Tzuen-Rong J. Tzeng

Distinguishing the viability of a single yeast cell with an ultra-sensitive radio frequency sensor

We propose and demonstrate a simple, ultra sensitive radio frequency (RF) sensor to detect a single yeast cell and distinguish its viability in a microfluidic channel. On-chip interference is used to cancel background probing signals to improve sensor sensitivity. Individual viable and nonviable yeast cells (approximately 5.83 +/- 0.85 microm in diameter) are measured with clear sensing and identification of these cells.

Microfluidic separation of live and dead yeast cells using reservoir-based dielectrophoresis

Separating live and dead cells is critical to the diagnosis of early stage diseases and to the efficacy test of drug screening, etc. This work demonstrates a novel microfluidic approach to dielectrophoretic separation of yeast cells by viability. It exploits the cell dielectrophoresis that is induced by the inherent electric field gradient at the reservoir-microchannel junction to selectively trap dead yeast cells and continuously separate them from live ones right inside the reservoir. This approach is therefore termed reservoir-based dielectrophoresis (rDEP). It has unique advantages as compared to existing dielectrophoretic approaches such as the occupation of zero channel space and the elimination of any mechanical or electrical parts inside microchannels. Such an rDEP cell sorter can be readily integrated with other components into lab-on-a-chip devices for applications to biomedical diagnostics and therapeutics.

Electrokinetic focusing and filtration of cells in a serpentine microchannel

Focusing cells into a single stream is usually a necessary step prior to counting and separating them in microfluidic devices such as flow cytometers and cell sorters. This work presents a sheathless electrokinetic focusing of yeast cells in a planar serpentine microchannel using dc-biased ac electric fields. The concurrent pumping and focusing of yeast cells arise from the dc electrokinetic transport and the turn-induced acdc dielectrophoretic motion, respectively. The effects of electric field (including ac to dc field ratio and ac field frequency) and concentration (including buffer concentration and cell concentration) on the cell focusing performance were studied experimentally and numerically. A continuous electrokinetic filtration of E. coli cells from yeast cells was also demonstrated via their differential electrokinetic focusing in a serpentine microchannel.

Continuous dielectrophoretic separation of particles in a spiral microchannel

Particle separation is a fundamental operation in the areas of biology and physical chemistry. A variety of force fields have been used to separate particles in microfluidic devices, among which electric field may be the most popular one due to its general applicability and adaptability. So far, however, electrophoresis‐based separations have been limited primarily to batchwise processes. Dielectrophoresis (DEP)‐based separations require in‐channel micro‐electrodes or micro‐insulators to produce electric field gradients. This article introduces a novel particle separation technique in DC electrokinetic flow through a planar double‐spiral microchannel. The continuous separation arises from the cross‐stream dielectrophoretic motion of particles induced by the non‐uniform electric field inherent to curved channels. Specifically, particles are focused by DEP to one sidewall of the first spiral, and then dielectrophoretically deflected toward the other sidewall of the second spiral at a particle‐dependent rate, leading to focused particle streams along different flow paths. This DEP‐based particle separation technique is demonstrated in an asymmetric double‐spiral microchannel by continuously separating a mixture of 5/10 μm particles and 3/5 μm particles.

Effect of laser fluence in laser‐assisted direct writing of human colon cancer cell

Purpose – The purpose of this paper is to study the effect of laser fluence on the post‐transfer cell viability of human colon cancer cells (HT‐29) during a typical biofabrication process, matrix‐assisted pulsed‐laser evaporation direct‐write (MAPLE DW).Design/methodology/approach – The post‐transfer cell viability in MAPLE DW depends on various operation conditions such as the applied laser fluence. HT‐29 cell was selected as a model mammalian cell to investigate the effect of laser fluence on the post‐transfer cell viability. MAPLE DW‐based HT‐29 cell direct writing was implemented using an ArF excimer laser under a wide range of laser fluence. Trypan blue dye‐exclusion was used to test the post‐transfer cell viability.Findings – It has been observed that: the HT‐29 cell viability decreases from 95 to 78 percent as the laser fluence increases from 258 to 1,482 mJ/cm2; and cell injury in this study is mainly due to the process‐induced mechanical stress during the cell droplet formation and landing proces...

Three-dimensional magnetic focusing of particles and cells in ferrofluid flow through a straight microchannel

Focusing particles and cells into a tight stream is often required in order for continuous flow detection, counting and sorting. So far a variety of particle focusing methods have been developed in microfluidic devices, among which magnetic focusing is still relatively new. We?develop in this work an approach to embedding symmetrically two repulsive permanent magnets about a straight rectangular microchannel in a PDMS-based microfluidic device. The closest distance between the magnets is limited only by the sizes of the embedded and holder magnets involved in the fabrication process. The developed device is used to implement and investigate the three-dimensional magnetic focusing of polystyrene particles in ferrofluid microflow with both the top- and side-view visualizations. The effects of flow speed and particle size on the particle focusing effectiveness are studied. The developed device is also applied to magnetically focus yeast cells in ferrofluid, which proves to be biocompatible as verified by a cell viability test. In addition, an analytical model is developed and found to be able to predict the experimentally observed particle and cell focusing behaviors with reasonable agreement.

Integrated electrical concentration and lysis of cells in a microfluidic chip

Lysing cells is an important step in the analysis of intracellular contents. Concentrating cells is often required in order to acquire adequate cells for lysis. This work presents an integrated concentration and lysis of mammalian cells in a constriction microchannel using dc-biased ac electric fields. By adjusting the dc component, the electrokinetic cell motion can be precisely controlled, leading to an easy switch between concentration and lysis of red blood cells in the channel constriction. These two operations are also used in conjunction to demonstrate a continuous concentration and separation of leukemia cells from red blood cells in the same microchannel. The observed cell behaviors agree reasonably with the simulation results.

Exploiting magnetic asymmetry to concentrate diamagnetic particles in ferrofluid microflows

Concentrating particles and cells for measurement or removal is often essential in many chemical and biological applications. Diamagnetic particle concentration has been demonstrated in magnetic fluids using two repulsive or attracting magnets, which in almost all cases are symmetrically positioned on the two sides of the particle-flowing channel. This work studies the effects of magnet asymmetry on the pattern and flow rate of diamagnetic particle concentration in ferrofluid flow through a straight rectangular microchannel. Two attracting permanent magnets with a fixed distance are each embedded on one side of the microchannel with a symmetric or an asymmetric configuration. A pair of symmetric counter-rotating circulations of concentrated particles is formed in the microchannel with a symmetric magnet configuration, which is found to grow in size and progress up the flow. In contrast, the single asymmetric circulation of concentrated particles formed in the microchannel with an asymmetric magnet configu...

Cytotoxic and potent CYP1 inhibitors from the marine algae Cymopolia barbata

Background Extracts from the marine algae Cymopolia barbata have previously shown promising pharmacological activity including antifungal, antitumor, antimicrobial, and antimutagenic properties. Even though extracts have demonstrated such bioactivity, isolated ingredients responsible for such bioactivity remain unspecified. In this study, we describe chemical characterization and evaluations of biological activity of prenylated bromohydroquinones (PBQ) isolated from the marine algae C. barbata for their cytotoxic and chemopreventive potential. Methods The impact of PBQs on the viability of cell lines (MCF-7, HT29, HepG, and CCD18 Co) was evaluated using the MTS assay. In addition, their inhibitory impact on the activities of heterologously expressed cytochrome P450 (CYP) enzymes (CYP1A1, CYP1A2, CYP1B1, CYP2C19, CYP2D6, and CYP3A4) was evaluated using a fluorescent assay. Results 7-Hydroxycymopochromanone (PBQ1) and 7-hydroxycymopolone (PBQ2) were isolated using liquid and column chromatography, identified using 1 H and 13 C NMR spectra and compared with the spectra of previously isolated PBQs. PBQ2 selectively impacted the viability of HT29, colon cancer cells with similar potency to the known chemotherapeutic drug, fluorouracil (IC50, 19.82 ± 0.46 μM compared to 23.50 ± 1.12 μM, respectively) with impact toward normal colon cells also being comparable (55.65 ± 3.28 compared to 55.51 ± 3.71 μM, respectively), while PBQ1 had no impact on these cells. Both PBQs had potent inhibition against the activities of CYP1A1 and CYP1B1, the latter which is known to be a universal marker for cancer and a target for drug discovery. Inhibitors of CYP1 enzymes by virtue of the prevention of activation of carcinogens such as benzo-a-pyrene have drawn attention as potential chemopreventors. PBQ2 potently inhibited the activity of CYP1B1 (IC50 0.14 ± 0.04 μM), while both PBQ1 and PBQ2 potently inhibited the activity of CYP1A1 (IC50s of 0.39 ± 0.05 μM and 0.93 ± 0.26 μM, respectively). Further characterizations showed partial noncompetitive enzyme kinetics for PBQ2 with CYP1B1 with a Ki of 4.7 × 10–3 ± 5.1 × 10–4 μM and uncompetitive kinetics with CYP1A1 (Ki = 0.84 ± 0.07 μM); while PBQ1 displayed partial non competitive enzyme kinetics with CYP1A1 (Ki of 3.07 ± 0.69 μM), noncompetitive kinetics with CYP1A2 (Ki = 9.16 ± 4.68 μM) and uncompetitive kinetics with CYP1B1 (Ki = 0.26 ± 0.03 μM) . Conclusions We report for the first time, two isolated ingredients from C. barbata, PBQ1 and PBQ2, that show potential as valuable chemotherapeutic compounds. A hydroxyl moiety resident in PBQ2 appears to be critical for selectivity and potency against the cancer colon cells, HT29, in comparison to the three other malignant cell lines studied. PBQs also show potency against the activities of CYP1 enzyme which may be a lead in chemoprevention. This study, the first on isolates from these marine algae, exemplifies the value of searching within nature for unique structural motifs that can display multiple biological activities.

Development of luminescent pH sensor films for monitoring bacterial growth through tissue

Although implanted medical devices (IMDs) offer many benefits, they are susceptible to bacterial colonization and infections. Such infections are difficult to treat because bacteria could form biofilms on the implant surface, which reduce antibiotics penetration and generate local dormant regions with low pH and low oxygen. In addition, these infections are hard to detect early because biofilms are often localized on the surface. Herein, an optical sensor film is developed to detect local acidosis on an implanted surface. The film contains both upconverting particles (UCPs) that serve as a light source and a pH indicator that alters the luminescence spectrum. When irradiated with 980 nm light, the UCPs produce deeply penetrating red light emission, while generating negligible autofluorescence in the tissue. The basic form of the pH indicator absorbs more of upconversion luminescence at 661 nm than at 671 nm and consequently the spectral ratio indicates pH. Implanting this pH sensor film beneath 6-7 mm of porcine tissue does not substantially affect the calibration curve because the peaks are closely spaced. Furthermore, growth of Staphylococcus epidermidis on the sensor surface causes a local pH decrease that can be detected non-invasively through the tissue.