Cascade-Amplified Microfluidic Detection of Tumor Biomarkers via Metal-DNA Nanospheres Activating CRISPR-Cas12a Assay

Abstract

The development of ultrasensitive protein detection platforms is pivotal for early cancer diagnosis, yet existing methods are often constrained by inefficient “protein-to-signal” conversion and complex instrumentation. While CRISPR-Cas-based biosensors offer higher sensitivity for nucleic acids, their application in protein analysis is fundamentally limited by the low nucleic acid loading capacity and tedious functionalization of traditional inorganic signal carriers such as gold nanoparticles and metal–organic frameworks (MOFs). Herein, we report a highly efficient signal-transduction strategy utilizing Fe-coordinated DNA nanospheres (FDNSs)─a programmable, metal-nucleic acid assembly─as a high-capacity DNA reservoir for amplified biomarker sensing. Unlike conventional surface-modified inorganic nanoparticles, the solid-core FDNSs achieve an unprecedented DNA loading density and exhibit specific stimulus-responsive degradation. In this FACC (FDNS-activated CRISPR chip) platform, the recognition of tumor biomarkers triggers the rapid collapse of FDNSs, releasing a massive flux of barcode DNA that subsequently activates the CRISPR-Cas12a system within a microfluidic device. This dual-amplification architecture enables the ultrasensitive quantification of prostate-specific antigen (PSA) and carcinoembryonic antigen (CEA) with detection limits of 0.028 fg/mL and 0.0265 fg/mL, respectively. By replacing complex inorganic carriers with this programmable DNA assembly, our platform simplifies the sensing workflow and significantly enhances sensitivity, providing a robust and portable solution for cancer screening in resource-limited settings.