We report carbon nanotube network field-effect transistors (NTNFETs) that function as selective detectors of DNA immobilization and hybridization. NTNFETs with immobilized synthetic oligonucleotides have been shown to specifically recognize target DNA sequences, including H63D single-nucleotide polymorphism (SNP) discrimination in the HFE gene, responsible for hereditary hemochromatosis. The electronic responses of NTNFETs upon single-stranded DNA immobilization and subsequent DNA hybridization events were confirmed by using fluorescence-labeled oligonucleotides and then were further explored for label-free DNA detection at picomolar to micromolar concentrations. We have also observed a strong effect of DNA counterions on the electronic response, thus suggesting a charge-based mechanism of DNA detection using NTNFET devices. Implementation of label-free electronic detection assays using NTNFETs constitutes an important step toward low-cost, low-complexity, highly sensitive and accurate molecular diagnostics.hemochromatosis ͉ SNP ͉ biosensor T he development of nucleic acids diagnostics has become the subject of intense research, especially in the postgenome era. Current methods have mainly focused on optical detection using fluorescence-labeled oligonucleotides with dyes (1), quantum dots (2), or enhanced absorption of light by oligonucleotidemodified gold nanoparticles (3). On the other hand, label-free electronic methods promise to offer sensitivity, selectivity, and low cost for the detection of DNA hybridization (4). For example, microfabricated silicon field-effect sensors can monitor directly the increase in surface charge when DNA was hybridized on the sensor surface (5). Nanomaterials possess unique properties that are amenable to biosensor applications; they are one-dimensional structures that are extremely sensitive to electronic perturbations, readily functionalized with biorecognition layers, and compatible with many semiconducting manufacturing processes. Thus, one-dimensional silicon nanowires (6-8) and indium oxide nanowires (9) have shown promising performance, because their electronic conductance is more sensitive to DNA-associated charges as a result of their high surface-tovolume ratio. Using smaller nanowires with virtually all atoms on their surface, such as single-walled carbon nanotubes (SWNTs), will provide additional advantages in DNA detection. To date, there are several reports on electrochemical detection of DNA hybridization using multi-walled carbon nanotube electrodes (ref. 10 and references therein, and ref. 11). Whereas electrochemical methods rely on electrochemical behavior of the labels, measurement of direct electron transfer between SWNTs and DNA molecules paves the way for label-free DNA detection. SWNT-based field-effect transistors (12) have excellent operating characteristics (13), and they have already been explored for highly sensitive electronic detection of gases (14, 15) and biomolecules such as antibodies (16,17).Single-stranded DNA (ssDNA) has been recently demons...

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.

Microelectronic DNA chip devices that contain planar arrays of microelectrodes have been developed for multiplex DNA hybridization and a variety of genomic research and DNA diagnostic applications. These devices are able to produce almost any desired electric field configuration on their surface. This ability to produce well‐defined electric fields allows charged molecules (DNA, RNA, proteins, enzymes, antibodies, nanobeads, and even micron scale semiconductor devices) to be electrophoretically transported to or from any microlocation on the planar surface of the device. Of key importance to the device function is the permeation layer which overcoats the microelectrodes. The permeation layer is generally a porous hydrogel material that allows water molecules and small ions (Na+, CI−, etc.) to freely contact the microelectrode surface, but impedes the transport of the larger analytes (oligonucleotides, DNA, RNA, proteins, etc.). The permeation layer prevents the destruction of DNA at the active microelectrode surface, ameliorates the adverse effects of electrolysis products on the sensitive hybridization reactions, and serves as a porous support structure for attaching DNA probes and other molecules to the array. In order to maintain rapid transport of DNA molecules, facilitate hybriziation, and work within constrained current and voltage ranges, low conductance buffers and various electronic pulsing scenarios have also been developed. These active microelectronic array devices allow electrophoretic fields to be used to carry out accelerated DNA hybridization reactions and to improve selectivity for single nucleotide polymorphism (SNP), short tandem repeat (STR), and point mutation analysis.

A variety of electronic DNA array devices and techniques have been developed that allow electric field enhanced hybridization to be carried out under special low-conductance conditions. These devices include both planar microelectronic DNA array/chip devices as well as electronic microtiter plate-like devices. Such "active" electronic devices are able to provide controlled electric (electrophoretic) fields that serve as a driving force to move and concentrate nucleic acid molecules (DNA/RNA) to selected microlocation test-sites on the device. In addition to ionic strength, pH, temperature and other agents, the electric field provides another controllable parameter that can affect and enhance DNA hybridization. With regard to the planar microelectronic array devices, special low-conductance buffers were developed in order to maintain rapid transport of DNA molecules and to facilitate hybridization within the constrained low current and voltage ranges for this type of device. With regard to electronic microtiter plate type devices (which do not have the low current/voltage constraints), the use of mixed buffers (low conductance upper chamber/high conductance lower chamber) can be used in a unique fashion to create favorable hybridization conditions in a microzone within the test site location. Both types of devices allow DNA molecules to be rapidly and selectively hybridized at the array test sites under conditions where the DNA in the bulk solution can remain substantially denatured.

Microelectronic DNA chip devices that contain planar arrays of microelectrodes have been developed for multiplex DNA hybridization and a variety of genomic research and DNA diagnostic applications. These devices are able to produce almost any desired electric field configuration on their surface. This ability to produce well-defined electric fields allows charged molecules (DNA, RNA, proteins, enzymes, antibodies, nanobeads, and even micron scale semiconductor devices) to be electrophoretically transported to or from any microlocation on the planar surface of the device. Of key importance to the device function is the permeation layer which overcoats the microelectrodes. The permeation layer is generally a porous hydrogel material that allows water molecules and small ions (Na+, CI-, etc.) to freely contact the microelectrode surface, but impedes the transport of the larger analytes (oligonucleotides, DNA, RNA, proteins, etc.). The permeation layer prevents the destruction of DNA at the active microelectrode surface, ameliorates the adverse effects of electrolysis products on the sensitive hybridization reactions, and serves as a porous support structure for attaching DNA probes and other molecules to the array. In order to maintain rapid transport of DNA molecules, facilitate hybridization, and work within constrained current and voltage ranges, low conductance buffers and various electronic pulsing scenarios have also been developed. These active microelectronic array devices allow electrophoretic fields to be used to carry out accelerated DNA hybridization reactions and to improve selectivity for single nucleotide polymorphism (SNP), short tandem repeat (STR), and point mutation analysis.

We describe a method for the discrimination of short tandem repeat (STR) alleles based on active microarray hybridization. An essential factor in this method is electronic hybridization of the target DNA, at high stringency, in <5 min. High stringency is critical to avoid slippage of hybrids along repeat tracts at allelespecific test sites in the array. These conditions are attainable only with hybridization kinetics realized by electronic concentration of DNA. A sandwich hybrid is assembled, in which proper base stacking of juxtaposed terminal nucleotides results in a thermodynamically favored complex. The increased stability of this complex relative to non-stacked termini and/or base pair mismatches is used to determine the identification of STR alleles. This method is capable of simultaneous and precise identification of alleles containing different numbers of repeats, as well as mutations within these repeats. Given the throughput capabilities of microarrays our system has the potential to enhance the use of microsatellites in forensic criminology, diagnostics and genetic mapping.