Reaction Amplification Research Articles

CRISPR/Cas12a powered sandwich aptamer assay coupled with effective DNA self-assembly amplification for sensitive detection of immunoglobulin E

Sensitive and accurate detection of protein biomarkers is of significance for disease diagnosis. Recently, CRISPR/Cas system has emerged as a powerful signal amplifier in the field of sensing technology. We report a CRISPR/Cas12a powered sandwich aptamer assay (CAPSA) for protein analysis coupled with effective DNA self-assembly amplifications. The target protein is sandwiched between the capture antibody on the microplate and the aptamer detection probe. The aptamer is engineered with a specific initiator to trigger streptavidin (SA)-biotin-based dendritic amplification reaction, yielding a DNA polymer with multiple repeats of active DNA (acDNA) as Cas12a recognition sites. With the addition of Cas12a reporter system, the acDNA activates Cas12a to cleave DNA reporters, producing amplified fluorescence signals. Particularly, we also describe an innovative one-step DNA self-assembly amplification strategy to meet the demands for fast point-of-care testing, which can be directly integrated into the CAPSA without additional operation steps. The proposed methods were demonstrated by detecting immunoglobulin E (IgE), which plays a vital role in allergic reactions. Our proposed one-step DNA self-assembly amplified CAPSA enabled us to detect IgE as low as 0.6 pM, exhibiting high selectivity and satisfactory performance in complex sample analysis. Collectively, this method combines the efficient signal amplification of DNA self-assembly with the high trans-cleavage activity of Cas12a, offering a simple, universal, and double-amplified approach for sensitive analysis of protein biomarkers.

Development and application of a rapid visual detection technique for VanA gene in vancomycin-resistant Enterococcus faecium.

One of the key approaches to treating and controlling vancomycin-resistant Enterococcus faecium (VREFm) is an accurate and rapid diagnosis. To achieve this goal, a simple and rapid method must be constructed for immediate detection in the field. Multienzyme isothermal rapid amplification (MIRA) is an isothermal rapid amplification method that allows amplification reactions to be completed under room temperature conditions. When combined with lateral flow strips (LFSs), MIRA-LFS enables the rapid detection of pathogenic microorganisms. However, the MIRA method often produces false signals. These false signals are eliminated by using base mismatches introduced in primers and probes. The MIRA-LFS system was constructed with high specificity and sensitivity for the detection of VREfm, without the limitation of sophisticated instruments. This enables the prompt formulation of diagnostic and therapeutic decisions.

Multiplexed and Quantitative Imaging of Live‐Cell Membrane Proteins by a Precise and Controllable DNA‐Encoded Amplification Reaction

AbstractAmplifying DNA conjugated affinity ligands can improve the sensitivity and multiplicity of cell imaging and play a crucial role in comprehensively deciphering cellular heterogeneity and dynamic changes during development and disease. However, the development of one‐step, controllable, and quantitative DNA amplification methods for multiplexed imaging of live‐cell membrane proteins is challenging. Here, we introduce the template adhesion reaction (TAR) method for assembling amplifiable DNA sequences with different affinity ligands, such as aptamers or antibodies, for amplified and multiplexed imaging of live‐cell membrane proteins with high quantitative fidelity. The precisely controllable TAR enables proportional amplification of membrane protein targets with variable abundances by modulating the concentration ratios of hairpin templates and primers, thus allowing sensitive visualization of multiple membrane proteins with enhanced signal‐to‐noise ratios (SNRs) without disturbing their original ratios. Using TAR, we achieved signal‐enhanced imaging of six proteins on the same live‐cell within 1–2 h. TAR represents an innovative and programmable molecular toolkit for multiplexed profiling of membrane proteins in live‐cells.

Multiplexed and Quantitative Imaging of Live-Cell Membrane Proteins by a Precise and Controllable DNA-Encoded Amplification Reaction.

Amplifying DNA conjugated affinity ligands can improve the sensitivity and multiplicity of cell imaging and play a crucial role in comprehensively deciphering cellular heterogeneity and dynamic changes during development and disease. However, the development of one-step, controllable, and quantitative DNA amplification methods for multiplexed imaging of live-cell membrane proteins is challenging. Here, we introduce the template adhesion reaction (TAR) method for assembling amplifiable DNA sequences with different affinity ligands, such as aptamers or antibodies, for amplified and multiplexed imaging of live-cell membrane proteins with high quantitative fidelity. The precisely controllable TAR enables proportional amplification of membrane protein targets with variable abundances by modulating the concentration ratios of hairpin templates and primers, thus allowing sensitive visualization of multiple membrane proteins with enhanced signal-to-noise ratios (SNRs) without disturbing their original ratios. Using TAR, we achieved signal-enhanced imaging of six proteins on the same live-cell within 1-2 h. TAR represents an innovative and programmable molecular toolkit for multiplexed profiling of membrane proteins in live-cells.

Product catalysis dual entropy-driven amplification reaction strategy for miRNA-21 detection in glioblastoma

MicroRNA is taken as diagnostic tumor markers in clinical diagnosis of various cancers. However, the complexity system and low utilization rate of entropy-driven amplification reaction (EDAR) limit its application. In this work, a product catalysis dual EDAR amplification strategy is developed for miRNA-21 detection. This strategy allows miRNA-21 to simultaneously catalyze two EDARs, greatly increasing the limit of detection to 0.40 pM and enhancing amplification efficiency. The amplification efficiency was also significantly enhanced with a much shorter reaction time by increasing concentration of the catalyzer (EDAR product). The approach was successfully applied in clinical samples, showing potential for early screening and diagnosis.

Entropy-Driven Catalytic G-Quadruple Cycle Amplification Integrated with Ligases for Label-Free Detection of Single Nucleotide Polymorphisms.

G-Quadruplex/thioflavin (G4/THT) has become a very promising label-free fluorescent luminescent element for nucleic acid detection due to its good programmability and compatibility. However, the weak fluorescence efficiency of single-molecule G4/THT limits its potential applications. Here, we developed an entropy-driven catalytic (EDC) G4 (EDC-G4) cycle amplification technology as a universal label-free signal amplification and output system by properly programming classical EDC and G4 backbone sequences, preintegrated ligase chain reaction (LCR) for label-free sensitive detection of single nucleotide polymorphisms (SNPs). First, the positive strand LCR enabled specific transduction and preliminary signal amplification from single-base mutation information to single-strand information. Subsequently, the EDC-G4 cycle amplification reaction was activated, accompanied by the production of a large number of G4/THT luminophores to output fluorescent signals. The EDC-G4 system was proposed to address the weak fluorescence of G4/THT and obtain a label-free fluorescence signal amplification. The dual-signal amplification effect enabled the LCR-EDC-G4 detection system to accurately detect mutant target (MT) at concentrations as low as 22.39 fM and specifically identify 0.01% MT in a mixed detection pool. Moreover, the LCR-EDC-G4 system was further demonstrated for its potential application in real biological samples. Therefore, this study not only contributes ideas for the development of label-free fluorescent biosensing strategies but also provides a high-performance and practical SNP detection tool in parallel.

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

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

Single Methylation Sensitive Restriction Endonuclease-Based Cascade Exponential Amplification Assay for Visual Detection of DNA Methylation at Single-Molecule Level.

Function as a potential cancer biomarker, DNA methylation shows great significance in cancer diagnosis, prognosis, and treatment monitoring. While the lack of an ultrasensitive, specific, and accurate method at the single-molecule level hinders the analysis of the exceedingly low levels of DNA methylation. Herein, based on the outstanding recognition and digestion ability of methylation-sensitive restriction endonuclease (MSRE), we established a single MSRE-based cascade exponential amplification method, which requires only two ingeniously designed primers and only one recognition site of MSRE for the detection of DNA methylation. Differentiated by MSRE digestion, the cleaved unmethylated DNA is too short to induce any amplification reactions, while methylated DNA remains intact to trigger cascade exponential amplification and the subsequent CRISPR/Cas12a system. By integrating the two exponential amplification reactions, as low as 1 aM methylated DNA can be accurately detected, which corresponds to 6 molecules in a 10 μL system, indicating that our method is more sensitive than single amplification-based methods with the ability to detect DNA methylation at the single-molecule level. In addition, 0.1% methylated DNA can be effectively distinguished from large amounts of unmethylated DNA. Our method is further introduced to exploit the expression difference of DNA methylation among normal cells and cancer cells. Moreover, the visual detection of DNA methylation is also realized by the full hybridization between amplification products and the crRNA of CRISPR/Cas12a. Therefore, the proposed method has great potential to be a promising and robust bisulfite-free method for the detection of DNA methylation at the single-molecule level, which is of great importance for early diagnosis of cancer.

Enzyme-free fluorescent DNA detection based on nucleic acid-templated click reaction via controllable synthesis of Cu2O as heterogeneous nanocatalyst

In the field of nucleic acid amplification assays, developing enzyme-free, easy-to-use, and highly sensitive amplification approaches remains a challenge. In this work, we synthesized a heterogeneous Cu2O nanocatalyst (hnCu2O) with different particle sizes and shapes, which was used for developing enzyme- and label-free nucleic acid amplification methods based on the nucleic acid-templated azide–alkyne cycloaddition (AAC) reaction catalyzed by hnCu2O. The hnCu2O exhibited size- and shape-dependent catalytic activity, with smaller sizes and spherical-like shapes exhibiting superior activity. Spherical-like hnCu2O (61 ± 8 nm) not only achieved a ligation yield of up to 84.2 ± 3.9 % in 3 min but also exhibited faster kinetics in the nucleic acid-templated hnCu2O-catalyzed AAC reaction, with a high reaction rate of 0.65 min−1 and a half-life of 1.07 ± 0.09 min. Based on this result, we developed nucleic acid-templated click ligation linear amplification reaction (NA-CLLAR) and nucleic acid-templated click ligation exponential amplification reaction (NA-CLEAR) approach. By combining the recognition (complementary to the target sequence) and signal output (split G-quadruplex sequence) elements into a DNA probe, the NA-CLLAR and NA-CLEAR fluorescence assays achieved highly specific detection of target nucleic acids, with a detection limit of 2.8 aM based on G-quadruplex-enhanced fluorescence. This work is a valuable reference and will inspire researchers to design enzyme-free nucleic acid signal amplification strategies by developing different types of Cu(I) catalysts with improved catalytic activity.

ZnS/MXene/CdS heterostructure modified electrode for ultrasensitive miRNA-21 assay with signal amplified photoelectrochemical strategy

The early-stage detection of microRNA (miRNA) holds significant value for clinical diagnosis of tumor. In this study, a highly sensitive miRNA detection platform is developed using a photoelectrochemical sensing system based on layer-by-layer (LBL) assembled ZnS/MXene/CdS (ZMC) heterostructure electrode. By incorporating ultrathin and highly conductive MXene, the ZMC electrode exhibits robust photocurrent signals upon exposure to light. The electrode is further modified HS-H1 DNA via disulfide bonds.With the help of auxiliary H2 DNA, a target miRNA-21 triggered catalytic hairpin assembly (CHA) amplification reaction is realized on the electrode surface through complementary base pairing. Then, manganese porphyrin (MnPP) is introduced to the formed double-stranded DNA after CHA reaction to catalyze the conversion of 4-chloro-1-naphthol (4-CN) to benzo-4-chloro-hexanedione (4-CD) precipitate in the presence of hydrogen peroxide, which leads to a significant decrease in photocurrent signals. As a result, the sensor demonstrates a reliable linear correlation in miRNA-21 detection with detection limits as low as 0.36 fM. The recoveries of miRNA-21 in human serum samples ranged from 98.27 % to 101.35 % with RSD values below 3.67 % suggest the reliability and practicality of this method. Therefore it shows great potential for the construction and application of highly sensitive photoelectrochemical biosensors in clinical sample detection.

Sensitive detection of CaMV35S based on exponential rolling circle amplification reaction and CRISPR/Cas12a using a portable 3D-printed visualizer

The biosafety of genetically modified organisms (GMOs) is still a subject of debate among the public, so developing practical and accurate GMOs detection techniques is crucial. However, the existing testing methodologies for GMOs products do not possess the requisite sensitivity and flexibility necessary for effective GMOs surveillance. In this study, our exponential rolling cycle amplification with the CRISPR/Cas12a assay (E-RCA-CRISPR/Cas12a) has successfully enhanced the limit of detection (LOD) for the CaMV35S fragment to 10 aM in a total of 100 min. Rolling cycle amplification can be initiated by the target CaMV35S sequence, utilizing Nb.BbvCI endonuclease nicking activity for the exponential rolling cycle amplification and the trans-cleavage activity of Cas12a for the detection and signal amplification. Our research greatly improves the amplification efficiency through this cyclic process. To reduce the reliance on signal reading devices, we also build a custom 3D-printed visualization vessel and combine a color-reading app on the smartphone to read the fluorescent signals through for sample visualization and analysis. Under ideal circumstances, the limits of detection of our convenient device is 1 fM. We also integrate the exponential rolling circle amplification (E-RCA-CRISPR/Cas12a) assay with the lateral flow strip (LFA) to achieve reading device-free detection with the LOD of 50 fM. Testing the plant and soy sauce samples successfully demonstrated the method’s applicability. The sensing platform thus offers a prototype technique for quick detection of various nucleic acid targets and has optimum sensitivity, specificity, and on-site detection capability.

Field detection of genetically modified crops: Simple DNA extraction, rapid assay and automatic output on smartphone

Rapid and accurate detection of genetically modified crops (GMC) is significant for crop supervision and genetically modified product labeling. For field detection of GMC, the rapidity and simplicity of the sample pretreatment and detection process are very important. Simplifying the extraction process of DNA in crops and speeding up the detection process are the technical problems that need to be solved. Therefore, this paper provides a field detection method for GMC. First, the selected universal LAMP primers of the chloroplast gene can be used to detect reference genes in nine crops with the most transgenic detection needs. The universal primers of reference genes can not only improve the accuracy of detection, but also omit the design and synthesis of reference gene primers in different crops. Second, a high concentration of DNA can be obtained using simple and fast DNA extraction methods within 5 min. The specific concentration of GuHCl in lysis buffer can accelerate the subsequent nucleic acid amplification reaction. Third, a new probe-LAMP method was established, with high reaction speed and specificity. Using the dual probe-LAMP for transgenic and reference genes in a handheld nucleic acid detector, the detection of GMC can be completed within 25 min. Two different fluorescence signals generated from the LAMP reaction are processed through a special logic gate, and finally three kinds of results of "Positive", "Negative" and "Invalid" are output on a smartphone. This method from sample processing to intelligent result output can achieve accurate, rapid, simple, low-cost, pollution-free field detection of GMC.

Enzyme-Assisted Colorimetric Salivary miR-21 Detection by Functional Gold Nanoparticles

miRNA-21 (miR-21) is a potential biomarker for the monitoring of diseases through its expression levels. Simple, portable and sensitive miR-21 detection of is advantageous for health monitoring in Point of Care Testing (POCT). Gold nanoparticles (AuNP) as excellent colorimetric sensors are widely used in the POCT. However, their low sensitivity is a limitation of their clinical use. Herein, we developed an AuNPs-based miR-21 assay with enzyme-assisted amplification reaction to construct the colorimetric platform capable of detecting as low as 0.1[Formula: see text]nM. In this platform, template ssDNA as a signal molecule could hybridize with ssDNA-modified AuNPs to generate the color reaction. The target miR-21 specifically hybridized to the template ssDNA, which was then cleaved by exonuclease III (Exo III) to release the target miR-21. As a trigger, miR-21 catalyzed the degradation of the template ssDNA to amplify the signal by Exo III. By hybridizing miR-21 and template ssDNA in the presence of Exo III, R-21 induced a significant decrease in the level of template ssDNA to reduce the aggregation of AuNPs. There is a clear color difference in the presence/absence of miR-21 in the assay. In this assay, the optimal concentration of templated ssDNA and Exo III were 100[Formula: see text]nM and 0.06[Formula: see text]U/[Formula: see text]L in a 100[Formula: see text][Formula: see text]L detection system. The LoD for UV–Vis spectrum and colorimetric reaction were 0.1[Formula: see text]nM and 0.5[Formula: see text]nM, respectively. The detection system has good selectivity and can be used to detect miR-21 in the simulated saliva. It has great potential for application in biomedical research as well as in clinical diagnostics.

An exonuclease III-driven dual-mode aptasensor based on Au-Pd@Fc nanozyme and magnetic separation pretreatment for aminoglycoside antibiotics detection

A novel dual-mode aptasensor was constructed for aminoglycoside antibiotics (AAs) detection by using a broad-spectrum aptamer as a biorecognition element, and Au-Pd@Fc functionalized by signal DNA as nanoprobes. In electrochemical mode, the target-induced cyclic amplification reaction run under the action of exonuclease-III, which increased the number of nanoprobes on the electrode surface. AAs could be quantitatively detected with LOD of 0.0355 ± 0.00613 nM. In colorimetric mode, the Au-Pd@Fc nanozyme catalyzed the color reaction of 3,3′,5,5′-tetramethylbenzidine. The blue-shifted absorbance will be observed with the change of AAs concentration, and the LOD was 0.0458 ± 0.00572 nM. Furthermore, a magnetic molecular-imprinted material capable of specific adsorption of AAs was prepared on milk sample pretreatment. The aptasensor was used to detect 10 kinds of AAs in milk and the recoveries were 97.19 ± 4.41% ∼ 98.70 ± 4.45% and 96.38 ± 3.53%-97.54 ± 4.13% in electrochemical and colorimetric methods. This work provided a theoretical basis for the application of aptamers in simultaneous detection of antibiotics.

An ultrasensitive dual-mode stagey for 17β-estradiol assay: Photoelectrochemical and colorimetric biosensor based on a WSe2/TiO2-modified electrode coupled with nucleic acid amplification

BackgroundThe abuse of 17β-estradiol(E2) has aroused wide concern in environmental and biomedical fields, which severely affects the endocrine function of human and animals. Therefore, an ultrasensitive and accurate assay of E2 is critically important. Traditional chromatography or immunoassay techniques exhibited good sensitivity and selectivity, but expensive instruments and antibodies may pose cost and stability issues, as well as difficulties in meeting on-site detection requirements. Ultrasensitive, reliable, and on-site detection of E2 at trace level remains a challenge. Hence, developing a simple, ultrasensitive assay to simultaneously achieve accurate detection and rapid visual analysis of E2 is extremely crucial. ResultsWe developed a versatile dual-mode photoelectrochemical (PEC) and colorimetric biosensor based on isothermal nucleic acid amplification strategy for the ultrasensitive and accurate detection of E2. The method modified titanium dioxide (TiO2) with tungsten selenide (WSe2) nanoflowers to synthesize WSe2/TiO2 heterostructures as a substrate for signal amplification and nanoprobe modification. Isothermal nucleic acid amplification strategy has been proven to be a powerful tool for strong signal amplification. The presence of a target triggered the nucleic acid amplification reaction, and produced a large amount of tDNA that competed with G-quadruplex immobilized on the electrode surface. The remaining G-quadruplex/hemin catalyzed the 4-chloro-1-naphthol (4-CN) to form biocatalytic precipitation (BCP) and ABTS-H2O2 chromogenic reaction, thus, the dual-mode platform was capable of achieving PEC-colorimetric ultrasensitive detection based on the catalytic activity of G-quadruplex/hemin DNAzyme. Within optimal conditions, the dual-mode biosensor exhibited a remarkable detection limit as low as 0.026 pM. SignificanceBenefiting from the superior performance of WSe2/TiO2 and the power signal amplification of isothermal nucleic acid amplification strategy, this aptasensor achieved the ultrasensitive detection of E2. The independent transmission paths of photoelectrochemical and colorimetric provide mutual support and flexible switching, significantly enhancing the overall sensitivity and accuracy of the detection strategy, which can meet the needs for E2 precise quantification and rapid on-site detection.

HCR-Assisted RTF-EXPAR-Based Lateral Flow Analysis for Sensitive Detection of H1N1 Influenza Virus.

The H1N1 influenza virus is a significant pathogen responsible for seasonal influenza, and its frequent outbreaks pose substantial challenges to global public health. The present study successfully developed a lateral flow analysis platform that integrates reverse transcription-free exponential amplification reaction (RTF-EXPAR) and hybridization chain reaction (HCR) processes with functionalized quantum dots for the direct detection of H1N1 influenza virus RNA, eliminating the need for reverse transcription. The fluorescence signal on the band recorded with a smartphone can be utilized for the quantitative determination of the target. Interestingly, the dual signal amplification strategy exhibits high sensitivity with a remarkably low detection limit of 10 aM. Moreover, this platform exhibits excellent flexibility and universality, where the various pathogens can be determined by replacing the specific nucleic acid fragments in RTF-EXPAR. The aforementioned advantages reveal its huge potential in the early diagnosis of H1N1 influenza virus infection and developing point-of-care testing (POCT) equipment for nucleic acid analysis.

Development of a DNAzyme-Driven Fluorescent Light-Up Aptasensor for Label-Free Detection of Multiple lncRNAs.

Long noncoding RNAs (lncRNAs) act as the dynamic regulatory molecules that control the expression of genes and affect numerous biological processes, and their dysregulation is associated with tumor progression. Herein, we develop a fluorescent light-up aptasensor to simultaneously measure multiple lncRNAs in living cells and breast tissue samples based on the DNAzyme-mediated cleavage reaction and transcription-driven synthesis of light-up aptamers. When target lncRNAs are present, they can be recognized by template probes to form the active DNAzyme structures, initiating the T4 PNK-catalyzed dephosphorylation-triggered extension reaction to generate double-strand DNAs with the T7 promoter sequences. The corresponding T7 promoters can initiate the transcription amplification catalyzed by the T7 RNA polymerase to generate abundant Broccoli aptamers and malachite green aptamers, which can bind DFHBI-1T and MG to generate strong fluorescence signals. Taking advantage of the good selectivity of DNAzyme-mediated cleavage of lncRNAs, high amplification efficiency of T7 transcription-driven amplification reaction, and bright fluorescence of the RNA aptamer-fluorophore complex, this method exhibits high sensitivity with a detection limit of 21.4 aM for lncRNA HOTAIR and 18.47 aM for lncRNA MALAT1, and it can accurately measure multiple lncRNAs in both tumor cell lines and breast tissue samples, providing a powerful paradigm for biomedical research and early clinic diagnostics.

Portable Aptasensor Based on Parallel Rolling Circle Amplification for Tumor-Derived Exosomes Liquid Biopsy.

Here, a separation-free and label-free portable aptasensor is developed for rapid and sensitive analysis of tumor-derived exosomes (TEXs). It integrated a parallel rolling circle amplification (RCA) reaction, selective binding of metal ions or small molecules to nucleic acid-specific conformations, and a low-cost, highly sensitive handheld fluorometer. Lung cancer, for example, is targeted with two typical biomarkers (mucin 1 and programmed cell death ligand 1 (PD-L1)) on its exosomes. The affinity of aptamers to the targets modulated the amount of RCA products (T-Hg2+-T and cytosine (C)-rich single-stranded DNA), which in turn affected the fluorescence intensity of quantum dots (QDs) and methylene blue (MB). The results revealed that the limit of detection (LOD) of the handheld fluorometer for cell-derived exosomes can be as low as 30 particlesmL-1. Moreover, its specificity, sensitivity, and area under the curve (AUC) are 93% (14/15), 92% (23/25), and 0.956, as determined by the analysis of 40 clinical samples. Retesting 16 of these samples with the handheld fluorometer yielded strong concordance between the fluorometer results and those acquired from clinical computed tomography (CT) and pathology.

Bioreaction-Compatible Bivariate Lanthanide MOF Sensor Enables Stimulus-Multiresponsive Platform for ctDNA On-Site Detection.

Detection of circulating tumor DNA (ctDNA) in liquid biopsy is of great importance for tumor diagnosis but difficult due to its low amount in bodily fluids. Herein, a novel ctDNA detection platform is established by quantifying DNA amplification by-product pyrophosphate (PPi) using a newly designed bivariable lanthanide metal-organic framework (Ln-MOF), namely, Ce/Eu-DPA MOF (CE-24, DPA = pyridine-2,6-dicarboxylic acid). CE-24 MOF exhibits ultrafast dual-response (fluorescence enhancement and enzyme-activity inhibition) to PPi stimuli by virtue of host-guest interaction. The platform is applied to detecting colon carcinoma-related ctDNA (KARS G12D mutation) combined with the isothermal nucleic acid exponential amplification reaction (EXPAR). ctDNA triggers the generation of a large amount of PPi, and the ctDNA quantification is achieved through the ratio fluorescence/colorimetric dual-mode assay of PPi. The combination of the EXPAR and the dual-mode PPi sensing allows the ctDNA assay method to be low-cost, convenient, bioreaction-compatible (freedom from the interference of bioreaction systems), sensitive (limit of detection down to 101 fM), and suitable for on-site detection. To the best of our knowledge, this work is the first application of Ln-MOF for ctDNA detection, and it provides a novel universal strategy for the rapid detection of nucleic acid biomarkers in point-of-care scenarios.

Exonuclease III assisted exponential amplification reaction (EXPAR) for specific miRNA-155 analysis during post-anesthetic nursing

The persistent obstacle in precise and sensitive identification of microRNAs (miRNAs) pertains to the advancement of expeditious and effective isothermal amplification methodologies suitable for point-of-care environments and monitoring the cancer prognosis in patients receiving post-anesthetic nursing. The exponential amplification reaction (EXPAR) has attracted considerable interest due to its simplicity and ability to rapidly amplify signals. The practical application of the EXPAR is, nevertheless, severely hampered by the inability to differentiate closely related homologous sequences and to modify the designed templates to suit other targets. A loop-stem template for the EXPAR system was developed in this study to facilitate specific target recognition with the aid of exonuclease III (Exo III). This innovation effectively eliminated non-specific hybridization that could occur between the template and interfering sequences, thereby ensuring minimal background amplification of EXPAR. By modulating Exo III-based target recycling, EXPAR based chain amplification and G4/hemin based color reaction, this method facilitated the precise and sensitive examination of miRNA-155, yielding acceptable yields and a minimal detection limit of 0.43 fM. The approach expedites simple and expeditious molecular diagnostic applications involving short nucleic acids and offers an innovative method for enhancing the selectivity of EXPAR-based techniques, providing a robust tool for monitoring the expression level from patients receiving post-anesthetic nursing and guiding the treatment strategy.