Gelatin-Based Hybrid Hydrogels as Matrices for Organoid Culture.

The application of liver organoids is very promising in the field of liver tissue engineering; however, it is still facing some limitations. One of the current major limitations is the matrix in which they are cultured. The mainly undefined and murine-originated tumor matrices derived from Engelbreth-Holm-Swarm (EHS) sarcoma, such as Matrigel, are still the standard culturing matrices for expansion and differentiation of organoids toward hepatocyte-like cells, which will obstruct its future clinical application potential. In this study, we exploited the use of newly developed highly defined hydrogels as potential matrices for the culture of liver organoids and compared them to Matrigel and two hydrogels that were already researched in the field of organoid research [i.e., polyisocyanopeptides, enriched with laminin-entactin complex (PIC-LEC) and gelatin methacryloyl (GelMA)]. The newly developed hydrogels are materials that have a physicochemical resemblance with native liver tissue. Norbornene-modified dextran cross-linked with thiolated gelatin (DexNB-GelSH) has a swelling ratio and macro- and microscale properties that highly mimic liver tissue. Norbornene-modified chondroitin sulfate cross-linked with thiolated gelatin (CSNB-GelSH) contains chondroitin sulfate, which is a glycosaminoglycan (GAG) that is present in the liver ECM. Furthermore, CSNB-GelSH hydrogels with different mechanical properties were evaluated. Bipotent intrahepatic cholangiocyte organoids (ICOs) were applied in this work and encapsulated in these materials. This research revealed that the newly developed materials outperformed Matrigel, PIC-LEC, and GelMA in the differentiation of ICOs toward hepatocyte-like cells. Furthermore, some trends indicate that an interplay of both the chemical composition and the mechanical properties has an influence on the relative expression of certain hepatocyte markers. Both DexNB-GelSH and CSNB-GelSH showed promising results for the expansion and differentiation of intrahepatic cholangiocyte organoids. The stiffest CSNB-GelSH hydrogel even significantly outperformed Matrigel based on ALB, BSEP, and CYP3A4 gene expression, being three important hepatocyte markers.

Gelatin-Based Hybrid Hydrogel Scaffolds: Toward Physicochemical Liver Mimicry.

There exists a clear need to develop novel materials that could serve liver tissue engineering purposes. Those materials need to be researched for the development of bioengineered liver tissue as an alternative to donor livers, as well as for materials that could be applied for scaffolds to develop an in vitro model for drug-induced liver injury (DILI) detection . In this paper, the hydrogels oxidized dextran-gelatin (Dexox-Gel) and norbornene-modified dextran-thiolated gelatin (DexNB-GelSH) were developed, and their feasibility toward processing via indirect 3D-printing was investigated with the aim to develop hydrogel scaffolds that physicochemically mimic native liver tissue. Furthermore, their in vitro biocompatibility was assessed using preliminary biological tests using HepG2 cells. Both materials were thoroughly physicochemically characterized and benchmarked to the methacrylated gelatin (GelMA) reference material. Due to inferior properties, Dexox-gel was not further processed into 3D-hydrogel scaffolds. This research revealed that DexNB-GelSH exhibited physicochemical properties that were in excellent agreement with the properties of natural liver tissue in contrast to GelMA. In combination with an equally good biological evaluation of DexNB-GelSH in comparison with GelMA based on an MTS proliferation assay and an albumin quantification assay, DexNB-GelSH can be considered promising in the field of liver tissue engineering.

Passive antifouling and active self-disinfecting antiviral surfaces.

Viruses pose a serious threat to human health and society in general, as virus infections are one of the main causes of morbidity and mortality. Till May 2022, over 513 million people around the world have been confirmed to be infected and more than 6.2 million have died due to SARS-CoV-2. Although the COVID-19 pandemic will be defeated in the near future, we are likely to face new viral threats in the coming years. One of the important instruments to protect from viruses are antiviral surfaces, which are essentially capable of limiting their spread. The formulation of the concept of antiviral surfaces is relatively new. In general, five types of mechanism directed against virus spread can be proposed for antiviral surfaces; involving: direct and indirect actions, receptor inactivation, photothermal effect, and antifouling behavior. All antiviral surfaces can be classified into two main types - passive and active. Passive antiviral surfaces are based on superhydrophobic coatings that are able to repel virus contaminated droplets. In turn, viruses can become biologically inert (e.g., blocked or destroyed) upon contact with active antiviral surfaces, as they contain antiviral agents: metal atoms, synthetic or natural polymers, and small molecules. The functionality of antiviral surfaces can be significantly improved with additional properties, such as temperature- or pH-responsivity, multifunctionality, non-specific action on different virus types, long-term application, high antiviral efficiency and self-cleaning.

Luminescent PMMA Films and PMMA@SiO2 Nanoparticles with Embedded Ln3+ Complexes for Highly Sensitive Optical Thermometers in the Physiological Temperature Range*.

In recent years, luminescent materials doped with Ln3+ ions have attracted much attention for their application as optical thermometers based on both downshifting and upconversion processes. This study presents research done on the development of highly sensitive optical thermometers in the physiological temperature range based on poly(methyl methacrylate) (PMMA) films doped with two series of visible Ln3+ complexes (Ln3+ =Tb3+ , Eu3+ , and Sm3+ ) and SiO2 nanoparticles (NPs) coated with these PMMA films. The best performing PMMA film doped with Tb3+ and Eu3+ complexes was the PMMA[TbEuL1 tppo]1 film (L1 =4,4,4-trifluoro-1-phenyl-1,3-butadionate; tppo=triphenylphosphine oxide), which showed good temperature sensing of Sr =4.21 % K-1 at 313 K, whereas for the PMMA films doped with Tb3+ and Sm3+ complexes the best performing was the PMMA[TbSmL2 tppo]3 film (L2 =4,4,4-trifluoro-1-(4-chlorophenyl)-1,3-butadionate), with Sr =3.64 % K-1 at 313 K. Additionally, SiO2 NPs coated with the best performing films from each of the series of PMMA films (Tb-Eu and Tb-Sm) and their temperature-sensing properties were studied in water, showing excellent performance in the physiological temperature range (PMMA[TbEuL1 tppo]1@SiO2 : Sr =3.84 % °C at 20 °C; PMMA[TbSmL2 tppo]3@SiO2 : Sr =3.27 % °C at 20 °C) and the toxicity of these nanoparticles on human cells was studied, showing that they were nontoxic.

Discriminating Bacterial Phenotypes at the Population and Single-Cell Level: A Comparison of Flow Cytometry and Raman Spectroscopy Fingerprinting.

Investigating phenotypic heterogeneity can help to better understand and manage microbial communities. However, characterizing phenotypic heterogeneity remains a challenge, as there is no standardized analysis framework. Several optical tools are available, such as flow cytometry and Raman spectroscopy, which describe optical properties of the individual cell. In this work, we compare Raman spectroscopy and flow cytometry to study phenotypic heterogeneity in bacterial populations. The growth stages of three replicate Escherichia coli populations were characterized using both technologies. Our findings show that flow cytometry detects and quantifies shifts in phenotypic heterogeneity at the population level due to its high-throughput nature. Raman spectroscopy, on the other hand, offers a much higher resolution at the single-cell level (i.e., more biochemical information is recorded). Therefore, it can identify distinct phenotypic populations when coupled with analyses tailored toward single-cell data. In addition, it provides information about biomolecules that are present, which can be linked to cell functionality. We propose a computational workflow to distinguish between bacterial phenotypic populations using Raman spectroscopy and validated this approach with an external data set. We recommend using flow cytometry to quantify phenotypic heterogeneity at the population level, and Raman spectroscopy to perform a more in-depth analysis of heterogeneity at the single-cell level. © 2019 International Society for Advancement of Cytometry.

Classification of analytics, sensorics, and bioanalytics with polyelectrolyte multilayer capsules.

Polyelectrolyte multilayer (PEM) capsules, constructed by LbL (layer-by-layer)-adsorbing polymers on sacrificial templates, have become important carriers due to multifunctionality of materials adsorbed on their surface or encapsulated into their interior. They have been also been used broadly used as analytical tools. Chronologically and traditionally, chemical analytics has been developed first, which has long been synonymous with all analytics. But it is not the only development. To the best of our knowledge, a summary of all advances including their classification is not available to date. Here, we classify analytics, sensorics, and biosensorics functionalities implemented with polyelectrolyte multilayer capsules and coated particles according to the respective stimuli and application areas. In this classification, three distinct categories are identified: (I) chemical analytics (pH; K+, Na+, and Pb2+ ion; oxygen; and hydrogen peroxide sensors and chemical sensing with surface-enhanced Raman scattering (SERS)); (II) physical sensorics (temperature, mechanical properties and forces, and osmotic pressure); and (III) biosensorics and bioanalytics (fluorescence, glucose, urea, and protease biosensing and theranostics). In addition to this classification, we discuss also principles of detection using the above-mentioned stimuli. These application areas are expected to grow further, but the classification provided here should help (a) to realize the wealth of already available analytical and bioanalytical tools developed with capsules using inputs of chemical, physical, and biological stimuli and (b) to position future developments in their respective fields according to employed stimuli and application areas. Graphical abstract.

Inter-protein interactions govern protein loading into porous vaterite CaCO3 crystals.

The fast development of protein therapeutics has resulted in a high demand for advanced delivery carriers that can effectively host therapeutic proteins, preserve their bioactivity and release them on demand. Accordingly, vaterite CaCO3 crystals have attracted special attention as sacrificial templates for protein encapsulation in micro- and nanoparticles (capsules and beads, respectively) under mild biofriendly conditions. This study aimed to better understand the mechanism of protein loading into crystals as a primary step for protein encapsulation. The loading of three therapeutic proteins (250 kDa catalase, 5.8 kDa insulin, and 6.5 kDa aprotinin) was investigated for crystals with different porosities. However, unexpectedly, the protein loading capacity was not consistent with the protein molecular weight. It solely depends on the inter-protein interactions in the bulk solution in the presence of crystals and that inside the crystals. The smallest protein aprotinin aggregates in the bulk (its aggregate size is about 100 nm), which prohibits its loading into the crystals. Insulin forms hexamers in the bulk, which can diffuse into the crystal pores but tend to aggregate inside the pores, suppressing protein diffusion inward. Catalase, the largest protein tested, does not form any aggregates in the bulk and diffuses freely into the crystals; however, its diffusion into small pores is sterically restricted. These findings are essential for the encapsulation of protein therapeutics by means of templating based on CaCO3 crystals and for the engineering of protein-containing microparticles having desired architectures.

Enhancement of Biomimetic Enzymatic Mineralization of Gellan Gum Polysaccharide Hydrogels by Plant-Derived Gallotannins.

Mineralization of hydrogel biomaterials with calcium phosphate (CaP) is considered advantageous for bone regeneration. Mineralization can be both induced by the enzyme alkaline phosphatase (ALP) and promoted by calcium-binding biomolecules, such as plant-derived polyphenols. In this study, ALP-loaded gellan gum (GG) hydrogels were enriched with gallotannins, a subclass of polyphenols. Five preparations were compared, namely three tannic acids of differing molecular weight (MW), pentagalloyl glucose (PGG), and a gallotannin-rich extract from mango kernel (Mangifera indica L.). Certain gallotannin preparations promoted mineralization to a greater degree than others. The various gallotannin preparations bound differently to ALP and influenced the size of aggregates of ALP, which may be related to ability to promote mineralization. Human osteoblast-like Saos-2 cells grew in eluate from mineralized hydrogels. Gallotannin incorporation impeded cell growth on hydrogels and did not impart antibacterial activity. In conclusion, gallotannin incorporation aided mineralization but reduced cytocompatibility.

Coculturing Bacteria Leads to Reduced Phenotypic Heterogeneities.

Isogenic bacterial populations are known to exhibit phenotypic heterogeneity at the single-cell level. Because of difficulties in assessing the phenotypic heterogeneity of a single taxon in a mixed community, the importance of this deeper level of organization remains relatively unknown for natural communities. In this study, we have used membrane-based microcosms that allow the probing of the phenotypic heterogeneity of a single taxon while interacting with a synthetic or natural community. Individual taxa were studied under axenic conditions, as members of a coculture with physical separation, and as a mixed culture. Phenotypic heterogeneity was assessed through both flow cytometry and Raman spectroscopy. Using this setup, we investigated the effect of microbial interactions on the individual phenotypic heterogeneities of two interacting drinking water isolates. Through flow cytometry we have demonstrated that interactions between these bacteria lead to a reduction of their individual phenotypic diversities and that this adjustment is conditional on the bacterial taxon. Single-cell Raman spectroscopy confirmed a taxon-dependent phenotypic shift due to the interaction. In conclusion, our data suggest that bacterial interactions may be a general driver of phenotypic heterogeneity in mixed microbial populations.IMPORTANCE Laboratory studies have shown the impact of phenotypic heterogeneity on the survival and functionality of isogenic populations. Because phenotypic heterogeneity plays an important role in pathogenicity and virulence, antibiotic resistance, biotechnological applications, and ecosystem properties, it is crucial to understand its influencing factors. An unanswered question is whether bacteria in mixed communities influence the phenotypic heterogeneity of their community partners. We found that coculturing bacteria leads to a reduction in their individual phenotypic heterogeneities, which led us to the hypothesis that the individual phenotypic diversity of a taxon is dependent on the community composition.

High index contrast photonic platforms for on-chip Raman spectroscopy.

Nanophotonic waveguide enhanced Raman spectroscopy (NWERS) is a sensing technique that uses a highly confined waveguide mode to excite and collect the Raman scattered signal from molecules in close vicinity of the waveguide. The most important parameters defining the figure of merit of an NWERS sensor include its ability to collect the Raman signal from an analyte, i.e. "the Raman conversion efficiency" and the amount of "Raman background" generated from the guiding material. Here, we compare different photonic integrated circuit (PIC) platforms capable of on-chip Raman sensing in terms of the aforementioned parameters. Among the four photonic platforms under study, tantalum oxide and silicon nitride waveguides exhibit high signal collection efficiency and low Raman background. In contrast, the performance of titania and alumina waveguides suffers from a strong Raman background and a weak signal collection efficiency, respectively.

Hybrids of Polymeric Capsules, Lipids, and Nanoparticles: Thermodynamics and Temperature Rise at the Nanoscale and Emerging Applications.

The importance of thermodynamics does not need to be emphasized. Indeed, elevated temperature processes govern not only industrial scale production but also self-assembly, chemical reaction, interaction between molecules, etc. Not surprisingly, biological processes typically take place at a specific temperature. Here, we look at possibilities to raise the localized temperature by a laser around noble-metal nanoparticles incorporated into shells of layer-by-layer polyelectrolyte microcapsules-freely suspended delivery vehicles in an aqueous solution, developed in the Department of Interfaces, Max Planck Institute of Colloids and Interfaces, headed by Helmuth Möhwald. Understanding the mechanisms of localized temperature rise is essential, that is why we analyze the influence of incident intensity, nanoparticle size, their distribution and aggregation state, as well as thermodynamics at the nanoscale. This leads us to scrutinize "global" (used for thermal encapsulation) versus "local" (used for release of encapsulated materials) temperature rise. Similar analysis is extended to planar polymeric coatings, the lipid membrane system of vesicles and cells, on which nanoparticles are adsorbed. Insights are provided into the mechanisms of physicochemical and biological effects, the nature of which has always been profoundly, interactively, and engagingly discussed in the Department of Interfaces. This analysis is combined with recent developments providing outlook and highlighting a broad range of emerging applications.

Controlled Deposition of Nanosize and Microsize Particles by Spin-Casting.

The deposition of nanosize and microsize spherical particles on planar solid substrates by hydrodynamic-evaporative spin-casting is studied. The particles are dispersed in a volatile liquid, which evaporates during the process, and the particles are finally deposited on the substrate. Their coverage, Γ, depends on the processing parameters (concentration by weight, particles size, etc.). The behavior of the particles during the spin-casting process and their final Γ values are investigated. It is found that for up to particle diameters of a few micrometers, particle deposition can be described by a theoretical approach developed for the spin-casting of polymer solutions (Karpitschka, S.; Weber, C. M.; Riegler, H. Chem. Eng. Sci. 2015, 129, 243-248. Danglad-Flores, J.; Eickelmann, S.; Riegler, H. Chem. Eng. Sci. 2018, 179, 257-264). For large particles, this basic theory fails. The causes of this failure are analyzed, and a corrected, more general theoretical approach is presented. It takes into account particle size effects as well as particle sedimentation. In summary, we present new insights into the spin-cast process of particle dispersions, analyze the contributions affecting the final particle coverage, and present a theoretical approach which describes and explains the experimental findings.

Lactoferrin translocates to the nucleus of bovine rectal epithelial cells in the presence of Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a foodborne pathogen which causes illness in humans. Ruminants are the main reservoirs and EHEC predominantly colonizes the epithelium of the recto-anal junction of cattle. Immunosuppression by EHEC promotes re-infection of cattle. However, bovine lactoferrin (bLF) apparently can overrule the immunosuppression by inducing EHEC-specific IgA responses at the mucosal site. The IgA responses are significantly correlated with reduced EHEC shedding and the absence of colonization at the rectal mucosa following re-infection. Therefore, to examine the interaction between bLF and bovine rectal epithelial cells, we first developed a method to establish a primary cell culture of epithelial cells of the rectum of cattle. Furthermore, we used LC-MS/MS to demonstrate the presence of secreted lactoferrin in bovine milk and the absence of a "delta" isoform which is known to translocate to the nucleus of cells. Nevertheless, lactoferrin derived from bovine milk was internalized by rectal epithelial cells and translocated to the nuclei. Moreover, nuclear translocation of bLF was significantly enhanced when the epithelial cells were inoculated with EHEC, as demonstrated by confocal fluorescence microscopy and confirmed by Raman microscopy and 3D imaging.

Whey Protein Complexes with Green Tea Polyphenols: Antimicrobial, Osteoblast-Stimulatory, and Antioxidant Activities.

Polyphenols are known for their antimicrobial activity, whilst both polyphenols and the globular protein ß-lactoglobulin (bLG) are suggested to have antioxidant properties and promote cell proliferation. These are potentially useful properties for a tissue-engineered construct, though it is unknown if they are retained when both compounds are used in combination. In this study, a range of different microbes and an osteoblast-like cell line (human fetal osteoblast, hFOB) were used to assess the combined effect of: (1) green tea extract (GTE), rich in the polyphenol epigallocatechin gallate (EGCG), and (2) whey protein isolate (WPI), rich in bLG. It was shown that approximately 20-48% of the EGCG in GTE reacted with WPI. GTE inhibited the growth of Gram-positive bacteria, an effect which was potentiated by the addition of WPI. GTE alone also significantly inhibited the growth of hFOB cells after 1, 4, and 7 days of culture. Alternatively, WPI significantly promoted hFOB cell growth in the absence of GTE and attenuated the effect of GTE at low concentrations (64 µg/mL) after 4 and 7 days. Low concentrations of WPI (50 µg/mL) also promoted the expression of the early osteogenic marker alkaline phosphatase (ALP) by hFOB cells, whereas GTE inhibited ALP activity. Therefore, the antioxidant effects of GTE can be boosted by WPI, but GTE is not suitable to be used as part of a tissue-engineered construct due to its cytotoxic effects which negate any positive effect WPI has on cell proliferation.

Application of whey protein isolate in bone regeneration: Effects on growth and osteogenic differentiation of bone-forming cells.

Recently, milk-derived proteins have attracted attention for applications in the biomedical field such as tissue regeneration. Whey protein isolate (WPI), especially its main component ß-lactoglobulin, can modulate immunity and acts as an antioxidant, antitumor, antiviral, and antibacterial agent. There are very few reports of the application of WPI in tissue engineering, especially in bone tissue engineering. In this study, we tested the influence of different concentrations of WPI on behavior of human osteoblast-like Saos-2 cells, human adipose tissue-derived stem cells (ASC), and human neonatal dermal fibroblasts (FIB). The positive effect on growth was apparent for Saos-2 cells and FIB but not for ASC. However, the expression of markers characteristic for early osteogenic cell differentiation [type-I collagen (COL1) and alkaline phosphatase (ALP)] as well as ALP activity, increased dose-dependently in ASC. Importantly, Saos-2 cells were able to deposit calcium in the presence of WPI, even in a proliferation medium without other supplements that support osteogenic cell differentiation. The results indicate that, depending on the cell type, WPI can act as an enhancer of cell proliferation and osteogenic differentiation. Therefore, enrichment of biomaterials for bone regeneration with WPI seems a promising approach, especially due to the low cost of WPI.

On-chip surface-enhanced Raman spectroscopy using nanosphere-lithography patterned antennas on silicon nitride waveguides.

A hybrid integration of nanoplasmonic antennas with silicon nitride waveguides enables miniaturized chips for surface-enhanced Raman spectroscopy at visible and near-infrared wavelengths. This integration can result in high-throughput SERS assays on low sampling volumes. However, current fabrication methods are complex and rely on electron-beam lithography, thereby obstructing the full use of an integrated photonics platform. Here, we demonstrate the electron-beam-free fabrication of gold nanotriangles on deep-UV patterned silicon nitride waveguides using nanosphere lithography. The localized surface-plasmon resonance of these nanotriangles is optimized for Raman excitation at 785 nm, resulting in a SERS substrate enhancement factor of 2.5 × 105. Furthermore, the SERS signal excited and collected through the waveguide is as strong as the free-space excited and collected signal through a high NA objective.

Superresolution 4π Raman microscopy.

The advent of 4π microscopy broke the conventional optical resolution limit in the axial direction of the microscope. In combination with fluorescence microscopy, it broadened the knowledge of cell biology at the expense of perturbing the samples with extrinsic fluorescent labels. In contrast, Raman microscopy acquires the molecular fingerprint of the sample without the need of extrinsic labels, and therefore improving its resolution can make an even greater impact. Here, we take advantage of the improved axial resolution of a 4π configuration to form a 4π Raman microscope. With this microscope, we independently and simultaneously analyzed different nanolayers in a multilayer stack. We identified their chemical composition and retrieved their relative subwavelength optical separation with a precision of 6 nm.

Gold nanodome SERS platform for label-free detection of protease activity.

Surface-enhanced Raman scattering provides a promising technology for sensitive and selective detection of protease activity by monitoring peptide cleavage. Not only are peptides and plasmonic hotspots similarly sized, Raman fingerprints also hold large potential for spectral multiplexing. Here, we use a gold-nanodome platform for real-time detection of trypsin activity on a CALNNYGGGGVRGNF substrate peptide. First, we investigate the spectral changes upon cleavage through the SERS signal of liquid-chromatography separated products. Next, we show that similar patterns are detected upon digesting surface-bound peptides. We demonstrate that the relative intensity of the fingerprints from aromatic amino acids before and after the cleavage site provides a robust figure of merit for the turnover rate. The presented method offers a generic approach for measuring protease activity, which is illustrated by developing an analogous substrate for endoproteinase Glu-C.

Identification of Individual Exosome-Like Vesicles by Surface Enhanced Raman Spectroscopy.

Exosome-like vesicles (ELVs) are a novel class of biomarkers that are receiving a lot of attention for the detection of cancer at an early stage. In this study the feasibility of using a surface enhanced Raman spectroscopy (SERS) based method to distinguish between ELVs derived from different cellular origins is evaluated. A gold nanoparticle based shell is deposited on the surface of ELVs derived from cancerous and healthy cells, which enhances the Raman signal while maintaining a colloidal suspension of individual vesicles. This nanocoating allows the recording of SERS spectra from single vesicles. By using partial least squares discriminant analysis on the obtained spectra, vesicles from different origin can be distinguished, even when present in the same mixture. This proof-of-concept study paves the way for noninvasive (cancer) diagnostic tools based on exosomal SERS fingerprinting in combination with multivariate statistical analysis.

From Beetles in Nature to the Laboratory: Actuating Underwater Locomotion on Hydrophobic Surfaces.

The controlled wetting and dewetting of surfaces is a primary mechanism used by beetles in nature, such as the ladybird and the leaf beetle for underwater locomotion.1 Their adhesion to surfaces underwater is enabled through the attachment of bubbles trapped in their setae-covered legs. Locomotion, however, is performed by applying mechanical forces in order to move, attach, and detach the bubbles in a controlled manner. Under synthetic conditions, however, when a bubble is bound to a surface, it is nearly impossible to maneuver without the use of external stimuli. Thus, actuated wetting and dewetting of surfaces remain challenges. Here, electrowetting-on-dielectric (EWOD) is used for the manipulation of bubble-particle complexes on unpatterned surfaces. Bubbles nucleate on catalytic Janus disks adjacent to a hydrophobic surface. By changing the wettability of the surface through electrowetting, the bubbles show a variety of reactions, depending on the shape and periodicity of the electrical signal. Time-resolved (µs) imaging of bubble radial oscillations reveals possible mechanisms for the lateral mobility of bubbles on a surface under electrowetting: bubble instability is induced when electric pulses are carefully adjusted. This instability is used to control the surface-bound bubble locomotion and is described in terms of the change in surface energy. It is shown that a deterministic force applied normal can lead to a random walk of micrometer-sized bubbles by exploiting the phenomenon of contact angle hysteresis. Finally, bubble use in nature for underwater locomotion and the actuated bubble locomotion presented in this study are compared.