Scalable biomimetic sensing system with membrane receptor dual-monolayer probe and graphene transistor arrays.

Affinity-based biosensing can enable point-of-care diagnostics and continuous health monitoring, which commonly follows bottom-up approaches and is inherently constrained by bioprobes' intrinsic properties, batch-to-batch consistency, and stability in biofluids. We present a biomimetic top-down platform to circumvent such difficulties by combining a "dual-monolayer" biorecognition construct with graphene-based field-effect-transistor arrays. The construct adopts redesigned water-soluble membrane receptors as specific sensing units, positioned by two-dimensional crystalline S-layer proteins as dense antifouling linkers guiding their orientations. Hundreds of transistors provide statistical significance from transduced signals. System feasibility was demonstrated with rSbpA-ZZ/CXCR4QTY-Fc combination. Nature-like specific interactions were achieved toward CXCL12 ligand and HIV coat glycoprotein in physiologically relevant concentrations, without notable sensitivity loss in 100% human serum. The construct is regeneratable by acidic buffer, allowing device reuse and functional tuning. The modular and generalizable architecture behaves similarly to natural systems but gives electrical outputs, which enables fabrication of multiplex sensors with tailored receptor panels for designated diagnostic purposes.

Isolation and Characterization of Cell Envelope Fragments Comprising Archaeal S-Layer Proteins.

The outermost component of cell envelopes of most bacteria and almost all archaea comprise a protein lattice, which is termed Surface (S-)layer. The S-layer lattice constitutes a highly porous structure with regularly arranged pores in the nm-range. Some archaea thrive in extreme milieus, thus producing highly stable S-layer protein lattices that aid in protecting the organisms. In the present study, fragments of the cell envelope from the hyperthermophilic acidophilic archaeon Saccharolobus solfataricus P2 (SSO) have been isolated by two different methods and characterized. The organization of the fragments and the molecular sieving properties have been elucidated by transmission electron microscopy and by determining the retention efficiency of proteins varying in size, respectively. The porosity of the archaeal S-layer fragments was determined to be 45%. S-layer fragments of SSO showed a retention efficiency of up to 100% for proteins having a molecular mass of ≥ 66 kDa. Moreover, the extraction costs for SSO fragments have been reduced by more than 80% compared to conventional methods, which makes the use of these archaeal S-layer material economically attractive.

A New Method for Dispersing Pristine Carbon Nanotubes Using Regularly Arranged S-Layer Proteins.

Homogeneous and stable dispersions of functionalized carbon nanotubes (CNTs) in aqueous solutions are imperative for a wide range of applications, especially in life and medical sciences. Various covalent and non-covalent approaches were published to separate the bundles into individual tubes. In this context, this work demonstrates the non-covalent modification and dispersion of pristine multi-walled carbon nanotubes (MWNTs) using two S-layer proteins, namely, SbpA from Lysinibacillus sphaericus CCM2177 and SbsB from Geobacillus stearothermophilus PV72/p2. Both the S-layer proteins coated the MWNTs completely. Furthermore, it was shown that SbpA can form caps at the ends of MWNTs. Reassembly experiments involving a mixture of both S-layer proteins in the same solution showed that the MWNTs were primarily coated with SbsB, whereas SbpA formed self-assembled layers. The dispersibility of the pristine nanotubes coated with SbpA was determined by zeta potential measurements (-24.4 +/- 0.6 mV, pH = 7). Finally, the SbpA-coated MWNTs were silicified with tetramethoxysilane (TMOS) using a mild biogenic approach. As expected, the thickness of the silica layer could be controlled by the reaction time and was 6.3 +/- 1.25 nm after 5 min and 25.0 +/- 5.9 nm after 15 min. Since S-layer proteins have already demonstrated their capability to bind (bio)molecules in dense packing or to act as catalytic sites in biomineralization processes, the successful coating of pristine MWNTs has great potential in the development of new materials, such as biosensor architectures.

S-Layer Ultrafiltration Membranes.

Monomolecular arrays of protein subunits forming surface layers (S-layers) are the most common outermost cell envelope components of prokaryotic organisms (bacteria and archaea). Since S-layers are periodic structures, they exhibit identical physicochemical properties for each constituent molecular unit down to the sub-nanometer level. Pores passing through S-layers show identical size and morphology and are in the range of ultrafiltration membranes. The functional groups on the surface and in the pores of the S-layer protein lattice are accessible for chemical modifications and for binding functional molecules in very precise fashion. S-layer ultrafiltration membranes (SUMs) can be produced by depositing S-layer fragments as a coherent (multi)layer on microfiltration membranes. After inter- and intramolecular crosslinking of the composite structure, the chemical and thermal resistance of these membranes was shown to be comparable to polyamide membranes. Chemical modification and/or specific binding of differently sized molecules allow the tuning of the surface properties and molecular sieving characteristics of SUMs. SUMs can be utilized as matrices for the controlled immobilization of functional biomolecules (e.g., ligands, enzymes, antibodies, and antigens) as required for many applications (e.g., biosensors, diagnostics, enzyme- and affinity-membranes). Finally, SUM represent unique supporting structures for stabilizing functional lipid membranes at meso- and macroscopic scale.

Nanotechnology with S-layer Proteins.

Nanosciences are distinguished by the cross-fertilization of biology, chemistry, material sciences, and solid-state physics and, hence, open up a great variety of new opportunities for innovation. The technological utilization of self-assembly systems, wherein molecules spontaneously associate under equilibrium conditions into reproducible supramolecular structures, is one key challenge in nanosciences for life and non-life science applications. The attractiveness of such processes is due to their ability to build uniform, ultra-small functional units with predictable properties down to the nanometer scale. Moreover, newly developed techniques and methods open up the possibility to exploit these structures at meso- and macroscopic scale. An immense significance at innovative approaches for the self-assembly of supramolecular structures and devices with dimensions of a few to tens of nanometers constitutes the utilization of crystalline bacterial cell surface proteins. The latter have proven to be particularly suited as building blocks in a molecular construction kit comprising of all major classes of biological molecules. The controlled immobilization of biomolecules in an ordered fashion on solid substrates and their directed confinement in definite areas of nanometer dimensions are key requirements for many applications including the development of bioanalytical sensors, biochips, molecular electronics, biocompatible surfaces, and signal processing between functional membranes, cells, and integrated circuits.

CRISPR-mediated gene silencing reveals involvement of the archaeal S-layer in cell division and virus infection.

The S-layer is a proteinaceous surface lattice found in the cell envelope of bacteria and archaea. In most archaea, a glycosylated S-layer constitutes the sole cell wall and there is evidence that it contributes to cell shape maintenance and stress resilience. Here we use a gene-knockdown technology based on an endogenous CRISPR type III complex to gradually silence slaB, which encodes the S-layer membrane anchor in the hyperthermophilic archaeon Sulfolobus solfataricus. Silenced cells exhibit a reduced or peeled-off S-layer lattice, cell shape alterations and decreased surface glycosylation. These cells barely propagate but increase in diameter and DNA content, indicating impaired cell division; their phenotypes can be rescued through genetic complementation. Furthermore, S-layer depleted cells are less susceptible to infection with the virus SSV1. Our study highlights the usefulness of the CRISPR type III system for gene silencing in archaea, and supports that an intact S-layer is important for cell division and virus susceptibility.

Acoustic and hybrid 3D-printed electrochemical biosensors for the real-time immunodetection of liver cancer cells (HepG2).

This study presents an efficient acoustic and hybrid three-dimensional (3D)-printed electrochemical biosensors for the detection of liver cancer cells. The biosensors function by recognizing the highly expressed tumor marker CD133, which is located on the surface of liver cancer cells. Detection was achieved by recrystallizing a recombinant S-layer fusion protein (rSbpA/ZZ) on the surface of the sensors. The fused ZZ-domain enables immobilization of the anti-CD133 antibody in a defined manner. These highly accessible anti-CD133 antibodies were employed as a sensing layer, thereby enabling the efficient detection of liver cancer cells (HepG2). The recognition of HepG2 cells was investigated in situ using a quartz crystal microbalance with dissipation monitoring (QCM-D), which enabled the label-free, real-time detection of living cells on the modified sensor surface under controlled conditions. Furthermore, the hybrid 3D additive printing strategy for biosensors facilitates both rapid development and small-scale manufacturing. The hybrid strategy of combining 3D-printed parts and more traditionally fabricated parts enables the use of optimal materials: a ceramic substrate with noble metals for the sensing element and 3D-printed capillary channels to guide and constrain the clinical sample. Cyclic voltammetry (CV) measurements confirmed the efficiency of the fabricated sensors. Most importantly, these sensors offer low-cost and disposable detection platforms for real-world applications. Thus, as demonstrated in this study, both fabricated acoustic and electrochemical sensing platforms can detect cancer cells and therefore may have further potential in other clinical applications and drug-screening studies.

Impact of surface wettability on S-layer recrystallization: a real-time characterization by QCM-D.

Quartz crystal microbalance with dissipation monitoring (QCM-D) has been employed to study the assembly and recrystallization kinetics of isolated SbpA bacterial surface proteins onto silicon dioxide substrates of different surface wettability. Surface modification by UV/ozone oxidation or by vapor deposition of 1H,1H,2H,2H-perfluorododecyltrichlorosilane yielded hydrophilic or hydrophobic samples, respectively. Time evolution of frequency and dissipation factors, either individually or combined as the so-called Df plots, showed a much faster formation of crystalline coatings for hydrophobic samples, characterized by a phase-transition peak at around the 70% of the total mass adsorbed. This behavior has been proven to mimic, both in terms of kinetics and film assembly steps, the recrystallization taking place on an underlying secondary cell-wall polymer (SCWP) as found in bacteria. Complementary atomic force microscopy (AFM) experiments corroborate these findings and reveal the impact on the final structure achieved.

In Vitro Characterization of the Two-Stage Non-Classical Reassembly Pathway of S-Layers.

The recombinant bacterial surface layer (S-layer) protein rSbpA of Lysinibacillus sphaericus CCM 2177 is an ideal model system to study non-classical nucleation and growth of protein crystals at surfaces since the recrystallization process may be separated into two distinct steps: (i) adsorption of S-layer protein monomers on silicon surfaces is completed within 5 min and the amount of bound S-layer protein sufficient for the subsequent formation of a closed crystalline monolayer; (ii) the recrystallization process is triggered-after washing away the unbound S-layer protein-by the addition of a CaCl2 containing buffer solution, and completed after approximately 2 h. The entire self-assembly process including the formation of amorphous clusters, the subsequent transformation into crystalline monomolecular arrays, and finally crystal growth into extended lattices was investigated by quartz crystal microbalance with dissipation (QCM-D) and atomic force microscopy (AFM). Moreover, contact angle measurements showed that the surface properties of S-layers change from hydrophilic to hydrophobic as the crystallization proceeds. This two-step approach is new in basic and application driven S-layer research and, most likely, will have advantages for functionalizing surfaces (e.g., by spray-coating) with tailor-made biological sensing layers.

Inspired and stabilized by nature: ribosomal synthesis of the human voltage gated ion channel (VDAC) into 2D-protein-tethered lipid interfaces.

We present an elegant synthesis and reconstitution approach for functional studies of voltage responsive membrane proteins. For such studies, we propose a planar architecture of an S-layer-supported lipid membrane as a suitable matrix for presenting unmodified membrane protein species, and here we focus on the voltage-dependent anion channel (VDAC) from human mitochondria. The presented cell-free strategy, in which VDAC proteins are synthesized in bacterial cell lysate, into a membrane structure, offers a great advantage in the study of such subtle membrane proteins over the conventional, cell-based synthesis approach in terms of reproducibility. The material-assay combination is superior over cell-culture related synthesis and purification approaches as here we bypass by a one-step synthesis procedure the complex cell culture, and expression and purification endeavours, and, moreover, the protein of interest never has to be detergent solubilized and had been synthesized de novo. We provide here a detailed description from the all over procedure and our first results, describing in detail the cell-free synthesis and robustness of such a material-assay combination: functional VDAC protein species embedded in a planar membrane architecture and ready for electrochemical characterization.

Relevance of glycosylation of S-layer proteins for cell surface properties.

Elucidating the building principles and intrinsic features modulating certain water-associated processes (e.g., surface roughness in the nanometer scale, surface hydration and accompanied antifouling property, etc.) of surface structures from (micro)organisms is nowadays a highly challenging task in fields like microbiology, biomimetic engineering and (bio)material sciences. Here, we show for the first time the recrystallization of the wild-type S-layer glycoprotein wtSgsE from Geobacillus stearothermophilus NRS 2004/3a and its recombinantly produced non-glycosylated form, rSgsE, on gold sensor surfaces. Whereas the proteinaceous lattice of the S-layer proteins is forming a rigid layer on the sensor surface, the glycan chains are developing an overall soft, highly dissipative film. Interestingly, to the wtSgsE lattice almost twice the amount of water is bound and/or coupled in comparison with the non-glycosylated rSgsE with the preferred region being the extending glycan residues. The present results are discussed in terms of the effect of the glycan residues on the recrystallization, the adjoining hydration layer, and the nanoscale roughness and fluidic behavior. The latter features may turn out to be one of the most general ones among bacterial and archaeal S-layer lattices.

S-layer fusion protein as a tool functionalizing emulsomes and CurcuEmulsomes for antibody binding and targeting.

Selective targeting of tumor cells by nanoparticle-based drug delivery systems is highly desirable because it maximizes the drug concentration at the desired target while simultaneously protecting the surrounding healthy tissues. Here, we show a design for smart nanocarriers based on a biomimetic approach that utilizes the building principle of virus envelope structures. Emulsomes and CurcuEmulsomes comprising a tripalmitin solid core surrounded by phospholipid layers are modified by S-layer proteins that self-assemble into a two-dimensional array to form a surface layer. One significant advantage of this nanoformulation is that it increases the solubility of the lipophilic anti-cancer agent curcumin in the CurcuEmulsomes by a factor of 2700. In order to make the emulsomes specific for IgG, the S-layer protein is fused with two protein G domains. This S-layer fusion protein preserves its recrystallization characteristics, forming an ordered surface layer (square lattice with 13 nm unit-by-unit distance). The GG domains are presented in a predicted orientation and exhibit a selective binding affinity for IgG.

Emulsomes meet S-layer proteins: an emerging targeted drug delivery system.

Here, the use of emulsomes as a drug delivery system is reviewed and compared with other similar lipidic nanoformulations. In particular, we look at surface modification of emulsomes using S-layer proteins, which are self-assembling proteins that cover the surface of many prokaryotic organisms. It has been shown that covering emulsomes with a crystalline S-layer lattice can protect cells from oxidative stress and membrane damage. In the future, the capability to recrystallize S-layer fusion proteins on lipidic nanoformulations may allow the presentation of binding functions or homing protein domains to achieve highly specific targeted delivery of drug-loaded emulsomes. Besides the discussion on several designs and advantages of composite emulsomes, the success of emulsomes for the delivery of drugs to fight against viral and fungal infections, dermal therapy, cancer, and autoimmunity is summarized. Further research might lead to smart, biocompatible emulsomes, which are able to protect and reduce the side effects caused by the drug, but at the same time are equipped with specific targeting molecules to find the desired site of action.

Probing peptide and protein insertion in a biomimetic S-layer supported lipid membrane platform.

The most important aspect of synthetic lipid membrane architectures is their ability to study functional membrane-active peptides and membrane proteins in an environment close to nature. Here, we report on the generation and performance of a biomimetic platform, the S-layer supported lipid membrane (SsLM), to investigate the structural and electrical characteristics of the membrane-active peptide gramicidin and the transmembrane protein α-hemolysin in real-time using a quartz crystal microbalance with dissipation monitoring in combination with electrochemical impedance spectroscopy. A shift in membrane resistance is caused by the interaction of α-hemolysin and gramicidin with SsLMs, even if only an attachment onto, or functional channels through the lipid membrane, respectively, are formed. Moreover, the obtained results did not indicate the formation of functional α-hemolysin pores, but evidence for functional incorporation of gramicidin into this biomimetic architecture is provided.

Reassembly of S-layer proteins.

Crystalline bacterial cell surface layers (S-layers) represent the outermost cell envelope component in a broad range of bacteria and archaea. They are monomolecular arrays composed of a single protein or glycoprotein species and represent the simplest biological membranes developed during evolution. They are highly porous protein mesh works with unit cell sizes in the range of 3 to 30 nm, and pore sizes of 2 to 8 nm. S-layers are usually 5 to 20 nm thick (in archaea, up to 70 nm). S-layer proteins are one of the most abundant biopolymers on earth. One of their key features, and the focus of this review, is the intrinsic capability of isolated native and recombinant S-layer proteins to form self-assembled mono- or double layers in suspension, at solid supports, the air-water interface, planar lipid films, liposomes, nanocapsules, and nanoparticles. The reassembly is entropy-driven and a fascinating example of matrix assembly following a multistage, non-classical pathway in which the process of S-layer protein folding is directly linked with assembly into extended clusters. Moreover, basic research on the structure, synthesis, genetics, assembly, and function of S-layer proteins laid the foundation for their application in novel approaches in biotechnology, biomimetics, synthetic biology, and nanotechnology.

Biomimetic interfaces based on S-layer proteins, lipid membranes and functional biomolecules.

Designing and utilization of biomimetic membrane systems generated by bottom-up processes is a rapidly growing scientific and engineering field. Elucidation of the supramolecular construction principle of archaeal cell envelopes composed of S-layer stabilized lipid membranes led to new strategies for generating highly stable functional lipid membranes at meso- and macroscopic scale. In this review, we provide a state-of-the-art survey of how S-layer proteins, lipids and polymers may be used as basic building blocks for the assembly of S-layer-supported lipid membranes. These biomimetic membrane systems are distinguished by a nanopatterned fluidity, enhanced stability and longevity and, thus, provide a dedicated reconstitution matrix for membrane-active peptides and transmembrane proteins. Exciting areas in the (lab-on-a-) biochip technology are combining composite S-layer membrane systems involving specific membrane functions with the silicon world. Thus, it might become possible to create artificial noses or tongues, where many receptor proteins have to be exposed and read out simultaneously. Moreover, S-layer-coated liposomes and emulsomes copying virus envelopes constitute promising nanoformulations for the production of novel targeting, delivery, encapsulation and imaging systems.

The grab-and-drop protocol: a novel strategy for membrane protein isolation and reconstitution from single cells.

We present a rapid and robust technique for the sampling of membrane-associated proteins from the surface of a single, live cell and their subsequent deposition onto a solid-supported lipid bilayer. As a proof of principle, this method has been used to extract green fluorescent protein (EGFP) labelled K-ras proteins located at the inner leaflet of the plasma membrane of colon carcinoma cells and to transfer them to an S-layer supported lipid bilayer system. The technique is non-destructive, meaning that both the cell and proteins are intact after the sampling operation, offering the potential for repeated measurements of the same cell of interest. This system provides the ideal tool for the investigation of cellular heterogeneity, as well as a platform for the investigation of rare cell types such as circulating tumour cells.

S-layers: principles and applications.

Monomolecular arrays of protein or glycoprotein subunits forming surface layers (S-layers) are one of the most commonly observed prokaryotic cell envelope components. S-layers are generally the most abundantly expressed proteins, have been observed in species of nearly every taxonomical group of walled bacteria, and represent an almost universal feature of archaeal envelopes. The isoporous lattices completely covering the cell surface provide organisms with various selection advantages including functioning as protective coats, molecular sieves and ion traps, as structures involved in surface recognition and cell adhesion, and as antifouling layers. S-layers are also identified to contribute to virulence when present as a structural component of pathogens. In Archaea, most of which possess S-layers as exclusive wall component, they are involved in determining cell shape and cell division. Studies on structure, chemistry, genetics, assembly, function, and evolutionary relationship of S-layers revealed considerable application potential in (nano)biotechnology, biomimetics, biomedicine, and synthetic biology.

Fluorescent sensors based on bacterial fusion proteins.

Fluorescence proteins are widely used as markers for biomedical and technological purposes. Therefore, the aim of this project was to create a fluorescent sensor, based in the green and cyan fluorescent protein, using bacterial S-layers proteins as scaffold for the fluorescent tag. We report the cloning, expression and purification of three S-layer fluorescent proteins: SgsE-EGFP, SgsE-ECFP and SgsE-13aa-ECFP, this last containing a 13-amino acid rigid linker. The pH dependence of the fluorescence intensity of the S-layer fusion proteins, monitored by fluorescence spectroscopy, showed that the ECFP tag was more stable than EGFP. Furthermore, the fluorescent fusion proteins were reassembled on silica particles modified with cationic and anionic polyelectrolytes. Zeta potential measurements confirmed the particle coatings and indicated their colloidal stability. Flow cytometry and fluorescence microscopy showed that the fluorescence of the fusion proteins was pH dependent and sensitive to the underlying polyelectrolyte coating. This might suggest that the fluorescent tag is not completely exposed to the bulk media as an independent moiety. Finally, it was found out that viscosity enhanced the fluorescence intensity of the three fluorescent S-layer proteins.

Characterization of CurcuEmulsomes: nanoformulation for enhanced solubility and delivery of curcumin.

BACKGROUND: Curcumin is a polyphenolic compound isolated from the rhizomes of the plant Curcuma longa and shows intrinsic anti-cancer properties. Its medical use remains limited due to its extremely low water solubility and bioavailability. Addressing this problem, drug delivery systems accompanied by nanoparticle technology have emerged. The present study introduces a novel nanocarrier system, so-called CurcuEmulsomes, where curcumin is encapsulated inside the solid core of emulsomes. RESULTS: CurcuEmulsomes are spherical solid nanoparticles with an average size of 286 nm and a zeta potential of 37 mV. Encapsulation increases the bioavailability of curcumin by up to 10,000 fold corresponding to a concentration of 0.11 mg/mL. Uptaken by HepG2 human liver carcinoma cell line, CurcuEmulsomes show a significantly prolonged biological activity and demonstrated therapeutic efficacy comparable to free curcumin against HepG2 in vitro - with a delay in response, as assessed by cell viability, apoptosis and cell cycle studies. The delay is attributed to the solid character of the nanocarrier prolonging the release of curcumin inside the HepG2 cells. CONCLUSIONS: Incorporation of curcumin into emulsomes results in water-soluble and stable CurcuEmulsome nanoformulations. CurcuEmulsomes do not only successfully facilitate the delivery of curcumin into the cell in vitro, but also enable curcumin to reach its effective concentrations inside the cell. The enhanced solubility of curcumin and the promising in vitro efficacy of CurcuEmulsomes highlight the potential of the system for the delivery of lipophilic drugs. Moreover, high degree of compatibility, prolonged release profile and tailoring properties feature CurcuEmulsomes for further therapeutic applications in vivo.