ENGF0001 – INTEGRATED ENGINEERING
SUBTASK GUIDANCE DOCUMENT
SUBTASK PLANNING – IMPEDANCE PROBE
INTRODUCTION
Measuring the growth of cells is an important component in the successful laboratory experiment or manufacturing process. Batch cell growth can be monitored and divided into three discrete phases. These are the lag phase, exponential phase and stationary phase.
During lag phase very little growth is expected. In the second phase, exponential growth, the cells achieve their maximum growth rate. However, this growth will eventually come to a stop as the media runs out of carbon source and other essential growth factors There is a fourth stage, which can occur if the cells are left in stationary phase too long, and this is the death phase. In this phase the cells will begin to break down and die. It is optimal to harvest the cells just as the stationary phase starts, but the time at which this occurs varies between batches.
Cell growth is ideally measured by an automated method, which can distinguish between live and dead cells, as well as determining the mass of biological material present.
You have been tasked with assembling and calibrating an impedance probe for monitoring cell growth.
THEORY
Living cells can be modelled as a resister (Ri) and capacitor (C) in series, both in parallel with a 2nd resistor (Re)(Figure 1).
In this simplified model, Re can be considered to describe the media the cells are suspended in, C is related to the cell membranes composition and cell size, and Ri related to the impedance of the cells. Changes in any of these, or their proportions will also affect the impedance. The impedance is frequency dependant and varies with cell density, under controlled conditions. Impedance (Z) is a vector, it’s modulus (|Z|) is equivalent to resistance.
Figure 1 Living cells modelled as simple circuit
Biological impedance is typically determined by applying an alternating current of fixed amplitude to a pair of electrodes (drive) and measuring the resultant voltage difference across a pair of electrodes. One or both electrodes may be shared between the drive and measurement circuits and one or more frequencies of injected current may be used.
Figure 2 Diagram representation converting voltage supply to current supply
To change a voltage source (e.g. from myDAC) to a current source, put a large resistor (R1) in series with the load, i.e. the yeast solution. For this circuit the choice or R1 has been determined for you. Figure 2 depicts this equivalency.
The printed circuit board (PCB) provided to you contains a simple differential amplifier (figure 3a) along with a 50Hz Twin T notch filter (figure 4b). A simple differential amplifier has a gain =R3/R2. Resistances (i.e. the ‘load’) in series with each R2 will alter the gain unless load<
Figure 3 a: simple amplifier; b: twin T notch filter
If Current (I) is constant, and the amplifier within its optimal operating range, |Z| will be proportional to the amplitude of the output voltage (V) of the amplifier (Ohm’s law).
DESIGN METHODOLOGY
You will be required to assemble and calibrate your own impedance probe from the materials available, and use it for monitoring cell growth during the 24h run. Design decisions will be required with respect to the gain, frequency and electrode arrangement of the probe, taking into account the dimensions of the prototype rector. You will calibrate the probe using the 50g/l yeast solution provided, and the following serial dilutions of this (20g/l, 8g/l, 4g/l, 2g/l, 1g/l).
EQUIPMENT AND MATERIALS AVAILABLE
● Enclosed PCB of combined amplification, filter and drive circuits
● myDAQ & accessories, (requires Windows PC with NI Elvis software)
● Connecting wire (6 colours) & cutters/strippers
● Plastic cup
● Electrode holder set (top ring and posts)
● Phosphate Buffer Solution (PBS) or saline of equivalent impedance.
● Activated yeast (Saccharomyces cerevisiae) solution (20g/l, 50g/l).
DELIVERABLES
● Amplitude(V) vs. frequency plot for a) PBS and b) the 20g/l yeast solution provided
● Impedance probe operating parameters (electrode locations, constant current source: input voltage, frequency/frequencies, …)
● Calibration curve of output amplitude(Vout) vs. yeast concentration
● Brief justification for the final design/settings
● A plot from the 24hr bioreactor run showing yeast growth in the bioreactor over time
TIPS
● Start with a quick scan of the measurement range, for all parameters, to give you a
rough idea of your expected results, record a qualitative description of the trends and other observations, and make a first estimate of the probe operating parameters.
● Change only one variable at a time
● Consistency in electrode arrangement is critical to consistent readings
● Use the same electrode configuration, spacing and liquid height for all ‘deliverables’
SCALE-UP CONSIDERATIONS
By the end of this subtask you will have gone through the methodology required to characterise impedance, as a function of cell growth, within the bioreactor. However, this is for a virtual small-scale model. Speculate how impedance might change in a larger (e.g. 1,000 litre) reactor. How might your design change?
REFERENCES
Brown, B. H., R. H. Smallwood, D. C. Barber, P. V. Lawford and D. R. Hose (1999). Medical Physics and Biomedical Engineering. London, Institute of Physics Publishing
Soley, A., M. Lecina, X. Gámez, J. J. Cairó, P. Riu, X. Rosell, R. Bragós and F. Gòdia (2005). "On-line monitoring of yeast cell growth by impedance spectroscopy." Journal of Biotechnology 118(4): 398-405.