Single-Cell Nucleic Acid and Protein Detection via Proximity Extension
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Solution Overview
Problem
Current methods for detecting and quantifying nucleic acids and proteins from single cells are challenging due to the minute amounts of material present, and existing techniques like single molecule detection or mass spectrometry are expensive. The Proximity Extension Assay (PEA) provides sensitivity but requires improvement for practical application.
Innovation Solution
A method involving proximity probes with target binding and interacting segments that hybridize and extend upon binding to nucleic acids, followed by amplification and detection using qPCR, allowing for sensitive detection of nucleic acids and proteins in a single cell sample.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If single molecule detection techniques or mass spectrometry are used for single cell analysis, then detection sensitivity is improved, but cost increases significantly
Solution Approach 1:
The patent uses an intermediary amplification mechanism where target nucleic acids are first amplified using isothermal amplification methods (such as RPA or LAMP) before detection. This amplification step serves as a mediator that converts minute amounts of target material into detectable signals, achieving single-cell sensitivity without requiring expensive single-molecule detection equipment or mass spectrometry
Solution Approach 2:
The patent replaces expensive mechanical/detection systems (single molecule detection techniques and mass spectrometry) with a biochemical amplification system based on isothermal amplification and colorimetric detection. This substitution uses enzymatic reactions and optical detection instead of complex instrumentation, dramatically reducing cost while maintaining detection sensitivity
2Measurement precision
If Proximity Extension Assay is used for protein detection, then detection sensitivity is improved, but the method cannot be applied to single cell analysis due to material limitations
Solution Approach 1:
The patent merges two previously separate methodologies: isothermal nucleic acid amplification and proximity extension assay for protein detection. By combining these methods into a single integrated workflow, the patent enables both nucleic acid and protein analysis from the same single-cell lysate, effectively utilizing the limited material available while achieving high sensitivity for both analyte types
Solution Approach 2:
The patent creates a universal detection platform that can analyze multiple target types (nucleic acids and proteins) from a single sample. The methodology uses universal components such as proximity probes with oligonucleotide tags that can detect both nucleic acid targets directly and protein targets through antibody-conjugated probes, making the system multi-functional and applicable to diverse single-cell analysis needs
3Adaptability or versatility
If bulk samples are divided into portions for separate protein and nucleic acid analysis, then analysis completeness is improved, but this approach cannot be applied to single cells
Solution Approach 1:
The patent segments the detection process into distinct but integrated modules: nucleic acid extraction and amplification, protein extraction and detection via proximity extension assay, and parallel or sequential analysis of multiple targets. This segmentation allows comprehensive analysis of different biomolecule types from limited single-cell material while maintaining the ability to perform complete proteomic and genomic profiling
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 sensitive and cost-effective detection and quantification of nucleic acids and proteins in single cells, facilitating multiplex assays for simultaneous analysis of multiple targets, thereby overcoming the limitations of existing techniques.
Implementation Method 1
the I segment of the first probe hybridizes to the I segment of the second probe to form a duplex comprising the I segments of the first and second probe
Implementation Method 2
The addition of a DNA polymerase results in extension of the hybridized oligonucleotides
Implementation Method 3
amplifying the extended product, or a subregion thereof, in an amplification reaction mixture comprising a pair of amplification primers that amplify the first extended product
Data Source
AI summary
Methods, reagents, and kits for detection and analysis of nucleic acids are provided. The methods can be used in conjunction with a proximity extension assay for protein detection to provide a multiplex assay to detect both nucleic acids (e.g., RNA) and proteins.