Sebastian Neugebauer

Nano-porous electrode systems by colloidal lithography for sensitive electrochemical detection: fabrication technology and properties

A porous metal-insulator-metal sensor system was developed with the ultimate goal of enhancing the sensitivity of electrochemical sensors by taking advantage of redox cycling of electro active molecules between closely spaced electrodes. The novel fabrication technology is based on thin film deposition in combination with colloidal self-assembly and reactive ion etching to create micro- or nanopores. This cost effective approach is advantageous compared to common interdigitated electrode arrays (IDA) since it does not require high definition lithography technology. Spin-coating and random particle deposition, combined with a new sublimation process are discussed as competing strategies to generate monolayers of colloidal spheres. Metal-insulator-metal layer systems with low leakage currents 90%). We also discuss possible causes of sensor failure with respect to critical fabrication processes. Short circuits which could occur during or as a result of the pore etching process were investigated in detail. Infrared microscopy in combination with focused ion beam etching/SEM were used to reveal a defect mechanism creating interconnects and increased leakage current between the top and bottom electrodes. Redox cycling provides for amplification factors of >100. A general applicability for electrochemical diagnostic assays is therefore anticipated.

Acrylic Acid-Based Copolymers as Immobilization Matrix for Amperometric Biosensors

Abstract Libraries consisting of acrylic acid-based polymer resins were synthesized by copolymerizing acrylic acid with different alkyl acrylates and 1-vinyl imidazole concomitantly varying the relative concentrations and the number of monomers present in the reaction mixture. The obtained polymer resins are suitable for electrochemically-induced precipitation on electrode surfaces caused by the protonation of acidic side groups at the polymer chains induced by a local electrochemical formation of protons in the diffusion zone in front of the electrode. Addition of suitable biological recognition elements such as glucose oxidase to suspensions of these electrodeposition paints (EDP) led to the entrapment of the enzyme within the slowly precipitating polymer film, and hence to the nonmanual fabrication of amperometric biosensors. The suitability of the obtained copolymers was screened with respect to the performance of the resulting glucose sensors. It could be demonstrated that the chemical composition of the used EDP significantly influences the properties of the sensors in terms of sensitivity and linear range of their response to glucose. †This article is dedicated to Prof. Dr. Pierre Coulet on the occasion of his retirement.

Imaging Biocatalytic Activity of Enzyme−Polymer Spots by Means of Combined Scanning Electrochemical Microscopy/Electrogenerated Chemiluminescence

The purpose of this study was to develop a scanning electrochemical microscopy (SECM) and scanning electrogenerated chemiluminescence (SECL) setup to visualize the localized enzymatic activity using glucose oxidase as a model. Combination of SECM and electrogenerated chemiluminescence (ECL) was made possible by integrating a photomultiplier tube (PMT) within a SECM setup which is mounted on top of an inverted microscope. An enzyme-polymer spot formed on a glass slide and placed on top of the entrance window of the PMT was used as a model sample to evaluate the potential of the combined SECM/ECL setup. Hydrogen peroxide, which was locally generated by the glucose oxidase (GOx)-catalyzed reaction, reacted with oxidized luminol which was simultaneously electrochemically generated at the positioned SECM electrode tip. By using the phase-sensitive lock-in amplifier, the potential applied to the SECM tip was sinusoidally swept to invoke an associated oscillation of the ECL. Thus, sensitivity of SECL could be substantially enhanced. Images of the local immobilized enzyme activity obtained both by ECL and generator/collector (GC) mode of SECM were compared to elucidate the pathway in which the SECM and SECL signals are generated.

Scanning electrochemical microscopy (SECM) as a tool in biosensor research.

Scanning electrochemical microscopy (SECM) is discussed as a versatile tool to provide localized (electro)chemical information in the context of biosensor research. Advantages of localized electrochemical measurements will be discussed and a brief introduction to SECM and its operation modes will be given. Experimental challenges of the different detection modes of SECM and its applicability for different fields in biosensor research are discussed. Among these are the evaluation of immobilization techniques by probing the local distribution of biological activity, the visualization of diffusion profiles of reactants, cofactors, mediators, and products, and the elucidation of (local) kinetic parameters. The combination of SECM with other scanning-probe techniques allows to maximize the information on a given biosensing system. The potential of SECM as a tool in micro-fabrication aiming for the fabrication of microstructured biosensors will be shortly discussed.

Optimization of an electrochemical DNA assay by using a 48-electrode array and redox amplification studies by means of scanning electrochemical microscopy.

Sensible DNA: An electrochemical DNA assay based on specific Salmonella spp. capture probes and enzyme labeling with alkaline phosphatase was optimized by using a 48‐electrode microarray and scanning electrochemical microscopy (SECM). SECM was further used to evaluate potential amplification strategies due to redox cycling.

Electrochemical detection of synthetic DNA and native 16S rRNA fragments on a microarray using a biotinylated intercalator as coupling site for an enzyme label.

The direct electrochemical detection of synthetic DNA and native 16S rRNA fragments isolated from Escherichia coli is described. Oligonucleotides are detected via selective post-labeling of double stranded DNA and DNA-RNA duplexes with a biotinylated intercalator that enables high-specific binding of a streptavidin/alkaline phosphatase conjugate. The alkaline phosphatase catalyzes formation of p-aminophenol that is subsequently oxidized at the underlying gold electrode and hence enables the detection of complementary hybridization of the DNA capture strands due to the enzymatic signal amplification. The hybridization assay was performed on microarrays consisting of 32 individually addressable gold microelectrodes. Synthetic DNA strands with sequences representing six different pathogens which are important for the diagnosis of urinary tract infections could be detected at concentrations of 60 nM. Native 16S rRNA isolated from the different pathogens could be detected at a concentration of 30 fM. Optimization of the sensing surface is described and influences on the assay performance are discussed.

A microelectrochemical sensing system for the determination of Epstein–Barr virus antibodies

AbstractAn electrochemical method for the detection of Epstein–Barr virus (EBV) infections is described. The method relies on an immunoassay with electrochemical read-outs based on recombinant antigens. The antigens are immobilised on an Au electrode surface and used to complementarily bind antibodies from serum samples found during different stages of infection with EBV. Thiol chemistry under formation of self-assembled monolayers functions as a means to immobilise the antigens at the Au electrodes. A reporter system consisting of a secondary antibody labelled with alkaline phosphatase is used for electrochemical detection. The feasibility of the assay design is demonstrated and the assay performance is tested against the current gold standard in EBV detection. Close correlation is obtained for the results found for the developed electrochemical immunoassay and a standard line assay. Moreover, the electrochemical immunoassay is combined with a nanoporous electrode system allowing signal amplification by means of redox recycling. An amplification factor of 24 could be achieved. FigureAmplified immunoassay for the determination of anti-EBV antibodies in serum based on enzyme amplification coupled with electrochemical amplification by redox cycling in nanopore electrodes