Exploring new detection schemes for molecular detection, nucleic acid research, and the study of in situ cellular chemistry.

摘要

In complex and dynamic environments such as biological systems, new detection schemes that are quick, inexpensive, and non-invasive are necessary to provide analyses with high quality data. To this end, the research presented here is focused on exploring new detection schemes for molecular detection, nucleic acid research, and the study of in situ cellular chemistry. This research investigates the use of surface enhanced Raman spectroscopy (SERS) as the primary detection technique, starting first with experiments designed to broaden and improve the application of SERS within biomedical settings and then applying SERS to further the fundamental understanding of relevant biomedical topics such as DNA mutation and stem cell differentiation.;The first set of experiments to be discussed involves a methodology to produce solid state nanogap electrodes. In Chapter 2 a combination of oligonucleotide-based molecular lithography and traditional photolithography is used to create finely tunable nanogap electrodes and to explore the fabrication of Raman enhancing substrates. Solid state nanogaps represent a relevant application of nanolithography as they can be used in sensitive chemical and biological detection schemes and are invaluable for investigating molecular electronic candidates, for researching biological systems, and as substrates for surface enhanced Raman spectroscopic studies. Here, nanogaps are implemented for monitoring by using two detection schemes, one optical (SERS) and one electronic (current-voltage responses). Using the strong Raman enhancements created by the nanogap, Raman spectra and current voltage traces show that the oligonucleotides used as the molecular resist are degraded during processing, that some of the degraded oligonucleotides are removed, and that fresh oligonucleotides are adsorbed.;Next, a methodology is explored to enable the collection of statistically significant SERS data using single nucleotide polymorphisms as a model system. We report a method of using surface enhanced Raman spectroscopy to probe single stranded DNA for genetic markers. Single-stranded oligonucleotides functionalized with gold nanoparticles are hybridized with oligonucleotides adsorbed to photolithographically defined gold surfaces thus creating a surface enhanced Raman environment around the DNA duplex. With this design characteristic Raman spectra are analyzed for differences between DNA duplexes formed from complementary oligonucleotides, completely mismatched oligonucleotides, and those formed from oligonucleotides that have a mid-sequence single nucleotide mismatch. The results show that statistically significant differences in Raman intensity for characteristic peaks can be collected for the three cases. This method is then improved upon by analyzing unmodified genes of moderate length by introducing the genes into a surface enhanced Raman complex. With this design we are able to collect characteristic Raman spectra about the genes and to again detect genetic markers such as single-nucleotide polymorphisms but also a variety of additional polymorphic regions. Results show that strands containing one of three different types of polymorphism can be differentiated using statistically significant peak position differences and trends regarding Raman intensity.;Finally, SERS is utilized in a more dynamic biological environment---in living human stem cells. Living cells uptake gold nanoparticles and sequester these particles in the endosomal pathway. Once inside the endosome, nanoparticles aggregate into clusters that give rise to large spectroscopic enhancements that can be used to elucidate local chemical environments through the use of surface enhanced Raman spectroscopy. This research uses colloidal gold nanoparticles to create volumes of surface-enhanced Raman scattering (SERS) within living human adipose derived adult stem cells enabling molecular information to be monitored. We exploit this technique to spectroscopically observe chemical changes that occur during the adipogenic differentiation of human adipose derived stem cells over a period of 22 days, monitoring both the production of lipids and the complex interplay between lipids, proteins, and chemical messengers involved in adipogenesis.