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Issue 32, 2018
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An ultrasensitive flow cytometric immunoassay based on bead surface-initiated template-free DNA extension

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Abstract

Proteins lack the duplication mechanism like nucleic acids, so the connection of immunoassays with effective nucleic acid amplification techniques has become a powerful way for the detection of trace protein biomarkers in biological fluids. However, such immunoassays generally suffer from rather stringent DNA sequence design and complicated operations. Herein, we propose a simple but highly sensitive flow cytometric immunoassay (FCI) by employing on-bead terminal deoxynucleotidyl transferase (TdT)-initiated template-free DNA extension as an effective signal amplification pathway (TdT-FCI), and gold nanoparticles (AuNPs) co-functionalized with both the detection antibody and a 3′-OH oligonucleotide (ODN) as the transducer to bridge the immunoassay and subsequent TdT-mediated DNA amplification. The target antigen can sandwich with the capture antibody immobilized on the magnetic beads (MBs) and the detection antibody on the AuNPs to bring a lot of ODNs onto the surface of MBs. Each ODN on the MBs can be effectively elongated by TdT in a template-free manner to produce a long poly(T) tail, which will then bind to many 6-carboxyfluorescein (FAM)-labeled poly(A)25. Since each AuNP can carry multiple ODNs and each extended ODN can ultimately capture numerous FAM-poly(A)25, efficiently amplified fluorophore accumulation on the MBs can be achieved. The fluorescent MBs can be individually interrogated with a flow cytometer and thus quantitative analysis of the target antigen can be realized. Coupled with the powerful flow cytometry analysis, the simple but efficient TdT-based signal amplification mechanism has pushed the detection limit of prostate specific antigen (PSA) down to a low level of 0.5 pg mL−1. Furthermore, based on an elegant bead size-encoding principle, we have further advanced the TdT-FCI for multiplexed antigen detection in a single reaction. Sharing the unique merits of simple design and operation, efficient signal amplification, powerful signal readout and the capability for multiplexed analysis, this TdT-FCI provides a versatile tool for detecting trace antigen biomarkers towards clinical diagnosis as well as prognosis.

Graphical abstract: An ultrasensitive flow cytometric immunoassay based on bead surface-initiated template-free DNA extension

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Supplementary files

Article information


Submitted
22 Jun 2018
Accepted
20 Jul 2018
First published
23 Jul 2018

This article is Open Access
All publication charges for this article have been paid for by the Royal Society of Chemistry

Chem. Sci., 2018,9, 6605-6613
Article type
Edge Article

An ultrasensitive flow cytometric immunoassay based on bead surface-initiated template-free DNA extension

L. Zhu, D. Chen, X. Lu, Y. Qi, P. He, C. Liu and Z. Li, Chem. Sci., 2018, 9, 6605
DOI: 10.1039/C8SC02752H

This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Material from this article can be used in other publications provided that the correct acknowledgement is given with the reproduced material and it is not used for commercial purposes.

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    [Original citation] - Published by The Royal Society of Chemistry (RSC) on behalf of the European Society for Photobiology, the European Photochemistry Association, and RSC.
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    [Original citation] - Published by The Royal Society of Chemistry.

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