Palindrome sequence mediated target recycling integrating with self-priming assisted signal reaction for sensitive miRNA detection.

The development of a sensitive and isothermal technique with a greatly enhanced miRNA detection signal is still technically problematic due to the low abundance of miRNA and high sequence similarities with homologous miRNAs. Herein, we propose a novel fluorescence approach for sensitive and reliable miRNA detection by integrating the palindrome sequence mediated target recycling with self-priming assisted signal reaction. In this method, a dual toehold DNA nano-probe (HT) with two functional arms is designed to mediate specific target recognition and signal amplification. In the presence of target miRNA, it binds to the recognition module of HT probe, releasing the "2" sequence to initiate strand displacement amplification (SDA) and a self-priming-induced signal reaction. Based on the elegant design, the proposed method exhibits a wide linear response range exceeding five orders of magnitude and a low limit of detection of 0.96 fM according to the 3δ rule. The non-specific signal is below 5 % for non-target miRNA detection. Taking the merits of excellent sensitivity, desirable specificity, and superior anti-interference ability, the proposed approach shows a promising prospect for detecting miRNAs in complicated biological environments and early diagnosis of diseases.

Fluorescence biosensor to detect microRNAs via integrating DNA hairpins transition mediated strand displacement amplification with primer exchange reaction.

Herein, we constructed a fluorescence biosensor for the ultra-sensitive analysis of microRNAs (miRNAs) by combining DNA hairpins transition triggered strand displacement amplification (DHT-SDA) with primer exchange reaction (PER). Target miRNA initiated DHT-SDA to facilitate the generation of multiple single-stranded DNA (ssDNA) as PER primer, which was extended into a long ssDNA. The biosensor is successfully utilized in detecting miRNAs with high sensitivity (limit of detection for miRNA-21 was 58 fM) and a good linear relationship between 100 nM and 100 fM. By simply changing the DNA hairpin sequence, the constructed biosensor can be extended to analyze another miRNAs. Moreover, the biosensor has the feasibility of detecting miRNAs in real samples with satisfactory accuracy and reliability. Therefore, the fluorescent biosensor has great application potential in clinical diagnosis.

CRISPR/Cas12a-coupled multiplexed strand displacement amplification for miRNA155 one-tube detection: via a dual-cavity PCR tube.

A CRISPR/Cas12a-coupled multiplexed strand displacement amplification (CMSDA) for the detection of miR155 has been developed. Non-specific amplification was avoided by designing a single-stranded DNA template with a hairpin structure. The detection target miR155 was used as a primer to initiate a multiple-strand displacement reaction to produce abundant ssDNA. ssDNA was recognized by the Cas12a/CrRNA binary complex, activating the trans-cleaving activity of Cas12a. The multiple-strand displacement reaction is more efficiently detected compared with a single-strand displacement reaction. The detection range is from 250 pM to 1 nM, and the limit of the detection is 6.5 pM. The proposed method showed a good applicability in complex serum environments, indicating that the method has a broad prospect for disease detection and clinical application. In addition, we designed a dual-cavity PCR tube, which realized one-tube detection of miRNA155 and avoided open-cap contamination.

A universal CRISPR-Cas14a responsive triple-sensitized upconversion photoelectrochemical sensor.

It has recently been discovered that, like other members of the Cas family (12a and 13a), the clustered regularly interspaced short palindrome repeat CRISPR-Cas14a system not only mediates high-sensitivity detection with exceptionally strong gene editing ability but is also generally useful for DNA detection via fluorescence. Photoelectrochemical (PEC) sensors have been widely applied as efficient analytical tools. Measuring electrical signals is more cost-effective and the necessary equipment is more easily portable than fluorescence signal detectors, but their stability still needs to be improved. The high base resolution of CRISPR-Cas14a can compensate for such shortcomings. Therefore, electrical signals and fluorescence signals were combined, and the development of a universal CRISPR-Cas14a-responsive ultrasensitive upconversion PEC sensor is described in this paper. Moreover, strand displacement amplification (SDA) and a near-infrared (NIR) light source were utilized to further improve the stability and sensitivity of the photoelectric signals. At the same time, the modified working electrode (UCNPs-ssDNA-CdS@Au/ITO) on the three-electrode disposable sensor was used as the reporter probe, which cooperates with the trans-cleavage activity of Cas14a endonuclease. To verify the universality of this sensor, the UCNPs-Cas14a-based PEC sensor was applied for the detection of the small-molecule toxin T2 and protein kinase PTK7. Here, we report that the limit of detection of this reagent was within the fg range, successfully applied to the detection of T2 in oats and PTK7 in human serum. We propose that by combining PEC and CRISPR-14a, UCNPs-Cas14a-based PEC sensors could become powerful drivers for the extensive development of ultrasensitive, accurate and cost-effective universal sensors for detection and diagnosis.

Photothermal biosensor for HPV16 based on strand-displacement amplification and gold nanoparticles using a thermometer as readout.

Gold nanoparticles (AuNPs) in aggregated state have a strong near infrared region (NIR) absorption and the causes a much stronger photothermal effect than that of the dispersed AuNPs. Strand-displacement amplification (SDA) can produce large amount of single-stranded DNA (ssDNA), which in turn effectively prevent AuNPs from aggregation. In this study, these characteristics had been applied to design a photothermal biosensor for human papilloma virus (HPV and HPV16 were chosen as model target) detection. In the absence of HPV16, AuNPs was in the aggregated state and large temperature rise can be detected after the irradiation by 808 nm laser. The presence of HPV16 triggers the SDA reaction with the help of Bst DNA polymerase and Nt.BstNBI nicking endonuclease resulting in the production of large amounts of ssDNA; this protects unmodified AuNPs from salt-induced aggregation. Therefore, AuNPs was in a dispersed state and the temperature change was not significant after the irradiation of 808 nm laser. The difference of the temperature changing can be applied for the quantitative detection of HPV16 using a thermometer as readout. The linear response range is 1.0 fM ~ 50 pM with a detection limit of 0.3 fM. The proposed method has been applied to detect HPV16 in clinical cervical sample and is competent for clinical analysis.

Fluorescent Aptasensor for Highly Specific Detection of ATP Using a Newly Screened Aptamer.

Owing to the significant roles of adenosine triphosphate (ATP) in diverse biological processes, ATP level is used to research and evaluate the physiological processes of organisms. Aptamer-based biosensors have been widely reported to achieve this purpose, which are superior in their flexible biosensing mechanism, with a high sensitivity and good biocompatibility; however, the aptamers currently used for ATP detection have a poor ability to discriminate ATP from adenosine diphosphate (ADP) and adenosine monophosphate (AMP). Herein, an ATP-specific aptamer was screened and applied to construct a fluorescent aptasensor for ATP by using graphene oxide (GO) and strand displacement amplification (SDA). The fluorescence intensity of the sensor is linearly related to the concentration of ATP within 0.1 µM to 25 µM under optimal experimental conditions, and the detection limit is 33.85 nM. The biosensor exhibits a satisfactory specificity for ATP. Moreover, the experimental results indicate that the biosensor can be applied to determine the ATP in human serum. In conclusion, the screened aptamer and the biosensor have promising applications in the determination of the real energy charge level and ATP content in a complex biological system.

Rapid and sensitive detection of Mycobacterium tuberculosis based on strand displacement amplification and magnetic beads.

Tuberculosis is one of the main infectious diseases threatening public health, and the development of simple, rapid, and cost-saving methods for tuberculosis diagnosis is of profound importance for tuberculosis prevention and treatment. The bacterium Mycobacterium tuberculosis (MTB) is the pathogen that causes tuberculosis, and assaying for MTB is the only criterion for tuberculosis diagnosis. A new enzyme-free method based on strand displacement amplification and magnetic beads was developed for simple, rapid, and cost-saving MTB detection. Under optimum conditions, a good linear relationship could be observed between fluorescence and MTB specific DNA concentration ranging from 0.05 to 150 nM with a correlation coefficient of 0.993 (n = 8) and a detection limit of 47 pM (3σ/K). The present method also distinguished a one base mismatch from MTB specific DNA, showing great promise for MTB genome single base polymorphism analysis. MTB specific DNA content in polymerase chain reaction samples was successfully detected using the new method, and recoveries were 97.8-100.8%, indicating that the present method had high accuracy and shows good potential for the early diagnosis of tuberculosis.

Biosensors Based on Isothermal DNA Amplification for Bacterial Detection in Food Safety and Environmental Monitoring.

The easy and rapid spread of bacterial contamination and the risk it poses to human health makes evident the need for analytical methods alternative to conventional time-consuming laboratory-based techniques for bacterial detection. To tackle this demand, biosensors based on isothermal DNA amplification methods have emerged, which avoid the need for thermal cycling, thus facilitating their integration into small and low-cost devices for in situ monitoring. This review focuses on the breakthroughs made on biosensors based on isothermal amplification methods for the detection of bacteria in the field of food safety and environmental monitoring. Optical and electrochemical biosensors based on loop mediated isothermal amplification (LAMP), rolling circle amplification (RCA), recombinase polymerase amplification (RPA), helicase dependent amplification (HDA), strand displacement amplification (SDA), and isothermal strand displacement polymerisation (ISDPR) are described, and an overview of their current advantages and limitations is provided. Although further efforts are required to harness the potential of these emerging analytical techniques, the coalescence of the different isothermal amplification techniques with the wide variety of biosensing detection strategies provides multiple possibilities for the efficient detection of bacteria far beyond the laboratory bench.

A fluorescent aptasensor for Staphylococcus aureus based on strand displacement amplification and self-assembled DNA hexagonal structure.

A fluorescent aptasensor for Staphylococcus aureus (S. aureus) is designed, which takes advantage of strand displacement amplification (SDA) technology and unique self-assembled DNA hexagonal structure. In the presence of S. aureus, a partially complementary strand of S. aureus aptamer (cDNA) is competitively released from cDNA/aptamer duplex immobilized on magnetic beads due to the affinity of the aptamer for S. aureus. The addition of primer starts the SDA reaction. With the catalysis of Bsm DNA polymerase and Nb.bpu10I endonuclease, a large number of single-stranded DNA (ssDNA) is produced, which induces the opening of a hairpin probe and the subsequent self-assembly to form a hexagonal structure. The staining of the DNA hexagon with SYBR Green I excites the fluorescence signal, which is used for detection. The aptasensor exhibits a broad linear range from 7 to 7 × 107 CFU/mL, with a detection limit of 1.7 CFU/mL for S. aureus. The sensor shows negligible responses to other bacteria. Moreover, the aptasensor has been applied to detect S. aureus in milk samples, and the results demonstrate the general applicability of the sensor and its prospect in systematic detection of S. aureus in food safety control and medicine-related fields. Graphical abstract The presence of S. aureus can be converted to the formation of unique DNA hexagonal structure and subsequent fluorescent signal by the combination of SDA with self-assembly technology.

Label-free detection of microRNA: two-stage signal enhancement with hairpin assisted cascade isothermal amplification and light-up DNA-silver nanoclusters.

A method is described for the determination of microRNAs via two-stage signal enhancement. This is attained by combining hairpin (HP) assisted cascade isothermal amplification with light-up DNA-Ag nanoclusters. A rationally designed dual-functional HP is used, and microRNA-21 is chosen as a model analyte. At the first stage, upon the hybridization of the microRNA-21 with HP, microRNA recycling via polymerase-displacement reaction and a circulative nicking-replication process are achieved. This generates numerous G-abundant overhang DNA sequences. In the second stage, the above-released G-abundant overhang DNA sequences hybridize with the dark green Ag NCs, and this results in the appearance of bright red fluorescence. Thanks to the two signal enhancement processes, a linear dependence between the fluorescence intensity at 616 nm and the concentration of microRNA-21 is obtained in the range from 1 pM to 20 pM with a detection limit of 0.7 pM. The strategy clearly discriminates between perfectly-matched and mismatched targets. The method was applied to the determination of microRNA-21 in a spiked serum sample. Graphical abstractSchematic representation of microRNA detection by integrating hairpin assisted cascade isothermal amplification with light-up DNA Ag nanoclusters. With microRNA, G-abundant overhang DNA sequences from amplification reaction hybridize with dark green Ag nanoclusters to produce a concentration-dependent bright red fluorescence.

Fluorometric determination of the p53 cancer gene using strand displacement amplification on gold nanoparticles.

A fluorometric assay is described for the tumor suppressor gene p53. The method is based on strand displacement amplification on gold nanoparticles (GNPs). A FAM-labeled hairpin probe (HPP) is used that can hybridize to the GNP-confined linker strand, and the green fluorescence of the FAM label is quenched by the GNPs. In the presence of the p53 gene, it will hybridize with the HPP. This leads to fluorescence recovery. The primer then hybridizes with the opened HPP and induces the polymerization/displacement reactions. As a result, the hybridized p53 gene is released and, in turn, hybridizes with another HPP on the surface of the GNPs. This triggers the next round of hybridization/enzymatic polymerization/displacement reactions. This results in efficient strand displacement amplification and generates a substantially amplified signal. The method is referred to as GNP-HPP because it involves the use of GNPs and a HPP. The method allows the target DNA (p53) to be quantified down to 1.6 pM concentrations with a linear response in the 5 pM to 1 nM concentration range. In addition, mutant p53 genes can be easily distinguished from the wild-type gene. The method is highly sensitive, selective, and has a low background signal. Graphical abstract Schematic presentation of a chain hybridization signal amplification system (GNP-HPP) based on the use of gold nanoparticles (GNP) as a quenching source for the tumor suppressor gene p53 detection. The hairpin probe (HPP) having a 5'-end modified fluorophore was used as a signalling probe.

Circular exponential amplification of photoinduced electron transfer using hairpin probes, G-quadruplex DNAzyme and silver nanocluster-labeled DNA for ultrasensitive fluorometric determination of pathogenic bacteria.

The authors describe a fluorometric strategy for the detection of pathogenic bacteria with ultrasensitivity and high specificity. This strategy relies on the combination of target-modulated photoinduced electron transfer (PET) between G-quadruplex DNAzyme and DNA (labeled with silver nanoclusters) along with hairpin probe-based circular exponential amplification. The reaction system involves three hairpin probes (H1, H2 and H3). Probe H1 contains an aptamer against S. Typhimurium and the recognition sequence for nicking endonuclease. It is used to recognize S. Typhimurium and participates in polymerase-catalyzed target recycle amplification and secondary-target recycle amplification. Probe H2 contains an aptamer against hemin and is used to form the G-quadruplex DNAzyme in the presence of hemin and potassium ion. It acts as the electron acceptor and quenches the fluorescence of the labeled DNA. Fluorescence is best measured at excitation/emission wavelengths of 567/650 nm. Probe H3 contains the template sequence for the synthesis of AgNCs and the H2-annealing sequence. Both H2 and H3 are utilized to perform a strand displacement reaction and to achieve PET between G-quadruplex DNAzyme and DNA/AgNCs. To the best of our knowledge, this is the first example of a PET between G-quadruplex DNAzyme and DNA/AgNCs coupled with circular exponential amplification. The assay has an ultra-low detection limit 8 cfu·mL-1 of S. Typhimurium. The assay is rapid, accurate, inexpensive and simple. Hence, the strategy may represent a useful platform for ultrasensitive and highly specific detection of pathogenic bacteria as encountered in food analysis and clinical diagnosis. Graphical abstract The reaction system includes three hairpin probes (H1, H2 and H3), primer probe (P), Phi 29 DNA ploymerase (Phi 29) and nicking endonuclease Nt.AlwI (Nt.AlwI). Phi 29 and Nt.AlwI -assisted signal amplification leads to the recycling of target and produces numerous single stranded-DNAs (S). Strand displacement amplification leads to photoinduced electron transfer (PET) between G-quadruplex DNAzyme and DNA/AgNCs. HAP-based circular exponential amplification of PET results in an ultrasensitive fluorometric assay.

Target-Induced Catalytic Assembly of Y-Shaped DNA and Its Application for In†Situ Imaging of MicroRNAs.

DNA is a highly programmable material that can be configured into unique high-order structures, such as DNA branched junctions containing multiple helical arms converging at a center. Herein we show that DNA programmability can deliver in situ growth of a 3-way junction-based DNA structure (denoted Y-shaped DNA) with the use of three hairpin-shaped DNA molecules as precursors, a specific microRNA target as a recyclable trigger, and a DNA polymerase as a driver. We demonstrate that the Y-shaped configuration comes with the benefit of restricted freedom of movement in confined cellular environment, which makes the approach ideally suited for in situ imaging of small RNA targets, such as microRNAs. Comparative analysis illustrates that the proposed imaging technique is superior to both the classic fluorescence in situ hybridization (FISH) method and an analogous amplified imaging method via programmed growth of a double-stranded DNA (rather than Y-shaped DNA) product.

Homogenous assay for protein detection based on proximity DNA hybridization and isothermal circular strand displacement amplification reaction.

This work proposed a homogenous fluorescence assay for proteins, based on the target-triggered proximity DNA hybridization in combination with strand displacement amplification (SDA). It benefited from target-triggered proximity DNA hybridization to specifically recognize the target and SDA making recycling signal amplification. The system included a molecular beacon (MB), an extended probe (EP), and an assistant probe (AP), which were not self-assembly in the absence of target proteins, due to the short length of the designed complementary sequence among MB, EP, and AP. Upon addition of the target proteins, EP and AP are bound to the target proteins, which induced the occurrence of proximity hybridization between MB, EP, and AP and followed by strand displacement amplification. Through the primer extension, a tripartite complex of probes and target was displaced and recycled to hybridize with another MB, and the more opened MB enabled the detection signal to amplify. Under optimum conditions, it was used for the detection of streptavidin and thrombin. Fluorescence intensity was proportional to the concentration of streptavidin and thrombin in the range of 0.2-30 and 0.2-35 nmol/L, respectively. Furthermore, this fluorescent method has a good selectivity, in which the fluorescence intensity of thrombin was ~37-fold or even larger than that of the other proteins at the same concentration. It is a new and simple method for SDA-involved target protein detection and possesses a great potential for other protein detection in the future. Graphical abstract A homogenous assay for protein detection is based on proximity DNA hybridization and strand displacement amplification reaction.

Quantification of multiple microRNAs by microchip electrophoresis assisted by strand displacement amplification.

MicroRNAs (miRNAs) are increasingly recognized as potential biomarkers for the early diagnosis of cancer. However, the concurrent detection of multiple miRNAs in biological samples presents a significant challenge due to their high homogeneity and low abundance. This study introduced a novel approach combining strand displacement amplification (SDA) with microchip electrophoresis (MCE) for the simultaneous quantitation of trace levels of three miRNAs associated with cancer: miRNA-21, miRNA-145, and miRNA-221. Specifically designed probes were utilized to selectively capture the target miRNAs, thereby initiating the SDA process in a single solution without cross-interference. Under optimized conditions, the SDA-MCE method achieved the limit of detection (LOD) as low as 0.02 fM (S/N = 3) and the limit of quantitation (LOQ) as low as 0.1 fM across a broad linear range spanning from 0.1 fM to 1 pM. The SDA reaction was completed in approximately 1.5 h, and all target products were separated within 135 s through MCE. Application of this method for the simultaneous detection of these three miRNAs in human lung cancer cell samples yielded satisfactory results. Featuring high sensitivity, rapid analysis, minimal reagent consumption, and straightforward operation, the proposed MCE-SDA strategy holds considerable promise for multi-miRNAs detection applications.

Aptamer-mediated double strand displacement amplification with microchip electrophoresis for ultrasensitive detection of Salmonella typhimurium.

Rapid and quantitative detection of foodborne bacteria is of great significance to public health. In this work, an aptamer-mediated double strand displacement amplification (SDA) strategy was first explored to couple with microchip electrophoresis (MCE) for rapid and ultrasensitive detection of Salmonella typhimurium (S. Typhimurium). In double-SDA, a bacteria-identified probe consisting of the aptamer (Apt) and trigger sequence (Tr) was ingeniously designed. The aptamer showed high affinity to the S. Typhimurium, releasing the Tr sequence from the probe. The released Tr hybridized with template C1 chain, initiating the first SDA to produce numerous output strands (OS). The second SDA process was induced with the hybridization of the liberated OS and template C2 sequence, generating a large number of reporter strands (RS), which were separated and quantified through MCE. Cascade signal amplification and rapid separation of nucleic acids could be realized by the proposed double-SDA method with MCE, achieving the limit of detection for S. typhimurium down to 6 CFU/mL under the optimal conditions. Based on the elaborate design of the probes, the double-SDA assisted MCE strategy achieved better amplification performance, showing high separation efficiency and simple operation, which has satisfactory expectation for bacterial disease diagnosis.

Ultrasensitive miRNA detection: A novel DNA nanomachine using split-type molecular beacons-mediated cascade amplification for cancer diagnostics.

MicroRNAs (miRNAs) are crucial regulators in various pathological and physiological processes, and their misregulation is a hallmark of many diseases. In this study, we introduce an advanced DNA nanomachine using split-type molecular beacons (STMBs) for sensitive detection of miR-21, a key biomarker in cancer diagnostics. Utilizing an innovative STMB-mediated cascade strand displacement amplification (STMB-CSDA) technique, our approach offers a powerful means for the precise quantification of miRNAs, using miR-21 as a primary example. The system operates through target-induced linkage of STMBs, initiating a series of strand displacement amplifications resulting in exponential signal amplification. Coupled with the precision of T4 DNA ligase, this mechanism translates minimal miRNA presence into significant fluorescence signals, offering detection sensitivity as low as 5.96 pM and a dynamic range spanning five orders of magnitude. Characterized by its high specificity, which includes the ability to identify single-base mismatches, along with its user-friendly design, our method represents a significant leap forward in miRNA analysis and molecular diagnostics. Its successful application in examining total RNA from cancer cells and clinical serum samples demonstrates its immense potential as a groundbreaking tool for early cancer detection and gene expression studies, paving the way for the next generation of non-invasive diagnostics in personalized healthcare.

Programmable DNA tweezers-SDA for ultra-sensitive signal amplification fluorescence sensing strategy.

BACKGROUND: DNA tweezers, classified as DNA nanomachines, have gained prominence as multifunctional biosensors due to their advantages, including a straightforward structure, response mechanism, and high programmability. While the DNA tweezers demonstrate simultaneous, rapid, and stable responses to different targets, their detection sensitivity requires enhancement. Some small molecules, such as mycotoxins, often require more sensitive detection due to their extremely high toxicity. Therefore, more effective signal amplification strategies are needed to further enhance the sensitivity of DNA tweezers in biosensing. RESULTS: We designed programmable DNA tweezers that detect small-molecule mycotoxins and miRNAs through simple sequence substitution. While the DNA tweezers demonstrate simultaneous, rapid, and stable responses to different targets, their detection sensitivity requires enhancement. We introduced the Strand Displacement Amplification (SDA) technique to address this limitation, proposing a strategy of novel programmable DNA tweezers-SDA ultrasensitive signal amplification fluorescence sensing. We specifically investigate the effectiveness of this approach concerning signal amplification for two critical mycotoxins: aflatoxin B1 (AFB1) and zearalenone (ZEN). Results indicate that the detection ranges of AFB1 and ZEN via this strategy were 1-10,000 pg mL -1 and 10-100,000 pg mL -1, respectively, with corresponding detection limits of 0.933 pg mL -1 and 1.07 pg mL -1. Compared with the DNA tweezers direct detection method for mycotoxins, the newly constructed programmable DNA tweezers-SDA fluorescence sensing strategy achieved a remarkable 104-fold increase in the detection sensitivity for AFB1 and ZEN. SIGNIFICANCE: The constructed programmable DNA tweezers-SDA ultrasensitive signal-amplified fluorescence sensing strategy exhibits excellent detection performance for mycotoxins. The superb versatility of this strategy allows the developed method to be easily used for detecting other analytes by simply replacing the aptamer and cDNA, which has incredible potential in various fields such as food safety screening, clinical diagnostics, and environmental analysis.

A highly sensitive Lock-Cas12a biosensor for detection and imaging of miRNA-21 in breast cancer cells.

The expression levels of microRNA (miRNA) vary significantly in correlation with the occurrence and progression of cancer, making them valuable biomarkers for cancer diagnosis. However, their quantitative detection faces challenges due to the high sequence homology, low abundance and small size. In this work, we established a strand displacement amplification (SDA) approach based on miRNA-triggered structural "Lock" nucleic acid ("Lock" DNA), coupled with the CRISPR/Cas12a system, for detecting miRNA-21 in breast cancer cells. The "Lock" DNA freed the CRISPR-derived RNA (crRNA) from the dependence on the target sequence and greatly facilitated the extended detection of different miRNAs. Moreover, the CRISPR/Cas12a system provided excellent amplification ability and specificity. The designed biosensor achieved high sensitivity detection of miRNA-21 with a limit of detection (LOD) of 28.8 aM. In particular, the biosensor could distinguish breast cancer cells from other cancer cells through intracellular imaging. With its straightforward sequence design and ease of use, the Lock-Cas12a biosensor offers significant advantages for cell imaging and early clinical diagnosis.

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.