Anson Hatch

A rapid diffusion immunoassay in a T-sensor

We have developed a rapid diffusion immunoassay that allows measurement of small molecules down to subnanomolar concentrations in <1 min. This competitive assay is based on measuring the distribution of a labeled probe molecule after it diffuses for a short time from one region into another region containing antigen-specific antibodies. The assay was demonstrated in the T-sensor, a simple microfluidic device that places two fluid streams in contact and allows interdiffusion of their components. The model analyte was phenytoin, a typical small drug molecule. Clinically relevant levels were measured in blood diluted from 10- to 400-fold in buffer containing the labeled antigen. Removal of cells from blood samples was not necessary. This assay compared favorably with fluorescence polarization immunoassay (FPIA) measurements. Numerical simulations agree well with experimental results and provide insight for predicting assay performance and limitations. The assay is homogeneous, requires <1 μl of reagents and sample, and is applicable to a wide range of analytes.

A ferrofluidic magnetic micropump

A microfluidic pump is described that uses magnetic actuation to push fluid through a microchannel. Operation relies on the use of magnetically-actuated plugs of ferrofluid, a suspension of nanosize ferromagnetic particles. The ferrofluid contacts but is immiscible with the pumped fluid. The prototype circular design demonstrates continuous pumping by regenerating a translating ferrofluidic plug at the conclusion of each pumping cycle. The flow rate can be controlled by adjusting device dimensions or the velocity of an external permanent magnet that directs the motion of the ferrofluid. The ferrofluidic plugs also serve as valves; if the magnetic actuator is stopped, pressure can be maintained with no power consumption. Flow can also be reversed by switching the direction of actuation. The maximum flow rate achieved with minimal backpressure was 45.8 /spl mu/l/min. The maximum pressure head achieved was 135 mm water (1.2 kPa).

Diffusion-based analysis of molecular interactions in microfluidic devices

The study of molecular interactions in biological fluids is important as a research tool to elucidate molecular and biological function, for discovering or designing molecules that have a desirable function, and for measuring or detecting analytes for clinical diagnostic purposes. Microfluidics is an emerging technology that has proven useful for studying and controlling molecular interactions with the potential advantages of reduced sample and reagent volumes, short reaction times, portable instrumentation, and high throughput. At microscale dimensions, diffusive transport of many biologically relevant molecules can cover large fractions of a fluid channel in a short time (seconds to minutes). We have exploited this feature to analyze molecular interactions based on changes in the diffusive transport of one of the reacting components. Here, we present a new assay configuration for studying molecular binding interactions and report new developments in the fabrication and use of hydrogels for diffusion-based analysis. We also overview system configurations and analysis techniques that have proven useful for studying molecular interactions of biological analytes and discuss their ability to operate using complex fluids such as blood.

Microfluidic approaches to immunoassays

An immunoassay format is presented that takes advantage of the microfluidic properties of the H-FilterTM for measuring sample analyte concentration. The method relies on the diffusion of analyte particles into a region containing beads coated with specific antibody. Competitive binding of labeled analyte and sample analyte with a limited number of binding sites allows measurement of the concentration of sample analyte based on the fraction of labeled analyte bound. The fraction of labeled analyte bound can be determined with a microcytometer by measuring the bead fluorescence intensity on the microcytometer portion of an integrated microfluidic chip. It is not necessary to separate the beads from the mixture because the bead intensity can be determined above the background of unbound labeled antigens. Other advantages include the ability to eliminate large interfering particles from samples, continuous sample monitoring, and the ability to concentrate the beads. Microfluidic immunoassay formats also consume smaller volumes of costly reagents and sample.

Aptamers as Affinity Reagents in an Integrated Electrophoretic Lab-on-a-Chip Platform

Nucleic acid based affinity reagents (e.g., aptamers) offer several possible advantages over antibodies as specific recognition elements in biochemical assays. Besides offering improved cost and stability, aptamers are ideal for rapid electrophoretic analysis due to their low molecular weight and high negative charge. While aptamers have proven well-suited for affinity-shift electrophoretic analysis, demonstrating a fully integrated aptamer-based assay platform represents an important achievement toward low-cost point-of-care analysis, particularly for remote or resource poor settings where cost and ambient stability of reagents is a key consideration. Here we perform and evaluate the suitability of aptamer-based affinity assays for two clinically relevant target analytes (IgE using a known aptamer and NF-κB using a thio-modified aptamer) in an integrated electrophoretic gel-shift platform. Key steps of (i) mixing sample with aptamer, (ii) buffer exchange, and (iii) preconcentration of sample were successfully integrated on-chip upstream of a fluorescence-based gel-shift analysis step. This approach, utilizing a size-exclusion membrane optimized here for aptamer retention and preconcentration with sample, enables automated sample-to-answer for trace analytes in 10 min or less. We addressed notable nonspecific interference from serum proteins by adding similar nucleic acid competitors to suppress such interactions with the aptamer. Nanomolar sensitivities were demonstrated and integrated preconcentration of sample provides an important means of further improving detection sensitivities. Aptamers proved superior in many respects to antibody reagents, particularly with regard to speed and resolution of gel-shifts associated with specific binding to target.

Diffusion Immunoassay in Polyacrylamide Hydrogels

A modified format for conducting a diffusion immunoassay is described. Polyacrylamide hydrogels containing antibody are used to conduct the assay under non-flow conditions. Fluorescein-labeled biotin and biotin-specific antibody were used as a model system. This assay format shrinks the device area required, reduces the required reagent volumes, and greatly simplifies fluid handling.

Microscale isoelectric fractionation using photopolymerized membranes.

In this work, we introduce microscale isoelectric fractionation (μIF) for isolation and enrichment of molecular species at any desired location in a microfluidic chip. Narrow pH-specific polyacrylamide membranes are photopatterned in situ for customizable device fabrication; multiple membranes of precise pH are easily incorporated throughout existing channel layouts. Samples are electrophoretically driven across the membranes such that charged species, for example, proteins and peptides, are rapidly discretized into fractions based on their isoelectric points (pI) without the use of carrier ampholytes. This format makes fractions easy to compartmentalize and access for integrated preparative or analytical operations on-chip. We present and discuss the key design considerations and trade-offs associated with proper system operation and optimal run conditions. Efficient and reproducible fractionation of model fluorescent pI markers and proteins is achieved using single membrane fractionators at pH 6.5 and 5.3 from both buffer and Escherichia coli cell lysate sample conditions. Effective fractionation is also shown using a serial 3-membrane fractionator tailored for isolating analytes-of-interest from high abundance components of serum. We further demonstrate that proteins focused in pH specific bins can be rapidly and efficiently transferred to another location in the same chip without unwanted dilution or dispersive effects. μIF provides a rapid and versatile option for integrated sample prep or multidimensional analysis, and addresses the glaring proteomic need to isolate trace analytes from high-abundance species in minute volumes of complex samples.

Microfluidic digital isoelectric fractionation for rapid multidimensional glycoprotein analysis.

Here we present an integrated microfluidic device for rapid and automated isolation and quantification of glycoprotein biomarkers directly from biological samples on a multidimensional analysis platform. In the first dimension, digital isoelectric fractionation (dIEF) uses discrete pH-specific membranes to separate proteins and their isoforms into precise bins in a highly flexible spatial arrangement on-chip. dIEF provides high sample preconcentration factors followed by immediate high-fidelity transfer of fractions for downstream analysis. We successfully fractionate isoforms of two potential glycoprotein cancer markers, fetuin and prostate-specific antigen (PSA), with 10 min run time, and results are compared qualitatively and quantitatively to conventional slab gel IEF. In the second dimension, functionalized monolithic columns are used to capture and detect targeted analytes from each fraction. We demonstrate rapid two-dimensional fractionation, immunocapture, and detection of C-reactive protein (CRP) spiked in human serum. This rapid, flexible, and automated approach is well-suited for glycoprotein biomarker research and verification studies and represents a practical avenue for glycoprotein isoform-based diagnostic testing.

Analytical Devices Based on Transverse Transport in Microchannels

The manipulation of transport transverse to the flow direction has permitted development of microfluidic devices that allow continuous processing of samples. One of these is a rapid competition immunoassay that relies on apparent changes in the diffusivity of antigen due to binding of the antigen to relatively slowly diffusing antibody. It has also been possible to utilize isoelectric focusing to concentrate and separate different types of analytes into separate flowing streams.