Polymeric Carbon Nitrides for Photoelectrochemical Applications: Ring Opening-Induced Degradation.

Active and stable materials that utilize solar radiation for promoting different reactions are critical for emerging technologies. Two of the most common polymeric carbon nitrides were prepared by the thermal polycondensation of melamine. The scope of this work is to investigate possible structural degradation before and after photoelectrochemical testing. The materials were characterized using synchrotron radiation and lab-based techniques, and subsequently degraded photoelectrochemically, followed by post-mortem analysis. Post-mortem investigations reveal: (1) carbon atoms bonded to three nitrogen atoms change into carbon atoms bonded to two nitrogen atoms and (2) the presence of methylene terminals in post-mortem materials. The study concludes that polymeric carbon nitrides are susceptible to photoelectrochemical degradation via ring opening.

Paraffin-Multilayer Graphene Composite for Thermal Management in Electronics.

Multilayer graphene-paraffin composites with different contents of graphene (0-10 wt.%) were prepared using an ultra-high shear mixer. The aim is to improve the heat transfer in paraffin wax, which will lead to more-efficient thermal buffering in electronic applications. The multi-layer graphenes obtained by supercritical fluid exfoliation of graphite in alcohol were investigated by Raman spectroscopy, scanning electron microscopy and atomic force microscopy. Interesting morphological features were found to be related to the intercalation of paraffins between the multilayer graphene flakes. Thermal properties were also investigated in terms of phase change transition temperatures, latent heat by differential scanning calorimetry and thermal conductivity. It was found that the addition of graphene resulted in a slight decrease in energy storage capacity but a 150% improvement in thermal conductivity at the highest graphene loading level. This phase-change material is then used as a thermal heat sink for an embedded electronic processor. The temperature of the processor during the execution of a pre-defined programme was used as a performance indicator. The use of materials with multilayer graphene contents of more than 5 wt.% was found to reduce the processor operating temperature by up to 20%. This indicates that the use of such composite materials can significantly improve the performance of processors.

Magnetically-actuated microcages for cells entrapment, fabricated by laser direct writing via two photon polymerization.

The manipulation of biological materials at cellular level constitutes a sine qua non and provocative research area regarding the development of micro/nano-medicine. In this study, we report on 3D superparamagnetic microcage-like structures that, in conjunction with an externally applied static magnetic field, were highly efficient in entrapping cells. The microcage-like structures were fabricated using Laser Direct Writing via Two-Photon Polymerization (LDW via TPP) of IP-L780 biocompatible photopolymer/iron oxide superparamagnetic nanoparticles (MNPs) composite. The unique properties of LDW via TPP technique enabled the reproduction of the complex architecture of the 3D structures, with a very high accuracy i.e., about 90 nm lateral resolution. 3D hyperspectral microscopy was employed to investigate the structural and compositional characteristics of the microcage-like structures. Scanning Electron Microscopy coupled with Energy Dispersive X-Ray Spectroscopy was used to prove the unique features regarding the morphology and the functionality of the 3D structures seeded with MG-63 osteoblast-like cells. Comparative studies were made on microcage-like structures made of IP-L780 photopolymer alone (i.e., without superparamagnetic properties). We found that the cell-seeded structures made by IP-L780/MNPs composite actuated by static magnetic fields of 1.3 T were 13.66 ± 5.11 folds (p < 0.01) more efficient in terms of cells entrapment than the structures made by IP-L780 photopolymer alone (i.e., that could not be actuated magnetically). The unique 3D architecture of the microcage-like superparamagnetic structures and their actuation by external static magnetic fields acted in synergy for entrapping osteoblast-like cells, showing a significant potential for bone tissue engineering applications.

Self-Sustained Three-Dimensional Macroporous TiO2-Graphene Photocatalyst for Sunlight Decolorization of Methyl Orange.

The development of highly efficient sunlight-driven photocatalysts has triggered increased attention due to their merit in effluent treatment through a chemically green approach. To this end, we present herein the synthesis and characterization of the TiO2/3D-GF/Ni hybrid emphasizing the main structural and morphological properties and the photodegradation process of a highly resistant aromatic azo dye, methyl orange, under both UV light and simulated sunlight. Three-dimensional (3D) graphene was grown by the thermal CVD method on the nickel foam and subsequently coated with thin films of anatase employing the sol-gel method. Thereafter, it was gratifyingly demonstrated that the hybrid nanomaterial, TiO2/3D-GF-Ni, was able to bring about more than 90% decolorization of methyl orange dye after 30 min under simulated sunlight irradiance. Moreover, the efficiency of the methyl orange decolorization was 99.5% after three successive cycles. This high-performance photocatalyst which can effectively decolorize methyl orange will most likely make a great contribution to reducing environmental pollution by employing renewable solar energy.

Ofloxacin Removal from Aqueous Media by Means of Magnetoactive Electrospun Fibrous Adsorbents.

Functionalized electrospun polymer microfibrous membranes were fabricated by electrospinning and further surface-functionalized with magnetic iron oxide (FexOy) nanoparticles to yield magnetoactive nanocomposite fibrous adsorbents. The latter were characterized in respect to their morphology, mechanical properties and magnetic properties while they were further evaluated as substrates for removing Ofloxacin (OFL) from synthetic aqueous media and secondary urban wastewater (UWW) under varying physicochemical parameters, including the concentration of the pharmaceutical pollutant, the solution pH and the membranes' magnetic content. The magnetic-functionalized fibrous adsorbents demonstrated significantly enhanced adsorption efficacy in comparison to their non-functionalized fibrous analogues while their magnetic properties enabled their magnetic recovery and regeneration.

One A3B Porphyrin Structure-Three Successful Applications.

Porphyrins are versatile structures capable of acting in multiple ways. A mixed substituted A3B porphyrin, 5-(3-hydroxy-phenyl)-10,15,20-tris-(3-methoxy-phenyl)-porphyrin and its Pt(II) complex, were synthesised and fully characterised by 1H- and 13C-NMR, TLC, UV-Vis, FT-IR, fluorescence, AFM, TEM and SEM with EDX microscopy, both in organic solvents and in acidic mediums. The pure compounds were used, firstly, as sensitive materials for sensitive and selective optical and fluorescence detection of hydroquinone with the best results in the range 0.039-6.71 µM and a detection limit of 0.013 µM and, secondly, as corrosion inhibitors for carbon-steel (OL) in an acid medium giving a best performance of 88% in the case of coverings with Pt-porphyrin. Finally, the electrocatalytic activity for the hydrogen and oxygen evolution reactions (HER and OER) of the free-base and Pt-metalated A3B porphyrins was evaluated in strong alkaline and acidic electrolyte solutions. The best results were obtained for the electrode modified with the metalated porphyrin, drop-casted on a graphite substrate from an N,N-dimethylformamide solution. In the strong acidic medium, the electrode displayed an HER overpotential of 108 mV, at i = -10 mA/cm2 and a Tafel slope value of 205 mV/dec.

Laser Direct Writing of Dual-Scale 3D Structures for Cell Repelling at High Cellular Density.

The fabrication of complex, reproducible, and accurate micro-and nanostructured interfaces that impede the interaction between material's surface and different cell types represents an important objective in the development of medical devices. This can be achieved by topographical means such as dual-scale structures, mainly represented by microstructures with surface nanopatterning. Fabrication via laser irradiation of materials seems promising. However, laser-assisted fabrication of dual-scale structures, i.e., ripples relies on stochastic processes deriving from laser-matter interaction, limiting the control over the structures' topography. In this paper, we report on laser fabrication of cell-repellent dual-scale 3D structures with fully reproducible and high spatial accuracy topographies. Structures were designed as micrometric "mushrooms" decorated with fingerprint-like nanometric features with heights and periodicities close to those of the calamistrum, i.e., 200-300 nm. They were fabricated by Laser Direct Writing via Two-Photon Polymerization of IP-Dip photoresist. Design and laser writing parameters were optimized for conferring cell-repellent properties to the structures, even for high cellular densities in the culture medium. The structures were most efficient in repelling the cells when the fingerprint-like features had periodicities and heights of ≅200 nm, fairly close to the repellent surfaces of the calamistrum. Laser power was the most important parameter for the optimization protocol.

Silk ProteinsEnriched Nanocomposite Hydrogels Based on Modified MMT Clay and Poly(2-hydroxyethyl methacrylate-co-2-acrylamido-2-methylpropane Sulfonic Acid) Display Favorable Properties for Soft Tissue Engineering.

Due to their remarkable structures and properties, three-dimensional hydrogels and nanostructured clay particles have been extensively studied and have shown a high potential for tissue engineering as solutions for tissue defects. In this study, four types of 2-hydroxyethyl methacrylate/2-acrylamido-2-methylpropane sulfonic acid/montmorillonite (HEMA/AMPSA/MMT) hydrogels enriched with sericin, and fibroin were prepared and studied in the context of regenerative medicine for soft tissue regenerative medicine. Our aim was to obtain crosslinked hydrogel structures using modified montmorillonite clay as a crosslinking agent. In order to improve the in vitro and in vivo biocompatibility, silk proteins were further incorporated within the hydrogel matrix. Fourier transform infrared spectroscopy with attenuated total reflectance (FTIR-ATR) were performed to prove the chemical structures of the modified MMT and nanocomposite hydrogels. Swelling and rheological measurements showed the good elastic behavior of the hydrogels due to this unique network structure in which modified MMT acts as a crosslinking agent. Hydrogel biocompatibility was assessed by MTT, LDH and LIVE/DEAD assays. The hydrogels were evaluated for their potential to support adipogenesis in vitro and human stem cells isolated from adipose tissue were seeded in them and induced to differentiate. The progress was assessed by evaluation of expression of adipogenic markers (ppar-γ2, perilipin) evaluated by qPCR. The potential of the materials to support tissue regeneration was further evaluated on animal models in vivo. All materials proved to be biocompatible, with better results on the 95% HEMA 5% AMPSA enriched with sericin and fibroin material. This composition promoted a better development of adipogenesis compared to the other compositions studied, due the addition of sericin and fibroin. The results were confirmed in vivo as well, with a better progress of soft tissue regeneration after implantation in mice. Therefore, hydrogel 95% HEMA 5% AMPSA enriched with sericin as well as fibroin showed the best results that recommend it for future soft tissue engineering application.

Novel chitosan and bacterial cellulose biocomposites tailored with polymeric nanoparticles for modern wound dressing development.

Dressing biomaterials play a key role in wound management keeping a moisture medium and protecting against external factors. Natural and synthetic materials could be used as dressings where chitosan and bacterial cellulose is one of the most important solutions. These biopolymers have been used for wound dressing based on their non-toxic, biodegradable, and biocompatible features. In this study, biocomposites based on bacterial cellulose and chitosan membranes tailored with antimicrobial loaded poly(N-isopropylacrylamide)/polyvinyl alcohol nanoparticles were prepared. Core-shell polymeric nanoparticles, bacterial cellulose/chitosan membranes, and biocomposites were independently loaded with silver sulfadiazine, a well-known sulfonamide antibacterial agent used in the therapy of mild-to-moderate infections for sensitive organisms. The chemistry, structure, morphology, and size distribution were investigated by Fourier transformed infrared spectroscopy (FTIR-ATR), RAMAN spectroscopy, Scanning electron (SEM) and Transmission electron microscopy (TEM), and Dynamic light scattering (DLS). In vitro release behaviors of silver sulfadiazine from polymeric nanoparticles and biocomposites were investigated. The biological investigations revealed good biocompatibility of both the nanoparticles and the biocomposites in terms of human dermal fibroblasts viability and proliferation potential. Finally, the drug-loaded polymeric biomaterials showed promising characteristics, proving their high potential as an alternative support to develop a biocompatible and antibacterial wound dressing.

In Vitro Interaction of Doxorubicin-Loaded Silk Sericin Nanocarriers with MCF-7 Breast Cancer Cells Leads to DNA Damage.

In this paper, Bombyx mori silk sericin nanocarriers with a very low size range were obtained by nanoprecipitation. Sericin nanoparticles were loaded with doxorubicin, and they were considered a promising tool for breast cancer therapy. The chemistry, structure, morphology, and size distribution of nanocarriers were investigated by Fourier transformed infrared spectroscopy (FTIR-ATR), scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and dynamic light scattering (DLS). Morphological investigation and DLS showed the formation of sericin nanoparticles in the 25-40 nm range. FTIR chemical characterization showed specific interactions of protein-doxorubicin-enzymes with a high influence on the drug delivery process and release behavior. The biological investigation via breast cancer cell line revealed a high activity of nanocarriers in cancer cells by inducing significant DNA damage.

Laser Direct Writing via Two-Photon Polymerization of 3D Hierarchical Structures with Cells-Antiadhesive Properties.

We report the design and fabrication by laser direct writing via two photons polymerization of innovative hierarchical structures with cell-repellency capability. The structures were designed in the shape of "mushrooms", consisting of an underside (mushroom's leg) acting as a support structure and a top side (mushroom's hat) decorated with micro- and nanostructures. A ripple-like pattern was created on top of the mushrooms, over length scales ranging from several µm (microstructured mushroom-like pillars, MMP) to tens of nm (nanostructured mushroom-like pillars, NMP). The MMP and NMP structures were hydrophobic, with contact angles of (127 ± 2)° and (128 ± 4)°, respectively, whereas flat polymer surfaces were hydrophilic, with a contact angle of (43 ± 1)°. The cell attachment on NMP structures was reduced by 55% as compared to the controls, whereas for the MMP, a reduction of only 21% was observed. Moreover, the MMP structures preserved the native spindle-like with phyllopodia cellular shape, whereas the cells from NMP structures showed a round shape and absence of phyllopodia. Overall, the NMP structures were more effective in impeding the cellular attachment and affected the cell shape to a greater extent than the MMP structures. The influence of the wettability on cell adhesion and shape was less important, the cellular behavior being mainly governed by structures' topography.

Beyond Nitrogen in the Oxygen Reduction Reaction on Nitrogen-Doped Carbons: A NEXAFS Investigation.

Polymer electrolyte membrane fuel cells require cheap and active electrocatalysts to drive the oxygen reduction reaction. Nitrogen-doped carbons have been extensively studied regarding their oxygen reduction reaction. The work at hand looks beyond the nitrogen chemistry and brings to light the role of oxygen. Nitrogen-doped nanocarbons were obtained by a radio-frequency plasma route at 0, 100, 250, and 350 W. The lateral size of the graphitic domain, determined from Raman spectroscopy, showed that the nitrogen plasma treatment decreased the crystallite size. Synchrotron radiation photoelectron spectroscopy showed a similar nitrogen chemistry, albeit the nitrogen concentration increased with the plasma power. Lateral crystallite size and several nitrogen moieties were plotted against the onset potential determined from oxygen reduction reaction curves. There was no correlation between the electrochemical activity and the sample structure, as determine from Raman and synchrotron radiation photoelectron spectroscopy. Near-edge X-ray absorption fine structure (NEXAFS) was performed to unravel the carbon and nitrogen local structure. A difference analysis of the NEXAFS spectra showed that the oxygen surrounding the pyridinic nitrogen was critical in achieving high onset potentials. The work shows that there were more factors at play, other than carbon organization and nitrogen chemistry.

Impact of the magnetic field on 3T3-E1 preosteoblasts inside SMART silk fibroin-based scaffolds decorated with magnetic nanoparticles.

This paper reports the impact of the magnetic field on 3T3-E1 preosteoblasts within silk-fibroin scaffolds decorated with magnetic nanoparticles. Scaffolds were prepared from silk fibroin and poly(2-hydroxyethyl methacrylate) template in which magnetite nanoparticles were embedded. The presence of the magnetite specific peaks within scaffolds compositions was evidenced by RAMAN analysis. Structural investigation was done by XRD analysis and morphological information including internal structure was obtained through SEM analysis. Geometrical evaluation (size and shape), crystalline structure of magnetic nanoparticles and the morphology of the silk fibroin scaffolds were investigated by HR-TEM. Magnetic nanoparticles were distributed within scaffolds structure. Biomineralization of hydroxyapatite on silk fibroin scaffolds with and without magnetic nanoparticles was investigated by an alternate soaking process. SEM images showed that the magnetic scaffolds were covered in an almost continuously film, which has a phase with nanostructured characteristics. This phase, which has as main components Ca and P, is made of lamellar formations. The design of an original magnetic 3D cell culture setup allowed us to observe cellular modifications under the exposure to magnetic field in the presence of magnetic silk fibroin biomaterials. The cellular proliferation potential of 3T3-E1 cell line was found increased under the magnetic field, especially in the presence of the magnetite nanoparticles. In addition, we showed that the low static magnetic field positively impacts on the osteogenic differentiation potential of the cells inside the biomimetic magnetic scaffolds.

Grafting versus Crosslinking of Silk Fibroin-g-PNIPAM via Tyrosine-NIPAM Bridges.

This paper reports the synthesis and complex characterization of novel polymeric networks based on the crosslinking of Bombyx mori silk fibroin via poly(N-isopropylacrylamide) bridges generated by an ammonium cerium nitrate redox system. The research study gives an understanding of the polymerization mechanism in terms of the generation of radical sites, radical growth and termination reaction, as well as the involvement of modifications on silk fibroin structure and properties. The physico-chemical characterization was carried out by FTIR-ATR, X-ray photoelectron spectroscopy and RAMAN spectroscopy with unravelling the chemical modification. The structural characterization and spatial arrangement by secondary structure were carried out by X-ray diffraction and circular dichroism. The thermal behavior and thermal stability were evaluated by differential scanning calorimetry and thermogravimetric analysis. The novel complex polymer network is intended to be used in the field of smart drug delivery systems.

Bi1-xEuxFeO3 Powders: Synthesis, Characterization, Magnetic and Photoluminescence Properties.

Europium substituted bismuth ferrite powders were synthesized by the sol-gel technique. The precursor xerogel was characterized by thermal analysis. Bi1-xEuxFeO3 (x = 0-0.20) powders obtained after thermal treatment of the xerogel at 600 °C for 30 min were investigated by X-ray diffraction (XRD), scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), Raman spectroscopy, and Mössbauer spectroscopy. Magnetic behavior at room temperature was tested using vibrating sample magnetometry. The comparative results showed that europium has a beneficial effect on the stabilization of the perovskite structure and induced a weak ferromagnetism. The particle size decreases after the introduction of Eu3+ from 167 nm for x = 0 to 51 nm for x = 0.20. Photoluminescence spectroscopy showed the enhancement of the characteristic emission peaks intensity with the increase of Eu3+ concentration.

Novel Nanocomposites Based on Functionalized Magnetic Nanoparticles and Polyacrylamide: Preparation and Complex Characterization.

This paper reports the synthesis and complex characterization of nanocomposite hydrogels based on polyacrylamide and functionalized magnetite nanoparticles. Magnetic nanoparticles were functionalized with double bonds by 3-trimethoxysilyl propyl methacrylate. Nanocomposite hydrogels were prepared by radical polymerization of acrylamide monomer and double bond modified magnetite nanoparticles. XPS spectra for magnetite and modified magnetite were recorded to evaluate the covalent bonding of silane modifying agent. Swelling measurements in saline solution were performed to evaluate the behavior of these hydrogels having various compositions. Mechanical properties were evaluated by dynamic rheological analysis for elastic modulus and vibrating sample magnetometry was used to investigate the magnetic properties. Morphology, geometrical evaluation (size and shape) of nanostructural characteristics and the crystalline structure of the samples were investigated by SEM, HR-TEM and selected area electron diffraction (SAED). The nanocomposite hydrogels will be further tested for the soft tissue engineering field as repairing scaffolds, due to their mechanical and magnetization behavior that can stimulate tissue regeneration.

Photoluminescent Hydroxylapatite: Eu3+ Doping Effect on Biological Behaviour.

Luminescent europium-doped hydroxylapatite (EuXHAp) nanomaterials were successfully obtained by co-precipitation method at low temperature. The morphological, structural and optical properties were investigated by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier Transform Infrared (FT-IR), UV-Vis and photoluminescence (PL) spectroscopy. The cytotoxicity and biocompatibility of EuXHAp were also evaluated using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)) assay, oxidative stress assessment and fluorescent microscopy. The results reveal that the Eu3+ has successfully doped the hexagonal lattice of hydroxylapatite. By enhancing the optical features, these EuXHAp materials demonstrated superior efficiency to become fluorescent labelling materials for bioimaging applications.

Fluorescent electrospun PMMA microfiber mats with embedded NaYF4: Yb/Er upconverting nanoparticles.

Functional upconverting nanoparticles (UCNPs) can offer new possibilities in fluorescent applications as they exhibit desired characteristic properties like large shift between the fluorescent emission signal and the infrared excitation wavelength, multi- and narrow-band absorption and emission in visible and near infrared - Vis/NIR, together with excellent photostability and low toxicity as opposed to semiconducting quantum dots. The upconversion luminescence emission or quenching characteristics of UCNPs can be altered upon exposure to physical or chemical environmental factors providing thus a functionality that can be utilized for sensing or imaging. Furthermore their functionalization with suitable indicator dyes or recognition elements can extend the range of luminescence response and ratiometric sensing to specific analytes. Synergistically, electrospun nano- and microfibers offering large surface area can enhance the functionality of UCNPs by retaining the fluorescence efficiency and improving the overall responsivity due to dramatically increased surface. For the optimization of this hybrid material system the controllable incorporation of UCNPs is required especially at increased concentration conditions needed for high brightness. Herein, we report the fabrication, morphological and optical characterization of electrospun polymer-based nanocomposite fibers, consisting of poly(methyl methacrylate) (PMMA) and upconverting lanthanide doped nanoparticles of the type NaYF4 : 20% Yb3+/2% Er3+ @ NaYF4. Morphological studies regarding the uniformity and aggregation effects of the UCNP inclusion within the fibers have been implemented followed by upconversion emission characterization by pulsed near-infrared excitation. The study and optimization of such nanocomposite fibrous systems could provide useful insights for the development of efficient upconverting electrospun fiber mats for a number of imaging and sensing applications.

Poly(3-hydroxybutyrate-CO-3-hydroxyvalerate) PHBHV biocompatible nanocarriers for 5-FU delivery targeting colorectal cancer.

Aiming to address the issue of poor bioavailability of most anti-tumor medicines against colorectal cancer, we developed a targeted anticancer nanocarrier using biocarriers able to both bind and easily release their load in a controlled manner. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) carriers were obtained via the emulsification-diffusion method, loaded with 5-fluorouracil and then characterized in terms of particle morphology and size (SEM, DLS), drug uptake and release. The cytotoxic potential of the 5-fluorouracil-loaded polymer nanocarriers on human adenocarcinoma cells (HT-29 cell line) was investigated. The in vitro studies clearly demonstrated that while the nanocarriers themselves slightly alter HT-29 cell viability, when loaded with 5-fluorouracil they significantly decrease cell viability, suggesting that the polymer itself exhibits low cytotoxicity and the drug-loaded carrier acts in an efficient manner to kill HT-29 human adenocarcinoma cells.

Coating Dependent In Vitro Biocompatibility of New Fe-Si Nanoparticles.

Magnetic nanoparticles offer multiple utilization possibilities in biomedicine. In this context, the interaction with cellular structures and their biological effects need to be understood and controlled for clinical safety. New magnetic nanoparticles containing metallic/carbidic iron and elemental silicon phases were synthesized by laser pyrolysis using Fe(CO)5 vapors and SiH4 gas as Fe and Si precursors, then passivated and coated with biocompatible agents, such as l-3,4-dihydroxyphenylalanine (l-DOPA) and sodium carboxymethyl cellulose (CMC-Na). The resulting magnetic nanoparticles were characterized by XRD, EDS, and TEM techniques. To evaluate their biocompatibility, doses ranging from 0⁻200 µg/mL hybrid Fe-Si nanoparticles were exposed to Caco2 cells for 24 and 72 h. Doses below 50 μg/mL of both l-DOPA and CMC-Na-coated Fe-Si nanoparticles induced no significant changes of cellular viability or membrane integrity. The cellular internalization of nanoparticles was dependent on their dispersion in culture medium and caused some changes of F-actin filaments organization after 72 h. However, reactive oxygen species were generated after exposure to 25 and 50 μg/mL of both Fe-Si nanoparticles types, inducing the increase of intracellular glutathione level and activation of transcription factor Nrf2. At nanoparticles doses below 50 μg/mL, Caco2 cells were able to counteract the oxidative stress by activating the cellular protection mechanisms. We concluded that in vitro biological responses to coated hybrid Fe-Si nanoparticles depended on particle synthesis conditions, surface coating, doses and incubation time.