Department of Chemical and Biomolecular Engineering
Korea Advanced Institute of Science and Technology

About the Lab

Biochip & Biodevice Laboratory

The research of our group is focused on devising convenient, sensitive, and reliable strategies for the detection of biomolecules and, in particular, on developing DNA-based methods, including aptamer- or/and DNAzyme-utilized biosensor and DNA microarray for genetic diagnosis. With the goal of becoming an international leader in the field of nucleic acid bioengineering, we have developed unique capabilities of rationally designing DNA sequences, utilizing DNA analogues or/and mimic molecules, and freely manipulating many kinds of DNA-related enzymes. By innovatively integrating all of these DNA-based technological components, we have been able to create novel engineering tools in form of PCR, biosensor, microarray, and electrochemical devices.

 

Nucleic Acid Bioengineering

 

1. Illusionary polymerase activity triggered by metal ions

 We proved for the first time that DNA polymerase activity could be intentionally triggered by the presence of Hg2+ and Ag+ ions through their interaction with the respective mismatched base pairs T-T and C-C, consequently resulting in an unusual and unnatural amplification reaction. We termed this new activity “Illusionary polymerase activity” because it is derived from the illusion of DNA polymerase that the metal-ion-mediated base pair is perfectly matched. 

The paper describing this work was highlighted as a cover paper of Angewandte Chemie International Edition and featured by Nature.

 

2. DNAzyme-based biosensor
DNAzymes are widely used as signal transduction components of biosensors because one DNAzyme molecule is able to catalyze many cycles of a specific reaction generating amplified readout signals. Among DNAzymes acting as amplifying labels, a binary peroxidase DNAzyme attracts special efforts, which is composed of two single-stranded guanine-rich DNA strands self-assembling into G-quadruplex structure with hemin. By innovatively integrating specially designed DNAzyme or/and aptamer sequences into signal-amplifying strategies, we developed several new biosensors to detect protein and nucleic acid. Representatively, we developed DNAzyme-conjugated aptasensor associated with a very unique enzyme-mediated amplification strategy for ultra highly sensitive detection of target protein. As a result, this new colorimetric protein detection method possesses extremely high sensitivity with a detection limit of 0.1 fM for a model lysozyme, the highest sensitivity reported in the DNAzyme-based aptasensor field. 

 

 

3. Aptamer & DNA analog-based biosensor

We have developed new signal-on aptasensors for molecular recognition that employ unmodified DNA aptamers and signaling probes containing the fluorescent base analog, pyrrolo-dC (PdC). Based on the unique property of the PdC, associated with the fact that its fluorescence is strongly sensitive to DNA helix formation, two versions of signal-on sensors were developed. In addition, we devised a new technology that enables the highly selective and sensitive detection of silver ions. The method takes advantage of the unique fluorescence property of a mismatched pyrrolo-dC (PdC)-modified duplex DNA, which serves as the key detection component, and the specific interaction of this duplex with silver ions.

 


Novel PCR and Microarray Technology

 

1. Novel isothermal PCR technologies

 The invention of the PCR technique has made a tremendous impact on the areas of biological research and diagnostics since it can be used to generate quantities of nucleic acids needed for detection and measurements. However, the need for a temperature cycling instrument limits applications of PCR in point-of-care testing (POCT) environments. In order to overcome this limitation, we have developed a new isothermal nucleic acid amplification method, highly sensitive and specific system, called isothermal chain amplification (ICA). The system employs rationally designed primers (outer and DNA-RNA-DNA chimeric inner primers) and the enzymatic activities of a 5’ to 3’ exonuclease-deficient DNA polymerase and RNase H. By simultaneously utilizing the dual amplification powers of the target DNA and probe DNA, we have demonstrated that our method can be used to detect even a single copy of a model Chlamydia trachomatis gene. In addition, we developed a facile colorimetric method for real-time detection of target DNA in conjugation with gold nanoparticle cross-linking assay (GCA). This approach would be very promising for application in portable sensor systems such as micro-fluidic devices or POCT diagnostic kits.

 


2. A Sexually Transmitted Disease (STD) DNA chip

We have developed the first DNA chip for effective diagnosis of genitourinary infections, which cause sexually transmitted diseases (STD). Our STD DNA chip is designed to detect the most abundant 14 pathogens found in Koreans, as determined by using a full statistical analysis of over 35,000 clinical cases. We successfully demonstrated the diagnostic capability of the developed DNA chip by accurately detecting various STD infections for real clinical samples. This effort demonstrates that the strategy we have employed for the entire process from the statistical analysis of a large number of clinical cases to the final development of STD DNA chip, can be readily applied in the clinical diagnostic field. As a result, the technology for the STD chip has been transferred to a private bio-venture company for commercialization and the STD chip has been approved for manufacturing as a medical device by Korean FDA in 2014.

 


3. Microarray cell-based sensor
We have developed a solid-phase multiplexed amino acid array, which comprises 16 different amino acid E. coli auxotrophs yielding rapid, specific, and sensitive cell growth as a direct response to the concentration of corresponding amino acids. To obtain the resulting cell-based signal, bioluminescence-based measurement of cell density was employed. As a result, an amino acid concentration-dependent luminescence response is produced. Such an approach has also been extended to rapid cell-based sensing of homocysteine that are closely associated with several human disorders including Alzheimer’s and Parkinson’s disease, neural tube defects, pregnancy complications, and osteoporosis.

 

 

 

Electrochemical Biosensing Technology

 

1. Illusionary polymerase activity triggered by metal ions

 We developed for the first time a unique and innovative biomolecular detection platform that utilizes a capacitive touchscreen, which is a mass-produced input unit widely used in current mobile devices. By systematically utilizing both surface capacitive and projected capacitive touchscreens, we have demonstrated the detection capability of a new technology for detection and quantitation of DNA and also verified the potential of this technology in the biosensing area. Since the technology is ready to be integrated into the touchscreen-equipped smart phones or smart pads, it should truly accelerate the realization of personalized portable biosensors. Due to the academic significance and the innovative idea of the work, the paper describing the results was highlighted as a cover article of Angewandte Chemie International Edition in 2012.

 

 

 

 

 

 

 



2. Electrochemical detection of genetic mutations

We developed new electrochemical methods to identify DNA mutation and SNP genotype based on mismatched DNA-specific cleavage activity of CEL I endonuclease. Our method for the detection of DNA mutations relies on enzymatic mismatched DNA-specific cleavage and electrocatalytic signaling. The detection strategy was successfully used to detect various human mutations from breast cancer patients, including single-base insertion, deletion, and substitution in BRCA1 gene. Owing to its simplicity, the new strategy should be applicable to methods for the detection of mutations or SNP genotyping. 

 

 

3. Electrochemical real-time PCR

 Real-time PCR monitoring is a powerful technique for quantitative analysis of nucleic acids in SNP genotyping, gene expression profiling, or genetic diagnosis. Its utility in the diagnostic field is becoming increasingly important, particularly owing to the emergence of new viral variants threatening humans. However, the current techniques inevitably require not only specialized reagents but also relatively expensive and bulky instrumentation for monitoring fluorescence. This limits their widespread utility in facility-limited environments. Toward the goal to develop procedures to decentralize the utility of PCR, development of electrochemical real-time PCR systems has become an attractive endeavor. These systems are simple, portable and cost-effective. Therefore, to accelerate the development of methylene blue (MB)-based electrochemical real-time PCR, we have carried out a systematic investigation of the mechanism for the electrochemical signal change of MB during PCR amplification. In this work, we have successfully demonstrated the application of the electrochemical real-time PCR system to quantification of initial copy numbers of target Chlamydia trachomatis DNA templates on a patterned electrode chip. This work was highlighted as a front cover article of Analyst.

 

 

 

 

 

 

Nanobiotechnology

 

1. Nanobiosensor

 nzymes have been widely used in various fields of biotechnology. However, several unavoidable limitations (e.g., instability caused by structural unfolding) of natural enzymes have hindered its widespread applications. To overcome the limitations, we have developed nanostructured multi-catalyst system consisting of magnetic nanoparticles as peroxidase mimetics and oxidative enzyme entrapped in large pore sized mesoporous silica or carbon. The nanocomposite concept was successfully 

demonstrated by very conveniently detecting target glucose, cholesterol, galactose, or alcohol with excellent reusability and stability. We also developed a new synergistically integrated nanocomposite consisting of magnetic and platinum nanoparticles simultaneously entrapped in large pore sized mesocellular carbon, which serves as a promising enzyme mimetic to achieve ultrafast colorimetric immunoassays. Using the new assay system, clinically important target molecules, such as human epidermal growth factor receptor 2 (HER2) and diarrhea-causing rotavirus, were able to be detected in only 3 min at room temperature with high specificity and sensitivity. Since the nanocomposite has significantly enhanced ability to catalyze oxidation reactions, it can be employed in new assay methods for rapid, robust, and convenient identification of target pathogens as part of efficient point-of-care (POC) clinical diagnostic systems.


2. DNA-mediated nanobiosensor
We developed a novel strategy for the ultrasensitive colorimetric detection of nucleic acids that is based on photoinduced silver ion (Ag+) reduction around DNA bases. The assay system, which takes advantage of the metal ion-reducing capability of DNA induced by photoirradiation, is capable of directly detecting bacterial genomic DNA without the need for PCR amplification. The analytical capability of this simple but very powerful strategy was demonstrated by its utilization in detecting unamplified genomic DNA from a Salmonella typhimurium pathogen with an ultrahigh sensitivity as low as 67 pg/μL. In addition, we are developing various biosensing strategies using DNA-templated fluorescent AgNCs consisting of a few silver atoms that is synthesized on the basis of the specific interaction of silver ions with cytosine nucleobase.

3. Colorimetric biosensor
Toward the goal for the development of very convenient biosensors for the detection of biomolecules, we developed several colorimetric or optical biosensors to detect nucleic acids or biomolecules related with human diseases. Most representatively, we have developed a new and simple colorimetric strategy for the detection of nucleic acids, which relies on target DNA-induced shielding action against the peroxidase mimicking activity of magnetic nanoparticles (MNPs). The viability of this simple yet efficient colorimetric detection system has been demonstrated by its application to the diagnosis of Chlamydia trachomatis pathogen infection by using a human urine sample. This work has been recently published in Small and highlighted as a front cover article. In addition, various colorimetric methods employing polydiacetylene (PDA) liposome, gold nanoparticles and gold nanorods have been developed to detect nucleic acids and proteins, based on the principle of ligand-receptor interactions. 

 

 

Hyun Gyu Park (박현규) 
Professor


Office Phone: +82-42-350-3932
Fax: +82-42-350-3910
E-mail: hgpark@kaist.ac.kr
Homepage: http://bcbd.kaist.ac.kr

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ProfessorHyun Gyu Park

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