Attachment of Oligonucleotide Probes to Polymer Biochips and Its Application for the Detection of Point Mutations
This dissertation is on the fabrication of polymer-based microfluidic arrays for the detection of genetic mutations. Poly(methyl methacrylate) was chosen as one of the polymer substrate materials due to its low background noise, low adsorption of biomolecules, and low assembly temperature. The surface modification of polymer substrates for covalent attachment of oligonucleotide probes, the construction of fluidic channels/arrays, and hybridization kinetics will be covered. As an example of the application, point mutation detection using immobilized arrays constructed in microfluidic devices will be demonstrated.
The PMMA surface was derivatized with N-lithioethylenediamine solution to introduce amine groups, which were utilized for the covalent immobilization of terminal amino modified oligonucleotide probes via a homo-bi-functional linker molecule. The coupling bonds formed were stable enough to withstand multiple denaturation/rehybridization cycles. To overcome the drawbacks associated with conventional 2-D flat microarrays, such as long hybridization times and large sample consumption, oligonucleotide arrays were constructed into the microfluidic channels hot embossed into PMMA substrate. With the use of these fluidic channels we observed increased hybridization kinetics as compared to that on the flat arrays. Another benefit is that the channel-attached oligonucleotide probes allow the detection of target concentrations down to pM levels. As such, the specially designed oligonucleotide probes, which have similar melting temperatures, were constructed in microfluidic channels. Low-abundance point mutations in K-ras genes were successfully detected by using a ligase detection reaction (LDR) combined with the microfluidic hybridization. Near-IR laser induced fluorescence technique was used for the detection of surface conducted bioanalytical reactions and high detection sensitivity was obtained.
In addition, preliminary work was also conducted on direct photo-patterning of deep ultraviolet (UV) light for immobilizing oligonucleotides on poly(methyl methacrylate) and polycarbonate substrates. Deep UV patterning using a through-hole mask indicated that more oligonucleotide molecules were immobilized on the UV-exposed areas than the non-exposed area in the presence of EDC conjugating reagent. However, the proper dose of UV-exposure and the appropriate EDC concentration need to be optimized in future work to increase the contrast on the immobilization efficiency between the exposed regions and un-exposed regions.
Advisor:Steven A. Soper; Arthur L. Penn; Rober P. Hammer; Robin L. McCarley; Andrew W. Maverick
School:Louisiana State University in Shreveport
School Location:USA - Louisiana
Source Type:Master's Thesis
Date of Publication:01/16/2004