Electron beam patterning of biomolecules

The patterning of biomolecules on semiconducting surfaces is of central importance in the fabrication of novel biodevices. In the process of patterning, it is required that the biomolecule preserves its properties and the substrate is not damaged by the chemicals, the temperatures or the patterning beams involved in the procedure. Recently, both DUV and electron beam microlithography have been used in order to deposit protein layers in predefined patterns. Various approaches have been used, some involving photoresists. Contrast between exposed and unexposed regions, resolution of adjacent features and sensitivity to dose variation, are the key issues. The approach followed in this paper consists of a direct patterning of a biotin layer, deposited on an amino-silane primed silicon nitride surface, using an electron beam. After irradiation, the surface is covered by bovine serum albumin (BSA), which acts as a blocking material to protect the exposed areas from streptavidin adsorption. Using 20 keV e-beam energy and doses, in the range 100-1000 microC/cm(2), submicrometer dense lines of 1-microm pitch have been obtained. The results have been tested by fluorescence optical microscopy.     

Selective patterning and immobilization of biomolecules within precisely-defined micro-reservoirs

Herein, we present the fabrication of well-defined micro-reservoirs and a simple strategy to immobilize biomolecules selectively inside the reservoirs. The micro-reservoirs are fabricated using a photocurable prepolymer, which enables the formation of concrete structures with high-fidelity, so that the reservoirs are spatially-segregated from each other by rigid physical barriers. For the directed binding of the protein, two steps are involved. First, poly(ethylene glycol) (PEG) is contact-printed on those areas where the protein binding is not desired, and next, protein binding is promoted where desired via carbodiimide chemistry. Fluorescein-tagged albumin is successfully immobilized inside the micro-reservoirs and microchannel arrays with high sensitivity, regardless of the sizes of the reservoirs and channels. The proposed system can be used for constructing multi-functional biosensors by immobilizing individual bioorganisms specifically in each micro-reservoir or microchannel.     

Epoxy-silane linking of biomolecules is simple and effective for patterning neuronal cultures

Surface chemistry is one of the main factors that contributes to the longevity and compliance of cell patterning. Two to three weeks are required for dissociated, embryonic rat neuronal cultures to mature to the point that they regularly produce spontaneous and evoked responses. Though proper surface chemistry can be achieved through the use of covalent protein attachment, often it is not maintainable for the time periods necessary to study neuronal growth. Here we report a new and effective covalent linking approach using (3-glycidoxypropyl) trimethoxysilane (3-GPS) for creating long term neuronal patterns. Micrometer scale patterns of cell adhesive proteins were formed using microstamping; hippocampal neurons, cultured up to 1 month, followed those patterns. Cells did not grow on unmodified 3-GPS surfaces, producing non-permissive regions for the long-term cell patterning. Patterned neuronal networks were formed on two different types of MEA (polyimide or silicon nitride insulation) and maintained for 3 weeks. Even though the 3-GPS layer increased the impedance of metal electrodes by a factor of 2-3, final impedance levels were low enough that low noise extracellular recordings were achievable. Spontaneous neural activity was recorded as early as 10 days in vitro. Neural recording and stimulation were readily achieved from these networks. Our results showed that 3-GPS could be used on surfaces to immobilize biomolecules for a variety of neural engineering applications.     

A two-step patterning process increases the robustness of periodic patterning in the fly eye

Complex periodic patterns can self-organize through dynamic interactions between diffusible activators and inhibitors. In the biological context, self-organized patterning is challenged by spatial heterogeneities (‘noise’) inherent to biological systems. How spatial variability impacts the periodic patterning mechanism and how it can be buffered to ensure precise patterning is not well understood. We examine the effect of spatial heterogeneity on the periodic patterning of the fruit fly eye, an organ composed of ～800 miniature eye units (ommatidia) whose periodic arrangement along a hexagonal lattice self-organizes during early stages of fly development. The patterning follows a two-step process, with an initial formation of evenly spaced clusters of ～10 cells followed by a subsequent refinement of each cluster into a single selected cell. Using a probabilistic approach, we calculate the rate of patterning errors resulting from spatial heterogeneities in cell size, position and biosynthetic capacity. Notably, error rates were largely independent of the desired cluster size but followed the distributions of signaling speeds. Pre-formation of large clusters therefore greatly increases the reproducibility of the overall periodic arrangement, suggesting that the two-stage patterning process functions to guard the pattern against errors caused by spatial heterogeneities. Our results emphasize the constraints imposed on self-organized patterning mechanisms by the need to buffer stochastic effects. Author summary Complex periodic patterns are common in nature and are observed in physical, chemical and biological systems. Understanding how these patterns are generated in a precise manner is a key challenge. Biological patterns are especially intriguing, as they are generated in a noisy environment; cell position and cell size, for example, are subject to stochastic variations, as are the strengths of the chemical signals mediating cell-to-cell communication. The need to generate a precise and robust pattern in this ‘noisy’ environment restricts the space of patterning mechanisms that can function in the biological setting. Mathematical modeling is useful in comparing the sensitivity of different mechanisms to such variations, thereby highlighting key aspects of their design.We use mathematical modeling to study the periodic patterning of the fruit fly eye. In this system, a highly ordered lattice of differentiated cells is generated in a two-dimensional cell epithelium. The pattern is first observed by the appearance of evenly spaced clusters of ～10 cells that express specific genes. Each cluster is subsequently refined into a single cell, which initiates the formation and differentiation of a miniature eye unit, the ommatidium. We formulate a mathematical model based on the known molecular properties of the patterning mechanism, and use a probabilistic approach to calculate the errors in cluster formation and refinement resulting from stochastic cell-to-cell variations (‘noise’) in different quantitative parameters. This enables us to define the parameters most influencing noise sensitivity. Notably, we find that this error is roughly independent of the desired cluster size, suggesting that large clusters are beneficial for ensuring the overall reproducibility of the periodic cluster arrangement. For the stage of cluster refinement, we find that rapid communication between cells is critical for reducing error. Our work provides new insights into the constraints imposed on mechanisms generating periodic patterning in a realistic, noisy environment, and in particular, discusses the different considerations in achieving optimal design of the patterning network.     

A biomolecule friendly photolithographic process for fabrication of protein microarrays on polymeric films coated on silicon chips

The last years, there is a steadily growing demand for methods and materials appropriate to create patterns of biomolecules for bioanalytical applications. Here, a photolithographic method for patterning biomolecules onto a silicon surface coated with a polymeric layer of high protein binding capacity is presented. The patterning process does not affect the polymeric film and the activity of the immobilized onto the surface biomolecules. Therefore, it permits sequential immobilization of different biomolecules on spatially distinct areas on the same solid support. The polymeric layer is based on a commercially available photoresist (AZ5214) that is cured at high temperature in order to provide a stable substrate for creation of protein microarrays by the developed photolithographic process. The photolithographic material consists of a (meth)acrylate copolymer and a sulfonium salt as a photoacid generator, and it is lithographically processed by thermal treatment at temperatures 相似文献   

Meiotic DSB patterning: A multifaceted process

Meiosis is a specialized two-step cell division responsible for genome haploidization and the generation of genetic diversity during gametogenesis. An integral and distinctive feature of the meiotic program is the evolutionarily conserved initiation of homologous recombination (HR) by the developmentally programmed induction of DNA double-strand breaks (DSBs). The inherently dangerous but essential act of DSB formation is subject to multiple forms of stringent and self-corrective regulation that collectively ensure fruitful and appropriate levels of genetic exchange without risk to cellular survival. Within this article we focus upon an emerging element of this control—spatial regulation—detailing recent advances made in understanding how DSBs are evenly distributed across the genome, and present a unified view of the underlying patterning mechanisms employed.     

Do biomolecules process information differently than synthetic organic molecules?

This paper compares information/signal processing in synthetic and biological molecules. The role of conformation-based (shape-based) mechanisms and electrostatic interactions in molecular recognition is discussed. In biological electron transfer, the 'electron shuttle'-mediated mechanism is contrasted with the mechanism based on pre-formed 'electron wires'. While biological information processing is thought to be more distributed (less discrete), an example of molecular switch is presented: visual transduction. We further speculate that visual transduction may be implemented in the form of a switch based on electrostatic interactions. The concept of intelligent materials is discussed with the well-known Bohr effect of hemoglobin oxygenation. Based on these examples, we argue that there are no fundamental differences between synthetic and biological molecules in their mode of information processing. In the pursuit of novel paradigms of molecular information processing, we also perceive no conflicts in developing molecular devices that emulate the switching function of conventional microelectronic devices.     

Alumina-phosphate complexes for immobilization of biomolecules

Organic compounds containing the -PO3H2 function are strongly and specifically adsorbed by aluminum oxide in water within a large range of pH. The reversible character of the interaction allows the adsorbed organic phosphates to be displaced by inorganic phosphate buffers resulting in their purification by an affinity-like chromatographic procedure. The interaction between alumina and selected multifunctional compounds containing a phosphonate group yields a chemically activated alumina-phosphate complex onto which enzymes or other molecules can be immobilized. A number of proteases immobilized on alumina through such phosphate interactions proved to be active in the presence of organic solvents. As a consequence, enzyme-catalyzed peptide synthesis in a water-limited environment and optical resolution of amino acids in water-organic solvent emulsions can be accomplished.     

T-Pile--a package for thermodynamic calculations for biomolecules

Molecular dynamics and Monte Carlo, usually conducted in canonical ensemble, deliver a plethora of biomolecular conformations. Proper analysis of the simulation data is a crucial part of biophysical and bioinformatics studies. Sequence alignment problem can be also formulated in terms of Boltzmann distribution. Therefore tools for efficient analysis of canonical ensemble data become extremely valuable. T-Pile package, presented here provides a user-friendly implementation of most important algorithms such as multihistogram analysis and reweighting technique. The package can be used in studies of virtually any system governed by Boltzmann distribution. AVAILABILITY: T-Pile can be downloaded from: http://biocomp.chem.uw.edu.pl/services/tpile. These pages provide a comprehensive tutorial and documentation with illustrative examples of applications. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

An affinity-based method for the purification of fluorescently-labeled biomolecules

Due to the difficulty of separating mixtures of labeled and unlabeled biomolecules, a general new method for the affinity purification of modified proteins has been developed. A Sepharose-based solid support bearing beta-cyclodextrin groups was used to capture chromophore-modified proteins selectively, while unmodified proteins remained in solution. After isolation of the resin, the modified proteins were released by treating the sample with a competitive cyclodextrin binder, such as adamantane carboxylic acid. This procedure was demonstrated for several dyes displaying a wide range of spectral characteristics and diverse chemical structures. Preliminary studies have shown that this method can also be used to enrich modified peptide fragments present in proteolytic digests. This technique is anticipated to accelerate the development of new protein modification reactions and could provide a useful tool for proteomics applications.     

Fragment molecular orbital calculations for biomolecules

Exploring biomolecule behavior, such as proteins and nucleic acids, using quantum mechanical theory can identify many life science phenomena from first principles. Fragment molecular orbital (FMO) calculations of whole single particles of biomolecules can determine the electronic state of the interior and surface of molecules and explore molecular recognition mechanisms based on intermolecular and intramolecular interactions. In this review, we summarized the current state of FMO calculations in drug discovery, virology, and structural biology, as well as recent developments from data science.     

Strategies for the identification and purification of ligands for orphan biomolecules

Summary The isolation of related genes with evolutionary conserved motifs by the application of polymerase chain reaction-based molecular biology techniques, or from database searching strategies, has facilitated the identification of new members of protein families. Many of these protein molecules will be involved in protein-protein interactions (e.g. growth factors, receptors, adhesion molecules), since such interactions are intrinsic to virtually every cellular process. However, the precise biological function and specific binding partners of these novel proteins are frequently unknown, hence they are known as ‘orphan’ molecules. Complementary technologies are required for the identification of the specific ligands or receptors for these and other orphan proteins (e.g., antibodies raised against crude biological extracts or whole cells). We describe herein several alternative strategies for the identification, purification and characterisation of orphan peptide and protein molecules, specifically the synergistic use of micropreparative HPLC and biosensor techniques. These authors made equivalent contributions.     

Nanowire sensors for multiplexed detection of biomolecules

Nanowire-based detection strategies provide promising new routes to bioanalysis that could one day revolutionize the healthcare industry. This review covers recent developments in nanowire sensors for multiplexed detection of biomolecules such as nucleic acids and proteins. We focus on encoded nanowire suspension arrays and semiconductor nanowire-based field-effect transistors. Nanowire assembly and integration with microchip technology is emphasized as a key step toward the ultimate goal of multiplexed detection at the point of care using portable, low power, electronic biosensor chips.     

Strategies for the identification and purification of ligands for orphan biomolecules

The isolation of related genes with evolutionary conserved motifs by the application ofpolymerase chain reaction-based molecular biology techniques, or from database searchingstrategies, has facilitated the identification of new members of protein families. Many of theseprotein molecules will be involved in protein–protein interactions (e.g. growth factors,receptors, adhesion molecules), since such interactions are intrinsic to virtually every cellularprocess. However, the precise biological function and specific binding partners of these novelproteins are frequently unknown, hence they are known as orphan molecules.Complementary technologies are required for the identification of the specific ligands orreceptors for these and other orphan proteins (e.g., antibodies raised against crude biologicalextracts or whole cells). We describe herein several alternative strategies for the identification,purification and characterisation of orphan peptide and protein molecules, specifically thesynergistic use of micropreparative HPLC and biosensor techniques.