Target-initiated triplex signal amplification cascades for non-label and sensitive fluorescence sensing of microRNA.

The aberrant expression of microRNAs (miRs) in cells is closely linked to the initiation and progression of various diseases. Sensitive monitoring of their level is hence vital for biomedical research and disease diagnosis. Herein, a highly sensitive and non-label fluorescence sensor based on multiple recycling signal amplification cascades is constructed for the detection of miR-21 in human sera. The presence of miR-21 initiates the primer-fueled target recycling process for the generation of many primer/hairpin templates for the subsequent auto-cycling primer extension (APE) amplification cycles, which result in the formation of lots of long-stem hairpins. The enzyme-based cleavage of such hairpins via polymerization/excision cycles further leads to the generation of abundant G-quadruplex strands, which associate with the thioflavin T (ThT) dye to emit remarkably magnified fluorescence for detecting miR-21 in the range of 1 pM-100 nM with a 0.32 pM detection limit without labeling the probes. Besides, the proposed assay can selectively discriminate miR-21 against other control molecules and realize the sensing of low levels of miR-21 in diluted sera. With features of high sensitivity via the triplex signal amplification cycles and simplicity in a non-label homogeneous manner, our miR sensing protocol can be a robust means for detecting various nucleic acids for the early diagnosis of diseases.

An aptamer triple helix molecular switch for sensitive electrochemical assay of lipocalin 1 biomarker via dual signal amplifications.

Sensitively monitoring the concentration change of lipocalin 1 (LCN1) can provide data support for accurately diagnosing diabetic retinopathy and efficacy of treatment. Using a new aptamer triplex switch (ATS) probe and catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) dual signal amplifications, a highly sensitive electrochemical and enzyme-free biosensor for detecting LCN1 is reported. The ATS probes recognize and bind LCN1 to change its structure and release ssDNA sequences, which trigger the capture of methylene blue (MB)-tagged DNA on the sensor electrode via the CHA and HCR reactions. These MB tags are then subjected to electrochemical measurements to obtain highly amplified current outputs to sensitively detect LCN1 from 5 pM to 1 nM with a detection limit of 0.85 pM. This sensor also exhibits high interfering protein discrimination capability and can be employed to monitor low levels of LCN1 in diluted tear samples. Our established sensor significantly outperforms current major LCN1 detection methods based on immunoassays and thus holds promising potential for evaluating the severity of diabetic retinopathy and monitoring treatment response.

The synchronization of multiple signal amplifications for label-free and sensitive aptamer-based sensing of a protein biomarker.

The abnormal variation of the mucin 1 (MUC1) protein level is associated with the development of multiple cancers, and the monitoring of trace MUC1 can be useful for early disease diagnosis. Here, on the basis of the synchronization of DNA-fueled sequence recycling and dual rolling circle amplification (RCA), the establishment of a non-label and highly sensitive fluorescent aptamer-based detection strategy for the MUC1 protein biomarker is described. The target MUC1 binds the aptamer hairpin probe and causes its structure switching to release an ssDNA tail to trigger the recycling of the complex via two toehold-mediated strand displacement reactions under assistance of a fuel DNA. Such a recycling amplification leads to the formation of a partial dsDNA duplex with two primers at both ends, which cooperatively bind the circular DNA ring template to start the dual RCA to produce many G-quadruplex sequences. The protoporphyrin IX dye further associates with the G-quadruplex structures to show a dramatically elevated fluorescent signal for sensitively detecting MUC1 with a low detection limit of 0.5 pM. The established aptamer-based detecting strategy is also highly selective and can realize assay of MUC1 in diluted human serums, highlighting its potential for the detection of different protein biomarkers at low contents.

In Situ-Generated Multivalent Aptamer Network for Efficient Capture and Sensitive Electrochemical Detection of Circulating Tumor Cells in Whole Blood.

Monitoring circulating tumor cells (CTCs) in human blood can offer useful information for convenient metastasis diagnosis, prognosis, and treatment of cancers. However, it remains a substantial challenge to detect CTCs because of their particular scarcity in complex peripheral blood. Herein, we describe an in situ-generated multivalent aptamer network-modified electrode interface for efficiently capturing and sensitively detecting CTCs in whole blood by electrochemistry. Such an interface was fabricated via rolling circle amplification extension of the electrode-immobilized primer/circular DNA complexes for the yield of long ssDNA strands with many repeated aptamer segments, which could achieve efficient capture of rare CTCs in a multivalent cooperative manner. The antibody and horseradish peroxidase-functionalized gold nanoparticles further specifically associated with the surface-bound CTCs and generated electrocatalytically amplified current outputs for highly sensitive detection of CTCs with an attractive detection limit of five cells. Also, the multivalent aptamer network interface could successfully distinguish the target cells from other control cells and achieve CTC detection in whole blood, demonstrating its promising potential for monitoring different rare CTCs in human blood.

DNA branch migration amplification cascades for enzyme-free and non-label aptamer sensing of mucin 1.

The sensitive and quantitative analysis of mucin 1 (MUC1) is very important for the prevention and early diagnosis of cancers. In the present work, based on the mechanism of the four-way DNA branch migration cascades, we constructed a simple and effective signal amplification strategy for aptamer-based sensitive detection of MUC1. The specific binding of MUC1 to the aptamer sequence in the hairpin probe unfolds and switches its structure, triggering the formation of the DNA Holliday junction structure for cascaded branch migrations with the assistance of two fuel DNA duplexes. Importantly, a target analogue DNA complex can be generated in such processes for recycling the branch migration reactions for the production of substantial amounts of G-quadruplexes, which can bind the thioflavin T dye to show significantly intensified fluorescence for detecting MUC1 with a low detection limit of 2.8 nM without the involvement of any labels or enzymes. In addition, this detection strategy could be successfully applied to monitor the target MUC1 in diluted human serums with a high selectivity and acceptable accuracy to demonstrate its potential application for real samples with the advantages of simplicity and signal amplification capability.

Coupling strand extension/excision amplification with target recycling enables highly sensitive and aptamer-based label-free sensing of ATP in human serum.

Detection of aberrant ATP concentrations with high sensitivity and selectivity is of critical importance for monitoring many biological processes and disease stages. By coupling extension/excision amplification with target recycling, we have established an aptamer-based method for label-free fluorescence ATP detection in human serum with high sensitivity. The ATP target molecules associate with the aptamer-containing double hairpin probes and cause conformational changes of the probes to initiate the cyclic strand extension/excision processes in the presence of polymerase, endonuclease and assistance sequences for the recycling of ATP and the production of a large number of G-quadruplex sequences. The organic dye thioflavin T subsequently binds these G-quadruplex sequences to yield substantially enhanced fluorescence emission for achieving highly sensitive detection of ATP down to 2.2 nM in the range of 5 to 200 nM without using any labels. The developed aptamer sensing method also exhibits high selectivity and allows the monitoring of ATP at low concentrations in diluted real samples, which offers promising opportunities to establish effective signal magnification means for the detection of various biomolecules at trace levels.

Aptamer-Functionalized and Gold Nanoparticle Array-Decorated Magnetic Graphene Nanosheets Enable Multiplexed and Sensitive Electrochemical Detection of Rare Circulating Tumor Cells in Whole Blood.

The identification and monitoring of circulating tumor cells (CTCs) in human blood plays a pivotal role in the convenient diagnosis of different cancers. However, it remains a major challenge to monitor these CTCs because of their extremely low abundance in human blood. Here, we describe the synthesis of a new aptamer-functionalized and gold nanoparticle (AuNP) array-decorated magnetic graphene nanosheet recognition probe to capture and isolate rare CTCs from human whole blood. In addition, by employing the aptamer/electroactive species-loaded AuNP signal amplification probes, multiplexed electrochemical detection of these low levels of CTCs can be realized. The incubation of the probes with the sample solutions containing the target CTCs can lead to the efficient separation of the CTCs and result in the generation of two distinct voltammetric peaks on a screen-printed carbon electrode, whose potentials and current intensities, respectively, reflect the identity and number of CTCs for the multiplexed detection of the Ramos and CCRF-CEM cells with detection limits down to 4 and 3 cells mL-1. With the successful demonstration of the concept, further extension of the developed sensing strategy for the determination of various CTCs in human whole blood for the screening of different cancers can be envisioned in the near future.

DNA-Templated In Situ Synthesis of Highly Dispersed AuNPs on Nitrogen-Doped Graphene for Real-Time Electrochemical Monitoring of Nitric Oxide Released from Live Cancer Cells.

Dispersion promotion of nanomaterials can significantly enhance their catalytic activities. With a new DNA-templated in situ synthesis approach, we report the preparation of highly dispersed AuNPs on nitrogen-doped graphene sheets (NGS) with significantly improved electrocatalytic ability for the monitoring of nitric oxide (NO) released from live cancer cells. The template DNA is adsorbed on NGS via π-π stacking, and the Au precursor chelates along the DNA lattice through dative bonding. Subsequent introduction of the reducing agent leads to in situ nucleation and growth of AuNPs, eventually resulting in highly dispersed AuNPs on NGS. Because of the synergistic enhancement of the catalytic activities of AuNPs and NGS, as well as the high dispersion of AuNPs, such a nanocomposite shows significant electro-oxidation capability toward NO, leading to a highly sensitive subnanomolar detection limit for NO in vitro. More importantly, the laminin glycoproteins can be readily adsorbed on the surface of the nanomaterials to render excellent biocompatibility for the adhesion and proliferation of live cells, enabling the biointerface for electrochemical detection of NO released from live cancer cells.

Target-induced structure switching of aptamers facilitates strand displacement for DNAzyme recycling amplification detection of thrombin in human serum.

To monitor the thrombin concentration under the condition of abnormal blood coagulation is of clinical significance for the diagnosis of various diseases. Here, on the basis of the aptamer structure switching induced by the target molecules and the signal amplification strategy via recycling of metal-ion dependent DNAzymes, we have established a sensitive and simple fluorescent aptasensor for detecting thrombin in human serum. The thrombin target specifically binds to the aptamer sequence and causes a corresponding conformational structure switching, which leads to the formation of a toehold sequence to facilitate the strand migration displacement reaction for the generation of functional metal-ion dependent DNAzymes. These DNAzymes further cleave the fluorescently quenched hairpin substrates cyclically to yield substantially amplified fluorescence recovery for sensitively detecting thrombin in the dynamic range from 0.01 nM to 50 nM. Such an aptasensor shows a detection limit of 6.9 pM and can achieve the monitoring of thrombin in diluted human serum with high selectivity, offering a universal sensing strategy for the construction of various sensitive and simple aptasensors to detect different biomarker molecules.

Click chemistry-mediated cyclic cleavage of metal ion-dependent DNAzymes for amplified and colorimetric detection of human serum copper (II).

The determination of the level of Cu2+ plays important roles in disease diagnosis and environmental monitoring. By coupling Cu+-catalyzed click chemistry and metal ion-dependent DNAzyme cyclic amplification, we have developed a convenient and sensitive colorimetric sensing method for the detection of Cu2+ in human serums. The target Cu2+ can be reduced by ascorbate to form Cu+, which catalyzes the azide-alkyne cycloaddition between the azide- and alkyne-modified DNAs to form Mg2+-dependent DNAzymes. Subsequently, the Mg2+ ions catalyze the cleavage of the hairpin DNA substrate sequences of the DNAzymes and trigger cyclic generation of a large number of free G-quadruplex sequences, which bind hemin to form the G-quadruplex/hemin artificial peroxidase to cause significant color transition of the sensing solution for sensitive colorimetric detection of Cu2+. This method shows a dynamic range of 5 to 500 nM and a detection limit of 2 nM for Cu2+ detection. Besides, the level of Cu2+ in human serums can also be determined by using this sensing approach. With the advantages of simplicity and high sensitivity, such sensing method thus holds great potential for on-site determination of Cu2+ in different samples. Graphical abstract Sensitive colorimetric detection of copper (II) by coupling click chemistry with metal ion-dependentDNAzymes.

DNA Cleavage and Condensation Activities of Mono- and Binuclear Hybrid Complexes and Regulation by Graphene Oxide.

Hybrid complexes with N,N'-bis(2-benzimidazolylmethyl)amine and cyclen moieties are novel enzyme mimics and controlled DNA release materials, which could interact with DNA through three models under different conditions. In this paper, the interactions between plasmid DNA and seven different complexes were investigated, and the methods to change the interaction patterns by graphene oxide (GO) or concentrations were also investigated. The cleavage of pUC19 DNA promoted by target complexes were via hydrolytic or oxidative mechanisms at low concentrations ranging from 3.13 × 10(-7) to 6.25 × 10(-5) mol/L. Dinuclear complexes 2a and 2b can promote the cleavage of plasmid pUC19 DNA to a linear form at pH values below 7.0. Furthermore, binuclear hybrid complexes could condense DNA as nanoparticles above 3.13 × 10(-5) mol/L and partly release DNA by graphene oxide with π-π stacking. Meanwhile, the results also reflected that graphene oxide could prevent DNA from breaking down. Cell viability assays showed dinuclear complexes were safe to normal human hepatic cells at relative high concentrations. The present work might help to develop novel strategies for the design and synthesis of DNA controllable releasing agents, which may be applied to gene delivery and also to exploit the new application for GO.

Aptamer pseudoknot-functionalized electronic sensor for reagentless and single-step detection of immunoglobulin E in human serum.

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.

Target-promoted autocatalytic hairpin assembly of bivalent DNAzymes for sensitive and label-free electrochemical metallothionein assay.

Metallothionein (MT) has shown to be an important biomarker for environmental monitoring and various diseases, due to its significant binding ability to heavy metal ions. On the basis of such a characteristic and the Hg2+-stabilized DNA duplex (Hg2+-dsDNA) probe, as well as a new autocatalytic hairpin assembly (aCHA)/DNAzyme cascaded signal enhancement strategy, the construction of a highly sensitive and label-free electrochemical MT biosensor is described. Target MT molecules bind Hg2+ in Hg2+-dsDNA to disrupt the duplex structure and to release ssDNA sequences, which trigger subsequent aCHA for efficient production of mimic aCHA triggering strands and many bivalent DNAzymes. The signal hairpins on the electrode are then cyclically cleaved by DNAzyme amplification cascade to liberate plenty G-quadruplex sequences, which bind hemin and yield largely enhanced currents for sensitive assay of MT with a detection limit of 0.217 nM in a label-free approach. Such sensor also shows selective discrimination capability to MT against other interfering proteins and assay of MT in normal serums with dilution has also been verified, indicating its potential for highly sensitive detection of different heavy metal ion binding molecules for various application scenarios.

Highly catalytic sulfur-doped and bimetal-coordinated CoFe(CN)5NO nanoparticles coupled with PER/HCR amplification cascades for sensitive electrochemical aptamer luteinizing hormone assay.

Sensitive monitoring of luteinizing hormone (LH), a glycoprotein that regulates the synthesis of regulatory steroid hormones, can facilitate the diagnosis of various reproductive diseases. In this work, a new and highly catalytic Sulfur-doped and bimetal-coordinated CoFe(CN)5NO (denoted as S-CoFe(CN)5NO) nanoparticles are synthesized. Such material is further used to construct high performance sensing interface and coupled with primer exchange reaction (PER) and hybridization chain reaction (HCR) amplification cascades for sensitive electrochemical aptamer-based LH assay. Target LH molecules bind aptamer sequences in DNA duplex probes to liberate ssDNA strands, which initiate subsequent PER/HCR amplification cascades for the capture of many ferrocene (Fc)-tagged DNAs on sensing interface. S-CoFe(CN)5NO subsequently leads to catalytic oxidation of these Fc tags for yielding substantially magnified currents for realizing ultrasensitive assay of LH with the detection limit of 0.69 pM in range from 5 pM to 10 nM. Owing to the high specificity of aptamer, such sensor has high selectivity and can achieve low levels of LH assay in diluted serum samples. With the successful demonstration for detecting trace LH, such sensor can be easily extended as a universal aptamer-based electrochemical sensing method for monitoring various target analytes in the biomedical and biological fields.

Label-free and highly sensitive detection of microRNA from cancer cells via target-induced cascade amplification generation of lighting-up RNA aptamers.

The abnormal expression levels of miRNAs have been proven to be highly related to the generation of various diseases and are also closely associated with the stages and types of disease development. The novel RNA aptamers-based homogenous fluorescent methods were simple, with low background signal and high signal-to-noise ratio, but lacked effective signal amplification technology to achieve sensitive detection of trace miRNA markers. There is an urgent need for combining effective nucleic acid amplification technology with RNA aptamer to achieve highly sensitive and accurate detection of miRNA. For this purpose, a new DNA multi-arm nanostructure-based dual rolling circle transcription machinery for the generation of lighting-up MG RNA aptamers is constructed for label-free and highly sensitive sensing of miRNA-21. In this system, the target miRNA-21 induces a structural transformation of the DNA multi-arm nanostructure probe to recycle miRNA-21 and trigger two independent rolling circle transcription reactions to generate two long RNAs, which can partially hybridize with each other to generate large amounts of complete MG RNA aptamers. These RNA aptamers can associate with organic MG dye to produce significantly enhanced fluorescence signals to accomplish ultrasensitive miRNA-21 detection down to 0.9 fM. In addition, this method exhibits high selectivity to distinguish miRNA-21 even with single nucleotide mismatch, and also has potential application capability to monitor different expression levels of miRNA-21 from different cancer cells. The effective collaboration between MG RNA aptamer and rolling circle transcription reaction makes this fluorescent method show the significant advantages of low background signal, high signal-to-noise ratio and high detection sensitivity. It has great potential to be a promising means to achieve label-free and highly sensitive monitoring of other trace biological markers via a simple change of target sequence.

Coupling of background reduction with rolling circle amplification for highly sensitive protein detection via terminal protection of small molecule-linked DNA.

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.

Proximity binding-initiated DNA walker and CRISPR/Cas12a reaction for dual signal amplification detection of thrombin.

We report here a highly sensitive fluorescent thrombin biomarker sensing method by integrating the DNA walker and CRISPR/Cas12a system. The presence of thrombin causes the localization of DNA moving arms on AuNP tracks via their proximity bindings with the dye-labeled probes immobilized on AuNPs. With the assistance of the primer and DNA polymerase, the arm sequences move continuously on the AuNP tracks to generate many CRISPR/Cas12a-responsive dsDNAs, which push the dye to move away from AuNPs to restore its fluorescence. Moreover, the dsDNAs can be recognized and cut by the CRISPR/Cas12a to trigger its trans-cleavage activity for cyclically cleaving the fluorescently quenched signal probes on the AuNP tracks, which liberates the dye from AuNPs to further enhance the fluorescence restoration to achieve highly sensitive thrombin assay with detection limit of 29.5 fM. Selectively detecting thrombin against other interference proteins and in diluted serums by such sensing method has also been verified, making it an attractive approach for monitoring other protein biomarkers at low levels for the diagnosis of diseases.

Programmable bidirectional dynamic DNA nano-device for accurate and ultrasensitive fluorescent detection of trace MUC1 biomarker in serums.

Accurate and ultrasensitive detection of biomarkers is significance for the diagnosis of diseases at early stage. For this purpose, we herein develop a bidirectional dynamic DNA nano-device for amplified fluorescent detection of tumor marker of mucin 1 (MUC1). The nano-device is constructed by immobilizing two sets of DNA cascade catalytic probes on two opposite directions of a single-stranded DNA tracker to limit probe reactants to a compact space. Once target MUC1 binds to the aptamer sequence, the initiator DNA locked in the duplex DNA substrate can be released to induce DNA-initiated cascade hybridization reactions (DCHRs) simultaneously in two opposite directions along the tracker DNA, accompanying the displacement of two quencher labeled-DNA intermediate initiators to facilitate successively execution of DCHRs on the DNA nano-devices, which results in the separation of fluorophore (FAM) and quencher (Dabycl) to produce substantially recovered fluorescent signals for rapid and sensitive detection of MUC1 with a detection limit down to 0.18 pM. In addition, this strategy also exhibits high selectivity against other interfering proteins and potential application capacity in real serum samples, indicating its promising application prospects in disease diagnosis and treatment.

Lighting-up aptamer transcriptional amplification for highly sensitive and label-free FEN1 detection.

Specific and sensitive detection of flap endonuclease 1 (FEN1), an enzyme biomarker involved in DNA replications and several metabolic pathways, is of high values for the diagnosis of various cancers. In this work, a fluorescence strategy based on transcriptional amplification of lighting-up aptamers for label-free, low background and sensitive monitoring of FEN1 is developed. FEN1 cleaves the 5' flap of the DNA complex probe with double flaps to form a notched dsDNA, which is ligated by T4 DNA ligase to yield fully complementary dsDNA. Subsequently, T7 RNA polymerase binds the promoter region to initiate cyclic transcriptional generation of many RNA aptamers that associate with the malachite green dye to yield highly amplified fluorescence for detecting FEN1 with detection limit as low as 0.22 pM in a selective way. In addition, the method can achieve diluted serum monitoring of low concentrations of FEN1, exhibiting its potential for the diagnosis of early-stage cancers.

Label-free and sensitive fluorescent sensing of ten-eleven translocation enzyme via cascaded recycling signal amplifications.

The sensitive detection of ten-eleven translocation (TET) dioxygenase is of significance for understanding the demethylation mechanism of 5-methylocytosine (5mC), which is responsible for a wide range of biological functions that can affect gene expression in eukaryotic species. Here, a non-label and sensitive fluorescence biosensing method for TET assay using TET1 as the model target molecule is established on the basis of target-triggered Mg2+-dependent DNAzyme and catalytic hairpin assembly (CHA)-mediated multiple signal amplification cascades. 5mC sites in the hairpin DNA probe are first oxidized by TET1 into 5-carboxycytosine, which are further reduced by pyridine borane into dihydrouracil, followed by its recognition and cleavage by the USER enzyme to liberate active DNAzyme and G-quadruplex sequences from the probe. The DNAzyme further cyclically cleaves the substrate hairpins to trigger subsequent CHA reaction and DNAzyme cleavage cycles for yielding many G-quadruplex strands. Thioflavin T dye then intercalates into G-quadruplexes to cause a magnificent increase of fluorescence for high sensitivity assay of TET1 with 47 fM detection limit. And, application of this method for TET1 monitoring in diluted serum has also been confirmed.