Target-Triggered Enzymatic Cascade LF-NMR Biosensor for the Detection of Circulating Tumor Cells

Abstract

A target-triggered, enzymatic cascade-amplified low-field nuclear magnetic resonance (LF-NMR) sensor was developed for the detection of the circulating tumor cell (CTC) A549. A multifunctional two-dimensional bionanomaterial GDA@GOX&DNA1 was designed as the initiator, with Fe₃O₄@DNA2/Apt as the recognition unit and CaO₂@MnO₂ as the signal unit. When A549 was present, the aptamer (Apt) detached from the recognition unit, allowing the formation of GDA@GOX&DNA1-DNA2@Fe₃O₄ and triggering the following reactions: (1) glucose oxidase (GOX) catalyzed the reaction between the substrate glucose and oxygen (O₂) to produce gluconic acid and hydrogen peroxide (H₂O₂); (2) the generated acid and H₂O₂ reacted with MnO₂, producing signal probes Mn²⁺ and O₂; and (3) CaO₂ reacted with the acid, generating H₂O₂. These cyclic reactions brought the generation of massive Mn²⁺ and a decrease of the transverse relaxation time (T₂), resulting in a target-triggered, enzymatic cascade-amplified LF-NMR biosensing of CTCs. Under the optimal experimental conditions, the linear range and limit of detection (LOD) were 10–1.0 × 10⁶ and 6 cells/mL, respectively. The feasibility and reliability in practical applications were verified by using spiked whole blood samples containing A549 cells. This study represents the first successful demonstration of an LF-NMR biosensor for the detection of intact CTCs, providing a new tool for clinical testing and diagnosis.