A plastic total internal reflection photoluminescence device for enzymatic biosensing.

A total internal reflection photoluminescence (TIRPh) device employing an easily fabricated PMMA/PDMS waveguide system provides a detection limit comparable to the best reported results but without using an excitation filter. The optical mechanism is similar to total-internal-reflection-fluorescence (TIRF) but uses a ruthenium-based phosphorescent dye (Ru(dpp)3) deposited on the PMMA core, motivating the generalized term of photoluminescence to include both fluorescence and phosphorescence. An enzymatic hydrogen peroxide (H2O2) biosensor incorporating catalase was fabricated on the TIRPh platform without photolithography or etching. The O2-sensitive phosphorescence of Ru(dpp)3 was used as a transduction mechanism and catalase was used as a biocomponent for sensing. The H2O2 sensor exhibits a phosphorescence to scattered excitation light ratio of 76 ± 10 without filtering. The unfiltered device demonstrates a detection limit of (2.2 ± 0.6) µM with a linear range of 0.1 mM to 20 mM. The device is the first total internal reflection photoluminescence based enzymatic biosensor platform, and is promising for cost-effective, low excitation interference, field-portable sensing.

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 immunosensor with integrated liquid core waveguides for sensitive Mie scattering detection of avian influenza antigens in a real biological matrix.

This work presents the use of integrated, liquid core, optical waveguides for measuring immunoagglutination-induced light scattering in a microfluidic device, towards rapid and sensitive detection of avian influenza (AI) viral antigens in a real biological matrix (chicken feces). Mie scattering simulations were performed and tested to optimize the scattering efficiency of the device through proper scatter angle waveguide geometry. The detection limit is demonstrated to be 1 pg mL(-1) in both clean buffer and real biological matrix. This low detection limit is made possible through on-chip diffusional mixing of AI target antigens and high acid content microparticle assay reagents, coupled with real-time monitoring of immunoagglutination-induced forward Mie scattering via high refractive index liquid core optical waveguides in close proximity (100 µm) to the sample chamber. The detection time for the assay is <2 min. This device could easily be modified to detect trace levels of any biological molecules that antibodies are available for, moving towards a robust platform for point-of-care disease diagnostics.

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).