V13 Electrochemical and Electrical Impedance Spectroscopy (EIS) WS 23 PDF

V13 V 13 Electrochemical and Electrical Impedance Spectroscopy < EIS > Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 1 Contents V13 Contents 13.1 Microelectrodes 13.2 Electrolyte-Electrode Interface  Nern...

V13 V 13 Electrochemical and Electrical Impedance Spectroscopy < EIS > Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 1 Contents V13 Contents 13.1 Microelectrodes 13.2 Electrolyte-Electrode Interface  Nernst equation  Butler-Volmer equation  Mass transport at microelectrodes 13.3 EIS 13.3.1 Electrochemical Impedance Spectroscopy 13.3.2 Electrical Impedance Spectroscopy Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 2 Learning targets V13 Learning targets  Electrode-electrolyte interface  EIS  Equivalent electric circuitry: Randles model  Difference between electrical and electrochemical impedance spectroscopy  EIS sensing applications Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 3 V13 13.1 Microelectrodes Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 4 13.1 Microelectrodes V13 Microelectrodes in Microfluidic Systems Sensors  Temperature  pH value  Electrochemical interactions at surfaces (binding events and binding kinetics)  Recording of nerve signals  Recording dielectric properties between electrodes Actuators  Heating, cooling  Electrokinetics (→ V09 - V11)  Electrowetting (→ V07)  Surface acoustic waves (→ V12)  Stimulation of nerve cells Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 5 13.1 Microelectrodes Substrate (Glass, Si, Flex) V13 6 Deposition of metal layer Metal layer (Au) Lithography, Electroplating, photo resist strip, metal layer etch Electroplated Au Contact pad If necessary: Deposition of electrode top layer, lithography, lift-off Electrode layer Deposition of passivation layer, lithography Passivation layer Cross section Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 Top view 13.1 Microelectrodes V13 Examples of Microelectrodes doi:10.1016/j.snb.2009.12.015 idw-online.de/ de/news60068 http://arcade.stanford.edu/ journals/occasion/node/24 http://inhabitat.com/ Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 www.labo.de/ 7 V13 13.2 Electrolyte-Electrode Interface Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 8 13.2 Electrolyte – Electrode Interface Metal Electrode - Electrolyte Interface  Electrode surface is charged when in contact with an electrolyte (fluid)  Formation of an electrical double layer (EDL) Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 V13 9 13.2 Electrolyte – Electrode Interface Metal Working Electrode V13 10 Potential @ Metal Electrode Reference electrode (in equilibrium = without current)  Is the result of the electromotive force (charge separation) of an electrochemical cell built of two electrodes  Appears at the interface between an electrode and electrolyte due to  Specific adsorption of ions at the interface  Specific adsorption/orientation of polar molecules, including those of the High resistive voltage meter solvent Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 13.2 Electrolyte – Electrode Interface V13 11 Potential Eeq @ Metal Electrode Metal Working Electrode Reference electrode (in equilibrium = without current) High resistive voltage meter  Is given with respect to a reference electrode when the potential of reference electrode E0 to solution is known Nernst equation (1889) COx RT Eeq  E   ln ( ) nF C Red 0 Eeq Equilibrium potential E0 Standard potential of reference electrode: normal hydrogen electrode NHE (E0= 0 V) or Ag/AgCl electrode (E0 = 208 mV @ pH 7 and 298 K) R … Ideal gas constant n … Number of transferred electrons F … Faraday constant C … Concentration of oxidized and reduced forms of a molecule Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 13.2 Electrolyte – Electrode Interface V13 12 Metal Working Electrode Reference electrode Nernst Equation Eeq ;25C COx 0.059 E   ln ( ) n C Red 0 Walter Nernst (1864 - 1941) Equilibrium electrode potential depends on the concentrations of oxidized and reduced forms of a molecule High resistive voltage meter Ox  n  e  Red Oxidation … Electron delivery Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 Reduction... Electron acceptance 13.2 Electrolyte – Electrode Interface Eeq Is called  Equilibrium potential Eeq  Open circuit potential OCP  Rest potential  Is the potential at which there is no current flow through the interface Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 V13 13 13.2 Electrolyte – Electrode Interface V13 14 Metal Electrode - Electrolyte Interface When an additional electric potential is applied to a metallic electrode submerged in an electrolyte  Current is generated across the interface  Electric current is converted to ionic current and vice versa  Electrochemical transducer which exchanges electrons with ions  Without energy supply from outside no charge transfer across interface is possible Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 13.2 Electrolyte – Electrode Interface V13 15 Application of an External Potential at the Working Electrode Results in a Current through the Interface Working Electrode Ox + ne- ↔ Red @ Negative poled electrode: cathodic current  Positive ions are attracted to the electrode  Electrons will be delivered from the electrode (cathode, (-) pole) to the medium  Dissolved ions take over the electrons  Ions are reduced @ Positive poled electrode: anodic current  Negative ions are attracted to the electrode  Electrons will be collected by the electrode (anode, (+) pole)  Dissolved ions release electrons  Ions are oxidized Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 13.2 Electrolyte – Electrode Interface V13 16 Butler-Volmer equation nitum.wordpress.com Current Density at Metal Electrode  nF  (U U equ )   (1RT)nF (U U equ ) RT j  j0 e e    Describes the change of current density with change of applied voltage at the working electrode John A.V. Butler (1899 – 1977) de.wikipedia.org At metal electrode-electrolyte interface U = RI j … Electrode current density [A/m2] j0.. Exchange current density [A/m2] n … Number of electrons involved in the reaction F … Faraday constant α … Symmetry coefficient U …... Applied electrode potential [V] Uequ … Equilibrium potential [V] R … Universal gas constant T … Absolute Temperature [K] Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 Max Volmer (1885-1965) 13.2 Electrolyte – Electrode Interface V13 17 Current Density at Metal Electrode Butler-Volmer equation  nF  (U U equ )   (1RT)nF (U U equ ) RT j  j0 e e    Valid for:  Fast, reversible reactions  No diffusion effects of species to and away from electrode  No polarization effects* at electrodes * Polarization is a collective term for certain mechanical side-effects (of an electrochemical process) by which isolating barriers develop at the interface between electrode and electrolyte Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 13.2 Electrolyte – Electrode Interface Butler-Volmer equation V13 18  nF  (U U equ )   (1RT)nF (U U equ ) RT j  j0 e e    Symmetry factor α α < 0.5 α = 0.5 α > 0.5 Cathodic current No Anodic current preferred Preference preferred Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 13.2 Electrolyte – Electrode Interface V13 19 Anodic Current Butler-Volmer equation α = 1 α = 0,75 α = 0,5 Cathodic current chemie.uni-erlangen.de α = 0,25 α=0   E  Eeq Overvoltage Butler-Volmer equation is valid only for specific conditions. In most real electrochemical systems diffusion processes of ions are involved. Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 13.2 Electrolyte – Electrode Interface V13 20 Rs Solution / Electrolyte resistance Electrode-Electrolyte Interface Randles Model (1947) EDL Rs www.gamry.com Depends on     Ion concentration Type of ion Temperature Area of cell Cdl Electric Double-layer Capacitance Depends on       Electrode potential Temperature Ionic concentrations Type of ions Electrode roughness Adsorption of impurities Rct Charge transfer resistance Depends on  Kind of reaction  Temperature  Concentration of reaction products  Applied potentials J.E.B. Randles: Disc. Faraday Soc. 1 11-19 (1947) EDL Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 V13 21 13.3 EIS 13.3.1 Electrochemical 13.3.2 Electrical Impedance Spectroscopy Impedance Spectroscopy Measures current-voltage dependencies Measures current-voltage dependencies at an electrode between two electrodes Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 V13 22 13.3.1 Electrochemical Impedance Spectroscopy (EIS) Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 13.3.1 Electrochemical Impedance Spectroscopy V13 23 Electrochemical Impedance Spectroscopy (EIS) Objectives ► To measure current-voltage dependencies at an electrode ► To calculate Rs, Rct, Cdl  Apply a sinusoidal potential to an electrochemical cell  Measure the current through the cell  Analyze the frequency-dependent complex electrical resistance, called electrochemical impedance H.S. Magar et al.: Sensors 21 6578 (2021) Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 13.3.1 Electrochemical Impedance Spectroscopy V13 24 Working Principle U Excitation signal U (t )  U 0  sin t t Typically applied Response signal I (t )  I 0  sin(t   ) Amplitude: 1 - 10 mV I Frequency: 10-2 - 107 Hz t   2  f Phase shift depends on frequency Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 13.3.1 Electrochemical Impedance Spectroscopy V13 25 Attention !  Ohm’s law is not valid in chemie.uni-erlangen.de Butler-Volmer equation α = 1 α = 0,75 α = 0,5 electrochemical cells  Double voltage ≠ Double current α = 0,25 α=0  Impedance analysis of linear systems is much easier  When small AC signals (1-10 mV) are applied, electrochemical cells become pseudo-linear Typically, experiments are carried out at Vbias = OCP = Eeq Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 13.3.1 Electrochemical Impedance Spectroscopy DC signals U R I AC signals Z U (t ) U 0  sin t sin t   Z0 I (t ) I 0  sin(t   ) sin(t   ) Ohm‘s law Impedance Euler‘s relationship V13 26 Impedance Z U (t ) U 0  sin t sin t   Z0 I (t ) I 0  sin(t   ) sin(t   ) e j  cos   j sin  U (t )  U 0e jt Z ( )  I (t )  I 0e j (t  ) U (t )  Z 0  e j  Z 0 (cos   j sin  ) I (t ) Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23  Re Z  j Im Z 13.3.1 Electrochemical Impedance Spectroscopy V13 27 Z ( )  Re Z  j Im Z Real part Re Z Imaginary part Im Z (Ohmic) Resistance Reactance  Independent of frequency  Current oscillates out of phase with applied voltage  Depends on frequency Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 13.3.1 Electrochemical Impedance Spectroscopy V13 28 Nyquist Plot for Reaction-Controlled Electrochemical Reaction Equivalent Circuit Randles Model Negative imaginary part at Electrode-Electrolyte Interface Rct/2 Z ( )  Re Z  j Im Z www.dechema.de/kwi_media/Downloads/ec/7+Messmethoden.pdf Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 Ru+Rct Real part 13.3.1 Electrochemical Impedance Spectroscopy V13 29 Bode Plot for Reaction-Controlled Electrochemical Reaction at Electrode-Electrolyte Interface Cut-off frequency Equivalent circuit - 90 Randles model Z   Re Z  2   Im Z  2 Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 13.3.1 Electrochemical Impedance Spectroscopy V13 30 Nyquist Plot for Diffusion-Controlled Reactions at Electrode-Electrolyte Interface* Kinetic control ZW … Warburg impedance Fitting parameter Ru Gabriel Warburg (1846 – 1931) www.dechema.de/kwi_media/Downloads/ec/7+Messmethoden.pdf Diffusion control Ru  Rct * For details: see Appendix Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 https://de.wikipedia.org/wiki/Emil_Warburg 13.3.1 Electrochemical Impedance Spectroscopy V13 31 Bode Plot for Diffusion-Controlled Reactions at Electrode-Electrolyte Interface* H.S. Magar et al.: Sensors 21 6578 (2021) https://www.gamry.com/application-notes/EIS/basics-of-electrochemical-impedance-spectroscopy/ Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 13.3.1 Electrochemical Impedance Spectroscopy V13 32 For Rough and/or Heterogeneous Electrodes Cdl is replaced by Constant Phase Element CPE CPE  1 n A j  CPE reflects  Inhomogeneity of electrode  Deviation from Randles model is fitted by parameter n (0 < n < 1)  n = 1 … Only capacitive behavior  n = 0 … Only resistive behavior E. Katz et al.: Electroanalysis 15 (11) 913-947 (2003) Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 13.3.1 Electrochemical Impedance Spectroscopy V13 33 CPE in Nyquist-Plot Pure CPE (pink line)  Straight line (-Q-)  Angle to x-axis: n ∙ 90° Resistance parallel to CPE (green line)  Half circle  Centre of half circle declined by an angle of (1-n) ∙ 90° Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 13.3.1 Electrochemical Impedance Spectroscopy Sensor Applications Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 V13 34 13.3.1 Electrochemical Impedance Spectroscopy V13 35 EIS for Immunoassay Analysis Top view Cross section + Flow Channel wall Au - Electrodes Substrate Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 13.3.1 Electrochemical Impedance Spectroscopy V13 36 EIS for Immunoassay Analysis Top view Cross section A Y YY + A‘ A Flow Channel wall Y YY Au - Electrodes Substrate Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 A‘ 13.3.1 Electrochemical Impedance Spectroscopy Detail of Sensor Area Microfluidic EIS Sensor J. Lazar et al.: Macromol. Rapid Commun. 36 (16) 1472-1478 (2015) J. Lazar et al.: Anal. Chem. 88 (1) 682-687 (2016) T. Wagner, J. Lazar et al: ACS Applied Materials & Interfaces 8 (49) 27282-27290 (2016) Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 V13 37 V13 38 J. Lazar et al.: Macromol. Rapid Commun. 36 (16) 1472-1478 (2015) J. Lazar et al.: Anal. Chem. 88 (1) 682-687 (2016) T. Wagner, J. Lazar et al: ACS Applied Materials & Interfaces 8 (49) 27282-27290 (2016) Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 13.3.1 Electrochemical Impedance Spectroscopy EIS for Immunoassay Analysis  Detection of a marker for breast cancer (MUC1 antigen)  Capture molecule on sensor surface: antibody 214D4 developed by Stefan Barth’s group (Fraunhofer-IME, Aachen, Germany) J. Lazar et al.: Clinician and Technology Journal 45 (4) 105-109 (2015) Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 V13 39 13.3.1 Electrochemical Impedance Spectroscopy V13 40 (A) Pre-treatment in oxygen plasma (B) Rinse in 11-mercaptopropionic acid (MPA) for 12 hours (C) Application of EDC/NHS coupling crosslinking chemistry (D) Treatment with antibody 214D4 diluted in BSA/PBS (E) Channel blocked with BSA in PBS (F) Treatment with protein MUC1ex in BSA/PBS J. Lazar et al.: Clinician and Technology Journal 45 (4) 105-109 (2015) Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 13.3.1 Electrochemical Impedance Spectroscopy V13 41 Imaginary part of Impedance Z Nyquist Plot Rct Real part of Impedance Z J. Lazar et al.: Clinician and Technology Journal 45 (4) 105-109 (2015) Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 13.3.1 Electrochemical Impedance Spectroscopy V13 42 D J. Lazar et al: Clinician and Technology Journal 45 (4) 105-109 (2015) Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 E F V13 43 13.3.2 Electrical Impedance Spectroscopy (EIS) Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 13.3.2 Electrical Impedance Spectroscopy V13 44 Electrical Impedance Spectroscopy (EIS) Objectives ► To measure current-voltage dependencies between two electrodes ► To calculate the dielectric properties of the material between the electrodes  Apply a sinusoidal potential between two electrodes  Measure the current between the electrodes  Analyze the frequency-dependent complex electrical resistance, called electrical impedance of the material between the electrodes Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 13.3.2 Electrical Impedance Spectroscopy Electrical Impedance Analysis in Flow Cytometers Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 V13 45 13.3.2 Electrical Impedance Spectroscopy EIS in Flow Cytometry Electrical impedance spectroscopy on single cells in microfluidic channel instead of scattered light or fluorescence spectroscopy http://www.abcam.com/ Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 V13 46 13.3.2 Electrical Impedance Spectroscopy V13 47 Impedance in Flow Cytometry*  Characterization of single cells in suspension with EIS  Measuring the change of electrolyte impedance  Advantages  High sampling rates * Invented by W.H. Coulter: Means of counting particles suspended in a fluid, US2656508A, October 1953 H. Morgan et al.: J. Phys. D: Appl. Phys. 40 61-70 (2007) T. Sun et al: NANO: Brief Reports and Reviews: 3 (1) 55-63 (2008) Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 13.3.2 Electrical Impedance Spectroscopy V13 48 Electrode Cdl Rct Ccell Rcell Cdl Rct The larger the cell, the higher the change of Rs electrical impedance Electrode Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 13.3.2 Electrical Impedance Spectroscopy V13 49 https://www.youtube.com/watch?v=7TeGk5tuDR0 https://www.youtube.com/watch?v=S8Gkjg9GFJw Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 V13 50 Electrical Impedance Analysis of Oocytes Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 13.3.2 Electrical Impedance Spectroscopy V13 51 Assisted Reproductive Technology (ART) - Artificial Fertilization of Mammalian Oocytes - Mammalian Oocyte ICSI Intracytoplasmic sperm injection Zona pellucida (ZP) IVF In vitro fertilization Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 13.3.2 Electrical Impedance Spectroscopy V13 52 Window of Best Fertilization When Zona Pellucida is Soft Mouse oocyte Y. Murayama et al. (2006) Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 13.3.2 Electrical Impedance Spectroscopy V13 53 Young‘s Modulus EZP of Zona Pellucida Is Typically Determined with Micropipette Aspiration Technique Zona pellucida Mouse Oocyte L … Aspiration length p … Aspiration pressure ri.. Inner pipette radius EZP. Young‘s modulus of ZP νZP Poisson ratio of ZP = 0.04 M. Khalilian et al. (2010) L.G. Alexopoulos et al. (2003) Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 13.3.2 Electrical Impedance Spectroscopy V13 54 Micropipette Aspiration Technique EIS in Combination with µF     Observer-independent readout method Easy cell handling Without optical control With multiplexing capability     Only single cell characterization Challenging manual handling Time-consuming Microscopic control  observer-dependent  low resolution (curved glass surfaces)  bad contrast (transparent ZP) Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 13.3.2 Electrical Impedance Spectroscopy 1st Setup Microfluidic Channel-Based System Outlet Inlet A. El Hasni et al.: Sensors and Actuators B 248 419-429 (2017) Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 V13 55 13.3.2 Electrical Impedance Spectroscopy V13 56 Hydrodynamic Trapping Equivalent Electrical Circuit Rhyd.2 𝑅ℎ𝑦𝑑.2 > 𝑅ℎ𝑦𝑑.1 → 𝑄1 > 𝑄2 → 𝑄1 𝑄1 +𝑄2 ≈ 0.7 Rhyd.1 Q1 Q2 Rhyd. Fluid resistance Q … Flow rate Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 13.3.2 Electrical Impedance Spectroscopy V13 57 + Coating the electrodes with PPy:PSS Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 13.3.2 Electrical Impedance Spectroscopy V13 58 50 µm Outlet Inlet 35 mm Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 50 µm 13.3.2 Electrical Impedance Spectroscopy V13 59 Mouse Oocyte 70 CONFIG14 60 without zona pellucida 50 ΔZ / [%] with zona pellucida 1 4 2 3 40 30 20 10 0 101 102 103 104 105 106 107 108 Frequency / [Hz] Zona Pellucida is more conductive than surrounding medium Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 13.3.2 Electrical Impedance Spectroscopy 2nd Setup Microfluidic Aspiration-Assisted ElS (MAEIS) Y. Cao et al.: Micromachines 12 (6) 632 (2021) U. Schnakenberg et al.: Patent DE102020214862A1 (2022) Y. Cao et al.: Sensors & Actuators B 380 133316 (2023) Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 V13 60 13.3.2 Electrical Impedance Spectroscopy Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 V13 61 13.3.2 Electrical Impedance Spectroscopy Impedance Analyser V13 62 µfluidic Chip & Read-out circuit Raspberry Pi Programable syringe pump Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 2nd Setup – MAEIS – Video of Hydrodynamic Oocyte Trappig 13.3.2 Electrical Impedance Spectroscopy Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 V13 63 13.3.2 Electrical Impedance Spectroscopy V13 64 CPE … Constant Phase Element CE … Crosstalk capacitance C … Film capacitance CP … Cytoplasm capacitance RP … Cytoplasm resistance RS … Solution resistance R1,2,3 … Zona pellucida resistances RM … Cytomembrane resistance RPVS … Perivitelline space resistance RA … Aperture resistance R … R1+R2+R3+RA Simplified equivalent electrical circuit Only R = f(R2) depends on suction pressure, because Zona Pellucida is squeezed Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 13.3.2 Electrical Impedance Spectroscopy Nyquist plot Parameter: Aspiration pressure The resistive part increases with increasing suction pressure Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 V13 65 Bode diagram 13.3.2 Electrical Impedance Spectroscopy V13 66 Three Mouse Oocyte ( ø 100 µm) Genotypes were tested Wild type (WT) Fetuin-B single knock-out (KO)  MII (ZP normal)  MII (ZP hard) Fetuin-B/ovastacin double knock-out  MII (ZP soft) (DKO) Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 13.3.2 Electrical Impedance Spectroscopy V13 67 Fitting the EIS Spectra with Simplified Equivalent Electrical Circuit Only R = f(R2) depends on suction pressure R … Resistance with trapped mouse oocyte at aperture R0.. Resistance of non-occupied open aperture Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 13.3.2 Electrical Impedance Spectroscopy V13 68 Young‘s Modulus EZP 𝑬𝐙𝐏 = 𝐶 ∗ ℎZP 1 − 𝑣ZP 2 𝑅2,0 1 𝑟i + 𝑅0 𝑟C 1 𝑟C 2 + 2 𝑟C 𝑟C − 𝑟C 2 − 𝑟i 2 𝑅2 −1 𝑅2,0 ∆𝒑 ∆ 𝑹Τ𝑹𝟎 Cell type Young’s modulus (kPa) ZP Hardness KO 23.29 ± 4.15 Hard WT 3.58 ± 0.63 Normal DKO 1.19 ± 0.30 Soft Remark: With micropipette aspiration technique EZP p L / ri Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 (see Slide 66) L … Aspiration length p … Aspiration pressure ri.. Inner pipette radius EZP. Young‘s modulus of ZP R … Resistance at sealed aperture R0.. Resistance at empty aperture Conclusion V13 69 Conclusion  Microelectrodes play an important role in microfluidics for sensor applications  EIS is an important measurement technique  Impedance spectra can be fitted with equivalent electrical circuits. Each circuit element must correspond to a physical parameter Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 V13 70 End of the course  Thank you very much for your interest ! Please, recommend the course* to your colleagues and keep in touch ! All my best for your future, Uwe Schnakenberg and his team * Final course will take place in SS 2026 Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23 V13 71 One Minute Paper 1. What was the most important topic you understood? 2. What was the topic you didn‘t catch? Lecture „Microfluidic Systems - Bio-MEMS“ – EIS Prof. Dr.-Ing. Uwe Schnakenberg | Institute of Materials in Electrical Engineering 1 | WS 23