Electric-field-directed self-assembly of active enzyme-nanoparticle structures.

A method is presented for the electric-field-directed self-assembly of higher-order structures composed of alternating layers of biotin nanoparticles and streptavidin-/avidin-conjugated enzymes carried out on a microelectrode array device. Enzymes included in the study were glucose oxidase (GOx), horseradish peroxidase (HRP), and alkaline phosphatase (AP); all of which could be used to form a light-emitting microscale glucose sensor. Directed assembly included fabricating multilayer structures with 200 nm or 40 nm GOx-avidin-biotin nanoparticles, with AP-streptavidin-biotin nanoparticles, and with HRP-streptavidin-biotin nanoparticles. Multilayered structures were also fabricated with alternate layering of HRP-streptavidin-biotin nanoparticles and GOx-avidin-biotin nanoparticles. Results showed that enzymatic activity was retained after the assembly process, indicating that substrates could still diffuse into the structures and that the electric-field-based fabrication process itself did not cause any significant loss of enzyme activity. These methods provide a solution to overcome the cumbersome passive layer-by-layer assembly methods to efficiently fabricate higher-order active biological and chemical hybrid structures that can be useful for creating novel biosensors and drug delivery nanostructures, as well as for diagnostic applications.

Multiplexed protein detection using antibody-conjugated microbead arrays in a microfabricated electrophoretic device.

We report the development of a microfabricated electrophoretic device for assembling high-density arrays of antibody-conjugated microbeads for chip-based protein detection. The device consists of a flow cell formed between a gold-coated silicon chip with an array of microwells etched in a silicon dioxide film and a glass coverslip with a series of thin gold counter electrode lines. We have demonstrated that 0.4 and 1 µm beads conjugated with antibodies can be rapidly assembled into the microwells by applying a pulsed electric field across the chamber. By assembling step-wise a mixture of fluorescently labeled antibody-conjugated microbeads, we incorporated both spatial and fluorescence encoding strategies to demonstrate significant multiplexing capabilities. We have shown that these antibody-conjugated microbead arrays can be used to perform on-chip sandwich immunoassays to detect test antigens at concentrations as low as 40 pM (6 ng/mL). A finite element model was also developed to examine the electric field distribution within the device for different counter electrode configurations over a range of line pitches and chamber heights. This device will be useful for assembling high-density, encoded antibody arrays for multiplexed detection of proteins and other types of protein-conjugated microbeads for applications such as the analysis of protein-protein interactions.

Electric field directed assembly of high-density microbead arrays.

We report a method for rapid, electric field directed assembly of high-density protein-conjugated microbead arrays. Photolithography is used to fabricate an array of micron to sub-micron-scale wells in an epoxy-based photoresist on a silicon wafer coated with a thin gold film, which serves as the primary electrode. A thin gasket is used to form a microfluidic chamber between the wafer and a glass coverslip coated with indium-tin oxide, which serves as the counter electrode. Streptavidin-conjugated microbeads suspended in a low conductance buffer are introduced into the chamber and directed into the wells via electrophoresis by applying a series of low voltage electrical pulses across the electrodes. Hundreds of millions of microbeads can be permanently assembled on these arrays in as little as 30 seconds and the process can be monitored in real time using epifluorescence microscopy. The binding of the microbeads to the gold film is robust and occurs through electrochemically induced gold-protein interactions, which allows excess beads to be washed away or recycled. The well and bead sizes are chosen such that only one bead can be captured in each well. Filling efficiencies greater than 99.9% have been demonstrated across wafer-scale arrays with densities as high as 69 million beads per cm(2). Potential applications for this technology include the assembly of DNA arrays for high-throughput genome sequencing and antibody arrays for proteomic studies. Following array assembly, this device may also be used to enhance the concentration-dependent processes of various assays through the accelerated transport of molecules using electric fields.

Capture and enumeration of mRNA transcripts from single cells using a microfluidic device.

Accurate measurement of RNA transcripts from single cells will enable the precise classification of cell types and characterization of the heterogeneity in cell populations that play key roles in normal cellular physiology and diseases. As a step towards this end, we have developed a microfluidic device and methods for automatic hydrodynamic capture of single mammalian cells and subsequent immobilization and digital counting of polyadenylated mRNA molecules released from the individual cells. Using single-molecule fluorescence imaging, we have demonstrated that polyadenylated mRNA molecules from single HeLa cells can be captured within minutes by hybridization to polydeoxyribothymidine oligonucleotides covalently attached on the glass surface in the device. The total mRNA molecule counts in the individual HeLa cells are found to vary significantly from one another. Our technology opens up the possibility of direct digital enumeration of RNA transcripts from single cells with single-molecule sensitivity using a single integrated microfluidic device.

Microfluidic Device for Capture and Isolation of Single Cells.

We describe a microfluidic device capable of trapping, isolating, and lysing individual cells in parallel using dielectrophoretic forces and a system of PDMS channels and valves. The device consists of a glass substrate patterned with electrodes and two PDMS layers comprising of the microfluidic channels and valve control channels. Individual cells are captured by positive dielectrophoresis using the microfabricated electrode pairs. The cells are then isolated into nanoliter compartments using pneumatically actuated PDMS valves. Following isolation, the cells are lysed open by applying an electric field using the same electrode pairs. With the ability to capture and compartmentalize single cells our device may be combined with analytical methods for in situ molecular analysis of cellular components from single cells in a highly parallel manner.