Cell Surface Ion-Seq: Potassium Ion Monitoring in the Colorectal Cancer Cellular Microenvironment Based on Split G-quadruplex Probe

Potassium ions (K⁺) are vital for biological systems and essential cellular functions, making their homeostasis monitoring crucial for investigating electrolyte imbalance symptoms. Antibody-based high-throughput single-cell sequencing enables simultaneous phenotype and genotype profiling in individual cells; however, its application to ion monitoring encounters inherent limitations. The subnanometer scale of K⁺ ions precludes antibody-based detection, creating a significant technical barrier for correlating ionic activity with cellular phenotypes at single-cell resolution. In this study, we report a cell surface biosensor for single-cell ion sequencing (Ion-seq) to monitor K⁺ homeostasis in clinical samples. The design utilizes split G-quadruplex (G4), where two guanine-rich DNA oligonucleotides cofold into four-stranded noncanonical secondary structures upon K⁺ binding. Specifically, a lipid-labeled capture probe anchors to the cell membrane. Upon drug-stimulated release of K⁺ from cells, the free sensing probe is captured, forming a complete G-quadruplex with the capture probe. Sequencing the sensing probe then enables monitoring of potassium homeostasis at single-cell resolution, allowing ionic phenotyping within the context of cellular heterogeneity and function. Moreover, this biosensor holds potential for broader bioapplications in analyzing other ions at the single-cell resolution, advancing disease diagnosis and personalized medicine.