Colorimetric Dye Research Articles

Retroreflectors for remote readout of colorimetric sensors

We have developed a remote detection system consisting of commercially available retroreflective material coated with an analyte-specific colorimetric dye. Quantitative performance modeling predicts that, given the appropriate indicator dye, a system with a 10 cm optic and eye-safe illumination should be capable of detecting small droplets of contamination at kilometer ranges. We have synthesized new colorimetric dyes specific to organophosphate contamination and, with these dyes, demonstrated detection of 1 μl of liquid malathion at over 150 m with less than 20 mW of laser illumination.

Synthesis and Characterization of Colorimetric Metal Sensing Properties Based on Azo Chromophore Moiety

In this work, we have designed and synthesized a particular colorimetric dye sensor for the detection of heavy metal ions. Dye sensor, namely 4-hydroxy-3-(fluoro-phenyl-thiourea-N'-nitrilo-methylidynyl)-azobenzene was synthesized using 5-phenyldiazenyl salicylaldehyde and 4-phenylthiosemi carbazide. The dye sensor has promising properties which are applicable to detect and recognize the presence of heavy metal ions with colorimetric changing functions. Electron rich part in dye sensor structure, which can be utilized as metal binding unit, such as NH, N, S and OH (atom/group) provides the functions to coordinate metal ions. This metal ion detection property was determined by using UV-Vis absorption, 1H-NMR peak shift and computational calculation method analysis. Furthermore, we estimated the electrochemical properties of dye sensor by using cyclicvoltammetry. In addition, the formation type of metal binding complex was determined by Job's plot measurements.

Development and Initial Results of a Low Cost, Disposable, Point-of-Care Testing Device for Pathogen Detection

Development of small footprint, disposable, fast, and inexpensive devices for pathogen detection in the field and clinic would benefit human and veterinary medicine by allowing evidence-based responses to future out breaks. We designed and tested an integrated nucleic acid extraction and amplification device employing a loop-mediated isothermal amplification (LAMP) or reverse transcriptase-LAMP assay. Our system provides a screening tool with polymerase-chain-reaction-level sensitivity and specificity for outbreak detection, response, and recovery. Time to result is ∼90 min. The device utilizes a swab that collects sample and then transfers it to a disc of cellulose-based nucleic acid binding paper. The disc is positioned within a disposable containment tube with a manual loading port. In order to test for the presence of target pathogens, LAMP reagents are loaded through the tube's port into contact with the sample containing cellulose disc. The reagents then are isothermally heated to 63°C for ∼1 h to achieve sequence-specific target nucleic acid amplification. Due to the presence of a colorimetric dye, amplification induces visible color change in the reagents from purple to blue. As initial demonstrations, we detected methicillin resistant Staphylococcus aureus genomic DNA, as well as recombinant and live foot-and-mouth disease virus.

The optimisation of a paired emitter–detector diode optical pH sensing device

With recent improvements in wireless sensor network hardware there has been a concurrent push to develop sensors that are suitable in terms of price and performance. In this paper a low-cost gas sensor is detailed, and significant improvements in sensor characteristics have been achieved compared to previously published results. A chemical sensor is presented based on the use of low-cost LEDs as both the light source and photodetector, coupled with a sensor slide coated with a pH sensitive colorimetric dye to create a simple gas sensor. Similar setups have been successfully used to detect both acetic acid and ammonia. The goal of this work was to optimise the system performance by integration of the sensing technique into a purposely deigned flowcell platform that holds the colorimetric slide and optical detector in position. The reproducibility of the sensor has been improved through this arrangement and careful control of deposited film thickness. The enhanced reproducibility between sensors opens the potential of calibration-free measurement, in that calibration of one sensor can be used to model the characteristics of all sensors in a particular batch.

Highly selective colorimetric fluorescence sensor for Cu 2+: cation-induced ‘ switching on’ of fluorescence due to excited state internal charge transfer in the red/near-infrared region of emission spectra

A 1,8-naphthalimide-based colorimetric fluorescence perinone dye, receptor 1 is reported herein for the selective detection of Cu 2+ over the other heavy and transition metal ions. Receptor 1 shows a strong colorimetric change from orange to purple and a dramatic enhancement of fluorescence intensity due to cation-induced excited state internal charge transfer during the sensing event, that is, a dual optical response that would facilitate naked-eye detection of Cu 2+.

Quantitative proteomics: assessing the spectrum of in-gel protein detection methods.

Proteomics research relies heavily on visualization methods for detection of proteins separated by polyacrylamide gel electrophoresis. Commonly used staining approaches involve colorimetric dyes such as Coomassie Brilliant Blue, fluorescent dyes including Sypro Ruby, newly developed reactive fluorophores, as well as a plethora of others. The most desired characteristic in selecting one stain over another is sensitivity, but this is far from the only important parameter. This review evaluates protein detection methods in terms of their quantitative attributes, including limit of detection (i.e., sensitivity), linear dynamic range, inter-protein variability, capacity for spot detection after 2D gel electrophoresis, and compatibility with subsequent mass spectrometric analyses. Unfortunately, many of these quantitative criteria are not routinely or consistently addressed by most of the studies published to date. We would urge more rigorous routine characterization of stains and detection methodologies as a critical approach to systematically improving these critically important tools for quantitative proteomics. In addition, substantial improvements in detection technology, particularly over the last decade or so, emphasize the need to consider renewed characterization of existing stains; the quantitative stains we need, or at least the chemistries required for their future development, may well already exist.

Differences in MSU-induced Superoxide Responses by Neutrophils from Gout Subjects Compared to Healthy Controls and a Role for Environmental Inflammatory Cytokines and Hyperuricemia in Neutrophil Function and Survival

To determine whether monosodium urate (MSU) crystal-induced superoxide production is greater for neutrophils from patients with gout compared to asymptomatic hyperuricemic and healthy controls, and whether neutrophil functions are altered by an MSU crystal-induced inflammatory environment. Neutrophils were purified from the whole blood of study participants, restimulated with 500 mg MSU crystals ex vivo, and superoxide production measured using the colorimetric dye WST-1. Purified neutrophils were exposed to conditioned media from MSU crystal-activated blood monocyte cultures with and without neutralizing antibodies for interleukin 1ss (IL-1ss), IL-8 (CXCL8), IL-6, and tumor necrosis factor-alpha (TNF-alpha). Neutrophil superoxide production was measured and neutrophil apoptosis and IL-8 production were determined by flow cytometry. Serum samples were collected from participants and analyzed by Lincoplex bead array for IL-1ss, IL-8, IL-6, and TNF-alpha. Neutrophils from gout and asymptomatic hyperuricemic subjects produced higher levels of MSU crystal-induced superoxide, and a weak trend toward elevated serum cytokines was observed compared to healthy controls. A correlation between serum uric acid and elevated neutrophil superoxide production was also observed. Neutrophils exposed to media from MSU crystal-activated monocytes exhibited enhanced superoxide production to MSU-crystal stimulation, increased IL-8 production, and extended cell survival that was predominantly dependent on IL-8, TNF-alpha and IL-6, respectively. Neutrophils from gout and asymptomatic hyperuricemic individuals are primed for enhanced MSU crystal-induced superoxide production that may be driven by a subclinical inflammatory cytokine environment combined with hyperuricemia. This inflammatory environment likely contributes to elevated neutrophil IL-8 production and survival in the absence of direct crystal stimulation. Asymptomatic hyperuricemia is not associated with suppressed neutrophil function.

Synergistic Growth Inhibition with a PI3 Kinase/mTOR Inhibitor Plus Lapatinib.

Abstract The mammalian target of rapamycin, mTOR, is a member of the phosphatidylinositol 3-kinase (PI3-K) /Akt family and plays a key role in cell growth and survival. The PI3-K/mTOR pathway is activated by many growth factor receptors in human tumor cells. In addition, the PI3-K pathway is constitutively activated by mutation in one-third of breast cancers. Therefore, blockade of the PI3-K/mTOR pathway may sensitize breast cancers to HER-2/neu inhibition. BEZ235 (Novartis Pharma AG) is a potent, oral, dual PI3-K/mTOR inhibitor currently in Phase I clinical trials. The purpose of this study was to assess the in vitro effects of combining a dual PI3-K/mTOR inhibitor (BEZ235, Novartis Pharma AG) and a lapatinib-like dual inhibitor of EGFR and HER-2/neu (GW2974) on the growth of HER-2/neu-transfected MCF-7 human breast cancer cells. Cells were exposed to the lapatinib-like compound [GW2974 (0.05-5.0µM)] or the dual PI3-K/mTOR inhibitor [BEZ235 (0.5-3nM)] alone or in combination for 3 days (12 different dose combinations were tested). Cell growth was determined using the colorimetric MTT tetrazolium dye assay. The Chou-Talalay method of isobologram analysis was performed to determine the inhibitory effect. Dose-dependent growth inhibition was observed in HER-2/neu-transfected MCF-7 cells after 3 days exposure to GW2974 (IC50: 5.0 mM) or BEZ235 (IC50: 2.2 nM). Exposure to the combination of GW2974 (0.05-0.50 mM) and BEZ235 (0.50-1.0 nM) resulted in synergistic growth inhibition in HER-2/neu-transfected MCF-7 human breast cancer cells. In summary, the combination of lapatinib plus PI3-K/mTOR warrants further investigation as a therapeutic strategy for breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6114.

Growth Inhibition of Human Breast Cancer Cells with an Iron Chelator (Deferasirox) and Docetaxel.

Abstract Treatment of cells with an iron chelator results in G1/S arrest and apoptosis. Tumor cells appear to be more sensitive to iron chelation probably because they express higher levels of the iron containing enzyme, ribonucleotide reductase. Previously, our laboratory has shown that an iron chelator (deferoxamine) inhibited the growth of a variety of cancer cell lines and also prolonged survival in tumor bearing mice. Docetaxel is an antineoplastic agent belonging to the taxane family. Docetaxel induces bcl-2 phosphorylation and subsequent apoptosis and is effective in the treatment of patients with breast cancer. The purpose of this study was to assess the in vitro effects of combining an iron chelator, deferasirox (Exjade: Novartis Pharma AG), and docetaxel on the growth of MCF-7 and HER-2/neu-transfected MCF-7 human breast cancer cells. Cells were exposed to deferasirox or docetaxel alone or in combination for 3 days (9 different dose combinations were tested). Cell growth was determined using the colorimetric MTT tetrazolium dye assay. Dose-dependent growth inhibition was observed in both the MCF-7 and the HER-2/neu-tranfected MCF-7 cells after 3 days exposure to deferasirox (IC50: 1.5 and 2.3 μM, respectively) or docetaxel (IC50: 0.9 nM and 2.0 nM, respectively). Exposure to the combination of deferasirox (0.25 – 1.5 μM) and docetaxel (0.1 – 0.5 nM) resulted in synergistic growth inhibition in both the MCF-7 and HER-2/neu-transfected MCF-7 human breast cancer cells. Sequencing experiments with the HER-2/neu-transfected MCF-7 cells showed that simultaneous exposure of the cells to deferasirox and docetaxel was superior to pretreating the cells with either of the agents 6 or 24 hours prior to addition of the second agent. These results suggest this combination warrants further investigation as a therapeutic strategy for breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5075.

Anticancer Properties of Highly Purified l-Asparaginase from Withania somnifera L. against Acute Lymphoblastic Leukemia

Withania somnifera L. has been traditionally used as a sedative and hypnotic. The present study was carried out for the purification, characterization, and in vitro cytotoxicity of L-asparaginase from W. somnifera L. L-Asparaginase was purified from the fruits of W. somnifera L. up to 95% through chromatography. The purified L-asparaginase was characterized by size exclusion chromatography, polyacrylamide gel electrophoresis (PAGE), and 2D PAGE. The antitumor and growth inhibition effect of the L-asparaginase was assessed using [3-(4, 5-dimethyl-thiazol-2yl)-2, 5-diphenyl-tetrazolium bromide] (MTT) colorimetric dye reduction method. The purified enzyme is a homodimer, with a molecular mass of 72 +/- 0.5 kDa, and the pI value of the enzyme was around 5.1. This is the first report of the plant containing L-asparaginase with antitumor activity. Data obtained from the MTT assay showed a LD(50) value of 1.45 +/- 0.05 IU/ml. W. somnifera L. proved to be an effective and a novel source of L: -asparaginase. Furthermore, it shows a lot of similarity with bacterial L-asparaginases EC-2.

Confounding experimental considerations in nanogenotoxicology

The development of novel nanomaterials with unique physico-chemical properties is increasing at a rapid rate, with potential applications across a broad range of manufacturing industries and consumer products. Nanomaterial safety is therefore becoming an increasingly contentious issue that has intensified over the past 4 years, and in response, a steady stream of studies focusing on nanotoxicology are emerging. However, it is becoming increasingly evident that nanomaterials cannot be treated in the same manner as chemical compounds with regards to their safety assessment, as their unique physico-chemical properties are also responsible for unexpected interactions with experimental components that generate misleading data-sets. In this report, we focus on nanomaterial interactions with colorimetric and fluorometric dyes, components of cell culture growth medium and genotoxicity assay components, and the resultant consequences on test systems are demonstrated. Thus, highlighting some of the potential confounding factors that need to be considered in order to ensure that in vitro genotoxicity assays report true biological impacts in response to nanomaterial exposure.

Oxidase-like activity of polymer-coated cerium oxide nanoparticles.

Inorganic enzyme? Ceria nanoparticles exhibit unique oxidase-like activity at acidic pH values. These redox catalysts can be used in immunoassays (ELISA) when modified with targeting ligands (see picture; light blue and yellow structures are nanoparticles with attached ligands). This modification allows both for binding and for detection by the catalytic oxidation of sensitive colorimetric dyes (e.g. TMB).

Purification, Characterization and In vitro Cytotoxicity of L-asparaginase from Withania somnifera L. Against Acute Lymphoblastic Leukemia

Withania somnifera L. has been traditionally used as a sedative and hypnotic. Withania somnifera L. is reported to have anti-carcinogenic effects in animal and cell cultures by decreasing the expression of nuclear factor-kappaB, suppressing intercellular tumor necrosis factor, and potentiating apoptotic signalling in cancerous cell lines [1]. The present study was carried out on the purification, characterization and in vitro cytotoxicity of L-asparaginase from Withania somnifera L., a popular medicinal plant. L-asparaginase was purified from the crude extract of the fruits of Withania somnifera L. up to 95% through column chromatography. The purified L-asparaginase was characterized by size exclusion chromatography, PAGE and 2-D PAGE. The antitumor and growth inhibition effect of the L-asparaginase was assessed using MTT colorimetric dye reduction method. The purified enzyme is a homodimer, with a molecular mass of 72 ± 0.5 kDa, and pI value of the enzyme was around 5.1. It is the first report for plant L-asparaginase with antitumor activity. Data obtained from the MTT assay indicated that L-asparaginase significantly (P < 0.05) reduced the viability of lymphocyte cells in a dose-dependent manner, showing a LD50 value of 1.45 ± 0.05 IU/ml. Withania somnifera L. proved to be an effective and a novel source of L-asparaginase, furthermore it shows lot of similarity with bacterial L-asparaginases which have already been commercialized for the treatment of acute lymphoblastic leukemia.

Colorimetric Sensor Array for Qualitative Water Analysis

A chemosensor array comprising 45 off-the-shelf colorimetric dyes, dubbed the Singapore Tongue (SGT), that is capable of discriminating different brands of bottled water and waters of different geographical attribute is described. Twelve kinds of bottled waters were tested by the SGT, and changes of absorbance spectra were analyzed by unsupervised classification methods to validate the SGT system for water analysis. All 12 bottled waters were discriminated at 1 × concentration, and SGT could distinguish the identity of samples of the waters diluted up to 100 times, except distilled waters. Following the study of 63 tap waters in different mass rapid transit stations in Singapore, two distinct clusters were observed from a principal component analysis plot, which correspond to the origin of the tap water. The successful discrimination and identification of in this study demonstrates the practical application of the SGT as a simple tool for water analysis.

A new approach to the toxicity testing of carbon-based nanomaterials—The clonogenic assay

The cellular toxicity of three types of carbon nanoparticles, namely HiPco ® single-walled carbon nanotubes (SWCNT), arc discharge SWCNT and Printex 90 carbon black nanoparticles, was studied on three different cell models including the human alveolar carcinoma epithelial cell line (A549), the normal human bronchial epithelial cell line (BEAS-2B) and the human keratinocyte cell line (HaCaT) using the clonogenic assay. Carbon nanomaterials are known to interact with colorimetric indicator dyes frequently used in cytotoxicity assays. By employing the clonogenic assay, any such interactions could be avoided, allowing a more reliable method for the in vitro toxicity assessment of carbon-based nanoparticles. It could be shown that the toxicity of as produced SWCNT samples differs between cell lines and the SWCNT production method used, with HiPco ® SWCNT samples being more reactive compared to arc discharge produced SWCNT samples, both eliciting a stronger cytotoxic response than carbon black. Furthermore, it was possible to distinguish between effects on cell viability and cell proliferation by including colony size as an additional endpoint in the clonogenic assay. All three particle types were highly effective in inhibiting cell proliferation in all three cell lines, whereas only HaCaT and BEAS-2B cells also showed decreased cell viability.

Counterion Free Colorimetric Metal Cation Sensor Array

By a systematic ionic effect study in colorimetric dye arrays, we effectively suppressed the anion effect using the proper buffer condition and configured a universal cation sensor system. This result cast a warning for careful validation for other anion sensing systems in aqueous solution.

Calcium oxalate, and not other metabolites, is responsible for the renal toxicity of ethylene glycol

Ethylene glycol (EG) is nephrotoxic due to its metabolism. Many studies suggest that the toxicity is due to oxalate accumulation, but others have conversely suggested that toxicity results from effects of metabolites such as glycolaldehyde or glyoxylic acid on proximal tubule cells. In vivo studies have indicated that accumulation of calcium oxalate monohydrate (COM) corresponds closely with development of toxicity in renal tissue. The present studies were therefore designed to clarify the roles of various metabolites in the mechanism for EG toxicity in vitro by comparing the relative cytotoxicity of EG metabolites using three measures of cell death, ethidium homodimer uptake, lactate dehydrogenase (LDH) release and the conversion of the tetrazolium salt XTT to a colorimetric dye. Human proximal tubule cells in culture were incubated in physiologic buffers for 6 h at 37 °C with COM (147–735 μg/ml, an oxalate equivalence of 1–5 mM), glycolate (5–25 mM), glyoxylate (0.2–5 mM) and glycolaldehyde (0.2–2 mM). To assess the effects of acidity on the cytotoxicity, incubations were carried out at pH 6–7.4. The results show that COM dose-dependently increased LDH release and ethidium homodimer uptake, while the other metabolites did not. Conversely, COM had no effect on the XTT assay, while high concentrations of glycolaldehyde and glyoxylate decreased XTT activity, but the latter only at acidic pH. The correlation between the uptake of ethidium homodimer and the release of LDH suggest that COM is cytotoxic to human kidney cells in culture, while the XTT assay does not validly measure cytotoxicity in this system. These results indicate that COM, and not glyoxylate or glycolaldehyde, is the toxic metabolite responsible for the acute tubular necrosis and renal failure that is observed in EG-poisoned patients.

Inhibition of the Extracellular Signal-regulated Kinase/Mitogen-activated Protein Kinase Pathway Decreases DNA Methylation in Colon Cancer Cells

The extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK-MAPK) pathway is a critical intermediary for cell proliferation, differentiation, and survival. In the human colon cancer cell line SW1116, treatment with the DNA methyltransferase 1 (DNMT1) inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) or the ERK-MAPK inhibitors PD98059 or rottlerin, or transient transfection with the MAP/ERK kinase (MEK)1/2 small interfering RNA down-regulates DNMT1 and proliferating cell nuclear antigen levels. In this report, we found that drug treatment or small interfering RNA transfection of SW1116 cells induced promoter demethylation of the p16(INK4A) and p21(WAF1) genes, which up-regulated their mRNA and protein expression levels. Flow cytometry revealed that rottlerin treatment induced cell cycle arrest at phase G(1) (p < 0.05). Thus, the ERK-MAPK inhibitor treatment or siRNA-mediated knockdown of ERK-MAPK decreases DNA methylation via down-regulating DNMT1 expression and other unknown mediator(s) in SW1116 colon cancer cells.

Protein stains for proteomic applications:Which, when, why?

This review recollects literature data on sensitivity and dynamic range for the most commonly used colorimetric and fluorescent dyes for general protein staining, and summarizes procedures for the most common PTM-specific detection methods. It also compiles some important points to be considered in imaging and evaluation. In addition to theoretical considerations, examples are provided to illustrate differential staining of specific proteins with different detection methods. This includes a large body of original data on the comparative evaluation of several pre- and post-electrophoresis stains used in parallel on a single specimen, horse serum run in 2-DE (IPG-DALT). A number of proteins/protein spots are found to be over- or under-revealed with some of the staining procedures.

Synergistic inhibition of breast cancer cell lines with the combination of a dual inhibitor of EGFR/HER-2/neu and a Bcl-2 inhibitor

13100 Background: The epidermal growth factor receptor (EGFR; ErbB1) and HER-2/neu (ErbB2), members of the ErbB family of receptor tyrosine kinases, are overexpressed in a variety of human tumors and overexpression generally correlates with poor prognosis and decreased survival. Use of inhibitors of these receptors as monotherapies, e.g., trastuzumab, Iressa, and erlotinib, has led to advances in treatment, but many patients do not respond or develop resistance. The anti-apoptotic protein, Bcl-2, is also overexpressed in a number of human tumors. Inhibitors of Bcl-2 induce apoptosis and sensitize cancer cells to other therapies. This study assesses the effects of a combination of a reversible inhibitor of both EGFR and HER-2/neu that is similar to lapatinib (GW2974) and a pan inhibitor of the Bcl-2 family (GX15–070: Gemin X Biotechnologies, Inc.) on the growth of human breast cancer cells. Methods: The MCF-7 human breast cancer cell line transfected with a control vector, MCF/neo, and the HER-2/neu transfected MCF-7 cell line, MCF/18, were treated with various concentrations of GW2974 (0.25–10 μM) and/or the GX15–070 pan Bcl-2 inhibitor (50–500 nM). After a 3 day exposure, cell number was determined using the colorimetric MTT tetrazolium dye assay. Percent of control was normalized to corresponding concentrations of the solvent for both agents (DMSO). Results: Treatment with the GW2974 dual inhibitor or the GX15–070 pan Bcl-2 inhibitor resulted in dose-dependent growth inhibition in both the control and HER-2/neu transfected MCF-7 cell lines. The combination of both agents produced synergistic growth inhibition in both cell lines as confirmed by isobologram analysis. Conclusions: This study has demonstrated synergy with the combination of a dual inhibitor of EGFR and HER-2/neu and an inhibitor of Bcl-2 in control and HER-2/neu overexpressing MCF-7 human breast cancer cells. This finding warrants an evaluation of this combination in clinical trials for the treatment of patients with metastatic breast cancer. [Table: see text]