FIG. 2E. Detailed biocompatibility assay information is provided in Example 2.3.

[0122] Example 1.3 Mechanics analysis under different loading scenarios

[0123] The flexibility and stretchability of the LoCL are useful parameters for reducing the risks of mechanical or electrical failure during various operational conditions, such as handling, cleaning, storage, and fitting of the lens. FIGs. 3A-3E illustrate the deformation and strain distributions of the LoCL, as determined by both experimental observations and finite element analysis (FEA) under five distinct loading scenarios: i) flipping, ii) folding, iii) radial expanding (-10%), iv) uniaxial stretching (-20%), and v) twisting (180° and -180°). For a comprehensive overview of the FEA simulation, refer to Example 2.4. The data reveal that the electrodes and conductive traces within the LoCL sustain maximal principal strain (cmax) values under 1 % for all these conditions, indicating a low likelihood of structural failure in all assessed modes of deformation For example, in the flipping test (with applied pressure of 600 kPa), the LoCL demonstrated minimal deformation, with a maximum principal strain under 0.2% (FIG. 3A). In the folding test, where the LoCL was folded along its symmetrical axis, the strain was predominantly localized along the fold line, yet did not exceed 0.1% (FIG. 3B). During tests of uniaxial stretching and radial expansion, the maximum principal strain consistently remained below 20% (FIGs. 3C and 3D). The mechanical robustness of the LoCL was further validated in a twisting test, which confirmed that the maximum principal strain was maintained as low as 20% even when twisted to 180° or -180° (FIG. 3E and FIG. 16).

[0124] Example 1.4 In vitro quantitative assessment

[0125] The performance of the LoCL system in monitoring serotonin was evaluated through benchtop tests, as shown in FIGs. 17A-17D, using cyclic voltammetry (CV) under optimized conditions (5 scan cycles, 100 mV / s scan rate). Quantitative analysis of serotonin dynamics was based on the CV curves obtained. FIGs. 18A-19D and Examples 2.S-2.6 provide a detailed account of the optimization process. Before initiating serotonin monitoring, the redox reaction of ferrocene in the LoCL system was verified in buffer solutions containing electrolytes. The CV curves of the ferrocene / amine / TPSA-modified LoCL at various scan rates (20-500 mV) are detailed in FIG. 19C. The observed linear increase in redox peak currents in relation to scan rate (R2= 0.982 and 0.975, respectively) aligns with the Randles-Sevcik equation (FIG. 4A), indicating that the serotonin biosensing mechanism on the LoCL platform is diffusion-controlled.

[0126] Under the optimized conditions, the LoCL system demonstrated accurate serotonin detection capabilities. An oxidation peak current (Ipa) was observed at 0.53 V, which can be attributed to the electrochemical oxidation of FeC+coinciding with serotonin oxidation at this potential, as shown in FIG. 19D. The absence of an oxidation peak at 0.53V in buffer fluid, which serves as a negative control, underscores the system’s specificity to serotonin. In contrast, LoCL systems without functionalization showed no redox reactions in serotonin solutions at concentrations of 1, 10, and 100 microM, as evidenced in FIG. 18D.

[0127] FIG. 4B illustrates the LoCL’s high responsiveness to varying serotonin concentrations found in human tears, ranging from 5 nM to 300 nM. The system shows a linear increase in current (R2= 0.997, sensitivity: 3.97 microA / nM) within buffer solutions spanning from 1 pM to 0.5 microM. FIGs. 20A and 20B further demonstrate comparable linearity (R2=0.995) but with an enhanced sensitivity (21.6 microA / nM) in artificial tear fluids ranging from 1 pM to 1 microM, indicating a sensitivity nearly six times greater than that in buffer fluids. FIG. 4C underscores the efficient serotonin monitoring capability of the LoCL in both fluids, achieving a very low detection limit(LoD) of 1 pM. Moreover, the LoCL is capable of detecting serotonin across a wide concentration range, spanning seven orders of magnitude (1 pM to 10 microM). These results validate the efficacy of the LoCL in various settings, including the complex milieu of human tears.

[0128] The lab-on-a-contact lens (LoCL) platform also demonstrates a rapid response to serotonin, achieving stabilization in less than 40 seconds upon exposure to a 1 microM serotonin solution, as indicated in FIGs. 20A and 20B. FIG. 4D shows the LoCL’s response time over various concentrations (500 pM to 500 nM), effectively encompassing the typical serotonin levels in human tears. The system consistently shows prompt response times, measured in seconds, which aligns with the physiological fluctuation range of serotonin (pM to microM), thereby underscoring its practicality for real-world applications. In addition, FIG. 4E presents real-time current variations in response to increasing serotonin concentrations (500 pM to 500 nM). Also, the LoCL demonstrates no reaction to artificial tear fluids (contact lens solution) without serotonin and common lubricant eye drops, suggesting that regular storage conditions will not impair the functionality of the LoCL.

[0129] The selectivity of the LoCL was also investigated by examining its response to interfering molecules structurally similar to serotonin, such as urea, uric acid (UA), dopamine (DA), and ascorbic acid (AA), within their physiological concentration ranges in tears. The LoCL displayed minimal response to these non-target molecules in comparison to serotonin at a concentration of 0.1 microM, a level representative of its physiological presence in tears (FIG. 4F). This high selectivity is attributed to the unique peak potential for serotonin oxidation, which does not overlap with the oxidation potentials of these interferents. As a result, the common electroactive components in biological tear fluids had a negligible impact on the current measurement. Consequently, the LoCL demonstrated consistent and stable performance in sensing serotonin, even when introduced in a complete matrix environment, without necessitating any preliminary separation processes.

[0130] The influence of tear pH on the functionality of the LoCL system was also explored. Given that the normal pH range of tears is between 6.5 and 7.4, the LoCLs were subjected to four distinct pH environments (pH = 6.0, 6.5, 7.0, and 7.5) for durations of up to 12 hours. The LoCLs' responses to varying concentrations of serotonin (0.1, 0.5, and 1 microM) exhibited no reduction in sensitivity across these pH conditions, as depicted in FIG. 4G, FIG. 20A, and FIG. 20B. This indicates their robust stability and reliability. Furthermore, extended testing over a two-week period maintained consistent sensitivity, underscoring the long-term stability of the LoCL's biosensing interface, as shown in FIG. 4H.

[0131] The durability of the LoCL's core signal transduction mechanism was further assessed, specifically for serotonin monitoring, under repetitive mechanical deformations, as illustrated in FIG. 41. The electrode, designed in a serpentine pattern that accommodates matrix deformation through buckling, maintained its sensitivity to serotonin consistently, even after undergoing 1000 bending cycles (with a radius of curvature of 6 mm). This observation is consistent with the mechanical simulation findings. In addition, post- 1000 bending cycles, the LoCL showed no visible cracks or defects, thereby emphasizing its mechanical flexibility and stability, a feature further documented in FIG. 23.

[0132] Another feature of the LoCL system is its capability for reversible and resettable monitoring, a benefit arising from the integration of non-enzymatic biosensing coupled with reversible redox reactions. This reversibility attribute was evaluated through 100 washing cycles, as shown in FIGs. 4J-4K, and by testing 1000 samples each containing 100 nM serotonin, as depicted in FIG. 4L. The results demonstrated that the system retained 95% of its initial response capability even after 1000 uses. In addition, the system exhibited great long-term stability, maintaining almost identical responses over three consecutive days of daily testing, as illustrated in FIGs. 24A-24D. This performance is attributed to the reversible nature of the redox reactions and the robustness of the covalently bonded surface of the biosensing interface.

[0133] Example 1.5 Ex vivo study in human tears

[0134] Both physiological and psychosocial stressors are known to impact serotonin levels, often leading to a transient decrease. The ability to monitor serotonin levels in readily accessible peripheral biofluids opens new avenues for personalized, non-invasive digital healthcare. This approach is particularly valuable given the significant individual variations in stress response. Such monitoring holds immense promise for advancing digital healthcare and the field of precision medicine.

[0135] The Trier Social Stress Test (TSST), a well-recognized laboratory method known for effectively inducing stress in human subjects, was utilized to establish a serotonin response profile under stress conditions. The TSST encompassed several stages: (1) adaptation to the test environment, (2) a waiting period during which participants were briefed about the forthcoming task, (3) a stress-induction phase involving a speech and a challenging arithmetic task in front of two supervisors, followed by (4) a recovery phase (FIG. 5A). Tear samples were collected from 10 healthy volunteers (Male / Female ratio = 1 / 1) at three intervals: during the waiting period, 5 minutes during the stress, and 35 minutes post-stress. These samples were analyzed using the LoCL system. Further details on the TSST procedure are elaborated in Examples 2.7 and 2.8. FIG. 5B illustrates the serotonin response profile detected by the LoCL during the TSST, showing a decrease in Ipa during stress and a subsequent increase towards baseline levels during recovery. In FIG. 5C, a reduction in tear fluid serotonin levels was observed in response to stress. Clinical studies suggest a potential mechanism wherein stress and depression, marked by elevated circulating cortisol levels, may up-regulate the expression of the serotonin transporter, thereby reducing serotonin availability and leading to impaired serotonergic ncurotransmission (increased serotonin uptake). These levels returned to normal during the recovery period. To confirm these changes in serotonin concentration, the responses were recalculated using the linearity graph equation presented in FIG. 21D. This observation corroborates previous research that associates reduced serotonin levels with an intensified stress response.

[0136] The LoCL system also shows promise as an analytical tool for investigating the link between psychological stress and dry eye disease (DED). DED is a chronic, progressive condition that may initially be minimally symptomatic but tends to worsen over time. It is characterized by a reduction in tear quantity and quality, abnormal ocular surfaces, and discomforting symptoms. These symptoms, including sensations of a foreign body in the eye, itching, redness, ocular pain, discomfort, and blurred vision, may not be severe but significantly diminish the patient's quality of life and adversely affect their mood and mental health. Clinical studies have suggested that the severity of these symptoms is often correlated with individual pain perception and psychosomatic conditions such as stress, depression, and anxiety. While recent advances in DED diagnostics include questionnaires, chief complaint assessments, and laboratory tests (such as the Schirmer test, tear osmolarity, and biomarker assessments for metalloproteinase-9 or lactoferrin), the aspect of mental health in DED patients has been less explored. The LoCL is poised to fulfill a clinical need for standardized, reliable home-based systems that monitor the psychosomatic status (e.g., depression, stress, and anxiety) in DED patients by tracking changes in tear serotonin levels.

[0137] The 5-item Dry Eye Questionnaire (DEQ-5) was used to evaluate symptoms of ocular dryness and to differentiate among varying self-assessed severity levels and among patients diagnosed with DED, as detailed in Table 2. FIGs. 5E and 5F, along with FIGs. 25A-25C, reveal a negative correlation between serotonin concentrations and DEQ-5 scores. This suggests an increase in stress levels corresponding with higher DEQ-5 scores. These results underscore the effectiveness of the LoCL in tracking and quantifying stress and depression in DED patients by measuring tear serotonin dynamics. Previous clinical studies have suggested that tear serotonin levels could serve as a surrogate biomarker for corneal nociceptor sensitization, thus potentially relating to DED symptoms and signs. This study highlights the LoCL's potential as a non-invasive diagnostic tool for identifying and monitoring the mental health of individuals with chronic ocular and systemic diseases, such as DED. In addition, the LoCL's reversible sensing capabilities were validated through repeated measurements on both normal and DED tear samples. FIG. 26 demonstrates the system's consistent performance over multiple tests, indicating minimal variance even after repeated usage of the LoCL systems.

[0138] Example 1.6 In vivo study in pig eye

[0139] The in vivo sensing capabilities of the LoCL system were evaluated using an anesthetized pig's eye (Female; 6 months old), with details illustrated in FIG. 6A and further described in Example 2.9. The pig's eye was chosen due to its anatomical resemblance to the human eye, as shown in FIGs. 6B and 6C. FIG. 6D demonstrates the application protocol, successfully depicting the LoCL's conformal fit on the pig's ocular surface. Post-removal biomicroscopy of the pig's eye revealed no signs of infection or inflammation. Throughout the duration of the LoCL wear, the ocular surface temperature was continuously monitored using an infrared camera, as presented in FIGs. 6E and 6F. The temperature remained stable at around 31°C, with minimal fluctuations, underscoring the LoCL's non-inflammatory characteristics. In addition, no adverse effects on the pig's ocular or systemic health was observed one month after wearing the LoCL, further affirming the safety of the LoCL's on-eye operation.

[0140] In a further in vivo experiment, artificial tear solutions with serotonin concentrations of 50 nM and 100 nM were sequentially administered onto a pig's eye equipped with the LoCL. Prior to application, these solutions were carefully prepared with the specified serotonin concentrations to validate the LoCL system’s ability to detect fluctuating serotonin levels in vivo. As demonstrated in FIG. 6G, the serotonin measurements obtained with the LoCL correlated well with the concentrations in the prepared serotonin solutions. The amperometric response of the LoCL decreased when the serotonin solution concentration was reduced from 100 nM to 50 nM, indicating reversibility in a living organism. Furthermore, the LoCL displayed a near- zero response when returned to its initial state, confirming its reliability and desired functional characteristics.

[0141] Moreover, histological assessments confirmed that utilization of the LoCL does not provoke ocular inflammation or irritation (as detailed in Example 2.10). Detailed examination reveals an unblemished corneal epithelium devoid of any disruptions or fissures indicative of corneal abrasion, and an absence of lymphocytic infiltration within any corneal stratum (FIG. 6H). The iris is distinguished by its pigmented stromal constitution and a more substantial pigmented epithelial layer; it remains intact alongside the pupillary sphincter, which is discernible within the iris as an eosinophilic stratum containing basophilic, non-pigmented cell bodies (FIG. 61). Further, both the sclera and the ciliary muscle exhibit an eosinophilic appearance, free from any scarring or damage and devoid of lymphocytic infiltration (FIG 6J). The retina remains intact, backed with multiple strata of pigmented epithelium (FIG 6K). The preservation of normal histologic features in these tissues following multiple acute applications implies the safety of the LoCL.

[0142] Example 1.7 Discussion

[0143] This example introduces an innovative platform technology that transforms readily available disposable contact lenses into advanced LoCL systems, specifically designed for the non-invasive monitoring of mental health indicators. The scalable printing of functional electronics onto conventional contact lenses integrates sensing capabilities without significantly impacting the lenses' inherent properties, such as flexibility, biocompatibility, softness, oxygen permeability, optical transparency, and wettability. The application of an electrochemical anchoring approach significantly improves mechanical durability and chemical stability, fulfilling some requirements for lens fitting, handling, cleaning, and disinfection. Utilizing commercially available contact lenses as the base ensures that the device can seamlessly adapt to a variety of comeal shapes, just like standard commercial lenses, thereby offering superior comfort and safety for on-eye use compared to traditional methods of serotonin analysis.

[0144] The LoCL system exemplifies a reversible biosensing technology for the non-invasive monitoring of serotonin, with its efficacy validated through comprehensive in vitro studies, ex vivo human pilot experiments, and in vivo animal testing. It boasts an ultra-low detection limit of 1 pM and a wide detection concentration range (1 pM-100 microM) and retains functional integrity in realistic tear environments (pH: 6.0-8.0) for up to 14 days. Moreover, the system maintains stability and reliability even after 1000 cycles of repetitive use and withstands various mechanical stressors such as flipping, folding, expanding, stretching, and twisting. The costeffectiveness of screen printing facilitates efficient high-throughput production. This example presents a pioneering lab-on-a-contact lens system, which holds significant potential for customization to meet a wide array of clinical requirements, particularly in the realm of wearable mental health monitoring. This contact lenses in this example represents a step forward in the development of multifunctional wearable diagnostic tools. It not only facilitates detailed monitoring of tear physiology but also contributes significantly to the advancement of home-based telehealth. This work establishes a foundation for an array of wearable ocular instruments capable of providing rich data about human health, thereby paving the way for the emergence of smart contact lenses in diagnostic and therapeutic applications.

[0145] Example 1.8 Materials and Methods

[0146] Experimental design

[0147] In this example, the design of the LoCL system was developed using CorelDRAW 2020 software. Laser engraving (Universal Laser System, United States) was employed to create the masks necessary for the screen-printing process. The incorporation of electrodes and serpentine interconnects into the LoCL was achieved by printing with conductive graphene ink (Nanochemazon, Canada) and Ag / AgCl ink (ERCON, United States). The animal study protocol received approval from the Institutional Animal Care and Use Committee of the Lundquist Institute (IRB#22809-02). Moreover, the evaluation of human tear samples, involving both healthy subjects and patients diagnosed with DED and SAD, was conducted under protocol sanctioned by the institutional review board at Indiana University (IRB#2004308902).

[0148] Statistical analysis

[0149] All the experiments in this example were replicated more than three times. One-sided statistical assessments were conducted employing a one-way or two-way analysis of variance (ANOVA). These statistical analyses were carried out in OriginPro (2021, Northampton, MA).

[0150] Example 2. Supporting Information for Example 1.

[0151] Example 2.1. Fabrication protocol of the LoCL system

[0152] Materials and reagents

[0153] Serotonin, ferrocene carboxaldehyde (FeC-CHO), ascorbic acid, urea, uric acid, dopamine, potassium chloride (KC1), ferricyanide [Fe(CN)6]3’, ferrocyanide [Fe(CN)6]4, hydrochloric acid (HC1), sodium hydroxide (NaOH) solution, ethanol, methanol, and (3- Aminopropyl)triethoxysilane (APTES) were purchased from Sigma Aldrich (United States). Sylgard 184 silicone elastomer kit was purchased from Dow Coming (United States). Silver / silver chloride (Ag / AgCl) ink and graphene conductive ink were purchased from ERCON (E2414, United States) and Nanochemazon (NCZ-VA- 150 / 21 , Canada), respectively. 3- (tricthoxysilyl)propylsuccinic anhydride (TPSA) was purchased from TCI Chemicals (United States). A phosphate-buffered saline (lx PBS, pH 7.4) solution was purchased from Gibco. Distilled water (DW) generated by Milli-Q (United States) was used.

[0154] Fabrication protocol of contact lens

[0155] Silicone contact lens'. The Sylgard 184 silicone elastomer and curing agent were combined at a ratio of 10:1. This mixture was thoroughly blended and then degassed to remove bubbles. Following this preparation, it was poured into a mold formed by two concentric stainless- steel hemispheres. The assembly was then placed in a vacuum oven, where it underwent a curing process at 80 °C for 60 minutes.

[0156] PHEMA contact lens: The PHEMA contact lenses were created using a free -radical polymerization process, which involved UV cross-linking. The process began by preparing a homogeneous mixture comprising 3 mL of 2-hydroxyethyl methacrylate (HEMA, 98%, Sigma- Aldrich, MO USA), 25 microL of ethylene glycol dimethacrylate as the cross-linker (EGDMA, 98%, Sigma- Aldrich, MO USA), and 125 microL of 2-hydroxy-2-methylpropiophenone as the photoinitiator (Darocur 1173, 97%, Sigma- Aldrich, MO USA). This monomer blend was then poured into a contact lens-shaped polypropylene mold. The polymerization was achieved by exposing the mold to UV light at a wavelength of 365 nm for 60 minutes. Following polymerization, the contact lenses were fabricated by a punching process.

[0157] Printing of stretchable electrodes

[0158] Electrode and interconnect patterns were designed using the CorelDRAW 2020 software. Following this, a shadow mask with a thickness of 100 microM was fabricated using a laser engraver (Universal Laser System, United States). The lens was then coated with Ag / AgCl conductive ink (ERCON, USA) and underwent a curing process at 80 °C for 30 minutes. After this step, graphene ink (Nanochemazone, Canada) was printed to the lens surface using another shadow mask and then cured at 80 °C for 30 minutes.

[0159] Example 2.2. Surface functionalization of the LoCL system

[0160] The surface functionalization process of the LoCL system involved several steps:

[0161] (1) Initially, a 10% v / v solution of 3-triethoxysilyl propylsuccinic anhydride (TPSA) was prepared by mixing it with deionized water, followed by a 2-hour sonication. Then, 1.5 microL of this TPS A solution was drop-cast onto the working electrode of the biosensor and left to incubate overnight at 4 °C.

[0162] (2) Afterward, 1 microL of a 3% (w / v) solution of (3-Aminopropyl)triethoxysilane (APTES) was applied to the working electrode. The surface was then rinsed with ethanol and annealed at 80 °C for 1 hour.

[0163] (3) Post-surface modification, a 50 mM FeC-CHO solution (1 microL), prepared by dissolution in methanol and a 10-minute sonication, was uniformly drop-casted on the amine- modified working electrode and incubated at room temperature. The LoCL's upper lens layer, designed to encapsulate the serpentine structure and secure the wires to the sensor, was positioned over the bottom lens layer. An opening of 5 mm diameter was made in the upper lens layer to allow tear fluid to interact with the biosensing interface. The integration of both upper and bottom lenses was achieved through a precursor- and oxygen plasma-bonded encapsulation process, as detailed in previous works. This also included connecting the wires to the contact pad for instrumental interfacing.

[0164] Example 2.3. Cytocompatibility assay

[0165] The cytocompatibility assessment was conducted using human dermal fibroblast (HDF) cells (ATCC, USA) in the presence of UV-sterilized devices. HDF cells, maintained at a confluency of > 90%, were trypsinized using 0.25% trypsin-EDTA (Invitrogen, USA) and counted with an automated cell counter (Countess 3, Invitrogen, USA). A suspension was then prepared in DMEM, supplemented with 10% v / v fetal bovine serum and 1% v / v penicillinstreptomycin, and seeded in a 12-well plate at 5,000 cells / well. The cells were incubated at 37 °C with -95% humidity and 5% CO2 (Thermo Fisher Scientific, USA). After 24 hours, UV- sterilized devices were placed in wells containing 2 mL of culture media, with device-free wells serving as controls.

[0166] Cell metabolic activity and proliferation were assessed using a PrestoBlue cell viability assay after 1 and 7 days of culture. Following medium removal, each well received 1 mL of diluted PrestoBlue reagent (lOx dilution in DMEM), incubated for about 1.5 hours at 37 °C in the dark. Then, 100 microL of supernatant from each well was transferred to a 96-well plate for fluorescence measurement at 530 / 590 nm excitation / emission (BioTek Synergy 2, USA).

[0167] On day 7, the devices were removed, and cells were rinsed with DPBS. Staining was performed using a LIVE / DEAD viability / cytotoxicity kit (Invitrogen, USA), following the manufacturer's instructions. The staining solution, containing ethidium homodimer-1 and calccin-AM in DPBS, was added to each well and incubated in the dark for 10 minutes. Fluorescence images were captured using a Keyence (bz-x710) microscope, and cell viability (ratio of live to total cells) was quantified using ImageJ software.

[0168] Example 2.4. FEA simulation

[0169] The FEA simulation was conducted using commercial software ABAQUS. The LoCL device was meshed by 8-node 3D solid stress elements (C3D8R) with fine size (-90,000 elements used), ensuring at least 3 elements distributed along the width of the serpentine. The device was loaded by a displacement control method except in the flipping case, where the clamped parts were coupled to control points and displacements were assigned to them. For flipping, a normal pressure (600 kPa) was applied in the central circular zone. The Young’s moduli of PDMS, silver and graphene ink are EPDMS= 750 kPa, EAg= 20 GPa, EGp= 470 GPa, respectively. The Poisson’s ratio of PDMS, silver and graphene ink are vPDMS= 0.49, vAg= 0.4, vGp= 0.197, respectively.

[0170] Example 2.5. LoCL in vitro characterization

[0171] Surface analysis. The surface morphology of the biosensor was analyzed using X-ray high-resolution scanning electron microscopy (XHR-SEM). This analysis was conducted with the FE1 Magellan 400 XHR SEM system, operating at a voltage of 10 kV. To optimize the imaging quality, all samples were subjected to sputter coating with a 5 nm thick layer of Iridium (Ir) using an EMS150T Iridium sputter coater from Electron Microscopy Sciences. Elemental characterization at various stages of WE surface modification was confirmed via field emission scanning electron microscopy (FE-SEM) equipped with an integrated energy-dispersive X-ray spectrometer (EDS) on the TESCAN MIRA3 instrument, located in Brno. Additionally, X-ray photoelectron spectroscopy (XPS) analysis was carried out using a Kratos Analytical AXIS Ultra Delay-Line Detector (DLD) Imaging XPS system, featuring an Al Ka X-ray source (1486.6 eV) and a 165-mm mean radius electron energy hemispherical analyzer. The vacuum pressure during the acquisition process was maintained below 3xl0‘9torr.

[0172] Electrochemical characterization. The electrochemical properties of the LoCLs, including both unfunctionalized and functionalized versions, were examined using a CH Instruments electrochemical workstation. The influence of surface modification on the working electrode (WE) of the LoCL platform was investigated via cyclic voltammetry (CV). For optimization, buffer solutions (lx phosphate-buffered saline) at pH 7.4 containing 0.1 M KC1 and 5 mM [Fc(CN)6]3’ / 4‘ were used. The CV sensing performance parameters involved a scan range from -0.6 to 0.8 V to adequately observe the redox reactions on the electrode, a scan rate of 100 mV / s, and the procedure included 5 CV cycles. All the experimental protocols were carried out under ambient conditions.

[0173] Selectivity test. The assessment of selectivity involved examining the sensor's response to potential 50 pM interferents such as urea, uric acid (UA), dopamine (DA), ascorbic acid (AA), and artificial tears (AT). These responses were compared to the sensor’s response to 1 pM serotonin. In addition, the sensor's stability across various pH ranges was tested using buffer fluid solutions with pH values between 6.0 and 8.0. These pH levels were obtained by mixing HC1 and NaOH solutions with lx PBS solution (pH 7.4). The pH of these solutions was verified using a commercial pH meter (SevenCompact, Mettler Toledo) before starting the experiments. Serotonin solutions of three different concentrations (0.1, 0.5, and 1 pM) were prepared in buffer solutions at five distinct pH levels for testing. Furthermore, the long-term stability of the sensor was evaluated over a period of two weeks, where the sensors were stored at 4 °C before being used for serotonin detection. All experimental procedures were conducted at room temperature.

[0174] Mechanical flexibility. The evaluation of the sensor's mechanical flexibility was conducted using a computer-controlled actuating system (Instron 5943, USA). The sensor was mounted on a stationary stage at one end, while the other end was attached to a movable stage. Bending tests were performed, achieving a maximum curvature radius of 6 mm at a rate of 300 mm / minute. Each bending cycle comprises compressing and then elongating the sensor. This bending procedure was methodically repeated for a total of 1,000 cycles. To assess the sensor's mechanical stability, evaluations were carried out at regular intervals after 100, 300, 500, 800, and 1,000 cycles.

[0175] Example 2.6. In vitro evaluation of serotonin sensing performance of LoCL

[0176] Sensing performance of serotonin in buffer fluid. The LoCL system was employed for a quantitative evaluation of its serotonin sensing capabilities in buffer fluid (lx PBS, pH 7.4). Key performance parameters such as response time, sensitivity, linearity, selectivity, and stability were assessed. As shown in FIGs. 20A and 20B, the functionalized LoCL system demonstrated an exponential increase in electrical current over extended incubation times. During the optimization process, the LoCL system was incubated with various serotonin concentrations ranging from 0 to 100 pM. The resulting peak current (Ipa) at 0.53 V correlated with the serotonin concentration, displaying a highly linear relationship (R2= 0.997).

[0177] Sensing performance of serotonin in artificial tear fluid. The sensing proficiency of the LoCL system was rigorously tested using commercially available artificial tears, branded as HSKLOCK. This evaluation focused on monitoring serotonin concentrations within the artificial tears, spanning a range of 0 to 100 M.

[0178] Example 2.7. Collection of human tears

[0179] Tear' samples were collected from a group of 10 participants. The collection process involved the use of microcapillary tubes, which were carefully positioned at the corners of the subjects' eyes to utilize capillary action for gathering tears. These collected tear samples were then analyzed using the LoCL system to quantify serotonin levels. The comprehensive analysis of these human tear samples was conducted with adult subjects, strictly following a protocol approved by the Institutional Review Board at Indiana University (IRB#2 004 308 902).

[0180] Example 2.8. Trier Social Stress Test (TSST) protocol

[0181] The Trier Social Stress Test (TSST) was conducted with 10 healthy volunteers, consisting of an equal distribution of 5 males and 5 females, in a controlled laboratory setting at room temperature. Before beginning the TSST, the participants spent 30 minutes in a calm room to become accustomed to the test environment. Tear collection was initiated prior to the start of the TSST. The TSST, lasting for 15 minutes, involved inducing stress in participants through a speech task and verbal arithmetic challenges, all overseen by two supervisors. Immediately after the TSST, a second round of tear collection was conducted for each participant. This was followed by a 30-minute recovery period in a separate waiting room, after which a final tear collection session took place. To ensure the integrity of the samples, each tear sample was gathered in an individual microcapillary tube and securely sealed with parafilm to prevent contamination.

[0182] Example 2.9. In vivo validation of the LoCL on a porcine model

[0183] The study protocol received approval from the Animal Committee of Lundquist Institute. The LoCL was gently positioned on the pig's eye and fitted onto its cornea. The pig's eye underwent continuous monitoring using a high-resolution infrared camera (A300; FLIR) while the LoCL was in operation. In a separate trial, an artificial tear solution containing serotonin concentrations of 50 nM and 100 nM was instilled onto the contact lens following its placement on a live pig's eye. For this particular experiment, a custom serotonin solution was combined with a commercial artificial tear solution to achieve the desired concentrations, and the mixture was applied for 1 minute.

[0184] Example 2.10. Histology evaluation Post-mortem, porcine eyes were meticulously excised to evaluate the impact of the LoCL on ocular integrity. These specimens were subsequently preserved in a 10% neutral buffered formalin solution. After fixation, the eyes were embedded in paraffin, and sections of 10 pm thickness were carefully prepared on Fisherbrand Superfrost slides (Fisher Scientific #22-037- 246). These sections underwent a deparaffinization process using a graduated xylenes and ethanol series, and were then subjected to staining with Harris Hematoxylin (Poly Scientific #s212A) and Eosin Phloxine (Poly Scientific #s 176) for detailed histological examination. The final step involved rehydration of the slides in a graduated series of ethanol and xylene, followed by sealing with Permount mounting medium (Fisher Scientific #SP15-100) to prepare them for microscopic analysis.

[0185] Table 1. Comparison of serotonin biosensing devices Abbreviations: LoD, Limit of detection.

[0186] Table 2. Detailed information on the tear samples from healthy subjects and patients with DED.

[0187] Abbreviations: DED, Dry eye syndrome; DEQ-5 Score, 5-Item Dry Eye Questionnaire.

[0188] EQUIVALENTS AND SCOPE While several embodiments of the present disclosure have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the present disclosure. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the teachings of the present disclosure is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the disclosure may be practiced otherwise than as specifically described and claimed. The present disclosure is directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the scope of the present disclosure.

[0189] In cases where the present specification and a document incorporated by reference include conflicting and / or inconsistent disclosure, the present specification shall control. If two or more documents incorporated by reference include conflicting and / or inconsistent disclosure with respect to each other, then the document having the later effective date shall control.

[0190] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0191] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0192] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0193] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.”

[0194] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0195] When the word “about” is used herein in reference to a number, it should be understood that still another embodiment of the disclosure includes that number not modified by the presence of the word “about.”

[0196] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0197] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims

What is claimed is:CLAIMS1. A contact lens, comprising: a first polymer lens; a second polymer lens defining a hole, in contact with the first polymer lens; and an electrochemical sensor in fluidic communication with the hole in the second polymer lens.

2. The contact lens of claim 1, wherein the electrochemical sensor comprises a working electrode.

3. The contact lens of claim 2, wherein the working electrode comprises graphene.

4. The contact lens of claim 2 or 3, wherein the working electrode comprises gold.

5. The contact lens of any one of claims 2-4, wherein the working electrode comprises platinum.

6. The contact lens of any one of claims 2-5, wherein the working electrode comprises a 3-triethyoxysilyl propylsuccindyl-group.

7. The contact lens of any one of claims 2-6, wherein the working electrode comprises a 3-aminopropyl triethoxysilyl-group.

8. The contact lens of any one of claims 1-7, wherein the electrochemical sensor comprises a counter electrode.

9. The contact lens of claim 8, wherein the counter electrode comprises graphene.

10. The contact lens of claim 8 or 9, wherein the counter electrode comprises gold.

11. The contact lens of any one of claims 8-10 wherein the counter electrode comprises platinum.

12. The contact lens of any one of claims 1-11, wherein the electrochemical sensor comprises a reference electrode.

13. The contact lens of claim 12, wherein the reference electrode comprises silver.

14. The contact lens of claim 12 or 13, wherein the reference electrode comprises sliver chloride.

15. The contact lens of any one of claims 12-14, wherein the electrochemical sensor has an average thickness of between 45 microns and 55 microns.

16. The contact lens of any one of claims 12-15, wherein the electrochemical sensor has a total area of between 35 mm2and 40 mm2.

17. The contact lens of any one of claims 1-16, further comprising an electrochemical circuit.

18. The contact lens of claim 17, wherein the electrochemical circuit comprises the electrochemical sensor.

19. The contact lens of claim 18, wherein the electrochemical circuit comprises a potentiostat.

20. The contact lens of claim 19, wherein the potentiostat electrically connects the working electrode to the counter electrode.

21. The contact lens of claim 19 or 20, wherein the potentiostat is configured to apply an electric potential between the working electrode and the counter electrode.

22. The contact lens of any one of claims 17-21, wherein the electrochemical circuit comprises an ammeter.

23. The contact lens of claim 22, wherein the ammeter is electrically connected to the working electrode and the counter electrode.

24. The contact lens of claim 22 or 23, wherein the ammeter measures a current generated by the electrochemical circuit.

25. The contact lens of claim 7, wherein at least a portion of the working electrode comprises an electrochemically reactive compound.

26. The contact lens of any one of claims 25, wherein the electrochemically reactive compound comprises ferrocene.

27. The contact lens of any one of claims 25 or 26, wherein at a first electric potential the electrochemically reactive compound is in a reduced state.

28. The contact lens of claim 27, wherein the first electric potential is between 0 V and 0.5 V.

29. The contact lens of claim 25-28, wherein at a second electric potential the electrochemically reactive compound is electrochemically oxidized to an oxidized state.

30. The contact lens of any one of claims 25 or 26, wherein at the first electric potential, the electrochemically reactive compound is in the oxidized state.

31. The contact lens of any one of claims 30, wherein the first electric potential is between 0 V and 0.5 V.

32. The contact lens of any one of claims 30 or 31, wherein at the second electric potential the electrochemically reactive compound is in the reduced state.

33. The contact lens of claim 7, wherein the working electrode has a total area of between 10 mm2and 12 mm2.

34. The contact lens of claims 7 or 33, wherein the working electrode has an average thickness of between 45 microns and 55 microns.

35. The contact lens of claims 7 or 33-34, wherein the working electrode further comprises a stretchable interconnect.

36. The contact lens of claims 35, wherein at least a portion of the stretchable interconnect is serpentine.

37. The contact lens of claim 36, wherein the serpentine has an external diameter of between 1 mm and 2 mm.

38. The contact lens of claims 35 or 36, wherein the serpentine portion of the stretchable interconnect has an internal diameter of between 0.75 mm and 1.25 mm.

39. The contact lens of any one of claims 36-38, wherein the serpentine portion of the stretchable interconnect has a ribbon width of between 0.1 mm and 0.5 mm.

40. The contact lens of any one of claims 36-39, wherein the serpentine portion of the stretchable interconnect has a pitch of between 2 mm and 4 mm.

41. The contact lens of claim 11 wherein the counter electrode has a total area of between 10 mm2and 12 mm2.

42. The contact lens of claim 11 or 41, wherein the counter electrode has an average thickness of between 45 microns and 55 microns.

43. The contact lens of claim 11 or 41-42, wherein the counter electrode further comprises a stretchable interconnect.

44. The contact lens of claim 43, wherein at least at least a portion of the stretchable interconnect is serpentine.

45. The contact lens of claim 44, wherein the serpentine portion of the stretchable interconnect has an external diameter of between 1 mm and 2 mm.

46. The contact lens of claim 44 or 45, wherein the serpentine portion of the stretchable interconnect has an internal diameter of between 0.75 mm and 1.25 mm.

47. The contact lens of any one of claims 44-46, wherein the serpentine portion of the stretchable interconnect has a ribbon width of between 0.1 mm and 0.5 mm.

48. The contact lens of any one of claims 44-47, wherein the serpentine portion of the stretchable interconnect has a pitch of between 2 mm and 4 mm.

49. The contact lens of claim 14, wherein the reference electrode has a total area of between 10 mm2and 12 mm2.

50. The contact lens of claim 14 or 49, wherein the reference electrode has an average thickness of between 45 microns and 55 microns.

51. The contact lens of claim 14 or 49-50, wherein the reference electrode comprises a stretchable interconnect.

52. The contact lens of claims 51, wherein at least at least a portion of the stretchable interconnect is serpentine.

53. The contact lens of claim 52, wherein the serpentine portion of the stretchable interconnect has an external diameter of between 1 mm and 2 mm.

54. The contact lens of claim 52 or 53, wherein the serpentine portion of the stretchable interconnect has an internal diameter of between 0.75 mm and 1.25 mm.

55. The contact lens of any one of claims 52-54, wherein the serpentine portion of the stretchable interconnect has a ribbon width of between 0.1 mm and 0.5 mm.

56. The contact lens of any one of claims 52-55, wherein the serpentine portion of the stretchable interconnect has a pitch of between 2 mm and 4 mm.

57. The contact lens of any one of claims 1-56, wherein the first polymer lens comprises poly hdroxyethylmethy acrylate (polyHEMA).

58. The contact lens of any one of claims 1-57, wherein the first polymer lens comprise silicone.

59. The contact lens of any one of claims 1-58, wherein the first polymer lens comprises polydimethylsiloxane (PDMS).

60. The contact lens of any one of claims 1-59, wherein the first polymer lens is deformable.

61. The contact lens of any one of claims 1-60, wherein the second polymer lens comprises poly hdroxyethylmethy acrylate (polyHEMA).

62. The contact lens of any one of claims 1-61, wherein the second polymer lens comprises silicone.

63. The contact lens of any one of claims 1-62, wherein the second polymer lens comprises polydimethylsiloxane (PDMS).

64. The contact lens of any one of claims 1-63, wherein the second polymer lens is deformable.

65. The contact lens of any one of claims 1-64, wherein the contact lens has an elastic modulus of between 0.8 MPa and 1.0 MPa.

66. The contact lens of claim 65, wherein the contact lens has an elastic modulus of 0.9 MPa.

67. The contact lens of any one of claims 1-66, wherein the contact lens exhibits a maximal principal strain of less than 1% following exposure to 600 kPa applied pressure.

68. The contact lens of any one of claims 1-67, wherein the contact lens exhibits a maximal principal strain of less than 1% when folded along a symmetrical axis.

69. The contact lens of any one of claims 1-68, wherein the contact lens exhibits a maximal principal strain of less than 20% when uniaxially stretched.

70. The contact lens of any one of claims 1-69, wherein the contact lens exhibits a maximal principal strain of less than 20% when radially expanded.

71. The contact lens of any one of claims 1-70, wherein the contact lens exhibits a maximal principal strain of less than 20% when twisted between +180 degrees and -180 degrees along a symmetrical axis.

72. The contact lens of any one of claims 1-71, wherein the contact lens is configured to measure a current generated when the electrochemically oxidized electrochemically reactive compound oxidizes a target analyte.

73. The contact lens of claim 72, wherein the current generated is proportional to a concentration of the target analyte.

74. The contact lens of claim 72 or 73, wherein the contact lens is configured to detect the target analyte at a concentration of between 1 picoM and 100 microM.

75. The contact lens of any one of claims 72-74, wherein the contact lens has a response time of at least 40 seconds when exposed to the target analyte at a concentration of at least 1 microM.

76. The contact lens of any one of claims 1-75, wherein the contact lens has a sensitivity of 3.8 microAmps / nanoM.

77. A contact lens, comprising:a first polymer lens; a second polymer lens defining a hole, in contact with the first polymer lens; and an electrochemically reactive compound in fluidic communication with the hole.

78. The contact lens of claim 77, wherein the electrochemically reactive compound is an oxidizing agent.

79. The contact lens of claim 78, wherein the oxidizing agent is an electron acceptor.

80. The contact lens of claim 77-79, wherein the electrochemically reactive compound comprises an organometallic compound.

81. The contact lens of claim 80, wherein the organometallic compound comprises ferrocene.

82. The contact lens of any one of claim 77-81, wherein the electrochemically reactive compound comprises multi- walled carbon nanotubes (MWCNT).

83. The contact lens of any one of claims 77-82, wherein the electrochemically reactive compound comprises zinc oxide (ZnO).

84. The contact lens of any one of claims 77-83, wherein the electrochemically reactive compound comprises a porphyrin.

85. The contact lens of claim 84, wherein the porphyrin comprises 5,15- pentafluorophenyl- 10,20-p-aminophyenylprophyrin (P).

86. The contact lens of claim 84 or 85, wherein the porphyrin comprises meso-tetrakis (2- aminophenyl) porphyrin.

87. The contact lens of any one of claims 77-86, wherein the electrochemically reactive compound comprises graphene oxide (GO).

88. The contact lens of any one of claims 77-87, wherein the electrochemically reactive compound comprises graphite.

89. The contact lens of any one of claims 77-88, wherein the electrochemically reactive compound comprises nanodiamonds.

90. The contact lens of any one of claims 77-89, wherein the electrochemically reactive compound comprises gold nanoparticles.

91. The contact lens of any one of claims 77-90, wherein the electrochemically reactive compound comprises poly (bromocresol green).

92. The contact lens of any one of claims 77-91, wherein the electrochemically reactive compound comprises magnetic nanoparticles.

93. The contact lens of any one of claims 77-92, wherein the electrochemically reactive compound comprises polypyrrole.

94. The contact lens of any one of claims 77-93, wherein the contact lens further comprises an electrochemical circuit.

95. The contact lens of claim 94, wherein the electrochemical circuit comprises one or more screen printed electrodes.

96. The contact lens of claim 95, wherein the one or more screen printed electrodes comprise a working electrode.

97. The contact lens of claim 95 or 96, wherein the one or more screen printed electrodes comprise a counter electrode.

98. The contact lens of any one of claims 95-97, wherein the one or more screen printed electrodes comprises a reference electrode.

99. The contact lens of any one of claims 94-98, wherein the electrochemical circuit comprises a potentiostat.

100. The contact lens of claim 99, wherein the potentiostat electrically connects the working electrode to the counter electrode.

101. The contact lens of claim 99 or 100, wherein the potentiostat is configured to apply an electric potential between the working electrode and the counter electrode.

102. The contact lens of claim 101, wherein at a first electric potential the electrochemically reactive compound is in a reduced state.

103. The contact lens of claim 101 or 102, wherein at a second electric potential the electrochemically reactive compound is electrochemically oxidized to an oxidized state.

104. The contact lens of claim 103, wherein the electrochemical circuit is configured to measure a current generated when the electrochemically oxidized electrochemically reactive compound oxidizes a target analyte.

105. The contact lens of claim 104, wherein the current generated is proportional to a concentration of the target analyte.

106. The contact lens of claim 104 or 105, wherein the target analyte comprises serotonin.

107. The contact lens of any one of claims 104-106, wherein the target analyte comprises glucose.

108. The contact lens of any one of claims 104-107, wherein the target analyte comprises mucin.

109. The contact lens of any one of claims 104-108, wherein the target analyte comprises lipids.

110. The contact lens of any one of claims 104-109, wherein the lipids comprise cholesterol.

111. The contact lens of any one of claims 104-110, wherein the lipids comprise triglycerides.

112. The contact lens of any one of claims 104-111, wherein the target analyte comprises an enzyme.

113. The contact lens of claim 112, wherein the enzyme comprises lysozyme.

114. The contact lens of any one of claims 104-113, wherein the target analyte comprises lactoferrin.

115. The contact lens of any one of claims 104-114, wherein the target analyte comprises lipcalin.

116. The contact lens of any one of claims 104-115, wherein the target analyte comprises lacritin.

117. The contact lens of any one of claims 104-116, wherein the target analyte comprises an immunoglobulin.

118. The contact lens of any one of claims 104-117, wherein the target analyte comprises urea.

119. The contact lens of any one of claims 104-118, wherein the target analyte comprises sodium.

120. The contact lens of any one of claims 104-119, wherein the target analyte comprises potassium.

121. The contact lens of any one of claims 104-120, wherein the target analyte comprises cortisol.

122. The contact lens of claim 104, wherein the contact lens is configured to detect the target analyte in a fluid, wherein a concentration of the target analyte in the fluid is between 1 picoM and 100 microM.

123. The contact lens of claim 122, wherein the fluid is a fluid associated with an eye.

124. The contact lens of claim 122 or 123, wherein the fluid is a tear fluid.

125. A contact lens, comprising: a first polymer lens; a second polymer lens in contact with the first polymer lens; and a working electrode, a reference electrode, and a counter electrode, each positioned between the first polymer lens and the second polymer lens, wherein the working electrode comprises ferrocene.

126. A method, comprising: exposing a contact lens to fluid produced by an eye of a subject, wherein the fluid comprises an analyte; electrochemically reacting the analyte with an electrochemical compound; and determining a current produced from the electrochemical reaction.

127. The method of claim 126, wherein the contact lens comprises a first polymer lens.

128. The method of claim 126 or 127, wherein the contact lens comprises a second polymer lens defining a hole.

129. The method of claim 128, wherein the second polymer lens is in contact with the first polymer lens.

130. The method of any one of claims 129, wherein the contact lens further comprises an electrochemical circuit.

131. The method of claim 130, wherein the electrochemical circuit comprises an electrochemical sensor in fluidic communication with the hole in the second polymer lens.

132. The method of claim 131, wherein upon exposing the contact lens to the fluid, the fluid enters the hole in the second polymer lens and contacts the electrochemical sensor.

133. The method of claim 131 or 132, wherein the electrochemical sensor comprises a working electrode.

134. The method of claim 133, wherein the working electrode comprises the electrochemical compound.

135. The method of claim 134, wherein the electrochemical compound comprises an organometallic compound.

136. The method of claim 135, wherein the organometallic compound comprises ferrocene.

137. The method of any one of claims 134-136, wherein the electrochemical compound comprises multi-walled carbon nanotubes (MWCNT).

138. The method of any one of claims 134-137, wherein the electrochemical compound comprises a porphyrin.

139. The method of any one of claims 134-138, wherein the electrochemical compound comprises graphene oxide (GO).

140. The method of any one of claims 134-139, wherein the electrochemical compound comprises nanodiamonds.

141. The method of any one of claims 134-140, wherein the electrochemical compound comprises metal nanoparticles.

142. The method of any one of claims 134-141, wherein the electrochemically reactive compound comprises polypyrrole.

143. The method of any one of claims 131-142, wherein the electrochemical sensor comprises a counter electrode.

144. The method of any one of claims 131-143, wherein the electrochemical sensor comprises a reference electrode.

145. The method of any one of claims 130-144, wherein the electrochemical circuit comprises a potentiostat.

146. The method of claim 145, wherein the potentiostat electrically connects the working electrode to the counter electrode.

147. The method of claim 145 or 146, wherein the potentiostat applies a voltage between the working electrode and the counter electrode.

148. The method of claim 147, wherein applying a voltage to the working electrode causes the electrochemical compound to undergo a reduction-oxidation reaction.

149. The method of claim 148, wherein the applied voltage is between 0 V and 0.5 V.

150. The method of claim 148 or 149, wherein applying the voltage to the working electrode electrochemically oxidizes the electrochemical compound.

151. The method of claim 150, wherein the oxidized electrochemical compound comprises ferrocenium.

152. The method of claim 150, further allowing the oxidized electrochemical compound to further oxidize the analyte.

153. The method of claim 152, wherein upon oxidizing the analyte the electrochemical compound is reduced.

154. The method of claim 153, wherein the reduced electrochemical compound comprises ferrocene.

155. The method of claim 130, comprising using the electrochemical circuit to measure the current generated by the continuous reduction-oxidation reaction of the electrochemical compound.

156. The method of claim 155, wherein the current generated is proportional to the concentration of the analyte in the fluid.

157. The method of claim 156, wherein the analyte is serotonin.

158. The method of claim 156 or 157, wherein the analyte comprises glucose.

159. The method of any one of claims 156-158, wherein the analyte comprises mucin.

160. The method of any one of claims 156-159, wherein the analyte comprises lipids.

161. The method of claim 160, wherein the lipids comprise cholesterol.

162. The method of any one of claims 160 or 161, wherein the lipids comprise triglycerides.

163. The method of any one of claims 156-162, wherein the analyte comprises an enzyme.

164. The method of any one of claims 156-163, wherein the enzyme comprises lysozyme.

165. The method of any one of claims 156-164, wherein the analyte comprises lactoferrin.

166. The method of any one of claims 156-165, wherein the analyte comprises lipcalin.

167. The method of any one of claims 156-166, wherein the analyte comprises lacritin.

168. The method of any one of claims 156-167, wherein the analyte comprises an immunoglobulin .

169. The method of any one of claims 156-168, wherein the analyte comprises urea.

170. The method of any one of claims 156-169, wherein the analyte comprises sodium.

171. The method of any one of claims 156-170, wherein the analyte comprises potassium.

172. The method of any one of claims 156-171, wherein the analyte comprises cortisol.

173. A method, comprising: determining a concentration of an analyte within a fluid within an eye of a subject by determining an electrical current of an electrochemical sensor within a contact lens on the eye of the subject.

174. A method, comprising: determining a concentration of serotonin from a fluid within an eye of a subject, using a contact lens, by measuring a current produced from a reaction of ferrocene contained within the contact lens.

175. A deformable contact lens comprising an electrochemical sensor, wherein the deformable contact lens has an elastic modulus of between 0.1 MPa and 1.5 MPa.

176. The contact lens of any preceding claim, wherein the contact lens comprises a battery.

177. The contact lens of any preceding claim, wherein the contact lens comprises a wireless monitoring system.

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