Duplex Shiny app quantification of the sepsis biomarkers C-reactive protein and interleukin-6 in a fast quantum dot labeled lateral flow assay

Christoph Ruppert,Matthias Kohl,Lisa Johanna Jacob,Stefan Laufer,Hans-Peter Deigner,Lars Kaiser

doi:10.1186/s12951-020-00688-1

Christoph Ruppert, Matthias Kohl + Show 4 more

Open Access

https://doi.org/10.1186/s12951-020-00688-1

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Abstract

Fast point-of-care (POC) diagnostics represent an unmet medical need and include applications such as lateral flow assays (LFAs) for the diagnosis of sepsis and consequences of cytokine storms and for the treatment of COVID-19 and other systemic, inflammatory events not caused by infection. Because of the complex pathophysiology of sepsis, multiple biomarkers must be analyzed to compensate for the low sensitivity and specificity of single biomarker targets. Conventional LFAs, such as gold nanoparticle dyed assays, are limited to approximately five targets—the maximum number of test lines on an assay. To increase the information obtainable from each test line, we combined green and red emitting quantum dots (QDs) as labels for C-reactive protein (CRP) and interleukin-6 (IL-6) antibodies in an optical duplex immunoassay. CdSe-QDs with sharp and tunable emission bands were used to simultaneously quantify CRP and IL-6 in a single test line, by using a single UV-light source and two suitable emission filters for readout through a widely available BioImager device. For image and data processing, a customized software tool, the MultiFlow-Shiny app was used to accelerate and simplify the readout process. The app software provides advanced tools for image processing, including assisted extraction of line intensities, advanced background correction and an easy workflow for creation and handling of experimental data in quantitative LFAs. The results generated with our MultiFlow-Shiny app were superior to those generated with the popular software ImageJ and resulted in lower detection limits. Our assay is applicable for detecting clinically relevant ranges of both target proteins and therefore may serve as a powerful tool for POC diagnosis of inflammation and infectious events.

Highlights

Sepsis, a life-threatening syndrome following a dysregulated host response to infection, frequently leads to organ dysfunction; it is a major public health concern becauseRuppert et al J Nanobiotechnol (2020) 18:130 risk [2]
After binding of biotinylated targets to the corresponding antibody-QDconjugates, the complex was bound on the streptavidin test line (Fig. 2a)
We first intended to use the system as a competitive immunoassay, in which the added target proteins, C-reactive protein (CRP)/ IL-6 without a biotin label, would compete for antibody binding, decreasing the fluorescence signal intensity on the test line with increasing target concentration

Summary

Introduction

A life-threatening syndrome following a dysregulated host response to infection, frequently leads to organ dysfunction; it is a major public health concern becauseRuppert et al J Nanobiotechnol (2020) 18:130 risk [2]. A life-threatening syndrome following a dysregulated host response to infection, frequently leads to organ dysfunction; it is a major public health concern because. Distinguishing sepsis from systemic inflammatory response syndrome (SIRS), which is not caused by a microbial insult, remains difficult, but this distinction is essential to determine proper treatment. Sepsis leading to organ failure frequently involves the so-called cytokine storm, which leads to complications in patients with COVID-19 [4]. To achieve efficient therapeutic approaches, there is a major clinical need for biomarker assays with a fast turnaround time of ≤ 30 min to diagnose sepsis and guide therapy. No single biomarker can be used for the diagnosis of sepsis. Evidence suggests that combined determination of multiple biomarkers might compensate for the low sensitivity and specificity of single marker molecules [5]

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