Enzyme Translocator Sequencing With Electroactive Base Detection
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Solution Overview
Problem
Existing nucleic acid sequencing techniques require significant rebuilding of sequence from small reads or repeated runs to achieve fidelity, lacking single base resolution.
Innovation Solution
A system with immobilized translocating proteins positioned between electrodes, utilizing a dielectric member to create a sensing zone for electroactive molecule interaction, enabling electron transfer through an electroactive label covalently bonded to nucleotides, and detecting current versus time to determine nucleotide presence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional sequencing techniques are used, then sequence fidelity can be achieved through repeated runs, but the process requires significant rebuilding of sequence from small reads and multiple runs
Solution Approach 1:
The sensing zone is divided into two electrodes separated by a dielectric member, creating distinct functional regions for electron transfer detection. This segmentation enables precise measurement of individual nucleotide events without requiring complex signal processing from multiple runs
Solution Approach 2:
An electroactive label covalently bonded to each nucleotide serves as an intermediary that facilitates electron transfer between the two electrodes. This mediator enables direct detection of nucleotide identity and position, achieving single-base resolution fidelity without repeated sequencing runs
2Productivity
If electroactive labels with electron transfer capability are used, then rapid electron transfer and accurate sequence determination are achieved, but the system requires precise control of translocation speed and positioning
Solution Approach 1:
The translocating protein is pre-immobilized on the dielectric member in a specific orientation and position, ensuring that nucleotides are presented to the sensing zone at the correct location. This preliminary positioning eliminates the need for complex real-time control during translocation
Solution Approach 2:
The translocating protein naturally maintains a constant translocation rate as it moves the polynucleotide strand through the sensing zone. This self-regulating mechanism provides inherent speed control without requiring external feedback systems, enabling rapid and accurate sequencing
3Measurement precision
If a dielectric member with immobilized proteins is used to create a sensing zone, then single base resolution is achieved, but the device requires precise positioning between electrodes
Solution Approach 1:
The dielectric member with immobilized translocating proteins is positioned within the electrode assembly, creating a nested structure where the sensing zone is contained within the electric field region. This nesting ensures that the critical measurement region is automatically positioned within the electrodes without requiring separate alignment steps
Solution Approach 2:
The dielectric member creates a localized sensing zone with unique electrical properties between the two electrodes. This local region has enhanced electron transfer capability and is specifically optimized for nucleotide detection, achieving single-base resolution through localized measurement rather than requiring precision across the entire device
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 single base resolution nucleic acid sequencing by controlling translocation speed and reducing fabrication steps, achieving rapid electron transfer and accurate sequence determination.
Implementation Method 1
The dielectric member positioned between the first and second electrodes creates a sensing zone allowing an electroactive molecule to interact with both the first and the second electrodes to complete an electrical circuit
Implementation Method 2
Directing current through the first electrode and the second electrode and holding the first electrode at a first voltage and the second electrode at a second voltage enables electron transfer via an electroactive label covalently bonded to a nucleotide
Implementation Method 3
Each of the two or more proteins captures a polynucleotide strand, brings the polynucleotide strand within the sensing zone, and translocates the polynucleotide strand across the sensing zone at a constant rate one nucleotide at a time
Implementation Method 4
Once the two or more proteins are exposed to a sample including the polynucleotide strand, current versus time of the first electrode and of the second electrode is detected to determine when the nucleotide with the electroactive label is within the sensing zone
Data Source
AI summary
Systems, devices, and methods for nucleic acid sequencing are provided. A dielectric member with multiple attached translocating proteins positioned between a first and a second electrode creates a sensing zone allowing an electroactive molecule to interact with both electrodes to complete an electrical circuit. Each of the multiple proteins captures a polynucleotide strand, brings the polynucleotide strand within the sensing zone, and translocates the polynucleotide strand across the sensing zone at a constant rate one nucleotide at a time. Directing current through the first electrode and the second electrode and holding the first electrode at a first voltage and the second electrode at a second voltage enables electron transfer via an electroactive label covalently bonded to a nucleotide. Current versus time measurements of the first electrode and of the second electrode are detected to determine when a nucleotide with an electroactive label is within the sensing zone.