Ultrasensitive Detection Of Nucleic Acids Research Articles

Bimetallic interface on an optical microfiber nucleic acid sensor: Early detection in body fluids

Nucleic acid detection, which is not limited by the “window period”, is an efficacious approach for detecting human immunodeficiency virus (HIV) infection at an early time. Here, method with a short turn-around time, portability, connectivity and cost-effectiveness is developed to suppress HIV disease in the early stage, especially in remote areas. Specifically, an optical microfiber sensor enhanced by a bimetallic interface is developed for the HIV-related DNA detection in body fluids. A bimetallic interface is constructed on the optical microfiber surface to ensure that target nucleic acid hybridization occurs between two gold nanostructures where the evanescent field is strongest. The selective enhancement of the evanescent field at the binding site prevents nonspecific amplification of the whole fiber surface, which may lead to false-positive results. With the enhancement in the bimetallic interface, the sensor can detect HIV-related DNA at concentrations from 1 aM to 0.1 pM, with a limit of detection (LOD) of 0.3 aM. The portability, flexibility and selectivity of the sensor enable its application in detecting in several body fluids, such as whole serum and artificial saliva, with LODs of 1.15 aM and 1.64 aM, respectively. This work provides a powerful tool for diagnosing HIV in early stages. Ultrasensitive nucleic acid detection in artificial saliva also provides a possibility for noninvasive detection.

Ultrasensitive detection of nucleic acid with a CRISPR/Cas12a empowered electrochemical sensor based on antimonene

Ultra-sensitive nucleic acid detection is important for rapid prevention of infectious diseases. Recently, clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) systems with great target specificity and programmability have demonstrated amazing capabilities in the field of nucleic acid detection. However, most CRISPR/Cas systems-based strategies still rely on pre-amplification of nucleic acid, which limits their clinical application in point of care detection. Here, we designed a novel electrochemical CRISPR/Cas biosensor (called E-Sb CRISPR) that utilizes antimonene nanosheets (Sb NSs) modifications. Due to the strong interaction between Sb NSs and single stranded DNA (ssDNA), E-Sb CRISPR exhibited specific nucleic acid detection capabilities with a detection limit of 100 aM within 35 min. Notably, the sensor showed excellent selectivity to target DNA in serum in the presence of nucleus acid extracted from other viruses. The excellent stability (8 weeks) and reproducibility (50 cycles) of this sensor were observed. By developing microcircuits and software systems, rapid acquisition of detection results on mobile electronic devices is possible. Therefore, the E-Sb CRISPR showed great promise as a scalable nucleic acid detection platform for early diagnosis.

Postamplifying Cas12a Activation through Hybridization Chain Reaction-Triggered Fluorescent Nanocluster Formation for Ultrasensitive Nucleic Acid Detection.

CRISPR/Cas12a-based biosensing is advancing rapidly; however, achieving sensitive and cost-effective reporting of Cas12a activation remains a challenge. In response, we have developed a label-free system capable of postamplifying Cas12a activation by integrating hybridization chain reaction (HCR) and DNA-copper nanoclusters (DNA-CuNCs). The trans-cleavage of Cas12a triggers a silenced HCR, leading to the in situ assembly of fluorescent DNA-CuNCs, allowing for the turn-on reporting of Cas12a activation. Without preamplification, this assay can detect DNA with a detection limit of 5 fM. Furthermore, when coupled with preamplification, the system achieves exceptional sensitivity, detecting the monkeypox virus (MPXV) plasmid at 1 copy in human serum. In a MPXV pseudovirus-based validation test, the obtained results are in agreement with those obtained by qPCR, reinforcing the robustness of this method. Our study represents the first effort to manipulate DNA-CuNC formation on HCR for highly sensitive and cost-effective reporting of Cas12a, resulting in an efficient synthetic biology-enabled sensing platform for biosafety applications.

Rapid ultra-sensitive nucleic acid detection using plasmonic fiber-optic spectral combs and gold nanoparticle-tagged targets

Nucleic acid (NA) is a widely-used biomarker for viruses. Accurate quantification of NA can provide a reliable basis for point-of-care diagnosis and treatment. Here, we propose a tilted fiber Bragg grating (TFBG)-based plasmonic fiber-optic spectral comb for fast response and ultralow limit NA detection. The TFBG is coated with a gold film which enables excitation of surface plasmon resonance (SPR), and single-stranded probe NAs with known base sequences are assembled on the gold film. To enhance sensitivity of refractive index (RI) for sensing a chosen combination of probe and target NAs around the TFBG surface, gold nanoparticles (AuNPs) are bonded to the target NA molecules as “RI-labels”. The NA combination-induced aggregation of AuNPs induces significant spectral responses in the TFBG that would be below the detection threshold for the NAs in the absence of the AuNPs. The proposed TFBG-SPR NA sensor shows a fast response time of 30 s and an ultra-wide NA detection range from 1 × 10−18 mol/L to 1 × 10−7 mol/L. In the NA concentration range of 1 × 10−12 mol/L (1 pM) to 105 pM, an ultra-high sensitivity of 1.534 dB/lg(pM) is obtained. The sensor achieves an ultra-low limit of detection down to 1.0 × 10−18 mol/L (1 aM), which is more than an order of magnitude lower than the previous reports. The proposed sensor not only shows potentials in practical applications of NA detection, but also provides a new way for TFBG-SPR biochemical sensors to achieve higher RI sensitivity.

Ultrasensitive detection of nucleic acid based on a novel isothermal amplification

The present study describes a novel technology referred to as reciprocal two single-stranded DNA (ssDNA) probe-mediated isothermal amplification (TPIA) that enables target nucleic acid detection. TPIA extends the ssDNA probe A into ultralong ssDNA products with tandem repeats of hairpin and ssDNA probe B to form double-stranded DNA hairpins of varying lengths via continuously repeated polymerization and displacement. The entire TPIA process was supervised in real time with the assistance of fluorescence techniques. By exploiting this new mechanism, the system can be used as an autocephalous module to provide a versatile tool for a biosensing program. The ssDNA probe B was relationally designed to form a stem-loop structure (HP), which can only unwind through specific recognition by the target nucleic acid. The HP stem region was then exposed and HP was rearranged to serve as the ssDNA probe B in the TPIA system after HP unfolding. The novel design strategy was exploited to specifically assay different target microRNAs (miR-21 and miR-221) with a low limit of detection on the 10 and 1 fM level, respectively. This method has great potential for nucleic acid detection in biomedical research and clinical diagnosis by rationally redesigning the template.

Key role of an alpha-helical lid in driving the target DNA toward catalysis in CRISPR-Cas12a.

CRISPR-Cas12a is an emerging gene editing system that came to the forefront with innovative applications such as rapid and ultrasensitive nucleic acid detection. At the core of this molecular complex, the RNA-guided Cas12a enzyme cleaves double-stranded DNA using a single catalytic cleft in the RuvC domain. This opens an overarching question on how the spatially distant DNA target strand (TS) traverses toward the RuvC domain for its accommodation and subsequent cleavage in the catalytic core. Here, continuous tens of microsecond-long molecular dynamics and free-energy simulations reveal that an α-helical lid, located within the RuvC domain, plays a pivotal role in the traversal of the TS by anchoring the crRNA:TS hybrid and elegantly guiding the TS toward the RuvC core, as also corroborated by DNA cleavage experiments. In this mechanism, the Rec2 domain drives the DNA target strand toward the RuvC catalytic cleft, owing to concerted motions with the Nuc domain. While the REC2 domain pushes the TS inward into the core of the complex with its short alpha helices, the Nuc domain aids the bending and accommodation of the TS within the RuvC core by bending inward. The identified intermediates provide information on the critical residues involved in the biophysical process, holding promises for future engineering strategies aimed at improving the overall Cas12a activity.

A point-of-care microfluidic biosensing system for rapid and ultrasensitive nucleic acid detection from clinical samples.

Rapid and ultrasensitive point-of-care RNA detection plays a critical role in the diagnosis and management of various infectious diseases. The gold-standard detection method of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is ultrasensitive and accurate yet limited by the lengthy turnaround time (1-2 days). On the other hand, an antigen test offers rapid at-home detection (typically ~15 min) but suffers from low sensitivity and high false-negative rates. An ideal point-of-care diagnostic device would combine the merits of PCR-level sensitivity and rapid sample-to-result workflow comparable to antigen testing. However, the existing detection platforms typically possess superior sensitivity or rapid sample-to-result time, but not both. This paper reports a point-of-care microfluidic device that offers ultrasensitive yet rapid detection of viral RNA from clinical samples. The device consists of a microfluidic chip for precisely manipulating small volumes of samples, a miniaturized heater for viral lysis and ribonuclease inactivation, a Cas13a-electrochemical sensor for target preamplification-free and ultrasensitive RNA detection, and a smartphone-compatible potentiostat for data acquisition. As demonstrations, the devices achieve the detection of heat-inactivated SARS-CoV-2 samples with a limit of detection down to 10 aM within 25 minutes, which is comparable to the sensitivity of RT-PCR and rapidness of an antigen test. The platform also successfully distinguishes all nine positive unprocessed clinical SARS-CoV-2 nasopharyngeal swab samples from four negative samples within 25 minutes of sample-to-result time. Together, this device provides a point-of-care solution that can be deployed in diverse settings beyond laboratory environments for rapid and accurate detection of RNA from clinical samples. The device can potentially be expandable to detect other viral targets, such as human immunodeficiency virus self-testing and Zika virus, where rapid and ultrasensitive point-of-care detection is required.

Engineered LwaCas13a with enhanced collateral activity for nucleic acid detection.

Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 13 (Cas13) has been rapidly developed for nucleic-acid-based diagnostics by using its characteristic collateral activity. Despite the recent progress in optimizing the Cas13 system for the detection of nucleic acids, engineering Cas13 protein with enhanced collateral activity has been challenging, mostly because of its complex structural dynamics. Here we successfully employed a novel strategy to engineer the Leptotrichia wadei (Lwa)Cas13a by inserting different RNA-binding domains into a unique active-site-proximal loop within its higher eukaryotes and prokaryotes nucleotide-binding domain. Two LwaCas13a variants showed enhanced collateral activity and improved sensitivity over the wild type in various buffer conditions. By combining with an electrochemical method, our variants detected the SARS-CoV-2 genome at attomolar concentrations from both inactive viral and unextracted clinical samples, without target preamplification. Our engineered LwaCas13a enzymes with enhanced collateral activity are ready to be integrated into other Cas13a-based platforms for ultrasensitive detection of nucleic acids.

Nanozyme-strip for rapid and ultrasensitive nucleic acid detection of SARS-CoV-2

The coronavirus disease 2019 (COVID-19) pandemic has created a huge demand for sensitive and rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The current gold standard for SARS-CoV-2 detection is reverse transcription-polymerase chain reaction (RT-PCR)-based nucleic acid amplification. However, RT-PCR is time consuming and requires specialists and large instruments that are unattainable for point-of-care testing (POCT). To develop POCT for SARS-CoV-2, we combined recombinase polymerase amplification (RPA) and FeS2 nanozyme strips to achieve facile nucleic acid amplification and subsequent colorimetric signal enhancement based on the high peroxidase-like activity of the FeS2 nanozymes. This method showed a nucleic acid limit of detection (LOD) for SARS-CoV-2 of 200 copies/mL, close to that of RT-PCR. The unique catalytic properties of the FeS2 nanozymes enabled the nanozyme-strip to amplify colorimetric signals via the nontoxic 3,3′,5,5′-tetramethylbenzidine (TMB) substrate. Importantly, the detection of clinical samples of human papilloma virus type 16 (HPV-16) showed 100% agreement with previous RT-PCR results, highlighting the versatility and reliability of this method. Our findings suggest that nanozyme-based nucleic acid detection has great potential in the development of POCT diagnosis for COVID-19 and other viral infections.

Integrating CRISPR and isothermal amplification reactions in single-tubes for ultrasensitive detection of nucleic acids: the SARS-CoV-2 RNA example

<p indent="0mm">Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (CRISPR-Cas) systems have advanced rapidly for the detection of nucleic acids and molecular diagnoses. The sensitivity of techniques directly using CRISPR-Cas systems for target recognition and signal generation is limited by the kinetics of <italic>trans</italic>-cleavage. Thus, CRISPR-Cas systems have been coupled with isothermal amplification techniques. One strategy for integrating CRISPR-Cas and amplification reactions into a single-tube is to place reagents in separate locations within the tube, maintaining optimum conditions for each reaction. A more challenging strategy is to mix all reagents and allow nucleic acid amplification and CRISPR-based detection to proceed in a homogeneous solution. This desirable approach requires substantial understanding of the compatibility of enzymatic reactions, systematic optimization, and appropriate adjustments of the integrated reactions to ensure high sensitivity. Ultrasensitive techniques have been developed for the detection of SARS-CoV-2 in single-tubes. In this review, we highlight the principle, research needs, and challenges of ultrasensitive single-tube RNA detection using CRISPR technology. We stress the importance of understanding the kinetics of <italic>trans</italic>-cleavage activity of CRISPR-Cas systems.

CRISPR/Cas12a-Triggered Chemiluminescence Enhancement Biosensor for Sensitive Detection of Nucleic Acids by Introducing a Tyramide Signal Amplification Strategy.

CRISPR-based biosensors have attracted increasing attention in accurate and sensitive nucleic acid detection. In this work, we report a CRISPR/Cas12a-triggered chemiluminescence enhancement biosensor for the ultrasensitive detection of nucleic acids by introducing tyramide signal amplification for the first time (termed CRICED). The hybrid chain DNA (crDNA) formed by NH2-capture DNA (capDNA) and biotin-recognition DNA (recDNA) was preferentially attached to the magnetic beads (MBs), and the streptavidin-HRP was subsequently introduced to obtain MB@HRP-crDNA. In the presence of the DNA target, the activated CRISPR/Cas12a is capable of randomly cutting initiator DNA (intDNA) into vast short products, and thus the fractured intDNA could not trigger the toehold-mediated DNA-strand displacement reaction (TSDR) event with MB@HRP-crDNA. After the addition of tyramine-AP and H2O2, abundant HRP-tyramine-AP emerges through the covalent attachment of HRP-tyramine, exhibiting enhanced chemiluminescence (CL) signals or visual image readouts. By virtue of this biosensor, we achieved high sensitivity of synthetic DNA target and amplified DNA plasmid using recombinase polymerase amplification (RPA) as low as 17 pM and single-copy detection, respectively. Our proposed CRICED was further evaluated to test 20 HPV clinical samples, showing a superior sensitivity of 87.50% and specificity of 100.00%. Consequently, the CRICED platform could be an attractive means for ultrasensitive and imaging detection of nucleic acids and holds a promising strategy for the practical application of CRISPR-based diagnostics.

Enhanced chemiluminescence imaging sensor for ultrasensitive detection of nucleic acids based on HCR-CRISPR/Cas12a

CRISPR/Cas systems have ignited increasing attention in accurate and sensitive nucleic acids detection. In this work, we proposed the first CRISPR/Cas12a-based chemiluminescence enhancement biosensor by employing HCR amplifying strategy (CLE-CRISPR) for nucleic acids detection, which shows the advantages of high sensitivity and specificity, low-cost, visual imaging by comparison to reported biosensors. Upon the DNA target recognition, the activated CRISPR/Cas12a enabled randomly cutting initiator DNA (intDNA) into vast short products, which could not trigger the toehold-mediated DNA-strand displacement reaction (TSDR) with MB@crDNA. Thereby, the terminus of crDNA induced the hybridization chain reaction (HCR) with the coexistence of two hairpins (H1 and H2), forming a long double-stranded DNA framework. The attached streptavidin-AP yielded a conspicuous CL signal or visual imaging directly related to the DNA target concentration. The proposed CLE-CRISPR platform exhibited excellent sensitivity, with a relatively low detection limit at 3 pM for synthetic DNA target and single copy detection for plasmid by combining recombinase polymerase amplification (RPA) kit. We further validated the practical application of this platform using HPV clinical samples, achieving superior sensitivity and specificity of 88.89% and 100%, respectively. We believe that this work not only extends the application scope of CRISPR/Cas12a, but also devotes a new approach for clinical diagnosis.

DropCRISPR: A LAMP-Cas12a based digital method for ultrasensitive detection of nucleic acid

Since their discovery, CRISPR/Cas systems have been extensively exploited in nucleic acid biosensing. However, the vast majority of contemporary platforms offer only qualitative detection of nucleic acid, and fail to realize ultrasensitive quantitative detection. Herein, we report a digital droplet-based platform (DropCRISPR), which combines loop-mediated isothermal amplification (LAMP) with CRISPR/Cas12a to realize ultrasensitive and quantitative detection of nucleic acids. This is achieved through a novel two-step microfluidic system which combines droplet LAMP with a picoinjector capable of injecting the required CRISPR/Cas12a reagents into each droplet. This method circumvents the temperature incompatibilities of LAMP and CRISPR/Cas12a and avoids mutual interference between amplification reaction and CRISPR detection. Ultrasensitive detection (at fM level) was achieved for a model plasmid containing the invA gene of Salmonella typhimurium (St), with detection down to 102 cfu/mL being achieved in pure bacterial culture. Additionally, we demonstrate that the DropCRISPR platform is capable of detecting St in raw milk samples without additional nucleic acid extraction. The sensitivity and robustness of the DropCRISPR further demonstrates the potential of CRISPR/Cas-based diagnostic platforms, particularly when combined with state-of-the-art microfluidic architectures.

DNA Hairpins and Dumbbell-Wheel Transitions Amplified Walking Nanomachine for Ultrasensitive Nucleic Acid Detection.

Nucleic acids, including circulating tumor DNA (ctDNA), microRNA, and virus DNA/RNA, have been widely applied as potential disease biomarkers for early clinical diagnosis. In this study, we present a concept of DNA nanostructures transitions for the construction of DNA bipedal walking nanomachine, which integrates dual signal amplification for direct nucleic acid assay. DNA hairpins transition is developed to facilitate the generation of multiple target sequences; meanwhile, the subsequent DNA dumbbell-wheel transition is controlled to achieve the bipedal walker, which cleaves multiple tracks around electrode surface. Through combination of strand displacement reaction and digestion cycles, DNA monolayer at the electrode interface could be engineered and target-induced signal variation is realized. In addition, pH-assisted detachable intermolecular DNA triplex design is utilized for the regeneration of electrochemical biosensor. The high consistency between this work and standard quantitative polymerase chain reaction is validated. Moreover, the feasibilities of this biosensor to detect ctDNA and SARS-CoV-2 RNA in clinical samples are demonstrated with satisfactory accuracy and reliability. Therefore, the proposed approach has great potential applications for nucleic acid based clinical diagnostics.

Single-Molecule Detection of Nucleic Acids via Liposome Signal Amplification in Mass Spectrometry.

A single-molecule detection method was developed for nucleic acids based on mass spectrometry counting single liposome particles. Before the appearance of symptoms, a negligible amount of nucleic acids and biomarkers for the clinical diagnosis of the disease were already present. However, it is difficult to detect extremely low concentrations of nucleic acids using the current methods. Hence, the establishment of an ultra-sensitive nucleic acid detection technique is urgently needed. Herein, magnetic beads were used to capture target nucleic acids, and liposome particles were employed as mass tags for single-particle measurements. Liposomes were released from magnetic beads via photocatalytic cleavage. Hence, one DNA molecule corresponded to one liposome particle, which could be counted using mass spectrometric measurement. The ultrasensitive detection of DNA (10–18 M) was achieved using this method.

Multi-microsecond molecular dynamics unveils the mechanism of DNA traversal within CRISPR-Cas12a

CRISPR-Cas12a is an emerging gene editing system that recently came to the forefront with innovative applications such as rapid and ultrasensitive nucleic acid detection. At the core of this molecular complex, the RNA-guided Cas12a enzyme cleaves its target double-stranded DNA using a single catalytic cleft in the RuvC domain. Despite extensive experimental research, it remains highly elusive how the DNA target strand is accommodated within the RuvC core for cleavage, making the “DNA traversal” a burgeoning question.

A CRISPR-Cas12a integrated SERS nanoplatform with chimeric DNA/RNA hairpin guide for ultrasensitive nucleic acid detection

Background: CRISPR-Cas12a has been integrated with nanomaterial-based optical techniques, such as surface-enhanced Raman scattering (SERS), to formulate a powerful amplification-free nucleic acid detection system. However, nanomaterials impose steric hindrance to limit the accessibility of CRISPR-Cas12a to the narrow gaps (SERS hot spots) among nanoparticles (NPs) for producing a significant change in signals after nucleic acid detection.Methods: To overcome this restriction, we specifically design chimeric DNA/RNA hairpins (displacers) that can be destabilized by activated CRISPR-Cas12a in the presence of target DNA, liberating excessive RNA that can disintegrate a core-satellite nanocluster via toehold-mediated strand displacement for orchestrating a promising “on-off” nucleic acid biosensor. The core-satellite nanocluster comprises a large gold nanoparticle (AuNP) core surrounded by small AuNPs with Raman tags via DNA hybridization as an ultrabright Raman reporter, and its disassembly leads to a drastic decrease of SERS intensity as signal readouts. We further introduce a magnetic core to the large AuNPs that can facilitate their separation from the disassembled nanostructures to suppress the background for improving detection sensitivity.Results: As a proof-of-concept study, our findings showed that the application of displacers was more effective in decreasing the SERS intensity of the system and attained a better limit of detection (LOD, 10 aM) than that by directly using activated CRISPR-Cas12a, with high selectivity and stability for nucleic acid detection. Introducing magnetic-responsive functionality to our system further improves the LOD to 1 aM.Conclusion: Our work not only offers a platform to sensitively and selectively probe nucleic acids without pre-amplification but also provides new insights into the design of the CRISPR-Cas12a/SERS integrated system to resolve the steric hindrance of nanomaterials for constructing biosensors.

CRISPR/Cas13-Based Approaches for Ultrasensitive and Specific Detection of microRNAs

MicroRNAs (miRNAs) have a prominent role in virtually every aspect of cell biology. Due to the small size of mature miRNAs, the high degree of similarity between miRNA family members, and the low abundance of miRNAs in body fluids, miRNA expression profiling is technically challenging. Biosensors based on electrochemical detection for nucleic acids are a novel category of inexpensive and very sensitive diagnostic tools. On the other hand, after recognizing the target sequence, specific CRISPR-associated proteins, including orthologues of Cas12, Cas13, and Cas14, exhibit collateral nonspecific catalytic activities that can be employed for specific and ultrasensitive nucleic acid detection from clinically relevant samples. Recently, several platforms have been developed, connecting the benefits of enzyme-assisted signal amplification and enzyme-free amplification biosensing technologies with CRISPR-based approaches for miRNA detection. Together, they provide high sensitivity, precision, and fewer limitations in diagnosis through efficient sensors at a low cost and a simple miniaturized readout. This review provides an overview of several CRISPR-based biosensing platforms that have been developed and successfully applied for ultrasensitive and specific miRNA detection.

Target dual-recycling-induced bipedal DNA walker and Bi2WO6/Bi2S3 cascade amplification strategy in photoelectrochemical biosensor for TP53 detection

A novel photoelectrochemical (PEC) biosensor was proposed for the ultrasensitive detection of nucleic acids, which coupled with an amplification strategy the bipedal DNA walker triggered dual recycle and Bismuth tungstate/Bismuth sulfide composite material (Bi2WO6/Bi2S3) as an excellent photoelectric layer on the surface of electrode. Ag2S quantum dots (QDs) as tags conjugated with DNA strand 2 (S2) were introduced on the electrode to increase the photocurrent response. With the participation of exonuclease III (Exo Ⅲ), the triple helix conformation of DNA hairpin 1, 2 (H1, H2) and target was disassembled, and the double bipedal DNA walkers were created to participate in next recycle. Catalyzed by Exo Ⅲ, the photocurrent signal dropped as the Ag2S QDs were forced away from the electrode surface. Based on the bipedal DNA walker triggered dual recycle cascade amplification strategy, the proposed PEC biosensor was used to detect target DNA TP53. Under optimized conditions, the photocurrent responded to target concentration variation from 10 fM to 1 μM with a detection limit of 5.83 fM. This work provides an effective method for the detection of nucleic acids biomarkers.

Ultrasensitive nucleic acid detection based on phosphorothioated hairpin-assisted isothermal amplification

Herein, we describe a phosphorothioated hairpin-assisted isothermal amplification (PHAmp) method for detection of a target nucleic acid. The hairpin probe (HP) is designed to contain a 5′ phosphorothioate (PS)-modified overhang, a target recognition site, and a 3′ self-priming (SP) region. Upon binding to the target nucleic acid, the HP opens and the SP region is rearranged to serve as a primer. The subsequent process of strand displacement DNA synthesis recycles the bound target to open another HP and produces an extended HP (EP) with a PS-DNA/DNA duplex at the end, which would be readily denatured due to its reduced thermal stability. The trigger then binds to the denatured 3′ end of the EP and is extended, producing an intermediate double-stranded (ds) DNA product (IP). The trigger also binds to the denatured 3′ end of the IP, and its extension produces the final dsDNA product along with concomitant displacement and recycling of EP. By monitoring the dsDNA products, the target nucleic acid can be identified down to 0.29 fM with a wide dynamic range from 1 nM to 1 fM yielding an excellent specificity to discriminate even a single base-mismatched target. The unique design principle could provide new insights into the development of novel isothermal amplification methods for nucleic acid detection.