Potential of using qFibrosis analysis to predict recurrent and survival outcome of patients with hepatocellular carcinoma after hepatic resection.

BACKGROUND: There remains a lack of studies addressing the stromal background and fibrosis features and its prognostic value in liver cancer. qFibrosis can identify, quantify and visualize the fibrosis features from biopsy samples. In this study, we aim to demonstrate the prognostic value of histological features by using qFibrosis analysis in liver cancer patients. METHODS: Liver specimen from 201 patients with hepatocellular carcinoma underwent curative resection were imaged and assessed using qFibrosis system, and generated a total of 33 and 156 collagen parameters from tumor part and non-tumor liver tissue, respectively. We used these collagen parameters on patients to build two combined indexes, RFS-index and OS-index, in order to differentiate patients with early recurrence and early death, respectively. The models were validated using leave-one-out method. RESULTS: Both combined indexes had significant prediction value of patients' outcome. The RFS-index of 0.52 well differentiates patients with early recurrence (p < 0.001), and the OS-index of 0.73 well differentiates patients with early death during follow-up (p = 0.02). CONCLUSIONS: Combined index calculated with qFibrosis from digital readout of fibrotic status of peri-tumor liver specimen in patients with HCC have prediction values for their disease and survival outcomes. These results demonstrated the potentials to transform histopathological features into quantifiable data that could be used to correlate with clinical outcome.

Tropifexor plus cenicriviroc combination versus monotherapy in nonalcoholic steatohepatitis: Results from the phase 2b TANDEM study.

BACKGROUND AND AIMS: With distinct mechanisms of action, the combination of tropifexor (TXR) and cenicriviroc (CVC) may provide an effective treatment for NASH. This randomized, multicenter, double-blind, phase 2b study assessed the safety and efficacy of TXR and CVC combination, compared with respective monotherapies. APPROACH AND RESULTS: Patients (N = 193) were randomized 1:1:1:1 to once-daily TXR 140 µg (TXR 140 ), CVC 150 mg (CVC), TXR 140 µg + CVC 150 mg (TXR 140 + CVC), or TXR 90 µg + CVC 150 mg (TXR 90 + CVC) for 48 weeks. The primary and secondary end points were safety and histological improvement, respectively. Rates of adverse events (AEs) were similar across treatment groups. Pruritus was the most frequently experienced AE, with highest incidence in the TXR 140 group (40.0%). In TXR and combination groups, alanine aminotransferase (ALT) decreased from baseline to 48 weeks (geometric mean change: -21%, TXR 140 ; -16%, TXR 140 + CVC; -13%, TXR 90 + CVC; and +17%, CVC). Reductions in body weight observed at week 24 (mean changes from baseline: TXR 140 , -2.5 kg; TXR 140 + CVC, -1.7 kg; TXR 90 + CVC, -1.0 kg; and CVC, -0.1 kg) were sustained to week 48. At least 1-point improvement in fibrosis stage/steatohepatitis resolution without worsening of fibrosis was observed in 32.3%/25.8%, 31.6%/15.8%, 29.7%/13.5%, and 32.5%/22.5% of patients in the TXR 140 , CVC, TXR 140 + CVC, and TXR 90 + CVC groups, respectively. CONCLUSIONS: The safety profile of TXR + CVC combination was similar to respective monotherapies, with no new signals. TXR monotherapy showed sustained ALT and body weight decreases. No substantial incremental efficacy was observed with TXR + CVC combination on ALT, body weight, or in histological end points compared with monotherapy.

Second-Harmonic Generated Quantifiable Fibrosis Parameters Provide Signatures for Disease Progression and Regression in Nonalcoholic Fatty Liver Disease.

Introduction: The current ordinal fibrosis staging system for nonalcoholic steatohepatitis (NASH) has a limited dynamic range. The goal of this study was to determine if second-harmonic generated (SHG) quantifiable collagen fibrillar properties (qFP) and their derived qFibrosis score capture changes in disease progression and regression in a murine model of NASH, in which disease progression can be induced by a high fat sugar water (HFSW) diet and regression by reversal to chow diet (CD). Methods: DIAMOND mice were fed a CD or HFSW diet for 40 to 52 weeks. Regression related changes were studied in mice with diet reversal for 4 weeks after 48 to 60 weeks of a HFSW diet. Results: As expected, mice on HFSW developed steatohepatitis with stage 2 to 3 fibrosis between weeks 40 and 44. Both the collagen proportionate area and the qFibrosis score based on 15 SHG-quantified collagen fibrillar properties in humans were significantly higher in mice on HFSW for 40 to 44 weeks compared to CD fed mice. These changes were greatest in the sinusoids (Zone 2) with further increase in septal and portal fibrosis related scores between weeks 44 and 48. Diet reversal led to decrease in qFibrosis, septal thickness, and cellularity with greatest changes in Zone 2. Specific qFPs associated with progression only, regression only, or both processes were identified and categorized based on direction of fibrosis change. Conclusion: Complementing recent human studies, these findings support the concept that changes of disease progression and regression can be assessed using SHG-based image quantification of fibrosis related parameters.

Machine learning liver histology scores correlate with portal hypertension assessments in nonalcoholic steatohepatitis cirrhosis.

BACKGROUND AND AIMS: In cirrhotic nonalcoholic steatohepatitis (NASH) clinical trials, primary efficacy endpoints have been hepatic venous pressure gradient (HVPG), liver histology and clinical liver outcomes. Important histologic features, such as septa thickness, nodules features and fibrosis area have not been included in the histologic assessment and may have important clinical relevance. We assessed these features with a machine learning (ML) model. METHODS: NASH patients with compensated cirrhosis and HVPG ≥6 mm Hg (n = 143) from the Belapectin phase 2b trial were studied. Liver biopsies, HVPG measurements and upper endoscopies were performed at baseline and at end of treatment (EOT). A second harmonic generation/two-photon excitation fluorescence provided an automated quantitative assessment of septa, nodules and fibrosis (SNOF). We created ML scores and tested their association with HVPG, clinically significant HVPG (≥10 mm Hg) and the presence of varices (SNOF-V). RESULTS: We derived 448 histologic variables (243 related to septa, 21 related to nodules and 184 related to fibrosis). The SNOF score (≥11.78) reliably distinguished CSPH at baseline and in the validation cohort (baseline + EOT) [AUC = 0.85 and 0.74, respectively]. The SNOF-V score (≥0.57) distinguished the presence of varices at baseline and in the same validation cohort [AUC = 0.86 and 0.73, respectively]. Finally, the SNOF-C score differentiated those who had >20% change in HVPG against those who did not, with an AUROC of 0.89. CONCLUSION: The ML algorithm accurately predicted HVPG, CSPH, the development of varices and HVPG changes in patients with NASH cirrhosis. The use of ML histology model in NASH cirrhosis trials may improve the assessment of key outcome changes.

Efficacy and Safety of a Botanical Formula Fuzheng Huayu for Hepatic Fibrosis in Patients with CHC: Results of a Phase 2 Clinical Trial.

Background: Hepatitis C virus (HCV) is a common cause of progressive hepatic fibrosis, cirrhosis, and hepatocellular carcinoma worldwide. Despite the availability of effective direct-acting antivirals, patients often have significant hepatic fibrosis at the time of diagnosis due to delay in diagnosis and comorbidities which promote fibrogenesis. Thus, antifibrotic agents represent an attractive adjunctive therapy. Fuzheng Huayu (FZHY), a traditional Chinese medicine botanical formulation, has been used as an antifibrotic agent in chronic HBV infection. Our aim was to assess FZHY in patients with HCV infection and active viremia. Method: We randomized 118 patients with active viremia from 8 liver centers in the U.S. to receive oral FZHY (n = 59) or placebo (n = 59) for 48 weeks. Efficacy was assessed by histopathologic changes at the end of therapy. A subset of biopsies was further analyzed using qFibrosis to detect subtle changes in fibrosis in different zones of the hepatic lobules. Results: FZHY was well tolerated and safe. Patients with baseline Ishak fibrosis stages F3 and F4 had better response rates to FZHY than patients with baseline F0-F2 (p=0.03). qFibrosis zonal analysis showed significant improvement in fibrosis in all zones in patients with regression of the fibrosis stage. Conclusions: FZHY produced antifibrotic effects in patients with baseline Ishak F3 and F4 fibrosis stages. Reduction in fibrosis severity was zonal and correlated with the severity of inflammation. Based on its tolerability, safety, and efficacy, FZHY should be further investigated as a therapy in chronic liver diseases because of its dual anti-inflammatory and antiibrotic properties. Lay Summary. This is the first US-based, multicenter and placebo-controlled clinical trial that shows statistically significant reduction in fibrosis in patients with active HCV using an antifibrotic botanical formula. This has important implications as there is an immediate need for effective antifibrotic agents in treating many chronic diseases including NASH that lead to scarring of the liver. With artificial intelligence-based methodology, qFibrosis, we may provide a more reliable way to assess the FZHY as a therapy in chronic liver diseases because of its dual anti-inflammatory and antifibrotic properties.

Digital pathology with artificial intelligence analyses provides greater insights into treatment-induced fibrosis regression in NASH.

BACKGROUND & AIMS: Liver fibrosis is a key prognostic determinant for clinical outcomes in non-alcoholic steatohepatitis (NASH). Current scoring systems have limitations, especially in assessing fibrosis regression. Second harmonic generation/two-photon excitation fluorescence (SHG/TPEF) microscopy with artificial intelligence analyses provides standardized evaluation of NASH features, especially liver fibrosis and collagen fiber quantitation on a continuous scale. This approach was applied to gain in-depth understanding of fibrosis dynamics after treatment with tropifexor (TXR), a non-bile acid farnesoid X receptor agonist in patients participating in the FLIGHT-FXR study (NCT02855164). METHOD: Unstained sections from 198 liver biopsies (paired: baseline and end-of-treatment) from 99 patients with NASH (fibrosis stage F2 or F3) who received placebo (n = 34), TXR 140 µg (n = 37), or TXR 200 µg (n = 28) for 48 weeks were examined. Liver fibrosis (qFibrosis®), hepatic fat (qSteatosis®), and ballooned hepatocytes (qBallooning®) were quantitated using SHG/TPEF microscopy. Changes in septa morphology, collagen fiber parameters, and zonal distribution within liver lobules were also quantitatively assessed. RESULTS: Digital analyses revealed treatment-associated reductions in overall liver fibrosis (qFibrosis®), unlike conventional microscopy, as well as marked regression in perisinusoidal fibrosis in patients who had either F2 or F3 fibrosis at baseline. Concomitant zonal quantitation of fibrosis and steatosis revealed that patients with greater qSteatosis reduction also have the greatest reduction in perisinusoidal fibrosis. Regressive changes in septa morphology and reduction in septa parameters were observed almost exclusively in F3 patients, who were adjudged as 'unchanged' with conventional scoring. CONCLUSION: Fibrosis regression following hepatic fat reduction occurs initially in the perisinusoidal regions, around areas of steatosis reduction. Digital pathology provides new insights into treatment-induced fibrosis regression in NASH, which are not captured by current staging systems. LAY SUMMARY: The degree of liver fibrosis (tissue scarring) in non-alcoholic steatohepatitis (NASH) is the main predictor of negative clinical outcomes. Accurate assessment of the quantity and architecture of liver fibrosis is fundamental for patient enrolment in NASH clinical trials and for determining treatment efficacy. Using digital microscopy with artificial intelligence analyses, the present study demonstrates that this novel approach has greater sensitivity in demonstrating treatment-induced reversal of fibrosis in the liver than current systems. Furthermore, additional details are obtained regarding the pathogenesis of NASH disease and the effects of therapy.

Complexity of ballooned hepatocyte feature recognition: Defining a training atlas for artificial intelligence-based imaging in NAFLD.

BACKGROUND & AIMS: Histologically assessed hepatocyte ballooning is a key feature discriminating non-alcoholic steatohepatitis (NASH) from steatosis (NAFL). Reliable identification underpins patient inclusion in clinical trials and serves as a key regulatory-approved surrogate endpoint for drug efficacy. High inter/intra-observer variation in ballooning measured using the NASH CRN semi-quantitative score has been reported yet no actionable solutions have been proposed. METHODS: A focused evaluation of hepatocyte ballooning recognition was conducted. Digitized slides were evaluated by 9 internationally recognized expert liver pathologists on 2 separate occasions: each pathologist independently marked every ballooned hepatocyte and later provided an overall non-NASH NAFL/NASH assessment. Interobserver variation was assessed and a 'concordance atlas' of ballooned hepatocytes generated to train second harmonic generation/two-photon excitation fluorescence imaging-based artificial intelligence (AI). RESULTS: The Fleiss kappa statistic for overall interobserver agreement for presence/absence of ballooning was 0.197 (95% CI 0.094-0.300), rising to 0.362 (0.258-0.465) with a ≥5-cell threshold. However, the intraclass correlation coefficient for consistency was higher (0.718 [0.511-0.900]), indicating 'moderate' agreement on ballooning burden. 133 ballooned cells were identified using a ≥5/9 majority to train AI ballooning detection (AI-pathologist pairwise concordance 19-42%, comparable to inter-pathologist pairwise concordance of between 8-75%). AI quantified change in ballooned cell burden in response to therapy in a separate slide set. CONCLUSIONS: The substantial divergence in hepatocyte ballooning identified amongst expert hepatopathologists suggests that ballooning is a spectrum, too subjective for its presence or complete absence to be unequivocally determined as a trial endpoint. A concordance atlas may be used to train AI assistive technologies to reproducibly quantify ballooned hepatocytes that standardize assessment of therapeutic efficacy. This atlas serves as a reference standard for ongoing work to refine how ballooning is classified by both pathologists and AI. LAY SUMMARY: For the first time, we show that, even amongst expert hepatopathologists, there is poor agreement regarding the number of ballooned hepatocytes seen on the same digitized histology images. This has important implications as the presence of ballooning is needed to establish the diagnosis of non-alcoholic steatohepatitis (NASH), and its unequivocal absence is one of the key requirements to show 'NASH resolution' to support drug efficacy in clinical trials. Artificial intelligence-based approaches may provide a more reliable way to assess the range of injury recorded as "hepatocyte ballooning".

MoS2 exhibits stronger toxicity with increased exfoliation.

MoS2 belong to a class of inorganic 2D nanomaterials known as transition metal dichalcogenides (TMDs) which have recently attracted a renewed and growing interest due to their interesting electronic and catalytic properties when scaled down to single or few layer sheets. Although exfoliated MoS2 nanosheets have been proposed for numerous energy-related and biosensing applications, little is known about the toxicological impacts of using MoS2 nanosheets. Here, we report about the in vitro toxicity of MoS2 nanosheets that have been chemically exfoliated with different lithium intercalating agents and compared their respective cytotoxic influence. Methyllithium (Me-Li), n-butyllithium (n-Bu-Li) and tert-butyllithium (t-Bu-Li) were used for the exfoliation of bulk MoS2 and we found the t-Bu-Li and n-Bu-Li exfoliated MoS2 nanosheets to be more cytotoxic than MoS2 exfoliated by Me-Li. t-Bu-Li and n-Bu-Li provide more efficient exfoliation over Me-Li, and we establish that the extent of exfoliation that MoS2 undergo is a factor influencing their toxicity. Specifically, the more exfoliated the MoS2 nanosheets, the stronger its cytotoxic influence, which may be due to an increase in surface area and active edge sites. The potential toxicity of these MoS2 nanosheets should be taken into account before their employment in real world applications and we have shown the effect the amount of exfoliation can have on the toxicity of MoS2 nanosheets, representing the first step towards a better understanding of their toxicological properties.

Graphene oxide nanoribbons exhibit significantly greater toxicity than graphene oxide nanoplatelets.

Graphene oxide (GOs) has emerged in recent years as a versatile nanomaterial, demonstrating tremendous potential for multifunctional biomedical applications. GOs can be prepared by the top-down or bottom-up approach, which leads to a great variability of GOs being produced due to the different procedures and starting carbon sources adopted. This will have an effect on the physiochemical properties of GOs and their resultant toxic behavior. In this study, we examined the cytotoxicity of graphene-oxide nanoribbons (GONRs; ∼310 × 5000 nm) and graphene-oxide nanoplatelets (GONPs; 100 × 100 nm), prepared from the oxidative treatment of multi-walled carbon nanotubes (MWCNTs; ∼100 × 5000 nm) and stacked graphene nanofibers (SGNFs; 100 × 5000 nm), respectively. In vitro assessments revealed that the GONRs exhibited a much stronger cytotoxicity over the GONPs, and we correlated that observation with characterization data that showed GONRs to have a greater amount of carbonyl groups as well as greater length. Therefore, we put forward that the stronger toxic behavior of GONRs is a result of the synergistic effect between these two factors, and the type of carbon source used to prepare GOs should be carefully considered in any future bioapplications.

Cytotoxicity of exfoliated transition-metal dichalcogenides (MoS2 , WS2 , and WSe2 ) is lower than that of graphene and its analogues.

Studies involving transition-metal dichalcogenides (TMDs) have been around for many decades and in recent years, many were focused on using TMDs to synthesize inorganic analogues of carbon nanotubes, fullerene, as well as graphene and its derivatives with the ultimate aim of employing these materials into consumer products. In view of this rising trend, we investigated the cytotoxicity of three common exfoliated TMDs (exTMDs), namely MoS2 , WS2 , and WSe2 , and compared their toxicological effects with graphene oxides and halogenated graphenes to find out whether these inorganic analogues of graphenes and derivatives would show improved biocompatibility. Based on the cell viability assessments using methylthiazolyldiphenyl-tetrazolium bromide (MTT) and water-soluble tetrazolium salt (WST-8) assays on human lung carcinoma epithelial cells (A549) following a 24 h exposure to varying concentrations of the three exTMDs, it was concluded that MoS2 and WS2 nanosheets induced very low cytotoxicity to A549 cells, even at high concentrations. On the other hand, WSe2 exhibited dose-dependent toxicological effects on A549 cells, reducing cell viability to 31.8 % at the maximum concentration of 400 µg mL(-1) ; the higher cytotoxicity displayed by WSe2 might be linked to the identity of the chalcogen. In comparison with graphene oxides and halogenated graphenes, MoS2 and WS2 were much less hazardous, whereas WSe2 showed similar degree of cytotoxicity. Future in-depth studies should be built upon this first work on the in vitro cytotoxicity of MoS2 and WS2 to ensure that they do not pose acute toxicity. Lastly, nanomaterial-induced interference control experiments revealed that exTMDs were capable of reacting with MTT assay viability markers in the absence of cells, but not with WST-8 assay. This suggests that the MTT assay is not suitable for measuring the cytotoxicity of exTMDs because inflated results will be obtained, giving false impressions that the materials are less toxic.

Cytotoxicity profile of highly hydrogenated graphene.

Graphene and its graphene-related counterparts have been considered the future of advanced nanomaterials owing to their exemplary properties. An increase in their potential applications in the biomedical field has led to serious concerns regarding their safety and impact on health. To understand the toxicity profile for a particular type of graphene utilized in a given application, it is important to recognize the differences between the graphene-related components and correlate their cellular toxicity effects to the attributed physiochemical properties. In this study, the cytoxicity effects of highly hydrogenated graphene (HHG) and its graphene oxide (GO) counterpart on the basis of in vitro toxicological assessments are reported and the effects correlated with the physiochemical properties of the tested nanomaterials. Upon 24 h exposure to the nanomaterials, a dose-dependent cellular cytotoxic effect was exhibited and the HHG was observed to be more cytotoxic than its GO control. Detailed characterization revealed an extensive C-H sp(3) network on the carbon backbone of HHG with few oxygen-containing groups, as opposed to the presence of large amounts of oxygen-containing groups on the GO. It is therefore hypothesized that the preferential adsorption of micronutrients on the surface of the HHG nanomaterial by means of hydrophobic interactions resulted in a reduction in the bioavailability of nutrients required for cellular viability. The nanotoxicological profile of highly hydrogenated graphene is assessed for the first time in our study, thereby paving the way for further evaluation of the toxicity risks involved with the utilization of various graphene-related nanomaterials in the real world.

Towards biocompatible nano/microscale machines: self-propelled catalytic nanomotors not exhibiting acute toxicity.

Recent advances in nanotechnology have led to the evolution of self-propelled, artificial nano/microjet motors. These intelligent devices are considered to be the next generation self-powered drug delivery system in the field of biomedical applications. While many studies have strived to further improve the various properties of these devices such as their efficiency, performance and power, little attention has been paid to the actual biocompatibility of nanojets in vivo. In this paper, we will present for the first time the investigation of the toxicity effects of nanojets on the viability of human lung epithelial cells (A549 cells). From the 24 h and 48 h post-exposure studies, it is clearly shown that the nanojets we used in our work has negligible influence on the cell viability across all the concentrations tested. As such, the toxicity profile of our nanojets have been shown to be neither dose- nor time-dependent. This is strongly indicative of the benign nature of our nanojets, which is of paramount significance as it is the first step towards the applications of nano/micromotors in real-world practical medical devices.

Cytotoxicity of halogenated graphenes.

Graphene and its family of derivatives possess unique and remarkable physicochemical properties which make them valuable materials for applications in many areas like electronics, energy storage and biomedicine. In response to the possibility of its large-scale manufacturing as commercial products in the future, an investigation was conducted to determine the cytotoxicity of one particular family of graphene derivatives, the halogenated graphenes, for the first time. Halogenated graphenes were prepared through thermal exfoliation of graphite oxide in gaseous chlorine, bromine or iodine atmospheres to yield chlorine- (TRGO-Cl), bromine- (TRGO-Br) and iodine-doped graphene (TRGO-I) respectively. 24 h exposure of human lung carcinoma epithelial cells (A549) to the three halogenated graphenes and subsequent cell viability assessments using methylthiazolyldiphenyl-tetrazolium bromide (MTT) and water-soluble tetrazolium salt (WST-8) assays revealed that all the halogenated graphenes examined are rather cytotoxic at the concentrations tested (3.125 µg mL(-1) to 200 µg mL(-1)) and the effects are dose-dependent, with TRGO-Cl reducing the cell viability to as low as 25.7% at the maximum concentration of 200 µg mL(-1). Their levels of cytotoxicity can be arranged in the order of TRGO-Cl > TRGO-Br > TRGO-I, and it is suggested that the amount of halogen present in the graphene material is the determining factor for the observed trend. Control experiments were carried out to test for possible nanomaterial-induced interference as a consequence of reaction between the halogenated graphenes and the viability markers (MTT/WST-8 reagent) or binding of the formazan products under cell-free conditions. The data obtained eliminate the probability of significant influence by these interferents as the change in the normalized percentage of formazan formed is relatively small and thorough washings were performed prior to the viability assessments to reduce the amount of halogenated graphenes that could eventually interact with the MTT/WST-8 assays. More studies need to be carried out in the future to complement the results obtained in this initial study in an attempt to develop a better understanding of the health hazards that the halogenated graphenes pose.

The toxicity of graphene oxides: dependence on the oxidative methods used.

Graphene, a class of two-dimensional carbon nanomaterial, has attracted extensive interest in recent years, with a significant amount of research focusing on graphene oxides (GOs). They have been primed as potential candidates for biomedical applications such as cell labeling and drug delivery, thus the toxicity and behavior of graphene oxides in biological systems are fundamental issues that need urgent attention. The production of GO is generally achieved through a top-down route, which includes the usage of concentrated H2SO4 along with: 1) concentrated nitric acid and KClO3 oxidant (Hoffmann); 2) fuming nitric acid and KClO3 oxidant (Staudenmaier); 3) concentrated phosphoric acid with KMnO4 (Tour); or 4) sodium nitrate for in-situ production of nitric acid in the presence of KMnO4 (Hummers). It has been widely assumed that the properties of these four GOs produced by using the above different methods are roughly similar, so the methods have been used interchangeably. However, several studies have reported that the toxicity of graphene-related nanomaterials in biological systems may be influenced by their physiochemical properties, such as surface functional groups and structural defects. In addition, considering how GOs are increasingly used in the field of biomedicine, we are interested to see how the oxygen content/functional groups of GOs can impact their toxicological profiles. Since in-vitro testing is a common first step in assessing the health risks related with engineered nanomaterials, the cytotoxicity of the GOs prepared by the four different oxidative treatments was investigated by measuring the mitochondrial activity in adherent lung epithelial cells (A549) by using commercially available viability assays. The dose-response data was generated by using two assays, the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and the water-soluble tetrazolium salt (WST-8). From the viability data, it is evident that there is a strong dose-dependent cytotoxic response resulting from the four GO nanomaterials tested after a 24 h exposure, and it is suggested that there is a correlation between the amounts of oxygen content/functional groups of GOs with their toxicological behavior towards the A549 cells.

Purification of carbon nanotubes by high temperature chlorine gas treatment.

Carbon nanotubes (CNTs) have a tremendous amount of potential to become useful components for future practical applications that may become a part of everyday life. While the sp(2) carbon itself is a rather chemically inert material, the issue of residual metal nanoparticle catalysts remains a prominent barrier in the utilization of CNTs in many areas due to the strong influence of these metallic impurities on the redox chemistry of biomarkers. Even with a standard purification procedure, CNTs have been shown to still contain residual metal nanoparticle catalysts. As such, presented in this paper is an improved purification technique for treating the CNTs with the highly reactive Cl2 gas at an elevated temperature of 1000 °C for 10 min, which would result in the vaporization of the metallic impurities as MxCly, leading to a large decrease in the amount of metallic nanoparticle impurities within the CNTs. By means of electrochemistry and X-ray fluorescence analysis, we demonstrate that the behaviour of such Cl2 treated CNTs showed a significant shift towards that of high purity CNTs, with a dramatic decrease in the influence of the residual metallic impurities on the electrochemical behaviour of CNTs. Therefore it is suggested that the Cl2 treatment of carbon nanotubes is a highly promising route towards the production of pure CNTs.

The hydrogen-bonded dianion of vitamin K1 produced in aqueous-organic solutions exists in equilibrium with its hydrogen-bonded semiquinone anion radical.

When the quinone, vitamin K1 (VK1), is electrochemically reduced in aqueous-acetonitrile solutions (CH3CN with 7.22 M H2O), it undergoes a two-electron reduction to form the dianion that is hydrogen-bonded with water [VK1(H2O)y(2­)]. EPR and voltammetry experiments have shown that the persistent existence of the semiquinone anion radical (also hydrogen-bonded with water) [VK1(H2O)x(­â€¢)] in aqueous or organic­aqueous solutions is a result of VK1(H2O)y(2­) undergoing a net homogeneous electron transfer reaction (comproportionation) with VK1, and not via direct one-electron reduction of VK1. When 1 mM solutions of VK1 were electrochemically reduced by two electrons in aqueous-acetonitrile solutions, quantitative EPR experiments indicated that the amount of VK1(H2O)x(­â€¢) produced was up to approximately 35% of all the reduced species. In situ electrochemical ATR-FTIR experiments on sequentially one- and two-electron bulk reduced solutions of VK1 (showing strong absorbances at 1664, 1598, and 1298 cm(­1)) in CH3CN containing <0.05 M H2O led to the detection of VK1(­â€¢) with strong absorbances at 1710, 1703, 1593, 1559, 1492, and 1466 cm(­1) and VK1(H2O)y(2­) with strong absorbances at 1372 and 1342 cm(­1).

Nanographite impurities in carbon nanotubes: their influence on the oxidation of insulin, nitric oxide, and extracellular thiols.

There has been growing interest in the use of modified-carbon-nanotube electrodes in applications such as the electrochemical detection of biologically significant compounds, owing to their apparent "electrocatalytic" properties and ability to enhance oxidative signals. In spite of their salient properties, little work has been done to further examine the reasons for these reported characteristics. In this report, we present clear evidence that the presence of nanographite impurities within carbon nanotubes (CNTs) is responsible for providing the previously reported enhanced electrochemical response. We have demonstrated this effect on homocysteine, N-acetyl-L-cysteine, nitric oxide, and insulin, which are important biological agents in the body. Moreover, we also showed that the influence of nanographite impurities on the electrochemistry of carbon nanotubes is prevalent among a variety of CNTs, such as single-walled CNTs, double-walled CNTs, and few-walled CNTs. Our findings will have a profound influence upon the biomedical applications of CNTs.

Voltammetric method for determining the trace moisture content of organic solvents based on hydrogen-bonding interactions with quinones.

Voltammetry experiments were performed on the natural quinone, vitamin K(1) (VK(1)), in a range of organic solvents of varying dielectric constant that are commonly used for electrochemical measurements [dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), acetonitrile (MeCN), propionitrile (EtCN), butyronitrile (PrCN), 1,2-dichloroethane (DCE), dichloromethane (DCM), and 1,1,2,2-tetrachloroethane (TCE)]. The water content of the solvents was accurately measured using Karl Fischer (KF) coulometric titrations, and the voltammetric data were used to estimate the degree of hydrogen-bonding interactions between the reduced forms of VK(1) and variable levels of water, thereby allowing a ranking of water-substrate interactions in the different solvents. The voltammetric data were analyzed based on interactions that occur between reduced forms of VK(1) and the water, the solvent, and the supporting electrolyte. Calibration data were obtained that are independent of the nature of the reference electrode and allow the water content of the solvents to be calculated by performing a single voltammetric scan in the presence of VK(1) and 0.2 M supporting electrolyte (Bu(4)NPF(6)).