Device and methods of using device for detection of hyperammonemia
Inventors
Ayyub, Omar Bilal • Behrens, Adam Michael • Kofinas, Peter • Summar, Marshall Lynn • Cabrera-Luque, Juan Manuel • Cunningham, Gary • Simeonov, Anton • Marugan, Juan
Assignees
Childrens National Medical Center Inc • University of Maryland College Park • US Department of Health and Human Services
Publication Number
US-9952199-B2
Publication Date
2018-04-24
Expiration Date
2034-09-02
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Abstract
The present disclosure relates to a bio-sensor capable of measuring the total concentration of one or a plurality of ammonia or ammonium ions with the use of indophenol reagents in the presence of an ionomer. In some embodiments, the biosensor comprises a perflurinated membrane that comprises an ionomer in contact with an alkaline buffer in a vessel configured to receive a sample, such as whole blood. The disclosure also relates to a method of detecting or quantifying the ammonia or ammonium ion concentration in whole blood in a point of care biosensor without reliance on gas chromatography or any measurement that takes more than about twenty minutes.
Core Innovation
The invention relates to a biosensor capable of measuring the total concentration of ammonia or ammonium ions in a sample, utilizing indophenol reagents in the presence of an ionomer comprising a perfluorinated membrane. The biosensor comprises at least a first vessel and a second vessel separated by a membrane functionalized to permit ion exchange, enabling ammonia to diffuse from a biological or environmental sample into a reagent compartment where the indophenol reaction occurs, producing a color change indicative of ammonia concentration. This detection can be quantified using photodiodes, spectrophotometers, voltmeters, or amperometers within a point of care setting without the need for gas chromatography or prolonged laboratory analysis.
The invention addresses the problem of diagnosing hyperammonemia rapidly and accurately at the point of care. Current methods such as fluorometry and tandem mass spectrometry require costly, bulky equipment and centralized labs, leading to delays of multiple days in diagnosis. Hyperammonemia is a serious condition often associated with liver cirrhosis and urea cycle disorders, especially in neonates, where delays in treatment degrade neurological outcomes. The disclosed biosensor enables rapid, cost-effective, and stable ammonia detection in whole blood and other fluids, facilitating immediate diagnosis and treatment monitoring that reduce morbidity and mortality.
The biosensor system utilizes a cation exchange membrane, such as Nafion, allowing selective passage of ammonia while excluding proteins and other interfering molecules. Reagents for the indophenol (Berthelot's) reaction, including phenolic compounds (e.g., 2-phenylphenol), hypohalite, sodium hydroxide, and catalysts like sodium nitroprusside, are prepared in a separate vessel where colorimetric changes upon reaction with ammonia are measured. The device includes configurations for quantitative measurement via optical or electrochemical means, including electrodes functionalized with metabolic enzymes to detect amino acid levels and convert enzymatic products into electrical signals for quantification. Microfluidic cartridges and modular wells are also disclosed, supporting integration of reagents and sample flow for practical use.
Claims Coverage
The claims include seven main inventive features focused on biosensor structure, reagent composition, functional membrane, and integrated measurement systems.
Biosensor comprising divided vessels separated by an ionomer membrane
A biosensor comprising at least a first and second vessel separated by a fluid exchange opening positioned with a membrane comprising an ionomer. The biosensor includes at least one conduit to receive a sample volume ranging from about 5 μL to about 100 μL. The first or second vessel individually or combined contains a hypohalite, an alkali buffer, and at least one phenolic reagent or indophenol related compound.
Inclusion of hypohalite, catalyst, and phenolic reagents in vessels
Either the first or second vessel individually comprises hypohalite, an alkali buffer, a catalyst, and at least one phenolic reagent or indophenol related compound, supporting the indophenol reaction for ammonia detection.
Integration of electrically conductive supports with biosensor
The biosensor further comprises at least a first electrically conductive support in fluid communication with the second vessel, operably connected via wire to instruments such as an amperometer, voltmeter, spectrophotometer, or combinations thereof.
Hydrogel and metabolic enzyme functionalization on electrically conductive support
The electrically conductive support further comprises a hydrogel incorporating at least one metabolic enzyme or functional fragment thereof, enabling electrochemical detection of ammonia or amino acid related metabolic products.
Exclusion of specific components ensuring functional and stable biosensor
The biosensor excludes any of: uricase or fragments, hydrogels comprising dextran or derivatives, bacterial cells, electronic dipoles for electrophoresis, 3,4-dihydroxybenzoic acid, and vaporizers, gas chromatographs, or heating elements for gas conversion of ammonia, ensuring a stable and practical design.
Biosensor operational independence from external stimuli
The biosensor is not functionally dependent upon exposure to any external stimulus to activate the detection or measurement process.
System integration and data display capabilities
The biosensor includes an electrical circuit and digital display connected to a processor configured to receive digital signals from detection components like a spectrophotometer and present the concentration values of ammonia, ammonium ions, and/or amino acids to the user.
The claims collectively cover a biosensor system employing vessels divided by an ionomer membrane, inclusion of specific reagents for an indophenol reaction, integration with conductive electrodes functionalized with enzymes and hydrogels, exclusion of interference factors, operational independence, and electronic processing and display systems, enabling rapid, point of care quantitative detection of ammonia and amino acids in samples.
Stated Advantages
Enables rapid, point of care quantification of ammonia or ammonium ion concentration in whole blood or other samples without need for gas chromatography or complex instrumentation.
Provides stability of reagents at room temperature for at least 50 days, improving shelf life and usability.
Minimizes interference from proteins and small molecules in complex biological fluids through use of ionomer membranes like Nafion and optimized reagent concentrations.
Reduces cost and complexity compared to centralized lab-based systems, facilitating bedside or home testing.
Enables detection of amino acids related to metabolic disorders by enzymatic conversion generating measurable ammonia, supporting diagnosis of aminoacidopathies.
Documented Applications
Diagnosis and monitoring of hyperammonemia in subjects at point of care using whole blood samples.
Detection and quantification of ammonia or ammonium ion in water, wastewater, environmental samples, and aquariums.
Diagnosis and treatment monitoring of aminoacidopathies and metabolic diseases via amino acid quantification with enzyme-functionalized biosensors.
Use as a portable, rapid testing device analogous to glucose meters for home or clinical settings.
Use in kits comprising biosensors, test strips, and computational systems for clinical or environmental ammonia and amino acid analysis.
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