Target Recycling Signal Amplification Research Articles

Isocarbophos determination using a nanozyme-catalytic photoelectrochemical fuel cell-based aptasensor

In the present work, we developed a nanozyme-catalytic photoelectrochemical fuel cell (PNFC)-based aptasensor to determine isocarbophos in food samples, in which TNA/g-C3N4/ZIF-67 photoanode was prepared by in-situ assembly of ZIF-67 nanozyme with excellent glucose dehydrogenase-mimic performance on g-C3N4 modified TiO2 nanotube arrays (TNA/g-C3N4), ITO/Fe-N-C/cDNA/apt-SH cathode was prepared by successively assembling captured DNA (cDNA) and mercapto-modified anti-isocarbophos aptamer (apt-SH) onto the indium tin oxide conductive glass (ITO) coated with Fe-N-C nanozyme. Due to the specific recognition of isocarbophos by its aptamer and high output power of PNFC, the PNFC-based aptasensor could determine isocarbophos based on catalytic mercapto-inhibition effect and exonuclease I-assisted target recycling signal amplification, which had a quantitative range of 0.01–100 ng mL−1, low detection limit of 3.5 pg mL−1 and good selectivity for isocarbophos determination, being applied for real food sample analysis with good precision of the relative standard deviation less than 5.4% and good accuracy of the recoveries from 96.2% to 108.0%. What’s more, the portable PNFC-based aptasensor did not need additional energy supply and other targets could be selectively determined only by replacing cDNA and aptamer.

Confined catalysis of MOF-818 nanozyme and colorimetric aptasensing for cardiac troponin I

Understanding the catalytic performance of nanozymes assembled in confined environment is an interesting topic. Herein, a three-dimensional nanozyme-catalytic nanoreactor was constructed by confining MOF-818 nanozyme in the pore of macroporous tungsten trioxide (p-WO3). The catalytic activity of MOF-818 assembled in-situ for the oxidation of 3,5-Di-tert-butylcatechol (3,5-DTBC) could be regulated by changing the pore size of p-WO3. Only when being confined in the pores of p-WO3 with an appropriate pore size, MOF-818 could exhibit high affinity towards 3,5-DTBC, and excellent catalytic activity for 3,5-DTBC oxidation, the catalytic rate constant kcat and Michaelis constant Km were determined to be 31.47 s−1 and 1.42 mM, respectively, and the maximum yield of 3,5-DTBC oxidation reached 95.2%. Furthermore, the as-constructed nanozyme-catalytic nanoreactor could be designed to construct a colorimetric aptasensor for selective determining cardiac troponin I based on the enzymatic inhibition effect and the exonuclease I-assisted target recycling signal amplification, which exhibited a good linear range of 50 fg mL−1 - 100 ng mL−1, low detection limit of 18 fg mL−1, and was applied for human serum analysis with RSD less than 5.2% and the recoveries ranged from 95% to 107%.

Non-enzymatic signal amplification-powered point-of-care SERS sensor for rapid and ultra-sensitive assay of SARS-CoV-2 RNA

The development of rapid and ultra-sensitive detection technology of SARS-CoV-2 RNA for shortening the diagnostic window and achieving early detection of virus infections is a huge challenge to the efficient prevention and control of COVID-19. Herein, a novel ultra-sensitive surface-enhanced Raman spectroscopy (SERS) sensor powered by non-enzymatic signal amplification is proposed for rapid and reliable assay of SARS-CoV-2 RNA based on SERS-active silver nanorods (AgNRs) sensing chips and a specially designed smart unlocking-mediated target recycling signal amplification strategy. The SERS sensing was carried out by a one-pot hybridization of the lock probes (LPs), hairpin DNAs and SERS tags with SARS-CoV-2 RNA samples on an arrayed SERS sensing chip to achieve the recognition of SARS-CoV-2 RNA, the execution of nuclease-free unlocking-mediated target recycling signal amplification, and the combination of SERS tags to generate SERS signal. The SERS sensor for SARS-CoV-2 RNA can be achieved within 50 min with an ultra-high sensitivity low to 51.38 copies/mL, and has good selectivity in discriminating SARS-CoV-2 RNA against other respiratory viruses in representative clinical samples, which is well adapted for rapid, ultra-sensitive, multi-channel and point-of-care testing of viral nucleic acids, and is expected to achieve detection of SARS-CoV-2 infection in earlier detection windows for efficient COVID-19 prevention and control.

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.

Photoelectrochemical aptasensor for kanamycin determination based on exonuclease I-assisted target signal amplification and photoexcited electron transfer strategy

• Effective assembly of β-glucosidase with the design of capture DNA and hairpin DNA. • Recovery of photocurrent of TNA/g-C 3 N 4 due to catalytic hydrolysis of β-glucosidase. • Sensitive PEC aptasensing for kanamycin with Exo I-assisted recycling amplification. In the present work, graphitic carbon nitride (g-C 3 N 4 ) was generated in situ on the TiO 2 nanotube arrays (TNAs) to form TNA/g-C 3 N 4 , the capture DNA (c-DNA) was immobilized on the TNA/g-C 3 N 4 and the anti-kanamycin aptamer was assembled by the complementary base pairing to construct a TNA/g-C 3 N 4 /c-DNA-apatmer photoelectrochemical (PEC) aptasensor for kanamycin determination, which had obvious visible-light photocurrent response, and the photocurrent would decrease when immersing the aptasensor in Cu(II) solution due to the photoexcited electron transfer from g-C 3 N 4 to Cu(II). In the presence of kanamycin, the anti-kanamycin aptamer left and more β-glucosidase labeled at 3′-end of hairpin DNA was assembled on the TNA/g-C 3 N 4 with the assistance of recycling cleavage of exonuclease I (Exo I) to single-stranded aptamer. Because of the catalytic hydrolysis of linolenin by β-glucosidase, the released cyanide ion coordinated with Cu(II), hindering the photoexcited electron transfer from g-C 3 N 4 to Cu (II), and leading to the increase of photocurrent. Therefore, a simple, rapid and sensitive PEC aptasensor was constructed for kanamycin determination with good reproducibility and stability based on the Exo I-assisted target recycling signal amplification and photoexcited electron transfer strategy, in which the increased photocurrent value of the aptasensor was linear with the concentration of kanamycin in the range of 0.1–150 nM, and the limit of detection was 0.03 nM with the signal-to-noise ratio of 3, which has been successfully applied for the real food sample analysis with good precision of RSD less than 5.6% and good accuracy of the recoveries ranged from 90% to 120%.

A signal-on electrochemical DNA biosensor based on exonuclease III-assisted recycling amplification.

DNA electrochemical detection technology has attracted tremendous interest in recent years. However, a facile and sensitive method for the detection of the disease indicators or genes is still waiting. Herein, we constructed a signal-on electrochemical platform for detecting the manganese superoxide dismutase (MnSOD) gene by incorporating a redox electrochemical signal probe (methylene blue) and exonuclease III-assisted target recycling signal amplification strategy. The sensor was prepared by self-assembly of a capture DNA probe of thiol-modified on GCE with gold electrodeposition. In the presence of target DNA, the exonuclease III can cleave the duplexes formed by the target DNA and the redox-labeled hairpin probes, release the target DNA and produce a residual sequence. The target DNA can continue to hybridize with the hairpin probe for the next cycle of amplification. The residual sequence hybridized with the surface-immobilized capture probes on AuNPs-modified GCE to generate a significantly amplified redox current. In particular, the redox current value of the resultant sensor showed a linear relationship with MnSOD gene concentration in the range of 1-104 pM with the detection limit as low as 0.3 pM. Furthermore, the sensor has excellent specificity and can distinguish single-base mismatch from perfectly matched target DNA. The sensor is fast in operation, and simple in design for detecting different DNA sequences or DNA identification by selecting the appropriate probe sequence, thus shedding light on a good promising application when encountering disease outbreaks or for the early clinical diagnosis of gene-related diseases.

Plasmonic Ag nanocube enhanced SERS biosensor for sensitive detection of oral cancer DNA based on nicking endonuclease signal amplification and heated electrode

Surface-enhanced Raman scattering (SERS) biosensing has attracted great attention due to its multiplexed capability, high sensitivity, and rapid readout. In this study, plasmonic Ag nanocube (AgNC) enhanced SERS biosensing platform was proposed by coupling nicking endonuclease-assisted target recycling signal amplification with heated electrode for sensitive detection of DNA species related to oral cancer. SERS tags were manufactured by signal DNA (sDNA) conjugating onto the surfaces of AgNCs, and 4-mercaptobenzoic acid (4-MBA) assembled on the surfaces of AgNC/sDNA as a Raman reporter. The sharp feature of edges and corners of AgNCs with high electromagnetic field provided SER hot spots with high quality, and stronger SERS signal could be achieved. In addition, the activity of nicking endonuclease Nt.BstNBI could be greatly enhanced by elevating the temperature of the surface of the heated Au electrode (HAuE) during Nt.BstNBI-assisted target recycling amplification process, resulting in dramatic amplification of target DNA detection. All of these contributed to the high sensitivity of the developed SERS biosensor. A limit of detection (LOD) of 3.1 fM (S/N = 3) could be obtained with an electrode temperature of 45 °C. This proposed SERS biosensor was anticipated to act as a promising biosensing platform for early clinical diagnosis of oral cancer.

Nuclease-assisted target recycling signal amplification strategy for graphene quantum dot-based fluorescent detection of marine biotoxins.

Saxitoxin (STX) is a major marine toxin from shellfish, and it is responsible for paralytic shellfish poisoning (PSP). In this study, a highly sensitive and rapid aptamer assay was developed for STX detection by combining fluorescence resonance energy transfer (FRET) and nuclease-assisted target recycling signal amplification. The aptamer STX-41 conjugated with graphene quantum dots (GQDs) was adsorbed on magnetic reduced graphene oxide (MRGO) to establish a fluorescence quenching system. Then, the binding between STX and aptamer induced the desorption of GQD-aptamer from MRGO and the restoring of fluorescence for the fluorescent determination of STX. The digestion of the target bound aptamer by DNase I could release the target for recycling thus achieving signal amplification. Under the optimized conditions, the aptamer assay showed a wide detection range (0.1-100ng·mL-1), low detection limit (LOD of 0.035ng·mL-1), high specificity, good recovery (86.75-94.08% in STX-spiked clam samples) and repeatability (RSD of 4.27-7.34%). Combined with fluorescent detection technology, signal amplification technology, and magnetic separation technology, the proposed method can be used to detect STX in seafood products successfully.

Paper-Based Constant Potential Electrochemiluminescence Sensing Platform with Black Phosphorus as a Luminophore Enabled by a Perovskite Solar Cell.

Exploring efficient luminophores in the electrochemiluminescence (ECL) system is highly desired to pursue a sensitive ECL sensing platform. Herein, the black phosphorus nanosheets (BP NSs) with excellent ECL properties are investigated and serve as the luminophore with the coreactant of peroxydisulfate (S2O82-) solution. Moreover, owing to the overlapping of emission and absorbance spectra, effective resonance energy transfer (RET) is realized between the BP NSs and the introduced Au nanoparticles. In order to achieve the portable and miniaturized developing trends for the paper-based ECL sensing platform, a paper-based perovskite solar cell (PSC) device is designed to act as the power source to replace the commonly utilized expensive and cumbersome electrochemical workstation. Benefiting from that, a PSC driven paper-based constant potential ECL-RET sensing platform is constructed, thereby realizing sensitive microRNAs (miRNAs) detection. What's more, to attain the preferable analytical performance, the duplex-specific nuclease (DSN) is also introduced to assist the target recycling signal amplification strategy. Based on this, highly sensitive detection of miRNA-107 with a range from 0.1 pM to 15 nM is achieved by this designed sensing platform. Most importantly, this work not only pioneers a precedent for developing a high-sensitivity PSC triggered ECL sensing platform but also explores the application prospect of BP nanomaterial in the field of bioanalysis.

A novel signal‐on fluorescent aptasensor for ochratoxin A detection based on RecJf exonuclease‐induced signal amplification

AbstractThis article describes a simple and homogeneous fluorescent aptasensor for the detection of ochratoxin A (OTA). With its high specificity and simplicity; RecJf exonuclease is used to cleave DNA strand of the FAM‐aptamer/OTA complex and realize target recycling signal amplification. In order to avoid the loss of reaction system, magnetic beads (MBs) are added only once at the last experimental step. This proposed fluorescent aptasensor showed the higher sensitivity in the range of 0.1–100 ng/mL with LOD of 0.056 ng/mL, and the good selectivity against other interfering toxins. The feasibility of the prepared aptasensor was studied by detecting OTA in spiked liquor and cereal samples. The obtained average recoveries ranged from 92% to 115%. This study provides a promising application with convenience and rapidness in the aptasensor fabrication for food safety analysis.

An aptamer based fluorometric assay for amyloid-β oligomers using a metal-organic framework of typeRu@MIL-101(Al) and enzyme-assisted recycling.

Amyloid-beta (Aβ) oligomers causing neuron damage are regarded as potential therapeutic targets and diagnostic markers for Alzheimer's disease (AD). A homogeneous turn-on fluorometric aptasensor is described for Aβ oligomers. It is highly selective and non-invasive and based on (a) the use of a luminescent metal-organic framework carrying aptamer-modifiedAuNPs (L-MOF/Apt-Au) as tracking agent, and (b) enzyme-assisted target recycling signal amplification. The tracking agent doesnot emit fluoresce by fluorescence resonance energy transfer (FRET) between theluminescent MOF as donor and Apt-Au as the acceptor under the excitation wavelength of 466nm. When Aβ oligomers are added to the tracking agent solution, the Apt-Au on tracking agent can preferentially bind with Aβ oligomers and then be released. This turns the "off" signal of theluminescent MOF tracer to the"on" state. The enzyme (Rec Jf exonuclease) added into the supernatant further improves sensitivity due to enzyme-assisted target-recycling signal amplification. The assay has anexcellent linear response to Aβ oligomers from 1.0pM to 10nM, with a detection limit of 0.3pM. This homogeneous turn-on fluorometric method is expected to have potential and applications in clinical diagnosis. Graphical abstractSchematic representation of fluorometric assay for amyloid-β oligomers based on luminescence metal-organic framework nanocomposites as tracking agent with exonuclease-assisted target recycling.

Switchable DNA tweezer and G-quadruplex nanostructures for ultrasensitive voltammetric determination of the K-ras gene fragment.

Voltammetric detection of the K-ras gene fragment was accomplished through the combined application of (a) a switchable DNA nanostructure, (b) the use of hairpin probe and exonuclease III (Exo III)-assisted signal amplification, (c) a split G-quadruplex, and (d) by exploiting the redox activity of DNAzyme. Three assistant oligonucleotides were designed to construct a DNA tweezer on a gold electrode. It is in "open state" in the absence of K-ras DNA. Then, a hairpin probe was introduced, whose stem-loop structure can be opened through hybridization with the K-ras DNA. Exo III is added which hydrolyzes the complementary region of the hairpin sequence to release a single-stranded rest fragment. The ssDNA hybridizes with the DNA tweezer on the electrode which thereby is switched to the "closed state". This leads to the formation of G-quadruplex due to the shortened distance of the split G-quadruplex-forming sequences in the tweezer. The voltammetric signal of the G-quadruplex-hemin complex, with a peak near -0.3V vs. Ag/AgCl, is used as the signal output. Under the optimal conditions, the current response in differential pulse voltammetry (DPV) increases linearly with the concentration of K-ras DNA in the range of 0.01-1000 pM, and the detection limit is 2.4 fM. The assay can clearly discriminate K-ras DNA from a single-base mutation. The method has excellent selectivity and was applied to the determination of K-ras DNA in (spiked) serum samples. Graphical abstractSchematic representation ofa method for the determination of the K-ras gene fragment through a combination of switchable DNA tweezer, split G-quadruplex, and exonuclease III (ExoIII)-assisted target recycling signal amplification.

A highly sensitive and selective fluorescence biosensor for hepatitis C virus DNA detection based on δ-FeOOH and exonuclease III-assisted signal amplification

Developing the high selectivity and sensitivity strategy for nucleic acid detection is crucial for early diagnosis and therapy of diseases. In this work, a novel low back-ground fluorescent sensor platform for the detection of nucleic acid has been developed based on δ-FeOOH nanosheets integrating with exonuclease III-assisted target-recycling signal amplification. Because of the strong binding ability between the single-strand DNA (ssDNA) and the δ-FeOOH nanosheets, the dye-labeled ssDNA probe would be quenched by δ-FeOOH nanosheets through fluorescence resonance energy transfer (FRET). By using magnetic separate properties of δ-FeOOH, the background signal was separated from the sensor system, and the low background sensor system was obtained. After adding the target DNA, a double-strand DNA complex (dsDNA) would be formed between the target DNA and dye-labeled ssDNA probe. Then, the dye-labeled ssDNA probe in the dsDNA complex would be stepwise hydrolyzed into short fragments from 3′-terminus by Exonuclease III, and the fluorescence signal was recovered due to the weak bind affinity between the short fragments and δ-FeOOH nanosheets. By using the fluorescence quenching ability of δ-FeOOH nanosheets and enzyme-assisted target-recycling signal amplification, this strategy could show an excellent selectivity toward hepatitis C virus DNA with a low detection limit of 10 pM. By simply changing the dye-labeled ssDNA probe sequence, this sensing platform can be developed as a universal approach for the simple, sensitive, and selective detection of different target DNA.

Fluorometric determination of mercury(II) using positively charged gold nanoparticles, DNA-templated silver nanoclusters, T-Hg(II)-T interaction and exonuclease assisted signal amplification.

The authors describe a method for detection of Hg2+ by using positively charged gold nanoparticles ((+)AuNPs) as a quencher of the fluorescence of DNA-capped silver nanoclusters (DNA-AgNCs) which are negatively charged. In the presence of Hg2+, a DNA duplex is formed through T-Hg2+-T coordination chemistry. The duplex can be digested by exonuclease III to form smaller DNA fragments. This leads to the release of the AgNCs and the recovery of fluorescence, best measured at excitation/emission wavelengths of 460/530nm. The (+)AuNPs and Hg2+ are also released and can be reused for target recycling signal amplification. Based on these findings, a method is worked out for the determination of Hg2+ that works in the 5.0 pM to 10nM concentration range and has a detection limit as low as 2.3 pM. It is highly selective because of the highly specific formation of T-Hg2+-T bonds. Graphical abstract By using ultrastable and positively charged gold nanoparticles as fluorescence quenchers andexonuclease assisted signal amplification, a method is developed for the sensitive and selective detection of Hg2+ in water samples.

A fluorescent biosensor for cardiac biomarker myoglobin detection based on carbon dots and deoxyribonuclease I-aided target recycling signal amplification.

A sensitive biosensor using carbon dots and deoxyribonuclease I-aided target recycling signal amplification has been developed to detect myoglobin (MB), which is an important cardiac biomarker and plays a major role in the diagnosis of acute myocardial infarction (AMI). Here, in the absence of MB, the MB aptamer (Ap) is absorbed on the surface of carbon dots (CDs) through π–π stacking interactions, resulting in quenching of the fluorescent label by forming CD–aptamer complexes. Upon adding MB, the Ap sequences could be specifically recognized by MB, leading to the recovery of quenched fluorescence. Thus, quantitative evaluation of MB concentration has been achieved in a broad range from 50 pg mL−1 to 100 ng mL−1, and the detection limit is as low as 20 pg mL−1. This strategy is capable of specific and sensitive detection of MB in human serum, urine, and saliva and can be used for the diagnosis of AMI in the future.

A simple, supersensitive and highly selective electrochemical aptasensor for Microcystin-LR based on synergistic signal amplification strategy with graphene, DNase I enzyme and Au nanoparticles

As cyanobacteria with high potential hepatotoxicity, Microcystin-LR (MC-LR) in water sample is highly concerned, but is very hard to directly detect by convenient electrochemistry technique due to its lack of electrochemical activity. In this work, an effective signal-on electrochemical aptasensing platform towards MC-LR has been proposed by designing a synergistic signal amplification system subtly and simply controlled by graphene bi-functional assembly. MC-LR binding aptamers were combined onto graphene surface, obtaining aptamer-graphene complex, and used as recognition element. Au nanoparticles (NPs) were modified onto Au electrode (Au NPs/Au) to act as the probe substrate. When it was functionalized by alkylate thiol, the electron transfer (eT) on Au NPs/Au could be greatly blocked, which could only be recovered by free graphene, while not by aptamer-graphene complex. According to this property, graphene was herein used bi-functionally, serving as not only the binder to recognition element, aptamer, by π-π stacking, but also the eT tunnel regulator to give a concentration-dependent response “turn-on” signal to MC-LR. DNase I enzyme, which can selective cleavage the aptamers bound with MC-LR, was simultaneously added to the system to release MC-LR and to give additional target recycling signal amplification. At the optimized condition, the dose-response curve of MC-LR and the current signal was established with a wide linear range of 1.0–100 pM, and a low detection limit of 0.8 pM, better than many reported literature. Moreover, this aptasensor system shows good selectivity towards MC-LR in the presence of 100-fold other contaminants. The potential sensing principle and recognition mechanism were discussed. A promising and convenient monitoring platform for MC-LR and other organic pollutants has thus been provided.

Ultrasensitive fluorescent aptasensor for MUC1 detection based on deoxyribonuclease I-aided target recycling signal amplification.

A novel sensing strategy for sensitive detection of mucin 1 protein (MUC1) based on deoxyribonuclease I-aided target recycling signal amplification was proposed. In this paper, in the absence of MUC1, the MUC1 aptamer is absorbed on the surface of graphene oxide (GO) via π-stacking interactions. This results in quenching of the fluorescent label and no fluorescence signal is observed. Upon adding MUC1, the probe sequences could be specifically recognized by MUC1, leading to an increase in the fluorescence intensity. The detection limit is as low as 10 pg mL−1, and a linear range from 50 pg mL−1 to 100 ng mL−1. The assay is specific and sensitive, and successfully applied to the determination of MUC1 in spiked human serum, urine and saliva. Importantly, the proposed aptasensing strategy has great potential in detecting various protein and even cancer cells.

Aptamer-involved fluorescence amplification strategy facilitated by directional enzymatic hydrolysis for bioassays based on a metal-organic framework platform: Highly selective and sensitive determination of thrombin and oxytetracycline

The authors describe a fluorescence amplification strategy for selective and sensitive fluorescent assays based on aptamer-triggered directional hydrolysis and on the use of metal organic frameworks (MOFs) of type MIL-101. The method is implemented by mixing MIL-101, fluorescein-labeled DNA probes, exonuclease of type RecJf, and targets. A smart design of the three-adenine bulge on the DNA probe facilitates exonuclease-assisted directional hydrolysis, making the strategy universal for determination of both proteins and small molecules as well. Good selectivity is accomplished due to the use of MIL-101 protected aptamers, while high sensitivity resulted from exonuclease-assisted target-recycling signal amplification. The power of the method is demonstrated by analyzing the two model analytes thrombin (a fairly large protein) and oxytetracycline (OTC; a small molecule antibiotic). The limits of detection are 15 pM for thrombin and 4.2 nM for OTC. This is two orders of magnitude lower than that of conventional 1:1 homogeneous fluorescence assays. The strategy was successfully applied to the analysis of thrombin and OTC in real samples. In our perception, the strategy presented here has a wide scope for selective and sensitive detection of trace analytes for which appropriate DNA probes can be identified.

Nuclease-free target recycling signal amplification for ultrasensitive multiplexing DNA biosensing

Ultrasensitive biosensing technologies without gene amplification held great promise for direct detection of DNA. Herein we report a novel biosensing method, combining target recycling signal-amplification strategy and a homemade electrochemical device. Especially, the target recycling was achieved by a strand displacement process, no needing the help of any nucleases. In the presence of target DNA, the recycling system could be activated to generate a cascade of assembly steps with three hairpin DNA segments. Each recycling process were accompanied by a disassembly step that the last hairpin DNA segment displaces target DNA from the complex at the end of each circulation, freeing targets to activate the self-assembly of more trefoil DNA structures. This biosensing method could detect target DNA at aM level and can distinguish target DNA from interfering DNAs, demonstrating its high sensitivity and high selectivity. Importantly, the biosensing method could work well with serum samples.

Sensitive detection of microRNA in complex biological samples by using two stages DSN-assisted target recycling signal amplification method

MicroRNA (miRNA) has become an important biomarker candidate for cancer diagnosis, prognosis, and therapy. In this study, we have developed a novel fluorescence method for sensitive and specific miRNA detection via duplex specific nuclease (DSN) signal amplification and demonstrated its practical application in biological samples. Malachite green (MG) was employed as a “label-free" signal transducer since fluorescence of MG could be enhanced by 100-fold when MG were binding to a G-quadruplex structure formed within the d(G2T)13G sequence. The proposed signal amplification strategy is an integrated “biological circuit” designed to initiate a cascade of enzymatic reactions in order to detect, amplify, and measure a specific miRNA sequence by using the isothermal cleavage property of a DSN. The circuit is composed of two molecular switches operating in series: the amplification reaction activated by a specific miRNA and the strand-displacement polymerization reaction designed to initiate molecular beacon-assisted amplification and signal transduction by using MG/G-quadruplex complex. The hsa-miR-141 (miR141) was chosen as a target miRNA because its level specifically abnormal in a wide range of common human cancers including breast, lung, colon, and prostate cancer. The proposed method allowed quantitative sequence-specific detection of miR141 (with a detection limit of 1.03pM) in a dynamic range from 1pM to 10μM, with an excellent ability to discriminate differences in miRNAs. Moreover, the detection assay was applied to quantify miR141 in cancerous cell lysates. On the basis of these findings, we believe that this proposed sensitive and specific assay has great potential as a miRNA quantification method for use in biomedical research and clinical diagnosis.