Method for calibrating biomass sensors operating with impedance spectroscopy and use of a suspension for carrying out such a method

Abstract

A method is provided for calibrating impedance-spectroscopic biomass sensors that are embodied to detect information regarding the quantity and/or size of living cells in a biomass by means of an electric field having a periodically changing field direction A calibration suspension encompasses an electrically conductive viscously flowable or viscoelastic carrier substance and electrically conductive solid particles and/or solid semiconductor particles received therein. An electric field is generated having a periodically changing field direction, which acts on the calibration suspension. At least one permittivity value is detected, respectively representing a permittivity of the calibration suspension, in a context of at least two electric fields having different field direction change frequencies. A difference value is ascertained that represents a difference between the detected permittivity values. The difference value is compared with a reference value associated with the calibration suspension.

Claims

1. A method for calibrating an impedance-spectroscopic biomass sensor that is embodied to detect information regarding the quantity and/or size of living cells in a biomass by means of an electric field having a periodically changing field direction, comprising the following steps: furnishing a calibration suspension, which is devoid of biological constituents in a form of living or dead cells and comprising an electrically conductive viscously flowable or viscoelastic carrier substance having an electrical conductivity in the range of 1 to 50 mS/cm, and electrically conductive solid particles and/or solid semiconductor particles received therein; generating an electric field, having a periodically changing field direction at a frequency in the range of 50 kHz to 50 MHz, which acts on the calibration suspension; detecting at least one permittivity value, respectively representing a permittivity of the calibration suspension, for each of at least two electric fields having different field direction change frequencies; ascertaining a difference value representing a difference between the detected permittivity values; and comparing the difference value with a reference value associated with the calibration suspension.

2. The calibration method according to claim 1, further comprising, before or during generation of the electric field, contact between the calibration suspension and at least a portion of the impedance-spectroscopic biomass sensor.

3. The calibration method according to claim 2, wherein contact between the calibration suspension and at least a portion of the impedance-spectroscopic biomass sensor includes wetting at least one sensor electrode of the impedance-spectroscopic biomass sensor with calibration suspension.

4. The calibration method according to claim 2, wherein contact between the calibration suspension and at least a portion of the biomass sensor encompasses wetting all sensor electrodes of the impedance-spectroscopic biomass sensor with calibration suspension.

5. The calibration method according to claim 1, further comprising a step of homogenizing the calibration suspension, by stirring and/or shaking the calibration suspension.

6. The calibration method according to claim 5, wherein the homogenizing step is performed before detection of the permittivity values.

7. The calibration method according to claim 5, wherein the homogenizing step is performed immediately before detection of the permittivity values.

8. The calibration method according to claim 1, further comprising adjusting the impedance-spectroscopic biomass sensor, or a data processing apparatus coupled to the impedance-spectroscopic biomass sensor in signal-transferring fashion, in accordance with a result of the comparison of the difference value with the reference value.

9. A method for impedance-spectroscopic detection of information regarding the quantity and/or size of living cells in a biomass, comprising the steps of: performing the calibration method according to claim 1; subsequently thereto: cleaning of the impedance-spectroscopic biomass sensor; subsequently thereto: impedance-spectroscopic detection of information regarding the quantity and/or size of living cells in the biomass, by means of the impedance-spectroscopic biomass sensor and an electric field, generated thereby, having a periodically changing field direction.

10. The method for impedance-spectroscopic detection of information regarding the quantity and/or size of living cells in a biomass according to claim 9, further comprising subsequently thereto: performing the calibration method according to claim 1.

11. A calibration arrangement, comprising: an impedance-spectroscopic biomass sensor that is embodied to detect information regarding the quantity and/or size of living cells in a biomass by means of an electric field having a periodically changing field direction at a frequency in the range of 50 kHz to 50 MHz; a calibration suspension being devoid of biological constituents in a form of living or dead cells and comprising an electrically conductive viscously flowable or viscoelastic carrier substance having an electrical conductivity in a range from 1 to 50 mS/cm and electrically conductive solid particles and/or solid semiconductor particles received therein, to calibrate the impedance-spectroscopic biomass sensor; and a data processing device that is embodied to process sensor signals delivered operationally by the impedance-spectroscopic biomass sensor, and that is embodied in particular to carry out the method according to claim 1.

12. The calibration method according to claim 1, wherein the calibration suspension comprises an electrically conductive viscously flowable or viscoelastic carrier substance and electrically conductive solid particles and/or solid semiconductor particles received therein, to calibrate the impedance-spectroscopic biomass sensor that is embodied to detect information regarding the quantity and/or size of living cells in a biomass by means of an electric field having a periodically changing field direction.

Description

(1) The present invention will be explained in further detail below with reference to the appended drawings, in which:

(2) FIG. 1 schematically depicts a correlation of the frequency dependence of the permittivity of a biomass; and

(3) FIG. 2 schematically depicts the frequency-dependent permittivity of the biomass in the beta-dispersion range that is of particular interest.

(4) FIG. 1 depicts, very generally and merely schematically, the frequency dependence of the permittivity of a biomass, i.e. for example of a liquid containing biological cells. Solid line 10 shows the real part of the permittivity ε as a function of frequency f; dashed line 12 shows the imaginary part of the frequency-dependent permittivity ε.

(5) The graphs are obtained by applying to a biomass an electric field whose field direction is periodically changed or reversed. The frequency f of the field direction change is plotted, in units of Hz, along the abscissa of the diagram of FIG. 1. The permittivity ε, in units of pF/cm, is plotted in the ordinate direction.

(6) When the frequency f is gradually elevated over a wide frequency range, different polarization effects occur in the biomass as a result of the constituents contained therein, and result in a change in the permittivity (and also capacitance) between the field-generating electrodes. The individual recognizable steps in the permittivity/frequency curve of a biomass are referred to, after H. P. Schwan, as alpha, beta, and gamma dispersions. The alpha dispersion (see alpha dispersion range 14 in FIG. 1) occurs at frequencies in the millihertz to kilohertz range. As currently understood, the alpha dispersion is produced by the movement and deposition of charge carriers on the surface of cells. The alpha dispersion is also influenced by active cell membrane effects and active ionic membrane channels.

(7) In FIG. 1, frequency range 14 of the alpha dispersion comprises two sub-dispersions alpha1 indicated by range 16, and alpha2 indicated by range 18.

(8) Frequency range 20 of the beta dispersion is in the range from a few kilohertz to approximately 100 MHz, and derives from the capacitive properties of cell membranes, for example because fats and proteins form a high-impedance structure, and further derives from the intracellular organelles and membrane structures where interfacial polarization occurs in the aforesaid frequency range.

(9) The gamma dispersion occurs in frequency range 22 from approximately 0.1 to 100 GHz. It is brought about by dipolar mechanisms of polar media such as water and the proteins that are also contained in biomass. This is an orientation polarization, produced by large molecules that have a dipole moment and align themselves at high frequencies.

(10) The real part 10 of permittivity ε exhibits, at the characteristic frequencies of the respective dispersions, an inflection point that is usually located between two plateau-like frequency ranges. The imaginary part of the permittivity ε exhibits a local maximum at the respective characteristic frequency of a dispersion.

(11) Whereas the gamma dispersion in frequency range 22 can be utilized for determination of the water content of biological specimens because of the polarization effects that occur in it, the beta dispersion range in frequency range 20 is of great interest for the assessment of biomass, since the permittivity ε, in particular its real part, in beta dispersion range 20 provides information regarding cell activity, cell size, and the number of living cells contained in the biomass.

(12) In FIG. 2, the real part of a frequency-dependent permittivity ε of a biomass is plotted in entirely qualitative fashion.

(13) The following information can be obtained qualitatively from curve 10 for the real part of the permittivity as a function of frequency f: FIG. 2 shows qualitatively that a frequency f.sub.Ch that is characteristic for beta dispersion range 20 is preceded by a plateau region 22 in which permittivity c changes very little with frequency as compared with the region around the characteristic frequency f.sub.Ch, and that the characteristic frequency f.sub.Ch is followed by a further plateau region 24 which is different from plateau region 22 preceding characteristic frequency f.sub.Ch and in which the permittivity ε, once again as compared with the region around characteristic frequency f.sub.Ch, likewise does not change greatly with frequency.

(14) If a permittivity value ε.sub.1 representing the permittivity ε (more precisely its real part) is therefore detected at a frequency f.sub.1 in plateau region 22, as well as a permittivity value ε.sub.2 at a frequency f.sub.2 in plateau region 24, it is then possible to ascertain, from the permittivity values ε.sub.1 and ε.sub.2 ascertained respectively at the two frequencies f.sub.1 and f.sub.2, a difference value Δε between the two permittivity values, difference value Δε being an indication of the number of living cells contained in the biomass. The alternative permittivity curve 30, indicated with two dots and three dashes, would result in a Δε having a greater magnitude at the respective measurement frequencies f.sub.1 and f.sub.2, which allows the conclusion that the biomass for which permittivity curve 30 was obtained comprises more living cells in the same volume than the biomass on which permittivity curve 10 is based.

(15) In the interest of completeness, be it noted that a change in the characteristic frequency f.sub.Ch indicates a change in the size of the cells or in their physiology. A permittivity curve 32 having two dots and a dash has a higher characteristic frequency in FIG. 2.

(16) The slope of the permittivity curve at the location of its characteristic frequency f.sub.Ch is an indication of the cell size distribution: an increasing slope indicates a more highly heterogeneous cell size distribution, and profiles of permittivity curve 10 which become flatter at the location of characteristic frequency f.sub.Ch signify more-homogeneous cell size distributions.

(17) If the beta dispersion region is known to some degree for a biomass that is to be detected instrumentally, frequencies f.sub.1 and f.sub.2 are known as measurement points.

(18) The calibration suspension described in this Application supplies, at the respective measurement frequencies f.sub.1 and f.sub.2, defined different permittivity values ε.sub.1 and ε.sub.2 that result in a likewise defined difference value Δε.

(19) Because the calibration suspension is known in terms of the materials used (carrier substance and solid particles) and in terms of the quantitative ratios used in producing the suspension, it can be reproduced with high accuracy.

(20) Reference values that can comprise a value pair, for example measurement frequency and permittivity value measured at that frequency, can thus be associated with the calibration suspension by way of corresponding measurements of permittivity values at different frequencies. The reference values can be statistically confirmed by repeating the measurements several times. The variability of the reference values around a reference value mean can therefore also be known.

(21) It is thus possible to ascertain from the associated reference values, for a predetermined calibration suspension at predetermined different measurement frequencies f.sub.1 and f.sub.2, the reference difference value of the permittivity values which is to be utilized for calibration. Carrying out a calibration measurement method using the biomass sensors that are to be calibrated, but using the defined calibration suspension instead of the biomass, thus allows a difference value of the permittivity values to be detected sensorially and compared with the reference difference value. Based on the degree of agreement between the reference difference value and the difference value detected in the calibration method, it is then possible to infer the functionality of the biomass sensors used in the calibration method; or the biomass sensors can be correspondingly adjusted so that the difference value of the calibration permittivity values which it supplies under defined calibration conditions agrees with the corresponding reference difference value.

(22) The calibration method, optionally followed by the adjustment method, can be carried out at regular intervals or can be carried out every time before sensorial detection of a biomass is carried out.

(23) The calibration method can also be carried out after sensorial detection of a biomass in order to verify that the functionality of the biomass sensors has not changed during the measurement method.

(24) A variety of calibration suspensions, which differ in terms of carrier substance and/or solid particles and/or in terms of the quantity of solid particles received in the carrier substance, can be furnished for the calibration of biomass sensors. It is thereby possible to perform a calibration using an appropriately selected calibration suspension that is as similar as possible to the measurement method carried out for sensorial detection of the biomass. The informative value of the calibration method for a measurement method carried out using the same biomass sensors can thereby advantageously be increased.