The Development and Characterization of a Surface Enhanced Raman Spectroscopy Biosensor Utilizing an Oligonucleotide

Citrulline has shown potential as a biomarker. It can be used for a number of clinical applications including diagnosis in the clinic, as a research tool, to accurately indicate the amount of radiation absorbed by the body, and as a point of care device. In particular, for this last application, rapid assessment of radiation exposure is critically important at the point-of-care (POC) for the proper triage and treatment of large populations exposed to ionizing radiation. Currently, to quantify the amount of citrulline in the body, a blood sample must be sent to a central lab where it is analyzed using bulky and expensive instruments, such as high-performance liquid chromatography (HPLC) or liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography-tandem mass spectrometry (LC/MS/MS). This process is very time consuming and prolongs the time until effective treatment can be administered. Therefore, a point-of-care surface enhanced Raman spectroscopy (SERS) biosensor was proposed to address the problems mentioned above and quantitatively detect citrulline. SERS is a method that uses a metallic surface or nanoparticle metal colloids to enhance the inelastic Raman scattering of photons. By providing vibrational energy information about molecules adsorbed to the metal surface, including nanoparticles, SERS can be used for trace analysis due to the substantial enhancement factor from the metallic substrate. SERS has the ability to analyze molecules in the micro- to nanomolar concentration range with some reports reaching picomolar levels in solution. SERS also has the potential for multiplexing multiple biomarkers because of their narrow spectral line widths, making such labels a promising analytical approach for biosensing. Moving towards a POC sensor for citrulline detection, four types of Raman reporter functionalized nanoparticles in a micro-to-nanochannel were characterized in this work. Further, a non-aggregating binding chemistry was created and characterized on silver (Ag) nanocubes for citrulline detection. Lastly, two citrulline SERS sensors were developed. The first sensor used a competitive binding approach and the second sensor used Ag nanocubes functionalized with citrulline aptamers. These sensors were then analyzed to test the feasibility of a future, quantitative, POC SERS biosensor