Method and Device for Measuring Process Parameters in Liquid Cultures

Content Provider	The Lens
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.
Related Links	https://www.lens.org/lens/patent/010-464-004-979-841/frontpage
Language	English
Publisher Date	2019-07-18
Access Restriction	Open
Content Type	Text
Resource Type	Patent
Jurisdiction	United States of America
Date Applied	2017-08-16
Applicant	Rheinisch Westfaelisch Technische Hochschule Rwth Aachen
Application No.	201716329364
Claim	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 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, the excitation wavelength of which is modified step by step, 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 wavelengths and depicts the emission spectrum fanned out on a sensor matrix of the at least one measuring device with photosensitive sensors, 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 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. 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. 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. 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. 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. 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. 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. 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. The method according to claim 8 , wherein the 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. 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. The method according to claim 1 , wherein the step of introducing the excitation light is interrupted depending on the position of the orbital shaker. The method according to claim 11 , wherein the position of the orbital shaker is determined by a position sensor. The method according to claim 1 , wherein the step of introducing includes masking the excitation wavelength in the emission spectrum. The method according to claim 13 , wherein the step of masking includes selectively modifying the position of the optical element so that the region of the emission spectrum having a wavelength less than or equal to the excitation wavelength is guided past the sensor matrix. The method according to claim 13 , wherein the excitation wavelength is masked by a moveable screen disposed between the optical element and the sensor matrix. 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. 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. 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. 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. 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, and automatically adding at least one of substances and liquids to the liquid culture at different times by the pipetting robot. The method according to claim 20 , 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. The method according to claim 1 , wherein in the plurality of microreactors, cultivations of liquid cultures are carried out under the same conditions, in each of the above-mentioned liquid cultures, 2D fluorescence spectra are recorded offset in time, and the respective 2D fluorescence spectra recorded offset in time in the plurality of microreactors are brought together in such a way that the fluorescence spectra from the above-mentioned microreactors are measured over a time vector. The method according to claim 1 , wherein cultivations in the liquid cultures are carried out in the plurality of microreactors under the same conditions, wherein the initial values of the process parameters to be measured in the liquid cultures in the microreactors are different, and the effect of the different initial values on the recorded 2D fluorescence spectra is used to develop chemometric models. The method according to claim 1 , wherein cultivations in the liquid cultures are carried out in the plurality of microreactors under the same conditions, wherein at different times, at least one of a substance and a liquid is added to individual microreactors of the plurality of microreactors, said at least one of a substance and a liquid modifying the process parameter to be measured in the liquid cultures in a defined manner, and the effect of the modifying on the recorded 2D fluorescence spectra is used to develop chemometric models. The method according to claim 1 , wherein: a functional relationship on which the modification of a process parameter in one of the liquid cultures is based is described by a mechanistic/mathematical model, model parameters for the mathematical model are assumed at the beginning of the cultivation, the process parameters determined based on the mathematical model are compared with the 2D fluorescence spectra recorded at different times during cultivation of this liquid culture, and the model parameters are corrected depending on the comparison. The method according to claim 21 , wherein at least one process parameter is determined using a 2D fluorescence spectrum recorded from a liquid culture using the chemometric models. A device for measuring process parameters using 2D fluorescence spectroscopy, comprising: a microreactor platform connected to an orbital shaker on which at least one microtiter plate with a plurality of microreactors is arranged, the microreactors configured to hold liquid cultures and the orbital shaker configured to agitate the liquid cultures by moving the at least one microtiter plate in an agitating motion, a light source, an automatically tunable monochromator for spectral isolation of different wavelengths from the incident light of the light source, configured to produce monochromatic excitation light, the excitation wavelength of which is modified step by step, a beam guidance system comprising an optical coupler that is configured for transferring the excitation light from the monochromator to the liquid culture and for transferring the emission spectrum from the liquid culture to an optical element, 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 a section of the microreactor that is permeable to electromagnetic radiation and wherein the optical element decomposes the emission spectrum for each excitation wavelength into the different wavelengths and fans it out, and a sensor matrix with photosensitive sensors, the optical element depicting the fanned-out emission spectrum on the sensor matrix, wherein the sensor matrix is configured to record a 2D fluorescence spectrum by measuring the intensity of the different wavelengths for each emission spectrum. The device according to claim 27 , further comprising a positioning unit configured to move the optical coupler between the microreactors of the at least one microtiter plate. The device according to claim 27 , wherein the at least one measuring device comprises a plurality of measuring devices that each comprise a light source, an automatically tunable monochromator, a beam guidance system, an optical element and a sensor matrix, the 2D fluorescence spectra of the liquid cultures in different microreactors being measured at the same time by the plurality of measuring devices. The device according to claim 29 , wherein the optical couplers of the plurality of measuring devices are moveable between the microreactors of the at least one microtiter plate by a common positioning unit. The device according to claim 27 , further comprising a shutter arranged in the optical path of the excitation light that is configured to interrupt the excitation light depending on the position of the orbital shaker. The device according to claim 31 , further comprising a position sensor for measuring the position of the orbital shaker arranged on the orbital shaker and a controller configured for processing the measured position signals of the position sensor and for interrupting the excitation light by the shutter depending on the position signa The device according to claim 27 , further comprising a moveable screen for masking the excitation wavelength arranged between the optical element and the sensor matrix. The device according to claim 27 , further comprising a lens for collimation or focusing of the excitation light arranged on the coupler. The device according to claim 27 , wherein the at least one measuring device comprises a photosensitive sensor that is configured to measure the backscattering of the excitation light irradiated into the liquid culture. The device according to claim 27 , wherein the beam guidance system comprises separate optical waveguides or a y-shaped optical waveguide with separate fibers. The device according to claim 27 , wherein the beam guidance system comprises a semitransparent mirror and an optical waveguide with only one fiber as optical components, wherein the optical components are arranged relative to each other in such a way that the excitation light is deflected by the semitransparent mirror and introduced via the optical waveguide into the microbial liquid culture, and the emission spectrum is transferred through the optical waveguide and the semitransparent mirror to the optical element. The device according to claim 27 , further comprising a pipetting robot configured to at least one of automatically take samples of the microbial liquid culture from a microreactor and add liquids at different times during cultivation.
CPC Classification	Investigating Or Analysing Materials By Determining Their Chemical Or Physical Properties Mixing; E.G. Dissolving; Emulsifying Or Dispersing APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY;APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS; FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS; i.e. BIOREACTORS OR FERMENTERS
Extended Family	018-902-427-645-542 107-258-160-233-291 144-554-145-516-267 082-597-170-619-615 199-008-982-918-850 166-638-025-361-113 018-427-138-088-620 098-666-170-886-411 077-172-827-144-227 188-252-108-321-842 149-657-283-887-449 010-464-004-979-841 179-931-849-954-178
Patent ID	20190219508
Inventor/Author	Büchs Jochen Ladner Tobias Wandrey Georg Paquet-durand Oliver Hitzmann Bernd
IPC	G01N21/64 C12M1/34 C12M3/06 G01N1/28
Status	Active
Owner	Rheinisch-westfälisch Technische Hochschule (rwth) Aachen
Simple Family	018-902-427-645-542 107-258-160-233-291 144-554-145-516-267 082-597-170-619-615 199-008-982-918-850 166-638-025-361-113 018-427-138-088-620 098-666-170-886-411 077-172-827-144-227 188-252-108-321-842 149-657-283-887-449 010-464-004-979-841 179-931-849-954-178
CPC (with Group)	G01N21/6452 B01F31/22 C12M27/16 C12M41/46 G01N21/253 G01N2021/6419 G01N2021/6421 C12M3/00 G01N1/286 G01N2201/08 G01N2201/1293
Issuing Authority	United States Patent and Trademark Office (USPTO)
Kind	Patent Application Publication