Solid-state nanopore detection of protein biomarkers for early sepsisdiagnosis

摘要 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，是一种 全球医疗保健问题具有高发病率和高死亡率，占全球死亡人数的20%。在……里面 在美国，败血症是住院患者最常见的死亡原因， 据估计，每年治疗败血症的费用超过240亿美元。脓毒症死亡率高的部分原因是 由于其最初的非典型和非特异性症状以及缺乏早期诊断和治疗而延误诊断和治疗 和敏感的诊断试验。该项目的目标是开发一种超敏感、高度选择性和 便携式固态纳米孔传感平台用于分析临床样本中的一组脓毒症蛋白生物标志物 而不是目前在临床环境中使用的一个特定的生物标记物，以提供更全面的 为早期准确诊断脓毒症和监测治疗预后提供参数。 目的1：以降钙素原(PCT)为模型蛋白，论证其应用的可行性 提出的固态纳米孔传感策略，它利用磁珠的组合， 夹心免疫分析、DNA报告探针级联和放大以及DNA功能化 金纳米颗粒(AuNPs)作为一种灵敏、准确检测蛋白质的有效通用方法 在临床样本中。为了优化传感器的灵敏度，考虑了孵化时间、 变性温度、AuNPs直径、DNA报告探针长度、纳米孔尺寸等对PCT检测的影响 将通过使用氮化硅纳米孔进行检查。此外，我们还将构建剂量-效应曲线 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，开发 一种基于纳米孔的多重脓毒症同时检测和定量传感平台 蛋白质生物标志物。一个由七个氮化硅纳米孔传感器组成的阵列将被构建并用于 定量检测PCT、C反应蛋白(CRP)、白介素1(IL-1)、IL-6、前列环素(可溶性CD14亚型)、 肿瘤坏死因子-α和脂多糖在不同浓度的蛋白质混合物中。 目的3：对临床血清标本进行分析。为了评估利用我们开发的固体材料的有效性- 国家纳米孔传感平台，用于准确诊断和预测脓毒症，多路纳米孔传感器 在AIM 2中开发的阵列将用于分析不同时期脓毒症患者的120份临床血清样本 分期和健康对照，以及接受抗菌治疗的脓毒症患者。这个 测定血清中PCT、C反应蛋白、IL-1、IL-6、前列环素、肿瘤坏死因子-α和内毒素的浓度。 相比之下，这些样本将另外使用酶联免疫吸附检测试剂盒进行分析。

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.