Overcoming the Thermodynamic Diffusion Barrier in DNA Cascade Amplifiers via Spatially Confined Entropy Reduction: A Versatile Kinetic Engineering Framework.

The reaction efficiency of solution-phase DNA cascade amplifiers is fundamentally constrained by stochastic Brownian motion, creating a thermodynamic diffusion bottleneck that limits sensitivity and speed. To overcome this, we present a general kinetic engineering framework based on spatially confined entropy reduction. By integrating a DNA tetrahedron scaffold with a cascade amplification circuit, we successfully transition the reaction from a stochastic dilute-phase regime to a deterministic pseudosolid-phase regime. For the first time, we employ phase-space kinetic mapping to visualize the entropy-driven acceleration in DNA nanomachines. This comprehensive analysis reveals that this architecture increases the effective local concentration by 4 orders of magnitude by prepaying the entropic cost of molecular collision. Using targets spanning small molecules (BPA), macromolecules (Thrombin), and nucleic acids (miRNA), the system achieved a detection limit of 0.1 pM with rapid kinetics. This work establishes a theoretical blueprint. Crucially, the phase-space kinetic mapping framework proposed here serves as a universal toolkit for the community, offering a new dimension to evaluate and optimize next-generation DNA cascade amplifiers.