SABRE Hyperpolarized Multichannel ¹⁹ F NMR for Sensitive Detection of Multiple Disease Marker Enzymes on a Benchtop NMR

Abstract The unique multichannel capability of 19 F nuclear magnetic resonance (NMR) stands as a pivotal and rapidly advancing frontier in chemistry, biology, and medical imaging. However, its inherent low sensitivity limits its widespread applications. While hyperpolarization significantly enhances 19 F signals, simultaneously hyperpolarizing multiple 19 F‐containing substrates, which is essential for unlocking the multichannel potential of 19 F NMR, remains a key challenge. Here, we introduce an approach that achieves effective and simultaneous hyperpolarization of different 19 F nuclei from several molecular probes through parahydrogen‐based signal amplification by reversible exchange (SABRE). Our strategy utilizes multiple 19 F‐labeled substrates for SABRE, acting as co‐ligands for simultaneous enhancement of their 19 F NMR signal intensities. The synergistic effect among different 19 F‐labeled co‐substrates is evidenced by the much higher signal enhancement, compared to the systems containing only one 19 F‐labeled substrate. The highest enhancement of the 19 F NMR signal reached over 9600‐fold on a benchtop NMR (1.4 T), corresponding to a 4.3% polarization level. On this basis, we successfully implemented multichannel quantitative detection of enzymatic biomarkers at µM levels. By utilizing three 19 F‐labeled pyridine‐based substrates, each bearing a specific enzyme‐responsive moiety, we demonstrate the capability of high‐speed, cost‐efficient, and highly sensitive SABRE‐polarized 19 F NMR for applications in biomarker detection. Our work paves the way for future applications of highly sensitive multichannel 19 F NMR analysis.