Elsevier

Analytica Chimica Acta

Volume 806, 2 January 2014, Pages 1-7
Analytica Chimica Acta

A novel non-invasive electrochemical biosensing device for in situ determination of the alcohol content in blood by monitoring ethanol in sweat

https://doi.org/10.1016/j.aca.2013.09.020Get rights and content

Highlights

  • New electrochemical biosensing device for determining the blood's ethanol content (BAC).

  • Prototype based on bienzyme amperometric composite biosensors.

  • Determination of BAC by amperometric monitoring of ethanol in sweat.

  • BAC determination in single measurement or in continuous modes.

  • Successful validation with 40 volunteers.

Abstract

A non-invasive, passive and simple to use skin surface based sensing device for determining the blood's ethanol content (BAC) by monitoring transdermal alcohol concentration (TAC) is designed and developed. The proposed prototype is based on bienzyme amperometric composite biosensors that are sensitive to the variation of ethanol concentration. The prototype correlates, through previous calibration set-up, the amperometric signal generated from ethanol in sweat with its content in blood in a short period of time. The characteristics of this sensor device permit determination of the ethanol concentration in isolated and in continuous form, giving information of the BAC of a subject either in a given moment or its evolution during long periods of time (8 h). Moreover, as the measurements are performed in a biological fluid, the evaluated individual is not able to alter the result of the analysis. The maximum limit of ethanol in blood allowed by legislation is included within the linear range of the device (0.0005–0.6 g L−1). Moreover, the device shows higher sensitivity than the breathalyzers marketed at the moment, allowing the monitoring of the ethanol content in blood to be obtained just 5 min after ingestion of the alcoholic drink. The comparison of the obtained results using the proposed device in the analysis of 40 volunteers with those provided by the gas chromatographic reference method for determination of BAC pointed out that there were no significant differences between both methods.

Introduction

The measurement of ethanol for clinical and safety purposes in fluids other than blood has become important because of major demands for non-invasive analysis [1]. In this context, sweat constitutes an attractive alternative because it is easier to get personal agreement for sampling, and its testing offers advantages over urine and blood regarding the easiness of collection under certain circumstances, such as the monitoring of drivers or individuals in safety-related work [1], [2].

Besides enzymatic degradation, orally administered ethanol is also eliminated through skin perspiration [3], [4], [5]. In fact, some publications suggest using human perspiration, instead of blood, for estimating ethanol concentration [4], [6], [7], [8] or other clinically relevant analytes such as lactate [9]. In these works, concentration of ethanol in the sweat patch was measured by the aspiration of an equilibrated head-space sample into an electrochemical detector [6], [7] or a gas chromatography system [4], [8]. The amount of ethanol leaving the skin after single liquor consumption was determined using ion mobility spectrometry [5]. Kamei et al. proposed a novel instrumentation for the estimation of ethanol concentration in sweat consisting of a sampling probe attached directly to the skin surface, a sweat rate meter, a cold trap and capillary gas chromatography. The accuracy of the measurement of ethanol in blood was 0.1 mg mL−1 [1]. These authors demonstrated for the first time the existence of a clear correlation between ethanol concentrations in sweat and blood during its consumption in the human body. Later, Buono et al. concluded that ethanol concentration in sweat was approximately 19% more than in whole blood. They measured ethanol concentrations using an enzymatic technique and the slope of the blood vs. sweat ethanol concentration plot was 0.81 [10].

Currently BAC is estimated by measuring breath alcohol concentration (BrAC), the different generations of breath alcohol testing instruments used for BrAC detection since 1930 having been reviewed by Wigmore et al. [11]. Breathalyzers, at least as they are currently used in practice, are not calibrated to each individual subject. These instruments estimate BAC via an idealized linear stoichiometric calculation based on Henry's law. However, the relationship between BAC and BrAC is not so simple and actually may vary from instrument to instrument and individual to individual [12].

Transdermal sensing systems are touch-sensors that can continuously monitor the analyte level in a person. In the particular case of BAC monitoring, traces of alcohol are present in the person's sweat when alcohol is consumed. Therefore, transdermal BAC devices measure BAC based on how much alcohol is present in perspiration. The only wearable transdermal alcohol sensor reported until now was the Giner WrisTAS V, developed primarily to monitor alcohol abstinence. Placed on the skin surface, the sensor oxidizes ethanol in a continuous manner, generating a current that is linearly related to the local alcohol concentration. It responds to 10  200 mg dL−1 BAC with a lag time of ∼30 min to plateau [12]. Recently, Dumett et al. have developed a calibrated model for estimating (breath measured) blood alcohol concentration from measurements of transdermal alcohol produced by the Giner WrisTAS V alcohol sensor. However, a number of unknown subject and device specific physical and physiological parameters must be estimated before the model can be used [12].

In this work a prototype for in situ and real time determination of ethanol content in blood through measurement of the amperometric signal obtained from the monitoring of ethanol in sweat, collected following pilocarpine iontophoresis, is described. The prototype comprises a sensing system based on an electrochemical alcohol oxidase/horseradish peroxidase (AOD/HRP) biosensor, a miniaturized potentiostat and a microprocessor that transduces the analytical signal obtained in perspiration into the BAC. As the proposed method uses non-invasive sampling, it may be applicable to determine BAC instead of the gas chromatography conventional method using blood sample. The goal for this prototype is to compete with the breathalyzers that are currently commercialized, as measurement of ethanol is performed directly in a biological fluid (sweat) hardly altered by the user. The proposed instrumentation may be very useful in checking drivers’ ethanol consumption or to protect workers from risk due to the residual effects of ethanol digestion.

Section snippets

Apparatus and electrodes

Composite bienzyme electrodes were fabricated in the form of cylindrical pellets as reported earlier [13]. Graphite (ultra F purity; Carbon of America, Bay City, USA), AOD (EC 1.1.3.13, from Pichia pastoris, activity 1430 U mL−1, Sigma, St. Louis, MO, USA), HRP (EC 1.11.1.7, type II, activity 240 U mg−1 of solid, Sigma), ferrocene (Fluka, Buchs, Switzerland) as redox mediator, and Teflon powder (Aldrich) were used to construct the pellets. A final Teflon percentage of 70% was employed. Several 3.0 

Results and discussion

The biochemical principle on which the biodevice behavior relies is the measurement of ethanol in sweat by means of a composite graphite-Teflon-AOD-HRP-ferrocene electrode. This biosensor responded to ethanol which is catalytically oxidized by AOD in the presence of oxygen, producing H2O2. The enzyme reaction with HRP mediated by ferrocene was employed to monitor the process at the low potential value of 0.00 V vs. the Ag/AgCl reference electrode. The measured current corresponded to the

Conclusions

A novel electrochemical biosensing device for determining the BAC by monitoring ethanol content in sweat has been developed. Very rapid and repeated analyses are possible by using the exclusive biodevice which permits BAC determination in single measurement or in continuous modes, giving information in real time and in a non-invasive way of the BAC for a subject in a particular moment or during long periods of time. The biodevice was successfully validated with 40 volunteers who drank different

Acknowledgements

The financial supports of Bialcon Project founded by Intecsa Inarsa S.A. and of the Spanish Ministerio de Economía y Competitividad Research Project CTQ2012-34238 are gratefully acknowledged. M. Gamella and S. Campuzano acknowledge their contracts to the Intecsa Insarsa S.A. Company.

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These authors have equally contributed to this work.

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