Excitation/emission-enhanced heterostructure photonic crystal array synergizing with "DD-A" FRET entropy-driven circuit for high-resolution and ultrasensitive analysis of ctDNA.

Circulating tumor DNA (ctDNA) is an emerging biomarker of liquid biopsy for cancer. But it remains a challenge to achieve simple, sensitive and specific detection of ctDNA because of low abundance and single-base mutation. In this work, an excitation/emission-enhanced heterostructure photonic crystal (PC) array synergizing with entropy-driven circuit (EDC) was developed for high-resolution and ultrasensitive analysis of ctDNA. The donor donor-acceptor FÖrster resonance energy transfer ("DD-A" FRET) was integrated in EDC based on the introduction of simple auxiliary strand, which exhibited higher sensitivity than that of traditional EDC. The heterostructure PC array was constructed with the bilayer periodic nanostructures of nanospheres. Because the heterostructure PC has the adjustable dual photonic band gaps (PBGs) by changing nanosphere sizes, and the "DD-A" FRET can offer the excitation and emission peak with enough distance, it helps the successful matches between the dual PBGs of heterostructure PC and the excitation/emission peaks of "DD-A" FRET; thus, the fluorescence from EDC can be enhanced effectively from both of excitation and emission processes on heterostructure PC array. Besides, high-resolution of single-base mutation was obtained through the strict recognition of EDC. Benefiting from the specific spectrum-matched and synergetic amplification of heterostructure PC and EDC with "DD-A" FRET, the proposed array obtained ultrasensitive detection of ctDNA with LOD of 12.9 fM, and achieved the analysis of mutation frequency as low as 0.01%. Therefore, the proposed strategy has the advantages of simple operation, mild conditions (enzyme-free and isothermal), high-sensitivity, high-resolution and high-throughput analysis, showing potential in bioassay and clinical application.

A quadratic isothermal amplification fluorescent biosensor without intermediate purification for ultrasensitive detection of circulating tumor DNA.

Circulating tumor DNA (ctDNA) is an auspicious tumor biomarker released into the bloodstream by tumor cells, offering abundant information concerning cancer genes. It plays a crucial role in the early diagnosis of cancer. However, due to extremely low levels in body fluids, achieving a simple, sensitive, and highly specific detection of ctDNA remains challenging. Here, we constructed a purification-free fluorescence biosensor based on quadratic amplification of ctDNA by combining nicking enzyme mediated amplification (NEMA) and catalytic hairpin assembly (CHA) reactions. After double isothermal amplification, this biosensor achieved an impressive signal amplification of nearly 107-fold, enabling it to detect ctDNA with ultra-sensitivity. And the detection limit of this biosensor is as low as 2 aM. In addition, we explored the influence of human serum on the performance of the biosensor and found that it showed favorable sensitivity in the presence of serum. This biosensor eliminates the need for an intermediate purification step, resulting in enhanced sensitivity and convenience. Thus, our purification-free fluorescent biosensor exhibits ultra-high sensitivity when compared to other biosensors and has the potential to serve as an effective diagnostic tool for early detection of cancer.