Method and apparatus for multiplex flow cytometry analysis of diverse mixed analytes from bodily fluid samples
First Claim
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1. A reagent for measuring target analytes in a test sample, said reagent comprising at least one type of analyte sensor particle selected from each of the following classes (a) and (b):
- (a) ion-sensors comprising a plurality of sample-insoluble particles having associated therewith a target ionophore adapted to interact with a target ion in the sample, and a first emitted fluorescent signal following interaction of the target ionophore with a target ion;
(b) metabolite-sensors comprising a plurality of sample-insoluble particles having associated therewith a ligand adapted to interact with a target metabolite in the sample to produce a second fluorescent signal following interaction of the ligand with the target metabolite; and
at least one type of analyte sensor particle selected from the following classes (c), (d) and (e);
(c) enzyme-sensors comprising a plurality of sample-insoluble particles having associated therewith a fluorogenic substrate adapted to interact with a target enzyme in the sample to produce a third fluorescent signal;
(d) antigen- or antibody-sensors comprising a plurality of sample-insoluble particles having associated therewith an immobilized pair member adapted to interact to form a complex with a complementary target antigen or a complementary antibody and to produce a fourth fluorescent signal following interaction of the pair member with the complementary target antigen or complementary antibody; and
(e) nucleotide sequence-sensors comprising a plurality of sample-insoluble particles having associated therewith a polynucleotide molecule complementary to a target nucleotide sequence and capable of hybridizing with the target nucleotide sequence under hybridizing conditions, and a fluorescent signal material that produces a fifth fluorescent signal upon hybridization between the complementary polynucleotide molecule and the target nucleotide sequence;
wherein the reagent mixture includes sensor particles which interact specifically with each of the following analytes;
(A) at least one analyte selected from the group consisting of alkali metal ions, alkaline earth metal ions, ammonium, halide ions, oxygen, pH, carbon dioxide;
(B) at least one analyte selected from the group consisting of saccharides, ammonia, urea, uric acid, cholesterol, triglycerides, ethanol, lactate, salicylate, acetaminophen, bilirubin and creatinine;
enzymes, antibodies, antigens and polynucleotide sequences.
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Abstract
This invention relates to a reagent mixture, as well as to a method and apparatus, for carrying out simultaneous, automated analysis of multiple analytes in a test sample, particularly a bodily fluid. The invention is especially relevant to the field of general clinical chemistry, but may find application in other fields of use.
37 Citations
21 Claims
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1. A reagent for measuring target analytes in a test sample, said reagent comprising at least one type of analyte sensor particle selected from each of the following classes (a) and (b):
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(a) ion-sensors comprising a plurality of sample-insoluble particles having associated therewith a target ionophore adapted to interact with a target ion in the sample, and a first emitted fluorescent signal following interaction of the target ionophore with a target ion;
(b) metabolite-sensors comprising a plurality of sample-insoluble particles having associated therewith a ligand adapted to interact with a target metabolite in the sample to produce a second fluorescent signal following interaction of the ligand with the target metabolite; and
at least one type of analyte sensor particle selected from the following classes (c), (d) and (e);
(c) enzyme-sensors comprising a plurality of sample-insoluble particles having associated therewith a fluorogenic substrate adapted to interact with a target enzyme in the sample to produce a third fluorescent signal;
(d) antigen- or antibody-sensors comprising a plurality of sample-insoluble particles having associated therewith an immobilized pair member adapted to interact to form a complex with a complementary target antigen or a complementary antibody and to produce a fourth fluorescent signal following interaction of the pair member with the complementary target antigen or complementary antibody; and
(e) nucleotide sequence-sensors comprising a plurality of sample-insoluble particles having associated therewith a polynucleotide molecule complementary to a target nucleotide sequence and capable of hybridizing with the target nucleotide sequence under hybridizing conditions, and a fluorescent signal material that produces a fifth fluorescent signal upon hybridization between the complementary polynucleotide molecule and the target nucleotide sequence;
wherein the reagent mixture includes sensor particles which interact specifically with each of the following analytes;
(A) at least one analyte selected from the group consisting of alkali metal ions, alkaline earth metal ions, ammonium, halide ions, oxygen, pH, carbon dioxide;
(B) at least one analyte selected from the group consisting of saccharides, ammonia, urea, uric acid, cholesterol, triglycerides, ethanol, lactate, salicylate, acetaminophen, bilirubin and creatinine;
enzymes, antibodies, antigens and polynucleotide sequences. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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13. A method for assaying multiple analytes in a test sample, said method comprising the steps of:
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(1) admixing a test sample containing multiple analytes to be measured with a reagent mixture comprising a plurality of types of sensor particles, each type of sensor particle comprising coding indicia which confer uniquely identifying optical properties on that type of particle and a measurement substrate which specifically interacts with an analyte of interest such that measurement optical properties of said substrate are varied, said reagent mixture including particles which interact specifically with each of the following analytes;
(A) at least one analyte selected from the group consisting of alkali metal ions, alkaline earth metal ions, ammonium, halide ions, oxygen, pH, carbon dioxide;
(B) at least one analyte selected from the group consisting of saccharides, ammonia, urea, uric acid, cholesterol, triglycerides, ethanol, lactate, salicylate, acetaminophen, bilirubin and creatinine;
(C) at least one analyte selected from the group consisting of enzymes, antibodies, antigens, and polynucleotide sequences, (2) allowing the resulting admixture to incubate for a period of time sufficient for each type of sensor particle to interact with the analyte with which it specifically interacts to vary the measurement optical properties of the sensor particle;
(3) transferring the admixture to a reading device and reading both the coding and the measurement optical properties of each sensor particle individually;
(4) storing the measured optical properties of each sensor particle type according to the coding optical properties read from the particles; and
(5) processing the stored measurements for each sensor particle type to obtain an assay result for the analyte associated with each type of sensor particle;
whereby a complete chemical analysis of the test sample is obtained. - View Dependent Claims (14, 15, 16, 17, 18)
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19. An apparatus for assaying multiple analytes in a test sample, said apparatus comprising:
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(A) an inlet for a test sample which is to be analyzed;
(B) an inlet for a reagent mixture containing a plurality of types of sensor particles, each type of sensor particle being designed to specifically interact with an analyte of interest to vary the optical properties of an associated fluorescent indicator;
(C) an incubation chamber or vessel in which the sample and reagent mixture are allowed to interact;
(D) a flow cytometer read cell adapted to read coding indicia on each sensor particle passed through the cell to identify the type of sensor particle and the analyte of interest with which the particle interacts, and to read the associated fluorescent indicator to obtain a measured value indicative of whether the sensor particle has interacted with the respective analyte of interest; and
(E) a controller adapted to store measured values read for each sensor particle according to the type of sensor particle and the specific analyte with which that type of sensor particle interacts, and to process the stored measured values for each sensor particle type to obtain an assay result for the analyte associated with each type of sensor particle. - View Dependent Claims (20)
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21. An apparatus for assaying multiple analytes in a test sample, said apparatus comprising:
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(A) means for introducing a test sample which is to be analyzed;
(B) means for introducing a reagent mixture containing a plurality of types of sensor particles, each type of sensor particle being designed to specifically interact with an analyte of interest to vary the optical properties of an associated fluorescent indicator;
(C) an incubation chamber or vessel in which the sample and reagent mixture are allowed to interact;
(D) a flow cytometer read cell adapted to read coding indicia on each sensor particle passed through the cell to identify the type of sensor particle and the analyte of interest with which the particle interacts, and to read the associated fluorescent indicator to obtain a measured value indicative of whether the sensor particle has interacted with the respective analyte of interest; and
(E) a controller adapted to store measured values read for each sensor particle according to the type of sensor particle and the specific analyte with which that type of sensor particle interacts, and to process the stored measured values for each sensor particle type to obtain an assay result for the analyte associated with each type of sensor particle.
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Specification