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

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

Features of Ru(II) complex.

<p><b>A.</b> The chemical structure of Ru(II) complex, 4-bromophenanthroline bis-2,2′-dipyridine Ruthenium bis (hexafluorophosphate). <b>B.</b> The absorbance and emission spectra of Ru(II) complex. The absorbance spectrum (blue dot-dashed line) was scanned from OD₂₀₀ to OD₆₀₀. The major absorption peak was at 289 nm and minor peak was at 452 nm. Emission spectra were detected from 500 nm to 800 nm and excited at 452 nm (black solid line) and 289 nm (red dashed line), respectively. The Ru(II) complex using 289 nm excitation wavelength showed weak fluorescent signal. On the other hand, the Ru(II) complex using 452 nm excitation wavelength showed large fluorescent intensity and the emission peak wavelength of Ru(II) complex was at 602 nm. <b>C.</b> Fluorescence decay curve of Ru(II) complex.</p

IgG binding assay of Ru(II)-protein G conjugates.

<p><b>A.</b> Schematic of IgG-binding assays of Ru(II)-protein G conjugates. Normal sheep IgG was immobilized on the 96 well plate, and then Ru(II)-protein G conjugates bound to the Fc region of IgG. <b>B.</b> Effect of molar ratios of SATA to Protein G for conjugation were tested: 10, 15 and 25-fold. Negative control: Ru(II) complex without Protein G.</p

SDS-PAGE of protein G and Ru(II)-protein G conjugates.

<p>The Ru(II) complex was successfully conjugated to the SATA modified protein G and higher molar ratio of SATA to protein G provided higher conjugation efficiency. SATA (+) represents 25-fold molar ratio to protein G. SATA (++) represents 50-fold molar ratio to protein G. Negative control: conjugation without protein G (lane F and G). SATA-Ru(II) complex was expected to form in the negative control.</p

Detection of purified recombinant proteins by Ru(II)-protein G conjugates.

<p><b>A.</b> Schematic of Ru(II)-protein G conjugates for detecting histidine-tagged protein. The purified histidine-tagged protein BasR was first immobilized on the 96 well plate and then recognized by anti-His antibody. Finally, the Ru(II)-protein G conjugates bound to the Fc region of anti-His antibody. <b>B.</b> Comparison of Ru(II)-protein G conjugates and Ru(II) complex for detecting histidine-tagged protein BasR. Ru(II)-protein G conjugates showed approximate 8-fold fluorescent signal compared with Ru(II) complexes (negative control) in the assay. <b>C.</b> Dose response of the histidine-tagged recombinant protein. The linear dynamic range of was from 0 to 10 µg/ml (R² = 0.96).</p

Schematics of conjugation between Ru(II) complex and protein G.

<p>The succinimide group of SATA reacted to primary amines of protein G and forms SATA modified protein G. Then, the SATA modified protein G was deacetylated by hydroxylamine. The resulting sulfhydryl modified protein G was conjugated with Ru(II) complex to form Ru(II)-protein G conjugates.</p