Rodolphe Marie, Jonas N. Pedersen, David L. V. Bauer, Kristian H. Rasmussen, Mohammed Yusuf, Emanuela Volpi, Henrik Flyvbjerg, Anders Kristensen, and Kalim U. Mir, Integrated view of genome structure and sequence of a single DNA molecule in a nanofluidic device, PNAS, (March 2013).
Abstract: We show how a bird’s-eye view of genomic structure can be obtained at ∼1-kb resolution from long (∼2 Mb) DNA molecules extracted from whole chromosomes in a nanofluidic laboratory-on-a-chip. We use an improved single-molecule denaturation mapping approach to detect repetitive elements and known as well as unique structural variation. Following its mapping, a molecule of interest was rescued from the chip; amplified and localized to a chromosome by FISH; and interrogated down to 1-bp resolution with a commercial sequencer, thereby reconciling haplotype-phased chromosome substructure with sequence.
Peter Friis Østergaard, Marco Matteucci, Walter Reisner, and Rafael Taboryski, DNA Barcoding via Counterstaining with AT/GC Sensitive Ligands in Injection-Molded All-Polymer Nanochannel Devices, Analyst, 138(4):1249-55 (February 2013).
Abstract: Nanochannel technology, coupled with a suitable DNA labeling chemistry, is a powerful approach for performing high-throughput single-molecule mapping of genomes. Yet so far nanochannel technology has remained inaccessible to the broader research community due to high fabrication cost and/or requirement of specialized facilities/skill-sets. In this article we show that nanochannel-based mapping can be performed in all polymer chips fabricated via injection molding: a fabrication process so inexpensive that the devices can be considered disposable. Fluorescent intensity variations can be obtained from molecules extended in the polymer nanochannels via chemical counterstaining against YOYO-1. In particular, we demonstrate that the counterstaining induced fluorescent intensity variations to a large degree appear to be proportional to the theoretically computed sequence-maps of both local AT and GC variation along DNA sequences.
M. Matteucci, T. L. Christiansena, S. Tanzia, P. F. Østergaard, and R. Taboryskia, Fabrication and characterization of injection molded multi level nano and microfluidic systems, Microelectronic engineering, (February 2013).
Abstract: We here present a method for fabrication of multi-level all-polymer chips by means of silicon dry etching, electroplating and injection molding. This method was used for successful fabrication of microfluidic chips for applications in the fields of electrochemistry, cell trapping and DNA elongation. These chips incorporate channel depths in the range between 100 nm and 100 lm and depth to width aspect ratios between 1/200 and 2. Optimization of the sealing process of all-polymer COC microfluidic chips by means of thermal bonding is also presented. The latter includes comparing the bonding strength of UV-treated foils and presentation of a simple model for estimating the delamination pressure. With UV surface treatments, foils of 100 lm thickness were found to withstand pressures up to 9 atm in Ø4 mm cylindrical inlets when thermally bonded to micropatterned substrates of 2 mm thickness.
David L. V. Bauer, Rodolphe Marie, Kristian H. Rasmussen, Anders Kristensen, and Kalim U. Mir, DNA catenation maintains structure of human metaphase chromosomes, Nucleic Acids Research, 2012, 1-7, (September 2012).
Abstract: Nanochannel technology, coupled with a suitable DNA labeling chemistry, is a powerful approach for performing high-throughput single-molecule mapping of genomes. Yet so far nanochannel technology has remained inaccessible to the broader research community due to high fabrication cost and/or requirement of specialized facilities/skill-sets. In this article we show that nanochannel-based mapping can be performed in all polymer chips fabricated via injection molding: a fabrication process so inexpensive that the devices can be considered disposable. Fluorescent intensity variations can be obtained from molecules extended in the polymer nanochannels via chemical counterstaining against YOYO-1. In particular, we demonstrate that the counterstaining induced fluorescent intensity variations to a large degree appear to be proportional to the theoretically computed sequence-maps of both local AT and GC variation along DNA sequences.
Simon T. Larsen, Michael L. Heien, and Rafael Taboryski: Amperometric Noise at Thin Film Band Electrodes, accepted for publication by Analytical Chemistry, Anal. Chem. 84 (2012) 7744-7749, (August 2012).
Abstract: Background current noise is often a significant limitation when using constant-potential amperometry for biosensor application such as amperometric recordings of transmitter release from single cells through exocytosis. In this paper, we fabricated thin-film electrodes of gold and conductive polymers and measured the current noise in physiological buffer solution for a wide range of different electrode areas. The noise measurements could be modeled by an analytical expression, representing the electrochemical cell as a resistor and capacitor in series. The studies revealed three domains; for electrodes with low capacitance, the amplifier noise dominated, for electrodes with large capacitances, the noise from the resistance of the electrochemical cell was ominant, while in the intermediate region, the current noise scaled with electrode capacitance. The experimental results and the model presented here can be used for choosing an electrode material and dimensions and when designing chip-based devices for low-noise current measurements.
Rodolphe Marie, and Anders Kristensen, Nanofluidic devices towards single DNA molecule sequence Mapping, Journal of Biophotonics, (July 2012).
Abstract: Nanofluidics enables the imaging of stretched single molecules with potential applications for single molecule sequence mapping. Lab-on-a-chip devices for single cell trapping and lyzing, genomic DNA extraction from single cells, and optical mapping of genomic length DNA has been demonstrated separately. Yet the pursuit for applying DNA optical mapping to solve real genomics challenges is still to come. We review lab-on-a-chip devices from literature that could be part of a complete system for the sequence mapping of single DNA molecules.
Robert L. Welch, Robert Sladek, Ken Dewar, and Walter W. Reisner, Denaturation Mapping of Saccharomyces cerevisiae, Lab Chip. 12(18):3314-21, (July 2012).
Abstract: Optical mapping of DNA provides large-scale genomic information that can be used to assemble contigs from next-generation sequencing, and to detect rearrangements between single cells. A recent optical mapping technique called denaturation mapping has the advantage of using physical principles rather than the action of enzymes to probe genomic structure. Denaturation mapping uses fluorescence microscopy to image the pattern of partial melting along a DNA molecule extended in a channel of cross-section 120 nm at the heart of a nanofluidic device. We used denaturation mapping to locate single DNA molecules on the yeast genome (12.1 Mbp) by comparing images to a computationally predicted map for the entire genome sequence. By locating 84 molecules we assembled an optical map of the yeast genome with > 50% coverage.
Simon T. Larsen, Richard F. Vreeland, Michael L. Heien, and Rafael Taboryski: Characterization of Poly(3,4-ethylenedioxythiophene):tosylate conductive polymer microelectrodes for transmitter detection, Analyst. 137 (2012) 1831-1836. ,(January 2012).
Abstract: In this paper we investigate the physical and electrochemical properties of micropatterned Poly(3,4-ethylenedioxythiophene):tosylate (Pedot:tosylate) microelectrodes for neurochemical detection. Pedot:tosylate is a promising conductive polymer electrode material for chip-based bioanalytical applications such as capillary electrophoresis, high-performance liquid chromatography, and constant potential amperometry at living cells. Band electrodes with widths down to 3 µm where fabricated on polymer substrates using UV lithographic methods. The electrodes are electrochemically stable in a range between -200 mV and 700 mV vs. Ag/AgCl and show a relatively low resistance. A wide range of transmitters are shown to oxidize readily on the electrodes. Kinetic rate constants and half wave potentials are reported. The capacitance pr area was found to be high 1670 ± 130 µF/cm2 compared to other thin film microelectrode materials. Finally, we use constant potential amperometry to measure the release of transmitters from a group of PC 12 cells. The results show how the current response decreases for a series of stimulations with high K+ buffer.
Simone Tanzi, Peter Friis Østergaard, Marco Matteucci, Thomas Lehrmann Christiansen, Jiri Cech, Rodolphe Marie, and Rafael Taboryski: Fabrication of combined-scale nano- and microfluidic polymer systems using a multilevel dry etching, electroplating and molding process, Journal of Micromechanics and Microengineering, 22 (11) 115008, (2012).
Abstract: Microfabricated single-cell capture and DNA stretching devices have been produced by injection molding. The fabrication scheme employed deep reactive ion etching in a silicon substrate, electroplating in nickel and molding in cyclic olefin polymer. This work proposes technical solutions to fabrication challenges associated with chip sealing and demolding of polymer high-volume replication methods. UV-assisted thermal bonding was found to ensure a strong seal of the microstructures in the molded part without altering the geometry of the channels. In the DNA stretching device, a low aspect ratio nanoslit (1/200) connecting two larger micro-channels was used to stretch a 168.5 kbp DNA molecule, while in the other device single-HeLa cells were captured against a micro-aperture connecting two larger microfluidic channels. Different dry etching processes have been investigated for the master origination of the cell-capture device. The combination of a modified Bosch process and an isotropic polysilicon etch was found to ensure the ease of demolding by resulting in slightly positively tapered sidewalls with negligible undercut at the mask interface.
Simon T. Larsen, and Rafael Taboryski: All polymer chip for amperometric studies of transmitter release from large groups of neuronal cells, Analyst, 137, 5057, (2012).
Abstract: We present an all polymer electrochemical chip for simple detection of transmitter release from large groups of cultured PC 12 cells. Conductive polymer PEDOT:tosylate microelectrodes were used together with constant potential amperometry to obtain easy-to-analyze oxidation signals from potassium-induced release of transmitter molecules. The nature of the resulting current peaks is discussed, and the time for restoring transmitter reservoirs is studied. The relationship between released transmitters and potassium concentration was found to fit to a sigmoidal dose–response curve. Finally, we demonstrate how the presented device can be used for simple drug screening purposes, by measuring the increase of transmitter release due to short-term treatment with L-DOPA.
Conferences
Thor Christian Hobæk, Henrik Pranov, Inge Marie Svane, and Niels Bent Larsen: Micro- and Nanopatterning Polymers for On-Chip Cell Selection in Toulouse France (September 2012).
Anders Kristensen, Christopher James Lüscher, Anil Thilsted, Johan Eriksen, Lasse Højlund Thamdrup, Jonas Nyvold Pedersen, Henrik Flyvbjerg, and Rodolphe Marie: Optothermally Actuated Nanofluidics for DNA Analysis in Toulouse France (September 2012).
M. Calaon, H.N. Hansen, G. Tosello, Jørgen Garnæs, C. Ravn, and P.T. Tang: Characterization of large area nanostructured surfaces using AFM measurements (June 2012).
Abstract: A surface characterisation study has been developed to validate an innovative tool making solution for nano patterning large areas via anodizing of aluminium (Al) and subsequent nickel electroforming. A surface topography characterization through atomic force microscopy (AFM) indicated a decreased magnitude of the 3D surface amplitude parameters chosen for the analysis, when increasing the Al purity from 99,5% to 99,999%. AFM was then employed to evaluate the periodical arrangements of the nano structured cells. Image processing was used to estimate the average areas value, the height variation relative to an average plane and the coefficient of variation of the fitted features curvature radius.
M. Calaon, G. Tosello, H. N. Hansen, C. Ravn, and A. Islam: 1, Packing parameters effect on injection molding of polypropylene nanostructured surfaces in Florida, USA. (April 2012).
Abstract: In today´s industry, applications involving surface patterning of sub-μm to nanometer scale structures have shown a high growth potential. To investigate the injection molding capability of replicating sub-μm surface texture on a large scale area, a 30x80 mm2 tool insert with surface structures having a diameter of 500 nm was employed. The tool insert surface was produced using chemical-based-batch techniques such aluminum anodization and nickel electroplating. During the injection molding process, polypropylene (PP) was employed as material and packing phase parameters (packing time, packing pressure) were investigated. The replicated surface topographies were quantitatively characterized by atomic force microscopy using specific three-dimensional surface parameters and qualitatively inspected by scanning electron microscopy. Results showed that the degree of replication from the toll to the polymer part was mainly influenced by packing pressure level and distance from the gate
Peter Østergaard, Marco Mateucci, Rodolphe Marie, Anders Kristensen, and Rafael Taboryski: All polymer, injection molded nanoslits, fabricated through two-level UV-LIGA processes in California, USA, (2012).
Simon Tylsgaard Larsen, Marco Matteucci, and Rafael Taboryski: Conductive Polymer Microelectrodes for on-chip measurement of transmitter release from living cells in California, USA, (2012).