Solid-state nanopore detection of protein biomarkers for early sepsisdiagnosis

用于脓毒症早期诊断的蛋白质生物标志物的固态纳米孔检测

• 批准号: 10841313
• 负责人: Xiyun Guan
• 金额: $ 31.01万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-15 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10841313
• 关键词: Antibodies; Area; Bacteria; Biological Assay; Biological Markers; Biosensing Techniques; C-reactive protein; CD14 gene; Cause of Death; Cessation of life; Clinical; DNA; Detection; Diagnosis; Diagnostic Sensitivity; Diagnostic tests; Diameter; Dimensions; Dose; Drug Industry; Early Diagnosis; Enzyme-Linked Immunosorbent Assay; Functional disorder; Goals; Grant; Healthcare; Hospital Costs; Hospitalization; Human; Immune response; Immunoassay; Incidence; Incubated; Individual; Infection; Interleukin-1; Interleukin-6; Investigation; Ionic Strengths; Length; Life; Lipopolysaccharides; Measurement; Medicine; Methodology; Modeling; Molecular Biology; Monitor; Monoclonal Antibodies; Nanotechnology; Organ; Patients; Persons; Preparation; Prognosis; Proteins; Reporter; Resolution; Sampling; Sepsis; Septic Shock; Series; Serum; Single-Stranded DNA; Societies; Sodium Chloride; Symptoms; System; TNF gene; Temperature; Thick; Time; Training; United States; Work; accurate diagnosis; antibody conjugate; antimicrobial; biomarker panel; clinical diagnosis; cost; detection limit; ds-DNA; early detection biomarkers; effectiveness evaluation; global health; improved; magnetic beads; microbial; mortality; multiplex assay; nanoGold; nanopore; portability; procalcitonin; programs; protein biomarkers; response; sensor; septic patients; silicon nitride; solid state; specific biomarkers; success; tool; voltage

Summary Sepsis is life-threatening organ dysfunction due to a dysregulated host response to microbial infection, and is a global healthcare problem with high incidence and mortality rates, responsible for 20% of deaths worldwide. In the USA, sepsis is the most common cause of death among hospitalized patients and the total hospital costs of treating sepsis are estimated at more than $24 billion annually. The high mortality rate of sepsis is due in part to delays in diagnosis and management, as a result of its initial atypical and nonspecific symptoms and lack of early and sensitive diagnostic test. The goal of this program is to develop an ultra-sensitive, highly selective, and portable solid-state nanopore sensing platform to profile a panel of sepsis protein biomarkers in clinical samples instead of one specific biomarker as currently used in the clinical setting to provide more comprehensive parameters for accurate diagnosis of sepsis at the early stage and monitoring the treatment prognosis. Aim 1: Utilize procalcitonin (PCT) as a model protein to demonstrate the feasibility of utilizing our proposed solid-state nanopore sensing strategy, which takes advantage of a combination of magnetic beads, sandwich immunoassay, DNA reporter probe cascade and amplification, and DNA-functionalized gold nanoparticles (AuNPs), as an effective generic approach for the sensitive and accurate detection of proteins in clinical samples. To optimize the sensor sensitivity, the effects of various factors such as incubation time, denature temperature, AuNPs diameter, DNA reporter probe length, nanopore dimension, etc. on PCT detection will be examined by using a silicon nitride nanopore. Furthermore, we will construct dose-response curve for PCT, and perform selectivity study & simulated serum sample analysis. Aim 2: Build on the nanopore-based PCT detection methodology developed in aim #1 to develop a nanopore-based multiplexing sensing platform for simultaneous detection and quantification of multiple sepsis protein biomarkers. An array of seven silicon nitride nanopore sensors will be constructed and used to quantitatively detect PCT, C-reactive protein (CRP), interleukin-1 (IL-1), IL-6, presepsin (soluble CD14 subtype), tumour necrosis factor-α (TNF-α) and lipopolysaccharide (LPS) in protein mixtures at various concentrations. Aim 3: Analyze clinical serum samples. To evaluate the effectiveness of utilizing our developed solid- state nanopore sensing platform for accurate sepsis diagnosis and prognosis, the multiplexing nanopore sensor array developed in aim 2 will be used to analyze 120 clinical serum samples from sepsis patients at different stages and healthy controls, as well as from sepsis patients who received antimicrobial therapy. The concentrations of PCT, CRP, IL-1, IL-6, presepsin, TNF-α and LPS in these serum samples will be determined. In comparison, these samples will additionally be analyzed using ELISA detection kits.

总结 脓毒症是由于宿主对微生物感染的反应失调而导致的危及生命的器官功能障碍，并且是一种严重的疾病。 高发病率和高死亡率的全球医疗保健问题，占全球死亡人数的20%。在 在美国，败血症是住院患者中最常见的死亡原因， 治疗败血症的费用估计每年超过240亿美元。败血症的高死亡率部分原因是 延误诊断和管理，由于其最初的非典型和非特异性症状和缺乏早期诊断， 敏感的诊断测试。该计划的目标是开发一种超敏感，高选择性， 便携式固态纳米孔感测平台以分析临床样品中的一组脓毒症蛋白质生物标志物 而不是如目前在临床环境中使用的一种特定生物标志物以提供更全面的 为脓毒症的早期准确诊断和治疗预后的监测提供参考。 目的1：利用降钙素原（PCT）作为模型蛋白，以证明利用我们的 提出了固态纳米孔传感策略，其利用磁珠的组合， 夹心免疫测定、DNA报告探针级联和扩增以及DNA功能化 金纳米粒子（AuNPs），作为一种有效的通用方法，用于蛋白质的灵敏和准确检测 在临床样本中。为了优化传感器灵敏度，需要考虑各种因素的影响，例如孵育时间， PCT检测中的变性温度、AuNPs直径、DNA报告探针长度、纳米孔尺寸等 将通过使用氮化硅纳米孔来检查。此外，我们将构建剂量-反应曲线， PCT，并进行选择性研究和模拟血清样本分析。 目标2：在目标1中开发的基于纳米孔的PCT检测方法的基础上， 用于同时检测和定量多种脓毒症的基于纳米孔的多路传感平台 蛋白质生物标志物。七个氮化硅纳米孔传感器的阵列将被构建并用于 定量检测PCT、C反应蛋白（CRP）、白细胞介素-1（IL-1）、IL-6、前体蛋白（可溶性CD 14亚型）， 肿瘤坏死因子-α（TNF-α）和脂多糖（LPS）在不同浓度的蛋白混合物中。 目的3：分析临床血清样品。为了评估利用我们开发的固体- 国家纳米孔传感平台，用于准确的脓毒症诊断和预后，多路复用纳米孔传感器 在aim 2中开发的阵列将用于分析120份来自不同浓度脓毒症患者的临床血清样本。 阶段和健康对照，以及接受抗菌治疗的脓毒症患者。的 将测定这些血清样本中的PCT、CRP、IL-1、IL-6、presepsin、TNF-α和LPS浓度。 相比之下，将使用ELISA检测试剂盒对这些样本进行额外分析。