The CRISPR/Cas12a system enables precise and rapid nucleic acid recognition through the programmable targeting capability of CRISPR RNA (crRNA). However, the intrinsic instability of crRNA limits the robustness and sensitivity of CRISPR-based molecular diagnosis in practical applications. Herein, we present a simple and general enhancement strategy that suppresses RNase-mediated crRNA degradation. This strategy, termed RNase inhibitor (RI)-assisted CRISPR (RI-CRISPR), leverages RI to specifically prevent crRNA degradation, thereby improving its stability and enhancing the detection performance and anti-interference capability of the CRISPR system. Using influenza A virus (IAV) and mycobacterium tuberculosis (MTB) as model targets, RI-CRISPR improves detection sensitivity by nearly twentyfold compared to conventional CRISPR/Cas12a. Clinical validation using 40 MTB samples, combined with recombinase polymerase amplification (RPA), achieves 100% specificity and 96% sensitivity compared with the GeneXpert assay. Overall, this work provides a practical strategy to enhance sensitivity and robustness of CRISPR-based diagnostics, and is expected to promote further biomedical applications of CRISPR technology.

Selective and tunable regulation of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a activity enables on-demand control, yet current strategies remain hindered by nonspecific regulation and limited tunability. Inspired by proximity effect, we present a Cas-regulation-targeting chimera (CasTAC) strategy that employs CRISPR RNA (crRNA) as a proximity mediator to carry phosphorothioate regulators to interfere with catalytic or recognition domains of Cas12a and consequently suppress its activity. This crRNA-induced proximity approach can effectively eliminate nonspecific interaction between phosphorothioate regulators and proteins within complex multi-enzyme systems, thereby enabling selective control over CRISPR/Cas12a activity. Furthermore, CRISPR/Cas12a activity can be finely tuned to different inhibitory levels by varying the number of phosphorothioate regulators. The CasTAC strategy also improves nuclease resistance and single-nucleotide discrimination, offering potential advances in the sensitivity of molecular diagnostics and the accuracy of gene editing. Notably, the CasTAC balances the kinetics of nucleic acid amplification and CRISPR cleavage, facilitating efficient product accumulation and resolving compatibility issues in one-pot assays. As a proof of concept, we develop a one-pot, one-step recombinase polymerase amplification-CasTAC assay that achieves over 1000-fold higher detection sensitivity than the conventional one-pot recombinase polymerase amplification-CRISPR/Cas12a assay. The CasTAC strategy provides a versatile framework for fine-tuning Cas activity and advances CRISPR technology toward refined and context-adaptable functionality.

Chloramphenicol (CAP), a broad-spectrum antibiotic derived from Streptomyces, readily persists and accumulates in humans through pathways such as the food chain, leading to significant toxic effects. Herein, a novel electrochemiluminescence (ECL) aptasensor based on the signal “ON–OFF–ON” strategy is proposed for CAP detection in food. We utilized the immobilization capacity and enzyme-mimicking activity of iron-lanthanum doped prussian blue analogue (Fe-La PBA) to catalytically enhance the luminol–H2O2 luminescent system, thereby engineering the composite nanozyme-based luminophore (luminol-Au@Fe-La PBA). Moreover, polydopamine (PDA)-coordinated cadmium sulfide (CdS) decorated with gold nanoparticles (Au@CdS/PDA) serves as an energy acceptor to quench the signal from emitter luminol-Au@Fe-La PBA. Firstly, the luminol-Au@Fe-La PBA emitter was immobilized on a glassy carbon electrode (GCE), providing a strong initial ECL signal and serving as a platform for anchoring cDNA hybridized with the aptamer (Apt). Then the specific quenching probe (Apt-Au@CdS/PDA) was introduced, leading to substantial signal reduction. Upon the action of CAP, the dissociation of the Apt–cDNA hybrid duplex led to the detachment of the quenching probe from the electrode surface, thereby restoring the ECL signal intensity. The aptasensor demonstrated a linear response over a concentration range of 10 to 107 pg mL− 1, and LOD was 1.18 pg mL− 1, exhibiting satisfactory recoveries in various food samples and quality control samples.

Conventional receptor-targeted fluorescent probes have shown promise in tumor imaging, yet achieving a high tumor-to-normal (T/N) tissue ratio in vivo remains challenging due to limited biomarker density on tumor cell membranes. Here, we present an in situ assembly strategy of bioorthogonal-functionalized chimeric artificial receptors (BCARs) that locally constructs BCARs on tumor surfaces, which amplify fluorescence signals and enable high-contrast imaging. Rapid, selective membrane engineering under physiological conditions increases effective receptor density, enhancing fluorophore binding and tumor visualization. Mechanistic studies reveal that BCARs exhibit exceptional membrane retention and spatial precision, sustaining signal amplification in heterogeneous tumor microenvironments. In air-pouch and orthotopic bladder cancer models, BCARs notably improve the T/N imaging ratio and tumor boundary delineation. Translational validation with surgical specimens from 14 patients with bladder cancer confirms clinical feasibility. This work establishes a versatile platform for on-site receptor reprogramming and signal amplification, offering a powerful tool for high-contrast tumor margin detection.

The development of efficient and easily synthesized electrochemiluminescent materials is attractive but still challenging. Metal-organic gels (MOGs) are porous, soft hybrid supramolecular materials with unique optical properties. In this work, functional luminescent lanthanide organic gels (LEOGs) were synthesized by rapid coordination of lanthanide metal (Eu3+) with 2,2":6",2''-terpyridine-4'-carboxylic acid (TPY). The self-luminescence behavior of LEOGs arises from the realization of the antenna effect within the molecule. According to the antenna effect, the TPY ligand is able to sensitize the central lanthanide ion by energy transfer, resulting in luminescence. On this basis, a zirconium-based organic framework (CJLU-1/Au@Pt) functionalized with gold-platinum core-shell nanoparticles (Au@Pt NPs) was used as a signal controller to regulate the emission of luminescent gel LEOGs, and the mechanism of the quenching effect between them was analyzed. In addition, we designed an "on-off" bioimmunosensor using electrochemiluminescence (ECL) generated by LEOGs as a quantitative indicator for the detection of neuron-specific enolase (NSE), a marker of small cell lung cancer (SCLC). This ECL immunosensor has good detection performance and provides a strategy and detection technique for the improvement of electrochemiluminescent materials as well as the early diagnosis of SCLC.

Imatinib (IMA) is crucial for the treatment of chronic myeloid leukemia (CML) and gastrointestinal stromal tumors (GIST). However, achieving highly sensitive detection of IMA remains challenging due to the low signal levels of most existing detection platforms and the high cost of instrumentation. In this work, we report a signal-enhanced electrochemiluminescence (ECL) aptasensor based on a Cd-TBAPy metal-organic framework (MOF) composite for sensitive detection of IMA. The enhanced ECL performance of Cd-TBAPy is attributed to the rigid coordination environment and ordered arrangement of H4TBAPy ligands within the MOF framework, which restrict intramolecular motion and reduce non-radiative relaxation pathways. Specific binding of IMA to the aptamer induces dissociation of the aptamer from the surface-confined CP-Apt duplex(dsDNA), thereby exposing the immobilized capture probe. The exposed capture probe subsequently hybridizes with the Cd-TBAPy-labeled signal probe, generating an ECL signal that increases with increasing target concentration. The sensor exhibits a good linear response over the range of 1 nM to 50,000 nM, with a detection limit as low as 0.47 nM, providing a promising basis for further development toward therapeutic drug monitoring of IMA.

Optimizing the stability and affinity of peptides in vivo is critical for their development as alternatives to approved monoclonal antibodies. In recent years, efforts in academia and industry have focused on modifying Pep2-8, a classical antagonistic peptide targeting proprotein convertase subtilisin/kexin type 9 (PCSK9), to enhance its specificity and affinity. However, developing effective PCSK9 inhibitory peptides remains challenging, especially given the limited examples of their successful in vivo applications. Here, we designed transformable inhibitory peptide (TIP) against PCSK9 based on the modular structure of Pep2-8. Upon encountering PCSK9, TIP undergoes in situ self-assembly at the epidermal growth factor-like domain A (EGF-A) binding domain of PCSK9 to form artificial topological nanostructures (ATNs). The ATNs not only enhance peptide stability and prolong in vivo retention time but also strengthen PCSK9 binding through multivalent synergistic effects. We demonstrate that compared to Pep2-8, TIP forms ATNs which increasing its binding affinity for PCSK9 by approximately 18.7-fold in vitro. In high-fat diet mouse models, TIP significantly increase hepatic LDLR levels (2.0-fold) and reduced LDL-C and TC levels. We envision that the in situ formation of ATNs by peptides enhances in vivo stability and affinity, which offering an approach for development as antibody alternatives in clinical.

This study innovatively constructed a "dual-engine" electrochemical aptasensor, achieving highly sensitive and efficient detection of tobramycin (TOB). Firstly, the primary engine consists of a bimetallic MOF-on-MOF heterostructure (Sn-MOF-on-Ce-MOF) loaded with gold nanowire (AuNWs), followed by the addition of the signaling maker toluidine blue (Tb). It is achieved by precisely modulating the interfacial properties between SnMOF and Ce-MOF for synergistic signal amplification, as well as utilizing the unique structure of one-dimensional AuNWs to enrich more signaling probes (SP), thereby significantly enhancing signal response intensity. Secondly, another engine refers to the matrix material composed of acetylene black (AB) modified with polyethyleneimine (PEI) loading on gold nanoparticles (AuNPs), and the material exhibits excellent electrical conductivity and a large specific surface area. This dual-engine synergistic interaction endows the sensor with exceptional detection performance: a broad linear detection range of 1.0 x 10-5 to 1.0 x 102 ng mL- 1 and an ultra-low detection limit of 0.19 fg mL-1. Notably, the sensor shows excellent applicability and ideal recovery in animal-derived foods, providing a reliable new method for the detection of TOB residues in foods.

MicroRNA-155 (miR-155) is a critical biomarker implicated in various pathological processes, including cancer progression, immune response, and cardiovascular diseases. Consequently, accurate detection of miR-155 at low concentrations is essential for early diagnosis and effective treatment monitoring. To address this need, this study introduces a novel application of reduced graphene oxide/silver sulfide-gold (rGO/Ag₂S-Au) nanocomposites as signal tracers for the sensitive detection of miR-155. First, Ag₂S-Au nanoparticles were synthesized via in-situ reduction of HAuCl₄ on Ag₂S surfaces and then incorporated into rGO to form the nanocomposites. Subsequently, the composites were hybridized with signal probes to fabricate signal labels. In the detection mechanism, miR-155 triggers local catalytic hairpin assembly (L-CHA) to amplify signals. Finally, the products were immobilized on capture probe-modified electrodes, and electrochemical responses were measured by differential pulse voltammetry (DPV). As a result, the biosensor exhibits a linear detection range from 1 fM to 1 μM, with a detection limit as low as 0.17 fM. Overall, this work advances electrochemical biosensor technology and provides valuable insights for high-performance biosensor design in rapid clinical diagnostics.

The ever-expanding set of nanocluster-based sensor array and their programmability exhibit remarkable capacity in sensitive and multiplex quantification of heavy metal ions, especially in complex environments. However, this quantitative performance comes with an ever-present constraint: limited luminescence efficiency of nanoclusters due to the intramolecular vibration and rotation phenomena. Here, we pioneered the development of a fluorescent sensor array utilizing metal-organic framework-scaffold gold nanoclusters (AuNCs@MOFs) as sensing elements, enabling precise identification and sensitive quantification of eight heavy metal ions (Ni2+, Cr3+, Co2+, Pb2+, Cd2+, Ag+, Cu2+, and Zn2+). Three kinds of ligand-protected AuNCs (GSH-AuNCs, MPA-AuNCs, and [2MBA]-AuNCs) embedded in zeolitic imidazolate framework-8 (ZIF-8) exhibit significantly increased luminescence performance. We demonstrate that the fluorescence intensity of the AuNCs@MOFs can be modulated by heavy metal ions, thereby generating distinct fingerprints, which are further analyzed by the statistical analysis method. The developed sensor array identifies eight heavy metal ions accurately across concentrations ranging from 0.5 to 50 mu M. Furthermore, this sensor array recognizes multi-component mixtures of heavy metal ions, exhibiting excellent discriminatory capability in both tap water and traditional Chinese medicine samples. Overall, this study provides a straightforward paradigm to construct a nanocluster-based sensor array for sensitive and multiplex heavy metal ions quantification in a complex environment.

A novel aptasensor for the detection of kanamycin (KNM) was developed based on dual signal amplification through nanomaterials and DNA rolling machines. Firstly, a composite consisting of the covalent organic framework (COF), ketjen black (KB) and toluidine blue (Tb), was synthesized for signal generation. Tb molecules were loaded into the composite and combined with AuNPs to serve as tracer label. Secondly, the wDNA-loaded Fe3O4 magnetic beads (wDNA@MBs) as the DNA walking legs, with cleavage by Mg2+-driven DNAzyme, the DNA walking legs can move along the sDNA-decorated MBs surface, converting the target KNM into numerous output DNA strand, achieving sensitive amplification. With a low detection limit of 0.66 fM and a wide linear dynamic range from 10 fM to 100 nM, this aptasensor showed good results in the detection of KNM in real samples. Therefore, we envision it can provide a useful method for KNM detection in actual samples.

Bioorthogonal-based prodrug activation strategy holds great potential in reducing the severe side effects of chemotherapy and has been widely applied in the development of in situ personalized cancer vaccines. However, current strategies mainly focus on the caging and release of cytotoxic drugs, while the simultaneous enhancement of drug accumulation and penetration within tumors remains underexplored. Here, we modularly design a tetrazine (Tz)-modified peptide (TMP) capable of cascade responsiveness to the tumor microenvironment and biomarkers, leading to the in situ self-assembly of an artificial topological nanostructure (ATNs) catalytic system on the tumor cell surface. This system significantly increases the local concentration of bioorthogonal handles (Tz), thereby improving the activation efficiency of the prodrugs trans-cyclooctene (TCO)-doxorubicin (Dox) and TCO-imiquimod (IMQ). We demonstrated that activated Dox induced immunogenic cell death (ICD) by releasing tumor-associated antigens (TAAs), while activated IMQ further amplified immune stimulation, leading to tumor ablation. Additionally, ATNs inhibit tumor cell migration, regulate tumor tissue permeability, and ultimately promote the penetration of the activated drugs. In vivo results demonstrated that the ATNs catalytic system enhanced drug penetration by 7.8-fold in tumor tissues and exhibited excellent safety and selectivity, leading to markedly improved survival outcomes. Moreover, this system also showed promising therapeutic effects in preventing tumor recurrence. We postulate that the synergistic integration of in situ self-assembly and bioorthogonal catalysis offers great potential for the safe and effective development of in situ cancer vaccines and holds promise as a universal prodrug delivery platform for cancer therapy.

Despite the widespread application of the CRISPR-Cas12a system in vitro diagnostics due to its high programmability and distinctive trans-cleavage activity, the susceptibility of its crRNA component to degradation and sensitivity to storage and working conditions poses a significant challenge to improving the practical efficacy of these diagnostic systems. Here, we show that engineered crRNA with a covalently closed circular structure (C-crRNA) can replace traditional linear crRNA to form functional complexes with Cas12a protein, significantly enhancing the anti-interference ability of the CRISPR-Cas12a system while maintaining its sensitivity and specificity. Based on this finding, a circular crRNA-mediated CRISPR molecular diagnostic (CRCD) toolkit is developed and successfully integrated with a standard nucleic acid amplification technique to detect synthesized Human Papillomavirus type 16 (HPV-16) plasmids down to 10 aM sensitivity levels. Furthermore, the CRCD system is applied for ultrasensitive detection of 40 HPV-16 and 40 influenza A viruses in clinical samples, with results consistent with those from PANTHER detection and quantitative real-time polymerase chain reaction (qRT-PCR). In conclusion, this strategy introduces a novel paradigm for engineering crRNA to program Cas12a, which has the potential to revolutionize the use of crRNA in CRISPR-based molecular diagnostics.

Phosphate plays a crucial role in various physiological and pathological processes; however, effective strategies for analyzing diverse phosphates and monitoring phosphate hydrolysis remain limited. Herein, we develop a metal-organic framework wrapped Cu nanoclusters-based (CuNCs@MOF) fluorescent sensor array for high-throughput discrimination of phosphates and real-time monitoring of their enzymatic hydrolysis. Leveraging the confinement effect of zinc-based MOFs, the encapsulated CuNCs exhibited reduced intramolecular vibration and rotation, resulting in enhanced luminescence performance. Due to the distinct affinities of various phosphates toward zinc ions, the sensor array generates differentiated fluorescence responses for seven phosphates, including adenosine triphosphate (ATP), cytidine triphosphate, uridine triphosphate, adenosine diphosphate, adenosine monophosphate, pyrophosphate (PPi), and inorganic phosphate. Subsequently, the fluorescence response signals are processed using the pattern recognition algorithm to generate the unique fingerprint spectra of each phosphate. We demonstrate that the sensor array can not only identify seven distinct phosphates at concentrations as low as 5 mu M and effectively distinguish between their mixtures but also exhibit excellent anti-interference capability and enable dynamic monitoring of ATP and PPi hydrolysis. Notably, the sensor array achieved outstanding performance in complex real-world samples and cellular-level analyses, demonstrating 100% accuracy in blind sample identification. Overall, this work develops a strategy for high-throughput discrimination of phosphates and real-time monitoring of their enzymatic hydrolysis.

Clustered regularly interspaced short palindromic repeats-associated (CRISPR/Cas) proteins have been used for a growing class of in vitro molecular diagnostics due to their modularity and high specificity in targeting nucleic acid. However, the requirement of a protospacer adjacent motif (PAM) for Cas protein-catalyzed trans-cleavage poses a challenge for random nucleic acid detection. Here, we demonstrate that dithiothreitol (DTT) enables LbCas12a to adopt a relaxed preference for PAM base pairing, thereby expanding the target sequence space. Accordingly, we propose a DTT-mediated CRISPR/Cas12a toolbox (DTT-deCRISPR) that exhibits relaxed PAM specificity and is readily compatible with nucleic acid amplification techniques including recombinase polymerase amplification (RPA) and polymerase chain reaction (PCR). As a proof of concept, we integrate DTT-deCRISPR with frequently used PCR for sensitively and selectively detecting high-risk human papillomavirus (HPV) 16 and 18. The platform demonstrates the ability to detect synthesized HPV 16 and 18 plasmids down to 1 aM within 60 min. Based on the receiver operating characteristic curve analysis, the clinical sensitivities of the developed method for detecting HPV 16 and 18 are 93.75% and 80.00%, respectively. We further incorporate it into a lateral flow assay (LFA) for point-of-care detection, and the HPV 16 and HPV 18 abundances determined by LFA for clinical samples are consistent with the fluorescence analysis results. Together, this work uncovers an unexpected connection between DTT and PAM preferences of LbCas12a, promoting the universality and flexibility of CRISPR technology in molecular diagnostics.

The growing problem of antibiotic abuse has become a major peril to human health. Therefore, it is crucial to develop an accurate, extremely sensitive, and efficient detection method. This study synthesized PdSn alloys with enhanced peroxidase activity via p-d orbital hybridization and immobilized them on MIL101(Fe), fabricating a novel nanoenzyme (PdSn/MIL101(Fe)). Benefiting from synergistic interactions, the nanozyme exhibits enhanced electrical conductivity and remarkable peroxidase-like activity, enabling efficient electron transfer. We constructed an electrochemical aptasensor by combining PdSn/MIL101(Fe) with signal probes (SP) as a tracer label and achieved the detection of oxytetracycline (OTC) using cyclic voltammetry (CV) and amperometric measurement. The aptasensor had a broad linear range (100 fg mL-1 to 100 ng mL-1) and a low limit of detection (LOD) of 8.13 fg mL-1. These superior characteristics endowed the aptasensor with excellent capability for OTC detection in both real samples and quality control samples.

A novel ternary electrochemiluminescence (ECL) aptasensor was creatively proposed for florfenicol (FF) detection, on account of the manganese-nitrogen dual-doped carbon dots (Mn-NHCDs) emitter and the facilitation of triethylamine (TEA) by zinc oxide (ZnO). First, Mn-NHCDs with low excitation potentials and excellent electrochemical luminescence properties were synthesized. Then, a newly formulated ZnO with an enlarged specific surface area and an appropriate mesoporous structure was utilized to load Mn-NHCDs, thereby diminishing the leakage of Mn-NHCDs and stabilizing the luminescence within the system. More importantly, ZnO can serve as a new co-reaction accelerator for TEA to improve the reaction rate of Mn-NHCDs with TEA efficiently. Furthermore, owing to the overlap of spectra, we employed an ECL resonance energy transfer (ECL-RET) strategy, with the Mn-NHCDs/ZnO as the donor and black hole quencher (BHQ) as the acceptor. The ECL biosensor illustrates an extremely selective and sensitive determination of FF from 1.00 fg mL⁻1 to 10.0 ng mL⁻1 and a detection Limit of 0.413 fg mL⁻1. We envision that our aptasensor will provide substantial assurance concerning food safety.

A novel electrochemiluminescence (ECL) immunosensor based on the resonance energy transfer (RET) effect between europium-based metal-organic framework (Eu-MOF, ETM) and GO-PEI/Au was developed for detecting neuron-specific enolase (NSE). First, using 4’,5’-Bis(4-carboxyphenyl)-[1,1’:2’,1”-terphenyl]-4,4”-dicarboxylic acid (TCPB) as the ligand, we synthesized an efficient and stable ECL emitter (ETM) as the signaling substrate. Due to efficient spectral overlap between the ECL emission spectrum of ETM and the UV-vis absorption spectrum of GO-PEI/Au, the ECL emission signal of ETM can be expeditiously quenched. Consequently, the ECL varied with the quenching efficiency of the immunosensor, which correlated with the target analyte concentration, enabling quantitative detection of NSE. The developed straightforward and cost-effective immunosensor exhibited outstanding sensitivity and selectivity in identifying NSE within the 10 fg mL− 1 to 100 ng mL− 1 range with an impressively low limit of detection (LOD) of 1.26 fg mL− 1. The immunosensor was successfully applied to quantify NSE in clinical human serum samples. Furthermore, by substituting the quenching probe, this platform can be readily adapted for detecting diverse biomarkers, demonstrating significant potential utility. To sum up, based on the ECL-RET strategy, a highly sensitive ECL immunosensor for NSE detection was constructed. It incorporated a novel ECL emitter (ETM) and established a new energy donor-acceptor pair, which demonstrated effective preliminary analysis of clinical samples.

Non-sputum-based diagnostic tests for tuberculosis (TB) are critical to global TB control. Herein, we developed an electrochemical aptasensor for detecting the ESAT-6 antigen of Mycobacterium tuberculosis (MTB) in human serum, using NiMn-layered double hydroxides@palladium nanoparticles (NiMn-LDHs@PdNPs) as a high-performance signal enhancer. NiMn-LDHs@PdNPs were synthesized via spontaneous in situ reduction, which requires no toxic reagents or complex equipment. This design not only addresses the limitations of insufficient active sites and slow electron transfer in layered double hydroxides (LDHs) but also mitigates the agglomeration tendency of palladium (Pd) nanoparticles, thereby enabling synergistic peroxidase-like activity and, importantly, relative spatial decoupling of recognition sites from catalytic sites. Functionalization with aptamers targeting the MTB ESAT-6 antigen did not significantly impair its catalytic activity. Employing a classic sandwich configuration, the aptasensor exhibited a wide linear detection range of 75 pg/mL to 10 ng/mL with a low limit of detection (LOD) of 0.629 pg/mL, alongside excellent specificity, stability, and reproducibility. In clinical validation, it successfully distinguished 14 TB patients from 14 healthy donors with 100

The development of electrochemiluminescence (ECL) sensors for the sensitive detection of circulating tumor DNA (ctDNA) associated with non-small cell lung cancer (NSCLC) offers a promising approach for early diagnosis; however, the lack of robust and efficient luminophore remains a key limitation to the analytical performance of ECL sensors. Herein, a ECL biosensor is developed using a novel His@ZIF-8/Fe-TCPP (HZTCP) luminophore for the sensitive detection of NSCLC-related ctDNA. The HZTCP luminophore, synthesized using a histidine imidazole framework (His@ZIF-8) as the precursor and tetra-(4-carboxyphenyl) porphyrin ferric chloride (Fe-TCPP) as the luminescent ligand, integrates the photoelectrochemical activity of porphyrin with the porous structure of the MOF, achieving excellent ECL performance. In addition, polyethyleneimine and gold nanoparticle-functionalized covalent organic frameworks (P-COF-AuNPs) serve as interfacial materials to significantly enhance the effective area and conductivity of the sensing interface, increase the solid-state loading of the capture probe, and improve the biosensor's sensitivity. The ECL biosensor achieves a wide detection range of 1 fM to 100 nM with a limit of detection of 0.35 fM, enabling the differentiation of NSCLC patients from healthy individuals through ctDNA detection. This work provides a straightforward approach for designing efficient ECL luminophores and presents a promising method for the rapid detection of non-small cell lung cancer-related ctDNA.