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WO2025064143A1 - Procédé de réalisation de dosages biologiques - Google Patents

Procédé de réalisation de dosages biologiques Download PDF

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Publication number
WO2025064143A1
WO2025064143A1 PCT/US2024/043448 US2024043448W WO2025064143A1 WO 2025064143 A1 WO2025064143 A1 WO 2025064143A1 US 2024043448 W US2024043448 W US 2024043448W WO 2025064143 A1 WO2025064143 A1 WO 2025064143A1
Authority
WO
WIPO (PCT)
Prior art keywords
light
intensities
knowledge base
light generating
bioassays
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2024/043448
Other languages
English (en)
Inventor
Mohiudeen AZHAR
Ryan Martin
Sai PATIBANDLA
Ishita Chakraborty
Kratika Gupta
Divya KHANDIGE
Nivedita MITRA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Siemens Healthcare Diagnostics Inc
Original Assignee
Siemens Healthcare Diagnostics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Siemens Healthcare Diagnostics Inc filed Critical Siemens Healthcare Diagnostics Inc
Publication of WO2025064143A1 publication Critical patent/WO2025064143A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/251—Colorimeters; Construction thereof
    • G01N21/253—Colorimeters; Construction thereof for batch operation, i.e. multisample apparatus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
    • G01N21/272—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration for following a reaction, e.g. for determining photometrically a reaction rate (photometric cinetic analysis)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/12—Circuits of general importance; Signal processing
    • G01N2201/129—Using chemometrical methods
    • G01N2201/1296—Using chemometrical methods using neural networks

Definitions

  • the present disclosure relates to a method of performing bioassays. More specifically, the present disclosure relates to a performing multiple bioassays on a single test sample.
  • BACKGROUND Bioassays are performed using a bioassay system to determine presence and/or concentration of substances in a living culture.
  • a bioassay system is an instrument that measures very low levels of light intensities generated upon combination of a test sample, such as blood sample taken from a patient with light generating reagents in a reaction vessel, such as a cuvette.
  • the light generating reagents bond with analytes present in the test sample and emit light.
  • Such chemical reaction is commonly known as Chemiluminescence (CL).
  • CL Chemiluminescence
  • the light emitted by such process is received and measured by a light detecting device, such as a Photomultiplier Tube (PMT) present in the bioassay system.
  • the amount of light detected by the bioassay system corresponds to a quantity of chemical constituent present in the test sample.
  • the present disclosure relates to a method of performing bioassays.
  • the method of performing bioassays may comprise determining a cumulative value of an intensity of light generated from a reaction between plurality of the light generating reagents and a test sample.
  • Each of the plurality of light generating reagent may correspond to a specific bioassay to be performed using the test samples.
  • a presence and/or concentration of substances corresponding to multiple bioassays may be determined.
  • the knowledge base may be prepared by determining intensities of light generated from multiple reactions between different concentrations of the plurality of light generating reagents and test samples of a human body.
  • the knowledge base may include values of slopes at different time instances. The values of slopes being obtained from a time plot of the intensities of light.
  • the knowledge base may also include values of the intensities of light when a time instance is zero in the time plot.
  • the knowledge base may further include a total value of the intensities of light at different time instances.
  • the knowledge base may be implemented as a machine learning model for determining presence and/or concentration of substances corresponding to the multiple bioassays.
  • the values of slopes may be stored as a matrix.
  • the intensities of light and the cumulative value of an intensity of light may be measured in terms of Relative Light Units (RLUs).
  • RLUs Relative Light Units
  • the intensities of light and the cumulative value of an intensity of light may be captured by a photomultiplier tube configured to receive light.
  • the test sample may be one of a blood sample, a urine sample, or a bodily fluid extracted from the human body.
  • Fig.1 illustrates a top view of a bioassay system, in accordance with an embodiment
  • Fig.2 illustrates a flow chart of a method of preparing the knowledge base and performing bioassays, in accordance with an embodiment
  • Figs.3(a) and 3(b) illustrate time varying chemiluminescence plots obtained on using an HIV1 light generating reagent and using an HIV2 light generating reagent, in accordance with an embodiment
  • Fig.4 illustrates a time varying chemiluminescence plot obtained on using a mixture of the HIV1 light generating reagent and HIV2 light generating reagent, in accordance with an embodiment
  • Fig.5 illustrates a time varying chemiluminescence plot obtained on using mixtures of HIV1 and HIV2 antibodies, in accordance with an embodiment
  • Fig.6 illustrates a plot of RLUs obtained on using mixtures of light
  • Fig. 1 illustrates a top view of a bioassay system 100, in accordance with an embodiment.
  • the bioassay system 100 may include an incubation ring 102 containing a plurality of cuvette holders 104.
  • the incubation ring 102 may be a circular insulated track rotatable in a predefined direction.
  • Each cuvette holder of the plurality of cuvette holders 104 may discretely accommodate a cuvette.
  • Each cuvette holder may discretely accommodate a cuvette 106.
  • a cuboidal shape cuvette is illustrated in Fig.1, the cuvette 106 may be of any other shape, such as a cylindrical shape.
  • the cuvette 106 may be placed inside a cuvette holder at an entry position 108 of the incubation ring 102 and may be removed from the cuvette holder at an exit position 110 of the incubation ring 102.
  • a cuvette loader may be used to place the cuvette 106 inside the cuvette holder and a cuvette remover may be used to remove the cuvette 106 from the cuvette holder.
  • the incubation ring 102 may advance the position of the plurality of cuvette holders 104. In one implementation, the incubation ring 102 may be rotated in a circular direction.
  • the cuvette 106 may be moved to a sample probe area 112 where a test sample is dispensed into the cuvette 106.
  • the test sample may be a blood sample, a urine sample, or a bodily fluid.
  • the incubation ring 102 may move the cuvette 106 from the sample probe area 112 to a reagent probe area 114.
  • the bioassay system 100 may comprise a PMT 116 placed in front of an opening of the incubation ring 102.
  • a photodetector in the PMT 116 may be configured to receive a cumulative value of the intensity of light emitted from the luminescent reactions between the plurality of light generating reagents and the test sample in the cuvette 106.
  • the bioassay system 100 may also include at least one memory and processing unit (or processor).
  • the memory may store values of intensities of light captured by the photodetector.
  • the memory may further store a knowledge base.
  • the knowledge base may include a repository of data related to determining presence and/or concentration of substances corresponding to multiple bioassays.
  • the processing unit may obtain the cumulative value of intensity of light captured by the photodetector in real-time or may obtain the cumulative value of intensity of light stored in the memory.
  • the processing unit may utilize the knowledge base to process the cumulative value of intensity of light to determine presence and/or concentration of substances in the test sample. The presence and/or concentration of substances corresponding to multiple bioassays may thus be determined by the processing unit in a single measurement of the test sample.
  • the substance may include a protein, sugar, antibodies, antigens, and oxygen present in the test sample.
  • the knowledge base may be implemented as a machine-learning model trained to determine presence and/or concentration of substances in the test sample corresponding to multiple bioassays. Method of development of the knowledge base and determining the presence and/or concentration of substances utilized by the processing unit has been described in detail henceforth. [0027] To prepare the knowledge base, different concentrations of a plurality of light generating reagents may be mixed with a test sample, in the bioassay system 100. The plurality of light generating reagents may be mixed with a small batch of the test sample to form multiple mixtures.
  • Fig.2 illustrates a method of preparing the knowledge base and performing bioassays, in accordance with an embodiment.
  • Different concentrations of light generating reagents may have different chemical kinetics, resulting in different time-varying chemiluminescence profile for each mixture of the multiple mixtures.
  • the chemiluminescence may be measured in terms of Relative Light Units (RLUs).
  • RLUs Relative Light Units
  • the RLUs generated by the light generating reagents at different concentrations may be measured.
  • a plot of RLUs at different concentrations may be determined.
  • values of slope of different concentrations of each of the plurality of light generating reagents at different time instances may be computed. The values of slope may be arranged in a coefficient matrix.
  • values of the intensities of light when a time instance is zero in the time plot may be computed.
  • the values of the intensities of light when the time instance is zero indicates a y-intercept of the time plot.
  • a total value of the intensities of light of the different concentrations of the plurality of the light generating reagents at different time instances may be computed.
  • the total value may be computed by adding values of the intensities of light of each of the plurality of the light generating reagents.
  • the knowledge base may be calibrated based on the total value of the intensity of light, the values of the y-intercepts and the coefficient matrix.
  • the knowledge base may be calibrated to determine the presence and/or concentration of different substances in a single measurement of the test sample, at a time of performing bioassay.
  • the time dependent chemiluminescence profile of two individual light generating reagents were plotted.
  • the two light generating reagents were Human Immunodeficiency Virus type 1 (HIV1) and Human Immunodeficiency Virus type 2 (HIV2).
  • Figs.3(a) and 3(b) illustrate a time varying chemiluminescence plot of HIV1 light generating reagent and HIV2 light generating reagent, in accordance with an embodiment.
  • Fig.3(a) depicts chemiluminescence profile of low concentration of HIV1 indicated by (i), chemiluminescence profile of medium concentration of HIV1 indicated by (ii), and chemiluminescence profile of high concentration of HIV1 indicated by (iii).
  • a low, medium, and high concentration of the HIV1 were obtained by diluting the HIV1 with water in ratios of 1:250, 1:25, and 1:2.5 respectively.
  • Fig.3(b) depicts chemiluminescence profile of low concentration of HIV2 indicated by (iv), chemiluminescence profile of medium concentration of HIV2 indicated by (v), and chemiluminescence profile of high concentration of HIV2 indicated by (vi).
  • a low, medium, and high concentration of HIV2 were obtained by diluting the HIV2 with water in ratios of 1:200, 1:20, and 1:2, respectively. It was observed that there was a difference in chemical kinetics of the HIV1 and HIV2 light generating reagents at different concentrations.
  • Fig.4 illustrates a time varying chemiluminescence plot of a mixture of the HIV1 light generating reagent and the HIV2 light generating reagent, in accordance with an embodiment.
  • the time varying chemiluminescence plot of mixture of the HIV1 light generating reagent and the HIV2 light generating reagent is indicated by (i).
  • Fig.5 illustrates a time varying chemiluminescence plot of mixtures of HIV1 and HIV2 antibodies, in accordance with an embodiment.
  • a plot of HIV1 antibodies is indicated by (i) and a plot of HIV2 antibodies is indicated by (ii).
  • Plots (i) and (ii) may be used to determine concentration of the HIV1 antibodies and the HIV2 antibodies.
  • Thyroid Stimulating Hormone 3-Ultra (TSH3UL) and Troponin I (TNIH) light generating reagents were prepared and the time-varying chemiluminescence profile of a mixture of the TSH3UL and the TNIH light generating reagents was plotted. Concentrations of the mixtures are represented in below mentioned Table 1 and Table 2: Table 1 Table 2 [0044] Fig.6 illustrates a plot of RLUs of all the mixtures against time instances, in accordance with an embodiment. Replicates of all the mixtures were taken.
  • the knowledge base may be developed and updated through training on data of the coefficient matrix, values of the y-intercepts, and the total value of the RLUs of all the mixtures at different time instances obtained from different concentrations of the light generating reagents. Some portion of the data may be used for testing the knowledge base for predicting presence and/or concentrations of the substances detectable by each of the light generating reagents.
  • Fig.7(a) through 7(e) illustrate time varying chemiluminescence plots of five mixtures, in accordance with an embodiment.
  • the time varying chemiluminescence plots were prepared via a simulation for each of the five mixes and an expected value of the concentration of the TSH3UL and the TNIH in the mixture was determined.
  • Five replicates R1, R2, R3, R4, and R5 were taken for the Mix-9, and it was observed from the Fig.7(a) that deviation in RLUs of the TSH3UL and the TNIH in the replicates R1 and R3 were correctly predicted by the trained knowledge base. The deviation may be due to pipetting or other wet- lab related errors during preparation of the five mixes.
  • the trained knowledge base was also able to predict low concentration combinations of three replicates R1, R2, and R3 of the Mix-13 with good precision as depicted in Fig.7(e).
  • Accuracy of the knowledge base may be improved by incorporating zero concentration RLUs for the light generating reagents in the mixture and by using the best fit slopes in the coefficient matrix for training the knowledge base.
  • the present disclosure provides an improved method for performing bioassays on the test sample in a single measurement. The method may be implemented on existing systems for performing the bio-medical tests, without any need for changed in hardware of the system.
  • all changes, modifications, and variations within the meaning and range of equivalency are considered within the scope and spirit of the disclosure. It is to be understood that the aspects and embodiment of the disclosure described above may be used in any combination with each other. Several of the aspects and embodiment may be combined together to form a further embodiment of the disclosure.

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  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Pathology (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Theoretical Computer Science (AREA)
  • Mathematical Physics (AREA)
  • Plasma & Fusion (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

La présente divulgation concerne un procédé de réalisation de dosages biologiques. Le procédé consiste à déterminer une valeur cumulée d'une intensité de lumière générée par une réaction entre une pluralité de réactifs générateurs de lumière et un échantillon de test. En se basant sur une base de connaissances, il est possible de déterminer la présence et/ou la concentration de substances correspondant à de multiples dosages biologiques. La base de connaissances est préparée par la détermination des intensités de lumière générées par de multiples réactions entre différentes concentrations de la pluralité de réactifs générateurs de lumière et des échantillons de test d'un corps humain. La base de connaissances comprend des valeurs de pentes à différents instants obtenues à partir d'une courbe temporelle des intensités de lumière. La base de connaissances comprend également des valeurs des intensités de lumière lorsqu'un instant correspond à une valeur de zéro sur la courbe temporelle et une valeur totale des intensités de lumière à différents instants.
PCT/US2024/043448 2023-09-20 2024-08-22 Procédé de réalisation de dosages biologiques Pending WO2025064143A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN202311063249 2023-09-20
IN202311063249 2023-09-20

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WO2025064143A1 true WO2025064143A1 (fr) 2025-03-27

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090150106A1 (en) * 2007-06-05 2009-06-11 Ecolab Inc. Method of calibration for nonlinear optical sensor

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090150106A1 (en) * 2007-06-05 2009-06-11 Ecolab Inc. Method of calibration for nonlinear optical sensor

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
AGUILAR-CABALLOS M P ET AL: "Simultaneous determination of benzoic acid and saccharin in soft drinks by using lanthanide-sensitized luminescence", ANALYST, ROYAL SOCIETY OF CHEMISTRY, UK, vol. 124, no. 7, 1999, pages 1078 - 1084, XP002508140, ISSN: 0003-2654, DOI: 10.1039/A902402F *
LI ET AL: "Simultaneous determination of three organophosphorus pesticides residues in vegetables using continuous-flow chemiluminescence with artificial neural network calibration", TALANTA, ELSEVIER, AMSTERDAM, NL, vol. 72, no. 1, 23 March 2007 (2007-03-23), pages 223 - 230, XP022343992, ISSN: 0039-9140, DOI: 10.1016/J.TALANTA.2006.10.023 *
MURILLO J A ET AL: "Resolution of Ofloxacin-Ciprofloxacin and Ofloxacin-Norfloxacin Binary Mixtures by Flow-Injection Chemiluminescence in Combination with Partial Least Squares Multivariate Calibration", JOURNAL OF FLUORESCENCE, KLUWER ACADEMIC PUBLISHERS-PLENUM PUBLISHERS, NE, vol. 17, no. 5, 19 June 2007 (2007-06-19), pages 481 - 491, XP019529674, ISSN: 1573-4994, DOI: 10.1007/S10895-007-0198-9 *
NAVAS DIAZ A ET AL: "NONLINEAR MULTICOMPONENT KINETIC ANALYSIS FOR THE SIMULTANEOUS STOPPED-FLOW DETERMINATION OF CHEMILUMINESCENCE ENHANCERS", ANALYTICAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 66, no. 7, 1 April 1994 (1994-04-01), pages 988 - 993, XP000575321, ISSN: 0003-2700, DOI: 10.1021/AC00079A010 *
PULGARIN ET AL: "Simultaneous stopped-flow determination of morphine and naloxone by time-resolved chemiluminescence", TALANTA, ELSEVIER, AMSTERDAM, NL, vol. 74, no. 5, 18 January 2008 (2008-01-18), pages 1539 - 1546, XP022426376, ISSN: 0039-9140, DOI: 10.1016/J.TALANTA.2007.09.032 *
YANG ET AL: "An ImmunoChip prototype for simultaneous detection of antiepileptic drugs using an enhanced one-step homogeneous immunoassay", ANALYTICAL BIOCHEMISTRY, ACADEMIC PRESS, AMSTERDAM, NL, vol. 365, no. 2, 10 May 2007 (2007-05-10), pages 222 - 229, XP022077171, ISSN: 0003-2697, DOI: 10.1016/J.AB.2007.03.019 *

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