Aptamer nano-flares for molecular detection in living cells

Abstract

We demonstrate a composite nanomaterial, termed an aptamer nano-flare, that can directly quantify an intracellular analyte in a living cell. Aptamer nano-flares consist of a gold nanoparticle core functionalized with a dense monolayer of nucleic acid aptamers with a high affinity for adenosine triphosphate (ATP). The probes bind selectively to target molecules and release fluorescent reporters which indicate the presence of the analyte. Additionally, these nanoconjugates are readily taken up by cells where their signal intensity can be used to quantify intracellular analyte concentration. These nanoconjugates are a promising approach for the intracellular quantification of other small molecules or proteins, or as agents that use aptamer binding to elicit a biological response in living systems.

Figure 1

(a) Aptamer nano-flares are gold nanoparticles functionalized with thiol-terminated aptamer sequences hybridized to a short complementary Cy5-labeled reporter strand. The reporter is capable of being displaced by a conformation change in the aptamer that is induced by the target molecule. (b) Oligonucleotide sequences used to prepare the aptamer nano-flares.

Figure 2

(a) Solution fluorescence spectra of aptamer nano-flares treated with ATP (2 mM) and UTP, GTP and CTP. (b) Fluorescence spectra of aptamer nano-flares in solutions with increasing concentrations of ATP (0.1 – 3.0 mM). Inset shows the peak intensity as a function of ATP concentration.

Figure 3

(a) Fluorescence microscopy images of HeLa cells incubated with aptamer nano-flares and control particles for 2 hrs. The red channel is Cy5 fluorescence associated with activated aptamer nano-flares, the green channel is fluorescence associated taxol-Alexa 488 and specific for tubulin, and the blue channel is fluorescence associated with Hoechst 3342 and specific for the nucleus. Scale bar is 20μm. (b) Cell-associated fluorescence (Cy5) of populations treated with aptamer nano-flares and control particles as determined by flow cytometry. Inset numbers indicate the mean fluorescence value of the population.

Figure 4

(a) Fluorescence of cell populations treated with doses of oligomycin followed by aptamer nano-flares (red) or control particles (black). The error bars represent standard deviation from at least 3 independent replicates. (b) Cell-associated fluorescence of living cells plotted as a function of cellular ATP concentration as bulk measurement by a commercial ATP detection kit. (see Supporting Information for experimental details) The error bars along the X- and Y-axis represent standard deviations from at least 3 replicates in cellular ATP concentrations and cell-associated fluorescence as measured by flow cytometry, respectively.