Whole cell biochips – Issues in Nano Bio Interfacing

1. Presented at N2L meeting, Lund, October 2007 Whole cell biochips – Issues in Nano Bio Interfacing Yosi Shacham-Diamand The Bernard L. Schwartz chair for nano Scale information Technologies The Dept. of Physical Electronics , School of EE , Faculty of Engineering Tel-Aviv University, Israel & The Dept. of Applied Chemistry Waseda University, Tokyo, Japan

2. Biological Recognition Hierarchy Antibody Specificity Enzyme Complexity Hierarchy DNA Whole cell Physiological effect Tissue

3. Why integrating live cells ? • Multi-cells: • Functional response • Emulating “real” life behavior • Emulating complex systems characteristics • Study cell behavior • Single cells: • All the above + cell sorting

4. “Canary in a cage” concept Photograph from the "Welsh Coal Mines" Collection from the National Museum of Wales

5. Whole cell Bio-Chip • Prokaryotic – Bacteria, • Sensors for acute toxicity in water, • Detecting toxicity of drugs, cosmetics etc. • Eukaryotic – • Mammalian cells - cancer therapy, stem cells characteristics

6. Interfacing cell biology & MEMS • Optical • Luminescence – photo luminescence, bio-luminescence • Electrical • Electrochemical – active or passive electrodes • Impedance spectroscopy • Mechanical • Resonators, deflection sensors

7. Electrochemical whole cell bio-chips

8. Why Electrochemistry? • Simple. • Sensitive. • Monitoring in turbid solutions. • Simultaneous measurements of several samples. • Electrical output- convenient to handle and analyze. • Easily scaled down.

9. Substrate Bacteria Promoter Reporter Plasmid Toxicant Genetically Engineered Bacteria b-gal b-gal b-gal

10. PAPG Genetically Engineered Bacteria Bacteria Enzyme Sabstrate Product PAP+ b-gal

11. PAPG PAP PAP PAP PAP PAP PAP Genetically Engineered Bacteria Bacteria Enzyme Sabstrate Product + b-gal

12. Chip Process

13. 1 cm Chip Process

14. Chip Process

15. Plastic Platform

16. Portable BioChip System Array of nano liter volume electrochemical cells Multiplexer Pocket PC Potentiostat a single chamber (Magnified)

17. Physiological Response To Phenol On-chip Control

18. Personalized MedicineHigh-throughput Detection Of Human Cancer Cells

19. Goal Evaluation of cancer cells response to different drugs. Bio-chips for differential therapy

20. Introduction Current Therapeutic Strategies: • Surgery • Chemotherapy • Irradiation Differentiation Therapy: • Cancerous cells are being induced to behave like normal cells • It restrains their growth • Differentiation agents tend to have less toxicity than conventional cancer treatments

21. How can we evaluate the efficiency of the drug? According to the enzymatic activity level of the treated cancer cells. • Normal enzymatic activity denotes that the cells become 'healthy‘, • Lack of enzymatic activity denotes ineffectual drug treatment for the particular cancer tumor and for the particular patient.

22. Experimental • Human colon cancer cells were treated with different differentiation therapy drug agents. • The cells were placed in each one of the electrochemical-cells in the array, while each chamber was treated with different drug type. • p-APP substrate was added. • The generated current signal was measured. • Cells number was counted under the microscope.

23. Results

24. Enzymatic activities vs. cancer cell number

25. Correlation between HT-29 colon cancer cell number and the induced alkaline phosphatase enzymatic activity (DI/Dt). (amperometric signal at 220mV).

26. Optical whole cell bio-chips

27. Photo luminescent bio-chip Emission (green) Photodiode Excitation chip (Blue) Cell’s container Bio chip

28. Bio-luminescent sensor Emission (green) Photodiode Cell’s container Bio chip

29. Reporting element gene promoter luxCDABE Sensing element Engineering live cells for the detection of toxicants The fusion of two genetic elements: • Sensing element: A promoter of a geneinvolved in the response to the desired target • Reporting element:Fluorescence or bioluminescence genes The final construct emits a dose-dependent signal in response to the presence of the target chemicals Light

30. Bioluminescent Prokaryote cell-based biochip • Comprised of: • (a) biochip sensor for optical/electrochemical measurement. • (b) microfluidic elements for delivery of samples and media. • A nano-patterning technique for spotting bacteria onto a platform is being developed. • The biochip functions in a “plug-&-play” mode of action to facilitate insertion into the microfluidic platform.

31. System outline Prokaryote cell biochip layout Schematic view of the four photo-diodes array

32. New setup – with 4 PV detectors/ mechanical scan Biochip platform (on the left) and its 3D model (on the right).

34. Sigmoid Cross-correlation: 10 ppm = 0.631 5 ppm = 0.647

35. Verhulst

36. Integrated heterodyne detection with Whole cell biochips

37. More complicated systemsBio-MEMS Lab-on-Chip • Using MEMS technology integrating low-light emitting whole-cell sensors, and VLSI devices. • Micromechanical shutters for luminescent bio-chips – modulates the light Optical Sensor Modulation Shutters Luminescence Whole cell Biochip

38. More complicated systemsBio-MEMS Lab-on-Chip • Using MEMS technology integrating low-light emitting whole-cell sensors, and VLSI devices. • Micromechanical shutters for luminescent bio-chips – modulates the light Optical Sensor Modulation Shutters Luminescence Whole cell Biochip

39. More complicated systemsBio-MEMS Lab-on-Chip • Using MEMS technology integrating low-light emitting whole-cell sensors, and VLSI devices. • Micromechanical shutters for luminescent bio-chips – modulates the light Optical Sensor Modulation Shutters Luminescence Whole cell Biochip

40. More complicated systemsBio-MEMS Lab-on-Chip • Using MEMS technology integrating low-light emitting whole-cell sensors, and VLSI devices. • Micromechanical shutters for luminescent bio-chips – modulates the light Optical Sensor Modulation Shutters Luminescence Whole cell Biochip

41. Integrated heterodyne detection with Whole cell biochips Heterodyne detection Output Optical Sensor Shutters Modulator ~1kHz Luminescence Whole cell Biochip • Converts low frequency biological signal to high frequency signal, • Reduces 1/f noise  improves the S/N ratio.

42. 500 μm 200 μm Fabrication Results Shutters, Springs, Comb-drives Backbone, Shutters, Shutter-Windows

43. MEMS Fabrication Array of resonators as band-pass filters Array of comb-drive actuators

44. Fabrication Results Released actuators Cross-section of electrically isolated device

45. Fabrication Results: Backside Characterization Goal: Characterize Deep Silicon backside etch of the shutter windows using the Bosch Process using windows with varying gaps Gap: 65 μm Gap: 60 μm Gap: 70 μm Gap: 50 μm Gap: 45 μm Gap: 55 μm

46. Cross-section sketch showing the components of the experimental set-up. Light emitted from the bio chip

47. Frequency response of the device. In air In vacuum Noel Elman, PhD thesis , TAU 2006

48. Integrated Heterodyne MEMS Response vs. Concentration Response vs. time

49. Key issues • Cell storage on the chip • Cell revival • Signal level – very low, the microbes emit ~ 0.1 – 10 photons/ sec. • Operation under flowing liquid • Detection in air – extracting onto water • Producing arrays

50. Acknowledgements Thanks to all my students, especially to Dr. Rachela Popovtzer (Graduated 2006) , Dr. Noel Elman (Graduated 2006), Dr. Ronen Almog (Post Doc), Arthur Rabner (2009), Hadar Ben-Yoav (2009), Sefi Wornick (2009), Amit Ron (2009), Amit Livneh (2007), Hila Einati (2009) and Hila Dagan (2008) Special thanks to Prof. Shimshon Belkin from the Hebrew University of Jerusalem (HUJI) Prof. Judith Rishpon and Prof. Eliora Ron from Tel Aviv University (TAU) Dr. Slava Krylov (TAU) and Dr. Marek Sternhaim for their help with the MEMS modeling