So Yeon Yi

Detection of mutant p53 using field-effect transistor biosensor.

We assessed the abilities of wild p53 and mutant p53 proteins to interact with the consensus DNA-binding sequence using a MOSFET biosensor. This is the first report in which mutant p53 has been detected on the basis of DNA-protein interaction using a FET-type biosensor. In an effort to evaluate the performance of this protocol, we constructed the core domain of wild p53 and mutant p53 (R248W), which is DNA-binding-defective. After the immobilization of the cognate DNA to the sensing layer, wild p53 and mutant p53 were applied to the DNA-coated gate surface, and subsequently analyzed using a semiconductor analyzer. As a consequence, a significant up-shift in drain current was noted in response to wild p53, but not mutant p53, thereby indicating that sequence-specific DNA-protein interactions could be successfully monitored using a field-effect-based biosensor. These data also corresponded to the results obtained using surface plasmon resonance (SPR) measurements. Taken together, our results show that a FET-type biosensor might be promising for the monitoring of mutant p53 on the basis of its DNA-binding activity, providing us with very valuable insights into the monitoring for diseases, particularly those associated with DNA-protein binding events.

Directed immobilization of DNA-binding proteins on a cognate DNA-modified chip surface.

Here we describe a useful method for the site-directed immobilization of proteins with a DNA-binding domain (DNA-BD) on the cognate DNA-coated gold surface for surface plasmon resonance (SPR) imaging analyses. In order to assess the performance of this procedure, we utilized two DNA-BDs, yeast GAL4 DNA-BD, and bacterial LexA DNA-BD. After the immobilization of the cognate double-stranded DNAs (dsDNAs) to a gold chip surface with a monolayer of poly(l-lysine) for sequence-specific DNA-protein interaction, purified recombinant GAL4 DNA-BD:EGFP and LexA DNA-BD:RFP fusion proteins were applied to a dsDNA-spotted gold chip, and were subsequently analyzed using an SPR imaging system. Consequently, the recombinant DNA-binding proteins, GAL4 DNA-BD:EGFP and LexA DNA-BD:RFP, were shown to bind selectively to their cognate DNA sequences on the gold chip. Collectively, our results revealed that sequence-specific dsDNA microarray approach could prove useful in performing the site-directed immobilization of DNA-binding proteins onto a gold thin film in a parallel format, and thereby potentially allowing for the analysis of transcription factor binding profiling as well as for the monitoring of protein-protein interactions between target proteins with DNA-binding domain as a fusion tag and their binding partners.

SPR Imaging-Based Monitoring of Caspase-3 Activation

The activation of caspase-3 plays an important role in the apoptotic process. In this study, we describe a novel method by which caspase-3-dependent proteolytic cleavage can be monitored, using a surface plasmon resonance (SPR) imaging protein chip system. To the best of our knowledge, this is the first report regarding the SPR imaging-based monitoring of caspase-3 activation. In order to evaluate the performance of this protocol, we constructed a chimeric caspase-3 substrate (GST:DEVD:EGFP) comprised of glutathione S transferase (GST) and enhanced green fluorescent protein (EGFP) with a specialized linker peptide harboring the caspase-3 cleavage sequence, DEVD. Using this reporter, we assessed the cleavage of the artificial caspase-3 substrate in response to caspase-3 using an SPR imaging sensor. The purified GST:DEVD:EGFP protein was initially immobilized onto a glutathionylated gold chip surface, and subsequently analyzed using an SPR imaging system. As a result, caspase-3 activation predicated on the proteolytic properties inherent to substrate specificity could be monitored via an SPR imaging system with a detection performance similar to that achievable by the conventional method, including fluorometric assays. Collectively, our data showed that SPR imaging protein chip system can be effectively utilized to monitor the proteolytic cleavage in caspase-3, thereby potentially enabling the detection of other intracellular protease activation via the alteration of the protease recognition site in the linker peptides.

Detection of conformationally changed MBP using intramolecular FRET

The principal objective of this study was to explore protein conformational changes using fluorescence resonance energy transfer (FRET) technology. Maltose binding protein (MBP) was adopted as a target model, due to its well-characterized structure and ligand specificity. To the best of our knowledge, this is the first report to provide information regarding the biological distance between the two lobes of MBP upon maltose binding. For the FRET pair, ECFP and EYFP were used as the donor and the acceptor, and were linked genetically to the C-terminal and N-terminal regions of MBP (ECFP:MBP:EYFP), respectively. After the FRET reaction, maltose-treated MBP was shown to exhibit a considerable energy transfer (FRET efficiency (E)= approximately 0.11, Distance (D)= approximately 6.93 nm) at the ensemble level, which was regarded as reflective of the increase in donor quenching and the upshift in acceptor emission intensity, thereby suggesting that the donor and the acceptor had been brought close together as the result of structural alterations in MBP. However, upon glucose treatment, no FRET phenomenon was detected, thereby implying the specificity of interaction between MBP and maltose. The in vitro FRET results were also confirmed via the acceptor photobleaching method. Therefore, our data showed that maltose-stimulated conformational changes of MBP could be measured by FRET, thereby providing biological information, including the FRET efficiency and the intramolecular distance.

Facile and oriented antibody immobilization on α-cyclodextrin-modified sensors surfaces

Abstract

Facile and sensitive detection of influenza viruses using SERS antibody probes

It is highly important to identify influenza viruses rapidly and accurately for the prevention of future pandemic outbreaks. We developed an easy and sensitive influenza virus detection method using surface-enhanced Raman scattering (SERS) antibody probes. The SERS antibody probes can be prepared easily by mixing Au nanoparticles, gold binding peptide-protein G, and antibodies without complicated chemical or biological reactions. They also retain the optimal conformation for the antibodys interaction with Influenza A/CA/07/2009 (pH1N1), allowing us to detect pH1N1 sensitively and selectively. This method provides a detection limit of 4.1 × 103 TCID per mL and is highly selective for pH1N1. We anticipate that this method can be useful in a wide range of immunoassays.

Novel application of surface plasmon resonance biosensor chips for measurement of advanced glycation end products in serum of Zucker diabetic fatty rats.

Advanced glycation end products (AGEs) have been implicated in diabetic complications. To measure AGEs, especially N(epsilon)-(carboxymethyl)lysine (CML), in sera from Zucker diabetic fatty rats (ZDF) and Zucker lean rats (ZL), we used a novel method of protein chip and surface plasmon resonance imaging (SPRI). Serum samples were obtained from male ZDF and ZL rats at 20 weeks of age. Antibodies to AGEs or CML were immobilized on a gold surface, which was modified by cysteine-tagged, protein-G constructs. The gold chip upon which the serum was spotted was optically coupled with a prism coupler. The reflected images from the gold chip were obtained using a charge-coupled device (CCD) camera. The direct analysis of the glycated proteins and products using SPRI showed that AGEs and CML levels were elevated in ZDF serum, compared with ZL serum. The lowest detection limit of AGEs was 10 ng/ml, with a working range covering the physiological range. These results indicate that the protein chip and SPRI system is very suitable for the measurement of glycated proteins and end products in serum samples. This system offers high sensitivity without any fluorescent or other labeling of the components and saves a substantial amount of time, resources, and labor. Our results suggest that SPRI systems can be used as a tool to diagnose diabetic complications.

A potent reporter applicable to the monitoring of caspase-3-dependent proteolytic cleavage.

In this study, we developed a chimeric caspase-3 substrate (GST:DEVD:EGFP) comprised of glutathione-S transferase (GST) and enhanced green fluorescent protein (EGFP) with a specialized linker peptide harboring the caspase-3 cleavage sequence, DEVD. Using this reporter, we assessed the proteolytic cleavage of the artificial caspase-3 substrate for caspase-3. The common feature of this approach is that the presence of the DEVD sequence between GST and EGFP allows for caspase-3-dependent cleavage after the Asp (D) residue, resulting in the elimination of EGFP from the GST:DEVD:EGFP reporter. To the best of our knowledge, this study reports the first application employing a chimeric protein substrate, with the similar accuracy level compared to the conventional methods such as fluorometric assays. As a result, using this GST:DEVD:EGFP reporter, caspase-3 activation based on proteolytic properties could be monitored via a variety of bioanalytical techniques such as immunoblot analysis, glutathione-agarose bead assay, and on-chip visualization, providing both technical and economical advantages over the extensively utilized fluorogenic peptide assay. Our results convincingly showed that this versatile reporter (GST:DEVD:EGFP) constitutes a useful system for the monitoring of caspase-3 activation, potentially enabling the monitoring of the proteolytic activities of different intra-cellular proteases via the substitution of the cleavage sequence within the same schematic construct.

A Portable Surface Plasmon Resonance Biosensor for Rapid Detection of Salmonella typhimurium

Here, the rapid detection of Salmonella typhimurium by a portable surface plasmon resonance (SPR) biosensor in which the beam from a diode laser is modulated by a rotating mirror is reported. Using this system, immunoassay based on lipopolysaccharides (LPS)-specific monoclonal anti-Salmonella antibody was performed. For the purpose of orientation-controlled immobilization of antibodies on the SPR chip surface, the cysteine-mediated immobilization method, which is based on interaction between a gold surface and a thiol group (-SH) of cysteine, was adopted. As a result, using the portable SPR-based immunoassay, we detected S. typhimurium in the range from 10^7 CFU/mL to 10^9 CFU/mL within 1 hour. The results indicate that the portable SPR system could be potentially applied for general laboratory detection as well as on-site monitoring of foodborne, clinical, and environmental agents of interest.

Multivalent Antibody–Nanoparticle Conjugates To Enhance the Sensitivity of Surface-Enhanced Raman Scattering-Based Immunoassays

Multivalent immunoprobes can improve the sensitivity of biosensors because increased valency can strengthen the binding affinity between the receptor and target biomolecules. Here, we report surface-enhanced Raman scattering (SERS)-based immunoassays using multivalent antibody-conjugated nanoparticles (NPs) for the first time. Multivalent antibodies were generated through the ligation of Fab fragments fused with Fc-binding peptides to immunoglobulin G. This fabrication method is easy and fast because of the elimination of heterologous protein expression, high degrees of antibody modifications, and covalent chemical ligation steps. We constructed multivalent antibody-NP conjugates (MANCs) and employed them as SERS immunoprobes. MANCs improved the sensitivity of SERS-based immunoassays by 100 times compared to standard antibody-NP conjugates. MANCs will increase the feasibility of practical SERS-based immunoassays.