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Research Goal
We are aiming to develop three nanobio-platform systems based upon nanomaterials biomaterials or their hybrids in conjunction with targeted libraries (aptamer or DNAzyme):
- Nanobiosensor (Nanoparticle-protein or nanoparticle-aptamer hybrid)
- Biochip (Peptide, antibody and other affinity-based chip)
- Imaging Probe (BRET/FRET-based imaging probe)
Through these designed platform systems, three important directions are being pursued to study nanobio interconncected issues in our group:
- Analysis of Post-Translational Modifications and Protein Interactions
: Development of BRET/FRET probes through engineered luciferases or intein-mediated process
for the detection of phosphorylation and protease activity
: Development of protein-DNA or protein-ligand interactions using smart sensors or biochips
- Biomedical Theranostics: Therapeutic and Diagnostic of Diseases
: Study on cancer diagnostics and photothermal therapy study
: Development of bioassays targeting ncRNAs and small molecules using DNAzyme-based probes
: Development of cell-specific aptamers for monitoring immune cells and stem cells
- Environmental Diagnostics
: Development of rapid detection of endocrine disruptors (EDCs) using nano/biosensors
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Representative Research Achievement
I. Assaying matrix metalloproteinases (MMPs) activity based upon nanoprobe and energy transfer system (나노프로브와 에너지 전이를 이용한 기질분해효소의 활성 측정)
1) Overview
• A subset of matrix metalloproteinases (MMPs) are implicated in a wide range of biological processes in normal and pathological events. Activity-based assay of the MMPs is a key issue to understand the contribution of MMPs to tumor progression.
• Novel energy transfer systems between biomolecules and nanoparticles bring new insight to detect biomolecular change with higher sensitivity and flexibility, compared to the traditional set (e.g. FRET and BRET).
2) Scientific Specialty and Levels
• Although MMP expression profiles have been reported to be useful in explicating various disease processes, a practical issue in quantifying levels of MMPs in clinical samples remains formidable because only difference in total MMP expression where the correlation might not necessarily be related to disease level. Therefore, emphasis should be placed on the importance of active MMPs rather than total MMP levels in tumor.
• Our designed probes include the sensing part (peptide substrate) and detection part (fluorescence, bioluminescence and/or their energy transferred couplers). Nanoparticles (gold, quantum dot, or polymer-based nanoparticles) have been combined with this system to enhance the sensitivity and other optical properties. This nanohybrid probe can serve as a biosensor and/or an imaging probe for targeting active MMP.
3) Expected Contributions & Future Direction
• MMP-related cancer metastasis study based on in vivo diagnostic/imaging of MMP activity
• Protease activity profiling to address biological function of proteases in vitro and in vivo
4) Related References
• Chem. Commun. 2010, 46, 76
• Biosens. Bioelectron. 2009, 24(5), 1189
• Anal. Chem. 2008, 80(13), 5094
• Anal. Chem. 2008, 80(12), 4634
Left. Chip-based multiplexed protease assay using quantum dot and gold nanoparticle. Right top. Bioluminescence-quenched nanosensor for detecting MMP activity using luciferase-conjugated gold nanoparticle. Right down. In vivo MMP activity imaging in the presence and absence of MMP inhibitor (SB-3CT).
II. Development of biochip system to detect protein kinase activity on peptide-conjugated nanoparticles (펩타이드 결합 나노입자 기반의 kinase의 활성 측정용 biochip system 개발)
1) Overview
• Collaboration with KRISS
• A number of kinases have long been considered the major targets for drug discovery
• Higher sensitivity and specificity are demanded to discriminate minor change in phosphorylation
2) Scientific Specialty and Levels
• Most studies to assay the kinases and their inhibitors have relied on radioactivity- or fluorescence-labeled techniques which are labor-intensive and time-consuming.
• Matrix-assisted laser desorption/ ionization (MALDI) has been employed to determine the kinase activity and screen their inhibitors, but it usually uses the matrix, leading to less reproducibility on surface à As a label-free approach, we developed matrix-free time-of-flight secondary ion mass spectrometry (TOF-SIMS)-based kinase assay system using peptide-conjugated gold nanoparticle on a surface.
• Based on the observation that secondary ion mass signal of peptides is highly enhanced on AuNPs, this system was applied to the assaying of both the protein kinase and its inhibition with high detection sensitivity (~fmol/mm2) of the mass change of peptide substrates in a kinase reaction.
• In addition to mass spectrometry, peptide-conjugated AuNP on a surface can also serve as a biochip platform system for assaying phosphorylation and antibody-antigen interactions with high detection sensitivity when employed in florescence-based detection.
3) Expected Contributions & Future Directions
• Therapeutic applications for drug development by screening various PTM-related enzymes and their inhibitors with high sensitivity in a high-throughput manner
4) Related References
• Anal. Chem. 2006, 78, 1913
• Angew. Chem. Int. Ed. 2007, 46, 6816
• Biosens. Bioelectron. 2008, 23(7), 980
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III. Bioluminescence resonance energy transfer (BRET) system for studying protein-protein interactions and for analyzing biomarkers (단백질-단백질 상호작용 연구 및 biomarker 분석을 위한 BRET system)
1) Overview:
• Collaboration with Stanford Univ.
• Unlike fluorescence-based methods, which generally encounter high-background signals from interfering species present in biological samples, bioluminescence-based detection provides great sensitivity due to its extremely low background.
• The general utility of BRET methods has not been fully tested for studying multiple protein interactions that may exist within larger protein complexes (e.g. SUMOylation and ubiqutinylation)
2) Scientific Specialty and Levels
• Full potential of FRET methods are often limited due to photobleaching, autofluorescence and high residual excitation of the acceptor fluorophore, complications intrinsic to FRET systems. In contrast, BRET methods do not require external excitation and therefore have relatively low background signal intensities, allowing for more sensitive detection of energy transfer during experiments
• In particular, quantum dots (qdot) offer several advantages when used in BRET system: full spectral separation (minimizing crosstalk between the BRET pair), increasing BRET efficiency and multiplexed assay with single luciferase
•BRET and bioluminescence imaging is expected to be useful for in vivo analysis of protein-protein interactions
3) Expected Contributions & Future Directions
4) Related References
• Biochem. Biophys. Res. Commun. 2009, 382(3), 530
• Chem. Commun. 2010, 46, 76
• Nat. Biotechnol. 2006, 24, 339 (Prof. Jianghong Rao's group at Stanford)
• Developing new BRET system with high efficiency and red-shifted property, which could be useful for studying P-P interactions and for tracking a panel of molecular biomarkers in cells and in vivo animal imaging.
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Research Area
The above objectives will be contributing to one or more of the following multidisciplinary areas:
1. Biomedicine
- In vitro diagnosis and in vivo imaging of cancer/disease biomarkers with sensitively designed probes
- Study on the physiological function of enzyme activity related to tumor biogenesis and drug resistance
- Screening system to study protein-protein interactions and cell-to-cell communications
2. Environmental Sciences
- Environmental sensor: detection of pathogenic bacteria, small molecules, and other toxic materials
- Marine biotechnology: develop new transformation system and nanosensor to marine microalgae
- Green energy: nanoparticle-actuated biofuel cell system
3. Nanobiotechnology
- Understanding of material science, chemistry, physics, and its correlation to life science
- Finding the biological applications through nanobio toolkits and novel nanoscale devices
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