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Nature Protocols | Protocol PrintEmailShare/bookmark Cite U LikeFacebookTwitterDeliciousDiggGoogle+LinkedInRedditStumbleUponPrevious articleNature Protocols | Protocol Monitoring in vivo reversible cysteine oxidation in proteins using ICAT and mass spectrometry Next articleNature Protocols | Protocol In vivo introduction of transgenes into mouse sciatic nerve cells in situ using viral vectors Synthesis, labeling and bioanalytical applications of a tris(2,2′-bipyridyl)ruthenium(II)-based electrochemiluminescence probe Xiaoming Zhou,1, Debin Zhu,1, Yuhui Liao,1, Weipeng Liu,1, Hongxing Liu,1, Zhaokui Ma1, & Da Xing1, AffiliationsContributionsCorresponding author Journal name: Nature Protocols Volume: 9, Pages: 1146–1159 Year published: (2014)DOI: doi:10.1038/nprot.2014.060 Published online17 April 2014 Article toolsFull text Synthesis, labeling and bioanalytical applications of a tris(2,2′-bipyridyl)ruthenium(II)-based electrochemiluminescence prob... Download as PDF (921 KB) View interactive PDF in ReadCube CitationReprintsRights & permissionsArticle metrics Abstract Abstract• References• Author information• Supplementary information 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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