Copper Nanosheet-Based Wash-Free Fluorescence Imaging of Cancer Cells.

Near-infrared fluorescence (NIRF) probes greatly facilitate in vivo imaging of various biologically important species. However, there are several significant limitations such as consuming washing steps, photobleaching, and low signal intensity. Herein, we synthesized fluorescent copper nanosheets templated with DNA scaffolds (DNS/CuNSs). We employ them and Cy5.5 of the fluorescence resonance energy transfer (FRET) system, which have a larger Stokes shift (∼12-fold) than the traditional NIRF dye Cy5.5. Based on their excellent fluorescence properties, we employ DNS/CuNSs-Cy5.5 for fluorescence probes in cancer cell imaging. Compared with the free Cy5.5 fluorescence probe, the novel fluorescence imaging probe implements wash-free imaging and exhibits enhanced anti-photobleaching ability (∼5.5-fold). Moreover, the FRET system constructed by DNS/CuNSs has a higher signal amplification ability (∼4.17-fold), which is more similar to that of Cu nanoclusters prepared with DNA nanomonomers as a template. This work provides a new idea for cancer cell MCF-7 imaging and is expected to promote the development of cancer cell fluorescence imaging.

Self-assembly Induced Enhanced Electrochemiluminescence of Copper Nanoclusters Using DNA Nanoribbon Templates.

Copper nanoclusters (CuNCs) are attractive electrochemiluminescence (ECL) emitters as Cu is comparatively inexpensive, nontoxic, and highly abundant. However, their ECL yield is relatively low. Herein, we report that orderly self-assembly of CuNCs using DNA nanoribbon as the template (DNR/CuNCs) conferred the CuNCs with improved ECL properties compared with individual CuNCs in both annihilation and co-reactant processes. The DNR/CuNCs resulted in a high ECL yield of 46.8 % in K2 S2 O8 , which was ≈68 times higher than that of individual CuNCs. This strategy was successfully extended to other ECL emitters, such as gold nanoclusters and the Ru(bpy)3 2+ /TPrA system. Furthermore, as an application of DNR/CuNCs, a DNR/CuNC-based ECL biosensor with higher sensitivity was constructed for dopamine determination (two orders of magnitude lower than that previously reported), showing that DNR/CuNCs have a potential for application in ECL bioanalysis as a new type of superior luminophore candidate.

Hexaconazole-Cu complex improves the salt tolerance of Triticum aestivum seedlings.

Hexaconazole is one of the triazole complexes that are broadly used as systemic fungicides with non-traditional plant growth regulator properties. Hexaconazole-Cu complex (Hex-Cu) is a new triazole derivative, and the biological effect of Hex-Cu has been rarely studied. In this work, we investigated the functions of Hex-Cu in regulating growth and the response to salt stress in the seedlings of Triticum aestivum. Pretreated with 60µmolL(-1) Hex-Cu, the seedling plants got increased root/shoot ratio by 42.0%, and the contents of chlorophyll and soluble protein were also increased by 38.1% and 27.9%, respectively. Furthermore, Hex-Cu alleviated the growth inhibition caused by salt stress, enabled the seedlings to maintain a higher proline content and lower malondialdehyde accumulation. The functions of Hex-Cu in regulating the expression of proline synthetase (P5CS and P5CR) genes were investigated by quantitative real-time PCR (qPCR). Under 100mmolL(-1) NaCl stress, the expression of P5CS and P5CR in the seedlings by Hex-Cu pretreatment were significantly up-regulated. It attributed to the enhanced salt tolerance in plants.

Thermodynamic Allosteric Switch-Actuated 3D DNA Nanomachine for Ultrasensitive Electrochemical/Fluorescent Dual-Mode Biosensing of a Transcription Factor.

Herein, we reported an innovative thermodynamic allosteric switch-actuated 3D DNA nanomachine for selective, sensitive, and accurate electrochemical (EC)/fluorescent (FL) dual-mode biosensing of a microphthalmia-associated transcription factor (MITF). The thermodynamic allosteric switch was ingeniously customized as a hairpin probe (HP) that was in dynamic equilibrium but rapidly interconverting conformations. At the "inactive state", the MITF-binding region and the switch part were "sequestered". Upon the introduction of MITF, an MITF-HP complex promptly formed, and the equilibrium of HP thermodynamically inclined from the "inactive state" toward the "active state" conformation. Immediately, the exposed switch on HP effectively actuated the 3D DNA nanomachine and synchronously produced the restriction site for Nb.BbvCI nicking endonuclease. After the autonomous conveying of the 3D DNA nanomachine by means of the high-efficiency circularly nicking endonuclease signal amplification (NESA), not only was MB-S1 in the supernatant used for FL measurements but also MB-SP/MNs/S2 in the precipitate was adapted for EC analysis, significantly improving the utilization of output products derived from the 3D DNA nanomachine. Accordingly, benefiting from the efficient DNA nanomachine signal amplification manner and the self-calibration function of a dual-mode bioassay, the constructed biosensor exhibits superior sensitivity and accuracy for MITF determination.

Self-assembly of Copper Nanoclusters Using DNA Nanoribbon Templates for Sensitive Electrochemical Detection of H2O2 in Live Cells.

The excessive secretion of H2O2 within cells is closely associated with cellular dysfunction. Therefore, high sensitivity in situ detection of H2O2 released from living cells was valuable in clinical diagnosis. In the present work, a novel electrochemical cells sensing platform by synthesizing copper nanoclusters (CuNCs) at room temperature based on DNA nanoribbon (DNR) as a template (DNR-CuNCs). The tight and ordered arrangement of nanostructured assemblies of DNR-CuNCs conferred the sensor with superior stability (45 days) and electrochemical performance. The MUC1 aptamer extending from the DNR template enabled the direct capture MCF-7 cells on electrode surface, this facilitated real-time monitoring of H2O2 release from stimulated MCF-7 cells. While the captured MCF-7 cells on the electrode surface significantly amplified the current signal of H2O2 release compared with the traditional electrochemical detection H2O2 released signal by MCF-7 cells in PBS solution. The approach provides an effective strategy for the design of versatile sensors and achieving monitored cell release of H2O2 in long time horizon (10 h). Thereby expanding the possibilities for detecting biomolecules from live cells in clinical diagnosis and biomedical applications.

DNA Nanoribbon-Assisted Intracellular Biosynthesis of Fluorescent Gold Nanoclusters for Cancer Cell Imaging.

Metal nanoclusters (NCs) have emerged as a promising class of fluorescent probes for cellular imaging due to their high resistance to photobleaching and low toxicity. Nevertheless, their widespread use in clinical diagnosis is limited by their unstable intracellular fluorescence. In this study, we develop an intracellularly biosynthesized fluorescent probe, DNA nanoribbon-gold NCs (DNR/AuNCs), for long-term cellular tracking. Our results show that DNR/AuNCs exhibit a 4-fold enhancement of intracellular fluorescence intensity compared to free AuNCs. We also investigated the mechanism underlying the fluorescence enhancement of AuNCs by DNRs. Our findings suggest that the higher synthesis efficiency and stability of AuNCs in the lysosome may contribute to their fluorescence enhancement, which enables long-term (up to 15 days) fluorescence imaging of cancer cells (enhancement of ∼60 times compared to free AuNCs). Furthermore, we observe similar results with other metal NCs, confirming the generality of the DNR-assisted biosynthesis approach for preparing highly bright and stable fluorescent metal NCs for cancer cell imaging.

[Tissue culture of Vinca minor and determination of vincamine].

OBJECTIVE: To study tissue culture of Vinca minor and determine the content of vincamine. METHOD: Leaf blades, stalks, root segment of V. minor were used as explants to study the effect of 2, 4-D,6-BA,NAA on its callus induction and vincamine contents in the orthogonal design experiment. In the peak period of callus formation, vincamine content in callus of V. minor and sterile plants was determined by HPLC. The experimental data was statistically analyzed. RESULT: The content of 6-BA and NAA had no significant effect on its callus induction. But the content of 2, 4-D had significant effect on its callus induction. Within 20,40,60 d, the content of vincamine in sterile plant was (0.015 +/- 0.003)%, (0.097 +/- 0.001)% , (0.113 +/- 0.06)%, respectively. In the peak period of callus formation, vincamine content in callus of leaf blades, stalks, root segment was (0.024 +/- 0.0025)%, (0.016 +/- 0.0015)%, (0.010 +/- 0.0015)%, respectively. To 30 days of subculture, vincamine content in callus of leaf blades, stalks, root segment was (0.041 +/- 0.002)%, (0.019 +/- 0001)%, (0.016 +/- 0.002)%, respectively. CONCLUSION: The optimal hormone combination for callus initiation was MS +2, 4-D 1.0 mg x L(-1) +6-BA 0.5 mg x L(-1) + NAA 0.5 mg x L(-1). In different growth periods, vincamine content in sterile plants is significantly different. From different explants in callus vincamine content is different, in which leaves callus is significantly higher than that of stems, roots produced callus organization.

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.

Snowflake-like DNA crystals templated Cu clusters as a fluorescent turn-on probe for sensing actin.

Herein, we synthesized snowflake-like DNA crystals (SDC) via hybridization chain reaction and used it for the first time in the synthesis of copper nanoclusters with enhanced fluorescence. Atomic force microscopy (AFM) and laser confocal microscopy characterization confirmed that SDC/CuNCs are self-assembled successfully on SDC. Aggregation induced emission allows SDC/CuNCs to exhibit better stability and stronger emission intensity. Thus, we developed the "turn-on" label-free fluorescence detection method of actin based on SDC/CuNCs which offer simplicity, low cost, good selectivity, and high sensitivity. The detection limit was determined to be 0.0124 µg mL-1, which was an order of magnitude lower than that of reported fluorescent methods (0.12 µg mL-1). Compared with previous method, the linear range is also much wider. We also performed standard recovery experiments in actual samples for evaluating the practicality of this strategy and proved that the capability of the proposed approach for the determination of actin is feasible and the interference from complex biological samples is negligible. These results indicate that SDC/CuNCs are expected to play a more important role in the field of biosensors.

NH2-Ni-MOF electrocatalysts with tunable size/morphology for ultrasensitive C-reactive protein detection via an aptamer binding induced DNA walker-antibody sandwich assay.

Here we report a general approach for synthesizing size/morphology-controlled NH2-Ni-MOFs by optimizing the solvent composition in the reaction system, and their correlating catalytic performances are clearly explored for the first time. Interestingly, the electrocatalytic performance was found to increase 2.7 fold when the particle size is reduced from 1.5 µm for NH2-Ni-MOF(a) to 300 nm for NH2-Ni-MOF(c). Subsequently, this work exhibits an example of a high-performance NH2-Ni-MOF(c) electrocatalyst for application in constructing an electrochemical aptasensor to achieve sensitive C-reactive protein (CRP) detection based on an aptamer binding induced DNA walker-antibody sandwich assay. The proposed aptasensor shows a wide linear range from 0.1 pg mL-1 to 100 ng mL-1 with a low detection limit of 0.029 pg mL-1. The work presented here can thus offer an atypical approach to size- and morphology-controlled MOFs in electrocatalysis and biosensing.

A C2H2 zinc-finger protein OsZFP213 interacts with OsMAPK3 to enhance salt tolerance in rice.

Improvement of salt tolerance is one of the major targets in rice breeding. Here, we report that the zinc-finger protein (ZFP) OsZFP213 functions in enhancing salt tolerance in rice. OsZFP213 is localized in the nucleus and has transactivation activity. Transgenic rice overexpressing OsZFP213 showed enhanced salt tolerance compared with wild type and OsZFP213 RNAi plants. Furthermore, OsZFP213 overexpression plants showed higher transcription levels of antioxidant system genes and higher catalytic activity of scavenging enzymes of reactive oxygen, such as superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and glutathione reductase (GR), and a lower level of ROS accumulation than that in wild type and OsZFP213 RNAi plants under salt treatment. Yeast two-hybrid, pull-down, and BiFC analysis showed that OsMAPK3 is a direct partner of OsZFP213, and this interaction enhanced the transactivation activity of OsZFP213. Taken together, these results suggest that OsZFP213 cooperates with OsMAPK3 in the regulation of rice salt stress tolerance by enhancing the ability of scavenging reactive oxygen.

Combining padlock exponential rolling circle amplification with CoFe2O4 magnetic nanoparticles for microRNA detection by nanoelectrocatalysis without a substrate.

In this report, an ultrasensitive electrochemical biosensor for miR-21 detection was designed on the basis of a padlock exponential rolling circle amplification (P-ERCA) assay and CoFe2O4 magnetic nanoparticles (CoFe2O4 MNPs) by nanoelectrocatalysis without a substrate for signal amplification. Here, to improve catalytic efficiency, nanocatalyst (CoFe2O4 MNPs) and redox molecule (Tb) were co-immobilized onto the graphene (Gra) surface and formed Au@CoFe2O4/Tb-Gra probe. The obtained probe exhibits high-performance Tb catalysis by reducing the interaction distance between CoFe2O4 MNPs and Tb, and importantly, the detection sensitivity is significantly improved even in the absence of substrate (H2O2). Simultaneously, to further improve the sensitivity and specificity of the biosensor, P-ERCA assay was introduced. After multiple polymerization and nicking reactions, plenty of P-ERCA amplification product was produced and circular exponential signal amplification was achieved, giving an extreme sensitivity for miR-21 assay. The as-prepared biosensor shows a wide dynamic range of 1 fM to 2 nM with a low detection limit of 0.3 fM for miR-21 detection and exhibits high specificity and sensitivity.

Effects of sludge pretreatments and organic acids on hydrogen production by anaerobic fermentation.

Using apple pomace (AP) as substrate, a series of batch experiments were conducted to investigate the effects of activated sludge pretreated by ultraviolet and ultrasonic on the anaerobic fermentative bio-H(2) production. The results show that a maximum cumulative H(2) production (CHP(m)) of 107.0mlg(-1)-TS and an average H(2) production rate (AHPR) of 15.0mlg(-1)-TSh(-1) were obtained by using the pretreated sludge that was irradiated by ultraviolet lamp (25w) for 15min as inoculant. Based on the optimal sludge pretreatment method, the experiments of bio-H(2) production under the optimal substrate pretreatment condition (soaked in the ammonia liquor of 6% for 24h) were further carried out, and the CHP(m) of 138.8mlg(-1)-TS was achieved, increased by 80.6% compared to the un-pretreated group (76.8mlg(-1)-TS). The effects of the various organic acids on the fermentative H(2) production were also investigated under the above optimal sludge pretreatment condition.

[Genetic transformation of Nicotiana tabacum L. by Agrobacterium tumefaciens carrying genes in the melatonin biosynthesis pathway and the enhancement of antioxidative capability in transgenic plants].

Arylalkylamine N-acetyltransferase (AANAT) and Hydroxyindole O-methyltransferase(HIOMT) are the key regulation enzymes in the melatonin biosynthesis pathway in mammals. The AANAT and HIOMT genes were constructed into a binary plant expression vector YXu55. Using leaf strips as the recipiences, we efficiently transformed tobacco (Nicotiana tabacum) variety qinyan 95 by the Agrobacterium mediated method. After gradient selection with gentamycin, a number of transgenic plants were regenerated. Southern blot and RT-PCR analyses showed that the AANAT-HIOMT genes were integrated into the genome of the transgenic plants and the target genes could express at the level of RNA transcription. By RP-HPLC, we measured the melatonin contents in transgenic plants. The results showed that the melatonin level in YXu55 (containing the gentamycin-resistance gene, the AANAT gene and HIOMT gene) transgenic plants were much higher than those in pZP122 (control containing only the gentamycin-resistance gene) transgenic plants and nontransgenic plants. The content of melatonin in pZP122 transgenic plants was nearly the same as that in nontransgenic plants. Physiological determination of antioxidative characteristics demonstrated that 1) the capacity of total antioxidation, 2) the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) and 3) the content of glutathione (GSH) were increased in YXu55 transgenic plants containing the AANAT-HIOMT genes as compared to the control plants (pZP122 or nontransgenic plants). At the same time, malonaldehyde (MDA) content did not appear remarkably difference between transgenic plants and nontransgenic plants. The above mentioned facts indicate enhancement of melatonin levels in YXu55 transgenic plants might help to reduce damage by oxidative stress.

[Hairy root induction and plant regeneration of crownvetch (Coronilla varia L.) transformed by Agrobacterium rhizogenes].

An efficient system of genetic transformation and plant regeneration via somatic embryogenesis was established in crownvetch (Coronilla varia L.) by infecting the segments of cotyledons and hypocotyls of 15d-old seedlings with Agrobacterium rhizogenes strain 15834. Hairy roots were produced directly from the wounded surface of the explants or via calluses on hormone-free Murashige and Skoog (MS) medium after infection by A. rhizogenes. Transformed roots grew rapidly either on solid or liquid MS medium, and exhibited typical hairy root phenotypes. The highest transformation frequency (87.4%) was achieved by preculturing cotyledons for 2d and pre-treating the A. rhizogenes with suitable concentration of acetosyringone at logarithmic phase (OD600 = 0.8). The embryogenic calluses with 100% induction frequency were induced from hairy roots on MS medium containing 0.2mg/L 2,4-D, 0.5mg/L NAA and 0.5mg/L KT. Globular-, heart-, torpedo-, and cotyledon shaped somatic embryos were produced orderly and developed into plantlets when transferred the embryogenic calluses on MS medium supplemented with 0.5mg/L KT, 0.2mg/L IBA and 300mg/L proline. The transformed plants did not show differences in morphology except abundant lateral root branches compared to the non-transformed plants. However, the contents of 3-nitropropanic acid in hairy roots and leaves of one of 5 transformed clones were 57.68% and 58.17% in roots and leaves of untransformed plants, respectively. Opine paper electrophoresis revealed the integration and expression of TR-DNA. PCR analysis confirmed that the TL-DNA including 654 bp rol B sequence was inserted into the genome of transformed hairy roots and their regenerated plants.