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Bio Micro and Nanosystems Conference, 2006. BMN '06

Date 15-18 Jan. 2006

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  • Biological complexity and robustness

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    This talk will describe qualitatively in as much detail as time allows these features of biological systems and their parallels in technology, using hopefully familiar and concrete examples. The aim is to be accessible to biologists, and not to depend critically on the mathematical framework. A crucial insight is that both evolution and natural selection or engineering design must produce high robustness to uncertain environments and components in order for systems to persist. Yet this allows and even facilitates severe fragility to novel perturbations, particularly those that exploit the very mechanisms providing robustness, and this "robust yet fragile" (RYF) feature must be exploited explicitly in any theory that hopes to scale to large systems. Time permitting, the mathematical research implications will be sketched of this view of "organized complexity" in biology, technology, and mathematics. This view contrasts sharply with that of "emergent complexity" popular in other areas of science in a way that can now be made mathematically precise View full abstract»

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  • Biomolecular mechanism of silica synthesis opens novel routes to low-temperature nanofabrication of semiconductors and other advanced materials

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    Summary form only given. Biological systems fabricate multifunctional, high-performance materials at low temperatures and near-neutral pH with a precision of three-dimensional nanostructural control that exceeds the capabilities of present human engineering. We discovered the mechanism governing the nanofabrication of silica in a biological system, and translated this mechanism to develop a new low-temperature route for the synthesis of a wide range of nano structured metal oxide, -hydroxide and -phosphate semiconductor thin films without the use of organic templates. As a first proof of principle, we have used this process for the low-temperature synthesis of a strongly photo conductive cobalt hydroxide-based thin film material never before attainable through conventional or high-temperature methods. This new biologically inspired synthesis method yields exceptionally pure inorganic semiconductors, and thus is potentially integrable with conventional manufacturing methods. In research leading to these developments, we discovered that the silicateins, a family of enzyme proteins we found occluded within the silica needles made by a marine sponge, can catalyze and structurally direct the polymerization of silica, silsesquioxanes, organometallics and a wide range of metal oxide semiconductors from the corresponding molecular precursors at neutral pH and low temperature. These were the first reported examples of enzyme-catalyzed, nano structure-directed synthesis of semiconductors. Interaction with the template-like protein surface stabilizes polymorphs of these materials (e.g., the anatase form of titanium dioxide and the spinel polymorph of gallium oxide) otherwise not formed at low temperatures. This observation and the preferential alignment of the Ga2O3 nanocrystallites suggested a pseudo-epitaxial relationship between the mineral crystallites and specific functional groups on the templating protein surface. As described above, we have used this biologically inspi- - red process for the low-temperature synthesis of a large number of metal-oxides, -hydroxides and -phosphates, producing many in forms that could not be attained by conventional syntheses. The electronic properties of these novel materials suggest strong advantages for energy applications View full abstract»

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  • Positional characteristics of fluorophores influencing signal output of a DNA nanoswitch

    Page(s): 3 - 6
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    The Holliday junction (HJ) structure, consisting of four DNA double helices with a central branch point, is capable of switching between conformational states upon ion binding. The HJ nanoswitch described here comprises a long, dual labeled cloverleaf oligonucleotide and a short, unlabeled oligonucleotide. Fluorescent labeling with donor and acceptor dyes placed on the HJ arms of the cloverleaf strand allows the ion induced conformational switch to be detected optically using fluorescence resonance energy transfer (FRET). The influence of donor and acceptor dye location on the detection of conformational switching has been investigated using two distinct HJ structures. In addition, the effect of increasing HJ arm length in order to increase donor and acceptor dye separation has been evaluated. We report that a preferential HJ nanoswitch structure can be determined, capable of efficient detection of ion induced conformational switching View full abstract»

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  • Nanocluster epitope presentation

    Page(s): 7
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    Summary form only given. A fundamental goal at the intersection of nanotechnology, immunological function and nanomedicine is the design of biologically active interfaces. Recently, we have developed both linear and conformational loop peptides on nanoclusters that mimic immunological epitopes. The nanocluster-epitope interface was studied using a quartz crystal microbalance to examine the detailed kinetic and thermodynamic binding parameters of the nanocluster-peptide interface with antibodies. These results demonstrate that the conformational loop epitopes effectively recapitulate the native protein loop conformations, paving the way for new methods of epitope screening, monitoring of immunological challenge and the design of novel nano-based multivalent vaccines View full abstract»

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  • Biodegradable, antioxidant-loaded nanoparticles: a first step toward attenuating oxidative injury in vitro

    Page(s): 8
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    Summary form only given. The relentless progression and devastating ramifications of neurodegenerative diseases make symptomatic and protective treatment of these ailments particularly critical and daunting. Parkinson's Disease (PD) is characterized by severe motor impairments, significant oxidative injury and degeneration of the nigrostriatal dopaminergic neurons. Although symptomatic PD therapy is initially effective, antioxidant treatments are more promising because they may decelerate and even prevent dopaminergic neurodegeneration. Therefore, we are developing antioxidant-loaded, biodegradable nanoparticles-drug delivery platforms that are advantageous as an antioxidant therapy to slow neurodegeneration. Innovations in bionanotechnology and drug delivery-areas that integrate bio engineering, materials science and cell biology-promise to revolutionize the treatment of neurodegenerative diseases like PD. In particular, drug-loaded, biodegradable poly(lactide-co-glycolide) (PLGA) nanoparticles are promising drug delivery devices because they protect unstable and poorly soluble drugs from the biological milieu, facilitate sustained drug levels in the vicinity of the nanoparticle and can be internalized by cells. We have synthesized novel Coenzyme Q10 (CoQ10)-loaded biodegradable PLGA nanoparticles with ester- and carboxylic acid- end groups by nanoprecipitation and characterized their physical properties with dynamic light scattering (DLS) and atomic force microscopy (AFM). We have consistently modulated the diameters of the nanoparticles between 100 nm and 200 nm by varying the PLGA mass in the solvent and the solvent/non-solvent volumetric ratio. These nanoparticle formulations were customarily monodisperse, with polydispersity indices <0.1. The CoQ10 incorporation efficiency was calculated spectrophotometrically by UV absorbance at 275 nm, and our preliminary data indicate successful encapsulation of CoQ10 within the nanoparticles. These results motivate fu- - rther investigation of the CoQ10-loaded nanoparticles as drug delivery devices in a simple yet appropriate PD cell model. Thus, we are concurrently developing an oxidative injury model with dopaminergic PC12 cells in order to test the antioxidant effects of these CoQ10-loaded PLGA nanoparticles. Our CoQ10-loaded biodegradable PLGA nanoparticles may significantly enhance the prospects for neuronal drug therapy by attenuating dopaminergic oxidative damage and slowing the neurodegeneration associated with PD View full abstract»

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  • Targeted delivery of therapeutic agents with controlled bacterial carriers in the human blood vessels

    Page(s): 9
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    Summary form only given. The treatment of cancer is one of the most challenging tasks of modern medicine and secondary toxicity remains a critical issue. Although intra-arterial chemotherapy or chemo-embolization provides interesting success, the release of drug in the systemic circulation prevents high intra-tumoral drug concentration to be sustained. Hence, targeting specifically the tumor cells becomes a major goal of modern oncology. As such, providing means of carrying microparticles for specific endovascular drug delivery or radioisotopes at the site of the tumor mass would be extremely attractive. Although we have shown experimentally that a clinical magnetic resonance imaging (MRI) system can propel a ferromagnetic core in a human cardiovascular network through an induced force generated by the same magnetic gradients used for MR-imaging, it becomes technological very challenging to apply this method to navigate particles in a 3D space in order to reach the tumor cells through an anarchic arteriolocapillar network stimulated by tumoral angiogenesis. Since the induction of a propulsion force decreases significantly for much smaller particles, propulsion in capillaries would require gradient amplitudes that are technologically and practically not an alternative considering the size and cooling issues of an additional gradient coils systems embedded in the MRI bore. Our proposed concept consists of using magnetotactic bacteria to push microbeads being coated with therapeutic agents in the capillary network. We have shown that the displacement path of a magnetotactic bacterium pushing a microbead can be modified by changing the orientation of the lines of a magnetic field. Preliminary experimental results also showed that magnetotactic bacteria of type MC-1 could swim efficiently in human blood and that a swarm of these bacteria could potentially be detected by MR-imaging. Although the DC magnetic field of clinical MRI systems complicates the directional control- - of such bacteria compared with an X-ray system that could be upgraded with peripheral permanent or electro-magnets, the use of an MRI system has many advantages in term of imaging modalities and the lack of radiation. We report preliminary studies related to local perturbations of the DC magnetic field of a clinical MRI system time multiplexed with imaging sequences that could allow the target delivery of therapeutic agents through microbeads being pushed by magnetotactic bacteria operating under computer control View full abstract»

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  • Engineering with life: new tools for the 21st century

    Page(s): 10
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    Integrative technology, the merging of nanotechnology, biotechnology and informatics offers an opportunity for realizing true advances in the manner in which technology interacts with humanity. Using the power of nanotechnology to manipulate matter, that is the placing of molecule's where we want, when we want, to perform functions that we want. Using the inspiration of biotechnology both to co-opt the tools of molecular manufacturing and to provide a baseline understanding of the way nature manipulates matter and information. And finally, using informatics to create a robust framework for transforming the information implicit in molecular and larger scale interactions to engineer complex adaptive systems that exhibit embedded higher-order behavior. Collectively these technologies established the basis for integrative technology, a new IT with emergent capabilities resulting from defined interactions within the system. The first examples of the implementation of integrated technology are manifested in the synthesis of a new class of smart materials. These materials have the potential to emulate much of the functionality associated with living systems such as the active transport and transformation of matter and information and, the transduction of energy into different forms. We will present the details of the technological demands and the results of efforts associated with the production of these new functional materials. Elements of the discussion will include the genetic engineering of active biological molecules into engineering building blocks, the precision assembly of these molecules into a stable, "active" material and, the promise of embedding intelligent behavior into the matrix of the assembled matter to a new class of intelligent therapeutics View full abstract»

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  • Gene network shaping of inherent noise spectra

    Page(s): 11
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    Recent work demonstrates that stochastic fluctuations in molecular populations have gene regulation consequences. Previous experiments focused on noise sources or noise propagation through gene networks by measuring noise magnitudes. However, in theoretical analysis we showed that noise frequency content is determined by the underlying gene circuits, leading to a mapping between gene circuit structure and the noise frequency range. An intriguing prediction was that negative autoregulation shifts noise to higher frequencies where it is more easily filtered out by gene networks, a property that may contribute to the prevalence (e.g. found in regulation of ~40% of E. coli genes) of autoregulation motifs. Here we measure noise frequency content in growing cultures of E. coli and verify the link between gene circuit structure and noise spectra by demonstrating the negative autoregulation-mediated spectral shift. We further demonstrate that noise spectral measurements provide mechanistic insights into gene regulation as perturbations of gene circuit parameters are discernible in the measured noise frequency ranges. These results suggest that noise spectral measurements could facilitate the discovery of novel regulatory relationships View full abstract»

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  • Monitoring dynamics of single-cell gene expression over multiple cell cycles

    Page(s): 12
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    Recent progress in reconstructing gene regulatory networks has established a framework for a quantitative description of the dynamics of many important cellular processes. Such a description will require novel experimental techniques that enable the generation of time-series data for the governing regulatory proteins in a large number of individual living cells. Here, we utilize microfabrication to construct a Tesla microchemostat that permits single-cell fluorescence imaging of gene expression over many cellular generations. The device is used to capture and constrain asymmetrically dividing or motile cells within a trapping region and to deliver nutrients and regulate the cellular population within this region. We illustrate the operation of the microchemostat with Saccharomyces cerevisiae and explore the evolution of single-cell gene expression and cycle time as a function of generation. Our findings highlight the importance of novel assays for quantifying the dynamics of gene expression and cellular growth, and establish a methodology for exploring the effects of gene expression on long-term processes such as cellular aging View full abstract»

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  • Cell mimics created from the controlled synthesis and directed assembly of carbon nanofibers

    Page(s): 13
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    Biological cells represent an engineering ideal. They are the fundamental unit of all biological systems, perform virtually any function, and operate with efficiencies unmatched by man-made creations. These capabilities result from the optimized arrangement of chemical and physical features across nanometer and micron lengths. Emerging capabilities to control synthesis and direct assembly of synthetic structures over similar length scales present the opportunity to mimic these natural structures. Vertically aligned carbon nanofibers (VACNFs) have a number of features that make them particularly well suited to the construction of cell mimics. VACNFs can be synthesized with nanometer scale dimensions, can be electrically addressed and can be deterministically grown in desired locations. Dense arrays of carbon nanofibers form semi-permeable barriers (pseudo-membranes) that can be integrated within fluidic structures or arranged as small volume (sub-nanoliter) containers. Size-dependent transport perpendicular to the orientation of the fibers can be controlled based on the wall-to-wall spacing of the individual nanofibers. Recent progress in VACNF device fabrication will be presented, focusing on efforts to tailor the effective membrane pore size, through oxide deposition and electropolymerization of polypyrrole, and to derivatize the surface of VACNFs for attachment of biomolecules such as proteins and DNA. By combining these modifications in permeability and chemical features, biologically inspired cell mimics of increasing sophistication can be constructed with the ultimate objective of emulating the structure, function, and organization of natural cells, while also providing unique capabilities in chemical recognition and separations View full abstract»

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  • Bacterial nanowires: electrically conductive filaments and their implications for energy transformation and distribution in natural and engineered systems

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    Bacteria, ranging from oxygenic photosynthetic cyanobacteria to heterotrophic sulfate reducing bacteria, produce electrically-conductive appendages referred to as bacterial nanowires. Dissimilatory metal reducing bacteria, including Shewanella oneidensis and Geobacter sulfurreducens, produce electrically conductive nanowires in direct response to electron acceptor limitation and facilitate electron transfer to solid phase iron oxides. Nanowires produced by S. oneidensis strain MR-1, which served as our primary model organism, are functionalized by decaheme cytochromes MtrC and OmcA that are distributed along the length of the nanowires. Mutants deficient in MtrC and OmcA produce nanowires that were poorly conductive. These mutants also differ from wild type cells in their ability to reduce solid phase iron oxides, to produce electrical current in a mediator less microbial fuel cell, and to form complex biofilms at air liquid interfaces. Although currently less completely characterized, conductive nanowires produced by other organisms reveal a strategy of energy/electron distribution that is conserved across a broad metabolic spectrum. This presentation will target the implications of bacterial nanowire for energy distribution and communication in biofilms and other natural microbial communities, bioelectrical coupling of electron donors with poorly accessible electron acceptors, and applications for alternative energy (microbial fuel cells) and nanoelectronic technologies View full abstract»

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  • Microbial synthesis of noble metal nanoparticles using the Fe(III)-reducing bacterium shewanella algae

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    Summary form only given. Noble metals nanoparticles can be applied to a wide range of functions such as catalysis, optics and biosensing. Although chemical and physical synthetic routes to noble metal nanoparticles have been extensively developed, another possibility is synthesis by bioreduction of noble metal ions using microorganism. For microbial synthesis of noble metal nanoparticles, we focused on the Fe(III)-reducing bacterium Shewanella algae, which is able to transfer electrons to Fe3+ ions. Intracellular synthesis of gold nanoparticles was achieved at 25degC and pH 7 using S. algae (ATCC 51181) with H2 as the electron donor. The reductive deposition of gold by S. algae was a fast process: 1 mol/m3 AuCl4 ions were completely reduced to insoluble gold within 30 min. Speciation of gold in bacterial cultures of S. algae by X-ray Absorption Near-Edge Spectroscopy (XANES) showed that the trivalent gold ions were rapidly reduced to gold metal in the bacterial cells. Transmission electron microscopy (TEM) of thin sections of S. algae cells revealed that the biogenic gold nanoparticles of 10-20 nm were located in the periplasm. When the pH of the HAuCl4 solution was decreased from 7 to 1, many of the biogenic gold particles were synthesized extracellularly. The gold particles synthesized at pH 1 were typically 50 nm and 500 nm, and some were angular in shape and single crystal. This suggests that at pH 1, a gold-reducing enzyme is released from the periplasmic space into the aqueous solution, and the enzyme catalyzes the reduction of gold ions in the aqueous solution. We can therefore conclude that the solution pH is an important factor in controlling the morphology of biogenic gold particles and location of particle formation View full abstract»

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  • Quantification of accessible DNA on vertically aligned carbon nanofibers in cellular delivery systems

    Page(s): 30
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    Vertically aligned carbon nanofibers with tethered plasmids have been developed as a novel tool for the direct physical introduction of nucleic acids (tethered genes) into living cells. Immobilization of DNA to a scaffold, or "tethering", can influence the accessibility and transcriptional activity of the DNA template. Therefore, it is necessary to determine the number of accessible gene copies on each nanofiber. In this investigation, polymerase chain reaction (PCR) and quantitative polymerase chain reaction (qPCR) were used as cell-free tools to quantify the number of gene copies tethered to a nanofiber. Using chips of nanofiber arrays with bound template (pd2EYFP-N1 vector), PCR was conducted with a primer set designed to amplify the CMV promoter and eYFP gene region (1603 bp). Using a non-specific binding protocol of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) condensation of DNA base amines to nanofiber carboxylic acids, amplification yields of the promoter/gene region of bound DNA were quantified and compared to controls with no EDC. During the first round of quantification, approximately 3.1times107 gene copies were amplified from each nanofiber chip in the presence of EDC, while 1.6times107 gene copies were amplified from control nanofiber chips in the absence of EDC. In subsequent quantification steps of the same nanofiber chips, DNA yields decreased dramatically (2.4times104 gene copies) on control chips, while chips incubated in the presence of EDC retained DNA (1.1times107 gene copies). We did not expect such a high number of gene copies from the first round of quantification on nanofiber chips in the absence of EDC. The subsequent decrease in gene copies from these samples suggests that perhaps this DNA was non-specifically adsorbed to the nanofibers and can be removed during thermal cycling. Investigations with other tethering strategies are being addressed to evaluate and optimize DNA binding strategies View full abstract»

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  • Origins of extrinsic variability in eukaryotic gene expression

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    Summary form only given. Variable gene expression within a clonal population of cells has been implicated in a number of important processes including mutation and evolution, determination of cell fates and the development of genetic disease. Recent studies have demonstrated that a significant component of expression variability arises from extrinsic factors thought to influence multiple genes in concert, yet the biological origins of this extrinsic variability have received little attention. Here we combine computational modeling with fluorescence data generated from multiple promoter-gene inserts in {em Saccharomyces cerevisiae} to identify two major sources of extrinsic variability. One unavoidable source arising from the coupling of gene expression with population dynamics leads to a ubiquitous noise floor in expression variability. A second source originating from a common upstream transcription factor exemplifies how regulatory networks can convert intrinsic or extrinsic noise in regulator expression into extrinsic noise at the output of a target gene. These results highlight the importance of the interplay of gene regulatory networks with population heterogeneity for understanding the origins of cellular diversity View full abstract»

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  • Photonic interactions in biomolecular micro and nano systems

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    The focus of this talk is the presentation of data with novel electronic devices that are comprised of biological components. We have extracted Photosystem I reaction centers from spinach leaves. Using the combined techniques of tapping-mode atomic force and scanning surface probe microscopies, we have demonstrated that PSI retains its molecular photovoltaic properties. PSI has been used to impart photoactivity to tissue cultures of retinoblastoma cells. Photoactive mammalian cells are novel hybrid biological structures that may have application in the field of artificial sight. We are also interested in the general problems of biomimetic photosynthesis, molecular "wiring" and the interfacial properties of protein-metal interfaces. Metallic platinum can be photoprecipitated at the point of electron emergence from the PSI reaction center in such a way that electric contact is made between protein and metal nanocatalyst. If platinization is performed on the stromal side of thylakoid membranes, it's possible to observe the simultaneous photoevolution of hydrogen and oxygen. This biomimetic reaction is a direct analog of natural photosynthesis in which the energy-rich product is molecular hydrogen rather than a carbon dioxide fixation compound. We have demonstrated that plastocyanin and PSI can be molecularly wired to enhance electron transfer between these two biomolecular electron transport proteins. In addition, we have performed spectroscopy and photochemistry of spinach PSI entrapped and stabilized in a hybrid organosilicate glass View full abstract»

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  • Coating of fuel cells using carbohydrate solutions

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    The current popular technique of preservation using cryogenic storage has several disadvantages especially in terms of the specialized equipment required. Preservation at ambient temperatures would eliminate the need for cryogenic equipment and the associated costs. Preservation of Geobacter Sulfurreducens (the bacterial fuel cell) at ambient temperatures would be particularly useful given its potential applications in technology. This has led to preliminary experiments for the preservation of the Geobacter Sulfurreducens in carbohydrate-based glasses using inositol, trehalose and sucrose. Geobacter was suspended in various carbohydrate solutions prepared from a stock solution of 4% D 2O in PBS and deposited on CaF2 slides in the form of small (400nl) droplets and dried isothermally at ambient temperatures. FTIR (Fourier Transform Infrared Spectroscopy) analysis of droplets containing the carbohydrate solutions and the Geobacter were performed to determine the desiccation characteristics of the carbohydrate solutions, their coating efficiency and the desiccation induced damage in Geobacter as a function of desiccation conditions. D 2O in the experimental solutions provided a unique peak around 2500 cm-1 in the IR spectra, which is used to quantify the water content of the sample. Optical analysis of the desiccated solutions showed heterogeneous conditions within the drying droplets. Comparison of the IR spectra at the center and the periphery of the droplets showed significant differences. This is thought to be a result of the bacteria clumping in the center regions and rejecting the solutes in the carbohydrate solution toward the circumference of the droplet. It was also observed that the peak magnitudes of the trehalose, sucrose and inositol were comparatively larger as compared to the amide-I peak (showing the bacteria concentration) at the circumference than at the center. This is in agreement with the optical observat- - ions showing glassy areas forming at the circumference during drying for the solutions containing sucrose and trehalose. These initial experiments demonstrated the heterogeneities occuring in sessile droplets during desiccation. Research is under way to develop methods that will enable homogeneous desiccation and vitrification of the biopreservation solutions coating the Geobacter. Additionally, mechanisms of desiccation-induced damage in Geobacter, the protection mechanisms of carbohydrates and the thermodynamics of the carbohydrate-based solutions are examined View full abstract»

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