Nanopore Analyte Detection via Fluorescence
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Solution Overview
Problem
Current methods for detecting small biomolecules, such as proteins or peptides, face challenges in providing accurate, efficient, and high-throughput measurements due to limitations in kinetic information, nonspecific binding, and spatial resolution, especially when using nanopore sensing and optical techniques.
Innovation Solution
A method combining nanopore sensing with single-molecule fluorescence spectroscopy, utilizing carrier nucleic acid molecules and detection elements with fluorophores and quenchers, allows for simultaneous electrochemical and optical measurements to detect analytes by monitoring time-dependent current responses and fluorescence emissions, providing synchronized signals indicative of analyte binding.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If nanopore sensing is used for detecting small biomolecules, then label-free detection is achieved, but spatial resolution and capture rate are insufficient
Solution Approach 1:
The patent combines nanopore sensing with single-molecule fluorescence spectroscopy into a unified detection system. The nanopore provides label-free detection capability while the fluorescence component adds spatial resolution through optical detection. The carrier molecule serves as a common platform that links both detection modalities, allowing simultaneous electrochemical and optical measurements of the same molecular event.
2Reliability
If conventional single molecule FCS is used, then real-time characterization is achieved, but spatial resolution is limited due to three-dimensional diffusion
Solution Approach 1:
The patent transitions from three-dimensional diffusion-based detection to one-dimensional translocation through a nanopore. By confining molecular motion to a linear path through the pore, the system achieves precise spatial positioning along the translocation axis while maintaining real-time detection capability. The nanopore acts as a spatial filter that restricts diffusion and enhances positional information.
3Measurement precision
If labelled methods are used for detection, then sensitivity is improved, but nonspecific binding interference increases
Solution Approach 1:
The patent introduces a carrier molecule as an intermediary that indirectly detects the target analyte. Instead of directly labeling the target molecule, the carrier binds to the target and then translocates through the nanopore, where its translocation event is detected. This indirect detection approach maintains sensitivity while reducing nonspecific binding interference, as the carrier serves as a standardized detection vehicle.
4Productivity
If smaller nanopore size is used, then capture rate increases, but translocation speed increases making detection more difficult
Solution Approach 1:
The patent employs fluorescent labeling on the carrier molecule to continuously monitor translocation events in real-time. The fluorescence signal provides continuous information about the position and state of the carrier during translocation, allowing detection even at higher speeds. This continuous optical monitoring complements the electrochemical signal and maintains detection capability despite increased translocation velocity.
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 detection of small biomolecules by reducing false positives, improving spatial resolution, and enabling multiplex detection without the need for labeling, thus providing accurate and efficient analysis of biomolecular interactions.
Implementation Method 1
irradiating the nanopore with radiation that excites the fluorophore and monitoring radiation emissions of the fluorophore over time
Implementation Method 2
at least one quencher that modifies spectroscopic detection of the fluorophore; in the absence of the analyte the fluorophore is quenched by the fluorescence quencher
Implementation Method 3
translocating the carrier nucleic acid/detection element/analyte complex through the nanopore via voltage-driven translocation and monitoring time-dependent current response
Data Source
AI summary
The invention relates to methods of detecting and/or quantifying analytes in a sample, as well as methods of detecting mutations and/or polymorphisms in nucleic acid molecules. The methods include: providing at least one carrier nucleic acid molecule comprising at least one single-stranded region; providing at least one detection element comprising: at least one fluorophore, at least one fluorescence quencher that quenches spectroscopic detection of the fluorophore; at least one analyte-binding moiety; and at least one nucleic acid moiety that binds to a single stranded region on the carrier nucleic acid molecule; wherein the detection element is configured such that in the absence of the analyte the fluorophore is quenched by the fluorescence quencher and upon analyte binding to the analyte-binding moiety fluorescence is restored; binding these with an analyte to form a complex; translocating the complex through a nanopore via voltage-driven translocation and monitoring time-dependent current response; irradiating the nanopore with radiation that excites the fluorophore and monitoring radiation emissions of the fluorophore over time; and comparing the signals from time-dependent current response and emission over time.