1. Fabrication of a Microelectrode Array Biosensor Based on a Modified Enzyme-Chitosan Biocomposite Lorenzo D’Amico October 1, 2008

2.  Amgen Scholars, 10 week summer research program at UCLA funded by the Amgen Foundation  Harold Monbouquette’s Biotechnology laboratory in the Chemical and Biomolecular Engineering Department Our ghetto Faraday cage (Amgen did not pay for equipment)

3. Immobilized oxidase Analyte H2O2H2O2 O 2 + H 2 0 Byproduct 2 e - ~0.4 – 0.7 V vs. Ag/AgCl Electrode surface Current Electroenzymatic detection: general concept Amperometric biosensors convert a biochemical signal into an electric signal Hydrogen peroxide is produced by the immobilized enzyme in the presence of analyte and is subsequently oxidized at the electrode surface, resulting in an electron flow.

4.  Using MEMS technology, sensors are mass produced and manufacturing costs dramatically reduced (~200 probes per wafer);  Probe shaft is ~200 µm wide, 9 mm long;  At the probe tip is the microelectrode array. Each platinum site has a surface area of ~.00005 cm 2.  This design offers an opportunity to make the sensor capable of detecting multiple analytes, or using one site as the reference electrode. Developing a fabrication strategy with good spatial control is a current obstacle for researchers working in this area. Micromachined Probe Design Credit: Monbouquette et al.

5. Current Neuroscience Studies Using This Microelectrode Biosensor Design Glutamate detection in a freely moving rat Platinum electrode surfaces are functionalized with glutamate oxidase and modified with polymers which act to block interferents like dopamine and ascorbic acid. The devices are implanted into brain tissue of a freely moving rat and extracellular glutamate levels are continuously monitored. Sensors are capable of detecting increases in glutamate concentrations (spikes in the graph) induced by pinching the rat’s tail. Credit: Monbouquette et al. Pt electrode e - + Nafion + _ Glutamate oxidase 0.7 V vs. Ag/AgCl H2O2H2O2 Glutamate Polypyrrole Dopamine Ascorbic Acid + _ (analyte) I = I H2O2

6. Native glucose oxidase Nanoencapsulated glucose oxidase *Image provided by the Lu group, Chemical and Biomolecular Engineering Department at UCLA Nanoencapsulation of glucose oxidase  The original motivation to modify the enzyme was to increase the electrophoretic ability of the molecule;  By making the molecule more negative, it was thought that its deposition could be guided by applying positive voltage to target electrode sites;  Preliminary tests of this hypothesis did not seem promising. However, the modified enzyme was applied to a biofabrication process developed by the Bentley research group at the University of Maryland. *

7. Chitosan Solution Bulk pH is low, chitosan is soluble Electrode Surface pH gradient 1/2H 2 H+H+ Apply voltage Signal Directed Assembly of Chitosan pH = pK a  The deposited chitosan film offers a scaffold on to which enzyme can adsorbed;  If the electrodeposition of chitosan can be limited to target electrode sites, then indirect spatial control over enzyme deposition could be achieved.

8. Overview of Biofabrication Process  Probe tip immersed in enzyme solution Probes are removed from wafer, packaged and chemically cleaned in preparation for surface modification Chitosan is electrodeposited to target electrode sites, -2V for 4 min Tips are immersed in modified glucose oxidase solution overnight Control probes, -2V for 4 min in PBS instead of chitosan solution Tips are immersed in native glucose oxidase solution overnight Probes are tested for glucose detection in vitro

9. Results In vitro glucose detection tests entailed injecting glucose solutions of increasing concentration, ending with an injection of hydrogen peroxide. The 4 channels (red, green, blue and black) represent data collected from the four electrode sites on the probe. The figure above depicts the current response of a sensor that did not have chitosan electrochemically deposited to electrode surfaces. There seems to be little more than noise resulting from glucose injections, suggesting that enzyme function is absent. Experiments much like this demonstrated the need for an immobilization mediator in fabricating these biosensors. Ch 1 Ch 3 Ch 4 Ch 2 Current response of sensor without chitosan during in vitro glucose test

10. Results, cont’d In these particular set of experiments, one channel (control) was not charged during the chitosan deposition. The current response of one such probe is depicted above. Control channels in many of these probes exhibited a significantly lower current response to glucose injections in vitro. However, although the response is lower in the control, the channel is generating an electrical signal; Ideally, this would not occur. Evaluation of the spatial control achieved during biofabrication with chitosan and modified enzyme

11. Results, cont’d Experiments were designed to compare the analytical capabilities of sensors fabricated with modified and native glucose oxidase. At the right, the current response of a sensor functionalized with native enzyme (top) and that of a sensor with modified enzyme (bottom) are shown. Clearly, the signal is higher for the electrode surfaces containing chitosan and modified enzyme. Current response of MEA biosensors during In vitro glucose tests

12. Results, cont’d A direct comparison of the device calibration curves further demonstrates that using nanoencapsulated glucose oxidase improves analytical capabilities of the MEA biosensor design. MEA biosensors functionalized with modified enzyme exhibited greater sensitivity to glucose injections on average during in vitro tests. Calibration curves of sensors fabricated with modified and native glucose oxidase

13. Conclusion Encapsulating glucose oxidase in a negatively charged polymer was insufficient in addressing the spatial control issues associated with electrochemical fabricating MEA biosensors; Using Chitosan in a two step biofabrication process mediated the immobilization of both native and modified glucose oxidase to the electrode surfaces on the probes, as well as granted some degree of spatial control over enzyme deposition; Use of nanoencapsulated glucose oxidase increased device sensitivity There remains a need to optimize several steps in the overall biofabrication process. Amgen Foundation University Research Center, Center for Academic and Research Excellence Dr. Harold Monbouquette and Vanessa Tolosa The UCLA Amgen Scholars site coordinators: Dr. Tama Hasson and Dr. Patricia Phelps and Michael Flaxman Acknowledgements