Jost Goettert

Fabrication and Preliminary Results for LiGA Fabricated Nickel Micro Gas Chromatograph Columns

High aspect ratio nickel microfluidic columns were fabricated using the LiGA technique. The 2-m-long 50-mum-wide high aspect ratio columns will be the separation component of a handheld gas chromatograph device for detecting semivolatile and volatile compounds. As a first step, 600-mum-deep electrodeposited nickel columns were fabricated. The serpentine columns were sealed and pressure-flow rate characteristics compared with the theoretical values. The response of the sealed columns was studied by running methane gas plugs through uncoated columns with a flame ionization detector at the exit. Negligible flow-induced dispersion was observed in the sealed metal columns. Unretained peak widths of ~15 ms were measured, and the experimental pressure and flow rate distributions matched those predicted by established analytical models within plusmn2.5%. Columns were coated with OV-1 stationary phase using static coating methods. A mixture of four hydrocarbons C6, C8, C10, and C12 was separated in a coated 50 mum by 600 mum by 0.5 m column in less than 2 s at 70 degC

Simple replication methods for producing nanoslits in thermoplastics and the transport dynamics of double-stranded DNA through these slits

Mixed-scale nano- and microfluidic networks were fabricated in thermoplastics using simple and robust methods that did not require the use of sophisticated equipment to produce the nanostructures. High-precision micromilling (HPMM) and photolithography were used to generate mixed-scale molding tools that were subsequently used for producing fluidic networks into thermoplastics such as poly(methyl methacrylate), PMMA, cyclic olefin copolymer, COC, and polycarbonate, PC. Nanoslit arrays were imprinted into the polymer using a nanoimprinting tool, which was composed of an optical mask with patterns that were 2-7 µm in width and a depth defined by the Cr layer (100 nm), which was deposited onto glass. The device also contained a microchannel network that was hot embossed into the polymer substrate using a metal molding tool prepared via HPMM. The mixed-scale device could also be used as a master to produce a polymer stamp, which was made from polydimethylsiloxane, PDMS, and used to generate the mixed-scale fluidic network in a single step. Thermal fusion bonding of the cover plate to the substrate at a temperature below their respective T(g) was accomplished by oxygen plasma treatment of both the substrate and cover plate, which significantly reduced thermally induced structural deformation during assembly: ∼6% for PMMA and ∼9% for COC nanoslits. The electrokinetic transport properties of double-stranded DNA (dsDNA) through the polymeric nanoslits (PMMA and COC) were carried out. In these polymer devices, the dsDNA demonstrated a field-dependent electrophoretic mobility with intermittent transport dynamics. DNA mobilities were found to be 8.2 ± 0.7 × 10(-4) cm(2) V(-1) s(-1) and 7.6 ± 0.6 × 10(-4) cm(2) V(-1) s(-1) for PMMA and COC, respectively, at a field strength of 25 V cm(-1). The extension factors for λ-DNA were 0.46 in PMMA and 0.53 in COC for the nanoslits (2-6% standard deviation).

Fabrication of a cyclic olefin copolymer planar waveguide embedded in a multi-channel poly(methyl methacrylate) fluidic chip for evanescence excitation

The fabrication and characterization of a novel cyclic olefin copolymer (COC) waveguide embedded in a poly(methyl methacrylate), PMMA, fluidic chip configured in a multi-channel format with an integrated monolithic prism for evanescent fluorescence excitation are reported. The fabrication approach allowed the embedded waveguide to be situated orthogonal to a series of fluidic channels within the PMMA wafer to sample fluorescent solutions in these channels using the evanescence properties of the waveguide. Construction of the device was achieved using several fabrication techniques including high precision micromilling, hot embossing and stenciling of a polymer melt to form the waveguide and coupling prism. A waveguide channel was fabricated in the fluidic chips cover plate, also made from PMMA, and was loaded with a COC solution using a pre-cast poly(dimethylsiloxane), PDMS, stencil containing a prism-shaped recess. The PMMA substrate contained multiple channels (100 microm wide x 30 microm deep with a pitch of 100 microm) that were situated orthogonal to the waveguide to allow penetration of the evanescent field into the sampling solution. The optical properties of the waveguide in terms of its transmission properties and penetration depth of the evanescent field in the adjacent solution were evaluated. Finally, the device was used for laser-induced fluorescence evanescent excitation of a dye solution hydrodynamically flowing through multiple microfluidic channels in the chip and processed using a microscope equipped with a charge-coupled device (CCD) for parallel readout. The device and optical system were able to image 11 channels simultaneously with a limit-of-detection of 7.1 x 10(-20) mol at a signal-to-noise ratio of 2. The waveguide was simple to manufacture and could be scaled to illuminate much higher channel numbers making it appropriate for high-throughput measurements using evanescent excitation.

Fully Integrated Thermoplastic Genosensor for the Highly Sensitive Detection and Identification of Multi‐Drug‐Resistant Tuberculosis

Infectious diseases are a major global health burden accounting for approximately 15 million deaths annually, many from drug resistant pathogenic agents, with a significant number of cases occurring in developing countries.[1–7] In particular, the resurgence of tuberculosis (TB) has been accompanied by the rapid spread of multi-drug resistance TB (MDR-TB) resulting from Mycobacterium tuberculosis (Mtb) strains that fail to respond to the first-line drugs, rifampin and isoniazid. Currently, <5% of ~0.5 million MDR-TB cases estimated globally are appropriately diagnosed and treated due in part to the long assay turnaround time associated with conventional culture-based drug susceptibility testing.[8]

Use of protein cross-linking and radiolytic footprinting to elucidate PsbP and PsbQ interactions within higher plant Photosystem II

Significance In higher plant Photosystem II, the PsbP and PsbQ proteins provide critical support for oxygen evolution at physiological calcium and chloride concentrations. The locations of these components within the photosystem, however, are unclear. Our findings that (i) the N terminus of PsbP, which is unresolved in the current high-resolution structure of this subunit, forms a compact structure and associates with the C-terminal domain of the protein and (ii) PsbP and PsbQ directly interact to form a framework for understanding the organization of these subunits within the higher plant photosystem. Protein cross-linking and radiolytic footprinting coupled with high-resolution mass spectrometry were used to examine the structure of PsbP and PsbQ when they are bound to Photosystem II. In its bound state, the N-terminal 15-amino-acid residue domain of PsbP, which is unresolved in current crystal structures, interacts with domains in the C terminus of the protein. These interactions may serve to stabilize the structure of the N terminus and may facilitate PsbP binding and function. These interactions place strong structural constraints on the organization of PsbP when associated with the Photosystem II complex. Additionally, amino acid residues in the structurally unresolved loop 3A domain of PsbP (90K–107V), 93Y and 96K, are in close proximity (≤11.4 Å) to the N-terminal 1E residue of PsbQ. These findings are the first, to our knowledge, to identify a putative region of interaction between these two components. Cross-linked domains within PsbQ were also identified, indicating that two PsbQ molecules can interact in higher plants in a manner similar to that observed by Liu et al. [(2014) Proc Natl Acad Sci 111(12):4638–4643] in cyanobacterial Photosystem II. This interaction is consistent with either intra-Photosystem II dimer or inter-Photosystem II dimer models in higher plants. Finally, OH• produced by synchrotron radiolysis of water was used to oxidatively modify surface residues on PsbP and PsbQ. Domains on the surface of both protein subunits were resistant to modification, indicating that they were shielded from water and appear to define buried regions that are in contact with other Photosystem II components.

Size evolution of gold nanoparticles in a millifluidic reactor.

The size evolution of gold nanoparticles in a millifluidic reactor is investigated using spatially resolved transmission electron microscopy (TEM). The experimental data is supported by numerical simulations, carried out to study the residence-time distribution (RTD) of tracers that have the same properties as Au ions. Size and size distribution of the particles within the channels are influenced by the mixing zones as well as the RTD. However, the Au nanoparticles obtained show a broader size distribution even at the shortest investigated residence time of 3.53 s, indicating that in addition to surface growth reaction kinetics also plays an important role. The comparison of time resolved particle growth within the millifluidic channel with flask-based reactions reveals that the particle size can be controlled better within millifluidic channels. Overall, the results indicate potential opportunities to utilize easy to fabricate millifluidic reactors for the synthesis of nanoparticles, as well as as for carrying out time resolved kinetic studies.

Tensile, creep and fatigue properties of LIGA nickel structures

Elevated temperature tensile, creep and high cycle fatigue properties of electro-deposited LIGA Ni structures have been measured and are being used to predict the reliability of LIGA based MEMS structures. Microsamples with dimensions of 100s of microns have been LIGA fabricated and characterized in terms of their elevated temperature tensile and creep strength and their high-cycle fatigue performance. The strength of these LIGA Ni structures was found to decrease dramatically at temperatures above 200/spl deg/C. At stresses significantly below the yield strength, substantial creep deformation was also observed at moderately elevated temperatures. The fatigue life of the LIGA Ni microsamples increased with decreasing stress amplitude in a manner comparable to what has been reported for wrought Ni. An apparent fatigue limit was observed for the LIGA Ni microsamples, but the importance of component geometry on the fatigue life was also highlighted.

SU-8-based deep x-ray lithography/LIGA

Poly-methylmethacrylate (PMMA), a positive resist, is the most commonly used resist for deep X-ray lithography (DXRL)/LIGA technology. Although PMMA offers superior quality with respect to accuracy and sidewall roughness but it is also extremely insensitive. In this paper, we present our research results on SU-8 as negative resist for deep X-ray lithography. The results show that SU-8 is over two order of magnitude more sensitive to X-ray radiation than PMMA and the accuracy of the SU-8 microstructures fabricated by deep X-ray lithography is superior to UV-lithography and comparable to PMMA structures. The good pattern quality together with the high sensitivity offers rapid prototyping and direct LIGA capability. Moreover, the combinational use of UV and X-ray lithography as well as the use of positive and negative resists made it possible to fabricate complex multi-level 3D microstructures. The new process can be used to fabricate complex multi-level 3D structures for MEMS, MOEMS, Bio-MEMS or other micro-devices.

Radiolytic Mapping of Solvent-Contact Surfaces in Photosystem II of Higher Plants EXPERIMENTAL IDENTIFICATION OF PUTATIVE WATER CHANNELS WITHIN THE PHOTOSYSTEM

Background: Substrate water must reach the buried Mn4O5Ca cluster in Photosystem II. Results: OH• produced by radiolysis modified buried amino acid residues. These were mapped onto the PS II crystal structure. Conclusion: Two groups of oxidized residues were identified which form putative pathways to the Mn4O5Ca cluster. Significance: Identification of water and oxygen channels is crucial for our understanding of Photosystem II function. Photosystem II uses water as an enzymatic substrate. It has been hypothesized that this water is vectored to the active site for water oxidation via water channels that lead from the surface of the protein complex to the Mn4O5Ca metal cluster. The radiolysis of water by synchrotron radiation produces amino acid residue-modifying OH• and is a powerful technique to identify regions of proteins that are in contact with water. In this study, we have used this technique to oxidatively modify buried amino acid residues in higher plant Photosystem II membranes. Fourier transform ion cyclotron resonance mass spectrometry was then used to identify these oxidized amino acid residues that were located in several core Photosystem II subunits (D1, D2, CP43, and CP47). While, as expected, the majority of the identified oxidized residues (≈75%) are located on the solvent-exposed surface of the complex, a number of buried residues on these proteins were also modified. These residues form groups which appear to lead from the surface of the complex to the Mn4O5Ca cluster. These residues may be in contact with putative water channels in the photosystem. These results are discussed within the context of a number of largely computational studies that have identified putative water channels in Photosystem II.

Polymer-based microfluidic devices for biomedical applications

Two types of Microfluidic bioanalytical systems were designed and fabricated in polymer substrates using the LIGA process. A continuous flow polymerase chain reaction (CFPCR) Microfluidic device was fabricated in polycarbonate (PC), which utilized isothermal zone and shuttling the sample through each zone to achieve amplification. A 20-cycle PCR amplification of a fragment of a plasmid DNA template was achieved in 5.3 min. The results were comparable to those obtained in commercial laboratory-scale PCR system. The second system consisted of a microchip contating a low-density array assembled into the Microfluidic channel, which was hot-embossed in poly(methyl methacrylate) (PMMA). The detection of low-abundant mutations in gene fragments (K-ras) that carry point mutations with high diagnostic value for colorectal cancer was successfully performed. The array accessed microfluidics in order to enhance the kinetic associated with hybridization.