Nicole Marmé

Single-molecule detection on a protein-array assay platform for the exposure of a tuberculosis antigen

Based on a single-molecule sensitive fluorescence-linked immunosorbent assay, an analytical platform for the detection of lipoarabinomannan (LAM), a lipopolysaccharide marker of tuberculosis, was established that is about 3 orders of magnitude more sensitive than comparable current ELISA assays. No amplification step was required. Also, no particular sample preparation had to be done. Since individual binding events are detected, true quantification was possible simply by counting individual signals. Utilizing a total internal reflection configuration, unprocessed biological samples (human urine and plasma) to which LAM was added could be analyzed without the requirement of sample purification or washing steps during analysis. Samples containing about 600 antigen molecules per microliter produced a distinct signal. The methodology developed can be employed for any set of target molecules for which appropriate antibodies exist.

Identification of single-point mutations in mycobacterial 16S rRNA sequences by confocal single-molecule fluorescence spectroscopy

We demonstrate the specific identification of single nucleotide polymorphism (SNP) responsible for rifampicin resistance of Mycobacterium tuberculosis applying fluorescently labeled DNA-hairpin structures (smart probes) in combination with single-molecule fluorescence spectroscopy. Smart probes are singly labeled hairpin-shaped oligonucleotides bearing a fluorescent dye at the 5′ end that is quenched by guanosine residues in the complementary stem. Upon hybridization to target sequences, a conformational change occurs, reflected in a strong increase in fluorescence intensity. An excess of unlabeled (‘cold’) oligonucleotides was used to prevent the formation of secondary structures in the target sequence and thus facilitates hybridization of smart probes. Applying standard ensemble fluorescence spectroscopy we demonstrate the identification of SNPs in PCR amplicons of mycobacterial rpoB gene fragments with a detection sensitivity of 10−8 M. To increase the detection sensitivity, confocal fluorescence microscopy was used to observe fluorescence bursts of individual smart probes freely diffusing through the detection volume. By measuring burst size, burst duration and fluorescence lifetime for each fluorescence burst the discrimination accuracy between closed and open (hybridized) smart probes could be substantially increased. The developed technique enables the identification of SNPs in 10−11 M solutions of PCR amplicons from M.tuberculosis in only 100 s.

DNA-probes for the highly sensitive identification of single nucleotide polymorphism using single-molecule spectroscopy

This article presents a new, highly sensitive method for the identification of single nucleotide polymorphisms (SNPs) in homogeneous solutions using fluorescently labeled hairpin‐structured oligonucleotides (smart probes) and fluorescence single‐molecule spectroscopy. While the hairpin probe is closed, fluorescence intensity is quenched due to close contact between the chromophore and several guanosine residues. Upon hybridization to the respective target SNP sequence, contact is lost and the fluorescence intensity increases significantly. High specificity is achieved by blocking sequences containing mismatch with unlabeled oligonucleotides. Time‐resolved single‐molecule fluorescence spectroscopy enables the detection of individual smart probes passing a small detection volume. This method leads to a subnanomolar sensitivity for this single nucleotide specific DNA assay technique.

Self-quenching DNA probes based on dye dimerization for identification of mycobacteria

This paper presents new self-quenching DNA probes that exploit the efficient fluorescence quenching by the formation of dye dimers. The probes consist of a hairpin-structured oligonucleotide that is labeled with two identical fluorescence dyes that are able to form non-fluorescent H-type dimers while the hairpin is closed. We used the oxazine derivative MR 121 that has a sufficient dimerization tendency and can be excited by a pulsed diode laser emitting at 635 nm. Upon hybridization to the target DNA, the dyes are separated and a 12-fold increase of the fluorescence intensity occurs. The probe was used for the specific detection of Mycobacterium xenopi in a model system. Specific target DNA and a control target, differing by six nucleotides were amplified by polymerase chain reaction (PCR). A confocal fluorescence microscope was used to observe the fluorescence bursts of individual probe molecules in the presence of target PCR product and controls. By experiment and by the respective simulation we demonstrated that the secondary structure of the target DNA hinders the hybridization to the DNA probe at room temperature. Based on these data a successful hybridization procedure was developed and allowing the detection of nanomolar concentrations of Mycobacterium xenopi specific target at room temperature, using single-molecule detection techniques.

Recent patents on self-quenching DNA probes.

In this review, we report on patents concerning self-quenching DNA probes for assaying DNA during or after amplification as well as for direct assaying DNA or RNA, for example in living cells. Usually the probes consist of fluorescently labeled oligonucleotides whose fluorescence is quenched in the absence of the matching target DNA. Thereby the fluorescence quenching is based on fluorescence resonance energy transfer (FRET), photoinduced electron transfer (PET), or electronically interactions between dye and quencher. However, upon hybridization to the target or after the degradation during a PCR, the fluorescence of the dye is restored. Although the presented probes were originally developed for use in homogeneous assay formats, most of them are also appropriate to improve surface-based assay methods. In particular we describe patents for self-quenching primers, self-quenching probes for TaqMan assays, probes based on G-quartets, Molecular Beacons, Smart Probes, and Pleiades Probes.

Probing conformational dynamics by photoinduced electron transfer

We demonstrate how photoinduced electron transfer (PET) reactions can be successfully applied to monitor conformational dynamics in individual biopolymers. Single-pair fluorescence resonance energy transfer (FRET) experiments are ideally suited to study conformational dynamics occurring on the nanometer scale, e.g. during protein folding or unfolding. In contrast, conformational dynamics with functional significance, for example occurring in enzymes at work, often appear on much smaller spatial scales of up to several Angströms. Our results demonstrate that selective PET-reactions between fluorophores and amino acids or DNA nucleotides represent a versatile tool to measure small-scale conformational dynamics in biopolymers on a wide range of time scales, extending from nanoseconds to seconds, at the single-molecule level under equilibrium conditions. That is, the monitoring of conformational dynamics of biopolymers with temporal resolutions comparable to those within reach using new techniques of molecular dynamic simulations. We present data about structural changes of single biomolecules like DNA hairpins and peptides by using quenching electron transfer reactions between guanosine or tryptophan residues in close proximity to fluorescent dyes. Furthermore, we demonstrate that the strong distance dependence of charge separation reactions on the sub-nanometer scale can be used to develop conformationally flexible PET-biosensors. These sensors enable the detection of specific target molecules in the sub-picomolar range and allow one to follow their molecular binding dynamics with temporal resolution.

New hairpin-structured DNA probes : alternatives to classical molecular beacons

In this article we report on two different classes of self-quenching hairpin-structured DNA probes that can be used as alternatives to Molecular Beacons. Compared to other hairpin-structured DNA probes, the so-called smart probes are labeled with only one extrinsic dye. The fluorescence of this dye is efficiently quenched by intrinsic guanine bases via a photo-induced electron transfer reaction in the closed hairpin. After hybridization to a target DNA, the distance between dye and the guanines is enlarged and the fluorescence is restored. The working mechanism of the second class of hairpin DNA probes is similar, but the probe oligonucleotide is labeled at both ends with an identical chromophore and thus the fluorescence of the closed hairpin is reduced due to formation of non-fluorescent dye dimers. Both types of probes are appropriate for the identification of single nucleotide polymorphisms and in combination with confocal single-molecule spectroscopy sensitivities in the picomolar range can be achieved.

Novel fluorescently labeled enzyme substrates for the sensitive detection of HIV-protease

In this paper we applied the efficient fluorescence quenching of the red-absorbing oxazine derivative MR121 by the amino acid tryptophan to develop a new fluorescence based enzyme assay that can be used for detection of exopeptidases and endopeptidases. Therefore, we developed peptide substrates labeled with only one chromophore, which is quenched by a neighbored tryptophan residue via photoinduced electron transfer. The specific cleavage site for the target enzyme is located between the chromophore and the tryptophan residue. After digestion of the substrate the contact formation between tryptophan and fluorescent dye is precluded and a significant increase in fluorescence intensity occurs. To demonstrate the new assay technique for exopeptidases, a substrate for the Carboxypeptidase A was designed and a detection limit below the picomolar range (~10-13 M) was achieved with standard fluorescence spectrometry. The primary objective was the detection of the HIV-protease, which is an endopeptidase digesting substrates containing seven specific amino acids in the cleavage site. We designed a substrate, which enables the detection of 10-9 M HIV-protease, whereas the continuous monitoring of the fluorescence signal also allows kinetic studies.

„Zwerge“ aus dem Minilabor

Die Nanotechnologie in all ihren Facetten wird fur die moderne Gesellschaft immer wichtiger und gilt als eine der Schlusseltechnologien des 21. Jahrhunderts. In diesem Artikel werden schulgerechte Synthesen von Gold- und Silber-Nanopartikeln mit Hilfe eines Minilabors vorgestellt, die im auserschulischen Lernort, im Schulerpraktikum oder auch im Rahmen groserer Projekte eigenstandig durchgefuhrt werden konnen. Auserdem wird ein schulerzentriertes Konzept fur eine Nanotechnologie-Unterrichtseinheit vorgestellt, welches auf den Erwerb von den in den Bildungsplanen geforderten Schlusselkompetenzen zielt und den Schulerinnen und Schulern einen Einblick in den Wissenschaftsalltag und in wissenschaftliche Arbeits- und Vorgehensweisen geben soll. Nanotechnology and its broad range of applications are becoming more and more important for modern society. It is said to be one of the key technologies of the current century. This article shows ways of implementing nanotechnology in schools. The presented syntheses of gold and silver nanoparticles were carried out in a miniaturized lab and adapted for use in class. They are appropriate to be carried out by the pupils themselves in school or in science centers, in brief or in longer projects. Furthermore, a student-centered teaching concept for a complete nanotechnology unit focused on key qualifications as claimed by the national curricula is presented. Students can learn about scientific work methods and get an insight view of scientific daily routine.

New HIV-protease assays applying self-quenching peptide substrates in combination with time-resolved fluorescence single-molecule spectroscopy

This work describes the optimization and adoption of an assay system for the Human Immunodeficiency Virus (HIV)-protease, whose inhibition plays a central role in HIV therapy. The HIV-protease, which is an essential enzyme during viral maturation, has a specific cleavage site of eight amino acid residues (SQNY*PIV). Adding two amino acid residues at the N-terminus and enclosing the resulting sequence by a dye-labelled lysine residue and a tryptophan residue leads to the substrate (K(dye)CGSQNY*PIVW) in which the fluorescence of the fluorophore is efficiently quenched by the intrinsic tryptophan due to a photoinduced electron transfer reaction. After cleavage of the substrate by the target enzyme, the dye and the tryptophan residue are separated, effecting a significant increase in fluorescence intensity. Measuring the fluorescence versus time enables an online-monitoring of the enzyme activity. With this method, a HIV-PR concentration of 10−9 M is detectable within minutes, which is comparable with commercially available assays using doubly labelled substrates based on a fluorescence resonance energy transfer. We were able to further increase the sensitivity to the subnanomolar range by using confocal single-molecule spectroscopy.