Microfluidic Devices for Performing Multiplexed Immunoassays and Nucleic Acid Tests Public Deposited

Downloadable Content

Download PDF
Last Modified
  • March 22, 2019
Creator
  • Oblath, Emily Anna
    • Affiliation: College of Arts and Sciences, Department of Chemistry
Abstract
  • This work describes the development of microfluidic devices to perform multiplexed immunoassays and nucleic acid tests for point-of-care (POC) diagnostics. Diagnostic testing is usually performed at centralized laboratories, imposing a significant delay in treatment. In contrast, POC testing is performed by the primary healthcare provider, and the results can be used to implement proper treatment immediately. Saliva can be an ideal sample for POC diagnostics since it is easily collected and contains many disease biomarkers. This work focuses on using saliva to monitor and diagnose pulmonary diseases. Cytokine biomarkers are measured as early indicators for asthma or cystic fibrosis, while DNA is characterized to identify bacteria that can cause respiratory infections. The first device described uses sandwich immunoassays to measure cytokines. The polydimethylsiloxane (PDMS)-glass hybrid chip integrated microfluidic channels with a 900-well microarray. The wells were loaded with antibody-functionalized microspheres from a random mixture. An encoding strategy was used for multiplexed assays so that microspheres functionalized with antibodies for one analyte could be distinguished from microspheres for another analyte. Optimization of the assay for reduced analysis time and a low limit of detection is described for two cytokines, VEGF and IL-8. The optimized assay required less than one hour, and the theoretical limits of detection were found to be well below physiological levels reported in the literature. A second device was developed to integrate DNA extraction and polymerase chain reaction (PCR) amplification for the detection of bacteria found in saliva. Sample extraction is a particularly challenging problem for POC, PCR-based diagnostics. In this device, DNA extraction was accomplished by filtering samples through an aluminum oxide membrane (AOM) integrated with a PDMS-glass channel structure. Parallel reaction wells located above the AOM were used to perform multiplexed analyses. Several designs and detection strategies were explored as the device was optimized. The final format incorporated 7 reaction wells and real-time detection with fluorescent probes or an intercalating dye. Detection of as little as 8-12 copies of purified template DNA was achieved. The successful identification of bacteria and spiked genomic DNA in saliva is also described.
Date of publication
Keyword
DOI
Resource type
Rights statement
  • In Copyright
Advisor
  • Ramsey, J. Michael
Degree
  • Doctor of Philosophy
Graduation year
  • 2012
Language
Publisher
Parents:

This work has no parents.

Items