Exponential Amplification Reaction Research Articles

A Novel CRISPR/Cas12a-Mediated Ratiometric Dual-Signal Electrochemical Biosensor for Ultrasensitive and Reliable Detection of Circulating Tumor Deoxyribonucleic Acid.

Circulating tumor DNA (ctDNA) holds great promise as a noninvasive biomarker for cancer diagnosis, treatment, and prognosis. However, the accurate and specific quantification of low-abundance ctDNA in serum remains a significant challenge. This study introduced, for the first time, a novel exponential amplification reaction (EXPAR)-assisted CRISPR/Cas12a-mediated ratiometric dual-signal electrochemical biosensor for ultrasensitive and reliable detection of ctDNA. To implement the dual-signal strategy, a signal unit (ssDNA-MB@Fc/UiO-66-NH2) was prepared, consisting of methylene blue-modified ssDNA as the biogate to encapsulate ferrocene signal molecules within UiO-66-NH2 nanocarriers. The presence of target ctDNA KRAS triggered EXPAR amplification, generating numerous activators for Cas12a activation, resulting in the cleavage of ssDNA-P fully complementary to the ssDNA-MB biogate. Due to the inability to form a rigid structure dsDNA (ssDNA-MB/ssDNA-P), the separation of ssDNA-MB biogate from the UiO-66-NH2 surface was hindered by electrostatic interactions. Consequently, the supernatant collected after centrifugation exhibited either no or only a weak presence of Fc and MB signal molecules. Conversely, in the absence of the target ctDNA, the ssDNA-MB biogate was open, leading to the leakage of Fc signal molecules. This clever ratiometric strategy with Cas12a as the "connector", reflecting the concentration of ctDNA KRAS based on the ratio of the current intensities of the two electroactive signal molecules, enhanced detection sensitivity by at least 60-300 times compared to single-signal strategies. Moreover, this strategy demonstrated satisfactory performance in ctDNA detection in complex human serum, highlighting its potential for cancer diagnosis.

On-site detection of methicillin-resistant Staphylococcus aureus (MRSA) utilizing G-quadruplex based isothermal exponential amplification reaction (GQ-EXPAR)

Methicillin-resistant Staphylococcus aureus (MRSA) has a high incidence in infectious hospitals and communities, highlighting the need for early on-site detection due to its resistance to methicillin antibiotics. The present study introduces a highly sensitive detection system for mecA, a crucial methicillin marker, utilizing an RCA-based isothermal exponential amplification reaction. The G-quadruplex-based isothermal exponential amplification reaction (GQ-EXPAR) method designs probes to establish G-quadruplex secondary structures incorporating thioflavin T for fluorescence. The system, unlike conventional genetic detection methods, works with portable isothermal PCR devices (isoQuark), facilitating on-site detection. A detection limit of 0.1 fmol was demonstrated using synthetic DNA, and effective detection was proven using thermal lysis. The study also validated the detection of targets swabbed from surfaces within bacterial 3D nanostructures using the GQ-EXPAR method. After applying complementary sequences to the padlock probe for the target, the GQ-EXPAR method can be used on various targets. The developed method could facilitate rapid and accurate diagnostics within MRSA strains.

Coupling Exponential to Linear Amplification for Endpoint Quantitative Analysis.

Exponential DNA amplification techniques are fundamental in ultrasensitive molecular diagnostics. These systems offer a wide dynamic range, but the quantification requires real-time monitoring of the amplification reaction. Linear amplification schemes, despite their limited sensitivity, can achieve quantitative measurement from a single end-point readout, suitable for low-cost, point-of-care, or massive testing. Reconciling the sensitivity of exponential amplification with the simplicity of end-point readout would thus break through a major design dilemma and open a route to a new generation of massively scalable quantitative bioassays. Here a hybrid nucleic acid-based circuit design is introduced to compute a logarithmic function, therefore providing a wide dynamic range based on a single end-point measurement. CELIA (Coupling Exponential amplification reaction to LInear Amplification) exploits a versatile biochemical circuit architecture to couple a tunable linear amplification stage - optionally embedding an inverter function - downstream of an exponential module in a one-pot format. Applied to the detection of microRNAs, CELIA provides a limit of detection in the femtomolar range and a dynamic range of six decades. This isothermal approach bypasses thermocyclers without compromising sensitivity, thereby opening the way to applications in various diagnostic assays, and providing a simplified, cost-efficient, and high throughput solution for quantitative nucleic acid analysis.

Protein-to-DNA Converter with High Signal Gain.

DNA isothermal amplification techniques have been applied extensively for evaluating nucleic acid inputs but cannot be implemented directly on other types of biomolecules. In this work, we designed a proximity activation mechanism that converts protein input into DNA barcodes for the DNA exponential amplification reaction, which we termed PEAR. Several design parameters were identified and experimentally verified, which included the choice of enzymes, sequences of proximity probes and template strand via the NUPACK design tool, and the implementation of a hairpin lock on the proximity probe structure. Our PEAR system was surprisingly more robust against nonspecific DNA amplification, which is a major challenge faced in existing formats of the DNA-based exponential amplification reaction. The as-designed PEAR exhibited good target responsiveness for three protein models with a dynamic range of 4-5 orders of magnitude down to femtomolar input concentration. Overall, our proposed protein-to-DNA converter module led to the development of a stable and robust configuration of the DNA exponential amplification reaction to achieve high signal gain. We foresee this enabling the use of protein inputs for more complex molecular evaluation as well as ultrasensitive protein detection.

Three-way junction structure-mediated reverse transcription-free exponential amplification reaction for pathogen RNA detection.

Since RNA is an important biomarker of many infectious pathogens, RNA detection of pathogenic organisms is crucial for disease diagnosis and environmental and food safety. By simulating the base mismatch during DNA replication, this study presents a novel three-way junction structure-mediated reverse transcription-free exponential amplification reaction (3WJ-RTF-EXPAR) for the rapid and sensitive detection of pathogen RNA. The target RNA served as a switch to initiate the reaction by forming a three-way junction (3WJ) structure with the ex-trigger strand and the ex-primer strand. The generated trigger strand could be significantly amplified through EXPAR to open the stem-loop structure of the molecular beacon to emit fluorescence signal. The proofreading activity of Vent DNA polymerase, in combination with the unique structure of 2+1 bases at the 3'-end of the ex-primer strand, could enhance the role of target RNA as a reaction switch to reduce non-specific amplification and ensure excellent specificity to differentiate target pathogen from those causing similar symptoms. Furthermore, detection of target RNA showed a detection limit of 1.0×104 copies/mL, while the time consumption was only 20 min, outperforming qRT-LAMP and qRT-PCR, the most commonly used RNA detection methods in clinical practice. All those indicates the great application prospects of this method in clinical diagnostic.

Specific quantitative detection of N6-methyladenosine at single-base resolution by extension-based isothermal exponential amplification reaction (E-IEXPAR)

BackgroundN6-methyladenosine (m6A) is a common modification in RNA, crucial for various cellular functions and associated with human diseases. Quantification of m6A at single-base resolution is of great significance for exploring its biological roles and related disease research. However, existing analysis techniques, such as polymerase chain reaction (PCR) or loop-mediated isothermal amplification (LAMP), face challenges like the requirement for thermal cycling or intricate primer design. Therefore, it is urgent to establish a simple, non-thermal cycling and highly sensitive assay for m6A. ResultsLeveraging the inhibitory effect of m6A on primer elongation and uncomplicated feature of the isothermal exponential amplification reaction (IEXPAR), we have developed an extension-based IEXPAR (E-IEXPAR). This approach requires just a single extension primer and one template, simplifying the design process in comparison to the more complex primer requirements of the LAMP methods. The reactions are conducted at constant temperatures, therby elimiating the use of thermal cycling that needed in PCR methods. By combining IEXPAR with an extension reaction, E-IEXPAR can identify m6A in RNA concentrations as low as 4 fM. We have also introduced a new analytical model to process E-IEXPAR results, which can aid to minimize the impact of unmodified adenine (A) on m6A measurements, enabling accurate m6A quantification in small mixed samples and cellular RNA specimens. Significance and noveltyE-IEXPAR streamlines m6A detection by eliminating the need for intricate primer design and thermal cycling, which are common in current analytical methods. Its utilization of an extension reaction for the initial identification of m6A, coupled with a novel calculation model tailored to E-IEXPAR outcomes, ensures accurate m6A selectivity in mixed samples. As a result, E-IEXPAR offers a reliable, straightforward, and potentially economical approach for specifically assaying m6A in both biological function studies and clinical research.

Integration of Demethylation-Activated DNAzyme with a Single Quantum Dot Nanosensor for Sensitive Detection of O6-Methylguanine DNA Methyltransferase in Breast Tissues.

O6-Methylguanine-DNA-methyltransferase (MGMT) is a demethylation protein that dynamically regulates the O6-methylguanine modification (O6 MeG), and dysregulated MGMT is implicated in various malignant tumors. Herein, we integrate demethylation-activated DNAzyme with a single quantum dot nanosensor to sensitively detect MGMT in breast tissues. The presence of MGMT induces the demethylation of the O6 MeG-caged DNAzyme and the restoration of catalytic activity. The activated DNAzyme then specifically cleaves the ribonucleic acid site of hairpin DNA to expose toehold sequences. The liberated toehold sequence may act as a primer to trigger a cyclic exponential amplification reaction for the generation of enormous signal strands that bind with the Cy5/biotin-labeled probes to form sandwich hybrids. The assembly of sandwich hybrids onto 605QD obtains 605QD-dsDNA-Cy5 nanostructures, inducing efficient FRET between the 605QD donor and Cy5 acceptor. Notably, the introduction of a mismatched base in hairpin DNA can greatly minimize the background and improve the signal-to-noise ratio. This nanosensor achieves a dynamic range of 1.0 × 10-8 to 0.1 ng/μL and a detection limit of 155.78 aM, and it can screen MGMT inhibitors and monitor cellular MGMT activity with single-cell sensitivity. Moreover, it can distinguish the MGMT level in tissues of breast cancer patients and healthy persons, holding great potential in clinical diagnostics and epigenetic research studies.

Elimination of Unspecific Amplification in the Exponential Amplification Reaction Using Polyethylene Glycol 200 (PEG 200) as a Cosolute

Exponential amplification reaction (EXPAR) enables extremely rapid nucleic acid measurements. However, the nonspecific amplification in EXPAR limits its application, especially for low abundance target nucleic acids. Herein, polyethylene glycol 200 (PEG200), a widely available small-molecular cosolute, is described to suppress this nonspecific amplification. This cosolute generates molecular crowding and reduces the solution water activity, thereby weakening the template-template interactions and catalysis of enzymes, the major causes of nonspecific amplification. The additional of PEG200 greatly reduces the background and thereby improves the sensitivity of EXPAR by 4 orders of magnitude without extending the analysis time. Hence, the determination of target miRNAs at 1 aM was obtained within 20 min, outperforming most conventional strategies. This work provides an efficient and affordable strategy of applying water soluble cosolutes to eliminate the nonspecific amplification in EXPAR, which is expected to be widely employed to eliminate the background in nicking enzyme assisted amplification (NEAA).

Ultrasensitive detection assay for Cronobacter sakazakii based on nucleic acid-driven aggregation-induced emission of gold nanoclusters and cascaded signal amplification

Cronobacter sakazakii (C. sakazakii), a foodborne pathogen frequently present in contaminated powdered infant formula (PIF), poses a substantial threat to food safety. Herein, a novel fluorescent biosensor based on gold nanoclusters (AuNCs) that exhibit aggregation-induced emission (AIE) when triggered by nucleic acids was developed. This biosensor integrates the exponential amplification reaction (EXPAR) with hybridization chain reaction (HCR) signal amplification (EXHCR), enabling ultrasensitive detection of C. sakazakii. The aggregations of glutathione protected AuNCs assembled by G-rich sequences (HP1, and HP2) was affected by the EXHCR reaction initiated with C. sakazakii, forming G-quadruplex nanowires, leading to the dis-aggregation of AuNCs and a turn-off response of fluorescence, thereby realizing the detection of C. sakazakii. The changes are discernible on cellulose paper with the naked eyes. The biosensor demonstrates exceptional selectivity due to the integration of an aptamer and a dual signal amplification strategy. Under optimized conditions, it achieves a remarkable detection limit of 1.10×100 CFU/mL, and a broad linear range from 1.10×100 to 1.10×107 CFU/mL. Furthermore, the biosensor’s capability to identify C. sakazakii in artificially contaminated milk powder with recoveries ranging from 97.8% to 107%, underscores its reliability and accuracy. Overall, this study offers a promising approach for nucleic acid and AIE-based detection, exhibiting considerable potential for developing highly efficient bacterial detectors.

Exponential Amplification-Induced Activation of CRISPR/Cas9 for Sensitive Detection of Exosomal miRNA.

As an important component of highly heterogeneous exosomes, exosomal microRNAs (miRNAs) have great potential as noninvasive biomarkers for cancer diagnosis. Therefore, a sensitive and simple sensor is the key for its clinical application. Herein, we designed an exponential amplification reaction (EXPAR) to induce the reactivation of the CRISPR-associated protein 9/small guide RNA (Cas9/sgRNA) complex, thus achieving sensitive and visual exosomal miRNAs-21 (miR-21) fluorescence sensing. In this design, we inactivated the sgRNA by hybridizing sgRNA and blocker DNA. Then, we used a trigger DNA to hybridize with miR-21 and produced a lot of activated DNA by EXPAR. Those activated DNA further hybridized with blocker DNA and released the free sgRNA to form the activated Cas9/sgRNA complex. Based on the quick cleavage of activated Cas9/sgRNA complex, the reporter DNA labeled by SYBR Green I was released from the surface of the magnetic nanoparticles (MNPs) into the supernatant, and thus was used to sensitively quantify the miRNAs concentration with a limit of detection of 3 × 103 particles/mL. In addition, this fluorescence sensor has also been successfully employed to distinguish healthy people and cancer patients by naked-eye observation of the fluorescence, thus demonstrating its great potential for accurate and point-of-care cancer diagnosis.

A Simple and Robust Exponential Amplification Reaction (EXPAR)-Based Hairpin Template (exp-Hairpin) for Highly Specific, Sensitive, and Universal MicroRNA Detection.

Specific and sensitive detection of microRNAs continues to encounter significant challenges, especially in the development of rapid and efficient isothermal amplification strategies for point-of-care settings. The exponential amplification reaction (EXPAR) has garnered significant attention owing to its simplicity and rapid amplification of signals within a short period. However, a substantial loss of amplification efficiency, difficulty in distinguishing closely related homologous sequences, and adapting the designed templates to other targets seriously hamper the practical application of the EXPAR. In this work, a hairpin template tailored for the EXPAR system (exp-Hairpin) was constructed by adding identical trigger sequences and enzyme cleavage sites on two arms of the hairpin, achieving theoretically more than 2n amplification efficiency and minimal background amplification of EXPAR. Modulating the stability of the exp-Hairpin template by increasing the stem length, the specificity of detecting target miRNA in highly homologous sequences could be significantly improved. Using miRNA let-7a as a target model, the exp-Hairpin with 8 bp stem length for EXPAR amplification curves could effectively distinguish target let-7a and nontarget let-7b/7c/7f/7g/7i homologous sequences. This strategy enabled the sensitive and accurate analysis of let-7a in diluted human serum with satisfactory recoveries. By simply replacing the loop recognition sequence of exp-Hairpin, the specific detection of miR-200b was also achieved, demonstrating the universality of this strategy. The exp-Hairpin EXPAR accelerates simple and rapid molecular diagnostic applications for short nucleic acids.

CRISPR/Cas12a cleavage-mediated isothermal amplification lights up the dimeric G-quadruplex signal unit for ultrasensitive and label-free detection of circulating tumor DNA

Circulating tumor DNA (ctDNA) is a valuable and potential cancer biomarker for real-time monitoring, and plays a vital role in cancer early diagnosis. In this work, we developed an ultrasensitive and label-free fluorescent biosensor for ctDNA detection. A well-designed RNA-DNA chimeric primer (RD) with phosphate groups modified at the 3-terminal was cleaved by target-initiated CRISPR/Cas12a, and then the subsequent exponential amplification reaction (EXPAR) was started to produce numerous G-rich sequences, which could be assembled with Thioflavin T to form a dimeric G-quadruplex (dimer-G4/ThT) and significantly enhance the fluorescence. Due to the innovative combination of the specificity of CRISPR/Cas12a and RD primers, the high amplification efficiency of EXPAR, and the strong fluorescence response of dimer-G4/ThT, this strategy demonstrated excellent sensitivity at the aM level. In addition, the sensing system showed satisfactory anti-interference ability and reliable accuracy in complex serum environment and tumor cell lysate. More importantly, this strategy avoided the complicated and expensive labeling process of traditional signal probes, making the construction of fluorescent biosensor simpler and more practical, offering practical insights for molecular diagnostics and clinical applications.

One-tube B7-H3 detection based on isothermal exponential amplification and dendritic hybridization chain reaction.

We have developed a one-tube fluorescence strategy for the detection of B7-H3 based on a proximity hybridization-mediated protein-to-DNA signal transducer, isothermal exponential amplification (EXPAR), and dendritic hybridization chain reaction (D-HCR). In this assay, a protein signal transducer was employed to convert the input protein to output single-stranded DNA with a nicking site. Antibody-conjugated DNA1 was first hybridized with the output DNA (DNA3). The binding of antibodies conjugated DNA1 and DNA2 to the same protein was able to increase the local concentrations, resulting in strand displacement between DNA3 and DNA2. DNA3 with a nicking endonuclease recognition sequence at the 5' end then hybridized with hairpin probe 1 to mediate EXPAR in the presence of nicking endonuclease and DNA polymerase. A large number of single-strand DNA were produced in the circle of nicking, polymerization, and strand displacement. The resulting ssDNA products were further amplified by D-HCR to produce many large-molecular concatemers. The resulting DNA products can be monitored in real-time fluorescence signaling. Our proposed assay can realize one-tube detection due to the same reaction temperature of the protein-to-DNA signal transducer, EXPAR, and DHCR. This assay has a linear range from 100 fg mL-1 to 1 μg mL-1 with a detection limit down to 100 fg mL-1. This work shows a good performance in clinical specimen detection.

A one-pot CRISPR-RCA strategy for ultrasensitive and specific detection of circRNA.

Accurate and precise detection of circular RNA (circRNA) is imperative for its clinical use. However, the inherent challenges in circRNA detection, arising from its low abundance and potential interference from linear isomers, necessitate innovative solutions. In this study, we introduce, for the first time, the application of the CRISPR/Cas12a system to establish a one-pot, rapid (30 minutes to 2 hours), specific and ultrasensitive circRNA detection strategy, termed RETA-CRISPR (reverse transcription-rolling circle amplification (RT-RCA) with the CRISPR/Cas12a). This method comprises two steps: (1) the RT-RCA process of circRNA amplification, generating repeat units containing the back-splicing junction (BSJ) sequences; and (2) leveraging the protospacer adjacent motif (PAM)-independent Cas12a/crRNA complex to precisely recognize target sequences with BSJ, thereby initiating the collateral cleavage activity of Cas12a to generate a robust fluorescence signal. Remarkably, this approach exhibits the capability to detect circRNAs at a concentration as low as 300 aM. The sensor has been successfully employed for accurate detection of a potential hepatocellular carcinoma biomarker hsa_circ_0001445 (circRNA1445) in various cell lines. In conclusion, RETA-CRISPR seamlessly integrates the advantages of exponential amplification reaction and the robust collateral cleavage activity of Cas12a, positioning it as a compelling tool for practical CRISPR-based diagnostics.

Programmable DNA barcode-encoded exponential amplification reaction for the multiplex detection of miRNAs.

Multiple analysis of miRNAs is essential for the early diagnosis and monitoring of diseases. Here, a programmable, multiplex, and sensitive approach was developed for one-pot detection of miRNAs by melting temperature encoded sequences and exponential isothermal amplification (E-EXPAR). In the presence of target miRNAs, the corresponding templates initiate the cycles of nicking and polymerization/displacement, generating numerous barcode strands with unique encoding sequences. Subsequently, generated barcode strands hybridize with fluorescent probes and quench the fluorophore by a triplet of G base through a photo-induced electron transfer mechanism. Finally, a melting curve analysis is performed to quantify miRNAs by calculating the rate of fluorescence change at the corresponding melting temperature. Based on this, miRNA-21, miRNA-9, and miRNA-122 were detected with the detection limits of 3.3 fM, 2.9 fM, and 1.7 fM, respectively. This E-EXPAR was also employed to simultaneously detect three miRNAs in biological samples, showing consistent results with RT-qPCR. Overall, this study provides a programmable and universal platform for multiplex analysis of miRNAs, and holds great promise as an alternative to the multiplex analysis in clinical diagnostics and prognostics for nucleic acid detection.

Smartphone-Based Fluorescent Profiling of Quaternary MicroRNAs in Urine for Rapid Diagnosis of Urological Cancers Using a Multiplexed Isothermal Exponential Amplification Reaction.

Urological cancers such as bladder or prostate cancer represent one of the most malignant tumors that accounts for an extremely high mortality. However, conventionally standard diagnostics for urological cancers are hardly available in low-resource settings. We developed herein a hand-held fluorescent imaging platform by integrating a multiplexed isothermal exponential amplification reaction (EXPAR) with a microgel-enriched methodology for sensitive profiling of quaternary microRNAs (miRNAs) in urine and quick diagnosis of urological cancers at the early stage. The target miRNA mixtures in the urine underwent four parallel EXPARs without cross-reactivity, followed by surface concentration and hybridization by the encoded polyacrylamide microgels. This mix-and-read strategy allowed for one-pot analysis of several key miRNAs simultaneously and provided 5-fold enhancement in fluorescent detection sensitivities compared to the individual EXPAR-based assays. Four urinary miRNAs (let-7a, miRNA-155, -223, and -143) could be quantitatively determined in a wide linear range from 50 fM to 30 nM, with the limits of detection at femtomolar levels. Using a smartphone-based imaging microreader, healthy and cancerous cohorts with prostate, bladder, and renal cell cancers could be discriminated in 30 min with the accuracy >83% using linear discriminant analysis. The developed detection platform has proven to be a portable, noninvasive, and useful complement to the toolbox for miRNA-based liquid biopsies, which holds immense potential and advantage for regular and large-scale applications in early cancer diagnosis.

Novel fluorescence nano-orbital biosensor for highly sensitive microRNA detection

BackgroundMicroRNAs play an important role in regulating cell function and gene expression. Early prevention and clinical diagnosis of diseases have high requirements for high-sensitivity detection of microRNAs. Due to the limitations of tedious operation and large sample size, miRNA with small molecular weight and low expression abundance cannot be accurately detected in traditional miRNA detection. To improve the sensitivity and accuracy of detection, we established a novel biosensor based on nucleic acid circuit of signal amplification, which converted miRNA recognition into a fluorescence signal for amplification. ResultsWe designed a biosensor based on an exponential amplification reaction with cascaded HCR and DNAzyme nucleic acid circuit (named E-NOF biosensor) by amplicon sub-fragments to trigger the construction of fluorescence nano-orbitals (NOF), which could be used to detect miRNA ultrasensitively. By modifying two fluorophores (Cy3 and Cy5) on the chain of constructing nano-orbitals, when the amplicon triggered the construction of nano-orbitals, fluorescence resonance energy transfer (FRET) occurred between Cy3 and Cy5, and then two fluorescence signals with different trends could be observed. Therefore, through the ratio of the two signals, we could quantitatively and quickly detect the miRNA from 1 fM to 100 nM, and the E-NOF biosensor detection limit was as low as 0.129 fM. Furthermore, the HCR nucleic acid circuit cascaded with DNAzyme could enrich the fluorophores on the nano-orbitals and significantly enhance the fluorescence signal by accelerating the reaction rate. SignificanceAccording to our understanding, the E-NOF biosensor is the first strategy to cascade EXPAR with HCR and DNAzyme nucleic acid circuit for miRNA-1246 detection. Accurate results can be obtained in only 120 min. Compared with the traditional HCR system, the sensitivity of the new E-NOF biosensor is increased by 1 × 109 times. Furthermore, the biosensor can also detect biomarkers in human serum samples. It has great potential in miRNA detection and identification.

Engineering of a Chimeric Template Triggers RNase H-Based Isothermal Amplification Approach for Sensitive Detection of Pathogen RNA.

RNA-based detection of pathogenic organisms is an emerging field of research that is crucial for disease diagnosis and environmental and food safety. By rationally engineering an RNA-DNA tandem (RDT) structural template, we proposed a novel RNase H-based isothermal exponential amplification (RH-IEA) reaction to rapidly identify long-stranded RNA. In this strategy, the rigid and compact RDT template selectively recognized the target RNA and formed a stable hybrid with it. Upon site-specific cleavage of RNase H, the 3' overhang of the target RNA was cut off, and a free hydroxyl end at the hydrolysis site was generated to trigger an exponential amplification reaction (EXPAR). This method maintained the high efficiency and rapid amplification kinetics of EXPAR. As a result, the RH-IEA strategy was able to sensitively and specifically detect the characteristic sequence of Escherichia coli O157:H7 RNA, with a detection sensitivity of 1 fg/μL. Besides, the RDT template can be used as an RNA protector to prevent specific segments of the target RNA from being degraded by RNase enzymes, allowing the sample to be stored at room temperature for a long time. With this advantage, the practicality of RH-IEA will be more flexible than the reverse transcription polymerase chain reaction. It was successfully applied in the identification of E. coli O157:H7 in milk with a minimum detection concentration of 1.0 × 102 CFU/mL. Therefore, the RH-IEA method will serve as a powerful tool for detecting long-stranded RNA and will also shed light on the pathogen detection in food safety and molecular diagnosis.

A label-free fluorescence sensing strategy based on GlaI-assisted EXPAR for rapid and accurate quantification of human methyltranferase activity

DNA methylation plays an important role in epigenetic modification. DNA methyltransferase (DNMT) is essential in the DNA methylation process, and its abnormal expression is closely related to cancer. In this study, we propose a novel biosensor platform (DS-GlaI-EXPAR) that combines hemi-methylated double-stranded DNA (dsDNA) as the substrate for DNMT1 with GlaI-assisted isothermal exponential amplification reaction (EXPAR) for rapid, simple, and sensitive detection of DNMT1 activity. The hemi-methylated dsDNA is fully methylated by DNMT1, and GlaI recognizes and cleaves the fully methylated sequence, generating terminal fragments that trigger EXPAR for efficient signal amplification. Whereas hemi-methylated dsDNA without DNMT1 will keep intact and cannot initiate EXPAR. DNMT1 activity can therefore be sensitively quantified by the real-time fluorescence signal of the DS-GlaI-EXPAR platform. The high-efficiency amplification of EXPAR and the recognition of GlaI enable the platform to overcome the inherent cumbersome and time-consuming shortcomings of traditional methods while meeting specificity and sensitivity. This DS-GlaI-EXPAR platform offers an impressively low limit of detection of 0.86 pg/μL and the entire detection process can be completed in a short time of 2.5 h in a single tube. Furthermore, DNMT1 activity detected by this platform in MCF-7 cells was significantly higher than that of HEK293 cells, and the inhibition of Apt. #9 was verified. This DNMT1 activity detection platform is very convenient and effective for the discovery of inhibitors and early cancer diagnosis.

Low background self-primer EXPAR coupled with colorimetric and lateral flow assay for rapid and sensitive point-of-care detection of miRNA

MicroRNAs (miRNAs) have emerged as powerful biomarkers for various diseases diagnosis, yet their clinical application is greatly plagued by the lack of reliable and rapid detection methods. Here, a simple, fast, and accurate point-of-care testing (POCT) platform for miRNA-155 detection was developed by interfacing self-primer exponential amplification reaction (SPEXPAR) with colorimetric and POCT lateral flow assay (LFA). In this platform, a hairpin template was designed for exponential amplification reaction, which could effectively avoid nonspecific template interactions and eventually effectively avoid undesirable background amplification; the colorimetric assay is based on the SPEXPAR amplicons induced gold nanoparticles (AuNPs) color change; and the LFA detection principle involves the amplicons hybridization with designed probes. Under the optimized conditions, the colorimetric assay could detect miRNA-155 as low as 0.41 pM in less than 35 min; and LFA could detect as low as 100 pM in less than 40 min. Furthermore, the method was adopted to detect miRNA-155 in human serum samples with satisfactory recoveries. In summary, this study offered a promising analytical tool for molecular diagnostic testing application.