Daisuke Niwa

Immunosensor with fluid control mechanism for salivary cortisol analysis

The purpose of this research is to demonstrate a new design for a cortisol immunosensor for the noninvasive and quantitative analysis of salivary cortisol. We propose a cortisol immunosensor with a fluid control mechanism which has both a vertical flow and a lateral flow. The detected current resulting from a competitive reaction between the sample cortisol and a glucose oxidase (GOD)-labeled cortisol conjugate was found to be inversely related to the concentration of cortisol in the sample solution. A calibration curve using the relative detected current showed a R(2)=0.98 and CV=14% for a range of standard cortisol solutions corresponding to the concentrations of native salivary cortisol (0.1-10 ng/ml). The measurement could be accomplished within 35 min and the cortisol immunosensor could be reused. These results show promise for realizing an on-site and easy-to-use biosensor for cortisol. Used for evaluation of human salivary cortisol levels, the cortisol immunosensor measurement corresponded closely with commercially available ELISA method (R(2)=0.92). Our results indicate the promise of the new cortisol immunosensor for noninvasive, point of care measurement of human salivary cortisol levels.

Point-of-use measurement of salivary cortisol levels

The objective is to develop a novel methodology to analyze cortisol levels in saliva as an index of neuroendocrine response. In order to realize a portable, rapid and hand held biosensor of cortisol, we proposed an immuno-chromatographic test-strip based biosensor consisting of a disposable test-strip and a monitor. A fabricated disposable type test-strip has a size of 5 × 1.5 × 50 mm3. In order to realize an immuno-chromatographic test-strip, a glucose oxidase (GOD)-cortisol conjugate was newly synthesized, because the speed and selectivity of immunological reactions is particularly attractive as a strategy for molecular recognition of cortisol. The synthesized molecule was identified as the GOD-cortisol conjugate by both its molecular weight and specific detection of cortisol. Three min after addition of GOD enzyme assay solution, a color change reaction at a test line was observed. The R2 value for the calibration curve of the cortisol test strip was 0.95, and the relationship between its reflectance, r, and the cortisol level was r = 0.629 (1−0.232e(−1.78 × cortisol)). The fabricated cortisol biosensor enabled us to analyze cortisol concentrations between 1 – 10 ng/ml within 25 min of dropping a cortisol solution on the test strip. Thus, it is suggested that the cortisol biosensor may possibly be used for point-of-use measurements in hospitals.

Automated-immunosensor with centrifugal fluid valves for salivary cortisol measurement

Point-of-care measurement of the stress hormone cortisol will greatly facilitate the timely diagnosis and management of stress-related disorders. We describe an automated salivary cortisol immunosensor, incorporating centrifugal fluid valves and a disposable disc-chip that allows for truncated reporting of cortisol levels (<15 min). The performance characteristics of the immunosensor are optimized through select blocking agents to prevent the non-specific adsorption of proteins; immunoglobulin G (IgG) polymer for the pad and milk protein for the reservoirs and the flow channels. Incorporated centrifugal fluid valves allow for rapid and repeat washings to remove impurities from the saliva samples. An optical reader and laptop computer automate the immunoassay processes and provide easily accessible digital readouts of salivary cortisol measurements. Linear regression analysis of the calibration curve for the cortisol immunosensor showed 0.92 of coefficient of multiple determination, R2, and 38.7% of coefficient of variation, CV, for a range of salivary cortisol concentrations between 0.4 and 11.3 ng/mL. The receiver operating characteristic (ROC) curve analysis of human saliva samples indicate potential utility for discriminating stress disorders and underscore potential application of the biosensor in stress disorders. The performance of our salivary cortisol immunosensor approaches laboratory based tests and allows noninvasive, quantitative, and automated analysis of human salivary cortisol levels with reporting times compatible with point-of-care applications.

Rapid hormone immunosensor with fluid control mechanism

The objective is to develop a novel methodology to analyze cortisol levels as an index of stress-related diseases. In order to realize a quick and easy-to-use biosensor of cortisol, we proposed a cortisol immunosensor using an electrochemical reaction (cortisol sensor). In order to realize it, a fluid control mechanism was newly designed because the sensitivity of an immunoassay is limited by the presence of unreacted labeled conjugate in the sample solution. In this paper, we report on the detected current characteristics of the fabricated cortisol sensor. A competitive reaction between the sample cortisol and a glucose oxidase -labeled conjugate was observed; the detected current was inversely proportional to the concentration of cortisol in the sample solution. The absolute value of maximum detected current reached 200 nA, which was enough to use as an electrochemical sensor. The fabricated cortisol biosensor enabled the analysis of cortisol between 0 – 10 ng/ml, which covers the concentration range in saliva samples.