A low-cost genomic sensor for ocean-observing systems and infectious disease detection

155590-Thumbnail Image.png
Description
Many environmental microorganisms such as marine microbes are un-culturable; hence, they should be analyzed in situ. Even though a few in situ ocean observing instruments have been available to oceanographers, their applications are limited, because these instruments are expensive and…

Many environmental microorganisms such as marine microbes are un-culturable; hence, they should be analyzed in situ. Even though a few in situ ocean observing instruments have been available to oceanographers, their applications are limited, because these instruments are expensive and power hungry.

In this dissertation project, an inexpensive, portable, low-energy consuming, and highly quantitative microbiological genomic sensor has been developed for in situ ocean-observing systems. A novel real-time colorimetric loop-mediated isothermal amplification (LAMP) technology has been developed for quantitative detection of microbial nucleic acids. This technology was implemented on a chip-level device with an embedded inexpensive imaging device and temperature controller to achieve quantitative detection within one hour. A bubble-free liquid handling approach was introduced to avoid bubble trapping during liquid loading, a common problem in microfluidic devices. An algorithm was developed to reject the effect of bubbles generated during the reaction process, to enable more accurate nucleic acid analysis. This genomic sensor has been validated at gene and gene expression levels using Synechocystis sp. PCC 6803 genomic DNA and total RNA. Results suggest that the detection limits reached 10 copies/μL and 100 fg/μL, respectively. This approach was highly quantitative, with linear standard curves down to 103 copies/μL and 1 pg/μL, respectively. In addition to environmental microbe characterization, this genomic sensor has been employed for viral RNA quantification during an infectious disease outbreak. As the Zika fever was spreading in America, a quantitative detection of Zika virus has been performed. The results show that the genomic sensor is highly quantitative from 10 copies/μL to 105 copies/μL. This suggests that the novel nucleic acid quantification technology is sensitive, quantitative, and robust. It is a promising candidate for rapid microbe detection and quantification in routine laboratories.

In the future, this genomic sensor will be implemented in in situ platforms as a core analytical module with minor modifications, and could be easily accessible by oceanographers. Deployment of this microbial genomic sensor in the field will enable new scientific advances in oceanography and provide a possible solution for infectious disease detection.
Date Created
2017
Agent

Simulation and Experimental Characterization of Microscopically Accessible Hydrodynamic Microvortices

128661-Thumbnail Image.png
Description

Single-cell studies of phenotypic heterogeneity reveal more information about pathogenic processes than conventional bulk-cell analysis methods. By enabling high-resolution structural and functional imaging, a single-cell three-dimensional (3D) imaging system can be used to study basic biological processes and to diagnose…

Single-cell studies of phenotypic heterogeneity reveal more information about pathogenic processes than conventional bulk-cell analysis methods. By enabling high-resolution structural and functional imaging, a single-cell three-dimensional (3D) imaging system can be used to study basic biological processes and to diagnose diseases such as cancer at an early stage. One mechanism that such systems apply to accomplish 3D imaging is rotation of a single cell about a fixed axis. However, many cell rotation mechanisms require intricate and tedious microfabrication, or fail to provide a suitable environment for living cells. To address these and related challenges, we applied numerical simulation methods to design new microfluidic chambers capable of generating fluidic microvortices to rotate suspended cells. We then compared several microfluidic chip designs experimentally in terms of: (1) their ability to rotate biological cells in a stable and precise manner; and (2) their suitability, from a geometric standpoint, for microscopic cell imaging. We selected a design that incorporates a trapezoidal side chamber connected to a main flow channel because it provided well-controlled circulation and met imaging requirements. Micro particle-image velocimetry (micro-PIV) was used to provide a detailed characterization of flows in the new design. Simulated and experimental results demonstrate that a trapezoidal side chamber represents a viable option for accomplishing controlled single cell rotation. Further, agreement between experimental and simulated results confirms that numerical simulation is an effective method for chamber design.

Date Created
2012-06-15
Agent

Microbe Observation and Cultivation Array (MOCA) for Cultivating and Analyzing Environmental Microbiota

129002-Thumbnail Image.png
Description

Background: The use of culture-independent nucleic acid techniques, such as ribosomal RNA gene cloning library analysis, has unveiled the tremendous microbial diversity that exists in natural environments. In sharp contrast to this great achievement is the current difficulty in cultivating the…

Background: The use of culture-independent nucleic acid techniques, such as ribosomal RNA gene cloning library analysis, has unveiled the tremendous microbial diversity that exists in natural environments. In sharp contrast to this great achievement is the current difficulty in cultivating the majority of bacterial species or phylotypes revealed by molecular approaches. Although recent new technologies such as metagenomics and metatranscriptomics can provide more functionality information about the microbial communities, it is still important to develop the capacity to isolate and cultivate individual microbial species or strains in order to gain a better understanding of microbial physiology and to apply isolates for various biotechnological applications.

Results: We have developed a new system to cultivate bacteria in an array of droplets. The key component of the system is the microbe observation and cultivation array (MOCA), which consists of a Petri dish that contains an array of droplets as cultivation chambers. MOCA exploits the dominance of surface tension in small amounts of liquid to spontaneously trap cells in well-defined droplets on hydrophilic patterns. During cultivation, the growth of the bacterial cells across the droplet array can be monitored using an automated microscope, which can produce a real-time record of the growth. When bacterial cells grow to a visible microcolony level in the system, they can be transferred using a micropipette for further cultivation or analysis.

Conclusions: MOCA is a flexible system that is easy to set up, and provides the sensitivity to monitor growth of single bacterial cells. It is a cost-efficient technical platform for bioassay screening and for cultivation and isolation of bacteria from natural environments.

Date Created
2013-01-09
Agent

Single cell RT-qPCR on 3D cell spheroids

155112-Thumbnail Image.png
Description
A single cell is the very fundamental element in an organism; however, it contains the most complicated and stochastic information, such as DNA, RNA, and protein expression. Thus, it is a necessity to study stochastic gene expression in order to…

A single cell is the very fundamental element in an organism; however, it contains the most complicated and stochastic information, such as DNA, RNA, and protein expression. Thus, it is a necessity to study stochastic gene expression in order to discover the biosignatures at the single-cell level. The heterogeneous gene expression of single cells from an isogenic cell population has already been studied for years. Yet to date, single-cell studies have been confined in a fashion of analyzing isolated single cells or a dilution of cells from the bulk-cell populations. These techniques or devices are limited by either the mechanism of cell lysis or the difficulties to target specific cells without harming neighboring cells.

This dissertation presents the development of a laser lysis chip combined with a two-photon laser system to perform single-cell lysis of single cells in situ from three-dimensional (3D) cell spheroids followed by analysis of the cell lysate with two-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The 3D spheroids were trapped in a well in the custom-designed laser lysis chip. Next, each single cell of interest in the 3D spheroid was identified and lysed one at a time utilizing a two-photon excited laser. After each cell lysis, the contents inside the target cell were released to the surrounding media and carried out to the lysate collector. Finally, the gene expression of each individual cell was measured by two-step RT-qPCR then spatially mapped back to its original location in the spheroids to construct a 3D gene expression map.

This novel technology and approach enables multiple gene expression measurements in single cells of multicellular organisms as well as cell-to-cell heterogeneous responses to the environment with spatial recognition. Furthermore, this method can be applied to study precancerous tissues for a better understanding of cancer progression and for identifying early tumor development.
Date Created
2016
Agent

Design of miniaturized underwater vehicle with propulsions for deep-sea research applications

152949-Thumbnail Image.png
Description
The ocean is vital to the health of our planet but remains virtually unexplored. Many researchers seek to understand a wide range of geological and biological phenomena by developing technologies which enable exploration of the deep-sea. The task…

The ocean is vital to the health of our planet but remains virtually unexplored. Many researchers seek to understand a wide range of geological and biological phenomena by developing technologies which enable exploration of the deep-sea. The task of developing a technology which can withstand extreme pressure and temperature gradients in the deep ocean is not trivial. Of these technologies, underwater vehicles were developed to study the deep ocean, but remain large and expensive to manufacture. I am proposing the development of cost efficient miniaturized underwater vehicle (mUV) with propulsion systems to carry small measurement devices and enable deep-sea exploration. These mUV's overall size is optimized based on the vehicle parameters such as energy density, desired velocity, swimming time and propulsion performance. However, there are limitations associated with the size of the mUV which leads to certain challenges. For example, 2000 m below the sea level, the pressure is as high as 3000 psi. Therefore, certain underwater vehicle modules, such as the propulsion system, will require pressure housing to ensure the functionality of the thrust generation. In the case of a mUV swimming against the deep-sea current, a thrust magnitude is required to enable the vehicle to overcome the ocean current speed and move forward. Therefore, the size of the mUV is limited by the energy density and the propeller size. An equation is derived to miniaturize underwater vehicle while performing with a certain specifications. An inrunner three-phase permanent magnet brushless DC motor is designed and fabricated with a specific size to fit inside the mUV's core. The motor is composed of stator winding in a pressure housing and an open to water ring-propeller rotor magnet. Several ring-propellers are 3D printed and tested experimentally to determine their performances and efficiencies. A planer motion optimal trajectory for the mUV is determined to minimize the energy usage. Those studies enable the design of size optimized underwater vehicle with propulsion to carry small measurement sensors and enable underwater exploration. Developing mUV's will enable ocean exploration that can lead to significant scientific discoveries and breakthroughs that will solve current world health and environmental problems.
Date Created
2014
Agent

Single cell RT-qPCR based ocean environmental sensing device development

152297-Thumbnail Image.png
Description
This thesis research focuses on developing a single-cell gene expression analysis method for marine diatom Thalassiosira pseudonana and constructing a chip level tool to realize the single cell RT-qPCR analysis. This chip will serve as a conceptual foundation for future…

This thesis research focuses on developing a single-cell gene expression analysis method for marine diatom Thalassiosira pseudonana and constructing a chip level tool to realize the single cell RT-qPCR analysis. This chip will serve as a conceptual foundation for future deployable ocean monitoring systems. T. pseudonana, which is a common surface water microorganism, was detected in the deep ocean as confirmed by phylogenetic and microbial community functional studies. Six-fold copy number differences between 23S rRNA and 23S rDNA were observed by RT-qPCR, demonstrating the moderate functional activity of detected photosynthetic microbes in the deep ocean including T. pseudonana. Because of the ubiquity of T. pseudonana, it is a good candidate for an early warning system for ocean environmental perturbation monitoring. This early warning system will depend on identifying outlier gene expression at the single-cell level. An early warning system based on single-cell analysis is expected to detect environmental perturbations earlier than population level analysis which can only be observed after a whole community has reacted. Preliminary work using tube-based, two-step RT-qPCR revealed for the first time, gene expression heterogeneity of T. pseudonana under different nutrient conditions. Heterogeneity was revealed by different gene expression activity for individual cells under the same conditions. This single cell analysis showed a skewed, lognormal distribution and helped to find outlier cells. The results indicate that the geometric average becomes more important and representative of the whole population than the arithmetic average. This is in contrast with population level analysis which is limited to arithmetic averages only and highlights the value of single cell analysis. In order to develop a deployable sensor in the ocean, a chip level device was constructed. The chip contains surface-adhering droplets, defined by hydrophilic patterning, that serve as real-time PCR reaction chambers when they are immersed in oil. The chip had demonstrated sensitivities at the single cell level for both DNA and RNA. The successful rate of these chip-based reactions was around 85%. The sensitivity of the chip was equivalent to published microfluidic devices with complicated designs and protocols, but the production process of the chip was simple and the materials were all easily accessible in conventional environmental and/or biology laboratories. On-chip tests provided heterogeneity information about the whole population and were validated by comparing with conventional tube based methods and by p-values analysis. The power of chip-based single-cell analyses were mainly between 65-90% which were acceptable and can be further increased by higher throughput devices. With this chip and single-cell analysis approaches, a new paradigm for robust early warning systems of ocean environmental perturbation is possible.
Date Created
2013
Agent

Underwater optical sensorbot for in situ pH monitoring

151156-Thumbnail Image.png
Description
Continuous underwater observation is a challenging engineering task that could be accomplished by development and deployment of a sensor array that can survive harsh underwater conditions. One approach to this challenge is a swarm of micro underwater robots, known as…

Continuous underwater observation is a challenging engineering task that could be accomplished by development and deployment of a sensor array that can survive harsh underwater conditions. One approach to this challenge is a swarm of micro underwater robots, known as Sensorbots, that are equipped with biogeochemical sensors that can relay information among themselves in real-time. This innovative method for underwater exploration can contribute to a more comprehensive understanding of the ocean by not limiting sampling to a single point and time. In this thesis, Sensorbot Beta, a low-cost fully enclosed Sensorbot prototype for bench-top characterization and short-term field testing, is presented in a modular format that provides flexibility and the potential for rapid design. Sensorbot Beta is designed around a microcontroller driven platform comprised of commercial off-the-shelf components for all hardware to reduce cost and development time. The primary sensor incorporated into Sensorbot Beta is an in situ fluorescent pH sensor. Design considerations have been made for easy adoption of other fluorescent or phosphorescent sensors, such as dissolved oxygen or temperature. Optical components are designed in a format that enables additional sensors. A real-time data acquisition system, utilizing Bluetooth, allows for characterization of the sensor in bench top experiments. The Sensorbot Beta demonstrates rapid calibration and future work will include deployment for large scale experiments in a lake or ocean.
Date Created
2012
Agent

Optimizing micro-vortex chamber for living single cell rotation

Description
Single cell phenotypic heterogeneity studies reveal more information about the pathogenesis process than conventional bulk methods. Furthermore, investigation of the individual cellular response mechanism during rapid environmental changes can only be achieved at single cell level. By enabling the study…

Single cell phenotypic heterogeneity studies reveal more information about the pathogenesis process than conventional bulk methods. Furthermore, investigation of the individual cellular response mechanism during rapid environmental changes can only be achieved at single cell level. By enabling the study of cellular morphology, a single cell three-dimensional (3D) imaging system can be used to diagnose fatal diseases, such as cancer, at an early stage. One proven method, CellCT, accomplishes 3D imaging by rotating a single cell around a fixed axis. However, some existing cell rotating mechanisms require either intricate microfabrication, and some fail to provide a suitable environment for living cells. This thesis develops a microvorterx chamber that allows living cells to be rotated by hydrodynamic alone while facilitating imaging access. In this thesis work, 1) the new chamber design was developed through numerical simulation. Simulations revealed that in order to form a microvortex in the side chamber, the ratio of the chamber opening to the channel width must be smaller than one. After comparing different chamber designs, the trapezoidal side chamber was selected because it demonstrated controllable circulation and met the imaging requirements. Microvortex properties were not sensitive to the chambers with interface angles ranging from 0.32 to 0.64. A similar trend was observed when chamber heights were larger than chamber opening. 2) Micro-particle image velocimetry was used to characterize microvortices and validate simulation results. Agreement between experimentation and simulation confirmed that numerical simulation was an effective method for chamber design. 3) Finally, cell rotation experiments were performed in the trapezoidal side chamber. The experimental results demonstrated cell rotational rates ranging from 12 to 29 rpm for regular cells. With a volumetric flow rate of 0.5 µL/s, an irregular cell rotated at a mean rate of 97 ± 3 rpm. Rotational rates can be changed by altering inlet flow rates.
Date Created
2011
Agent
Subscribe to Chao, Shih-hui