Gerhard Hartwich

Electrically detected displacement assay (EDDA): a practical approach to nucleic acid testing in clinical or medical diagnosis

This paper introduces the electrically detected displacement assay (EDDA), a electrical biosensor detection principle for applications in medical and clinical diagnosis, and compares the method to currently available microarray technologies in this field. The sensor can be integrated into automated systems of routine diagnosis, but may also be used as a sensor that is directly applied to the polymerase chain reaction (PCR) reaction vessel to detect unlabeled target amplicons within a few minutes. Major aspects of sensor assembly like immobilization procedure, accessibility of the capture probes, and prevention from nonspecific target adsorption, that are a prerequisite for a robust and reliable performance of the sensor, are demonstrated. Additionally, exemplary results from a human papillomavirus assay are presented.

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.

Amplified detection of DNA hybridization using post-labelling with a biotin-modified intercalator

A 32-electrode microelectrode array modified with a self-assembled monolayer of a thiolated DNA capture strand and 11-mercapto-l1-undecanol was used for the detection of multi-resistant Staphylococcus aureus (MRSA) upon hybridization of the complementary target DNA. In the proposed assay strategy the obtained double-stranded DNA (dsDNA) is at first non-covalently labeled by intercalation of a proflavine derivative which is functionalized via a flexible spacer with biotin moieties. Subsequent to this epost-labelling a avidin/alkaline phosphatase conjugate is bound to the biotin moieties thus introducing a reporter group at sites bearing dsDNA. Hybridization and hence the presence of MRSA DNA is detected via oxidation ofp-aminophenol enzymatically generated from p-aminophenylphosphate. The assay strategy was carefully evaluated using ferrocene-modified target strands. An increase in sensitivity of the proposed label-free DNA assays based on a careful design of the sensing interface and the implemented enzymatic amplification was achieved.

Amperometric Enzyme Sensors based on Direct and Mediated Electron Transfer

Publisher Summary The simplest design of an amperometric biosensor is the direct measurement of either an enzymatically generated product or of an electron transfer (ET) mediator naturally involved in the biocatalytic process. For a principal understanding of the ET processes underlying the functionality of all amperometric biosensors, the theoretical background is used as it was developed for the elucidation of ET processes that play a vital role in a variety of biological reactions or in artificial “donor–acceptor system”. These donor–acceptor reactions can be very rapid even when the reactants are separated over longer distances. This chapter presents a brief summary of the Marcus-theory of ET and also focuses on the fabrication of suitable biosensor architectures facilitating electrochemical communication between the immobilized redox proteins and the electrode surface. The chosen immobilization technique and its impact on the biological recognition element affects, significantly, the overall biosensor performance and determines the selectivity, sensitivity, specificity, dynamic range, response time, and reliability of the biosensor. Several techniques for the immobilization of redox protein on electrode surfaces have been described in detail. In order to control the fabrication and later, function of an amperometric biosensor to its largest extent, the development of the so-called “reagentless biosensors” is of increasing importance. For the development of a reagentless biosensor, the most appropriate methods are the use of an enzyme with tightly bound redox centers and an ET pathway either by direct ET or via securely immobilized redox relays.

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