Fluorescent labeling can improve the detection sensitivity in capillary electrophoretic (CE) separations down to attomolar concentrations. However, most fluorescent labels are not compatible with CE because their fluorescence properties and charge states are pH-dependent, they are often hydrophobic and they have a tendency to significantly change the properties of the analytes after labeling. A group of fluorescent labeling reagents have been prepared whose fluorophores have properties that are optimized for CE separations. These fluorophores have fluorescence properties and charge states that are independent of pH in the 2 < pH < 11 range. Their excitation maxima are also compatible with the 488 nm line of the Argon ion laser. A mono-cationic acridine-based fluorescent label was prepared and was found to not shift the pI of a labeled model protein in capillary isoelectric focusing separation (cIEF). Lower loading, due to increased sensitivity, led to better resolution of closely spaced isoform peaks having a pI = 0.05. A tri-anionic pyrene-based fluorescent labeling reagent was also synthesized and was used in the sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) separation of proteins. The fluorophore led to an LOQ in the nM range, and did not alter the migration behavior of proteins in the sieving matrix. A third fluorescent labeling reagent was developed as a solid phase reagent (SPR) where the fluorophore was immobilized on a solid surface through a cleavable anchor. The fluorophore is di-anionic and is based on pyrene. The SPR was designed to allow the simultaneous capture and labeling of an analyte and the efficient release of the label-analyte conjugate under mild acidic conditions. The use of the SPR allowed the labeling of a diamine whose concentration was in the low nanomolar range. The SPR opens up the possibility for mono-labeling and proportional multiple labeling of proteins.

Global threats to treatment of bacterial infections due to antibiotic resistance (AR) have been on the rise in recent years. Current diagnostic tests identify bacteria by using blood culture, which takes more than 24 hours. This study focuses on the fluorescent labeling of DNA derived from bacterial AR genes (KPC & VIM) and other model DNAs using oligreen dye (OG) and molecular beacons (MB). A NanoDrop 3300 fluorospectrometer was used to take fluorescence measurements. Linear dynamic range and labeling efficiency were dependent on the following optimized conditions: dilution factor of OG (200 fold), buffer (20 mM Tris HCl, pH 8), and heat treatment of 95 °C for 15 min.Fluorescence analysis of a target DNA with a designed MB showed signal-to-background of 10 with our buffer only and 20 with our buffer and 25% ethanol. I also demonstrated a simple microfluidic device capable of detecting AR genes using model DNAs, magnetic beads, and designed MBs for assays of µ50 L volume. This study provides a first step towards detecting MB-DNA complexes by a simple, low cost, and fast non-amplified method, which may be used to detect AR genes in clinical samples in the future.