br Materials and methods br Results br Discussion The techni

Materials and methods
Results
Discussion The technical improvements to the salivary cortisol immunosensor platform and the incorporation of centrifugal fluid valves eliminate many of the sample preprocessing steps associated with a previous version of our salivary cortisol immunosensor [27]. The performance of the disc-chip is optimized by choosing the appropriate blocking agents. Our evaluation of the effects of different surface pre-treatments reveal that IgG polymer for the pad and the milk protein for the reservoirs (Type I-M) and the flow AMN-107 was the most suitable combination of blocking agents to prevent the non-specific adsorption of proteins (Table 1 and Fig. 5). The performance characteristics of the salivary cortisol immunosensor were evaluated using cortisol standard solution measured by the salivary cortisol ELISA. Comparison of the calibration curves of the salivary cortisol immunosensor with different combinations of the blocking agents, showed that the results obtained with Type I-M agent had a high correlation with those obtained by the commercial ELISA assay (R2=0.93) and had a low CV=12.3% for the range of cortisol standards used (1–10ng/mL, Fig. 6A). Additionally, the dynamic range of the cortisol immunosensor (1–10ng/mL) was broad enough to cover the range of salivary cortisol concentrations (1–8ng/mL) reported by Aardal and Holm [1] in healthy adults (1ng/mL=0.1μg/dL=2.76nmol/L). Thus, the flow can be controlled just by changing the speed and number of rotations to determine which centrifugal fluid valve will open. Using a set of centrifugal fluid valves, valves A and B, enables the setting of any number of washing times to remove impurities. This mechanism allows for automated-analyses which makes possible to shorten the sampling-reporting cycle less than 15min. Analyzed by means of ROC curves, the salivary cortisol was shown to have good discriminating power (0.88) in discriminating of psychological stress (Fig. 7). Thus, our early testing of the salivary cortisol immunosensor using ROC curve analysis of human saliva samples indicates potential utility for near-real time assessment of stress reactions based on salivary cortisol levels.
Conclusions The analytical performance of our salivary cortisol immunosensor, incorporating centrifugal fluid valves and a disposable disc-chip, allows for automated-analyses of human salivary cortisol levels within a shortened timespan (<15min) compatible with “point-of-care” applications. The performance characteristics of the salivary cortisol immunosensor are optimized though select blocking agents to prevent the non-specific adsorption of proteins; IgG polymer for the pad and milk protein for the reservoirs and the flow channels. Incorporated centrifugal fluid valves allow multiple washings to remove impurities. The optical reader and laptop computer automate the immunoassay processes and provide easily accessible digital readouts of salivary cortisol measurements. Our early ROC curve analysis of human saliva samples indicate good utility for discriminating stress disorders and underscore potential application for point-of-care measurement of cortisol
Conflict of interest
Acknowledgements This research was supported in part by Grants from the ‘Ministry of Education, Culture, Sports, Science, and Technology – Japan’, Research Project of Recovery Supporting Technology from Disaster Stress Based on Psychosomatic Functional Evidence (P.I.-M. Yamaguchi), Japan, and Grant No. 25350517 from the ‘Japan Society for the Promotion of Science – Japan’, Proposal for Fluid control mechanism Based on Wettability and Establishment of Quantitative and Automated-biosensor (P.I.-M. Yamaguchi), Japan.
Introduction Metallic nanostructures have been recognized for their unique optical properties owing to their ability to support surface plasmons, which are oscillations of free electrons in the metals that are bound by nanoparticle geometry [20,6]. Plasmonic properties sensitively depend on geometric parameters, such as nanoparticle size, shape, crystal face, surface roughness, and interparticle spacing [23,24,13]. These properties have been widely explored in various applications that include surface-enhanced Raman scattering (SERS) [2,5]. Unlike fluorescence-based detection methods, SERS shows high sensitivity (up to single-molecule detection), narrow spectral width, multiplex capability, and insensitivity to quenching [19]. To achieve maximal enhancement of the Raman signal, various SERS-active materials have been developed [1]. High Raman enhancements are generally obtained by increasing the number of “hot spots” at the junctions of particle to particle boundaries. Thus, aggregates of silver or gold nanoparticles have emerged as effective SERS substrates [11]. Aggregations of metal nanoparticles have been conventionally induced by the addition of surface charge altering agents, such as poly-(l-lysine), spermine hydrochloride, and sodium chloride [22]. However, the precise and reproducible formation of metal clusters is not easily achieved, and this interferes with the reproducibility of SERS experiments. There was a lack of control over both the size of resulting aggregates and the gaps between SERS aggregates using these approaches [21,12].