Name: highly sensitive and rapid fluorescence detection with a portable fret analyzer
Uploaded: 2016-10-01T13:00:00
Description: Watch this Scientific Journal Video about Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer at JoVE.com

This research was supported by grants from the Intelligent Synthetic Biology Center of Global Frontier Project (2011-0031944) and the KRIBB Research Initiative Program.

1. Preparation of Biosensor
<ol>
	<li>Construct the plasmid pET21a(+)-CFP-MBP-YFP-His6 by following the previously-established protocol2.</li>
	<li>Inoculate 5 ml of Luria broth (LB) with a single colony of an Escherichia coli DE3 strain and incubate at 37 &#176;C for 16 hr with shaking.</li>
	<li>Transfer 1 ml of the O/N culture into a 500-ml flask containing 100 ml LB and incubate at 37 &#176;C in a shaking incubator until the optical density at 600 nm (OD600) reaches 0.5 (about 3 hr).<...

<ol>
	<li>Deuschle, K., Okumoto, S., Fehr, M., Looger, L. L., Kozhukh, L., Frommer, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+optimization+of+a+family+of+genetically+encoded+metabolite+sensors+by+semirational+protein+engineering.">Construction and optimization of a family of genetically encoded metabolite sensors by semirational protein engineering.</a> Protein Sci. 14 (9), 2304-2314 (2005).</li><li>Ha, J. S., Song, J. J., Lee, Y. M., Kim, S. J., Sohn, J. H., Shin, C. S., Lee, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+application+of+highly+responsive+fluorescence+resonance+energy+transfer+biosensors+for+detection+of+sugar+in+living+Saccharomyces+cerevisiae+cells.">Design and application of highly responsive fluorescence resonance energy transfer biosensors for detection of sugar in living Saccharomyces cerevisiae cells.</a> Appl. Environ. Microbiol. 73 (22), 7408-7414 (2007).</li><li>Nagai, T., Yamada, S., Tominaga, T., Ichikawa, M., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanded+dynamic+range+of+fluorescent+indicators+for+Ca(2+)+by+circularly+permuted+yellow+fluorescent+proteins.">Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins.</a> Proc. Natl. Acad. Sci. U.S.A. 101 (29), 10554-10559 (2004).</li><li>Okumoto, S., Looger, L. L., Micheva, K. D., Reimer, R. J., Smith, S. J., Frommer, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+glutamate+release+from+neurons+by+genetically+encoded+surface-displayed+FRET+nanosensors.">Detection of glutamate release from neurons by genetically encoded surface-displayed FRET nanosensors.</a> Proc. Natl. Acad. Sci. U.S.A. 102 (24), 8740-8745 (2005).</li><li>Merzlyakov, M., Li, E., Casas, R., Hristova, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+F&#246;rster+resonance+energy+transfer+detection+of+protein+interactions+in+surface-supported+bilayers.">Spectral F&#246;rster resonance energy transfer detection of protein interactions in surface-supported bilayers.</a> Langmuir. 22 (16), 6986-6992 (2006).</li><li>Zhang, J., Campbell, R. E., Ting, A. Y., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creating+new+fluorescent+probes+for+cell+biology.">Creating new fluorescent probes for cell biology.</a> Nat. Rev. Mol. Cell Biol. 3 (12), 906-918 (2002).</li><li>Kim, H., Kim, H. S., Ha, J. S., Lee, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+portable+FRET+analyzer+for+rapid+detection+of+sugar+content.">A portable FRET analyzer for rapid detection of sugar content.</a> Analyst. 140 (10), 3384-3389 (2015).</li><li>Gam, J., Ha, J. -. S., Kim, H., Lee, D. -. H., Lee, J., Lee, S. -. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ratiometric+analyses+at+critical+temperatures+can+magnify+the+signal+intensity+of+FRET-based+sugar+sensors+with+periplasmic+binding+proteins.">Ratiometric analyses at critical temperatures can magnify the signal intensity of FRET-based sugar sensors with periplasmic binding proteins.</a> Biosens. Bioelectron. 72, 37-43 (2015).</li><li>Hessels, A. M., Merkx, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically-encoded+FRET-based+sensors+for+monitoring+Zn2++in+living+cells.">Genetically-encoded FRET-based sensors for monitoring Zn2+ in living cells.</a> Metallomics. 7 (2), 258-266 (2015).</li><li>Song, Y., Yang, M., Wegner, S. V., Zhao, J., Zhu, R., Wu, Y., He, C., Chen, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetically+encoded+FRET+sensor+for+intracellular+heme.">A genetically encoded FRET sensor for intracellular heme.</a> ACS Chem. Biol. 10 (7), 1610-1615 (2015).</li><li>. Fluorescent Protein Guide: Biosensors Available from: <a target="_blank" href="https://www.addgene.org/fluorescent-proteins/biosensors/">https://www.addgene.org/fluorescent-proteins/biosensors/</a> (2015)</li><li>Rajendran, R., Rayman, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Point-of-care+blood+glucose+testing+for+diabetes+care+in+hospitalized+patients:+an+evidence-based+review.">Point-of-care blood glucose testing for diabetes care in hospitalized patients: an evidence-based review.</a> J. Diabetes Sci. Technol. 8 (6), 1081-1090 (2014).</li><li>Vyas, N. K., Vyas, M. N., Quiocho, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sugar+and+signal-transducer+binding+sites+of+the+Escherichia+coli+galactose+chemoreceptor+protein.">Sugar and signal-transducer binding sites of the Escherichia coli galactose chemoreceptor protein.</a> Science. 242, 1290-1295 (1988).</li><li>Leermakers, E. T. M., Felix, J. F., Erler, N. S., &#266;erimagi&#263;, A., Wijtzes, A. I., Hofman, A., Raat, H., Moll, H. A., Rivadeneira, F., Jaddoe, V. W., Franco, O. H., Kiefte-de Jong, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sugar-containing+beverage+intake+in+toddlers+and+body+composition+up+to+age+6+years:+The+Generation+R+Study.">Sugar-containing beverage intake in toddlers and body composition up to age 6 years: The Generation R Study.</a> Eur. J. Clin. Nutr. 69 (3), 314-321 (2015).</li><li>Shilts, M., Styne, D., Drake, C., Aden, C., Townsend, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+food,+fat+and+sugar+sweetened+beverage+items+are+related+to+children's+dietary+energy+density.">Fast food, fat and sugar sweetened beverage items are related to children's dietary energy density.</a> FASEB J. 29 (1), 731-736 (2015).</li><li>Larsson, S. C., &#197;kesson, A., Wolk, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sweetened+beverage+consumption+is+associated+with+increased+risk+of+stroke+in+women+and+men.">Sweetened beverage consumption is associated with increased risk of stroke in women and men.</a> J Nutr. 144 (6), 856-860 (2014).</li><li>Melkko, S., Neri, D., Vaillancourt, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calmodulin+as+an+affinity+purification+tag.">Calmodulin as an affinity purification tag.</a> E. coli Gene Expression Protocols. , 69-77 (2003).</li></ol>

This protocol allows rapid and efficient quantification of the sugar content in beverage samples, using a custom-made FRET analyzer7 at an optimal temperature for FRET sensors. The analyzer was designed with a recently-developed, inexpensive 405-nm band ultraviolet-LED as the light source and two photodetectors with a silicon photodiode. This device is more cost-effective than other comparable fluorometers. The device showed high detection sensitivity, specifically when measuring the ratio of two emission wave...

F&#246;rster resonance energy transfer (FRET) has been widely used as a biometric sensor to detect small molecules such as sugars, calcium ions, and amino acids1-4. FRET biosensors contain fluorescent proteins, cyan fluorescent proteins (CFPs), and yellow fluorescent proteins (YFPs), which are fused to both ends of periplasmic-binding proteins (PBPs). Sugars bind to PBPs located in the middle of the FRET sensor, causing structural changes to the sensor that subsequently alter the distance and transition dipole orientation of the two fluorescent proteins at either end of the PBPs. This change enables quantitative analysis of sugar content by measuring the ratio of the emission wavelengths of EYFP (530 nm) and ECFP (480 nm). Owing to the high sensitivity, specificity, real-time monitoring capacity, and fast response time of FRET biosensors, these sensors are widely used in environmental, industrial, and medical applications5. Moreover, ratiometric measurement using FRET biosensors has important practical benefits, as it can be used to measure components in complex biological samples where the sensor concentration cannot be easily controlled and background fluorescence is always present.
Despite these advantages of FRET-based sensors for quantitative visualization, small structural changes with incomplete domain motion-transfer to the fluorescent proteins produce inherently weak signal intensity. This weak signal limits the application of FRET-based sensors for in vitro or in vivo analysis6. Consequently, most FRET biosensors require the use of expensive and highly sensitive equipment. Previously, we developed an inexpensive and portable FRET analyzer with capabilities similar to those of the existing fluorescence analyzers7. In this device, inexpensive 405-nm band ultraviolet light-emitting diode (LED) was used as the light source to cause excitation of the fluorescence signal, replacing an expensive lamp or laser. The detection system of the analyzer efficiently focuses the dissipating fluorescence signal onto two photodetectors with a silicon photodiode. In a more recent study, we showed that optimization of detection temperature at 50 - 55 &#176;C could significantly magnify the ratiometric FRET signal8. This temperature-specific signal enhancement, along with the custom-made FRET analyzer, enables the use of FRET sensors in more general diagnostic applications with rapid and high sensitivity.
In this protocol, we demonstrated the general applicability of the FRET analyzer under optimal FRET temperature conditions by quantifying the sugar content of commercially-available beverages. This protocol provides the details of the FRET device operation, as well as a brief description of sensor and sample preparation. We anticipate that this report will promote the potential application of the portable analyzer in small-scale laboratory environments and provide a foundation for further development of an inexpensive on-site diagnostic device with FRET-based biosensors.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">LB</td>
			<td style="width:191px;">BD</td>
			<td style="width:119px;">#244620</td>
			<td style="width:760px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">isopropyl &#946;-D-thiogalactoside (IPTG)</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:119px;">I6758</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Ampicillin</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:119px;">A9518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Tri-HCl</td>
			<td style="width:191px;">Bioneer</td>
			<td style="width:119px;">C-9006-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">PMSF</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:119px;">78830</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">EDTA</td>
			<td style="width:191px;">Bioneer</td>
			<td style="width:119px;">C-9007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">DTT</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:119px;">D0632</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">NaCl</td>
			<td style="width:191px;">Junsei</td>
			<td style="width:119px;">19015-0350</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:227px;">phosphate-buffered saline (PBS)</td>
			<td style="width:191px;">Gibco</td>
			<td style="width:119px;">70011-044</td>
			<td>0.8% NaCl, 0.02% KCl, 0.0144% Na2HPO4, 0.024% KH2OP4, pH 7.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">SOC</td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td>2% tryptone, 0.5% Yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MGCl2, 20 mM Glucose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Resource Q</td>
			<td>Amersham Biosciences</td>
			<td style="width:119px;">17-1177-01</td>
			<td>6 &#215; 30 mm anion-exchange chromatography column&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">HisTrap HP1</td>
			<td>Amersham Biosciences</td>
			<td style="width:119px;">29-0510-21</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Quartz cuvette</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:119px;">Z802875</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">AK&#196;KTAFPLC</td>
			<td>Amersham Biosciences</td>
			<td style="width:119px;">18-1900-26</td>
			<td>a fast protein liquid chromatography (FPLC)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cary Eclipse</td>
			<td>VarianInc</td>
			<td style="width:119px;"></td>
			<td>a fluorescence spectrophotometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">VICTOR&#160;&#160;</td>
			<td style="width:191px;">PerkinElmer</td>
			<td style="width:119px;">2030-0050</td>
			<td>a multilabel plate reader</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">E. coli JM109 (DE3)</td>
			<td style="width:191px;">Promega</td>
			<td style="width:119px;"></td>
			<td>Electrocompetent cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">A (Beverage)</td>
			<td style="width:191px;">Korea Yakult Co. (Korea)</td>
			<td style="width:119px;">Birak</td>
			<td>Fermented drinks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">B (Beverage)</td>
			<td style="width:191px;">Lotte Foods (Korea)</td>
			<td style="width:119px;">Epro</td>
			<td>Soft drink</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">C (Beverage)</td>
			<td style="width:191px;">Lotte Foods (Korea)</td>
			<td style="width:119px;">Getoray</td>
			<td>Sports drink</td>
		</tr>
	</tbody>
</table>

highly sensitive and rapid fluorescence detection with a portable fret analyzer

Recent improvements in F&#246;rster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and amino acids. However, the innate weak signal intensity of FRET sensors is a major challenge that prevents their application in various fields and makes the use of expensive, high-end fluorometers necessary. Previously, we built a cost-effective, high-performance FRET analyzer that can specifically measure the ratio of two emission wavelength bands (530 and 480 nm) to achieve high detection sensitivity. More recently, it was discovered that FRET sensors with bacterial periplasmic binding proteins detect ligands with maximum sensitivity in the critical temperature range of 50 - 55 &#176;C. This report describes a protocol for assessing sugar content in commercially-available beverage samples using our portable FRET analyzer with a temperature-specific FRET sensor. Our results showed that the additional preheating process of the FRET sensor significantly increases the FRET ratio signal, to enable more accurate measurement of sugar content. The custom-made FRET analyzer and sensor were successfully applied to quantify the sugar content in three types of commercial beverages. We anticipate that further size reduction and performance enhancement of the equipment will facilitate the use of hand-held analyzers in environments where high-end equipment is not available.

To perform quantitative analysis of sugar content using the FRET analyzer, it is necessary to build a fitted curve estimating the target sugar concentration from the observed FRET ratio. Let r define the ratio of the emission intensity of CFP at 480 nm and the emission intensity of YFP generated at 530 nm (Eq. 1).
<img alt="Equation 1" src="/files/ftp_upload/54144/54144equation1.jpg" />
<p class="jove_content" fo:ke...

Watch this Scientific Journal Video about Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer at JoVE.com

Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer

This protocol describes the rapid and highly sensitive quantification of F&#246;rster resonance energy transfer (FRET) sensor data using a custom-made portable FRET analyzer. The device was used to detect maltose within a critical temperature range that maximizes detection sensitivity, enabling practical and efficient assessment of sugar content.

Recent improvements in F&#246;rster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and ...

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