A non-invasive nanotechnology-based skin patch for multiplexed diagnostics

描述（由申请人提供）：汗液是一种未开发的生物液体，可以提供丰富的诊断信息。我们提出了一种用于床边或现场的新技术：一种诊断皮肤贴片，它可以收集、浓缩和稳定来自皮肤渗出液或汗液的一组生物标志物。虽然药物输送贴片是常规使用，但这里提出的技术具有完全相反的功能：收集诊断标记。使用质谱法，我们已经鉴定出228种蛋白质和多肽，这些蛋白质和多肽以前不知道存在于人类汗液中。为了开发这类新的分析物，我们提出了一种新的亲和性诱饵纳米粒子，结合在一个粘附的皮肤贴片内。这项提议的技术是革命性的，因为它克服了所有阻碍使用这种生物液体进行诊断测试的主要生理障碍。汗液疾病生物标志物a)由于汗液和正常皮肤菌群中存在的蛋白酶而容易快速降解，b)丰度极低，远低于标准分析平台的检测灵敏度。收获水凝胶纳米颗粒是用化学高亲和力诱饵设计的，因此它们隔离了低丰度的目标分析物，并保护它们免受无限期降解。我们建议将纳米颗粒整合到皮肤贴片的织物中。一旦贴在皮肤上，贴片中的纳米颗粒每分钟都在收集，并防止降解，所有候选分析物都在贴片下的汗液中。核壳诱饵纳米颗粒是一种全新的技术，可以将生物标志物检测的灵敏度提高100倍。没有其他技术具有类似的产量、浓缩能力和稳定功能。一旦收集完成，贴片可以简单地在室温下邮寄到诊断实验室。接收后，纳米颗粒捕获的感兴趣的分析物可以从贴片中洗脱，用于使用任何平台进行常规测量。我们的可行性研究表明，在模型汗液中，低丰度白介素的捕获率和洗脱率几乎达到100%。我们将设计纳米粒子，并构建测试贴片装置。测试贴片将在动物模型中进行评估，以验证没有皮肤刺激。我们将在IRB批准下，使用FDA批准的用于电解质测量的离子电泳取样装置收集健康志愿者的汗液。我们将在体外收集点将收集到的汗液应用到新型纳米颗粒贴片上。质谱法将用于发现新的汗液生物标志物，这些生物标志物已经被浓缩并保存在贴片中。从纳米颗粒中收集的低丰度不稳定汗液生物标志物将通过临床免疫分析来测量，以验证其灵敏度和精度。所得的汗液蛋白清单将成为汗液生物标志物测试领域的重要基础。该技术特别适合于评估神经系统疾病，因为它是非侵入性的，完全可以作为常规筛查程序。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.