Review on the Significant Interactions between Ultrafine Gas Bubbles and Biological Systems.

Having sizes comparable with living cells and high abundance, ultrafine bubbles (UBs) are prone to inevitable interactions with different types of cells and facilitate alterations in physiological properties. The interactions of four typical cell types (e.g., bacterial, fungal, plant, and mammalian cells) with UBs have been studied over recent years. For bacterial cells, UBs have been utilized in creating the capillary force to tear down biofilms. The release of high amounts of heat, pressure, and free radicals during bubble rupture is also found to affect bacterial cell growth. Similarly, the bubble gas core identity plays an important role in the development of fungal cells. By the proposed mechanism of attachment of UBs on hydrophobin proteins in the fungal cell wall, oxygen and ozone gas-filled ultrafine bubbles can either promote or hinder the cell growth rate. On the other hand, reactive oxygen species (ROS) formation and mass transfer facilitation are two means of indirect interactions between UBs and plant cells. Likewise, the use of different gas cores in generating bubbles can produce different physical effects on these cells, for example, hydrogen gas for antioxidation against infections and oxygen for oxidation of toxic metal ions. For mammalian cells, the importance of investigating their interactions with UBs lies in the bubbles' action on cell viability as membrane poration for drug delivery can greatly affect cells' survival. UBs have been utilized and tested in forming the pores by different methods, ranging from bubble oscillation and microstream generation through acoustic cavitation to bubble implosion.

Crucial Role of Surfactants in Improving the Extraction Efficiency of ß-Amyrin from Dischidia major Using Ultrasound-Assisted Extraction Coupled with Gas Bubble Flotation.

Ultrasound-assisted extraction coupled with gas bubble flotation was developed as a green method for extracting ß-amyrin fromDischidia major. The solvent system was water:ethanol (9:1). To improve the adsorption of ß-amyrin onto the air/liquid interface during flotation, surfactants were employed; however, the positive effect was only observed with cationic surfactants. High-performance liquid chromatography with spectrophotometric detection (HPLC-PDA) was, for the first time, applied to quantify the ß-amyrin content in D. major and its extracts. With the assistance of surfactants, the foam layer collected from flotation showed high selectivity toward ß-amyrin. The product content was notably increased after surfactants had been removed from the solution.

New Evidence of Head-to-Tail Complex Formation of SDS-DOH Mixtures Adsorbed at the Air-Water Interface as Revealed by Vibrational Sum Frequency Generation Spectroscopy and Isotope Labelling.

Details about the molecular structures of surfactant mixtures adsorbed at the air-water interface have been controversial. Using sum frequency generation vibrational spectroscopy (SFG) and isotope labeling, we show here for the first time that mixtures of dodecanol (DOH) and sodium dodecyl sulfate (SDS) adsorb at the air-water interface with the formation of a head-to-tail complex. We observed this complex formation to occur first in the aqueous subphase, followed by complex adsorption onto the interface. This new piece of evidence for the head-to-tail complex conformation contradicts the conjectured tail-to-tail adsorption of the surfactant mixtures. The SFG data also show the dominating adsorption of the SDS-DOH complex over the single molecules of SDS and DOH at the air-water interface. The interfacial DOH-to-SDS molecular ratio of approximately 2.2:1 at a DOH-to-SDS bulk concentration ratio of 10 µM/2 mM was determined by isotope labeling of the surfactants. In addition to a smaller number of gauche defects, the DOH-SDS complex was found to adopt a higher level of orderliness than the adsorbed single surfactants. These findings provide important insights into the descriptions and interpretation of DOH-SDS adsorption at the air-water interface and its properties.

Effects of Ultrafine Bubbles on Gram-Negative Bacteria: Inhibition or Selection?

Ultrafine bubbles exist in all liquids and are naturally stable. As their properties are not entirely known, it is unclear how they impact the surrounding solution and comparable-sized particles within it. It is essential to further investigate the properties of ultrafine bubbles in order to expand their industrial application. In this regard, the effect of ultrafine bubbles on bacterial development is of particular interest. Our current study, using optical density measurements and fluorescence microscopic images has demonstrated that ultrafine gas bubbles impact the morphology and phenotype of Escherichia coli and Pseudomonas aeruginosa. Specifically, Fourier transform infrared spectroscopic measurements indicated a thickening of bacterial membranes in samples exposed to ultrafine bubbles. The study also confirmed that ultrafine bubbles can inhibit bacterial cell growth. This study signifies the role of surface phenomena in bacterial culture, which is crucial in the upstream processes of recombinant DNA technology applications.

Combined Sum Frequency Generation and Thin Liquid Film Study of the Specific Effect of Monovalent Cations on the Interfacial Water Structure.

Some salts have been recently shown to decrease the sum frequency generation (SFG) intensity of the hydrogen-bonded water molecules, but a quantitative explanation is still awaited. Here, we report a similar trend for the chloride salts of monovalent cations, that is, LiCl, NaCl, and CsCl, at low concentrations. Specifically, we revealed not only the specific adsorption of cations at the water surface but also the concentration-dependent effect of ions on the SFG response of the interfacial water molecules. Our thin-film pressure balance (TFPB) measurements (stabilized by 10 mM of methyl isobutyl carbinol) enabled the determination of the surface potential that governs the surface electric field affecting interfacial water dipoles. The use of the special alcohol also enabled us to identify a remarkable specific screening effect of cations on the surface potential. We explained the concentration dependency by considering the direct ion-water interactions and water reorientation under the influence of surface electric field as the two main contributors to the overall SFG signal of the hydrogen-bonded water molecules. Although the former was dominant only at the low-concentration range, the effect of the latter intensified with increasing salt concentration, leading to the recovery of the band intensity at medium concentrations. We discussed the likelihood of a correlation between the effect of ions on reorientation dynamics of water molecules and the broad-band intensity drop in the SFG spectra of salt solutions. We proposed a mechanism for the cation-specific effect through the formation of an ionic capacitance at the solution surface. It explains how cations could impart the ion specificity while they are traditionally believed to be repelled from the interfacial region. The electrical potential of this capacitance varies with the charge separation and ion density at the interface. The charge separation being controlled by the polarizability difference between anions and cations was identified using the SFG response of the interfacial water molecules as an indirect probe. The ion density being affected by the absolute polarizability of ions was tracked through the measurement of the surface potentials and Debye-Hückel lengths using the TFPB technique.

In Situ Investigation of Peptide-Lipid Interaction Between PAP248-286 and Model Cell Membranes.

Sum frequency generation vibrational spectroscopy (SFG) was utilized to investigate the interaction between PAP248-286 and the two lipid bilayer systems. The present study also provides spectroscopic evidence to confirm that, although PAP248-286 is unable to penetrate into the hydrophobic core of the lipid bilayers, it is capable of interacting more intimately with the fluid-phase POPG/POPC than with the gel-phase DPPG/DPPC lipid bilayer. The helical structure content of lipid-bound PAP248-286 was also observed to be high, in contrast to the results previously reported using nuclear magnetic resonance (NMR). Collectively, our SFG data suggest that lipid-bound PAP248-286 actually resembles its structure in 50 % 2,2,2-trifluoroethanol better than the structure when the peptide binds to SDS micelles. This present study questions the use of SDS micelles as the model membrane for NMR studies of PAP248-286 due to its protein denaturing activity.

A sum-frequency generation spectroscopic study of the Gibbs analysis paradox: monolayer or sub-monolayer adsorption?

The Gibbs adsorption isotherm (GAI) has been considered as the foundation of surfactant adsorption studies for over a century; however, its application in determining the limiting surface excess has recently been intensively discussed, with contradictory experimental evidence either supporting or refuting the theory. The available arguments are based on monolayer adsorption models. In this paper, we experimentally and intellectually propose and validate the contribution of sub-monolayer adsorption to the GAI paradox. We utilize a powerful intrinsically surface-sensitive technique, vibrational sum-frequency generation spectroscopy (SFG), complementing with conventional tensiometric measurements to address these controversies both quantitatively and qualitatively. Our SFG results revealed that the precipitous decrease in surface tension directly corresponds to surface occupancy by adsorbates. In addition, the Gibbs analysis was successfully applied to the soluble monolayer of a surface-active alcohol to full saturation. However, the full saturation of the topmost monolayer does not necessarily mean that the surface adsorption was completed because the adsorption was observed to continuously occur in the sub-monolayer region soon after the topmost monolayer became saturated. Nonetheless, the Gibbs isotherm failed to account for the excess of alcohol adsorbed in this sub-monolayer region. This new concept of surface excess must therefore be treated thermodynamically.

Suppressing interfacial water signals to assist the peak assignment of the N⁺-H stretching mode in sum frequency generation vibrational spectroscopy.

Amines are one of the common functional groups of interest due to their abundant presence in natural proteins, surfactants and other chemicals. However, their accurate spectral assignment of vibrational modes, critical to interpreting SFG signals for characterizing various bio-interfaces such as protein-membrane interaction and surfactant adsorption, still remains elusive. Herein we present a systematic study to identify and justify the correct peak assignment of the N(+)-H stretching mode at the air-water interface. We used three special surfactants: hexadecylamine (a primary amine without counterions), dodecylamine hydrochloride (a primary amine with counterions) and hexadecyltrimethylammonium bromide as a control (the N(+)-H stretching mode is absent in this quarternary amine). We suppressed the SFG interfacial water signals using saturated NaCl solutions. Our designed experiments resolved the current controversy and concluded that the 3080 cm(-1) peak is from the N(+)-H vibrations, while the 3330 cm(-1) peak is not due to ammonium species but rather originates from the interfacial water vibrational modes or the backbone amide modes.

In situ investigation of halide co-ion effects on SDS adsorption at air-water interfaces.

Co-ions are believed to have a negligible effect on surfactant adsorption, but we show here that they can significantly affect the surfactant adsorption at the air-water interface. Sum frequency generation vibrational spectroscopy (SFG) was employed to examine the effects of three halides (Cl(-), Br(-) and I(-)) on the adsorption of an anionic surfactant, sodium dodecyl sulphate (SDS), at the air-water interface. The SFG spectral features of both the interfacial water molecules and the C-H vibrations of the adsorbed surfactant alkyl chains were analysed to characterize the surfactant adsorption. We demonstrate and compare the effects of the three halides, as well as explain the unusual effect of Br(-), on the interfacial SDS and water molecules at the air/aqueous solution interface. It was observed that each of the three co-ions has a unique effect on the adsorption and conformation of the interfacial surfactant molecules at low halide concentrations of 10-50 mM, when the effect of halides on the interfacial water structure is believed to be negligible. This discovery implies that not only do they influence surfactant adsorption indirectly via the interfacial water network but also that there is an interaction occurring between these co-ions and the negatively charged head-groups at the interface via hydration by the interfacial water molecules. Even though this interaction/competition is likely to occur only between the surfactant head-groups and the halides, the surfactant hydrophobic tail was also observed to be influenced by the co-ions. These observed behavioural differences between the co-ions cannot be explained by the variation of charge densities. Therefore, further studies are required to determine the mode of action of halides influencing the adsorption of surfactant which gives Br(-) such a unique effect.

Interactions between halide anions and interfacial water molecules in relation to the Jones-Ray effect.

The Jones-Ray effect is shown to be governed by a different mechanism to enhanced anion adsorption. Halide ions at sub-molar concentrations are not exposed to the vapour phase; instead their first-solvating shell intimately interacts with the outmost water layer. Our novel proposal opens challenges for predicting related interfacial phenomena consistently.

Investigating the effects of ultrafine bubbles on bacterial growth.

Several previous studies have considered ultrafine bubbles as a potential research target because their properties can be applied in many different research areas. In particular, the interaction between UFBs and microorganisms has always been one of the aspects that receives much attention due to the high difficulty in controlling a living system. The properties of UFBs, as mobile air-water interfaces, are greatly determined by their gas cores which play a critical role in regulating microbial growth. This study aims to investigate the effects of ultrafine bubbles on bacterial growth. Two well-studied organisms were chosen as models - Escherichia coli and Staphylococcus aureus. Their growing behavior was examined based on the growth rate, phenotype and biomass. Three types of Luria-Bertani cultures were tested, including a standard culture containing distilled water, an air ultrafine bubble culture, and a hydrogen ultrafine bubble culture. The UFBs were generated via ultrasonic cavitation and stabilized by 50 µM SDS, which was proven to have negligible effects on bacterial growth. By comparing among the three cultivation conditions, the bacterial growth rates were observed to be the highest in exposure to HUFBs. The results also signified that UFBs had an enhancement on cell proliferation. On the other hand, while proposing an increase in cell density, bacteria cultured in HUFB media have their sizes decreased uniformly and significantly (p-value < 0.05). This study confirmed that bacterial growth was promoted by UFBs; and better effects recorded were due to the HUFB present in the culture media. However, the average morphological size of bacteria was in negative correlation with their population size.

Probing the spontaneous membrane insertion of a tail-anchored membrane protein by sum frequency generation spectroscopy.

In addition to providing a semipermeable barrier that protects a cell from harmful stimuli, lipid membranes occupy a central role in hosting a variety of biological processes, including cellular communications and membrane protein functions. Most importantly, protein-membrane interactions are implicated in a variety of diseases and therefore many analytical techniques were developed to study the basis of these interactions and their influence on the molecular architecture of the cell membrane. In this study, sum frequency generation (SFG) vibrational spectroscopy is used to investigate the spontaneous membrane insertion process of cytochrome b(5) and its mutants. Experimental results show a significant difference in the membrane insertion and orientation properties of these proteins, which can be correlated with their functional differences. In particular, our results correlate the nonfunctional property of a mutant cytochrome b(5) with its inability to insert into the lipid bilayer. The approach reported in this study could be used as a potential rapid screening tool in measuring the topology of membrane proteins as well as interactions of biomolecules with lipid bilayers in situ.

Orientation difference of chemically immobilized and physically adsorbed biological molecules on polymers detected at the solid/liquid interfaces in situ.

A surface sensitive second order nonlinear optical technique, sum frequency generation vibrational spectroscopy, was applied to study peptide orientation on polymer surfaces, supplemented by a linear vibrational spectroscopy, attenuated total reflectance Fourier transform infrared spectroscopy. Using the antimicrobial peptide Cecropin P1 as a model system, we have quantitatively demonstrated that chemically immobilized peptides on polymers adopt a more ordered orientation than less tightly bound physically adsorbed peptides. These differences were also observed in different chemical environments, for example, air versus water. Although numerous studies have reported a direct correlation between the choice of immobilization method and the performance of an attached biological molecule, the lack of direct biomolecular structure and orientation data has made it difficult to elucidate the relationship between structure, orientation, and function at a surface. In this work, we directly studied the effect of chemical immobilization method on biomolecular orientation/ordering, an important step for future studies of biomolecular activity. The methods for orientation analysis described within are also of relevance to understanding biosensors, biocompatibility, marine-antifouling, membrane protein functions, and antimicrobial peptide activities.

Hydrophobizing cellulose surfaces via catalyzed transesterification reaction using soybean oil and starch.

Biodegradable modified natural polymers have great potential in curbing the threat of plastic pollution, but are still uncompetitive to petrochemical-based plastic. In this study, starch was hydrophobized by treating starch-dimethyl sulfoxide solutions with soybean oil at high temperature in the presence of sodium carbonate, then spray-coated on paper. The modified starch was evaluated by Fourier-transform infrared spectroscopy analysis and contact angle value measurement of coated paper. FTIR analysis confirmed the substitution of hydroxyl groups with fatty acid ester and provided an estimate of the degree of substitution. The contact angle value of starch-coated paper surfaces was 121°, and was 111° after 10 min, demonstrating the high hydrophobicity and potential of the modified starch coating as a water-resistant treatment. The high hydrophobicity of the coated paper was due to formation of a textured surface with two levels of roughness, caused by the deposition of rough hydrophobic starch particles on paper fibers.

In situ molecular level studies on membrane related peptides and proteins in real time using sum frequency generation vibrational spectroscopy.

Sum frequency generation (SFG) vibrational spectroscopy has been demonstrated to be a powerful technique to study the molecular structures of surfaces and interfaces in different chemical environments. This review summarizes recent SFG studies on hybrid bilayer membranes and substrate-supported lipid monolayers and bilayers, the interaction between peptides/proteins and lipid monolayers/bilayers, and bilayer perturbation induced by peptides/proteins. To demonstrate the ability of SFG to determine the orientations of various secondary structures, studies on the interactions between different peptides/proteins (melittin, G proteins, alamethicin, and tachyplesin I) and lipid bilayers are discussed. Molecular level details revealed by SFG in these studies show that SFG can provide a unique understanding on the interactions between a lipid monolayer/bilayer and peptides/proteins in real time, in situ and without any exogenous labeling.

Molecular interactions between magainin 2 and model membranes in situ.

In this paper, we investigated the molecular interactions of magainin 2 with model cell membranes using sum frequency generation (SFG) vibrational spectroscopy and attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR). Symmetric 1-palmitoyl-2-oleoyl-sn-glycero-3-[Phospho-rac-(1-glycerol)] (POPG) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers, which model the bacterial and mammalian cell membranes, respectively, were used in the studies. It was observed by SFG that magainin 2 orients relatively parallel to the POPG lipid bilayer surface at low solution concentrations, around 200 nM. When increasing the magainin 2 concentration to 800 nM, both SFG and ATR-FTIR results indicate that magainin 2 molecules insert into the POPG bilayer and adopt a transmembrane orientation with an angle of about 20 degrees from the POPG bilayer normal. For the POPC bilayer, even at a much higher peptide concentration of 2.0 microM, no ATR-FTIR signal was detected. For this concentration on POPC, SFG studies indicated that magainin 2 molecules adopt an orientation nearly parallel to the bilayer surface, with an orientation angle of about 75 degrees from the surface normal. This shows that SFG has a much better detection limit than ATR-FTIR and can therefore be applied to study interfacial molecules with a much lower surface coverage. This magainin 2 orientation study and further investigation of the lipid bilayer SFG signals support the proposed toroidal pore model for the antimicrobial activity of magainin 2.

Orientation determination of protein helical secondary structures using linear and nonlinear vibrational spectroscopy.

In this paper, we systematically presented the orientation determination of protein helical secondary structures using vibrational spectroscopic methods, particularly, nonlinear sum frequency generation (SFG) vibrational spectroscopy, along with linear vibrational spectroscopic techniques such as infrared spectroscopy and Raman scattering. SFG amide I signals can be collected using different polarization combinations of the input laser beams and output signal beam to measure the second-order nonlinear optical susceptibility components of the helical amide I modes, which are related to their molecular hyperpolarizability elements through the orientation distribution of these helices. The molecular hyperpolarizability elements of amide I modes of a helix can be calculated based on the infrared transition dipole moment and Raman polarizability tensor of the helix; these quantities are determined by using the bond additivity model to sum over the individual infrared transition dipole moments and Raman polarizability tensors, respectively, of the peptide units (or the amino acid residues). The computed overall infrared transition dipole moment and Raman polarizability tensor of a helix can be validated by experimental data using polarized infrared and polarized Raman spectroscopy on samples with well-aligned helical structures. From the deduced SFG hyperpolarizability elements and measured SFG second-order nonlinear susceptibility components, orientation information regarding helical structures can be determined. Even though such orientation information can also be measured using polarized infrared or polarized Raman amide I signals, SFG has a much lower detection limit, which can be used to study the orientation of a helix when its surface coverage is much lower than a monolayer. In addition, the combination of different vibrational spectroscopic techniques, for example, SFG and attenuated total reflectance Fourier transform infrared spectroscopy, provides more measured parameters for orientation determination, aiding in the deduction of more complicated orientation distributions. In this paper, we discussed two types of helices, the alpha-helix and 3-10 helix. However, the orientation determination method presented here is general and thus can be applied to study other helices as well. The calculations of SFG amide I hyperpolarizability components for alpha-helical and 3-10 helical structures with different chain lengths have also been performed. It was found that when the helices reached a certain length, the number of peptide units in the helix should not alter the data analysis substantially. It was shown in the calculation, however, that when the helix chain is short, the SFG hyperpolarizability component ratios can vary substantially when the chain length is changed. Because 3-10 helical structures can be quite short in proteins, the orientation determination for a short 3-10 helix needs to take into account the number of peptide units in the helix.

An electronically enhanced chiral sum frequency generation vibrational spectroscopy study of lipid-bound cytochrome c.

Electronically enhanced chiral SFG spectroscopy was employed to study the lipid bound cyt c in situ. It was directly observed that upon interacting with anionic phospholipids, the amino acid residues around the heme adopted the ß-sheet conformation. In addition, the orientation of this newly formed ß-sheet structure was found to be sensitive to the bulk pH.

Orientation determination of interfacial beta-sheet structures in situ.

Structural information such as orientations of interfacial proteins and peptides is important for understanding properties and functions of such biological molecules, which play crucial roles in biological applications and processes such as antimicrobial selectivity, membrane protein activity, biocompatibility, and biosensing performance. The alpha-helical and beta-sheet structures are the most widely encountered secondary structures in peptides and proteins. In this paper, for the first time, a method to quantify the orientation of the interfacial beta-sheet structure using a combined attenuated total reflectance Fourier transformation infrared spectroscopic (ATR-FTIR) and sum frequency generation (SFG) vibrational spectroscopic study was developed. As an illustration of the methodology, the orientation of tachyplesin I, a 17 amino acid peptide with an antiparallel beta-sheet, adsorbed to polymer surfaces as well as associated with a lipid bilayer was determined using the regular and chiral SFG spectra, together with polarized ATR-FTIR amide I signals. Both the tilt angle (theta) and the twist angle (psi) of the beta-sheet at interfaces are determined. The developed method in this paper can be used to obtain in situ structural information of beta-sheet components in complex molecules. The combination of this method and the existing methodology that is currently used to investigate alpha-helical structures will greatly broaden the application of optical spectroscopy in physical chemistry, biochemistry, biophysics, and structural biology.

Interactions of alamethicin with model cell membranes investigated using sum frequency generation vibrational spectroscopy in real time in situ.

Structures of membrane-associated peptides and molecular interactions between peptides and cell membrane bilayers govern biological functions of these peptides. Sum frequency generation (SFG) vibrational spectroscopy has been demonstrated to be a powerful technique to study such structures and interactions at the molecular level. In this research, SFG has been applied, supplemented by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), to characterize the interactions between alamethicin (a model for larger channel proteins) and different lipid bilayers in the absence of membrane potential. The orientation of alamethicin in lipid bilayers has been determined using SFG amide I spectra detected with different polarization combinations. It was found that alamethicin adopts a mixed alpha-helical and 3(10)-helical structure in fluid-phase lipid bilayers. The helix (mainly alpha-helix) at the N-terminus tilts at about 63 degrees versus the surface normal in a fluid-phase 1,2-dimyristoyl-d54-sn-glycero-3-phosphocholine-1,1,2,2-d4-N,N,N-trimethyl-d9 (d-DMPC)/1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayer. The 3(10)-helix at the C-terminus (beyond the Pro14 residue) tilts at about 43 degrees versus the surface normal. This is the first time to apply SFG to study a 3(10)-helix experimentally. When interacting with a gel-phase lipid bilayer, alamethicin lies down on the gel-phase bilayer surface or aggregates or both, which does not have significant insertion into the lipid bilayer.