WO2015173443A1

WO2015173443A1 - Bioimpedance measuring system for wirelessly monitoring cell cultures in real time, based on cmos circuits and electrical modelling

Info

Publication number: WO2015173443A1
Application number: PCT/ES2015/000064
Authority: WO
Inventors: Alberto OLMO FERNÁNDEZ; Gloria HUERTAS SÁNCHEZ; Alberto YÚFERA GARCÍA
Original assignee: Universidad De Sevilla
Priority date: 2014-05-13
Filing date: 2015-05-12
Publication date: 2015-11-19
Also published as: ES2551249A1; ES2551249B1

Abstract

The invention relates to a novel bioimpedance measuring system for wirelessly monitoring cell cultures in real time. The system uses a two-dimensional (2D) array of electrodes as bioimpedance sensors and implements the measuring circuit with CMOS technology, using electrical modelling for image reconstruction.

Description

Title

Bioimpedance measurement system for real-time and wireless monitoring of cell cultures based on CMOS circuits and electrical modeling

Object of the invention

The invention patent refers to a new bioimpedance measurement system for real-time and wireless monitoring of cell cultures. The system uses a two-dimensional (2D) array of electrodes as bioimpedance sensors and implements the measurement circuit with CMOS technology, using electrical modeling for image reconstruction.

The invention is framed within the measure of electrical impedance of biological material. It also refers to a sensor microelectronic device for carrying out said measurement.

State of the art

Many biological parameters and processes can be detected and controlled by measuring their bioimpedance, with the advantage of being a non-invasive and relatively cheap technique. Cell growth, changes in cell composition or changes in cell location are just a few examples of processes that can be detected by microelectrodes through impedance changes [1-4].

This technique was invented by Ivar Giaever and Charles Keese in 1993 [5], registering in a patent an apparatus for cell culture monitoring, based on a series of wells where cell culture is performed, each with an array of microelectrodes through which an alternating current is introduced, measuring the resulting electrical impedance. This initial patent was completed with a series of related patents, applied to the subject of the study of cell mobility [6] or metastatic activity of cancer cells [7] ·

Another bioimpedance measurement system was registered in 2005 by B. Rubinsky et al. [8]. This system uses two electrodes between which a potential difference is applied and a dielectric membrane with micro-holes through which the passage of electric current is forced. In Spain methods have also been registered for the simultaneous determination and visualization of electrical bioimpedance signals in biological material at various frequencies [9], using a treatment of the excitation and response signal as two independent functions in the time domain, and applying signal processing techniques ( cross correlation and Fourier transformation) for best results.

In general, for the problem of measuring a given impedance Z _x , of magnitude Z _x0 and phase φ, several methods have been described, which require excitation and processing circuits. The excitation is usually implemented with alternating current (AC), while the processing is based on the principle of coherent demodulation [10] or synchronous sampling [11], [12]. In both, the circuit processing must be synchronized with the excitation signals, as a requirement for the technique to work, obtaining the best noise performance when the appropriate filter functions (High-Pass (HP) or Low-Pass are incorporated) (LP)).

This paper presents a new impedance measurement system for biological samples useful for obtaining 2D images of a cell culture in real time and wirelessly, unlike other systems found in the literature. It is based on the use of a two-dimensional array of electrodes as bioimpedance sensors, CMOS technology for the implementation of the measurement circuit and the novel use of electrical modeling for image reconstruction, another characteristic that gives the system a greater precision in the task of reconstruction based on bioimpedance.

RELEVANT DOCUMENTS

[1] I. Giaever et al., "Use of Electric Fields to Monitor the Dynamical Aspect of Cell Behavior in Tissue Culture," IEEE Transaction on Biomedical Engineering, vol BME-33, No. 2, pp: 242-247, Feb. 1986.

[2] S. M. Radke and E. C. Alocilja, "Design and Fabrication of a Microimpedance Biosensor for Bacterial Detection," IEEE Sensor Journal, vol 4, n ° 4, pp: 434-440, Aug. 2004.

[3] DA Borkholder: "Cell-Based Biosensors Using Microelectrodes," PhD Thesis, Stanford University. Nov. 1998. [4] A. Yúfera et al., "A Tissue Impedance Measurement Chip for Myocardial Ischemia Detection". IEEE transaction on Circuits and Systems: Part I. vol.52, no .: 12, pp: 2620-2628. Dec. 2005.

[5] I. Giaever, C. R. Keese, Cell susbstrate electrical impedance sensor with multiple electrode array,, US 5,187,096, Feb. 16, 1993.

[6] I. Giaever, C. R. Keese, Electrical wounding assay for cells in vitro, US 7,332,313 Feb. 19, 2008.

[7] I. Giaever, C. R. Keese, Real-time impedance assay to follow the invasive activities of metastatic cells in culture, US 7,399,631, July 15, 2008.

[8] B. Rubinsky, Y. Huang, Cell viability detection using electrical measurements, US 6,927,049 B2, Aug. 9, 2005.

[9] P. Owen Whiters, Method and apparatus for displaying bio-impedance at multiple frequencies, ES 2 1 18 133 T3.

[10] J.J. Ackmann, Complex bioelectric impedance measurement system for the frequency range from 5-1 MHz, Annals of Biomedical Engineering 21 (1993) 135-146.

[1 1] R.PalIzs, J.G. Webbster, Bioelectric impedance measurements using synchronous sampling, IEEE Transactions on Biomedical Engineering 40 (8) (1993) 824-829.

[12] M. Min, A. Kink, R. Land and T. Parve, Method and device for measurement of electrical bioimpedance, US 7,706,872 B2, Apr 27, 2010.

[13] X. Huang et al., "Simulation of Microelectrode Impedance Changes Due to Cell Growth," IEEE Sensors Journal, vol.4, n ° 5, pp: 576-583. 2004

[14] N. Joye, et al., "An Electrical Model of the Cell-Electrode Interface for High-density Microelectrode Arrays," IEEE EMBS pp: 559-562. 2008 Description of the content of the figures

Figure 1. Scheme of the complete system architecture. The system consists of a 2D array of electrodes, on which cell culture is performed, an excitation and impedance measurement circuit, a radio frequency transmitter circuit for wireless data transmission, and software for decoding and reconstruction. of the image, based on electrical modeling.

Figure 2. Block diagram of the excitation circuit and impedance measurement. The main components of the circuit are: an instrumentation amplifier (Al), the AC - DC converter or rectifier, the error amplifier (AE) and the current oscillator with the programmable current output

Figure 3. Temporal evolution of a culture of MCF-7, in which the initial growth is observed, introduction of a toxic dose (protease inhibitor) and a final process (from minute 4369) of washing, from which The cell culture grows again.

Figure 4. a) Selection of 8x8 matrix in MCF-7 cell culture b) Fill factor obtained by electric modeling for the 8x8 matrix, defining the fill factor as the percentage of area occupied in each pixel by cells of the culture, varying from ff = 0, if the presence of any cell is not detected, up to ff = 1, with the entire area occupied by cells.

Description of the invention

The system object of the present invention is composed of a 2D array of electrodes, on which the cell culture is carried out, an excitation and impedance measurement circuit, a radio frequency transmitter circuit for wireless data transmission, and software for decoding and reconstruction of the image, based on electrical modeling. The scheme of the system architecture is shown in Figure 1.

Each cell in the electrode array will consist of two electrodes, a central electrode (e-ι) and one of a larger area called a reference electrode (e ₂ ), among which an AC current is established at a given frequency, and an impedance Z _x is measured. The electrical modeling, later described, allows the measured impedance, Z _x , to correspond with the area covered by the cell culture in the AC electrodes. The electrodes can be manufactured with CMOS technology.

The block diagram of the circuit proposed for the excitation and impedance measurement is in a closed loop and is shown in Figure 2. For the measurement of the magnitude of impedance, Z _x , it is considered that the excitation signal is AC current, at a given frequency ω. The circuits are designed to work at a constant amplitude on the V _x sensor, which is known as the P _sta i condition.

The main components of the circuit are: an instrumentation amplifier (Al), the AC - DC converter or rectifier, the error amplifier (AE) and the current oscillator with the programmable current output. The voltage gain of the instrumentation amplifier in its bandpass is _ai . The rectifier functions as a full-wave peak detector, to measure the largest (and smallest) voltage amplitude of V ₀ . Its output is a DC voltage, directly proportional to the amplitude of the output voltage of the instrumentation amplifier, with a gain of _dc . The error amplifier, with a gain at _ea , compares the DC signal with a reference V _ref , to amplify the difference. The current generator generates the AC current to excite the sensor. It consists of an external AC voltage source (V _s ), a transconductance amplifier (OTA) with transconductance g _m and a four quadrant voltage multiplier of constant K. The voltage generated by V _s is multiplied by V _m and is converted current by the OTA. A simple analysis of the complete system gives the approximate expression for the voltage amplitude in V _x :

V., (1)

V _r

a, „ ^■ a when the condition that

Z-GL -a. a, a «de» 1 (2) the voltage V _x remains constant in equation 1 and independent of the load Z _x . Considering the relationship between the current i _x and the voltage V _m (¡ _x = G _m V _m ), the magnitude of the impedance can be expressed as:

G V

Equation 3 allows the calculation of the magnitude of impedance Z _x from the voltage V _m , since V _x and G _m are known from equation 1 and the design parameters. The impedance phase can also be measured from ν _Φι as shown in Figure 2. In particular, the use of CMOS 0.35μιη technology is proposed for circuit implementation, with a supply voltage of 3V.

The radio frequency signal transmitter and receiver circuit will emit, in digital or analog form, the bioimpedance signal. This allows the wireless monitoring of the cell culture, without the need to extract the samples from the incubator or to interfere with the processes of the cell culture. Likewise, this radio frequency signal transmitter and receiver circuit allows wireless programming of the excitation and bioimpedance measurement circuit, parameters such as the sampling frequency, amplitude of the excitation current and other described parameters being remotely established. previously.

This radio frequency signal transmitter and receiver circuit may be implemented so that the data is transmitted at a frequency of 2.4 Ghz or other available bands, and so that it is compatible with 802.11, 802.15 or similar standards.

The monitoring software obtains several graphs that allow monitoring the behavior of the cell culture, among which are the temporal evolution of the absolute value of the impedance and the phase, for different frequencies and for each of the electrodes of the matrix. Likewise, the software will integrate an electrical modeling for the characterization of the electrode - cell interface in each electrode, which will allow advanced functionalities in the reconstruction of the image based on bioimpedance. The cell-electrode electrical models are keys for the correspondence between simulations and real behavior of the systems, and therefore, for the correct decoding of the results obtained experimentally, which is generally known as the reconstruction problem. [3, 13, 14].

The impedance of the electrodes in ionic liquids has been extensively studied. When a solid (including metals, semiconductors and insulators) is immersed in an ionic solution or electrolyte, the ions in the solution can react with the electrode and the solid ions of the electrode can be added to the solution. The result is a complex, electrified or double layer interface. This complex system can be modeled using passive circuit elements, as has been described in numerous biomedicine and electrochemical texts. The Helmholtz-Gouy-Chapman-Stern model is the commonly accepted model for electrically describing the distribution of charges at the electrode interface, being able to approximate by the following expression for the double layer:

{2nf) ^V2 _{ j {2nf) (4)

KK where σ _{ύ \} and Edi are the conductivity and the dielectric permittivity, t is the thickness of the region, Q is the capacity of the interface per unit area, which consists of the serial combination of the Helmholtz layer and the double diffuse layer, and K is a constant related to Warburg impedance (associated with mass diffusion processes in the interface).

We model the electrode-cell space (eiectrode) by means of a region of thickness t with a _gap conductivity value, with the following value:

t cell-electrode (5) '

medium gap

We model the charging region (also called the electrical double layer) that is form in the electrolyte in the inferred with the cell with a capacity C _hd , defined as the series of three capacities [14]: ε0 ^£ 1ΗΡ (6)

l IHP

C ^ε 0 ^£ ΟΗΡ

"OHP ^~ " IHP

c _{d =} qj2s ₀ s _d KTz ² n ° N

KT where A _ce is the surface of the attached membrane, ε ₀ is the dielectric permittivity of free space; £ | _H p and ε ₀ ΗΡ are, respectively, interior and exterior of Helmholtz relative plane dielectric constant; d | _H p is the distance from the inner plane of Helmholtz to the membrane; d ₀ HP is the distance from the outer plane of Helmholtz to the membrane; £ d is the relative dielectric constant diffuse layer; K is Boltzmann's constant; T is the absolute temperature; q is the charge of the electron; z is the valence of the ions in solution; N ₀ is the mass concentration of ions in solution, and N is Avogadro's number.

Finally, the equivalent circuit of the membrane is modeled as a resistance R _m in parallel with a capacity of C _m , in a manner similar to that reported by Joye et al. [14].

R- = ^■ ^■ A

Where A is the area of the membrane, g _mem is the conductivity of the membrane and C _mem the capacity of the membrane per unit area.

Therefore, by integrating the electrical modeling described in a finite element analysis software, in which we describe the geometry of the electrodes, we can study the correspondence between simulations and real behavior of the systems, and therefore, correctly decode the results obtained experimentally. Similarly, as mentioned above, the control software allows remote selection of parameters such as the sampling frequency or the output current level, wirelessly programming the excitation circuit and bioimpedance measurement.

Embodiment of the invention

Example. Analysis of cell culture of MCF-7 from an 8x8 electrode array.

In this example the particularization of the system for the measurement of impedance in a cell culture of MCF-7 cancer epithelial cells, through an 8x8 electrode array is presented. The size of the selected pixel is 50 μιη x 50 μιη, similar to the dimensions of the cell. The excitation frequency of the electrodes varies from 10 kHz to 100 kHz, obtaining for each frequency an impedance measurement in each pixel of the electrode array.

The monitoring software allows to obtain wireless signals that show the evolution of cell culture over time, for different sampling frequencies. Figure 3 shows an example of the history recorded for a culture of MCF-7 cells.

Through electrical modeling we can obtain an estimate of the area occupied in each pixel by crop cells. We can use the parameter fill factor (ff), as the percentage of area occupied in each pixel by cells of the culture, varying from ff = 0, if the presence of any cell is not detected, up to ff = 1, with the entire area occupied by cells. Figure 4 shows the fill factor obtained for each pixel of the 8x8 matrix.

With the wireless sending of the impedance data for each pixel, the growth of the cell culture can be monitored in real time, without the need for a visual inspection of the culture, with the consequent saving of time and with the possibility of implementing automatic alarm signals to unexpected changes. Similarly, automation in obtaining the information in digital form through a 2D matrix allows further processing of the data of each pixel for a more advanced study of the evolution of the crop.

Claims

1 - Bioimpedance measurement system for real-time and wireless monitoring of cell cultures based on CMOS circuits and electrical modeling, characterized by understanding;

a) a matrix or 2D vector of electrodes; a central electrode (e ^ and one of greater area called reference electrode (e ₂ ),

b) a closed loop bioimpedance excitation and measurement circuit, c) a radio frequency signal transmission and reception circuit compatible with 802.11, 802.15 or similar standards

d) and a monitoring and programming software based on electrical modeling.

2 - Bioimpedance measurement system for real-time and wireless monitoring of cell cultures based on CMOS circuits and electrical modeling, according to claim 1, characterized by the use of technology based on CMOS processes for the implementation of the array or vector of microelectrodes .

3 - Bioimpedance measurement system for real-time and wireless monitoring of cell cultures based on CMOS circuits and electrical modeling, according to previous claims, characterized by the use of technology based on CMOS processes for the implementation of integrated circuits.

4 - Bioimpedance measurement system for real-time and wireless monitoring of cell cultures based on CMOS circuits and electrical modeling, according to previous claims, characterized by the use of an instrumentation amplifier, an AC-DC converter or rectifier, an amplifier error and a current oscillator with the programmable current output for the excitation circuit and bioimpedance measurement.

5 - Bioimpedance measurement system for real-time and wireless monitoring of cell cultures based on CMOS circuits and electrical modeling, according to previous claims, characterized by the use of the fill factor parameter (ff, percentage of area occupied in each pixel by cells of the culture) for the decoding of the experimental results and the reconstruction of image.