Autoenhanced Raman Spectroscopy via Plasmonic Trapping for Molecular Sensing.

As a label-free and sensitive biosensor, surface-enhanced Raman spectroscopy (SERS) is a rapidly emerging technique. However, because SERS spectra are obtained in the area of light excitation and the enhancement effect can be varied depending on the position of a substrate, it is important to match the enhanced area with an illuminated spot. Here, in order to overcome such difficulty, we demonstrated a new technique combining SERS with plasmonic trapping. By plasmonic trapping, we can collect gold nanoparticles (GNPs) in the middle of initially fabricated nanobowtie structures where a laser is excited. As a result of trapping GNPs, hot-spots are formed at that area. Because SERS is measured in the area irradiated by a laser, hot-spot can be simultaneously coincided with a detection site for SERS. By using this, we detected Rhodamine 6G to 100 pM. To further verify and improve the reproducibility of our technique, we also calculated the electric field distribution, trapping force and trapping potential.

In-situ nanospectroscopic pH monitoring by plasmon resonance energy transfer (PRET).

Real-time detection of pH value in a living cell is in central importance for research about cells and diseases. In spite of the great advances in science and technology, pH measurement in a living cell is still limited in spatial resolution, in-situ detection, and intracellular monitoring. Here, we designed a nanoscale pH meter by Plasmon Resonance Energy Transfer (PRET). In order to highly sensitively measure pH with nanoscale spatial resolution, we choose 80 nm spherical gold nanoparticle (GNP) and phenol red which is commonly used in cell media for pH determination. The resonance energy of GNP is transferred to phenol red because the scattering intensity of GNP is overlapped with the second absorption intensity of phenol red at near 560 nm. Meanwhile, the absorption intensity of phenol red molecules is changing with pH value of the solution. For that reason, the intensity of PRET from GNP to phenol red molecules also changes by the acidity of phenol red solution. Then we can detect pH values with nanoscale spatial resolution through the Rayleigh scattering intensity of GNP. As we changed pH value from 6.0 to 9.0, the scattering intensity of GNP is decreased because the absorbance of phenol red at 560 nm wavelength is increased with increasing pH value. The Gaussian peak of a difference in Rayleigh scattering spectra of GNP between pH 6.0 and pH 9.0 indicates exactly the same as UV-vis spectral difference between basic and acidic phenol red solution. We expect that this pH measuring technique has a significant impact on the pH detection of living cells with nanoscale, and it can make possibility to image the cell structure by pH variation.

Precise nanoinjection delivery of plasmid DNA into a single fibroblast for direct conversion of astrocyte.

Direct conversion is a powerful approach to safely generate mature neural lineages with potential for treatment of neurological disorders. Astrocytes play a crucial role in neuronal homeostasis and their dysfunctions contribute to several neurodegenerative diseases. Using a single-cell approach for precision, we describe here a robust method using optimized DNA amounts for the direct conversion of mouse fibroblasts to astrocytes. Controlled amount of the reprogramming factors Oct4, Sox2, Klf4 and cMyc was directly delivered into a single fibroblast cell. Consequently, 2500 DNA molecules, no more or less, were found to be the optimal amount that dramatically increased the expression levels of the astrocyte-specific markers GFAP and S100b and the demethylation gene TET1, the expression of which was sustained to maintain astrocyte functionality. The converted astrocytes showed glutamate uptake ability and electrophysiological activity. Furthermore, we demonstrated a potential mechanism whereby fibroblast was directly converted into astrocyte at a single-cell level; this was achieved by activating BMP2 pathway through direct binding of Sox2 protein to BMP2 gene. This study suggests that nanotechnology for directly injecting plasmid DNAs into cell nuclei may help understand such a conversion at single-cell level.

Spatio-temporally controlled transfection by quantitative injection into a single cell.

Transfection-based cellular control has been widely used in biology; however, conventional transfection methods cannot control spatio-temporal differences in gene expression or the quantity of delivered materials such as external DNA or RNA. Here, we present a non-viral and spatio-temporally controlled transfection technique of a quantitative injection into a single cell. DNA was quantitatively injected into a single cell at a desired location and time, and the optimal gene delivery and expression conditions were determined based on the amount of the delivered DNA and the transfection efficacy. Interestingly, an injection of 1500 DNAs produced an about average 30% gene expression efficiency, which was the optimal condition, and gene expression was sustained for more than 14 days. In a single cell, fluorescent intensity and polymerase chain reaction (PCR) results were compared for the quantity of gene expression. The high coincidence of both results suggests that the fluorescence intensity can reveal gene expression level which was investigated by PCR. In addition, 3 multiple DNA genes were successfully expressed in a single cell with different ratio. Overall, these results demonstrate that spatio-temporally controlled transfection by quantitative transfection is a useful technique for regulating gene expression in a single cell, which suggests that this technique may be used for stem cell research, including the creation of induced pluripotent stem (iPS) cells.