Pilot-scale co-precipitation synthesis of a novel active ingredient made of ultrasmall iron (oxyhydr)oxide nanoparticles for the treatment of hyperphosphatemia.

Due to its simplicity, co-precipitation is the most commonly used method for producing iron (oxyhydr)oxide nanoparticles. However, it is reported to be sensitive to changes in process parameters, which complicates scale-up and is why only volumes up to 1.2 L have been described in the literature. This study aims to demonstrate the scale-up of a co-precipitation synthesis to 100 L using the example of a new phosphate-binding active ingredient based on iron (oxyhydr)oxide. The synthesis was shown to be very robust to changes in synthesis parameters and stirrer geometries. The in vitro phosphate-binding efficacy and the yield were maintained in all five scales tested. Only the content of the components in the nanoparticles varied slightly. However, Mössbauer spectroscopy, dynamic light scattering (DLS), and attenuated total reflection Fourier transform infrared spectroscopy (FT-IR) revealed no evidence of structural changes, but a reduction in the size of the iron (oxyhydr)oxide cores and the total core-shell nanoparticle sizes. Overall, this study has successfully demonstrated that ultrasmall iron (oxyhydr)oxide nanoparticles can be produced on a pilot scale by co-precipitation with a yield of >40 g L-1.

Direct Affinity Ligand Immobilization onto Bare Iron Oxide Nanoparticles Enables Efficient Magnetic Separation of Antibodies.

Magnetic separation is a promising alternative to chromatography for enhancing the downstream processing (DSP) of monoclonal antibodies (mAbs). However, there is a lack of efficient magnetic particles for successful application. Aiming to fill this gap, we demonstrate the suitability of bare iron oxide nanoparticles (BION) with physical site-directed immobilization of an engineered Protein A affinity ligand (rSpA) as an innovative magnetic material. The rSpA ligand contains a short peptide tag that enables the direct and stable immobilization onto the uncoated BION surface without commonly required laborious particle activation. The resulting BION@rSpA have beneficial characteristics outperforming conventional Protein A-functionalized magnetic particles: a simple, fast, low-cost synthesis, a particle size in the nanometer range with a large effective specific surface area enabling large immunoglobulin G (IgG) binding capacity, and a high magnetophoretic velocity advantageous for fast processing. We further show rapid interactions of IgG with the easily accessible rSpA ligands. The binding of IgG to BION@rSpA is thereby highly selective and not impeded by impurity molecules in perfusion cell culture supernatant. Regarding the subsequent acidic IgG elution from BION@rSpA@IgG, we observed a hampering pH increase caused by the protonation of large iron oxide surfaces after concentrating the particles in 100 mM sodium acetate buffer. However, the pH can be stabilized by adding 50 mM glycine to the elution buffer, resulting in recoveries above 85% even at high particle concentrations. Our work shows that BION@rSpA enable efficient magnetic mAb separation and could help to overcome emerging bottlenecks in DSP.

Development of a New Affinity Gold Polymer Membrane with Immobilized Protein A.

New and highly selective stationary phases for affinity membrane chromatography have the potential to significantly enhance the efficiency and specificity of therapeutic protein purification by reduced mass transfer limitations. This work developed and compared different immobilization strategies for recombinant Protein A ligands to a gold-sputtered polymer membrane for antibody separation in terms of functionalization and immobilization success, protein load, and stability. Successful, functionalization was validated via X-ray photoelectron spectroscopy (XPS). Here, a recombinant Protein A ligand was coupled by N-hydroxysuccinimide (NHS)/N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) chemistry to carboxy-functionalized, gold-sputtered membranes. We achieved a binding capacity of up to 104 ± 17 mg of the protein ligand per gram of the gold-sputtered membrane. The developed membranes were able to successfully capture and release the monoclonal antibody (mAb) Trastuzumab, as well as antibodies from fresh frozen human blood plasma in both static and dynamic setups. Therefore, they demonstrated successful functionalization and immobilization strategies. The antibody load was tested using bicinchoninic acid (BCA), ultraviolet-visible spectroscopy (UV-vis) measurements, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The outcome is a fully functional affinity membrane that can be implemented in a variety of different antibody purification processes, eliminating the need for creating individualized strategies for modifying the surface to suit different substrates or conditions.

Design and characterization of an electrochemically-modulated membrane chromatography device.

Membrane separations offer a compelling alternative to traditional chromatographic methods by overcoming mass transport limitations. We introduce an additional degree of freedom in modulating membrane chromatography by using metalized membranes in a potential-driven process. Investigating the impact of a gold coating on membrane characteristics, the sputtered gold layer enhances the surface conductivity with stable electrochemical behavior. However, this comes at the expense of reduced permeability, wettability, and static binding capacity (∼ 474 µg g-1 of maleic acid). The designed device displayed a homogenous flow distribution, and the membrane electrodes exhibit predominantly capacitive behavior during potential application. Modulating the electrical potential during the adsorption and desorption phase strongly influenced the binding and elution behavior of anion-exchange membranes. Switching potentials between ±1.0 V vs. Ag/AgCl induces desorption, confirming the process principle. Elution efficiency reaches up to 58 % at -1.0 V vs. Ag/AgCl in the desorption phase without any alteration of the mobile phase. Increasing the potential perturbation ranging from +1.0 V to -1.0 V vs. Ag/AgCl resulted in reduced peak width and improved elution behavior, demonstrating the feasibility of electrochemically-modulated membrane chromatography. The developed process has great potential as a gentle and sustainable separation step in the biotechnological and chemical industry.

Iron Oxide Nanoparticles with Supramolecular Ureido-Pyrimidinone Coating for Antimicrobial Peptide Delivery.

Antimicrobial peptides (AMPs) can kill bacteria by disrupting their cytoplasmic membrane, which reduces the tendency of antibacterial resistance compared to conventional antibiotics. Their possible toxicity to human cells, however, limits their applicability. The combination of magnetically controlled drug delivery and supramolecular engineering can help to reduce the dosage of AMPs, control the delivery, and improve their cytocompatibility. Lasioglossin III (LL) is a natural AMP form bee venom that is highly antimicrobial. Here, superparamagnetic iron oxide nanoparticles (IONs) with a supramolecular ureido-pyrimidinone (UPy) coating were investigated as a drug carrier for LL for a controlled delivery to a specific target. Binding to IONs can improve the antimicrobial activity of the peptide. Different transmission electron microscopy (TEM) techniques showed that the particles have a crystalline iron oxide core with a UPy shell and UPy fibers. Cytocompatibility and internalization experiments were carried out with two different cell types, phagocytic and nonphagocytic cells. The drug carrier system showed good cytocompatibility (>70%) with human kidney cells (HK-2) and concentration-dependent toxicity to macrophagic cells (THP-1). The particles were internalized by both cell types, giving them the potential for effective delivery of AMPs into mammalian cells. By self-assembly, the UPy-coated nanoparticles can bind UPy-functionalized LL (UPy-LL) highly efficiently (99%), leading to a drug loading of 0.68 g g-1. The binding of UPy-LL on the supramolecular nanoparticle system increased its antimicrobial activity against E. coli (MIC 3.53 µM to 1.77 µM) and improved its cytocompatible dosage for HK-2 cells from 5.40 µM to 10.6 µM. The system showed higher cytotoxicity (5.4 µM) to the macrophages. The high drug loading, efficient binding, enhanced antimicrobial behavior, and reduced cytotoxicity makes ION@UPy-NH2 an interesting drug carrier for AMPs. The combination with superparamagnetic IONs allows potential magnetically controlled drug delivery and reduced drug amount of the system to address intracellular infections or improve cancer treatment.

Application of magnetic iron oxide nanoparticles: Thrombotic activity, imaging and cytocompatibility of silica-coated and carboxymethyl dextrane-coated particles.

Coated iron oxide nanoparticles (IONs) are promising candidates for various applications in nanomedicine, including imaging, magnetic hyperthermia, and drug delivery. The application of IONs in nanomedicine is influenced by factors such as biocompatibility, surface properties, agglomeration, degradation behavior, and thrombogenicity. Therefore, it is essential to investigate the effects of coating material and thickness on the behavior and performance of IONs in the human body. In this study, IONs with a carboxymethyl dextran (CMD) coating and two thicknesses of silica coating (TEOS0.98, and TEOS3.91) were screened and compared to bare iron oxide nanoparticles (BIONs). All three coated particles showed good cytocompatibility (>70%) when tested with smooth muscle cells over three days. To investigate their potential long term behavior inside the human body, the Fe2+ release and hydrodynamic diameters of silica-coated and CMD (carboxymethyl dextrane)-coated IONs were analyzed in simulated body fluids for 72 h at 37 °C. The ION@CMD showed moderate agglomeration of around 100 nm in all four simulated fluids and dissolved faster than the silica-coated particles in artificial exosomal fluid and artificial lysosomal fluid. The particles with silica coating agglomerated in all tested simulated media above 1000 nm. Increased thickness of the silica coating led to decreased degradation of particles. Additionally, CMD coating resulted in nanoparticles with the least prothrombotic activity, and the thick silica coating apparently decreased the prothrombotic properties of nanoparticles compared to BIONs and ION@TEOS0.98. For magnetic resonance applications, ION@CMD and ION@TEOS3.91 showed comparatively high relaxation rates R2 values. In magnetic particle imaging experiments ION@TEOS3.91 yielded the highest normalized signal to noise ratio values and in magnetic hyperthermia studies, ION@CMD and ION@TEOS0.98 showed similar specific loss power. These findings demonstrate the potential of coated IONs in nanomedicine and emphasize the importance of understanding the effect of coating material and thickness on their behavior and performance in the human body.

Superparamagnetic iron oxide nanoparticles for their application in the human body: Influence of the surface.

Iron oxide nanoparticles (IONs) are of great interest in nanomedicine for imaging, drug delivery, or for hyperthermia treatment. Although many research groups have focused on the synthesis and application of IONs in nanomedicine, little is known about the influence of the surface properties on the particles' behavior in the human body. This study analyzes the impact of surface coatings (dextran, polyvinyl alcohol, polylactide-co-glycolide) on the nanoparticles' cytocompatibility, agglomeration, degradation, and the resulting oxidative stress induced by the particle degradation. All particles, including bare IONs (BIONs), are highly cytocompatible (>70%) and show no significant toxicity towards smooth muscle cells. Small-angle X-ray scattering profiles visualize the aggregation behavior of nanoparticles and yield primary particle sizes of around 20 nm for the investigated nanoparticles. A combined experimental setup of dynamic light scattering and phenanthroline assay was used to analyze the long-term agglomeration and degradation profile of IONs in simulated body fluids, allowing fast screening of multiple candidates. All particles degraded in simulated endosomal and lysosomal fluid, confirming the pH-dependent dissolution. The degradation rate decreased with the shrinking size of particles leading to a plateau. The fastest Fe2+ release could be measured for the polyvinyl-coated IONs. The analytical setup is ideal for a quick preclinical study of IONs, giving often neglected yet crucial information about the behavior and toxicity of nanoparticles in the human body. Moreover, this study allows for the development and evaluation of novel ferroptosis-inducing agents.

Interaction and mechanisms in the phosphate-binding of iron(oxyhydr)oxide core-shell nanoparticles.

HYPOTHESIS: The high binding affinity of iron(oxyhydr)oxides for phosphate has recently been used in medicine to treat hyperphosphatemia, an abnormally elevated phosphate concentration in the blood. For iron(oxyhydr)oxide nanoparticles, the composition of the organic shell has a more significant influence on their interaction with phosphate than is often assumed. This study shows different mechanisms in phosphate binding, using the example of two similar new phosphate-binding agents. EXPERIMENTS: We characterized the phosphate-binding behavior of two iron(oxyhydr)oxide-based nanomaterials with similar composition and particle properties and investigated their binding mechanisms by spectroscopic methods. FINDINGS: For the often prescribed Velphoro, we demonstrated a phosphate binding capacity of>210 mg/g. A similar active ingredient named C-PAM binds over 573 mg/g. Spectroscopic measurements highlighted differences in the binding mechanism. While Velphoro binds phosphate via surface complexation independent of pH and adsorbent concentration, C-PAM shows a strong concentration dependence. At low concentrations, phosphate is bound via complexation reactions. The iron(oxyhydr)oxide structure was dissolved at higher phosphate concentrations and formed various iron phosphate species. The substances behave differently upon interaction with phosphate, although being very similar in composition and crystal structure. Thus, we demonstrated a crucial influence of the ligands in the shell on the binding mechanism.

Competition at the Bio-nano Interface: A Protein, a Polysaccharide, and a Fatty Acid Adsorb onto Magnetic Nanoparticles.

Magnetic nanoparticles are an attractive bioseparation tool due to their magnetic susceptibility and high adsorption capacity for different types of molecules. A major challenge for separation is to generate selectivity for a target molecule, or for a group of molecules in complex environments such as cell lysates. It is crucial to understand the factors that determine the targets' adsorption behavior in mixtures for triggering intended interactions and selectivity. Here we use a model system containing three molecules, each of them a common representative of the more abundant types of macromolecules in living systems: sodium oleate (SO), a fatty acid; bovine serum albumin (BSA), a protein; and dextran, a polysaccharide. Our results show that (a) the BSA adsorption capacity on the iron oxide material depends markedly on the pH, with the maximum capacity at the pI of the protein (0.39 g gMNP-1 ); (b) sodium oleate, a strongly negatively charged molecule, an organic anion, renders a maximum adsorption capacity of 0.40 g gMNP-1, even at pHs at which oleate as well as the nanoparticle surface are negatively charged; (c) the adsorbed masses of dextran, a neutral sugar, are lower than for the other two molecules, between 0.09 and 0.13 g gMNP-1, regardless of the system's pH. We observe an unexpected behavior in mixtures: SO completely prevents the adsorption of BSA, and dextran decreases the adsorption of the other competitors, SO and BSA, while adsorbing at the same capacities, unaffected by either the presence of the other two molecules or the pH. BSA does not decrease the oleate adsorption capacity. We demonstrate the essential role of pH in the adsorption of BSA (a protein) and SO (a fatty acid), as well as its impact in the structural organization of the oleate molecules in water. Moreover, we present exciting data on the adsorption of the molecules in competition, revealing the need to focus on interaction studies in more complex environments. This study attempts to open the scope of the current research of bio-nano interactions to not only proteins but also to mixtures, and generally to molecules with other physicochemical characteristics. Furthermore, we contribute to the understanding of multicomponent systems with the vision set in enhancing biomass exploitation and biofractionation processes.

Carboxymethyl-Dextran-Coated Superparamagnetic Iron Oxide Nanoparticles for Drug Delivery: Influence of the Coating Thickness on the Particle Properties.

Carboxymethyl-dextran (CMD)-coated iron oxide nanoparticles (IONs) are of great interest in nanomedicine, especially for applications in drug delivery. To develop a magnetically controlled drug delivery system, many factors must be considered, including the composition, surface properties, size and agglomeration, magnetization, cytocompatibility, and drug activity. This study reveals how the CMD coating thickness can influence these particle properties. ION@CMD are synthesized by co-precipitation. A higher quantity of CMD leads to a thicker coating and a reduced superparamagnetic core size with decreasing magnetization. Above 12.5−25.0 g L−1 of CMD, the particles are colloidally stable. All the particles show hydrodynamic diameters < 100 nm and a good cell viability in contact with smooth muscle cells, fulfilling two of the most critical characteristics of drug delivery systems. New insights into the significant impact of agglomeration on the magnetophoretic behavior are shown. Remarkable drug loadings (62%) with the antimicrobial peptide lasioglossin and an excellent efficiency (82.3%) were obtained by covalent coupling with the EDC/NHS (N-ethyl-N'-(3-(dimethylamino)propyl)carbodiimide/N-hydroxysuccinimide) method in comparison with the adsorption method (24% drug loading, 28% efficiency). The systems showed high antimicrobial activity with a minimal inhibitory concentration of 1.13 µM (adsorption) and 1.70 µM (covalent). This system successfully combines an antimicrobial peptide with a magnetically controllable drug carrier.

Peptide adsorption on silica surfaces: Simulation and experimental insights.

The understanding of interactions between proteins with silica surface is crucial for a wide range of different applications: from medical devices, drug delivery and bioelectronics to biotechnology and downstream processing. We show the application of EISM (Effective Implicit Surface Model) for discovering the set of peptide interactions with silica surface. The EISM is employed for a high-speed computational screening of peptides to model the binding affinity of small peptides to silica surfaces. The simulations are complemented with experimental data of peptides with silica nanoparticles from microscale thermophoresis and from infrared spectroscopy. The experimental work shows excellent agreement with computational results and verifies the EISM model for the prediction of peptide-surface interactions. 57 peptides, with amino acids favorable for adsorption on Silica surface, are screened by EISM model for obtaining results, which are worth to be considered as a guidance for future experimental and theoretical works. This model can be used as a broad platform for multiple challenges at surfaces which can be applied for multiple surfaces and biomolecules beyond silica and peptides.

Current research approaches in downstream processing of pharmaceutically relevant proteins.

Biopharmaceuticals and their production are on the rise. They are needed to treat and to prevent multiple diseases. Therefore, an urgent need for process intensification in downstream processing (DSP) has been identified to produce biopharmaceuticals more efficiently. The DSP currently accounts for the majority of production costs of pharmaceutically relevant proteins. This short review gathers essential research over the past 3 years that addresses novel solutions to overcome this bottleneck. The overview includes promising studies in the fields of chromatography, aqueous two-phase systems, precipitation, crystallization, magnetic separation, and filtration for the purification of pharmaceutically relevant proteins.

Direct capture and selective elution of a secreted polyglutamate-tagged nanobody using bare magnetic nanoparticles.

BACKGROUND: The secretion and direct capture of proteins from the extracellular medium is a promising approach for purification, thus enabling integrated bioprocesses. MAJOR RESULTS: We demonstrate the secretion of a nanobody (VHH) to the extracellular medium (EM) and its direct capture by bare, non-functionalized magnetic nanoparticles (MNPs). An ompA signal peptide for periplasmic localization, a polyglutamate-tag (E8 ) for selective MNP binding, and a factor Xa protease cleavage site were fused N-terminally to the nanobody. The extracellular production of the E8 -VHH (36 mg L-1 ) was enabled using a growth-decoupled Escherichia coli-based expression system. The direct binding of E8 -VHH to the bare magnetic nanoparticles was possible and could be drastically improved up to a yield of 88% by adding polyethylene glycol (PEG). The selectivity of the polyglutamate-tag enabled a selective elution of the E8 -VHH from the bare MNPs while raising the concentration factor (5x) and purification factor (4x) significantly. CONCLUSION: Our studies clearly show that the unique combination of a growth-decoupled E. coli secretion system, the polyglutamate affinity tag, non-functionalized magnetic nanoparticles, and affinity magnetic precipitation is an innovative and novel way to capture and concentrate nanobodies.

Purification of a peptide tagged protein via an affinity chromatographic process with underivatized silica.

Silica is widely used for chromatography resins due to its high mechanical strength, column efficiency, easy manufacturing (i.e. controlled size and porosity), and low-cost. Despite these positive attributes to silica, it is currently used as a backbone for chromatographic resins in biotechnological downstream processing. The aim of this study is to show how the octapeptide (RH)4 can be used as peptide tag for high-purity protein purification on bare silica. The tag possesses a high affinity to deprotonated silanol groups because the tag's arginine groups interact with the surface via an ion pairing mechanism. A chromatographic workflow to purify GFP fused with (RH)4 could be implemented. Purities were determined by SDS-PAGE and RP-HPLC. The equilibrium binding capacity of the fusion protein GFP-(RH)4 on silica is 450 mg/g and the dynamic binding capacity around 3 mg/mL. One-step purification from clarified lysate achieved a purity of 93% and a recovery of 94%. Overloading the column enhances the purity to >95%. Static experiments with different buffers showed variability of the method making the system independent from buffer choice. Our designed peptide tag allows bare silica to be utilized in preparative chromatography for downstream bioprocessing; thus, providing a cost saving factor regarding expensive surface functionalization. Underivatized silica in combination with our (RH)4 peptide tag allows the purification of proteins, in all scales, without relying on complex resins.

Insights on Alanine and Arginine Binding to Silica with Atomic Resolution.

Interactions of biomolecules with inorganic oxide surfaces such as silica in aqueous solutions are of profound interest in various research fields, including chemistry, biotechnology, and medicine. While there is a general understanding of the dominating electrostatic interactions, the binding mechanism is still not fully understood. Here, chromatographic zonal elution and flow microcalorimetry experiments were combined with molecular dynamic simulations to describe the interaction of different capped amino acids with the silica surface. We demonstrate that ion pairing is the dominant electrostatic interaction. Surprisingly, the interaction strength is more dependent on the repulsive carboxy group than on the attracting amino group. These findings are essential for conducting experimental and simulative studies on amino acids when transferring the results to biomolecule-surface interactions.

Visualization of USPIO-labeled melt-electrowritten scaffolds by non-invasive magnetic resonance imaging.

Melt electrowriting (MEW) is a high-resolution fiber-forming technology for the digital fabrication of complex micro-structured scaffolds for tissue engineering, which has convincingly shown its potential in in vitro and in vivo animal studies. The clinical translation of such constructs to the patient requires the capability to visualize them upon implantation with clinically accepted methods such as magnetic resonance imaging (MRI). To this end, this work presents the modification of polycaprolactone (PCL) scaffolds with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles to render them visualizable by MRI. Composite scaffolds containing up to 0.3 weight % USPIOs were 3D printed by MEW and could be sensitively detected in vitro using T2- and T2*-weighted MRI. At the same time, USPIO incorporation did not affect the usability of PCL for tissue engineering applications as demonstrated by the mechanical and cytocompatibility evaluation. Concentrations up to 0.2% caused small to no decrease in the ultimate tensile strength and Young's modulus. Cytocompatibility tests resulted in excellent cell viability, with proliferating cells adhering to all the scaffolds. This work contributes to the materials library for MEW and opens the possibility of using MRI for longitudinal monitoring of MEW grafts.

DNA Binding to the Silica: Cooperative Adsorption in Action.

The adsorption and desorption of nucleic acid to a solid surface is ubiquitous in various research areas like pharmaceutics, nanotechnology, molecular biology, and molecular electronics. In spite of this widespread importance, it is still not well understood how the negatively charged deoxyribonucleic acid (DNA) binds to the negatively charged silica surface in an aqueous solution. In this article, we study the adsorption of DNA to the silica surface using both modeling and experiments and shed light on the complicated binding (DNA to silica) process. The binding agent mediated DNA adsorption was elegantly captured by cooperative Langmuir model. Bulk-depletion experiments were performed to conclude the necessity of a positively charged binding agent for efficient DNA binding, which complements the findings from the model. A profound understanding of DNA binding will help to tune various processes for efficient nucleic acid extraction and purification. However, this work goes beyond the DNA binding and can shed light on other binding agent mediated surface-surface, surface-molecule, molecule-molecule interaction.

Bare Iron Oxide Nanoparticles as Drug Delivery Carrier for the Short Cationic Peptide Lasioglossin.

New drug delivery systems are a potential solution for administering drugs to reduce common side effects of traditional methods, such as in cancer therapy. Iron oxide nanoparticles (IONs) can increase the drugs' biological activity through high binding efficiency and magnetically targeted drug delivery. Understanding the adsorption and release process of a drug to the carrier material plays a significant role in research to generate an applicable and controlled drug delivery system. This contribution focuses on the binding patterns of the peptide lasioglossin III from bee venom on bare IONs. Lasioglossin has a high antimicrobial behavior and due to its cationic properties, it has high binding potential. Considering the influence of pH, the buffer type, the particle concentration, and time, the highest drug loading of 22.7% is achieved in phosphate-buffered saline. Analysis of the desorption conditions revealed temperature and salt concentration sensitivity. The nanoparticles and peptide-ION complexes are analyzed with dynamic light scattering, zeta potential, and infrared spectroscopy. Additionally, cytotoxicity experiments performed on Escherichia coli show higher antimicrobial activity of bound lasioglossin than of the free peptide. Therefore, bare IONs are an interesting platform material for the development of drug-delivery carriers for cationic peptides.

Detection of targeted bacteria species on filtration membranes.

The detection of pathogens in aquatic environments issues a time-consuming challenge, but it is an essential task to prevent the spread of diseases. We have developed a new point-of-care (POC) method for the fast and efficient detection of Legionella pneumophila in water. The method consists first of the generation of immunocomplexes of bacteria species with its corresponding targeted fluorescence-labelled serogroup-specific antibodies, and second a concentration step of pathogens with a membrane filter. Third, on the filtration membrane, our method can detect the fluorescence intensity corresponding to the pathogen concentration. Thus selective and efficient evidence for the presence of bacteria can be evaluated. We tested our system on fluorescent Escherichia coli bacteria and were able to reach an accurate determination of 1000 cells. The technique was furthermore tested on Legionella pneumophila cells, which were labelled with fluorescence-labelled antibodies as a proof of principle. Furthermore, we were able to verify this method in the presence of other bacteria species. We were able to detect bacteria cells within half an hour, a substantial advancement compared to the prevailling state of the art detection method based on the cultivation of Legionella pneumophila. Hence, this system represents the basis for future developments in analysis of pathogens.