Attenuated total reflectance-based biosensor for conformation and secondary structure analysis
Inventors
GERWERT, Klaus • Wiltfang, Jens • OLLESCH, Julian • Nabers, Andreas • SCHARTNER, Jonas • KÖTTING, Carsten
Assignees
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Publication Number
US-12031907-B2
Publication Date
2024-07-09
Expiration Date
Abstract
Provided herein is a biosensor for conformation and secondary structure analysis, notably for the direct non-invasive qualitative secondary structure analysis of a single selected protein within a complex mixture, as e.g. a body fluid, by vibrational spectroscopic methods. For the analysis it is not required that the selected substance be isolated, concentrated, or pretreated by a special preparative procedure.
Core Innovation
The invention relates to an ATR-based IR biosensor and a method for determining, in a body fluid, an average secondary structure composition of a protein undergoing conformational transitions associated with disease progression. The method conducts an IR measurement in an IR cell using an IR sensor element functionalized with at least one receptor grafted onto a functionalized surface, where the receptor is capable of specific and conformationally independent binding to the protein. The body fluid is used as a flux comprising the protein without pretreatment to isolate or concentrate the protein.
An IR beam is submitted through the cell to obtain an IR spectrum from the protein in the body fluid. The determining of the average secondary structure composition is based on detecting an amide I band maximum of the protein in the obtained IR spectrum. The sensor is configured to detect different conformations of the secondary structure of the protein present in the body fluid.
In representative implementations, the receptor-functionalized sensor is an ATR device with a germanium internal reflection element, including multi-reflection geometries such as trapezoid or parallelogram forms. The sensor surface is functionalized with antibodies immobilized via short silane or thiol linkers, and remaining reactive amines are blocked using a non-cross-reacting blocking substance such as casein. Captured biomarker proteins are analyzed by IR spectroscopy to quantify conformational transitions using the amide I band maximum, with optional parallel fluorescence detection.
Claims Coverage
Independent coverage is provided by one independent claim describing a full method for determining an average secondary structure composition in a body fluid using IR amide I band measurements with a conformationally independent, receptor-functionalized IR sensor and without isolating or concentrating the protein.
Disease-progression secondary-structure determination in an untreated body fluid by receptor-functionalized IR amide I detection
A method for determining, in a body fluid, an average secondary structure composition of a protein undergoing conformational transitions associated with disease progression by conducting, in an IR cell comprising an IR sensor element having a functionalized surface with at least one receptor grafted onto the functionalized surface and capable of specific and conformationally independent binding to the protein, a flux of body fluid comprising the protein but not pretreated to isolate or concentrate the protein; submitting an IR beam through the cell and obtaining an IR spectrum; and detecting an amide I band maximum of the protein to determine the average secondary structure composition present in the body fluid.
Germanium internal reflection element receptor grafted via short linkers with blocking of remaining reactive groups
The method of claim 1, wherein the IR sensor element uses an internal germanium reflection element configured for IR sensing, with receptor grafting onto the germanium element by silanization or thiolation using short silane or short thiol linkers (not more than 20 atoms) reacted with amine-reactive linker groups, followed by blocking of remaining amine-reactive groups using a non-cross-reacting blocking substance.
Across the independent claim set, the core inventive concept is detecting disease-progressing conformational transitions via amide I band maximum measurements from a receptor-functionalized IR ATR sensor, while using untreated body fluid (no isolation or concentration) and, in refined independent coverage, using germanium multi-reflection ATR geometry with receptor immobilization via short linkers and non-cross-reacting blocking.
Stated Advantages
Enables direct determination of an average secondary structure composition of a protein in a body fluid without isolating or concentrating the protein.
Provides detection of different conformations of the secondary structure of the protein in the body fluid via an IR spectrum by amide I band maximum.
Allows receptor functionalization with specific and conformationally independent binding to the protein, intended to support conformational-transition measurements in complex body fluids.
Documented Applications
Discrimination or determination of Alzheimer’s disease progression using amyloid-beta peptides by detecting an amide I band maximum threshold.
Discrimination or determination of Parkinson’s disease progression using alpha-synuclein by detecting an amide I band maximum threshold.
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