Acoustofluidic Pipette for Rapid Serodiagnosis of Candida Infection

用于念珠菌感染快速血清诊断的声流控移液器

• 批准号: 10388272
• 负责人: Charles Wyatt Shields IV
• 金额: $ 19.4万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-09 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10388272
• 关键词: Acoustics; Address; Amphotericin B; Antibiotics; Antibodies; Antibody Response; Antifungal Agents; Antigens; Automation; Bar Codes; Benchmarking; Biological Assay; Biological Markers; Biosensing Techniques; Biosensor; Blood; Blood specimen; Buffers; Candida; Candida albicans; Candida auris; Candidiasis; Clinical; Color; Cytolysis; Dangerousness; Detection; Development; Devices; Diagnosis; Disease; Early Diagnosis; Early treatment; Elasticity; Elements; Enzyme-Linked Immunosorbent Assay; Equation; Erythrocytes; Flow Cytometry; Fluorescence; Fluorescent Antibody Technique; Flushing; Fructosediphosphate Aldolase; Future; Glucans; Goals; Health; Histopathology; Immobilization; Infection; Label; Lasers; Liquid substance; Measures; Mechanics; Methods; Microscope; Microscopy; Modeling; Multi-Drug Resistance; Mycoses; Outcome; Particle Size; Patients; Pharmaceutical Preparations; Phase; Physiological; Radiation; Reader; Research; Resistance; Resistant candida; Risk Factors; Sampling; Sensitivity and Specificity; Serodiagnoses; Serology; Serology test; Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stains; System; Techniques; Technology; Testing; Therapeutic Intervention; Time; Work; antibody detection; antigen detection; beta-Glucans; commensal microbes; cost; culture plates; detector; dysbiosis; enolase; fungus; head-to-head comparison; improved; innovation; instrument; multiplex assay; multiplex detection; particle; particle chamber; pathogen; point of care; pressure; processing speed; prototype; specific biomarkers; targeted biomarker; tool; wasting

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.

抽象的 侵入性真菌疾病（IFD）代表了美国常规方法的大问题 检测IFD包括血液培养和组织病理学，需要两到三天才能完成。作为 预防性测量，患者通常会被给予广谱抗生素，而对IFD的无效 并可能损害患者的共生微生物群，导致严重的营养不良和相关的健康问题。到 解决这个问题，已经提出了几种血清学测定法以检测抗原或抗体反应 血。但是，由于成本高，复杂性和 最重要的是，敏感性和/或特异性有限，尤其是在感染的早期阶段。我们的中心 假设是可以捕获和 纯化多种生物标志物将根本上提高血清诊断的价值。我们的广阔 目的是开发一个手持式声学移液器，以快速有效地隔离真菌生物标志物和 准备这些生物标志物进行下游分析。这项探索性研究的结果将是一种多功能 多重血清学检测的原型，可自动化。这个乐器将是 与其他IFD生物传感技术通过创新使用负面的对比度区别 颗粒（NACP）。由于其弹性，NACP可以固定在声学的压力抗in 站立波。这将使我们能够捕获特定的生物标志物（例如抗体，抗原），声学陷阱 它们在设备中，将血液碎屑作为废物冲洗。然后，将捕获的生物标志物被混合并标记 使用微结构的液体室与一次性弹药筒中的二级荧光抗体一起使用 在移液器中。一旦将生物标志物洗涤并染色，NACP将从设备中弹出 通过标准分析技术进行分析，例如显微镜或流式细胞仪。我们专注于白色念珠菌作为 由于其独特且特征良好的生物标志物，该提案中的IFD病原体模型。我们提出了三个 具体目标。 AIM 1将开发一种声学移液器，用于快速的NACP混合，捕获和洗涤。 AIM 2意志 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，抗体 FBA1抗体和ENO抗原。 AIM 3将开发多重测定法以同时隔离和 使用条形码的NACP的白色念珠菌的独特多个生物标志物。 AIM 3还将扩展此多路复用 筛选L. prourificans（一种对两性霉素B具有抗性的真菌）的概念，以告知早期治疗 抗真菌药物的决定。如果成功，这项技术将大大缩短 分离和检测生物标志物不仅可能影响念珠菌病的诊断，而且还会影响更大的致命性和 符合人体工程学和半自动化设备中的IFD。一旦得到验证，未来的工作将推进 通过在线荧光检查模块的整合到完整POC仪器的原型。

项目成果

Magnetically locked Janus particle clusters with orientation-dependent motion in AC electric fields.

磁锁 Janus 粒子簇在交流电场中具有方向相关的运动。

• DOI: 10.1039/d3nr03744d
• 发表时间: 2023
• 期刊: Nanoscale
• 影响因子: 6.7
• 作者: Lee,JinGyun;Thome,CooperP;Cruse,ZoeA;Ganguly,Arkava;Gupta,Ankur;Shields4th,CWyatt
• 通讯作者: Shields4th,CWyatt