Lanthanide coordination polymers@CuO nanoparticles: Enhanced self-cascade nanoenzyme activity and ratiometric fluorescence assay of glutathione.

Tandem enzyme can catalyze some cascade reactions with high efficiency, and some few tandem enzyme-like mimics have been discovered recently. Further improving the catalytic efficiency of tandem nanoenzymes with facile method may undoubtedly promote and broaden their applications in various fields. In this work, cupric oxide nanoparticles (CuO NPs) with dual-functional enzyme mimics were synthesized using the rapid deposition method in advance, which simultaneously combined with lanthanide infinite coordination polymers (Ln ICPs) during the self-assemble of Tb3+, guanine-5'-triphosphate (GTP) and auxiliary ligand terephthalic acid (TA). Excitingly, the obtained Tb-GTP/TA@CuO ICPs, not only displayed obviously enhanced tandem catalytic activity compared with pure CuO NPs, but also provided a versatile ratiometric platform for ultrahigh selective and sensitive detection of glutathione (GSH) under single-wavelength excitation. A good linear relationship between the ratio signal and the GSH concentration was spanning from 0.001 to 20 µM with an impressive detection limit of 0.50 nM. This study opens a new and universal avenue for preparing integrated multifunctional probes by coupling of nanoenzyme catalytic activity with superior luminescent Ln ICPs through facile method.

Developing Isomeric Peptides for Mimicking the Sequence-Activity Landscapes of Enzyme Evolution.

Enzymes catalyze almost all material conversion processes within living organisms, yet their natural evolution remains unobserved. Short peptides, derived from proteins and featuring active sites, have emerged as promising building blocks for constructing bioactive supramolecular materials that mimic native proteins through self-assembly. Herein, we employ histidine-containing isomeric tetrapeptides KHFF, HKFF, KFHF, HFKF, FKHF, and FHKF to craft supramolecular self-assemblies, aiming to explore the sequence-activity landscapes of enzyme evolution. Our investigations reveal the profound impact of peptide sequence variations on both assembly behavior and catalytic activity as hydrolytic simulation enzymes. During self-assembly, a delicate balance of multiple intermolecular interactions, particularly hydrogen bonding and aromatic-aromatic interactions, influences nanostructure formation, yielding various morphologies (e.g., nanofibers, nanospheres, and nanodiscs). Furthermore, the analysis of the structure-activity relationship demonstrates a strong correlation between the distribution of the His active site on the nanostructures and the formation of the catalytic microenvironment. This investigation of the sequence-structure-activity paradigm reflects how natural enzymes enhance catalytic activity by adjusting the primary structure during evolution, promoting fundamental research related to enzyme evolutionary processes.

Cascade signal amplification strategy for the electrochemical aptasensing of nucleic acid: Combination of dual-output toehold-mediated DNA strand displacement, DNA walker and Exo III.

BACKGROUND: Sensitive and selective analysis of low content nucleic acid sequences plays an important role in pathogen analysis, disease diagnosis and biomedicine. The electrochemical biosensor based on toehold-mediated strand displacement reaction (TMSD) is highly attractive in nucleic acid detection due to their improved sensitivity and rapid response. But the traditional TMSD carried out on the electrode always with low displacement efficiency and complicated electrode operation, resulting in compromised sensing performance. There is a great need to construct a novel TMSD based electrochemical detection strategy to overcome such challenges in nucleic acid detecting. RESULT: Herein, a triple signal amplification electrochemical aptasensor was developed for ultrasensitive detection of CYFRA21-1 DNA. The dual-output toehold mediated strand displacement reaction (dTMSD) can convert one input to two strands output within one strand displacement cycle. So that it possesses a higher efficiency for improving the sensitivity in comparison with the single-output TMSD. And the fuel strand was configured with a tail to realize successive DNA circuits through self-propelling as a DNA walker. All the above processes were carried out on magnetic beads, which is conducive to achieving effective sample purification and minimizing the background signals. Besides, Exonuclease III was further amplified signal. As a result, through the cascade use of above three technologies, the proposed biosensing strategy realized sensitive detection of target DNA with a low detection limit of 0.35 fM (S/N = 3) and wide linear range (0.5 fM-500 pM). SIGNIFICANCE: The proposed novel dTMSD combining multiple signal amplification strategies for electrochemical detection of CYFRA21-1 DNA with easy operation not only possesses excellent sensitivity and selectivity, but also has potential application value for monitoring DNA in serum. Meanwhile, the development of highly sensitive and specific CYFRA21-1 DNA detection methods is very important for the prevention and treatment of lung cancer.

Survivin as a potential biomarker in the diagnosis of bladder cancer: A systematic review and meta-analysis.

Early detection, diagnosis, and treatment take on critical significance in preventing and treating bladder cancer. As indicated by numerous studies, survivin can serve as a biomarker of bladder cancer, whereas the results of a wide variety of studies have been controversial. This paper is to assess the accuracy of survivin in the diagnosis of bladder cancer by a meta-analysis. The studies regarding the diagnosis of bladder cancer using survivin were systematically retrieved from the CNKI, WanFang, CBM, VIP, Web of science, cochrane library and pubmed were extracted, and the literature quality was assessed. Meta-analysis was conducted using STATA 16.0 MP. 2,082 relevant studies were searched, and 40 studies were finally covered for meta-analysis. The pooled specificity and pooled sensitivity of survivin mRNA was 0.95 (95%CI: 0.91, 0.97) and 0.94 (95%CI: 0.88, 0.97). The pooled specificity and pooled sensitivity of survivin protein reached 0.95 (95%CI: 0.90, 0.97) and 0.87 (95%CI: 0.78, 0.92). The pooled positive likelihood ratio, pooled negative likelihood ratio, the area under the curve, and diagnostic odds ratio for survivin mRNA reached 17.7 (95%CI: 10.3, 30.6), 0.07 (95%CI: 0.04, 0.12), 0.98 (95%CI: 0.97, 0.99) and 266 (95%CI: 114, 621), respectively. For survivin protein was 16.4 (95%CI: 7.9, 33.9), 0.14 (95%CI: 0.08, 0.24), 0.97 (95%CI: 0.95, 0.98) and 117 (95%CI: 38, 357), respectively. Survivin takes on great significance in diagnosing bladder cancer. However, due to some limitations in the number and quality of covered studies, this conclusion should be validated through additional higher quality clinical studies.

Influence of Nitrogen Supply on Growth, Antioxidant Capacity and Cadmium Absorption of Kenaf (Hibiscus cannabinus L.) Seedlings.

Kenaf (Hibiscus cannabinus L.) is considered suitable for the remediation of cadmium (Cd)-contaminated farmlands, because of its large biomass and resistance to Cd stress. The addition of nitrogen (N) fertilizer is an important measure used to increase crop yields, and it may also affect Cd accumulation in plants. To clarify the effects of different forms and concentrations of N on plant growth and Cd absorption in kenaf, a hydroponic experiment was conducted using three N forms (NH4+-N, NO3--N and urea-N) at four concentrations (0, 2, 4 and 8 mM, 0 mM as control) under Cd stress (30 µM). The plant growth, the antioxidant enzyme activity and the Cd contents of various parts of the kenaf seedlings were measured. The results showed that the N form had the greatest impact on the growth of the kenaf and the absorption and transport of the Cd, followed by the interaction effect between the N type and the concentration. Compared to the control, the addition of N fertilizer promoted the growth of kenaf to varying degrees. Among all the treatments, the use of 2 mM of NO3--N enhanced the biomass and Cd accumulation to the greatest extent compared to CK from 2.02 g to 4.35 g and 341.30 µg to 809.22 µg per plant, respectively. The NH4+-N significantly reduced the Cd contents of different parts but enhanced the translocation factors of Cd stem to root (TF S/R) and leaf to stem (TF L/S) by 34.29~78.57% and 45.10~72.55%, respectively. The peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT) enzyme activities of the kenaf increased with the N treatments, especially with NH4+-N. Overall, applying low concentrations of NO3--N can better promote the extraction of Cd by kenaf.

Noncoding RNAs as a potential biomarker for the prognosis of bladder cancer: a systematic review and meta-analysis.

OBJECTIVE: The relationship between noncoding RNAs and the prognosis of bladder cancer (BC) is still controversial. The purpose of this study is to evaluate the relationship between noncoding RNAs and prognosis by meta-analysis. METHODS: Comprehensive retrieval of PubMed, Embase, the Cochrane Library, the Web of Science, CNKI, and WanFang databases is related to the correlation between noncoding RNAs and the prognosis of BC. Data were extracted, and the literature quality was evaluated. STATA16.0 served for the meta-analysis. RESULTS: 1. CircRNAs: High circ-ZFR expression led to poor overall survival (OS) of BC. 2. LncRNAs: Low lnc-GAS5 expression predicted poor OS of BC, high lnc-TUG1 expression predicted poor OS of BC. 3. MiRNAs: High miR-21 expression predicted poor OS of BC, high miR-222 expression led to poor OS of BC, high miR-155 expression predicted poor progression-free survival (PFS) of BC, high miR-143 expression caused poor PFS of BC, low miR-214 expression could result in poor recurrence-free survival (RFS) of BC. CONCLUSIONS: High circ-ZFR, lnc-TUG1, miR-222, and miR-21 expressions were correlated with poor OS of BC; high miR-155 and miR-143 expression predicted poor PFS of BC; low lnc-GAS5 expression predicted poor OS of BC; low miR-214 expression predicted poor RFS of BC.

Iron-based passivator mitigates the coupling process of anaerobic methane oxidation and arsenate reduction in paddy soils.

Arsenic (As) is a toxic metalloid that is ubiquitous in paddy soils, where passivation is the most widely used method for remediating As contamination. Recently, anaerobic methane oxidation coupled with arsenate (As(V)) reduction (AOM-AsR) has been shown to act as a critical driver for As release in paddy fields. However, the effect and mechanism of the passivators on the AOM-AsR process remain unclear. In this study, we incubated arsenate-contaminated paddy soils under anaerobic conditions. Using isotopically labelled methane and different passivators, we found that an iron-based passivator containing calcium sulfate and iron oxide (9:1, m/m) named IBP showed a much better performance than the other passivators. Adding IBP decreased the arsenite (As(III)) concentration in the soil solution by 78% and increased the AOM rate by 55%. Furthermore, we employed high-throughput sequencing and real-time quantitative polymerase chain reaction (qPCR) to investigate the ability of IBP to control As release mediated by AOM-AsR in paddy fields, as well as its underlying mechanism. Our results showed that IBP addition significantly increased anaerobic methanotrophic (ANME) archaea (ANME-2a-c, ANME-2d, and ANME-3) by 91%, and increased the methane-oxidizing bacterium Methylobacter by 262%. Similarly, IBP addition significantly increased the Fe(III) concentration in soil solution by 39% and increased the absolute abundance of Fe(III)-reducing bacteria (Geobacteraceae) by 21 times in soil. Adding IBP may significantly promote AOM coupled with Fe(III) reduction, significantly reducing electron transfer from AOM to As(V) reduction. Hence, IBP may be used as an efficient passivator to remediate As-contaminated soil using an active AOM-AsR process. These results provide a novel insight into controlling soil As release by regulating an active and critical As mobilization pathway in the environment.

An ingenious electrochemical aptasensor for detection of CYFRA 21-1 based on dual-output toehold mediated strand displacement reaction.

Herein, a "signal-on" electrochemical aptasensor based on dual-output toehold mediated strand displacement reaction (TMSDR) was developed to detect cytokeratin fragment antigen 21-1 (CYFRA 21-1). First, the methylene blue (MB) labeled signal probe (SP) was combined with a long template strand (TS) to form the three-strands duplex, which was immobilized on magnetic beads. The sequence of TS was innovatively designed with two toehold zones. Thus, with the presence of target DNA and Fuel strand (F), double SP and target were released through TMSDR. Besides, the released target could participate in the next cycle, achieving the further amplification. The released SP was selected by magnetic separation, then added to the gold electrode (GE) to hybridize with the hairpin DNA. Ultimately, the target can be detected by recording the current response of the MB. Under optimized conditions, the response peak current was linear with the concentration of CYFRA 21-1 in range from 10 pM to 1 µM with detection limit of 8.079 pM. The proposed signal amplification strategy was enzyme-free in homogenous solution and the using of magnetic beads improved the recognition efficiency and simplified the experimental steps, which endowed the sensor with super-sensitivity.

Dual function metal-organic frameworks based ratiometric electrochemical sensor for detection of doxorubicin.

Cancer is one of the main diseases threatening human health in the world. Doxorubicin (DOX) is a common anti-cancer drug that can be used for chemotherapy to extend a cancer patient's life. It is our common wish that treatment process of cancer is efficient and secure. Therefore, it is of great significance to develop sensitive, rapid and accurate techniques for anti-cancer drug doxorubicin (DOX) detection. Herein, in our work, a ratiometric electrochemical sensor for DOX detection was designed, which based on MB@MWCNTs/UiO-66-NH2 composites. The porous materials UiO-66-NH2 magically shoulder double function in our ratiometric electrochemical strategy, which can reduce the interior error caused by the various complex materials. Specifically, on the one hand, it can be used to catalyze DOX, which can provide a great current signal to be detected, on the other hand, its special property of absorbing molecules was utilized to load materials as internal reference. Consequently, we chose methylene blue (MB) as the substance that can generate an internal reference signal, because it is a specific and stable electroactive substance. Then, we added MWCNTs as a part of the material modified on the ratiometric electrochemical platform to enhance the signal of the target due to its feature of good electrical conductivity. Under the optimized conditions, the ratiometric electrochemical sensor displayed a wide linear detection with the range from 0.1 µM to 75 µM and the limit of detection (LOD) of 0.051 µM. The applicability of this method in the analysis of actual human saliva samples has been confirmed by the results of selectivity, stability, and reproducibility tests, which was prospective in clinical application.

An ultrasensitive carcinoembryonic antigen electrochemical aptasensor based on 3D DNA nanoprobe and Exo III.

In this study, a highly ordered three dimensional (3D) DNA nanostructure was self-assembled by label-free DNA nanotweezers, which was used as recognized probe to interact with target. Once the target was recognized by the 3D DNA nanoprobe (3D DNT), DNA nanotweezers opened to release target analog (T1). This recognition process was proceeded in homogeneous solution, which can avoid complex electrode modification and improve reaction efficiency. Then these target analogs were captured by the signal DNA probes (E1) modified on the electrode. In the assistance of Exo III, E1 was digested and the T1 was released to participate in the next cycle to realize signal amplification. Finally, an ultrasensitive carcinoembryonic antigen (CEA) electrochemical biosensing with a detection limit of 4.88 fg mL-1 was developed.

Ratiometric fluorescence detection of ciprofloxacin using the terbium-based coordination polymers.

Herein, a facile self-assembly through adenosine monophosphate (AMP) and luminol with Tb3+ was employed to construct a dual-ligand coordinated AMP-Tb-luminol coordination polymers (CPs), which emitted the typical fluorescence of luminol. Based on the sensitization effect of ciprofloxacin (CIP) on the luminescence of Tb3+, a ratiometric sensor was fabricated using the fluorescence of luminol as an inert reference. The fluorescent intensity ratios of Tb3+ to that of luminol enhanced linearly with the CIP concentration in the range from 5 nM to 2.5 µM with a lower limit of detection of 2 nM. In addition, the proposed ratiometric fluorescent sensor exhibited high selectivity for CIP, which could also be used to detect CIP in human blood serum (HBS) with satisfactory results. To our knowledge, this is the first demonstration of using dual-ligand coordination lanthanide (Ln)-based CPs for ratio-metric CIP assay, and this straightforward strategy may open up a new platform for designing the ratio-metric sensors based on the Ln CPs.

A Cooperatively Activatable DNA Nanoprobe for Cancer Cell-Selective Imaging of ATP.

DNA-based nanoprobes have attracted extensive interest in the field of bioanalysis. Notably, engineered DNA nanoprobes that can respond to multiple pathological parameters are desirable to detect targets precisely. Here we design a split aptamer/DNAzyme (aptazyme)-based DNA probe for fluorescence detection of ATP and further develop a cooperatively activatable DNA nanoprobe for tumor-specific imaging of ATP in vivo. The DNA nanoprobes comprising split aptazyme-coated MnO2 nanovectors have high stability and are synergistically activated by multiple biomarkers, GSH and ATP. Upon stimuli by overexpressed GSH in tumor cells, this DNA nanoprobe can release the aptazyme and self-supply cofactor Mn2+ of the DNAzyme. Sequentially, intracellular ATP induces the proper folding of the split ATP aptamer and Mn2+-dependent DNAzyme, which activates the specific cleavage of substrate and generates the optical readout signal. This nanoprobe exhibits remarkable resistance to enzymatic degradation, satisfactory biosafety, identifies ATP specifically within cancer cells, and selectively lights up solid tumors. Our research provides a reliable method for ATP imaging in cancer cells and opens a new avenue for biochemical research and highly accurate disease diagnosis.

Novel preparation method of bipedal DNA walker based on hybridization chain reaction for ultrasensitive DNA biosensing.

In this work, a novel strategy for preparation of bipedal DNA walker (BDW) based on hybridization chain reaction (HCR) with the assistance of Exonuclease III (Exo III) was proposed. Based on this strategy, an electrochemical biosensor was constructed to achieve sensitive detection of CYFRA 21-1 DNA. Firstly, target recognition and circulation were achieved through a one-step catalytic hairpin assembly (CHA) reaction. For further amplification, hybridization chain reaction (HCR) was employed to form duplex-stranded DNA (dsDNA) nanostructure in homogeneous solution. In particular, the elongated single strand of the hairpin DNA for HCR was designed as the Mg2+ DNAzyme sequence. With the assistance of Exo III, dsDNA nanostructure can be digested and transformed into large amounts of BDW. These BDW can cleave the signal probe driven by Mg2+, which was modified on the electrode surface and thus achieved "signal-off" detection of target. This BDW preparation method based on HCR with the digestion of Exo III converted one target input into large amount of BDW. Coupled with the walking cleavage of BDW, a series of cascade amplification endowed high sensitivity with this biosensor and realized ultrasensitive detection of target DNA with the detection limit as low as 3.01 aM.

A Deoxyribozyme-Initiated Self-Catalytic DNA Machine for Amplified Live-Cell Imaging of MicroRNA.

Functional DNA nanostructures have been widely used in various bioassay fields. Yet, the programmable assembly of functional DNA nanostructures in living cells still represents a challenging goal for guaranteeing the sensitive and specific biosensing utility. In this work, we report a self-catalytic DNA assembly (SDA) machine by using a feedback deoxyribozyme (DNAzyme)-amplified branched DNA assembly. This SDA system consists of catalytic self-assembly (CSA) and DNAzyme amplification modules for recognizing and amplifying the target analyte. The analyte initiates the CSA reaction, leading to the formation of Y-shaped DNA that carries two RNA-cleaving DNAzymes. One DNAzyme can then successively cleave the corresponding substrate and generate numerous additional inputs to activate new CSA reactions, thus realizing a self-catalytic amplification reaction. Simultaneously, the other DNAzyme is assembled as a versatile signal transducer for cleaving the fluorophore/quencher-modified substrate, leading to the generation of an amplified fluorescence readout. By incorporating a flexible auxiliary sensing module, the SDA system can be converted into a universal sensing platform for detecting cancerous biomarkers, e.g., a well-known oncogene microRNA-21 (miR-21). Moreover, the SDA system realized the precise intracellular miR-21 imaging in living cells, which is attributed to the reciprocal amplification property between CSA reactions and DNAzyme biocatalysis. This compact SDA amplifier machine provides a universal and facile toolbox for the highly efficient identification of cancerous biomarkers and thus holds great potential for early cancer diagnosis.

Enhanced oxidase-mimicking activity of Ce4+ by complexing with nucleotides and its tunable activity for colorimetric detection of Fe2.

Complexing with adenosine-5'-monophosphate (AMP) was proven to be a facile way to enhance the oxidase-mimicking activity of Ce4+, and enabled nanoenzyme recovery and reuse. Additionally, the oxidase-mimicking activity of AMP-Ce4+ infinite coordination polymers (ICPs) could be specifically inhibited by Fe2+. Based on this finding, we developed a simple and highly selective colorimetric assay to detect Fe2+.

An ultrasensitive disposable sandwich-configuration electrochemical immunosensor based on OMC@AuNPs composites and AuPt-MB for alpha-fetoprotein detection.

Early finding and diagnosis are critical for prevention and treatment of hepatocellular carcinoma (HCC). Alpha-fetoprotein (AFP) is a typical biomarker of HCC. Since AFP level can reflect the severity of HCC, it is essential to ensure the accurate detection of AFP. In this study, through a combination of the advantages exhibited by ordered mesoporous carbon (OMC)@gold nanoparticles (AuNPs) composites and AuPt-methylene blue (AuPt-MB), a disposable ultrasensitive sandwich-configuration electrochemical immunosensor for determination of AFP was designed. Characterized by excellent conductivity, highly ordered pore distribution and great surface area, OMC can be effective in promoting electron transfer and loading a large number of AuNPs. In the meantime, AuNPs can also immobilize AFP-Ab1 through Au-N bonds. As a new redox-active species, rod-like AuPt-MB demonstrates high conductivity, uniform morphology and excellent biocompatibility, which makes it capable not only to fix AFP-Ab2, but also to release electrochemical signals. A wide linearity of 10 fg mL-1-100 ng mL-1 and a low detection limit of 3.33 fg mL-1 (S/N = 3) were obtained. Moreover, the proposed immunosensor exhibited acceptable selectivity, high stability and reproducibility. The excellent performance in detecting serum samples endows the proposed immunosensor with broad prospects of extensive application in the detection of disease biomarkers.

Novel electrochemical biosensor based on Exo III-assisted digestion of dsDNA polymer from hybridization chain reaction in homogeneous solution for CYFRA 21-1 DNA assay.

A novel electrochemical biosensing strategy was proposed to detect cytokeratin fragment antigen 21-1 (CYFRA 21-1) DNA based on Exo III-assisted digestion of dsDNA polymer (EADDP) from hybridization chain reaction (HCR). Primarily, the presence of target can drive a catalytic hairpin assembly (CHA) reaction, which was aimed to achieve target recognition and circulation. Then the HCR can be triggered for further signal amplification and generate long dsDNA polymer with signal tags. Subsequently, the introduction of Exo III can digest the long dsDNA polymer to produce large amounts of double signal fragments (DSFs). The above experiments were all carried out in homogeneous solution. Finally, the released DSF can be captured onto the electrode directly by capture probe (CP) and a highly amplified electrochemical signal can be detected. The EADDP in homogeneous solution circumvented complex solid-liquid interface reaction and tedious operation steps on electrode. Besides, one target can be converted into abundant DSFs, which greatly improved the sensitivity. This biosensor exhibited a low detection limit (0.0348 fM) and wide linear range (5 fM âˆ¼ 50 nM) for CYFRA 21-1 DNA biosensing with reliable specificity and stability.

Ratiometric fluorescence sensing of glutathione by using the oxidase-mimicking activity of MnO2 nanosheet.

In this work, a facile ratiometric fluorescence sensor for GSH measurement was designed based on MnO2 nanosheet (NS), carbon dots (CDs), as well as a simple substrate o-phenylenediamine (OPD). Herein, MnO2 NS played triple essential roles in the sensing system. First, it could be reduced by GSH through a special reaction, and therefore served as GSH recognizer. Second, it played as a fluorescence nanoquencher to strongly quench the fluorescence of CDs. Third, it could directly oxidize OPD to yield a luminescent product 2, 3-diaminophenazine (DAP) via the intrinsic oxidase-like activity. It revealed that MnO2 NS could be reduced to Mn2+ in the presence of GSH. Thus its oxidase-like activity and fluorescence quenching abilities were inhibited, which then restricted the generation of DAP and recovered the fluorescence of CDs. Based on this phenomenon, a novel ratiometric fluorescence sensor for GSH determination was fabricated by measuring the ratio of fluorescent intensity of DAP to that of CDs. Besides, the constructed ratiometric fluorescent sensor, which could be facilely operated with single-wavelength excitation, exhibited high sensitivity and selectivity with a wider linear range and a lower detection limit.

[Toxicological research and safety consideration of coumarins].

Coumarin is an important class of natural organic compounds, which widely exists in a variety of plants and microorganisms. Coumarins have many biological activities and wide clinical applications, such as anti-tumor, anti-HIV, anti-bacterial, anti-inflammatory, anti-oxidation, anti-coagulation, but they have obvious toxic effects in rodents. It was found that the toxicity of coumarins in different animals and organs was significantly different, and high dose oral administration was more likely to produce toxic reactions. Based on the research and analysis of domestic and foreign literatures in recent 60 years, this paper mainly summarized the hepatotoxicity and pulmonary toxicity induced by coumarins, and probed into their possible mechanisms. It was found that the toxicity of coumarins had metabolic differences and species differences. The liver of rats and lungs of mice were more susceptible to coumarins. Toxic reactions occurred mainly in the second metabolic pathway of coumarin metabolism in vivo. In order to put forward safety considerations and evaluate the impact of coumarin on human body, it was found that coumarin is unlikely to produce hepatotoxicity at normal exposure level. It was also suggested that species differences due to different metabolic patterns in model animals should be carefully considered when assessing coumarin toxicity, in order to provide reference for clinical research and rational use of coumarins and improve the rational use of coumarins.

A "signal-on" electrochemical biosensor based on DNAzyme-driven bipedal DNA walkers and TdT-mediated cascade signal amplification strategy.

In this work, a dual amplified signal enhancement approach based on coupling deoxyribozyme (DNAzyme)-driven bipedal DNA walkers (BDW) and terminal deoxynucleotidyl transferase (TdT)-mediated DNA elongation signal amplifications has been developed for highly sensitive and label-free electrochemical detection of thrombin in human serums. In presence of thrombin, the BDW complex, which is comprised from the target thrombin and two DNAzyme-containing probes, can exhibit autonomous cleavage behavior on the surface of the substrate DNA (SD) modified electrode, and remove the cleaved DNA fragment from the electrode surface. Subsequently, the TdT can catalyze the elongation of the SD with free 3'-OH termini and formation of many G-quadruplex sequence replicates with the presence of 2'-deoxyaguanosine-5'-triphosphate (dGTP) and adenosine 5'-triphosphate (dATP) at a molar ratio of 6:4. These G-quadruplex sequences bind hemin and generate drastically amplified current response for sensitive detection of thrombin in a "signal-on" and completely label-free fashion. Under optimized conditions, the response peak current was linear with the concentration of thrombin in the range from 0.5 pM to 100000 pM with detection limit of 0.31 pM. This research provides us a sustainable idea for the hyphenated multiple amplification strategies and a stable and effective method for the detection of protein biomarkers.