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The accurate detection of flap endonuclease 1 (FEN1) activity is crucial for early cancer diagnosis and therapy. Herein, we have established a "target-substrate double-enhanced" DNA electrochemical biosensor by integrating a cleavage-ligation logic transcription circuit-activated DNAzyme amplifier with the transition-metal carbides-gold nanoparticles (MXene-Au) composites for the accurate detection of FEN1 activity. The FEN1 can be translated into a stable transcription hairpin DNA (TH), incorporating the catalytic strand of the DNAzyme via the logic transcription circuit. Then, the TH hybridizes with the substrate strand of DNAzyme and the DNAzyme amplifier can be activated, thus producing a significant quantity of single-stranded DNA (sDNA) and achieving the efficient amplification of the FEN1. Finally, the sDNA loaded with electroactive substances is immobilized on the capture DNA-anchored MXene-Au modified electrode, significantly enhancing the electrochemical signal intensity for highly sensitive FEN1 activity detection. The excellent selectivity and sensitivity of the proposed electrochemical biosensor can facilitate the accurate detection of FEN1 activity in biological samples and aid in screening FEN1-releated drugs. Additionally, three-input concatenated logic gates (AND-AND, INHIBIT-AND, and AND-OR) are constructed for FEN1 activity detection. Particularly, the AND-AND gate achieves logic detection of FEN1 activity in complex samples. Consequently, this bidirectionally enhanced electrochemical biosensor offers a novel analytical method for FEN1 activity detection, showing substantial promise for early diagnosis and drug discovery of FEN1-related disease. • The cleavage-ligation logic transcription circuit ensures the specificity. • DNAzyme amplifier facilitates the transcription product amplification. • MXene-Au improves the conductibility for increasing the signal intensity. • The bidirectional-enhanced DNA electrochemical biosensor for FEN1 sensitive detection. [ABSTRACT FROM AUTHOR]

Accurate and rapid nucleic acid testing for pathogen diagnostics is central to controlling the spread of infectious diseases. Here, we report an assay for rapid bacterial DNA detection via PAM (protospacer adjacent motif)-free Cas12f biosensor. The assay, which is named LSD12f (LAMP-driven strand displacement for Cas12f detection), involved multiplexed loop-mediated isothermal amplification (LAMP) followed by isothermal strand displacement reaction and Cas12f-mediated cleavage of a quenched fluorophore. The core of LSD12f was engineered with forward/reverse inner primers, which made LAMP products recognized and cut by enzyme digestion, triggering single-stranded DNA (ssDNA) production. Moreover, CRISPR-Cas12f could especially recognize target ssDNA region without PAM sites to reduce false-positive amplification. LSD12f could detect Streptococcus pyogenes in blood and flesh samples within 60 min with 100 % specificity and 100 % accuracy when validated by quantitative PCR (qPCR). LSD12f showed impressive specificity and sensitivity as a rapid and adaptive toolkit to broaden the use of molecular diagnostics. • LAMP-driven strand displacement integrated CRISPR-Cas12f (LSD12f) for pathogen DNA detection without PAM site restriction. • LSD12f detection could be achieved at constant temperature within an hour. • LAMP-mediated ssDNA production for CRISPR-Cas12f detection could perform in one single tube. • LSD12f accurate detection for flesh and whole blood samples demonstrated prospective POCT applications. [ABSTRACT FROM AUTHOR]

Developing appropriate protein assay methods is necessary, as protein assays play an important role in molecular diagnosis, drug screening, and biomedical research. This article presents a universal homogeneous method based on the CRISPR/Cas12a system and the terminal protection strategy for protein assays. Initially, a single-stranded DNA (ssDNA) labeled with a small molecule at its 3'-end was designed to respond to the target protein and serve as an activator of Cas12a. In the absence of the target protein, small-molecule-linked ssDNA was effectively cleaved using exonuclease I (Exo I). The lack of the activator resulted in the trans -cleavage inactivity of Cas12a, leading to low fluorescence in the detection solution. However, in the presence of the target protein, the specific binding of the target protein to the small molecules on the activator protected the activator against digestion by Exo I. The remaining intact activator triggered the trans -cleavage activation of Cas12a, producing strong fluorescence. Based on this principle, our method achieved fluorescence enhanced detection of the target protein. After experimental condition optimization, our method achieved highly sensitive and specific protein detection in two models: folate receptor and streptavidin. The excellent performance of the fluorescence method highlights the applicability and effectiveness of the CRISPR/Cas12a system in protein assays, and broadening its application scope. [Display omitted] • A universal homogeneous method for protein assays was developed. • This method integrated the terminal protection strategy with the CRISPR/Cas12a system. • Sensitive detection of two target proteins was achieved. • The practicability of the proposed method was demonstrated. [ABSTRACT FROM AUTHOR]

The fat mass and obesity-associated protein (FTO) is vital for modulating dynamic reversible process of N6-methyladenosine (m6A) level in the nucleus and the abnormal expression of FTO correlates with generation and development of Alzheimer's disease, obesity and several cancers. The sensitive detection of FTO is therefore of significance for diagnosis of FTO-related diseases. In this work, on the basis of the demethylation enzyme capability of FTO and the dual Mg2+-dependent DNAzyme recycled signal magnification strategy, establishment of a highly sensitive electrochemical FTO biosensor is described. The target FTO molecules catalyze the demethylation of m6A-modified sequences to form effective cleavage substrates for the Mbo I endonuclease, leading to the release of active DNAzyme sequences to trigger subsequent dual DNAzyme recycling cycles. Many methylene blue-tagged ssDNA sequences can thus be yielded via the DNAzyme amplifications and captured on the sensor electrode to generate highly amplified currents for detecting FTO in range of 0.04 pM to 10 nM with 6.11 fM detection limit. Additionally, the sensor demonstrates high selectivity and is able to be used for detecting cell lysate FTO, showing its potential for the diagnosis and study of FTO-related diseases for various application purposes. • A highly sensitive electrochemical FTO biosensor is developed. • Target activation of dual DNAzyme recycling leads to significant signal amplification. • Such sensor can achieve sensitive detection of low levels of FTO in cell lysate. [ABSTRACT FROM AUTHOR]

False positives and high background are key problems in nucleic acid amplification-based methods. Herein, a triple amplification and low background sensing platform is developed, termed double CRISPR/Cas12a-drived hyperbranched rolling circle amplification (D-Cas12a/HRCA). By introducing two kinds of CRISPR/Cas12a, we effectively avoid background signal and improve sensitivity. The target miRNA is firstly recognized by a padlock probe to trigger the rolling circle amplification (RCA), which generates mass of long ssDNA and achieves the first amplification. The ssDNA can unlock the cis- and trans-cleavage activity of CRISPR/Cas12a (Cas12a/crRNA-1). Therefore, the cis-cleaved ssDNA and trans-cleaved primer 2 (neighboring C3 inhibitor modification) are used as template and primer for the next hyperbranched rolling circle amplification (HRCA, secondary amplification), respectively. The above amplification products (dsDNA, ssDNA) are recognized again by the CRISPR/Cas12a (third amplification) and output fluorescence or visualization signals. Compared with traditional RCA/Cas12a or HRCA/Cas12a, D-Cas12a/HRCA shows higher sensitivity with lower background. This assay achieves sensitive detection of miRNA-21 low to 10.02 fM. The lateral flow detection strips enable miRNA-21 detection with a LOD of 100 fM, showing the potential of CRISPR/Cas-based technology in POCT detection. More importantly, the platform is consistent with RT-qPCR in detecting miRNA-21 extracted from different cell lines. • D-Cas12a/HRCA is a triple amplification with high sensitivity. • The two kinds of CRISPR/Cas12a enable detection with ultra-low background. • Fluorescence or visualization signals are achieved. • The actual detection results were consistent with RT-qPCR. [ABSTRACT FROM AUTHOR]

The present study describes the development of a fluorescent biosensor for the identification of the pathogenic bacteria Escherichia coli O157:H7. The biosensor is based on a catalytic hairpin assembly (CHA) and λ-Exonuclease cleavage reaction, which enables rapid and precise detection. This approach uses a hairpin H0 that is intended to open when it binds to the target pathogenic bacterium. When the hairpin H0 was opened, the activator domain within the hairpin H0 stem region was revealed, triggering numerous CHA cycles, generating many H1/H2 double-stranded DNAs. λ-Exonuclease cleaved the 5′-phosphorylated H2 strand in the H1/H2 double-stranded DNA to release single-stranded DNA H1, which hybridized with another hairpin H2 to start a new cycle. In both amplification reactions, a significant quantity of hairpin H2 was opened and cleaved, resulting in a fluorescent signature that could be watched in real time. By employing this innovative design philosophy, we were able to successfully identify Escherichia coli O157:H7 within a broad range of concentrations (101-108 CFU/mL), with a detection limit of 10 CFU/mL. This strategy's useful diagnostic ability was also effectively proven for identifying other target pathogenic bacteria by logically redesigning the hairpin H0. • λ-Exonuclease and CHA were used to constructed a fluorescent biosensor. • Escherichia coli O157:H7 was detected without nucleic acid extraction. • The fluorescent biosensor exhibited a lower limit of detection. (10 CFU/mL). [ABSTRACT FROM AUTHOR]

In recent years, electrochemical sensors for nucleic acid testing based on CRISPR/Cas12a technology have attracted widespread interest due to their ease of operation and high detection sensitivity. Here, a label-free electrochemical sensor amplified by primer exchange reaction (PER), hybridization chain reaction (HCR) and CRISPR/Cas12a system was developed for analyzing miRNA- 155 and PCB 77. In the presence of targets, PER was initiated, whose products could be used to trigger the trans-cleavage activity of Cas12a for the single-stranded DNA (ssDNA) on the electrode. Afterwards, with the amplification of HCR on the electrode and electrostatic adsorption of Ru(NH 3) 6 3+, the concentration of the targets could be indicated by the DPV intensity with a low background signal. The detection limits for miRNA- 155 and PCB 77 were estimated to be 67 aM and 6.9 pg/L, respectively. This work provides a versatile method for sensitive detection of different targets using the same crRNA and can be used in various biochemical investigations. • A label-free electrochemical sensor was used for the detection of two targets. • PER, CRISPR/Cas system and HCR were applied for signal amplification. • CRISPR/Cas system can decrease the background signal without the usage of PAN. • The same crRNA can be used to detect different targets. [ABSTRACT FROM AUTHOR]

Aflatoxin B1 (AFB1) leads to considerable pollution to grains and causes harmful effects on human health. Herein, we present a regenerable AFB1 aptasensor, based on electro-oxidation conducted host-guest dissociation for natural AFB1 detection. Molybdenum disulfide-Au nanobipyramids (MoS 2 -AuNBPs) serve as signal carriers and are equipped with indicative thionine (Thi) and semi-complementary chains to AFB1 aptamer (ssDNA). The electrode surface is modified with gold nanoparticles (AuNPs) and β-cyclodextrin (β-CD). Aptamer-ferrocene (Apt-Fc) can be recognized in cavity of β-CD through host-guest interaction. The signal probe, self-assembled with AFB1 Apt via hybridization, can be easily expelled by AFB1, bringing a ratiometric readout Δ I Thi / I Fc. Particularly, regeneration process is realized with synergy of AFB1-conducted Apt-ssDNA dissociation and electro-oxidation conducted host (β-CD)-guest (Fc) dissociation. Modifications and regeneration behaviors are morphologically and electrochemically validated. Regenerations are dependably performed in repetitive cycles (N = 7), which exhibit considerable performance in analyses. Electro-oxidation time is experimentally optimized (6 min). This method exhibits wide range from 1.0 pg/mL to 80.0 ng/mL, and limit of detection is 5.0 × 10−1 pg/mL, to target AFB1. During natural AFB1 determinations, proposed readouts are validated with certified HPLC and recovery test. Relative errors and recovery rates are calculated as 2.04–7.22% and 94.41–106.54%, respectively. [Display omitted] • MoS 2 -AuNBPs is synthesized as a high-performance signal carrier for ssDNA and Thi. • Thi signal negatively answers AFB1 change, due to competitive expellee of ssDNA. • Electro-oxidation regeneration between host (β-CD) and guest (Fc) is clarified. • Proposed sensor is soundly validated in natural sample analysis against interference. [ABSTRACT FROM AUTHOR]

DNA glycosylase assumes a pivotal role in the preservation of genome integrity by recognizing and excising damaged bases. Herein, we develop a single primer/self-template-powered cascade rolling circle amplification (RCA) and apurinic/apyrimidinic endonuclease (APE1)-mediated signal amplification for simultaneous detection of human alkyladenine DNA glycosylase (hAAG) and uracil DNA glycosylase (UDG). The uniqueness of this approach lies in the use of custom-designed hairpin probes to establish a prerequisite for RCA: the conformational change of hairpin probe to circular template, which effectively suppress undesired non-specific amplification. We design two hairpin probes featuring elongated stem structure and 3′-phosphate (3′-PO 4) modifications. The stems of hairpin probes are modified with glycosylase substrates and BER process can cleave hairpin probes, resulting in the generation of single-stranded DNAs (ssDNAs) with 3′-hydroxyl (3′-OH) ends. These ssDNAs can function as padlock probes to form circular template upon ligation by ligase. Ultimately, the self-template-initiated RCA and APE1-mediated signal amplification cascade occurs, generating a substantial number of free Cy3 and Cy5 fluorophores that can be simply visualized by single-molecule fluorescence detection. This method demonstrates excellent specificity and sensitivity, with a limit of detection (LOD) of 3.42 × 10-12 U/μL for hAAG and 2.41 × 10-12 U/μL for UDG. This method also can be used for screening of potential inhibitor and kinetic parameter measurement. Most importantly, this method enables accurate profiling of glycosylases expression levels in various cancer cell lines and detection of glycosylase activities at the single-cell level, offering a bright future in cancer detection, medical research, and precision medicine. • Simultaneous detection of multiple glycosylases at ultra-low concentrations. • Conformation-switchable hairpin probes and self-templated amplification reduce non-specific amplification. • One primer probe can simultaneously trigger two isothermal amplifications. • Accurate assessment of DNA glycosylase expression in different cancer cell lines. [ABSTRACT FROM AUTHOR]

The expression levels of microRNAs (miRNA) can provide insights into the occurrence, progression, and treatment efficacy in tumors, highlighting the need for precise and easily controllable detection methodologies. The reasonable design of carbon dots enables the precise manipulation of luminescence and surface characteristics. Herein, fluorine doped carbon dots (F-CDs) were designed and prepared to establish a reasonable control of the interaction force between DNA and F-CDs. F-CDs were synthesized through a hydrothermal reaction involving tetrafluoroterephthalic acid and tetraethylene pentaamine, resulting in noteworthy optical and amphiphilic properties. F-CDs proficiently interact with single-stranded DNA (ssDNA) through hydrophobic interaction, leading to the quenching of dye labeled ssDNA, while maintaining self-fluorescence stability. The quenching mechanism was elucidated. The complementary DNA sequence induced nearly 100% desorption of ssDNA from F-CDs. Assisted by the target cycling signal amplification from duplex specific nuclease (DSN) and the self-fluorescence of F-CDs, an effective ratiometric strategy utilizing the adsorption system of F-CDs and probe DNA for let-7a was established for detecting let-7a, a critical miRNA in the assessment of tumor development. Under the optimized conditions, this method demonstrated high sensitivity and reproducibility in detecting let-7a with the linear range from 1.0 fM to 10.0 nM and a low limit of detection (LOD) of 3.0 fM. The recovery of let-7a in plasma and the evaluation of let-7a levels in seven cell types underscored the method's potential for efficient applications in monitoring of miRNA levels in real samples. • Fluorine doped carbon dots (F-CDs) with amphiphilic behavior were prepared. • F-CDs exhibited direct, stable, and reversible adsorption of DNA probe via hydrophobic interaction. • Dye labeled ssDNA was adsorbed and quenched by F-CDs effectively. • The adsorption and quenching mechanism of F-CDs as carrier were clarified. • A ratiometric approach based on F-CDs as reference accurately monitored let-7a in plasma and cells. [ABSTRACT FROM AUTHOR]

Herein, an ultrasensitive self-powered genosensor is constructed by modifying a model hairpin capture DNA (cDNA) on a nanostructured n-type CdS modified ITO (n-CdS|ITO) photoanode of a photofuel cell. Then, the modified hairpin cDNA is transformed into a single-stranded DNA (ssDNA) by a target DNA (tDNA) and λ-exonuclease. Afterwards, a triple-quenching tag is specially designed by the in situ growth of p-type Cu 2 O nanoparticles on ultrathin (a few layers) Ti 3 C 2 T x nanosheets to form a p-Cu 2 O/Ti 3 C 2 T x composite. This is then introduced onto the photoanode surface via strong chelation between the exposed Ti ions on Ti 3 C 2 T x and the ssDNA on n-CdS|ITO. The introduction of p-Cu 2 O/Ti 3 C 2 T x dramatically decreases the open-circuit voltage (E ocv) of the genosensor and is based on a p-n type semiconductor quenching strategy. This strategy simplifies the complex and expensive aptamer chain design and modification process for the introduction of p-type semiconductors in other biosensing platforms based on a similar quenching mechanism. Meanwhile, both p-Cu 2 O and Ti 3 C 2 T x exhibit good peroxidase-like catalytic activity, which results in precipitation and steric hindrance on n-CdS|ITO to further quench the output signals. Furthermore, the large Ti 3 C 2 T x nanosheets partially blocked the light absorption of n-CdS. Due to this triple-quenching effect, a wide linear range (1 fM ∼ 100 pM) and a low detection limit (0.230 fM, S/N = 3) are obtained by the genosensor for detecting the tDNA, in this case a biomarker for chronic myelogenous leukemia. • A self-powered biosensor is made by modifying hairpin capture DNA on n-type CdS|ITO. • cDNA is transformed to single-stranded DNA (ssDNA) by target DNA and λ-exonuclease. • A quenching tag is made by growing p-type Cu 2 O nanoparticles on Ti 3 C 2 T x nanosheets. • Chelation of Ti on Ti 3 C 2 T x with ssDNA on n-CdS|ITO enables p - n quenching effect. • Peroxidase-like p -Cu 2 O and Ti 3 C 2 T x produce precipitation quenching on n -CdS|ITO. [ABSTRACT FROM AUTHOR]

Surface-enhanced Raman spectroscopy (SERS) has exhibited great potential in deoxyribonucleic acid (DNA) analysis. Rapid and label-free identification of single-base mutation in DNA is still limited by the sensitivity and reproducibility of SERS substrates. In this work, we develope plasmonic SiNW arrays decorated with Ag nanoparticles (SiNW@AgNP arrays) via hexadecyltrimethylammonium bromide (CTAB)-assisted galvanic reaction to address these challenges, which show excellent sensitivity and reproducibility. Under optimized experimental conditions, plasmonic SiNW@AgNP arrays enable detection of DNA bases of adenine (A), guanine (G), cytosine (C), and thymine (T) in solution at concentration down to pM-μM range, and multiple detection ability with mixture of DNA bases. More significantly, our study demonstrates the SiNW@AgNP arrays may serve as excellent SERS substrates in discriminating DNA single-base mutation between sickle cell anemia gene and normal hemoglobin gene in serum samples, which can be exploited in a wide range of plasmonic enhanced sensing applications for a fast, real-time, label-free and cost-effective clinical diagnosis. • A simple and efficient strategy has been developed to prepare SiNW@AgNP arrays platform via CTAB-assisted galvanic reaction. • The prepared SERS platform exhibits excellent sensitivity (LOD down to 0.1 fM) and reproducibility (RSD of 3.4 %). • As-prepared SERS platform can discriminate DNA single-base mutation in sickle cell anemia in serum samples. [ABSTRACT FROM AUTHOR]

The aquatic foodborne pathogen-induced infectious disease is becoming one of the killers to people's health worldwide, attributed to its long latent period and high infectivity. It is urgently desiderated to develop simple, rapid, sensitive and universal detection techniques for early diagnosis and timely treatment of pathogens. Here, we present a novel visible CRISPR biosensing pathogen detection system named integrating competitive annealing mediated isothermal amplification (CAMP) -mediated CRISPR-Cas14a isothermal detection platform (CCIP) through CAMP with CRISPR-Cas14a. Initially, in the presence of target gene, CAMP was conducted at constant temperature to produce folded CAMP amplicon with abundant single-stranded DNA (ssDNA) target sites. When Cas14a-small guide RNA (sgRNA) binary complex specially recognized target sequences, it would trigger the cleavage collateral activity on nonspecific ssDNA fluorescence probes to produce optical signal with a quite high turnover. Furthermore, the results could be easily observed by naked eyes in an hour with high specificity and sensitivity of 102 aM. Furthermore, the method was verified with a pathogen detection result of 100% accuracy for 15 whole blood samples. Thus, CCIP is a promising point-of-care testing (POCT) technique for practical pathogen diagnosis. • Isothermal amplification integrated CCIP detected pathogen DNA without PAM site restriction. • CCIP detection could be achieved at constant temperature within an hour. • The diagnosis signal could be either real-time fluorescence or visual fluorescence readout. • CCIP accurate detection for whole blood samples demonstrated prospective POCT applications. [ABSTRACT FROM AUTHOR]

Heavy metal ions (HMIs), including Pb2+, Cu2+, and Hg2+, has shown great threat to the environment and public health worldwide owing to their high toxic at trace levels. However, conventional laboratory-based methods for analyzing HMIs require expensive instrumentation and complex operations limit their further application. In this study, we present a novel "two-step" approach based on entropy-driven multicolor DNA nanoflowers (DNFL) for the simultaneous ultrasensitive detection of three typical HMIs (Pb2+, Cu2+, and Hg2+). Through specific cleavage/competition reactions initiated by the target metal ions, a large number of single-stranded DNA (ssDNA) are produced, triggering the blooming of DNFL. Subsequently, with the assistance of fuel strands, an entropy-driven strand displacement reaction (ESDR) will further accelerate the conformational change of DNFL within one hour. Different fluorophores (FAM, ROX, and Cy5) attached to the opened DNFL serve as indicator molecules for the respective HMIs. After reasonable design and optimization, the developed fluorescence analysis method demonstrates excellent analytical performance (with limit of detection, 0.13 nM, 3.5 pM, 4.6 pM for Pb2+, Cu2+, and Hg2+, respectively) and can be applied to the simultaneous and rapid detection of multiple HMIs in water environment. • An entropy-driven DNA sensor based on a multicolor DNA nanoflower was developed. • The biosensor can realize the detection of multiple HMIs in water through a simple "two-step" approach. • The flexible architecture and precise probe arrangement achieve stable output of fluorescence signals. • The method exhibits satisfactory performance for rapid detection of multiple HMIs in water environments. [ABSTRACT FROM AUTHOR]

Aptamer-based biosensors capable of rapid one-step detection of viruses provide new capabilities for large-scale detection of COVID-19. Here we report a combined aptamer-based electrochemical and computational approach to characterize the interaction between a 60 base single stranded (ss)DNA aptamer and the viral S protein using molecular docking and bioinformatics tools. To construct the biosensor, the DNA aptamer was immobilized to the surface of a gold nanoparticles (AuNPs) modified electrode and methylene blue (MB) was used as redox indicator. Based on thermodynamic calculations for the aptamer sequence as well as several runs in APBS (Adaptive Poisson-Boltzmann Equation), we found that S protein GLN104-ASP115 pocket is the candidate for DNA 30–39(GGGAGTGGTT) sequence interaction. In the presence of S-protein, the target induced conformational changes in the aptamer's structure, generating an increase in the MB redox signal, with a linear range between 10 pM to 6 nM and a limit of detection of 91.1 pM using differential pulse voltammetry (DPV). The aptasensor was investigated in spiked human saliva and serum samples, demonstrating performance in both media. This study demonstrates the potential of the MB-labeled electrochemical aptasensor for direct one-step detection of viral proteins, COVID-19 and potentially other viral agents. [Display omitted] • An electrochemical aptasensor with S Protein Binding affinity is reported. • Bioinformatics offers insights into the molecular docking of the S protein to ssDNA. • Sensitive detection of S protein between 10 pM and 6 nM is demonstrated. • Experimental-computational investigation of viral protein binding for virus detection. [ABSTRACT FROM AUTHOR]

A simple and low-cost paper-based microfluidic biosensor has been developed for on-site detection of Pb(II) by using CRISPR-Cas12a assisted smartphone device. Pb(II) induced G-quadruplex can inhibit the activation of the CRISPR-Cas12a system and subsequently prevent the cleavage of single-stranded DNA around gold nanoparticles. Thus, the resultant dispersed gold nanoparticles will flow into the detection zone and be subsequently captured by DNA nano-flowers in the detection zone of the paper-based microfluidic chip, resulting in the corresponding color change. The R/G values analyzed through our established SmartIons APP, correlate well with Pb(II) concentrations with a linear detection range of 0.1 μM - 10 μM and a lowest detection limit of 18.3 nM. The paper-based microfluidic biosensor exhibits high specificity, good anti-interference ability, stability and applicability. In view of low cost, easy customization and multiple channel, paper-based microfluidic biosensor has great potential for the on-site detection of Pb(II). [Display omitted] • For the first time, the inhibition of CRISPR-Cas12a system by Pb(II) has been studied. • Smartphone detection of paper-based microfluidic biosensor has been developed. • The biosensor can sensitively detect Pb(II) with a lowest detection limit of 18.3 nM. • The biosensor can be applied to on-site detect Pb(II) in environmental samples. [ABSTRACT FROM AUTHOR]

The present study describes a novel technology referred to as reciprocal two single-stranded DNA (ssDNA) probe-mediated isothermal amplification (TPIA) that enables target nucleic acid detection. TPIA extends the ssDNA probe A into ultralong ssDNA products with tandem repeats of hairpin and ssDNA probe B to form double-stranded DNA hairpins of varying lengths via continuously repeated polymerization and displacement. The entire TPIA process was supervised in real time with the assistance of fluorescence techniques. By exploiting this new mechanism, the system can be used as an autocephalous module to provide a versatile tool for a biosensing program. The ssDNA probe B was relationally designed to form a stem-loop structure (HP), which can only unwind through specific recognition by the target nucleic acid. The HP stem region was then exposed and HP was rearranged to serve as the ssDNA probe B in the TPIA system after HP unfolding. The novel design strategy was exploited to specifically assay different target microRNAs (miR-21 and miR-221) with a low limit of detection on the 10 and 1 fM level, respectively. This method has great potential for nucleic acid detection in biomedical research and clinical diagnosis by rationally redesigning the template. • A simple and novel isothermal amplification technology was established. • It's a simple nucleic acid diagnostic system. • This novel strategy can be used to specifically different target microRNA. • This biosensor had acceptable reproducibility in the complicated matrix samples. [ABSTRACT FROM AUTHOR]

A novel electrochemiluminescence (ECL) aptasensor based on tetrahedral DNA nanostructures (TDN) and CdTe QDs was fabricated for the detection of acetamiprid residues. Three TDNs with different sizes were successfully designed and synthesized. The unique and rigid tetrahedral structure of TDN can ensure that the biological probe always maintains the orientation perpendicular to the sensing interface, greatly reducing the background noise and effectively improving the signal-to-noise ratio. It has great significance to improve the order of the probe on the sensing interface. The ECL signals of TDN with different sizes were compared with the conventional aptasensors (c-DNA) modified by single-stranded DNA (ssDNA). The results displayed that TDN can effectively amplify the sensor's ECL signal, the size of TDN was negatively correlated with the amplification coefficient and hybridization time of aptamer. Compared various parameters of the c-DNA sensor and TDN 13 sensor which with the highest signal. The linear range of the TDN 13 sensor is 0.100 pM∼100 nM, the detection limit is 34.0 fM. The linear range of the c-DNA sensor is 1.00 pM∼10.0 nM, the detection limit is 178 fM. TDN 13 sensor was applied to measure acetamiprid residues in Chinese cabbage samples with satisfactory results. • TDN can improve the signal-to-noise ratio of c-DNA sensor. • The ECL intensity of TDN sensor is greater than that of c-DNA sensor. • Explored relationship between TDN's size and ECL signals' amplification coefficient. • The introduction of TDN could greatly shorten the time of sensor's construction. [ABSTRACT FROM AUTHOR]

Methicillin-resistant Staphylococcus aureus (MRSA) strains are prevalent foodborne superbugs and pose significant pathogens for both humans and animals. The sensitive and specific diagnosis of MRSA holds paramount importance for its prevention and treatment. Herein, we develop a T 7 exonuclease-assisted PAM-free D ual CRISPR- C as12a B iosensor (TDCB) targeting the antibiotic resistance gene MecA for ultrasensitive and highly specific MRSA detection. After generation of single-stranded DNA (ssDNA) obtained with T7 exonuclease digestion of amplified MRSA genome, the trans -cleavage activity of dual-Cas12a-crRNA complexes was initiated without the requirement of protospacer adjacent motifs (PAM). The fluorescent signal obtained with TDCB biosensor was stronger, compared with the single-Cas12a biosensor. TDCB detects the MRSA genome and cells with a high sensitivity of 100 ag/mL and 1 cfu/mL, respectively. Moreover, TDCB can distinguish MRSA from tested 6 non-MRSA bacterial species from each other. We further validated TDCB using MRSA-infected tilapia fishes and obtained results consistent with the qPCR assay. This TDCB technology, a novel Cas12a-based biosensor that detects genomic regions without reliance on PAM motifs, can have very broad utilization in hospitals, food and feed industry, and fish and dairy farming. [Display omitted] • A dual-CRISPR-Cas12a biosensor was prepared. • The biosensor is PAM motifs-free for detection of MecA gene. • The diagnostic result of MRSA-infected tilapia is consistent with qPCR. • The biosensor has high sensitivity with 1 cfu/mL. [ABSTRACT FROM AUTHOR]

Thrombin played important roles for the diagnosis of neurodegenerative and cardiovascular diseases. Herein, we successfully constructed a rapid and sensitive fluorescence and colorimetric dual-signal sensing strategy for the detection of thrombin by taking advantage of the DNA-enhanced peroxidase like activity FeCo-ONSs and magnetic separation. In this work, single-stranded DNA S1 with thrombin aptamer was used to enhance the catalytic activity of FeCo-ONSs, and single-stranded DNA S2 with different thrombin aptamer was modified on the surface of magnetic beads (MBs) to construct MBs-S2 for subsequent magnetic separation. In the presence of thrombin, S1 and MBs-S2 quickly combined with thrombin and the amount of S1 decreased in the solution after magnetic separation. The enhancement of peroxidase like activity of S1/FeCo-ONSs was weakened, resulting in the inhibition of the catalytic oxidation process of substrate TMB. The formation of oxTMB and the fluorescence quenching efficiency of MoS 2 QDs were further affected. Therefore, the quantitative detection of thrombin can be achieved by measuring the absorption signal of oxTMB and the fluorescence signal of MoS 2 QDs. The limits of detection for thrombin were 0.67 pM and 2.36 pM by the outputting fluorometric and colorimetric dual signals, respectively. This sensing strategy not only has high specificity, but also can be expanded as a more universal platform for sensitive detection of different proteins by changing the aptamer probe pairs. • The hydrophilic MoS 2 QDs with emission of 420 nm were prepared by a facile one-step approach. • The analysis of thrombin activity was realized though a new biosensor by DNA-enhanced peroxidase like activity of FeCo-ONSs. • The application of magnetic separation technology can eliminate interferences in complex samples. • The dual-signal biosensing system showed good detection limit down to 0.67 pM and 2.36 pM, respectively. [ABSTRACT FROM AUTHOR]

Quantity analysis of bacterial 16 S rDNA gene is still a challenge. Here we successfully achieved bacterial 16 S rDNA gene SERS analysis with target aided self-assembly of DNA hyperbranched nanostructures. Catalytic hairpin assembly (CHA) was first introduced for self-assembly of branched nanostructures, including target bacterial DNA served as trigger probe and three hairpin probes for further target activated cross opening. Rolling cycle amplification (RCA) was then introduced by using a circular DNA as template and single stranded branches of the formed branched nanostructures as primer, forming a concatemer containing multiple copies of tandem repeating segments with same sequences complementary to the circular templates. After that, thousands of Raman-Au nanoprobe would hybridize this long single stranded DNAs for DNA-AuNPs nanoaggretates assembly. By using SERS signal collection, target bacterial DNAs would be successfully analyzed with ultra-sensitivity as low as 15.0 fM. Furthermore, selectivity test for targets with mismatched DNA sequences and other bacterial 16 S rDNA genes show good results. This sensing method was extended to quantify target genes in human plasma samples, showing its excellent applicability in complex samples. • Target aided self-assembly of DNA hyperbranched nanostructures was first formed. • Coupled with DNA hyperbranched nanostructures, this approach exhibits excellent sensitivity of 15 fM. • This approach describes high selectivity and its satisfactory complex targets sensing ability of enduring the interference. [ABSTRACT FROM AUTHOR]

A novel sensitive electrochemistry aptasensor was constructed for the detection of patulin (PAT) using AuNPs-CS-ZnO NFs as signal amplification and hierarchically porous metal organic framework@methylene blue connection aptamer as signal tags. • A novel electrochemical aptasensor strategy for the ultrasensitive detection of PAT is constructed. • ZnO NFs for electrode sensing platform preparation are used for signal amplification. • Aminated hierarchically porous Zr-MOFs labelled methylene blue are used as signal tags. • The protocol possesses accepted specificity and can be applied in apple juice sample. In this work, the feasibility of a novel sensitive electrochemistry aptasensor for the detection of patulin (PAT) using hierarchically porous metal organic framework@methylene blue connection aptamer as signal tags was demonstrated. The substrate electrode of the aptasensor was prepared by electrodeposited gold nanoparticles (AuNPs) on chitosan-zinc oxide nano flower-modified electrode. Subsequently, the complementary single-stranded DNA was attached to the surface of the modified electrode by Au-S bond. Signal tags self-assemble on the surface of modified electrodes by aptamer-cDNA binding, while methylene blue as mediator for electrochemical detection. Thanks to the large specific surface area provided by zinc oxide nano flower, the electrode can load more signal tags and AuNPs to achieve signal amplification. In presence of PAT, Apt preferentially binds to PAT, causing dissociation of some Apt-cDNA and releasing some signal tags leading, resulting in decreased electrical signals. Under optimal conditions, the change in electrochemical signal was proportional to the logarithm of PAT concentration, from 5 × 10−8-5 × 10-1 μg mL−1, and the detection limit was estimated to be 1.46 × 10-8 μg mL-1. The aptasensor exhibited aceptable reproducibility, renewability, long-term stability and specificity. Moreover, aptasensor was reliable in determination of PAT in spiked apple juice samples. [ABSTRACT FROM AUTHOR]

• A label-free and dye-free strategy for sensitive DNA detection has been developed based on self-fluorescent G-quadruplex. • The method allows quantification of DNA without dyes, making the analysis economically and environmentally friendly. • The proposed signal output owns potential for real sample analysis for its biocompatibility and resistance to DNAzyme. The intrinsic fluorescence of the interlocked bimolecular DNA G-quadruplex (93del4T) was utilized to achieve label-free and non-toxic detection of target DNA. The designed probe consists of a single stranded DNA template that hybridizes with the sequence of 93del4T at 5′ end forming partial complementary duplex and leaving single strand overhang at 3′ terminus to recognize target DNA molecule. Once the target DNA hybridizes with the probe to form another part of duplex at 3′ terminus, exonuclease Ⅲ will catalyze the stepwise removal of nucleotides from the blunt 3′ terminus, consecutively liberating the target DNA and the sequence of 93del4T. The released target DNAs can recycle to bind more probes and release more 93del4T that can fold into G-quadruplexes to amplify fluorescence signals. As a result, quantitative detection of the target DNA in human serum has been achieved according to fluorescent signal intensities. In comparison to other methods, this strategy is simple, rapid, inexpensive and toxic free. [ABSTRACT FROM AUTHOR]

Graphical abstract Herein, we develop an innovative, oxygen independent cathodic PEC bioassay on the basis of the largely augmented photocurrent of bismuth oxyiodide (BiOI) by potassium ferricyanide (K 3 Fe(CN) 6) under aerobic conditions. The K 3 Fe(CN) 6 is found to be a more efficient electron acceptor than dissolved oxygen for promoting the photocurrent of BiOI, and the enhancement of K 3 Fe(CN) 6 for BiOI is almost unaffected by dissolved oxygen. This may be attributed to the fast charge transfer rate of K 3 Fe(CN) 6 , which renders it more competitive than dissolved oxygen for capturing the photo generated electrons of BiOI. Since the detection takes place in ambient air and is not interfered from the atmospheric oxygen, this facile and robust cathodic PEC biosensing system displays promising applicability for on-site and real-time diagnosis. The developed cathodic PEC assay possessed excellent analytical performances in terms of sensitivity, specificity and reproducibility. Highlights • The integration of the K 3 Fe(CN) 6 with bioreactions for PEC detection is shown for the first time. • K 3 Fe(CN) 6 was found to enhance the cathodic photocurrent of flower-like BiOI for the first time. • The first example of oxygen-independent cathodic PEC bioassay. • A "signal-on" and "label-free" versatile platform assisted by the signal amplification strategy. Abstract Most of the current cathodic photoelectrochemical (PEC) bioassays either rely on dissolved oxygen as a signal reporting molecule or are interfered by atmospheric oxygen (if using other molecules than oxygen as the signaling molecule), which leads to their monotonous detection mechanism, vulnerable/nonadjustable performance, complicated instrument and time consuming operation. Herein, we develop an innovative, oxygen independent cathodic PEC bioassay with distinct advantages of robust performance, simple instrument/facile operation, as well as sensitive and prompt readout. Specifically, taking the ochratoxin A (OTA) as a model analyte, the detection combines the largely augmented photocurrent of flower-like bismuth oxyiodide (BiOI) microspheres by potassium ferricyanide (K 3 [Fe(CN) 6 ]) under aerobic conditions, the highly selective aptamer recognition as well as the powerful exonuclease-catalyzed target recycling amplification. Removal of the surface immobilized DNA under the cooperative effects of OTA induced double-stranded DNA (dsDNA) dissociation and RecJ f exonuclease mediated digestion of the released single-stranded DNA results in enhanced flux of K 3 [Fe(CN) 6 ] to the photoelectrode, enables a "signal-on" and "label-free" mode for OTA detection with high sensitivity and good selectivity. It also shows acceptable results for the detection of OTA spiked in wine samples. Since the detection takes place in ambient air and is not interfered by the atmospheric oxygen, this facile and robust cathodic PEC biosensing system displays promising applicability for on-site and real-time diagnosis. More significantly, it is extensible as a general strategy for versatile cathodic PEC bioanalysis. [ABSTRACT FROM AUTHOR]

Highlights • AβO specific thiol-terminated ssDNA aptamer receptors were tethered to gold electrodes. • Target detection was characterized by electrochemical impedance spectroscopy. • Aptasensor exhibited a selectivity towards different Aβ species. • Aptasensor was used for monitoring the aggregation process of amyloid-β proteins. • AFM was carried out to determine the morphologies of Aβ species during incubation. Abstract Amyloid-β oligomers (AβO) are important diagnostic markers for Alzheimer's disease (AD) and potential therapeutic targets for treating it. Herein, a simple and label-free electrochemical biosensor is presented for the specific recognition of AβO based on the binding of these biomarkers to ssDNA aptamer receptors. The ssDNA aptamer self-assembles to gold rod electrodes through Au-S interactions and binds specifically to AβO. The novel aptasensor shows a wide linear concentration range from 0.1 to 500 nM with a low detection limit of 0.03 nM as derived from electrochemical impedance spectroscopy invesastigations. Furthermore, owing to the high selectivity among Aβ species, this label-free sensor was used to monitor the process of Aβ protein aggregation, which was validated by atomic force microscope analysis. In addition, the aptasensor can be used to detect AβO in artificial CSF with satisfying accuracy. To our knowledge, it is first label-free aptasensor for an AβO assay based on EIS that works in artificial CSF and can be used for monitoring Aβ protein aggregation. These features, together with its easy fabrication, operation convenience, and effective regeneration, make our new aptasensor a promising tool for the early detection of AD and drug development by monitoring Aβ plaques degradation. [ABSTRACT FROM AUTHOR]

Highlights • This is the first report on the peroxidase-like activity of CoOOH nanoflakes for the detection of MTase activity. • The peroxidase-like activity of CoOOH nanoflakes can be improved by adsorbing ssDNA. • The colorimetric sensor with a low detection limit of 0.069 U/mL for M.SssI MTase activity detection. • The colorimetric sensor allows detection of M.SssI MTase in cell lysates of lung cancer cells (A549). Abstract A label-free colorimetric assay for MTase activity based on the nanozyme activity of cobalt oxyhydroxide (CoOOH) nanoflakes and the difference in affinity of the nanoflakes with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) has been developed. In this strategy, ssDNA can enhance the nanozyme activity of CoOOH nanoflakes, which could be attributed to the electrostatic attraction and π-π stacking interaction between 3,3′,5,5′-tetramethylbenzidine (TMB) and ssDNA. The proposed method presents a wide linear range from 0.08 to 50 U/mL and a low detection limit (LOD) of 0.069 U/mL. Additionally, this colorimetric sensor also has been proved to detect MTase in the cell lysates of lung cancer cells (A549) with good recovery and reproducibility. Furthermore, the novel strategy can be applied for high-throughput evaluation and screening the inhibitors for M.SssI MTase by using representative anticancer drugs as model inhibitors, therefore, the colorimetric sensor can not only achieve the purpose of sensitively detecting the activity of MTase, but also has the potential in clinical diagnostics and drug discovery. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Highly specific ssDNA aptamers for SMZ were selected for the first time. • The selected aptamers bind SMZ with affinity in the nanomolar range. • The high specificity of the aptamers was determined by molecular simulation. • An aptasensor was developed which had remarkable specificity and sensitivity. • The aptasensor was used to detect SMZ in milk showing high accuracy and precision. Abstract Antibody-based sensors are the most widely used for screening antibiotic residues in food samples. However, these sensors always suffer from cross-reacting antibiotics which are able to bind the employed antibodies in a manner similar to the target, thus resulting in inaccurate quantification. In this study, we developed a highly specific and sensitive chemiluminescence aptasensor for sulfamethazine (SMZ) by employing the supernormal selectivity of a new selected aptamer. Four SMZ-specific aptamers (K d ranging from 79 to 274 nM) were selected in vitro by MB-SELEX with an intentional counter selection to avoid the recognition other sulfonamides, which is impossible to achieve in the corresponding procedure of antibody production. The binding mechanism of aptamers for SMZ was further analyzed and clarified with molecular simulation and docking. Under optimum conditions, the chemiluminescence aptasensor based on the highest affinity aptamer named SA07 provided a limit of detection of 0.92 ng/mL with negligible cross-reactivity with the 27 tested sulfonamides. The developed aptasensor was applied to milk samples, and achieved good accuracy and precision. These results demonstrated that the integration of an aptamer and chemiluminescence in a sensor platform is a promising method for the sensitive, specific, rapid and accurate detection of antibiotic residues of interest in food samples. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Monitoring of platelet-derived growth factor-BB (PDGF-BB) can be used in different types of cancer diagnosis at early stages. • A simple and sensitive aptamer-based fluorescent biosensor was fabricated for ultrasensitive detection of PDGF-BB protein. • The aptasensor was based on split complementary strand of aptamer (CS), streptavidin-coated magnetic beads and picogreen. • The presences of both magnetic beads and split CS in the designed biosensor improved the sensitivity of the aptasensor. • The sensor showed a linear response to concentrations of protein from 0.2 to 100 nM with detection limit as low as 0.13 nM. Abstract In this work, a simple and sensitive aptamer-based fluorescent biosensor was fabricated for ultrasensitive detection of platelet-derived growth factor-BB (PDGF-BB) protein, which is known as a potential biomarker for diagnosis of different cancers. The developed aptasensor was based on PDGF-BB aptamer (Apt), a split complementary strand of aptamer (CS), magnetic beads and picogreen (PG) as a sensitive double-stranded DNA (dsDNA) fluorescent dye. Presence of magnetic beads with the capability of sample condensation and split CS in the fabricated biosensor improved the analytical performance of the aptasensor. In the absence of PDGF-BB protein, dsDNA (CS1-CS2/Apt) is formed on the surfaces of magnetic beads. Therefore, after addition of PG to the sample, a strong fluorescence signal is recorded. On the contrary, in the presence of PDGF-BB protein only CS2 as a very short single-stranded DNA (ssDNA) remains on the surfaces of magnetic beads, leading to a weak fluorescence response following the addition of PG. Under optimized condition, the designed biosensor showed a linear response to concentrations of protein from 0.2 to 100 nM with detection limit as low as 0.13 nM. The biosensor was further employed to detect PDGF-BB protein in serum samples and demonstrated satisfied recovery, ranging from 103.6 to 105.2%. [ABSTRACT FROM AUTHOR]

Recently, CRISPR/Cas associated biosensors have been shown to have great potential in sensing applications due to their high sensitivity and high base resolution. However, the signal reporter system containing two organic fluorescent dye pair is limited by high cost and less stability. In contrast, functional nanomaterials exhibit robust stability, excellent optical properties and low preparation cost, making them suitable reporters. In this study, a MoS 2 nanosheets (NSs) improved CRISPR/Cas12a-based biosensing platform was constructed for the first time to detect food-borne parasites. MoS 2 NSs were used as fluorescence quenchers and single-stranded DNA (ssDNA) discriminated carrier to construct CRISPR/Cas signal reporter system. The combination of recombinase polymerase amplification with MoS 2 NSs modulated CRISPR/Cas12a helped achieve attomolar sensitivity for nucleic acid detection within 35 min. Moreover, the results were obtained using a portable apparatus, enabling visual detection at the point of care. The practical applicability of this biosensing platform was successfully achieved through the detection of anisakis in real samples. This study provides novel insights into exploring the feasibility of two-dimensional nanomaterials based reporter in the CRISPR/Cas12a system, as well as offers a reliable tool for on-site monitoring of parasites. [Display omitted] • MoS 2 NSs as fluorescence quencher and ssDNA discriminated carrier in CRISPR/Cas assay. • The MoS 2 NSs-ssDNA FRET-based sensor was compatible with CRISPR/Cas12a system. • MoS 2 NSs was firstly introduced into Cas12a system for on-site visual detection of parasite. • Attomolar sensitivity for nucleic acid detection within 35 min was achieved. [ABSTRACT FROM AUTHOR]

Exosomes are extracellular vesicles that inherit molecular markers from their parent cells for the transfer and delivery of membrane and cytosolic molecules between cells. Exosome detection and protein profiling provide noninvasive access to disease diagnosis and treatment. The nanozyme designed in this paper contains polydopamine-functionalization of graphene oxide (rGO@PDA) units that can adsorb ssDNA aptamers and CeO 2 metal oxide nanosheet units with peroxidase activity. As auxiliary regulator, adenosine triphosphate (ATP) enhances peroxidase activity of rGO@PDA@CeO 2 under a wide range of pH (4–7.4), which breaks through the limitation that traditional nanozymes can only exhibit peroxidase activity under acidic conditions. The ssDNA aptamers, which are recognition probes of exosome, can be adsorbed on the surface of rGO@PDA by aromatic stacking, competing with ATP molecules. Thus, the ssDNA aptamer acts as the switch for colorimetric sensor to flexibly regulate the peroxidase activity of the rGO@PDA@CeO 2 under physiological conditions, and realizes sensitive detection of exosomes. This assay achieves a detection linear range of 0.46 × 107–108 particle/mL and a limit down to 3.81 × 105 particles/mL. In addition, this aptasensor sensitively profiles six exosomal protein across five cell types and clinical samples, suggesting their potential in clinic diagnosis. [Display omitted] • rGO@PDA@CeO 2 was a complex nanozyme, which CeO 2 provided peroxidase activity and rGO@PDA adsorbed ssDNA. • ATP enhanced the peroxidase activity of rGO@PDA@CeO 2 under a wide range of pH (4–7.4). • DNA aptamers were involved in regulating peroxidase activity as competitors for ATP. • The aptasensor sensitively profiled six exosomal protein across five cell types. [ABSTRACT FROM AUTHOR]

Flap endonuclease 1 (FEN1) is a structure-specific nuclease that may act as a valuable biomarker for cancer diagnosis. Herein, we construct a dual signal amplification-integrated single-molecule fluorescent biosensor for ultrasensitive detection of FEN1 in breast cancer tissues. We design a triplex DNA containing a flap structure for both recognizing and amplifying FEN1 signal. When FEN1 is present, flap is specifically cleaved by FEN1 and released from triplex DNA to form a short ssDNA strand with free 3' OH terminus, and the resultant ssDNA can serve as the primer and binds to circular template in triplex DNA to trigger rolling circle amplification (RCA), generating a long ssDNA product with abundant repeated activator sequences. The hybridization of the resultant activators with crRNAs subsequently activates Cas12a to catalyze trans-cleavage of Cy5- and BHQ-labeled probes to restore Cy5 fluorescence that can be quantified by single-molecule detection. Notably, only a single triplex DNA is required for both initiating and implementing RCA, greatly simplifying the reaction system and eliminating nonspecific amplification. This biosensor exhibits ultrahigh sensitivity with a detection limit of 2.24 × 10-5 U/μL, facilitating accurate detection of endogenous FEN1 activity with single-cell sensitivity and discrimination of different FEN1 levels in clinical breast cancer tissues. • We construct a single-molecule biosensor for FEN1 activity assay. • Dual signal amplification is introduced to achieve high sensitivity. • This biosensor can screen FEN1 inhibitors. • This biosensor can measure endogenous FEN1 activity at single-cell level. • This biosensor can detect FEN1 activity in breast cancer tissues. [ABSTRACT FROM AUTHOR]

Thiol-gold (Au) conjugation is perhaps the most routinely applied surface chemistry approach to immobilize receptors within Au-based molecular sensing platforms. However, no previous studies have investigated in situ immobilization in a nanofluidic systems. In this work, flowthrough receptor immobilization is demonstrated, using a nanoslit induced depletion zone to enable direct real-time visualization for characterizing the effect of sodium chloride (NaCl) concentrations during immobilization of thiolated single-stranded DNA on Au. The results show that the amount of fixed-DNA on Au, as well as the non-specific adsorption of DNA on fused silica surface, strongly depend on NaCl levels. Furthermore, the non-specific interaction at high NaCl levels reduces the DNA available for immobilization on Au, but enriches the DNA molecules in the channel under wash by deionized water. By adding Tween-20 to decrease the non-specific adsorption and increasing the driving pressure to increase the DNA flux under high-salt level, the immobilization time can be further decreased to 10 min or less, which is at least an order of magnitude faster than recently proposed bioconjugation methods for thiolated-DNA–Au conjugation. Moreover, this work demonstrates the capability of the platform for DNA- and aptamer-based sensing assays, thereby opening up new strategies for developing biosensors using nanofluidic platforms. [Display omitted] • Real-time visualization of DNA immobilization on Au surface in nanochannels. • The amounts of immobilized DNA on Au surface depend on the NaCl levels. • The adsorption of DNA on the fused silica surface depends on the NaCl levels. • Ultrarapid DNA immobilization on Au surface in the nanochannel (∼ 10 minutes). • Wash-free, real-time visualization for gene and protein detections [ABSTRACT FROM AUTHOR]

Mild traumatic brain injury (mTBI) may cause increasing risk of long-term sequelae during brain development among children and adolescents. Recent investigations have declared that salivary microRNAs (miRNAs) in mTBI were reliable diagnostic markers. Herein, a versatile biosensor, namely, self-primer exponential amplified Cas12a tandem (SP-ExACT) reaction for quantifying mTBI-related miRNAs was established by integrating CRISPR-Cas12a with copper(I) 1,2,4-triazolate coordination polymer ([Cu(tz)]) nanosheets. Unlike conventional strand displacement amplification, we designed DNA hairpins with integrated primer and template sequences to generate multiple single-stranded DNA (ssDNA) identical to targets. They could trigger the following rolling circle amplification together and then activate Cas12a to indiscriminately cleave the intact fluorophores-labeled ssDNA that absorbed on [Cu(tz)] nanosheets. The corresponding fluorescence signal would recover for the desorption of cleaved short ssDNA from nanosheets. Owing to the remarkable performance of SP-ExACT-[Cu(tz)] reaction, this biosensor could detect let-7a, miR-30e and miR-21 with a LOD of 11.6, 5.4, 7.4 fM, respectively. This study explored a novel fluorescence biosensor based on [Cu(tz)] nanosheets combined with CRISPR-Cas12a to reduce initial background signal, and combined multiple signal amplification strategies to achieve ultra-sensitive detection. Moreover, multiple mTBI-related miRNAs detection could avoid false positive/negative results, and a universal detection platform was constructed through changing corresponding sequence. • A SP-ExACT-[Cu(tz)] system to detect mTBI-related miRNAs in human saliva was explored. • Target-induced hairpin intramolecular directed strand displacement amplification. • Ingenious integration of dual signal amplification strategies and CRISPR system. • [Cu(tz)] nanosheets for DNA adsorption and fluorescence quenching was applied to reducing background signals. [ABSTRACT FROM AUTHOR]

Apolipoprotein E (APOE) genotyping is important for assessing the risk of late-onset Alzheimer's disease. We developed a DNA amplification-free method to perform APOE genetic analysis based on the high sensitivity of fiber optic particle plasmon resonance (FOPPR) biosensor and the specificity of ligase reaction. The method employed the dual-functional gold-iron oxide core-satellite hybrid nanoparticles (HNPs), which was modified with a single-stranded DNA (ssDNA) probe-P1, and a ssDNA probe-P2 modified with biotin to form a nanoplasmonic detection probe. Ligation of a ssDNA target complementary to the P1–P2 pair results in a ligation product with the HNP at one end and the biotin group at the other end, which can be captured by a streptavidin-functionalized sensor fiber, leading to an increase of nanoplasmonic absorption with magnitude useful for APOE genotype determination. Using synthetic ssDNA targets as standards, we achieved limits of detection in the range of 16–38 fM and the sensor responses from the mimic homozygous samples were about two times of that from mimic heterozygous samples at the same ssDNA concentration. For proof-of-principle, three genomic samples extracted from blood were analyzed and the results favorably agree with that by DNA sequencing. • Amplification-free biosensor for genotyping of Apolipoprotein E (APOE) using ligase reaction for specificity. • High sensitivity with detection limits in the range of 16–38 fM. • Allow discrimination between homozygous DNA and heterozygous DNA. • Good correlation with DNA sequencing for genomic samples extracted from blood. • Require very small amount of sample and thus provide an opportunity to develop a POCT device for APOE genotyping. [ABSTRACT FROM AUTHOR]

Nanozyme technique with efficient modulating strategies is a promising tool for biocatalytic and biosensing applications, yet insights into target-responsive controllability of oxidase nanozymes remain largely unknown. In this contribution, we report a state-of-art oxidase-like and ssDNA-tunable nanozyme for biomolecular recognition. In detail, the spinel-type manganese cation substituted cobalt oxide (MnCo 2 O 4 ) submicrospheres can imitate oxidase for efficiently catalytic oxidation of colorimetric substrate, superior to current metal oxide-based oxidase nanozymes. More importantly, the nanozyme activity of spinel MnCo 2 O 4 can be reversibly inhibited by the attachment/detachment of aptamer strands on MnCo 2 O 4 surface via the aptamer-target binding event, enabling a new colorimetric access for detecting biomolecules. Taking highly toxic ochratoxin A (OTA) as model molecule, we can selectively determine OTA in maize samples with a limit of detection (3δ/ S ) of 0.08 ng/mL. The unique nanozyme property of spinel MnCo 2 O 4 , together with the principle of our approach, reveals the potential of ssDNA-tailorable oxidase nanozymes in biocatalytic and biosensing fields. [ABSTRACT FROM AUTHOR]

Despite the major role of cancer antigen 125 (CA125) in cancer progression, its structural diversity makes antibody-based immunoassay of this protein difficult. Here we report the selection of an anti-CA125 ssDNA aptamer and its application to sensitive detection of CA125 when used together with a CA125-specific antibody immobilized on a three-dimensional network of carbon nanotubes (3DN-CNTs). With exceptional stability and easy modifications over antibodies, the selected ssDNA aptamer (rCAA-8) showed high binding affinity (166 nM) for recombinant CA125, which enabled CA125-specific imaging in ovarian cancer cells (OVCAR-3). Furthermore, when compared to other fluorescent assays based on graphene oxide or flat surface and traditional enzyme-linked immunosorbent assay, a chip-based assay using a 3DN-CNT surface and anti-CA125 antibody-aptamer pair resulted in higher sensitivity and broader dynamic range as a function of CA125 concentration, due to high target specificity and high surface loading density. Our on-chip aptamer-based assay will facilitate sensitive and specific monitoring of CA125 in the biological and clinical fields. [ABSTRACT FROM AUTHOR]

A resonance Rayleigh scattering (RRS) method for determination of Hg 2+ concentration using positively charged gold nanorods (GNRs) and thymine-rich single-stranded DNA (ssDNA) was developed. Hg 2+ can induce a conformation change of ssDNA from a random coil to a hairpin structure via thymine–Hg 2+ –thymine coordination. Hairpin shaped DNA possesses a high density of negative charges, therefore it caused in a drastic aggregation of GNRs. This resulted in an enhancement of RRS intensity. In this work, a change in RRS intensity proportional to the concentration of Hg 2+ was monitored by spectrofluorometer at excitation and RRS wavelengths of 550 nm (λ ex = λ RRS ). This method offered a detection limit of 0.23 nM, which is lower than the maximum mercury contaminant level specified by the US Environmental Protection Agency. Good precision of analysis was obtained (3.1% relative standard deviation). Furthermore, the method was simple, involving in-situ mixing and sensing in a one-pot solution. Detection was accomplished in 5 min. This makes the method advantageous in terms of convenience and rapidity of analysis. The developed method was successfully applied for detection of Hg 2+ in tap water samples. Recoveries were found in range of 100.6–119.0%. [ABSTRACT FROM AUTHOR]

A novel ratiometric fluorescent aptasensor was constructed utilizing ZIF-8-encapsulated palladium nanoclusters (ZIF-8 @PdNCs) as reference signals that emit blue fluorescence, and DNA-templated silver nanoclusters (DNA-AgNCs) containing aptamer fragments as response signals that emit yellow fluorescence. In the absence of tobramycin (TOB), the aptamers of DNA-AgNCs bind to ZIF-8 @PdNCs via π-π stacking, resulting in the quenching of the fluorescent signals of DNA-AgNCs. The presence of TOB can weaken the π-π stacking interaction between ZIF-8 @PdNCs and single-stranded DNA (ssDNA) aptamers, which can lead to the desorption of DNA-AgNCs from the surface of ZIF-8 @PdNCs. As a result, the fluorescence signals of DNA-AgNCs are restored. The developed fluorescent sensor exhibits a linear range between 50 nM and 500 nM, with a limit of detection determined to be 15.3 nM. Exposure to UV light triggers a visible change in fluorescent color from blue to yellow as the concentration of TOB increases. To build a miniaturized and portable device for detecting TOB concentration, we employed 3D printing technology to create an RGB (Red Green Blue) instrument recognition framework with ultraviolet lamp beads and RGB recognition components. The RGB standard curve and programming in Python can be used to directly read the TOB concentration. • Constructing a ratiometric fluorescent sensor improves the accuracy of detection. • The encapsulation of ZIF-8 improves the anti-interference ability of Met-PdNCs. • This strategy provides a method for the visual detection of tobramycin. • A rapid detection device was developed based on fluorescence RGB colorimetry. [ABSTRACT FROM AUTHOR]

As a next-generation biosensing technology, the application of Argonaute for analytical science research has attracted considerable attention. A Thermus thermophilus Argonaute (Tt Ago)-based sensor is presented for detecting T4 polynucleotide kinase (PNK) activity considering that Tt Ago's multiple-turnover catalytic activity can be activated by binding to a 5′-phosphate termini of single-stranded DNA (ssDNA) for the cleavage of complementary strand. PNK catalyzes the phosphorylation of 5′-hydroxyl termini of ssDNA, and the phosphorylation product termed guide can activate the Tt Ago-mediated complementary ssDNA reporter cleavage to release fluorescence signal. For the PNK activity monitoring, the developed method shows good analytic performance with a linear range of 0.0010–2 U mL−1 and a detection limit of 0.0010 U mL−1 without the use of a preamplification reaction. This method is successfully applied for the analysis of HeLa cells at a single-cell level. The developed method shows several advantages, including high sensitivity, low cost, facile operation, and has remarkable potential application for early disease diagnosis, drug screening, and clinical treatment. • Tt Ago-based biosensor for PNK activity detection. • Multiple-turnover cleavage activity of Tt Ago provides post-amplification capacity. • The method realizes detection of HeLa cells at single-cell level. • The method is simple, rapid, low-cost, and sensitive. • Potential application in on-site monitoring. [ABSTRACT FROM AUTHOR]

We report the use of graphitic carbon nitride, g-­C 3 N 4 (g­-CN) based electrochemical genosensor for the ultrahigh sensitive detection of DNA, taking breast cancer (BRCA1) gene as a model gene. The sensor uses the traditional sandwich detection strategy, in which the capture probe DNA (DNA­-c) was immobilized on g-­CN modified glassy carbon electrode (GCE) surface. Use of a gold nanoparticle cluster (AuNPC) as electrochemical label in reporter probe DNA and g-­CN as immobilization platform for capture probe DNA in the sensor design leads to highly sensitive electrochemical detection of BRCA1 gene. The sensor could detect down to 10 attomolar target DNA. Endonuclease reaction provided high selectivity to the mismatched DNA and the sensor was found to be stable and reproducible. [ABSTRACT FROM AUTHOR]

This paper reports a new assay platform that performed in polar organic solutions for ultrasensitive and highly selective detection of isocarbophos pesticide. The sensing system just contains hemin molecules and random ssDNAs. Free hemin molecules have a strong absorption peak at Soret band in polar organic solvents, and they can assemble into few small particles with random ssDNAs via their special π–π stacking interaction. However, the subsequent addition of isocarbophos into above sensing system can aggregate hemin molecules to form many larger particles, which result in a great decrease in absorbance value of sensing solution. UV/Vis absorption spectra and scanning probe microscope (SPM) were used to characterize hemin aggregation. Additionally, the effects of some parameters like type of solvents, hemin amount and random ssDNAs on the sensing platform were also investigated. The present assay platform has a detection limit as low as 0.2 μg/L, a dynamic range of 0.5–500 μg/L, and high selectivity over other competitive pesticides. Moreover, the assay platform also displays excellent stability and accuracy in the detection of spiked real samples, suggesting that such sensing platform will play an important role in biological and environmental detection. [ABSTRACT FROM AUTHOR]

CRISPR-Cas12a system opens up a new avenue for biosensing due to its flexible programmability and excellent target recognition accuracy. In this work, a novel label-free fluorescent biosensor for highly sensitive detection of bleomycin (BLM) was developed based on BLM-mediated inactivation of CRISPR-Cas12a and thioflavin T (ThT)-induced G-quadruplex formation. We designed a single-stranded DNA (ssDNA) probe containing the scission site of BLM as a DNA activator for Cas12a-crRNA. In the absence of BLM, the DNA activator binds to and activates the Cas12a, the activated CRISPR-Cas12a system trans-cleaves the ThT-induced G-quadruplex formation. Upon the addition of BLM, the DNA activator undergoes an irreversible cleavage event at the scission site via the oxidative effect of BLM with Fe(Ⅱ) as a cofactor. As a result, the trans-cleavage activity of Cas12a cannot be activated so the G-quadruplex-ThT complex remains intact, resulting in a significant increase of fluorescence signal. Attributing to the specific recognition of BLM and the excellent cleavage activity of Cas12a on DNA G-quadruplexes, the fluorescent biosensor exhibits high sensitivity and selectivity for detection of BLM in human serum samples, illustrating a promising tool for BLM assay in biomedical research. [Display omitted] • A novel label-free fluorescent strategy is developed based on CRISPR-Cas12a and G-quadruplex-ThT complex. • The fluorescent sensing strategy exhibits high sensitivity for BLM detection with a detection limit down to 45 pM. • The proposed strategy can be applied for the determination of BLM in human serum samples. • This work broadens the applications of CRISPR/Cas-based sensing systems in biomedical research. [ABSTRACT FROM AUTHOR]

DNA walker has been widely used in various types of biosensors for signal-amplification detection of low abundance analytes. However, the usual design of unipedal walker provides limited signal amplification effect with poor sensitivity. Here, we designed an electrochemical biosensor based on bipedal DNA walker allowing for highly sensitive detection of apurinic/apyrimidinic endonuclease 1 (APE 1), a significant base excision repair enzyme, which is over-expressed in multiple cancer cells emerging as a promising biomarker for cancer diagnostics. The bipedal DNA walker can be released after APE1 cleaving apurinic/apyrimidinic (AP) sites, and then perform the catalytic hairpin assembly (CHA) process, triggering the downstream hybridization chain reaction (HCR) to achieve a dual-amplified current signal. This bipedal walker presents dramatically increased catalytic efficiency in the upstream CHA process, and finally contributes almost 5 times synergistic enhanced current signal compared to unipedal walker. We successfully realized the highly sensitive detection of APE 1 with a wide linear range from 0.001 U mL−1 to 1 U mL−1 reaching a detection limit of 0.001 U mL−1. This biosensor showed good specificity in discrimination of APE1 from other interfering enzymes. It was successfully applied to investigate APE 1 expression level in cell lysate, demonstrating its potential diagnostics applications. • A bipedal-walker electrochemical biosensor for highly sensitive detection of APE 1. • A cascade synergistic amplification system combining bipedal walker-catalyzed CHA and HCR. • Bipedal-walker nanodevice presents 5 times higher current signal than unipedal walker. • This biosensor has good discrimination ability of Hela cells from normal cells. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) is one of the leading cause of infection-related deaths worldwide. Conventional diagnostic methods for TB require infectious samples and are time-consuming. In this study, we aimed to develop an aptamer-based qPCR (Apta-qPCR) assay to diagnose TB safely and rapidly. The assay uses a single-stranded DNA aptamer with affinity for a TB biomarker, ESAT6, and molecular beacon capable of hybridizing to the aptamer. Apta-qPCR enables aptamer-mediated detection of ESAT6 via qPCR, wherein the aptamer serves as both a target recognition agent and template for quantification. This diagnostic method achieved a desirable detection limit of 2.03 nM in buffer and 2.56 nM in serum-spiked conditions with high reproducibility and reliability. In addition, the diagnostic performance was verified in a clinical application using human serum samples from normal controls and patients with active TB. The assay showed 50.0% sensitivity, 91.7% specificity, and 78.1% accuracy, similar to sputum smear microscopy, which is currently the most used method for TB diagnosis. Importantly, this assay has an advantage over smear microscopy in that it reduces the risk of infection by avoiding the use of sputum-containing infectious pathogens. [Display omitted] • An aptmer-based qPCR (Apta-qPCR) assay was developed to diagnose tuberculosis. • Apta-qPCR can safely detect a protein biomarker ESAT6 from non-infectious serum samples. • The assay shows high clinical sensitivity, specificity and accuracy. • Apta-qPCR can be used to detect various protein biomarkers. [ABSTRACT FROM AUTHOR]

Human single-stranded-selective monofunctional uracil glycosylase (hSMUG1) and alkyladenine glycosylase (hAAG) are important base-excision repair (BER) glycosylases responsible for deamination and alkylation repair. Their dysfunctional activities have strong association with various multifactorial diseases and cancers. Herein, by integrating three-dimensional (3D) DNA walker with single-molecule technique, we construct bidirectional strand displacement-driven 3D DNA walkers for single-molecule monitoring of multiple DNA glycosylases. In the presence of hSMUG1 and hAAG, the damaged U and I in bifunctional dumbbell probe are removed by APE1, activating bidirectional self-priming strand displacement amplification (SP-SDA) to produce two trigger DNAs. Trigger DNAs hybridize with Cy5/Alexa Fluor 488 signal probes and subsequently serve as the walker DNAs to induce cyclic APE1-powered cleavage of two signal probes, liberating abundant Cy5 and Alexa Fluor 488 from AuNPs. Due to high precision of intracellular BER mechanisms, high efficiency of bidirectional SP-SDA-directed 3D DNA walkers and ultrahigh signal-to-noise ratio of single-molecule imaging, this nanosensor exhibits a detection limit of 8.14 × 10−10 U/μL for hSMUG1 and 4.50 × 10−9 U/μL for hAAG. Importantly, it can measure kinetic parameters, identify potential inhibitors, discriminate cancer from normal cells, and quantify glycosylases activities in various cancer cells at single-cell level, with promising potentials in biomedical and clinical applications. • We construct bidirectional strand displacement-driven 3D DNA walkers. • This nanosensor enables single-molecule monitoring of multiple human glycosylases. • This nanosensor can measure kinetic parameters and screen potential inhibitors. • This nanosensor can discriminate cancer cells from normal cells. [ABSTRACT FROM AUTHOR]

Digital analysis is an effective single-molecule detection method and has attracted extensive attention in the field of bioassays. However, most digital assays require microchambers for signal compartmentalization. Herein, we developed a microchamber-free and enzyme-free digital assay by labeling paramagnetic beads directly with ultrabright fluorescent microspheres. In this assay, a DNA sandwich analysis was firstly performed on the bead to label a fluorescent microsphere. Then, the beads were loaded on the glass slide to form a monolayer film for signal readout. The whole analysis process does not require the participation of enzymes and the preparation of microchambers, which greatly simplifies the experimental steps and saves the costs. Furthermore, by introducing non-enzymatic hybridization chain reaction (HCR) and biotinylated DNA-conjugated gold nanoparticles (Au NPs-Bio), the capture efficiency and analytical sensitivity were improved. As a proof of concept, single-stranded DNA (ssDNA) of SARS-CoV-2 fragment was chosen as a model, and a detection limit of 1.5 fM was achieved. Spiked and recovery experiments on human serum and saliva samples validated the good performance of the proposed digital assay in real biological samples. The proposed assay provides a facile way of signal generation and readout for digital analysis. • A microchamber-free digital assay was developed. • A digital assay that does not require the involvement of enzymes was achieved. • The discrimination between positive and negative beads in digital assay was improved. • A facile way of signal generation and readout for digital analysis was proposed. [ABSTRACT FROM AUTHOR]

Exploring loop-mediated isothermal amplification (LAMP)-based electrochemical assay for short-stranded DNA biomarkers is intriguing. Herein, a HIV-specific DNA segment (tDNA) was introduced to implement a variant LAMP reaction, where the generated by-products H+, v LAMP(H+), acted as signal transducers for developing a sensitive electrochemical biosensor. As proof-of-concept, the tDNA-recognizable complement was programmed in a template hairpin (TH) that was ended with a 3′-terminus phosphate group to avoid non-specific elongation. Upon TH simultaneously hybridizing with tDNA and a precursor hairpin (PH), the PH underwent a loop-mediated extension with Bst polymerase and dNTPs, forming a new stem-loop hairpin in another terminus. Then the elongated product containing a double stem-loop dumbbell-like DNA structure (d DDS) was displaced and released. Using d DDS as an initiator, the v LAMP reaction occurred via consecutively cycled strand displacement for self-primed DNA incorporation. The resulting tDNA-dependent v LAMP(H+) was introduced in the modified electrode surface to guide the pH-sensitive i-motif (iM) folding. Thus, a signaling hairpin labeled with electroactive ferrocene (Fc) was dehybridized and closed, thereby bringing Fc close to the electrode interface for increased current signal readout that was highly sensitive to tDNA down to 0.034 fM. Our electrochemical biosensor would be applicable for detecting various short-stranded DNA or miRNA. [Display omitted] ● A variant LAMP-integrated electrochemical biosensor was developed for detecting short-stranded DNAs. ● A HIV-specific DNA segment of 21-nt induced loop-mediated extension of a precursor hairpin. ● A double dumbbell-like structure (d DDS) was formed and acted as template initiator to guide v LAMP. ● The generated v LAMP(H+) was implemented to switch i-motif folds for dependent signal transduction. ● The electrochemical readout of redox tag was highly target-sensitive and specific, simple and rapid. [ABSTRACT FROM AUTHOR]

CRISPR/Cas12a has been widely used in molecular diagnostic due to its excellent trans-cleavage activity. However, its trans-cleavage efficiency on G-quadruplex (G4) is highly correlated with the stability of G4, which causes long cutting time and limits the wide application of G4-probing CRISPR/Cas12a assays. To address these challenges, we utilize the G-quadruplex molecular beacon (G4MB) with the stem-loop structure incorporating split G-quadruplex as the reporter to construct a CRISPR/Cas12a-based biosensor. In comparison with the previously reported G4 reporter, the cleavage speed of Cas12 on G4MB reporter is faster because of the high susceptibility of Cas12a to ssDNA (ssDNA). More importantly, the formation or disassembly of G4MB can be regulated directly by its stem-loop structure, thus the trans-cleavage rate of Cas12a on G4MB was not affected by the stability of G-quadruplex, offering great extent of freedom to choose suitable G4 sequence in CRISPR/Cas12a system. In addition, this biosensor is label-free by the utilization of G4 binding dye (ThT) and has high selectivity due to the high fidelity of Cas12a. This work can broaden the application range of G4 in CRISPR/Cas12a-based biosensors and hold enormous promise in future research on medical diagnoses and biology science. • The hairpin loop design of G4MB improved the cleavage speed of Cas12a on G4 and broadened the application range of G4. • The introduction of G-quadruplex binding dye avoided the complex modification and reduced the cost. • This biosensor has high selectivity and could be applied for the detection of ctDNA in human blood samples. [ABSTRACT FROM AUTHOR]

A growing number of studies suggest that miRNAs play a vital role in the diagnosis and treatment of several diseases, especially for cancers. However, miRNA detection still remains great challenges because of its small size, low abundance and easy degradation. In this contribution, we demonstrate an intelligent DNA machine for the detection of miRNA-21 via designing a multiple palindrome-mediated strand displacement amplification (called MPSDA) strategy combined with fluorescence measurement. Essentially, the nano-machine is a scarecrow-shaped DNA nanoprobe (S-nanoprobe) assembled from only three single-stranded DNAs (ssDNAs) each with a palindromic fragment at the 3′-end. In the presence of target, S-nanoprobe can be dissembled into two parts, leading to the release of palindromic ends that can execute MPSDA under DNA polymerase. Benefiting from the high efficiency of MPSDA, S-nanoprobe can be used to detect down to 1 fM of target and exhibit a high specificity. Target miRNA can be 100% discriminated from homologous miRNAs and can also be sensitively detected in complex biological samples. More excitingly, a high recovery rate, desirable blind test results and precise target assay in real samples demonstrate the huge application potential of S-nanoprobe in the detection of tumor-associated biomarkers in early diagnosis. • Target-Induced Multiple Palindrome-Mediated SDA (MPSDA) for miR-21 detection. • The miR-21 could be detected down to 1 fM with a wide dynamic range. • This strategy displayed an excellent selectivity. • The nanoprobe possessed enhanced resistance to nuclease degradation. • Suitable for the detection of target in a complicate biological milieu. [ABSTRACT FROM AUTHOR]

A growing number of studies suggest that miRNAs play a vital role in the diagnosis and treatment of several diseases, especially for cancers. However, miRNA detection still remains great challenges because of its small size, low abundance and easy degradation. In this contribution, we demonstrate an intelligent DNA machine for the detection of miRNA-21 via designing a multiple palindrome-mediated strand displacement amplification (called MPSDA) strategy combined with fluorescence measurement. Essentially, the nano-machine is a scarecrow-shaped DNA nanoprobe (S-nanoprobe) assembled from only three single-stranded DNAs (ssDNAs) each with a palindromic fragment at the 3′-end. In the presence of target, S-nanoprobe can be dissembled into two parts, leading to the release of palindromic ends that can execute MPSDA under DNA polymerase. Benefiting from the high efficiency of MPSDA, S-nanoprobe can be used to detect down to 1 fM of target and exhibit a high specificity. Target miRNA can be 100% discriminated from homologous miRNAs and can also be sensitively detected in complex biological samples. More excitingly, a high recovery rate, desirable blind test results and precise target assay in real samples demonstrate the huge application potential of S-nanoprobe in the detection of tumor-associated biomarkers in early diagnosis. • Target-Induced Multiple Palindrome-Mediated SDA (MPSDA) for miR-21 detection. • The miR-21 could be detected down to 1 fM with a wide dynamic range. • This strategy displayed an excellent selectivity. • The nanoprobe possessed enhanced resistance to nuclease degradation. • Suitable for the detection of target in a complicate biological milieu. [ABSTRACT FROM AUTHOR]