Method and device for measuring process parameters in liquid cultures

Patent metadata

Field	Value
Publication number	US-11635381-B2
Application number	US-201716329364-A
Country	US
Kind code	B2
Filing date	Aug 16, 2017
Priority date	Sep 1, 2016
Publication date	Apr 25, 2023
Grant date	Apr 25, 2023

Abstract

A method for measuring process parameters in liquid cultures in a plurality of microreactors of at least one microtiter plate includes continuously agitating the liquid cultures using an orbital agitator at least until the reaction is completed in all the microreactors. In order to allow process parameters also of such substances which themselves do not have any fluorescence activity to be measured with relatively low complexity and within a short time, 2D fluorescence spectra are recorded in a plurality of in particular different liquid cultures in the microreactors of agitated microplates. A device for carrying out the method is also disclosed.

First claim

The invention claimed is:

1. A method for determining process parameters using 2D fluorescence spectroscopy in liquid cultures using a device having a plurality of microreactors of at least one microtiter plate, the liquid cultures being held in the microreactors, an orbital shaker configured to agitate the liquid cultures by moving the at least one microtiter plate in an agitating motion at least until completion of cultivation in all of the microreactors, and at least one measuring device configured to record 2D fluorescence spectra of the liquid cultures during the cultivation, the at least one measuring device being decoupled from the agitating motion of the microtiter plate, the method comprising the following steps:
1.1 producing monochromatic excitation light, an excitation wavelength of which is modified step by step so that the excitation light is produced with different excitation wavelengths,
1.2 successively introducing the excitation light with different excitation wavelengths into the liquid culture in one of the microreactors,
1.3 guiding emission spectra from the liquid culture in the one of the microreactors to an optical element that decomposes the emission spectrum for each excitation wavelength into the different individual wavelengths and depicts the emission spectrum fanned out on a sensor matrix of the at least one measuring device with photosensitive sensors to form bands on the sensor matrix for the individual wavelengths,
1.4 recording, using the sensor matrix of the at least one measuring device, a 2D fluorescence spectrum by measuring an intensity of the different individual wavelengths of each emission spectrum for each excitation wavelength successively introduced in the liquid culture in the one of the microreactors, and
1.5 using steps 1.1-1.4 to record 2D fluorescence spectra of the liquid cultures in further microreactors of the at least one microtiter plate,
wherein the step of guiding includes selectively modifying a position of the optical element so that a region of the emission spectrum having a wavelength less than or equal to the excitation wavelength is guided past the sensor matrix.

2. The method according to claim 1 , wherein the step of introducing the excitation light and the step of guiding the emission spectra are carried out through a surface on the underside of each microreactor that is transparent for the excitation light and the emission spectra.

3. The method according to claim 1 , wherein the excitation light is generated by an automatically tunable monochromator for spectral isolation of different wavelengths from the incident light of a light source.

4. The method according to claim 3 , wherein the step of introducing the excitation light from the monochromator to the liquid culture and the step of guiding the emission spectrum from the liquid culture to the optical element are carried out by a beam guidance system comprising an optical coupler, wherein the optical coupler for introducing the excitation light into the liquid culture and for coupling the emission spectrum into the beam guidance system is oriented with respect to the microreactor containing the liquid culture.

5. The method according to claim 4 , wherein the optical coupler is not moved during recording of the 2D fluorescence spectrum, so that the agitated microreactors move relative to the optical coupler.

6. The method according to claim 4 , wherein the optical coupler, following the step of recording of the 2D fluorescence spectrum, is moved by a positioning unit between the microreactors of the at least one microtiter plate.

7. The method according to claim 4 , wherein an agitation diameter of the orbital shaker is adjusted in such a way that at least two microreactors of the plurality of microreactors, during one rotation of the orbital shaker, successively circle above the optical coupler of a measuring device of the at least one measuring device, and the recorded fluorescence spectra are assigned to the at least two microreactors circling above the optical coupler.

8. The method according to claim 4 , wherein the excitation light and the emission spectrum in the beam guidance system are transferred via separate optical waveguides or a y-shaped optical waveguide with separate fibers for the excitation light and the emission spectrum.

9. The method according to claim 4 , wherein in the beam guidance system,
the excitation light is deflected by a semitransparent mirror and introduced into the liquid culture via an optical waveguide with only one fiber, and
the emission spectrum is transferred through the optical waveguide and the semitransparent mirror to the optical element.

10. The method according to claim 3 , wherein an agitation diameter of the orbital shaker is adjusted in such a way that the excitation light during recording of the fluorescence spectrum is introduced exclusively into the liquid culture of one of the microreactors and the emission spectrum of this liquid culture is exclusively introduced into the optical coupler.

11. The method according to claim 1 , wherein the at least one measuring device includes a plurality of measuring devices and the 2D fluorescence spectra of the liquid cultures in different microreactors are recorded simultaneously by the plurality of measuring devices.

12. The method according to claim 11 , the step of introducing the excitation light from the monochromator to the liquid culture and the step of guiding the emission spectrum from the liquid culture to the optical element are carried out by a beam guidance system comprising a plurality of optical couplers corresponding to the plurality of measuring devices, wherein the plurality of optical couplers of the plurality of measuring devices are movable by a common positioning unit between the microreactors of the at least one microtiter plate.

13. The method according to claim 1 , wherein the step of introducing the excitation light is interrupted depending on the position of the orbital shaker.

14. The method according to claim 13 , wherein the position of the orbital shaker is determined by a position sensor.

15. The method according to claim 1 , wherein the region of the emission spectrum having the wavelength less than or equal to the excitation wavelength is excluded from the measurement of the emission spectrum .

16. The method according to claim 1 , further comprising the step of at least one of:
collimating or focusing the excitation light before the step of introducing, and
concentrating the emission spectrum.

17. The method according to claim 1 , further comprising the step of measuring backscattering of the excitation light irradiated into the liquid culture using a separate photosensitive sensor of the measuring device.

18. The method according to claim 1 , wherein the device includes a pipetting robot and the method further includes at least one of:
during cultivation, automatically taking samples of the liquid culture from one of the microreactors at different times by the pipetting robot and analyzing the samples offline with respect to specified process parameters of the process parameters, and
automatically adding at least one of substances and liquids to the liquid culture at different times by the pipetting robot.

19. The method according to claim 18 , wherein the process parameters of the samples analyzed offline and the 2D fluorescence spectra recorded at the different sampling times are used to prepare chemometric models.

20. The method according to claim 19 , wherein at least o

Assignees

• Rwth Aachen
• Rheinisch Westfaelisch Technische Hochschule Rwth Aachen

Inventors

• Büchs Jochen
• Ladner Tobias
• Wandrey Georg
• Paquet-Durand Oliver
• Hitzmann Bernd

Classifications

• G01N2021/6419

  Excitation at two or more wavelengths · CPC title

• G01N21/6452 Primary

  Individual samples arranged in a regular 2D-array, e.g. multiwell plates · CPC title

• G01N21/253

  for batch operation, i.e. multisample apparatus (analytical automats G01N35/00) · CPC title

• G01N1/286

  involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising (microtomes G01N1/06; pulverising in general B02C; mixing in general B01F) · CPC title

• C12M41/46

  of cellular or enzymatic activity or functionality, e.g. cell viability · CPC title