Transmission of poly(dT60 ) single-stranded DNA through polycarbonate track-etched ultrafiltration membranes.

The objective of this study was to examine membrane filtration of a single stranded DNA (ssDNA) with 60 thymine nucleotides, and to elucidate the variables controlling its transmission across track-etched porous membranes. Dead end filtration measurements were performed using different pore size membranes (10, 15, and 30 nm) at different transmembrane pressures in solutions with ionic strength ranging from 0 to 1000 mM NaCl. The diffusivity of the ssDNA was determined using fluorescence recovery after photobleaching, yielding hydrodynamic radii ranging from 1.6 to 2.8 nm, with values decreasing with increasing solution ionic strength. Despite the small ssDNA/membrane pore size, nearly 100% rejection was observed for measurements performed with the 10 and 15 nm pore size membranes under low-ionic strength conditions. These high rejections can be attributed to strong repulsive electrostatic ssDNA-membrane interactions. With increasing ionic strength, electrostatic interactions as well as the effective size of the ssDNA decreases and the flexibility of the ssDNA increases, leading to a reduction in ssDNA rejection. A design of experiments approach was used to plan filtration experiments that adequately covered the variable space with a manageable number of experiments. The results yielded an empirical expression relating ssDNA rejection to pore size, solution ionic strength and transmembrane pressure. There was evidence of flow induced elongation at high-transmembrane pressures in the 30 nm pore size membranes, but not in the smaller pore size membranes. These results are consistent with critical flux estimates developed using a free draining model for the ssDNA.

Can Hindered Transport Models for Rigid Spheres Predict the Rejection of Single Stranded DNA from Porous Membranes?

In this paper, predictions from a theoretical model describing the rejection of a rigid spherical solute from porous membranes are compared to experimental results for a single stranded DNA (ssDNA) with 60 thymine nucleotides. Experiments were conducted with different pore size track-etched membranes at different transmembrane pressures and different NaCl concentrations. The model includes both hydrodynamic and electrostatic solute-pore wall interactions; predictions were made using different size parameters for the ssDNA (radius of gyration, hydrodynamic radius, and root mean square end-to-end distance). At low transmembrane pressures, experimental results are in good agreement with rejection predictions made using the hard sphere model for the ssDNA when the solute size is described using its root mean square end-to-end distance. When the ssDNA size is characterized using the radius of gyration or the hydrodynamic radius, the hard sphere model under-predicts rejection. Not surprisingly, the model overestimates ssDNA rejection at conditions where flow induced elongation of the DNA is expected. The results from this study are encouraging because they mean that a relatively simple hindered transport model can be used to estimate the rejection of a small DNA from porous membranes.

Free Diffusivity of Icosahedral and Tailed Bacteriophages: Experiments, Modeling, and Implications for Virus Behavior in Media Filtration and Flocculation.

The aqueous bulk diffusivities of several near-spherical (icosahedral) and nonspherical (tailed) bacterial viruses were experimentally determined by measuring their flux across large pore membranes and using dynamic light scattering, with excellent agreement between values measured using the two techniques. For the icosahedral viruses, good agreement was also found between measured diffusivity values and values predicted with the Stokes-Einstein equation. However, when the tailed viruses were approximated as spheres, poor agreement was found between measured values and Stokes-Einstein predictions. The shape of the tailed organisms was incorporated into two modeling approaches used to predict diffusivity. Model predictions were found to be in good agreement with measured values, demonstrating the importance of the tail in the diffusive transport of these viruses. Our calculations also show that inaccurate estimates of virus diffusion can lead to significant errors when predicting diffusive contributions to flocculation and to single collector efficiency in media filtration.

Effect of electrostatic interactions on rejection of capsular and spherical particles from porous membranes: theory and experiment.

HYPOTHESES: Particle rejection from porous membranes will increase when particle and membrane carry like charges. The influence of charge on particle rejection can be modeled by first solving the Poisson-Boltzmann equation for the electrostatic particle-pore wall interaction energy, enabling one to predict the cross sectional particle concentration in a pore. Rejection coefficients can then be predicted by combining the Boltzmann factor with a hydrodynamic lag coefficient. EXPERIMENTS: Rejection experiments were conducted with three different spherical colloidal silica particles, a spherical virus (PRD1) and gold nanorods of two different aspect ratios (ratio of length to diameter). Track-etched polycarbonate microfiltration and ultrafiltration membranes having nearly parallel pores of cylindrical cross-section were used. Experiments were conducted under conditions where both particle and membrane carried a negative charge as well as under conditions where surface charges had minimal impact. Experiments were designed to cover a broad range of dimensionless particle sizes under conditions when convection dominated particle transport. FINDINGS: Model predictions and experimental measurements demonstrate that particle rejection can be enhanced significantly when particle and pore carry like charges.

Quartz crystal microbalance (QCM) with immobilized protein receptors: comparison of response to ligand binding for direct protein immobilization and protein attachment via disulfide linker.

Results from an investigation of the frequency response resulting from ligand binding for a genetically engineered hormone-binding domain of the alpha-estrogen receptor immobilized to a piezoelectric quartz crystal are reported. Two different approaches were used to attach a genetically altered receptor to the gold electrode on the quartz surface: (1) the mutant receptor containing a single solvent-exposed cysteine was directly attached to the crystal via a sulfur to gold covalent bond, forming a self-assembled protein monolayer, and (2) the N-terminal histidine-tagged end was utilized to attach the receptor via a 3,3-dithiobis[N-(5-amino-5-carboxypentyl)propionamide-N',N'-diacetic acid] linker complexed with nickel. Previous studies have shown that these engineered constructs bind 17beta-estradiol and are fully functional. Exposure of the receptor directly attached to the piezoelectric crystal to the known ligand 17beta-estradiol resulted in a measurable frequency response, consistent with a change in conformation of the receptor with ligand binding. However, no response was observed when the receptor immobilized via the linker was exposed to the same ligand. The presence of the linker between the quartz surface and the protein receptor does not allow the crystal to sense the conformational change in the receptor that occurs with ligand binding. These results illustrate that the immobilization strategy used to bind the receptor to the sensor platform is key to eliciting an appropriate response from this biosensor. This study has important implications for the development of QCM-based sensors using protein receptors.

A biosensor for estrogenic substances using the quartz crystal microbalance.

This article describes a biosensor that detects estrogenic substances using a quartz crystal microbalance with a genetically engineered construct of the hormone-binding domain of the alpha-estrogen receptor. The receptor was immobilized to a piezoelectric quartz crystal via a single exposed cysteine, forming a uniform orientation on the crystal surface. Our results illustrate that this sensor responds to a variety of ligands that are known to bind to the estrogen receptor. No response was observed for nonbinding substances such as testosterone and progesterone. The sensitive response of this biosensor to estrogenic substances results from changes in the structural rigidity of the immobilized receptor that occurs with ligand binding. Agonist and antagonist show different responses.

A piezoelectric quartz crystal biosensor: the use of two single cysteine mutants of the periplasmic Escherichia coli glucose/galactose receptor as target proteins for the detection of glucose.

We have examined the potential utility of a glucose biosensor that employs the glucose/galactose receptor of Escherichia coli with a quartz crystal microbalance (QCM). Two different genetically engineered mutant proteins were utilized, each involving the incorporation of a single cysteine into the amino acid sequence of the protein. The proteins were immobilized on the surface of a piezoelectric crystal by a direct sulfur-gold linkage. Since the cysteines were located at different positions in the sequence, the receptors attach to the surface with different orientations. Considering only mass effects, the target sugars for this receptor are predicted to be too small to be detectable with a QCM. However, our sensors indicated measurable and reproducible frequency responses when immobilized receptor was exposed to sugar. This unexpectedly large frequency response occurs because the protein film is transformed from a viscous layer to a more rigid nondissipative film. The QCM can detect these changes because of the direct linkage of the proteins to the surface. Calculations of the frequency response expected for a viscoelastic film with different rheological characteristics support this hypothesis. This study is significant because it illustrates a widened applicability for the QCM methodology to protein systems that bind small molecules and undergo ligand-induced conformational changes.