Development of liquid biopsy for screening colorectal cancer through the combination of an antibody microarray-based metal-enhanced sandwich immunofluorescent assay of cytokines with machine learning

Abstract

The simultaneous determination of the expression levels of multiple inflammation-associated cytokines in blood holds great promise for the early screening of cancer including colorectal cancer (CRC). Herein, an antibody microarray-based sandwich metal-enhanced immunofluorescent assay (AMSMEIFA) is developed for the quantitative measurement of five cytokines simultaneously through the fabrication of an antibody microarray on a slide coated with a poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) layer and modified with gold nanorods (GNR@P(GMA-HEMA) slide). Benefiting from the metal-enhanced fluorescence (MEF) property and abundant antibody immobilization sites in the GNR@P(GMA-HEMA) slide, the newly developed AMSMEIFA enables the selective measurement of five pro-inflammatory soluble cytokines (interleukins (IL-1β, IL-2 and IL-6), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ)) with low limits of detection (LODs) at the sub-pg mL⁻¹ level. The practicability of AMSMEIFA is demonstrated by the simultaneous determination of five cytokines in 35 clinical serum samples, which are obtained from 25 CRC patients and 10 healthy donors (HDs). After analyzing the expression levels of five pro-inflammatory soluble cytokines using a machine learning (ML) model based on the least absolute shrinkage and selector operator (LASSO) regression, the area under the receiver operator characteristic curve (AUC) of CRC is as high as 0.92, demonstrating that ML-assisted AMSMEIFA could be used as a liquid biopsy for screening CRC with reasonable accuracy.