A Disposable Biosensor Based On Screen-Printed Electrode

Blood alcohol determination plays an important role in the forensic medicine and laboratory medicine for several reasons. First drunk driving is a very serious problem in the modern world, which results in lots of fatal accidents every year. Blood alcohol concentration (BAC) values served as the"gold standard"in which drivers were recognized as drunk driving by police officers. It is also necessary for clinical laboratories to detect the acute alcoholism and some syndromes related alcohol abuse. Nowadays, many methods have been used for alcohol measurement, such as spectrometric and chromatographic analysis or breathalyzer where the alcohol concentration or refractivity was detected. However, these methods are time consuming and complex to perform laborious sample pre-treatment .In addition, the expensive analytical apparatus is necessary. Thus, there is an increasing requirement for rapid, accurate and inexpensive methods for alcohol determination.The disposable amperometric biosensor should be readily applicable to alcohol determination, since biosensors have such favorable analytical characteristics as portability, low cost and potential for fabrication. Several alcohol biosensors have been reported to control the fermentation process in the food and beverages industries, based on immobilization of Alcohol Oxidase (AOD) or Alcohol Dehydrogenase (ADH). In an AOD-based biosensor, O2 consumption or H2O2 production is determined. However, this kind of biosensor is oxygen dependent and low selectivity for alcohol. Concerning ADH-based biosensor, it is possible to directly detect reduced form of Nicotinamide Adenine Dinucleotid (NADH) produced in the enzymatic reaction of alcohol with Nicotinamide Adenine Dinucleotide (NAD+) under catalysis of ADH. However, the electrochemical oxidation of NADH involves the use of a high overpotential at which a few oxidizable substances in the real samples would be oxidized, thus increasing the likelihood of interferences. For this purpose many electron transfer mediators have been used for electrochemical oxidation of NADH, which could be detected at a low oxidizing potential. Among them, Meldola's Blue (MB) shows some of the most promising characteristics. However, it has been found that MB could not be absorbed stably on the electrode, meaning that the electrode is unstable for practical applications. To improve MB attachment to the electrode, a few methods have been used. However, these methods are complicated. Nafion, a kind of ion exchanger, can absorb MB through ion exchange.Screen printing technology, due to its low cost and mass production, is a versatile tool for the inexpensive, easy and highly reproducible production of disposable biosensors. Up to now, most of disposable biosensors based on screen printing technology are applied in the forensic medicine and laboratory diagnosis, since it could resolve such critical problems as low cost, fast response and portability.This paper describes the development of disposable alcohol biosensor based on screen-printed electrode (SPE). The biosensor response for alcohol is investigated in terms of pH, buffer solution, temperature and some interferents. It presents the good specificity, reproducibility, stability, accuracy and provides a fast response. The biosensor is applied for measuring serum alcohol and satisfactory result is obtained.Our research is mainly divided into two parts as follows:1. Development of the disposable alcohol biosensor based on the cross-linking method to immobilize ADH and NAD+ on the Nafion-MB modified screen-printed electrode. Methods:1 ) Preparation of screen-printed electrode. A polyvinyl chloride (PVC) film is selected as the support of SPE. The biosensor comprises a 3-mm diameter working area, the insulation layer and an electrical contact site. The biosensors are printed onto the PVC sheet. The different layers including silver conducting basal track, carbon working area and insulation layer are printed one after the other. After every step the film is left to dry for 2 hours in an oven at 40℃to drive off the solvents from the applied ink. 2)Preparation of Nafion-MB modified electrode. The SPE is ultrasonically cleaned with ethanol and ultrapure water for 10 min respectively, and dried at room temperature. A 5% (w/w) Nafion-117 solution is diluted to 1% (w/w) with absolute ethanol. 5μL of 1% (w/w) Nafion is dropped onto the SPE and dried at room temperature. The pretreated electrode is scanned by cyclic voltammetry in the 0.1mol L-1 (pH8.0) phosphate buffer solution (PBS) with 1mmol L-1 MB at room temperature. Eventually, a Nafion-MB modified electrode is ready for use. 3)Preparation of alcohol biosensor. 4mg of ADH,5mg of NAD+ and 10mg of BSA are dissolved in 200μL of 0.1 mol L-1 PBS (pH8.0), then 20μL of 2.5% (w/w) glutaraldehyde solution is added to the mixed enzyme solution with vigorous stirring. 5μL of the mixed solution described above is dropped onto the working area of the electrode and allowed to dry in air at room temperature for 12 hours. The alcohol biosensor is prepared and kept dry in a refrigerator at 4℃before use. Results : All electrochemical measurements are carried out in a conventional three electrode cell which is composed of saturated calomel reference electrode (SCE), a platinum wire counter electrode and a modified working electrode. The maximum response current for alcohol is obtained in 0.1mol L-1 PBS (pH 8.0) at the working potential of -0.17V (vs. SCE), at 25℃. The detection limit of the biosensor is estimated to be 1.1×10-5mol L-1 alcohol at a signal to noise ration of 3. The linear response range of the biosensor to alcohol can be extended at least to 5mmol L-1. The biosensor response time is very short, reaching 95% of the steady-state current within 30s. The reproducibility of the identical biosensor is examined in 0.1mol L-1 PBS (pH8.0) with 1mmol L-1 alcohol at the working potential of -0.17V (vs. SCE), at 25℃. The relative standard deviation is 3.6% for 10 successive assays. 5 electrodes are randomly selected from 5 batches of screen-printed electrodes to fabricate the alcohol biosensors independently. The acceptable batch reproducibility with a relative standard deviation of 4.3% is obtained. The storage stability of biosensor toward the response for alcohol is examined as well. After being stored at 4℃for 10,20,30 days, the response current decreased by 3.7%,5.2%,8.4% of the initial response, respectively. The electroactive substances commonly present in the real samples, such as methanol, glucose, lactic acid, ascorbic acid, all can not influence on the biosensor response for alcohol. Conclusion: The biosensor presents the good specificity, reproducibility, stability, accuracy and provides a fast response. The proposed biosensor may provide a useful screening procedure for the determination of alcohol.2. Development of the disposable alcohol biosensor based on Nafion combined with gold nanoparticles (GNPs) to immobilize ADH and NAD+ on the MB modified screen-printed electrode. Methods:1)Preparation of gold nanoparticles. Gold nanoparticles are prepared by adding sodium citrate solution to a boiling HAuCl4 aqueous solution. The solution is stored in brown glass bottles at 4℃refrigerator. The average particle diameter is 20nm as determined by transmission electron microscopy. 2)Preparation of MB modified screen-printed electrode. The polyester is selected as the support of the biosensor. The biosensor comprises a 3-mm diameter working area, the insulation layer and an electrical contact site. The biosensors are printed onto the polyester sheet. The different layers including silver ink, carbon ink and UV adhesives are printed one after the other. After printing of silver and carbon ink the polyester sheet is heated for 2h in an oven at 40℃to drive off the solvents from the applied ink. After printing of UV adhesives the ultraviolet curing of UV adhesives is followed. 3)Preparation of alcohol biosensor. The MB modified SPE is ultrasonically cleaned with absolute ethanol and ultrapure water for 10 min respectively, and dried at room temperature. Enzymes solution is obtained by dissolving 4mg of ADH and 6mg of NAD+ in 200μL of 0.1mol/L (pH8.0) phosphate buffer solution (PBS), then 100μL of enzymes solution and 100μL of 5% (w/w) Nafion solution is mixed with vigorous stirring. 100μL of gold nanoparticles solution is added to this mixed solution with vigorous stirring. 10μL of the freshly prepared mixed solution is dropped onto the working area of the SPE and allowed to dry in air at room temperature for 12h. The alcohol biosensor is prepared and kept dry in a refrigerator at 4℃before use. Results:All electrochemical measurements are carried out in a conventional three electrode cell which is composed of saturated calomel reference electrode, a platinum wire counter electrode and a modified working electrode. The maximum response current for alcohol is obtained in 0.1mol L-1 PBS (pH 8.0) at the working potential of 0.0 (vs. SCE), at 25℃. The detection limit of the biosensor is estimated to be 1.6×10-5 mol/L alcohol at a signal to noise ration of 3. The linear response range of the biosensor to alcohol could be extended at least to 8mmol/L. The biosensor response time is very short, reaching 95% of the steady-state current within 40s. The two different alcohol concentrations (1 and 5mmol/L) are detected using the identical biosensor for 10 successive assays within one day respectively. The relative standard deviations are 5.49 and 1.44%, respectively. 10 electrodes are randomly selected from 10 batches of screen-printed electrodes to fabricate the alcohol biosensors independently. 1 and 5mmol/L alcohol concentrations are detected using the ten different biosensors respectively. The relative standard deviations are 6.98 and 2.49%, respectively. The storage stability of biosensor toward the response for alcohol is examined as well. After being stored at 4℃for 10,20,30 days, the response current for 1mmol/L alcohol decreased by 3.1%,4.6%,7.7% of the initial response, respectively. The electroactive substances commonly present in the real samples, such as methanol, glucose, lactic acid, ascorbic acid, all can not influence on the biosensor response for alcohol. Conclusion: The biosensor presents the good specificity, reproducibility, stability, accuracy and provides a fast response. The proposed biosensor may provide a useful screening procedure for the determination of alcohol.