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).</li>
	<li>Harvest the cells in a 50-ml conical tube by centrifugation at 1,000 &#215; g for 20 min at 4 &#176;C.</li>
	<li>Resuspend the pellet quickly in each tube with 50 ml ice-cold distilled water (DW) and centrifuge at 1,000&#160;&#215; g for 20 min at 4 &#176;C.</li>
	<li>Resuspend the pellet in 50 &#956;l of ice-cold DW with 10% (v/v) glycerol by gently swirling until the solution (electrocompetent cells) reaches an OD600 of 100.</li>
	<li>Place the mixture of electrocompetent cells (50 &#956;l of the cells at an OD600 of 100) and 10 ng of the plasmid pET21a(+)-CFP-MBP-YFP-His6 in an ice-cold electroporation cuvette in an electroporation device and electroporate the mixture (18 kV/cm, 25 &#181;F).</li>
	<li>Quickly add 1 ml SOC medium to the cuvette and resuspend the cells gently, followed by recovery at 37 &#176;C for 1 hr with gentle shaking in a 15 ml round-bottom tube.</li>
	<li>Spread the cells on an LB plate containing 100 &#181;g/ml ampicillin and incubate at 37 &#176;C for 12 hr.</li>
	<li>Isolate a single colony using a loop and inoculate the colony in 10 ml of LB containing 100 &#181;g/ml ampicillin at 37 &#176;C in a shaker for 12 hr.</li>
	<li>Add 5 ml of the seed culture to 500 ml of LB containing 100 &#181;g/ml ampicillin and incubate the culture in a 37 &#176;C shaking incubator.</li>
	<li>Add 0.5 mM isopropyl &#946;-d-thiogalactoside (IPTG) when the OD600 reaches 0.5 and incubate the culture in a 37 &#176;C shaking incubator for 24 hr.</li>
	<li>Centrifuge the cells at 4,500 &#215; g for 20 min (4 &#176;C) and gently remove supernatant.</li>
	<li>Resuspend the pellet in 5 mL binding buffer (20 mM Tris-HCl, pH 8.0, 1 mM PMSF, 0.5 mM EDTA, and 1 mM DTT).</li>
	<li>Sonicate the cells on ice with six 10-sec bursts at 200-300 W, following each burst with 10 sec of cooling.</li>
	<li>Centrifuge the lysate at 10,000 &#215; g for 30 min at 4 &#176;C to pellet the cellular debris. Transfer supernatant (soluble protein) into a new collection tube.</li>
	<li>To achieve affinity purification of the FRET sensor proteins, load 4 ml of the cleared cell lysate onto a Ni-NTA affinity column (5-ml volume) and perform a chromatography assay using fast protein liquid chromatography (FPLC)18.</li>
	<li>Wash the column once with five column volumes of wash buffer I (50 mM phosphate buffer, 300 mM sodium chloride, 10 mM imidazole, pH 7.0).</li>
	<li>Repeat the wash step with five column volumes of wash buffer II (50 mM phosphate buffer, 300 mM sodium chloride, 20 mM imidazole, pH 7.0).</li>
	<li>Elute the sensor protein with five column volumes of elution buffer (50 mM phosphate buffer, 300 mM sodium chloride, 500 mM imidazole, pH 7.0).</li>
	<li>To concentrate and desalt the eluted sample, fill concentrator (membrane size of 10,000 MW) with up to 20 ml of sample and centrifuge for 10 min at 3,000 &#215; g. Refill concentrator with 0.8% phosphate-buffered saline (PBS). Repeat this step twice, first filling the concentrator with 20 ml of sample, and then refilling with PBS.</li>
	<li>Recover the concentrated and de-salted sensor protein and store it at -80 &#176;C.</li>
</ol>
2. Measurement of Sugar Content using the FRET Analyzer
NOTE: The details of the FRET analyzer construction were described in our previous work7.
<ol>
	<li>Prepare a detection solution of 0.8% PBS containing 0.2 &#181;M of the sensor proteins.</li>
	<li>Turn on the FRET analyzer. Press the &#34;UP&#34; button for 2 sec to calibrate the optimal temperature. Set the temperature to 53 &#176;C using the &#34;UP&#34; and &#34;DOWN&#34; buttons and press the &#34;SET&#34; button.</li>
	<li>For the calibration, press and hold the &#34;UP&#34; and &#34;DOWN&#34; buttons simultaneously for 2 sec. Confirm that the LED panel displays &#34;CALIB&#34; and press the &#34;SET&#34; button.</li>
	<li>Place a 12.5 &#215; 12.5 &#215; 45 mm (length &#215; width &#215; height) rectangular parallelepiped vessel (cuvette) containing only PBS buffer into a cuvette holder of the analyzer and press the &#34;SET&#34; button.</li>
	<li>Replace the cuvette with one containing only the detection solution (see 2.1) without sugar (maltose/sucrose) and press the &#34;SET&#34; button to calibrate the baseline.</li>
	<li>Replace the cuvette with one containing the detection solution with 10 mM sugar and press the &#34;SET&#34; button.</li>
	<li>To determine the sugar content of a beverage sample, put 1 ml beverage sample in a 1.5-ml microcentrifuge tube and centrifuge at 16,000 &#215; g for 1 min. 
	NOTE: FRET sensor-based fluorescence measurement has the advantage of not requiring special pre-treatment of the sample because only 1% (v/v) of the sample is included in the total volume. However, we recommend removing any material that may affect the fluorescence measurement (e.g., cells, insoluble particles, lipid, fat, or any material with autofluorescence). In addition, if a strong acid, strong base, cleaning agent (detergent), or emulsifying agent (emulsifier) is present at a high concentration and may affect the properties of the FRET biological sensor, it should be removed using an organic solvent or a neutralizer. For example, when dairy fat and emulsifiers are eliminated from frozen snacks, the samples are centrifuged in a microfuge tube at 16,000 &#215; g for 30 min, and the liquid between the bottom sediment and the top layer of dairy fat is extracted. An equal amount of hexane is then added, followed by centrifugation at 15,000 &#215; g for 30 min to eliminate lipids.</li>
	<li>Remove the supernatant with a 1-ml syringe and filter it through a syringe filter (pore size 0.2 &#181;m).</li>
	<li>Place 0.1 ml filtered beverage sample in a 1.5-ml microcentrifuge tube containing 0.9 ml PBS and vortex gently. 
	NOTE: It is critical to dilute the beverage sample properly. In this case, 1,000-fold dilution was performed so that the sugar concentration would fall within the dynamic range of the device. We recommend estimating the target sugar concentration in advance by referring to the sugar content in the label of the beverage.</li>
	<li>Add 5 &#181;l of the diluted beverage sample (1%, v/v) to a cuvette containing 0.495 ml of the detection solution.</li>
	<li>Place the cuvette in a cuvette holder of the FRET analyzer and preheat the sample solution to 53 &#176;C.</li>
	<li>Press the &#34;SET&#34; button to measure the sugar content. 
	Note It is possible to evaluate the FRET measurement using a multilabel plate reader or a fluorescence spectrophotometer equipped with a Peltier device for temperature control by reading the ratio at 488/535 nm7,8. For sucrose detection, follow the steps from 1.1 to 2.12 with a CSY-LH sensor2.</li>
</ol>

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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. 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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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The authors have nothing to disclose.

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 wavelength bands (530 nm and 480 nm) in an optimal temperature range for FRET sensors. Its sensitivity and intensity in detecting various sugars were superior to those of a fluorescence spectrophotometer device7.
The main goal of this protocol is to support the wide applicability of the FRET-based sensors with the custom-made FRET analyzer. While the analyzer indirectly measures sugar content via FRET sensors, it is clear that the device incorporates a number of the benefits of FRET sensors, including widely extendable genetically engineered ligand specificity, modular design, sensor concentration-independent signals, and accurate targeting of subcellular small molecules. FRET sensors are actually used to detect a wide range of small molecules, including ions9, heme10, and others. Moreover, more than 20 types of FRET constructs can be easily found and ordered through the nonprofit depository AddGene11.
Despite the wide applicability of the FRET analyzer, there are two main issues with the operation of the device. First, because operation of the device is relatively simple, sample preprocessing is the critical step that affects quality of detection, except in cases of device malfunction. In this protocol, one step (sample dilution) was sufficient to process liquid samples that were clearly transparent and contained no insoluble particles. However, other samples may require additional processing to remove insoluble materials, such as cellular or lipid components. Any autofluorescent particles that can affect the FRET signal should also be removed, as noted following Step 2.7. Secondly, quality control and connectivity interfacing with hospital information systems need to be addressed, as with all types of point-of-care testing (POCT) tools12. Since the signal quality of the FRET analyzer largely depends on the quality of the FRET sensor and on the preprocessing steps, regular quality control checks are required to ensure that measurements remain within the standard signal range for regular quality control data analysis. The FRET sensor stability and storage period, both of which are essential for further reliable applications, should be investigated during quality control checks. Creating guidelines and developing appropriate software can also address the connectivity limitation. Current versions of the FRET analyzer are equipped with RS232 connectivity for remote command line control, but wireless communication may be a feature of the next version of the analyzer, which will have an improved interface for hospital information systems.
However, FRET sensors have been engineered for substrate specificity, an approach that normally allows broader specificity2. Consequently, the FRET signal may encounter unintended interference from other ingredients, including other types of sugars in commercial beverages. Further investigations should explore how FRET sensors respond to various sugar mixtures to accurately quantify the amount of sugar. Collaboration with companies that produce the beverages will help confirm the sugar content to calibrate of the FRET analyzer.
It is anticipated that the proposed portable FRET device with various FRET sensors will be used in POCT applications. POCT is used for assessing pregnancy, blood glucose levels, biomarker proteins, infectious bacteria, and infectious viruses. POCT methods have rapid turnaround times and generally exhibit low error rates owing to the small number of processing steps. These are important advantages of POCT over the central laboratory testing approach. Hand-held portable POCT devices, such as the device described herein, have attracted increasing attention because of their potential applications in food assessment and blood sugar monitoring. In particular, glucose monitoring of blood samples in patients with diabetes requires a rapid, accurate, and cost-effective POCT method13. After the Ames research team developed the first blood glucose test strip in 1965 (using a strip that contains glucose oxidase), several technologies were proposed for blood glucose monitoring purposes12. The FRET analyzer is also available to detect glucose in blood samples with appropriate preprocessing of blood and periplasmic glucose-binding protein (MglB)14-based FRET protein.
Simple, rapid methods for food quality assessment are needed. The consumption of sugar-containing beverages is associated with a variety of diseases and syndromes, such as increased body mass index in childhood15, pediatric obesity16, and risk of stroke17. Understanding this connection necessitates accurate measurement of sugar components in beverages. Therefore, the glucose and fructose concentrations of beverages are of interest to scientists concerned with human health. This protocol demonstrates the highly sensitive performance of the FRET analyzer with optimal temperature control. The device may be used with various FRET sensors to detect various small molecules- including glucose and fructose14,15. Our portable and rechargeable device, which has a battery life of 10 - 20 hr, depending on the heating protocol, is applicable for POCT. Its simple operational protocol makes the device easy to use and eliminates the need for complicated staff training. With technical improvements, including reduction of equipment size, minimization of pretreatment steps, and identification of practical requirements for field use, this device will promote FRET-based research development in small-scale laboratory environments.

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><tbody><tr><td>LB</td><td>BD</td><td>#244620</td><td></td></tr><tr><td>isopropyl &amp;beta;-D-thiogalactoside (IPTG)</td><td>Sigma</td><td>I6758</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma</td><td>A9518</td><td></td></tr><tr><td>Tri-HCl</td><td>Bioneer</td><td>C-9006-1</td><td></td></tr><tr><td>PMSF</td><td>Sigma</td><td>78830</td><td></td></tr><tr><td>EDTA</td><td>Bioneer</td><td>C-9007</td><td></td></tr><tr><td>DTT</td><td>Sigma</td><td>D0632</td><td></td></tr><tr><td>NaCl</td><td>Junsei</td><td>19015-0350</td><td></td></tr><tr><td>phosphate-buffered saline (PBS)</td><td>Gibco</td><td>70011-044</td><td>0.8% NaCl, 0.02% KCl, 0.0144% Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 0.024% KH&lt;sub&gt;2&lt;/sub&gt;OP&lt;sub&gt;4, &lt;/sub&gt;pH 7.4</td></tr><tr><td>SOC</td><td></td><td></td><td>2% tryptone, 0.5% Yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MGCl&lt;sub&gt;2&lt;/sub&gt;, 20 mM Glucose</td></tr><tr><td>Resource Q</td><td>Amersham Biosciences</td><td>17-1177-01</td><td>6 &amp;times; 30 mm anion-exchange chromatography column&amp;nbsp;</td></tr><tr><td>HisTrap HP1</td><td>Amersham Biosciences</td><td>29-0510-21</td><td></td></tr><tr><td>Quartz cuvette</td><td>Sigma</td><td>Z802875</td><td></td></tr><tr><td>AK&amp;Auml;KTAFPLC</td><td>Amersham Biosciences</td><td>18-1900-26</td><td>a fast protein liquid chromatography (FPLC)</td></tr><tr><td>Cary Eclipse</td><td>VarianInc</td><td></td><td>a fluorescence spectrophotometer</td></tr><tr><td>VICTOR&amp;nbsp;&amp;nbsp;</td><td>PerkinElmer</td><td>2030-0050</td><td>a multilabel plate reader</td></tr><tr><td>&lt;i&gt;E. coli&lt;/i&gt; JM109 (DE3)</td><td>Promega</td><td></td><td>Electrocompetent cells</td></tr><tr><td>A (Beverage)</td><td>Korea Yakult Co. (Korea)</td><td>Birak</td><td>Fermented drinks</td></tr><tr><td>B (Beverage)</td><td>Lotte Foods (Korea)</td><td>Epro</td><td>Soft drink</td></tr><tr><td>C (Beverage)</td><td>Lotte Foods (Korea)</td><td>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" />
The dose-response curve of the FRET biosensor (CMY-BII at 53 &#176;C) can be generated by observing the FRET ratio, r, at different sugar concentrations. The curve can then be expressed as an S-shaped sigmoidal curve as follows:
<img alt="Equation 2" src="/files/ftp_upload/54144/54144equation2.jpg" />
where rmax and rmin represent the signal ratio with sugar concentrations of 0 and saturated (1,000 &#956;M), respectively; x0 represents the sugar concentration at 50% response; and p represents the slope of the response, which is close to 1 or -1. In the present study, rmax, rmin, x0, and p are 4.256, 2.672, 71.779, and 1, respectively. The concentration range from 1 &#181;M to 1000 &#181;M was used in the model fitting.
Using Equations 1 and 2, the sugar content of commercially- available beverages was quantified with the FRET analyzer. Two maltose FRET sensors were examined to test the signal, r, depending on various temperatures2,8. The first FRET sensor, CMY-0, is a basic FRET-based sensor consisting of CFP, maltose-binding protein (MBP), and YFP, with no linker peptides. The second sensor, CMY-BII, has a Ser-Arg linker between MBP and the two fluorescence proteins2. As Figure 1A shows, CMY-0 is not observed at measurement temperatures below 50 &#176;C, as there is no signal difference between 0 and 1 mM maltose concentration. The signal differences of both FRET sensors were maximized between 50 and 55 &#176;C (Figure 1)8. In order to quantify the sugar content of the three types of commercially-available beverages, a dose-response curve of the CMY-BII sensor at 53-&#176;C was generated (Figure 2A) and the maltose content of the three samples was identified by converting the FRET ratio into the maltose concentration.
As sample A is made of grains such as rice and barley, which are important maltose sources, the sample was expected to contain relatively high maltose content (average 11.892 g/235 ml) (Figure 2A). In contrast, sample C is a sports drink that had the lowest maltose (0.29 g/250 ml) among the three beverages. These results suggest that the FRET analyzer can be used at optimal temperatures to maximize the quantification of sugar content, eliminating the need for an expensive high-end FRET-detection device.
<img alt="Figure 1" src="/files/ftp_upload/54144/54144fig1.jpg" /> 
Figure 1. FRET Signal Difference Between 0 and 1 mM Maltose using the FRET Analyzer at Various Temperatures. (A) The CMY-0 sensor showed no signal difference at different maltose concentrations at temperatures below 50 &#176;C. (B) The CMY-BII sensor was able to distinguish the FRET signal difference between 0 and 1 mM maltose in a wide range of temperatures. In both cases, the signal difference dramatically increased in a specific temperature range (50 - 55 &#176;C). The error bars represent standard deviation. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54144/54144fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54144/54144fig2.jpg" /> 
Figure 2. Maltose Content Quantification in Three Commercially-available Beverages. (A) A dose-response curve for CMY-BII. (B) Maltose content of three beverage samples was quantified. Note that &#34;Total sugar&#34; indicates the quantity of all sugar (including maltose) reported by the beverage manufacturer on the beverage label. The error bar indicates the standard deviation. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54144/54144fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

highly-sensitive-rapid-fluorescence-detection-with-portable-fret

Nanyang Technological University

Research

JoVE Journal

Biochemistry

9.0K Views.  Korea Research Institute of Bioscience and Biotechnology.  Dit protocol beschrijft een snelle en gevoelige kwantificatie van Förster resonantie energieoverdracht (FRET) sensorgegevens met een maat draagbare FRET analyzer. De inrichting werd gebruikt voor maltose binnen een kritisch temperatuurtraject dat detectiegevoeligheid maximaliseert, zodat praktische en efficiënte evaluatie van het suikergehalte detecteren. 

Video: Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, such as nucleic acids and proteins. Single molecule FRET (sm-FRET) measurements on immobilized molecules permit long observations of the system -effectively until both dyes photobleach- resulting in time-traces that report on biomolecular dynamics with a broad range of timescales from milliseconds to minutes. To facilitate the acquisition of large number of traces for statistical analyses, the process must be automated and the sample environment should be tightly controlled over the entire measurement time (~12 hours). This is accomplished using an automated scanning confocal microscope that allows the interrogation of thousands of single molecules overnight, and a microfluidic cell that permits the controlled exchange of buffer, with restricted oxygen content and maintains a constant temperature throughout the entire measuring period. Here we show how to assemble the microfluidic device and how to activate its surface for DNA immobilization. Then we explain how to prepare a buffer to maximize the photostability and lifetime of the fluorophores. Finally, we show the steps involved in preparing the setup for the automated acquisition of time-resolved single molecule FRET traces of DNA molecules.

In this article we describe how we obtain FRET traces from individual DNA molecules immobilized to a surface using an automated scanning confocal microscope.

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, ...

Biology

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called mechanotransduction. A crucial step in mechanotransduction is the transmission of forces between the external and internal environments. To transmit forces, there must be a sustained, unbroken physical linkage created by a series of protein-protein interactions. For a given protein-protein interaction, mechanical load can either have no effect on the interaction, lead to faster disassociation of the interaction, or even stabilize the interaction. Understanding how molecular load dictates protein turnover in living cells can provide valuable information about the mechanical state of a protein, in turn elucidating its role in mechanotransduction. Existing techniques for measuring force-sensitive protein dynamics either lack direct measurements of protein load or rely on the measurements performed outside of the cellular context. Here, we describe a protocol for the F&#246;rster resonance energy transfer-fluorescence recovery after photobleaching (FRET-FRAP) technique, which enables the measurement of force-sensitive protein dynamics within living cells. This technique is potentially applicable to any FRET-based tension sensor, facilitating the study of force-sensitive protein dynamics in variety of subcellular structures and in different cell types.

Here, we present a protocol for the simultaneous use of F&#246;rster resonance energy transfer-based tension sensors to measure protein load and fluorescence recovery after photobleaching to measure protein dynamics enabling the measurement of force-sensitive protein dynamics within living cells.

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called ...

Bioengineering

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Automated Two-dimensional Spatiotemporal Analysis of Mobile Single-molecule FRET Probes

Single-molecule F&#246;rster resonance energy transfer (smFRET) is a versatile technique reporting on distances in the sub-nanometer to nanometer range. It has been used in a wide range of biophysical and molecular biological experiments, including the measurement of molecular forces, characterization of conformational dynamics of biomolecules, observation of intracellular colocalization of proteins, and determination of receptor-ligand interaction times. In a widefield microscopy configuration, experiments are typically performed using surface-immobilized probes. Here, a method combining single-molecule tracking with alternating excitation (ALEX) smFRET experiments is presented, permitting the acquisition of smFRET time traces of surface-bound, yet mobile probes in plasma membranes or glass-supported lipid bilayers. For the analysis of recorded data, an automated, open-source software collection was developed supporting (i) the localization of fluorescent signals, (ii) single-particle tracking, (iii) determination of FRET-related quantities including correction factors, (iv) stringent verification of smFRET traces, and (v) intuitive presentation of the results. The generated data can conveniently be used as input for further exploration via specialized software, e.g., for the assessment of the diffusional behavior of probes or the investigation of FRET transitions.

This article presents a method for spatiotemporal analysis of mobile, single-molecule F&#246;rster resonance energy transfer (smFRET)-based probes using widefield fluorescence microscopy. The newly developed software toolkit allows the determination of smFRET time traces of moving probes, including the correct FRET efficiency and the molecular positions, as functions of time.

Single-molecule F&#246;rster resonance energy transfer (smFRET) is a versatile technique reporting on distances in the sub-nanometer to nanometer range. ...

Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins such as GFP to study their expression, localization and function. However, GFP and its derivatives are significantly larger and less photostable than organic fluorophores generally used for in vitro experiments, and this can limit the scope of investigation. 
We recently introduced a straightforward, versatile and high-throughput method based on electroporation, allowing the internalization of biomolecules labeled with organic fluorophores into living microorganisms. Here we describe how to use electroporation to internalize labeled DNA fragments or proteins into Escherichia coli and Saccharomyces cerevisi&#230;, how to quantify the number of internalized molecules using fluorescence microscopy, and how to quantify the viability of electroporated cells. Data can be acquired at the single-cell or single-molecule level using fluorescence or FRET. The possibility of internalizing non-labeled molecules that trigger a physiological observable response in vivo is also presented. Finally, strategies of optimization of the protocol for specific biological systems are discussed.

Studies of biomolecules in vivo are crucial for understanding molecular function in a biological context. Here we describe a novel method allowing the internalization of fluorescent biomolecules, such as DNA or proteins, into living microorganisms. Analysis of in vivo data recorded by fluorescence microscopy is also presented and discussed.

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in &#181;Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in &#181;Manager and data analysis is undertaken in FLIMfit.

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...

Real-Time Monitoring of Aurora kinase A Activation using Conformational FRET Biosensors in Live Cells

Epithelial cancers are often hallmarked by the overexpression of the Ser/Thr kinase Aurora A/AURKA. AURKA is a multifunctional protein that activates upon its autophosphorylation on Thr288. AURKA abundance peaks in mitosis, where it controls the stability and the fidelity of the mitotic spindle, and the overall efficiency of mitosis. Although well characterized at the structural level, a consistent monitoring of the activation of AURKA throughout the cell cycle is lacking. A possible solution consists in using genetically-encoded F&#246;rster&#8217;s Resonance Energy Transfer (FRET) biosensors to gain insight into the autophosphorylation of AURKA with sufficient spatiotemporal resolution. Here, we describe a protocol to engineer FRET biosensors detecting Thr288 autophosphorylation, and how to follow this modification during mitosis. First, we provide an overview of possible donor/acceptor FRET pairs, and we show possible cloning and insertion methods of AURKA FRET biosensors in mammalian cells. Then, we provide a step-by-step analysis for rapid FRET measurements by fluorescence lifetime imaging microscopy (FLIM) on a custom-built setup. However, this protocol is also applicable to alternative commercial solutions available. We conclude by considering the most appropriate FRET controls for an AURKA-based biosensor, and by highlighting potential future improvements to further increase the sensitivity of this tool.

The activation of the multifunctional Ser/Thr kinase AURKA is hallmarked by its autophosphorylation on Thr288. Fluorescent probes relying on FRET can discriminate between its inactive and activated states. Here, we illustrate some strategies for probe engineering, together with a rapid FRET protocol to follow the kinase activation throughout mitosis.

Epithelial cancers are often hallmarked by the overexpression of the Ser/Thr kinase Aurora A/AURKA. AURKA is a multifunctional protein that activates upon ...

Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission

F&#246;rster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the distance and orientation of the molecules as well as the extent of overlap between the donor emission and acceptor absorption spectra. FRET permits to study the interaction of proteins in the living cell over time and in different subcellular compartments. Different intensity-based algorithms to measure FRET using microscopy have been described in the literature. Here, a protocol and an algorithm are provided to quantify FRET efficiency based on measuring both the sensitized emission of the acceptor and quenching of the donor molecule. The quantification of ratiometric FRET in the living cell not only requires the determination of the crosstalk (spectral spill-over, or bleed-through) of the fluorescent proteins but also the detection efficiency of the microscopic setup. The protocol provided here details how to assess these critical parameters.

F&#246;rster Resonance Energy Transfer (FRET) between two fluorophore molecules can be used for studying protein interactions in the living cell. Here, a protocol is provided as to how to measure FRET in live cells by detecting sensitized emission of the acceptor and quenching of the donor molecule using confocal laser scanning microscopy.

F&#246;rster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the ...

Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer

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