Wiktor P.,Engineering Arts, Llc |
Wiktor P.,Arizona State University |
Brunner A.,Engineering Arts, Llc |
Kahn P.,Engineering Arts, Llc |
And 5 more authors.
Scientific Reports | Year: 2015
We report a device to fill an array of small chemical reaction chambers (microreactors) with reagent and then seal them using pressurized viscous liquid acting through a flexible membrane. The device enables multiple, independent chemical reactions involving free floating intermediate molecules without interference from neighboring reactions or external environments. The device is validated by protein expressed in situ directly from DNA in a microarray of ∼10,000 spots with no diffusion during three hours incubation. Using the device to probe for an autoantibody cancer biomarker in blood serum sample gave five times higher signal to background ratio compared to standard protein microarray expressed on a flat microscope slide. Physical design principles to effectively fill the array of microreactors with reagent and experimental results of alternate methods for sealing the microreactors are presented.
Song L.,Arizona State University |
Wallstrom G.,Arizona State University |
Yu X.,Beijing Institute of Radiation Medicine |
Hopper M.,Arizona State University |
And 13 more authors.
Molecular and Cellular Proteomics | Year: 2017
Better and more diverse biomarkers for the development of simple point-of-care tests for active tuberculosis (TB), a clinically heterogeneous disease, are urgently needed. We generated a proteomic Mycobacterium tuberculosis (Mtb) High-Density Nucleic Acid Programmable Protein Array (HD-NAPPA) that used a novel multiplexed strategy for expedited high-throughput screening for antibody responses to the Mtb proteome. We screened sera from HIV uninfected and coinfected TB patients and controls (n = 120) from the US and South Africa (SA) using the multiplex HD-NAPPA for discovery, followed by deconvolution and validation through single protein HD-NAPPA with biologically independent samples (n = 124). We verified the top proteins with enzyme-linked immunosorbent assays (ELISA) using the original screening and validation samples (n = 244) and heretofore untested samples (n = 41). We identified 8 proteins with TB biomarker value; four (Rv0054, Rv0831c, Rv2031c and Rv0222) of these were previously identified in serology studies, and four (Rv0948c, Rv2853, Rv3405c, Rv3544c) were not known to elicit antibody responses. Using ELISA data, we created classifiers that could discriminate patients' TB status according to geography (US or SA) and HIV (HIV- or HIV+) status. With ROC curve analysis under cross validation, the classifiers performed with an AUC for US/HIV- at 0.807; US/HIV+ at 0.782; SA/HIV- at 0.868; and SA/HIV+ at 0.723. With this study we demonstrate a new platform for biomarker/antibody screening and delineate its utility to identify previously unknown immunoreactive proteins. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
PubMed | University of Florida, Queen's University of Belfast, Arizona State University, University of Ulster and Engineering Arts, Llc
Type: Comparative Study | Journal: Proteomics | Year: 2015
Viral infections elicit antiviral antibodies and have been associated with various chronic diseases. Detection of these antibodies can facilitate diagnosis, treatment of infection, and understanding of the mechanisms of virus-associated diseases. In this work, we assayed antiviral antibodies using a novel high-density nucleic acid programmable protein array (HD-NAPPA) platform. Individual viral proteins were expressed in situ directly from plasmids encoding proteins in an array of microscopic reaction chambers. Quality of protein display and serum response was assured by comparing intra- and inter-array correlation within or between printing batches with average correlation coefficients of 0.91 and 0.96, respectively. HD-NAPPA showed higher signal-to-background ratio compared with standard NAPPA on planar glass slides and ELISA. Antibody responses to 761 antigens from 25 different viruses were profiled among patients with juvenile idiopathic arthritis and type 1 diabetes. Common and unique antibody reactivity patterns were detected between patients and healthy controls. We believe HD-viral-NAPPA will enable the study of host-pathogen interactions at unprecedented dimensions and elucidate the role of pathogen infections in disease development.
Arizona Board Of Regents and Engineering Arts, Llc | Date: 2012-10-24
Biomolecule arrays on a substrate are described which contain a plurality of biomolecules, such as coding nucleic acids and/or isolated polypeptides, at a plurality of discrete, isolated, locations. The arrays can be used, for example, in high throughput genomics and proteomics for specific uses including, but not limited molecular diagnostics for early detection, diagnosis, treatment, prognosis, monitoring clinical response, and protein crystallography.
Takulapalli B.R.,Arizona State University |
Qiu J.,Arizona State University |
Magee D.M.,Arizona State University |
Kahn P.,Engineering Arts, Llc |
And 12 more authors.
Journal of Proteome Research | Year: 2012
Proteomics aspires to elucidate the functions of all proteins. Protein microarrays provide an important step by enabling high-throughput studies of displayed proteins. However, many functional assays of proteins include untethered intermediates or products, which could frustrate the use of planar arrays at very high densities because of diffusion to neighboring features. The nucleic acid programmable protein array (NAPPA) is a robust in situ synthesis method for producing functional proteins just-in-time, which includes steps with diffusible intermediates. We determined that diffusion of expressed proteins led to cross-binding at neighboring spots at very high densities with reduced interspot spacing. To address this limitation, we have developed an innovative platform using photolithographically etched discrete silicon nanowells and used NAPPA as a test case. This arrested protein diffusion and cross-binding. We present confined high density protein expression and display, as well as functional protein-protein interactions, in 8000 nanowell arrays. This is the highest density of individual proteins in nanovessels demonstrated on a single slide. We further present proof of principle results on ultrahigh density protein arrays capable of up to 24000 nanowells on a single slide. © 2012 American Chemical Society.
PubMed | Arizona State University and Engineering Arts, Llc
Type: | Journal: Scientific reports | Year: 2015
We report a device to fill an array of small chemical reaction chambers (microreactors) with reagent and then seal them using pressurized viscous liquid acting through a flexible membrane. The device enables multiple, independent chemical reactions involving free floating intermediate molecules without interference from neighboring reactions or external environments. The device is validated by protein expressed in situ directly from DNA in a microarray of ~10,000 spots with no diffusion during three hours incubation. Using the device to probe for an autoantibody cancer biomarker in blood serum sample gave five times higher signal to background ratio compared to standard protein microarray expressed on a flat microscope slide. Physical design principles to effectively fill the array of microreactors with reagent and experimental results of alternate methods for sealing the microreactors are presented.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 149.80K | Year: 2014
DESCRIPTION (provided by applicant): Project Summary / Abstract Engineering Arts (EA) proposes the development of a detachable nozzle piezoelectric dispense system for use in Cryo-TEM (Cold - Transmission Electron Microscopy) sample preparation instrumentation. Cryo-TEM is an important tool for studying the structure of biological macromolecules in their native aqueous state. The challenging and laborious process of preparing 'thin-ice' frozen aqueous samples for Cryo-TEM has remained essentially unchangedfor decades. Typically a single aqueous biological sample (~3 l) is manually applied onto a thin porous Electron Microscopy (EM) grid which is then blotted to remove excess sample and then plunged into liquid-ethane for flash freezing. This process hopefully leaves enough regions on the grid with the proper vitrified 'thin-ice', typically 50 to 200nm thickness, required for EM imaging. The detachable piezoelectric nozzle dispense system proposed here is a key innovation that will lead to productivity,
Agency: Department of Health and Human Services | Branch: | Program: STTR | Phase: Phase II | Award Amount: 1.79M | Year: 2012
DESCRIPTION (provided by applicant): Among the currently available techniques for high throughput proteomics, protein microarrays have the greatest prospects to revolutionize molecular diagnostics for early detection, diagnosis, treatment, prognosis and monitoring clinical response. However, protein microarrays have yet to reach their full potential as a research or clinical molecular diagnostics tool due to difficulties associated with their manufacture. Currently protein microarrays are manufactured by expressing and purifying thousands of proteins, which are then stored until they are printed using pin-spotters, a process flow with many inherent logistical problems. Furthermore, many proteins are unstable so these steps must all be maintained at cold temperature. Problems associated with pin spotters include: relatively slow printing speeds, poor spot morphology, pin biofouling issues, variable spot sizes, limited microarray densities and others. Thus, there are compelling needs for better and less expensive manufacturing methods for protein microarrays. In this grant we will combine two successful technologies to develop an innovative method for mass production of faster, better and cheaper protein microarrays. One technology is based on our advanced highspeed piezoelectric pipettes to print arrays of cDNA templates and the other is to express proteins in situ directly on the microarray surface. Engineering Arts specializes in providing microarray production solutions based on its proprietary piezoelectricpipetting technology. Dr. LaBaer is the co-inventor of nucleic acid programmable protein arrays (NAPPA): the very first method to express proteins in situ directly in a microarray format. Engineering Arts will install one of its production-scale piezoelectric microarray machines (POC2) in Dr. LaBaer's Center for Personalized Diagnostics (CPD), Biodesign Institute, Arizona State University. We will develop tools, protocols and process controls required to manufacture production-scale, commercial-grade, high-density, customizable protein microarrays making them readily accessible to the broad proteomics research and clinical diagnostics communities. This grant directly addresses the call to develop a broadly applicable research tool that addresses a core technical challenge in proteomics. By making high quality protein microarrays more readily assessable, this grant will help unlock their true potential for research and clinical applications. This grant brings together world-class piezoelectric pipettes and electronics developed at Engineering Arts, over ten years experience in developing commercial automated production-scale piezoelectric microarraying manufacturing capabilities for high-density whole-genome gene expression microarrays; world class production-scale automation process manufacturing equipment from an established Singapore based semiconductor production equipment manufacturer, Dr. LaBaer's unique and patented NAPPA technology together in his CPD to develop, characterize and validate the nextgeneration of commercial protein microarrays. PUBLIC HEALTH RELEVANCE: Nearly all diagnostics and therapeutics act through proteins, which are the working machines of biology. The study of proteins, both their activities and their dysfunction in disease, has been historically managed one- protein-at-a-time; however, this will be dramatically accelerated through the use of protein microarrays, which microscopically display thousands of functional proteins. This grant will develop technology to mass produce better and less expensive protein microarrays, making them more readily accessible to the broad research and health care communities.
Scripps Research Institute and Engineering Arts, Llc | Date: 2013-01-14
The invention provides methods and devices for preparing frozen vitrified samples for transmission electron microscopy. By reducing the volume of sample from microliter scale to picoliter scale, the requirement for blotting of excess fluid is minimized or eliminated.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 149.80K | Year: 2012
DESCRIPTION (provided by applicant): Every gene has its own set of small RNA molecules, known as small interfering RNA (siRNA), that inhibit expression of the gene's proteins. The siRNA molecules are part of an ancient natural mechanism of gene regulation,known as RNA interference (RNAi) that has been evolutionarily conserved since the earliest eukaryotic cells. More recently researchers have exploited RNAi to systematically knockdown, one-at-a-time, every single gene in living cell-cultures. These sets ofexperiments, known as cell-based genomic high throughput screening (HTS) provide insight into gene function especially as it relates to diseases and their treatments. Currently genomic HTS is typically done in 96 or 384-well microplates. A set of 100 plates is required to run HTS on the whole human genome. These large screens are now only done at well-funded institutions using rooms full of expensive automated liquid and microplate handling equipment. Our collaborator on this grant, Dr. Saez, has co-invented a novel HTS gene function platform, called, 'electroporation-ready- microwell-arrays', that will allow whole human genome screens on a single plate that is ready for cell culture and electroporation. The platform consists of a micro-machined array of electrically conductive micorwells that enable simultaneous electroporation of cultured-cells with thousands of different siRNA. This platform will enable genomic HTS to be routinely performed in smaller research labs which will dramatically increase the rate of discovery of new molecular pathways related to disease with corresponding impact on novel treatments and public health. As proof of concept, in this grant, we will perform a smaller screen for the human kinome, a set of 518 genes for kinase enzymes that are key controllers of cell activity and have great pharmaceutical significance. Engineering Arts has developed proprietary non-contact piezoelectric inkjet dispensing technology for microarraying applications. We have developed a high-speed microarraying instrument capable of on-the-fly dispensing of thousands of different sub-nanoliter sized reagents onto 36 individual microscope- slide substrates. Engineering Arts has already manufactured a handful of proof-of-concept electroporation- ready-microwell-arrays platforms for Saez's lab with encouraging preliminary results. Under this grant we will develop the additional manufacturing technology required to automatically align the relative positions of the microscopic features of Saez's platform within +/-20 um. We will also increase high-speed dispensing volume to ~10 nanoliter per microwell. Together these innovations will allow manufacturing production rates of 36 electroporation-ready-microwell-arrays platforms in 8 hours. After manufacturing the platforms, they will be tested in Saez's lab at the SCRIPPS Institute in La Jolla California and Pedro Aza's lab at Burnham, who ran a similar screen using conventional 384-well plates. The novel alignment technology and dispensing technology developed under this grant will enable many other biomedical applications that require the capability to deliver small fluid volumes precisely to microscopic features on 'biochips'. PUBLIC HEALTH RELEVANCE: New miniature test platforms are being developed with thousands of microscopic features to do thousands of biology experiments simultaneously. In this grant, Engineering Arts will develop the critical manufacturing technology to rapidly and cost-effectively deliver thousands of different nanoliter-sized drops ofbiological molecules or other chemicals to those features. The value of the manufacturing technology will be demonstrated on a platform that enables the simultaneous study of the function of thousands of genes in living cells.