Ratiometric QD-FRET Sensing of Aqueous H2S in Vitro

Abstract

We report a platform for the ratiometric fluorescent sensing of endogenously generated gaseous transmitter H2S in its aqueous form (bisulfide or hydrogen sulfide anion) based on the alteration of Förster resonance energy transfer from an emissive semiconductor quantum dot (QD) donor to a dithiol-linked organic dye acceptor. The disulfide bridge between the two chromophores is cleaved upon exposure to bisulfide, resulting in termination of FRET as the dye diffuses away from the QD. This results in enhanced QD emission and dye quenching. The resulting ratiometric response can be correlated quantitatively to the concentration of bisulfide and was found to have a detection limit as low as 1.36 ± 0.03 μM. The potential for use in biological applications was demonstrated by measuring the response of the QD-based FRET sensor microinjected into live HeLa cells upon extracellular exposure to bisulfide. The methodology used here is built upon a highly multifunctional platform that offers numerous advantages, such as low detection limit, enhanced photochemical stability, and sensing ability within a biological milieu.

Figure 1

(A) Normalized emission of the coupled QD/bisulfide-reactive carboxyrhodamine dye (compound 8) sensor (3.4 × 10^-8 M) upon exposure to HS^-. Also the ratio of the integrated emission of the QD donor over the dye acceptor as a function of HS^- concentration reveals a 21.6 ± 0.4 μM detection limit. (B) The same for the QD/bisulfide-reactive carboxyrhodamine dye sensor at 10× dilution (3.4 × 10^-9 M) reveals a lower detection limit of 1.36 ± 0.03 μM.

Figure 2

Fluorescence intensity ratio change in response to various relevant analytes (25 μM). The data are normalized against the response to bisulfide.

Figure 3

Two sets of fluorescence images of HS^- detection in HeLa cells. The cells are imaged with optical filters to separately measure the donor QD vs. dye acceptor emissions as labeled in the figures. Before: Image prior to sodium sulfide addition. After: The same after exposure to sodium sulfide solution.

Scheme 1

Mechanism of the ratiometric response of the sensor based on FRET modulation.

Scheme 2

Synthesis of the bisulfide-reactive carboxyrhodamine B dye, and subsequent conjugation to water-soluble QDs.