Brian C. Heinze

Single cell level detection of Escherichia coli in microfluidic device

Detection of Escherichia coli K-12 in phosphate buffered saline (PBS) was demonstrated in a Y-channel polydimethylsiloxane (PDMS) microfluidic device through optical fiber monitoring of latex immunoagglutination. The latex immunoagglutination assay was performed for serially diluted E. coli solutions using 0.92-microm highly carboxylated polystyrene particles conjugated with polyclonal anti-E. coli. Pre-treatments such as cell lysis or culturing to enhance the signal were not used. Proximity optical fibers around the view cell of the device were used to quantify the increase in 45 degrees forward light scattering of the immunoagglutinated particles. In order to reduce false positive signals caused by antibodies binding to non-viable E. coli cells or free antigens in solution, target solutions were washed three times, and then the results were compared to non-washing treatments. The detection limit was found to be less than 10 cfu ml(-1) (1 cfu per device) without PBS washing (thus detecting non-viable cells and free antigens), or less than 40 cfu ml(-1) (4 cfu per device) with PBS washing (thus detecting viable E. coli cells only).

Nanoparticle immunoagglutination Rayleigh scatter assay to complement microparticle immunoagglutination Mie scatter assay in a microfluidic device

In this work, particle immunoagglutination assays for pathogen detection, utilizing light scattering measurements at a fixed angle from incident light delivery, are explored in both Rayleigh and Mie scatter regimes through scatter intensity simulations and compared to experimental results. The average size of immunoagglutinated particles obtained from microscope images correspond to the particle size parameter from simulations. Mie scatter measurements yield a greater signal increase with increasing pathogen concentration than Rayleigh scatter measurements, but with a non-monotonic relationship that is not observed in the Rayleigh scatter regime. These two similar yet distinctly different sources of information could easily be integrated into a single device through fabrication of a simple microfluidic device containing two y-channels, each for performing the respective light scattering measurement. Escherichia coli was used as a representative target, and detected in a microfluidic device down to a concentration of 1 colony forming units (CFU) per mL.

Optofluidic device monitoring and fluid dynamics simulation for the spread of viral pathogens in a livestock environment.

Rapid monitoring of the spreads of porcine reproductive and respiratory syndrome virus (PRRSV) was attempted using samples collected from nasal swabs of pigs and air samplers within an experimental swine building. An optofluidic device containing liquid-core waveguides was used to detect forward Mie light scattering caused by the agglutination of anti-PRRSV-conjugated submicron particles, with enhanced sensitivity, signal reproducibility, and reusability (reusable up to 75 assays). These results were compared with reverse transcription polymerase chain reaction (RT-PCR) assays (35 cycles) and showed excellent agreements to them. Each assay took less than 10 min including all necessary sample pre-processing, while the RT-PCR assays took up to 4 h including sample pre-processing and gel imaging for PCR products. A 3-D computational fluid dynamics (CFD) simulation was utilized to track the transport and distribution of PRRSV (from the mouths of pigs to the exhaust fans) within a swine building, and compared with the readings from an optofluidic device. Simulation results corresponded well with the experimental data, validating our 3-D CFD model for the spread of viral pathogens in a livestock environment. The developed optofluidic device and 3-D CFD model can serve as a good model for monitoring the spread of influenza A (swine and avian) within animal and human environments.

Microfluidic device detection of waterborne pathogens through static light scattering of latex immunoagglutination using proximity optical fibers

Microfluidic device detections of E. coli K12 in deionized (DI) water and E. coli in field water sample were demonstrated through static light scattering of latex immunoagglutination using proximity optical fibers. This method is a fully-automated, one-step detection, and requires neither sample pre-treatment nor cell culturing often required in many on-chip detections. We have used highly carboxylated polystyrene submicron latex particles without surfactants to enhance diffusional mixing and prevent non-specific bindings towards successful demonstration of latex immunoagglutination in microfluidic device. Detection of E. coli was performed by taking microscopic images from the view cell of a microfluidic device and counting the fractions of non-agglutinated and agglutinated particles. The limit of detection (LOD) was ca. 150 CFU ml-1 with this method for both E. coli K12 in DI water and E. coli in field water sample, indicating no non-specific bindings. Improved LOD of < 4.3 CFU ml-1 was achieved by measuring forward static light scattering from microfluidic device, using proximity optical fibers and a USB-powered miniature spectrometer. The total assay time for sample preparation (mostly dilutions) and on-chip assay (mostly injections and short incubation time) was < 10 min.

Magnetic particle enhanced microcantilever biosensor for rapid and Sensitive E. Coli detection

This paper presents a novel concept for integrating a magnetic microparticle sandwich assay onto a microcantilever beam, towards rapid and sensitive detection of E. Coli. The gold coated surface of the microcantilever beam and the surface of superparamagnetic microparticles are functionalized with anti-E. coli IgG so that they bind only in presence of and via the E. coli. An external magnetic field produced by an electromagnet is used to enhance the microcantilever deflection, and an optical lever method is used to sensitively detect it.

Real-Time Monitoring of Fibrinogen Cross-Linking on Model Biomaterial Surfaces with Quartz Crystal Microbalance

A quartz crystal microbalance was used for real-time monitoring of fibrinogen cross-linking on three model biomaterial surfaces. Fibrinogen adsorbs slowly and forms a less rigid multi-layer on hydrophobic surfaces, while it ad- sorbs quickly, forming a single mono-layer on hydrophilic surfaces. The extent of fibrinogen cross-linking is greater on hydrophobic surfaces. Fibrinogen cross-linking can also rigidify the relatively soft coatings of poly(methyl methacrylate) and dodecanethiol self-assembled monolayer.

Latex Immunoagglutination Assay for Bovine Viral Diarrhea Virus Utilizing Forward Light Scattering in a Microfluidic Device

We have investigated the utilization of particle agglutination assays using forward light scattering measurements in a microfluidic device towards detecting viral particles. The model viral target was bovine viral diarrhea virus (BVDV). Highly carboxylated polystyrene microspheres (510 nm) were coated with anti-BVDV monoclonal antibodies. This solution was in turn used to detect live modified BVDV. This assay was first performed in a two well slide for proof of concept and then in a simple y-channel microfluidic device with optical fibers arranged in a close proximity setup. Particle immunoagglutination was detected through static light scattering measurements taken at 45° to incident light. In the microfluidic device, modified live BVDV was detected with a detection limit of 0.5 TCID50 mL-1.

Detection of avian influenza antigens in proximity fiber, droplet and optical waveguide microfluidics

Virus antigens of avian influenza subtype H3N2 were detected on two different microfluidic platforms: microchannel and droplet. Latex immunoagglutination assays were performed using 920-nm highly carboxylated polystyrene beads that are conjugated with antibody to avian influenza virus. The bead suspension was merged with the solutions of avian influenza virus antigens in a Y-junction of a microchannel made by polydimethylsiloxane soft lithography. The resulting latex immunoagglutinations were measured with two optical fibers in proximity setup to detect 45° forward light scattering. Alternatively, 10 μL droplets of a bead suspension and an antigen solution were merged on a superhydrophobic surface (water contact angle = 155°), whose movement was guided by a metal wire, and 180° back light scattering is measured with a backscattering optical probe. Detection limits were 0.1 pg mL-1 for both microchannel with proximity fibers and droplet microfluidics, thanks to the use of micro-positioning stages to help generate reproducible optical signals. Additionally, optical waveguide was tested by constructing optical waveguide channels (filled with mineral oil) within a microfluidic device to detect the same light scattering. Detection limit was 0.1 ng mL-1 for an optical waveguide device, with a strong potential of improvement in the near future. The use of optical waveguide enabled smaller device setup, easier operation, smaller standard deviations and broader linear range of assay than proximity fiber microchannel and droplet microfluidics. Total assay time was less than 10 min.

Microfluidic Device Monitoring of Waterborne Pathogens in Model Water Distribution Systems

Microfluidic device detections of E. coli K12 in deionized (DI) water and E. coli in field water sample were demonstrated through static light scattering of latex immunoagglutination using proximity optical fibers. This method is a fully-automated, one-step detection, and requires neither sample pre-treatment nor cell culturing often required in many on-chip detections. We have used highly carboxylated polystyrene submicron latex particles without surfactants to enhance diffusional mixing and prevent non-specific bindings towards successful demonstration of latex immunoagglutination in microfluidic device. Detection of E. coli was performed by taking microscopic images from the view cell of a microfluidic device and counting the fractions of non-agglutinated and agglutinated particles. The limit of detection (LOD) was ca. 150 CFU ml-1 with this method for both E. coli K12 in DI water and E. coli in field water sample, indicating no non-specific bindings. Improved LOD of ca. 5 CFU ml-1 was achieved by measuring forward static light scattering from microfluidic device, using proximity optical fibers and a USB-powered miniature spectrometer. The total assay time for sample preparation (mostly dilutions) and on-chip assay (mostly injections and short incubation time) was < 10 min. Devices made out of polydimethylsiloxane (PDMS) were reusable up to 4 times, while those made out of polyethylene glycol (PEG) showed improved reusability of up to 20 times.