Development of a toxi n screening multi-parameter on-line bio chip system.

1. Development of new devices for toxin detectionCarmen Moldovan, Rodica Iosub, Bogdan Firtat, Daniel Necula, *Eric Moore, *Gheorghe MarinIMT –Bucharest*Tyndall National Institute, Cork Bucharest, 6th of December “Cooperation in FP7 Biomedical applications of micro and nanotechnologies”

2. Development of a toxin screening multi-parameter on-line biochip system. ToxiChip Proposal No. 27900 Bucharest, 6th of December “Cooperation in FP7 Biomedical applications of micro and nanotechnologies”

3. Toxichip Objective • Alternative in-vitro testing methods for the monitoring of toxichemicals • A eukaryote cell based biochip that examines the effects of toxichemicals on cell impedance, morphology, distribution and pH • A panel of genetically engineered bacterial strains that optically report on the presence of toxichemicals that normally affect eukaryotic cells • A prokaryote based biochip that uses optical analysis to measure the signal (fluorescence, bioluminescence) emitted by the bacteria in the presence of different toxichemicals • Develop a microfluidic system with temperature control and pH sensor Bucharest, 6th of December “Cooperation in FP7 Biomedical applications of micro and nanotechnologies”

4. Inlet Temperature sensor pH sensor Connector Outlet Dipstick O-ring Solid Support Impedance sensor Eukaryote cell-based biochip • Comprise of finger electrodes composed of indium tin oxide (ITO) • ITO will be used as an impedance sensor that allows real time non-invasive in-vitro analysis • Biochip will function in a “plug-in-play” mode that will facilitate its insertion into the microfluidic platform Bucharest, 6th of December “Cooperation in FP7 Biomedical applications of micro and nanotechnologies”

5. Further Objectives • specialised interfaces that links the biochip to a PC, that record data and provides on-line continuous information on microfluidics, electro-chemical signals, temperature and pH • Develop a management software for project experiments, such as data acquisition, parameters control and data mining   • Performance evaluation of the biochips will be done for the various integrated sensing capabilities - optical and electrochemical detection • Characterise/Validate the biochips by using a combination of cellular bioassays and evaluating the biological effects of high doses and low doses, the impacts on DNA and endocrine perturbations on the bioassays. Bucharest, 6th of December “Cooperation in FP7 Biomedical applications of micro and nanotechnologies”

6. Bucharest, 6th of December “Cooperation in FP7 Biomedical applications of micro and nanotechnologies”

7. National Institute for Research and Development in Microtechnologies (Romania) • Microsystems research • Simulation, design, technology, biosensors, electrodes for biological sensors, microprobes for recording of electrical activity of cells • Technological development: • -Micromachining techniques for silicon, glass and ceramics, • -Immobilization technique of enzymes on electrodes • -Spin deposition of thin film sensitive polymers Bucharest, 6th of December “Cooperation in FP7 Biomedical applications of micro and nanotechnologies”

8. Schematic of Toxichip Operating System Electrode Surface Chemistry Cells + + PC EIS Biochip + PlatformInterface Inverted Phase-contrast/Fluorescent Microscope coupled with CCD camera Bucharest, 6th of December “Cooperation in FP7 Biomedical applications of micro and nanotechnologies”

9. Development of temperature sensor integrated with the microfluidic platform • Simulation and design of the temperature sensor together with the microfluidic part where the sensor is integrated. • Study of the compatibility of the sensor with the chemical aggressive working environment. • Biocompatibility of the microsensor materials with the biological media. • Experiments, manufacturing design and testing. Development of pH sensors integrated with the microfluidic platform • Development of the pH sensor together with the microfluidic part where the sensor is positioned. • Development of materials, manufacturing steps, experiments and testing. Bucharest, 6th of December “Cooperation in FP7 Biomedical applications of micro and nanotechnologies”

10. Three channels1mmx1mm each Bucharest, 6th of December “Cooperation in FP7 Biomedical applications of micro and nanotechnologies”

11. 1 channel flow Bucharest, 6th of December “Cooperation in FP7 Biomedical applications of micro and nanotechnologies”

12. 3 channels flow Bucharest, 6th of December “Cooperation in FP7 Biomedical applications of micro and nanotechnologies”

13. 2 mm x 2mm channels with enlarged areas (4 mmx2mm) hosting sensors and Cells (6mm exhaust) Mesh: Bucharest, 6th of December “Cooperation in FP7 Biomedical applications of micro and nanotechnologies”

14. Flow analysis Bucharest, 6th of December “Cooperation in FP7 Biomedical applications of micro and nanotechnologies”

15. Flow analysis(details) Bucharest, 6th of December “Cooperation in FP7 Biomedical applications of micro and nanotechnologies”

16. Analysis of the flow rate by square section channels Bucharest, 6th of December “Cooperation in FP7 Biomedical applications of micro and nanotechnologies”

17. Analysis of the flow rate by square section channels (one active entry – detail) Bucharest, 6th of December “Cooperation in FP7 Biomedical applications of micro and nanotechnologies”

18. Flow rate analysisconsidering a round shape ofthe enlarged area hosting ITO electrode (three entries in the exhaust channel) Bucharest, 6th of December “Cooperation in FP7 Biomedical applications of micro and nanotechnologies”

19. Flow rate analysisconsidering a round shape ofthe enlarged area hosting ITO electrode (detail) Bucharest, 6th of December “Cooperation in FP7 Biomedical applications of micro and nanotechnologies”

20. Flow rate analysisconsidering a round shape ofthe enlarged area hosting ITO electrode (one entry in the exhaust channel) Bucharest, 6th of December “Cooperation in FP7 Biomedical applications of micro and nanotechnologies”

21. Flow rate analysisconsidering a round shape ofthe enlarged area hosting ITO electrode (one entry in the exhaust channel) - detail Bucharest, 6th of December “Cooperation in FP7 Biomedical applications of micro and nanotechnologies”

22. Flow rate analysisconsidering a round shape ofthe enlarged area hosting ITO electrode Bucharest, 6th of December “Cooperation in FP7 Biomedical applications of micro and nanotechnologies”

23. Flow rate analysis Bucharest, 6th of December “Cooperation in FP7 Biomedical applications of micro and nanotechnologies”

24. Technological flow – microfluidic channels • Material – PDMS • Patterning Laser machining Goal: study, set-up and optimisation of the microfluidic channels issues: design, technology, connections, integration into the platform Bucharest, 6th of December “Cooperation in FP7 Biomedical applications of micro and nanotechnologies”

25. Microfluidic channels Bucharest, 6th of December “Cooperation in FP7 Biomedical applications of micro and nanotechnologies”

26. Microfluidic channels Bucharest, 6th of December “Cooperation in FP7 Biomedical applications of micro and nanotechnologies”

27. Sensors cell Sensors Unit pH Signal Processing Unit Reference Temperature sensor Bucharest, 6th of December “Cooperation in FP7 Biomedical applications of micro and nanotechnologies”

28. Results: • Design of the microfluidic platform • Study of sensors, materials, technological versions; test structure utilisation • Design of the sensors • Interconnection layer design - draft Bucharest, 6th of December “Cooperation in FP7 Biomedical applications of micro and nanotechnologies”

29. Biosensors for neurotoxic substances detection The biosensors for neurotoxic substances will be developed as ISFET-type biosensors. The ISFET structure is represented by a concentration-potential transducer, with a biosensitive layer deposited on the gate (Acetylcholinesterases, immobilised on chitosane), which generates an interface potential on the gate. The enzymatic ISFET structure is developed in CMOS technology and the sensor’s response characteristics depend mainly on the AChE enzyme immobilisation mode. Optical photography of the sensor chip Bucharest, 6th of December “Cooperation in FP7 Biomedical applications of micro and nanotechnologies”

30. ISFET and chemoresistive sensors Principle: The change in conductivity at the sensitive layer surface, deposited on top of the sensor . Characterisation The enzyme electrode is a combination of any electrochemical probe (amperometric, potentiometric or conductometric) with a thin layer (10200mm) of immobilized enzyme. In these devices, the function of the enzyme is providing selectivity by virtue of its biological affinity for a particular substrate molecule. For example, an enzyme is capable of catalyzing a particular reaction of a given substrate even though other isomers of that substrate or similar substrates may be present Bucharest, 6th of December “Cooperation in FP7 Biomedical applications of micro and nanotechnologies”

31. H H H O H O -O N H C 2 O H ) ( H O O H O n O H SiO2 O -O N H H C H 2 H AChE O N SiO2 H H H Bio integration I. Surface Chemistry Functionalization The silicon or glass substrates are functionalized as hydrophobic, hydrophilic, biocompatible surfaces Available procedures: Cleaning procedures Chemical Vapor Deposition: CVD, PSG, BPSG Polymer Adlayers: SU-8, Organosilanes (Rn-Si-X(4-n)) Passivation (treatment in N2 and H2) Immobilization technique Ex: After surface functionalization , the immobilised AChE enzyme within a chitosan biolayer laid on the sensor structure Bucharest, 6th of December “Cooperation in FP7 Biomedical applications of micro and nanotechnologies”

32. Field effect gas sensors Field effect gas sensors are based on metal-insulator-semiconductor structures used to detect chemical quantities. Examples are biological and medical applications. The surface field effect is a desirable mechanism for a generating potential that provides high chemical selectivity and sensitivity. Using micromachining techniques we manufactured an ISFET device with posibility of integrating an area of sensing devices and the electronics on the same chip; ISFET sensors use the field effect transistors to detect very small quantities (10-3 g). Simulation and Layout of a FET sensor on the tip of a microprobe Bucharest, 6th of December “Cooperation in FP7 Biomedical applications of micro and nanotechnologies”

33. II. Deposition and characterization of biological materials Deposition –Plotter (OmniGrid Micro): Biological materials deposition for biosensors manufacturing Immobilized enzyme on gold electrodes Enzyme based biosensor Characterization – Scanner (GeneTAC UC4 Microarray scanner) Enzyme based biosensor (fluorescence) Bucharest, 6th of December “Cooperation in FP7 Biomedical applications of micro and nanotechnologies”

34. III.Biosensors - Devices design and fabrication including masks processing • Microelectrodes to be deposited with biomaterials IMT can provide a wide range of microelectrodes design and manufacturing, for biomaterials applications. The microelectrodes can be deposited and configured on silicon substrates in our technological facilities. • ISFET sensor for ions detection in biological media The surface field effect is a desirable mechanism for a generating potential that provides high chemical selectivity and sensitivity. The ISFET is essentially an extended gate field effect transistor with the surface of the transistor and the reference electrode. Example of microelectrodes layout, for biomaterials applications ISFET sensor placed on a thin tip to be inserted in small liquid media Bucharest, 6th of December “Cooperation in FP7 Biomedical applications of micro and nanotechnologies”

35. Microfluidics devices development: design, simulations, manufacturing Design and simulations for microfluidics devices, including general flow, thermal, fluid mixing, electrokinetic, chemical reactions, etc. Bucharest, 6th of December “Cooperation in FP7 Biomedical applications of micro and nanotechnologies”

36. THANK YOU! • The paper is presenting the result of two projects: Toxichip, IST, STREP Toxisystem, Romanian Security Programme Bucharest, 6th of December “Cooperation in FP7 Biomedical applications of micro and nanotechnologies”