Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer

Summary

This protocol describes the rapid and highly sensitive quantification of Förster 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.

Abstract

Recent improvements in Förster 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 °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.

Introduction

Förster resonance energy transfer (FRET) has been widely used as a biometric sensor to detect small molecules such as sugars, calcium ions, and amino acids^1-4. 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 °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.

Protocol

1. Preparation of Biosensor

1. Construct the plasmid pET21a(+)-CFP-MBP-YFP-His6 by following the previously-established protocol².
2. Inoculate 5 ml of Luria broth (LB) with a single colony of an Escherichia coli DE3 strain and incubate at 37 °C for 16 hr with shaking.
3. Transfer 1 ml of the O/N culture into a 500-ml flask containing 100 ml LB and incubate at 37 °C in a shaking incubator until the optical density at 600 nm (OD₆₀₀) reaches 0.5 (about 3 hr).
4. Harvest the cells in a 50-ml conical tube by centrifugation at 1,000 × g for 20 min at 4 °C.
5. Resuspend the pellet quickly in each tube with 50 ml ice-cold distilled water (DW) and centrifuge at 1,000 × g for 20 min at 4 °C.
6. Resuspend the pellet in 50 μl of ice-cold DW with 10% (v/v) glycerol by gently swirling until the solution (electrocompetent cells) reaches an OD₆₀₀ of 100.
7. Place the mixture of electrocompetent cells (50 μl 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 µF).
8. Quickly add 1 ml SOC medium to the cuvette and resuspend the cells gently, followed by recovery at 37 °C for 1 hr with gentle shaking in a 15 ml round-bottom tube.
9. Spread the cells on an LB plate containing 100 µg/ml ampicillin and incubate at 37 °C for 12 hr.
10. Isolate a single colony using a loop and inoculate the colony in 10 ml of LB containing 100 µg/ml ampicillin at 37 °C in a shaker for 12 hr.
11. Add 5 ml of the seed culture to 500 ml of LB containing 100 µg/ml ampicillin and incubate the culture in a 37 °C shaking incubator.
12. Add 0.5 mM isopropyl β-d-thiogalactoside (IPTG) when the OD₆₀₀ reaches 0.5 and incubate the culture in a 37 °C shaking incubator for 24 hr.
13. Centrifuge the cells at 4,500 × g for 20 min (4 °C) and gently remove supernatant.
14. 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).
15. Sonicate the cells on ice with six 10-sec bursts at 200-300 W, following each burst with 10 sec of cooling.
16. Centrifuge the lysate at 10,000 × g for 30 min at 4 °C to pellet the cellular debris. Transfer supernatant (soluble protein) into a new collection tube.
17. 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.
18. 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).
19. 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).
20. 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).
21. 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 × 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.
22. Recover the concentrated and de-salted sensor protein and store it at -80 °C.

2. Measurement of Sugar Content using the FRET Analyzer

NOTE: The details of the FRET analyzer construction were described in our previous work⁷.

1. Prepare a detection solution of 0.8% PBS containing 0.2 µM of the sensor proteins.
2. Turn on the FRET analyzer. Press the "UP" button for 2 sec to calibrate the optimal temperature. Set the temperature to 53 °C using the "UP" and "DOWN" buttons and press the "SET" button.
3. For the calibration, press and hold the "UP" and "DOWN" buttons simultaneously for 2 sec. Confirm that the LED panel displays "CALIB" and press the "SET" button.
4. Place a 12.5 × 12.5 × 45 mm (length × width × height) rectangular parallelepiped vessel (cuvette) containing only PBS buffer into a cuvette holder of the analyzer and press the "SET" button.
5. Replace the cuvette with one containing only the detection solution (see 2.1) without sugar (maltose/sucrose) and press the "SET" button to calibrate the baseline.
6. Replace the cuvette with one containing the detection solution with 10 mM sugar and press the "SET" button.
7. 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 × 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 × 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 × g for 30 min to eliminate lipids.
8. Remove the supernatant with a 1-ml syringe and filter it through a syringe filter (pore size 0.2 µm).
9. 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.
10. Add 5 µl of the diluted beverage sample (1%, v/v) to a cuvette containing 0.495 ml of the detection solution.
11. Place the cuvette in a cuvette holder of the FRET analyzer and preheat the sample solution to 53 °C.
12. Press the "SET" 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 nm^7,8. For sucrose detection, follow the steps from 1.1 to 2.12 with a CSY-LH sensor².

Results

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).

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
LB	BD	#244620
isopropyl β-D-thiogalactoside (IPTG)	Sigma	I6758
Ampicillin	Sigma	A9518
Tri-HCl	Bioneer	C-9006-1
PMSF	Sigma	78830
EDTA	Bioneer	C-9007
DTT	Sigma	D0632
NaCl	Junsei	19015-0350
phosphate-buffered saline (PBS)	Gibco	70011-044	0.8% NaCl, 0.02% KCl, 0.0144% Na2HPO4, 0.024% KH2OP4, pH 7.4
SOC			2% tryptone, 0.5% Yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MGCl2, 20 mM Glucose
Resource Q	Amersham Biosciences	17-1177-01	6 × 30 mm anion-exchange chromatography column
HisTrap HP1	Amersham Biosciences	29-0510-21
Quartz cuvette	Sigma	Z802875
AKÄKTAFPLC	Amersham Biosciences	18-1900-26	a fast protein liquid chromatography (FPLC)
Cary Eclipse	VarianInc		a fluorescence spectrophotometer
VICTOR	PerkinElmer	2030-0050	a multilabel plate reader
E. coli JM109 (DE3)	Promega		Electrocompetent cells
A (Beverage)	Korea Yakult Co. (Korea)	Birak	Fermented drinks
B (Beverage)	Lotte Foods (Korea)	Epro	Soft drink
C (Beverage)	Lotte Foods (Korea)	Getoray	Sports drink