Download or read book Single Molecule Delivery and Detection Into Crowded Environment with a Solid state Nanopore written by Chi Cheng Chau and published by . This book was released on 2021 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Trapping Single Molecules with a Solid State Nanopore written by Marc Herman Gershow and published by . This book was released on 2008 with total page 147 pages. Available in PDF, EPUB and Kindle. Book excerpt: I describe a system for measuring and manipulating single molecules using a solid state nanopore, a small hole in an insulating film fashioned using solid-state Silicon processing techniques. In this work, pores less than 10 nanometers in diameter in 20-30 nm thick Silicon Nitride membranes are used to join two reservoirs of salt water. DNA molecules added to one reservoir are forced through the nanopore by an applied voltage bias. Single molecules of DNA are detected passing through the pore by a blockage of the ionic current between the reservoirs. The depth and duration of the current blockages reveal information about the molecule's geometry while the shape of the blockage depends on the molecule's conformation. If the driving voltage is reversed soon after a molecule has passed through the nanopore, the same molecule can be "recaptured," induced to pass through the pore a second time. This process reveals information about the dynamics of the molecule outside of and its interaction with the nanopore. The recapture process can be repeated multiple times to form a single molecule trap.

Download or read book The Study of Single Molecule Protein Biophysics Using a Solid State Nanopore written by Kevin J. Freedman and published by . This book was released on 2013 with total page 442 pages. Available in PDF, EPUB and Kindle. Book excerpt: The kinetics of protein folding and binding has been studied for several decades and continues to reveal links to overall cellular health,cell functionality, and responses to therapeutic agents. Despite numerous methods to purify and detect proteins, there is still a growing need to develop technology that can enhance sensing and reveal potentially hidden properties of proteins. This is important not only from a scientific perspective but also in practically achieving the goals of personalized healthcare. Next generation sensors should ideally have three main characteristics: (1) high resolution sensing, (2) high-throughput sensing, and (3) the potential to be automated and used by untrained personnel. Future devices will revolutionize the healthcare industry by decreasing both the time and cost to do basic scientific research, diagnostic testing, and drug development. A likely candidate for next-generation protein sensing is solid-state nanopores. The pores developed here are fabricated in a 50 nm thick silicon nitride membrane and a single nanopore is drilled using a focused ion beam or a focused electron beam. The detection method employed is largely based on resistive pulse sensing where analytes are electrokinetically transported through a pore and identified by their unique modulation of ionic current (i.e. an ionic blockade). Since the dimensions of the nanopore are on the same scale as the molecule being sensed, only a single molecule can enter the pore allowing individual protein kinetics to be probed. Traditionally proteins are detected by ensemble averaging which hides important kinetics and sub-populations of molecules that may be important to understanding the early stages of a disease or detect a disease early. In the first section of this study, the prominent issue of protein adsorption onto the sensing device (i.e. the nanopore) is addressed and resolved by using a modified voltage protocol. The rationale behind the new sensing scheme is explained in terms of the interplay of diffusive and entropic (barrier-dominated) forces on a protein. In the second section, we discovered that the voltage which drives the protein through the pore also has denaturing effects. The unfolding data supports a gradual unfolding mechanism rather than the cooperative transition observed by classical urea denaturation experiments. Lastly it is shown that the voltage-mediated unfolding is a function of the stability of the protein by comparing two mutationally destabilized variants of the protein. In the final section of this study, voltage-mediated unbinding of a single protein complex is studied. We argue that determining the unbinding forces between two proteins adds an additional level of specificity which is needed for eventual use as a diagnostic tool. In this study, nanopores are developed not only as a sensor but also a single molecule or protein complex manipulator that can locally unfold or unbind molecules.

Download or read book Single Molecule Sensing Beyond Fluorescence written by Warwick Bowen and published by Springer Nature. This book was released on 2022-03-01 with total page 426 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book provides an interesting snapshot of recent advances in the field of single molecule nanosensing. The ability to sense single molecules, and to precisely monitor and control their motion is crucial to build a microscopic understanding of key processes in nature, from protein folding to chemical reactions. Recently a range of new techniques have been developed that allow single molecule sensing and control without the use of fluorescent labels. This volume provides an overview of recent advances that take advantage of micro- and nanoscale sensing technologies and provide the prospect for rapid future progress. The book endeavors to provide basic introductions to key techniques, recent research highlights, and an outlook on big challenges in the field and where it will go in future. It is a valuable contribution to the field of single molecule nanosensing and it will be of great interest to graduates and researchers working in this topic.

Download or read book Integrating Solid State Nanopore Sensors Within Various Microfluidic Arrays for Single Molecule Detection written by Radin Tahvildari and published by . This book was released on 2017 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: The miniaturization afforded by the integration of microfluidic technologies within lab-on-a-chip devices has greatly enhanced analytical capabilities in several key applications. Microfluidics has been utilized in a wide range of areas including sample preparation and analysis, DNA microarrays, cell detection, as well as environmental monitoring. The use of microfluidics in these applications offer many unique advantages: reduction in the required sample size, reduction in analysis time, lowered cost through batch fabrication, potentially higher throughput and the vision of having such devices used in portable systems. Nanopore sensors are a relatively new technology capable of detection and analysis with single-molecule sensitivity, and show promise in many applications related to the diagnosis and treatment of many diseases. Recently, some research groups demonstrated the integration of nanopores within microfluidic devices to increase analytical throughput. This thesis describes a methodology for integrating nanopore sensors within microfluidic devices with the aim of enhancing the analytical capabilities required to analyze biomolecular samples. In this work, the first generation of an integrated nanopore-microfluidic device contained multiple independently addressable microfluidic channels to fabricate an array of nanopore sensors using controlled breakdown (CBD). Next, for the second generation, we added pneumatic microvalves to manipulate electrical and fluidic access through connected microfluidic channels. As a proof-of-concept, single molecules (single- and double-stranded DNA, proteins) were successfully detected in the devices. It is also demonstrated that inclusion of the microfluidic via (microvia) limited the exposed area of the embedded silicon nitride membrane to the solution. This helped in localizing nanopore formation by confining the electric field to specific regions of the insulating membrane while significantly reducing high frequency noise in the ionic current signal through the reduction of chip capacitance. The devices highlighted in this thesis were designed and fabricated using soft lithography techniques which are available in most biotechnology laboratories. The core of this thesis is based on two scientific articles (Chapters 3 and 4), which are published in peer-reviewed scientific journals. These chapters are preceded by an introductory chapter and another chapter detailing the experimental setup and the methods used during the course of this study.

Download or read book Optimizing the Single Molecule Counting Process of Solid State Nanopores written by Martin Charron and published by . This book was released on 2020 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Due to their intrinsic single-molecule resolution and now easy fabrication and microfluidic integration, solid-state nanopores show great potential of becoming flexible low-cost, point-of-need, ultra-sensitive biomarker detection sensors. Since nanopores are still limited by the arrival time of the analyte to the sensor, reaching ultra-low concentration levels (fM) in a reasonable measurement time remains a challenge. Before approaches solving this problem become possible, one subject, not often discussed, needs to be addressed: The reliability and uncertainty of capture-based nanopore measurements. Counting with nanopores is often accomplished through measuring translocation frequencies. However, limitations of nanofabrication techniques tend to produce solid-state nanopores with, at the atomic scale, different geometries or chemical structures. A question then arises: How different will the measured capture rate be for two seemingly identical pores, i.e. pores intended to have same diameter and thickness within the fabrication capabilities? One solution to circumvent this problem is to use calibration curves by measuring the capture frequency of different concentrations, which prompts a follow up question: Does a single nanopore capture frequency change over time during the course of an experiment? This thesis investigates these two questions and experimentally shows that intra-pore and inter-pore capture variations can be significant, thus reducing the precision of simple nanopore counting to determine concentration of an analyte. To address these complications, a solution involving an internal calibrator is presented and is demonstrated to increase the sensitivity of concentration measurements.

Download or read book New Approach in Fabrication of Solid State Nanopore for Bio Sensing Applications written by Wing Hei Harold Kwok and published by . This book was released on 2015 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Solid state Nanopore Sensors for Nucleic Acid Analysis written by Bala Murali K. Venkatesan and published by . This book was released on 2011 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Nanopore DNA analysis is an emerging technique that involves electrophoretically driving DNA molecules through a nano-scale pore in solution and monitoring the corresponding change in ionic pore current. This versatile approach permits the label-free, amplification-free analysis of charged polymers (single stranded DNA, double stranded DNA and RNA) ranging in length from single nucleotides to kilobase long genomic DNA fragments with subnanometer resolution. Recent advances in nanopores suggest that this low-cost, highly scalable technology could lend itself to the development of third generation DNA sequencing technologies, promising rapid and reliable sequencing of the human diploid genome for under $1000. Here, we report the development of versatile, nano-manufactured Al2O3 solid-state nanopores and nanopore arrays for rapid, label-free, single-molecule detection and analysis of DNA and protein. This nano-scale technology has proven to be reliable, affordable, and mass producible, and allows for integration with VLSI processes. A detailed characterization of nanopore performance in terms of electrical noise, mechanical robustness and materials analysis is provided, and the functionality of this technology in experimental DNA biophysics is explored. A framework for the application of this technology to medical diagnostics and sequencing is also presented. Specifically, studies involved the detection of DNA-protein complexes, a viable strategy in screening methylation patterns in panels of genes for early cancer detection, and the creation of lipid bilayer coated nanopore sensors, useful in creating hybrid biological/solid-state nanopores for DNA sequencing applications. The concept of a gated nanopore is also presented with preliminary results. The fabrication of this novel system has been enabled by the recent discovery of graphene, a highly versatile material with remarkable electrical and mechanical properties. Direct modulation of the nanopore conductance was observed through the application of potentials to the graphene gate. These exciting results suggest this technology could potentially be useful in slowing down or trapping a DNA molecule in the pore, thereby enabling solid-state nanopore sequencing.

Download or read book Precise Size Control and Noise Reduction of Solid state Nanopores for the Detection of DNA protein Complexes written by Eric Beamish and published by . This book was released on 2012 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Over the past decade, solid-state nanopores have emerged as a versatile tool for the detection and characterization of single molecules, showing great promise in the field of personalized medicine as diagnostic and genotyping platforms. While solid-state nanopores offer increased durability and functionality over a wider range of experimental conditions compared to their biological counterparts, reliable fabrication of low-noise solid-state nanopores remains a challenge. In this thesis, a methodology for treating nanopores using high electric fields in an automated fashion by applying short (0.1-2 s) pulses of 6-10 V is presented which drastically improves the yield of nanopores that can be used for molecular recognition studies. In particular, this technique allows for sub-nanometer control over nanopore size under experimental conditions, facilitates complete wetting of nanopores, reduces noise by up to three orders of magnitude and rejuvenates used pores for further experimentation. This improvement in fabrication yield (over 90%) ultimately makes nanopore-based sensing more efficient, cost-effective and accessible. Tuning size using high electric fields facilitates nanopore fabrication and improves functionality for single-molecule experiments. Here, the use of nanopores for the detection of DNA-protein complexes is examined. As proof-of-concept, neutravidin bound to double-stranded DNA is used as a model complex. The creation of the DNA-neutravidin complex using polymerase chain reaction with biotinylated primers and subsequent purification and multiplex creation is discussed. Finally, an outlook for extending this scheme for the identification of proteins in a sample based on translocation signatures is presented which could be implemented in a portable lab-on-a-chip device for the rapid detection of disease biomarkers.

Download or read book Hybrid Solid state Nanopores for Stochastic Single molecule Detection written by and published by . This book was released on 2015 with total page 110 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Error Analysis and Parameter Estimation for Nanopore Based Molecular Detection written by Christopher R. O0́9Donnell and published by . This book was released on 2018 with total page 92 pages. Available in PDF, EPUB and Kindle. Book excerpt: Nanopores are powerful tools for measuring and probing single molecules. A nanopore is a nanometer-sized opening in a membrane that separates two chambers filled with buffered ionic solution. By applying a voltage and measuring the ionic current through the nanopore, it is possible to detect the presence of individual DNA, RNA and proteins as they pass through the pore, and even read the sequence of individual nucleobases that make up a single strand of DNA. However, the speed with which molecules translocate and the size of the sensing region have presented challenges for using nanopores to sequence DNA. Most nanopore-based DNA sequencing research focuses on using biological nanopores paired with an enzyme to slow down the passage of DNA through the pore, but recent advances in solid-state fabrication technology have made it possible to create artificial solid-state nanopores in insulating membranes, typically made of silicon. These pores can be made in a larger range of sizes, are more durable, and are more amenable to large scale fabrication than their biological counterparts. In order to control the rate of molecular translocation through solid-state nanopores, researchers are developing a two-pore architecture, which utilizes time-varying voltage patterns to enable rereading of individual molecules to gain confidence in feature sensing. This thesis presents a numerical study that provides an error analysis of an idealized nanopore sequencing method in which ionic current measurements are used to sequence intact single-stranded DNA in the pore while an enzyme controls DNA motion. This analysis presents examples of systematic and random errors associated with this method of sequencing and demonstrates the necessity of rereading sequences at least 140 times to achieve 99.99% accuracy. Two different methods of parameter estimation are then presented that overcome the problem of contamination of the measured ionic current by capacitive elements in the system and facilitate active control with the two-pore architecture.

Download or read book A Preconcentrating Lab on a Chip Device Targeted Towards Nanopore Sensors written by Kaitlyn Kean and published by . This book was released on 2020 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Continuous progress in the nanotechnology field has allowed for the emergence of powerful, nanopore-based detection technology. Solid-state nanopores were developed for next-generation sequencing and single-molecule detection. They are advantageous over their biological counterpart because they offer robustness, stability, tunable pore size and the ability to be integrated within a microfluidic device. With all of these attractive attributes, solid-state nanopores are a top contender for point-of-care diagnostic technologies. However, hindering their performance is an inability to distinguish between small molecules, pore-clogging, and the detection rate's dependence on sample concentration. The concentration-dependent detection rate becomes particularly evident at low sample concentrations (

Download or read book Self induced Back Action Actuated Nanopore Electrophoresis SANE Sensor for Molecular Detection and Analysis written by Sai Santosh Sasank Peri and published by . This book was released on 2021 with total page 107 pages. Available in PDF, EPUB and Kindle. Book excerpt: We fabricated a novel single molecule nanosensor by integrating a Solid-State Nanopore (SSNP) and a Double Nanohole (DNH) nanoaperture. The nanosensor employs Self-Induced Back-Action (SIBA) for optical trapping and enables SIBA-Actuated Nanopore Electrophoresis (SANE) for concurrent acquisition of bimodal optical and electrical signatures of molecular interactions. We demonstrated the potential utility of the SANE sensor by trapping and translocating 20 nm silica and gold nanoparticles. The electrical translocation time of the nanoparticles was extended by four orders of magnitude due to opposing electrical and optical forces acting on the nanoparticle, causing high frequency oscillations or bobbing in the electrical signal. Using frequency analysis, we were able to show that bobbing can be used as a signature to distinguish between single and multiple trapping. These promising results enabled us to pursue biomolecular detection with SANE sensor. We used high affinity T-cell receptor-like antibodies (TCRmAbs), and tested their binding to specific peptide-presenting Major Histocompatibility Complex (pMHC) ligands. We used irrelevant TCRmAbs, targeting the same pMHCs as control experiments. We were able to distinguish between individual molecules and their specific and non-specific mixtures. The optical-electrical metrics enabled measurement of increased bound fraction of the antibody-ligand complexes at lower concentrations than bulk solution equilibrium binding constant (KD). In addition, we detected low affinity ligand-receptor interactions between soluble heterodimer receptors and pMHC ligands. We used irrelevant pMHCs to target the same receptor as a control experiment. We discriminated the optical-electrical signatures for specific and non-specific binding of receptor-ligand interactions, and were able to quantify the dissociation rate constant (koff) of the receptor-ligand binding comparable to the commercial technologies. The measurement koff value can be correlated to the receptor-ligand binding time required for activation of immune response in vivo. Therefore, we demonstrated the utility of SANE sensor as a potential screening tool in cancer immunotherapy.

Download or read book Engineered Nanopores for Bioanalytical Applications written by Joshua B. Edel and published by William Andrew. This book was released on 2013-03-19 with total page 189 pages. Available in PDF, EPUB and Kindle. Book excerpt: Engineered Nanopores for Bioanalytical Applications is the first book to focus primarily on practical analytical applications of nanopore development. These nanoscale analytical techniques have exciting potential because they can be used in applications such as DNA sequencing, DNA fragment sizing, DNA/protein binding, and protein/protein binding.This book provides a solid professional reference on nanopores for readers in academia, industry and engineering and biomedical fields. In addition, the book describes the instrumentation, fabrication, and experimental methods necessary to carry out nanopore-based experiments for developing new devices. - Includes application case studies for detection, identification and analysis of biomolecules and related functional nanomaterials - Introduces the techniques of manufacturing solid state materials with functional nanopores - Explains the use of nanopores in DNA sequencing and the wider range of applications from environmental monitoring to forensics

Download or read book Solid state Nanopore Fabrication and Sensing Towards Integrated Nucleic Acid Testing written by Zifan Tang and published by . This book was released on 2022 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Regular, accurate, rapid, and inexpensive self-testing for infectious diseases is urgently needed to optimize clinical care and guide infection control to limit disease spread. Nucleic Acid Amplification Test (NAAT) is the most sensitive and specific method, thus becoming the gold-standard technique for diagnosis. However, laboratory-based NAAT requires highly trained personnel, dedicated facilities, and instrumentations, delaying testing results and limiting testing capacity. Existing self-testing methods usually rely almost exclusively on rapid antigen tests. Typically, the sensitivity of antigen tests is 30% to 40% lower than the nucleic acid amplification testing (NAAT), which could miss a significant portion of infected patients. Therefore, a self-testing NAAT device for diagnosis is strongly needed to optimize clinical care and guide infection control to limit disease spread. This thesis mainly focuses on exploring the possibility of developing a solid-state nanopore-based NAAT device for a new form of ultracompact, rapid, and label-free nucleic acid self-testing. We demonstrated the nucleic acid amplification coupled nanopore counting method for qualitative positive/negative nucleic acid testing. Due to its intrinsic single molecule sensitivity, the nanopore sensor could make a faster positive/negative call than bulk optical methods. To further explore a more reliable and integratable method for nanopore fabrication, we developed the laser-assisted breakdown method for single nanopore fabrication. We theoretically and experimentally demonstrated that combining a high laser power and a low electric field is statistically favorable for forming a single nanopore at a programmed location. Furthermore, we developed a fully integrated sample-in answer-out NAT device for SARS-CoV-2 detection using a self-collected saliva sample. This system can automatically handle the complexity of heat-inactivated sample preparation, pressure-driven sample dispensing, real-time RT-LAMP reaction and detection, and data processing and storage. Using an optical sensor, we achieved a limit of detection (LoD) of 5 virus particles/[mu]l of saliva sample in 45 minutes. The final amplicons from the developed prototype were also detected by nanopore counting methods. Therefore, the successful completion of this project will pave the way for ultracompact, rapid, and affordable nanopore-based nucleic acid testing.

Download or read book Improved Single Molecule Detection of Native Proteins Using Hydrogel Backed Nanopores written by Reyhaneh Nazarian and published by . This book was released on 2021 with total page 128 pages. Available in PDF, EPUB and Kindle. Book excerpt: Abstract Accurate identification and quantification of proteins in a solution using nanopores is technologically challenging in part because of the large fraction of missed translocation events due to short event times and limitations of conventional current amplifiers. Previously, we have shown that a nanopore interfaced with PEG(1000)-DMA hydrogel with an average mesh size of 3.1 nm significantly enhances protein residence time inside the nanopore, reducing the number of missed events. Following up on our previous work, here, we explored measurement limits, sensitivity, and further characterization capabilities of our proposed hydrogel-backed nanopore system. We demonstrated the ability of the hydrogel-backed nanopores to sense unlabeled proteins as small as 5.5 kD in size and 10 fM in concentration, without a major restriction on the nanopore size or the experimental setup. Also, we showed that the frequency of protein translocation events scales linearly with the bulk concentration over a wide range of concentrations, and an unknown protein concentration can be determined from an interpolation of the frequency-concentration calibration curve with less than 10% error. Further, we precisely determined protein volumes from measurement data, and we employed an iterative method to determine a protein's volume when its diameter is comparable to nanopore diameter. We investigated possible mechanisms for detection enhancement enabled by the presence of the hydrogel; we found that the possible gap between the pore mouth and the hydrogel indicates the sensitivity of the hydrogel-backed nanopores. Moreover, we demonstrated that hydrogel-backed nanopores can serve as an effective, reliable, ultra-sensitive, non-destructive, reproducible, and easy-to-operate substitute for commonly used UV-Vis detectors in fast protein liquid chromatography. The hydrogel-backed nanopores resolved protein fractions at much lower concentrations than the minimum concentration detectable by the standard UV-Vis detector. They also measured protein fractions with a higher selectivity and provided a more informative analysis of proteins' physical properties than the UV-Vis detector. Additionally, we integrated the nanopore with PDMS microchannels to create a fluidic circuit between a chromatographic column and the nanopore to facilitate the continuous and live measurement of column effluents. Finally, we demonstrated that integrating lipid-bilayer coated nanopores with a hydrogel is a suitable platform for acquiring artifact-free and long protein translocation events to analyze a single protein translocation event accurately. Using hydrogel-backed lipid-bilayer coated nanopores, we determined the volumes of IgG, Ovalbumin, and gold nanoparticles (5 nm diameter) from individual single translocation events in agreement with reference values. Further, we observed that higher applied voltages increased the probability of IgG alignment. We determined the volume and the length-to-diameter ratio of IgG molecules at different applied voltages and noticed an expansion in the conformation of IgG molecules with an increase in the voltage; this is likely due to IgG's flexibility and an intense electric field's ability to expand the IgG hinges from one another.

Download or read book Sensing Native and Unfolded Single Protein Molecules with Solid state Nanopores written by Bradley Thomas Ledden and published by . This book was released on 2011 with total page 262 pages. Available in PDF, EPUB and Kindle. Book excerpt: