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. 2023 Feb 22;24(5):4347.
doi: 10.3390/ijms24054347.

Label-Free Multiplexed Microfluidic Analysis of Protein Interactions Based on Photonic Crystal Surface Mode Imaging

Galina Nifontova  1 , Irina Petrova  2 , Evgeniia Gerasimovich  2 , Valery N Konopsky  3 , Nizar Ayadi  4 , Cathy Charlier  5 , Fabrice Fleury  4 , Alexander Karaulov  6 , Alyona Sukhanova  1 , Igor Nabiev  1   2   6
Affiliations

Label-Free Multiplexed Microfluidic Analysis of Protein Interactions Based on Photonic Crystal Surface Mode Imaging

Galina Nifontova et al. Int J Mol Sci. .

Abstract

High-throughput protein assays are crucial for modern diagnostics, drug discovery, proteomics, and other fields of biology and medicine. It allows simultaneous detection of hundreds of analytes and miniaturization of both fabrication and analytical procedures. Photonic crystal surface mode (PC SM) imaging is an effective alternative to surface plasmon resonance (SPR) imaging used in conventional gold-coated, label-free biosensors. PC SM imaging is advantageous as a quick, label-free, and reproducible technique for multiplexed analysis of biomolecular interactions. PC SM sensors are characterized by a longer signal propagation at the cost of a lower spatial resolution, which makes them more sensitive than classical SPR imaging sensors. We describe an approach for designing label-free protein biosensing assays employing PC SM imaging in the microfluidic mode. Label-free, real-time detection of PC SM imaging biosensors using two-dimensional imaging of binding events has been designed to study arrays of model proteins (antibodies, immunoglobulin G-binding proteins, serum proteins, and DNA repair proteins) at 96 points prepared by automated spotting. The data prove feasibility of simultaneous PC SM imaging of multiple protein interactions. The results pave the way to further develop PC SM imaging as an advanced label-free microfluidic assay for the multiplexed detection of protein interactions.

Keywords: immunoassay; label-free biosensing; multiplexed detection; photonic crystal surface mode imaging; protein array.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
A schematic diagram of the photonic crystal chip with proteins and peptides deposited and possible interactions between the deposited spots and injected analytes. (a) The layout of spotting of model proteins and peptides. (b) The possible interactions between the proteins. The arrow in panel A indicates an increasing number of drops per spot. Abbreviations: Ab, antibody; Ab-AF680, secondary antibody conjugated with Alexa Fluor 680; Ab-IRD800, secondary antibody conjugated with IRDye 800CW; Y315, Y315 peptide; pY315, phosphorylated Y315 peptide; RAD51, DNA repair protein; phosphotyrosine BSA, bovine serum albumin phosphorylated at tyrosine residues; BSA, bovine serum albumin; anti-phosphotyrosine Ab, antibody for the detection of phosphorylated proteins; anti-RAD51 Ab, antibody for the detection of the DNA repair protein; anti-pY315 Ab, antibody for the detection of the phosphorylated Y315 peptide; anti-His tag Ab, anti-polyhistidine antibody.
Figure 2
Figure 2
The overview of the chip surface with applied proteins and peptides. (a) A black-and-white 2D image of the chip placed in front of the CCD camera before antibody running. (b) A layout consisting of 96 regions of interest (ROIs) corresponding to the deposited spot duplicates. The protein spotting pattern corresponds to the scheme shown in Figure 1. Abbreviations: Ab, antibody; Ab-AF680, secondary antibody conjugated with Alexa Fluor 680; Ab-IRD800, secondary antibody conjugated with IRDye 800CW; Y315, Y315 peptide; pY315, phosphorylated Y315 peptide; RAD51, DNA repair protein; phosphotyrosine-BSA, bovine serum albumin phosphorylated at tyrosine residues; BSA, bovine serum albumin; anti-phosphotyrosine Ab, antibody for the detection of phosphorylated proteins; anti-RAD51 Ab, antibody for the detection of the DNA repair protein; anti-pY315 Ab, antibody for the detection of the phosphorylated Y315 peptide; anti-His tag Ab, anti-polyhistidine antibody.
Figure 3
Figure 3
The sensorgrams of interaction of proteins, peptides, and injected antibody analytes in the selected spots. The start of the injection of each antibody analyte run through the microfluidic cell is indicated with an arrow. Each resultant sensorgram is an average of two sensorgrams recorded in duplicate. Abbreviations: da, adlayer thickness; Ab, antibody; Ab-AF680, secondary antibody conjugated with Alexa Fluor 680; Ab-IRD800, secondary antibody conjugated with IRDye 800CW; phosphotyrosine-BSA, bovine serum albumin phosphorylated on tyrosine residues; anti-RAD51 Ab, antibody for the detection of the DNA repair protein; anti-phosphotyrosine Ab, antibody for the detection phosphorylated protein and peptides; anti-His tag Ab, anti-polyhistidine antibody; anti-rabbit Ab, antibody for the detection of rabbit antibodies.
Scheme 1
Scheme 1
The concept of the label-free microfluidic sensor of multiple protein interactions based on photonic crystal surface mode imaging. Abbreviations: 1D PC, one-dimensional photonic crystal; Ab, antibody; p, polarizer; A, association of an antibody analyte to a protein spot; S, saturation of a protein spot with an antibody analyte; D, dissociation of an antibody–protein complex.
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