DNA Nanodevice Signal Amplification for Multiplex Biomolecule Detection
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Solution Overview
Problem
Current methods for detecting and quantifying biomolecules, particularly in single-cell analysis, face limitations in sensitivity, throughput, and multiplexity, with technologies like mass cytometry struggling to profile low-abundance proteins and transcription factors due to sensitivity constraints.
Innovation Solution
The method involves generating concatemers of nucleic acid sequences on target-binding ligands, which are then hybridized with labeled probes, and cross-linked to amplify the signal, enabling detection with single-molecule sensitivity using techniques such as mass cytometry and flow cytometry.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If mass cytometry is used to detect proteins, then multiplex capability is improved, but sensitivity deteriorates for low-abundance proteins
Solution Approach 1:
The detection system is segmented into multiple independent channels, each detecting a specific protein target with its own antibody-conjugate-probe complex. This allows simultaneous multiplexed detection while maintaining high sensitivity for each individual target through dedicated signal amplification in each channel.
Solution Approach 2:
The patent implements a nested structure where antibodies are conjugated to oligonucleotide primers, which are then extended to form concatemers that serve as templates for probe hybridization. This nested arrangement (antibody→primer→concatemer→probe) enables signal amplification while maintaining the ability to detect multiple targets simultaneously.
2Device complexity
If conventional antibody labeling is used, then simplicity is maintained, but signal amplification is insufficient for single-molecule detection
Solution Approach 1:
The method performs preliminary actions by conjugating oligonucleotide primers to antibodies before the detection step. This pre-prepared antibody-primer conjugate then undergoes template-directed extension to form concatemers, which subsequently serve as templates for probe hybridization. This preliminary conjugation enables signal amplification without adding complexity to the actual detection step.
Solution Approach 2:
The patent introduces an intermediary system where oligonucleotide primers and their extended concatemer forms act as mediators between the antibody (which binds the target) and the detectable probe signal. This intermediary mechanism enables signal amplification while keeping the antibody-probe interaction simple and specific.
3Measurement precision
If single-cell analysis methods are used, then resolution is improved, but throughput deteriorates
Solution Approach 1:
The patent uses template-directed DNA extension to create multiple copies (concatemers) of the oligonucleotide primer sequence attached to each antibody. These copied sequences then serve as multiple binding sites for detectable probes, amplifying the signal from each single-cell target without requiring analysis of multiple cells, thus maintaining single-cell resolution while improving detectability.
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 approach enhances the sensitivity and multiplex capability of single-cell analysis, allowing for the detection of low-abundance proteins and transcription factors, and improves the resolution and throughput of methods like mass cytometry and flow cytometry.
Implementation Method 1
contacting the conjugated oligonucleotide primer with a single-stranded extender template and a template-dependent nucleic acid polymerase under conditions permitting hybridization and extension
Implementation Method 2
hybridization and extension of the oligonucleotide primer using the single-stranded extender template
Implementation Method 3
template-dependent nucleic acid polymerase under conditions permitting hybridization and extension
Implementation Method 4
extending the oligonucleotide primer to comprise a concatemer of sequence comprised by the oligonucleotide primer
Implementation Method 5
the repeated sequences provide hybridization sites for a plurality of labeled nucleic acid probes per target-binding ligand molecule
Implementation Method 6
cross-linking the hybridized probe nucleic acid molecules to the extended oligonucleotide primer
Data Source
AI summary
The methods, compositions and kits described herein provide signal amplification approaches for the detection of target biomolecules that significantly increase the sensitivity of detection using target-binding ligand molecules. The methods include cyclic addition of nucleic acid repeats to, e.g., target-binding molecules, thereby providing multiple landing pads for labeled probes. The methods are well-suited to performance in multiplex, thereby permitting the sensitive detection of multiple targets in a single assay. These methods and compositions can be applied to, among other things, imaging, flow cytometry, and mass cytometry/Cy TOF. providing additional tools for research and diagnostic purposes.