We have demonstrated that controlled electric fields can be used to regulate transport, concentration, hybridization, and denaturation of single-and doublestranded oligonucleotides. Discrimination among oligonucleotide hybrids with widely varying binding strengths may be attained by simple adjustment of the electric field strength. When this approach is used, electric field denaturation control allows single base pair mismatch discrimination to be carried out rapidly (<15 sec) and with high resolution. Electric field denaturation takes place at temperatures well below the melting point of the hybrids, and it may constitute a novel mechanism of DNA denaturation.Combinations of the disciplines of microfabrication, chemistry, and molecular biology have allowed the generation of large oligonucleotide probe arrays which may facilitate rapid multiplex analysis of nucleic acid samples. Previous efforts have demonstrated the successful application of miniaturization technology, array formats, microfabrication techniques, and highly sensitive detection technology to obtain such genetic analysis on a chip (1-7). However, those models have used passive hybridization in which the reaction rate is limited by diffusion. In an attempt to circumvent this, we have investigated the effect of electric fields on biomolecular reactions.We have developed a microscopic format which contains an electronically addressable electrode array that provides direct electric field control over a variety of biomolecular reactions. The electric field facilitates two interactions: transport of charged molecules to selected microlocations and hence concentration over an immobilized substrate. Subsequent reversal of the field may be used to selectively repulse those molecules with reduced affinity for the substrate. In the case of nucleic acids, regulation of the electric field strength allows adjustment of hybridization stringency for homologous interactions. MATERIALS AND METHODSMicrofabrication. The devices were fabricated on thermally oxidized silicon substrates by using standard microelectronics techniques (8). Aluminum was initially sputtered onto the substrates, and was then coated with 1 m of positive photoresist. The photoresist was patterned in a proximity mask aligner in such a manner as to open holes in the resist over the desired electrode locations. A 20-nm Cr adhesion layer and a 500-nm Pt electrode layer were then sequentially deposited on the wafer by electron-beam evaporation. A solvent was used to remove the remaining photoresist, which lifted off the Cr-Pt layer, leaving only Cr-Pt in the electrode locations. The underlying Al layer was then chemically etched, using the Cr-Pt layer as a mask to complete the electrode fabrication.Two micrometers of low-stress silicon nitride was then deposited on the wafer by plasma-enhanced chemical vapor deposition. The silicon nitride was again coated with photoresist, exposed, and developed to open holes above the electrodes, and the nitride was etched down to the electrodes, using pla...

Escherichia coli were separated from a mixture containing human blood cells by means of dielectrophoresis and then subjected to electronic lysis followed by proteolytic digestion on a single microfabricated bioelectronic chip. An alternating current electric field was used to direct the bacteria to 25 microlocations above individually addressable platinum microelectrodes. The platinum electrodes were 80 microns in diameter and had center-to-center spacings of 200 microns. After the isolation, the bacteria were lysed by a series of high-voltage pulses. The lysate contained a spectrum of nucleic acids including RNA, plasmid DNA, and genomic DNA. The lysate was further examined by electronically enhanced hybridization on separate bioelectronic chips. Dielectrophoretic separation of cells followed by electronic lysis and digestion on an electronically active chip may have potential as a sample preparation process for chip-based hybridization assays in an integrated DNA/RNA analysis system.

No abstract

Tumour necrosis factor (TNF) is a cytokine belonging to a family of trimeric proteins; it has been shown to be a key mediator in autoimmune diseases such as rheumatoid arthritis and Crohn’s disease. While TNF is the target of several successful biologic drugs, attempts to design small molecule therapies directed to this cytokine have not led to approved products. Here we report the discovery of potent small molecule inhibitors of TNF that stabilise an asymmetrical form of the soluble TNF trimer, compromising signalling and inhibiting the functions of TNF in vitro and in vivo. This discovery paves the way for a class of small molecule drugs capable of modulating TNF function by stabilising a naturally sampled, receptor-incompetent conformation of TNF. Furthermore, this approach may prove to be a more general mechanism for inhibiting protein–protein interactions.

The separation and subsequent isolation of the metastatic human cervical carcinoma cell line (HeLa cells) from normal human peripheral blood cells has been achieved by exploiting their differential dielectric properties. The isolation process is carried out on a silicon chip containing a five-by-five array of microlocations. These microlocations contain underlying circular platinum electrodes with 80-micron diameters and center-to-center spacing of 200 microns. The surfaces of the electrodes and nonmetallized areas have been coated with a permeation layer to prevent the direct contact of cells with the electrode and also to minimize the nonspecific adhesion of the cells to the chip surface. An inhomogenous ac field is applied to the electrodes to create the conditions for dielectrophoretic separation of cells. Cell separation using dielectrophoresis as well as electronic lysis on a silicon chip would provide essential sample-processing steps which may be combined with a later multiplex electronic hybridization step in an integrated assay system.