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

Massively Parallel Microfluidic Cell-Pairing Platform for the Statistical Study of Immunological Cell-Cell Interactions

Variability in cell-cell interactions is ubiquitous and particularly relevant for the immune system, where the reliability of cell-cell interactions is critical for the prevention of disease. This variability is poorly understood mainly due to the limitations of current methods. We have therefore designed a highly parallel microfluidic cell-pairing device and optimized its pairing efficiency using fluids modeling. The optimized device can hydrodynamically pair hundreds of primary mouse immune-cells at an efficiency of ~50%. We measured T cell activation dynamics of ~130 primary mouse T cells paired with B cells. Our findings represent the first time that variation has been observed in T cell activation dynamics.National Institutes of Health (U.S.) (NIH (EB008550))Singapore-MIT Allianc

CENTRIFUGAL LABTUBE FOR FULLY AUTOMATED DNA EXTRACTION & LAMP AMPLIFICATION BASED ON AN INTEGRATED, LOW-COST HEATING SYSTEM

In this paper, we introduce a disposable battery-driven heating system for loop-mediated isothermal DNA amplification (LAMP) inside a centrifugally-driven DNA-extraction platform (LabTube). We demonstrate fully automated, fully closed extraction of as little as 100 DNA copies of verotoxin-producing (VTEC) Escherichia coli lysate in water, milk and apple juice in a standard laboratory centrifuge, followed by subsequent automatic LAMP amplification with an overall time-to-result of 1.5hrs. The system is disposable, fully closed and automated, requiring only a single pipetting step. The microcontroller-driven heating system is low-cost (<1$) and it can be easily parallelized. Because the heated LabSystem runs within a standard laboratory centrifuge, it is suitable for DNA extraction and amplification in low-resource areas, at production sites or sales locations

LOW-COST BACTERIAL DETECTION SYSTEM FOR FOOD SAFETY BASED ON AUTOMATED DNA EXTRACTION, AMPLIFICATION AND READOUT

To ensure food, medical and environmental safety and quality, rapid, low-cost and easy-to-use detection methods are desirable. Here, the LabSystem is introduced for integrated, automated DNA purification and amplification. It consists of a disposable, centrifugally-driven DNA purification platform (LabTube) and the subsequent amplification in a low-cost UV/vis-reader (LabReader). In this paper, food safety was chosen as the first sample application with pathogenic verotoxin-producing (VTEC) Escherichia coli (E.coli) in water and milk, and the product-spoiler Alicyclobacillus acidoterrestris (A acidoterrestris) in apple juice as sample organisms. DNA was amplified qualitatively using isothermal loop-mediated DNA amplification (LAMP) and quantitatively using real-time PCR. By optimizing manual purification protocols inside the LabTube, as little as 45 inserted DNA copies were extracted from E.coli and A.acidoterrestris lysates in real samples (milk, juice and water). To run isothermal DNA amplification (LAMP) and PCR inside the LabReader, temperature control as well as data analysis methods were implemented. Combined detection limits for DNA purification and amplification from bacteria lysates in real samples at 102-103 inserted copies were achieved. The demonstrated LabSystem runs with standard laboratory equipment and reduces hands-on times from 30min to 3min

Rapid and Robust Detection Methods for Poison and Microbial Contamination

Real-time on-site monitoring of analytes is currently in high demand for food contamination, water, medicines, and ingestible household products that were never tested appropriately. Here we introduce chemical methods for the rapid quantification of a wide range of chemical and microbial contaminations using a simple instrument. Within the testing procedure, we used a multichannel, multisample, UV–vis spectrophotometer/fluorometer that employs two frequencies of light simultaneously to interrogate the sample. We present new enzyme- and dye-based methods to detect (di)ethylene glycol in consumables above 0.1 wt % without interference and alcohols above 1 ppb. Using DNA intercalating dyes, we can detect a range of pathogens (E. coli, Salmonella, V. Cholera, and a model for Malaria) in water, foods, and blood without background signal. We achieved universal scaling independent of pathogen size above 10[superscript 4] CFU/mL by taking advantage of the simultaneous measurement at multiple wavelengths. We can detect contaminants directly, without separation, purification, concentration, or incubation. Our chemistry is stable to ±1% for >3 weeks without refrigeration, and measurements require <5 min.CIMIT: Center for Integration of Medicine and Innovative TechnologyLegatum Center for Development & Entrepreneurship (Massachusetts Institute of Technology)MIT Department of Physics Pappalardo Progra

Robust and economical multi-sample, multi-wavelength UV/vis absorption and fluorescence detector for biological and chemical contamination

We present a portable multi-channel, multi-sample UV/vis absorption and fluorescence detection device, which has no moving parts, can operate wirelessly and on batteries, interfaces with smart mobile phones or tablets, and has the sensitivity of commercial instruments costing an order of magnitude more. We use UV absorption to measure the concentration of ethylene glycol in water solutions at all levels above those deemed unsafe by the United States Food and Drug Administration; in addition we use fluorescence to measure the concentration of d-glucose. Both wavelengths can be used concurrently to increase measurement robustness and increase detection sensitivity. Our small robust economical device can be deployed in the absence of laboratory infrastructure, and therefore may find applications immediately following natural disasters, and in more general deployment for much broader-based testing of food, agricultural and household products to prevent outbreaks of poisoning and disease.Center for Integration of Medicine and Innovative TechnologyLegatum Center for Development & Entrepreneurship (Massachusetts Institute of Technology)NVIDIA Corporation (Professor Partnership Program