Volker Nock

A Simple Approach to Patterned Protein Immobilization on Silicon via Electrografting from Diazonium Salt Solutions

A highly versatile method utilizing diazonium salt chemistry has been developed for the fabrication of protein arrays. Conventional ultraviolet mask lithography was used to pattern micrometer sized regions into a commercial photoresist on a highly doped p-type silicon (100) substrate. These patterned regions were used as a template for the electrochemical grafting of the in situ generated p-aminobenzenediazonium cation to form patterns of aminophenyl film on silicon. Immobilization of biomolecules was demonstrated by coupling biotin to the aminophenyl regions followed by reaction with fluorescently labeled avidin and visualization with fluorescence microscopy. This simple patterning strategy is promising for future application in biosensor devices.

Droplet actuation induced by coalescence: Experimental evidences and phenomenological modeling

This paper considers the interaction between two droplets placed on a substrate in immediate vicinity. We show here that when the two droplets are of different fluids and especially when one of the droplet is highly volatile, a wealth of fascinating phenomena can be observed. In particular, the interaction may result in the actuation of the droplet system, i.e. its displacement over a finite length. In order to control this displacement, we consider droplets confined on a hydrophilic stripe created by plasma-treating a PDMS substrate. This controlled actuation opens up unexplored opportunities in the field of microfluidics. In order to explain the observed actuation phenomenon, we propose a simple phenomenological model based on Newton’s second law and a simple balance between the driving force arising from surface energy gradients and the viscous resistive force. This simple model is able to reproduce qualitatively and quantitatively the observed droplet dynamics.

Surface Patterning Using Two-Phase Laminar Flow and In Situ Formation of Aryldiazonium Salts**

Patterned nanoscale molecular films will be a key componentof advanced devices such as novel chemical and biologicalsensors and nanoelectronic systems. For long-establishedsurface modification strategies such as the use of self-assembledmonolayers ofalkanethiolsat noblemetal surfacesandreactionsofsilanesatoxidesurfaces,patterningstrategieshave reached a high level of sophistication.

Bioimprinted polymer platforms for cell culture using soft lithography.

BackgroundIt is becoming recognised that traditional methods of culture in vitro on flat substrates do not replicate physiological conditions well, and a number of studies have indicated that the physical environment is crucial to the directed functioning of cells in vivo. In this paper we report the development of a platform with cell-like features that is suitable for in vitro investigation of cell activity. Biological cells were imprinted in hard methacrylate copolymer using soft lithography. The cell structures were replicated at high nanometre scale resolution, as confirmed by atomic force microscopy. Optimisation of the methacrylate-based co-polymer mixture for transparency and biocompatibility was performed, and cytotoxicity and chemical stability of the cured polymer in cell culture conditions were evaluated. Cells of an endometrial adenocarcinoma cell line (Ishikawa) were cultured on bioimprinted substrates.ResultsThe cells exhibited differential attachment on the bioimprint substrate surface compared to those on areas of flat surface and preferentially followed the pattern of the original cell footprint.ConclusionsThe results revealed for the first time that the cancer cells distinguished between behavioural cues from surfaces that had features reminiscent of themselves and that of flat areas. Therefore the imprinted platform will lend itself to detailed studies of relevant physical substrate environments on cell behaviour. The material is not degraded and its permanency allows reuse of the same substrate in multiple experimental runs. It is simple and does not require expensive or specialised equipment. In this work cancer cells were studied, and the growth behaviour of the tumour-derived cells was modified by alterations of the cells’ physical environment. Implications are also clear for studies in other crucial areas of health, such as wound healing and artificial tissues.

Spatially Resolved Measurement of Dissolved Oxygen in Multistream Microfluidic Devices

Dissolved oxygen (DO) is an important parameter with significant effect on cellular development and function. Micron-scale laminar flow and hydrodynamic focusing provide ideal tools for the generation of controlled chemical micro-environments and their application as stimuli to cells. In this paper, we demonstrate the generation and characterization of multistream laminar flow and hydrodynamically focused sample streams with defined dissolved oxygen concentrations on chip. A solid-state oxygen sensor layer was integrated into PDMS-based microchannels and calibrated. Several combinations of sample and buffer streams with concentrations ranging from 0 to 34 mg/l DO were generated and measured for up to three independent parallel flow streams. In addition, diffusion-based stream broadening measured with the sensor was used to determine the coefficient of diffusion of O2 in the flow medium. The devices have the potential to provide novel insights into cell biology and improve the relevance of in vitro cell assays.

Oxygen Control For Bioreactors And In‐vitro Cell Assays

Dissolved oxygen (DO) is an important parameter in biomedical and cell‐culture applications. Several studies have found cell survival and function to be intimately linked to oxygen concentration. Laminar flow, as observed in microfluidic devices, provides an ideal environment to manipulate and control concentration gradients. In this paper we demonstrate the first characterization of integrated fluorescence‐based oxygen sensors for DO measurement within a cell‐culture bioreactor device. Solid‐state PtOEPK/PS sensor patterns were integrated into the PDMS‐based bioreactor and calibrated for detection of DO concentration with a superimposed layer of collagen and Ishikawa human endometrial cancer cells. The sensor signal of the layer subjacent to the cells was found to follow a Stern‐Volmer model and the intensity ratio was measured to I0/I100 = 3.9 after 3 days in culture. The device provides a novel tool for the control and spatially‐resolved measurement of oxygen levels in cellular assays and cell‐culture ...

The use of substrate materials and topography to modify growth patterns and rates of differentiation of muscle cells.

Cells are cultured on platforms made of a variety of materials with selected topographies during studies of cell response and behavior. Understanding the effects of substrates is essential for such applications as developing effective interfaces between body cells and implanted materials and devices. In this study, the effects of substrate surface properties on cell differentiation and alignment on C2C12 myoblasts cultured on conventional or fabricated polymeric cell culture substrates were investigated. Comparisons were made between cells cultured on tissue culture grade polystyrene (TCPS), glass, Permanox, and cured polydimethylsiloxane (PDMS) substrates. Fluorescent immunohistochemistry of cell markers was used to analyse the extent of differentiation. Alignment and guidance of cell growth and spread were studied using patterned platforms. Gratings were made on polystyrene (PS) and PDMS and differentiation was facilitated after 5 days by media exchange. Differences in cell morphology were observed between cells cultured on TCPS and PDMS substrates. Fully differentiated myotubes were observed in highest numbers on TCPS substrates and were non-detectable on PDMS substrates in the time frame of 144 h. Muscle cell alignment and their differentiation followed along the grating patterns on PS and elongated along the pattern length. On the other hand, on PDMS cells formed sheets of tissue and peeled from the substrate. We have revealed the potential for the combinations of surface materials and topography on cell behavior to induce accelerated differentiation and coordinated alignment. The results demonstrate that culture environment can be designed or engineered to modify or regulate muscle cell functions.

Micro-patterning of polymer-based optical oxygen sensors for lab-on-chip applications

Soft-lithography and plasma etching with reactive ions were used to fabricate a polymer microfluidic cell-culture bioreactor with integrated optical oxygen sensor. Platinum (II) octaethylporphyrin ketone (PtOEPK) suspended in a microporous polystyrene (PS) matrix was spin-coated to form sensor films of variable thickness from 1.1 μm to 400 nm on glass substrates. Sensor films were found to be smooth and well adhered. Arbitrary patterns with a minimum feature size of 25 μm could be routinely replicated in the PtOEPK/PS layer using polydimethylsiloxane (PDMS) elastomer stamps as etch masks in a reactive ion etcher. No effect of plasma patterning and sensor integration by plasma bonding on the sensor signal could be observed. Detection of different gaseous and dissolved oxygen concentrations with the patterned sensor followed linear Stern-Volmer behavior. Dynamic measurement of sensor intensity as a function of different oxygen concentration showed good reproducibility and a nearly instantaneous response to gas changes. For gaseous and dissolved oxygen measurement with a patterned 400 nm thick film I0/I100 ratios of 3.2 and 2.7 were found, respectively.

An integrated platform enabling optogenetic illumination of Caenorhabditis elegans neurons and muscular force measurement in microstructured environments

Optogenetics has been recently applied to manipulate the neural circuits of Caenorhabditis elegans (C. elegans) to investigate its mechanosensation and locomotive behavior, which is a fundamental topic in model biology. In most neuron-related research, free C. elegans moves on an open area such as agar surface. However, this simple environment is different from the soil, in which C. elegans naturally dwells. To bridge up the gap, this paper presents integration of optogenetic illumination of C. elegans neural circuits and muscular force measurement in a structured microfluidic chip mimicking the C. elegans soil habitat. The microfluidic chip is essentially a ∼1 × 1 cm(2) elastomeric polydimethylsiloxane micro-pillar array, configured in either form of lattice (LC) or honeycomb (HC) to mimic the environment in which the worm dwells. The integrated system has four key modules for illumination pattern generation, pattern projection, automatic tracking of the worm, and force measurement. Specifically, two optical pathways co-exist in an inverted microscope, including built-in bright-field illumination for worm tracking and pattern generation, and added-in optogenetic illumination for pattern projection onto the worm body segment. The behavior of a freely moving worm in the chip under optogenetic manipulation can be recorded for off-line force measurements. Using wild-type N2 C. elegans, we demonstrated optical illumination of C. elegans neurons by projecting light onto its head/tail segment at 14 Hz refresh frequency. We also measured the force and observed three representative locomotion patterns of forward movement, reversal, and omega turn for LC and HC configurations. Being capable of stimulating or inhibiting worm neurons and simultaneously measuring the thrust force, this enabling platform would offer new insights into the correlation between neurons and locomotive behaviors of the nematode under a complex environment.

Fabrication of polymeric substrates with micro- and nanoscale topography bioimprinted at progressive cell morphologies

This work introduces a novel process for the fabrication of polymer cell culture substrates containing physical topography based on timepoint specific cell phenotype replicas. Bioimprinting of human nasal chondrocyte at different cell adhesion time points was used to demonstrate the nanoscale replication process. Atomic force microscopy confirmed morphology progression at 1, 6, 12, and 24 h timepoints corresponding to dedifferentiation of the chondrocytes to fibroblast-like phenotype. Topographical analysis of the imprinted substrates yielded an inverse relationship between surface coverage, increasing from 11% to 87%, and maximum average pattern depth, decreasing from 1.2 μm to 430 nm. Methacrylate bioimprint features were successively replicated into a transitional polydmethylsiloxane mold used as an intermediate for further replication into polystyrene by a high-throughput embossing method. Substrates fabricated with this process have applications in stem cell engineering, regenerative medicine, and im...