Microfluidic-based devices have allowed miniaturization and increased parallelism of many common functions in biological assays; however, development of a practical technology for microfluidic-based fluorescence-activated cell sorting has proved challenging. Although a variety of different physical on-chip switch mechanisms have been proposed, none has satisfied simultaneously the requirements of high throughput, purity, and recovery of live, unstressed mammalian cells. Here we show that optical forces can be used for the rapid (2-4 ms), active control of cell routing on a microfluidic chip. Optical switch controls reduce the complexity of the chip and simplify connectivity. Using all-optical switching, we have implemented a fluorescence-activated microfluidic cell sorter and evaluated its performance on live, stably transfected HeLa cells expressing a fused histone-green fluorescent protein. Recovered populations were verified to be both viable and unstressed by evaluation of the transcriptional expression of two genes, HSPA6 and FOS, known indicators of cellular stress.

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...

Selection and adjustment of proper physical parameters enables rapid DNA transport, site selective concentration, and accelerated hybridization reactions to be carried out on active microelectronic arrays. These physical parameters include DC current, voltage, solution conductivity and buffer species. Generally, at any given current and voltage level, the transport or mobility of DNA is inversely proportional to electrolyte or buffer conductivity. However, only a subset of buffer species produce both rapid transport, site specific concentration and accelerated hybridization. These buffers include zwitterionic and low conductivity species such as: d- and l-histidine; 1- and 3-methylhistidines; carnosine; imidazole; pyridine; and collidine. In contrast, buffers such as glycine, beta-alanine and gamma-amino-butyric acid (GABA) produce rapid transport and site selective concentration but do not facilitate hybridization. Our results suggest that the ability of these buffers (histidine, etc.) to facilitate hybridization appears linked to their ability to provide electric field concentration of DNA; to buffer acidic conditions present at the anode; and in this process acquire a net positive charge which then shields or diminishes repulsion between the DNA strands, thus promoting hybridization.

No abstract

No abstract