A Microfluidic Device for Impedance Spectroscopy

Recently, microfluidics has become a versatile tool to investigate cellular biology and to build novel biomedical devices. Dielectric spectroscopy, on the other hand, allows non-invasive probing of biological cells. Information on the cell membrane, cytoplasm, and nucleus can be obtained by dielectric spectroscopy provided that appropriate tools are used in specific frequency ranges. This dissertation includes fabrication, characterization, and testing of a simple microfluidic device to measure cell dielectric properties. The dielectric measurements are performed on human T-cell leukemia (Jurkat), mouse melanoma (B16), mouse hepatoma (Hepa), and human costal chondrocyte cells. Dielectric measurements consist of measuring the complex impedance of cell suspensions as a function of frequency. Physical models are fitted to raw impedance data to obtain parameters for cell compartments. The dielectric measurements are further supported by dielectrophoresis (DEP) experiments. Crossover frequency, which is the applied frequency when the DEP force is equal to zero, is recorded for cells by changing buffer conductivity. Cell membrane properties are also estimated from the crossover frequency measurements. Sensing capability of the microfluidic device to external stimuli is tested with Jurkat, chondrocyte, and Hepa cells. Jurkat and chondrocyte cells are suspended in buffers with changing osmolarity, and cell membrane properties are probed. Results indicate osmotic swelling of Jurkat cells. Interestingly similar changes were not observed in chondrocyte cells. Ion efflux from Hepa cells is quantified by conductivity measurements, and ionic flux from an average cell is calculated. Finally, a separability parameter is introduced and plotted for Jurkat and B16 cells pair. The separability parameter is based on the difference of two cells\u27 Clausius-Mossotti factors, which is a function of the dielectric parameters of the cells, field frequency, and buffer conductivity. Using the separability maps one can choose the optimum conditions for cell separation using DEP

Electrical System for Bioelectric Impedance using AD5933 Impedance Converter

Benchtop lab equipment, such as the HP4194A Precision Impedance Analyzer, offer accuracy and functionality but are often expensive and bulky. Although integrated circuits offer less functionality than benchtop equivalents, they can be a cheaper alternative for smaller applications. In this study, the AD5933 Impedance Converter was investigated as a low-cost option for impedance analysis of the bicep brachii. The AD5933 evaluation board was properly calibrated to measure human muscle by using an equivalent parallel circuit consisting of a 5 nF capacitor and a 1 kOhm resistor. Next, the impedance of the bicep was measured using the AD5933 as the experimental device and the HP4194A as the control device. A 2 volt peak-to- peak signal was generated for a 10 kHz to 100 kHz frequency range and the resulting impedance of the bicep was measured. The AD5933 recorded impedance curves qualitatively and quantitatively similar to those recorded by the HP4194A. Thus, the AD5933 was considered to be a low- cost alternative for impedance analysis in small-scale medical applications

Dielectric Characterization of Coastal Cartilage Chondrocytes

BACKGROUND: Chondrocytes respond to biomechanical and bioelectrochemical stimuli by secreting appropriate extracellular matrix proteins that enable the tissue to withstand the large forces it experiences. Although biomechanical aspects of cartilage are well described, little is known of the bioelectrochemical responses. The focus of this study is to identify bioelectrical characteristics of human costal cartilage cells using dielectric spectroscopy. METHODS: Dielectric spectroscopy allows non-invasive probing of biological cells. An in house computer program is developed to extract dielectric properties of human costal cartilage cells from raw cell suspension impedance data measured by a microfluidic device. The dielectric properties of chondrocytes are compared with other cell types in order to comparatively assess the electrical nature of chondrocytes. RESULTS: The results suggest that electrical cell membrane characteristics of chondrocyte cells are close to cardiomyoblast cells, cells known to possess an array of active ion channels. The blocking effect of the non-specific ion channel blocker gadolinium is tested on chondrocytes with a significant reduction in both membrane capacitance and conductance. CONCLUSIONS: We have utilized a microfluidic chamber to mimic biomechanical events through changes in bioelectrochemistry and described the dielectric properties of chondrocytes to be closer to cells derived from electrically excitably tissues. GENERAL SIGNIFICANCE: The study describes dielectric characterization of human costal chondrocyte cells using physical tools, where results and methodology can be used to identify potential anomalies in bioelectrochemical responses that may lead to cartilage disorders

Enhanced Killing Effect of Nanosecond Pulse Electric Fields on PANC1 and Jurkat Cell Lines in the Presence of Tween 80

We investigated the effects of nanosecond pulse electric fields (nsPEFs) on Jurkat and PANC1 cells, which are human carcinoma cell lines, in the presence of Tween 80 (T80) at a concentration of 0.18% and demonstarted an enhanced killing effect. We used two biological assays to determine cell viability after exposing cells to nsPEFs in the presence of T80 and observed a significant increase in the killing effect of nsPEFs. We did not see a toxic effect of T80 when cells were exposed to surfactant alone. However, we saw a synergistic effect when cells exposed to T80 were combined with the nsPEFs. Increasing the time of exposure for up to 8 h in T80 led to a significant decrease in cell viability when nsPEFs were applied to cells compared to control cells. We also observed cell type-specific swelling in the presence of T80. We suggest that T80 acts as an adjuvant in facilitating the effects of nsPEFs on the cell membrane; however, the limitations of the viability assays were addressed. We conclude that T80 may increase the fragility of the cell membrane, which makes it more susceptible to nsPEF-mediated killing

Probing Nanoparticle Interactions in Cell Culture Media

Nanoparticle research is often performed in vitro with little emphasis on the potential role of cell culture medium. In this study, gold nanoparticle interactions with cell culture medium and two cancer cell lines (human T-cell leukemia Jurkat and human pancreatic carcinoma PANC1) were investigated. Gold nanoparticles of 10, 25, 50, and 100 nm in diameter at fixed mass concentration were tested. Size distributions and zeta potentials of gold nanoparticles suspended in deionized (DI) water and Dulbecco\u27s Modified Eagle\u27s Media (DMEM) supplemented with fetal calf serum (FCS) were measured using dynamic light scattering (DLS) technique. In DI water, particle size distributions exhibited peaks around their nominal diameters. However, the gold nanoparticles suspended in DMEM supplemented with FCS formed complexes around 100 nm, regardless of their nominal sizes. The DLS and UV-vis spectroscopy results indicate gold nanoparticle agglomeration in DMEM that is not supplemented by FCS. The zeta potential results indicate that protein rich FCS increases the dispersion quality of gold nanoparticle suspensions through steric effects. Cellular uptake of 25 and 50 nm gold nanoparticles by Jurkat and PANC1 cell lines were investigated using inductively coupled plasma-mass spectroscopy. The intracellular gold level of PANC1 cells was higher than that of Jurkat cells, where 50 nm particles enter cells at faster rates than the 25 nm particles

Microfluidic Impedance Spectroscopy as a Tool for Quantitative Biology and Biotechnology

A microfluidic device that is able to perform dielectric spectroscopy is developed. The device consists of a measurement chamber that is 250 μm thick and 750 μm radius. Around 1000 cells fit inside the chamber assuming average quantities for cell radius and volume fraction. This number is about 1000 folds lower than the capacity of conventional fixtures. A T-cell leukemia cell line Jurkat is tested using the microfluidic device. Measurements of deionized water and salt solutions are utilized to determine parasitic effects and geometric capacitance of the device. Physical models, including Maxwell-Wagner mixture and double shell models, are used to derive quantities for sub-cellular units. Clausius-Mossotti factor of Jurkat cells is extracted from the impedance spectrum. Effects of cellular heterogeneity are discussed and parameterized. Jurkat cells are also tested with a time domain reflectometry system for verification of the microfluidic device. Results indicate good agreement of values obtained with both techniques. The device can be used as a unique cell diagnostic tool to yield information on sub-cellular units. (C) 2012 American Institute of Physics

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P < 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

Electrical System for Bioelectric Impedance using AD5933 Impedance Converter

Benchtop lab equipment, such as the HP4194A Precision Impedance Analyzer, offer accuracy and functionality but are often expensive and bulky. Although integrated circuits offer less functionality than benchtop equivalents, they can be a cheaper alternative for smaller applications. In this study, the AD5933 Impedance Converter was investigated as a low-cost option for impedance analysis of the bicep brachii. The AD5933 evaluation board was properly calibrated to measure human muscle by using an equivalent parallel circuit consisting of a 5 nF capacitor and a 1 kOhm resistor. Next, the impedance of the bicep was measured using the AD5933 as the experimental device and the HP4194A as the control device. A 2 volt peak-to- peak signal was generated for a 10 kHz to 100 kHz frequency range and the resulting impedance of the bicep was measured. The AD5933 recorded impedance curves qualitatively and quantitatively similar to those recorded by the HP4194A. Thus, the AD5933 was considered to be a low- cost alternative for impedance analysis in small-scale medical applications