Cyclic Enzymatic Amplification Research Articles

Multiplex aptamer cluster detection platform and systems toxicology study for 17β-estradiol, bisphenol A, and diethylstilbestrol

Intake of 17β-estradiol (E2), bisphenol A (BPA), and diethylstilbestrol (DES) from food can contribute to endocrine disorders. Therefore, developing a sensitive method for the simultaneous detection of E2, BPA, and DES and understanding their combined effects on endocrine disruption are crucial. We developed a fluorescence aptasensing platform utilizing DNase I–assisted cyclic enzymatic signal amplification in conjunction with an aptamer/graphene oxide complex. Using PEG 20000 as a surface-blocking agent, the aptasensor achieved ultralow detection limits of 2.643, 0.3039, and 0.6996 for E2, BPA, and DES, respectively. The sensor demonstrated accurate detection in plastic bottled water at spiked levels of 10 and 100 ng/mL. Systems toxicology revealed 30 potential targets for mixture-induced endocrine disruption. Molecular docking showed binding affinities of E2, BPA, and DES for ESR1 of −9.94, −8.29, and − 8.98 kcal/mol, respectively. These results highlight the effectiveness of the aptasensor and provide valuable insights into endocrine disruption mechanisms.

Cyclic Enzymatic Signal Amplification-Driven DNA Logic Nanodevices on Framework Nucleic Acid for Highly Sensitive Electrochemiluminescence Detection of Dual Myocardial miRNAs.

MicroRNAs (miRNAs) have emerged as promising biomarkers for acute myocardial infarction (AMI). There is an urgent imperative to develop analytical methodologies capable of intelligently discerning multiple circulating miRNAs. Here, we present a dual miRNA detection platform for AMI using DNA logic gates coupled with an electrochemiluminescence (ECL) response. The platform integrates DNA truncated square pyramids as capture probes on gold-deposited electrodes, enabling precise quantification of miRNA associated with AMI. The cyclic enzymatic signal amplification principle of strand displacement amplification enhances the miRNA detection sensitivity. AND and OR logic gates have been successfully constructed, enabling intelligent identification of miRNAs in AMI. Calibration curves show strong linear correlations between ECL intensity and target miRNA concentration (10 fM to 10 nM), with excellent stability in consecutive measurements. When applied to clinical serum samples, the biosensor exhibits consistent performance, underscoring its reliability for clinical diagnostics. This innovative approach not only demonstrates DNA nanotechnology's potential in biosensing but also offers a promising solution for improving AMI diagnosis and prognosis through precise miRNA biomarker detection.

Mix-and-Detection Assay with Multiple Cyclic Enzymatic Repairing Amplification for Rapid and Ultrasensitive Detection of Long Noncoding RNAs in Breast Tissues.

Long noncoding RNAs (lncRNAs) are valuable biomarkers and therapeutic targets, and they play essential roles in various pathological and biological processes. So far, the reported lncRNA assays usually suffer from unsatisfactory sensitivity and time-consuming procedures. Herein, we develop a mix-and-read assay based on multiple cyclic enzymatic repairing amplification (ERA) for sensitive and rapid detection of mammalian metastasis-associated lung adenocarcinoma transcript 1 (lncRNA MALAT1). In this assay, we design two three-way junction (3WJ) probes including a 3WJ template and a 3WJ primer to specifically recognize lncRNA MALAT1, and the formation of a stable 3WJ structure induces cyclic ERA to generate triggers. The resulting triggers subsequently hybridize with a free 3WJ template and act as primers to initiate new rounds of cyclic ERA, generating abundant triggers. The hybridization of triggers with signal probes forms stable double-stranded DNA duplexes that can be specifically cleaved by apurinic/apyrimidinic endonuclease 1 to produce a high fluorescence signal. This assay can be carried out in a mix-and-read manner within 10 min under an isothermal condition (50 °C), which is the rapidest and simplest method reported so far for the lncRNA MALAT1 assay. This method can sensitively detect lncRNA MALAT1 with a limit of detection of 0.87 aM, and it can accurately measure endogenous lncRNA MALAT1 at the single-cell level. Moreover, this method can distinguish lncRNA MALAT1 expression in breast cancer patient tissues and their corresponding healthy adjacent tissues. Importantly, the extension of this assay to different RNAs detection can be achieved by simply replacing the corresponding target recognition sequences.

Dual Detection of Hemagglutinin Proteins of H5N1 and H1N1 Influenza Viruses Based on FRET Combined With DNase I.

Influenza A viruses (IAV) are classified based on their surface proteins hemagglutinin (HA) and neuraminidase (NA). Both pandemic H1N1 (pH1N1) and highly pathogenic avian influenza (HPAI) H5N1 viruses pose a significant threat to public health. Effective methods to simultaneously distinguish H1N1 and H5N1 are thus of great clinical value. In this study, a protocol for detection of HA proteins of both H1N1 and H5N1 was established. Specifically, we designed an aptasensor for HA using fluorescence resonance energy transfer (FRET) strategy combined with DNase I-assisted cyclic enzymatic signal amplification. HA aptamers of H1N1 and H5N1 IAVs labeled with various fluorescent dyes were used as probes. Graphene oxide (GO) acted as a FRET acceptor for quenching the fluorescence signal and protected aptamers from DNase I cleavage. The fluorescence signal was recovered owing to aptamer release from GO with HA protein. DNase I-digested free aptamers and HA proteins were able to further interact with more fluorescent aptamer probes, resulting in increased signal amplification. The limits of detection (LOD) of H5N1 HA and H1N1 HA were 0.73 and 0.43 ng/ml, respectively, which were 19 and 27 times higher than LOD values obtained with the DNase I-free system. The recovery rate of HA protein in human serum samples ranged from 88.23 to 117.86%, supporting the accuracy and stability of this method in a complex detection environment. Our rapid, sensitive, and cost-effective novel approach could be expanded to other subtypes of IAVs other than H1N1 and H5N1.

Label-free and highly sensitive detection of CRP based on the combination of nicking endonuclease-assisted signal amplification and capillary electrophoresis-UV assay

Elevated C-reactive protein (CRP) levels are linked with bacterial infection, local inflammation in osteoarthritis and the increased risk of developing cardiovascular disease. Here, a sensitive and label-free CRP assay is developed by combining cyclic enzymatic signal amplification and capillary electrophoresis (CE) with UV detection. This assay is constructed of base pairing and target recognition. Thereinto, nicking endonuclease (NEase) can recognize the specific nucleotide sequences in double-stranded DNA (dsDNA), which is formed by a CRP aptamer and its complementary DNA (cDNA). Sequentially, NEase cleaves only cDNA to produce signal DNAs. Therefore, a large number of signal DNAs are generated through continuous enzyme cleavage. In the presence of CRP, the aptamer recognizes and binds to CRP with high affinity and selectivity, which results in a decrease in signal DNAs, and thus the UV absorption value of CE significantly decreases, too. A wide linear range was obtained between 0.0125 and 15 μg mL−1 (0.11–130.5 nM) in 1% human serum with a detection limit of 4 ng mL−1 (35 pM). Additionally, the proposed method is universal and can be applied to analyze other similar substances by altering the matched aptamer.

In Situ Reduction of Gold Nanoparticle-Decorated Ti3C2 MXene for Ultrasensitive Electrochemical Detection of MicroRNA-21 with a Cascaded Signal Amplification Strategy

Sensitive detection of miRNA-21 provides remarkable results for the diagnosis of early breast cancer. Here, we fabricated a novel electrochemical biosensor for the ultrasensitive detection of miRNA-21 via in situ reduction of gold nanoparticles (AuNPs)-decorated Ti3C2 MXene, combined with a cascaded signal amplification strategy, that is, strand displacement of DNA walker-induced multi-DNA (product DNA) release and cyclic enzymatic signal amplification. First, in situ reduction of AuNPs on a Ti3C2 MXene layer was not only used as a carrier of capture DNA (C-DNA) and a way to make DNA hybridization accessible but also to provide a predominant (111) facet with high electrocatalytic activity of AuNPs that significantly ameliorated the electrochemical signal, in which MXene acted as both reductant and stabilizer. In addition, in the presence of miRNA-21, multi-DNA fragments (p-DNA) that were produced by strand displacement of the DNA walker could be captured by C-DNA on the fabricated biosensor and trigger Exo III cyclic digestion for further amplification of electrochemical signals. By cascaded signal amplification, the changes in peak signal currents (ΔI) using differential pulse voltammetry were amplified. Under optimal conditions, the electrochemical biosensor achieved a detection limit of 50 aM (S/N = 3) with a linear range from 100 aM to 1.0 nM. With its excellent analytical performance, this biosensor may have the potential to be used in early diagnosis and biomedical applications.

Dual-Aptamer-Assisted AND Logic Gate for Cyclic Enzymatic Signal Amplification Electrochemical Detection of Tumor-Derived Small Extracellular Vesicles.

Small extracellular vesicles (sEVs), often referred to as exosomes, are potential biomarkers for noninvasive cancer diagnosis. However, because of their phenotype heterogeneity, precise detection of tumor-derived sEVs is a great challenge. Herein, a dual-aptamer-assisted AND logic gate was fabricated for sensitive electrochemical detection of tumor-derived sEVs based on a cyclic enzymatic signal amplification strategy. Four different tumor-derived sEVs were used to verify the feasibility of the AND logic gate, and CCRF-CEM sEVs were successfully detected by this assay. The electrochemical assay shows a good linear response from 4 × 103 to 8 × 107 particles/μL, with a detection limit of 920 particles/μL, for CCRF-CEM sEVs, indicating potential application in accurate cancer diagnostics.

Simple Mix-and-Read Assay with Multiple Cyclic Enzymatic Repairing Amplification for Rapid and Sensitive Detection of DNA Glycosylase.

Human 8-oxoguanine DNA glycosylase (hOGG1) can initiate base excision repair of genomic 8-oxoguanine (8-oxoG), and it can locate and remove damaged 8-oxoG through extrusion and excision. Sensitive detection of hOGG1 is critical for clinical diagnosis. Herein, we develop a simple mix-and-read assay for the sensitive detection of DNA glycosylase using multiple cyclic enzymatic repairing amplification. The hOGG1 can excise the 8-oxoG base of the DNA substrate to produce an apurinic/apyrimidinic (AP) site, and then, the AP site can be cleaved by apurinic/apyrimidic endonuclease 1 (APE1), producing the substrate fragment with a free 3'-OH terminus. Subsequently, the substrate fragment can initiate cyclic enzymatic repairing amplification, generating two triggers. The resultant two triggers can function as the primers to induce three cyclic enzymatic repairing amplification, respectively, producing more and more triggers. We experimentally verify the occurrence of each cyclic enzymatic repairing amplification and uracil DNA glycosylase (UDG)-mediated exponential amplification. The amplification products can be simply detected using SYBR Green II as the fluorescent dye. This mix-and-read assay is very simple and rapid (within 40 min) without the requirement of any extra primers and modification/separation steps. This method can sensitively measure hOGG1 with a detection limit of 2.97 × 10-8 U/μL, and it can be applied for the screening of inhibitors and the monitoring of cellular hOGG1 activity at the single-cell level, providing an adaptive and flexible tool for clinical diagnosis and drug discovery.

Dual-Mode FEN1 Activity Detection Based on Nt.BstNBI-Induced Tandem Signal Amplification.

Flap endonuclease 1 (FEN1) is a structure-specific nuclease that cleaves the 5' single-stranded protrusion (also known as 5' flap) during Okazaki fragment processing. It is overexpressed in various types of human cancer cells and has been considered as an important biomarker for cancer diagnosis. However, conventional methods for FEN1 assay usually suffer from complicated platform and laborious procedures with a limited sensitivity. Here, we developed a dual-signal method for sensitive detection of FEN1 on the basis of duplex-specific nuclease actuated cyclic enzymatic repairing-mediated signal amplification. Once the 5' flap of the double-flap DNA substrate was cleaved by target FEN1, the cleaved 5' flap initiated strand-displacement amplification to produce plenty of G-rich DNA (G) sequences. These G sequences that self-assembled into G-quadruplexes in the presence of hemin revealed horseradish-peroxidase-like catalytic activities as well as fluorescence enhancement of thioflavin T. The UV-vis signal showed a good linear relationship with the logarithm of FEN1 activity ranging from 0.03 to 1.5 U with a detection limit of 0.01 U. The fluorescence signal correlated linearly with the logarithm of FEN1 activity ranging from 0.001 to 1.5 U with a detection limit of 0.75 mU. In addition, FEN1 can be visualized not only by colorimetry but also by fluorescence (under ice-water mixture conditions). This reliable, accurate, and convenient method would be a potential powerful tool in point-of-care testing applications and therapeutic response assessment.

A carbon nanoparticle and DNase I-Assisted amplified fluorescent biosensor for miRNA analysis

Nucleic acid-based biosensors have become powerful tools in biomedical applications. But the stability issue seriously limits their wide applications. Fortunately, the emergence of carbon nanoparticles (CNPs), which can effectively protect DNA probes from enzymatic digestion and unspecific protein binding, provides a good solution. In this work, a DNase I-aided cyclic enzymatic amplification method (CEAM) for microRNA analysis has been developed based on the coupling use of nucleic acid probes with specific molecular recognition ability as well as CNPs with excellent biostability. The method is simple and sensitive, with a detection limit down to 3.2 pM. Furthermore, satisfactory results are achieved for miRNA analysis in breast cancer cell lysate, demonstrating the applicability in disease diagnosis. The ingenious combination of CNPs and nucleic acid probes can open a new chapter in the development of versatile analytical strategies that holds great potentials for clinical diagnosis, food safety, and environmental monitoring.

A AuNP-capped cage fluorescent biosensor based on controlled-release and cyclic enzymatic amplification for ultrasensitive detection of ATP.

Gold nanodevices have attracted extensive interest in the detection of specific targets within cells. However, constructing gold sensing devices that can be activated by the simulation of remote applications remains a huge challenge. Here, we report a Au nanoparticle (AuNP)-capped cage fluorescent biosensor based on controlled-release and Exonuclease III (Exo III) assisted cyclic enzymatic amplification that can be activated by adenosine triphosphate (ATP). In the system, AuNPs were used as the building blocks to cap the pores of Au nanocages (AuNCs) loaded with Rhodamine B (RhB) molecules through the hybridization of DNA. The RhB fluorescent molecules were finally released with the help of Exo III in the presence of ATP for detection purposes. Ultimately, the biosensor leads to a wide linear ATP detection range from 1.0 × 10-9 to 1.0 × 10-7 M with a limit of detection (LOD) down to 0.88 nM. In addition, it also has good selectivity for ATP to distinguish between ATP and ATP analogues such as cytidine triphosphate (CTP), guanosine triphosphate (GTP), and uridine triphosphate (UTP). Therefore, as a convenient and sensitive biosensor, it is expected to be widely used in the biomedical field.

Ratiometric fluorescence sensor based on carbon dots as internal reference signal and T7 exonuclease-assisted signal amplification strategy for microRNA-21 detection

The expression level of miRNA-21 is closely related to the occurrence and development of cancer, especially in gastrointestinal cancer. Monitoring miRNA-21 has clinical application in the diagnosis and evaluation of gastrointestinal cancer. A turn-on ratiometric fluorescence bioassay based on the T7 exonuclease-mediated cyclic enzymatic amplification method was developed for miRNA-21 determination by using carbon dots (CDs) and FAM-labeled ssDNA as the signal source. CDs demonstrated the triple functions of built-in internal fluorescence, probe carrier, and quencher in this strategy. In the absence of miRNA-21, FAM-labeled ssDNA would be adsorbed and quenched by CDs. The addition of miRNA-21 induced cycle hydrolysis from the 5′ end by the T7 exonuclease and then released the short-cleaved FAM-labeled oligonucleotides. Then, the increased FAM signal (FFAM) and the stable CD signal (FCDs) would be tested through a ratiometric routine for the quantification of miRNA-21. The FFAM/FCDs value showed a good linear relationship with the concentration of miRNA-21 in the range of 0.05–10 nM, and the detection limit for miRNA-21 was 1 pM with excellent selectivity and reproducibility. Furthermore, this sensor successfully evaluated the expression level of miRNA-21 in clinical blood samples from healthy individuals and gastrointestinal cancer patients, and the results were highly consistent with those of qRT-PCR, suggesting the great clinical application value in the diagnosis of cancer associated with miRNA-21 expression levels.

Bioinspired sensor chip for detection of miRNA-21 based on photonic crystals assisted cyclic enzymatic amplification method

Cancer, as the most invasive disease in the world, has led to an increasing amount of death year by year, so it is highly desired to develop a portable device to monitor the aberrant expression of biomarker in cancer patient. Here, we present a bio-photonic periodic nanostructures sensor chip assisted cyclic enzymatic amplification method to detect miRNA-21 with a detection limit of 55 fM. By employing biocompatible polydopamine nanospheres (PDANs) and DNaseⅠto construct an target-recycling amplification process on the photonic crystals, the output fluorescence signal can be strengthened selectively and short amplification time is needed. Benefiting from the synergy of the enhancement of photonic crystals and enzymatic cycle amplification, we realize high sensitivity detection of miRNA-21 with a detection range of 1 pM–10 nM and a detection limit of about four orders of magnitudes lower than the method employs no amplification, showing an expectable prospect in the early diagnosis of cancer.

Double signal amplification strategy for ultrasensitive electrochemical biosensor based on nuclease and quantum dot-DNA nanocomposites in the detection of breast cancer 1 gene mutation

Rapid and efficient detection of microRNA (miRNA) of breast cancer 1 gene mutation (BRCA1) at their earliest stages is one of the crucial challenges in cancer diagnostics. In this study, a highly-sensitive electrochemical DNA biosensor was fabricated by double signal amplification (DSA) strategy for the detection of ultra-trace miRNA of BRCA1. In the presence of target miRNA of BRCA1, the well-matched RNA-DNA duplexes were specifically recognized by double-strand specific nuclease (DSN), and the DNA part of the duplexes were then cleaved and miRNAs were released to trigger another following cycle, which produced a primarily amplified signal by such a cyclic enzymatic signal amplification (CESA). Then triple-CdTe quantum dot labelled DNA nanocomposites (3-QD@DNA NC) was selectively hybridized with the cleaved DNA probe on the electrode and produced multiply amplified signals. The biosensor exhibited a high sensitivity for the detection of miRNA of BRCA1 in concentrations ranging from 5 aM to 5 fM, and its detection limit of 1.2 aM was obtained, which is two or three orders of magnitude lower than those by single signal amplification strategy such as CESA or QD-labeled DNA probes. The as-prepared biosensor was successfully used to detect the miRNA of BRCA1 in human serum samples with acceptable stability, good reproducibility, and good recovery. The proposed DNA biosensor based on double signal amplification strategy provided a feasible, rapid, and sensitive platform for early clinical diagnosis and practical applications.

Aptasensor for multiplex detection of antibiotics based on FRET strategy combined with aptamer/graphene oxide complex

The development of a multiplexed sensing platform is necessary for highly selective, sensitive, and rapid screening of specific antibiotics. In this study, we designed a novel multiplex aptasensor for antibiotics by fluorescence resonance energy transfer (FRET) strategy using DNase I-assisted cyclic enzymatic signal amplification (CESA) method combined with aptamer/graphene oxide complex. The aptamers specific for sulfadimethoxine, kanamycin, and ampicillin were conjugated with Cyanine 3 (Cy3), 6-Carboxyfluorescein (FAM), and Cyanine 5 (Cy5), respectively, and graphene oxide (GO) was adopted to quench the fluorescence of the three different fluorophores with the efficiencies of 94.36%, 93.94%, and 96.97% for Cy3, FAM, and Cy5, respectively. CESA method was used for sensitive detection, resulting in a 2.1-fold increased signal compared to those of unamplified method. The aptasensor rapidly detected antibiotics in solution with limit of detection of 1.997, 2.664, and 2.337 ng/mL for sulfadimethoxine, kanamycin, and ampicillin, respectively. In addition, antibiotics dissolved in milk were efficiently detected with similar sensitivities. Multiplexed detection test proved that the fluorescently modified aptamers could work separately from each other. The results indicate that the aptasensor offers high specificity for each antibiotic and enables simultaneous and multicolor sensing for rapid screening of multiple antibiotics at the same time.

Cyclic enzymatic amplification method for highly sensitive detection of nuclear factor-kappa B

Effective detection of the intracellular expression level of transcription factors is important for biological research and medical diagnosis. This study proposes a rapid and simple fluorescence sensing strategy for highly sensitive detection of a transcription factor, nuclear factor-kappa B (NF-κB). NF-κB binds to double-stranded DNA duplex, one of which is then protected from Exonuclease III (Exo III) digestion. An Exo III-mediated hydrolysis cycle on TaqMan probes is then triggered to achieve highly sensitive detection of NF-κB. This method can detect NF-κB with concentration as low as 45.6 pM. Furthermore, sequence-specific binding of NF-κB to DNA provides good selectivity. This method can be used for the direct quantification of nuclear proteins extracted from cells. More importantly, by simply replacing the sequence of the probe binding site, this method can also be used for reliable quantification of other DNA-binding proteins and is thus a universal sensing protocol. This strategy can be a powerful technology in the areas of disease diagnosis and pharmaceutical research.

Colorimetric and visual mercury(II) assay based on target-induced cyclic enzymatic amplification, thymine-Hg(II)-thymine interaction, and aggregation of gold nanoparticles.

A colorimetric biosensor and visual test is described for the determination of mercury(II). It relies on the specific thymine-Hg(II)-thymine (T-Hg2+-T) interaction which induces a cyclic amplification process (caused by the enzyme exonuclease III) and the aggregation of gold nanoparticles. These results in a color change from red to violet. Under optimized conditions, this colorimetric assay (best performed at 524nm) has a detection limit as low as 0.9nM with a detection range over 4 orders of magnitude (from 1nM to 10μM). Graphical abstract Schematic of a colorimetric method for determination ofmercury ions (Hg2+) based on the thymine-Hg2+-thymine interaction-triggered cyclic enzymatic amplification and aggregation of gold nanoparticles with the aid of exonuclease III(Exo III).

Ultrasensitive detection of long non-coding RNAs based on duplex-specific nuclease-actuated cyclic enzymatic repairing-mediated signal amplification.

We develop a new fluorescent method for sensitive detection of long noncoding RNAs (lncRNAs) on the basis of duplex-specific nuclease-actuated cyclic enzymatic repairing-mediated signal amplification. This method exhibits high sensitivity with a detection limit of 0.081 fM, and it can accurately detect the endogenous lncRNA HOTAIR in cancer cells, providing a new approach to study the physiological function of lncRNAs in human diseases.

Quantification of MicroRNAs by Coupling Cyclic Enzymatic Amplification with Microfluidic Voltage-Assisted Liquid Desorption Electrospray Ionization Mass Spectrometry.

Quantitative assay of microRNAs (miRNAs) with mass spectrometric detection currently suffers from two major disadvantages, i.e., being insufficient in sensitivity and requiring an extraction or chromatographic separation prior to MS detection. In this work, we developed a facile and sensitive assay of targeted miRNAs based on the combination of cyclic enzymatic amplification (CEA) with microfluidic voltage-assisted liquid desorption electrospray ionization tandem mass spectrometry (VAL-DESI-MS/MS). The single-stranded DNA (ssDNA) probe was designed to have a sequence complementary to the miRNA target with an extension of a two-base nucleotide fragment (i.e., CpC) at the 3'-position as MS signal reporter, thus being easy to prepare and high in stability. In the proposed CEA-VAL-DESI-MS/MS assay, an ssDNA probe was added to a sample solution, forming a DNA-miRNA hybrid. Duplex-specific nuclease (DSN) was then added to cleave specifically the DNA probe in the heteroduplex strands. As the hybridization-cleavage cycle repeated itself for many rounds, a large quantity of CpC molecules was produced that was quantified by VAL-DESI-MS/MS with accuracy and specificity. miRNA-21 was tested as the model target. The assay had a linear calibration equation in the range from 2.5 pM to 1.0 nM with a limit of detection of 0.25 pM. Determination of miRNA-21 in cellular samples was demonstrated. miRNA-21 was found to be 95.3 ± 13.95 amol ( n = 3) in 100 mouse peritoneal macrophages with a recovery of 94.2 ± 2.6% ( n = 3). Interestingly, analysis of exosomes secreted from these cells revealed that exposure of the cells to chemical stimuli caused a 3-fold increase in exosomal level of miRNA-21. The results suggest that the proposed assay may provide an accurate and cost-effective means for quantification of targeted miRNAs in biomedical samples.

Highly Sensitive Electrochemical Detection of Tumor Exosomes Based on Aptamer Recognition-Induced Multi-DNA Release and Cyclic Enzymatic Amplification.

Sensitive and specific detection of tumor exosomes is of great significance for early cancer diagnosis. In this paper, we report an aptamer strategy for exosome detection based on aptamer recognition-induced multi-DNA release and cyclic enzymatic amplification. First, we use aptamer-magnetic bead bioconjugates to capture tumor exosomes derived from LNCaP cells, leading to the release of three kinds of messenger DNAs (mDNAs). After magnetic separation, the released mDNAs hybridized with the probe DNAs immobilized on a gold electrode. Electroactive Ru(NH3)63+ was used as the signal reporter because of its electrostatic attraction to DNA. Subsequent Exo III cyclic digestion caused the electrochemical signal to "turn off". Because the electrochemical signal reflects the concentration of Ru(NH3)63+ and the concentration of Ru(NH3)63+ is correlated with the mDNA concentration, which is correlated with the exosome concentration, the tumor exosomes can be detected by examining the decrease in the peak current of Ru(NH3)63+. In this paper, the signal was amplified by the numerous mDNAs released from the magnetic bead and the Exo III-assisted mDNA recycling. Under the optimal conditions, a detection limit down to 70 particles/μL was achieved, which is lower than the LODs of most currently available methods. Furthermore, this assay can be used to detect tumor exosomes in complex biological samples, demonstrating potential application in real sample diagnosis.