Encoded Nanopore Sensor Multiplex Nucleic Acid Detection
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Solution Overview
Problem
Current nanopore detection systems face challenges in accurately and efficiently detecting multiple miRNAs simultaneously, which is necessary for specific disease diagnostics, as they require sensitive, speedy, and cost-effective methods for multiplex analysis.
Innovation Solution
The use of sets of probe molecules with capture domains complementary to target nucleic acids and polymer labels of different lengths, allowing for independent detection of multiple target nucleic acids in a nanopore system by generating distinct signature conductance blocks when hybridized and subjected to an applied voltage.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional nanopore detection systems are used for multiplex miRNA detection, then the system structure remains simple, but the detection precision and ability to differentiate multiple targets simultaneously deteriorates
Solution Approach 1:
The probe molecule is segmented into distinct functional domains: a capture domain for target recognition and a terminal extension with polymer label for detection. This segmentation allows each domain to perform its specific function independently, enabling multiplex detection while maintaining probe simplicity
Solution Approach 2:
Different regions of the probe molecule are assigned different properties - the capture domain has sequence specificity for target binding, while the terminal extension contains polymer labels with distinct physical properties (hydrophilicity, length) that generate unique conductance signatures. This local differentiation enables multiplex detection precision
2Adaptability or versatility
If multiple probe molecules with different polymer labels are used for multiplex detection, then the ability to detect and differentiate multiple target nucleic acids improves, but the complexity of probe design and manufacturing increases
Solution Approach 1:
The probe design uses universal structural elements (capture domain with complementarity sequence, terminal extension) that can be combined with different polymer labels to detect multiple targets. This modular universality allows the same basic probe structure to serve multiple detection functions
Solution Approach 2:
The polymer labels differ in physical parameters such as hydrophilicity and length, which directly affect their interaction with the nanopore and generate distinct conductance signatures. By changing these physical parameters rather than the fundamental probe structure, manufacturing remains relatively simple
3Reliability
If polymer labels with distinct properties are attached to probe molecules, then the signal differentiation and detection sensitivity improve, but the complexity of data analysis and signature interpretation increases
Solution Approach 1:
The polymer labels generate distinct electrical conductance signatures analogous to different colors in optical detection. Each polymer label (varying in hydrophilicity and length) produces a characteristic conductance block pattern that serves as a unique identifier for the corresponding target nucleic acid, simplifying signal interpretation
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, selective, and direct detection and differentiation of distinct single-strand oligonucleotides, including miRNAs, in a multiplex format, facilitating non-invasive and cost-effective early diagnosis and continuous monitoring of markers in patients' samples.
Implementation Method 1
subjected to an applied voltage in a nanopore system by an unzipping process... drive translocation of the hybridized probes and target nucleic acids through a nanopore
Implementation Method 2
Individual target molecules passing through the pore characteristically block the pore conductance, resulting in a signature for both target identification and quantization
Data Source
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AI summary
The present invention provides a new and improved multiplexed oligonucleotide detection method based on the nanopore technology with one or more probes containing a sequence with complementarity to the target oligonucleotide, a terminal extension at the probe's 3' terminus, 5' terminus, or both termini and a label attached to the terminus. The improved probes and probe sets enable sensitive, selective, and direct multiplex detection, differentiation and quantification of distinct target oligonucleotides such as miRNAs. The inventive detection method may also be employed as a non-invasive and cost-effective diagnostic method based on miRNA levels in the patient's tissue sample.