• Bulletin of the Korean Chemical Society
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• 제33권11호
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• pp.3681-3685
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• 2012

We developed a simple and effective method for the SERS-based detection of protein-small molecule complexes and label-free proteins using avidin-induced silver aggregation. Upon excitation with light of the appropriate wavelength (633 and 532 nm), the aggregated silver nanoparticles generate a strong electric field that couples with the resonance of the molecules (atto610 and cytochrome c), increasing the characteristic signals of these molecules and resulting in sensitive detection. The detection limit of biotin with the proposed method is as low as 48 ng/mL. The most important aspect of this method is the induction of silver aggregation by a protein (avidin), which makes the silver more biocompatible. This technique is very useful for the detection of protein-small molecule complexes.

• 한국농업기계학회:학술대회논문집
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• 한국농업기계학회 2003년도 하계 학술대회 논문집
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• pp.421-426
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• 2003

The Immunosensor based on antigen-antibody binding have been developed for detecting several analytes including antigen, small molecules, and cell. This method can be rapid and show very good detection limits. For Implementation of immunosensor, technologies for immobilization of antibody onto solid surface and detection of protein-protein binding must be developed. (an ellipsis)

• BMB Reports
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• 제38권6호
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• pp.683-689
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• 2005

Antibody to hepatitis B surface antigen (HBsAb) is the important serological marker of the hepatitis B virus (HBV) infection. Conventionally, the hepatitis B surface antigen (HBsAg) obtained from the plasma of HBV carriers is used as the diagnostic antigen for detection of HBsAb. This blood-origin antigen has some disadvantages involved in high cost, over-elaborate preparation, risk of infection, et al. In an attempt to explore the suitable recombinant HBsAg for the diagnostic purpose, the HBV S gene was expressed in Pichia pastoris and the product was applied for detection of HBsAb. Hepatitis B virus S gene was inserted into the yeast vector and the expressed product was analyzed by sodium dodecyl sulphate polyacrolamide gel electrophoresis (SDS-PAGE), immunoblot, electronic microscope and enzyme linked immunosorbent assay (ELISA). The preparations of synthesized S protein were applied to detect HBsAb by sandwich ELISA. The S gene encoding the 226 amino acid of HBsAg carrying ahexa-histidine tag at C terminus was successfully expressed in Pichia pastoris. The His-Tagged S protein in this strain was expressed at a level of about 14.5% of total cell protein. Immunoblot showed the recombinant HBsAg recognized by monoclonal HBsAb and there was no cross reaction between all proteins from the host and normal sera. HBsAb detection indicated that the sensitivity reached 10 mIu (micro international unit)/ml and the specificity was 100% with HBsAb standard of National Center for Clinical Laboratories. A total of 293 random sera were assayed using recombinant S protein and a commercial HBsAb ELISA kit (produced by blood-origin HBsAg), 35 HBsAb positive sera and 258 HBsAb negative sera were examined. The same results were obtained with two different reagents and there was no significant difference in the value of S/CO between the two reagents. The recombinant HBV S protein with good immunoreactivity and specificity was successfully expressed in Pichia pastoris. The reagent for HBsAb detection prepared by Pichia pastoris-derived S protein showed high sensitivity and specificity for detection of HBsAb standard. And a good correlation was obtained between the reagent produced by recombinant S protein and commercial kit produced by blood-origin HBsAg in random samples.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVI)
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• pp.18-18
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• 2005

Photoinduced electron transport process in nature such as photoelectric conversion and long-range electron transfer in photosynthetic organisms are known to occur not only very efficiently but also unidirectionally through the functional groups of biomolecules. The basic principles in the development of new functional devices can be inspired from the biological systems such as molecular recognition, electron transfer chain, or photosynthetic reaction center. By mimicking the organization of the biological system, molecular electronic devices can be realized $artificially^{1)}$. The nano-fabrication technology of biomolecules was applied to the development of nano-protein chip for simultaneously analyzing many kinds of proteins as a rapid tool for proteome research. The results showed that the self-assembled protein layer had an influence on the sensitivity of the fabricated bio-surface to the target molecules, which would give us a way to fabricate the nano-protein chip with high sensitivity. The results implicate that the biosurface fabrication using self-assembled protein molecules could be successfully applied to the construction of nanoscale bio-photodiode and nano-protein chip based on electrical detection.

• Biotechnology and Bioprocess Engineering:BBE
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• 제11권5호
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• pp.455-461
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• 2006

In this study, we have described a method for the fabrication of a protein chip on silicon substrate using hydrophobic thin film and microfluidic channels, for the simultaneous detection of multiple targets in samples. The use of hydrophobic thin film provides for a physical, chemical, and biological barrier for protein patterning. The microfluidic channels create four protein patterned strips on the silicon surfaces with a high signal-to-noise ratio. The feasibility of the protein chips was determined in order to discriminate between each protein interaction in a mixture sample that included biotin, ovalbumin, hepatitis B antigen, and hepatitis C antigen. In the fabrication of the multiplexed assay system, the utilization of the hydrophobic thin film and the microfluidic networks constitutes a more convenient method for the development of biosensors or biochips. This technique may be applicable to the simultaneous evaluation of multiple protein-protein interactions.

• 대한전기학회논문지:전기물성ㆍ응용부문C
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• 제54권8호
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• pp.378-383
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• 2005

We report the development of miniature fluorescence detection systems that employ miniature prism, mirrors and low coat CCD camera to detect the fluorescence emitted from 40 fluorescently-labeled protein patterns without scanner. This kind of miniature fluorescence detection system can be used in point of care. We introduce two systems, one uses prism+mirror block and the other uses prism and two mirrors. A large NA microscope eyepiece and low cost CCD camera are used. We fabricated protein chip containing multi-pattern BSA labeled with Cy5, using MEMS technology and modified the surface chemically to clean and to immobilize proteins. The measurements show that the combination of prism and mirrors can homogenize elliptical excitation light over the sample with higher optical efficiency, and increase the separation between excitation and fluorescence light at the CCD to give higher signal intensity and higher signal to noise ratio. The measurements also show that protein concentrations ranging from 10 ng/ml to 1000 ng/ml can be assayed with very small error. We believe that the proposed fluorescence detection system can be refined to build a commercially valuable hand-held or miniature detection device.

• Journal of Microbiology and Biotechnology
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• 제11권6호
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• pp.979-985
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• 2001

A fluorometric detection technique for HDL (High Density Lipoprotein) and LDL (Low Density Lipoprotein) was developed for application in a fiber-optic immunosensor using a protein A Langmuir-Blodgget (LB) film. For the fluorescence immunoassay, antibodies specific to HDL or LDL were imobilied on the protein A LB film, and a fluorescence amplification method was developed to overcome their weak fluorescence. The deposition of protein A using the LB technique was monitored using a surface pressure-are $({\pi}-A)$ curve, and the antibody immobilization of the protein A LB film was experimentally verified. The immobilized antibody was used to separate only HDL and LDL from a sample, then the fluorescence of he separated HDL or LDL was amplified. The amount of LDL or HDL was measured using the developed fiber optic fluorescence detection system. The optical properties resulting from the reaction of HDL or LDL with o-phtaldialdehyde, detection range, response time, and stability of the immunoassay were all investigated. The respective detection ranges for HDL and LDL were sufficient to diagnose the risk of coronary heart disease. The amplification step increased the sensitivity, while selective separation using the immobilized antibody led to linearity in the sensor signal. The regeneration of the antibody-immobilized substrate could produce a stable and reproducible immunosensor.

• 한국식물병리학회지
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• 제14권4호
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• pp.314-318
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• 1998

Using the reverse transcription polymerase chain reaction (RT-PCR), a rapid and sensitive assay method for the detection and identification of barley yellow mosaic virus (BaYMV) and barley mild mosaic virus (BaMMV) was adapted. Two units of primers from each virus were selected and used for the determination of two different viruses. PCR fragments of BaYMV (ca. 0.9kb) and BaMMV (ca. 0.8kb) were obtained from the designed method for the assay of BaYMV and BaMMV coat protein. PT-PCR fragments were cloned using vector pT7 Blue and the sequences of the selected clones were analyzed. coat protein of BaYMV and that of BaMMV consisted of 297 amino acids (891 nucleotides) and 251 amino acids (753 nucleotides), respectively. The snalysis of coat protein genes from these two viruses showed that 45.6% of nucleotides sequence ad 34.9% of amino acid in BaYMV were homologous to those in BaMMV.

• 한국전기전자재료학회논문지
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• 제17권4호
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• pp.404-409
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• 2004

We fabricated photo-sensor for fluorescence detection in LOC. LOC is high throughput screening system. Our LOC screens biochemical reaction of protein using the immunoassay, and converts biochemical reaction into electrical signal using LIF(Laser Induced Fluorescence) detection method. Protein is labeled with rhodamine intercalating dye and finger PIN photodiode is used as photo-sensor We measured fluorescence emission of rhodamine dye and analyzed tendency of fluorescence detection, according to photo-sensor size, light intensity, and rhodamine concentration. Detection current was almost linearly proportional to two parameters, intensity and concentration, and was inversely proportional to photo-sensor size. Integrated LOC consists of optical-filter deposited photo-sensor and PDMS microchannel detected 50 (pg/${mu}ell$) rhodamine. For integrated LOC including light source, we used green LED as the light source and measured emitted fluorescence.

• Journal of Microbiology and Biotechnology
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• 제26권1호
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• pp.38-43
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• 2016

Novel and specific recognition elements are of central importance in the development of a pathogen detection method. Here, we describe a simple method for identifying the cell-wall binding domain (CBD) from a sequenced bacterial genome employing homology search for phage lysin genes. A putative CBD (CPF369_CBD) was identified from a genome of Clostridium perfringens type strain ATCC 13124, and its function was studied with the CBD-GFP fusion protein recombinantly expressed in Escherichia coli. Fluorescence microscopy showed the specific binding of the fusion protein to C. perfringens cells, which demonstrates the potential of this method for the identification of novel bioprobes for specific detection of pathogenic bacteria.