Transplantation of insulin-producing cells derived from human MSCs to treat diabetes in a non-human primate model.

BACKGROUND: Islet cell transplantation is an emerging therapy in the treatment of diabetes mellitus. Differentiation of islet cells from mesenchymal stem cells (MSCs) is a potential solution to the challenge of insufficient donor sources. This study investigated whether human umbilical cord-derived MSCs could effectively differentiate into insulin-producing cells (IPCs) and evaluated the therapeutic efficacy of IPCs in treating diabetes. METHODS: IPCs were induced from MSCs by a two-step protocol. IPC expression products were evaluated by western blot and real-time PCR. IPC insulin secretion was evaluated by ELISA. The viability of IPCs was measured by FDA/PI and dithizone staining. The non-human primate tree shrew was used as a diabetes model. After a single STZ induction into a diabetes model, a single intraportal transplantation of IPCs, MSCs, or normal saline was performed (n = 6 per group). Blood glucose was monitored for 3 weeks, then the animals were euthanized and the distribution of IPCs in the liver was examined pathologically. RESULTS: After about 3 weeks of in vitro induction, IPCs formed microspheres of 100-200 µm, with >95% viable cells that were dithizone stain positive. IPCs expressed islet-related genes and proteins and secreted high levels of insulin whether stimulated by low or high levels of glucose. After transplantation of IPCs into diabetic tree shrews, blood glucose levels decreased rapidly to near normal and were significantly lower than the MSC or saline groups for 3 weeks thereafter. CONCLUSION: We present the novel discovery that IPCs derived from human umbilical cord MSCs exert a therapeutic effect in a non-human primate model of diabetes. This study provides a preliminary experimental basis for the use of autologous MSC-derived IPCs in the treatment of human diabetes.

Paneth cells protect intestinal stem cell niche to alleviate deoxynivalenol-induced intestinal injury.

Deoxynivalenol (DON) is a common toxin in grains and feeds, and DON exposure triggers severe small intestinal injury and inflammation, which harms the health of humans and livestock. DON treatment leads to a decrease in Paneth cells, whereas the role of Paneth cells in DON-induced intestinal injury is poorly understood. We utilized dithizone (40 mg/kg) to keep murine Paneth cell number at a low level. The results showed that dithizone-mediated long-term disruption of Paneth cells aggravated intestinal injury, intestinal stem cell (ISC) loss, and microbiota disorder in DON (2 mg/kg)-treated mice. Unexpectedly, the number of goblet cells and proliferative cells was boosted in mice treated with dithizone and DON. After dithizone and DON treatments, the Firmicutes/Bacteroidetes (F/B) ratio was reduced, and the increased abundance of Dubosiella and the decreased abundance of Lactobacillus were observed in mice. The functional recovery of Paneth cells by lysozyme (200 U/day) supplementation improved intestinal injury and ISC loss in mice after DON challenge. In addition, lysozyme also promoted the growth and ISC activity of intestinal organoids. Taken together, these results demonstrate the protective role of Paneth cells in DON-induced intestinal injury. Our study raises a novel target, Paneth cell, for the treatment of DON exposure.

Dithizone-functionalized C18 online solid-phase extraction-HPLC-ICP-MS for speciation of ultra-trace organic and inorganic mercury in cereals and environmental samples.

A simple and efficient dithizone-functionalized solid-phase extraction (SPE) procedure, online coupled with high-performance liquid chromatography (HPLC)-inductively coupled plasma mass spectrometry, was developed for the first time for enrichment and determination of ultra-trace mercury (Hg) species (inorganic divalent Hg (Hg(II)), methylmercury (CH3Hg(II)) and ethylmercury (C2H5Hg(II)) in cereals and environmental samples. In the proposed method, functionalization of the commercial C18 column with dithizone, enrichment, and elution of the above Hg species can be completed online with the developed SPE device. A simple solution of 2-mercaptoethanol (1% (V/V)) could be used as an eluent for both the SPE and HPLC separation of Hg species, significantly simplifying the method and instrumentation. The online SPE method was optimized by varying dithizone dose, 2-mercaptoethanol concentration, and sample volume. In addition, the effect of pH, coexisting interfering ions, and salt effect on the enrichment was also discussed. Under the optimized conditions, the detection limits of Hg species for 5 mL water sample were 0.15 ng/L for Hg(II), 0.07 ng/L for CH3Hg(II), and 0.04 ng/L for C2H5Hg(II) with recoveries in the range of 85%-100%. The developed dithizone-functionalized C18 SPE column can be reused after a single functionalization, which significantly simplifies the enrichment step. Moreover, the stability of Hg species enriched on the SPE column demonstrated its suitability for field sampling of Hg species for later laboratory analysis. This environment-friendly method offers a robust tool to detect ultra-trace Hg species in cereals and environmental samples.

A sensitive fluorescent probe based on dithizone-capped ZnS quantum dots for quercetin determination in biological samples.

A simple turn on/off fluorescence approach based on dithizone-capped ZnS quantum dots (ZnS@DZ QDs) with the help of lead ions as a fluorescent probe for the quantitative determination of quercetin is reported. The interaction of lead ions with dithizone led to the formation of a rigid structure on the surface of ZnS@DZ QDs and turned on the fluorescence intensity of the QDs. After addition of quercetin to this probe and interaction with lead ions, the fluorescence emission turned off. Concerning the quenching fluorescence intensity of ZnS@DZ QDs/Pb2+ QDs probe induced by the target, under the optimum conditions, the probe enabled detection of quercetin in the concentration range from 0.54 µM to 21.7 µM with a correlation coefficient of 0.993 and detection limit of 0.25 µM. The present probe was applied successfully to the determine quercetin as a nutritional biomarker in human serum and 24-h urine samples.

Cysteine Prevents the Development of Experimental Diabetes Induced by Zinc-Binding Substances.

In experimental rabbits, cysteine injected intravenously in a dose of 1000 mg/kg temporarily bound zinc in ß cells and prevented the formation of chelate zinc complexes in response to subsequent injection of diabetogenic zinc-binding substances that induce cell destruction. Injection of cysteine to animals was associated with a sharply negative reaction to zinc in ß cells, which attests to blockade of zinc ions. Injection of cysteine few minutes after dithizone and formation of zinc-dithizone complex was followed by displacement of dithizone from the complex and prevented the development of diabetes in most animals. The most plausible mechanism of preventive effect of cysteine is the formation of 2:1 zinc-cysteine complex in ß cells with possible fixation of Zn atom between sulfur atoms from SH groups of two cysteine molecules.

Paneth Cell Ablation Aggravates Pancreatic and Intestinal Injuries in a Rat Model of Acute Necrotizing Pancreatitis after Normal and High-Fat Diet.

We previously reported that acute necrotizing pancreatitis (ANP) after normal or high-fat diet is associated with a decreased number of Paneth cells in ileal crypts. Here, we ablated Paneth cells in a rat model of ANP after normal and high-fat diet to investigate the effects on disease symptoms. Adult male Sprague-Dawley rats received standard rat chow or a high-fat diet for 2 weeks, after which they were treated with dithizone to deplete Paneth cells. Six hours later, ANP was established by retrograde injection of sodium taurocholate into the biliopancreatic duct. Rats were sacrificed at 6, 12, and 24 h for assessment. We found dithizone aggravated ANP-associated pathological injuries to the pancreas and ileum in rats on high-fat or standard diets. Lysozyme expression in ileal crypts was decreased, while serum inflammatory cytokines (TNFα, IL-1ß, and IL-17A) and intestinal permeability (serum DAO activity and D-lactate) were increased. Expression of tight junction proteins (claudin-1, zo-1, and occludin) was decreased. Using high-throughput 16S rRNA sequencing, we found dithizone reduced microbiota diversity and altered microbiota composition in rats on high-fat or standard diets. Dithizone decreased fecal short-chain fatty acids (SCFAs) in rats on high-fat or standard diets. Changes in intestinal microbiota correlated significantly with SCFAs, lysozyme, DAO activity, D-lactate, inflammatory cytokines, and pathological injury to the pancreas and ileum in rats on high-fat or standard diets. In conclusion, ablation of Paneth cells exacerbates pancreatic and intestinal injuries in ANP after normal and high-fat diet. These symptoms may be related to changes in the intestinal microbiota.

In situ clinical evidence that zinc levels are decreased in breast invasive ductal carcinoma.

PURPOSE: Altered zinc levels in malignant cells versus their normal cells have important implications in the development and progression of several cancers. Prostate, pancreatic, and hepatocellular carcinomas exhibit consistent marked zinc decrease in situ in the malignant cells, and other cancers (such as kidney, lung, and thyroid) also exhibit decreased tissue zinc levels. However, zinc levels are increased in breast cancer tissue compared to breast normal tissue, and the contemporary dominant view is that zinc is increased in invasive ductal carcinoma. This has important implications regarding the role and effects of zinc in breast malignancy compared to other cancers, which caused us to initiate this study to either confirm or challenge the contemporary view of an increased zinc level in the invasive ductal malignant cells. METHODS: We employed dithizone staining of breast tissue sections and tissue cores to determine the relative in situ cellular zinc levels specifically in the invasive ductal malignant cells as compared to normal ductal epithelium. This approach had not been employed in any of the reported breast studies. RESULTS: The results revealed that the zinc levels are consistently and markedly decreased in the ductal malignant cells as compared with higher prominent zinc levels in the normal ductal epithelium. Decreased zinc is evident in Grade 1 well-differentiated malignancy and in Grade 2 and Grade 3 carcinomas. Among the twenty-five cancer cases in this study, none exhibited increased zinc in the invasive ductal carcinoma compared to the zinc level in the normal ductal epithelium. CONCLUSIONS: The decreased zinc levels in breast invasive ductal carcinoma is consistent with prostate, pancreatic, and liver carcinomas in which the decrease in zinc is a required event in the development of malignancy to prevent cytotoxicity that would result from the higher zinc levels in the normal cells. This new understanding requires a redirection in elucidating the mechanisms and factors regarding the regulation of zinc in breast cancer, its potential translational applications as possible biomarkers, and for treatment of breast invasive ductal carcinoma.

Zinc-Dithizone Complex Engineered Upconverting Nanosensors for the Detection of Hypochlorite in Living Cells.

Current chemo/biosensors for hypochlorous acid or hypochlorite detections are usually limited to the submicromolar level because of their insufficient sensitivity, which is a problem because the concentrations in biological matrices is generally on the nanomolar scale or even lower. Developing a probe with a high enough sensitivity remains a challenge. Using the minimal background fluorescence of upconversion nanocrystals to our advantage, we herein report on an energy-transfer mechanism-based upconversion luminescent nanosensor for the sensitive and selective detection of hypochlorite in aqueous solution. In this nanosensor water-dispersible upconversion nanoparticles act as the energy donor and a novel hypochlorite-responsive coordination complex Zn(DZ)3 is employed as the energy acceptor. The quenched upconversion luminescence, induced by the Zn(DZ)3 complex, can be efficiently recovered after addition of hypochlorite through the selective oxidative breakage of the Zn-S-C bonds in the Zn(DZ)3 complex, which was verified by mass spectrometry. The detection limit for hypochlorite of this sensing system is as low as 3 nM. Furthermore, this newly coordination-complex engineered upconversion nanosensor is successfully applied to image different amounts of exogenous hypochlorite in living HeLa cells.

Cadmium uptake, localization and stress-induced morphogenic response in the fern Pteris vittata.

Cadmium uptake, tissue localization and structural changes induced at cellular level are essential to understand Cd tolerance in plants. In this study we have exposed plants of Pteris vittata to different concentrations of CdCl2 (0, 30, 60, 100 µM) to evaluate the tolerance of the fern to cadmium. Cadmium content determination and its histochemical localization showed that P. vittata not only takes up, but also transports and accumulates cadmium in the aboveground tissues, delocalizing it mainly in the less bioactive tissues of the frond, the trichomes and the scales. Cadmium tolerance in P. vittata was strictly related to morphogenic response induced by the metal itself in the root system. Adaptive response regarded changes of the root apex size, the developmental pattern of root hairs, the differentiation of xylem elements and endodermal suberin lamellae. All the considered parameters suggest that, in our experimental conditions, 60 µM of Cd may represent the highest concentration that P. vittata can tolerate; indeed this Cd level even improves the absorbance features of the root and allows good transport and accumulation of the metal in the fronds. The results of this study can provide useful information for phytoremediation strategies of soils contaminated by Cd, exploiting the established ability of P. vittata to transport, delocalize in the aboveground biomass and accumulate polluting metals.

Ultrasound-assisted surfactant-enhanced emulsification microextraction for the determination of Cd and Ni in tea and water samples.

A microextraction method based on ultrasound-assisted surfactant-enhanced emulsification using solidification of a floating organic droplet (UASEME-SFO) was evaluated for simultaneous determination of Cd and Ni in water and tea samples followed by flame atomic absorption spectrometry. In the UASEME-SFO technique, Triton X-100 was used as an emulsifier to accelerate the emulsification of the extraction solvent into a sample solution and hasten the mass transfer of the analytes. Analytes form a complex and are extracted into 1-dodecanol which was used as an extraction solvent. Some parameters such as type and volume of the extraction solvent, the type and concentration of the surfactant, ultrasound extraction time, reagent concentration, centrifuge conditions and salt concentration were investigated. Under optimum conditions, calibration curves were linear in the range of 0.3-100 and 0.6-180 microg L(-1) with detection limits of 0.11 and 0.20 microg L(-1) for Cd and Ni, respectively. The accuracy of the method was confirmed by parallel analyses using the certified reference material of water and tea samples. The recoveries of the analytes in tea leaves, tea infusions and water samples were in the range of 96.5-105.1%.

Differentiation of pancreatic stem and progenitor ß-cells into insulin secreting cells in mice with diabetes mellitus.

We studied in vitro differentiation of pancreatic stem and progenitor cells into insulin secreting cells in the model of streptozotocin-induced diabetes in C57Bl/6 mice. Streptozotocin was shown to increase the population of pancreatic oligopotent ß-cell precursors (CD45(-), TER119(-), CD133(+), and CD49f(low)) and did not affect multipotent (stem) progenitor cells (CD45(-), TER119(-), CD17(-), CD309(-)). During long-term culturing, diabetic multipotent progenitor cells showed high capacity for self-renewal. A population of dithizone-positive (insulin secreting cells) mononuclear cells was obtained releasing insulin after prolonged culturing in suspension enriched with diabetic CD45(-), TER119(-), CD17(-), and CD309(-) cells. The rate of generation of "new" insulin-producing cells and insulin release in the samples of experimental group considerably exceeded activity of the corresponding processes in the control group.

[Cloud Point extraction for determination of mercury in Chinese herbal medicine by hydride generation atomic fluorescence spectrometry with optimization using Box-Behnken design].

Cloud point extraction (CPE) is proposed as a pre-concentration procedure for the determination of Hg in Chinese herbal medicine samples by hydride generation-atomic fluorescence spectrometry (HG-AFS). Hg2+ was reacted with dithizone to form hydrophobic chelate under the condition of pH. Using Triton X-114, as surfactant, chelate was quantitatively extracted into small volume of the surfactant-rich phase by heating the solution in a water bath for 15 min and centrifuging. Four variables including pH, dithizone concentration, Triton X-114 concentration and equilibrium temperature (T) showed the significant effect on extraction efficiency of total Hg evaluated by single-factor experiment, and Box-Behnken design and response surface method- ology were adopted to further investigate the mutual interactions between these variables and to identify their optimal values that would generate maximum extraction efficiency. The results showed that the binomial was used to fit the response to experimental levels of each variable. ALL linear, quadratic terms of four variables, and interactions between pH and Trion X-114, pH and di- thizone affected the response value(extraction efficiency) significantly at 5% level. The optimum extraction conditions were as follows: pH 5.1, Triton X-114 concentration of 1.16 g x L(-1), dithizone concentration of 4.87 mol x L(-1), and T 58.2 degrees C, the predicted value of fluorescence was 4528.74 under the optimum conditions, and the experimental value had only 2.1% difference with it. Under the conditions, fluorescence was linear to mercury concentration in the range of 1-5 microg x L(-1). The limit of detection obtained was 0.01247 microg x L(-1) with the relative standard deviations (R.S.D.) for six replicate determinations of 1.30%. The proposed method was successfully applied to determination of Hg in morindae Radix, Andrographitis and dried tangerine samples with the recoveries of 95.0%-100.0%. Apparently Box-Behnken design combined with response surface analysis method was considered to be well used for optimization of the cloud point extraction.

Colorimetric response of dithizone product and hexadecyl trimethyl ammonium bromide modified gold nanoparticle dispersion to 10 types of heavy metal ions: understanding the involved molecules from experiment to simulation.

A new kind of analytical reagent, hexadecyl trimethyl ammonium bromide (CTAB), and dithizone product-modified gold nanoparticle dispersion, is developed for colorimetric response to 10 types of heavy metal ions (M(n+)), including Cr(VI), Cr(3+), Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), Cd(2+), Hg(2+), and Pb(2+). The color change of the modified gold nanoparticle dispersion is instantaneous and distinct for Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), Cd(2+), Hg(2+), and Pb(2+). The color change results from the multiple reasons, such as electronic transitions, cation-π interactions, formation of coordination bonds, and M(n+)-induced aggregation of gold nanoparticles (AuNPs). The different combining capacity of heavy metal ions to modifiers results in the different broadening and red-shifting of the plasmon peak of modified AuNPs. In addition, Cr(VI), Cu(2+), Co(2+), Ni(2+), and Mn(2+) cause the new UV-vis absorption peaks in the region of 360-460 nm. The interactions between the modifiers and AuNPs, and between the modifiers and M(n+), are investigated by using Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. The results confirm that AuNPs are modified by CTAB and dithizone products through electrostatic interactions and Au-S bonds, respectively, and the M(n+)-N bonds form between M(n+) and dithizone products. Furthermore, the experimental and density functional theory calculated IR spectra prove that dithizone reacts with NaOH to produce C6H5O(-) and [SCH2N4](2-). The validation of this method is carried out by analysis of heavy metal ions in tap water.

Differentiation of mesenchymal stem cells derived from human bone marrow and subcutaneous adipose tissue into pancreatic islet-like clusters in vitro.

Although stem cells are present in various adult tissues and body fluids, bone marrow has been the most popular source of stem cells for treatment of a wide range of diseases. Recent results for stem cells from adipose tissue have put it in a position to compete for being the leading therapeutic source. The major advantage of these stem cells over their counterparts is their amazing proliferative and differentiation potency. However, their pancreatic lineage transdifferentiation competence was not compared to that for bone marrow-derived stem cells. This study aims to identify an efficient source for transdifferentiation into pancreatic islet-like clusters, which would increase potential application in curative diabetic therapy. The results reveal that mesenchymal stem cells (MSC) derived from bone marrow and subcutaneous adipose tissue can differentiate into pancreatic islet-like clusters, as evidenced by their islet-like morphology, positive dithizone staining and expression of genes such as Nestin, PDX1, Isl 1, Ngn 3, Pax 4 and Insulin. The pancreatic lineage differentiation was further corroborated by positive results in the glucose challenge assay. However, the results indicate that bone marrow-derived MSCs are superior to those from subcutaneous adipose tissue in terms of differentiation into pancreatic islet-like clusters. In conclusion, bone marrow-derived MSC might serve as a better alternative in the treatment of diabetes mellitus than those from adipose tissue.

Sonic hedgehog inhibition induces mouse embryonic stem cells to differentiate toward definitive endoderm.

In the experimental group (shh inhibited group), there were significant decreases in the expression of Oct4, Nanog, Shh, GATA4, Brachyury and Goosecoid, while increases were observed for TAT and Pdx1. The expression of Sox17 did not differ between two control and experimental groups. In experimental group, the amount of GSC positive cells was somehow lower but it seems that there was no difference for Sox17. Shh inhibition induces ESCs to differentiate toward definitive endoderm by committing mesendodermal lineages.

Ultra-trace determination of cadmium in water and food samples by a thin-film microextraction using a supported liquid membrane combined with smartphone-based colorimetric detection.

A mixture of n-octanol and dithizone was introduced as an effective and novel extraction agent in a thin-film microextraction technique for the pre-concentration of cadmium ions. The extraction agent was immobilized on small pieces of porous polypropylene flat membrane as a supported liquid membrane. The analyte extraction was performed by immersing the modified film in the sample solution, and via a complex formation between the immobilized dithizone on the film and cadmium ions. After the thin-film microextraction process, the colored cadmium-dithizone complex was directly measured by a smartphone colorimetric analysis. Under optimized conditions, the linear dynamic range, the limit of detection, and the limit of quantification were 0.5-300.0, 0.1, and 0.4 µg L-1, respectively. The developed technique was successfully employed to quantify cadmium ions in water and food samples. The high relative recovery values (95.0-103.0%) along with relative standard deviations of less than 2.5% were obtained for the spiked samples.

Plasma jet desorption atomization-atomic fluorescence spectrometry and its application to mercury speciation by coupling with thin layer chromatography.

A novel plasma jet desorption atomization (PJDA) source was developed for atomic fluorescence spectrometry (AFS) and coupled on line with thin layer chromatography (TLC) for mercury speciation. An argon dielectric barrier discharge plasma jet, which is generated inside a 300 µm quartz capillary, interacts directly with the sample being analyzed and is found to desorb and atomize surface mercury species rapidly. The effectiveness of this PJDA surface sampling technique was demonstrated by measuring AFS signals of inorganic Hg(2+), methylmercury (MeHg), and phenylmercury (PhHg) deposited directly on TLC plate. The detection limits of the proposed PJDA-AFS method for inorganic Hg(2+), MeHg, and PhHg were 0.51, 0.29, and 0.34 pg, respectively, and repeatability was 4.7%, 2.2%, and 4.3% for 10 pg Hg(2+), MeHg, and PhHg. The proposed PJDA-AFS was also successfully coupled to TLC for mercury speciation. Under optimized conditions, the measurements of mercury dithizonate (Hg-D), methylmercury dithizonate (MeHg-D), and phenylmercury dithizonate (PhHg-D) could be achieved within 3 min with detection limits as low as 8.7 pg. The combination of TLC with PJDA-AFS provides a simple, cost-effective, relatively high-throughput way for mercury speciation.

Enhancement of olfaction by femtomolar concentrations of zinc ions.

Zinc is recognized as an important element for olfaction. Zinc nanoparticles enhance olfaction in response to odors; however, the mechanisms underlying this action remain unknown. Herein, the effect of zinc on olfactory receptors was deduced using electro-olfactogram (EOG) responses recorded from the isolated olfactory mucosae of bullfrogs (Rana catesbeiana) following the administration or chelation of zinc ions. Menthone and n-amyl acetate were used as odorants, whereas forskolin (an adenylate cyclase activator) and cholera toxin (a Gαolf activator) were used as intracellular signal transduction activators. The EOG responses provoked by the odorants and cholera toxin were suppressed by dithizone-mediated zinc ion chelation, and the EOG responses were recovered by administering non-chelated zinc. However, the EOG response to forskolin was not suppressed by dithizone. In contrast, the addition of femtomolar concentrations of zinc ions enhanced the EOG responses. The above-mentioned effects on EOG responses were examined by changing the concentration of zinc ions but not zinc nanoparticles. The results of this study suggest that Gαolf alone or both olfactory receptors and Gαolf likely require zinc ions for their activation.

In vitro differentiation of human pancreatic duct-derived PANC-1 cells into ß-cell phenotype using Tinospora cordifolia.

Type 1 diabetes mellitus is an autoimmune disorder leading to loss of beta cells. There is a dire need to inhibit apoptosis and induce regeneration of new beta cells. There are plants in the Indian medicine system having the potential for rejuvenation. In the present study, we have attempted to evaluate the capacity of aqueous extract of Tinospora cordifolia to regenerate beta cells from PANC-1 ductal cells. After differentiation, the characterization of ß-cell phenotype was carried out using dithizone and Gomori's staining and further confirmed by mRNA expression study of insulin, Pdx-1, and carbonic anhydrase-9. Insulin production was estimated with ELISA. Aqueous extract of Tinospora cordifolia at 15 µg/ml concentration can effectively induce differentiation of PANC-1 cells into beta cells. The morphological observations showed brownish-colored dithizone and purple-colored Gomori's staining. The ß-cells demonstrated significant mRNA expression of insulin and Pdx-1 and downregulation of carbonic anhydrase-9. The functionality of beta cells was demonstrated by 1.5-fold increase in insulin secretion in response to high glucose. Tinospora cordifolia has potential to differentiate PANC-1 ductal cells into functional beta cells and can be a lead towards non-invasive treatment of type 1 diabetes mellitus.

Environmental monitoring of trace metal pollutants using cellulosic-paper incorporating color change of azo-chromophore.

An essential requirement for colorimetric paper-sensor is to allow the target analytes (heavy metal ions) to access the chromophore while maintaining strong chromophore immobilization on the porous substrate surface. This work evaluates the selection of sensitive chromophores (dithizone, 1-(2-pyridylazo) 2-naphthol and 4-(2-pyridylazo)-resorcinol) and their immobilization strategies on paper sensors. Dithizone (DTz) are capable of producing a significant color transition at unadjusted pH, observed by UV-Vis absorption spectroscopy and visible recognition. After immobilizing DTz on a paper substrate (cellulose acetate/chitosan substrate), the DTz-paper sensor showed a distinctive color change from blue-green to peach-pink upon reaction with Pb2+ ions, and the color intensity was proportional to the metal concentration. Quantitative analysis using RGB (R:Red; G:Green; B:Blue) plots showed that increasing DTz concentration on the CA/CS paper sensor increases the difference in total color intensity (∆IT) and the difference in red code intensity (∆IR). This is due to the formation of more DTz-Pb2+ complexes on the CA/CS paper substrate. The CA/CS paper strips immobilized with 100 ppm DTz showed practical potential for rapid detection of heavy metal ions. The DTz-CA/CS paper sensor showed significant color change when detecting spiked heavy metals ions (0.1 ppm Pb2+, 2.0 ppm Zn2+, and 0.2 ppm Cu2+) in river water samples that prepared at the maximum permissible limit for industrial effluent in Malaysia.