Molecular Probes with DNA Barcodes for Nanopore Protein Detection
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Solution Overview
Problem
Current protein detection and quantification methods lack the ability to provide ultra-sensitive, multiplexed, and cost-effective analysis of single analytes, particularly in clinical diagnostics, due to limitations in sensitivity, multiplexing capabilities, and high costs associated with existing techniques such as Mass Spectrometry and immunoassays.
Innovation Solution
A method and system utilizing molecular probes with identifier biopolymers, such as DNA barcodes, conjugated to binding molecules, which are translocated through nanopore sensors to generate unique identification signals, enabling highly sensitive and rapid detection and quantification of multiple analytes without the need for amplification steps.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If Mass Spectrometry is used for protein detection, then multiple proteins can be quantified simultaneously, but high abundance requirements and expensive equipment limit its application
Solution Approach 1:
The system segments the detection process into two independent modules: (1) binding molecules (antibodies, aptamers, or DNA binders) that provide analyte-specific recognition, and (2) identifier biopolymers (DNA barcodes with structural elements) that provide detectable signals. This segmentation allows each module to be optimized independently, enabling simultaneous detection of multiple analytes with single-molecule sensitivity.
Solution Approach 2:
The invention introduces an intermediary identifier biopolymer that bridges the binding molecule and the detection system. The biopolymer carries structural elements (hairpins, G-quadruplexes, i-motifs) that generate unique identification signals when translocated through nanopores, while the binding molecule provides specific analyte recognition. This intermediary enables multiplexed detection without compromising sensitivity.
2Measurement precision
If traditional immunoassays are used, then sensitivity is improved, but they are restricted to single analyte detection and have calibration curve dependencies
Solution Approach 1:
The system employs universal identifier biopolymers with structural elements that can be conjugated to different binding molecules, enabling a single detection platform to analyze multiple analytes simultaneously. The structural elements (hairpins, G-quadruplexes, i-motifs) serve universal functions of signal generation, while the binding molecules provide analyte-specific recognition, achieving both sensitivity and multiplexing capability.
Solution Approach 2:
The invention changes the detection parameter from signal intensity (which requires calibration curves) to translocation time of structured biopolymers through nanopores. The structural elements create characteristic time signatures that are inherently quantitative, eliminating calibration curve dependencies while enabling multiplexed detection through distinct temporal patterns.
3Adaptability or versatility
If bead array-based immunoassays are used, then multiplexed analysis is enabled, but sensitivity constraints prevent use in high-multiplex scenarios
Solution Approach 1:
The invention replaces the mechanical/optical bead array system with an electrical nanopore detection system. Instead of measuring light scattering or fluorescence from beads, the system measures ionic current blockades as structured biopolymers translocate through nanopores. This substitution enables single-molecule sensitivity while maintaining high multiplexing capability through the use of diverse structural elements.
4Adaptability or versatility
If nucleic acid-based immunoassays are used, then high multiplexing capabilities are achieved, but sensitivity is limited and they require time-intensive qPCR amplification
Solution Approach 1:
The invention extracts and eliminates the qPCR amplification step from the detection workflow. By using structural elements on biopolymers that generate intrinsic identification signals during nanopore translocation, the system achieves direct detection without amplification. This extraction of the amplification step reduces detection time while maintaining single-molecule sensitivity and high multiplexing capability.
Solution Approach 2:
The invention uses structural conformational changes in biopolymers (formation of hairpins, G-quadruplexes, i-motifs) as detectable signals analogous to color changes. These structural elements create unique translocation time signatures that serve as identification codes, enabling multiplexed detection without requiring amplification or additional reagents.
5Measurement precision
If PEA and NULISA are paired with Next Generation Sequencing, then detection capability is improved, but cost and time intensity make them unsuitable for routine diagnostics
Solution Approach 1:
The invention replaces the complex NGS sequencing workflow with a simpler nanopore-based electrical detection system. Instead of synthesizing and sequencing DNA fragments, the system directly measures ionic current blockades as structured biopolymers pass through nanopores. This substitution maintains detection accuracy through unique structural signatures while dramatically reducing detection time and cost for routine diagnostic applications.
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 achieves single molecule resolution with high multiplexing capacity, providing accurate and cost-effective detection and quantification of proteins, suitable for clinical diagnostics and personalized treatments, without the limitations of existing methods.
Implementation Method 1
The one or more structural element is configured such as to generate a unique identification signal when translocating through a pore of a nanopore sensor
Implementation Method 2
a binding molecule able to bind to a target analyte
Data Source
Figure 1A~1B
Figure 2A~2G
Figure 3A~3E
AI summary
This invention concerns a molecular probe comprising an identifier biopolymer conjugated with a binding molecule able to bind to a target analyte, wherein the identifier biopolymer comprises one or more structural elements having a defined nanostructure, said nanostructure being defined by the primary sequence of the identifier biopolymer, wherein said structural element is configured to generate a unique identification signal when translocating through a nanopore sensor. The invention further concerns a method and a system for detecting and/or quantifying a target analyte in a sample, which uses this molecular probe.