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PubMed2003

Cell and organ printing 1: protein and cell printers.

We have developed several devices for positioning organic molecules, molecular aggregates, cells, and single-cell organisms onto solid supports. These printers can create stable, functional protein arrays using an inexpensive technology. The cell printer allows us to create cell libraries as well as cellular assemblies that mimic their respective position in organs. The printers are derived from commercially available ink-jet printers that are modified to dispense protein or cell solutions instead of ink. We describe here the modifications to the print heads, and the printer hardware and software that enabled us to adapt the ink-jet printers for the manufacture of cell and protein arrays. The printers have the advantage of being fully automated and computer controlled, and allow for the high-throughput manufacture of protein and cell arrays.

Animals↗
PubMed2004

Peptide arrays with designed secondary structures for protein characterization using fluorescent fingerprint patterns.

To realize a practical high-throughput protein-detection system, novel peptide arrays have been constructed using designed peptide libraries with loop, alpha-helix, or beta-strand structures. Here, we describe the overview of the reported designed peptide arrays with loop and alpha-helix structures and the new results of those with beta-strand structures. Initially, several model peptides known to interact with model structured proteins were selected to establish the present strategy for high-throughput detection of proteins. The fluorescent probes and suitable scaffolds of peptides were examined for the effective detection of proteins. The detection methods were established in solution and in an immobilized manner using the model systems. In the case of alpha-helix peptide, the response of a peptide with fluorescent resonance energy transfer between two probes at both termini was several times higher than that of a peptide with a single probe. In the cases of peptides with other structures, however, proteins were effectively detectable even by the fluorescent change of one probe. Furthermore, structurally focused libraries consisting of a total of ca. 250 different peptides based on the model peptides with secondary and/or tertiary structures were constructed with systematic replacement of residues. Using these libraries, various proteins were characterized effectively to give their own fluorescent "protein fingerprint" patterns. The resulting protein fingerprints correlated with the recognition properties of the proteins. These studies demonstrate that arrays with peptide libraries based on designed structures can be promising tools for detecting the target proteins. Designed synthetic peptides play roles as the capturing agents to be developed for practical protein chips.

Amino Acid Sequence↗
PubMed2004

Detection of biomolecular interaction between biotin and streptavidin on a self-assembled monolayer using magnetic nanoparticles.

For developing a magnetic bioassay system, an investigation to determine the presence of a specific biomolecular interaction between biotin and streptavidin was done using magnetic nanoparticles and a silicon substrate with a self-assembled monolayer. Streptavidin was immobilized on the magnetic particles, and biotin was attached to the monolayer-modified substrate. The reaction of streptavidin-modified magnetic particles on the biotin-modified substrate was clearly observed under an optical microscope. The magnetic signals from the particles were detected using a magnetic force microscope. The results of this study demonstrate that the combination of a monolayer-modified substrate with biomolecule-modified magnetic particles is useful for detecting biomolecular interactions in medical and diagnostic analyses.

Binding Sites↗
PubMed2005

High-affinity chelator thiols for switchable and oriented immobilization of histidine-tagged proteins: a generic platform for protein chip technologies.

Protein micro-/nanoarrays are becoming increasingly important in systematic approaches for the exploration of protein-protein interactions and dynamic protein networks, so there is a high demand for specific, generic, stable, uniform, and locally addressable protein immobilization on solid supports. Here we present multivalent metal-chelating thiols that are suitable for stable binding of histidine-tagged proteins on biocompatible self-assembled monolayers (SAMs). The architectures and physicochemical properties of these SAMs have been probed by various surface-sensitive techniques such as contact angle goniometry, ellipsometry, and infrared reflection-absorption spectroscopy. The specific molecular organization of proteins and protein complexes was demonstrated by surface plasmon resonance, confocal laser scanning, and atomic force microscopy. In contrast to the mono-NTA/His6 tag interaction, which has major drawbacks because of its low affinity and fast dissociation, drastically improved stability of protein binding by these multivalent chelator surfaces was observed. The immobilized histidine-tagged proteins are uniformly oriented and retain their function. At the same time, proteins can be removed from the chip surface under mild conditions (switchability). This new platform for switchable and oriented immobilization should assist proteome-wide wide analyses of protein-protein interactions as well as structural and single-molecule studies.

Biosensing Techniques↗
PubMed2003

Prostate carcinoma tissue proteomics for biomarker discovery.

BACKGROUND: The advent of the prostate-specific antigen (PSA) test has had a profound impact on the diagnosis and treatment of prostate carcinoma. However, the use of PSA levels alone for screening for prostate carcinoma was compromised by the variations in the amount of PSA produced by the benign prostatic tissue specimens. Proteins were involved in various pathways that determine the behavior of a cell. Therefore, information regarding proteins may reveal drug targets and/or markers for early detection. METHODS: The authors used surface-enhanced laser desorption/ionization time-of-flight mass spectrometry to determine the protein profiles from fresh tissues of the prostate. Laser capture microdissection was performed to isolate pure populations of cells. RESULTS: The authors identified a protein with an average m/Z of 24,782.56 +/- 107.27 that was correlated with the presence of prostate carcinoma. Furthermore, using laser capture microdissection, they demonstrated that the origin of this protein, which the authors designated PCa-24, was derived from the epithelial cells of the prostate. PCa-24 expression was detected in 16 of 17 (94%) prostate carcinoma specimens but not in paired normal cells. In addition, this protein was not expressed in any of the 12 benign prostatic hyperplasia specimens that were assayed. CONCLUSIONS: PCa-24 may be useful a marker for prostate carcinoma.

Adult↗
PubMed2006

DNA tile based self-assembly: building complex nanoarchitectures.

DNA tile based self-assembly provides an attractive route to create nanoarchitectures of programmable patterns. It also offers excellent scaffolds for directed self-assembly of nanometer-scale materials, ranging from nanoparticles to proteins, with potential applications in constructing nanoelectronic/nanophotonic devices and protein/ligand nanoarrays. This Review first summarizes the currently available DNA tile toolboxes and further emphasizes recent developments toward self-assembling DNA nanostructures with increasing complexity. Exciting progress using DNA tiles for directed self-assembly of other nanometer scale components is also discussed.

Base Composition↗
PubMed2003

Cell detection based on protein array using modified glass slides.

A protein array for cell detection was fabricated by spotting different antibodies on modified glass slides. Glass slides were modified to allow antibodies to be immobilized on it and to selectively bind antigens. Antibodies were specially selected with the cells to be detected as targets, which permitted target cells in samples to bind specifically to the array with little nonspecific binding. Results can be obtained by directly putting the samples onto the array for 1 h or a little longer to let the cells specifically interact with the antibodies. After washing the unbound samples away, images were observed with a microscope and captured with a CCD camera. The assessment of antibody-cell binding was evaluated by capturing red blood cells (RBCs) in human blood with blood group antibodies (anti-A and anti-B). Blood group antibodies were spotted on the modified glass slide and kept at 4<.deg> degrees C overnight for immobilization. Human blood samples diluted to different concentrations were used to examine the sensitivity and specificity of the method.

ABO Blood-Group System↗
PubMed2005

Fast prototyping of hydrophobic disposable polymer support arrays for matrix-assisted laser desorption/ionization-time of flight-mass spectrometry of proteins by atmospheric molding.

A fast protocol for prototyping hydrophobic disposable poly(alkyl methacrylate-co-methyl methacrylate) copolymer sample support arrays for matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) of proteins by atmospheric molding is introduced. The sample support arrays were replicated by molding prepolymer alkyl methacrylate solutions into sandwich molds containing a micromachined silicon master, an aluminum spacer, and glass cover plates, followed by UV-initiated in situ polymerization under atmospheric pressure. The fabrication procedure enables a simultaneous fabrication/modification of single-use polymer arrays by a targeted selection of functional groups of the copolymerized monomers during molding. The one-step modification during the fabrication is demonstrated for enhanced protein adsorption to the modified materials by introduction of hydrophobic butyl-, dodecyl-, and octadecyl groups to the polymer backbone without a need for additional surface coating or derivatization. The MALDI-MS performance of the new polymer chips was tested for spectral measurements of bovine pancreas insulin, horse heart myoglobin, and bovine serum albumin. The protein adsorption to the new hydrophobic copolymer chips was studied for bovine pancreas trypsinogen; the sample desalting parameters, such as time and volume, were optimized for myoglobin as model proteins. A significant signal increase was achieved after efficient desalting of an insect Delta11-desaturase membrane protein fragment from a complex elution buffer (100 mM phosphate, 10 mM tris(hydroxyethyl)aminomethane, 0.5 M NaCl, and 10 mM ethylenediamine tetraacetic acid) on the poly(butyl methacrylate-co-methyl methacrylate) copolymer chip (monomer ratio 8:2 v/v) by simply washing the target zones. The new chips offer reduced sample manipulation and device fabrication times as well as simple operation.

Atmosphere↗
PubMed2004

New directions of miniaturization within the biomarker research area.

An overview of the current trends within protein expression profiling is given where multidimensional separation of both gel and liquid phase techniques linked to mass spectrometry is viewed as a major route in the global proteome mapping. A clear trend in these biochemical developments is the effort to sequence and identify low-abundant protein expressions where assay miniaturization and integrated sample processing play a central role. Two areas of miniaturization within the proteomics field are addressed: (i) sample cleanup and enrichment, and (ii) silicon microstructure developments for protein chip microarrays.

Animals↗
PubMed2006

A chip-based miniaturized format for protein-expression profiling: the exploitation of comprehensively produced antibodies.

Numerous antibodies have been developed and validated in recent years, and show promise for use in novel functional protein assays. Such assays would be an alternative to pre-existing comprehensive assays, such as DNA microarrays. Antibody microarrays are thought to represent those functional protein assays. While a variety of attempts have been made to apply DNA microarray technology to antibody microarrays, a fully optimized protocol has not been established. We have been conducting a project to comprehensively produce antibodies against mouse KIAA ("KI" stands for "Kazusa DNA Research Institute" and "AA" are reference characters) proteins. Using our library of antibodies, we established a novel antibody microarray format that utilizes surface plasmon resonance (SPR) technology. A label-free real-time measurement of protein expression in crude cell lysates was achieved by direct readout of the bindings using SPR. Further refinement of the antibody microarray format enabled us to detect a smaller quantity of target proteins in the lysate without the bulk effect. In this review, we first summarize available antibody array formats and then describe the above-mentioned format utilizing updated SPR technology.

Animals↗