Device and methods of using device for detection of aminoacidopathies
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-10392646-B2
Publication Date
2019-08-27
Expiration Date
2033-10-17
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Abstract
The present invention relates to a biosensor capable of measuring the total concentration of one or a plurality of amino acids with the use of a reagentless system comprising an electrode modified by hydrogel that comprises at least one enzyme that oxidizes at least one substrate that is at least one amino acid. In some embodiments, the biosensor comprises a hydrogel comprising alginate. In some embodiments, the biosensor comprises use of a thermophilic bacterial metabolic enzyme immobilized or attached to the hydrogel.
Core Innovation
The invention provides a biosensor capable of measuring the total concentration of one or a plurality of amino acids in a sample using a reagentless system comprising an electrode modified by a hydrogel that includes at least one enzyme that oxidizes at least one amino acid substrate. The biosensor typically comprises a hydrogel containing alginate and uses a thermophilic bacterial metabolic enzyme immobilized or attached to the hydrogel.
The device includes at least a first electrically conductive surface for measurement and a second electrically conductive surface as a counter electrode, wherein the first surface incorporates mediators and enzymes that selectively use specific amino acids as substrates. The enzymes produce reaction products from the amino acids which generate electron flow that is measured as current or voltage differential, enabling determination of amino acid concentration through calibration.
The problem addressed arises from metabolic disorders such as aminoacidopathies and hyperammonemia which involve elevated metabolites measurable only by specialized hospital-based mass spectrometry. Current monitoring methods require hospital visits for testing, making management difficult and costly. The invention aims to provide a point-of-care, real-time sensor to conveniently detect and quantify amino acids in bodily fluids without need for reagents or external stimuli, thereby facilitating better management and treatment of metabolic diseases.
Claims Coverage
The patent features two independent claims encompassing biosensors and systems for detecting amino acids using hydrogel-modified electrodes with immobilized enzymes from thermophilic bacteria, as well as related components and configurations.
Biosensor with hydrogel comprising phenylalanine dehydrogenase
A biosensor comprising at least one electrically conductive support attached to a hydrogel that includes at least one electron mediator, at least one reduction agent, and at least one metabolic enzyme derived from a thermophilic bacterial cell or functional fragment thereof, wherein the hydrogel comprises alginate. The enzyme is phenylalanine dehydrogenase or functional fragment with at least about 70% sequence identity to specified sequences. The biosensor is operably connected to an amperometer and/or voltmeter and can include multiple conductive supports such as silver and silver chloride wires. The biosensor exhibits biological activity after storage and is not dependent on UV light exposure or external stimuli.
System comprising the biosensor with computer interface
A system comprising the disclosed biosensor operably connected to at least one computer storage memory and a computer processor, further including a digital display configured to show amino acid concentration values measured by the sensor when in contact with a sample. The system measures current or voltage differentials resulting from enzymatic oxidation of amino acids immobilized within the hydrogel on the electrically conductive support.
Biosensor with enzymes having specified sequence homology
A biosensor comprising at least one electrically conductive support attached to a hydrogel containing at least one electron mediator, at least one reduction agent, and at least one metabolic enzyme or functional fragment having at least 70% homology to specified enzyme sequences, operably connected to an amperometer and/or voltmeter.
The claims broadly cover biosensors and systems utilizing hydrogel-coated electrodes with immobilized thermophilic bacterial enzymes, particularly phenylalanine dehydrogenase, to electrochemically detect and quantify amino acid concentrations in bodily fluids, along with associated hardware and software components enabling real-time measurement and display.
Stated Advantages
Allows real-time, point-of-care detection and quantification of amino acid metabolites without requiring reagents or external stimuli.
Improves patient quality of life and reduces healthcare costs by enabling immediate metabolite level assessment outside hospital settings.
The alginate hydrogel filter selectively excludes interfering ions and proteins, improving measurement accuracy and signal-to-noise ratio.
Use of thermophilic bacterial enzymes enhances enzyme stability and biosensor longevity, retaining significant activity after prolonged storage.
Provides a portable, reagentless biosensor system capable of diagnosing and monitoring aminoacidopathies and related metabolic diseases efficiently.
Documented Applications
Diagnosing subjects with aminoacidopathies by detecting, identifying, and quantifying amino acid concentrations in bodily fluids.
Monitoring concentrations of amino acids in subjects over time to manage metabolic diseases like phenylketonuria, hyperammonemia, and maple syrup urine disease.
Sorting and cataloguing mixtures of bodily fluid samples based on amino acid concentration profiles.
Determining patient responsiveness to therapies by comparing amino acid concentration values to threshold values associated with metabolic diseases.
Use in test strips and portable devices for point-of-care measurement of amino acids directly from human or non-human blood samples without preprocessing.
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