Low-cost optical lifetime assisted ratiometric glutamine sensor based on glutamine binding protein

Abstract

Here we report a reagentless fluorescence sensing technique for glutamine in the submicromolar range based on the glutamine binding protein (QBP). The S179C mutant is labeled with the short-lived acrylodan (lifetime<5ns) and the long-lived tris(dibenzoylmethane) mono(5-amino-1,10-phenanthroline)europium(III) (lifetime > 300 micros) at the -SH and the N-terminal positions, respectively. In the presence of glutamine the fluorescence of acrylodan is quenched, while the fluorescence of europium complex remains constant. In this report we describe an innovative technique, the so called lifetime assisted ratiometric sensing to discriminate the two fluorescence signals using minimal optics and power requirements. This method exploits the large difference between the fluorescence lifetimes of the two fluorophores to isolate the individual fluorescence from each other by alternating the modulation frequency of the excitation light between 300 Hz and 10 kHz. The result is a ratiometric optical method that does not require expensive and highly attenuating band pass filters for each of the dyes, but only one long pass filter for both. Thus, the signal to noise ratio is enhanced, and at the same time, the optical setup is simplified. The end product is a simple sensing device suitable for low-cost applications such as point-of-care diagnostics or in-the-field analysis.

Figure 1

Principle of glutamine sensing. QBP is labeled with the polarity sensitive dye acrylodan and the insensitive reference dye. The complexation of glutamine causes the transition from the open to the close conformation which changes the microenvironment of the acrylodan molecule. This results in a fluorescence decrease.

Figure 2

Two-step process to couple the eu(tdap) complex to the QBP protein. Two separate steps are required in order to prevent glutaraldehyde from crosslinking two proteins which may deactivate the proteins.

Figure 3

Experimental setup for the glutamine measurement based on LARS. The LED is driven and modulated by the lockin amplifier which also recovers the signal from the noise. The fluorescence is measured in 90° angle to excitation light path. The sample is held in a standard cuvette for fluorescence measurement.

Figure 4

Excitation and emission spectra of acrylodan and eu(tdap) dissolved in tetrahydrofuran (THF). The concentrations of the solutions are 1.2 and 1.4 µM respectively. The emission spectrum of acrylodan is obtained by exciting the solution at 390nm and the eu(tdap) - at 370nm. The spectra are taken with the Carey Eclipse spectrofluorimeter at room temperature. The control voltage of the spectrofluorimeter is set at 700.

Figure 5

a) Titration of the 1.8 µM dual labeled QBP solution (PBS buffer, 20mmolar, pH7.5) with a buffered glutamine solution (PBS buffer, 20mmolar, pH7.5). As shown, the fluorescence intensity of acrylodan decreases with increasing glutamine concentration. In contrast, the fluorescence of the eu(tdap) complex is not affected. b) Response of the protein attached acrylodan and eu(tdap) on the glutamine concentration. Each data point on the fluorescence curve of acrylodan and eu(tdap) represents the integral of the emission curve in figure 5a. The integration interval of the acrylodan peak and the eu(tdap) peak is set from 450 to 575nm and 600 to 640nm respectively.

Figure 6

Modulation frequency scan of dual-labeled QBP solution between 100Hz and 20 kHz. The setup for this experiment is depicted in figure 4. The theoretical curve is calculated with the eq.6. The difference between the two curves at the start results from the interference with the 60 Hz.

Figure 7

LARS based measurement of glutamine with the 1.8 µM dual-labeled QBP dissolved in 20mM phosphate buffer. The measurement is carried out by using the experimental setup (figure 3) at room temperature.