Protein Microarray Technology

Dr. Dev Kambhampati has an extensive background in the areas of chemical engineering, materials science and bioanotechnology. In his current position as a Research Development Officer at the University of California, he is widely involved in initiating strategic university industry partnerships in the areas of biotechnology, materials science and nanotechnology. Prior to this, during his tenure in industry, he was extensively involved in both research and product development activities concerning biochips and instrumentation. At Thermo Hybaid Dev Kambhampati was responsible for DNA, protein and glycochip product development that resulted in numerous publications and innovative products, while at Xomics he was instrumental in managing the R&D activities involving bio MEMS, biochips and nano assemblies. Dev Kambhampati has wide ranging international experience and is a frequent speaker and consultant at leading industry and research events. In addition to his current interests, he is also working on topical projects related to homeland security and biological systems and devices that have the potential to revolutionize the next generation of biological sensors. His focus has constantly been on developing biocompatible screening platforms and advanced detection technologies some of the concepts described in great detail in the book. Dev Kambhampati's note worthy educational accomplishments include receiving the prestigious Max Planck Society Fellowhip and the Riotech Environmental Excellence Award.

List of Authors. Colour Plates. 1. Protein Microarrays: From Fundamental Screening to Clinical Diagnostics (Dev Kambhampati). 1.1 Potential Need for Protein Microarrays. 1.1.1 Protein Therapeutics. 1.1.2 Clinical Diagnostics. 1.1.3 National Security. 1.2 Current Applications of Protein Microarrays. 1.3 Problems and Challenges. 1.3.1 Sample Preparation and Handling (Probe and Target). 1.3.2 Microarray Platform. 1.3.3 Detection Technologies. 1.3.4 Data Analysis. 1.4 Potential Solutions: Enabling Technologies and Advancements. References. 2. Protein Microarray Surface Chemistry and Coupling Schemes (Michael Schaeferling and Dev Kambhampati). 2.1 Introduction. 2.1.1 Background and Current State of Biomolecule Libraries. 2.2 Microarray Based of Class Substrates. 2.2.1 Surface Modification. 2.2.2 Current State of Glass Based Protein Microarrays. 2.3 M icroarrays based of Gold Substrates. 2.3.1 Surface Modifications. 2.3.2 Current State of Gold based Protein Microarrays. 2.4 Microarrays based on Polymer Substrates. 2.4.1 Surface Modifications. 2.5 Special Formats: Microfluidic Devices and Integrated Semiconductor Chips. 2.6 Chemical Immobilization Techniques for Proteins. 2.6.1 Covalent Chemical Coupling. 2.6.1 Photochemical Cross Coupling. 2.6.3 Tagged Proteins. 2.6.4 Site Specific Immobilization of Antibodies. 2.7 Conclusions. References. 3. Optimization of a Protein Microarray Platform Based on a Small molecule Chemical Affinity System (Karin A. Hughes, Lisa R. Booth, Robert J. Kaiser, Kevin P. Lund and Douglas A. Spicer). 3.1 Introduction. 3.2 Experimental. 3.2.1 Reagents and Materials. 3.2.2 Comparison of the Intrinsic Fluorescence and Non Specific Protein Binding of 2 D and 3 D SHA Coated Glass Slides. 3.2.2.1 Preparation of 2D SHA Coated Glass Surfaces. 3.2.2.2 Preparation of PDBA modified Bovine Serum Albumin. 3.2.2.3 Preparation of PDBA modified Human IgG. 3.2.2.4 Printing and Development of 2 D and 3 D SHA coated Glass Slides 44 3.2.2.5 Comparison of the Intrinsic Flurescence of Unmodified, 2 D and 3 D SHA ccoated Glass Slides. 3.2.2.6 Determination of the Non Spcific Protein Binding of 2 D and 3 D SHA coated Glass Slides. 3.2.3 Immunoassay Using a 3 D SHA coated Glass Slide. 3.2.3.1 Preparation of PDBA Cy3 modified Bovine Serum Albumin, PDBA Human IgG, PDBA Goat anti Human IgG. 3.2.3.2 Printing the Array and Analyzing the Data. 3.2.4 Stability of the PDBA Protein Conjugates Immobilized on 3 D SHA coated Glass Slides. 3.2.4.1 Preparation of PDBA modified Goat Anti rabbit Fc specific IgG, PDBA modified Goat Anti rabbit Fc specific F(ab)2, PDBA modified Goat Anti human F(ab)2, and PDBA modified Goat Anti mouse Fca specific F(ab)2. 3.2.4.2 Printing and Reading the Array. 3.3 Results and Discussion. 3.3.1 Comparison of the Intrinsic Fluorescence and Non specific Protein Binding of 2 D and 3 D SHA coated Glass Slides. 3.3.2 Immunoassay on a 3 D SHA coated Slide. 3.3.3 Stability of PDBA Protein Conjugates Immobilized on 3 D SHA coated Glass Slides. 3.4 Conclusions. References. 4. Seein Beneath the Surface of Biomolecular Interactions: Real time Characterization of Label free Binding Interactions using Biacore's Optical Biosensors (Gary Franklin and Alan McWhirter). 4.1 Introduction. 4.1.1 The Proteomics Revolution. 4.2 Biacore Technology. 4.2.1 Surface Plasmon Resonance. 4.2.2 The Sensor Surface and Immobilization Chemistry. 4.2.2.1 Sensor chips. 4.2.2.2 Immobilization Chemistry. 4.2.2.3 General Capture Methods. 4.2.2.4 Specialized Capture Methods. 4.2.2.5 Lipids and Membrane Proteins. 4.2.3 The Microfluidics System. 4.2.4 Biacore Assay Basics. 4.2.4.1 Correlation of SPR Response with Surface Binding. 4.2.4.2 Assay Design: Assay Formats. 4.2.4.3 Assay Design: Surface Preparation. 4.2.4.4 Regeneration. 4.2.5 Theoretical and Practical Implications of Technology Design for Biacore Assays. 4.2.5.1 Label free Detection. 4.2.5.2 The Optical Detection System and Bulk Effects. 4.2.5.3 Surface based Versus Solution based Analysis. 4.2.5.4 The Microfluidic Design and Mass Transport Effects. 4.3 Biacore Applications in Basic and Clinical Research. 4.3.1 Introduction. 4.3.2 Applications of Biacore in Cancer Research. 4.3.3 Applications of Biacore in Clinical Research. 4.3.4 Applications of Biacore in Neuroscience. 4.3.5 Plasma Protein Interactions. 4.3.6 Drug: DNA Interactions. 4.3.7 Nuclei Acid Structure and Analysis. 4.3.8 Protein: RNA Interactions. 4.4 Current Developments and Future Perspectives. 4.4.1 SPR MS. 4.4.2 The Challenges of High throughput Protein Analysis and Array Technologies. References. 5. Surface Plasmon Resonance Imaging Measurements of DNA, RNA, and Protein Interactions to Biomolecular Arrays (Greta J. Wegner, Hye Jin Lee and Robert M. Corn). 5.1 Introduction. 5.2 Surface Plasmon Resonance Imaging. 5.3 Surface Attachment Chemistries. 5.3.1 SSMCC Attachment Chemistry. 5.3.2 SATP Attachment Chemistry. 5.4 SPR Imaging Experiments Using Photopatterned Arrays. 5.4.1 DNA DNA Hybridization. 5.4.2 Mismatch Binding Protein, MutS. 5.4.3 Bacterial Response Regulators, OmpR and VanR. 5.5 SPR Imaging Experiments of Arrays Created by Microfluidic Stencils. 5.5.1 1 D Peptide Array for Antibody Binding Measurements. 5.5.2 2 D DNA Array for RNA Hybridization. 5.5.3 2 D Peptide Array for Antibody Binding Measurements. 5.5.4 2 D Protein Array. 5.6 Conclusions. References. 6. Surface Plasmon Fluorescence Spectroscopy for Protein Binding Studies (Fang Yu, Bjorn Persson, Stefan Lofas and Wolfgang Knoll). 6.1 Introduction. 6.2 Fluorescence Profile at the Interface. 6.3 Instrumentation. 6.4 SPR sSignal Conversion. 6.5 Sandwich Detection. 6.6 LOD Evalution. 6.7 Conclusions. References. 7. The Use of Proteinchip Arrays for Deciphering Biological Pathways (Lee O. Lomas). 7.1 Introduction. 7.2 Methods to Study Protein Interactions. 7.2.1 Genomic Approaches. 7.2.2 Proteomic Approaches Leading to the Development of Protein Microarrays. 7.2.3 Protein Biochips Utilizing Surface Enhanced Laser Desorption/Ionization (SELD) ProteinChip System Methodologies. 7.2.3.1 EDM. 7.2.3.2 IDM. 7.2.4 The Use of Biochips in Mechanistic Studies. 7.3 Conclusions and Outlook. References. 8. Production of Protein Microarrays (Christopher J. Mann, Sarah K. Stephens and Julian F. Burke). 8.1 Introduction. 8.2 From DNA Arrays to Protein Arrays. 8.3 Overview of Protein Microarray Spotting. 8.4 Types of Protein Microarrays. 8.5 Protein Arrayers. 8.6 Surface Chemistry. 8.6.1 Derivatized Glass Slides. 8.6.1.1 Amine coated or Poly L Lysine coated Slides. 8.6.1.2 Aldehyde coated Slides. 8.6.1.3 Epoxy coated Slides. 8.6.1.4 Bovine Serum Albumin: N Hydroxy Succinimide (BSA NHS) Slides. 8.6.2 Oriented Surfaces for Tagged Proteins. 8.6.2.1 Nickel coated Slides. 8.6.2.2 Streptavidin coated Slides. 8.6.3 Three dimensional Surfaces. 8.6.3.1 Polyacrylamide coated Slides. 8.6.3.2 Agarose coated Slides. 8.6.3.3 Nitrocellulose Slides. 8.7 The Arraying Process. 8.8 Detection Issues. 8.8.1 Labeled Proteins. 8.8.1.1 Chemical Labeling. 8.8.1.2 Radiolabeling. 8.8.1.3 Fluorescent Fusion Proteins. 8.8.2 Sandwich Assays. 8.8.3 Direct Measurement. 8.8.3.1 Mass Spectrometry. 8.8.3.2 Biosensor technology. 8.8.4 Immunoassay Amplification. 8.9 Validation of Results. 8.10 Stability of Protein Microarrays. 8.11 Future Perspectives and Challenges. 8.12 Conclusion. References. 9. Nanomechanical Cantilever Sensors for Microarrays (Marko K. Baller and Jurgen Fritz). 9.1 Introduction. 9.2 Basic Technology and Instrumentation. 9.2.1 Cantilever Design and Properties. 9.2.2 Measuring Cantilever Bending. 9.2.3 Differential Detection and Noise. 9.2.4 Mechanism of Cantilever Bending. 9.3 Cantilever Functionalization. 9.4 Experiments. 9.4.1 Early Surface Stress Experiments. 9.4.2 Nucleic Acids. 9.4.3 Proteins. 9.5 Conclusion and Outlook. References. 10. Image Analysis Issues and Solution for High Density Arrays (Anton Petrov and Soheil Shams). 10.1 Introduction. 10.2 Image Alignment, Grid Placement, and Spot Location. 10.2.1 Image Alignment. 10.2.2 Manual Spot Finding. 10.2.3 Automatic Spot Finding. 10.3 Spatial Segmentation of Signal and Background Pixels. 10.3.1 Pure Space based Signal Segmentation. 10.3.2 Pure Intensity based Signal Segmentation. 10.3.3 Mann Whitney Segmentation. 10.3.4 The Method of Trimmed Measurements. 10.3.5 Integrating Spatial and Intensity Information for Signal Segmentation. 10.4 Data Quantification. 10.5 Quality Control. 10.5.1 Background Contamination. 10.5.2 Signal Contamination. 10.5.3 Position Offset. 10.5.4 Percentage of Ignored Pixels. 10.5.4.1 Percentage with an Open Perimeter. 10.5.4.2 Shape regularity. 10.6 Batch Automation. 10.7 Experimental Results. 10.7.1 Signal Estimation: Segmentation Method. 10.7.2 Signal Estimation: Quantification Method. 10.7.3 Background Estimation: Segmentation Method. 10.7.4 Background Estimation:; Quantification Method. 10.7.5 Ratio Estimation: Quantification Method. 10.7.6 Quality Control. 10.8 Conclusion. References. Index.