A Sulfhydryl-Reactive Ruthenium (II) Complex and Its Conjugation to Protein G as a Universal Reagent for Fluorescent Immunoassays

Jing-Tang Lin,Jean-François Biellmann,Po-Chung Chen,Chien-Sheng Chen,Tzu-Chau Lin,Thirumani Venkatshwar Goud,Bor-Rong Huang,Mark Cobbold

doi:10.1371/journal.pone.0036086

Jing-Tang Lin, Jean-François Biellmann + Show 6 more

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https://doi.org/10.1371/journal.pone.0036086

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Abstract

To develop a fluorescent ruthenium complex for biosensing, we synthesized a novel sulfhydryl-reactive compound, 4-bromophenanthroline bis-2,2′-dipyridine Ruthenium bis (hexafluorophosphate). The synthesized Ru(II) complex was crosslinked with thiol-modified protein G to form a universal reagent for fluorescent immunoassays. The resulting Ru(II)-protein G conjugates were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The emission peak wavelength of the Ru(II)-protein G conjugate was 602 nm at the excitation of 452 nm which is similar to the spectra of the Ru(II) complex, indicating that Ru(II)-protein G conjugates still remain the same fluorescence after conjugation. To test the usefulness of the conjugate for biosensing, immunoglobulin G (IgG) binding assay was conducted. The result showed that Ru(II)-protein G conjugates were capable of binding IgG and the more cross-linkers to modify protein G, the higher conjugation efficiency. To demonstrate the feasibility of Ru(II)-protein G conjugates for fluorescent immunoassays, the detection of recombinant histidine-tagged protein using the conjugates and anti-histidine antibody was developed. The results showed that the histidine-tagged protein was successfully detected with dose-response, indicating that Ru(II)-protein G conjugate is a useful universal fluorescent reagent for quantitative immunoassays.

Highlights

Biomolecule detection plays an important role in the biological research
We described here the synthesis of a novel sulfhydryl-reactive fluorescent ruthenium complex: 4-bromophenanthroline bis-2,29dipyridine Ruthenium bis, and its conjugation to protein G as a universal reagent for fluorescent immunoassays
The fluorescent peak wavelength of Ru(II)-protein G conjugates was at 602 nm, which is similar to the Ru (II) complex. These results indicated that Ru(II)-protein G conjugates was still a feasible fluorophore after conjugation

Summary

Introduction

Biomolecule detection plays an important role in the biological research. Biosensing which uses biorecognition elements for detection is a rapid and easy method for biomolecule detection. The bioconjugation between detectable reagent and biorecognition elements is commonly used because of its higher sensitivity compared with label-free detecting system [1]. These detectable reagents include not exhaustively: fluorescence [2,3,4], chemiluminescence [5], radioactive isotopes [6,7,8], enzymes [9], nanocrystals [10] and liposomes [11]. We described here the synthesis of a novel sulfhydryl-reactive fluorescent ruthenium complex: 4-bromophenanthroline bis-2,29dipyridine Ruthenium bis (hexafluorophosphate), and its conjugation to protein G as a universal reagent for fluorescent immunoassays. The detection of antibody and recombinant histidine-tagged protein using Ru(II)-protein G conjugates were demonstrated, respectively

Results and Discussion

Conclusion

Materials and Methods