• Title/Summary/Keyword: FRET

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FRET을 이용한 근접장 현미경 개발

  • Park, Chung-Hyeon;Lee, Gyu-Seung;Jo, Yong-Hun
    • Proceedings of the Korean Vacuum Society Conference
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    • 2010.02a
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    • pp.352-352
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    • 2010
  • 일반적인 근접장 현미경은 광섬유 팁 (tip) 끝에 수십 nm 크기의 구멍을 이용하여 근접장을 발생시키거나 측정한다. 근접장은 전파되는 빛보다 미세한 구조의 정보를 반영하게 되는데 수십 나노미터의 구멍대신 FRET (fluorescence resonance energy transfer)이라는 현상을 근접장 현미경에 적용하고자 한다. 10 nm 이내의 거리에서 상호작용을 하는 이 현상을 이용하여 광학적 분해능을 향상시킬 수 있다. FRET 현상의 도우너(donor)로서는 양자점을 사용하였으며 억셉터(acceptor) 로서는 Cy5 염료를 사용하였다. 팁으로는 광섬유를 에칭한 팁에 Cy5 염료를 코팅한 팁과 광섬유의 코어(core) 부근에 양자점이 포함된 광 폴리머를 응고시켜서 만들어진 팁을 사용하였다. 팁에 위치한 도우너와 시료로 사용되는 억셉터를 FRET 상호 작용 거리 내로 접근시키기 위하여 tuning fork를 이용한 shear force control을 사용하였다. 한 점에서의 접근 과정에서 FRET의 현상의 특징으로서 도우너인 양자점의 형광이 약해지고 Cy5의 형광이 강해지는 것을 측정하였다. 또한, 양자점을 Cy5 염료에 근접시켰을 때 발광 생존시간 (lifetime)이 짧아지는 것을 관찰하여 FRET 현상을 재확인 하였다.

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Fabrication of Photoluminescent Dye Embedded PMMA Nanofiber and its Fluorescence Resonance Energy Transfer

  • Lee, Kyung-Jin;Oh, Joon-Hak;Kim, Young-Geun;Jang, Jyong-Sik
    • Proceedings of the Polymer Society of Korea Conference
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    • 2006.10a
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    • pp.314-314
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    • 2006
  • The FRET property has been extensively studied from the theoretical view points to the practical applications. In case that the donor and acceptor are confined in nanodimension, the FRET effectively occurs, because of their distant dependent characteristic. However, there are no reports concerning FRET with one dimensional (1D) nanomaterial. We have successfully prepared the PMMA nanotubes using vapor deposition polymerization as the platform of FRET. The dye-PMMA composite nanofiber has also been produced without phase separation and any deterioration of properties of the dyes. The PMMA 1D nanocomposite doped two dyes with great spectral overlap between donor and acceptor displayed FRET property.

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Development of a FRET-based High-Throughput Screening System for the Discovery of Hsp90 Inhibitors

  • Oh, Sang-Mi;Ko, Yeon-Jin;Lee, Han-Jae;Kim, Jong-Hoon;Chung, Young-Sun;Park, Seung-Bum
    • Bulletin of the Korean Chemical Society
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    • v.32 no.9
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    • pp.3229-3232
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    • 2011
  • A FRET-based high-throughput screening system was developed for the discovery of competitive smallmolecule Hsp90 inhibitors. The biarsenical fluorescein derivative FlAsH and dabcyl-conjugated Hsp90 inhibitor GM were employed as the FRET donor and quencher, respectively. The spatial proximity perturbation between FlAsH-labeled Hsp90N and GM-dabcyl upon treatment of a small molecule led to changes in the FRET-induced fluorescence, monitored in a high-throughput fashion.

An Ultrasensitive FRET-based DNA Sensor via the Accumulated QD System Derivatized in the Nano-beads

  • Yang, Lan-Hee;Ahn, Dong June;Koo, Eunhae
    • BioChip Journal
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    • v.12 no.4
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    • pp.340-347
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    • 2018
  • $F{\ddot{o}}rster$ resonance energy transfer (FRET) is extremely sensitive to the separation distance between the donor and the acceptor which is ideal for probing such biological phenomena. Also, FRET-based probes have been developing for detecting an unamplified, low-abundance of target DNA. Here we describe the development of FRET based DNA sensor based on an accumulated QD system for detecting KRAS G12D mutation which is the most common mutation in cancer. The accumulated QD system consists of the polystyrene beads which surface is modified with carboxyl modified QDs. The QDs are sandwich-hybridized with DNA of a capture probe, a reporter probe with Texas-red, and a target DNA by EDC-NHS coupling. Because the carboxyl modified QDs are located closely to each other in the accumulated QDs, these neighboring QDs are enough to transfer the energy to the acceptor dyes. Therefore the FRET factor in the bead system is enhancing by the additional increase of 29.2% as compared to that in a single QD system. These results suggest that the accumulated nanobead probe with conjugated QDs can be used as ultrasensitive DNA nanosensors detecting the mutation in the various cancers.

FRET-Based Quantitative Discrimination of Bisulfite-Untreated DNA from Bisulfite-Treated DNA

  • Lee, Eun Jeong;Cho, Yea Seul;Song, Seongeun;Hwang, Sang-Hyun;Hah, Sang Soo
    • Bulletin of the Korean Chemical Society
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    • v.35 no.5
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    • pp.1455-1459
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    • 2014
  • We report a sensitive and reliable FRET-based nanotechnology assay for efficient detection and quantification of bisulfite-unmodified or modified DNA. Bisulfite-untreated DNA or bisulfite-treated DNA is subjected to PCR amplification with biotin-conjugated primers so that the amounts of bisulfite-untreated and treated DNA can be differentiated. Streptavidin-coated quantum dots (QDs) are used to capture biotinylated PCR products intercalated with SYBR Green, enabling FRET measurement. Key features of our method include its low intrinsic background noise, high resolution, and high sensitivity, enabling detection of as little as 1.75 ng of bisulfite-untreated DNA in the presence of an approximately 1,000-fold excess of bisulfite-untreated DNA compared to bisulfate-treated DNA, with the use of PCR reduced (as low as 15 cycles). SYBR Green as an intercalating dye as well as a FRET acceptor allows for a single-step preparation without the need for primers or probes to be chemically conjugated to an organic fluorophore. Multiple acceptors per FRET donor significantly enhance the signal-to-noise ratio as well. In consideration of the high relevance of bisulfite treatment to DNA methylation quantitation, our system for FRET measurement between QDs and intercalating dyes can be generally utilized to analyze DNA methylation and to potentially benefit the scientific and clinical community.

A FRET Assay for Celiac Disease

  • Lee, Sae A;Cho, Chul Min;Jang, Il Ho;Kang, Jung Sook
    • Biomedical Science Letters
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    • v.22 no.4
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    • pp.160-166
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    • 2016
  • To provide a basis for a homogeneous fluorescence resonance energy transfer (FRET) immunoassay for celiac disease, we carried out a FRET experiment using guinea pig tissue transglutaminase (tTG) and antibodies to tTG (anti-tTG) purified from rat serum. Fluorescein was utilized as the probe, and a nonfluorescent dye, QSY 7 served as the quencher. We labeled anti-tTG and tTG with fluorescein isothiocyanate and QSY 7 succinimidyl ester, respectively. Fluorescein-labeled anti-tTG was the donor, and QSY 7-labeled tTG was the acceptor of the FRET experiment. When we titrated fluorescein-labeled anti-tTG with QSY 7-labeled tTG, we observed a large decrease in the steady-state fluorescence intensity, which was due to strong FRET from fluorescein-labeled anti-tTG to QSY 7-labeled tTG. Using time-resolved fluorescence spectroscopy, we could also observe a decrease in the fluorescence lifetime, which confirms the steady-state data. We expect that these results might be useful in the development of a novel fluorescence immunoassay for an easy screening and follow-up of celiac patients.

A Possible Merge of FRET and SPR Sensing System for Highly Accurate and Selective Immunosensing

  • Lee, Jae-Beom;Chen, Hongxia;Lee, Jae-Wook;Sun, Fangfang;Kim, Cheol-Min;Chang, Chul-Hun L.;Koh, Kwang-Nak
    • Bulletin of the Korean Chemical Society
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    • v.30 no.12
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    • pp.2905-2908
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    • 2009
  • Immuno-sensing for high accurate and selective sensing was performed by fluorescence spectroscopy and surface plasmon resonance (SPR), respectively. Engineered assembly of two fluorescent quantum dots (QDs) with bovine serum albumin (BSA) and anti-BSA was fabricated in PBS buffer for fluorescence analysis of fluorescence resonance energy transfer (FRET). Furthermore, the same bio-moieties were immobilized on Au plates for SPR analysis. Naturally-driven binding affinity of immuno-moieties induced FRET and plasmon resonance angle shift in the nanoscale sensing system. Interestingly, the sensing ranges were uniquely different in two systems: e.g., SPR spectroscopy was suitable for highly accurate analysis to measure in the range of 10$^{-15{\sim}-10$ng/mL while the QD fluorescent sensing system was relatively lower sensing ranges in 10$^{-10{\sim}-6$ng/mL. However, the QD sensing system was larger than the SPR sensing system in terms of sensing capacity per one specimen. It is, therefore, suggested that a mutual assistance of FRET and SPR combined sensing system would be a potentially promising candidate for high accuracy and reliable in situ sensing system of immune-related diseases.

Development of an Open Sandwich Fluoroimmunoassay Based on FRET (FRET에 기반한 Open Sandwich Fluoroimmunoassay)

  • Wei, Quande;Lee, Moon-Kwon;Seong, Gi-Hun;Choo, Jae-Bum;Lee, Eun-Kyu
    • KSBB Journal
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    • v.22 no.6
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    • pp.426-432
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    • 2007
  • We have developed a sensitive, one-step, homogeneous open sandwich fluoroimmunoassay (OsFIA) based on fluorescence resonance energy transfer (FRET) and luminescent semiconductor quantum dots (QDs). In this FRET assay, estrogen receptor-$\beta$ (ER-$\beta$) antigen was incubated with QD-labeled anti-ER-$\beta$ monoclonal antibody and AF (Alexa Fluoro)-labeled anti-ER polyclonal antibody for 30 minutes, followed by FRET measurement. The dye separation distance was estimated to be between $80\sim90\;{\AA}$. The present method is rapid, simple and highly sensitive, and did not require the bound/free reagent separation steps and solid-phase carriers. A concentration as low as 0.05 nM (2.65 ng/ml) receptor was detected with linearity ($R^2$ > 0.990). In addition, the assay was performed with commercial antibodies. This assay provides a convenient alternative to conventional, laborious sandwich immunoassays.

Comparative Analysis of Src Activity in Plasma Membrane Subdomains via Genetically Encoded FRET Biosensors (유전적으로 암호화된 FRET 바이오센서를 통한 세포막 하위 도메인의 Src 활성 비교 분석)

  • Gyuho Choi;Yoon-Kwan Jang;Jung-Soo Suh;Heonsu Kim;Sanghyun Ahn;Tae-Jin Kim
    • Journal of Life Science
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    • v.33 no.2
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    • pp.191-198
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    • 2023
  • As a member of the focal adhesion complex of the plasma membrane, Src is a nonreceptor tyrosine kinase that controls cell adhesion and motility. However, how Src activity is regulated in the plasma membrane microdomain in response to components of the extracellular matrix (ECM) remains unclear. This study compared and investigated the activity of Src in response to three representative ECM proteins: collagen type 1, fibronectin, and laminin. Genetically encoded FRET-based Src biosensors for plasma membrane subdomains were used. FRET-based biosensors allow the real-time analysis of protein activity in living cells based on their high spatiotemporal resolution. The results showed that Src activity was maintained at a high level under all ECM conditions of the lipid raft, and there was no significant difference between the ECM conditions. In contrast, Src activity was maintained at a low level in the non-lipid raft membrane. In addition, the Src activity of lipid rafts remained significantly higher than that of non-lipid raft regions under the same ECM conditions. In conclusion, this study demonstrates that Src activity can be controlled differently by lipid rafts and non-lipid raft microdomains.

CdS Nanoparticles as Efficient Fluorescence Resonance Energy Transfer Donors for Various Organic Dyes in an Aqueous Solution

  • Ock, Kwang-Su;Ganbold, Erdene-Ochir;Jeong, Sae-Ro-Mi;Seo, Ji-Hye;Joo, Sang-Woo
    • Bulletin of the Korean Chemical Society
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    • v.32 no.10
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    • pp.3610-3613
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    • 2011
  • CdS nanoparticles (NPs) were synthesized in an aqueous phase in order to investigate their spectral behaviors as efficient fluorescence resonance energy transfer (FRET) donors for various organic dye acceptors. Our prepared CdS NPs exhibiting strong and broad emission spectra between 480-520 nm were able to transfer energy in a wide wavelength region from green to red fluorescence dyes. Rhodamine 6G (Rh6G), rhodamine B (RhB), and sulforhodamine 101 acid (Texas red) were tested as acceptors of the energy transfer from the CdS NPs. The three dyes and synthesized CdS NPs exhibited good FRET behaviors as acceptors and donors, respectively. Energy transfers from the CdS NPs and organic Cy3 dye were compared to the same acceptor Texas red dye at different concentrations. Our prepared CdS NPs appeared to exhibit better FRET behaviors comparable to those of the Cy3 dye. These CdS NPs in an aqueous solution may be efficient FRET donors for various organic dyes in a wide wavelength range between green and red colors.