Rutin-modified silver nanoparticles as a chromogenic probe for the selective detection of Fe3+ in aqueous medium

The development of nanoprobes for selective detection of metal ions in solution has attracted great attention due to their impact on living organisms. As a contribution to this field, this paper reports the synthesis of silver nanoparticles modified with rutin in the presence of ascorbic acid and their successful use as a chromogenic probe for the selective detection of Fe³⁺ in aqueous solution. Limits of detection and quantification were found to be 17 nmol L⁻¹ and 56 nmol L⁻¹, respectively. The sensing ability is proposed to proceed via an iron-induced nanoparticle growth/aggregation mechanism. A practical approach using image analysis for quantification of Fe³⁺ is also described.

The use of rutin-modified silver nanoparticles for selective detection and sensitive quantification of Fe³⁺ in aqueous solution is described.

Metal ions are key species in nature due to their essential functions in living organisms.^1,2 On the other hand, heavy metals as well as essential metals at abnormally high levels are toxic.² Iron, for instance, in addition to its popular use in industry and construction, is essential to the human body and active in biological processes. Although the trivalent form of iron is particularly important for oxygen transport in blood and the mitochondrial respiratory chain, high levels of this cation are associated with important pathologies.^3,4 The detection of metal ions in aqueous solution is traditionally performed by methods including atomic absorption spectrometry,⁵ electrochemical measurements,^6,7 and inductively coupled plasma techniques,⁸ among others. However, these techniques have important drawbacks, notably the need for sophisticated instrumentation, in addition to being time-consuming and requiring laborious procedures. To overcome these issues, the development of chromogenic and fluorogenic chemosensors for the selective detection of metal-targets has attracted great attention, especially due to the possibility of fast, sensitive and non-expensive analysis.^9,10 In the last decade, nanoscaled materials have been reported as selective probes for metal ions, including Fe³⁺.^11–24Silver nanoparticles (AgNPs) are of particular interest because of the affordable price of starting materials, ease of controlling size and morphology, possibility to functionalize their surface with organic molecules, and optical properties that enable detection of a variety of analytes via simple UV-vis spectroscopy and digital image analysis. Furthermore, applications of AgNPs are also biotechnologically relevant due to the possibility of green synthetic protocols, including the use of plant extracts,²⁵ natural sources,²⁶ glycerol,²⁷ among others.Flavonoids are secondary metabolites naturally found in fruits and other vegetables with relevant roles due to their nutritional, pharmaceutical and medicinal properties.²⁸ Because of their adequate structural features, flavonoids are candidates to be employed in the synthesis of AgNPs.²⁶ Rutin (RU), a sugar-based flavonoid, may be employed as reducing agent in the synthesis of AgNPs along with a stabilizer such as polyvinylpyrrolidone (PVP)²⁶ or used as crude plant extract component.^29,30This paper reports the use of RU-modified AgNPs (RU-AgNPs) as a chromogenic probe for Fe³⁺ in aqueous medium in the presence of ascorbic acid (AA). Sensing ability of RU-AgNPs for the selective detection of Fe³⁺ toward other metal cations was investigated with UV-vis spectroscopy analysis. These data and transmission electronic microscopy (TEM) results allowed a mechanistic proposal involved in the selective detection of Fe³⁺ by RU-AgNPs. Furthermore, a practical approach based on correlation of images of solutions obtained with a conventional smartphone and chemometrics was employed for a simpler quantification of Fe³⁺ in aqueous medium.Initially, the order of reagent combination was investigated in the synthesis of RU-AgNPs. Concentration of RU ranged from 0.10 to 0.50 mmol L⁻¹, while concentrations of other components were fixed at 0.20 mmol L⁻¹ AgNO₃, 0.10 mmol L⁻¹ AA and 0.10 M NaOH. Water was used as solvent in all cases. Narrower surface plasmon resonance (SPR) bands were obtained from adding a solution of AA and NaOH to a solution containing RU and AgNO₃ (Fig. 1a) against the addition of RU, AA, and NaOH to AgNO₃ solution (Fig. S1a – ESI^†), or the addition of RU and NaOH to a solution of AA and AgNO₃ (Fig. S1b^†). RU-AgNPs obtained from the condition presented in Fig. 1a are small (4.1 nm average diameter) and considerably polydisperse (standard deviation of 4.7 nm), however, presenting only one population (Fig. 1b and S2^†). A study of the influence of RU concentration (0.10 to 0.50 mmol L⁻¹) on the stability of RU-AgNPs over time indicated that 0.10 mmol L⁻¹ RU generates more stable RU-AgNPs (Fig. S3^†). Next, a study on the influence of pH indicated that RU-AgNPs are only stable under strong alkaline conditions (pH 12.5 or higher) (Fig. S4^†).Open in a separate windowFig. 1(a) UV-vis analysis of RU-AgNPs under strong alkaline condition (pH > 12.5) based on the order of adding reagents (RU, 0.10 to 0.50 mmol L⁻¹; AgNO₃, 0.20 mmol L⁻¹; AA, 0.10 mmol L⁻¹; NaOH, 0.10 mmol L⁻¹); (b) TEM image of RU-AgNPs under the selected condition.The ability of RU-AgNPs to sense metal cations was investigated by both naked-eye and UV-vis spectroscopy analysis (Fig. 2). The separate addition of 10 μmol L⁻¹ of several metal cations (Fe³⁺, Co²⁺, Zn²⁺, Sr²⁺, Cu²⁺. Al³⁺, Ba²⁺, Cd²⁺, Pb²⁺, Ni²⁺, Mg²⁺, Hg²⁺, Cu⁺ and Cr³⁺) to solutions of RU-AgNPs (prepared according to Fig. 1a) indicated that only Fe³⁺ induces a significant colorimetric change in the final aspect of solution after 50 minutes (Fig. 2a).Open in a separate windowFig. 2Naked-eye (a) and UV-vis (b) analysis of RU-AgNPs in absence (control, C) and presence of 10 μmol L⁻¹ of selected metal cations (Fe³⁺, Co²⁺, Zn²⁺, Sr²⁺, Cu²⁺. Al³⁺, Ba²⁺, Cd²⁺, Pb²⁺, Ni²⁺, Mg²⁺, Hg²⁺, Cu⁺ and Cr³⁺) after 50 min; (c) calibration curve obtained by the addition of different amounts (0 to 10 μmol L⁻¹) of Fe³⁺ to solutions of RU-AgNPs; (d) TEM image of RU-AgNPs after addition of Fe³⁺ (10 μmol L⁻¹). In all experiments, RU-AgNPs were prepared in the presence of ascorbic acid.The results presented in Fig. 2a are consistent with the UV-vis spectroscopy analysis (Fig. 2b). Co²⁺ ions also induce some change in the system, however at a considerably smaller extension than Fe³⁺. In this study, AA played a crucial role in the selective detection of Fe³⁺ by the referred nanoprobe. In the absence of AA, Co²⁺ (mainly) as well as other cations induce stronger colorimetric and spectral changes (Fig. S5a and b,^† respectively) in the analysis of solutions of RU-AgNPs when compared to the system containing AA. This selectivity may arise from two possible reasons: (i) preservation of RU by avoiding its oxidation in the reduction of Ag⁺ ions; (ii) coordination of ascorbate anion to cations other than Fe³⁺.Interaction of RU-AgNPs and Fe³⁺ (10 μmol L⁻¹) stabilizes after approximately 40 minutes (Fig. S6^†). Next, a calibration curve was built from the direct relationship between the absorbance at 396 nm and the concentration of Fe³⁺ (Fig. 2c), presenting a good correlation (R² = 0.9929). The limits of detection and quantification were found to be 17 nmol L⁻¹ and 56 nmol L⁻¹, respectively, which is very satisfactory.^13,14 The influence of other cations in the detection of Fe³⁺ was investigated by UV-vis spectroscopy. Fig. S7^† clearly demonstrates that there are only small changes when a second cation (30 μmol L⁻¹) is added together with Fe³⁺ to the RU-AgNPs solution.Mechanistically, the detection of Fe³⁺ by RU-AgNPs in aqueous medium proceeds via a growth/aggregation-combined process. This proposal is first evidenced by UV-vis analysis due the suppression of SPR band (Fig. 2b), a behavior consistent with the literature.^31,32 Interestingly, TEM analysis clearly shows a growth in AgNPs size after addition of Fe³⁺ to the solution (Fig. 2d), resulting in a final single AgNPs population with average diameter of 14.7 nm ± 8.9 nm. Due to the strong alkaline medium, the main specie responsible for the behavior of NPs is likely to be Fe(OH)₃. A schematic illustration of the mechanism involving aggregation of RU-AgNPs induced by the addition of Fe³⁺ is presented in Fig. 3. AgNPs are initially formed by adsorption of anionic RU to the silver surface via deprotonated 5-hydroxychromen-4-one moiety. This is supported by the literature³³ and confirmed by alteration in the 1800–1500 cm⁻¹ region of the RU infrared spectra before and after coordination with silver (Fig. S9^†). Afterwards, the addition of Fe³⁺ induces the formation of a coordination complex through an anionic catechol group, in which at least 2 : 1 ligand–Fe³⁺ stoichiometry is required for an aggregated effect.Open in a separate windowFig. 3Mechanistic proposal for growth/aggregation of RU-AgNPs in the presence of Fe³⁺. Insert: binding model for RU-AgNPs.Due to increasing interest in image processing as an analytical tool for many purposes,^34,35 Multiple Linear Regression was employed to verify the capacity of the RU-AgNPs to probe Fe³⁺ standards at distinct concentrations, as presented in Fig. 4. The curve was obtained by plotting the color absorbances RGB-based values versus the concentrations of Fe³⁺ standards after RU-AgNPs interaction. Predicted iron is a vector based on RGB values that were then extracted from the filtered images and inserted in the equation described by Beer–Lambert law in order to generate the absorbances for the construction of the analytical curve. A linear behavior between the predicted response and the measured concentrations was observed (R² = 0.9806).Open in a separate windowFig. 4Calibration curve for Fe³⁺ analysis showing the predicted iron (RGB) vs. iron(iii) concentration (1 to 8 μmol L⁻¹). Adjusted R² = 0.9806.Regression coefficients of the calibration model (using the R, G and B channels simultaneously) obtained by MLR method are shown in eqn (1):[Fe³⁺] = 44.5R − 4.9G − 16.4B + 5.11where Fe³⁺ concentration is the dependent variable (Predicted Iron), 5.1 is the intercept (β₀), 44.5, −4.9 and −16.4 are the regression coefficients of the independent variables (R, G and B channels, respectively).It is possible to observe an interesting performance of the method using the three RGB color channels allied with MLR to quantify the Fe³⁺ content, which presented reasonable deviations in its responses considering that it is simple and low-cost. The good linearity is similar to other colorimetric methods such as sodium determination in seawater and coconut water (R² > 0.91) by Moraes and coauthors,³⁶ or iron(ii) in simulated seawater (R² = 0.9993) by Gasparotto et al.³⁷Second order regression was applied to the dataset to obtain a better adjustment, resulting in an adjusted R-squared of 0.9955. RGB values were then inserted in eqn (2) in order to generate the construction of the correlation:[Fe³⁺] = 14.4R + 30.7G − 23.4B − 398RG − 348RB − 0.8BG − 978R² + 695G² + 26.9B² + 4.52This paper reports the use of AgNPs functionalized with RU as nanoprobes for selective detection and sensitive quantification of Fe³⁺ in aqueous solution. The synthesis of RU-AgNPs is reproducible, easily performed and requires no stabilizer agent other than RU. AA has a crucial role in the selectivity by either the avoidance of oxidation of RU by silver and/or coordination of ascorbate with other cations. The literature brings relevant examples of chromogenic and fluorogenic chemosensors for selective detection of Fe³⁺ in solution. Many of these artificial organic receptors present high selectivity and relevant limits of detection, requiring, however, very specific reagents and laborious synthetic procedures.^38–41 Metal-based nanoparticles have emerged as potential probes for detection of Fe³⁺.^11–16 Although effective in Fe³⁺ sensing, the synthesis of these nanoprobes require some toxic reagents, such as NaBH₄ or PVP, use plant extracts, which may lead to some drawbacks, such as the understanding of the sensing mechanism. In contrast, our method is based on commercially available, nontoxic, low-cost reagents. Fe³⁺ sensing performed satisfactorily in the 1–10 μmol L⁻¹ range, and the limit of detection obtained with this method (17 nmol L⁻¹) is comparable to the most sensitive methods reported in literature. A mechanism for the detection of Fe³⁺ by RU-AgNPs involves a combined growth/aggregation of the NPs. There is a still limited number of nanoscaled systems reported as being selective and sensitive in the detection of Fe³⁺, which reinforces the relevance of the method reported herein. The linearity range obtained by both UV-vis spectroscopy and image analysis comprises the maximum of residual Fe³⁺ in drinking water according to the European and US legislations.^15,42     

Diagnosing diabetic foot osteomyelitis

Bone involvement during an infection of the diabetic foot represents a serious complication associated with a high risk of amputation, prolonged antibiotic treatment and hospitalization. Diabetic foot osteomyelitis (DFOs) require a multidisciplinary approach given the usual complexity of these situations. DFO should be suspected in most cases especially in the most severe forms of soft tissue diabetic foot infections (DFIs) where the prevalence of bone infection may be up to 60%. Suspicion is based on clinical signs in particular a positive probe‐to‐bone (PTB) test, elevated inflammatory biomarkers especially erythrocyte sedimentation rate and abnormal imaging assessment using plain X‐ray as a first‐line choice. The combination of PTB test with plain X‐ray has proven effective in the diagnosis of DFO. The confirmation (definite) diagnosis of DFO is based on the results of a bone sample examination obtained by either surgical or percutaneous biopsy. Sophisticated imaging examinations such as Magnetic Resonance Imaging (MRI) and nuclear imaging techniques are useful where doubt persists after first‐line imaging assessment. These techniques may also help localize the bone infection site and increase the diagnostic performance of percutaneous bone biopsy. The quality of the microbiological documentation of DFO is likely to improve the adequacy of the antimicrobial therapy especially when medical (ie, no surgical resection of the infected bone tissues) is considered. The use of new (molecular) techniques for the identification of the bone pathogens have not yet proven superiority on classic cultural techniques for the management of such patients.     

Circadian regulation of olfaction and an evolutionarily conserved,nontranscriptional marker in Caenorhabditis elegans

Circadian clocks provide a temporal structure to processes from gene expression to behavior in organisms from all phyla. Most clocks are synchronized to the environment by alternations of light and dark. However, many organisms experience only muted daily environmental cycles due to their lightless spatial niches (e.g., caves or soil). This has led to speculation that they may dispense with the daily clock. However, recent reports contradict this notion, showing various behavioral and molecular rhythms in Caenorhabditis elegans and in blind cave fish. Based on the ecology of nematodes, we applied low-amplitude temperature cycles to synchronize populations of animals through development. This entrainment regime reveals rhythms on multiple levels: in olfactory cued behavior, in RNA and protein abundance, and in the oxidation state of a broadly conserved peroxiredoxin protein. Our work links the nematode clock with that of other clock model systems; it also emphasizes the importance of daily rhythms in sensory functions that are likely to impact on organism fitness and population structure.     

Influence of HTLV-1 on the clinical, microbiologic and immunologic presentation of tuberculosis

ABSTRACT: BACKGROUND: HTLV-1 is associated with increased susceptibility to Mycobacterium tuberculosis infection and severity of tuberculosis. Although previous studies have shown that HTLV-1 infected individuals have a low frequency of positive tuberculin skin test (TST) and decreasing in lymphoproliferative responses compared to HTLV-1 uninfected persons, these studies were not performed in individuals with history of tuberculosis or evidence of M. tuberculosis infection. Therefore the reasons why HTLV-1 infection increases susceptibility to infection and severity of tuberculosis are not understood.The aim of this study was to evaluate how HTLV-1 may influence the clinical, bacteriologic and immunologic presentation of tuberculosis. METHODS: The study prospectively enrolled and followed 13 new cases of tuberculosis associated with HTLV-1 (cases) and 25 patients with tuberculosis without HTLV-1 infection (controls). Clinical findings, bacterial load in the sputum, x-rays, immunological response and death were compared in the two groups. RESULTS: There were no differences in the demographic, clinical and TST response between the two study groups. IFN-gamma and TNF-alpha production was higher in unstimulated cultures of mononuclear cells of case than in control patients (p < 0.01). While there was no difference in IFN-gamma production in PPD stimulated cultures, TNF-alpha levels were lower in cases than in controls (p = 0.01). There was no difference in the bacterial load among the groups but sputum smear microscopy results became negative faster in cases than in controls. Death only occurred in two co-infected patients. CONCLUSION: While the increased susceptibility for tuberculosis infection in HTLV-1 infected subjects may be related to impairment in TNF-alpha production, the severity of tuberculosis in co-infected patients may be due to the enhancement of the Th1 inflammatory response, rather than in their decreased ability to control bacterial growth.     

Amyloid-β25–35 induces a permanent phosphorylation of HSF-1, but a transitory and inflammation-independent overexpression of Hsp-70 in C6 astrocytoma cells

Two hallmarks of Alzheimer diseases are the continuous inflammatory process, and the brain deposit of Amyloid b (Aβ), a cytotoxic protein. The intracellular accumulation of Aβ_25–35 fractions, in the absence of Heat Shock proteins (Hs?s), could be responsible for its cytotoxic activity. As, pro-inflammatory mediators and nitric oxide control the expression of Hs?s, our aim was to investigate the effect of Aβ_25–35 on the concentration of IL-1β, TNF-α and nitrite levels, and their relation to pHSF-1, Hsp-60, -70 and -90 expressions, in the rat C6 astrocyte cells. Interleukin-specific ELISA kits, immunohistochemistry with monoclonal anti-Hsp and anti pHSF-1 antibodies, and histochemistry techniques, were used. Our results showed that Aβ_25–35 treatment of C6 cells increased, significantly and consistently the concentration of IL-1β, TNF-α and nitrite 3 days after initiating treatment. The immunoreactivity of C6 cells to Hsp-70 reached its peak after 3 days of treatment followed by an abrupt decrease, as opposed to Hsp-60 and -90 expressions that showed an initial and progressive increase after 3 days of Aβ_25–35 treatment. pHSF-1 was identified throughout the experimental period. Nevertheless, progressive and sustained cell death was observed during all the treatment times and it was not caspase-3 dependent. Our results suggest that Hsp-70 temporary expression serves as a trigger to inhibit casapase-3 pathway and allow the expression of Hsp-60 and -90 in C6 astrocytoma cells stimulated with Aβ_25–35.