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

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.

SUBMITTER: Ruppert C

PROVIDER: S-EPMC7481553 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Charge transfer processes with semiconductor quantum dots (QDs) have generated much interest for potential utility in energy conversion. Such configurations are generally nonbiological; however, recent studies have shown that a redox-active ruthenium(II)-phenanthroline complex (Ru2+-phen) is particularly efficient at quenching the photoluminescence (PL) of QDs, and this mechanism demonstrates good potential for application as a generalized biosensing detection modality since it is aqueous compatible. Multiple possibilities for charge transfer and/or energy transfer mechanisms exist within this type of assembly, and there is currently a limited understanding of the underlying photophysical processes in such biocomposite systems where nanomaterials are directly interfaced with biomolecules such as proteins. Here, we utilize redox reactions, steady-state absorption, PL spectroscopy, time-resolved PL spectroscopy, and femtosecond transient absorption spectroscopy (FSTA) to investigate PL quenching in biological assemblies of CdSe/ZnS QDs formed with peptide-linked Ru2+-phen. The results reveal that QD quenching requires the Ru2+ oxidation state and is not consistent with Förster resonance energy transfer, strongly supporting a charge transfer mechanism. Further, two colors of CdSe/ZnS core/shell QDs with similar macroscopic optical properties were found to have very different rates of charge transfer quenching, by Ru2+-phen with the key difference between them appearing to be the thickness of their ZnS outer shell. The effect of shell thickness was found to be larger than the effect of increasing distance between the QD and Ru2+-phen when using peptides of increasing persistence length. FSTA and time-resolved upconversion PL results further show that exciton quenching is a rather slow process consistent with other QD conjugate materials that undergo hole transfer. An improved understanding of the QD-Ru2+-phen system can allow for the design of more sophisticated charge-transfer-based biosensors using QD platforms.

Dataset Information

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

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