Eszter Gabriella Holczer

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.

Optimized Simulation and Validation of Particle Advection in Asymmetric Staggered Herringbone Type Micromixers

This paper presents and compares two different strategies in the numerical simulation of passive microfluidic mixers based on chaotic advection. In addition to flow velocity field calculations, concentration distributions of molecules and trajectories of microscale particles were determined and compared to evaluate the performance of the applied modeling approaches in the proposed geometries. A staggered herringbone type micromixer (SHM) was selected and studied in order to demonstrate finite element modeling issues. The selected microstructures were fabricated by a soft lithography technique, utilizing multilayer SU-8 epoxy-based photoresist as a molding replica for polydimethylsiloxane (PDMS) casting. The mixing processes in the microfluidic systems were characterized by applying molecular and particle (cell) solutions and adequate microscopic visualization techniques. We proved that modeling of the molecular concentration field is more costly, in regards to computational time, than the particle trajectory based method. However, both approaches showed adequate qualitative agreement with the experimental results.

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.

Design, realisation and validation of microfluidic stochastic mixers integrable in bioanalytical systems using CFD modeling

In this work we present the design aspects of special microfluidic structures applicable to dilute and transport analyte solutions (such as whole blood) to the sensing area of biosensors. Our goal is to design and realise a reliable microfluidic system which is applicable for effective sample transport and can accomplish simple sample preparation functions such as mixing to ensure homogeneous concentration distribution of the species along the fluidic channel. The behaviour of different chaotic mixers were analysed by numerical modeling and experimentally to determine their efficiency. At first we used the concentration distribution method, however because of numerical diffusion this required higher mesh resolutions. Using the particle tracing method is more efficient according to the experimental results and requires lower computational effort. The microstructures were realised by micro-fabrication in polydimethylsiloxane (PDMS) and integrated into a real microfluidic transport system. The functional performance was verified by biological analyte.

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.

Modelling and Characterisation of Droplet Generation and Trapping in Cell Analytical Two-Phase Microfluidic System

Present study analyses the influence of flow characteristics of special water-oil two-phase microfluidic systems regarding the droplet generation, cell encapsulation and trapping processes. Water droplets were dispersed in oil continuous phase with the requirement of precise size distribution to enable effective cell entrapment. The evolving droplet size and the number of encapsulated cells were examined considering the applied flow rate ratios of the two phases. The hydrodynamic behaviour of the microfluidic system was modelled by Finite Element Method (FEM) coupled with particle trajectory calculation applying COMSOL Multiphysics code. The experimental results were compared to the simulation and the applicability of our droplet based cell encapsulating and trapping microfluidic system was characterised.