鈴木 誠一

Previous investigations into DNA orientation under an electrostatic field used either a low-intensity or a pulsed field, and the measurements were made in the region where the degree of orientation was relatively low, This was because applying high-intensity steady fields to aqueous solutions resulted in a temperature rise and caused turbulence or boiling, which interfered with orientation, In this paper, microfabricated electrodes are used to obtain stationary ac electric fields in excess of 1 MV/m, Planar microelectrodes with either 15- or 60-mu m gaps, depending on the length of sample DNA to be used, are fabricated with planar technique on glass substrates. Because the high-field region in the gap is small and has a high surface-to-volume ratio, joule heat is efficiently removed, so that a very high-intensity field can be created without excessive temperature rise, A high-sensitivity detection method is required for measurements with microelectrodes, due to the small number of molecules involved, For this purpose, a fluorescent dye is intercalated into the bases of the DNA, and the optical polarization of emitted fluorescence is measured, The polarization components of the emitted light, both parallel and perpendicular to the applied electrostatic field, are measured independently, and fluorescent anisotropy, the ratio of the difference between parallel and perpendicular polarization to total emission, is used as an index of DNA orientation, The field-intensity dependence of fluorescence anisotropy is measured using pUC18 (2.7-kb DNA), and the result is compared with an analytical model, The measured polarization factor is found to be several orders of magnitude larger than that of a conducting ellipsoid with the same dimension, This can be explained by assuming a "swelling" of electrical equivalent diameter of DNA by 20 nm, comparable to the Debye length, i.e., the thickness of a counterion cloud, The counterion concentration is varied by changing pH of the medium, keeping its conductivity constant, As pH increases, an increase in the anisotropy is observed, in particular between pH 5 and 6, This is attributed to the dissociation of phosphate groups, by which the negative charge density on the DNA backbone is increased, The multivalent cations Ca2+, Mg2+, Zn2+, and Al3+ are expected to bind to DNA backbone and reduce polarization, However, no significant change of anisotropy is observed in the concentration range 10(-4)-10(-6) M under our experimental conditions. Further increases in concentration were prevented by conductivity constraints.

The authors have previously reported that the electrostatic orientation and the dielectrophoresis (DEP) of DNA occur under approximate to 1 MHz, > 1 x 10(6) V/m field, by which DNA strands are stretched straight along field lines and positioned onto electrode edges, This paper presents some application of this stretch-and-positioning method to genetic engineering, It is shown that the DNA size distribution, as well as the activities of nuclease, can be determined by the measurement of the apparent length of stretched DNA. Several methods are developed to immobilize stretched DNA onto a substrate, including: 1) immobilization onto a conducting substrate for observations with the scanning tunneling microscopy, 2) anchoring onto a substrate only at the both ends of DNA using special electrode configuration, and/or molecular binding between avidin and biotin, The DNA can be held without contact to the substrate in the latter method, so that it does not cause steric hindrances to the DNA-binding enzymes. A novel Fluid Integrated Circuit (FIC) device is proposed in which stretched DNA is cut by laser beam for the successive sequencing. A method to obtain unidirectionally oriented DNA is developed. The spatial resolution, and the small number of molecules required, are the advantages of the assays and measurements using electrostatic DNA manipulations over conventional biochemical methods. It is hoped that the methods may open a way to a novel category of ''molecular biochemistry with spatial resolution.''

A black membrane is a biological-membrane analogue, i.e. a phospholipid bilayer membrane, artificially formed on an orifice immersed in water. It is used to investigate the properties of the membrane itself and channels embedded therein. In this paper, microfabrication techniques are applied to fabricate the orifice, and a glass substrate is isotropically etched to define the orifice geometry. The periphery of the orifice was patterned with aminosilane to anchor the membrane. The remainder part was coated with fluorosilane to make the surface hydrophobic and to prevent adsorption of channel-forming molecules. We demonstrated experimentally that a stable and reproducible membrane is easily obtainable using the orifice.