Surface Plasmon Resonance (SPR) spectroscopy is a potentially valuable tool for measuring protein-protein interactions and protein levels <i>in vitro</i> and <i>in vivo</i>. Fiber-optic based SPR allows for rapid quantitative measurement of disease markers such as the truncated (exons 1-7) survival of motor neuron (SMN) protein. Unlike micro fluidics-based SPR systems, sample loss is eliminated in fiber-optic SPR and the small size of the fiber optic probes (400μm or smaller) facilitates the potential for use <i>in vivo</i>. Recombinant SMN protein overexpressed in <i>E. Coli</i> as well as native SMN from cultured HeLa cells has been successfully measured using fiber-optic SPR.
Detection of multiple biologically relevant molecules was accomplished at sub-ng/mL levels in highly fouling media using fiber- optic based surface plasmon resonance sensors. Myocardial infarction markers, myoglobin and cTnI, were quantified in full serum with limits of detection below 1 ng/mL. Biologically relevant levels are between 15-30 ng/mL and 1-5 ng/mL for myoglobin and cTnI respectively. Cytokines involved in chronic wound healing, Interleukin 1, Interleukin 6, and tumor necrosis factor α, were detected at around 1 ng/mL in cell culture media. Preliminary results in monitoring these cytokines in cell cultures expressing the cytokines were obtained. The protein diagnostic of spinal muscular atrophy, survival motor neuron protein, was quantified from cell lysate. To obtain such results in complex media, the sensor's stability to non-specific protein adsorption had to be optimized. A layer of the N-hydroxysuccinimide ester of 16-mercaptohexadecanoic acid is attached to the sensor. This layer optimizes the antibody attachment to the sensor while minimizing the non-specific signal from serum proteins.
Multiple layers were attached to the gold surface of surface plasmon resonance (SPR) sensor to maximize the antibody loading and the specific signal of an antigen, while minimizing non-specific signal from full serum proteins. A three-fold improvement of the specific signal from myoglobin and a three-fold decrease of non-specific signal from serum were observed using the N-hydroxysuccinimide ester of 16-mercaptohexadecanoic acid (NHS-MHA) compared to the currently commercially available carboxymethylated dextran. Self-assembled monolayers were attached to the gold surface. 2-mercaptoacetic acid, 3-mercaptopropionic acid, 4,4'-dithiodibutyric acid, 11- mercaptoundecanoic acid, and 16-mercaptohexadecanoic acid were investigated. The covalent attachment of the layers was monitored using SPR and GATR-FTIR. Antibodies to human myoglobin were covalently attached to the sensor using EDC / NHS chemistry and detection of 25 ng/mL myoglobin solution was monitored for the specific signal. Exposing the sensors with the layers to full bovine serum, protein concentration of 72 mg/mL, monitored non-specific signal. NHS-MHA was used to quantify proteins cell culture media with limits of detection below 1 ng/mL.