Oligonucleotide-Coded Probes for 1000-Target Protein Quantification
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Solution Overview
Problem
Existing immunohistochemical methods are limited to the simultaneous detection of six to ten protein targets, with three to four being typical, and in situ hybridization methods are limited to fewer than ten nucleic acid targets, necessitating a need for improved probes and methods for multiplexed detection and quantification of protein and nucleic acid expression in user-defined regions of tissues, cells, and subcellular structures.
Innovation Solution
The development of probes and methods that allow for the simultaneous, multiplexed detection and quantification of multiple protein and nucleic acid targets by contacting cells or tissues with probes containing target-binding domains and signal oligonucleotides, applying a force to release the oligonucleotides, and collecting and identifying them to detect specific locations within user-defined regions, using techniques such as nCounter® systems and digital mirror devices for high-resolution imaging and signal collection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If standard immunohistochemical methods are used, then detection of protein targets is achieved, but the number of simultaneously detectable targets is limited to six to ten
Solution Approach 1:
The detection system is segmented into multiple independent probe sets, each targeting specific protein markers. Each probe contains a unique oligonucleotide sequence that can be individually detected and quantified, allowing simultaneous measurement of many targets without increasing overall method complexity
Solution Approach 2:
Oligonucleotide sequences serve as intermediaries between protein targets and detection systems. Probes conjugate oligonucleotides to antibody targets, enabling indirect detection that allows multiplexing beyond the limitations of direct fluorescent labeling while maintaining quantification capability
2Measurement precision
If standard immunohistochemical methods are used, then protein detection is achieved, but quantification precision and dynamic range are limited
Solution Approach 1:
The system replaces traditional optical fluorescence detection with oligonucleotide-based detection and counting. This substitution enables digital quantification with superior precision and a dynamic range of at least 5 logs, while maintaining high throughput through automated analysis
Solution Approach 2:
The invention creates digital copies of protein target information through oligonucleotide sequences. Each detected protein target is represented by a specific oligonucleotide sequence that can be copied, counted, and quantified digitally, enabling precise measurement without the limitations of analog fluorescent signal detection
3Quantity of substance
If multiplexed detection of 1000 targets is achieved, then detection capability is improved, but probe and method complexity increases
Solution Approach 1:
The system uses oligonucleotide sequence parameters (unique base sequences) to differentiate between thousands of targets. By changing the nucleotide sequence parameter rather than using complex protein structures or large molecules, the invention enables high multiplexing with relatively simple probe construction and manufacturing
Solution Approach 2:
The probe design uses universal components (oligonucleotide sequences, antibody conjugation methods, detection protocols) that can be applied across all 1000+ targets. This universal approach allows the same basic methodology to detect any number of targets by simply changing the specific oligonucleotide sequences used, rather than requiring unique complex procedures for each target
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
Enables the detection and quantification of up to 1000 protein or nucleic acid targets with high precision and reliability, allowing for comparisons across multiple centers and providing a linear dynamic range of greater than or equal to 5 logs, surpassing the limitations of standard methods.
Implementation Method 1
a probe comprising a target-binding domain and a signal oligonucleotide
Implementation Method 2
providing a force to a location of the tissue sample sufficient to release the signal oligonucleotide
Data Source
AI summary
The present invention relates to, among other things, probes, compositions, methods, and kits for simultaneous, multiplexed detection and quantification of protein expression in a user-defined region of a tissue, user-defined cell, and/or user-defined subcellular structure within a cell.