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Chip-based impedance measurement on single cells for monitoring sub-toxic effects on cell membranes
Biosensors and Bioelectronics 26 (2011) 3405–3412
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Chip-based impedance measurement on single cells for monitoring sub-toxic effects on cell membranes Christian M. Kurz a , Heiko Büth a , Adam Sossalla a , Vincent Vermeersch b , Veska Toncheva b , Peter Dubruel b , Etienne Schacht b , Hagen Thielecke a,∗ a b
Fraunhofer Institute for Biomedical Engineering, Ensheimer Str. 38, 66386 St. Ingbert, Germany Ghent University, Krijgslaan 281 S4, 9000 Ghent, Belgium
a r t i c l e
i n f o
Article history: Received 6 October 2010 Received in revised form 18 December 2010 Accepted 10 January 2011 Available online 19 January 2011 Keywords: Electrical biosensor Single cell impedance Membrane integrity Polymer/polymer–DNA complex Finite element method
a b s t r a c t There is a lack of methods suitable for generation of data about the dynamics of effects on cell membranes with a high sensitivity. Such methods are urgently needed to support the optimisation of interaction of substances, particles or materials with cell. The goal of this article is to use an improved microhole chip system to monitor the alterations of cells due to the interactions of polymer–DNA complexes. This should demonstrate exemplarily that subtoxic effect of biological relevant particles or substances at relevant concentrations can be monitored for several hours. By using a microhole cell chip and a microfluidic unit single cells can be electrically interfaced via microholes and the use of small electrodes with high impedances is not necessary. For separation and positioning of the cells onto the hole negative pressure is applied on the reverse side of the chip. Under cell culture conditions the cell starts to spread on the biocompatible insulating chip membrane resulting in a stable interface to an adherent growing cell. After the spreading process is finished, the polymer/polyplex solution is added and the impedance is measured with respect to time. To illustrate the cellular parameter which can affect the measured impedance a simple simulation based on the finite element method (FEM) is performed. It was shown for the first time that the impedance-based method predicated on the microhole chip can be used for biological relevant substances at relevant concentrations and that it is more sensitive than the well-established biological marker. © 2011 Elsevier B.V. All rights reserved.
1. Introduction For many applications in cell-based biotechnology the optimisation of interaction of substances, particles or materials with cell is of high interest. One prominent example is non-viral DNA transfection by using polymer–DNA (Pack et al., 2005). However, there is a lack of methods suitable for generation of data about the dynamics of effects on cell membranes with a high sensitivity. Since the electrical impedance of biological cells is mainly determined by the cell membrane and since cells can be handled and interfaced by Micro-Electro-Mechanical Systems (MEMS, Huang et al., 2007; Johnstone et al., 2010; Narakathu et al., 2010; Xiang et al., 2007) the combination of impedance spectroscopy with technology (MEMS technology) is promising for the development of systems for monitoring cell membrane alterations.
∗ Corresponding author. Tel.: +49 6894 980 162; fax: +49 6894 980 185. E-mail address: [email protected] (H. Thielecke). URL: http://www.ibmt.fhg.de (H. Thielecke). 0956-5663/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2011.01.012
In the early 20th century it was experimentally demonstrated that cell membrane damage can be monitored by impedance measurements (Höber, 1910, 1912, 1913). The effect of membrane disruptions on dielectrical properties of biological cells has also been theoretically studied by three-dimensional finite-difference method (3D-FDM) simulations (Asami, 2006). Diverse MEMS has been developed in order to characterise single cells by impedance spectroscopy or by the patch clamp technique. Some known examples are microfluidic channels with integrated electrodes (Schade-Kampmann et al., 2007), microfabricated patch clamping sensor systems (Han and Frazier, 2006) and cell-filters (Bao et al., 1993). These systems are engineered for instance for label-free cell discrimination, ion channel characterisation and for basic studies of electrical and dielectrical properties of biological cells, respectively. However, until now, it has not been shown that subtoxic effect of biological relevant particles or substances at relevant concentrations on single cells can be monitored for several hours. For sensitive characterisation of membrane alterations on single cells on the one hand, impedance based systems for analysing cell membrane interactions have to be highly susceptible in the
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lower frequency range (Pething, 1987), while on the other hand the electrode sizes have to be reduced for interfacing single cells. The reduction of electrode size results in a higher electrode polarisation impedance which has an impact on the sensitivity of impedance measurement in the lower frequency range (de Boer and van Osterom, 1978). This contradiction is one main challenge to deal with when building a sensing system for monitoring dynamic membrane interactions. Cho and Thielecke (2007) analysed the impedance of single cells by using a microhole chip. The goal of this article is to use an improved microhole chip system to monitor the alterations of cells due to the interactions of polymer–DNA complexes. This should demonstrate exemplarily that subtoxic effect of biological relevant particles or substances at relevant concentrations can be monitored for several hours. By using a microhole cell chip and a microfluidic unit single cells can be electrically interfaced via microholes and the use of small electrodes with high impedances is not necessary. For separation and positioning of the cells onto the hole negative pressure is applied on the reverse side of the chip. Under cell culture conditions the cell starts to spread on the biocompatible insulating chip membrane resulting in a stable interface to an adherent growing cell. After the spreading process is finished, the polymer/polyplex solution is added and the impedance is measured with respect to time. To illustrate the cellular parameter which can affect the measured impedance a simple simulation based on the finite element method (FEM) is performed.
2. Methodology 2.1. Experimental system For this measurement, the microhole cell chip (Thielecke et al., 1999) and a microfluidic unit were used. In brief, this unit consisted of one fluidic connection nanoport as well as two gold electrodes (2-terminal measurement). The gold wires (Alfa Aesar, Karlsruhe, Germany) had a purity of 99.99% and a diameter of 0.5 mm and were integrated into the upper- and lower chamber through small holes in the adapter and fixed by a silicone adhesive. The electrodes were electrically linked to an impedance analyser (Solartron 1260 and Solartron 1294, Solartron Analytical, Farnborough, UK). The applied input potential was set to 5 mV, and the impedance was measured at 1 kHz with a sampling rate of 5 s. The fluidic controller consisted of a low range pressure regulator (Marsh BelloFram, Newell, USA), which controlled the positive pressure of the compressed-air outlet. This pressure was converted into a suction pressure by a venturi injector (Festo, Esslingen-Berkheim, Germany) and monitored by a pressure sensor (SensorTechnics, Puchheim, Germany). To realise cell culture conditions, the complete unit was put into a cultivation chamber combined with an integrated inverse microscope (Olympus IX81, Hamburg, Germany). This microscope was used for taking phase contrast and fluorescent pictures. The temperature, humidity and CO2 were adjusted to 37 ◦ C, 80% and 5%, respectively. The schematic of the measurement setup is shown in Fig. 1a.
Fig. 1. Schematic shows the single cell impedance setup. (a) Experimental setup with the single cell kept on the microhole. The suction pressure was generated and monitored by a fluidic controller and the impedance was measured by two electrodes connected to an impedance analyser. For the optical monitoring an inverse microscope was used. (b) Simulation setup of the occupied microhole cell chip with a disc-shaped cell. The cell parameters rcell-hole , rcell , dcell-membrane , g and hcell represents the cell membrane hole radius, cell radius, membrane thickness, cell/substrate gap and the height of the cell, respectively. The chip membrane height and the chip hole diameter were fixed to 1.6 m and 5 m.
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After the cell suspension (∼100 l) was added to the upper chamber of the system, a gentle and controlled negative pressure was applied to the reverse side of the chip to position the single cell on the hole. For the cell positioning a differential negative pressure of around 0.05 mbar was used. For reducing the cell’s movement on the microhole, this value was increased to 0.2 mbar after the cell spreading process. Extra cells, which were located on the chip membrane in addition to the positioned cell, were not rinsed away. 2.2. Fabrication of microhole cell chip For the fabrication of the microhole cell chip a two-sided polished 4 in. (1 0 0) silicon wafer with a thickness of 525 ± 5 m was used. A 1 m thick silicon nitride layer (Si3 N4 ) was deposited on both sides by plasma-enhanced chemical vapour deposition (PECVD). The holes with diameters of 5 m were patterned by photolithography and dry etching into one of the silicon nitride layers. On the other side the Si3 N4 was opened (835 m × 835 m) above the microhole by the same process technology. This opening was used as an etch mask for the anisotropic etching of the silicon layer in a potassium hydroxide (KOH) solution. After this wet etching process, a Si3 N4 membrane window (92.5 ± 7 m × 92.5 ± 7 m) with the centred microhole was achieved. For the electrical isolation of the exposed silicon areas, a 600 nm thick Si3 N4 layer was deposited at the cavity sides. Therefore the resulting membrane thickness was 1.6 m. 2.3. Polymer synthesis The polymers used in this work (VT01 and VT09) were guanidine derivatives of polyethyleneimine (PEI). VT01 and VT09 were prepared by reacting part of the primary amine groups in PEI with 1-H-pyrazole-1-carboxamidine·HCl in MeOH/H2 O. VT01 was prepared as follows: 2.0 g (0.0465 mol aminecontaining units) of PEI (MW 25,000 Da) were dissolved in a mixture of 16 ml methanol and 2 ml water. 2.3 g (0.016 mol) of 1-Hpyrazole-1-carboxamidine·HCl was added. After dissolving, 4.4 ml (0.027 mol) of diisopropylethylamine was added and the solution was stirred overnight. The product was then precipitated in isopropanol, filtered, and reprecipitated from methanol/isopropanol. The polymer was further purified by preparative GPC (Sephadex G25, eluent water) and isolated by lyophilisation. VT09 was prepared using the same method but a different ratio between the components. The extent of guanidinylation of the polymer was quantified by determination of the remaining free amine groups on the polymer using the ninhydrin method (Yeh et al., 1994). It was taken into account that the branched PEI contained 25 mol% primary amine groups. Ninhydrin analysis showed that VT01 contained 5 mol% unreacted primary amine groups and VT09 contained 11 mol% primary amine groups. This led to the conclusion that VT01 contained 20 mol% guanidine and 5 mol% primary amine groups and VT09 contained 14 mol% guanidine and 11 mol% primary amine groups. The presence of the guanidine groups was confirmed by 1 H NMR (D2 O) as well. 2.4. Formation of polymer/DNA complexes (polyplexes) The DNA applied for physico-chemical characterisation of polyplexes is calf thymus DNA sodium salt (Sigma–Aldrich Chemie GmbH, Schnelldorf, Germany). For every polymer–DNA complex, the charge ratio (positive to negative or polymer to DNA) is defined using the mutual relation of mass/charge between the macroions, expressed in the following equation: z
X Y
y = x,
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where X is the mass/charge of the polycation (Da/mol), Y is the mass/charge of the polyanion (Da/mol), y is the amount of polyanion added (g), x is the amount of polycation added (g) and z is the integer multiple of the excess of polycation over polyanion (where z = 1 for an equal charge ratio, 1/1). Polyplexes were obtained in 2 ml solutions via a specific order of mixing: 16 l of DNA (2.5 mg/ml stock solution) was added to an empty vessel. Next, demineralised water was added. Subsequently, an amount of polymer (x), correspondent to the intended charge ratio (in this work: charge ratio was 2/1), was added from a 1 mg/ml stock solution. This solution was vortexed for 5 s at medium speed (∼1500 rpm) and stored at 4 ◦ C until usage. In this way a polyplex solution with 20 g/ml DNA was used for the experiments. 2.5. Cell culture, Propidium Iodide (PI) staining and polymer/polyplex application For the experiment, Arpe-19 (human retinal pigment epithelial cell line) cells were used. The cells were cultivated in an incubator (Heraeus BBD6220, Hanau, Germany), where the temperature, humidity and CO2 were adjusted to 37 ◦ C, 95% and 5%, respectively. The Arpe-19 cells were trypsinised and resuspended in fresh culture medium and put into a falcon tube. After 2 h staying in the tube the cells were used for the experiments. The culture medium consisted of DMEM/F12 (Invitrogen/GIBCO, Karlsruhe, Germany), 10% FCS “Gold” (PAA Laboratories GmbH, Cölbe, Germany) and 1% penicillin/streptomycin (Invitrogen/GIBCO). Each polymer/polyplex was mixed with the culture medium and put into 1.5 ml safe lock tubes. The control was just cell culture medium with the entrapped cell for the impedance measurements. The polymer/polyplex application was done by the replacement of all culture medium above the positioned cell by the polymer/polyplex solution. PI (Sigma–Aldrich Chemie GmbH, Schnelldorf, Germany) was used in the cell culture medium with a 1% concentration for the staining. The PI was applied to the upper chamber directly after the polymer/polyplex application. 2.6. Simplified modelling and simulation It was assumed that the cell has a disc shape, is positioned in the middle of the microhole and that no part of the cell is inserted into the hole (Fig. 1b). Further actin filaments and adhesion proteins from the cell to the chip surface were neglected for simplicity. The spread out disc shape cell was modelled with a height of hcell = 3.5 m and a membrane thickness of dcell-membrane = 10 nm. The radius rcell and the cell/substrate gap g were set to the values 7, 8.5 and 10 m and 25, 50 and 75 nm, respectively and the simulated impedance magnitude was investigated at 1 kHz. Membrane permeabilisation was simplified by two holes in the cell membrane directly above the microhole. The electrical properties for the different subdomains in Fig. 1b are taken out of the literature (Elshabini and Barlow, 1998; Grimnes and Martinsen, 2000; Malmivuo and Plonsey, 1995). The conductivity (S/m) for the cytoplasm, cell membrane, culture medium and the chip membrane is 0.5, 10−7 , 1.6 and 10−12 , where the relative permittivity εr is 80, 11.3, 80 and 5.5, respectively. For the subdomains it was assumed that the conductivity and the relative permittivity are linear and have an isotropic distribution. The computer based calculations were predicated using the assumption that a current density occurs if an electrical field is applied to a dielectric material (Harrington, 1961): = J = ( + jωε0 εr ) E, ∇ ·H the magnetic field, E the electric where J is the current density, H field, ω the angular frequency, ε0 the permittivity of free space and
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√ j = −1. On the basis of the reciprocal lead field Jreci and excitation current density Jcc the general transfer impedance Zt is (Grimnes and Martinsen, 2008):
1 J · J dv cc reci
Zt = v
This equation is simplified in a two electrode arrangement where the forward and reciprocal current densities are identical to:
1 2 J dv
Z= v
where J is the unit current density [1/m2 ]. The impedance was computed in a cylindrical symmetry model by FEM simulation (used software COMSOL Multiphysics).
3. Results 3.1. Electrical characterisation of the assembled unit without cell For the electrical characterisation the assembled microhole cell chip unit was filled with cell culture medium and the impedance was measured in the frequency range from 0.1 Hz to 1 MHz (Fig. 2a). In the lower frequency range (1–0.1 Hz) the impedance magnitude increased from nearly 200 k to above 1 M, which was caused by the electrode impedance. This was modelled by the constant phase element CPEelectrode . In the frequency range from 1 Hz to 1 kHz the impedance magnitude showed a nearly stable value. In the higher frequency range (above 1 kHz) the impedance magnitude decreased to almost 1 k at 1 MHz. The impedance progression between 1 Hz and 1 MHz was electrically modelled by a parallel circuit of the resistance Rchip-hole with the constant phase element CPEchip-membrane . For the circuit parameters, the frequency band from 0.1 Hz up to 10 kHz was fitted best by the designed electrical circuit (Fig. 2b). For the parameters of the circuit the values were fitted using ZView, where the impedance of a constant phase element was defined as ZCPE (ω) = 1/[(jω)n Q◦ ]. The values for 0 0 Rchip-hole , Qchip-membrane , nchip-membrane , Qelectrode and nelectrode were 156.13 ± 12.03 k, 1.476 ± 0.297 nF · snchip-membrane −1 , 0.890 ± 0.014, 1.480 ± 0.073 F · snelectrode −1 and 0.918 ± 0.006, respectively. The resistance of the microhole can be calculated analytically using (Malmivuo and Plonsey, 1995; Newman, 1966): 1 dchip-membrane 1 1 +2· · · Achip-hole 4 rchip-hole 1 1 dchip-membrane 1 · · = + 2 2 rchip-hole · rchip-hole
Rchip-hole =
=
1 · rchip-hole
dchip-membrane · rchip-hole
1 + 2
For the chip parameters (dchip-membrane = 1.6 m, rchip-hole = 2.5 m) and a medium conductivity = 1.6 S/m, the hole resistance should result in Rchip-hole = 175.93 k. The variation of the fitted hole resistance value to the calculated one could be directly connected to variations in the chip geometries (Fig. 2c). For this examination, one parameter was fixed and the other was calculated for Rchip-hole = 156.13 ± 12.03 k. For a fixed membrane thickness of dchip-membrane = 1.6 m the hole radius is rchip-hole = 2.76 ± 0.17 m. If the hole radius was fixed at 2.5 m, dchip-membrane results in 0.98 ± 0.38 m.
Fig. 2. Electrical and analytical characterisation of the measurement setup without positioned cell. (a) Measured and fitted data in the Bode plot of the assembled system. The impedance magnitude |Z| is labelled by the opened circle; the impedance phase ϕ is labelled by a filled circle, respectively. (b) Simplified equivalent electrical circuit for the assembled system in the frequency range from 0.1 Hz to 10 kHz. CPEelectrode describes the electrode impedance (constant phase element), Rchip-hole is the hole resistance and CPEchip-membrane is the interface impedance between the chip membrane and the saline solution (constant phase element). (c) Analytically calculated influence on the hole resistance Rchip-hole of the microhole cell chip by the variation of the hole radius rchip-hole and the membrane thickness dchip-membrane .
3.2. Influence of the captured cell on the measurement The measured impedance described the cell positioning on the hole and attachment to the chip membrane surface (Fig. 3). During the first 3 min the impedance magnitude value was almost constant (∼120 k). This impedance value described the unoccupied microhole. The steep increase in the magnitude value from 120 k to nearly 180 k was induced by the cell arriving over the hole and the blocking of the hole. After the cell was positioned on the hole the magnitude increased because of the cell spreading on the chip membrane surface (Fig. 3b). The steady increase in the
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Fig. 3. The positioning and attachment process of the single cell. (a) The impedance magnitude |Z| is labelled by the opened circle; the impedance phase ϕ is labelled by a filled circle, respectively. The abrupt rise at 3.25 min belongs to the cell positioning on the microhole. The further increase in the magnitude is caused by the cell spreading on the chip membrane surface. (b) The phase contrast picture taken directly after positioning shows still a round cell, where the positioned cell after 10 min seems to be attached onto the chip membrane surface (scale bar = 25 m).
impedance magnitude ended nearly 8 min after the cell positioning at an impedance magnitude around 350 k.
the normalised impedance magnitude by approximately −0.14 or roughly 0.12, respectively.
3.3. Application of the polymers and polyplexes
4. Discussion
Modified PEI with guanidine functional groups is believed to improve cell-internalisation while diminishing the toxic effects of typical polymer delivery systems (Nguyen et al., 2008). The normalised impedance magnitude after polyplex VT01 + DNA application showed almost no changes compared to the control (Fig. 4a). The control is just cell culture medium with a trapped cell on the hole. Also the Propidium Iodide (PI) staining showed no cell membrane perforation. The normalised impedance magnitude exhibited decay 85 min after VT09 application (Fig. 4b). The decline ended at 110 min at an almost stable value of around 0.7. An increase in the standard deviation 110 min after the VT09 application occurred. In the first 50 min after the polyplex VT09 + DNA (Fig. 4c) application the measured impedance data showed a higher impedance magnitude compared to the control, where a maximum was reached after 30 min. Above 50 min the normalised impedance magnitude decreased against the control level. Also a higher standard deviation resulted after 50 min. A stable level of around 0.85 was observed from 85 min until 125 min after the application. After 125 min the impedance magnitude showed another decay to a level of 0.55. The measurement results from the polymer VT09 and the complex VT09 + DNA were correlated to PI. The microscope pictures after VT09 application (Fig. 5a) showed no positive PI stained cell before 100 min whereas after 115 min cells on the chip membrane as well as the captured cell on the hole exhibited a PI stained nucleus. The images of VT09 + DNA (Fig. 5b) showed PI stained nuclei cells after 128 min. The captured cell on the hole exhibited a PI stained nucleus after 135 min of the polyplex application.
It was shown that the microhole cell chip unit can be used for the single cell manipulation as well as the electrical recording of membrane integrity effects. The manipulation of the cell by a suction pressure was adjusted manually and monitored by a pressure sensor. The positioning pressure was around 0.05 mbar. This pressure was increased to 0.2 mbar after the cell spread onto the microhole to reduce cells movement. These values were adjusted and established, so that during the positioning and the holding of the cell on the microhole neither membrane nor cell damages due to hydrodynamic stress were observed by the use of fluorescent markers (results not shown). For the electrical characterisation, the assembled unit without captured cell was measured and then fitted by an electrical circuit. It was found that the frequency range between 1 Hz and 10 kHz in the 2-terminal measurement can be directly connected to the resistance of the microhole in parallel to the non-ideal capacitance (CPE) of the chip membrane. The capacitance breakdown of the silicon nitride membrane limited the suitable frequency range, because it shortened the current through the microhole. Therefore the upper bound for the frequency range in the microhole chip arrangement was around 1 kHz. The variation of the fitted hole resistance Rchip-hole = 156.13 ± 12.03 k to the calculated value might originated from the deviation during the chip-microfabrication process, where the two parameters rchip-hole and dchip-membrane were not exactly adjusted to the specified values. In order to determine if an effect was caused by the cell response to a reagent a stable impedance level had to be guaranteed. During the cell spreading process the impedance magnitude at 1 kHz increased steadily by more than 194% in the first 8 min after the cell positioning. This might be based on the enlargement of the cell covered chip membrane surface and by the reducing of the cell/substrate gap (see simulations), which resulted in a higher resistance for the current mostly flowing between the cell and the chip membrane at this frequency. Therefore a suitable starting time for all measurements was greater or equal to 8 min after the cell positioning. In this work the starting time was chosen to be 14 min after cell positioning. The fast cell spreading could be based on the fact that the cells were standing for nearly 2 h in the falcon tube, where the adhesion proteins are still expressed, before they were used. By the use of FEM simulations the influence from cell parameters like the cell size, the cell/substrate gap without cell membrane holes as well as with cell membrane disruption on the impedance
3.4. Simulation results The theoretical investigations showed that for smaller cell/substrate gaps and larger cell radius the impedance magnitude possessed a sharper decline for increasing cell membrane holes (Fig. 6). If there was no cell membrane disruption (see rcell-hole = 0 nm) the simulation results illustrated how strong the cell parameters could affect the impedance data. If the cell/substrate gap increased from 50 to 75 nm or reduced from 50 to 25 nm, the normalised impedance magnitude reduced by almost 0.29 or increased by nearly 0.76, respectively. A variation in the cell radius from 8.5 to 7 m or from 8.5 to 10 m affected
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Fig. 4. The normalised impedance magnitude data measured with respect to time after polymer/polyplex application. (a) The measured data after polyplex VT01 + DNA application has a similar curve progression as the control. (b) The measured data after polymer VT09 application exhibits a decay at 85 min to a value of 0.7 compared to the control. (c) The measured data after polyplex VT09 + DNA application exhibits a maximum at 30 min, a first decay after 50 min until 80 min and a second decay after 125 min compared to the control.
was investigated. This results might be used to relate the measured data to the cell membrane hole size and cell parameters. For long-term observations of polymer complexes, cells had to be kept as long as possible on the microhole. The results showed that cells could be kept for nearly 3 h on the hole. The normalised mean impedance magnitude values varied in a range of 3.12%. This variation could not be completely reduced because of the cell’s movement and morphological changes on the hole. Consequently impedance magnitude variations in this range were not based on the cell responses to special reagents. The system was verified for several polymer/polyplexes at biological relevant concentrations. The polymer/polyplex application was done by exchanging the whole cell culture medium above the microhole chip with the polymer-/polyplex solution. This solution replacement had no direct influence on the impedance magnitude, because all measured data showed no variations compared to the control directly after application. The normalised impedance magnitude after application of the complex VT01 + DNA exhibited no relevant changes in relation to the control over the whole monitoring time. The correlation with the Propidium Iodide (PI) staining displayed no positively stained cell nucleus 95 min after the application. PI can be used as a marker for visualising cell membrane perforations. Therefore the polyplex VT01 + DNA did not permeate the cell membrane, which was shown by the impedance data and the PI staining. The data for the polymer VT09 showed no difference with respect to the control values in the first 80 min. In addition the standard deviations compared to the control were almost in the same range, and the fluorescent combined phase contrast pictures showed no stained cell nucleus in this time range. This suggests that nothing happened to the cell membrane of the captured cell during the first 80 min. Between 85 min and 100 min after the application the normalised impedance magnitude dropped down from 1 to a value of nearly 0.9. This decay is not arisen from membrane disruption (no PI-staining at 100 min), but from a shrinking in the cell size or a detaching from the chip membrane. A cell shrinking is visible in the microscope images taken at 55 min and 100 min (from nearly rcell = 10–8.5 m). The simulation showed that such a cell shrinking (rcell = 10–8.5 m) at a cell/substrate gap of 50 nm shows a difference of 0.12 in the normalised impedance magnitude with no cell membrane disruption. The phase contrast/fluorescent image taken at 115 min after VT09 application showed stained cell nuclei. Therefore cell membrane perforations occurred in the range between 100 min and 115 min, which could be recognised by the steep slope in the impedance magnitude. From the simulation a decrease of 0.2 in the impedance can be related to a membrane hole radius of 82 nm at a cell/substrate gap of 25 nm and a cell radius of 8.5 m. Above 115 min the cell membrane was disrupted the cell did not show any further reactions and therefore a stable normalised impedance magnitude was observed. The increase in the standard deviation after 100 min could be based on fast cell membrane alterations. In the case of the polyplex VT09 + DNA the normalised impedance magnitude showed an increase in the first 50 min compared to the control, where a maximum of 1.05 was reached after 30 min. This might be based on a swelling of the cell in the first 30 min caused by the polyplex application. This swelling was reversed in the time from 30 min until 50 min, because the normalised impedance magnitude reached the starting value of 1 again. Between 50 min and 85 min the normalised magnitude decreased compared to the control almost linearly and showed also an increase in the standard deviation. This effect was caused not by cell membrane perforations, due to the fact that the captured cell showed no PI stained nuclei. This decreasing in the impedance might be based on a small cell shrinking (significant changing in cell size is not visible in the microscope images) combined with a cell detachment from the chip membrane. During the second decline in the normalised impedance magnitude from 125 min until 135 min cell membrane
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Fig. 5. Phase contrast pictures superimposed with the fluorescent pictures. (a) Pictures after VT09 application. After 115 min the captured cell exhibits a stained nucleus. (b) Pictures after VT09 + DNA application. The captured cell on the hole shows a stained nucleus after 135 min (scale bars = 25 m).
Fig. 6. Simulation results for the normalised impedance magnitude values. The impedance magnitude is plotted against the cell membrane hole radius for different cell/substrate gaps g where the cell radius was fixed to 8.5 m as well as cell radii rcell with a constant cell/substrate gap of 50 nm.
disruption occurred, because the phase contrast/fluorescent picture taken at 135 min showed a PI stained nucleus of the captured cell. This decline of 0.3 in the normalised impedance can be related to the simulated membrane hole radius of around 125 nm if the cell/substrate gap is roughly 25 nm and the cell radius is around 10 m.
Another also interesting part can be the identification of biological relevant substances or particles, because the measured curves showed a completely different progression. Therefore possible application areas could be to improve or select cell membrane crossing vehicles, measure effects on single cells and the identification or characterising from different polymer/polyplexes.
5. Conclusion
References
The Micro-Electrical-Impedance-Spectroscopy System (EIS) based on a microhole chip can be used for impedance monitoring on single cells. It was shown first time that the chip setup is sensitive and can be used to measure subtoxic effects of biological relevant particles or substances at relevant concentrations. With the correlation from the microscope images, the simulation results and the impedance data it is possible to give a statement about changes from cell parameters like the cell size, the cell/substrate gap and the cell membrane disruption, which can be an identification for subtoxic effects on cells, whereas the PI-staining only can visualise cell membrane perforations.
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Chip-based impedance measurement on single cells for monitoring sub-toxic effects on cell membranes Christian M. Kurz a , Heiko Büth a , Adam Sossalla a , Vincent Vermeersch b , Veska Toncheva b , Peter Dubruel b , Etienne Schacht b , Hagen Thielecke a,∗ a b
Fraunhofer Institute for Biomedical Engineering, Ensheimer Str. 38, 66386 St. Ingbert, Germany Ghent University, Krijgslaan 281 S4, 9000 Ghent, Belgium
a r t i c l e
i n f o
Article history: Received 6 October 2010 Received in revised form 18 December 2010 Accepted 10 January 2011 Available online 19 January 2011 Keywords: Electrical biosensor Single cell impedance Membrane integrity Polymer/polymer–DNA complex Finite element method
a b s t r a c t There is a lack of methods suitable for generation of data about the dynamics of effects on cell membranes with a high sensitivity. Such methods are urgently needed to support the optimisation of interaction of substances, particles or materials with cell. The goal of this article is to use an improved microhole chip system to monitor the alterations of cells due to the interactions of polymer–DNA complexes. This should demonstrate exemplarily that subtoxic effect of biological relevant particles or substances at relevant concentrations can be monitored for several hours. By using a microhole cell chip and a microfluidic unit single cells can be electrically interfaced via microholes and the use of small electrodes with high impedances is not necessary. For separation and positioning of the cells onto the hole negative pressure is applied on the reverse side of the chip. Under cell culture conditions the cell starts to spread on the biocompatible insulating chip membrane resulting in a stable interface to an adherent growing cell. After the spreading process is finished, the polymer/polyplex solution is added and the impedance is measured with respect to time. To illustrate the cellular parameter which can affect the measured impedance a simple simulation based on the finite element method (FEM) is performed. It was shown for the first time that the impedance-based method predicated on the microhole chip can be used for biological relevant substances at relevant concentrations and that it is more sensitive than the well-established biological marker. © 2011 Elsevier B.V. All rights reserved.
1. Introduction For many applications in cell-based biotechnology the optimisation of interaction of substances, particles or materials with cell is of high interest. One prominent example is non-viral DNA transfection by using polymer–DNA (Pack et al., 2005). However, there is a lack of methods suitable for generation of data about the dynamics of effects on cell membranes with a high sensitivity. Since the electrical impedance of biological cells is mainly determined by the cell membrane and since cells can be handled and interfaced by Micro-Electro-Mechanical Systems (MEMS, Huang et al., 2007; Johnstone et al., 2010; Narakathu et al., 2010; Xiang et al., 2007) the combination of impedance spectroscopy with technology (MEMS technology) is promising for the development of systems for monitoring cell membrane alterations.
∗ Corresponding author. Tel.: +49 6894 980 162; fax: +49 6894 980 185. E-mail address: [email protected] (H. Thielecke). URL: http://www.ibmt.fhg.de (H. Thielecke). 0956-5663/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2011.01.012
In the early 20th century it was experimentally demonstrated that cell membrane damage can be monitored by impedance measurements (Höber, 1910, 1912, 1913). The effect of membrane disruptions on dielectrical properties of biological cells has also been theoretically studied by three-dimensional finite-difference method (3D-FDM) simulations (Asami, 2006). Diverse MEMS has been developed in order to characterise single cells by impedance spectroscopy or by the patch clamp technique. Some known examples are microfluidic channels with integrated electrodes (Schade-Kampmann et al., 2007), microfabricated patch clamping sensor systems (Han and Frazier, 2006) and cell-filters (Bao et al., 1993). These systems are engineered for instance for label-free cell discrimination, ion channel characterisation and for basic studies of electrical and dielectrical properties of biological cells, respectively. However, until now, it has not been shown that subtoxic effect of biological relevant particles or substances at relevant concentrations on single cells can be monitored for several hours. For sensitive characterisation of membrane alterations on single cells on the one hand, impedance based systems for analysing cell membrane interactions have to be highly susceptible in the
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lower frequency range (Pething, 1987), while on the other hand the electrode sizes have to be reduced for interfacing single cells. The reduction of electrode size results in a higher electrode polarisation impedance which has an impact on the sensitivity of impedance measurement in the lower frequency range (de Boer and van Osterom, 1978). This contradiction is one main challenge to deal with when building a sensing system for monitoring dynamic membrane interactions. Cho and Thielecke (2007) analysed the impedance of single cells by using a microhole chip. The goal of this article is to use an improved microhole chip system to monitor the alterations of cells due to the interactions of polymer–DNA complexes. This should demonstrate exemplarily that subtoxic effect of biological relevant particles or substances at relevant concentrations can be monitored for several hours. By using a microhole cell chip and a microfluidic unit single cells can be electrically interfaced via microholes and the use of small electrodes with high impedances is not necessary. For separation and positioning of the cells onto the hole negative pressure is applied on the reverse side of the chip. Under cell culture conditions the cell starts to spread on the biocompatible insulating chip membrane resulting in a stable interface to an adherent growing cell. After the spreading process is finished, the polymer/polyplex solution is added and the impedance is measured with respect to time. To illustrate the cellular parameter which can affect the measured impedance a simple simulation based on the finite element method (FEM) is performed.
2. Methodology 2.1. Experimental system For this measurement, the microhole cell chip (Thielecke et al., 1999) and a microfluidic unit were used. In brief, this unit consisted of one fluidic connection nanoport as well as two gold electrodes (2-terminal measurement). The gold wires (Alfa Aesar, Karlsruhe, Germany) had a purity of 99.99% and a diameter of 0.5 mm and were integrated into the upper- and lower chamber through small holes in the adapter and fixed by a silicone adhesive. The electrodes were electrically linked to an impedance analyser (Solartron 1260 and Solartron 1294, Solartron Analytical, Farnborough, UK). The applied input potential was set to 5 mV, and the impedance was measured at 1 kHz with a sampling rate of 5 s. The fluidic controller consisted of a low range pressure regulator (Marsh BelloFram, Newell, USA), which controlled the positive pressure of the compressed-air outlet. This pressure was converted into a suction pressure by a venturi injector (Festo, Esslingen-Berkheim, Germany) and monitored by a pressure sensor (SensorTechnics, Puchheim, Germany). To realise cell culture conditions, the complete unit was put into a cultivation chamber combined with an integrated inverse microscope (Olympus IX81, Hamburg, Germany). This microscope was used for taking phase contrast and fluorescent pictures. The temperature, humidity and CO2 were adjusted to 37 ◦ C, 80% and 5%, respectively. The schematic of the measurement setup is shown in Fig. 1a.
Fig. 1. Schematic shows the single cell impedance setup. (a) Experimental setup with the single cell kept on the microhole. The suction pressure was generated and monitored by a fluidic controller and the impedance was measured by two electrodes connected to an impedance analyser. For the optical monitoring an inverse microscope was used. (b) Simulation setup of the occupied microhole cell chip with a disc-shaped cell. The cell parameters rcell-hole , rcell , dcell-membrane , g and hcell represents the cell membrane hole radius, cell radius, membrane thickness, cell/substrate gap and the height of the cell, respectively. The chip membrane height and the chip hole diameter were fixed to 1.6 m and 5 m.
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After the cell suspension (∼100 l) was added to the upper chamber of the system, a gentle and controlled negative pressure was applied to the reverse side of the chip to position the single cell on the hole. For the cell positioning a differential negative pressure of around 0.05 mbar was used. For reducing the cell’s movement on the microhole, this value was increased to 0.2 mbar after the cell spreading process. Extra cells, which were located on the chip membrane in addition to the positioned cell, were not rinsed away. 2.2. Fabrication of microhole cell chip For the fabrication of the microhole cell chip a two-sided polished 4 in. (1 0 0) silicon wafer with a thickness of 525 ± 5 m was used. A 1 m thick silicon nitride layer (Si3 N4 ) was deposited on both sides by plasma-enhanced chemical vapour deposition (PECVD). The holes with diameters of 5 m were patterned by photolithography and dry etching into one of the silicon nitride layers. On the other side the Si3 N4 was opened (835 m × 835 m) above the microhole by the same process technology. This opening was used as an etch mask for the anisotropic etching of the silicon layer in a potassium hydroxide (KOH) solution. After this wet etching process, a Si3 N4 membrane window (92.5 ± 7 m × 92.5 ± 7 m) with the centred microhole was achieved. For the electrical isolation of the exposed silicon areas, a 600 nm thick Si3 N4 layer was deposited at the cavity sides. Therefore the resulting membrane thickness was 1.6 m. 2.3. Polymer synthesis The polymers used in this work (VT01 and VT09) were guanidine derivatives of polyethyleneimine (PEI). VT01 and VT09 were prepared by reacting part of the primary amine groups in PEI with 1-H-pyrazole-1-carboxamidine·HCl in MeOH/H2 O. VT01 was prepared as follows: 2.0 g (0.0465 mol aminecontaining units) of PEI (MW 25,000 Da) were dissolved in a mixture of 16 ml methanol and 2 ml water. 2.3 g (0.016 mol) of 1-Hpyrazole-1-carboxamidine·HCl was added. After dissolving, 4.4 ml (0.027 mol) of diisopropylethylamine was added and the solution was stirred overnight. The product was then precipitated in isopropanol, filtered, and reprecipitated from methanol/isopropanol. The polymer was further purified by preparative GPC (Sephadex G25, eluent water) and isolated by lyophilisation. VT09 was prepared using the same method but a different ratio between the components. The extent of guanidinylation of the polymer was quantified by determination of the remaining free amine groups on the polymer using the ninhydrin method (Yeh et al., 1994). It was taken into account that the branched PEI contained 25 mol% primary amine groups. Ninhydrin analysis showed that VT01 contained 5 mol% unreacted primary amine groups and VT09 contained 11 mol% primary amine groups. This led to the conclusion that VT01 contained 20 mol% guanidine and 5 mol% primary amine groups and VT09 contained 14 mol% guanidine and 11 mol% primary amine groups. The presence of the guanidine groups was confirmed by 1 H NMR (D2 O) as well. 2.4. Formation of polymer/DNA complexes (polyplexes) The DNA applied for physico-chemical characterisation of polyplexes is calf thymus DNA sodium salt (Sigma–Aldrich Chemie GmbH, Schnelldorf, Germany). For every polymer–DNA complex, the charge ratio (positive to negative or polymer to DNA) is defined using the mutual relation of mass/charge between the macroions, expressed in the following equation: z
X Y
y = x,
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where X is the mass/charge of the polycation (Da/mol), Y is the mass/charge of the polyanion (Da/mol), y is the amount of polyanion added (g), x is the amount of polycation added (g) and z is the integer multiple of the excess of polycation over polyanion (where z = 1 for an equal charge ratio, 1/1). Polyplexes were obtained in 2 ml solutions via a specific order of mixing: 16 l of DNA (2.5 mg/ml stock solution) was added to an empty vessel. Next, demineralised water was added. Subsequently, an amount of polymer (x), correspondent to the intended charge ratio (in this work: charge ratio was 2/1), was added from a 1 mg/ml stock solution. This solution was vortexed for 5 s at medium speed (∼1500 rpm) and stored at 4 ◦ C until usage. In this way a polyplex solution with 20 g/ml DNA was used for the experiments. 2.5. Cell culture, Propidium Iodide (PI) staining and polymer/polyplex application For the experiment, Arpe-19 (human retinal pigment epithelial cell line) cells were used. The cells were cultivated in an incubator (Heraeus BBD6220, Hanau, Germany), where the temperature, humidity and CO2 were adjusted to 37 ◦ C, 95% and 5%, respectively. The Arpe-19 cells were trypsinised and resuspended in fresh culture medium and put into a falcon tube. After 2 h staying in the tube the cells were used for the experiments. The culture medium consisted of DMEM/F12 (Invitrogen/GIBCO, Karlsruhe, Germany), 10% FCS “Gold” (PAA Laboratories GmbH, Cölbe, Germany) and 1% penicillin/streptomycin (Invitrogen/GIBCO). Each polymer/polyplex was mixed with the culture medium and put into 1.5 ml safe lock tubes. The control was just cell culture medium with the entrapped cell for the impedance measurements. The polymer/polyplex application was done by the replacement of all culture medium above the positioned cell by the polymer/polyplex solution. PI (Sigma–Aldrich Chemie GmbH, Schnelldorf, Germany) was used in the cell culture medium with a 1% concentration for the staining. The PI was applied to the upper chamber directly after the polymer/polyplex application. 2.6. Simplified modelling and simulation It was assumed that the cell has a disc shape, is positioned in the middle of the microhole and that no part of the cell is inserted into the hole (Fig. 1b). Further actin filaments and adhesion proteins from the cell to the chip surface were neglected for simplicity. The spread out disc shape cell was modelled with a height of hcell = 3.5 m and a membrane thickness of dcell-membrane = 10 nm. The radius rcell and the cell/substrate gap g were set to the values 7, 8.5 and 10 m and 25, 50 and 75 nm, respectively and the simulated impedance magnitude was investigated at 1 kHz. Membrane permeabilisation was simplified by two holes in the cell membrane directly above the microhole. The electrical properties for the different subdomains in Fig. 1b are taken out of the literature (Elshabini and Barlow, 1998; Grimnes and Martinsen, 2000; Malmivuo and Plonsey, 1995). The conductivity (S/m) for the cytoplasm, cell membrane, culture medium and the chip membrane is 0.5, 10−7 , 1.6 and 10−12 , where the relative permittivity εr is 80, 11.3, 80 and 5.5, respectively. For the subdomains it was assumed that the conductivity and the relative permittivity are linear and have an isotropic distribution. The computer based calculations were predicated using the assumption that a current density occurs if an electrical field is applied to a dielectric material (Harrington, 1961): = J = ( + jωε0 εr ) E, ∇ ·H the magnetic field, E the electric where J is the current density, H field, ω the angular frequency, ε0 the permittivity of free space and
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√ j = −1. On the basis of the reciprocal lead field Jreci and excitation current density Jcc the general transfer impedance Zt is (Grimnes and Martinsen, 2008):
1 J · J dv cc reci
Zt = v
This equation is simplified in a two electrode arrangement where the forward and reciprocal current densities are identical to:
1 2 J dv
Z= v
where J is the unit current density [1/m2 ]. The impedance was computed in a cylindrical symmetry model by FEM simulation (used software COMSOL Multiphysics).
3. Results 3.1. Electrical characterisation of the assembled unit without cell For the electrical characterisation the assembled microhole cell chip unit was filled with cell culture medium and the impedance was measured in the frequency range from 0.1 Hz to 1 MHz (Fig. 2a). In the lower frequency range (1–0.1 Hz) the impedance magnitude increased from nearly 200 k to above 1 M, which was caused by the electrode impedance. This was modelled by the constant phase element CPEelectrode . In the frequency range from 1 Hz to 1 kHz the impedance magnitude showed a nearly stable value. In the higher frequency range (above 1 kHz) the impedance magnitude decreased to almost 1 k at 1 MHz. The impedance progression between 1 Hz and 1 MHz was electrically modelled by a parallel circuit of the resistance Rchip-hole with the constant phase element CPEchip-membrane . For the circuit parameters, the frequency band from 0.1 Hz up to 10 kHz was fitted best by the designed electrical circuit (Fig. 2b). For the parameters of the circuit the values were fitted using ZView, where the impedance of a constant phase element was defined as ZCPE (ω) = 1/[(jω)n Q◦ ]. The values for 0 0 Rchip-hole , Qchip-membrane , nchip-membrane , Qelectrode and nelectrode were 156.13 ± 12.03 k, 1.476 ± 0.297 nF · snchip-membrane −1 , 0.890 ± 0.014, 1.480 ± 0.073 F · snelectrode −1 and 0.918 ± 0.006, respectively. The resistance of the microhole can be calculated analytically using (Malmivuo and Plonsey, 1995; Newman, 1966): 1 dchip-membrane 1 1 +2· · · Achip-hole 4 rchip-hole 1 1 dchip-membrane 1 · · = + 2 2 rchip-hole · rchip-hole
Rchip-hole =
=
1 · rchip-hole
dchip-membrane · rchip-hole
1 + 2
For the chip parameters (dchip-membrane = 1.6 m, rchip-hole = 2.5 m) and a medium conductivity = 1.6 S/m, the hole resistance should result in Rchip-hole = 175.93 k. The variation of the fitted hole resistance value to the calculated one could be directly connected to variations in the chip geometries (Fig. 2c). For this examination, one parameter was fixed and the other was calculated for Rchip-hole = 156.13 ± 12.03 k. For a fixed membrane thickness of dchip-membrane = 1.6 m the hole radius is rchip-hole = 2.76 ± 0.17 m. If the hole radius was fixed at 2.5 m, dchip-membrane results in 0.98 ± 0.38 m.
Fig. 2. Electrical and analytical characterisation of the measurement setup without positioned cell. (a) Measured and fitted data in the Bode plot of the assembled system. The impedance magnitude |Z| is labelled by the opened circle; the impedance phase ϕ is labelled by a filled circle, respectively. (b) Simplified equivalent electrical circuit for the assembled system in the frequency range from 0.1 Hz to 10 kHz. CPEelectrode describes the electrode impedance (constant phase element), Rchip-hole is the hole resistance and CPEchip-membrane is the interface impedance between the chip membrane and the saline solution (constant phase element). (c) Analytically calculated influence on the hole resistance Rchip-hole of the microhole cell chip by the variation of the hole radius rchip-hole and the membrane thickness dchip-membrane .
3.2. Influence of the captured cell on the measurement The measured impedance described the cell positioning on the hole and attachment to the chip membrane surface (Fig. 3). During the first 3 min the impedance magnitude value was almost constant (∼120 k). This impedance value described the unoccupied microhole. The steep increase in the magnitude value from 120 k to nearly 180 k was induced by the cell arriving over the hole and the blocking of the hole. After the cell was positioned on the hole the magnitude increased because of the cell spreading on the chip membrane surface (Fig. 3b). The steady increase in the
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Fig. 3. The positioning and attachment process of the single cell. (a) The impedance magnitude |Z| is labelled by the opened circle; the impedance phase ϕ is labelled by a filled circle, respectively. The abrupt rise at 3.25 min belongs to the cell positioning on the microhole. The further increase in the magnitude is caused by the cell spreading on the chip membrane surface. (b) The phase contrast picture taken directly after positioning shows still a round cell, where the positioned cell after 10 min seems to be attached onto the chip membrane surface (scale bar = 25 m).
impedance magnitude ended nearly 8 min after the cell positioning at an impedance magnitude around 350 k.
the normalised impedance magnitude by approximately −0.14 or roughly 0.12, respectively.
3.3. Application of the polymers and polyplexes
4. Discussion
Modified PEI with guanidine functional groups is believed to improve cell-internalisation while diminishing the toxic effects of typical polymer delivery systems (Nguyen et al., 2008). The normalised impedance magnitude after polyplex VT01 + DNA application showed almost no changes compared to the control (Fig. 4a). The control is just cell culture medium with a trapped cell on the hole. Also the Propidium Iodide (PI) staining showed no cell membrane perforation. The normalised impedance magnitude exhibited decay 85 min after VT09 application (Fig. 4b). The decline ended at 110 min at an almost stable value of around 0.7. An increase in the standard deviation 110 min after the VT09 application occurred. In the first 50 min after the polyplex VT09 + DNA (Fig. 4c) application the measured impedance data showed a higher impedance magnitude compared to the control, where a maximum was reached after 30 min. Above 50 min the normalised impedance magnitude decreased against the control level. Also a higher standard deviation resulted after 50 min. A stable level of around 0.85 was observed from 85 min until 125 min after the application. After 125 min the impedance magnitude showed another decay to a level of 0.55. The measurement results from the polymer VT09 and the complex VT09 + DNA were correlated to PI. The microscope pictures after VT09 application (Fig. 5a) showed no positive PI stained cell before 100 min whereas after 115 min cells on the chip membrane as well as the captured cell on the hole exhibited a PI stained nucleus. The images of VT09 + DNA (Fig. 5b) showed PI stained nuclei cells after 128 min. The captured cell on the hole exhibited a PI stained nucleus after 135 min of the polyplex application.
It was shown that the microhole cell chip unit can be used for the single cell manipulation as well as the electrical recording of membrane integrity effects. The manipulation of the cell by a suction pressure was adjusted manually and monitored by a pressure sensor. The positioning pressure was around 0.05 mbar. This pressure was increased to 0.2 mbar after the cell spread onto the microhole to reduce cells movement. These values were adjusted and established, so that during the positioning and the holding of the cell on the microhole neither membrane nor cell damages due to hydrodynamic stress were observed by the use of fluorescent markers (results not shown). For the electrical characterisation, the assembled unit without captured cell was measured and then fitted by an electrical circuit. It was found that the frequency range between 1 Hz and 10 kHz in the 2-terminal measurement can be directly connected to the resistance of the microhole in parallel to the non-ideal capacitance (CPE) of the chip membrane. The capacitance breakdown of the silicon nitride membrane limited the suitable frequency range, because it shortened the current through the microhole. Therefore the upper bound for the frequency range in the microhole chip arrangement was around 1 kHz. The variation of the fitted hole resistance Rchip-hole = 156.13 ± 12.03 k to the calculated value might originated from the deviation during the chip-microfabrication process, where the two parameters rchip-hole and dchip-membrane were not exactly adjusted to the specified values. In order to determine if an effect was caused by the cell response to a reagent a stable impedance level had to be guaranteed. During the cell spreading process the impedance magnitude at 1 kHz increased steadily by more than 194% in the first 8 min after the cell positioning. This might be based on the enlargement of the cell covered chip membrane surface and by the reducing of the cell/substrate gap (see simulations), which resulted in a higher resistance for the current mostly flowing between the cell and the chip membrane at this frequency. Therefore a suitable starting time for all measurements was greater or equal to 8 min after the cell positioning. In this work the starting time was chosen to be 14 min after cell positioning. The fast cell spreading could be based on the fact that the cells were standing for nearly 2 h in the falcon tube, where the adhesion proteins are still expressed, before they were used. By the use of FEM simulations the influence from cell parameters like the cell size, the cell/substrate gap without cell membrane holes as well as with cell membrane disruption on the impedance
3.4. Simulation results The theoretical investigations showed that for smaller cell/substrate gaps and larger cell radius the impedance magnitude possessed a sharper decline for increasing cell membrane holes (Fig. 6). If there was no cell membrane disruption (see rcell-hole = 0 nm) the simulation results illustrated how strong the cell parameters could affect the impedance data. If the cell/substrate gap increased from 50 to 75 nm or reduced from 50 to 25 nm, the normalised impedance magnitude reduced by almost 0.29 or increased by nearly 0.76, respectively. A variation in the cell radius from 8.5 to 7 m or from 8.5 to 10 m affected
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Fig. 4. The normalised impedance magnitude data measured with respect to time after polymer/polyplex application. (a) The measured data after polyplex VT01 + DNA application has a similar curve progression as the control. (b) The measured data after polymer VT09 application exhibits a decay at 85 min to a value of 0.7 compared to the control. (c) The measured data after polyplex VT09 + DNA application exhibits a maximum at 30 min, a first decay after 50 min until 80 min and a second decay after 125 min compared to the control.
was investigated. This results might be used to relate the measured data to the cell membrane hole size and cell parameters. For long-term observations of polymer complexes, cells had to be kept as long as possible on the microhole. The results showed that cells could be kept for nearly 3 h on the hole. The normalised mean impedance magnitude values varied in a range of 3.12%. This variation could not be completely reduced because of the cell’s movement and morphological changes on the hole. Consequently impedance magnitude variations in this range were not based on the cell responses to special reagents. The system was verified for several polymer/polyplexes at biological relevant concentrations. The polymer/polyplex application was done by exchanging the whole cell culture medium above the microhole chip with the polymer-/polyplex solution. This solution replacement had no direct influence on the impedance magnitude, because all measured data showed no variations compared to the control directly after application. The normalised impedance magnitude after application of the complex VT01 + DNA exhibited no relevant changes in relation to the control over the whole monitoring time. The correlation with the Propidium Iodide (PI) staining displayed no positively stained cell nucleus 95 min after the application. PI can be used as a marker for visualising cell membrane perforations. Therefore the polyplex VT01 + DNA did not permeate the cell membrane, which was shown by the impedance data and the PI staining. The data for the polymer VT09 showed no difference with respect to the control values in the first 80 min. In addition the standard deviations compared to the control were almost in the same range, and the fluorescent combined phase contrast pictures showed no stained cell nucleus in this time range. This suggests that nothing happened to the cell membrane of the captured cell during the first 80 min. Between 85 min and 100 min after the application the normalised impedance magnitude dropped down from 1 to a value of nearly 0.9. This decay is not arisen from membrane disruption (no PI-staining at 100 min), but from a shrinking in the cell size or a detaching from the chip membrane. A cell shrinking is visible in the microscope images taken at 55 min and 100 min (from nearly rcell = 10–8.5 m). The simulation showed that such a cell shrinking (rcell = 10–8.5 m) at a cell/substrate gap of 50 nm shows a difference of 0.12 in the normalised impedance magnitude with no cell membrane disruption. The phase contrast/fluorescent image taken at 115 min after VT09 application showed stained cell nuclei. Therefore cell membrane perforations occurred in the range between 100 min and 115 min, which could be recognised by the steep slope in the impedance magnitude. From the simulation a decrease of 0.2 in the impedance can be related to a membrane hole radius of 82 nm at a cell/substrate gap of 25 nm and a cell radius of 8.5 m. Above 115 min the cell membrane was disrupted the cell did not show any further reactions and therefore a stable normalised impedance magnitude was observed. The increase in the standard deviation after 100 min could be based on fast cell membrane alterations. In the case of the polyplex VT09 + DNA the normalised impedance magnitude showed an increase in the first 50 min compared to the control, where a maximum of 1.05 was reached after 30 min. This might be based on a swelling of the cell in the first 30 min caused by the polyplex application. This swelling was reversed in the time from 30 min until 50 min, because the normalised impedance magnitude reached the starting value of 1 again. Between 50 min and 85 min the normalised magnitude decreased compared to the control almost linearly and showed also an increase in the standard deviation. This effect was caused not by cell membrane perforations, due to the fact that the captured cell showed no PI stained nuclei. This decreasing in the impedance might be based on a small cell shrinking (significant changing in cell size is not visible in the microscope images) combined with a cell detachment from the chip membrane. During the second decline in the normalised impedance magnitude from 125 min until 135 min cell membrane
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Fig. 5. Phase contrast pictures superimposed with the fluorescent pictures. (a) Pictures after VT09 application. After 115 min the captured cell exhibits a stained nucleus. (b) Pictures after VT09 + DNA application. The captured cell on the hole shows a stained nucleus after 135 min (scale bars = 25 m).
Fig. 6. Simulation results for the normalised impedance magnitude values. The impedance magnitude is plotted against the cell membrane hole radius for different cell/substrate gaps g where the cell radius was fixed to 8.5 m as well as cell radii rcell with a constant cell/substrate gap of 50 nm.
disruption occurred, because the phase contrast/fluorescent picture taken at 135 min showed a PI stained nucleus of the captured cell. This decline of 0.3 in the normalised impedance can be related to the simulated membrane hole radius of around 125 nm if the cell/substrate gap is roughly 25 nm and the cell radius is around 10 m.
Another also interesting part can be the identification of biological relevant substances or particles, because the measured curves showed a completely different progression. Therefore possible application areas could be to improve or select cell membrane crossing vehicles, measure effects on single cells and the identification or characterising from different polymer/polyplexes.
5. Conclusion
References
The Micro-Electrical-Impedance-Spectroscopy System (EIS) based on a microhole chip can be used for impedance monitoring on single cells. It was shown first time that the chip setup is sensitive and can be used to measure subtoxic effects of biological relevant particles or substances at relevant concentrations. With the correlation from the microscope images, the simulation results and the impedance data it is possible to give a statement about changes from cell parameters like the cell size, the cell/substrate gap and the cell membrane disruption, which can be an identification for subtoxic effects on cells, whereas the PI-staining only can visualise cell membrane perforations.
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