Target Recycling Amplification Strategy Research Articles

A sandwich-type electrochemical biosensing platform for microRNA-21 detection using carbon sphere-MoS2 and catalyzed hairpin assembly for signal amplification

In this work, a sandwich-type electrochemical biosensing platform is developed based on carbon sphere-MoS2 (CS-MoS2) and target recycling amplification of catalyzed hairpin assembly (CHA) strategy for ultrasensitive determination of microRNA (miRNA). The CS-MoS2 is prepared and used as sensing substrate, which shows high specific surface area, enhanced stability and good dispersibility. The capture probe is first modified on Au nanoparticles/CS-MoS2 modified glassy carbon electrode through Au-S band, and hairpin DNA1 (H1) then reacts with it and unfolds its loop domain by hybridizing with target miRNA-21. The opened H1 subsequently assembles with biotin labeled hairpin DNA 2 (H2) to displace the target. The liberated miRNA-21 is free and backs to the original solution to proceed next circulation, resulting in the generation of large amounts of H1–H2 duplex. Massive signal indicators horseradish peroxidase are then immobilized on electrode, and catalyze hydrogenperoxide + hydroquinone (H2O2 + HQ) system to produce a strong electrochemical response. The proposed assay benefits from advantages of sandwich-type structure, favorable electrochemical properties of CS-MoS2 and CHA target recycling amplification strategy. Therefore, it exhibits a wide linear relation between 1 × 10−16 and 1 × 10−10 M with a low detection limit of 1.6 × 10−17 M for miRNA-21 detection.

Enzyme-free Target Recycling and Double-Output Amplification System for Electrochemiluminescent Assay of Mucin 1 with MoS2 Nanoflowers as Co-reaction Accelerator.

In this work, a sensitive electrochemiluminescent assay of mucin 1 (MUC1) was developed with the advantages of target recycling amplification strategy and effective MoS2 nanoflower (MoS2 NF)-based signal probe. Briefly, the target MUC1 triggered enzyme-free recycling and a double-output amplification process was executed to acquire masses of single-stranded DNA as a mimic target, which further participated in the catalytic hairpin assembly process for signal amplification. Meanwhile, MoS2 NFs were prepared as an effective co-reaction accelerator, which not only possessed excellent catalytic performance for H2O2 decomposition to largely enhance the luminous intensity of N-(aminobutyl)- N-(ethylisoluminol) (ABEI)-H2O2 electrochemiluminescence system but also offered a desirable platform for ABEI-functionalized Ag nanoparticles (ABEI-Ag complexes) loading via Ag-S binding. The experimental results showed the proposed aptasensor had a good linear relationship in the range of 1 fg/mL to 10 ng/mL for MUC1 detection and the limit of detection was 0.58 fg/mL (S/N = 3). In addition, the aptasensor had nice stability and selectivity and huge potential to be applied in clinical research.

Heating enhanced sensitive and selective electrochemical detection of Hg2+ based on T-Hg2+-T structure and exonuclease III-assisted target recycling amplification strategy at heated gold disk electrode

A sensitive and selective electrochemical Hg2+ sensor was developed based on T-Hg2+-T structure and exonuclease (Exo) III -assisted target recycling amplification at heated gold disk electrode (HAuDE). First, a DNA signal probe P1 was for the first time designed and labeled with ferrocene (Fc) near the attached SH-5′-end, so as to shorten the distance between Fc and the electrode and enhance the initial current of Fc compared with that labeled at the 3′-end far from the electrode. Then the signal amplification was achieved by Exo III-assisted Hg2+ recycling. Briefly, the P1 was complementary to the assistant DNA P2 except the T-T mismatches. In the presence of Hg2+, the P1 self-assembled on the HAuDE could hybridize with P2 and form DNA duplex with blunt end at the 3′- terminus, triggering Exo III to stepwise digest mononucleotides from the 3′-terminus of P1, ultimately liberating Hg2+ and P2, which could be “recycled”, resulting in the digestion of a large amount of P1 and significantly decrease the amount of Fc. The electrochemical signal difference before and after digestion was proportional to the Hg2+ concentration. Furthermore, during the digestion period, the Exo III activity could be significantly increased by elevating the electrode temperature, great improving the sensitivity and efficiency for Hg2+ detection. A detection limit of 6.2 pM (S/N = 3) could be obtained with an electrode temperature of 40°C during 60min digestion period, which was lower ca. two magnitudes than that at 0°C and one magnitude than that at 25°C.

Multiplexed fluorescence detection of microRNAs based on novel distinguishable quantum dot signal probes by cycle amplification strategy

MicroRNA (miRNA) is a biomarker that has great potential for the diagnosis and prediction of different cancers. Exploring a rapid and specific method for detecting miRNA has become a research hotspot. In this study, a novel strategy for simultaneous fluorescence detection of dual target miRNAs (miRNA-141 and miRNA-21) based on two distinguishable CdSe@ZnS and CdTe quantum dot (QD) probes by target recycling amplification strategy was reported. The hairpin (HP) DNA was firstly linked to the magnetic bead@Au (MB@Au) complex, then the present target miRNAs hybridized with HP, which then initiated the exonuclease III-aided target recycling process, leading to the generation of numerous intermediate DNA sequences (s1) on MB@Au. Subsequently, a large number of QDs fluorescence probes were introduced to hybridize with s1, which will further trigger the scission with addition of Nb.BbvCI. Through convenient magnetic separation, much amplified fluorescence signal of QDs was detected, which has a quantitative relation with the target miRNAs. Importantly, two kinds of distinguishable fluorescent QDs probes were applied to the simultaneous detection of dual miRNAs by cycling amplification strategy. Thus, the proposed fluorescence method could be used to rapidly detect miRNA-141 and miRNA-21 with high sensitivity, and effectively distinguished single-base-mismatched miRNA sequences with high specificity. Therefore, this strategy holds great potential for sensitive, efficient and convenient detection of various tumor markers in clinical application.

A novel silver nanocluster in situ synthesized as versatile probe for electrochemiluminescence and electrochemical detection of thrombin by multiple signal amplification strategy

In this work, a novel silver nanoclusters (AgNCs) were in situ synthesized and used as versatile electrochemiluminescence (ECL) and electrochemical (EL) signal probes for thrombin detection by using DNAzyme-assisted target recycling and hybridization chain reaction (HCR) multiple amplification strategy. The presence of target thrombin firstly opened the hairpin DNA, followed by DNAzyme-catalytic recycling cleavage of excess substrates, which could generate large number of substrate fragments (s1). Then these s1 fragments were captured by SH–DNA on the Au nanoparticle-modified electrode, which further triggered the subsequent HCR of the hairpin DNA probes (H1 and H2) to form the long dsDNA. The numerous AgNCs were thus in situ synthesized by incubation the dsDNA template (with cytosine-rich loop)-modified electrode in solution with AgNO3 and sodium borohydride. By integrating the DNAzyme recycling and HCR dual amplification strategy, the amount of AgNCs is dramatically enhanced, leading to substantially amplified ECL and electrochemical signals for sensitive thrombin detection. Importantly, this design introduces the novel AgNCs into versatile ECL and EL bioassays by multiple amplification strategy, thus it is promising to provide a highly sensitive platform for various target biomolecules.

Exonuclease-assisted target recycling amplification for label-free chemiluminescence assay and molecular logic operations.

A versatile exonuclease III (Exo III)-assisted target recycling amplification (Exo-TRA) strategy is demonstrated to achieve label-free and homogeneous chemiluminescence (CL) detection of target DNA. Inspired by this principle, a series of logic gates are constructed using DNA molecules as "inputs" to activate the Exo-TRA system.

A label-free electrochemical biosensor for microRNA detection based on catalytic hairpin assembly and in situ formation of molybdophosphate

A label-free electrochemical biosensor for sensitive detection of miRNA-155 was presented by coupling enzyme-catalyzed in situ generation of electronic mediator for signal introduction with catalytic hairpin assembly (CHA) induced target recycling amplification strategy. In this work, alkaline phosphatase (ALP) was adopted to hydrolyze inactive substrate 1-naphthyl phosphate (NPP) to produce phosphate ion (PO43−), which could further react with acidic molybdate to form abundant molybdophosphate anion (PMo12O403−) on the surface of electrode. The produced PMo12O403− could directly generate a strong and stable electrochemical signal for quantitative detection of targets. In addition, CHA induced the cyclic reuse of the target was also employed as an effective strategy for improving the sensitivity of biosensor. This electrochemical method for miRNA-155 detection had achieved a good linear relationship ranging from 10fM to 1nM with a detection limit of 1.64fM. With this assay successfully applied in tumor cell lysates, it holds great potential for early cancer diagnosis by employing miRNA as the effective biomarker.

Fluorescent trimethyl-substituted naphthyridine as a label-free signal reporter for one-step and highly sensitive fluorescent detection of DNA in serum samples

A facile label-free sensing method is developed for the one-step and highly sensitive fluorescent detection of DNA, which couples the specific C-C mismatch bonding and fluorescent quenching property of a trimethyl-substituted naphthyridine dye (ATMND) with the exonuclease III (Exo III) assisted cascade target recycling amplification strategy. In the absence of target DNA, the DNA hairpin probe with a C-C mismatch in the stem and more than 4 bases overhung at the 3' terminus could entrap and quench the fluorescence of ATMND and resist the digestion of Exo III, thus showing a low fluorescence background. In the presence of the target, however, the hybridization event between the two protruding segments and the target triggers the digestion reaction of Exo III, recycles the initial target, and simultaneously releases both the secondary target analogue and the ATMND caged in the stem. The released initial and secondary targets take part in another cycle of digestion, thus leading to the release of a huge amount of free ATMND for signal transducing. Based on the fluorescence recovery, the as-proposed label-free fluorescent sensing strategy shows very good analytical performances towards DNA detection, such as a wide linear range from 10pM to 1μM, a low limit of detection of 6pM, good selectivity, and a facile one-step operation at room temperature. Practical sample analysis in serum samples indicates the method has good precision and accuracy, which may thus have application potentials for point-of-care screening of DNA in complex clinical and environmental samples.

Signal-Switchable Electrochemiluminescence System Coupled with Target Recycling Amplification Strategy for Sensitive Mercury Ion and Mucin 1 Assay.

In the present work, we first found that mercury ion (Hg(2+)) has an efficient quenching effect on the electrochemiluminescence (ECL) of N-(aminobutyl)-N-(ethylisoluminol) (ABEI). Since we were inspired by this discovery, an aptamer-based ECL sensor was fabricated based on a Hg(2+) triggered signal switch coupled with an exonuclease I (Exo I)-stimulated target recycling amplification strategy for ultrasensitive determination of Hg(2+) and mucin 1 (MUC1). Concretely, the ECL intensity of ABEI-functionalized silver nanoparticles decorated graphene oxide nanocomposite (GO-AgNPs-ABEI) was initially enhanced by ferrocene labeled ssDNA (Fc-S1) (first signal switch "on" state) in the existence of H2O2. With the aid of aptamer, assistant ssDNA (S2) and full thymine (T) bases ssDNA (S3) modified Au nanoparticles (AuNPs-S2-S3) were immobilized on the sensing surface through the hybridization reaction. Then, via the strong and stable T-Hg(2+)-T interaction, an abundance of Hg(2+) was successfully captured on the AuNPs-S2-S3 and effectively inhibited the ECL reaction of ABEI (signal switch "off" state). Finally, the signal switch "on" state was executed by utilizing MUC1 as an aptamer-specific target to bind aptamer, leading to the large decrease of the captured Hg(2+). To further improve the sensitivity of the aptasensor, Exo I was implemented to digest the binded aptamer, which resulted in the release of MUC1 for achieving target recycling with strong detectable ECL signal even in a low level of MUC1. By integrating the quenching effect of Hg(2+) to reduce the background signal and target recycling for signal amplification, this proposed ECL aptasensor was successfully used to detect Hg(2+) and MUC1 sensitively with a wide linear response.

Self-Enhanced Ultrasensitive Photoelectrochemical Biosensor Based on Nanocapsule Packaging Both Donor-Acceptor-Type Photoactive Material and Its Sensitizer.

In this work, a self-enhanced ultrasensitive photoelectrochemical (PEC) biosensor was established based on a functionalized nanocapsule packaging both donor-acceptor-type photoactive material and its sensitizer. The functionalized nanocapsule with self-enhanced PEC responses was achieved first by packaging both the donor-acceptor-type photoactive material (poly{4,8-bis[5-(2-ethylhexyl)thiophen-2-yl]benzo[1,2-b:4,5-b']dithiophene-2,6-diyl-alt-3-fluoro-2-[(2-ethylhexyl)carbonyl]thieno[3,4-b]thiophene-4,6-diyl}, PTB7-Th) and its sensitizer (nano-C60, fullerene) in poly(ethylene glycol) (PEG) to form a nanocapsule, which significantly enhanced PEC signal and stability of the PEC biosensor. Moreover, a quadratic enzymes-assisted target recycling amplification strategy was introduced to the system for ultrasensitive determination. Compared with other established PEC biosensors, our proposed self-enhanced approach showed higher effectivity, accuracy, sensitivity, and convenience without any addition of coreactant or sensitizers into the testing electrolyte for photocurrent amplification and performed excellent analytical properties for microRNA estimation down to femtomole level with microRNA-141 as a model. Additionally, the proposed PEC biosensor was employed for estimation of microRNA in different cancer cells and pharmacodynamic evaluation in cancer cells. This self-enhanced PEC strategy has laid the foundation for fabrication of simple, effective, and ultrasensitive PEC diagnostic devices, leading to the possibility for early diagnosis, timely stage estimation, and accurate prognosis judgment of disease.

Wavelength-resolved simultaneous photoelectrochemical bifunctional sensor on single interface: A newly in vitro approach for multiplexed DNA monitoring in cancer cells

Currently, the photoelectrochemical (PEC) strategies can just achieve single analyte detection on a single interface with limited detection efficiency. It is highly valuable but full of challenge to develop a PEC biosensor for multiple analytes evaluation on a single interface. For this point, the wavelength-selective photoactive materials, which could generate separated photocurrents under excitation lights with certain wavelengths, were mainly important to overcome this challenge. Herein, these wavelength-selective photoactive materials were successfully synthesized and served as signal indicators to construct a novel PEC biosensor for multiple analytes evaluation on a single interface for the first time. Moreover, an enzyme-assisted target recycling amplification strategy was introduced for ultrasensitive monitoring. As a result, the proposed PEC biosensor showed excellent analytical performance for both oral cancer (ORVOA 1) gene and p53 gene down to attomolar level. In addition, the fabricated PEC biosensor was employed to evaluate ORVOA 1 gene and p53 gene in Hela cells. This assay has laid the foundation for fabrication of simple, ultrasensitive and economical PEC diagnostic devices to detect multiple analytes in cells, which paved a new avenue for early diagnosis of cancer with higher efficiency and accuracy.

An autocatalytic DNA machine with autonomous target recycling and cascade circular exponential amplification for one-pot, isothermal and ultrasensitive nucleic acid detection.

An autonomous target recycling and cascade circular exponential amplification strategy was proposed to construct an autocatalytic DNA machine, which afforded one-pot, isothermal and ultrasensitive detection of nucleic acids. A low detection limit of 0.61 fM toward the target DNA with an excellent selectivity could be obtained.

Au nanoparticles fluorescence switch-mediated target recycling amplification strategy for sensitive nucleic acid detection

A sensitive strategy based on Au nanoparticles fluorescence switch-mediated target recycling amplification for nucleic acid detection.

Label-free electrochemical nucleic acid biosensing by tandem polymerization and cleavage-mediated cascade target recycling and DNAzyme amplification

Owing to the intrinsic importance of nucleic acid as bio-targets, the achievement of its simple and sensitive detection with high confidence is very essential for biological studies and diagnostic purposes. Herein, a label-free, isothermal, and ultrasensitive electrochemical detection of target DNA was developed by using a tandem polymerization and cleavage-mediated cascade target recycling and DNAzyme releasing amplification strategy. Upon sensing of the nucleic acid analyte for the assembled hairpin-like probe DNA on the electrode, the DNA polymerase guided the target recycling and simultaneously triggered the lambda exonuclease cleavage, accompanied by the cascade recycling of the released new complementary strand and the amplified liberation of the G-rich sequence of the HRP-mimicking DNAzyme. The electrocatalytic reduction of H2O2 by the generated hemin/G-quadruplex DNAzyme was used for the signal readout and further amplification toward target response. Such tandem functional operation by DNA polymerase, lambda exonuclease and DNAzyme endows the developed biosensor with a high sensitivity and also a high confidence. A low detection limit of 5fM with an excellent selectivity toward target DNA could be achieved. It also exhibits the distinct advantages of simplicity in probe design and biosensor fabrication, and label-free electrochemical detection, thus may offer a promising avenue for the applications in disease diagnosis and clinical biomedicine.

Fluorometric detection of mutant DNA oligonucleotide based on toehold strand displacement-driving target recycling strategy and exonuclease III-assisted suppression.

We describe here a fluorometric assay for sensitive detection of oligonucleotides, based on a target recycling amplification strategy driven by toehold-mediated strand displacement reaction and on exonuclease III (Exo Ш)-assisted fluorescence background suppression strategy. The network consists of a pair of partially complementary DNA hairpins (HP1 and HP2) with 3' overhang ends, between which the spontaneous hybridization is kinetically hindered by the stems. The target DNA is repeatedly used to trigger a recycling progress between the hairpins, generating numerous HP1-HP2 duplex complexes. Exo III was then employed to digest the double strand parts of the residual hairpins and the intermediate products. The fluorescent dye, SYBR Green I, binds to the double-strand DNA products and emits strong fluorescence to achieve sensitive detection of the target DNA with the detection limit of 5.34 pM. Moreover, this proposed strategy showed high discrimination efficiency towards target DNA against mismatched DNA and was successfully applied in the analysis of human serum sample.

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.

Amplified electrochemical detection of mecA gene in methicillin-resistant Staphylococcus aureus based on target recycling amplification and isothermal strand-displacement polymerization reaction

Rapid detection of drug-resistant bacteria can facilitate the early diagnosis of various infections and provide essential guidance for appropriate usage of antibiotics. An ultrasensitive electrochemical DNA (E-DNA) sensor based on isothermal strand-displacement polymerization reaction (ISDPR) for detection of mecA gene in methicillin-resistant Staphylococcus aureus (MRSA) was proposed in this study. Methylene blue (MB)-labeled hairpin probes were self-assembled on a gold electrode to confine MB molecules close to the electrode surface for efficient electron transfer. The hairpin probes were then hybridized with complementary target DNA and undergone conformational changes, which led to a decreased electrochemical response. The primers annealed with the opened stems of hairpin probes were extended by DNA polymerase, which in turn released the target DNA to trigger the next polymerization cycle. Finally, each target DNA underwent through many rounds, resulting in a mass of MB molecules moved away from the electrode surface, which generated significant amplified current suppression for mecA gene detection. With this target recycling amplification strategy, the proposed DNA biosensor could offer excellent analytical performance for the detection of mecA gene and provide a new electrochemical method for the early diagnosis of drug-resistant bacteria.

An exonuclease-assisted amplification electrochemical aptasensor for Hg(2+) detection based on hybridization chain reaction.

In this work, a novel electrochemical aptasensor was developed for Hg(2+) detection based on exonuclease-assisted target recycling and hybridization chain reaction (HCR) dual signal amplification strategy. The presence of Hg(2+) induced the T-rich DNA partly folded into duplex-like structure via the Hg(2+) mediated T-Hg(2+)-T base pairs, which triggered the activity of exonuclease III (Exo III). Exo III selectively digested the double-strand DNA containing multiple T-Hg(2+)-T base pairs from its 3'-end, the released Hg(2+) participated analyte recycle. With each digestion cycle, a digestion product named as help DNA was obtained, which acted as a linkage between the capture DNA and auxiliary DNA. The presence of help DNA and two auxiliary DNA collectively facilitated successful HCR process and formed long double-stranded DNA. [Ru(NH3)6](3+) was used as redox indicator, which electrostatically bound to the double strands and produced an electrochemical signal. Exo III-assisted target recycling and HCR dual amplification significantly improved the sensitivity for Hg(2+) with a detection limit of 0.12 pM (S/N=3). Furthermore, the proposed aptasensor had a promising potential for the application of Hg(2+) detection in real aquatic sample analysis.

A dual target-recycling amplification strategy for sensitive detection of microRNAs based on duplex-specific nuclease and catalytic hairpin assembly.

A dual target-recycling amplification strategy for sensitive detection of microRNAs based on duplex-specific nuclease and catalytic hairpin assembly was reported for the first time.