Lab-on-a-chip models of the blood-brain barrier: evolution, problems, perspectives.

A great progress has been made in the development and use of lab-on-a-chip devices to model and study the blood-brain barrier (BBB) in the last decade. We present the main types of BBB-on-chip models and their use for the investigation of BBB physiology, drug and nanoparticle transport, toxicology and pathology. The selection of the appropriate cell types to be integrated into BBB-on-chip devices is discussed, as this greatly impacts the physiological relevance and translatability of findings. We identify knowledge gaps, neglected engineering and cell biological aspects and point out problems and contradictions in the literature of BBB-on-chip models, and suggest areas for further studies to progress this highly interdisciplinary field. BBB-on-chip models have an exceptional potential as predictive tools and alternatives of animal experiments in basic and preclinical research. To exploit the full potential of this technique expertise from materials science, bioengineering as well as stem cell and vascular/BBB biology is necessary. There is a need for better integration of these diverse disciplines that can only be achieved by setting clear parameters for characterizing both the chip and the BBB model parts technically and functionally.

Microsecond All-Optical Modulation by Biofunctionalized Porous Silicon Microcavity.

We successfully created a composite photonic structure out of porous silicon (PSi) microcavities doped by the photochromic protein, photoactive yellow protein (PYP). Massive incorporation of the protein molecules into the pores was substantiated by a 30 nm shift of the resonance dip upon functionalization, and light-induced reflectance changes of the device due to the protein photocycle were recorded. Model calculations for the photonic properties of the device were consistent with earlier results on the nonlinear optical properties of the protein, whose degree of incorporation into the PSi structure was also estimated. The successful proof-of-concept results are discussed in light of possible practical applications in the future.

The Use of Sensors in Blood-Brain Barrier-on-a-Chip Devices: Current Practice and Future Directions.

The application of lab-on-a-chip technologies in in vitro cell culturing swiftly resulted in improved models of human organs compared to static culture insert-based ones. These chip devices provide controlled cell culture environments to mimic physiological functions and properties. Models of the blood-brain barrier (BBB) especially profited from this advanced technological approach. The BBB represents the tightest endothelial barrier within the vasculature with high electric resistance and low passive permeability, providing a controlled interface between the circulation and the brain. The multi-cell type dynamic BBB-on-chip models are in demand in several fields as alternatives to expensive animal studies or static culture inserts methods. Their combination with integrated biosensors provides real-time and noninvasive monitoring of the integrity of the BBB and of the presence and concentration of agents contributing to the physiological and metabolic functions and pathologies. In this review, we describe built-in sensors to characterize BBB models via quasi-direct current and electrical impedance measurements, as well as the different types of biosensors for the detection of metabolites, drugs, or toxic agents. We also give an outlook on the future of the field, with potential combinations of existing methods and possible improvements of current techniques.

Multifunctional microfluidic chips for the single particle inductively coupled plasma mass spectrometry analysis of inorganic nanoparticles.

This study aimed at exploiting the so far unexploited potential of carrying out on-line sample pretreatment steps on microfluidic chips for single particle inductively coupled plasma mass spectrometry (spICP-MS) measurements, and demonstrating their ability to practically facilitate most of the simpler tasks involved in the spICP-MS analysis of nanoparticles. For this purpose, polydimethylsiloxane microfluidic chips, capable of high-range dilution and sample injection were made by casting, using high-precision, 3D-printed molds. Optimization of their geometry and functions was done by running several hydrodynamic simulations and by gravimetric, fluorescence enhanced microscope imaging and solution-based ICP-MS experiments. On the optimized microfluidic chips, several experiments were done, demonstrating the benefits of the approach and these devices, such as the determination of nanoparticle concentration using only a few tens of microliters of sample, elimination of solute interferences by dilution, solution-based size calibration and characterisation of binary nanoparticles. Due to the unique design of the chips, they can be linked together to extend the dilution range of the system by more than a magnitude per chip. This feature was also demonstrated in applications requiring multiple-magnitude dilution rates, when two chips were sequentially coupled.

Penetration of the SARS-CoV-2 Spike Protein across the Blood-Brain Barrier, as Revealed by a Combination of a Human Cell Culture Model System and Optical Biosensing.

Since the outbreak of the global pandemic caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), several clinical aspects of the disease have come into attention. Besides its primary route of infection through the respiratory system, SARS-CoV-2 is known to have neuroinvasive capacity, causing multiple neurological symptoms with increased neuroinflammation and blood-brain barrier (BBB) damage. The viral spike protein disseminates via circulation during infection, and when reaching the brain could possibly cross the BBB, which was demonstrated in mice. Therefore, its medical relevance is of high importance. The aim of this study was to evaluate the barrier penetration of the S1 subunit of spike protein in model systems of human organs highly exposed to the infection. For this purpose, in vitro human BBB and intestinal barrier cell-culture systems were investigated by an optical biosensing method. We found that spike protein crossed the human brain endothelial cell barrier effectively. Additionally, spike protein passage was found in a lower amount for the intestinal barrier cell layer. These observations were corroborated with parallel specific ELISAs. The findings on the BBB model could provide a further basis for studies focusing on the mechanism and consequences of spike protein penetration across the BBB to the brain.

All-Optical Switching Demonstrated with Photoactive Yellow Protein Films.

Integrated optics (IO) is a field of photonics which focuses on manufacturing circuits similar to those in integrated electronics, but that work on an optical basis to establish means of faster data transfer and processing. Currently, the biggest task in IO is finding or manufacturing materials with the proper nonlinear optical characteristics to implement as active components in IO circuits. Using biological materials in IO has recently been proposed, the first material to be investigated for this purpose being the protein bacteriorhodopsin; however, since then, other proteins have also been considered, such as the photoactive yellow protein (PYP). In our current work, we directly demonstrate the all-optical switching capabilities of PYP films combined with an IO Mach-Zehnder interferometer (MZI) for the first time. By exploiting photoreactions in the reaction cycle of PYP, we also show how a combination of exciting light beams can introduce an extra degree of freedom to control the operation of the device. Based on our results, we discuss how the special advantages of PYP can be utilized in future IO applications.

Transendothelial Electrical Resistance Measurement across the Blood-Brain Barrier: A Critical Review of Methods.

The blood-brain barrier (BBB) represents the tightest endothelial barrier within the cardiovascular system characterized by very low ionic permeability. Our aim was to describe the setups, electrodes, and instruments to measure electrical resistance across brain microvessels and culture models of the BBB, as well as critically assess the influence of often neglected physical and technical parameters such as temperature, viscosity, current density generated by different electrode types, surface size, circumference, and porosity of the culture insert membrane. We demonstrate that these physical and technical parameters greatly influence the measurement of transendothelial electrical resistance/resistivity (TEER) across BBB culture models resulting in severalfold differences in TEER values of the same biological model, especially in the low-TEER range. We show that elevated culture medium viscosity significantly increases, while higher membrane porosity decreases TEER values. TEER data measured by chopstick electrodes can be threefold higher than values measured by chamber electrodes due to different electrode size and geometry, resulting in current distribution inhomogeneity. An additional shunt resistance at the circumference of culture inserts results in lower TEER values. A detailed description of setups and technical parameters is crucial for the correct interpretation and comparison of TEER values of BBB models.

Nonlinear Optical Investigation of Microbial Chromoproteins.

Membrane-bound or cytosolic light-sensitive proteins, playing a crucial role in energy- and signal-transduction processes of various photosynthetic microorganisms, have been optimized for sensing or harvesting light by myriads of years of evolution. Upon absorption of a photon, they undergo a usually cyclic reaction series of conformations, and the accompanying spectro-kinetic events assign robust nonlinear optical (NLO) properties for these chromoproteins. During recent years, they have attracted a considerable interest among researchers of the applied optics community as well, where finding the appropriate NLO material for a particular application is a pivotal task. Potential applications have emerged in various branches of photonics, including optical information storage and processing, higher-harmonic and white-light continuum generation, or biosensorics. In our earlier work, we also raised the possibility of using chromoproteins, such as bacteriorhodopsin (bR), as building blocks for the active elements of integrated optical (IO) circuits, where several organic and inorganic photonic materials have been considered as active components, but so far none of them has been deemed ideal for the purpose. In the current study, we investigate the linear and NLO properties of biofilms made of photoactive yellow protein (PYP) and bR. The kinetics of the photoreactions are monitored by time-resolved absorption experiments, while the refractive index of the films and its light-induced changes are measured using the Optical Waveguide Lightmode Spectroscopy (OWLS) and Z-scan techniques, respectively. The nonlinear refractive index and the refractive index change of both protein films were determined in the green spectral range in a wide range of intensities and at various laser repetition rates. The nonlinear refractive index and refractive index change of PYP were compared to those of bR, with respect to photonics applications. Our results imply that the NLO properties of these proteins make them promising candidates for utilization in applied photonics, and they should be considered as valid alternatives for active components of IO circuits.

A chip device to determine surface charge properties of confluent cell monolayers by measuring streaming potential.

Cell surface charge is an important element of the function of biological barriers, but no chip device has been described to measure cell surface charge properties of confluent barrier cell monolayers. The aim of this study was the design and fabrication of a dynamic lab-on-a-chip (LOC) device which is suitable to monitor transcellular electrical resistance, as well as streaming potential parallel to the surface of cell layers. We successfully measured the streaming potential of a biological barrier culture model with the help of our previously published versatile lab-on-a-chip device equipped with two Ag/AgCl electrodes. The inclusion of these "zeta electrodes", a voltage preamplifier and an oscilloscope in our set-up made it possible to successfully record signals describing the surface charge properties of brain endothelial cell monolayers, used as a barrier model in our experiments. Data obtained on the new chip device were verified by comparing streaming potential results measured in the LOC device and zeta potential results by the commonly used laser-Doppler velocimetry (LDv) method and model simulations. Changes in the negative surface charge of the barrier model by treatments with neuraminidase enzyme modifying the cell membrane glycocalyx or lidocaine altering the lipid membrane charge could be measured by both the upgraded LOC device and LDv. The new chip device can help to gain meaningful new information on how surface charge is linked to barrier function in both physiological and pathological conditions.

Lidocaine turns the surface charge of biological membranes more positive and changes the permeability of blood-brain barrier culture models.

The surface charge of brain endothelial cells forming the blood-brain barrier (BBB) is highly negative due to phospholipids in the plasma membrane and the glycocalyx. This negative charge is an important element of the defense systems of the BBB. Lidocaine, a cationic and lipophilic molecule which has anaesthetic and antiarrhytmic properties, exerts its actions by interacting with lipid membranes. Lidocaine when administered intravenously acts on vascular endothelial cells, but its direct effect on brain endothelial cells has not yet been studied. Our aim was to measure the effect of lidocaine on the charge of biological membranes and the barrier function of brain endothelial cells. We used the simplified membrane model, the bacteriorhodopsin (bR) containing purple membrane of Halobacterium salinarum and culture models of the BBB. We found that lidocaine turns the negative surface charge of purple membrane more positive and restores the function of the proton pump bR. Lidocaine also changed the zeta potential of brain endothelial cells in the same way. Short-term lidocaine treatment at a 10 µM therapeutically relevant concentration did not cause major BBB barrier dysfunction, substantial change in cell morphology or P-glycoprotein efflux pump inhibition. Lidocaine treatment decreased the flux of a cationic lipophilic molecule across the cell layer, but had no effect on the penetration of hydrophilic neutral or negatively charged markers. Our observations help to understand the biophysical background of the effect of lidocaine on biological membranes and draws the attention to the interaction of cationic drug molecules at the level of the BBB.

[Integrated optical sensor for rapid analysis of biological samples]. / Integrált optikai szenzor biológiai minták gyors analíziséhez.

INTRODUCTION: In the medical diagnostics of bacteria, the rapid detection of pathogenic microorganisms from body fluids is one of the most important tasks. The majority of the modern measuring techniques are based on specific labels bound to the bacteria. However, this strategy usually assumes a rather time-consuming procedure involving several steps (e.g., the widely used enzyme-linked immunosorbent assay normally consists of 5 consecutive steps). Hence, there is an urgent need for the elaboration of rapid, "label-free" techniques, that are often based on Lab-on-a-chip devices. AIM: In this paper, the authors report on the development of a biosensor based on a miniature, integrated optical Mach-Zehnder interferometer. METHOD: Functionalization of the measuring arm of the sensor by antibodies, made the rapid and specific label-free detection of pathogens feasible. RESULTS: Using the combination of the interferometer with a microfluidic system, the device was able to detect Escherichia coli bacteria at concentrations as low as 10(6) colony forming unit/ml within minutes. CONCLUSIONS: This makes the newly developed biosensor a promising device for a wide range of applications, not only in medical microbiology, but microbial forensics, criminal investigations, bio-terrorism threats and in environmental studies as well.

Microfluidic study of the chemotactic response of Escherichia coli to amino acids, signaling molecules and secondary metabolites.

Quorum sensing and chemotaxis both affect bacterial behavior on the population level. Chemotaxis shapes the spatial distribution of cells, while quorum sensing realizes a cell-density dependent gene regulation. An interesting question is if these mechanisms interact on some level: Does quorum sensing, a density dependent process, affect cell density itself via chemotaxis? Since quorum sensing often spans across species, such a feedback mechanism may also exist between multiple species. We constructed a microfluidic platform to study these questions. A flow-free, stable linear chemical gradient is formed in our device within a few minutes that makes it suitable for sensitive testing of chemoeffectors: we showed that the amino acid lysine is a weak chemoattractant for Escherichia coli, while arginine is neutral. We studied the effect of quorum sensing signal molecules of Pseudomonas aeruginosa on E. coli chemotaxis. Our results show that N-(3-oxododecanoyl)-homoserine lactone (oxo-C12-HSL) and N-(butryl)-homoserine lactone (C4-HSL) are attractants. Furthermore, we tested the chemoeffector potential of pyocyanin and pyoverdine, secondary metabolites under a quorum sensing control. Pyocyanin is proved to be a weak attractant while pyoverdine are repellent. We demonstrated the usability of the device in co-culturing experiments, where we showed that various factors released by P. aeruginosa affect the dynamic spatial rearrangement of a neighboring E. coli population, while surface adhesion of the cells is also modulated.

Stretching of red blood cells using an electro-optics trap.

The stretching stiffness of Red Blood Cells (RBCs) was investigated using a combination of an AC dielectrophoretic apparatus and a single-beam optical tweezer. The experiments were performed at 10 MHz, a frequency high enough to avoid conductivity losses, but below the second turnover point between positive and negative dielectrophoresis. By measuring the geometrical parameters of single healthy human RBCs as a function of the applied voltage, the elastic modulus of RBCs was determined (µ = 1.80 ± 0.5 µN/m) and compared with similar values of the literature got by other techniques. The method is expected to be an easy-to-use, alternative tool to determine the mechano-elastic properties of living cells, and, on this basis, to distinguish healthy and diseased cells.

High-speed integrated optical logic based on the protein bacteriorhodopsin.

The principle of all-optical logical operations utilizing the unique nonlinear optical properties of a protein was demonstrated by a logic gate constructed from an integrated optical Mach-Zehnder interferometer as a passive structure, covered by a bacteriorhodopsin (bR) adlayer as the active element. Logical operations were based on a reversible change of the refractive index of the bR adlayer over one or both arms of the interferometer. Depending on the operating point of the interferometer, we demonstrated binary and ternary logical modes of operation. Using an ultrafast transition of the bR photocycle (BR-K), we achieved high-speed (nanosecond) logical switching. This is the fastest operation of a protein-based integrated optical logic gate that has been demonstrated so far. The results are expected to have important implications for finding novel, alternative solutions in all-optical data processing research.

Optical tweezers with tips grown at the end of fibers by photopolymerization.

We present a method to build an optical tip at the end of a single-mode optical fiber. The tip is grown by a self-writing process: photopolymerization by the light coming from the optical fiber. We developed a technique to produce a flat end surface on the tip. The good optical quality of the tip and the output laser beam was demonstrated by the fact that a counterpropagating optical trap could be constructed by using the tips with parameters comparable to regular fiber traps. Because of the small size of the tips, the tweezers require a much smaller space than regular fiber traps.

Integrated optical switching based on the protein bacteriorhodopsin.

According to our earlier pioneering study, a dry film containing native bacteriorhodopsin (bR) shows unique nonlinear optical properties (refractive index change, controllable by light of different colors, greater than 2 x 10(-3)) that are in many respects superior to those of the materials presently applied in integrated optics. Here, we report on the first integrated optical application based on a miniature Mach-Zehnder interferometer (see Figs. 1 and 2) demonstrating a real switching effect by bR (efficiency higher than 90%) due to the M-state. Our results also imply that the refractive index change of the K-state (9 x 10(-4)) is high enough for fast switching.

Parallel photopolymerisation with complex light patterns generated by diffractive optical elements.

Photopolymerisation by scanning a focused laser beam is a powerful method to build structures of arbitrary complexity with submicrometer resolution. We introduce parallel photopolymerisation to enhance the efficiency. Instead of multidimensional scanning of a single focus, the structure is generated simultaneously with diffractive patterns. We used fixed diffractive optical elements (DOEs), kinoforms, and Spatial Light Modulators (SLMs). The possibilities of photopolymerisation using SLM were investigated: the added flexibility using the programmable device is demonstrated. By using these DOEs, straight and helical cross shaped columns were produced with a single scan at a rate about an order of magnitude faster than by simple scanning. The produced helical structures could be rotated by optical tweezers.

Integrated optical motor.

A light-driven micrometer-sized mechanical motor is created by laser-light-induced two-photon photopolymerization. All necessary components of the engine are built upon a glass surface by an identical procedure and include the following: a rigid mechanical framework, a rotor freely rotating on an axis, and an integrated optical waveguide carrying the actuating light to the rotor. The resulting product is a most practical stand-alone system. The light introduced into the integrated optical waveguide input of the motor provides the driving force: neither optical tweezers or even a microscope are needed for the function. The power and efficiency of the motor are evaluated. The independent unit is expected to become an important component of more complex integrated lab-on-a-chip devices.