Design, Construction and Application of Nanostructured Electrochemical Biosensor Based on Aptamers for Diagnosis Some of the Pathogenic Bacteria

Ranjbar, Saba | 2019

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  1. Type of Document: Ph.D. Dissertation
  2. Language: Farsi
  3. Document No: 52328 (03)
  4. University: Sharif University of Technology
  5. Department: Chemistry
  6. Advisor(s): Shahrokhian, Saeed
  7. Abstract:
  8. Since that pathogenic bacteria have a predominant role in different aspects of human life such as clinical analysis and spread of contagious disease, food quality control and monitoring of environmental microbial infections, the aim of these studies is design and construction of electrochemical and electro-optical biosensors based on nanomaterials employing specific aptamer and antibody for detection of different bacteria in various real samples. An impedimetric biosensor based on nanoporous gold (NPG) was presented in the first part of the thesis for detection of Salmonella typhimurium as one of the main food- borne pathogenic bacterium. Three-dimensional structure of NPG was synthesized electrochemically by selective removal of Cu atoms from Au-Cu film in H2SO4 media and rearrangement of gold atoms. Due to large surface area and high binding energy, NPG used as an effective platform to immobilize thiolated specific salmonella aptamer via self-assemble monolayers (SAMs) formation. Electrochemical impedance spectroscopy was conducted to translate aptamers- bacteria interactions and detection of bacteria based on increasing steric hindrance. The purposed aptasensor is capable to detect salmonella in egg as a food sample in a wide linear dynamic range 6.5×102 to 6.5×108 CFU mL−1. In the second part, according to high electrical conductivity, high porosity and large surface area, a new organic-inorganic nanocomposite based on gold nanoparticles/carbon nanoparticles (AuNPs/CNPs) was synthesized to immobilize staphylococcus aureus specific aptamer with thiol functional group via SAMs. To better adhesion of AuNPs/CNPs layer at the surface of the electrode and improve the stability of aptasensor, AuNPs/CNPs were prepared in cellulose nanofiber (CNFs) suspension. The fabricated aptasensor based on AuNPs/CNPs/CNFs was used to detection of staphylococcus with electrochemical impedance spectroscopy in the range of 1.2×101 to 1.2×108 CFU mL−1with low LOD. Finally, the designed aptasensor was applied to the analysis of human blood serum samples with high precision and accuracy. In the third part, based on the importance of Escherichia coli O157:H7 in the quality of water resources, a novel aptasensor was developed using metal–organic frameworks (MOFs) as new generations of organic–inorganic hybrid composites. Despite of high porosity, effective surface area and thermal and chemical stabilities, MOFs suffer from low conductivity. To address this challenge, the film of polyaniline was used to cover the surface of MOF crystals and provide the conductive bridge between each crystal. The prepared polyaniline/MOFs were used as a modifier of glassy carbon electrode to fabricate Escherichia coli O157:H7 specific aptasensor via glutaraldehyde as a cross-linking agent to attachment of the amine-modified aptamer to the polyanilinated MOFs. Monitoring of Escherichia coli and quantitation of the interaction between the aptamer and bacteria was followed by signal-off strategy with differential pulse voltammetry technique using methylene blue as an electrochemical indicator. Changes in the reduction peak current of MB in the presence of Escherichia coli was recorded as an analytical signal and indicated a relationship with the logarithm of the bacteria concentration in the range of 2.1×101 to 2.1×107 CFU mL−1. The fabricated electrochemical aptasensor displayed good recovery values for the detection of Escherichia coli in different water resources. The fourth part of the thesis has been devoted to develop a reliable electrochemical biosensor based on aptamer immobilized in zeolitic imidazolate framework-8 (ZIFs-8) for detection of Pseudomonas aeruginosa. ZIF-8 crystals were chemically etched in tannic acid to synthesize hollow porous ZIFs-8 (HZIFs-8) with a thin shell containing carboxylic acid groups. The obtained HZIFs-8 was applied to immobilize specific Pseudomonas aptamer functionalized with amine groups via EDC-NHS chemistry. With respect to unique π−π interactions between aptamer and graphene oxide (GO), the differential pulse voltammetry technique was conducted with ferrocene-graphene oxide (Fc-GO) as an electroactive indicator for the detection of Pseudomonas. In the presence of bacteria, the configuration of the aptamer showed a change and Fc-GO was released from the electrode surface. On the basis of the signal-off strategy, the proposed biosensor exhibits a wide linear dynamic range (1.2×101 to 1.2×107 CFU mL−1) and the results reveal that the fabricated aptasensor has high potential applicability in the field of monitoring disease therapy and controlling the safety of the clinical sites. The last part of this thesis reports the results of design and fabrication a novel electro-optical immunosensor against Escherichia coli by considering the advantages of analytical capabilities of electrochemical sensors with simple optical read out. By electro-polymerization the thin film of polyaniline on the indium tin oxide screen-printed electrode (ITO SPE), not only the multicolor electrochromic behavior was observed but also, a stable platform with abundant amine groups obtained to modify the electrode surface with antibodies via glutaraldehyde as a cross-linking agent to visually detection of Escherichia coli. Applying a constant potential to the polyaniline- modified ITO SPE induces a change in the oxidation states, which in turn generates a color change on the electrode surface. The electrochromic behavior of polyaniline was stable in the absence of bacteria and the color of the electrode changed from yellow to purple. The presence of bacteria created the resistance related to antibody-antigen complex that disrupted the electrochromicity of polyaniline and blue, green and yellow color obtained for the low, medium and high concentration of Escherichia coli. The proposed strategy provided a smart device for monitoring the quality of water resources from bacteria contaminations in the range of 101 to 106 CFU mL−1with high precisions
  9. Keywords:
  10. Pathogenic Bacterias ; Electrochemical Biosensors ; Electrochromic Biosensors ; Aptamer ; Antibady ; Modified Electrodes ; Carbon/Metal/Polymer Nanostructures ; Metal-Organic Framework ; Electrochromic ; Differential Pulse Voltammetry

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