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.
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.
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.
Abstract:In this work, advances in the fabrication technology and functional analysis of a polymer microfluidic system -as a significant part of a developed polymer photonic biosensor -are reported. Robust and cost-effective microfluidics in PDMS including sample preparation functions is designed and realized by using SU-8 moulding replica. Surface modification strategies using Triton X-100 and PDMS-PEO and their effect on device sealing and non-specific protein adsorption are investigated by contact angle measurement and in situ fluorescence microscopy.Response to Reviewers: Dear Editors and Reviewers, First of all, thank You for the effort in improving our paper. Regarding the reviewer comments, we made the following modifications regarding the latest version of the manuscript submitted. The modifications and the required supplements are submitted in pdf format with the revised manuscript.Thank You again for the efforts to improve our manuscript.
Regards, Péter Fürjes
The material aspects of a polymer based microfluidic structure were characterised considering the compatibility of the system with bioanalytical applications. The polydimethylsiloxane (PDMS) based channel system is to be integrated in a full polymer photonic biosensor device developed within the European Union project P3SENS (FP7-ICT4-248304).This work is intended to define a modified material composition, which is appropriate to improve both the wettability and the non-specific protein binding characteristics of the PDMS significantly. Triton X-100 (Sigma-Aldrich) surfactant was added to the raw PDMS before polymerisation. The influence of the tenside was studied considering the polymerisation reaction, the surface characteristics and the functional applicability. To test the hydrodynamic behaviour and non-specific protein adsorption on the surfaces, phosphate buffered saline (PBS) solution and fluorescent labelled human serum albumin (HSA) was applied in a microfluidic capillary system.
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.
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.
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