Target Recycling Amplification Strategy Research Articles

Bioinspired superwettable electrode towards sensitive detection of myocardial infarction-specific miRNA

The development of a reliable and sensitive method for the detection of myocardial infarction (MI)-specific miRNAs is of great significance in the diagnosis and management of MI. Electrochemical biosensors have emerged as a promising tool for the detection of biomolecules due to their high sensitivity, selectivity, simplicity of operation, and miniaturization. In order to further improve the sensitivity and selectivity of DNA electrochemical biosensors and meet the requirements of microRNA detection in biological samples, we fabricated a patterned superwettable gold electrode (PS-Au electrode) by mimicking the superwettable micropatterns of the desert beetle. Combining this bioinspired electrode with a DSN-assisted target recycling amplification strategy, we achieved ultrasensitive detection of myocardial infarction-specific miRNA-483 ranging from 10 aM to 100 fM with a low detection limit of 5.32 aM for miRNA-483. The electrochemical biosensor based on PS-Au electrode and DSN-assisted target recycling amplification (PSE-DTRA-EB) has good stability and specificity. Our study provides a new method for the ultrasensitive detection of MI-specific miRNAs, which may have important clinical applications in the diagnosis and prognosis of MI.

Label-free and ultrasensitive electrochemical detection of nucleic acids based on an exonuclease III-assisted target recycling amplification strategy using a heated gold disk electrode.

The present work demonstrates a label-free, rapid and ultrasensitive electrochemical sensor for specific DNA detection with an exonuclease III (Exo III)-assisted target recycling amplification strategy and elevated electrode temperature at a heated gold disk electrode (HAuDE). The proposed electrochemical DNA (E-DNA) sensor was designed such that in the presence of the target DNA, the electrode self-assembled capture probe hybridizes with the target DNA to form a duplex structure, which triggers Exo III to specifically recognize this structure and selectively digest the capture probe, while the released target DNA underwent recycling to hybridize with a new capture probe, leading to the gradual digestion of a large amount of capture probes. It was found that during the digestion period, the activity of Exo III could be significantly improved by elevating electrode temperature, thus promoting the digestion reaction and improving the sensitivity for target DNA detection. Furthermore, an electrochemical indicator ([Ru(NH3)6]3+) was electrostatically bound to the capture probe, leading to a significant square wave voltammetry (SWV) response, which directly related to the amount and length of the capture probes remaining in the electrode and provided a quantitative measure for target DNA detection. The proposed strategy realized the highly sensitive detection of the target DNA with a detection limit of 26 aM (S/N = 3) at an electrode temperature of 40 °C during the digestion period, which was about two magnitudes lower than that at 24 °C.

A cascade signal-amplified fluorescent biosensor combining APE1 enzyme cleavage-assisted target cycling with rolling circle amplification.

A cascade signal-amplified fluorescent biosensor was developed for miRNA-21 detection by combining APE1 enzyme-assisted target recycling and rolling circle amplification strategy. A key feature of this biosensor is its dual-trigger mechanism, utilizing both tumor-endogenous miRNA-21 and the APE1 enzyme in the initial amplification step, followed by a second rolling circle amplification reaction. This dual signal amplification cascade significantly enhanced sensitivity, achieving a detection limit of 3.33 pM. Furthermore, this biosensor exhibited excellent specificity and resistance to interference, allowing it to effectively distinguish and detect the target miRNA-21 in the presence of multiple interfering miRNAs. Moreover, the biosensor maintained its robust detection capabilities in a 10% serum environment, demonstrating its potential for clinical disease diagnosis applications.

LuMA-Functionalized Thermosensitive Hydrogel: A Versatile and Robust Dopamine-Triggered Platform for Diverse Biomolecules Sensing.

It is of great significance for the analysis of multiple biomarkers because a single biomarker is difficult to accurately achieve early diagnosis, disease course monitoring, and prognosis evaluation. Herein, a luminescence thermosensitive hydrogel was synthesized by radical polymerization using a methacrylic acid derivative monomer of luminol (LuMA) as luminescent, N-isopropylacrylamide (NIPAM) as thermosensitive monomer, and acrydite-oligonucleotides [dopamine (DA) aptamer, DNA C1, and DNA C2] as recognition elements. The combined DA based on the affinity interaction between the DA and the aptamer on the hydrogel polymer chain was electrochemically oxidized to dopamine quinone during the electrochemiluminescence (ECL) scanning, which effectively quenched the ECL signal of LuMA due to the resonance energy transfer (RET). In addition, the thermosensitive hydrogel showed swelling-collapse characteristics when the temperature was below and above the volume phase transition temperature. Undergoing the collapse process initiated by the temperature, the RET efficiency was further enhanced due to the shortened distance between the energy donor and acceptor, showing a 1.4 times signal amplification and achieving sensitive detection of DA with a limit of detection (LOD) of 1.7 × 10-10 M. For a proof of concept application, coupled with the target-induced release of DA from the DNA-magnetic beads bioconjugations based on duplex-specific nuclease (DSN)-assisted target recycling amplification strategy and DNAzyme cleavage reaction, this ECL-RET approach was successfully used to evaluate multiple targets including miRNA-141 and MUC1 with the LOD of 2.5 aM and 1.6 fg/mL, respectively. The excellent performances of the versatile and robust ECL-RET hydrogel in multiple target sensing showed potential applications in clinical diagnosis and disease therapeutic assay.

Heating promoted super sensitive electrochemical detection of p53 gene based on alkaline phosphatase and nicking endonuclease Nt.BstNBI-assisted target recycling amplification strategy at heated gold disk electrode

An ultrasensitive electrochemical biosensor for detecting p53 gene was fabricated based on heated gold disk electrode coupling with endonuclease Nt.BstNBI-assisted target recycle amplification and alkaline phosphatase (ALP)-based electrocatalytic signal amplification. For biosensor assembling, biotinylated ssDNA capture probes were first immobilized on heated Au disk electrode (HAuDE), then combined with streptavidin-alkaline phosphatase (SA-ALP) by biotin-SA interaction. ALP could catalyze the hydrolysis of ascorbic acid 2-phosphate (AAP) to produce ascorbic acid (AA). While AA could induce the redox cycling to generate electrocatalytic oxidation current in the presence of ferrocene methanol (FcM). When capture probes hybridized with p53, Nt.BstNBI would recognize and cleave the duplexes and p53 was released for recycling. Meanwhile, the biotin group dropt from the electrode surface and subsequently SA-ALP could not adhere to the electrode. The signal difference before and after cleavage was proportional to the p53 gene concentration. Furthermore, with electrode temperature elevated, the Nt.BstNBI and ALP activities could be increased, greatly improving the sensitivity and efficiency for p53 detection. A detection limit of 9.5 × 10−17 M could be obtained (S/N = 3) with an electrode temperature of 40 °C, ca. four magnitudes lower than that at 25 °C.

A highly sensitive competitive aptasensor for AFB1 detection based on an exonuclease-assisted target recycling amplification strategy.

Aflatoxin B1 (AFB1) is a typical mycotoxin found in agricultural products, and poses a huge threat to both humans and animals. Accurate and rapid measurement of AFB1 is essential for environmental analysis and food safety. Based on molecular docking simulation design and exonuclease-assisted target recycling amplification, we designed a competitive fluorescence aptasensor to detect AFB1 rapidly and sensitively. According to the molecular docking simulations, a complementary strand (cDNA) was designed by searching for potential binding sites of the aptamer, which had the lowest binding energy. Magnetic beads modified with biotin-Apt were used as the capture probe, while FAM-labeled cDNA acted as the reporter probe. By using EXO I for target recycling amplification, this aptasensor was highly sensitive and selective for AFB1. The detection limit of the suggested aptasensor under optimal conditions was 0.36 ng mL-1 (S/N = 3) in the range of 1-1000 ng mL-1 (R2 = 0.991). The developed aptasensor was successfully used to analyze AFB1 in oil samples.

Ratiometric electrochemical aptasensor for the sensitive detection of carcinoembryonic antigen based on a hairpin DNA probe and exonuclease I-assisted target recycling

A novel ratiometric electrochemical aptasensor was constructed for the detection of carcinoembryonic antigen (CEA) based on a hairpin DNA (hpDNA) probe and exonuclease Ⅰ (Exo Ⅰ)-assisted target recycling amplification strategy. A thiolated methylene blue (MB)-labeled hpDNA as the internal control element was fixed on the surface of the gold nanoparticles (AuNPs)-modified gold electrode (AuE) through Au–S bonds. A ferrocene (Fc)-modified aptamer DNA (Fc-Apt) was partially hybridized with hpDNA to form a Fc-Apt/hpDNA duplex. Due to the specific recognition of Fc-Apt to CEA, the presence of CEA caused dissociation of Fc-Apt from the duplex, and further triggered the degradation process of Exo Ⅰ and recycling of CEA. Hence, the Fc tags were released from the electrode surface and the oxidation peak current of Fc (IFc) decreased while that of MB (IMB) remained stable owing to the distance between MB tags and the electrode unchanged. A linear relationship was observed between IFc/IMB and the logarithm of CEA concentration from 10 pg mL−1 to 100 ng mL−1 with a detection limit of 1.9 pg mL−1. Moreover, the developed aptasensor had been applied to detect CEA in diluted human serum with satisfactory results, indicating its great potential in practical applications.

A new ratiometric electrochemical aptasensor for the sensitive detection of carcinoembryonic antigen based on exonuclease I-assisted target recycling and hybridization chain reaction amplification strategy

In this work, a novel ratiometric electrochemical aptasensor was designed on the basis of dual-amplification mechanism by using exonuclease I (Exo I)-assisted target recycling and hybridization chain reaction (HCR). The thiol-modified capture DNA (Cp-DNA) was fixed on the surface of the gold electrode (AuE) modified with gold nanoparticles (AuNPs) through Au-S bonds. The ferrocene (Fc)-labeled DNA (Fc-DNA) hybridized with Cp-DNA to form Fc-DNA/Cp-DNA duplex. The presence of carcinoembryonic antigen (CEA) led to the dissociation of Fc-DNA from Fc-DNA/Cp-DNA duplex and initiated Exo Ⅰ-assisted target recycling to digest Fc-DNA, so numerous Fc left away from the electrode surface and resulting in the “signal off” of Fc. Moreover, Cp-DNA was exposed to bind trigger DNA and initiate HCR with the presence of H1 and H2 probes, resulting in the intercalating of numerous methylene blue (MB) molecules and the “signal-on” of MB. This dual signal strategy significantly amplified the decrease of Fc signal and the increase of MB signal, achieving a linear range of 1 pg mL−1 to 100 ng mL−1 with a detection limit of 0.479 pg mL−1 (S/N = 3). Besides, the developed aptasensor exhibited high specificity, good stability and reproducibility. More importantly, the aptasensor had been applied in detection of CEA in diluted human serum and displayed satisfactory anti-interference property. By changing the corresponding aptamer sequences, the designed strategy can be employed to detect other targets, which holds promising application in clinical diagnosis.

Highly sensitive label-free multi-signal sensing platform for Kras gene detection by exonuclease assisted target recycling amplification based on AIZS QDs

In this study, a new multi-channel biosensor was constructed with exonuclease assisted target recycling signal amplification to sensitively determine Kras gene. The hairpin DNA (h-DNA) contained a recognition sequence to target Kras gene and can specifically hybridized with Kras gene. In the presence of Kras gene, G-quadruplex could be produced through exonuclease III (Exo III) assisted target recycling amplification strategy and then Kras gene was used as the trigger to initiate the next recycling. When hemin was introduced, a large quantity of hemin/G-quadruplex DNAzymes were formed to catalyze the oxidation of 2,4-dichlorophenol (2,4-DP). 4-aminoantipyrine (4-AP) could couple with the oxidation product of 2,4-DP to produce a red adduct (quinone imine) with a characteristic absorbance peak at 506 nm, resulting in the fluorescence quenching of AIZS QDs. Based on the changes of fluorometric and colorimetric dual-signals, Kras gene activity was sensitively detected with the limits of detection as low as 0.12 pM and 0.85 pM, respectively. Meanwhile, our sensing system could realize the visual detection through the color change of the reaction solution. Furthermore, the sensing platform showed satisfactory results for measuring the Kras gene in human serum and showed great potential in diagnostic analysis.

Ultrasensitive ratiometric fluorescent probes for Hg(II) and trypsin activity based on carbon dots and metalloporphyrin via a target recycling amplification strategy.

A convenient and ultrasensitive ratiometric fluorescent probe was innovatively developed for Hg(II) detection and trypsin activity evaluation based on carbon dots (CDs) and tetraphenylporphyrin tetrasulfonic acid (TPPS) using bovine serum albumin (BSA) as the substrate of trypsin. The ratiometric fluorescence signal arises from CDs (λem = 506 nm) and TPPS (λem = 645 nm) via an inner filter effect. Hg2+ can trigger the formation of TPPS-Mn2+ metalloporphyrin for target Hg2+ recycling amplification, while both TPPS-Hg2+ and TPPS-Mn2+ metalloporphyrins do not affect the fluorescence of CDs. Small amino acids and peptide fragments, which are the products of BSA under the digestion of trypsin, bind stronger with Hg2+ than with TPPS. The decomposition of both TPPS-Hg2+ and TPPS-Mn2+ metalloporphyrins leads to a variation in the ratiometric fluorescence signal. Under optimized conditions, this probe provided an inspiring detection limit of 0.086 nM for Hg2+ and 0.013 ng mL-1 for trypsin, which possessed acceptable sensitivity for Hg2+ detection and trypsin activity evaluation in authentic samples. This unprecedented CD-based ratiometric fluorescence proposal for ultrasensitive quantification of Hg2+ concentration and selective assessment of trypsin activity gives a new insight for designing metal ion assays or enzymatic activity bioassays under different enzymatic substrates in the near future.

A programmatic electrochemical nanosensor based on robust bipedal DNA walkers fueled by catalytic hairpin assembly

In this paper, a robust and highly integrated DNA bipedal walker based electrochemical biosensor was designed on single nanoelectrodes for ultrasensitive miRNA-21 detection through click chemistry, enzyme-free target recycling amplification strategy and programmed catalysis hairpin assembly. Triggered by target miRNA-21, the DNA bipedal walker was built through hybridization and chemical ligation of two hairpin DNA probes (W1 and W2), accompanied with the releasing of target. Then driven by MB-labeled hairpin DNA (MB-FS), the DNA bipedal walker was activated to run continuously along the DNA track and attach a large number of signal molecules MB on the nanoelectrode surface, resulting in cascade signal amplification. With the target recycling amplification strategy and DNA walker, the biosensor showed high analysis performance in detection of miRNA-21 including wide detection range (0.1 fM-50 pM), low detection limit (60 aM) and high selectivity. What’s more, owing to the proximity-dependent hybridization and programmed catalysis hairpin assembly, the nanosensor was one-incubated and regenerative which showed a promising approach for miRNA biomarker expression in cancer cells.

Controlling surface nanoarchitectures of DNA modified electrodes for improved label-free electrochemical detection of p53 gene

Combined superb electron transfer ability of gold nanoparticles with target recycling amplification strategy for label-free electrochemical detection of p53 gene. • An efficient p53 gene biosensor was developed by controlling surface nanoarchitectures of DNA modified electrodes. • Nicking endonuclease combined with gold nanoparticles was employed for improved sensing behavior. • How the different status of DNA strands on electrode surfaces affect GNPs mediated ET process was studied. • The proposed biosensor showed high performance with a detection limit of 0.0835 fM and a linear calibration range of 0.1 fM ~ 1 fM. P53 gene plays a vital role in regulating cellular processes and its accurate quantification is of great significance for the early cancer screening. Herein, by controlling surface nanoarchitectures of DNA modified electrode, we designed a new biosensing interface for the detection of p53 gene. In this method, gold nanoparticles (GNPs) were used to mediate electron transfer (ET) across different DNA monolayers modified electrode surfaces, and NESA was incorporated into the bio-sensing system. The proposed simple and label-free biosensor exhibited high performance with a detection limit of 0.0835 fM and a linear calibration range of 0.1 fM ~ 1 fM for p53 DNA sensing. Moreover, the method shows good sequence selectivity and reproducibility, demonstrating this strategy has potential applications in clinical diagnosis and biomedical research.

Construction of a Sensing Platform Based on DNA-Encoded Magnetic Beads and Copper Nanoclusters for Viral Gene Analysis with Target Recycling Amplification.

The rapid and accurate monitoring of viral genes plays an important role in the area of disease diagnosis, biomedical research, and food safety. Herein, we successfully designed a sensing system that combined the technologies of target DNA recycling amplification, magnetic separation, and in situ formation of fluorescent copper nanoclusters (CuNCs) for viral DNA analysis. In the presence of target viral DNA (tDNA), a large quantity of output DNA (oDNA) was produced from hairpin DNA (hDNA) through an exonuclease III-assisted target recycling amplification strategy. Magnetic beads (MBs) labeled with capture DNA (cDNA) were hybridized with oDNA, and the partially complementary oDNA served as a bridge that could link AT-rich dsDNA on the surface of MBs, which led to a decrease of AT-rich dsDNA in solution after magnetic separation. On account of the lack of AT-rich dsDNA as a template in solution, in situ formation of fluorescent CuNCs was blocked, which resulted in a decrease in the fluorescence intensity at 590 nm. Therefore, taking advantage of one-step magnetic separation and in situ formation of CuNCs, the target viral DNA was sensitively and specifically detected in a linear range from 5 pM to 5 nM with a detection limit of 1 pM. The MB-based platform was not only reusable but also achieved magnetic separation, which could eliminate interferences in complex samples. The assay combining the MB-based probe with fluorescent CuNCs provided a universal, label-free, and reusable platform for viral DNA detection.

Dual-color graphene quantum dots and carbon nanoparticles biosensing platform combined with Exonuclease III-assisted signal amplification for simultaneous detection of multiple DNA targets

Sensitive and simultaneous detection of multiple biomarkers such as target DNA or proteins using biocompatible materials with good analysis performance remains an important challenge. Herein, we successfully developed a signal “off-on” highly sensitive multiplex detection platform based on the combination of dual-color graphene quantum dots (blue GQDs and green GQDs) modified DNA probes with carbon nanoparticles (CNPs), which is a cheap, effective nonfluorescent quencher to simultaneously quench the fluorescence of both GQDs-DNA probes. The Exo III-assisted sequence-independent target recycling and signal amplification strategy was integrated into this sensing platform, which endows it with high sensitivity towards the multiplex detection of targets DNA. The detection limits of 6.6 pM for HIV and 9.5 pM for HBV were achieved respectively, which is about 60-fold lower than that of traditional unamplified homogeneous fluorescent assay methods. Our proposed multiplex detecting platform is advantageous in both respective and simultaneous detection of multiple targets and can also discriminate perfectly matched targets from mismatched targets in both PBS buffer and 1% human serum samples, demonstrating its potential to be a reliable strategy for highly sensitive simultaneous detection of multiple target genes in practical diagnosis applications.

Highly sensitive label-free fluorescence determination of lymphotropic virus DNA based on exonuclease assisted target recycling amplification and in-situ generation of fluorescent copper nanoclusters

Specific and sensitive detection of target DNA plays a significant role in clinical diagnosis and biomedical research. Herein, a sensitive ratio fluorescence biosensor was constructed based on in-situ generation of fluorescent copper nanoclusters (CuNCs) on the DNA modified graphene quantum dots (GQDs) for the detection of HTLV-I DNA. Firstly, large amount of AT rich single DNA (o-DNA) can be released from the designed hairpin DNA in the presence of trace target DNA via exonuclease III assisted target recycling amplification strategy. Then, the released o-DNA hybridized with the complementary DNA1 which was modified on the surface of GQDs, and the formed hybridized DNA with blunt 3′-hydroxylated terminus that couldn’t be digested by exonuclease I could act as the template for the generation of fluorescent CuNCs. While in the absence of target DNA, the o-DNA couldn’t be released, thus the DNA1 with 3′overhang ends could be degraded by exonuclease I, which resulted in the in-situ generation of fluorescent CuNCs on the surface of GQDs was blocked owing to the lack of DNA template. The GQD fluorescence in the GQD-CuNC nanohybrid was almost uninfluenced during the whole process. Therefore, with the GQD serving as the reference and CuNC acting as the reporter signal, there was an excellent linear relationship between the change of fluorescence intensity ratio (F595/F445) and the concentration of HTLV-I DNA in the range of 20 pM-12 nM with a detection limit of 10 pM. Furthermore, the biosensor exhibited satisfactory results for monitoring the presence of HTLV-I DNA in human serum.

Highly Sensitive Detection of Multiple MicroRNAs by High-Performance Liquid Chromatography Coupled with Long and Short Probe-Based Recycling Amplification.

This report demonstrated the utility of high-performance liquid chromatography (HPLC)-fluorescence detection for selective separation and sensitive quantification of multiple microRNAs (miRNAs). A duplex specific nuclease (DSN)-assisted target recycling amplification strategy was developed to enhance the signals of miRNAs, which alleviates the low sensitivity of conventional HPLC to nucleic acids. To separate the signals of different miRNAs, DNA probes with different lengths and base sequences were immobilized on magnetic beads. The application of an effective magnetic separation minimized the background signal and extended the dynamic range. This assay achieved a limit of detection of 0.39 fM for miRNA-122, 0.30 fM for miRNA-155, and 0.26 fM for miRNA-21, respectively. The proposed assay was successfully applied to detect simultaneously miRNA-122, miRNA-155, and miRNA-21 in serum samples from healthy persons and cervical cancer patients, and the results were then compared with those of quantitative real-time-polymerase chain reaction amplification.

Ultrasensitive Detection of MicroRNA via a Au@Ag Nanosnowman.

MicroRNAs (miRNAs) play key roles in many serious diseases, such as cancer. As a consequence, miRNAs are of great interest as biomarkers in clinical diagnostics. Simple, fast, selective, and sensitive detection of miRNAs, however, is challenged by their short length, homogeneous sequence, susceptibility to degradation, and low abundance in human serum. Here, we present a new strategy for highly sensitive and selective detection of miRNA based on the formation of a plasmonic Au@Ag nanosnowman. When triggered by miRNA-21, bimetallic nanoparticles with an asymmetric Au@Ag head-body structure were formed with significant red shift of the localized surface plasmon resonance (LSPR) scattering wavelength and clear color change from green to red. When combined with exonuclease III (Exo III)-assisted target recycling and hybridization chain reaction (HCR) amplification strategy, the proposed bioassay showed excellent selectivity toward miRNA-21 with a proportional band from 1 fM to 100 pM and ultrahigh sensitivity with a limit of detection of 0.60 fM under dark-field microscopy. The proposed strategy is universal, which shows good application prospects in clinical analysis.

Ultrasensitive aptamer biosensor for arsenic (III) detection based on label-free triple-helix molecular switch and fluorescence sensing platform

Arsenic ion is a well-known harmful heavy element widely existing in the environment. Arsenic pollution occurring frequently has become increasing a serious worldwide threat to human health and the environment. The development of sensitive and reliable methods to detect As3+ in water is of great importance to biochemical research and monitoring applications. Herein, a label-free fluorescence sensing platform was elaborately designed for As3+ monitoring using exonuclease III (Exo III)-assisted cascade target recycling amplification strategy. The triple-helix molecular switch was employed as the sensing element and 2-amino-5,6,7-trimethyl-1,8-naphthyridine was used as the signal indicator. The resulting biosensor is simple, ultrasensitive, and exhibits a limit of detection of 5 ng/L with high selectivity. Meanwhile, the proposed sensor is successfully applied to determination of As3+ in practical sample analysis (tap water, lake water and pond water). The results shown herein have important implications in the development of new fluorescent sensors for the fast, easy, and selective detection and quantification of As3+ in water samples. More importantly, the proposed platform can be extended to detect other heavy metal ions with newly designed triple-helix molecular switch, as well as pesticide residue, antibiotic residues, and biomarkers by using aptamer sequences.

Highly Efficient Target Recycling-Based Netlike Y-DNA for Regulation of Electrocatalysis toward Methylene Blue for Sensitive DNA Detection

In this work, the highly efficient target recycling-based netlike Y-shaped DNA (Y-DNA), which regulated the electrocatalysis of Fe3O4@CeO2-Pt nanoparticles (Fe3O4@CeO2-PtNPs) toward methylene blue (MB) for signal amplification, was developed to prepare a sensitive DNA biosensor for detecting the DNA associated with oral cancer. Specifically, with the help of highly efficient enzyme-assisted target recycling (EATR) amplification strategy, one target DNA input was converted to corresponding plenty of DNA strands S1-Fe3O4@CeO2-Pt and S2-MB output, which could be employed to interact with HP2 immobilized on the electrode surface to form stable netlike Y-DNA without any waste of recycling products. Meanwhile, the formation of netlike Y-DNA could regulate electrocatalytic efficiency of Fe3O4@CeO2-PtNPs, inducing the proximity of Fe3O4@CeO2-PtNPs to MB and significantly enhancing electrochemical signal. Further, the signal could also be amplified by Fe3O4@CeO2-PtNPs modified on the electrode surface. By virtue of this ingenious design, a novel netlike Y-DNA structure based on highly efficient EATR was simply constructed and successfully applied to an electrochemical DNA biosensor along with electrocatalysis of Fe3O4@CeO2-PtNPs, achieving the sensitive detection of target DNA ranging from 10 fM to 50 nM with a detection limit of 3.5 fM. Impressively, the biosensor here demonstrates an admirable method for regulating the electrocatalysis of NPs toward substrates to enhance signal, and we believe that this biosensor is a potential candidate for the sensitive detection of target DNA or other disease-related nucleic acids.

Homogeneous electrochemical aptasensor for mucin 1 detection based on exonuclease I-assisted target recycling amplification strategy

A sensitive and homogeneous electrochemical aptasensor was fabricated for the detection of mucin 1 (MUC1) by combining a well-designed DNA bulge-loop (L-DNA) structure with high-efficient exonuclease I (Exo I)-assisted target recycling amplification strategy. The L-DNA probe was constructed via the hybridization of the MUC1 aptamer and methylene blue (MB) labeled complementary DNA (cDNA) (cDNA-MB) and hence could not diffuse freely to the negatively charged ITO electrode surface due to the strong electrostatic repulsion, so small electrochemical signal was detected. The addition of MUC1 caused the dissociation of L-DNA structure due to the specificity between aptamer and MUC1. Then Exo I was implemented to digest the released cDNA-MB into mononucleotides and then produced short MB-labeled mononucleotides fragments (MB-MFs). As the MB-MFs contained few negative charges, it diffused easily to the negatively charged ITO electrode surface and resulted in the enhanced electrochemical signal. Meanwhile, the MUC1-aptamer complex was also specifically digested by Exo I, resulting in the liberation of MUC1 and hence realized the target recycling and then caused the amplification of the electrochemical signal. The enhanced electrochemical signal has a good linear relationship with logarithm of MUC1 concentration in the range of 1.0 pg mL−1–50 ng mL−1 with a limit of detection of 0.40 pg mL−1 (S/N = 3). Additionally, the fabricated aptasensor has been successfully applied to detect MUC1 in serum samples with satisfactory results and thereby it exhibits great potential in the practical application of clinical diagnosis.