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/CXCR4 ^QTY -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.

Bacterial surface layers (S-layers) have been observed as the outermost cell envelope component in a wide range of bacteria and most archaea. S-layers are monomolecular lattices composed of a single protein or glycoprotein species and have either oblique, square or hexagonal lattice symmetry with unit cell dimensions ranging from 3 to 30 nm. They are generally 5 to 10 nm thick (up to 70 nm in archaea) and represent highly porous protein lattices (30–70% porosity) with pores of uniform size and morphology in the range of 2 to 8 nm. Since S-layers can be considered as one of the simplest protein lattices found in nature and the constituent units are probably the most abundantly expressed proteins on earth, it seems justified to briefly review the different S-layer lattice types, the need for lattice imperfections and the discussion of S-layers from the perspective of an isoporous protein network in the ultrafiltration region. Finally, basic research on S-layers laid the foundation for applications in biotechnology, synthetic biology, and biomimetics.

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.

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.

Carbon nanotubes (CNTs) have already been considered for medical applications due to their small diameter and ability to penetrate cells and tissues. However, since CNTs are chemically inert and non-dispersible in water, they have to be chemically functionalized or coated with biomolecules to carry payloads or interact with the environment. Proteins, although often only randomly bound to the CNT surface, are preferred because they provide a better biocompatibility and present functional groups for binding additional molecules. A new approach to functionalize CNTs with a closed and precisely ordered protein layer is offered by bacterial surface layer (S-layer) proteins, which have already attracted much attention in the functionalization of surfaces. We could demonstrate that bacterial S-layer proteins (SbpA of Lysinibacillus sphaericus CCM 2177 and the recombinant fusion protein rSbpA_31-1068GG comprising the S-layer protein and two copies of the IgG binding region of Protein G) can be used to disperse and functionalize oxidized multi walled CNTs. Following a simple protocol, a complete surface coverage with a long-range crystalline S-layer lattice can be obtained. When rSbpA_31-1068GG was used for coating, the introduced functionality could be confirmed by binding gold labeled antibodies via the IgG binding domain of the fusion protein. Since a great variety of functional S-layer fusion proteins has already been described, our new technology has the potential for a broad spectrum of functionalized CNTs.

The critical role of divalent ions (M²⁺) in the self-assembly of SbpA S-layer proteins (from Lysinibacillus sphaericus CCM 2177) into crystalline structures has been reported in several studies. Hence, ions such as magnesium, barium, nickel and, most commonly, calcium (Ca²⁺) have proven to trigger both protein-protein and protein-substrate interactions involved in the two-stage non-classical pathway recrystallization followed by SbpA units. As a result, two dimensional, crystalline nanometric sheets in a highly ordered tetrameric state (p4) can be formed on top of different surfaces. The use of iron in its ferrous state (Fe²⁺) as self-assembly inducing candidate has been omitted so far due to its instability under aerobic conditions, tending to natural oxidation to the ferric (Fe³⁺) state. In this work, the potentiality of assembling fully functional S-layers from iron (II) salts (FeCl₂ and FeSO₄) is described for the first time. A combination of chemical (oxidation retardants) and electrical (−1 V potential) factors has been applied to effectively act against such an oxidizing trend. Formation of the respective crystalline films has been followed by means of Electrochemical Quartz Crystal Microbalance with Dissipation (EQCM-D) measurements and complementary Atomic Force Microscopy (AFM) topography studies, which prove the presence of squared lattice symmetry at the end of the recrystallization process. Both techniques, together with additional electrochemical tests performed over the ion permeability of both types of S-layer coatings formed, show the influence of the counterion chosen (chloride vs. sulphate) in the final packing and performance of the S-layer. The presence of an underlying Secondary Cell Wall Polymer (SCWP) as in the natural case contributes to pair both systems, due to the high lateral motility freedom provided by this biopolymer to SbpA units in comparison to uncoated substrates.