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

• 批准号: 10427339
• 负责人: JONATHAN D FLAX
• 金额: $ 38.82万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 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/10427339
• 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; Emergency Department patient; Ensure; Enzyme-Linked Immunosorbent Assay; FDA approved; Geometry; Glial Fibrillary Acidic Protein; Gold; Hour; Hybrids; IL8 gene; Immunoassay; Immunotherapy; Injury; Interleukin-2; Interleukin-6; Label; Life; Liquid substance; 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; System; TNF gene; Techniques; Translating; Traumatic Brain Injury; UCHL1 gene; Ultraviolet Rays; Urine; Work; base; biomarker panel; cancer immunotherapy; cancer therapy; clinically relevant; cost; cost effective; cytokine; design; detector; diagnostic platform; digital; electronic sensor; enhancing factor; head-to-head comparison; instrument; invention; magnetic beads; miniaturize; molecular diagnostics; nanoGold; nanomembrane; nanoparticle; nanopore; next generation; non-muscle invasive bladder cancer; novel; operation; pandemic disease; point of care; point-of-care diagnostics; portability; predicting response; predictive marker; protein biomarkers; response; sensor; silicon nitride; single molecule; solid state

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迹象 威胁条件，但在超低浓度下很难检测到。而超敏感蛋白检测 使用数字（即分子计数）ELISA（DELISA）平台（例如Quanterix的Simoa）来实现 这些仪器需要专业且复杂的光学元件进行单分子检测，这很难 小型化。固态纳米孔（SSNP）现在为蛋白质提供了替代的数字传感机会 生物标志物遵循我们打算受控介电故障（CBD）作为廉价方法的生物标志物 单纳米孔制造。与Delisa平台的光学读数不同，SSNP可以完全提供 用于将超敏诊断的低成本，护理仪器的电子解决方案 资源设置低。我们的建议汇集了一支具有SSNP传感器专业知识的成就团队， 分离，微流体和分子诊断，以解决剩余的技术挑战 超敏感的基于SSNP的灵敏度。我们将通过与之进行正面比较来确定可行性 Quanterix Simoa。 信号扩增的技术挑战将通过组合转导的新免疫测定法解决 每个靶蛋白生物标志物中的血清或尿液中的每个靶蛋白生物标志物，至数百只100个DNA代理，耦合到60 nm Gold 纳米颗粒（AUNP）。然后，该信号将使用超薄纳米孔集中在微流体平台中 氮化硅（NPN）机制具有可靠的能力捕获和集中大小高达10,000的能力 折叠。代理将用紫外线从NPN释放，并将与DNA纳米结构迅速杂交 在仅位于几百微米的SSNP传感器中，这给出了强大的信号。组合106- 生物标志物浓度的倍数增加将使SSNP能够从FM浓度的蛋白质处理信号 生物标志物在几分钟内。这款新颖的乐器，我们称其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记面板上进行验证，以增加 增强因素：1）预测膀胱癌免疫疗法效率的尿液生物标志物；和2） 血清中的生物标志物用于检测脑损伤。尿液生物标志物将从Cyprit nomargram中获取 预测膀胱癌（BC）对接种芽孢杆菌（BC）的反应的面板。 该面板的阈值在低PM/高FM范围内。我们更挑战的应用是低FM 血清中TBI生物标志物（UCHL1，GFAP）的水平检测。两项研究的表现将基准测试 反对来自Quanterix的Simoa HD-1Analyzer。