Shirley S. Chan

Electrodeless Dielectrophoresis of Single- and Double-Stranded DNA

Dielectrophoretic trapping of molecules is typically carried out using metal electrodes to provide high field gradients. In this paper we demonstrate dielectrophoretic trapping using insulating constrictions at far lower frequencies than are feasible with metallic trapping structures because of water electrolysis. We demonstrate that electrodeless dielectrophoresis (EDEP) can be used for concentration and patterning of both single-strand and double-strand DNA. A possible mechanism for DNA polarization in ionic solution is discussed based on the frequency, viscosity, and field dependence of the observed trapping force.

Dynamics of carbon monoxide binding to protoheme

Protoheme rebinding of carbon monoxide after photodissociation has been observed at temperatures from 5 to 340 K for times from 2 μs to 1 ks. Below 80 K, binding is nonexponential in time and CO‐concentration independent, above 230 K exponential and the rate is CO‐concentration proportional. A model is proposed in which the carbon monoxide, moving from the solvent to the binding site at the ferrous heme iron, encounters two successive barriers. The outer is formed by the solvent, the inner is a property of the heme and probably connected to the motion of the iron from the spin‐2 deoxy to the spin‐0 carbon monoxide state. The temperature dependence of the two processes yields all activation enthalpies and entropies for the two barriers. The nonexponential rebinding observed at low temperatures implies that the inner barrier possesses distributed activation enthalpy and entropy. The enthalpy spectrum and the entropy spread are determined. The spectrum demonstrates that heme exists in many different conformational states. At low temperatures, these states are frozen; above about 230 K, rapid conformational relaxation renders rebinding exponential. Below 15 K, quantum‐mechanical molecular tunneling dominates. The tunneling rate yields the width of the innermost barrier. Earlier experiments on carbon monoxide binding to myoglobin had provided evidence for four barriers. The present results imply that the innermost barrier in myoglobin is caused by the heme, the outermost by the solvent, and the two intermediate ones by the globin.

Transient analyzer with logarithmic time base

Many transient phenomena extend over more than two orders of magnitude in time. Such processes are most efficiently observed with systems that possess logarithmic time bases. A digital analyzer is described that records smoothly and in one sweep transient processes over more than eight orders of magnitude in time. A typical sweep covers the time range from 2 μsec to 5 min.

Activation and Sorting of Human White Blood Cells

We demonstrate a novel activation behavior of human leucocyte adhesion under physiological flow conditions in a microfabricated silicon array of channels with length scales similar to those of human capillaries. Vital nuclei stains and cell specific, flourochrome labeled antibodies reveal that the equilibrium distribution of stuck cells in the arrays displays a strong dependence on cell type and nuclear morphology, and there is eventual separation of the two cell types in the array. The distortion of the cells is the same as they experience in vivo and the response of the granulocytes is consistent with a model describing adhesion as a function of the distortion of the cell by its environment; in other words, activated adhesion. We propose that this complex non-random behavior is due to a deformation activated change in the cells relevant to observed in vivo behavior.

Fast reactions in carbon monoxide binding to heme proteins.

Using fast flash photolysis, we have measured the binding of CO to carboxymethylated cytochrome c and to heme c octapeptide as a function of temperature (5 degrees-350 degreesK) over an extended time range (100 ns(-1) ks). Experiments used a microsecond dye laser (lambda = 540 nm), and a mode-locked frequency-doubled Nd-glass laser (lambda = 530 nm). At low temperatures (5 degrees-120 degreesK) the rebinding exhibits two components. The slower component (I) is nonexponential in time and has an optical spectrum corresponding to rebiding from an S = 2, CO-free deoxy state. The fast component (I*) is exponential in time with a lifetime shorter than 10 mus and an optical spectrum different from the slow component. In myoglobin and the separated alpha and beta chains of hemoglobin, only process I is visible. The optical absorption spectrum of I* and its time dependence suggest that it may correspond to recombination from an excited state in which the iron has not yet moved out of the heme plane. The temperature dependences of both processes have been measured. Both occur via quantum mechanical tunneling at the lowest temperatures and via over-the-barrier motion at higher temperatures.

Sizing, Fractionation and Mixing of Biological Objects Via Microfabricated Devices

One aspect of micro and nanofabrication that has not been exploited a great deal has been the ability to make structures to probe and separate complex mixtures using designed environments. We will give three brief examples of such second-generation uses of microfabrication, as opposed to simply shrinking the size of the vessels or tubes used in macroscopic lab environments. The three examples chosen are blood cell fractionation and cell sorting, asymmetric brownian motion fractionation and ultra-high speed fluid mixing.

Microfabricated arrays for fractionation of large DNA molecules via pulsed field electrophoresis

Novel microfabricated devices promise to accomplish fractionation of chromosomal size DNA more quickly, more accurately, at lower cost, and by using smaller sample amounts. Chromosomes are released by lysing cells directly in the device. The chromosomal DNA is further concentrated on a platinum wire in a 10 μm wide band using the phenomenon of dielectric trapping in AC fields. The DNA is then electrophoretically driven into a microfabricated array of posts arranged in a hexagonal lattice. Under electric fields whose direction periodically changes by 120°, the longer DNA molecules move at lower speeds than the shorter ones, and separation according to size is achieved. This technique allows application of electric fields as large as 1000 V/cm and, thus, promises to reduce considerably separation times compared to the presently used technique of pulsed-field gel electrophoresis.

Polymer dynamics and fluid flow in microfabricated devices

We will discuss two recent directions of our work: (1) The influence of submicron length scales on polymer dynamics, (2) Ultra-rapid mixing via sub-micron hydrodynamic focusing. (1) Polymer dynamics at sub-micron length scales. We have explored the changes in the dynamics of long polymers as the thickness of the quasi-2 dimensional space is varied from 0.09 microns to 10 microns. We will show how the thickness of this space, scaled with the persistence length of the polymer, changes the dynamics of the polymer. The consequences of this qualitative change in polymer dynamics is quite important, since it controls the elongation of the polymer at a given force field and hence the ability of he array to fractionate the polymer. (2) Mixing at the sub- micron length scale cannot be tubulent but only diffusive in nature. We will show how it is possible using hydrodynamics to produce liquid jets of width under 20 nanometers which can mix fluids in under 1 microsecond times.

Sequence Matters: The Influence of Basepair Sequence on DNA-protein Interactions

The sequencing of the human genome, along with the 200-odd other genomes that have been sequenced, does not represent the solution to a puzzle but rather the necessary introduction to a bigger puzzle. That puzzle is how all the 30,000-odd some genes in the human genome are expressed and controlled in a proper sequence for a cell to function. We can hardly address this enormous problem in this brief review, but instead wish to concentrate on one very small but very important aspect of this problem: physical aspects to how proteins are able to achieve base-pair specific recognition. By “physical aspects” we mean that the proteins distort (strain) the DNA helix when they bind, and if this strain is a function of the sequence of the distorted region then the basepair dependent free energy associated with the strain provides a way to discriminate amongst the basepairs.

Hydrodynamic activation and sorting of white blood cells in a microfabricated lattice

We demonstrate a novel hydrodynamic shear activation of leucocyte adhesion, using physiological flow conditions and a microfabricated array of channels with length scales similar to those of human capillaries. Vital chromosome stains and cell specific fluorochrome labeled antibodies reveal that the eventual adhesion of the leukocytes to the silicon array displays a strong dependence on cell type and nuclear morphology, with granulocytes activating more rapidly with distance and penetrating a smaller distance than lymphocytes. Further, the granulocytes interact with the lymphocytes in a self-exclusionary manner under shearing flow with the eventual separation of the two cell types in the array. Such arrays of microfabricated obstacles thus have an interesting potential for sorting white blood cells by type from a 10 microliter drop of whole blood.