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

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.

[1]  Ling Zhang,et al.  Label-free supersandwich electrochemiluminescence assay for detection of sub-nanomolar Hg2+. , 2011, Chemical communications.

[2]  Y. Pommier,et al.  Novel high-throughput electrochemiluminescent assay for identification of human tyrosyl-DNA phosphodiesterase ( Tdp 1 ) inhibitors and characterization of furamidine ( NSC 305831 ) as an inhibitor of Tdp 1 , 2007 .

[3]  A. Campagnari,et al.  Detection of biological threat agents by immunomagnetic microsphere-based solid phase fluorogenic- and electro-chemiluminescence. , 2000, Biosensors & bioelectronics.

[4]  Jinghua Yu,et al.  Three-dimensional paper-based electrochemiluminescence immunodevice for multiplexed measurement of biomarkers and point-of-care testing. , 2012, Biomaterials.

[5]  H Szmacinski,et al.  Metal-ligand complexes as a new class of long-lived fluorophores for protein hydrodynamics. , 1995, Biophysical journal.

[6]  Paolo Bertoncello,et al.  Nanostructured materials for electrochemiluminescence (ECL)-based detection methods: recent advances and future perspectives. , 2009, Biosensors & bioelectronics.

[7]  Chwan-Chuen King,et al.  Detection of Dengue Viral RNA Using a Nucleic Acid Sequence-Based Amplification Assay , 2001, Journal of Clinical Microbiology.

[8]  Yafeng Wu,et al.  A novel electrochemiluminescence immunosensor via polymerization-assisted amplification. , 2010, Chemical communications.

[9]  Dik-Lung Ma,et al.  Luminescent detection of DNA-binding proteins , 2011, Nucleic acids research.

[10]  Guobao Xu,et al.  Environmentally friendly and highly sensitive ruthenium(II) tris(2,2'-bipyridyl) electrochemiluminescent system using 2-(dibutylamino)ethanol as co-reactant. , 2007, Angewandte Chemie.

[11]  Q. Gao,et al.  Double covalent coupling method for the fabrication of highly sensitive and reusable electrogenerated chemiluminescence sensors. , 2010, Analytical chemistry.

[12]  J. Kiel,et al.  In vitro selection of DNA aptamers to anthrax spores with electrochemiluminescence detection. , 1999, Biosensors & bioelectronics.

[13]  Da Xing,et al.  Nano-magnetic primer based electrochemiluminescence-polymerase chain reaction (NMPE-PCR) assay. , 2012, Biosensors & bioelectronics.

[14]  D. Xing,et al.  Rapid and sensitive immunomagnetic-electrochemiluminescent detection of p53 antibodies in human serum. , 2004, Journal of immunological methods.

[15]  M. Richter,et al.  The effects of nonionic surfactants on the tris(2,2'-bipyridyl)ruthenium(II)--tripropylamine electrochemiluminescence system. , 2000, Analytical chemistry.

[16]  Signal-on electrochemiluminescence biosensor for thrombin based on target-induced conjunction of split aptamer fragments. , 2010, Chemical communications.

[17]  K. Eidne,et al.  Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET) , 2006, Nature Methods.

[18]  Jonathan K. Leland,et al.  Electrogenerated Chemiluminescence: An Oxidative‐Reduction Type ECL Reaction Sequence Using Tripropyl Amine , 1990 .

[19]  D. van Strijp,et al.  A one-tube quantitative HIV-1 RNA NASBA nucleic acid amplification assay using electrochemiluminescent (ECL) labelled probes. , 1994, Journal of virological methods.

[20]  Won-Yong Lee,et al.  Tris (2,2′-bipyridyl)ruthenium(II) electrogenerated chemiluminescence in analytical science , 1997 .

[21]  M. Richter Electrochemiluminescence (ECL). , 2004, Chemical reviews.

[22]  J. Link,et al.  Rapid, non-separation electrochemiluminescent DNA hybridization assays for PCR products, using 3'-labelled oligonucleotide probes. , 1992, Molecular and cellular probes.

[23]  A. Bard,et al.  Electrogenerated Chemiluminescence. 55. Emission from Adsorbed Ru(bpy)32+ on Graphite, Platinum, and Gold , 1994 .

[24]  Da Xing,et al.  Magnetic beads based rolling circle amplification-electrochemiluminescence assay for highly sensitive detection of point mutation. , 2010, Biosensors & bioelectronics.

[25]  A. Bard,et al.  Electrogenerated chemiluminescence. 67. Dependence of light emission of the tris(2,2')bipyridylruthenium(II)/tripropylamine system on electrode surface hydrophobicity. , 2001, Analytical chemistry.

[26]  A. Bard,et al.  Electrogenerated Chemiluminescence 69 : The Tris ( 2 , 2 ′-bipyridine ) ruthenium ( II ) , ( Ru ( bpy ) 32 + ) / Trin-propylamine ( TPrA ) System Revisited s A New Route Involving TPrA , 2002 .

[27]  Neso Sojic,et al.  Multiplexed sandwich immunoassays using electrochemiluminescence imaging resolved at the single bead level. , 2009, Journal of the American Chemical Society.

[28]  R Lejeune,et al.  Chemiluminescence as diagnostic tool. A review. , 2000, Talanta.

[29]  C. Rossi,et al.  Rapid and sensitive immunomagnetic-electrochemiluminescent detection of staphyloccocal enterotoxin B. , 2000, Journal of immunological methods.

[30]  Da Xing,et al.  Magnetic bead and nanoparticle based electrochemiluminescence amplification assay for direct and sensitive measuring of telomerase activity. , 2009, Analytical chemistry.

[31]  R. A. Kamin,et al.  Electrochemiluminescence detection for development of immunoassays and DNA probe assays for clinical diagnostics. , 1991, Clinical chemistry.

[32]  J. Landers,et al.  Quenching of the electrochemiluminescence of tris(2,2'-bipyridine)ruthenium(II) by ferrocene and its potential application to quantitative DNA detection. , 2006, Journal of the American Chemical Society.

[33]  J. Link,et al.  Rapid electrochemiluminescence assays of polymerase chain reaction products. , 1991, Clinical chemistry.

[34]  Ka-Ho Leung,et al.  Label-Free Luminescent Oligonucleotide-Based Probes , 2013 .

[35]  J. Weel,et al.  A Highly Sensitive Assay for Detection and Quantitation of Human Cytomegalovirus DNA in Serum and Plasma by PCR and Electrochemiluminescence , 1999, Journal of Clinical Microbiology.

[36]  E. Wang,et al.  Solid-state electrochemiluminescence of tris(2,2′-bipyridyl) ruthenium , 2008 .

[37]  W. Miao Electrogenerated chemiluminescence and its biorelated applications. , 2008, Chemical reviews.

[38]  D. Xing,et al.  Rapid and highly sensitive detection of mercury ion (Hg2+) by magnetic beads-based electrochemiluminescence assay. , 2010, Biosensors & bioelectronics.

[39]  P. Thullier,et al.  Comparison of an electrochemiluminescence assay in plate format over a colorimetric ELISA, for the detection of ricin B chain (RCA-B). , 2007, Journal of immunological methods.

[40]  Jun Wang,et al.  Amplified voltammetric detection of DNA hybridization via oxidation of ferrocene caps on gold nanoparticle/streptavidin conjugates. , 2003, Analytical chemistry.

[41]  Ka-Ho Leung,et al.  Detection of base excision repair enzyme activity using a luminescent G-quadruplex selective switch-on probe. , 2013, Chemical communications.

[42]  R. Forster,et al.  Electrogenerated chemiluminescence. , 2009, Annual review of analytical chemistry.

[43]  Da Xing,et al.  Electrochemiluminescence biobarcode method based on cysteamine--gold nanoparticle conjugates. , 2010, Analytical chemistry.

[44]  Bin Su,et al.  Imaging latent fingerprints by electrochemiluminescence. , 2012, Angewandte Chemie.

[45]  J. Lakowicz,et al.  Fluorescence polarization immunoassay of a high-molecular-weight antigen based on a long-lifetime Ru-ligand complex. , 1995, Analytical biochemistry.

[46]  Da Xing,et al.  A method to quantitatively detect H-ras point mutation based on electrochemiluminescence. , 2004, Biochemical and biophysical research communications.

[47]  E. Wang,et al.  Analytical applications of the electrochemiluminescence of tris (2,2'-bipyridyl) ruthenium and its derivatives , 2004 .

[48]  Xiaoying Wang,et al.  A solid-state electrochemiluminescence biosensing switch for detection of thrombin based on ferrocene-labeled molecular beacon aptamer. , 2009, Biosensors & bioelectronics.

[49]  O. Attrée,et al.  Comparison of electrochemiluminescence assay and ELISA for the detection of Clostridium botulinum type B neurotoxin. , 2005, Journal of immunological methods.

[50]  Yan Zhang,et al.  Magnetic beads-based electrochemiluminescence immunosensor for determination of cancer markers using quantum dot functionalized PtRu alloys as labels. , 2012, The Analyst.

[51]  G. Robertson,et al.  Multilabeling biomolecules at a single site. 1. Synthesis and characterization of a dendritic label for electrochemiluminescence assays. , 2003, Analytical chemistry.

[52]  Guobao Xu,et al.  Applications and trends in electrochemiluminescence. , 2010, Chemical Society reviews.

[53]  Fengwang Li,et al.  Quenching of the electrochemiluminescence of tris(2,2'-bipyridine)ruthenium(II)/tri-n-propylamine by pristine carbon nanotube and its application to quantitative detection of DNA. , 2013, Analytical chemistry.

[54]  Allen J. Bard,et al.  Electrogenerated Chemiluminescence. 30. Electrochemical Oxidation of Oxalate Ion in the Presence of Luminescers in Acetonitrile Solutions , 1977 .

[55]  Da Xing,et al.  PCR-free quantitative detection of genetically modified organism from raw materials. An electrochemiluminescence-based bio bar code method. , 2008, Analytical chemistry.

[56]  Yves Pommier,et al.  Novel high-throughput electrochemiluminescent assay for identification of human tyrosyl-DNA phosphodiesterase (Tdp1) inhibitors and characterization of furamidine (NSC 305831) as an inhibitor of Tdp1 , 2007, Nucleic acids research.