Verification of a Stability of Polymer Matrix for the Salivary-Nitrate-ISFETS
Authors: Osaki Shuto , Kintoki Takuya , Moriuchi Takayo , Kitamura Kenichi , Wakida Shin-ichi 
Published: February 2020

[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.