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

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

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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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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Materials

Basic Protocol 1: Saliva Collection and Storage

Materials

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

Materials

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 H₂ O (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, http://www.ciencia.com/)
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

Materials

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 H₂ O (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 H₂ O (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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Figures

Figure 18.16.1 Indirect fluorescent immunoassay using grating‐coupled surface plasmon‐coupled emission (GCSPCE). In this GCSPCE assay diagram, capture antibodies have been printed on a gold sensor chip with a holographic diffraction grating. Analyte capture was detected by recirculating an analyte‐specific biotinylated secondary antibody and streptavidin‐Alexa Fluor 647 over the chip. Fluorophores on the chip were then excited using an LED light source, and a CCD camera was used to detect the resulting fluorescence. In addition to the standard fluorescent excitation shown above, the interaction of the surface plasmon increases the directional fluorescent emission which amplifies this fluorescent signal ∼80‐fold.

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Figure 18.16.2 GCSPCE microarray image using a saliva sample. The GCSPCE microarray image was taken after exposure to human saliva doped with 14 recombinant human protein biomarkers of interest and a cocktail of fluorescently labeled secondary antibodies. Six cytokines were not included in the saliva sample to provide negative controls. Regions of interest (ROIs) are printed in groups of three for each biomarker. Isotype controls, diluent control (PBS), and positive control ROIs are included in the image.

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Figure 18.16.3 Saliva does not affect the IL‐2 limit of detection by GCSPCE. Recombinant human IL‐2 was diluted in protease inhibitor‐treated saliva and PBS (1:1) (•) or PBS alone (▴) and detected using an indirect sandwich assay. Points indicate the mean fluorescence detected from 3 ROI microspots. Error bars indicate standard deviation.

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