Nucleic Acid Engineering (2017 spring)
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1. Biosensors

Biosensors are devices that consist of a physical or chemical transducer and a biological sensing element. Such systems could be applied to the analysis of biomolecular recognition at cellular membranes, ligand-receptor binding events, and other processes having biotechnological significance. Since the first biosensor was invented, many studies for creating new biosensing devices havebeen performed. The development of biosensors covers electrochemical sensors, quartz crystal microbalance (QCM) sensors, optical sensors that are surface plasmon resonance (SPR) sensors, microfluidic chips that include capillary electrophoresis (CE) chips, and chip array for nucleic acids and proteins, etc.

Especially, parallel development of colorimetric biosensors can allow rapid screening of large number of chemical and biological compounds without a costly equipment. Examples of colorimetric detection include colorimetric noble metal nanoparticles (e.g., gold and silver) and a colorimetric polydiacetylene (PDA) biosensor, etc.

Supramolecular chemical assemblies composed of polydiacetylene (PDA) exhibit rapid colorimetric transitions upon specific interactions with a variety of biological analytes in aqueous solutions. Among the analytes that give rise to the unique blue?red color changes are lipophilic enzymes, antibacterial peptides, ions, antibodies, and membrane penetration enhancers. The chemical assemblies include conjugated PDA, responsible for the chromatic transitions, and the molecular recognition elements, which are either chemically or physically associated with the PDA. Thus, by incorporation of specific recognition elements, the system can be designed in ways allowing for highly selective identification of analytes. In particular, receptors can be incorporated within the sensor assembly, which then determine the specificity of the colorimetric transitions. The PDA-based molecular assemblies are robust and can be readily applied to diagnosis of physiological molecules and for rapid screening of chemical and biological libraries

PDA-based biosensors for the detection of biologically important molecules have been intensively investigated due to the unique stimuli-responsive color-changing properties. Closely packed and properly designed diacetylene lipids can undergo polymerization via 1,4-addition reaction to form an ene-yne alternating polymer chain upon UV irradiation at 254 nm. Vesicles containing PDA have been shown to undergo dramatic blue to red color changes following interactions with various biological analytes, for examples, proteins, viruses, lipophilic enzymes, antibacterial peptides, ions, antibodies, and pharmacologically active compounds. An application currently explored in our laboratory involves the utilization of a sort of protein-protein interactions.

An advantage of using nanostructured PDAs as biosensors is that such a striking color change from blue to red occurs upon the specific interactions between a surface bound ligand and its complementary receptor. Biotin/STA interactions were one of the most specific biological recognition processes of the ligand-receptor system. The highly conjugated blue polymer can be converted into the less conjugated red polymer when STA is bound to a specifically designed biotin that is linked to the diacetylene surface. This scheme provides a useful model to demonstrate molecular recognition between the protein STA and the biotin-polymer matrix.

2. DNA chip PNA chip

[Introduction]


Our research field include DNA chip technology for diagnosis of genetic mutations. The completion of human genome project offers new possibillites for exploring many genetic diseases or molecular pathogene of infectious diseases. Our group intensively try to enhance the performance of DNA chip for much higher sensitivity and accuracy. Recently, we introduce PNA(Peptide Nucleic Acids) for a new microarray and also developed immobilization method using multi-epoxy compound.


[DNA chip?]


DNA chip is a solid substrate, such as glass or silicon, which contain DNA fragments or oligonucleotides on its surface. Theses nucleic acids are complementary to thousands of genes of known or unknown function. Although, hybridization method is not new, DNA chip have opened the new way for the parallel detection and analysis of gene expression in a single experiment. They also allow for the detection of subtle difference that are much harder to detect with subtraction or other molecular methods.


[DNA chip을 이용한 질병진단]

[PNA chip]

Our laboratory try to develop a new DNA chip using PNA. PNA is a DNA mimics in which sugar-phosphate backbone is replaced with peptide bond. Due to the PNA backbone is electrically neutral, there is no charge repulsion between PNA-DNA, and PNA can bind to DNA or RNA with higher affinity and selectivity even in low salt concentration. PNA also has high resistance to chemical, enzyme. Using these superior feature of PNA, we already developed PNA zip-code microarray and demonstrated for the detection of HNF-1α mutations.

 
DNA
PNA
Stronger binding H-bonding/charge repulsion H-bonding/no charge repulsion
Hybridization affinity - 1℃ higher per base
Hybridization rate - 100~50,000 times higher
salt concentration highly dependent independent
Tim for single mismatch(15mer) 4~16℃ lower(11℃) 8~20℃ lower(15℃)
Typical length for DNA chip 15~25 bases 8~15 bases
Chemical stability depurination with strong acid stable
Water solubility high low
Max base length no limit 18 bases due to self-aggregation
Cost low high
Cost established difficult

3. Lab-on-a chip

There have been many experiments to make small units that can perform complex biochemical analyses because they have many advantages compared to conventional sized units, which are as follows: small amount of reagent, low operating costs, short reaction time, and increased throughput. These small systems have been described as laboratories-on-a-chip (LOC).

We integrated microfluidic devices for multi-sample injection, reaction, washing and analysis. The ‘multisample-multicapture-testing’ chip offers cost and time-effective alternative, particularly for applications in which a large number of samples needs to be tested for the same biological materials. The chip was designed to fabricate matrix form photopolymer monolith containing proteins. With the strategy of immobilization, this simple manual procedure for manufacturing PDMS-based immunoassay may be applied to the fields of other biochip.

4. Nanomanipulation and immobilization of biocatalysts on nanostructured matrices

[Purpose]

This research focuses on the development of enzyme immobilization technology into nanoporous materials in order to dramatically enhance the stability and loading of the enzymes.

With these strategies, we can pave the way to the ideal enzyme system for various enzyme-based applications such as chemical conversions, chiral resolution, biosensing, and bioremediation using the combined technology of nanotechnology and biotechnology.

[Contents]

Several conventional approaches have been taken to improve the catalytic stability of enzymes:
① Enzyme Immobilization ② Enzyme Modification ③ Genetic Modification

1. We will develop a novel strategy to immobilize enzymes into various nanoporous materials. The nanoporous materials have attracted great attention for their controlled and sufficiently large pores to utilize inside of the pore for enzyme adsorption. As an immobilization substrate of enzymes, various nano-structured silicas and carbons will be synthesized and utilized. Cross-linked enzyme aggregates (CLEAs) method will be used for the immobilization. We will approach a unique and effective way to immobilize enzymes using various nanostructured matrices.

2. We will extend the above approach to develop enzyme sensors with high activity, selectivity, and stability and/or the enzymatic synthesis for chiral drug intermediates in non-aqueous organic solvents.

3. Proper nanomanipulation of enzymes using various nanostructured matrices will greatly enhance the enzyme stability, reduce the cost for enzymes in the enzymatic process, and consequently revitalize the biotechnology industries using enzymes. Furthermore, this unique nano-biotechnological approach will expand the areas using enzymes.


5. Nanoparticle conjugated biomaterial detection field

Metal nanoparticles have shown very promising applications, such as lubricants, catalysts, and magnetic recording media. Silver and gold nanoparticles in particular, are of great interest because of their ability to efficiently interact with light by virtue of plasmon resonances, which are the collective oscillations of the conduction electrons in the metal. Silver and gold nanoparticles certainly have the potential to be the building blocks of future photonic and plasmonic devices as the field of nanotechnology matures.

One of the major challenges is still the large-scale synthesis of nanoparticles with a narrow size distribution. Various size of silver and gold nanopartilcle were prepared by chemical or gamma-irradiation method in our lab. Size provides important control over many of the physical and chemical properties of nanoscale materials, including luminescence (due to its colorful reason), conductivity, and catalytic activity. Colloid chemists have achieved excellent control over particle size for several spherical metal.

Of all the noble metals, silver and gold have received the most attention in connection with biomolecular conjugates because their nanoparticle-related chemistry has been most extensively studied. Gold nanoparticles have long been conjugated with antibodies and other proteins (e.g., streptavidin) for use in protein and DNA detection assays, and such chemistry has resulted in the commercial development of some systems, primarily for proteins. Labeling DNA targets with metal nanoparticle rather than fluorophore probes substantially alters the melting probes of the targets from an array substrate. when used a silver enhancement with gold labeling DNA, the signal will be amplified 100 times or ten thousand times and more.


Conjugation of thiol group-containing biomolecules, such as cysteine, glutathione and penicillamine, with silver nanoparticles was bound by chemisorption. Those conjugated molecules also aggregated by hydrogen bonding which generated between carbonyl group and amino acid group among the biomolecules. These phenomena are very important in the nanoparticle handled biomolecular detection field. We found that chiral biomolecules conjugated with Ag nanoparticles can lead to new CD signals in the near-UV region, whereas there was no change in the CD signals for Au nanoparticles. The CD signal-generating phenomena of chiral biomolecules could provide a novel strategy for the chiral detection or molecular interaction analysis of biomolecules.