WO2025064143A1 - Method of performing bioassays - Google Patents

Abstract

The present disclosure relates to a method of performing bioassays. The method comprises determining a cumulative value of an intensity of light generated from a reaction between plurality of the light generating reagents and a test sample. Based on a knowledge base, presence and/or concentration of substances corresponding to multiple bioassays may be determined. The knowledge base is 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 includes values of slopes at different time instances obtained from a time plot of the intensities of light. The knowledge base also include values of the intensities of light when a time instance is zero in the time plot and a total value of the intensities of light at different time instances.

Description

A SYSTEM AND A METHOD OF PERFORMING BIOASSAYS [0001] The present patent document claims the benefit of Indian Patent Application No. 202311063249, filed September 20, 2023, which is hereby incorporated by reference in its entirety. TECHNICAL FIELD [0002] 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 [0003] 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). 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. [0004] To conduct multiple bioassays on a single test sample filter-wheels, for example, optical filters are used. Each filter-wheel allows a light of particular wavelength to pass through itself and reach the PMT for detection. However, it is not possible to perform multiple bioassays in a single cuvette without making the above-mentioned hardware changes in the bioassay system. [0005] Thus, there is a need for an easy and effective method of performing multiple bioassays on a single test sample without making hardware changes in the bioassay system. SUMMARY [0006] An objective of the present disclosure is to provide a method for performing multiple bioassays on a single test sample. [0007] Another objective of the present disclosure is to provide a method for performing multiple bioassays on a single test sample without making changes in hardware of existing bioassay systems. [0008] Yet another objective of the present disclosure is to provide a low-cost method for performing multiple bioassays. [0009] Other aspects and advantages of the disclosure become apparent from the following description, taken in conjunction with the accompanying drawings, illustrating by way of example the principles of the disclosure. [0010] 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. Based on a knowledge base, 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. [0011] In one aspect, the knowledge base may be implemented as a machine learning model for determining presence and/or concentration of substances corresponding to the multiple bioassays. [0012] In one aspect, the values of slopes may be stored as a matrix. [0013] In one aspect, the intensities of light and the cumulative value of an intensity of light may be measured in terms of Relative Light Units (RLUs). [0014] In one aspect, the intensities of light and the cumulative value of an intensity of light may be captured by a photomultiplier tube configured to receive light. [0015] In one aspect, the test sample may be one of a blood sample, a urine sample, or a bodily fluid extracted from the human body. BRIEF DESCRIPTION OF THE DRAWINGS [0016] The accompanying drawings constitute a part of the description and are used to provide further understanding of the present disclosure. Such accompanying drawings illustrate the embodiments that are used to describe the principles of the present disclosure. The embodiments are illustrated by way of example and not by way of limitation in the figures of the accompanying drawings in which like references indicate similar elements. It should be noted that references to “an” or “one” embodiment in this disclosure are not necessarily to the same embodiment, and they mean at least one. In the drawings: [0017] Fig.1 illustrates a top view of a bioassay system, in accordance with an embodiment; [0018] Fig.2 illustrates a flow chart of a method of preparing the knowledge base and performing bioassays, in accordance with an embodiment; [0019] 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; [0020] 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; [0021] Fig.5 illustrates a time varying chemiluminescence plot obtained on using mixtures of HIV1 and HIV2 antibodies, in accordance with an embodiment; [0022] Fig.6 illustrates a plot of RLUs obtained on using mixtures of light generating reagents against time instances, in accordance with an embodiment; and [0023] Fig.7(a) through 7(e) illustrate time varying chemiluminescence plots obtained on using five mixtures of light generating reagents, in accordance with an embodiment. DETAILED DESCRIPTION [0024] The detailed description set forth below in connection with the appended drawings is intended as a description of various embodiments and is not intended to represent the only embodiments in which the present disclosure may be practiced. Each embodiment described in this disclosure is provided merely as an example or illustration of the present disclosure and should not necessarily be construed as preferred or advantageous over other embodiments. The detailed description includes specific details for the purpose of providing a thorough understanding of the present disclosure. However, it will be apparent to those skilled in the art that the present disclosure may be practiced without these specific details. [0025] The present disclosure relates to a method of performing multiple bioassays. 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. Although 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. In one implementation, 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. At the reagent probe area 114, multiple light generating reagents may be dispensed into the cuvette 106 containing the test sample. The light generating reagents may react with the test sample to emit the light through the cuvette 106. 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. [0026] 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. In one embodiment, 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). [0028] At step 202, the RLUs generated by the light generating reagents at different concentrations may be measured. [0029] At step 204, a plot of RLUs at different concentrations may be determined. [0030] At step 206, from the plot, 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. [0031] At step 208, 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. Thereupon, 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. [0032] At step 210, the total value may be computed by adding values of the intensities of light of each of the plurality of the light generating reagents. [0033] At step 212, 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. [0034] In one implementation, 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. Similarly, 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. [0035] At an interval of 2 milliseconds (time instances T1, T2, and T3), slopes ( ^^^^ _{^^^^ ^^^^ ^^^^1}( ^^^^) ^^^^ ^^^^ ^^^^ ^^^^ _{^^^^ ^^^^ ^^^^2}( ^^^^)) of the HIV1 and HIV2 light generating reagents and y-intercepts _{( ^^^^ ^^^^ ^^^^ ^^^^1} ⁽ _^^^^ ⁾ _{& ^^^^ ^^^^ ^^^^ ^^^^2} ⁽ _^^^^ ⁾ _{) were obtained at each timepoint as shown in Figs. 3(a) and 3(b). The} slopes were used to populate the coefficient matrix of the HIV1 and HIV2 light generating reagents, as given by below mentioned Matrix 1:

[0036] RLUs at the time instances T1, T2, and T3 were determined using the slope and the y-intercept, given by below mentioned equations 1, 2, and 3, respectively.

……. (equation 1) ^_{^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^1} ⁽ _^^^^2 ⁾ _{= ^^^^ ^^^^ ^^^^ ^^^^1} ⁽ _^^^^2 ⁾ _{. ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^1 + ^^^^ ^^^^ ^^^^ ^^^^1(T2) ……. (equation 2) ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^1} ⁽ _^^^^3 ⁾ _{= ^^^^ ^^^^ ^^^^ ^^^^1} ⁽ _^^^^3 ⁾ _{. ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^1 + ^^^^ ^^^^ ^^^^ ^^^^1(T3) …….. (equation 3)} [0037] Similarly, using the slope and the y-intercept at the time instances T1, T2, and T3 for a particular concentration of the HIV2 light generating reagent, RLUs at the time instances T1, T2, and T3 were determined, given by below mentioned equations 4, 5, and 6, respectively: ^^^^ ^^^^ ^^^^ _{^^^^ ^^^^ ^^^^2}( ^^^^1) = ^^^^ _{^^^^ ^^^^ ^^^^2}( ^^^^1). ^^^^ ^^^^ ^^^^ ^^^^ _{^^^^ ^^^^ ^^^^2} + ^^^^ _{^^^^ ^^^^ ^^^^2}(T1) ……. (equation 4) ^^^^ ^^^^ ^^^^ _{^^^^ ^^^^ ^^^^2}( ^^^^2) = ^^^^ _{^^^^ ^^^^ ^^^^2}( ^^^^2). ^^^^ ^^^^ ^^^^ ^^^^ _{^^^^ ^^^^ ^^^^2} + ^^^^ _{^^^^ ^^^^ ^^^^2}(T2) ……... (equation 5) ^^^^ ^^^^ ^^^^ _{^^^^ ^^^^ ^^^^2}( ^^^^3) = ^^^^ _{^^^^ ^^^^ ^^^^2}( ^^^^3). ^^^^ ^^^^ ^^^^ ^^^^ _{^^^^ ^^^^ ^^^^2} + ^^^^ _{^^^^ ^^^^ ^^^^2}(T3) ……... (equation 6) [0038] From the RLUs of the HIV1 and the HIV2 light generating reagents, RLUs of a mixture of the HIV1 and the HIV2 light generating reagents was determined, as given by below mentioned Equation 7: ^_{^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^} ⁽ _^^^^ ⁾ _{= ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^1 + ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^2 = ^^^^ ^^^^ ^^^^ ^^^^1} ⁽ _^^^^ ⁾ _{. ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^1 + ^^^^ ^^^^ ^^^^ ^^^^2} ⁽ _^^^^ ⁾ _{. ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^2 + ^^^^ … (equation 7) [0039] In equation 7, c is computed}

[0040] 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). A matrix for RLUs of the mixture of the HIV1 and the HIV2 light generating reagents was determined, by below mentioned Equation 8: … (equation 8) [0041] Upon calibration of the coefficient matrix, the y-intercepts, and values of the RLUs from the mixture, parameters of the plurality of bio-medical tests such as concentrations of unknown mixtures of HIV1 and HIV2 antibodies may be derived from the below mentioned Equation 9: … (equation 9)

[0042] Fig.5 illustrates a time varying chemiluminescence plot of mixtures of HIV1 and HIV2 antibodies, in accordance with an embodiment. In Fig.5, 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. [0043] In one implementation, five mixes of dilution of each of 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. An expected value of concentrations of the TSH3UL and the TNIH was determined upon reaction of the replicates of each of the mixture with a test sample. The plot of the RLUs of all the five mixes were used for development of the knowledge base, using machine learning techniques. [0045] 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. [0046] 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. Three replicates R1, R2, and R3 were taken for the Mix-10, and it was observed from the Fig.7(b) that deviation in RLUs of the TSH3UL and the TNIH in the replicates R1, R2, and R3 were correctly predicted by the trained data mode. Tailing ends of plots of replicates R1, R2, and R3 of the Mix-10 seemed to indicate possible wet-lab errors in preparation of Mix-10. Three replicates R1, R2, and R3 were taken for the Mix-11 and Mix-12 respectively in Fig.7(c) and Fig.7(d). It was observed from the Fig.7(c) and Fig.7(d) that the trained knowledge base showed good accuracy and precision across each of the three replicates of the Mix-11 and the Mix-12. 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). [0047] 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. [0048] 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. [0049] In view of the present disclosure, 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.

Claims

CLAIMS 1. A method of performing bioassays, the method comprising: determining a cumulative value of an intensity of light generated from a reaction between a plurality of the light generating reagents and a test sample, wherein each light generating reagent of the plurality of light generating reagents corresponds to a specific bioassay to be performed using the test sample; and determining a presence and/or a concentration of substances corresponding to multiple bioassays based on a knowledge base, wherein the knowledge base is 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, and wherein the knowledge base comprises: values of slopes at different time instances, the values of slopes being obtained from a time plot of the intensities of light; values of the intensities of light when a time instance is zero in the time plot; and a total value of the intensities of light at different time instances.

2. The method as claimed in claim 1, wherein the knowledge base is implemented as a machine learning model for determining the presence and/or the concentration of the substances corresponding to the multiple bioassays.

3. The method as claimed in claim 1, wherein the values of slopes are stored as a matrix.

4. The method as claimed in claim 1, wherein the intensities of light and the cumulative value of the intensity of light are measured in terms of Relative Light Units (RLUs).

5. The method as claimed in claim 1, wherein the intensities of light and the cumulative value of an intensity of light are captured by a photomultiplier tube configured to receive light.

6. The method as claimed in claim 1, wherein the test sample is a blood sample, a urine sample, or a bodily fluid extracted from the human body.