Wednesday, 4 October 2017
Prince George's Exhibit Hall D/E (Gaylord National Resort and Convention Center)
The sensing of biomarkers in the body is very effective for diagnosis of disease and monitoring of human health condition. However, blood sampling of mental stress markers is stressful for those who do not feel serious health concerns when they want to check only their own stress level. We need to measure stress markers noninvasively in our daily lives. Saliva is an ideal biological sample to be taken noninvasively. α-amylase and cortisol are known as stress markers in saliva. Since individual differences in stress response are large, it is important to simultaneously detect multiple markers and evaluate the stress level. In this study, we attempted to fabricate two field effect transistor (FET) biosensors using aptamers against α-amylase and cortisol as indexes of stress. Our purpose was to investigate the characteristics of sensing two types of biomarkers, a protein and a steroid molecule, using aptamer-immobilized FET biosensors. The aptamer against α-amylase and the one against cortisol were obtained after eight rounds of selection by SELEX method from an initial DNA library. The gate insulator (SiO2) of FET was modified with 3-aminopropyltriethoxysilane, followed by addition of glutaraldehyde. Each of two aptamers, which are terminated with amino group, was immobilized on the thus-prepared surface of insulator. We detected α-amylase and cortisol in phosphate buffer using the aptamer against α-amylase and the one against cortisol respectively. An increase in the magnitude of response was observed with an increase of α-amylase concentration in the range between 1 nmol/dm3 and 100 nmol/dm3. FET sensor responded as a gate voltage shift to the change in charge on the gate insulator surface. The observed response was suggested the binding of negatively charged α-amylase (isoelectric point = 6.34) to the aptamer. Though cortisol is an electrically neutral molecule, an increase in the magnitude of response was observed with an increase of cortisol concentration in the range between 1 μmol/dm3 and 1 mmol/dm3. It was a similar behavior toward the increase of negative charge or the decrease of positive charge. The phosphate groups of DNA aptamers and their counter ions have an electrical charge on the gate insulator. This suggests that the cortisol aptamer contracted and its negative charge penetrated Debye length or that the counter ion bound to the aptamer beforehand was removed after the association of cortisol. The concentration of α-amylase in saliva is 1 - 3 μmol/dm3. The sensitivity of this α-amylase sensor is at a practical level. The concentration of cortisol in saliva is 3 - 11 nmol/dm3. This cortisol FET sensor requires optimization of aptamer and refinement of the device for practical application. The quantitative detection of α-amylase and cortisol using aptamer-immobilized FET sensor was achieved. This sensor can be sensitive to a structural change of aptamer induced by the target binding. It was demonstrated that the detection of the neutral target molecule is possible.
Acknowledgement
This work is supported by Adaptable and Seamless Technology transfer Program through targetdriven R&D (A-STEP), and the Center of Innovation S&T-Based Radical Innovation & Entrepreneurship Program (COI-STREAM), both from the Japan Science and Technology Agency (JST) and the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.