Dieter Heindl

Gold nanostoves for microsecond DNA melting analysis.

In traditional DNA melting assays, the temperature of the DNA-containing solution is slowly ramped up. In contrast, we use 300 ns laser pulses to rapidly heat DNA bound gold nanoparticle aggregates. We show that double-stranded DNA melts on a microsecond time scale that leads to a disintegration of the gold nanoparticle aggregates on a millisecond time scale. A perfectly matching and a point-mutated DNA sequence can be clearly distinguished in less than one millisecond even in a 1:1 mixture of both targets.

A Convergent Strategy for the Modification of Peptide Nucleic Acids: Novel Mismatch‐Specific PNA‐Hybridization Probes

Coupling of nonnatural nucleobases to the orthogonally protected backbone 1 on the solid phase provided access to novel peptide nucleic acid (PNA) conjugates 2, which are difficult to synthesize by standard routes. Hybridization probes containing the thiazolorange dye might allow DNA sequence analysis in real time. B-CH(2)CO=modified nucleobase, fluorescent dye, etc; Boc, Fmoc=protecting groups.

Competitive homogeneous digoxigenin immunoassay based on fluorescence quenching by gold nanoparticles

We report on a competitive, homogeneous immunoassay for the detection of the hapten digoxigenin. The assay is based on competitive fluorescence quenching by gold nanoparticles. Digoxigenin is indirectly labeled with the fluorophore Cy3B through bovine serum albumin and used as a marker. Gold nanoparticles functionalized with anti-digoxigenin antibodies serve as fluorescence quenchers. Free digoxigenin molecules in the analyte solution compete with the labeled markers for antibodies on the gold nanoparticles. The fluorescence signal depends linearly on the free digoxigenin concentration within a range of concentration from 0.5 to 3 ng mL(-1). The limit of detection is estimated as 0.2 ng mL(-1) and the limit of quantitation is estimated as 0.6 ng mL(-1). The method can be used to detect digoxin, a drug used to cure cardiac arrhythmia.

Polymerase/DNA interactions and enzymatic activity: multi-parameter analysis with electro-switchable biosurfaces

The engineering of high-performance enzymes for future sequencing and PCR technologies as well as the development of many anticancer drugs requires a detailed analysis of DNA/RNA synthesis processes. However, due to the complex molecular interplay involved, real-time methodologies have not been available to obtain comprehensive information on both binding parameters and enzymatic activities. Here we introduce a chip-based method to investigate polymerases and their interactions with nucleic acids, which employs an electrical actuation of DNA templates on microelectrodes. Two measurement modes track both the dynamics of the induced switching process and the DNA extension simultaneously to quantitate binding kinetics, dissociation constants and thermodynamic energies. The high sensitivity of the method reveals previously unidentified tight binding states for Taq and Pol I (KF) DNA polymerases. Furthermore, the incorporation of label-free nucleotides can be followed in real-time and changes in the DNA polymerase conformation (finger closing) during enzymatic activity are observable.

Easy method for the synthesis of labeled oligonucleotides.

We developed a new strategy for labeling oligonucleotides. Labels bearing an acceptor substituted azide moiety, e.g. a sulfonyl azide substituent are used during oligonucleotide synthesis instead of conventional dye phosphoramidites. Azides are well known to react with trivalent phosphor compounds to phosphor amidates and therefore they could be used instead of an oxidizer during oligonucleotide synthesis. Because N-Alkyl or N-Aryl phosphor amidates are hydrolyzed especially under acidic conditions, we used acceptor substituted azides as reactants, which results in remarkable stabilization of the corresponding amidates. This method is suitable to introduce labels at any internucleosidic linkage of an oligonucleotide and could be used for synthesis of any kind of labeled or polylabeled detection probes. Probes synthesized with these new labeling reagents are evaluated in Real Time PCR. They show the same performance like probes synthesized by conventional means. Since the labeling reagents could be easily synthesized and since excess reagent could be recycled and used for further labeling reactions, this method represents a very cost effective way for the synthesis of labeled oligonucleotides.

Overview of Colorimetric, Chemiluminometric, and Fluorimetric Detection Systems

A large number of detection systems for biomolecules have been described in the literature; therefore, only a brief overview of the most important nonradioactive methods will be given here. More details can be found in the other chapters of this book or in the cited literature.

Abstract A156: Anti-p53 auto-antibody serum profiling using high-density peptide arrays

Cancer is a result of a number of genetic alterations that disturb normal, controlled cell growth and differentiation. Mutational events leading to the activation of oncogenes or the inactivation of tumor-suppressor genes have been linked causally to the formation of tumors. p53 is one of the most important regulators of transcription, cellular cycle, DNA repair and apoptosis detected to date. Anti-p53 antibodies have been detected in the serum of cancer patients. This immune response is probably due to a self-immunization process linked to the strong immunogenicity of the p53 protein, and is associated predominantly with p53 missense mutation and p53 accumulation in the tumor. Auto-antibodies have also been proposed as potential diagnostic biomarkers for early stage diagnosis of cancers, since an increase in serum levels of certain auto-antibodies has been shown to precede the development of disease symptoms and correlate with cancer incidence for various cancers including breast and lung cancer. Here we systematically evaluate reactivity of antibodies in p53 positive serum samples and identify reactive epitopes to normal and mutant peptides using a high-density (2.9 Million) peptide microarray. We assess the effect of linker length, peptide length, and flanking serines on antibody detection. We propose peptide array design parameters that can be applied to a whole proteome level to enable biomarker discovery and validation of novel auto-antibody epitopes associated with cancer. Citation Format: Ken C. Lo, John C. Tan, Eric Sullivan, Ryan Bannen, Todd Richmond, Florian Grupp, Stefan Weiser, Dieter Heindl, Klaus-Peter Stengele, Albert Thomas. Anti-p53 auto-antibody serum profiling using high-density peptide arrays. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A156.

Chemiluminescent Detection with Horseradish Peroxidase and luminol

Principle and The chemiluminescence (CL) of luminol and related compounds is a well-applications known reaction and has been studied intensively (Gundermann, 1974). The mechanism of the CL reaction of the so-called diacyl hydrazides is very complex and depends on several conditions (Gundermann and McCapra, 1987), e.g., whether the reaction is carried out in water or in aprotic solvents. In DMSO luminol exists as an activated aminophthalic acid, which gives off a bright blue-green light in the presence of a strong base together with hydrogen peroxide.

Indigo/Tetrazolium Dyes

One of the most sensitive and widespread colorimetric detection systems in matrix-based application formats is the redox couple 5-bromo-4-chloro-indolyl phosphate (BCIP)/nitroblue tetrazolium chloride (NBT). It is used for the detection and localisation of alkaline phosphatase activity especially in Western and Southern blots or in immunohisto/cytochemistry applications.Indolyl derivatives of type (I) are usually stable over a wide pH range, but after enzymatically catalyzed hydrolysis to the indolyl derivative (II) they rapidly form an indigoid dye (III) through atmospheric or chemical oxidation (see Figure 1).

Labeling of Biomolecules with Fluorophores

Coumarins, fluorescein, and resorufin derivatives are three important fluorescence labels which can be used for labeling of biomolecules. During the last years a wide range of new fluorescence labels have been published and are commercially available, especially covering the long wavelength/ NIR range. Among these are new rhodamine and oxazine derivatives (Arden-Jakob et al., 1997) cyanine derivatives (Thompson, 1994) and also BODIPY fluorophores (Haugland, 1996). The development was especially driven by the upcoming cheap laser diodes (Wersig et al., 1993) and the demand to reduce background fluorescence.