Spatially isolated reactions in a complex array: using magnetic beads to purify and quantify nucleic acids with digital and quantitative real-time PCR in thousands of parallel microwells.

Quantitative real-time PCR (qPCR) has been the standard for nucleic acid quantification as it has a large dynamic range and good sensitivity. Digital PCR is rapidly supplanting qPCR in many applications as it provides excellent quantitative precision. However, both techniques require extensive sample preparation, and highly multiplexed assays that quantify multiple targets can be difficult to design and optimize. Here we describe a new nucleic acid quantification method that we call Spatially Isolated Reactions in a Complex Array (SIRCA), a highly parallel nucleic acid preparation, amplification, and detection approach that uses superparamagnetic microbeads in an array of thousands of 100 fL microwells to simplify sample purification and reduce reagent dispensing steps. Primers, attached to superparamagnetic microbeads through a thermo-labile bond, capture and separate target sequences from the sample. The microbeads are then magnetically loaded into a microwell array such that wells predominately contain a single bead. Master mix, lacking primers, is added before sealing the reaction wells with hydrophobic oil. Thermocycling releases the primer pair from the beads during PCR amplification. At low target concentrations, most beads capture, on average, less than one target molecule, and precise, digital PCR quantification can be derived from the percentage of positive reactions. At higher concentrations, qPCR signal is used to determine the average number of target molecules per reaction, significantly extending the dynamic range beyond the digital saturation point. We demonstrate that SIRCA can quantify DNA and RNA targets using thousands of parallel reactions, achieving attomolar limits of detection and a linear dynamic range of 105. The work reported here is a first step towards multiplexed SIRCA assays.

High resolution separations of charge variants and disulfide isomers of monoclonal antibodies and antibody drug conjugates using ultra-high voltage capillary electrophoresis with high electric field strength.

Ultra-high voltage capillary electrophoresis with high electric field strength has been applied to the separation of the charge variants, drug conjugates, and disulfide isomers of monoclonal antibodies. Samples composed of many closely related species are difficult to resolve and quantify using traditional analytical instrumentation. High performance instrumentation can often save considerable time and effort otherwise spent on extensive method development. Ideally, the resolution obtained for a given CE buffer system scales with the square root of the applied voltage. Currently available commercial CE instrumentation is limited to an applied voltage of approximately 30kV and a maximum electric field strength of 1kV/cm due to design limitations. The instrumentation described here is capable of safely applying potentials of at least 120kV with electric field strengths over 2000V/cm, potentially doubling the resolution of the best conventional CE buffer/capillary systems while decreasing analysis time in some applications. Separations of these complex mixtures using this new instrumentation demonstrate the potential of ultra-high voltage CE to identify the presence of previously unresolved components and to reduce analysis time for complex mixtures of antibody variants and drug conjugates.

Use of Ice-Nucleating Proteins To Improve the Performance of Freeze-Thaw Valves in Microfluidic Devices.

Currently, reliable valving on integrated microfluidic devices fabricated from rigid materials is confined to expensive and complex methods. Freeze-thaw valves (FTVs) can provide a low cost, low complexity valving mechanism, but reliable implementation of them has been greatly hindered by the lack of ice nucleation sites within the valve body's small volume. Work to date has required very low temperatures (on the order of -40 °C or colder) to induce freezing without nucleation sites, making FTVs impractical due to instrument engineering challenges. Here, we report the use of ice-nucleating proteins (INPs) to induce ice formation at relatively warm temperatures in microfluidic devices. Microfluidic channels were filled with buffers containing femtomolar INP concentrations from Pseudomonas syringae. The channels were cooled externally with simple, small-footprint Peltier thermoelectric coolers (TECs), and the times required for channel freezing (valve closure) and thawing (valve opening) were measured. Under optimized conditions in plastic chips, INPs made sub-10 s actuations possible at TEC temperatures as warm as -13 °C. Additionally, INPs were found to have no discernible inhibitory effects in model enzyme-linked immunosorbent assays or polymerase chain reactions, indicating their compatibility with microfluidic systems that incorporate these widely used bioassays. FTVs with INPs provide a much needed reliable valving scheme for rigid plastic devices with low complexity, low cost, and no moving parts on the device or instrument. The reduction in freeze time, accessible actuation temperatures, chemical compatibility, and low complexity make the implementation of compact INP-based FTV arrays practical and attractive for the control of integrated biochemical assays.

An automated integrated platform for rapid and sensitive multiplexed protein profiling using human saliva samples.

During the last decade, saliva has emerged as a potentially ideal diagnostic biofluid for noninvasive testing. In this paper, we present an automated, integrated platform useable by minimally trained personnel in the field for the diagnosis of respiratory diseases using human saliva as a sample specimen. In this platform, a saliva sample is loaded onto a disposable microfluidic chip containing all the necessary reagents and components required for saliva analysis. The chip is then inserted into the automated analyzer, the SDReader, where multiple potential protein biomarkers for respiratory diseases are measured simultaneously using a microsphere-based array via fluorescence sandwich immunoassays. The results are read optically, and the images are analyzed by a custom-designed algorithm. The fully automated assay requires as little as 10 µL of saliva sample, and the results are reported in 70 min. The performance of the platform was characterized by testing protein standard solutions, and the results were comparable to those from the 3.5 h lab bench assay that we have previously reported. The device was also deployed in two clinical environments where 273 human saliva samples collected from different subjects were successfully tested, demonstrating the device's potential to assist clinicians with the diagnosis of respiratory diseases by providing timely protein biomarker profiling information. This platform, which combines noninvasive sample collection and fully automated analysis, can also be utilized in point-of-care diagnostics.

Microfluidic amperometric sensor for analysis of nitric oxide in whole blood.

Standard photolithographic techniques and a nitric oxide (NO) selective xerogel polymer were utilized to fabricate an amperometric NO microfluidic sensor with low background noise and the ability to analyze NO levels in small sample volumes (~250 µL). The sensor exhibited excellent analytical performance in phosphate buffered saline, including a NO sensitivity of 1.4 pA nM(-1), a limit of detection (LOD) of 840 pM, and selectivity over nitrite, ascorbic acid, acetaminophen, uric acid, hydrogen sulfide, ammonium, ammonia, and both protonated and deprotonated peroxynitrite (selectivity coefficients of -5.3, -4.2, -4.0, -5.0, -6.0, -5.8, -3.8, -1.5, and -4.0, respectively). To demonstrate the utility of the microfluidic NO sensor for biomedical analysis, the device was used to monitor changes in blood NO levels during the onset of sepsis in a murine pneumonia model.

A microfluidic chip integrating DNA extraction and real-time PCR for the detection of bacteria in saliva.

A microfluidic chip integrating DNA extraction, amplification, and detection for the identification of bacteria in saliva is described. The chip design integrated a monolithic aluminum oxide membrane (AOM) for DNA extraction with seven parallel reaction wells for real-time polymerase chain reaction (rtPCR) amplification of the extracted DNA. Samples were first heated to lyse target organisms and then added to the chip and filtered through the nanoporous AOM to extract the DNA. PCR reagents were added to each of the wells and the chip was thermocycled. Identification of Streptococcus mutans in a saliva sample is demonstrated along with the detection of 300 fg (100-125 copies) of both methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) genomic DNA (gDNA) spiked into a saliva sample. Multiple target species and strains of bacteria can be simultaneously identified in the same sample by varying the primers and probes used in each of the seven reaction wells. In initial tests, as little as 30 fg (8-12 copies) of MSSA gDNA in buffer has been successfully amplified and detected with this device.

Laser-based directed release of array elements for efficient collection into targeted microwells.

A cell separation strategy capable of the systematic isolation and collection of moderate to large numbers (25-400) of single cells into a targeted microwell is demonstrated. An array of microfabricated, releasable, transparent micron-scale pedestals termed pallets and an array of microwells in poly(dimethylsiloxane) (PDMS) were mated to enable selective release and retrieval of individual cells. Cells cultured on a pallet array mounted on a custom designed stage permitted the array to be positioned independently of the microwell locations. Individual pallets containing cells were detached in a targeted fashion using a pulsed Nd:YAG laser. The location of the laser focal point was optimized to transfer individual pallets to designated microwells. In a large-scale sort (n = 401), the accuracy, defined as placing a pallet in the intended well, was 94% and the collection efficiency was 100%. Multiple pallets were observed in only 4% of the targeted wells. In cell sorting experiments, the technique provided a yield and purity of target cells identified by their fluorescence signature of 91% and 93%, respectively. Cell viability based on single-cell cloning efficiency at 72 h post collection was 77%.

High electric field strength two-dimensional peptide separations using a microfluidic device.

New instrumentation has been developed to improve the resolution, efficiency, and speed of microfluidic 2D separations using MEKC coupled to high field strength CE. Previously published 2D separation instrumentation [Ramsey, J. D. et al., Anal. Chem. 2003, 75, 3758-3764] from our group was limited to a maximum potential difference of 8.4 kV, resulting in an electric field strength of only approximately 200 V/cm in the first dimension. The circuit described in this report has been designed to couple a higher voltage supply with a rapidly switching, lower voltage supply to utilize the best features of each. Voltages applied in excess of 20 kV lead to high electric field strength separations in both dimensions, increasing the separation resolution, efficiency, and peak capacity while reducing the required analysis time. Detection rates as high as six peptides per second (based on total analysis time) were observed for a model protein tryptic digest separation. Additionally, higher applied voltages used in conjunction with microfluidic chips with longer length channels maintained higher electric field strengths and produced peak capacities of over 4000 for some separations. Total separation time in these longer channel devices was comparable to that obtained in short channels at low field strength; however, resolving power improved approximately threefold.

Ultra-high voltage capillary electrophoresis >300 kV: recent advances in instrumentation and analyte detection.

Instrumentation has been developed for the implementation of ultra-high voltage capillary electrophoresis (UHVCE) with potentials up to and exceeding 300 kV. Several separations have been used to demonstrate the utility of higher applied voltages for improving the resolution of peptide, protein, and nucleic acid separations. Previously reported instrumentation was limited to 120 kV and required submersion in a bath of transformer oil to prevent corona and high voltage arcing between the components of the instrument [Hutterer, 1999, 2000, 2005] [1-3]. A modular design that uses plastic dielectric materials to overcome these obstacles enabling simplified operation of the instrument in air is described here in detail. A forced air system developed to control the temperature of the instrument to within a few degrees over a range of 25-60 °C for use with ultra-high voltage capillary gel electrophoresis is also described. UHVCE instrumentation and its applications with UV absorption and laser induced fluorescence detection are further developed, and the first demonstration of UHVCE coupled to electrospray ionization-mass spectrometry is shown.

Fabrication of microfluidic devices containing patterned microwell arrays.

A rapid fabrication and prototyping technique to incorporate microwell arrays with sub-10 µm features within a single layer of microfluidic circuitry is presented. Typically, the construction of devices that incorporate very small architecture within larger components has required the assembly of multiple elements to form a working device. Rapid, facile production of a working device using only a single layer of molded polydimethylsiloxane (PDMS) and a glass support substrate is achieved with the reported fabrication technique. A combination of conventional wet-chemical etching for larger (≥20 µm) microchannel features and focused ion beam (FIB) milling for smaller (≤10 µm) microwell features was used to fabricate a monolithic glass master mold. PDMS/glass hybrid chips were then produced using simple molding and oxygen plasma bonding methods. Microwell structures were loaded with 3 µm antibody-functionalized dye-encoded polystyrene spheres, and a sandwich immunoassay for common cytokines was performed to demonstrate proof-of-principle. Potential applications for this device include highly parallel multiplexed sandwich immunoassays, DNA/RNA hybridization analyses, and enzyme linked immunosorbent assay (ELISA). The fabrication technique described can be used for rapid prototyping of devices wherever submicrometer- to micrometer-sized features are incorporated into a microfluidic device.

Monolithic integration of two-dimensional liquid chromatography-capillary electrophoresis and electrospray ionization on a microfluidic device.

A microfluidic device capable of two-dimensional reversed-phase liquid chromatography-capillary electrophoresis with integrated electrospray ionization (LC-CE-ESI) for mass spectrometry (MS)-based proteomic applications is described. Traditional instrumentation was used for the LC sample injection and delivery of the LC mobile phase. The glass microfabricated device incorporated a sample-trapping region and an LC channel packed with reversed-phase particles. Rapid electrokinetic injections of the LC effluent into the CE dimension were performed at a cross-channel intersection. The CE separation channel terminated at a corner of the square device, which functioned as an integrated electrospray tip. In addition to LC-CE-ESI, this device was used for LC-ESI without any instrumental modifications. To evaluate the system, LC-MS and LC-CE-MS analyses of protein digests were performed and compared.

Flow counterbalanced capillary electrophoresis using packed capillary columns: resolution of enantiomers and isotopomers.

A method with the ability to increase greatly both the resolution and efficiency of a given capillary electrophoretic system is described. This method differs from traditional capillary electrophoresis (CE) in that a counterflow is induced in the direction opposite to the electrokinetic migration of the analyte. This has the effect of extending not only the time the analytes migrate in the electric field but also the effective length and the effective applied voltage of the system. Previous work in our group with flow counterbalanced capillary electrophoresis has utilized an open tube of small inner diameter to reduce peak broadening caused by hydrodynamic flow. Narrow-diameter capillaries (5-10 microm) restricted analysis to fluorescent analytes and laser-induced fluorescence detection. The method described here uses a capillary of much larger inner diameter (75 microm) that has been packed with nonporous silica particles. The packing material reduces the amount of band broadening caused by pressure-induced flow relative to that experienced in an open tube. A larger diameter capillary allows the detection of analytes by UV absorption, not only eliminating the need to tag analytes with fluorescent tags but also allowing for the detection of a much broader range of analytes. The system was evaluated by studying the separations of several enantiomers using only beta-cyclodextrin as the chiral selector. The system was also used to resolve the two naturally occurring isotopes of bromine and to resolve phenylalanine from phenylalanine-d8. Relative to traditional CE, large improvements in resolution and separation efficiency have been achieved with this method.