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Abstract INTRODUCTION:
In 2023, the global medical devices market was estimated at $518 billion USD; the 2023 global paper diagnostics market, which includes volumetric absorptive microsampling devices [Within which dried matrix spots (DMS) are situated], was valued at $15.5 billion USD, which equates to ~3% of the overall medical devices market. With DMS, a fixed volume of sample, generally ~10 microliters, is volumetrically absorbed into a substrate; DMS methods have been noted as clinically-relevant, cost-effective, simple, and reproducible. Though blood microsampling is perhaps the most commonly-recognized DMS application, other samples include saliva and urine for a range of analyte scenarios. Regardless of the sample, DMS often rely on at least tens-of-microliter volumes, and DMS preparative/sampling methods usually involve direct transfer of the biofluid onto a sample storage medium, which is oftentimes filter paper. This transfer step has several inherent pitfalls, including variance in sample volume being applied and/or uneven lateral distribution of the sample. Furthermore, most analytical techniques cannot be directly integrated with the storage medium, i.e., the biofluid spot must be extracted offline from the substrate for complex and solvent-intensive analytical methods. Additionally, DMS methods have not yet been thoroughly supplemented by artificial intelligence/computer vision.
OBJECTIVES:
Given the market interests in paper-based devices, we have combined a simple hydrophobic surface treatment with laser-micromachining to modify a paper substrate with surface energy traps (SETs) to precisely confine, dry-down, and directly analyze DMS with liquid microjunction - surface sampling probe - mass spectrometry (LMJ-SSP-MS) [Note that the LMJ-SSP enables a combined (automatable) workflow of sample extraction and subsequent introduction of analyte(s) to the MS]. Additionally, we are creating and optimizing an object-based detection program to leverage computer vision to aid the sampling process. Taken together, we anticipate that this work will impact: 1) The manner by which DMS are prepared and then 2) the analytical process(es) through which DMS are interrogated.
METHODS:
The modified (paper) substrates are rendered hydrophobic by dip-coating with a commercially available coating formulation, Aculon (San Diego, California USA). After the surface hydrophobicity treatment, the substrate(s) is/are micromachined with an Oxford Lasers A Series picosecond compact laser-micromachining system. Note that the laser-micromachining system is used to both excise the (paper) substrates from the parent Whatman filter paper sheet and to machine the circular SETs, typically less than 1 mm diameter, and the reticules for visual targeting. SETs are visually assessed with various aqueous dye solutions and a combination of optical and scanning electron microscopies, as well as goniometry. Lastly, SETs are qualitatively/quantitatively assessed using various aqueous and artificial biofluid solutions and LMJ-SSP-MS, in particular a model analyte recovery study. In-house, Python-based computer programs have been developed to control the 3D-printer chassis's xyz movement, as well as the camera's ability to recognize the targeting reticules via computer vision.
RESULTS:
Previously, we have observed analyte signal reductions (Up to 32-fold) when sampling from a porous substrate. Here, laser-micromachining enables the creation of superficial-yet-functional SETs, meaning that by tuning the laser power and speed, the SETs are prepared such that the liquid is confined within and dries-down on the SET without penetrating deeply into the substrate; this superficiality maximizes sample recovery/introduction via LMJ-SSP-MS. After the surface hydrophobicity treatment, the modified paper substrate achieves a nearly superhydrophobic water contact angle, roughly 145 degrees, given the surface roughness afforded by the cellulose fiber network in conjunction with the Aculon coating. After laser-micromachining, a one microliter droplet is neatly retained within the SET and dries-down within the laser-micromachined area with minimal lateral diffusion (< 5%) and practically imperceptible transverse diffusion. Preliminary results using a bilirubin standard in artificial urine show promise that near-quantitative recoveries could potentially be achieved, though in this study we are using the absence/presence of bilirubin in an artificial urine standard solution as a binary "Yes/No" proof-of-principle example, i.e. a rudimentary model (hyper)bilirubinemia system. Moreover, under certain dimensional parameters, Version 1 of the computer vision program can detect a model SET's center within an average of 0.023 mm (2.3% error) (n = 3).
DISCUSSION/CONCLUSION:
Especially on a paper substrate, SETs can be difficult to visualize for sampling, so the targeting indicators, or reticules, that we have prepared around the SETs for visualization are poised to be a marked improvement to the design and use of (paper) substrates for DMS preparation and sampling. Moreover, the introduction of novel substrates like polymeric materials for DMS preparation could further increase the appeal of DMS as a diagnostic tool. As this project progresses, we plan to include additional demonstrations using analyte(s) of interest in artificial blood and saliva solutions. Lastly, in keeping with trends towards laboratory automation and high throughput assay(s)/screening(s), we are working to ensure that our early-stage object detection program that recognizes the reticules on the various substrates is accurate, precise, and robust, i.e., integrable with other xyz locomotion systems. |
= Discovery stage. (53.14%, 2025)
= Translation stage. (22.33%, 2025)
= Clinically available. (24.53%, 2025)
