Microfluidics for Electrical Biochip Technology

Transcription

1 Fraunhofer Institute for Silicon Technology (ISIT) Microfluidics for Electrical Biochip Technology 8th Workshop Low Flows in Medical Technology, September 24th 2014 Lars Blohm, Department Biotechnical Microsystems Fraunhofer ISIT Biotechnical Microsystems Continuous Enzyme Sensors Glucose and Lactate Monitoring Multi-Pore Membrane Chips Bioprocess Analytics Electrical Biochip Arrays Protein and DNA Detection Chromatography On-Chip Small Molecule Detection Mass Flow Sensors

2 Fraunhofer ISIT Research and Production at one Location Microelectronics and Microsystems Technology IC Technology and Power Electronics Micro Electrical Mechanical System (MEMS) IC Design Biotechnical Microsystems Packaging Technology for Microsystems and Microelectronics Quality and Reliability of Electronic Assemblies Integrated Power Systems Electronic detection of lactate (ELaN) Continuous monitoring of lactate in sweat during exercise for sports medicine

3 Motivation for continuous monitoring of lactate in sweat Individual check of endurance performance Non-invasive measuring of lactate during exercise Recognition of anaerobic threshold for continuous training control Bio-compatible disposable sensor system for monitoring Low production costs by printed electronics Evaluation of fitness status via Smartphone, PC or internet portal Concept of the project (ELaN) Data evaluation: Smartphone, Server, PC NFC Label / Plaster Data transmission Data preparation Memory Display Lactate sensor chip Measurement electronics Energy supply

4 Principle of the enzyme sensor Diffusion of the analyte through the pore membran Enzymatic reaction (e.g. lactate lactate oxidase) Amperometric detection of hydrogen peroxide - G. Piechotta et al., Novel micromachined silicon sensor for continuous glucose monitoring, Biosensors and Bioelectr. 21 (2005) Layout and dimensions of the current lactate chips Chip size: 6 mm x 7 mm Pore membrane: 0,65 mm x 0,65 mm Pore size: 5 µm or 10 µm Amount of pores: 9, 25, 100

5 Layout and dimensions of the current lactate chips Counter electrode Working electrodes Reference elcectrode Temperature sensor Conductivity electrodes Heating resistor Contact pads Lactate chip and pore membrane with 100 Pores (ᴓ10µm) 650 µm

6 Concentration series with lactate chip in a flow through cell Differential measurements for compensation of interfering substances Flow rates ~ 50µl / min Lactate chip with 25 pores (5 µm diameter) Pt-working electrode: 500 mv in relation to IrOx-reference electrode 40 mm 50 mm current [n na] mm 30 mm mm 20 5 mm 1 mm 0 mm reference signal (cavity without enzyme) time [s] Sweat collection by microfluidic cartridge ASIC (SO16) NFC (SO28) CR2032 FTS X- DV CLP X-D Sweat collector prototype

7 Comparison of different measuring methods in an Ergometer test Increase in steps of 50 W Interval 3 min. blood: photometer saliva: photometer saliva: silicon sensor - C.G.J. Schabmueller et al., «Micromachined sensor for lactate monitoring in saliva, Biosensors and Bioelectronics 21 (2006) Cell-Free Bioproduction Fraunhofer Lighthouse Project (IBMT, IGB, IME, IPA, IPK, ISiT, ISI, IZM)

8 Cell-free protein production with integrated energy supply Scheme of pore membrane for ATP synthesis ATP-Synthase is just active in lipid double layer Micro-pores in silicon membranes and integrated micro electrodes Chips with 4 and 16 pores on a Wafer Membrane with 16 pores / electrodes µm --- Pore (ᴓ 4 µm) Membrane (1.8 µm thick) on the bottom of a cavity Electrode (ᴓ 50 µm)

9 Technical membrane with16 Pores on a silicon chip Impedimetric detection of lipid double layer Electrochemical impedance spectroscopy Pore n-goldchip-pbs03nze lle_01.m pr Z, log spacing vs. freq, log spacing Phase(Z) vs. freq, log spacing # Z /Ohm, log spaci ing Phase(Z)/deg freq/hz, log spacing Formation of lipid bilayer on membrane chips with 4 pores TEOS-Chip-4Poren+DiphPC-Grube-vorne Warnerzelle2+Alubox-Schirm ung 10m V 33Werte-Sek-ohneFilter-AgCl-PBS_01.m pr I vs. time First brushing of lipid in solvent Lipid layer on 1. pore Lipid layer on 2. pore I/pA TEOS-Chip-4Poren+DiphPC+PC-Layer-Grube-vorne Warnerzelle2+Alubox-Schirm ung 10mV 33Werte-Sek-ohneFilter-AgCl-PBS_01.m pr I vs. time Second brushing of lipid in solvent I/pA Lipid layer on 3. pore mv; ~0.1 pa => > 100 GΩ time/h Lipid layer on 4. pore time/s

10 ph-measurement in bioreactors for cell-free bio production ph-sensitive iridium oxide microelectrodes Integration of the sensor chips in microfluidic bioreactors Measuring of proton gradient on micro pore membranes Evaluation of IrOx-microelectrodes for ph-measurement on micro pore membranes 0, µm² 0, Ewe/V 0,2 0,19 0,18 0,17 0,16 0, time/s 0,28 0, µm² 0,26 0,25 Ewe/V 0,24 0,23 0,22 0,21 0, time/s

11 Biotechnische Mikrosysteme Electrical Array-Biochips Technology of the electrical array biochips Principle: Sandwich-ELISA on gold electrode arrays. Detection via single electrode redox-cycling. array positions size: 8 x 10 mm²

12 Position specific dispensing of biological components spotter tips 25 droplets of 400 pl silicon dioxide µm silicon 350 µm position 1 position 2 passivation layer (hydrophobic) gold (hydrophilic) Piezoelectric nanodispensing device

13 Immunoassay on an electrical biochip Biochip spottet with different capture molecules 1. Adding a diluted serum or whole blood sample 2. Adding the enzyme conjugate 3. Adding the substrate and electrical read out current signal redox cycling Pump and flow rates for assay procedure

14 Hepatitis-C (HCV) detection with the electrical biochip system slope [na/min] negative sample positive control Core NS NS4A negative control slope [na/min] assay time: <15 min. positive control positive sample Core NS3 NS4A negative control Comparison of the biochip system with standard ELISA by HCV-Assay 32 positive HCV samples 39 negative HCV samples Standard ELISA Biochip Correctness of the test 88,7% 94,4% Durationof the test 4h 15 min Universal platform for protein detection High sensitivity and selectivity Small volume whole blood analysis Fast, user friendly and reproducible system Portable device for on-site detection L.Blohm et al., Rapid detection of different human anti-hcv immunoglobulins on electrical biochips, Antibody Technology Journal 2014:

15 Biotechnical Microsystems Microsystem for mobile analysis of biochemical molecules MEMS-Chromatography Development of a portable analytical system on the basis of liquid chromatographic separation processes Separation column with very large surface (porous material) Compatible with silicon technology Components for detection of biochemical substances Electrochemical detection methods Integration of 3D-Column structures and detection in a microsystem Process-and module integration on wafer level

16 3D-MEMS process for porous separation column top view cross section column structure Column chip and test cartridge Integrated column column chip flow through cell detection chip fluidic fittings

17 Electrochemical detection of three different antibiotics with a commercial column and gold microelectrodes Au electrodes 600mV vs. IrOx reference Microsystem Technology Micro Electrical Mechanical Systems (MEMS) Mass Flow Sensors

18 Temperature compensated Hot-Wire Mass Flow sensor with recognition of direction Suitability for Gases and Fluids Recognition of Flow Direction Fields of Application Automotive Public Water Supply Medical Technology Principle and test of the mass flow sensor Flow Sensor Concepts 2 Heaters 2 Temperature Sensors Response function of flow rate in volume per minute Wide dynamic range Independence of T (ambient)

19 Acknowledgments Thanks to all involved project partner and colleagues from ISIT. Department Biotechnical Microsystems Eric Nebling, Gundula Piechotta, Jörg Albers, Denise Rühmann, Simone Holz Department Microsystem Technology Lutz-Martin Buchmann, Thomas Lisec, Peter Lange Thank you for your attention!