Microfluidic array chip for highly parallel genomic and transcriptomic applications

The Polymerase Chain Reaction (PCR) is an enzyme-based technique to amplify a single or few copies of Deoxyribose Nucleic Acid (DNA) in an exponential process, generating millions of copies of a known DNA sequence. The technique relies on thermal cycling; consisting of repeated cycles of heating and...

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Main Author: Naveen Ramalingam
Other Authors: Gong Haiqing, Thomas
Format: Theses and Dissertations
Language:English
Published: 2012
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Online Access:https://hdl.handle.net/10356/49499
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Institution: Nanyang Technological University
Language: English
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spelling sg-ntu-dr.10356-494992023-03-11T17:50:47Z Microfluidic array chip for highly parallel genomic and transcriptomic applications Naveen Ramalingam Gong Haiqing, Thomas Thomas Lufkin School of Mechanical and Aerospace Engineering DRNTU::Engineering::Mechanical engineering The Polymerase Chain Reaction (PCR) is an enzyme-based technique to amplify a single or few copies of Deoxyribose Nucleic Acid (DNA) in an exponential process, generating millions of copies of a known DNA sequence. The technique relies on thermal cycling; consisting of repeated cycles of heating and cooling of a reaction for DNA melting, target identification and enzymatic replication of the target DNA. The PCR technique is widely used for diagnosis, prognosis of diseases and enumeration of pathogen loads. Conventional PCR instruments are bulky, limiting its use in the field, but rapid development of miniaturized total analysis systems (μTAS) enables commercialization of point-of-care devices. In resource-limited settings, it is impractical to get access to a diagnostic laboratory having sophisticated instruments and in such settings, it is desirable to use disposable point-of-care high-throughput diagnostic chips that do not require liquid handling or pumping instruments for sample distribution among an array of reactors. In addition to the pump-less sample loading method, the challenge to seal an array of reactors without the use of microvalves or mechanical parts persists. Implementation of microvalve array adds complexity to the chip fabrication and operation processes, and reduces the space on the microchip. The current point-of-care, PCR-based biochips are limited by its capability to test multiple samples or different gene targets. In this doctoral work, a high-throughput, primer pair pre-loaded quantitative PCR chip platform for parallel analyses of multiple gene targets is developed. The PCR mixture distribution among an array of microreactors and subsequent isolation of the reactors were solely realized by a two-step surface tension-based microfluidic scheme, which eliminates the use of pumps, valves and liquid handling instruments. Sample loading and reactor sealing is achieved by implementing an unbalanced Young’s force achieved by fabricating poly(dimethylsiloxane) (PDMS) and glass surface’s with different wettability. The reactor-to-reactor diffusion of the pre-loaded primer pairs is investigated to eliminate the possibility of primer cross-contamination. Key technical issues such as primer pair degradation due to chip bonding conditions and bubble generation due to different PDMS-glass bonding methods are investigated. The capability of the developed PCR array chip is demonstrated by simultaneous (parallel) amplification of twelve different gene targets against cDNA template for risk prediction of human hepatocellular carcinoma and simultaneous detection of multiple water-borne pathogens. In addition, a microfluidic device harboring an array of open/unsealed reactors was developed for isothermal amplification and detection of SARS cDNA template. Another version of high-throughput PCR array chip was developed as PDMS matrix chip. Sample loading and microwell sealing in this chip was achieved by vacuum and positive-pressure based loading of liquid PDMS prepolymer. Finally, the PDMS matrix chip was applied to solve an important clinical application. A microfluidic digital PCR was developed on the PDMS matrix chip for absolute quantification of live Methicillin-Resistant Staphylococcus aureus (MRSA) in mixed bacterial samples. DOCTOR OF PHILOSOPHY (MAE) 2012-05-21T03:51:57Z 2012-05-21T03:51:57Z 2011 2011 Thesis Naveen, R. (2011). Microfluidic array chip for highly parallel genomic and transcriptomic applications. Doctoral thesis, Nanyang Technological University, Singapore. https://hdl.handle.net/10356/49499 10.32657/10356/49499 en 226 p. application/pdf
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
content_provider NTU Library
collection DR-NTU
language English
topic DRNTU::Engineering::Mechanical engineering
spellingShingle DRNTU::Engineering::Mechanical engineering
Naveen Ramalingam
Microfluidic array chip for highly parallel genomic and transcriptomic applications
description The Polymerase Chain Reaction (PCR) is an enzyme-based technique to amplify a single or few copies of Deoxyribose Nucleic Acid (DNA) in an exponential process, generating millions of copies of a known DNA sequence. The technique relies on thermal cycling; consisting of repeated cycles of heating and cooling of a reaction for DNA melting, target identification and enzymatic replication of the target DNA. The PCR technique is widely used for diagnosis, prognosis of diseases and enumeration of pathogen loads. Conventional PCR instruments are bulky, limiting its use in the field, but rapid development of miniaturized total analysis systems (μTAS) enables commercialization of point-of-care devices. In resource-limited settings, it is impractical to get access to a diagnostic laboratory having sophisticated instruments and in such settings, it is desirable to use disposable point-of-care high-throughput diagnostic chips that do not require liquid handling or pumping instruments for sample distribution among an array of reactors. In addition to the pump-less sample loading method, the challenge to seal an array of reactors without the use of microvalves or mechanical parts persists. Implementation of microvalve array adds complexity to the chip fabrication and operation processes, and reduces the space on the microchip. The current point-of-care, PCR-based biochips are limited by its capability to test multiple samples or different gene targets. In this doctoral work, a high-throughput, primer pair pre-loaded quantitative PCR chip platform for parallel analyses of multiple gene targets is developed. The PCR mixture distribution among an array of microreactors and subsequent isolation of the reactors were solely realized by a two-step surface tension-based microfluidic scheme, which eliminates the use of pumps, valves and liquid handling instruments. Sample loading and reactor sealing is achieved by implementing an unbalanced Young’s force achieved by fabricating poly(dimethylsiloxane) (PDMS) and glass surface’s with different wettability. The reactor-to-reactor diffusion of the pre-loaded primer pairs is investigated to eliminate the possibility of primer cross-contamination. Key technical issues such as primer pair degradation due to chip bonding conditions and bubble generation due to different PDMS-glass bonding methods are investigated. The capability of the developed PCR array chip is demonstrated by simultaneous (parallel) amplification of twelve different gene targets against cDNA template for risk prediction of human hepatocellular carcinoma and simultaneous detection of multiple water-borne pathogens. In addition, a microfluidic device harboring an array of open/unsealed reactors was developed for isothermal amplification and detection of SARS cDNA template. Another version of high-throughput PCR array chip was developed as PDMS matrix chip. Sample loading and microwell sealing in this chip was achieved by vacuum and positive-pressure based loading of liquid PDMS prepolymer. Finally, the PDMS matrix chip was applied to solve an important clinical application. A microfluidic digital PCR was developed on the PDMS matrix chip for absolute quantification of live Methicillin-Resistant Staphylococcus aureus (MRSA) in mixed bacterial samples.
author2 Gong Haiqing, Thomas
author_facet Gong Haiqing, Thomas
Naveen Ramalingam
format Theses and Dissertations
author Naveen Ramalingam
author_sort Naveen Ramalingam
title Microfluidic array chip for highly parallel genomic and transcriptomic applications
title_short Microfluidic array chip for highly parallel genomic and transcriptomic applications
title_full Microfluidic array chip for highly parallel genomic and transcriptomic applications
title_fullStr Microfluidic array chip for highly parallel genomic and transcriptomic applications
title_full_unstemmed Microfluidic array chip for highly parallel genomic and transcriptomic applications
title_sort microfluidic array chip for highly parallel genomic and transcriptomic applications
publishDate 2012
url https://hdl.handle.net/10356/49499
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