A standard procedure for rapid toxicity evaluation of carbon dots both in vitro and in vivo.

Carbon dots (CDs) are an emerging class of fluorescent quantum dot nanomaterials that have attracted considerable scientific attention for biomedical or bioimaging applications due to their physicochemical and biochemical properties. With the emergence of massive novel synthetic CDs applying to biomedical fields of science, evaluating their biosafety before any biological application is essential. However, there is no universal protocol or routine procedures for toxicity detection and biosafety assessment of CDs in general biological environments. Herein, we provide an ideal and fast operating system to detect the biotoxicity of CDs, which has been preliminary practiced. Briefly, the obtained CDs will be evaluated by in vitro cytotoxicity assay using cell counting kit-8, lactate dehydrogenase assay kit, and flow cytometry. Meanwhile, the model creature zebrafish is employed to perform in vivo evaluation by measuring body length, hatching rate, heart rate, and morphological observation. Our operating procedure condenses previous scattered biosafety detection methods into a rapid standard evaluation protocol that can be applied to early biotoxicity screening of CDs. This protocol will accelerate CDs biological exploitation and guide future industrialized biosafety assessment in large-scale applications.

Atmospheric pollutant black carbon induces ocular surface damage in mice.

The threats of air pollution to human health have been gradually discovered, including its effects on eyes. The purpose of the study is to investigate the potential correlation between ocular surface exposure to black carbon and ocular surface structural damage as well as tear film dysfunction. To achieve this goal, 60 6-8-week-aged male BALB/C mice were randomly divided into 4 groups (n = 15). 0.5 mg/ml (group A), 1 mg/ml (group B), 5 mg/ml (group C) black carbon suspension droplets and PBS solution (group D) were used in the right eyes, 4 µl per time of three times per day. Tear break-up time, corneal fluorescein staining scores, and tear volume were assessed before treatment (day 0) and on days 4, 7, 10, and 14 after treatment. On day 14, the mice were sacrificed, and corneal and conjunctival tissues were collected for histological analysis. As the exposure time increased, there were no significant changes in the measured parameters from PBS-treated group of mice (P > 0.05). However, in the black carbon-treated group, there were significant decreases in tear film break-up time, significant increases in corneal fluorescein staining scores, and significant reductions in tear secretion (all P < 0.05). After 14 days, H&E staining of the corneal epithelium showed that in the PBS-treated group of mice, the corneal epithelial cells were neatly arranged, with no inflammatory cell infiltration, while in the black carbon-treated group, the corneal epithelium was significantly thickened, the basal cell arrangement was disrupted, the number of cell layers increased, and there was evidence of inflammatory cell infiltration. In the ultrastructure of the corneal epithelium, it could be observed that the black carbon-treated group had an increased amount of corneal epithelial cell detachment compared to the PBS-treated group, at the same time, the intercellular connections were looser, and there was a decrease in the number of microvilli and desmosomes in the black carbon-treated group. The results indicate that the ocular surface exposure to black carbon can result in a decrease in tear film stability and tear secretion in mice. Moreover, it can induce alterations in the corneal structure.

The discovery of regional neurotoxicity-associated metabolic alterations induced by carbon quantum dots in brain of mice using a spatial metabolomics analysis.

BACKGROUND: Recently, carbon quantum dots (CQDs) have been widely used in various fields, especially in the diagnosis and therapy of neurological disorders, due to their excellent prospects. However, the associated inevitable exposure of CQDs to the environment and the public could have serious severe consequences limiting their safe application and sustainable development. RESULTS: In this study, we found that intranasal treatment of 5 mg/kg BW (20 µL/nose of 0.5 mg/mL) CQDs affected the distribution of multiple metabolites and associated pathways in the brain of mice through the airflow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI) technique, which proved effective in discovery has proven to be significantly alerted and research into tissue-specific toxic biomarkers and molecular toxicity analysis. The neurotoxic biomarkers of CQDs identified by MSI analysis mainly contained aminos, lipids and lipid-like molecules which are involved in arginine and proline metabolism, biosynthesis of unsaturated fatty acids, and glutamine and glutamate metabolism, etc. as well as related metabolic enzymes. The levels or expressions of these metabolites and enzymes changed by CQDs in different brain regions would induce neuroinflammation, organelle damage, oxidative stress and multiple programmed cell deaths (PCDs), leading to neurodegeneration, such as Parkinson's disease-like symptoms. This study enlightened risk assessments and interventions of QD-type or carbon-based nanoparticles on the nervous system based on toxic biomarkers regarding region-specific profiling of altered metabolic signatures. CONCLUSION: These findings provide information to advance knowledge of neurotoxic effects of CQDs and guide their further safety evaluation.

Synthesis Processes, Photoluminescence Mechanism, and the Toxicity of Amorphous or Polymeric Carbon Dots.

Fluorescence is the emission of light following photon absorption. This optical phenomenon has many applications in daily life, such as in LED lamps, forensics, and bioimaging. Traditionally, small-molecule fluorophores were most common, but the types of molecules and particles with compelling fluorescence properties have expanded. For example, green fluorescent protein (GFP) was isolated from jellyfish and won the Nobel prize in 2008 due to its significant utility as a fluorescent biomarker. Using the intrinsic fluorescence of GFP, many previously invisible biological processes and substances can now be observed and studied. Other fluorescent materials have also been developed, greatly expanding the potential applications. Semiconductor quantum dots (QDs), which have bright fluorescence and a narrow bandwidth, are a popular choice for display technologies. However, QDs are made of heavy metal elements such as Cd and Se, which pose potential safety concerns to the environment and human health. Thus, new fluorescent organic materials are being developed to mitigate the toxicological concerns while maintaining the QD advantages.One type of new material attracting great attention as an environmentally friendly substitute for semiconductor QDs is carbon dots (CDs). CDs have been developed with strong fluorescence, good photostability, and low toxicity using a variety of precursors, and some synthesis processes have good potential for scale-up. However, since they are made of a variety of materials and through different methods, the structure and properties of CDs can differ from preparation to preparation. There are three major types of CDs: graphene quantum dots (GQDs), carbon quantum dots (CQDs), and amorphous or polymeric carbon dots (PCDs). This Account focuses on PCDs and their unique properties by comparing it with other types of CDs. The synthesis processes, fluorescence properties, fluorescence mechanisms, and toxicity are discussed below with an emphasis on the distinct attributes of PCDs.PCDs can be synthesized from small molecules or polymers. They have an amorphous or cross-linked polymer structure with bright fluorescence. This fluorescence is possibly due to cross-link-enhanced emission or clusteroluminescence that arises from the through-space interactions of heteroatomic-rich functional groups. Other fluorescence mechanisms of CDs, including distinct contributions from the carbon core and surface states, may also contribute. The toxicological profiles of CDs are influenced by the chemical composition, surface functionalization, and light illumination. CDs are generally thought to be of low toxicity, and this can be further improved by removing toxic byproducts, functionalizing the surface, and reducing light exposure to minimize the generation of reactive oxygen species.

Placental-fetal distribution of carbon particles in a pregnant rabbit model after repeated exposure to diluted diesel engine exhaust.

BACKGROUND: Airborne pollution particles have been shown to translocate from the mother's lung to the fetal circulation, but their distribution and internal placental-fetal tissue load remain poorly explored. Here, we investigated the placental-fetal load and distribution of diesel engine exhaust particles during gestation under controlled exposure conditions using a pregnant rabbit model. Pregnant dams were exposed by nose-only inhalation to either clean air (controls) or diluted and filtered diesel engine exhaust (1 mg/m3) for 2 h/day, 5 days/week, from gestational day (GD) 3 to GD27. At GD28, placental and fetal tissues (i.e., heart, kidney, liver, lung and gonads) were collected for biometry and to study the presence of carbon particles (CPs) using white light generation by carbonaceous particles under femtosecond pulsed laser illumination. RESULTS: CPs were detected in the placenta, fetal heart, kidney, liver, lung and gonads in significantly higher amounts in exposed rabbits compared with controls. Through multiple factor analysis, we were able to discriminate the diesel engine exposed pregnant rabbits from the control group taking all variables related to fetoplacental biometry and CP load into consideration. Our findings did not reveal a sex effect, yet a potential interaction effect might be present between exposure and fetal sex. CONCLUSIONS: The results confirmed the translocation of maternally inhaled CPs from diesel engine exhaust to the placenta which could be detected in fetal organs during late-stage pregnancy. The exposed can be clearly discriminated from the control group with respect to fetoplacental biometry and CP load. The differential particle load in the fetal organs may contribute to the effects on fetoplacental biometry and to the malprogramming of the fetal phenotype with long-term effects later in life.

Rational design of a novel acryl-modified CQDs fluorescent probe for highly selective detection and imaging of cysteine in vitro and in vivo.

A novel fluorescent nanoprobe CQDs-O-Acryl has been designed and synthesized to directly and accurately identify Cys over other biothiols in PBS (10 mM, pH 7.4) buffer. The carbon quantum dots (CQDs-OH) (λex/em maxima = 495/525 nm) were fabricated by a solvothermal method using resorcinol as the carbon source. The CQDs-O-Acryl was achieved through covalently grafting the acryloyl group on the surface of carbon quantum dots by nuclear reaction based on static quenching. The structure and morphology of CQDs-OH and CQDs-O-Acryl have been characterized by transmission electron microscopy, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, and UV-vis absorption spectroscopy. Upon the addition of Cys, the ester bond of CQDs-O-Acryl has been broken, and the free CQDs were released by conjugated addition and cyclization reactions successively, emitting strong green fluorescence at 525 nm (λex = 495 nm). Under the optimized conditions, CQDs-O-Acryl exhibited good sensing of Cys within the range 0.095-16 µM (the LOD of 0.095 µM). Due to the high sensitivity, reliability, fast fluorescence response (10 min), and low toxicity of CQDs-O-Acryl, it was successfully applied to fluorescence imaging of Cys in A549 cells and zebrafish.

Microbial Fuel Cell Biosensor with Capillary Carbon Source Delivery for Real-Time Toxicity Detection.

A microbial fuel cell (MFC) biosensor with an anode as a sensing element is often unreliable at low or significantly fluctuating organic matter concentrations. To remove this limitation, this work demonstrates capillary action-aided carbon source delivery to an anode-sensing MFC biosensor for use in carbon-depleted environments, e.g., potable water. First, different carbon source delivery configurations using several thread types, silk, nylon, cotton, and polyester, are evaluated. Silk thread was determined to be the most suitable material for passive delivery of a 40 g L-1 acetate solution. This carbon source delivery system was then incorporated into the design of an MFC biosensor for real-time detection of toxicity spikes in tap water, providing an organic matter concentration of 56 ± 15 mg L-1. The biosensor was subsequently able to detect spikes of toxicants such as chlorine, formaldehyde, mercury, and cyanobacterial microcystins. The 16S sequencing results demonstrated the proliferation of Desulfatirhabdium (10.7% of the total population), Pelobacter (10.3%), and Geobacter (10.2%) genera. Overall, this work shows that the proposed approach can be used to achieve real-time toxicant detection by MFC biosensors in carbon-depleted environments.

Sensitive visual detection of intracellular zinc ions based on signal-on polydopamine carbon dots.

The concentration of intracellular zinc ions is a significant clinical parameter for diagnosis. However, it is still a challenge for direct visual detection of zinc ions in cells at single-cell level. To address this issue, herein, water-soluble amino-rich polydopamine carbon quantum dots (PDA-CQDs) were successfully synthesized, with strong blue-green fluorescence as the probes for zinc ions detection in cells. The structure and properties of PDA-CQDs were confirmed by transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transformed infrared (FT-IR), UV-visible spectrophotometry (UV-vis), and fluorescence spectroscopy. Importantly, by successfully linking salicylaldehyde (SA) to PDA-CQDs via nucleophilic reaction, the FL quenching and Zn ions induced FL-recovering system was built up, thus offering a signal-on platform for the detection of zinc ions. This PDA-CQDs-SA nanoprobe can be applied for the detection of Zn2+with a detection limit of 0.09µM, with good biocompatibility confirmed using cytotoxicity assay. Of significance, the results of fluorescence bioimaging showed that PDA-CQDs-SA is able to detect Zn2+in single-cell visually, with the detection limit of Zn ions in cells as low as 0.11µM per cell, which was confirmed using flow cytometry. Therefore, this work offers a potential probe for Zn2+detection in cells at single-cell level, towards the precise diagnosis of zinc ions related diseases.

Sustainable agronomic response of carbon quantum dots on Allium sativum: Translocation, physiological responses and alternations in chromosomal aberrations.

The revolutionary growth in the usage of carbon quantum dots (CQDs) in different areas have ultimately directed their discharge in the environment and further augmented the exposure of agricultural crops to these released particles. Therefore, the aim of current study is to evaluate the uptake, translocation and phytotoxicity of blue emissive CQDs on Allium sativum plant. The genotoxicity and cytotoxicity assessment of CQDs towards Allium sativum roots was estimated as function of three different concentrations. Considering the role of CQDs in promoting seed germination at 50 ppm concentration, a greenhouse experiment was performed to evaluate their effect on plant growth. Systematic investigations have shown the translocation of CQDs and their physiological response in terms of increased shoot length wherein P-CQDs exhibited more accumulation into Allium sativum parts. Our investigations unfold the opportunity to utilize Aegle marmelos fruit derived CQDs as a growth regulator in variety of other food plants.

TFEB-lysosome pathway activation is associated with different cell death responses to carbon quantum dots in Kupffer cells and hepatocytes.

BACKGROUND: Carbon dot has been widely used in biomedical field as a kind of nanomaterial with low toxicity and high biocompatibility. CDs has demonstrated its unique advantages in assisted drug delivery, target diagnosis and targeted therapy with its small size and spontaneous fluorescence. However, the potential biosafety of CDs cannot be evaluated. Therefore, we focused on the study of liver, the target organ involved in CDs metabolism, to evaluate the risk of CDs in vitro. METHODS AND RESULTS: Liver macrophage KUP5 cells and normal liver cells AML12 cells were incubated in CDs at the same concentration for 24 h to compare the different effects under the same exposure conditions. The study found that both liver cell models showed ATP metabolism disorder, membrane damage, autophagosome formation and lysosome damage, but the difference was that, KUP5 cells exhibited more serious damage than AML12 cells, suggesting that immunogenic cell type is particularly sensitive to CDs. The underlying mechanism of CDs-induced death of the two hepatocyte types were also assessed. In KUP5 cells, death was caused by inhibition of autophagic flux caused by autophagosome accumulation, this process that was reversed when autophagosome accumulation was prevented by 3-MA. AML12 cells had no such response, suggesting that the accumulation of autophagosomes caused by CDs may be specific to macrophages. CONCLUSION: Activation of the TFEB-lysosome pathway is important in regulating autophagy and apoptosis. The dual regulation of ERK and mTOR phosphorylation upstream of TFEB influences the death outcome of AML12 cells. These findings provide a new understanding of how CDs impact different liver cells and contribute to a more complete toxicological safety evaluation of CDs.

Red-emitting carbon dots aggregates-based fluorescent probe for monitoring Cu2.

R-CDAs have been synthesized in a one-pot solvothermal procedure starting from 3,4-diaminobenzoic acid in an acidic medium. Transmission electron microscopy (TEM) revealed that R-CDAs nanoparticles exhibited a much larger diameter of 7.2-28.8 nm than traditional monodisperse carbon dots. X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FT-IR) revealed the presence of polar functional groups (hydroxyl, amino, carboxyl) on the surface of R-CDAs. Upon excitation with visible light (550 nm), R-CDAs emit stable, red fluorescence with a maximum at 610 nm. Under the optimum conditions, Cu2+ ions quench the fluorescence of this probe, and the signal is linear in a concentration range of copper ions between 5 and 600 nM with the detection limit of only 0.4 nM. Recoveries from 98.0 to 105.0% and relative standard deviations (RSD) from 2.8 to 4.5% have been obtained for detection of Cu2+ in real water samples. Furthermore, the R-CDAs fluorescent probe showed negligible cytotoxicity toward HeLa cells and good bioimaging ability, suggesting its potential applicability as a diagnostic tool in biomedicine.

Carbon quantum dots with pH-responsive orange-/red-light emission for fluorescence imaging of intracellular pH.

N-doped carbon quantum dots (N-CDs) with polyaminobenzene hydrazine as precursor were prepared by solvothermal method for the monitoring of pH fluctuation in HeLa cells via fluorescence imaging. The N-CDs show two emission wavelengths at 582 and 640 nm under different pH with two excitation wavelengths. The fluorescence intensity at 640 nm (λex = 520 nm) and the ratio of F582/F640 (λex = 470 nm) linearly increase with pH in the range of 2.4 ~ 3.6 (R2 = 992) and 5.6 ~ 7.6 (R2 = 0.987), respectively. The sensor exhibits high sensitivity and reversibility and anti-interference capability, thus enabling sensing pH change in intracellular environment in real time, as demonstrated by successful monitoring of intracellular pH fluctuation during H2O2 stimulation in HeLa cells.

An insight into the potentials of carbon dots for in vitro live-cell imaging: recent progress, challenges, and prospects.

Carbon dots (CDs) are a strong alternative to conventional fluorescent probes for cell imaging due to their brightness, photostability, tunable fluorescence emission, low toxicity, inexpensive preparation, and chemical diversity. Improving the targeting efficiency by modulation of the surface functional groups and understanding the mechanisms of targeted imaging are the most challenging issues in cell imaging by CDs. Firstly, we briefly discuss important features of fluorescent CDs for live-cell imaging application in this review. Then, the newest modulated CDs for targeted live-cell imaging of whole-cell, cell organelles, pH, ions, small molecules, and proteins are elaborately discussed, and their challenges in these fields are explained.

Milk-Derived Carbon Quantum Dots: Study of Biological and Chemical Properties Provides Evidence of Toxicity.

Carbon dots (CDs) are carbon-based zero-dimensional nanomaterials that can be prepared from a number of organic precursors. In this research, they are prepared using fat-free UHT cow milk through the hydrothermal method. FTIR analysis shows C=O and C-H bond presence, as well as nitrogen-based bond like C-N, C=N and -NH2 presence in CDs, while the absorption spectra show the absorption band at 280 ± 3 nm. Next, the Biuret test was performed, with the results showing no presence of unreacted proteins in CDs. It can be said that all proteins are converted in CDs. Photo luminance spectra shows the emission of CDs is 420 nm and a toxicity study of CDs was performed. The Presto Blue method was used to test the toxicity of CDs for murine hippocampal cells. CDs at a concentration of 4 mg/mL were hazardous independent of synthesis time, while the toxicity was higher for lower synthesis times of 1 and 2 h. When the concentration is reduced in 1 and 2 h synthesized CDs, the cytotoxic effect also decreases significantly, ensuring a survival rate of 60-80%. However, when the synthesis time of CDs is increased, the cytotoxic effect decreases to a lesser extent. The CDs with the highest synthesis time of 8 h do not show a cytotoxic effect above 60%. The cytotoxicity study shows that CDs may have a concentration and time-dependent cytotoxic effect, reducing the number of viable cells by 40%.

Density of surface charge is a more predictive factor of the toxicity of cationic carbon nanoparticles than zeta potential.

BACKGROUND: A positive surface charge has been largely associated with nanoparticle (NP) toxicity. However, by screening a carbon NP library in macrophages, we found that a cationic charge does not systematically translate into toxicity. To get deeper insight into this, we carried out a comprehensive study on 5 cationic carbon NPs (NP2 to NP6) exhibiting a similar zeta (ζ) potential value (from + 20.6 to + 26.9 mV) but displaying an increasing surface charge density (electrokinetic charge, Qek from 0.23 to 4.39 µmol/g). An anionic and non-cytotoxic NP (NP1, ζ-potential = - 38.5 mV) was used as control. RESULTS: The 5 cationic NPs induced high (NP6 and NP5, Qek of 2.95 and 4.39 µmol/g, respectively), little (NP3 and NP4, Qek of 0.78 and 1.35 µmol/g, respectively) or no (NP2, Qek of 0.23 µmol/g) viability loss in THP-1-derived macrophages exposed for 24 h to escalating NP dose (3 to 200 µg/mL). A similar toxicity trend was observed in airway epithelial cells (A549 and Calu-3), with less viability loss than in THP-1 cells. NP3, NP5 and NP6 were taken up by THP-1 cells at 4 h, whereas NP1, NP2 and NP4 were not. Among the 6 NPs, only NP5 and NP6 with the highest surface charge density induced significant oxidative stress, IL-8 release, mitochondrial dysfunction and loss in lysosomal integrity in THP-1 cells. As well, in mice, NP5 and NP6 only induced airway inflammation. NP5 also increased allergen-induced immune response, airway inflammation and mucus production. CONCLUSIONS: Thus, this study clearly reveals that the surface charge density of a cationic carbon NP rather than the absolute value of its ζ-potential is a relevant descriptor of its in vitro and in vivo toxicity.

One-step synthesis of nitrogen-doped multi-emission carbon dots and their fluorescent sensing in HClO and cellular imaging.

Tunable multicolor carbon dots (CDs) with a quantum yield reach up to 35% were generated directly from rhodamine and urea via one-step hydrothermal approach and purified through silica gel column chromatography. Transmission electron microscopy images reveal that the as-prepared CDs possess a small size distribution below 10 nm with bright blue, green, and yellow color emission, designated as b-CDs, g-CDs, and y-CDs, respectively. The in-depth investigations reveal that the multicolor emission CDs with different fraction displays fluorescence emission wavelength ranges from 398 nm (b-CDs), 525 nm (g-CDs), to 553 nm (y-CDs) which could be well modulated by controlling the amount of heteroatom nitrogen especially amino nitrogen onto their surface structures. Further experiments verify the important role of nitrogen content by using rhodamine solely or substituting urea with sulfur containing compounds as precursors to produce corresponding CDs since the performance is lower than that of urea incorporation. Theoretical calculation results also reveal that the increasing amount of amino nitrogen into their surface structures of b-CDs, g-CDs to y-CDs is responsible for reduced band gaps energy, which result in the redshifted wavelength. Benefiting from the excellent photoluminescence properties, wide pH variation range, high photo stability, and low toxicity, these CDs were employed for HClO sensing at 553 nm within the range 5 to 140 µM with a limit of detection (LOD) of 0.27 ± 0.025 µM (n = 3) and multicolor cellular imaging in HeLa cells. Tunable multicolor carbon dots (CDs) were generated directly from rhodamine and urea via one-step hydrothermal approach and purified through silica gel column chromatography. The as-prepared CDs exhibit bright blue, green, and yellow color emission which could be well modulated by controlling the increasing incorporation of heteroatom nitrogen especially amino nitrogen into their surface structures. These CDs were employed for HClO sensing and demonstrated to multicolor cellular imaging in HeLa cells.

Nanoplatform based on GSH-responsive mesoporous silica nanoparticles for cancer therapy and mitochondrial targeted imaging.

Mitochondria, as the energy factory of most cells, are not only responsible for the generation of adenosine triphosphoric acid (ATP) but also essential targets for therapy and diagnosis of various diseases, especially cancer. The safe and potential nanoplatform which can deliver various therapeutic agents to cancer cells and mitochondrial targeted imaging is urgently required. Herein, Au nanoparticles (AuNPs), mesoporous silica nanoparticles (MSN), cationic ligand (triphenylphosphine (TPP)), doxorubicin (DOX), and carbon nanodots (CDs) were utilized to fabricate mitochondrial targeting drug delivery system (denoted as CDs(DOX)@MSN-TPP@AuNPs). Since AuNPs, as the gatekeepers, can be etched by intracellular glutathione (GSH) via ligand exchange induced etching process, DOX can be released into cells in a GSH-dependent manner which results in the superior GSH-modulated tumor inhibition activity. Moreover, after etching by GSH, the CDs(DOX)@MSN-TPP@AuNPs can serve as promising fluorescent probe (λex = 633 nm, λem = 650 nm) for targeted imaging of mitochondria in living cells with near-infrared fluorescence. The induction of apoptosis derived from the membrane depolarization of mitochondria is the primary anti-tumor route of CDs(DOX)@MSN-TPP@AuNPs. As a kind of GSH-responsive mitochondrial targeting nanoplatform, it holds great promising for effective cancer therapy and mitochondrial targeted imaging. The mitochondrial targeting drug delivery system was fabricated by AuNPs, MSN, TPP, and CDs. The nanoplatform can realize redox-responsive drug delivery and targeted imaging of mitochondria in living cells to improve the therapeutic efficiency and security.

Toxicity of Carbon Nanomaterials-Towards Reliable Viability Assessment via New Approach in Flow Cytometry.

The scope of application of carbon nanomaterials in biomedical, environmental and industrial fields is recently substantially increasing. Since in vitro toxicity testing is the first essential step for any commercial usage, it is crucial to have a reliable method to analyze the potentially harmful effects of carbon nanomaterials. Even though researchers already reported the interference of carbon nanomaterials with common toxicity assays, there is still, unfortunately, a large number of studies that neglect this fact. In this study, we investigated interference of four bio-promising carbon nanomaterials (graphene acid (GA), cyanographene (GCN), graphitic carbon nitride (g-C3N4) and carbon dots (QCDs)) in commonly used LIVE/DEAD assay. When a standard procedure was applied, materials caused various types of interference. While positively charged g-C3N4 and QCDs induced false results through the creation of free agglomerates and intrinsic fluorescence properties, negatively charged GA and GCN led to false signals due to the complex quenching effect of the fluorescent dye of a LIVE/DEAD kit. Thus, we developed a new approach using a specific gating strategy based on additional controls that successfully overcame all types of interference and lead to reliable results in LIVE/DEAD assay. We suggest that the newly developed procedure should be a mandatory tool for all in vitro flow cytometry assays of any class of carbon nanomaterials.

Supported Biofilms on Carbon-Oxide Composites for Nitrate Reduction in Agricultural Waste Water.

Escherichia coli colonies were grown on different supports for the removal of nitrates from water. A carbon material and different commercial metal oxides, such as SiO2, TiO2 and Al2O3, and their corresponding carbon-metal oxide composites were studied. The physicochemical properties were analyzed by different techniques and the results were correlated with their performance in the denitrification process. Developed biofilms effectively adhere to the supports and always reach the complete reduction of nitrates to gaseous products. Nevertheless, faster processes occur when the biofilm is supported on mesoporous and non-acid materials (carbon and silica).

Carbon Dots Conjugated with Vascular Endothelial Growth Factor for Protein Tracking in Angiogenic Therapy.

One of the challenges of using growth factors for tissue regeneration is to monitor their biodistributions and delivery to injured tissues for minimally invasive detection. In the present study, tracking of human vascular endothelial growth factor (VEGF) was achieved by chemically linking it to photoluminescent carbon dots (CDs). Carbon dots were synthesized by the hydrothermal method and, subsequently, conjugated with VEGF using carbodiimide coupling. ELISA and western blot analysis revealed that VEGF-conjugated CDs preserve the binding affinity of VEGF to its antibodies. We also show that VEGF-conjugated CDs maintain the functionality of VEGF for tube formation and cell migration. The VEGF-conjugated CDs were also used for in vitro imaging of human umbilical vein endothelial cells. The results of this work suggest that cell-penetrating VEGF-conjugated CDs can be used for growth factor protein tracking in therapeutic and tissue engineering applications.