Jörg Opitz

Selective targeting of green fluorescent nanodiamond conjugates to mitochondria in HeLa cells

Fluorescent cellular biomarkers play a prominent role in biosciences. Most of the available biomarkers have some drawbacks due to either physical and optical or cytotoxic properties. In view of this, we investigated the potential of green fluorescent nanodiamonds as biomarkers in living cells. Nanodiamonds were functionalized by attaching antibodies that target intracellular structures such as actin filaments and mitochondria. Then, the nanodiamond conjugates were transfected into HeLa cells. Transfections were mediated by 4(th)-generation dendrimers, cationic liposomes and protamine sulfate. Using fluorescence microscopy, we confirmed successful transfections of the nanodiamonds into HeLa cells. Nanodiamond fluorescence could be easily differentiated from cellular autofluorescence. Furthermore, nanodiamonds could be targeted selectively to intracellular structures. Therefore, nanodiamonds are a promising tool for intracellular assays.

Schottky barrier-based silicon nanowire pH sensor with live sensitivity control

We demonstrate a pH sensor based on ultrasensitive nanosize Schottky junctions formed within bottom-up grown dopant-free arrays of assembled silicon nanowires. A new measurement concept relying on a continuous gate sweep is presented, which allows the straightforward determination of the point of maximum sensitivity of the device and allows sensing experiments to be performed in the optimum regime. Integration of devices into a portable fluidic system and an electrode isolation strategy affords a stable environment and enables long time robust FET sensing measurements in a liquid environment to be carried out. Investigations of the physical and chemical sensitivity of our devices at different pH values and a comparison with theoretical limits are also discussed. We believe that such a combination of nanofabrication and engineering advances make this Schottky barrier-powered silicon nanowire lab-on-a-chip platform suitable for efficient biodetection and even for more complex biochemical analysis.

Patterned biochemical functionalization improves aptamer-based detection of unlabeled thrombin in a sandwich assay.

Here we propose a platform for the detection of unlabeled human α-thrombin down to the picomolar range in a fluorescence-based aptamer assay. In this concept, thrombin is captured between two different thrombin binding aptamers, TBA1 (15mer) and TBA2 (29mer), each labeled with a specific fluorescent dye. One aptamer is attached to the surface, the second one is in solution and recognizes surface-captured thrombin. To improve the limit of detection and the comparability of measurements, we employed and compared two approaches to pattern the chip substrate-microcontact printing of organosilanes onto bare glass slides, and controlled printing of the capture aptamer TBA1 in arrays onto functionalized glass substrates using a nanoplotter device. The parallel presence of functionalized and control areas acts as an internal reference. We demonstrate that both techniques enable the detection of thrombin concentrations in a wide range from 0.02 to 200 nM with a detection limit at 20 pM. Finally, the developed method could be transferred to any substrate to probe different targets that have two distinct possible receptors without the need for direct target labeling.

Site-specific binding and stretching of DNA molecules at UV-light-patterned aminoterpolymer films

Site-specific deposition of nanoparticles and DNA onto micro-patterned aminoterpolymer films is reported. The chemical patterning of the film surface is accomplished by an easy-to-handle, one-step procedure. Photolabile protection groups are locally removed by applying UV light through an optical mask. This causes exposure of amino groups to the surface to which charged nanoparticles can then associate. End-specific binding of DNA at the surface-exposed amino groups is obtained at optimum pH conditions. This allows a site-specific attachment and stretching of single DNA molecules at the patterned polymer film surface.

Dielectrophoretic growth of platinum nanowires: concentration and temperature dependence of the growth velocity.

The growth velocity of platinum nanowires in an aqueous solution of K(2)PtCl(4) is investigated as a function of the metal complex concentration and temperature. The solution is specially prepared to provide mainly the neutral complex cis-[PtCl(2)(H(2)O)(2)] for growing nanowires by dielectrophoresis. The measured growth velocities indicate diffusion-limited nanowire growth at low concentration and high temperature in qualitative agreement with a theoretical analysis that includes the diffusion of metal complexes and the dielectrophoretic force on the complexes. At concentrations greater than 100 μM and low temperature, different behavior is observed, suggesting the growth rate to be limited by the deposition reaction of platinum at the nanowire tip. The enhancement of the K(+) concentration is found to support nanowire growth. Possible reasons for a rate limitation and for the difference between observed and calculated nanowire growth velocities are discussed.

Biotechnological hydrogen production by photosynthesis

Microbiological photosynthesis is a promising tool for producing hydrogen in an ecologically friendly and economically efficient way. Certain microorganisms (e.g. algae and bacteria) can produce hydrogen using hydrogenase and/or nitrogenase enzymes. However, their natural capacity to produce hydrogen is relatively low. Thus, there is a need to optimize their core photosynthetic processes as well as their cultivation, for more efficient hydrogen production. This review aims to provide a holistic overview of the recent technological and research developments relating to photobiological hydrogen production and downstream processing. First we cover photobiological hydrogen synthesis within cells and the enzymes that catalyze the hydrogen production. This is followed by strategies for enhancing bacterial hydrogen production by genetic engineering, technological development, and innovation in bioreactor design. The remaining sections focus on hydrogen as a product, that is, quantification via (in‐process) gas analysis, recent developments in gas separation technology. Finally, a discussion of the sociological (market) barriers to future hydrogen usage is provided as well as an overview of methods for life cycle assessment that can be used to calculate the environmental consequences of hydrogen production.

Light‐field‐characterization in a continuous hydrogen‐producing photobioreactor by optical simulation and computational fluid dynamics

Externally illuminated photobioreactors (PBRs) are widely used in studies on the use of phototrophic microorganisms as sources of bioenergy and other photobiotechnology research. In this work, straightforward simulation techniques were used to describe effects of varying fluid flow conditions in a continuous hydrogen‐producing PBR on the rate of photofermentative hydrogen production (rH2) by Rhodobacter sphaeroides DSM 158. A ZEMAX optical ray tracing simulation was performed to quantify the illumination intensity reaching the interior of the cylindrical PBR vessel. 24.2% of the emitted energy was lost through optical effects, or did not reach the PBR surface. In a dense culture of continuously producing bacteria during chemostatic cultivation, the illumination intensity became completely attenuated within the first centimeter of the PBR radius as described by an empirical three‐parametric model implemented in Mathcad. The bacterial movement in chemostatic steady‐state conditions was influenced by varying the fluid Reynolds number. The “Computational Fluid Dynamics” and “Particle Tracing” tools of COMSOL Multiphysics were used to visualize the fluid flow pattern and cellular trajectories through well‐illuminated zones near the PBR periphery and dark zones in the center of the PBR. A moderate turbulence (Reynolds number = 12,600) and fluctuating illumination of 1.5 Hz were found to yield the highest continuous rH2 by R. sphaeroides DSM 158 (170.5 mL L−1 h−1) in this study. Biotechnol. Bioeng. 2015;112: 2439–2449.

Detonation nanodiamonds biofunctionalization and immobilization to titanium alloy surfaces as first steps towards medical application.

Summary Due to their outstanding properties nanodiamonds are a promising nanoscale material in various applications such as microelectronics, polishing, optical monitoring, medicine and biotechnology. Beyond the typical diamond characteristics like extreme hardness or high thermal conductivity, they have additional benefits as intrinsic fluorescence due to lattice defects without photobleaching, obtained during the high pressure high temperature process. Further the carbon surface and its various functional groups in consequence of the synthesis, facilitate additional chemical and biological modification. In this work we present our recent results on chemical modification of the nanodiamond surface with phosphate groups and their electrochemically assisted immobilization on titanium-based materials to increase adhesion at biomaterial surfaces. The starting material is detonation nanodiamond, which exhibits a heterogeneous surface due to the functional groups resulting from the nitrogen-rich explosives and the subsequent purification steps after detonation synthesis. Nanodiamond surfaces are chemically homogenized before proceeding with further functionalization. Suspensions of resulting surface-modified nanodiamonds are applied to the titanium alloy surfaces and the nanodiamonds subsequently fixed by electrochemical immobilization. Titanium and its alloys have been widely used in bone and dental implants for being a metal that is biocompatible with body tissues and able to bind with adjacent bone during healing. In order to improve titanium material properties towards biomedical applications the authors aim to increase adhesion to bone material by incorporating nanodiamonds into the implant surface, namely the anodically grown titanium dioxide layer. Differently functionalized nanodiamonds are characterized by infrared spectroscopy and the modified titanium alloys surfaces by scanning and transmission electron microscopy. The process described shows an adsorption and immobilization of modified nanodiamonds on titanium; where aminosilanized nanodiamonds coupled with O-phosphorylethanolamine show a homogeneous interaction with the titanium substrate.

Combinatorial approaches to evaluate nanodiamond uptake and induced cellular fate

Nanodiamonds (NDs) are an emerging class of engineered nanomaterials that hold great promise for the next generation of bionanotechnological products to be used for drug and gene delivery, or for bio-imaging and biosensing. Previous studies have shown that upon their cellular uptake, NDs exhibit high biocompatibility in various in vitro and in vivo set-ups. Herein we hypothesized that the increased NDs biocompatibility is a result of minimum membrane perturbations and their reduced ability to induce disruption or damage during cellular translocation. Using multi-scale combinatorial approaches that simulate ND-membrane interactions, we correlated NDs real-time cellular uptake and kinetics with the ND-induced membrane fluctuations to derive energy requirements for the uptake to occur. Our discrete and real-time analyses showed that the majority of NDs internalization occurs within 2 h of cellular exposure, however, with no effects on cellular viability, proliferation or cellular behavior. Furthermore, our simulation analyses using coarse-grained models identified key changes in the energy profile, membrane deformation and recovery time, all functions of the average ND or ND-based agglomerate size. Understanding the mechanisms responsible for ND-cell membrane interactions could possibly advance their implementation in various biomedical applications.

High yield formation of lipid bilayer shells around silicon nanowires in aqueous solution

The combination of nanoscaled materials and biological self-assembly is a key step for the development of novel approaches for biotechnology and bionanoelectronic devices. Here we propose a route to merge these two subsystems and report on the formation of highly concentrated aqueous solutions of silanized silicon nanowires wrapped in a lipid bilayer shell. We developed protocols and investigated the dynamics of lipid films on both planar surfaces and silicon nanowires using fluorescence recovery after photobleaching, demonstrating fully intact and fluid bilayers without the presence of a lipid molecule reservoir. Finally, the experimental setup allowed for in situ observation of spontaneous bilayer formation around the nanowire by lipid diffusion from a vesicle to the nanowire. Such aqueous solutions of lipid coated nanowires are a versatile tool for characterization purposes and are relevant for newly emerging bioinspired electronics and nanosensorics.