Method to determine the decolorization potential of persistent dyes by white rot fungi by colorimetric assays.

Decolorization assays allow to assess the ability of white rot fungi to degrade persistent organic molecules such as textile dyes and can contribute to discover microorganisms that can be used for bioremediation. The decolorization can be overlayed by the absorption from metabolites that are produced by fungi during screening, which interfere with the results. To compensate for this interference a method was developed by using different controls to subtract interfering signals. The method was designed for simple screening in multiwell plates that can be operated with a plate reader. It was applied to four different textile dyes (Reactive Black 5, Reactive Blue 4, Reactive Green 19, and Reactive Orange 16) that were degraded by the white rot fungus Phanerochaete velutina. The four textile dyes showed different results with a different degree of interference. The controls allow to compensate for interfering signals and to calculate kinetic parameters for the decolorization reaction and the enzymatic degradation.•Determine the non-enzymatic degradation of the dyes in experiments without fungi.•Determine the absorbance of metabolites and subtract it from the decolorization data to obtain the degradation of the dye.•Determine kinetic parameters of the degradation to compare the efficiency of the enzymes towards dyes.

Screening for Best Neuronal-Glial Differentiation Protocols of Neuralizing Agents Using a Multi-Sized Microfluidic Embryoid Body Array.

Stem cell technology and embryonic stem cell models are of great interest in biomedical research since they provide deeper insights into, e.g., neurogenesis and early mammalian brain development. Despite their great scientific potential, the reliable establishment of three-dimensional embryoid bodies (EBs) remains a major challenge, and the current lack of standardization and comparability is still limiting a broader application and translation of stem cell technology. Among others, a vital aspect for the reliable formation of EBs is optimizing differentiation protocols since organized differentiation is influenced by soluble inducers and EB size. A microfluidic biochip array was employed to automate cell loading and optimize directed neuronal and astrocytic differentiation protocols using murine P19 embryoid bodies to facilitate reliable embryonic stem cell differentiation. Our gravity-driven microfluidic size-controlled embryoid body-on-a-chip system allows (a) the robust operation and cultivation of up to 90 EBs in parallel and (b) the reproducible generation of five increasing sizes ranging from 300 µm to 1000 µm diameters. A comparative study adds two differentiation-inducers such as retinoic acid and EC23 to size-controlled embryoid bodies to identify the optimal differentiation protocol. Our study revealed a 1.4 to 1.9-fold higher neuron and astrocyte expression in larger embryoid bodies (above 750 µm) over smaller-sized EBs (below 450 µm), thus highlighting the importance of EB size in the establishment of robust neurodevelopmental in vitro models.

A Microfluidic Multisize Spheroid Array for Multiparametric Screening of Anticancer Drugs and Blood-Brain Barrier Transport Properties.

Physiological-relevant in vitro tissue models with their promise of better predictability have the potential to improve drug screening outcomes in preclinical studies. Despite the advances of spheroid models in pharmaceutical screening applications, variations in spheroid size and consequential altered cell responses often lead to nonreproducible and unpredictable results. Here, a microfluidic multisize spheroid array is established and characterized using liver, lung, colon, and skin cells as well as a triple-culture model of the blood-brain barrier (BBB) to assess the effects of spheroid size on (a) anticancer drug toxicity and (b) compound penetration across an advanced BBB model. The reproducible on-chip generation of 360 spheroids of five dimensions on a well-plate format using an integrated microlens technology is demonstrated. While spheroid size-related IC50 values vary up to 160% using the anticancer drugs cisplatin (CIS) or doxorubicin (DOX), reduced CIS:DOX drug dose combinations eliminate all lung microtumors independent of their sizes. A further application includes optimizing cell seeding ratios and size-dependent compound uptake studies in a perfused BBB model. Generally, smaller BBB-spheroids reveal an 80% higher compound penetration than larger spheroids while verifying the BBB opening effect of mannitol and a spheroid size-related modulation on paracellular transport properties.

Monitoring tissue-level remodelling during inflammatory arthritis using a three-dimensional synovium-on-a-chip with non-invasive light scattering biosensing.

Rheumatoid arthritis is a chronic, systemic joint disease in which an autoimmune response translates into an inflammatory attack resulting in joint damage, disability and decreased quality of life. Despite recent introduction of therapeutic agents such as anti-TNFα, even the best current therapies fail to achieve disease remission in most arthritis patients. Therefore, research into the mechanisms governing the destructive inflammatory process in rheumatoid arthritis is of great importance and may reveal novel strategies for the therapeutic interventions. To gain deeper insight into its pathogensis, we have developed for the first time a three-dimensional synovium-on-a-chip system in order to monitor the onset and progression of inflammatory synovial tissue responses. In our study, patient-derived primary synovial organoids are cultivated on a single chip platform containing embedded organic-photodetector arrays for over a week in the absence and presence of tumor-necrosis-factor. Using a label-free and non-invasive optical light-scatter biosensing strategy inflammation-induced 3D tissue-level architectural changes were already detected after two days. We demonstrate that the integration of complex human synovial organ cultures in a lab-on-a-chip provides reproducible and reliable information on how systemic stress factors affect synovial tissue architectures.

Effect of Spheroidal Age on Sorafenib Diffusivity and Toxicity in a 3D HepG2 Spheroid Model.

The enhanced predictive power of 3D multi-cellular spheroids in comparison to conventional monolayer cultures makes them a promising drug screening tool. However, clinical translation for pharmacology and toxicology is lagging its technological progression. Even though spheroids show a biological complexity resembling native tissue, standardization and validation of drug screening protocols are influenced by continuously changing physiological parameters during spheroid formation. Such cellular heterogeneities impede the comparability of drug efficacy studies and toxicological screenings. In this paper, we demonstrated that aside from already well-established physiological parameters, spheroidal age is an additional critical parameter that impacts drug diffusivity and toxicity in 3D cell culture models. HepG2 spheroids were generated and maintained on a self-assembled ultra-low attachment nanobiointerface and characterized regarding time-dependent changes in morphology, functionality as well as anti-cancer drug resistance. We demonstrated that spheroidal aging directly influences drug response due to the evolution of spheroid micro-structure and organo-typic functions, that alter inward diffusion, thus drug uptake.

Optimized alamarBlue assay protocol for drug dose-response determination of 3D tumor spheroids.

The assessment of drug-dose responses is vital for the prediction of unwanted toxicological effects in modern medicine. Three-dimensional (3D) cell cultures techniques can provide in vivo-like spheroids and microtissues that resemble natural tumor function. However, formation of necrotic core and diffusion limitation of chemical compounds within these models can reduce the reproducibility and precision of standard bioassay protocols used to test two-dimensional (2D) cell cultures. Nonetheless, the accurate prediction of detrimental effects of test compounds based on functional bioassays is essential for the development of new efficient therapeutic strategies. For instance, alamarBlue® is a widely-used commercially available redox indicator dye that can evaluate metabolic activity and cellular health status in a single-step procedure however, suitability and optimization of this bioassay must be determined for each individual application scenario. Here, we optimized the standard alamarBlue® proliferation/viability protocol for tumor spheroid cultures to enhance assay precision during toxicological drug screening. We optimized the original protocol of alamarBlue® assay that usually suggests an incubation time of 2-4 hours. The key modifications of the protocol for spheroid cultures are as follows: •Aspiration of cell culture medium before drug exposure.•Replacement of drug-supplemented medium with 10% (v/v) alamarBlue® reagent mixed with culture medium.•Increase of incubation period to 24 h at 37 °C protected from light.

A Self-Assembled Antifouling Nano-Biointerface for the Generation of Spheroids.

Several techniques have been established over the last decades to produce three-dimensional (3D) cellular spheroids and each method has its advantages and limitations. The unique self-assembly properties of surface layer (S-layer) proteins have already been applied to a broad range of life science applications. The bacterial S-layer protein SbpA displays a strong antifouling behavior when recrystallized on planar surfaces and offers the opportunity to induce 3D cell aggregation. In this chapter, an S-layer nanointerface is presented as novel ultralow attachment material for the formation of functional spheroids of reproducible sizes. The system is compatible with standard microwell plates and enables long-term 3D cell culture and in situ monitoring of cellular viability. Moreover, this facile and stable biointerface has potential for use in toxicity screening assays and represents an alternative to conventional materials like polyethylene glycol (PEG), agarose, or hydrogel surfaces.

Development of a Multifunctional Nanobiointerface Based on Self-Assembled Fusion-Protein rSbpA/ZZ for Blood Cell Enrichment and Phenotyping.

We present a multifunctional nanobiointerface for blood cell capture and phenotyping applications that features both excellent antifouling properties and high antibody activity. Multifunctionality is accomplished by modifying polymeric materials using self-assembled S-layer fusion-protein rSbpA/ZZ to immobilize high density antibodies at the two protein A binding sites of the rSbpA/ZZ nanolattice structure. Controlled orientation and alignment of the antibodies reduced antibody consumption 100-fold and increased cell capture efficiency 4-fold over standard methodologies. Cell analysis in complex samples was made possible by the remarkable antifouling properties of the rSbpA domain, while at the same time reducing unspecific binding and forgoing tedious blocking procedures. An automated microfluidic in situ cell analysis platform for isolation and phenotyping of primary peripheral blood mononuclear cells was developed as practical application. Results obtained using our automated microfluidic cell analysis platform showed that the multifunctional nanobiointerface can discriminate among T helper and cytotoxic T cells, and thymocytes. Additionally, on-chip cell capture under flow conditions using a high affinity CD 3 selective nanobiointerface preferentially isolated cells with strong surface marker expression. This means that our dynamic microfluidic cell purification method allows the enrichment of 773 CD 8 positive cytotoxic T cells out of a total blood cell population of 7728 PBMCs, which is an increase in cell enrichment of 8-fold with a purity of 85%.

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.

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.

Liquid crystal based sensors monitoring lipase activity: a new rapid and sensitive method for cytotoxicity assays.

In this work we present liquid crystal (LC) based sensor devices to monitor cell viability. The sensing layer is composed by the LC and a planar monolayer of phospholipids. In the presence of minute traces of phospholipases, which hydrolyze enzymatically phospholipids, the LC-lipid interface is disintegrated. This event causes a change in orientation of the LC, which was followed in a polarized microscope. The lipase activity can be used to measure the cell viability, since members of this enzyme family are released by cells, as they undergo necrosis. The described sensor was used to monitor the presence of the lipases released from three different cell lines, which were either exposed to highly cytotoxic model compounds (sodium azide and paracetamol) or subjected to freeze-thaw cycles to induce cell death by a non-chemical based inducer for apoptosis, such as temperature. Finally, the comparison of lipase activity detected by a state-of-the-art fluorescence assay to the LC based system resulted in the superiority of the LC system concerning incubation time and sensitivity.

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.

Exploitation of S-layer anisotropy: pH-dependent nanolayer orientation for cellular micropatterning.

We have developed a tunable, facile, and reliable cell patterning method using a self-assembled crystalline protein monolayer that, depending on its orientation, can exhibit either cell adhesive (cytophilic) or cell repulsive (cytophobic) surface properties. Our technique exploits, for the first time, the inherent biological anisotropy of the bacterial cell wall protein SbpA capable of interacting with its cytophilic inner side with components of the cell wall, while its outer cytophobic side interacts with the environment. By simply altering the recrystallization protocol from a basic to an acidic condition, the SbpA-protein layer orientation and function can be switched from preventing unspecific protein adsorption and cell adhesion to effectively promote cell attachment, spreading, and proliferation. As a result, the same protein solution can be used to form cell adhesive and repulsive regions over large areas on a single substrate using a simple pH-dependent self-assembly procedure. The reliable establishment of cytophobic and cytophilic SbpA layers allows the generation of well-defined surface patterns that exhibit uniform height (9-10 nm), p4 lattice symmetry with center-to-center spacing of the morphological units of 12 nm, as well as similar surface potential and charge distributions under cell culture conditions. The pH-dependent "orientation switch" of the SbpA protein nanolayer was integrated with micromolding in capillaries (MIMIC) technology to demonstrate its application for cell patterning using a variety of cell lines including epithelial, fibroblast and endothelial cells.

S-layer coated emulsomes as potential nanocarriers.

The present study introduces a novel nanocarrier system comprising lipidic emulsomes and S-layer (fusion) proteins as functionalizing tools coating the surface. Emulsomes composed of a solid tripalmitin core and a phospholipid shell are created reproducibly with an average diameter of approximately 300 nm using temperature-controlled extrusion steps. Both wildtype (wt) and recombinant (r) S-layer protein SbsB of Geobacillus stearothermophilus PV72/p2 are capable of forming coherent crystalline envelope structures with oblique (p1) lattice symmetry, as evidenced by transmission electron microscopy. Upon coating with wtSbsB, positive charge of emulsomes shifts to a highly negative zeta potential, whereas those coated with rSbsB become charge neutral. This observation is attributed to the presence of a negatively charged glycan, the secondary cell wall polymer (SCWP), which is associated only with wtSbsB. The present study shows for the first time the ability of recombinant and wildtype S-layer proteins to cover the entire surface of emulsomes with its characteristic crystalline lattice. Furthermore, in vitro cell culture studies reveal that S-layer coated emulsomes can be uptaken by human liver carcinoma cells (HepG2) without showing any significant cytotoxicity over a wide range of concentrations. The utilization of S-layer fusion proteins equipped in a nanopatterned fashion by identical or diverse functions may lead to further development of emulsomes in nanomedicine, especially for drug delivery and targeting.

Nanobiotechnology advanced antifouling surfaces for the continuous electrochemical monitoring of glucose in whole blood using a lab-on-a-chip.

In the current work we have developed a lab-on-a-chip containing embedded amperometric sensors in four microreactors that can be addressed individually and that are coated with crystalline surface protein monolayers to provide a continuous, stable, reliable and accurate detection of blood glucose. It is envisioned that the microfluidic device will be used in a feedback loop mechanism to assess natural variations in blood glucose levels during hemodialysis to allow the individual adjustment of glucose. Reliable and accurate detection of blood glucose is accomplished by simultaneously performing (a) blood glucose measurements, (b) autocalibration routines, (c) mediator-interferences detection, and (d) background subtractions. The electrochemical detection of blood glucose variations in the absence of electrode fouling events is performed by integrating crystalline surface layer proteins (S-layer) that function as an efficient antifouling coating, a highly-oriented immobilization matrix for biomolecules and an effective molecular sieve with pore sizes of 4 to 5 nm. We demonstrate that the S-layer protein SbpA (from Lysinibacillus sphaericus CCM 2177) readily forms monomolecular lattice structures at the various microchip surfaces (e.g. glass, PDMS, platinum and gold) within 60 min, eliminating unspecific adsorption events in the presence of human serum albumin, human plasma and freshly-drawn blood samples. The highly isoporous SbpA-coating allows undisturbed diffusion of the mediator between the electrode surface, thus enabling bioelectrochemical measurements of glucose concentrations between 500 µM to 50 mM (calibration slope δI/δc of 8.7 nA mM(-1)). Final proof-of-concept implementing the four microfluidic microreactor design is demonstrated using freshly drawn blood. Accurate and drift-free assessment of blood glucose concentrations (6. 4 mM) is accomplished over 130 min at 37 °C using immobilized enzyme glucose oxidase by calculating the difference between autocalibration (10 mM glc) and background measurements. The novel combination of biologically-derived nanostructured surfaces with microchip technology constitutes a powerful new tool for multiplexed analysis of complex samples.

Rapid liposome quality assessment using a lab-on-a-chip.

Although liposomes have many outstanding features such as biocompatibility, biodegradability, low toxicity and structural diversity, and are successfully applied in many areas of chemistry and biotechnology, a lack of characterization standards and quality control tools are still inhibiting the translation of liposome technology into clinical routine. The greatest obstacle to clinical scale commercialization is the inability to ensure liposome formulation stability because small size variations or altered surface chemistries can significantly influence in vivo distribution and excretion kinetics that could in turn lead to unpredictable therapy outcomes. To enhance the product development process we have developed a microfluidic biochip containing embedded dielectric microsensors capable of providing quantitative results on formulation composition and stability based on the monitoring of the unique electric properties of liposomes. Computational fluid dynamic (CFD) simulations confirmed that microfluidics offer reproducible and well-defined measurement conditions where a moving liposome suspension within a microchannel behaves like a bulk material. Results of this study demonstrate the ability of microfluidics, in combination with dielectric spectroscopy and multivariate data analysis methods, to identify nine different liposomes. We also show that various liposome modifications such as membrane-bound surface proteins, lipid bilayer soluble drugs, as well as protein and dye encapsulations, can be detected in the absence of any labels or indicators. Since shelf-life stability of a liposome formulation is regarded of prime importance for regulatory approval and clinical application, we further provide a possible practical application of the developed liposome analysis platform as a high-throughput tool for industrial quality insurance purposes.

Novel biocatalysts based on S-layer self-assembly of Geobacillus stearothermophilus NRS 2004/3a: a nanobiotechnological approach.

The crystalline cell-surface (S) layer sgsE of Geobacillus stearothermophilus NRS 2004/3a represents a natural protein self-assembly system with nanometer-scale periodicity that is evaluated as a combined carrier/patterning element for the conception of novel types of biocatalyst aiming at the controllable display of biocatalytic epitopes, storage stability, and reuse. The glucose-1-phosphate thymidylyltransferase RmlA is used as a model enzyme and chimeric proteins are constructed by translational fusion of rmlA to the C-terminus of truncated forms of sgsE (rSgsE (131-903), rSgsE(331-903)) and used for the construction of three principal types of biocatalysts: soluble (monomeric), self-assembled in aqueous solution, and recrystallized on negatively charged liposomes. Enzyme activity of the biocatalysts reaches up to 100 % compared to sole RmlA cloned from the same bacterium. The S-layer portion of the biocatalysts confers significantly improved shelf life to the fused enzyme without loss of activity over more than three months, and also enables biocatalyst recycling. These nanopatterned composites may open up new functional concepts for biocatalytic applications in nanobiotechnology.

Fluorescence emission properties of S-Layer enhanced green fluorescent fusion protein as a function of temperature, pH conditions, and guanidine hydrochloride concentration.

The fluorescent properties of the S-layer enhanced green fluorescent fusion protein (rSbpA31-1068/EGFP) were investigated as a function of temperature, pH conditions, and guanidine hydrochloride concentration. These results were compared to the fluorescent properties of the recombinant enhanced green fluorescent protein (EGFP) and an equimolar mixture of the S-layer protein rSbpA and EGFP. The intensity of the fluorescence emission of the EGFP at 510 nm, after excitation at 490 nm, is not affected by the presence of rSbpA, either as a fusion partner or as a free protein in solution. In each of the three protein systems, the emission intensity at 510 nm reaches its maximum value between pH 7 and 9 at 20 degrees C and at 0 M guanidine hydrochloride. No fluorescence could be measured at pH 4 and 6 M guanidine hydrochloride. These results show that the S-layer fusion protein (rSbpA31-1068/EGFP) is a suitable candidate for future applications in nanobiotechonology at a wide range of pH, temperature, and guanidine hydrochloride concentrations.

Recrystallization of bacterial S-layers on flat polyelectrolyte surfaces and hollow polyelectrolyte capsules.

Polyelectrolyte multilayer (PE) deposition and S-layer technology have been combined to make novel robust biomimetic surfaces and membranes. Isolated subunits of the bacterial cell surface layer from Bacillus sphaericus CCM2177 SbpA was self-assembled on PE multilayer supports, with the composition of the multilayer playing a crucial role in determining the structure of the resulting supported protein layers. Flat substrates were studied using atomic force microscopy and neutron reflectometry; protein on suitable PE combinations showed a crystalline structure with lattice constants equal to those found in vivo on bacterial surfaces. The mechanical stability of the S-layer is higher when recrystallized on PEs than directly on silicon supports. The recrystallization process was subsequently used to coat colloidal particles, permitting the determination of zeta potentials before and after coating. Hollow capsules could also be coated in the same way, as proven by various techniques. Our results suggest that electrostatic interactions via divalent cations are important for the assembly process. The results also demonstrate that the versatility of the PE multilayer membranes can be successfully combined with the well-defined surface chemistry and structure of 2D protein crystals.

A functional chimaeric S-layer-enhanced green fluorescent protein to follow the uptake of S-layer-coated liposomes into eukaryotic cells.

The chimaeric gene encoding a C-terminally truncated form of the S-layer protein SbpA of Bacillus sphaericus CCM 2177 and the EGFP (enhanced green fluorescent protein) was ligated into plasmid pET28a and cloned and expressed in Escherichia coli. Just 1 h after induction of expression an intense EGFP fluorescence was detected in the cytoplasm of the host cells. Expression at 28 degrees C instead of 37 degrees C resulted in clearly increased fluorescence intensity, indicating that the folding process of the EGFP moiety was temperature sensitive. To maintain the EGFP fluorescence, isolation of the fusion protein from the host cells had to be performed in the presence of reducing agents. SDS/PAGE analysis, immunoblotting and N-terminal sequencing of the isolated and purified fusion protein confirmed the presence of both the S-layer protein and the EGFP moiety. The fusion protein had maintained the ability to self-assemble in suspension and to recrystallize on peptidoglycan-containing sacculi or on positively charged liposomes, as well as to fluoresce. Comparison of fluorescence excitation and emission spectra of recombinant EGFP and rSbpA(31-1068)/EGFP revealed identical maxima at 488 and 507 nm respectively. The uptake of liposomes coated with a fluorescent monomolecular protein lattice of rSbpA(31-1068)/EGFP into HeLa cells was studied by confocal laser-scanning microscopy. The major part of the liposomes was internalized within 2 h of incubation and entered the HeLa cells by endocytosis.