The detection of specific intracellular microRNAs (miRNAs) in living cells can potentially provide insight into the causal mechanism of cancer metastasis and invasion. However, because of the characteristic nature of miRNAs in terms of small sizes, low abundance, and similarity among family members, it is a great challenge to monitor miRNAs in living cells, especially those with much lower expression levels. In this work, we describe the establishment of a DNA-fueled and catalytic molecule machinery in cell signal amplification approach for monitoring trace and under-expressed miRNAs in living cells. The presence of the target miRNA releases the hairpin sequences from the dsDNA (containing the fluorescence resonance energy transfer (FRET) pair-labeled and unfolded hairpin sequences)-conjugated gold nanoparticles (dsDNA-AuNPs), and the DNA fuel strands assist the recycling of the target miRNA sequences via two cascaded strand displacement reactions, leading to the operation of the molecular machine in a catalytic fashion and the release of many hairpin sequences. As a result, the liberated hairpin sequences restore the folded hairpin structures and bring the FRET pair into close proximity to generate significantly amplified signals for detecting trace miRNA targets. Besides, the dsDNA-AuNP nanoprobes have good nuclease stability and show low cytotoxicity to cells, and the application of such a molecular system for monitoring trace and under-expressed miRNAs in living cells has also been demonstrated. With the advantages of in cell signal amplification and reduced background noise, the developed method thus offers new opportunities for detecting various trace intracellular miRNA species.

The development of convenient and sensitive methods without involving any enzymes or complex nanomaterials for the monitoring of proteins is of great significance in disease diagnostics. In this work, we describe the validation of a new aptamer/protein proximity binding-triggered molecular machinery amplification strategy for sensitive electrochemical assay of thrombin in complex serum samples. The sensing interface is prepared by self-assembly of three-stranded DNA complexes on the gold electrode. The association of two distinct functional aptamers with different sites of thrombin triggers proximity binding-induced displacement of one of the short single-stranded DNAs (ssDNAs) from the surface-immobilized three-stranded DNA complexes, exposing a prelocked toehold domain to hybridize with a methylene blue (MB)-tagged fuel ssDNA strand (MB-DNA). Subsequent toehold-mediated strand displacement by the MB-DNA leads to the release and recycling of the aptamer/protein complexes and the function of the molecular machine. Eventually, a large number of MB-DNA strands are captured by the sensor surface, generating drastically amplified electrochemical responses from the MB tags for sensitive detection of thrombin. Our signal amplified sensor is completely enzyme-free and shows a dynamic range from 5 pM to 1 nM with a detection limit of 1.7 pM. Such sensor also has a high specificity for thrombin assay in serum samples. By changing the affinity probe pairs, the developed sensor can be readily expanded as a more general platform for sensitive detection of different types of proteins.

•Dam MTase-induced methylation of dsDNA probes inactivates endonuclease activity.•The protected dsDNAs inhibit the formation of supersandwich DNAzyme structures.•Label-free and amplified ECL detection of Dam MTase activity is developed.•The method shows high sensitivity and the capability to screen anti-cancer drugs.Sensitive detection of DNA methyltransferase (MTase) activity plays important roles for the diagnosis of many types of genetic disorder diseases and human malignancies. In this work, we describe the development of a label-free, sensitive and signal-on electrochemiluminescence (ECL) method for detecting MTase activity based on a restriction enzyme inactivation strategy. The sequence-specific double stranded DNA (dsDNA) probes on the sensing electrode can be cleaved by the restriction enzyme to initiate hybridization chain reaction (HCR) formation of the supersandwich DNAzyme structures, which catalyze the reduction and depletion of the dissolved oxygen to significantly quench the ECL of the oxygen/persulfate (O2/S2O82−) system. The methylation of the dsDNA probes by DNA adenine methyltransferase (Dam MTase) can inactivate the restriction enzyme activity and inhibit subsequent HCR formation of the DNAzymes, leading to recovery of the ECL signal for signal-on detection of Dam MTase in a dynamic range from 0.01 U mL−1 to 50 U mL−1 with the detection limit of 6.4 × 10−3 U mL−1. The inhibition of Dam MTase activity by different drugs is also evaluated, offering this method new opportunity for highly sensitive detection of Dam MTase activity and for screening potential drugs for cancer therapy as well.Download high-res image (96KB)Download full-size image

•CHA and TdT-mediated polymerization are integrated to achieve signal amplification.•Sensitive and label-free electrochemical detection of thrombin can be realized.•The sensor shows high selectivity and can be used to detect thrombin in human serums.Specific and sensitive detection of protein biomarkers is of great importance in biomedical and bioanalytical applications. In this work, a dual amplified signal enhancement approach based on the integration of catalytic hairpin assembly (CHA) and terminal deoxynucleotidyl transferase (TdT)-mediated in situ DNA polymerization has been developed for highly sensitive and label-free electrochemical detection of thrombin in human serums. The presence of the target thrombin leads to the unfolding and capture of a significant number of hairpin signal probes with free 3′-OH termini on the sensor electrode. Subsequently, TdT can catalyze the elongation of the signal probes and formation of many G-quadruplex sequence replicates with the presence of dGTP and dATP at a molar ratio of 6:4. These G-quadruplex sequences bind hemin and generate drastically amplified current response for sensitive detection of thrombin in a completely label-free fashion. The sensor shows a linear range of 0.5 pM–10.0 nM and a detection limit of 0.12 pM for thrombin. Moreover, the developed sensor can selectively discriminate the target thrombin against other non-target proteins and can be employed to monitor thrombin in human serum samples.

The monitoring of intracellular microRNAs plays important roles in elucidating the biological function and biogenesis of miRNAs in living cells. However, because of their sequence similarity, low abundance, and small size, it is a great challenge to detect intracellular miRNAs, especially for those with much lower expression levels. To address this issue, we have developed an in cell signal amplification approach for monitoring down-regulated miRNAs in living cells based on biodegradable MnO2 nanosheet-mediated and target-triggered assembly of hairpins. The MnO2 nanosheets can adsorb and exhibit an excellent quenching effect to the dye labeled hairpin probes. Besides, due to their biodegradability, the MnO2 nanosheets feature highly reduced cytotoxicity to the target cells. Upon entering cells, the surface-adsorbed FAM- and Tamra (TMR)-conjugated hairpins can be released due to the displacement reactions by other proteins or nucleic acids and the degradation of the MnO2 nanosheets by cellular GSH. Subsequently, the down-regulated target miRNA-21 triggers cascaded assembly of the two hairpins into long dsDNA polymers, which brings the fluorescence resonance energy transfer (FRET) pair, FAM (donor), and TMR (acceptor) into close proximity to generate significantly enhanced FRET signals for detecting trace miRNA-21 in living cells. By carefully tailoring the sequences of the hairpins, the developed method can offer new opportunities for monitoring various trace intracellular miRNA targets with low expression levels in living cells.Keywords: living cells; manganese oxide nanosheets; microRNA; self-assembly; signal amplification;

Based on an endonuclease-assisted, cross-triggered and cascaded recycling amplification strategy, the construction of a simple electrochemical sensing platform for the ultrasensitive detection of the mutant p53 gene in human serum is described. Using this new signal amplification approach, the sub-femtomolar level of the mutant p53 gene can be selectively detected.

Based on a new target-triggered aptamer molecular machine, a label-free and non-enzymatic target recycling amplification strategy for sensitive fluorescence detection of ATP in human serums is described. The presence of the target ATP together with the DNA fuel strand initiates the operation of the aptamer machine and leads to cyclic reuse of ATP and the release of many G-quadruplex sequences, which associate with a fluorescent dye to generate significantly amplified fluorescence signals to achieve sensitive detection of ATP.

The presence of target microRNA triggers cascaded and catalytic self-assembly of two DNA motifs into DNA nanostructures, which serves as a remarkable signal amplification means for the highly sensitive monitoring of the target microRNA and the detection of low numbers of tumor cells.

Despite the widespread utilization of gold nanoparticles and graphene for in vivo applications, complex steps for the preparation and functionalization of these nanomaterials are commonly required. In addition, the cytotoxicity of such materials is currently still under debate. In this work, by taking the significant advantages of DNA in terms of biocompatibility, nontoxicity, and controllability as building blocks for DNA nanostructures, we describe the construction of a reconfigurable, multicolor-encoded DNA nanostructure for multiplexed monitoring of intracellular microRNAs (miRNAs) in living cells. The DNA nanostructure nanoprobes containing two fluorescently quenched hairpins can be obtained by simple thermal annealing of four ssDNA oligonucleotides. The presence of the target miRNAs can unfold the hairpin structures and recover fluorescent emissions at distinct wavelengths to achieve multiplexed detection of miRNAs. Importantly, the DNA nanostructure nanoprobes exhibit significantly improved stability over conventional DNA molecular beacon probes in cell lysates and can steadily enter cells to realize simultaneous detection of two types of intracellular miRNAs. The demonstration of the self-assembled DNA nanostructures for intracellular sensing thus offers great potential application of these nanoprobes for imaging, drug delivery and cancer therapy in vivo.

Thrombin plays important roles for the diagnosis of neurodegenerative and cardiovascular diseases. By integrating proximity binding-induced strand displacement and metal ion-dependent DNAzyme recycling amplification, we demonstrate here the development of a simple and sensitive strategy for the detection of thrombin in human serums. The binding of the two distinct aptamers to the thrombin targets increases the local concentration of the aptamers and facilitates the release of the enzymatic sequences through proximity binding-induced strand displacement. The liberated enzymatic sequences further hybridize with the G-quadruplex containing and hairpin-structured substrate sequences on the sensor electrode to form the metal-ion dependent DNAzymes. Subsequently, the metal ions catalyze the cleavage of the substrate sequences to unlock the G-quadruplex forming sequences and to release the enzymatic sequences to trigger another cleavage cycle. Such metal ion-dependent DNAzyme recycling amplification leads to the formation of many active G-quadruplex forming sequences, which associate with hemin to form G-quadruplex/hemin complexes on the electrode surface. Direct electron transfer of hemin to the electrode during the potential scan can thus generate significantly amplified current for sensitive detection of thrombin at the low picomolar level. The work demonstrated here can thus offer new opportunities for the development of convenient signal amplification strategies for detecting various protein targets.

•Amplified and sensitive detection of microRNA from tumor cells is achieved.•Signal amplification is realized by two cascaded strand displacement reactions.•The developed sensor is selective and label-free without involving any enzymes.The monitoring of microRNA (miRNA) expression levels is of great importance in cancer diagnosis. In the present work, based on two cascaded toehold-mediated strand displacement reactions (TSDRs), we have developed a label- and enzyme-free target recycling signal amplification approach for sensitive electronic detection of miRNA-21 from human breast cancer cells. The junction probes containing the locked G-quadruplex forming sequences are self-assembled on the senor surface. The presence of the target miRNA-21 initiates the first TSDR and results in the disassembly of the junction probes and the release of the active G-quadruplex forming sequences. Subsequently, the DNA fuel strand triggers the second TSDR and leads to cyclic reuse of the target miRNA-21. The cascaded TSDRs thus generate many active G-quadruplex forming sequences on the sensor surface, which associate with hemin to produce significantly amplified current response for sensitive detection of miRNA-21 at 1.15 fM. The sensor is also selective and can be employed to monitor miRNA-21 from human breast cancer cells.

•DNAzyme nanostructures can be prepared by simple thermal annealing of ssDNAs.•The nanostructures are encoded with different colors.•The DNAzyme nanostructures exhibit improved stability in cell lysates.•Multiplexed imagining of intracellular metal ions in living cells can be realized.The detection of intracellular metal ions is of great importance in understanding metal homeostasis in cells and related diseases, and yet it remains a significant challenge to achieve this goal. Based on a new self-assembled and dual-color encoded DNAzyme nanostructure, we describe here an approach for multiplexed sensing of UO22+ and Pb2+ in living cells. The fluorescently quenched nanoprobes can be prepared by simple thermal annealing of four ssDNAs containing the metal ion-dependent enzymatic and substrate sequences. The self-assembly formation of the nanostructures are verified by native polyacrylamide gel electrophoresis. The target metal ions can cleave the substrate sequences in the DNAzyme nanostructures to recover fluorescent emissions at different wavelengths for sensitive and selective in vitro multiplexed detection of UO22+ and Pb2+ with the detection limits of 0.6 nM and 3.9 nM, respectively. Importantly, we demonstrate that these nanoprobes are stable in cell lysates and can enter cells without the aid of any transfection agents for simultaneous imaging intracellular UO22+ and Pb2+. Moreover, the nanoprobes offer excellent biocompatibility and non-cytotoxicity. With these unique features, the dual-color encoded nanostructures presented here can thus offer new opportunities for multiplexed detection of specific intracellular species.

•A new small molecule epitope-modified dsDNA is used as probe for the detection of antibody.•The binding of the target antibody to the dsDNA probe lowers its melting temperature.•Simple and sensitive electronic detection of antibodies in human serum can be realized.We describe here the development of a sensitive and convenient electronic sensor for the detection of antibodies in human serums. The sensor is constructed by self-assembly formation of a mixed monolayer containing the small molecule epitope conjugated double stranded DNA probes on gold electrode. The target antibody binds the epitope on the dsDNA probe and lowers the melting temperature of the duplex, which facilitates the displacement of the antibody-linked strand of the duplex probe by an invading methylene blue-tagged single stranded DNA (MB-ssDNA) through the strand displacement reaction and leads to the capture of many MB-ssDNA on the sensor surface. Subsequent electrochemical oxidation of the methylene blue labels results in amplified current response for sensitive monitoring of the antibodies. The antibody assay conditions are optimized and the sensor exhibits a linear range between 1.0 and 25.0 nM with a detection limit of 0.67 nM for the target antibody. The sensor is also selective and can be employed to detect the target antibodies in human serum samples. With the advantages of using small molecule epitope as the antibody recognition element over traditional antigen, the versatile manipulability of the DNA probes and the unique properties of the electrochemical transduction technique, the developed sensor thus hold great potential for simple and sensitive detection of different antibodies and other proteins in real samples.

The construction of DNA nanostructures with various sizes and shapes has significantly advanced during the past three decades, yet the application of these DNA nanostructures for solving real problems is still in the early stage. On the basis of microRNA-triggered, catalytic self-assembly formation of the functional “DNAzyme ferris wheel” nanostructures, we show here a new signal amplification platform for highly sensitive, label-free and non-enzyme colorimetric detection of a small number of human prostate cancer cells. The microRNA (miR-141), which is catalytically recycled and reused, triggers isothermal self-assembly of a pre-designed, G-quadruplex sequence containing hairpin DNAs into “DNAzyme ferris wheel”-like nanostructures (in association with hemin) with horseradish peroxidase mimicking activity. These DNAzyme nanostructures catalyze an intensified color transition of the probe solution for highly sensitive detection of miR-141 down to 0.5 pM with the naked eye, and the monitoring of as low as 283 human prostate cancer cells can also, theoretically, be achieved in a colorimetric approach. The work demonstrated here thus offers new opportunities for the construction of functional DNA nanostructures and for the application of these DNA nanostructures as an effective signal amplification means in the sensitive detection of nucleic acid biomarkers.

The construction of a restriction enzyme (Nt.AlwI)-powered DNA walking machine and its application for highly sensitive detection of DNA are described. DNA nanostructure tracks containing four overhang sequences with electrochemiluminescence (ECL) labels and complementary to the walker (target DNA) are self-assembled on the sensing electrode. The walker hybridizes with the complementary sequences on the tracks and forms specific recognition sites for Nt.AlwI, which cleaves the overhang sequences, releases the ECL labels and enables directional movement of the walker along the tracks. The formation of the nanostructure tracks and the Nt.AlwI-assisted cleavage of the overhang sequences in the presence of the walker are verified by using polyacrylamide gel electrophoresis analysis and cyclic voltammetry. The successive movement of the walker on the nanostructure tracks leads to continuous removal of massive ECL labels from the sensing electrode, which results in a significantly amplified suppression of the ECL emission for highly sensitive detection of sequence-specific DNA down to 0.19 pM. Results show that this DNA walking machine can also offer single-base mismatch discrimination capability. The successful application of the DNA walking machine for sequence-specific DNA detection can thus offer new opportunities for molecular machines in biosensing applications.

On the basis of the use of silver nanoclusters (AgNCs) in situ synthesized by cytosine (C)-rich loop DNA templates as signal amplification labels, the development of a label-free and highly sensitive method for electrochemical detection of microRNA (miRNA-199a) is described. The target miRNA-199a hybridizes with the partial dsDNA probes to initiate the target-assisted polymerization nicking reaction (TAPNR) amplification to produce massive intermediate sequences, which can be captured on the sensing electrode by the self-assembled DNA secondary probes. These surface-captured intermediate sequences further trigger the hybridization chain reaction (HCR) amplification to form dsDNA polymers with numerous C-rich loop DNA templates on the electrode surface. DNA-templated synthesis of AgNCs can be realized by subsequent incubation of the dsDNA polymer-modified electrode with AgNO3 and sodium borohydride. With this integrated TAPNR and HCR dual amplification strategy, the amount of in situ synthesized AgNCs is dramatically enhanced, leading to substantially amplified current response for highly sensitive detection of miRNA-199a down to 0.64 fM. In addition, the developed method also shows high selectivity toward the target miRNA-199a. Featured with high sensitivity and label-free capability, the proposed sensing scheme can thus offer new opportunities for achieving sensitive, selective, and simple detection of different types of microRNA targets.Keywords: electrochemical; label-free; MicroRNA; serum; signal amplification; silver nanoclusters

Catalytic self-assembly of DNA nanostructures triggered by microRNA 21 (miR-21) is achieved through isothermal toe-hold strand displacement reactions. The miR-21 is autonomously recycled during the self-assembly process, which makes the generation of the DNA nanostructures proceed in a catalytic fashion. The miR-21-triggered self-assembly of DNA nanostructures can also serve as a remarkable signal amplification platform to achieve ultrasensitive detection of miR-21 from as low as 10 MCF-7 human breast cancer cells.

Chemically reduced Cu2+ triggers the ligation of alkynyl- and azido-modified DNA via click chemistry. Subsequently, the ligated DNA initiates cyclic assembly of two fluorescently quenched hairpin DNAs and generates significantly amplified fluorescence signals for highly sensitive detection of Cu2+ in human serum samples.

The presence of the microRNA-141 target molecules activates the DNA molecular machine powered by the DNA fuel strands, leading to non-enzymatic target cyclic reuse of microRNA-141 and significantly amplified fluorescent signals for sensitive monitoring of microRNA-141 from low numbers of human prostate cancer cells.

The development of electronic sensors with minimized usage of reagents and washing steps in the sensing protocols will significantly facilitate the detection of biomolecules. In this work, by using a new pseudoknot design of the aptamer probes, the construction of an electronic sensor for reagentless and single-step detection of immunoglobulin E (IgE) in human serum is described. The pseudoknot aptamer probes are self-assembled on the disposable electrode surface. The association of IgE with the aptamer probes leads to conformational changes of the pseudoknot aptamer structures and brings the redox-tags in close proximity to the electrode, resulting in amplified current response for monitoring IgE. The effects of the pseudoknot structure and the immobilization concentration of the aptamer probes on the sensor performance are evaluated. Under optimal conditions, the detection limit for IgE is estimated to be 60 pM. The sensor is also selective and can be employed to detect IgE in human serum samples. The developed sensor can achieve reagentless, washing-free and low-cost (with the disposable electrode) electrochemical detection of proteins, making this device a convenient sensing platform for the monitoring of different biomarkers when coupled with the appropriate aptamer probes.

The variations in microRNA (miRNA) expression levels can be useful biomarkers for the diagnosis of different cancers. In this work, on the basis of a new miRNA-triggered molecular machine for enzyme-free target recycling signal amplification, the development of a simple electronic sensor for highly sensitive detection of miRNA-21 from human breast cancer cells is described. The three-stand DNA duplex probes are self-assembled on the gold electrode surface to fabricate the sensor. The miRNA-21 target binds to the terminal toehold region of the probes, displaces one of the short strands through toehold-mediated strand displacement reactions, and exposes the secondary toehold region for subsequent hybridization with the methylene blue (MB)-modified DNA fuel strand, which further displaces both the miRNA-21 target and the other short strand to activate the operation of the molecular machine. As a result, the miRNA-21 target is cyclically reused, and many MB-DNA fuel strands are attached to the sensor surface, leading to a significantly amplified current response for sensitive detection of miRNA-21 down to 1.4 fM. The developed sensor also shows high sequence discrimination capability and can be used to monitor miRNA-21 expression levels in cancer cells. Moreover, this sensor avoids the involvement of any enzymes for target recycling amplification and features with highly minimized background noise for miRNA detection, which makes this method hold great potential for convenient monitoring of different miRNA biomarkers for early diagnosis of various cancers.

•A new class of signal amplification tag is employed to simple detect MicroRNA and can be applied for real sample analysis.•The signal acquisition way by QDs intercalation into the RNA/DNA hybrids can overcome the drawbacks of labeling and embedding.•The method shows high selectivity toward the target microRNA and can be applied for real sample analysis.Herein, we report on the development of a simple and sensitive biosensor for electrochemiluminescent (ECL) detection of microRNAs (miRNA) based on the intercalation of doxorubicin-conjugated quantum dot nanoparticles (Dox-QDs) into the DNA/RNA hybrids as the new signal acquisition and amplification platform. The thiolated DNA capture probes are self-assembled onto gold electrodes via   the formation of Au–S bonds. The sensing surface is then incubated in a target miRNA-containing buffer solution to form the double-stranded duplexes. In this case, massive Dox-QDs can intercalate into the base pairs of the hybrid duplexes, resulting in amplified ECL emissions due to their reactions with the coreactant S2O82−S2O82− and the dissolved oxygen in the detection buffer. The increase in ECL intensity proportional to the amount of target miRNA in the testing samples serves as the quantitative basis. Different from traditional QDs-based methods such as labeling and embedding, our sensor involves the employment of the intercalation of the Dox-QDs as the signal acquisition and amplification platform. The combination of the QDs intercalation amplification with the high sensitivity of the ECL technique enables us to detect miRNA down to the low femtomolar level. Moreover, our method is also coupled with acceptable selectivity in discriminating the target miRNA and against its family members as well as other interference sequence, and can monitor miRNAs from human prostate carcinoma (22Rv1) cell lysates.

By coupling target DNA-induced reconfiguration of the dsDNA probes with enzyme-assisted target recycling amplification, we describe the development of a signal-on electrochemical sensing approach for sensitive detection of hereditary tyrosinemia type I gene. The dsDNA probes are self-assembled on the sensing electrode, and the addition of the target DNA reconfigures and switches the dsDNA probes into active substrates for exonuclease III, which catalytically digests the probes and leads to cyclic reuse of the target DNA. The target DNA recycling and the removal of one of the ssDNA from the dsDNA probes by exonuclease III result in the formation of many hairpin structures on the sensor surface, which brings the electroactive methylene blue labels into proximity with the electrode and produces a significantly amplified current response for sensitive detection of the target gene down to 0.24 pM. This method is also selective to discriminate single-base mismatch and can be employed to detect the target gene in human serum samples. With the demonstration for the detection of the target gene, we expect the developed method to be a universal sensitive sensing platform for the detection of different nucleic acid sequences.

Small molecule/protein interactions have a key role in drug discovery, clinic diagnosis and protein–metabolite interactions in biology. By using the specific interaction between folic acid (FA) and folate receptor (FR) as a model, the development of a label-free and sensitive colorimetric approach for the detection of the FR biomarker is described. The sensing approach relies on the coupling of the FR-induced terminal protection of FA-linked ssDNA strategy with significant signal amplification by self-assembled DNAzyme polymers. The FR binds to the FA-ssDNA and protects the FR/FA-ssDNA from digesting by exonuclease I. The terminal protected ssDNA further triggers autonomous self-assembly of two G-quadruplex sequence-containing hairpin DNAs into DNAzyme polymers, which result in intensified color change of the probe solution for label-free and highly sensitive colorimetric detection of FR. The terminal protection mechanism and the self-assembly formation of the DNAzyme polymers are characterized by using polyacrylamide gel electrophoresis, and the sensing parameters are optimized as well. Under optimal experimental conditions, the detection limit of 0.35 pM for FR can be obtained by using a UV-Vis spectrophotometer and the presence of as low as 5 pM of FR can be directly visualized by the naked eye. The developed method is also selective and can be applied to detect FR in serum samples, which makes this approach a sensitive platform for sensing different types of small molecule/protein interactions.

•Label-free and homogeneous detection of PDGF-BB is realized.•Detection signal is based on aptamer proximity binding formation of G-quadruplexes.•Sensitive and selective detection of PDGF-BB at low nanomolar level is achieved.•This assay can be used to monitor PDGF-BB in diluted human serums.By using the aptamer proximity binding assay strategy, the development of a label-free and homogeneous approach for fluorescent detection of human platelet-derived growth factor BB (PDGF-BB) is described. Two G-quadruplex forming sequence-linked aptamers bind to the PDGF-BB proteins, which leads to the increase in local concentration of the aptamers and promotes the formation of the G-quadruplex structures. Subsequently, the fluorescent dye, N-methylmesoporphyrin IX, binds to these G-quadruplex structures and generates enhanced fluorescence emission signal for sensitive detection of PDGF-BB. The association of the aptamers to the PDGF-BB proteins is characterized by using native polyacrylamide gel electrophoresis. The experimental conditions are optimized to reach an estimated detection limit of 3.2 nM for PDGF-BB. The developed method is also selective and can be applied for monitoring PDGF-BB in human serum samples. With the advantages of label-free and homogeneous detection, the demonstrated approach can be potentially employed to detect other biomarkers in a relatively simple way.

•Non-enzyme and label-free fluorescent detection of thrombin at the low picomolar level is realized.•Signal amplification is achieved by toehold strand displacement-driven assembly of multiple G-quadruplex DNA.•The binding of the fluorescent dye to the G-quadruplex DNA generates significantly amplified signal output.Based on a new signal amplification strategy by the toehold strand displacement-driven cyclic assembly of G-quadruplex DNA, the development of an enzyme-free and non-label aptamer sensing approach for sensitive fluorescent detection of thrombin is described. The target thrombin associates with the corresponding aptamer of the partial dsDNA probes and liberates single stranded initiation sequences, which trigger the toehold strand displacement assembly of two G-quadruplex containing hairpin DNAs. This toehold strand displacement reaction leads to the cyclic reuse of the initiation sequences and the production of DNA assemblies with numerous G-quadruplex structures. The fluorescent dye, N-Methyl mesoporphyrin IX, binds to these G-quadruplex structures and generates significantly amplified fluorescent signals to achieve highly sensitive detection of thrombin down to 5 pM. Besides, this method shows high selectivity towards the target thrombin against other control proteins. The developed thrombin sensing method herein avoids the modification of the probes and the involvement of any enzyme or nanomaterial labels for signal amplification. With the successful demonstration for thrombin detection, our approach can be easily adopted to monitor other target molecules in a simple, low-cost, sensitive and selective way by choosing appropriate aptamer/ligand pairs.

•RNA-regulated “on-and-off” switching of DNA tweezers are demonstrated.•The constructed DNA tweezers can achieve sensitive fluorescent detection of microRNA-141.•The DNA tweezer sensor is selective and can be employed to monitor microRNA-141 from cancer cell lysate.We describe here the construction of the DNA self-assembled molecular tweezers and the application of the tweezers for the monitoring of microRNA (miR-141) from human prostate cancer cells. The self-assembly formation of the DNA tweezers and the regulation of the tweezers upon alternative addition of the fuel miR-141 and the anti-fuel strands are characterized by native polyacrylamide gel electrophoresis. The addition of miR-141 to the DNA tweezers turns “off” the tweezers, while subsequent introduction of the anti-fuel strands switches the tweezers back to the “on” state, which verifies the regulatory ability of the tweezers. The miR-141-regulated DNA tweezers are concentration dependent and can be employed for sensitive detection of miR-141 down to 0.6 pM. The DNA tweezers also show high selectivity toward the fuel strand and can be used to monitor miR-141 expression in cancer cells, which provides new opportunities for the application of the dynamic DNA devices in clinical diagnostics.

The self-assembled DNA nanostructure has been one of the most interesting research areas in the field of nanoscience, and the application of the DNA self-assembled nanostructures in biosensing is still in the early stage. In this work, based on the target-induced autonomous disassembly of the aptamer–DNAzyme supersandwich nanostructures, we demonstrated a highly sensitive strategy for electrochemiluminescent (ECL) detection of ochratoxin A (OTA). The aptamer–DNAzyme supersandwich nanostructures, which exhibited significant ECL quenching effect toward the oxygen/persulfate (O2/S2O82−) system, were self-assembled on the gold electrode surface. The presence of the target OTA and the exonuclease (RecJf) resulted in autonomous disassembly of the nanostructures and cyclic reuse of OTA, leading to efficient recovery of the ECL emission and highly sensitive detection of OTA. Our developed method also showed high selectivity against other interference molecules and can be applied for the detection of OTA in real red wine samples, which offers the proposed method opportunities for designing new DNA-based nanostructures for biosensing applications.

The association of the target thrombin with the corresponding aptamer leads to structure switching of the dsDNA probes and the formation of nicking sites for exonuclease III, which causes cyclic cleavage of the dsDNA probes and highly reduced intercalation of the electrochemiluminescent signal indicators for label-free and sensitive detection of thrombin at the femtomolar level.

The employment of DNAzyme probes for visual biodetections has received increasing interest recently due to the simple nature of this type of assay. However, achieving high sensitivity and detecting targets beyond nucleic acids remain two major challenges in DNAzyme-based visual detections. In this work, based on a new quadratic amplification strategy, we developed a sensitive and visual detection method for cytokines by using hairpin aptamer DNAzyme probes. The target cytokine, interferon γ (IFN-γ), associates with the aptamer sequences and unfolds the hairpin structure of the probes, leading to simultaneous recycling of the target IFN-γ (assisted by Bst-polymerase) and the DNA sequences (aided by λ exonuclease) to achieve quadratic amplification. This quadratic amplification results in the generation of numerous peroxidase-mimicking DNAzymes, which cause significantly intensified color change of the probe solution for highly sensitive detection of IFN-γ by the naked eye down to 50 pM. The proposed visual sensing method shows also high selectivity toward the target IFN-γ and can be performed in homogeneous solutions with using completely unmodified, synthetic aptamer DNAzyme probes. These distinct advantages of our developed assay protocol make it a potential platform for detecting various types of biomolecules with careful probe designs.

The detection of microRNA expression profiles plays an important role in early diagnosis of different cancers. On the basis of the employment of redox labels with distinct potential positions and duplex specific nuclease (DSN)-assisted target recycling signal amplifications, we have developed a multiplexed and convenient electronic sensor for highly sensitive detection of microRNA (miRNA)-141 and miRNA-21. The sensor is constructed by self-assembly of thiol-modified, redox species-labeled hairpin probes on the gold sensing electrode. The hybridizations between the target miRNAs and the surface-immobilized probes lead to the formation of RNA/DNA duplexes, and DSN subsequently cleaves the redox-labeled hairpin probes of the RNA/DNA duplexes to recycle the target miRNAs and to generate significantly amplified current suppression at different potentials for multiplexed detection of miRNA-141 and miRNA-21 down to 4.2 and 3.0 fM, respectively. The sensor is also highly selective toward the target miRNAs and can be employed to monitor miRNAs from human prostate carcinoma (22Rv1) and breast cancer (MCF-7) cell lysates simultaneously. The sensor reported here thus holds great potential for the development of multiplexed, sensitive, selective, and simple sensing platforms for simultaneous detection of a variety of miRNA biomarkers for clinic applications with careful selection of the labels.

•Completely un-modified aptamers and DNAzymes are employed as probes.•The colorimetric signal output is intensified by Exo III-assisted ATP recycling.•Selective and sensitive colorimetric detection of ATP at sub-nanomolar is achieved.Based on target recycling amplification, the development of a new label-free, simple and sensitive colorimetric detection method for ATP by using un-modified aptamers and DNAzymes is described. The association of the model target molecules (ATP) with the corresponding aptamers of the dsDNA probes leads to the release of the G-quadruplex sequences. The ATP-bound aptamers can be further degraded by Exonuclease III to release ATP, which can again bind the aptamers of the dsDNA probes to initiate the target recycling amplification process. Due to this target recycling amplification, the amount of the released G-quadruplex sequences is significantly enhanced. Subsequently, these G-quadruplex sequences bind hemin to form numerous peroxidase mimicking DNAzymes, which cause substantially intensified color change of the probe solution for highly sensitive colorimetric detection of ATP down to the sub-nanomolar (0.33 nM) level. Our method is highly selective toward ATP against other control molecules and can be performed in one single homogeneous solution, which makes our sensing approach hold great potential for sensitive colorimetric detection of other small molecules and proteins.

In this work, by integrating multiple signal enhancement approaches, a new cascade signal amplification strategy is described to achieve highly sensitive electrochemical DNA detection. The presence of the target DNA leads to the unfolding of the biotin-modified hairpin probes on the sensor surface. With the addition of the primer sequences and polymerase, the target DNA is recycled and reused through isothermal strand-displacement polymerase reactions (ISDPR) to unfold a large number of the probes, which offer numerous binding sites to capture alkaline phosphatase (ALP)-loaded nanoparticle labels. These surface captured ALP enzymes catalyze the conversion of p-aminophenylphosphate to p-aminophenol, which generates amplified catalytic current responses due to the redox-recycling process during the potential sweep in the presence of the co-reactant NADH. With the synergistic signal amplifications by ISDPR-assisted target recycling, multi-ALP enzyme labels and redox-recycling, the proposed method offers highly sensitive detection of DNA down to 0.1 fM with single-base discrimination capability. Due to the significantly high sensitivity, the developed cascade signal amplification strategy can be potentially extended to detect various DNA targets at ultralow levels for early diagnoses of different diseases.

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.

•A label-free approach for fluorescent detection of folate receptor is established.•The detection mechanism is based on terminal protection of the folate-linked ssDNA.•SYBR Gold fluorescent dye is used as the signal indicator.•Selective and sensitive detection of 30 pM folate receptor is achieved.In this work, based on terminal protection of folate-linked ssDNA (FA-ssDNA) and the SYBR Gold fluorescent dye, we describe the development of a label-free fluorescent strategy for the detection of folate receptors (FRs). The binding between the target FR and the FA moiety of the FA-ssDNA protects the FR bound FA-ssDNA from digesting by Exo I. The binding of SYBR Gold to the terminal protected, un-digested FA-ssDNA leads to enhanced fluorescent emission for the monitoring of FR with a detection limit of 30 pM. Besides, the developed method also shows high selectivity toward FR against other control proteins. Moreover, our approach avoids the labeling of the probes with fluorescent tags and achieves label-free detection of FR. With these advantages, the proposed method thus holds promising potential for the development of simple and convenient strategies for the detection of other proteins by using different small molecule receptor/protein ligand pairs.

•Sensitive and visual detection of HIV DNA down to the low picomolar level is realized by a new quadratic signal amplification strategy.•Quadratic signal amplification is achieved by two exonuclease III-assisted DNA recycling cycles.•The employment of the un-modified, G-quadruplex sequence-integrated hairpin DNA probes leads to label-free detection of HIV DNA.•The proposed method can be applied for visual detection of HIV DNA in diluted serum samples.Visual detections have attracted great research attentions recently due to their convenient monitoring of the target analytes without using any advanced instruments. However, achieving visual detection of trace amounts of biomolecules with PCR-like sensitivity remains a major challenge. In current work, we describe a new quadratic signal amplification strategy for sensitive visual detection of HIV DNA biomarkers based on exonuclease III (Exo III)-assisted DNA recycling amplification and DNAzymes. The presence of the target HIV DNA leads to two independent and simultaneous DNA recycling processes to achieve quadratic signal amplification with the assistance of Exo III. This quadratic signal amplification results in catalytic cleavage of the G-quadruplex sequence-locked hairpin probes to release numerous active G-quadruplex sequences, which further associate with hemin to form DNAzymes and cause significantly intensified color change for sensitive and visual detection of HIV DNA down to 2.5 pM. The proposed visual detection method employs un-modified hairpin DNA as probes, avoids using any complex and expensive instruments for signal transduction and is essentially simple. This method also shows single-base mismatch discrimination capability as well. All these features make our developed DNA detection method holds great potential for visual monitoring of various DNA biomarkers at ultralow levels with careful and proper probe designs.

•Target recycling and RCA are integrated into one assay protocol to achieve dual signal amplification.•The hybrid, dual signal amplification strategy leads to the generation of numerous G-quadruplex/hemin complexes.•Direct electron transfer from hemin to the sensing electrode results in significantly amplified analytical current response.•The proposed method offers excellent single-base mismatch discrimination capability.Based on nicking endonuclease (NEase)-assisted target recycling and rolling circle amplification (RCA) for in situ generation of numerous G-quadruplex/hemin complexes, we developed a new, dual amplified and ultrasensitive electrochemical biosensor for mutant human p53 gene. The target mutant DNA hybridizes with the loop portion of a dithiol-modified hairpin probe (HP) self-assembled on a gold sensing electrode and forms nicking site for the NEase, which cleaves the HP and releases the target DNA. The released target DNA again hybridizes with the intact HP and initiates the DNA recycling process with the assistance of the NEase, leading to the cleavage of a large number of the HPs and the generation of numerous primers for RCA. With rationally designed, G-quadruplex complementary sequence-encoded RCA circular template, subsequent RCA results in the formation of long DNA sequences with massive tandem-repeat G-quadruplex sequences, which further associate with hemin and generate significantly amplified current response for highly sensitive DNA detection down to 0.25 fM. The developed method also exhibits high specificity for the target DNA against single-base mismatched sequence. With the ultrahigh sensitivity feature induced by the dual signal amplification, the proposed method can thus offer new opportunities for the detection of trace amounts of DNA.

•Un-modified hairpin aptamer probes and SYBR Green I dye are employed to achieve label-free fluorescent detection of ATP.•The presence of ATP leads to re-configuration of the hairpin aptamer probes.•Exonuclease III cleaves the re-configured hairpin aptamer probes to release the target ATP and initiate target recycling amplification.•The proposed method offers selective and sensitive detection of ATP down to the low nanomolar level.In this work, we described the development of a new label-free, simple and sensitive fluorescent ATP sensing platform based on exonuclease III (Exo III)-catalyzed target recycling (ECTR) amplification and SYBR Green I indicator. The hairpin aptamer probes underwent conformational structure switching and re-configuration in the presence of ATP, which led to catalytic cleavage of the re-configured aptamers by Exo III to release ATP and to initiate the ECTR process. Such ECTR process resulted in the digestion of a significant number of the hairpin aptamer probes, leading to much less intercalation of SYBR Green I to the hairpin stems and drastic suppression of the fluorescence emission for sensitive ATP detection down to the low nanomolar level. Due to the highly specific affinity bindings between aptamers and ATP, the developed method exhibited excellent selectivity toward ATP against other analogous molecules. Besides, our ATP sensing approach used un-modified aptamer probes and could be performed in a “mix-and-detect” fashion in homogenous solutions. All these distinct advantages of the developed method thus made it hold great potential for the development of simple and robust sensing strategies for the detection of other small molecules.

•A highly sensitive aptasensor for the detection of Ochratoxin A is developed.•Ochratoxin A leads to the inhibition of the electron transfer of the CdTe QDs.•The inhibition is significantly enhanced by Ochratoxin A recycling amplification.•The method is successfully employed to detect Ochratoxin A in red wine samples.Based on the recovery of the quantum dot (QD) electrochemiluminescence (ECL) and exonuclease-catalyzed target recycling amplification, the development of a highly sensitive aptasensor for Ochratoxin A (OTA) detection is described. The duplex DNA probes containing the biotin-modified aptamer are immobilized on a CdTe QD composite film-coated electrode. The presence of the OTA target leads to effective removal of the biotin–aptamers from the electrode surface via exonuclease-catalyzed recycling and reuse of OTA, which prevents the attachment of streptavidin–alkaline phosphatase (STV–ALP) through biotin–STV interaction. The electron transfer (ET) from the excited state CdTe QD ([CdTe]⁎) to the electro-oxidized species of the enzymatic product of ALP during the potential scan is thus inhibited and the QD ECL emission is restored for quantitative OTA detection. Due to the exonuclease-catalyzed target recycling amplification, the inhibition effect of ET is significantly enhanced to achieve sensitive detection of OTA down to 0.64 pg mL−1. The proposed method is selective for OTA and can be used to monitor OTA in real red wine samples. Our developed ECL recovery-based aptasensor thus offers great potential for the development of new ECL sensing platforms for various target analytes.

Positively charged gold nanoparticles ((+)AuNPs) are electrostatically adsorbed on the negatively charged aptamers and the solution displays a blue color due to the aggregation of the (+)AuNPs, while the binding of the target lysozyme with the aptamers reduces the charge of the aptamers, which stabilizes the (+)AuNPs and the (+)AuNPs solution remains red in color.

A highly sensitive electrochemical sequence-specific DNA detection strategy is demonstrated by coupling N.BstNB I (a nicking endonuclease)-assisted target recycling amplification with DNA supersandwich assembly signal enhancement. The proposed method avoids any extra chemical labeling steps and offers high selectivity against single-base mismatch sequences and a low detection limit down to 0.36 fM.

•Ligase chain reaction amplification (LCR) is employed to sensitively detect single nucleotide polymorphisms.•During LCR, the mutant target gene is recycled and duplicated exponentially to achieve dramatic signal amplification.•The method shows a selectivity factor of 103 toward the mutant target gene against the interfering wild target gene.Single nucleotide polymorphisms are the most common type of genetic variations among human beings and can serve as biomarkers for various types of diseases. In this work, based on ligase chain reaction amplification for the production of massive hemin/G-quadruplex DNAzymes to quench the electrochemiluminescent (ECL) emission of quantum dots (QDs), a universal and sensitive single nucleotide polymorphism detection method is described. During the ligase chain reaction process, the mutant K-ras target gene is recycled and exponentially duplicated, leading to the attachment of numerous G-rich sequences on the QD-embedded sensing surface. Upon the addition of the assistant sequences and hemin, numerous hemin/G-quadruplex DNAzymes are formed, which consume the dissolved oxygen in the detection buffer and result in significant quenching of QD ECL emission for sensitive single nucleotide polymorphism determination. The developed method shows a linear range of 50 fM to 50 pM and an estimated detection limit of 45 fM for the mutant K-ras gene. The proposed strategy also exhibits high selectivity towards the mutant K-ras gene against the co-existence of 103-fold excess of the wild-type K-ras gene, which makes our method a useful addition to the alternatives for single nucleotide polymorphism monitoring.

In this work, by coupling background current reduction with rolling circle amplification (RCA), we describe the development of an ultrasensitive electrochemical sensing method for protein detection based on a small molecule-linked DNA terminal protection strategy. Our detection platform employs a typical streptavidin (STV)–biotin interaction system. Biotin-linked single-stranded DNA (SH–ssDNA–biotin) is self-assembled on a gold electrode to capture the target protein, STV. The binding of STV with the biotin small molecule recognition element protects the SH–ssDNA–biotin against hydrolysis by exonuclease I (Exo I), while the unbound SH–ssDNA–biotin is effectively hydrolyzed and removed from the electrode surface. The bound STV further interacts with long, RCA-amplified biotin DNAs to facilitate the adsorption of numerous electroactive reporters, hexaammineruthenium(III) chloride (RuHex) via electrostatic interactions, which results in significantly amplified signals for the quantitative determination of STV. Moreover, the removal of the unbound SH–ssDNA–biotin probes from the sensing electrode obviates the accumulation of RuHex and leads to a highly minimized background current. The simultaneous RCA signal amplification and background current reduction is expected to significantly enhance the signal-to-noise ratio and to achieve ultrahigh sensitivity. The results reveal that the developed strategy provides a low detection limit of 0.4 pM with high selectivity.

In this work, we describe a new sensitive strategy for electrochemical detection of protein via small molecule/protein interactions. This assay is based on a terminal protection mechanism that small molecule-linked single-stranded DNA (ssDNA) is protected against hydrolysis by exonuclease I when the target protein is captured by the corresponding small molecule recognition element. Positively charged gold nanoparticles (AuNPs) are attached to the termini-protected and negatively charged ssDNA through electrostatic interactions. Subsequent AuNP-catalyzed silver enhancement followed by a highly characteristic and sensitive solid-state Ag/AgCl process is introduced to the sensing platform to amplify the signal output. By combining the amplification ability resulting from the silver deposition on the surface-captured AuNPs with the inherent high sensitivity of the electrochemical solid-state Ag/AgCl process, our method expands its range to the detection of macromolecules that bind to specific small molecules and enables low picomolar detection of protein. As a model of biotin/streptavidin interaction, a detection limit of 10 pM for streptavidin is readily achieved with desirable sensitivity and specificity, which indicates that the terminal protection assay coupled with the electrochemical solid-state Ag/AgCl process can offer a promising platform for the determination of various of types of proteins or small molecule-protein interactions.Highlights► Small molecule/protein interaction is interrogated by using a terminal protection strategy. ► The association of protein with small molecule-linked ssDNA protects ssDNA from digesting by exonuclease I. ► Small molecule/protein interaction is amplified by a highly characteristic solid-state Ag/AgCl electrochemical process. ► The proposed method offers selective and sensitive detection of protein down to the low picomolar level.

A facile and universal aptamer-based label-free approach for selective and sensitive fluorescence detection of proteins and small biomolecules by using the SYBR Green I (SGI) dye is developed. This robust versatile biosensing strategy relies on fluorescence turn-off changes of SGI, resulting from target-induced structure switching of aptamers. Upon binding with the targets, the aptamers dissociate from the respective cDNA/aptamer duplexes, leading to the release of the dsDNA-intercalated SGI into solution and the quenching of the corresponding fluorescence intensities. Such target-induced conformational changes and release of aptamers from the DNA duplexes essentially lead to the change in the fluorescence signal of the SGI and thus constitute the mechanism of our aptamer-based label-free fluorescence biosensor for specific target analyses. Under optimized conditions, our method exhibits high sensitivity and selectivity for the quantification of ATP and thrombin with low detection limits (23.4 nM and 1.1 nM, respectively). Compared with previous reported methods for aptamer-based detection of ATP and thrombin, this label-free approach is selective, simple, convenient and cost-efficient without any chemical labeling of the probe or the target. Therefore, the present strategy could be easily applicable to biosensors that target a wide range of biomolecules.Highlights► Label-free aptamer-based detection of biomolecules is achieved by using the commercially available SYBR Green I dye. ► Our method exempts from labeling of any aptamer probe or the target. ► Developed detection strategy is universal for both ATP and thrombin monitoring. ► One step detection of ATP or thrombin can be performed in a homogeneous solution.

The commercially available glucometer has been the most successful point-of-care (POC) sensor up to date. However, the glucometer only responds to glucose rather than other species. Extending the use of the glucometer for monitoring different types of targets would potentially revolutionize the applicability of the glucometer. Here we report a new sensing strategy for sensitive and selective detection of Cu2+ based on multi-invertase conjugated magnetic bead signal amplification labels and a glucometer transducer. The Cu2+ is in situ reduced to Cu+ by sodium ascorbate, which catalyzes the click linking between the alkynyl-DNA immobilized on a disposable screen printed carbon electrode and the azido-DNA attached to the invertase/magnetic bead conjugates. The numerous invertase on the magnetic bead labels through Cu+-catalyzed click chemistry reaction convert sucrose to glucose, which is monitored by the glucometer and offers amplified digital readings for Cu2+ detection. By employing the multi-invertase signal amplification, as low as 10 nM Cu2+ can be detected. Our method also shows high selectivity for Cu2+ against other metal ions owing to the highly specific Cu+-catalyzed click chemistry reaction, and is applicable for monitoring Cu2+ in real river samples. Our strategy can be easily expanded for the monitoring of a wide range of targets when coupled with various recognition events.Highlights► A commercial glucometer is used as the signal transducer to achieve sensitive detection of copper(II). ► The copper(II) detection is based on highly specific Cu(I)-catalyzed click chemistry through in situ reduction of copper(II). ► Multi-enzyme-loaded microbeads are employed as labels to amplify the signal output to achieve nanomolar detection of copper(II).

The hybridizations between the HIV target DNA and the capture probes as well as the signal probes conjugated to the multi-invertase/nanoparticle composites lead to the conversion of sucrose to glucose, which is monitored by the personal glucometer and provides quantitative digital readings for point-of-care diagnosis of HIV DNA fragments.

Early POC diagnosis of cancer is demonstrated by using multi-invertase conjugated microsphere labels and a personal glucose sensor (PGS) transducer. The invertase, which catalyzes the hydrolysis of sucrose to glucose, enables the PGS to detect target analytes beyond glucose, and the numerous invertase labels involved in each antibody–antigen binding event lead to significantly amplified PGS readings for sensitive protein detection.

By using exonuclease I and biobarcode nanoparticles, we describe a novel background current reduction strategy for amplified electrochemical detection of uropathogen specific sequences at ultralow concentrations.

In this work, by incorporating a specific DNAzyme sequence into a hairpin aptamer probe, we describe a label-free and sensitive method for electrochemical detection of cytokines using recombinant human IFN-γ as the model analyte. The hairpin aptamer probes are immobilized on a gold electrode through self-assembly. The presence of IFN-γ opens the hairpin structure and forms the hemin/G-quadruplex peroxidase-mimicking DNAzyme with subsequent addition of hemin. The peroxidase-mimicking DNAzyme catalyzes the electro-reduction of H2O2 and amplifies the current response for IFN-γ detection, which enables the monitoring of IFN-γ at the sub-nanomolar level. The proposed sensor also shows high selectivity towards the target analyte. Our strategy thus opens new opportunities for label-free and amplified detection of different types of cytokines.

We demonstrated a new strategy for highly sensitive electrochemical detection of cocaine by using two engineered aptamers in connection to redox-recycling signal amplification. The graphene/AuNP nanocomposites were electrochemically deposited on a screen printed carbon electrode to enhance the electron transfers. The cocaine primary binding aptamers were self-assembled on the electrode surface through sulfur–Au interactions. The presence of the target cocaine and the biotin-modified secondary binding aptamers leads to the formation of sandwich complexes on the electrode surface. The streptavidin-conjugated alkaline phosphatases (ALPs) were used as labels to generate quantitative signals. The addition of the ALP substrate and the co-reactant NADH results in the formation of a redox cycle between the enzymatic product and the electrochemically oxidized species and the signal is thus significantly amplified. Because of the effective modification of the sensing surface and signal amplification, low nanomolar (1 nM) detection limit for cocaine is achieved. The proposed aptamer-based sandwich sensing approach for amplified detection of cocaine thus opens new opportunities for highly sensitive determination of other small molecules.Highlights► Modification of electrode surface with graphene/AuNP nanocomposite to enhance electron transfers. ► The use of two engineered aptamers to form sandwich format for cocaine detection. ► Dual signal amplification by catalytic redox-recycling. ► Highly sensitive detection of cocaine down to the low nanomolar level.

A dual signal amplified strategy for sensitive EC detection of pathogenic DNA sequences based on poly[G]20-conjugated biobarcode nanoparticles and carboxyl functionalized graphene is described. The capture probes are covalently linked to the graphene modified electrode. The addition of the target sequences and the biobarcode amplification labels leads to the formation of sandwich structures on the electrode surface. The presence of Ru(bpy)32+ in the detection buffer catalyzes the electro-oxidation of the numerous guanine bases captured on the electrode surface and generates significantly amplified current response for quantitation. Moreover, graphene can offer excellent heterogeneous electron transfer during electro-catalytic oxidation of guanine bases. This dual signal amplification strategy thus results in the determination of pathogenic DNA sequences down to the low picomolar level (1 pM). Our approach also exhibits excellent discrimination between the target and single-base mismatch sequences, which provides great potential for highly sensitive and selective detection of different nucleic acid sequences in general.

One-spot signal-on and simultaneous electronic detection of lysozyme and adenosine is achieved based on target-induced release of aptamers and back-filling hybridization of the resulting single stranded DNAs with redox-tags conjugated aptamers.

The preparation and use of a new class of signal amplification label, the CdTe quantum dot layer-by-layer assembled polystyrene microbead composite, for amplified ultrasensitive electrochemiluminescent detection of thrombin is described.

The presence of exonuclease III leads to direct recycling and reuse of the target DNA, which in turn results in substantial signal amplification for highly sensitive, label-free impedimetric detection of specific DNA sequences.

Reagentless, sensitive and multiplexed analysis of gyrB and K-rasgene biomarkers is achieved based on the proximity changes of two different redox-tags to the electrode surface upon DNA hybridizations, and the presence of the two gene biomarkers also acts as inputs and activates the logic gate.

A novel strategy for “signal on” and sensitive one-spot simultaneous detection of multiple small molecular analytes based on electrochemically encoded barcode quantum dot (QD) tags is described. The target analytes, adenosine triphosphate (ATP) and cocaine, respectively, are sandwiched between the corresponding set of surface-immobilized primary binding aptamers and the secondary binding aptamer/QD bioconjugates. The captured QDs yield distinct electrochemical signatures after acid dissolution, whose position and size reflect the identity and level, respectively, of the corresponding target analytes. Due to the inherent amplification feature of the QD labels and the “signal on” detection scheme, as well as the sensitive monitoring of the metal ions released upon acid dissolution of the QD labels, low detection limits of 30 nM and 50 nM were obtained for ATP and cocaine, respectively, in our assays. Our multi-analyte sensing system also shows high specificity to target analytes and promising applicability to complex sample matrix, which makes the proposed assay protocol an attractive route for screening of small molecules in clinical diagnosis.

We report an ultrasensitive electrochemical approach for the detection of uropathogen sequence-specific DNA target. The sensing strategy involves a dual signal amplification process, which combines the signal enhancement by the enzymatic target recycling technique with the sensitivity improvement by the quantum dot (QD) layer-by-layer (LBL) assembled labels. The enzyme-based catalytic target DNA recycling process results in the use of each target DNA sequence for multiple times and leads to direct amplification of the analytical signal. Moreover, the LBL assembled QD labels can further enhance the sensitivity of the sensing system. The coupling of these two effective signal amplification strategies thus leads to low femtomolar (5 fM) detection of the target DNA sequences. The proposed strategy also shows excellent discrimination between the target DNA and the single-base mismatch sequences. The advantageous intrinsic sequence-independent property of exonuclease III over other sequence-dependent enzymes makes our new dual signal amplification system a general sensing platform for monitoring ultralow level of various types of target DNA sequences.

An electronic DNAzyme sensor for highly sensitive detection of Pb2+ is demonstrated by coupling the significant signal enhancement of the layer-by-layer (LBL) assembled quantum dots (QDs) with Pb2+ specific DNAzymes. The presence of Pb2+ cleaves the DNAzymes and releases the biotin-modified fragments, which further hybridize with the complementary strands immobilized on the gold substrate. The streptavidin-coated, QD LBL assembled nanocomposites were captured on the gold substrate through biotin–streptavidin interactions. Subsequent electrochemical signals of the captured QDs upon acid dissolution provide quantitative information on the concentrations of Pb2+ with a dynamic range from 1 to 1000 nM. Due to the dramatic signal amplification by the numerous QDs, subnanomolar level (0.6 nM) of Pb2+ can be detected. The proposed sensor also shows good selectivity against other divalent metal ions and thus holds great potential for the construction of general DNAzyme-based sensing platform for the monitoring of other heavy metal ions.

The construction of a reagentless, sensitive, disposable and multiplexed electronic sensing platform for one-spot simultaneous determination of biomolecules with significant difference in size (proteins and small molecules) is described. The sensing surface is fabricated by the hybridization of two types of redox-tags conjugated aptamers with the corresponding complementary DNAs, which are self-assembled on a gold nanoparticle-modified screen printed carbon electrode. The presence of the target analytes leads to the release of the tagged signaling aptamers from the sensing surface, and the surface-remained tags exhibit well-resolved peaks, whose positions and sizes reflect the identities and concentrations of the target analytes, respectively. The application of the proposed sensing platform for molecular logic gate operations is also demonstrated.

A sensitive electrochemical immunoassay system for the detection of a protein tumor biomarker through a dual amplified strategy was reported. Firstly, this protocol involves in the electropolymerization of o-aminobenzoic acid (o-ABA) on a glass carbon electrode (GCE). Subsequently, capture anti-CEA (Ab1) is covalently linked to poly(o-ABA) (PAB) film, via N-(3-dimethylamminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysulfosuccinimid sodium salt (NHS) activation of the carboxyl groups and surface blocking with ethanolamine. Later, the target, carcinoembryonic antigen (CEA), is sandwiched between an electrode surface confined Ab1 and the alkaline phosphatase-labeled signal anti-CEA antibodies conjugated with gold nanoparticles (Ab2-ALP-AuNP bioconjugates). The dual biocatalytic signal amplification for CEA monitoring is achieved by coupling the numerous enzymes loaded on the AuNPs with redox-recycling of the enzymatic products in the presence of the secondary enzyme and the corresponding substrate. The novel dramatic signal amplification strategy, exhibits a good linearity at the studied concentration range from 0.005 to 50 ng mL−1 towards CEA with a detection limit of 2 pg mL−1 (S/N=3). There is a 5–100-fold improvement in detection limit compared to other similar studies. The developed dual signal amplified strategy shows good selectivity, regeneration, stability and acceptable reproducibility. Therefore, the signal amplification approach holds great potential applications in detection of ultra-trace protein biomarkers.

A convenient aptamer-based competitive electrochemical biosensor for a small biomolecule, adenosine, was described. The sensing surface was fabricated by self-assembly of an aptamer/mercaptohexanol monolayer on a gold disk electrode. The principle of this aptasensor is based on the competition between an adenosine target molecule and a ferrocene-conjugated signaling DNA strand for the aptamer binding site on the sensing surface. Due to the competitive nature of this assay, the electrochemical responses of the surface captured ferrocene are inversely proportional to log[adenosine] in the range from 0.05 to 3.2 μM, with a detection limit of 25 nM. Moreover, the aptasensor also shows high selectivity for adenosine. The proposed aptasensor thus holds great potential for the detection of other small biomolecules.

Reproducible electrochemically encoded quantum dot (QD) barcodes were prepared using the reverse-micelle synthetic approach. The encoding elements, Zn2+, Cd2+, and Pb2+, were confined within a single QD, which eliminates the cumbersome encapsulation process used by other common nanoparticle-based barcode preparation schemes. The distinct voltammetric stripping patterns of Zn2+, Cd2+ and Pb2+ at distinguishable potentials with controllable current intensities offer excellent encoding capability for the prepared electrochemical (EC) QDs. Additionally, the simultaneous modification of the QD barcode surface with organic ligands during the preparation process make them potentially useful in biomedical research. For proof of concept of their application in bioassays, the EC QD barcodes were further employed as tags for an immunoassay of a cancer marker, carcinoembryonic antigen (CEA). The voltammetric stripping response of the dissolved bardcode tags was proportional to log[CEA] in the range from 0.01 to 80 ng mL−1, with a detection limit of 3.3 pg mL−1. The synthesized EC QD barcodes hold considerable potential in biodetection, encrypted information, and product tracking.

We present an ultrasensitive aptasensor for the electronic monitoring of proteins through a dual amplified strategy in this paper. The target protein thrombin is sandwiched between an electrode surface confined aptamer and an aptamer–enzyme–carbon nanotube bioconjugate. The analytical signal amplification is achieved by coupling the signal amplification nature of multiple enzymes with the biocatalytic signal enhancement of redox-recycling. Our novel dramatic signal amplification strategy, with a detection limit of 8.3 fM, shows about 4 orders of magnitude improvement in the sensitivity for thrombin detection compared to other universal single enzyme-based assay. This makes our approach an attractive alternative to other common PCR-based signal amplification in ultralow level of protein detection.

•The binding of DNA-linked antibodies to antigen leads to the formation of DNAzymes.•Cyclic cleavage of DNAzymes generates significantly amplified detection signals.•Enzyme-free amplified detection of proteins in human serums can be realized.Based on target-induced immuno-proximity binding and metal ion-dependent DNAzyme recycling signal amplification, we describe the development of a homogeneous and sensitive fluorescent method for the detection of protein cancer biomarkers by using α-fetoprotein (AFP) as the model target. Two DNA strands with short complementary regions are conjugated to the AFP antibodies to prepare the recognition probes. The hybridization of the two DNAs in the absence of the AFP target molecules is inhibited due to the low melting temperature (Tm) of the complementary regions. The binding of the antibody-linked DNAs to the AFP target molecules can increase the local effective concentrations of the two DNAs and facilitate their hybridization with significantly increased Tm to form the catalytic cores of the metal ion-dependent DNAzymes. The fluorescently quenched hairpin substrate sequences further bind the catalytic cores to form the DNAzymes, in which the substrate sequences are cyclically cleaved by the corresponding metal ions to generate remarkably enhanced fluorescent signals for sensitive detection of AFP in the dynamic range from 2 to 500 pM with the detection limit of 1.8 pM. The sensing approach also possesses high selectivity toward the target AFP over other interfering proteins, and can be employed to detect AFP in human serum samples. The significant signal amplification in our approach avoids the involvement of any enzyme or nanomaterial labels and sensitive protein detection can be realized in homogeneous solution, which makes the developed method holds great potential for convenient and sensitive detection of various protein biomarkers for early disease diagnosis.

•A sensitive fluorescent method for detecting Dam MTase activity is developed.•Signal amplification is achieved by the integration of HCR with DNAzyme recycling.•The method is convenient and shows high sensitivity.•The application of this method for anti-cancer drug screening is also demonstrated.Aberrant DNA methylation originated from changes in DNA methyltransferase activity can lead to many genetic diseases and tumor types, and the monitoring of methyltransferase activity is thus of great importance in disease diagnosis and drug screening. In this work, by combing hybridization chain reaction (HCR) and metal ion-dependent DNAzyme recycling, we have developed a convenient enzyme-free signal amplification strategy for highly sensitive detection of DNA adenine methyltransferase (Dam MTase) activity and its inhibitors. The Dam MTase-induced methylation and subsequent cleavage of the methylated hairpin DNA probes by DpnI endonuclease lead to the release of ssDNA triggers for HCR formation of many Mg2+-dependent DNAzymes, in which the fluorescently quenched substrate sequences are catalytically and cyclically cleaved by Mg2+ to generate remarkably amplified fluorescent signals for highly sensitive detection of Dam MTase at 7.23 × 10−4 U/mL. In addition, the inhibition of different drugs to Dam MTase activity can also be evaluated with the developed method. With the advantages of simplicity and significant signal amplification over other common methods, the demonstrated biosensing approach thus offers great potential for highly sensitive detection of various methyltransferases and provides a convenient platform for drug screening for therapeutic applications.Figure optionsDownload full-size imageDownload as PowerPoint slide

Based on an endonuclease-assisted, cross-triggered and cascaded recycling amplification strategy, the construction of a simple electrochemical sensing platform for the ultrasensitive detection of the mutant p53 gene in human serum is described. Using this new signal amplification approach, the sub-femtomolar level of the mutant p53 gene can be selectively detected.

Chemically reduced Cu2+ triggers the ligation of alkynyl- and azido-modified DNA via click chemistry. Subsequently, the ligated DNA initiates cyclic assembly of two fluorescently quenched hairpin DNAs and generates significantly amplified fluorescence signals for highly sensitive detection of Cu2+ in human serum samples.

Catalytic self-assembly of DNA nanostructures triggered by microRNA 21 (miR-21) is achieved through isothermal toe-hold strand displacement reactions. The miR-21 is autonomously recycled during the self-assembly process, which makes the generation of the DNA nanostructures proceed in a catalytic fashion. The miR-21-triggered self-assembly of DNA nanostructures can also serve as a remarkable signal amplification platform to achieve ultrasensitive detection of miR-21 from as low as 10 MCF-7 human breast cancer cells.

By using exonuclease I and biobarcode nanoparticles, we describe a novel background current reduction strategy for amplified electrochemical detection of uropathogen specific sequences at ultralow concentrations.

The hybridizations between the HIV target DNA and the capture probes as well as the signal probes conjugated to the multi-invertase/nanoparticle composites lead to the conversion of sucrose to glucose, which is monitored by the personal glucometer and provides quantitative digital readings for point-of-care diagnosis of HIV DNA fragments.

A highly sensitive electrochemical sequence-specific DNA detection strategy is demonstrated by coupling N.BstNB I (a nicking endonuclease)-assisted target recycling amplification with DNA supersandwich assembly signal enhancement. The proposed method avoids any extra chemical labeling steps and offers high selectivity against single-base mismatch sequences and a low detection limit down to 0.36 fM.

Positively charged gold nanoparticles ((+)AuNPs) are electrostatically adsorbed on the negatively charged aptamers and the solution displays a blue color due to the aggregation of the (+)AuNPs, while the binding of the target lysozyme with the aptamers reduces the charge of the aptamers, which stabilizes the (+)AuNPs and the (+)AuNPs solution remains red in color.

Early POC diagnosis of cancer is demonstrated by using multi-invertase conjugated microsphere labels and a personal glucose sensor (PGS) transducer. The invertase, which catalyzes the hydrolysis of sucrose to glucose, enables the PGS to detect target analytes beyond glucose, and the numerous invertase labels involved in each antibody–antigen binding event lead to significantly amplified PGS readings for sensitive protein detection.

One-spot signal-on and simultaneous electronic detection of lysozyme and adenosine is achieved based on target-induced release of aptamers and back-filling hybridization of the resulting single stranded DNAs with redox-tags conjugated aptamers.

The presence of exonuclease III leads to direct recycling and reuse of the target DNA, which in turn results in substantial signal amplification for highly sensitive, label-free impedimetric detection of specific DNA sequences.

Based on a new steric hindrance inhibition of the DNA strand displacement strategy, we report the design of a robust fluorescence signal-on method for homogeneous and sensitive detection of antibodies from human serum samples. Such a steric hindrance effect leads to sensitive detection of the target antibodies with a detection limit of 5.6 nM. In addition, the developed sensing approach shows high selectivity against other interference proteins and the detection of the target antibodies in human sera by this method is also verified.

Reagentless, sensitive and multiplexed analysis of gyrB and K-rasgene biomarkers is achieved based on the proximity changes of two different redox-tags to the electrode surface upon DNA hybridizations, and the presence of the two gene biomarkers also acts as inputs and activates the logic gate.

The preparation and use of a new class of signal amplification label, the CdTe quantum dot layer-by-layer assembled polystyrene microbead composite, for amplified ultrasensitive electrochemiluminescent detection of thrombin is described.

The association of the target thrombin with the corresponding aptamer leads to structure switching of the dsDNA probes and the formation of nicking sites for exonuclease III, which causes cyclic cleavage of the dsDNA probes and highly reduced intercalation of the electrochemiluminescent signal indicators for label-free and sensitive detection of thrombin at the femtomolar level.

Based on a new target-triggered aptamer molecular machine, a label-free and non-enzymatic target recycling amplification strategy for sensitive fluorescence detection of ATP in human serums is described. The presence of the target ATP together with the DNA fuel strand initiates the operation of the aptamer machine and leads to cyclic reuse of ATP and the release of many G-quadruplex sequences, which associate with a fluorescent dye to generate significantly amplified fluorescence signals to achieve sensitive detection of ATP.

The presence of the microRNA-141 target molecules activates the DNA molecular machine powered by the DNA fuel strands, leading to non-enzymatic target cyclic reuse of microRNA-141 and significantly amplified fluorescent signals for sensitive monitoring of microRNA-141 from low numbers of human prostate cancer cells.

The presence of target microRNA triggers cascaded and catalytic self-assembly of two DNA motifs into DNA nanostructures, which serves as a remarkable signal amplification means for the highly sensitive monitoring of the target microRNA and the detection of low numbers of tumor cells.