You-Ming Hsu

Dielectrophoretic chip with multilayer electrodes and microcavity arrays for trapping and programmable releasing of single cells

Cell characterization analysis usually involves a sequence of steps such as culture, separation, trapping, examination and recollection. In general, it is difficult to recover the identified cells and achieve a multi-run examination on a single chip for clinical samples. In the present study, a dielectrophoresis (DEP) micro-device was developed for multi-step manipulations of cells at the single-cell level. The structure of the DEP chip consisted of an indium tin oxide (ITO) top electrode, a flow chamber, a middle electrode on an SU-8 surface, a micro-cavity array of SU-8 and distributed electrodes at the bottom of the micro-cavities. The purpose of the three-layer-electrode design was threefold. First, cells could be trapped into the micro-cavities by negative DEP between the top and middle electrodes. After cells were trapped, cell analysis at the single-cell level could potentially be performed. This could include, for example, drug treatment or biomedical sensing on the chip without applying voltage. Once identified, the target cells could be individually released by controlling the bottom distributed electrodes. Finally, the rest of the trapped cells could be pulled out by a positive DEP force between the top and middle electrodes and flushed away for the next run of cell analysis. The multi-step manipulations of human bladder cancer cells (TSGH8301) were successfully demonstrated and discussed, providing an excellent platform technology for a lab-on-a-chip (LOC).

The effects of nanoparticles uptaken by cells on electrorotation

Electrorotation (ER) has become a very powerful diagnostic technique for the measurement of dielectric properties of cells. However, only a few papers have investigated the electric‐induced rotation of particles in a stationary alternating (AC) electric field instead of a rotating electric field. In this study, a microchip composed of a top‐grounded electrode, flow chamber and bottom chess‐type electrode arrays was used to construct a stationary non‐uniform AC electric field for the manipulation of cells by dielectrophoretic force. We focused on the effects of metal and dielectric nanoparticles uptaken by cells under ER, by using human promyelocytic leukemia cells (HL‐60), 13 nm Au and 19 nm SiO2 nanoparticles. As revealed by the experimental results, both the percentage of cells in rotation and the range of rotational (ROT) frequency for the uptake of Au nanoparticle cells were higher and wider than in the case of SiO2 nanoparticles. In addition, the rotation of lone cells and pearl‐chain cells under non‐uniform and uniform electric field were quantitatively investigated, respectively. The membrane capacitance and membrane conductance of HL‐60 cells can be extracted from the ROT spectra as 10.18±1.92 mF/m2 and 1500±321 S/m2, respectively. In general, the ER of cells in a stationary AC electric field can be attributed to the highly non‐uniform electric field and non‐uniform dispersion of nanoparticles within cells; therefore, the electrical properties of uptaken nanoparticles and the aggregation phenomenon have significant influences on the resulting electrical torque.

The effects of microstructures on a dielectrophoretic chip for trapping particles

A dielectrophoretic (DEP) chip with an SU‐8 microcavity array for trapping single particle/cell is designed, fabricated, and quantitatively examined by simulations and experiments. The particles can be easily trapped in or pulled out of the microcavity based on negative or positive DEP force, respectively. The nonuniform electric field is formed in relation to the configuration of the microcavity array, i.e. its diameter and spacing, as described in the simulation results. In order to investigate the effects of the microcavity, two maximal flow rates for trapping particles in the microcavity and washing them away from the microcavity under different DEP voltages are determined by experiments. As the experimental results show, the extrastationary effects, provided as a particle is trapped in the microcavity, mean that the trapped particle can sustain a low flow rate even without applying DEP voltage. Consequently, this DEP chip is suitable for long‐term monitoring of trapped cells by supplying the subsisting buffer at a low flow rate without the damage or heating effect caused by DEP voltage.

Multilayer Electrodes DEP Chip for Single-cell Level Impedance Measurement

The authors demonstrated the capability of cell trapping with combination of dielectrophoresis (DEP) and 3D microstructures array in the prior work (Chen-Hsin Chuang, 2006). In the present study, in order to measure the variation of electric properties for a single cell under biochemical reactions, a novel design of multilayer electrodes is utilized to achieve both DEP positioning and impedance measurement with single-cell resolution. In this chip, three electrodes are separated by flow chamber and SU-8 holes array, respectively. Firstly, bioparticles can be trapped in SU-8 holes array by applying AC power on top ITO electrode and middle Au electrode on SU-8 surface. According to experimental results, the trapped bioparticles will be constrained in stationary due to microstructures for a long period of time without applying DEP force. Sequentially, the impedance measurement for bioparticles or cells can be performed by LCR meter. As the results shown, the resulting impedance can be varied by the size of trapped bioparticle. Therefore, this study not only provides an efficient way for trapping cell but also an alternative way to identify the size of particles by impedance measurement.

Depth effects of DEP chip with microcavities array on impedance measurement for live and dead cells

We demonstrated the capability of cell trapping with combination of dielectrophoresis (DEP) and 3D microstructures array and also designed a multi-layer electrodes for impedance measurement of particles in the prior work. In the present study, in order to discriminate live and dead cells based on impedance measurement, a DEP chip consisted of microcavities array and three-layer electrodes was designed to trap cells and further impedance measurement. By introducing microstructure to DEP chip, the capability of positioning and immobilization of single cell could be achieved, however, some problems also accompanied along as impedance measurement, such as noise signal and less difference between different samples. Therefore, the depth effects on DEP force and electrical properties measurement were analyzed by numerical simulation. According to the numerical results, the microcavity with 10 mum in depth was the optimal design for this multi-functional DEP chip. In addition, two kinds of suspension cell lines, NB4 and HL-60, were utilized to examine the identification of live and dead cells by in vitro experiments. In our impedance measurement, the operation voltage is 0.2 V and the scan frequency is from 1 KHz to 3 MHz. The difference of impedance magnitude between live and dead cells was larger in the low frequency range; therefore, this microchip not only provides an efficient way to immobilization cells in the microcavity for a long period of time without applying DEP force but also easily identifies the live and dead cells based on impedance measurement.

Electrorotation of HL-60 cells uptake of metal and dielectric nanoparticles in a stationary AC electric field

Electrorotation has become a very powerful diagnostic technique for measurement of dielectric properties of cells. However, only few papers investigated in the electric-induced rotation of particles in a stationary alternating (AC) electric field instead of a rotating electric field. In this study, a microchip comprised top grounded electrode, flow chamber and bottom chess-type electrode arrays to construct a stationary nonuniform AC electric field for manipulation of cells by dielectrophoretic (DEP) force. We aimed at the discrepancy on electrorotation between metal (Au) and dielectric (SiO2) nanoparticles uptaken by the human promyelocytic leukemia cells (HL- 60). As experimental results, both the percentage of cells in rotation and the range of rotational frequency for uptaken-Au-nanoparticles cells were higher and wider than the case of SiO2 nanoparticles, respectively. In addition, the cell membrane for a continued rotating cell could be deformed and broken after 30 to 40 minutes. According to the rotational spectra (ROT), the membrane capacitance and membrane conductance of HL-60 cells can be extracted, 9.95 mF/m2 and 1460 S/m2, respectively. In general, the electrorotation of cells in a stationary AC electric field can be attributed to the highly nonuniform electric field and nonuniform dispersion of nanoparticles within cells; therefore, the electrical properties of uptaken nanoparticles and the aggregation phenomenon have significant influences on resulting electrical torque, the membrane capacitance and membrane conductance of HL-60 cells can be calculated in this method without a rotating electric field excited by poly-phase AC voltages.

The Study of Microfluidic Chip with Micro Cylindrical Post Array for Separating Particles

This study presents the design, simulation, fabrication and measurement of a microfluidic chip with micro cylindrical post array for separating particles. The structure of microfluidic chip is consisted of top glass, a micro channel with cylindrical post array, two Au end electrodes and bottom glass. Both in-line and staggered arrays of micro cylindrical posts were designed and made of SU-8 inside the micro channel by MEMS technology. The simulated results showed that the applied DC voltage at the end electrodes would generate a non-uniform electric field within the array of cylindrical posts, and particles would be attracted there. From the results of experiment, the minimum required DC voltages to drive micro particles flowing into the micro channel by electrokinetic effect were 15V and 30V, respectively, at the electrode spacing of 8mm and 25mm for both array types. Under the dielectrophoretic effect created by the non-uniform electric field, the latex particles could be concentrated around micro cylindrical posts with electric field intensity minima and separated from flowing fluid. This phenomenon is in agreement with the simulation.

Dielectrophoretic Frequency Effect on Purification and Field Emission of Carbon Nanotubes

This paper presents the design, fabrication and test of a field emission chip with carbon nanotubes (CNTs). The objective of this study was to utilize positive dielectrophoresis (DEP) to purify CNTs on parallel microelectrodes and then to perform a field emission test on the CNT film. Study parameters include the electrode width and gap, and the frequency of DEP. To understand the locations where the CNTs might be accumulated, a computer simulation was used to analyze the distribution of gradient of the square of electric field, VE, around the electrodes under the electric field. Three combinations of parallel electrodes, with various width and gap, were designed for the purification and field emission of CNTs. A test chip consisting of three parallel micro electrodes and a micro channel, formed by polydimethyl siloxane (PDMS), was manufactured by photolithography. The medium, also served as cathode surfactant and dispersant, was prepared by sodium dodecyl sulfate (SDS), and the medium with CNTs was fulfilled into the micro channel by syringe pump. By applying different DEP frequencies on the electrodes, the experiment on trapping and purification of CNTs was observed and analyzed. The results showed the CNTs would be accumulated around the edges of electrodes where higher values of VE existed. This trend was in good agreement with the simulation. After the CNTs were purified, Raman spectroscopy showed that the relative intensity ratio of G-band to D-band increased from 0.36 to 0.56 for the samples prepared by DEP at frequencies from 1 to 25 MHz, while the value of non-purified CNTs was 0.29. Therefore, higher frequency of DEP resulted in better purification of CNTs. Followed by the field emission test on the CNT film under voltage ranging from 0 to 1100 V. Results revealed that the CNT film prepared at higher frequency of DEP had a lower threshold of electric field for field emission.

Impedance Analysis of HL-60 Cells Uptake of Au Nanoparticles by Using DEP Chip with Multi-layer Electrodes and SU-8 Microstructures

In this study, a DEP chip with multi-layer electrodes and microcavities array was designed for trapping cells and further impedance measurement. The capability of trapping cells can be achieved single-cell level and without overlapping due to the effects of SU-8 microcavities. However, the depth of microcavity also could affect the results of impedance measurement owing to the insulation layer of SU-8. Two kinds of DEP chip with different thickness of SU-8 layer were, therefore, fabricated and performed to investigate the microstructural effects on impedance measurement. By using this microchip, we can easily identify whether cells (HL-60) are uptaken of Au nanoparticles or not according to the impedance spectrum. Consequently, this method provides an alternative way to separate normal cells and cells uptake of nanoparticles for further inspection.