Detection of Cortisol in Saliva with a Flow-Filtered, Portable Surface Plasmon Resonance Biosensor System

Abstract

Saliva provides a useful and non-invasive alternative to blood for many biomedical diagnostic assays. The level of the hormone cortisol in blood and saliva is related to the level of stress. We present here the development of a portable surface plasmon resonance (SPR) biosensor system for detection of cortisol in saliva. Cortisol-specific monoclonal antibodies were used to develop a competition assay with a 6-channel portable SPR biosensor designed in our laboratory. The detection limit of cortisol in laboratory buffers was 0.36 ng/ml (1.0 nM). An in-line filter based on diffusion through a hollow fiber hydrophilic membrane served to separate small molecules from the complex macromolecular matrix of saliva prior to introduction to the sensor surface. The filtering flow cell provided in-line separation of small molecules from salivary mucins and other large molecules with only a 29% reduction of signal compared with direct flow of the same concentration of analyte over the sensor surface. A standard curve for detection of cortisol in saliva was generated with a detection limit of 1.0 ng/ml (3.6 nM), sufficiently sensitive for clinical use. The system will also be useful for a wide range of applications where small molecular weight analytes are found in complex matrices.

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

“…Previous initial studies showed that the AuNP assay with 15-1 responded to the stress biomarker cortisol, but not to two other markers of stress, epinephrine and norepinephrine, or cholic acid, a marker of liver disease structurally similar to cortisol 24 . The response was observed in the normal range of free cortisol reported in human serum (~150-600 nM) 6 , validating that the aptamer selected for a structural change upon cortisol binding produces a response in the AuNP assay. In this current work, characterization of the system was taken one step further by adjusting the coverage (density) of 15 .…”

“…This implies that the detection range for the target of interest can be adjusted based on optimizing the DNA surface coverage in the AuNP assay. This could be beneficial when comparing, for example, the range of free cortisol in human serum (~150-600 nM) versus human saliva (~5-25 nM) 6,37 . While physiological fluids are far more complex than this buffer assay, the results provide an extension on the proof-of-principle work demonstrating that AuNP conditions can be adjusted depending on the application, and that further work towards broadening the medium to biological fluids would be of interest to the biosensor community.…”

“…Various small molecules have also been suggested as biomarkers indicative of physiological conditions including stress 2,3 , fatigue 4 , and disease 5 . For example, elevated cortisol levels correlate with stress which may result in decreased physiological performance and other health conditions [6][7][8] . Likewise, variations in the ratios of certain peptides in saliva are predictive of fatigue, where physiological manifestations include lack of concentration, impaired reaction time, and reduced cognitive function 4 .…”

“…Historically, small molecule detection has been performed by labor-intensive separation techniques or by antibody based recognition 6,7 . More recently, nucleic acid aptamers 9,10 have emerged as recognition elements that possess distinct advantages over antibodies in specific applications.…”

Label-Free Detection of Tobramycin in Serum by Transmission-Localized Surface Plasmon Resonance

“…Because our system is label-free and, thus, prone to be sensitive to nonspecific binding, FBS was filtered with a 3 kDa cutoff membrane in order to remove most of the proteins in the serum (see Experimental Section). 10 Tobramycin concentrations within the clinical analytical range (10,20,40,60, and 80 μM; clinical range of 0.5−80 μM) were then spiked in FBS, filtered, and injected at 10 μL/min over the sensing areas in order to be measured with the T-LSPR measurement system (Figure 8). The data were normalized for baseline drift and response variations by the signal corresponding to the regeneration injection (NaCl 1M, entirely bulk effect).…”