Development of Alcohol Sensor for Pharmaceutical Products

Development of Alcohol Sensor for Pharmaceutical Products 1.0Â  Title Development of alcohol sensor for detection of alcohol content in pharmaceutical products 2.0Â  Introduction Over the years, alcohol is being used widely in various number of industries. Alcohol poisoning and inflammation may happen whenever the alcohol concentration exceeds the toxic level for the living creatures. Nowadays, alcohol abuse is one of the worldwide social problems and has become a public sanitation issue. Hence, the preventative pathways must be taken so that the toxicological and psychological effects can be avoided. It is essential to come with a safer, low cost, fast, sensitive and selective analytical method in order to determine the amount of alcohol content in any samples tested. As pharmaceutical products are the essential part in our daily life nowadays, hence, designing the analytical methods for detection of alcohol content in this products is very important, especially for the prevention of consuming the chemically unsafe products. Various of analytical methods have been used to determine the alcohol concentration during the years. Some techniques may come with their own advantages and even drawbacks. The discovered disadvantages can be overcome by producing a utilization of enzymatic methods. The enzymes will catalyses almost all the transformation of the chemical that exist during the cell metabolism. Moreover, the chemical analysis can be done more efficiently as the nature and specificity of the enzyme catalytic activities. The external addition of the cofactor also helps a lot with the alcohol sensor designed. 3.0Â  Literature Review 3.1Â  Alcohol dehydrogenase Alcohol dehydrogenase is being applied as the bioselective compounds in alcohol biosensors. This enzyme is essential in catalyzing the reversible oxidation process occurred for the primary aliphatic and the aromatic alcohols, but not for methanol. The process that took place is based on the Equation (1). RCH2OH + NAD+ ↔ ADHRCHO + NADH + H+ (1) When compared with the alcohol oxidase-based alcohol biosensor, alcohol dehydrogenase-based are more stable and explicit. However, the exterior addition of the co-enzyme nicotinamide adenine dinucleotide (NAD+) is needed by this type of biosensors. Furthermore, the added cofactor requires to be close to the enzyme and it must not irreversibly combined or entrapped (Azevedo et al., 2005). The combination of electrogenerated chemiluminescence ethanol biosensor and the alcohol oxidase enzymatic reaction is practised for detecting the ethanol in the several subject materials tested (Jia et al., 2009). Hence, it is essential for a biosensor to exhibiting significant reproducibility and stability. It is a requirement for the alcohol biosensor having a great potential for the usage in other biological assays and able to determine variety of substrates. 3.2 Alcohol Oxidase Known as an oligomeric enzyme, alcohol oxidases have eight identical monomers arranged in a quasi-cubic arrangement. Each sub-units of this arrangement are holding a strongly bounded cofactor which is flavin-adenine dinucleotide (FAD) molecule (Vonck van Bruggen, 1990). This enzyme is associated in the methanol oxidation pathway of methylotrophic yeasts. Besides involving in the methanol oxidation, alcohol oxidase also oxidises the short-chain alcohols like ethanol, propanol and butanol (Azevedo et al., 2005). Going through the oxidase-catalysed reaction, the ways to follow the reaction is by measuring the decline in O2 tension or the elevation in H2O2 concentration. Alcohol oxidation catalysed by this enzyme is an irreversible mechanism as O2 has a strong oxidising characteristic. The reaction requires alcohol oxidase and utilizing molecular oxygen (O2) as an electron acceptor, referring to Equation (2) (Azevedo et al., 2005). Alcohol Oxidase RCH2OH + O2 RCHO + H2O2(2) The characteristics of this form alcohol biosensor acts as a finer alternative to other determination methods in detecting the ethanol for various types of sample material tested, such as in pharmaceutical products nowadays (Kuswandi et al., 2014). It will be a great improvement if the enzymatic reaction occurred is able to be monitored optically so that the alcohol presence can be detected by the naked eye. Hence, a better quality of alcohol biosensor can be made. 3.3O2 Detection According to the Equation (1), the consumption of oxygen can be monitored by the alcohol oxidase sensors. The monitoring is done subject to the electrochemical detection principles and by the optical detection (Azevedo et al., 2005). The enzyme-catalysed reaction will be able to generate the optical or visual determination of alcohol based on the oxidation or reduction of H2O2. Hence, the use of optical membrane or a film is required so that it will be more efficient to monitor the reaction. Next, the O2 tension can be detected readily. 3.3.1Electrochemical detection Monitoring of O2 is generally done based on a Clark-type O2 electrode, which consists of a platinum cathode and a reference electrode, sunk in an electrolyte solution and a semi-permeable membrane covering it, so that O2 will be able to diffuse through (Azevedo et al., 2005). Equation (4) shows an example of the reduction process of oxygen while proportionally producing a current. Ag anode, 4Ag + 4Cl → 4AgCl + 4e(3) Pt cathode, O2 + 4H+ + 4e → 2H2O(4) Commonly, oxygen probes-based ethanol sensors have a membrane covering the Clark-type electrode, where alcohol oxidase is immobilised. The difference between the base oxygen level and the level after oxygen concentration decreases due to the enzymatic reaction will be shown as the electrode signal output. It is clearly showed that there will be no electrochemical interference comes from other sample elements. Nevertheless, the accuracy and reproducibility of the sensor may be lessened due to its oxygen dependency basis of the measurement. Hence, a low response is obtained, causes by the high value of the minimum detectable concentration of the oxygen due to the high background signal (Bott, 1998). However, the new alternative can be utilized to overcome the flaws is by using H2O2 detection. 3.3.2Â  Optical detection The developing of alcohol sensors has built up the fluorescence-based sensors. It works based on the enhancement of the fluorescence or other certain compounds quenching, including malachite green, fluorescent dyes and even ligands towards the alcohol disclosure. Besides that, the immobilisation of alcohol oxidase onto the oxygen sensor coated with an oxygen sensitive ruthenium organic complex is done to construct an optical bio-sniffer for ethanol vapours. Moreover, co-immobilisation of alcohol oxidase and oxygen sensitive dyes is designed to assemble the other optical sensors (Azevedo et al., 2005). 3.4Â  Detection of H2O2 3.4.1Â  Electrochemical methods 3.4.1.1Â  Amperometric detection H2O2 which is formed by alcohol oxidase enzymatic reaction can be identified electrochemically with amperometric electrodes. The detection is done either by measuring the anodic or cathodic response, which showing the oxidation and reduction of H2O2 at the working electrode surface correspondingly. As shown by Equation (5), the enzymatic reaction will result the oxidation of H2O2. H2O2 → O2 + 2H+ + 2e(5) Nonetheless, H2O2 is electroactive too at the negative potentials, according to Equation (6). H2O2 + 2e + 2H+ → 2H2O (6) These reactions are not discovered on oxygen probes based on the Clark electrode, due to the electrode surface is covered by an oxygen membrane, which is not permeable to H2O2 and mostly other compounds. Lately, by using other immobilisation procedures, carbon paste electrodes (CPE) and screen-printed electrodes are being developed (Azevedo et al., 2005). The most significant advantage of H2O2 electrode based sensor is easy to construct the sensor in small size besides having a high upper linearity and a wider linear range. In contrast, the presence of reducing compounds in any real sample matrices will be oxidised too, hence will causes the electrochemical interference to occur. Besides that, slower responses are observed too. Finally, the electrode with an electrocatalyst species is needed to be modified for both the reduction and oxidation of H2O2 so that the required applied potential can be decreased. 3.4.1.2Â  Potentiometric detection The potentiometric biosensor is constructed by co-immobilising alcohol oxidase and horseradish peroxidase in the surface of a fluoride-sensitive electrolyte isolator semiconductor capacitor chip. The capacitance will change if there is any presence of ethanol and p-fluoraniline (Menzel et al., 1995). Si/SiO2/Si3N4/LaF3 layers utilized in the fluoride-sensitive biosensor, are able to determine the ethanol concentration in the time of the on-line monitoring of different bioprocesses, according to reactions (7) and (8) (Azevedo et al., 2005). Alcohol Oxidase Ethanol + O2 Acetaldehyde + H2O2 (7) Horseradish Peroxidase H2O2 + p-fluoraniline F + H2O + aniline-derivative polymers(8) 3.4.2Â  Spectroscopic methods To detect the H2O2 production by alcohol oxidase during the ethanol oxidation, few methods can be benefited. Colorimetric methods which are based on the chromogen substrate conversion into a coloured product will absorb in the visible spectral region. Next, fluorescent methods are due to the production of fluorophore product and being stimulated with a shorter wavelength radiation before emitting a visible light. Then, chemiluminescence works by the emission of visible light upon chemical reaction (Azevedo et al., 2005). The methods being chosen must be fast, cheap, sensitive, reliable, stable and undergo continuous analysis methods with a high sample. The numbers of variety types of analytical techniques are flow analysis, segmented flow analysis, flow-injection analysis and liquid chromatographic analysis. 3.5Â  Immobilisation techniques Many ways are being implemented to immobilise enzymes while designing the biosensors. The enzymes are able to be immobilised by physical adsorption or covalently attached to the insoluble matrices, by cross-linking which employing the bifunctional reagent or by entrapment into the membranes or polymeric films. 3.5.1Â  Enzyme modified electrodes There are numbers of approaches in order to implement the physical combination of immobilised enzymes and the electrodes. 3.5.1.1 Membrane electrodes Immobilising the enzymes on a membrane is the most popular techniques being used for the biosensors. This cannot be beaten by other methods since it is easy to construct and its simplicity. Enzyme immobilisation is done by sandwiching the particular enzyme between the electrode and the membrane. The improved procedures used may lead to a higher enzyme activity and a greater stability (Nanjo Guilbault, 1975). As a protective retention layer, a membrane prevents electrochemically interfering compounds from touching the electrode surface. This is due to the presence of the charged groups on the membrane surface and the exclusion of size. Besides that, the covered electrode are protected because the membrane used is impermeable to most substances (Boujtita et al., 2000). Furthermore, covering the enzyme electrode with a membrane has variety of purposes such as producing the diffusion barrier between enzyme and the substrate. This also enables the prevention of a swamping effect whenever the substrate concentration is high. At the same time, a linear response to the concentration is also allowed. 3.5.1.2Â  Carbon paste electrodes By mixing an electrically conducting graphite or carbon powder with a pasting liquid, the carbon paste electrodes is able to be prepared. The examples of pasting liquid being used are mineral oil, silicon oil paraffin oil. Enzymes involved are incorporated within the paste or previously immobilised on the graphite powder by adsorption or covalently bonded. In addition, some additives are added to the paste so that the sensitivity and the storage and operational stability of the sensor can be improved (Azevedo et al., 2005). 3.5.1.3Â  Self-assembled monolayers This form of technique is implemented by sequentially self-depositing the transducing and biocatalytic modules by adsorption through electrostatic interactions. Two different catalytic layers which consist of alcohol oxidase, a modified horseradish peroxidase and electrochemical interface are sequentially and rationally deposited. Next, supramolecular structures are produced and connect catalytic reactions, substrate and product diffusion and heterogeneous electron transfer steps readily (Azevedo et al., 2005). 3.5.1.4Â  Screen-printed electrodes Screen-printed electrodes consist of a polyester substrate and a three electrodes system. The electrodes also containing fabrication of alcohol oxidase immobilized in a poly(carbamoyl)sulfonate hydrogel using poly(ethylene glycol)diglycidyl ether (Patel et al.,2001). This type of electrode system is a low cost screen-printed electrode. 3.5.2Â  Immobilised enzyme reactors The most significant benefit of the usage of enzyme immobilized reactor is producing the great quantity of enzyme that are able to be immobilise and even in micro reactors. This will allow the equilibrium of the reaction that occurred to be attained besides capable of completing the substrate conversion. Moreover, the operational stability of the sensor is enhanced. It is stated that any small alterations in flow rate, temperature, pH, ionic strength and the activators and inhibitors presence will deflate the effect on final signal (Gorton et al., 1991). Bioreactors that are used with immobilised alcohol oxidase exist in different types, generally packed bed, rotating bioreactor and open-tube reactors. Usually the packed bed reactor is implemented with immobilised alcohol oxidase. This form of bioreactor integrates a flow analysis system with electrochemical or spectrometric detection (KÃ ¼nnecke Schmid, 1990). Commonly, rotating bioreactor also used immobilised alcohol oxidase with the electrochemical detection of H2O2. The rotation will enable the presence of circumvent diffusional constrains in the low-dimensional spaces like around the active sites of the enzymes (Matsumoto Waki, 1999). For covalently immobilised enzyme, controlled pore glass is usually utilized for solid support purpose. It is a macro-porous high-silica glass acquired from the alkali-borosilicate glass. Alkali-borosilicate glass is came with the fine mechanical properties and is able to designed with broader porosities and pore size range. Furthermore, it can be adjusted with several reagents so that other functionalities can be proposed (Azevedo et al., 2005). Currently, the optimisation of enzyme immobilisation is done to produce better stability to the controlled pore glass preparations of alcohol oxidase. 4.0Â  Problem Statements The purpose of this study is to determine the right method for detection of alcohol concentration in the pharmaceutical product samples. Alcohol is the substance that present in more than 500 medication products and is found in concentrations up to 68 percent. This may causes negative effects towards the patients that are consuming the products, such as for the patient under treatment with central nervous system depressants or other substances that interact with alcohol. Drug addictionand habituationmay happen and it is formerly known that all drugs haveside effects. It is an essential step to developing a right and efficient method in determining the alcohol content in these pharmaceutical products before approving the production of them into the industry range. 5.0Â  Objectives These are few objectives that have been identified in order to deal up with the problem statement and carried out the research on alcohol content detection content in the pharmaceutical products. Study of methods in determining the alcohol concentration in the samples tested. Study of enzymatic reactions involve during the alcohol detection analysis. Study of advantages and disadvantages for different techniques in the alcohol content determination.