Ultrasensitive Detection of Urine LAM for Point-of-Care Diagnosis of All Forms of Tuberculosis

项目摘要 结核病(TB)仍然是最具挑战性的疾病之一，有1000万新病例和200万新病例 每年死亡，以及因诊断不足和检测不足而出现的严重问题。医疗点(PoC)测试 对于所有敏感、快速、简单、不以痰为基础的结核病是迫切需要的。最有希望的 实现这一点的方法是一种基于结核生物标记物脂阿拉伯甘露聚糖(LAM)的免疫分析。 S的长期目标是开发一套超灵敏、与目标无关、低复杂性的新型 基于光流体单分子技术的诊断仪器，将集成光学和 基于单芯片系统的微流控技术。该项目的目标是开发第一个兼容PoC的 与FIND(创新创新基金会)合作建立用于结核病诊断的尿液LAM检测系统 诊断)，世界领先的非营利性组织，为贫困相关疾病的诊断。这个测试 将使用单蛋白检测来实现PCR的灵敏度，同时相对简单和快速的 免疫分析。第一阶段将介绍项目核心的创新元素，并产生 演示PoC测试的关键性能参数。 我们的目标将通过完成以下具体目标来实现，这些目标将引入新的创新 概念：(1)超灵敏LAM分析的演示；(2)在 标准的光流控体系结构；以及(3)优化的样品制备过程的集成。在.之下 首先，我们将证明LAM可以用单分子分析灵敏度进行鉴定和定量。 具体地说，我们将开发一种基于珠子的固相萃取分析方法，使用同类中最好的抗体(提供 FIND)和明亮的荧光纳米颗粒记者。1 pg/m L的分析下限和动力学 将展示5个对数的范围-比唯一可用的LAM分析(Alere LF-LAM)的灵敏度高500倍。 第二个目的是介绍一种利用光栅耦合将光传输到光流控芯片的新方法。这 该方法针对大规模制造以及PoC仪器的复杂性和成本进行了优化。我们将适配当前的芯片 集成了光栅耦合器的制造工艺，并展示了20%的光耦合效率。在.之下 第三，我们将推出一种一次性预备盒，用于一步提取/分离尿液LAM 通过流体-流体界面，具有至少90%的捕获效率。 这项拟议的工作意义重大，因为它将导致首个用于结核病诊断的超灵敏LAM试验。 在PoC的尿液。该检测将具有PCR检测的特异性和敏感度，但复杂性和 免疫分析的费用，以及诊断各种结核病的费用。然后，这一概念将成为 更广泛的单分子诊断仪器，用于病原体和生物标志物的快速检测。 1

Project Summary Tuberculosis (TB) remains one of the most challenging diseases with ten million new cases and two million deaths yearly, and severe problems due to underdiagnosis and inadequate testing. A point-of-care (PoC) test for all kinds of TB that is sensitive, quick, simple, and not sputum-based is urgently needed. The most promising avenue to accomplish this is an immunoassay based on the TB biomarker Lipoarabinomannan (LAM). Fluxus Inc.’s long-term goal is to develop a novel suite of ultrasensitive, target-agnostic, low-complexity diagnostic instruments based on optofluidic single-molecule technology that combines integrated optics and microfluidics on a single chip-based system. The objective of this project is to develop the first PoC compatible system for detection of LAM in urine for TB diagnosis in collaboration with FIND (Foundation for Innovative New Diagnostics), the world’s leading non-profit organization for diagnostics for poverty-related diseases. The test will use single protein detection to achieve the sensitivity of PCR with the relative simplicity and speed of an immunoassay. Phase I will introduce the innovative elements at the core of the project and result in the demonstration of the critical performance parameters for a PoC test. Our objectives will be accomplished by completing the following specific aims that introduce new innovative concepts: (1) Demonstration of an ultrasensitive LAM assay; (2) Introduction of grating coupled waveguides on the standard optofluidic architecture; and (3) Integration of an optimized sample-preparation process. Under the first Aim, we will show that LAM can be identified and quantitated with single-molecule analytical sensitivity. Specifically, we will develop a bead-based solid-phase extraction assay using best-in-class antibodies (provided by FIND) and bright fluorescent nanoparticle reporters. An analytical limit of detection of 1 pg/mL and a dynamic range of 5 logs will be demonstrated – 500x more sensitive than the only available LAM assay (Alere LF-LAM). The second Aim introduces a new approach using a grating couple to deliver light into the optofluidic chip. This method is optimized for mass fabrication and PoC instrument complexity and cost. We will adapt the current chip fabrication process to incorporate the grating coupler and demonstrate 20% light coupling efficiency. Under the third Aim, we will introduce a disposable pre-prep cartridge for single-step urine LAM extraction/separation across a fluid-fluid interface with at least 90% capture efficiency. The proposed work is significant because it will lead to the first ultrasensitive LAM assay for TB diagnosis from urine at the PoC. The test will have the specificity and sensitivity of PCR detection, but at the low complexity and cost of an immunoassay, and for diagnosis of all kinds of TB. This concept will then serve as the blueprint for a broader class of single-molecule diagnostic instruments for rapid pathogen and biomarker detection. 1