Contact lens-based lysozyme detection in tear using a mobile sensor

Abstract

We report a method for sensing analytes in tear-fluid using commercial contact lenses (CLs) as sample collectors for subsequent analysis with a cost-effective and field-portable reader. In this study we quantify lysozyme, the most prevalent protein in tear fluid, non-specifically bound to CLs worn by human participants. Our mobile reader uses time-lapse imaging to capture an increasing fluorescent signal in a standard well-plate, the rate-of-change of which is used to indirectly infer lysozyme concentration through the use of a standard curve. We empirically determined the best-suited CL material for our sampling procedure and assay, and subsequently monitored the lysozyme levels of nine healthy human participants over a two-week period. Of these participants who were regular CL wearers (6 out of 9), we observed an increase in lysozyme levels from 6.89 ± 2.02 μg/mL to 10.72 ± 3.22 μg/mL (mean ± SD) when inducing an instance of digital eye-strain by asking them to play a game on their mobile-phones during the CL wear-duration. We also observed a lower mean lysozyme concentration (2.43 ± 1.66 μg/mL) in a patient cohort with Dry Eye Disease (DED) as compared to the average monitoring level of healthy (no DED) human participants (6.89 ± 2.02 μg/mL). Taken together, this study demonstrates tear-fluid analysis with simple and non-invasive sampling steps along with a rapid, easy-to-use, and cost-effective measurement system, ultimately indicating physiological differences in human participants. We believe this method could be used in future tear-fluid studies, even supporting multiplexed detection of a panel of tear biomarkers toward improved diagnostics and prognostics as well as personalized mobile-health applications.

Graphical Abstract

A rapid and cost-effective method for monitoring proteins in tear-fluid is reported, which enables biomarker monitoring using contact lenses toward personalized mobile-health applications.

INTRODUCTION

There have been major advances in sensing bio-fluids previously considered inaccessible. For example, wristbands and wearable patches have been used to detect key metabolites and biomarkers from sweat in real-time.^1–3 Contact lens-based sensing has also been demonstrated for monitoring biomarkers in tear fluid such as glucose, lactate, and potassium ions.^4–8 Wearable sensing systems such as these provide an unprecedented look at the human body and its interactions, enabling frequent and remote monitoring of various symptoms. Additionally, because these new platforms monitor non-traditional bio-fluids such as sweat or tear, they can enable discovery of novel diagnoses or therapies and can help pave the way for personalized medicine. Furthermore, systems that are cost-effective and portable are especially attractive as they can be distributed to low-resource clinics or even in the home, providing an affordable and convenient means of health monitoring.

Tear-fluid, specifically, holds significant potential for sensing. The lacrimal glands produce nearly 1 mL of basal tear-fluid in a day, replenishing the aqueous layer of the tear film which serves as a barrier between the eye and external environment. Tear contains a characteristically high concentration of proteins, as well as many of the same biomarkers present in blood, such as glucose, metabolites like lactate and potassium, and even viruses such as Herpes and HIV.^9–12 Additionally, due to the absence of red bloods cells, tear fluid provides a relatively ‘clean’ sensing background compared to blood. Therefore, if enabled by the appropriate sensing technologies, tear-fluid could be widely used for diagnostics, detecting the presence of ocular diseases along with other underlying disease states.^10,13–15

Established methods of extracting and accessing tear fluid for in-vitro analysis include the use of Schirmer Strips, ophthalmic sponges, the eye-flush method, as well as direct collection with a micro-pipette.^13,16–19 Schirmer Strips, for example, are placed inside the lower eyelid in order to directly contact the tear film and extract fluid via the capillary forces in the paper.²⁰ They are routinely used in clinical practice to diagnose Dry Eye Disease by assessing the volume of an individual’s tear production, and have also been employed to extract tear fluid for subsequent protein analysis^19,21–23. However, Schirmer Strips are considered mildly irritating and uncomfortable, consequently collecting a mixture of basal tear fluid and reflex tears which have been shown to have different protein compositions.^23–26 Direct collection of basal tear fluid with a micro-pipette is therefore the preferred method in a number of clinical studies, as it can be more precise and avoid sampling reflex tears ^14,27–29. However, collection with a pipette must be performed by a trained clinician in order to mitigate the risk of damage the cornea, and can result in low extraction volumes of sample fluid (e.g., <5 μL) which can be inadequate for collecting fluid from patients with a dry-eye condition.^13,17,30 Due to the intense interest in analyzing tear-fluid, comparative studies between these sample collection methods have been performed, reporting that the protein composition and the measured concentrations depend on the specific method that is used.^19,30–33

Contact lenses (CLs), on the other hand, present an attractive alternative substrate for accessing tear fluid. They can be worn for extended durations with minimal irritation and are already widely adopted, in part, due to their comfort, ease-of-use and low-cost. The FDA has even approved CLs for therapeutic use, beyond improving vision, for patients recovering from surgery or corneal damage. New sensing systems can therefore leverage CLs as minimally invasive sample collectors, and analyze the biomarkers adsorbed onto their surface (specifically and/or non-specifically) in-vivo or in-vitro for diagnostic or prognostic applications.³⁴ Although not focused on the applications of CLs for sensing, earlier studies investigated protein deposition onto CL materials,^35–39 aiming to understand the clinical implications of protein and pathogen build-up on CLs in terms reduced comfort and medical complications such as conjunctivitis. In contrast, this pilot study addresses the feasibility of CLs as non-specific sample collectors for analysis with a point-of-care reader to enable regular monitoring of potential tear-fluid biomarkers in e.g., an ophthalmological clinic or at home.

Here we demonstrate a simple mobile sensing workflow, where commercially available CLs are used to collect biomarkers in tear-fluid for potential point-of-care diagnostics applications. A previously developed mobile-phone based well-plate reader⁴⁰ is employed for rapid analysis, where time-lapse imaging is used to capture an increasing fluorescent signal produced by a commercial enzymatic assay performed in a 96 well-plate. The calculated rate-of-change in the detected fluorescence is then used to indirectly infer the enzyme activity by comparison to a standard curve. Using this method, we quantified lysozyme, an important anti-microbial enzyme that constitutes roughly 25% of the total proteins in tear fluid. Among many other proteins in tear, lysozyme has been studied as a potential biomarker for a number of ocular diseases including the Dry Eye Disease (DED), glaucoma, Sjögren’s syndrome, and even other underlying disease states such as cancer and multiple sclerosis. ^{8,10,13,14,41–47} It is also important to note that there is no clinically available test capable of reliably differentiating DED patients from non-DED patients.²² As a result, protein analysis of tear fluid has been proposed as an alternative means of diagnostic testing which could result in higher sensitivity and specificity compared to current clinical methods that have limited performance.

In this work, we report the results of an IRB-approved participant study, where we used our CL-based mobile sensing approach to monitor the lysozyme levels of a healthy participant cohort over a two-week period. Differences in monitoring measurements were observed between regular CL wearers and non-CL wearers, and a mean increase for the CL wearers’ lysozyme concentration measurements (N=6) from 6.89 ± 2.02 μg/mL to 10.72 ± 3.22 μg/mL (mean ± SD) was observed when they played a mobile-phone game during the wear-duration, inducing an instance of digital eye-strain. Additionally, a statistically significant difference (p < 0.01) in lysozyme levels was observed between the healthy cohort (N=6) when compared to a patient cohort diagnosed with DED (N=6).

Taken together, we demonstrate the use of CLs along with a field-portable and cost-effective reader for simple and rapid biomarker quantification in tear fluid. Such an approach could enable new diagnostic tests for DED, Sjögren’s syndrome, or viral conjunctivitis, among others, and could even be used for detecting the presence of pathogens such as Staphylococcus Aureus and Acanthamoeba.^21,34 In addition to enabling new tear-fluid based diagnostics for ocular diseases, we believe that the presented simple mobile workflow can lead to different point-of-care sensing platforms which can be used for advanced health monitoring and personalized medicine related applications.

MATERIALS AND METHODS

Participant study

Two cohorts of participants were recruited for this study, and gave informed, written consent under IRB approval (IRB#16–000841). The first cohort of participants included 9 subjects (3 male, 6 female) aged 21–22 years, and all self-reported as having no known ocular conditions including DED. 6 were regular CL wearers (CLWs) and the remaining 3 were non-CL wearers (NCLWs). The second cohort was comprised of 6 participants (1 male, 5 female) aged 29 to 48 (mean ± SD: 34.7 ± 7.3 years) recruited through the Stein Eye Institute at UCLA, and were all NCLWs at the time of the measurement. Because the previous studies reported statistically significant differences in lysozyme levels of individuals over the age of 60, we limited the inclusion criterion in both cohorts to individuals under the age of 50^48,49. Importantly, in contrast to the first cohort, all the participants in the second cohort had either signs or symptoms of DED confirmed by a board certified cornea specialist. Because DED is considered a multifactorial disease, where aqueous deficient and evaporative dry eye exists as a spectrum for individuals, a number of symptoms and tests were used for diagnosis.⁵⁰ Specifically, all the participants in the second cohort had OSDI (ocular surface disease index) score of ≥ 13 and evidence of loss of homeostasis in the following forms: non-invasive tear break up time < 10 seconds, osmolarity ≥ 308 mOsm/L or a difference in osmolarity between the left and right eye > 8 mOsm/L, ocular surface staining of > 5 corneal spots, ≥ 9 conjunctival spots, or lid margin staining ≥ 2 mm or 25% width of lid margin.

The first cohort participated in lysozyme measurements over six separate visits/days spanning a two-week period. For each lysozyme measurement, the participants first washed their hands, personally inserted CLs (PureVision 2, 0 power) into the left and right eye, and wore them for a 15 minute period in the designated waiting room. During the first 5 days of the measurements, the participants were asked to not look at their mobile-phones or any other screens, and on the last day of the measurements, participants were asked to play a game on their mobile-phones with full screen brightness during the 15 minute wear duration in order to induce digital eye strain. The lysozyme measurements were fixed to between 6 and 7 pm every experiment day to help standardize the measurements by avoiding possible diurnal fluctuations.^49,51

The second cohort of participants (the DED patients) underwent a single lysozyme measurement, under the same conditions of the first cohort with the exception that the insertion and removal was performed by an ophthalmologist at the Stein Eye Institute. The participant/patient availability was considerably lower among this second cohort, which made it prohibitive to perform a monitoring study, therefore only single-day measurements of the DED cohort were performed for this pilot study. The measurement times were also not fixed between 6 and 7 pm, taking place after 12 pm and before 5 pm. The lysozyme assay read-out was performed using our mobile-phone based well-plate reader within one hour of each sampling event for both cohorts.

CL tear-fluid sampling protocol

Figure 1a depicts the CL tear-fluid sampling process. After CLs are worn for a set duration of 15 minutes, they are immediately placed in individual capped 1.5 mL collection tubes containing 600 μL of the assay reaction buffer (0.1 M sodium phosphate, 0.1 M NaCl, pH 7.5, containing 2 mM sodium azide as a preservative). After placement in the collection tubes, the CL and reaction buffer solution are mixed by carefully rotating the tubes upside down and right-side up 15 times in succession. The CLs are then removed from the collection tubes and discarded, and the remaining solution is used for subsequent analysis.

Figure 1.

Overview of the contact lens sensing process and mobile-phone based well-plate reader. (a) (i) Commercial contact lenses are worn under normal conditions for 15 min before being (ii) removed and placed in a collection tube filled with a reaction buffer. (iii) The contact lenses are then washed in the reaction tube before they are removed and discarded. (iv) 100 μL of the washed solution is mixed with 50 μL of the fluorescent Micrococcus lysodeikticus cell solution in an ELISA well and monitored over time. (b) The mobile-phone based well-plate reader schematic and (c) picture with contact lens cases.

Fluorescent lysozyme assay

The fluorescent lysozyme assay (EnzCheck Lysozyme assay kit, ThermoFisher, E22013) implemented in this work quantifies the lysozyme activity on the cell-walls of Micrococcus lysodeikticus cells. When the lysozyme in the sample acts on the cell walls, fluorescein previously bounded to the cell wall is unquenched, increasing the fluorescent signal in the sample at a rate proportional to the lysozyme activity (Figure 2). For this study, the assay protocol followed the standard operating instructions using 100 μL sample volume and 50 μL of 50 μg/mL fluorescent Micrococcus lysodeikticus cell solution per well. Every measurement of unknown lysozyme activity was measured with a corresponding standard curve comprised of 5 wells containing known human lysozyme (Sigma Aldrich, L6876) concentrations at 1000, 500, 250, 125 and 0 Units/mL. The lysozyme used for the standard curve has a specific activity of ≥40,000 Units per milligram, which was used as a conversion factor in our experiments such that the active lysozyme levels per patient can be reported in μg/mL. It is important to note that this enzymatic assay was chosen for this study due to its simple, two-step protocol and rapid time to results. For this study, the first 10 minutes of the reaction kinetics were measured producing a repeatable response in the standard curve (Figure 2b). In contrast, the more widely used Enzyme-Linked Immunosorbent Assay (ELISA) based methods require lengthy protocols and incubation steps exceeding e.g., >60 minutes for testing, and is thus not conducive to the point-of-care nature of the tear-fluid sampling.

Figure 2.

Green channel image from the mobile-phone based well-plate reader showing the fiber bundle which samples the ELISA wells with three fibers per well (left). The graph (right) shows the increasing fluorescent signal recorded over time (10 min), where the dotted black line represents the linear best fit for the signal of the three fibers. (b) Example standard curve of the fluorescent well-plate assay where the intersection of the red horizontal and vertical lines represent the limit of detection (1.99 μg/mL, as determined by the x-axis) defined by three times the standard deviation plus the mean of the negative control. The error bars represent the standard deviation of four successive standard curve measurements.

Mobile-phone based well-plate reader and image processing

Our mobile-phone based well-plate reader uses an array of Light Emitting Diodes (LEDs) (λ = 475 nm) passed through a bandpass excitation filter (FF01–465/30 nm, Semrock) to illuminate a standard fluorescent ELISA well-plate (Corning 96-well-plate, clear flat bottom wells, black) (Figure 1b). The fluorescent light emission is transmitted through a bandpass emission filter (530F30nm, OD 6, Omega) before being sampled by the optical fibers that are bundled together on the opposite end within the field of view of a mobile-phone camera (Nokia Lumia, 1020) coupled with an external lens (6 mm diameter, 8 mm focal distance) (see Fig. 1b). This device is based upon a design reported in our previous work,⁴⁰ however, for this study we implemented an alternative fiber bundle design with a non-random mapping between the ELISA well-plate and the compact bundle imaged by the mobile-phone, in order to reduce non-uniformities in bending and mechanical stress of the ensemble of fibers (see Fig. S1). Glass-core (400μm) optical fibers (Thorlabs, FT400UMT) were incorporated for sampling the ELISA wells, with three fibers sampling each well in a 5×5 grid (i.e., 75 fibers sampling 25 wells of the 96 well-plate). Given this 25 well subset, up to 10 independent CL measurements can be made in parallel, assuming duplicate measurements and the allocation of 5 wells for the standard curve. Additionally, each 96 well-plate can be used twice by first inserting the front end of the well-plate into the reader and then rotating the plate 180^o and inserting the back-end, easily culminating in 20 independent CL measurements per well-plate (see Fig S1). All the fibers used to sample the well-plate were cut with rotating diamond cutter and subsequently inspected with a 4x objective lens to identify and remove any fibers with fractures or damage.

To monitor the fluorescent assay, we used a time-lapse application (ProShot) which took 48 MP Jpeg images (4 second exposure time, daylight white-balance setting, and ISO of 100) with a period of 1 min over the first 10 minutes of the assay reaction. After the completion of the assay, the images were transferred to a standard desktop for processing where the image-stack was registered and the average pixel intensity of the fibers were automatically segmented and sampled at 70% of their area. A linear model was then fit to each fiber’s change of fluorescence over time, and the average slope of the three fibers per-well was interpolated onto the standard curve, recovering the lysozyme activity (Figure 2a). The standard curve is comprised of the change in the fluorescence signal of 5 wells, each with a known lysozyme concentration, and is generated for every plate measurement (i.e., every time-lapse measurement). It should be noted that for potential applications in the field, images can be sent over the cellular network and processed on a remote server or local workstation for processing, eliminating the need for manual transfer of data.⁵²

Figure 2b shows the assay standard curve which is used to establish the calibration between the rate of change of fluorescence and lysozyme concentration. It is important to note the saturation characteristic of the enzymatic assay. The activity of the lysozyme along with the concentration of the Micrococcus lysodeikticus cells in the fluorescent assay limit the change in fluorescence over time at high lysozyme concentrations, therefore the accuracy of the assay declines towards this saturation point, resulting in large standard deviations at high concentrations. This effect can be mitigated by adequately diluting the tear fluid during the washing step before the in-vitro measurements are made, and empirically determining a dilution factor that will avoid saturation while ensuring that the measured concentrations fall above the detection limit.

In-vitro incubation study

For in-vitro incubation studies, hemispherical lens cases were first submerged in 1% BSA blocking solution for 30 minutes to prevent non-specific binding of lysozyme to the case itself. The cases were then washed with DI water and dried, before adding 200 μL of lysozyme solution in phosphate buffered saline (PBS) pH = 7.4. PureVision 2 CLs were soaked for 15 minutes in the lysozyme solutions with different concentrations (0 to 2.4 mg/L). The CLs were then removed and contacted with an absorbent clean-room wipe to remove excess solution, before undergoing the CL tear-fluid sampling protocol outlined above.

RESULTS & DISCUSSION

Selection of contact lenses for efficient sampling of tear-fluid

A number of CL materials exist on the market, differing in surface charge, pore size, and water content which taken together can influence their surface interactions with proteins.^35–38 As a result, each CL material has a different protein capture property which must be considered before being used in a sampling and sensing application. We therefore empirically compared the relative protein adsorption of two silicone hydrogel polymers; Senofilicon A (Acuvve Oaysis) and Balafilicon A (Pure Vision 2), and one FDA group IV material; Etafilicon A (Acuvue 2). Three human participants gave informed and written consent under IRB and wore each CL type for a duration of 15 minutes under normal wear conditions, replicating the tear-fluid sampling protocol for the larger participant study (see the Materials and Methods). The average protein concentrations measured after the sample collection procedure were found to be 1.79, 6.43, and 29.94 μg/mL for the Senofilicon A, Balafilicon A, and Etafilicon A, respectively, matching previous studies with respect to their relative levels (Figure 3a).^35,37 For our CL-based tear sampling approach, the Balafilicon A material exhibited protein adsorption properties which resulted in the best match to the dynamic range of our assay (i.e., 2– 15 μg/mL). Therefore, PureVision2 CLs were chosen for the subsequent human participant study. The 15 minute wear-duration was also compared to extended wear durations (up to 3 hours), showing no benefits of longer wear duration in terms of increased protein concentration (see Figure S2). Additionally, to further standardize the sample collection methods, CL with 0 power were used. It is important to note that this specific PureVision 2, 0 power lens, was recently FDA-approved for therapeutic use and can therefore also potentially be leveraged for standardizing sampling and sensing even for individuals without a vision prescription.⁵³ Taken together, any CL and lysozyme assay can be used in principle for the tear-fluid sampling method discussed herein, provided an adequate amount of lysozyme is recovered such that the measurement exhibits a signal within the dynamic range of the assay.

Figure 3.

(a) Comparison of the lysozyme levels measured by our CL sampling method using three different common CL materials worn by three human participants, each for a 15 minute wear duration. The error bars represent the standard deviation between the right and left CL measurements. (b) Incubated lysozyme concentration versus the lysozyme levels measured by our CL sampling method during an in-vitro incubation with PureVision2 CLs. The red horizontal and vertical lines represent the limit of detection of the measured lysozyme levels (0.952 μg/mL) and corresponding incubated lysozyme concentration (35.9 μg/mL), respectively. Here the limit of detection is defined by three times the standard deviation plus the mean of the negative control. The error bars represent the standard deviation between two separate CLs soaked in lysozyme solution.

The in-vitro incubation study results (Fig. 3b) show the protein capture rate of the Balafilicon A lens material from spiked solutions of lysozyme at different concentrations. These data established a detection limit of 0.952 μg/mL for the CL-sampled lysozyme concentration, which corresponds to a 35.9 μg/mL limit of detection for the incubated lysozyme concentration, well-below the typical physiological values in human tear fluid (2.46 ± 0.44 mg/mL).²⁹ Additionally, it can be seen that quantitative information can be gathered through this sampling and sensing process with an average protein transfer rate of 2.2% defined as the ratio of the CL-sampled active lysozyme concentration to the incubated lysozyme concentration. This empirically determined protein transfer rate is similar to the 4.8% transfer rate reported in other work detailing lysozyme deposition on Balafilicon A CLs during in-vitro soaking experiments, despite some differences in the protein extraction procedures used.⁵⁴ It is important to emphasize that this artificial incubation does not exactly mimic the physiology of the human eye, instead soaking the CL in a relatively large amount of fluid when compared to normal basal tear-fluid volume (~ 7 μL).³⁰ Therefore this transfer rate is most likely an over-estimate of the true transfer rate between the CL and basal tear fluid in the human eye, as evidenced by the lower average lysozyme concentration measurements from the human participants. Nevertheless, this in-vitro incubation experiment confirms that it is feasible to acquire quantitative information from tear-fluid with contact lenses as passive, non-specific, sample collectors using simple and rapid handling, alongside point-of-care sensing systems.

Human experiments

The lysozyme monitoring results from a cohort of healthy human participants are summarized in Figures 4a and 4b, with a full table of results shown in Supplementary Table 1. The CLWs (i.e., participants A through F, Fig. 4a) have a smaller mean deviation from their 5-day monitoring average (23.4%) when compared to the NCLWs (49.2%) (i.e., participants G through I, Fig 4b). The average standard deviation between the left and right eye lysozyme measurements is also lower for the CLWs (1.56 μg/mL) compared to the NCLWs (4.84 μg/mL). These discrepancies could be due to the irritation and excessive handling time associated with the lack of experience inserting and removing the CL shared by the NCLWs. As a result, they might experience greater variability in the amount of overall tear fluid interacting with the lens, in this case both basal and reflex tears, leading to larger variations.

Figure 4.

CL based lysozyme measurements of human participants using the mobile-phone based well-plate reader. (a) The lysozyme monitoring results of the CLW participants and (b) the NCLW participants in the healthy cohort (i.e. without the DED diagnosis) with six measurement days over a two-week period. The dotted black line defines the average of the first five days of measurements, and the pink star indicates the measurement day where participants were asked to play a game on their mobile-phones during the 15 min wear duration (i.e., the ‘Gameday’). (c) All of the measurements (N = 30) over the 5-day monitoring period from the CLWs without DED (color-coded to reveal different participants A-F, according to the letter colors in (a)) are compared against the one time measurements of lysozyme concentration from the participants with DED (N = 6). Here the red horizontal lines in each box represents the median, and the bottom and top edges of the box represent the 25^th and 75^th percentile, respectively. The whiskers that extend beyond the box represent the most extreme data points of the given participant’s measurements.

On the sixth day of the monitoring period, participants were asked to play a game on their mobile-phones during the 15-minute wear duration in order to induce a common instance of eye-strain, which has been observed to alter tear protein levels.⁵⁵ Accounting for the CLWs only, an increase in average lysozyme levels was found between the sixth day (mean ± SD, 10.72 ± 3.22 μg/mL) and the 5-day average monitoring measurements (6.89 ± 2.02 μg/mL). Excessive tearing is a symptom of digital eye-strain, and could form a hypothesis for the mean lysozyme concentration increase due to the extra tear volume that is sampled by the CLs.^56–58 However, other dry eye-like symptoms of digital eye strain could also motivate a hypothesis for decreased lysozyme levels. Therefore, we tested the statistical significance of the observed increase in lysozyme against the null hypothesis of no mean change (i.e. either an increase or decrease) using a two-tailed t-test assuming unequal variances. Based on this analysis, no statistical significance, t(5) = 2.37, p = 0.064, was found for the change in lysozyme levels on game-day.

The second cohort of participants with DED underwent a one-time CL lysozyme measurement as a comparative study (see Supplementary Table 2). A significantly smaller mean lysozyme level was found for the DED patients (2.43 ± 1.66 μg/mL) when compared to the non-DED CLWs (6.89 μg/mL ± 2.02 μg/mL), t(10) = 2, p <0.01, via a one-tailed Mann-Whitney U-test. ⁵⁹ This result is in agreement with other studies which assess lysozyme concentrations in tear fluid between DED and non-DED individuals ^10,14,60,61. However, it is important to note that this decrease is also in agreement with a study by Stuchell et al. citing population differences between regular CLWs and NCLWs.²⁵ The NCLWs in cohort 1 were not used for comparison with the participants in cohort 2 due to the large standard deviations in their measurements and overall higher average monitoring concentrations, which is most likely due to the irritation and influx of reflex tears onto the CL as previously discussed. Though none of the DED patients were CL wearers at the time of measurement, a trained ophthalmologist was able to perform the CL insertion resulting in an average standard deviation between the left and right eye measurements of 1.04 μg/mL over the six DED participants. This observed variability is closer to the standard deviation observed for the healthy CLWs (1.56 μg/mL) compared to the healthy NCLWs (4.84 μg/mL), and potentially suggests a mitigation of irritation and excessive handling due to the physician’s training. Additionally, it is important to note that due to the relatively small cohort size in this pilot study, over interpretation of these results in terms of diagnostic performance should be avoided, and larger-scale studies with more extensive monitoring periods should be conducted before making strong conclusions.

Though we did not assess DED diagnostic performance in this study due to the limited sample size, a number of other studies have showed protein measurements of tear-fluid biomarkers can lead to diagnostic accuracies for DED as high as 96%.^14,61 Similar diagnostic studies for Sjögren’s Syndrome have also reported success via tear protein assays in contrast to existing diagnostic methods, e.g., via the Schirmer Strip test.^42,44,62 By using the standard ELISA well-plate format with a cost-effective mobile reader, our platform could multiplex measurements to analyze a panel of key tear-fluid biomarkers such as lysozyme, lactoferrin, and IgA among others, which could collectively help improve the diagnostic capabilities. Furthermore, it is important to note that for this study we intentionally limited the CL handling steps, forgoing specialized lab equipment needed for sonication, centrifugation, or vortexing in order to more realistically address the feasibility in a point-of-care setting. However, due to a number of factors that can interfere with obtaining accurate results such as non-uniform interaction and handling during the insertion and removal, as well as variations in the CL washing technique and duration, future studies are needed to further standardize the CL-sampling method described in this work. Additionally, a direct comparison between the CL sampling method and other widely used methods such as collection via Schirmer Strip and micropipette should be performed to establish the relative sampling error and its implications for diagnostics²⁷. To enable widespread use as a diagnostic method, special attention must remain directed toward keeping the protocol simple low-risk, timely, and equipment-free.

Taken together, we reported a rapid, cost-effective, and minimally invasive method for analyzing tear-fluid. We demonstrated the feasibility of this method to obtain quantitative information, revealing statistical differences in-line with previous reports. We believe this tear-fluid sampling approach alongside point-of-care sensing systems can enable new tear-fluid based diagnostics for ocular diseases together with other underlying disease states toward the goal of advanced health monitoring and personalized medicine.