Integrated biosensor systems for ethanol analysis.

Different integrated systems with a bi-enzymatic biosensor, working with two different methods for ethanol detection--flow injection analysis (FIA) or sequential injection analysis (SIA)--were developed and applied for ethanol extracted from gasohol mixtures, as well as for samples of alcoholic beverages and fermentation medium. A detection range of 0.05-1.5 g ethanol/l, with a correlation coefficient of 0.9909, has been reached when using FIA system, working with only one microreactor packed with immobilized alcohol oxidase and injecting free horseradish peroxidase. When using both enzymes, immobilized separately in two microreactors, the detection ranges obtained varied from 0.001 to 0.066 g ethanol/l, without on-line dilution to 0.010-0.047 g ethanol/l when a 1:7,000 dilution ratio was employed, reaching correlation coefficients of 0.9897 and 0.9992, respectively. For the integrated biosensor SIA system with the stop-flow technique, the linear range was 0.005-0.04 g/l, with a correlation coefficient of 0.9922.

Enzymatic microreactors for the determination of ethanol by an automatic sequential injection analysis system.

A sequential injection analysis system with two enzymatic microreactors for the determination of ethanol has been designed. Alcohol oxidase and horseradish peroxidase were separately immobilized on glass aminopropyl beads, and packed in 0.91-mL volume microreactors, working in line with the sequential injection analysis system. A stop flow of 120 s was selected for a linear ethanol range of 0.005-0.04 g/L +/- 0.6% relative standard deviation with a throughput of seven analyses per hour. The system was applied to measure ethanol concentrations in samples of distilled and nondistilled alcoholic beverages, and of alcoholic fermentation with good performance and no significant difference compared with other analytical procedures (gas chromatography and high-performance liquid chromatography).

A sequential enzymatic microreactor system for ethanol detection of gasohol mixtures.

A sequential enzymatic double microreactor system with dilution line was developed for quantifying ethanol from gasohol mixtures, using a colorimetric detection method, as a new proposal to the single micro reactor system used in previous work. Alcohol oxidase (AOD) and horseradish peroxidase (HRP) immobilized on glass beads, one in each microreactor, were used with phenol and 4-aminophenazone and the red-colored product was detected with a spectrophotometer at 555 nm. Good results were obtained with the immobilization technique used for both AOD and HRP enzymes, with best retention efficiencies of 95.3 +/- 2.3% and 63.2 +/- 7.0%, respectively. The two microreactors were used to analyze extracted ethanol from gasohol blends in the range 1-30 % v/v (10.0-238.9 g ethanol/L), with and without an on-line dilution sampling line. A calibration curve was obtained in the range 0.0034-0.087 g ethanol/L working with the on-line dilution integrated to the biosensor FIA system proposed. The diluted sample concentrations were also determined by gas chromatography (GC) and high-pressure liquid chromatography (HPLC) methods and the results compared with the proposed sequential system measurements. The effect of the number of analysis performed with the same system was also investigated.

Development and application of an integrated system for monitoring ethanol content of fuels.

An automated flow injection analysis (FIA) system for quantifying ethanol was developed using alcohol oxidase, horseradish peroxidase, 4-amino-phenazone, and phenol. A colorimetric detection method was developed using two different methods of analysis, with free and immobilized enzymes. The system with free enzymes permitted analysis of standard ethanol solution in a range of 0.05-1.0 g of ethanol/L without external dilution, a sampling frequency of 15 analyses/h, and relative SD of 3.5%. A new system was designed consisting of a microreactor with a 0.91-mL internal volume filled with alcohol oxidase immobilized on glass beads and an addition of free peroxidase, adapted in an FIA line, for continued reuse. This integrated biosensor-FIA system is being used for quality control of biofuels, gasohol, and hydrated ethanol. The FIA system integrated with the microreactor showed a calibration curve in the range of 0.05-1.5 g of ethanol/L, and good results were obtained compared with the ethanol content measured by high-performance liquid chromatography and gas chromatography standard methods.