Target Recycling Research Articles

Prussian blue nanoparticle-labeled aptasensing platform on graphene oxide for voltammetric detection of α-fetoprotein in hepatocellular carcinoma with target recycling.

An enzyme-free electrochemical aptasensing platform based on a graphene oxide nanosheet-modified gold-disk electrode was developed for the voltammetric detection of alpha-fetoprotein (AFP) in hepatocellular carcinoma by using a Prussian blue nanoparticle (PBNP)-labeled aptamer. The electroactive PBNP, a typical signal-generation tag, was utilized for the labeling of the aminated AFP aptamer by using covalent conjugation. The electrochemical sensing platform was prepared in a simple manner on the basis of a π-π stacking reaction between the immobilized graphene oxide and the PBNP-labeled AFP aptamer. Upon target AFP introduction, the analyte reacted with the aptamer, thus resulting in the dissociation of the PBNP from the nanosheets. In the presence of DNase I, the newly formed AFP/aptamer-PBNP complex was cleaved to release target AFP, which could react again with the aptamer on the nanosheets, thereby causing target recycling. During this process, the cleaved PBNP-aptamer was far away from the electrode to decrease the voltammetric signal. Under optimum conditions, the voltammetric peak current of the modified electrode decreased with the increment of the target AFP concentration within the linear range of 0.01-300 ng mL-1 at a low detection limit of 6.3 pg mL-1. The precision and reproducibility of the aptasensing protocol were acceptable (CV: <15% for intra-assay and inter-assay). Other possible nontarget biomarkers did not interfere significantly with the voltammetric signal of this system. Human serum samples containing target AFP were assayed with electrochemical aptasensing and a commercial human AFP ELISA kit, and gave well-matched results from these two methods. Importantly, our strategy provides a new horizon for the determination of disease-related proteins.

A novel fluorescent assay for the ultrasensitive detection of miRNA-21 with the use of G-quadruplex structures as an immobilization material for a signal indicator.

In this work, a novel fluorescent assay was proposed for the ultrasensitive detection of microRNA-21 (miRNA-21) based on the efficient immobilization of protoporphyrin IX (PPIX) as signal indicators in massive G-quadruplex structures obtained by target recycling, three-dimensional DNA walker and a rolling circle amplification (RCA) coupled cascade nucleic acid amplification strategy.

An enzyme-free FRET nanoprobe for ultrasensitive ketamine detection based on ATP-fueled target recycling.

Ketamine is a commonly abused drug due to its stimulant, dissociative and hallucinogenic effects. An overdose of ketamine has been found to cause a variety of side effects. Therefore, the identification and quantification of ketamine are of significant importance for clinical purposes and drug seizing. However, conventional methods for ketamine detection possess some disadvantages such as sophisticated procedures, expensive instruments and low sensitivity. Herein, we develop a novel fluorescent nanoprobe for ultrasensitive ketamine detection with signal amplification based on Adenosine Triphosphate (ATP)-fueled target recycling and FRET (fluorescence resonance energy transfer) occurring between the FAM (Fluorescein, tagged with Y-shape DNA) and AuNPs. Based on the combination of FRET and signals circle amplification, the gold nanospheres functionalized with Y-motif DNA (Y@AuNPs) nanoprobe was utilized for effective ketamine detection with the limit of detection (LOD) down to 3 pg mL−1, which was lower than previously reported. Furthermore, the high sensitivity of Y@AuNPs facilitated quantitative analysis in biological media and practical samples.

A ratiometric electrochemical biosensor for ultrasensitive and highly selective detection of the K-ras gene via exonuclease III-assisted target recycling and rolling circle amplification strategies

A ratiometric electrochemical biosensor for ultrasensitive and highly selective detection of the K-ras gene via Exo III-assisted target recycling and RCA strategies.

A design for the photoelectrochemical detection of miRNA-221 based on a tungsten diselenide-cysteine-dopamine nanoprobe coupled with mismatched catalytic hairpin assembly target recycling with ultra-low background noise.

A strategy for the photoelectrochemical detection of miRNA with ultra-low background noise was developed using tungsten diselenide-cysteine-dopamine (WSe2/Cys/DA) as a nanoprobe coupled with mismatched catalytic hairpin assembly target recycling. A superior detection limit of 3.3 aM toward miRNA-221 was achieved.

A split molecular beacon for specific identification of cancer-related single nucleotide polymorphism

A highly selective and sensitive split molecular beacon (SMB)-based SNP genotyping biosensing system was developed by combining the selectivity of ligation detection reaction (LDR) with the efficient signal amplification of target recycling.

MicroRNA detection based on duplex-specific nuclease-assisted target recycling and gold nanoparticle/graphene oxide nanocomposite-mediated electrocatalytic amplification

DNA technology based bio-responsive nanomaterials have been widely studied as promising tools for biomedical applications. Gold nanoparticles (AuNPs) and graphene oxide (GO) sheets are representative zero- and two-dimensional nanomaterials that have long been combined with DNA technology for point-of-care diagnostics. Herein, a cascade amplification system based on duplex-specific nuclease (DSN)-assisted target recycling and electrocatalytic water-splitting is demonstrated for the detection of microRNA. Target microRNAs can form DNA: RNA heteroduplexes with DNA probes on the surface of AuNPs, which can be hydrolyzed by DSN. MicroRNAs are preserved during the reaction and released into the suspension for the digestion of multiple DNA probes. After the DSN-based reaction, AuNPs are collected and mixed with GO to form AuNP/GO nanocomposite on an electrode for the following electrocatalytic amplification. The utilization of AuNP/GO nanocomposite offers large surface area, exceptional affinity to water molecules, and facilitated mass diffusion for the water-splitting reaction. For let-7b detection, the proposed biosensor achieved a limit detection of 1.5 fM in 80 min with a linear detection range of approximately four orders of magnitude. Moreover, it has the capability of discriminating non-target microRNAs containing even single-nucleotide mismatches, thus holding considerable potential for clinical diagnostics.

Multiplexed electrochemical aptasensor for antibiotics detection using metallic-encoded apoferritin probes and double stirring bars-assisted target recycling for signal amplification

Simultaneous and sensitive detection of various antibiotic residues in one sample is essential to evaluation of food safety status. Herein, a multiplexed electrochemical aptasensor for multiplex antibiotics detection, with kanamycin (KANA) and ampicillin (AMP) as representative analytes, was designed by using metal ions encoded apoferrtin probes and double stirring bars-assisted target recycling for signal amplification. The encoded probes were prepared by apoferritin loading Cd2+ and Pb2+ ions and labeling with duplex DNAs (aptamers corresponding to KANA and AMP hybrid with its complementary DNA sequence), respectively. In the presence of KANA and AMP, the targets can recurrently react with the probes on the bars, and then replace a lot of Apo-Mencoded signal tags into supernatant. The peak currents of Cd2+and Pb2+from the tags corresponding with the concentrations of KANA and AMP were detected by square wave voltammetry in one run. As a result, KANA and AMP can be detected simultaneously within the range from 0.05 pM to 50 nM. And the detection limits were 18 fM KANA and 15 fM AMP (S/N = 3). The assay was testified to detect KANA and AMP residues with consistent results of ELISA in samples, e.g. milks and fishes. The assay was highly-sensitive, selective, cost-effective and easy-to-operate due to Apo-M encoded probes with high loading capacity of signal source substances. Moreover, double stirring bar-assisted target recycling, which was enzyme-free and could overcome matrix interference, was fabricated for signal amplification. Thus, the assay showed potential advantages for sensitively screening of antibiotic residues in foods.

Building a Fluorescent Aptasensor Based on Exonuclease-Assisted Target Recycling Strategy for One-Step Detection of T-2 Toxin

In this work, a rapid and accurate assay was successfully developed for T-2 toxin detection based on exonuclease-catalyzed target recycling strategy. Upconversion nanoparticles (UCNPs) were conjugated with T-2 aptamer and used as signal probes, while magnetic nanoparticles (MNPs) were conjugated with the complementary DNA of T-2 aptamer (cDNA) and used as capture probes. The results reveled that good linear correlation (R2 = 0.9988) was achieved for T-2 toxin detection over the concentration range of 0.1–100 ng/mL with a detection limit as low as 0.035 ng/mL (S/N = 3). In addition, the reliability of the proposed method was also applied to the determination of T-2 toxin contents in real food samples and the average recoveries ranged from 95.97 to 104.00%. The sensing platform developed in our study demonstrated great potential for simple and sensitive detection of T-2 toxin contents in food samples.

DNA-Functionalized Porous Fe3O4 Nanoparticles for the Construction of Self-Powered miRNA Biosensor with Target Recycling Amplification.

Herein, we have developed an ultrasensitive self-powered biosensor for miRNA assay based on biofuel cells. The system is composed of indium tin oxide cathode and graphene oxide/gold nanoparticle/glucose oxidase anode. Redox probe of [Fe(CN)6]3- is entrapped inside porous Fe3O4 nanoparticles by DNA. However, in the presence of target miRNA, hybridization reaction occurs between miRNA and DNA, which initiates the release of [Fe(CN)6]3-. Moreover, duplex specific nuclease is further employed to trigger target recycling amplification. As a result, much more redox probes are released and the open circuit voltage is significantly increased. A "signal-on" self-powered biosensor for miRNA quantification is thus developed. The detection range is from 10 aM to 10 fM; meanwhile, the limit of detection is as low as 1.4 aM, which is superior to that in most reported methods. Therefore, the proposed biosensor is expected to be a powerful point-of-care tool for miRNA diagnostics, which may have wide applications in the future.

Affinity-Modulated Molecular Beacons on MoS2 Nanosheets for MicroRNA Detection.

DNA-functionalized layered two-dimensional transition-metal dichalcogenides have attracted tremendous interest for constructing biosensors in recent years. In this work, we report diblock molecular beacons with poly-cytosine (polyC) tails anchored on molybdenum disulfide (MoS2) nanosheets as probes for microRNA detection. The polyC block is adsorbed on MoS2 and the molecular beacon block is available for hybridization to the target; duplex-specific nuclease provides signal amplification by target recycling. By changing the length of polyC, we regulate the density of probes on MoS2 and inhibit the adsorption of enzyme-cleaved oligonucleotides, thereby leading to higher quenching efficiency. PolyC-mediated molecular beacons on MoS2 have very low background signal, ultrahigh sensitivity (limit of detection ∼3.4 fM), specificity to detect a single nucleotide mismatch, and selectivity to detect target microRNA from serum samples. This detection platform holds great potential for quantitative analysis of miRNAs in clinical diagnosis and biomedical research.

A fluorometric aptasensor for patulin based on the use of magnetized graphene oxide and DNase I-assisted target recycling amplification.

A fluorometric patulin (PAT)assayis presented that is based on theuse of magnetic reducedgrapheneoxide (rGO) and DNase I. The fluorescence of the PAT aptamer labelled with6-carboxyfluorescein (FAM) is quenched by magnetized reduced graphene oxide (rGO-Fe3O4) due to fluorescence resonance energy transfer (FRET). However, in the presence of PAT, the labelled aptamer is stripped offfrom rGO-Fe3O4. The rGO-Fe3O4 is then magnetically separated so that the fluorescence of freelabelled PAT aptameris restored. DNase I cannot hydrolyze the aptamer on rGO-Fe3O4, but it can cleave the free aptamer-PAT complex. This will release FAM and PAT which can undergo a number of additional cycles to trigger the cleavage of abundant aptamer. Recycling of DNase I-assisted target therefore leads to a strong amplification of fluorescence and consequently to an assay with low limit of detection. The detection limit for PAT is as low as 0.28μgL-1 which is about 13 times lower than that without using DNase I. The method offers a new approach towards rapid, sensitive and selective detection based on anaptamer. Conceivably, it has a wide scope in that it may be applied to numerous other analytes if appropriate aptamers are available. Abstract Schematic of a fluorometric assay based on theuse of magnetic grapheneoxide and DNase I. It was applied to the determination of patulin. DNase I was introduced for recycling amplification. The detection limit is about 13 times lower than that without usingDNase I. Figure a contains poor quality of text in image. Otherwise, please provide replacement figure file.Thank you. I will provide the figure file.

Low Background Cascade Signal Amplification Electrochemical Sensing Platform for Tumor-Related mRNA Quantification by Target-Activated Hybridization Chain Reaction and Electroactive Cargo Release.

Herein a low background cascade signal amplification electrochemical sensing platform has been proposed for the ultrasensitive detection of mRNA (mRNA) by coupling the target-activated hybridization chain reaction and electroactive cargo release from mesoporous silica nanocontainers (MSNs). In this sensing platform, the 5'-phosphate-terminated DNA (5'-PO4 cDNA) complement to target mRNA is hybridized with the trigger DNA and anchor DNA on the surface of the MSNs, aiming at forming a double-stranded DNA gate molecule and sealing the methylene blue (MB) in the inner pores of the MSNs. In the presence of target mRNA, the 5'-PO4 cDNA is displaced from the MSNs and competitively hybridizes with mRNA, which led to the liberation of the trigger DNA and the opening of the MSNs pore. The liberated trigger DNA can be then immobilized onto the electrode surface through hybridization with the capture DNA, triggering HCR on the electrode surface. At the same time, the MB released from the MSNs will selectively intercalate into the HCR long dsDNA polymers, giving rise to significant electrochemical response. In addition, due to the λ-exonuclease (λ-Exo) cleavage reaction-assisted target recycling, more amounts of trigger DNA will be liberated and trigger HCR, and numerous MB are uncapped and intercalate into the HCR products. As proof of concept, thymidine kinase 1 (TK1) mRNA was used as a model target. Featured with amplification efficiency, label-free capability, and low background signal, the strategy could quantitatively detect TK1 mRNA down to 2.0 aM with a linear calibration range from 0.1 fM to 1 pM. We have also demonstrated the practical application of our proposed sensing platform for detecting TK1 mRNA in real samples, opening up new avenues for highly sensitive quantification of biomarkers in bioanalysis and clinical diagnosis.

Electrochemical detection of tobramycin based on enzymes-assisted dual signal amplification by using a novel truncated aptamer with high affinity

An aptamer with the length of only 15 nucleotides specific for tobramycin was obtained through rationally designed truncation from a previously reported long sequence. The structural and binding properties of the aptamer were characterized. The dissociation constant (Kd) was determined to be 42.12 nM, indicating high affinity of the aptamer for tobramycin. Then an electrochemical sensor based on this aptamer was developed, which employed an enzymes-assisted dual signal amplification cycle through target recycling and strand-displacement DNA polymerization. A hairpin probe containing the aptamer sequence was designed and used to start the production cycle of a short ssDNA fragment in the presence of tobramycin, with the help of phi29 DNA polymerase and nicking endonuclease Nt.AlwI. The ssDNA fragment was captured by a signal transduction probe modified on gold electrode to form a triple-helix structure. With the help of [Ru(NH3)6]3+, a significant electrochemical signal was observed in differential pulse voltammetry (DPV). Under the optimal conditions, the current in DPV is linearly related with the concentration of tobramycin in the range of 10–200 nM, and the detection limit is 5.13 nM. The electrochemical sensor showed high specificity for tobramycin when it was challenged by other antibiotics. In addition, the constructed sensor was used to detect tobramycin in milk and water samples, and showed satisfactory performance. Therefore, the screened aptamer as well as the developed sensor has great application prospects in the fields of food safety control, medical test and environment monitoring.

Tungsten disulfide (WS2) nanosheet-based photoelectrochemical aptasensing of chloramphenicol.

A method is described for photoelectrochemical determination of chloramphenicol (CLOA). It is based on the use of (a) aptamers protected with photoactive WS2 nanosheets, and (b) DNase I-assisted target recycling. The DNA aptamer without label was employed for recognition of CLOA. In the absence of CLOA, the aptamer is adsorbed on the surface of WS2. This leads to a decrease of photocurrent due to the steric-hindrance effect of aptamer DNA. The adsorption of WS2 also protects the aptamer from digestion by DNase. In the presence of CLOA, the aptamer will be desorbed from the WS2 surface due to formation of an aptamer/CLOA conjugate. This results in an increased photocurrent due to a decreased amount of aptamer DNA on the electrode surface. The increase of photocurrent can be further improved by applying DNase triggered catalytic recycling of CLOA. Under optimal experimental conditions, the response is linear 10 pM - 10nM CLOA concentration range, with a 3.6 pM lower detection limit (at 3σ). This method is acceptably selective, accurate and stable. It was applied to the determination of CLOA in spiked milk samples and gave satisfactory results. Graphical abstract A simple and sensitive photoelectrochemical apta-biosensor was fabricated for chloramphenicol detection. In this work, WS2 nanosheets were employed as photoactive material, and DNase I catalytic chloramphenicol recycling strategy was adopted to amplify the detection signal.

MicroRNA Detection through DNAzyme-Mediated Disintegration of Magnetic Nanoparticle Assemblies.

DNA-assembled nanoparticle superstructures offer numerous bioresponsive properties that can be utilized for point-of-care diagnostics. Functional DNA sequences such as deoxyribozymes (DNAzymes) provide novel bioresponsive strategies and further extend the application of DNA-assembled nanoparticle superstructures. In this work, we describe a microRNA detection biosensor that combines magnetic nanoparticle (MNP) assemblies with DNAzyme-assisted target recycling. The DNA scaffolds of the MNP assemblies contain substrate sequences for DNAzyme and can form cleavage catalytic structures in the presence of target DNA or RNA sequences, leading to rupture of the scaffolds and disintegration of the MNP assemblies. The target sequences are preserved during the cleavage reaction and release into the suspension to trigger the digestion of multiple DNA scaffolds. The high local concentration of substrate sequences in the MNP assemblies reduces the diffusion time for target recycling. The concentration of released MNPs, which is proportional to the concentration of the target, can be quantified by a 405 nm laser-based optomagnetic sensor. For the detection of let-7b in 10% serum, after 1 h of isothermal reaction at 50 °C, we found a linear detection range between 10 pM and 100 nM with a limit of detection of 6 pM. For the quantification of DNA target in buffer solution, a limit of detection of 1.5 pM was achieved. Compared to protein enzyme-based microRNA detection methods, the proposed DNAzyme-based biosensor has an increased stability, a reduced cost and a possibility to be used in living cells, all of which are valuable features for biosensing applications.

Catalytic hairpin assembly-mediated surface charge density on the electrode for sensitive potentiometric detection of microRNA-21 in IgA-nephropathy

Methods based on electrochemical biosensors have been developed for detection of microRNA-21 (miRNA-21), but most involve complicated labeling or stripping procedures and are unsuitable for routine use in diagnosis/treatment of IgA-nephropathy. Herein we report on the proof-of-concept of simple and sensitive potentiometric DNA biosensor for specific detection of miRNA-21 coupling and catalytic hairpin assembly (CHA) with target recycling. This system consists of two hairpin DNA probes and one single-stranded DNA (capture probe). To decrease the background signal, the thiolated capture probe is covalently conjugated onto a cleaned gold electrode through typical Au-S bond. Upon target miRNA-21 introduction, the analyte opens hairpins DNA in turn to propagate the CHA reaction between two alternating hairpins accompanying the release of target miRNA-21 for recycling, thereby resulting in formation of numerous nicked double-helixes. Meanwhile, the newly produced CHA product hybridizes with the immobilized capture DNA probe on the electrode, thus causing the change in the surface charge density for potentiometric measurement. Under optimum conditions, CHA-based DNA biosensor presents good potentiometric responses for determination of target miRNA-21 at a level as low as 43 fM. An intermediate precision of <10% is accomplished with the batch-to-batch identification. The specificity and stability of CHA-based biosensor are satisfactory. Importantly, CHA-based potentiometric biosensor offers promise for the label-free, rapid, simple, cost-effective analysis of biological samples.

Duplex-Specific Nuclease-Amplified Detection of MicroRNA Using Compact Quantum Dot-DNA Conjugates.

Advances in nanotechnology have provided new opportunities for the design of next-generation nucleic acid biosensors and diagnostics. Indeed, combining advances in functional nanoparticles, DNA nanotechnology, and nuclease-enzyme-based amplification can give rise to new assays with advantageous properties. In this work, we developed a microRNA (miRNA) assay using bright fluorescent quantum dots (QDs), simple DNA probes, and the enzyme duplex-specific nuclease. We employed an isothermal target-recycling mechanism, where a single miRNA target triggers the cleavage of many DNA signal probes. The incorporation of DNA-functionalized QDs enabled a quantitative fluorescent readout, mediated by Förster resonance energy transfer (FRET)-based interaction with the DNA signal probes. Our approach splits the reaction in two, performing the enzyme-mediated amplification and QD-based detection steps separately such that each reaction could be optimized for performance of the active components. Target recycling gave ca. 3 orders of magnitude amplification, yielding highly sensitive detection with a limit of 42 fM (or 1.2 amol) of miR-148, with excellent selectivity versus mismatched sequences and other miRNAs. Furthermore, we used an alternative target (miR-21) and FRET pair for direct and absolute quantification of miR-21 in RNA extracts from human cancer and normal cell lines.

Novel 2D-DNA-Nanoprobe-Mediated Enzyme-Free-Target-Recycling Amplification for the Ultrasensitive Electrochemical Detection of MicroRNA.

In this work, on the basis of a new 2D DNA nanoprobe (DNP) and an enzyme-free-target-recycling amplification, an electrochemical biosensor is developed for the ultrasensitive detection of microRNA-21 (miRNA-21). Herein, two ferrocene-labeled bipedal DNPs, which show small steric hindrance and strong stability, are prepared on the basis of the mechanism of the proximity-ligation assay (PLA), improving the space utilization. In the presence of the target, miRNA-21, and a hairpin DNA strand, the DNP will collapse, and then two ferrocene-labeled DNA strands and the miRNA-21 will be simultaneously released from the electrode surface through toehold-mediated strand-displacement reactions (TSDRs), leading to a decrease in the electrochemical signal and realization of enzyme-free target recycling. As a result, the one input target, miRNA-21, could release 2 N ferrocene-labeled DNA strands, achieving a dramatic decrease in the electrochemical signal. Combining DNPs and enzyme-free target recycling, this proposed biosensor showed a linear dependence with miRNA-21 concentration, ranging from 1.0 fM to 10 nM with a detection limit of 0.31 fM. In addition, it is worth mentioning that this biosensor can be regenerated through incubating with three assistant-DNA strands, realizing the reuse of raw materials. Surprisingly, the elaborated biosensor provides a novel strategy for building controllable DNA nanoprobes for the sensitive detection of various biomarkers.

A microchip electrophoresis-based assay for ratiometric detection of kanamycin by R-shape probe and exonuclease-assisted signal amplification

Excessive intake of kanamycin (KANA) can cause some serious drug-resistant diseases, so it is urgent to develop some accurate and rapid analytical methods for monitor KANA residues in foodstuffs with complex matrix. Recently, many ratiometric assays were reported to be capable of overcoming matrix interference. Herein, a ratiometric and homogeneous assay for KANA detection based on microchip electrophoresis (MCE) was developed. First, by one single strand DNA (S-DNA) and one hairpin DNA (H-DNA), a novel R shape DNA probe (R-DNA) was prepared. After the probe was incubated with KANA, the S-DNA-KANA complex was formed, and H-DNA was released. Moreover, in the presence of exonuclease I (Exo-I), S-DNA-KANA complex would be digested to release the captured KANA for triggering target recycling and signal amplification. With the reaction going on, the fluorescence intensity of H-DNA (IH) increased and that of R-DNA (IR) decreased. They can be separated at different voltage intensities and converted to fluorescent signals for signal readout by MCE. The signal ratio of IH/IR was found to be linear toward target from 0.5 pg mL−1 to 10 ng mL−1, and the limit of detection was 150 fg mL−1. Moreover, it was successfully employed for KANA detection in milk and fish samples with consistent results of enzyme linked immune sorbent assay (ELISA). The R-DNA probe can quantitatively convert the amount of target to the intensity of DNA without label by MCE, and achieved exonuclease assisted signal amplification in homogenous solution. It was valuable to detect antibiotics residues in foodstuff with complex matrix. This approach broadened the application field of MCE to detect antibiotics without derivatization, which provided a promising platform for rapid screening of antibiotic residues in food.