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>
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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. 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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.

<table border="0" cellpadding="0" cellspacing="0" width="1297">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<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 ...

The overall goal of this protocol is to demonstrate the general applicability of the FRET analyzer under optimal conditions by quantifying the sugar content of commercially available beverages. This method can help answer key questions in the sensing and monitoring field of small molecules, such as sugar. The main advantage of this technique is that it can efficiently monitor a fresh signal responsing to the small molecules without expensive, high-end fluorometers.Demonstrating the procedure will be Dr.Han from our laboratory. First, prepare the maltose or sucrose-specific cyan and yellow fluorescent biosensor plasmid. Then innoculate five milliliters of Luria broth with a single colony of DE3 E.Coli, and incubate the culture at 37 degrees Celsius in a shaking incubator for 16 hours.Next, place one millileter of the culture into a flask containing 100 milliliters of LB.Incubate the flask at 37 degrees Celsius in a shaking incubator until the optical density at 600 nanometers is about 0.5. Centrifuge the culture for 20 minutes at four degrees Celsius. Quickly resuspend the pellet in 50 milliliters of ice-cold distilled water, and centrifuge for 20 minutes again.Resuspend the pellet in 50 microliters of a 10 volume percent solution of glycerol and ice-cold distilled water. Gently swirl the mixture until the optical density at 600 nanometers reaches 100. In an ice-cold electroporation cuvette, combine the mixture with 10 nanograms of CMY plasmid.Electroporate the mixture. In the cuvette, resuspend the cells in one millileter of SOC medium, and then transfer them to a 15 milliliter round-bottom tube. Recover the cells by gently shaking at 37 degrees for one hour.Spread the cells on an LB agar plate with 100 micrograms per a milliliter ampicillin. Incubate the cells at 37 degrees for 12 hours. Using a loop, transfer a single colony to 10 milliliters of LB containing 100 micrograms per milliliter ampicillin.Incubate the mixture at 37 degrees while shaking for 12 hours to create the seed culture. Transfer five milliliters of the seed culture to 500 milliliters of LB with ampicillin, and incubate the cells at 37 degrees in a shaking incubator. Once the optical density at 600 nanometers reaches 0.5, add IPTG to make a final concentration of 0.5 millimolar.After incubating the culture for 24 hours, centrifuge the cells for 20 minutes. Carefully remove the supernatant and resuspend the pellet in five milliliters of binding buffer. Sonicate the cells on ice for 10 seconds, and then cool the cells for 10 seconds.Repeat the sonication and cooling five more times. Centrifuge the lysate for 30 minutes. Transfer the supernatant to another collection tube.Purify the supernatant on a nickel NTA affinity column. Fill a 10, 000 milliwatt concentrator with 20 milliliters of the purified sample. Centrifuge the sample for 10 minutes, and then refill the concentrator with 0.8%phosphate buffered saline.Repeat this process with the rest of the sample in this way, 20 milliliters at a time, until all of the protein has been concentrated and desalted. Store the purified FRET biosensor at 80 degrees Celsius. Prepare a 0.2 micromolar solution of CMY biosensor protein and 0.8%PBS as the detection solution.Turn on a handheld FRET analyzer. Set the analysis temperature to 53 degrees Celsius. Then set the instrument to calibration mode.Fill a cuvette with 0.8%PBS as a background. Preheat the sample to 53 degrees Celsius in the analyzer, and then set the background. Next, fill a cuvette with detection solution.Place the cuvette into the analyzer and set the baseline. Replace the cuvette with one containing detection solution and one millimolar sugar. Set the intensity at saturation to finish the calibration.To begin preparing a beverage sample for sugar content analysis, using a 1.5 milliliter microcentrifuge tube, centrifuge one milliliter of the beverage. Remove the supernatant with a one milliliter syringe and filter the supernatant though a 0.2 micrometer syringe filter. Combine 0.1 milliliters of filtrate and 0.9 milliliters of 0.8%PBS in a 1.5 milliliter microcentrifuge tube.Gently vortex the mixture to ensure complete dilution. Place five microliters of the diluted beverage sample and 495 microliters of detection solution in a cuvette. Place the cuvette in the analyzer and preheat the sample to 53 degrees Celsius.Press the set button to measure the sugar content of the sample. In this method, the FRET efficiency is measured by the ratio of the emission intensity of the yellow fluorescent protein to the cyan fluorescent protein in the biosensor. The optimal measurement temperature for these CMY biosensors is between 50 and 55 degrees Celsius, as the difference in the emission intensity ratio between a control solution and a saturated solution of maltose is greatest.The CMY-BII biosensor was used to evalute the maltose content of three beverages at 53 degrees. A dose response curve is generated from obtaining this ratio at various maltose concentrations at a given temperature. The emission ratio is related to the maltose concentration using the values obtained during calibration.The measured maltose contents are shown with the total sugar contents reported by the beverage manufacturers. Beverage A contained maltose sources such as rice and barley. Correspondingly, the observed amount of maltose accounts for about half of the total reported sugar content.Beverage C, a sports drink, had a very low maltose content. While attempting this procedure, it's important to remember to operate the device and FRET sensors between 50 and 55 degrees Celsius. After watching this video, you should have a good understanding of how to quantify such a small molecules with a custom-made handheld FRET analyzer.

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Research

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Biochemistry

8.9K 次观看.  Korea Research Institute of Bioscience and Biotechnology. The overall goal of this protocol is to demonstrate the general applicability of the FRET analyzer under optimal conditions by quantifying the sugar content of commercially available beverages. This method can help answer key questions in the sensing and monitoring field of small molecules, such as sugar. The main advantage of this technique is that it can efficiently monitor a fresh signal responsing to the small molecules without expensive, high-end fluorometers.Demonstrating the pro...