Lab-on-a-chip optical immunosensor for pathogen detection.

摘要

This dissertation develops technology for microfluidic point-of-care (POC) immunoassay devices, divided into three papers, and explores the use of a quartz crystal microbalance for real time monitoring of blood coagulation in a fourth paper. The concept of POC testing has been well established around the world. With testing conveniently brought to the vicinity of the patient or testing site, results can be obtained in a much shorter time. There has been a global push in recent years to develop POC molecular diagnostics devices for resource-limited regions where well equipped centralized laboratories are not readily accessible. POC testing has applications in medical/veterinary diagnostics, environmental monitoring, as well as defense related testing.;Immunoassay techniques take advantage of the high specificity and sensitivity exhibited by antibody-antigen interactions, enabling for detection and/or quantification of target analytes in complex chemical matrices. Fields of application of immunoassays vary from clinical diagnostics to environmental analysis, to food safety assessment, and the target analytes cover the widest range of molecular weight, from small organic molecules to biomolecules of hundreds of kilodaltons.;In the first paper, we demonstrated the use of latex immunoagglutination assays within a microfluidic chip to be an effective and sensitive method for detecting the bovine viral diarrhea virus. This assay has been shown to be more sensitive than conventional BVDV detection protocols such as RT-PCR, detecting the viral particles down to a concentration of 10 TCID50 mL-1; while RT-PCR performed by this lab on the same viral sample showed a detection limit of 103 TCID50 mL -1. Literature sources site detecting the virus down to 10 1.5 TCID50 mL-1 (not in a microfluidic chip format) under carefully optimized protocols, instruments, and conditions. The total assay time for virus detection on this platform is less than 5 min, and the chip has the potential to become portable, demonstrating the possibility for development of a truly rapid point of care detection device. While BVDV was used as the model pathogenic target in this experiment, it should be noted that the target of the assay can easily be changed by adsorbing the appropriate antibodies to the microparticles, such that this platform could theoretically be used to detect many different pathogens in parallel.;In the second paper the feasibility and general ease of integrating liquid core optical components onto a microfluidic lab-on-a-chip type device, for point-of-care AI diagnosis is demonstrated. Integration of liquid core optical waveguides provides a method for further miniaturization of the device towards a truly POC sensor. The extremely low device detection limit of 1 pg /mL for AI antigens in both clean buffer and real biological matrix, coupled with the potential portability, make this device a good candidate for a point-of-care diagnostic tool for early AI detection.;In the third paper particle agglutination assays, utilizing light scattering measurements at a fixed angle from incident light delivery, for pathogen detection are explored in both Rayleigh and Mie scatter regimes through scatter intensity simulations and compared to experimental results. This work demonstrates the feasibility of utilizing light scatter measurements of particle immunoassays in both Rayleigh and Mie regimes as complementary information towards a robust platform for pathogen detection that is both highly sensitive and quantitative. 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.;In the fourth paper a quartz crystal microbalance was used for real-time monitoring of fibrinogen cross-linking on three model biomaterial surfaces. We found that fibrinogen adsorbs slowly and forms a less rigid multi-layer on hydrophobic surfaces, while it adsorbs 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. This type of analysis could prove useful for testing the blood compatibility of biomaterial candidates.