Aligned Immobilization of Proteins Using AC Electric Fields

Abstract

Protein molecules are aligned and immobilized from solution by AC electric fields. In a single-step experiment, the enhanced green fluorescent proteins are immobilized on the surface as well as at the edges of planar nanoelectrodes. Alignment is found to follow the molecules' geometrical shape with their longitudinal axes parallel to the electric field. Simultaneous dielectrophoretic attraction and AC electroosmotic flow are identified as the dominant forces causing protein movement and alignment. Molecular orientation is determined by fluorescence microscopy based on polarized excitation of the proteins' chromophores. The chromophores' orientation with respect to the whole molecule supports X-ray crystal data.

AC electrokinetic immobilization of organic dye molecules

“…Thus, AC electrophoresis might not only contribute to migration of charged molecules towards electrode surfaces; even more important, the concept of AC electrophoresis provides an explanation for the permanent character of the immobilization of molecules. The hypothesis of migrating molecules by AC electrophoresis is further supported by our observation of gas bubbles at the electrodes at frequencies below 5 kHz [24,42] caused by water electrolysis, which has often been mentioned in the literature [22,28,[43][44][45]. Another interesting result was reported by Ying et al for the accumulation of DNA fragments and nucleotide triphosphate at the tip of a nanopipette, conducted at a frequency of 0.5 Hz [46].…”

“…The electrophoretic mobility of porphyrins amounts to about 2.7 × 10 −8 m 2 V −1 s −1 [47]. According to our numerical field simulations, the maximal field strength in the electrode gap is 12 MV/m [22]. This value quickly decreases with height, as has been reported by Green et al [48].…”

“…The fact that no fluorescence had been detected from these large substance accumulations is attributed to a very high local fluorophore density, which can cause fluorescence quenching. In general, the molecules' functionality is assumed to be maintained after the electrokinetic immobilization process, as has already been published for several other molecules [22][23][24][25].…”

“…Even if the Stokes radius of a molecule or ion is considered instead of the dimensions of the intrinsic molecule or ion, the difference in radii is too small to explain the observed attraction of small dye molecules. From simulations of the electric field and its gradient for the present electrode design [22], however, it follows that Brownian motion could be overcome with the electric field parameters used, even for molecules of diameters below 1 nm. The crucial parameter is a sufficiently high electric field gradient ∇|E| 2 , which is calculated to around 3 × 10 21 V 2 /m 3 .…”

“…Interdigitated electrodes (IDE) made of tungsten with a configuration similar to that described in [22] were used. Electrode chips with sizes of 10 mm × 10 mm were produced according to a standard 0.25-μm complementary metal-oxidesemiconductor (CMOS) process on 8 in.…”