Adaptive Escape of Pseudomonas aeruginosa by Application of Low-Amplitude Electric Pulses.

Marine antibiofouling using low-amplitude electric pulses (EP) is an energy-efficient and eco-friendly approach, but potential mechanisms for preventing biofouling remain unclear. In the present study, the 3D adhesion dynamics of a model microorganism─Pseudomonas aeruginosa (PAO1)─under low-amplitude cathodic EP were examined as a function of applying voltage and its duration (td). The results demonstrated that adhered bacteria escaped from the electrode surface even when EP was removed. The escaped bacteria ratio, induction period of escape, and duration of the detachment were influenced profoundly by EP amplitude but slightly by td when td ≥ 5 min. The acceleration of escaped PAO1 from the surface indicated that their flagellar motor was powered by EP. Particularly, EP enabled swimming bacteria to have adaptive motions that were sustainable and regulated by the gene rsmA. As a result, they had less accumulation near the surface. The propulsion of adhered bacteria and adaptive escape of swimming bacteria were enhanced in response to low-amplitude EP. Hence, low-amplitude and short-duration EP is promising for sustainable antibiofouling applications.

Occurrence, Spatial Distribution, and Bioaccumulation of Dissolved Synthetic Musks in Freshwaters across China.

Commercial chemicals, such as synthetic musks, are of global concern, but data on their occurrence and spatial distribution in aquatic environments of large scale are scarce. Two sampling campaigns were conducted in the present study to measure freely dissolved synthetic musks in freshwaters across China using passive samplers, along with biological coexposure at selected sites. Polycyclic musks (PCMs) dominated synthetic musks, with a detection frequency of 95%. Higher concentrations of PCMs were observed in densely populated Mid, East, and South China compared to less populated regions, indicating the significance of anthropogenic activities for synthetic musks in water. The concentration ratios of galaxolide (HHCB)/tonalide (AHTN) were significantly higher in low-latitude areas than in high-latitude areas from June to September, suggesting that solar radiation played an important role in the degradation of HHCB/AHTN. Significant correlations were found between dissolved concentrations of HHCB and AHTN and their lipid-normalized concentrations in coexposed fish and clam. The estimated hazard quotients for HHCB and AHTN in freshwater fish consumed by humans were less than 0.01 at all sampling sites except the Yangtze River Basin. These results help to understand the environmental fate and ecological risks of synthetic musks on a large geographical scale.

Ionic-Wind-Enhanced Raman Spectroscopy without Enhancement Substrates.

Raman spectra are often masked by strong fluorescence, which severely hinders the applications of Raman spectroscopy. Herein, for the first time, we report ionic-wind-enhanced Raman spectroscopy (IWERS) incorporated with photobleaching (PB) as a noninvasive approach to detect fluorescent and vulnerable samples without a substrate. In this study, ionic wind (IW) generated by needle-net electrodes transfers charges to the sample surface in air on the scale of millimeters rather than nanometers in surface-enhanced Raman spectroscopy. Density functional theory calculations reveal that the ionic particles in IW increase the susceptibility of the sample molecules, thus enhancing the Raman signals. Meanwhile, the incorporation of IW with PB yields a synergistic effect to quench fluorescence. Therefore, this approach can improve the signal-to-noise ratio of Raman peaks up to three times higher than that with only PB. At the same time, IWERS can avoid sample pollution and destruction without substrates as well as high laser power. For archeological samples and a red rock as an analogue to Mars geological samples, IWERS successfully identified weak but key Raman peaks, which were masked by strong florescence. It suggests that IWERS is a promising tool for characterizations in the fields of archeology, planetary science, biomedicine, and soft matter.

Role of Surface Coverage of Sessile Probiotics in Their Interplay with Pathogen Bacteria Investigated by Digital Holographic Microscopy.

The adhesion of probiotics plays an important role in the gastrointestinal tract. Understanding the effect of the coverage of colonized probiotics on enteric pathogens is critical for the design of effective probiotic therapies. In the present work, we have investigated the adaptive behaviors of the intestinal pathogenic bacteria Enterobacter sakazakii (ES) near the surfaces coated with a probiotic─Lactobacillus rhamnosus GG (LGG) as a function of surface coverage ratio (CRLGG) by using a home-setup digital holographic microscopy. It shows that ES cells can adaptively sense LGG within a distance of 4.2 µm, even at CRLGG values as low as 0.05%. The growth inhibition of ES cells slightly varies with CRLGG, but the near-surface acceleration and accumulation of ES cells have much dependence on CRLGG. As CRLGG increases from 0.05 to 24.6%, the percentage of actively swimming ES, the motion bias, the acceleration, and the interplay duration do not linearly vary with CRLGG. Instead, each of them shows an extreme at CRLGG of 13.4%, corresponding to the chemotaxis behaviors of ES cells induced by diffusing stimuli (organic acids, bacteriocins, etc.) released from LGG, which showed an extreme concentration gradient at CRLGG = 13.4% by simulations. Our study clearly demonstrates that surface coverage of sessile probiotics profoundly influences their interplay with pathogen bacteria, which should be taken into account in designing probiotic therapies.

Mechanism Insights of Antibacterial Surfaces Coated with Dead Probiotics.

To understand the antimicrobial effect of surfaces fabricated with dead probiotics, we prepared surfaces decorated with dead probiotics Lactobacillus rhamnosus GG (LGG) with varied inactivation methods and explored their inhibitory interactions with Pseudomonas aeruginosa (PAO1). By combining several techniques, i.e., digital holographic microscopy (DHM), atomic force microscopy (AFM), RNA sequencing, and metabolomic analysis, we studied the three-dimensional (3D) swimming behaviors, surface adhesion, biofilm formation, and adaptive responses of PAO1 near such surfaces. The results show that planktonic PAO1 decreases their flick and reverse motions by downregulating the chemotaxis pathway and accelerates with less accumulation near dead LGG surfaces by upregulating the flagellar assembly pathway and decreasing cyclic adenosine monophosphate. Distinct from live siblings, the surfaces decorated with dead LGG show a significant reduction in adhesion strength with PAO1 and inhibit biofilm formation with more downregulated genes in the Pseudomonas quinolone signal and biofilm formation pathway. We demonstrate that the antibacterial ability of such surfaces stems from the gradually released lysate from the dead LGG that is unfavorable to PAO1 in close proximity. The releasing rate and order depend on the cell membrane integrity, which closely relates to the inactivation methods.

Fluorescent Visualization of Chemical Profiles across the Air-Water Interface.

Chemical transfer across the air-water interface is one of the most important geochemical processes of global significance. Quantifying such a process has remained extremely challenging due to the lack of suitable technologies to measure chemical diffusion across the air-water microlayer. Herein, we present a fluorescence optical system capable of visualizing the formation of the air-water microlayer with a spatial resolution of 10 µm and quantifying air-water diffusion fluxes using pyrene as a target chemical. We show for the first time that the air-water microlayer is composed of the surface microlayer in water (∼290 ± 40 µm) and a diffusion layer in air (∼350 ± 40 µm) with 1 µg L-1 of pyrene. The diffusion flux of pyrene across the air-water interface is derived from its high-resolution concentration profile without any pre-emptive assumption, which is 2 orders of magnitude lower than those from the conventional method. This system can be expanded to visualize diffusion dynamics of other fluorescent chemicals across the air-water interface and provides a powerful tool for furthering our understanding of air-water mass transfer of organic chemicals related to their global cycling.

Cation-amino acid interactions: Implications for protein destabilization.

The mechanism for protein stabilization or destabilization has long been an open quest. In the present study, we have studied the interactions between amino acids and guanidinium (Gdm+)/ammonium (NH4+) ions by using low field nuclear magnetic resonance (LF-NMR), where Gdm+ and NH4+ are denaturant and stabilizer for proteins, respectively. It shows that Gdm+ favors to bind to the thiol group or the hydroxyl group on the side chain but weakly interacts with the α-carboxyl group. In contrast, NH4+ prefers to bind to the α-carboxyl group but slightly interacts with the thiol group or the hydroxyl group on the side chain of amino acids. 1HNMR reveals the hydrogen bonding between NH4+ and the α-carboxyl group, which is not involved in the interactions between Gdm+ and cysteine. Our study demonstrates that the strong interactions between the denaturant and the sulfur atom or the disulfide bond promote the direct binding of the denaturant toward proteins, leading to the destabilization.

Method for 3D tracking behaviors of interplaying bacteria individuals.

Behaviors of platonic bacteria individuals are profoundly influenced by their interplay. However, probing such interplay still remains a challenge since identification and tracking of bacterial individuals becomes difficult as they come close and interact with each other. Herein, we report 3D tracking of the motions of multiple bacteria by using digital holographic microscopy (DHM), where the subtle 3D behaviors can be characterized as bacteria approach and run away from each other. An algorithm was developed to identify and recover the gap between 3D trajectory segments raising by the interruption from other bacteria through lateral image recognition and axial loalization utilizing cost function. We value the performance of the algorithm in terms of the statistics in trajectory length and correct rate. The study clearly shows how the interplaying Escherichia coli alter their motions.

Investigation of Formation of Bacterial Biofilm upon Dead Siblings.

Biocides can effectively kill bacteria; however, whether the dead bacterial cells left on the surface influence the later growth of biofilm is unknown. In this study, we have cultured Pseudomonas aeruginosa (PAO1) biofilm on their dead siblings and have investigated their evolution by using magnetic force modulation atomic force microscopy (MF-AFM). The time dependence of the biofilm thickness indicates that the deposited dead siblings can slow down the growth of PAO1 biofilm. The biofilm growing on dead bacteria layers is softer in comparison with those upon alive siblings, as reflected by the static elastic modulus ( E) and dynamic stiffness ( kd) scaled to the disturbing frequency ( f) as kd = kd,0 fγ, where kd,0 is the scaling factor and γ is the power-law exponent. We reveal that the smaller population instead of the variation of extracellular polymeric substances (EPS) within the biofilm upon the dead siblings is responsible for the softer biofilm. The present study provides a better understanding of the biofilm formation, thus, making it significant for designing antimicrobial medical materials and antifouling coatings.

Three-Dimensional Bacterial Motions near a Surface Investigated by Digital Holographic Microscopy: Effect of Surface Stiffness.

Surface stiffness plays a critical role in bacterial adhesion, but the mechanism is unclear since the bacterial motion before adhesion is overlooked. Herein, the three-dimensional (3D) motions of Escherichia coli and Pseudonomas sp. nov 776 onto poly(dimethylsiloxane) (PDMS) surfaces with varying stiffness before adhering were monitored by digital holographic microscopy (DHM). As Young's modulus (E) of the PDMS surface decreases from 278.1 to 3.4 MPa, the adhered E. coli and Pseudonomas sp. decrease in number by 40.4 and 34.9%, respectively. Atomic force microscopy (AFM) measurements show that the adhesion force of bacteria to the surface declines with the decreased surface stiffness. In contrast, a nontumbling mutant of adhered E. coli (HCB1414 with the adaptive function being partially deficient) decreases much less (by 18.4%). On the other hand, the tumble frequency (Ft) of E. coli HCB1 and flick frequency (Ff) of Pseudomonas sp. increase as the surface stiffness decreases, and the motion bias (Bθ) of Pseudomonas sp. also increases. These facts clearly indicate that the bacteria have adapted responses to the surface stiffness. RNA sequencing (RNA-seq) reveals that the downregulated Cph2 and CsrA as well as the upregulated GcvA of swimming E. coli HCB1 in bulk near the softer surface promote the bacterial motility.

Improving axial resolution for holographic tracking of colloids and bacteria over a wide depth of field by optimizing different factors.

Improving the axial resolution for multiparticle three-dimensional (3D) holographic tracking is crucial but challenging. Here we study the impacts of incident light power, uniformity of the illumination as well as image pixel size on the axial tracking resolution for digital holographic microscopy (DHM). We demonstrate that the resolution highly depends on the image pixel size and the uniformity of the illumination. A 3D localization algorithm based on local-intensity-maxima searching and a Gaussian fit to the integrated intensity of the reconstructed lateral images along the axial direction proves a robust strategy to enhance the axial resolution for colloids and bacteria within a wide depth of field over several tens of micrometers.

Near-surface microrheology reveals dynamics and viscoelasticity of soft matter.

The development of experimental techniques able to probe the microscale viscoelastic properties of soft matter over a broad time scale is essential to uncover the physics that govern their behavior. Herein, we report the development of a microrheology technique that can determine the near-surface dynamics and viscoelastic behaviors of soft matter like polymer solution/gels and colloidal dispersions. Our approach combines a magnetic-field-induced stimulator with total internal reflection microscopy (TIRM) to apply mechanical loading (∼pN) to a micro-sized probe particle and capture its axial displacement near the surface with nano-scaled sensitivity. We demonstrate the use of this technique to measure the detachment of a colloid to a solid substrate and identify three quantitatively different regimes of mechanical coupling that differ in colloid-surface separation and interaction: exclusion, aging, and non-exclusion. We also apply it to study a physical gelation process of a volume-phase transition in thermosensitive microgels and a chemically cross-linked sol-gel transition of 4-arm star polymers by monitoring the evolution of complex modulus near solid surface with frequency, time, and separation distance. In contrast to passive microrheology techniques that rely on particle tracking, we can probe the viscoelastic behavior over five orders of magnitude in stiffness, from 10-3 to 102 Pa, providing excellent coverage for dynamics and heterogeneous samples. We expect this technique will stimulate the development of new experimental methods to explore the complex microscale rheology of macromolecular networks, soft materials, and living cytoplasm.

A mobile precursor determines protein resistance on nanostructured surfaces.

Biomaterials are often engineered with nanostructured surfaces to control interactions with proteins and thus regulate their biofunctions. However, the mechanism of how nanostructured surfaces resist or attract proteins together with the underlying design rules remains poorly understood at a molecular level, greatly limiting attempts to develop high-performance biomaterials and devices through the rational design of nanostructures. Here, we study the dynamics of nonspecific protein adsorption on block copolymer nanostructures of varying adhesive domain areas in a resistant matrix. Using surface plasmon resonance and single molecule tracking techniques, we show that weakly adsorbed proteins with two-dimensional diffusivity are critical precursors to protein resistance on nanostructured surfaces. The adhesive domain areas must be more than tens or hundreds of times those of the protein footprints to slow down the 2D-mobility of the precursor proteins for their irreversible adsorption. This precursor model can be used to quantitatively analyze the kinetics of nonspecific protein adsorption on nanostructured surfaces. Our method is applicable to precisely manipulate protein adsorption and resistance on various nanostructured surfaces, e.g., amphiphilic, low-surface-energy, and charged nanostructures, for the design of protein-compatible materials.

Landing Dynamics of Swimming Bacteria on a Polymeric Surface: Effect of Surface Properties.

Landing of bacteria for adhesion on a surface is a common phenomenon in our life. However, how surface properties are involved in this process remains largely unclear. Using digital holographic microscopy, we investigated the three-dimensional motions of flagellate Escherichia coli swimming near polymeric surfaces with different properties in aqueous solution before adhesion. We monitored the bacteria landing dynamics, which shows that the density distribution, the probability, and the orientation for collisions of the bacteria are determined by their motility but are slightly affected by the surface properties. However, surface hydrophobicity reduces the near-wall velocity of the bacteria through collisions and slightly increases the collision duration. This promotes the landing and adhesion of bacteria. By contrast, most bacteria collide with the surface using their flagella, which resist adhesion.

Three-Dimensional Bacterial Behavior near Dynamic Surfaces Formed by Degradable Polymers.

Understanding the behavior of bacteria near biodegradable surfaces is critical for the development of biomedical and antibiofouling materials. By using digital holographic microscopy (DHM), we investigated the three-dimensional (3D) behavior of Escherichia coli and Pseudomonas sp. in lipase-containing aquatic environments near dynamic surfaces constructed by biodegradable poly(ε-caprolactone) (PCL)-based polymers in real time. As the enzymatic degradation rate increases, the percentage of near-surface subdiffusive bacteria and consequently, the irreversible adhesion decreases. Atomic force microscopy (AFM) measurements reveal that the adhesion force between bacteria and the surfaces decreases with an increasing degradation rate. In addition, the degradation products elicit a negative chemotactic response in E. coli, further driving them away from the dynamic surfaces through more frequent tumbling motion. Our study clearly demonstrates that bacterial adhesion can be reduced on dynamic surfaces formed by degradable polymers.

Measuring the Surface-Surface Interactions Induced by Serum Proteins in a Physiological Environment.

In this work, we applied total internal reflection microscopy (TIRM) to directly measure the interactions between three different kinds of macroscopic surfaces: namely bare polystyrene (PS) particle and bare silica surface (bare-PS/bare-silica), PS particle and silica surfaces both coated with bovine serum albumin (BSA) (BSA-PS/BSA-silica), and PS particle and silica surfaces both modified with polyethylene glycol (PEG) (PEG-PS/PEG-silica) polymers, in phosphate buffer solution (PBS) and fetal bovine serum (FBS). Our results showed that in PBS, all the bare-PS, BSA-PS, and PEG-PS particles were irreversibly deposited onto the bare silica surface or surfaces coated either with BSA or PEG. However, in FBS, the interaction potentials between the particle and surface exhibited both free-diffusing particle and stuck particle profiles. Dynamic light scattering (DLS) and elliposmeter measurements indicated that there was a layer of serum proteins adsorbed on the PS particle and silica surface. TIRM measurement revealed that such adsorbed serum proteins can mediate the surface-surface interactions by providing additional stabilization under certain conditions, but also promoting bridging effect between the two surfaces. The measured potential profile of the stuck particle in FBS thus was much wider than in PBS. These quantitative measurements provide insights that serum proteins adsorbed onto surfaces can regulate surface-surface interactions, thus leading to unique moving behavior and stability of colloidal particles in the serum environment.

Surface Roughness Modulates Diffusion and Fibrillation of Amyloid-ß Peptide.

The presence of surfaces influences the kinetics of amyloid-ß (Aß) peptide fibrillation. Although it has been generally recognized that the fibrillation process can be assisted or accelerated by surface chemistry, the impact of surface topography, i.e., roughness, on peptide fibrillation is relatively little understood. Here we study the role of surface roughness on surface-mediated fibrillation using polymer coatings of varying roughness as well as polymer microparticles. Using single-molecule tracking, atomic force microscopy, and the thioflavin T fluorescence technique, we show that a rough surface decelerates the two-dimensional (2D) diffusion of peptides and retards the surface-mediated fibrillation. A higher degree of roughness that presents an obstacle to peptide diffusion is found to inhibit the fibrillation process.

Measurements of long-range interactions between protein-functionalized surfaces by total internal reflection microscopy.

Understanding the interaction between protein-functionalized surfaces is an important subject in a variety of protein-related processes, ranging from coatings for biomedical implants to targeted drug carriers and biosensors. In this work, utilizing a total internal reflection microscope (TIRM), we have directly measured the interactions between micron-sized particles decorated with three types of common proteins concanavalin A (ConA), bovine serum albumin (BSA), lysozyme (LYZ), and glass surface coated with soy proteins (SP). Our results show that the protein adsorption greatly affects the charge property of the surfaces, and the interactions between those protein-functionalized surfaces depend on solution pH values. At pH 7.5-10.0, all these three protein-functionalized particles are highly negatively charged, and they move freely above the negatively charged SP-functionalized surface. The net interaction between protein-functionalized surfaces captured by TIRM was found as a long-range, nonspecific double-layer repulsion. When pH was decreased to 5.0, both protein-functionalized surfaces became neutral and double-layer repulsion was greatly reduced, resulting in adhesion of all three protein-functionalized particles to the SP-functionalized surface due to the hydrophobic attraction. The situation is very different at pH = 4.0: BSA-decorated particles, which are highly charged, can move freely above the SP-functionalized surfaces, while ConA- and LYZ-decorated particles can only move restrictively in a limited range. Our results quantify these nonspecific kT-scale interactions between protein-functionalized surfaces, which will enable the design of surfaces for use in biomedical applications and study of biomolecular interactions.

Tuning the particle-surface interactions in aqueous solutions by soft microgel particles.

Due to the softness and deformability, interaction between colloidal surfaces induced by soft particles varies in a more complex way than for solid particles and thus has attracted much attention in recent years. In the present study, we use total internal reflection microscopy (TIRM) to directly measure the interaction between polystyrene (PS) microparticles and a flat glass surface in a poly(N-isopropylacrylamide) (PNIPAM) microgel dispersion with concentration varying from dilute (0.1 wt %) to highly concentrated regime (7.5 wt %). Our result shows that the PS particle-surface interactions mediated by the soft microgels are greatly affected by the particle concentration, the configuration of those microgels adsorbed on the surfaces, and the structure and packing of microgels in bulk solution. With increasing the microgel concentration (Cmicrogel), the interaction between the PS particle and surface turned from bridging attraction to steric repulsion, and then depletion attraction, which were mainly governed by the adsorption amount and configuration of microgels on the two surfaces. By further increasing Cmicrogel to condensed situation, structural force with oscillated energy wells was detected. The variation of interactions induced by the soft microgels was further confirmed by optical imaging. Crystallization of the PS microparticles appeared at moderate Cmicrogel; however, crystallization was hindered at higher Cmicrogel where the microgels are highly packed in the bulk solution. Furthermore, using TIRM, microgel packing with local energy well (0.1-1.0 kBT) at the highly condensed state (7.5 wt %) was resolved from the interaction profiles. Therefore, the shear force and modulus generated by such microgel packing can be determined as ∼0.2 pN and tens of mPa, respectively, which are much weaker than data given by conventional active methods.

Copper-catalyzed one-pot trifluoromethylation/aryl migration/carbonyl formation with homopropargylic alcohols.

A novel copper-catalyzed one-pot functionalization of homopropargylic alcohols that involves trifluoromethylation, aryl migration, and formation of a carbonyl moiety has been developed. This reaction constitutes the first direct conversion of homopropargylic alcohols into CF3-containing 3-butenal or 3-buten-1-one derivatives in a regioselective manner. Mechanistic studies indicate that the 1,4-aryl migration proceeds through a radical pathway.