Multiplexed Fluorescent Microarray for Human Salivary Protein Analysis Using Polymer Microspheres and Fiber-optic Bundles

Summary

We describe a procedure for profiling salivary proteins using multiplexed microsphere-based antibody arrays. Monoclonal antibodies were covalently linked to fluorescent dye-encoded 4.5 μm polymer microspheres using carbodiimide chemistry. The modified microspheres were deposited in fiber-optic microwells to measure protein levels in saliva using fluorescence sandwich immunoassays.

Abstract

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array technology employed combines the advantages of microsphere-based suspension array fabrication with the use of fluorescence microscopy. As described in the video protocol, commercially available 4.5 μm polymer microspheres were encoded into seven different types, differentiated by the concentration of two fluorescent dyes physically trapped inside the microspheres. The encoded microspheres containing surface carboxyl groups were modified with monoclonal capture antibodies through EDC/NHS coupling chemistry. To assemble the protein microarray, the different types of encoded and functionalized microspheres were mixed and randomly deposited in 4.5 μm microwells, which were chemically etched at the proximal end of a fiber-optic bundle. The fiber-optic bundle was used as both a carrier and for imaging the microspheres. Once assembled, the microarray was used to capture proteins in the saliva supernatant collected from the clinic. The detection was based on a sandwich immunoassay using a mixture of biotinylated detection antibodies for different analytes with a streptavidin-conjugated fluorescent probe, R-phycoerythrin. The microarray was imaged by fluorescence microscopy in three different channels, two for microsphere registration and one for the assay signal. The fluorescence micrographs were then decoded and analyzed using a homemade algorithm in MATLAB.

Introduction

Since the first microarray reported by Mark Schena and coworkers in the mid-1990s, this powerful tool has been utilized in many fields of biological research¹. Antibody microarrays capable of simultaneously detecting multiple proteins in diagnostic fluids, such as blood, have important applications in clinical diagnostics and biomarker screening^2-10. Saliva, containing many of the same analytes as blood, has been considered as a preferable alternative to blood because saliva collection is safe, noninvasive, and can be carried out by minimally-trained medical personnel^11-13. Currently, multiplexed protein analysis using saliva samples i....

Protocol

Figure 1. Workflow for applying fiber-optic microsphere antibody array to saliva profiling. (1) Microspheres are internally encoded with two fluorescent dyes; (2) the encoded microspheres are externally modified with protein-specific monoclonal antibodies; (3) the multiplexed microspheres are mixed, and (4) randomly deposited in microwells etc....

Results

Fluorescence images from three channels showing a small section of the fiber-optic bundle are shown in Figures 2A-C. These images were analyzed using an algorithm written in MATLAB (as described in more detail in the Discussion section). The analysis employs both information from the Eu-TTA encoding image (Figure 2A) and the C30 encoding image (Figure 2B) to decode the microspheres, and the fluorescence intensities of different microspheres in the signal image (F.......

Discussion

Researchers should pay extra attention to the following steps: for better decoding accuracy, it is necessary to verify the microspheres were homogeneously suspended in all incubation and wash steps during the microspheres encoding procedure. In addition, the encoded microspheres need to be protected from light throughout the entire experiment. Following proper encoding and storage procedures, we found that overall decoding accuracy was above 99%. The encoded microspheres should be stored at 4 °C. Avoid freez.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Eu-TTA dye	Fisher Scientific	AC42319-0010
THF	Sigma-Aldrich	34865-100ML
Amber glass vial	Fisher Scientific	03-339-23B
Coumarin 30 dye	Sigma-Aldrich	546127-100MG
Microspheres	Bangslabs	PC05N/6698
1.5 ml microcentrifuge tubes	Fisher Scientific	05-408-129
PBS 10x concentrate	Sigma-Aldrich	P5493-1L
Water	Sigma-Aldrich	W4502-1L
Methanol	Sigma-Aldrich	34860-100ML
Tw-20	Sigma-Aldrich	P7949-100 ml
BupH MES buffered saline	Thermo Scientific	28390
SDS	Sigma-Aldrich	05030-500ML-F
NaOH solution	Fisher Scientific	SS256-500
Safe-lock microcentrifuge tube	VWR labshop	53511-997
EDC	Thermo Scientific	22980
Sulfo-NHS	Thermo Scientific	24510
Human VEGF capture antibody	R&D Systems	MAB293
Human IP-10 capture antibody	R&D Systems	MAB266
Human IL-8 capture antibody	R&D Systems	MAB208
Human EGF capture antibody	R&D Systems	MAB636
Human MMP-9 capture antibody	R&D Systems	MAB936
Human IL-1β capture antibody	R&D Systems	MAB601
Mouse IgG1 isotype control antibody	R&D Systems	MAB002
StartingBlock (TBS) buffer	Thermo Scientific	37542
HCl standard solution 1.0 N	Sigma-Aldrich	318949-500 ml
0.5 ml microcentrifuge tubes	Fisher Scientific	05-408-120
Protein-free (PBS) buffer	Thermo Scientific	37572
Recombinant human VEGF 165	R&D Systems	293-VE
Recombinant human IP-10	R&D Systems	266-IP
Recombinant human IL-8	R&D Systems	208-IL
Recombinant human EGF	R&D Systems	236-EG
Recombinant human MMP-9	R&D Systems	911-MP
Recombinant human IL-1β	R&D Systems	201-LB
StartingBlock T20 (PBS) buffer	Thermo Scientific	37539
Blocker BSA in PBS	Thermo Scientific	37525
Biotinylated VEGF detection antibody	R&D Systems	BAF293
Biotinylated IP-10 detection antibody	R&D Systems	BAF266
Biotinylated IL-8 detection antibody	R&D Systems	BAF208
Biotinylated EGF detection antibody	R&D Systems	BAF236
Biotinylated MMP-9 detection antibody	R&D Systems	BAF911
Biotinylated IL-1β detection antibody	R&D Systems	BAF201
Streptavidin, R-phycoerythrin	Invitrogen	S-21388
Ethanol (200 proof)	Sigma-Aldrich	E7023-500ML