DE102015015816A1 - Sensory device for the optical detection of the particle concentration in suspensions of microscopic particles - Google Patents

Sensory device for the optical detection of the particle concentration in suspensions of microscopic particles

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Publication number
DE102015015816A1
DE102015015816A1 DE102015015816.3A DE102015015816A DE102015015816A1 DE 102015015816 A1 DE102015015816 A1 DE 102015015816A1 DE 102015015816 A DE102015015816 A DE 102015015816A DE 102015015816 A1 DE102015015816 A1 DE 102015015816A1
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Germany
Prior art keywords
light source
sensory
photodiode
light
suspension
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Pending
Application number
DE102015015816.3A
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German (de)
Inventor
Robert Lehmann
Vincent Rüsike
Lars Bähr
Original Assignee
Lars Bähr
Robert Lehmann
Vincent Rüsike
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Application filed by Lars Bähr, Robert Lehmann, Vincent Rüsike filed Critical Lars Bähr
Priority to DE102015015816.3A priority Critical patent/DE102015015816A1/en
Publication of DE102015015816A1 publication Critical patent/DE102015015816A1/en
Pending legal-status Critical Current

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/06Investigating concentration of particle suspensions
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS 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
    • C12M1/00Apparatus for enzymology or microbiology
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS 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
    • C12M41/00Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/36Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
    • G01N21/17Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/59Transmissivity
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N2015/0042Investigating dispersion of solids
    • G01N2015/0053Investigating dispersion of solids in liquids, e.g. trouble
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N2015/0065Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials biological, e.g. blood
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/06Investigating concentration of particle suspensions
    • G01N2015/0693Investigating concentration of particle suspensions by optical means, e.g. by integrated nephelometry

Abstract

A sensory device is provided for optically detecting particle concentration in dense suspensions of microscopic particles. The device comprises a light source 1, which generates a photon flux through the suspension on a transmission path. In addition, it contains at least two photodiodes sensitive to the emitted photons, which are arranged on the vessel with the suspension so that they can be reached for parts of the emitted light. The photodiodes differ by their different position to the light source and the transmission path. One of the photodiodes, the reference photodiode 2, is arranged so that the reference signal formed by it upon activation of the light source is proportional to the light intensity at the beginning of the transmission path. The sensory photodiode (s) 3 is / are arranged such that the sensory signal formed by it upon activation of the light source is proportional to the light intensity at the end of a defined transmission path. The invention further includes an electronic device which activates the light source for defined time intervals and an electronic evaluation unit. The evaluation unit displays the attenuation ratio R increasing with the particle concentration between the reference signal and the sensory signal generated in the same time interval or sets R to calculate a parameter proportional to the particle concentration.

Description

  • A common task in biotechnological research and bioprocess engineering is the measurement of cell density or biomass concentration. It is usually achieved either by measuring the attenuation of light (attenuation) by scattered light losses or by detecting scattered light signals. The two optical principles mentioned have a broad field of application in the measurement of the concentration of microscopic artificial or natural solid particles. Light with wavelengths> 600 nm or infrared light up to a wavelength of 1000 nm is usually used to detect the scattered light losses or scattered light signals, thereby minimizing the absorptively induced light attenuation and thus the influence of the pigmentation of the particles. For optical detection of scattered light or stray light losses in suspensions of cyanobacteria or microalgae, the wavelength of light must be> 730 nm due to the absorption of the light by chlorophyll.
  • It is often desirable to use external sensors for determining the concentration in reaction or flow-through vessels for detecting the particle concentration. Mobile external sensors are advantageous, for example, for measuring the increase in the concentration of microorganisms or cells during growth in culture vessels.
  • Recently, commercially available devices are used for this purpose, which detect the cell concentration according to the principle of backscattering of an incident light pulse by one or more photodiodes and cover a wide range of particle concentration. Since the particle concentration is calculated by a complex analysis of several scattered light intensities, these devices are subject to a certain susceptibility to disturbing factors such as the variation of the vessel wall thickness and the refraction of light on the vessel walls, the need for complicated calibration procedures.
  • While the principle of measuring multiple stray light intensities with external sensory devices ( US 6573991 B1 . US 8405033B2 . US 8603772B2 ), also in combination with transmitted light measurements ( US 4,193,692 ) can be used for a very broad concentration range of the light-scattering particles, measurements of scattered light loss in the transmission of light by a suspension for concentration measurement with external sensors are currently only in the very limited range of particle concentration, at which the optical density depends strongly on the particle concentration. carried out. In photometry of colored solutions, the term optical density (OD) is used as a synonym for the extinction E (the negative decadic logarithm of the transmittance). In real solutions of a light-absorbing absorbent, E is a suitable measure of the concentration of the dissolved absorbent. It is proportional to the concentration of the absorbent according to Lambert-Beer's law. In transmitted light measurements on particle suspensions, however, E is proportional to the particle concentration in the suspension only if E falls below a certain value, which depends on the particle size (eg 0.25 with a particle size of 1-2 μm). Therefore, when measuring the scattered light loss in particle suspensions of higher concentration C, it is usual to dilute the suspension to determine a normalized optical density (OD *) in the concentration range of the linear relationship between E and C by measuring the scattered light loss in a transmitted light measurement ( Myers J, Curtis, BS, Curtis WR, 2013, BMC Biophysics 6: 4, 1-15 ). The thus standardized optical density (OD *) then results as the product of the dilution factor with the measured value. OD * is proportional to particle concentration or biomass concentration even in very dense suspensions. The time course of the OD * values reflects microbial growth and can be used directly to capture the specific growth rate. OD *, after suitable dilution under standard conditions with a photometer, can also be determined for very concentrated particle concentrations or microorganism cultures. However, this requires the removal of samples from the culture vessel. The extinction measured in a culture vessel with an external optical sensor is only of limited suitability for the quantitative description of the growth. For their interpretation, at higher concentrations, the nonlinear dependence of the absorbance on the cell density must be taken into account by means of nonlinear calibration curves. This makes sense only in a very limited measuring range. Measurements of the extinction> 1.5 express the dependence of the transmitted light signal on the concentration of the light-scattering particles only in a very unsatisfactory manner, because the relative change in the extinction at a relative change in the concentration C, dEC / dCE , at high values of E is very low. To solve this problem, the transmission distance in the extinction measurement can be greatly shortened ( Myers et al., 2013 ), so that the relationship between E and C is linear even at higher cell densities. However, this requires, for each culture vessel or each approach to be examined, a hydraulic system which allows the suspension to flow through the measurement cell.
  • The object of the invention is to provide an external sensory device based on light attenuation (attenuation) by scattered light losses for measuring the concentration of suspended microscopic Particles in sealed vessels, for example of microorganisms in shaken or stirred culture vessels. The external sensory device should enable a measurement of particle concentrations up to OD * values of 80 without sampling or dilution on a relatively long transmission distance of, for example, 10 to 20 mm and thus be widely used in biotechnology research and bioprocessing. The object of the invention is achieved by a sensory device according to claim 1. In the dependent claims advantageous embodiments of the invention are shown.
  • The device according to the invention ( 1 ) includes a light source 1 which generates a photon flux on a transmission path through the suspension and at least two photodiodes sensitive to the emitted photons 2 and 3 , The latter are arranged on the vessel containing the suspension so that they can be reached for parts of the emitted light, but differ in their position relative to the light source and to the transmission path. One of the photodiodes, the reference photodiode 2 , generates a reference signal which is proportional to the intensity of the light emitted by the light source at the beginning of the transmission path, while the signal (s) generated by the sensory photodiode (s) corresponds to the intensity of the emitted light at the end of a defined Transmission path is proportional / are. The device according to the invention further comprises an electronic device which activates the light source for defined time intervals and an electronic evaluation unit. The latter shows the attenuation ratio increasing with the particle concentration R = A / B between the signal A generated by activation of the light source in a reference photodiode and the signal B generated in the same time interval in a sensory photodiode and / or uses this signal to calculate the particle concentration.
  • The reference signal is formed when the light source is active for a short time interval. For its detection, part of the photon flux emanating from the light source is directed from the light source to the reference photodiode, while another part of the same photon flux is directed through a suspension of defined length through the suspension onto the sensory photocell. The photons detected by the reference photocell take a reference path that does not pass through the suspension or only a comparatively short distance through the suspension. The reference signal does not correspond to the blank value customary with photometers, which reflects the photon flux on the transmission path in the particle-free transparent suspension medium. It only represents a portion of the photons emitted by the light source, which does not depend on the particle concentration, or does so to a lesser extent than the fraction responsible for the sensory signal.
  • The electronic evaluation unit turns the signals A and B into the attenuation ratio R = A / B educated. When R is formed by a device according to the invention, its size is independent of variations in the intensity of the light source as well as photons not originating from the light source. The value R can therefore be determined very accurately with the help of sensitive photodiodes even at high particle concentrations, even if the light intensity at the end of the transmission line reaches a very small value, which is two to three orders of magnitude below the light intensity at the beginning of the transmission line.
  • 1 shows an embodiment of the invention, in which the of the light source 1 outgoing photon flux before entering the particle suspension on the reference photodiode 2 is steered. In this case, the reference signal A is independent of the particle concentration C, while that on the sensory photocell 3 formed sensory signal decreases with increasing particle concentration. Therefore, the ratio between the reference signal A and the sensory signal B, the attenuation ratio Q, increases with increasing particle concentration. It is obvious that the relative change of the attenuation ratio with a change of the particle concentration (dQ / dQC) is independent of the absolute value of the reference signal.
  • 2 shows an embodiment of the invention, in which a portion of the photon flux is directed to the reference photodiode after a relatively small distance in the suspension by lateral particle scattering and after a comparatively larger distance in the suspension by lateral particle scattering another part of the sensory photocell , In this case, with sufficient particle concentration, the reference signal is a measure of the light intensity near the reference photodiode and the sensory signal is a measure of the light intensity near the sensory photodiode. Because of the additional path through the suspension, in this case the sensory signal B is weakened more strongly by an increase in the particle concentration C than the reference signal A: dB / BdC> dA / AdC
  • In this case too, the attenuation ratio decreases R = A / B with increasing particle concentration too. The relative change of R with a change in concentration (dR / RdC ) increases with the length of the transmission path. Results are presented elsewhere. In advantageous Embodiments of the invention, the light source and the photodiodes are mounted on a holder with opaque walls, which can be adapted to the vessels or tubes in which the particle suspension are located. It is advantageous for the reproducibility of the measurement when the light flow outside the vessel with the suspension through a transparent medium ( 4 . 1 ), which has a similar refractive index as the medium in which the particles are suspended.
  • 3 Fig. 12 shows measured values of R as a function of the particle concentration obtained with devices on relatively concentrated suspensions of baker's yeast (Saccharomyces cerevisiae).
  • 3a shows the function R = f (OD *), which results from measured values of the device 1 results.
  • 3b the function R = f (OD *) is derived from measured values of the device 2 results. The distance of the sensory photodiode from the location of the entry of the emitted radiation into the suspension or the distance between the sensory photodiode of the reference photodiode was about 10 mm. The strong and continuous dependence of the attenuation ratio R on OD * allows an accurate numerical determination of OD * by binomial regression. The correlation coefficients of corresponding regressions are close to 1. The relative slope (dOR * R / OD * dR) lies at the in 3a shown function in the entire calibration range near 1 and at in 3b illustrated calibration function in the entire calibration range near 0.5. The investigated external sensors according to the invention are accordingly for accurate detection of the cell concentration of Saccharomyces cerevisiae in a range of OD * from 0 to 80 ( 3a ) or 10 to 80 ( 3b ) suitable. According to the conversion factor determined for the yeast suspensions by Myers et al. 2013 ) can be used for accurate determination of the biomass concentration up to at least 50 g dry matter per liter. In the 2 illustrated embodiment has the advantage of a particularly simple construction. How out 3b it is different from the one in 1 illustrated embodiment (see. 3a ) for the numerical determination of the biomass concentration with the help of the indicated regression only at OD *> 10 suitable. At lower concentrations (OD * from 0 to 10) another function (not shown here) should be used which takes into account the dependence of the reference signal on the particle concentration.
  • LIST OF REFERENCE NUMBERS
  • 1
    light source
    2
    Reference photodiode
    3
    sensory photodiode
    4
    transparent medium
  • Consider patent specifications
  • Quoted literature
    • Myers J, Curtis, BS, Curtis: Improving accuracy of cell and chromophore concentration measurements using optical density. WR BMC Biophysics 6: 4 (2013) 1-15
  • QUOTES INCLUDE IN THE DESCRIPTION
  • This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.
  • Cited patent literature
    • US 6573991 B1 [0004, 0014]
    • US 8405033 B2 [0004, 0014]
    • US 8603772 B2 [0004, 0014]
    • US 4193692 [0004, 0014]
  • Cited non-patent literature
    • Myers J, Curtis, BS, Curtis WR, 2013, BMC Biophysics 6: 4, 1-15 [0004]
    • Myers et al., 2013 [0004]
    • Myers et al. 2013 [0014]

Claims (9)

  1. Sensory device for the optical detection of the particle concentration in suspensions of microscopic particles, comprising • a light source which generates a photon flux on a transmission path through the suspension, • at least two photodiodes sensitive to the emitted photons, which are arranged on the vessel containing the suspension such that They are accessible to parts of the emitted light, but differ by their position to the light source and the transmission path, wherein one of the photodiodes, the reference photodiode 2 , generates a reference signal which is proportional to the intensity of the light emitted by the light source at the beginning of the transmission path, while the signal (s) generated by the sensory photodiode (s) defines the intensity of the light at the end of a transmission path Length is proportional / are. An electronic device which activates the light source for defined time intervals and an electronic evaluation unit, which increases the attenuation ratio which increases with the particle concentration R = A / B between the signal A generated by activating the light source in the reference photodiode and the signal B generated in a sensory photodiode at the same time interval or used to calculate the particle concentration.
  2. Sensory device according to claim 1, having a holder for the photodiodes, which has opaque walls with perforations for mounting the light source and the photodiodes, wherein the walls of the holder can be adapted to the vessels or pipe connections in which the particle suspensions are located from the outside in that during the activation of the light source, a photon flux passing through the suspension is generated, from which a defined portion reaches the sensory photodiode.
  3. Sensory device according to Claim 2, in which at least a part of the perforations is / are designed as a diaphragm (s) for influencing the portion of the photon flux emitted by the light source falling on the photodiodes.
  4. Sensory device according to one of the preceding claims, wherein the photon flux outside of the vessel or tube containing the particle suspension through a transparent medium 4 is passed, which has a similar refractive index as the liquid medium of the suspension.
  5. Sensory device according to one of the preceding claims, comprising a light source which emits light of a wavelength of 750 to 950 nm.
  6. Sensory device according to one of the preceding claims, wherein the transmission distance is 5 to 50 mm.
  7. Sensory device according to one of the preceding claims, comprising a holding device with two opaque flat walls approximately perpendicular to each other, the first and second walls, which can be adapted to the walls of the vessels containing the suspensions from the outside, the light source at a bore in the first wall is arranged and the sensory photodiode / s are arranged in a bore / holes in the second wall.
  8. A sensor device according to claim 7, wherein the support means has an axis of rotation for accurately adjusting the approximately perpendicular angle between the first and second walls to the approximately perpendicular angle between two planar vessel walls.
  9. A sensor device according to claim 7 or claim 8, wherein The light source is aligned to produce a photon flux approximately parallel to the second wall, • the reference photodiode and the sensory photodiode (s) in the second wall lie on a line which is approximately parallel to the photon flux, and • the reference photodiode has a distance of less than 10 mm from the first wall, while the distance between the sensory photodiode / s and the reference photodiode is between 10 and 30 mm.
DE102015015816.3A 2015-12-07 2015-12-07 Sensory device for the optical detection of the particle concentration in suspensions of microscopic particles Pending DE102015015816A1 (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4193692A (en) 1978-06-07 1980-03-18 Monitek, Inc. Method and apparatus for the optical measurement of the concentration of a particulate in a fluid
US6573991B1 (en) 2000-04-26 2003-06-03 Martin Paul Debreczeny Self-compensating radiation sensor with wide dynamic range
US8405033B2 (en) 2010-07-30 2013-03-26 Buglab Llc Optical sensor for rapid determination of particulate concentration
US8603772B2 (en) 2007-07-28 2013-12-10 Bug Lab LLC Particle sensor with wide linear range

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4193692A (en) 1978-06-07 1980-03-18 Monitek, Inc. Method and apparatus for the optical measurement of the concentration of a particulate in a fluid
US6573991B1 (en) 2000-04-26 2003-06-03 Martin Paul Debreczeny Self-compensating radiation sensor with wide dynamic range
US8603772B2 (en) 2007-07-28 2013-12-10 Bug Lab LLC Particle sensor with wide linear range
US8405033B2 (en) 2010-07-30 2013-03-26 Buglab Llc Optical sensor for rapid determination of particulate concentration

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
2) MYERS, John A, ; CURTIS, Brandon S. ; CURTIS, Wayne R.: Improving accuracy of cell and chromophore concentration measurements using optical density. In: BMC Biophysics, Vol. 6, 2013, No. 4, 15 S. – ISSN 2046-1682 *
2) MYERS, John A, ; CURTIS, Brandon S. ; CURTIS, Wayne R.: Improving accuracy of cell and chromophore concentration measurements using optical density. In: BMC Biophysics, Vol. 6, 2013, No. 4, 15 S. – ISSN 2046-1682
Myers et al. 2013
Myers et al., 2013
Myers J, Curtis, BS, Curtis WR, 2013, BMC Biophysics 6: 4, 1–15
Myers J, Curtis, BS, Curtis: Improving accuracy of cell and chromophore concentration measurements using optical density. WR BMC Biophysics 6: 4 (2013) 1–15

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