BIO INSTRUMENT FOR DETERMINING THE VIABILITY OF CEL LS ON THE BASIS OF THE IMPEDANCE MEASUREMENT

A. Rösner, Th. Frank, I. Tobehn, A. Steinke

CiS research institute for microsensorical technology and photovoltaics, D-99084 Erfurt, Germany

Abstract – The creation of new cosmetic, pharmaceutical and other chemical substances puts the material screening to the proof. According to the DIN ISO 10993-5 norm, every substance needs to be tested in respect of their biocompatibility. To supplement the widely used optical methods, a new sensorial system has been created, which is based on an impedance spectroscopy. The basis of this system is the adhesion of certain cells on the sensorial surface, thus giving information about the vitality of cell cultures according to the biocompatibility of active substances, materials and consumables.

Keywords : impedance spectroscopy, AD5933,

biocompatibility, cellular adhesion I - Introduction The testing of materials in terms of their

biocompatibility is a central task in research and economy nowadays. Before a new substance can be placed on the market, the compatibility of these fabricates needs to be checked[1]. For this reason cell counts will be taken out to classify the mortality of cell cultures as a function of the biocompatibility of the examined materials according to the DIN ISO 10993-5 norm [1].

For a quantitative analysis of the number of vital cells in a nutrition medium, especially histological methods are used. In the course of these processes, cytochemical dyestuffs are used to mark cells, leading to a better level of diversity of living and death cells, which is needed for a properly done cell counting. There are several ways to do such counting procedures. One procedure is, for example the so called resazurin-resorufin-test (short RR-test [2]), another one are the automatically working counter systems.

A problem of this method is that it is not possible to gain information about the lethality of the examined cell populations until a histological treatment and the subsequent cell counting took place. In order to acquire predictions about the biocompatibility, the new system should make it possible to determine changes in the cell vitality while the cell probes stay in the incubator.

The base of this project is the galvanical interconnection of the cells and the sensorial elements due to the cellular adhesion. Because of the impedances dependency from this interconnection, it is possible to make assertions, regarding the vitality of cell probes.

Figures 1 and 2 illustrates a trial measurement from the Kurt-Schwabe-institution[7]. The metering shows

L929 cells at 37°C in a nutrition fluid of the type RPMI 1640 with 2 g/l NaHCO3 and 5 mg/l phenol red with 10% FCS and 5% CO2 in an incubator. Living cells behave like a hollow sphere in an electric field, containing two regions with different electrical properties. In addition, the cell membrane interfaces with the sensorial surface through adhesion, thus building a galvanic coupling to the scaffolds surface.

In the course of death, the cell membrane severs and the adhering junctions are falling apart. The cell is no longer an insulator, which leads to a changing value in the impedance. Alterations of that kind make it possible, to define statements about the growth or decaying of cell cultures through an impedance spectroscopy. Furthermore, these observations leads to predictions about the biocompatibility of the substances which have been mixed with the nutrient fluid.

There is an equivalent circuit diagram of the measuring system shown in figure 3. The schematics of a cell conforms with a series circuit consisting of only passive elements. The inner components of a cell is thereby simulated as a pure ohmic resistance, while the lipid bilayer of the cells membrane is modelled as a paralell circuit, containing a resistance and capacitive element [3]. To prevent death cells from falsifying the measurement, they will be cleared away from the scaffolds. An schematic diagram is shown in figure 4, which demonstrates the effect of cell adhesion on the impedance spectroscopy.

It should be mentioned, that there will be no impedance spectroscopy on a single cell, but a measurement of the whole cell probe, which is located on the electrodes surface of the sensorial elements. On the basis of these values, predictions about the biocompatibility will be made.

Figure 1: behaviour of the impedance of dying cells (sensor electrodes gapwidth 5µm) [7]

0

10

20

30

40

50

60

70

80

0 12 24 36 48 60time/h

Z/k

Oh

m 0,2 kHz

1,0 kHz

2,0 kHz

Figure 2: recording of a stereomicroscope, showing a

dying cell culture on the sensorial elements (sensor electrodes gapwidth 3µm) [7]

Figure 3: schematic circuit of the measurement system in the

case of a capacitive coupling; Displayed units: R1 - resistance of nutrient solution R2 - resistance of inner cell components R3 - substrate resistance R4 - membrane resistance C1 - capacity of passivation layer C2 - capacity of oxyd layer C3 - external capacity C4 - membrane capacity

Figure 4: schematic diagram of the expected changes in

the impedance value

II – System Construction The measuring arrangement is containing 3 main

components: the control board USB51-868, the adapter board 3D Cellsens, and a PCI-board containing the sensorial elements with additional ports for a temperature diode, which is used for monitoring the nutrition fluid.

Thereby this construction allows a parallel measurement of 16 cell tanks.

A. Control Board The control board USB51-868 [4] manages the

communication between the hardware components of the system and a personal computer. Thereby is this connection established via USB. Furthermore, it is possible to use a wireless data tranfer.

A microcontroller is transmitting commands to the demanded impedance converter for measuring specific tanks. Previous to this step, the needed arguments for an impedance measurement are transformed into hex code by this controller. After an impedance measurement is done, the microcontroller reads out the values and transforms them to decimal numbers, which are needed for the Graphical User Interface to work.

B. Adapter Board 3D Cellsens and PCI-board The function of the adapter board is to establish a

connection between the USB51-868 module and the intermountable PCI-board. In addition, the impedance converting devices of the type AD5933 are placed on this adapter board. The range for a frequency sweep of these integrated circuits reaches from 1 kHz up to 100 kHz, while the voltage-dependent resolution lies between 20 kOhm up to 10 GOhm [5]. The range of measurement that arises out of the voltage alignment is shown in table 1, referring to a pure capacitive reactance. Another important feature of the Analog Device components is the minimum frequency increment, which is 10 Hz.

To realise the connection of intermountable PCI-boards, the link between the components is realised as a plug contact, which ensures a tight fit of the measuring plates, that is preventing them from unleashing e.g. by vibrations.

metering

voltage

[V]

impedance

range

[kOhm]

Range of pure capacitive

reactance

(1 kHz / 100 kHz)

0.2 20 – 10,000 7.96 nF – 15.9 pF

/ 79.6 pF – 0.16 pF

0.4 40 – 10,000 3.98 nF – 15.9 pF

/ 39.8 pF – 0.16 pF

1 100 –

10,000

1.59 nF – 15.9 pF

/ 15.9 pF – 0.16 pF

2 200 –

10,000

0.79 nF – 15.9 pF

/ 7.96 pF – 0.16 pF

Table 1: Range of measurement of an AD5933

C. Sensorial Elements

The sensorial elements can be delivered in different realizations:

- for a galvanic coupling, the sensors may be ordered with different electrode materials in the absence of a passivation layer

- in the case of a capacitive linkage, the interdigital structures will be furnished with a oxide film

The basic construction of the sensors is defined through an interdigital structure, as seen in figure 5. Therefore a scatter field will be created, which is projected into the nutrient fluid. The fundamental principle of the sensorial elements is used to detect the presence and due to that the quantitative value of a cell colonies impedance. Again, this is possible due to the different electrical behaviour of cells and the nutrition medium.

The interdigital structures got an active surface area of 1630 µm x 1950 µm. By picking TiN or MoSi2 as the contact material, electrode distances between 1.1 µm and 75 µm are possible, whereby the minimum electrode distance is limited to the actual technology bounds at CiS. The width of these electrodes is thereby always bigger than the distance by a factor of 1,3. This relation results due to optimization processes via FEM programs, like Ansys or Comsol. For sensorial elements, which are used without a passivation layer, the minimum distance is limited by technical bounds to 3 µm.

The sensorial elements may be connected to a substrate by a surface layer, which is build through a combination of a SiO2 and SixNy film. Due to that layer, the DUT and the interdigital structure are capacitive coupled in a way, that the thickness of the surface layer will determine the electrical behaviour of the sensor. In contrast to that, the dry capacity of not passivated gold electrodes on glass substrate depends on the distance of the electrodes and as a result to that got small values. For electrodes, which are preferably produced with MoSi2-technology and perhaps are covered with a passivation layer, the interdigital structure is produced on a siliconchip.

Figure 5: Layout of the sensorial element consisting of a

interdigital structure and a temperature diode

III – Evaluation Program A. Memory Control and Calibration A Graphical User Interface has been programmed

with LabView to aid the user by creating, transforming and controlling the commands, that are presented to the system. Furthermore it is supporting the manipulation and evaluation of the gained data.

The user will be enabled to determine the range for a frequency sweep, the increment in frequency, as well as the metering voltage of the system and of course the tanks, which should be measured. The values which were gained after a measurement with the selected arguments, are visualized by 2 x-y plotters. In these diagrams, the impedance and the phase angle will be plotted against the frequency in kHz. A trial measurement with empty sensorial elements is given by figure 6.

To prevent an overstuffed internal memory and due to that a decreasing processing speed, all measurement series will be automatically saved after a complete frequency sweep. Both the folder in which the files are saved and the file names themselves are created by the system and can be reloaded to the program for data manipulation and evaluation purposes. In addition this feature prohibits the loss of data, which has been gained so far in the measuring process.

Concerning the wish for reliable results, all AD5933 are calibrated separate. Furthermore this calibration is carried out with a bigger effort than the calibration which is described in the data sheet of the AD5933 modules [5]. Whereas the characterisation uses a single-frequency or two-point calibration with the assumption of linear changes in the gain and offset values, our system calibrates itself for every integral frequency point. This procedure is used, because the gain and offset values are not increasing linearly but exponential in an inhomogeneous manner. To reduce the memories occupancy load, the collected values of the calibration will be used for a polynomial fitting, thus reducing the needed data for a calibration of measured data to 2 %.

In addition, the system is able to do a recalibration, if for example one of the AD5933 modules must be exchanged while the residual parts are alright. The possibility of recalibrating just the new module while leaving the other values untouched is an time-saving gadget. Another benefit is the opportunity to fix wrong calibration values which could have been occurred due to a broken wire or false settings.

B. Measurements and Data Manipulation The excitation of the USB51-868 board with the

required parameters is done by the LabView program. After transforming the selected arguments, the impedance spectroscopy of the picked tank starts and determines the values of the complex resistance step by step at every frequency point. After the calibration and a first manipulation of the real and imaginary data, which the

impedance converters have measured, the values will be shown to the plots on the user interface, after they were transformed into polar coordinates. The internal measurement process of the AD5933 modules is carried out through a DFT, with a measurement error of +/- 5% [5].

Besides the digital presentation of the impedance and phase angle, it is possible to switch the plots into showing either the real and imaginary parts of the complex resistance or a equivalent electrical circuit containing of a parallel connection between a pure ohmic resistance and a capacity against the frequency.

Considering the fact, that for measures of biocompatibility the time-dependent changes of a cell populations vitality is an important process, the system is able to do measurements automatic. To do so, an interval can be defined, after which a new measurement will start by itself. This could be done without a time limit, until the user stops the measurements, or with a well defined amount of measures which are to be made. Because of this feature, the measurements do not need to be monitored by a user through the whole time, leaving the possibility to do other work.

After several measures have been done with the help of automatic measurements due to an interval, it is possible to let the personal computer do a analysis of the gathered data. Therefore, the first measured row will be taken as the initial state where the cell vitality is assumed to be 100%. Following this step, every measure will be compared with the initial state, leading to a time-dependent history of the cell probes. Once the comparison is done, the system plots the calculated values to a histogram, showing the time-dependent vitality of the examined samples. Due to this, first assumptions can be made, before a histological treatment of the cells has been done.

Besides the frequency sweep, it is possible to let the system run an automatic measurement with the purpose to find a range, where the changes in the impedance of the cell sample are at a maximum. With the detected values, frequency sweeps can be done, which are only considering this range of interest.

Figure 6: GUI of the measurement program

IV – Advantages of the System The big advantage of this system is that the data

manipulation is done mainly by the hardware, which reduces the amount of noise that influences the data. Besides that there are already systems on the market, which are using an impedance spectroscopy for the purpose of testing substances for biocompatibility, we did not managed to find an other product which got as small dimensions as our system. The size of a conventional system is about 25 x 30 x 20 cm while our system only requires about 8 x 10 x 4 cm. This reduction in size offers the chance to take out more measurements of cellular samples at the same time.

The PCI-boards will be, like by other systems, exchangeable cell containers. Furthermore is the impedance of the used sensorial elements within a low-ohmic range, which results in a little difference of the measured impedance. Besides does the fine structures of the impedimetric sensors, which were made using thin-film technology, enable a better sensitivity regarding the resolution of electrode gaps and thereby creating a good detection of cell layers.

Another advantage is that this measuring system does not need any new hardware to be installed on the system. All that is needed to do measurements is an version of the LabView runtime engine version 11f3 or higher and of course the application.

References

[1] DIN ISO 10993 [2] www.imtek.uni-freiburg.de [3] S. Grimnes und O.G. Martinsen: Bioimpedance

and Bioelectricity Basics; Academic Press; ISBN 0-12-303260; 2000

[4] http://www.tekmodul.de/shop-fertiggeraete _loesungen.html?&tt_products[cat]=14%2C15% 2C16%2C17%2C18&tt_products[begin_at]=30& tt_products[product]=13&cHash=62422f4465fd34d790a76b6b72bde1db

[5] http://www.analog.com/static/imported-files/ data_sheets/AD5933.pdf

[6] http://www.cismst.org/loesungen/ impedimetrische-sensorik/

[7] Thomas Frank, Arndt Steinke, Ingo Tobehn, Sandra Päßler, Wolfgang Fichtner, Impedimetric biosensor for cell viability, Proceedings, IMCS 2012 – The 14th International Meeting on Chemical Sensors, May 20 – 23, 2012, Nuremberg, Germany, ISBN 978-3-9813484-2-2