Oligonucleotide Marker Blocking for Low-Background Fluorescence Imaging
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Solution Overview
Problem
Background fluorescence in fluorescence imaging, including autofluorescence and nonspecific binding of fluorescent markers, reduces signal-to-noise ratio and compromises image quality, particularly in high-plex applications.
Innovation Solution
A marker system comprising affinity reagents with oligonucleotides and labels with complementary and partially complementary blocking oligonucleotides to reduce nonspecific binding and background fluorescence, ensuring specific and stable binding of labels to target analytes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorescent markers are used to detect target analytes, then detection capability is improved, but background fluorescence increases due to nonspecific binding
Solution Approach 1:
The patent introduces an oligonucleotide mediator that serves as an intermediary between the fluorescent marker and the target analyte. The marker is conjugated to an oligonucleotide that specifically hybridizes to a complementary sequence on the target, rather than binding directly to the target. This intermediary oligonucleotide reduces nonspecific binding and background fluorescence while maintaining detection capability through specific hybridization.
Solution Approach 2:
The patent employs composite fluorescent markers consisting of multiple components: a fluorescent dye, a linker, and an oligonucleotide sequence. This composite structure allows the marker to maintain fluorescent properties while the oligonucleotide component provides specific binding through hybridization, reducing nonspecific interactions and background fluorescence.
2Adaptability or versatility
If a large number of fluorescent markers are used for high-plex applications, then multiplexing capability is improved, but background fluorescence and nonspecific binding increase
Solution Approach 1:
The patent segments the detection system into multiple independent oligonucleotide-based markers, each with a unique oligonucleotide sequence specific to its target analyte. This segmentation allows each marker to operate independently with high specificity, reducing cross-reactivity and nonspecific binding even when multiple markers are used simultaneously in high-plex applications.
3Measurement precision
If marker concentration is increased to improve detection sensitivity, then signal intensity is improved, but nonspecific binding and background fluorescence increase
Solution Approach 1:
The patent replaces direct affinity binding (mechanical/chemical interaction) with oligonucleotide hybridization (nucleic acid base pairing). This substitution provides more specific and controllable binding through complementary base pairing, allowing higher marker concentrations to be used without proportionally increasing nonsspecific binding, as the hybridization mechanism is inherently more specific.
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
The system enhances image contrast and accuracy by minimizing background fluorescence, allowing for precise detection and quantification of target analytes even in complex biological samples with multiple markers.
Implementation Method 1
the only at least partially complementary blocking sequence of the at least one blocking oligonucleotide enables blocking of either the barcode sequence or the complementary barcode sequence
Implementation Method 2
Background fluorescence is a significant disadvantage in fluorescence imaging because it can obscure the signal from the target fluorescent molecules
Data Source
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AI summary
In a first aspect a marker (100, 200) for analysing a biological sample is provided. The marker (100, 200) comprises an affinity reagent (104) comprising an affinity reagent oligonucleotide (108) with a barcode sequence (110). The marker (100, 200) further comprises a label (112) comprising a label oligonucleotide (114) with a complementary barcode sequence (116) at least partially complementary to the barcode sequence (110) of the affinity reagent oligonucleotide (108). The marker (100, 200) further comprises at least one blocking oligonucleotide (102, 202) with a blocking sequence (118, 204) that is only partially complementary to the barcode sequence (110) of the affinity reagent oligonucleotide (108) or to the complementary barcode sequence (116) of the label oligonucleotide (114).