1. Dynamic Thermoelectric Glucose Sensing with Layer-by-Layer Glucose Oxidase Immobilization Siva Mahesh Tangutooru1 V.L. Kopparthy, R. Gumma, G.G. Nestorova, E.J. Guilbeau Center for Biomedical Engineering and Rehabilitation Science Louisiana Tech University

2. Goal To develop a microfluidic glucose calorimeter by immobilizing glucose oxidase using layer-by-layer self-assembly.

3. Presentation Summary Principle of thermoelectricity Calorimeter design and measurement system Layer-by-layer self-assembly procedure Results Conclusion Future work

4. Thermoelectric detection of glucose 𝐺𝑙𝑢𝑐𝑜𝑠𝑒+𝑂2+𝐻2𝑂𝑔𝑙𝑢𝑐𝑜𝑠𝑒 𝑜𝑥𝑖𝑑𝑎𝑠𝑒𝐺𝑙𝑢𝑐𝑜𝑛𝑖𝑐 𝑎𝑐𝑖𝑑+𝐻2𝑂2+79 𝑘𝐽/𝑚𝑜𝑙

5. Outlet Inlet1 Device fabrication Inlet2 Thermopile Microfluidic Device Microfluidic Calorimeter PDMS inlet & outlet connectors Microscope glass slide Kapton® tape Microscope glass coverslip (a) (a) (b) (c) (d)

6. Experimental measurement system

7. Hydrodynamic focusing G. G. Nestorova and E. J. Guilbeau, "Thermoelectric method for sequencing DNA," Lab on a Chip, vol. 11, pp. 1761-1769.

8. PEI Layer-by-layer self assembly PSS Glucose Oxidase PEI Layer-by-layer electrostatic adsorption mechanism. General adsorption procedure of ployelectrolytes on a substrate. Layer-by-layer assembly of glucose oxidase on the substrate. Structure of polyelectrolytes used for LbL assembly. N+ H2 SO3- Na+ Polyelectrolyte Glucose Oxidase Immobilization procedure for LbL assembly. K. Ariga, Y. Lvov “Self-Assembly of Functional Protein Multilayers: From Flat Films to Microtemplate Encapsulation”, “Biopolymers at Interfaces” Ed. M. Malmsten, M. Dekker Publ., 2003, NY, p.367-391. (a) PSS PEI (c) (b)

9. Our novel glucose calorimeter Calorimeter Self-generating signal No external power requirement Relatively inexpensive Small in size and light weight High rejection of common mode thermal signals Relatively simple to manufacture Rugged and durable Layer-by-layer Immobilization Multiple layers are physically adsorbed. Easy preparation and high bond strength.

10. Sensor response for glucose concentration 75 mg/dL 100 mg/dL Flow rates: 100 µlmin-1 and 25 µlmin-1 No. of Immobilized glucose oxidase layers: 2

11. Effect of number of immobilized layers on sensor response Flow rates: 100 µlmin-1 and 25 µlmin-1

12. Effect of flow rates on sensor response No. of Immobilized glucose oxidase layers: 2

13. Conclusion Successfully immobilized multiple layers of glucose oxidase using LbL self-assembly. Multiple layers of glucose oxidase had little effect because oxygen limited the reaction. Increasing the ratio of inlet flow rates improved calorimeter response. Future work This thermoelectric method can be employed to detect the enthalpy produced by enzymatic reactions. glutamate concentration detection in the flow stream by immobilizing glutamate oxidase using layer-by-layer self-assembly.

14. Thank you Questions ???

15. M. J. Muehlbauer, E. J. Guilbeau, and B. C. Towe, "Applications and stability of a thermoelectric enzyme sensor," Sensors & Actuators: B. Chemical, vol. 2, pp. 223-232, 1990.