Easy Surface Functionalization and Bioconjugation of Peptides as Capture Agents of a Microfluidic Biosensing Platform for Multiplex Assay in Serum.

Concetta Di Natale,Gabriele Pitingolo,Narayana Reddy,Filippo Causa,Vincenzo Lettera,Raffaele Vecchione,Paolo Antonio Netti,Edmondo Battista

doi:10.1021/acs.bioconjchem.1c00146

Abstract

The development of assays for protein biomarkers in complex matrices is a demanding task that still needs implementation of new approaches. Antibodies as capture agents have been largely used in bioassays but their low stability, low-efficiency production, and cross-reactivity in multiplex approaches impairs their larger applications. Instead, synthetic peptides, even with higher stability and easily adapted amino acid sequences, still remain largely unexplored in this field. Here, we provide a proof-of-concept of a microfluidic device for direct detection of biomarker overexpression. The multichannel microfluidic polydimethylsiloxane (PDMS) device was first derivatized with PAA (poly(acrylic acid)) solution. CRP-1, VEGF-114, and ΦG6 peptides were preliminarily tested to respectively bind the biomarkers, C-reactive protein (CRP), vascular endothelial growth factor (VEGF), and tumor necrosis factor-alpha (TNF-α). Each PDMS microchannel was then respectively bioconjugated with a specific peptide (CRP-1, VEGF-114, or ΦG6) to specifically capture CRP, VEGF, and TNF-α. With such microdevices, a fluorescence bioassay has been set up with sensitivity in the nanomolar range, both in buffered solution and in human serum. The proposed multiplex assay worked with a low amount of sample (25 μL) and detected biomarker overexpression (above nM concentration), representing a noninvasive and inexpensive screening platform.

Highlights

In the past decades, the development of microassays able to detect and simultaneously monitor the levels and activities of a large number of proteins is becoming one of the hot topics in the biotechnological field.[1]
The microfluidic chip was fabricated by coupling micromilling and soft lithography technology based on polydimethylsiloxane (PDMS)
The fabrication of PDMS microchannels is straightforward. They can be replicated from poly(methyl methacrylate) (PMMA) negative master molds (Figure 1a)

Summary

INTRODUCTION

The development of microassays able to detect and simultaneously monitor the levels and activities of a large number of proteins is becoming one of the hot topics in the biotechnological field.[1]. Peptides can be produced through an economically affordable methodology and purified in large quantities, with an efficient quality control;[9−13] they possess good stability that does not need particular environmental conditions such as temperature/pH variations, presence/absence of water molecules, or protease/ nuclease degradation.[10] they can be isolated from combinatorial libraries and are able to bind target proteins with high affinity.[10,14,15] In addition, thanks to the possibility to change/design their native amino acid sequence, they can be synthesized with a common sequence able to bind to the activated surface in order to avoid cross-reactivity phenomena in multiplex assays or to generate a more uniform deposition.[16] Based on these statements, we selected three different peptides as model ligands of various inflammatory-cancer biomarkers: tumor necrosis factor-α (TNF-α), vascular endothelial growth factor (VEGF), and C-reactive protein (CRP). To the best of our knowledge, few examples of microfluidic peptide-based biosensors are reported in the literature, and none of these can be compared with the robustness of the gold standard methods such as ELISA.[35,36] Anyway, with an appropriate level of improvement for their user-friendly nature, they can have a major impact on clinical diagnostics creating a novel-based generation of biosensors for specific biomolecule targeting.[37]

RESULTS AND DISCUSSION

EXPERIMENTAL SECTION

CONCLUSIONS

■ REFERENCES