Fluorescence-doped silica nanoparticles for ultrasensitive detection of prostate specific antigen
- Authors: Msutu, Tumelo
- Date: 2024-10-11
- Subjects: Nanoparticles , Ferric oxide , Prostate-specific antigen , Prostate Cancer Diagnosis , Fluorescence microscopy
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/464543 , vital:76520
- Description: Prostate cancer (PCa) is the topmost diagnosed cancer in males in South Africa according to the National Cancer Registry 2020, affecting both adolescent and young adult men. Currently, PCa is diagnosed using the dreaded digital rectal examination (DRE) followed by tissue biopsy, and prostate-specific antigen (PSA) test for confirmation. DRE is an invasive method and can detect PCa in late stages when the tumour has formed. Therefore, there is a need for early diagnosis technology, that can detect PCa during carcinogenesis. PSA test offers this opportunity even though it is non-specific. This test is a non-invasive, well-established, and conventional method used as a confirmatory test. However, the test suffers from shortfalls like the misdiagnosis of benign prostatic hyperplasia (BPH) or prostatitis as prostate cancer. In this work, we have developed an ultrasensitive fluorescence-based PSA test based on the oriented immobilization of anti-PSA monoclonal and polyclonal antibodies. Anti-PSA polyclonal antibodies (anti-PSA-pAb) were bioconjugated via boronate ester onto the fluorescein-5-isothiocyanate (FITC) doped silica nanoparticles for detection. Anti-PSA monoclonal antibodies were used as capture antibodies on dark fluorescence microplate or magnetic nanoparticles. A sandwich immunoassay was used for the detection of PSA. The anti-PSA-pAb was bioconjugated on the surface of the FITC-doped silica nanoparticles. In the design, the FITC-(3-aminopropyl)triethoxysilane (APTES) organosilane precursor was encapsulated into the silica nanoparticles to form fluorescent silica nanoparticles (FITC@SiO2NPs). The silica shell prevented dye leakage and promoted fluorescent signal amplification. Optimization of the as-prepared fluorescent silica nanoparticles was investigated at altered FITC-dye loadings of 3.0% and 6.0% (for high loading). The fluorescent silica nanobioconjugates exhibited a strong excitation-dependent emission property at 518 nm and was photostable over time. The fluorescent silica nanoparticles exhibited 65% dye loading for both 3.0% and 6.0% silica nanoparticles respectively for 30 ± 3 nm and 46 ± 2 nm. The fluorescent nanobioconjugates’ performance was evaluated using black 96-well microplates. A fluorescence sandwich-type immunoreaction was achieved via the antibody-antigen reaction of the FITC@SiO2-prAmPBA-anti-PSA-pAb/glucose nanobioconjugates binding to the captured PSA analyte. NaOH was used for alkali hydrolysis of the sensing nanobioconjugates to release the FITC-APTES molecules, leading to fluorescence signal amplification. The proposed fluorescence immunobiosensor exhibited a linear relationship between the fluorescent signals and the concentration of PSA obtained in the range of 2.0 pg.mL-1 to 50 ng.mL-1, with excellent limit of detection and limit of quantification both in the pg.mL-1 range in PBS buffer (pH 7.4) and PSA in spiked serum samples. The immunosensor was based on the immunometric sandwich protocol, using nanomagnetic-silica antibody bioconjugates for capture and fluorescent nanobioconjugates as sensing probes. The use of nanomagnetic-silica antibody bioconjugates allowed concentration of PSA as the analyte and ease of separation using a magnet. Alkali hydrolysis of the sensing fluorescent nanobioconjugates is achieved using NaOH, resulting in the release of FITC molecules and an amplified fluorescence detection signal. The analytical performance of the proposed fluorescence immunobiosensor exhibited a linear relationship between the fluorescent signals and the concentration of PSA obtained in the range of 2.0 pg.mL-1 to 100 ng.mL-1, with excellent limit of detection and limit of quantification both in the pg.mL-1 range in PBS buffer (pH 7.4) and PSA in spiked serum samples. The immunosensor also exhibited good specificity and selectivity for PSA with 94.8% - 102.5% recovery rates. The proposed fluorescence immunoassay exhibited high selectivity and specificity for the detection of PSA. , Thesis (MSc) -- Faculty of Science, Chemistry, 2024
- Full Text:
- Date Issued: 2024-10-11
- Authors: Msutu, Tumelo
- Date: 2024-10-11
- Subjects: Nanoparticles , Ferric oxide , Prostate-specific antigen , Prostate Cancer Diagnosis , Fluorescence microscopy
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/464543 , vital:76520
- Description: Prostate cancer (PCa) is the topmost diagnosed cancer in males in South Africa according to the National Cancer Registry 2020, affecting both adolescent and young adult men. Currently, PCa is diagnosed using the dreaded digital rectal examination (DRE) followed by tissue biopsy, and prostate-specific antigen (PSA) test for confirmation. DRE is an invasive method and can detect PCa in late stages when the tumour has formed. Therefore, there is a need for early diagnosis technology, that can detect PCa during carcinogenesis. PSA test offers this opportunity even though it is non-specific. This test is a non-invasive, well-established, and conventional method used as a confirmatory test. However, the test suffers from shortfalls like the misdiagnosis of benign prostatic hyperplasia (BPH) or prostatitis as prostate cancer. In this work, we have developed an ultrasensitive fluorescence-based PSA test based on the oriented immobilization of anti-PSA monoclonal and polyclonal antibodies. Anti-PSA polyclonal antibodies (anti-PSA-pAb) were bioconjugated via boronate ester onto the fluorescein-5-isothiocyanate (FITC) doped silica nanoparticles for detection. Anti-PSA monoclonal antibodies were used as capture antibodies on dark fluorescence microplate or magnetic nanoparticles. A sandwich immunoassay was used for the detection of PSA. The anti-PSA-pAb was bioconjugated on the surface of the FITC-doped silica nanoparticles. In the design, the FITC-(3-aminopropyl)triethoxysilane (APTES) organosilane precursor was encapsulated into the silica nanoparticles to form fluorescent silica nanoparticles (FITC@SiO2NPs). The silica shell prevented dye leakage and promoted fluorescent signal amplification. Optimization of the as-prepared fluorescent silica nanoparticles was investigated at altered FITC-dye loadings of 3.0% and 6.0% (for high loading). The fluorescent silica nanobioconjugates exhibited a strong excitation-dependent emission property at 518 nm and was photostable over time. The fluorescent silica nanoparticles exhibited 65% dye loading for both 3.0% and 6.0% silica nanoparticles respectively for 30 ± 3 nm and 46 ± 2 nm. The fluorescent nanobioconjugates’ performance was evaluated using black 96-well microplates. A fluorescence sandwich-type immunoreaction was achieved via the antibody-antigen reaction of the FITC@SiO2-prAmPBA-anti-PSA-pAb/glucose nanobioconjugates binding to the captured PSA analyte. NaOH was used for alkali hydrolysis of the sensing nanobioconjugates to release the FITC-APTES molecules, leading to fluorescence signal amplification. The proposed fluorescence immunobiosensor exhibited a linear relationship between the fluorescent signals and the concentration of PSA obtained in the range of 2.0 pg.mL-1 to 50 ng.mL-1, with excellent limit of detection and limit of quantification both in the pg.mL-1 range in PBS buffer (pH 7.4) and PSA in spiked serum samples. The immunosensor was based on the immunometric sandwich protocol, using nanomagnetic-silica antibody bioconjugates for capture and fluorescent nanobioconjugates as sensing probes. The use of nanomagnetic-silica antibody bioconjugates allowed concentration of PSA as the analyte and ease of separation using a magnet. Alkali hydrolysis of the sensing fluorescent nanobioconjugates is achieved using NaOH, resulting in the release of FITC molecules and an amplified fluorescence detection signal. The analytical performance of the proposed fluorescence immunobiosensor exhibited a linear relationship between the fluorescent signals and the concentration of PSA obtained in the range of 2.0 pg.mL-1 to 100 ng.mL-1, with excellent limit of detection and limit of quantification both in the pg.mL-1 range in PBS buffer (pH 7.4) and PSA in spiked serum samples. The immunosensor also exhibited good specificity and selectivity for PSA with 94.8% - 102.5% recovery rates. The proposed fluorescence immunoassay exhibited high selectivity and specificity for the detection of PSA. , Thesis (MSc) -- Faculty of Science, Chemistry, 2024
- Full Text:
- Date Issued: 2024-10-11
Aptamer-based biosensor for prostate specific antigen detection using cobalt phthalocyanine-exfoliated graphite composites
- Authors: Benise, Emihle
- Date: 2024-04-04
- Subjects: Aptamer , Exfoliated graphite nano-platelets , Phthalocyanines , Impedance spectroscopy , Prostate-specific antigen
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/434850 , vital:73110
- Description: The work focuses on the development of biosensors and their use for the detection of prostate specific antigen (PSA). Four cobalt phthalocyanines (CoPcs) complexes: (1) cobalt tetra pyridyloxy phthalocyanine, (2) cobalt tetra acetamidophenoxy phthalocyanine, (3) cobalt tris(acetamidophenoxy) mono benzoic acid phthalocyanine, and (4) cobalt tris(acetamidophenoxy) mono propionic acid phthalocyanine, an exfoliated graphite (EG), and aptamer are used to make probes for PSA detection. Each complex is π-π stacked onto the EG to form EG-CoPc(π-π) hybrid which was used to modify a glassy carbon electrode (GCE). EG and CoPc were also used to modify the GCE sequential (seq) with CoPc on top to give GCE-EG-CoPc(seq). For the detection PSA, PSA specific aptamer was either sequential added or covalently linked to complexes 3 and 4 on the modified electrodes and was only sequentially added onto complexes 1 and 2 modified electrodes. Electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) were the techniques used for the detection of PSA. The electrodes were found to be selective in bovine serum albumin, glucose and cysteine and stable when 50 DPV scans were run. Electrodes gave good % recovery when human serum was spiked with different PSA concentrations. , Thesis (MSc) -- Faculty of Science, Chemistry, 2024
- Full Text:
- Date Issued: 2024-04-04
- Authors: Benise, Emihle
- Date: 2024-04-04
- Subjects: Aptamer , Exfoliated graphite nano-platelets , Phthalocyanines , Impedance spectroscopy , Prostate-specific antigen
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/434850 , vital:73110
- Description: The work focuses on the development of biosensors and their use for the detection of prostate specific antigen (PSA). Four cobalt phthalocyanines (CoPcs) complexes: (1) cobalt tetra pyridyloxy phthalocyanine, (2) cobalt tetra acetamidophenoxy phthalocyanine, (3) cobalt tris(acetamidophenoxy) mono benzoic acid phthalocyanine, and (4) cobalt tris(acetamidophenoxy) mono propionic acid phthalocyanine, an exfoliated graphite (EG), and aptamer are used to make probes for PSA detection. Each complex is π-π stacked onto the EG to form EG-CoPc(π-π) hybrid which was used to modify a glassy carbon electrode (GCE). EG and CoPc were also used to modify the GCE sequential (seq) with CoPc on top to give GCE-EG-CoPc(seq). For the detection PSA, PSA specific aptamer was either sequential added or covalently linked to complexes 3 and 4 on the modified electrodes and was only sequentially added onto complexes 1 and 2 modified electrodes. Electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) were the techniques used for the detection of PSA. The electrodes were found to be selective in bovine serum albumin, glucose and cysteine and stable when 50 DPV scans were run. Electrodes gave good % recovery when human serum was spiked with different PSA concentrations. , Thesis (MSc) -- Faculty of Science, Chemistry, 2024
- Full Text:
- Date Issued: 2024-04-04
Development of biosensor systems for the detection of anti-cancer drugs and prostate cancer
- Authors: Mwanza, Daniel
- Date: 2022-10-14
- Subjects: Prostate Cancer , Cancer Treatment , Cancer Early detection , Biosensors , Methotrexate , Prostate-specific antigen
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365929 , vital:65803 , DOI 10.21504/10962/365929
- Description: Rapid, low-cost and accurate point-of-care analytical devices are highly required for the detection of cancer biomarkers for the early diagnosis and determination of anti-cancer drugs for monitoring of cancer-related diseases. This thesis focused on the development of biosensor systems for the detection of anti-cancer drug, methotrexate (MTX) and prostate cancer biomarker, prostate-specific antigen (PSA). Ultrasensitive electrochemical immunosensors were fabricated by covalent immobilization of polyclonal anti-MTX and monoclonal anti-PSA antibodies onto glassy carbon, screen-printed carbon and gold electrodes which were pre-modified with isophthalic acid (IPA) thin monolayer film. A methodology based on the steric hindrance by 1,3-substituted aryldiazonium salt was adopted to enable the electrografting of IPA thin monolayer film on electrode surfaces. The antibodies were immobilized onto the IPA thin monolayer film via carbodiimide chemistry to form a sensing or analyte capture surface. For the detection of MTX, the analytical performance of the non-Faradaic electrochemical impedance spectroscopy (EIS) immunosensor was validated using singular value decomposition (SVD). The non-Faradaic EIS detection of PSA was validated using Nyquist plots of total capacitance, complex capacitance calculated from the imaginary part of impedance, and the capacitance acquired from the circuit fitting. The detection of PSA was further studied using colorimetric sensing platform which was developed by forming a sandwich immunoassay. For the immunoassay detection, the anti-PSA captured monoclonal antibodies were immobilized onto the microwell plates. The sensing signal was obtained from bioconjugating the anti-PSA antibody polyclonal onto glucose-encapsulating nanoliposomes (GENLs-anti-PSA-pAb). The preparation of glucose -encapsulated nanoliposomes were evaluated for their potential to release glucose in a controlled manner using Triton-X 100 or acidic phosphate buffer saline (PBS, pH 5.0). The detection of PSA (in a sandwich manner) was correlated to the concentration of glucose quantified using horseradish peroxidase (HRP), Pd|PdO nanoparticles, and personal glucose meter. The immunosensors developed in this work exhibited high stability, selectivity, low detection limits, and a wide linear range which was suitable for screening of both PSA and MTX. , Thesis (PhD) -- Faculty of Science, Chemistry, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Mwanza, Daniel
- Date: 2022-10-14
- Subjects: Prostate Cancer , Cancer Treatment , Cancer Early detection , Biosensors , Methotrexate , Prostate-specific antigen
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365929 , vital:65803 , DOI 10.21504/10962/365929
- Description: Rapid, low-cost and accurate point-of-care analytical devices are highly required for the detection of cancer biomarkers for the early diagnosis and determination of anti-cancer drugs for monitoring of cancer-related diseases. This thesis focused on the development of biosensor systems for the detection of anti-cancer drug, methotrexate (MTX) and prostate cancer biomarker, prostate-specific antigen (PSA). Ultrasensitive electrochemical immunosensors were fabricated by covalent immobilization of polyclonal anti-MTX and monoclonal anti-PSA antibodies onto glassy carbon, screen-printed carbon and gold electrodes which were pre-modified with isophthalic acid (IPA) thin monolayer film. A methodology based on the steric hindrance by 1,3-substituted aryldiazonium salt was adopted to enable the electrografting of IPA thin monolayer film on electrode surfaces. The antibodies were immobilized onto the IPA thin monolayer film via carbodiimide chemistry to form a sensing or analyte capture surface. For the detection of MTX, the analytical performance of the non-Faradaic electrochemical impedance spectroscopy (EIS) immunosensor was validated using singular value decomposition (SVD). The non-Faradaic EIS detection of PSA was validated using Nyquist plots of total capacitance, complex capacitance calculated from the imaginary part of impedance, and the capacitance acquired from the circuit fitting. The detection of PSA was further studied using colorimetric sensing platform which was developed by forming a sandwich immunoassay. For the immunoassay detection, the anti-PSA captured monoclonal antibodies were immobilized onto the microwell plates. The sensing signal was obtained from bioconjugating the anti-PSA antibody polyclonal onto glucose-encapsulating nanoliposomes (GENLs-anti-PSA-pAb). The preparation of glucose -encapsulated nanoliposomes were evaluated for their potential to release glucose in a controlled manner using Triton-X 100 or acidic phosphate buffer saline (PBS, pH 5.0). The detection of PSA (in a sandwich manner) was correlated to the concentration of glucose quantified using horseradish peroxidase (HRP), Pd|PdO nanoparticles, and personal glucose meter. The immunosensors developed in this work exhibited high stability, selectivity, low detection limits, and a wide linear range which was suitable for screening of both PSA and MTX. , Thesis (PhD) -- Faculty of Science, Chemistry, 2022
- Full Text:
- Date Issued: 2022-10-14
The systematic assembly of prostate specific antigen electrochemical sensors based on asymmetric Co(II) phthalocyanines, graphitic quantum dots and an aptamer
- Authors: Nxele, Siphesihle Robin
- Date: 2022-04-08
- Subjects: Prostate-specific antigen , Electrochemical sensors , Phthalocyanines , Quantum dots , Co(II) phthalocyanines , Aptamer
- Language: English
- Type: Doctoral thesis , text
- Identifier: http://hdl.handle.net/10962/232893 , vital:50035 , DOI 10.21504/10962/232893
- Description: The need for low-cost, efficient and simple diagnostic tools has led to more research going into this subject, with the aim of making such medical devices more accessible where they are needed. This has led to more researchers developing point-of-care devices for this purpose worldwide, by sensor fabrication. This thesis focuses on electrochemical sensor development for the early diagnosis of prostate cancer. It is common knowledge that prostate cancer is one of the most prevalent carcinomas that have claimed lives due to late diagnosis where even the most invasive treatments have failed. For this reason, development of early detection devices that can even be used in the comfort of home is necessary and quite crucial. Electrochemical sensors have gained much attention due to their ease of fabrication, cost effectiveness, simplicity, ease of use and high efficiency. Using nanocomposites as modifiers has also become popular as they provide greater stability and improve detection limits when used together with biomolecules. With that said, the work reported herein has combined nanocomposites of graphenebased quantum dots, gold nanoparticles, phthalocyanines and an aptamer in order to fabricate aptasensors for the electrochemical detection of prostate cancer biomarker. The aptamer is specifically designed to bind to the biomarker, and the nanocomposites are expected to enhance current output thus lowering detection limits and increasing stability and efficiency. Reproducible results are also expected. Prior to the detection of the prostate cancer biomarker, the quantum dots-phthalocyanine nanohybrids were used to detect L-cysteine, which is an amino acid, in order to verify the synergistic effects as electrode modifiers that lead to the enhancement of current output. This increase in current output is then v exploited for the improvement of aptasensor functionality upon incorporation of the aptamer, for the detection of prostate specific antigen. The research in this thesis has been carried out with the intention of contributing to the world of medical research, more so because of the ever-increasing need for medical care to become accessible to all and not only to those who can afford expensive technologies and treatments. , Thesis (PhD) -- Faculty of Science, Chemistry, 2022
- Full Text:
- Date Issued: 2022-04-08
- Authors: Nxele, Siphesihle Robin
- Date: 2022-04-08
- Subjects: Prostate-specific antigen , Electrochemical sensors , Phthalocyanines , Quantum dots , Co(II) phthalocyanines , Aptamer
- Language: English
- Type: Doctoral thesis , text
- Identifier: http://hdl.handle.net/10962/232893 , vital:50035 , DOI 10.21504/10962/232893
- Description: The need for low-cost, efficient and simple diagnostic tools has led to more research going into this subject, with the aim of making such medical devices more accessible where they are needed. This has led to more researchers developing point-of-care devices for this purpose worldwide, by sensor fabrication. This thesis focuses on electrochemical sensor development for the early diagnosis of prostate cancer. It is common knowledge that prostate cancer is one of the most prevalent carcinomas that have claimed lives due to late diagnosis where even the most invasive treatments have failed. For this reason, development of early detection devices that can even be used in the comfort of home is necessary and quite crucial. Electrochemical sensors have gained much attention due to their ease of fabrication, cost effectiveness, simplicity, ease of use and high efficiency. Using nanocomposites as modifiers has also become popular as they provide greater stability and improve detection limits when used together with biomolecules. With that said, the work reported herein has combined nanocomposites of graphenebased quantum dots, gold nanoparticles, phthalocyanines and an aptamer in order to fabricate aptasensors for the electrochemical detection of prostate cancer biomarker. The aptamer is specifically designed to bind to the biomarker, and the nanocomposites are expected to enhance current output thus lowering detection limits and increasing stability and efficiency. Reproducible results are also expected. Prior to the detection of the prostate cancer biomarker, the quantum dots-phthalocyanine nanohybrids were used to detect L-cysteine, which is an amino acid, in order to verify the synergistic effects as electrode modifiers that lead to the enhancement of current output. This increase in current output is then v exploited for the improvement of aptasensor functionality upon incorporation of the aptamer, for the detection of prostate specific antigen. The research in this thesis has been carried out with the intention of contributing to the world of medical research, more so because of the ever-increasing need for medical care to become accessible to all and not only to those who can afford expensive technologies and treatments. , Thesis (PhD) -- Faculty of Science, Chemistry, 2022
- Full Text:
- Date Issued: 2022-04-08
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