A dual-amplification aptasensor for highly sensitive detection of thrombin based on the functionalized graphene-Pd nanoparticles composites and the hemin/G-quadruplex

In this work, an advanced sandwich-type electrochemical aptasensor for thrombin was proposed by integrating hemin/G-quadruplex with functionalized graphene-Pd nanoparticles composites (PdNPs-RGs). The hemin/G-quadruplex formed by intercalating hemin into thrombin binding aptamer (TBA), firstly acted as a NADH oxidase, assisting the oxidation of NADH to NAD⁺ accompanying with the generation of H₂O₂ in the presence of dissolved O₂. Subsequently, the hemin/G-quadruplex acted as HRP-mimicking DNAzyme that rapidly bioelectrocatalyze the reduction of the produced H₂O₂. At the same time, the Pd nanoparticles supported on p-iodoaniline functionalized graphene were also adopted to catalyze the reduction of H₂O₂. Thus, with the dual catalysis, a dramatically amplified electrochemical signal could be obtained. Besides, the avidin–biotin system for binding aptamer sequences on electrodes not only improved the sensitivity of thrombin analysis but also obtained an acceptable repeatability of the aptasensor. With several factors mentioned above, a wide linear ranged from 0.1 pM to 50 nM was acquired with a relatively low detection limit of 0.03 pM (defined as S/N = 3). These excellent performances provided our approach a promising way for ultrasensitive assay in electrochemical aptasensors.     

A pseudo triple-enzyme electrochemical aptasensor based on the amplification of Pt–Pd nanowires and hemin/G-quadruplex

Our present work aimed at developing a pseudo triple-enzyme cascade electrocatalytic electrochemical aptasensor for determination of thrombin with the amplification of alcohol dehydrogenase (ADH)-Pt–Pd nanowires bionanocomposite and hemin/G-quadruplex structure that simultaneously acted as NADH oxidase and HRP-mimicking DNAzyme. With the addition of ethanol to the electrolyte, the ADH immobilized on the Pt–Pd nanowires catalyzed ethanol to acetaldehyde accompanied by NAD⁺ being converted to NADH. Then the hemin/G-quadruplex firstly served as NADH oxidase, converting the produced NADH to NAD⁺ with the concomitant local formation of high concentration of H₂O₂. Subsequently, the hemin/G-quadruplex acted as HRP-mimicking DNAzyme, bioelectrocatalyzing the produced H₂O₂. At the same time, the Pt–Pd nanowires employed in our strategy not only provided a large surface area for immobilizing thrombin binding aptamer (TBA) and ADH, but also served as HRP-mimicking DNAzyme which rapidly bioelectrocatalyzed the reduction of the produced H₂O₂. Thus, such a pseudo triple-enzyme cascade electrochemical aptasensor could greatly promote the electron transfer of hemin and resulted in the dramatic enhancement of electrochemical signal. As a result, a wide dynamic concentration linear range from 0.2 pM to 20 nM with a low detection limit of 0.067 pM for thrombin (TB) determination was obtained. The excellent performance indicated that our strategy was a promising way for ultrasensitive assays in electrochemical aptasensors.     

A sensitive electrochemical aptasensor based on palladium nanoparticles decorated graphene–molybdenum disulfide flower-like nanocomposites and enzymatic signal amplification

In the present study, with the aggregated advantages of graphene and molybdenum disulfide (MoS₂), we prepared poly(diallyldimethylammonium chloride)–graphene/molybdenum disulfide (PDDA–G–MoS₂) nanocomposites with flower-like structure, large surface area and excellent conductivity. Furthermore, an advanced sandwich-type electrochemical assay for sensitive detection of thrombin (TB) was fabricated using palladium nanoparticles decorated PDDA–G–MoS₂ (PdNPs/PDDA–G–MoS₂) as nanocarriers, which were functionalized by hemin/G-quadruplex, glucose oxidase (GOD), and toluidine blue (Tb) as redox probes. The signal amplification strategy was achieved as follows: Firstly, the immobilized GOD could effectively catalyze the oxidation of glucose to gluconolactone, coupling with the reduction of the dissolved oxygen to H₂O₂. Then, both PdNPs and hemin/G-quadruplex acting as hydrogen peroxide (HRP)-mimicking enzyme could further catalyze the reduction of H₂O₂, resulting in significant electrochemical signal amplification. So the proposed aptasensor showed high sensitivity with a wide dynamic linear range of 0.0001 to 40 nM and a relatively low detection limit of 0.062 pM for TB determination. The strategy showed huge potential of application in protein detection and disease diagnosis.     

A homogeneous hemin/G-quadruplex DNAzyme based turn-on chemiluminescence aptasensor for interferon-gamma detection via in-situ assembly of luminol functionalized gold nanoparticles,deoxyribonucleic acid,interferon-gamma and hemin

A homogeneous hemin/G-quadruplex DNAzyme (HGDNAzyme) based turn-on chemiluminescence aptasensor for interferon-gamma (IFN-γ) detection is developed, via dynamic in-situ assembly of luminol functionalized gold nanoparticles (lum-AuNPs), DNA, IFN-γ and hemin. The G-quadruplex oligomer of the HGDNAzyme was split into two halves, which was connected with the complementary sequence of P1 (IFN-γ-binding aptamer) to form the oligonucleotide P2. P2 hybridized with IFN-γ-binding aptamer and meanwhile assembled onto lum-AuNPs through biotin–streptavidin specific interaction. When IFN-γ was recognized by aptamer, P2 was released into the solution. The two lateral portions of P2 combined with hemin to yield the catalytic hemin/G-quadruplex DNAzyme, which amplified the luminol oxidation for a turn-on chemiluminescence signaling. Based on this strategy, the homogeneous aptasensor enables the facile detection of IFN-γ in a range of 0.5–100 nM. Moreover, the aptasensor showed high sensitivity (0.4 nM) and satisfactory specificity, pointing to great potential applications in clinical analysis.     

3,4,9,10-Perylenetetracarboxylic dianhydride functionalized graphene sheet as labels for ultrasensitive electrochemiluminescent detection of thrombin

A novel tracer, 3,4,9,10-perylenetetracarboxylic dianhydride (PTCDA) functionalized graphene sheet (GS) composite (GS–TCDA), is employed to label the secondary anti-thrombin aptamer (TBA) to construct an ultrasensitive electrochemiluminescent sandwich-type aptasensor. The GS provided large surface area for loading abundant PTCDA and TBA with good stability and biocompatibility. Because of the excellent electroconductivity of GS and the desirable optical properties of PTCDA, the as-formed Apt II bioconjugate considerably amplified the electrochmiluminescence (ECL) signal of peroxydisulfate (S₂O₈²⁻) and worked as the desirable label for Apt II. On the basis of the considerably amplified ECL signal and sandwich format, an extremely wide range from 1 fM to 1 nM with an ultralow detection limit of 0.33 fM for thrombin was obtained. Additionally, the selectivity and stability of the proposed aptasensor were also excellent. Thus, this procedure has great promise for detection of thrombin present at ultra-trace levels during early stage of diseases.     

A novel electrochemical aptasensor for highly sensitive detection of thrombin based on the autonomous assembly of hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme nanowires

In this work, a new signal amplified strategy was constructed based on isothermal exponential amplification reaction (EXPAR) and hybridization chain reaction (HCR) generating the hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme (HRP-mimicking DNAzyme) nanowires as signal output component for the sensitive detection of thrombin (TB). We employed EXPAR’s ultra-high amplification efficiency to produce a large amount of two hairpin helper DNAs within a minutes. And then the resultant two hairpin helper DNAs could autonomously assemble the hemin/G-quadruplex HRP-mimicking DNAzymes nanowires as the redox-active reporter units on the electrode surface via hybridization chain reaction (HCR). The hemin/G-quadruplex structures simultaneously served as electron transfer medium and electrocatalyst to amplify the signal in the presence of H₂O₂. Specifically, only when the EXPAR reaction process has occurred, the HCR could be achieved and the hemin/G-quadruplex complexes could be formed on the surface of an electrode to give a detectable signal. The proposed strategy combines the amplification power of the EXPAR, HCR, and the inherent high sensitivity of the electrochemical detection. With such design, the proposed assay showed a good linear relationship within the range of 0.1 pM–50 nM with a detection limit of 33 fM (defined as S/N = 3) for TB.     

An exonuclease-assisted amplification electrochemical aptasensor of thrombin coupling “signal on/off” strategy

In this work, a dual-signaling electrochemical aptasensor based on exonuclease-catalyzed target recycling was developed for thrombin detection. The proposed aptasensor coupled “signal-on” and “signal-off” strategies. As to the construction of the aptasensor, ferrocene (Fc) labeled thrombin binding aptamer (TBA) could perfectly hybridize with the methylene blue (MB) modified thiolated capture DNA to form double-stranded structure, hence emerged two different electrochemical signals. In the presence of thrombin, TBA could form a G-quadruplex structure with thrombin, leading to the dissociation of TBA from the duplex DNA and capture DNA formed hairpin structure. Exonuclease could selectively digest single-stranded TBA in G-quadruplex structure and released thrombin to realize target recycling. As a consequence, the electrochemical signal of MB enhanced significantly, which realized “signal on” strategy, meanwhile, the deoxidization peak current of Fc decreased distinctly, which realized “signal off” strategy. The employment of exonuclease and superposition of two signals significantly improved the sensitivity of the aptasensor. In this way, an aptasensor with high sensitivity, good stability and selectivity for quantitative detection of thrombin was constructed, which exhibited a good linear range from 5 pM to 50 nM with a detection limit of 0.9 pM (defined as S/N = 3). In addition, this design strategy could be applied to the detection of other proteins and small molecules.     

Polyethyleneimine‐Functionalized Platinum Nanoparticles with High Electrochemiluminescence Activity and Their Applications to Amplified Analysis of Biomolecules

Polyethyleneimine‐functionalized platinum nanoparticles (PtNPs) with excellent electrochemiluminescence (ECL) properties were synthesized and applied to the amplified analysis of biomolecules. These particles were prepared at room temperature, with hyperbranched polyethyleneimine (HBPEI) as the stabilizer. The UV/Vis absorption spectra and transmission electron microscopy images clearly confirmed the formation of monodisperse PtNPs. Such particles proved to possess high stability against salt‐induced aggregation, enabling them to be employed even under high‐salt conditions. Owing to the existence of many tertiary amine groups, these particles exhibited excellent ECL behavior in the presence of tris(2,2′‐bipyridyl)ruthenium(II). An HBPEI‐coated particle possessed an ECL activity that was at least 60 times higher than that of a tripropylamine molecule. Furthermore, these particles could be immobilized on the 3‐aminopropyltriethoxysilane‐treated quartz substrates to amplify the binding sites for carboxyl groups. Through this approach, PtNPs were applied to the amplified analysis of the hemin/G‐quadruplex DNAzyme by using the luminol/H₂O₂ chemiluminescence method.     

An electrochemical aptasensor for thrombin detection based on the recycling of exonuclease III and double-stranded DNA-templated copper nanoparticles assisted signal amplification

In this paper, we report an improved electrochemical aptasensor based on exonuclease III and double-stranded DNA (dsDNA)-templated copper nanoparticles (CuNPs) assisted signal amplification. In this sensor, duplex DNA from the hybridization of ligated thrombin-binding aptamer (TBA) subunits and probe DNA can act as an effective template for the formation of CuNPs on the electrode surface, so copper ions released from acid-dissolution of CuNPs may catalyze the oxidation of ο-phenylenediamine to produce an amplified electrochemical response. In the presence of thrombin, a short duplex domain with four complementary base pairs can be stabilized by the binding of TBA subunits with thrombin, in which TBA subunit 2 can be partially digested from 3′ terminal with the cycle of exonuclease III, so the ligation of TBA subunits and the subsequent formation of CuNPs can be inhibited. By electrochemical characterization of dsDNA-templated CuNPs on the electrode surface, our aptasensor can display excellent performances for the detection of thrombin in a broad linear range from 100 fM to 1 nM with a low detection limit of 20.3 fM, which can also specially distinguish thrombin in both PBS and serum samples. Therefore, our aptasensor might have great potential for clinical diagnosis of biomarkers in the future.     

A Sandwich‐type Electrochemiluminescence Aptasensor for Thrombin Based on Functional Co‐polymer Electrode Using Ru(bpy)32+ Doped Nanocomposites as Signal‐amplifying Tags

Herein, a signal‐on sandwich‐type electrochemiluminescence (ECL) aptasensor for the detection of thrombin (TB) was proposed. The graphene (GR) doped thionine (TH) was electropolymerized synchronously on the bare glassy carbon electrode (GCE) to form co‐polymer (PTG) electrode. The gold nanoparticles (AuNPs) were decorated on the surface of the PTG by in‐situ electrodeposition, and the functional co‐polymer (PTG‐AuNPs) electrode was utilized as sensing interface. Then, TB binding aptamer I (TBA I) as capture probes were modified on the PTG‐AuNPs electrode to capture TB, and Ru(bpy)₃²⁺/silver nanoparticles doped silica core‐shell nanocomposites‐labeled TB binding aptamer II (RuAg/SiO₂NPs@TBA II) were used as signal probes to further bind TB, resulting in a sandwich structure. With the assistant of silica shell and AgNPs, the enrichment and luminous efficiency of Ru(bpy)₃²⁺ were significantly improved. Under the synergy of PTG‐AuNPs and RuAg/SiO₂NPs, the ECL signal was dramatically increased. The proposed ECL aptasensor displayed a wide linear range from 2 fM to 2 pM with the detection limit of 1 fM, which is comparable or better than that in reported ECL aptasensors for TB using Ru(bpy)₃²⁺ and its derivatives as the luminescent substance. The excellent sensitivity makes the proposed aptasensor a promising potential in pharmaceutical and clinical analysis.     

Detection of thrombin using electrogenerated chemiluminescence based on Ru(bpy)3(2+)-doped silica nanoparticle aptasensor via target protein-induced strand displacement

A sensitive and selective aptasensor using tri(2,2′-bipyridyl)ruthenium(II)-doped silica nanoparticles (Ru(bpy)₃²⁺-doped SNPs) as DNA tags for detection of thrombin is developed based on the target protein-induced strand displacement of the DNA probe. For the proposed aptasensor, the aptamer was assembled on the surface of the Au electrode through Au-S binding. The hybridization event between the DNA probe labeled by the Ru(bpy)₃²⁺-doped SNPs and the aptamer was evaluated by electrogenerated chemiluminescence (ECL) measurements. Then, the DNA probe was displaced by thrombin and the binding event between the thrombin and the aptamer was monitored by ECL measurements again. The difference of ECL intensity (ΔI_ECL) of the two events could be used to quantify the thrombin. Other proteins, such as bovine serum albumin and bovine hemoglobin, had almost negligible ΔI_ECL. Under the optimal conditions, the ΔI_ECL was linearly related to the concentration of the thrombin in the range of 10 fM to 10 pM and the detection limit was down to 1.0 fM since SNPs containing a large number of Ru(bpy)₃²⁺ molecules were labeled on the DNA probe.     

Highly sensitive luminol electrochemiluminescence immunosensor based on ZnO nanoparticles and glucose oxidase decorated graphene for cancer biomarker detection

In this work, we reported a sandwiched luminol electrochemiluminescence (ECL) immunosensor using ZnO nanoparticles (ZnONPs) and glucose oxidase (GOD) decorated graphene as labels and in situ generated hydrogen peroxide as coreactant. In order to construct the base of the immunosensor, a hybrid architecture of Au nanoparticles and graphene by reduction of HAuCl₄ and graphene oxide (GO) with ascorbic acid was prepared. The resulted hybrid architecture modified electrode provided an excellent platform for immobilization of antibody with good bioactivity and stability. Then, ZnONPs and GOD functionalized graphene labeled secondary antibody was designed for fabricating a novel sandwiched ECL immunosensor. Enhanced sensitivity was obtained by in situ generating hydrogen peroxide with glucose oxidase and the catalysis of ZnONPs to the ECL reaction of luminol–H₂O₂ system. The as-prepared ECL immunosensor exhibited excellent analytical property for the detection of carcinoembryonic antigen (CEA) in the range from 10 pg mL⁻¹ to 80 ng mL⁻¹ and with a detection limit of 3.3 pg mL⁻¹ (S N⁻¹ = 3). The amplification strategy performed good promise for clinical application of screening of cancer biomarkers.     

Sensitive Electrochemical Aptasensor for Thrombin Detection with Platinum Nanoparticles,Blocking Reagent‐Horseradish Peroxidase and Graphene Oxide as Enhancers

A highly sensitive thrombin electrochemical aptasensor with Pt nanoparticles, blocking reagent‐horseradish peroxidase (HRP) and inert graphene oxide (GO) as enhancers was successfully fabricated. Firstly, Pt nanoparticles with high surface to volume ratio could increase the amount of the immobilized redox probe hexacyanoferrate nanoparticles (NiHCFNPs) and effectively enhance the electron transfer. Secondly, HRP and Pt nanoparticles with high catalytic activity extremely amplify the electrochemical signal of NiHCFNPs toward H₂O₂. Lastly, inert graphene oxide (GO) labeled TBA could be used for enlarging the steric hindrance of thrombin. As a result, the aptasensor showed a high sensitivity with a detection limit of 500 fM.     

A novel label-free electrochemical aptasensor for thrombin based on the {nano-Au/thionine}n multilayer films as redox probes

Herein, a novel label-free electrochemical aptasensor based on direct immobilization of the redox probes on an electrode surface was reported. Gold electrode coated Nafion was firstly modified with redox probe-thionine (Thi) through ion exchange adsorption. Then, with the help of chemisorption and electrostatic adsorption, negatively charged nano-Au and positively charged Thi were layer-by-layer (LBL) self-assembled onto the modified electrode surface, which formed {nano-Au/Thi⁺}_n multilayer films for improving the amount of redox probes and immobilizing thiolated thrombin aptamers (TBA). In the presence of target thrombin (TB), the TBA on the multilayer film could catch the TB onto the electrode surface, which resulted in a barrier for electro-transfer, leading to decrease of the current. The proposed method avoided the cubsome redox probe labeling process, increased the amount of redox probe and reduced the distance between the redox probe and electrode surface. Thus, the approach showed a high sensitivity and a wider linearity to TB in the range from 0.12 nM to 46 nM with a detection limit of 40 pM.     

Sensitive pseudobienzyme electrocatalytic DNA biosensor for mercury(II) ion by using the autonomously assembled hemin/G-quadruplex DNAzyme nanowires for signal amplification

Herein, a novel sensitive pseudobienzyme electrocatalytic DNA biosensor was proposed for mercury ion (Hg²⁺) detection by using autonomously assembled hemin/G-quadruplex DNAzyme nanowires for signal amplification. Thiol functionalized capture DNA was firstly immobilized on a nano-Au modified glass carbon electrode (GCE). In presence of Hg²⁺, the specific coordination between Hg²⁺ and T could result in the assembly of primer DNA on the electrode, which successfully triggered the HCR to form the hemin/G-quadruplex DNAzyme nanowires with substantial redox probe thionine (Thi). In the electrolyte of PBS containing NADH, the hemin/G-quadruplex nanowires firstly acted as an NADH oxidase to assist the concomitant formation of H₂O₂ in the presence of dissolved O₂. Then, with the redox probe Thi as electron mediator, the hemin/G-quadruplex nanowires acted as an HRP-mimicking DNAzyme that quickly bioelectrocatalyzed the reduction of produced H₂O₂, which finally led to a dramatically amplified electrochemical signal. This method has demonstrated a high sensitivity of Hg²⁺ detection with the dynamic concentration range spanning from 1.0 ng L⁻¹ to 10 mg L⁻¹ Hg²⁺ and a detection limit of 0.5 ng L⁻¹ (2.5 pM) at the 3S_blank level, and it also demonstrated excellent selectivity against other interferential metal ions.     

Carbon nanospheres enhanced electrochemiluminescence of CdS quantum dots for biosensing of hypoxanthine

This work developed a novel method to greatly enhance the electrochemiluminescence (ECL) of CdS quantum dots (QDs). The ECL amplification was achieved by the assembly of QDs on poly (diallyldimethylammonium chloride)-functionalized carbon nanospheres (PFCNSs), and successfully employed for sensitive ECL biosensing of oxidase substrates. The carbon nanospheres were prepared by a “green” method, and the high loading of QDs on carbon nanospheres led to a 4-times increased ECL intensity with dissolved O₂ as the coreactant. Using xanthine oxidase (XOD) as a model, an ECL biosensor was fabricated by immobilizing the enzyme on the mixing membrane of PFCNSs and QDs. The ECL biosensor showed a fast response to hypoxanthine with a linear concentration range from 2.5 × 10⁻⁸ to 1.4 × 10⁻⁵ M. The limit of detection was 5 nM at a signal-to-noise ratio of 3. The assay results of hypoxanthine in fish samples were in a good agreement with the reference values by amperometric technique. This facile approach to prepare the PFCNSs/QDs system for ECL biosensing could be of promising application in bioanalysis and electronic device.     

G‐Quadruplex‐Based DNAzymes Aptasensor for the Amplified Electrochemical Detection of Thrombin

A novel G‐quadruplex‐based DNAzymes aptasensor for the amplified electrochemical detection of thrombin has been described. The aptasensor utilized a combination of hemin and guanine‐rich thrombin‐binding aptamer (TBA) to form horseradish peroxidase (HRP)‐mimicking DNAzymes with peroxidase catalytic activity. In the presence of thrombin, the enzyme activity could be extensively promoted, thereby providing the amplified electrochemical readout signals for detecting thrombin. This aptasensor exhibited high sensitivity and selectivity for thrombin determination, which enabled the analysis of thrombin with a detection limit of 6×10^–11 M. On the basis of results, this method could have broad applications in the detection of proteins and other biomolecules.     

Highly sensitive lable-free electrochemical aptasensor for thrombin detection with cobalt hexacyanoferrate as the electrochemical probe

A highly sensitive label-free electrochemical aptasensor has been constructed for the electrochemical detection of thrombin (TB), where two layers of cobalt hexacyanoferrate (CoHCF) redox probes sandwiched with carbon nanotubes–Nafion were directly immobilized on the electrode surface by electrodeposition. Through the strong interaction between CN^? (CoHCF) and gold nanoparticles (GNPs), GNPs were assembled on the CoHCF-modified electrode for the immobilization of thiolated thrombin aptamers (TBA). In the presence of target TB, TBA on the electrode surface could catch TB to form TBA–TB complex, which made a barrier for the electron transfer, resulting in a greater decrease in CoHCF redox probe signals. Thus, the proposed aptasensor showed a high sensitivity and a much wider linearity to TB in the range of 1.0 pg/mL?～?1.0 μg/mL with a detection limit of 0.28 pg/mL.     

Quantum dots based electrochemiluminescent immunosensor by coupling enzymatic amplification for ultrasensitive detection of clenbuterol

An ultrasensitive electrochemiluminescence (ECL) immunosensor based on CdSe quantum dots (QDs) has been designed for the detection of clenbuterol. The immunosensor was fabricated by layer by layer and characterized with atomic force microscopic images (AFM) and electrochemical impedance spectra (EIS). In oxygen-saturated pH = 9.0 Tris-HCl buffer, a strong ECL emission of QDs could be observed during the cathodic process due to the H₂O₂ product from electrochemical reduction of dissolved oxygen. Upon the formation of immunocomplex, the second antibody labeled with horseradish peroxidase was simply immobilized on the electrode surface. The ECL emission decreased since steric hindrance of the immunocomplex slowed down the electron-transfer speed of dissolved oxygen, and also could be greatly amplified by an enzymatic cycle to consume the self-produced coreactant. Using clenbuterol as model analyte, the ECL intensity was determined by the concentration of competitive immunoassay of clenbuterol with a wide calibration in the range of 0.05 ng mL⁻¹ to 1000 ng mL⁻¹, and a low detection limit was 0.02 ng mL⁻¹. The immunosensor shows good stability and fabrication reproducibility. It was applied to detecting practical samples with the satisfactory results. This immunosensing strategy opens a new avenue for detection of residue and application of QDs in ECL biosensing.     

Advanced Pt hollow nanospheres/rubrene nanoleaves coupled with M-shaped DNA walker for ultrasensitive electrochemiluminescence bioassay

Herein, an intense electrochemiluminescence (ECL) was achieved based on Pt hollow nanospheres/rubrene nanoleaves (Pt HNSs/Rub NLs) without the addition of any coreactant, which was employed for ultrasensitive detection of carcinoembryonic antigen (CEA) coupled with an M-shaped DNA walker (M-DNA walker) as signal switch. Specifically, in comparison with platinum nanoparticles (Pt NPs), Pt HNSs revealed excellent catalytic performance and pore confinement-enhanced ECL, which could significantly amplify ECL intensity of Rub NLs/dissolved O₂ (DO) binary system. Then, the tracks and M-DNA walker were confined on the Pt HNSs simultaneously to promote the reaction efficiency, whose M-structure boosted the interaction sites between walking strands and tracks and reduced the rigidity of their recognition. Once the CEA approached the sensing interface, the M-DNA walker was activated based on highly specific aptamer recognition to recover ECL intensity with the assistance of exonuclease Ⅲ (Exo Ⅲ). As proof of concept, the “on-off-on” switch aptasensor was constructed for CEA detection with a low detection limit of 0.20 fg/mL. The principle of the constructed ECL aptasensor also enables a universal platform for sensitive detection of other tumor markers.