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All on size-coded single bead set: a modular enrich-amplify-amplify strategy for attomolar level multi-immunoassay.

ABSTRACT: Ultrasensitive protein analysis is of great significance for early diagnosis and biological studies. The core challenge is that many critical protein markers at extremely low aM to fM levels are difficult to accurately quantify because the target-induced weak signal may be easily masked by the surrounding background. Hence, we propose herein an ultrasensitive immunoassay based on a modular Single Bead Enrich-Amplify-Amplify (SBEAA) strategy. The highly efficient enrichment of targets on only a single bead (enrich) could confine the target-responsive signal output within a limited tiny space. Furthermore, a cascade tyramide signal amplification design enables remarkable in situ signal enhancement just affixed to the target. As a result, the efficient but space-confined fluorescence deposition on a single bead will significantly exceed the background and provide a wide dynamic range. Importantly, the SBEAA system can be modularly combined to meet different levels of clinical need regarding the detection sensitivity from aM to nM. Finally, a size-coded SBEAA set (SC-SBEAA) is also designed that allows ultrasensitive multi-immunoassay for rare samples in a single tube.

SUBMITTER: Chen D

PROVIDER: S-EPMC8943839 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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All on size-coded single bead set: a modular enrich-amplify-amplify strategy for attomolar level multi-immunoassay.

Chen Desheng D Zhang Xiaobo X Zhu Liping L Liu Chenghui C Li Zhengping Z

Chemical science 20220217 12

Ultrasensitive protein analysis is of great significance for early diagnosis and biological studies. The core challenge is that many critical protein markers at extremely low aM to fM levels are difficult to accurately quantify because the target-induced weak signal may be easily masked by the surrounding background. Hence, we propose herein an ultrasensitive immunoassay based on a modular Single Bead Enrich-Amplify-Amplify (SBEAA) strategy. The highly efficient enrichment of targets on only a s ...[more]

PMID: 35432875

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Project description:Colorectal cancer (CRC) is the second leading cause of cancer deaths in the US and Western world. Despite increased screening and advances in treatment, the mortality rate (ca. 50,000/year) and high national health-care burden for CRC are likely to remain high unless an effective non-invasive screening test for CRC is instituted for a large segment of the population. Blood-based protein biomarkers hold great promise for early disease diagnosis and personalized medicine; yet robust and reproducible multiplexing platforms and methodologies have lagged behind their genomic counterparts. Here, we report the development of a novel, multiplexed, hybrid immunoassay for CRC that is formatted on barcoded VeraCode™ micro-beads, which have until now only been used for genomic assays. The method combines a sandwich immunoassay format for detection of serum protein biomarkers with an antigen assay for autoantibody detection. The serum protein biomarkers CEA and GDF15 as well as autoantibodies to the p53 tumor associated antigen (TAA) were used to exemplify the method. This multiplex biomarker panel was configured to run on Illumina's holographically barcoded VeraCode™ micro-bead platform, which is capable of measuring hundreds of analytes simultaneously in a single well from small volumes of blood (<50 ?L) using a 96-well industry standard microtiter plate. This novel use of the VeraCode™ micro-bead platform translates into a potentially low volume, high throughput, multiplexed assay for CRC, for the purposes of biomarker validation, as well as patient screening, diagnostics and prognostics. In an evaluation of a 186 patient sera training set (CRC and normal), we obtained a diagnostic sensitivity of 54% and a specificity of 98%. We anticipate that by expanding and refining the biomarkers in this initial panel, and performing more extensive clinical validations, such an assay could ultimately provide a basis for CRC population screening to complement the more invasive, expensive and low throughput (but highly sensitive and specific) colonoscopy.

Dataset Information

All on size-coded single bead set: a modular enrich-amplify-amplify strategy for attomolar level multi-immunoassay.

Publications

All on size-coded single bead set: a modular enrich-amplify-amplify strategy for attomolar level multi-immunoassay.

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