Electronic gene sequencing : a novel method for DNA sequencing based on direct heat or pH measurement
Abstract/Contents
- Abstract
- The Human Genome Project was accomplished by a reduction in the cost of DNA sequencing by three orders of magnitude. Further cost reductions are required for sequencing to become a standard tool in clinical medicine and to enable personalized medicine via individual genome sequencing. The current cost varies between $50k to $100k over a period of months; depend on the technology, accuracy and read-length. It is desired to reduce the cost to $1000 per genome to enable profiling of individuals genome. To achieve this goal, a highly integrated platform with simplified chemistry is required. In this dissertation, we introduce a novel method for DNA sequencing based on electrical detection of polymerization reaction, called "Thermo/pH sequencing". Our proposed method is based on the direct measurement of the heat release or the pH modulation (change of H+ ion concentration in the solution) during DNA extension. For high throughput DNA sequencing, DNA strands are immobilized to small micron-size beads in a microfluidic platform. The DNA-beads are in a reaction mixture in contact with an array of sensitive micro-machined heat or pH sensors, which detect the electrical signature from incorporation of a complementary base (dNTP) in the presence of appropriate reagents (DNA polymerase, and polymerase reaction buffer). This results to a label-free, long-read and fast chemistry; 10x reduction in reagent cost with 10x increase in throughput can potentially yield to significant improvement in the cost of genome sequencing to less than $1000. In addition, substituting optical detection set-up with microelectronic sensor reduces the capital cost of sequencing instruments from $500k to less than $50k. We demonstrate the proof of concept for this technology at large scale. Then we describe the development of an appropriate microfluidic platform and two micromachined electrical biosensors that employ electrical detection for heat or pH detection. Both versatile platforms can be multiplexed and have the potential of providing rapid and inexpensive measurements without any compromise in the sensitivity, making them good potential candidates for use in the clinical setting. We report a chip-based integrated differential microfluidic nanocalimeters with on-chip injection and multiplexing unit, capable of characterizing the heat of reaction with unprecedented 2-nW resolution in 1 Hz bandwidth for nanoliter scale samples. We successfully demonstrate DNA Thermosequencing with sequential injection of different nucleotides into the integrated microfluidic calorimeter device. In addition, the device can serve as a powerful tool to characterize a variety of the biomedical processes, such as metabolic activities of microorganisms, living cells and catalyzed reactions. We also present a microfabricated device in microfluidics for pH sequencing, called nanoneedle biosensor. The key element for this device is a 10nm wide gap on the end of the needle of total diameter about 100nm. Any change in the population of molecules in this gap results in a change of impedance across the gap; single molecule detection should be possible. In addition, DNA-beads can be allocated iv near the sensors to measure the pH change during DNA extension. The design, fabrication, testing, optimization and a modified structure of the device for higher signal to noise ratio are presented. Toward an integrated sequencer platform, automation and reduced labor cost, higher throughput, accuracy and efficiency for genomics and proteomics analysis; further integration and optimization of the presented systems are required. We envision the integration of our CMOS-compatible devices with a CMOS integrated circuitry into a high throughput gene sequencer or proteomics system. The proteomics system enables multiplex analysis using an array of micro-channels for probing clinically relevant samples such as the human serum for various protein and nucleic acid biomarkers for cancer detection, and also the detection of pathogenic bacteria in solution.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2010 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Esfandyarpour, Hesaam | |
|---|---|---|
| Associated with | Stanford University, Department of Electrical Engineering | |
| Primary advisor | Davis, Ronald W. (Ronald Wayne), 1941- | |
| Primary advisor | Pease, R. (R. Fabian W.) | |
| Thesis advisor | Davis, Ronald W. (Ronald Wayne), 1941- | |
| Thesis advisor | Pease, R. (R. Fabian W.) | |
| Thesis advisor | Pianetta, Piero | |
| Advisor | Pianetta, Piero |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Hesaam Esfandyarpour. |
|---|---|
| Note | Submitted to the Department of Electrical Engineering. |
| Thesis | Ph. D. Stanford University 2010 |
| Location | electronic resource |
Access conditions
- Copyright
- © 2010 by Hesaam Esfandyarpour
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