Special Feature: Mini Review

Received: 11 October 2010, Accepted: 04 January 2011, Published online in Wiley Online Library: 14 March 2011

(wileyonlinelibrary.com) DOI 10.1002/bio.1279

Electrochemiluminescence of
tris(2,2′‐bipyridyl)ruthenium and its
applications in bioanalysis: a review
Hui Wei† and Erkang Wang*
ABSTRACT: Electrochemiluminescence (ECL) of tris(2,2’‐bipyridyl)ruthenium(II) [Ru(bpy)32+] is an active research area and
includes the synthesis of ECL‐active materials, mechanistic studies and broad applications. Extensive research has been
focused on this area, due to its scientific and practical importance. In this mini‐review we focus on the bio‐related
applications of ECL. After a brief introduction to Ru(bpy)32+ ECL and its mechanisms, its application in constructing an
effective bioassay is discussed in detail. Three types of ECL assay are covered: DNA, immunoassay and functional nucleic acid
sensors. Finally, future directions for these assays are discussed. Copyright © 2011 John Wiley & Sons, Ltd.

Keywords: electrochemiluminescence; tris(2,2′‐bipyridyl)ruthenium (II); applications

Introduction (bpy)32+ (and its analogues) is used as the ECL luminophore and
tri‐n‐propylamine (TPrA, and its alternatives) is widely used as
Electrochemiluminescence (or electrogenerated chemilumines- the co‐reactant. Here we focus on the Ru(bpy)32+–TPrA system
cence, ECL) is a means to emit measurable luminescent signals in order to elucidate the 'oxidative–reductive' reaction mechanism.
by converting electrochemical energy into radiative energy via As shown in equations 1– 4, when a potential is applied to the
an electrochemical reaction (1). Luminescent signals can be system, Ru(bpy)32+ is oxidized to Ru(bpy)33+ at the electrode
obtained when the excited state of ECL‐active species surface. Then Ru(bpy)33+ can be further reduced with TPrA●
generated at electrode surfaces decays to the ground state. (which is from the oxidation of TPrA at the electrode surface) to
ECL has become a very active area of research in the past few produce the excited state Ru(bpy)32+*. When Ru(bpy)32+* decays
decades. Many materials have been explored to investigate their to the ground state, Ru(bpy)32+, a red light will be emitted
ECL behaviour and they can be classified into two types, around 620 nm (17):
based on their chemical nature: (a) transition metal complexes;
and (b) organic molecules and nanomaterials. Among these RuðbpyÞ32þ –e– →RuðbpyÞ33þ (1)
systems, the ECL of tris(2,2′‐bipyridyl)ruthenium(II) [Ru(bpy)32+]
(including its analogues) and its co‐reactants as a highly TPrA–e– →½TPrAþ● →TPrA● þ Hþ (2)
sensitive detection method has received considerable attention,
due to its advantages over existing systems in clinical testing
and biomolecule detection. To date, a comprehensive mono- RuðbpyÞ33þ þ TPrA● →RuðbpyÞ32þ * þ products (3)
graph and several excellent reviews have been published in this
area (1–16). RuðbpyÞ32þ *→RuðbpyÞ32þ þ hv (4)
In this minireview, we focus on the state of the art in ECL
based on Ru(bpy)32+ for bioanalytical applications. We discuss
As shown in the scheme above, the ECL luminophore Ru(bpy)32+
Ru(bpy)32+ based ECL mechanisms, its applications in DNA
is regenerated during the reaction, which results in enhanced
detection, immunoassay and functional nucleic acids sensors
sensitivity compared with chemiluminescent systems. Also, the ECL
and provide a summary and outlook for this technology.
signal intensity correlates with the concentrations of Ru(bpy)32+
Readers are referred to the monographs and reviews for more
detailed information (1–16).

* Correspondence to: Erkang Wang, State Key Laboratory of Electroanalytical
Mechanisms of Ru(bpy)32+‐based ECL Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of
Sciences, Changchun, Jilin 130022, People's Republic of China.
The mechanisms of Ru(bpy)32+ based ECL have been extensively E-mail: ekwang@ciac.jl.cn
studied by many researchers and are well summarized in several
reviews (1,7,13,15,16). Three different mechanisms have been † Current address: Department of Chemistry, University of Illinois at Urbana‐
proposed: the 'oxidative–reductive' co‐reactant pathway; the Champaign, Urbana, IL 61801, USA.
'reductive–oxidative' co‐reactant pathway; and the hot electron‐ State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of
induced pathway. Most of the bioapplications of Ru(bpy)32+‐ Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, People's
based ECL are based on 'oxidative–reductive' ECL, where Ru Republic of China
77

Luminescence 2011; 26: 77–85 Copyright © 2011 John Wiley & Sons, Ltd.

it into this category. the ECL intensity is state ECL and separation techniques‐coupled ECL detection fall positively related to the concentration of the co‐reactant. Luminescence 2011. DNA detection based on Ru(bpy)32+ with DNA‐binding intercalators as the co‐reactant (28). Schematic diagram of glassy carbon (GC) electrode modification and DNA ECL detection procedures (26). thus. and the co‐reactant. 26: 77–85 . Reproduced with copyright permission from Elsevier. Wang Figure 1. Wei and E.com/journal/luminescence Copyright © 2011 John Wiley & Sons. so ECL can be used to detect both. If the co‐reactant is in excess. Reproduced with copyright permission from Elsevier. the ECL 78 wileyonlinelibrary. If the could be used for determination of the reactant. Both the solid‐ concentration of Ru(bpy)32+ is constant. H. Figure 2. Ltd.

These analytes usually act as the alternative co‐reactant to TPrA.14.15. DNA detection intensity is positively related to the concentration of Ru(bpy)32+. 26: 77–85 Copyright © 2011 John Wiley & Sons. Most of the examples discussed below fall into this category. wide dynamic range. non‐co‐reactant analytes. amino acids. other types of label‐free ECL sensors (such as those based on interactions between DNA and its intercalators) have also been Figure 4. wide range of analytes and compatibility with separation techniques. forensic chemistry and mutated gene 79 Luminescence 2011. alcohol. drugs. such as extreme stability. have been successfully determined with low detection limits (7. Ru(bpy)32+‐based ECL for bioapplications Ru(bpy)32+‐based ECL has been widely used in analytical science due to its inherent advantages. In this case. such as most biomolecules. spatial and temporal controlla- bility. DNA detection based on a bar code and magnetic microparticle method (33).com/journal/luminescence . On the other hand. can be readily detected combined with Ru(bpy)32+ labelling. analogue as an ECL label.18–22). Reproduced with sensors for bioapplications are discussed in this section. however.ECL of Ru(bpy)32+ and its applications in bioanalysis Figure 3. Besides. instead of detection of Ru(bpy)32+. analyte of interest is detected via conjugation with Ru(bpy)32+ pathogen detection. including amines. high sensitivity. oxalate. glucose and certain biomolecules. Reproduced with copyright permission from ACS. wileyonlinelibrary. Many analytes of interest. Both labelled and label‐free Ru(bpy)32+‐based ECL as enhanced ECL tags for detection of cancer biomarker PSA (43). Ltd. the DNA detection is of great importance in clinical testing. good water solubility. copyright permission from RSC. Sandwich type of ECL sensor with Ru(bpy)32+‐doped silica nanoparticles fabricated.

Reproduced with copyright permission from ACS. Wang et al. BSA and IgG detection with DMBA as ECL tags and gold nanoparticles as amplifying elements (46).393 nmol/L.4 nmol/L. H. 26: 77–85 . 80 wileyonlinelibrary. based on the catalytic oxidation of guanine screened. Wang diagnosis associated with human diseases. Scheme for fabrication of a ECL aptasensor for cocaine detection (49). Reproduced with copyright permission from ACS. Ltd.com/journal/luminescence Copyright © 2011 John Wiley & Sons. with a low concentration of been widely used in DNA detection (23–36). daunorubicin and 4′.6‐ and adenine bases in nucleic acids (Figure 1) (26). Luminescence 2011. developed a label‐free ECL developed by using DNA‐binding intercalators as the co‐reactant approach for sensitive DNA detection with a Ru(bpy)32+‐ (Figure 2) (28). Certain double‐stranded DNA intercalators were modified electrode. p53 gene sequence segment was successfully realized at using a [Ru(bpy)2(PVP)10]2+‐modified electrode (23). doxorubicin. method also differentiated chemically damaged DNA and its An interesting label‐free ECL sensor for DNA detection was native counterpart. 30. Wei and E. Owing to its approach distinctly discriminated double‐stranded DNA and its excellent analytical performance. Their 0. Their diamidino‐2‐phenylindole (DAPI) were found to be good Figure 5. Further. Figure 6. a single‐base mismatch detection of a Rusling’s group reported the direct ECL detection of DNA. among them. Ru(bpy)32+‐based ECL has thermally denatured counterparts.

However. so ECL tags are required to label the biomolecules with Ru(bpy)32+ For most DNA sensors. 81 Luminescence 2011. Recently. ECL tags with enhanced sensitivity could be detection in human serum (33). producing strong ECL signals. The double‐stranded DNA quenched by ferrocene.35. by as efficient electrical communication pathways. Figure 8. thus. PSA in cell lysates employing a 'molecular beacon'‐like design. Aptamer‐based biosensor for the detection of lysozyme by increasing sensitivity with gold nanoparticle amplification (54). and ferrocene was labelled on one end of the labelling (46. Reproduced with copyright permission from ACS. To improve both the sensitivity and (bpy)32+ or its analogue. Immunoassay Based on this mechanism.ECL of Ru(bpy)32+ and its applications in bioanalysis co‐reactants for Ru(bpy)32+ ECL. ECL co‐reactants could also be used as tags for an electrode surface. the presence of target DNA interacted with these intercalators. cysteamine rather By encapsulating thousands of Ru(bpy)32+ molecules into micro‐ or than DNA was used as a bar code for single‐mismatched DNA nano‐structures. the probe strand was labelled with Ru derivatives (15. Further amplification protocols were biocompatibility. Ru(bpy)32+ was immobilized on Alternatively. As shown in Figure 3.47). Other detection of nucleic acid targets was reported using Ru(bpy)32+‐ interesting amplification methods were also developed (35. A PCR‐free quantitative used for multi‐labelling a biomolecule at a single site (39). would open the hairpin structure and thus restore the ECL intensity. Without target DNA. limited or zero ECL signals were obtained. 26: 77–85 Copyright © 2011 John Wiley & Sons. a Ru(bpy)32+ derivative of dendritic structure was developed to improve the sensitivity. For example. For example. a quantitative DNA detection nanoparticles as enhanced ECL tags and carbon nanotube forests method was reported by Cao and co‐workers (37). the sensing system sandwich type of ECL sensor for prostate‐specific antigen (PSA) had a detection limit as low as 100 fmol/L. Schematic for the principle of the ECL aptasensor for thrombin detection (64).39–45). was explored as an ECL tag for Figure 7.com/journal/luminescence . intercalated Ru(bpy)32+ ECL. However. the Ru(bpy)32+ ECL was (DMBA).40–45). was fabricated (38). the detection of a single nucleotide polymorphism for hepatitis C virus was accomplished with DAPI‐ For immunoassay. combined obtained and further used for immunoassay. 4‐(dimethylamino)butyric acid hairpin DNA. an analogue of TPrA. Using Ru(bpy)32+‐doped silica (bpy)32+ ECL with ferrocene. most of the biological targets are not ECL‐active. Also. a turn‐on DNA sensor and human serum samples was successfully detected (43). Ltd. In this design. single‐stranded DNA could not interact with these intercalators. wileyonlinelibrary. By quenching of Ru detection was fabricated (Figure 4). labelled DNA as a bar code (31). a with bar code and magnetic microparticles.

49–66). range from metal ions. Since their targets could switching aptasensor with a solid‐state ECL sensing platform was 82 wileyonlinelibrary. More over their direct immobilization on an electrode using DMBA than 10 years ago Bruno et al. employed ECL for selection of an labelling. electrode surface (Figure 6) (49). A highly sensitive and reusable ECL aptasensor for cocaine Functional nucleic acid sensors detection has been fabricated by immobilizing the Ru(bpy)32+‐ As functional nucleic acids. Ltd. heroin and caffeine. Combined with ECL detection. A structure‐ by exponential enrichment' (SELEX).com/journal/luminescence Copyright © 2011 John Wiley & Sons. H. of BSA and IgG in the presence of Ru(bpy)32+ solution. ECL‐based aptasensors have already been used to detect small molecules and proteins. small molecules. biological substance labelling and ECL detection (Figure 5) (46). ECL detection of Pb2+ based on the Pb2+‐dependent DNAzyme (65). many sensors based on six‐fold sensitivity enhancement was obtained for BSA and IgG aptamers (aptasensors) have been developed (16. Reproduced with copyright permission from RSC. 26: 77–85 . were used as models for ECL detection recognition elements to construct novel sensors. a 10‐ and targets. By further Aptamers are functional nucleic acids that can bind to specific employing gold nanoparticles as amplifying elements. The ‘as‐prepared’ aptasensor the highly‐selective aptamer (or DNAzyme) can be obtained from showed a very low detection limit of 10 pmol/L for cocaine and a test tubes via an approach called 'systematic evolution of ligands good selectivity toward cocaine. Wei and E. functional nucleic acids have been adopted as G (IgG). Wang Figure 9. bovine serum albumin (BSA) and anti‐immunoglobulin bacteria. Luminescence 2011. aptamers and DNAzymes are gaining attached cocaine aptamer on a modified paraffin impregnated more and more attention from researchers (48). For a given target. proteins to even cells and Specifically. tagged with DMBA. aptamer to anthrax spores (50).

Reproduced with copyright permission from RCS. such as CCD and fibre. (61). This will supply substrates. No. UTP) and a low detection limit of 100 nmol/L ATP. This phenomenon has been solution. For example. it showed good selectivity and sensitivity toward types of specific interaction between metal ions and DNA. For adenosine detection. the mismatch of T–T switched to stable T‐Hg2+–T detection. When ATP signal‐on ECL sensor was also constructed for Hg2+ detection apatamer was used as the probe. thymine–thymine mismatches in double‐stranded was reported by Xu et al. With gold nanoparticle amplifi. Using their temporal sensitive devices. a 100 fmol/L detection limit was obtained. Since the design was independent of conformational will be another interesting and promising research field. 1000 times when [Ru(bpy)2dppz]2+ widely used for Hg2+ sensing. KJCX2 YW HO9). immobilized on an electrode surface. which will not only reduce the cost of from fluorescent. A highly sensitive and selective was intercalated into the nucleic acid structure. colorimetric 2010CB933600) and the Chinese Academy of Sciences (Grant and electrical biosensors. the 973 Project (Grant Nos 2009CB930100 and 0. Most of these studies used either thrombin or lysozyme Conclusions and outlook as the model proteins. single‐cell and single‐bacterium‐based assay obtained. the ECL intensity of more strongly than [Ru(bpy)2dppz]2+. 83 Luminescence 2011. ECL detection of Hg2+ based on the T‐Hg2+–T pair (67). Several aptasensors for protein detection have also been reported. method. then the Ru(bpy)32+‐ tagged substrate strand was hybridized with the enzyme Acknowledgements strand to form double‐stranded DNA. The sensor was commercialized for immunoassays. The presence of lead (Pb2+) would release the cleaved substrate fragment and This work is supported by the National Natural Science Foundation of China (Grant Nos 20675078. dynamic range. etc. Second. so it could specifically affect the sensor was low because of the mismatch of T–T. As shown in Figure 10. Chen and co‐workers extended DNAzymes sensors current instrumentation. Multiplex aptasensor for thrombin detection. which was lower than most fluorescent.2 amol/L for thrombin was single‐molecule. 26: 77–85 Copyright © 2011 John Wiley & Sons. due to its high sensitivity. The enzyme strand was first technique to a bed‐side one. especially for in vivo DNAzymes are catalytic nucleic acids capable of catalysing imaging. An interesting label‐free ATP aptasensor example. Their design was based on the fact DNA could be selectively stabilized by forming thymine–Hg2+– that the ECL of [Ru(bpy)2dppz]2+ was negligible in aqueous thymine (T‐Hg2+–T) base pairs. Finally. a lysozyme aptasensor was Among the most ultrasensitive detection methods. Most of the DNAzymes sensors developed so far important insights into biological systems. without Hg2+.ECL of Ru(bpy)32+ and its applications in bioanalysis Figure 10. more have been based on metal ion‐dependent DNAzymes. colorimetric.1 nmol/L. it could be readily extended to targets whose aptamers will advance our fundamental understanding of these systems. selective and sensitive label‐free ECL an emerging area that will engage future research. which gave enhanced ECL signals. Yin and co‐ ical performance has been achieved. Although it was a Besides metal ion‐dependent DNAzymes. there are other signal‐off sensor. a mass detection limit of 0. using the intercalation of detection might be accomplished with highly spatial and Ru(phen)32+ into double‐stranded DNA (Figure 8) (64). ATP could bind to the aptamer (67). which changes. developed for the detection of adenosine (66). to clinical assay but also provide a chance to convert the bench an ECL type (Figure 9) (65). the exploration of ECL for imaging. Third. where native lysozyme had ECL has been widely used for both biological and non‐biological higher affinity towards its aptamer than the [Ru(bpy)2(dcbpy)NHS]‐ assays. The limit of detection for Pb2+ was 90713022). and increased ca. such as cleaving nucleic acid spatial and temporal) than simple biosensors. small sample volume and wide labelled lysozyme (Figure 7) (54). Although impressive analyt- successfully used in the detection of lysozyme in egg. however. The sensor had good selectivity (against CTP. Ltd. have no specific conformational changes. The related techniques have already been cation. Ru(bpy)32+ reported based on competition assay. will provide more bioanalytical information (both a broad range of reactions. the ECL of [Ru(bpy)2dppz]2+ and thus provide a method for ATP with Hg2+.com/journal/luminescence . 20735003. multiplex detection is still workers reported a simple. electrochemical types. GTP and base pairs. decrease the ECL signal. wileyonlinelibrary. attention should be focused on the miniaturization of the Recently.

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