Elsevier

Biosensors and Bioelectronics

Volume 21, Issue 7, 15 January 2006, Pages 1007-1014
Biosensors and Bioelectronics

Hand-held monitor of sympathetic nervous system using salivary amylase activity and its validation by driver fatigue assessment

https://doi.org/10.1016/j.bios.2005.03.014Get rights and content

Abstract

In order to realize a hand-held monitor of the sympathetic nervous system, we fabricated a completely automated analytical system for salivary amylase activity using a dry-chemistry system. This was made possible by the fabrication of a disposable test-strip equipped with built-in collecting and reagent papers and an automatic saliva transfer device. In order to cancel out the effects of variations in environmental temperature and pH of saliva, temperature- and pH-adjusted equations were experimentally determined, and each theoretical value was input into the memory of the hand-held monitor. Within a range of salivary amylase activity between 10 and 140 kU/l, the calibration curve for the hand-held monitor showed a coefficient with R2 = 0.97. Accordingly, it was demonstrated that the hand-held monitor enabled a user to automatically measure the salivary amylase activity with high accuracy with only 30 μl sample of saliva within a minute from collection to completion of the measurement. In order to make individual variations of salivary amylase activity negligible during driver fatigue assessment, a normalized equation was proposed. The normalized salivary amylase activity correlated with the mental and physical fatigue states. Thus, this study demonstrated that an excellent hand-held monitor with an algorithm for normalization of individuals’ differences in salivary amylase activity, which could be easily and quickly used for evaluating the activity of the sympathetic nervous system at any time. Furthermore, it is suggested that the salivary amylase activity might be used as a better index for psychological research.

Introduction

Human whole saliva contains not only pure saliva secreted from the salivary glands but also some body fluid derived from the glands and tissues of non-salivary origin, including gingival crevicular fluid (GCF) (Brill and Krasse, 1958, Lenander-Lumikari and Loimaranta, 2000) and bronchial secretion fluid. Pure saliva is considered to be a body fluid and to contain some chemical components similar to those in blood (Tenovuo, 1989). It is considered that the chemical components contained in pure saliva reflect the concentrations of electrolytes, enzymes, hormones, immunological molecules and administered drugs. Therefore, one of the most promising areas of biomarker research is the application of portable diagnostic technologies for detecting and analyzing components in saliva (Speirs, 1984, Guilbault and Palleschi, 1995, Streckfus and Bigler, 2002).

Saliva sampling has the advantage that it is noninvasive, making multiple sampling easy and stress free. In order to quantify psychological stress and to distinguish activated and inactivated physical states of the sympathetic activity, we have been investigating the establishment of a method that can quantify the concentration of alpha-amylase in saliva (salivary amylase activity, Yamaguchi et al., 2001, Takai et al., 2004). The activated states are ambiguously defined and diversely assessed (Friedman et al., 1985). An example of a person in an activated state might be a driver who struggles for long hours at his car constantly tapping his fingers, watching rearview mirror and front car at the same time and becomes highly irritated by heavy traffic or missing a turning/losing his way.

Alpha-amylase is one of the major salivary enzymes in humans, and is secreted from the salivary glands in response to sympathetic stimuli (Gallacher and Petersen, 1983). Chatterton et al., reported in 1996 that there was a good association between the salivary amylase activity and blood levels of cathecholamines. Currently, it is considered that measurement of this salivary amylase is a useful tool for evaluating the sympathetic activity of the sympathetic nervous-adrenomedullary (SAM) system (Walsh et al., 1999, Skosnik et al., 2000).

The authors previously reported that a flow-injection-type enzyme-based biosensor could be utilized for the continuous analysis of salivary amylase activity (Yamaguchi et al., 2003). If this biosensor is portable, it would allow us to assess more psychological research. It is considered that a dry-chemistry system may be more suitable for a portable biosensor than the flow-injection system. Since a specific system to control the reaction time was required to quantify the enzymatic activity using the dry-chemistry system, we proposed a saliva transfer mechanism and fabricated a monitor of salivary amylase activity with a dry-chemistry system in an experimental form (Yamaguchi et al., 2004). However, in order to take advantage of the merits of saliva sampling, that is, samples can be collected at any time, as quickly as desired and with ease, the saliva transfer mechanism needed to be automated for sampling and analyzing. The enzyme activity will change because of cooling down the collected sample saliva to environmental temperature and the pH of the individuals’ saliva. Moreover, because of relatively large individual variations in the basis value of salivary amylase activity, an algorithm to evaluate the activation or inactivation of the sympathetic nerve system as assessed by the salivary amylase activity was needed in order to develop a hand-held monitor of the sympathetic nervous system.

The purpose of this paper is to demonstrate a new design of a portable and rapid hand-held monitor of the sympathetic nervous system using salivary amylase activity, and to illustrate its feasibility for in vivo monitoring of sympathetic stimuli. Firstly, the structure and mechanism of a disposable test-strip that was fabricated to make an automatic saliva transfer mechanism was described. Then, temperature- and pH-characteristics of salivary amylase activity were investigated and their characteristic profiles were put into the system in order to adjust the variation of conditions. Finally, a driver fatigue assessment was conducted in order to demonstrate the validity of the algorithm that evaluated the sympathetic nerve activity using the measured salivary amylase activity. Recently, there have been extensive studies to monitor and assess the degree of fatigue during a driving task, in order to improve the safety of transportation systems (Sluiter et al., 1998, Saroj and Craig, 2002, Morrow and Crum, 2004, Belz et al., 2004). Based on these results, we discussed the usefulness of the hand-held monitor of the sympathetic nervous system.

Section snippets

Hand-held monitor of the sympathetic nervous system

A hand-held monitor of the sympathetic nervous system that we fabricated was the system to measure the enzymatic activity in a batch type, using a reagent paper containing 2-chloro-4-nitrophenyl-4-O-β-d-galactopyranosylmaltoside (Gal-G2-CNP, Toyobo Co. Ltd., Japan), a substrate for amylase. When Gal-G2-CNP is hydrolyzed by amylase, the hydrolyzed product (CNP) develops a yellow color with time in the following reaction equation (Yamaguchi et al., 2004).Gal-G2-CNPalpha-amylaseGal-G2+CNP(white

Temperature and pH characteristics

It was observed that salivary amylase activity reduced proportionally to a decrease in temperature (Fig. 4a). In particular, the salivary amylase activity at 10 °C decreased by approximately 80% as compared to that at 37 °C. The following equation (Eq. (3), R2 = 0.99) was obtained as a temperature-adjusted equation to normalize the amylase activity with regard to the standard measurement temperature conditions (NC-IUBMB, 1992, 37 °C).%AMY=0.048T2+0.59T+12.1(%)In order that measured results were

Conclusions

In order to realize a hand-held monitor of the sympathetic nervous system, we fabricated an automatic analytical system for salivary amylase activity using a dry-chemistry system. A system to quantify salivary amylase activity by the rate method was completely automated. This was made possible by the fabrication of a disposable test-strip equipped with both collecting and reagent papers and the automatic saliva transfer device. In order to cancel out the effects of variations in environmental

Acknowledgements

Part of this research was supported by the FY2003 Promotion Program of the Business–Academic–Public Sector Cooperation Centering on Universities by the Ministry of Education, Culture, Sports, Science and Technology of Japan. The authors wish to thank Mr. H. Yoshida, manager at Nipro Co., Research and Development Laboratory, for providing the chromogen used in these experiments; and Mr. Iwao Hamano at Tonami Transportation Co. Ltd. for providing their drivers.

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