Copper nanocluster-Ru(dcbpy)32+ as a cathodic ECL-RET probe combined with 3D bipedal DNA walker amplification for bioanalysis.

In this study, a cathodic intra-molecular electrochemiluminescence resonance energy transfer (ECL-RET) probe was exquisitely designed via the integration of an ECL donor (Cu NCs) with an acceptor (Ru(dcbpy)32+), and further employed the 3D bipedal DNA walker amplification strategy to monitor the platelet-derived growth factor BB (PDGF-BB). Specifically, blue emission Cu NCs with low consumption, biocompatibility and numerous resources, act as well-overlapped donors and significantly improve the ECL efficiency of Ru(dcbpy)32+. More impressively, the intra-molecular ECL-RET of Cu NC-Ru endowed a better and more stable ECL signal by reducing the electron-transfer distance and decreasing the energy loss. Furthermore, 3D bipedal DNA walker amplification was employed to efficiently convert the target PDGF-BB into numerous DNA strands, achieving sensitive target amplification. By virtue of such design, the constructed aptasensor exhibited a sensitive and selective assay for PDGF-BB with a detection range from 0.01 pM to 10 nM and a detection limit of 3.3 fM. The intramolecular ECL-RET and 3D bipedal DNA walker amplification strategy designed in this study will provide valuable insight into promising ultrasensitive ECL bioanalysis.

Core-shell Au@PtAg modified TiO2-Ti3C2 heterostructure and target-triggered DNAzyme cascade amplification for photoelectrochemical detection of ochratoxin A.

Efficient charge separation and utilization are critical factors to obtain a high initial signal in photoelectrochemical (PEC) aptasensor. Reports demonstrate that constructing metal/semiconductor Schottky junction can effectively improve the charge separation efficiency. Herein, a photoelectrode Au@PtAg/TiO2-Ti3C2 Schottky junction is successfully synthesized. Specifically, the Schottky junction between core-shell Au@PtAg and TiO2-Ti3C2 facilitates the efficiency of photogenerated electron transfer and enables the transfer of photogenerated electrons from TiO2-Ti3C2 to Au@PtAg. Noteworthy, the core-shell Au@PtAg acts as a photoelectron receiver to capture and store electrons, which further facilitates the separation of photogenerated electron-hole pairs, resulting enhanced photocurrent generation without sacrificial agents. Moreover, through the Mg2+-dependent DNAzyme cascade amplification, the sensitivity of the PEC aptasensor is further improved. Hence, we report an ultrasensitive PEC aptasensor for ochratoxin A (OTA) assy based on Au@PtAg/TiO2-Ti3C2 Schottky junction and Mg2+-dependent DNAzyme cascade amplification. As a result, the established PEC aptasensor exhibits excellent photocurrent performance in the range of 5 fg mL-1-10 ng mL-1 with a detection limit as low as 1.73 fg mL-1, showing high sensitivity, selectivity as well as stability. This strategy provides a versatile and promising avenue for the development of high-performance PEC aptasensor.

DNA coated CoZn-ZIF metal-organic frameworks for fluorescent sensing guanosine triphosphate and discrimination of nucleoside triphosphates.

Imidazole-based metal-organic frameworks (MOFs) are easy to prepare as well-dispersed nanoparticles, which have attracted a lot of interest in sensing. Metal substitution is an effective way to regulate the composition and performance of MOFs. Herein, by tuning the contents of Co and Zn, a series of homobimetallic CoxZn100-x-ZIF (x = 0-100) were synthesized. Using a fluorescently-labeled DNA oligonucleotide probe, guanosine triphosphate (GTP) can readily displace the adsorbed DNA from Co50Zn50-ZIF, resulting in over 30-fold fluorescence enhancement with 1 mM GTP. Co80Zn20-ZIF could specifically recognize adenosine triphosphate (ATP), whereas Co65Zn35-ZIF and Co20Zn80-ZIF responded to both ATP and GTP. For comparison, Co50Ni50-ZIF and Co50Cu50-ZIF were also prepared, but none of them were selective for any of the molecules, indicating a synergetic effect of cobalt and zinc in Co50Zn50-ZIF for the selective recognition of GTP. This system can sensitively detect GTP with a detection limit of 0.13 µM. Moreover, based on the varying binding affinities of these CoZn-ZIFs towards different nucleoside triphosphates (NTPs), a ZIF fluorescent sensor array was also designed for the discrimination of the four types of NTPs.

A sensor array based on DNA-wrapped bimetallic zeolitic imidazolate frameworks for detection of ATP hydrolysis products.

Most current biosensors were designed for the detection of individual analytes, or a group of chemically similar analytes. We reason that sensors designed to track both reactants and products might be useful for following chemical reactions. Adenosine triphosphate (ATP) is a key biomolecule that participates in various biochemical reactions, and its hydrolysis plays a fundamental role in life. ATP can be converted to adenosine diphosphate (ADP) and inorganic phosphate (Pi) via the dephosphorylation process. ATP can also be hydrolyzed to adenosine monophosphate (AMP) and pyrophosphate (PPi) through depyrophosphorylation, depending on where the bond is cleaved. The detection of ATP-related hydrolysates would enable a better understanding of the different reaction pathways with a high level of robustness and confidence. Herein, we prepared a fluorescent sensor array based on a series of bimetallic zeolite imidazole frameworks M/ZIF-8 (M = Ni, Mn, Cu) and ZIF-67 to discriminate ATP hydrolysis and detect ATP hydrolysis related analytes. A fluorescently-labeled DNA oligonucleotide was used for signaling. Interestingly, Cu/ZIF-8 exhibited an ultrahigh selectivity for recognizing pyrophosphate with a detection limit of 2.5 µM. Moreover, the practicality of this sensor array was demonstrated in fetal bovine serum, clearly discriminating ATP hydrolysis products.

Sensing ATP: Zeolitic Imidazolate Framework-67 Is Superior to Aptamers for Target Recognition.

In a typical biosensor, a biomolecule such as an aptamer is used for target recognition, and a nanomaterial is used for signal generation. Herein, we communicate a reverse system using a nanomaterial for target recognition and a DNA for signaling. We discovered that a classic metal-organic framework material, zeolitic imidazolate framework (ZIF)-67, has ultrahigh selectivity for recognizing adenosine triphosphate (ATP), allowing a fluorescently labeled DNA oligonucleotide to be used for signal generation. This sensor showed up to a 24-fold increase in fluorescence upon adding 1 mM ATP, while the fluorescence increase after adding adenosine or guanosine triphosphate was less than twofold. Its selectivity is much better than that of the ATP aptamer, which binds adenosine even better. Using isothermal titration calorimetry, the selective binding of ATP was independently verified. This sensor has a detection limit of 29 nM ATP and it can even detect ATP in serum. By replacing Co2+ with Zn2+ to form ZIF-8 or by using CoO, the selectivity for ATP was lost. Therefore, by sophisticated material design, ultrahigh selectivity for molecular recognition can be achieved.

Cu(I)-Catalyzed Click Reaction-Triggered 3D DNA Walker for Constructing an "OFF-ON" Fluorescent Biosensor for Cu2+ Detection.

Herein, a highly selective and sensitive "OFF-ON" fluorescent biosensor was designed for intracellular Cu2+ detection. Compared to the fluorescent Cu2+ biosensors reported so far, this work tackled the tricky issue of reliability of Cu2+, which mainly depends on the integration of the high selectivity of the Cu(I)-catalyzed click reaction with the ultrahigh sensitivity of a spherical nucleic acid-based 3D DNA walker. Typically, DNA track is carried out by coconjugating N3-S1 and Cy3-HP onto gold nanoparticles (AuNPs). On this state, fluorophore (Cy3) was close to the surface of AuNPs (as a nanoquencher), generating a quenched fluorescence and thus causing the initial "OFF" state. In the presence of Cu2+ and H2C2-swing arm, Cu+ was in situ generated quickly from the reduction of Cu2+ with the assistance of ascorbic acid, which could promptly and selectively trigger the Cu(I)-catalyzed click reaction-based 3D DNA walker between azide on N3-S1 and alkyne on the H2C2-swing arm. Sequentially, the activated H2C2-swing arm was able to hybridize with adjacent Cy3-HP and the 3D DNA walker was automatically driven by N.BstNBI to produce multiple Cy3-labeled DNA fragments away from the AuNP surface for signal amplification, performing a recovered fluorescence response (turning into the "ON" state). Accordingly, the ingenious integration of an efficient click reaction and smart 3D DNA walker endows the constructed fluorescent biosensor with superior selectivity and ultrahigh sensitivity. We further apply this platform for Cu2+ sensing in biological systems; this assay will provide a signal transduction strategy for evaluating intracellular Cu2+ at picomolar levels.

Cortex dictamni-induced liver injury in mice: The role of P450-mediated metabolic activation of furanoids.

Many furan containing compounds have been reported to be toxic resulted from the metabolic activation of the furan ring to reactive metabolite (RM). Cortex Dictamni (CD), a widely used herbal medicine, has been reported to cause severe even fatal hepatotoxicity. The injurious components and mechanism of CD-induced liver injury remain unclear. Our preliminary study showed that dictamnine, one major furanoid in CD, caused mouse liver injury via its reactive epoxide metabolite. Besides dictamnine, the major components of CD are series of bioactivation-alerting furanoids. Thus, we hypothesize that series of furanoids in CD may undergo metabolic activation and play a key role in CD-induced liver injury. Here, a single oral dose of 60 g/kg ethanol extract of CD (ECD) caused severe hepatocellular necrosis in mice at 24 h post-dose. ECD-induced liver injury showed a dose- and time-dependent manner. The hepatotoxic effects could be completely abolished by P450 nonselective inhibitor 1-aminobenzotriazole (ABT) and strongly modulated by other P450 modulators. The furanoids-concentrated fraction of ECD was responsible for the hepatotoxicity. At least ten furanoids with high abundance in ECD, such as obakunone, dictamnine, fraxinellone, limonin, were found to be metabolized to reactive epoxide or cis-enedione. The RM levels were consistent with the liver injury degree. Multiple furanoids, rather than single one, cooperatively contributed to the hepatotoxicity. ECD-induced liver injury could be reproduced by a mixture of pure furanoids. In summary, this study provides toxic component profiles of CD and demonstrates that P450-mediated bioactivation of multiple furanoids is responsible for CD-induced liver injury.

Simultaneous Quantitative Determination of Polyphenolic Compounds in Blumea balsamifera (Ai-Na-Xiang, Sembung) by High-Performance Liquid Chromatography with Photodiode Array Detector.

A high-performance liquid chromatography method was developed for simultaneous quantification of 18 polyphenolic compounds from the leaves of Blumea balsamifera, including 17 flavonoids and 1 phenylethanone. The B. balsamifera extraction was separated by a Kromasil C18 column (250 × 4.6 mm, 5 µm) with a binary gradient mobile phase consisting of acetonitrile and 0.2% aqueous acetic acid. A photodiode array detector (PDA) was used to record the signals of investigated constituents. The linearity, sensitivity, stability, precision, and accuracy of the established assay methods were assessed to meet the requirements of quantitative determination. Samples extracted by reflux in 25 mL of 80% methanol for 30 minutes were selected for the extraction method. The 18 compounds were accurately identified by comparing with the reference compounds. The purity of each peak was confirmed by the base peak in the mass spectrum. The contents of 18 compounds in Blumea samples from four different regions were successfully determined. The results also showed that 3,3',5,7-tetrahydroxy-4'-methoxyflavanone was the most abundant constituent, which could be used as a potential chemical marker for quality control of B. balsamifera and Chinese patent medications containing B. balsamifera herb.

Ultrasensitive aptasensing of insulin based on hollow porous C3N4/S2O82-/AuPtAg ECL ternary system and DNA walker amplification.

In this work, a high-efficiency electrochemiluminescence (ECL) ternary system was constructed for ultrasensitive assay of insulin based on hollow porous graphitic carbon nitride (HP-C3N4) as novel luminophore, S2O82- as coreactant and tri-metallic AuPtAg as coreaction accelerator. Specifically, in comparison with C3N4-based bulk nanomaterials, the as-prepared HP-C3N4 exhibits high luminous efficiency though decreased inner filter effect and minimized inactive ECL emitter. Noteworthy, tri-metallic AuPtAg, possessing the superiority of Au, Pt and Ag, was first used as coreaction accelerator to significantly enhance ECL intensity of HP-C3N4 and S2O82-. As a consequence, with the resultant ECL ternary (HP-C3N4/S2O82-/AuPtAg) system as aptasensing platform, a high-intense initial ECL signal was achieved. Subsequently, ferrocene-labeled quenching probe (Fc-HP2) as ECL quencher was used to quench the initial signal and achieve the low-background noise. Eventually, in the presence of insulin, the target-induced triple-helix molecular switch and Nb.BbvCI-assisted DNA walker amplification were executed to recover a strong ECL signal by releasing Fc-HP2 from the electrode surface. As expected, the constructed aptasensor presents an excellent sensitivity and selectivity for detecting insulin range from 0.05 pg mL-1 to 100 ng mL-1 with a detection limit of 17 fg mL-1. This work provides a new avenue for developing highly efficient HP-C3N4 based ECL ternary system as well as ultrasensitive ECL aptasensors for bioanalysis.

Gypenosides Altered Hepatic Bile Acids Homeostasis in Mice Treated with High Fat Diet.

Gypenosides extracted from Gynostemma pentaphyllum (Thunb.) Makino have significant role in reducing serum lipid level and treating fatty liver diseases, however, without clear mechanism. As gypenosides share the similar core structures with bile acids (the endogenous ligands of nuclear receptor FXR), we hypothesize that gypenosides may improve hypercholesterolemia via FXR-mediated bile acids signaling. The present study was designed to validate the role of gypenosides in reducing levels of serum total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C), as well as in regulating bile acids homeostasis and related gene expression levels. The C57BL/6 male mice were divided into four groups. Mice in groups ND and HFD were fed with normal diet and high fat diet for 38 weeks, respectively. In groups HFD+GP and HFD+ST, mice were fed with high fat diet for 38 weeks and treated with gypenosides and simvastatin (positive control) from weeks 16 to 38, respectively. Serum TC and LDL-C levels were assayed by commercially available kits. Expression levels of genes were tested by the quantitative real-time PCR. The LC-MS/MS was applied to quantify major bile acids in mice livers. Our results showed that gypenosides significantly decreased serum TC and LDL-C levels. The gene expression level of Shp was downregulated while the levels of Cyp7a1, Cyp8b1, Fxr, Lrh1, Jnk1/2, and Erk1/2 were upregulated by gypenosides. Indicated by LC-MS/MS technology, gypenosides increased the hepatic levels of several free bile acids and most taurine-conjugated bile acids while decreasing glycine-conjugated bile acids levels. In addition, gypenosides decreased the CA/CDCA ratio. Gypenosides may improve the abnormal lipid profile of HFD-fed mice via two pathways: (1) enhancing the bile acids biosynthesis from cholesterol; (2) decreasing the CA/CDCA ratio which is positively related to cholesterol absorption.

Development and validation of a rapid LC-MS/MS method for simultaneous quantification of arecoline and its two active metabolites in rat plasma and its application to a pharmacokinetic study.

Arecoline is the primary active and toxic constituent of areca nut. Arecaidine and arecoline N-oxide are two major active metabolites of arecoline. In this work, an accurate and simple high performance liquid chromatography tandem mass spectrometry method for simultaneous quantification of arecoline, arecaidine and arecoline N-oxide in rat plasma was developed and fully validated to study their pharmacokinetic behaviors in rats. After extracted from rat plasma by protein precipitation with methanol and then concentrated, the analytes were chromatographic separated on a Sepax Sapphire C18 analytical column. The mobile phase consisted of methanol and 2 mM ammonium acetate buffer solution containing 0.2% (v/v) formic acid (8:92, v/v) under isocratic elution. The analytes were detected by multiple reaction monitoring (MRM) with an electrospray ionization source in the positive ion mode. The transitions of m/z 156.2 → 53.2, m/z 142.2 → 44.2 and m/z 172.2 → 60.2 were selected for arecoline, arecaidine and arecoline N-oxide, respectively. The method was linear over the concentration range of 0.5-100 ng/mL for arecoline, 5-5000 ng/mL for arecaidine and arecoline N-oxide with no carry-over effect. The accuracies and intra- and inter-batch precisions were all within the acceptance limits. No matrix effect and potential interconversion between the analytes and other metabolites were observed in this method. The validated method was further employed to a preclinical pharmacokinetic study of arecoline, arecaidine and arecoline N-oxide after oral treatment with 20 mg/kg arecoline to rats.

Liquid chromatography-tandem mass spectrometric assay for determination of unstable arecoline in rat plasma and its application.

Arecoline, a predominant alkaloid in areca nut, shows several pharmacological and toxicological effects. In present study, a sensitive and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and fully validated for quantification of arecoline in rat plasma. Importantly, our group found that arecoline was highly unstable in rat plasma samples, which brought challenges to accurately quantify in vivo. The hydrolysis of ester moiety of arecoline by carboxylesterases was responsible for its instability, and arecoline was hydrolyzed to arecaidine. The degradation of arecoline was completely inhibited using 5% formic acid as a stabilizer, which was immediately added to freshly collected rat plasma samples. EDTA was adopted as the anticoagulant to also reduce the degradation during blood collection and plasma separation owing to its anti-esterase effect. The plasma sample was separated by a C18 analytical column with a mobile phase of 0.1% formic acid and methanol (95:5v/v) at an isocratic flow rate of 300µL/min. The analyte was monitored on a tandem mass spectrometer using the multiple reaction monitoring scan in a positive electrospray ionization mode. The method exhibited high sensitivity and a good linearity rang of 1-1000ng/mL. The developed analytical method was employed in a pilot pharmacokinetic study of arecoline in rats. Arecoline was rapidly eliminated within 45min in rats after oral treatment of 150mg/kg arecoline.

Separation and purification of five alkaloids from Aconitum duclouxii by counter-current chromatography.

C19 -diterpenoid alkaloids are the main components of Aconitum duclouxii Levl. The process of separation and purification of these compounds in previous studies was tedious and time consuming, requiring multiple chromatographic steps, thus resulted in low recovery and high cost. In the present work, five C19 -diterpenoid alkaloids, namely, benzoylaconine (1), N-deethylaconitine (2), aconitine (3), deoxyaconitine (4), and ducloudine A (5), were efficiently prepared from A. duclouxii Levl (Aconitum L.) by ethyl acetate extraction followed with counter-current chromatography. In the process of separation, the critical conditions of counter-current chromatography were optimized. The two-phase solvent system composed of n-hexane/ethyl acetate/methanol/water/NH3 ·H2 O (25%) (1:1:1:1:0.1, v/v) was selected and 148.2 mg of 1, 24.1 mg of 2, 250.6 mg of 3, 73.9 mg of 4, and 31.4 mg of 5 were obtained from 1 g total Aconitum alkaloids extract, respectively, in a single run within 4 h. Their purities were found to be 98.4, 97.2, 98.2, 96.8, and 96.6%, respectively, by ultra-high performance liquid chromatography analysis. The presented separation and purification method was simple, fast, and efficient, and the obtained highly pure alkaloids are suitable for biochemical and toxicological investigation.

Separation and purification of 9,10-dihydrophenanthrenes and bibenzyls from Pholidota chinensis by high-speed countercurrent chromatography.

Stilbenoids are the main components of leaves and stems of Pholidota chinensis. In the present investigation, high-speed counter-current chromatography was used for the separation and purification of two classes of stilbenoids, namely, bibenzyls and 9,10-dihydrophenanthrenes, on a preparative scale from whole plants of P. chinensis with different solvent systems after silica gel column chromatography fractionation. n-Hexane/ethyl acetate/methanol/water (1.2:1:1:0.8, v/v/v/v) was selected as the optimum solvent system to purify 1-(3,4,5-trimethoxyphenyl)-1',2'-ethanediol (1), coelonin (2), 3,4'-dihydroxy-5,5'-dimethoxybibenzyl (3), and 2,​7-​dihydroxy-​3,​4,​6-​trimethoxy-​9,​10-​dihydrophenanthrene (4). While 2,7-dihydroxy-3,4,6-trimethoxy-​9,​10-​dihydrophenanthrene (5), batatasin III (6), orchinol (7), and 3'-O-methylbatatasin III (8) were purified by n-hexane/ethyl acetate/methanol/water (1.6:0.8:1.2:0.4, v/v/v/v). After the high-speed counter-current chromatography isolation procedure, the purity of all compounds was over 94% assayed by ultra high performance liquid chromatography. The chemical structure identification of all compounds was carried out by mass spectrometry and (1)H and (13)C NMR spectroscopy. To the best of our knowledge, the current investigation is the first study for the separation and purification of bibenzyls and 9,10-dihydrophenanthrenes by high-speed counter-current chromatography from natural resources.