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.