Mining the Salivary Proteome with Grating‐Coupled Surface Plasmon Resonance Imaging and Surface Plasmon Coupled Emission Microarrays

Ryan D. Molony1, James M. Rice1, Jong Seol Yuk2, Vivek Shetty3, Dipak Dey4, David A. Lawrence5, Michael A. Lynes5

1 These authors contributed equally and should be considered co‐first authors, 2 Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut, 3 School of Dentistry, University of California, Los Angeles, Los Angeles, California, 4 Department of Statistics, University of Connecticut, Storrs, Connecticut, 5 Wadsworth Center, New York Department of Health, Albany, New York
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 18.16
DOI:  10.1002/0471140856.tx1816s53
Online Posting Date:  August, 2012
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Biological indicators have numerous and widespread utility in personalized medicine, but the measurement of these indicators also poses many technological and practical challenges. Blood/plasma has typically been used as the sample source with which to measure these indicators, but the invasiveness associated with sample procurement has led to increased interest in saliva as an attractive alternative. However, there are unique issues associated with the measurement of saliva biomarkers. These issues are compounded by the imperfect correlation between saliva and plasma with respect to biomarker profiles. In this manuscript, we address the technical challenges associated with saliva biomarker quantification. We describe a high‐content microarray assay that employs both grating‐coupled surface plasmon resonance imaging and surface plasmon–coupled emission modalities in a highly sensitive assay with a large dynamic range. This powerful approach provides the tools to map the proteome of saliva, which in turn should greatly enhance the utility of salivary biomarker profiles in personalized medicine. Curr. Protoc. Toxicol. 53:18.16.1‐18.16.19. © 2012 by John Wiley & Sons, Inc.

Keywords: saliva; biomarker; proteomics; surface plasmon resonance; SPR; emission microarray; GCSPR; GCSPCE; personalized medicine; biofluid; biomolecule

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Saliva Collection and Storage
  • Basic Protocol 2: GCSPR/GCSPCE Microarray Sensor Chip Preparation
  • Basic Protocol 3: Saliva Indirect Immunoassay Microarray Using GCSPCE
  • Alternate Protocol 1: Manual Chip Printing
  • Alternate Protocol 2: Direct Fluorescent Immunoassay Microarray Using GCSPCE
  • Alternate Protocol 3: Detection of Viral Particles from Saliva on a Microarray Using GCSPR Imaging
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Saliva Collection and Storage

  • Complete Protease Inhibitor Cocktail (Roche)
  • 15‐ml collection tubes
  • 1‐ml syringe
  • 0.22‐µm syringe filter (Fisher)

Basic Protocol 2: GCSPR/GCSPCE Microarray Sensor Chip Preparation

  • Blocking buffer (see recipe)
  • Other buffers used in procedure (as negative controls)
  • Capture antibody or protein (in PBS with no carrier protein)
  • Control antibody
  • Biotinylated protein for positive control
  • 95% ethanol
  • Ultrapure H 2O (18 MΩ)
  • DSP (Dithiobis(succinimidyl) propionate) (Thermo Scientific)
  • Dimethylsulfoxide (DMSO)
  • 384‐well low‐protein‐binding V bottom microplates (Thermo Scientific)
  • Foil covers for microplates
  • Centrifuge with microtiter plate carrier
  • Gold GCSPR/GCSPCE dual‐mode sensor chips (Ciencia,
  • Source of pressurized filtered air
  • Spotbot II Automated Spotting System (ArrayIt Microarray Technology)
  • Small brush for cleaning print head of spotting machine
  • Magnifying glass (10×)
  • Humidified incubator or other humid environment (80% humidity)
  • Desiccator

Basic Protocol 3: Saliva Indirect Immunoassay Microarray Using GCSPCE

  • Matched‐pair biotinylated detection antibodies
  • Reagent buffer: PBST (see recipe) containing 0.1% BSA
  • Streptavidin–Alexa Fluor 647 (Invitrogen)
  • PBS (see recipe)
  • PBST (see recipe)
  • Blocking buffer (see recipe)
  • Saliva sample ( protocol 1)
  • Ultrapure H 2O (18 MΩ)
  • Printed gold sensor chip ( protocol 2) (Ciencia)
  • Glass coverslip window with inlet/outlet ports (Ciencia)
  • Double‐sided adhesive gasket (Ciencia)
  • GCSPR/GCSPCE instrument (Ciencia)
  • Used sensor chip (for instrument set‐up)

Alternate Protocol 1: Manual Chip Printing

  • 96‐well low‐protein‐binding U‐bottom microplates
  • MicroCaster System 8‐pin manual arrayer (Schleicher & Schuell Bioscience, or other manual spotting system)

Alternate Protocol 2: Direct Fluorescent Immunoassay Microarray Using GCSPCE

  • Alexa Fluor 647‐NHS (Invitrogen)
  • Dimethylsulfoxide (DMSO)
  • Sample (saliva from protocol 1 or cell lysate)
  • 1 M sodium bicarbonate in ultrapure H 2O (18 MΩ)
  • M‐PER Mammalian Protein Extraction Reagent (Thermo Scientific)
  • Complete Protease Inhibitor Cocktail, 10× (Roche)
  • Sephadex G‐100 size‐exclusion resin (Pharmacia Fine Chemicals)
  • Column
  • Automated fraction collector (optional)
  • 96‐well plate, black
  • SpectraMax M2 Microplate Reader (Molecular Devices) or other fluorescent plate reader

Alternate Protocol 3: Detection of Viral Particles from Saliva on a Microarray Using GCSPR Imaging

  • Sample containing viral particles
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