Enhanced recognition of G-quadruplex DNA oxidative damage based on DNA-mediated charge transfer.

G-quadruplex (G4) DNA is present in human telomere oligonucleotide sequences. Oxidative damage to telomeric DNA accelerates telomere shortening, which is strongly associated with aging and cancer. Most of the current analyses on oxidative DNA damage are based on ds-DNA. Here, we developed a electrochemiluminescence (ECL) probe for enhanced recognition of oxidative damage in G4-DNA based on DNA-mediated charge transfer (CT), which could specifically recognize damaged sites depending on the position of 8-oxoguanine (8-oxoG). First, a uniform G4-DNA monolayer interface was fabricated; the G4-DNA mediated CT properties were examined using an iridium(III) complex [Ir(ppy)2(pip)]PF6 stacked with G4-DNA as an indicator. The results showed that G4-DNA with 8-oxoG attenuated DNA CT. The topological effects of oxidative damage at different sites of G4-DNA and their effects on DNA CT were revealed. The sensing platform was also used for the sensitive and quantitative detection of 8-oxoG in G4-DNA, with a detection limit of 28.9 fmol. Overall, these findings present a sensitive platform to study G4-DNA structural and stability changes caused by oxidative damage as well as the specific and quantitative detection of oxidation sites. The different damage sites in the G-quadruplex could provide detailed clues for understanding the function of G4-associated telomere functional enzymes.

Sensitive electrochemical measurement of nitric oxide released from living cells based on dealloyed PtBi alloy nanoparticles.

Nitric oxide (NO), as a vital signaling molecule related to different physiological and pathological processes in living systems, is closely associated with cancer and cardiovascular disease. However, the detection of NO in real-time remains a difficulty. Here, PtBi alloy nanoparticles (NPs) were synthesized, dealloyed, and then fabricated to NP-based electrodes for the electrochemical detection of NO. Transmission electron microscopy (TEM), small-angle X-ray scattering (SAXS), and nitrogen physical adsorption/desorption show that dealloyed PtBi alloy nanoparticles (dPtBi NPs) have a porous nanostructure. Electrochemical impedance spectroscopy and cyclic voltammetry results exhibit that the dPtBi NP electrode possesses unique electrocatalytic features such as low charge transfer resistance and large electrochemically active surface area, which lead to its excellent NO electrochemical sensing performance. Owing to the higher density of catalytical active sites formed PtBi bimetallic interface, the dPtBi NP electrode displays superior electrocatalytic activity toward the oxidation of NO with a peak potential at 0.74 V vs. SCE. The dPtBi NP electrode shows a wide dynamic range (0.09-31.5 µM) and a low detection limit of 1 nM (3σ/k) as well as high sensitivity (130 and 36.5 µA µM-1 cm-2). Moreover, the developed dPtBi NP-based electrochemical sensor also exhibited good reproducibility (RSD 5.7%) and repeatability (RSD 3.4%). The electrochemical sensor was successfully used for the sensitive detection of NO produced by live cells. This study indicates a highly effective approach for regulating the composition and nanostructures of metal alloy nanomaterials, which might provide new technical insights for developing high-performance NO-sensitive systems, and have important implications in enabling real-time detection of NO produced by live cells.

Single-Cell Metabolomics-Based Strategy for Studying the Mechanisms of Drug Action.

Studying the mechanisms of drug antitumor activity at the single-cell level can provide information about the responses of cell subpopulations to drug therapy, which is essential for the accurate treatment of cancer. Due to the small size of single cells and the low contents of metabolites, metabolomics-based approaches to studying the mechanisms of drug action at the single-cell level are lacking. Herein, we develop a label-free platform for studying the mechanisms of drug action based on single-cell metabolomics (sMDA-scM) by integrating intact living-cell electro-launching ionization mass spectrometry (ILCEI-MS) with metabolomics analysis. Using this platform, we reveal that non-small-cell lung cancer (NSCLC) cells treated by gefitinib can be clustered into two cell subpopulations with different metabolic responses. The glutathione metabolic pathway of the subpopulation containing 14.4% of the cells is not significantly affected by gefitinib, exhibiting certain resistance characteristics. The presence of these cells masked the judgment of whether cysteine and methionine metabolic pathway was remarkably influenced in the analysis of overall average results, revealing the heterogeneity of the response of single NSCLC cells to gefitinib treatment. The findings provide a basis for evaluating the early therapeutic effects of clinical medicines and insights for overcoming drug resistance in NSCLC subpopulations.

NaCl Micro-Crystal as a Molecular Mold for Enhanced Synthesis of Planar Phenazines and Their Applications on Chemosensing and a Full-Color Fluorescent Material.

NaCl has been successfully used as a template for the synthesis of 2D nanomaterials, but it is seldom used for the construction of flat small organic molecules. Herein, a simple, low-cost, and highly efficient synthesis of phenazines with planar main frames, such as 5-phenyl-5,14-dihydro-5,7,12,14-tetraazapentacene, in the presence of NaCl micro-crystal as a kind of molecular mold is described. The reactants were mixed with NaCl powder and heated to 320 °C for 5 min. Yields >90% were readily achieved after a simple precipitation in water. The effectiveness of NaCl crystal as a mold with HCl was confirmed by comparison with common inorganic salts, SiO2, and γ-Al2O3 with HCl together with combinations including NaNO3 + HNO3, Na2SO4 + H2SO4, NaH2PO4 + H3PO4, and NaH2PO4 + polyphosphoric acid. The mechanism was deduced with the aid of computer simulation, which confirms the stabilization of 5,14-dihydro-5,7,12,14-tetraazapentacene by the NaCl surface. DMSO solution of a product, 1,3-dihydro-imidazo[4,5-b]phenazin-2-one, showed enhanced fluorescence in H2O, and it was used as a fluorescent probe for pH and Hg2+. A full-color material was prepared by mixing precursors of epoxy resin and phenazines, and its fluorescent color could be adjusted by the ratio of phenazines.

Synergy in Au-CuO Janus Structure for Catalytic Isopropanol Oxidative Dehydrogenation to Acetone.

The controlled oxidation of alcohols to the corresponding ketones or aldehydes via selective cleavage of the ß-C-H bond of alcohols under mild conditions still remains a significant challenge. Although the metal/oxide interface is highly active and selective, the interfacial sites fall far behind the demand, due to the large and thick support. Herein, we successfully develop a unique Au-CuO Janus structure (average particle size=3.8 nm) with an ultrathin CuO layer (0.5 nm thickness) via a bimetal in situ activation and separation strategy. The resulting Au-CuO interfacial sites prominently enhance isopropanol adsorption and decrease the energy barrier of ß-C-H bond scission from 1.44 to 0.01 eV due to the strong affinity between the O atom of CuO and the H atom of isopropanol, compared with Au sites alone, thereby achieving ultrahigh acetone selectivity (99.3 %) over 1.1 wt % AuCu0.75 /Al2 O3 at 100 °C and atmospheric pressure with 97.5 % isopropanol conversion. Furthermore, Au-CuO Janus structures supported on SiO2 , TiO2 or CeO2 exhibit remarkable catalytic performance, and great promotion in activity and acetone selectivity is achieved as well for other reducible oxides derived from Fe, Co, Ni and Mn. This study should help to develop strategies for maximized interfacial site construction and structure optimization for efficient ß-C-H bond activation.

Controllable Fabrication of Small-Size Holding Pipets for the Nondestructive Manipulation of Suspended Living Single Cells.

The capture and manipulation of single cells are an important premise and basis for intracellular delivery, which provides abundant molecular and omics information for biomedical development. However, for intracellular delivery of cargos into/from small-size suspended living single cells, the capture methods are limited by the lack of small-size holding pipets, poor cell activity, and the low spatial accuracy of intracellular delivery. To solve these problems, a method for the controllable fabrication of small-size holding pipets was proposed. A simple, homemade microforge instrument including an imaging device was built to cut and melt the glass capillary tip by controlling the heat production of a nichrome wire. The controllable fabrication of small-size holding pipets was realized by observing the fabrication process in real time. Combined with an electroosmotic drive system and a micromanipulation system with high spatial resolution, the holding pipet achieved the active capture, movement, and sampling of suspended living single cells. Moreover, solid-phase microextraction was performed on captured single pheochromocytoma cells, and the extracted dopamine was successfully detected using an electrochemical method. The homemade microforge instrument overcame the limitations of traditional microforges, resulting in holding pipets that were sufficiently small for small-size suspended single living cells (5-30 µm). This proactive capture method overcame the shortcomings of existing methods to achieve the multiangle, high-precision manipulation of single cells, thereby allowing the intracellular delivery of small-size single cells in suspension with high spatiotemporal resolution.

Solid-phase microextraction integrated nanobiosensors for the serial detection of cytoplasmic dopamine in a single living cell.

Dopamine participates in many physiological and pathological processes. Dynamic monitoring of dopamine levels in the cytoplasm of a single living cell reflects not only the functional state of dopamine synthesis factors but also the processes of related neurodegenerative diseases. Due to the low content of cytoplasmic dopamine and the difficulty to keep cells alive during the operating process, the detection of cytoplasmic dopamine is still challenging. Herein, a solid-phase microextraction (SPME) technique integrated nanobiosensor was employed to trace and quantify dopamine concentration fluctuations in the cytoplasm of a single living cell. We designed a polypyrrole modified carbon fiber nanoprobe as a bifunctional nanoprobe that can extract cytoplasmic dopamine and then perform electrochemical detection. This bifunctional nanoprobe can detect 10 pmol/L extracted dopamine and detected a 60% decrease of the cytoplasmic dopamine concentration in a single living cell by K+ stimulation. This study allowed for the first time serially detecting cytoplasmic dopamine while keeping the target cell alive, which might yield a new method for research on dopamine neurotoxicity and the related drug action mechanisms for neurodegenerative disease.

Aggregation-Induced Electrochemiluminescence of the Dichlorobis(1,10-phenanthroline)ruthenium(II) (Ru(phen)2Cl2)/Tri-n-propylamine (TPrA) System in H2O-MeCN Mixtures for Identification of Nucleic Acids.

Aggregation-induced electrochemiluminescence (AIECL) of the dichlorobis(1,10-phenanthroline)ruthenium(II) (Ru(phen)2Cl2)/tri-n-propylamine (TPrA) system was systematically investigated in H2O-MeCN media. Up to a 120-fold increase in the ECL intensity was observed when the H2O fraction (v%) was changed from 30% to 70%, whereas only an approximately 5.7-fold increase in the corresponding aggregation-induced fluorescence emission was demonstrated. The gradual formation of clusters of Ru(phen)2Cl2 nanoaggregates along with the increase in the H2O fraction to MeCN, which was verified by dynamic light scattering (DLS), scanning electron microscopy (SEM), and transmission electron microscopy (TEM), was believed to be responsible for the remarkable ECL enhancement. Significantly, the above-mentioned AIECL behavior was found to be very sensitive to the types and sequences of nucleic acids present in solution, which provided an effective and novel strategy for distinguishing RNA from DNA and for differentiating different miRNAs. The present study could have a substantial impact in various research areas, such as molecular sensors, bioimaging probes, organelle-specific imaging, and tumor diagnosis.

Electrogenerated Chemiluminescence Biosensor with a Tripod Probe for the Highly Sensitive Detection of MicroRNA.

A novel probe for the highly sensitive detection of microRNA with enhanced helix accessibility and good assembling without backfilling was developed using a tripod structure fabricated by triplex DNA. A layer of triplex DNA assembled on electrodeposited reduced graphene oxide was used as the capture probe, and a subsequent hybridization chain reaction that promoted the efficient intercalation of the electrogenerated chemiluminescence (ECL) emitter [Ru(bpy)2(dppz)]2+ (bpy refers to 2,2'-bipyridine, and dppz refers to dipyrido[3,2- a:2',3'- c]phenazine) was used as an analytical-signal amplifier. The fabricated biosensor was examined with an anodic ECL mode using tri- n-propyl amine as the coreactant. The construction of the biosensor was systematically characterized with various techniques including atomic-force microscopy, gel electrophoresis, cyclic voltammetry, and electrochemical-impedance spectroscopy, and its performance was optimized under a variety of experimental conditions, especially the concentration of each reagent as well as the incubation time. Under the optimal experimental conditions, the reported biosensor showed a very low limit of detection of 0.10 fM (S/N = 3) and a wide linear dynamic range covering 0.50 fM to 100 pM toward microRNA-155 with excellent specificity, stability, and reproducibility. Finally, the biosensor was successfully applied to the detection of microRNA-155 extracted from the colon-cancer cell line DLD1, demonstrating its potential application in the sensitive detection of biological samples in the early diagnosis of diseases.

Rapid separation and sensitive determination of banned aromatic amines with plastic microchip electrophoresis.

Rapid analysis of trace amount of aromatic amines in environmental samples and daily necessities has attracted considerable attentions because some of them are strongly toxic and carcinogenic. In this study, fast and efficient electrophoretic separation and sensitive determination of 5 banned aromatic amines were explored for practical analysis using disposable plastic microchips combined with a low-cost laser-induced fluorescence detector. The effect of running buffer and its additive was systematically investigated. Under the selected condition, 5 fluorescein isothiocyanate labeled aromatic amines could be baseline separated within 90s by using a 10mmol/L borate buffer containing 2% (w/v) hydroxypropyl cellulose. Calibration curves of peak areas vs. concentrations were linear up to 40 or 120µmol/L for different analytes and limits of detection were in a range of 1-3nmol/L. Theoretical plate numbers of 6.8-8.5×10(5)/m were readily achieved. The method exhibited good repeatability, relative standard deviations (n=5) of peak areas and migration times were no more than 4.6% and 0.9%, respectively. The established method was successfully applied in the quantitative analysis of these banned aromatic amines in real samples of waste water and textile, recoveries of added standards were 85-110%.

Microfluidic cell culture and metabolism detection with electrospray ionization quadrupole time-of-flight mass spectrometer.

A novel method for the characterization of drug metabolites was developed by integrating chip-based solid-phase extraction (SPE) with an online electrospray ionization quadrupole time-of-fight mass spectrometer (ESI-Q-TOF-MS). The integrated microfluidic device was composed of circular chambers for cell culture and straight microchannels with shrink ends to pack the solid-phase material for sample cleanup and concentration prior to mass analysis. By connecting the two separated microchannels with polyethylene tubes, drug metabolism studies related to functional units, including cell culture, metabolism generation, sample pretreatment, and detection, were all integrated into the microfluidic device. To verify the feasibility of a drug metabolism study on the microfluidic device, the metabolism of vitamin E in human lung epithelial A549 cells was studied. The metabolites were successfully detected by online ESI-Q-TOF-MS with high sensitivity and short analysis time (8 min). By integrating several parallel channels, the desalting and concentration process could be simultaneously achieved. The total sample pretreatment time only needed about 15 min, and solvent consumption could be reduced to less than 100 microL. All this demonstrated that the developed microfluidic device could be a potential useful tool for cellular drug metabolism research.

A microfluidic approach for anticancer drug analysis based on hydrogel encapsulated tumor cells.

A novel method based on fluorescence detection of hydrogel encapsulated cells in microchannels was developed for anticancer drug analysis. In this work, human hepatoma HepG2 cells and human lung epithelial A549 cells were simultaneously immobilized inside two different shapes of three-dimensional hydrogel microstructures using photolithography approach on a same array. Microarrays of living cells offer the potential for parallel detection of many cells and thereby enable high-throughput assays. Using a photolithographic setup, we investigated the prepolymer composition and crosslinking parameters that influenced cell viability inside photocrosslinked hydrogels. The viability of cells encapsulated inside hydrogel microstructures was higher than 90% under optimized photocrosslinking conditions. The cells were further cultured under stable conditions and remained viable for at least three days that were able to carry out cell-based assays. Furthermore, we studied the variation of two intracellular redox parameters (glutathione and reactive oxygen species) in anticancer drug-induced apoptosis in HepG2 and A549 cells. Two anticancer drugs exhibited distinct effects on the levels of intracellular glutathione and reactive oxygen species, indicating the selectivity of these drugs on the disturbance of redox balance within cells. The established platform provides a convenient and fast method for monitoring the effect of anticancer drugs on tumor cells, which is very useful for fundamental biomedical research.