Pump It Up

Designers of microscale analysis chips have long drawn inspiration from cellular processes and substructures to develop sensors and miniaturized assays. Although these efforts have made clever use of biochemical reactions, cellular work in the form of physical movement has not yet been harnessed. In a recent report on the use of cultured cardiomyocytes, Tanaka et al., change all that. It is well known that cardiomyocytes pulse synchronously in culture; Tanaka et al., decided to use this spontaneous activity to move a simple pump in a microfabricated chip. The strategy is ingenious but surprisingly straightforward. The authors isolated primary neonatal rat cardiomyocytes and seeded them on a cell culture substrate that fully supports cell attachment at 37°C, but loses cell adhesive properties when cooled to 32°C. By allowing the cells to reach confluence before reducing the temperature, the cardiomyocytes are liberated as an intact sheet when they detach. This sheet—happily beating away—can then be transferred to a PDMS microchip, and the cells reattached to fibronectin-coated surfaces. In this case, the sheet was allowed to adhere in such a way that it draped over a push-bar: as the cells pulse in sync, the bar is forced down and displaces fluid in the adjoining chamber, resulting in liquid flow. The authors monitored liquid movement with polystyrene tracking particles; amazingly, this bio-pump could keep up its work for 1 week in culture and could even be regulated in a crude fashion by temperature control (the higher the temperature, the faster the beat frequency). Under directional pumping conditions, a flow rate of 2 nL/min could be produced. This bio-actuator offers a tantalizing glimpse of the possibilities afforded by integration of whole cells into lab-on-a-chip devices, and the likelihood that glucose and oxygen may be able to substitute for electrical power in some microfluidics systems. –ND

Reproduced with permission. © 2006 The Royal Society of Chemistry.

Tanaka et al. 2006. An actuated pump on-chip powered by cultured cardiomyocytes. Lab on a Chip 6(3):362-368.

Designer Genes

As genetic engineering has advanced, researchers have moved from cutting and splicing using restriction sites to making more subtle single base changes by point mutation, and now, to creating completely synthetic genes and even chromosomes. The in silico design and construction of synthetic genes is a labor-intensive task, frequently requiring the use of a number of software packages and extensive sequence manipulation, all of which can increase the chance of errors creeping in. To address this problem, Jef Boeke and his group at Johns Hopkins in Baltimore have introduced a single, complete web-based software suite—aptly named GeneDesign—that they assure will alleviate some of the more tedious and error-prone steps in drawing up synthetic gene blueprints. The web interface is very easy to use, and the authors have provided sufficiently detailed information to help you through the process, plus an online manual for those so inclined. Best of all, each of the six modules can be used either consecutively or piecemeal, and not necessarily for gene design purposes, antithetically making the sum of the parts more than the whole. The program is also able to generate a very useful final report that details all modifications made (such as the addition/deletion of restriction endonuclease sites), as well as including a list of all overlapping synthetic oligonucleotides needed to construct the gene, ready for ordering. A number of companies offer synthetic gene design and synthesis, and some commercial software packages are available. However, the GeneDesign suite is publicly available for free on the web, providing a low cost solution for those wishing to be more hands-on in this process. In its essence, it is very similar to the open source GeMS software, but does provide some additional functionality, including the design of longer oligonucleotides and the ability to adjust the annealing temperature when designing the overlapping oligonucleotides for PCR. Take a look now at slam.bs.jhmi.edu/gd. –SS

Richardson et al. 2006. GeneDesign: rapid, automated design of multikilobase synthetic genes. Genome Research [Epub ahead of print, February 15, 2006].

Something From Nothing

Far from delivering empty promises, zero-mode waveguides (ZMW) allow single-molecule analysis of reactions performed using physiologically relevant concentrations. This is notable for two reasons: (i) many interesting molecular interactions occur at micromolar concentrations and (ii) extreme dilution (pico- and nanomolar levels) are required to separate molecules adequately so that they can be measured at the individual level by fluorescence correlation spec-troscopy (FCS). The ZMW works not by limiting the number of molecules in the observation chamber, but by severely restricting the reaction volume that is illuminated by the excitation light. This follows from the properties of the ZMW: a waveguide is generally an opening that only allows light of wavelengths equal to or less than its aperture to pass through. The "zero-mode" label means a waveguide in which the hole is smaller than the light's wavelength. It stands to reason that no light should be able to penetrate, but in actual fact, the light does enter 10–15 nm into the hole before being entirely blocked. That illumination zone provides an exceedingly small observation window that allows single molecule monitoring even at relatively high molecule concentrations. To date ZMWs have been used to observe polymerization along a single DNA molecule and protein oligomerization kinetics. In new work, Harold Craighead's lab at Cornell shows that lipid bilayers are compatible with ZMW-mediated analysis. Samiee et al., were able to find conditions under which membranes would invaginate into the nanoscale holes of the ZMW. As proof of principle, the authors show results recording the interaction of fluorescently labeled tetanus toxin and membrane-contained G_T1b ganglioside by FCS in ZMWs. Although further refinement of diffusion models is still required, the approach represents an attractive opportunity for fast, single-molecule analysis of the ligand binding kinetics of membrane receptors. –ND

Samiee et al. 2006. Zero mode waveguides for single molecule spectroscopy on lipid membranes. Biophysical Journal [Epub ahead of print, February 3, 2006].

Diseases On Display

The search for human disease markers that can be targeted by small molecule or biologics therapy is an ongoing, perhaps never-ending, endeavor. A battery of techniques are available, both on the genomic and proteomic level, to identify disease-related genes and their protein products; only some are high-throughput and many are time-intensive. A group from the Netherlands recently presented a novel combination of techniques that offers a slightly different approach to disease target identification, using a two-step selection method that brings together phage display utilizing a mixture of libraries from patients with autoimmune diseases, and Western blot detection to provide a facile means to identify protein epitopes associated with disease. These epitopes can subsequently be used as potential targets for therapeutics. The protocol, which is described in exceptional detail, begins by the removal of those phages recognizing non-apoptotic proteins using two rounds of subtractive selection against non-apoptotic HeLa cell extracts. The aforementioned extracts were previously covalently labeled with biotin to enable easy selection with magnetic streptavidin beads in solution. The third round of selection uses identically labeled extract, this time from HeLa cells in which apoptosis has been induced, enabling the isolation of phages reacting strongly with apoptosis-specific epitopes. These phages are then further selected by 2-D Western immunoblotting, in which apoptotic extract from HeLa cells separated in two dimensions is probed using the previously selected phages. Following the application of refining selection criteria, the scFv inserts of phages obtained from excised spots are His-tagged, expressed, immobilized on beads, and used to immunoprecipitate their antigens, which can then be identified by mass spectrometry. Although a system of fairly low complexity was used to demonstrate the efficacy of the method, only a few antigens were identified; while clearly a promising combination of techniques, further development and refinement to increase the sensitivity appears necessary to make it sufficiently powerful for routine high-throughput screening. –SS

Reprinted with permission. © 2006 The American Society for Biochemistry and Molecular Biology.

Hof et al. 2006. A novel subtractive antibody phage display method to discover disease markers. Molecular & Cellular Proteomics 5:245-255.