Highly sensitive biological and chemical sensors based on reversible fluorescence quenching in a conjugated polymer

Abstract

The fluorescence of a polyanionic conjugated polymer can be quenched by extremely low concentrations of cationic electron acceptors in aqueous solutions. We report a greater than million-fold amplification of the sensitivity to fluorescence quenching compared with corresponding "molecular excited states." Using a combination of steady-state and ultrafast spectroscopy, we have established that the dramatic quenching results from weak complex formation [polymer(-)/quencher(+)], followed by ultrafast electron transfer from excitations on the entire polymer chain to the quencher, with a time constant of 650 fs. Because of the weak complex formation, the quenching can be selectively reversed by using a quencher-recognition diad. We have constructed such a diad and demonstrate that the fluorescence is fully recovered on binding between the recognition site and a specific analyte protein. In both solutions and thin films, this reversible fluorescence quenching provides the basis for a new class of highly sensitive biological and chemical sensors.

Figure 1

Chemical structures for the molecules in this study.

Figure 2

(A) Amplification of fluorescence quenching sensitivity. (B) Biosensor application.

Figure 3

Absorption and fluorescence spectra (excited at 500 nm) of MPS-PPV (1.7 × 10⁻⁵ M in monomer repeat units) in water in the presence (dotted line) and absence (solid line) of MV²⁺ (1 × 10⁻⁷ M).

Figure 4

Stern-Volmer plot for quenching of the fluorescence of 1.2 × 10⁻⁵ M (repeat units) MPS-PPV by MV²⁺ in aqueous solution.

Figure 5

(A Inset) TA spectra in 1.5 × 10⁻³ M MPS-PPV solution at zero time delay (circles) and 2-ps delay (triangles), showing SE peak (500 nm) and PA peak (720 nm), together with difference spectra showing secondary PA peak caused by excimer formation in the aggregated polymer. (A) SE decay (circles) and excimer PA growth (squares) showing complementary dynamics and 1.5-ps decay/growth time. (B)SE decay in 5 × 10⁻⁴ M MPS-PPV solution before (circles) and after addition of 1 × 10⁻⁵ M MV²⁺, with equilibration of solution (squares) and after agitation (triangles).

Figure 6

Fluorescence spectra from aqueous solution of MPS-PPV (1.7 × 10⁻⁵ M in monomer repeat units) excited at 500 nm in water alone (solid line), after addition of 2 × 10⁻⁶ M B-MV (dash-dot line), after addition of 1 × 10⁻⁷ M avidin (dash line), and after addition of 2 × 10⁻⁷ M avidin (dot line).

Figure 7

Partial quenching of fluorescence of MPS-PPM (10⁻⁵ M in “repeat units”) by B-MV (Upper) and MV⁺ (Lower) (quencher concentration 3.2 × 10⁻⁷ M in each case). In the upper plot, the fluorescence quenching is reversed by addition of 1.2 × 10⁻⁸ M and 3 × 10⁻⁸ M avidin whereas addition of similar amounts to the MV⁺-quenched sample (Lower) shows no increase.

Figure 8

Fluorescence of a solid film of MPS-PPV excited at 400 nm on exposure of dinitrotoluene vapor as a function of time before exposure (A) and at 10 s (B), 30 s (C), and 60 s (D).