Spatiotemporal Visualization of Paraquat Distribution, Toxicokinetics, and Its Detoxification in Zebrafish Using Matrix-Assisted Laser Desorption Ionization Mass Spectrometry Imaging.

Paraquat is a highly toxic quaternary ammonium herbicide. It can damage the functions of multiple organs and cause irreversible pulmonary fibrosis in the human body. However, the toxicological mechanism of paraquat is not yet fully understood, and due to the lack of specific antidotes, the clinical treatment of paraquat intoxication is still a great medical challenge. In-depth research on its toxicity mechanism, toxicokinetics, and effective antidotes is urgently demanded. A new molecular imaging technique, matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI), can simultaneously achieve quantitative and spatial analysis and offer an alternative, distinct, and useful technique for paraquat intoxication and consequent detoxication. Here, we visualized the spatial-temporal distribution and conducted toxicokinetic research on paraquat in zebrafish by using stable isotope-labeled internal-standard-aided MALDI-MSI for the first time. The results indicated that paraquat had a fast absorption rate and was widely distributed in different organs, such as the brain, gills, kidneys, and liver in zebrafish. Its half-life was long, and the elimination rate was slow. Paraquat reached its peak at 30 min and was mainly distributed in kidneys and intestines and then showed a tendency of declining first but mildly rising later at 6 h, accompanied by a wide distribution in kidneys and intestines again. It suggested that entero-systemic recirculation might lead to the observed secondary peaks, and perhaps it extended the residence time of paraquat in the body. In addition, we validated the potential detoxification effect of sodium salicylate as a potential antidote for paraquat from both the dimensions of distribution and quantification. In conclusion, MALDI-MSI conveniently provided the distinct and quantitative spatial-temporal distribution information on paraquat in the whole body of zebrafish; it will promote the understanding of its toxicokinetic characteristics and provide more valuable information for clinical treatment.

A spatially-controlled DNA triangular prism nanomachine for AND-gated intracellular imaging of ATP in acidic microenvironment.

A DNA triangular prism nanomachine (TPN)-based logic device for intracellular AND-gated imaging of adenosine triphosphate (ATP) has been constructed. By using i-motif sequences and ATP-binding aptamers as logic control units, the TPN logic device is qualified to respond to the acidic environment and ATP in cancer cell lysosomes. Once internalized into the lysosome, the specific acidic microenvironment in lysosome causes the i-motif sequence to fold into a tetramer, resulting in compression of DNA tri-prism. Subsequently, the split ATP aptamer located at the tip of the collapsed triangular prism binds stably to ATP, which results in the fluorescent dyes (Cy3 and Cy5) modified at the ends of the split aptamer being in close proximity to each other, allowing Förster Resonance Energy Transfer (FRET) to occur. The FRET signals are excited at a wavelength of 543 nm and can be collected within the emission range of 646-730 nm. This enables the precise imaging of ATP within a cell. We also dynamically operate AND logic gates in living cells by modulating intracellular pH and ATP levels with the help of external drugs. Owing to the AND logic unit on TPN it can simultaneously recognize two targets and give corresponding intelligent logic judgment via imaging signal output. The accuracy of molecular diagnosis of cancer can be improved thus eliminating the false positive signal of single target-based detection. Hence, this space-controlled TPN-based logical sensing platform greatly avoids sensitivity to extracellular targets during the cell entry process, providing a useful tool for high-precision imaging of the cancer cell's endogenous target ATP.

Ultrasensitive Uracil-DNA Glycosylase Activity Assay and Its Inhibitor Screening Based on Primer Remodeling Jointly via Repair Enzyme and Polymerase.

The development of isothermal nucleic acid amplification techniques has great significance for highly sensitive biosensing in modern biology and biomedicine. A facile and robust exponential rolling circle amplification (RCA) strategy is proposed based on primer-remodeling amplification jointly via a repair enzyme and polymerase, and uracil-DNA glycosylase (UDG) is selected as a model analyte. Two kinds of complexes, complex I and complex II, are preprepared by hybridizing a circular template (CT) with a uracil-containing hairpin probe and tetrahydrofuran abasic site mimic (AP site)-embedded fluorescence-quenched probe (AFP), respectively. The target UDG specifically binds to complex I, resulting in the generation of an AP site, followed by cleavage via endonuclease IV (Endo IV) and the successive trimming of unmatched 3' terminus via phi29 DNA polymerase, thus producing a useable primer-CT complex that actuates the primary RCA. Then, numerous complex II anneal with the first-generation RCA product (RP), generating a complex II-RP assembly containing AP sites within the DNA duplex. With the aid of Endo IV and phi29, AFP, as a pre-primer in complex II, is converted into a mature primer to initiate additional rounds of RCA. So, countless AFPs are cleaved, releasing remarkably strong fluorescent signals. The biosensor is demonstrated to enable rapid and accurate detection of the UDG activity with an improved detection limit as low as 4.7 × 10-5 U·mL-1. Moreover, this biosensor is successfully applied for UDG inhibitor screening and complicated biological samples analysis. Compared to the previous exponential RCA methods, our proposed strategy offers additional advantages, including excellent stability, optional design of CT, and simplified operating steps. Therefore, this proposed strategy may create a useful and practical platform for ultrasensitive detection of low levels of analytes in clinical diagnosis and fundamental biomedicine research.

DNAzyme-controlled plasmonic coupling for SERS-based determination of Salmonella typhimurium using hybridization chain reaction self-assembled G-quadruplex.

A facile and rapid SERS strategy for S. typhimurium detection based on hybridization chain reaction (HCR) self-assembled G-quadruplex DNAzyme (GQH DNAzyme)-controlled plasmonic coupling was developed. GQH DNAzyme is introduced as a biocatalyst to catalyze the oxidation of L-cysteines to cysteines (thiols to disulfides) to assist SERS signal transduction. This is the first time that the self-assembled split GQH DNAzyme-controlled plasmonic coupling is integrated with SERS sensing. The results reveal the proposed SERS strategy can quantify S. typhimurium with a wide linear range (5 to 105 cfu mL-1) and a low detection limit (4 cfu mL-1; n = 5, mean ± standard deviation) and RSD of 7%. The method exhibited preeminent detection performance in spiked samples with recoveries of 93.1-117%. The proposed strategy has great potential for being a versatile SERS platform for detecting a wide spectrum of analytes by replacing them with the corresponding recognition elements. Therefore, this study not only creates a practical platform for pathogenic bacteria identification and related food safety testing and environmental monitoring, but also provides a new paradigm for building SERS sensor. A facile and rapid SERS strategy for S. Typhimurium detection based on hybridization chain reaction (HCR) self-assembled G-quadruplex DNAzyme-controlled plasmonic coupling.

Accurate and Nonpurified Identification of Extracellular Vesicles Using Dual-Binding Recognition Mode.

Circulating extracellular vesicles (EVs) are promising biomarkers for the early diagnosis and prognosis of cancer in a non-invasive manner. However, the rapid and accurate identification of EVs in complex biological samples is technically challenging, which is attributed to the requirement of extensive sample purification and unsatisfactory detection accuracy due to the disturbance of interfering proteins. Herein, a simultaneous binding of double-positive EV membrane protein-based recognition mode (DRM) is proposed. By the combination of DRM-mediated toehold activation and G-quadruplex DNAZyme-catalyzed etching of Au@Ag nanorods (Au@Ag NRs), we have developed an accurate, non-purified, low-cost, and visual strategy for EV identification. The synchronous binding of double-positive proteins on EV membranes is validated by confocal laser scanning microscopy analysis. This approach exhibits excellent specificity and sensitivity toward EVs ranging from 1.0 × 105 to 1.0 × 109 particles/mL with a detection limit of 6.31 × 104 particles/mL. Moreover, we have successfully realized non-purified EV quantification in complex biological media. In addition, target-initiated catalyzed hairpin assembly (CHA) is integrated with G-quadruplex DNAZyme-catalyzed color variation of Au@Ag NRs; thus, low-background EV detection can be achieved by the naked eye. Furthermore, our strategy is easy to adapt to high-throughput formats by using an automatic microplate reader, which could be expected to meet the requirements for high-throughput detection of clinical samples. With its capacities of rapidness, portability, affordability, high throughput, non-purification, and visual detection, this strategy could provide a practical tool for accurate identification of EVs and early diagnosis of cancer.

A three-dimensional dynamic DNA walker-mediated branching hybridization chain reaction for the ultrasensitive fluorescence sensing of ampicillin.

In this study, a novel, rapid and ultrasensitive fluorescence strategy using the three-dimensional (3D) dynamic DNA walker (DW)-induced branched hybridization chain reaction (bHCR) has been proposed for the detection of ampicillin (AMP). The sensing system was composed of an Nt·Bbvcl-powered DNA walker blocked by an AMP aptamer, hairpin-shaped DNA track probe (TP) and four kinds of metastable hairpin probes as the substrates of bHCR, which triggered the formation of the split G-quadruplex as the signal molecule. Due to the reasonable design, the specific binding between AMP and its aptamer activated the DW, and the DW moved on the surface of the gold nanoparticles (AuNPs) with the help of Nt·Bbvcl to produce primer probes (PPs), which induced bHCR. The products of the bHCR gathered two split G-quadruplex sequences together to form one complete G-quadruplex. The formed G-quadruplex emitted a strong fluorescence signal in the presence of thioflavin-T (ThT) to achieve the purpose of detecting AMP. The sensitivity of this method was greatly improved by the use of the 3D DNA walker and bHCR. The split G-quadruplex enhanced the signal-to-noise ratio (SNR). Under the optimal experimental conditions, a good correlation was obtained between the fluorescence intensity of the sensing system and the concentration of AMP ranging from 5 pM to 500 nM with a limit of detection (LOD) of 3.68 pM. Simultaneously, the method has been applied to the detection of antibiotics in spiked milk samples with satisfactory results.

Target-swiped DNA lock for electrochemical sensing of miRNAs based on DNAzyme-assisted primer-generation amplification.

As an extremely important post-transcriptional regulator, miRNAs are involved in a variety of crucial biological processes, and the abnormal expressions of miRNAs are closely related to a variety of diseases. In this work, for the first time, we designed a nucleic acid lock nanostructure for specific detection of miRNA-21, which changes the self-structure to "active conformation" by binding the target, in order to generate triggers to initiate the subsequent reaction. Emphatically, this flexible nucleic acid lock is capable of self-cleaving without the assistance of external component, overcoming the disadvantages of the complex design and requiring protease assistance in traditional nanostructure. Moreover, the combination of DNAzyme and RCA technology not only greatly improves the efficiency of signal amplification but also enables primer generation to simultaneous cascade RCA amplification. Additionally, the electrochemical detection technology based on silver nanoclusters overcomes the shortcomings of traditional detection methods such as low sensitivity and complex operation. The detection limit achieved was 9.3 aM with a wide dynamic response ranging from 10 aM to 100 pM (at the DPV peak of - 0.5 V), which is comparable to most of the reported studies. Therefore, our work provided an ultra-sensitive way for the detection of miRNAs using nanostructures and revealed an effective means for disease theranostics and cancer diagnosis. In this work, for the first time, we designed a nucleic acid lock nanostructure based on its self-structural transformation for the specific detection of miRNA. And the combination of DNAzyme and cascade RCA reaction greatly improved the signal amplification efficiency.

Comparisons of urea or ammonium on growth and fermentative metabolism of Saccharomyces cerevisiae in ethanol fermentation.

This work was mainly about the understanding of how urea and ammonium affect growth, glucose consumption and ethanol production of S. cerevisiae, in particular regarding the basic physiology of cell. The basic physiology of cell included intracellular pH, ATP, NADH and enzyme activity. Results showed that fermentation time was reduced by 19% when using urea compared with ammonium. The maximal ethanol production rate using urea was 1.14 g/L/h, increasing 30% comparing with the medium prepared with ammonium. Moreover, urea could decrease the synthesis of glycerol from glucose by 26% comparing with ammonium. The by-product of acetic acid yields decreased from 40 mmol/mol of glucose (with urea) to 24 mmol/mol of glucose (with ammonium). At the end of ethanol fermentation, cell number and pH were greater with urea than ammonium. Comparing with urea, ammonium decreased the intracellular pH by 14% (from 7.1 to 6.1). Urease converting urea into ammonia resulted in a more than 50% lower of ATP when comparing with ammonium. The values of NADH/DCW were 0.21 mg/g and 0.14 mg/g respectively with urea and ammonium, suggesting a 33% lower NADH. The enzyme activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was 0.0225 and 0.0275 U/mg protein respectively with urea and ammonium, which was consistent with the yields of glycerol.

AKT/FOXO1 axis links cross-talking of endothelial cell and pericyte in TIE2-mutated venous malformations.

BACKGROUND: Venous malformations (VMs), most of which associated with activating mutations in the endothelial cells (ECs) tyrosine kinase receptor TIE2, are characterized by dilated and immature veins with scarce smooth muscle cells (SMCs) coverage. However, the underlying mechanism of interaction between ECs and SMCs responsible for VMs has not been fully understood. METHODS: Here, we screened 5 patients with TIE2-L914F mutation who were diagnosed with VMs by SNP sequencing, and we compared the expression of platelet-derived growth factor beta (PDGFB) and α-SMA in TIE2 mutant veins and normal veins by immunohistochemistry. In vitro, we generated TIE2-L914F-expressing human umbilical vein endothelial cells (HUVECs) and performed BrdU, CCK-8, transwell and tube formation experiments on none-transfected and transfected ECs. Then we investigated the effects of rapamycin (RAPA) on cellular characteristics. Next we established a co-culture system and investigated the role of AKT/FOXO1/PDGFB in regulating cross-talking of mutant ECs and SMCs. RESULTS: VMs with TIE2-L914F mutation showed lower expression of PDGFB and α-SMA than normal veins. TIE2 mutant ECs revealed enhanced cell viability and motility, and decreased tube formation, whereas these phenotypes could be reversed by rapamycin. Mechanically, RAPA ameliorated the physiological function of mutant ECs by inhibiting AKT-mTOR pathway, but also facilitated the nuclear location of FOXO1 and the expression of PDGFB in mutant ECs, and then improved paracrine interactions between ECs and SMCs. Moreover, TIE2 mutant ECs strongly accelerated the transition of SMCs from contractile phenotype to synthetic phenotype, whereas RAPA could prevent the phenotype transition of SMCs. CONCLUSIONS: Our data demonstrate a previously unknown mechanistic linkage of AKT-mTOR/FOXO1 pathway between mutant ECs and SMCs in modulating venous dysmorphogenesis, and AKT/FOXO1 axis might be a potential therapeutic target for the recovery of TIE2-mutation causing VMs. Video Abstract.

Target-activated DNA nanomachines for the ATP detection based on the SERS of plasmonic coupling from gold nanoparticle aggregation.

The self-assembly of plasmonic nanoparticles provides a powerful approach to generate surface-enhanced Raman scattering (SERS), which promotes the actual applications in chemical and biomolecular analyses. Herein, we developed a facile SERS sensing strategy for an ATP assay with a 3-D DNA nanomachine that walks by the Exo III cleavage, leading to the formation of AuNP aggregates, which resulted in the enhancement of the electromagnetic field. Depending on the target-activated Exo III cleavage, the 3-D nanomachine can walk along the 3-D track on the surface of AuNPs and generate self-assembled hot-spots to enhance the SERS signal of a Raman dye, allowing a homogenous assay of the ATP concentration with high sensitivity and reproducibility. Under optimized experimental conditions, the biosensor detected ATP with a widened dynamic range from 1 pM to 1 × 105 pM with a limit of detection of up to 0.29 pM. Hence, the novel strategy provides a useful and practical platform for the SERS assay of ATP with high sensitivity and repeatability. Besides, this platform shows great potential for applications in high-throughput assays for drug screening and clinical diagnostics.

Efficient strand displacement amplification via stepwise movement of a bipedal DNA walker on an electrode surface for ultrasensitive detection of antibiotics.

DNA walkers, one of the artificial molecular machines which are constructed via smart synthetic DNA, have attracted rapidly growing attention from researchers in the biosensing field. In this work, we design an Exonuclease III (Exo III)-aided target-aptamer binding recycling (ETBR) activated bipedal DNA machine for highly sensitive electrochemical detection of antibiotics. To the best of our knowledge, this is the first time that a bipedal DNA machine has been applied in electrochemical sensing for antibiotics. On the one hand, the bipedal DNA walker exceeds the conventional single swing arm DNA walker in terms of walking efficiency and stability. On the other hand, the ETBR strategy, along with efficient strand displacement amplification via stepwise movement of a bipedal DNA walker significantly promotes the signal amplification efficiency. Under optimal conditions, this bipedal DNA machine possesses a detection limit of 7.1 fM within a linear detection range from 10 fM to 100 pM. Moreover, this electrochemical biosensor is expected to detect a wide variety of analytes using the corresponding target recognition probes. Thus, our proposed strategy offers a highly efficient, stable and practical platform for small molecule analysis.

Highly efficient fluorescence sensing of kanamycin using Endo IV-powered DNA walker and hybridization chain reaction amplification.

An ultrasensitive fluorescence sensing strategy for kanamycin (KANA) determination using endonuclease IV (Endo IV)-powered DNA walker, and hybridization chain reaction (HCR) amplification was reported. The sensing system consists of Endo IV-powered 3D DNA walker using for the specific recognition of KANA and the formation of the initiators, two metastable hairpin probes as the substrates of HCR and a tetrahydrofuran abasic site (AP site)-embeded fluorescence-quenched probe for fluorescence signal output. On account of this skilled design of sensing system, the specific binding between KANA and its aptamer activates DNA walker, in which the swing arm can move autonomously along the 3D track via Endo IV-mediated hydrolysis of the anchorages, inducing the formation of initiators that initiates HCR and the following Endo IV-assisted cyclic cleavage of fluorescence reporter probes. The use of Endo IV offers the advantages of simplified and accessible design without the need of specific sequence in DNA substrates. Under the optimal experimental conditions, the fluorescence biosensor shows excellent sensitivity toward KANA detection with a detection limit as low as 1.01 pM (the excitation wavelength is 486 nm). The practical applicability of this strategy is demonstrated by detecting KANA in spiked milk samples with recovery in the range of 98 to 102%. Therefore, this reported strategy might create an accurate and robust fluorescence sensing platform for trace amounts of antibiotic residues determination and related safety analysis. Graphical abstract Highly efficient fluorescence sensing of kanamycin using Endo IV-powered DNA Walker and hybridization chain, reaction amplification, Xiaonan Qu, Jingfeng Wang, Rufeng Zhang, Yihan Zhao, Shasha Li, Yu Wang, Su Liu*, Jiadong Huang, and Jinghua Yu, an ultrasensitive fluorescence sensing strategy for kanamycin determination using endonuclease IV-powered DNA walker, and hybridization chain reaction amplification is reported.

Triple-helix molecular-switch-actuated exponential rolling circular amplification for ultrasensitive fluorescence detection of miRNAs.

We have formulated a rapid and high-efficiency fluorescent biosensing platform based on a target-activated triple-helix molecular switch (THMS)-conformation-change-induced exponential rolling circular amplification (RCA) strategy for the ultrasensitive detection of miR-21. In this strategy, there are several aspects that are worthwhile. First, the functionalized THMS, comprising a typical triplex structure (T-A·T), specific recognition sequence for nicking endonuclease, complementary sequence for miR-21, and RCA product-annealing sequence, was concurrently used to perform signal transduction with one fluorophore and one quencher. As compared to the traditional double-helix molecular switches or molecular beacons, one of the biggest differentiating factors is that the properties of THMSs are independent of any specific binding sequence that they may contain. As far as we know, this is the first time that an ingeniously designed THMS not only contains the primer for exponential RCA, but also functions as the tracer for fluorescence assay. In the presence of miR-21, targets can induce conformation changes in THMS with the release of the trapped DNA segment (P), which, in turn, can activate the first run of the RCA process. Meanwhile, the RCA reaction is also initiated by the formation of a similar primer (SP) as the trapped DNA through a continuous "extension-nicking" reaction. Secondly, the resultant first-generation RCA product consists of numerous tandem repeated regions that can attach to countless THMSs, resulting in the release of the trapped DNA segment (P) for initiating the second run of the RCA reaction. Significantly, a large amount of THMSs were continuously consumed to yield a remarkably strong fluorescent signal. In addition, this biosensor was demonstrated to exhibit improved sensitivity owing to the high efficiency and rapid amplification kinetics of the exponential RCA and high selectivity toward miR-21 with a limit of detection as low as 1.1 aM. Hence, the target-mediated THMS-conformation-change-initiated exponential RCA strategy presents an optimal detection performance toward analytes for potential applications in related fundamental research and clinical diagnosis.

Robust and highly specific fluorescence sensing of Salmonella typhimurium based on dual-functional phi29 DNA polymerase-mediated isothermal circular strand displacement polymerization.

A simple and robust fluorescence sensing strategy has been developed for the detection of pathogenic bacteria by the combination of the dual functionality of phi29 DNA polymerase with isothermal circular strand displacement polymerization (ICSDP). The strategy relies on target-triggered formation of a mature primer that initiates the cyclic strand displacement polymerization reaction with the aid of dual functional phi29; thus, amplified detection of the target can be achieved. To our knowledge, this work is the first report where dual functional phi29-assisted ICSDP has been employed for fluorescence sensing of pathogenic bacteria. It is worth noting that a hairpin pre-primer is introduced that can be trimmed into a mature primer for initiating ICSDP via the 3' → 5' proofreading exonuclease activity of phi29, which contributes to the ultrahigh specificity of the strategy owing to the elimination of the unwished nonspecific extension. On the basis of the present amplification strategy, our biosensor exhibits excellent specificity and sensitivity toward S. typhimurium with an excellent detection limit as low as 1.5 cfu mL-1. In addition, the strategy offers the advantages of a simplified operation, shortened analysis time, and highly sensitive detection of pathogens with only a one-step reaction. Furthermore, by redesigning the corresponding binding molecules, the proposed strategy can be easily extended for the detection of a wide spectrum of analytes. Hence, the dual functional phi29-assisted ICSDP strategy indeed creates a robust and convenient fluorescence sensing platform for the identification of pathogenic bacteria and related food safety analysis.

A facile signal-on electrochemical DNA sensing platform for ultrasensitive detection of pathogenic bacteria based on Exo III-assisted autonomous multiple-cycle amplification.

A facile signal-on electrochemical DNA biosensor has been developed for ultrasensitive detection of pathogenic bacteria using an Exo III-assisted autonomous multiple-cycle amplification strategy. The strategy relies on pathogens and aptamer binding-initiated release of a trigger, which combines with the 3'-protruding terminus of the hairpin probe 1, leading to the formation of double-stranded DNA with a blunt 3' terminus which starts the Exo III-assisted multiple signal amplification reaction. In addition, hairpin probe 2 labeled with an electroactive reporter at the middle of the loop region is ingeniously designed to contain a short hairpin-embedded segment, which can fold into a hairpin structure via an Exo III-assisted cleavage reaction, thus bringing the redox molecule in proximity to the electrode surface for "signal-on" sensing. Under optimal conditions, this biosensor exhibits a very low detection limit as low as 8 cfu mL-1 and a wide linear range from 1.0 × 101 to 1.0 × 107 cfu mL-1 of target pathogenic bacteria. As far as we know, this is the first time that the Exo III-assisted autonomous multiple-cycle amplification strategy has been used for signal-on electrochemical sensing of pathogenic bacteria. In addition, the proposed sensor can also be used for highly sensitive detection of other targets by changing the aptamer sequence, such as nucleic acids, proteins and small molecules. Therefore, the proposed signal-on electrochemical sensing strategy might provide a simple and practical new platform for detection of pathogenic bacteria and related biological analysis, food safety inspection and environmental monitoring.

DNA three-way junction-actuated strand displacement for miRNA detection using a fluorescence light-up Ag nanocluster probe.

A rapid and label-free fluorescence biosensing strategy for highly sensitive detection of microRNA-122 (miR-122) has been developed by the combination of DNA three-way junction (TWJ)-actuated strand displacement and a fluorescence light-up Ag nanocluster (AgNC) probe. In the presence of target miR-122, the attachment of miR-122 to its complementary DNA results in the unblocking of the toehold and branch migration domains in the TWJ, activating the strand displacement reaction (SDR) accompanied by the proximity between the G-rich DNA probe and DNA-AgNC probe; thus a remarkably enhanced fluorescence signal of AgNCs can be obtained owing to the G-rich fluorescence enhancement mechanism. The results reveal that this biosensor exhibits superb specificity and high sensitivity toward miR-122 with a detection limit of 0.030 nM. In addition, the practicality of the biosensor is demonstrated by analyzing miR-122 in three cell lines with satisfactory results. Furthermore, by the utilization of the toehold-mediated SDR and DNA-AgNC conjugates, this proposed strategy offers the advantages of rapidness, convenience, low cost, and simplified operation without the need for biological labeling and the addition of enzymes. Thus, the constructed biosensor might provide a valuable and practical tool for detecting miRNA and the related clinical diagnosis and fundamental biomedicine research.

Primer remodeling amplification-activated multisite-catalytic hairpin assembly enabling the concurrent formation of Y-shaped DNA nanotorches for the fluorescence assay of ochratoxin A.

DNA can be configured into unique high-order structures due to its significantly high programmability, such as a three-way junction-based structure (denoted Y-shaped DNA), for further applications. Herein, we report a label-free fluorescent signal-on biosensor based on the target-driven primer remodeling rolling circle amplification (RCA)-activated multisite-catalytic hairpin assembly (CHA) enabling the concurrent formation of Y-shaped DNA nanotorches (Y-DNTs) for ultrasensitive detection of ochratoxin A (OTA). Two kinds of masterfully-designed probes, termed Complex I and II, were pre-prepared by the combination of a circular template (CT) with an OTA aptamer (S1), a substrate probe (S2) and hairpin probe 1 (HP1), respectively. Target OTA specifically binds to Complex I, resulting in the release of the remnant element in S2 and successive remodeling into a mature primer for RCA by phi29 DNA polymerase, thus a usable primer-CT complex is produced, which actuates primary RCA. Then, numerous Complex II probes can anneal with the first-generation RCA product (RP) with multiple sites to activate the CHA process. With the participation of endonuclease IV (Endo IV) and phi29, HP1 as a pre-primer containing a tetrahydrofuran abasic site mimic (AP site) in Complex II is converted into a mature primer to initiate additional rounds of RCA. So, countless Y-DNTs are formed concurrently containing a G-quadruplex structure that enables the N-methylmesoporphyrin IX (NMM) to be embedded, generating remarkably strong fluorescence signals. The biosensor was demonstrated to enable rapid and accurate highly efficient and selective detection of OTA with an improved detection limit of as low as 0.0002 ng mL-1 and a widened dynamic range of over 4 orders of magnitude. Meanwhile, this method was proven to be capable of being used to analyze actual samples. Therefore, this proposed strategy may be established as a useful and practical platform for the ultrasensitive detection of mycotoxins in food safety testing.

A label-free electrochemical platform for the detection of antibiotics based on cascade enzymatic amplification coupled with a split G-quadruplex DNAzyme.

Herein, a split G-quadruplex DNAzyme as a signal reporter was integrated into an electrochemical sensing platform for the detection of antibiotics with specificity and sensitivity. To improve the signal-to-noise ratio, two G-rich oligonucleotide sequences (G1 and G2) were blocked into two different hairpin probes, preventing the two segments from assembling into a spilt G-quadruplex structure. Moreover, we designed a double-arch probe, consisting of an aptamer as the recognition element and two-step enzymatic signal amplification. Concretely, the first is the Nt.BbvCI-assisted nicking cyclic reaction activated by target-aptamer binding, and the second is exonuclease III-aided cyclic amplification for generating abundant G1 and G2. The modified capture probe on the electrode was used to combine G1 and G2 to form the spilt G-quadruplex/hemin when K+ and hemin were present. This complex plays the role of DNAzyme with superior horseradish peroxidase activity in catalyzing the decomposition of H2O2. Under optimal conditions, this biosensor showed an excellent performance for sensing kanamycin with a detection limit of 83 fM for kanamycin concentrations ranging from 100 fM to 1 nM. Hence, the proposed strategy has potential as an efficient and actual platform for small molecule analysis.

Colorimetric and visual mercury(II) assay based on target-induced cyclic enzymatic amplification, thymine-Hg(II)-thymine interaction, and aggregation of gold nanoparticles.

A colorimetric biosensor and visual test is described for the determination of mercury(II). It relies on the specific thymine-Hg(II)-thymine (T-Hg2+-T) interaction which induces a cyclic amplification process (caused by the enzyme exonuclease III) and the aggregation of gold nanoparticles. These results in a color change from red to violet. Under optimized conditions, this colorimetric assay (best performed at 524 nm) has a detection limit as low as 0.9 nM with a detection range over 4 orders of magnitude (from 1 nM to 10 µM). Graphical abstract Schematic of a colorimetric method for determination of mercury ions (Hg2+) based on the thymine-Hg2+-thymine interaction-triggered cyclic enzymatic amplification and aggregation of gold nanoparticles with the aid of exonuclease III (Exo III).

A triply amplified electrochemical lead(II) sensor by using a DNAzyme and via formation of a DNA-gold nanoparticle network induced by a catalytic hairpin assembly.

An amplified electrochemical biosensing scheme is described for lead(II) ions. It is making use of DNAzyme-assisted target recycling and catalytic hairpin assembly (CHA). The hairpin strand (substrate probe for the Pb2+-based DNAzyme; referred to as SP) is composed of trigger probe (TP) and a capture probe 1 attached to gold nanoparticles (AuNP). In the presence of the enzyme probe that partially hybridizes with SP, the introduction of Pb2+ triggers target recycling and drives the highly amplified translation of target Pb(II) to TP. The CHA reaction is further initiated by TP. The modified AuNP act as the connecting unit, and this leads to the formation of a 3D DNA-AuNP network on the electrode (which is the third amplification step). It can bind the positively charged redox mediator RuHex via electrostatic interaction for electrochemical detection. This biosensor has a low detection limit (95 pM) and any analytical range that covers the 100 pM to 5 µM Pb(II) concentration range. It is selective over other divalent metal ions. It was applied to the determination of Pb2+ in spiked real-world samples. Graphical abstract Schematic presentation of the electrochemical biosensor. The triply amplified electrochemical assay is based on the use of DNAzyme-assisted target recycling with catalytic hairpin assembly (CHA) reaction for sensitive and selective determination of lead ion (Pb2+). AuNP: gold nanoparticles; SP: substrate probe; EP: enzyme probe.