Nanobioelectrochemistry (Applied) electrochemistry at nanoscale Dr. Karel Lacina lacinak@chemi.muni.cz Nanobioelectrochemistry • Electrochemistry at nanoscale • Broad field of application • Nanobiosensors • Nanopores and Nanoscale field effect devices DNA sequencing • Biological inspiration and Study of electrochemical processes at nanoscale • Biofuel cells • SECM Literature • Nanobioelectrochemistry, From Implantable Biosensors to Green Power Generation – ed. F.N. Crespilho (2013, Springer) • Internet… Electrochemistry with Nanoparticles (NPs) • Au (most used), Ag, Pt, Pd, Cu, Co… • Nanocrystals – Prussian blue, • Synthesis, enhancement of function, stabilisation • Carbon nanomaterials • Conductive! Liu, Liang, Theranostics 2 (2013) 235 Electrochemistry at nanoscale • Problem – Noise distorts the accuracy of the measurements – fA or pA and lower values of current are measured (x10-15 or x10-12) • Instrument demanding • Macroscale smoothing of electrochemical signal can not be simply used! • Possible solutions are sought - numerical modeling, filters, etc. Electrodes • (Macro)electrodes with nanostructures – On polished flat surface in mm range – Au, Pt, C … • Microelectrodes – lithography for production of defined structures Varshney, Li, Biosensor Bioelectronic 24 (2009) 2951 Yu, Wilson, Faraday Discuss 116 (2000) 305 For illustration Microelectrode Array - MEA Chip • Cell culture experiments Bucher, Schubert, Kern, Nisch, Microelectron Eng 57-58 (2001) 705 Yakushenko, Mayer, Buitenhuis, Offenhäusser, Wolfrum, Lab Chip 14 (2014) 602 Nanostructured electrodes • Enhancement of (electro)active x sensing surface – NOT ONLY! • Incubation (min) – (a) 0 – (b) 5 – (c) 15 – (d) 120 • Valid for all nanoparticles attached to the surface! Surface modification • Langmuir-Blodgett films – Well defined monolayers – Co-deposition of polyanions and polycations • Layer-by-layer • Nanostructured modifications of electrodes – 0D quantum dots, nanoparticles – 1D nanowires and carbon nanotubes – 2D metallic platelets and graphene sheets • Depends on particular case of study Electrochemistry at nanoscale • Electrode modifications • Current (today’s) functional schemes improved by employment of “nano” • Nano utilisation results in novel functional schemes • Electrochemical biosensors using nanoparticles – Enhancement of active surface Biosensors at nanoscale • Usual biosensing schemes using nanotechnology • Improvement in – Sensitivity – New functional schemes – Selectivity ? – Instrumental simplicity ? – Low cost ? http://www.micruxfluidic.com /technology.html Biosensing schemes using Electrochemistry • Enzymatic biosensors • Immunoassays http://www.ysilifesciences.com www.lifetechnologies.com Enzymatic biosensors • Enhancement of electroactive surface area – higher signal (also non-specific) • Direct electron transfer GOD O2 H2 O2 eβ−β−β−β−D-glucose D-glucono-1,5-lactone Enzymatic biosensors Wiring of enzymes • Connection of enzyme redox (active) site and electrode • Interference free • Improved electrode kinetics in comparison with • Employed also in biofuel cell technology www.rsc.org GOD O2 H2 O2 eβ−β−β−β−D-glucose D-glucono-1,5-lactone Electrochemical immunoassays using nanomaterials • Stemming from ELISA (Enzyme-linked immunosorbent assay) • Employment of – Nanostructured electrodes – Nanoparticle labels – Magnetic nanoparticles • Protein cancer markers www.lifetechnologies.com Possible amplification of signal for electrochemical immunoassay Nanostructured electrodes in immunoassay • Bigger surface: • Enabling the attachment of a large number of capture antibodies on the sensor surface • Better access of protein analytes to these antibodies – carbon nanotubes (single-, multi- walled CN) – gold nanoparticles – electrodepositing gold Nano-Immunoassay • Nanoparticles firstly used in immunoassay in 2000 by Dequaire et. al. • Amplification by nanoparticles – Dissolution to electroactive salts – Multi-enzyme NP – Quantum dots – etc. • LOD in range of pico (10-12) to femto (10-15) molar concentration Nanopores - principle • Monitoring of ionic current • Blockage = generation of signal • Nanopore = diameter in range of nm Nanopores Sensing • Nanopore modified with e.g. – Antigen – DNA polymerase Fabrication of nanopores • Crucial parameter Gyurcsanyi, TRAC-Trend Anal Chem 27 (2008) 627 Nanopores • Affinity reactions – equilibrium (Ab-Ag high binding constant) • “Pulse”-like signal • Binding and release etc…. • Estimation of affinity Nanopores DNA sequencing • Several sensing schemes: • Membrane and pore – Solid (ion beam milling) – Protein pore in bilipid layer • Detection – Charge of single base (A,G,C,T) – Number of H+ released upon DNA polymerisation Real-time DNA sequencing Example Derrington, Butler, Collins, Manrao, Pavlenok, Niederweis, Gundlach, PNAS 107 (2015) 16060 Field-effect transistor (FET) • Semiconductor micro/nanotechnology • Part of Field Effect Devices category – ISFET (ion-sensitive field-effect transistors) – EGFET (extended gate field effect transistors) – capacitive EIS sensor (electrolyte-insulator- semiconductor) – LAPS (light-addressable potentiometric sensors) • Transistor – Electronic element – Amplification of signal Field-effect transistor • Principle – electric field creates regions of excess charge in a semiconductor substrate FET sensor principle of operation • Signal generation (electric field modulation) – pH – changes of ion- concentration – changes of ion-species through enzymatic reaction – adsorption of macromolecules – affinity binding of molecules (Ab-Ag, DNA hybridization) – Changes due to the living systems (e.g. metabolic processes) http://lsi.epfl.ch Types of Field Effect Devices (FED) • ISFET – Bergveld, 1970 • EIS – Simplest senor based on FEDs • LAPS – Modulation of/by photocurrent FED based sensors FET based sensors using nanotechnology • FET coupled with biorecognition element (e.g. enzyme, antibody) • SWCNT – Uniform and enhanced adsorption of enzyme – Enhanced porosity facilitates the ion permeation http://lsi.epfl.ch Field-effect transistor (FET) Matsumoto, Miyahara, Nanoscale 5 (2013) 10702 Combination of FET with Nanopores DNA sequencing Matsumoto, Miyahara, Nanoscale 5 (2013) 10702 Biological (nano)sensors • Biological demand • Not only human development – e.g. plants, bacteria • Feedback loop regulation • Regulation of metabolism Sousa, Tuckerman, Gonzalez, Gilles-Gonzalez, Protein Sci 16 (2007) 1708–1719 Allosteric regulation • Hemoglobin – pH dependent O2 binding and its release Combined techniques • Electrochemical methods combined with – AFM (Atomic Force Microscopy) – SPR (Surface Plasmon Resonance) • Complementary and additional information Electrochemistry and AFM • Possible modes: – Discrete AFM and electrochemical characterisation – EAFM – Electrochemical AFM, AFM tip is conductive and used as working electrode (similar to SECM - overlap) Macpherson, Unwin, Anal Chem 72 (2000) 276 Electrochemistry and SPR • Simultaneous information about the (electrochemical) processes at the surface • Nanometer range layer • antigen–antibody, nucleic acids, cells, enzymes, microorganisms, etc. • Characterisation of – self-assembly and electro-polymerisation – ultra-thin film and conducting polymers – redox transformations – electrochemically catalysed processes Biofuel cell Biofuel cells • Enzymatic fuel cell • Interface between redox enzymes and electrical circuitry • efficient immobilisation and wiring of enzymes – carbon nanotubes, inorganic and polymer nanoparticles Fuel cell • transforming chemical energy into electrical energy BioFuel cell • “Fuel cell” using the bio-catalytic reaction of enzymes or living organisms http://www.toto.co.jp Biofuel cell • Redox enzymes • Oxidation at anode – sugars, alcohol • Reduction at cathode – O2, H2O2 Biofuel cell • Firstly described in 1964 • Advantages – “ecofrendly” – No metal catalyst (platinum, palladium, iridium,…) – Biodegradable • Motivation – Power supply for pacemakers ,micro machines, micro-pumps, sensors – Utilisation of glucose and O2 – present in our body fluids Biofuel cell Principle of function • Majority – oxidation of glucose by glucose oxidase – Reduction of oxygen by laccase (bilirubin and ascorbate oxidase) • Mediated or direct electron transfer – Between enzyme redox centre (active site) and electrode Biofuel cell Principle of function www.rsc.org • Mediated ET – Electron mediator (ferrocene, Os(bipy)) • Direct ET – Wiring of enzymes Kavanagh, Leech, Phys Chem Chem Phys 15 (2013) 4859 Biofuel cell performance • Maximum power density • Maximum current density • Open circuit potential • Operational stability (time characteristic of power supply by biofuel cell) • Storage stability • Measured by polarization curves Biofuel cells using nanotechnology • Nanoparticles for enhanced immobilisation of biomolecules (increased surface coverage) and wiring of enzymes • Carbon nanotubes – nanowire morphology – biocompatibility – excellent conductivity – well described functionalization • Graphene (carbon nanomaterials) • Clay nanomaterials • Metal nanoparticles (gold) • Polymer materials (functionalization, enzyme entrapment) Clay Scanning electrochemical microscopy (SECM) • Belongs to Scanning Probe Microscopies (STM, AFM, SNOM etc.) • Probe = microelectrode 1 dimension in the range of micrometer (x10-6 m) Bipotentiostat Electrochemistry Piezo- or stepPositioning system SECM AFM SNOM Microelectrodes Electrode kinetics Brownson, Kampouris, Banks, Chem Soc Rev 41 (2012) 6944 Microelectrodes • Melting of Pt wire (25 µm) into glass capillary • Electronic connection • Grinding and polishing of the tip depending of the application – feedback mode – G/C mode (generation/collection) – in-vivo measurement Wei, Bailey, Andrew, Ryhanen, Lab Chip 9 (2009) 2123 Microelectrodes - litography • NOT USABLE for SECM technology SECM - fundamentals • Positive feedback • Negative feedback • Approaching curve Normalised distance Normalisedcurrent SECM - fundamentals Edwards, Martin, Whitworth, Macpherson, Unwin, Physiol Meas 27 (2006) 63 SECM Measuring applications • Surface topography and chemical properties • Activity of protein (enzymes, mediators) • Permeability of membranes and channels • Activity of individual cells and cell cultures • In-vivo measurements • … • Surfaces can be modified!!! W. Schumann et. al. F. Laforge 2 4 6 8 10 2 4 6 8 10 0,0009 0,0010 0,0011 0,0012 0,0013 0,0014 0,0015 0,0016 0,0017 2 46 8 10 2 4 6 8 10 -0,0045 -0,0040 -0,0035 -0,0030 -0,0025 X Axis Y A xis (a) Current/µA (b) Activity of immobilised enzymes Glucose oxidase • Glucose oxidase (GOD) immobilised • 50 mM glucose • Production of H2O2 or consumption of O2 Lacina, Skladal, Nagy, Chem Listy 106 (2012) 253