A methyl-β-D-galactopyranoside breath probe for early detection of hepatocellular carcinoma

A non-isotopic methanol-releasing probe detects HCC early via breath. • Selectively cleaved by β-galactosidase in HCC for tumor-specific signal. • A low-cost dual-sensor array enables real-time exhaled methanol detection. • Distinguishes early-stage tumor-bearing mice from controls (< 2 mm). • Offers a portable, affordable screening tool for primary healthcare. Early detection is crucial for improving therapeutic outcomes in hepatocellular carcinoma (HCC), yet the widespread implementation of low-cost, accessible screening for high-risk populations remains a significant challenge. To address this issue, we investigated the β-galactosidase–methanol signaling axis using a hydrophilic, cell-membrane-impermeable probe, methyl-β-D-galactopyranoside (MGP). This probe is selectively hydrolyzed by β-galactosidase, which is overexpressed in the HCC microenvironment, releasing methanol—an easily volatile, non-isotopic, and metabolically inert volatile organic compounds (VOCs). Methanol levels in exhaled breath were quantified using a custom-designed sensor array. Through in vitro enzymatic assays, cellular studies, and animal experiments, we demonstrated that methanol concentrations in tumor-bearing mice (with tumor diameters <2 mm) were significantly elevated compared to healthy controls, and these levels positively correlated with tumor volume. Our findings highlight the potential of Me-β-Gal in addressing critical barriers to early HCC detection. With further validation, this approach could potentially improve clinical management for high-risk populations, including patients with liver cirrhosis.