Sensing and Actuation in Miniaturized Systems FABRICATION PROCESS OF INTEGRATED MULTI-ANALYTE BIOCHIP SYSTEM FOR IMPLANTABLE APPLICATION Good afternoon everyone, my name is Chen Han-Yi. The topic I will present today is Mass-production-oriented Ionic Polymer Actuator Based on Engineered Material Structure. The authors are come from Sony Corporation in Tokyo, JAPAN. Author: NY. -C. Tsai, N.-F. Chiu, P.-C. Liu, Y.-C Ou, H.-H. Liao, Y.-J. Yang, L.-J. Yang, U. Lei, F.-S. Chao, S.-S. Lu, C.-W. Lin, P.-Z. Chang, and W. -P. Shih Professor: Dr. Cheng-Hsien Liu (劉承賢教授) Student: Han-Yi Chen (陳翰儀) Date: 2009.12.29 1

Biocompatible package Control and wireless chip Outline Introduction Implantable multi-analyte biochip system Review Design Biocompatible package PDMS micro-channel Control and wireless chip Battery and RF power Blood inlet and outlet Fabrication Electrode/glass chip PDMS microchannel Biochip integration Test result and discussion DEP micropump test Cyclic voltammetric measurement Current Response Measurement Conclusions References

Introduction

Implantable Multi-analyte Biochip System Purpose: Monitor the physiological parameters (such as glucose concentration) continuously and simultaneously. stable and accurate implantable biosensor systems Thermal metabolic sensor integrated with microfluidics Silicon nanochannel biological field effect transistors Flexible biocompatible polymer glucose sensors Micromachining techniques used for biosensors Amperometric biochip Flexible polymer tube lab-chip integrated with microsensors Not been miniaturized Smart microcatheter

Implantable Multi-analyte Biochip System Issues: The incompatibility between the biomaterials and the MEMS processes System miniaturization Schematic of the wireless implantable biochip system Coated by parylene-C: biocompatibility. Circular shape: avoid the tissue injury during implantation. Biocompatible package PDMS micro-channel Control and wireless chip Battery and RF power Blood inlet and outlet Connected to blood vessels for in-situ biological detections: increase the performance of the measurement Control module: (1) drive the DEP micropump (2) supply the working voltage to the electrochemical electrodes Wireless sub-module: microcontroller unit (MCU), amplifier, and RF transmission section for signal transmission and process Supplies a 3.7 V voltage The programmatic target is to fabricate the microchannel which contains the micropump and the biosensors to apply implantable multi-analyte biochip system.

Implantable Multi-analyte Biochip System The DEP micropump and electrochemical electrodes are embedded in the microchannel Schematic of the PDMS microchannel which contains dielectrophoresis electrodes and chemical electrodes Dielectrophoresis electrodes for separation of erythrocyte and plasma Outlet Electrochemistry detection electrodes Inlet The motion of the electrolytes can urge the blood flow in the microchannel GOD enzyme is coated on the gold working electrode Pt & Ag improve the sensor lifetime and accuracy.

Fabrication

Dielectrophoresis electrodes for separation of erythrocyte and plasma Electrode/Glass Chip The electrodes are deposited by e-beam evaporation and patterned by using lift-off process. Dielectrophoresis electrodes: Number: 36 gold electrodes Thickness: 180 nm Area: 500 μm*15 μm Gap between adjacent DEP electrodes: 15 μm Dielectrophoresis electrodes for separation of erythrocyte and plasma Outlet Electrochemistry detection electrodes Inlet Electrochemistry electrodes: Area: 500 μm*500 μm Gap: 500 μm Thickness: Au180 nm, Pt 200 nm, Ag 250 nm Adhesion layer: 10 nm chromium

Dielectrophoresis electrodes for separation of erythrocyte and plasma PDMS Microchannel 500 µm Dielectrophoresis electrodes for separation of erythrocyte and plasma Outlet Electrochemistry detection electrodes Inlet 12 mm 40 µm SU-8 mold on a silicon substrate is patterned by photolithography. Then PDMS is applied and cured on the SU-8 mold. The cured PDMS is peeled off the SU-8 mold and then bonded on electrode/glass substrate. The length, width, and height of the PDMS microchannel are 12mm, 500μm, and 40μm, respectively. The inlet/outlet section in the microchannel has a large circular area in which the interconnection can be easily implemented.

Biochip Integration Chip assembly process: (a) Fixing the electrode/glass chip on the PCB by AB glue. (d) Bonding the PDMS microchannel by oxygen plasma. (b) Wire-bonding and applying AB glue on the wires for protection. (e) Connecting the tubes to the microchannel and then welding ICs on the PCB. (c) Placing the PDMS lump on the working and reference electrodes, respectively.

Pictures Pictures of the electrochemistry detection system and the DEP control module. (a) Front side: micro-channel and DEP control ICs. (b) Back side: circuit for electrochemical detection. The integration of the microchannel electrodes and the control ICs enables the multi-analyte detection on a single chip and has made the implantable system practicable.

Test Result and Discussion

Dielectrophoresis electrodes for separation of erythrocyte and plasma DEP Micropump Test Optical images of the blood flow driven by the four-phase DEP micropump Human blood is used to test the transport capability of the DEP micropump. Dielectrophoresis electrodes for separation of erythrocyte and plasma Outlet Electrochemistry detection electrodes Inlet The driving signal of the DEP micropump is a 25 MHz, 5 V four-phase sine wave. The measured blood flow velocity is 14 μm/s. This driving condition requires low electric voltage so that the low power consumption of the DEP micropump can be achieved and be suitable for the implantable biochip system.

Cyclic Voltammetric Measurement Cyclic voltammetric measurement result of different glucose concentrations The phosphate buffer solution (PBS) and different glucose concentration solution are injected into microchannel individually for measurement. 0.25 V The measured peak potential for different glucose concentration is about 0.25 V. The current response increases with the increasing glucose concentration at 0.25 V.

Current Response Measurement Measurement result of the current response after injecting 50mM potassium ferrocyanide Constant voltage 0.25 V An current response about 50 nA is obtained after the glucose and 50 mM potassium ferrocyanide mediator (as a means of shuttling electrons between the immobilized glucose oxidase enzyme and the electrode surface) is injected into the microchannel. 50nA Then the phosphate buffer solution is injected to the microchannel, the current level returns to its initial value. The current response decreases with time due to the decreasing concentration.

Conclusions

Conclusions (1) The integration of an implantable multi-analyte system for continuously in-vivo monitoring important physiological signals has been proposed and demonstrated. By protecting the biomaterials during the oxygen plasma bonding, multiple biosensors can be easily integrated in the microchannel. The proposed process sequences of the implantable biosensor system can overcome the difficulty in the system miniaturization. The glucose detection is achieved by using electrochemical method, and the function of the DEP micropump is verified.

Picture of the wireless implanted biochip system Conclusions (2) The electrochemistry and DEP control circuits are integrated with the inductive power coupling and wireless communication modules. A fully packaged miniature system with the total volume of 13 c.c. is achieved. In addition, the GOD enzyme can be replaced by other enzymes or other bio-marker for different implantable applications. Picture of the wireless implanted biochip system

References (1) [1] H. Kudo, T. Sawada, E. Kazawa, H. Yoshida, Y. Iwasaki, K. Mitsubayashi, “A flexible and wearable glucose sensor based on functional polymers with Soft-MEMS techniques”, Biosensors and Bioelectronics, vol. 22, pp. 558-562, 2006. [2] K. Mitsubayashi, S. Iguchi, T. Endo, S. Tanimoto, D. Murotomi, “Flexible glucose sensors with a film-type oxygen electrode by microfabrication techniques”, in Digest Tech. Papers Transducers‘03 Conference, Boston, June 8-12, 2003, pp. 1201-1204. [3] L. Wang, D. M. Sipe, Y. Xu, Q. Lin, “A MEMS thermal biosensor for metabolic monitoring applications“, Journal of Microelectromechanical Systems, vol. 17, no. 2, pp. 318-327, 2008. [4] X. Wang, Y. Chen, K. A. Gibney, S. Erramilli, P. Mohanty, “Silicon-based nanochannel glucose sensor”, Applied Physics Letters, vol. 92, 013903, 2008. [5] J. Wu, J. Suls, W. Sansen, “The glucose sensor integratable in the microchannel”, Sensors and Actuators B, vol. 78, pp. 221-227, 2001. [6] A. Guiseppi-Elie, S. Brahim, G. Slaughter, K. R. Ward, “Design of a subcutaneous implantable biochip for monitoring of glucose and lactate”, IEEE Sensors Journal, vol. 5,no. 3, pp. 345-355, 2005.

References (2) [7] M. Pepper, N. S. Palsandram, P. Zhang, M. Lee, H. J. Cho, “Interconnecting fluidic packages and interfaces for micromachined sensors”, Sensors and Actuators A, vol. 134, pp. 278-285, 2007. [8] C. Li, P.-M. Wu, J. Han, C. H. Ahn, “A flexible polymer tube lab-chip integrated with microsensors for smart microcatheter“, Biomed Microdevices, vol. 10, pp. 671-679, 2008. [9] C. Y. Yang, U. Lei, “Dielectrophoretic force and torque on an ellipsoid in an arbitrary time varying electric field”, Applied Physics Letters, vol. 90, 153901, 2007. [10] P. A. Fiorito, S. I. Cordoba de Torresi, “Glucose amperometric biosensor based on the Co-immobilization of glucose oxidase (GOx) and Ferrocene in poly(pyrrole) generated from ethanol/water mixtures”, J. Braz. Chem. Soc., vol. 12, no. 6, pp. 729-733, 2001. 207

Thank you for your attention!! 21

Back-up Foil Biochip Integration The process for fabricating the implantable biosensor system should be carefully designed to resolve the incompatibility of the biomaterials and to achieve the system　miniaturization. The electrode/glass chip is fixed on the printed circuit board (PCB) by AB glue (Figure 3(a)). The electrodes on the glass substrate are connected to the corresponding pads on the PCB by applying wire-bonding. The wires are then covered by the AB glue which serves as the protection layer in the following process steps (Figure3(b)). The wire-bonding process should be carried out prior to bonding the PDMS microchannel on the glass substrate. Otherwise, the PDMS microchannel would hinder the view for aligning the electrodes with the bonding pads in the wire-bonding process. The AB glue must not cover the electrodes on the electrode/glass chip for the success of bonding the PDMS microchannel. Before bonding the PDMS microchannel, the Ag/AgCl reference electrode is chloridized. The enzyme polymerization on the Au working electrode should also be carried out prior to bonding the PDMS microchannel because it is difficult to proceed in the microchannel. The electroplating method is used to chloridize the Ag/AgCl reference electrode. A 0.1M NaCl solution is applied on the electrode/glass chip. Meanwhile, the platinum and silver electrodes are protected. In the electroplating process, the current density is 40μA/cm2. The processing time is 10 minutes. The polypyrrole (from Merck) and GOD mixture is electrochemically polymerized on the working electrode by controlling the voltage from 0V to 1.2V in a cyclic voltammetry. After the GOD polymerization, the working electrode becomes dark brown.

Back-up Foil To bond the PDMS microchannel onto the glass substrate, the bonding surfaces should be modified by using oxygen plasma. It should be noted that the working electrode and the Ag/AgCl reference electrode should be covered for avoiding direct contact with the oxygen plasma (Figure 3(c)). Otherwise, the enzyme on the working electrode will be damaged, and the Ag/AgCl reference electrode will be oxidized. The microchannel which is bonded on the glass substrate is illustrated in Figure 3(d). Finally, the tubes are connected with PDMS microchannel. The interconnection is sealed by AB glue (Figure 3(e)). The polyethylene tube has 0.86mm inner diameter and 1.27mm outer diameter. In order to make the compact interconnection, the “L” shape passage of 1.25mm diameter is used. The diameter of the passage is slightly smaller than the outer diameter of the tube. The elastic PDMS passage can tightly clamp the tube and avert the AB glue from permeating into the microchannel by capillary force.