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

The unique multichannel capability of ¹⁹F 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 ¹⁹F signals, simultaneously hyperpolarizing multiple ¹⁹F-containing substrates, which is essential for unlocking the multichannel potential of ¹⁹F NMR, remains a key challenge. Here, we introduce an approach that achieves effective and simultaneous hyperpolarization of different ¹⁹F nuclei from several molecular probes through parahydrogen-based signal amplification by reversible exchange (SABRE). Our strategy utilizes multiple ¹⁹F-labeled substrates for SABRE, acting as co-ligands for simultaneous enhancement of their ¹⁹F NMR signal intensities. The synergistic effect among different ¹⁹F-labeled co-substrates is evidenced by the much higher signal enhancement, compared to the systems containing only one ¹⁹F-labeled substrate. The highest enhancement of the ¹⁹F 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 ¹⁹F-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 ¹⁹F NMR for applications in biomarker detection. Our work paves the way for future applications of highly sensitive multichannel ¹⁹F NMR analysis.