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Designing and Fabrication of Microfluidic Biosensor by DNA-Directed Immobilization

Esmaeili, Elaheh | 2017

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  1. Type of Document: Ph.D. Dissertation
  2. Language: Farsi
  3. Document No: 49564 (46)
  4. University: Sharif University of Technology
  5. Department: Institute for Nanoscience and Nanotechnology
  6. Advisor(s): Vossoughi, Manouchehr; Soleimani, Masoud; Shamloo, Amir
  7. Abstract:
  8. The present study is aimed at the development of a novel approach based on the magnetically improvement of DNA-directed immobilization to prepare a highly efficient sensor for prostate diagnosis. The novelty of this work is in the use of antibody conjugated magnetic nanoparticles via DDI. DNA-modified magnetic nanoparticles are added in solution to capture DNA-conjugated, fluorescently-labeled immunocomplexes formed in solution free of steric constraints. The DDI-based nanoconstructs are then concentrated and immobilized using a magnetic field. Compared to a process in which the immunocomplex directly forms on the sensing surface, the proposed approach provides higher mass transfer and lower equilibration time. At first, the binding capacity of antibody based on the magnetically enhanced DNA-directed immobilization on the poly-l-lysine coated surface has been studied. To compare, similar experiments was carried out lacking MNPs in the diffusion-limited regime, on the PLL and plasma treated surfaces. PLL coated surface is advantageous over the ammonia plasma treated surface as it leads to a three-fold increase in the absorbance, which reflects a more efficient surface immobilization of the antibody. This aspect can reflect higher ssDNA loading onto the PLL-coated surface compared to ammonia plasma-treated surface. Also, the utilization of MNPs prompts immobilization efficiency of the anti-PSMA-containing constructs considerably higher than (two-fold) in the corresponding, diffusion-limited analogs. Thereafter, the binding capacity of the immunocomplex based on the magnetically enhanced DNA-directed immobilization on the streptavidin surface has been studied. We used two complementary approaches to capture protein biomarkers in solution, to permit detection at high sensitivity. Both are based on DDI and primarily differ in the type of biomolecular recognition that is responsible for surface immobilization. In one DDI approach, hybridization between two ssDNA molecules, one of which is immobilized on the sensing surface, and the other is incorporated in the biomarker-containing molecular construct, has been exploited. In the second, surface immobilization involves the association between a biotin moiety (incorporated in the molecular construct) and streptavidin attached on the surface. Control experiments were included lacking MNPs (diffusion-limited regime). It is shown that immunocomplex immobilization is more efficient using MNPs compared to diffusion limited regime by a factor of 7 and 4 using the first and second approach respectively. In addition, the first approach is advantageous over the second as it leads to a two-fold increase in fluorescence intensity. The higher fluorescence intensity reflects a more efficient surface immobilization of the PSMA-containing construct. Then, the detection limit of our sensing approach using the first approach has been studied for PSMA antigen in the range 0.5-70 nM. The detection limit is 1 nM. The sensing area can be efficiently reused with a simple washing step which releases the immunocomplex from the surface. Finally, a hybrid magnetic-DNA directed immobilization approach is presented to diminish the usage of expansive immunoreaganets on a microfluidic platform. Finite element analysis was used to study the magnetic configuration design and improve it. A magnetic set-up composed of cubic permanent magnets around a microchannel was designed and implemented to efficiently concentrate the nanoparticles only over a defined area of the microchannel. This in turn, led to the fluorescence emission localization and the searching area reduction. Immunocomplexes are formed in the solution and concentrated on the specified region using a magnetic field. Different PSMA protein dilutions in the range of 0.7-20 nM have been evaluated. The set-up is capable of detecting prostate-specific membrane antigen with concentrations down to 0.7 nM. Our findings suggest that the proposed approach with proper specificity and sensitivity holds a great promise for applications in clinical assays and disease diagnosis
  9. Keywords:
  10. Fluorescence ; Prostate Cancer ; Microfluidic System ; DNA-directed Immobilization ; Magnetic Nanodot ; Immunoassay

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