(1) University of Jember, Jember, Indonesia
(2) Universiti Sains Islam Malaysia, Negeri Sembilan, Malaysia
* Corresponding author Email:
A biosensor is an analytical tool that comprises two essential components, an immobilized bio-component, in intimate contact with a transducer that converts a biological signal into a measurable electrical signal. Both electrochemical and optical transducers are mainly transduction methods that are employed in biosensor developments. This review summarises the studies carried on ethanol determination based on enzyme biosensors, using alcohol dehydogenase (ADH), alcohol oxidase (AOX) or bi-enzyme system, the various techniques of immobilisation, the transducers used and analytical characteristics for biosensor development are described. Almost all enzyme based ethanol biosensors developed are based on the monitoring of NADH in the case of ADH based biosensor, O2 consumption or H2O2 production in the case of AOX biosensor and H2O2 production in the case of thebi-enzyme system. Underlying the importance of this review is the fact that alcohol istoxic above certain concentrations and its continuous real time monitoring in clinical, environmental and food related environments is of utmost interest.
Ethanol can be efficiently determined using enzyme based biosensors, which are simple to assemble and operate. Both electrochemical and optical methods can be exploited, using either modified electrodes or modified optodes. The most promising ethanol biosensors developed up to now are the bi-enzymatic system based on immobilisation of AOX coupled with HRP. Other applications that require fast and reliable methods for alcohol determination will certainly benefit with the commercial development of more enzyme based biosensors.
According to a proposed IUPAC definition, a biosensor is an analyticaldevice which is capable of providing specific quantitative or semi quantitative analytical information using a biological recognition element (biochemical receptor) which is in direct spatial contact with a transducer element. Generally, the analytical information is a quantifiable electrical signal. Therefore, this integration of biotechnology and electronics has raiseda technology called biosensor technology.[2,3] There are two types of biosensors, i.e. catalytic and affinity biosensors. The catalytic biosensor uses mainly enzymes as the biological component, catalysing a signalling biochemical reaction. The affinity biosensor, designed to monitor the binding event itself, uses specific binding proteins, lectins, receptors, nucleic acids, whole cells, antibodies or antibody-related substances for bio-molecular recognition.[5,6,7] Table 1 shows the main types of transducer usually used in the development of biosensors. The transducers can be classified into four main groups, i.e. optical, electrochemical, mass-sensitive, and thermometric.8 Each group can be further sub-classified into different types, due to the variety of methods used to monitoranalyte-bioreceptor interactions.
The main types of biological components and transducers used in the biosensor development.
Enzymes are highly specific, it was found in many specific applications in biosensors. Most of the enzymesemployed in biosensors have been isolated from micro-organisms;therefore themicroorganisms have also been used as microbial biosensors. Furthermore, cell membranes and organelles have been used for a cell based biosensor, tissues were also used as a tissue based biosensor. Antibodies and antigen interactions have been exploited asimmunosensors, while DNA hybridisation and intercalation have been used in DNA biosensor constructions.
Since, IUPAC definition stated that a biosensor should be clearly distinguished from a bio-analytical system, which requires additional processing steps, such as reagent addition1. This additional processing step usually called immobilization of the bio-reagent, several types of immobilization methods are used. These are adsorption, entrapment, encapsulation, covalent binding, and cross-linking.[12,13] Table 2 enlists some of the advantages and disadvantages of these methods. In general, the choice of method depends on the nature of the biological component, the transducer used, the physicochemical properties of the analyte and the operating conditions in which the biosensor is to be used. All these considerations are the necessity for the biological component to exhibitmaximum activity in its immobilised microenvironment. The bio-component, like enzyme, specifically recognizes the analyte and the physiochemical changes caused by the interaction between the enzyme and the analyte such as change of light absorption or electrical charge etc. are indicated by the transducer. Thus, the key part of the biosensor is the transducer which makes use of the physicochemical change accompanying the bio-reaction. Therefore, the fabrication of a successful biosensor is dependent on characterization of bioassay principle in immobilized form and its coupling with a suitable transducer.
Methods of immobilisation of the biological component
In an alcohol biosensor, two enzymes have been extensively used in the determination of alcohols, namely alcohol dehydrogenase (ADH) and alcohol oxidase (AOX). This is due to the ability of ADH or AOX as a single enzyme molecule to catalyse the reaction of alcohol as a substrate molecule and provides an amplification effect, which enhances the sensitivity of the analysis. Furthermore, the most ADH or AOX catalysed reactions can be followed by simple, widely available transducers e.g. opticalor electrochemical methods as discussed in this review. Therefore, many alcohol biosensors have been applied in various fields ranging from clinical analysis (e.g. blood, serum, saliva, urine, breath and sweat) to food and alcoholic beverages (wine, beer and spirits). The aim of this review was to discuss recent progress in alcohol biosensors.
The authors have referenced some of their own studies in this review. The protocols of these studies have been approved by the relevant ethics committees related to the institutions in which these studies were performed.
Alcohol dehydrogenase based biosensor
Alcohol dehydrogenase (ADH; Alcohol:NAD+oxidoreductase, EC 220.127.116.11) catalyses the reversible oxidation of primary aliphatic and aromatic alcohols other than methanol, according to Eq. (1).
Ethanol biosensors based on ADH have been reported to be more stable and specific than those based on AOX. Nevertheless, ADH biosensors require the external addition of the co-enzyme NAD+. Moreover, the cofactor needs to be close to the enzyme, without becoming irreversibly entrapped or linked.
The common ways to monitor an ADH catalysed reaction have been performed by detection of the products, i.e. the increase in NADH concentration. Therefore, in many biosensor developments NADH have been extensively used as sensing scheme, for instance, an optical biosensor based on fluorescence of the cofactor (NADH) as shown in Figure 2 has been developed for sensing of short-chained alcohols. Since, the free yeast ADH has been used previously in a solution for alcohol that takes advantage of NADH fluorescence.
The scheme of an optical biosensor based on fluorescence of the cofactor (NADH)
NAD+ and its reduced form (NADH), a product of the reaction between NAD+ and primary alcohols, are the key central charge carriers in living cells. NAD+ is a very important cofactor, since it participates in enzymatic catalysis of more than 300 dehydrogenase enzymes.[19,20] In the electrochemical biosensor, the NAD+/NADH redox couple have been used as a sensing scheme as shown in Figure 3 in various electrodes. However, only a limited number of electrochemical biosensors based on ADHs were reported because of the need for cofactors for regeneration.
The electrochemical biosensor scheme in the NAD+/NADH redox couple
The electrochemical oxidation of NADH to the corresponding oxidized form NAD+ at a bare electrode surface is highly irreversible and requires high over-potentials, many studieshave been focused on the development of new efficient electrode materials.[23,24,25,26] In order to reduce the high over-potentials and to minimize the side reactions, various redox mediators immobilized on the electrode surface have been reported in the literature.[27,22,28] Generally, NAD+ was produced as a result of the reaction between an oxidised mediator (Medox) and NADH. Then, as a result of the reaction between NAD+ and ADH, ethanol was converted to acetaldehyde. The redox reactionoccurred on the electrode surface according to the following mechanism:
Medox + NADH à NAD[+] + Medred(2)
MedredàMedox + 2ē (4)
Catechols, ferrocene, phenoxazines, phenothiazines,[30,32] and conducting polymers33 are examples of immobilised redox mediators that have been investigated extensively. The use of such mediators immobilized on an electrode coupled with ADHs and their applications to alcohol biosensor development are also described.[23,34,35]
Alcohol oxidase based biosensor
Alcohol oxidase (AOX; Alcohol:O2 oxidoreductase, EC 18.104.22.168) is an oligomeric enzyme consisting of eight identical sub-units arranged in a quasi-cubic arrangement, each containing a strongly bound cofactor, flavin adenine dinucleotide (FAD) molecule. Commercially available AOX isolated from Candida sp., Hansenula sp. or Pichiapastoris. AOX is the firstenzyme involved in the methanol oxidation, however, it is also able to oxidise other short-chain alcohols, such as ethanol, propanol and butanol. Thus, AOX is responsible for the oxidation of low molecular weight alcohols to the corresponding aldehyde, using molecular oxygen (O2) as the electron acceptor, according to Eq. (5). The oxidation of alcohols by AOX is irreversible, due to the strong oxidising character of O2.
RCH2OH + O2→RCHO + H2O2(5)
The classical ways to monitor an oxidase-catalysed reaction have been performed by measuring either the consumption of O2 or the production of H2O2.
The AOX biosensors that monitor the consumption of O2 are commonly used electrochemical methods, although optical methods can also be used. The most common method of monitoring O2 is based on a Clark-type O2 electrode. The first AOX electrode consisted of a platinum electrode on the surface of which the immobilised AOX was mountedand secured with a nylon cloth and an O-ring. In fact, most ethanol sensors based on O2 probes consist of an electrode covered by a membrane onto which AOX was immobilised.[37,38] The signal output of the electrode is the difference between the base oxygen level (100% saturation) and the level attained as a result of oxygen depletion by the enzymatic reaction. Figure 1 shows a typical schematic diagram of such anO2electrode. The oxygen-based sensor offers the advantage of no electrochemical interference from other sample constituents. However, their practical limitations are slow in response, and lower the accuracy and reproducibility due to oxygen dependency. In addition, due to high in the background signal, the limit of detection is achieved in the ppm range.
The schematic diagram of the proposed biosensor for alcohol detection
In the optical method, fluorescence-based biosensors for alcohols have been reported in literature. For instance, an optical bio-sniffer for ethanol vapours was constructed by immobilising AOX onto a tip of a fibre optic oxygen sensor coated with an oxygen sensitive ruthenium complex. The bio-sensing principle is based on quenching of the ruthenium complex, in the presence of O2 molecules (both liquid and gas-phases). Other optical biosensors were constructed by co-immobilisation of AOX and O2 sensitive dyes, e.g. ruthenium complex derivative on a PVC membrane.41 Due to detection of O2 it has limitations particularly in low accuracy and reproducibility. The detection of H2O2 is the most commonly used alternative to overcome these drawbacks.
AOX biosensors that rely on the production of H2O2 can use the optical or electrochemical method. In the electrochemical method, H2O2 produced by AOX can be detected with amperometric electrodes, either by measuring the anodic or cathodic response, due to the oxidation or reduction of H2O2 at the surface of the working electrode, respectively. The detection of H2O2 by AOX electrodes can be employed in two sensing schemes, i.e. direct and indirect H2O2detection as shown in Figure 4.
The detection of H2O2 by AOX electrodes can be employed in two sensing schemes (a) direct and (b) indirect H2O2 detection
The most important advantages of the H2O2 sensor over other types of sensors are the relative ease of fabrication and miniaturisation. In addition, the biosensors are usually characterised by high upper linearity and wider linear range. However, the high potential necessary to oxidise H2O2 poses a problem of electrochemical interference, due to the presence of reducing compounds present in real sample matrices (such as ascorbic acid and uric acid),which are also oxidised at the same potential. Furthermore, slower responses are observed. In order to overcome the drawbacks encountered with the direct H2O2 detection, alternative routes to follow the production of H2O2 have been proposed. One of the ways to decrease the necessary applied potential is to modify the electrode with an electrocatalyst species (mediators) for either the reduction or oxidationof H2O2. Prussian Blue[43,44] ,Meldola blue, Ferrocyanide,[44,45], ferrocene, cobalt ion and Co-phthalocyanine 44,48 have been proposed asredox mediators.
In the optical biosensor, a fibre-optic chemiluminescence method for the determination of ethanol in beverages has been proposed. In this method, the H2O2 produced was determined by measuring the luminescence of the oxidation of luminol, by H2O2, using K3FeIII(CN)6 as a catalyst. Recently, other methods have also been reported to follow the oxidation of alcohols by AOX. In this method H2O2 produced has been detected using PANI, where H2O2oxidized the PANI film, by changing the colour from green to blue. The biosensor was constructed as a dip stick format for visual and simple use.
Alcohol oxidase-peroxidase based biosensor
In order to circumvent the problem with H2O2 detection is the useof a bi-enzyme system, i.e. ADH and peroxidase (POD). Commonly, POD used is horseradish peroxidase (HRP). The reaction involves oxidation of alcohols by AOX that produce H2O2, which further reduces to H2O by HRP as shown in Figure 5, where the redox reaction can be monitored electrochemically or optically.
The reaction of alcohols oxidation by AOX that produce H2O2, which further reduces to H2O by HRP
Generally many amperometric bi-enzyme electrodes with AOX and HRP(or other peroxidases) use either direct or mediated electron transfer as shown in Figure 6,  Frequently, unmediated enzyme electrodes are characterised by low sensitivities and high detection limits. Hence mediated electrodes have been used to make the electron transfer between the electrode and HRP much faster, thereby increasing the sensitivity and lowering the detection limit.
The scheme of bi-enzyme electrodes with AOX and HRP use either (a) direct and (b) mediated electron transfer
H2O2 detection by peroxidase
Typically, in bi-enzyme systems, a POD is used to efficiently reduce enzymatically generated H2O2 at low working potentials. The biosensors were built based on two different approaches, direct and mediated electron transfer. Unmediated electrodes were constructed by incorporating carbodiimide, glutaraldehyde and polyethyleneimine into the carbon paste to immobilise both enzymes. The surface of the electrodes was protected first by electrode positinga layer of o-phenylenediamine, followed by deposition of a cation exchange membrane. Mediated electrodes were built in the same way butan osmium complex was used to bind electrostatically to HRP.
In the optical based biosensor, colorimetric, chemiluminescent and fluorescent methods can be used to detect the production of H2O2 by AOX during the oxidation of ethanol. Colorimetric methods are based on the conversion of a chromogen substrate into a coloured product, which absorbs in the visible spectral region. Fluorescent methods are based on the formation of a fluorophore product, which emits visible light after being stimulated with a shorter wavelength radiation. Finally, chemiluminescence consists in the emission of visible light upon chemical reaction. Most of the optical methods reported in the literature for the determination of ethanol are based on a bi-enzymatic system, comprising AOX and a POD, which further reduces H2O2 to H2O at the expense of hydrogen donor molecules. A wide range of substrates have been used, including chromogenic, fluorigenic and luminogenic substrates. One of the most popular substrates used is 2,2-azino-di(3-ethylbenzthiazoline-6-sulfonate), ABTS. The oxidised form of ABTS has a bluish green colour and is usually detected around 415 nm. In this method AOX and HRP were immobilised on the inside surface of a 96-well microtitre plate.
Ethanol containing samples were added to the AOX microtitre plate and after the reaction (30 min), a sample was transferred to the HRP plate, which already contained the chromogen, ABTS. A linear response was obtained for ethanol concentrations in the range 0.1–1 mM and can be used for 20 assays. Another currently used chromogen is 4-aminoantipyrine (4-AAP), which is used in combination with another reducing substrate such as phenol, 4-hydroxybenzosulfonate, and 8-hydroxyquinoline.
The need for rapid, low-cost, sensitive and continuous analysis methods with a high sample throughput has been led to the application of enzyme based biosensors into flow system based analysis. Different types of analytical measurements can be considered, namely, continuous flow analysis, segmented flow analysis and flow-injection analysis. The combination of reliable and fast flow systems with sensitive and stable immobilised bioreactors is becoming increasingly popular. Packed bed, and rotating bioreactors  have been commonly used to support the immobilised AOX.A flow system based on the detection of H2O2 using HRP-catalysed chemiluminescence of luminol has also been proposed.
Ethanol can be efficiently determined using enzyme based biosensors, i.e. ADH, AOX and AOX-POD, which are simple to assemble and operate. The detection could be based on NADH produced in the case of the ADH based biosensor, O2 consumption or H2O2 produced in the case of AOX based biosensor and effective detection of H2O2 produced in the case of AOX-POD. Both electrochemical and optical methods can be employed, either using modified electrodes and modified optode, where the bio-sensing scheme can be used as a direct detection or indirect detection of those analytes (NADH, O2 or H2O2).
Currently, the most promising ethanol biosensors proposed are the bi-enzymatic biosensors based on immobilisation of AOX coupled with HRP that can make use of the direct electron transfer effectively between HRP and the electrode or the expense of hydrogen donor molecules in the optical method. Other applications, such as flow system, that require fast and reliable methods for alcohol determination in various fields, will certainly benefit with the commercial development of more enzyme based biosensors that are currently available in the market.
The authors gratefully thank the DP2M, Higher Education, Ministry of National Education, Republic of Indonesia for supporting this work via the International Research Collaboration Program 2014.
ADH, alcohol dehydrogenase; AOX, alcohol oxidase; NAD+, nicotinamide adenine dinucleotide cation; NADH reduced form of NAD; FADH, flavin adenine dinucleotide, POD, peroxidase; HRP, horseradish peroxidase; ABTS, 2,2_-azino-di(3-ethylbenzthiazoline-6-sulfonate).
19. Ramesh P, Sivakumar P, Sampath S. Renewable surface electrodes based on dopamine functionalized exfoliated graphite NADH oxidation and ethanol biosensing. J. Electroanal. Chem. 2002 June; 528 (1-2): 82-92.
20. ZareHR,NasirizadehN, GolabiSM, NamazianM, ArdakaniMM, NematollahiD. Electrochemical evaluation of coumestan modified carbon paste electrode: Study on its application as a NADH biosensor in presence of uric acid. Sens. Actuat. B. 2006 April;114 (2): 610-617.
21. SerbanS.MurrNE. Synergetic effect for NADH oxidation of ferrocene and zeolite in modified carbon paste electrodes: New approach for dehydrogenase based biosensors. Biosens. Bioelectron. 2004 Sept;20(2): 161-166.
23. SantosAS, Freire RS, KubotaLT. Highly stable amperometric biosensor for ethanol based on Meldola’s blue adsorbed on silica gel modified with niobium oxide. J. Electroanal. Chem. 2003 Feb;547(2): 135-142.
26. Salimi F, NegahdaryM,MazaheriG, Akbari-dastjerdi H, Ghanbari-kakavandiY, Javadi S, etal.A novel alcohol biosensor based on alcohol dehydrogenase and modified electrode with ZrO2 Nanoparticles.Int. J. Electrochem. Sci. 2012 Mar; 7(3): 7225 – 7234.
27. MalinauskasA,RuzgasT, GortonL. Electrochemical study of the redox dyes Nile Blue and Toluidine Blue adsorbed on graphite and zirconium phosphate modified graphite. J. Electroanal. Chem. 2000 April; 484(1): 55-63.
28. MunteanuFD, OkamotoY, GortonL. Electrochemical and catalytic investigation of carbon paste modified with Toluidine Blue O covalently immobilised on silica gel. Anal. Chim. Acta. 2003 Jan: 476(1): 43-54.
29. JaegfeldtH, TorstenssonABC, GortonL, JohanssonG. Catalytic oxidation of reduced nicotinamide adenine dinucleotide by graphite electrodes modified with adsorbed aromatics containing catechol functionalities. Anal. Chem.1981 Nov; 53(13): 1979-1982.
30. MatsueT, SudaM, Kato IUT,AkibaU, OsaT. Electrocatalytic oxidation of NADH by ferrocene derivatives and the influence of cyclodextrincomplexation. J. Electroanal. Chem.1987 Sept; 234(1-2): 163-173.
31. ChiQJ, DongSJ.A comparison of electrocatalytic ability of various mediators adsorbed onto paraffin impregnated graphite electrodes for oxidation of reduced nicotinamide coenzymes. J. Mol. Catal. A.1996 Mar;105(3): 193-201.
32. HajizadehK, Tang HT, HalsallHB, Heineman WR. Chemical cross-linking of a redox mediator thionin for electrocatalytic oxidation of reduced β-nicotinamide adenine dinucluotide. Anal. Lett. 1991 Oct; 24(8): 1453-1469.
33. LoboMJ, MirandeAJ, FonsecaJML, TunonP. Electrocatalytic detection of nicotinamide coenzymes by poly(o-aminophenol)- and poly(o-phenylenediamine)-modified carbon paste electrodes. Anal. Chim. Acta. 1996 May; 325(1-2) 33-42.
35. YaoQ, Yabuki A, MizutaniF. Preparation of a carbon paste/alcohol dehydrogenase electrode using polyethylene glycol-modified enzyme and oil-soluble mediator. Sens. Actuat. B. 2000 June:65(1-3): 147-149.
36.Vonck J, van Bruggen EF. Electron microscopy and image analysisof two-dimensional crystals and single molecules of alcohol oxidasefrom Hansenula polymorpha. Biochim. Biophys. Acta. 1990 Jan; 1038(1): 74–79.
38. Akyilmaz E, Dinckaya E.An amperometric microbial biosensordevelopment based on Candida tropicalis yeast cells for sensitivedetermination of ethanol. Biosens. Bioelectron. 2005 Des; 20(6): 1163–1269.
43. ChiuJ-Y, YuC-M, YenM-J, ChenL-C.A novel hemin-based organic phase artificial enzyme electrode and its application in different hydrophobicity organic solventsBiosens. Bioelectron. 2009 March; 24(7): 2015–2020.
44. Ramaa EC, Biscaya J, Garcíaa MBG, Reviejo AJ, CarrazónJMP, Garcíaa AC. Comparative study of different alcohol sensors based on Screen-Printed Carbon ElectrodesAnal. Chim. Acta. 2012 May; 728 (4) 69-76.
47. Boujtita M, Hart JP, Pittson R. Development of a disposableethanol biosensor based on a chemically modified screen-printed electrodecoated with alcohol oxidase for the analysis of beer. Biosensor.Bioelectron. 2000 May; 15(5–6), 257–263.
48. Sehlotho N, Griveau S, Ruillé N, Boujtita M, Nyokong T, Bedioui F. Electro-catalyzed oxidation of reduced glutathione and 2-mercaptoethanol by cobalt phthalocyanine-containing screen printed graphite electrodes Mater. Sci. Eng. C. 2008 July; 28(5-6): 606–612.
50. KuswandiB, IrmawatiT, HidayatMA, Jayus, AhmadM.A simple visual ethanol biosensor based on alcohol oxidase immobilised onto polyaniline film for halal verification of fermented beverage samples. Sensors. 2014 Jan; 14(1): 2135-2149.
51. Johansson K, Jonsson-Pettersson G, Gorton L, Marko-Varga, G, Csoregi E. A reagentless amperometric biosensor for alcoholdetection in column liquid chromatography based on co-immobilizedperoxidase and alcohol oxidase in carbon paste. J. Biotechnol. 1993 Mar; 31(3): 301–316.
52. Liden H, Vijayakumar AR, Gorton L, Marko-Varga G. Rapidalcohol determination in plasma and urine by column liquid chromatographywith biosensor detection. J. Pharm. Biomed. Anal. 1998 June; 17(6–7):1111–1128.
59. Marshall RW, Gibson TD. Determination of sub-nanomoleamounts of hydrogen peroxide using an immobilized enzyme flowcell. Application to the determination of ethanol. Anal. Chim. Acta. 1992 Sept; 266(2): 309–315.
The main types of biological components and transducers used in the biosensor development.
(Purified or crude mixtures; single or multiple enzyme)
Catalytic transformation of analyte into a detectable product
The analyte inhibits the enzyme activity
Enzyme characteristic change upon reaction with analyte
Surface Acoustic Wave
High affinity and specific binding between antibody-antigen
Specific DNA sequences by hybridisation
Micro-organisme (Bacteria, Fungi)
Analyte (pollutant) dependent increase in micro-organisme respiration
Analyte (pollutant) dependent decrease in micro-organisme respiration
Extracelullar, membrane or intra-celullar transport protein
RNA or DNA aptamers
Genetically engineered molecules
Molecular imprinted polymers
Methods of immobilisation of the biological component
No modification of bio-component
Gentle treatment of bio-component
Matrix can be regenerated
Maximal retention of activity
Very weak bonds
Susceptible to change in pH, temperature & ionic strength
Entrapment and encapsulation
Only physical confinement
No direct chemical modification
High diffusion barrier
Continuous loss of activity
Low diffusional resistance
Strong binding force between bio-component and matrix
Stable under adverse condition
May involve harsh/toxic chemicals
Matrix not regenerated
Often occurring loss of activity
Loss of biocatalyst at minimum
Harsh treatment by toxic chemicals