Shin-Ichi Wakida

From our preceding studies, we disclosed the characteristics of the under-development device, called salivary NO 3 ⁻ Ion-Sensitive Field-Effect Transistors (NO 3 ⁻ -ISFETs). These transistors have evaluated the navigators’ mental workload in maneuvering a ship through the ship simulator experiments which can arrange the uniform experimental circumstances at the different simulator’s scenario. The NO 3 ⁻ -ISFETs give the quantitative evaluation of the mental workload, using NO 3 ⁻ in onedrop saliva. The NO 3 ⁻ -ISFETs are supposed to be used for the navigators working anywhere in a ship around the clock. Therefore, our goal is to complete the NO 3 ⁻ -ISFETs, which have the durability for mucin in saliva when they measure the salivary NO 3 ⁻ long time for the purpose of the prevention of navigator’s human factors in their duties. This time, we tried to carry out another ship-simulator experiment with new NO 3 ⁻ -ISFETs changed the polymer which is used at the device’s sensor part. We can confirm the instrumental difference between conventional NO 3 ⁻ -ISFETs and new ones, while we evaluate the variability of navigators’ mental workload.

[Introduction] Mental stress is closely connected with our physical and mental wellness. In particular, depression may be associated with stress. Therefore, stress measurement can contribute to assess our lifestyle and increase our quality of life. However, objective stress-measurement is not established. To establish objective stress-measurement, an individual must indicate the stress level by obtaining numerous data continuously throughout the day. Therefore, the measurement method which can be suitable for this condition is needed. In this paper, we detect the secretory Immunoglobulin A (sIgA), which is the candidate of salivary stress-markers, with original electrochemical immunoassay: Gold-Linked Electrochemical Immuno Assay (GLEIA). This biosensor is based on a sandwich-type immunosensor, and adopt the electrochemical method to detect the reduction peak from Au nanoparticles linked to the secondary antibody. GLEIA is convenient and cost-effective that only requires low sample volume (10 µL). In addition, all devices that GLEIA needs are minimal such as note PC and portable potentiostat and printed electrode. Therefore, GLEIA method shows various advantages suitable for stress measurement as compared with other sIgA detection methods. In this paper, we aim to the on-site and continuous measurement of salivary IgA in our daily life, to determine the various concentration of sIgA in standard solution and artificial saliva using GLEIA. [Experimental] Primary Anti-IgA Antibody solution was dropped onto the working electrode for immobilizing the antibody by physical adsorption. After, the Au nanoparticle solution was mixed with the phosphate buffer (50 mM, pH=8.3). The anti-IgA to dissolve the 5 mM of phosphate buffer add the Au nanoparticle solution. For the preparing of secondary antibody modified Au nanoparticle, the antibody was conjugated with Au nanoparticle under the pH=8.3 condition and kept for 10 min at room temperature. Then, 10% BSA in PBS buffer and 1% PEG in PBS buffer were added in preparing Au solution. Finally, the concentration was adjusted for OD520=6.0 by centrifuge and exclude the supernatant. The Au Anti-IgA conjugate was diluted by 3 times with 50 w/v of trehalose (OD520=2) and dripping 5µL of this solution in the immunomodule. Then the immunomodule was dried in vacuum condition for 5 minutes. Dried immunomodule was stored at 4 ℃ until use. Different concentrations of the IgA antigen solution (0, 10, 60, 150, 300 ng/mL) were made by diluting in PBS. For the detection of Antibody-Antigen reaction, the IgA solutions were mixed in prepared immunomodule, After the 1 minute, and 1.4 µL of the solution was placed on immunosensor to incubate for 1 hour, after rinsing with PBS, and eliminate the PBS solution with the air gun. The direct redox reaction was performed in 2M KCl solution (20 µL) covering the entire three-electrode zone at room temperature. [Results and Discussion] We obtained the linear response to relate the concentration of sIgA (10-300 ng/mL) in D-PBS buffer with the artificial-saliva which includes salivary inorganic salt and typically glycoprotein (mucin). Figure 1 shows the differential pulse voltammogram of GLEIA with IgA in artificial saliva (a) and calibration curve (b). In addition, the calibration curve shows the linear range within 10-300 ng/mL and the correlation coefficient (R2) of the calibration curve for this relationship was 0.963. The peak current intensity is decreased from in standard solution. It may prevent the Antigen-Antibody reaction from artificial saliva which pH is nearly 8.5. Generally, the antibody shows the best affinity in pH= 6.5~8.4 condition. However, these results show that GLEIA is suitable for salivary IgA detection because the salivary pH is mainly neutral. Furthermore, we evaluate the selectivity of the immunosensor by comparing it with the reduction peak between IgA= 300ng/mL or 0 ng/mL and interference protein such as α-amylase, human serum albumin, immunoglobulin G (IgG), lysozyme and mucin. We acknowledge GLEIA shows the signal increasing within the existence of interference proteins. This may be the affinity of antibody and non-specific reaction between antibody and the proteins. More importantly, we recognize that the reduction peak is increasing with the concentration of sIgA in the existence of interference proteins. Therefore, GLEIA can be applied for stress measurement using salivary sIgA that can be used in daily life anywhere and anytime whenever necessary. In the future, we try to detect the sIgA in real saliva for on-site stress measurement using GLEIA and to integrate the various immunosensors for stress-markers in saliva.nd anytime whenever necessary. In the future, we try to detect the sIgA in real saliva for on-site stress measurement using GLEIA and to integrate the various immunosensors for stress-markers in saliva. Figure 1

[Introduction] We have studied the stress measurement by making use of salivary nitrate, which can be a candidate of stress markers. Ion-selective Field-Effect Transistors (ISFETs) are suitable for the on-site single-drop analysis of salivary nitrate within 10 seconds. The ISFETs are based on field-effect transistors (FET) combined with an ion-selective membrane (ISM). The ISFETs are characterized by the ISM which consists of ionophore, plasticizer and polymer matrix. An ionophore and a plasticizer of the ISM are optimized to determine salivary nitrate from our researches. However, the performance of the polymer matrix is not enough to do so. Hence, the ISFETs have a problem for salivary nitrate which is called the initial drift. The initial drift makes it difficult to determine salivary nitrate. Fogt, et al. considered that the cause of the initial drift is the aqueous layer on between the ISM and the ISFETs’ gate (gate)-interface generating the pH change. This pH change is concerned with CO₂ gas diffusion which approaches the aqueous layer through the ISM. The gate is made of pH-sensitive material (Ta₂O₅, SiO₂, and Si₃N₄), hereinafter pH-sensitive ISFETs called pH-ISFETs. Other previous researches attempted to insert the inner layer: pHEMA-gel saturated buffer, Ag/AgCl and H⁺ selective membrane in the aqueous layer. On the other hand, Abramova, et al. considered that the cause of initial drift is the protein adsorption to the ISM surface. Photocurable polyurethane as a matrix membrane is effective to measure the electrolyte of blood electrolyte. However, there is no report that aims to suppress drift in saliva. In this research, we verified polymer stability of ISM and introduce the novel polyurethane matrix. Actually we used three polyurethanes as polymer matrix: a novel polyurethane A and a conventional B and C. The polyurethane is widely used as a catheter in the medical field. Hence, polyurethane is expected to suppress the adsorption of glycoproteins in saliva. Furthermore, the ISM based on the novel polyurethane A is adhesiveness and it can be closely adhered to the gate to restrict the appearance of the aqueous layer. [Method] Nitrate selective ISFETs (NO₃-ISFETs) are prepared by the following procedure. We dissolve completely and make the THF solution which forms the nitrate-selective membrane, containing 5 wt% of the nitrate ionophore ([Cu(bcp)₂]NO₃), 65 wt % of the plasticizer (NPDDE) and 30 wt% of the polymer matrixes. We cast the THF solution onto the gate part of the pH-ISFETs. After this casting, THF solution on the gate part is evaporated in a clean space, and this manner repeats several times. After all, the nitrate-selective membrane is completely dried half a day. The thickness of the nitrate selective membrane is ca. 0.2 mm. The NO3-ISFETs was conditioned in 1 mM (mol/L) KNO₃ solution (standard solution) for 3 hours. Its calibration curve and selectivity coefficients are determined using the Nicolsky-Eisenman’s equation. The evaluation of initial drift is performed by looking at the output values of ISFETs immersed in various solution about 8 hours. Main measurement target is human saliva. Two types of human saliva are prepared for the sampling method. The one is obtained from the saliva-collection kit (Salivette^®) and another is obtained from a metal spoon. For comparison of the initial drift, we use four kinds of solutions: standard solution, artificial saliva, the standard solution with 0.5 wt% of Mucin (mucin solution), and standard solution with CO₂ bubbling (CO₂ solution). Artificial saliva is the aqueous solution reproducing human’s salivary electrolytes. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the saliva sample experiments were approved by the Ethics Committee of National Institute of Advanced Industrial Science and Technology (AIST). [Results and Discussion] The calibration curves and selectivity the coefficients show that potential determinants are not polymer matrix but ionophore and plasticizer. The drift characteristics of ISFETs based on the novel polyurethane A is more stable than other ISFETs based on the conventional polyurethanes B and C. These results may be due to the adhesiveness of the novel polyurethane A. On the other hand, we hypothesize that the urethane bond captures H⁺ and OH^- in the ISM. However, this hypothesis is not applied for the initial drift in conventional polyurethane membrane. Therefore, this hypothesis is under investigation and we would like to introduce and discuss the details on the day of my presentation.

We have investigated human-stress monitoring by making use of salivary nitrate, which can be a candidate for stress markers, with ion-selective field-effect transistors (ISFETs). ISFETs are suitable for on-site single-drop analysis of salivary nitrate within 10 s. However, when ISFETs are used for salivary nitrate, ISFETs have a problem that is called the initial drift. The initial drift makes accurate nitrate monitoring difficult. Thus, the purpose of this study is to prevent the initial drift and to search for a new, simple polymer to possess a better performance of sensor responses than conventional matrix membranes, such as PVC. In this research, we investigated ISFETs using specific matrix membranes, for example KP-13, Pellethane^®­_­, and P7281-PU. The initial drift was evaluated from the fluctuations of the response values generated by the ISFETs when immersed in saliva or aqueous solution. As a result, P7281-PU showed a prevention effect on the initial drift, both in the whole saliva and in various solutions. Furthermore, the cause of drift may be H⁺ diffusion, and the drift prevention effect of P7281-PU may be affected by urethane bond capturing H⁺ in the ion-selective membrane. This result suggests that a continuous nitrate monitoring is feasible and may be applied to wearable sensors.

We have studied on the stress measurement by making use of salivary nitrate, which can be a candidate of stress markers, with ion-selective field-effect transistors (ISFETs). ISFETs are suitable for on-site single-drop analysis of salivary nitrate within 10 seconds. However, when ISFETs are used for salivary nitrate, ISFETs have a problem which is called the initial drift. The initial drift makes it difficult for determination of an accurate nitrate monitoring. Thus, the purpose of this study is to suppress an initial drift and to search for new easy polymer to possess more performance of sensor responses than conventional matrix membrane such as PVC. In this research, we investigated ISFETs using specific matrix membrane for example, KP-13, Pellethane® and P7281-PU. The initial drift was evaluated from the fluctuation of the response values generated by ISFETs which are immersed in saliva or aqueous solution. As a result, P7281-PU showed its suppression effect for the initial drift in the whole saliva and various solutions. Furthermore, the cause of drift may be H+ diffusion, and drift suppression effect of P7281-PU may be affected by urethane bond capturing H+ in ion-selective membrane. This result suggests a continuous nitrate monitoring and development of wearable sensors.

The goal of our research is to quantitatively assess the mental workload of port-coordinators, who perform radio communication duties for port and harbor, by using salivary NO ₃ ^- . We confirmed that salivary NO ₃ ^- which is one of the physiological indices responds to mental workload, but it is not clear how it responds to various events [1]. It is necessary to examine the relationship between the response from salivary NO ₃ ^- and various events to make how salivary NO ₃ ^- responds to various events clear. Therefore, we utilize the duties list for port-coordinators which can classify the events to the duty items of that. The events are the points where the port-coordinators receive heavy mental workload. However, we confirmed that the existing duties list is not appropriate to examine the relationship between mental workload and various events. This research aims to construct a new duties list for port-coordinators to assess the mental workload. In this research, we measure salivary NO3 ^- of the port-coordinators and improved the items of the duties list on the basis of heavy mental workload events and the existing duties list. We inspect the correspondence between heavy mental workload events and the items of the duties list. Through this research, we could construct a new duties list for port-coordinators which can examine the relationship between the response from salivary NO ₃ ^- and various events. We have become able to classify various events of port-coordinators as their duty contents by using the new duties list. We will be able to clarify how salivary NO ₃ ^- responds to various duties of port-coordinators. Therefore, we will be able to assess quantitative their mental workload.

This paper aims to acknowledge the trend of mental workload of a newly-appointed marine pilot compared with that of a veteran pilot, using the physiological index, salivary NO ₃ ^- and LF/HF values, and confirm the effectiveness of salivary NO ₃ ^- as a physiological index. This paper also aims to confirm that the pilots and port-coordinators' trends of mental workload respond risky situations including collision with other vessels in their duty. Pilots handle vessels for the captain in a specific port or harbor and pilots contribute the safety of the sea area. That is why to verify pilots' mental workload is essential for the purpose of preventing the risky situations. On the other hand, port-coordinators play a role of sea-safe controller at a port, and their advices by wireless radio support pilot's duty. To confirm and verify port-coordinators' mental workload is also essential. From this experiment we confirm the salivary NO ₃ ^- and LF/HF values are useful to evaluation pilots' mental workload, and are also useful for a newly-appointed pilot's drill on board.

Herein, we report on the fabrication of an extended-gated organic field-effect transistor (OFET)-based immunosensor and its application in the detection of human chromogranin A (hCgA). The fabricated OFET device possesses an extended-gate electrode immobilized with an anti-CgA antibody. The titration results of hCgA showed that the electrical changes in the OFET characteristics corresponded to the glycoprotein recognition ability of the monoclonal antibody (anti-CgA). The observed sensitivity (detection limit: 0.11 µg/mL) and selectivity indicate that the OFET-based immunosensor can be potentially applied to the rapid detection of the glycoprotein concentration without any labeling.