Analyte Detection via Oligonucleotide Tagged Probes
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for detecting active analytes and determining the binding ability of substances to their active sites in biological samples face challenges such as interference from heterophilic antibodies, low sensitivity, and inability to accurately quantify proteins like PSA and enzymes like PSMA and CA-IX, leading to false positives and false negatives, especially in complex matrices like serum and plasma.
Innovation Solution
A method involving immobilization of analytes on a solid carrier with a detection probe that includes a low molecular weight compound and an oligonucleotide tag, allowing for selective binding to the active site, followed by quantitative PCR to determine the amount of bound probe, which is proportional to the analyte concentration, and assessing the binding ability of tested substances by comparing probe binding in their presence and absence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If ELISA methods are used for detecting analytes in biological samples, then the detection can be performed with standard procedures, but the sensitivity is insufficient and false positives occur due to interfering heterophilic antibodies
Solution Approach 1:
The patent introduces an oligonucleotide tag as an intermediary between the analyte and the detection system. The detection probe consists of a ligand bound to an oligonucleotide tag, which allows detection through PCR amplification rather than traditional ELISA readout. This intermediary approach enables highly specific detection that is not affected by heterophilic antibodies, as the oligonucleotide tag provides a unique molecular identifier that can be amplified with high specificity through PCR, eliminating cross-reactivity issues inherent in antibody-based detection
Solution Approach 2:
The patent replaces the enzymatic amplification system used in ELISA with a nucleic acid-based PCR amplification system. Instead of using enzyme-conjugated antibodies that produce colorimetric signals and are susceptible to antibody interference, the invention uses ligand-probe complexes with oligonucleotide tags that are detected through PCR. This substitution of the detection mechanism provides exponential amplification capability and eliminates the interference from heterophilic antibodies that plague traditional ELISA methods
2Measurement precision
If conventional detection methods are used, then the procedure is simple, but the detection limit is insufficient for early cancer detection requiring attogram-level sensitivity
Solution Approach 1:
The patent employs preliminary action by first immobilizing the analyte on a solid support and then introducing the detection probe with the oligonucleotide tag before PCR amplification. This sequential preparation ensures that only analyte-bound probes are amplified, eliminating background noise from free probes. The method also includes preliminary blocking steps to prevent non-specific binding, and uses a sandwich assay format where capture antibodies are pre-immobilized on the plate, ensuring high specificity before the detection step
Solution Approach 2:
The invention implements a nested structure where the ligand is bound to the oligonucleotide tag to form a detection probe, which then binds to the analyte in a sandwich format with capture antibodies. The oligonucleotide tag is nested within the probe structure, and the entire probe-analyte complex is nested within the PCR amplification system. This nested architecture allows each component to perform its function while being protected by the surrounding layers, achieving high sensitivity through multiple levels of signal amplification and specificity
3Measurement precision
If high probe concentrations are used in binding assays, then the signal is strong, but the dynamic range for determining binding constants is limited
Solution Approach 1:
The patent implements dynamics by enabling the detection system to adapt to different analyte concentrations through PCR amplification. The oligonucleotide tag allows the system to detect a wide range of probe amounts through exponential amplification, where even tiny differences in initial probe binding are magnified into detectable signal differences. This dynamic response allows the assay to maintain sensitivity across a broad concentration range, from low-abundance analytes requiring high probe concentrations to high-abundance analytes where lower probe concentrations suffice
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This method achieves high sensitivity, with detection limits reaching ten attograms for PSMA, enabling early cancer detection and accurate quantification of analytes in complex biological matrices, reducing false results and requiring minimal sample volume, and allowing for precise determination of binding constants across a wide dynamic range.
Implementation Method 1
The amount of the bound probe is determined, which is directly proportional to the amount of immobilized analyte; preferably the determination is performed by detection of oligonucleotide tags in quantitative polymerase chain reaction (qPCR)
Implementation Method 2
a detection probe is selectively bound to the analyte. The detection probe consists of a compound for selective binding to the active site of the analyte
Data Source
AI summary
A method for detection of active form of analytes in a sample and/or for determination of ability of tested substances to bind to the active site of these analytes has the following steps: a) analyte or group of analytes from the sample is immobilized on the surface of a solid carrier; b) analyte or group of analytes is incubated with a detection probe; c) then the solid carrier is washed to remove unbound detection probe; and subsequently, the amount of bound detection probe is determined.