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

基本信息

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
项目类别：
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

项目摘要

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.

概括由于宿主对微生物感染的反应失调，败血症是威胁生命的器官功能障碍，并且是一种全球医疗保健问题与高事件和死亡率高，造成全球20％的死亡。在美国，败血症是住院患者中最常见的死亡原因，医院总费用为治疗败血症每年估计超过240亿美元。败血症的高死亡率部分是由于由于其最初的非典型和非特异性症状以及缺乏早期的诊断和管理延迟和敏感的诊断测试。该计划的目的是开发超敏感，高度选择性，并且便携式固态纳米孔传感平台，以介绍临床样品中败血症蛋白生物标志物的小组而不是当前在临床环境中使用的特定生物标志物来提供更全面的早期败血症准确诊断并监测治疗预后的参数。 AIM 1：利用procalcitonin（PCT）作为模型蛋白来证明利用我们的可行性提出的固态纳米孔感应策略，利用磁珠组合的优势三明治免疫测定，DNA报告基因探测级联和扩增以及DNA官能化金纳米颗粒（AUNP），作为敏感和准确检测蛋白质的有效通用方法在临床样品中。为了优化传感器敏感性，各种因素（例如孵化时间）的影响，变性温度，Aunps直径，DNA报告探头长度，纳米孔维度等。将使用氮化硅纳米孔检查。此外，我们将构建剂量响应曲线 PCT并进行选择性研究和模拟血清样品分析。目标2：建立在AIM＃1中开发的基于纳米孔的PCT检测方法基础上基于纳米孔的多重灵敏度平台，用于简单检测和定量多种败血症蛋白质生物标志物。将构建七个氮化硅纳米孔传感器的阵列，并用于定量检测PCT，C反应蛋白（CRP），白介素-1（IL-1），IL-6，Presepsin（可溶性CD14亚型），蛋白质混合物中各种浓度的肿瘤坏死因子-α（TNF-α）和脂多糖（LPS）。目标3：分析临床血清样品。评估使用我们开发的固体的有效性用于精确败血症诊断和预后的状态纳米孔传感器平台，多重纳米孔传感器在AIM 2中开发的阵列将用于分析不同的败血症患者的120个临床血清样品阶段和健康对照，以及接受抗菌治疗的败血症患者。这些血清样品中的PCT，CRP，IL-1，IL-6，Presepsin，TNF-α和LP的浓度将确定。相比之下，将使用ELISA检测试剂盒对这些样品进行分析。