Meng-Hua Yen

Crack-free direct-writing on glass using a low-power UV laser in the manufacture of a microfluidic chip

Glass is an excellent material for use as a microfluidic chip substrate because it has great chemical and thermal stability. This work describes a flexible platform for the rapid prototyping of microfluidic chips fabricated from glass. A debris-free laser direct-writing technology that requires no photomask generation is developed. A 266 nm laser with a high repetition rate is employed in laser-induced backside wet etching (LIBWE) for glass machining. A microfluidic pattern is designed using computer drawing software and then automatically translated into computer numerical control motion so that the microtrench is directly fabricated on the glass chip. The overall machining speed can be increased by increasing the repetition rate to ~6 kHz. Without a clean room facility or the highly corrosive acid, HF, the overall development time is within hours. Trenches with complex structures that are hard to fabricate by photolithography were easily produced by laser direct-writing. An integrated microreactor/concentrator is demonstrated. The crack-free and debris-free surface was characterized by SEM and a surface profiler. Various effective etching chemicals for the LIBWE process were investigated to understand the etching mechanism. The minimal laser power used for glass etching was approximately 20 mW for a 6 µm wide microtrench. Several new compounds have been demonstrated to be effective in ablation. The etch threshold is minimum and does not decrease further as the unit length absorbance increases above 8000 in acetone solution.

Electrotaxis of lung cancer cells in a multiple-electric-field chip

We report a microfluidic cell culture chip that was used for long-term electrotaxis study on a microscope. The cellular response under three different electric field strengths was studied in a single channel microfluidic chip. Electric field (EF) inside the microchamber was numerically simulated and compared to the measured value. Lung cancer cell lines with high and weak metastasis potential, CL1-5 and CL1-0, respectively, were used to demonstrate the function of the multi-field chip (MFC). The two cell lines exhibited greatly different response under the applied EF of E=74-375 mV/mm. CL1-5 cells migrated toward the anode while CL1-0 cells did not show obvious response. Under the applied EF, cell orientation was observed accompanying the cell migration. Judging from the different temporal responses of the orientation and the migration, it is proposed that the two EF-induced responses may involve different signaling pathways.

A transparent cell-culture microchamber with a variably controlled concentration gradient generator and flow field rectifier

Real-time observation of cell growth provides essential information for studies such as cell migration and chemotaxis. A conventional cell incubation device is usually too clumsy for these applications. Here we report a transparent microfluidic device that has an integrated heater and a concentration gradient generator. A piece of indium tin oxide (ITO) coated glass was ablated by our newly developed visible laser-induced backside wet etching (LIBWE) so that transparent heater strips were prepared on the glass substrate. A polymethylmethacrylate (PMMA) microfluidic chamber with flow field rectifiers and a reagent effusion hole was fabricated by a CO(2) laser and then assembled with the ITO heater so that the chamber temperature can be controlled for cell culturing. A variable chemical gradient was generated inside the chamber by combining the lateral medium flow and the flow from the effusion hole. Successful culturing was performed inside the device. Continuous long-term (>10 days) observation on cell growth was achieved. In this work the flow field, medium replacement, and chemical gradient in the microchamber are elaborated.

Rapid cell-patterning and microfluidic chip fabrication by crack-free CO2 laser ablation on glass

This paper uses a widely available CO2 laser scriber (λ = 10.6 µm) to perform the direct-writing ablation of quartz, borofloat and pyrex substrates for the development of microfluidic chips and cell chips. The surface quality of the ablated microchannels and the presence of debris and distortion are examined by scanning electron microscopy, atomic force microscopy and surface profile measurement techniques. The developed laser ablation system provides a versatile and economic approach for the fabrication of glass microfluidic chips with crack-free structures. In the laser writing process, the desired microfluidic patterns are designed using commercial computer software and are then transferred to the laser scriber to ablate the trenches. This process eliminates the requirement for corrosive chemicals and photomasks, and hence the overall microchip development time is limited to less than 24 h. Additionally, since the laser writing process is not limited by the dimensions of a photomask, the microchannels can be written over a large substrate area. The machining capability and versatility of the laser writing system are demonstrated through its application to the fabrication of a borofloat microfluidic chip and the writing of a series of asymmetric trenches in a microwell array. It is shown that the minimum attainable trench width is 95 µm and that the maximum trench depth is 225 µm. The system provides an economic and powerful means of rapid glass microfluidic chip development. A rapid cell-patterning method based on this method is also demonstrated.

The enhancement of dermal papilla cell aggregation by extracellular matrix proteins through effects on cell-substratum adhesivity and cell motility.

Generally, cells tend to aggregate on a substratum with lower cell adhesivity. However, it also leads to compromised cell growth and higher cell loss after seeding. This study is aimed at tackling this dilemma by extracellular matrix (ECM) protein coating of a lower adhesive substratum poly(ethylene-co-vinyl alcohol) (EVAL) that has been shown to facilitate hair follicle dermal papilla (DP) spheroid formation. We found that coating with either fibronectin (Fn), collagen I, or collagen IV yields higher adhesivity and cell growth than that with laminin. However, cells can only aggregate on uncoated or Fn-coated EVAL. Quantitatively, Fn coating increases the number of spheroids by 67%. Analysis of cell migration reveals that collagen I, collagen IV and laminin coatings reduce cell motility, while Fn coating keeps cells highly motile. Inhibition of cell migration hinders spheroid formation. In addition, disruption of Fn function does not significantly compromise intercellular adhesion. Hence, Fn enhances cell aggregation by enhancing cell attachment, cell growth and cell motility. Our study demonstrates that intercellular organization as spheroids or flat monolayers is switchable by specific ECM protein coating and preserving cell motility is vital to cell aggregation. In addition to generation of spheroidal DP microtissues for hair follicle regeneration and large-scale production of aggregates of other cells, this strategy can help to regulate the tissue-substrate adhesivity and tissue spreadability on the surface of implantable materials.

Crack-free micromachining on glass using an economic Q-switched 532 nm laser

Laser-induced backside wet etching (LIBWE) is an effective method for crack-free etching of transparent materials such as glass and quartz. Traditionally, LIBWE is performed using ultraviolet (UV) laser sources. However, this study describes the use of an economic Q-switched 532 nm green laser in the LIBWE microfabrication of sodalime glass substrates. Using a common organic dye (Rose Bengal) as the photoetchant, crack-free microstructures with a minimum feature size of 18 µm are obtained. The typical etch rate is approximately 10 to 70 nm/pulse and the maximum attainable depth is found to be approximately 65 µm. The etch threshold is 5.7 J cm−2. The surface quality of the micro-trenches produced by the visible LIBWE source is comparable to that obtained in the traditional UV LIBWE process. Microtrenching in sodalime is demonstrated to show the feasibility of microfluidic chip development using visible LIBWE.

Gene Expression of Human Lung Cancer Cell Line CL1-5 in Response to a Direct Current Electric Field

Background Electrotaxis is the movement of adherent living cells in response to a direct current (dc) electric field (EF) of physiological strength. Highly metastatic human lung cancer cells, CL1–5, exhibit directional migration and orientation under dcEFs. To understand the transcriptional response of CL1–5 cells to a dcEF, microarray analysis was performed in this study. Methodology/Principal Findings A large electric-field chip (LEFC) was designed, fabricated, and used in this study. CL1–5 cells were treated with the EF strength of 0mV/mm (the control group) and 300mV/mm (the EF-treated group) for two hours. Signaling pathways involving the genes that expressed differently between the two groups were revealed. It was shown that the EF-regulated genes highly correlated to adherens junction, telomerase RNA component gene regulation, and tight junction. Some up-regulated genes such as ACVR1B and CTTN, and some down-regulated genes such as PTEN, are known to be positively and negatively correlated to cell migration, respectively. The protein-protein interactions of adherens junction-associated EF-regulated genes suggested that platelet-derived growth factor (PDGF) receptors and ephrin receptors may participate in sensing extracellular electrical stimuli. We further observed a high percentage of significantly regulated genes which encode cell membrane proteins, suggesting that dcEF may directly influence the activity of cell membrane proteins in signal transduction. Conclusions/Significance In this study, some of the EF-regulated genes have been reported to be essential whereas others are novel for electrotaxis. Our result confirms that the regulation of gene expression is involved in the mechanism of electrotactic response.

ITO patterning by a low power Q-switched green laser and its use in the fabrication of a transparent flow meter

A new ITO patterning method is reported and then applied for fabricating a transparent gas flow meter. Laser-induced backside wet etching using a low power green laser (visible-LIBWE) was used for direct-write ITO patterning. The obtained ablation result shows a smoother surface and less debris than that obtained from front-side laser ablation. Surface quality was examined by SEM, energy dispersive spectroscopy, electron spectroscopy for chemical analysis and surface profiler, and was compared to the etching result by UV and IR lasers. Electrical isolation was obtained using this new ITO ablation method. The method was then utilized in fabricating a transparent gas flow meter. The gas flow was measured by monitoring the resistance change of ITO micro strips by a multimeter. The flow meters detection limit was estimated to be 0.24 ml min−1 and its response time was 2.6 s.

Programmable Laser-Assisted Surface Microfabrication on a Poly(Vinyl Alcohol)-Coated Glass Chip with Self-Changing Cell Adhesivity for Heterotypic Cell Patterning

Organs are composed of heterotypic cells with patterned architecture that enables intercellular interaction to perform specific functions. In tissue engineering, the ability to pattern heterotypic cells into desired arrangement will allow us to model complex tissues in vitro and to create tissue equivalents for regeneration. This study was aimed at developing a method for fast heterotypic cell patterning with controllable topological manipulation on a glass chip. We found that poly(vinyl alcohol)-coated glass showed a biphasic change in adhesivity to cells in vitro: low adhesivity in the first 24 h and higher adhesivity at later hours due to increased serum protein adsorption. Combining programmable CO2 laser ablation to remove poly(vinyl alcohol) and glass, we were able to create arrays of adhesive microwells of adjustable patterns. We tested whether controllable patterns of epithelial-mesenchymal interaction could be created. When skin dermal papilla cells and fibroblasts were seeded respectively 24 h apart, we were able to pattern these two cells into aggregates of dermal papilla cells in arrays of microwells in a background of fibroblasts sheet. Seeded later, keratinocytes attached to these mesenchymal cells. Keratinocytes contacting dermal papilla cells started to differentiate toward a hair follicle fate, demonstrating patternable epithelial-mesenchymal interaction. This method allows fast adjustable heterotypic cell patterning and surface topology control and can be applied to the investigation of heterotypic cellular interaction and creation of tissue equivalent in vitro.

Crack-free laser direct-writing on quartz and glass for microfluidic chip development

We present a flexible microfluidic channel fabrication platform that can be used to develop microfluidic chips. A DPSS (diode pumped solid state) frequency quadrupled (λ = 266 nm, the UV system) Nd:YAG laser and a CO2 laser (λ = 10.6 μm, the IR system) are compared for their ablation capability on quartz and glass. We have also compared their performance in developing microfluidic chips. The resultant surface quality, including microcracking, debris, and distortion, is examined by SEM and a surface profiler. In these systems, users design microfluidic patterns by commercial software. The pattern is then transferred to a CNC stage for trenching. The microfabrication process can be completed in several minutes. Without the need to fabricate photomask for patterning, the development time can be reduced from weeks to hours. In addition, the substrate size is not limited by the dimension of the photomask. Asymmetric trenches demonstrating the machining capability of these systems have been fabricated by these systems. The minimal feature for the IR system and the UV system is 140 μm and 5 μm, respectively. These systems are very powerful for rapid glass microfluidic chip development.