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Selection and Characterization of ssDNA Aptamers for Salivary Peptide Histatin 3 and Their Application Towards Assay and Point-of-Care Biosensing.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Selection and Characterization of ssDNA Aptamers for Salivary Peptide Histatin 3 and Their Application Towards Assay and Point-of-Care Biosensing./
作者:	Ojha, Yagya Raj.
出版者:	Ann Arbor : ProQuest Dissertations & Theses, : 2019,
面頁冊數:	231 p.
附註:	Source: Dissertations Abstracts International, Volume: 82-05, Section: B.
Contained By:	Dissertations Abstracts International82-05B.
標題:	Nanotechnology. -
電子資源:	https://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=28274258
ISBN:	9798691218125

Selection and Characterization of ssDNA Aptamers for Salivary Peptide Histatin 3 and Their Application Towards Assay and Point-of-Care Biosensing.
Ojha, Yagya Raj.

Selection and Characterization of ssDNA Aptamers for Salivary Peptide Histatin 3 and Their Application Towards Assay and Point-of-Care Biosensing. - Ann Arbor : ProQuest Dissertations & Theses, 2019 - 231 p.

Source: Dissertations Abstracts International, Volume: 82-05, Section: B.

Thesis (Ph.D.)--The University of Toledo, 2019.

The development of detection methods for the novel biomarkers can have a significant impact on the research and clinical applications such as drug discovery, disease diagnosis, and treatment monitoring. Histatin 3 (H3) is an antimicrobial salivary peptide that possesses the capability of being a therapeutic agent against oral candidiasis and has recently been linked to acute stress as a potential novel biomarker. Stress biomarkers reflect the physical and cognitive performance of an individual, and their monitoring in real-time is of vital importance for the high-risk jobs, including military, pilot, and surgeon, where higher vigilance is required for an extended period. The salivary levels of H3 also have been correlated with the HIV-infection and associated oral candidiasis. Therefore, monitoring H3 levels in human saliva can provide essential information about an individual's health status, including HIV-infection, oral candidiasis, and acute stress. Additionally, H3 detection could serve as therapeutic drug monitoring if H3 can be established as an alternative therapeutic agent.The currently available detection techniques for H3 are gel chromatography, high-performance liquid chromatography (HPLC), mass spectrometry (MS), and antibody-based immunoassays. The Chromatographic and mass-spectroscopic methods are laborious, utilize expensive instrumentation, require trained personnel, and time-consuming. Whereas antibody-based immunoassays are not widely validated, expensive, sensitive to temperature, and have a short lifespan. This void in analytical methods is not just for H3 but also applies to several other biomarkers in saliva. Even though saliva is considered as an optimal biofluid, several limitations are impeding its use in diagnostic and research. The major hurdles include the deficient concentration of biomarkers, need of laboratory-based preprocessing to remove mucin and interfering particulate matters, and lack of standard sample collection methods. Also, due to the absence of easily accessible and cost-effective analytical methods, it is challenging to figure out the circadian variation of biomolecules and consequently establish biomolecules as biomarkers. All these necessities mandate the development of alternative techniques that offer more accessible, cost-effective, rapid, real-time, and probably the point-of-care (POC) testing of salivary biomarkers. The aptamers, synthetic oligonucleotides functionally similar to antibodies but with several advantages, have shown promising possibilities to fulfill the requirements in such applications. However, no aptamer for H3 and several other salivary biomarkers exist in literature. This dissertation presents a widely applicable aptamer selection method for the novel biomarkers and the application of the identified aptamers with various platforms for the detection application in human saliva. In order to identify H3 aptamers, a library immobilization version of an iterative in vitro process known as the systematic evolution of ligands by exponential enrichment (SELEX) was established. This method does not require target immobilization and offers the isolation of signaling aptamers for a target in its unmodified form. Also, the signaling, structure-switching upon target binding, is a highly desired attribute of the aptamers for the biosensing application. Within the SELEX process, counter-selection steps were instituted to stop the evolution of nonspecific sequences that can bind to the molecules closely related to the target. Through the repetitive rounds of selection and counter-selection, four unique aptamer candidates sharing a consensus sequence were identified. The obtained aptamers were analyzed via online servers to predict the stability and structural conformations. A superior candidate in terms of stability and structural complexity was picked for the visual confirmation of binding. The magnetic bead-based affinity capture combined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), confirmed the selective binding between the selected aptamer candidate and target H3, indicating the successful aptamer selection process.Following the preliminary studies, more detailed characterizations were performed by developing a direct format enzyme-linked aptamer sorbent assay (ELASA). The aptamers were compared for their binding affinity and specificity to the target molecule H3. Also, the effect of primer binding sites on the binding affinity was probed. Selected clones exhibited affinity and specificity to H3, and the sequence with the highest affinity exhibited enhanced binding following the removal of primer binding sites. Based on these results, the truncated aptamer sequence was chosen for further application with other assay platforms. Next, the signaling feature of the aptamer was employed to explore the prospect of gold nanoparticles (AuNPs)-based colorimetric assays. Two different formats of the colorimetric assays were developed. After optimizing various parameters, both assay schemes were able to detect H3 within a salivary level of a healthy individual. Besides, with the assay-format-2, the overall assay time was reduced to just 12 minutes. Furthermore, surface-enhanced Raman spectroscopy (SERS)-based magnetic aptasensor was developed for the ultrasensitive detection of H3. By optimizing various parameters, SERS-based atpasensor was established. Additionally, the lateral flow test strip to support POC measurements of H3 was demonstrated by utilizing the optical properties of the AuNPs and the specificity of the identified H3 aptamer. The captured AuNPs on the test line of the lateral flow device provided qualitative analysis within 5 minutes for visual observation. Finally, human saliva and the artificial saliva samples were tested with the developed assay formats to investigate the challenges and the complications associated with the saliva-based measurements.In conclusion, the work presented in this dissertation lays the foundation for the identification of the signaling aptamers and their subsequent implementation in several platforms for the detection of salivary biomarkers. Further improvements and optimizations of the assay methods will eventually lead to a fast, reliable, cost-effective, ultrasensitive, and POC detection methods for the salivary biomarkers.

ISBN: 9798691218125Subjects--Topical Terms:

526235
Nanotechnology.
Subjects--Index Terms:

Aptamer

Selection and Characterization of ssDNA Aptamers for Salivary Peptide Histatin 3 and Their Application Towards Assay and Point-of-Care Biosensing.
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Additionally, H3 detection could serve as therapeutic drug monitoring if H3 can be established as an alternative therapeutic agent.The currently available detection techniques for H3 are gel chromatography, high-performance liquid chromatography (HPLC), mass spectrometry (MS), and antibody-based immunoassays. The Chromatographic and mass-spectroscopic methods are laborious, utilize expensive instrumentation, require trained personnel, and time-consuming. Whereas antibody-based immunoassays are not widely validated, expensive, sensitive to temperature, and have a short lifespan. This void in analytical methods is not just for H3 but also applies to several other biomarkers in saliva. Even though saliva is considered as an optimal biofluid, several limitations are impeding its use in diagnostic and research. The major hurdles include the deficient concentration of biomarkers, need of laboratory-based preprocessing to remove mucin and interfering particulate matters, and lack of standard sample collection methods. Also, due to the absence of easily accessible and cost-effective analytical methods, it is challenging to figure out the circadian variation of biomolecules and consequently establish biomolecules as biomarkers. All these necessities mandate the development of alternative techniques that offer more accessible, cost-effective, rapid, real-time, and probably the point-of-care (POC) testing of salivary biomarkers. The aptamers, synthetic oligonucleotides functionally similar to antibodies but with several advantages, have shown promising possibilities to fulfill the requirements in such applications. However, no aptamer for H3 and several other salivary biomarkers exist in literature. This dissertation presents a widely applicable aptamer selection method for the novel biomarkers and the application of the identified aptamers with various platforms for the detection application in human saliva. In order to identify H3 aptamers, a library immobilization version of an iterative in vitro process known as the systematic evolution of ligands by exponential enrichment (SELEX) was established. This method does not require target immobilization and offers the isolation of signaling aptamers for a target in its unmodified form. Also, the signaling, structure-switching upon target binding, is a highly desired attribute of the aptamers for the biosensing application. Within the SELEX process, counter-selection steps were instituted to stop the evolution of nonspecific sequences that can bind to the molecules closely related to the target. Through the repetitive rounds of selection and counter-selection, four unique aptamer candidates sharing a consensus sequence were identified. The obtained aptamers were analyzed via online servers to predict the stability and structural conformations. A superior candidate in terms of stability and structural complexity was picked for the visual confirmation of binding. The magnetic bead-based affinity capture combined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), confirmed the selective binding between the selected aptamer candidate and target H3, indicating the successful aptamer selection process.Following the preliminary studies, more detailed characterizations were performed by developing a direct format enzyme-linked aptamer sorbent assay (ELASA). The aptamers were compared for their binding affinity and specificity to the target molecule H3. Also, the effect of primer binding sites on the binding affinity was probed. Selected clones exhibited affinity and specificity to H3, and the sequence with the highest affinity exhibited enhanced binding following the removal of primer binding sites. Based on these results, the truncated aptamer sequence was chosen for further application with other assay platforms. Next, the signaling feature of the aptamer was employed to explore the prospect of gold nanoparticles (AuNPs)-based colorimetric assays. Two different formats of the colorimetric assays were developed. After optimizing various parameters, both assay schemes were able to detect H3 within a salivary level of a healthy individual. Besides, with the assay-format-2, the overall assay time was reduced to just 12 minutes. Furthermore, surface-enhanced Raman spectroscopy (SERS)-based magnetic aptasensor was developed for the ultrasensitive detection of H3. By optimizing various parameters, SERS-based atpasensor was established. Additionally, the lateral flow test strip to support POC measurements of H3 was demonstrated by utilizing the optical properties of the AuNPs and the specificity of the identified H3 aptamer. The captured AuNPs on the test line of the lateral flow device provided qualitative analysis within 5 minutes for visual observation. Finally, human saliva and the artificial saliva samples were tested with the developed assay formats to investigate the challenges and the complications associated with the saliva-based measurements.In conclusion, the work presented in this dissertation lays the foundation for the identification of the signaling aptamers and their subsequent implementation in several platforms for the detection of salivary biomarkers. Further improvements and optimizations of the assay methods will eventually lead to a fast, reliable, cost-effective, ultrasensitive, and POC detection methods for the salivary biomarkers.
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