2008 — 2009 |
Soh, Hyongsok Tom |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Rapid Generation of High Affinity Protein Sensor Elements @ University of California Santa Barbara
DESCRIPTION (provided by applicant): Effective molecular recognition and highly specific chemical interactions between the receptor (sensor) and ligand (target molecule) determine the performance of biosensors;in other words, the usefulness of a biosensor is governed by the quality of the affinity reagent. The invention of hybridoma monoclonal antibody (mAb) technology opened the paradigm of affinity reagents, and since then, there have been many significant breakthroughs including bacteriophage display, cell-surface display, mRNA display and aptamers to name a few. However, the current technology to obtain affinity reagents for new molecular targets continue to be limited by the high cost, lengthy development time, limited availability, and often inadequate biochemical properties (e.g. affinity, specificity, stability, durability) for many applications. This rings especially true in advanced diagnostic and proteomic applications, where the demand for new reagents far exceeds the rate at which they can be produced. Aptamers are functional binding molecules composed of nucleic acids which are selected from combinatorial nucleic acid libraries by in vitro selection through a process known as Systematic Evolution of Ligands by EXponential enrichment (SELEX) 1, 2. Unlike traditional reagents (i.e. mAb), aptamers provide a significant advantages as biosensors because they are chemically synthesized, can be evolved for affinity as well as specificity, and they undergo reversible de-naturation, thus making them potentially ideal for field use. However, current methods of SELEX usually require intensive manual labor (8-15 rounds of selection) and time (multiple weeks). Technically, the challenge in isolating high affinity binders originates from multiple factors;first, due to the fact that the starting library contains significantly fewer high affinity binders compared to non- and low-affinity binders, they "out-compete" the high-affinity binders due to mass-action, especially during the crucial, early rounds of selection. Secondly, the non specific binding of the aptamers to the molecular target, the solid support or the separation apparatus degrades the efficiency of molecular partitioning. Finally, using traditional methods of selection, such as affinity columns and nitrocellulose membranes, the partition efficiency in separating the high-affinity binders from non/low-binders is limited, necessitating many rounds of selection. Thus, in order to accelerate the affinity reagent generation, a fundamentally new technology platform to screen molecular libraries must be developed that can overcome these key challenges. Toward this end, our laboratory recently developed a novel system for high performance magnetophoretic separation called CMACS where we use micropatterned ferromagnetic materials in microfluidic channels to generate a highly controlled magnetophoretic force field that separates superparamagnetically labeled molecules from complex backgrounds with unprecedented efficiency. This device allows the use of very small number of magnetic beads (<106 per selection) and target proteins (down to ~ 20 attomoles) enabling exceptional stringency such that low-affinity binders, do not "out-compete" the high-affinity binders due to mass-action. Furthermore, the partition efficiency (PE) of CMACS to be 1-2 orders of magnitude higher than those achieved by capillary electrophoresis (CE). These advantages, combined with our bead conjugation chemistry, which inhibits non-specific binding have enabled us to generate aptamers with low nanomolar affinities to protein targets, in a single round of selection within 30 minutes of separation. Building on these foundations, the main goal of this work is to: 1) optimize the M-SELEX process and verify that affinities of the aptamers can be further improved with more rounds of selection, and 2) test the generalizability of the M-SELEX system by generating aptamers for multiple protein targets. The success of this project will have significant impact in multiple areas of biotechnology because it will enable the generation of high performance affinity reagents at an unprecedented speed with minimal labor and cost. PUBLIC HEALTH RELEVANCE Detection of target viruses with traditional surface marker-based immunoassays (i.e., ELISA) using monoclonal antibodies (mAb) suffer from major drawbacks because 1) a new mAb typically takes more than 6 months to generate, 2) many of the antibodies cross-react across species and subtypes, thereby compromising the detection specificity, and 3) it is extremely costly to develop arrays of antibodies that can provide a composite signature of the surface proteins. Aptamers are functional binding molecules composed of nucleic acids which are selected from combinatorial libraries by in vitro selection. Unlike mAbs, aptamers provide a significant advantages as biosensors because they are chemically synthesized, can be evolved for affinity as well as specificity, and they undergo reversible de-naturation, thus making them ideal for field use. Expanding on our previous work on ultrahigh performance Microfluidic-SELEX, this proposal seeks to develop the Microfluidic Viral SELEX (MVX) technology which will be capable of generating a set of high affinity aptamers which are specific to the target virus, so that a composite signature of viral surface proteins can be generated on demand - rapidly, efficiently, reproducibly, and in disposable microfluidic devices. We envision that, when fully developed, the proposed MVX technology will be applicable to almost all viruses. However, in this R21 project, in close collaboration with a leading virology lab at UCLA, we propose to use two closely related herpes simplex viruses (HSV) as a model system. HSVs have complex surface structures and they will serve as a challenging, yet realistic models. If successful, the proposed MVX technology has the potential to fundamentally revolutionize the way we generate affinity ligands, and perform viral diagnostics.
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1 |
2009 — 2010 |
Soh, Hyongsok Tom |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Micromagnetic Aptamer Pcr System For Ultrasensitive Multiplexed Protein Detection @ University of California Santa Barbara
DESCRIPTION (provided by applicant): For many diseases, effective diagnosis and prognosis depends on the ability to quantitatively measure protein biomarkers present at low concentrations in clinical samples. For example, altered serum concentrations of cytokines, such as platelet-derived growth factor (PDGF) can point to tumor angiogenesis, while virus-related proteins, like hemagglutinin can indicate the extent of viral pathogen infection. Although blood serum is a rich source of diagnostic information, the analytical challenge in utilizing serum biomarkers arises from the fact that it contains thousands of proteins whose concentrations range over 12 orders of magnitude. Albumin, for example, constitutes approximately half of the serum proteins and is present at 30-50 mg/mL (0.5-0.8 mM), while many important biomarkers exist at concentrations at 1 pg/mL (10-100 fM). Due to this wide dynamic range, specific and quantitative detection of protein biomarkers in a single assay has been particularly difficult. Therefore, an innovative protein detection technologies with the following capabilities are critically needed: I) Integrated sample preparation - the sensor should have the capability to directly process clinical samples (i.e. whole blood or blood serum), II) the sensor should provide quantitative results of multiple markers that provides actionable diagnostic value, III) the sensor must have high sensitivity (femtomolar detection limits) and broad dynamic range, IV) the detection system must produce reproducible results and have low rates of false positives and negatives, V) the sensor should have a short total assay time and most importantly, VI) the total cost of the diagnostic information must be low. In order to address these challenges, a number of recent immunological methods such as ImmunoPCR, Proximity Ligation, and the Bio-Barcode assays have been developed to achieve detection performances beyond the capabilities of the enzyme-linked immunosorbent assay (ELISA), which as been the gold-standard for over three decades. However, such assays suffer from the fact that the reaction chemistry is hindered by the background serum proteins and other contaminants in the clinical samples. As a solution to this problem, we propose to integrate sample preparation, amplification and detection in a single disposable system such that we effectively transform a homogenous binding assay into a heterogeneous detection system. We propose to develop the the Micro-Magnetic Separation - Quantitative Polymerase Chain Reaction (MMS-QPCR) system wherein we integrate chip-based, high-gradient micro-magnetic separation with aptamer-based quantitative PCR to achieve quantitative, multiplexed protein detection with femtomolar detection sensitivities directly from undiluted serum. The project will be organized in three sections: first, we will design multiple sets of antibody - aptamer pairs to capture and label the target proteins with high affinity and specificity. Second, we will develop the micro-magnetic separation (MMS) chip to purify the target proteins from background serum for downstream detection. The use of magnetic particles will significantly improve the reaction kinetics, reduce incubation times, and eliminate extensive washing steps. The MMS chip will be directly interfaced as a front-end to a QPCR detection system. Thirdly, using the primer sequences imbedded in the aptamers, we will perform QPCR to achieve quantitative, multiplexed detection of target proteins at femtomolar concentrations. As a model system we will demonstrate simultaneous detection of three cancer markers, platelet derived growth factor (PDGF), hepatocyte growth factor (HGF), and thyroid transcription factor (TTF1), at femtomolar concentration levels directly from serum. From the preliminary results, we expect the limit of detection to be ~ low fM (5 orders of magnitude higher than traditional ELISA technique), with a wide dynamic range spanning 6 orders of magnitude (femto molar to nanomolar concentrations). PUBLIC HEALTH RELEVANCE: For many diseases, effective diagnosis and prognosis depends on the ability to quantitatively measure protein biomarkers which are present at low concentrations in clinical samples. Although blood serum is a rich source of such information, the challenge arises from the fact that it contains thousands of proteins whose concentrations range over 12 orders of magnitude (micromolar to femtomolar), making the analysis of rare protein markers extremely difficult. As a solution to this important problem, we propose to combine chip-based, high-gradient micro-magnetic separation technology with aptamer- based quantitative PCR to achieve quantitative, multiplexed protein detection with femtomolar detection sensitivities directly from undiluted serum samples.
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1 |
2009 — 2013 |
Roberts, Richard W [⬀] Roberts, Richard W [⬀] Soh, Hyongsok Tom |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Polypeptide Design With Proteomic Scope Via Microfluidic Mrna Display @ University of Southern California
DESCRIPTION (provided by applicant): In this project, we propose to combine mRNA display (a protein design/evolution method) and high efficiency microfluidic sorting to create a new technology-microfluidic mRNA display-for the purpose of enabling design of peptides and proteins that can be used as protein capture reagents. We will develop and apply this powerful new technology toward creating a comprehensive reagent set aimed at the Hepatitis C virus (HCV) proteome. In this section, we begin by describing the existing state-of-the-art in 1) mRNA display-based peptide and protein design, 2) bead-based micromagnetic separations, and 3) give an introduction to the proteins expressed by the HCV that will be the targets of this work. This is followed by our description of how we will integrate these technologies to achieve our goal of high-throughput development of new protein capture reagents.
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0.976 |
2009 — 2012 |
Soh, Hyongsok Tom |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Strain Specific Detection of Influenza At the Point-of-Care @ University of California Santa Barbara
DESCRIPTION (provided by applicant): Strain Specific Detection of Influenza at the Point-of-Care 1. PROJECT SUMMARY Influenza viruses cause significant morbidity and mortality worldwide; World Health Organization (WHO) estimates 250,000 ~ 500,000 yearly deaths. In particular, a highly pathogenic strain - avian influenza virus H5N1 (15, 56) - poses a threat of a possible pandemic because it can rapidly mutate to acquire the ability to transmit among humans (23, 49). The detection of minimal human infectious dose (~100 viral particles) (65) of Influenza A from patient samples (e.g. nasal swabs or mucus) is a significant analytical challenge. Detection by viral culture is sensitive and specific; however, this method is slow (3-10 days) and cannot be performed at POC. Surface marker-based detection (e.g. ELISA) provide a simple and rapid testing, however, it suffers from a poor limit of detection (~ 105 viral particles/ml) (9, 21, 59). Currently, polymerase chain reaction (PCR) offers the highest sensitivity in a much shorter turn-around time (2-4 hours) (29, 61, 67). Nevertheless, due to the difficulties in POC operation, patient samples are currently analyzed at central laboratories which typically requires ~2-3 days. Currently in the field, there is no automated technology solution that can perform specific influenza detection directly from patient samples with high sensitivity and specificity, and the WHO has specifically emphasized the critical need to fill this important technological gap (75). Towards a solution to this important problem, this group of three PI's with complementary skill sets (influenza virology, aptamer biochemistry and microfluidic device engineering) propose to develop a powerful, field- portable, microfluidic platform for Influenza detection at POC. This effort is truly unique in many facets; first, to circumvent the problem posed by rapid mutation of the coat protein, we propose to generate novel DNA aptamer reagents which will specifically bind to the nucleoprotein complex of the virus. Currently, there are no reported aptamers for the nucleoprotein complex, and due to the fact that the nucleoprotein is conserved, it will serve as a universal tag to label Influenza A. Secondly, the aptamer reagent will be conjugated to magnetic beads to purify the nucleoprotein complex from nasal swab samples using our unique micromagnetics technology. Thirdly, by leveraging our expertise in miniaturized genetic analysis systems, we will develop the IMED chip capable of 1) integrated micromagnetic sample purification, 2) reverse transcription-PCR amplification and 3) sequence-specific electrochemical detection in a single monolithic device. The successful completion of this project will allow a quantitative sample-to-answer capability - the input into the system will be unprocessed patient samples, and the output will be an electrochemical signal that will be directly proportional to the number of copies of specific influenza RNA sequences in the sample. Such powerful combination of novel affinity reagents with highly integrated disposable devices will enable the development of critically needed effective POC diagnostics systems. PUBLIC HEALTH RELEVANCE: Strain Specific Detection of Influenza at the Point-of-Care Due to the fact that the envelope of influenza A virus differs among subtypes and evolves continuously, there is an urgent need for a field-portable genetic detection platform that can rapidly identify pathogenic strain of viral pathogens from unprocessed samples with high sensitivity and specificity. Towards an effective solution, the three PI's with complementary skill sets (influenza virology, aptamer biochemistry and microfluidic device engineering) propose to develop an effective point-of-care diagnostic system for influenza by 1) generating specific, high affinity DNA aptamers to tag the conserved regions of Influenza A, and 2) developing a highly integrated microfluidic system capable of magnetic sample preparation, RT-PCR amplification and sequence specific electrochemical detection in a single chip. By integrating the functions in a monolithic chip, the success of this project will yield a novel POC analysis system with unprecedented capabilities which will have a significant impact for Influenza A detection as well as many other applications in food safety, environmental monitoring and homeland security. 1
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1 |
2011 — 2013 |
Smith, Lloyd M (co-PI) [⬀] Soh, Hyongsok Tom Stewart, Ron Thomson, James Alexander (co-PI) [⬀] |
U54Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These differ from program project in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes, with funding component staff helping to identify appropriate priority needs. |
Qpass: Quantitative Parallel Aptamer Selection System @ University of California Santa Barbara
Nucleic acid aptamers possess many useful features as affinity reagents, including facile chemical synthesis, reversible folding, thermal stability and low cost, making them a powerful alternative to antibodies and other protein-based reagents. However, over the past two decades, aptamers have suffered from the fact that 1) the conventional method of aptamer generation (SELEX) is lengthy, labor intensive and often does not yield aptamers with sufficient affinity (< 1 nM) and specificity; 2) there is no standard protocol that can be generally applied to most protein targets to generate aptamers; and 3) the characterization steps to measure the affinity and specificity of candidate aptamers are lengthy and resource-intensive, because each aptamer must be measured individually. We believe that these challenges arise from deficiencies in the conventional methodology of performing the selection, which has not changed significantly since its initial description 20 years ago. We also believe that these problems can be solved, by systematically taking fundamentally different approaches towards the three central stages of the process - selection, analysis and characterization of the aptamers. We propose here the development of such a system. We will combine three distinctly novel technologies -microfluidic selection, next-generation aptamer sequencing, and SPR Imaging - to develop the Quantitative Parallel Aptamer Selection System (QPASS) platform. The QPASS platform will generate specific aptamers with sub-nanomolar affinities (Kd) for a wide range of protein targets within 3 rounds of selection, identify a pool of the best candidates by next generation DNA sequencing and bioinformatic analysis, and home in on the optimal aptamer sequence by the parallel synthesis and measurement of the affinities of thousands of aptamer candidates. Individually, each component represents a significant technological advance. Combined this integrated approach offers an opportunity to revolutionize the process of aptamer generation.
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1 |
2017 — 2021 |
Demirci, Utkan (co-PI) [⬀] Soh, Hyongsok Tom |
R25Activity Code Description: For support to develop and/or implement a program as it relates to a category in one or more of the areas of education, information, training, technical assistance, coordination, or evaluation. |
Canary Cancer Research Education Summer Training (Canary Crest) Program
PROJECT SUMMARY/ABSTRACT: The Canary Center at Stanford for Cancer Early Detection (?Canary Center?) is a world-class facility with the mission to foster interdisciplinary research leading to the development of blood tests and molecular imaging approaches to detect and localize early cancers by integrating research in in vivo and in vitro diagnostics to deliver these tests. Embedded within this mission is the need to formally present new and innovative approaches to scientific communities at all levels. The Canary Cancer Research Education Summer Training (Canary CREST) Program fulfills an educational mission by introducing students to research education and new career paths. The overall goal of the program is to train a new generation of interdisciplinary scientists in early cancer detection by offering an integrative hands-on research experience at an early stage of their scientific education. The Canary CREST Program is a 10-week instructional summer program for 25 undergraduate students in the biological, engineering, mathematical, or physical sciences, and offers a structured research experience with a focus on early cancer detection. The program will be administered by the Canary Center and brings together a multidisciplinary group of 28 faculty whose research groups are dedicated to the field of early cancer detection using experimental and computational approaches in biochemistry, bioengineering, bioinformatics, molecular imaging, and cancer biology. Proposed Canary CREST Program activities include: (1) Mentor-directed research in one of six investigative areas, namely, development of devices for cancer diagnostics, cancer biomarker discovery and validation, cancer biology, molecular imaging of cancer, clinical imaging of cancer, and cancer bioinformatics; (2) Specially-designed classroom sessions to provide a conceptual framework of the field of early cancer detection; (3) Seminars in scientific research; (4) A comprehensive professional development component that includes career talks, student presentations, workshops on communication skills and career opportunities; and (5) Participation in an Ethics Forum. The key aspect of this program is to offer participants the opportunity to conduct mentor-directed research while developing an understanding of hypothesis-driven studies with critical interpretation of results and analysis of data. The long-term objective of this educational research program is to support the growing need of specialized researchers who will have a significant impact in the rapidly-expanding area of cancer early detection.
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0.954 |
2018 — 2021 |
Soh, Hyongsok Tom |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Integrated Instrument For Non-Natural Aptamer Generation
Project Summary DNA and RNA aptamers are a useful class of synthetic affinity reagents. However, their performance can be greatly improved through the site-specific incorporation of chemically modified, ?non-natural? nucleotides that provide a greater chemical repertoire to enable superior aptamer affinity and specificity. Because a broad spectrum of chemical functional groups can be incorporated, non-natural aptamers offer the exciting potential for targeting molecules for which the generation of monoclonal antibodies remains difficult, such as small- molecule drugs, metabolites and carbohydrates. Unfortunately, the access to non-natural aptamers is severely limited. This is because the process of generating non-natural aptamers is technically challenging and limited to a few specialized laboratories. The goal of this project is to develop an integrated instrument, the Non-Natural Aptamer Array (N2A2) that eliminates these bottlenecks and enable rapid and facile non-natural aptamer discovery at virtually any research laboratory. The N2A2 will be built on a modified version of a benchtop commercial sequencer (Illumina MiSeq), and will perform every stage of non-natural aptamer discovery? including sequencing, screening and binding measurements?as part of a single work-flow. There are three main innovative aspects of our N2A2 system. First, our approach will entirely eliminate the need for polymerase engineering, and thus allows us to incorporate virtually any chemical functional group through click chemistry. Second, N2A2 will enable us to directly obtain the binding affinity (Kd) of ~10^7 aptamers directly in complex samples (e.g. cell lysate or serum), thereby resulting in aptamers with high-specificity. Finally, we will develop a machine-learning (ML) approach to identify key motifs (?k-mers?) and predict novel sequences with potentially higher affinity and specificity that can be tested using the N2A2 instrument. We believe this powerful combination of massively parallel, sequence-linked binding measurements with ML-based predictions will allow us to explore sequence space that is currently inaccessible to traditional in vitro selection methods, and enable us to discover aptamers with superior performance. The success of this project will produce an integrated instrument that greatly streamlines and accelerates the discovery of non-natural aptamers for a wide range of targets in complex media. The instrument is based on a commercially available sequencer and we will make all software available to the public. In this way, we believe the N2A2 instrument could broadly expand access to robust, high quality, custom affinity reagents for biomedical research and clinical diagnostics.
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0.954 |
2018 — 2021 |
Soh, Hyongsok Tom |
OT2Activity Code Description: A single-component research award that is not a grant, cooperative agreement or contract using Other Transaction Authorities |
Real-Time Biosensor For Mapping the Function of the Pancreas
Project Summary: The capability to manipulate pancreatic function through peripheral nerve stimulation offers exciting therapeutic potential, but will require a deeper understanding of underlying molecular biology as well as inter-organ signaling and communication. Such processes are difficult to study in vivo because they occur on time-scales of seconds to minutes?far faster than can be measured by conventional assays. The primary goal of this project is to develop real-time in vivo biosensors that can continuously measure the physiological concentrations of glucose, insulin and glucagon in specific areas of the pancreas of freely moving animals. Our biosensors will detect these three biomarkers with high sensitivity (pM~nM), spatial resolution (~10 ?m) and temporal resolution (~1 minute), and thereby offer unprecedented insights into the molecular biology of the pancreas. We will be building upon aptamer biosensor technology previously developed by our group, with which we have already demonstrated the first continuous measurement of small molecule drugs in vivo as well as closed-loop feedback control in live animals. This is a ?platform? technology that can be readily adapted to measure a wide range of biomarkers by swapping the aptamer probes, as we have demonstrated previously. Upon successful development of this real-time biosensor for the pancreas, we will expand the range of targets to 20 other biomarkers, including neuromodulators and cytokines that play important roles in peripheral nerve stimulation. To make these biosensor systems available to other researchers, a startup company (RT Biotech) will refine the prototypes and produce them at low volumes for other groups within the SPARC program within 3 years.
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0.954 |