Sweat is an unexploited biological fluid that can provide a wealth of diagnostic information. We propose a novel technology to be used at the bedside or in the field: a diagnostic skin patch which harvests, concentrates, and stabilizes a panel of biomarkers derived from skin transudate or sweat. While drug delivery patches are routinely used, the technology proposed here has exactly the opposite function: the harvesting of diagnostic markers. Using mass spectrometry we have identified 228 proteins and peptides that were not previously known to exist in human sweat. In order to exploit this new class of analytes we propose to create novel affinity bait nanoparticles, bound within an adhesive skin patch. The proposed technology is transformative because it overcomes all the major physiological barriers that have prevented the use of this biologic fluid for diagnostic testing. Sweat disease biomarkers a) are subject to rapid degradation due to proteases present in sweat and normal skin bacterial flora, and b) exist in extremely low abundance, far below the detection sensitivity of standard analysis platforms. Harvesting hydrogel nanoparticles are engineered with chemical high affinity baits so that they sequester the low abundance target analytes, and protect them from degradation indefinitely. We propose to integrate the nanoparticles into the fabric of an adhesive skin patch. Once applied to the skin, the nanoparticles in the patch harvest minute by minute, and protect from degradation, all candidate analytes in the sweat underneath the patch. The core shell bait nanoparticles are a completely novel technology that can amplify the sensitivity of biomarker detection by 100 fold. No other technology exists that has a similar yield, concentration ability, and stabilization function. Once the collection is complete, the patch can simply be mailed to the diagnostic lab at room temperature. Upon receipt, the nanoparticle-captured analytes of interest can be eluted from the patch for routine measurement using any platform. Our feasibility studies demonstrate virtually 100 percent capture and 100 percent elution yield of low abundance interleukins in model sweat solutions. We will engineer the nanoparticles, and construct test patch devices. The test patches will be evaluated in animal models to verify lack of skin irritation. We will collect sweat from healthy volunteers under IRB approval using an FDA approved iontophoresis sampling device used for electrolyte measurement. We will apply the collected sweat to the novel nanoparticle patch ex vivo at the point of collection. Mass spectrometry will be used to discover novel sweat biomarkers that have been concentrated and preserved in the patch. Low abundance labile sweat biomarkers harvested from the nanoparticles will be measured by clinical immunoassays to verify sensitivity and precision. The derived list of eccrine sweat proteins will be an important deliverable as a foundation for the general field of sweat biomarker testing. The technology is especially suited to the evaluation of neurological disorders as it is non invasive and would be fully acceptable as a routine screening procedure.
The low abundance and low molecular weight proteins and metabolites present in human sweat provide great promise as a source of new biomarkers for neurological disease diagnosis. The nanotechnology proposed in this grant will greatly reduce the preanalytical variability associated with collection and storage of biological fluids for clinical analysis and at the same time allow for detection of low abundance and labile analytes otherwise not possible.
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