Integrated filtration bioanalyzer
Inventors
Putnam, David L. • Putnam, Jason A. • Hubbard, Todd W. • van Zee, James A.
Assignees
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Publication Number
US-9068976-B2
Publication Date
2015-06-30
Expiration Date
Abstract
The present invention relates to an in vitro assay method and device that provides for detection and measurement of entities in a fluid sample that can be captured and concentrated in a unitized self-contained enclosed filter apparatus that is analyzed in an optical detection instrument for indications of the entity. It provides for analysis of biological material including cells, their enzymes, or other constituents thereof, that can be identified based on an indicator-generating means. The analysis provided for include detection of the presence of the entity, and changes in the entity over time, such as associated with growth and increasing metabolic activity with an expanding population of cells, or decreasing metabolic activity, for example, due to presence of inhibitory or toxic agents.
Core Innovation
The integrated filter assay system provides an in vitro method for determining the presence and/or activity of biological material in a liquid sample by keeping the complete sequence within an enclosed filter apparatus. A liquid sample is filtered through an enclosed filter apparatus that traps the biological material, and the retained biological material is processed without removing it from the enclosed filter apparatus during subsequent steps.
A reaction fluid is administered into and/or through the filter apparatus to support and/or affect the biological activity of the retained biological material and to provide an indicator means for indicating the presence and/or activity of the biological material. The filter apparatus, retained biological material, and reaction fluid are incubated in a measurement apparatus, while one or more optical measurements of the indicator means and/or the biological material are performed concurrent with incubation.
The measurements are analyzed to determine activity of the biological material, using time-kinetic derivative analysis of optical reporter activity to distinguish true growth from background enzyme activity and to reduce false negatives and false positives. The approach includes unitized enclosed cartridges with optical readers and reporter substrates based on enzyme activity, including fluorogenic and/or chromomeric indicator means such as resorufin-b2-glucuronide and related b2-galactosidase and b2-glucuronidase substrates, with measurement algorithms based on derivative signal analysis.
Claims Coverage
The independent claim covers an in vitro integrated method where filtering, reaction-fluid administration with an indicator means, incubation, concurrent optical measurement, and analysis are performed within an enclosed filter apparatus. Dependent claims further refine indicator chemistry, filter retention capability, enclosure features, and time-based quantitative estimation using detection time.
Enclosed filter apparatus trapping and retained processing
Filtering a liquid sample through an enclosed filter apparatus capable of trapping said biological material, whereby said biological material present in said liquid sample is retained in said filter apparatus, and performing steps (i)-(v) within the enclosed filter apparatus.
Reaction fluid with activity-supporting components and indicator means
Administering a reaction fluid into and/or through said filter apparatus, said reaction fluid comprising components to support and/or affect the biological activity of said biological material and an indicator means for indicating the presence and/or activity of said biological material.
Concurrent incubation with optical measurement using a measurement apparatus
Incubating said filter apparatus, said biological material retained and said reaction fluid in a measurement apparatus, and performing one or more measurements by optical means of said indicator means and/or said biological material concurrent with incubation in said filter apparatus.
Analyzing optical measurements to determine biological activity
Performing analysis of said measurements of said indicator means and/or said biological material to determine activity of said biological material.
Fluorogenic and chromomeric substrate reporter indicator means
Using an indicator means that is a fluorogenic and/or chromomeric substrate reporter for b2-galactosidase, with reporter molecules including resorufin, 4-methylumbelliferyl, ethyl-7-hydroxycourmarin-4-carboxylate, fluorescein, 6-chloro-3-indoxyl, 5-bromo-4-chloro-3-hydroxyindoxyl-, and 5-bromo-6-chloro-3-indoxyl-.
b2-glucuronidase substrate reporter for E. coli
Using an indicator substrate reporter of b2-glucuronidase to detect E. coli.
Filter element with pore or retention capability
Including a filter element that retains the biological material with a pore or retention capability of at least 0.2 microns.
Housing with closed chamber and fluid-handling features
Specifying that the housing includes a closed chamber, an inlet, a reservoir sized to hold filter means and growth media, an outlet, and a vent that permits gas flow while retaining liquids within the housing.
Estimating amount using detection time and reference-based model or table
Determining the amount of biological material in a liquid sample during filtration by detecting a change in a characteristic indicator means, comparing the detection time to that in known reference biological material, and extrapolating the amount from a table or mathematical model using the detection time.
The claim set is anchored by the enclosed-filter, reaction-fluid-with-indicator, concurrent optical measurement during incubation, and measurement analysis to determine biological activity. Dependent coverage narrows indicator chemistry, filter retention, enclosure features, and quantification through detection-time comparison and model or table extrapolation.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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