Electrochemical Biosensors Based on Membrane-Bound Enzymes in Biomimetic Configurations.

In nature, many enzymes are attached or inserted into the cell membrane, having hydrophobic subunits or lipid chains for this purpose. Their reconstitution on electrodes maintaining their natural structural characteristics allows for optimizing their electrocatalytic properties and stability. Different biomimetic strategies have been developed for modifying electrodes surfaces to accommodate membrane-bound enzymes, including the formation of self-assembled monolayers of hydrophobic compounds, lipid bilayers, or liposomes deposition. An overview of the different strategies used for the formation of biomimetic membranes, the reconstitution of membrane enzymes on electrodes, and their applications as biosensors is presented.

PM-IRRAS Study on the Effect of Phytantriol-Based Cubosomes on DMPC Bilayers as Model Lipid Membranes.

The effect of phytantriol (PT)-based liquid-crystalline nanoparticles, cubosomes, on the lipid bilayer membranes has been investigated using the combined Langmuir-Blodgett/Langmuir-Schaefer (LB-LS) technique to form an h-1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) monolayer at the air-water interface and transfer the lipid bilayer onto the Au(111) substrate. Changes of the compression isotherms confirmed incorporation of cubosomes dispersed in the subphase into the h-DMPC monolayer at the air-water interface. The photon polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) measurements of the gold electrode covered by the transferred DMPC bilayer showed for the first time how the incorporation of cubosome material affects the orientation and conformation of lipid molecules in the membrane. Exposure to cubosomes affected the packing of d54-DMPC bilayers and introduced disorder of chains by increasing the contribution of gauche conformation. The decrease of the tilt angle of the acyl chains of adsorbed DMPC in the whole range of potentials applied to the gold electrode confirmed that incorporation of cubosome material results in a more tightly packed bilayer. The presence of phytantriol molecules within the d63-DMPC matrix was confirmed by PM-IRRAS studies of the PT-related bands. The LB and PM-IRRAS studies demonstrated in a convincing way that PT-based cubosomes change the organization of model lipid layers leading to structural changes of the membranes which have to be taken into consideration when PT-cubosomes are employed as drug carriers.

Electric-Field-Driven Molecular Recognition Reactions of Guanine with 1,2-Dipalmitoyl-sn-glycero-3-cytidine Monolayers Deposited on Gold Electrodes.

Monolayers of 1,2-dipalmitoyl-sn-glycero-3-cytidine were incubated with guanine in a 0.1 M NaF electrolyte at the surface of a Langmuir trough and transferred to gold (111) electrodes using the Langmuir-Schaefer technique. Chronocoulometry and photon polarization modulation infrared reflection absorption spectroscopy were employed to investigate the influence of the static electric field on the orientation and conformation of the cytidine nucleolipid molecules on the metal surface in the presence of guanine and to monitor the molecular recognition of guanine with the cytosine moiety. When the monolayer is exposed to guanine solutions, the cytosine moiety binds to the guanine residue in either a Watson-Crick complex at positively charged electrode surfaces or a noncomplexed state at negative surface charges. The positive electrostatic field causes the cytosine moiety and the cytosine-guanine complex to adopt a nearly parallel orientation with respect to the plane of the monolayer with a measured tilt angle of ∼10°. The parallel orientation is stabilized by the interactions between the permanent dipole of the cytosine moiety or the Watson-Crick complex and the static electric field. At negative charge densities, the tilt of the cytosine moiety increases by ∼15-20°, destabilizing the complex. Our results demonstrate that the static electric field has an influence on the molecular recognition reactions between nucleoside base pairs at the metal-solution interface and can be controlled by altering the surface charge at the metal.

Effects of Amiloride, an Ion Channel Blocker, on Alamethicin Pore Formation in Negatively Charged, Gold-Supported, Phospholipid Bilayers: A Molecular View.

The effects of amiloride on the structure and conductivity of alamethicin ion pore formation within negatively charged, gold-supported, 1,2-dimyristoyl- sn-glycero-3-phosphocholine/Egg-PG membranes were investigated with the help of electrochemical impedance spectroscopy (EIS), photon polarization modulation-infrared reflection spectroscopy (PM-IRRAS), and atomic force microscopy (AFM). The EIS results indicate that ion conductivity across negatively charged phospholipid bilayers containing alamethicin decreases by an order of magnitude when amiloride is introduced to the system. Despite the reduction in ion conductivity, the PM-IRRAS data shows that amiloride does not inhibit ion channel formation by alamethicin peptides. High-resolution AFM images revealed that amiloride enlarges and distorts the shape of alamethicin ion pores when introduced to the system, indicating that it is inserting itself into the mouth of the alamethicin pores. This effect is driven by electrostatic interactions between positively charged amiloride molecules and the negative charge on the membrane.

Spectroelectrochemical Characterization of 1,2-Dipalmitoyl- sn-glycero-3-cytidine Diphosphate Nucleolipid Monolayer Supported on Gold (111) Electrode.

The effect of the electrode potential on the orientation and conformation of the 1,2-dipalmitoyl- sn-glycero-3-cytidine monolayer deposited on a gold (111) electrode surface was described. The potential of zero free charge ( Epzc) for the monolayer-covered electrode was determined to be -0.2 V vs SCE. The differential capacitance and charge density data indicated that the monolayer is stable at the electrode surface when ( E - Epzc) > 0.0 V. At negative rational potentials, a progressive detachment (electrodewetting) of the monolayer occurs. The monolayer is fully detached from the electrode surface at ( E - Epzc) < -0.6 V. The conformation and orientation of the acyl chains and the orientation of the cytosine moiety were determined with the help of photon polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS). The IR measurements demonstrate that the acyl chains are predominantly in the gel phase in the adsorbed state and tilted at an angle of ∼30° with respect to the electrode surface normal. The tilt angle of the acyl chains increases when the film is detached from the gold surface, indicating that the monolayer becomes more disordered. At ( E - Epzc) > 0.0 V, the plane of the cytosine moiety assumes a small angle of ∼20° with respect to the surface. At negative potentials, the tilt angle of the cytosine fragment increases and rotates. With the help of DFT calculations, these changes were explained by the repulsion of the positive pole of the cytosine permanent dipole moment by the positively charged gold surface and its attraction to the metal surface at negative electrode potentials. This work provides unique information for the future development of sensors based on the molecular recognition of nucleoside targets.

Pore Forming Properties of Alamethicin in Negatively Charged Floating Bilayer Lipid Membranes Supported on Gold Electrodes.

Electrochemical impedance spectroscopy (EIS), atomic force microscopy (AFM), and photon polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) were employed to investigate the formation of alamethicin pores in negatively charged bilayers composed of a mixture of 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) and egg-PG floating at gold (111) electrode surfaces modified by self-assembled monolayers of 1-thio-ß-d-glucose (ß-Tg). The EIS data showed that the presence of alamethicin decreases the membrane resistivity by about 1 order of magnitude. PM-IRRAS measurements provided information about the tilt angles of peptide helical axis with respect to the bilayer normal. The small tilt angles obtained for the peptide helical axis prove that the alamethicin molecules were inserted into the DMPC/egg-PG membranes. The tilt angles decreased when negative potentials were applied, which correlates with the observed decrease in membrane resistivity, indicating that ion pore formation is assisted by the transmembrane potential. Molecular resolution AFM images provided visual evidence that alamethicin molecules aggregate forming hexagonal porous 2D lattices with periodicities of 2.0 ± 0.2 nm. The pore formation by alamethicin in the negatively charged membrane was compared with the interaction of this peptide with a bilayer formed by zwitterionic lipids. The comparison of these results showed that alamethicin preferentially forms ion translocating pores in negatively charged phospholipid membranes.

Transforming an esterase into an enantioselective catecholase through bioconjugation of a versatile metal-chelating inhibitor.

Metal complexes introduced into protein scaffolds can generate versatile biomimetic catalysts endowed with a variety of catalytic properties. Here, we synthesized and covalently bound a bipyridinyl derivative to the active centre of an esterase to generate a biomimetic catalyst that shows catecholase activity and enantioselective catalytic oxidation of (+)-catechin.

Bioelectrocatalytic Activity of W-Formate Dehydrogenase Covalently Immobilized on Functionalized Gold and Graphite Electrodes.

The decrease of greenhouse gases such as CO2 has become a key challenge for the human kind and the study of the electrocatalytic properties of CO2-reducing enzymes such as formate dehydrogenases is of importance for this goal. In this work, we study the covalent bonding of Desulfovibrio vulgaris Hildenborough FdhAB formate dehydrogenase to chemically modified gold and low-density graphite electrodes, using electrostatic interactions for favoring oriented immobilization of the enzyme. Electrochemical measurements show both bioelectrocatalytic oxidation of formate and reduction of CO2 by direct electron transfer (DET). Atomic force microscopy and quartz crystal microbalance characterization, as well as a comparison of direct and mediated electrocatalysis, suggest that a compact layer of formate dehydrogenase was anchored to the electrode surface with some crosslinked aggregates. Furthermore, the operational stability for CO2 electroreduction to formate by DET is shown with approximately 100% Faradaic yield.

How do lipid nanocarriers - Cubosomes affect electrochemical properties of DMPC bilayers deposited on gold (111) electrodes?

Cubosome nanocarriers are promising biomimetic drug delivery systems used in particular for highly toxic drugs in cases where decreasing unwanted side effects is especially important. The properties of electrode supported lipid bilayer prepared by the combined Langmuir-Blodgett and Langmuir-Schaefer techniques were studied using electrochemical techniques following exposure of the film - covered electrode to a solution containing phytantriol - based cubosomes. The inclusion of the carrier in the model membrane under different experimental conditions was probed and the modifications induced in the lipid organization were for the first time inferred by quantitative analysis of the responses of cyclic voltammetry (CV), AC voltammetry and Electrochemical Impedance Spectroscopy (EIS) as well as blocking assays using a redox probe in the solution. Exposure of a preformed DMPC bilayer to cubosome solution resulted in the improved barrier properties of the film reflecting disintegration of cubosomes and formation of additional phytantriol/Pluronic F-108 polymer layer on the top of the DMPC bilayer. On the other hand, formation of the layer in the presence of cubosomes in the subphase lead to an increased capacitance of the film since penetration of the lipid layers by the cubosomal phytantriol increased the porosity of the film.

Molecular recognition between guanine and cytosine-functionalized nucleolipid hybrid bilayers supported on gold (111) electrodes.

A hybrid bilayer lipid membrane (hBLM), constructed with a 1-hexadecanethiol self-assembled interior leaflet and a 1,2-dipalmitoyl-sn-glycero-3-cytidine nucleolipid exterior leaflet, was deposited at the surface of a gold (111) electrode. This system was used to investigate the molecular recognition reaction between the cytosine moieties of the lipid head group with guanine molecules in the bulk electrolyte solution. Electrochemical measurements and photon polarization modulation infrared reflection absorption spectroscopy (PMIRRAS) were employed to characterize the system and determine the extent of the molecular recognition reaction. The capacitance of the hBLM-covered gold electrode was very low (~1 µF cm-2), therefore the charge density at the gold surface was small. Changing the electrode potential had a minimal effect on the complexation between the cytosine moieties and guanine molecules due to small changes in the static electric field across the membrane. This behavior favored the formation of the guanine-cytosine complex.

Potentiometric detection of ATP based on the transmembrane proton gradient generated by ATPase reconstituted on a gold electrode.

Adenosine triphosphate (ATP) is a key molecule as energy vector for living organisms, therefore its detection reveals the presence of microbial colonies. Environments where the existence of microbial pathogens suppose a health hazard can benefit from real time monitoring of such molecule. We report a potentiometric biosensor based on ATP-synthase from Escherichia coli reconstituted in a floating phospholipid bilayer over gold electrodes modified with a 4-aminothiophenol self-assembled monolayer. The use of a pH-dependent redox probe on the electrode surface allows a simple, specific and reliable on site determination of ATP concentration from 1 µM to 1 mM. The broad range ATP biosensor can offer an alternative way of measuring in a few minutes the presence of microbial contamination.