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

摘要 血清和尿液中的蛋白质可作为早期癌症、创伤性脑损伤和其他生命的诊断指标。 具有威胁性的条件，但在超低浓度下很难检测到。而超灵敏的蛋白质检测 已经使用诸如Quanterix的SiMoA的数字(即分子计数)ELISA(DeLISA)平台， 这些仪器需要专门和复杂的光学元件来进行单分子检测，这是很难做到的 小型化。固态纳米孔(SsNPs)现在为蛋白质提供了一种替代的数字传感机会 生物标志物在我们发明受控介电击穿(CBD)作为一种廉价的 单一纳米孔的制造。与DeLisa平台的光学读出不同，ssNPs可以提供完全 电子解决方案，适用于将超灵敏诊断带到 资源设置较低。我们的提案汇集了一支在SSNP传感器方面拥有专业知识的成熟团队， 分离、微流控和分子诊断，以解决剩余的技术挑战 基于SSNP的超灵敏传感。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分析来解决 血清或尿液中的每个靶蛋白生物标志物与数百个50nt100个DNA代用品偶联到60 nm金 纳米颗粒(AuNP)。然后，该信号将在使用超薄纳米孔的微流控平台中浓缩 氮化硅(NPN)膜，经证实具有捕获和浓缩高达10,000个AuNPs的能力 收牌。这些代理将在紫外光下从NPN中释放出来，并将快速与DNA纳米结构杂交 这在仅几百微米外的SSNP传感器中提供了强大的信号。加起来有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 生物标记物在几分钟内。这个新的工具，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需要增加的生物标志物小组上进行验证 增强因素：1)预测膀胱癌免疫治疗疗效的尿液生物标志物； 血清中的生物标记物用于检测脑损伤。尿液生物标志物将从CyPRIT诺模图中提取 预测膀胱癌(BC)对接种卡介苗(BCG)的反应的小组。 此面板的阈值在低PM/高FM范围内。我们更具挑战性的应用将是低调频 血清脑损伤生物标志物(UCHL1、GFAP)水平检测。这两项研究的表现都将作为基准 与Quanterix的SiMoA HD-1分析仪进行对比。

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.