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

摘要 血清和尿液中的蛋白质提供了早期癌症、创伤性脑损伤和其他生命体征的诊断指征。 威胁条件，但在超低浓度下难以检测。当超灵敏的蛋白质检测 已经使用数字（即分子计数）ELISA（dELISA）平台如Quanterix的SiMoA， 这些仪器需要用于单分子检测的专门且复杂的光学器件， 把它藏起来。固态纳米孔（ssNPs）现在为蛋白质提供了另一种数字传感机会 在我们发明受控介电击穿（CBD）作为一种廉价的方法之后， 单纳米孔制造。与dELISA平台的光学读出不同，ssNP可以提供完全的免疫荧光。 电子解决方案的低成本，即时护理仪器，需要带来超灵敏的诊断， 低资源设置。我们的提案汇集了一个在ssNP传感器方面具有专业知识的成功团队， 分离，微流体和分子诊断，以解决剩余的技术挑战， 基于ssNP的超灵敏感测。我们将通过与 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， 血清或尿液中的每个靶蛋白生物标志物与数百个50 nt 100 DNA代理物偶联到60 nm金 纳米颗粒（AuNP）。然后，该信号将被集中在微流体平台中， 氮化硅（NPN）膜，具有捕获和浓缩高达10，000 AuNP的能力 折代理将从NPN中释放出紫外光，并将迅速与DNA纳米结构杂交 在距离只有几百微米的ssNP传感器中提供强大的信号。共106- 生物标志物浓度的成倍增加将使ssNP能够处理来自fM浓度的蛋白质的信号 生物标志物（分钟）这种新颖的工具，我们称之为代理增强的捕获和释放 ssNP（CRePE-ssNP）将在两个临床相关的生物标志物组上进行验证， 增强因子：1）预测膀胱癌免疫治疗功效的尿生物标志物;和2） 用于检测脑损伤的血清生物标志物。将从CyPRIT列线图中提取尿液生物标志物 预测膀胱癌（BC）对接种卡介苗（BCG）的反应。 该面板的阈值在低pM/高fM范围内。我们更具挑战性的应用将是低fM 血清中TBI生物标志物（UCHL 1、GFAP）的水平检测。将对两项研究的性能进行基准测试 对比Quanterix的SiMoA HD-1分析仪。

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.