Daniel J. Throckmorton

Microfluidic immunoassays as rapid saliva-based clinical diagnostics

At present, point-of-care (POC) diagnostics typically provide a binary indication of health status (e.g., home pregnancy test strip). Before anticipatory use of diagnostics for assessment of complex diseases becomes widespread, development of sophisticated bioassays capable of quantitatively measuring disease biomarkers is necessary. Successful translation of new bioassays into clinical settings demands the ability to monitor both the onset and progression of disease. Here we report on a clinical POC diagnostic that enables rapid quantitation of an oral disease biomarker in human saliva by using a monolithic disposable cartridge designed to operate in a compact analytical instrument. Our microfluidic method facilitates hands-free saliva analysis by integrating sample pretreatment (filtering, enrichment, mixing) with electrophoretic immunoassays to quickly measure analyte concentrations in minimally pretreated saliva samples. Using 20 μl of saliva, we demonstrate rapid (<10 min) measurement of the collagen-cleaving enzyme matrix metalloproteinase-8 (MMP-8) in saliva from healthy and periodontally diseased subjects. In addition to physiologically measurable indicators of periodontal disease, conventional measurements of salivary MMP-8 were used to validate the microfluidic assays described in this proof-of-principle study. The microchip-based POC diagnostic demonstrated is applicable to rapid, reliable measurement of proteinaceous disease biomarkers in biological fluids.

Reversed-phase electrochromatography of amino acids and peptides using porous polymer monoliths

Efficient and rapid separation of minute levels of amino acids and bioactive peptides is of significant importance in the emerging field of proteomics as well as in the clinical and pharmaceutical arena. We have developed novel UV-initiated acrylate-based porous polymer monoliths as stationary phases for capillary- and chip-electrochromatography of cationic, anionic, and neutral amino acids and peptides, followed by absorbance or laser-induced fluorescence detection. The rigid monoliths are cast-to-shape and are tunable for charge and hydrophobicity. For separations at low pH, monoliths containing quaternary amine moieties were used to achieve high electroosmotic flow, and for high pH separations monoliths with acidic sulfonic acid groups were employed. Efficient and reproducible separations of phenylthiohydantoin-labeled amino acids, native peptides, and amino acids and peptides labeled with naphthalene-2,3-dicarboxaldehyde (NDA) were achieved using both negatively- and positively-charged polymer monoliths in capillaries. Separation efficiencies in the range of 65,000-371,000 plates/m were obtained with capillary electrochromatography. Buffer composition and the degree of column hydrophobicity were studied systematically to optimize separations. The monoliths were also cast in the microchannels of glass chips and electrochromatographic separation followed by laser-induced fluorescence detection of three NDA-labeled bioactive peptides was obtained.

Integrated microfluidic platform for oral diagnostics.

Abstract:  While many point‐of‐care (POC) diagnostic methods have been developed for blood‐borne analytes, development of saliva‐based POC diagnostics is in its infancy. We have developed a portable microfluidic device for detection of potential biomarkers of periodontal disease in saliva. The device performs rapid microfluidic chip‐based immunoassays (<3–10 min) with low sample volume requirements (10 μL) and appreciable sensitivity (nM–pM). Our microfluidic method facilitates hands‐free saliva analysis by integrating sample pretreatment (filtering, enrichment, mixing) with electrophoretic immunoassays to quickly measure analyte concentrations in minimally pretreated saliva samples. The microfluidic chip has been integrated with miniaturized electronics, optical elements, such as diode lasers, fluid‐handling components, and data acquisition software to develop a portable, self‐contained device. The device and methods are being tested by detecting potential biomarkers in saliva samples from patients diagnosed with periodontal disease. Our microchip‐based analysis can readily be extended to detection of biomarkers of other diseases, both oral and systemic, in saliva and other oral fluids.

Rapid Microchip-Based Electrophoretic Immunoassays For The Detection Of Swine Influenza Virus.

Towards developing rapid and portable diagnostics for detecting zoonotic diseases, we have developed microchip-based electrophoretic immunoassays for sensitive and rapid detection of viruses. Two types of microchip-based electrophoretic immunoassays were developed. The initial assay used open channel electrophoresis and laser-induced fluorescence detection with a labeled antibody to detect influenza virus. However, this assay did not have adequate sensitivity to detect viruses at relevant concentrations for diagnostic applications. Hence, a novel assay was developed that allows simultaneous concentration and detection of viruses using a microfluidic chip with an integrated nanoporous membrane. The size-exclusion properties of the in situ polymerized polyacrylamide membrane are exploited to simultaneously concentrate viral particles and separate the virus/fluorescent antibody complex from the unbound antibody. The assay is performed in two simple steps--addition of fluorescently labeled antibodies to the sample, followed by concentration of antibody-virus complexes on a porous membrane. Excess antibodies are removed by electrophoresis through the membrane and the complex is then detected downstream of the membrane. This new assay detected inactivated swine influenza virus at a concentration four times lower than that of the open-channel electrophoresis assay. The total assay time, including device regeneration, is six minutes and requires <50 microl of sample. The filtration effect of the polymer membrane eliminates the need for washing, commonly required with surface-based immunoassays, increasing the speed of the assay. This assay is intended to form the core of a portable device for the diagnosis of high-consequence animal pathogens such as foot-and-mouth disease. The electrophoretic immunoassay format is rapid and simple while providing the necessary sensitivity for diagnosis of the illness state. This would allow the development of a portable, cost-effective, on-site diagnostic system for rapid screening of large populations of livestock, including sheep, pigs, cattle, and potentially birds.

A Novel Miniaturized Protein Preconcentrator Based on Electric Field-Addressable Retention and Release

We report a novel technique for concentration of proteins from dilute solutions using electrokinetic trapping [1] where charged macromolecules are reversibly trapped in a microchannel packed with porous silica particles under an applied electric field. Electrokinetic trapping is electric field-addressable and reversible; the trapped proteins can be recovered quantitatively by removing the electric field. The concentration of proteins is carried out in two steps -1) electrokinetic injection of proteins in channels packed with porous silica particles, and 2) elution by applying pressure (using a mechanical pump) in the absence of applied voltage (Figure 1). A model protein, ovalbumin, could be concentrated by over two orders of magnitude using electrokinetic trapping. Electrokinetic trapping provides a simple, on-column method of preconcentration of proteins and other charged macromolecules that can be easily integrated with a miniaturized separation device. Furthermore, it does not require change of solvents (or buffers) or change of flow direction for elution of concentrated protein and hence, has advantages over filtration-based, electrolyte bridge-based [2] or adsorption-based [3] miniaturized concentrators. We demonstrate that the electrokinetic trapping of proteins is quantitative, reproducible and works for several proteins. Electrokinetic trapping can also be used for applications including selective modification of trapped proteins, using trapped proteins as immobilized catalysts, and concentration of DNA.

Rapid Separation of Peptides and Amino Acids in Glass Microchips by Reversed-Phase Electrochromatography

We have developed microfabricated glass chips for reversed-phase separation followed by laser-induced fluorescence detection of peptides and amino acids. Hydrophobic acrylate-based porous polymer monoliths were cast in the channels by photopolymerization. Use of UV-light for initiation of polymerization allows for selective patterning of stationary phase in the channels for optimal design of injection and detection regions. Charged functionalities such as either sulfonic acids or quaternary amines were incorporated during polymerization for generation of electroosmotic flow. These monoliths can be cast in situ in less than 10 minutes, are very reproducible with respect to separation characteristics, and allow easy manipulation of separation parameters such as charge, hydrophobicity, and pore size. Moreover, the solvent used to cast the polymer is chosen so as to allow electroosmotic flow to flush the channels thereby obviating any need for application of high pressures for conditioning. Rapid (50 seconds) and efficient (up to 600,000 plates/m) separations of peptides and amino acids were achieved in the microchip.

Bubble masks for time-encoded imaging of fast neutrons

Time-encoded imaging is an approach to directional radiation detection that is being developed at SNL with a focus on fast neutron directional detection. In this technique, a time modulation of a detected neutron signal is induced-typically, a moving mask that attenuates neutrons with a time structure that depends on the source position. An important challenge in time-encoded imaging is to develop high-resolution two-dimensional imaging capabilities; building a mechanically moving high-resolution mask presents challenges both theoretical and technical. We have investigated an alternative to mechanical masks that replaces the solid mask with a liquid such as mineral oil. Instead of fixed blocks of solid material that move in predefined patterns, the oil is contained in tubing structures, and carefully introduced air gaps-bubbles-propagate through the tubing, generating moving patterns of oil mask elements and air apertures. Compared to current moving-mask techniques, the bubble mask is simple, since mechanical motion is replaced by gravity-driven bubble propagation; it is flexible, since arbitrary bubble patterns can be generated by a software-controlled valve actuator; and it is potentially high performance, since the tubing and bubble size can be tuned for high-resolution imaging requirements. We have built and tested various single-tube mask elements, and will present results on bubble introduction and propagation for different tube sizes and cross-sectional shapes; real-time bubble position tracking; neutron source imaging tests; and reconstruction techniques demonstrated on simple test data as well as a simulated full detector system.

Studies of Phosphorylation During Innate Immune Signaling using On-Chip Cell Preparation and Downstream Flow Cytometry

Fine temporal resolution is required for monitoring protein phosphorylation events key to cellular signaling pathways. With the goal of teasing apart the kinetics of phosphorylation cascades central to the human innate immune response to pathogen invasion, our group has developed a microfluidic device that integrates flow cytometry with required upstream cell preparation steps. Streamlined cell preparation and analysis allows monitoring of events with kinetic resolution on the order of minutes - not tens of minutes to hours. The planar microfluidic device contains 50 mm long spiral mixers, porous polymer monoliths for selective exchange of reagents, and incubation chambers (~1000 cells per chamber) where macrophage cells (RAW264.7) are challenged with a chemical signal of Gram-negative bacteria (lipopolysaccharide) and subsequently labeled with fluorescent immunoreagents. Finally, on the same device, the labeled macrophage cells are analyzed using two-color flow cytometry. Such an integrated self-contained microfluidic platform promises to be of widespread use to host- pathogen studies in infectious disease laboratories.

Electrophoretic immunoassays for oral diagnostics

Analysis of oral fluid (e.g., saliva) has the potential to aid diagnosis of both oral and systemic diseases. Oral fluids offer advantages over other bodily fluids, including quick, non-invasive, and inexpensive collection in both clinical and non-clinical settings. Nevertheless, current assay techniques using saliva are time-consuming, require large sample volumes, and are not amenable to automation or portability. We report on a robust microfluidic platform that performs rapid immunoassay-based analysis (<3 minutes) of saliva with low sample volume requirements (10 /spl mu/L) and appreciable sensitivity (10/sup -12/ M) with a high-degree of autonomy. Immunoassays for the detection of the systemic disease biomarker, C-reactive protein, and the cytokines interleukin-6 and tumor necrosis factor-/spl alpha/ were conducted in spiked human saliva using a microfluidic chip. The immunoassay developed is based on binding of fluorescently-labeled reporter antibodies to analytes followed by electrophoretic separation and quantitation of bound and unbound reporter. The separation selectivity was achieved using native polyacrylamide gel electrophoresis in a microchip containing in situ photolithographically patterned cross-linked polyacrylamide. Native electrophoresis conditions maintained the integrity of fragile immune complexes and provided rapid separations of complex samples. On-going work focuses on integration of the on-chip immunoassay with miniaturized hardware towards developing a portable prototype.