BIOFABRICATION OF MEA GLUCOSE SENSORS Dry in air Ready for in vitro glucose detection (B) Chitosan biopolymer is electrodeposited on target electrode sites; (C) MEA tip is immersed in enzyme solution overnight. During this stage, enzyme is adsorbed into the chitosan film and retained by London forces. (A) Probes are removed from wafer and packaged for modification; Chemical cleaning GLUCOSE DETECTION IN VITRO, CONTINUED In figure B, the responses to glucose and hydrogen peroxide injections by sensor channels functionalized with chitosan-modified glucose oxidase are shown. A dramatic increase in current response is seen when compared to the native enzyme biosensor response depicted in figure A..5 mM glucose 2.5 mM glucose 4.8 mM glucose 9.6 mM glucose 9.8 mM H 2 O 2.5 mM glucose 2.5 mM glucose 4.8 mM glucose 9.6 mM glucose 9.8 mM H 2 O 2 ELECTRODEPOSITION OF CHITOSAN Chitosan is a linear β-1,4-linked aminopolysaccharide (like cellulose) that is derived from the partial deacetylation of chitin. It has unique properties ideal for electroenzymatic biosensor manufacturing; The probe tip is immersed in a slightly acidic chitosan solution. Under acidic conditions, chitosan’s amine groups are protonated and the molecule becomes soluble; Applying a negative voltage to the target electrode surface creates a local pH gradient; At the surface of the electrode, where the pH is sufficiently high, chitosan’s amine groups become deprotonated and a thin, porous matrix is formed on the target electrode site. Chitosan Solution pH gradient pH = pK a 1/2H 2 H+H+ Chitosan is soluble in low pH MICROMACHINED PROBE DESIGN Probe shaft is ~200 μm wide, 9 mm long Using MEMS technology, sensors can be mass produced and manufacturing costs dramatically reduced (~200 sensors per wafer) Design features four platinum electrode sites ~5000 μm 2 in area; multiple sites can potentially allow for simultaneous detection of multiple analytes. ABSTRACT A novel microelectrode array (MEA) biosensor was fabricated through a simple, two step process that entails electrodeposition of chitosan and subsequent adsorption of a modified form of glucose oxidase enzyme. Modification involves the nanoencapsulation of enzymes in a negatively charged, porous polymer matrix. The aminopolysaccharide chitosan can be directed to assemble a thin, porous matrix in response to an applied electrical signal that provides a scaffold onto which enzyme can be adsorbed. Use of the chitosan-modified glucose oxidase biocomposite in MEA glucose sensor fabrication displays improved analytical capabilities. Sensors produced using modified glucose oxidase exhibited higher sensitivity to glucose ( nA mM -1 cm -2 ) than those fabricated with native enzyme (998.8 nA mM -1 cm -2 ). In addition, the detection limit was lower for the modified enzyme biocomposite (250 μM) than that of the native enzyme biocomposite (655 μM). Furthermore, the signal directed assembly of chitosan on target electrode surfaces was shown to grant some degree of spatial control over enzyme deposition, though there remains a need for optimization of this technique. Modifying specific electrode sites of the array may prove to be a critical step in the development of a biosensor capable of near real-time recording of multiple analytes simultaneously. Fabrication of a Microelectrode Array Biosensor Based on a Chitosan-Modified Enzyme Biocomposite Lorenzo D’Amico 1, Vanessa Tolosa 2, Harold Monbouquette 2 2 Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, CA 1 Department of Bioengineering, University of California, San Diego, CA MODIFICATION OF GLUCOSE OXIDASE Glucose oxidase is encapsulated by a novel process developed by the Lu group at UCLA* in a negatively charged, porous polymer matrix. Native function is preserved while the stability and strength of interaction with the biopolymer chitosan are increased. Native glucose oxidase Modification FUTURE STUDIES Optimize spatial control over enzyme deposition by improving the biofabrication process at three stages:  Rearrange the experimental set up during electrodeposition to uniformly distribute the biopolymer over all target sites;  Vary chitosan concentration and electrodeposition time to determine optimal values for assembling the immobilizing film;  Determine an enzyme concentration during the adsorption stage that maximizes the amount of protein adsorbed by the chitosan film. Electrode Surface ELECTROENZYMATIC SENSING PRINCIPLE Glucose oxidase is immobilized on electrode via chitosan biopolymer; O 2 serves as the electron transfer mediator; The oxidation current of H 2 O 2 reflects the glucose concentration. CONCLUSIONS Chitosan was demonstrated as an adequate medium for enzyme immobilization on the platinum microelectrode surface; The modified enzyme-chitosan biocomposite exhibited higher sensitivity to glucose and a lower detection limit than sensors produced using native enzyme; Some degree of spatial control was achieved over enzyme deposition using the described biofabrication process. ACKNOWLEDGMENTS Amgen Foundation and the Amgen Scholar’s program at UCLA, URC CARE at UCLA The Lu research group in Chemical and Biomolecular Engineering Department at UCLA * for providing the modified enzyme GLUCOSE DETECTION IN VITRO Encapsulated glucose oxidase GlucOx ?D-glucose D-glucono-1,5-lactone 2e - Electrode O 2 + H 2 O H 2 O 2 0.7Vvs Ag/AgCl GlucOx βD-glucose D-glucono-1,5-lactone 2e - Electrode O 2 + H 2 O H 2 O 2 Current 0.7Vvs. * * The glucose response of sensors functionalized with chitosan-modified enzyme (red, black and green). Chitosan was not deposited on the control (blue). As predicted, the current measured by the control is significantly lower, suggesting that at this site enzymatic activity is absent. The steps that do appear in the control channel reveal a need for optimization of this fabrication protocol..5 mM glucose 2.5 mM glucose 4.8 mM glucose 9.6 mM glucose 9.8 mM H 2 O 2 In figure A, the current responses of MEA sensor channels modified with chitosan-native enzyme biocomposite are shown. The amplitude of the steps are relatively low compared to those in figure B. A B