Dean-Flow Affected Lateral Focusing and Separation of Particles and Cells in Periodically Inhomogeneous Microfluidic Channels.

The purpose of the recent work is to give a better explanation of how Dean vortices affect lateral focusing, and to understand how cell morphology can alter the focusing position compared to spherical particles. The position and extent of the focused region were investigated using polystyrene fluorescent beads with different bead diameters (Ø = 0.5, 1.1, 1.97, 2.9, 4.8, 5.4, 6.08, 10.2, 15.8, 16.5 µm) at different flow rates (0.5, 1, 2 µL/s). Size-dependent focusing generated a precise map of the equilibrium positions of the spherical beads at the end of the periodically altering channels, which gave a good benchmark for focusing multi-dimensional particles and cells. The biological samples used for experiments were rod-shaped Escherichia coli (E. coli), discoid biconcave-shaped red blood cells (RBC), round or ovoid-shaped yeast, Saccharomyces cerevisiae, and soft-irregular-shaped HeLa cancer-cell-line cells to understand how the shape of the cells affects the focusing position at the end of the channel.

Geometry-Dependent Efficiency of Dean-Flow Affected Lateral Particle Focusing and Separation in Periodically Inhomogeneous Microfluidic Channels.

In this study, inertial focusing phenomenon was investigated, which can be used as a passive method for sample preparation and target manipulation in case of particulate suspensions. Asymmetric channel geometry was designed to apply additional inertial forces besides lift forces to promote laterally ordered particles to achieve sheathless focusing or size-dependent sorting. The evolving hydrodynamic forces were tailored with altered channel parameters (width and height), and different flow rates, to get a better understanding of smaller beads' lateral migration. Fluorescent beads (with the diameter of 4.8 µm and 15.8 µm) were used to distinguish the focusing position in continuous flow, and experimental results were compared to in silico models for particle movement prediction, made in COMSOL Multiphysics. The focusing behaviour of the applied microfluidic system was mainly characterised for particle size in the range close to blood cells and bacteria.

Development and In-Depth Characterization of Bacteria Repellent and Bacteria Adhesive Antibody-Coated Surfaces Using Optical Waveguide Biosensing.

Bacteria repellent surfaces and antibody-based coatings for bacterial assays have shown a growing demand in the field of biosensors, and have crucial importance in the design of biomedical devices. However, in-depth investigations and comparisons of possible solutions are still missing. The optical waveguide lightmode spectroscopy (OWLS) technique offers label-free, non-invasive, in situ characterization of protein and bacterial adsorption. Moreover, it has excellent flexibility for testing various surface coatings. Here, we describe an OWLS-based method supporting the development of bacteria repellent surfaces and characterize the layer structures and affinities of different antibody-based coatings for bacterial assays. In order to test nonspecific binding blocking agents against bacteria, OWLS chips were coated with bovine serum albumin (BSA), I-block, PAcrAM-g-(PMOXA, NH2, Si), (PAcrAM-P) and PLL-g-PEG (PP) (with different coating temperatures), and subsequent Escherichia coli adhesion was monitored. We found that the best performing blocking agents could inhibit bacterial adhesion from samples with bacteria concentrations of up to 107 cells/mL. Various immobilization methods were applied to graft a wide range of selected antibodies onto the biosensor's surface. Simple physisorption, Mix&Go (AnteoBind) (MG) films, covalently immobilized protein A and avidin-biotin based surface chemistries were all fabricated and tested. The surface adsorbed mass densities of deposited antibodies were determined, and the biosensor;s kinetic data were evaluated to divine the possible orientations of the bacteria-capturing antibodies and determine the rate constants and footprints of the binding events. The development of affinity layers was supported by enzyme-linked immunosorbent assay (ELISA) measurements in order to test the bacteria binding capabilities of the antibodies. The best performance in the biosensor measurements was achieved by employing a polyclonal antibody in combination with protein A-based immobilization and PAcrAM-P blocking of nonspecific binding. Using this setting, a surface sensitivity of 70 cells/mm2 was demonstrated.

Membrane-Based In Situ Mid-Infrared Spectroscopic Ellipsometry: A Study on the Membrane Affinity of Polylactide-co-glycolide Nanoparticulate Systems.

Mid-infrared (IR) ellipsometry of thin films and molecule layers at solid-liquid interfaces has been a challenge because of the absorption of light in water. It has been usually overcome by using configurations utilizing illumination through the solid substrate. However, the access to the solid-liquid interface in a broad spectral range is also challenging due to the limited transparency of most structural materials in the IR wavelength range. In this work, we propose a concept of a microfabricated analysis cell based on an IR-transparent Si membrane with advantages of a robust design, flexible adaptation to existing equipment, small volume, multiple-angle capabilities, broad wavelength range, and opportunities of multilayer applications for adjusted ranges of high sensitivity. The chamber was prepared by 3D micromachining technology utilizing deep reactive ion etching of a silicon-on-insulator wafer and bonded to a polydimethylsiloxane microfluidic injection system resulting in a cell volume of approximately 50 µL. The mechanical stability of the 2 and 5 µm-thick membranes was tested using different "backbone" reinforcement structures. It was proved that the 5 µm-thick membranes are stable at lateral cell sizes of 5 mm by 20 mm. The cell provides good intensity and adjustment capabilities on the stage of a commercial mid-IR ellipsometer. The membrane configuration also provides optical access to the sensing interfaces at a broad range of incident angles, which is a significant advantage in many potential sensing structure configurations, such as plasmonic, multilayer, 2D, or metamaterial applications.

Controlled Focused Ion Beam Milling of Composite Solid State Nanopore Arrays for Molecule Sensing.

Various nanoscale fabrication techniques are elaborated to form artificial nanoporous/nanochannel membranes to be applied for biosensing: one of the most prevalent is the micro-electromechanical systems (MEMS) compatible focused ion beam (FIB) milling. This technique can be easily adopted in micro- and nanomachining process sequences to develop composite multi-pore structures, although its precision and reproducibility are key points in the case of these thick multi-layered membranes. This work is to demonstrate a comprehensive characterisation of FIB milling to improve the reliability of the fabrication of solid state nanopore arrays with precisely predetermined pore geometries for a targeted molecule type to be recognised. The statistical geometric features of the fabricated nanopores were recorded as the function of the process parameters, and the resulting geometries were analysed in detail by high resolution scanning electron microscope (SEM), transmission electron microscope (TEM) and ion scanning microscopy. Continuous function of the pore diameter evolution rate was derived from the experimental results in the case of different material structures, and compared to former dissentient estimations. The additional metal layer was deposited onto the backside of the membrane and grounded during the ion milling to prevent the electrical charging of dielectric layers. The study proved that the conformity of the pore geometry and the reliability of their fabrication could be improved significantly. The applicability of the developed nanopore arrays for molecule detection was also considered by characterising the pore diameter dependent sensitivity of the membrane impedance modulation based measurement method.

Preparation and Characterization of Perforated SERS Active Array for Particle Trapping and Sensitive Molecular Analysis.

A gold-coated array of flow-through inverse pyramids applicable as substrate for entrapment and immobilization of micro-objects and for surface enhanced Raman spectroscopic measurements was fabricated using bulk micromachining techniques from silicon. Surface morphology, optical reflectance, immobilization properties, and surface enhanced Raman amplification of the array were modelled and characterized. It was found that the special perforated periodic 3D structure can be used for parallel particle and cell trapping and highly sensitive molecular analysis of the immobilized objects.

Tilted pillar array fabrication by the combination of proton beam writing and soft lithography for microfluidic cell capture Part 2: Image sequence analysis based evaluation and biological application.

As a continuation of our previously published work, this paper presents a detailed evaluation of a microfabricated cell capture device utilizing a doubly tilted micropillar array. The device was fabricated using a novel hybrid technology based on the combination of proton beam writing and conventional lithography techniques. Tilted pillars offer unique flow characteristics and support enhanced fluidic interaction for improved immunoaffinity based cell capture. The performance of the microdevice was evaluated by an image sequence analysis based in-house developed single-cell tracking system. Individual cell tracking allowed in-depth analysis of the cell-chip surface interaction mechanism from hydrodynamic point of view. Simulation results were validated by using the hybrid device and the optimized surface functionalization procedure. Finally, the cell capture capability of this new generation microdevice was demonstrated by efficiently arresting cells from a HT29 cell-line suspension.

Detection of red blood cell surface antigens by probe-triggered cell collision and flow retardation in an autonomous microfluidic system.

Microfluidic devices exploit combined physical, chemical and biological phenomena that could be unique in the sub-millimeter dimensions. The current goal of development of Point-of-Care (POC) medical devices is to extract the biomedical information from the blood. We examined the characteristics of blood flow in autonomous microfluidic devices with the aim to realize sensitive detection of interactions between particulate elements of the blood and the appropriately modified surfaces of the system. As a model experiment we demonstrated the fast analysis of the AB0 blood group system. We observed that the accumulation of red blood cells immobilized on the capillary wall leads to increased lateral movement of the flowing cells, resulting in the overall selective deceleration of the red blood cell flow column compared to the plasma fraction. We showed that by monitoring the flow rate characteristics in capillaries coated with blood type reagents it is possible to identify red blood cell types. Analysis of hydrodynamic effects governing blood flow by Finite Element Method based modelling supported our observations. Our proof-of-concept results point to a novel direction in blood analysis in autonomous microfluidic systems and also provide the basis for the construction of a simple quantitative device for blood group determination.

Potentiometric sensing of nucleic acids using chemically modified nanopores.

Unlike the overwhelming majority of nanopore sensors that are based on the measurement of a transpore ionic current, here we introduce a potentiometric sensing scheme and demonstrate its application for the selective detection of nucleic acids. The sensing concept uses the charge inversion that occurs in the sensing zone of a nanopore upon binding of negatively charged microRNA strands to positively charged peptide-nucleic acid (PNA) modified nanopores. The initial anionic permselectivity of PNA-modified nanopores is thus gradually changed to cationic permselectivity, which can be detected simply by measuring the nanoporous membrane potential. A quantitative theoretical treatment of the potentiometric microRNA response is provided based on the Nernst-Planck/Poisson model for the nanopore system assuming first order kinetics for the nucleic acid hybridization. An excellent correlation between the theoretical and experimental results was observed, which revealed that the binding process is focused at the nanopore entrance with contributions from both in pore and out of pore sections of the nanoporous membrane. The theoretical treatment is able to give clear guidelines for further optimization of potentiometric nanopore-based nucleic acid sensors by predicting the effect of the most important experimental parameters on the potential response.

Automated single cell isolation from suspension with computer vision.

Current robots can manipulate only surface-attached cells seriously limiting the fields of their application for single cell handling. We developed a computer vision-based robot applying a motorized microscope and micropipette to recognize and gently isolate intact individual cells for subsequent analysis, e.g., DNA/RNA sequencing in 1-2 nanoliters from a thin (~100 µm) layer of cell suspension. It can retrieve rare cells, needs minimal sample preparation, and can be applied for virtually any tissue cell type. Combination of 1 µm positioning precision, adaptive cell targeting and below 1 nl liquid handling precision resulted in an unprecedented accuracy and efficiency in robotic single cell isolation. Single cells were injected either into the wells of a miniature plate with a sorting speed of 3 cells/min or into standard PCR tubes with 2 cells/min. We could isolate labeled cells also from dense cultures containing ~1,000 times more unlabeled cells by the successive application of the sorting process. We compared the efficiency of our method to that of single cell entrapment in microwells and subsequent sorting with the automated micropipette: the recovery rate of single cells was greatly improved.

Tilted pillar array fabrication by the combination of proton beam writing and soft lithography for microfluidic cell capture: Part 1 Design and feasibility.

Design, fabrication, integration, and feasibility test results of a novel microfluidic cell capture device is presented, exploiting the advantages of proton beam writing to make lithographic irradiations under multiple target tilting angles and UV lithography to easily reproduce large area structures. A cell capture device is demonstrated with a unique doubly tilted micropillar array design for cell manipulation in microfluidic applications. Tilting the pillars increased their functional surface, therefore, enhanced fluidic interaction when special bioaffinity coating was used, and improved fluid dynamic behavior regarding cell culture injection. The proposed microstructures were capable to support adequate distribution of body fluids, such as blood, spinal fluid, etc., between the inlet and outlet of the microfluidic sample reservoirs, offering advanced cell capture capability on the functionalized surfaces. The hydrodynamic characteristics of the microfluidic systems were tested with yeast cells (similar size as red blood cells) for efficient capture.

[Lab-on-a-chip systems in the point-of-care diagnostics]. / Lab-on-a-chip rendszerek a betegágy melletti diagnosztikában.

The need in modern medicine for near-patient diagnostics being able to accelerate therapeutic decisions and possibly replacing laboratory measurements is significantly growing. Reliable and cost-effective bioanalytical measurement systems are required which - acting as a micro-laboratory - contain integrated biomolecular recognition, sensing, signal processing and complex microfluidic sample preparation modules. These micro- and nanofabricated Lab-on-a-chip systems open new perspectives in the diagnostic supply chain, since they are able even for quantitative, high-precision and immediate analysis of special disease specific molecular markers or their combinations from a single drop of sample. Accordingly, crucial requirements regarding the instruments and the analytical methods are the high selectivity, extremely low detection limit, short response time and integrability into the healthcare information networks. All these features can make the hierarchical examination chain shorten, and revolutionize laboratory diagnostics, evolving a brand new situation in therapeutic intervention.

Computational fluid dynamics-based design of a microfabricated cell capture device.

A microfluidic cell capture device was designed, fabricated, evaluated by numerical simulations and validated experimentally. The cell capture device was designed with a minimal footprint compartment comprising internal micropillars with the goal to obtain a compact, integrated bioanalytical system. The design of the device was accomplished by computational fluid dynamics (CFD) simulations. Various microdevice designs were rapidly prototyped in poly-dimethylsiloxane using conventional soft lithograpy technique applying micropatterned SU-8 epoxy based negative photoresist as moulding replica. The numerically modeled flow characteristics of the cell capture device were experimentally validated by tracing and microscopic recording the flow trajectories using yeast cells. Finally, we give some perspectives on how CFD modeling can be used in the early stage of microfluidics-based cell capture device development.

Calibration-less sizing and quantitation of polymeric nanoparticles and viruses with quartz nanopipets.

The feasibility of using quartz nanopipets as simple and cost-effective Coulter counters for calibration-less quantitation and sizing of nanoparticles by resistive pulsing sensing (RPS) was investigated. A refined theory was implemented to calculate the size distribution of nanoparticles based on the amplitude of resistive pulses caused by their translocation through nanopipets of known geometry. The RPS provided diameters of monodisperse latex nanoparticles agreed within the experimental error with those measured by using scanning electron microscopy (SEM), dynamic light scattering (DLS), and nanoparticle tracking analysis (NTA). The nanopipet-based counter, by detecting individual nanoparticles, could resolve with similar resolution as SEM mixtures of monodisperse nanoparticles having partially overlapping size distributions, which could not be discriminated by DLS or NTA. Furthermore, by calculating the hydrodynamic resistance of the nanopipets and consequently the volume flow through the tip enabled for the first time the calibration-less determination of nanoparticle concentrations with nanopipets. The calibration-less methodology is applied to sizing and quantitation of inactivated poliovirus of ~26 nm diameter, which is the smallest size spherical shape virus ever measured by resistive pulse sensing.