Label-free fluorescent aptamer sensor based on regulation of malachite green fluorescence

Abstract

We report a label-free fluorescent aptamer sensor for adenosine based on the regulation of malachite green (MG) fluorescence, with comparable sensitivity and selectivity to other labeled adenosine aptamer-based sensors. The sensor consists of free MG, an aptamer strand containing an adenosine aptamer next to an MG aptamer, and a bridging strand that partially hybridizes to the aptamer strand. Such a hybridization prevents MG from binding to MG aptamer, resulting in low fluorescence of MG in the absence of adenosine. Addition of adenosine causes the adenosine aptamer to bind adenosine, weakening the hybridization of the aptamer strand with the bridging strand, making it possible for MG to bind to the aptamer strand and exhibit high fluorescence intensity. Since this design is based purely on nucleic acid hybridization, it can be generally applied to other aptamers for the label-free detection of a broad range of analytes.

Figure 1

Scheme showing the regulation of the fluorescence of malachite green by adenosine. Without adenosine, the affinity of the aptamer strand (in red-blue) is inhibited by the bridging strand (in brown). With adenosine, the bridging strand separates from the aptamer strand, which then binds malachite green, leading to an enhancement of fluorescence.

Figure 2

Sensor performance with different sequences of the bridging strands. 1 µM aptamer strand, bridging strand and MG were used as the sensor. 20 mM Tris (pH 7.4), 140 mM KCl, 5 mM NaCl and 5 mM MgCl₂ were used as buffer. 5 mM adenosine was added at t = 25 s and fluorescence versus time was recorded and plotted

Figure 3

Optimization of the ratio of the aptamer and bridge 9-6 strands and MG. The nucleic strands were kept 1 µM and MG concentration varied (A); the aptamer strands and MG were kept 1 µM and 0.6 µM respectively and the concentration of bridge 9-6 varied.

Figure 4

(A) The fluorescence enhancement of the optimized sensor upon the addition of adenosine; (B) the saturated fluorescence of malachite green with various concentrations of adenosine. The inset shows the fluorescence response at low concentrations of adenosine and the red line shows a linear fitting of the data. (C) selectivity of the sensor towards other nucleosides. Cytidine and uridine did not increase the fluorescence of MG.