Gold Nanoparticle‑Based Immunofluorescence Enables Rapid, Sensitive Detection of Inflammation‑Associated Cancer Biomarkers

Abstract

Early cancer detection hinges on identifying inflammatory cues that precede malignancy. We evaluated primary antibody‑conjugated gold nanoparticles (AuNPs) as targeted contrast agents for chronic inflammation in ulcerative colitis (UC) and steatohepatitis models, conditions that predispose to colorectal and liver cancer. AuNPs (7.1 nm) were synthesized via a cost‑effective, eco‑friendly route and conjugated with anti‑COX‑2, anti‑MIF, and Alexa Fluor® 488 (ALEXA). Three immunofluorescence (IF) protocols—30‑min and overnight incubation, plus an amplification protocol—were compared to the standard ALEXA IF workflow. Flow cytometry (FC) used AuNPs to label M2 macrophages. Conjugation was achieved by direct adsorption, confirmed by comparable fluorescence intensity to ALEXA. The 30‑min AuNP protocol reduced total IF time from 23 h to 5 h and FC time from 4 h to 1 h, offering a labor‑saving, clinically viable alternative for inflammation‑driven cancer diagnostics.

Introduction

Gold nanoparticles (AuNPs) have emerged as versatile probes for optical imaging, offering size‑tunable optical properties, high biocompatibility, and ease of functionalisation. Antibody‑AuNP conjugates enable real‑time detection of gold uptake in living cells, facilitating precise quantification of nanoparticle distribution in disease states. Historically, colloidal gold has been employed for electron microscopy of bacterial antigens, and later extended to diverse biomedical applications, including genomics, biosensing, and photothermal therapy. In oncology, AuNPs enhance fluorescence signals and X‑ray attenuation, improving tumour imaging sensitivity.

Building on our prior work demonstrating AuNP‑conjugated antibodies for colorectal carcinoma imaging, this study introduces three IF protocols that replace conventional fluorophores with AuNPs, thereby accelerating staining and reducing reagent consumption. We also validate AuNPs in FC for labeling M2 macrophages, key players in tumour‑associated inflammation.

Methods

Aim

To demonstrate that AuNPs can replace conventional fluorophores in IF and FC, providing faster, equally reliable staining for inflammation‑driven cancer biomarkers.

Reagents

AuCl₃, PVP (MW 10 000), NaOH, glycerol, PBS, BSA (5 %), anti‑COX‑2, anti‑MIF, Alexa Fluor® 488 secondary antibody, and Fluoroshield with DAPI.

AuNP Synthesis and Characterisation

Spherical AuNPs (7.1 nm) were prepared by reducing AuCl₃ in the presence of PVP, glycerol, and NaOH, yielding a deep‑red colloid. UV–vis spectra and fluorescence measurements confirmed monodispersity and surface plasmon resonance. Antibodies were adsorbed directly onto AuNPs, achieving stable conjugates.

Animal Models

Acetic acid‑induced UC in female Wistar rats (n = 5) and alcohol‑induced steatohepatitis in male Wistar rats (n = 5) were established per standard protocols. Tissues were fixed, embedded, and sectioned (4 µm) for IF.

Immunofluorescence Protocols

Three AuNP‑based protocols were evaluated:

• NP1 (30 min): Primary antibody (1:500 COX‑2, 1:400 MIF) incubated 30 min, followed by 30 min AuNP addition.
• NP2 (overnight): Primary antibody incubated 18 h, then 1 h AuNP incubation.
• NP3 (amplification): ALEXA diluted (1:200–1:800) mixed with AuNPs; 1 h incubation.

Standard ALEXA protocols (A1 overnight, A2 30 min) served as controls. Sections were imaged on a Zeiss Observer z.1 with quantitative pixel‑wise fluorescence analysis.

M2 Macrophage Labeling and Flow Cytometry

RAW 264.7 cells were polarized to M2 with IL‑4 (20 ng mL⁻¹) for 48 h. Cells were incubated with COX‑2/MIF primary antibodies and labelled with either ALEXA or AuNPs (N1 protocol). After fixation, samples were analysed on a BD FACSCanto II; data were processed with FlowJo.

Statistical Analysis

ANOVA with Bonferroni post‑hoc (IF) and Dunn’s test (FC) assessed significance; p < 0.05 considered significant.

Results

Microscopy

H&E images confirmed expected inflammatory pathology. Fluorescence intensity of NP1 and NP2 matched A1, and NP3 amplified signal, allowing two‑fold ALEXA dilution without loss of brightness. Short‑time (30 min) protocols (A2, NP1) produced comparable staining to overnight standards, demonstrating feasibility for rapid diagnostics.

Flow Cytometry

IL‑4‑polarised RAW cells exhibited elevated CD163 (M2 marker) and unchanged CD86 (M1). AuNP‑labelled COX‑2/MIF produced fluorescence intensities comparable to ALEXA, confirming efficient surface binding and minimal reagent usage.

Fluorescence Spectroscopy

AuNPs exhibited emission at 650–900 nm upon 320 nm excitation. Antibody adsorption increased fluorescence by ~12 a.u., indicating successful conjugation and enhanced signal due to reduced oxygen quenching.

Discussion

AuNPs possess intrinsic fluorescence and act as signal amplifiers by blocking oxygen access to surface electrons, thereby enhancing surface plasmon resonance. Conjugation via direct adsorption provides a simple, scalable approach, avoiding covalent chemistry and preserving antibody activity. The NP1 protocol reduces total IF workflow by 18 h, while FC time drops from 4 h to 1 h, offering substantial workflow efficiencies. Importantly, AuNPs retain sensitivity for COX‑2 and MIF, key biomarkers in UC‑associated colorectal cancer and alcohol‑induced steatohepatitis‑related hepatocellular carcinoma. Given the high prevalence of chronic inflammation in cancer risk, rapid, cost‑effective detection methods are essential.

Conclusions

AuNP‑based IF and FC protocols deliver fluorescence intensities on par with conventional fluorophores, while drastically cutting incubation times and reagent consumption. These findings open avenues for clinical adoption of AuNPs in diagnostic pathology and potentially in therapeutic targeting of M2 macrophages.

Abbreviations

A1

ALEXA standard protocol

A2

ALEXA modified protocol

ALD

Alcoholic liver disease

ALEXA

Alexa Fluor® 488®

AuNP

Gold nanoparticles

BSA

Bovine serum albumin

COX‑2

Cyclooxygenase‑2

FC

Flow cytometry

FI

Fluorescence imaging

IF

Immunofluorescence

MDA

Malondialdehyde

MIF

Macrophage migration inhibitory factor

NP1

Nanoparticles protocol 1

NP2

Nanoparticles protocol 2

NP3

Nanoparticles protocol 3

SPR

Surface plasmon resonance

UC

Ulcerative colitis