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Carbon nanotubes are promising materials for biomedical applications like delivery systems and tissue scaffolds. In this paper, magnetic carbon nanotubes (M-CNTs) covered with bovine serum albumin (M-CNTs-BSA) or functionalized with hydrophilic monomers (M-CNTs-HL) were synthesized, characterized, and evaluated concerning their interaction with Caco-2 cells. There is no comparison between these two types of functionalization, and this study aimed to verify their influence on the material's interaction with the cells. Different concentrations of the nanotubes were applied to investigate cytotoxicity, cell metabolism, oxidative stress, apoptosis, and capability to cross biomimetic barriers. The materials showed cytocompatibility up to 100 µg mL-1 and a hemolysis rate below 2 %. Nanotubes' suspensions were allowed to permeate Caco-2 monolayers for up to 8 h under the effect of the magnetic field. Magnetic nanoparticles associated with the nanotubes allowed estimation of permeation through the monolayers, with values ranging from 0.50 to 7.19 and 0.27 to 9.30 × 10-3 µg (equivalent to 0.43 to 6.22 and 0.23 to 9.54 × 10-2 % of the initially estimated mass of magnetic nanoparticles) for cells exposed and non-exposed to the magnets, respectively. Together, these results support that the developed materials are promising for applications in biomedical and biotechnological fields.

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Quercetin (Q) dietary supplements exhibit poor oral bioavailability because of degradation throughout gastrointestinal digestion (GD), which may be overcome using mesoporous silica particles (MSPs) as an oral delivery system (ODS). This study aimed to elucidate the effect of the functionalization of MSPs with amine-(A-MSP), carboxyl-(C-MSP), or thiol-(T-MSP) groups on their efficiency as a quercetin ODS (QODS). The type and degree of functionalization (DF) were used as factors in an experimental design. The Q-loaded F-MSP (F-MSP/Q) was characterized by gas physisorption analysis, loading capacity (LC), and dynamic light scattering and kinetics of Q release at gastric and intestinal pHs. Antioxidant capacity and Q concentration of media containing F-MSP/Q were evaluated after simulated GD. A-MSP showed the highest LC (19.79 ± 2.42%). C-MSP showed the lowest particle size at pH 1.5 or 7.4 (≈200 nm). T-MSP exhibited the maximum Q release at pH 7.4 (11.43%). High DF of A-MSP increased Q retention, regardless of pH. A-MSP preserved antioxidant capacity of Q-released gastric media (58.95 ± 3.34%). Nonetheless, MSP and F-MSP did not protect antioxidant properties of Q released in intestinal conditions. C-MSP and T-MSP showed essential features for cellular uptake and Q release within cells that need to be assessed.

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This study presents a new optical sensor for tartrazine (TAR) quantification developed using a molecularly imprinted polymer (MIP) as the recognition element, with optical fiber serving as the supporting substrate. The fiber surface was functionalized with 3-(trimethoxysilyl)propyl methacrylate (MPS), and the fiber was coated with MIP using the precipitation polymerization method. The analysis of MIP immobilization on the functionalized optical fiber (FF) was conducted through the use of scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) techniques. Experimental parameters, such as contact time and fiber length, were adjusted in order to obtain the highest sensitive response signal for the functionalized optical fiber (FF-MIP). The fiber sensor, FF-MIP, exhibited a relatively higher response signal for tartrazine compared to other interfering dyes. The rapid and total desorption of the analyte from FF-MIP allowed the immediate reemployment of FF-MIP, which also presented an acceptable repeatability for the reflectance signal. The imprinting factors for the studied dyes were between 0.112 and 0.936 in front of TAR, 1.405, and selectivity factors were between 1.501 and 12.545, confirming the sensor selectivity. The FF-MIP sensor was successfully applied for tartrazine quantification in real water samples, where it yielded satisfactory results comparable to those of the HPLC reference method.

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Molecular diagnostic methods to detect and quantify viral RNA in clinical samples rely on the purification of the genetic material prior to reverse transcription polymerase chain reaction (qRT-PCR). Due to the large number of samples processed in clinical laboratories, automation has become a necessity in order to increase method processivity and maximize throughput per unit of time. An attractive option for isolating viral RNA is based on the magnetic solid phase separation procedure (MSPS) using magnetic microparticles. This method offers the advantage over other alternative methods of making it possible to automate the process. In this study, we report the results of the MSPS method based on magnetic microparticles obtained by a simple synthesis process, to purify RNA from oro- and nasopharyngeal swab samples of patients suspected of COVID-19 provided by three diagnostic laboratories located in the Buenos Aires Province, Argentina. Magnetite nanoparticles of Fe3O4 (MNPs) were synthesized by the coprecipitation method and then coated with silica (SiO2) produced by hydrolysis of tetraethyl orthosilicate (TEOS). After preliminary tests on samples from the A549 human lung cell line and swabs, an extraction protocol was developed. The quantity and purity of the RNA obtained were determined by gel electrophoresis, spectrophotometry, and qRT-PCR. Tests on samples from naso- and oropharyngeal swabs were performed in order to validate the method for RNA purification in high-throughput SARS-CoV-2 diagnosis by qRT-PCR. The method was compared to the spin columns method and the automated method using commercial magnetic particles. The results show that the method developed is efficient for RNA extraction from nasal and oropharyngeal swab samples, and also comparable to other extraction methods in terms of sensitivity for SARS-CoV-2 detection. Of note, this procedure and reagents developed locally were intended to overcome the shortage of imported diagnostic supplies as the sudden spread of COVID-19 required unexpected quantities of nucleic acid isolation and diagnostic kits worldwide.

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Encapsulating drugs into functionalized nanoparticles (NPs) is an alternative to reach the specific therapeutic target with lower doses. However, when the NPs are in contact with physiological media, proteins adsorb on their surfaces, forming a protein corona (PC) biomolecular layer, acquiring a distinct biological identity that alters their interactions with cells. Itraconazole (ITZ), an antifungal agent, is encapsulated into PEGylated and/or functionalized NPs with high specificity for macrophages. It is evaluated how the PC impacts their cell uptake and antifungal effect. The minimum inhibitory concentration and colony-forming unit assays demonstrate that encapsulated ITZ into poly(ethylene glycol) (PEG) NPs improves the antifungal effect compared with NPs lacking PEGylation. The improvement can be related to the synergistic effect of the encapsulated ITZ and NPs composition and the reduction of PC formation in PEG NPs. Functionalized NPs with anti-F4/80 and anti-MARCO antibodies, or mannose without PEG and treated with PC, show an improved uptake but, in the presence of PEG, significantly reduce the endocytosis, dominating the stealth effect from PEG. Therefore, the PC plays a crucial role in the nanosystem uptake and antifungal effects, which suggests the need for in vivo model studies to evaluate the effect of PC in the specificity and biodistribution.

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Glioblastoma multiforme is the most common and aggressive malignant tumor that affects the central nervous system, with high mortality and low survival. Glioblastoma multiforme treatment includes resection tumor surgery, followed by radiotherapy and chemotherapy adjuvants. However, the drugs used in chemotherapy present some limitations, such as the difficulty of crossing the bloodbrain barrier and resisting the cellular mechanisms of drug efflux. The use of polymeric nanoparticles has proven to be an effective alternative to circumvent such limitations, as it allows the exploration of a range of polymeric structures that can be modified in order to control the biodistribution and cytotoxic effect of the drug delivery systems. Nanoparticles are nanometric in size and allow the incorporation of targeting ligands on their surface, favoring the transposition of the blood-brain barrier and the delivery of the drug to specific sites, increasing the selectivity and safety of chemotherapy. The present review has described the characteristics of chitosan, poly(vinyl alcohol), poly(lactic-coglycolic acid), poly(ethylene glycol), poly(ß-amino ester), and poly(ε-caprolactone), which are some of the most commonly used polymers in the manufacture of nanoparticles for the treatment of glioblastoma multiforme. In addition, some of the main targeting ligands used in these nanosystems are presented, such as transferrin, chlorotoxin, albumin, epidermal growth factor, and epidermal growth factor receptor blockers, explored for the active targeting of antiglioblastoma agents.

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This research evaluated H2TiO7 nanotubes (TiNTs) functionalized with 1 (1TiCN), 5 (2TiCN), and 10 (3TiCN) wt.% of chitosan for the removal of clonazepam by an adsorption/photocatalysis-conjugated method. The samples were immobilized on glass, and their mechanical stability was tested by washings. The functionalization of the samples was verified by the FTIR and DRS techniques. SEM images displayed nanotubes in the samples and thickness of 4.24 µm for the 2TiCN coating. The chemical composition of the 2TiCN coating was obtained by EDS. The XRD patterns evidenced chitosan and titanate phases in the functionalized samples. Furthermore, the 2TiCN coating was evaluated in the removal of clonazepam, reaching 80.79% (4.38 and 49.64% more than the TiNT and commercial TiO2 powders, respectively) after 240 min and being 6.88% more efficient after 4 reuses than the 2TiCN powders. OH- ions were the main oxidizing species found by scavenger tests. The surface area of 2TiCN (168.6 m2/g) was 2 times higher than that of TiNTs, and its bandgap (2.95 eV) was the lowest. Therefore, the 2TiCN coating is an excellent alternative to remove clonazepam.

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This research aims to analyze the effect of functionalized nanosilica (NSF) with different levels of amine groups in the formation of hydration products. Four cement pastes were investigated, one reference with Portland cement and three replacing 1% of Portland cement by nanosilica (NS), NSF with a low content of amine groups, and NSF with a high content of amine groups. The heat of hydration of the pastes was evaluated up to 7 days of hydration, the amount of calcium hydroxide (CH) and hydrated phases by means of the thermogravimetric analysis (TGA) test and compressive strength at 2, 7, and 28 days, and porosity through tests of mercury intrusion porosimetry and computed tomography at 28 days of hydration. It was possible to observe that the NSF directly influenced the hydration kinetics of the pastes, delaying the hydration of the Portland cement; however, it demonstrated a similar mechanical performance to the paste with NS at 2 days of hydration and an increase of 10% at 28 days of hydration due to the improvement in the hydration process. Thus, it is possible to conclude that the functionalization of NSF with a low 3-aminopropyltriethoxysilane (APTES) content is promising for use in cementitious materials and may improve hydration and mechanical performance at more advanced ages compared to NS.

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Currently, there are many studies on the application of nanotechnology in therapy. Metallic nanoparticles are promising nanomaterials in cancer therapy; however, functionalization of these nanoparticles with biomolecules has become relevant as their effect on cancer cells is considerably increased by photothermal and photodynamic therapies, drug nanocarriers, and specificity by antibodies, resulting in new therapies that are more specific against different types of cancer. This review describes studies on the effect of functionalized palladium, gold, silver and platinum nanoparticles in the treatment of cancer, these nanoparticles themselves show an anticancer effect. This effect is further enhanced when the NPs are functionalized with either antibodies, DNA, RNA, peptides, proteins, or folic acid and other molecules. These NPs can penetrate the cell and accumulate in the tumor tissue, resulting in a cytotoxic effect through the generation of ROS, the induction of apoptosis, cell cycle arrest, DNA fragmentation, and a photothermal effect. NP-based therapy is a new strategy that can be used synergistically with chemotherapy and radiotherapy to achieve more effective therapies and reduce side effects.

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Cisplatin (CDDP) is a potent antitumor drug used in first-line chemotherapy against several solid tumors, including breast cancer. However, toxicities and drug resistance limit its clinical application. Thermosensitive liposome (TSL) functionalized with hyaluronic acid (HA) containing cisplatin (TSL-CDDP-HA) was developed by our research group aiming to promote the release of CDDP in the tumor region under hyperthermia conditions, as well as to decrease toxicity. Thus, this study aimed to evaluate this new formulation (HA-coated TSL-CDDP) concerning in vitro behavior and in vivo toxicity compared to non-coated TSL-CDDP and free CDDP. Cytotoxicity assays and nuclear morphology were carried out against triple-negative breast cancer cells (MDA-MB-231), while an in vivo toxicity study was performed using healthy Swiss mice. The results showed an increase (around 3-fold) in cytotoxicity of the cationic formulation (non-coated TSL-CDDP) compared to free CDDP. On the other hand, TSL-CDDP treatment induced the appearance of 2.5-fold more senescent cells with alteration of nuclear morphology than the free drug after hyperthermia condition. Furthermore, the association of liposomal formulations treatment with hyperthermia increased the percentage of apoptotic cells compared to those without heating. The percentage of apoptotic cells was 1.7-fold higher for TSL-CDDP-HA than for TSL-CDDP. For the in vivo toxicity data, the TSL-CDDP treatment was also toxic to healthy cells, inducing nephrotoxicity with a significant increase in urea levels compared to the saline control group (73.1 ± 2.4 vs. 49.2 ± 2.8 mg/mL). On the other hand, the HA-coated TSL-CDDP eliminated the damages related to the use of CDDP since the animals did not show changes in hematological and biochemical examinations and histological analyses. Thus, data suggest that this new formulation is a potential candidate for the intravenous therapy of solid tumors.

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Biocides are employed to prevent biodeterioration in waterborne paints. In the present study, we used zinc oxide nanoparticles (obtained from spent alkaline batteries) as biocide for indoor waterborne paint at 1.5% of the total solid content in paint. Two different zinc oxides synthesized from spent alkaline batteries, which showed photocatalyst activity, were employed as an antimicrobial agents. After leaching the anode of alkaline batteries, zinc was precipitated from the leachate liquor by introducing oxalic acid (O-ZnO) or sodium carbonate (C-ZnO). The antimicrobial properties of the prepared oxides were tested against Staphylococcus aureus (bacteria), Chaetomium globosum, and Aspergillus fumigatus (fungi) using agar well diffusion method. C-ZnO inhibited the growth of all the strains studied and presented enhanced activity than O-ZnO. The better performance as antimicrobial agent of C-ZnO compared to O-ZnO was attributed to its lower crystallite size, higher amount of oxygen monovacancies, and to its lower band gap energy. The oxide with the best performance in antimicrobial activity, C-ZnO, was employed for the formulation of waterborne acrylic paints. It was observed that 1.5% C-ZnO improved the antifungal properties and antibacterial properties compared to the control sample.

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Diabetic foot ulcers (DFUs) are considered one of the most severe chronic complications of diabetes and can lead to amputation in severe cases. In addition, bacterial infections in diabetic chronic wounds aggravate this scenario by threatening human health. Wound dressings made of polymer matrices with embedded metal nanoparticles can inhibit microorganism growth and promote wound healing, although the current clinical treatments for diabetic chronic wounds remain unsatisfactory. In this view, this research reports the synthesis and characterization of innovative hybrid hydrogels made of carboxymethyl cellulose (CMC) and poly(vinyl alcohol) (PVA) chemically crosslinked by citric acid (CA) functionalized with silver nanoparticles (AgNPs) generated in situ using an eco-friendly aqueous process. The results assessed through comprehensive in vitro and in vivo assays demonstrated that these hybrid polymer hydrogels functionalized with AgNPs possess physicochemical properties, cytocompatibility, hemocompatibility, bioadhesion, antibacterial activity, and biocompatibility suitable for wound dressings to support chronic wound healing process as well as preventing and treating bacterial infections. Hence, it can be envisioned that, with further research and development, these polymer-based hybrid nanoplatforms hold great potential as an important tool for creating a new generation of smart dressings for treating chronic diabetic wounds and opportunistic bacterial infections.

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Infectious diseases caused by intracellular microorganisms such as Histoplasma capsulatum represent a significant challenge worldwide. Drug encapsulation into functionalized nanoparticles (NPs) is a valuable alternative to improving drug solubility and bioavailability, preventing undesirable interactions and drug degradation, and reaching the specific therapeutic target with lower doses. This work reports on Itraconazole (ITZ) encapsulated into core-shell-like polymeric NPs and functionalized with anti-F4/80 antibodies for their targeted and controlled release into macrophages. Uptake assay on co-culture showed significant differences between the uptake of functionalized and bare NPs, higher with functionalized NPs. In vitro assays showed that F4/80-NPs with 0.007 µg/mL of encapsulated ITZ eliminated the H. capsulatum fungus in co-culture with macrophages effectively compared to the bare NPs, without any cytotoxic effect on macrophages after 24 h interaction. Furthermore, encapsulated ITZ modulated the gene expression of anti and pro-inflammatory cytokines (IL-1, INF-Y, IL-6 and IL-10) on macrophages. Additionally, the anti-F4/80 antibody-coating enhanced natural and adequate antifungal response in the cells, exerting a synergistic effect that prevented the growth of the fungus at the intracellular level. Functionalized NPs can potentially improve macrophage-targeted therapy, increasing NPs endocytosis and intracellular drug concentration.

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Functionalized platinum nanoparticles have been of considerable interest in recent research due to their properties and applications, among which they stand out as therapeutic agents. The functionalization of the surfaces of nanoparticles can overcome the limits of medicine by increasing selectivity and thereby reducing the side effects of conventional drugs. With the constant development of nanotechnology in the biomedical field, functionalized platinum nanoparticles have been used to diagnose and treat diseases such as cancer and infections caused by pathogens. This review reports on physical, chemical, and biological methods of obtaining platinum nanoparticles and the advantages and disadvantages of their synthesis. Additionally, applications in the biomedical field that can be utilized once the surfaces of nanoparticles have been functionalized with different bioactive molecules are discussed, among which antibodies, biodegradable polymers, and biomolecules stand out.

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The contamination of water resources with heavy metals is a very serious concern that demands prompt and effective attention due to the serious health risks caused by these contaminants. The synthesis and ring-opening metathesis polymerization (ROMP) of norbornene dicarboximides bearing thiol pendant groups, specifically, N-4-thiophenyl-exo-norbornene-5,6-dicarboximide (1a), N-4-(methylthio)phenyl-exo-norbornene-5,6-dicarboximide (1b) and N-4-(trifluoromethylthio)phenyl-exo-norbornene-5,6-dicarboximide (1c), as well as their assessment for the removal of heavy metals from aqueous systems, is addressed in this work. The polymers were characterized by NMR, SEM and TGA, among others. Single and multicomponent aqueous solutions of Pb2+, Cd2+ and Ni2+ were employed to perform both kinetic and isothermal adsorption studies taking into account several experimental parameters, for instance, the initial metal concentration, the contact time and the mass of the polymer. In general, the adsorption kinetic data fit the pseudo-second-order model more efficiently, while the adsorption isotherms fit the Freundlich and Langmuir models. The maximum metal uptakes were 53.7 mg/g for Pb2+, 43.8 mg/g for Cd2+ and 29.1 mg/g for Ni2+ in the SH-bearing polymer 2a, 46.4 mg/g for Pb2+, 32.9 mg/g for Cd2+ and 27.1 mg/g for Ni2+ in the SCH3-bearing polymer 2b and 40.3 mg/g for Pb2+, 35.9 mg/g for Cd2+ and 27.8 mg/g for Ni2+ in the SCF3-bearing polymer 2c, correspondingly. The better performance of polymer 2a for the metal uptake was ascribed to the lower steric hindrance and higher hydrophilicity imparted by -SH groups to the polymer. The results show that these thiol-functionalized polymers are effective adsorbents of heavy metal ions from aqueous media.

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Chemical modification in surface of cellulose nanofibrils CNFs (20 nm) from an endemic and non-significant value-added, Argentine bamboo, was developed. The modification in the CNFs was carried out with three simple routes using a low molecular weight polylactic acid synthesized in our laboratory (PLA1). The first step comprises of protection of the hydroxyl groups of PLA1 through a benzoylation (PLA1Bz). The next step consisted of the activation of carboxyl groups using thionyl chloride and the last reaction was the grafting of the modified PLA onto the CNFs (PLA1Bz-g-CNF). The covalently functionalization is confirmed by spectroscopically techniques as well as PLA1Bz-g-CNFs were characterized by thermal analyses. The PLA1Bz-g-CNFs were taken up such as nanocharges to improve properties of compatibilization and changing surface properties in films based on PLA. The comparison between the films with PLA1Bz-g-CNFs with respect to the physic mixture of the components (PLA1Bz/CNF), shows an improvement in the thermal, mechanical, and surface properties of the material, particularly when 5% of PLA1Bz-g-CNFs was added. The dispersive (γS D) component of film is increased in 36.1 mN/m respect to 29.3 mN/m from the films obtained with the physic mixture nanofibrils without modification and a plasticizing effect was noticed in the final material.

Assuntos

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The following data provide evidence of the green functionalization process of a cellulose substrate by gamma radiation to be used as template in the preparation of photocatalyst composites. Functionalized cellulose, by gamma radiation treatment, improved its stability in water and exhibited a reduced size. Our data showed an intensification of carbonyl groups signal and a decrease in the thermal stability of the cellulose as result of the gamma radiation dose. Infrared and thermal data of the treated cellulose provide evidence of bond scission and the formation of functional groups that improved it is application as template. Finally, the conductive polymer poly(3,4-ethylenedioxythiophene) was deposited on the gamma irradiated cellulose to be used as photo-catalyze in the treatment of contaminated water with pharmaceutical compounds.

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This work presents, for the first time, a fast and highly sensitive electrochemical method for determination of three organophosphorus compounds (OPs), diazinon (DZN), malathion (MLT), and chlorpyrifos (CLPF), using a modified pyrolytic graphite electrode (PGE) coupled to batch injection analysis system with multiple pulse amperometric detection (BIA-MPA). The PGE was modified by a nanocomposite based on functionalized carbon nanotubes (CNTf) and silver nanoparticles (AgNPs). The OPs samples were directly analyzed on the modified working electrode surface by BIA-MPA system in Britton-Robinson (BR) buffer 0.15 mol L-1 at pH 6.0. The MPA detection of DZN, MLT and CLPF was performed using two potential pulses, which were sequentially applied on modified PGE at -1.3 V (100 ms) and +0.8 V (100 ms) for selective determination of these three OPs and working electrode cleaning, respectively. Under optimized conditions, the sensor presented a linear range of 0.1-20 µmol L-1 for DZN, 1.0-30 µmol L-1 for MLT and from 0.25 to 50 µmol L-1 for CLPF. The limits of detection (LOD) and quantification (LOQ) of 0.35 and 1.18 µmol L-1 for DZN, 0.89 and 2.98 µmol L-1 for MLT, and 0.53 and 1.78 µmol L-1 for CLPF were obtained. The proposed method exhibited high sensitivity of 0.068, 0.030 and 0.043 mA L µmol-1 for DZN, MLT and CLPF detection, respectively. Furthermore, the BIA-MPA system provided an analytical frequency of 71 determinations per hour for direct determination of these OPs in water and food samples. The modified PGE coupled to BIA-MPA system showed a high stability of electrochemical response for OPs detection with relative standard deviation (RSD) of 1.60% (n = 20). The addition-recovery studies of the proposed method were carried out in tap water, orange juice, and apple fruit real samples, which showed suitable recovery values between 77 and 124%. The analytical performance of the developed sensor provides an attractive alternative method for OPs determination with great potential for a fast and sensitive application in contaminated samples with these pesticides.

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The current work focuses on the facile and effective synthesis of a new nanocomposite based on multiwalled carbon nanotubes (MWCNT) decorated with magnetic core-shell Fe3O4@SiO2 and functionalized with 3-mercaptopropyltrimethoxysilane (3-MPTS) used in the vortex-assisted dispersive magnetic solid-phase extraction (VA-DMSPE) of Cd(II) ions in environmental and food samples. The nanocomposite was characterized and the parameters that influenced the VA-DMSPE were optimized through a fractional factorial design 25-1. The proposed method provided a preconcentration factor of 33.14 times, detection and quantification limits of 0.090 µg L-1 and 0.302 µg L-1, respectively, and a linearity range of 0.001-40.0 µg L-1. The developed method was effectively applied to preconcentrate and determine Cd(II) in water, tobacco, green tea leaves, ginkgo biloba, carrots, and rice samples, and its accuracy was evaluated using GF AAS.

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Novel functionalized indolines were synthesized from 2-(((N-aryl)amino)methyl)acrylates and formamides under ultrasonic irradiation for the first time. Aiming to develop a straightforward and easy-to-implement methodology for the synthesis of indolines, an instrumentation setup was designed, including ultrasound (US) equipment (Ultrasonic Horn; tip diameter of 12.7 mm, 20 kHz, maximum power of 400 W), an open reaction flask, and an inexpensive and green catalyst (1 mol%; FeSO4·7H2O; CAS: 7782-63-0) without the need for anhydrous conditions. The use of the sono-Fenton process in the presence of formamides and 2-(((N-aryl)amino)methyl)acrylates afforded a broad range of functionalized indolines within 60 s in high yields. Several experimental parameters of the ultrasound-assisted reaction were evaluated, such as amplitude (40-80%), sonication time (15-60 s), and pulsed ultrasonic irradiation. A 60 s silent reaction did not produce the desired indoline. The optimized conditions for US-mediated reactions allowed the production of functionalized indolines in high isolated yields (up to 99%, 60 s reaction, pulse ration 1 s:1 s, US amplitude 60 %).