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Synthesis, labeling and bioanalytical applications of a tris(2,2′-bipyridyl)ruthenium(II)-based electrochemiluminescence probe

Nature Protocols volume 9, pages 1146–1159 (2014)Cite this article

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Abstract

Assays using probes labeled with electrochemiluminescent moieties are extremely powerful analytical tools that are used in fields such as medical diagnostics, environmental analysis and food safety monitoring, in which sensitive, reliable and reproducible detection of biomolecules is a requirement. The most efficient electrochemiluminescence (ECL) reaction to date is based on tris(2,2′-bipyridyl)ruthenium(II) (Ru(bpy)32+) with tripropylamine (TPrA) as the co-reactant. Here we present a detailed protocol for preparing Ru(bpy)32+ probes and their bioanalytical applications. This protocol includes (i) the synthesis of a biologically active Ru(bpy)32+-N-hydroxysuccinimide (NHS) ester, (ii) its covalent labeling with both antibodies and DNA probes and (iii) the detection and quantification of ECL in a microfluidic system with a paramagnetic microbead solid support. In our magnetic bead–based ECL system, two probes are required: a capture probe (labeled with biotin to be captured by a streptavidin-coated magnetic bead) and a detector probe (labeled with Ru(bpy)32+). The complex consisting of the analyte, the capture probe, the detector probe and the magnetic bead is brought into contact with the electrode by using a magnetic field. The Ru(bpy)32+ reacts with TPrA in solution to generate the ECL signal. The full protocol, including the synthesis and labeling of the bioactive Ru(bpy)32+, requires 5–6 d to complete. ECL immunoassays or nucleic acid tests only require 1.5–2 h, including the sample preparation time.

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Figure 1: Magnetic bead–based ECL measurement system.
Figure 2
Figure 3
Figure 4: Schematic of the sandwich immunoassay and nucleic acid assays.
Figure 5: The reaction in progress.
Figure 6: Spectral characterization of the Ru(bpy)32+-NHS ester.
Figure 7: Assay performance of protein detection by current ECL technique.
Figure 8: Assay performance of nucleic acid detection by the current ECL technique.

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Acknowledgements

This research is supported by the National Basic Research Program of China (2010CB732602), the National Natural Science Foundation of China (NSFC) (81101121), the Key Program of the NSFC-Guangdong Joint Funds of China (U0931005) and the Program of the Pearl River Young Talents of Science and Technology in Guangzhou, China (2013J2200021).

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Authors and Affiliations

  1. Ministry of Education Key Laboratory of Laser Life Science and Institute of Laser Life Science, South China Normal University, Guangzhou, China

    Xiaoming Zhou, Debin Zhu, Yuhui Liao, Weipeng Liu, Hongxing Liu, Zhaokui Ma & Da Xing

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Contributions

X.Z. and D.X. conceived and designed the study, supervised the work and wrote the manuscript. X.Z., Y.L., Z.M., D.Z. and W.L. conducted the experiments and the data analysis. X.Z., W.L. and H.L. designed and prepared all figures.

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Correspondence to Da Xing.

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Integrated supplementary information

Supplementary Figure 1 ESI-MS of [Ru(bpy)2(dcbpy)(PF6)2].

Supplementary Figure 2 1H NMR of [Ru(bpy)2(dcbpy)(PF6)2] in DMSO at 400 MHz.

Supplementary Figure 3 Characterization of the labeling of DNA by LC/MS.

LC/MS chromatogram of (A) DNA probes not labelled with Ru(bpy)32+-NHS, and (B) DNA probes labeled with Ru(bpy)32+-NHS. The mass of the unlabeled DNA probes was 6872.4, and the mass of the Ru(bpy)32+-labeled DNA probes was 7510.7. A mass increase of 638.3 after labeling indicated only a single probe labeled each DNA molecule. (Note that another NHS group unanticipated in the labeling reaction would hydrolyze to a carboxyl group).

Supplementary information

Supplementary Figure 1

ESI-MS of [Ru(bpy)2(dcbpy)(PF6)2]. (PDF 67 kb)

Supplementary Figure 2

1H NMR of [Ru(bpy)2(dcbpy)(PF6)2] in DMSO at 400 MHz. (PDF 166 kb)

Supplementary Figure 3

Characterization of the labeling of DNA by LC/MS. (PDF 167 kb)

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Zhou, X., Zhu, D., Liao, Y. et al. Synthesis, labeling and bioanalytical applications of a tris(2,2′-bipyridyl)ruthenium(II)-based electrochemiluminescence probe. Nat Protoc 9, 1146–1159 (2014). https://doi.org/10.1038/nprot.2014.060

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