Assisted Target Recycling Amplification Strategy Research Articles

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.

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.

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.

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.

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.

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.

Ultrasensitive Electrochemical Biosensor for HIV Gene Detection Based on Graphene Stabilized Gold Nanoclusters with Exonuclease Amplification.

Because human immunodeficiency virus (HIV) has been one of the most terrible viruses in recent decades, early diagnosis of the HIV gene is of great importance for all scientists around the world. In our work, we developed a novel electrochemical biosensor based on one-step ultrasonic synthesized graphene stabilized gold nanocluster (GR/AuNC) modified glassy carbon electrode (GCE) with an exonuclease III (Exo III)-assisted target recycling amplification strategy for the detection of HIV DNA. It is the first time that GR/AuNCs have been used as biosensor platform and aptamer with cytosine-rich base set as capture probe to construct the biosensor. With the combination of cytosine-rich capture probe, good conductivity and high surfaces of GR/AuNCs, and Exo III-assisted target recycling amplification, we realized high sensitivity and good selectivity detection of target HIV DNA with a detection limit of 30 aM (S/N = 3). Furthermore, the proposed biosensor has a promising potential application for target detection in human serum analysis.

Ultrasensitive Electrochemical Detection of Nucleic Acids Based on the Dual-Signaling Electrochemical Ratiometric Method and Exonuclease III-Assisted Target Recycling Amplification Strategy.

Because of the intrinsic importance of nucleic acids as biotargets, the simple and sensitive detection of nucleic acids is very essential for biological studies and medical diagnostics. In this work, a novel, simple, and selective electrochemical DNA biosensor for the sensitive detection of target DNA (T-DNA) has been developed based on the dual-signaling electrochemical ratiometric method and exonuclease III (Exo III)-assisted target recycling amplification strategy. The assay strategy includes both "signal-on" and "signal-off" elements. The stem-loop (hairpin) DNA capture probe (HP), which was labeled by thiolated methylene blue (MB) at the 3'-protruding termini and ferrocene (Fc) in the middle of the loop, first self-assembled on the gold electrode surface via a Au-S bond. In the presence of T-DNA, the T-DNA hybridized with HP, which triggered the Exo III cleavage process and accompanied the release of T-DNA. As a result, the MB tags were away from and the Fc tags close to the gold electrode surface, leading to the decrease of the oxidation peak current of MB (I(MB)) and the increase of that of Fc (I(Fc)). The value of ΔI(Fc)/|ΔI(MB)| (ΔI(Fc) and ΔI(MB) are the change values of the oxidation peak currents of Fc and MB, respectively) is linear with the concentration of T-DNA from 0.01 pM to 0.8 pM. The detection limit (4.16 fM) of the developed method is much lower than that of the most reported electrochemical DNA biosensors. This strategy provides a simple and sensitive approach for the detection of T-DNA and has promising applications in bioanalysis, disease diagnostics, and clinical biomedicine.

An electrochemical biosensor for the sensitive detection of specific DNA based on a dual-enzyme assisted amplification

In this work, a simple and ultrasensitive electrochemical DNA biosensor was developed based on the horseradish peroxidase (HRP)-catalyzed electrochemical process and exonuclease III (Exo III)-assisted target recycling amplification strategy. A biotin tag labeled molecular beacon (MB) with hairpin structure was ingeniously designed and assembled on an electrode as a recognition element. The 3’ overhang end of the MB labeled with biotin would bind streptavidin (SA)-HRP to give a strong initial signal. Upon target DNA sensing, the MB probe would be stepwise removed by the Exo III accompanied by the release of the target DNA for the successive hybridization and cleavage process. Simultaneously, numerous biotin labeled mononucleotides were liberated, leading to a decrease in the binding of the biotin and SA-HRP polymer on the electrode. As a result, the amplified electrochemical current significantly decreased. Because of the autocatalytic target recycling amplification and the HRP-catalyzed electrochemical process, the dual enzyme-based strategy provided an ultrasensitive approach (down to the 10fM level) for the electrochemical detection of DNA, and discriminated mismatched DNA from perfectly matched target DNA, which has great potential for early diagnosis in gene-related diseases.

Graphene nanosensor for highly sensitive fluorescence turn-on detection of Hg2+based on target recycling amplification

The development of sensitive and selective methods for the monitoring of toxic heavy metal ions is highly demanded because of their threats to the environment and human health. Based on a new exonuclease III (Exo III)-assisted target recycling amplification strategy, a highly sensitive fluorescence turn-on nanosensor for Hg2+ detection using graphene oxide (GO)-quenched, thymine-rich FAM-ssDNA nanoprobes is developed. The target Hg2+ ions bind and fold the GO-adsorbed FAM-ssDNA into duplex structures through the formation of T–Hg2+–T base pairing, leading to the release of the FAM-ssDNA from the surface of GO and recovery of the fluorescent signal. Besides, the released and folded duplex can be digested by Exo III to liberate the bound Hg2+ ions, which can again associate with the GO-quenched FAM-ssDNA nanoprobes and trigger the target recycling process to cause cyclic cleavage of the GO-adsorbed FAM-ssDNA. This target recycling process therefore results in the release of numerous FAM labels back into the solution and significantly amplified fluorescent signal is obtained for highly sensitive detection of Hg2+ down to the sub-nanomolar level. The developed nanosensor also exhibits high selectivity against non-specific ions and can be potentially employed to monitor other toxic heavy metal ions at ultralow levels.

Label-Free and Ultrasensitive Electrochemical Detection of Nucleic Acids Based on Autocatalytic and Exonuclease III-Assisted Target Recycling Strategy

In this work, a very simple, label-free, isothermal, and ultrasensitive electrochemical DNA biosensor has been developed on the basis of an autocatalytic and exonuclease III (Exo III)-assisted target recycling amplification strategy. A duplex DNA probe constructed by the hybridization of a quadruplex-forming oligomer with a molecular beacon is ingeniously designed and assembled on the electrode as recognition element. Upon sensing of the analyte nucleic acid, the strand of molecular beacon in the duplex DNA probe could be stepwise removed by Exo III accompanied by the releasing of target DNA and autonomous generation of new secondary target DNA fragment for the successive hybridization and cleavage process. Simultaneously, numerous quadruplex-forming oligomers are liberated and folded into G-quadruplex-hemin complexes with the help of K(+) and hemin on the electrode surface to give a remarkable electrochemical response. Because of this autocatalytic target recycling amplification and the specifically catalyzed formation of G-quadruplex-hemin complexes, this newly designed protocol provides an ultrasensitive electrochemical detection of DNA down to the 10 fM level, can discriminate mismatched DNA from perfectly matched target DNA, and holds a great potential for early diagnosis in gene-related diseases. It further could be developed as a universal protocol for the detection of various DNA sequences and may be extended for the detection of aptamer-binding molecules.