The application of gold nanoshells (NS) as a surface-enhanced Raman (SER) platform for intracellular sensing in NIH-3T3 fibroblast cells was studied by using a near-infrared Raman system. To show the feasibility of using these 151 +/- 5 nm sized solution-stable nanoparticles inside living cells, we investigated the uptake, cellular response, and the health of the cell population. We show that NS are taken up voluntarily and can be found in the cytosol by transmission electron microscopy (TEM), which also provides detailed information about location and immediate surrounding of the NS. The internalization into cells has been found to be independent of active cellular mechanisms, such as endocytosis, and can be suggested to be of passive nature. Uptake of NS into cells can be controlled, and cells show no increase in necrosis or apoptosis as a result; we show that NS-based intracytosolic SER spectra can be measured on biological samples using short acquisition times and low laser powers. We demonstrate its application using 4-mercaptobenzoic acid (4-MBA)-functionalized nanoshells as a pH sensor.
We present a new type of DNA switch, based on the Holliday junction, that uses a combination of binding and conformational switching to enable specific label-free detection of DNA and RNA. We show that a single RNA oligonucleotide species can be detected in a complex mixture of extracted cellular RNA and demonstrate that by exploiting different aspects of the switch characteristics we can achieve 30-fold discrimination between single-nucleotide mismatches in a DNA oligonucleotide.
DNA nanoswitches can be designed to detect unlabelled nucleic acid targets and have been shown to discriminate between targets which differ in the identity of only one base. This paper demonstrates that the fluorescent base analogue 2-aminopurine (AP) can be used to discriminate between nanoswitches with and without targets and to discriminate between matched and mismatched targets. In particular, we have used both steady-state and time-resolved fluorescence spectroscopy to determine differences in AP environment at the branchpoint of nanoswitches assembled using complementary targets and targets which incorporate single base mismatches.
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