Exponential Amplification Research Articles

Highly Sensitive One-Pot Isothermal Assay Combining Rolling Circle Amplification and CRISPR/Cas12a for Aflatoxin B1 Detection.

Occurrences of mycotoxins in cereals are widespread throughout the world. However, the lack of efficient and ultrasensitive tests has largely impeded the identification of these substances in actual samples. Herein, a novel one-pot isothermal assay that integrates rolling-circle amplification (RCA) and CRISPR/Cas12a to detect aflatoxin B1 (AFB1) is reported. Upon addition of AFB1 to the magnetic bead functionalized with a duplex of the AFB1 aptamer and its complementary DNA (cDNA), the specific recognition of AFB1 by the aptamer causes the release of cDNA to activate the RCA reaction. Subsequently, the RCA amplicon initiates both trans-cleavage and cis-cleavage activities of the endonuclease Cas12a. The synergistic coupling of RCA and CRISPR/Cas12a enables exponential amplification of cDNA, which further promotes CRISPR/Cas12a to nonspecifically cleave the single-stranded DNA reporters with enhanced detection signals. Remarkably, the CRISPR/Cas12a-assisted one-pot isothermal assay can not only achieve ultrasensitive quantitative detection through fluorescence detection, but also achieve visual detection through a lateral flow strip, which improves accessibility to mycotoxin detection in resource-limited regions. The limit of detection was 0.016 and 0.408 ng/mL, respectively. The proposed assay successfully applies in real samples with satisfactory recoveries from 90 to 114%. This study presents a powerful and versatile method for reliable and ultrasensitive detection of mycotoxins in various applications.

An ultrasensitive fluorescent strategy for 17β-estradiol based on aptamer and isothermal exponential amplification reaction

The abuse of 17β-estradiol (E2) in breeding and the excretion into the environment by human beings result in exposure of E2 to humans. Excessive levels of E2 can cause an adverse effect on the human endocrine systems. Therefore, an efficient and sensitive method for the analysis of E2 is required. Herein, based on the specific capture of aptamer and the digestion od Exonuclease III, efficient amplification of isothermal exponential amplification reaction (EXPAR), a highly sensitive and selective strategy is established for E2 analysis. E2 binds to the aptamer due to their high affinity, preventing the complementary DNA (cDNA) of the aptamer from hybridizing with it. Thus, the cDNA remains unbound and freely available in the solution. Then, Exonuclease III digests the cDNA-aptamer complex, leaving the free cDNA intact. cDNA serves as primers to trigger EXPAR with the help of Bst 2.0 WarmStart DNA polymerase and Nt.BstNBⅠ nicking enzyme, achieving sensitive detection of E2. Without E2, cDNA hybridizes with the aptamer to form double-stranded DNA, which is degraded by Exonuclease III. As a result, the amplification reaction fails to initiate. The approach shows a linear range of 100 aM-10 pM, spanning 5 orders of magnitude, with the limit of detection as low as 36 aM. In addition, the selectivity and reproducibility are great, and the recoveries in tap water and milk ranged from 99.4% to 117.3%, suggesting good practicability. This method may also provide a new possibility for highly sensitive detection of other small-molecule targets via using the corresponding aptamers.

Cross-chiral exponential amplification of an RNA enzyme.

An RNA ligase ribozyme that catalyzes the joining of RNA molecules of the opposite chiral handedness was optimized for the ability to synthesize its own enantiomer from two component fragments. The mirror-image D- and L-ligases operate in concert to provide a system for cross-chiral replication, whereby they catalyze each other's synthesis and undergo mutual amplification at constant temperature, with apparent exponential growth and a doubling time of about 1 h. Neither the D- nor the L-RNA components alone can achieve autocatalytic self-replication. Cross-chiral exponential amplification can be continued indefinitely through a serial-transfer process that provides an ongoing supply of the component D- and L-substrates. Unlike the familiar paradigm of semiconservative nucleic acid replication that relies on Watson-Crick pairing between complementary strands, cross-chiral replication relies on tertiary interactions between structured nucleic acids "across the mirror." There are few examples, outside of biology, of autocatalytic self-replication systems that undergo exponential amplification and there are no prior examples, in either biological or chemical systems, of cross-chiral replication enabling exponential amplification.

Self-Replicating Catalytic Hybridization Assembly of Bipedal DNAzyme Walkers for Enhanced Electrochemiluminescence Bioanalysis.

Dynamic DNA nanodevices, particularly DNA walkers, have proven to be versatile tools for target recognition, signal conversion, and amplification in biosensing. However, their ability to detect low-abundance analytes in complex biological samples is often compromised by limited amplification depth and severe signal leakage. To address these challenges, we developed a simple yet highly efficient strategy to engineer a self-replicating bipedal DNAzyme (SEDY) walker for sensitive and selective electrochemiluminescence (ECL) bioanalysis. Unlike conventional DNA walkers that are typically constructed by catalytic DNA assembly in a single direction, the SEDY walker integrates a self-replicating feedback mechanism that greatly enhances both the selectivity and sensitivity of bioanalysis. First, the SEDY walker is assembled through a target-triggered, enzyme-free, self-replicating catalytic approach, minimizing the risk of undesired side reactions and signal leakage by simplifying reactant complexity. Furthermore, the SEDY walker features newly exposed trigger sequences that facilitate its autonomous replication, leading to a robust and exponential amplification of its products. Our experiments demonstrate that the SEDY walker can sensitively and selectively detect acetamiprid by navigating specific probes within cross-shaped DNA orbits. The ECL biosensor offers a linear detection range from 1 × 10-15 M to 1 × 10-9 M, with a limit of detection as low as 5.8 × 10-16 M. We anticipate that the SEDY walker will be a powerful tool for detecting various analytes in biological applications.

Electrochemiluminescence biosensor for MMP-2 determinationusing CRISPR/Cas13a and EXPAR amplification: a novel approach for anti-aging research.

Matrix metalloproteinase-2 (MMP-2) plays a pivotal role in anti-aging research. Developing advanced detection platforms for MMP-2 with high specificity, sensitivity, and accessibility is crucial. This study introduces a novel electrochemiluminescence (ECL) biosensor for MMP-2 determination, leveraging the CRISPR/Cas13a system and Exponential Amplification Reaction (EXPAR). The biosensor operates by utilizing the T7 RNA polymerase to transcribe RNA from a DNA template upon MMP-2 interaction. This RNA activates Cas13a, leading to signal amplification and ECL detection. The incorporation of the "photoswitch" molecule [Ru(phen)2dppz]2+ streamlines the process by eliminating the need for extensive electrode modification and cleaning. Under optimized conditions, the biosensor achieved an impressive detection limit of 12.8 aM for MMP-2. The platform demonstrated excellent selectivity, reproducibility, and stability, making it highly suitable for detecting MMP-2 in complex biological samples. This innovative approach shows great potential for applications in molecular diagnostics and anti-aging research.

Effect of an external axial magnetic field on thermal convection of an electric conducting fluid in a rotating spherical shell

We investigated the interaction between the thermal convection structure in a rotating spherical shell and a uniformly applied magnetic field acting externally toward the axis of rotation. We considered a system that the inner and outer spherical boundaries are heated and cooled both isothermally and the effects of induced magnetic fields are neglected. The convection structure of this system is characterized by the generation of travelling waves around the axis of rotation. We numerically investigated how this is affected under the magnetic field. The characteristics of the disturbance (i.e., growth rate and phase velocity) were estimated from the exponential amplification period using three-dimensional (3D) simulations and compared with those obtained from linear stability analyses (LSA). In particular, the effects of the magnetic field on the kinetic energy and Nusselt number were investigated for a quasi-steady state using 3D simulations. Our numerical results revealed that the applied magnetic field destabilizes the flow field and enhances heat transfer. This is noteworthy, contrary to the common sense that magnetic field stabilizes the flow field and reduces the heat transfer coefficient. However, the magnetic field strengths at which the kinetic energy and heat transfer coefficient took their maximum values differ slightly.

A-251 Simplification of Molecular Diagnostics for Sexually Transmitted Infection Point-of-Care Testing

Abstract Background HPV-caused cancers contribute to a large disease burden, accounting for 5% of all cancers. It is estimated that over 700,000 people develop HPV-related cancers each year with the majority being cervical cancer. The burden of this disease is disproportionately carried by low-income countries with the highest rates of HPV cervical infections primarily found in sub-Saharan Africa, Latin America, Eastern Europe, and South-East Asia. Many regions in low-resource countries lack adequate access to sensitive point-of-care screening tools, preventing timely diagnosis and treatment. Current low-cost methods, such as Pap smears and visual inspection using acetic acid suffer from low sensitivity, specificity, and reproducibility which limit their effectiveness as a screening modality. Many countries use nucleic acid amplification tests (NAATs) to identify key HPV infections which may progress towards cervical cancer. However, the expenditure for NAAT-capable equipment and reagents are cost-prohibitive for rural and underserved communities. To reduce screening barriers, we developed an isothermal HPV molecular test that can detect all 14 cancer-causing HPV strains using a novel amplification methodology combined with G-quadruplex DNAzyme colorimetric detection. Methods We developed a novel isothermal amplification technique that is related to loop-mediated isothermal amplification (LAMP) but uses modified looped primers to enable exponential amplification at 60°C for 1 hour. The modified primers only require two binding sites compared to 4-6 in LAMP. To detect the amplified products, we used G-quadruplex peroxidase-like DNAzyme probes which reduce ABTS to produce a visually interpreted result. Analytical validation evaluated the method’s limit of detection, specificity, and accuracy. As proof of concept in a relevant matrix, clinical cervical brushings collected ThinPrep Pap media were tested with the new method and compared to an FDA-approved reference qPCR test. Results Repeated experiments demonstrate that the lowest detectable HPV DNA concentration was approximately 1000 genomic copies/µL with no amplification with other pathogens. As proof of concept, the comparison to the reference standard demonstrated high accuracy. Conclusions The application of our assay is intended as a near-patient screening tool with further evaluation by a clinician for confirmation. With an accurate and rapid screening tool, we envision low-resource regions across the world will have the tools to rapidly detect and treat cervical cancers in their early stages.

Advanced design of target-driven self-powered sensor assisted by cascade catalytic strategy

In this work, a self-powered microsensor platform based on enzyme biofuel cells (EBFCs) was developed for intelligent monitoring of disease markers miRNA-451. The cascade catalysis system constructed by using the strategy of enzyme-like ZIF-8 nanocapsule incorporation with biological enzymes, which could simultaneously take into account the specificity of biological enzymes and the high activity of nano-enzymes, significantly promoted the electron transfer between glucose and the bio-anode surface, and improved the sensitivity and stability of the sensing system. Meanwhile, the target-triggered hybridisation chain reaction (HCR) amplification strategy to achieve exponential signal amplification based on accurate recognition, and jointly improve the detection sensitivity. As expected, the micro-sensor platform has a wide linear range of 0.5-1.0 fmol/L with a low limit of detection (LOD) of 0.13 fmol/L (S/N=3) and exhibits excellent selectivity, reproducibility and stability in interference assays under optimal detection conditions. The designed self-powered system is simple to construct, easy to transport and the data transmission mode is intelligent and controllable, which is expected to be used in basic biochemical research, clinical diagnosis and environmental monitoring.

A dual-mode colorimetric and surface-enhanced Raman strategy for chloramphenicol detection based on the Au@Pt nanozyme and EXPAR

The misuse of chloramphenicol (CAP) has resulted in an escalation of antibiotic-resistant bacterial strains, posing a significant threat to human health. Through the fabrication of magnetic multi-“hotspot” nanoflower particles as SERS substrates and combine bimetallic peroxidase Au@Pt, dual-mode detection of CAP was achieved. The preparation of magnetic nanoparticles Fe3O4@Au facilitated rapid aggregation, enriching a SERS signal. Integrated with the exponential amplification reaction (EXPAR) strategy, under isothermal conditions, a highly rate increase of amplicon production was achieved, thereby binding more nanozyme Au@Pt. In the presence of the target CAP, due to specific binding with the aptamer, the EXPAR was activated, generating a substantial amount of amplicons that formed a stable “Y-shaped” structure with magnetic nanoparticle probes and nanozyme probes. With the addition of TMB, the excellent peroxidase-like activity of the nanozyme oxidized it to oxTMB, resulting in changes in both color and Raman signals. This strategy achieved dual-mode ultra-trace detection of CAP. The SERS detection ranged from 1.0 × 10-12 M to 1.0 × 10-6 M, with a detection limit of 4.96 × 10-13 M; the colorimetric method ranged from 2.5 × 10-7 M to 1.0 × 10-8 M, with a detection limit of 9.23 × 10-9 M. This method offers a new approach and insights for the ultra-trace detection of antibiotics.

3D DNAzyme Motor Nanodevice With Self-Powered FRET Amplifier and Self-Supplied H2O2 for Enhancing Human Neutrophil Elastase Profiling and Chemodynamic Therapy in Lung Tumor.

The development of theragnostic nanosystems integrating FRET (fluorescence resonance energy transfer) imaging and chemodynamic therapy (CDT) for accurate diagnosis and effective treatment of lung tumors is still a big challenge. Herein, a peptide-assembled 3D DNAzyme motor nanodevice is engineered for a self-powered FRET amplifier profiling human neutrophil elastase (HNE) and self-supplied H2O2 enhancing CDT. The nanodevice is prepared by depositing AuNPs on ZIF-8, in which ZIF-8 co-loaded the lysosomal targeting peptide-modified copper peroxides (PCPs) and hairpins (H1, H2, and H3), AuNPs are co-labeled by DNAzyme-peptide (DP) conjugate and H3. In the tumor micro-environment, HNE driven 3D DNAzyme walker followed by an exponential amplification constructed by a synergistic cross-activation between hybridization chain reaction and DNAzyme, generating a self-powered FRET amplifier. The FRET amplifier specifically measures HNE with a sensitivity of 0.026pM, and successfully images exogenous HNE in living cells and monitors HNE in mouse models. Moreover, the PCPs can target lysosomes, reducing lysosome escape. The self-supplying H2O2 undertaken by PCPs improves the Cu (II)-catalyzed Fenton-like reaction, effectively causing cell apoptosis to inhibit tumor growth. Significantly, the nanodevice successfully screens inhibitors and discriminates the HNE level in normal and lung cancer tissues, suggesting that the nanodevice provides an effective tool for the diagnosis and treatment of lung tumors.

Highly sensitive detection of RNase H via a novel DNA/RNA heteroduplex combining isothermal exponential amplification strategy

Ribonuclease H (RNase H) plays a crucial role in a variety of cellular processes and is emerging as an essential therapeutic target for many diseases. Various methods have been constructed to assay RNase H activity, but these methods are either time-consuming or have poor sensitivity. In this study, we developed a novel strategy that combined a specially designed DNA/RNA chimeric substrate with exponential amplification reaction (EXPAR) for rapid and sensitive detection of RNase H activity. In the presence of RNase H, the RNA strand of the DNA/RNA heteroduplex was specifically degraded, forming a 3′-hydroxyl group in the locking primer for recognition and extension by DNA polymerase. This ultimately triggered EXPAR and produced a large number of single-stranded DNA, which was monitored in real-time with fluorescent dye. Under optimized conditions, the proposed strategy can detect as low as 6 × 10−10 U/μL of RNase H, which was at least 1000 times more sensitive than several reported methods. Furthermore, we demonstrated the usage of this method for RNase H inhibitor analysis and practical application in complex biological samples, including serum and tumor cell extracts. Therefore, these results suggested that the developed method is a promising tool for highly sensitive detection of RNase H and RNase H-associated disease diagnosis.

Immuno-Rolling Circle Amplification (Immuno-RCA): Biosensing Strategies, Practical Applications, and Future Perspectives.

In the rapidly evolving field of life sciences and biomedicine, detecting low-abundance biomolecules, and ultraweak biosignals presents significant challenges. This has spurred a rapid development of analytical techniques aiming for increased sensitivity and specificity. These advancements, including signal amplification strategies and the integration of biorecognition events, mark a transformative era in bioanalytical precision and accuracy. A prominent method among these innovations is immuno-rolling circle amplification (immuno-RCA) technology, which effectively combines immunoassays with signal amplification via RCA. This process starts when a targeted biomolecule, such as a protein or cell, binds to an immobilized antibody or probe on a substrate. The introduction of a circular DNA template triggers RCA, leading to exponential amplification and significantly enhanced signal intensity, thus the target molecule is detectable and quantifiable even at the single-molecule level. This review provides an overview of the biosensing strategy and extensive practical applications of immuno-RCA in detecting biomarkers. Furthermore, it scrutinizes the limitations inherent to these sensors and sets forth expectations for their future trajectory. This review serves as a valuable reference for advancing immuno-RCA in various domains, such as diagnostics, biomarker discovery, and molecular imaging.

Enzyme-Mediated Duplex Exponential Amplification: A New Platform for Rapid Screening of <i>Hylurgus ligniperda</i>

As the world's second largest timber importer, wood demand in China has been growing extremely rapidly, leading to an increase of 163% from 2009 to 2018. The plant quarantine pest <i>H. ligniperda Fabricius, 1787</i> is an invasive species frequently intercepted at ports. <i>H. ligniperda</i> causes damage mainly to pine and spruce. To improve the efficiency of on-site inspection and the efficacy of early detection, tight quarantine in ports, time-effective identification, and a national surveillance program for high-risk invasive bark beetles are in urgent need. In this study, a simple, fast and accurate classification method for <i>H. ligniperda </i>is established based on the enzyme-mediated duplex exponential amplification (EmDEA) technique. Partial region from <i>inhibitor of apoptosis 2</i> (<I>IAP2)</I> gene was selected as the target and 6 primer/probe combinations were designed. Through selection, the combination of 3-HY-F3, 3-HY-R2 and 3-HY-RNA5 was chosen as the final primer-probe set, as it showed the lowest Ct with highest final fluorescence signal. Method validation and specificity test using 6 other beetle species living on coniferous wood showed that this method is result reliable and specific. Through parameter analysis with positive plasmid, the detection limit was calculated to be 13.6 copies/μL (9×10<sup>-7</sup> ng DNA/ reaction), much higher than conventional molecular methods such as PCR. The whole process including isothermal amplification, data analysis, and result output can be finished in 30 min, which is highly time-effective. Besides, the operation is simple and little training is needed for non-professionals. The application prospects of this rapid screening system include customs screening in ports, wild survey in non-lab situations and early warning system development. The new analysis platform EmDEA, can also be implemented in rapid detection and identification of other forestry pests.

Robust Sequence Design Space for the Isothermal Exponential Amplification of Short Oligonucleotides.

Advances in isothermal amplification techniques have accelerated development in biosensing applications and the design of complex molecular devices. The exponential amplification reaction technique, or EXPAR, is uniquely positioned to process molecular information from short oligonucleotide strands (≈10nucleotides length) typically encountered in molecular computing or microRNA detection. Despite its conceptual simplicity (requiring only a template strand and two enzymes), the issue of nonspecific background amplification has hindered broader adoption. In this work, a new system configuration is established at 37 °C to achieve significantly improved performance. Critical sequence motifs responsible for the excellent signal-to-background profile are identified and generalized as a universal adapter design framework. Orthogonal template sequences generated from the framework are implemented for a triplex reaction and successfully evaluated mixtures of multiple-target inputs in a single-step, one-pot format without the need for exogenous agents.

Single Methylation Sensitive Restriction Endonuclease-Based Cascade Exponential Amplification Assay for Visual Detection of DNA Methylation at Single-Molecule Level.

Function as a potential cancer biomarker, DNA methylation shows great significance in cancer diagnosis, prognosis, and treatment monitoring. While the lack of an ultrasensitive, specific, and accurate method at the single-molecule level hinders the analysis of the exceedingly low levels of DNA methylation. Herein, based on the outstanding recognition and digestion ability of methylation-sensitive restriction endonuclease (MSRE), we established a single MSRE-based cascade exponential amplification method, which requires only two ingeniously designed primers and only one recognition site of MSRE for the detection of DNA methylation. Differentiated by MSRE digestion, the cleaved unmethylated DNA is too short to induce any amplification reactions, while methylated DNA remains intact to trigger cascade exponential amplification and the subsequent CRISPR/Cas12a system. By integrating the two exponential amplification reactions, as low as 1 aM methylated DNA can be accurately detected, which corresponds to 6 molecules in a 10 μL system, indicating that our method is more sensitive than single amplification-based methods with the ability to detect DNA methylation at the single-molecule level. In addition, 0.1% methylated DNA can be effectively distinguished from large amounts of unmethylated DNA. Our method is further introduced to exploit the expression difference of DNA methylation among normal cells and cancer cells. Moreover, the visual detection of DNA methylation is also realized by the full hybridization between amplification products and the crRNA of CRISPR/Cas12a. Therefore, the proposed method has great potential to be a promising and robust bisulfite-free method for the detection of DNA methylation at the single-molecule level, which is of great importance for early diagnosis of cancer.

Capillarity-powered and CRISPR/Cas12a-responsive DNA hydrogel distance sensor for highly sensitive visual detection of HPV DNA

The rapid and specific identification and sensitive detection of human papillomavirus (HPV) infection is critical for preventing cervical cancer, particularly in resource-limited regions. In this work, we hope to propose a capillarity-powered and CRISPR/Cas12a-responsive DNA hydrogel distance sensor for point-of-care (POC) DNA testing. Using the thermal reversibility of DNA hydrogel and capillarity, the novel DNA hydrogel distance sensor can be rapidly and simply constructed by loading an ultra-thin CRISPR/Cas12a-responsive DNA-crosslinked hydrogel film at the end of the capillary tube. The target DNA-specific recombinase polymerase reaction (RPA) amplicons activate the trans-cleavage activity of the Cas12a enzyme, cleaving the crosslinked DNA in hydrogel film, and causing an increase of hydrogel's permeability. As a result, a sample solution containing target DNA travels into the capillary tube at a longer distance compared to the negative samples. Reading the solution traveling distance in capillary tubes, the novel sensor realizes target DNA detection without any special equipment. Benefiting from the exponential target amplification of RPA and multiple turnover response of trans-cleavage of CRISPR/Cas12a, the developed sensor can visually and specifically detect as low as 1 aM HPV 16 DNA within 30 min. These outstanding features, including exceptional sensitivity and specificity, simple and portable design, mild measurement conditions, quick turnaround time, and user-friendly read-out, make the novel distance sensor a promising option for POC diagnostic applications.

Enzyme-free fluorescent DNA detection based on nucleic acid-templated click reaction via controllable synthesis of Cu2O as heterogeneous nanocatalyst

In the field of nucleic acid amplification assays, developing enzyme-free, easy-to-use, and highly sensitive amplification approaches remains a challenge. In this work, we synthesized a heterogeneous Cu2O nanocatalyst (hnCu2O) with different particle sizes and shapes, which was used for developing enzyme- and label-free nucleic acid amplification methods based on the nucleic acid-templated azide–alkyne cycloaddition (AAC) reaction catalyzed by hnCu2O. The hnCu2O exhibited size- and shape-dependent catalytic activity, with smaller sizes and spherical-like shapes exhibiting superior activity. Spherical-like hnCu2O (61 ± 8 nm) not only achieved a ligation yield of up to 84.2 ± 3.9 % in 3 min but also exhibited faster kinetics in the nucleic acid-templated hnCu2O-catalyzed AAC reaction, with a high reaction rate of 0.65 min−1 and a half-life of 1.07 ± 0.09 min. Based on this result, we developed nucleic acid-templated click ligation linear amplification reaction (NA-CLLAR) and nucleic acid-templated click ligation exponential amplification reaction (NA-CLEAR) approach. By combining the recognition (complementary to the target sequence) and signal output (split G-quadruplex sequence) elements into a DNA probe, the NA-CLLAR and NA-CLEAR fluorescence assays achieved highly specific detection of target nucleic acids, with a detection limit of 2.8 aM based on G-quadruplex-enhanced fluorescence. This work is a valuable reference and will inspire researchers to design enzyme-free nucleic acid signal amplification strategies by developing different types of Cu(I) catalysts with improved catalytic activity.

Dual-Function DNA Nanowires with Self-Feedback Amplification and Efficient Signal Transduction for Intracellular Imaging of MicroRNA-155.

Intracellular detection and imaging of microRNAs (miRNAs) with low expression usually face the problem of unsatisfactory sensitivity. Herein, a novel dual-function DNA nanowire (DDN) with self-feedback amplification and efficient signal transduction was developed for the sensitive detection and intracellular imaging of microRNA-155 (miRNA-155). Target miRNA-155 triggered catalytic hairpin assembly (CHA) to generate plenty of double-stranded DNA (dsDNA), and a trigger primer exposed in dsDNA initiated a hybridization chain reaction (HCR) between four well-designed hairpins to produce DDN, which was encoded with massive target sequences and DNAzyme. On the one hand, target sequences in DDN acted as self-feedback amplifiers to reactivate cascaded CHA and HCR, achieving exponential signal amplification. On the other hand, DNAzyme encoded in DDN acted as signal transducers, successively cleaving Cy5 and BHQ-2 labeled substrate S to obtain a significantly enhanced fluorescence signal. This efficient signal transduction coupling self-feedback amplification greatly improved the detection sensitivity with a limit of detection of 160 aM for miRNA-155, enabling ultrasensitive imaging of low-abundance miRNA-155 in living cells. The constructed DDN creates a promising fluorescence detection and intracellular imaging platform for low-expressed biomarkers, exhibiting tremendous potential in biomedical studies and clinical diagnosis of diseases.

High‐Performance Cooperative DNA Nanodevice Enables Sensitive Circular RNA Imaging and Precise Tumor Growth Suppression

The utility of circular RNAs (circRNAs) as emerging biomarkers and regulatory factors in medical diagnostics and therapeutics is hampered by the challenges associated with their sensitive detection and precise modulation. Herein, a high‐performance cooperative DNA nanodevice (HCDN) based on DNA tetrahedron‐confined catalytic DNA assembly reaction (DT‐CDA) that enables both imaging and regulation of circRNAs is developed. Activation of the DT‐CDA is contingent upon the presence of the target circRNA, which, together with a replicative fuel probe, catalyzes the sequential opening of additional DT‐CDAs. This cooperative exponential signal amplification with negligible background interference allows HCDN to effectively detect minute quantities of circRNAs. Employing circSATB2 as a model, the HCDN demonstrates substantial downregulation of Cyclin D1 (CCND1) mRNA and protein levels in cellular and in vivo models, thereby inhibiting tumor growth. The innovative design of HCDN sets the stage for a powerful methodology conducive to enhanced clinical diagnostics and biomolecule manipulation, thereby advancing the capabilities and applications of DNA nanotechnology.

Protein-Induced DNA Dumbbell Amplification (PINDA) and its applications to food hazards detection

Quantification of trace amounts of proteins is technically challenging because proteins cannot be directly amplified like nucleic acids. To improve the analytical sensitivity and to complement conventional protein analysis methods, we developed a highly sensitive and homogeneous detection strategy called Protein-Induced DNA Dumbbell Amplification (PINDA). PINDA combines protein recognition with exponential nucleic acid amplification by using protein binding probes made of DNA strands conjugated to protein affinity ligands. When a pair of probes bind to the same target protein, complementary nucleic acid sequences that are conjugated to each probe are brought into close proximity. The increased local concentration of the probes results in the formation of a stable dumbbell structure of the nucleic acids. The DNA dumbbell is readily amplifiable exponentially using techniques such as loop-mediated isothermal amplification. The PINDA assay eliminates the need for washing or separation steps, and is suitable for on-site applications. Detection of the model protein, thrombin, has a linear range of 10 fM-100 pM and detection limit of 10 fM. The PINDA technique is successfully applied to the analysis of dairy samples for the detection of β-lactoglobulin, a common food allergen, and Salmonella enteritidis, a foodborne pathogenic bacterium. The PINDA assay can be easily modified to detect other targets by changing the affinity ligands used to bind to the specific targets.