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FUNCTIONALIZED NANOSCALE MATERIALS FOR PROTEIN BIOMARKER DETECTION
Title
FUNCTIONALIZED NANOSCALE MATERIALS FOR PROTEIN BIOMARKER DETECTION
Creator
Zhang, Youwen
Date
2020
Description
Proteins are vital biomolecules in living organisms which function as the working element for many aspects of life. An abnormal expression of...
Show moreProteins are vital biomolecules in living organisms which function as the working element for many aspects of life. An abnormal expression of proteins or expression of unique proteins is often associated with certain disease. Accordingly, proteins have become valuable biomarkers for disease diagnosis and prognosis. So far, numerous methods have been developed for detections of protein biomarkers. However, most of them suffer from the lack of accuracy, sensitivity, and specificity for clinical diagnostic applications. With the rapid advancement in nanotechnology, functional nanoscale materials, which could overcome the biocompatibility and biological recognition ability, have been widely used to develop sensitive and selective biosensors.In this dissertation, two kinds of functionalized nanoscale materials-based sensing strategies are investigated for protein biomarker detection. One strategy takes advantages of graphene oxide (GO) and utilizes fluorescence resonance energy transfer (FRET) for fast and sensitive protease detection by covalent attaching fluorescently labeled protease substrate peptide to the GO surface. This type of GO-based fluorescence sensor is highly sensitive (with a detection limit of picomolar concentration) and selective (other structure similar proteases does not interfere with the target analyte detection). In addition, it could accurately analyze serum samples. With this strategy, we have successfully achieved the detection of the HIV-1 PR (HIV-1 protease, a significant biomarker for AIDS) and ADAMs (a disintegrin and metalloproteinases, a biomarker for human cancers). It could be visualized that this GO platform could be utilized to detect various proteases by only changing the peptide substrate and solution pH. In addition, by coupling multiple substrate peptides on the GO surface, we developed a multiplex GO sensing system for simultaneously profiling of the activities of a panel of MMPs/ADAMs. Under the assistance of joint entropy and programming, our sensor could identify up to 5 types of human cancers, and offers the potential to detect other cancer types by changing biomarkers.The other strategy is to utilize nanopore stochastic sensing to detect proteins, which involves measuring the ionic current modulation generated by analytes’ electro-osmotic flow through a chemical functionalized nanoscale sized pore. As a sensitive and label-free technique, nanopores have been highly recognized as one of the emerging techniques to detect analytes at the single-molecule level. Unlike DNA molecules which are uniformly charged, proteins are an isotropically charged molecules, which have low translocation probability through a nanopore. Since the protein pore-based sensing system is not suitable as deployable tools for detection of proteins due to the size limitation and fragile nature of the biological membranes. In this project, we fabricated solid-state nanopores using PET membranes followed by chemical functionalization of their inner surfaces. The modified- PET nanopore was sensitive and could detect HIV-1 protease at picomolar concentration. More importantly, the modified-nanopore sensor was selective, and could differentiate the target protein from others such as Trypsin, BSA and HSA. Furthermore, the modified PET nanopore strategy developed in this work provide a general platform for exploring fundamental protein dynamics and rapid detection of proteins at the single-molecule level
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Nanopore Detection of Heavy Metal Ions
Title
Nanopore Detection of Heavy Metal Ions
Creator
MohammadiRoozbahani, Golbarg
Date
2019
Description
Nanopore sensing is an emerging analytical technique for measuring single molecules. Under an applied potential bias, analyte molecules are...
Show moreNanopore sensing is an emerging analytical technique for measuring single molecules. Under an applied potential bias, analyte molecules are transported through the nanopore and cause ionic current modulations. Accordingly, the fingerprint of the analyte is reflected in the signature of the current blockage events. Due to its advantages such as lable-free and multi-analyte detection, nanopore sensing technology has been utilized as an attractive versatile tool to study a variety of topics, including biosensing of different species, such as DNA, RNA, proteins, peptides, anions, and metal ions.Metal ions play a crucial role in human health and environmental safety. Although metal ions are essential for numerous biological processes, the presence of the wrong metal, or even the essential metals in the wrong concentration or location, can lead to undesirable results and serious health concerns, including antibiotic resistance, metabolic disorders, mental retardation, and even cancer. Therefore, it is still of prime importance to develop highly sensitive and selective sensors for metal ions.In this dissertation, various nanopore sensing strategies to detect metal ions will first be discussed. These include: a) construction of metal ion binding sites in the nanopore inner surface; b) utilization of a biomolecule as a ligand probe; and c) employing enzymatic reactions. Then, three projects will be summarized. Among them, two projects are involved with detection of non-essential metal ions: uranyl and thorium ions, while the other is targeted at essential element, zinc ion. To be more specific, uranyl and thorium ions are detected by taking advantage of peptide molecules as ligand probes. In this case, the event signatures of peptide molecules in the nanopore are significantly different in the absence and presence of metal ions, which might be attributed to the conformational change of the biomolecules induced by the metal ion-biomolecule interaction. On the other hand, zinc ion is detected based on enzymatic reaction: without Zn2+, ADAM17 (a zinc dependent protease) is inactive and cannot cleave peptide substrate molecules; in contrast, with Zn2+ ion in the solution, the enzyme was activated, and its cleavage of the peptide substrate produced new types of blockage events with smaller residence time and amplitude values than those the peptide substrate.
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Nanopore sensing for environmental and biomarker analysis
Title
Nanopore sensing for environmental and biomarker analysis
Creator
Arora, Pearl
Date
2024
Description
Nanopore stochastic sensing is a powerful analytical tool for detecting target molecules through a nanoscale pore. The analyte and electrolyte...
Show moreNanopore stochastic sensing is a powerful analytical tool for detecting target molecules through a nanoscale pore. The analyte and electrolyte ions are subjected to a voltage bias which drives them to translocate through the nanopore, resulting in disruptions in the ionic current. These disruptions are translated to blockage events which can serve as a signature of the analyte. Owing to its unique features of single-molecule and label-free sensing, nanopore technique has been exploited in a wide array of applications such as detection of metal ions, proteins, DNA, microRNA, toxic agents etc. In this dissertation, projects showcasing nanopore’s sensing capability of different biomarkers and in the detection of a wide range of target molecules based on non-covalent interactions are presented. Particularly in the first two projects, nanopore detection of ferric ions relevant to environmental regulation as well as a biomarker for human health and a miRNA-based biomarker for oral cancer and oral related diseases are summarized. Ferric ions, which are benign if present in balanced quantities but can be toxic otherwise, are detected by using an engineered multifunctional nanopore and a chelating organophosphonic acid ligand. The chelate complex formed after ferric ions bind to ligand gives significantly different event signatures than the free ligand in the solution enabling ferric ion detection. Even in the presence of interfering ions, the ferric ions could be recognized easily because of the conformational changes brought in the nanopore lumen by the interaction of the interfering metal ions with the His-tags of the nanopore which in turn resulted in variations in the characteristics of blocking events. In the second project, miR31, an oral cancer biomarker, is selectively detected with the help of an engineered nanopore, and a DNA based probe. Several probes with variations in length, composition and position of the overhangs or probes with no overhangs were compared and studied as the probes play a crucial role in capturing the target of interest with high specificity. Our strategically designed probe emerged as the most effective in capturing the target even in presence of large background from human saliva samples and enhanced the sensitivity of the system. In the first two projects, nanopores are utilized for selective and specific detection of certain target molecules. However, in order to analyze diverse range of analytes, numerous sensing systems have to be constructed which can be a time-consuming and challenging task. To circumvent this limitation, in the third project, diverse recognition sites based on various non-covalent interactions are incorporated into the α-hemolysin protein pore to achieve detection of not just a single analyte but broad category of molecules such as cations, anions, aromatic and hydrophobic compounds.
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Nanopore sensing for environmental and biomarker analysis
Title
Nanopore sensing for environmental and biomarker analysis
Creator
Arora, Pearl
Date
2024
Description
Nanopore stochastic sensing is a powerful analytical tool for detecting target molecules through a nanoscale pore. The analyte and electrolyte...
Show moreNanopore stochastic sensing is a powerful analytical tool for detecting target molecules through a nanoscale pore. The analyte and electrolyte ions are subjected to a voltage bias which drives them to translocate through the nanopore, resulting in disruptions in the ionic current. These disruptions are translated to blockage events which can serve as a signature of the analyte. Owing to its unique features of single-molecule and label-free sensing, nanopore technique has been exploited in a wide array of applications such as detection of metal ions, proteins, DNA, microRNA, toxic agents etc. In this dissertation, projects showcasing nanopore’s sensing capability of different biomarkers and in the detection of a wide range of target molecules based on non-covalent interactions are presented. Particularly in the first two projects, nanopore detection of ferric ions relevant to environmental regulation as well as a biomarker for human health and a miRNA-based biomarker for oral cancer and oral related diseases are summarized. Ferric ions, which are benign if present in balanced quantities but can be toxic otherwise, are detected by using an engineered multifunctional nanopore and a chelating organophosphonic acid ligand. The chelate complex formed after ferric ions bind to ligand gives significantly different event signatures than the free ligand in the solution enabling ferric ion detection. Even in the presence of interfering ions, the ferric ions could be recognized easily because of the conformational changes brought in the nanopore lumen by the interaction of the interfering metal ions with the His-tags of the nanopore which in turn resulted in variations in the characteristics of blocking events. In the second project, miR31, an oral cancer biomarker, is selectively detected with the help of an engineered nanopore, and a DNA based probe. Several probes with variations in length, composition and position of the overhangs or probes with no overhangs were compared and studied as the probes play a crucial role in capturing the target of interest with high specificity. Our strategically designed probe emerged as the most effective in capturing the target even in presence of large background from human saliva samples and enhanced the sensitivity of the system. In the first two projects, nanopores are utilized for selective and specific detection of certain target molecules. However, in order to analyze diverse range of analytes, numerous sensing systems have to be constructed which can be a time-consuming and challenging task. To circumvent this limitation, in the third project, diverse recognition sites based on various non-covalent interactions are incorporated into the α-hemolysin protein pore to achieve detection of not just a single analyte but broad category of molecules such as cations, anions, aromatic and hydrophobic compounds.
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