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.

Target-responsive triplex aptamer nanoswitch enables label-free and ultrasensitive detection of antibody in human serum via lighting-up RNA aptamer transcriptions.

Accurate and sensitive monitoring of the concentration change of anti-digoxigenin (Anti-Dig) antibody is of great importance for diagnosing infectious and immunological diseases. Combining a novel triplex aptamer nanoswitch and the high signal-to-noise ratio of lighting-up RNA aptamer signal amplification, a label-free and ultrasensitive fluorescent sensing approach for detecting Anti-Dig antibodies is described. The target Anti-Dig antibodies recognize and bind with the nanoswitch to open its triplex helix stem structure to release Taq DNA polymerase and short ssDNA primer simultaneously, which activates the Taq DNA polymerase to initiate downstream strand extension of ssDNA primer to yield specific dsDNA containing RNA promoter sequence. T7 RNA polymerase recognizes and binds to these promoter sequences to initiate RNA transcription reaction to produce many RNA aptamer sequences. These aptamers can recognize and bind with Malachite Green (MG) dye specifically and produce highly amplified fluorescent signal for monitoring Anti-Dig antibodies from 50 pM to 50 nM with a detection limit down to 33 pM. The method also exhibits high selectivity for Anti-Dig antibodies and can be used to discriminate trace Anti-Dig antibodies in diluted serum samples. Our method is superior to many immunization-based Anti-Dig antibody detection methods and thus holds great potential for monitoring disease progression and efficacy.

Simultaneous and Sensitive Sensing of Intracellular MicroRNA and mRNA for the Detection of the PI3K/AKT Signaling Pathway in Live Cells.

Simultaneous detection of the concentration variations of microRNA-221 (miRNA-221) and PTEN mRNA molecules in the PI3K/AKT signaling pathway is of significance to elucidate cancer cell migration and invasion, which is useful for cancer diagnosis and therapy. In this work, we show the biodegradable MnO2 nanosheet-assisted and target-triggered DNAzyme recycling signal amplification cascaded approach for the specific detection of the PI3K/AKT signaling pathway in live cells via simultaneous and sensitive monitoring of the variation of intracellular miRNA-221 and PTEN mRNA. Our nanoprobes enable highly sensitive and multiplexed sensing of miRNA-221 and PTEN mRNA with low detection limits of 23.6 and 0.59 pM in vitro, respectively, due to the signal amplification cascades. Importantly, the nanoprobes can be readily delivered into cancer cells and the MnO2 nanosheets can be degraded by intracellular glutathione to release the Mn2+ cofactors to trigger multiple DNAzyme recycling cycles to show highly enhanced fluorescence at different wavelengths to realize sensitive and multiplexed imaging of PTEN mRNA and miRNA-221 for detecting the PI3K/AKT signaling pathway. Moreover, the regulation of PTEN mRNA expression by miRNA-221 upon stimulation by various drugs can also be verified by our method, indicating its promising potentials for both disease diagnosis and drug screening.

Hg2+-triggered cascade strand displacement assisted CRISPR-Cas12a for Hg2+ quantitative detection using a portable glucose meter.

CRISPR-Cas12a is a powerful and programmable tool that has revolutionized the field of biosensing. However, the construction of a CRISPR-Cas12a-mediated portable system for on-site and quantitative detection of mercury ion (Hg2+) has yet to be explored. By integrating a target-triggered cascade toehold-mediated strand displacement reaction (TSDR) and CRISPR-Cas12a, we herein construct a portable on-site biosensor for the quantitative, sensitive, and selective detection of Hg2+ with a glucose meter. The Hg2+ initiates two cascade TSDRs through the T-Hg2+-T interaction to produce multiple double-stranded DNAs that can activate Cas12a's trans-cleavage activity. The Cas12a cleaves the sucrase-modified DNA on the electrode, resulting in the liberation of sucrase into the solution. The freed sucrase can catalyze sucrose to generate glucose, which can be quantitatively monitored by a glucometer. The developed portable biosensor provides a dynamic range of 5 orders of magnitude with a detection limit of 40 fM. This biosensor also displays excellent selectivity and stability for detecting Hg2+. Moreover, environmental water samples are utilized to further verify the robustness and effectiveness of the developed biosensor, highlighting its potential application in environmental monitoring and food safety analysis.

Targeted Delivery of DNA Framework-Encapsulated Native Therapeutic Protein into Cancer Cells.

A protein-based therapy is significantly challenged by the successful delivery of native proteins into the targeted cancer cells. We address this challenge here using an all-sealed divalent aptamer tetrahedral DNA framework (asdTDF) delivery platform, in which the protein drug is encapsulated inside the cavity of the framework stoichiometrically via a reversible chemical bond. The ligase-assisted seal of the nicks results in highly enhanced TDF stability of the against nuclease digestion to effectively protect the therapeutic protein from degradation. In addition, the divalent aptamer sequences incorporated into the framework favor it with a target-specific and efficient delivery capability. Importantly, upon being readily delivered into the targeted cancer cells, endogenous glutathione can trigger the release of the native therapeutic protein from the TDF in a traceless fashion by cleaving the reversible chemical bond, thereby leading to effective apoptosis of the specific cancer cells.

Lighting-up RNA aptamer transcription synchronization amplification for ultrasensitive and label-free imaging of microRNA in single cells.

Sensitive imaging of intracellular microRNAs (miRNAs) in cells is of great significance in clinical diagnoses and disease treatments, and it remains a major challenge to achieve this goal. Herein, we report a new in situ rolling circle transcription synchronization machinery (RCTsm) of lighting-up RNA aptamer strategy for highly sensitive imaging and selective differentiation of miRNA expression levels in cells. Such a RCTsm approach utilizes a DNA promoter to recycle the target miRNAs to trigger the initiation of multiple RCT process for the yield of many lighting-up RNA aptamers. The malachite green dye further binds these aptamers to show significantly enhanced fluorescence for completely label-free detection of the target miRNAs with a high sensitivity in vitro with a low femtomolar detection limit. More importantly, sensitive detection of under-expressed miRNAs in cells and distinct differentiation of the miRNA expression variations in different cells can also be realized with this RCTsm approach in a washing-free format, making it a versatile and useful tool for imaging trace miRNAs in single cells with the great potential for early cancer diagnosis as well as biomedical research.

Efficient and Exponential Rolling Circle Amplification Molecular Network Leads to Ultrasensitive and Label-Free Detection of MicroRNA.

MicroRNAs (miRNAs) are useful biomarkers for the diagnosis of a variety of cancers. However, it is a major challenge to detect miRNAs, considering their high sequence similarity, low concentration, and small size nature. With the establishment of an efficient rolling circle amplification (RCA) molecular network by target-driven polymerization/nicking reactions, we present here an exponential amplification strategy for detecting miRNA in a label-free way with ultrahigh sensitivity. The target miRNA sequences can bind two ssDNA probes to form a junction structure to initiate a dual polymerization/nicking cyclic reaction for the production of many primers, which further trigger multiple RCA reactions in a drastically amplified sequence replication and extension mode for the yield of substantial dsDNAs with various sizes. The SYBR Green I then binds these dsDNAs to induce significantly magnified fluorescence emission for detecting the target miRNA sequences with a detection limit down to 0.86 fM in the linear range between 1 fM and 10 pM. Because of the involvement of the presynthesized circular DNA template, the RCA efficiency is further improved, and such a method can also be used for detecting miRNA in diluted human serum samples, demonstrating its great potential and universality for detecting different nucleic acid sequences for biochemical research and clinical diagnosis applications.

Target-mediated base-mismatch initiation of a non-enzymatic signal amplification network for highly sensitive sensing of Hg2.

Because of its adverse environmental effects, the establishment of convenient methods for monitoring Hg2+ with ultrahigh sensitivity is important to human health. With a new target-mediated base-mismatch initiation of a signal amplification network strategy, we describe the development of a simple fluorescence sensing approach for detecting Hg2+ in water samples with high sensitivity. The assistant DNA probes trigger the catalytic hairpin assembly (CHA) of two elaborately designed hairpins for the formation of many Mg2+-dependent DNAzymes via T-Hg2+-T base mismatch hybridization. Subsequently, the fluorescence-quenched signal probes are cyclically cleaved by these DNAzymes to recover fluorescence and to release lots of secondary target sequences, which synchronously trigger the CHA of the two hairpins to form a signal amplification network to yield drastically enhanced fluorescence for detecting Hg2+ with high sensitivity at 7.9 pM. Moreover, two mismatched bases are incorporated into the hairpin probes to reduce the background noise to further enhance sensitivity. The developed sensing method exhibits excellent selectivity toward Hg2+ and works well for real water samples. The successful implementation of our amplification strategy for the detection of Hg2+ can make this sensing method a non-enzymatic and convenient signal amplification means for detecting other biomolecules.

Cascaded multiple recycling amplifications for aptamer-based ultrasensitive fluorescence detection of protein biomarkers.

Highly sensitive detection of molecular biomarkers plays a significant role in diagnosing various types of diseases at the early stage. We demonstrated in this paper an ultrasensitive aptamer-based fluorescence method for detecting mucin 1 (MUC1) in human serum via a cascaded multiple recycling signal amplification strategy. The MUC1 target molecules present in the samples cause structure switching of the hairpin aptamer probes, which initiates three cascaded recycling cycles for the cleavage of the fluorescently quenched signal probes to recover significant fluorescence for highly sensitive detection of MUC1. The developed method has a linear range from 100 fM to 1 nM for MUC1 detection. Besides, owing to the substantial signal amplification by the integrated and cascaded recycling cycles, a low detection limit of 35 fM is achieved with high selectivity. Moreover, the monitoring of trace MUC1 in human serum can also be realized with such a method, indicating its great potential for highly sensitive detection of different disease biomarkers.

Netlike hybridization chain reaction assembly of DNA nanostructures enables exceptional signal amplification for sensing trace cytokines.

The monitoring and detection of molecular biomarkers play crucial roles in disease diagnosis and treatment. In this work, we proposed a target-responsive netlike hybridization chain reaction (nHCR) DNA nanostructure construction method, which can offer an exceptional signal enhancement, for highly sensitive fluorescence detection of cytokine, interferon-gamma (IFN-γ). The presence of the target cytokine can lead to the conformational change of the aptamer recognition hairpin probes and the liberation of the nHCR initiator strands, which further trigger the nHCR process between two dye-labeled and double hairpin-structured probes to form netlike DNA nanostructures. The formation of the DNA nanostructures brings the dyes into close proximity, resulting in significantly amplified fluorescence resonance energy transfer signals for sensitive and enzyme-free detection of IFN-γ. The present method has a detection limit of 1.2 pM and a dynamic linear range of 5 to 1000 pM for IFN-γ detection. Besides, with the high specificity of the aptamer probe and the significant signal amplification of the nHCR, such an IFN-γ detection strategy shows excellent selectivity and high sensitivity, which can be potentially applied to detect IFN-γ in human serums. With such a demonstration of the detection of IFN-γ, this proposed method can be extended for detecting different types of biomolecules.

Hairpin/DNA ring ternary probes for highly sensitive detection and selective discrimination of microRNA among family members.

The detection and quantification of microRNA (miRNA) plays essential roles in clinical and biomedical research. Yet, it is of major challenge to sense miRNA with high degree of selectivity and sensitivity due to its unique characteristics of short length, similarity of sequence among family members and low abundance. Here, with the design of a new hairpin/DNA ring ternary probe, we describe the development of a rolling circle amplification (RCA) method for sensitively and selectively sensing miRNA from cancer cells. The target miRNA binds the hairpin/DNA ring probes through toehold-mediated strand displacement (TSD) to form the ternary structures, in which the bound miRNA and DNA ring are respectively used as the primer and template to realize RCA, leading to the generation of many repeated metal ion-dependent DNAzyme sequences. The fluorescently quenched hairpin signal probes can be cyclically cleaved by these DNAzyme sequences with co-existence of the corresponding metal ions in buffer to show drastically enhanced fluorescence recovery for highly sensitive sensing of miRNA in the range between 10 fM and 10 nM with a detection limit of 1.51 fM. Besides, owing to the high base variation discrimination ability of TSD, selective detection of the target miRNA among the corresponding family members can be achieved by this method. Moreover, such a method can also be employed to differentiate miRNA expression variations in cancer cells for screening potential therapeutic drugs.

Programmable DNA Ring/Hairpin-Constrained Structure Enables Ligation-Free Rolling Circle Amplification for Imaging mRNAs in Single Cells.

Self-assembled functional DNA structures have proven to be excellent materials for designing and implementing a variety of nanoscale devices. We demonstrate here that a rationally designed and programmable DNA ring/hairpin-constrained structure can achieve in situ ligation-free rolling circle amplification (RCA), which further leads to highly specific, sensitive, and multicolor imaging of mRNA molecules in single cells. Such a structure aims at addressing current challenges in terms of simplicity, sensitivity, and multiplexing capability related to the detection and imaging of intracellular mRNA sequences. With this new DNA ring/hairpin-RCA approach, we are able to detect the target mRNAs with high sensitivity at the subpicomolar levels in vitro. Besides, the multiplexing capability of the DNA structures can be readily realized by barcoding the DNA rings and hairpins with distinct sequences. Due to the excellent sequence recognition ability of the hairpins, the DNA structures exhibit single-base variation discrimination capability for the target mRNA and can be used to image trace amounts of down-expressed mRNAs in single cells. Moreover, drug-dependent mRNA expression variations can also be clearly differentiated by these DNA structures, highlighting the great potential of such structures for early disease diagnosis and for screening possible therapeutic drugs.

Silver ion-stabilized DNA triplexes for completely enzyme-free and sensitive fluorescence detection of transcription factors via catalytic hairpin assembly amplification.

Transcription factors play important roles in gene regulation and have been identified as promising biomarkers for disease diagnosis. On the basis of a new Ag+-stabilized DNA triplex probe and catalytic hairpin assembly (CHA) signal amplification, we have established a completely enzyme-free and sensitive method for simple fluorescence detection of NF-κB p50 (nuclear factor-kappa B), a transcription factor. We found that the employment of Ag+ to stabilize the DNA triplex structure could effectively reduce the background noise. The association of the target NF-κB p50 with the recognition hairpin in the Ag+-stabilized DNA triplex leads to the release of a single stranded DNA, which is used as the trigger to initiate subsequent CHA between a fluorescently quenched signal hairpin and the recognition hairpin in the triplex DNA structure, thereby resulting in drastically amplified fluorescence recovery for sensitive detection of NF-κB p50. This assay method shows a dynamic concentration range of 5 to 150 pM and a detection limit of 1.5 pM for the detection of NF-κB p50. Besides, the presence of the target molecules can also be selectively discriminated from other non-specific proteins. Moreover, the presence of low concentrations of NF-κB p50 in human serum samples could be monitored with this approach. With the successful demonstration for NF-κB p50, such a method can be potentially extended to detecting other transcription factors in a convenient and sensitive manner without using any enzymes for signal amplification.

Bio-cleavable nanoprobes for target-triggered catalytic hairpin assembly amplification detection of microRNAs in live cancer cells.

The monitoring and imaging of intracellular microRNAs (miRNAs) with specific sequences plays a vital role in cell biology as it can potentially elucidate many cellular processes and diseases related to miRNAs in living cells with accurate information. However, the detection of trace amounts of under-expressed intracellular miRNAs in living cells represents one of the current major challenges. In an effort to address this issue, we describe the establishment of an in cell catalytic hairpin assembly (CHA) signal amplification strategy for imaging under-expressed intracellular miRNAs in this work. Gold nanoparticles functionalized with FAM- and TAMRA-labeled hairpins with disulfide bonds in the stems are readily delivered into cells via endocytosis. Glutathione with evaluated concentrations in cancer cells cleaves the disulfide bonds in the hairpins by reduction to release the hairpins, and the target miRNAs further trigger CHA between the two hairpins to form many DNA duplexes, which bring the FAM and TAMRA labels into close proximity to generate apparently enhanced fluorescence resonance energy transfer (FRET) for the sensitive monitoring of low amounts of under-expressed miRNAs in live cancer cells. Using CHA to amplify the signal output and FRET to reduce the background noise, a significantly enhanced signal-to-noise ratio, thereby high sensitivity, over conventional fluorescence imaging can be realized, making our method particularly suitable for monitoring low levels of intracellular species.

A catalytic and dual recycling amplification ATP sensor based on target-driven allosteric structure switching of aptamer beacons.

Abnormal concentrations of ATP are associated with many diseases and cancers, and quantitative detection of ATP is thus of great importance for disease diagnosis and prognosis. In the present work, we report a new dual recycling amplification sensor integrated with catalytic hairpin assembly (CHA) to achieve high sensitivity for fluorescent detection of ATP. The association of the target ATP with the aptamer beacons causes the allosteric structure switching of the aptamer beacons to expose the toehold regions, which hybridize with and unfold the fluorescently quenched hairpin signal probes (HP1) to recycle the target ATP and to trigger CHA between HP1 and the secondary hairpin probes (HP2) to form HP1/HP2 duplexes. Due to the recycling amplification, the presence of ATP leads to the formation of many HP1/HP2 duplexes, generating dramatically amplified fluorescent signals for sensitive detection of ATP. Under optimal experimental conditions, our sensor linearly responds to ATP in the range from 25 to 600nM with a calculated detection limit of 8.2nM. Furthermore, the sensor shows a high selectivity and can also be used to detect ATP in human serums to realize its application for real samples. With the distinct advantage of significant signal amplification without the involvement of any nanomaterial and enzyme, the developed sensor thus holds great potential for simple and sensitive detection of different small molecules and proteins.

A target-responsive autonomous aptamer machine biosensor for enzyme-free and sensitive detection of protein biomarkers.

Highly sensitive and selective detection of protein disease markers is crucial for fundamental research and disease diagnosis. In this work, based on a new target-triggered autonomous aptamer machinery amplification approach, we developed a simple and sensitive sensing platform for the detection of a cancer biomarker, platelet-derived growth factor BB (PDGF-BB), in human sera. The target PDGF-BB binds the fluorescently quenched hairpin aptamer probes and causes structural and conformational changes in the aptamers to recover their fluorescence. The presence of partial duplex DNA fuel strands further leads to the recycling of the target proteins and the functioning of the aptamer machine autonomously to amplify the fluorescence emission significantly, thereby resulting in sensitive detection of PDGF-BB down to the low picomolar level. Besides, due to the high specificity of the aptamer, such a protein detection method shows high selectivity and can be employed to detect PDGF-BB in human sera. Featuring the advantages of being enzyme-free and nanomaterial-free for signal amplification, as well as homogeneous detection, the developed approach thus holds great potential for the construction of various aptasensors for the convenient and sensitive detection of different 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.

A DNA-Fueled and Catalytic Molecule Machine Lights Up Trace Under-Expressed MicroRNAs in Living Cells.

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.

Biodegradable MnO2 Nanosheet-Mediated Signal Amplification in Living Cells Enables Sensitive Detection of Down-Regulated Intracellular MicroRNA.

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.

Metal-ion dependent DNAzyme recycling amplification for sensitive and homogeneous immuno-proximity binding assay of α-fetoprotein biomarker.

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 500pM with the detection limit of 1.8pM. 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.