Activity Imaging of Immobilized Enzymes

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Prof. Dr. Gunther Wittstock
Group Leader

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+49 (0) 441 798 3970

+49 (0) 441 798 3979

Mailing Address

University of Oldenburg
School of Mathematics
   and Science
Institute of Chemistry
Wittstock Group
D-26111 Oldenburg
Germany

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University of Oldenburg
Campus Wechloy
Carl-von-Ossietzky Street 9-11
Building W3, 1st floor
D-26129 Oldenburg
Germany

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Activity Imaging of Immobilized Enzymes

Binding of Proteins to Surfaces

For some fundamental studies but also for applications in sensors, biochemical functional carriers such as enzymes or antibodies need to be attached (immobilized) to surfaces while maintaining their special properties. In many cases, there is also an interest in converting the activity or binding events on these proteins into an electrical or optical signal. For this purpose, signal converters (transducers) are used

We focus on the generation of electrical signals for the course of catalytic (enzymatic) reactions. In this case the transducer is an electrode. This can happen, for example, when products of the enzymatic reaction are oxidized or reduced at an electrode. To ensure that as many as possible of the particles generated by the enzyme also reach the electrode, the biomolecules must be immobilized at a dense distance from the electrode. The interfaces for immobilization and electrochemical conversion can be identical (Figure 1), intertwined (Figure 2) or spatially separated (Figure 3).

Scheme enzyme electrode
Figure 1. Schematic cross-section of a surface in which the carrier and transducer surfaces are identical.
Scheme microkompatimentalization
Figure 2. Schematic cross-section of a surface in which the carrier and transducer surfaces are interleaved (microcompartmentalized).
Schema arrangement in channel
Figure 3a
Scheme scanning electrochemical microscopya elektrochemische Rastermikroskopie
Figure 3b

Figure 3. Schematic cross-section of a surface in which the carrier and transducer surfaces are spatially separated; a) in a microfluidic channel array; b) in a scanning electrochemical microscope.

Functional Coupling of Enzymatic Reactions with Electrodes

Electron transfer at the transducer surface can occur via oxidation of a reaction product of the enzymatic reaction (Figure 4) or via electrochemical regeneration of a redox mediator (Redoxmediator, Figure 5) or in special cases via direct electron transfer between electrode and protein (Figure 6).

Scheme cross section enzyme electrode
Figure 4. Schematic cross-section of a Pber surface with detection of a product of the enzymatic reaction. The electode is a carrier and transducer.
Scheme cross section mediator electrode
Figure 5. Schematic cross-section of an electrode with electron transfer via a redox mediator.
Scheme cross section of electrode with direkt electron transfer
Figure 6. Schematic cross-section of an electrode with direct electron transfer.

Molecular Design of Interfaces

Depending on which transduction principles are to be used, different supramolecular interface architectures can be considered (Figures 7-12).

Scheme coupling to a self-assembled monolayer
Figure 7. Schematic cross-section of an electrode with covalent binding of an enzyme to a self-assembled monolayer.
Scheme enbedding in polymer network
Figure 8. Schematic cross-section of an electrode with embedding of a protein in a gel or a polymer network.
Scheme embedding in conducting polymer network
Figure 9. Schematic cross-section of an electrode with integration of proteins in polymer network, which can also conduct the electrons (intrinsically conductive polymers or polymers with redox-active side chains).
Scheme adsorption of proteins
Figure 10. Schematic cross-section of an electrode with adsorption of a protein on surfaces.
Scheme cross-linking
Figure 11. Schematic cross-section of an electrode with cross-linking of proteins via bifunctional reagents.
Scheme embedding in lipid bilayer
Figure 12. Schematic cross-section of an electrode with integration of a protein into a supported artificial biomembrane.

Reactivity Imaging of Immobilized Enzymes with the Scanning Electrochemical Microscope

Imaging of the activity of locally immobilized enzymes on surfaces can be performed with scanning electrochemical microscopy (SECM) in different ways.

Scheme of the generation-collection mode
Figure 13. Principle of the generation-collection mode.

Imaging in Substrate-Generation/Tip Collection Mode

In this case, a redox active product formed by the enzyme is oxidized or reduced at the microelectrode of the SECM (Figure 13). The advantage of the method is a very high sensitivity and the possibility to measure not only oxidoreductases but also the activity of other important enzymes such as alkaline phosphatase or galactosidase. The disadvantage is the low resolution of the method and specific requirements to make these measurements quantitative.

Example. Mapping the activity of galactosidase (Figure 14) takes advantage of the fact that p-aminopehnol can be oxidized at the detection potential. However, the parent compound cannot. Figure 15 shows a raster image of three modified surface areas with a different amount of the enzyme galactosidase. A curve fit to a cut line in  Figure 15 can be used for quantitative evaluation.

Scheme for the detection of the activity of galactosidase
Figure 14. Schematic representation of activity mapping of galactosidase (not to scale).
Example data for galactosidase activity imaging
Figure 15. Examples of activity imaging on surfaces locally modified with galactosidase.
Profilline
Figure 16. Example for activity imaging of the enzyme galactosidase. Curve fitting to a section line.

Imaging in Feedback Mode

In this case, the function of a natural cosubstrate of the enzyme (such as O2 for gluocose oxidase) is taken over by an artificial redox mediator (such as ferrocene derivatives for glucose oxidase). At the microelectrode of the scanning electrochemical microscope, the mediator (R in Figure 17) is continuously converted to its oxidized form. Current amplification occurs when the mediator is regenerated by the enzymatic reaction to be detected at the surface by the mediator accepting electrons from the enzyme.

The advantage of the method is the highly predictable quantitative relationship between the current at the microelectrode and that of the activity of the immbilized enzyme. The disadvantage is the lower sensitivity compared to the generator-collector mode.

Schema des Feedback-Modus
Figure 17. Schematic representation of the reactions involved in imaging the activity of glucose oxidase (GOx).

Example: In the schematic diagram of the coupled reactions in Figure 17, it can be seen that the oxidation of glucose at the enzyme glucose oxidase (Gox) only occurs when the ferrocene derivative oxidized at the microelectrode is available. In the enzymatic reaction, the original form of the mediator is regenerated and is again available for oxidation at the microelectrode, thus increasing the current. In the example measurement in Figure 18 and in the profile line, it can be seen that the edges are much sharper (higher lateral resolution) than in Figure 15 and 16 under roughly comparable conditions.

Example data for local measurement of glucose oxidase activity in the feedback mode
Figure 18. Example of activity imaging of glucose oxidase; left) two-dimensional imaging; right) sectional view of two-dimensional imaging.

Imaging in redox competition mode

In cases where the reaction product of the enzymatic reaction is water, i.e., the solvent, the generator-collector mode cannot be used. In these cases, the redox competition mode can be applied.

Scheme of reaction at the enzyme horse radish peroxidase
Figure 19: Schematic representation of the reactions on the enzyme horseradish peroxidase (HRP).

Example: The enzyme horseradish peroxidase (HRP) converts hydrogen peroxide H2O2 to water. The electrons can be supplied by an artificial electron donor (here ferrocene methanol, Fc, Figure 19). The activity of the enzyme can be measured either in generation-collection mode by detecting the oxidized form of ferrocene (ferrocenium, Fc+, Figure 20). This results in a reduction current at the microelectrode (plotted donwards). Another variant is the use of the redox-competition mode (Figure 21). Here the enzyme at the substrate and the microelectrode compete for the co-substrate ferrocene (Fc). The lower the local current, the more Fc is consumed by the enzyme. Figures 20 and 21 show approach curves. These are plots of the current at the microelectrode versus the distance between microelectrode and sample. The steeper the curve, the better the resulting resolution in two-dimensional images. Approach curves can also be used very well for mechanistic studies.

Example data for generation-collection mode
Figure 20. Approach curve in generator-collector mode. Fc+ is detected at the microelectrode by reduction (current plotted downards).
Example data for redox competition mode
Figure 21. Approach curve in redox competition mode (red). The consumption of H2O2 by the enzyme is detected. H2O2 is oxidized at the microelectrode (plotted upwards). The lower the current, the less H2O2 reaches the microelectrode. For comparison, the curve recorded over a surface without immobilized horseradish peroxidase is shown in blue.

Own Contributions to the Area

Review Paper and Book Chapters

  • B. R. Horrocks, G. Wittstock
    Biotechnological Applications in Scanning Electrochemical Microscopy
    in Scanning Electrochemical Microscopy, 3rd ed. (A. J. Bard, M. V. Mirkin, Eds.), CRC Press, Boca Raton 2022, Chap. 10, pp. 243-296. Abstract & Link
  • G. Wittstock, M. Burchardt, S. E. Pust, Y. Shen, C. Zhao
    Scanning Electrochemical Microscopy for Direct Imaging of Reaction Rates
    Angew. Chem. Int. Ed. 2007, 46, 1584-1617. Abstract & Link
  • T. Wilhelm, G. Wittstock
    Analysis of Interaction in Patterned Multienzyme Layers by Using Scanning Electrochemical Microscopy
    Angew. Chem. Int. Ed. 2003, 42, 2247-2250. Abstract & Link
  • T. Wilhelm, G. Wittstock
    Analyse von Wechselwirkungen in gemusterten Multi-Enzymschichten mit elektrochemischer Rastermikroskopie (SECM)
    Angew. Chem. 2003, 115, 2350-2353. Abstract & Link
  • G. Wittstock
    Modification and characterization of artificially patterned enzymatically active surfaces by scanning electrochemical microscopy
    Fresenius J. Anal. Chem. 2001, 370, 303-315. Abstract & Link

Study of Systems with Two Interacting Emzymes

  • M. Burchardt, G. Wittstock,
    Micropatterned Multienzyme Devices with Adjustable Amounts of Immobilized Enzymes,
    Langmuir 2013, 29 (48), 15090-15099, Abstract & Link
  • C. Zhao, G. Wittstock
    An SECM detection scheme with improved sensitivity and lateral resolution: Detection iof galactosidase activity with signal amplification by glucose dehydrogenase
    Angew. Chem. Int. Ed. 2004, 43, 4170-4172. Abstract & Link
  • C. Zhao, G. Wittstock
    Ein SECM-Detektionsmodus mit verbesserter Empfindlichkeit und lateraler Auflösung: Detektion von Galactosidaseactivität mit Signalverstärkung durch Glucosedehydrogenase
    Angew. Chem. 2004, 116, 4264-4267. Abstract & Link
  • G. Wittstock, W. Schuhmann
    Formation and Imaging of Microscopic Enzymatically Active Spots on an Alkanethiolate-Covered Gold Electrode by Scanning Electrochemical Microscopy
    Anal. Chem. 1997, 69, 5059-5066. Abstract & Link

Activity Imaging of Immobilized, Biotechnologically Relevant Enzymes

  • Nogala, A. Celebanska, G.Wittstock, M.Opallo
    Bioelectrocatalytic Carbon Ceramic Gas Electrode for Reduction of Dioxygen and Its Application in a Zinc–Dioxygen Cell
    Fuel Cells 2010, 6, 1157-1163. Abstract & Link
  • Nogala, K. Szot, M. Burchardt, F. Roelfs, J. Rogalski, M. Opallo, G. Wittstock
    Feedback mode SECM study of laccase and bilirubin oxidase immobilised in a sol–gel processed silicate film
    Analyst 2010, 135, 2051–2058. Abstract & Link
  • Nogala, A. Celebanska, K. Szot, G. Wittstock, M. Opallo
    Bioelectrocatalytic mediatorless dioxygen reduction at carbon ceramic electrodes modified with bilirubin oxidase
    Electrochim. Acta 2010, 55, 5719–5724. Abstract & Link
  • W. Nogala, K. Szot, M. Burchardt, M. Joensson-Niedziolka, J. Rogalski, G. Wittstock, M. Opallo
    Scanning electrochemical microscopy activity mapping of electrodes modified with laccase encapsulated in sol–gel processed matrix
    Bioelectrochemistry 2010, 79, 101-107. Abstract & Link
  • C. Nunes Kirchner, M. Träuble, G. Wittstock
    Study of Diffusion and Reaction in Microbead Agglomerates
    Anal. Chem. 2010, 82, 2626-2635. Abstract & Link
  • M. Burchardt, M. Träuble, G. Wittstock
    Digital Simulation of Scanning Electrochemical Microscopy Approach Curves to Enzyme Films with Michaelis-Menten Kinetics
    Anal. Chem. 2009, 81, 4857–4863. Abstract & Link
  • P. C. Chen, R. L. C. Chen, T. J. Cheng, G. Wittstock
    Localized Deposition of Chitosan as Matrix for Enzyme Immobilization
    Electroanalysis 2009, 21, 804-810. Abstract & Link
  • K. Szot, W. Nogala, J. Niedziolka-Jönssona, M. Jönsson-Niedziolka, F. Marken, J. Rogalski, C. Nunes Kirchner, G. Wittstock, M. Opallo
    Hydrophilic carbon nanoparticle-laccase thin film electrode for mediatorless dioxygen reduction SECM activity mapping and application in zinc-dioxygen battery
    Electrochim. Acta 2009, 54, 4620–4625. Abstract & Link
  • M. J. W. Ludden, J. K. Sinha, G. Wittstock, D. N. Reinhoudt, J. Huskens
    Control over binding stoichiometry and specificity in the supramolecular immobilization of cytochrome c on a molecular printboard
    Org. Biomol. Chem. 2008, 6, 1553-1557. Abstract & Link
  • W. Nogala, M. Burchardt, J. Rogalski, M. Opallo, G. Wittstock
    Scanning electrochemical microscopy study of laccase within a sol-gel processed silicate film
    Bioelectrochemistry 2008, 72, 174-182. Abstract & Link
  • M. Burchardt, G. Wittstock
    Kinetic studies of glucose oxidase in polyelectrolyte multilayer films by means of scanning electrochemical microscopy (SECM)
    Bioelectrochemistry 2008, 72, 66-76. Abstract & Link
  • C. Nunes Kirchner, S. Szunerits, G. Wittstock
    Scanning electrochemical microscopy (SECM) based detection of oligonucleotide hybridization and simultaneous determination of the surface concentration of immobilized oligonucleotides on gold
    Electroanalysis 2007, 19, 1258-1267. Abstract & Link
  • C. Nunes Kirchner, C. Zhao, G. Wittstock
    Analysis of the activity of beta-galactosidase from Escherichia coli by scanning electrochemical microscopy (SECM)
    Comprehensive Analytical Chemistry 2007, 49, e371-e379. Abstract & Link
  • C. Nunes Kirchner, G. Wittstock
    Kinetic analysis of titanium nitride thin films by scanning electrochemical microscopy
    Comprehensive Analytical Chemistry 2007, 49, e363-e370. Abstract & Link
  • G. Wittstock, M. Burchardt, C. Nunes Kirchner
    Scanning electrochemical microscopy in biosensor research
    Comprehensive Analytical Chemistry 2007, 49, 907-939. Abstract & Link
  • M. Zhang, G. Wittstock, Y. Shao, H. H. Girault
    Scanning Electrochemical Microscopy as a Readout Tool for Protein Electrophoresis
    Anal. Chem. 2007, 79, 4833-4839. Abstract & Link
  • O. Sklyar, M. Träuble, C. Zhao, G. Wittstock
    Modeling Steady-State Experiments with a Scanning Electrochemical Microscope Involving Several Independent Diffusing Species Using the Boundary Element Method
    J. Phys. Chem. B 2006, 110, 15869-15877. Abstract & Link
  • C. Zhao, G. Wittstock
    Scanning electrochemical microscopy for detection of Biosensor and biochip surfaces with immobilized pyrroloquinone (PQQ)-dependent glucose dehydrogenase as enzyme label
    Biosens. Bioelectron. 2005, 20, 1277-1284. Abstract & Link
  • C. Zhao, J. Sinha, C. A. Wijayawardhana, G. Wittstock
    Monitoring beta-Galactosidase Activity by Means of Scanning Electrochemical Microscopy
    J. Electroanal. Chem. 2004, 561, 83-91. Abstract & Link
  • C. Zhao, G. Wittstock
    Scanning Electrochemical Microscopy of Quinoprotein Glucose Dehydrogenase
    Anal. Chem. 2004, 76, 3145-3154. Abstract & Link
  • T. Wilhelm, G. Wittstock
    Generation of Periodic Enzyme Patterns by Soft Lithography and Activity Imaging by Scanning Electrochemical Microscopy.
    Langmuir 2002, 18, 9486-9493. Abstract & Link
  • G. Wittstock, T. Wilhelm
    Characterization and Manipulation of Microscopic Biochemically Active Regions by Scanning Electrochemical Microscopy (SECM)
    Anal. Sci. 2002, 18, 1199-1204. Abstract & Link
  • T. Wilhelm, G. Wittstock
    Patterns of functional proteins formed by local electrochemical desorption of self-assembled monolayers
    Electrochim. Acta 2001, 47, 275-281. Abstract & Link
  • G. Wittstock, T. Wilhelm, S. Bahrs, P. Steinrücke
    SECM feedback imaging of enzymatic activity on agglomerated microbeads
    Electroanalysis 2001, 13, 669-675. Abstract & Link
  • C. A. Wijayawardhana, N. J. Ronkainen-Matsuno, S. M. Farrel, G. Wittstock, H. B. Halsall, W. R. Heineman
    Microspot Enzyme Assays with Scanning Electrochemical Microscopy
    Anal. Sci. 2001, 17 Supplement, 535-538. Abstract & Link
  • J. Zaumseil, G. Wittstock, S. Bahrs, P. Steinrücke
    Imaging the activity of nitrate reductase by means of a scanning electrochemical microscope
    Fresenius J. Anal. Chem. 2000, 367, 346-351. Abstract & Link
  • C. A. Wijayawardhana, G. Wittstock, H. B. Halsall, W. R. Heineman
    Electrochemical Immunoassay with Microscopic Immunomagnetic Bead Domains and Scanning Electrochemical Microscopy
    Electroanalysis 2000, 12, 640-644. Abstract & Link
  • C.A. Wijayawardhana, G. Wittstock, H.B. Halsall, W.R. Heineman
    Spatially Addressed Deposition and Imaging of Biochemically Active Bead Microstructures by Scanning Electrochemical Microscopy
    Anal. Chem. 2000, 72, 333-338. Abstract & Link
  • T. Wilhelm, G. Wittstock, R. Szargan
    Scanning electrochemical microscopy of enzymes immobilized on structured glass-gold substrates
    Fresenius J. Anal. Chem. 1999, 365, 163- 167. Abstract & Link
  • C. Kranz, G. Wittstock, H. Wohlschläger, W. Schuhmann
    Imaging of Microstructured Biochemically Active Surfaces with Scanning Electrochemical Microscopy
    Electrochim. Acta 1997, 42, 3105-3111. Abstract & Link
  • G. Wittstock, R. Hesse, W. Schuhmann
    Patterned Self-Assembled Alkanethiolate Monolayers on Gold. Patterning and Imaging by Means of Scanning Electrochemical Microscopy
    Electroanalysis 1997, 9, 746-750. Abstract & Link
  • G. Wittstock, K. Yu, H. B. Halsall, T. H. Ridgway, W. R. Heineman
    Imaging of Immobilized Antibody Layers with Scanning Electrochemical Microscopy
    Anal. Chem. 1995, 67, 3578-3582. Abstract & Link
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