Yaoping Liu

A microfluidic chip for highly efficient cell capturing and pairing.

This paper examined the feasibility of a microfluidics chip for cell capturing and pairing with a high efficiency. The chip was fabricated by the polydimethylsiloxane-based soft-lithography technique and contained two suction duct arrays set in parallel on both sides of a main microchannel. Cells were captured and paired by activating two sets of suction ducts one by one with the help of syringe pumps along with switching the cell suspensions inside the main microchannel correspondingly. The effects of suction flow rate and the dimensions of suction channels on the cell capturing and pairing efficiency were characterized. The present chip was capable of creating 1024 pairs of two different cell populations in parallel. The preliminary experimental results showed that the cell capturing efficiency was 100% and the pairing one was 88% with an optimal suction rate of 5 μl/min in the chip in the 2 μm-sized suction duct chip. The cell viability after capture inside the microfluidic device was 90.0 ± 5.3%. With this cell capturing and pairing chip, interaction between cells in a single pair mode can be studied. The ability to create cell pairs has a number of biological applications for cell fusion, cell-cell interaction studies, and cell toxicity screening.

Restraining non-specific adsorption of protein using Parylene C-caulked polydimethylsiloxane.

Non-specific adsorption (NSA) of proteins on surface is a critical issue in polydimethylsiloxane (PDMS)-based microfluidics, which may either considerably decrease the efficiency of a continuous flow reaction or cause a large background noise in a heterogeneous sensing. This work introduced a new method to restrain NSA of protein by caulking PDMS with Parylene C, i.e., forming a Parylene C-caulked PDMS (pcPDMS) surface. The caulking depth of Parylene C inside PDMS matrix was characterized by laser scanning confocal microscopy based on a detectable autofluorescence intensity difference between Parylene C and PDMS after being annealed at 270 °C for 2 h in nitrogen. NSA of bovine serum albumin (BSA) on the inner surfaces of PDMS and pcPDMS microchannels was experimentally compared. The results indicated that the adsorbed BSA on the pcPDMS surface were 35.2% of that on the pristine PDMS surface after the BSA solution flowing through the microchannels at a flow rate of 2000 nL/min, a typical scenario of the continuous flow reaction. In a case mimicking the heterogeneous sensing, after a 60 min washing of phosphate buffered saline flow on a pre-saturated BSA adsorbed surface, the residual BSA on the pcPDMS surface was only 4.5% of that on the pristine PDMS surface. Adsorption/desorption coefficients of BSA on the PDMS and the pcPDMS surfaces were extracted from the experimental results based on the first-order Langmuir model, which indicated that the pcPDMS has a lower adsorption coefficient (Ka ) and a higher desorption coefficient (Kd ), compared to those of the pristine PDMS. A preliminary experiment also indicated that Taq polymerase kept 93.0% activity after flowing through a pcPDMS microchannel, while only 28.9% activity was left after passing a pristine PDMS microchannel under the same operation condition.

Chromatic Confocal Imaging based mechanical test platform for micro porous membrane

This paper reports a mechanical test platform for micropore-arrayed membrane with a Chromatic Confocal Imaging to measure the deflection. A demonstration of the measurement is presented by testing a Parylene C filtration membrane. The test result is analyzed by a classical model of thin plate deflection and the deformation of the membrane in filtration process is estimated.

Mechanical strength of 2.5D parylene C micropore-arrayed filtration membrane

This work experimentally studied the mechanical strength of the previously reported Parylene C based micropore-array membrane with a high porosity. The results indicated that the membrane, though with a high porosity, is strong enough and has a negligible pore size variation (<30 nm) during an up-to 150 mL/min filtration. The equivalent Youngs modulus of the 2.5D filtration membrane were extracted preliminarily based on the experimental measurement, compared with theoretical prediction.

Regulation of cell arrangement using a novel composite micropattern

Micropatterning technique has been used to control single cell geometry in many researches, however, this is no report that it is used to control multicelluar geometry, which not only control single cell geometry but also organize those cells by a certain pattern. In this work, a composite protein micropattern is developed to control both cell shape and cell location simultaneously. The composite micropattern consists of a central circle 15 μm in diameter for single-cell capture, surrounded by small, square arrays (3 μm × 3 μm) for cell spreading. This is surrounded by a border 2 μm wide for restricting cell edges. The composite pattern results in two-cell and three-cell capture efficiencies of 32.1% ± 1.94% and 24.2% ± 2.89%, respectively, representing an 8.52% and 9.58% increase, respectively, over rates of original patterns. Fluorescent imaging of cytoskeleton alignment demonstrates that actin is gradually aligned parallel to the direction of the entire pattern arrangement, rather than to that of a single pattern. This indicates that cell arrangement is also an important factor in determining cell physiology. This composite micropattern could be a potential method to precisely control multi-cells for cell junctions, cell interactions, cell signal transduction, and eventually for tissue rebuilding study.

Caulking polydimethylsiloxane molecular networks by thermal chemical vapor deposition of Parylene-C

Surface functionalization of polydimethylsiloxane (PDMS) is important in developing high-performance microfluidic devices. This work applied the thermal chemical vapor deposition (t-CVD) of Parylene-C onto PDMS to caulk the molecular network while retaining the original surface properties for the oxygen plasma bonding. The very low deposition rates (for example, a nominal rate of 0.12 Å min-1 at 135 °C) of Parylene-C at elevated substrate temperatures enabled the reactive Parylene-C monomers to penetrate into the PDMS matrix up to 4.6 ± 0.1 μm (135 °C), which was verified for the first time by a scanning electron microscope with an energy dispersive X-ray analysis (SEM-EDAX). The Parylene-C caulked in the molecular network of PDMS matrix guaranteed an excellent resistance to small molecule permeations. Meanwhile, only discrete nucleation islands were formed on the top surface rather than a continuous Parylene-C layer as observed under the AFM scan, which made the processed PDMS surface ready for device assembly. This surface functionalization method has better long-term stability than the other wet-type rivals. The barrier for oxygen plasma bonding in previously reported dry surface treatments was also avoided, thereby, facilitating the device assembly. The present work successfully developed a novel pcPDMS (Parylene-C caulked PDMS) technique, which overcame the bonding difficulty in the previous works but retained the low small molecule permeability as before. Caulking a molecular network through the t-CVD of Parylene-C also demonstrated a new strategy of functionalizing polymer surfaces and preparing new hybrid materials for wide lab-on-a-chip applications.

2.5-Dimensional Parylene C micropore array with a large area and a high porosity for high-throughput particle and cell separation

Large-area micropore arrays with a high porosity are in high demand because of their promising potential in liquid biopsy with a large volume of clinical sample. However, a micropore array with a large area and a high porosity faces a serious mechanical strength challenge. The filtration membrane may undergo large deformation at a high filtration throughput, which will decrease its size separation accuracy. In this work, a keyhole-free Parylene molding process has been developed to prepare a large (>20 mm × 20 mm) filtration membrane containing a 2.5-dimensional (2.5D) micropore array with an ultra-high porosity (up to 91.37% with designed pore diameter/space of 100 μm/4 μm). The notation 2.5D indicates that the large area and the relatively small thickness (approximately 10 μm) of the fabricated membranes represent 2D properties, while the large thickness-to-width ratio (10 μm/ < 4 μm) of the spaces between the adjacent pores corresponds to a local 3D feature. The large area and high porosity of the micropore array achieved filtration with a throughput up to 180 mL/min (PBS solution) simply driven by gravity. Meanwhile, the high mechanical strength, benefiting from the 2.5D structure of the micropore array, ensured a negligible pore size variation during the high-throughput filtration, thereby enabling high size resolution separation, which was proven by single-layer and multi-layer filtrations for particle separation. Furthermore, as a preliminary demonstration, the prepared 2.5-dimensional Parylene C micropore array was implemented as an efficient filter for rare cancer cell separation from a large volume, approximately 10 cells in 10 mL PBS and undiluted urine, with high recovery rates of 87 ± 13% and 56 ± 13%, respectively.Membranes: Steep walls speed up separationsPolymer films that detect cancer cells in blood or urine samples using size-based filtrations can benefit from a strategy for improving the strength and throughput rate of micropore arrays. Yaoping Liu from Peking University in China and colleagues first fabricated a silicon wafer of close-packed micropillars, and then coated this template with the polymer film Parylene C. The team used reactive ion etching to carve out a hexagonal array of microscale openings in the polymer with unique dimensions—smaller-than normal pore separations for high flow rates, and thickness more than twice the width of the sidewall to give exceptional mechanical stability. Experiments proved the new array structure could selectively trap particles ranging from microbeads to cancer cells with throughputs hundreds of times faster than conventional filtering devices.

An annealing process for mechanical strength improvement of Parylene micropore-arrayed membrane

This paper used an effective annealing process to improve the performance of Parylene C micropore-arrayed membranes fabricated with Parylene C moulding strategy. The results indicated that the annealing treatment at 320°C for 2h in nitrogen sealed the keyholes, which were unavoidably generated in the conventional Parylene C moulding process, because of melting and reflow at high temperature. A chromatic confocal imaging (CCI) based platform was established to test the mechanical properties of the prepared Parylene C micropore-arrayed membranes after annealing treatment, with the one without annealing as comparison. The mechanical testing results demonstrated that the annealing treated Parylene C micropore-arrayed membranes presented 13.4% higher mechanical strength compared to the unannealed one.

Designable regulation of cell filtration through a micropore-arrayed filter

Micropore-arrayed filter has been considered as the only technique for rare cell separation from a large volume of clinical sample (>10 mL) with a high recovery rate. This work reports a designable regulation of cell filtration through the micropore-arrayed filter by tuning the cell deformability with cytochalasin D. Cytochalasin D was thought to have defined effects on cytoskeleton depolymerization. Cells treated with cytochalasin D had an increased deformability and passed through the micropore even if which is of smaller size. The effects of concentrations (0.5, 1.0, 2.0, 5.0, 10 µg/ml for 30 min) and durations (5, 10, 20, 30, 40 min at 5.0 µg/ml) on Hela cells were studied. Besides, the viability of cytochalasin D-treated cells was tested with trypan blue staining and were higher than 94%. This paper demonstrated a designable regulation of cell filtration through micropore-arrayed filter, which will be useful to establish an actively modulating method in cell filtration.