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This work is focused on a simple method for fingerprint visualization on problematic metal substrates for criminology by the application of a non-traditional polymer prepared from neutral red using cyclic voltammetry (CV). Experiments have shown that, under optimal electrochemical conditions, a latent fingerprint on a Pt and brass substrates can be quickly made significantly more visible. Using poly(neutral red), which has not been yet studied in this area, it was even possible to observe details of the secondary (bifurcation) and tertiary (pores) levels of the fingerprint papillary lines using optical microscopy.

Carbon nanodots modified with Neutral Red covalently inserted in the nanostructure (NR-CDs) have been prepared by a simple synthesis method based on microwave irradiation under controlled temperature and pressure. The synthetized NR-CDs have been characterized by different techniques, demonstrating the covalent bonding of Neutral Red molecules to the carbon dots nanostructure. Fluorescence activity of the prepare NR-CDs has been explored showing different interaction pathways with singled and doubled stranded DNA. These studies have been successfully applied to develop a new fluorescence DNA hybridization assay to the detection of a specific DNA sequence of Escherichia coli bacteria. Graphical abstract: [Figure not available: see fulltext.], Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work has been supported by the Spanish Ministerio de Ciencia, Innovación y Universidades (CTQ2017- 84309-C2-1-R; RED2018-102412-T) and Comunidad Autónoma de Madrid (SI3/PJI/2021–00341, P2018/NMT-4349 TRANSNANOAVANSENS Program)

Harsh conditions within the tumor microenvironment, such as hypoxia and extracellular acidic pH (pH(e)), inactivate some chemotherapies, which results in limited or no cytotoxicity. Standard MTT, ATPlite and protease assays that are used to determine the potency of newly developed drugs often give erroneous results when applied under hypoxic or acidic conditions. Therefore, development of a cytotoxicity assay that does not yield false positive or false negative results under circumstances of both hypoxia and acidic pH(e) is needed. We evaluated currently used cell viability assays as well as neutral red staining to assess viability of ovarian and pancreatic cancer cells grown in an acidic pH(e) microenvironment after treatment with carboplatin, gemcitabine or chloroquine. We validated cell viability using western blotting of pro-caspase-9 and cleaved-caspase-9, and LC3-I and –II. Standard cell viability assays indicated cell viability accurately at pH(e) 7.4, but was not correlated with induction of apoptosis or autophagy at acidic pH(e). By contrast, our modified neutral red assay detected cell viability accurately over a range of pH(e) as demonstrated by its correlation with induction of apoptosis and autophagy. Neutral red staining is effective for evaluating the effect of chemotherapeutic agents on cell viability under acidic pH(e) or hypoxic conditions.

The photodynamic activity of Neutral Red and the new monobrominated Neutral Red was studied in suspensions of Staphylococcus aureus. The effect of mannitol and sodium azide in the presence of 25 μm photosensitizer on lethal photosensitization were investigated. The results of the mechanistic evaluation of Neutral Red showed that both mannitol and sodium azide produced a completed protective effect after irradiation without significant differences between them. The evaluation of monobrominated Neutral Red also showed a protective effect of microorganisms with the addition of mannitol. Although sodium azide produced a protective effect of the photoinactivation, it was incomplete and less than that exhibited by mannitol. The results indicate that the starting reagent, Neutral Red, is a producer of radical species, acting through a type I mechanism, whereas the halogenated derivative of Neutral Red produced reactive oxygen species and a contribution of singlet molecular oxygen cannot be discarded in the photoinactivation of Staphylococcus aureus cells. These results, analyzed together with the previously evaluated properties of the dyes, allow us to explain the differences observed in the photoinactivation of Staphylococcus aureus mediated by both azine photosensitizers.

Copper is essential for numerous physiological functions, and copper compounds may display therapeutic as well as cytotoxic effects. The MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay is a standard test largely used in cytotoxicity studies. This report shows that low micromolar levels of copper compounds such as Cu(II)Urea2, Cu(II)Ser2 and CuCl2 can interfere with the MTT assay making improper the detection of formazan product of MTT reduction. Comparatively, the Neutral Red assay appears to be sensitive and showing no interference with these compounds. The lactate dehydrogenase alternative assay cannot be used because of inhibitory effect of these copper compounds on the enzyme activity.

Mycobacterium tuberculosis protein Rv0577 is a prominent antigen in tuberculosis patients, the component responsible for neutral red staining of virulent strains of M. tuberculosis, a putative component in a methylglyoxal detoxification pathway, and an agonist of toll-like receptor 2. It also has an amino acid sequence that is 36% identical to that of Streptomyces coelicolor AfsK-binding protein A (KbpA), a component in the complex secondary metabolite pathways in the Streptomyces genus. To gain insight into the biological function of Rv0577 and the family of KpbA kinase regulators, the crystal structure for Rv0577 was determined to a resolution of 1.75 A, binding properties with neutral red and deoxyadenosine were surveyed, backbone dynamics were measured, and thermal stability was assayed by circular dichroism spectroscopy. The protein is composed of four approximate repeats with a βαβββ topology arranged radially in consecutive pairs to form two continuous eight-strand β-sheets capped on both ends with an α-helix. The two β-sheets intersect in the center at roughly a right angle and form two asymmetric deep "saddles" that may serve to bind ligands. Nuclear magnetic resonance chemical shift perturbation experiments show that neutral red and deoxyadenosine bind to Rv0577. Binding to deoxyadenosine is weaker with an estimated dissociation constants of 4.1 ± 0.3 mM for saddle 1. Heteronuclear steady-state {1H}-15N nuclear Overhauser effect, T1, and T2 values were generally uniform throughout the sequence with only a few modest pockets of differences. Circular dichroism spectroscopy characterization of the thermal stability of Rv0577 indicated irreversible unfolding upon heating with an estimated melting temperature of 56 °C.

The host-guest interactions and the consequent modulation in the prototropic equilibrium of a phenazine dye, neutral red, with p-sulfonatocalix[4]arene (SCX4) and p-sulfonatocalix[6]arene (SCX6) macrocyclic hosts have been investigated. Both the neutral (NR) and cationic (NRH+) forms of dyes formed inclusion complexes with SCX6, with a larger binding constant for the latter (K = 8.6 × 105 M-1versus 4.8 × 103 M-1) due to the cation receptor behavior of the calixarenes. The distinct differences in the binding constant of NR and NRH+ provided a finite tuning of pKa between 6.5 and 8.8, through a competitive binding with metal ions. Importantly, the fluorescence quenching observed in the SCX-neutral red interactions stands in contrast to the fluorescence enhancement observed with other macrocyclic hosts, such as β-cyclodextrin and cucurbit[7]uril. This is due to the unique portal stacking interaction of NRH+ with the SCXs, compared to the axial inclusion geometry documented for the other macrocycles. The electron transfer from the SCX to the neutral red dye is adjudged to be the effective excited-state relaxation pathway leading to fluorescence quenching. In combination with the metal-ion induced fluorescence regeneration and tuning the pKa value, the SCX-neutral red system finds potential applications in drug delivery, photodynamic therapy, catalysis, and sensor applications.

As alternative microorganisms for butanol production with high butanol tolerant and productivity are in high demand, one excellent butanol-tolerant bacterium, S10, was isolated and identified as Clostridium acetobutylicum S10. In order to enhance the performance of butanol production, organic acids and neutral red were added during butanol fermentation. Synergistic effects were exhibited in the combinations of organic acids and neutral red to promote butanol production. Consequently, the optimal concentrations of combined acetate, butyrate, and neutral red were determined at sodium acetate 1.61 g/L, sodium butyrate 1.88 g/L, and neutral red 0.79 g/L, respectively, with the butanol yield of 6.09 g/L which was 20.89 % higher than that in control. These results indicated that combination of adding organic acid and neutral red is a potential effective measure to improve butanol production.

We report a specific light-up fluorescent probe for i-motif DNA for the first time. Compared with the previously reported probes, neutral red could selectively interact with an i-motif and show a significant increase in its fluorescence. This feature makes it advantageous for designing label-free fluorescent sensing systems.

Voltammetric DNA sensor has been proposed on the platform of glassy carbon electrode covered with carbon black with adsorbed pillar[5]arene molecules. Electropolymerization of Neutral Red performed in the presence of native or oxidatively damaged DNA resulted in formation of hybrid material which activity depended on the DNA conditions. The assembling of the surface layer was confirmed by scanning electron microscopy and electrochemical impedance spectroscopy. The influence of DNA and pillar[5]arene on redox activity of polymeric dye was investigated and a significant increase of the peak currents was found for DNA damaged by reactive oxygen species generated by Cu2+/H2O2 mixture. Pillar[5]arene improves the electron exchange conditions and increases the response and its reproducibility. The applicability of the DNA sensor developed was shown on the example of ascorbic acid as antioxidant. It decreases the current in the concentration range from 1.0 &mu, M to 1.0 mM. The possibility to detect antioxidant activity was qualitatively confirmed by testing tera infusion. The DNA sensor developed can find application in testing of carcinogenic species and searching for new antitumor drugs.

The aim of this work was to compare the effects of electrosynthesis on different bacterial species. The effects of neutral red-mediated electrosynthesis on the metabolite profiles of three microorganisms: Escherichia coli, Klebsiella pneumoniae, and Zymomonas mobilis, were measured and compared and contrasted. A statistically comprehensive analysis of neutral red-mediated electrosynthesis is presented using the analysis of end-product profiles, current delivered, and changes in cellular protein expression. K. pneumoniae displayed the most dramatic response to electrosynthesis of the three bacteria, producing 93% more ethanol and 76% more lactate vs. control fermentation with no neutral red and no electron delivery. Z. mobilis showed no response to electrosynthesis except elevated acetate titers. Stoichiometric comparison showed that NAD(+) reduction by neutral red could not account for changes in metabolites during electrosynthesis. Neutral red-mediated electrosynthesis was shown to have multifarious effects on the three species.

Neutral red is an electron transport compound that is used in bacteria as a mediator for decolorization, degradation of different organic compounds and in electricity production using microbial fuel cells (MFCs). In this study, bacterial electron transport was followed by monitoring electricity production in a double chamber microbial fuel cell, and measured as current (mA). To enhance electron transfer and consequently increase electricity production, neutral red was added as a mediator. The optimal concentration that had minimal effect on bacterial growth was 15 mM. Spiking the culture with this concentration resulted in a maximal current of 1.36 mA and a power intensity that reached 467.6 mW/m2over 120 h, as compared to 0.74 mA and 209.5 mW/m2 over a period of just 24 h as a control. This was also confirmed by the increase in initial bacterial conductivity measured in the presence of neutral red as compared to low initial conductivity in the absence of a mediator. To prove that the process of electricity production is correlated to electron transfer, azole and azide were both used to inhibit membrane cytochrome complexes. The results showed a sharp decrease in electrical current production within less than 24 h, suggesting that the process takes place via membrane protein cytochromes. The predominant microorganism present under all previously mentioned conditions was Buttiauxella agrestis SW-1 (GenBank ID KP770022) as identified using 16S rRNA phylogenetic analysis, this bacterium was also studied morphologically to detect micropilli. Protein profiles of the membrane showed a distinct band close to that reported for cytochrome c; nevertheless, further investigation is required to highlight both its nature and its role.

The neutral red uptake assay is a cell viability assay that allows in vitro quantification of xenobiotic-induced cytotoxicity. The assay relies on the ability of living cells to incorporate and bind neutral red, a weak cationic dye, in lysosomes. As such, cytotoxicity is expressed as a concentration-dependent reduction of the uptake of neutral red after exposure to the xenobiotic under investigation. The neutral red uptake assay is mainly used for hazard assessment in in vitro toxicology applications. This method has also been introduced in regulatory recommendations as part of 3T3-NRU-phototoxicity-assay, which was regulatory accepted in all EU member states in 2000 and in the OECD member states in 2004 as a test guideline (TG 432). The present protocol describes the neutral red uptake assay using the human hepatoma cell line HepG2, which is often employed as an alternative in vitro model for human hepatocytes. As an example, the cytotoxicity of acetaminophen and acetyl salicylic acid is assessed.

Forster resonant energy transfer (FRET) process can be monitored by observing the quenching of the donor luminescence in the presence of an acceptor particle. In a different way, the present study demonstrates such process in a pair system represented by CdSe/ZnS quantum dots (QDs) and Neutral Red dyes through a spatially resolved micro-photoluminescence (μ-PL) technique. The possibility of their use in biological applications can be explored, such as mimicking sensitive chemical-biological sensors. In order to obtain the signature of the nonradiative transfer FRET, the donor-acceptor intermolecular distance was changed by the acceptors concentration. A macroscopic point of view of the system was obtained by measures of the photon diffusion lengths as a function of the donor-acceptor distances. Both the QD donor fluorescence quenching and the fall of the spatial photon distribution with the acceptor concentration evidenced the FRET process. The results also confirmed that the chloroform solvent was clearly more appropriate for enhancing the FRET in the QD-NR system.

The acridine orange (AO) and neutral red (NR) dyes, commonly used as probes to measure the internal pH in acidic vesicles, are compared in this article. The comparison between the two dyes (arising from calculations taking into account their analytical constants) illustrated that the use of AO is preferential to that of NR because the AO response is sensitive over the whole pH range between 4.0 and 7.4, whereas the NR response is effective only between pHs 4.0 and 6.0. In addition, it became evident from the mitochondrial respiration response that NR, unlike AO, is a protonophore. When taken into consideration, these two properties suggest that AO is more suitable than NR as an indicator of toxicity measurements in water samples because the environmental toxic compounds induce pH changes in the acidic vesicles of biological structures that are used as environmental biosensors.

Biosensing of NADH on bare electrodes has drawbacks such as high over-potential and poisoning during the oxidation reaction. To overcome this challenge a different approach has been undertaken by incorporating neutral red (NR) in Al doped ZnO (AZO) thin films using one-pot chemical bath deposition (CBD). The surface morphology of the films was hexagonal nanorods along the c-axis, perpendicular to the substrate. The thickness of the thin films were ranging from 400 to 3000 nm varying dependent on time of deposition (30 to 150 min). The average diameter of the nanorods was larger in the presence of neutral red (NR-AZO) with ~ 300 nm in contrast to its absence (AZO) with ~ 200 nm. The density of the packing of nanorods was dependent on the citrate concentration used during deposition. Control over the dopant concentration in the films was achieved by varying the area of Al foil used in the deposition solution. The selected area diffraction (SAED) and X-ray diffraction (XRD) indicated 002 plane of orientation in the nanorods. FTIR and FT-Raman analysis revealed conserved structure of NR and AZO. Chronoamperometric (CA) analysis showed a sensitivity of 0.45 μA cm− 2 mM− 1 and LoD of 22 μM within the range 0.075–4 mM of NADH. The biological sensing of NADH was validated by physical adsorption of NAD+ dependent-lactate dehydrogenase (LDH) on NR-AZO. CA showed sensitivity of 0.56 μA cm− 2 mM− 1 and LoD for lactate was 27 μM in the range of 0.1–1 mM of lactate. Further validation with real-time serum sample shows that LDH/NR-AZO correlates with the clinical values. The distinction in this study is that the organic mediator like neutral red has been incorporated into the grain structure of the ZnO thin film whereas other study with the mediators have only attempted surface functionalization. This article is part of a Special Issue entitled "Recent Advances in Bionanomaterials" Guest Editor: Dr. Marie-Louise Saboungi and Dr. Samuel D. Bader.

A simple method to immobilize poly(neutral red) (PNR) and flavin adenine dinucleotide (FAD) hybrid film (PNR/FAD) by cyclic voltammetry is proposed. The PNR/FAD hybrid film can be easily prepared on an electrode surface involving electropolymerization of neutral red (NR) monomers and the electrostatic interaction between the positively charged PNR and the negatively charged FAD. It exhibits electroactive, stable, surface-confined, pH-dependent, nano-sized, and compatible properties. It provides good electrocatalytic properties to various species. It shows a sensitivity of 5.4 μA mM(-1) cm(-2) and 21.5 μA mM(-1) cm(-2) for hydrogen peroxide (H(2)O(2)) and nicotinamide adenine dinucleotide (NADH) with the linear range of 0.1 μM-39 mM and 5 × 10(-5) to 2.5 × 10(-4) M, respectively. It shows another linear range of 48.8-355.5 mM with the sensitivity of 12.3 μA mM(-1) cm(-2) for H(2)O(2). In particular, the PNR/FAD hybrid film has potential to replace some hemoproteins to be a cathode of biofuel cells and provide the biosensing system for glucose and ethanol.

Whole mount preparations of mouse mammary glands are useful for evaluating overall changes in growth and morphology, and are essential for detecting and evaluating focal or regionally-localized phenotypes that would be difficult to detect or analyze using other techniques. We present three newly-developed methods for preparing whole mounts of mammary glands from genetically-engineered mice expressing fluorescent proteins, as well as using either neutral red or a variety of fluorescent dyes. Unlike traditional hematoxylin- or carmine-stained preparations, neutral red-stained, and some fluorescent preparations, can be used for several common downstream analyses.

The interaction between the drug, zidovudine and calf thymus DNA (CT-DNA) in physiological buffer (pH 7.4) was investigated using neutral red (NR) and methylene blue (MB) dyes as a spectral probes by UV–vis absorption and fluorescence spectroscopy, as well as circular dichroism (CD) spectroscopy. The experimental results showed that the conformational changes in DNA helix induced by zidovudine are the reason for the fluorescence quenching of the DNA-NR system. In addition, by increasing zidovudine to DNA-MB solution, the fluorescence has no change. From the experimental results, it was found that zidovudine can cause structural changes on CT-DNA and bind with DNA via groove binding mode. At the same time, the paper proved that conformational changes of DNA can also lead to the fluorescence decrease of DNA-probe systems.

A novel fluorescence sensor of NR-β-CD@AuNPs was prepared for the trace detection of nitrite in quantities as low as 4.25 × 10−3 μg∙mL−1 in an aqueous medium. The fluorescence was due to the host-guest inclusion complexes between neutral red (NR) molecules and gold nanoparticles (AuNPs), which were modified by per-6-mercapto-beta-cyclodextrins (SH-β-CDs) as both a reducing agent and a stabilizer under microwave radiation. The color of the NR-β-CD@AuNPs changed in the presence of nitrite ions. A sensor was applied to the determination of trace nitrites in environmental water samples with satisfactory results.

The interaction between Ni 2+ and calf thymus DNA (ctDNA) was investigated in simulated physiological buffer (pH 7.4) using the Neutral Red (NR) dye as a spectral probe by UV–vis absorption and fluorescence spectroscopy, as well as CD spectra. The experimental results showed that the conformational changes in DNA helix induced by Ni 2+ are the reason for the fluorescence quenching of the DNA–NR system. From the experimental results, conclusion can be drawn that Ni 2+ can cause structural changes of ctDNA and bind with DNA by electrostatic interaction. At the same time, the paper proved that conformation changes of DNA can also lead to the fluorescence decrease of DNA–probe systems.

The neutral red retention time (NRRT) assay is useful for detecting decreased lysosomal membrane stability in haemocytes sampled from bivalves, a phenomenon often associated with exposure to environmental pollutants including nanomaterials. Bivalves are popular sentinel species in ecotoxicology and use of NRRT in study of species in the genus Mytilus is widespread in environmental monitoring. The NRRT assay has been used as an in vivo test for toxicity of carbon nanoparticles (Moore MN, Readman JAJ, Readman JW, Lowe DM, Frickers PE, Beesley A. 2009. Lysosomal cytotoxicity of carbon nanoparticles in cells of the molluscan immune system: An in vivo study. Nanotoxicology . 3 (1), 40–45). We here report application of this assay adapted to a microtitre plate format to a panel of metal and metal oxide nanoparticles (2 ppm). This showed that copper, chromium and cobalt nanoparticles are toxic by this criterion while gold and titanium nanoparticles are not. As the former three nanoparticles are often reported to be cytotoxic while the latter two are thought to be non-cytotoxic, these data support use of NRRT as a general in vitro assay in nanotoxicology.

In this article the interaction of neutral red with heparin was characterized by linear sweep voltammetry on the hanging mercury drop electrode (HMDE) and further used for the determination of heparin. In pH 1.5 Britton‐Robinson (B‐R) buffer solution, neutral red (NR) has a well‐defined second‐order derivative linear sweep voltammetric reductive wave at −0.24 V (vs. SCE). After the addition of heparin into the NR solution, the reductive peak current decreases without the movement of the peak potential and the appearances of new peaks in the scanning potential range. Based on the decrease of the reductive peak current, a new voltammetric method for the determination of heparin is established. The conditions for the interaction and the electrochemical detection are optimized, and the interference substances for detection are investigated. Under the optimal conditions, the decrease of reductive peak current of NR is proportional to heparin concentration in the range of 2.0–10.0 mg/L with the linear ...

Neutral red (NR) functioned as an electronophore or electron channel enabling either cells or membranes purified from Actinobacillus succinogenes to drive electron transfer and proton translocation by coupling fumarate reduction to succinate production. Electrically reduced NR, unlike methyl or benzyl viologen, bound to cell membranes, was not toxic, and chemically reduced NAD. The cell membrane of A. succinogenes contained high levels of benzyl viologen-linked hydrogenase (12.2 U), fumarate reductase (13.1 U), and diaphorase (109.7 U) activities. Fumarate reductase (24.5 U) displayed the highest activity with NR as the electron carrier, whereas hydrogenase (1.1 U) and diaphorase (0.8 U) did not. Proton translocation by whole cells was dependent on either electrically reduced NR or H 2 as the electron donor and on the fumarate concentration. During the growth of Actinobacillus on glucose plus electrically reduced NR in an electrochemical bioreactor system versus on glucose alone, electrically reduced NR enhanced glucose consumption, growth, and succinate production by about 20% while it decreased acetate production by about 50%. The rate of fumarate reduction to succinate by purified membranes was twofold higher with electrically reduced NR than with hydrogen as the electron donor. The addition of 2-( n -heptyl)-4-hydroxyquinoline N -oxide to whole cells or purified membranes inhibited succinate production from H 2 plus fumarate but not from electrically reduced NR plus fumarate. Thus, NR appears to replace the function of menaquinone in the fumarate reductase complex, and it enables A. succinogenes to utilize electricity as a significant source of metabolic reducing power.

The connection between the mode of toxic action and the genetic response caused by the type I photosensitizer and photosynthesis inhibitor neutral red (NR) and the type II photosensitizer rose bengal (RB) was investigated in the green alga Chlamydomonas reinhardtii. For both photosensitizers, a light intensity-dependent increase in toxicity and expression of the glutathione peroxidase homologous gene (Gpxh) was found. The toxicity of RB was reduced by the singlet oxygen (1O2) quenchers 1,4-diazabicyclo[2.2.2]octane and L-histidine, and the RB-induced Gpxh expression was stimulated in deuterium oxide-supplemented growth medium. These observations clearly indicate the involvement of 1O2 in both toxicity and the genetic response caused by RB. NR up-regulated the expression of typical oxidative and general stress response genes, probably by a type I mechanism, and also strongly induced the Gpxh expression. The stimulating effect of deuterium oxide in the growth medium suggested the involvement of 1O2 also in the NR-induced response. Indeed, an increased 1O2 formation was detected with EPR-spin trapping in NR-treated spinach thylakoids. However, none of the 102 quenchers could reduce the light-dependent toxicity of NR in C. reinhardtii, indicating that NR has a different mode of toxic action than RB.

Glucose oxidase (GOx) was immobilized onto glassy carbon electrode (GCE) that modified by reduced graphene oxide-gold nanoparticles- poly neutral red (RGO/AuNPs/PNR) nanocomposite. The composite was analyzed by scanning electron microscope (SEM), energy dispersive x-ray (EDX) spectroscopy, atomic force microscopy (AFM), attenuated total reflectance (ATR), cyclic voltammetry (CV), chronoamperometry and electrochemical impedance spectroscopy (EIS). SEM/EDX analysis showed the morphological of the nanocomposite. AFM results showed the morphology and structure of the RGO/AuNPs and RGO surfaces. The covalent bonding between glucose oxidase and composite was confirmed by ATR technique. The electrochemical experiments were done in 100 mM phosphate buffer at pH 7 and temperature of 25 °C with three electrodes including Ag/AgCl, platinum wire and the modified GCE as the reference electrode, the auxiliary electrode and working electrode respectively. The electrochemical results confirmed the activity and direct electron transfer of immobilized enzyme. The immobilized electroactive GOx concentration was estimated 3.06 × 10−11 mol cm−2. The results showed the immobilized enzyme had a good stability and maintained 90% of its performance after two weeks. The nanocomposite bioanode in an air-birthing biofuel cell and 100 mM glucose concentration showed 176 μWcm−2 Power density. This strategy could be used for GOx-based biofuel cells.

Neutral red fluorescence has been used to monitor neuronal activity. Local changes in either pH or hydrophobicity are reported to increase neutral red fluorescence, but the mechanisms underlying the increases remain unclear. In this study, the pH-dependent fluorescence changes in the basic and acidic forms of neutral red preloaded in rat cerebellar slices were separately measured with two excitation wavelengths. Bath application of kainate, domoate or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) at 1 mM increased fluorescence of the acidic form and decreased that of the basic form irreversibly, consistent with a pH-dependent mechanism. In contrast, application of lower concentrations of AMPA, (S)-3,5-dihydroxyphenylglycine or KCl increased fluorescence of both forms transiently, suggesting that a pH-independent mechanism may also increase neutral red fluorescence. Electrical stimulation in the molecular layer also increased fluorescence of both forms. The response to weak electrical stimulation decayed in about 1 s, while response to intense stimulation lasted longer than 1 min. Neutral red binding to phospholipids was also detected as fluorescent spots on thin-layer chromatography, suggesting that interaction with phospholipids enhances neutral red fluorescence. Thus, the short-lasting signal of neutral red suggests its usage as a hydrophobic probe for neuronal activity.

Aporrectodea caliginosa is the most common endogeic (topsoil) earthworm in New Zealand and, because of its habitat, is potentially vulnerable to surface-applied pesticides. Lysosomal damage to earthworms, which can be visualized by the use of the neutral red retention assay (NRRA) has been evaluated in this species as a biomarker of organophosphate exposure. Earthworms were exposed in the laboratory to sub-lethal concentrations of chlorpyrifos and diazinon. In a semi-field experiment, earthworms were placed in mesocosms in a field sprayed with these pesticides at the rate recommended for a vegetable crop. In the laboratory, the neutral red retention time (NRRT) was significantly reduced following exposure to both pesticides compared with controls. In the semi-field experiment, earthworm NRRT was significantly reduced by both pesticides. These experiments have shown that the NRRA is very sensitive to exposure to chlorpyrifos and diazinon even at field rates. It therefore shows promise as a potential biomarker of contamination of soil by organophosphates.

Thrombocytes are multifunctional, nucleated blood cells. Morphological changes in thrombocytes have been used as a physiological indicator of a stress response. This study investigated the effects of in vitro heat stress (HS) on the neutral red uptake activity of chicken thrombocytes. Chicken thrombocytes (98% pure) were prepared from adult Barred Plymouth Rock and Rhode Island Red males. The isolated thrombocytes were preincubated at normal (41 C) or HS (45 C) temperatures for 30 min before either a 30- or 90-min incubation with neutral red at these same temperatures. After incubation the cells were washed, lysed, and the internal neutral red concentrations analyzed. There was no difference in thrombocyte numbers or their uptake of neutral red in samples from males of these two chicken breeds. At 41 C thrombocytes actively took up neutral red over the 90-min incubation period. However, at 45 C thrombocyte internalization of neutral red was significantly reduced. At both time periods (30 and 90 min), thrombocytes at 41 C took up significantly (P < or = 0.05) more neutral red than their counterparts at 45 C. This lack of neutral red uptake was not due to cell death as monitored by Trypan blue exclusion. Following the 30-min incubation there was no difference in viability between thrombocytes at the two temperatures. Although there was a significant (P < or = 0.05) increase in cell death at 90 min for thrombocytes kept at 45 C, the livability difference was of a much smaller magnitude than the difference in neutral red uptake when comparing to cells cultured at 41 C. Neutral red uptake is a rapid, inexpensive and repeatable technique for the study of thrombocyte function.

The pH sensitive dye, Neutral Red, was used with optical imaging techniques to map intracellular pH shifts elicited by cortical surface stimulation of the rat cerebellum. In the in vivo rat cerebellar cortex stained with Neutral Red, a brief stimulus train (three stimuli at 33 Hz) evoked a longitudinal beam of increased fluorescence (acidic shift) running parallel to the long axis of the folium within 100 ms of stimulation onset. A 5-10 s stimulus train (5-20 Hz) produced a biphasic optical response consisting of a beam of increased fluorescence (acidic shift) which returned to baseline in approximately 60 s, followed by a beam of decreased fluorescence (alkaline shift) for up to 120 s. A close spatial correspondence was observed between electrophysiological and optical maps of the response to surface stimulation. Application of acetazolamide enhanced the optical signals, acetabenzolamide-phenoxyethene had no effect, and the glutamate antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, decreased the optical signals. Increased fluorescence was produced by superfusion of the cerebellar cortex with acidic Ringer solutions and a decrease in fluorescence by basic solutions. These fluorescence changes also occurred in the presence of several ion channel/receptor blockers. Increased fluorescence resulted from superfusion with Ringer solution containing sodium propionate and decreased fluorescence with the transition from 5% carbon dioxide to nominally carbon dioxide-free Ringer solution. Recovery from acid loading with ammonium chloride was prevented by amiloride, an inhibitor of the Na+/H+ transporter. Application of Ringer solution with high potassium concentration produced an increase in fluorescence but only a decrease in fluorescence was detected when neuronal blockers were present, an effect consistent with a glial contribution. This decrease in fluorescence was blocked by adding barium. No epifluorescent optical signals were obtained from unstained preparations or preparations stained with cell-permeant fluorescence markers, suggesting little contribution from activity-dependent volume changes and other intrinsic signals. These results demonstrate that the Neutral Red optical signals evoked by cerebellar surface stimulation are primarily pH based and include a significant component related to intracellular pH shifts. The large amplitudes of these optical signals are particularly useful for mapping neuronal activity. Furthermore, this technique provides a novel tool for the study of pH changes in vivo at both high spatial and temporal resolution.

The formation of the inclusion complexes of neutral red (NR) with cyclodextrins (CDs) including β-cyclodextrin (β-CD), hydroxypropyl-β-cyclodextrin (HP-β-CD) and sulfobutylether-β-cyclodextrin (SBE-β-CD) was studied by fluorescence and UV-Vis absorption spectroscopy and the binding constants ( K ) of the inclusion complexes were obtained by steady-state fluorescence measurements. Experimental conditions including the concentrations of various CDs and media acidity were investigated in detail. The results suggested that NR exists in two molecular forms in aqueous solution (the acidic form and the neutral form). CDs were most suitable for inclusion of the neutral form of NR and could cause enhanced fluorescence emission and absorption by NR. Moreover, the effect of the charged CD (SBE-β-CD) was quite different from that of the uncharged CDs (β-CD and HP-β-CD). A mechanism is proposed to explain the inclusion process.

A novel sensitive, simple and fast kinetic method for the determination of trace amounts of resorcinol (0.50 — 3.00 μg ml−1) based on its inhibitory effect on the formaldehyde catalyzed oxidation reaction of neutral red by bromate ion in acidic media is reported. The reaction is monitored spectrophotometrically by measuring the decrease in the absorbance of neutral red in an inhibited reaction at 524 nm using a variable - time method. The detection limit is 0.38 μg ml−1 and the relative standard deviation of ten replicate measurements for 1 μg ml−1 of resorcinol is 4.2%. The procedure was successfully applied to the determination of resorcinol in resorcinol - S topical cream.

Hybridization of poly(luminol) (PLM) and poly(neutral red) (PNR) has been successfully performed and further enhanced by a conductive and steric hybrid nanotemplate using graphene oxide (GO) and multi-walled carbon nanotubes (MWCNTs). The morphology of the PLM-PNR-MWCNT-GO mycelium-like nanocomposite is studied by SEM and AFM and it is found to be electroactive, pH-dependent, and stable in the electrochemical system. It shows electrocatalytic activity towards NADH with a high current response and low overpotential. Using amperometry, it has been shown to have a high sensitivity of 288.9 μA mM(-1) cm(-2) to NADH (Eapp. = +0.1 V). Linearity is estimated in a concentration range of 1.33 × 10(-8) to 1.95 × 10(-4) M with a detection limit of 1.33 × 10(-8) M (S/N = 3). Particularly, it also shows another linear range of 2.08 × 10(-4) to 5.81 × 10(-4) M with a sensitivity of 151.3 μA mM(-1) cm(-2). The hybridization and activity of PLM and PNR can be effectively enhanced by MWCNTs and GO, resulting in an active hybrid nanocomposite for determination of NADH.

With the measurement of molecular absorption, the interaction of neutral red (NR) with double-stranded DNA in large excess was investigated. It was found that the interaction of NR, existing in the acidic state (HNR) at pH 4.56, with double-stranded structure DNA displays different spectral features depending on the molar ratio of HNR/DNA, R. If R>1.33, the binding process is characterized by a binding constant at the 10(6) level with each nucleotide residue of double-stranded DNA binding one HNR molecule. If R

Neutral red is a lysosomal probe and a biological pH indicator. In aqueous solutions, the protonated (NRH) and neutral (NR) forms of monomeric neutral red exhibit distinct absorption maxima (535 and 450 nm, respectively) but have the same fluorescence with a maximum at 637 nm and a quantum yield of 0.02. The similarity of the fluorescence spectra at acidic and basic pH suggests deprotonation of cationic species in the first singlet excited state. The NR fluorescence strongly depends on the solvent polarity as shown by addition of increasing amounts of water to pure dioxane, which gradually shifts the fluorescence maximum from 540 nm in pure dioxane to 637 nm in water. The fluorescence quantum yield increases from 0.17 in dioxane to 0.3 upon addition of 7% water and then decreases, reaching 0.02 in pure water. Immediately after incubation of human skin fibroblasts with neutral red, excitation with 435 nm light produces a fluorescence whose maximum is recorded at 575 nm. This fluorescence is located in the perinuclear region and originates from large fluorescent intracytoplasmic spots, suggesting staining of the endoplasmic reticulum-Golgi complex. At longer times, this fluorescence is shifted to 606 nm, suggesting slow diffusion of the lysosomotropic dye toward the more hydrated and acidic interior of lysosomes. Addition of a lysosomotropic detergent to cells previously incubated with neutral red shifts the fluorescence to the blue. Thus, in complex biological systems, this probe cannot be a good pH indicator but is a very sensitive probe of lysosomal microenvironments.

Two methodologies used in vitro to estimate cytotoxicity in cell culture systems were compared: these were the neutral red uptake assay (NRU), which is used to measure toxicity caused by an extended (48-hr) exposure to the test material, and the neutral red release assay (NRR), which is used to measure toxicity caused by a short-term (1-min) exposure to the test material. Both methodologies used the normal human epidermal keratinocyte (NHEK)-based NeutralRed Bioassay supplied by Clonetics Corporation (San Diego, CA, USA). 10 materials (paracetamol, acetylsalicylic acid, ferrous sulphate, diazepam, amitriptyline, digoxin, ethylene glycol, methanol, ethanol and isopropanol), which are part of the Multicenter Evaluation of In Vitro Cytotoxicity (MEIC) panel, were tested. NRU50 values for the 10 compounds covered more than an eight-log range from 0.004 μ m (digoxin) to 1.0 × 106 μ m (methanol). Because of solubility limits, NRR50 values for diazepam, digoxin, ferrous sulphate and paracetamol could not be determined. NRR50 values for the remaining six compounds covered approximately a three-log range from 3.2 × 103 to 7.1 × 106 μ m . When compared with documented values for either the human acute oral lethal dose or the human acute lethal blood concentration, the NRU assay was found to be much more useful in predicting human acute toxicity than was the NRR assay.

INTRODUCTIONFor almost 100 years, neutral red has been used to stain living cells and fixed tissue. It can be used as a general-purpose stain, a pH indicator (turning from red to yellow, as the medium becomes alkaline), or a nuclear stain. In this protocol, neutral red is used to stain plant vacuoles.

By combining three separate toxic assays, the neutral red (NR), MTT and crystal violet staining assays (CVS), we developed a convenient assay method, the NMC assay, in which the NR, MTT and CVS assays are performed consecutively on the same cultured HeLa cells. The NMC assay is performed as follows: the cultured HeLa cells are first treated with NR, and thereafter with MTT; the NR extracted with 30% methanol and the MTT extracted with isopropanol are measured spectrophotometrically (NMC-NR and NMC-MTT assays); the residual cells adhered on the microplate are then stained with crystal violet (NMC-CVS assay). The absorbance values in each of the NMC assay correlated with the viable cell count, as when the assays were performed individually. The EC(50) values of sodium lauryl sulfate (SLS) obtained by the NMC-NR, NMC-MTT and NMC-CVS assays in the HeLa cells were also in agreement with that obtained by each individual assay. Some chemicals, namely monensin, diisopropanol amine and antimycin A, gave different dose-cell viability curves in the NMC-NR, NMC-MTT and NMC-CVS assays depending on their specific mechanisms of action.

This paper reports our recent research about poly(neutral red) film modified glass carbon electrodes and their electrocatalytic oxidation reactions for both dopamine and ascorbic acid. The catalytic currents are linearly related to both dopamine and ascorbic acid concentrations in the ranges 5.0×10 −6 –2.0×10 −4 and 2.5×10 −5 –1.0×10 −1 mol l −1 , respectively. Moreover, the dopamine and ascorbic acid concentrations can be determined simultaneously by using the difference of the catalytic currents. This method has been used for determining dopamine and ascorbic acid concentrations in real samples with satisfactory results.

We have adapted the neutral red uptake assay for quantitative assessment of injury to fibroblast cultures by potential phototoxins. Tetracycline derivatives, quinolone derivatives, and chlorpromazine were used as model compounds for development of the assay. Human fibroblasts were incubated with potential phototoxins, the cell cultures irradiated with UV, and the capacity for neutral red uptake determined. Demeclocycline and doxycycline, two known photosensitizers, showed a 94% and 95% decrease of neutral red uptake, respectively, indicating photo-induced cytotoxicity. Minocycline, a non-photosensitizing tetracycline derivative, showed no decrease in uptake. Tetracycline, a weak phototoxin, showed minor (10%) decrease at equivalent concentrations (20 micrograms/ml). Microscopic observation of neutral red uptake and cell damage paralleled the spectrophotometric findings. Chlorpromazine, a non-tetracycline phototoxin, showed 91% decrease. An additional group of phototoxic drugs, quinolone antibacterials, were studied. Nalidixic acid, ofloxacin, ciprofloxacin, and norfloxacin all demonstrated phototoxicity, with nalidixic acid showing the greatest decrease in neutral red uptake. This methodology may provide a useful rapid method to quantify phototoxic potential of new drugs or suspected phototoxins.

A technique is described which uses the lipid fluorochrome neutral red as a cytochemical probe to detect the hydrophobic domain of the ligno-suberin matrix in native and wound periderm of potato tuber. Toluidine blue O is used as a counterstain to quench autofluorescence. The neutral red technique appears to be specific for the hydrophobic/lipid domain of suberin and is significantly more sensitive than Sudan III and IV. The fluorochrome was extensively used on paraffin-embedded tissue with excellent results but also worked on freehand sections of fresh periderm tissue. In tuber tissue undergoing wound-healing, the pattern of suberin fluorescence obtained with the neutral red probe was identical in specificity to the color pattern obtained with Sudan III/IV, but somewhat different than that observed when berberine was used. Results obtained with the neutral red probe and berberine probe visually demonstrated that during ligno-suberin biosynthesis, the depositions of hydrophobic/lipid and phenolic/lignin-like components in potato tuber periderm were separate processes. The deposition of these components does not necessarily require their simultaneous presence because the fluorescence from these probes showed that the components were not consistently present together on the cell walls.

Electrochemical biosensors have been developed for the determination of cadmium, cobalt, and copper cations through their inhibitory effect on glucose oxidase activity, using cobalt hexacyanoferrate or poly(neutral red) as redox mediators. Cobalt hexacyanoferrate and poly(neutral red) were used to modify carbon film electrodes by electrodeposition and glucose oxidase was then immobilized on the electrode by crosslinking with glutaraldehyde. A comparison between the two redox mediated biosensors for the determination of the metal ions was performed under the same experimental conditions. In fixed-potential amperometry at −0.35 V vs. the saturated calomel electrode, the detection limits for the metal cations studied were in the low micromolar range, lower at glucose oxidase/cobalt hexacyanoferrate/carbon film electrode, and the lowest achieved with glucose oxidase inhibition measurements. The linear dynamic ranges were wider at the glucose oxidase/poly(neutral red)/carbon film electrode for all three metal ...

A new three-dimensional human skin model consisting of several layers of actively dividing and metabolically active human neonatal foreskin-derived fibroblasts and epidermal keratinocytes grown on nylon mesh has been used to assess the in vitro toxicity of test agents from various classes. Utilizing a slight modification of the published neutral red viability assay for endpoint determination, we have assayed and obtained dose-dependent toxicity curves for test agents from the following classes: detergents (n = 15), alcohols (n = 5), metal chlorides (n = 10), perfumes and colognes (n = 5), shampoos (n = 4), conditioners (n = 3), moisturizers (n = 3), pesticides (n = 3), and antimicrobial preservatives (n = 4). Limited comparisons to in vivo ocular irritancy data with alcohols and detergents are encouraging. We have demonstrated the utility of this metabolically active dermal substrate containing naturally secreted collagen and other extracellular matrix proteins along with the neutral red viability assay for assessing the toxicity of a number of test agents from a variety of different classes with broad industrial applications.

Neutral red (Nr) is relatively non-toxic and is widely used as indicator dye in many biological test systems. It absorbs visible light and is known to act as a photosensitizer, involving the generation of reactive oxygen species (type-I reaction) and singlet oxygen (type-II reaction). The mutagenicity of Nr was determined in the Ames test (with Salmonella typhimurium strains TA1535, TA97, TA98, TA98NR, TA100, and TA102) with and without metabolic activation, and with and without photo-activation on agar plates. Similarly to the situation following metabolic activation, photo-mutagenicity of Nr was seen with all Salmonella strains tested, albeit with different effects between these strains. To our knowledge, Nr is the only photo-mutagen showing such a broad action. Since the effects are also observed in strains not known to be responsive to ROS, this indicates that ROS production is not the sole mode of action that leads to photo-genotoxicity. The reactive species produced by irradiation are short-lived as pre-irradiation of an Nr solution did not produce mutagenic effects when added to the bacteria. In addition, mutagenicity in TA98 following irradiation was stronger than in the nitroreductase-deficient strain TA98NR, indicating that nitro derivatives that are transformed by bacterial nitroreductase to hydroxylamines appear to play a role in the photo-mutagenicity of Nr. Photo-genotoxicity of Nr was further investigated in the comet assay and micronucleus test in L5178Y cells. Concentration-dependent increases in primary DNA damage and in the frequency of micronuclei were observed after irradiation.