Biomedical subjects

Bethany A Staggemeier

Publications and source records attributed to Bethany A Staggemeier.

3 recordsLinked to original sources

PubMed2005

High-throughput screening of protein surface activity via flow injection analysis-pH gradient-dynamic surface tension detection.

Using flow injection analysis (FIA), a pH gradient is blended in real time with a protein sample as the pH-dependent protein surface activity is measured by a dynamic surface tension detector (FIA-pH-DSTD). This instrumental system was developed as a high-throughput method for the screening of protein surface activity at the air/liquid interface as a function of pH. This method utilizes the continuous flow, drop-based dynamic surface tension detector in combination with flow injection sample introduction and blending of a steady-state concentration of protein sample with a pH gradient ranging from pH 2.0 to pH 11.5. Dynamic surface tension is measured through the differential pressure across the air/liquid interface of repeatedly growing and detaching drops. Continuous surface tension measurement is achieved for each eluting drop of 2-s length (2 muL), providing insight into both the kinetic and thermodynamic behaviors of molecular orientation processes at the liquid/air interface. Three-dimensional data are obtained, with surface tension first converted to surface pressure, which is collected as a function of elution time versus drop time. In FIA-pH-DSTD, a commercial pH probe is used to measure pH during elution time, enabling surface pressure throughout drop time to be subsequently plotted as a function of eluting pH. An automated DSTD calibration procedure and data analysis method is applied, which allows simultaneous use of two different solvents, permitting real-time dynamic surface tension data to be obtained. The method was applied to the analysis of 14 commercial purified proteins, yielding characteristic features of surface activity as a function of pH. The reproducibility of the measurement and selectivity advantage of the DSTD was shown for the analysis of serum albumins from various mammalian sources. Several applications were also suggested and discussed in order to show the potential of the method for protein and food chemistry studies and in the study of protein-polymer interactions.

Journal Article↗

PubMed2003

Determination, by dynamic surface-tension analysis, of the molar mass of proteins denatured in guanidine thiocyanate.

A drop-based dynamic surface-tension detector (DSTD) has been used to study the dynamic surface tension behavior of proteins denatured in guanidine thiocyanate (GndSCN). The dynamic surface tension at the air-liquid interface is obtained by measuring the internal pressure of drops that grow and detach at a specified rate. In the method the sample of interest is injected and subsequently flows to the DSTD-sensing capillary tip. For this work, a novel DSTD calibration procedure utilizing two distinct mobile phases is applied. Here, the mobile phases are aqueous with different constituents, for example GndSCN and phosphate buffer, either added or omitted. The dual-mobile phase calibration procedure gives the analyst the capability of making protein measurements in a GndSCN-phosphate buffer mobile phase, while measuring a calibration standard in another mobile phase, such as water, in which the surface tension of the calibration standard is readily available. Results are presented with drop volumes of either 2 microL (i.e. 2-s drops) or 7 microL (i.e. 7-s drops) for proteins varying in molar mass from 12,000 to 330,000 g mol(-1). We demonstrate that the DSTD can be used to determine the molar mass of proteins denatured in GndSCN. The method applies a regime where the denatured protein is detected by surface-active properties, and selectivity with regard to molar mass is contained in the dynamic component of the DSTD signal. The dynamic surface pressure signals of the denatured proteins suggest that diffusion plays a large role in the kinetics of the surface activity. The limit of detection for the denatured proteins studied ranged from 3 mg L(-1) to 14 mg L(-1). The DSTD, coupled with the novel dual-mobile phase calibration procedure, can be used to investigate the fundamental properties of proteins. Insight into the behavior at the air-liquid interface for native and denatured proteins is achieved; this is a novel tool for studying protein denaturation, complementary to other common approaches such as spectroscopy and calorimetry. Furthermore, the reported method could be widely applied to the study of effects on the interfacial properties of proteins after a variety of chemical and physical modifications that are possible with the dual-mobile phase calibration procedure.

Calibration↗

PubMed2003

Diffusion coefficient measurement in a microfluidic analyzer using dual-beam microscale-refractive index gradient detection. Application to on-chip molecular size determination.

We report a microchip-based detection scheme to determine the diffusion coefficient and molecular mass (to the extent correlated to molecular size) of analytes of interest. The device works by simultaneously measuring the refractive index gradient (RIG) between adjacent laminar flows at two different positions along a microchannel. The device, referred to as a microscale molecular mass sensor (micro-MMS), takes advantage of laminar flow conditions where the mixing of two streams occurs essentially by diffusion across the boundary between the two streams. Two flows merge on the microchip, one containing solvent only, referred to as the mobile phase stream and one which contains the analyte(s) of interest in the solvent, i.e. the sample stream. As these two streams merge and flow parallel to each other down the microchannel a RIG is created by the concentration gradient. The RIG is further influenced by analyte diffusion from the sample stream into the mobile phase stream. Measuring the RIG at a position close to the merging point (upstream signal) and simultaneously a selected distance further down the microchannel (downstream signal) provides real-time data related to the extent a given analyte has diffused, which can be readily correlated to analyte molecular mass by taking the ratio of the downstream-to-upstream signals. For the dual-beam RIG measurements, a diode laser output is coupled to a single mode fiber optic splitter with two output fibers. Light from each fiber passes through a graded refractive index (GRIN) lens forming a collimated beam that then passes through the microchannel and then on to a position sensitive detector (PSD). The RIG at both detection positions deflects the two collimated probe beams. The deflection angle of each beam is then measured on two separate PSDs. The micro-MMS was evaluated using polyethylene glycols (PEGs), sugars, and as a detector for size-exclusion chromatography (SEC). Peak purity can be readily identified using the micro-MMS with SEC. The limit of detection was 0.9 ppm (PEG at 11 840 g/mol) at the upstream detection position corresponding to a RI limit of detection (LOD) (3sigma) of 7-10(-8) RI. The pathlength for the RIG measurement was 200 microm and the angular LOD was 0.23 micro(rad) with a detection volume of 8 nl at both positions. The average molecular mass resolution was 9% (relative standard deviation) for a series of PEGs ranging in molecular mass from 106 to 22 800 g/mol. With this excellent mass resolution, small molecules such as monosaccharides, disaccharides, and so on, are readily distinguished. The sensor is demonstrated to readily determine unknown diffusion coefficients.

Chromatography, Gel↗