Biomedical subjects

M R Shortreed

Publications and source records attributed to M R Shortreed.

5 recordsLinked to original sources

PubMed2001

Real-time dynamics of single-DNA molecules undergoing adsorption and desorption at liquid-solid interfaces.

The conformational dynamics and adsorption/desorption behavior of individual lambda-DNA molecules at liquid-solid interfaces were monitored by imaging within the evanescent field layer using total internal reflection fluorescence microscopy. At a fused-silica surface, molecular conformation and adsorption behavior were found to depend on both pH and buffer composition. A histogram of individual lambda-DNA adsorption durations measured by hydrodynamically flowing molecules along the interface exhibited asymmetry nearly identical to that of the corresponding elution peaks found in capillary liquid chromatography and capillary electrophoresis. The accessibility of the surface to the molecules, which is proportional to the capillary surface area-to-volume ratio, can be correlated with the capacity factor and the relative adsorption factor. At a C18 surface, the dynamics of individual DNA molecules changed with the addition of organic solvent as well as with pH. Hydrophobic interaction rather than electrostatic interaction was the major driving force for adsorption of individual DNA molecules.

Adsorption↗

PubMed2001

Single-molecule immunoassay and DNA diagnosis.

Many assays relevant to disease diagnosis are based on electrophoresis, where the migration velocity is used for distinguishing molecules of different size or charge. However, standard gel electrophoresis is not only slow but also insensitive. We describe a single-molecule imaging procedure to measure the electrophoretic mobilities of up to 100000 distinct molecules every second. The results correlate well with capillary electrophoresis (CE) experiments and afford confident discrimination between normal (16.5 kbp) and abnormal (6.1 kbp) mitochondrial DNA fragments, or beta-phycoerythrin-labeled digoxigenin (BP-D) and its immunocomplex (anti-D-BP-D). This demonstrates that virtually all electrophoresis diagnostic protocols from slab gels to CE should be adaptable to single-molecule detection. This opens up the prossibility of screening single copies of DNA or proteins within single biological cells for disease markers without performing polymerase chain reaction (PCR) or other biological amplification.

Antigen-Antibody Complex↗

PubMed2000

High-throughput single-molecule spectroscopy in free solution.

A high-speed high-throughput single-molecule imaging technique for identifying molecules in free solution based on differences in their fluorescence emission spectra is presented. Unlike previous reports, the entire spectrum, rather than selected wavelengths through optical filters, is recorded. Furthermore, the millisecond data acquisition time means that the molecules do not need to be immobilized or spatially confined. In one example, individual lambdaDNA molecules labeled with YOYO-I, POPO-III, or a combination of the two dyes can be distinguished from one another. In another example, biotinylated 2.1-kb DNA labeled with YOYO-I was reacted with avidin-conjugated R-phycoerythrin. The two different reactant molecules and the product molecule can be simultaneously imaged and identified by their spectroscopic characteristics. This technique can therefore be used for screening single molecules for disease markers and for monitoring individual molecular interactions at a rate of thousands of molecules per second.

Biotinylation↗

PubMed2000

High-throughput single-molecule DNA screening based on electrophoresis.

In electrophoresis, the migration velocity is used for sizing DNA and proteins or for distinguishing molecules based on charge and hydrodynamic radius. Many protein and DNA assays relevant to disease diagnosis are based on such separations. However, standard protocols are not only slow (minutes to hours) but also insensitive (many molecules in a detectable band). We successfully demonstrated a high-throughput imaging approach that allows determination of the individual electrophoretic mobilities of many molecules at a time. Each measurement only requires a few milliseconds to complete. This opens up the possibility of screening single copies of DNA or proteins within single biological cells for disease markers without performing polymerase chain reaction or other biological amplification. The purpose is not to separate the DNA molecules but to identify each one on the basis of the measured electrophoretic mobility. We developed three different procedures to measure the individual molecular mobilities. The results correlate well with capillary electrophoresis (CE) experiments for the same samples (2-49 kb dsDNA) under identical separation conditions. The implication is that any electrophoresis protocols from slab gels to CE should be adaptable to single-molecule screening for disease diagnosis.

Buffers↗

PubMed1997

Utilization of lipophilic ionic additives in liquid polymer film optodes for selective anion activity measurements.

Lipophilic anionic and cationic additives are investigated as components for use in liquid polymer film-based optodes. These ionic additives are known to strongly influence the selectivity behavior of anion-selective electrodes. They also make it possible to construct ion coextraction optodes that can measure anion activities. Theoretical, thermodynamic anion-optode equilibria formalisms were derived to help understand the influence these additives have on the overall optode response and on the selectivity behavior. Neutral and charged anion carrier film configurations are described and tested for two anion ionophores with known modes of action, ruthenium(II) octaethylporphyrin and a vitamin B12 derivative (cyanoaquacobyrinic acid heptakis(2-phenylethyl ester)). These film configurations were further tested using a less well-understood ionophore, indium(III) octaethylporphyrin. Notable is the indium(III) octaethylporphyrin optode, which has a dynamic range appropriate for physiological measurements of chloride at neutral pH values. Also of interest is the optode with the vitamin B12 derivative ionophore, which has a dynamic range appropriate for some physiological measurements of nitrite at neutral pH values.

Anions↗