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Eingeladener Vortrag
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In this work we report a novel paper-based analytical device read-out via LED-induced fluorescence detection (FPAD) for the quantification of the emerging pollutant ethinylestradiol (EE2) in river water samples. The PAD was used as a reaction platform for a competitive enzyme immunoassay. For the PAD development, microzones of filter paper, printed by a wax printing method, were modified with amino-functionalized SBA-15 and subsequently, anti-EE2 specific antibodies were covalently immobilized. The determination of EE2 in water was
carried out by adding a fixed concentration of EE2 conjugated with the enzyme horseradish peroxidase (HRP) to samples and standards. Then, the FPAD were added and incubated for 10 min. Finally, the detection was performed by the reaction of 10-acetyl-3,7-dihydroxyphenoxazine (ADHP) whose oxidation is catalyzed by HRP in the presence of H2O2, obtaining the highly fluorescent resorufin (R). Resorufin was detected by LED excitation at 550 nm, observing emission at 585 nm. The EE2 concentration in the samples was inversely proportional to the relative fluorescence obtained from the enzymatic reaction products. The FPAD assay showed a detection Limit (LOD) of 0.05 ng L−1 and coefficients of variation (CV) below 4.5% within-assay and below 6.5% between-assay, respectively. The results obtained show the potential suitability of our FPAD for the selective and sensitive quantification of EE2 in river water samples. In addition, it has the PADs advantages of being disposable, easy to
apply and inexpensive.
Decoration of trastuzumab with short oligonucleotides: synthesis and detailed characterization
(2017)
Trastuzumab (Herceptin) is an FDA-approved therapeutic antibody currently employed in the treatment of metastatic stages of breast cancer. Herein, we propose a simple, fast and cost-effective methodology to conjugate trastuzumab with 22-mer 5' thiol-modified oligonucleotides using a bifunctional crosslinker. The conjugates were successfully characterized by MALDI-ToF MS and SDS-PAGE, obviating the need for enzymatic digestion and difficult chromatographic separations. Furthermore, ELISA was performed to ensure that trastuzumab activity is not affected by oligonucleotide conjugation.
We report a novel and innovative electrochemical paper-based immunocapture assay (EPIA) to address the need for ultrasensitive detection of emerging pollutants without regulatory status and whose effects on environment and human health are not completely yet understood. In particular, we present the application of this system toward highly sensitive detection of the emerging pollutant ethinyl estradiol (EE2). The EPIA approach is based on the use of paper microzones modified with silica nanoparticles (SNs) and anti-EE2 specific antibodies for capture and preconcentration of EE2 from river water samples. After the preconcentration procedure, the paper microzones are placed onto a screen-printed carbon electrode modified with electrochemically reduced graphene (RG). The bound EE2 is subsequently desorbed adding a diluted solution of sulfuric acid on the paper microzones. Finally, recovered EE2 is electrochemically detected by OSWV. The proposed novel methodology showed an appropriate LOD and linear range for the quantification of EE2 for water samples with different origins. The nonsophisticated equipment required, the adequate recovery values obtained (from 97% to 104%, with a RSD less than 4.9%), and the appropriate LOD and linear range value (0.1 ng L−1 and 0.5−120 ng L−1, respectively) achieved by our immunocapture sensor present significant analytical figures of merit, particularly when the routine quantification of EE2 is considered. In addition, our System was based on electrochemical paper-based technology, which allows obtainment of portable, easy-to-use, inexpensive, and disposable devices. The EPIA can also serve as a general-purpose immunoassay platform applicable to quantitation of other drugs and emerging pollutants in environmental samples.
Im Rahmen eines von der Kulturstiftung des Bundes und der Kulturstiftung der Länder geförderten
Projektes zum Erhalt naturkundlicher Nasssammlungen wurden 1) der Zustand der Sammlungen bewertet
(Profiling; Analyse der Vergällungsmittel), 2) über Workshops Expertenwissen in das Projekt gebracht,
3) neue archivbeständige Materialien und Managementmaßnahmen eingeführt (Borosilikat-Gläser und -
Röhrchen, Augenwatte, Tefloneinlagen, Japanpapier, archivbeständige Kleber und Papier, Etikettenaufbewahrung;
digitales Dichtemessgerät, Alcomon-Indikator, Kontrollintervalle), 4) Erhaltungsmaßnahmen
zur langfristigen Konservierung in großem Umfange durchgeführt (Messen der Alkoholkonzentration,
Umsetzen von Präparaten in geeignetere Gefäße, Wiederverschluss von Schaugläsern) und 5) Erfahrungen
und Diskussionsergebnisse zusammengestellt, nicht ohne auf Wissenslücken und wünschenswerte
Forschungsaspekte hinzuweisen. Das Projekt profitierte besonders von der Expertise aus den Materialwissenschaften
und der Papierrestaurierung. Das Museum für Naturkunde Berlin kooperierte eng mit
dem Berliner Medizinhistorischen Museum der Charité, der Zoologischen Staatssammlung München und
dem Forschungsinstitut Senckenberg in Frankfurt. Letzteres Museum führte ein eigenes, themenverwandtes
KUR-Projekt Restauration der Sammlungen 'Vergleichende Anatomie, Embryologie und Histologie'
des Naturmuseums Senckenberg durch.
In this work, we report an electrochemical immunosensor to detect ethinylestradiol in water samples, using electrochemical impedance spectroscopy (EIS) as a detection technique. For the development of this immunosensor, the direct modification of the working electrode of a screen-printed carbon electrode was carried out. First, to reduce the resistance of the electrode, electroreduced graphene was incorporated on the surface.
Second, a porous gold structure was electrodeposited on reduced graphene by electrodeposition and the dynamic hydrogen bubble template assisted method. Thus, a marked increase in surface area was obtained for anti-EE2 antibodies immobilization. Subsequently, the specific anti-EE2 antibodies were covalently immobilized using α-lipoic acid for attaching them to the gold surface.
The electrode modified with the antibodies was incubated for 30 min in the samples containing EE2, producing the specific Antigen antibody binding. As the charge transfer resistance of a redox probe in the electrode surface is governed by the surface blocking effects, the charge transfer resistance was related to the amount of EE2 captured to realize a quantitative determination. For this, the EIS measurements were performed in a 4 mM [Fe(CN)6]4−/3− solution in 0.1 M KCl. The obtained Nyquist diagrams were adjusted using the Randles circuit as an equivalent circuit to obtain the corresponding resistances. The developed methodology showed good selectivity, precision, and sensitivity; although the LOD obtained was higher than those presented in other published articles, it turned out to be an alternative that allows the determination of ethinylestradiol using a simple disposable electrode.
A lateral flow immunoassay (LFIA) using latex particles labeled with antibody to BSAclarithromycin (CLA) was developed for the rapid simultaneous group determination of six macrolide antibiotics. Optimization of antigen spotting on the membrane and latex probe loading allowed improving visual detectability (vLOD) 100 times, which was 1, 1, 10, 10, 50, and 1000 ng/mL for CLA, roxithromycin, erythromycin, dirithromycin, azithromycin, and oleandomycin in buffer, respectively. The calculated limits of instrumental detection (cLOD) were respectively 0.12, 0.15, 1.4, 2.1, 2.4, and 3.3 ng/mL. To avoid a strong influence of breast milk of a very diverse and variable composition, a sample pretreatment is proposed. The six macrolides mentioned can be visually detected in breast milk after 20 minutes pretreatment at concentrations of 10-1000 ng / ml or instrumentally with cLOD of 4.0, 2.5, 30, 42, 42 and 180 ng/mL. The recovery rate from the spiked samples carried out using a strip scanner device ranged from 71% to 110%, and precision expressed as relative standard deviation was between 3-14%. The described rapid on-site diagnostic assay format can be useful for monitoring the content of antibiotics in breast milk during macrolide treatment to ensure safe breastfeeding of infants.
The effects of long-term application of different organic amendments, as compared to mineral fertilizer, on Zn, Cu and Pb content and leachability in a luvisol derived from loess were assessed. The organic fertilizers, applied since 1962, were compost (COM) – from green organic household waste, sewage sludge (SLU) – from municipal water treatment facilities, farmyard manure (FYM) and the doses applied since 1997 were 90 t ha-1, 10 t ha-1 and 9 t ha-1, once in 3 years, respectively. The kinetics of metals extraction with 0.05 mol dm-3 EDTA at pH 6.0 has been studied. The two first-order reactions model was fitted to the kinetic data and allowed to distinguish two pools for each metal: a 'labile' fraction (Q1), quickly extracted with a rate constant k1, and a 'moderately labile' fraction (Q2), more slowly extracted, with a rate constant k2. Simultaneously, the pseudo-total metal contents in the soil samples were determined after digestion with aqua regia (3:1 HCl + HNO3). The obtained parameters Q1, k1, Q2, k2, for the kinetics of extraction of each metal in the three replicates of each fertilization mode, as well as the pseudo-total metal contents, were statistically analysed. COM and SLU application resulted in an increase of the total contents of Pb, Zn and Cu in soil. Further, the percentage of labile Zn and Pb also increased in consequence of the application of those amendments, particularly COM. The increase was more noticeable for Zn. FYM, despite not increasing the total content of Pb, Zn or Cu, did also have an effect on the leachability of Zn and Pb, increasing their labile fraction in soil. These results point to a potential risk of increasing metals mobility in soil, mainly Zn, associated to the use of organic amendments, particularly COM or SLU.
The toxicity induced in non-target organisms by pharmaceutical drugs has been the focus of several studies. In the aquatic environment, most of the studies have been devoted to fish and bivalves, while little is known on the impacts induced in polychaetes. The present study evaluated the impacts of carbamazepine and caffeine on the regenerative capacity of Diopatra neapolitana, a polychaete species with high ecological and economic relevance. Under laboratory controlled conditions polychaetes were exposed, during 28 days, to carbamazepine (Ctl-0.0; 0.3; 3.0; 6.0; 9.0 mg/L) and caffeine (Ctl-0.0; 0.5; 3.0; 18.0 mg/L). During the experiment, at days 11, 18, 25, 32, 39 and 46 after amputation, for each specimen, the percentage of the body width regenerated was determined and the number of new segments was counted. The regenerative capacity was assessed considering the number of days needed to achieve full regeneration and the total number of new segments. The obtained results revealed that with the increase of drugs concentrations organisms regenerated less new segments and took longer to completely regenerate.
Ocean acidification and increasing discharges of pharmaceutical contaminants into aquatic systems are among key and/or emerging drivers of environmental change affecting marine ecosystems. A growing body of evidence demonstrates that ocean acidification can have direct and indirect impacts on marine organisms although combined effects with other stressors, namely with pharmaceuticals, have received very little attention to date. The present study aimed to evaluate the impacts of the pharmaceutical drug Carbamazepine and pH 7.1, acting alone and in combination, on the clamScrobicularia plana. For this, a long-termexposure (28 days)was conducted and a set of oxidative stress markers was investigated. The results obtained showed that S. plana was able to develop mechanisms to prevent oxidative damage when under low pH for a long period, presenting higher survival when exposed to this stressor compared to CBZ or the combination of CBZ with pH 7.1. Furthermore, the toxicity of CBZ on S. plana was synergistically increased under ocean acidification conditions (CBZ + pH 7.1): specimens survival was reduced and oxidative stress was enhanced when compared to single exposures. These findings add to the growing body of evidence that ocean acidification will act to increase the toxicity of CBZ to marine organisms,which has clear implications for coastal benthic ecosystems suffering chronic pollution from pharmaceutical drugs.
In the last decade studies have reported the presence of several pharmaceutical drugs in aquatic environments worldwide and an increasing effort has been done to understand the impacts induced on wildlife. Among the most abundant drugs in the environment is caffeine, which has been reported as an effective chemical anthropogenic marker. However, as for the majority of pharmaceuticals, scarce information is available on the adverse effects of caffeine on marine benthic organisms, namely polychaetes which are the most abundant group of organisms in several aquatic ecossystems. Thus, the present study aimed to evaluate the biochemical alterations induced by environmentally relevant concentrations of caffeine on the polychaete species Diopatra neapolitana and Arenicola marina. The results obtained demonstrated that after 28 days exposure oxidative stress was induced in both species, especially noticed in A. marina, resulting from the incapacity of antioxidant and biotransformation enzymes to prevent cells from lipid peroxidation. The present study further revealed that D. neapolitana used glycogen and proteins as energy to develop defense mechanisms while in A. marina these reserves were maintained independently on the exposure concentration, reinforcing the low capacity of this species to fight against oxidative stress.