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Recent findings on CRISPR-Cas enzymes with collateral DNAse/RNAse activity have led to new and innovative methods for pathogen detection. However, many CRISPR-Cas assays necessitate DNA pre-amplification to boost sensitivity, restricting their utility for point-of-care applications. Achieving higher sensitivity without DNA pre-amplification presents a significant challenge. In this study, we introduce a Terminal deoxynucleotidyl Transferase (TdT)-based amplification loop, creating a positive feedback mechanism within the CRISPR-Cas12a pathogen detection system. Upon recognizing pathogenic target DNA, Cas12a triggers trans-cleavage of a FRET reporter and a specific enhancer molecule oligonucleotide, indicated by the acronym POISER (Partial Or Incomplete Sites for crRNA recognition). POISER comprises half of a CRISPR-RNA recognition site, which is subsequently elongated by TdT enzymatic activity. This process, involving pathogen recognition-induced Cas12a cleavage and TdT elongation, results in a novel single-stranded DNA target. This target can subsequently be recognized by a POISER-specific crRNA, activating more Cas12a enzymes. Our study demonstrates that these POISER-cycles enhance the signal strength in fluorescent-based CRISPR-Cas12a assays. Although further refinement is desirable, POISER holds promise as a valuable tool for the detection of pathogens in point-of-care testing, surveillance, and environmental monitoring. • This study introduces a Terminal deoxynucleotidyl Transferasebased amplification loop within a CRISPR-Cas12a DNA detection system. By creating a positive feedback mechanism, this innovative approach enhances the signal strength in fluorescent-based CRISPR-Cas12a assays without the need for DNA pre-amplification. • The POISER system employs an enhancer oligonucleotide containing half of a crRNA recognition site. Combined with TdT activity, this system generates a novel single-stranded DNA target that enhances the signal intensity in CRISPR-Cas12a FRET assays. [added extra bullet point] By leveraging the POISER system, this study demonstrates increased signals in DNA detection assays, facilitating the development of more efficient diagnostic tools. [ABSTRACT FROM AUTHOR]

To address the limitations of the CRISPR/Cas12f1 system in clinical diagnostics, which require the complex preparation of single-stranded DNA (ssDNA) or in vitro transcripts (RNA), we developed a fluorescent biosensor named PDTCTR (PAM-dependent dsDNA Target-activated Cas12f1 Trans Reporter). This innovative biosensor integrates Recombinase Polymerase Amplification (RPA) with the Cas12f_ge4.1 system, facilitating the direct detection of double-stranded DNA (dsDNA). PDTCTR represents a significant leap forward, exhibiting a detection sensitivity that is a hundredfold greater than the original Cas12f1 system. It demonstrates the capability to detect Mycoplasma pneumoniae (M. pneumoniae) and Hepatitis B virus (HBV) with excellent sensitivity of 10 copies per microliter (16.8 aM) and distinguishes single nucleotide variations (SNVs) with high precision, including the EGFR (L858R) mutations prevalent in non-small cell lung cancer (NSCLC). Clinical evaluations of PDTCTR have demonstrated its high sensitivity and specificity, with rates ranging from 93%–100% and 100%, respectively, highlighting its potential to revolutionize diagnostic approaches for infectious diseases and cancer-related SNVs.This research underscores the substantial advancements in CRISPR technology for clinical diagnostics and its promising future in early disease detection and personalized medicine. [ABSTRACT FROM AUTHOR]

The isolation and identification of pathogenic bacteria from a variety of samples are critical for controlling bacterial infection-related health problems. The conventional methods, such as plate counting and polymerase chain reaction-based approaches, tend to be time-consuming and reliant on specific instruments, severely limiting the effective identification of these pathogens. In this study, we employed the specificity of the cell wall-binding (CBD) domain of the Staphylococcus aureus bacteriophage 80 alpha (80α) endolysin towards the host bacteria for isolation. Amidase 3-CBD conjugated magnetic beads successfully isolated as few as 1 × 102 CFU/mL of S. aureus cells from milk, blood, and saliva. The cell wall hydrolyzing activity of 80α endolysin promoted the genomic DNA extraction efficiency by 12.7 folds on average, compared to the commercial bacterial genomic DNA extraction kit. Then, recombinase polymerase amplification (RPA) was exploited to amplify the nuc gene of S. aureus from the extracted DNA at 37 °C for 30 min. The RPA product activated Cas12a endonuclease activity to cleave fluorescently labeled ssDNA probes. We then converted the generated signal into a fluorescent readout, detectable by either the naked eye or a portable, self-assembled instrument with ultrasensitivity. The entire procedure, from isolation to identification, can be completed within 2 h. The simplicity and sensitivity of the method developed in this study make it of great application value in S. aureus detection, especially in areas with limited resource supply. [ABSTRACT FROM AUTHOR]

Fumonisin (FB) is a pervasive hazardous substance in the environment, presenting significant threats to human health and ecological systems. Thus, the selective and sensitive detection of fumonisin B1 (FB1) is crucial due to its high toxicity and wide distribution in corn, oats, and related products. In this work, we developed a novel and versatile fluorescent aptasensor by combining enzyme-assisted dual recycling amplification with 2D δ-FeOOH-NH 2 nanosheets for the determination of FB1. The established CRISPR/Cas12a system was activated by using activator DNA (aDNA), which was released via a T7 exonuclease-assisted recycling reaction. Additionally, the activated Cas12a protein was utilized for non-specifically cleavage of the FAM-labeled single-stranded DNA (ssDNA-FAM) anchored on δ-FeOOH-NH 2 nanosheets. The pre-quenched fluorescence signal was restored due to the desorption of the cleaved ssDNA-FAM. Due to the utilization of this T7 exonuclease-Cas12a-δ-FeOOH-NH 2 aptasensor for signal amplification, the detection range of FB1 was expanded from 1 pg/mL to 100 ng/mL, with a limit of detection (LOD) as low as 0.45 pg/mL. This study not only provides novel insights into the development of fluorescence biosensors based on 2D nanomaterials combined with CRISPR/Cas12a, but also exhibits remarkable applicability in detecting other significant targets. [ABSTRACT FROM AUTHOR]

The detection of circulating tumor DNA (ctDNA), as a practical liquid biopsy technique, was of great significance for the study of cancer diagnosis and prognosis. However, reported methods for detection ctDNA still have some limitations, such as tedious process and high cost. In this study, CsPbBr 3 nanosheet (CsPbBr 3 NS) with high water stability was prepared by etching, and its fluorescence intensity could be stably stored for 1 year. The Ti 3 C 2 T x possessed high quenching efficiency for CsPbBr 3 NS and the HOMO-LUMO orbital study revealed that the PET mechanism was responsible for fluorescence quenching. And the Ti 3 C 2 T x showed stronger affinity towards single-stranded DNA (ssDNA), as compared with double-stranded DNA (dsDNA). The probe ssDNA could be adsorbed on the surface of Ti 3 C 2 T x through π-π stacking. After the targets were recognized by probe ssDNA to form dsDNA, its affinity with Ti 3 C 2 T x decreased and the active site of Ti 3 C 2 T x recovered, causing a high quenching efficiency on CsPbBr 3 NS. Based on this, a label-free fluorescent biosensor was designed for the sensitive detection of ctDNA (EGFR 19 Dels for non-small cell lung cancer, NSCLC). Under the optimal experimental conditions, this biosensor exhibited a detection limit of 180 fM and a linear range of 50 pM–350 pM with amplification of magnetic beads through strand displacement reaction. In addition, this sensor was applied to the detection of ctDNA in serum samples and cells lysates. This method for ctDNA detection was expected to have great potential for biomarker detection in the field of liquid biopsy. [ABSTRACT FROM AUTHOR]

With the globalization and complexity of the food supply chain, the market is becoming increasingly competitive and food fraudulent activities are intensifying. The current state of food detection faced two primary challenges. Firstly, existing testing methods were predominantly laboratory-based, requiring complex procedures and precision instruments. Secondly, there was a lack of accurate and efficient quantitative detection methods. Taking cow's milk as an example, this study introduced a novel method for nucleic acid quantification in dairy products, based on lateral flow strips (LFS). The core idea of this method is to design single-stranded DNA (ssDNA) probes to hybridize with mitochondrial genes, which are abundant, stable, and species-specific in dairy products, as detection targets. Drawing inspiration from the principles of nucleic acid amplification, this research innovatively established a new DNA hybridization method, named LAMP-Like Hybridization (HybLAMP-Like). Leveraging the denaturation and DNA polymerization functions of the bst enzyme, efficient binding of the probe and template strand was achieved. This method eliminated the need for nucleic acid amplification, simplifying the procedure and mitigating aerosol contamination, thereby ensuring the accuracy of the detection results. The method exhibited exceptional sensitivity, capable of detecting extremely low to 12.5 ng in visual inspection and 3.125 ng when using a reader. In terms of practicality, it could achieve visual detection of cow's milk content as low as 1% in adulterated dairy products. When combined with a portable LFS reader, it also enabled precise quantitative analysis of milk adulteration. [Display omitted] [ABSTRACT FROM AUTHOR]

This study presents a straightforward efficient technique for extracting circulating tumor cells (CTCs) and a rapid one-step electrochemical method (45 min) for detecting lung cancer A549 cells based on the specific recognition of mucin 1 using aptamers and the modulation of Cu2+ electrochemical signals by biomolecules. The CTCs separation and enrichment process can be completed within 45 min using lymphocyte separation solution (LSS), erythrocyte lysis solution (ELS), and three centrifugations. Besides, the influence of various biomolecules on Cu2+ electrochemical signals is comprehensively discussed, with DNA nanospheres selected as the medium. Three single-stranded DNA sequences were hybridized to form Y-shaped DNA (Y-DNA), creating DNA nanospheres. Upon specific capture of mucin 1 by the aptamer, most DNA nanospheres could form complexes with Cu2+ (DNA nanosphere-Cu2+), significantly reducing the concentration of free Cu2+. Our approach yielded the limit of detection (LOD) of 2 ag/mL for mucin 1 and 1 cell/mL for A549 cells. 39 clinical blood samples were used for further validation, yielding results closely correlated with pathological, computed tomography (CT) scan findings and folate receptor-polymerase chain reaction (FR-PCR) kits. The receiver operating characteristic (ROC) curve displayed an area under the curve (AUC) value of 0.960, demonstrating 100% specificity and 93.1% sensitivity for the assay. Taken together, our findings indicate that this straightforward and efficient pretreatment and rapid, highly sensitive electrochemical assay holds great promise for liquid biopsy-based tumor detection using CTCs. [ABSTRACT FROM AUTHOR]

A homogeneous rapid (45 min) one-pot electrochemical (EC) aptasensor was established to quantitatively detect circulating tumor cells (CTCs) in lung cancer patients using mucin 1 as a marker. The core of this study is that the three single-stranded DNA (Y1, Y2, and Y3) could be hybridized to form Y-shaped DNA (Y-DNA) and further self-assemble to form DNA nanosphere. The aptamer of mucin 1 could be complementary and paired with Y1, thus disrupting the conformation of the DNA nanosphere. When mucin 1 was present, the aptamer combined specifically with mucin 1, thus preserving the DNA nanosphere structure. Methylene blue (MB) acted as a signal reporter, which could be embedded between two base pairs in the DNA nanosphere to form a DNA nanosphere-MB complex, reducing free MB and resulting in a lower electrochemical signal. The results demonstrated that the linear ranges for mucin 1 and A549 cells were 1 ag/mL-1 fg/mL and 1–100 cells/mL, respectively, with minimum detectable concentrations were 1 ag/mL and 1 cell/mL, respectively. The quantitative analysis of CTCs in 44 clinical blood samples was performed, and the results were consistent with the computerized tomography (CT) images, pathological findings and folate receptor-polymerase chain reaction (FR-PCR) kits. The receiver operating characteristic (ROC) curve exhibited an area under the curve (AUC) value of 0.970. The assay revealed 100% specificity and 94.1% sensitivity. It is believed that this electrochemical aptasensor could provide a new approach to detect CTCs. [ABSTRACT FROM AUTHOR]

Exosomal microRNA (miRNA) are important biomarkers for liquid biopsy, and display clinical molecular signatures for cancer diagnosis. Although advanced detection methods have been established to detect exosomal miRNAs, they are faced with certain challenges. Therefore, we aimed to establish a dual amplification-based electrochemical method for detecting exosomal miRNA. This method combined a two-hairpins-based ternary hybridization structure (thTHS)-initiated single-stranded DNA (ssDNA) amplification reaction (ssDAR) and sodium perchlorate (NaClO 4)-assisted electrocatalytic cycle. Two DNA hairpins were designed to hybridize with target miRNA, forming thTHS. Next, ssDAR was triggered by thTHS to produce long ssDNA on magnetic beads. The long ssDNA, complementary to the signal probes, was subsequently released onto a methylene blue (MB)-labeled double-stranded DNA-modified electrode for strand displacement reaction. This led to a quantitative change in MB and a change in electrocatalytic reduction current from the electrocatalytic cycle of MB-ferricyanide. An amplified electrocatalytic reduction current was produced by adding NaClO 4 to the electrocatalytic system, which substantially improved the signal response range and detection sensitivity. Ultimately, exosomal miRNA detection was achieved by recording changes in the electrocatalytic reduction current before and after miRNA addition. This electrochemical method exhibited a sensitive concentration response with a detection limit of 45 aM and selective miRNA recognition, and successfully used to detect exosomal miRNA derived from cells and serum. Additionally, this method exhibited better discrimination ability between patients with breast cancer (BC) and those people without BC (patients with benign breast disease and healthy people), providing a promising strategy for detecting and monitoring cancer biomarkers. [ABSTRACT FROM AUTHOR]

Toehold-mediated strand displacement (TMSD) is widely employed in constructing a wide range of chemical reaction networks. In TMSD, single-stranded DNA or RNA can fold back upon itself to form a local short double-strand structure often hindering bimolecular hybridization. Here, based on series and parallel circuits, we introduce two mechanisms: series toehold-mediated strand displacement (STMSD) and parallel toehold-mediated strand displacement (PTMSD). These mechanisms can be highly effective when the target area is blocked by a secondary structure. In addition, these systems allow regulating the reaction rates spanning three to five orders of magnitude by adjusting the length of the two toeholds with the added advantage of multifunctional regulation and selectivity. To demonstrate the impressive function of this approach, a logic operation system based on STMSD was constructed to simulate the signal processing of a half-adder. We believe that the introduction of series and parallel toeholds will provide design flexibility contributing to the development of molecular computers, molecular robotics, and DNA-based biosensors. [Display omitted] [ABSTRACT FROM AUTHOR]

A suspended carbon nanotube (SCNT)-based field effective transistor (SCNT-FET), which was fabricated by utilizing the surface tension of liquid silver to suspend a CNT between two Pd electrodes, was proposed for the detection of DNA hybridization. Benefits from the separation between the CNT and the substrates could be observed; namely, the conductivity of a SCNT-FET was much higher (two orders of magnitude) than that of a FET based on an unsuspended CNT and about 50% sensing surface of CNT was freed from substrate. The Slater-Koster tight-binding method was adopted for geometry optimization and transport property calculation of the SCNT bound with DNA. The result showed that the conductance (G = 1/R) of the SCNT decreased in order with the binding of single-stranded DNA (SSDNA, probe DNA) and double-stranded DNA (DSDNA) and that the ability of DSDNA to weaken the conductivity of the SCNT was several times higher than that of SSDNA. SEM and Raman spectroscopy were used to demonstrate that DNA could be bound successfully onto the SCNT using a 1-pyrenebutanoic acid succinimidyl ester (PBASE) as a linkage. Ultra-high sensitivity detection of DNA [with a limit of detection (LOD) as low as 10 aM] was obtained using such an SCNT-FET, which showed a lower value than that of a previously reported FET DNA biosensor whose sensing materials were in direct contact with the substrate. [ABSTRACT FROM AUTHOR]

DNAzyme-based fluorescent probes have provided valuable protocols for detecting uranium, one of the most common radioactive contaminants in the environment, with ultra-high selectivity and sensitivity. Designing novel DNAzyme beacons to update the mode of fluorescence reporting and/or quenching will continuously enhance "turn-on" sensing performance as well as promote actual application of the biological probes. In this work, we developed a novel quencher-free DNAzyme beacon by embedding fluorescent 2-aminopurine for rapid detection of uranyl ion. 2-aminopurine is able to substitute adenine and keep strong fluorescence in single-stranded DNA whereas being quenched in the hybridized double-stranded DNA by the base-stacking interaction. The combination of such trait of 2-aminopurine and cleavage reaction of DNAzyme in the presence of target co-factors possesses two main advantages for ion sensing: simplicity for avoidance of extra quencher groups and high performance because of superiority of DNAzyme essence. The experimental conditions including embedding site, pH and salt concentration of buffer solutions, and the amount ratio of enzyme strand to substrate strand used to form DNAzymes were systematically optimized to inspire the highest performance of the biological beacon. Thus, a detection limit of 9.6 nM, a wide linear range from 5 nM to 400 nM (R2 = 0.997), and selectivity of more than 400 000-fold over other metal ions were achieved by the novel DNAzyme probes. The highly sensitive, selective and quencher-free DNAzyme probes accommodated a simple and cost-efficient alternative to current fluorescent counterparts, holding a great potential for further application in practical ion assay. • Fluorescence of 2-aminopurine (2-AP) can emit in ss -DNA but vanish in ds -DNA. • Embedding 2-AP into DNAzyme can sense metal ions without quenchers. • High-performance detection was achieved due to the superiority of DNAzyme. • Ambient conditions related to DNAzyme performance were systematically optimized. [ABSTRACT FROM AUTHOR]

An electrochemical genosensor for the detection and quantification of Karlodinium armiger is presented. The genosensor exploits tailed primers and ferrocene labelled dATP analogue to produce PCR products that can be directly hybridised on a gold electrode array and quantitatively measured using square wave voltammetry. Tailed primers consist of a sequence specific for the target, followed by a carbon spacer and a sequence specifically designed not to bind to genomic DNA, resulting in a duplex flanked by single stranded binding primers. The incorporation of the 7-(ferrocenylethynyl)-7-deaza-2′-deoxyadenosine triphosphate was optimised in terms of a compromise between maximum PCR efficiency and the limit of detection and sensitivity attainable using electrochemical detection via hybridisation of the tailed, ferrocene labelled PCR product. A limit of detection of 277aM with a linear range from 315aM to 10 fM starting DNA concentration and a sensitivity of 122 nA decade−1 was achieved. The system was successfully applied to the detection of genomic DNA in real seawater samples. • Direct electrochemical detection of tailed, ferrocene labelled PCR amplicons. • PCR incorporation of ferrocene labelled nucleotides. • Micrcofabricated gold electrode array. [ABSTRACT FROM AUTHOR]

A simple "signal-on" photoelectrochemical (PEC) aptasensor is constructed for Aflatoxin B1 (AFB1) detection based on electrochemically reduced graphene oxide/poly(5-formylindole)/Au (erGO/P5FIn/Au) nanocomposites. The nanocomposites are synthesized by simple electrochemical deposition method and show good photoelectrochemical performance. Poly(5-formylindole) (P5FIn) can generate electron-hole pairs under light irradiation, leading to the formation of robust cathode photocurrent. Au can be acted as signal amplifier due to the high conductivity. The erGO is used to immobilize AFB1 aptamer chain by π-π stacking interaction between the carbon six-membered ring in graphene and the C-N heterocyclic ring in nucleobases of ssDNA. After the insulating AFB1 aptamer chain is fixed to the electrode, the signal of PEC sensor is "OFF". In the process of AFB1 detection, the aptamer chain detaches from the surface of erGO, which results in "ON" of the sensor signal. Based on this design, this constructed PEC aptasensor shows a high sensitivity for AFB1 with a wide linear detection range (LDR) from 0.01 ng mL−1 to 100 ng mL−1. The limit of detection (LOD) is 0.002 ng mL−1. This PEC sensor also exhibits good stability, selectivity, specificity, and satisfactory practical sample analysis ability. This work may provide a new promising PEC platform for AFB1 detection as well as some other small molecules analysis. • The erGO/P5FIn/Au nanocomposites are firstly prepared by simple electrochemical deposition method. • A "signal-on" PEC aptasensor for AFB1 detection based on this nanocomposites is fabricated. • This PEC aptasensor exhibits satisfactory sensitivity, reproducibility, stability and high specificity for AFB1. [ABSTRACT FROM AUTHOR]

An efficient and new electrochemical biosensor for detection of DNA damage, induced by the interaction of the hybrid anti-cancer compound (7ESTAC01) with DNA, was studied by differential pulse voltammetry (DPV). The biosensor consists of a Stem-Loop DNA (SL-DNA) probe covalently attached to the gold electrode (GE) surface that hybridizes to a complementary DNA strand (cDNA) to form a double-stranded DNA (dsDNA). The interaction and DNA damage induced by 7ESTAC01 was electrochemically studied based on the oxidation signals of the electroactive nucleic acids on the surface of the GE by DPV. As a result, the SL-DNA/GE and dsDNA/GE were tested with the reduced 7ESTAC01, showing the voltammetric signal of guanine and adenine, increase in the presence of 7ESTAC01. Under optimum conditions, the dsDNA/GE biosensor exhibited excellent DPV response in the presence of 7ESTAC01. The bonding interaction between 7ESTAC01 and calf thymus DNA (ctDNA) was confirmed by UV–Vis absorption spectroscopy, dynamic simulations (performed to investigate the DNA structure under physiological conditions), and molecular docking. Theoretical results showed the presence of hydrogen bonding and intercalation in the minor groove of DNA, involving hydrophobic interactions. • A sensitive electrochemical Stem-Loop DNA biosensor is proposed to detect DNA-hybrid cancer drug intercalation. • A novel hybrid anti-cancer drug, acridine and thiophene, intercalate into the DNA as a dominant mode. • This biosensor can be applied as DNA oxidative damage evaluation method for other cancer drugs candidates. [ABSTRACT FROM AUTHOR]

Chemiresistive platforms are best suited for developing DNA hybridization detection systems, owing to their ease of fabrication, simple detection methodology and amenability towards electronics. In this work, we report development of a generic, robust, electrospun nanofiber based interdigitated chemiresistive platform for DNA hybridization detection. The platform comprises of interdigitated metal electrodes decorated with electrospun nanofibers on the top. Two approaches viz., drop casting of graphene doped Mn 2 O 3 nanofibers (GMnO) and direct electrospinning of polyaniline/polyethylene oxide (PANi/PEO) composite nanofibers, have been utilized to decorate these electrodes. In both approaches, inter-device variability, a key challenge for converting this proof-of-concept into a tangible prototype/product, has been addressed using a shadow masking technique. Consequently, the relative standard deviation for multiple PANi/PEO nanofiber based chemiresistors has been brought down from 17.82% (without shadow masking) to 4.41% (with shadow masking). The nanofibers are further modified with single-stranded probe DNAs, to capture a desired hybridization event. To establish the generic nature of the platform, detection of multiple target DNAs has been successfully demonstrated. These targets include dengue virus specific consensus primer (DENVCP) and four DNAs corresponding to Staphylococcus aureus specific genes, namely nuc, mecA, vanA and protein A. The chemiresistive detection of DENVCP has been performed in the concentration range of 10 fM – 1 µM, whereas the detection of the other targets has been carried out in the range of 1 pM – 1 µM. Using a 3σ method, we have estimated the limit of detection for the chemiresistive detection of DENVCP to be 1.9 fM. • Novel method to minimize inter-device variability of electrospun nanofibers. • Electrospun nanofiber based chemiresistive platform with two distinct schemes. • Sub-femtomolar DNA hybridization detection for multiple targets. • Targeted Application in disease diagnosis and food analysis. [ABSTRACT FROM AUTHOR]

Carbon dots have been extensively used for the development of fluorescent based molecular affinity sensors. However, label free DNA sensing by electrochemical method is not reported so far. Herein, we report carbon dots stabilized silver nanoparticles (CD-AgNPs) lipid nano hybrids as a sensitive and selective platform for label free electrochemical DNA sensing. The CD-AgNPs were synthesized by wet chemical method and then characterized by UV-visible, Fourier-transform Infra-red (FT-IR), dynamic light scattering (DLS) and high resolution transmission electron microscopy (HR-TEM) techniques. These CD-AgNPs were used for decorating the binary lipid 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine (DOPE) and N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP) surface (named as lipid) and tethered on self-assembled monolayer of 3-mercaptopropionic acid (MPA) (MPA-lipid-CD-AgNPs). The formation of array of MPA-lipid-CD-AgNPs on Au electrode was confirmed by atomic force microscopy (AFM). Electrochemical behavior of MPA- lipid-CD-AgNPs was monitored in the presence of 1 mM potassium ferri/ferrocyanide (K 3 /K 4 [Fe(CN) 6 ]). The formation of layer-by-layer MPA-lipid-CD-AgNPs is indicated by increased anodic and cathodic peak (ΔE p) separation with decreased redox peak current of K 3 /K 4 [Fe(CN) 6 ]. Short chain DNA (30 mer oligonucleotide, representing the lung cancer) was used as a model system for label free DNA sensing. Un-hybridized (single stranded DNA), hybridized (complementary hybridized), single, double and triple base mismatched target DNA hybridized surfaces were efficiently discriminated at 1 µM target DNA concentration at the Au/MPA-lipid-CD-AgNPs electrode by change in the charge transfer resistance from impedance technique. Further, the modified electrode was successfully used to determine target DNA in a wide linear range from 10-16 to 10-11 M. The present work open doors for the utilization of CDs in molecular affinity based electrochemical sensor design and development. fx1 • Economically viable and less toxic carbon dots stabilized silver nanoparticle is decorated on the liposome vesicle. • Carbon dot stabilized metal-lipid nano hybrids are used for electrochemical DNA detection for the first time. • The sensor showed a wide linear range from 10-16 to 10-11 M with a lowest detection limit of 10-16 M for cDNA. [ABSTRACT FROM AUTHOR]

Abstract An early and accurate diagnosis of a specific DNA mutations has a decisive role for effective treatment. Especially, when an immediate decision on treatment most needs to be made, the rapid and precise confirmation of clinical findings is vital. Herein, we show a new strategy for the gene mutation (BRAF c.1799T>A; p. V600E) identification using highly SERS-active and reproducible SERS substrate (photo-etched GaN covered with a thin layer of sputtered gold) and surface enhanced Raman scattering (SERS) spectroscopy. The detection is based on the conformation change (gauche → trans) of the alkanethiol linker modifying the capture DNA during the hybridization process. The value of the intensity ratio of the ν(C–S) bands of the trans and gauche conformer higher than 1.0 indicated the presence of mutation. The demonstrated new DNA SERS (bio)sensor is characterized by the low detection limit at the level of pg μL–1, wide analytical range from 6.75 pg μL–1 to 67.5 ng μL–1 and high selectivity. The proposed bioactive platforms, based on nanostructured GaN substrates modified with thiolated ssDNA (single stranded DNA) can be successfully used in the analysis of clinical samples. Highlights • Highly SERS–active and reproducible SERS substrate: photo–etched GaN covered with a thin layer of sputtered gold. • Gene mutation detection based on the conformation change of the alkanethiol DNA linker (gauche → trans). • Gene mutation (BRAF , c.1799T>A; p.600E) limit of detection was 0.17 pg μL–1. • Analysis of gene mutation (BRAF , c.1799T>A; p.600E) in clinical samples. [ABSTRACT FROM AUTHOR]

Abstract In this work, an aptamer was used as the target capturing agent and a trimetallic signal amplification strategy based on Ce-MOF@Au and AuPtRu NPs was demonstrated for the sensitive detection of TSP-1. Herein, the synthesized AuPtRu nanocomposite (AuPtRu NPs) not only acts as the catalyst for catalyzing hydrogen peroxide but also acts as a nanocarrier for capturing the -NH 2 termination single strand DNA (S1) to obtain the signal probe (SP, AuPtRu nanocomposite/S1). Then, SP was efficiently linked into TSP-1 aptamers with the addition of complementary linking strands to form M1 (SP/aptamer). The Ce-MOF@Au nanocomposites were obtained by in situ reduction and used as GCE electrode modification materials. The -NH 2 -modified capture probe (CP) DNA was immobilized on the surface of Ce-MOF@Au nanocomposites for hybridizing SP. In the presence of the target TSP-1, the aptamer recognizes the target and binds strongly so that SP is released from the prepared M1 and then hybridized with CP. When the detection solution contains an electrochemical matrix of H 2 O 2 , AuPtRu NPs can oxidize H 2 O 2 to obtain an enhanced signal. Furthermore, the proposed aptasensor has a very low LOD of 0.13 fg mL−1 TSP-1 in the detection range of 1 fg mL−1 to 10 ng mL−1. Moreover, the proposed platform also has application implications for other potential targets. Highlights • Aptasensor for TSP-1 detection in the human serum samples can be achieved. • Aptamer and complement recognition as a bispecific recognition mechanism. • Trimetallic AuPtRu NPs was synthesized and showed excellent catalytic ability. • Synthesizing Ce-MOF@Au by one-pot method used as electrode modification materials. [ABSTRACT FROM AUTHOR]

Abstract The integrating of high fluorescent quenching capability of two-dimensional nanomaterials with dye-labelled ssDNA as nanoprobes has attracted increasing interest for biosensing applications. However, this absolute intensity-dependent single fluorescent signal may be influenced by target concentration-independent factors. To overcome this challenge, a ratiometric nanoprobe was developed by utilizing green fluorescent phenyl-doped carbon nitride (PDCN) nanosheets as an internal reference and quenching platform. 5-carboxy-X-rhodamine-labelled anti-adenosine aptamer was used as a signal probe. PDCN nanosheets quenched the fluorescence of the absorbed signal probe while kept their own fluorescence constantly. Upon addition of adenosine, the formation of adenosine-aptamer complexes led to desorption of the signal probe from the surface of PDCN nanosheets, resulted in the fluorescent recovery of the signal probe. The ratio of the fluorescent enhancement of the signal probe to the inherent fluorescence of PDCN nanosheets was used to quantitatively measure adenosine. The limit of detection for adenosine was 6.86 μM. Finally, the ratiometric nanoprobe was applied to determine adenosine in serum samples. Highlights • PDCN nanosheets used as both an internal reference and a quenching platform. • Sensitive, accurate and reliable detection of adenosine. • Expand the general scope of 2D nanomaterials-based biosensing applications. [ABSTRACT FROM AUTHOR]

In recent years, the demand for rapid, sensitive, and simple methods for diagnosing deoxyribonucleic acid (DNA) has grown due to the increase in the variation of infectious diseases. This work aimed to develop a flash signal amplification method coupled with electrochemical detection for polymerase chain reaction (PCR)-free tuberculosis (TB) molecular diagnosis. We exploited the slightly miscible properties of butanol and water to instantly concentrate a capture probe DNA, a single-stranded mismatch DNA, and gold nanoparticles (AuNPs) to a small volume to reduce the diffusion and reaction time in the solution. In addition, the electrochemical signal was enhanced once two strands of DNA were hybridized and bound to the surface of the gold nanoparticle at an ultra-high density. To eliminate non-specific adsorption and identify mismatched DNA, the self-assembled monolayers (SAMs) and Muts proteins were sequentially modified on the working electrode. This sensitive and specific approach can detect as low as attomolar levels of DNA targets (18 aM) and is successfully applied to detecting tuberculosis-associated single nucleotide polymorphisms (SNPs) in synovial fluid. More importantly, as this biosensing strategy can amplify the signal in only a few seconds, it possesses a great potential for point-of-care and molecular diagnosis applications. [Display omitted] [ABSTRACT FROM AUTHOR]

The coronavirus disease (COVID-19) pandemic has highlighted the importance of rapid and accurate diagnosis, and loop-mediated isothermal amplification (LAMP) has become a popular method because of its powerful amplification ability using a simple instrument such as a heater or water bath. However, LAMP has limitations such as the complexity of primer design and the difficulty of designing sequence-specific probes, leading to non-specific amplicons and false-positive results. To overcome these limitations, we developed a novel isothermal amplification system called the Ex tension-mediated self-folding I sothermal amplification T echnology (ExIT). ExIT uses a newly designed, self-folding primer (SP) with two key functions. Hairpin structures are formed when the extended strand of the SP hybridizes, exposing the priming site for continuous binding of the new SP. This results in exponential amplification with only two primers, unlike conventional LAMP primer systems. Additionally, an unnatural base was introduced into the SP, which terminated the extension of polymerase and generated a ssDNA amplicon. This makes it easier to design and apply probes, reducing the possibility of false-positive results even if non-specific amplicons are produced. Through this strategy, we confirmed a sensitivity of 90 copies (3.6 copies/μL) and verified the specificity by testing for the presence or absence of non-complementary targets. Therefore, the validation of the ExIT was completed. In conclusion, ExIT will be key to solving the complexity of conventional LAMP design and offers great potential for successfully introducing sequence-specific probes to improve false positives. [ABSTRACT FROM AUTHOR]

The Systematic Evolution Ligands by Exponential Enrichment (SELEX) is common used for selection of high affinity single-stranded DNA (ssDNA) aptamer with target protein. However, we do not know what the most stable configuration of the selected aptamer bound with target protein is. Therefore, a systematic search process using the stochastic tunneling-basin hopping (STUN-BH) method is proposed to find the most stable configuration of the ssDNA aptamer specific for vascular endothelial growth factor (VEGF) capture (Apt VEGF ; 5′-TGTGGGGGTGGACGGGCCGGGTAGA-3′). After the most stable configuration was obtained by the STUN-BH method, molecular dynamics (MD) simulation was carried out to investigate the thermal stability of Apt VEGF /VEGF at 300 K in both vacuum and water. All molecular simulations were conducted with the large-scale atomic/molecular massively parallel simulator (LAMMPS), and the AMBER99SB force field was used to describe the atomic interactions for the current Apt VEGF /VEGF system. The three most stable Apt VEGF /VEGF configurations obtained by the STUN-BH method indicated that Apt VEGF residues exhibit greater affinity for VEGF surface loop fragments as compared with surface alpha helix and beta sheet fragments. Results indicated that after the first Apt VEGF (Apt VEGF I) occupies most of the VEGF loop fragment, the second Apt VEGF (Apt VEGF II) is adsorbed by the rest of the VEGF loop fragment and the VEGF Chain B beta sheet fragment, resulting in a 24.8% reduction in binding strength as compared to that of Apt VEGF I. Furthermore, when Apt VEGF I and Apt VEGF II chains were stably adsorbed by VEGF, the third Apt VEGF (Apt VEGF III) chain can only partially attach to VEGF, as confirmed by real Apt VEGF -VEGF binding experiments. Lastly, we demonstrated that the aptasensor constructed according to MD simulation is highly sensitive for VEGF with a linear detection range of 10 pg/mL–10 ng/mL. [ABSTRACT FROM AUTHOR]

The development of nucleic acid-based portable platforms for the real-time analysis of diseases has attracted considerable scientific and commercial interest. Recently, 2D layered molybdenum sulfide (2D MoS 2 from here on) nanosheets have shown great potential for the development of next-generation platforms for efficient signal transduction. Through combination with DNA as a biorecognition medium, MoS 2 nanostructures have opened new opportunities to design and construct highly sensitive, specific, and commercially viable sensing devices. The use of specific short ssDNA sequences like aptamers has been proven to bind well with the unique transduction properties of 2D MoS 2 nanosheets to realize aptasensing devices. Such sensors can be operated on the principles of fluorescence, electro-cheumuluminescence, and electrochemistry with many advantageous features (e.g., robust biointerfacing through various conjugation chemistries, facile sensor assembly, high stability with regard to temperature/pH, and high affinity to target). This review encompasses the state of the art information on various design tactics and working principles of MoS 2 /DNA sensor technology which is emerging as one of the most sought-after and valuable fields with the advent of nucleic acid inspired devices. To help achieve a new milestone in biosensing applications, great potential of this emerging technique is described further with regard to sensitivity, specificity, operational convenience, and versatility. [ABSTRACT FROM AUTHOR]

The difference of electrostatic interaction between free Ru(phen) 3 2+ and Ru(phen) 3 2+ embedded in double strand DNA (dsDNA) to the negatively charged indium tin oxide (ITO) electrode has been applied to develop a homogeneous and label-free electrochemiluminescence (ECL) aptasensor for the first time. Ochratoxin A (OTA) has been chosen as the model target. The OTA aptamer is first hybridized with its complementary single strand DNA (ssDNA) to form dsDNA and then interacted with Ru(phen) 3 2+ via the grooves binding mode to form dsDNA-Ru(phen) 3 2+ complex, which remains negatively charged feature as well as low diffusion capacity to the negatively charged ITO electrode surface owing to the electrostatic repulsion. Meanwhile, the intercalated Ru(phen) 3 2+ in the grooves of dsDNA works as an ECL signal reporter instead of the labor-intensive labeling steps and can generate much more ECL signal than that from the labeling probe. In the presence of target, the aptamer prefers to form an aptamer-target complex in lieu of dsDNA, which induces the releasing of Ru(phen) 3 2+ from the dsDNA-Ru(phen) 3 2+ complex into the solution. With the assistance of RecJ f exonuclease (a ssDNA specific exonuclease), the released ssDNA and the aptamer in the target-complex were digested into mononucleotides. In the meantime, the target can be also liberated from OTA-aptamer complex and induce target cycling and large amount of free Ru(phen) 3 2+ present in the solution. Since Ru(phen) 3 2+ contains positive charges, which can diffuses easily to the ITO electrode surface because of electrostatic attraction, causing an obviously enhanced ECL signal detected. Under the optimal conditions, the enhanced ECL of the system has a linear relationship with the OTA concentration in the range of 0.01–1.0 ng/mL with a detection limit of 2 pg/mL. This innovative system not only expands the immobilization-free sensors in the electrochemiluminescent fields, but also can be developed for the detection of different targets easily with the same strategy by changing the aptamer used. [ABSTRACT FROM AUTHOR]

A high-performance sensing platform based on poly-xanthurenic acid (PXA) film functionalized MoS 2 nanosheets was developed for electrochemical detection of circulating tumor DNA in peripheral blood. The MoS 2 nanosheets were obtained using a simple ultrasonic method from bulk MoS 2 . The physical adsorption between MoS 2 and aromatic XA monomers effectively improved the electropolymerization efficiency, accompanied with an increased electrochemical response of PXA. The obtained PXA/MoS 2 nanocomposite not only served as a substrate for DNA immobilization but also reflected the electrochemical transduction originating from DNA immobilization and hybridization without any complex labelling processes or outer indicators. The immobilization of the probe ssDNA was achieved via noncovalent assembly due to the π–π interaction between PXA and DNA bases. After the hybridization of the probe ssDNA with the target DNA, the formation of helix structure induced the resulted dsDNA to be released from the surface of the PXA/MoS 2 nanocomposite. The detection limit of this constructed DNA biosensor was calculated in the linear target DNA concentrations range from 1.0 × 10 –16 mol/L to 1.0 × 10 -10 mol/L and it was found to be 1.8 × 10 –17 mol/L. [ABSTRACT FROM AUTHOR]

A simple and sensitive electrochemical biosensor was developed for microRNA-196a detection, which is of important diagnostic significance for pancreatic cancer. It was based on cyclic enzymatic signal amplification (CESA) and template-free DNA extension reaction. In the presence of microRNA-196a, duplex-specific nuclease (DSN) catalyzed the digestion of the 3′-PO 4 terminated capture probe (CP), resulting in the target recycling amplification. Meanwhile, the 3′-OH terminal of CP was exposed. Then, template-free DNA extension reaction was triggered by terminal deoxynucleotidyl transferase (TdT), producing amounts of single-stranded DNA (ssDNA). After ssDNA absorbed numerous methylene blue (MB), an ultrasensitive electrochemical readout was obtained. Based on this dual amplification mechanism, the proposed biosensor exhibited a high sensitivity for detection of microRNA-196a down to 15 aM with a linear range from 0.05 fM to 50 pM. This biosensor displayed high specificity, which could discriminate target microRNAs from one base mismatched microRNAs. It also showed good reproducibility and stability. Furthermore, it was successfully applied to the determination of microRNA-196a in plasma samples. In conclusion, with the excellent analytical performance, this biosensor might have the potential for application in clinical diagnostics of pancreatic cancer. [ABSTRACT FROM AUTHOR]

Early screening of biomarkers benefits therapy and prognosis of cancers. MiRNAs encapsulated in tumor-derived exosomes are emerging biomarkers for early diagnosis of cancers. Nevertheless, traditional methods suffer certain drawbacks, which hamper their wide applications. In this contribution, we have developed a convenient electrochemical approach for quantification of exosomal miRNA based on the assembly of DNA triangular pyramid frustum (TPF) and strand displacement amplification. Four single-stranded DNA helps the formation of primary DNA triangle with three thiols for gold electrode immobilization at the bottom and three amino groups on overhangs for the capture of silver nanoparticles. On the other hand, target miRNA induced strand displacement reaction produces abundant specific DNA strands, which help the DNA structural transition from triangle to TPF. Amino groups are thus hidden and the declined silver stripping current can be used for the evaluation of target miRNA concentration. This biosensor exhibits excellent analytical performances and successfully achieves analysis of exosomal miRNAs from cells and clinical serum samples. • An ultrasensitive electrochemical biosensor for the detection of exosomal miRNAis developed. • DNA nanostructural transition from triangle to triangular pyramid frustum is utilized for signal blocking. • miRNA induced strand displacement reaction produces single-stranded DNA as fuel strands for triangular pyramid frustum assembly. • This biosensor achieves excellent analytical performances and practical utility for clinical samples is demonstrated. [ABSTRACT FROM AUTHOR]

Sensitive quantification of multiple analytes of interest is of great significance for clinical diagnosis. CRISPR Cas platforms offer a strategy for improving the specificity, sensitivity, and speed of nucleic acid-based diagnostics, while their multiplex analysis capability is still limited and challenging. Herein, we develop a novel DNA Tetrahedron (DTN)-supported biosensor based on the spatially separated CRISPR Cas self-amplification strategy and multiple-metal-nanoparticle tagging coupled with inductively coupled plasma mass spectrometry (ICP-MS) detection to improve the sensitivity and feasibility of the platform for multiplex detection of HPV-DNA (HPV-16, HPV-18 and HPV-52). Given target DNA induces robust trans-cleavage activity of the Cas12a/crRNA duplex, and the surrounding corresponding single-stranded DNA (ssDNA) linker are cleaved into short fragments that are unable to bond metal-nanoparticle probes (197Au, 107Ag, 195Pt) onto DTN modified magnetic beads probe (MBs-DTN), resulting in obvious ICP-MS signal change. Of note, compared with ssDNA functionalized MBs, a higher Signal-to-Noise Ratio was obtained by using MBs-DTN in our system, further amplifying the signal by regulating probes on the surface of MBs. As expected, the HPV-DNA could be detected with detection limits as low as 218 fM and be multiplexed assayed at one test with high accuracy and specificity by this proposed strategy. Furthermore, we demonstrated that the HPV-DNA in cervical swab samples could be detected, showing high consistency with DNA sequencing results. We believe that this work provides a promising option in designing CRISPR based multiplex detection system for high sensitivity, good specificity, and clinical molecular diagnostics. [ABSTRACT FROM AUTHOR]

Novel fluorescent DNA quantum dots (QDs) were synthesized by hydrothermal treatment of G-/T-rich ssDNA at relatively low reaction temperature. The obtained DNA QDs demonstrate unique optical properties, maintain the basic structure and biological activities of ssDNA precursors, which makes the DNA QDs able to specifically bind with arsenite, driving the (GT) 29 region suffer conformation evolution and form well-ordered assembly rather than random aggregations. We speculate that the strong inter-molecule interaction and efficient stacking of base pairs stiffen the assembly structure, block the nonradiative relaxation channels, populate the radiative decay, and thus making the assembly be highly emissive as a new fluorescence center. The arsenite-induced specific fluorescence enhancement facilitates DNA QDs as light-up probes for arsenite sensing. Under optimal conditions, a linear relationship between the increased fluorescence intensity of DNA QDs and the logarithmic values of arsenite concentration in the range of 1–150 ppb with a detection limit of 0.2 ppb (3σ) was obtained. The nanosensor shows excellent selectivity for “turn on” arsenite determination and arsenate does not show any interference, facilitating its application in complex real water analysis. [ABSTRACT FROM AUTHOR]

We describe the use of ssDNA functionalized silver nanoparticle (AgNP) probes for quantitative investigation of biorecognition and real time detection of biomolecular targets using nano-impact electrochemistry. The method is based on measurements of the individual collision events between ssDNA aptamer-functionalized AgNPs and a carbon fiber miroelectrode (CFME). Specific binding events of target analyte induced collision frequency changes enabling ultrasensitive detection of the aptamer target in a single step. These changes are assigned to the surface coverage of the NP by the ssDNA aptamers and subsequent conformational changes of the aptamer probe which affect the electron transfer between the NP and the electrode surface. The method enables sensitive and selective detection of ochratoxin A (OTA), chosen here as a model target, with a limit of detection of 0.05 nM and a relative standard deviation of 4.9%. The study provides a means of characterizing bioconjugation of AgNPs with aptamers and assessing biomolecular recognition events with high sensitivity and without the use of exogenous reagents or enzyme amplification steps. This methodology can be broadly applicable to other bioconjugated systems, biosensing and related bioanalytical applications. [ABSTRACT FROM AUTHOR]

Rapid, sensitive, and one-pot diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays an extremely important role in point-of-care testing (POCT). Herein, we report an ultra-sensitive and rapid one-pot enzyme-catalyzed rolling circle amplification-assisted CRISPR/FnCas12a assay, termed OPERATOR. OPERATOR employs a single well-designed single-strand padlock DNA, containing a protospacer adjacent motif (PAM) site and a sequence complementary to the target RNA which procedure converts and amplifies genomic RNA to DNA by RNA-templated DNA ligation and multiply-primed rolling circle amplification (MRCA). The MRCA amplicon of single-stranded DNA is cleaved by the FnCas12a/crRNA complex and detected via a fluorescence reader or lateral flow strip. OPERATOR presents outstanding advantages including ultra-sensitivity (1.625 copies per reaction), high specificity (100%), rapid reaction speed (∼30 min), easy operation, low cost, and on-spot visualization. Furthermore, we established a POCT platform by combining OPERATOR with rapid RNA release and a lateral flow strip without professional equipment. The high performance of OPERATOR in SARS-CoV-2 tests was confirmed using both reference materials and clinical samples, and the results suggest that is readily adaptable for point-of-care testing of other RNA viruses. [ABSTRACT FROM AUTHOR]

As an essential biomarker associated with various diseases, Uracil-DNA Glycosylase (UDG) detection is vital for disease diagnosis, treatment selection, and prognosis assessment. In recent years, the signal amplification effect of the CRISPR-Cas12a trans-cleaved single-stranded DNA probe has provided an available strategy for constructing highly sensitive biosensors. However, its superior trans-cleavage activity has become a "double-edged sword" for building biosensors that can amplify the target signal while also amplifying the leakage signal, causing out of control. Therefore, the construction of structurally simple, extremely low-background, highly sensitive CRISPR-Cas12a-based biosensors is an urgent bottleneck problem in the field. Here, we applied CRISPR-Cas12a with a DNA hybridization reaction to develop a simple, rapid, low background, and highly sensitive method for UDG activity detection. It has no PAM restriction and the detection limit is as low as 2.5 × 10−6 U/mL. As far as we know, this method is one of the most sensitive methods for UDG detection. We also used this system to analyze UDG activity in tumor cells (LOD: 1 cell/uL) and to evaluate the ability to screen for UDG inhibitors. Furthermore, we verified the possibility of intracellular UDG activity imaging by transfecting the biosensors to the cells. We believe this novel sensor has good clinical application prospects and will effectively broaden the application space of CRISPR-Cas12a. • A novel sensor based on CRISPR-Cas12a coupled with DNA hybridization reaction for UDG detection. • Toehold-mediated CRISPR-Cas12a system activation without the need of PAM. • Ultra-sensitive biosensor with 2.5x10-6 U/mL of LOD in UDG detection. • Short reaction time, simple operation, without extra amplification steps. • Robust biosensor for complex bio-sample detection at the single-cell level, inhibitor screening, and intercellular imaging. [ABSTRACT FROM AUTHOR]

In this work, we tactfully constructed a novel label-free electrochemical aptasensor for rapid and facile detection of thrombin using graphene oxide (GO) and thrombin binding aptamer (TBA). The strategy relies on the preferential adsorption of single-stranded DNA (ssDNA) to GO over aptamer-target complexes. The TBA-thrombin complex formation was monitored by differential pulse voltammetry (DPV) using the guanine oxidation signal. In the absence of thrombin, the aptamers adsorbed onto the surface of GO leading to a strong background guanine oxidation signal. Conversely, in the presence of thrombin, the conformational transformation of TBA after incubating with the thrombin solution and formation of the aptamer-thrombin complexes which had weak binding ability to GO, leads to the desorption of TBA-thrombin complex from electrode surface and significant oxidation signal decrease. The selectivity of the biosensor was studied using other biological substances. The biosensor's signal was proportional to the thrombin concentration from 0.1 to 10 nM with a detection limit of 0.07 nM. Particularly, the proposed method could be widely applied to the aptamer-based determination of other target analytes. [ABSTRACT FROM AUTHOR]

Toehold-mediated strand displacement (TMSD) reactions between double-stranded DNA and single-stranded oligonucleotides, which use a toehold structure as a switch, enable DNA rehybridization in a fast, base-to-base, programmable and controlled manner. TMSD offers numerous advantages, as reactions are enzyme-free, highly efficient, isothermal and cost-effective. Consequently, TMSD-based reaction systems have attracted great interest from researchers in the last two decades as they become increasingly diverse, versatile and robust, and have been widely used in biochemical sensing and medical diagnosis. In this review, we provide a comprehensive overview of TMSD reactions and their applications in biosensors. The six current TMSD reaction types are generalized according to their various modes of action and nucleic acid conformations. Reaction mechanisms via three major thermodynamic driving forces are explained, along with their dynamic affecting factors, and seven toehold-mediated biosensing strategies are summarized. Furthermore, TMSD-reaction biodetection and bioimaging applications are discussed and, finally, several current challenges and perspectives in the field are considered. [ABSTRACT FROM AUTHOR]

Flap endonuclease 1 (FEN1) is an endonuclease that specially removes 5′ single-stranded overhang of branched duplex DNA (5′ flap). While FEN1 is essential in various DNA metabolism pathways for preventing the malignant transformation of cells, an unusual expression of FEN1 is often associated with tumor progression, making it a potential biomarker for cancer diagnosis and treatment. Here we report a multimodal detection of FEN1 activity based on CRISPR/Cas12a trans-cleavage of single-strand DNA oligonucleotides (ssDNA). A dumbbell DNA structure with a 5' flap was designed, which can be cleaved by the FEN1 and the dumbbell DNA is subsequently ligated by T4 DNA ligase. The resulting closed duplex DNA contains a specific protospacer adjacent motif (PAM) that activates trans-cleavage of ssDNA after binding to CRISPR/Cas12a-crRNA. The trans-cleavage is activated only once and is independent to length or sequence of the ssDNA, which allows efficient signal amplification and multimodal signals such as fluorescence or cleaved connection between magnetic microparticles (MMPs) and polystyrene microparticles (PMPs) that alters solution turbidity after magnetic separation. In addition, by loading the particle solution into a microfluidic chip, unconnected PMPs escaping from a magnetic separator are amassed at the particle dam, enabling a visible PMP accumulation length proportional to the FEN1 activity. This multimodal detection is selective to FEN1 and achieves a low limit of detection (LOD) with only 40 min of reaction time. Applying to cell lysates, higher FEN1 activity was detected in breast cancer cells, suggesting a great potential for cancer diagnosis. [ABSTRACT FROM AUTHOR]

Development of simple, low-cost and highly sensitive electrochemiluminescence (ECL) sensors for various targets is urgent but remains challenging. It is because most of emitters were required to be dissolved in organic solvent or immobilized at electrode's surface to display ECL emission, and suffered from complicated tagging procedures and short emission wavelength. Herein, we synthesize an iridium (III) complex (Ir-ECL) and applied it as a ECL emitter for improved target sensing. ECL emission of Ir-ECL originated from the sensitization of N-heterocyclic ligands on Ir (III). Impressively, Ir-ECL exhibited ECL emission wavelength at 590 nm, and displayed a superior intercalation ability into G-quadruplex DNA against single-stranded DNA and double-stranded DNA (dsDNA). Using such properties, Ir-ECL was applied to enzyme-free, label-free, sensitive and homogeneous ECL analysis of pesticide acetamiprid (Ace) based on aptamer-target recognition-driven hybridization chain reaction (HCR). The recognition of H1 by Ace switched HCR of H2 and H3 to generate a long-chain dsDNA with abundant G-quadruplex DNAs, in which large numbers of Ir-ECL were locked, resulting in falling diffusion toward electrode, declining ECL signal and eventually improving Ace ECL sensing. [ABSTRACT FROM AUTHOR]

Based on the remarkable difference between the interactions of carbon nanoparticles (CNPs) oxide with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), and the fact that fluorescence of DNA-stabilized silver nanoclusters (AgNCs) can be quenched by CNPs oxide, DNA-functionalized AgNCs were applied as label-free fluorescence probes and a novel fluorescence resonance energy transfer (FRET) sensor was successfully constructed for the detection of human immunodeficiency virus (HIV) DNA sequences. CNPs oxide were prepared with the oxidation of candle soot, hence it is simple, time-saving and low-cost. The strategy of dual AgNCs probes was applied to improve the detection sensitivity by using dual- probe capturing the same target DNA in a sandwich mode and as the fluorescence donor, and using CNPs oxide as the acceptor. In the presence of target DNA, a dsDNA hybrid forms, leading to the desorption of the ssDNA-AgNCs probes from CNPs oxide, and the recovering of fluorescence of the AgNCs in a HIV-DNA concentration-dependent manner. The results show that HIV-DNA can be detected in the range of 1–50 nM with a detection limit of 0.40 nM in aqueous buffer. The method is simple, rapid and sensitive with no need of labeled fluorescent probes, and moreover, the design of fluorescent dual-probe makes full use of the excellent fluorescence property of AgNCs and further improves the detection sensitivity. [ABSTRACT FROM AUTHOR]

A label-free DNA biosensor based on microfiber-assisted Mach-Zehnder interferometer (MAMZI) for in-situ real-time DNA hybridization kinetics detection has been proposed and experimentally demonstrated. A microfiber of hundreds of microns in length is fabricated by tapering a segment of standard single-mode fiber (SMF) to construct the U-shaped microcavity between the lead-in and lead-out SMFs. Thanks to the mode field mismatching between the SMF and microfiber, the incident guided mode light would separate into two beams that respectively propagate in the air microcavity and the microfiber. Consequently, interference between different light modes would occur at the joint between the microfiber and the lead-out SMF. Experimental results indicate that owing to the participation of opening cavity modes in the modal interference process, the interferometric spectrum of our proposed microcavity sensor is highly sensitive to the variation of environmental refractive index (RI), especially for the RI range around 1.34 which is useful for most biological applications. The microfiber functionalization is achieved by stepwise modifying the microfiber with monolayer Poly- l -lysine (PLL) and single-stranded DNA (ssDNA) probes to produce the sensitive surface that could uniquely attach specific target ssDNAs. The fiber surface functionalization as well as DNA hybridization processes have been experimentally investigated for different target ssDNA solutions in real time. The interferometric transmission spectrum shows large wavelength shift for different biological phases, and a detection limit conservatively down to 0.0001 pmol/μL has been acquired by employing the U-shaped microcavity of 176.88 μm in length. Our proposed DNA biosensor possesses several advantages such as compact size, ease of fabrication, and strong response for DNA hybridization, which make it a promising candidate for potential applications in such rapidly expanding areas as medical diagnosis, cancer screenings, medicine examination and environmental engineering, etc. [ABSTRACT FROM AUTHOR]

A novel fluorescence turn-on strategy for the alkaline phosphatase (ALP) assay is developed based on the preferential binding of graphene oxide (GO) to single-stranded DNA (ssDNA) over double-stranded DNA (dsDNA) coupled with λ exonuclease (λ exo) cleavage. Specifically, in the absence of ALP, the substrate-dsDNA constructed by one oligonucleotide with a fluorophore at the 3′-end (F-DNA) and its complementary sequence modified with a 5′-phosphoryl termini ( p -DNA), is promptly cleaved by λ exo, and the resulting F-DNA is adsorbed on GO surface, allowing fluorescence quenching. Whereas the introduction of ALP leads to the hydrolysis of the P-DNA, and the yielding 5′-hydroxyl end product hampers the λ exo cleavage, inducing significant fluorescence enhancement due to the weak binding of dsDNA with GO. Under the optimized conditions, the approach exhibits high sensitivity and specificity to ALP with a detection limit of 0.19 U/L, and the determination of ALP in spiked human serum samples has also been realized. Notably, this new approach not only provides a novel and sensitive platform for the ALP activity detection but also promotes the exploitation of the GO-based biosensing for the detection of the protein with no specific binding element, and thus extending the GO-based sensing applications into a new field. [ABSTRACT FROM AUTHOR]

Electrochemical aptamer-based (E-AB) sensors employing electrode-immobilized, redox-tagged aptamer probes have emerged as a promising platform for the sensitive and quick detection of target analytes ranging from small molecules to proteins. Signal generation in this class of sensor is linked to change in electron transfer efficiency upon binding-induced change in flexibility/conformation of the aptamer probe. Because of this signaling mechanism, signal gains of these sensors can be improved by employing a displacement-based recognition system, which links target binding with a large-scale flexibility/conformation shift from the aptamer-DNA duplex to the single-stranded DNA or the native aptamer. Despite the relatively large number of displacement-based E-AB sensor samples, little attention has been paid to the structure variation of the aptamer-DNA duplex probe. Here we detail the effects of complementary length and position of the aptamer-DNA duplex probe on the performance of a model displacement-based E-AB sensor for ATP. We find that, greater background suppression and signal gain are observed with longer complementary length of the aptamer-DNA duplex probe. However, sensor equilibration time slows monotonically with increasing complementary length; and with too many target binding sites in aptamer sequence being occupied by the complementary DNA, the aptamer-target binding does not occur and no signal gain observed. We also demonstrate that signal gain of the displacement-based E-AB sensor is strongly dependent on the complementary position of the aptamer-DNA duplex probe, with complementary position located at the electrode-attached or redox-tagged end of the duplex probe, larger background suppression and signal increase than that of the middle position are observed. These results highlight the importance of rational structure design of the aptamer-DNA duplex probe and provide new insights into the optimization of displacement-based E-AB sensors. [ABSTRACT FROM AUTHOR]

The need for sensitive detection of DNA is growing as more specific DNA sequences are being correlated to gene markers for disease diagnosis, food safety and other security related applications. Detection in hybridization-based assays is usually achieved with target-specific ssDNA probes conjugated directly to enzyme labels like HRP that provide signal amplification or with nanoparticles functionalized with DNA and multiple HRP molecules. In order to overcome some of the drawbacks presented by these approaches, we developed a unique DNA sensing platform based on an HRP–DNA binding protein tag conjugate and a hybrid ssDNA–dsDNA detection probe. Specifically, in this work we describe the preparation and characterization of an HRP conjugate with scCro DNA binding protein tag and its application for the detection of a model ssDNA target sequence. By using the HRP–scCro conjugate together with a hybrid detection probe containing three scCro‐specific dsDNA binding sites, we demonstrate an improvement by over 3-fold in both sensitivity and limit of detection of high-risk human papillomavirus (HPV16), compared to the standard ssDNA–HRP conjugate. These results show that the HRP–DNA binding protein tag conjugate can be used as an alternative and universal tool for signal enhancement in enzyme-linked assays suitable for integration in point-of-care systems. [ABSTRACT FROM AUTHOR]

A simple, environmentally friendly hydrothermal method was used to prepare strongly luminescent, nitrogen-doped carbon dots (NCDs) with the use of Chinese yams as a source of carbon and nitrogen. Such NCDs have an average size of 2.7±1.4 nm; they emit blue light at 420 nm and have a quantum yield of up to 9.3%. Thus, carboxyfluorescein (FAM)-DNA macro-molecules were assembled on the surfaces of the NCDs, and stabilised by strong π–π stacking; the so formed hybrid nano-sensors were found to have an ultra-sensitive response to 6-mercaptopurine (6-MP). A strong emission and enhancement of yellow radiation was observed from FAM. Furthermore, due to the specific interactions between DNA and Hg 2+ , which resulted in the formation of the T–Hg 2+ –T (T: thymine base) complex – a large, conjugated system, which formed between NCDs, DNA and 6-MP, was broken up. Thus, the fluorescence from FAM was quenched. The detection limits for 6-MP and Hg 2+ were 0.67 and 1.26 nM, respectively. The proposed method was applied for the determination of 6-MP in human serum and Hg 2+ in water samples with satisfactory results. [ABSTRACT FROM AUTHOR]

Transcription factors (TFs) are key substances in regulating the transcription, replication and expression of genes, and the detection of TFs can provide valuable information to diagnose a variety of diseases. By integrating hybridization chain reaction (HCR)-activated Cas12a enzyme with bio-responsive DNA hydrogels, we propose a dual amplification and label-free homogeneous electrochemical detection method to realize sensitive nuclear factor-kappa B p50 (NF-κB p50) detection. The presence of the target molecules protects the DNA duplex probes from digesting by exonuclease III and initiates HCR to generate long double stranded DNAs that can activate the activity of RNA-guided Cas12a enzymes. The single-stranded region of the DNA linkers that crosslink the DNA hydrogels can be cleaved by the activated Cas12a to release a large number of electroactive substances embedded in the gels, which exhibit highly enhanced electrochemical signals for detecting target molecules at the detection limit of 54.1 fM. In addition, the successful interrogation of NF-κB p50 spiked into lysate of HeLa cells by such method is also verified. The established method thus shows new opportunities for sensitive and convenient monitoring of other transcription factors and biomarkers. [ABSTRACT FROM AUTHOR]

To develop various biosensors, several 2D nanomaterials adsorb DNA probes (aptamers) via π – π stacking interactions. However, interference from DNA displacement by external non-targeted ligands has precluded their practical applications for specific detection and imaging at high protein concentrations. Metal coordination is an attractive strategy for biomolecular crosslinking and functional molecular self-assembly. Herein, a robust 2D biosensor nanoplatform was developed to enhance DNA adsorption and affinity using Mn2+-modified black phosphorus nanosheets (BPNS@Mn2+) via metal coordination. The Mn2+ can simultaneously coordinate with the lone pair electrons (π bonds) of the BPNS and nucleotide bases to provide binding sites for DNA nucleobases on the BPNS surface, which greatly enhances the stability of the inner BPNS and improves DNA adsorption and affinity. The DNA adsorption mechanism of BPNS@Mn2+ was also characterized, and is extensively discussed. Without any further modification, this BPNS@Mn2+/DNA biosensor specifically detected single-stranded DNA (linear range: 10–200 nM, detection limit: 5.76 nM) and thrombin (linear range: 20–180 nM, detection limit: 2.39 nM) in 100 nM bovine serum albumin solution. The nonspecific ligands in the environment did not affect the detection performance of the robust biosensor. In addition, the expression levels of microRNA-21 can be imaged and analyzed in living cells using this biosensor, which is consistent with the results of the polymerase chain reaction. This study highlights the potential of metal coordination in surface modification and provides new opportunities for biomedical applications of 2D nanomaterials with superior DNA-adsorption capacity, facilitating the development of biosensor design and nucleic acid/drug delivery. [Display omitted] [ABSTRACT FROM AUTHOR]

Rational detection of syndrome coronavirus 2 (SARS-CoV-2) is crucial to prevention, control, and treatment of disease. Herein, a dual-wavelength ratiometric electrochemiluminescence (ECL) biosensor based on resonance energy transfer (RET) between g-C 3 N 4 nanosheets and Ru–SiO 2 @folic acid (FA) nanomaterials was designed to realize ultrasensitive detection of SARS-CoV-2 virus (RdRp gene). Firstly, the unique g-C 3 N 4 nanosheets displayed very intense and stable ECL at 460 nm, then the triple helix DNA was stably and vertically bound to g-C 3 N 4 on electrode by high binding affinity between ssDNA and g-C 3 N 4. Meanwhile, trace amounts of target genes were converted to a large number of output by three-dimensional (3D) DNA walker multiple amplification, and the output bridged a multifunctional probe Ru–SiO 2 @FA to electrode. Ru–SiO 2 @FA not only showed high ECL at 620 nm, but also effectively quenched g-C 3 N 4 ECL. As a result, ECL decreased at 460 nm and increased at 620 nm, which was used to design a rational ECL biosensor for detection of SARS gene. The results show that the biosensor has excellent detection sensitivity for RdRp gene with a dynamic detection range of 1 fM to 10 nM and a limit of detection (LOD) of 0.18 fM. The dual-wavelength ratio ECL biosensor has inestimable value and application prospects in the fields of biosensing and clinical diagnosis. [ABSTRACT FROM AUTHOR]

Flap endonuclease 1 (FEN1) is a structure-specific nuclease, which catalyzes the removal of 5′ overhanging DNA flap from a specific DNA structure. FEN1 has been considered as an important biomarker for cancer diagnosis since it is over-expressed in various types of human tumor cells and closely related to cancer development. Nanoprobes gradually become basic tools for analyzing biomarkers variations in vivo. Here, we utilized aminoated mesoporous silica nanoparticles (NH 2 -MSNs) with a rich porous structure as the fluorescence nanoprobes to entrap the rhodamine 6G (Rh6G) molecules. Then gold nanoparticles linked specific single-stranded DNA (AuNPs-ssDNA) as a molecular gate was used to coat the NH 2 -MSNs surface. The fluorescence signal was weak when the fluorescence molecules were blocked by the AuNPs-ssDNA. In the presence of FEN1, it recognized and cleaved the specific ssDNA to release the Rh6G from NH 2 -MSNs, which resulted in recovered fluorescence signals. Thus, the sensitive detection of FEN1 activity was realized by controlled-release of Rh6G. The fluorescence signal showed a good linear relationship with the logarithm of FEN1 activity ranging from 0.05 to 1.75 U with a detection limit of 0.03 U. Moreover, confocal imaging demonstrated that the proposed biosensor could distinguish tumor cells from normal cells. Therefore, this technique contributes to clinical diagnostic and therapeutic monitoring. [ABSTRACT FROM AUTHOR]

Identification and detection of extreme rare circulating tumor cells (CTCs) in peripheral blood can precisely monitor cancer recurrence and metastasis, however, how to ultra-sensitively and reliably detect CTCs is a big challenge. In this work, a ratiometric surface-enhanced Raman spectroscopy (SERS)-based strategy for ultra-sensitively and nondestructively detecting CTCs was proposed via CTCs-triggered DNA walker-assisted assembly of plasmonic nanostructure networks consisting of Walker probes and SERS tags. The Walker probes were prepared by modifying Fe 3 O 4 @SiO 2 @Au nanoparticles (GMNPs) with ROX-labeled EpCAM aptamer-blocked Zn2+-specific DNAzyme and hairpin-structured single-stranded DNAs H1, and the SERS tags were constructed by co-labelling hairpin-structured single-stranded DNAs H2 and Raman molecules (DTNB) on Au NPs. The aptamers can recognize EpCAM-positive CTCs via the specific binding to EpCAM, so that the activity of DNAzymes is activated with the assistance of Zn2+ to launch the DNA walker to move around for the cleavage of H1 on GMNPs. The residual fragments of H1 on GMNPs can hybridize with H2 on SERS tags and result in the formation of Walker probe-SERS tag network nanostructures (Nw NSs) with rich SERS hot spots. The reliable SERS detection of CTCs is achieved by the stable ratiometric SERS signals of DTNB and ROX generated from the Nw NSs, and a good linear relation between ratiometric SERS signal and MCF-7 cells concentration was obtained with the detection limit low to 1 cell/mL. The recovery rate of MCF-7 cells in peripheral blood is in the range of 94.0%–104.5%, which indicates a good application prospect of the novel ratiometric SERS cytosensor in the clinic detection of EpCAM-positive CTCs. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) play an essential role in cancer diagnosis and prognosis. Developing a new method for sensitive detection of miRNA is constantly in demand. CRISPR/Cas12a system can nonspecifically cleave single-stranded DNA after specific recognition of target DNA, showing tremendous potential in molecular diagnostics. However, CRISPR-based detection methods require synthesizing different crRNAs for detecting different targets, which limit their widespread application. Herein, we design a versatile and sensitive miRNA detection platform based on CRISPR/Cas12a system combined with a hybridization chain reaction (HCR) circuit. In this design, the HCR circuit as the signal transducer converts each miRNA into multiple DNA duplexes, which act as the activators to activate the trans -cleavage activity of Cas12a for further signal amplification. More importantly, this platform can sensitively detect different miRNAs without changing the spacer sequence of crRNA due to the fixed activators formed by HCR. In addition, the consistency between the proposed platform and RT-qPCR in miRNA detection extracted from different cell lines validated its practicability, demonstrating the potential in clinical diagnosis of cancers and monitoring therapy. [ABSTRACT FROM AUTHOR]

Cocaine is a strong central nervous system stimulant and one of the most commonly abused drugs. In this study, an electrochemical aptasensor was designed for sensitive and selective detection of cocaine, based on single-walled carbon nanotubes (SWNTs), gold electrode and complimentary strand of aptamer (CS). This electrochemical aptasensor inherits properties of SWNTs and gold such as large surface area and high electrochemical conductivity, as well as high affinity and selectivity of aptamer toward its target and the stronger interaction of SWNTs with single-stranded DNA (ssDNA) than double-stranded DNA (dsDNA). In the absence of cocaine, a little amount of SWNTs bind to Aptamer-CS-modified electrode, so that the electrochemical signal is weak. In the presence of cocaine, aptamer binds to cocaine, leaves the surface of electrode. So that, a large amount of SWNTs bind to CS-modified electrode, generating to a strong electrochemical signal. The designed electrochemical aptasensor showed good selectivity toward cocaine with a limit of detection (LOD) as low as 105 pM. Moreover, the fabricated electrochemical aptasensor was successfully applied to detect cocaine in serum with a LOD as low as 136 pM. [ABSTRACT FROM AUTHOR]