Detection of proteins and pathogens in complex matrices using a novel cantilever design

Detection of biological molecules at low concentrations has a variety of applications in medicine, food processing, and biothreat detection. Immunoassays that are currently available or under development require some sample preparation or labeled reagents and are limited by low sensitivity (pg/mL to ng/mL) and poor specificity in complex matrices. The aim of the research presented in this dissertation was to develop a novel cantilever sensor geometry that demonstrates a high degree of sensitivity and specificity for detecting biological species in complex matrices. Sensor fabrication was explored using computer simulation, analytical calculations, and applied experimentation. The new cantilever design showed mass-change sensitivities of 300 ag/Hz to 1.5 fg/Hz and was used to detect pathogens, toxins, and proteins in a variety of practical applications, including the following results. The pathogenic bacterium, Escherichia coli O157:H7, was detected at less than 10 cells/ml in a ground beef wash in ∼ 10 minutes. Staphylococcus enterotoxin B (SEB) was detected in apple juice and milk at 10 and 100 fg (effective concentrations of 2.5 and 25 fg/mL). A method was developed for detecting and quantifying a prostate cancer biomarker (α-methylacyl-CoA racemase; AMACR) in patient urine at fg/mL concentrations. A novel amplification strategy, using label-free additions of secondary and tertiary antibody molecules, was used to detect an ovarian cancer biomarker (CA-125) in human serum at 5 to 50 ag (effective concentrations of 1 and 10 ag/mL). All successful detections were accomplished without sample preparation or the use of labeled reagents. The results demonstrate that the new cantilever sensor geometry can reliably detect low concentrations (ag/mL to fg/mL) of specific biomolecules in real systems containing varying levels of contaminant particles.