Ultrasensitive detection of colorectal cancer biomarkers in exhaled breath using superhydrophobic HCPs-2Br-based SPME-GC-MS with a two-step pre-enrichment strategy.

[BACKGROUND] Exhaled breath has emerged as a promising biospecimen for disease diagnosis, particularly for non-invasive screening. However, the detection of disease biomarkers in exhaled breath presents significant challenges due to their ultra-trace levels and the complex, high-humidity composition of the breath matrix. Consequently, there is a pressing need to develop innovative pretreatment technologies to enable the sensitive detection of colorectal cancer (CRC)-related biomarkers for early non-invasive screening.

[RESULTS] In this study, a superhydrophobic dibromodibenzothiophene-based hypercrosslinked polymer (HCPs-2Br) was developed as a novel solid-phase microextraction (SPME) fiber coating and established a two-step pre-enrichment strategy to overcome these challenges. Target biomarkers were first pre-concentrated using Tenax TA adsorption tubes, followed by methanol elution, and subsequently enriched using HCPs-2Br-SPME. Coupled with GC-MS, this method enabled ultrasensitive and selective quantification of six CRC-related biomarkers in human exhaled breath. The HCPs-2Br coating demonstrated excellent affinity and selectivity, achieving enrichment factors ranging from 1498 to 29,078. The limits of detection were as low as 0.4-14.8 pg mL, with strong linearity (r ≥ 0.9987), recoveries between 86.3 % and 117.3 %, and relative standard deviations below 12.4 %. The method was applied to exhaled breath samples from patients and volunteers, confirming its robustness, reproducibility, and clinical potential.

[SIGNIFICANCE] This work presents a novel pretreatment framework for the analysis of disease-related volatile organic compounds in exhaled breath. It provides a simple, cost-effective, and highly sensitive platform for non-invasive CRC screening, laying the foundation for broader applications of breath-based biomarker detection in clinical diagnostics and metabolic research.