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 侵袭性真菌病（IFD）是美国面临的一个日益严重的大问题。 检测 IFD 包括血培养和组织病理学，需要两到三天才能完成。 作为预防措施，患者通常会服用广谱抗生素，但这些抗生素对 IFD 无效 并可能损害患者的共生微生物群，导致严重的菌群失调和相关的健康问题。 为了解决这个问题，已经提出了几种血清学测定法来检测抗原或抗体反应 然而，由于成本高、复杂性和稳定性差，这些方法未能取代临床主流。 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。 假设是开发一种简单而灵敏的护理点 (POC) 设备，可以捕获和 同时纯化多种生物标志物将从根本上提高血清诊断的价值。 目标是开发一种手持式声移液器，用于快速有效地分离真菌生物标志物和 准备这些生物标志物用于下游分析。这项探索性研究的结果将是多用途的。 该仪器将用于多重血清学检测的原型。 通过创新性地使用负声学对比，与其他 IFD 生物传感技术区分开来 由于其弹性，NACP 可以抵抗声波的压力波腹而固定不动。 这将使我们能够捕获特定的生物标志物（例如抗体、抗原）、声学捕获。 将它们放入设备中，并将血液碎片作为废物冲洗掉，然后将捕获的生物标记物混合并标记。 带有二次荧光抗体，使用一次性盒中的微结构流体室 一旦生物标记物被清洗和染色，NACP 就会从设备中弹出。 通过标准分析技术（如显微镜或流式细胞术）进行分析，我们重点关注白色念珠菌。 由于其独特且特征明确的生物标志物，我们在本提案中提出了模型 IFD 病原体。 具体目标 目标 1 将开发用于快速 NACP 混合、捕获和清洗的声学移液器。 确定检测白色念珠菌生物标志物的敏感性测定和特异性：抗 Eno 抗体、抗 Aim 3 将开发一种多重检测方法来同时分离和检测 Fba1 抗体和 Eno 抗原。 使用条形码 NACP 区分白色念珠菌的多个生物标志物 Aim 3 也将扩展这种多重检测。 筛查繁殖乳杆菌（一种对两性霉素 B 完全耐药的真菌）的概念，为早期治疗提供信息 如果成功，这项技术将大大缩短抗真菌药物决策所需的时间。 分离和检测生物标志物不仅可能影响念珠菌病的诊断，而且可能影响更致命和更严重的疾病。 一旦经过验证，未来的工作将推动这一点。 通过集成在线荧光检测模块将原型转变为完整的 POC 仪器。

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