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.

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.

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.

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.

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.

Self-assembly of protein-DNA hybrids dedicated to an accelerated and self-primed strand displacement amplification for reinforced serum microRNA probing.

BACKGROUND: High-efficiency and highly reliable analysis of microRNAs (miRNAs) in bodily fluids highlights its significance to be extensively utilized as candidates for non-invasive "liquid biopsy" approaches. DNA biosensors based on strand displacement amplification (SDA) methods have been successfully designed to detect miRNAs given the efficiently amplified and recycled of the target sequences. However, the unpredictable DNA framework and heavy reliance on free diffusion or random reactant collisions in existing approaches lead to delayed reaction kinetics and inadequate amplification. Thus, it is crucial to create a modular probe with a controlled structure, high local concentration, and ease of synthesis. RESULTS: Inspired by the natural spatial-confinement effect based on a well-known streptavidin-biotin interaction, we constructed a protein-DNA hybrid, named protein-scaffolded DNA tetrads (PDT), which consists of four biotinylated Y-shaped DNA (Y-DNA) surrounding a streptavidin protein center via a streptavidin-biotin bridge. The streptavidin-biotin recognition system significantly increased the local concentration and intermolecular distance of the probes to achieve enhanced reaction efficiency and kinetics. The PDT-based assay starts with the target miRNA binding to Y-DNA, which disassembles the Y-DNA structures into three types of hairpin-shaped structures via self-primed strand displacement amplification (SPSDA) and generates remarkable fluorescence signal that is proportional to the miRNA concentration. Results demonstrated that PDT enabled a more efficient detection of miRNA-21 with a sensitivity of 1 fM. Moreover, it was proven reliable for the detection of clinical serum samples, suggesting great potential for advancing the development of rapid and robust signal amplification technologies for early diagnosis. SIGNIFICANCE: This simple yet robust system contributes to the early diagnosis of miR-21 with satisfactory sensitivity and specificity, and display a significantly improved nuclease resistance owing to their unique structure. The results suggested that the strategy is expected to provide a promising potential platform for tumor diagnosis, prognosis and therapy.

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.

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.

Multiple signal amplification strategy induced by biomarkers of lung cancer: A self-powered biosensing platform adapted for smartphones.

Lung cancer is a major malignant cancer with low survival rates, and early diagnosis is crucial for effective treatment. Herein, a biosensing platform that is self-powered derived from a capacitor-coupled EBFC has been developed for ultra-sensitive real-time identification of microRNA-21 (miRNA-21) with the assistance of a mobile phone. The flexible substrate of the platform is prepared on a carbon paper modified with graphdiyne and gold nanoparticles. The biosensor employs DNAzyme-mediated dual strand displacement amplification, which enhances the signal output intensity of the EBFC and improves selectivity. The coupling of the capacitor with the EBFC significantly amplifies the sensing signal, causing a 10.6-fold surge in current respond and further improving the sensitivity of the sensing platform. The established detection approach demonstrates a linear relationship varied from 0.0001 to 10,000 pM, with a sensitivity down to 32.3 aM as the minimum detectable limit, which has been effectively utilized for detecting miRNA-21 in practical samples. This sensing system provides strong support for the construction of portable detection devices, and the strategy of the platform construction provides an effective method for ultra-sensitive and accurate detection of miRNA, holding great potential in clinical diagnosis, prognosis evaluation, and drug screening for cancer.

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.

A universal nucleic acid detection platform combing CRISPR/Cas12a and strand displacement amplification with multiple signal readout.

Rapid and sensitive detection of nucleic acids has become crucial in various fields. However, most current nucleic acid detection methods can only be used in specific scenarios, such as RT-qPCR, which relies on fluorometer for signal readout, limiting its application at home or in the field due to its high price. In this paper, a universal nucleic acid detection platform combing CRISPR/Cas12a and strand displacement amplification (CRISPR-SDA) with multiple signal readout was established to adapt to different application scenarios. Nucleocapsid protein gene of SARS-CoV-2 (N gene) and hepatitis B virus (HBV) DNA were selected as model targets. The proposed strategy achieved the sensitivity of 53.1 fM, 0.15 pM, and 1 pM for N gene in fluorescence mode, personal glucose meter (PGM) mode and lateral flow assay (LFA) mode, respectively. It possessed the ability to differentiate single-base mismatch and the presence of salmon sperm DNA with a mass up to 105-fold of the targets did not significantly interfere with the assay signal. The general and modular design idea made CRISPR-SDA as simple as building blocks to construct nucleic acid sensing methods to meet different requirements by simply changing the SDA template and selecting suitable signal report probes, which was expected to find a breadth of applications in nucleic acids detection.

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.

Strand displacement amplification triggered 3D DNA roller assisted CRISPR/Cas12a electrochemiluminescence cascaded signal amplification for sensitive detection of Ec-16S rDNA.

BACKGROUND: Escherichia coli can cause gastrointestinal infection, urinary tract infection and other infectious diseases. Accurate detection of Escherichia coli 16S rDNA (Ec-16S rDNA) in clinical practice is of great significance for the identification and treatment of related diseases. At present, there are various types of sensors that can achieve accurate detection of Ec-16S rDNA. Electrochemiluminescence (ECL) has attracted considerable attention from researchers, which causes excellent performance in bioanalysis. Based on the previous research, it is significance to develop a novel, sensitive and efficient ECL biosensor. RESULTS: In this work, an ECL biosensor for the detection of Ec-16S rDNA was constructed by integrating CRISPR/Cas12a technology with the cascade signal amplification strategy consisting of strand displacement amplification (SDA) and dual-particle three-dimensional (3D) DNA rollers. The amplification products of SDA triggered the operation of the DNA rollers, and the products generated by the DNA rollers activated CRISPR/Cas12a to cleave the signal probe, thereby realizing the change of the ECL signal. The cascade amplification strategy realized the exponential amplification of the target signal and greatly improved the sensitivity. Manganese dioxide nanoflowers (MnO2 NFs) as a co-reaction promoter effectively enhanced the ECL intensity of tin disulfide quantum dots (SnS2 QDs). A new ternary ECL system (SnS2 QDs/S2O82-/MnO2 NFs) was prepared, which made the change of ECL intensity of biosensor more significant. The proposed biosensor had a response range of 100 aM-10 nM and a detection limit of 27.29 aM (S/N = 3). SIGNIFICANCE AND NOVELTY: Herein, the cascade signal amplification strategy formed by SDA and dual-particle 3D DNA rollers enabled the ECL biosensor to have high sensitivity and low detection limit. At the same time, the cascade signal amplification strategy was integrated with CRISPR/Cas12a to enable the biosensor to efficiently detect the target. It can provide a new idea for the detection of Ec-16S rDNA in disease diagnosis and clinical analysis.

Highly specific detection of Neisseria gonorrhoeae based on recombinase polymerase amplification-initiated strand displacement amplification.

Neisseria gonorrhoeae is the only pathogen that causes gonorrhea, and can have serious consequences if left untreated. A simple and accurate detection method for N. gonorrhoeae is essential for the diagnosis of gonorrhea and the appropriate prescription of antibiotics. The application of isothermal recombinase polymerase amplification (RPA) to detect this pathogen is advantageous because of its rapid performance, high sensitivity, and minimal dependency on equipment. However, this simplicity is offset by the risk of false-positive signals from primer-dimers and primer-probe dimers. In this study, RPA-initiated strand displacement amplification (SDA) was established for the detection of N. gonorrhoeae, and eliminated false-positive signals from primer-dimers and primer-probe dimers. The developed biosensor allows for the reduced generation of nonspecific RPA amplification through the design of enzyme cleavage sites on primers, introduction of SDA, and detection of the final product using a molecular beacon (MB). Using this system, the DNA double strand is transformed into single-stranded DNA following SDA, thereby providing a more suitable binding substrate and improving the efficiency of MB detection. Amplification can be conducted below 37 °C, and the process can be completed within 90 min. The limit of detection was determined to be 0.81 copies/µL. This system is highly specific for N. gonorrhoeae and exhibits no cross-reactivity with other common urogenital pathogens. The results of this study are consistent with those of real-time PCR performed on clinical specimens of urogenital secretions. In summary, the biosensor is a simple and specific detection method for N. gonorrhoeae.

Fluorometric Detection of SARS-CoV-2 Single-Nucleotide Variant L452R Using Ligation-Based Isothermal Gene Amplification.

Since the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant was first discovered, several variants showing different infectivity and immune responses have emerged globally. As the conventional method, whole-genome sequencing following polymerase chain reaction (PCR) is currently used for diagnosis of SARS-CoV-2 mutations. However, these conventional PCR-based direct DNA sequencing methods are time-consuming, complicated, and require expensive DNA sequencing modules. Here, we developed a fluorometric method for the accurate detection of a single missense mutation of U to G in the spike (S) gene that changes leucine to arginine (L452R) in SARS-CoV-2 genomic RNA. Our method for the detection of single-nucleotide mutations (SNM) in the viral RNA genome includes RNA sequence-dependent DNA ligation and tandem isothermal gene amplification methods, such as strand displacement amplification (SDA) and rolling circle amplification (RCA) generating G-quadruplex (GQ). In the presence of SNM in the viral RNA, ligation of both ends of the probe DNAs occurs between 5'-phosphorylated hairpin DNA and linear probe DNA that can discriminate a single base mismatch. The ligated DNAs were then extended to generate long-stem hairpin DNAs that are subjected to the first isothermal gene amplification (SDA). SDA produces multitudes of short ssDNA from the long-stem hairpin DNAs, which then serve as primers by annealing to circular padlock DNA for the second isothermal gene amplification (RCA). RCA produces a long stretch of ssDNA containing GQ structures. Thioflavin T (ThT) is then intercalated into GQ and emits green fluorescence, which allows the fluorometric identification of SARS-CoV-2 variants. This fluorometric analysis sensitively distinguished SNM in the L452R variant of SARS-CoV-2 RNA as low as 10 pM within 2 h. Hence, this fluorometric detection method using ligation-assisted tandem isothermal gene amplification can be applied for the diagnosis of SARS-CoV-2 SNM variants with high accuracy and sensitivity, without the need for cumbersome whole-genome DNA sequencing.

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.

CRISPR/Cas12a-based fluorescence aptasensor integrated with two-dimensional cobalt oxyhydroxide nanosheets for IFN-γ detection.

Cytokine storm (CS) is a risky immune overreaction accompanied by significant elevations of pro-inflammatory cytokines including interferon-γ (IFN-γ), interleukin and tumor necrosis factor. Sensitive detection of cytokine is conducive to studying CS progress and diagnosing infectious diseases. In this study, we developed a tandem system combining aptamer, strand displacement amplification (SDA), CRISPR/Cas12a, and cobalt oxyhydroxide nanosheets (termed Apt-SCN tandem system) as a signal-amplified platform for IFN-γ detection. Owing to the stronger affinity, target IFN-γ bound specifically to the aptamer from aptamer-complementary DNA (Apt-cDNA) duplex. The cDNA released from the Apt-cDNA duplex initiated SDA, resulting in the generation of double-stranded DNA products that could activate the trans-cleavage activity of CRISPR/Cas12a. The activated CRISPR/Cas12a further cleaved FAM-labeled single-stranded DNA probe, preventing it from adhering to the cobalt oxyhydroxide nanosheets and recovering the fluorescence signal. Sensitive fluorometric analysis of IFN-γ was successfully performed with detection limit as low as 0.37 nM. Unlike traditional protein analysis methods, Apt-SCN tandem system incorporates multiple signal amplification techniques and may also be applicable for other cytokines assay. This study was the initial study to utilize SDA and CRISPR/Cas12a to detect IFN-γ, showing great potential for cytokines clinical assay and CS prevention.

Identification, cleavage, and amplification (ICA): A versatile strategy for highly sensitive detection of miRNA.

MicroRNAs (miRNAs) are small RNA molecules that can play important roles as diagnostic/prognostic biomarkers and therapeutic targets for cancers and other diseases. Herein, an identification-cleavage-amplification (ICA) strategy for highly sensitive and versatile detection of miRNA has been proposed, and successfully applied to miR-155 and miR-21 assays. It combines an aligner-target mediated cleavage with strand displacement amplification (ATMC-SDA) to achieve the ICA process. During the identification process, a DNA-aligner (DA) and a DNA-amplicon (DM) can bind together with the help of target miRNA, forming a T-junction structure. Then, a nicking endonuclease (NEase), binding on the recognition sequence at the stem part of DA, can make a cleavage on DM, and the cleaved DM (CDM) can serve as an initiator to trigger the SDA reaction for signal amplification. Sharing the same set of enzymes and primers, the proposed ATMC-SDA can serve as a versatile ICA strategy for highly sensitive detection of various miRNAs, without the requirement of reverse transcription. Results show that the limits of detection (LOD) for miR-155 and miR-21 are 5.4 aM and 6.8 aM, respectively, with a dynamic range from 10.0 aM to 10.0 pM. The compatibility of ATMC-SDA with biological samples has also been tested by using human serum, indicating a promising potential for a wide variety of applications.

Trigger-activated autonomous DNA machine for amplified liver cancer biomarker microRNA21 imaging.

MicroRNA-21 (miRNA-21) is a kind of RNA that exists in biological fluids such as blood, urine and saliva. It has over expression in liver cancer and has different expression in different stages of cancer. However, due to the characteristics of small base number, short length, low abundance and easy degradation of miRNA-21, the detection of miRNA-21 is a challenging subject. Visualization, sensitive, specific and stable detection of tumor suppressor or oncogene microRNAs (miRNAs) remains challenging and is highly significant for clinical diagnostics. To solve this problem, we have developed a target-triggered hybridization assembly DNA machine for intracellular miRNA imaging based on strand displacement amplification (SDA) and branched hybridization chain reaction (B-HCR). In this approach, the target miRNA could hybridize with the template probe to trigger the SDA, resulting in the formation of nicked fragments (NFs) that hybridized with hairpin probe1 (HP1). The opened HP1 could hybridize with hairpin probe2 (HP2), leading to the self-assembly of hyperbranched DNA nanostructures through B-HCR. As expected, the newly developed method exhibits a detection limit down to 11.3 pM miRNA-21 and achieves high selectivity toward miRNA-21 against other interfering miRNAs. Due to its superior sensitivity and selectivity, our method can be further used to detect miRNA-21 in human serum samples. By taking advantage of intelligent design, the proposed method was also used for image miRNA-21 expression levels in different cell lines. This method shows a broad application in clinical diagnosis.

Ultrasensitive detection of exosomes by microchip electrophoresis combining with triple amplification strategies.

The analysis of exosomes is significant as they can be used for various pathophysiological processes, especially cancer related intercellular communication. Therefore, a convenient, reliable, and sensitive detection method is urgently needed. Strand displacement amplification (SDA) and catalytic hairpin assembly (CHA) are two kinds of effective isothermal nucleic acid amplification methods. In this article, an efficient quantitative MCE method for detecting human breast cancer cell (MCF-7) exosomes assisted by triple amplification strategies combining cholesterol probe (Chol-probe) with SDA-CHA was first developed. CD63 aptamer was immobilized on the avidin magnetic beads to specifically capture exosomes and then Chol-probe with high affinity was spontaneously inserted into the exosome membrane, which was the first step of amplification strategy to improve detection sensitivity. After magnetic separation, Chol-probe could complement ssDNA and trigger SDA, producing a large number of DNA sequences (Ta) to trigger CHA, achieving SDA-CHA amplification. Under optimal conditions, the detection limit (LOD) for MCF-7 exosomes was as low as 26 particle/µL (S/N = 3). This method provides an effective approach for sensitive and accurate quantification of tumor exosomes, and can be expected to detect exosomes in clinical samples.