Jürgen Fritz

Electronic detection of DNA by its intrinsic molecular charge.

We report the selective and real-time detection of label-free DNA using an electronic readout. Microfabricated silicon field-effect sensors were used to directly monitor the increase in surface charge when DNA hybridizes on the sensor surface. The electrostatic immobilization of probe DNA on a positively charged poly-l-lysine layer allows hybridization at low ionic strength where field-effect sensing is most sensitive. Nanomolar DNA concentrations can be detected within minutes, and a single base mismatch within 12-mer oligonucleotides can be distinguished by using a differential detection technique with two sensors in parallel. The sensors were fabricated by standard silicon microtechnology and show promise for future electronic DNA arrays and rapid characterization of nucleic acid samples. This approach demonstrates the most direct and simple translation of genetic information to microelectronics.

A Systematic In Vitro Study of Nucleoprotein Complexes Formed by Bacterial Nucleoid-Associated Proteins Revealing Novel Types of DNA Organization

Bacterial nucleoid is a dynamic entity that changes its three-dimensional shape and compaction depending on cellular physiology. While these changes are tightly associated with compositional alterations of abundant nucleoid-associated proteins implicated in reshaping the nucleoid, their cooperation in regular long-range DNA organization is poorly understood. In this study, we reconstitute a novel nucleoprotein structure in vitro, which is stabilized by cooperative effects of major bacterial DNA architectural proteins. While, individually, these proteins stabilize alternative DNA architectures consistent with either plectonemic or toroidal coiling of DNA, the combination of histone-like protein, histone-like nucleoid structuring protein, and integration host factor produces a conspicuous semiperiodic structure. By employing a bottom-up in vitro approach, we thus characterize a minimum set of bacterial proteins cooperating in organizing a regular DNA structure. Visualized structures suggest a mechanism for nucleation of topological transitions underlying the reshaping of DNA by bacterial nucleoid-associated proteins.

Fabrication and characterization of a micromechanical sensor for differential detection of nanoscale motions

We have micromachined a mechanical sensor that uses interferometry to detect the differential and absolute deflections of two adjacent cantilevers. The overall geometry of the device allows simple fluidic delivery to each cantilever to immobilize molecules for biological and chemical detection. We show that differential sensing is 50 times less affected by ambient temperature changes than the absolute, thus enabling a more reliable differentiation between specific cantilever bending and background effects. We describe the fabrication process and show results related to the dynamic characterization of the device as a differential sensor. The root-mean-squared (r.m.s.) sensor noise in water and air is /spl sim/1 nm over the frequency range of 0.4-40 Hz. We also find that in air, the deflection resolution is limited only by the cantilevers thermomechanical noise level of 0.008 /spl Aring//Hz/sup 1/2/ over the frequency range of 40-1000 Hz.

Function and disruption of DNA Methyltransferase 3a cooperative DNA binding and nucleoprotein filament formation

The catalytic domain of Dnmt3a cooperatively multimerizes on DNA forming nucleoprotein filaments. Based on modeling, we identified the interface of Dnmt3a complexes binding next to each other on the DNA and disrupted it by charge reversal of critical residues. This prevented cooperative DNA binding and multimerization of Dnmt3a on the DNA, as shown by the loss of cooperative complex formation in electrophoretic mobility shift assay, the loss of cooperativity in DNA binding in solution, the loss of a characteristic 8- to 10-bp periodicity in DNA methylation and direct imaging of protein–DNA complexes by scanning force microscopy. Non-cooperative Dnmt3a-C variants bound DNA well and retained methylation activity, indicating that cooperative DNA binding and multimerization of Dnmt3a on the DNA are not required for activity. However, one non-cooperative variant showed reduced heterochromatic localization in mammalian cells. We propose two roles of Dnmt3a cooperative DNA binding in the cell: (i) either nucleofilament formation could be required for periodic DNA methylation or (ii) favorable interactions between Dnmt3a complexes may be needed for the tight packing of Dnmt3a at heterochromatic regions. The complex interface optimized for tight packing would then promote the cooperative binding of Dnmt3a to naked DNA in vitro.

The nanomechanical NOSE

We present a novel chemical sensor based on a microfabricated array of silicon cantilevers. Individual cantilevers are sensitized for the detection of analytes using metal coatings. Analyte molecules chemisorbing or physisorbing on the cantilever coating and chemical reactions produce a change in interfacial stress between analyte molecules and cantilever. This leads to a nanomechanical response of the cantilever, i.e. bending. The bending is read out using a time-multiplexed optical beam-deflection technique. From magnitude and temporal evolution of the bending, quantitative information on analyte species and concentration is derived. Here, we demonstrate the detection of ethene and water vapor with such a nanomechanical nose.

Biomass-Adsorbent Adhesion Forces as an Useful Indicator of Performance in Expanded Beds

Biomass deposition onto an adsorbent matrix can severely affect early downstream bioprocessing performance e.g., during expanded bed adsorption. Cell deposition phenomena are sensitive to the nature of the interacting cells and matrix bodies and to the solution chemistry, but also depend on the exerted hydrodynamic shear forces. Strong adhesion forces require high hydrodynamic shear for cell detachment, e.g., ≈1400 pN would be needed to detach a yeast cell from a DEAE Sepharose bead. Both adhesion and detachment forces can be reduced by spontaneous coverage of the adsorbent surface with polyvinyl pyrrolidone. For comparison, only ≈270 pN would be required to remove such a cell deposited onto a Chelating-Cu2+ bead. First examples of corroborating calculated XDLVO interaction energies by direct force measurements with an atomic force microscopy are presented. Evaluating interfacial forces at the nanoscale can allow for an optimized bioprocess and adsorbent design.

Transient Grating Studies of Femtosecond Processes in Ultra‐Thin Layers of PTCDA

Elementary processes like energy transfer, charge transport, and exciton diffusion in thin films occur on time scales of femtoseconds. Time-resolved photo-electron spectroscopy, a technique limited to ultra-high vacuum environment and the proper choice of a substrate, has been used to study ultrafast processes in sub-nanometer thin films so far. Herein we show that a transient (population) grating created by the interference of laser pulses can be used to study ultrafast processes in such films under ambient conditions. Our investigations of exciton dynamics in 1.4±0.2 nm and 0.4±0.2 nm thin films, formed by nanocrystals of 3,4,9,10-Perylenetetracarboxylic dianhydride (PTCDA) on glass and mica, show that the dynamics differ with the crystal size, possibly due to the confinement induced changes in the electronic structure. The technique is sensitive enough to investigate the dynamics in systems, where only 20 % of the surface is covered by nano-crystals. We expect such an optical technique that is sensitive enough to study dynamics in few to sub-nanometer thin layers under ambient conditions to become important in investigating ultrafast dynamics on surfaces, interfaces, functionalized materials, organic semiconductors, and quantum phenomena in ordered structures of reduced dimensions, such as quantum dots and graphene sheets.