Strand Displacement Reaction Research Articles

Dissociation Constant (Kd) Measurement for Small-Molecule Binding Aptamers: Homogeneous Assay Methods and Critical Evaluations.

Since 1990, numerous aptamers have been isolated and discovered for use in various analytical, biomedical, and environmental applications. This trend continues to date. A critical step in the characterization of aptamer binding is to measure its binding affinity toward both target and non-target molecules. Dissociation constant (Kd) is the most commonly used value in characterizing aptamer binding. In this article, homogenous assays are reviewed for aptamers that can bind small-molecule targets. The reviewed methods include label-free methods, such as isothermal titration calorimetry, intrinsic fluorescence of target molecules, DNA staining dyes, and nuclease digestion assays, and labeled methods, such as the strand displacement reaction. Some methods are not recommended, such as those based on the aggregation of gold nanoparticles and the desorption of fluorophore-labeled DNA from nanomaterials. The difference between the measured apparent Kd and the true Kd of aptamer binding is stressed. In addition, avoiding the titration regime and paying attention to the time required to reach equilibrium are discussed. Finally, it is important to include mutated non-binding sequences as controls.

Close Proximity of Cholesterol Anchors in Membrane Induces the Dissociation of Amphiphilic DNA Strand from Membrane Surface.

Dynamic DNA nanotechnology is appealing for membrane surface engineering due to their versatility and programmability. To modulate the dynamic interactions between the DNA functional units immobilized on membrane surface, membrane-anchored DNA functional units often come into close proximity each other due to DNA base pairing, which also leads to the close contact of the hydrophobic anchors in membrane. However, whether the close contact of hydrophobic anchors induces the dissociation of amphiphilic DNA structures from membrane surface is not concerned. Herein, we utilized cholesterol-labeled single-stranded DNA (ssDNA) as a simplified amphiphilic DNA structure to investigate the stability of membrane anchored DNA strands upon the closely contact of cholesterol anchors. The close contact of cholesterol-labeled ssDNA molecules driven by toe-hold mediated strand displacement reaction leads to approximately 41 % membrane anchored ssDNA dissociation from membrane surface, indicating the proximal cholesterol anchors in membrane could reduce the anchoring stability of cholesterol-modified DNA strands. This work enhances our understanding of the interactions between amphiphilic DNA and membranes, and provides valuable insights for the design of future DNA constructs intended for applications involving dynamic DNA reactions on membrane surface.

Construction of an efficient microRNA sensing platform based on terminal deoxynucleotidyl transferase-mediated synthesis of copper nanoclusters

A conceptual innovative electrochemical sensing platform was constructed for microRNA (miRNA) detection based on terminal deoxynucleotidyl transferase (TdT)-mediated synthesis of copper nanoclusters. In principle, the substrate strand immobilized on working electrode acted as the template for the TdT-mediated DNA extension reaction, while the DNAzyme probe would be activated in the absence of miRNA 21, which led to the cleavage of the substrate strand to produce the 5’ phosphate terminus and inhibited the TdT-mediated DNA extension reaction. On the other hand, the presence of miRNA 21 initiated the toehold-mediated strand displacement reaction, causing the disassembly of the DNAzyme structure and preventing the substrate strand from being cleaved. This enabled the recycle amplification of target miRNA 21, and TdT catalyzed the DNA extension reaction on the 3’ hydroxyl terminus of the substrate strand, producing a large number of poly thymine (polyT) sequences. These sequences bound with copper ions for in-situ synthesis of copper nanoclusters on the sensing electrode, and the increased electrochemical signal of copper was recorded by differential pulse stripping voltammetry (DPSV), which paved the way for sensitive determination of miRNA 21 extracted from cancer cells with a detection limit of 36 aM. Due to its high sensitivity, user-friendly operation, and cost-effectiveness, this method is helpful for the fundamental research of sensing mechanism as well as the diagnosis of related diseases.

Directing Nanoparticle Organization in Response to Diverse Chemical Inputs.

Signaling cascades are crucial for transducing stimuli in biological systems, enabling multiple stimuli to regulate a downstream target with precisely controlled timing and amplifying signals through a series of intermediary reactions. Developing a robust signaling system with such capabilities would be pivotal for programming complex behaviors in synthetic DNA-based molecular devices. However, although "software" such as nucleic acid circuits could potentially be harnessed to relay signals to DNA-based nanostructure hardware, such explorations have been limited. Here, we develop a platform for transducing a variety of stimuli via messenger-mediated reactions to regulate the release and reloading of gold nanoparticles (AuNPs) in a 3D DNA framework. In the first step, an in vitro transcription circuit is engineered to sense and amplify chemical stimuli, including arbitrary DNA sequences and proteins, producing RNA. In the second step, the RNA releases the DNA-coated AuNPs from the DNA framework via a strand displacement reaction. AuNP reloading is controlled by a separate step driven by degradation of the RNA. Our platform holds promise for applications requiring dynamic multiagent control over DNA-based devices, offering a versatile tool for advanced molecular device engineering.

Single nucleotide polymorphism discrimination and genotyping based on cascade strand displacement reaction mediated label-free Cas12a system

Single nucleotide polymorphism (SNP) discrimination plays an important role in precision medicine by providing insights into an individual’s genetic information. The low abundance of the mutant target and the minimal impact caused by SNP on the whole nucleic acid sequence remain a challenge during discrimination. Herein, a cascade strand displacement reaction mediated label-free Cas12a sensing platform is established for SNP analysis. Split G-quadruplex (G4) motif is recruited as label-free signal output for Cas12a sensing system, and hence overcomes relying of fluorescence labeled substrates like conventional method. The established platform exhibits excellent single base difference discrimination ability attributing to the cascade strand displacement process, during which, single-base mismatch will affect the strand-exchange rate significantly. The limit of detection reaches 1 copy / test after integration with isothermal preamplification. The proposed method can discriminate as low as 0.1 % single base variation and perform robustly in biological matrixes. Human buccal swab samples are successfully genotyped with high accuracy.

Efficient Multidriven Strand Displacement Reaction for Biosensing.

A key challenge for achieving high-efficient DNA strand displacement reaction (SDR) with existing technologies is the inferior kinetic performance due to the alternately cumbersome conjunction and dissociation of dsDNA. In this work, a novel multidriven SDR collaborated by toehold initiator, strand towing, and click chemistry is engineered. The invasion strand (O) endows the hybridization with a basal strand (M) in dsDNA for releasing a displacement strand (P), which can be significantly boosted by the towing of a helper strand and impetus from the click reaction. Accordingly, the hybridization rate and dissociation extent of P can be largely improved and showed a desiring displacement rate close to 6-fold compared with the traditional method, providing a newly high-efficient SDR strategy for potential application in biosensing, clinical diagnostics, and DNA nanotechnology. In view of this, a practical biosensing platform by combining the multidriven SDR (MSDR) with waste-free DNA multi-cycle amplification is constructed for the rapid and ultrasensitive electrochemical detection of cancer-related miRNA-21. The substantial output DNA as an invasion strand (O) from target-triggered waste-free DNA multicycle can high-efficiently release a signal probe (Fc)-labeled displacement strand (P) on an electrode by using the proposed MSDR, obtaining a low detection limit below 106.8 aM.

Activated single nucleotide mismatch determination through toehold-embedded hairpin-mediated strand displacement reaction alongside CRISPR-Cas12a for detection of TP53 point mutation

The CRISPR-Cas12a-based diagnostic platform is renowned for its high sensitivity and, when coupled with the toehold-mediated strand displacement (TMSD) reaction, enables the detection of single nucleotide mismatch (SNM). However, conventional TMSD with the CRISPR-Cas12a system suffers from signal leakage due to overlapping activator sequences and low displacement efficiency. Here, we propose a toehold-embedded hairpin-mediated strand displacement (THMSD) method and leverage the magnesium attenuation effect of CRISPR-Cas12a for selective single-point mutation detection in the TP53 gene. This approach effectively mitigates signal leakage as the hairpin DNA retains partial crRNA targeting sequences within its loop, preventing sequence overlap with the trigger and direct Cas12a activation. Additionally, the magnesium attenuation effect reduces background signal and enhances selectivity. We investigated toehold length and mismatch position to detect SNM with a high discrimination factor (DF). This high DF allows the THMSD/CRISPR-Cas12a system to differentiate between TP53 R273H mutant and TP53 wildtype genes for cancer screening at low abundances, down to 0.1 % with a concentration of 20 pM. When used for screening between H1975 and WI38 cells, the statistically significant difference was attainable with polymerase chain reaction (PCR) template loading as low as 4 ng. These results pave the way for developing CRISPR-Cas12a-based methods to effectively detect TP53 mutations as lung cancer biomarkers.

Dual mature microRNA-responsive logic biosensing platform based on CRISPR/Cas12a and DNA nanocage

Mature microRNAs play crucial roles in tumorigenesis and progression. However, their potential as cancer biomarkers is limited by the sequence interference of precursor microRNAs and the occurrence of false positive signals mediated by single microRNAs. Herein, we reported a dual mature microRNA-responsive second-order (YES-AND) logic biosensing platform for accurate cancer diagnosis. Specifically, DNA nanocages were conceived as the first stage of “YES” gates, capable of signal transduction through strand displacement reactions, and realizing size-selective discrimination of mature microRNAs and pre-microRNAs. Subsequently, CRISPR/Cas12a system served as the second stage of “AND” gate, wherein dual activators cooperatively triggered trans-cleavage. As a proof-of-concept, this second-order logic biosensing platform was successfully applied to detect non-small cell lung cancer-related mature microRNA in clinical serum, and showed remarkable sensitivity (Lod = 100 pM) and trueness (recovery ≥ 90%). Our study represents a significant step forward in the development of intelligent biosensors capable of performing complex computations within pathological networks, and opens up broader possibilities for applications in biological science study and clinic disease diagnosis.

Design of a portable bioassay system based on highly efficent nanozyme and Au-DNA nanocluster: Toward current/colorimetric dual-signal detection of sugarcane smut

Sugarcane smut is a major agricultural pest affecting sugarcane yield and quality, posing a significant threat to the sustainable and healthy development of the global sugar industry. The best strategy for controlling Sugarcane smut is early and precise diagnosis. An intelligent mobile bio-platform is constructed based on Au-Co/Zn ZIF nanozymes and Au-DNA nanoclusters for current/colorimetric dual signal detection of the sugarcane smut pathogen. Efficient and specific cycling of the specific gene fragment (bE4′) for Sugarcane smut is achieved by strand displacement reaction. The output strand then forms Au-DNA nanoclusters with AuNPs-DNA, and the bioconjugate is captured by H2 probe, which results in the formation of more double-stranded structures, causing methylene blue (MB) to adsorb on electrode. The anodic nanozyme efficiently catalyzes the oxidation of glucose, generating a large number of electrons, and MB in the system is therefore reduced to MBH, causing a color change. This results in distinct current/colorimetric dual output signals, significantly enhancing the accuracy. A capacitor is further coupled with the system to improve the sensitivity. The detection signals are read in real-time by a smartphone. The developed method for sugarcane smut genes shows good linearity within a concentration range of 0.0001–10000 pM, with a detection limit as low as 23.59 aM (S/N = 3). This work integrates advanced technologies from various fields to create a portable sensing device with extremely high sensitivity and precision for detecting sugarcane smut. It provides a reliable new option for early and precise diagnosis of the disease and offers valuable theoretical and technical insights for early prevention, intelligent detection, and real-time monitoring of other plant diseases.

Titanium Carbide-Based Spatiotemporally Selectable-Activated Entropy-Driven DNA Nanoplatform for Amplified MicroRNA Imaging and Photothermal Therapy In Vivo.

Engineering an elaborate nanotheranostic platform that can achieve spatiotemporally selective microRNA (miRNA) imaging and imaging-guided therapy in time is critical for precise cancer diagnosis and efficient treatment, yet remains a challenge. Herein, we present an on-site-activatable nanotheranostic platform (Ti3C2-NEDR) that engineers a photothermal-activated entropy-driven strand displacement reaction (NEDR) module on a photothermal conversion module (Ti3C2) for achieving spatiotemporally controlled miRNA-21 imaging in vivo and imaging-guided photothermal therapy only by varying the power of the near-infrared (NIR) laser. The upstream NIR photothermal conversion module, Ti3C2, can act not only as a DNA circuit carrier to deliver the NEDR module but also as a photothermal agent to activate the downstream NEDR module in low-power NIR laser irradiation. Once the NEDR module is activated by the NIR laser, the entropy-driven strand displacement reaction can be innated by intracellular miRNA-21 to generate an amplified fluorescence signal for the spatiotemporally selective imaging of miRNA-21 in vivo. Thereafter, the imaging-guided in vivo photothermal therapy can be achieved in time only by switching to the high-power NIR laser. It is envisioned that this strategy of NIR light-activated spatiotemporally selective miRNA imaging and imaging-guided on-demand therapy may expand the nanotheranostic platform for precise cancer diagnosis and personalized therapy in time, providing a remarkable prospect in biomedical diagnosis and therapy.

Ultrasensitive miRNA-let-7a detection based on AgInS2 quantum dots co-sensitization and CRISPR/Cas12a induced strand displacement reaction for signal amplification

The evolution of breast cancer is usually accompanied by abnormal expression of miRNA-let-7a. Sensitive gauging of miRNA-let-7a is crucial for breast cancer monitoring and prognosis. Herein, a novel photoelectrochemical (PEC) biosensor was served as miRNA-let-7a detection on the basis of the coupling of co-sensitization and CRISPR/Cas12a-mediated strand displacement amplification (SDA). In short, AgInS2 quantum dots (QDs), characterized by a wide visible light absorption range and a stable photocurrent response, have an obvious sensitizing effect on TiO2/Bi2MoO6. Under the condition of without miRNA-let-7a, the AgInS2 fixed DNA probe was located near the surface of the TiO2/Bi2MoO6 electrode, forming a co-sensitive structure. During the detection process, hairpin DNA2 (HP2) specifically recognized miRNA-let-7a through base complementary pairing, triggering the SDA reaction to convert massive double-stranded DNA (dsDNA). The dsDNA product then triggered CRISPR/Cas12a lateral cleavage activity, causing AgInS2-sensitized DNA to leave the electrode surface and the co-sensitization effect to disappear, further reducing the photocurrent. Using a dual effect for signal amplification, the biosensor platform had linear detection capability within the scope of 0.01–10 nM, and the detection limit was 3.35 pM. The biosensor which was fabricated effectively provided an excellent platform for the effective monitoring of various tumor biomarkers in clinical detection.

On-Demand Elongation of Peptide Nanofibrils at Cellular Interfaces to Modulate Cell-Cell Interactions.

Natural cells can achieve specific cell-cell interactions by enriching nonspecific binding molecules on demand at intercellular contact faces, a pathway currently beyond synthetic capabilities. We are inspired to construct responsive peptide fibrils on cell surfaces, which elongate upon encountering target cells while maintaining a short length when contacting competing cells, as directed by a strand-displacement reaction arranged on target cell surfaces. With the display of ligands that bind to both target and competing cells, the contact-induced, region-selective fibril elongation selectively promotes host-target cell interactions via the accumulation of nonspecific ligands between matched cells. This approach is effective in guiding natural killer cells, the broad-spectrum effector lymphocytes, to eliminate specific cancer cells. In contrast to conventional methods relying on target cell-specific binding molecules for the desired cellular interactions, this dynamic scaffold-based approach would broaden the scope of cell combinations for manipulation and enhance the adjustability of cell behaviors for future applications.

DNA Origami Vesicle Sensors with Triggered Single-Molecule Cargo Transfer.

Interacting with living systems typically involves the ability to address lipid membranes of cellular systems. The first step of interaction of a nanorobot with a cell will thus be the detection of binding to a lipid membrane. Utilizing DNA origami, we engineered a biosensor with single-molecule Fluorescence Resonance Energy Transfer (smFRET) as transduction mechanism for precise lipid vesicle detection and cargo delivery. The system hinges on a hydrophobic ATTO647N modified single-stranded DNA (ssDNA) leash, protruding from a DNA origami nanostructure. In a vesicle-free environment, the ssDNA coils, ensuring high FRET efficiency. Upon vesicle binding to cholesterol anchors on the DNA origami, hydrophobic ATTO647N induces the ssDNA to stretch towards the lipid bilayer, reducing FRET efficiency. As the next step, the sensing strand serves as molecular cargo that can be transferred to the vesicle through a triggered strand displacement reaction. Depending on the number of cholesterols on the displacer strands, we either induce a diffusive release of the fluorescent load towards neighboring vesicles or a stoichiometric release of a single cargo-unit to the vesicle on the nanosensor. Ultimately, our multi-functional liposome interaction and detection platform opens up pathways for innovative biosensing applications and controllable stoichiometric loading of vesicles with single-molecule control.

Nucleic acid-functionalized upconversion luminescence biosensor based on strand displacement-mediated signal amplification for the detection of trivalent chromium ions

BackgroundRapid industrial development has generated serious pollution, including the presence of toxic and harmful heavy metal ions. Among them, trivalent chromium ion (Cr3+) is a very important element that poses a threat to life and health in our industrial wastewater pollution. Thus, it is important to develop efficient fluorescence methods for Cr3+ detection. In this study, an upconversion luminescence biosensor for detecting Cr3+ was constructed based on a DNAzyme, strand displacement reaction (SDR), and DNA-functionalized upconversion nanoparticles (UCNPs). ResultsThe sulfonate-rich poly (sodium 4-styrene sulfonate) (PSS) was modified onto the surface of UCNPs, forming UCNPs@PSS. Then, NH2-Capture probe DNA (NH2-Cp) was further modified onto the UCNPs@PSS surface through sulfonylation, resulting in UCNPs@PSS@NH2-Cp. The DNAzyme activated by Cr3+ triggered the release of the primer probe (Pp), which initiated the SDR system cycle, thereby releasing a tetramethylrhodamine (TAMRA)-modified signal probe (TAMRA-Sp). Finally, UCNPs@PSS@NH2-Cp bound to TAMRA-Sp through complementary base pairing, causing UCNPs and TAMRA to approach each other. Because of the luminescence resonance energy transfer (LRET) mechanism, the upconversion luminescence (UCL) signal of the UCNPs was quenched by TAMRA, enabling the detection of Cr3+ by the change of I585/I545 ratio. This biosensor has good stability, selectivity, and sensitivity, with a linear range of 0.5–75 nM and a detection limit of 0.135 nM for Cr3+. Significance and noveltyFirstly, based on LRET between UCNPs and TAMRA, the quantitative analysis of Cr3+ is achieved through the changes of ratio fluorescence. Secondly, the specificity of the biosensor is improved by utilizing the specific recognition of DNA enzymes. Thirdly, the signal amplification technology of the SDR cycle greatly improves the sensitivity of biosensor. This biosensor will be useful for future environmental safety monitoring and biopsy of biological fluids.

Ingenious dual-circle DNA walker-mediated electrochemical biosensor for rapid and ultrasensitive detection of microRNA

In this work, an ingenious dual-circle DNA walker (DCDW) with pretty fast walking speed and high amplification efficiency was developed for rapid and ultrasensitive electrochemical detection of microRNA-221 (miRNA-221) related to liver cancer, combined with the toehold-mediated strand-displacement reactions (TSDRs). Impressively, compared with the traditional DNA walker, the DCDW with unique double-stranded interlocked DNA nanostructure not only possesses higher stability, flexibility, and anti-entanglement ability, but also enables more functional domain in a smaller area, thereby enhancing the local concentration, which can greatly improve the working efficiency. As a validation, the electrochemical biosensor realized rapid and ultrasensitive detection of miRNA-221 with a reaction time of 15 min and detection limit down to 1.9 aM, and had been applied in MHCC97L and HeLa cancer cell lysates, thus providing an innovative insight to design intelligent functional interlocked DNA walkers for ultimate application in the construction of biosensing platform and miRNA detection in biological sample.

Dimeric-molecular beacon based intramolecular strand displacement amplification enables robust analysis of miRNA

Given the critical role of miRNAs in regulating gene expression and their potential as biomarkers for various diseases, accurate and sensitive miRNA detection is essential for early diagnosis and monitoring of conditions such as cancer. In this study, we introduce a dimeric molecular beacon (Di-MB) based isothermal strand displacement amplification (ISDA) system (Di-MB-ISDA) for enhanced miRNA detection. The Di-MB system is composed of two monomeric MBs (Mono-MBs) connected by a double-stranded DNA linker with single-stranded sequences in the middle, facilitating binding with the flexible arms of the Mono-MBs. This design forms a compact, high-density structure, significantly improving biostability against nuclease degradation. In the absence of target miRNA, the Di-MB maintains its stable structure. When target miRNA is present, it binds to the stem-loop regions, causing the hairpin structure to unfold and expose the stem sequences. These sequences serve as templates for the built-in primers, triggering DNA replication through an intramolecular recognition mechanism. This spatial confinement effect accelerates the strand displacement reaction, allowing the target miRNA to initiate additional reaction cycles and amplify the detection signal. The Di-MB-ISDA system addresses key challenges such as poor biostability and limited sensitivity seen in traditional methods. By enhancing biostability and optimizing reaction conditions, this system demonstrates robust performance for miRNA detection with a detection limit of 100 pM. The findings highlight the potential of Di-MB-ISDA for sensitive and accurate miRNA analysis, paving the way for its application in biomedical study and disease diagnosis in complex biological samples.

Rapid LAMP-driven strand displacement for PAM-free CRISPR-based pathogen diagnostics

Accurate and rapid nucleic acid testing for pathogen diagnostics is central to controlling the spread of infectious diseases. Here, we report an assay for rapid bacterial DNA detection via PAM (protospacer adjacent motif)-free Cas12f biosensor. The assay, which is named LSD12f (LAMP-driven strand displacement for Cas12f detection), involved multiplexed loop-mediated isothermal amplification (LAMP) followed by isothermal strand displacement reaction and Cas12f-mediated cleavage of a quenched fluorophore. The core of LSD12f was engineered with forward/reverse inner primers, which made LAMP products recognized and cut by enzyme digestion, triggering single-stranded DNA (ssDNA) production. Moreover, CRISPR-Cas12f could especially recognize target ssDNA region without PAM sites to reduce false-positive amplification. LSD12f could detect Streptococcus pyogenes in blood and flesh samples within 60 min with 100 % specificity and 100 % accuracy when validated by quantitative PCR (qPCR). LSD12f showed impressive specificity and sensitivity as a rapid and adaptive toolkit to broaden the use of molecular diagnostics.

Construction of scalable multi-channel DNA nanoplatform for the combined detection of ctDNA biomarkers of ovarian cancer.

Single-level biomarker detection has the limitation of insufficient accuracy in cancer diagnosis. Therefore, the strategy of developing highly sensitive, multi-channel biosensors for high-throughput ctDNAdetermination is critical to improve the accuracy of early diagnosis of clinical tumors. Herein, in order to achieve efficient detection of up to ten targets for early diagnosis of ovarian cancer, a DNA-nanoswitch-based multi-channel (DNA-NSMC) biosensor was built based on the multi-module catalytic hairpin assembly-mediated signal amplification (CHA) and toehold-mediated DNA strand displacement (TDSD) reaction. Only two different fluorescence signals were used as outputs, combined with modular segmentation strategy of DNA-nanoswitch-based reaction platform; the multi-channel detection of up to ten targets was successfully achieved for the first time. The experimental results suggest that the proposed biosensor is a promising tool for simultaneously detecting multiple biomarkers for the early diagnosis of ovarian cancer, offering new strategies for the early screening, diagnosis, and treatment not only for ovarian cancer but also for other cancers.

Electrochemical Biosensing Platform Based on Toehold-Mediated Strand Displacement Reaction and DSN Enzyme-Assisted Amplification for Two-Target Detection.

Simultaneous detection of multiple targets is of great significance for accurate disease diagnosis. Herein, based on duplex-specific nuclease (DSN) assisted signal amplification and the toehold-mediated strand displacement reaction (TSDR), we constructed an electrochemical biosensor with high sensitivity and high specificity for dual-target detection. MiRNA-141 and miRNA-133a were used as the targets, and ferrocene (Fc) and methylene blue (MB) with significant peak potential differentiation were used as the electrochemical signal probes. The elaborately designed hairpin probe H1, which was fixed on the electrode surface, could be hybridized with the target miRNA-141 to perform signal amplification by the DSN-assisted enzyme cleavage cycle; thus, miRNA-141 could be detected by Fc signal changes at 0.41 V. The hairpin H1 can also combine with the MB-labeled signal probe (SP) output from miRNA-133a-induced TSDR, and the detection of miRNA-133a can be realized according to the response signal generated by MB at -0.26 V. The two sensing lines are independent of each other, and there is no mutual interference in the detection process. Therefore, two independent detection lines could be connected in series, and the simultaneous detection of two targets can be achieved on a single electrode. This novel detection strategy provides a new way to simultaneously detect different biomarkers.

A self-powered sensor based on hollow bimetallic sulfide and double-strand TSDR amplification achieves sensitive miRNA-21 detection

MicroRNA (miRNA) sensitive detection is significant for cancer diagnosis. Currently, self-powered biosensors in clinical miRNA detection show the advantages of no external power supply, mild reaction conditions, low cost and portability. In this work, the bimetallic sulfide heterojunction hollow nanocubes are prepared as the substrate material, which can significantly improve the electrochemical reaction rate and increase the enzyme load. Meanwhile, we design the independent duplex-chain assisted toehold mediated strand displacement reaction (TSDR) strategy. The two substable double chains trigger a continuous chain displacement reaction, and the current signal amplification is realized based on molecular recognition. The constructed sensor has a linear response range of 0.05–10000 fM and a detection limit (LOD) of 8.3 aM. In addition, we use commercial apps to present signals, fundamentally changing the signal output mode of electrochemical sensors, and facilitating the construction of a portable real-time sensor platform. The self-powered biosensor has the characteristics of good biocompatibility, real-time monitoring and economic efficiency in miRNA detection, which provides a new idea for sensitive detection of miRNA. Moreover, it has far-reaching reference significance for the development of other sensing technologies, which is expected to extend from the medical field to environmental monitoring, food safety and other fields.