(Nano) Biosensors are hybrid devices that transform chemical information into an analytically useful signal by means of a biochemical mechanism. Biosensors consist of a receptor system, in which a biological component interacts specifically with a given analyte, and a coupled physicochemical transducer that amplifies the signal resulting from such interaction from molecular up to macroscopical level. The purpose of these constructs is the identification, quantification and eventual screening of specific molecules, as present in complex mixtures from moderate to very low concentrations. Therefore, biosensors have utility in analytical research but also in clinical diagnosis, food and pharmaceutical industry, environmental control and process monitoring. The interest in biosensor development has partly arisen from the need of fast and routine analysis of a large number of samples, what requires robustness, sensitivity and reproducibility. Essentially, these instruments can generate either optical or electric signals which are very convenient for instrument development, and the commercial application of many biosensors in research. Work on biosensors has focused mostly on enzyme based sensors, receptor based sensor, nucleic acid based sensors, immunosensors, and microbial sensors.
In Chapter 2, we examined the possibility of in situ enzymatic reaction around the SWNTs. As a model reaction system, we selected the enzyme, cyclodextrin glucanotransferase (CGTase), which allows synthesis of cyclodextrins (CDs) from starch. CDs are able to form inclusion complexes with hydrophobic insoluble molecules via host-guest recognition and thereby solubilizing them in aqueous solution. Therefore, we decided to examine whether in situ CD formation around SWNTs from starch by CGTase will result in solubilization of SWNTs. Here, we report successful in situ CGTase reaction on the surface of SWNTs in aqueous solution, and subsequent formation of interesting macrocyclic supramolecules around the SWNTs resulting in the solubilization of SWNTs. The diameters of macrocyclic CD rings were much larger than those expected if the rings were formed around single SWNTs. It was suggested that wrapping of CDs leads to increase the solubilization and dispersion of SWNTs. Hence, an increasing of the CGTase concentration results in dramatic enhancement of the solubility of SWNTs at shorter time.
In Chapter 3, hybridization occurs only when there is a perfect match between the target oligonuleotide and the specific probe of the encoded GFP displayed spore-biotinylated antibody, whereas no hybridization occurs in cases where there is a mismatch. After addition of Streptavidin-conjugate, SpectraMax® M2 microplate reader analysis is used simultaneously to determine the extent of hybridization. Each encoded GFP displayed spore-biotinylated antibody is identified by its unique fluorescence profile of green (488 nm) and red (607 nm) emitted light. A hybridization signal emitted as red fluorescence (607 nm) by the Qdot Streptavidin Conjugates is present only on those encoded GFP displayed spore-biotinylated antibody to which biotinylated probes have hybridized to specific oligonucleotides. Homozygous and heterozygous SNP genotypes are then determined based on the relative proportions of the paired alleles present in the genomic DNA.
In Chapter 4-1, bacterial surface layer (S-layer) protein production, purification and characterization from Geobacillus stearothermophilus KCTC 2107, which is the thermophile bacteria were demonstrated. For the large scale production of S-layer protein, fed-batch culture of Geobacillus stearothermophilus was carried out by DO-stat strategy. By high-cell density cultivation, cells grew to 10 g dry cell weight $1^{-1}$ in 65 h and the S-layer proteins were achieved up to 40% of total proteins. The S-layer proteins produced were purified to electrophoretic homogeneity and the molecular mass was estimated to about 105 kDa by SDS-PAGE. After recrystallization of purified S-layer proteins, the formation of two-dimensional oblique lattice was observed by transmission electron microscopy (TEM), demonstrating that it could be used as protein template for immobilization molecular array and/or biosensor applications.
In Chapter 4-2, a simple procedure is described for patterning biotin on a glass substrate and then selectively immobilizing proteins of interest onto the biotin-patterned surface. Microcontact printing (μCP) was used to generate the micropattern of biotin and to demonstrate the selective immobilization of proteins by using enhanced green fluorescent protein (EGFP) as a model protein, of which the C-terminus was fused to a core streptavidin (cSA) gene of Streptomyces avidinii. Confocal fluorescence microscopy was used to visualize the pattern of the immobilized protein (EGFP-cSA), and surface plasmon resonance was used to characterize biological activity of the immobilized EGFP-cSA. The results suggest that this strategy, which consists of a combination of μCP and cSA-fused proteins, is an effective way for fabricating biologically active substrates that are suitable for a wide variety of applications, one such being the use in protein-protein assays.
In Chapter 5-1, two presentative bacteria pathogens, Salmonella typhimurium and Pseudomonas aeruginosa PAO1 are the major focus on this study. As shown in text, compound of mannose tethered Qdot binds specifically to bacterial Type1 pili, showing that stronger interaction with FimH than free mannose dose in the competition assay. The spherical mannose-tethered Qdots with an average diameter of ~20 nm were observed by TEM and no aggregation was found in the images. The fluorescence intensity results showed that mannose-tethered Qdots specifically bound the pili of the Salmonella tiphymirium and Pseudomonas aeruginosa PAO1, demonstrating that specific binding of mannose-tethered Qdots to Fim protein.
In Chapter 5-2, we expand this technology to establish new method by means of the display of target molecules on the surface of bacterial spores for studying the carbohydrate-protein interactions using solution-based analysis methods. As expected, cells in the absence of streptavidin have the same mean fluorescence as the unlabeled cells, indicating that there is no nonspecific binding of the cells by ConA, Texas $Red^®$ conjugate. These results confirm that a cell surface that displays streptavidin retained native biological activity for the binding to biotinylated mannose. Consequently, these results suggested that this feature permits the quantitative evaluation of ConA binding affinity in solution-based analysis tools. Interestingly, the specific and strong fluorescence signals were only detected on the spots corresponding to immobilized spore chips, while no fluorescence was visualized with the spots that the negative controls.
In Chapter 6, we investigate β-galactosidae expression in a small number of bacterial spores cells by utilizing an eletrochemical microdevice with on-chip incubation and on-chip monitoring. In our case, for the activity of expressed β-galactosidase can be determined by usng p-aminophenyl β-D-galactopyranoside (PAPG) as s substrate. Clearly, the oxidation current of PAP increased with incubation time, the increase in current corresponds to the β-galactosidase acitivity. The oxidation current of PAP produced from the spore cells in the microstructure mainly depends on the metabolic activity and the cell population.
In Chapter 7, the SWNTs formed on the silicon wafer are aligned along the direction approximately horizontal to the surface of the substrate. Most of carbon nanotubes are straight, some are curled due to the defects formed at SWNTs. Together with the SWNTs, some impurities and graphite fragments were also observed. It should be note that the relatively shorter SWNTs that are also formed in the parallel film. Also, the SWNTs align parallel to the applied magnetic field. Two-dimensional crossed-networks of SWNTs can be fabricated using this approach.
생물공학 (biotechnology) 와 나노테크놀러지 (nanotechnology) 기법들을 결합하여 생물공학적 및 환경모니터링을 위한 나노바이오센서 (nanobiosensor) 개발연구에 관한 것이다.
첫째로, 효소학적 반응 (in situ enzymatic reaction)을 통해 탄수화물이 랩핑된 수용성 탄소나노튜브 (carbon nanotubes: CNTs)를 제조방법을 구축하고 이를 AFM (atomic force microscopy), TEM (transmission electron microscopy) 이용하여 확인하였다. 또한, 이 방법에 의하여 제조된 탄수화물이 랩핑된 수용성 CNTs는 용해성 (solubility) 이 증가된 것으로 확인되었으며 이는 표적 바이오 물질과 결합하는 바이오 리셉터를 선택적으로 부착하여 얻어지는 바이오센서로서의 응용가능성이 기대된다.
둘째로, 탄소나노튜브에 박테리아 유래 생물 자기조립 (self-assembly)의 물질, 에스레이어 단백질 (s-layer protein, SLP)을 랩핑 (wrapping)하는 기능성 탄소나노튜브의 제조방법 개발하였으며 이 방법에 의해서 제조된 기능성 탄소나노튜브는 단백질 템플레이트 (protein templates) 로서 응용이 가능하며 이를 TEM (transmission electron microscopy) 을 통해서 확인하였다.
셋째로, optical detection method와 live bacterial spores를 메트릭스 (matrix)로 이용하여 DNA sensor를 개발하였다. 외부환경에 대해서 강한 내성을 가지고 있는 bacterial spores를 cell-surface display engineering 기법을 이용하여 형광단백질 (GFP)를 표면 발현하고 antibody reaction 을 통해서 DNA hybridization 조건을 확립하였으며 이 방법을 통해서 single nucleotide polyporphism (SNP) genotyping 을 수행하였다. 또한 대장균 유래의 DNA repair 에 관여하는 DNA binding protein (MutS)를 통해서 mismatched DNA 를 검색할 수 있었다.
넷째로, quantum dot nanoparticles을 이용하여 두 가지 대료적인 pathogen bateria, Salmonella tiphymurium 과 Pseudomonas aeruginosa 를 optical detection method를 이용하여 간단한 방법으로 검색할 수 있었다.
다섯째로, streptavidin 을 bacterial spores에 표면 발현을 통해서 carbohydrate sensor 를 개발하였다. 이는 탄수화물에 특이적으로 결합하는 lectin 단백질인 concanavalin A (ConA)를 이용하였다. FACS 를 통해서 표면 발현 여부를 확인하였으며 이를 microarray 에 적용함으로서 탄수화물에 특이적으로 작용하는 저해제 (inhibitors) 을 검색하기 위한 high-throughput screening 기법을 제공한 데 의의가 있을 수 있다 하겠다.
여섯째로, microfluidic chip에서 reporter gene 의 gene expression profiling 을 전기화학적인 방법으로 구현하였다. Reporter gene의 모델로서는 β-galactosidase 을 encoding하는 lacZ를 선택하였으며 p-aminophenyl-D-galactopyranoside (PAPG)를 기질로 사용하였다. lacZ를 가지는 bacterial spore를 정제하고 on-chip 상에서 gene expression 여부를 cyclic voltammography를 통해서 확인하였다. 이 방법을 통해서 reporter gene의 발현여부를 전기화학적인 방법을 제공하며, 기타 유전공학적인 실험에 간접적으로 이용할 수 있다.
마지막으로, 탄소나노튜브 (carbon nanotubes; CNTs)에 자기성 물질 (magnetic particles)을 결합시키고, 여기에 자기성 물질이 고정된 CNT를 기질 상에 위치시킨 다음, 수직 또는 수평 방향으로 자기장을 걸어주어CNT를 기질상에 align 하는 방법을 개발하였다. 이는 TEM (transmission electron microscopy) 이용하여 확인하였다. TEM 분석에 의하면 수직 또는 수평으로 정렬된 CNT 어레이의 제조가 가능함을 제시하고 있으며 본 연구개발을 통해서 CNT 어레이에 바이오 리셉터가 결합된 CNT-바이오칩 또는 어레이의 제조방법도 함께 구현할 수 있다.
