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MicroRNAs (miRNAs) are currently recognized as important biomarkers for the early diagnosis and prognostic treatment of cancer. Herein, we developed a simple and label-free method for the multiplex detection of miRNAs, based on entropy-driven circuit (EDC) amplification and non-gel sieving capillary electrophoresis-LED induced fluorescence detection (NGCE-LEDIF) platform. In this system, three different lengths of fuel chains were designed to catalyze three EDC, targeting miRNA-21, miRNA-155, and miRNA-10b, respectively. In the presence of target miRNA, the EDC cycle amplification reaction was triggered, generating numerous stable double-strands products (F-DNA/L-DNA). Since the three miRNAs correspond to three different lengths of F-DNA/L-DNA, they can be easily isolated and detected by NGCE. This strategy has good sensitivity, with detection limits of 68 amol, 292.2 amol, and 394 amol for miRNA-21, miRNA-155, and miRNA-10b, respectively. Additionally, this method has good specificity and can effectively distinguish single-base mismatches of miRNA. The recoveries of the three miRNAs in deproteinized healthy human serum ranged from 91.28 % to 108.4 %, with a relative standard deviation (RSD) of less than 7.9 %. This method was further applied to detect cellular miRNAs in human breast cancer (MCF-7) cell extracts, revealing an up-regulation of miRNA-21, miRNA-155, and miRNA-10b in MCF-7 cells. The successful spiked recovery in human serum and RNA extraction from MCF-7 cells underscores the practicality of this method. Therefore, this strategy has broad application prospects in biomedical research.

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Sensitive monitoring of human 8-oxyguanine DNA glycosylase (hOGG1) activity in living cells is helpful to understand its function in damage repair and evaluate its role in disease diagnosis. Herein, a functional DNA-Zn2+ coordination nanospheres was proposed for sensitive imaging of hOGG1 in living cells. The nanospheres were constructed through the coordination-driven self-assembly of the entropy driven reaction (EDR) -deoxyribozyme (DNAzyme) system with Zn2+, where DNAzyme was designed to split structure and assembled into the EDR system. When the nanospheres entered the cell, the competitive coordination between phosphate in the cell and Zn2+ leaded to the disintegration of the nanospheres, releasing DNA and some Zn2+. The released Zn2+ acted as a cofactor of DNAzyme. In the presence of hOGG1, the EDR was completed, accompanied by fluorescence recovery and the generation of a complete DNAzyme. With the assistance of Zn2+, DNAzyme continuously cleaved substrates to produce plenty of fluorescence signals, thus achieving sensitive imaging of hOGG1 activity. The nanospheres successfully achieved sensitive imaging of hOGG1 in human cervical cancer cells (HeLa), human non-small cell lung cancer cells and human normal colonic epithelial cells, and assayed changes in hOGG1 activity in HeLa cells. This nanospheres may provide a new tool for intracellular hOGG1 imaging and related biomedical studies.

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Circulating tumor DNA (ctDNA) is an emerging biomarker of liquid biopsy for cancer. But it remains a challenge to achieve simple, sensitive and specific detection of ctDNA because of low abundance and single-base mutation. In this work, an excitation/emission-enhanced heterostructure photonic crystal (PC) array synergizing with entropy-driven circuit (EDC) was developed for high-resolution and ultrasensitive analysis of ctDNA. The donor donor-acceptor FÖrster resonance energy transfer ("DD-A" FRET) was integrated in EDC based on the introduction of simple auxiliary strand, which exhibited higher sensitivity than that of traditional EDC. The heterostructure PC array was constructed with the bilayer periodic nanostructures of nanospheres. Because the heterostructure PC has the adjustable dual photonic band gaps (PBGs) by changing nanosphere sizes, and the "DD-A" FRET can offer the excitation and emission peak with enough distance, it helps the successful matches between the dual PBGs of heterostructure PC and the excitation/emission peaks of "DD-A" FRET; thus, the fluorescence from EDC can be enhanced effectively from both of excitation and emission processes on heterostructure PC array. Besides, high-resolution of single-base mutation was obtained through the strict recognition of EDC. Benefiting from the specific spectrum-matched and synergetic amplification of heterostructure PC and EDC with "DD-A" FRET, the proposed array obtained ultrasensitive detection of ctDNA with LOD of 12.9 fM, and achieved the analysis of mutation frequency as low as 0.01%. Therefore, the proposed strategy has the advantages of simple operation, mild conditions (enzyme-free and isothermal), high-sensitivity, high-resolution and high-throughput analysis, showing potential in bioassay and clinical application.

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BACKGROUND: It is estimated that over 50 % of human cancers are caused by mutations in the p53 gene. Early sensitive and accurate detection of the p53 gene is important for diagnosis of cancers in the early stage. However, conventional detection techniques often suffer from strict reaction conditions, or unsatisfied sensitivity, so we need to develop a new strategy for accurate detection of p53 gene with smart designability, multiple signal amplification in mild reaction conditions. RESULTS: In this study, CRISPR/Cas system is exploited in entropy-driven catalysis (EDC) and hybridization chain reaction (CHA) dual signal amplification sensing strategies. The products of both reactions can efficiently and separately activate CRISPR/Cas12a which greatly amplifies the fluorescent signal. The method has good linearity in p53 detection with the concentration ranged from 0.1 fM to 0.5 pM with ultra-low detection limit of 0.096 fM. It also showed good performance in serum, offering potentials for early disease detection. SIGNIFICANCE: The designed dual amplification dynamic DNA network system exhibits an ultra-sensitive fluorescence biosensing for p53 gene identification. The method is simple to operate and requires only one buffer for the experiment, and meanwhile shows smart designability which could be used for a wide range of markers. Thus, we believe the present work will provide a potential tool for the construction and development of sensitive fluorescent biosensors for diseases.

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Polymeric supramolecular hydrogels (PSHs) leverage the thermodynamic and kinetic properties of non-covalent interactions between polymer chains to govern their structural characteristics. As these materials are formed via endothermic or exothermic equilibria, their thermal response is challenging to control without drastically changing the nature of the chemistry used to join them. In this study, we introduce a novel class of PSHs utilizing the intercalation of double-stranded DNA (dsDNA) as the primary dynamic non-covalent interaction. The resulting dsDNA intercalating supramolecular hydrogels (DISHs) can be tuned to exhibit both endothermically or exothermically driven binding through strategic selection of intercalators. Bifunctional polyethylene glycol (MW ~ 2000 Da) capped with intercalators of varying hydrophobicity, charge, and size (acridine, psoralen, thiazole orange, and phenanthridine) produced DISHs with comparable moduli (500 - 1000 Pa) but unique thermal viscoelastic response. Notably, acridine-based cross-linkers displayed invariant and even increasing elasticity with temperature, suggesting an endothermic binding mechanism. This methodology expands the set of structure-properties available to biomass-derived DNA biomaterials and promises a new material system where a broad set of thermal and viscoelastic responses can be obtained due to the sheer number and variety of intercalating molecules.

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The most trivial example of self-assembly is the entropy-driven crystallization of hard spheres. Past works have established the similarities and differences in the phase behavior of monomers and chains made of hard spheres. Inspired by the difference in the melting points of the pure components, we study, through Monte Carlo simulations, the phase behavior of athermal mixtures composed of fully flexible polymers and individual monomers of uniform size. We analyze how the relative number fraction and the packing density affect crystallization and the established ordered morphologies. As a first result, a more precise determination of the melting point for freely jointed chains of tangent hard spheres is extracted. A synergetic effect is observed in the crystallization leading to synchronous crystallization of the two species. Structural analysis of the resulting ordered morphologies shows perfect mixing and thus no phase separation. Due to the constraints imposed by chain connectivity, the local environment of the individual spheres, as quantified by the Voronoi polyhedron, is systematically more spherical and more symmetric compared to that of spheres belonging to chains. In turn, the local environment of the ordered phase is more symmetric and more spherical compared to that of the initial random packing, demonstrating the entropic origins of the phase transition. In general, increasing the polymer content reduces the degree of crystallinity and increases the melting point to higher volume fractions. According to the present findings, relative concentration is another determining factor in controlling the phase behavior of hard colloidal mixtures based on polymers.

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MicroRNA (miRNA) is a pivotal biomarker in the diagnosis of various cancers, including bladder cancer (BCa). Despite their significance, the low abundance of miRNA presents a substantial challenge for sensitive and reliable detection. We introduce an innovative, highly sensitive assay for miRNA expression quantification that is both enzyme-free and portable. This method leverages the synergy of target recycling and entropy-driven assembly (EDA) for enhanced sensitivity and specificity. The proposed method possesses several advantages, including i) dual signal amplification through target recycling and EDA, which significantly boosts sensitivity with a lower limit of detection of 2.54 fM; ii) elimination of enzyme requirements, resulting in a cost-effective and stable signal amplification process; and iii) utilization of a personal glucose meter (PGM) for signal recording, rendering the method portable and adaptable to diverse settings. In summary, this PGM-based approach holds promising potential for clinical molecular diagnostics, offering a practical and efficient solution for miRNA analysis in cancer detection.

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Electrolyte engineering plays a crucial role in enhancing the performance of lithium metal batteries (LMBs) featuring high-voltage cathodes and limited lithium anodes, thereby unlocking their potential for high-energy electrochemical storage. Herein, an entropy-driven hybrid gel electrolyte with enhanced diversity in Li-ion solvation structures is designed by incorporating substantial amounts of insoluble LiPO2F2 and LiNO3 salts into LiPF6-based carbonate electrolytes, followed by in situ thermal polymerization. Specifically, the Li+ solvation structures are modulated via ionophilic NO3- and PO2F2- to generate an anion-rich solvation sheath and thus promote anion reduction at the electrode-electrolyte interface. The interfaces enriched in anion-derived inorganic components facilitate rapid ionic transport, thus enabling smooth and dense Li morphology and ultimately enhancing the electrochemical performance of LMBs. As a result, this high-hybrid gel electrolyte confers LMBs employing high-voltage NCM cathodes, as demonstrated by sustained performance in both coin-cell (500 cycles at 4.5 V) and Ah-level pouch cell configurations under practical conditions (60 cycles, N/P: 1.92, and E/C: 2.0 g Ah -1).

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DNA Methyltransferase 1 (DNMT1) serves as a crucial biomarker associated with various diseases and is essential for evaluating DNA methylation levels, diagnosing diseases, and evaluating prognosis. As a result, a convenient, quantitative, and sensitive assay for detecting DNMT1 is in high demand. However, current techniques for DNMT1 detection struggle to balance accuracy, low cost, and high sensitivity, limiting their clinical usefulness. To address this challenge, we have developed a DNMT1 detection method (CAED), which combines aptamer-specific recognition with a highly programmable Entropy-driven catalysis DNA network and is further integrated with the CRISPR-Cas12a system. This innovative approach achieves a detection limit as low as 90.9 fmol/L. To demonstrate the clinical applicability and significance of our CAED method, we successfully measured DNMT1 levels in 10 plasma samples 10 cervical tissue samples. These results underscore the potential of our method as an accurate, affordable, and ultra-sensitive tool for evaluating DNMT1 levels. This innovative method offers a potent means for assessing DNMT1 levels and significantly advances disease diagnosis and health risk prediction. Plus, it establishes an innovative design framework for CRISPR-Cas12a-based biosensors, tailored explicitly for enzyme content quantification.

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BACKGROUND: Kanamycin is an antibiotic that can easily cause adverse side effects if used improperly. Due to the extremely low concentrations of kanamycin in food, quantitative detection of kanamycin becomes a challenge. As one of the DNA self-assembly strategies, entropy-driven strand displacement reaction (EDSDR) does not require enzymes or hairpins to participate in the reaction, which greatly reduces the instability of detection results. Therefore, it is a very beneficial attempt to construct a highly sensitive and specific fluorescence detection method based on EDSDR that can detect kanamycin easily and quickly while ensuring that the results are effective and stable. RESULTS: We created an enzyme-free fluorescent aptamer sensor with high specificity and sensitivity for detecting kanamycin in milk by taking advantage of EDSDR and the high specific binding between the target and its aptamer. The specific binding can result in the release of the promoter chain, which then sets off the pre-planned EDSDR cycle. Fluorescent label modification on DNA combined with the fluorescence quenching-recovery mechanism gives the sensor impressive fluorescence response capabilities. The research results showed that within the concentration range of 0.1 nM-50 nM, there was a good relationship between the fluorescence intensity of the solution and the concentration of kanamycin. Specificity experiments and actual sample detection experiments confirmed that the biosensor could achieve highly sensitive and specific detection of trace amounts of kanamycin in food, with a detection limit of 0.053 nM (S/N = 3). SIGNIFICANCE: To our knowledge, this is the first strategy to combine EDSDR with fluorescence to detect kanamycin in food. Accurate results can be obtained in as little as 90 min with no enzymes or hairpins involved in the reaction. Furthermore, our enzyme-free biosensing method is straightforward, highly sensitive, and extremely specific. It has many possible applications, including monitoring antibiotic residues and food safety.

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The construction of efficient methods for highly sensitive and rapid detection of disease markers is essential for the early diagnosis of serious diseases. In this paper, taking advantage of the UiO-66-NH2 signal molecule in combination with a waste-free entropy-driven DNA machine, a novel homogeneous electrochemical ratiometric platform is developed to detect MircoRNA (miRNA). Metal-organic framework materials (UiO-66-NH2 MOF) and ferrocene were utilized as electrochemical signal tags and reference probes, respectively. The target-initiated waste-free three-dimensional (3D) entropy-driven DNA nanomachine is activated in the presence of miRNA, resulting in DNA-labeled-UiO-66-NH2 falling off from the electrode, leading to a decrease in the signal of UiO-66-NH2 at 0.83V. Our strategy can mitigate false positive responses induced by the DNA probes immobilized on electrodes in traditional distance-dependent signal adjustment ratiometric strategies. The proposed ratiometric platform demonstrates superior sensitivity (a detection limit of 9.8 fM), simplified operation, high selectivity, and high repeatability. The ratiometric biosensor is also applied to detect miRNA content in spiked serum samples.

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Accurate microRNA (miRNA) detection is pivotal in the diagnosis and monitoring of cancer. Entropy-driven catalysis (EDC) has attracted widespread attention as an enzyme-free, isothermal technique for miRNA detection owing to its inherent simplicity and reliability. However, conventional EDC is a single-output mode, limiting the efficiency of signal amplification. In this study, a novel EDC dual-output mode was employed in conjunction with DNAzyme, resulting in the development of an EDC dual-end DNAzyme (EDC-DED) approach for highly sensitive miRNA detection. In this system, miRNA-21 initiated the EDC reaction, producing a large amount of catalytically active dual-end Mg2+-dependent DNAzyme. The DNAzyme further cleaved the reporter cyclically, generating a notably amplified fluorescence signal. The proposed method achieved a low detection limit of 2 pM. Compared with the traditional EDC single-end DNAzyme (EDC-SED) strategy, the present method exhibited superior amplification efficiency, enhancing detection sensitivity by approximately 46.5-fold. Furthermore, this platform demonstrated ideal specificity, satisfactory reproducibility and acceptable detection capabilities in clinical serum samples. Therefore, the straightforward and convenient strategy is a potential tool for miRNA analysis, which may provide a new perspective for biological analysis and clinical application.

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MicroRNAs (miRNAs) are crucial biomarkers for the early detection and monitoring of disease progression of chronic obstructive pulmonary disease (COPD). Herein, we have devised a method for detecting miRNA using a combination of colorimetric and graphene oxide-based fluorescent techniques. The target miRNA in our design could precisely activate the trans-cleavage activity of the CRISPR-Cas13a system. The activated Cas13a enzyme cuts the "rUrU" section in the P1 probe, generating a nicking site to induce entropy-driven amplification (EDA). One of the available EDA products has the capability to unfold the hairpin probe, thereby initiating the catalytic hairpin assembly, exposing the G-quadruplex structure, facilitating the subsequent color response. The fuel strand labeled with Cy3 successfully established a double-stranded DNA structure with DNA3, and consequently the Cy3 would not be quenched by graphene oxide (GO). The implementation of the dual-mode technique in this method yields greater benefits in terms of improving the precision and consistency of the miRNA measurements. The developed method has the capability to fluorescently measure miRNA-21 levels down to a concentration of 5.8 fM. In addition, the analysis of miRNA targets from clinical samples using this method demonstrates its promising utility in the fields of biomedical research of COPD.

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With the emergence of microRNA (miRNA) as a promising biomarker in cancer diagnosis, it is significant to develop multiple analyses of miRNAs. However, it still faces difficulties in ensuring the sensitivity and accuracy during multiplex detection owing to the low abundance and experimental deviation of miRNAs. In this work, a flexible-arranged biomimetic array integrated with parallel entropy-driven circuits (EDCs) was developed for ultrasensitive, multiplex, reliable, and high-throughput detection of miRNAs. The biomimetic array was fabricated by arrangement of various photonic crystals (PCs) for adjustable photonic band gaps (PBGs) and specific fluorescence enhancement. Meanwhile, two cancer-related miRNAs and one reference miRNA were introduced as multiple analytes as a proof-of-concept. The parallel EDCs with negligible crosstalk were designed based on the modular property. Because of the one-to-one match between the emitted fluorescence of parallel EDCs and the PBGs of the flexible-arranged biomimetic array, the generated fluorescence signal triggered by target miRNAs can be enhanced on the corresponding domain of the array. Furthermore, the amplified signal of the array was detected with high-throughput scanning, which could reveal specific information on cancer-related miRNAs as well as reference miRNA, enhancing the abundance and reliability of the analysis. The proposed array has the merits of a modular design, flexible deployment, simple operation (nonenzymatic and isothermal), improved accuracy, high sensitivity, and multiplex analysis, showing potential in disease diagnosis.

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Na4Fe3(PO4)2(P2O7) (NFPP) is regarded as a promising cathode material for sodium-ion batteries (SIBs) owing to its low cost, easy manufacture, environmental purity, high structural stability, unique three-dimensional Na-ion diffusion channels, and appropriate working voltage. However, for NFPP, the low conductivity of electrons and ions limits their capacity and power density. The generation of NaFeP2O7 and NaFePO4 inhibits the diffusion of sodium ions and reduces reversible capacity and rate performance during the manufacturing process in synthesis methods. Herein, we report an entropy-driven approach to enhance the electronic conductivity and, concurrently, phase purity of NFPP as the superior cathode in sodium-ion batteries. This approach was realized via Ti ions substituting different ratios of Fe-occupied sites in the NFPP lattice (denoted as NTFPP-X, T is the Ti in the lattice, X is the ratio of Ti-substitution) with the configurational entropic increment of the lattice structures from 0.68 R to 0.79 R. Specifically, 5% Ti-substituted lattice (NTFPP-0.05) inducing entropic augmentation not only improves the electronic conductivity from 7.1 × 10-2 S/m to 8.6 × 10-2 S/m but also generates the pure-phase of NFPP (suppressing the impure phases of the NaFeP2O7 and NaFePO4) of the lattice structure, which is validated by a series of characterizations, including powder X-ray diffraction (XRD), Fourier transform infrared spectra (FT-IR), X-ray photoelectron spectroscopy (XPS), and density functional theory (DFT). Benefiting from the Ti replacement in the lattice, the optimal NTFPP-0.05 composite shows a high first discharge capacity (118.5 mAh g-1 at 0.1 C), superior rate performance (70.5 mAh g-1 at 10 C), and excellent long cycling life (1200 cycles at 10 C with capacity retention of 86.9%). This research proposes a new entropy-driven approach to improve the electrochemical performance of NFPP and reports a low-cost, ultrastable, and high-rate cathode material of NTFPP-0.05 for SIBs.

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Exosomal proteins from the parental cells are considered to be promising biomarker sets for precise tumor diagnostics and monitoring. However, the accurate quantitative analysis of low-abundance exosomal proteins remains challenging due to the heterogeneity of clinical samples. Here, we standardized the exosomal concentration with a fluorogenic membrane probe and developed an aptamer-bivalent-cholesterol-mediated Proximity Entropy-driven Exosomal Protein Reporter (PEEPR). The proposed PEEPR enables the in-situ analysis of multiple exosomal proteins by integrating bivalent cholesterol anchor (exosomal lipid bilayer) and aptamer (exosomal proteins) with a proximity entropy-driven circuit. Based on this strategy, we successfully achieved detection limits of 3.9 pg/mL exosomal GPC-3 and 3.4 pg/mL exosomal PD-L1. Notably, the standardization of exosome concentrations is designed to avoid errors due to biological heterogeneity. The results showed that evaluating the levels of exosomal GPC-3 and PD-L1 in clinical samples via this strategy could accurately differentiate healthy individuals, hepatitis B patients, and hepatocellular carcinoma patients. In summary, PEEPR is a promising clinical diagnostic strategy for the quantitative analysis of a variety of tumor-associated exosomal proteins for the precise diagnosis and personalized treatment monitoring of tumors.

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Alzheimer's disease (AD) is a degenerative disease of the brain worldwide. Currently, there is no effective cure. But accurate and early diagnosis of AD is critical to the development of patient care and future treatments. MiRNA-16 has been considered as an effective diagnostic biomarker for AD because of its regulatory effect on key proteins of AD. Herein, a colorimetric lateral flow assay (LFA) was developed for sensitive detection of miRNA-16 based on entropy-driven catalysis (EDC) amplification strategy. MiRNA-16 triggered EDC and released more linker DNAs (LDNA) of sandwich structure. Thus, AuNPs were enriched at the T-line to enhance the colorimetric signal and improve the sensitivity of visual assay. It showed good specificity and sensitivity for detecting miRNA-16 with a detection limit of 1.01 pM. The practical detection of miRNA-16 in human serum obtained satisfactory result. Significantly, EDC achieved signal amplification in homogeneous solution without enzyme and DNA labeling, leading to a cheap and easy detection of miRNA-16. Therefore, it provided a portable and rapid assay for AD-related nucleic acid, which holds a potential for point-of-care testing (POCT) of AD.

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It is of great interest and necessity to develop a nonenzymatic, simple but highly sensitive biosensor for early diagnosis of oral cancer. Present here is an electrochemical DNA biosensor which integrates a target-triggered, entropy-driven, nonenzymatic and isothermal amplification strategy with gold nanoparticles/zeolitic imidazolate frameworks-8 (AuNPs@ZIF-8) nanocomposites for ultra-sensitive detection of oral cancer-related biomarker (ORAOV 1) in saliva. It is worth noting that the nuclease is not involved in the whole reaction process, which is simple and flexible in design only using a series of linear single-stranded DNA, avoiding undesired secondary structure interference. Meanwhile, due to the synergistic effect of AuNPs and ZIF-8, AuNPs@ZIF-8 nanocomposites display high stability, excellent electrical conductivity and exceptional electrocatalytic activity, further enhancing the electrochemical signal and avoiding labeling electrochemical signal probes. Experimental results demonstrate that this electrochemical DNA biosensor has a wide linear range (1 fM ∼1 nM), a low limit of detection (163 aM), excellent specificity, superior reproducibility and stability to ORAOV 1. More importantly, the actual application of the newly developed electrochemical biosensor is exemplified in human saliva with satisfactory recoveries. Therefore, the newly developed electrochemical biosensor has a broad application prospect in the nondestructive and early screening of oral cancer.

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In this work, an intelligent and versatile electrochemical biosensor was constructed to detect two types of biomarkers by utilizing "off-on-off" switching. Firstly, human apurinic/apyrimidinic endonuclease1(APE1) mediated specific cleavage of the AP site, initiating activation DNAzyme and entropy-driven catalytic (EDC) reaction. Subsequently, large amounts of ferrocene labeled single-stranded DNA was released and captured with a remarkable electrochemical signal, achieving "off-on" state. In the presence of microRNA 21(miRNA-21), the DNA/RNA heteroduplexes were formed and cleaved by duplex-specific nuclease (DSN) with recovery the target miRNA-21, causing the current suppression in an "on-off" state. This sensor achieved highly sensitive detection of APE1 and miRNA-21 with a detection limit of 2.5 mU·mL-1 and 1.33 × 10-20 M, respectively, and also exhibited good selectivity, reproducibility and stability. Moreover, this proposed biosensor made it possible to realize analysis of multiple types of biomarkers on a single sensor, which improved utilization and analysis efficiency compared to traditional sensors. This study might open a new avenue to design multifunctional sensing platform for biological research and early disease diagnosis.

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An electrochemical biosensor is reported for controlling CRISPR/Cas12a activity through the utilization of entropy-driven reactions, alongside the construction of a highly sensitive biosensor for B-type natriuretic peptide (BNP) detection. In the biosensor, entropy-driven reactions are employed to regulate the activity of CRISPR/Cas12a - a gene editing tool - capable of nonspecific cleavage of single-stranded DNA (ssDNA). The biosensor architecture encompasses an electrode that is modified with ssDNA probes designed to hybridize with target BNP aptamers. These aptamers, furnished with labeled ssDNA triggers, facilitate the activation of CRISPR/Cas12a through interaction with its guide RNA. Upon the presence of BNP, it associates with the aptamers, subsequently liberating the triggers that instigate the entropy-driven reactions. As a consequence of these reactions, more stable duplexes emerge between the triggers and guide RNA, thereby activating CRISPR/Cas12a. The activated CRISPR/Cas12a subsequently executes cleavage of ssDNA probes residing on the electrode surface, culminating in the generation of an electrochemical signal directly (the calibration plots of differential pulse voltammetric detection were acquired at a working potential of 0.2 V (vs. ref. electrode)) proportional to the BNP concentration. Validation of the biosensor's performance is undertaken, wherein BNP detection is demonstrated in both buffer and human serum samples. Evident in the findings is the biosensor's discernible sensitivity and specificity for BNP detection, exemplified by a detection limit of 13.53 fM and a lack of interference originating from other cardiac biomarkers, respectively. Furthermore, the biosensor's potential to discriminate between healthy individuals and those afflicted by heart failure, predicated on distinctive BNP levels, is illustrated.

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