Ji-Yen Cheng

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 planar interdigitated ring electrode array via dielectrophoresis for uniform patterning of cells

Uniform patterning of cells is highly desirable for most cellular studies involving cell-cell interactions but is often difficult in an in vitro environment. This paper presents the development of a collagen-coated planar interdigitated ring electrode (PIRE) array utilizing positive dielectrophoresis to pattern cells uniformly. Key features of the PIRE design include: (1) maximizing length along the edges where the localized maximum in the electric field exists; (2) making the inner gap slightly smaller than the outer gap in causing the electric field strength near the center of a PIRE being generally stronger than that near the outer edge of the same PIRE. Results of human hepatocellular carcinoma cells, HepG2, adhered on a 6x6 PIRE array show that cells patterned within minutes with good uniformity (48+/-6 cells per PIRE). Cell viability test revealed healthy patterned cells after 24h that were still confined to the collagen-coated PIREs. Furthermore, quantification of fluorescence intensity of living cells shows an acceptable reproducibility of cell viability among PIREs (mean normalized intensity per PIRE was 1+/-0.138). The results suggest that the PIRE array would benefit applications that desire uniform cellular patterning, and improve both response and reproducibility of cell-based biosensors.

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.

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.

Magnetic nanoparticle-enhanced SPR on gold nanoslits for ultra-sensitive, label-free detection of nucleic acid biomarkers

We have demonstrated a detection method for the ultra-sensitive detection of an mRNA biomarker. The method utilizes functionalized magnetic nanoparticles (MNPs) for signal enhancement in conjunction with surface plasmon resonance (SPR) on gold nanoslits. The approach for detection includes double hybridization at two different specific locations in two steps. First, the biomarker target molecule is captured with MNPs, and second, MNPs carrying the target molecule are introduced to the SPR chip to hybridize with probes immobilized on the gold nanoslits. In this work, MNPs were applied for a dual purpose: to isolate the target molecule from the sample matrix to prevent non-specific binding and to enhance the SPR response. Gold nanoslits that provide SPR sensing were fabricated by nanoimprinting lithography on polycarbonate (PC) film. The film was integrated with a microliter volume microfluidic chip to form the SPR detection chip. This detection method was used to detect mRNA heterogeneous nuclear ribonucleoproteins (hnRNP B1) in two cancer cell lines, CL1-0 and CL1-5. hnRNP B1 is an mRNA biomarker that is overexpressed in lung cancer tissue in the early stage of cancer and can be found in the serum and plasma of lung cancer patients. A synthetic target molecule and extracted total RNA from the cell lines were used as samples. Without amplification and labeling of the target molecule, the SPR results demonstrate a specific and sensitive method for the detection of hnRNP B1 mRNA in extracted RNA from the two selected cell lines. The method is capable of measuring down to 30 fM of the target molecule in a 7 μl sample (corresponding to 1.26 × 10(5) molecules) without amplification and labeling of the target molecule.

Enhanced localized plasmonic detections using partially-embedded gold nanoparticles and ellipsometric measurements

A cost-effective, stable and ultrasensitive localized surface plasmon resonance (LSPR) sensor based on gold nanoparticles (AuNPs) partially embedded in transparent substrate is presented. Partially embedded AuNPs were prepared by thermal annealing of gold thin films deposited on glass at a temperature close to the glass transition temperature of the substrate. Annealed samples were optically characterized by using spectroscopic ellipsometry and compare with theoretical modeling to understand the optical responses from the samples. By combining the partially-embedded AuNPs substrate with a microfluidic flow cell and dove prism in an ellipsometry setup, an ultrasensitive change in the LSPR signal can be detected. The refractive index sensitivity obtained from the phase measurement is up to 1938 degrees/RIU which is several times higher than that of synthesized colloidal gold nanoparticles. The sample is further used to investigate the interactions between primary and secondary antibodies. The bio-molecular detection limit of the LSPR signal is down to 20 pM. Our proposed sensor is label free, non-destructive, with high sensitivity, low cost, and easy to fabricate. These features make it feasible for commercialization in biomedical applications.

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.

Evaluation of EGFR and RTK Signaling in the Electrotaxis of Lung Adenocarcinoma Cells under Direct-Current Electric Field Stimulation

Physiological electric field (EF) plays a pivotal role in tissue development and regeneration. In vitro, cells under direct-current electric field (dcEF) stimulation may demonstrate directional migration (electrotaxis) and long axis reorientation (electro-alignment). Although the biophysical models and biochemical signaling pathways behind cell electrotaxis have been investigated in numerous normal cells and cancer cells, the molecular signaling mechanisms in CL1 lung adenocarcinoma cells have not been identified. Two subclones of CL1 cells, the low invasive CL1-0 cells and the highly invasive CL 1-5 cells, were investigated in the present study. CL1-0 cells are non-electrotactic while the CL 1-5 cells are anodally electrotactic and have high expression level of epidermal growth factor receptor (EGFR), in this study, we investigated the generally accepted hypothesis of receptor tyrosine kinase (RTK) activation in the two cell lines under dcEF stimulation. Erbitux, a therapeutic drug containing an anti-EGFR monoclonal antibody, cetuximab, was used to investigate the EGFR signaling in the electrotaxis of CL 1-5 cells. To investigate RTK phosphorylation and intracellular signaling in the CL1 cells, large amount of cellular proteins were collected in an airtight dcEF stimulation device, which has advantages of large culture area, uniform EF distribution, easy operation, easy cell collection, no contamination, and no medium evaporation. Commercial antibody arrays and Western blotting were used to study the phosphorylation profiles of major proteins in CL1 cells under dcEF stimulation. We found that electrotaxis of CL 1-5 cells is serum independent and EGFR independent. Moreover, the phosphorylation of Akt and S6 ribosomal protein (rpS6) in dcEF-stimulated CL1 cells are different from that in EGF-stimulated cells. This result suggests that CL1 cells’ response to dcEF stimulation is not through EGFR-triggered pathways. The new large-scale dcEF stimulation device developed in the present work will aid the sample preparation for protein-based experiments.