Acoustofluidic Pipette for Rapid Serodiagnosis of Candida Infection

摘要 侵袭性真菌病(IFDS)在美国是一个巨大且日益严重的问题。传统的治疗方法 IFDS检测包括血培养和组织病理学，需要两到三天的时间才能完成。作为一名 预防措施，病人经常被给予广谱抗生素，但对IFDS无效。 并会损害患者的共生微生物区系，导致严重的生物失调和相关的健康问题。至 为了解决这个问题，已经提出了几种血清学检测方法来检测猪瘟的抗原或抗体反应。 血。然而，这些方法由于高成本、复杂性和高成本而未能取代临床支柱。 最重要的是，敏感性和/或特异性有限，特别是在感染的早期阶段。我们的中央 假设是开发一种简单而灵敏的护理点(POC)设备，该设备可以捕获和 同时纯化多个生物标志物将从根本上提高血清诊断的价值。我们的大妈 目的是研制一种手持式声学移液管，用于快速有效地分离真菌生物标志物和 为下游分析准备这些生物标记物。这项探索性研究的结果将是多才多艺的 适用于自动化的多重血清学检测的原型。这台仪器将会是 通过创新地使用负声学对比度，使其有别于其他IFD生物传感技术 粒子(NACP)。由于它们的弹性，NACPs可以固定在声学的压力抗力点上。 驻波。这将使我们能够捕获特定的生物标记物(例如，抗体、抗原)，从声音上捕获 并将血液碎片作为废物冲走。然后，捕获的生物标志物将被混合和标记 在容纳的一次性盒中使用微结构流体室进行二次荧光抗体 在移液管内。一旦生物标记物被清洗和染色，NACP将从设备中排出用于 通过标准分析技术进行分析，如显微镜或流式细胞仪。我们把白色念珠菌作为一种 由于其独特的和特征良好的生物标志物，本提案中的模型IFD病原体。我们提出三个建议 明确的目标。AIM 1将开发一种声学移液管，用于快速混合、捕获和清洗NACP。目标2将 确定检测白色念珠菌生物标志物的敏感性和特异性：抗ENO抗体、抗-ENO抗体 Fba1抗体和eNO抗原。AIM 3将开发一种多重检测方法，同时分离和 使用条形码NACP区分白色念珠菌的多个生物标志物。AIM 3也将扩展这种多路传输 概念筛选高产乳杆菌，一种对两性霉素B完全抗药性的真菌，以告知早期治疗 抗真菌药物的决定。如果成功，这项技术将极大地缩短 分离和检测生物标志物不仅可能影响念珠菌病的诊断，而且更具致命性和 人体工程学半自动装置中的模糊IFDS。一旦得到验证，未来的工作将推动这一点 通过集成在线荧光检测模块，将样机转化为完整的POC仪器。

ABSTRACT Invasive fungal diseases (IFDs) represent a large and growing problem in the U.S. Conventional methods for detecting IFDs include blood culture and histopathology, which require two to three days to complete. As a precautionary measure, patients are often given broad-spectrum antibiotics, which are ineffective against IFDs and can damage patients’ commensal microbiota, leading to severe dysbiosis and related health issues. To address this issue, several serological assays have been proposed to detect antigens or antibody responses in blood. However, these methods have failed to supplant clinical mainstays due to high cost, complexity, and most of all, limited sensitivity and/or specificity, especially during the early phases of infection. Our central hypothesis is that development of a simple and sensitive point-of-care (POC) device that can capture and purify multiple biomarkers simultaneously will radically improve the value of serodiagnoses. Our broad objective is to develop a handheld acoustic pipette for the rapid and efficient isolation of fungal biomarkers and prepare those biomarkers for downstream analysis. The outcome of this exploratory research will be a versatile prototype for multiplexed serological detections that is amenable to automation. This instrument will be distinguished from other IFD biosensing technologies through its innovative use of negative acoustic contrast particles (NACPs). Due to their elasticity, NACPs can immobilize against the pressure antinodes of an acoustic standing wave. This will allow us to capture specific biomarkers (e.g., antibodies, antigens), acoustically trap them within the device, and flush blood debris as waste. Then, captured biomarkers will be mixed and labeled with secondary fluorescent antibodies using microstructured fluidic chambers in a disposable cartridge housed within the pipette. Once biomarkers have been washed and stained, NACPs will be ejected from the device for analysis by standard analytical techniques, like microscopy or flow cytometry. We focus on C. albicans as a model IFD pathogen in this proposal due to its unique and well-characterized biomarkers. We propose three Specific Aims. Aim 1 will develop an acoustic pipette for rapid NACP mixing, trapping and washing. Aim 2 will determine the assay sensitivity and specificity for detecting C. albicans biomarkers: anti-Eno antibodies, anti- Fba1 antibodies and Eno antigens. Aim 3 will develop a multiplexed assay to simultaneously isolate and distinguish multiple biomarkers for C. albicans using barcoded NACPs. Aim 3 will also extend this multiplexing concept to screen for L. prolificans, a fungus that is fully resistant to amphotericin B, to inform early treatment decisions for antifungal medications. If successful, this technology will dramatically shorten the time needed to isolate and detect biomarkers that may impact not only the diagnosis of candidiasis, but also more lethal and obscure IFDs in an ergonomic and semi-automated device. Once validated, future work will advance this prototype to a full POC instrument by the integration of an in-line fluorescence inspection module.