12 research outputs found
Magnetic microsphere-based methods to study the interaction of teicoplanin with peptides and bacteria
Teicoplanin (teic) from Actinoplanes teichomyceticus is a glycopeptide antibiotic used to treat many Gram-positive bacterial infections. Glycopeptide antibiotics inhibit bacterial growth by binding to carboxy-terminal D-Ala-D-Ala intermediates in the peptidoglycan of the cell wall of Gram-positive bacteria. In this paper we report the derivatization of magnetic microspheres with teic (teic-microspheres). Fluorescence-based techniques have been developed to analyze the binding properties of the microspheres to two D-Ala-D-Ala terminus peptides. The dissociation constant for the binding of carboxyfluorescein-labeled D-Ala-D-Ala-D-Ala to teic on microspheres was established via fluorimetry and flow cytometry and was determined to be 0.5x10^-6 and 3.0x10^-6 mol L^-1, respectively. The feasibility of utilizing microparticles with fluorescence methods to detect low levels (the limit of bacterial detection was determined to be 30 colon-forming units; cfu) of Gram-positive bacteria has been demonstrated. A simple microfluidic experiment is reported to demonstrate the possibility of developing microsphere-based affinity assays to study peptide-antibiotic interaction
Fate, Transformation, and Toxicological Impacts of Pharmaceutical and Personal Care Products in Surface Waters
With the growth of the human population, a greater quantity of pharmaceutical and personal care products (PPCPs) have been released into the environment. Although research has addressed the levels and the impact of PPCPs in the environment, the fate of these compounds in surface waters is neither well known nor characterized. In the environment, PPCPs can undergo various transformations that are critically dependent on environmental factors such as solar radiation and the presence of soil particles. Given that the degradation products of PPCPs are poorly characterized, these “secondary residues” can be a significant environmental health hazard due to their drastically different toxicologic effects when compared with the parent compounds. To better understand the fate of PPCPs, we studied the degradation of selected PPCPs, including ibuprofen and clofibric acid, in aqueous solutions that contained kaolinite clay and were irradiated with a solar simulator. The most abundant degradation products were identified and assessed for their toxicologic impact on selected microorganisms. The degraded mixtures showed lower toxicity than the starting compounds; however, as these degradation products are capable of further transformation and interaction with other PPCPs in natural waters, our work highlights the importance of additionally characterizing the PPCP degradation products
Multinode Acoustic Focusing for Parallel Flow Cytometry
Flow cytometry can simultaneously measure and analyze
multiple
properties of single cells or particles with high sensitivity and
precision. Yet, conventional flow cytometers have fundamental limitations
with regards to analyzing particles larger than about 70 μm,
analyzing at flow rates greater than a few hundred microliters per
minute, and providing analysis rates greater than 50 000 per
second. To overcome these limits, we have developed multinode acoustic
focusing flow cells that can position particles (as small as a red
blood cell and as large as 107 μm in diameter) into as many
as 37 parallel flow streams. We demonstrate the potential of such
flow cells for the development of high throughput, parallel flow cytometers
by precision focusing of flow cytometry alignment microspheres, red
blood cells, and the analysis of a CD4+ cellular immunophenotyping
assay. This approach will have significant impact toward the creation
of high throughput flow cytometers for rare cell detection applications
(e.g., circulating tumor cells), applications requiring large particle
analysis, and high volume flow cytometry
Elastomeric Negative Acoustic Contrast Particles for Affinity Capture Assays
This report describes the development of elastomeric
capture microparticles
(ECμPs) and their use with acoustophoretic separation to perform
microparticle assays via flow cytometry.We have developed simple methods
to form ECμPs by cross-linking droplets of common commercially
available silicone precursors in suspension followed by surface functionalization
with biomolecular recognition reagents. The ECμPs are compressible
particles that exhibit negative acoustic contrast in ultrasound when
suspended in aqueous media, blood serum, or diluted blood. In this
study, these particles have been functionalized with antibodies to
bind prostate specific antigen and immunoglobulin (IgG). Specific
separation of the ECμPs from blood cells is achieved by flowing
them through a microfluidic acoustophoretic device that uses an ultrasonic
standing wave to align the blood cells, which exhibit positive acoustic
contrast, at a node in the acoustic pressure distribution while aligning
the negative acoustic contrast ECμPs at the antinodes. Laminar
flow of the separated particles to downstream collection ports allows
for collection of the separated negative contrast (ECμPs) and
positive contrast particles (cells). Separated ECμPs were analyzed
via flow cytometry to demonstrate nanomolar detection for prostate
specific antigen in aqueous buffer and picomolar detection for IgG
in plasma and diluted blood samples. This approach has potential applications
in the development of rapid assays that detect the presence of low
concentrations of biomarkers in a number of biological sample types