Integrated Microfluidic Bioprocessors for Infectious Disease Detection

The emergence of micro-Total Analysis Systems has revolutionized the world of molecular diagnostics by enabling sensitive multi-step fluidic processes to be performed and reliably integrated in a single platform. In particular, microfluidic systems now provide the tools and components to enable quantitative detection of biomarkers relevant to pathogen identification and disease characterization. In this thesis, these advances are exploited to develop an integrated microfluidic platform for automated, rapid and sensitive genetic identification of infectious food-borne bacterial and respiratory viral pathogens.My first goal was the integration of improved sample purification, preconcentration and injection technology with a polymerase chain reaction-capillary electrophoresis (PCR-CE) microdevice. By introducing an in-line affinity capture system utilizing an in situ photopolymerized oligonucleotide capture gel, double-stranded PCR amplicons generated in an integrated PCR reactor were selectively captured, purified and injected with 100% efficiency for high resolution CE separation. The superior performance of this integrated platform was demonstrated in a quantitative genetic analysis of E. coli. This integrated system exhibits a six- fold improvement in resolution of a multiplex analysis of Escherichia coli O157/E. coli K12 and is able to detect E. coli O157 in a 500-fold higher background of E. coli K12.To enable the parallel detection of multiple infectious pathogens, an improved purification method relying on biotin-streptavidin interaction was developed for universal product capture. This technique has the advantage of eliminating the complications associated with designing sequence-specific oligonucleotide capture probes for multiple targets. This process was integrated into a new 4-unit array PCR-CE microchip designed for automated product amplification, capture, and analysis. Coupled with a portable laser-induced fluorescence rotary scanner, the system can simultaneously detect as few as ten copies per reactor of influenza A & B, human metapneumovirus (hMPV), and coronavirus samples from cloned plasmid standards within 2.5 hours. Furthermore, the ability of the system to process RNA samples was demonstrated by performing RT-PCR analyses of an influenza B/hMPV co-infection model case, with respective detection limits of 50 and 100 copies/reactor.This thesis concludes with a discussion of proposed methods for nucleic acid isolation from biological samples that will provide a complete sample-in to answer-out diagnostic device and method for pathogen detection. When fully developed, this technology will be a significant advancement in infectious disease detection and surveillance both inside and outside clinical settings

Implementation of microfluidic sandwich ELISA for superior detection of plant pathogens.

Rapid and economical screening of plant pathogens is a high-priority need in the seed industry. Crop quality control and disease surveillance demand early and accurate detection in addition to robustness, scalability, and cost efficiency typically required for selective breeding and certification programs. Compared to conventional bench-top detection techniques routinely employed, a microfluidic-based approach offers unique benefits to address these needs simultaneously. To our knowledge, this work reports the first attempt to perform microfluidic sandwich ELISA for Acidovorax citrulli (Ac), watermelon silver mottle virus (WSMoV), and melon yellow spot virus (MYSV) screening. The immunoassay occurs on the surface of a reaction chamber represented by a microfluidic channel. The capillary force within the microchannel draws a reagent into the reaction chamber as well as facilitates assay incubation. Because the underlying pad automatically absorbs excess fluid, the only operation required is sequential loading of buffers/reagents. Buffer selection, antibody concentrations, and sample loading scheme were optimized for each pathogen. Assay optimization reveals that the 20-folds lower sample volume demanded by the microchannel structure outweighs the 2- to 4-folds higher antibody concentrations required, resulting in overall 5-10 folds of reagent savings. In addition to cutting the assay time by more than 50%, the new platform offers 65% cost savings from less reagent consumption and labor cost. Our study also shows 12.5-, 2-, and 4-fold improvement in assay sensitivity for Ac, WSMoV, and MYSV, respectively. Practical feasibility is demonstrated using 19 real plant samples. Given a standard 96-well plate format, the developed assay is compatible with commercial fluorescent plate readers and readily amendable to robotic liquid handling systems for completely hand-free assay automation

Specificity determination.

<p>Specificity of the 11E5/MPC, 2D6/MYSV6, and 5E7/MYSV6 antibody pairs was tested for Ac, WSMoV, and MYSV, respectively. The antibody pairs were tested against bacteria (Ac, SQB, Pf, and DAc) and viral (TYLCV) protein standards (n = 3). The data shows averaged S/N values with error bars representing standard deviations. The dotted horizontal line indicates the threshold (or the cutoff value) of S/N = 2 (twice of values obtained from negative controls).</p

Study of repetitive sample loading.

<p>Effect of repetitive sample loading on assay dynamic range was investigated, using Ac as a model. The plot indicates that multiple loading helps increase assay sensitivity (n = 3). Error bars indicate ± standard deviations.</p

Schematic workflow depicting sequential molecular binding events of the sandwich ELISA.

<p>Within each reaction chamber, the capture antibody is adsorbed on the reactive surface followed by surface passivation by a blocking buffer. Upon target binding to the capture antibody, alkaline phosphates (AP)-tagged detection antibody specific to the antigen is added. Addition of fluorescent substrate (PNPP or <i>p</i>-nitrophenyl phosphate for the traditional well format, and Attophos for the micofluidic format) activated by AP generates detectable fluorescent signal, indicating successful binding events.</p

Coating and blocking buffer selection.

<p>A. Different coating buffers (Optibind A-L) were tested for maximum surface binding of 11E5, 2D6, and 5E7 (n = 3). B. Blocking buffers (2% BSA, 3% skim milk, 1% casein, and Optiblock solution) were tested for Ac, WSMoV, and MYSV detection (n = 3). An ideal blocking buffer resulted in highest S/N ratios. Error bars indicate ± standard deviations.</p

Sensitivity determination of the microfluidic platform.

<p>Comparison of assay dynamic range for Ac (A), WSMoV (B), and MYSV (C) detection between protein standards and spiked plant extracts (n = 3) by the microfluidic platform. Error bars indicate ± standard deviations.</p

Optimization of antibody concentration.

<p>Nine different conditions for each disease panel were tested on the microfluidic platform using combinations of three concentrations of capture Ab (11E5, 2D6, and 5E7) and three concentrations of detection Ab (MPC-AP, MYSV6-AP). Panel A–C show results for Ac, WSMoV, and MYSV detection, respectively (n = 4). The S/N ratios were plotted for each of the conditions tested. Error bars indicate ± standard deviations.</p