David J. Graves

The biophysics of DNA hybridization with immobilized oligonucleotide probes

A mathematical model based on receptor-ligand interactions at a cell surface has been modified and further developed to represent heterogeneous DNA-DNA hybridization on a solid surface. The immobilized DNA molecules with known sequences are called probes, and the DNA molecules in solution with unknown sequences are called targets in this model. Capture of the perfectly complementary target is modeled as a combined reaction-diffusion limited irreversible reaction. In the model, there are two different mechanisms by which targets can hybridize with the complementary probes: direct hybridization from the solution and hybridization by molecules that adsorb nonspecifically and then surface diffuse to the probe. The results indicate that nonspecific adsorption of single-stranded DNA on the surface and subsequent two-dimensional diffusion can significantly enhance the overall reaction rate. Heterogeneous hybridization depends strongly on the rate constants for DNA adsorption/desorption in the non-probe-covered regions of the surface, the two-dimensional (2D) diffusion coefficient, and the size of probes and targets. The model shows that the overall kinetics of DNA hybridization to DNA on a solid support may be an extremely efficient process for physically realistic 2D diffusion coefficients, target concentrations, and surface probe densities. The implication for design and operation of a DNA hybridization surface is that there is an optimal surface probe density when 2D diffusion occurs; values above that optimum do not increase the capture rate. Our model predicts capture rates in agreement with those from recent experimental literature. The results of our analysis predict that several things can be done to improve heterogeneous hybridization: 1) the solution phase target molecules should be about 100 bases or less in size to speed solution-phase and surface diffusion; 2) conditions should be created such that reversible adsorption and two-dimensional diffusion occur in the surface regions between DNA probe molecules; 3) provided that 2) is satisfied, one can achieve results with a sparse probe coverage that are equal to or better than those obtained with a surface totally covered with DNA probes.

Powerful tools for genetic analysis come of age.

Microscopic arrays of oligonucleotides or cDNA containing up to several hundred thousand different sequences are starting to influence methodologies and paths to discovery in genomics. Gene polymorphisms and mutations can be found and gene expression measured with unprecedented speed and parallelism. The principles of this modern technology and some of the problems awaiting further study are discussed.

STRUCTURAL STUDIES OF A LIQUID-FLUIDIZED MAGNETICALLY STABILIZED BED

As part of an effort to apply the magnetically stabilized fluidized bed (MSFB) to bioseparations, we have carried out a number of studies aimed at understanding its structure and properties. These included liquid-phase dispersion, conductivity (a measure of particle-particle contact), and studies with two different orientations of the applied magnetic field (parallel and transverse to the direction of liquid flow). The dispersion studies showed that the MSFB behaves much like a packed bed and is very different from an ordinary fluidized bed. The direction in which the magnetic field is applied is quite important, in one case causing a contraction and in the other an expansion of the bed compared to the height of a bed which is fluidized under identical conditions without a field.

Kinetics of heterogeneous hybridization on indium tin oxide surfaces with and without an applied potential

The rate of hybridization of oligonucleotide target sequences to chemically immobilized oligonucleotide probes has been studied both with and without an electrical field. The probe size was 20–24 nucleotides (nt) while the target size ranged from 157 to 864 nt. In agreement with previous studies, complete hybridization under normal conditions required 10–30 hours, depending on target size. The kinetics were characterized by a characteristic lag time followed by an asymptotic rise to the final value. In contrast, with an applied electrical field, all but the largest target hybridized in about 10 min while the longest hybridized within 1 h. Deleterious electrode reactions were avoided by close spacing of the anode and cathode and application of very small voltages. Our results suggest that probes and targets orient flat on the surface. A model is suggested to explain the kinetics observed that involves a series of surface states between initial target arrival and final hybridized state. Our results show that the electric field accelerated hybrid capture of solution‐phase targets by surface‐bound probes. This approach may have implications for enhancing array‐based hybrid capture for mutation detection, copy number determination and/or gene expression profiling.

Opportunities for near-infrared thermal ablation of colorectal metastases by guanylyl cyclase C-targeted gold nanoshells

Colorectal cancer is the third most common malignancy and the second most common cause of cancer-related mortality worldwide. While surgery remains the mainstay of therapy, approximately 50% of patients who undergo resection develop parenchymal metastatic disease. Unfortunately, current therapeutic regimens offer little improvement to the survival of patients with parenchymal metastases in the liver and lung. In that context, there is a significant unrealized opportunity at the intersection of engineering and biology for the development of novel targeted therapeutic approaches to colorectal cancer metastases. This opportunity exploits the discovery that an intestinal receptor, guanylyl cyclase C, which mediates diarrhea induced by bacterial heat-stable enterotoxins (STs), is over-expressed by metastatic colorectal tumors only. Moreover, it leverages recent advances in the fabrication of metal nanoshells with defined thicknesses absorb near-infrared (NIR) light, resulting in resonance and transfer of thermal energies of more than 40 degrees C. Thus, the conjugation of ST to gold nanoshells, which can undergo resonance excitation by NIR light and emit heat, represents a previously unrecognized approach for the targeted therapy of parenchymal colorectal cancer metastases, specifically to the liver and lung. This article discusses the potential of ST-targeted nanoshells for NIR thermal ablation of metastatic colorectal tumors and highlights the significant challenges and solutions linked to the translation of this emerging technology to patient care.

Diffusion and reversible reaction in a sphere: a numerical study using IMN approximants

Abstract Transient diffusion in a sphere, where reversible adsorption reaction takes place, was studied by a method of inverting Laplace transforms based on I MN approximants. The simplicity, speed, and reliability of this method make it especially suitable for numerical analysis of linear partial differential equations governing such processes as diffusion. Results show that in certain types of diffusion-adsorption processes, it may take a long time for equilibrium to be reached. The diffusive mass flux initially behaves as though there were no reaction taking place. This behavior is followed by a period where both reaction and diffusion determine the value of the flux and in fact the flux is almost constant during this period. After this follows a period during which the reaction appears to be very much faster than diffusion and therefore is essentially at equilibrium at any point within the sphere.

Response of Cutaneous Laser Velocimetry to a Temperature Change: Normal and Dysvascular Patients Compared

In previous studies of skin blood flow using a helium flux technique, we found a linear relationship between blood flow and skin temperature. In this paper we compared the helium flux method to laser Doppler velocimetry (LDV) over a range of temperatures in 5 normal healthy volunteers. In a separate normal group we measured the increase in LDV signal to a temperature change in both the volar aspect of the forearm and the dorsum of the foot. We then compared the LDV temperature response data from the normal subjects with data from a group of 20 patients with known peripheral vascular disease of the lower extremities and found a highly significant reduction in LDV output vs temperature in the patient groups. We concluded that the LDV temperature response data provide a convenient method for comparison of LDV data be tween patients and between sites on the same patient.

Affinity Chromatography of Mushroom Tyrosinase

Abstract The purification by column chromatography of a phenol-oxidizing enzyme, mushroom tyrosinase, was investigated using solid phase adsorbents designed to have specific affinity for the enzyme. Sepharose 4B, aminophenyl-bearing porous glass, and p-aminobenzylcellulose were chemically modified to introduce phenolic, catecholic, or benzoic groups on the polymer surface. The resulting preparations were tested for their effectiveness in separating tyrosinase from an impure protein mixture. The phenolic and benzoic polymers displayed no specific affinity for tyrosinase. Aminophenyl glass, with or without an attached phenolic group, adsorbed appreciable quantities of protein nonspecif-ically, thus complicating studies of its tyrosinase affinity properties. Dopamine, a dihydroxyphenyl derivative, was bound to Sepharose and was found to be effective in retaining tyrosinase at pH 5.5; elution of the enzyme by washing at pH 8.8 resulted in its purification by a factor of 10 to 14. Enzymatic oxidation of the ad...

Pulmonary capillary permeability in man and a canine model of chemical pulmonary edema.

Abstract Pulmonary capillary permeability-surface products (PSP) for sucrose and water have been calculated in normal, awake man and in anesthetized dogs using multiple indicator dilution data and a numerical deconvolution technique to estimate maximal extraction. In man and control animals the PSP for sucrose was found to be 9.1 and 7.1 cm 3 /sec, respectively, while PSPs for water were found to be 34.0 and 30.9 cm 3 /sec. The effects of flow and choice of the reference tracer (red blood cells or albumin) were examined. With the exception of water in man, the PSPs for sucrose and water did not change significantly with an increased cardiac output, regardless of the reference tracer chosen. Pulmonary capillary damage induced by the infusion of 75 mg/kg of alloxan in dogs increased the PSPs for sucrose and water by 35 and 52%, respectively, when red cells formed the reference tracer material. This change was eliminated for sucrose when albumin formed the reference tracer and reduced the magnitude of the change for water from 52 to 34%. The permeability increases in the alloxan damaged dogs occurred in the presence of normal pulmonary artery and wedge pressures. We conclude that the exchange of water is not flow-limited at physiologic or higher rates of perfusion and that albumin is misleading as a reference tracer in multiple indicator dilution studies of pulmonary edema.

Application of magnetically stabilized fluidized beds to bioseparations

Abstract Magnetically stabilized fluidized beds (MSFBs) have many advantages over current column separation techniques. The backmixing of solids seen in normal fluidized beds is eliminated, making countercurrent solids/fluid contact possible. Compared to the fixed bed, the MSFB allows continuous processing of the feed material while operating at a low bed pressure drop. Although other methods are available for carrying out continuous chromatographic separations, no other methods that we are aware of allow continuous bed treatment or replacement and few achieve a system which is as simple to operate. We have tested one application of MSFBs in bioprocessing: continuous affinity chromatography. The experimental results obtained using human serum albumin, Cibacron Blue F3GA®, and dried calcium alginate / magnetite beads agree with our mathematical model.