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MicroRNAs (miRNAs) and short RNA fragments (18-25 nt) are crucial biomarkers in biological research and disease diagnostics. However, their accurate and rapid detection remains a challenge, largely due to their low abundance, short length, and sequence similarities. In this study, we report on a highly sensitive, one-step RNA O-circle amplification (ROA) assay for rapid and accurate miRNA detection. The ROA assay commences with the hybridization of a circular probe with the test RNA, followed by a linear rolling circle amplification (RCA) using dUTP. This amplification process is facilitated by U-nick reactions, which lead to an exponential amplification for readout. Under optimized conditions, assays can be completed within an hour, producing an amplification yield up to the microgram level, with a detection limit as low as 0.15 fmol (6 pM). Notably, the ROA assay requires only one step, and the results can be easily read visually, making it user-friendly. This ROA assay has proven effective in detecting various miRNAs and phage ssRNA. Overall, the ROA assay offers a user-friendly, rapid, and accurate solution for miRNA detection. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-024-00140-0.

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The high toxicity and widespread contamination of ochratoxin A (OTA) make it urgent to develop a sensitive method to detect trace OTA in complex food matrices. Herein, an indirect competitive enzyme-linked immunosorbent assay (icELISA)-based on the CRISPR/Cas12a system is described. DNA amplicons with multiple activation sequences of the CRISPR/Cas12a system were pre-prepared to improve detection sensitivity. In the absence of OTA, streptavidin-mediated biotinylated DNA amplicons were captured by the biotinylated secondary antibody on the microplate. The captured DNA amplicons activated the CRISPR/Cas12a system, which thereby effectively cleaved the reporter DNA, producing strong fluorescence. The presence of OTA led to a decrease in DNA amplicons on the microplate, resulting in a decrease in activated Cas12a and ultimately a drop in fluorescence intensity. OTA in food matrices at nanogram per milliliter levels can be detected. Therefore, the new method has great potential in monitoring OTA.

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Precise identification of small extracellular vesicles (sEVs) is crucial for improving disease diagnosis and treatments, such as bladder cancer. However, accurate isolation and simultaneously quantification of sEVs remain a huge challenge. We have introduced a new technique that combines immobilization with aptamer-assisted dual cycle amplification to isolate and analyze sEVs with high sensitivity. In this method, the CD9 protein antibody is attached to the plate's surface for the initial identification of sEVs, while an aptamer probe is used to detect the exosomal surface protein CD63. We have created an sEVs-surface method that combines target recognition initiated signal recycling and rolling circle amplification (RCA) for signal amplification. This approach allows for the "AND" logic analysis of dual biomarkers, enabling both sEVs quantification and tracing. The proposed approach has a broad detection range and a low limit of detection. Moreover, the established method showed good stability in detecting sEVs with a low coefficient of variation. Our method can effectively isolate certain sEVs and accurately identify them, making it suitable for many uses in biological science, biomedical engineering, and personalized medicine.

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Chronic wound management is affected by three primary challenges: bacterial infection, oxidative stress and inflammation, and impaired regenerative capacity. Conventional treatment methods typically fail to deliver optimal outcomes, thus highlighting the urgency to develop innovative materials that can address these issues and improve efficacy. Recent advances in DNA nanotechnology have garnered significant interest, particularly in the field of functional nucleic acid (FNA) nanomaterials, owing to their exceptional biocompatibility, programmability, and therapeutic potential. Among them, FNAs with unique nanostructures have garnered considerable attention. First, they inherit the biological properties of FNAs, including biocompatibility, reactive oxygen species (ROS)-scavenging capabilities, and modulation of cellular functions. Second, based on a precise design, these nanostructures exhibit superior physical properties, stability, and cellular uptake. Third, by leveraging the programmability of DNA strands, FNA nanostructures can be customized to accommodate therapeutic nucleic acids, peptides, and small-molecule drugs, thereby enabling a stable and controlled drug delivery system. These unique characteristics enable the use of FNA nanostructures to effectively address the major challenges in chronic wound management. This review focuses on various FNA nanostructures, including tetrahedral framework nucleic acids (tFNAs), DNA hydrogels, DNA origami, and rolling-circle amplification (RCA) DNA assembly. Additionally, a summary of recent advancements in their design and application for chronic wound management as well as insights for future research in this field are provided.

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There is an urgent need to accurately quantify microRNA (miRNA)-based Alzheimer's disease (AD) biomarkers, which have emerged as promising diagnostic biomarkers. In this study, we present a rapid and universal approach to establishing a target miRNA-triggered rolling circle amplification (RCA) detection strategy, which achieves ultrasensitive detection of several targets, including miR-let7a-5p, miR-34a-5p, miR-206-3p, miR-9-5p, miR-132-3p, miR-146a-5p, and miR-21-5p. Herein, the padlock probe contains three repeated signal strand binding regions and a target miRNA-specific region. The target miRNA-specific region captures miRNA, and then the padlock probe is circularized with the addition of T4 DNA ligase. Subsequently, an RCA reaction is triggered, and RCA products containing multiple signal strand binding regions are generated to trap abundant fluorescein-labeled signal strands. The addition of exonuclease III (Exo III) causes signal strand digestion and leads to RCA product recycling and liberation of fluorescein. Ultimately, graphene oxide (GO) does not absorb the liberated fluorescein because of poor mutual interaction. This method exhibited high specificity, sensitivity, repeatability, and stability toward let-7a, with a detection limit of 19.35 fM and a linear range of 50 fM to 5 nM. Moreover, it showed excellent applicability for recovering miRNAs in normal human serum. Our strategy was applied to detect miRNAs in the plasma of APP/PS1 mice, demonstrating its potential in the diagnosis of miRNA-associated disease and biochemical research.

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Foodborne illnesses caused by Salmonella and Staphylococcus aureus are a significant public health concern, leading to societal and economic repercussions. It is important to develop a simple and straightforward bacteria detection and identification method. A triple-probe multiplex rolling circle amplification technique has been developed to simultaneously detect Salmonella Typhimurium and S. aureus. This method utilizes fluorophore-labeled long padlock probes targeting S. Typhimurium invA and S. aureus glnA specific genes, along with a pH-based detection approach for direct visual identification. The multiplex hyperbranched saltatory rolling circle amplification assay at 30 °C has showed promising results with synthetic targets within 30 min and real bacteria within 2 h after establishing the detection settings. The assay is specific for S. aureus and S. Typhimurium, with a limit of detection of 39 µM for fluorescence and 78 µM for colorimetric. In the simulative test of this method for the detection of S. Typhimurium and S. aureus in milk, the limit of detection for the fluorescence signal after 2 h of amplification was 10 CFU/mL and 5 CFU/mL, respectively. The detection method was evaluated to be stable enough to detect pathogen for 3.29 months. Consequently, this triple-probe-multiplex rolling circle amplification method displays notable specificity, sensitivity, as well as ease of interpretation when testing food samples for harmful pathogens.

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Visualization of multiple targets in living cells is important for understanding complex biological processes, but it still faces difficulties, such as complex operation, difficulty in multiplexing, and expensive equipment. Here, we developed a nanoplatform integrating a nucleic acid aptamer and DNA nanotechnology for living cell imaging. Aptamer-based recognition probes (RPs) were synthesized through rolling circle amplification, which were further self-assembled into DNA nanoflowers encapsulated by an aptamer loop. The signal probes (SPs) were obtained by conjugation of multicolor emission carbon quantum dots with oligonucleotides complementary to RPs. Through base pairing, RPs and SPs were hybridized to generate aptamer sgc8-, AS1411-, and Apt-based imaging systems. They were used for individual/simultaneous imaging of cellular membrane protein PTK7, nucleolin, and adenosine triphosphate (ATP) molecules. Fluorescence imaging and intensity analysis showed that the living cell imaging system can not only specifically recognize and efficiently bind their respective targets but also provide a 5-10-fold signal amplification. Cell-cycle-dependent distribution of nucleolin and concentration-dependent fluorescence intensity of ATP demonstrated the utility of the system for tracking changes in cellular status. Overall, this system shows the potential to be a simple, low-cost, highly selective, and sensitive living cell imaging platform.

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Mercury ion (Hg2+), a highly toxic metal pollutant, is widely found in the environment and can enter the human body through the food chain, causing various health issues. Sensitive and accurate methods for monitoring Hg2+ are highly desirable for ensuring food safety. Herein, we propose a self-sustainable multiple amplification system (MAS) for Hg2+ determination through the reciprocal activation between catalytic hairpin assembly (CHA) and rolling circle amplification (RCA). The thymine-encoded recognition element specifically recognizes Hg2+, triggering the exposure of the initiator. The initiator then motivates the mutual activation of CHA and RCA to accelerate the production of an exponentially amplified signal. The MAS method achieved a low detection limit of 11 pM. Due to its reliable target recognition and robust amplification efficiency, the MAS circuit facilitated the highly efficient and accurate analysis of low-abundance Hg2+ in milk and snakehead samples, thus providing a potentially new tool for food safety control.

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A novel miRNA detection technique named Dumbbell probe initiated multi-Rolling Circle Amplification assisted CRISPR/Cas12a (DBmRCA) was developed relying on the ligation-free dumbbell probe and the high-sensitivity CRISPR/Cas12a signal out strategy. This DBmRCA assay streamlines miRNA quantification within a mere 30-min timeframe and with exceptional analytical precision. The efficacy of this method was validated by assessing miRNA levels in clinical samples, revealing distinct expression panel of miR-200a and miR-126 in lung cancer/adjacent/normal tissue specimens. Moreover, a predictive model was established to classify benign and malignant tumor. Due to its time efficiency, enhanced sensitivity, and streamlined workflow, this assay would be a reliable tool for miRNA analysis in clinical settings, offering potential guidance for early diagnosis and treatment of lung cancer.

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A novel functional nucleic acid (FNA) nanomaterial based on hybrid chain reaction (HCR) nanoscaffolds is proposed to solve the problem of time superposition and repeated primer design in sensitive miRND detection using cascade amplification technique. Rolling circle amplification (RCA) was cascaded with the prepared FNA nanomaterials for miRNA let-7a (as a model target) sensitive detection by lateral flow assay (LFA). Under the optimal conditions, the proposed RCA-FNA-LFA assay demonstrated the specificity and accuracy for miRNA let-7a detection with a detection limit of 1.07 pM, which increased sensitivity by nearly 20 times compared with that of RCA -LFA assay. It is worth noting that the non-target-dependent self-assembly process of HCR nanoscaffolds does not take up the whole detection time, thus, less time is taken than that of the conventional cascaded method. Moreover, the proposed assay does not need to consider the system compatibility between two kinds of isothermal amplification techniques. As for detection of different miRNAs, only the homologous arm of the padlock probe of RCA needs to be changed, while the FNA nanomaterial does not need any change, which greatly simplifies the primer design of the cascaded amplification techniques. With further development, the proposed RCA-FNA-LFA assay might achieve more sensitive and faster results to better satisfy the requirements of clinical diagnosis combing with more sensitive labels or small strip reader.

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MicroRNA (miRNA) has garnered considerable attention due to its diagnostic capabilities, such as in hypoxic cognitive impairment and cancers. However, the existing miRNA detection methods are commonly criticized for the drawbacks of low sensitivity and false-positive detection derived from interfering molecules. Here, we provide a novel, sensitive and portable method for miRNA detection by combining target identification based cyclization of padlocks, immobilized primer-based signal amplification and a personal glucose meter. The proposed method exhibits several advantages, including precise identification of specific sites, exceptional sensitivity and instrument-free feature. These attributes hold great promise for the diagnosis and clinical investigation of various diseases, such as cancer and hypoxic cognitive impairment, enabling a deeper understanding of their pathological and physiological aspects.

With miRNA-155 as detective target, the feasibility of the method has been demonstrated. The padlock sequences are cyclized by miRNA-155, which subsequently hybridize with primer sequence that is immobilized on the surface of a 96-well plate, and the interfering molecules are removed. This DNA polymerase triggers a chain extension process on the terminus of primer sequence, activating DNAzyme based cleavage. Consequently, a multitude of linker sequences are generated to facilitate the formation of the 'e/linker/f/sucrase' on magnetic bead, thereby enabling the catalysis of sucrose into glucose. This enzymatic reaction may be identified and measured using the personal glucose meter.

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The emergence of antibiotic resistance has become a global health crisis, and everyone must arm themselves with wisdom to effectively combat the "silent tsunami" of infections that are no longer treatable with antibiotics. However, the overuse or inappropriate use of unnecessary antibiotics is still routine for administering them due to the unavailability of rapid, precise, and point-of-care assays. Here, a rapid antimicrobial-resistance point-of-care identification device (RAPIDx) is reported for the accurate and simultaneous identification of bacterial species (genotype) and target enzyme activity (phenotype). First, a contamination-free active target enzyme is extracted via the photothermal lysis of preconcentrated bacteria cells on a nanoplasmonic functional layer on-chip. Second, the rapid, precise identification of pathogens is achieved by the photonic rolling circle amplification of DNA on a chip. Third, the simultaneous identification of bacterial species (genotype) and target enzyme activity (phenotype) is demonstrated within a sample-to-answer 45 min operation via the RAPIDx. It is believed that the RAPIDx will be a valuable method for solving the bottleneck of employing on-chip nanotechnology for antibiotic-resistant bioassay and other infectious diseases.

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The presence of viable pathogenic bacteria in food can lead to serious foodborne diseases, thus posing a risk to human health. Here, we develop a digital rolling circle amplification (dRCA) assay that enables the precise and sensitive quantification of viable foodborne pathogenic bacteria. Directly targeting pathogenic RNAs via a ligation-based padlock probe allows for precisely discriminating viable bacteria from dead one. The one-target-one-amplicon characteristic of dRCA enables high sensitivity and a broad quantitative detection range, conferring a detection limit of 10 CFU/mL and a dynamic range of 6 orders. dRCA can detect rare viable bacteria, even at a proportion as low as 0.1%, which is 50 times more sensitive than the live/dead staining method. The high sensitivity for detecting viable bacteria accommodates dRCA for assessing sterilization efficiency. Based on the assay, we found that, for pasteurization, slightly elevating the temperature to 68 °C can reduce the heating time to 10 min, which may minimize nutrient degradation caused by high-temperature exposure. The assay can serve as a precise tool for estimating the contamination by viable pathogenic bacteria and assessing sterilization, which facilitates food safety control.

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DNA hydrogels hold significant promise for biomedical applications and can be synthesized through enzymatic Rolling Circle Amplification (RCA). Due to the exploratory nature of this emerging field, standardized RCA protocols specifying the impact of reaction parameters are currently lacking. This study varied template sequences and reagent concentrations, evaluating RCA synthesis efficiency and hydrogel mechanical properties through quantitative PCR (qPCR) and indentation measurements, respectively. Primer concentration and stabilizing additives showed minimal impact on RCA efficiency, while changes in polymerase and nucleotide concentrations had a stronger effect. Concentration of the circular template exerted the greatest influence on RCA productivity. An exponential correlation between hydrogel viscosity and DNA amplicon concentration was observed, with nucleobase sequence significantly affecting both amplification efficiency and material properties, particularly through secondary structures. This study suggests that combining high-throughput experimental methods with structural folding prediction offers a viable approach for systematically establishing structure-property relationships, aiding the rational design of DNA hydrogel material systems.

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In the past few years, the COVID-19 pandemic, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) seriously threatens global public health security due to its high contagiousness. It remains of vital importance to develop a rapid and sensitive assay for SARS-CoV-2. In this work, we proposed a sandwich-type assay based on poly(N-isopropylacrylamide) (PNIPAM), allowing efficient detection of the SARS-CoV-2 S1 protein in the homogeneous solution. Firstly, a direct sandwich-type assay was established with a linear range of 0.2-2 µg/mL and a limit of detection (LOD) of 0.11 µg/mL, which could realize rapid detection in about 1 h. Furthermore, the sandwich-type assay coupled with rolling circle amplification (RCA) obtained an increase in sensitivity of 5.9 × 104 folds with a wide linear range of 0.01 - 100 ng/mL and a LOD of 1.88 pg/mL. The average recoveries in unpretreated saliva were 90 %-113.0 %, indicating the potential of the developed method for application in practical samples. Given the high selectivity and sensitivity of the developed method, it has a significant potential for rapid and early detection of SARS-CoV-2.

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Automation of liquid handling is indispensable to improve throughput and reproducibility in biochemical assays. However, the incorporation of automated systems into laboratory workflows is often hindered by the high cost and complexity associated with building robotic liquid handlers. Here, we report a 3D-printed liquid handler based on a fluidic manifold, thereby obviating the need for complex robotic mechanisms. The fluidic manifold, termed a dispensing and aspirating (DA) device, comprises parallelized multi-pipette structures connected by distribution and aspiration channels, enabling the precise supply and removal of reagents, respectively. Leveraging the versatility of 3D printing, the DA device can be custom-designed and printed to fit specific applications. As a proof-of-principle, we engineered a 3D-printed liquid handler dedicated for 3D digital rolling circle amplification (4DRCA), an advanced biochemical assay involving multiple sample preparation steps such as antibody incubation, cell fixation, nucleic acid amplification, probe hybridization, and extensive washing. We demonstrate the efficacy of the 3D-printed liquid handler to automate the preparation of clinical samples for the simultaneous, in situ analysis of oncogenic protein and transcript markers in B-cell acute lymphoblastic leukemia cells using 4DRCA. This approach provides an effective and accessible solution for liquid handling automation, offering high throughput and reproducibility in biochemical assays.

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RNA in situ hybridization reveals the abundance and location of gene expression in cells or tissues, providing a technical basis for the clinical diagnosis of diseases. In this chapter, we show a "V" shape probe-mediated single-molecule chromogenic in situ hybridization (vsmCISH) technique for bright-field visualization of individual RNA molecules. In our method, several pairs of target hybridization probes are hybridized to RNA molecules and each probe pair forms a "V" shape overhang. The overhang oligonucleotides then mediated the proximity ligation to form DNA circles, followed by rolling circle amplification for signal enhancement and enzyme-catalyzed chromogenic reaction-based readout. The colorimetric assay avoids problems such as photobleaching and autofluorescence of current fluorescent in situ hybridization-based single-molecule RNA detection techniques. Furthermore, the relatively straightforward protocol makes the method useful for biological research and clinical diagnosis applications.

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We present a powerful method for direct mRNA detection based on ligation-based recognition and in situ amplification, capable of single-cell imaging mRNA at single-nucleotide and single-molecule resolution. Attributed to the use of Splint R ligase that can ligate padlock probe with RNA as target template, this method can efficiently detect mRNA in the absence of reverse transcription. This method enables spatial localization and correlation analysis of gene expression in single cells, which helps us to elucidate gene function and regulatory mechanisms.

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Targeted and stimuli-responsive drug delivery enhances therapeutic efficacy and minimizes undesirable side effects of cancer treatment. Although cellulose nanocrystals (CNCs) are used as drug carriers because of their robustness, spindle shape, biocompatibility, renewability, and nontoxicity, the lack of programmability and functionality of CNCs-based platforms hampers their application. Thus, high adaptability and the capacity to form dynamic 3D nanostructures of DNA may be advantageous, as they can provide functionalities such as target-specific and stimuli-responsive drug release. Using DNA nanotechnology, the functional polymeric form of DNA nanostructures can be replicated using rolling circle amplification (RCA), and the biologically and physiologically stable DNA nanostructures may overcome the challenges of CNCs. In this study, multifunctional polymeric DNAs produced with RCA were strongly complexed with surface-modified CNCs via electrostatic interactions to form polymeric DNA-decorated CNCs (pDCs). Particle size, polydispersity, zeta potential, and biostability of the nanocomplexes were analyzed. As a proof of concept, the dynamic structural functionalities of DNA nanostructures were verified by observing cancer-targeted intracellular delivery and pH-responsive drug release. pDCs showed anticancer properties without side effects in vitro, owing to their aptamer and i-motif functionalities. In conclusion, pDCs exhibited multifunctional anticancer activities, demonstrating their potential as a promising hybrid nanocomplex platform for targeted cancer therapy.

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Multimeric aptamers have gained more attention than their monomeric counterparts due to providing more binding sites for target analytes, leading to increased affinity. This work attempted to engineer the surface-based generation of multimeric aptamers by employing the room temperature rolling circle amplification (RCA) technique and chemically modified primers for developing a highly sensitive and selective electrochemical aptasensor. The multimeric aptamers, generated through surface RCA, are hybridized to modified spacer primers, facilitating the positioning of the aptamers in the proximity of sensing surfaces. These multimeric aptamers can be used as bio-receptors for capturing specific targets. The surface amplification process was fully characterized, and the optimal amplification time for biosensing purposes was determined, using SARS-CoV-2 spike protein (SP). Interestingly, multimeric aptasensors produced considerably higher response signals and affinity (more than 10-fold), as well as higher sensitivity (almost 4-fold) compared to monomeric aptasensors. Furthermore, the impact of surface structures on the response signals was studied by utilizing both flat working electrodes (WEs) and nano-/microislands (NMIs) WEs. The NMIs multimeric aptasensors showed significantly higher sensitivity in buffer and saliva media with the limit of detection less than 2 fg/ml. Finally, the developed NMIs multimeric aptasensors were clinically challenged with several saliva patient samples.

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