Research Article Fabrication of an Amperometric Flow-Injection Microfluidic Biosensor Based on Laccase for In Situ Determination of Phenolic Compounds Juan C. Gonzalez-Rivera and Johann F. Osma CMUA, Department of Electrical and Electronics Engineering, University of Los Andes, Cra 1 E No. 19 A-40, Bogota, Colombia Correspondence should be addressed to Johann F. Osma; jf.osma43@uniandes.edu.co Received 18 February 2015; Revised 30 April 2015; Accepted 1 May 2015 Academic Editor: Jinsong Ren Copyright © 2015 J. C. Gonzalez-Rivera and J. F. Osma. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. We aim to develop an in situ microfuidic biosensor based on laccase from Trametes pubescens with fow-injection and amperometry as the transducer method. Te enzyme was directly immobilized by potential step chronoamperometry, and the immobilization was studied using cyclic voltammetry and electrochemical impedance spectroscopy. Te electrode response by amperometry was probed using ABTS and syringaldazine. A shif of interfacial electron transfer resistance and the electron transfer rate constant from 18.1 kΩ to 3.9 MΩ and 4.6 × 10 −2 cm s −1 to 2.1 × 10 −4 cm s −1 , respectively, evidenced that laccase was immobilized on the electrode by the proposed method. We established the optimum operating conditions of temperature (55 ∘ C), pH (4.5), injection fow rate (200 L min −1 ), and applied potential (0.4 V). Finally, the microfuidic biosensor showed better lower limit of detection (0.149 M) and sensitivity (0.2341 nA M −1 ) for ABTS than previous laccase-based biosensors and the in situ operation capacity. 1. Introduction Phenols are employed in several industries in the manu- facture of plastics and plasticizers, resins, explosives, drugs, detergents, paper, fungicides, preservatives, dyes, and lubri- cants [1, 2]. Most phenolic compounds are toxic, noxious, and mutagenic and have carcinogenic activity [2] that accumulate in the environment and are found in food, potable water, sediments, and soil. Currently, many organizations have established proce- dures using colorimetry, gas chromatography, liquid chro- matography, capillary electrophoresis, and their variations [3]. Even though these methods attain accurate results for a wide range of phenolic compounds, conventional approaches are time-consuming and cost-intensive and require large volumes of organic solvents. Consequently, a market demand exists for a reliable, portable, simple, and cost-efective detection method of phenolic compounds. Both enzymatic-based biosensors and microfuidic biosensors have attracted increasing among the diferent confgurations of biosensors [4–8]. Microfuidic biosensors combine the advantages of fuidic microsystems, such as low cost, short analysis time, less consumption of sample and reagents, and portability, with the advantages of biosensors such as selectivity, moderate operational potentials, high sensitivity, specifcity, and easiness to be miniaturized and integrated [3, 6, 9, 10]. Terefore they have potential in environmental safety, food, and clinic analysis. Te immobilization method is a key parameter for the design and fabrication of microfuidic biosensors [11]. Te bioreceptor and the sensor elements can be coupled together with several methods, such as physical adsorption, entrap- ment, cross-linking, and covalent bonding [12, 13]. However, enzyme degradation and surface inaccessibility arise with the enzyme immobilization inside a microchannel. Tus, we propose the direct electrochemical immobilization of laccase afer the sensor sealing since this technique enables an easier immobilization than traditional techniques. Amperometry is the most common transducer tech- nique in biosensors because it ofers detection in real time [14, 15]. When this technique is coupled with convec- tive mass transport, the resulting technique—hydrodynamic Hindawi Publishing Corporation BioMed Research International Volume 2015, Article ID 845261, 9 pages http://dx.doi.org/10.1155/2015/845261