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Biomedical subjects

E V Anslyn

Publications and source records attributed to E V Anslyn.

10 recordsLinked to original sources
PubMed2001

Characterization of multicomponent monosaccharide solutions using an enzyme-based sensor array.

We report the development of a sensor for rapidly and simultaneously measuring multiple sugars in aqueous samples. In this strategy, enzyme-based assays are localized within an array of individually addressable sites on a micromachined silicon chip. Microspheres derivatized with monosaccharide-specific dehydrogenases are distributed to pyramidal cavities anisotropically etched in a wafer of silicon (100) and are exposed to sample solution that is forced through the cavities by a liquid chromatography pumping system. Production of fluorescent reporter molecules is monitored under stopped-flow conditions when localized dehydrogenase enzyme systems are exposed to their target sugars. We demonstrate the capability of this analysis strategy to quantify beta-D-glucose and beta-D-galactose at low micromolar to millimolar levels, with no detectable cross-talk between assay sites. Analysis is achieved either through fluorescence detection of an initial dehydrogenase product (NADH, NADPH) or by production of a secondary fluorescent product created by hydride transfer from the reduced nicotinamide cofactor to a fluorogenic reagent. The array format of this sensor provides capabilities for redundant analysis of sugars and for monitoring levels of other solution components known to affect the activity of enzymes. The use of this strategy to normalize raw fluorescence signals is demonstrated by the determination of glucose and pH on a single chip. Alternatively, uncertainties in the activity of an immobilized enzyme can be accounted for using standard additions, an approach used here in the determination of serum glucose.

Biosensing Techniques↗
PubMed2001

pK(a) values and geometries of secondary and tertiary amines complexed to boronic acids-implications for sensor design.

[structure in text] The pK(a) values and the geometries of secondary and tertiary amines adjacent to boronic acids were determined using potentiometric and (11)B NMR titrations. The studies showed that the secondary ammonium ion has a pK(a) similar to that of the tertiary ammonium species, which leads to the formation of tetrahedral boron centers at pH values above approximately 5.5. Therefore, secondary amines as well as tertiary amines, when placed proximal to boron centers, can be used to create tetrahedral boronic acids at neutral pH for diol complexation.

Amines↗
PubMed2001

Development of multianalyte sensor arrays composed of chemically derivatized polymeric microspheres localized in micromachined cavities.

The development of a chip-based sensor array composed of individually addressable polystyrene-poly(ethylene glycol) and agarose microspheres has been demonstrated. The microspheres are selectively arranged in micromachined cavities localized on silicon wafers. These cavities are created with an anisotropic etch and serve as miniaturized reaction vessels and analysis chambers. A single drop of fluid provides sufficient analysis media to complete approximately 100 assays in these microetch pits. The cavities possess pyramidal pit shapes with trans-wafer openings that allows for both fluid flow through the microreactors/analysis chambers and optical access to the chemically sensitive microspheres. Identification and quantitation of analytes occurs via colorimetric and fluorescence changes to receptor and indicator molecules that are covalently attached to termination sites on the polymeric microspheres. Spectral data are extracted from the array efficiently using a charge-coupled device allowing for the near-real-time digital analysis of complex fluids. The power and utility of this new microbead array detection methodology is demonstrated here for the analysis of complex fluids containing a variety of important classes of analytes including acids, bases, metal cations, metabolic cofactors, and antibody reagents.

Chemistry Techniques, Analytical↗
PubMed2001

Artificial receptors involved in enolization and pK(a) shifts.

Abiotic receptors used to enolize carbonyl compounds or to shift substrate pK(a) values are reviewed. These systems exhibit disparate frameworks and several approaches to binding and anion stabilization. Detailed emphasis is placed on a bicyclic cyclophane that induces pK(a) shifts in active methylene compounds through NH-pi hydrogen bonding with the resultant enolates.

Catalysis↗
PubMed2001

Teaching old indicators new tricks.

Most synthetic sensors are designed with covalent attachment between a receptor and a reporter moiety. In this report, we describe the current progress of our use of noncovalently attached indicators to signal binding of analytes. With these systems, analyte binding leads to indicator displacement from the binding cavity, which in turn yields an optical signal modulation. We include previous examples, the strategies involved in our development, and the advantages as well as disadvantages of this method. Finally, our latest research in this field is briefly presented.

Binding Sites↗
PubMed1999

Anion recognition: synthetic receptors for anions and their application in sensors.

Important contributions to the field of anion sensing include electrochemical lipophilic uranyl salophene receptors incorporated into membranes that act as fluoride-selective potentiometric microsensors. A promising optical-based sensor, selective for cyclic AMP, involves a preorganized, molecularly imprinted polymer employing an intrinsic fluorophore. Competition methods using ensembles of recognition units and external indicators have been used to sense citrate in highly competitive media and micromolar concentrations of inositol(tris)phosphate in water. In addition, DNA dendrimers immobilized on a quartz-crystal microbalance acted as an elegant biosensor for Cryptosporidium DNA. These designs display the varied methods of anion detection currently being pursued.

Anions↗
PubMed1997

Using guanidinium groups for the recognition of RNA and as catalysts for the hydrolysis of RNA.

The guanidinium functional group is commonly used in nature to recognize and bind anions through ion pairing and hydrogen bonding. Specific hydrogen-bonding patterns can be found in crystal structures of simple guanidinium salts. Analysis of these simple salts reveals a variety of features which are found in natural systems. These features have been applied to a series of artificial phosphodiesterases for RNA. These receptors incorporate guanidinium groups positioned to mimic the hydrogen-bonding patterns found in simple guanidinium salts and natural enzymes. This paper outlines general guanidinium hydrogen-bonding patterns. Next, the complexation of phosphodiesters with a series of artificial receptors are analyzed in terms of counterions, solvent mixtures, and cavity flexibility. In addition, strategies to enhance catalysis through a pKa analysis of phosphoranes are addressed. Next, we describe how our findings were incorporated into second generation receptors/catalysts. Finally, our future work is discussed.

Catalysis↗
PubMed1994

Radioactive end labeling to determine hydrolytic rates of nuclease mimics.

Radioactive end labeling can be used to determine the hydrolytic rates of nuclease mimics on moderate to long lengths of RNA or DNA. However, the reliability of end labeling as an assay can vary depending on how well the unincorporated label is removed from the labeled RNA or DNA products. Therefore, gel filtration, acid precipitation, membrane diafiltration, and paper chromatography were tested to determine which technique was the most effective at such separation. The results in order of decreasing contamination by [gamma-32P]ATP were gel filtration (40%), acid precipitation (5%), diafiltration (2%), and paper chromatography (1%); and, in order of decreasing loss of RNA, were acid precipitation (30%), diafiltration (11%), gel filtration (10%), and paper chromatography (1%). In order for the resultant radioactive counts to be linearly proportional to the number of cleavage sites, the total ATP in the end-labeling reaction should be in excess of 5'-hydroxyl ends by a factor of 10 or more. Interference by nuclease mimics in the end-labeling reaction should be accounted for by including the mimics when developing a standard curve based on known concentrations of 5'-hydroxyl ends.

Chromatography, Gel↗