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Biomedical subjects

John D Brennan

Publications and source records attributed to John D Brennan.

At least 19 recordsLinked to original sources
PubMed2006

Entrapment of horseradish peroxidase in sugar-modified silica monoliths: toward the development of a biocatalytic sensor.

A miniaturized HRP-entrapped bioreactor was prepared by a one-step enzyme immobilization method using a biocompatible sol-gel processing method employing either diglycerylsilane (DGS) or sodium silicate (SS) as precursors and a covalently tethered sugar, N-(3-triethoxysilylpropyl)gluconamide (GLS) as a silica modifier. Factors such as leaching, catalytic efficiency and long-term stability were examined to assess the role of the precursor and modifier in influencing enzyme performance. The results showed that sodium silicate derived materials modified with covalently bound sugars at a level of 10 mol% were optically transparent and provided the highest catalytic turnover rate for entrapped HRP. The stability and reusability of the entrapped HRP was found to be satisfactory for at least 1 month in the GLS-doped SS materials, and the entrapped HRP was able to respond linearly to the presence of peroxide over the concentration range of 0-750 microM with a detection limit of 6 microM, demonstrating the potential of this material for the development of a reusable optical biosensor.

Biosensing Techniques↗
PubMed2006

Two-site ionic labeling with pyranine: implications for structural dynamics studies of polymers and polypeptides by time-resolved fluorescence anisotropy.

Time-resolved fluorescence anisotropy (TRFA) is widely used to study dynamic motions of biomolecules in a variety of environments. However, depolarization due to rapid side chain motions often complicates the interpretation of anisotropy decay data and interferes with the accurate observation of segmental motions. Here, we demonstrate a new method for two-point ionic labeling of polymers and biomolecules that have appropriately spaced amino groups using the fluorescent probe 8-hydroxyl-1,3,6-trisulfonated pyrene (pyranine). TRFA analysis shows that such labeling provides a more rigid attachment of the fluorophore to the macromolecule than the covalent or single-point ionic labeling of amino groups, leading to time-resolved anisotropy decays that better reflect the backbone motion of the labeled polymer segment. Optimal coupling of pyranine to biomolecule dynamics is shown to be obtained for appropriately spaced Arg groups, and in such cases the ionic binding is stable up to 150 mM ionic strength. TRFA was used to monitor the behavior of pyranine-labeled poly(allylamine) (PAM) and poly-d-lysine (PL) in sodium silicate derived sol-gel materials and revealed significant restriction of backbone motion upon entrapment for both polymers, an observation that was not readily apparent in a previous study with entrapped fluorescein-labeled PAM and PL. The implications of these findings for fluorescence studies of polymer and biomolecule dynamics are discussed.

Journal Article↗
PubMed2006

Quantifying surface coverage of colloidal silica by a cationic peptide using a combined centrifugation/time-resolved fluorescence anisotropy approach.

Recent experimental studies have shown that time-resolved fluorescence anisotropy (TRFA) is a promising methodology for in situ characterization of the surface modification of aqueous silica nanocolloids. Here we provide a more fundamental insight into the principle of this approach and discuss how the adsorption parameters for a cationic peptide, Lys-Trp-Lys (denoted using the standard shortform KWK), onto Ludox nanoparticles (NPs) are linked to the rotational dynamics of rhodamine 6G (R6G) dispersed in the KWK/Ludox mixture. First, the adsorption isotherm of KWK on hydrophilic controlled pore glass (CPG-3000) was obtained using the traditional centrifugation method, which provides the total molar amount of KWK per unit surface area of the silica. Assuming that both CPG and Ludox particles possess identical surface properties when suspended in the same aqueous buffer, both materials should also have identical adsorption properties. Thus, the adsorbed amount of KWK per unit area at a given total KWK concentration, as determined by the centrifugation method, can be plotted against the fractions of R6G anisotropy decay components at the same KWK concentration to relate the anisotropy components to the absolute surface coverage. Using this approach, it was determined that the concentration of KWK at which the CPG surface was saturated corresponded to the condition g = 0 in the R6G decay, where g is the fraction of the nondecaying anisotropy component. This condition means that there is no R6G bound to the fraction of Ludox NPs with a radius R > 2.5 nm at maximum KWK coverage, consistent with the adsorbed peptide forming a continuous layer on the Ludox surface. Hence, the g value obtained from TRFA analysis can be used to assess the absolute surface coverage of monolayer coatings on colloidal nanoparticles.

Centrifugation↗
PubMed2006

Quenching of fluorophore-labeled DNA oligonucleotides by divalent metal ions: implications for selection, design, and applications of signaling aptamers and signaling deoxyribozymes.

Recent years have seen a dramatic increase in the use of fluorescence-signaling DNA aptamers and deoxyribozymes as novel biosensing moieties. Many of these functional single-stranded DNA molecules are either engineered to function in the presence of divalent metal ion cofactors or designed as sensors for specific divalent metal ions. However, many divalent metal ions are potent fluorescence quenchers. In this study, we first set out to examine the factors that contribute to quenching of DNA-bound fluorophores by commonly used divalent metal ions, with the goal of establishing general principles that can guide future exploitation of fluorescence-signaling DNA aptamers and deoxyribozymes as biosensing probes. We then extended these studies to examine the effect of specific metals on the signaling performance of both a structure-switching signaling DNA aptamer and an RNA-cleaving and fluorescence-signaling deoxyribozyme. These studies showed extensive quenching was obtained when using divalent transition metal ions owing to direct DNA-metal ion interactions, leading to combined static and dynamic quenching. The extent of quenching was dependent on the type of metal ion and the concentration of supporting monovalent cations in the buffer, with quenching increasing with the number of unpaired electrons in the metal ion and decreasing with the concentration of monovalent ions. The extent of quenching was independent of the fluorophore, indicating that quenching cannot be alleviated simply by changing the nature of the fluorescent probe. Our results also show that the DNA sequence and the local secondary structure in the region of the fluorescent tag can dramatically influence the degree of quenching by divalent transition metal ions. In particular, the extent of quenching is predominantly determined by the fluorophore location with respect to guanine-rich and duplex regions within the strand sequence. Examination of the effect of both the type and concentration of metal ions on the performance of a fluorescence-signaling aptamer and a signaling deoxyribozyme confirms that judicious choice of divalent transition metal ions is important in maximizing signals obtained from such systems.

Aptamers, Nucleotide↗
PubMed2005

Nanovolume kinase inhibition assay using a sol-gel-derived multicomponent microarray.

We report on the development of a new class of kinase microarrays based on the coimmobilization of both kinase and substrate components within a single pin-printed sol-gel microarray element and the use of such arrays for nanovolume inhibition assays. We successfully immobilized the alpha-catalytic subunit of cAMP-dependent protein kinase (PKA) and the peptide substrate kemptide within sol-gel-derived microarrays for the purpose of monitoring phosphorylation and inhibition. Using Pro-Q Diamond stain as an end-point indicator of phosphorylation, we demonstrate the selective detection of phosphoproteins over nonphosphorylated controls and the ability to detect phosphorylated proteins over a 500-fold concentration range. Limits of detection for the phosphoprotein beta-casein were 7.5 pg, and the detectable signal remained linear up to 3.75 ng of protein per array spot. PKA is demonstrated to be active when coentrapped with two different substrates, and inhibition assays for PKA with the inhibitors H7 and H89 demonstrate the ability to detect kinase inhibition as well as derive IC50 plots from a single array using an overprinting method to deliver approximately 0.6 nL of reagent per array element, or a total of 72 nL of reagents to generate a full, 12-point IC50 curve in pentuplicate.

Caseins↗
PubMed2005

Inhibitor screening using immobilized enzyme reactor chromatography/mass spectrometry.

We describe the coupling of capillary-scale monolithic enzyme reactor columns directly to a tandem mass spectrometer for screening of enzyme inhibitors. A two-channel nanoLC system is used to continuously infuse substrate or substrate/inhibitor mixtures through the column, allowing continuous variation of inhibitor concentration by simply altering the ratio of flow from the two pumps. In the absence of inhibitor, infusion of substrate leads to formation of product, and both substrate and product ions can be simultaneously monitored in a quantitative manner by MS/MS. The presence of inhibitor leads to a decrease in product and an increase in substrate concentration in the column eluent. Knowing the product/substrate ratio and the total analyte concentration (P + S), the concentration of product eluting, and hence the relative enzyme activity, can be determined. Both IC50 and KI values can then be obtained by direct MS detection of the effect of inhibitors on relative activity. Inhibitor screening is demonstrated using reusable, sol-gel derived, monolithic capillary columns containing adenosine deaminase, directly interfaced to ESI-MS/MS. On-column enzyme activity was assessed by monitoring inosine and adenosine elution. It is shown that the method can be used for automated screening of the effects of compound mixtures on ADA activity and to determine the KI value of the known inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine, even when the compound is present within a mixture.

Adenosine Deaminase↗
PubMed2005

Characterizing the secondary structure and identifying functionally essential nucleotides of pH6DZ1, a fluorescence-signaling and RNA-cleaving deoxyribozyme.

pH6DZ1 is a synthetic deoxyribozyme that is able to couple catalysis with fluorescence signal generation. This deoxyribozyme has the ability to cleave itself at a lone ribonucleotide that is present between a pair of deoxyribothymidines, one modified with a fluorophore (fluorescein) and the other with a quencher (DABCYL). Herein, we report on the sequence truncation and secondary structure characterization of pH6DZ1 as well as the identification of functionally important nucleotides within this deoxyribozyme. Our data indicate that pH6DZ1 has a four-way, junction-like secondary structure comprised of four short duplexes, three hairpin loops, and three interhelical unpaired elements. Ten nucleotides, all located in two separate single-stranded regions, were identified as functionally indispensable nucleotides (complete loss of the catalytic function was obtained upon mutation). Nine nucleotides, most of which are also distributed in three single-stranded DNA elements, were identified as functionally vital nucleotides (at least a 1000-fold activity reduction was obtained upon mutation). Our study has shown that pH6DZ1 has a secondary structure that is more complex than those reported for other RNA-cleaving deoxyribozymes. The identification of functionally important nucleotides lays the foundation for future mechanistic studies on this DNAzyme. The elucidation of the secondary structure of pH6DZ1 should facilitate the future exploration of this unique DNAzyme for the development of DNAzyme-based biosensors.

Base Sequence↗
PubMed2005

Capillary-scale monolithic immunoaffinity columns for immunoextraction with in-line laser-induced fluorescence detection.

A bimodal meso/macroporous monolithic silica capillary column containing an entrapped antibody was prepared by a biocompatible sol-gel process and used for nanoflow immunoaffinity chromatography and immunoextraction studies. Stationary phases were prepared by combining the protein-compatible silane precursor diglycerylsilane with an aqueous solution containing 10,000 Da poly(ethylene glycol) and the antibody. An analytical method was developed that was capable of determining both the dissociation constant and binding site content for the anti-fluorescein antibody within the stationary phase. The assay showed that while the antibody residing in macropores was easily removed, approximately 20% of initially loaded antibody remained active and accessible after several washes, consistent with the antibody being entrapped within the mesopores of the sol-gel matrix. The dissociation constants for fluorescein binding to the anti-fluorescein antibody were similar in solution and in the meso/macroporous silica, indicating that the entrapped antibody retained its native conformation within such a matrix. The mixture was loaded into a 250-microm-i.d. fused-silica capillary where the polymer phase separated from the silica followed by gelation of the silica. The capillary-scale immunoaffinity columns could be operated at low back pressure using a syringe pump and were capable of performing chromatographic separations that were dependent on the presence of the antibody within the stationary phase. Such columns could also be operated using in-line laser-induced fluorescence detection. The use of the capillary-scale monolithic columns for on-column immunoextraction and preconcentration is also demonstrated.

Journal Article↗
PubMed2005

Entrapment of fluorescent signaling DNA aptamers in sol-gel-derived silica.

We report on the first successful immobilization of a DNA aptamer, in particular, a fluorescence-signaling DNA aptamer, within a sol-gel-derived matrix. The specific aptamer examined in this study undergoes a structural switch in the presence of adenosine triphosphate (ATP) to release a dabcyl-labeled nucleotide strand (QDNA), which in turn relieves the quenching of a fluorescein label that is also present in the aptamer structure. It was demonstrated that aptamers containing a complementary QDNA strand along with either a short complimentary strand bearing fluorescein (tripartite structure) or a directly bound fluorescein moiety (bipartite structure) remained intact upon entrapment within biocompatible sol-gel derived materials and retained binding activity, structure-switching capabilities, and fluorescence signal generation that was selective and sensitive to ATP concentration. Studies were undertaken to evaluate the properties of the immobilized aptamers that were either in their native state or bound to streptavidin using a terminal biotin group on the aptamer, including response time, accessibility, and leaching. Furthermore, signaling abilities were optimized through evaluation of different QDNA constructs. These studies indicated that the aptamers remained in a state that was similar to solution, with moderate leaching, only minor decreases in accessibility to ATP, and an expected reduction in response time due to diffusional barriers to mass transport of the analyte through the silica matrix. Entrapment of the aptamer also resulted in protection of the DNA against degradation from nucleases, improving the potential for use of the aptamer for in vivo sensing. This work demonstrates that sol-gel-derived materials can be used to successfully immobilize and protect DNA-based biorecognition elements and, in particular, DNA aptamers, opening new possibilities for the development of DNA aptamer-based devices, such as affinity columns, microarrays, and fiber-optic sensors.

Adenosine Triphosphate↗
PubMed2005

Direct and indirect monitoring of peptide-silica interactions using time-resolved fluorescence anisotropy.

The present work extends the application of time-resolved fluorescence anisotropy (TRFA) of a cationic probe rhodamine 6G (R6G) in aqueous Ludox to in situ monitoring of peptide adsorption onto the silica particles. Steady-state anisotropy and TRFA of R6G in Ludox sols were measured to characterize the extent of the ionic binding of the probe to silica particles in the presence of varying levels of tripeptides of varying charge, including Lys-Trp-Lys (KWK), N-acetylated Lys-Trp-Lys (Ac-KWK), Glu-Trp-Glu (EWE), and N-acetylated Glu-Trp-Glu (Ac-EWE). The results were compared to those obtained by direct observation of peptide adsorption using the steady-state anisotropy of the intrinsic tryptophan residue. Ionic binding of the peptides to Ludox particles produced an increase in the steady-state Trp anisotropy that was dependent on the number of cationic groups present, but the limiting anisotropy values were relatively low, indicating significant rotational freedom of the indole residue in the adsorbed peptides. On the other hand, R6G showed significant decreases in anisotropy in the presence of cationic peptides, consistent with the cationic peptides blocking the adsorption of the dye to the silica surface. Thus, R6G is able to indirectly report on the binding of peptides to Ludox particles. It was noteworthy that, while there were similar trends in the data obtained from steady-state anisotropy and TRFA studies of R6G, the use of steady-state anisotropy to assess binding of peptides overestimated the degree of peptide adsorption relative to the value obtained by TRFA. The study shows that the competitive binding method can be used to assess the binding of various biologically relevant compounds onto silica surfaces and demonstrates the potential of TRFA for probing peptide-silica and protein-silica interactions.

Adsorption↗
PubMed2005

Capillary-scale frontal affinity chromatography/MALDI tandem mass spectrometry using protein-doped monolithic silica columns.

Frontal affinity chromatography (FAC) interfaced with electrospray mass spectrometry (ESI-MS) has been reported as a potential method for screening of compound mixtures against immobilized target proteins. However, the interfacing of bioaffinity columns to ESI-MS requires that the eluent that passes through the protein-loaded column have a relatively low ionic strength to produce a stable spray. Such low ionic strength solvents can cause serious problems with protein stability and may also affect binding constants and lead to high nonspecific binding to the column. Herein, we report on the interfacing of bioaffinity columns to matrix-assisted laser desorption/ionization (MALDI) MS/MS as a new platform for FAC/MS studies. Capillary columns containing a monolithic silica material with entrapped dihydrofolate reductase were used for frontal affinity chromatography of small-molecule mixtures. The output from the column was combined with a second stream containing alpha-cyano-hydoxycinnamic acid in methanol and was deposited using a nebulizer-assisted electrospray method onto a conventional MALDI plate that moved relative to the column via a computer-controlled x-y stage, creating a semipermanent record of the FAC run. The use of MALDI MS/MS allowed for buffers with significantly higher ionic strength to be used for FAC studies, which reduced nonspecific binding of ionic compounds and allowed for better retention of protein activity over multiple runs. Following deposition, MALDI analysis required only a fraction of the chromatographic run time, and the deposited track could be rerun multiple times to optimize ionization parameters and allow signal averaging to improve the signal-to-noise ratio. Furthermore, high levels of potential inhibitors could be detected via MALDI with limited ion suppression effects. Both MALDI- and ESI-based analysis showed similar retention of inhibitors present in compound mixtures when using identical ionic strength conditions. The results show that FAC/MALDI-MS should provide advantages over FAC/ESI-MS for high-throughput screening of compound mixtures.

Automation↗
PubMed2005

Evidence for rigid binding of rhodamine 6G to silica surfaces in aqueous solution based on fluorescence anisotropy decay analysis.

Strong ionic binding of the cationic probe rhodamine 6G (R6G) to the anionic surface of silica particles in water provides a convenient labeling procedure to study both particle growth kinetics and surface modification by time-resolved fluorescence anisotropy (TRFA). The decays for R6G dispersed in diluted Ludox silica sols usually fit to a sum of picosecond and nanosecond decay components, along with a significant residual anisotropy component. The origin of the nanosecond decay component (phi2) is not fully understood, and has been ascribed to wobbling of the probe on the silica surface, the presence of a subpopulation of small nanoparticles in the Ludox sol, or rapid exchange between free and bound R6G. To elucidate the physical meaning of phi2, measurements were performed in various silica-based colloidal systems using different concentrations of silica. We found that the fraction of phi2 was generally higher in Ludox than in aqueous sodium silicate and decreased with increasing silica concentration; phi2 vanished upon gelation of sodium silicate at pH 7 leading to a total loss of R6G depolarization (r(t) = const). These results rule out the presence of local R6G wobbling when bound ionically to colloidal silica and support the rigid sphere model to describe the TRFA decays for R6G-Ludox. This conclusion is entirely supported by steady-state anisotropy data and structural considerations for the R6G molecule and the silica surface.

Journal Article↗
PubMed2005

Characterization of a fluorescence-signaling and RNA-cleaving deoxyribozyme.

DNA enzymes (deoxyribozymes or DNAzymes) are single-stranded DNA molecules with catalytic function. Previously, we isolated several RNA-cleaving deoxyribozymes with fluorescence-signaling properties. These special DNA molecules are capable of cleaving an RNA linkage embedded within a DNA sequence and flanked by a pair of deoxyribothymidines modified with a fluorophore (fluorescein) and a quencher (dabcyl). Here we report on the sequence truncation and secondary structure characterization of one such deoxyribozyme known as pH6DZ1 as well as the identification of functionally important nucleotides within this deoxyribozyme. Our data indicates that pH6DZ1 has a four-way junction-like secondary structure comprising four short duplexes, three hairpin loops, and three inter-helical unpaired elements.

Base Sequence↗
PubMed2004

Entrapment of highly active membrane-bound receptors in macroporous Sol-Gel derived silica.

The immobilization of membrane-associated proteins remains a challenging task. Herein, we report on the entrapment of two classes of membrane-bound receptors into sol-gel derived silica. Both nicotinic acetylcholine receptor (nAChR), a ligand-gated ion channel, and dopamine D(2Short) receptor (D2R), a G-protein coupled receptor, were entrapped into a series of sol-gel derived nanocomposite materials. In cases where the silica had a bimodal pore size distribution wherein both mesopores and macropores were present, the two receptors showed 40-80% of solution activity over periods of at least 1 month. Furthermore, the dissociation constants of entrapped nAChR and D2R for binding to known agonists and antagonists were very close to the values obtained for free receptors in solution. These results indicate that membrane-bound receptors entrapped into bimodal meso/macroporous silica should provide a useful platform for the development of bioanalytical devices such as bioaffinity columns or microarrays, which could aid in diagnosis and high-throughput drug screening.

Animals↗
PubMed2004

Bridging the gap between in vitro and in vivo imaging: isostructural Re and 99mTc complexes for correlating fluorescence and radioimaging studies.

A bifunctional ligand that is capable of forming Re and 99mTc complexes as complementary fluorescent and radioactive probes was developed. The tridentate bis(quinoline) amine ligand, which is referred to as the SAACQ system, was prepared in a single step from Fmoc protected lysine in high yield. Reaction of the SAACQ ligand with [Re(CO)3Br3]2- resulted in the formation of the SAACQ-(Re(CO)3)+complex which exhibits favorable fluorescence properties including a long lifetime and a large Stoke's shift. Because the SAACQ ligand is derived from an amino acid, it can readily be linked to or incorporated within peptides as a means of targeting the probe to specific receptors. To demonstrate this feature, the SAACQ ligand and the SAACQ-Re complex were incorporated into fMLFG, a peptide that binds to the formyl peptide receptor (FPR). Uptake of the fMLF[(SAACQ-Re(CO)3)+]G conjugate into human leukocytes in vitro was visualized by fluorescence microscopy, and the observed distribution of the peptide was similar to that of a well-established fluorescent FPR probe. The corresponding Tc complex, fMLF[(SAACQ-99mTc(CO)3)+]G, was prepared in excellent yield from [99mTc(CO)3(OH2)3]+, which affords the opportunity to correlate the results of the microscopy experiments with in vivo radioimaging studies because the probes are isostructural.

Fluorescent Dyes↗