In this letter, we investigate the fabrication of Silicon nanostructure patterned on lightly doped (10(15) cm(-3)) p-type silicon-on-insulator by atomic force microscope nanolithography technique. The local anodic oxidation followed by two wet etching steps, potassium hydroxide etching for silicon removal and hydrofluoric etching for oxide removal, are implemented to reach the structures. The impact of contributing parameters in oxidation such as tip materials, applying voltage on the tip, relative humidity and exposure time are studied. The effect of the etchant concentration (10% to 30% wt) of potassium hydroxide and its mixture with isopropyl alcohol (10%vol. IPA ) at different temperatures on silicon surface are expressed. For different KOH concentrations, the effect of etching with the IPA admixture and the effect of the immersing time in the etching process on the structure are investigated. The etching processes are accurately optimized by 30%wt. KOH +10%vol. IPA in appropriate time, temperature, and humidity.
Amyloid fibers are classified as a new generation of tunable bionanomaterials that exhibit new functions related to their distinctive characteristics, such as their universality, tunability, and stiffness. Here, we introduce the catalytic residues of serine protease into a peptide catalyst (PC) via an enzyme-mimic approach. The rational design of a repeating pattern of polar and nonpolar amino acids favors the conversion of the peptides into amyloid-like fibrils via self-assembly. Distinct fibrous morphologies have been observed at different pH values and temperatures, which indicates that different fibril packing schemes can be designed; hence, fibrillar peptides can be used to generate efficient artificial catalysts for amidolytic activities at mild pH values. The results of atomic force microscopy, Raman spectroscopy, and wide-angle X-ray scattering analyses are used to discuss and compare the fibril structure of a fibrillar PC with its amidolytic activity. The pH of the fibrillation reaction crucially affects the pKa of the side chains of the catalytic triads and is important for stable fibril formation. Temperature is another important parameter that controls the self-assembly of peptides into highly stacked and laminated morphologies. The morphology and stability of fibrils are crucial and represent important factors for demonstrating the capability of the peptides to exert amidolytic activity. The observed amidolytic activity of PC4, one of the PCs, was validated using an inhibition assay, which revealed that PC4 can perform enzyme-like amidolytic catalysis. These results provide insights into the potential use of designed peptides in the generation of efficient artificial enzymes.
This article reports on the studies of structural and optical properties of nanoporous GaN prepared by Pt assisted electro chemical etching. The porous GaN samples were investigated by scanning electron microscopy (SEM), atomic force microscopy (AFM), and optical transmission (OT). SEM images liang indicated that the density of the pores increased with etching duration, however, the etching duration has no significant effect on the size and shape of the pores. AFM measurements exhibited that the surface roughness was increased with etching durations, however, for long etching duration, the increase of the surface roughness became insignificant. OT measurements revealed that the increase of pore density would lead to the reduction of light transmission. The studies showed that the porosity could influence the structural and optical properties of the GaN.
Successful use of tissue expanders depends on the quality of expanded tissue. This study evaluates the impact of anisotropic self-inflating tissue expander (SITE) on the biomechanics of skin. Two different SITE were implanted subcutaneously on sheep scalps; SITE that requires 30days for maximum expansion (Group A; n=5), and SITE that requires 21days for maximum expansion (Group B; n=5). Control animals (n=5) were maintained without SITE implantation. Young's Modulus, D-periodicity, overlap and gap region length, diameter, and height difference between overlap and gap regions on collagen fibrils were analyzed using atomic force microscopy. Histology showed no significant differences in dermal thickness between control and expanded skin of groups A and B. Furthermore, most parameters of expanded skin were similar to controls (p>0.05). However, the height difference between overlap and gap regions was significantly smaller in group B compared to both control and group A (p<0.01). Strong correlation was observed between Young's Modulus of overlap and gap regions of the control and group A, but not group B. Results suggest that a relatively slower SITE can be useful in reconstructive surgery to maintain the biomechanical properties of expanded skin.
Recently, drug functionalized biodegradable polymers have been appreciated to be imperative to fabricate multi-drug delivery nanosystems for sustainable drug release. In this work, amphiphilic chitosan-grafted-(cetyl alcohol-maleic anhydride-pyrazinamide) (CS-g-(CA-MA-PZA)) was synthesized by multi-step reactions. The incorporation of rifampicin (RF) and entrapment of silver nanoparticles (Ag NPs) on CS-g-(CA-MA-PZA) polymer was carried out by dialysis technique. From the FT-IR experiment, the polymer modification, incorporation of drugs and the entrapment of Ag NPs on micelles were confirmed. The surface morphology of Ag NPs, polymeric system and drug loaded micelles was described by SEM, TEM and AFM techniques. In addition, the controlled release behaviour of CS-g-(CA-MA-PZA) micelles was studied by UV-Vis spectroscopy. In vitro cell viability, cell apoptosis and cellular uptake experiments shows that multi-drug delivery system could enhance the biocompatibility and higher the cytotoxicity effect on the cells. Since the prepared amphiphilic polymeric micelles exhibit spotty features and the system is a promising strategy for a novel candidate for immediate therapeutically effects for alveolar macrophages.
Fabrication of functional DNA nanostructures operating at a cellular level has been accomplished through molecular programming techniques such as DNA origami and single-stranded tiles (SST). During implementation, restrictive and constraint dependent designs are enforced to ensure conformity is attainable. We propose a concept of DNA polyominoes that promotes flexibility in molecular programming. The fabrication of complex structures is achieved through self-assembly of distinct heterogeneous shapes (i.e., self-organised optimisation among competing DNA basic shapes) with total flexibility during the design and assembly phases. In this study, the plausibility of the approach is validated using the formation of multiple 3×4 DNA network fabricated from five basic DNA shapes with distinct configurations (monomino, tromino and tetrominoes). Computational tools to aid the design of compatible DNA shapes and the structure assembly assessment are presented. The formations of the desired structures were validated using Atomic Force Microscopy (AFM) imagery. Five 3×4 DNA networks were successfully constructed using combinatorics of these five distinct DNA heterogeneous shapes. Our findings revealed that the construction of DNA supra-structures could be achieved using a more natural-like orchestration as compared to the rigid and restrictive conventional approaches adopted previously.
The creation of an appropriate thin film is important for the development of novel sensing surfaces, which will ultimately enhance the properties and output of high-performance sensors. In this study, we have fabricated and characterized zinc oxide (ZnO) thin films on silicon substrates, which were hybridized with gold nanoparticles (AuNPs) to obtain ZnO-Aux (x = 10, 20, 30, 40 and 50 nm) hybrid structures with different thicknesses. Nanoscale imaging by field emission scanning electron microscopy revealed increasing film uniformity and coverage with the Au deposition thickness. Transmission electron microscopy analysis indicated that the AuNPs exhibit an increasing average diameter (5-10 nm). The face center cubic Au were found to co-exist with wurtzite ZnO nanostructure. Atomic force microscopy observations revealed that as the Au content increased, the overall crystallite size increased, which was supported by X-ray diffraction measurements. The structural characterizations indicated that the Au on the ZnO crystal lattice exists without any impurities in a preferred orientation (002). When the ZnO thickness increased from 10 to 40 nm, transmittance and an optical bandgap value decreased. Interestingly, with 50 nm thickness, the band gap value was increased, which might be due to the Burstein-Moss effect. Photoluminescence studies revealed that the overall structural defect (green emission) improved significantly as the Au deposition increased. The impedance measurements shows a decreasing value of impedance arc with increasing Au thicknesses (0 to 40 nm). In contrast, the 50 nm AuNP impedance arc shows an increased value compared to lower sputtering thicknesses, which indicated the presence of larger sized AuNPs that form a continuous film, and its ohmic characteristics changed to rectifying characteristics. This improved hybrid thin film (ZnO/Au) is suitable for a wide range of sensing applications.
Mechanical stretch-induced tyrosine phosphorylation in the proline-rich 306-residue substrate domain (CasSD) of p130Cas (or BCAR1) has eluded an experimentally validated structural understanding. Cellular p130Cas tyrosine phosphorylation is shown to function in areas without internal actomyosin contractility, sensing force at the leading edge of cell migration. Circular dichroism shows CasSD is intrinsically disordered with dominant polyproline type II conformations. Strongly conserved in placental mammals, the proline-rich sequence exhibits a pseudo-repeat unit with variation hotspots 2-9 residues before substrate tyrosine residues. Atomic-force microscopy pulling experiments show CasSD requires minimal extension force and exhibits infrequent, random regions of weak stability. Proteolysis, light scattering and ultracentrifugation results show that a monomeric intrinsically disordered form persists for CasSD in solution with an expanded hydrodynamic radius. All-atom 3D conformer sampling with the TraDES package yields ensembles in agreement with experiment when coil-biased sampling is used, matching the experimental radius of gyration. Increasing β-sampling propensities increases the number of prolate conformers. Combining the results, we conclude that CasSD has no stable compact structure and is unlikely to efficiently autoinhibit phosphorylation. Taking into consideration the structural propensity of CasSD and the fact that it is known to bind to LIM domains, we propose a model of how CasSD and LIM domain family of transcription factor proteins may function together to regulate phosphorylation of CasSD and effect machanosensing.
Zinc oxide eugenol (ZOE) cements are generally made up of 80%-90% ZnO powder while the remaining content consists of eugenol bonding resin. ZnO structure plays a major role in the morphology and mechanical properties of ZOE. In this study, we investigated the effects of different particle sizes/shapes of ZnO particles on the surface and mechanical properties of ZOE. Three samples were prepared namely ZnO-Ax, ZnO-B and ZnO-K. The crystallite sizes calculated from XRD were 37.76 nm (ZnO-Ax), 39.46 nm (ZnO-B) and 42.20 nm (ZnO-K) while the average particle sizes obtained by DLS were 21.11nm (ZnO-Ax), 56.73 nm (ZnO-B) and 2012 nm (ZnO-K). Results revealed that the compressive strengths of ZOE-Ax and ZOE-B were improved by 87.92% and 57.16%, respectively, relative to that of commercial ZOE-K. Vickers hardness test demonstrated that the hardness of ZOE-Ax and ZOE-B also increased by 74.9% and 31.1%, respectively. The ZnO-Ax nanostructure possessed a small average particle size (21.11 nm), a homogeneous size distribution (DLS) and an oxygen-rich surface (from EDS and elemental mapping). Meanwhile, ZnO-B exhibited a slightly larger average particle size of 56.73 nm compared with that of other samples. Sample ZnO-Ax demonstrated the highest compressive strength which was attributed to its large particle surface area (21.11 nm particle size) that provided a large contact area and greater interfacial (or interlock) bonding capability if compared to that of ZnO-K sample (2012 nm particle size).
In this study, the antifouling properties of polyethersulfone (PES) membranes blended with different amounts of ZnO nanoparticles and a fixed ratio of N-methyl-2-pyrrolidone (NMP)-acetone mixture as a solvent were investigated. The properties and performance of the fabricated membranes were examined in terms of hydrophilicity, porosity, pore size, surface and cross-section image using scanning electron microscopy (SEM), surface roughness using atomic force microscopy (AFM), pure water flux, and humic acid filtration. Addition of ZnO as expected was found to improve the hydrophilicity as well as to encourage pore formation. However, the agglomeration of ZnO at a higher concentration cannot be avoided even when dissolved in a mixed solvent. The presence of highly volatile acetone contributed to the tight skin layer of the membrane which shows remarkable antifouling ability with the highest flux recovery ratio and negligible irreversible fouling. ZnO NPs in acetone/NMP mixed solvent shows an improvement in flux and rejection, but, the fouling resistance was moderate compared to the pristine membrane.
Acanthamoeba spp. are the causative agent of Acanthamoeba keratitis and granulomatous amoebic encephalitis (GAE). The current options to treat Acanthamoeba infections have limited success. Silver nanoparticles show antimicrobial effects and enhance the efficacy of their payload at the specific biological targets. Natural folk plants have been widely used for treating diseases as the phytochemicals from several plants have been shown to exhibit amoebicidal effects. Herein, we used natural products of plant or commercial sources including quercetin (QT), kolavenic acid (PGEA) isolated from plant extracts of Polyalthia longifolia var pendula and crude plant methanolic extract of Caesalpinia pulcherrima (CPFLM) as antiacanthamoebic agents. Furthermore, these plant-based materials were conjugated with silver nanoparticles (AgNPs) to determine the effects of the natural compounds and their nanoconjugates against a clinical isolate of A. castellanii from a keratitis patient (ATCC 50492) belonging to the T4 genotype. The compounds were conjugated with AgNPs and characterized by using ultraviolet visible spectrophotometry and atomic force microscopy. Quercetin coated silver nanoparticles (QT-AgNPs) showed characteristic surface plasmon resonance band at 443 nm and the average size distribution was found to be around 45 nm. The natural compounds alone and their nanoconjugates were tested for the viability of amoebae, encystation and excystation activity against A. castellanii. The natural compounds showed significant growth inhibition of A. castellanii while QT-AgNPs specifically exhibited enhanced antiamoebic effects as well as interrupted the encystation and excystation activity of the amoebae. Interestingly, these compounds and nanoconjugates did not exhibit in vitro cytotoxic effects against human cells. Plant-based compounds and extracts could be an interesting strategy in development of alternative therapeutics against Acanthamoeba infections.
Brain-eating amoebae cause devastating infections in the central nervous system of humans, resulting in a mortality rate of 95%. There are limited effective therapeutic options available clinically for treating granulomatous amoebic encephalitis and primary amoebic meningoencephalitis caused by Acanthamoeba castellanii (A. castellanii) and Naegleria fowleri (N. fowleri), respectively. Here, we report for the first time that guanabenz conjugated to gold and silver nanoparticles has significant antiamoebic activity against both A. castellanii and N. fowleri. Gold and silver conjugated guanabenz nanoparticles were synthesized by the one-phase reduction method and were characterized by ultraviolet-visible spectrophotometry and atomic force microscopy. Both metals were facilely stabilized by the coating of guanabenz, which was examined by surface plasmon resonance determination. The average size of gold nanoconjugated guanabenz was found to be 60 nm, whereas silver nanoparticles were produced in a larger size distribution with the average diameter of around 100 nm. Guanabenz and its noble metal nanoconjugates exhibited potent antiamoebic effects in the range of 2.5 to 100 μM against both amoebae. Nanoparticle conjugation enhanced the antiamoebic effects of guanabenz, as more potent activity was observed at a lower effective concentration (2.5 and 5 μM) compared to the drug alone. Moreover, encystation and excystation assays revealed that guanabenz inhibits the interconversion between the trophozoite and cyst forms of A. castellanii. Cysticdal effects against N. fowleri were also observed. Notably, pretreatment of A. castellanii with guanabenz and its nanoconjugates exhibited a significant reduction in the host cell cytopathogenicity from 65% to 38% and 2% in case of gold and silver nanoconjugates, respectively. Moreover, the cytotoxic evaluation of guanabenz and its nanoconjugates revealed negligible cytotoxicity against human cells. Guanabenz is already approved for hypertension and crosses the blood-brain barrier; the results of our current study suggest that guanabenz and its conjugated gold and silver nanoparticles can be repurposed as a potential drug for treating brain-eating amoebic infections.
Plant mediated synthesis of nanoparticles has been considered as green route and a reliable technique for the synthesis of nanoparticles due to its eco-friendly approach. In this study, we report a simple and eco-friendly approach for the synthesis of silver nanoparticles (AgNPs) using methanolic Momordica cymbalaria fruit extract as reducing agent. The fruit extract of M. cymbalaria exposed to AgNO3 solution showed the change in color from green to light yellow at room temperature within 1h of incubation confirms the synthesis of AgNPs. UV-vis spectra analysis revealed that the synthesized AgNPs had a sharp surface plasmon resonance at around 450 nm, while, the X-ray Diffraction (XRD) patterns confirmed distinctive peaks indices to the crystalline planes of the face centered cubic silver. The Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM) analysis results confirmed the presence of spherical shaped AgNPs by a huge disparity in the particle size distribution with an average size of 15.5 nm. The synthesized AgNPs showed strong antibacterial activity against all the tested multidrug resistant human pathogenic bacterial strains and also exhibited highest free radical scavenging activity (74.2%) compared to fruit extract (60.4%). Moreover, both fruit extract and the synthesized AgNPs showed the cytotoxicity towards Rat L6 skeletal muscle cell line at different concentrations, but the highest inhibition percentage was recorded for AgNPs at concentration of 100 μg/ml.
Frictional and nanomechanical properties of nanostructured polymer surfaces are important to their technological and biomedical applications. In this work, poly(ethylene terephthalate) (PET) surfaces with a periodic distribution of well-defined nanopillars were fabricated through an anodization/embossing process. The apparent surface energy of the nanopillared surfaces was evaluated using the Fowkes acid-base approach, and the surface morphology was characterized using scanning electron microscope (SEM) and atomic force microscope (AFM). The normal and lateral forces between a silica microparticle and these surfaces were quantified using colloidal probe atomic force microscopy (CP-AFM). The friction-load relationship followed Amonton's first law, and the friction coefficient appeared to scale linearly with the nanopillar height. Furthermore, all the nanopillared surfaces showed pronounced frictional instabilities compared to the smooth sliding friction loop on the flat control. Performing the stick-slip amplitude coefficient (SSAC) analysis, we found a correlation between the frictional instabilities and the nanopillars density, pull-off force and work of adhesion. We have summarised the dependence of the nanotribological properties on such nanopillared surfaces on five relevant parameters, i.e. pull-off force fp, Amontons' friction coefficient μ, RMS roughness Rq, stick-slip amplitude friction coefficient SSAC, and work of adhesion between the substrate and water Wadh in a radar chart. Whilst demonstrating the complexity of the frictional behaviour of nanopillared polymer surfaces, our results show that analyses of multiparametric nanotribological properties of nanostructured surfaces should go beyond classic Amontons' laws, with the SSAC more representative of the frictional properties compared to the friction coefficient.
This research work demonstrates compositional engineering of an organic-inorganic hybrid nano-composites for modifying absolute threshold of humidity sensors. Vanadyl-2,9,16,23-tetraphenoxy-29H,31H-phthalocyanine (VOPcPhO), an organic semiconductor, doped with Titanium-dioxide nanoparticles (TiO2NPs) has been employed to fabricate humidity sensors. The morphology of the VOPcPhO:TiO2nano-composite films has been analyzed by atomic force microscopy (AFM) and field emission scanning electron microscopy (FESEM). The sensors have been examined over a wide range of relative humidity i.e. 20-99% RH. The sensor with TiO2(90nm) shows reduced sensitivity-threshold and improved linearity. The VOPcPhO:TiO2(90nm) nano-composite film is comprised of uniformly distributed voids which makes the surface more favorable for adsorption of moisture content from environment. The VOPcPhO:TiO2nano-composite based sensor demonstrates remarkable improvement in the sensing parameter when equated with VOPcPhO sensors.
Considering the important factor of bioactive nanohydoxyapatite (nHA) to enhance osteoconductivity or bone-bonding capacity, nHA was incorporated into an electrospun polycaprolactone (PCL) membrane using electrospinning techniques. The viscosity of the PCL and nHA/PCL with different concentrations of nHA was measured and the morphology of the electrospun membranes was compared using a field emission scanning electron microscopy. The water contact angle of the nanofiber determined the wettability of the membranes of different concentrations. The surface roughness of the electrospun nanofibers fabricated from pure PCL and nHA/PCL was determined and compared using atomic force microscopy. Attenuated total reflectance Fourier transform infrared spectroscopy was used to study the chemical bonding of the composite electrospun nanofibers. Beadless nanofibers were achieved after the incorporation of nHA with a diameter of 200-700 nm. Results showed that the fiber diameter and the surface roughness of electrospun nanofibers were significantly increased after the incorporation of nHA. In contrast, the water contact angle (132° ± 3.5°) was reduced for PCL membrane after addition of 10% (w/w) nHA (112° ± 3.0°). Ultimate tensile strengths of PCL membrane and 10% (w/w) nHA/PCL membrane were 25.02 ± 2.3 and 18.5 ± 4.4 MPa. A model drug tetracycline hydrochloride was successfully loaded in the membrane and the membrane demonstrated good antibacterial effects against the growth of bacteria by showing inhibition zone for E. coli (2.53 ± 0.06 cm) and B. cereus (2.87 ± 0.06 cm).
Scaffolds supplemented with naturally derived materials seem to be a good choice in bone tissue engineering. This study aims to develop polyurethane (PU) nanofibers added with ylang ylang (YY) and zinc nitrate (ZnNO3) using the electrospinning method. Field emission scanning electron microscopy (FESEM) images showed that the diameter of the PU nanofibers (869 ± 122 nm) was reduced with the addition of YY and ZnNO3 (PU/YY-467 ± 132 nm and PU/YY/ZnNO3-290 ± 163 nm). Fourier transform infrared (FTIR), a thermal gravimetric analysis (TGA) and an X-ray diffraction (XRD) analysis confirmed the interactions between PU with YY and ZnNO3. In addition, a thermal gravimetric analysis (TGA) study revealed the improved thermal stability for PU/YY and a slight reduction in the thermal stability for PU/YY/ZnNO3. A tensile test indicated that the addition of YY and ZnNO3 (PU/YY-12.32 MPa and PU/YY/ZnNO3-14.90 MPa) improved the mechanical properties of the pristine PU (6.83 MPa). The electrospun PU/YY (524 nm) and PU/YY/ZnNO3 (284 nm) showed a reduced surface roughness when compared with the pristine PU (776 nm) as depicted in the atomic force microscopy (AFM) analysis. The addition of YY and ZnNO3 improved the anticoagulant and biocompatibility nature of the pristine PU. Furthermore, the bone mineralization study depicted the improved calcium deposition in the fabricated composites (PU/YY-7.919% and PU/YY/ZnNO3-10.150%) compared to the pristine PU (5.323%). Hence, the developed composites with desirable physico-chemical properties, biocompatibility and calcium deposition can serve as plausible candidates for bone tissue engineering.
Label-free-based detection is pivotal for real-time monitoring of biomolecular interactions and to eliminate the need for labeling with tags that can occupy important binding sites of biomolecules. One simplest form of label-free-based detection is ultraviolet-visible-near-infrared (UV-vis-NIR) spectroscopy, which measure changes in reflectivity as a means to monitor immobilization and interaction of biomolecules with their corresponding partners. In biosensor development, the platform used for the biomolecular interaction should be suitable for different molecular recognition elements. In this study, gold (Au)-coated polycarbonate was used as a platform and as a proof-of-concept, erythropoietin (EPO), a doping substance widely abused by the athletes was used as the target. The interaction of EPO with its corresponding molecular recognition elements (anti-EPO monoclonal antibody and anti-EPO DNA aptamer) is monitored by UV-vis-NIR spectroscopy. Prior to this, to show that UV-vis-NIR spectroscopy is a suitable method for measuring biomolecular interaction, the interaction between biotin and streptavidin was demonstrated via this strategy and reflectivity of this interaction decreased by 25%. Subsequent to this, interaction of the EPO with anti-EPO monoclonal antibody and anti-EPO DNA aptamer resulted in the decrease of reflectivity by 5% and 10%, respectively. The results indicated that Au-coated polycarbonate could be an ideal biosensor platform for monitoring biomolecular interactions using UV-vis-NIR spectroscopy. A smaller version of the Au-coated polycarbonate substrates can be derived from the recent set-up, to be applied towards detecting EPO abuse among atheletes.
Biodegradable polymeric films, obtained from chitosan/natural rubber latex (CS/NRL) blends with different compositions, have been prepared by wetting process. The blends were characterized by dynamic mechanical thermal analysis (DMTA) and found that the CS/NRL blends are thermodynamically incompatible. This is evident from the presence of two glass transitions, corresponding to CS and NRL phases in the blend. The mechanical properties of the CS/NRL blends were improved with increasing the amount of chitosan and after surface treatment with sulphuric acid due to the sulfonate ionic interaction. The dielectric properties was determined using Precision LCR meter in the frequency range 75 kHz up to 30 MHz. After CS/NRL surface treatment with sulphuric acid at high content of chitosan showed the highest dielectric constant. The surface properties of the CS/NRL blend films before and after surface treatment were confirmed by atomic force microscopy (AFM), respectively.
In this research, silver nanoparticles (AgNPs) were synthesized in chitosan (Cts), Cts/gelatin and gelatin suspensions using a chemical reducing agent. Cts and gelatin were used as natural stabilizers and solid support, whereas AgNO(3) was used as the silver precursor. Sodium borohydride (NaBH(4)) was used as the reducing agent. The properties of AgNPs in Cts, Cts/gelatin and gelatin bionanocomposites (BNCs) were studied in terms of their surface plasmon resonance, crystalline structure, average diameter size, particle distributions, surface topography and functional groups. All the samples were characterized by UV-visible spectroscopy, powder X-ray diffraction, transmission electron microscopy, atomic force microscopy and Fourier transform infrared spectroscopy.