• 미생물학회지
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• 제37권1호
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• pp.72-79
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• 2001

폐수 처리장의 방류수에 페놀을 첨가한 후 terminal restriction fragment length polymorphism (T-RFLP) 방법을 이용하여 세균군집의 구조와 변화를 조사하였다. 시료로부터 얻은 16S rRNA gene은 eubacterial primer로 증폭하였으며, 한 primer는 5'말단에 biotin을 부착하였다. 증폭된 product는 HaeIII와 AluI으로 각각 절단하였고, 절단된 단편 중에서 terminal restriction fragment (T-RF)를 streptavidin paramagnetic particle을 이용하여 분리하였다. 분리된 T-RF는 전기영동과 silver staining을 통하여 확인하였다. 본 실험의 유용성을 검증하기 위하여 표준 균주 10 균주를 대상으로 실험하였고, 균주마다 특징적인 T-RF를 가지는 것과 그 크기가 Ribosomal database project (RDP) 자료로부터 계산된 결과와 일치하는 것을 확인할 수 있었다. 한편, 대조군으로 사용된 페놀을 첨가하지 않은 방류수 시료에서는 Acinetobacter, Bacillus, Pseudomonas 속 등이 우점종을 차지하고 있었고, 페놀 (최종농도 250mg.$l^{-1}$)을 첨가한 방류수 시료에서는 Acinetobacter, Comamonas, Cytophaga, Pseudomonas 속 등이 우점종을 차지함을 알 수 있었다. Gel에서 분리한 Acinetobacter와 Cytophaga에 해당되는 T-RF는 재증폭 및 염기 서열 분석이 가능하였는데, database의 염기서열과 비교한 결과 Acinetobacter junii와 유연관계가 가깝다는 것을 확인하였다.

• KSBB Journal
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• 제18권3호
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• pp.190-196
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• 2003

임신이나 배란진단과 같이 가정에서 직접 사용할 수 있는 형태의 membrane strip 크로마토그래피 방법을 이용하여 식중독 균 DNA 분석시스템을 개발하였다. 분석물질로서는 빈번하게 발생하고 있는 식중독 미생물 중에서 S. typhimurium을 선택하였으며, Salmonella 종에 특이한 유전자 부위인 invA 유전자 분석을 목표로 하였다. 우선 이 유전자는 본 실험실 내에서 설계한 primer 쌍을 사용한 PCR 공정을 통하여 증폭되었다. 이렇게 증폭한 산물을 본 연구자들이 설계한 DNA probe와의 hybridization을 통하여 분석함으로써 전통적으로 사용하고 있는 전기영동 분석법의 단순히 분자크기에 의한 분리법과 비교하여 특이한 분석을 할 수 있었다. 이때 PCR 후 과량으로 잔존하는 primer를 별도로 제거하지 않고 hybridization을 수행할 수 있도록 특별하게 DNA probe를 설계하였다. 또한, probe가 증폭된 DNA와 hybridization 하였을 때 고체표면에 의한 간섭효과가 최소화되도록 설계 시 반영되었고 더욱이 streptavidin-비오틴의 결합을 이용하여 probe를 고정함으로써 고상에서의 상호작용이 더욱 용이하도록 배려하였다. 이러한 분석방법을 이용하여 분석한 결과 시료첨가 후 20-40분 정도에 최소 $10^3$cfu/mL (10 cells/system) 농도의 박테리아를 분석할 수 있었다. 이러한 결과는 일반적으로 사용하는 전기영동법보다 약 10배정도 더 민감한 결과일 뿐만 아니라 실험실 기기를 사용하지 않고 분석을 수행함으로써 분석시료가 제공되는 현장에서도 식중독 균의 탐지가 가능하게 되었다.

• Journal of electromagnetic engineering and science
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• 제12권4호
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• pp.227-233
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• 2012

This paper describes a novel and compact biosensing platform using an RF active system. The proposed sensing system is based on the oscillation frequency deviation due to the biomolecular binding mechanism on a resonator. The impedance variation of the resonator, which is caused by a specific biomolecular interaction results in a corresponding change in the oscillation frequency of the oscillator so that this change is used for the discrimination of the biomolecular binding, along with concentration variation. Also, a Surface Acoustic Wave (SAW) filter is utilized in order to enhance the biosensing performance of our system. Because the oscillator operates at the skirt frequency range of the SAW filter, a small amount of oscillation frequency deviation is transformed into a large variation in the output amplitude. Next, a power detector is used to detect the amplitude variation and convert it to DC voltage. It was also found that the frequency response of the biosensing system changes linearly with three streptavidin concentrations. Therefore, we expect that the proposed RF biosensing system can be applied to bio/medical applications capable of detecting a nano-sized biomolecular interaction.

• Journal of Microbiology and Biotechnology
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• 제27권12호
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• pp.2173-2179
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• 2017

The intracytoplasmic membrane of Rhodobacter sphaeroides readily vesiculates when cells are lysed. The resulting chromatophore membrane vesicle (CMV) contains the photosynthetic machineries to synthesize ATP by ATPase. The light-dependent ATPase activity of CMV was lowered in the presence of $O_2$, but the activity increased to the level observed under anaerobic condition when the reaction mixture was supplemented with ascorbic acid (${\geq}0.5mM$). Cell lysis in the presence of biotinyl cap phospholipid (bcp) resulted in the incorporation of bcp into the membrane to form biotinylated CMV (bCMV), which binds to streptavidin resin at a ratio of approximately $24{\mu}g$ bacteriochlorophyll a/ml resin. The ATPase activity of CMV was not affected by biotinylation, but approximately 30% of the activity was lost by immobilization to resin. Interestingly, the remaining 70% of ATPase activity stayed constant during 7-day storage at $4^{\circ}C$. On the contrary, the ATPase activity of bCMV without immobilization gradually decreased to approximately 40% of the initial level in the same comparison. Thus, the ATPase activity of CMV is sustainable after immobilization, and the immobilized bCMV can be used repeatedly as an ATP generator.

• 대한세포병리학회지
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• 제12권2호
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• pp.89-95
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• 2001

The cytological distinction of carcinoma cells from reactive mesothelial cells in serous effusions nay be difficult or imposslble based on morphology alone, especially In specimens containing reactive mesothelial cells which form glandular or ball- or papillary-shaped conglomerates or which mimic malignant nuclear features. Calretinin is a newly reported immunocytochemical marker for mesothelial cells, which can potentially be utilized for facilitating this distinction. This study evaluated the usefulness of calretinin for the discrimination between reactive mesothelial and metastatic carcinoma cells in serous effusion. Immunocytochemical staining was undertaken on 33 benign reactive and 87 malignant serous effusion specimens with histologically confirmed diagnoses. The specimens including smears and cell blocks were stained with polyclonal antibody to calretinin by labelled streptavidin-biotin method. The positive expression of calretinin was noted In 32(97.0%) of 33 benign reactive effusions and 9(10.3%) of 87 malignant effusions. The sensitivity and specificity of the calretinin immunostaining for reactive mesothelial cells was 97.0% and 89.7%, respectively. In conclusion, calretinin is a useful marker for distinguishing between reactive mesothelial cells and carcinoma cells in serous effusions.

• 동의생리병리학회지
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• 제18권4호
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• pp.1154-1158
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• 2004

The classical ABC (avidin-biotin peroxidase complex) method for immunohistochemistry in the paraffin-embedded tissues bring into being disadvantage such as low sensitivity of antigen detection and highly background. The biotinyl-tyramide conjugation recently introduced for sensitive immunohistochemistry was applied to light microscopy in paraffin-embedded pancreatic and liver tissues. The protocol consists of an indirect method in which 4-5㎛ tissue sections are reacted successively within a specific primary antibody, followed by a biotinylated secondary antibody, streptavidin-horseradich peroxidase (HRP), and then finally with biotinyl-tyramide. The labeling obtained for insulin and collagen antigen tested in pancreatic and liver tissues, respectively, was found to be highly specific with the labeling for each antigen confined to its particular cellular compartment. In this study, fish (flounder) serum was specially applied to remove nonspecific binding. Background levels and nospecific deposition of the staining were negligible. This results suggest that super-signal induction method by biotinyl-tyramide conjugate can readily applied to antigen detection of the paraffin-embedded tissues.

• Macromolecular Research
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• 제17권4호
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• pp.259-264
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• 2009

Chemical modification of magnetic nanoparticles(MNPs) with functional polymers has recently gained a great deal of attention because of the potential application of MNPs to in vivo and in vitro biotechnology. The potential use of MNPs as capturing agents and sensitive biosensors has been intensively investigated because MNPs exhibit good separation-capability and binding-specificity for biomolecules after suitable surface functionalization processes. In this work, we demonstrate an efficient method for the surface modification of MNPs, by combining surface-initiated polymerization and the subsequent conjugation of the biologically active molecules. The polymeric shells of non-biofouling poly(poly(ethylene glycol) methacrylate)(pPEGMA) were introduced onto the surface of MNPs by surface-initiated, atom transfer radical polymerization(SI-ATRP). With biotin as a model of biologically active compounds, the polymeric shells underwent successful post-functionalization via activation of the polymeric shells and bioconjugation of biotin. The resulting MNP hybrids showed a biospecific binding property for streptavidin and could be separated by magnet capture.

• BMB Reports
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• 제32권5호
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• pp.497-501
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• 1999

Biotinylation of recombinant proteins is a powerful tool for the detection and analysis of proteins of interest in a large variety of assay systems. The recent development of in vivo biotinylation techniques in E. coli has opened new possibilities for the production of site-specifically biotinylated proteins without the need for further manipulation after the isolation of the recombinantly expressed proteins. In the present study, a novel vector set was generated which allows the convenient cloning and expression of proteins of interest fused with an N-terminal in vivo biotinylated thioredoxin (TRX) protein. These vectors were derived from the previously reported pBIOTRX vector into which was incorporated part of the pBluescript II+phagemid multiple cloning site (MCS), amplified by PCR using a pair of sophisticated oligonucleotide primers. The functionality of these novel vectors was examined in this system by recombinant expression of rat transforming growth factor-$\beta$. Western-blot analysis using TRX-specific antibodies or peroxidase-conjugated streptavidin confirmed the successful induction of the fusion protein and the in vivo conjugation of biotin molecules, respectively. The convenience of molecular subcloning provided by the MCS and the effective in vivo biotinylation of proteins of interest makes this novel vector set an interesting alternative for the production of biotinylated proteins.

• 대한기계학회논문집A
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• 제28권9호
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• pp.1429-1434
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• 2004

Immunoassay is one of the important analytical methods for clinical diagnoses and biochemical studies, but needs a long time, troublesome procedures and expensive reagents. In this study, therefore, we propose the micro filter chip with microbeads for immunoassay, which has pillar structures. The advantage of the proposed micro filter chip is to use simple fabrication process and cheap materials. The mold was made by the photolithography technique with Si wafer and negative photoresist SU-8. The replica was made of PDMS, bonded on the pyrex glass. The micro filter chip consists of inlet channel, filter chamber and outlet channel. HBV (Hepatitius B virus) monoclonal antibody (Ag1) labeled with biotin were immobilized onto streptavidin coated beads of 30∼50 $\mu$m size. Fluorescein isothiocyanate (FITC)-labeled HBV monoclonal antibody (Ag8) was used to detect HBsAg (Hebatitis B virus surface Antigen), and fluorescence intensity was monitored by epi-fluorescence microscope. In this study, the immune response of less than 30 min was obtained with with the use of 100 $m\ell$ of sample.

• Asian Pacific Journal of Cancer Prevention
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• 제15권16호
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• pp.6787-6790
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• 2014

Background: The aim of this study was to evaluate cyclooxygenase-2 (COX-2) immunoreactivity in colorectal adenocarcinomas and to find correlations with different pathological features. Materials and Methods: This study included 35 cases of colorectal carcinoma foir which surgical colectomy specimens were collected. Immunohistochemical staining of COX-2 (cyclooxygenase-2) is done by using the Streptavidin-biotin technique. Results: This work reveals that COX-2 is positive in most cases of colorectal carcinoma and negative in normal colon tissue with statistically non significant relations between COX-2 immunostaining and different pathological features. Conclusions: Our data suggest over expression of COX-2 protein in colorectal carcinoma in contrast to normal mucosa, with a possible role in cell proliferation in carcinogenesis.