Design of a microfabricated device for Ligase Detection Reaction (LDR)

The Ligase Detection Reaction (LDR) is a mutation detection technique used to identify point mutations in deoxyribonucleic acid (DNA). Developed by Francis Barany and associates at Cornell University it is used to find specific low abundant point mutations that may lead to colorectal cancer in the early stages of disease development. The research objective was to design and manufacture a microscale Ligase Detection Reaction (LDR) device in polycarbonate. The LDR module will be incorporated with other microdevices such as: Continuous Flow Polymerase Chain Reaction (CFRCR) and Capillary Electrophoresis (CE) in modular lab-on-a-chip technology. In making the microdevice, the duration of original reaction had to be scaled down from the current 2½ hours for 20 cycles for the macroscale reaction. It was found that an excess of primers in relation to PCR product was needed for efficient ligation. By changing the concentrations, volumes and time for the process the current time is down to 40 minutes for 20 cycles with indications that further time reductions are possible on the microscale. There are two mixing stages involved in the reaction. Micromixers were simulated in Fluent (v5.4, Lebanon, NH) and several test geometries selected for fabrication. Passive diffusion mixing was used based on obtaining high aspect ratios, 7 to 20. The mixers were made by SU-8 lithography, LIGA, laser ablation, and micromilling to characterize each fabrication method. It was found that LIGA was best for making the micromixers, but was the longest process. The micromixers are fabricated and tested using chemi-luminescence technique. For a successful reaction, temperatures of 0°C, 95°C and 65°C were needed. A stationary chamber was used for thermal cycling in which the sample sits while the temperature is cycled. Finite element analysis showed uniform temperatures in the rectangular 1.5μl chambers and that air slits can effectively separate the thermal cycle zone from the 0°C cooling zone and also isolate the mixing region. A test device was laid out and micromilled with the temperature zones maintained and fluid flow controlled. A commercial thin film heater and a thermoelectric module were used with PID controls to obtain the required process temperatures. Heating from 65°C to 95°C took 10 seconds, while cooling from 95°C to 65°C also took 10 seconds. The residence times at the required temperatures can be adapted to changes in the LDR

THERMAL CYCLING DESIGN ALTERNATIVES FOR THE POLYMERASE CHAIN REACTION

PCR (polymerase chain reaction) is a process by which a small amount of DNA is amplified many times to yield an easily detectable amount. This process is widely used for the detection of bacterial pathogens for biodefense, in basic research, criminal identification, and disease detection in humans. The reaction mixture must be cycled repeatedly between three different temperature levels in PCR. The reaction mixture is first heated at 94 °C, cooled to 54 °C, and then heated to 72 °C. This cycle is repeated 20-40 times. The main objective of the work reported here is to evaluate alternative heating/cooling schemes for PCR with the ultimate goal of speeding up a PCR reaction. A secondary goal is to arrive at a design that is consistent with battery operation to allow for a portable PCR device. Insight is gained about interactions between the PCR reaction and the engineering system

Virtual reaction chambers as a tool for polymerase chain reaction and protein thermal shift assays

IIn the frame of this thesis Virtual reaction chambers (VRCs) were analysed as a tool for polymerase chain reaction and protein analysis. VRCs are digital microfluidic reactors, consisting of a aqueous sample volume (100nL - 300 nL) encapsulated by an oil droplet ( 1microL). Firstly a portable system for the temperature manipulation and fluorescence measurement of VRCs was constructed for this thesis. Subsequently the device was used to perform polymerase chain reactions (PCR) from DNA as well as reverse transcription PCR from RNA. Furthermore two methods of multiplexed PCR were developed, one of which specifcally exploits the advantages of VRCs. Thus it can be concluded that virtual reaction chambers are a suitable tool for polymerase chain reaction of DNA and RNA especially for point-of-care applications. Furthermore a mathematical model based on chain growth was constructed to describe the PCR, with particular focus on the extension step. Additionally the heating device and fluorescence measurement were used to measure the thermal stability of proteins. Here advantage was taken of the fact that VRCs are easily superheatable. Thus proteins still stable at temperatures above 100°C can be measured using this system. This and the low sample consumption makes VRCs an ideal tool for screening purposes in protein analysis.Im Rahmen dieser These wurden Virtual Reaction Chambers (VRCs) als Werkzeug für die Polymerase Kettenreaktion (PCR) und die Analyse von Proteinen getestet. VRCs sind digitale, mikrofluidische Reaktionsvolumen die aus einem wässrigen Probevolumen (100 - 300nL) eingeschlossen von einem Öltropfen ( 1µL) bestehen. In einem ersten Schritt wurde ein tragbares System für die Temperaturmanipulation sowie Flureszensmessung von VRCs konstruiert. Im Folgenden wurde mit dem Gerät die Polymerase Kettenreaktion von DNA, sowie reverse transkriptions PCR von RNA durchgeführt. Im Weiteren wurden zwei Methoden zur multiplex PCR entwickelt, von denen eine die Vorteile von Virtual Reaction Chambers nutzt. Aus den Ergebnissen kann gefolgert werden, dass VRCs ein geeignetes Werkzeug für die PCR von DNA und RNA sind, im Besonderen für Point-of-Care Anwendungen. Außerdem wurde ein mathematisches Modell zur Beschreibung der Reaktion geschrieben. Das Modell basiert auf Chain-Growth Prozessen und ist im Besonderen auf den Elongination-Schritt der Reaktion fokussiert. In einem zweiten Teil wurde das entwickelte Gerät benutzt, um die Temperatur-Stabilität von Proteinen zu messen. Hier wurde ausgenutzt, dass VRCs eine einfache Möglichkeit zum Superheating von Flüssigkeiten bieten. Dadurch konnten Proteine gemessen werden, welche auch noch bei 100°C stabil sind. Dies und der niedrige Probenverbrauch machen aus VRCs ein geeignetes Werkzeug zur Analyse von Proteinen, insbesondere im Rahmen von Screening-Versuchen

Virtual reaction chambers as a tool for polymerase chain reaction and protein thermal shift assays

IIn the frame of this thesis Virtual reaction chambers (VRCs) were analysed as a tool for polymerase chain reaction and protein analysis. VRCs are digital microfluidic reactors, consisting of a aqueous sample volume (100nL - 300 nL) encapsulated by an oil droplet ( 1microL). Firstly a portable system for the temperature manipulation and fluorescence measurement of VRCs was constructed for this thesis. Subsequently the device was used to perform polymerase chain reactions (PCR) from DNA as well as reverse transcription PCR from RNA. Furthermore two methods of multiplexed PCR were developed, one of which specifcally exploits the advantages of VRCs. Thus it can be concluded that virtual reaction chambers are a suitable tool for polymerase chain reaction of DNA and RNA especially for point-of-care applications. Furthermore a mathematical model based on chain growth was constructed to describe the PCR, with particular focus on the extension step. Additionally the heating device and fluorescence measurement were used to measure the thermal stability of proteins. Here advantage was taken of the fact that VRCs are easily superheatable. Thus proteins still stable at temperatures above 100°C can be measured using this system. This and the low sample consumption makes VRCs an ideal tool for screening purposes in protein analysis.Im Rahmen dieser These wurden Virtual Reaction Chambers (VRCs) als Werkzeug für die Polymerase Kettenreaktion (PCR) und die Analyse von Proteinen getestet. VRCs sind digitale, mikrofluidische Reaktionsvolumen die aus einem wässrigen Probevolumen (100 - 300nL) eingeschlossen von einem Öltropfen ( 1µL) bestehen. In einem ersten Schritt wurde ein tragbares System für die Temperaturmanipulation sowie Flureszensmessung von VRCs konstruiert. Im Folgenden wurde mit dem Gerät die Polymerase Kettenreaktion von DNA, sowie reverse transkriptions PCR von RNA durchgeführt. Im Weiteren wurden zwei Methoden zur multiplex PCR entwickelt, von denen eine die Vorteile von Virtual Reaction Chambers nutzt. Aus den Ergebnissen kann gefolgert werden, dass VRCs ein geeignetes Werkzeug für die PCR von DNA und RNA sind, im Besonderen für Point-of-Care Anwendungen. Außerdem wurde ein mathematisches Modell zur Beschreibung der Reaktion geschrieben. Das Modell basiert auf Chain-Growth Prozessen und ist im Besonderen auf den Elongination-Schritt der Reaktion fokussiert. In einem zweiten Teil wurde das entwickelte Gerät benutzt, um die Temperatur-Stabilität von Proteinen zu messen. Hier wurde ausgenutzt, dass VRCs eine einfache Möglichkeit zum Superheating von Flüssigkeiten bieten. Dadurch konnten Proteine gemessen werden, welche auch noch bei 100°C stabil sind. Dies und der niedrige Probenverbrauch machen aus VRCs ein geeignetes Werkzeug zur Analyse von Proteinen, insbesondere im Rahmen von Screening-Versuchen

Virtual reaction chambers as a tool for polymerase chain reaction and protein thermal shift assays

IIn the frame of this thesis Virtual reaction chambers (VRCs) were analysed as a tool for polymerase chain reaction and protein analysis. VRCs are digital microfluidic reactors, consisting of a aqueous sample volume (100nL - 300 nL) encapsulated by an oil droplet ( 1microL). Firstly a portable system for the temperature manipulation and fluorescence measurement of VRCs was constructed for this thesis. Subsequently the device was used to perform polymerase chain reactions (PCR) from DNA as well as reverse transcription PCR from RNA. Furthermore two methods of multiplexed PCR were developed, one of which specifcally exploits the advantages of VRCs. Thus it can be concluded that virtual reaction chambers are a suitable tool for polymerase chain reaction of DNA and RNA especially for point-of-care applications. Furthermore a mathematical model based on chain growth was constructed to describe the PCR, with particular focus on the extension step. Additionally the heating device and fluorescence measurement were used to measure the thermal stability of proteins. Here advantage was taken of the fact that VRCs are easily superheatable. Thus proteins still stable at temperatures above 100°C can be measured using this system. This and the low sample consumption makes VRCs an ideal tool for screening purposes in protein analysis.Im Rahmen dieser These wurden Virtual Reaction Chambers (VRCs) als Werkzeug für die Polymerase Kettenreaktion (PCR) und die Analyse von Proteinen getestet. VRCs sind digitale, mikrofluidische Reaktionsvolumen die aus einem wässrigen Probevolumen (100 - 300nL) eingeschlossen von einem Öltropfen ( 1µL) bestehen. In einem ersten Schritt wurde ein tragbares System für die Temperaturmanipulation sowie Flureszensmessung von VRCs konstruiert. Im Folgenden wurde mit dem Gerät die Polymerase Kettenreaktion von DNA, sowie reverse transkriptions PCR von RNA durchgeführt. Im Weiteren wurden zwei Methoden zur multiplex PCR entwickelt, von denen eine die Vorteile von Virtual Reaction Chambers nutzt. Aus den Ergebnissen kann gefolgert werden, dass VRCs ein geeignetes Werkzeug für die PCR von DNA und RNA sind, im Besonderen für Point-of-Care Anwendungen. Außerdem wurde ein mathematisches Modell zur Beschreibung der Reaktion geschrieben. Das Modell basiert auf Chain-Growth Prozessen und ist im Besonderen auf den Elongination-Schritt der Reaktion fokussiert. In einem zweiten Teil wurde das entwickelte Gerät benutzt, um die Temperatur-Stabilität von Proteinen zu messen. Hier wurde ausgenutzt, dass VRCs eine einfache Möglichkeit zum Superheating von Flüssigkeiten bieten. Dadurch konnten Proteine gemessen werden, welche auch noch bei 100°C stabil sind. Dies und der niedrige Probenverbrauch machen aus VRCs ein geeignetes Werkzeug zur Analyse von Proteinen, insbesondere im Rahmen von Screening-Versuchen

Energy: A continuing bibliography with indexes, supplement 16, January 1978

This bibliography lists 1287 reports, articles, and other documents introduced into the NASA scientific and technical information system from October 1, 1977 through December 31, 1977

NASA Tech Briefs, December 1997

Topics: Design and Analysis Software; Electronic Components and Circuits; Electronic Systems; Physical Sciences; Materials; Computer Software; Mechanics; Manufacturing/Fabrication; Mathematics and Information Sciences; Books and Reports

Planetary Science Vision 2050 Workshop : February 27–28 and March 1, 2017, Washington, DC

This workshop is meant to provide NASA’s Planetary Science Division with a very long-range vision of what planetary science may look like in the future.Organizer, Lunar and Planetary Institute ; Conveners, James Green, NASA Planetary Science Division, Doris Daou, NASA Planetary Science Division ; Science Organizing Committee, Stephen Mackwell, Universities Space Research Association [and 14 others]PARTIAL CONTENTS: Exploration Missions to the Kuiper Belt and Oort Cloud--Future Mercury Exploration: Unique Science Opportunities from Our Solar System’s Innermost Planet--A Vision for Ice Giant Exploration--BAOBAB (Big and Outrageously Bold Asteroid Belt) Project--Asteroid Studies: A 35-Year Forecast--Sampling the Solar System: The Next Level of Understanding--A Ground Truth-Based Approach to Future Solar System Origins Research--Isotope Geochemistry for Comparative Planetology of Exoplanets--The Moon as a Laboratory for Biological Contamination Research--“Be Careful What You Wish For:” The Scientific, Practical, and Cultural Implications of Discovering Life in Our Solar System--The Importance of Particle Induced X-Ray Emission (PIXE) Analysis and Imaging to the Search for Life on the Ocean Worlds--Follow the (Outer Solar System) Water: Program Options to Explore Ocean Worlds--Analogies Among Current and Future Life Detection Missions and the Pharmaceutical/ Biomedical Industries--On Neuromorphic Architectures for Efficient, Robust, and Adaptable Autonomy in Life Detection and Other Deep Space Missions