Presence Of 2-mercaptoethanol Research Articles

An efficient spectrofluorimetric method adopts doxazosin, terazosin and alfuzosin coupling with orthophthalaldehyde: Application in human plasma

A new, selective and sensitive spectrofluorimetric method was designed for the quantitation of doxazosin (DOX), terazosin (TER) and alfuzosin (ALF) in their dosage forms and human plasma. The method adopts efficient derivatization of the studied drugs with ortho-phthalaldehyde (OPA), in the presence of 2-mercaptoethanol in borate buffer (pH9.7) to generate a highly fluorescent isoindole derivatives, which can strongly enhance the fluorescence intensities of the studied drugs, allowing their sensitive determination at 430nm after excitation at 337nm. The fluorescence-concentration plots were rectilinear over the ranges (10.0–400.0) ng/mL. Detection and quantification limits were found to be (0.52–3.88) and (1.59–11.76) ng/mL, respectively. The proposed method was validated according to ICH guidelines, and successfully applied for the determination of pharmaceutical preparations of the studied drugs. Moreover, the high sensitivity of the proposed method permits its successful application to the analysis of the studied drugs in spiked human plasma with % recovery (96.12±1.34–100.66±0.57, n=3). A proposal for the reaction mechanism was presented.

Characterization of Proanthocyanidin Oligomers of Ephedra sinica.

Ephedra sinica, an important plant in Chinese traditional medicine, contains a complex mixture of proanthocyanidin oligomers as major constituents; however, only the minor components have been chemically characterized. In this study, oligomers with relatively large molecular weights, which form the main body of the proanthocyanidin fractions, were separated by adsorption and size-exclusion chromatography. Acid-catalyzed degradation in the presence of mercaptoethanol or phloroglucinol led to the isolation of 18 fragments, the structures of which were elucidated from their experimental and TDDFT-calculated ECD spectra. The results indicated that (−)-epigallocatechin was the main extension unit, while catechin, the A-type epigallocatechin–gallocatechin dimer, and the A-type epigallocatechin homodimer, were identified as the terminal units. Among the degradation products, thioethers of gallocatechin with 3,4-cis configurations, a B-type prodelphinidin dimer, a prodelphinidin trimer with both A- and B-type linkages, and a prodelphinidin dimer with an α-substituted A-type linkage were new compounds. In addition, a phloroglucinol adduct of an A-type prodelphinidin dimer, a doubly-linked phloroglucinol adduct of epigallocatechin, and a unique product with a flavan-3-ol skeleton generated by the rearrangement of the aromatic rings were also isolated.

The influence of antioxidants in the thiyl radical induced lipid peroxidation and geometrical isomerization in micelles of linoleic acid

The biomimetic model of micelles of linoleic acid containing 2-mercaptoethanol and the antioxidant was examined under gamma irradiation up to 400 Gy in aerobic or deoxygenated conditions where thiyl radicals are the main reactive species. Lipid peroxidation was retarded by ascorbic acid and α-tocopherol, whereas this process was strongly inhibited by resveratrol as effectively as the ascorbic acid/α-tocopherol mixture. Furthermore, antioxidants have a much stronger inhibitory effect on the peroxidation in the presence of 2-mercaptoethanol, and at the same time show protective properties of the double bond, decreasing the cis–trans isomerization. Under anaerobic conditions, cis–trans isomerization occurred and antioxidants efficiency increased along the series: resveratrol < α-tocopherol < ascorbic acid. This result is explained taking into account the double bond localization in the hydrophobic core of the micelle and the need of co-localization of the antioxidant in order to get an anti-isomerizing activity and protection of the natural lipid geometry.

Synthesis of novel high primary hydroxyl functionality polyol from sunflower oil using thiol-yne reaction and their application in polyurethane coating

In this study, novel bio-based polyurethanes that have characteristics of both thermosets and thermoplastics were prepared. The polyol functionalized by a high number of hydroxyl groups was synthesized and its application in polyurethanes, a well-known polymer in coating industry, was investigated. Sunflower oil was decorated for the first time with propargyl groups by ring-opening of epoxied groups using boron trifluoride diethyl etherate as catalyst. The polyol is obtained by photochemical thiol-yne coupling functionalization of propargylated oil in presence of mercaptoethanol in an economical, and environmental friendly procedure. The chemical structure of this sunflower based polyol and its intermediates were determined by 1H NMR and FT-IR analysis. A series of polyurethanes containing various NCO/OH molar ratio were synthesized by a one-step procedure. The properties of the polyurethanes were studied by performing tensile testing, differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). A facile and green method for the synthesis of bio-based polyurethanes and their application as a transparent coating made them exponentially advantageous.

The functional properties of a xyloglucanase (GH12) of Aspergillus terreus expressed in Aspergillus nidulans may increase performance of biomass degradation.

Filamentous fungi are attractive hosts for heterologous protein expression due to their capacity to secrete large amounts of enzymes into the extracellular medium. Xyloglucanases, which specifically hydrolyze xyloglucan, have been recently applied in lignocellulosic biomass degradation and conversion in many other industrial processes. In this context, this work aimed to clone, express, and determine the functional properties of a recombinant xyloglucanase (AtXEG12) from Aspergillus terreus, and also its solid-state (SSF) and submerged (SmF) fermentation in bioreactors. The purified AtXEG12 showed optimum pH and temperature of 5.5 and 65°C, respectively, demonstrating to be 90% stable after 24h of incubation at 50°C. AtXEG12 activity increased in the presence of 2-mercaptoethanol (65%) and Zn+2 (45%), while Cu+2 and Ag+ ions drastically decreased its activity. A substrate assay showed, for the first time for this enzyme's family, xylanase activity. The enzyme exhibited high specificity for tamarind xyloglucan (K M 1.2mgmL-1) and V max of 17.4μmolmin-1mg-1 of protein. The capillary zone electrophoresis analysis revealed that AtXEG12 is an endo-xyloglucanase. The heterologous xyloglucanase secretion was greater than the production by wild-type A. terreus cultivated in SmF. On the other hand, AtXEG12 activity reached by SSF was sevenfold higher than values achieved by SmF, showing that the expression of recombinant enzymes can be significantly improved by cultivation under SSF.

Zinc sulfide quantum dots as powerful and efficient nanophotocatalysts for the removal of industrial pollutant

In this work, a simple, efficient and powerful photocatalytic methodology based on application of pure zinc sulfide (ZnS) quantum dots (QDs) is presented for the removal of naphthalene, as a model molecule of industrial pollutants. A facile room-temperature precipitation method was applied for the synthesis of ZnS QDs, in the presence of 2-mercaptoethanol as capping agents. The characterization of the prepared QDs was carried out by X-ray diffraction (XRD), transmission electron microscopy, high-resolution scanning electron microscopy, scanning tunneling microscopy, fluorescence, and UV–Vis absorption spectroscopies. The XRD investigations indicated that the prepared ZnS QDs are crystalline, with cubic zinc blend structure, with the average particle diameters of 1.2 nm. The fluorescence and UV–Vis absorption investigations showed a narrow and sharp emission band at 424 nm with a broad adsorption band around 235 nm. In addition, the prepared ZnS QDs were used as nanophotocatalysts, for the removal of naphthalene, as a model molecule. The influence of the experimental parameters on the removal efficiency of the ZnS QDs was studied. According to the results, the highest photocatalytic degradation of naphthalene in the presence of ZnS QDs (>95 %) was obtained at alkaline pH, in the presence of 10 mg of QDs, after a reasonable reaction time.

Molecular cloning and characterization of a galectin-1 homolog in orange-spotted grouper, Epinephelus coioides.

As a member of animal lectin family, galectin has the functions of pathogen recognition, anti-bacteria and anti-virus. In the present study, a galectin-1 homolog (EcGel-1) from grouper (Epinephelus coioides) was cloned and its possible role in fish immunity was analyzed. The full length cDNA of EcGel-1 is 504bp, including a 408bp open reading frame (ORF) which encodes 135 amino acids with a molecular mass of 15.19kDa. Quantitative real-time PCR analysis indicated that EcGel-1 was constitutively expressed in all analyzed tissues of healthy grouper. The expression of EcGel-1 in the spleen of grouper was differentially up-regulated challenged with Singapore grouper iridovirus (SGIV), poly (I:C), and LPS. EcGel-1 was abundantly distributed in the cytoplasm in GS cells. Recombinant EcGel-1(rEcGel-1) protein can make chicken erythrocyte aggregation, and combine with gram negative bacteria and gram positive bacteria in the presence of 2-Mercaptoethanol (β-ME). Taken together, the results showed that EcGel-1 may be an important molecule involved in pathogen recognition and pathogen elimination in the innate immunity of grouper.

Lipid modification processes induced by thiyl radicals

Polyunsaturated fatty acid (PUFA) oxidation by thiyl radicals (RS•) is believed to be responsible for some of the biological radiation damage. At the same time, RS• can cause isomerization of PUFA double bonds with the formation of trans isomers. The aim of this study was to better understand the competition between lipid peroxidation and geometrical isomerization processes in biomimetic model system of linoleic acid in the presence of 2-mercaptoethanol using irradiation as a method for free radicals generation. In air-equilibrated conditions the propagation of lipid peroxidation was dominant up to the dose of 400Gy, after which at higher doses up to 10kGy the termination occurred with the predominance of geometrical isomerization. This study revealed that undesirable and permanent lipid modifications are possible at higher irradiation doses which should be considered in the planning of irradiation treatment of foods and feeds with high content of lipids and sulfur compounds.

Preparation of silver nanowires coated with TiO2 using chemical binder and their applications as photoanodes in dye sensitized solar cell

In this study, the core–shell structured Ag@TiO2 wire was prepared for application to dye-sensitized solar cell (DSSC). The Ag nanowire, having an excellent electrical conductivity, was synthesized by using the facile microwave-assisted polyol reduction process. The diameter and length of Ag wires were 40–50nm and 20–30μm, respectively, and the face-centered cubic silver crystal structure was obtained. In the presence of 2-mercaptoethanol as a chemical binder, the entire surface of Ag wire was coated with the TiO2 shell, which has thickness of 20nm, through solvothermal method. The crystalline structure of TiO2 shell was the anatase phase possessing an advantage to achieve the high efficiency in DSSC. The core–shell structured Ag@TiO2 wire exhibited the high thermal stability. The high conversion efficiency (5.56%) in fabricated device with Ag@TiO2 electrode, which is about 10% higher than reference cell, was achieved by enhancement of short-current density (Jsc) value. The core–shell structured Ag@TiO2 wire could effectively reduce the charge recombination through the contribution to electron shortcut for improvement in the electron transfer rate and lifetime.

The first trimeric Galanthus nivalis agglutinin-related lectin of Orchidaceae was found in Dendrobium pendulum: purification, characterization, and effects of stress factors.

Trimeric Galanthus nivalis agglutinin-related lectin of Orchidaceae with two conformational forms was first studied in Dendrobium pendulum . It was highly expressed by stress factors. Using mannan-agarose column chromatography, a mannose-binding protein was purified from Dendrobium pendulum Roxb. pseudobulb. After heating in the presence of sodium dodecyl sulfate (SDS) with or without 2-mercaptoethanol, the protein showed one band with molecular mass of 14.0 kDa on SDS-polyacrylamide gel electrophoresis (PAGE). Without heating, three bands were found at positions of 14.0, 39.4, and 41.5 kDa, but a higher amount of 39.4 and 41.5 kDa protein bands were seen in the presence of 2-mercaptoethanol. Liquid chromatography-tandem mass spectrometry and database search indicated that the 14.0 kDa protein band contained three peptide fragments identical to parts of a lectin precursor from Dendrobiu m findleyanum Parish & Rchb.f. Native-PAGE and Ferguson plot showed that the purified protein had two native forms with molecular masses of 44.2 and 45.3 kDa, indicating three 14.0 kDa polypeptide subunits. The purified protein exhibited the agglutination activity with trypsinized chicken erythrocytes. It was then recognized as a Galanthus nivalis agglutinin-related lectin and named D. pendulum agglutinin (DPA). Using reverse transcription-polymerase chain reaction and DNA sequencing, the deduced amino acid sequence of DPA precursor showed the highest homology (96.4%) with a lectin precursor of D. findleyanum and contained three mannose-binding sites. Greater amounts of DPA were found when the pseudobulbs were treated with stress factors including ultraviolet light, abscisic acid, hydrogen peroxide, and acetylene gas.

Controlled degradation of disulfide-based epoxy thermosets for extreme environments

The burgeoning field of smart materials for oil exploration and production (E&P) demands robust polymers that remain stable in extreme conditions, but readily respond to specific chemical cues. Here, disulfide-based epoxy polymers are designed to withstand harsh, simulated oil reservoir conditions while simultaneously retaining the ability to degrade from thiol compounds. Degradable epoxy thermosets are prepared by using Bisphenol-F diglycidyl ether (BFDGE) in combination with blends of p,p′-diaminodicyclohexylmethane (PACM) and 4-aminophenyl disulfide (4APDS). These polymer systems retain their mechanical properties after exposure to extreme conditions (e.g., two weeks, 69 MPa, 100 °C, pH 12), but degrade in the presence of 2-mercaptoethanol (2-ME). Parameters that influence polymer degradation are detailed including temperature, concentration of disulfide groups, and monomer stoichiometry. Formulations that entirely dissolve after exposure to 2-ME are utilized further for release of prototype tracers. Disulfide-based epoxy thermosets provide a unique platform for applications in harsh environments requiring robust materials capable of on-demand degradation.

Synthesis, Dynamic Combinatorial Chemistry, and PCR Amplification of 3′–5′ and 3′–6′ Disulfide‐linked Oligonucleotides

AbstractDisulfide dithymidines linked 3′–5′ or 3′–6′ were synthesized and incorporated into oligonucleotides through a combined phosphotriester and phosphoramidite solid‐phase oligonucleotide synthesis approach. The disulfide links are cleaved and formed reversibly in the presence of thiols and oligonucleotides. This link was shown to be sequence‐adaptive in response to given templates in the presence of mercaptoethanol. The artificial 3′–5′ and 3′–6′ disulfide link was tolerated by polymerases in the polymerase chain reaction (PCR). By using sequencing analysis, we show that single mutations frequently occurred randomly in the amplification products of the PCR.

Synthesis, dynamic combinatorial chemistry, and PCR amplification of 3'-5' and 3'-6' disulfide-linked oligonucleotides.

Disulfide dithymidines linked 3'-5' or 3'-6' were synthesized and incorporated into oligonucleotides through a combined phosphotriester and phosphoramidite solid-phase oligonucleotide synthesis approach. The disulfide links are cleaved and formed reversibly in the presence of thiols and oligonucleotides. This link was shown to be sequence-adaptive in response to given templates in the presence of mercaptoethanol. The artificial 3'-5' and 3'-6' disulfide link was tolerated by polymerases in the polymerase chain reaction (PCR). By using sequencing analysis, we show that single mutations frequently occurred randomly in the amplification products of the PCR.

A high-performance liquid chromatography-based assay of glutathione transferase omega 1 supported by glutathione or non-physiological reductants

The unusual glutathione S-transferase GSTO1 reduces, rather than conjugates, endo- and xenobiotics, and its role in diverse cellular processes has been proposed. GSTO1 has been assayed spectrophotometrically by measuring the disappearance of its substrate, S-(4-nitrophenacyl)glutathione (4-NPG), in the presence of 2-mercaptoethanol that regenerates GSTO1 from its mixed disulfide. To assay GSTO1 in rat liver cytosol, we have developed a high-performance liquid chromatography (HPLC)-based procedure with two main advantages: (i) it measures the formation of the 4-NPG reduction product 4-nitroacetophenone, thereby offering improved sensitivity and accuracy, and (ii) it can use glutathione, the physiological reductant of GSTO1, which is impossible to do with the spectrophotometric procedure. Using the new assay, we show that (i) the GSTO1-catalyzed reduction of 4-NPG in rat liver cytosol also yields 1-(4-nitrophenyl)ethanol, whose formation from 4-nitroacetophenone requires NAD(P)H; (ii) the two assays measure comparable activities with 2-mercaptoethanol or tris(2-carboxyethyl)phosphine used as reductant; (iii) the cytosolic reduction of 4-NPG is inhibited by GSTO1 inhibitors (KT53, 5-chloromethylfluorescein diacetate, and zinc), although the inhibitory effect is strikingly influenced by the type of reductant in the assay and by the sequence of reductant and inhibitor addition. Characterization of GSTO1 inhibitors with the improved assay provides better understanding of interaction of these chemicals with the enzyme.

Quantitative analysis for estimating injury effects of metal-catalyzed oxidation on human erythrocytes

ObjectiveTo investigate whether human erythrocyte proteins were susceptible to oxidative effects of pharmacological doses of iron and whether resulting damages affect their structure. MethodsConformational changes in hemoglobin were indicated by spectrophotometric analysis from 300 to 650 nm. Carbonyl assay was performed for estimating the protein oxidation in erythrocytes. Oxidative injury in erythrocyte membrane was investigated by evaluation of the structural changes in cytoskeleton proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis in presence of 2-mercaptoethanol and staining with Coomassie briliant blue G-250. ResultsA significant increase in absorbance at 630 nm represented the formation of methemoglobin. Increase in absorbance at 340 nm was indicated by interaction between globin and heme group, which predicted for low oxygen affinity. A decrease in absorbance at 420 nm showed the conversion of oxygen hemoglobin to methemoglobin and significant decrease in oxygen hemoglobin concentration. There was marked elevation in hemichrome compared with control group. Of interest, a positive correlation was observed between iron concentration and hemoglobin absorbance at 340 nm. Elevated levels of carbonyl groups confirmed the oxidative damage to erythrocyte proteins. Analysis of membrane proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis, showed molecular aggregates in the range of 150 to 180 kDa and slight decrease in the intensity of α-spectrin band. ConclusionsIt is possible to predict the situation of everyone who exposed to oxidant agent via a simple blood analysis. In this way, contents of oxidative products in blood samples would be assessed by this method.

Analysis of amino acid composition in proteins of animal tissues and foods as pre-column o-phthaldialdehyde derivatives by HPLC with fluorescence detection.

Studies of protein nutrition and biochemistry require reliable methods for analysis of amino acid (AA) composition in polypeptides of animal tissues and foods. Proteins are hydrolyzed by 6M HCl (110°C for 24h), 4.2M NaOH (105°C for 20 h), or proteases. Analytical techniques that require high-performance liquid chromatography (HPLC) include pre-column derivatization with 4-chloro-7-nitrobenzofurazan, 9-fluorenyl methylchloroformate, phenylisothiocyanate, naphthalene-2,3-dicarboxaldehyde, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, and o-phthaldialdehyde (OPA). OPA reacts with primary AA (except cysteine or cystine) in the presence of 2-mercaptoethanol or 3-mercaptopropionic acid to form a highly fluorescent adduct. OPA also reacts with 4-amino-1-butanol and 4-aminobutane-1,3-diol produced from oxidation of proline and 4-hydroxyproline, respectively, in the presence of chloramine-T plus sodium borohydride at 60°C, or with S-carboxymethyl-cysteine formed from cysteine and iodoacetic acid at 25°C. Fluorescence of OPA derivatives is monitored at excitation and emission wavelengths of 340 and 455 nm, respectively. Detection limits are 50 fmol for AA. This technique offers the following advantages: simple procedures for preparation of samples, reagents, and mobile-phase solutions; rapid pre-column formation of OPA-AA derivatives and their efficient separation at room temperature (e.g., 20-25°C); high sensitivity of detection; easy automation on the HPLC apparatus; few interfering side reactions; a stable chromatography baseline for accurate integration of peak areas; and rapid regeneration of guard and analytical columns. Thus, the OPA method provides a useful tool to determine AA composition in proteins of animal tissues (e.g., skeletal muscle, liver, intestine, placenta, brain, and body homogenates) and foods (e.g., milk, corn grain, meat, and soybean meal).

Lipidic polyols using thiol‐ene/yne strategy for crosslinked polyurethanes

ABSTRACTOleic acid and α,ω‐diacid were converted into propargylic esters followed by thiol‐ene/yne coupling (TEC/TYC) functionalization in presence of mercaptoethanol. The multiradical addition on fatty esters leads to the formation of lipidic polyols (OH1 and OH2), as judged by 1H NMR and mass spectroscopies as well as by size exclusion chromatography. The crosslinking reaction between TEC/TYC‐based polyols and 4,4′‐methylene bis(phenylisocyanate) isocyanate reactant was monitored by FTIR experiment and reaction parameters were optimized. By differential scanning calorimetry, relatively high glass transitions are measured corresponding to structure with little or without dangling chain. Moreover, the thermal stability of the resulting plant oil‐based polyurethane materials (PU1 and PU2) were found to be fully consistent with that of other lipidic PUs respecting a three‐step process. Thanks to TYC methodology, fatty α,ω‐diacid produces lipidic polyol without dangling chain and lipidic thermoset PU with relatively high Tg. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014, 52, 1597–1606

A sequential-injection reversed-phase chromatography method for fluorimetric determination of glyphosate and aminomethylphosphonic acid

Glyphosate (GLYPH) is one of the most used herbicides around the world. In soil and waters, GLYPH is metabolized into aminomethylphosphonic acid (AMPA). Because of their high water solubility and half-life, both GLYPH and AMPA have been detected long after application. The present paper describes the development of a low cost Sequential Injection Chromatography (SIC) method for determination of GLYPH and AMPA based on pre-column oxidation of GLYPH to glycine (GLY) by hypochlorite, exploiting the stopped flow approach, followed by reaction with the fluorogenic reagent o-phthaldialdehyde in the presence of 2-mercaptoethanol (OPA-2ME) in borate buffer (pH > 9) to produce the fluorescent 1-(2′-hydroxyethylthio)-2-N-alkylisoindole. While GLYPH is being converted into GLY, the determination of AMPA is made by pre-column derivatization with OPA-2ME. Isocratic elution of both GLYPH and AMPA derivatives was done using a mobile phase composed of 20:80 (v/v) methanol:10 mM phosphate buffer (pH 6.8) at a flow rate of 30 μL s−1. The proposed method solved the mutual fluorescence interference of AMPA and GLY derivatives, and the interference of humic substances. A linear response was observed for concentrations up to 5.0 μM for both compounds, with limits of detection and quantification of 0.11 and 0.35 μM (GLYPH), and 0.06 and 0.20 μM (AMPA), respectively. Application of the method to spiked natural waters gave recoveries between 78 and 134%, suggesting that the method has potential to be applied for water sampling trials.

Thiol-Mediated Reduction of &amp;lt;i&amp;gt;Staphylococcus aureus&amp;lt;/i&amp;gt; Tellurite Resistance

The goal of this work was to enhance tellurite toxicity against Gram positive bacteria, especially Staphyloccocus aureus. Using a combination of assays (growth inhibition zones, growth curves, and minimal inhibitory concentrations), tellurite toxicity against this bacterium was tested in the presence of various SH- and OH-containing compounds. Results showed that the noxiousness of tellurite was strongly enhanced in the presence of 2-mercaptoethanol or dithiothreitol. The potentiating effect was observed in S. aureus ATCC 6538 (the model organism of this study) and in other Gram positive bacteria such as Bacillus subtillis ATCC 6051 and the methicillin-resistant S. aureus strain 622-4. No enhancing effect was observed in Gram negative bacteria such as Escherichia coli BW25113, Salmonella Typhimurium LT2 and Klebsiella pneumoniae ATCC 11388. These results open new perspectives for studying the treatment of multidrug-resistant Gram positive bacteria, which are responsible for numerous nosocomial infections.

Innovative self-etching dental adhesives formulated using reactive nanogel additives

Purpose: Enzymatic degradation of poorly resin-infiltrated collagen and hydrolysis of the resin-based polymeric matrix are interfacial degradation mechanisms. However, since the depth of demineralization and resin infiltration in self-etching adhesives (SEAs) occurs simultaneously, the hydrolysis of the hydrophilicmonomers requires further investigation. The aim of this study was to evaluate the microtensile bond strength (mTBS) and the confocal interfacial ultra-morphology of innovative nanogel-modified two-step SEAs bonded to dentin. Methods and materials: Nanogel copolymers were synthesized using isobornyl-methacrylate, urethane-dimethacrylate (UDMA, Esstech, USA)in presence of 2-Mercaptoethanol as a chain-transfer agent and photo-polymerized (2h) in toluene solution with bis(2,4,6-trimethylbenzoyl)phenylphosphine oxide (320-500nm, 2500 mW/cm(2)). A two-step self-etching adhesive was prepared using UDMA, 2-hydroxyethyl methacrylate and 10-methacryloxydecyldihydrogen-phosphate (40/25/15wt%). Camphorquinone and N,N-ethyl-dimethylaminobenzoate were added (0.3 and 0.7 wt%, respectively). Ethanol (14wt%) and water (5wt%) were also included to solvate the resin blend to create a control primer, while non-solvated resin was used as the adhesive. The nanogels were added to create an experimental primer (15wt%) and adhesive (25wt%). Clearfil SE-Bond (CSE; Kuraray, Japan) was also used as control SEA. Several bonding primer/adhesive combinations were applied on middle dentin (5 specimens each group), light cured (20s) and resin composite (N’Durance, Septodont, USA) build-ups (5mm) were created and light-cured (40s each 1-mm layer). The resin-bonded specimens beams (0.9mm(2)) were submitted to mTBS after 24h or 6 months of water aging. The results were statistically analysed by two-way ANOVA and Student–Newman–Keuls test used (p 0.05) after 24h. However, only the control nanogel-free primer/adhesive and the primer/adhesive both containing nanogels showed significant mTBS reduction (p<0.05) after 6 months. The greatest resistance to water aging was attained when the nanogels where present only in the adhesive. The CLSM analysis showed nanogels infiltration within the hybrid layer and low micropermeability both after 24h and 3 months of water aging. Conclusion: The use of nanogels within the composition of two-step SEAs improved their long-termdurability due to infiltration within the hybrid layer, so making the resin-dentine interface hydrophobic andmore resistant to hydrolytic degradation.