Dye-labeled Probes Research Articles

DNA-PAINT Probe Modifications Support High-Resolution Imaging with Shorter Binding Domains.

DNA-based Points Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) is an effective super resolution microscopy technique, and its optimization is key to improve nanoscale detection. The state-of-the-art improvements that are at the base of this optimization have been first routinely validated on DNA nanostructure devices before being tested on biological samples. This allows researchers to finely tune DNA-PAINT imaging features in a more controllable in vitro environment. Dye-labeled oligonucleotide probes with short hybridization domains can expand DNA-PAINT's detection by targeting short nucleotide sequences and improving resolution, speed, and multiplexing. However, developing these probes is challenging as their brief bound state makes them difficult to capture under routine imaging conditions. To extend dwell binding times and promote duplex stability, we introduced structural and chemical modifications to our imager probes. The modifications included mini-hairpins and/or Bridged Nucleic Acids (BNA); both of which increase the thermomechanical stability of a DNA duplex. Using this approach we demonstrate DNA-PAINT imaging with approximately 5 nm resolution using a 4-nucleotide hybridization domain that is 43% shorter than previously reported probes. Imager probes with such short hybridization domains are key for improving detection on DNA nanostructure devices because they have the capability to target a larger number of binding domains per localization unit. This is essential for metrology applications such as Nucleic Acid Memory (NAM) where the information density is dependent on the binding site length. The selected imager probes reported here present imaging resolution equivalent to current state-of-the-art DNA-PAINT probes, creating a strategy to image shorter DNA domains for nanoscience and nanotechnology alike.

Development of a multiplex one-tube loop-mediated isothermal amplification method for rapidly simultaneous detection of the mecA and nuc genes in methicillin-resistant Staphylococcus aureus

This study developed a multiplex one-tube loop-mediated isothermal amplification method using fluorescent dye-labeled DNA probes (mfLAMP) for the rapid simultaneous detection of the nuc (S. aureus-specific marker) and mecA genes (PBP2a-specific marker) in methicillin-resistant Staphylococcus aureus (MRSA). In the mfLAMP method, a graphene oxide (GO) was used as an effective adsorbent to eliminate free fluorescent dye-labeled DNA probes (mecAf-probe: Cy5-ssDNA-Biotin and nucf-probe: FAM-ssDNA-Biotin) present in negative results. The samples were then measured using a developed program that enable simultaneous detection of fluorescein (FAM) and Cyanine5 (Cy5). Green fluorescence represents the detection of the nuc gene, red fluorescence indicates the detection of the mecA gene, and yellow fluorescence signifies the detection of MRSA. The mfLAMP was specific to MRSA and could detect 10°CFU/mL of MRSA in standard buffer and artificially inoculated irrigation water. The recovery rate from contaminated irrigation water ranged from 100.41 to 109.85 %. This study is the first to report the mfLAMP using GO, which could be point-of-care testing for the rapid and simultaneous detection of MRSA's nuc and mecA genes. The results demonstrate that the mfLAMP will be a novel tool for simultaneously detecting more than two target genes or bacteria within 1 h without an enrichment step.

Signal-On Fluorescence Biosensor for Detection of miRNA-21 Based on ROX labeled Specific Stem-Loop Probe.

The abnormal expression of microRNA (miRNA) influences RNA transcription and protein translation, leading to tumor progression and metastasis. Today, reliably identifying aberrant miRNA expression remains challenging, especially when employing quick, simple, and portable detection methods. This study aimed to diagnose and detect the miR-21 biomarker with high sensitivity and specificity. Our detection approach involves immobilizing ROX dye-labeled single-stranded DNA probes (ROX-labeled ssDNA) onto MWCNTs to detect target miRNA-21. Initially, adsorbing ROX-labeled ssDNA onto MWCNTs causes fluorescence quenching of ROX. Subsequently, introducing its complementary DNA (cDNA) forms double-stranded DNA (dsDNA), which results in the desorption and release from MWCNTs, thus restoring ROX fluorescence. The study examined changes in fluorescence intensities before and after hybridization with miRNA-21. The fluorescence emission intensities responded linearly to increases in miR-21 concentration from 10-9 to 3.2 × 10-6 M. The developed fluorescence sensor exhibited a detection limit of 1.12 × 10-9 M. This work demonstrates that using a nano-biosensor based on carbon nanotubes offers a highly sensitive method for the early detection of colorectal cancer (CRC), supplementing existing techniques.

High Fidelity Machine-Learning-Assisted False Positive Discrimination in Loop-Mediated Isothermal Amplification Using Nanopore-Based Sizing and Counting.

Loop-mediated isothermal amplification (LAMP) is a rapid, sensitive, and cost-effective method for developing point-of-care nucleic acid testing due to its isothermal nature. Yet, LAMP can suffer from the issue of false positives, which can compromise the specificity of the results. LAMP false positives typically arise due to contamination, nonspecific amplification, and nonspecific signal reporting (intercalating dyes, colorimetric, turbidity, etc.). While dye-labeled primers or probes have been introduced for multiplexed detection and enhanced specificity in LAMP assays, they carry the risk of reaction inhibition. This inhibition can result from the labeled primers with fluorophores or quenchers and probes that do not fully dissociate during reaction. This work demonstrated a nanopore-based system for probe-free LAMP readouts by employing amplicon sizing and counting, analogous to an electronic version of gel electrophoresis. We first developed a model to explore LAMP kinetics and verified distinct patterns between true and false positives via gel electrophoresis. Subsequently, we implemented nanopore sized counting and calibrated the event charge deficit (ECD) values and frequencies to ensure a fair analysis of amplicon profiles. This sized counting method, integrated with machine learning, achieved 91.67% accuracy for false positive discrimination, enhancing LAMP's reliability for nucleic acid detection.

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.

Evaluation of DNA extraction protocols and real-time PCR-based methods for efficient investigation of pig traces in foods

Industrially processed foods are composed of a complex mixture of molecules combined under specific chemical and physical conditions. Besides their native interactions, most of the ingredients included in processed foods are highly transformed through extreme heat variations, grinding, freezing, pH, and pressure fluctuations in order to reach the desired final product. Due to their complex structure and high level of degradation, processed foods are difficult to analyse. Undeclared components are often detected in processed foods, and accurate diagnostic testing is required to protect those with health, cultural, and religious restrictions. Molecular biology techniques involving PCR are most frequently used for determining the authenticity of foods containing derivatives of living organisms. In the present work, we investigated four different DNA extraction protocols of three commercial kits, two different quantitative PCR (qPCR) techniques, and six different primer pairs. We analysed 96 extracts (12 samples from each of the eight products) by SYBR Green-based qPCR using the two most specific and sensitive primer pairs, and compared these results to those obtained with standard commercial kits that use dual dye-labelled probes. Adopting high-efficiency DNA extraction protocols, our findings highlighted the importance of targeting several small regions of the mitochondrial genome to effectively detect small traces of porcine products, and reduce the risk of false-negative results. Adopting these will ensure that consumers can make accurate and informed choices.

2D MOF Nanosensor-Integrated Digital Droplet Microfluidic Flow Cytometry for In Situ Detection of Multiple miRNAs in Single CTC Cells.

Current circulating tumor cells (CTCs) detection strategies based on surface epithelial markers suffer from low specificity in distinguishing between CTCs and epithelial cells in hematopoietic cell population. Tumor-associated miRNAs within CTCs are emerging as new biomarkers due to their high correlation with tumor development and progress. However, in-situ simultaneous analysis of multiple miRNAs in single CTC cell is still challenging. To overcome this limitation, a digital droplet microfluidic flow cytometry based on biofunctionalized 2D metal-organic framework nanosensor (Nano-DMFC) is developed for in situ detection of dual miRNAs simultaneously in single living breast cancer cells. Here, 2D MOF-based fluorescent resonance energy transfer (FRET) nanosensors are established by conjugating dual-color fluorescence dye-labeled DNA probes on MOF nanosheet surface. In the Nano-DMFC, 2D MOF-based nanoprobes are precisely microinjected into each single-cell encapsulated droplets to achieve dual miRNA characterization in single cancer cell. This Nano-DMFC platform successfully detects dual miRNAs at single-cell resolution in 10 mixed positive MCF-7 cells out of 10 000 negative epithelial cells in serum biomimic samples. Moreover, this Nano-DMFC platform shows good reproductivity in the recovery experiment of spiked blood samples, which demonstrate the high potential for CTC-based cancer early diagnosis and prognosis.

A Dendrimer-Based Time-Gated Concentric FRET Configuration for Multiplexed Sensing

Förster resonance energy transfer (FRET) is widely used for the development of biological probes and sensors. In this context, the norm for multiplexed detection is deployment of multiple probes, each a discrete donor-acceptor pair. Concentric FRET (cFRET) probes enable multiplexed sensing with a single vector but, to date, have only been developed around semiconductor quantum dots, which may limit the scope of biological applications for such probes. Here, we demonstrate that dendrimers labeled with a luminescent terbium complex (Tb) are a viable and advantageous alternative platform for cFRET probes. Polyamidoamine dendrimers were functionalized with Tb, biotin, NeutrAvidin, and three types of dye-labeled oligonucleotide probes to establish a network of competitive and sequential Tb-to-dye and dye-to-dye FRET pathways. These probes were characterized physically and photophysically, and a time-gated multiplexed assay for DNA targets was demonstrated. The time-gating offered by the Tb allowed the rejection of background autofluorescence from serum. More broadly, this dendrimer-based architecture shows that cFRET is a general concept and is an important step toward a new generation of probes for biological sensing.

Selective Detection of an Infection Biomarker by an Osteo-Friend Scaffold: Development of a Multifunctional Artificial Bone Substitute.

Developments in three-dimensional (3D) printing technologies have led to many potential applications in various biomedical fields, especially artificial bone substitutes (ABSs). However, due to the characteristics of artificial materials, biocompatibility and infection remain issues. Here, multifunctional ABSs have been designed to overcome these issues by the inclusion of a biochemical modality that allows simultaneous detection of an infection biomarker by osteo-friend 3D scaffolds. The developed multifunctional scaffolds consist of calcium-deficient hydroxyapatite (CDHA), which has a similar geometric structure and chemical composition to human bone, and gold nanoparticles (Au NPs), which assists osteogenesis and modulates the fluorescence of labels in their microenvironment. The Au NPs were subsequently conjugated with fluorescent dye-labeled probe DNA, which allowed selective interaction with a specific target biomarker, and the fluorescent signal of the dye was temporally quenched by the Au NP-derived Förster resonance energy transfer (FRET). When the probe DNA unfolded to bind to the target biomarker, the fluorescence signal was recovered due to the increased distance between the dye and Au NPs. To demonstrate this sensing mechanism, a microbial oligonucleotide was selected as a target biomarker. Consequently, the multifunctional scaffold simultaneously facilitated osteogenic proliferation and the detection of the infection biomarker.

Fluorescence Detection of miRNA-21 Using Au/Pt Bimetallic Tubular Micromotors Driven by Chemical and Surface Acoustic Wave Forces.

In this study, surface acoustic wave (SAW) systems are described for the removal of molecules that are unbound to micromotors, thereby lowering the detection limit of the cancer-related biomarker miRNA-21. For this purpose, in the first step, mass production of the Au/Pt bimetallic tubular micromotor was performed with a simple membrane template electrodeposition. The motions of catalytic Au/Pt micromotors in peroxide fuel media were analyzed under the SAW field effect. The changes in the micromotor speed were investigated depending on the type and concentration of surfactants in the presence and absence of SAW streaming. Our detection strategy was based on immobilization of probe dye-labeled single-stranded probe DNA (6-carboxyfluorescein dye-labeled-single-stranded DNA) to Au/Pt micromotors that recognize target miRNA-21. Before/after hybridization of miRNA-21 (for both w/o SAW and SAW streaming conditions), the changes in the speed of micromotors and their fluorescence intensities were studied. The response of fluorescence intensities was observed to be linearly varied with the increase of the miRNA-21 concentration from 0.5 to 5 nM under both w/o SAW and with SAW. The resulting fluorescence sensor showed a limit of detection of 0.19 nM, more than 2 folds lower compared to w/o SAW conditions. Thus, the sensor and behaviors of Au/Pt tubular micromotors were improved by acoustic removal systems.

Multiplexed Single-Molecule Experiments Reveal Nucleosome Invasion Dynamics of the Cas9 Genome Editor.

Single-molecule measurements provide detailed mechanistic insights into molecular processes, for example in genome regulation where DNA access is controlled by nucleosomes and the chromatin machinery. However, real-time single-molecule observations of nuclear factors acting on defined chromatin substrates are challenging to perform quantitatively and reproducibly. Here we present XSCAN (multiplexed single-molecule detection of chromatin association), a method to parallelize single-molecule experiments by simultaneous imaging of a nucleosome library, where each nucleosome type carries an identifiable DNA sequence within its nucleosomal DNA. Parallel experiments are subsequently spatially decoded, via the detection of specific binding of dye-labeled DNA probes. We use this method to reveal how the Cas9 nuclease overcomes the nucleosome barrier when invading chromatinized DNA as a function of PAM position.

Droplet Microarray Based on Nanosensing Probe Patterns for Simultaneous Detection of Multiple HIV Retroviral Nucleic Acids.

Multiplexed detection of viral nucleic acids is important for rapid screening of viral infection. In this study, we present a molybdenum disulfide (MoS2) nanosheet-modified dendrimer droplet microarray (DMA) for rapid and sensitive detection of retroviral nucleic acids of human immunodeficiency virus-1 (HIV-1) and human immunodeficiency virus-2 (HIV-2) simultaneously. The DMA platform was fabricated by omniphobic–omniphilic patterning on a surface-grafted dendrimer substrate. Functionalized MoS2 nanosheets modified with fluorescent dye-labeled oligomer probes were prepatterned on positively charged amino-modified omniphilic spots to form a fluorescence resonance energy transfer (FRET) sensing microarray. With the formation of separated microdroplets of sample on the hydrophobic–hydrophilic micropattern, prepatterned oligomer probes specifically hybridized with the target HIV genes and detached from the MoS2 nanosheet surface due to weakening of the adsorption force, leading to fluorescence signal recovery. As a proof of concept, we used this microarray with a small sample size (<150 nL) for simultaneous detection of HIV-1 and HIV-2 nucleic acids with a limit of detection (LOD) of 50 pM. The multiplex detection capability was further demonstrated for simultaneous detection of five viral genes (HIV-1, HIV-2, ORFlab, and N genes of SARS-COV-2 and M gene of Influenza A). This work demonstrated the potential of this novel MoS2-DMA FRET sensing platform for high-throughput multiplexed viral nucleic acid screening.

In Situ Imaging of Pathological Collagen by Electrostatic Repulsion-Destabilized Peptide Probes.

The development of robust collagen assays is crucial in the diagnosis and treatment of various pathological conditions. Peptide probes composed of the (Gly-Pro-Hyp)n sequences have received extensive attention for their remarkable collagen-targeting capability, which unfortunately has been severely impaired by their high triple helical stability. Herein, we report an efficient strategy to reduce the triple helical propensity of the (Gly-Pro-Hyp)n sequences by electrostatic repulsion. A series of peptides consisting of the (Gly-Pro-Hyp)7 sequence and a number of charged amino acid Asp have been investigated, indicating that the presence of six additional Asp pronouncedly weakened the triple helical stability of peptide probe FAM-PCTP-D6 under physiological conditions (pH 7.4). FAM-PCTP-D6 could be directly applied without any pretreatment to recognize denatured collagen with high selectivity, whereas another dye-labeled peptide probe ROX-PCTP-D6 specifically targeted pathological collagen in various connective tissues of animal disease models and human patients. The inclusion of extra charged natural amino acids has been demonstrated as a convenient approach to create biocompatible collagen-targeting peptide probes with much weaker triple helical stability. Without the need for preheating treatment, these electrostatic repulsion-driven peptide probes provide a handy tool for histopathology staining, showing promising applications in collagen-involved diseases.

Femtosecond Laser Pulse Excitation of DNA-Labeled Gold Nanoparticles: Establishing a Quantitative Local Nanothermometer for Biological Applications.

Femtosecond (fs) laser pulsed excitation of plasmonic nanoparticle (NP)-biomolecule conjugates is a promising method to locally heat biological materials. Studies have demonstrated that fs pulses of light can modulate the activity of DNA or proteins when attached to plasmonic NPs; however, the precision over subsequent biological function remains largely undetermined. Specifically, the temperature the localized biomolecules "experience" remains unknown. We used 55 nm gold nanoparticles (AuNPs) displaying double-stranded (ds) DNA to examine how, for dsDNA with different melting temperatures, the laser pulse energy fluence and bulk solution temperature affect the rate of local DNA denaturation. A universal "template" single-stranded DNA was attached to the AuNP surface, and three dye-labeled probe strands, distinct in length and melting temperature, were hybridized to it creating three individual dsDNA-AuNP bioconjugates. The dye-labeled probe strands were used to quantify the rate and amount of DNA release after a given number of light pulses, which was then correlated to the dsDNA denaturation temperature, resulting in a quantitative nanothermometer. The localized DNA denaturation rate could be modulated by more than threefold over the biologically relevant range of 8-53 °C by varying pulse energy fluence, DNA melting temperature, and surrounding bath temperature. With a modified dissociation equation tailored for this system, a "sensed" temperature parameter was extracted and compared to simulated AuNP temperature profiles. Determining actual biological responses in such systems can allow researchers to design precision nanoscale photothermal heating sources.

Palindromic probe-mediated strand displacement amplification for highly sensitive and selective microRNA imaging

MicroRNAs (miRNAs) are involved in a variety of biological processes, and the accurate detection of miRNAs is of great importance for early diagnosis of various cancers. Herein, we have developed a highly sensitive method for the intracellular imaging of miRNAs based on a palindromic probe-induced strand displacement amplification (pSDA). The sensing element is a partly complementary hybrid consisting of two DNA components: one fluorescent dye-labeled signaling probe containing a palindromic sequence and loop-based target recognition site and one quencher moiety-attached locking probe. In the presence of target miRNA, the target species can hybridize with the loop site and release the terminal palindromic fragment, initiating the pSDA reaction. Thus, a considerable amount of fluorescent moieties are spatially separated from the quenchers, generating a dramatically enhanced fluorescence signal. As a result, the target miRNAs can be quantified down to 25 pM with the linear response range over four orders of magnitude. The detection specificity is high enough to eliminate the interference from nontarget miRNAs and other biospecies co-existing in samples, and thus the diseased cells are easily distinguished from healthy cells. Strikingly, the pSDA-based system possesses the desirable capability to discriminate tumor cells from healthy cells, indicating a promising diagnostic tool for the detection of cancers and other diseases in early stage.

A RuO2 Nanosheet as a Novel Quencher-free Platform for the Detection of Nucleic Acids in a Homogeneous Solution.

A fluorescent dye-labeled DNA probe was adsorbed and quenched on the monolayer of RuO2 nanosheets. Significant fluorescent recovery was observed upon the addition of complementary DNA due to desorption of the probe from the surface of the RuO2 nanosheet through duplex formation. The efficiency of fluorescence recovery was higher than that for graphene oxide, which was known as a quencher-free platform for the detection of nucleic acids in a homogeneous solution.

DNA branched junctions induced the enhanced fluorescence recovery of FAM-labeled probes on rGO for detecting Pb2.

The reduced graphene oxide (rGO) could strongly adsorb and quench the fluorescence of dye-labeled single-stranded DNA (ssDNA); thus, it is widely applied in fluorescent sensors. However, these sensors may suffer from a limited sensitivity due to the low fluorescence recovery when adding the complementary DNA (cDNA) sequence. In this work, the powerful DNA branched junctions were constructed to improve the fluorescence recovery of FAM-labeled probe on rGO. In the presence of target Pb2+, the ribonucleotide (rA) in the substrate was cleaved specifically and the catalytic hairpin assembly of three metastable hairpins was further initiated, accompanied by the formation of DNA branched junctions. Then, the liberated Pb2+ could be recyclable. Impressively, the DNA branched junctions not only hybridize with the FAM-labeled probes with a high efficiency, but also are significantly undesirable for the rGO. Thus, a high fluorescence recovery of FAM-labeled probe on rGO was expected. The integration of the high fluorescence recovery and dual-cycle signal amplification endows the sensing strategy with a good performance for Pb2+ detection, including low detection limit (0.17nM), good selectivity, and satisfactory practical applicability. The proposed DNA branched junctions offer a novel avenue to improve the fluorescence recovery of the dye-labeled probes on rGO for biological analysis.

Rational Design of Conformation-Specific Antibodies for Tau Oligomers

Antibodies are powerful tools for biological research and clinical applications owing to their ability to bind targets with high affinity and specificity. To exploit them against neurodegeneration, we are developing antibodies to target most toxic early stage aggregates of the protein tau, whose deposition in the brain is linked with Alzheimer's disease and other tauopathies. We generated a panel of single-domain antibodies targeting selected epitopes within tau, using the ‘cascade method’. Briefly, we designed complementary peptides targeting linear epitopes within the sequence of tau using a library of beta-sheet fragments from the protein data bank. We then grafted each peptide into the CDR3 loop of a VH human scaffold. Using this approach, we produced a library of fourteen antibodies covering systematically the length of tau. We will use these antibodies to understand which epitopes are specifically exposed in toxic tau aggregates. In particular, we will employ them in super-resolved imaging, such as DNA points accumulation for imaging in nanoscale topography (DNA PAINT), of protein aggregates. DNA PAINT enables visualization of structures with resolutions below the diffraction limit using short dye-labelled oligonucleotide probes. Transient binding of a dye-labelled ‘imager strand’ to a ‘docking strand’ (DS), which we conjugated to the C-terminus of DesAbs creates a blinking effect. Hence, a highly resolved image can be reconstructed from plotting the measured positions of the hydrolyzed DNA state. Using this technique to image protein aggregates in human cerebrospinal fluid and serum will provide novel insights into the composition, structure, size and number of aggregates present in those samples.

Exonuclease III-Regulated Target Cyclic Amplification-Based Single Nucleotide Polymorphism Detection Using Ultrathin Ternary Chalcogenide Nanosheets

Herein, we report that the ternary chalcogenide nanosheet exhibits different affinity toward oligonucleotides with different lengths and efficiently quenches the fluorescence of dye-labeled DNA probes. Based on these findings, as a proof-of-concept application, the ternary chalcogenide nanosheet is used as a target cyclic amplification biosensor, showing high specificity in discriminating single-base mismatch. This simple strategy is fast and sensitive for the single nucleotide polymorphism detection. Ultralow detection limit of unlabeled target (250 fM) and high discrimination ratio (5%) in the mixture of perfect match (mutant-type) and single-base mismatch (wild-type) target are achieved. This sensing method is extensively compatible for the single nucleotide polymorphism detection in clinical samples, making it a promising tool for the mutation-based clinical diagnostic and genomic research.

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

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