Zongwen Jin

Controlled and oriented immobilization of protein by site-specific incorporation of unnatural amino acid.

Immobilization of proteins in a functionally active form and proper orientation is crucial for effective surface-based analysis of proteins. Here we present a general method for controlled and oriented immobilization of protein by site-specific incorporation of unnatural amino acid and click chemistry. The utility and potential of this method was demonstrated by applying it to the analysis of interaction between a pathogenic protein DrrA of Legionella pneumophila and its binding partner Rab1 of human. Kinetic analysis of Rab1 binding onto the DrrA-immobilized surfaces using surface plasmon resonance revealed that immobilization of site-specifically biotinylated DrrA results in about 10-fold higher sensitivity in binding assay than the conventional immobilization of DrrA with random orientation. The present method is expected to find wide applications in the fields of the surface-based studies of protein-protein (or ligand) interactions, drug screening, biochip, and single molecule analysis.

Sensitive and high-fidelity electrochemical immunoassay using carbon nanotubes coated with enzymes and magnetic nanoparticles

We demonstrate a highly sensitive electrochemical immunosensor based on the combined use of substrate recycling and carbon nanotubes (CNTs) coated with tyrosinase (TYR) and magnetic nanoparticles (MNP). Both TYR and MNP were immobilized on the surface of CNTs by covalent attachment, followed by additional cross-linking via glutaraldehyde treatment to construct multi-layered cross-linked TYR-MNP aggregates (M-EC-CNT). Magnetically capturable, highly active and stable M-EC-CNT were further conjugated with primary antibody against a target analyte of hIgG, and used for a sandwich-type immunoassay with a secondary antibody conjugated with alkaline phosphatase (ALP). In the presence of a target analyte, a sensing assembly of M-EC-CNT and ALP-conjugated antibody was attracted onto a gold electrode using a magnet. On an electrode, ALP-catalyzed hydrolysis of phenyl phosphate generated phenol, and successive TYR-catalyzed oxidation of phenol produced electrochemically measurable o-quinone that was converted to catechol in a scheme of substrate recycling. Combination of highly active M-EC-CNT and substrate recycling for the detection of hIgG resulted in a sensitivity of 27.6 nA ng(-1) mL(-1) and a detection limit of 0.19 ng mL(-1) (1.2 pM), respectively, representing better performance than any other electrochemical immunosensors relying on the substrate recycling with the TYR-ALP combination. The present immunosensing system also displayed a long-term stability by showing a negligible loss of electrochemical detection signal even after reagents were stored in an aqueous buffer at 4°C for more than 6 months.

Magnetophoretic position detection for multiplexed immunoassay using colored microspheres in a microchannel.

This paper demonstrates a new magnetophoretic position detection method for multiplexed immunoassay using colored microspheres as an encoding tool in a microchannel. Colored microspheres conjugated with respective capture molecules are incubated with a mixture of target analytes, followed by reaction with the probe molecules which had been conjugated with superparamagnetic nanoparticles (SMNPs). Under the magnetic field gradient, the resulting microspheres are deflected from their focused streamlines in a microchannel, and respective colored microspheres are detected using color charge-coupled device (CCD) in a specific detection region of the microchannel. The color and position of respective colored microspheres are automatically decoded and analyzed by MATLAB program, and the position was correlated with the concentration of corresponding target analytes. As a proof-of-concept, we attempted to assay simultaneously three types of biotinylated immunoglobuline Gs (IgGs), such as goat, rabbit and mouse IgGs, using colored microspheres (red, yellow and blue, respectively). As the capture molecules, corresponding anti-IgGs were employed and target analytes were probed using streptavidin-modified superparamagnetic nanoparticles. As a result, three analytes were simultaneously assayed using colored microspheres with high accuracy, and detection limits of goat IgG, rabbit IgG and mouse IgG were estimated to be 10.9, 30.6 and 12.1fM, respectively. In addition, with adjustment of the flow rate and detection zone, the dynamic range could be controlled by more than one order of magnitude.

Analysis of in vitro SUMOylation using bioluminescence resonance energy transfer (BRET)

We demonstrated in vitro small ubiquitin-like modifier (SUMO)-mediated modification (SUMOylation) of RanGTPase activating protein-1 (RanGAP1) by using bioluminescence resonance energy transfer (BRET) for studying protein interactions. Renilla luciferase (Rluc) was fused to SUMO, and RanGAP1, the binding partner of SUMO, was fused to enhanced yellow fluorescence protein (EYFP). Upon binding of SUMO and RanGAP1, BRET was observed between EYFP (donor) and Rluc (acceptor) in the presence of E1 (Aos1/Uba2) and E2 (Ubc9) enzymes, whereas mutation (K524A) of RanGAP1 at its SUMO binding site prevented significant energy transfer. Comparing BRET and fluorescence resonance energy transfer (FRET) efficiencies using this in vitro model system, we observed that BRET efficiency was 3-fold higher than FRET efficiency, due to the lower background signal intensity of EYFP in the BRET system. Consequently, BRET system is expected to be useful for in vitro analysis of SUMOylation as well as studying other protein interactions.

Magnetic nanoclusters for ultrasensitive magnetophoretic assays.

Assays of metabolites and disease biomarkers with high sensitivity are most demanding in the fields of medical and biological sciences. Toaccomplish this goal, theuseofmagnetic particles (MPs) has been attractive mainly due to their distinct advantages, including facile control by magnetism, high biocompatibility, and high detection sensitivity. In particular, integration with microfluidic systems endowed the MPbased assay with significantly enhanced sensitivity and selectivity in detecting target analytes. In addition to the combination of a microfluidic system, modulations in the structure and shape of MPs are expected to confer enhanced analytical performance on theMP-based assay. It was reported that multimeric form or self-assembly of magnetic nanoparticles (MNPs) can improve the sensitivity of the bioassay due to the amplified transverse relaxation time.

Size-controlled construction of magnetic nanoparticle clusters using DNA-binding zinc finger protein.

Nanoparticle clusters (NPCs) have attracted significant interest owing to their unique characteristics arising from their collective individual properties. Nonetheless, the construction of NPCs in a structurally well-defined and size-controllable manner remains a challenge. Here we demonstrate a strategy to construct size-controlled NPCs using the DNA-binding zinc finger (ZnF) protein. Biotinylated ZnF was conjugated to DNA templates with different lengths, followed by incubation with neutravidin-conjugated nanoparticles. The sequence specificity of ZnF and programmable DNA templates enabled a size-controlled construction of NPCs, resulting in a homogeneous size distribution. We demonstrated the utility of magnetic NPCs by showing a three-fold increase in the spin-spin relaxivity in MRI compared with Feridex. Furthermore, folate-conjugated magnetic NPCs exhibited a specific targeting ability for HeLa cells. The present approach can be applicable to other nanoparticles, finding wide applications in many areas such as disease diagnosis, imaging, and delivery of drugs and genes.