DOCSLIB.ORG

Low-Density Protein Microarray on Nitrocellulose Membrane for the Detection of Human Cytokines

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Low-Density Protein Microarray on Nitrocellulose Membrane for the Detection of Human Cytokines C. Y. Yu, M. L. Liu, I. C. Kuan, et al Low-Density Protein Microarray on Nitrocellulose Membrane for the Detection of Human Cytokines Chi-Yang Yu, Mao-Lung Liu, I-Ching Kuan and Shiow-Ling Lee Department of Bioengineering, Tatung University, Taipei, Taiwan, Republic of China A low-cost, low-density protein microarray on nitrocellulose membrane was developed for the de- tection of human cytokines. The microarray was derived from chemiluminescent sandwich-type immunoassay. Anti-human IL-2 and anti-human IL-4 polyclonal antibodies were arrayed and im- mobilized on the membrane. After the membrane was blocked and then incubation with human cytokines IL-2 and IL-4, biotinylated anti-human IL-2 and anti-human IL-4 monoclonal antibodies were added to form complexes with the cytokines and the immobilized polyclonal antibodies. Streptavidin-horseradish peroxidase conjugate was applied to detect the level of biotin. Finally, substrates for peroxidase were added to initiate chemiluminescence. The printed spots were uni- form and consistent in size. Under optimized conditions, the limits of detection for human IL-2 and IL-4 were 0.5 and 0.125 ng/ml, respectively. The linear range of the calibration curves spanned over two orders of magnitude. The application of 10 ng/ml streptavidin-peroxidase polymer was found to facilitate the analysis process. Key words: Chemiluminescence, cytokine, microarray, nitrocellulose tions [5]. Human cytokines IL-2 and IL-4 were selected Introduction as model analytes to compare our system with others [6,7]. The microarray was based on chemiluminescent Protein microarray has become an important research sandwich-type immunoassay. Anti-human IL-2 and tool in proteomics. While the combination of two- anti-human IL-4 polyclonal antibodies were printed and dimensional gel electrophoresis and mass spectrometry immobilized on nitrocellulose membranes with a manual is very useful for discovery-oriented proteomics, protein microarrayer. The membrane was then blocked with bo- microarray is well suited for the study of a collection of vine serum albumin (BSA). After the membrane was related proteins [1]. The most important advantage of incubated with human cytokines, biotinylated anti-human protein microarray is the ability to perform multiplexed IL-2 and anti-human IL-4 monoclonal antibodies were analysis. Other advantages include small sample vol- added to form sandwiched complexes with the cytokines umes, low reagent consumption, low limit of detection and the polyclonal antibodies immobilized on the mem- (LOD), and high sensitivity [2]. brane. The biotin label was detected by adding strepta- Although protein microarray is a powerful research vidin-horseradish peroxidase conjugate (SA-HRP). The tool, one major disadvantage is its high cost. The cost of peroxidase emitted chemiluminescence when its sub- a commercial system ranges from 25,000 to 220,000 strate was added. A digital camera originally designed USD [3]. Colorimetric detection has been applied to for fluorescence microscopy was converted to a simple protein microarray as a low-cost alternative [4]. How- detector for the chemiluminescence. ever, the LOD remains high compared to fluorescence or chemiluminescence. Materials and Methods We developed a low-cost, low-density protein mi- croarray system suitable for protein-detecting applica- Substrate Preparation and Printing Received: June 1, 2006 Revised: July 31, 2006 Accepted: September 11, 2006 Address for correspondence: Chi-Yang Yu, 40 Chung-Shan N. Rd., Sec. 3, Taipei, Taiwan 104, R.O.C., Tel: 886-2-25925252-3315-27 Fax: 886-2-25854735 E-mail: [email protected] J Biomed Lab Sci 2006 Vol 18 No 1~4 15 Protein microarray for cytokine detection Protran® nitrocellulose membrane (Schleicher & Schuell, streptavidin-peroxidase polymer (Sigma, St. Louis, USA). Dassel, Germany) was cut into 2.2×6 cm. Two pieces of 2.2×0.3 cm double-sided tapes were attached to the 2.2 cm sides of the membrane. The membrane was then at- Results and Discussion tached to a standard glass slide using the tapes; the 2.2 cm side of the membrane was flushed with one of the Printing Quality of the Microarray short sides of the slide. The margins from the 6 cm sides of the membrane to the long sides of the slide were kept MicroCaster hand–held microarray system has a maxi- × equal. Pins of the MicroCaster hand–held microarray mal printing density of 768 spots (24 32; x-axis pitch= μ μ × system (Schleicher & Schuell, Dassel, Germany) were 750 m and y-axis pitch=1125 m) in a 1.8 3.6 cm area. coated with the supplier’s pin conditioner according to The capillary action of the porous membrane may their protocol. enlarge the printed spots. To examine this effect, we Enzymes, antibodies, and recombinant human cyto- printed 0.3 % methylene blue on the membrane. Maxi- kines were purchased from Pierce (Rockford, USA). mal printing density was obtained without overlapping PBS buffer was printed as negative control; 0.1 mg /ml spots. The spots were almost circular under microscope × of horseradish peroxidase and 0.5 μg /ml of biotinylated (4 10 magnification). Using ocular micrometer, the av- anti-IL-4 monoclonal antibody were printed as positive erage diameter of the spots was 634.6±12.3 μm (CV= controls. For the detection of cytokines, 0.1 and 1 mg/ml 1.9 %, n=60). The arrayer has eight printing pins. We polyclonal antibody were used. used average diameter from each grid (spots printed by the same pin, n=8) to determine the variation in diameter Detection of Cytokines caused by individual printing pins. The CV between grids was only 0.5 %, suggesting the pins were almost The membrane was detached from the slide after print- identical in terms of printing size. ing and placed in a small zip-locked plastic bag. All in- The radial diffusion of the chemiluminescent reac- cubation steps were performed at room temperature on tion intermediates often results in a larger spot image an orbital shaker. The membrane was blocked in 1 ml of than the actual printed size. This effect may cause larger PBS containing 1 % (w/v) BSA for 1 h. After blocking, variation in spot diameter and decrease the practical ar- the membrane was incubated with 0.5 ml of cytokine raying density. We printed 25 μg/ml horseradish peroxi- standard (mixture of IL-2 and IL-4) prepared in the dase onto the membrane and initiated the chemilumi- blocking solution for 2 h. The membrane was then nescence reaction by incubating the membrane with Su- washed three times with 15 ml of PBS containing 0.1 % perSignal West Femto substrate for 5 min. The diameter (v/v) Tween 20 in a petri dish for 5 min. One milliliter of of the chemiluminescent spot determined in pixel num- 62.5 ng/ml biotinylated antibody (mixture of anti-human bers using Scion Image software (www.scioncorp.com) IL-2 and anti-human IL-4) in PBS was added and incu- was uniform (CV=3 %, n=60). Maximal printing density bated for 2 h, and then the membrane was washed as (768=24×32) could not be achieved due to spot overlap- previously described. After incubation with 1 ml of 10 ping. The highest printing density was decreased to 192 ng/ml SA-HRP in PBS for 1 h, the membrane was (12×16; x-axis pitch=1500 μm and y-axis pitch=2250 washed again, followed by incubation with 0.5 ml of μm). A 10×10 pixel area within the spot was selected to SuperSignal West Femto substrate (Pierce, Rockford, determine the uniformity of chemiluminescence. The CV USA) for 5 min. The membrane was covered in Saran of the intensity ranged from 1.5 to 5 % (n=60), suggest- wrap and placed in the detector. ing the printed spots were uniform. The simple chemiluminescence detector was based The complete system can be built for less than on a Penguin 600CLM digital camera (Pixera, Los Gatos, 10,000 USD. The cost of the printing substrate is under 1 USA) that is originally designed for fluorescence mi- USD, which is at least ten times lower than most com- croscopy imaging. The camera was first attached to a mercial products [3]. macro video zoom lens (Optem, Fairport, USA) and the assembly was mounted on a dark box. The exposure Optimization of Cytokine Detection time was set to 60 s (2 integrated frames, ISO=400). The images were analyzed using ScanAlyze software The specificity of the antibodies was examined by incu- (rana.lbl.gov/EisenSoftware.htm). For signal amplifica- bating the microarrays with 12.5 ng/ml IL-2 or IL-4 in tion, SA-HRP was replaced by various concentrations of PBS containing 1 % (w/v) BSA. No cross-reaction be- 16 J Biomed Lab Sci 2006 Vol 18 No1~4 C. Y. Yu, M. L. Liu, I. C. Kuan, et al tween the antibody pairs was observed when compared antibody pairs. The faint spots from 0.1 mg/ml poly- to the blank. clonal antibody also made the analysis difficult since the To optimize the detection, 0.1 or 1 mg/ml polyclonal ScanAlyze software requires the user visually identify antibody, 20, 62.5, or 200 ng/ml biotinylated monoclonal the locations and contours of the spots. We therefore antibody, 2 or 10 ng/ml SA-HRP were tested. Using 0.1 conclude that 1 mg/ml polyclonal antibody was optimal. mg/ml polyclonal antibody resulted in poor response to Similar result was also reported by others [7]. the cytokines (Fig. 1, grid 3 and 4). For IL-2, the chemi- Although the membrane was blocked in PBS con- luminescence increased slightly, compared to the blank taining 1 % (w/v) BSA for 1 h, the background increased when the highest concentration of cytokine standard was significantly as the concentration of the biotinylated an- applied.
Load more

Recommended publications
  • Protein Function Microarrays: Design, Use and Bioinformatic Analysis in Cancer Biomarker Discovery and Quantitation
    Chapter 3 Protein Function Microarrays: Design, Use and Bioinformatic Analysis in Cancer Biomarker Discovery and Quantitation Jessica Duarte , Jean-Michel Serufuri , Nicola Mulder , and Jonathan Blackburn Abstract Protein microarrays have many potential applications in the systematic, quantitative analysis of protein function, including in biomarker discovery applica- tions. In this chapter, we review available methodologies relevant to this fi eld and describe a simple approach to the design and fabrication of cancer-antigen arrays suitable for cancer biomarker discovery through serological analysis of cancer patients. We consider general issues that arise in antigen content generation, microar- ray fabrication and microarray-based assays and provide practical examples of experimental approaches that address these. We then focus on general issues that arise in raw data extraction, raw data preprocessing and analysis of the resultant preprocessed data to determine its biological signi fi cance, and we describe compu- tational approaches to address these that enable quantitative assessment of serologi- cal protein microarray data. We exemplify this overall approach by reference to the creation of a multiplexed cancer-antigen microarray that contains 100 unique, puri fi ed, immobilised antigens in a spatially de fi ned array, and we describe speci fi c methods for serological assay and data analysis on such microarrays, including test cases with data originated from a malignant melanoma cohort. Keywords Protein microarrays • Cancer–testis antigens • Cancer biomarker discovery • Bioinformatic analysis • Pipeline J. Duarte • J.-M. Serufuri • N. Mulder • J. Blackburn , D.Phil (*) Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences , University of Cape Town , N3.06 Wernher Beit North Building, Anzio Road, Observatory , Cape Town 7925 , South Africa e-mail: [email protected] X.
    [Show full text]
  • Development of a Novel, Quantitative Protein Microarray Platform for the Multiplexed Serological Analysis of Autoantibodies to Cancer-Testis Antigens
    IJC International Journal of Cancer Development of a novel, quantitative protein microarray platform for the multiplexed serological analysis of autoantibodies to cancer-testis antigens Natasha Beeton-Kempen1,2*, Jessica Duarte1*, Aubrey Shoko3, Jean-Michel Serufuri1, Thomas John4,5, Jonathan Cebon4,5 and Jonathan Blackburn1 1 Institute of Infectious Disease and Molecular Medicine/Division of Medical Biochemistry, University of Cape Town, Cape Town, South Africa 2 Biosciences Division, Council for Scientific and Industrial Research, Pretoria, South Africa 3 Centre for Proteomic and Genomic Research, Cape Town, South Africa 4 Ludwig Institute for Cancer Research, Melbourne, Australia 5 Joint Ludwig-Austin Oncology Unit, Austin Health, Victoria, Australia The cancer-testis antigens are a group of unrelated proteins aberrantly expressed in various cancers in adult somatic tissues. This aberrant expression can trigger spontaneous immune responses, a phenomenon exploited for the development of disease markers and therapeutic vaccines. However, expression levels often vary amongst patients presenting the same cancer type, and these antigens are therefore unlikely to be individually viable as diagnostic or prognostic markers. Nevertheless, patterns of anti- gen expression may provide correlates of specific cancer types and disease progression. Herein, we describe the development of a novel, readily customizable cancer-testis antigen microarray platform together with robust bioinformatics tools, with which to quantify anti-cancer testis antigen autoantibody profiles in patient sera. By exploiting the high affinity between autoantibodies and tumor antigens, we achieved linearity of response and an autoantibody quantitation limit in the pg/mL range—equating to a million-fold serum dilution. By using oriented attachment of folded, recombinant antigens and a polyethylene glycol microarray surface coating, we attained minimal non-specific antibody binding.
    [Show full text]
  • PREPARATION of MICROARRAY for DISEASE DETECTION - a SURVEY on DIFFERENT METHODS Kavitha B1, Manjusha G Y2, Sachin D’Souza3 & Hemalatha N4
    International Journal of Latest Trends in Engineering and Technology Special Issue SACAIM 2017, pp. 088-090 e-ISSN:2278-621X PREPARATION OF MICROARRAY FOR DISEASE DETECTION - A SURVEY ON DIFFERENT METHODS Kavitha B1, Manjusha G Y2, Sachin D’Souza3 & Hemalatha N4 Abstract- Microarray is a pattern of ssDNA probes which are immobilized on a surface called a chip or a slide.It is used to detect the expression of thousands of gene at the same time. Microarrays (biochip) plays an important role in the drug discovery. The biochip is used to monitor changes in gene expression in response to drug treatments and also used to examine the response of the host against pathogen. The oligonucleotide microarrays provides a rapid, specific and high throughput means for the detection and identification of the food-borne pathogens. In this paper we have described microarray-based tests or methods for detecting the various kinds of diseases. By using several methods and tools like allergen microarray one can screen the serum IgE reactivity. cDNA microarrays has been developed for analysing estrogen responsive genes and in detecting anti hormone therapy. Evaluation of the potential of pathogenicity is determined by detection of a range of virulence factors and serotype determination. Micro RNA identifier array used for the detection of various type of micro RNA in human or for simultaneous detection and genotyping of different virus types in a single reaction. Keywords- Microarray, DNA, RNA,Gene 1. INTRODUCTION Microarray is multiplex lab on a chip. It is a pattern of ssDNA probes which are immobilized on a surface called chip or a slide.
    [Show full text]
  • Protein Microarray Technology: Assisting Personalized Medicine in Oncology (Review)
    WORLD ACADEMY OF SCIENCES JOURNAL 1: 113-124, 2019 Protein microarray technology: Assisting personalized medicine in oncology (Review) MONICA NEAGU1-3, MARINELA BOSTAN1,4 and CAROLINA CONSTANTIN1,2 1Department of Immunology, ‘Victor Babes’ National Institute of Pathology, 050096 Bucharest; 2Research Core of The Pathology Department, Colentina University Hospital, Bucharest 020125; 3Faculty of Biology, University of Bucharest, 050095 Bucharest; 4Immunology Center, ‘Stefan Nicolau’ Institute of Virology, 030304 Bucharest, Romania Received April 23, 2019; Accepted June 11, 2019 DOI: 10.3892/wasj.2019.15 Abstract. Among proteomics technologies, protein microarray, into the future. We present personalized medicine goals and over the past last years, has gained an increased momentum discuss how protein microarray can aid in these goals, with an in the biomarkers discovery domain. The characteristics of emphasis on several oncological diseases. We also discuss how protein microarray, namely that it is a high-throughput tool, it protein technology has been used in diseases, such as lung, provides a high specificity and only requires a minute amount breast cancers, as well as in other diseases that, over the past of biological samples, render it a suitable tool for searching, last years, have taken advantage of this proteomic endeavor. quantifying and validating biomarkers in various pathologies. Protein microarray is based on the specific antigen‑antibody reaction, such as any enzyme-linked immunosorbent assay, Contents the specific reaction occurring on a miniaturized support (chip or slide), thus having the advantage of simultaneous eval- 1. Introduction uation of tens to thousands of molecules in small samples with 2. Personalized medicine: A step forward in improving a highly specific recognition for the detection system.
    [Show full text]
  • Biomolecular Applications for Biomems Primary Knowledge Participant Guide
    Biomolecular Applications for bioMEMS Primary Knowledge Participant Guide Description and Estimated Time to Complete This learning module is an overview of biomolecules, what are they, types of biomolecules, and how microtechnology is using biomolecules or exploiting their functions for micro and nano-sized transducers, sensors and actuators. Activities provide the opportunity to better understand the function of biomolecules, their scale and why they are so important for micro and nanotechnologies. This unit discusses the characteristics and phenomena of biomolecules that make them attractive components for bioMEMS devices. It provides information that helps you understand how biological molecules can be used as working devices within bioMEMS. Estimated Time to Complete Allow at least 30 minutes to complete. Introduction A MEMS cantilever coated with a monolayer of biological molecules (probe surface layer) can bind with and capture other molecules (analytes) of complementary shapes Southwest Center for Microsystems Education (SCME) Page 1 of 26 App_BioMEM_PK36s_PG_August2017.docx Biomolecular Applications of bioMEMS PK As MEMS devices become smaller and smaller, the use of biomolecules as a MEMS component becomes more attractive. But how can biomolecules be used as a MEMS component? • Biomolecules can self-assemble into predictable and precise structures in the nano range. This offers a vast new repertoire of structures and functions to MEMS devices. • Many of these molecules’ functions in living organisms can be harnessed to perform the same functions in a bioMEMS device. This provides a wealth of materials and applications that can be applied to bioMEMS design. To understand how biomolecules can be used in bioMEMS, it is useful to learn about the types of biomolecules and how they associate with each other in functional units.
    [Show full text]
  • Lec-29-Handout
    NPTEL VIDEO COURSE – PROTEOMICS PROF. SANJEEVA SRIVASTAVA HANDOUT LECTURE-29 CELL-FREE SYNTHESIS BASED PROTEIN MICROARRAYS Slide 1: In today’s lecture we will talk about protein microarrays based on cell-free synthesis. So cell-free synthesis based protein microarrays provides a high throughput, versatile platform for large-scale analysis of functional proteins. These microarrays are used for various applications for eg, antibody profiling, biomarker discovery, enzyme-substrate identification, protein-protein interaction etc. The traditional cell based methods which were used for making the protein microarrays involve protein expression in heterologous system such as E. coli. But the protein purification is a very laborious process. It involves various steps and of the protein purity, protein integrity, its stability and the functionality. All of these remain major issues for protein purification. So if we have to generate high throughput, large number of proteins which is required for performing protein microarray studies, it is very tedious, because one need to purify large number of proteins in the scale of thousands and then maintain the functionality and keep them properly folded. So these limitations of traditional protein purification and protein microarrays generated by these purified proteins have been the major motivation for cell-free expression based microarray field. The cell-free expression based systems have overcome various limitations of protein purification and they perform in situ transcription and translation. Slide 2: I will give you an overview and the principle of various cell-free expression based protein microarrays. We will talk about protein in situ arrays (PISA), nucleic acid programmable protein arrays (NAPPA), multiple spotting technique (MIST), DNA array to protein array (DAPA) and HaloTag array.
    [Show full text]
  • Applications of High Content Antibody Microarrays for Biomarker Discovery and Tracking Cel- Lular Signaling
    Advances in Proteomics and Bioinformatics Yue L and Pelech S. Adv Proteomics Bioinform: APBI-107. Review Article DOI: 10.29011/APBI -107. 100007 Applications of High Content Antibody Microarrays for Bio- marker Discovery and Tracking Cellular Signaling Lambert Yue1, Steven Pelech1,2* 1Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada 2Kinexus Bioinformatics Corporation, Vancouver, British Columbia, Canada *Corresponding author: Steven Pelech, Department of Medicine, University of British Columbia, Kinexus Bioinformatics Corpo- ration, Vancouver, British Columbia, Canada. Tel: +16043232547; Fax: +16043232548; Email: [email protected] Citation: Yue L, Pelech S (2018) Applications of High Content Antibody Microarrays for Biomarker Discovery and Tracking Cel- lular Signaling. Adv Proteomics Bioinform: APBI-107. DOI: 10.29011/APBI -107. 100007 Received Date: 26 June, 2018; Accepted Date: 05 July, 2018; Published Date: 12 July, 2018 Abstract Fuelled by advances in our understanding of the human genome and proteome and the increasing availability of pan- and phosphosite-specific antibodies, antibody microarrays have emerged as powerful tools for interrogating protein expression and post-translational modifications, as well as for uncovering interactions with other proteins and small molecules. This economi- cal platform permits ultra-sensitive semi-quantitative measurements of hundreds of proteins simultaneously using only minute amounts of biofluid, tissue and cell specimens. Recent innovations in the design and fabrication of antibody microarrays and in sample preparation have permitted further refinements of the technology to yield significant improvements in data quality. In this review, we described the applications of antibody microarrays for disease biomarker discovery, highlighting how biologi- cal complexity and sample handling have compromised earlier work, and how this technology can be exploited for tracking the expression, phosphorylation and ubiquitination of proteins in crude cell and tissue lysate samples.
    [Show full text]
  • Protein Microarrays As Tools for Functional Proteomics
    ics om & B te i ro o P in f f o o r l m a Journal of a n t r i Lueong et al., J Proteomics Bioinform 2013, S7 c u s o J DOI: 10.4172/jpb.S7-004 ISSN: 0974-276X Proteomics & Bioinformatics ReviewResearch Article Article OpenOpen Access Access Protein Microarrays as Tools for Functional Proteomics: Achievements, Promises and Challenges Smiths S Lueong, Jörg D Hoheisel and Mohamed Saiel Saeed Alhamdani* Division of Functional Genome Analysis, Deutsches Krebsforschungszentrum (DKFZ), Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany Abstract The quest for a better understanding of organisms, and the human body in particular, at a comprehensive level has stimulated the development of techniques and processes that permit the analysis and assessment of biomedical information at high throughput. This has had substantial impact, not only by merely gathering knowledge but also by making scientists aware of the necessity to view organisms as complex biological systems rather than an assembly of individual biochemical pathways. Proteomics is still lagging behind genomics in this holistic analysis, however, because of the much higher degree of complexity that needs to be dealt with. Protein arrays, among other techniques, offer the prospect of advancing global protein analysis, similar to the impact of arrays in genomics. In basic research, protein arrays already contributed substantially, permitting the identification of many protein interactions and providing information on expression variations and structural changes, for example. Beside the fact of high throughput, arrays require relatively small reaction volumes, which is critical in view of the lack of means for in vitro protein amplification and beneficial for good assay sensitivity.
    [Show full text]
  • Proteomic Applications of Surface Plasmon Resonance Biosensors: Analysis of Protein Arrays
    EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 37, No. 1, 1-10, February 2005 Proteomic applications of surface plasmon resonance biosensors: analysis of protein arrays Jong Seol Yuk1 and Kwon-Soo Ha1,2 (Zhu and Snyder, 2003). Several techniques for the analysis of protein interactions on protein arrays have 1 been reported based on various methods such as Department of Molecular and Cellular Biochemistry fluorescence detection, surface-enhanced laser de- Nano-bio Sensor Research Center sorption/ionization (SELDI) mass spectrometry, atomic Kangwon National University School of Medicine force microscopy (AFM), and surface plasmon re- Chunchon, Kangwon-do 200-701, South Korea sonance (SPR). 2Coressponding author: Tel, 82-33-250-8833; SPR has been used as an optical detection te- Fax, 82-33-250-8807; E-mail, [email protected] chnology since Lidberg et al. (1983) demonstrated its potential to use as biosensors in 1983 (Yuk et al., Accepted 4 February 2005 2004a). SPR-based biosensors have several advant- ages in the analysis of protein interactions compared to other sensors. SPR spectroscopy allows real-time Abbreviations: AFM, atomic force microscopy; MALDI, matrix- measurement of biomolecular interactions without la- assisted laser desorption/ionization; SELDI, surface-enhanced la- beling and with simple optical system device (Zhu and ser desorption/ionization; SPR, surface plasmon resonance Snyder, 2003). There are several examples in which SPR biosensors have been used to characterize the details of biomolecular interactions, and thus the SPR biosensors have emerged as one of the most powerful Abstract tools in biochemical and proteomics researches (My- Proteomics is one of the most important issues szka and Rich, 2000).
    [Show full text]
  • Microfluidic Methods for Protein Microarrays
    Microfluidic Methods for Protein Microarrays Michael Hartmann Doctoral Thesis School of Chemical Science and Engineering Department of Chemistry Division of Analytical Chemistry KTH, Royal Institute of Technology Stockholm, Sweden, 2010 AKADEMISK AVHANDLING som med tillstånd av Kungliga Tekniska Högskolan i Stockholm framlägges till offentlig granskning för avläggande av teknologie doktorsexamen 2010-11-19 i sal F3, Lindstedtsvägen 26, KTH, Stockholm. Avhandlingen presenteras på engelska. ii Microfluidic Methods for Protein Microarrays Michael Hartmann Thesis for the degree of PhD of Technology in Chemistry KTH, Royal Institute of Technology School of Chemical Science and Engineering Department of Chemistry Division of Analytical Chemistry SE-10044-Stockholm, Sweden. ISBN 978-91-7415-761-1 TRITA-CHE Report 2010:45 ISSN 1654-1081 Copyright © Michael Hartmann, 2010 All rights reserved for the summary part of this thesis, including all pictures and figures. No part of this publication may be reproduced or transmitted in any form or by any means, without prior permission in writing from the copyright holder. The copyright for the appended journal papers as well as some figures in the thesis belongs to the publishing houses of the journals concerned, and permission has been granted to reproduce this material in the thesis. Printed by E-Print iii Abstract Protein microarray technology has an enormous potential for in vitro diagnostics (IVD) 1. Miniaturized and parallelized immunoassays are powerful tools to measure dozens of parameters from minute amounts of sample, whilst only requiring small amounts of reagent. Protein microarrays have become well-established research tools in basic and applied research and the first diagnostic products are already released on the market.
    [Show full text]
  • Fundamentals of Microfluidics with Applications in Biological Analysis and Discovery
    Fundamentals of Microfluidics with Applications in Biological Analysis and Discovery Harvard Extension School E155 Dr. Anas Chalah 03.29.16 1 Microfluidics Microfluidics refers to the set of technologies used to control and manipulate the flow of few microliters of a fluid sample (liquid or gas) in a miniaturized microfluidic device 2 Microfluidic Devices by Defini2on: Architecture: 3D network of channels and other components (pumps, valves, heaters, reservoirs, ..etc) Dimension: Has one or more flow channel component with at least one dimension less than 1mm Function: Provide a high level of fluid control required for a certain application 3 Microarray Microarrays are broadly defined as tools for paralleled ligand binding assays Biomolecules (oligonucleo3des, protein fragments) are placed on a solid support (glass, PDMS slides) at high density Goal: recognizing and analyzing a complex mixture of target molecules 4 Microarray Design In general, A microarray is a DEVICE that consists of different PROBES chemically attached to a SUBSTARTE and designed to detect a biological target molecule 5 Microarray Technology Microarray technology allows for: (1) Estimation of target abundance (2) Detection of biological interactions Both at the molecular or cellular level 6 Building a Microarray Chip Design Thinking Application à Components à Fabrication à Redesign ß Testing 7 Design Factors Device: Fabricaon - Design - Signal - Interface Probe: Bio materials (DNA, RNA, Protein, Cells, Tissue) Substrate: Chemical Plaorm (Polymers: Glass, Silicon, PDMS)
    [Show full text]
  • Omics-Scale Bioinformatics Technology and Methods: from Data to Information
    Omics-Scale Bioinformatics Technology and Methods: from Data to Information A Dissertation Presented to Faculty of the Department of Biology and Biochemistry University of Houston In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy By Haosi Chen December 2014 Omics-Scale Bioinformatics Technology and Methods: from Data to Information ------------------------------------- Haosi Chen APPROVED -------------------------------------- Dr. Xiaolian Gao, Chair of the Committee -------------------------------------- Dr. James M. Briggs -------------------------------------- Dr. Cecilia M. Williams -------------------------------------- Dr. Fuli Yu -------------------------------------- Dean, College of Natural Sciences and Mathematics II Acknowledgements I am using this opportunity to express my sincere appreciation to my advisor, Dr. Xiaolian Gao, for her inspiring guidance and for the tremendous learning opportunities she provided for my Ph.D. studies at the University of Houston. I also appreciate the rest of my committee, Drs. James Briggs, Cecilia Williams and Fuli Yu, for their invaluable constructive criticism and friendly advice during my graduate studies. I am very grateful to all my collaborators: Ms. Ruijuan Zhu, she made great contributions for the miRFocus project; Ms. Chengping Sui, she collected the data for the miRNA-miRNA relationship database in the miRFocus project; Ms. Wen Wan, a student in University of Science and Technology of China, she performed the experiments of the Microchip-synthesized DNA project and generously shared the data with me; Dr. Qi Zhu, senior scientist at LC Sciences, he sequenced the nucleotides for the Microchip-synthetic DNA project; Dr. Bing Zhu, he performed the experiment of the Histone PepArrary chip project; Dr. Ailing Hong, senior chemist at LC Sciences, she made the PepArrary chips for the Histone PepArray project.
    [Show full text]