Interferometric detection and enumeration of viral particles using Si-based microfluidics

Single-particle interferometric reflectance imaging sensor enables optical visualization and characterization of individual nanoparticles without any labels. Using this technique, we have shown end-point and real-time detection of viral particles using laminate-based active and passive cartridge configurations. Here, we present a new concept for low-cost microfluidic integration of the sensor chips into compact cartridges through utilization of readily available silicon fabrication technologies. This new cartridge configuration will allow simultaneous detection of individual virus binding events on a 9-spot microarray, and provide the needed simplicity and robustness for routine real-time operation for discrete detection of viral particles in a multiplex format.This work was supported in part by a research contract with the ASELSAN Research Center, Ankara, Turkey, and in part by the European Union's Horizon 2020 FET Open program under Grant 766466-INDEX. (ASELSAN Research Center, Ankara, Turkey; 766466-INDEX - European Union's Horizon 2020 FET Open program)First author draf

Robust visualization and discrimination of nanoparticles by interferometric imaging

Single-molecule and single-nanoparticle biosensors are a growing frontier in diagnostics. Digital biosensors are those which enumerate all specifically immobilized biomolecules or biological nanoparticles, and thereby achieve limits of detection usually beyond the reach of ensemble measurements. Here we review modern optical techniques for single nanoparticle detection and describe the single-particle interferometric reflectance imaging sensor (SP-IRIS). We present challenges associated with reliably detecting faint nanoparticles with SP-IRIS, and describe image acquisition processes and software modifications to address them. Specifically, we describe a image acquisition processing method for the discrimination and accurate counting of nanoparticles that greatly reduces both the number of false positives and false negatives. These engineering improvements are critical steps in the translation of SP-IRIS towards applications in medical diagnostics.R01 AI096159 - NIAID NIH HHSFirst author draf

Instrument design and optimization of interferometric reflectance imaging sensors for in vitro diagnostics

Thesis (Ph.D.)--Boston UniversityIn the field of drug discovery and disease diagnostics, protein microarrays have generated much enthusiasm for their high-throughput monitoring of biomarkers; however, this technology has yet to translate from research laboratories to commercialization. The hindrance is the considerable uncertainty and skepticism regarding data obtained. The disparity in results from different laboratories performing identical tests is attributed to a lack of assay quality control. Unlike DNA microarrays, protein microarrays have a higher level of bioreceptor immobilization variability and non-specific binding because of the more complex molecular structure and broader physiochemical properties. Traditional assay detection modalities, such as fluorescence microscopy and surface plasmon resonance, are unable to overcome both of these sources of variation. This dissertation describes the hardware and software design and biological validation of three complementary platforms that overcome bioreceptor variability and non-specific binding for diagnostics. In order to quantify the bioreceptor quality, a label-free, nondestructive, low cost, and high-throughput interferometric sensor has been developed as a quality control tool. The quality control tool was combined with a wide-field fluorescence imaging system to improve fluorescence experimental repeatability. Lastly, a novel high-throughput and label-free platform for quality control and specific protein microarray detection is described. This platform overcomes the additional complexities and time required with labeled assays by discriminating between specific and nonspecific detection by including sizing of individual binding events. Protein microarrays may one day emerge as routine clinical laboratory tests; however, it is important that the proper quality control procedures are in place to minimize erroneous results. These platforms provide reliable and repeatable protein microarray measurements for new advancements in disease diagnostics with the potential for drug discovery

Interferometric biosensing platform for multiplexed digital detection of viral pathogens and biomarkers

Thesis (Ph.D.)--Boston UniversityLabel-free optical biosensors have been established as proven tools for monitoring specific biomolecular interactions. However, compact and robust embodiments of such instruments have yet to be introduced in order to provide sensitive, quantitative, and high-throughput biosensing for low-cost research and clinical applications. Here we present the interferometric reflectance-imaging sensor (IRIS). IRIS allows sensitive label free analysis using an inexpensive and durable multi-color LED illumination source on a silicon based surface. IRIS monitors biomolecular interaction through measurement of biomass addition to the sensor's surface. We demonstrate the capability of this system to dynamically monitor antigen-antibody interactions with a noise floor of 5.2 pg/mm^2 and DNA single mismatch detection under isothermal melting conditions in an array format. Ensemble detection of binding events using IRIS did not provide the sensitivity needed for detection of infectious disease and biomarkers at clinically relevant concentrations. Therefore, a new approach was adapted to the IRIS platform that allowed the detection and identification of individual nanoparticles on the sensor's surface. The new detection method was te1med single-particle IRIS (SP-IRIS). We developed two detection modalities for SP-IRIS. The first modality is when the target is a nanoparticle such as a virus. We verified that SP-IRIS can accurately detect and size individual viral particles. Then we demonstrated that single nanoparticle counting and sizing methodology on SP-IRIS leads to a specific and sensitive virus sensor that can be multiplexed. Finally, we developed an assay for the detection of Ebola and Marburg. A detection limit of 3 x 10^3 PFU/ml was demonstrated for vesicular stomatitis virus (VSV) pseudotyped with Ebola or Marburg virus glycoprotein. We have demonstrated that virus detection can be done in human whole blood directly without the need for sample preparation. The second modality of SP-IRIS we developed was single molecule counting of biomarkers utilizing a sandwich assay with detection probes labeled with gold nanoparticles. We demonstrated the use of single molecule counting in a nucleic acid assay for melanoma biomarker detection. We showed that a single molecule counting assay can lead to detection limits in the attomolar range. The improved sensitivity of IRIS utilizing single nanoparticle detection holds promise for a simple and low-cost technology for rapid virus detection and multiplexed molecular screening for clinical applications

The Boston University Photonics Center annual report 2013-2014

This repository item contains an annual report that summarizes activities of the Boston University Photonics Center in the 2013-2014 academic year. The report provides quantitative and descriptive information regarding photonics programs in education, interdisciplinary research, business innovation, and technology development. The Boston University Photonics Center (BUPC) is an interdisciplinary hub for education, research, scholarship, innovation, and technology development associated with practical uses of light.This annual report summarizes activities of the Boston University Photonics Center in the 2013–2014 academic year.This has been a good year for the Photonics Center. In the following pages, you will see that the center’s faculty received prodigious honors and awards, generated more than 100 notable scholarly publications in the leading journals in our field, and attracted $14.5M in new research grants and contracts this year. Faculty and staff also expanded their efforts in education and training, through National Science Foundation–sponsored sites for Research Experiences for Undergraduates and for Teachers. As a community, we hosted a compelling series of distinguished invited speakers, and emphasized the theme of Innovations at the Intersections of Micro/Nanofabrication Technology, Biology, and Biomedicine at our annual Future of Light Symposium. We took a leadership role in running national workshops on emerging photonic fields, including an OSA Incubator on Controlled Light Propagation through Complex Media, and an NSF Workshop on Noninvasive Imaging of Brain Function. Highlights of our research achievements for the year include a distinctive Presidential Early Career Award for Scientists and Engineers (PECASE) for Assistant Professor Xue Han, an ambitious new DoD-sponsored grant for Multi-Scale Multi-Disciplinary Modeling of Electronic Materials led by Professor Enrico Bellotti, launch of our NIH-sponsored Center for Innovation in Point of Care Technologies for the Future of Cancer Care led by Professor Cathy Klapperich, and successful completion of the ambitious IARPA-funded contract for Next Generation Solid Immersion Microscopy for Fault Isolation in Back-Side Circuit Analysis led by Professor Bennett Goldberg. These three programs, which represent more than$20M in research funding for the University, are indicative of the breadth of Photonics Center research interests: from fundamental modeling of optoelectronic materials to practical development of cancer diagnostics, from exciting new discoveries in optogenetics for understanding brain function to the achievement of world-record resolution in semiconductor circuit microscopy. Our community welcomed an auspicious cohort of new faculty members, including a newly hired assistant professor and a newly hired professor (and Chair of the Mechanical Engineering Department). The Industry/University Cooperative Research Center—the centerpiece of our translational biophotonics program—continues to focus on advancing the health care and medical device industries, and has entered its fourth year of operation with a strong record of achievement and with the support of an enthusiastic industrial membership base

The Boston University Photonics Center annual report 2013-2014

This repository item contains an annual report that summarizes activities of the Boston University Photonics Center in the 2013-2014 academic year. The report provides quantitative and descriptive information regarding photonics programs in education, interdisciplinary research, business innovation, and technology development. The Boston University Photonics Center (BUPC) is an interdisciplinary hub for education, research, scholarship, innovation, and technology development associated with practical uses of light.This annual report summarizes activities of the Boston University Photonics Center in the 2013–2014 academic year.This has been a good year for the Photonics Center. In the following pages, you will see that the center’s faculty received prodigious honors and awards, generated more than 100 notable scholarly publications in the leading journals in our field, and attracted $14.5M in new research grants and contracts this year. Faculty and staff also expanded their efforts in education and training, through National Science Foundation–sponsored sites for Research Experiences for Undergraduates and for Teachers. As a community, we hosted a compelling series of distinguished invited speakers, and emphasized the theme of Innovations at the Intersections of Micro/Nanofabrication Technology, Biology, and Biomedicine at our annual Future of Light Symposium. We took a leadership role in running national workshops on emerging photonic fields, including an OSA Incubator on Controlled Light Propagation through Complex Media, and an NSF Workshop on Noninvasive Imaging of Brain Function. Highlights of our research achievements for the year include a distinctive Presidential Early Career Award for Scientists and Engineers (PECASE) for Assistant Professor Xue Han, an ambitious new DoD-sponsored grant for Multi-Scale Multi-Disciplinary Modeling of Electronic Materials led by Professor Enrico Bellotti, launch of our NIH-sponsored Center for Innovation in Point of Care Technologies for the Future of Cancer Care led by Professor Cathy Klapperich, and successful completion of the ambitious IARPA-funded contract for Next Generation Solid Immersion Microscopy for Fault Isolation in Back-Side Circuit Analysis led by Professor Bennett Goldberg. These three programs, which represent more than$20M in research funding for the University, are indicative of the breadth of Photonics Center research interests: from fundamental modeling of optoelectronic materials to practical development of cancer diagnostics, from exciting new discoveries in optogenetics for understanding brain function to the achievement of world-record resolution in semiconductor circuit microscopy. Our community welcomed an auspicious cohort of new faculty members, including a newly hired assistant professor and a newly hired professor (and Chair of the Mechanical Engineering Department). The Industry/University Cooperative Research Center—the centerpiece of our translational biophotonics program—continues to focus on advancing the health care and medical device industries, and has entered its fourth year of operation with a strong record of achievement and with the support of an enthusiastic industrial membership base

The Boston University Photonics Center annual report 2014-2015

This repository item contains an annual report that summarizes activities of the Boston University Photonics Center in the 2014-2015 academic year. The report provides quantitative and descriptive information regarding photonics programs in education, interdisciplinary research, business innovation, and technology development. The Boston University Photonics Center (BUPC) is an interdisciplinary hub for education, research, scholarship, innovation, and technology development associated with practical uses of light.This has been a good year for the Photonics Center. In the following pages, you will see that the center’s faculty received prodigious honors and awards, generated more than 100 notable scholarly publications in the leading journals in our field, and attracted $18.6M in new research grants/contracts. Faculty and staff also expanded their efforts in education and training, and were awarded two new National Science Foundation– sponsored sites for Research Experiences for Undergraduates and for Teachers. As a community, we hosted a compelling series of distinguished invited speakers, and emphasized the theme of Advanced Materials by Design for the 21st Century at our annual symposium. We continued to support the National Photonics Initiative, and are a part of a New York–based consortium that won the competition for a new photonics- themed node in the National Network of Manufacturing Institutes. Highlights of our research achievements for the year include an ambitious new DoD-sponsored grant for Multi-Scale Multi-Disciplinary Modeling of Electronic Materials led by Professor Enrico Bellotti, continued support of our NIH-sponsored Center for Innovation in Point of Care Technologies for the Future of Cancer Care led by Professor Catherine Klapperich, a new award for Personalized Chemotherapy Through Rapid Monitoring with Wearable Optics led by Assistant Professor Darren Roblyer, and a new award from DARPA to conduct research on Calligraphy to Build Tunable Optical Metamaterials led by Professor Dave Bishop. We were also honored to receive an award from the Massachusetts Life Sciences Center to develop a biophotonics laboratory in our Business Innovation Center

The Boston University Photonics Center annual report 2014-2015

This repository item contains an annual report that summarizes activities of the Boston University Photonics Center in the 2014-2015 academic year. The report provides quantitative and descriptive information regarding photonics programs in education, interdisciplinary research, business innovation, and technology development. The Boston University Photonics Center (BUPC) is an interdisciplinary hub for education, research, scholarship, innovation, and technology development associated with practical uses of light.This has been a good year for the Photonics Center. In the following pages, you will see that the center’s faculty received prodigious honors and awards, generated more than 100 notable scholarly publications in the leading journals in our field, and attracted $18.6M in new research grants/contracts. Faculty and staff also expanded their efforts in education and training, and were awarded two new National Science Foundation– sponsored sites for Research Experiences for Undergraduates and for Teachers. As a community, we hosted a compelling series of distinguished invited speakers, and emphasized the theme of Advanced Materials by Design for the 21st Century at our annual symposium. We continued to support the National Photonics Initiative, and are a part of a New York–based consortium that won the competition for a new photonics- themed node in the National Network of Manufacturing Institutes. Highlights of our research achievements for the year include an ambitious new DoD-sponsored grant for Multi-Scale Multi-Disciplinary Modeling of Electronic Materials led by Professor Enrico Bellotti, continued support of our NIH-sponsored Center for Innovation in Point of Care Technologies for the Future of Cancer Care led by Professor Catherine Klapperich, a new award for Personalized Chemotherapy Through Rapid Monitoring with Wearable Optics led by Assistant Professor Darren Roblyer, and a new award from DARPA to conduct research on Calligraphy to Build Tunable Optical Metamaterials led by Professor Dave Bishop. We were also honored to receive an award from the Massachusetts Life Sciences Center to develop a biophotonics laboratory in our Business Innovation Center

The Boston University Photonics Center annual report 2015-2016

This repository item contains an annual report that summarizes activities of the Boston University Photonics Center in the 2015-2016 academic year. The report provides quantitative and descriptive information regarding photonics programs in education, interdisciplinary research, business innovation, and technology development. The Boston University Photonics Center (BUPC) is an interdisciplinary hub for education, research, scholarship, innovation, and technology development associated with practical uses of light.This has been a good year for the Photonics Center. In the following pages, you will see that this year the Center’s faculty received prodigious honors and awards, generated more than 100 notable scholarly publications in the leading journals in our field, and attracted $18.9M in new research grants/contracts. Faculty and staff also expanded their efforts in education and training, and cooperated in supporting National Science Foundation sponsored Sites for Research Experiences for Undergraduates and for Research Experiences for Teachers. As a community, we emphasized the theme of “Frontiers in Plasmonics as Enabling Science in Photonics and Beyond” at our annual symposium, hosted by Bjoern Reinhard. We continued to support the National Photonics Initiative, and contributed as a cooperating site in the American Institute for Manufacturing Integrated Photonics (AIM Photonics) which began this year as a new photonics-themed node in the National Network of Manufacturing Institutes. Highlights of our research achievements for the year include an ambitious new DoD-sponsored grant for Development of Less Toxic Treatment Strategies for Metastatic and Drug Resistant Breast Cancer Using Noninvasive Optical Monitoring led by Professor Darren Roblyer, continued support of our NIH-sponsored, Center for Innovation in Point of Care Technologies for the Future of Cancer Care led by Professor Cathy Klapperich, and an exciting confluence of new grant awards in the area of Neurophotonics led by Professors Christopher Gabel, Timothy Gardner, Xue Han, Jerome Mertz, Siddharth Ramachandran, Jason Ritt, and John White. Neurophotonics is fast becoming a leading area of strength of the Photonics Center. The Industry/University Collaborative Research Center, which has become the centerpiece of our translational biophotonics program, continues to focus onadvancing the health care and medical device industries, and has entered its sixth year of operation with a strong record of achievement and with the support of an enthusiastic industrial membership base

Optimization of a microarray-based biosensor for detection of viral pathogens

Rapid and sensitive detection of viral infections is of significant importance for improving patient care and containing outbreaks that threaten public health. Although there has been an enormous effort to develop point-of-care biosensors for viral diagnostics applications, sensitive, robust and easily portable platforms have yet to be realized. This dissertation focuses on optimization of a multiplexed immunoassay platform for viral diagnostics applications using a label-free optical biosensor termed Single-Particle Interferometric Reflectance Imaging Sensor (SP-IRIS). SP-IRIS utilizes an antibody microarray that captures the target viruses and an optical instrument that allows visualization of individual captured virus particles. Since this platform relies on capture of whole viruses, it is crucial to identify high-affinity antibodies that are capable of recognizing intact virions. For this purpose, we screened various antibodies for their performance on the SP-IRIS platform. By screening 43 different antibodies for three different viruses, we demonstrated specific and sensitive detection of different viruses and different subtypes of the same virus. This work allowed us to assemble an antibody microarray capable of multiplexed detection that has been tested in our laboratory as well as at two separate high-containment facilities. Next, we adapted a different antibody immobilization technique, DNA-directed antibody immobilization (DDI), to the SP-IRIS platform as a means to improve the sensitivity and robustness of the assay. First, we characterized the elevation of the antibodies conjugated to a DNA sequence on a three-dimensional polymeric surface using a fluorescence axial localization technique, Spectral Self-Interference Fluorescence Microscopy, determining the optimal length of the DNA linkers for SP-IRIS substrates. We subsequently showed the specific detection of Vesicular Stomatitis Virus (VSV) expressing Ebola glycoprotein on SP-IRIS platform using the DDI approach. We showed that DNA-conjugated antibodies improve the capture efficiency resulting in over a ten-fold improvement in assay sensitivity compared to directly immobilized antibodies. To demonstrate the feasibility of the DDI technique for multiplexed virus detection utilizing SP-IRIS, we used VSVs expressing Ebola, Marburg or Lassa surface glycoproteins and successfully demonstrated specific and multiplexed detection using a DNA microarray surface. We also combined this approach with a passive microfluidic cartridge, demonstrating the feasibility of SP-IRIS as a rapid testing technique that is well suited for point-of-care applications