Effect of cell settlement on the electrochemical collision behaviors of single microbes.

Electrochemical collision technique has emerged as a powerful approach to detect the intrinsic properties of single entities. The diffusion model, together with migration and convection processes are generally used to describe the transport and collision processes of single entities. However, things become more complicated concerning microbes because of their relatively large size, inherent motility and biological activities. In this work, the electrochemical collision behaviors of four different microorganisms: Escherichia coli (Gram-negative bacteria), Staphylococcus aureus, Bacillus subtilis (Gram-positive bacteria) and Saccharomyces cerevisiae (fungus) were systematically detected and compared using a blocking strategy. By using K4Fe(CN)6 as redox probe, the downwards step-like signals were recorded in the collision process of all the three bacteria, whereas the collision of S. cerevisiae was rarely detected. To further investigate the underlying reason for the abnormal collision behavior of S. cerevisiae, the effect of cell settlement was discussed. The results indicated that ellipsoidal S. cerevisiae with a cell size larger than 2 µm exhibited a cell sedimentation rate of 261.759 nm s-1, which is dozens of times higher than the other three bacteria. By further enhanced convection near the microelectrode or positioned the microelectrode at the bottom of electrochemical cell, the collision signals of S. cerevisiae were successfully detected, indicating cell sedimentation is a nonnegligible force in large cell transport. This study fully addressed the effect of cell settlement on the transport of microbial cells and provided two strategies to counteract this effect, which benefit for the deeper understanding and further application of electrochemical collision technique in single-cell detection.

Feasibility investigation and development of microbial electrochemical biosensors for marine pollution monitoring.

Electrochemical biosensor, as a real-time and rapid detection method, has rarely been explored in marine monitoring. In present work, microbial electrochemical biosensors based on two design strategies: disperse system and integrated microbial electrode, were systematically discussed and their feasibility in marine biotoxicity assessment were investigated. An isolation method was initially investigated to eliminate the potential interference and detect the biological response accurately. The influence of water salinity on the current response was eliminated by adopting the salt-tolerant bacteria Staphylococcus aureus as test microorganism and buffer solution with sufficient ionic strength. The biotoxicity of heavy metal ions and pesticides were sensitively determined. Furthermore, a novel integrated microbial biosensor was designed by immobilizing S. aureus with a redox-active gel that consists of chitosan and poly (diallyl dimethyl ammonium chloride) mixture and confined potassium ferricyanide via electrostatic interaction. The IC50 values for Cu2+, Zn2+, Cr2O72- and Ni2+ were 3.01 mg/L, 1.34 mg/L, 7.64 mg/L and 9.41 mg/L, respectively. This work not only verified the feasibility of electrochemical biosensor in marine pollution monitoring, but also compared the pros and cons of two biosensor design strategies, which provide a guidance for the future development and application of marine monitoring devices based on electrochemical method.

Superhydrophobic Nanodiamond-Functionalized Melamine Sponge for Oil/Water Separation.

Fabrication of absorbent materials foroil/water separation is an important ecological pursuit for oil spill clean-up and organic pollutants' removal. In this study, nanodiamonds (NDs), a promising member of the carbon family, were functionally modified by the covalent linking of octadecylamine (ODA). Subsequently, the superhydrophobic sponge with hierarchical microstructures was fabricated by embedding ND-ODA into a melamine sponge (MS) skeleton via a simple immersion-drying process. The as-prepared sponge (ND-ODA@PDMS@MS) showed superhydrophobic properties with a water contact angle of 155 ± 2°. For various oils and organic solvents, ND-ODA@PDMS@MS possesses excellent absorption capacity (26.65-55.64 g/g) and oil/water separation efficiency (above 98.6%). Furthermore, the adsorption capacity to crude oil remained relatively stable in highly acidic, alkaline, and salty conditions, ensuring the application in the clean-up of industrial oily sewages and marine oil spills. Besides absorbing oil for a single time, ND-ODA@PDMS@MS also exhibited satisfactory performance in continuous oil/water separation. Therefore, this work provides a facile strategy to produce robust and efficient absorbent materials for oil/water separation in large-scale oil and organic solvent clean-ups.

Three-Mediator Enhanced Collisions on an Ultramicroelectrode for Selective Identification of Single Saccharomyces cerevisiae.

Selective detection of colliding entities, especially cells and microbes, is of great challenge in single-entity electrochemistry. Herein, based on the different cellular electron transport pathways between microbes and mediators, we report a three-mediator system [K3Fe(CN)6, K4Fe(CN)6, and menadione] to achieve redox activity analysis and selective identification of single Saccharomyces cerevisiae without the usage of antibodies. K4Fe(CN)6 in the three-mediator system will oxidize near the electrode surface and increase the local concentration of K3Fe(CN)6, which will promote the redox reaction of S. cerevisiae. The hydrophobic mediator─menadione─can selectively penetrate through the S. cerevisiae membrane and get access to its intracellular redox center and can further react with K3Fe(CN)6 in the bulk solution. In contrast, the mediator can only get access to the bacterial membranes of Escherichia coli and Staphylococcus aureus, which results in different electrochemical collision signals between the above microbes. In the three-mediator system, upward step-like collision signals were observed in S. cerevisiae suspension, which are related to their microbial redox activity. In comparison, E. coli or S. aureus only generated downward current steps because the blockage effect of mediator diffusion suppresses their redox activities. When S. cerevisiae co-existed with E. coli or S. aureus, transients generated by both blockage and redox activity were observed. The approach enables us to trace the collision behaviors of different microbes and distinguish their simultaneous collisions, which is the foundation for further application of electrochemical collision technique in the specific identification of single biological entities.

Photosensitizer Functionalized Nanodiamonds for Raman Imaging and Photodynamic Therapy of Cancer Cells.

One novel nanoplatform with multiple functions including Raman imaging and photodynamic therapy (PDT) capacities was constructed through modifying nanodiamonds (NDs) with photosensitizer chlorin e6 (Ce6). The NDs-Ce6 nanoparticles show enhanced singlet oxygen generation efficiency relative to free Ce6. Cytotoxicity tests indicate that NDs-Ce6 have negligible influence toward HeLa cells vitality under dark condition but enhanced photodynamic ablation upon 660 nm laser irradiation in comparison with free Ce6. In addition, the NDs-Ce6 could be used as Raman imaging probes toward HeLa cells. These results demonstrate that the NDs-Ce6 multifunctional nanoplatform have attractive features using for Raman imaging and PDT. Additionally, a new idea could be provided for designing the multifunctional platform from the work.

Nanodiamonds Inhibit Cancer Cell Migration by Strengthening Cell Adhesion: Implications for Cancer Treatment.

Nanodiamonds (NDs) are a type of biocompatible nanomaterial with easily modified surfaces and are considered as promising candidates in biomedicine. In this work, the inhibition of tumor cell migration by carboxylated nanodiamonds (cNDs) was investigated. AFM-based single cell adhesion and F-actin staining experiments demonstrated that cNDs treatment could enhance cell adhesion and impair assembly of the cytoskeleton. The mechanism analysis of the regulatory protein expression level also proved that cNDs could inhibit the migration of Hela cells by preventing the epithelial-mesenchymal transition (EMT) process through the transforming growth factor ß (TGF-ß) signaling pathway. The in vivo pulmonary metastasis model also showed that cNDs effectively reduced the metastasis of murine B16 melanoma cells. In summary, cNDs have been demonstrated to inhibit cancer cell migration in vitro and decrease tumor metastasis in vivo. Therefore, cNDs might have potential utility for specific cancer treatment.

Redox activity of single bacteria revealed by electrochemical collision technique.

This paper reports on an innovative strategy based on the electrochemical collision technique to quantify the redox activity of two bacterial species: the Gram-negative Escherichia coli and the Gram-positive Bacillus subtilis. Thionine (TH), as a redox mediator, was electrostatically adsorbed on bacterial surface and formed the bacterium-TH complexes. TH can receive electrons from bacterial metabolic pathways and be reduced. When a single bacterium-TH complex collides on the ultramicroelectrode, the reduced TH will be re-oxidized at certain potential and generate current spike. The frequency of the spikes is linearly proportional to the living bacteria concentration, and the redox activity of individual bacterium can be quantified by the charges enclosed in the current spike. The redox ability of Gram-negative E.coli to the TH mediator was 6.79 ± 0.26 × 10-18 mol per bacterial cell in 30 min, which is relatively more reactive than B. subtilis (3.52 ± 0.31 × 10-18 mol per cell). The spike signals, fitted by 3D COMSOL Multiphysics simulation, revealed that there is inherent redox ability difference of two bacterial strains besides the difference in bacterial size and collision position. This work successfully quantified the bacterial redox activity to mediator in single cells level, which is of great significance to improve understanding of heterogeneous electron transfer process and build foundations to the microorganism selection in the design of microbial electrochemical devices.

A portable instrument for monitoring acute water toxicity based on mediated electrochemical biosensor: Design, testing and evaluation.

A water quality early-warning instrument for evaluating acute water toxicity based on the electrochemical biosensor, Model ETOX18-01, was developed and manufactured with the features of low current detection (0.1 nA), precise thermostatic control, self-cleaning as well as remote data transmission. A sensitive integrated microbial electrode, made up of a glass carbon electrode that was modified by an active biofilm consisting of Escherichia coli, thionine, carbon nanodots and chitosan, has been fabricated as the biosensor. To validate the performance, multiple real water samples and artificial water samples were tested by Model ETOX18-01, and compared with ISO standardized luminescent bacterial test simultaneously. The correlation between the Model ETOX18-01 and luminescent bacterial test for these water samples showed good determination coefficient (R2 = 0.9827). In addition, Model ETOX18-01 is more sensitive to colored metal ionic samples. With its characteristics of high sensitivity, excellent repeatability and easy operation, the instrument Model ETOX18-01 provides a promising tool for large-scale water environmental assessment, and has a potential application in evaluating the water quality and early risk warning.

Development of an Escherichia coli-based electrochemical biosensor for mycotoxin toxicity detection.

Mycotoxin contamination in food and feed is a global concern because mycotoxin contamination can cause both acute and chronic health effects in humans and animals. In the present work, an Escherichia coli-based biosensor is described for the toxicity assessment of aflatoxin B1 (AFB1) and zearalenone (ZEN). In this electrochemical biosensor, E. coli is used as the signal recognition element, p-benzoquinone is used as the mediator, and a two-step reaction procedure has been developed to separate the mediator from the mycotoxins. The current value of the as-prepared microbial biosensor exhibits a linear decrease with concentrations of AFB1 and ZEN in the range of 0.01-0.3 and 0.05-0.5 µg/mL, with detection limits reaching 1 and 6 ng/mL, respectively. The IC25 values obtained by the present method are 0.25 and 0.40 µg/mL for AFB1 and ZEN, which shows that the cytotoxicity of AFB1 to E. coli is more severe than the cytotoxicity of ZEN to E. coli. The combined toxic effect of these two mycotoxins has also been explored, and synergistic biotoxicity has been observed. Moreover, the biosensor is successfully applied to the toxicity evaluation of mycotoxins in real samples, including peanut and corn oils. This work could provide new insight into mycotoxin and microorganism interactions and could establish a new approach for future mycotoxin detection.

Nanodiamond-Based Theranostic Platform for Drug Delivery and Bioimaging.

Nanodiamonds (NDs) are promising candidates for biomedical application due to their excellent biocompatibility and innate physicochemical properties. In this Concept article, nanodiamond-based theranostic platforms, which combine both drug delivery features and bioimaging functions, are discussed. The latest developments of therapeutic strategies are introduced and future perspectives for theranostic NDs are addressed.

Nanodiamonds conjugated upconversion nanoparticles for bio-imaging and drug delivery.

A multifunctional nanoplatform with simultaneous imaging and therapeutic capacities has been fabricated by covalently conjugating nanodiamonds (NDs) with upconversion NaYF4:Yb,Er nanoparticles (UCNPs). Green emission can be observed for the UCNP-NDs composites under 980 nm excitation, and in vitro imaging was carried out towards HeLa cells. The UCNP-NDs also shows good drug storage capability toward anticancer drug doxorubicin (Dox) and exhibits significant pH-dependent drug-release behavior. Dox-loaded UCNP-NDs shows higher therapy efficiency towards Hela cells than free Dox when the equivalent concentration of the Dox is more than 10 µg/mL. Thus, a favorable strategy may be provided to decrease the side effects of Dox, minimize drug dose, and improve its efficacy. These findings highlight the fascinating features of UCNP-NDs nanocomposites as upconversion fluorescence imaging contrast agents and doxorubicin loading carrier. In addition, this work may also provide a novel strategy for the design of multifunctional nanoplatforms.

Electrochemical synthesis of multicolor fluorescent N-doped graphene quantum dots as a ferric ion sensor and their application in bioimaging.

A novel electrochemical strategy for simple and facile synthesis of semicarbazide functionalized nitrogen-doped graphene quantum dots (N-GQDs) was reported, based on direct exfoliation and oxidation from graphite rods. The average diameter of the as-synthesized N-GQDs is about 20 nm, and their dispersion is bright yellow due to the rich nitrogen and oxygen functional groups on their surface. The N-GQD dispersion was further applied in the selective detection of ferric ions (Fe3+) based on the photoluminescence (PL) quenching of N-GQDs after adding Fe3+. The fluorescent sensor has a wide linear range of 0-200 µM and a detection limit of 0.87 µM, which is much lower than the maximum level (0.3 mg L-1, equivalent to 5.4 µM) of Fe3+ permitted in drinking water by the U.S. Environmental Protection Agency (EPA). Moreover, these novel N-GQDs exhibit much wider emission bands, which extend into the entire visible region, and emit three primary color fluorescence independently. This distinctive behavior of the as-prepared GQDs not only breaks the limitation that traditional reported GQDs only exhibit blue emission in the short-wavelength region, but may also provide a new research platform for further applications of GQDs in real environmental detection and biological imaging systems.

Toward the Detection and Identification of Single Bacteria by Electrochemical Collision Technique.

Fast and cost-efficient detection and identification of bacteria in food and water samples and biological fluids is an important challenge in bioanalytical chemistry. It was shown recently that bacteria can be detected by measuring the decrease in the diffusion current to the ultramicroelectrode caused by cell collisions with its surface. To add selectivity to the bacteria detection, herein we show the possibility of collision experiments with the signal produced by electrochemical activity of bacterial cells reducing (or oxidizing) redox species. The mediator oxidation/reduction rate can be used to identify different types of bacteria based on their specific redox activities. Here we report the analysis of electrochemical collision transients produced by two kinds of bacteria, Escherichia coli and Stenotrophomonas maltophilia. The effects of the charge and redox activity of bacterial cells on collision events are discussed. The current transients due to live cell collisions were compared to those produced by bacteria killed either by heavy metal ions (cobalt) or by an antibiotic (colistin). This approach is potentially useful for evaluating the effectiveness of antimicrobial agents. Finite-element simulations were carried out to model collision transients.

Simple preparation and highly selective detection of silver ions using an electrochemical sensor based on sulfur-doped graphene and a 3,3',5,5'-tetramethylbenzidine composite modified electrode.

A novel electrochemical sensor based on sulfur (S)-doped graphene (S-Gr) and a 3,3',5,5'-tetramethylbenzidine (TMB) composite (S-Gr-TMB) modified glassy carbon (GCE) electrode for highly selective quantitative detection of silver ions (Ag+) were fabricated. The S-Gr-TMB composite was first prepared via electrostatic interaction between TMB and S-Gr and then, the composite was coated on the surface of GCE. The resultant S-Gr-TMB/GCE electrode showed a significant voltammetric response to Ag+ at 0.3 V vs. Ag/AgCl due to the synergistic effect of S-Gr and TMB. The sensor showed good linearity from 50 µM to 400 µM with a detection limit of 2.15 µM towards the determination of Ag+. In addition, after the addition of Fe3+ and other metal ions, including Al3+, Ca2+, Cd2+, Co2+, Cu2+, K+, Mg2+, Na+, Ni2+, Pb2+ and Zn2+, in the same concentration, the current signal remained almost unchanged, revealing that the proposed electrochemical sensor exhibited a high selectivity for Ag+, which solves the nonselective problem of TMB as a spectral probe. This enhanced detection performance is attributed to two factors: (1) S-Gr has excellent electrical conductivity; (2) the coupling interactions between Ag-S are speculated to result in strengthened enrichment for Ag and good selective performance.

Problems analysis and new fabrication strategies of mediated electrochemical biosensors for wastewater toxicity assessment.

Conventional mediated electrochemical biosensors for toxicity assessment were almost based on 'one-pot' principle, i.e., mediators and the under-test chemicals were mixed together in the same vessel. In this process, the electron mediator is assumed to be merely an electron acceptor and cannot react with under-test toxicants. Actually，some under-test pollutants (such as metal ions) could react with the electron mediators, thus affecting the detection accuracy and sensitivity of the sensors. It was also found that at least two other interference factors have been ignored in present'one-pot' mediated electrochemical biosensor systems, i.e., (1) the electrochemical sensitivity of mediators to pH; and (2) the potential reactions between under-test chemicals and buffers and the consequent pH changes. In this study, the three ignored interference factors have been investigated systematically and demonstrated by significance tests. Moreover, a solving strategy, an isolation method, is proposed for fabrication of novel mediated electrochemical biosensor to avoid the interference factors existing at present mediated electrochemical biosensor. According to the testing results obtained from the isolation method, IC50 values of Cu2+, Cd2+, Zn2+, Fe3+, Ni2+ and Cr3+ were 21.3 mg/L, 3.7 mg/L, 26.7 mg/L, 4.4 mg/L and 10.7 mg/L, respectively. The detection results of four real water samples also suggested this method could be applied for the practical and complex samples.

Cetuximab-conjugated nanodiamonds drug delivery system for enhanced targeting therapy and 3D Raman imaging.

In this study, a multicomponent nanodiamonds (NDs)-based targeting drug delivery system, cetuximab-NDs-cisplatin bioconjugate, combining both specific targeting and enhanced therapeutic efficacy capabilities, is developed and characterized. The specific targeting ability of cetuximab-NDs-cisplatin system on human liver hepatocellular carcinoma (HepG2) cells is evaluated through epidermal growth factor receptor (EGFR) blocking experiments, since EGFR is over-expressed on HepG2 cell membrane. Besides, cytotoxic evaluation confirms that cetuximab-NDs-cisplatin system could significantly inhibit the growth of HepG2 cells, and the therapeutic activity of this system is proven to be better than that of both nonspecific NDs-cisplatin conjugate and specific EGF-NDs-cisplatin conjugate. Furthermore, a 3-dimensional (3D) Raman imaging technique is utilized to visualize the targeting efficacy and enhanced internalization of cetuximab-NDs-cisplatin system in HepG2 cells, using the NDs existing in the bioconjugate as Raman probes, based on the characteristic Raman signal of NDs at 1332 cm-1 . These advantageous properties of cetuximab-NDs-cisplatin system propose a prospective imaging and treatment tool for further diagnostic and therapeutic purposes.

A double-mediator based whole cell electrochemical biosensor for acute biotoxicity assessment of wastewater.

This work investigates the feasibility and sensitivity of a double-mediator based whole cell electrochemical biosensor to detect the acute biotoxicity of wastewater. The lipophilic mediator menadione was used to mediate the intracellular metabolic activities whereas hydrophilic potassium ferricyanide was employed as extracellular electron acceptor to transport the electron from the menadiol to anode. A chitosan hydrogel polymer film with boron-doped nanocrystalline diamond (BND) particles was electrodeposited onto a glassy carbon (GC) electrode to immobilize Saccharomyces cerevisiae cells and the mediators. The feasibility of the as-prepared biosensor was verified by determine the acute biotoxicity of four heavy metal ions(Cu2+, Cd2+, Ni2+, Pb2+), three phenol pollutants (3,5-dichlorophenol, 4-chlorophenol, phenol) and three real wastewater samples. The IC50 values for Cu2+, Cd2+, Ni2+, Pb2+ are 10.12mg/L,13.88mg/L, 17.06mg/L and 34.56mg/L. And the IC50 value is 16.48mg/L, 34.40mg/L and 44.55mg/L for 3,5-dichlorophenol, 4-chlorophenol and phenol, respectively. The results of this work indicate that the double-mediator based whole cell electrochemical biosensor could be applied into the acute toxicity assessment of real wastewater samples with excellent performance and highlight their merit as portable and sensitive, which may providing a reasonable and reliable way for wastewater toxicity online detection.

Development of a mediated whole cell-based electrochemical biosensor for joint toxicity assessment of multi-pollutants using a mixed microbial consortium.

Since most risk assessment for toxicants is based on individual single-species test, the deduction of such results to ecosystem evaluation is afflicted with uncertainties. Herein, we successfully developed a p-benzoquinone mediated whole-cell electrochemical biosensor for multi-pollutants toxicological analysis by co-immobilizing mixed strains of microorganism, including Escherichia coli (gram-negative bacteria), Bacillus subtilis (gram-positive bacteria) and Saccharomyces cerevisiae (fungus). The individual and combined toxicities of heavy metal ions (Cu(2+), Cd(2+)), phenol (3,5-dichlorophenol) and pesticides (Ametryn, Acephate) were examined. The experimental results showed that the order of toxicity for individual toxicant was ranked as Cu(2+) > 3,5-dichlorophenol (DCP) > Ametryn > Cd(2+) > Acephate. Then the toxic unit (TU) model was applied to determine the nature of toxicological interaction of the toxicants which can be classified as concentration additive (IC50mix = 1TU), synergistic (IC50mix < 1TU) and antagonistic (IC50mix > 1TU) responses. The binary combination of Cu(2+) + Cd(2+), Cu(2+) + DCP, Cu(2+) + Acephate, DCP + Acephate, Acephate + Ametryn were analyzed and the three kind of joint toxicity effects (i.e. additive, synergistic and antagonistic) mentioned above were observed according to the dose-response relationship. The results indicate that the whole-cell electrochemical biosensor based on mixed microbial consortium is more reasonable to reflect the joint biotoxicity of multi-pollutants existing in real wastewater, and combined effects of toxicants is extremely necessary to be taken into account in ecological risk assessment. Thus, present study has provided a promising approach to the quality assessment of wastewater and a reliable way for early risk warning of acute biotoxicity.

Adaptive use of a personal glucose meter (PGM) for acute biotoxicity assessment based on the glucose consumption of microbes.

In this study, a new method for acute biotoxicity assessment was proposed by measuring the glucose consumption of microbes with a personal glucose meter (PGM). To obtain an ideal biotoxicity assessment performance, an appropriate microbe was selected first, and then the relevant parameters, such as temperature and microbial concentration were optimized. Under the optimized parameters, the acute biotoxicity of four environmental pollutants (As(3+), Ni(2+), 4-chlorophenol, and 2,4-dichlorophenol), three wastewater samples and three soil samples were evaluated. This technology breakthrough will help us develop a low cost, easy to use water-environmental early-warning kit.

Correlation between band alignment and enhanced photocatalysis: a case study with anatase/TiO2(B) nanotube heterojunction.

It has long been known that efficient interfacial charge transfer between different phases of TiO2 is beneficial for enhanced photocatalysis. However, there has been considerable debate over the direction of the charge transfer across the interface of the different TiO2 phases. In this work, we study the case of TiO2 with a novel anatase/TiO2(B) heterojunction (ABHJ), wherein charge carrier transfer across the heterojunction interface is intensively investigated. The ABHJ is prepared by a two-step alkaline hydrothermal route and features nanotubes with a large surface area. Comprehensive analysis including UV-Vis-DRS, XPS, Mott-Schottky measurements, EPR and transient photovoltage techniques provides evidence for a type II band alignment in the ABHJ and migration of the photogenerated electrons from anatase to TiO2(B), which could effectively inhibit the recombination rate of photo-induced electron-hole pairs. Photocatalytic tests demonstrate that the as-obtained ABHJ shows higher activity than both the single phase and P25, not only for hydrogen production, but also for the photodegradation of gaseous acetaldehyde, which is due to the synergistic effect between an efficient charge separation at the interface and a high surface area.