A luminescence lifetime assisted ratiometric fluorimeter for biological applications

Abstract

In general, the most difficult task in developing devices for fluorescence ratiometric sensing is the isolation of signals from overlapping emission wavelengths. Wavelength discrimination can be achieved by using monochromators or bandpass filters, which often lead to decreased signal intensities. The result is a device that is both complex and expensive. Here we present an alternative system--a low-cost standalone optical fluorimeter based on luminescence lifetime assisted ratiometric sensing (LARS). This paper describes the principle of this technique and the overall design of the sensor device. The most significant innovation of LARS is the ability to discriminate between two overlapping luminescence signals based on differences in their luminescence decay rates. Thus, minimal filtering is required and the two signals can be isolated despite significant overlap of luminescence spectra. The result is a device that is both simple and inexpensive. The electronic circuit employs the lock-in amplification technique for the signal processing and the system is controlled by an onboard microcontroller. In addition, the system is designed to communicate with external devices via Bluetooth.

Figure 1

Optical setup of the fluorimeter.

Figure 2

Schematic block diagram of the electronic circuit of the fluorimeter.

Figure 3

Timing diagram of the output modulation in the phase and frequency corrected pulse width modulation mode.

Figure 4

FFT analysis at different stages of the signal processing steps. The measurement was carried out by a digital oscilloscope Tektronix TSD2000B.

Figure 5

Normalized fluorescence spectra of rhodamine B, Rubp, CdTe quantum dots, and Eu(tdap) in aqueous solution.

Figure 6

Modulation sweep carried out with the aqueous solutions of rhodamine B, CdTe quantum dots, Rubp, and Eu(tdap). These solutions contain only one fluorophore.

Figure 7

Wavelength scan of dual-fluorophores solutions consisting of Eu(tdap) and one of the fluorophores rhodamin B (Eu+Rho), Rubp (Eu+Ru), and CdTe quantum dots (Eu+Qu). The fluorescence peak of Eu(tdap) is centered at 612/3 nm

Figure 8

Modulation sweep carried out with the aqueous solutions of a mixture of Eu(tdap) with one of the following dyes rhodamine B (Eu+Rho), CdTe quantum dots (Eu+Qu), and Rubp (Eu+Ru). The concentration of Eu(tdap) in each of the mixture is varied to demonstrate signal discrimination.

Figure 9

Response of a 5 μM glutamine binding protein S179C labeled with acrylodan and Eu(tdap) in 20 mM phosphate buffer to glutamine. The measurement is carried out at room temperature. The detection range is found to be below 2 μM.

Figure 10

Response of a 7 μM solution of glucose binding protein S179C labeled with acrylodan and Eu(tdap) to glutamine. The measurement is carried out at room temperature. The detection range is found to be below 5 μM.