Sensors and Actuators B 118 (2006) 115–120
A micro-electrode array biosensor for impedance spectroscopy of human umbilical vein endothelial cells Abdur Rub Abdur Rahman a , Chun-Min Lo b , Shekhar Bhansali a,∗ a
BioMEMS and Microsystems Laboratory, Department of Electrical Engineering, University of South Florida, 4202 E Fowler Ave., Tampa, FL 33620, United States b Department of Physics, College of Arts and Science, University of South Florida, 4202 E Fowler Ave., Tampa, FL 33620, United States Available online 5 June 2006
Abstract We report on a novel, radial micro-electrode array biosensor for impedance spectroscopy (IS) of Human Umbilical Vein Endothelial Cells (HUVECs). The electrical parameters of the HUVEC monolayer, which are important indicators of physiological factors such as cell–cell interactions and cell motility, were measured using this micro-electrode biosensor. HUVEC monolayer was cultured directly on the surface of the micro-electrode array. Impedance modulus and the phase angle of HUVEC’s in the frequency range from 12.5 kHz to 500 kHz were recorded and studied. A modified Randles cell (MRC) equivalent circuit was used to extract the electrical parameters of transendothelial monolayer. Differential analysis of HUVEC and Hanks’ Balanced Salt Solution (HBSS) impedance data shows electrical resonance at 52.5 kHz. The value of capacitance at resonance was measured to be ∼25 F/cm2 . © 2006 Elsevier B.V. All rights reserved. Keywords: MEMS micro-electrode; Bioimpedance; Impedance spectroscopy; HUVEC; Transendothelial resistance
1. Introduction Bioimpedance can be broadly defined as the impedance of biological specimens ranging from the human body impedance to the impedance of DNA. Bioimpedance analysis enables detection of physiological changes in cell–cell, cell matrix interactions caused by the effect of viral and bacterial infections [1–4], environmental parameters [5], toxicity [6] and the effect of pharmaceutical compounds [7]. Electrical investigation of biological materials has been performed over the past century, using both conventional electrodes and microelectrodes. Micro-electrodes were mainly used for intracellular recordings such as patch-clamp studies to study the ionic channel currents and trans-membrane potential. Currently, micro-electrodes are increasingly being used for extracellular biophysical investigation of cells and monolayers as they offer several advantages over their conventional counterparts for impedemetric investigations. Microelectrodes have a very small I–R drop thereby allowing impedance spectroscopy (IS) of high resistivity samples such as biological fluids and specimens. Microelectrodes generally
∗
Corresponding author. Tel.: +1 813 974 3593; fax: +1 813 974 5250. E-mail address:
[email protected] (S. Bhansali).
0925-4005/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2006.04.060
allow small currents; hence, these electrodes are generally nondestructive to the solution and species under investigation. This advantage is significant in biological samples and for in vivo measurements, where such destruction should be eliminated [8]. A commonly used impedance measurement device in biophysical investigations is the Electrical Cell-substrate Sensing (ECSTM ) device [9]. The ECSTM impedance measurement device consists of a 250 m electrode and a counter electrode. Impedance changes due to the fractal motion of cells during their spreading and adhesion can be recorded as impedance changes [10–13]. A planar electrode array with electrodes of varying sizes and spacing was also demonstrated for measuring resistivities of stratified layers of thin biomaterials [14]. In this paper a multi-electrode array based biosensor which is capable of measuring transcellular impedance across 15 electrode pairs is presented. Fig. 1(a) schematically illustrates the layout of the biosensor. In this biosensor the electrode tracks are spaced 22.5◦ in a radial array. A radial array configuration offers the flexibility of measuring impedance in the close proximity of cells (adjacent electrodes) as well as across the confluence (diagonal electrodes). Multiple electrode recordings would allow for statistical data correlation in homogenous samples. For cells and tissues with anisotropic impedance distribution, this device can facilitate impedance data recording at several discrete angles
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Fig. 2. (a–e) The fabrication procedure of micro-electrode array bio-sensor.
Fig. 1. (a) A radial micro-electrode array with coplanar shielded signal conductors. Grounded shields confine electrostatic fields from individual conductors within their span, thereby reducing stray field interference. (b) A single coplanar electrode with symmetrically placed ground electrodes.
across the tissue. A version of this device has previously been demonstrated for characterization of tissue samples from the skin by analyzing its electrical impedance characteristics [15]. 2. Theory and design The biosensor described in this paper is modeled on a 16 electrode array Electrical Impedance Tomograph (EIT). Fig. 1(a) illustrates the biosensor design. The sensor consists of 16 electrodes radially arranged around the periphery of a circle of 125 m diameter. Fig. 1(b) illustrates the dimensions of individual electrodes as well as the accompanying ground electrodes. Individual electrodes measure 3622 m × 17 m, with a 426 m × 256 m electrical contact pad placed at the end to facilitate electrical contact for probing. The ground electrodes are symmetrically placed on either side of the electrode, similar to a coplanar wave guide. This ground electrode placement was designed to minimize inter-electrode interferences. The ground planes confine the electrostatic fields emanating from the conductor within the bounds of the ground planes, thus reducing electrostatic field interference between adjacent electrodes. 3. Fabrication of biosensor Fig. 2(a–e) illustrates the process flow for fabricating the micro-electrode array biosensor. The bio-sensor was fabricated
on a 250 m thick, 2-in. diameter Pyrex glass substrate. The choice of substrate was based on the need to minimize stray substrate capacitance which contributes to measurement noise. Glass has a lower dielectric constant than silicon, which leads to lower capacitive coupling between the device and substrate. A 30 nm layer of chromium (Cr) was evaporated on the substrate followed by 150 nm layer of gold (Au) (Fig. 2(a)). The wafer was lithographically patterned with the electrode mask, using AZ1813 photoresist, in an EVG620 aligner (Fig. 2(b)). Au electrodes were formed by electroplating the substrate in an electrochemical plating bath. TRANSENE 25ETM , a commercially available plating solution from TECHNIC Inc., was used to electroplate Au to a thickness of approximately 2 m (Fig. 2(c)). Subsequently, the photoresist was removed with acetone and methanol wash and seed layer was etched away in commercially available Au and Cr etching solutions (Fig. 2(d)). Another lithography step was then performed to open a 125 m diameter circle at the center of the device to expose a 10 m tip of all electrodes and the bond pads; the rest of the electrode area under photoresist was then hard cured at 110◦ C for 45 s, to stabilize the resist layer (Fig. 2(e)). After fabricating the biosensor, a 0.5 cm internal diameter and 1 cm high FISHERTM cloning cylinder was attached on to the biosensor using photoresist as adhesive to confine cells and fluids to the center region of the device. 4. Cell culture and growth HUVECs (Clonetics Corp., San Diego, CA) were cultured at 37 ◦ C and 5% CO2 in endothelial cell growth medium (EGM; Clonetics Corp.) which was supplemented with the following: 10 ng/ml human recombinant epidermal growth factor, 1 mg/ml hydrocortisone, 50 mg/ml getamicin, 50 ng/ml amphotericin B, 12 mg/ml bovine brain extract, and 2% fetal bovine serum
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(amounts indicate final concentration). HUVECs were subcultured when they were 70% confluent, and the medium was changed every 48 h thereafter. HUVECs passaged less than six times were used in experiments. Twenty-four hours after inoculating cells into electrode-containing cylindrical wells at 8 × 104 cm−2 , the normal medium was replaced by Hanks’ Balanced Salt Solution (HBSS; Mediatech, Inc., Herndon, VA) without phenol red. Impedance data was taken under room temperature at about 22 ◦ C. 5. Impedance spectroscopy procedure for HBSS and HUVECs Impedance measurement of HBSS was recorded by dispensing 30 l of HBSS into the cloning cylinder attached to the biosensor and measuring impedance across diagonal electrodes. HUVECs were transferred onto the biosensor. HUVECs, in the biosensor were placed overnight in a REVCO ELITE II series CO2 incubator under standard temperature, pH and CO2 concentrations for cell cultures. HUVECs formed a confluent monolayer on the electrode test area. HUVEC monolayer confluence was verified using Axiovert microscope before proceeding to electrical testing. Fig. 3 is the optical micrograph of cultured HUVECs on electrode array. Fig. 4 is a photograph of the impedance measurement set-up. The test set-up consists of a Cascade MicroChamberTM probe station with an Agilent 4294A Impedance Analyzer. The Agilent 4294A Impedance Analyzer is a pseudo 4-point probe which facilitates bipolar measurements. In it, the high current terminal is electrically shorted to the high voltage terminal and the low current terminal is electrically shorted with the low voltage terminal. Contact was made on two electrodes to record a data instead of four electrodes as is the case in true tetrapolar measurements. The impedance analyzer was calibrated to eliminate cable and fixture capacitances, using a 50 TRL calibration standard. The biosensor was vacuum mounted on the probe station chuck. The chuck region is electromagnetically shielded from the environment via a Faraday cage. The parameters of interest were impedance modulus (|Z|), phase angle (θ), series resistance (Rs ), and series capacitance (Cs ). In diagonal electrode configuration, measurements are
Fig. 3. The optical microphotograph of confluent monolayer of cultured HUVECs on micro-electrode array biosensor.
Fig. 4. The photograph of measurement set-up for impedance spectroscopy of HBSS and HUVECs. Impedance analyzer is connected to the probe station via two sets of triaxial cables. The probe station is equipped with micro-probes which contact the device pads for electrical testing.
recorded between electrodes which are placed diagonally opposite to each other, with a separation distance of 125 m between the tips. All measurements were performed in the frequency range of 12.5 kHz and 500 kHz. 6. Results and discussion 6.1. HBSS impedance spectrum Fig. 5 shows the impedance modulus and phase angle plot of HBSS and HUVEC. The HBSS measurement data is discussed in this section followed with HUVEC measurement data in the next section. The electrode–HBSS system is a typical electrode–electrolyte system. The basic elements of such a system are the solution resistance, the charge transfer resistance
Fig. 5. The impedance and phase angle of HUVEC and HBSS from 12.5 kHz to 500 kHz. Phase angle data indicates contribution of transendothelial resistance in HUVEC impedance response, whereas HBSS impedance is dominated by double layer and stern capacitances.
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and double layer capacitance. From the HBSS Bode diagram, it is seen that, at lower frequencies the double layer capacitance dominates the frequency spectrum, as evidenced by the large capacitive phase angle, up to 100 kHz. The impedance response indicates decreasing impedance with frequency, which is typical of a double layer capacitance in parallel with a charge transfer resistance. The frequency dependent capacitive reactance decreases with increasing frequency. At higher frequencies, the double layer capacitor would pass ac with minimal resistance and a plateau would be observed which corresponds to the solution resistance on the impedance axis of the Bode diagram. As mentioned above the electrode–electrolyte system comprises of the solution resistance, the charge transfer resistance and double layer capacitance. The Double layer capacitance is represented by a constant phase element (CPE). The CPE has a reactance, given by, 1/(jωC)α , where the factor α = 1 for a perfect capacitor and leads to a perfect semicircle in the Nyquist plane, however for real situations
