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Biomedical subjects

Christine D Keating

Publications and source records attributed to Christine D Keating.

11 recordsLinked to original sources
PubMed2006

Coupling molecular beacons to barcoded metal nanowires for multiplexed, sealed chamber DNA bioassays.

We have combined molecular beacon (MB) probes with barcoded metal nanowires to enable no-wash, sealed chamber, multiplexed detection of nucleic acids. Probe design and experimental parameters important in nanowire-based MB assays are discussed. Loop regions of 24 bases and 5 base pair stem regions in the beacon probes gave optimal performance. Our results suggest that thermodynamic predictions for secondary structure stability of solution-phase MB can guide probe design for nanowire-based assays. Dengue virus-specific probes with predicted solution-phase DeltaG of folding in 500 mM buffered NaCl of approximately -4 kcal/mol performed better than those with DeltaG > -2 or < -6 kcal/mol. Buffered 300-500 mM NaCl was selected after comparison of several buffers previously reported for similar types of assays, and 200-500 mM NaCl was found to be the optimal ionic strength for the hybridization temperatures (25 and 50 degrees C) and probe designs used here. Target binding to the surface as a function of solution concentration fit a Sips isotherm with Kd = 1.7 +/- 0.3 nM. The detection limit was approximately 100 pM, limited by incomplete quenching. Single base mismatches could be discriminated from fully complementary targets. Oligonucleotide target sequences specific for human immunodeficiency, hepatitis C, and severe acute respiratory viruses were assayed simultaneously in a no-wash, sealed chamber, multiplexed experiment in which each of three probe sequences was attached to a different pattern of encoded nanowires. Finally, we demonstrated that probe-coated nanowires retain their selectivity and sensitivity in a triplexed assay after storage for over 3 months.

DNA, Viral↗
PubMed2006

Distance-dependent emission from dye-labeled oligonucleotides on striped Au/Ag nanowires: effect of secondary structure and hybridization efficiency.

When fluorescently tagged oligonucleotides are located near metal surfaces, their emission intensity is impacted by both electromagnetic effects (i.e., quenching and/or enhancement of emission) and the structure of the nucleic acids (e.g., random coil, hairpin, or duplex). We present experiments exploring the effect of label position and secondary structure in oligonucleotide probes as a function of hybridization buffer, which impacts the percentage of double-stranded probes on the surface after exposure to complementary DNA. Nanowires containing identifiable patterns of Au and Ag segments were used as the metal substrates in this work, which enabled us to directly compare different dye positions in a single multiplexed experiment and differences in emission for probes attached to the two metals. The observed metal-dye separation dependence for unstructured surface-bound oligonucleotides is highly sensitive to hybridization efficiency, due to substantial changes in DNA extension from the surface upon hybridization. In contrast, fluorophore labeled oligonucleotides designed to form hairpin secondary structures analogous to solution-phase molecular beacon probes are relatively insensitive to hybridization efficiency, since the folded form is quenched and therefore does not appreciably impact the observed distance-dependence of the response. Differences in fluorescence patterning on Au and Ag were noted as a function of not only chromophore identity but also metal-dye separation. For example, emission intensity for TAMRA-labeled oligonucleotides changed from brighter on Ag for 24-base probes to brighter on Au for 48-base probes. We also observed fluorescence enhancement at the ends of nanowires and at surface defects where heightened electromagnetic fields affect the fluorescence.

DNA↗
PubMed2006

Nanoparticle conjugation increases protein partitioning in aqueous two-phase systems.

We describe the effect of bioconjugation to colloidal Au nanoparticles on protein partitioning in poly(ethylene glycol) (PEG)/dextran aqueous two-phase systems (ATPS). Horseradish peroxidase (HRP) was conjugated to colloidal Au nanoparticles by direct adsorption. Although HRP alone had very little phase preference, HRP/Au nanoparticle conjugates typically partitioned to the PEG-rich phase, up to a factor of 150:1 for conjugates of 15-nm colloidal Au. Other protein/Au nanoparticle conjugates exhibited partitioning of greater than 2000:1 to the dextran-rich phase, as compared with approximately 5:1 for the free protein. The degree of partitioning was dependent on polymer concentration and molecular weight, nanoparticle diameter, and in some instances, nanoparticle concentration in the ATPS. The substantial improvements in protein partitioning achievable by conjugation to Au nanoparticles appear to result largely from increased surface area of the conjugates and require neither chemical modification of the proteins or polymers with affinity ligands, increased polymer concentrations, nor addition of high concentrations of salts. Adsorption to colloidal particles thus provides an attractive route for increased partitioning of enzymes and other proteins in ATPS. Furthermore, these results point to ATPS partitioning as a powerful means of purification for biomolecule/nanoparticle conjugates, which are increasingly used in diagnostics and materials applications.

Cell Physiological Phenomena↗
PubMed2006

Temperature-programmed assembly of DNA:Au nanoparticle bioconjugates.

Temperature has been used to control the order of assembly events in a solution containing three types of particles to be linked by two different sets of complementary DNA. At higher temperatures, only the duplexes having higher thermal stability were able to form. By starting at a high temperature and then cooling the sample, these more stable sequences hybridized first, followed by the less stable sequences at lower temperatures. Because of the use of thiolated DNA on Au particles, some loss and exchange of the DNA strands occurred at elevated temperatures. However, since cooperativity favors the "correct" assemblies, Au-S bond lability did not appreciably impact the order of the assembly process. Temperature programming combines the selectivity of DNA-directed assembly with the ability to control the order in which several complementary strands hybridize in a common solution and could contribute to the synthesis of more complex nanostructured materials.

Base Pairing↗
PubMed2005

Partitioning and assembly of metal particles and their bioconjugates in aqueous two-phase systems.

The behavior of metal nanospheres and nanowires and their bioconjugates in aqueous two-phase systems (ATPS) is described. The ATPS used in this work comprised poly(ethylene glycol) (PEG), dextran, and water or aqueous buffer. Au and Ag nanospheres less than 100 nm in diameter partition between the PEG-rich and dextran-rich phases on the basis of their surface chemistry and can be separated on this basis. Larger Au nanospheres and wires accumulate at the interface between the two aqueous phases. The influence of polymer molecular weight and concentration on interfacial assembly of Au wires is described. DNA-derivatized nanowires at the aqueous/aqueous interface retain the ability to selectively bind to fluorescent complementary DNA. In addition, Au nanoparticles have been bound to Au wires via selective DNA hybridization at the ATPS interface. Transmission electron microscopy and thermal denaturation experiments confirm that DNA-driven assembly is responsible for the formation of the nanosphere/wire assemblies. These results demonstrate the biocompatibility of the two-phase interface and point to future use as scaffolding in biorecognition-driven assembly.

DNA↗
PubMed2005

Dynamic microcompartmentation in synthetic cells.

An experimental model for cytoplasmic organization is presented. We demonstrate dynamic control over protein distribution within synthetic cells comprising a lipid bilayer membrane surrounding an aqueous polymer solution. This polymer solution generally exists as two immiscible aqueous phases. Protein partitioning between these phases leads to microcompartmentation, or heterogeneous protein distribution within the "cell" interior. This model cytoplasm can be reversibly converted to a single phase by slight changes in temperature or osmolarity, such that local protein concentrations can be manipulated within the vesicle interior.

Base Sequence↗
PubMed2005

Batch preparation of linear Au and Ag nanoparticle chains via wet chemistry.

We present a simple, wet-chemical method for fabricating linear chains of Au and Ag nanoparticles by selectively etching alternating segments from striped metal nanowires. Nanowires composed of sacrificial Ni segments as well as Au and/or Ag segments were prepared by templated electrodeposition in the pores of alumina membranes. After removal from the membrane, wires were coated with SiO2, and selective etching revealed the nanoparticle chains. Extinction spectra are presented as a function of interparticle spacing.

Electrochemistry↗
PubMed2004

Effect of macromolecular crowding on DNA:Au nanoparticle bioconjugate assembly.

DNA:Au nanosphere bioconjugates have applications in biosensing and in the bottom-up assembly of materials. These bioconjugates can be selectively assembled into three-dimensional aggregates upon addition of complementary DNA oligonucleotides and can be dissociated by heating above a melting transition temperature at which the DNA duplexes are denatured. Herein we describe the impact of polymeric solutes on the thermal denaturation behavior of DNA:Au nanoparticle bioconjugate assemblies. Polymeric solutes can dramatically impact biochemical reactions via macromolecular crowding. Poly(ethylene glycol)s (PEGs) and dextrans of varying molecular weights were used as crowding reagents. While both PEG and dextran increased the stability of DNA:Au aggregates, melting transition temperatures in the presence of PEG were impacted more significantly. Polymer molecular weight was less important than polymer chemistry and weight percent in solution. For a high (15%) weight percent of PEG, aggregation was observed even in the absence of complementary oligonucleotides. These results underscore the importance of polymer chemistry in addition to physical volume exclusion in macromolecular crowding and point to the importance of understanding these effects when designing biorecognition-based nanoparticle assembly schemes in complex matrixes (i.e., any involving polymeric solutes).

Base Sequence↗
PubMed2002

Aqueous phase separation in giant vesicles.

We report the synthesis and initial characterization of approximately 10 mum diameter lipid vesicles that contain two distinct aqueous phases. The aqueous two-phase system is a dextran/poly(ethylene glycol) solution that exhibits temperature-dependent phase behavior. Vesicles were prepared above the phase transition temperature of the polymer solution. Upon cooling to room temperature, the polymer solution phase separated both within the vesicles and in the bulk solution. The location of poly(ethylene glycol)-rich and dextran-rich phases was determined by fluorescence microscopy. These structures are exciting in that they enable for the first time the interior volume of liposomes to be structured.

Dextrans↗
PubMed2002

Hybridization and enzymatic extension of au nanoparticle-bound oligonucleotides.

We have investigated the impact of steric effects on the hybridization and enzymatic extension of oligonucleotides bound to 12-nm colloidal Au particles. In these experiments, a nanoparticle-bound 12-mer sequence is hybridized either to its solution phase 12-mer complement or to an 88-mer template sequence. The particle-bound oligonucleotide serves as a primer for enzymatic extension reactions, in which covalent incorporation of nucleotides to form the complement of the template is achieved by the action of DNA polymerase. Primers were attached via-C(6)H(12)SH, -C(12)H(24)SH, and -TTACAATC(6)H(12)SH linkers attached at the 5' end. Primer coverage on the nanoparticles was varied by dilution with (5')HSC(6)H(12)AAA AAA(3'). Hybridization efficiencies were determined as a function of linker length, primer coverage, complement length (12-mer vs 88-mer), and primer:complement concentration ratio. In all cases, hybridization for the 88-mer was less efficient than for the 12-mer. Low primer surface coverage, greater particle-primer separation, and higher primer:complement ratios led to optimal hybridization. Hybridization efficiencies as high as 98% and 75% were observed for the 12-mer and 88-mer, respectively. Enzymatic extension of particle-bound primers was observed under all conditions tested; however, the efficiency of the reaction was strongly affected by linker length and primer coverage. Extension of primers attached by the longest linker was as efficient as the solution-phase reaction.

DNA↗