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Design and Fabrication of Microfluidic Biosensors based on Modified Electrospun Nanofibrous Membranes for Biomarkers Detection

Mahmoudifard, Matin | 2017

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  1. Type of Document: Ph.D. Dissertation
  2. Language: Farsi
  3. Document No: 50181 (48)
  4. University: Sharif University of Technology
  5. Department: Institute for Nanoscience and Nanotechnology
  6. Advisor(s): Vosoughi, Manuchehr; Soleimani, Masoud; Soudi, Sara
  7. Abstract:
  8. The accurate assessment of biomarkers likes proteins, hormones or other biomolecules that amount of them changes with state of disease or level of treatment and gives diagnostic information is a difficult duty. In these samples, pathogens exist in small quantity compared to other background molecules. Therefore, appropriate sampling process for concentrating and separating pathogens from the matrix should be developed in order to provide rapid and accurate pathogen detection test. In this thesis design and fabrication of microfluidic biosensors based on the modified nanofibrous membranes were investigated and explained. PES nanofibrous membrane with carboxyl functional groups for covalent attachment of antibodies were treated by EDC/NHS coupling agent. The quantity of antibody immobilization was measured by enzyme-linked immuno sorbent assay (ELISA) method. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) spectroscopy was performed to confirm the covalent immobilization of antibody on membrane. Atomic force microscopy, scanning electron microscopy and invert fluorescence microscopy were used to analyze the antibody distribution pattern on solid surfacesIt was found that the use of nanofibrous membrane causes the improved detection signal of ELISA based biosensors in comparison to the standard assay carried out in the 96-well microtiter plate. This method has the potential to improve the ELISA-based biosensor and we believe that this technique can be used in various biosensing methods.For example, when antigen immobilized through EDC/NHS on the plasma- treated substrate, the designed indirect- based ELISA biosensor's limit of detection tend to decreased from 78 ng/ml (for physical immobilization) to 0.3 ng/ml. in addition, it was found that the use of EDC/NHS can also enhance protein immobilization and their stability on the substrates without carboxyl and hydroxyl functional groups. For example, results showed that the LOD of fabricated biosensor based on the indirect ELISA enjoy enhancement from 7 ng/ml (for physical immobilization of protein) to 1 ng/ml (for protein immobilization through EDC/NHS) on the non- plasma substrates. Therefore, in addition to conventional mechanism for protein immobilization through EDC/NHS, we proposed new mechanism in which we hypotheses that EDC/NHS can adsorb through physical forces on the all substrates and after that with their functional groups can bind to functional group of proteins and in this way causes oriented immobilization of proteins on the substrates and signal enhancement of designed biosensor. Results showed plasma duration, nanofibrous membrane thickness, nanofiber diameter, polymer type of nanofibrous membrane, molar ratio of EDC to NHS and incubation time for EDC/NHS solution have significant effect on protein quantity immobilized on the substrate. It was also observed, due to high surface area, the use of nanofibrous membrane in microfluidic systems, can enhance the capacity of protein immobilization more than 2 order of magnitude compared to conventional substrate of microfluidic devices. Due to the solution leakage through micro-channels, the use of plasma treated membrane in microfluidic systems were impossible. Thus, the use of PLGA and PLLA nanofibrous membrane which contain carboxyl and hydroxyl functional group in their chemical structure was proposed. Again, EDC/NHS mechanism was used to immobilize protein on the nanofibrous membranes and results confirmed that PLGA and PLLA are more efficient in protein immobilization.
    In the last part of this thesis, ammonia plasma technique was applied to create amine functional group on PAN nanofibrous membrane. Glutaraldehyde was used as cross-linker to immobilized protein on the PAN substrates covalently. Similar to above section, it was concluded that protein immobilization through GA was more efficient and stable.
  9. Keywords:
  10. Immunoassay ; Biosensor ; Nanofibrous Membrane ; Microfluidic System ; Nanofibrous Scaffold ; Biomarker

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