Micromachined Coulter counter for dynamic impedance study of time sensitive cells
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This paper describes the design, modeling, fabrication and characterization MEMS Coulter counter that can detect and monitor the dynamic cell impedance changes in situ as a function of time after mixing isolated cell populations with different extracellular media within 0.3 s from the start of mixing. The novelty of this design is the use of multi-electrodes with vertical sidewalls to enable the measurements of time sensitive cells with significantly enhanced sensitivity as well as the integration of passive mixing, focusing of cells in line and impedance detection using the vertical electrodes on a single chip that is made mainly using multilayer of SU-8, which has not been reported before. The devices were tested with both fluidic and electrical functionality using yeast cells in cryoprotectant agent (diluted dimethyl sulfoxide), red blood cells, microbeads with different dimensions, and dyed fluids. The results demonstrate rapid changes of cell volume within the first 0.6 s after mixing followed by a stable and a fixed cell volume. The micromixer was initially simulated using COMSOL finite element tool. Image processing technique was used to quantitatively evaluate mixing efficiency by analyzing color intensities variation of captured images of 2 dyed fluids mixed in the channel at flow rates between 0.1–0.4 μl/min, the mixing efficiencies were between 87 %–95 %, respectively.
KeywordsCoulter counter Time sensitive cell Dynamic study of cell Red blood cell Yeast cells BioMEMS
Coulter counter is a standard diagnostic device for counting and sizing cells and particles widely used in laboratory medicine and pathology, clinical diagnostics, environmental monitoring, and food pathogen screening (Levin et al. 1980; Gast et al. 2006). These devices are used to perform rapid, accurate analysis of blood, colloidal particles, antigens, pollen and viruses, and other cells and tissues (England-Down 1975). Commercially available Coulter counters have a number of limitations. They are configured to require relatively large sample volume and severely limit measurement of time sensitive cell characteristics (e.g. changes in volume in response to changes in solute concentrations). The sample size and time constraints are detrimental to accurate dynamic volume measurements for some cell types. For example, red blood cells rapidly adjust volumetrically to anisosmotic environments making measurements of water and solute permeability parameters impossible with existing Coulter counter technology. Our counter is specifically designed to measure the cell impedance changes within 0.3 s from the start of mixing. This time delay is determined by the time scale of the water permeation process for specific cell types (Levin et al. 1980; Papanek 1978; Milgram-Solomon).
Several groups have successfully demonstrated miniaturized Coulter counters with various designs using micromachining technology (Larsen et al. 1997; Koch et al. 1999; Zhe et al. 2007; Ateya et al. 2008; Saleh and Sohn 2003). These miniaturized Coulter counters are designed to measure impedance of cells using one or two electrode pairs and thus may only be used for cell counting purposes and static cell sizing. In addition, these methods cannot accurately measure the impedance of cells with significantly different cell geometry such as platelets. Miniaturized Coulter counters provide many advantages including significantly reduced sample volume, low cost, low power consumption, and portability (Koch et al. 1999). Currently, several groups have addressed many issues related to Coulter’s channel clogging, detection techniques, sensitivity and throughput. Coulter counters generally employ a focusing mechanism to confine a small well- defined volume sample to the center of the channel to prevent clogging of the entrance of the Coulter channel, and an electrode pair for impedance measurement as a means for cell counting. The cells are focused horizontally into a tight stream, or liquid aperture, to the approximate size of the cell or beads by two high flow rate sheath fluid flows using two-dimensional hydrodynamic focusing (Scott et al. 2008; Tsai et al. 2008; Gawad et al. 2001; Sundararajan et al. 2004). Since the physical dimensions of the channel are much larger than the Coulter aperture, the use of hydrodynamic focusing prevents channel blocking, but there is the possibility of fluid diffusion. Another drawback is the need for an additional reservoir for the sheath flow medium which has to be kept free of dust and bacteria. To resolve this issue, other groups proposed to use air as a sheath fluid, which eliminates the complex maintenance of the liquid reservoirs (Ateya et al. 2008; Lin et al. 2004). However, this technique has a limited sensitivity since the top and bottom fluid path is not focused. 3-D hydrodynamic focusing solves this issue by vertically focusing the sample to a size comparable to the cell dimensions, thus preventing cell overlap in the vertical direction (Tsai et al. 2008; Sundararajan et al. 2004; Chang et al. 2007; Mao et al. 2007), and enhancing sensitivity. This technique enables the device to probe particles with a wide range of diameters. Dielectrophoresis (DEP) focusing the motion of the cells in the channel is regulated by the negative DEP force in the cross flow direction and combined with the hydrodynamic flow force in the flow direction (Holmes et al. 2006; Wang et al. 2006). This is more desirable because it does not need sheath flow.
Several cell and particle detection techniques including optical and electrical impedance sensing techniques were used. The former includes fluorescence detection using a fluorescent marker to facilitate their optical detection and counting (Chen and Wang 2009); fluorescence detection with micromachined fibers as a waveguide on the microchip (Bernini et al. 2006); and laser-induced fluorescence techniques (Chen and Wang 2008). The later technique is label-free and more adaptable to miniaturization. It includes DC impedance sensing, low frequency (100 kHz) AC impedance sensing and high frequency (above 100 kHz) AC impedance sensing (Zheng et al. 2008a, b, c). The DC impedance sensing was first invented by Wallace H. Coulter (Coulter 1953). This technique is accurate for electrodes with large size because it reduces the electrode-electrolyte impedance. In this case, the cell volume is the only factor that determines the measured system impedance. In the MEMS-based particle counter, electrodes are in the micrometer range and double-layer impedance must be taken into account since it is inversely proportional to the electrode area (Zheng et al. 2008a, b). Reducing the system sensitivity may also cause irreversible oxidation of electrodes (Zheng and Tai 2006). The AC measurement could solve oxidation and bubble problems. At low frequency, the impedance of the cell, which also includes the double layer capacitance, is mainly determined by its volume. It is important to note that the double layer capacitance decreases as the frequency increases. At higher frequency, the intracellular structure contributes to the overall measured impedance (Nieuwenhuis et al. 2004), and the double layer capacitance becomes comparable to the stray capacitance between the sensing electrodes. Thus, the operational frequency cannot go beyond an upper limit point and has to be selected carefully in order to obtain signal from the particle or cell (Nieuwenhuis et al. 2004; Gawad et al. 2004). In order words, the high frequency requires more consideration of the electrode design and signal processing. The capacitive measurement technique measures the AC capacitance instead of DC resistance when a particle passes the sensing electrodes. Thus, the capacitive measurement is particularly useful in the case of low electrical conductance liquids because it is very difficult to detect the resistance change resulted from the passage of a particle in an insulating solution. Other measurement techniques include inductive measurements (Du et al. 2010), metal-oxide-semiconductor field-effect transistor (MOSFET) which detects the particles by monitoring the MOSFET drain current modulation (Sridhar et al. 2008), radio frequency reflectometer, and capacitance measurements (Murali et al. 2008).
The objective of this paper is to develop a MEMS Coulter counter that can detect and monitor dynamic cell impedance changes and cellular volumetric changes as function of time and at various temperatures in response to mixing isolated cell populations with different extracellular media by using a sequence of ten electrode pairs. The cellular volumetric changes can be used to accurately determine cell-membrane permeability characteristics that can be used to significantly improve the efficacy of cryopreservation procedures and enhance the cell survival rates. We report the detection of yeast cells, red blood cells and microbeads with diameters between 7–20 μm using series of vertical electrodes and through impedance measurement. We also report cell mixing efficiency using a passive mixer, and focusing of cells using dielectrophoresis and hydrodynamic focusing.
2 Coulter design and modeling
2.1 Design of mixing region
2.2 Design of cell focusing region
2.3 Design of detection zone
3 Device fabrication
4 Testing and results
4.1 Experimental set up
4.2 Mixing of fluids in the channel
This value was selected in cell study measurements as a trade-off between mixing time and mixing efficiency. With this flow rate, we achieved sufficient mixing and a velocity of 3.4 m/s which correspond to a time span between the start of mixing and the first impedance measurement of 0.3 s. It is important to note that the use of T-shaped channel with narrower cell center channel have improved our mixing efficiency without compromising the mixing time. In future experiment, the outer channel will be made four times wider than the center channel, and the focusing region channel width will be reduced by 20 % to 80 μm. These modifications will increase the mixing efficiency to 100 % and decrease the mixing time to 0.2 s.
4.3 Focusing electrode testing
4.4 Electrical testing
Each sampling point of those set of experiments was consisted of 25 samples of microbeads or cells. After each experiment, T-test was conducted to assess whether the mean of two consecutive sampling points were statistically different from each other. All the calculated T-tests p-values were smaller than 0.05 which indicates their means were statistically different from each other.
A novel MEMS Coulter counter has been designed for measuring the dynamics of single cell volume in response to mixing with various agents. The device uses passive mixing, the phenomenon of dielectrophoresis to focus the cells to the center of the channel, and Coulter principle to detect cells based on the change in impedance when they pass through the sensing zone. The fluidic testing which include mixing and focusing demonstrate sufficient mixing and satisfactory focusing. The electrical testing were performed using latex microbeads with diameters of 5 μm, 10 μm and 15 μm, yeast cells and red blood cells with dynamic volumes changes, validate the performance of the device. The device has the potential to provide data enabling the development of a mathematical model to describe the reaction of CPA and cells in cryopreservation.
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