Aptamers Embedded in Polyacrylamide Nanoparticles: A Tool for in Vivo Metabolite Sensing Lise J. Nielsen, Lars F. Olsen, and Veli C. Ozalp* CelCom, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230, Odense M, Denmark M etabolite sensing is an impor- tant tool in understanding cell metabolism. However, with cur- rent technologies it is only possible to meas- ure a few metabolites in vivo without the need to first quench the cells and subse- quently extract the cell material for later analysis. In recent years there has been an interest in using optical nanosensors to monitor in vivo metabolite dynamics of a number of cell intermediates. Probes en- capsulated by biologically localized embed- ding (PEBBLE) defines a class of nanosen- sors designed for minimally invasive analyte monitoring in vivo. 1 Nanoparticles possess unique properties to develop better optical sensing mainly because of their large sur- face areas achieved by small size. Nanosensors are chemically stable nano- particles loaded with a sensing component. The sensing involves permeation of a small molecule target into the particle matrix where it interacts selectively with the sens- ing element leading to a measurable signal change. Polymeric nanosensor systems have been based on fluorescent indicator dyes or encapsulated target-specific bio- logical receptors such as DNA, proteins, or peptides as reviewed recently. 2 For ex- ample, a phosphate nanosensor was devel- oped by embedding fluorescent reporter proteins responsive to phosphate ions in polyacrylamide nanoparticles. In another example, horseradish peroxidase embed- ded in polyacrylamide nanoparticles was designed to measure reactive oxygen spe- cies (ROS). 3 All the previously reported nanosensors were designed for the target of interest by finding dyes or receptor molecules that in- teract specifically with the analyte of inter- est and this limits the development of new nanosensors. However, the embedding of aptamers might open up a new class of sen- sors that are more widely applicable, since it is possible to artificially select an aptamer for any metabolite of interest, and there- fore aptamer-based PEBBLE nanosensors can be a generic system for in vivo monitor- ing of small molecules. Aptamers are single-stranded oligonu- cleotides that can bind to specific targets. Nucleic acid aptamers may comprise un- modified DNA and RNA as well as chemi- cally modified forms. They have similar properties to antibodies in that they bind targets with high affinity and specificity. Furthermore, they possess additional prop- erties making them superior to antibodies: They are easy to synthesize chemically and they can be developed by artificial selec- tion. Therefore, they are considered to be powerful candidates for developing better sensors for diagnostic or therapeutic appli- cations. Aptamers are selected from a com- binatorial library through an artificial evolu- tion procedure called SELEX 4,5 and have *Address correspondence to cengiz@bmb.sdu.dk. Received for review October 23, 2009 and accepted July 15, 2010. Published online July 22, 2010. 10.1021/nn100635j © 2010 American Chemical Society ABSTRACT We describe a new type of aptamer-based optical nanosensor which uses the embedding of target responsive oligonucleotides in porous polyacrylamide nanoparticles to eliminate nuclease instability. The latter is a common problem in the use of aptamer sensors in biological environments. These aptamers embedded in nanoparticles (AptaNPs) are proposed as a tool in real-time metabolite measurements in living cells. The AptaNPs comprise 30 nm polyacrylamide nanoparticles, prepared by inverse microemulsion polymerization, which contain water-soluble aptamer switch probes (ASPs) trapped in the porous matrix of the nanoparticles. The matrix acts as a molecular fence allowing rapid diffusion of small metabolites into the particles to interact with the aptamer molecules, but at the same time it retains the larger aptamer molecules inside the nanoparticles providing protection against intracellular degradation. We tested the ability of the AptaNPs to measure the adenine- nucleotide content in yeast cells. Our results successfully demonstrate the potential for monitoring any metabolite of interest in living cells by selecting specific aptamers and embedding them in nanoparticles. KEYWORDS: nanoparticles · aptamers · metabolite monitoring · Saccharomyces cerevisiae · nanosensor · PEBBLE · molecular beacon ARTICLE www.acsnano.org VOL. 4 ▪ NO. 8 ▪ 4361–4370 ▪ 2010 4361