Evaluation of Digitally Encoded Layer‐by‐layer Coated Microparticles as Cell Carriers

To obtain more biologically relevant data there is a growing interest in the use of living cells for assaying the biological activity of unknown chemical compounds. Density ‘multiplex’ cell‐based assays, where different cell types are mixed in one well and simultaneously investigated upon exposure to a certain compound are beginning to emerge. To be able to identify the cells they should be attached to microscopic carriers that are encoded. This paper investigates how digitally encoded microparticles can be loaded with cells while keeping the digital code in the microcarriers readable. It turns out that coating the surface of the encoded microcarriers with polyelectrolytes using the layer‐by‐layer (LbL) approach provides the microcarriers with a ‘highly functional’ surface. The polyelectrolyte layer allows the growth of the cells, allows the orientation of the cell loaded microcarriers in a magnetic field, and does not hamper the reading of the code. It has further been shown that the cells growing on the polyelectrolyte layer can become transduced by adenoviral particles hosted by the polyelectrolyte layer. It is concluded that the digitally encoded microparticles are promising materials for use in biomedical and pharmaceutical in‐vitro research where cells are used as tools.     

Porous Nanofilm Biomaterials Via Templated Layer‐by‐Layer Assembly

Hydrogel‐like biomaterials are often too soft to support robust cell adhesion, yet methods to increase mechanical rigidity (e.g., covalent cross‐linking the gel matrix) can compromise bioactivity by suppressing the accessibility or activity of embedded biomolecules. Nanoparticle templating is reported here as a strategy toward porous, layer‐by‐layer assembled, thin polyelectrolyte films of sufficient mechanical rigidity to promote strong initial cell adhesion, and that are capable of high bioactive species loading. Latex nanoparticles are incorporated during layer‐by‐layer assembly, and following 1‐ethyl‐3‐[3‐dimethylaminopropyl]carbodiimide/N‐hydroxysulfosuccinimide (EDC‐NHS) cross‐linking of the polyelectrolyte film, are removed via exposure to tetrahydrofuran (THF). THF exposure results in only a partial reduction in film thickness (as observed by ellipsometry), suggesting the presence of internal pore space. The attachment, spreading, and metabolic activity of pre‐osteoblastic MC3T3‐E1 cells cultured on templated, cross‐linked films are statistically similar to those on non‐templated films, and much greater than those on non‐cross‐linked films. Laser scanning confocal microscopy and quartz crystal microgravimetry indicate a high capacity for bioactive species loading (ca. 10% of film mass) in nanoparticle templated films. Porous nanofilm biomaterials, formed via layer‐by‐layer assembly with nanoparticle templating, promote robust cell adhesion and exhibit high bioactive species loading, and thus appear to be excellent candidates for cell‐contacting applications.     

Layer‐by‐Layer Controlled Perovskite Nanocomposite Thin Films for Piezoelectric Nanogenerators

Perovskite nanoparticle‐based nanocomposite thin films strictly tailored using unconventional layer‐by‐layer (LbL) assembly in organic media for piezoelectric nanogenerators (NGs) are demonstrated. By employing sub‐20‐nm BaTiO₃ nanoparticles stabilized by oleic acid ligands (i.e., OA‐BTO_NPs) and carboxylic acid (COOH)‐functionalized polymers, such as poly(acrylic acid) (PAA), the resulting OA‐BTO_NP/PAA nanocomposite multilayers are prepared by exploiting the high affinity between the COOH groups of PAA and the BTO_NPs. The ferroelectric and piezoelectric performance of the (PAA/OA‐BTO_NP)_n thin films can be precisely controlled by altering the bilayer number, inserted polymer type, and OA‐BTO_NP size. It is found that the LbL assembly in nonpolar solvent media can effectively increase the quantity of adsorbed OA‐BTO_NPs, resulting in the dramatic enhancement of electric power output from the piezoelectric NGs. Furthermore, very low leakage currents are detected from the (PAA/OA‐BTO_NP)_n thin films for obtaining highly reliable power‐generating performance of piezoelectric NGs.     

A Hybrid Carrier System Based on Origami Nanostrucutures and Layer‐by‐Layer Microparticles

Recent progress in DNA nanotechnology allows the fabrication of 3D structures that can be loaded with a large variety of molecular cargos and even be responsive to external stimuli. This makes the use of DNA nanostructures a promising approach for applications in nanomedicine and drug delivery. However, their low stability in the extra‐ and intracellular environment as well as low cellular uptake rates and release rates from endosomes into the cytoplasm hamper the efficient and targeted use of DNA nanostructures in medical applications. Here, such major obstacles are overcome by integrating DNA origami nanostructures into superordinated layer‐by‐layer based microparticles made from biopolymers. The modular assembly of the polymer layer allows a high‐density incorporation of the DNA structures at different depth. This enables controllable protection of the DNA nanostructures over extended durations in a broad range of extra‐ and intracellular conditions without compromising the cell viability. Furthermore, by producing protein‐complexed DNA nanostructures it is demonstrated that molecular cargo can be conveniently integrated into the developed hybrid system. This work provides the basis for a new multistage carrier system allowing for an efficient and protected transport of active agents inside responsive DNA nanostructures.     

Layer‐By‐Layer Coating of Aminocellulose and Quorum Quenching Acylase on Silver Nanoparticles Synergistically Eradicate Bacteria and Their Biofilms

The emergence of antibiotic‐resistant bacteria and the failure of the existing antibacterial therapeutics call for development of novel treatment strategies. Furthermore, the formation of bacterial biofilms restricts drug penetration and efficiency, causing life‐threatening infections. Bacterial attachment and biofilm formation are regulated by the cell‐to‐cell communication phenomenon called quorum sensing (QS). In this work, antimicrobial silver nanoparticles (AgNPs) are decorated in a layer‐by‐layer fashion with the oppositely charged aminocellulose (AM) and acylase to generate hybrid nanoentities with enhanced antibacterial and antibiofilm activities as well as reduced cytotoxicity. Acylase, a quorum‐quenching enzyme that degrades the QS signals in the extracellular environment of bacteria, disrupts the bacterial QS process and together with the bactericidal AM synergistically lowers fourfold the minimum inhibitory concentration of the AgNPs templates toward Gram‐negative Pseudomonas aeruginosa (P. aeruginosa). The hybrid nanoparticles in eightfold‐lower concentration than the AgNPs inhibit 45% of the QS‐regulated virulence factors produced by the reporter Chromobacterium violaceum bacterial strain and reduce by 100% the P. aeruginosa biofilm formation. Moreover, the sequential deposition of antibacterial/antibiofilm active and biocompatible biopolymers onto the AgNPs allows the engineering of safe nanomaterials that do not affect the viability of human cells.     

Biomaterials by Design: Layer‐By‐Layer Assembled Ion‐Selective and Biocompatible Films of TiO2 Nanoshells for Neurochemical Monitoring

Titania nanoshells with an external diameter of 10–30 nm and a wall thickness of 3–5 nm were prepared by dissolving the silver cores of Ag@TiO₂ nanoparticles in a concentrated solution of ammonium hydroxide. The nanoshells were assembled layer‐by‐layer (LBL), with negatively charged poly(acrylic acid) (PAA) to produce coatings with a network of voids and channels in the interior of the film. The diameter of the channels in the titania shells was comparable to the thickness of the electrical double layer in porous matter (0.3–30 nm). The prepared nanoparticulate films demonstrated strong ion‐sieving properties due to the exclusion of some ions from the diffuse region of the electrical double layer. The permeation of ions could be tuned effectively by the pH and ionic strength of a solution between “open” and “closed” states. The ion‐separation effect was utilized for the selective determination of one of the most important neurotransmitters, dopamine, on a background of ascorbic acid. Under physiological conditions, the negative charge on the surface of TiO₂ facilitated the permeation of positively charged dopamine through the LBL film to the electrode, preventing the access of the negatively charged ascorbic acid. The deposition of the nanoshell/polyelectrolyte film resulted in a significant improvement to the selectivity of dopamine determination. The prepared nanoshell films were also found to be compatible with nervous tissue secreting dopamine. Although the obtained data demonstrated the potential of TiO₂ LBL films for implantable biomedical devices for nerve tissue monitoring, the problem of electrode poisoning by the by‐products of dopamine reduction has yet to be resolved.     

Design Principle of Conjugated Polyelectrolytes to Make Them Water‐Soluble and Highly Emissive

The correlation between the molecular design of a conjugated polyelectrolyte (CPE) and its aggregated structure and the emissive properties in water is systematically investigated by means of UV–vis spectrometry, fluorescence spectroscopy, and scanning/transmission electron microscopy. Five different and rationally designed CPEs having carboxylic acid side chains are synthesized. All five conjugated polyelectrolytes are seemingly completely soluble in water in visual observation. However, their quantum yields are dramatically different, changing from 0.45 to 51.4%. Morphological analysis by electron microscopy combined with fluorescence spectrophotometry reveals that the CPEs form self‐assembled aggregates at the nanoscale depending on the nature of their side chains. The feature of the self‐assembled aggregates directly determines the emissive property of the CPEs. The nature and the length of the spacer between the carboxylic acid group and the CPE backbone have a strong influence on the quantum yield of the CPEs. Our study demonstrates that bulky and hydrophilic side chains and spacers are required to achieve complete water‐solubility and high quantum yield of CPEs in water, providing an important molecular design principle to develop functional CPEs.     

Surface Imprinting in Layer‐by‐Layer Nanostructured Films

In this Full Paper, we develop a novel approach for the generation of stable molecularly imprinted sites in polymeric films by combining the layer‐by‐layer (LbL) technique and photochemical crosslinking of the layered structure. After photo‐crosslinking, the imprinted films show high reproducibility and rapid loading and unloading of imprinted sites by the template molecules. Moreover, the competitive adsorption of template molecules and redox labels into the imprinted film using electrochemical methods indicates that the imprinted film has higher affinity for template molecules. We believe this approach may have some advantages over traditional ways of preparing imprinted sites in polymer matrices and it may open a new avenue for the functionalization of LbL films.     

High‐Performance Dopamine Sensors Based on Whole‐Graphene Solution‐Gated Transistors

Solution‐gated graphene transistors with graphene as both channel and gate electrodes are fabricated for the first time and used as dopamine sensors with the detection limit down to 1 nM, which is three orders of magnitude better than that of conventional electrochemical measurements. The sensing mechanism is attributed to the change of effective gate voltage applied on the transistors induced by the electro‐oxidation of dopamine at the graphene gate electrodes. The interference from glucose, uric acid, and ascorbic acid on the dopamine sensor is characterized. The selectivity of the dopamine sensor is dramatically improved by modifying the gate electrode with a thin Nafion film by solution process. This work paves the way for developing many other biosensors based on the solution‐gated graphene transistors by specifically functionalizing the gate electrodes. Because the devices are mainly made of graphene, they are potentially low cost and ideal for high‐density integration as multifunctional sensor arrays.     

Inverted Layer‐By‐Layer Fabrication of an Ultraflexible and Transparent Ag Nanowire/Conductive Polymer Composite Electrode for Use in High‐Performance Organic Solar Cells

A highly flexible and transparent conductive electrode based on consecutively stacked layers of conductive polymer (CP) and silver nanowires (AgNWs) fully embedded in a colorless polyimide (cPI) is achieved by utilizing an inverted layer‐by‐layer processing method. This CP‐AgNW composite electrode exhibits a high transparency of >92% at wavelengths of 450–700 nm and a low resistivity of 7.7 Ω ?^?1, while its ultrasmooth surface provides a large contact area for conductive pathways. Furthermore, it demonstrates an unprecedentedly high flexibility and good mechanical durability during both outward and inward bending to a radius of 40 μm. Subsequent application of this composite electrode in organic solar cells achieves power conversion efficiencies as high as 7.42%, which represents a significant improvement over simply embedding AgNWs in cPI. This is attributed to a reduction in bimolecular recombination and an increased charge collection efficiency, resulting in performance comparable to that of indium tin oxide‐based devices. More importantly, the high mechanical stability means that only a very slight reduction in efficiency is observed with bending (<5%) to a radius of 40 μm. This newly developed composite electrode is therefore expected to be directly applicable to a wide range of high‐performance, low‐cost flexible electronic devices.     

Biocatalytic Nanoscale Coatings Through Biomimetic Layer‐by‐Layer Mineralization

Recent insight into the molecular mechanisms of biological mineral formation (biomineralization) has enabled biomimetic approaches for the synthesis of functional organic‐inorganic hybrid materials under mild reaction conditions. Here we describe a novel method for enzyme immobilization in thin (nanoscale) conformal mineral coatings using biomimetic layer‐by‐layer (LbL) mineralization. The method utilizes a multifunctional molecule comprised of a naturally‐occurring peptide, protamine (PA), covalently bound to the redox enzyme Glucose oxidase (GOx). PA mimics the mineralizing properties of biomolecules involved in silica biomineralization in diatoms, and its covalent attachment to GOx does not interfere with the catalytic activity. Highly efficient and stable incorporation of this modified enzyme (GOx‐PA) into nanoscale layers (～5–7 nm thickness) of Ti‐O and Si‐O is accomplished during protamine‐enabled LbL mineralization on silica spheres. Depending on the layer location of the enzyme and the type of mineral (silica or titania) within which the enzyme is incorporated, the resulting multilayer biocatalytic hybrid materials exhibit between 20–100% of the activity of the free enzyme in solution. Analyses of kinetic properties (V_max, K_M) of the immobilized enzyme, coupled with characterization of physical properties of the mineral‐bearing layers (thickness, porosity, pore size distribution), indicates that the catalytic activities of the synthesized hybrid nanoscale coatings are largely determined by substrate diffusion rather than enzyme functionality. The GOx‐PA immobilized in these nanoscale layers is substantially stabilized against heat‐induced denaturation and largely protected from proteolytic attack. The method for enzyme immobilization described here enables, for the first time, the high yield immobilization and stabilization of enzymes within continuous, conformal, and nanoscale coatings through biomimetic LbL mineralization. This approach will likely be applicable to a wide variety of surfaces and functional biomolecules. The ability to synthesize thin (nanoscale) conformal enzyme‐loaded layers is of interest for numerous applications, including enzyme‐based biofuel cells and biosensors.     

Tubular Titania Nanostructures via Layer‐by‐Layer Self‐Assembly

Nanostructured titania‐polyelectrolyte composite and pure anatase and rutile titania tubes were successfully prepared by layer‐by‐layer (LbL) deposition of a water‐soluble titania precursor, titanium(IV ) bis(ammonium lactato) dihydroxide (TALH) and the oppositely charged poly(ethylenimine) (PEI) to form multilayer films. The tube structure was produced by depositing inside the cylindrical pores of a polycarbonate (PC) membrane template, followed by calcination at various temperatures. The morphology, structure and crystal phase of the titania tubes were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X‐ray diffraction (XRD) and UV‐vis absorbance measurements. The as‐prepared anatase titania tubes exhibit very promising photocatalytic properties, demonstrated by the degradation of the azodye methyl orange (MO) as a model molecule. They are also easily separated from the reaction system by simple filtration or centrifugation, allowing for straightforward recycling. The reported strategy provides a simple and versatile technique to fabricate titania based tubular nanostructures, which could easily be extended to prepare tubular structures of other materials and may find application in catalysis, chemical sensing, and nanodevices.     

Cationic Polyfluorenes with Phosphorescent Iridium(III) Complexes for Time‐Resolved Luminescent Biosensing and Fluorescence Lifetime Imaging

The application of a time‐resolved photoluminescence technique and fluorescence lifetime imaging microscopy for biosensing and bioimaging based on phosphorescent conjugated polyelectrolytes (PCPEs) containing Ir(III) complexes and polyfluorene units is reported. The specially designed PCPEs form 50 nm nanoparticles with blue fluorescence in aqueous solutions. Electrostatic interaction between the nanoparticles and heparin improves the energy transfer between the polyfluorene units to Ir(III) complex, which lights up the red signal for naked‐eye sensing. Good selectivity has been demonstrated for heparin sensing in aqueous solution and serum with quantification ranges of 0–70 μM and 0–5 μM, respectively. The signal‐to‐noise ratio can be further improved through time‐resolved emission spectra, especially when the detection is conducted in complicated environment, e.g., in the presence of fluorescent dyes. In addition to heparin sensing, the PCPEs have also been used for specific labeling of live KB cell membrane with high contrast using both confocal fluorescent cellular imaging and fluorescence lifetime imaging microscopies. This study provides a new perspective for designing promising CPEs for biosensing and bioimaging applications.     

Spray Layer‐by‐Layer Electrospun Composite Proton Exchange Membranes

Polymer electrolyte films are deposited onto highly porous electrospun mats using layer‐by‐layer (LbL) processing to fabricate composite proton conducting membranes. By simply changing the assembly conditions for generation of the LbL film on the nanofiber mat substrate, three different and unique composite film morphologies can be achieved in which the electrospun mats provide mechanical support; the LbL assembly produces highly conductive films that coat the mats in a controlled fashion, separately providing the ionic conductivity and fuel blocking characteristics of the composite membrane. Coating an electrospun mat with the LbL dipping process produces composite membranes with “webbed” morphologies that link the fibers in‐plane and give the composite membrane in‐plane proton conductivities similar to that of the pristine LbL system. In contrast, coating an electrospun mat using the spray‐LbL process without vacuum produces a uniform film that bridges across all of the pores of the mat. These membranes have methanol permeability similar to free‐standing poly(diallyl dimethyl ammonium chloride)/sulfonated poly(2,6‐dimethyl 1,4‐phenylene oxide) (PDAC/sPPO) thin films. Coating an electrospun mat with the vacuum‐assisted spray‐LbL process produces composite membranes with conformally coated fibers throughout the bulk of the mat with nanometer control of the coating thickness on each fiber. The mechanical properties of the LbL‐coated mats display composite properties, exhibiting the strength of the glassy PDAC/sPPO films when dry and the properties of the underlying electrospun polyamide mat when hydrated. By combining the different spray‐LbL fabrication techniques with electrospun fiber supports and tuning the parameters, mechanically stable membranes with high selectivity can be produced, potentially for use in fuel cell applications.     

Layer‐by‐Layer Assembly of Light‐Responsive Polymeric Multilayer Systems

Layer‐by‐Layer (LbL) assembly is a simple and highly versatile method to modify surfaces and fabricate robust and highly‐ordered nanostructured coatings over almost any type of substrate. Such versatility enables the incorporation of a plethora of building blocks, including materials exhibiting switchable properties, in a single device through a multitude of complementary intermolecular interactions. Switchable materials may undergo reversible physicochemical changes in response to a variety of external triggers. Although most of the works in the literature have been focusing on stimuli‐responsive materials that are sensitive to common triggers such as pH, ionic strength, or temperature, much less has been discussed on LbL systems which are sensitive to non‐invasive and easily controlled light stimulus, despite its unique potential. This review provides a deep overview of the recent progresses achieved in the design and fabrication of light‐responsive LbL polymeric multilayer systems, their potential future challenges and opportunities, and possible applications. Many examples are given on light‐responsive polymeric multilayer assemblies built from metal nanoparticles, functional dyes, and metal oxides. Such stimuli‐responsive functional materials, and combinations among them, may lead to novel and highly promising nanostructured smart functional systems well‐suited for a wide range of research fields, including biomedicine and biotechnology.     

Freely Suspended Layer‐by‐Layer Nanomembranes: Testing Micromechanical Properties

Freely suspended nanocomposite layer‐by‐layer (LbL) nanomembranes composed of a central layer of gold nanoparticles sandwiched between polyelectrolyte multilayers are fabricated via spin‐assisted LbL assembly. The diameter of the circular membranes is varied from 150 to 600 μm and the thickness is kept within the range of 25–70 nm. The micro‐ and nanomechanical properties of these membranes are studied using a combination of resonance‐frequency and bulging tests, and point‐load nanodeflection experiments. Our results suggest that these freely suspended nanomembranes, with a Young's modulus of 5–10 GPa are very robust and can sustain multiple significant deformations. They are very sensitive to minor variations in pressure, surpassing ordinary semiconductor and metal membranes by three to four orders of magnitude and therefore have potential applications as pressure and acoustic microsensors.     

Lubricant‐Infused Nanoparticulate Coatings Assembled by Layer‐by‐Layer Deposition

Omniphobic coatings are designed to repel a wide range of liquids without leaving stains on the surface. A practical coating should exhibit stable repellency, show no interference with color or transparency of the underlying substrate and, ideally, be deposited in a simple process on arbitrarily shaped surfaces. We use layer‐by‐layer (LbL) deposition of negatively charged silica nanoparticles and positively charged polyelectrolytes to create nanoscale surface structures that are further surface‐functionalized with fluorinated silanes and infiltrated with fluorinated oil, forming a smooth, highly repellent coating on surfaces of different materials and shapes. We show that four or more LbL cycles introduce sufficient surface roughness to effectively immobilize the lubricant into the nanoporous coating and provide a stable liquid interface that repels water, low‐surface‐tension liquids and complex fluids. The absence of hierarchical structures and the small size of the silica nanoparticles enables complete transparency of the coating, with light transmittance exceeding that of normal glass. The coating is mechanically robust, maintains its repellency after exposure to continuous flow for several days and prevents adsorption of streptavidin as a model protein. The LbL process is conceptually simple, of low cost, environmentally benign, scalable, automatable and therefore may present an efficient synthetic route to non‐fouling materials.     

Layer‐by‐Layer Growth of Multicomponent Colloidal Crystals Over Large Areas

Self‐assembly of different sized colloidal particles into multicomponent crystals results in novel material properties compared to the properties of the individual components alone. The formation of binary and, for the first time, ternary colloidal crystals through a simple and inexpensive confined‐area evaporation‐induced layer‐by‐layer (LBL) assembly method is reported. The proposed method produces high quality multicomponent colloidal crystal films over a broad range of particle size‐ratios and large surface areas (cm²) from silica/polystyrene colloidal suspensions of low concentration. By adjusting the size‐ratio and concentration of the colloidal particles, complex crystals of tunable stoichiometries are fabricated and their structural characteristics are further confirmed with reported crystal analogues. In addition, complex structures form as a result of the interplay of the template layer effect, the surface forces exerted by the meniscus of the drying liquid, the space filling principle, and entropic forces. Thus, this LBL approach is a versatile way to grow colloidal crystals with binary, ternary, or more complex structures.     

Layer‐by‐Layer Assembly of Functional Nanoparticles for Hepatocellular Carcinoma Therapy

Cancer treatments with conventional approaches often result in limited clinical outcomes due to inefficient therapeutic efficacy and cumulative toxicity against normal tissue. Recently, most research has focused on combined therapeutic studies by functional carriers. In this study, functional nanoparticles (FNPs) are assembled in a layer‐by‐layer fashion. FNPs are loaded with two drugs (10‐hydroxycamptothecin and apoptin plasmid) with dual hepatocellular carcinoma‐targeting ligands (lactobionic acid and biotin) on the surface. Cytotoxicity studies and acute toxicity experiments in BAL b/c mice show that blank FNPs demonstrate good biocompatibility. Flow cytometry analysis and cytotoxicity studies demonstrate that the dual‐targeting FNPs allow for better specificity and selectivity of the tumor mass. FNPs can escape from endosomal/lysosomal compartments effectively, as is demonstrated using the Cell Navigator lysosome staining kit. When the drugs are released into the cytosol, the nuclear localization signal can enhance the nuclear delivery of 10‐hydroxycamptothecin loaded carriers and apoptin plasmids, as is demonstrated by confocal laser scanning microscopy. In vivo experiments show the circulation time and tissue distribution of FNPs, which greatly improve the therapeutic efficacy of BAL b/c nude mice with subcutaneous tumors. Taken together, the results suggest that FNPs are a promising candidate for hepatocellular carcinoma therapy.