Versatile, automated sample preparation and detection of contaminants and biological materials

Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, 2013.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Cataloged from student-submitted PDF version of thesis.Includes bibliographical references (pages xviii-xxxvi).Contamination of food, water, medicine and ingestible household products is a public health hazard that episodically causes outbreaks worldwide. Existing laboratory methods are often expensive, require a laboratory environment and/or trained staff to perform manual steps. The aim of this PhD thesis was to create and test methods and instruments for affordable diagnostic tests for contaminants and pathogens. To achieve this goal, the LabReader was introduced, which employs a LED-based detection scheme for four simultaneous fluorescence- and UV-measurements. Assays were developed to detect (di-)ethylene glycol in consumables ≥0.1wt% and alcohols ≥1ppb. Pathogens in water, foods and blood were detected at ≥104 CFU/ml using nonspecific intercalating dyes. To gain sensitivity and specificity for cell-based analysis, biochemical amplification methods had to be incorporated. To be deployable outside a laboratory, sample preparation needed to be automated. Automation was achieved by combining the LabReader with the already developed LabTube, a disposable platform for automated DNA extraction inside a standard centrifuge. Performing DNA amplification/readout in an external optical reader, made the LabSystem broadly deployable and flexible. DNA extraction of food bacteria (E.coli and Alicyclobacillus) was optimized inside the LabTube for 102-109 inserted DNA copies. The extracted DNA was amplified using the qualitative isothermal LAMP method and semi-quantitative, real-time PCR inside the LabReader. The combined extraction and amplification detection limit of the LAMP-LabSystem and the quantitation limit of the PCR-LabSystem were as low as 102 copies. Performing extraction and amplification inside the centrifuge/LabTube was also outlined, which may be preferable when contamination risks are high. After theoretically evaluating heating methods, a battery-driven heated LabTube was designed, in which 102-108 DNA copies of VTEC E.coli were extracted, LAMP-amplified and visually readout within 1.5 hrs. The major contribution of this thesis is the full system integration of versatile, automated sample preparation and detection systems. They offer great flexibility as they may be used with each other or in combination with other analytic methods, depending on the application. At the same time, they are frugal and deployable at low-to-medium throughput - even outside a traditional laboratory. Whilst the focus was put on food safety, the systems were also used for medical, environmental or consumer product quality applications, hence demonstrating their broad applicability.by Melanie Margarete Hoehl.Ph. D

MICROFLUIDIC APPROACHES FOR QUANTITATIVE ANALYSIS AND SCREENING OF SYNTHETICALLY ENGINEERED MICROBES

Department of Biomedical EngineeringRecently, our understanding of complex genomes, proteomes, bio-molecules, and even many metabolic pathways has been developed significantly as growing a fundamental knowledge of the biochemistry of life. These newly revealed findings have incredibly influenced in recent bio-technologies, such as synthetic biology having massive potential to solve the missing connected dots. To have better understanding of microbe, microfluidic approaches were innovatively introduced to the field with the potential to revolutionize high-throughput biological assays. In this study, suggested approach to address limitations of conventional microbiology is microfluidics integrated with synthetic biology. At first, the microbial biosensors will be introduced into microfluidic ratchet platform for a quantitative analysis of microbial bio-signal. The microfluidic device using microfabricated arrowhead-shaped ratchet structures has an intrinsic function that concentrates motile microbes in a microchamber array. Additionally, the ratchet structure provides the concentrated microbes to grow better in a continuous-feed mode. A continuous exposure of detection analytes leads the amplification of fluorescence signal from microbes in a microchamber. Therefore, it was noted that the substantial amplification of bio-signal was achieved from the microfluidic device and measured signals were analyzed in quantitative manner. As a second practical application of the microfluidic approach, for high-throughput screening (HTS) application, a fluid array will be developed by using immiscible character between water and oil for microbial incubation, analysis, selective extraction, recovery process and the demonstration of practical applications. From the characterization of the fluid array platform, HTS will be demonstrated based on two different categories: reporter-gene basis and growth complementation basis. The fluid array device showed not only demonstrations of high-throughput screening, but also advanced screening applications were also demonstrated with higher mutant library screening with 106 and C2C communication screening system. Outstanding mutants were sophisticatedly screened among 106 of a mutant library based on the hybrid type screening method. Also, the proposed C2C screening approach has enabled high-throughput compartmentalization and resulted in 10 possible mutants showing higher extracellular biomolecule secretion performance. The proposed microfluidic approaches can be practically useful combinations showing many advantages: 1) economical and reduced time requirement for real application without complex instruments, 2) facile potentials to enable a multiplex quantitative analysis in a high-throughput manner, and 3) selective, direct and convenient measurement without pre- or post-treatment of sample solutions in near future the entire processes could be fully automated. In this dissertation, different type of microfluidic devices was developed for various collaborative purposes for the bottleneck of conventional microbiology. Therefore, the microfluidic devices have knocked a new door for high-throughput screening application for synthetically engineered microorganisms and quantitative approaches for microbial biosensors. Thus, the research contributions in this doctoral dissertation are the microfluidic approaches to popularize and overcome conventional constraints from biological experimental tools by integration of total analysis system for synthetically engineered microbe cases.ope

Examination of Pseudomonas fluorescence as a Recombinant Expression Host: Cloning, Expression, and Chromatography

In an effort to expand the pool of bacterium useful for biotechnology applications, Pseudomonas fluorescens, a common gram negative microbe, was examined for its ability to function in a recombinant setting. P. fluorescens is ubiquitous in nature and was initially identified as a soil bacterium found in dirt and is typically associated with plant material. Past literature indicates that it shared characteristics common to Escherichia coli and Bacillus subtilis, including simple growth conditions and potential cloning vectors, providing motivation to look into both the upstream and downstream characteristics of this bacterium. First, it was demonstrated that P. fluorescens could be grown to acceptable cell densities in simple batch with cell weights on the order of 60 g/L in the absence of optimization. Lysates of cells were subjected to DEAE ion exchange chromatography to identify the subproteome of soluble proteins which are retained by this resin to guide cellular modifications that reduce the amount and number of host cell proteins (HCPs) encountered during bioseparation. Finally, cloning experiments with Green Fluorescent Protein and FC fragment of tetanus demonstrated both moderate- and large- recombinant DNA products may be obtained from this host

Experimental investigation of the combined effect of light and temperature on microalgae growth in milli-photobioreactors

openMicroalgae represent one of the most potential sustainable alternatives for producing energy and environmentally benign products. However, costs of production are still limitative for many applications and photobioreactors play important roles in these processes. The aim of this thesis is to develop a suitable model for large-scale cultivation for finding the best condition of light and temperature to optimize the productivity in a microalgal artificially illuminate system. For this purpose, several experiments with the microalga Acutodesmus obliquus 276-7 were conducted in a small-scale photobioreactor of 45 ml.Microalgae represent one of the most potential sustainable alternatives for producing energy and environmentally benign products. However, costs of production are still limitative for many applications and photobioreactors play important roles in these processes. The aim of this thesis is to develop a suitable model for large-scale cultivation for finding the best condition of light and temperature to optimize the productivity in a microalgal artificially illuminate system. For this purpose, several experiments with the microalga Acutodesmus obliquus 276-7 were conducted in a small-scale photobioreactor of 45 ml

Microreactor synthesis of labelled polyphenols : a route to antibacterial modes of action in important hospital pathogens

The aims of the PhD were to synthesize deuterium labelled epicatechin in a microreactor, discover if theaflavin and epicatechin possess antibacterial action against important hospital pathogens and to determine if synergy exists between polyphenols and between polyphenol and current antibiotics. Other aims were to determine the minimum inhibitory concentrations of theaflavin and a theaflavin:epicatechin combination against the bacteria Stenotrophomonas maltophilia, to synthesize theaflavin and deuterium labelled theaflavin in a microreactor and to link the continuous flow process to antimicrobial testing for future mode of action analysis.A 2-step microreactor synthesis using in a T-shaped microreactor successfully produced deuterium labelled epicatechin in position 8. Mass spectrometry and H-NMR confirmed deuterium labelled epicatechin had been produced. The 2- step synthesis produced a yield in excess of 90%. A 4-step micro reactor synthesis of deuterium labelled epicatechin, was found to be unsuccessful after step 2 of the synthesis. A 1-step method for the microreactor production of planar epicatechin was also shown to be unsuccessful in a microreactor.Antimicrobial testing of theaflavin, epicatechin and a 2:1 combination of theaflavin and epicatechin was performed against 4 clinical isolates of MRSA, 6 clinical isolates of Acinetobacter baumannii and 6 clinical isolates of Stenotrophomonas maltophilia. Results from the disc diffusion assay confirmed that epicatechin produced no antibacterial action and theaflavin produced strong antibacterial action. The combination of theaflavin and epicatechin produced higher antibacterial activity than theaflavin alone indicating synergy between the two polyphenols. Minimum inhibitory concentrations (MICs) of theaflavin and the theaflavin:epicatechin combination (2:1) were determined against 9 clinical isolates and one control isolate of Stenotrophomonas maltophilia using a microtiter assay. Results from the microtiter assay used indicated that the MIC for theaflavin was between 400 and 800 μg/mL. The MIC for the theaflavin:epicatechin combination (2:1) was between 200 and 400 μg/mL.A 1-step microreactor synthesis of theaflavin from epicatechin and epigallocatechin using extracted polyphenol oxidase was shown to be successful, producing high yields. Using the same methodology, the synthesis of deuterium labelled theaflavin was undertaken using epigallocatechin and the deuterium labelled epicatechin. However, this reaction was shown to be unsuccessful. The antibacterial action of microreactor synthesized theaflavin against Acinetobacter baumannii in a continuous flow process was investigated. Bacterial viability was tested using the resazurin indicator method. No viable cells were observed from bacterial samples exposed to greater than or equal to 4 hours of the continuous flow of theaflavin products. This indicated that the theaflavin produced antibacterial action after this time of exposure. At exposure times less than 4 hours, viable cells were detected

Multiplexed, affordable, and portable platform for real time quantification of counterfeit and substandard medicines

The World Health Organization estimates that about 10-30% of pharmaceuticals in the world are either substandard or counterfeit. The number is even higher in the developing countries. From a public health perspective, a key contributor to the development and proliferation drug resistant strains of infections, including tuberculosis (TB), malaria and other infections that are leading killers in resource limited settings is poor quality medicines. Most of the main causes are profit driven corruption in many pharmaceutical companies, the poor manufacture and quality control, and/or the inappropriate storage conditions. Poor quality drugs lead to loss of life, create morbidity, strain the financial structure of the health system and lead to long-term drug resistance that affects us all. The current technology for screening poor quality drugs can be divided into 2 categories: the high end, precise and high cost technologies (such as High Performance Liquid Chromatography) and lower cost and qualitative technologies (such as Thin-Layered Chromatography). The high-end methods can give a precise measurement of active pharmaceutical ingredient (API) concentration and the presence of impurities in the tablets, but require trained personnel, advanced machine and lab set up, not suitable for field testing where most of poor quality pharmaceuticals have been found. The lower cost techniques require little training and simple equipment to operate at a relatively inexpensive price, but only gives qualitative results. In addition, most of current methods do not look at the dissolution profile of the tablets simultaneously with the concentration of API. Therefore, we propose to develop an assay that can quantify the concentrations of multiple APIs simultaneously and measure dissolution rates. In order to address current gaps in knowledge, my research proposal has three main parts in the assay development: 1) Development of an fluorescent/luminescent assay for detection of counterfeit/substandard antimalarial using small-molecules-based methods and field testing in Ghana; 2) Development of a fluorescent assay for detection of water-soluble pharmaceuticals using SELEX; and 3) Design a detection platform using microfluidic chips for real time quantification of multiple active pharmaceutical ingredients. For proof-of-concept, an antimalarial drug (artesunate and amodiaquine) and antibacterial antibiotics (sulfamethoxazole and trimethoprim) are selected to demonstrate the probe development and test the chip performance. Overall, the assay will be rapid, robust, portable, inexpensive, multiplexed, quantitative, specific, and sensitive. At a big picture level, emphasizing drug quality and creating robust mechanisms of drug testing will improve health outcomes and enhance treatment efficacy in resource limited settings

Monitoring cell cultures in real time in a biochip

Drug screening is a very important procedure in the approval of drugs for cancer treatment. This process is generally carried out using in vitro or in vivo models that aren’t very efficient due to the non-reproducibility of the cellular and/or tissue microenvironment and ethical issues due to the use of animal models. Additionally, drug approval is a process that could last 10 to 15 years, too much time when therapy is required with urgency. Microfluidic structures can address such issues, decreasing the time per assay, as well as decreasing the quantity of reagents used and the volume of waste generated, thus decreasing the costs. Also, due to the generation of concentration gradients inside a microfluidic device, it mimics the microenvironment characteristic of conventional cell culture. In this work, a reproducible cell culture of HCT-116, a human colon cancer cell line, is successfully grown inside a microfluidic device for a posterior exposure to anti-cancer drugs. The cell viability, detected through staining the DNA with fluorophores, is on average 90%. To monitor the cell death via exposure to drugs, a specific cell death biomarker, adenylate kinase (AK), is detected inside a microfluidic device using a photomultiplier and a fluorescence microscope in a chip-based immunoassay. AK concentrations near the concentrations of the enzyme released by dead cells were detected with the immunoassay by concentrating the AK in packed agarose beads inside de microfluidic structure

Development and Application of Analytical Techniques for Evaluating Function in Pancreatic Islets of Langerhans.

Type 1 diabetes is caused by autoimmune destruction of insulin-secreting beta-cells found in the islets of Langerhans of the pancreas. Severe cases can be treated in a minimally invasive way by islet transplantation; however, islet transplantation has been limited by an inability to measure islet viability and potency prior to transplant. To address this need, we have developed a microfluidic platform to measure both intracellular calcium flux and insulin secretion, two important indicators of beta-cell function, at high temporal resolution during glucose treatment. Combining these measures on islets required methods for measuring fluorescence at two separate locations on a microfluidic system. To accomplish this objective, we used a 2-chip system in which perfusate was collected in fractions while intracellular calcium was measured using fluorescence imaging. The perfusate was subsequently analyzed for insulin by microchip electrophoresis with laser-induced fluorescence detection (MCE-LIF) using the same fluorescence microscope. We were able to distinguish first and second phase insulin secretion from batches of 8-10 islets with 80 s temporal resolution. Measured basal and peak first phase insulin secretion correlated well with previously reported results. Total analysis time using this system was <90 min. For an alternative approach to islet evaluation, we developed a metabolomic method to identify potential biomarkers of islet health for transplant. Using a miniaturized sample preparation method and HPLC-TOF-MS, we were able to identify 62 metabolites reliably in whole islet samples. To mimic damage that can occur during islet transplant, we induced oxidative stress in islets using hydrogen peroxide and measured their immediate metabolomic response as well as their response 1-4 h following stress removal. Increased concentrations of pentose phosphates, glucose-6-phosphate, and fructose bisphosphate in the immediate response corresponded to glycolysis blockage and possibly increased flux through the pentose phosphate pathway. Post-stress responses included increased levels of free fatty acids, phospholipids, long chain CoAs, and HMG-CoA as well blunted malonyl CoA concentrations, potentially relating to alterations in the glycerolipid/free fatty acid cycle and mevalonate pathway. These metabolites could comprise a metabolic signature of stressed cells for islet evaluation prior to transplantation.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/113374/1/ccipolla_1.pd