Monoliths for microfluidic devices in proteomics

We report here on the preparation of monolithic capillary columns in view to their integration in a microsystem for on-chip sample preparation before their on-line analysis by electrospray and mass spectrometry (ESI-MS). These monolithic columns are based on polymer materials and consist of reverse phases for peptide separation and/or desalting. They were prepared using lauryl methacrylate (LMA), ethylene dimethacrylate (EDMA) as well as a suitable porogenic mixture composed of cyclohexanol and ethylene glycol. The resulting stationary phases present thus a C12-functionality. The LMA-based columns were first prepared in a capillary format using capillary tubing of 75 microm i.d. and tested in nanoLC-MS experiments for the separation of a commercial Cytochrome C digest composed of 12 peptidic fragments whose isoelectric point values and hydrophobic character cover a wide range. The LMA-based columns were capable of separating the peptidic fragments and their performances were seen to be similar as those of standard commercial columns dedicated to proteomic purposes with calculated separation efficiencies up to 145 x 10(3) plates/m. Monolithic LMA-based phases were then successfully polymerized in microchannels fabricated using the negative photoresist SU-8. After the polymerization, the systems were seen to withstand the pressures applied during the nanoLC-MS separation tests that were carried out in the same conditions as for the monolithic capillary columns. The pressure drop during these tests of the in-microchannel monoliths was as high as 50 bar; however, the separation was not as good as for a capillary format which could be accounted for by the monolith dimensions.     

Contoured elastic-membrane microvalves for microfluidic network integration

A contoured elastic-membrane microvalve is presented that enables integrated microfluidic processing at the network level. This method takes advantage of two ideas to improve performance: flexible elastic membranes (which enable high-performance shutoff and reduced footprint), and three-dimensionally contoured valve geometries (which reduce dead volume, improve fluidic priming, and reduce susceptibility to cavitation at high fluid velocities). We describe the use of laser-induced etching for microfluidic manifold fabrication, discuss the nonlinear load-deflection behavior of elastic membranes that can occur below 30 psi, and present flow-rate data for microvalves under inlet pressures of 0-20 psi with zero applied membrane pressure. Valve-closure data for inlet pressures of 0-30 psi are presented for fully assembled microvalve structures. The microvalve structures under test were capable of turning off flows of > 20 microL/s. These flow rates were shown to be limited by inlet and outlet flow resistances and not by the valve structure itself, so that higher maximum flow rate capabilities should be readily achieved.     

Non-plasma bonding of PDMS for inexpensive fabrication of microfluidic devices

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding. In some cases it may be desirable to reversibly bond devices to the Corning No. 1 cover glass. This could be due, perhaps, to a plasma cleaner not being available. In other instances, it may be desirable to remove the device from the glass after the culturing of neurons for certain types of microscopy or for immunostaining, though it is not necessary to remove the device for immunostaining since the neurons can be stained in the device. Some researchers, however, still prefer to remove the device. In this case, reversible bonding of the device to the cover glass makes that possible. There are some disadvantages to non-plasma bonding of the devices in that not as tight of a seal is formed. In some cases axons may grow under the grooves rather than through them. Also, because the glass and PDMS are hydrophobic, liquids do not readily enter the device making it necessary at times to force media and other reagents into the device. Liquids will enter the device via capillary action, but it takes significantly longer as compared to devices that have been plasma bonded. The plasma cleaner creates temporary hydrophilic charges on the glass and device that facilitate the flow of liquids through the device after bonding within seconds. For non-plasma bound devices, liquid flow through the devices takes several minutes. It is also important to note that the devices to be used with non-plasma bonding need to be sterilized first, whereas plasma treated devices do not need to be sterilized prior to use because the plasma cleaner will sterilize them.     

HELM: searching for helix motifs within protein sequences

SUMMARY: HELM is a web tool designed to automate the analysis of protein sequences searching for alpha helix motifs. This analysis can be useful in protein engineering studies, aimed at the identification of regions to be modified in order to obtain more suitable features of local and/or global stability. AVAILABILITY: The tool is available to academic and commercial institutions at the URL http://crisceb.area.na.cnr.it/angelo/ PROTEIN_TOOLS/HELM/ CONTACT: angelo@crisceb.area.na.cnr.it     

Printable microfluidic systems using pressure sensitive adhesive material for biosensing devices

Background

In biosensors with a fluid analyte, the integration of a microfluidic system, which guides the analyte into the sensing area, is critical. Quicker and economical ways to build up microfluidic systems will make point of care diagnostics viable. Printing is a low-cost technology that is increasingly used in emerging organic and flexible electronics and biosensors. In this paper, we present printed fluidic systems on flexible substrates made with pressure sensitive adhesive materials.

Methods

Printable pressure sensitive adhesive materials have been used for making microfluidic systems. Flexible substrates have been used, and two types of adhesive materials, one thermally dried and another UV curable, have been tested. Top sealing layer was laminated directly on top of the printed microfluidic structure. Flow tests were done with deionized water.

Results

Flow tests with deionized water show that both adhesive materials are suitable for capillary flow driven fluidic devices. Flow test using water as dielectric material was also done successfully on a printed electrolyte gated organic field effect transistor with an integrated microfluidic system.

General significance

Due to its ease of process and low cost, printed microfluidic system is believed to find more applications in biosensing devices. This article is part of a Special Issue entitled Organic Bioelectronics—Novel Applications in Biomedicine.     

Micro-bioreactors for fed-batch fermentations with integrated online monitoring and microfluidic devices

In this study an array of micro-bioreactors based on the format of 48-well microtiter plates (MTP) is presented. The process parameters pH-value and biomass are monitored online by a combination of different sensors, the biolector measurement technology and conductance measurements. A microfluidic device dispenses two fluids individually into each well for controlling the pH-value of fermentations. The micro-bioreactor consists of four wells and two reservoirs. In each well a polyimide foil with platinum electrodes for conductance measurements is integrated. The microfluidic device is fabricated using softlithographic techniques and utilizes pneumatically actuated microvalves. The device is able to dispense volumes below 5nl. Finally, fermentations of Escherichia coli are carried out in the micro-bioreactor system. During the fermentation, the pH-value is measured optically and the biomass development is monitored by the scattered light signal. Meanwhile, the pH-value is controlled by dispensing sodium hydroxide and phosphoric acid. This micro-bioreactor demonstrates the possibility of online monitored and pH-controlled fermentations in micro-scale. The pH-value in the uncontrolled culture varies within the range of 6.46-8.83 whereas the pH-value in the controlled cultures can be kept within 6.85-7.07. This results in an increase in biomass in the pH-controlled culture compared to the nearly completely inhibited pH-uncontrolled culture.     

Monolithic microfluidic mixing–spraying devices for time-resolved cryo-electron microscopy

The goal of time-resolved cryo-electron microscopy is to determine structural models for transient functional states of large macromolecular complexes such as ribosomes and viruses. The challenge of time-resolved cryo-electron microscopy is to rapidly mix reactants, and then, following a defined time interval, to rapidly deposit them as a thin film and freeze the sample to the vitreous state. Here we describe a methodology in which reaction components are mixed and allowed to react, and are then sprayed onto an EM grid as it is being plunged into cryogen. All steps are accomplished by a monolithic, microfabricated silicon device that incorporates a mixer, reaction channel, and pneumatic sprayer in a single chip. We have found that microdroplets produced by air atomization spread to sufficiently thin films on a millisecond time scale provided that the carbon supporting film is made suitably hydrophilic. The device incorporates two T-mixers flowing into a single channel of four butterfly-shaped mixing elements that ensure effective mixing, followed by a microfluidic reaction channel whose length can be varied to achieve the desired reaction time. The reaction channel is flanked by two ports connected to compressed humidified nitrogen gas (at 50 psi) to generate the spray. The monolithic mixer-sprayer is incorporated into a computer-controlled plunging apparatus. To test the mixing performance and the suitability of the device for preparation of biological macromolecules for cryo-EM, ribosomes and ferritin were mixed in the device and sprayed onto grids. Three-dimensional reconstructions of the ribosomes demonstrated retention of native structure, and 30S and 50S subunits were shown to be capable of reassociation into ribosomes after passage through the device.     

Plastic biochannel hybridization devices: a new concept for microfluidic DNA arrays

A xanthine oxidase hydroxyl radical (.OH)-generating system was created for sustained in vitro production of *OH. This assay was coupled with microdialysis sampling to elucidate the factors that influence microdialysis calibration during radical trapping. A *OH trapping agent, 4-hydroxybenzoic acid, was included either in the microdialysis perfusion fluid or in the medium external to the microdialysis probe. Xanthine oxidase enzymatic activity was reproducible and had an average activity measured by UV absorbance of produced uric acid of 0.037 +/- 0.005 deltaAU/min (n = 5). A considerable amount of variance in the rate and amount of the product, 3,4-dihydroxybenzoic acid (3,4-DHBA), was observed when one microdialysis probe was placed in the reaction mixture. When two microdialysis probes were placed in the reaction mixture, a greater rate and amount of 3,4-DHBA was observed. Different concentrations of 3,4-DHBA were obtained between quiescent and stirred systems.     

Affinity separations using microfabricated microfluidic devices:<Emphasis Type="Italic">In situ</Emphasis> photopolymerization and use in protein separations

The use of microfabricated microfluidic devices offers significant advantages over current technologies including fast analysis time and small reagent requirements. In the context of proteomic research, the possibility of using affinity-based separations for prefractionation of samples using microfluidic devices has significant potential. We demonstrate the use of microscale devices to achieve affinity separations of proteins using a device fabricated from borosilicate glass wafers. Photolithography and wet etching are used to pattern individual glass wafers and the wafers are fusion bonded at 650°C to obtain enclosed channels. A polymer has been successfully polymerizedin situ and used either as a frit for packing beads or, when derivatized with Cibacron Blue 3GA, as a separation matrix. Both of these technologies are based onin situ UV photopolymerization of glycidyl methacrylate (GMA) and trimethylolpropane trimethacrylate (TRIM) in channels.     

Uniform integration of genome mapping data using intersection graphs

MOTIVATION: The methods for analyzing overlap data are distinct from those for analyzing probe data, making integration of the two forms awkward. Conversion of overlap data to probe-like data elements would facilitate comparison and uniform integration of overlap data and probe data using software developed for analysis of STS data. RESULTS: We show that overlap data can be effectively converted to probe-like data elements by extracting maximal sets of mutually overlapping clones. We call these sets virtual probes, since each set determines a site in the genome corresponding to the region which is common among the clones of the set. Finding the virtual probes is equivalent to finding the maximal cliques of a graph. We modify a known maximal-clique algorithm such that it finds all virtual probes in a large dataset within minutes. We illustrate the algorithm by converting fingerprint and Alu-PCR overlap data to virtual probes. The virtual probes are then analyzed using double-linkage intersection graphs and structure graphs to show that methods designed for STS data are also applicable to overlap data represented as virtual probes. Next we show that virtual probes can produce a uniform integration of different kinds of mapping data, in particular STS probe data and fingerprint and Alu-PCR overlap data. The integrated virtual probes produce longer double-linkage contigs than STS probes alone, and in conjunction with structure graphs they facilitate the identification and elimination of anomalies. Thus, the virtual-probe technique provides: (i) a new way to examine overlap data; (ii) a basis on which to compare overlap data and probe data using the same systems and standards; and (iii) a unique and useful way to uniformly integrate overlap data with probe data.     

Facile synthesis of chain-end functionalized glycopolymers for site-specific bioconjugation

A series of derivatized arylamine initiators were used to generate chain-end functionalized glycopolymers by cyanoxyl-mediated free-radical polymerization. Significant features of this strategy include the capacity to produce polymers of low polydispersity (PDI < 1.5) under aqueous conditions using unprotected monomers bearing a wide range of functional groups. In addition, the presence of a phenyl ring simplifies calculation of polymer saccharide content and molar mass by (1)H NMR. It is particularly noteworthy, however, that derivatized arylamine initiators in conjunction with the presence of a terminal cyanate group provide a convenient approach for synthesizing polymers with a variety of distinct functional groups at alpha and omega chain ends. In the process, the capacity to label glycopolymers or otherwise conjugate them to proteins or other molecules is greatly enhanced.     

Metabolic integration of organisms within symbiotic systems

Adaptation of organisms to coexisence in symbiotic systems is usually related to significant metabolic changes resulting in the integration of the biochemical pathways of the partners. In the symbioses between plants and nitrogen-fixing organisms, between heterotrophic and autotrophic organisms, as well as between animals and microorganisms providing the consumption of plant biomass, the systems of C- and N-metabolism, controlling the utilization of various sources of nitrogen (N2, organic and inorganic compounds, metabolic waste of the host) and carbon (CO2, plant polymers), of the partners are tightly integrated. Bilateral biochemical links between partners are typical to mutualistic symbioses (wherein biotrophic nutrition predominates, in some cases including necrotrophy of secondary origin). In antagonistic symbioses, unilateral links predominate, though active assimilation of the pathogen's secondary metabolites by the host is also possible. In most mutualistic symbioses, integrated metabolic ties have derived from trophic chains in biocenoses (syntrophic consortia, "predator-prey" systems), but not from the systems where the pathogens consume host metabolites. At the same time, molecular analysis of symbiotic interactions has shown that symbioses considerably differ from biocenoses, where the cycling of nutrients and energy implies no functional integration of the partner's genes.     

Two motifs within a transmembrane domain, one for homodimerization and the other for heterodimerization

Protein assembly is a critical process involved in a wide range of cellular events and occurs through extracellular and/or transmembrane domains (TMs). Previous studies demonstrated that a GXXXG motif is crucial for homodimer formation. Here we selected the TMs of ErbB1 and ErbB2 as a model since these receptors function both as homodimers and as heterodimers. Both TMs contain two GXXXG-like motifs located at the C and N termini. The C-terminal motifs were implicated previously in homodimer formation, but the role of the N-terminal motifs was not clear. We used the ToxR system and expressed the TMs of both ErbB1 and ErbB2 containing only the N-terminal GXXXG motifs. The data revealed that the ErbB2 but not the ErbB1 construct formed homodimers. Importantly, a synthetic ErbB1 TM peptide was able to form a heterodimer with ErbB2, by displacing the ErbB2 TM homodimer. The specificity of the interaction was demonstrated by using three controls: (i) Two single mutations within the GXXXG-like motif of the ErbB1 peptide reduced or preserved its activity, in agreement with similar mutations in glycophorin A. (ii) A TM peptide of the bacterial Tar receptor did not assemble with the ErbB2 construct. (iii) The ErbB1 peptide had no effect on the dimerization of a construct containing the TM-1 domain of the Tar receptor. Fluorescence microscopy demonstrated that all the peptides localized on the membrane. Furthermore, incubation with the peptides had no effect on bacterial growth and protein expression levels. Our results suggest that the N-terminal GXXXG-like motif of the ErbB1 TM plays a role in heterodimerization with the ErbB2 transmembrane domain. To our knowledge, this is the first demonstration of a transmembrane domain with two distinct recognition motifs, one for homodimerization and the other for heterodimerization.     

Towards molecular computing: co-development of microfluidic devices and chemical reaction media

Microfluidics provides a powerful technology for both the production of molecular computing components and for the implementation of molecular computing architectures. The potential commercial applications of microfluidics drive rapid progress in this field-but at the same time focus interest on materials that are compatible with physiological aqueous conditions. For engineering applications that consider a broader range of physico-chemical conditions the narrow set of established materials for microfluidics can be a challenge. As a consequence of the large surface to volume ratio inherent in microfluidic technology the material of the device can greatly affect the chemistry in the channels of the device. In practice it is necessary to co-develop the chemical medium to be used in the device together with the microfluidic devices. We describe this process for a molecular computing architecture that makes use of excitable lipid-coated droplets of Belousov-Zhabotinsky reaction medium as its active processing components. We identify fluoropolymers with low melting temperature as a suitable substrate for microfluidics to be used in conjunction with Belousov-Zhabotinsky droplets in decane.