Detection of Stress-Related Secretory IgA in Human Sweat Using Lectin-Immobilized Field Effect Transistor Biosensor

Abstract

A field effect transistor (FET) biosensor is a promising device for various applications such as medical diagnosis and environmental monitoring. Because characteristics of FET biosensors are directly influenced by the change of gate-insulator surface potential induced by the adsorption of charged molecules, FET biosensors could provide the rapid and label-free biomolecular detection. Recently, mental stress-related diseases, such as integration disorder syndrome and depression, affect people's health, resulting that simple stress monitoring is expected for early stage detection of the disease. Previously, the relation between concentration of stress markers and mental stress has been reported [1], and the monitoring of circadian concentration of the markers is found to be important for prediction of the stress condition. Especially, secretory immunoglobulin A (s-IgA), which is an immunity-related molecule present in the human mucus, is one of the candidates to be monitored as a stress marker. However, conventional methods for measuring concentration of s-IgA are restricted in daily use due to complex protocol, time-consuming and expensive equipment.Nowadays, we have investigated sensitive detection method for various targets by using the FET biosensor [2,3]. To achieve improvement of the sensitivity, small receptors have been applied to increase electrical responses owing to the effective use of a charge-recognition region from FET gate surface, Debye length [4,5]. In this study, we selected a small plant lectin, jacalin (66 kDa), which specifically binds glycan of hinge region of IgA, as a receptor. Additionally, jacalin was inexpensive compared with antibody due to the purification from jackfruits seeds. From these points, jacalin-immobilized FET biosensor was worth to be investigated to realize a simple, sensitive and low-cost stress monitoring device for stress marker. Thus, we investigated the usefulness of the jacalin as a FET receptor.The SiO2 gate insulator of the FET was exposed to O2 plasma (200 W for 1 min) for introduction of hydroxyl groups reacting with triethoxysilane groups of self-assembled monolayer (SAM). Then, the FET chip was immersed in toluene solvent including 1%(v/w) 3-aminopropyltriethoxysilane in an argon atmosphere (60ºC for 7 min.). Following by the cross-linking by glutaraldehyde, jacalin was immobilized on FET gate surfaces. Finally, ethanolamine capping was performed to prevent the non-specific adsorption of contaminating molecules in analyzed samples, resulting in the fabrication of the jacalin-immobilized FET biosensor. The FET characteristics were measured by sweeping the gate-voltage (V g) from -2.0 V to 0 V with 0.1 V drain voltage (V d) in 0.01 × phosphate buffered saline (pH 7.4). Then, the electrical responses (ΔV g) were analyzed from the FET characteristics before and after the addition of analyte to gate surface.To evaluate the specificity of jacalin-immobilized FET biosensor, ΔV g caused by the addition of s-IgA and human serum albumin (HSA) were measured. The FET charactristics was shifted in a positive direction (+53 mV) due to the adsorption of negative-charged s-IgA (Figure 1a), while the responses related with HSA addition were scarcely observed. Thus, specific capture of the s-IgA molecules by the jacalin-immobilized surface was indicated. Moreover, to evaluate the advantage of jacalin, we compared ΔV g with FET functionalized by antigen binding fragment (Fab), which was obtained by cleaving the anti-s-IgA antibody. An electrical response of Fab-immobilized FET was +24 mV (Figure 1b). The change in ΔV g values for these two FET sensors using jacalin or Fab could be discussed as follows. Jacalin could capture more s-IgA molecules within Debye length from the gate surface of FET. In addition, the jacalin-immobilized FET responded linearly to s-IgA in a concentration range from 0.1 μg/mL to 100 μg/mL. Finally, sweat samples collected from healthy persons were examined with the developed jacalin-immobilized FET biosensor, and clear responses were obtained. From these results, jacalin was found to be useful as a receptor for FET biosensors to achieve a sensitive, simple and non-invasive detection of s-IgA.[1] K. Obayashi, Clin. Chim. Acta, 425, 196-201 (2013).[2] S. Hideshima, M. Kobayashi, T. Wada, S. Kuroiwa, T. Nakanishi, N. Sawamura, T. Asahi, T. Osaka, Chem. Commun., 50, 3476-3479 (2014).[3] S. Hideshima, K. Fujita, Y. Harada, M. Tsuna, Y. Seto, S. Sekiguchi, S. Kuroiwa, T. Nakanishi, T. Osaka, Sens. Bio-Sens. Res., 7, 90–94 (2016).[4] S. Cheng, K. Hotani, S. Hideshima, S. Kuroiwa, T. Nakanishi, M. Hashimoto, Y. Mori, T. Osaka, Materials, 7, (4), 2490-2500 (2014).[5] S. Hideshima, H. Hayashi, H. Hinou, S. Nambuya, S. Kuroiwa, T. Nakanishi, T. Momma, S.-I. Nishimura, Y. Sakoda, T. Osaka, Sci. Rep., 9, 11616 (2019).Figure 1 V g-I d characteristics of (a) jacalin or (b) Fab-immobilized FET biosensor before and after the addition of 100 μg/mL s-IgA. Figure 1