Electrochemical Biosensor Research Articles

Sensitive electrochemical biosensor for rapid detection of sEV-miRNA based turbo-like localized catalytic hairpin assembly

Small extracellular vesicle-associated microRNAs (sEV-miRNAs) have emerged as critical biomarkers for cancer diagnosis, yet the rapid detection of these low-abundance molecules in clinical samples remains a formidable challenge. Herein, a simple turbo-like localized catalytic hairpin assembly (TL-CHA) was proposed for sEV-miR-1246 measurement. This electrochemical sensor achieves dual localization through the ingeniously use of AuNPs and DNA nanowires, which provides rich sites for CHA cascade amplification, significantly enhancing the effective reaction and amplify the detection response. Leveraging this innovative design, this biosensor demonstrated the ability to detect sEV-miRNA at concentrations as low as 5.24 aM in a time frame of 30 min. The precision of the measurements was validated through reverse transcription quantitative polymerase chain reaction. Furthermore, the sensor was used for analyzing plasma samples from gastric cancer patients yielded AUC values of 0.973 for all stages and 0.945 for early stages. This demonstrates the sensor's robust performance in both the staging diagnosis and early screening of gastric cancer. Therefore, this platform has great potential for the clinical cancer diagnosis.

Aptamer-based label-free electrochemical biosensors for the diagnosis of sickle cell anemia

Aptamer-based label-free electrochemical biosensors for the diagnosis of sickle cell anemia

A universal three-dimensional hydrogel electrode for electrochemical detection of SARS-CoV-2 nucleocapsid protein and hydrogen peroxide

Coronavirus disease 2019 (COVID-19) is a highly contagious illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulting in a global health crisis. The primary diagnostic method for COVID-19 is quantitative reverse transcription PCR, which is time-consuming and requires expensive instrumentation. Here, we developed an electrochemical biosensor for detecting SARS-CoV-2 biomarkers using a 3D porous polyacrylamide/polyaniline hydrogel (PPG) electrode prepared by UV photopolymerization and in situ polymerization. The electrochemical immunosensor for detecting SARS-CoV-2 N protein via the immune sandwich principle demonstrated a lower detection limit of 42 pg/mL and comparable specificity to commercial enzyme-linked immunosorbent assay, which was additionally validated in pseudoviruses. The electrochemical sensor for hydrogen peroxide showed a low detection limit of 0.5 μM and excellent selectivity, which was further confirmed in cancer cells under oxidative stress. The biomarkers of SARS-CoV-2 were successfully detected due to the signal amplification capability provided by 3D porous electrodes and the high sensitivity of the antigen-antibody specific binding. This study introduces a novel three-dimensional electrode with great potential for the early detection of SARS-CoV-2.

Microfluidic electrochemical biosensors: tools for advancing the sustainable development goals

New technologies can help to achieve the sustainable development goals (SDGs) of the United Nations. We discuss the contribution of microfluidic electrochemical biosensors to advancing the SDGs. These sensors can be applied in various fields given their low cost, self-powering ability, environmental compatibility, ease of use, and small sample volume requirements.

Reagent-Free Lactate Detection Using Prussian Blue and Electropolymerized-Molecularly Imprinted Polymers-Based Electrochemical Biosensors.

Sweat lactate, a promising biomarker for assessing physical performance and health conditions, calls for noninvasive, convenient, and affordable detection methods. This study leverages molecularly imprinted polymers (MIPs) as a synthetic biorecognition element for lactate detection due to their affordability and high stability. Traditional MIPs-based electrochemical sensors often require external redox probes such as ferricyanide/ferrocyanide in the solution to signal the binding between analytes and MIPs, which restricts their applicability. To address this, our study introduces an innovative approach utilizing a layer of Prussian blue (PB) nanoparticles as the internal redox probe on screen-printed carbon electrodes (SPCE), followed by a layer of electropolymerized MIP (eMIP) for molecular recognition, enabling reagent-free lactate detection. The real-time growth of eMIP and the processes of template elution and lactate rebinding were examined and validated using electrochemical surface plasmon resonance (EC-SPR) spectroscopy. The sensor's performance was thoroughly investigated using Differential Pulsed Voltammetry (DPV) and Electrochemical Impedance Spectroscopy (EIS) with samples spiked in 0.1 M KCl solution and artificial sweat. The developed sensors demonstrated a fast and selective response to lactate, detecting concentrations from 1 to 35 mM with a Limit of Detection (LOD) of 0.20 mM, defined by a signal-to-noise ratio of 3 in the DPV measurements. They also exhibited excellent reproducibility, reusability, and a shelf life of up to 10 months under ambient conditions. These eMIP/PB/SPCE-based lactate sensors show considerable potential as point-of-care (POC) devices for sweat lactate detection, and the technology could be adapted for reagent-free detection of a broad spectrum of molecules.

Sensitive detection of exosomes by an electrochemical aptasensor based on double nucleic acid amplifications

Exosomes are extracellular vesicles secreted by mammalian cells and recognized as a powerful non-invasive cancer marker because they carry the cellular components specific to parent cells. The development of a convenient, rapid and sensitive exosome detection method is very important for the early diagnosis of cancer. In this study, an electrochemical biosensor based on two signal amplification methods, primer exchange reaction (PER) and rolling circle amplification (RCA), was developed for the high sensitive detection of exosomes. First, exosomes were specifically captured by CD63 aptamer and DNA strands (T) were released through a chain replacement reaction. Then the T strands initiated the PER reaction, producing a large number of short strands of DNA (P). Afterwards, RCA reaction was carried out on the electrode surface. Finally, a large amount of horseradish peroxidase (HRP) was bound to the electrode surface to catalyze o-phenylenediamine (OPD) oxidation, and the reduction current signal was obtained to realize the quantitative detection of exosomes. Under the optimal experimental conditions, there was a good linear relationship between DPV signal and the logarithm of exosome concentration in the range of 88 ∼ 8.8 × 106 particle/μL with a low limit of detection of 22 particle/μL. This strategy could realize sensitive detection of exosomes and provide a new strategy for the detection of other tumor markers.

Electrochemical aptasensor for ultrasensitive cadmium detection through hybridization chain-amplified reaction

Detection of cadmium ion (Cd2+) is of great necessity in safeguarding public health by providing accurate information about its presence in food matrices. A signal amplification electrochemical biosensor system with DNA cascade reaction was designed for ultrasensitive determination of Cd2+, in which in-situ synthesized Ag nanoclusters (Ag NCs) were identified as signal reporters. The highly conductive electrode with large active area, produced by gold deposition on the screen-printed electrodes (SPE), was regarded as an electrochemical platform. Cd2+ aptamer (Apt) was immobilized on the Au NPs modified SPE (Au-SPE) through the Au-S bound to capture Cd2+ by conformational change. Meanwhile, the unoccupied Apt was able to be paired with H1 and H2 by base complementation to accelerate the hybridization chain reaction (HCR) cascade, and the in-situ synthesis of Ag NCs on cytosine-rich sequence of H1 to achieve the quantification of Cd2+ according to the linear sweep voltammetry (LSV). The constructed electrochemical aptasensor exhibited a linear range from 0.1 ng/mL to 100 ng/mL, and the detection limit was 0.088 ng/mL for Cd2+ under optimal conditions. This HCR-based electrochemical aptasensor allowed practical application to tea and vegetable samples with satisfactory accuracy, confirming its promising application for rapid detection of targets in complex systems. Therefore, this platform holds promise for the rapid detection of heavy metals.

Gold nanoparticles-doped MXene heterostructure for ultrasensitive electrochemical detection of fumonisin B1 and ampicillin.

Early transition metal carbides (MXene) hybridized by precious metals open a door for innovative electrochemical biosensing device design. Herein, we present a facile one-pot synthesis of gold nanoparticles (AuNPs)-doped two-dimensional (2D) titanium carbide MXene nanoflakes (Ti3C2Tx/Au). Ti3C2Tx MXene exhibits high electrical conductivity and yields synergistic signal amplification in conjunction with AuNPs leading to excellent electrochemical performance. Thus Ti3C2Tx/Au hybrid nanostructure can be used as an electrode platform for the electrochemical analysis of various targets. We used screen-printed electrodes modified with the Ti3C2Tx/Au electrode and functionalized with different biorecognition elements to detect and quantify an antibiotic, ampicillin (AMP), and a mycotoxin, fumonisin B1 (FB1). The ultralow limits of detection of 2.284pM and 1.617pg.mL-1, which we achieved respectively for AMP and FB1 are far lower than their corresponding maximum residue limits of 2.8nM in milk and 2 to 4mgkg-1 in corn products for human consumption set by the United States Food and Drug Administration. Additionally, the linear range of detection and quantification of AMP and FB1 were, respectively, 10pM to 500nM and 10pgmL-1 to 1µgmL-1. The unique structure and excellent electrochemical performance of Ti3C2Tx/Au nanocomposite suggest that it is highly suitable for anchoring biorecognition entities such as antibodies and oligonucleotides for monitoring various deleterious contaminants in agri-food products.

Intrinsically stretchable jellyfish-like gold nanowires film as multifunctional wearable chemical and physical sensors

We report on an intrinsically stretchable jellyfish-like gold nanowires film as a multifunctional wearable platform that can detect both chemical and physical biometrics. The film inherently exhibits multifunctionality, serving directly as an electrochemical electrode, strain sensor, temperature sensor, pressure sensor, and noncontact humidity sensor without the need of any additional materials. Our results highlight the excellent electrochemical properties, evidenced by a large electrochemical active surface area (EASA) of approximately 22.39 cm2 and the ability to withstand significant strain values up to 70 %. Leveraging these attributes, a stretchable electrochemical glucose biosensor was further designed with high sensitivity (45.40μA·mM−1·cm−2) and low limit of detection (10 μM). In addition, its sensing applications in strain, temperature, pressure and noncontact humidity were systematically explored. Impressively, the gold film is capable of monitoring multiple human motions and physiological signals, including joints bending, pulse motions, throat motions (e.g., drinking, speaking, swallowing and pronunciation recognition) and breathing. Furthermore, the film exhibited great versatility by integrating with 3D printing hydrogels, enabling customized patterning. The results demonstrate that our gold nanowires coating approach represent a new route to design multimodal soft wearable platforms for applications in personalized medicine and next-generation healthcare systems.

Electrochemical C-H Mono-/Multi-Bromination Regulation of N-Sulfonylanilines on a Cost-Effective Carbon Fiber Electrode and Its Prospective Electroactive Molecule Screening.

Electrochemical C-H mono/multi-bromination regulation of N-sulfonylanilines on the cost-effective CF electrode is described. This reaction proceeds smoothly under mild conditions with a broad substrate scope, affording diverse mono/multi-brominated anilines in moderate to good yields. Mechanism study reveals that this transformation involves anodic oxidation, aromatic electrophilic substitution, and deprotonation. Preliminary electroactive molecule screening results in its prospective application in electroactive MBs for electrochemical biosensors.

Electrochemical sensor for rapid diagnosis and early-stage detection of pancreatic cancer using Er-GQDs decorated MoS2 nanoflowers

In this study, for the first time, the erbium-doped graphene quantum dots (Er-GQDs) were decorated onto a three-dimensional MoS2 nanoflower (EGM) for electrochemically sensitive and selective detection of carbohydrate antigen, CA 19-9, in phosphate buffer saline (PBS) and biological fluids. The MoS2 nanoflowers (MoS2 NF) were hydrothermally prepared at 200 °C for 24 h, and then 2–8 nm of Er-GQDs were uniformly deposited onto 200 nm MoS2 NF. The MoS2 NF has a high electric conductivity for electrochemical performance, whereas Er-GQDs act as an effective anchor with anti-CA19-9 antibodies (CA19-9 Ab) for ultra-selectivity to CA19-9 antigen. The SPE||EGM/Ab immunosensor has substantial benefits by providing a long-lasting, ultra-sensitive, and highly selective platform for CA19-9 antigen detection. The dynamic ranges in PBS, human serum, and saliva are 5 × 10−5–250, 5 × 10−4–250, and 3 × 10−4–250 U mL−1, respectively, with limits of detection (LODs) of (0.18–2.95) × 10−4 U mL−1. Furthermore, the SPE||EGM/Ab immunosensor can detect CA19-9 in human serum with a recovery of 97–108 %. The non-invasive detection of CA19-9 in saliva is stably sensitive before and after 1 h of breakfast. Moreover, the SPE||EGM/Ab immunosensor has excellent selectivity toward CA19-9 over the other 17 interferences. Results elaborate that the SPE||EGM/Ab immunosensor is a promising electrochemical biosensing probe for detecting CA19-9 in human serum and saliva for clinical diagnosis and prognosis of pancreatic cancer in the early stage.

A novel electrochemical aptamer biosensor for lead (Ⅱ) ion detection utilizing GO/PAMAM @Au and Au/MB as dual signal amplifiers

A competitive electrochemical aptamer biosensor for the selective detection of Pb2+ was developed in this study. The PAMAM@Au hybrid materials and the bioprobe MB-GNPs-ssDNA were synthesized with chemical methods. Modified electrode with carboxylated graphene oxide was prepared via electrodeposition. PAMAM@Au was immobilized on the modified electrode using the EDC/NHS crosslinker, and aptamer (Apt) was bound to PAMAM@Au hybrid materials using the glutaraldehyde crosslinker. The assembly of MB-GNPs-ssDNA/Apt/PAMAM@Au/GO/SPCE biosensors was achieved through immobilizing bioprobe onto electrodes, based on the theory of aptamer-complementary chain reactions. The fabricated biosensor was characterized through electrochemical experiments, SEM, AFM, TEM, XPS and FTIR. The biosensor's performance was evaluated in a series of Pb2+ aqueous solutions by differential pulse voltammetry (DPV), and the results demonstrated this biosensor had excellent repeatability, stability, and specificity. The detection range spanned from 0.5 pM/L to 500 nM/L, with a detection limit of 5 fM/L. Consequently, the proposed biosensor can serve as a reliable analytical tool for the sensitive detection of lead ions.

A homogenous electrochemical biosensor for ultrasensitive ofloxacin detection based on AuNPs-enhanced synergistic amplification of enzymes

Antibiotic residue in food is still a serious health hazard to human with the various and severe adverse effects, so ultrasensitive detection of antibiotic residue becomes increasingly significant. Herein, a homogenous electrochemical biosensor is established for ultrasensitive detection of antibiotic ofloxacin (OFL) based on AuNPs-enhanced synergistic amplification of dual enzymes. Target OFL is ingeniously designed to initiate the synergistic effect of exonuclease III-catalyzed DNA cycle and terminal deoxynucleotidyl transferase-mediated polymerization, producing a great many polyA@AuNPs which subsequently adsorb onto the surface of gold electrode and finally lead to highly amplified electrochemical signal by binding with numerous electroactive indicator RuHex. Benefiting from the collaboratively multiple signal amplifications enhanced by AuNPs with good electrical conductivity and probe loading capacity, the constructed biosensor exhibits excellent quantitative detection performance for OFL with a linear range of 0.008 to 100 ng mL−1 and a limit of detection as low as 2.73 pg mL−1. Furthermore, the detection system accomplished in homogeneous solution without complex chemical modification and rebuilding of sensing architectures on the electrode endows the biosensor with the advantages of convenient operation, good reproducibility and stability. Moreover, the biosensor shows satisfactory analytical performance for OFL detection in real food samples, providing a potential approach for practical application.

Fenton reaction enhanced enzyme free electrochemical biosensor for rapid and highly sensitive detection of Salmonella

To fulfill the stringent demands of clinical Salmonella detection, this study employed the benefits of Ni-TCPP(Fe2+) and ConA to establish a novel approach that is culture-free, enzyme-free, secondary antibody-free, and ultra-sensitive. Our investigations revealed two key mechanisms that significantly bolster the signal-to-noise ratio (SNR) and enhance detection stability: firstly, the Fenton reaction and the mimic enzyme catalysis of Ni-TCPP(Fe2+) improved the sensitivity of the biosensor; secondly, the substitution of the secondary antibody with ConA led to increased binding of Ni-TCPP(Fe2+) due to the abundance of ConA receptors on the bacterial surface. With proper assembly, the developed biosensor exhibited high sensitivity in the range of 101–107 CFU/mL, along with a detection limit as low as 4 CFU/mL, and achieved rapid detection within a mere 2 h. Moreover, this biosensor has proven its efficacy in detecting Salmonella in clinical biological samples. Its advantages of being culture-free, secondary antibody-free, and ultra-sensitive establish a solid research foundation for the expedited detection of Salmonella in clinical settings and point-of-care testing (POCT).

Extended Review Concerning the Integration of Electrochemical Biosensors into Modern IoT and Wearable Devices

Electrochemical biosensors include a recognition component and an electronic transducer, which detect the body fluids with a high degree of accuracy. More importantly, they generate timely readings of the related physiological parameters, and they are suitable for integration into portable, wearable and implantable devices that are significant relative to point-of-care diagnostics scenarios. As an example, the personal glucose meter fundamentally improves the management of diabetes in the comfort of the patients’ homes. This review paper analyzes the principles of electrochemical biosensing and the structural features of electrochemical biosensors relative to the implementation of health monitoring and disease diagnostics strategies. The analysis particularly considers the integration of the biosensors into wearable, portable, and implantable systems. The fundamental aim of this paper is to present and critically evaluate the identified significant developments in the scope of electrochemical biosensing for preventive and customized point-of-care diagnostic devices. The paper also approaches the most important engineering challenges that should be addressed in order to improve the sensing accuracy, and enable multiplexing and one-step processes, which mediate the integration of electrochemical biosensing devices into digital healthcare scenarios.

Covalent organic frameworks (COFs) as carrier for improved drug delivery and biosensing applications.

Porous organic frameworks (POFs) represent a significant subclass of nanoporous materials in the field of materials science, offering exceptional characteristics for advanced applications. Covalent organic frameworks (COFs), as a novel and intriguing type of porous material, have garnered considerable attention due to their unique design capabilities, diverse nature, and wide-ranging applications. The unique structural features of COFs, such as high surface area, tuneable pore size, and chemical stability, render them highly attractive for various applications, including targeted and controlled drug release, as well as improving the sensitivity and selectivity of electrochemical biosensors. Therefore, it is crucial to comprehend the methods employed in creating COFs with specific properties that can be effectively utilized in biomedical applications. To address this indispensable fact, this review paper commences with a concise summary of the different methods and classifications utilized in synthesizing COFs. Second, it highlights the recent advancements in COFs for drug delivery, including drug carriers as well as the classification of drug delivery systems and biosensing, encompassing drugs, biomacromolecules, small biomolecules and the detection of biomarkers. While exploring the potential of COFs in the biomedical field, it is important to acknowledge the limitations that researchers may encounter, which could impact the practicality of their applications. Third, this paper concludes with a thought-provoking discussion that thoroughly addresses the challenges and opportunities associated with leveraging COFs for biomedical applications. This review paper aims to contribute to the scientific community's understanding of the immense potential of COFs in improving drug delivery systems and enhancing the performance of biosensors in biomedical applications.

Electrospun EU/HPMC nanofibers decorated by ZIF-8 nanoparticle as the advanced electrochemical biosensor modifier for sensitive and selective detection of c-MET cancer biomarker in human plasma sample

This research presents a selective and sensitive electrochemical biosensor for the detection of the mesenchymal–epithelial transition factor (c-MET). The biosensing is based on a modification of the SPCE (screen-printed carbon electrode) with the electrospun nanofiber containing eudragit (EU), hydroxypropyl methylcellulose (HPMC), and Zeolite imidazolate frameworks (ZIF-8) nanoparticles. EU/HPMC/ZIF-8 nanofibers have presented a high capability of electron transfer, and more active surface area than bare SPCE due to synergistic effects between EU, HPMC, and ZIF-8. On the other hand, EU/HPMC nanofibers provided high porosity, flexible structures, high specific surface area, and good mechanical strength. The presence of ZIF-8 nanoparticles improved the immobilization of anti-c-MET on the modified SPCE and also resulted in increasing the conductivity. By c-MET incubation on the modified SPCE, c-MET was connected to anti-c-MET, and consequently the electrochemical signal of [Fe(CN)6]3-/4- as the anion redox probe was reduced. In order to investigate the structural and morphological characteristics and elemental composition of electrospun nanofibers, various characterization methods including FE-SEM, XRD, FTIR, and EDS were used. Under optimum conditions with a working potential range −0.3−0.6 V (vs. Ag/AgCl), linear range (LR), correlation coefficient (R2), sensitivity, and limit of detection (LOD) were acquired at 100 fg/mL-100 ng/mL, 0.9985, 53.28 μA/cm2.dec, and 1.28 fg/mL, respectively. Moreover, the mentioned biosensor was investigated in a human plasma sample to determine c-MET and showed ideal results including reproducibility, stability, and good selectivity against other proteins.

Uricase conjugated nanostructured NiFe2O4/rGO modified flexible screen-printed carbon electrode based interface for long-range electrochemical determination of uric acid

Uric acid (UA) is a biomarker linked with gout and kidney stones that is harmful to the human body. Thus, this study explores the detection of UA using the differential pulse voltammetry (DPV) method. Initially synthesizing a highly active Nickel ferrite/reduced graphene oxide (NiFe2O4/rGO) nanocomposite ferrite material by simple surfactant-free low-temperature hydrothermal approach. The as-synthesized material was studied using various spectroscopic and microscopic techniques. The bioelectrode was fabricated on a screen-printed carbon electrode (SPCE), and the as-prepared Uricase/NiFe2O4/rGO/SPCE demonstrated improved electrochemical oxidation peak current (Ipa). While its electrochemical biosensing performance towards various UA concentrations shows a wide linear range from 5 to 900 μM, with a lower limit of detection (21·9 μM), high sensitivity (1·05×10−4 mA/μM cm2), good selectivity (RSD < 5·7 %), excellent stability, and repeatability. In addition, the fabricated bioelectrode was also explored to quantify the amount of UA in bovine serum albumin (BSA) real spiked samples. Hence, the proposed biosensor has potential applications in point-of-care-treatment of numerous disorders related to UA metabolism.

A low-fouling electrochemical biosensor based on BSA hydrogel doped with carbon black for the detection of cortisol in human serum

Electrochemical biosensors with high sensitivity can detect low concentrations of biomarkers, but their practical detection applications in complex biological environments such as human serum and sweat are severely limited by the biofouling. Herein, a conductive hydrogel based on bovine serum albumin (BSA) and conductive carbon black (CCB) was prepared for the construction of an antifouling biosensor. The BSA hydrogel (BSAG) was doped with CCB, and the prepared composite hydrogel exhibited good conductivity originated from the CCB and antifouling capability owing to the BSA hydrogel. An antifouling biosensor for the sensitive detection of cortisol was fabricated by drop-coating the conductive hydrogel onto a poly(3,4-ethylenedioxythiophene) (PEDOT) modified electrode and further immobilizing the cortisol aptamer. The constructed biosensor showed a linear range of 100 pg mL−1 - 10 μg mL−1 and a limit of detection of 26.0 pg mL−1 for the detection of cortisol, and it was capable of assaying cortisol accurately in complex human serum. This strategy of preparing antifouling and conductive hydrogels provides an effective way to develop robust electrochemical biosensors for biomarker detection in complex biological media.

A Novel CRISPR/Cas12a-Mediated Ratiometric Dual-Signal Electrochemical Biosensor for Ultrasensitive and Reliable Detection of Circulating Tumor Deoxyribonucleic Acid.

Circulating tumor DNA (ctDNA) holds great promise as a noninvasive biomarker for cancer diagnosis, treatment, and prognosis. However, the accurate and specific quantification of low-abundance ctDNA in serum remains a significant challenge. This study introduced, for the first time, a novel exponential amplification reaction (EXPAR)-assisted CRISPR/Cas12a-mediated ratiometric dual-signal electrochemical biosensor for ultrasensitive and reliable detection of ctDNA. To implement the dual-signal strategy, a signal unit (ssDNA-MB@Fc/UiO-66-NH2) was prepared, consisting of methylene blue-modified ssDNA as the biogate to encapsulate ferrocene signal molecules within UiO-66-NH2 nanocarriers. The presence of target ctDNA KRAS triggered EXPAR amplification, generating numerous activators for Cas12a activation, resulting in the cleavage of ssDNA-P fully complementary to the ssDNA-MB biogate. Due to the inability to form a rigid structure dsDNA (ssDNA-MB/ssDNA-P), the separation of ssDNA-MB biogate from the UiO-66-NH2 surface was hindered by electrostatic interactions. Consequently, the supernatant collected after centrifugation exhibited either no or only a weak presence of Fc and MB signal molecules. Conversely, in the absence of the target ctDNA, the ssDNA-MB biogate was open, leading to the leakage of Fc signal molecules. This clever ratiometric strategy with Cas12a as the "connector", reflecting the concentration of ctDNA KRAS based on the ratio of the current intensities of the two electroactive signal molecules, enhanced detection sensitivity by at least 60-300 times compared to single-signal strategies. Moreover, this strategy demonstrated satisfactory performance in ctDNA detection in complex human serum, highlighting its potential for cancer diagnosis.