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

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 ((90)K-(107)V), (93)Y and (96)K, are in close proximity (≤ 11.4 Å) to the N-terminal (1)E 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.

Use of Protein Cross-Linking and Radiolytic Labeling To Elucidate the Structure of PsbO within Higher-Plant Photosystem II.

We have used protein cross-linking with the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and radiolytic footprinting coupled with high-resolution tandem mass spectrometry, to examine the structure of higher-plant PsbO when it is bound to Photosystem II. Twenty intramolecular cross-linked residue pairs were identified. On the basis of this cross-linking data, spinach PsbO was modeled using the Thermosynechococcus vulcanus PsbO structure as a template, with the cross-linking distance constraints incorporated using the MODELLER program. Our model of higher-plant PsbO identifies several differences between the spinach and cyanobacterial proteins. The N-terminal region is particularly interesting, as this region has been suggested to be important for oxygen evolution and for the specific binding of PsbO to Photosystem II. Additionally, using radiolytic mapping, we have identified regions on spinach PsbO that are shielded from the bulk solvent. These domains may represent regions on PsbO that interact with other components, as yet unidentified, of the photosystem.

Radiolytic mapping of solvent-contact surfaces in Photosystem II of higher plants: experimental identification of putative water channels within the photosystem.

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.

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.

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 chip's 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.

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).

Hybrid integration of injector and detector functions for microchip gas chromatography.

Hybrid microchips containing high aspect ratio gas chromatograph (GC) columns with an integrated on-chip split injection and a flame ionization detector were developed. Two different column configurations, spiral and serpentine, both 1 m long by 50 µm wide and 500 µm tall, were fabricated out of electrodeposited nickel. The hybrid chip allowed injection plugs on the order of 1-2 ms, which lowered the height equivalent to theoretical plates (HETP) and allowed a comparison of system level band broadening between the two column configurations. The gas phase band broadening was estimated by measuring the flow characteristics and peak broadening of an unretained compound, and the results were compared with kinetic models. Experimental results show that both spiral and serpentine column layouts had similar flow and band broadening, suggesting that gas phase band broadening may be independent of column layout. The necessity for narrow injection bands for fast micro-chip chromatographic analysis was demonstrated, which emphasized the importance of component integration in designing powerful micro-analytical systems.

Microfluidic devices fabricated in poly(methyl methacrylate) using hot-embossing with integrated sampling capillary and fiber optics for fluorescence detection.

High-aspect-ratio microstructures have been prepared using hot-embossing techniques in poly(methyl methacrylate) (PMMA) from Ni-based molding dies prepared using LIGA (Lithographie, Galvanoformung, Abformung). Due to the small amount of mask undercutting associated with X-ray lithography and the high energy X-ray beam used during photoresist patterning, deep structures with sharp and smooth sidewalls have been prepared. The Ni-electroforms produced devices with minimal replication errors using hot-embossing at a turn around time of approximately 5 min per device. In addition, several different polymers (with different glass transition temperatures) could be effectively molded with these Ni-electroforms and many devices (>300) molded with the same master without any noticeable degradation. The PMMA devices consisted of deep and narrow channels for insertion of a capillary for the automated electrokinetic loading of sample into the microfluidic device and also, a pair of optical fibers for shuttling laser light to the detection zone and collecting the resulting emission for fluorescence analysis. Electrophoretic separations of double-stranded DNA ladders Phi X174 digested with Hae III) were performed with fluorescence detection accomplished using near-IR excitation. It was found that the narrow width of the channels did not contribute significantly to electrophoretic zone broadening and the plate numbers generated in the extended length separation channel allowed sorting of the 271/281 base pair fragments associated with this sizing ladder when electrophoresed in methylcellulose entangled polymer solutions. The dual fiber detector produced sub-attomole detection limits with the entire detector, including laser source, electronics and photon transducer, situated in a single box measuring 3'' x 10" x 14".

Polymer microfluidic chips with integrated waveguides for reading microarrays.

A microfluidic chip with an integrated planar waveguide was fabricated in poly(methyl methacrylate), PMMA, using a single-step, double-sided hot-embossing approach. The waveguide was embedded in air on three sides, the solution being interrogated on the fourth. DNA probes were covalently attached to the waveguide surface by plasma activating the PMMA and the use of carbodiimide coupling chemistry. Successful hybridization events were read using evanescent excitation monitored by an imaging microscope, which offered high spatial resolution (2 microm) and a large field-of-view (20 mm diameter field-of-view), providing imaging of the entire array without scanning. The application of the microfluidic/waveguide assembly was demonstrated by detecting low abundant point mutations; insertion C mutations in BRCA1 genes associated with breast cancer were analyzed using a universal array coupled to an allele-specific ligation assay. DNA probes consisting of amine-terminated oligonucleotides were printed inside the microfluidic channel using a noncontact microspotter. Mutant and wild-type genomic DNAs of BRCA1 were PCR (polymerase chain reaction) amplified, with the amplicons subjected to ligation detection reactions (LDRs). LDR solutions were allowed to flow over the microarray positioned on the polymer waveguide with successful ligation events discerned through fluorescence signatures present at certain locations of the array. The microfluidic/waveguide assembly could detect polymorphisms present at <1% of the total DNA content.

Multichannel microchip electrophoresis device fabricated in polycarbonate with an integrated contact conductivity sensor array.

A 16-channel microfluidic chip with an integrated contact conductivity sensor array is presented. The microfluidic network consisted of 16 separation channels that were hot-embossed into polycarbonate (PC) using a high-precision micromilled metal master. All channels were 40 microm deep and 60 microm wide with an effective separation length of 40 mm. A gold (Au) sensor array was lithographically patterned onto a PC cover plate and assembled to the fluidic chip via thermal bonding in such a way that a pair of Au microelectrodes (60 microm wide with a 5 microm spacing) was incorporated into each of the 16 channels and served as independent contact conductivity detectors. The spacing between the corresponding fluidic reservoirs for each separation channel was set to 9 mm, which allowed for loading samples and buffers to all 40 reservoirs situated on the microchip in only five pipetting steps using an 8-channel pipettor. A printed circuit board (PCB) with platinum (Pt) wires was used to distribute the electrophoresis high-voltage to all reservoirs situated on the fluidic chip. Another PCB was used for collecting the conductivity signals from the patterned Au microelectrodes. The device performance was evaluated using microchip capillary zone electrophoresis (mu-CZE) of amino acid, peptide, and protein mixtures as well as oligonucleotides that were separated via microchip capillary electrochromatography (mu-CEC). The separations were performed with an electric field (E) of 90 V/cm and were completed in less than 4 min in all cases. The conductivity detection was carried out using a bipolar pulse voltage waveform with a pulse amplitude of +/-0.6 V and a frequency of 6.0 kHz. The conductivity sensor array concentration limit of detection (SNR = 3) was determined to be 7.1 microM for alanine. The separation efficiency was found to be 6.4 x 10(4), 2.0 x 10(3), 4.8 x 10(3), and 3.4 x 10(2) plates for the mu-CEC of the oligonucleotides and mu-CZE of the amino acids, peptides, and proteins, respectively, with an average channel-to-channel migration time reproducibility of 2.8%. The average resolution obtained for mu-CEC of the oligonucleotides and mu-CZE of the amino acids, peptides, and proteins was 4.6, 1.0, 0.9, and 1.0, respectively. To the best of our knowledge, this report is the first to describe a multichannel microchip electrophoresis device with integrated contact conductivity sensor array.