Solid-state nanopores and silicon nanomembranes for ultrasensitive protein biomarker detection

用于超灵敏蛋白质生物标志物检测的固态纳米孔和硅纳米膜

基本信息

批准号：
10631966
负责人：
JONATHAN D FLAX
金额：
$ 41.42万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-06-15 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10631966
关键词：
Affinity Chromatography BCG Live Bacillus Benchmarking Binding Biological Assay Biological Markers Blood Brain Injuries Calibration Complex Coupled DNA Detection Development Devices Diagnosis Diagnostic Electron Microscopy Electronics Emergency Department patient Ensure Enzyme-Linked Immunosorbent Assay FDA approved Geometry Glial Fibrillary Acidic Protein Hour IL8 gene Immunoassay Immunotherapy Injury Interleukin-2 Interleukin-6 Label Life Magnetism Malignant Neoplasms Malignant neoplasm of urinary bladder Measurement Measures Membrane Methods Microfluidic Microchips Microfluidics Microspheres Modeling Molecular Nanoporous Nanostructures Nervous System Trauma Nomograms Optics Patients Performance Positioning Attribute Process Proteins Proxy Public Health Resource-limited setting Sampling Scheme Serum Signal Transduction Silicon Single-Stranded DNA System TNF gene Techniques Translating Traumatic Brain Injury UCHL1 gene Ultraviolet Rays Urine Work biomarker panel cancer immunotherapy cancer therapy clinically relevant cost cost effective current pandemic cytokine design detector diagnostic platform digital electronic sensor enhancing factor fabrication head-to-head comparison instrument invention magnetic beads meter miniaturize molecular diagnostics nanoGold nanomembrane nanoparticle nanopore next generation non-muscle invasive bladder cancer novel operation point of care point-of-care diagnostics portability predicting response predictive marker protein biomarkers response sensor silicon nitride single molecule solid state

项目摘要

Abstract Proteins in serum and urine provide diagnostic indications of early cancers, traumatic brain injury, and other life threatening conditions, but are difficult to detect at ultra-low concentrations. While ultrasensitive protein detection has been achieved using digital (i.e. molecular counting) ELISA (dELISA) platforms such as Quanterix’s SiMoA, these instruments require specialized and complex optics for single molecule detection, which is difficult to miniaturize. Solid-state nanopores (ssNPs) now offer an alternative digital sensing opportunity for protein biomarkers following our invention of Controlled Dielectric Breakdown (CBD) as an inexpensive method for single nanopore fabrication. Unlike the optical readout of dELISA platforms, ssNPs can provide a completely electronic solution for low-cost, point-of-care instruments that are needed to bring ultrasensitive diagnostics to low resource settings. Our proposal brings together an accomplished team with expertise in ssNP sensors, separations, microfluidics, and molecular diagnostics to solve the remaining technical challenges for ultrasensitive ssNP-based sensing. We will establish feasibility through a head-to-head comparison to the Quanterix SiMoA. The technical challenge of signal amplification will be solved by combining a new immunoassay that transduces every target protein biomarker in serum or urine to hundreds of 50nt 100 DNA proxies coupled to 60 nm gold nanoparticles (AuNP). This signal will then be concentrated in a microfluidic platform using ultrathin nanoporous silicon nitride (NPN) membranes that have a proven capacity to capture and concentrate AuNPs up to 10,000 fold. The proxies will be released from the NPN with UV light and will rapidly hybridize with DNA nanostructures that give robust signals in a ssNP sensor positioned only a few hundred micrometers away. The combined 106- fold increase in biomarker concentration will enable the ssNP to process signals from fM concentrations of protein biomarker in minutes. This novel instrument, which we’ve termed the catch and release for proxy enhancement ssNP (CRePE-ssNP), will be validated on two clinically relevant biomarker panels of requiring increasing enhancement factors: 1) urine biomarkers that predict bladder cancer immunotherapy efficacy; and 2) biomarkers in serum used to detect brain injury. Urine biomarkers will be drawn from the CyPRIT Nomogram panel which predict bladder cancer (BC) response to an inoculation with Bacillus Calmette-Guérin (BCG). Thresholds for this panel are in the low pM/high fM range. Our more challenging application will be the low fM level detection of TBI biomarkers (UCHL1, GFAP) in serum. Performance in both studies will be benchmarked against the SiMoA HD-1Analyzer from Quanterix.

抽象的血清和尿液中的蛋白质提供早期癌症、创伤性脑损伤和其他生命的诊断指示威胁条件，但在超低浓度下难以检测，而超灵敏的蛋白质检测。已通过使用数字（即分子计数）ELISA (dELISA) 平台（例如 Quanterix 的 SiMoA）实现，这些仪器需要专门且复杂的光学器件来进行单分子检测，这很难固态纳米孔（ssNP）现在为蛋白质提供了另一种数字传感机会。我们发明了受控介电击穿 (CBD) 作为一种廉价的方法与 dELISA 平台的光学读出不同，ssNP 可以提供完整的纳米孔制造。用于低成本、现场护理仪器的电子解决方案，这些仪器需要将超灵敏的诊断带到我们的建议汇集了一支在 ssNP 传感器方面具有专业知识的优秀团队，分离、微流体和分子诊断，以解决剩余的技术挑战我们将通过与基于 ssNP 的超灵敏传感进行头对头比较来确定其可行性。 Quanterix SiMoA。信号放大的技术挑战将通过结合一种新的免疫测定来解决，该免疫测定可转导血清或尿液中的每个目标蛋白生物标志物与数百个 50nt 100 DNA 代理耦合到 60 nm 金然后，该信号将被集中在使用超薄纳米多孔的微流体平台中。氮化硅 (NPN) 膜经证实能够捕获和浓缩高达 10,000 的 AuNP 代理将在紫外光的作用下从 NPN 中释放出来，并与 DNA 纳米结构快速杂交。在仅几百微米外的 ssNP 传感器中提供强大的信号组合的 106- 生物标志物浓度倍数增加将使 ssNP 能够处理来自 fM 蛋白质浓度的信号这种新颖的工具可以在几分钟内完成生物标记，我们将其称为代理增强的捕获和释放。 ssNP (CRePE-ssNP)，将在两个临床相关生物标志物组上进行验证，需要增加增强因素：1）预测膀胱癌免疫治疗效果的尿液生物标志物；2）用于检测脑损伤的血清中的生物标志物将从 CyPRIT 列线图中绘制。预测膀胱癌 (BC) 对接种卡介苗 (BCG) 的反应的小组。该面板的阈值处于低 pM/高 fM 范围内，我们更具挑战性的应用是低 fM。血清中 TBI 生物标志物（UCHL1、GFAP）的水平检测将对这两项研究的表现进行基准测试。与 Quanterix 的 SiMoA HD-1Analyzer 进行比较。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Digital immunoassay for biomarker concentration quantification using solid-state nanopores.

使用固态纳米孔进行生物标志物浓度定量的数字免疫测定。

DOI：
发表时间：
2021
期刊：
影响因子：
16.6
作者：
He, Liqun;Tessier, Daniel R;Briggs, Kyle;Tsangaris, Matthaios;Charron, Martin;McConnell, Erin M;Lomovtsev, Dmytro;Tabard
通讯作者：
Tabard

Analysis of Nanopore Data: Classification Strategies for an Unbiased Curation of Single-Molecule Events from DNA Nanostructures.

纳米孔数据分析：DNA 纳米结构单分子事件公正管理的分类策略。