Design of Immunobiosensors for Detection of Tumor-Associated Anti-P53 Autoantibodies: Method Development
- Authors: Adeniyi, Omotayo Kayode
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/162988 , vital:41002 , 10.21504/10962/162988
- Description: Introduction -- Experimental -- Label-Free Impedimetric Sensing of Anti-P53ab... -- Fluorescent detection of Anti-P53ab -- Peroxidase-like activity of Fe3O4@SiNP-APTES-Au@Pd... -- Colorimetric detection of Anti-P53ab , Thesis (PhD)--Rhodes University, Science Faculty, Department of Chemistry, 2020. , Detection and profiling of circulating tumor-associated autoantibodies (TAAbs) are useful for screening and early-stage diagnosis of asymptomatic lung cancer. Immunobiosensor technologies aimed to accomplish the highly sensitive, rapid and low-cost detection of TAAbs can improve the early-stage detection of lung cancer. Immunobiosensors for the detection of anti-P53-tumour associated autoantibodies have been developed in this work. The design of sensing interfaces with immobilized P53 protein (P53ag) as a sensing element layer on a solid interface was investigated. Several methods of detecting anti-P53-antibodies (anti-P53ab) were investigated. These methods are label-free detection using electrochemical impedance spectroscopy (EIS) and two label techniques. The label-free electrochemical techniques utilize gold electrode pre-modified with a conducting layer of electrochemically grafted phenylethylamine for covalent immobilization of P53ag. The limit of anti-P53ab detection with the label-free EIS was 103.0 pg.ml-1. The labeled technique developed utilizes fluorescent, and peroxidase-like nanomaterial labeled antibody as a detection probe. For the fluorescence detection, fluorescent silica nanoparticles were synthesized by overloading FITC into the silica matrix and conjugated to detection antibody (anti-IgG). The detection of the anti-P53ab was based on the dissolution of the silica nanoparticles to release the loaded dye as a signal amplification strategy. The fluorescence detection was carried out on a microplate, and magnetic bead modified P53-antigen platforms and limit of detection (LoD) were 42.0 fg.ml-1 and 3.3 fg.ml-1 for anti-P53ab; respectively. Fe3O4@SiNP-APTES-Au@Pd hybrid nanoparticles were synthesized, and their peroxidase-like activity and colorimetric detection were evaluated. The Fe3O4@SiNP-APTES-Au@Pd exhibited comparable activity to HRP. The Fe3O4@SiNP-APTES-Au@Pd was conjugated to protein-G-anti-IgG for the detection of anti-P53ab on a microplate and cellulose paper platforms. The LoD was 20.0 fg.ml-1 and 63.0 fg.ml-1 for the microplate and cellulose paper platform; respectively. The potential application of the designed immunobiosensor was evaluated in simulated serum samples. The developed sensors showed higher detection sensitivity, stability and had a lower detection limit for anti-P53ab when compared with the ELISA based detection. The results have provided alternative and effective quantification approaches to ELISA and a promising future for multiplexed detection of tumor-associated autoantibodies. The developed methodologies in this thesis could be applied for the detection of other autoantibodies in other cancer types and auto-immune diseases.
- Full Text:
- Authors: Adeniyi, Omotayo Kayode
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/162988 , vital:41002 , 10.21504/10962/162988
- Description: Introduction -- Experimental -- Label-Free Impedimetric Sensing of Anti-P53ab... -- Fluorescent detection of Anti-P53ab -- Peroxidase-like activity of Fe3O4@SiNP-APTES-Au@Pd... -- Colorimetric detection of Anti-P53ab , Thesis (PhD)--Rhodes University, Science Faculty, Department of Chemistry, 2020. , Detection and profiling of circulating tumor-associated autoantibodies (TAAbs) are useful for screening and early-stage diagnosis of asymptomatic lung cancer. Immunobiosensor technologies aimed to accomplish the highly sensitive, rapid and low-cost detection of TAAbs can improve the early-stage detection of lung cancer. Immunobiosensors for the detection of anti-P53-tumour associated autoantibodies have been developed in this work. The design of sensing interfaces with immobilized P53 protein (P53ag) as a sensing element layer on a solid interface was investigated. Several methods of detecting anti-P53-antibodies (anti-P53ab) were investigated. These methods are label-free detection using electrochemical impedance spectroscopy (EIS) and two label techniques. The label-free electrochemical techniques utilize gold electrode pre-modified with a conducting layer of electrochemically grafted phenylethylamine for covalent immobilization of P53ag. The limit of anti-P53ab detection with the label-free EIS was 103.0 pg.ml-1. The labeled technique developed utilizes fluorescent, and peroxidase-like nanomaterial labeled antibody as a detection probe. For the fluorescence detection, fluorescent silica nanoparticles were synthesized by overloading FITC into the silica matrix and conjugated to detection antibody (anti-IgG). The detection of the anti-P53ab was based on the dissolution of the silica nanoparticles to release the loaded dye as a signal amplification strategy. The fluorescence detection was carried out on a microplate, and magnetic bead modified P53-antigen platforms and limit of detection (LoD) were 42.0 fg.ml-1 and 3.3 fg.ml-1 for anti-P53ab; respectively. Fe3O4@SiNP-APTES-Au@Pd hybrid nanoparticles were synthesized, and their peroxidase-like activity and colorimetric detection were evaluated. The Fe3O4@SiNP-APTES-Au@Pd exhibited comparable activity to HRP. The Fe3O4@SiNP-APTES-Au@Pd was conjugated to protein-G-anti-IgG for the detection of anti-P53ab on a microplate and cellulose paper platforms. The LoD was 20.0 fg.ml-1 and 63.0 fg.ml-1 for the microplate and cellulose paper platform; respectively. The potential application of the designed immunobiosensor was evaluated in simulated serum samples. The developed sensors showed higher detection sensitivity, stability and had a lower detection limit for anti-P53ab when compared with the ELISA based detection. The results have provided alternative and effective quantification approaches to ELISA and a promising future for multiplexed detection of tumor-associated autoantibodies. The developed methodologies in this thesis could be applied for the detection of other autoantibodies in other cancer types and auto-immune diseases.
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Design of immunosensor for the detection of C-reactive protein using oriented antibody immobilization
- Authors: Adesina, Abiola Olanike
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/163080 , vital:41010 , https://dx.doi.org/10.21504/10962/163080
- Description: Thesis (PhD)--Rhodes University, Science Faculty, Department of Chemistry, 2020.
- Full Text:
- Authors: Adesina, Abiola Olanike
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/163080 , vital:41010 , https://dx.doi.org/10.21504/10962/163080
- Description: Thesis (PhD)--Rhodes University, Science Faculty, Department of Chemistry, 2020.
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Evaluating the potential of monometallic and bimetallic nanomaterials as horseradish peroxidase mimetics
- Authors: Mvango, Sindisiwe
- Date: 2017
- Subjects: Uncatalogued
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/65134 , vital:28694
- Description: This study presents the synthesis of citrate-capped gold nanoparticles (cit-AuNPs), copper oxide nanoparticles (CuONPs), glutathione-capped gold nanoparticles (GSH-AuNPs), 4- aminothiophenol-capped gold nanoparticles (4-ATP-AuNPs), 4-mercapto benzoic acid- capped gold nanoparticles (4-MBA-AuNPs) and copper oxide gold nanoalloys (CuO-Au nanoalloys). Microscopy and spectroscopy techniques were used to confirm the successful synthesis of these nanoparticles. The synthesized nanoparticles were studied their potential applications as horseradish peroxidase (HPR) enzyme mimetics and for the detection of glucose. The cit-AuNPs and GSH-AuNPs exhibited peroxidase-like activity towards hydrogen peroxide (H2O2) with high Michaelis-Menten (Km) values of 61.5 mM and 30.8 mM, respectively. The other nanoparticles, that is, 4-ATP-AuNPs, CuONPs and CuO-Au nanoalloys gave lower Km values of 4.74 mM, 1.92 mM and 4.05 mM, respectively. The obtained Km values were comparable to those of HRP enzymes which ranged from 0.214 - 3.70 mM with 4-ATP-AuNPs and CuO-Au nanoalloys slightly higher. These values were within the reasonable experimental values of the HRP enzyme. The studies showed that the gold nanoparticles had low adsorptive efficiency towards H2O2 compared to the copper-based nanoparticles (CuONPs and CuO-Au nanoalloys). The CuO-Au nanoalloys also showed the synergistic effect between the gold and copper nanoparticles with extended linear concentration range for the quantification of H2O2. The mechanism of catalysis was confirmed using UV-vis spectroscopy and electron paramagnetic resonance (EPR) in that the generation of reactive oxygen species was observed. The use of 1,3-diphenylisobenzofuran (DPBF) as radical quencher and 5,5- dimethyl-1-pyrroline N-oxide (DMPO) as a radical scavenger confirmed the production of reductive reactive oxygen species using UV-vis and EPR studies. The rate of production of reactive oxygen species in the gold-based nanoparticles was small compared to the copper-based nanoparticles, that is CuONPs and CuO-Au (bimetallic) nanoalloys. The synthesized nanoparticles were further studied their potential use in the colorimetric detection of glucose. The copper-based nanomaterials, CuONPs and CuO-Au nanoalloys, were excellent towards detection of glucose, with a limit of detection (LoD) of 9.34 pM for CuONPs and 6.75 pM for CuO-Au nanoalloys. The linear concentration (LCR) range of CuONPs was 0 to 70 pM and for CuO-Au nanoalloys the LCR was 0.0 - 30 pM. , Thesis (MSc) -- Faculty of Science, Chemistry, 2017
- Full Text:
- Authors: Mvango, Sindisiwe
- Date: 2017
- Subjects: Uncatalogued
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/65134 , vital:28694
- Description: This study presents the synthesis of citrate-capped gold nanoparticles (cit-AuNPs), copper oxide nanoparticles (CuONPs), glutathione-capped gold nanoparticles (GSH-AuNPs), 4- aminothiophenol-capped gold nanoparticles (4-ATP-AuNPs), 4-mercapto benzoic acid- capped gold nanoparticles (4-MBA-AuNPs) and copper oxide gold nanoalloys (CuO-Au nanoalloys). Microscopy and spectroscopy techniques were used to confirm the successful synthesis of these nanoparticles. The synthesized nanoparticles were studied their potential applications as horseradish peroxidase (HPR) enzyme mimetics and for the detection of glucose. The cit-AuNPs and GSH-AuNPs exhibited peroxidase-like activity towards hydrogen peroxide (H2O2) with high Michaelis-Menten (Km) values of 61.5 mM and 30.8 mM, respectively. The other nanoparticles, that is, 4-ATP-AuNPs, CuONPs and CuO-Au nanoalloys gave lower Km values of 4.74 mM, 1.92 mM and 4.05 mM, respectively. The obtained Km values were comparable to those of HRP enzymes which ranged from 0.214 - 3.70 mM with 4-ATP-AuNPs and CuO-Au nanoalloys slightly higher. These values were within the reasonable experimental values of the HRP enzyme. The studies showed that the gold nanoparticles had low adsorptive efficiency towards H2O2 compared to the copper-based nanoparticles (CuONPs and CuO-Au nanoalloys). The CuO-Au nanoalloys also showed the synergistic effect between the gold and copper nanoparticles with extended linear concentration range for the quantification of H2O2. The mechanism of catalysis was confirmed using UV-vis spectroscopy and electron paramagnetic resonance (EPR) in that the generation of reactive oxygen species was observed. The use of 1,3-diphenylisobenzofuran (DPBF) as radical quencher and 5,5- dimethyl-1-pyrroline N-oxide (DMPO) as a radical scavenger confirmed the production of reductive reactive oxygen species using UV-vis and EPR studies. The rate of production of reactive oxygen species in the gold-based nanoparticles was small compared to the copper-based nanoparticles, that is CuONPs and CuO-Au (bimetallic) nanoalloys. The synthesized nanoparticles were further studied their potential use in the colorimetric detection of glucose. The copper-based nanomaterials, CuONPs and CuO-Au nanoalloys, were excellent towards detection of glucose, with a limit of detection (LoD) of 9.34 pM for CuONPs and 6.75 pM for CuO-Au nanoalloys. The linear concentration (LCR) range of CuONPs was 0 to 70 pM and for CuO-Au nanoalloys the LCR was 0.0 - 30 pM. , Thesis (MSc) -- Faculty of Science, Chemistry, 2017
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