Rastislav Levicky - US grants

This emerging technology of "micro total analysis systems" (uTAS) is ideal for processing of small quantities of chemical or biological species in a miniaturized, chip-based format that is robust, superior in performance, economical, and portable. This proposal focuses on broad scientific and engineering issues needed for integrated optical/microfluidic devices, with a strong component addressing fundamental concepts relevant to processing of liquid-phase materials in confined spaces (e.g. DNA solutions). The program is foremost an effort to advance basic technologies and knowledge rather than a developmental project aimed at producing a specific device. The research topics include: (i) on-chip integration of microfluidic structures with relatively short-wavelength optical systems for light sources, detectors, and waveguide routing systems, (ii) investigation of the transport of simple fluids and macromolecular solutions in submicrometer channels, (iii) fabrication of novel integrated mechanical actuators, and (iv) development of simulation and design tools for integrated microfluidic/optical uTAS systems. Various test structures will be fabricated with regard to these goals, and the fabrication effort will vigorously pursue new materials and prototyping strategies for device production. The program involves an interdisciplinary group of faculty from Columbia University's Departments of Applied Physics, Electrical, Mechanical, and Chemical Engineering as well as collaboration with other universities.

The proposed research is a fundamental investigation of biological polyelectrolyte brushes and their interaction with polyelectrolytes in solution. In addition, the research aims to build an important bridge between biotechnology and the field of polymer science, which is increasingly contributing vital insights to biological systems. A huge past research effort has investigated synthetic polymer brushes, consisting of arrays of polymers tethered by one end to a surface. When these polymers are charged (polyelectrolyte brushes) many basic questions remain unanswered: theory has predicted a complex diagram of states but relatively little experiment exists. The research proposed here advances fundamental understanding of polyelectrolyte brushes by focusing on brushes of the single most important charged biological polymer, DNA. Experimental investigations of DNA brushes placed in contact with free DNA solutions will be carried out, with emphasis on measuring equilibrium and kinetic aspects of the penetration of the free DNA into the brush and its attachment ("hybridization") to complementary regions of the tethered DNA. Theory will be simultaneously developed to approach a comprehensive, molecular-based understanding of the mechanisms involved. If successful, this work will: (i) advance basic understanding of polyelectrolyte brushes, (ii) reveal to what extent theoretical frameworks developed for synthetic polyelectrolyte brushes may be carried over to this biopolymer system, (iii) establish how equilibrium and kinetics of the hybridization process reflect internal organization of the brush, and (iv) result in a systematic understanding of the mechanisms involved in the functioning of modern medical and biological technologies such as DNA microarrays. The primary experimental tool will be confocal fluorescence microscopy, with modulated ellipsometry, X-ray photoelectron spectroscopy, and X-ray reflectivity serving as key secondary characterization tools.
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DNA brushes are at the heart of a biotechnology of enormous importance: the processing of genetic information for disease studies and medical diagnosis by use of DNA chips and microarrays. Since these methods work on the principle of hybridization between surface-tethered and free nucleic acids, the planned research provides the fundamental guidance needed to reduce the current empiricism in their use and optimization. The research program also closely integrates with multiple educational initiatives, including: (1) stimulating intellectual growth by providing research opportunities for students from high school through graduate levels, (2) forging links with the broader educational community through development of a series of experimental units for high school science courses that demonstrate scientific and engineering principles using common "supermarket" materials, and (3) developing new pedagogical structure for the core undergraduate laboratory that will teach students to learn through research-type, objective-driven projects. Successful accomplishment of the research and educational goals of the proposed plan is supported through strategic partnerships, which include George Washington High School, an urban school in the Manhattan School District, and industrial and institutional research collaborations.

0428544
Shepard
This research addresses the development of multiplexed CMOS-based fluorescence sensors for sensing biomolecules (proteins and DNA). The goal is to develop generalized planar arrays with sensor densities on the order of ten thousand sensors per cm2. The investigators will explore both steady state and dynamic fluorescence. The sensor will contain an integrated interference filter consisting of quarter-wavelength layers for rejecting the excitation source. The investigators will consider many measurement issues, and are guided in part by their previous experience with the CMOS biochip labeled Imager F1. These issues include leveraging of economies of scale to provide low-cost devices; optimizing device self-sufficiency and miniaturization to afford maximum portability; developing parallelized designs for detection of multiple target analytes; enabling sensor-level logic for detection, calibration, and control; and optimizing dynamic range, sensitivity, and speed of detection. The existing technology is to do the detection chemistry on a glass slide, and then to carry out the detection via external sensors (or readers). This design while robust is meant for a laboratory setting. The proposed approach of integrating sensing chemistry with sensors has the potential of being compact and portable.

DESCRIPTION (provided by applicant): This research program seeks to integrate silicon microelectronics with genetic analyses to develop advanced diagnostic platforms with potential to address the information content of entire genomes. The proposed technology consists of fully-integrated microelectronic instruments that present an array of sensing sites or pixels, each of which is designed to detect a unique nucleic acid sequence or genetic marker. This format is compatible with the numerous, now established uses of high-throughput cDNA and oligonucleotide microarrays including gene expression studies, genotyping, mutation identification, and comparative genomics. The proposed devices differ from conventional "slide" microarrays in that analyte detection, analog-to-digital conversion, and other functions are integrated directly on-chip via industry-standard complementary-metal-oxide-semiconductor (CMOS) circuitry. Two types of detection technologies will be developed: fluorescent and electrical. Fluorescence detection on-chip eliminates need for macroscopic, external optics and maintains continuity with today's prevalent diagnostic protocols. CMOS microarrays for fluorescence-diagnostics will be capable of time-resolved measurements, to be exploited in two novel ways: (i) temporal separation of excitation pulse and data collection stages, thus allowing operation without integrated optical filters and, (ii) multicolor diagnostics driven by difference in fluorescence lifetimes between dyes. Electrical detection will advance label-free diagnostics that do not require analyte labeling and that can monitor samples in-situ and in real time. Two electrical approaches will be contrasted in an initial feasibility phase: (i) direct measurement of electrical impedance changes accompanying hybridization and, (ii) a field effect actuation method based on shifts in threshold voltage of a transistor realized in a CMOS compatible structure. A fully-featured CMOS instrument, with associated signal processing and data analysis circuits, will be subsequently fabricated based on the most promising approach. Moreover, the effort will explore prospects for on-chip electronic control of hybridization thermodynamics, with view to developing application-specific operational modes (e.g. for genotyping). To provide for future accessibility, device design will maintain maximal compatibility with low-cost CMOS fabrication. Application of inexpensive microelectronic technology to high-throughput genetic analyses, if realized at mass production scales, promises significant reductions in costs for clinical as well as research applications in genomics, pharmacogenomics, and related fields

This award by the Biomaterials Program in the Division of Materials Research to Polytechnic University and to Columbia University is to study "Mechanisms of Hybridization Kinetics in DNA Surface Layers." These collaborative proposals combined experimental and theoretical approaches, and will seek to understand the mechanisms governing competitive surface hybridization, where two different nucleic acid types, one a complementary and the other a mismatched sequence, are present in solution and compete for binding to a single type of immobilized strand on a solid surface. Through this process, the genomic and genetic information contained in the sample is identified and quantified for numerous applications: to study gene function, to identify the genotype of individuals, to perform forensic analysis, to carry out comparative genomic studies across species, or to support other genodiagnostic needs that require quantification of information from nucleic acid molecules. Understanding the complex, dynamic evolution of the competing hybridization reactions occurring during such a measurement is essential both to experimental design and to data analysis. Electrochemical techniques will be used to monitor kinetics of surface hybridization reactions. In parallel and in close interaction, a theoretical effort will develop kinetic models that integrate microscopic theories of reaction rate "constants" needed to analyze and interpret the experimental data. These prototypic studies will then be extended to more complex systems characterized by greater diversity of sequences. This program is expected to contribute to the development of a technologically and scientifically knowledgeable workforce by training individuals at different stages of learning, from high school through graduate school.

Presently the powerful, yet problematic, technologies of surface hybridization lack the firm footing required for clinical applications. The knowledge this project will provide will be especially crucial to healthcare applications such as personalized medicine, where accurate determination of a patient's genetic makeup (genotype) can provide vital lifesaving capability. The program will seek outstanding high school students for summer research in coordination with the New York Academy of Sciences and other local high school outreach programs. The program will also engage undergraduate and graduate students in collaborative research encompassing experiment and theory that will introduce team research skills, provide broad exposure to laboratory, modeling, and theoretical methods, and stimulate career interest at the highly critical interface between chemical, biomaterial, and surface engineering and biotechnology. Educational electronic "research stories" will be posted on the PIs' websites in a format accessible to broad audiences. The program will also enrich institutional curricula, including creation of a lecture-laboratory hybrid course in Biointerfacial Engineering at Polytechnic University.

[unreadable] DESCRIPTION (provided by applicant): Surface hybridization techniques, as widely practiced in DNA microarray and biosensor technologies, have penetrated into nearly every corner of fundamental and applied genomics. In these methods, the identity and/or the quantity of sample nucleic acid material are determined by its hybridization to complementary "probe" molecules immobilized on a solid support. Despite their widespread application, demonstrated versatility, and powerful diagnostic capabilities, surface hybridization measurements are subject to physical and experimental complexities that can seriously confound interpretation of results. These complexities arise in part because the final diagnostic signal is a cumulative reflection of the history of multiple sequential, nonequilibrium rate processes including denaturation of target sample, slow competitive surface hybridization, and washing of the reacted surface to improve sequence discrimination. This research seeks to eliminate the central uncertainties in the practice of surface hybridization by developing diagnostic technology based on Morpholino, instead of DNA, probes. Morpholinos hybridize nucleic acids according to the usual base-pairing rules, possess excellent solubility and chemical stability, and can be prepared at lengths sufficient for nearly all diagnostic applications. Their lack of charge and unique hybridization properties promise a number of advantages which will be adapted to diagnostic applications through the following Specific Aims: 1). Critical comparison of Morpholino and DNA microarrays for pathogen detection in conventional fluorescence as well as label-free detection formats, 2). Development of fundamental understanding of the kinetics and thermodynamics of Morpholino surface hybridization, and 3). Establishment of optimized microarray assay protocols to minimize cross-hybridization and interference from sample secondary structure. Because Morpholinos are not charged, they hybridize with nucleic acids even under low salt conditions. Low ionic strength diagnostics promise to eliminate much of the measurement complexity by providing continually denaturing, high stringency conditions during the assay with prospects for abolishing the need for dedicated processing steps. Moreover, uncharged probes allow for highly specific electrostatic communication between the solid support and sample nucleic acids, a feature that will be exploited to develop electronic methods for controlling the hybridization reaction. By reassessing the nature of the probe platform, the proposed program seeks to overcome key challenges associated with today's large scale surface hybridization technologies, to make their data interpretation more transparent and robust, and ultimately to advance the practice of these powerful tools. PUBLIC HEALTH RELEVANCE The goal of this program is to develop Morpholino probe technology for analysis of nucleic acids by surface hybridization, with applications to pathogen detection. The unique chemical properties of Morpholinos allow them to bind and thus identify nucleic acid sequences under low salt conditions where a markedly enhanced diagnostic response is realized compared to conventional approaches based on DNA probes, and where purely electronic (i.e. not requiring generation and measurement of light) detection of analyte and control of the assay are especially effective. Morpholino assays are expected to be highly suited to applications in gene expression and pathogen analysis, and a demonstration application aimed at identification of respiratorial pathogens is integrated as part of the project. [unreadable] [unreadable] [unreadable]

1126005
Gross

Funds are requested to purchase a 500 MHz NMR to replace the very old and Bruker DPX 300 MHz NMR at Polytechnic Institute of New York University?s (NYU-POLY). The instrument will have two radio-frequency channels, pulsed-field gradients, variable-temperature capability, two solution probes with automatic tune and match, and an autosampler. The current NMR needs replacement for the following reasons: i) it is very old, ii) its capabilities are severely limited, iii) the need for greater dispersion and sensitivity provided by higher field strength. The current NMR has no pulse field gradients and no ability to shape radio-frequency pulse and, as such, it cannot perform modern 2D experiments or modern solvent suppression echniques.Contemporary NMR methods are essential to research at NYU-POLY. Our current instrument is so old that spare parts are no longer available and the instrument will soon cease to function. Furthermore, its sensitivity is poor and the requested NMR will provide five times greater sensitivity. The proposed 500 MHz NMR with high dispersion and ability to perform modern 2D experiments will be an essential research tool to the proposal PI, Co-PI?s, their students (in total 9 High School, 18 undergrad, 21 MS, 27 Ph.D. and 5.5 postdoctoral fellows), and other faculty in the Polymer Research Institute (PRI), Departments of Chemical and Biological Sciences, Chemical and Biological Engineering, and Bioengineering.

ID: MPS/DMR/BMAT(7623) 1206754 PI: Levicky, Rastislav ORG: NYU Polytechnic Institute

Title: Thermodynamic Cycles and Relaxation Timescales in Surface Hybridization

INTELLECTUAL MERIT: Analysis of nucleic acids is becoming increasingly integrated into health care and clinical diagnostics. A technology that especially excels in combining throughput with affordability is multiplexed surface hybridization (SH). Clinical SH tests are becoming available for applications that include cancer diagnostics, cystic fibrosis prescreening, pathogen detection, and assessment of drug metabolism. These tests function by monitoring the extent of hybridization, or association, between two nucleic acid strands, one a "probe" immobilized on a solid material surface and the other a sample "target" sequence present in solution. Arrival of SH technologies in clinical diagnostics greatly intensifies the need for fundamental understanding, so that diagnostic performance can be optimized. This project addresses two outstanding challenges about the material surfaces used in SH applications: (1) the link between hybridization thermodynamics at the surface with those in solution, and (2) the timescales for approaching equilibrium when many target sequences compete for the probes. The first topic is essential for enabling decades of solution hybridization research to be applied to SH applications, for example, to the design of optimized probe sequences and the development of corrective algorithms for cross-hybridization. The second topic centers on identification of rate-limiting bottlenecks in competitive SH, when many target sequences compete for the probes, with the goal to advance strategies for minimizing kinetic biases and for enhancing sensitivity to lower copy target sequences. As part of these studies, benchmark thermodynamic data will be obtained on molecular interactions, including between probes, that affect hybridization reactions on densely modified material surfaces. By elucidating the thermodynamic connection between surface and solution hybridization, and identifying kinetic bottlenecks to equilibration, this project will formulate design principles central to material surfaces used in research and emergent clinical technologies based on surface hybridization.

BROADER IMPACTS: The complex molecular phenomena underlying SH have long hindered development of universal guidelines for diagnostic applications. Results from this project will therefore be immediately useful for design of probe-modified material surfaces as well as for interpretation of experiments performed in hundreds of research and, increasingly, clinical laboratories. The research effort will be integrated with an educational initiative on development of educational gaming software that will introduce general concepts from science and engineering through direct integration into game mechanics. The pedagogical strategy of the software is to develop qualitative familiarity with STEM concepts through an enjoyable gaming experience that can heighten interest in students before they encounter related concepts in formal coursework. The software will be developed in collaboration with the Game Innovation Laboratory at the Polytechnic Institute of NYU, at a level accessible to middle school students. The initial concept for the gaming software is to use fairy tale characters to solve problems through relying on molecular concepts. The software will introduce notions of molecules, of selective interactions between molecules (e.g. assembly of supramolecular structures), and of simple renditions of thermodynamic concepts through use of "molecular bricks" to build fortifications with different energy scores, all as an integral part of game mechanics.

PI: Levicky, Rastislav
Proposal Number: 1600584

The central aim is to advance biosensing of DNA, such as the human genome, in support of personalized medicine, clinical diagnostics, and basic life science research. A technology termed pulsed-field hybridization will be developed by combining electric field control of molecular behavior with optimized sensor designs, based on synthetic DNA analogues, to eliminate errors and to significantly shorten analysis time. The project also encompasses educational goals centered on design of learning software that illustrates molecular properties and on organization of publicly accessible science lectures and demonstrations.


Biosensing applications must handle complex sample matrices in which the analyte of interest needs to be distinguished against a background of other molecules. In nucleic acid diagnostics, background interference most often involves cross-hybridization from DNA or RNA molecules with a degree of similarity to the desired sequence. Cross-hybridization leads to multiple detrimental consequences including blocking binding of the perfectly matched analyte, markedly slowing down approach to equilibrium so that assay endpoints are defined by kinetic history rather than path-independent thermodynamics, and loss of precision due to contributions from the cross-hybridized species. These concerns become amplified for sequence-rich samples comprising larger genomes, mixtures of genetic material from multiple sources, or when the sequence of interest is one of several closely related sequences. This project proposes a bioanalytical method termed pulsed field hybridization (PFH) to solve these challenges. Through use of charge-neutral DNA analogues called morpholinos and modulated electric fields, PFH seeks highly efficient, field-based manipulation of hybridization to dramatically reduce equilibration timescales and to enable "distilling", through a stage-wise operation, of the purity of the hybridized product. The project will develop a thorough fundamental understanding of how surface fields influence thermodynamics and kinetics of hybridization on morpholino-derivatized sensors, and of the impact of analyte characteristics including excess nucleotides, unhybridized tails, and variously placed mismatches. These fundamental studies will be accompanied by application to complex sample matrices typical of clinical situations to demonstrate a fully functional, practical method. Project participants will in addition assist with research exhibitions open to the public, engage in organizing lectures on nanoscience, and collaborate on development of learning software to provide a fun, informative look into the behavior of molecules.