Multiplexed Imaging Using Enzyme-Amplified Oligonucleotide Labeling
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Solution Overview
Problem
Conventional immunohistochemistry methods do not provide adequate signal for weakly expressed or inefficiently targeted immunological targets, limiting detection capabilities.
Innovation Solution
Employing tyramide signal amplification (TSA) with enzyme-mediated deposition of oligonucleotide sequences and orthogonal oligonucleotide sequences to enhance detection, allowing for multiple rounds of amplification and multiplexed detection of targets using chromogenic or fluorescent dyes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional immunohistochemistry methods are used, then the detection process is simple, but the signal intensity is insufficient for weakly expressed targets
Solution Approach 1:
The patent introduces an intermediary enzyme (horseradish peroxidase) that bridges the antibody-target binding and the detectable signal. The enzyme catalyzes the deposition of numerous oligonucleotide-labeled dye molecules at the target site, amplifying the signal without requiring direct conjugation of large dye molecules to the antibody, thus improving detection sensitivity while maintaining procedural simplicity
Solution Approach 2:
The patent changes the detection parameter from direct antibody-dye binding to enzyme-catalyzed signal amplification. By using the enzyme's catalytic activity to generate multiple detectable molecules per target, the signal intensity parameter is significantly enhanced, enabling reliable detection of weakly expressed targets
2Adaptability or versatility
If multiple targets are detected simultaneously, then the multiplexing capability increases, but the signal differentiation becomes more difficult
Solution Approach 1:
The patent segments the detection process into distinct phases: first, multiple primary antibodies with different specificities are bound to their respective targets; second, a universal enzyme conjugate is applied to all targets; third, oligonucleotide-labeled dyes are deposited and can be differentiated by their unique nucleotide sequences or spectral properties. This segmentation allows multiple targets to be detected simultaneously with clear signal differentiation
Solution Approach 2:
The patent uses composite detection signals combining enzyme catalysis with oligonucleotide-dye conjugates. The composite nature of the signal system allows for multiplexing because different oligonucleotide sequences can be detected with different fluorescent dyes having distinct emission spectra, enabling precise differentiation of multiple targets in a single assay
3Reliability
If signal amplification is applied, then the detection sensitivity improves, but the background noise increases
Solution Approach 1:
The patent applies local quality by ensuring that the enzyme-catalyzed signal amplification occurs only at the specific locations where primary antibodies have bound to their targets. The oligonucleotide-labeled dye molecules are deposited locally through the enzyme's catalytic activity, creating concentrated signal at target sites while leaving background areas without signal, thus improving signal-to-noise ratio
Solution Approach 2:
The enzyme acts as a localized intermediary that confines the signal amplification reaction to specific target sites. By requiring the presence of both the enzyme conjugate and the oligonucleotide-labeled substrate for signal generation, the system ensures that amplification occurs only where targets are present, minimizing background noise while maximizing detection reliability
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
Achieves high-level multiplexing and enhanced detection of multiple targets by amplifying dye molecules relative to target molecules, enabling clear imaging of weakly expressed antigens.
Implementation Method 1
An enzyme such as horseradish peroxidase (HRP) is conjugated to countersense sequence Si′ and applied to the sample
Implementation Method 2
enzyme-mediated deposition of oligonucleotide sequences onto a sample using the TSA methodology
Implementation Method 3
One or more dyes labeled with countersense oligonucleotide sequences are introduced, and hybridize with the corresponding (i.e., complementary) TSA-deposited oligonucleotide sequences
Implementation Method 4
enhance detection of targets using chromogenic or fluorescent dyes
Data Source
AI summary
Methods for imaging an analyte in a sample include contacting the biological sample with a binding agent, where the binding agent includes a binding moiety that binds to the analyte and a first nucleotide sequence, contacting the biological sample with a catalytic agent, where the catalytic agent includes a second nucleotide sequence linked to an enzyme, and where the second nucleotide sequence hybridizes to the first nucleotide sequence, contacting the biological sample with a localization agent, where the localization agent includes a substrate complementary to the enzyme and a third nucleotide sequence linked to the substrate, and contacting the biological sample with a labeling agent, where the labeling agent includes a fourth nucleotide sequence linked to an optical label, where the fourth nucleotide sequence hybridizes to the third nucleotide sequence.