Design of Immunobiosensors for Detection of Tumor-Associated Anti-P53 Autoantibodies: Method Development
- Authors: Adeniyi, Omotayo Kayode
- Date: 2020
- Subjects: Lungs Cancer Early detection , Autoantibodies , Biosensors , Immunochemistry , Biochemical markers , p53 antioncogene
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/162988 , vital:41002 , 10.21504/10962/162988
- Description: 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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- Date Issued: 2020
Design of immunosensor for the detection of C-reactive protein using oriented antibody immobilization
- Authors: Adesina, Abiola Olanike
- Date: 2020
- Subjects: Immunochemistry , Biosensors , C-reactive protein , Immunoassay , Cardiovascular system Diseases
- 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: Early diagnosis of cardiovascular diseases (CVDs) has been a major challenge since CVDs are clinically silent. The current methods available for the diagnosis are not sensitive enough at the onset of the disease. Also, the use of sophisticated equipments and experts in the result interpretation has created a lot of barriers to the early diagnosis of CVDs. Biomarkers detection using electrochemical immunoassay offers great advantages in terms of sensitivity, miniaturization and low cost. This can be integrated into portable devices which can be made available in the remote areas for easy assessment of health care services. The fabrication of piezoelectric and electrochemical immunosensors for the detection of C-reactive protein (a cardiac biomarker) are presented in this thesis. The electrochemical immunosensor investigates the effect of linkers chain length on the analytical performance of the immunosensor. The fabricated immunosensors were based on two simple and sensitive label-free impedimetric assay. Oriented immobilization of anti-CRP monoclonal antibody (mAb) unto gold surface was achieved using carbohydrate specific boronic ester reaction for enhanced capture and specific detection of CRP protein. Quartz crystal microbalance with dissipation (QCM-D) was employed to establish the immunocomplex formation between the mAb and CRP antigen. This was achieved by forming a self-assembled monolayer (SAM) of 4-mercaptophenylboronic acid (MPBA) onto the quartz crystal surface. The limit of detection (LoD) for the direct and sandwich immunoassay was 5.45 and 3.65 ng mL-1, respectively. The Au-MPBA-mAb/glucose immunosensor with the shortest chain length of boronic acid was fabricated. The use of SAM of 4-mercaptophenylboronic acid (MPBA) afforded a thio phenylboronic acid functionalized gold surface (Au-MPBA SAM). The anti-CRP-mAb capture antibody was immobilized in an oriented manner onto gold thiophenylboronic acid to yield an Au-MPBA-mAb surface. The non-specific boronic surface was blocked using glucose to yield an Au-MPBA-mAb/glucose. The modified gold surface could detect CRP antigen. The limit of detection (LoD) was found to be 9.82 and 6.23 ng mL-1 for the direct and sandwich immunoassay; respectively. The Au-MBA-APBA-mAb/glucose immunosensor was designed by forming a SAM of 4-mercaptobenzoic acid (MBA) onto gold electrode surface. The terminal -COOH group of the MBA SAM reacted with an amino (NH2) group the 4-aminophenylboronic acid (APBA) using EDC/NHS coupling. This was followed by the immobilization of the capture antibody and the blocking of non-specific binding sites using glucose. Improved analytical parameters were obtained with LoD for the direct and sandwich immunoassays found to be 2.90 and 1.20 ng mL-1; respectively. A more stable immunosensor utilizing electrochemical grafting was investigated for the fabrication of Au-PEA-SA-APBA-mAb/glucose. The enhanced stability of the immunosensor was through the electrochemical reduction of 4-aminoethyl benzene diazonium (AEBD) salt. The surface was further derivatized with succinic anhydride to have a carboxylic derivatized surface. Carbodiimide chemistry was used to form a covalent linkage between the APBA amine group and the surface -COOH terminal group to yield an Au-PEA-SA-APBA surface. The immobilization of mAb and glucose resulted in Au-PEA-SA-APBA-mAb/glucose immunosensor. For the detection and signal enhancement, the magnetic nanoparticles conjugated with anti-CRP polyclonal antibody (pAb) was prepared. The sandwich immunoassay was used to detect CRP by the first capture at Au-PEA-SA-APBA-mAb/glucose. This was followed by signal amplification using magnetic nanoparticles coated with a silica shell and conjugated to pAb (MNP-SiO-APTES-PBA-pAb/glucose). The limit of detection was found to be 560 pg mL-1 and much lower than sandwich immunosensor fabricated using SAMS. The signal enhancement, lower detection limits and high sensitivity were obtained due to the nanoparticles for the sandwich immunoassay. The linear range for all the fabricated immunosensor ranges from 10 – 100 ng mL-1. The sensitivity obtained for Au-MPBA-mAb/glucose, Au-MBA-APBA-mAb/glucose, and Au-PEA-SA-APBA-mAb/glucose were 0.691, 0.885, and 11.08 kΩ.ng-1.ml.cm-2 for the sandwich immunoassay. The piezoelectric immunosensor was regenerated using 0.1 M HCl without affecting the immobilized capture antibody. The real sample analysis was carried out in 10 % serum in a recovery study for all the fabricated immunosensor. The percentage of recovery was very close to 100 %. , Thesis (PhD)--Rhodes University, Science Faculty, Department of Chemistry, 2020.
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- Date Issued: 2020