**Abstract**

Electrochemical biosensors for measuring ethanol were developed in this study. Silver nanoparticles were incorporated to increase sensibility. Firstly, the transducer element was prepared with silver nanoparticles/polyaniline/graphite/epoxy composite (AgNPs/PANI/GEC) where the enzymes *alcohol oxidase* (AOD) and *horseradish peroxidase* (HRP) were immobilized by adsorption. The composition of the immobilized solution was indicated by an experimental design (85% of the bi-enzymatic solution, 10% of albumin and 5% of glutaraldehyde). The immobilization method adopted in this study showed to be highly reproducible. The values of variance and standard deviation were low (0.003 and 0.053, respectively—means of three electrodes). The linear range was 0–30 g/L (R2 = 0.983) and the sensitivity 0.004 mA L/g. A second biosensor was made with a transducer prepared with AgNPs/PANI/GEC. A chitosan film was applied over the adsorbed enzymatic solution to avoid desorption of the immobilized enzymes. The AgNPs/PANI/GEC electrodes were characterized using cycle voltammetry and the composite surface by scanning electron microscopy. The calibration for ethanol samples with this second composite fitted in a range of 0.0–0.35 g/L (R2 = 0.984). Square rate voltammetry was the electrochemical analytical method used to obtain the amperometric calibration curves.

**Keywords:** alcohol oxidase, horseradish peroxidase, polyaniline, silver nanoparticles, electrochemical biosensor

### **1. Introduction**

Biosensors with electrochemical transducers are attractive from a commercial point of view due to its low expense and simplicity of construction and because it is easy to employ in a broad range of applications and portable [1, 2]. The voltammetric and amperometric technique analyses are a full implementation to measure low levels of concentration. Additionally, as an electrochemical surface method, it has the

advantage of generating a response signal working with small sample volumes, in real time. The working electrode can be prepared by surface immobilization of one or more enzymes, which are involved in recognition of the analyte. The enzymes could be regarded as the critical component of the electrode since they are related to the selectivity of the sensor to catalyze the formation of the electroactive detection product [3]. Additionally, the enzymes are ideal biological recognition elements in the construction of biosensors, because of their high specificity, which enables the development of analytical methods with high accuracy [1]. Biosensor technologies are recognized as an emerging science to produce analytical devices that can help to detect specific compounds in complex mixtures such as liquid waste residues and blood serum [4, 5]. Nowadays, ethanol analysis is fundamental for criminal justice systems, in clinical and toxicological diagnostic analyses such as blood, serum and urine analysis, monitoring medical conditions of HIV patients, as well as public safety issues regarding the pilots and drivers. In the food and beverage industries, the determination of alcohol content is critical for the control of the fermentation process and product quality. Besides these applications, determination of ethanol is also important in agricultural, biofuel and environmental analyses [6–8]. *Alcohol oxidase* (AOD) allows the qualitative and quantitative determination of ethanol or methanol, removal of alcohol or aldehyde and hydrogen peroxide production and removal of oxygen [9]. *Horseradish peroxidase* (HRP) uses the haem group and hydrogen peroxide substrate to oxidize a variety of organic and inorganic compounds [10]. The enzymatic mechanisms of these enzymes together facilitate the detection of AOD substrates by electrochemical and spectroscopic methods such as ethanol. The co-immobilization of the enzymes holds the potential to increase the selectivity and amplify the sensitivity of the biosensor improving the potential for quantitative ethanol detection [11, 12]. Polyaniline (PANI) has attracted the attention of the scientific community in the last two decades. PANI, a family of conductive semi-flexible polymers in a green protonated emeraldine form had high electrical conductivity and low production cost. PANI has been explored for various applications, including those in biosensors due to some useful features such as redox conductivity and polyelectrolyte characteristics, high surface area, chemical characteristics, long-term environmental stability and tunable properties enhancing the electron transfer flux ability and also the reversibility of the electrochemical response signal [12–14]. In the previous work, the electroconductivity of PANI-GEC was reported and verified that PANI is an attractive polymer compound to be applied in sensor interface transducer biosensors. A conductivity of 28 μS/cm for 30 w/w% PANI-GEC was measured and cyclic voltammograms for 10 mM potassium ferricyanide obtained working with a scan rate of 100 mV s<sup>−</sup><sup>1</sup> . The composite can act as an effective mediator in the transference of electrons in redox or enzymatic reactions [14]. With the use of nanomaterials, greater sensitivity and attachment of enzymes are achieved due to their high surface area as well as the physical, chemical and electronic properties. The literature reported that nanomaterial application had attracted much attention regarding the development of high-performance electrochemical biosensors [13]. The preparation of chemically modified electrodes with silver nanoparticles (AgNPs) has been applied to amplify the electrochemical response signal. Since silver is four times cheaper than gold and shows excellent catalytic activity and good electrical/heat conductivity, its application is very favorable in electroanalysis acting as a pre-concentrator of species of interest and/or mediating redox reactions [15]. The literature also reported an increase of 9.71% in the anodic peak current and 32.35% for the cathode peak current in the presence of AgNPs. The authors observed an increase in the reversibility of the voltammetric response signal in the composite based on AgNPs/PANI/GEC and the ratio of the anodic (Ipa) and cathodic (Ipc) peak currents | Ipa/Ipc | = 1.07 at 40 mV s<sup>−</sup><sup>1</sup> . Nevertheless, the ratio was of the order of 1.28 for composite without AgNPs [16]. From a point of view of analytical instruments,

**123**

detection levels.

*Development of an Ethanol Biosensor Based on Silver Nanoparticles/Polyaniline/Graphite…*

biosensors are used also for quality control, because they have important technical characteristics, such as low response time, high selectivity, stability under the conditions of the analysis, and reproducibility of the measurements. So, ethanol as a hydrous biofuel (Brazil, 5.3 volume % of water) or in the anhydrous form mixed in blends with gasoline (USA—10%, 15% and 85% of anhydrous ethanol, western Europe—5% of anhydrous ethanol and Brazil—27% of anhydrous ethanol) have being used in regular cars and flex fuel vehicles. Methods for monitoring the percentage of ethanol in the mixtures (product quality control) or in case of spilling of gasohol blends need to be develop. Many efforts for different potentiometric, amperometric, and spectrophotometric biosensors have been developed for ethanol analyses, with microorganisms like *Gluconobacter oxydans, Saccharomyces ellipsoideus,* or enzymes as alcohol dehydrogenase or alcohol oxidase were just reported in the literature [17]. In Brazil, there is a rigorous program for a strict control of the physicochemical characteristics of the gasohol blend and hydrated fuel alcohol to prevent adulteration or environmental contamination. Nowadays, the 4.0 industry claims for more robust and sensitive instruments for long-distance transmission and data transfer systems to an analytical central station in monitoring and process control program. Only a few biosensors are commercially available at present for analysis control and the integration of nanomaterials composites within these enzymatic biosensors brings new strategies for enhancing their analytical performances [16, 17]. The high ethanol solubility turns the assessment and analytical methods limited. Many road or pipeline accidents can spill fuel blends into the environment. Significant environmental impacts related to ethanol spills have been to surface water and fishes were killed several days after as a result of oxygen depletion. Spilled ethanol from the surface through soil to groundwater contamination is also of concern, and anaerobic biodegradation of ethanol in groundwater results in the production of methane [18]. The development of more selective and integrated systems for application in fast and high accuracy analysis needs further innovation and research investments. The enzyme immobilization methods represent an important step for the new technologies applied in bioinstrumentation techniques. The incorporation of silver nanoparticles in two different composites and the electrochemical response signals generated from each biosensor were investigated. An experimental design was used and statistic analysis to define the best condition for low cost enzymatic immobilization method. The AgNPs/ PANI/GEC biosensor with AOD and HRP immobilized enzymes was firstly prepared to detect ethanol. A second composite with only AgNPs/GEC was prepared and the immobilized AOD and HRP adsorbed enzymes covered with a chitosan film.

Voltammetry/amperometric techniques were applied to characterize the electrochemi-

cal transduction systems. Calibration curves were obtained for each composite electrode biosensor in order to evaluate the ethanol analytical ranges and

The composite used in the manufacture of the electrode for the biosensor comprised 40% of PANI, 35% epoxy and 25% enriched with graphite AgNPs as described in [16]. The mixture was inserted into the empty end of the Teflon support (rod of 50 mm length × 7 mm in outer diameter, recessed 3 mm diameter × 3 mm deep). The electrodes containing the composites were maintained in an incubator at 30°C for 24 h, and then, the end polished with 1200 mesh sandpaper. In this work two strategies of enzyme immobilization were studied, one by adsorption on the surface composite and a second way covering the bi-enzymatic solution with a film of chitosan polymer. **Figure 1** shows an illustration of the composite surface with

**2. Electrode construction and characterization**

*DOI: http://dx.doi.org/10.5772/intechopen.89359*

### *Development of an Ethanol Biosensor Based on Silver Nanoparticles/Polyaniline/Graphite… DOI: http://dx.doi.org/10.5772/intechopen.89359*

biosensors are used also for quality control, because they have important technical characteristics, such as low response time, high selectivity, stability under the conditions of the analysis, and reproducibility of the measurements. So, ethanol as a hydrous biofuel (Brazil, 5.3 volume % of water) or in the anhydrous form mixed in blends with gasoline (USA—10%, 15% and 85% of anhydrous ethanol, western Europe—5% of anhydrous ethanol and Brazil—27% of anhydrous ethanol) have being used in regular cars and flex fuel vehicles. Methods for monitoring the percentage of ethanol in the mixtures (product quality control) or in case of spilling of gasohol blends need to be develop. Many efforts for different potentiometric, amperometric, and spectrophotometric biosensors have been developed for ethanol analyses, with microorganisms like *Gluconobacter oxydans, Saccharomyces ellipsoideus,* or enzymes as alcohol dehydrogenase or alcohol oxidase were just reported in the literature [17]. In Brazil, there is a rigorous program for a strict control of the physicochemical characteristics of the gasohol blend and hydrated fuel alcohol to prevent adulteration or environmental contamination. Nowadays, the 4.0 industry claims for more robust and sensitive instruments for long-distance transmission and data transfer systems to an analytical central station in monitoring and process control program. Only a few biosensors are commercially available at present for analysis control and the integration of nanomaterials composites within these enzymatic biosensors brings new strategies for enhancing their analytical performances [16, 17]. The high ethanol solubility turns the assessment and analytical methods limited. Many road or pipeline accidents can spill fuel blends into the environment. Significant environmental impacts related to ethanol spills have been to surface water and fishes were killed several days after as a result of oxygen depletion. Spilled ethanol from the surface through soil to groundwater contamination is also of concern, and anaerobic biodegradation of ethanol in groundwater results in the production of methane [18]. The development of more selective and integrated systems for application in fast and high accuracy analysis needs further innovation and research investments. The enzyme immobilization methods represent an important step for the new technologies applied in bioinstrumentation techniques. The incorporation of silver nanoparticles in two different composites and the electrochemical response signals generated from each biosensor were investigated. An experimental design was used and statistic analysis to define the best condition for low cost enzymatic immobilization method. The AgNPs/ PANI/GEC biosensor with AOD and HRP immobilized enzymes was firstly prepared to detect ethanol. A second composite with only AgNPs/GEC was prepared and the immobilized AOD and HRP adsorbed enzymes covered with a chitosan film. Voltammetry/amperometric techniques were applied to characterize the electrochemical transduction systems. Calibration curves were obtained for each composite electrode biosensor in order to evaluate the ethanol analytical ranges and detection levels.
