One dimensional gold nano-structures for single cell analysis and biosensor applications

Abstract

Recently, Nanotechnology has been emerged as an important research area and studied by many researchers. For increasing understanding of nanosized world, necessity of nanosized electrode has been emphasized. One dimensional nanoelectrode means which has at least one of its critical dimensions is in nanometer scale. Due to its unique structural properties, nanoeletrodes can detect or providing small signal which macro sized probe cannot. Nanoelectrodes are widely used for analyzing biological event at submicron level and biochemical sensor. For detecting these microscopic changes, robust and reproducible nanoprobe is needed. We report vapor transport method for single crystalline and atomically flat gold nanostructure. Au nanoelectrode was fabricated with tungsten tip using micromanipulation. Then, insulation was conducted for linked part between gold nanostructure and tungsten tip. Firstly, fabricated Au nanoelectrodes ability as an electrode was verified through simple electrochemical experiment, then applicability as a sensor tool was also tested by arsenic ion detection experiment.

In chapter 2, Surface modification of Au nanowire electrode was carried out for hydrogen peroxide detection at single cell level. Reactive oxygen species (ROSs) are kinds of important biomolecules which concerns cell signaling process and homeostasis. Among various ROSs, $H_2O_2$ is a main molecule for oxygen involved metabolism. Overexpression of $H_2O_2$ can cause aging of cell, apoptosis and even transformation to cancer cell. Prussian blue, which is even called ‘artificial enzyme’, shows great properties as a $H_2O_2$ transducer. In this experiment, mechanical stimulation was given to living Hela cell, which cause oxidative stress to cell. Then, $H_2O_2$ are overexpressed and released from the cell, $H_2O_2$ release was detected selectively using Prussian blue modified Au nanowire electrode. Upon insertion of Au nanowire electrode doesn’t cause severe damage to cell, we could successfully detect $H_2O_2$ release inside and outside of cell. We anticipate that Au nanowire electrode platform can increase understanding of single cell metabolism.

In chapter 3, Au nanoprobe was employed for regenerative astaxanthin milking from Haematococcus pluvialis. Milking of microalgae, the process of reusing the biomass for continuous production of target compounds can strikingly overcome the time and cost constraints associated with biorefinery. This process can significantly improve production efficiency of highly valuable chemicals, for example, astaxanthin (AXT) from Haematococcus pluvialis. Detailed understanding of the biological process of cell survival and AXT reaccumulation after extraction would be of great help for successful milking. Here we report extraction of AXT from a single cell of H. pluvialis through incision of the cell wall by a gold nanoscalpel (Au-NS), which allows single-cell analysis of wound healing and reaccumulation of AXT. Interestingly, upon the Au-NS incision, the cell could reaccumulate AXT at a rate two times faster than the control cells. Efficient extraction as well as minimal cellular damage, keeping cells alive, could be achieved with the optimized shape and dimensions of Au-NS: a well-defined sharp tip, thickness under 300 nm, and 1-3 $\mu m$ of width. The demonstration of regenerative extraction of AXT at a single cell level hints toward the potential of a milking process for continuous recovery of target compounds from microalgae while keeping the cells alive.

In chapter 4, we report site specific delivery of GFP interfering siRNA using Au nanowire probe. siRNA, short interference Ribose nucleic acid, interfere specific target mRNA to prohibit transcript of protein, so that It has been emerged as a one of important materials for gene therapy. However, it is hard to deliver siRNA with high efficiency and targeted site because of endosomal escape in intracellular environment, so that not all RNA interference process is revealed. Here we use Au-S bond to modify surface of Au nanoprobe with GFP-suppressing siRNA, then through micro-manipulation which is based on its one dimensional structural strength, siRNA was delivered site specifically to nuclear, cytoplasm of hela cell. We followed change of cell fluorescent based on delivered site. We expect that Au nanoprobe based delivery platform can unveil the detailed RNAi process through direct delivery of target siRNA to various specific sites.

In the last chapter, surface enhanced Raman scattering (SERS) sensor was fabricated by Au nanowire for uranyl ion sensor. Uranium is an essential raw material in nuclear energy generation

however, its use raises concerns about the possibility of severe damage to human health and the natural environment. In this work, we report an ultrasensitive uranyl ion ($UO_2^{2+}$) detection method in natural water that uses a plasmonic nanowire interstice (PNI) sensor combined with a DNAzyme-cleaved reaction. ($UO_2^{2+}$) induces the cleavage of DNAzymes into enzyme strands and released strands, which include Raman-active molecules. A PNI sensor can capture the released strands, providing strong surface-enhanced Raman scattering signal. The combination of a PNI sensor and a DNAzyme-cleaved reaction significantly improves the ($UO_2^{2+}$) detection performance, resulting in a detection limit of 1 pM and high selectivity. More importantly, the PNI sensor operates perfectly, even in ($UO_2^{2+}$)-contaminated natural water samples. This suggests the potential usefulness of a PNI sensor in practical ($UO_2^{2+}$)-sensing applications. We anticipate that diverse toxic metal ions can be detected by applying various ion-specific DNA-based ligands to PNI sensors.