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At least 469 records · Page 26Linked to original sources
PubMed2006

PCR-free DNA detection using a magnetic bead-supported polymeric transducer and microelectromagnetic traps.

A fluorescent polymeric hybridization transducer supported on magnetic microbeads was investigated for the rapid, ultrasensitive, and sequence-specific detection of DNA. We show that the polymer derivative can be used to detect target DNA directly on magnetic particles by preparing "target-ready" microbeads grafted with the polymer and suitable DNA probes. A detection limit of approximately 200 target copies in a probed volume of 150 muL (1.4 copies/muL) was obtained for a DNA sequence specific to Candida albicans This detection scheme does not require the release of the hybridized target DNA prior to its detection or the labeling or amplification of the nucleic acids. Furthermore, we show that the fluorescence from these biosensing magnetic beads can be read while magnetically confined in a small volume by a microelectromagnetic trap, which offers the possibility of performing both the preconcentration and detection steps simultaneously on the same support. The combination of the fluorescent polymer biosensor with magnetic particle-assisted DNA preconcentration extends the application of this ultrasensitive biosensor to biological samples with complex matrixes and to integrated lab-on-a-chip platforms, where it could be used for fast multitarget DNA detection in point-of-care diagnostics and field analysis.

Base Sequence↗
PubMed2006

Electrohydrodynamic generation and delivery of monodisperse picoliter droplets using a poly(dimethylsiloxane) microchip.

We developed a drop-on-demand microdroplet generator for the discrete dispensing of biosamples into a bioanalytical unit. This disposable PDMS microfluidic device can generate monodisperse droplets of picoliter volume directly out of a plane sidewall of the microfluidic chip by an electrohydrodynamic mechanism. The droplet generation was accomplished without using either an inserted capillary or a monolithically built-in tip. The minimum droplet volume was approximately 4 pL, and the droplet generation was repeatable and stable for at least 30 min, with a typical variation of less than 2.0% of drop size. The Taylor cone, which is usually observed in electrospray, was suppressed by controlling the surface wetting property of the PDMS device as well as the surface tension of the sample liquids. A modification of the channel geometry right before the opening of the microchannel also enhanced the continuous droplet generation without applying any external pumping. A simple numerical simulation of the droplet generation verified the importance of controlling the surface wetting conditions for the droplet formation. Our microdroplet generator can be effectively applied to a direct interface of a microfluidic chip to a biosensing unit, such as AMS, MALDI-MS or protein microarray-type biochips.

Chemical Phenomena↗
PubMedDate not supplied

Synthesis and characterization of a photoactivatable glycoaryldiazirine for surface glycoengineering.

Biological systems make considerable use of specific molecular interactions. Many biomolecules involved in biorecognition are glycosylated, the carbohydrate moiety playing an essential role. Controlled surface glycoengineering is thus of crucial importance in biosensing, cell guidance, and biomedical applications. This study describes the synthesis of an aryldiazirine-derivatized galactose and the functionalization of surfaces by carbohydrates using photochemical immobilization techniques. A photoactivatable glycosylated reagent was synthesized by addition of thiogalactopyranose to the maleimide group of N-[m-[3-(trifluoromethyl)diazirin-3-yl]phenyl]-4-maleimidobutyr amide (MAD) to give N-[m-[3-(trifluoromethyl)diazirin-3-yl]phenyl]-4-[3-thio (1-D-galactopyranosyl)succinimidyl]butyramide (MAD-Gal). The structure of the newly synthesized molecule was confirmed by UV spectroscopy, photoactivation, 1H NMR, and 13C NMR. MAD-Gal was immobilized on thin diamond films by photoactivation of the diazirine function (350 nm). Surface modification was investigated by XPS (X-ray photoelectron spectroscopy) and ToF-SIMS (time-of-flight secondary ion mass spectrometry). Imaging ToF-SIMS was applied to detect glycopatterns generated by mask-assisted lithography.

Glycosides↗
PubMed2006

Patterned protein films on poly(lipid) bilayers by microcontact printing.

The use of polymerized lipid bilayers as substrates for microcontact printing (muCP) of protein films was investigated. We have previously shown that vesicle fusion of bis-SorbPC, a dienoate lipid, on glass and silica substrates, followed by redox-initiated radical polymerization, produces a planar supported lipid bilayer (PSLB) that is ultrastable(1a) [Ross, E. E.; Rozanski, L. J.; Spratt, T.; Liu, S.; O'Brien, D. F.; Saavedra, S. S. Langmuir 2003, 19, 1752] and highly resistant to nonspecific adsorption of dissolved proteins [Ross, E. E.; Spratt, T.; Liu, S.; Rozanski, L. J.; O'Brien, D. F.; Saavedra, S. S. Langmuir 2003, 19, 1766].(1b) Here we demonstrate that muCP of bovine serum albumin (BSA) onto a dried poly(bis-SorbPC) PSLB from a poly(dimethylsiloxane) (PDMS) stamp produces a layer of strongly adsorbed protein, comparable in surface coverage to films printed on glass surfaces. Immobilization of proteins on poly(PSLB)s has potential applications in biosensing, and this work shows that direct muCP of proteins is a technically simple approach to create immobilized monolayers, as well as multilayers of different proteins.

Biofilms↗
PubMedDate not supplied

Candida bombicola cells immobilized on patterned lipid films as enzyme sources for the transformation of arachidonic acid to 20-HETE.

Preparation of biocompatible surfaces for immobilization of enzymes and whole cells is an important aspect of biotechnology due to their potential applications in biocatalysis, biosensing, and immunological applications. In this report, patterned thermally evaporated octadecylamine (ODA) films are used for the immobilization of Candida bombicola cells. The attachment of the cells to the ODA film surface occurs possibly through nonspecific interactions such as hydrophobic interactions between the cell walls and the ODA molecules. The enzyme cytochrome P450 present in the immobilized yeast cells on the ODA film surface was used for the transformation of the arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE). The assembly of cells on the hydrophobic ODA surface was confirmed by quartz crystal microgravimetry (QCM), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). SEM images confirmed the strong binding of the yeast cells to the ODA film surface after biocatalytic reactions. Moreover, the biocomposite films could be easily separated from the reaction medium and reused.

Amines↗
PubMedDate not supplied

Hepatocyte viability and protein expression within hydrogel microstructures.

Poly(ethylene) glycol (PEG) hydrogels have been successfully used to entrap mammalian cells for potential high throughput drug screening and biosensing applications. To determine the influence of PEG composition on the production of cellular protein, mammalian hepatocytes were maintained in PEG hydrogels for 7 days. Total cell viability, total protein production, and the production of two specific proteins, albumin and fibronectin, were monitored. Studies revealed that while PEG composition has no effect on cell viability, increasing amounts of PEG in the hydrogel decrease the amount of protein production by the cells after 7 days from 1.0 x 10(5) +/- 1.7 x 10(4) to 5.2 x 10(3) +/- 1.3 x 10(3) g accumulated protein/mL/million cells. Additionally, cells entrapped in PEG hydrogels produce greater amounts of protein than traditional monolayer culture (1.5 x 10(3) +/- 1.9 x 10(2) g accumulated protein/mL/million cells after 7 days). The addition of the synthetic peptide RGD to 10% PEG hydrogels altered the production of the proteins albumin and fibronectin. Hydrogels with the RGD sequence produced 287 +/- 27 ng/mL/million cells albumin after 7 days, an order of magnitude greater than monolayer cultures, whereas cells in hydrogels without the RGD sequence produced undetectable levels of albumin. Conversely, cells entrapped in 10% PEG hydrogels without the RGD sequence produced 1014 +/- 328 ng/mL/million cells fibronectin after 7 days, whereas 10% PEG hydrogels with the RGD sequence produced 200 +/- 58 ng/mL/million cells fibronectin after 7 days.

Animals↗
PubMedDate not supplied

Functionalization of gold and glass surfaces with magnetic nanoparticles using biomolecular interactions.

Advances in nanotechnology have enabled the production and characterization of magnetic particles with nanometer-sized features that can be functionalized with biological recognition elements for numerous applications in biotechnology. In the present study, the synthesis of and interactions between self-assembled monolayers (SAMs) on gold and glass surfaces and functionalized magnetic nanoparticles have been characterized. Immobilization of 10-15 nm streptavidin-functionalized nanoparticles to biotinylated gold and glass surfaces was achieved by the strong interactions between biotin and streptavidin. Fluorescent streptavidin-functionalized nanoparticles, biotinylated surfaces, and combinations of the two were characterized by Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and electron and fluorescent microscopy to confirm that little or no functionalization occurred in nonbiotinylated regions of the gold and glass surfaces compared to the biotinylated sites. Together these techniques have potential use in studying the modification and behavior of functionalized nanoparticles on surfaces in biosensing and other applications.

Biotinylation↗
PubMedDate not supplied

Development of novel yeast cell surface display system for homo-oligomeric protein by coexpression of native and anchored subunits.

Streptavidin derived from Streptomyces avidinii was displayed on the cell surface of the yeast Saccharomyces cerevisiae by cell-surface engineering using two types of plasmid for the expression of a native subunit and an anchored subunit fused with the C-terminus of 318 amino acids of Flo1p containing a glycosylphosphatidylinositol anchor attachment signal. The displayed streptavidin had the binding ability for biotinylated compounds. This was confirmed by fluorescence microscopy after the adsorption of yeast cells displaying streptavidin and biotinylated fluorescein isothiocyanate. On the other hand, streptavidin produced by cells harboring only the plasmid for the expression of the anchored subunit showed a very low binding activity for biotinylated compounds. Cells displaying streptavidin may constitute novel whole-cell affinity adsorbents widely used for immunoassay and biosensing. This coexpression method will ensure that proteins, such as homo- and hetero-oligomeric proteins, are displayed on the cell surface in an active form.

Adsorption↗
PubMed2002

Self-assembled nanoparticle probes for recognition and detection of biomolecules.

Colloidal gold nanocrystals have been used to develop a new class of nanobiosensors that is able to recognize and detect specific DNA sequences and single-base mutations in a homogeneous format. At the core of this biosensor is a 2.5-nm gold nanoparticle that functions as both a nano-scaffold and a nano-quencher (efficient energy acceptor). Attached to this core are oligonucleotide molecules labeled with a thiol group at one end and a fluorophore at the other. This hybrid bio/inorganic construct is found to spontaneously assemble into a constrained arch-like conformation on the particle surface. Binding of target molecules results in a conformational change, which restores the fluorescence of the quenched fluorophore. Unlike conventional molecular beacons with a stem-and-loop structure, the nanoparticle probes do not require a stem, and their background fluorescence increases little with temperature. In comparison with the organic quencher Dabcyl (4,4'-dimethylaminophenyl azo benzoic acid), metal nanoparticles have unique structural and optical properties for new applications in biosensing and molecular engineering.

Base Sequence↗
PubMed2004

Reversible modulation of quantum dot photoluminescence using a protein- bound photochromic fluorescence resonance energy transfer acceptor.

Multiple copies ( approximately 20) of Escherichia coli maltose binding protein (MBP) were coordinated to luminescent semiconductor quantum dots (QDs) via a C-terminal oligohistidine segment. The MBP was labeled with a sulfo-N-hydroxysuccinimide-activated photochromic BIPS molecule (1',3-dihydro-1'-(2-carboxyethyl)-3,3-dimethyl-6-nitrospiro[2H-1-benzopyran-2,2'-(2H)-indoline]) at two different dye-to-MBP ratios; D/P = 1 and 5. The ability of MBP-BIPS to modulate QD photoluminescence was tested by switching BIPS from the colorless spiropyran (SP) to the colored merocyanine (MC) using white light (>500 nm) or UV light ( approximately 365 nm), respectively. QDs surrounded by MBP-BIPS with D/P = 1 were quenched on average approximately 25% with consecutive repeated switches, while QDs surrounded by MBP-BIPS with D/P = 5 were quenched approximately 60%. This result suggests a possible use of BIPS-labeled proteins in QD-based nanostructures as part of a threshold switch or other biosensing device.

Benzopyrans↗
PubMed2005

Enzymatic fabrication of DNA nanostructures: extension of a self-assembled oligonucleotide monolayer on gold arrays.

Nucleic acid nanostructures are useful as templates for bionanofabrication of composite molecular nanostructures in materials science, molecular electronics, and biosensing. Here, we demonstrate that terminal deoxynucleotidyl transferase, which repetitively adds mononucleotides to the 3' end of a short DNA initiator, can be used to rapidly fabricate DNA nanostructures up to 121 nm high with lateral dimensions from 0.1 to 4 mum in 2 h. These programmable scaffolds can potentially be employed to build more complex nanostructures consisting of natural or unnatural nucleotides with selective docking sites along the single-stranded DNA.

DNA↗
PubMed2006

Nanoparticle-based sensing of glycan-lectin interactions.

Here we present the first report on nanoparticle-based biosensing of glycan markers of diseases. The protocol relies on the competition between a nanocrystal (CdS)-tagged sugar and the target sugar for the binding sites of surface-confined lectin and monitoring the extent of competition through highly sensitive electrochemical detection of the captured nanocrystal. This development is expected to allow decentralized detection of carbohydrate moieties and lectin-carbohydrate interactions to be performed more rapidly, sensitively, inexpensively, and reliably.

Lectins↗
PubMed2006

Grafting and patterned grafting of block copolymer nanotubes onto inorganic substrates.

Chemical patterning of inorganic substrates by soft lithography has enabled various high-tech applications and cutting-edge fundamental research. In this paper, we report on methods for the grafting and patterned grafting of block copolymer nanotubes onto glass and mica surfaces. Under optimized conditions the density of such grafted nanotubes can be high, and most of the grafted tubes are in a standing position even after solvent evaporation. Surfaces modified with exotic reagents such as block copolymer nanofibers or nanotubes may find applications in biosensing, etc.

Journal Article↗
PubMed2005

Chemiluminescent reactions induced by gold nanoparticles.

The reaction of gold nanoparticles with a potassium periodate-sodium hydroxide-carbonate system undergoes chemiluminescence with three emission bands at 380-390, 430-450, and 490-500 nm, respectively. It was found that the light intensity increased linearly with the concentration of the gold nanoparticles, and the CL intensity increased dramatically when the citrate ions on the nanoparticle surface were replaced by SCN(-). The shape, size, and oxidation state of gold nanoparticles after the chemiluminescent reaction were characterized by UV-visible absorption spectrometry, transmission electron microscopy (TEM), and X-ray photoelectron spectrometry (XPS). Gold nanoparticles are supposed to function as a nanosized platform for the observed chemiluminescent reactions. A chemiluminescent mechanism has been proposed in which the interaction between free CO(3)(*-) and O(2)(*-) radicals generated by a KIO(4)-NaOH-Na(2)CO(3) system and gold nanoparticles results in the formation of emissive intermediate gold(I) complexes, carbon dioxide dimers, and singlet oxygen molecular pairs on the surface of the gold nanoparticles. This work is not only of great importance for gaining a better understanding of the unique optical and surface properties and chemical reactivity of nanoparticles but also of great potential for developing new biosensing and immunolabeling technologies.

Journal Article↗
PubMed2005

Semiconductor nanowires for subwavelength photonics integration.

This article focuses on one-dimensional (1D) semiconductor subwavelength optical elements and assesses their potential use as active and passive components in photonic devices. An updated overview of their optical properties, including spontaneous emission, ultrafast carrier dynamics, cavity resonance feedback (lasing), photodetection, and waveguiding, is provided. The ability to physically manipulate these structures on surfaces to form simple networks and assemblies is the first step toward integrating chemically synthesized nanomaterials into photonic circuitry. These high index semiconductor nanowires are capable of efficiently guiding light through liquid media, suggesting a role for such materials in microfluidics-based biosensing applications.

Journal Article↗
PubMed2006

Structure and reactivity of alkoxycarbonyl (ester)-terminated monolayers on silicon: sum frequency generation spectroscopy.

Carboxylic acid-terminated monolayers on crystalline silicon surfaces can be readily modified with biological macromolecules for the fabrication of semiconductor-based biosensing devices. They were prepared by acid-catalyzed hydrolysis of alkoxycarbonyl (ester)-terminated monolayers and studied by vibrational sum frequency generation (SFG) spectroscopy. The C-H vibration region of the SFG spectra consists of strong methyl bands with significant contributions from methylene stretching modes, indicating that these monolayers are generally ordered but with considerable gauche defects in the alkyl chains in comparison with n-alkyl monolayers. After hydrolysis, the methylene stretching modes prevail, with "residues" of the methyl bands, indicating incomplete hydrolysis and disruption of the monolayer structure. This work demonstrates that SFG is capable of providing quantitative information on structure-reactivity correlations in organic monolayers.

Journal Article↗
PubMed2004

On-line monitoring of polymer deposition for tailoring the waveguide characteristics of love-wave biosensors.

The Love-wave sensor is an acoustic sensing device which is particularly suitable for sensing in a liquid environment. The superior characteristics of the device are achieved by the use of an acoustic waveguide, consisting of a thin layer deposited on the surface of the substrate material. The exact thickness and material properties of the layer will not only determine sensitivity and sensing performance of the resulting device but can also be adjusted to generate higher-order Love modes. Thus, to obtain a sensing device with the desired specifications, precise control over the process of waveguide deposition is required. This has been realized by implementation of a vapor deposition polymerization system where the transmission curve (amplitude vs frequency) of one of the sensing devices is continuously monitored during deposition. As soon as the desired device specifications are reached, the deposition can be interrupted immediately. From the recorded transmission curves, information about the sensitivity of the device can be deduced, and the formation of higher-order Love modes can be visualized. The system has been used to produce biosensors based on various Love modes. It is shown that sensors operated on higher-order Love modes have a high mass sensitivity which, together with their excellent shielding properties, makes them advantageous for biosensing in conducting buffer solutions.

Journal Article↗
PubMed2005

Encapsulation of carbon nanotubes by self-assembling peptide amphiphiles.

We demonstrate the dispersion and noncovalent functionalization of carbon nanotubes in water using peptide amphiphiles each consisting of a short hydrophobic alkyl tail coupled to a more hydrophilic peptide sequence. The assembly of peptide amphiphile molecules on the surfaces of carbon nanotubes adds biofunctionality to these one-dimensional conductors and simultaneously eliminates the hydrophobic nanotube-water interface, thus dispersing them in the aqueous medium. This should occur without the degradation of their structural, electronic, and optical properties caused by covalent functionalization and without the need for specific peptide sequences designed to bind with nanotube surfaces. The encapsulation by peptide amphiphiles is confirmed using transmission electron microscopy and optical absorbance spectroscopy and may have significant future applications in biosensing or medicine.

Microscopy, Electron↗