Nanopore Sequencing Ratiometric Impedance Measurement

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Solution Overview

Problem

Current nanopore-based DNA and RNA sequencing technologies face challenges in achieving accurate and reliable sequence determination at single-nucleotide resolution and high-speed sequencing with nano-scale dimensions.

Innovation Solution

A device comprising three fluidic regions with a nanopore connecting them, using drive electrodes to transport molecules through the nanopore and measuring potential differences to detect the presence of molecules, along with a polymerase enzyme and nucleotide analogs with current blockade labels for real-time sequencing.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If nanopores are used for DNA sequencing, then sequence information can be detected, but single-nucleotide resolution and sequencing accuracy are insufficient

Engineering Contradiction:

Improvesingle-nucleotide resolutionVSAvoidsequencing accuracy

Core Design Contradiction:

Measurement precisionVSReliability

Solution Approach 1:

A polymerase enzyme is introduced as an intermediary component that binds to the nanopore and processes nucleic acid molecules. The polymerase contains a nucleotide-binding site where incoming nucleotides are incorporated, and this binding event causes a detectable change in ionic current through the nanopore, enabling single-nucleotide resolution detection

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent replaces traditional mechanical or optical sequencing methods with an electrical detection system. By measuring changes in ionic current through the nanopore as nucleotides are incorporated by the polymerase, the system achieves high-resolution sequencing through electrical signal detection rather than mechanical manipulation or optical imaging

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

2Productivity

If nanopore sequencing is implemented, then sequence determination is possible, but sequencing speed is limited

Engineering Contradiction:

Improvesequencing speedVSAvoidsequence determination accuracy

Core Design Contradiction:

ProductivityVSMeasurement precision

Solution Approach 1:

The polymerase enzyme remains bound to the nanopore throughout the sequencing process, continuously processing nucleic acid molecules as they pass through. This continuous configuration allows for rapid sequential detection of multiple nucleotides without repeated positioning or reconfiguration, maintaining both high speed and accuracy

Inventive Principle:

Principle #20Continuity of useful action

Solution Approach 2:

The polymerase enzyme is pre-positioned at the nanopore with its active site ready to accept nucleotides. This preliminary configuration eliminates the need for dynamic positioning during sequencing, allowing immediate detection of nucleotide incorporation events as molecules pass through the pore

Inventive Principle:

Principle #10Preliminary action

3Ease of manufacture

If traditional nanopore devices are used, then basic sequencing is possible, but device fabrication and manufacturing are difficult

Engineering Contradiction:

Improvedevice fabricationVSAvoidnanopore device structure

Core Design Contradiction:

Ease of manufactureVSDevice complexity

Solution Approach 1:

The patent integrates multiple functions into a single nanopore device structure: the nanopore serves as both the detection channel for ionic current and the binding site for the polymerase enzyme. This multi-functional design simplifies fabrication by eliminating the need for separate components for molecule transport, enzymatic reaction, and signal detection

Inventive Principle:

Principle #6Universality (Multi-functionality)

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 accurate and high-resolution sequencing of nucleic acids by detecting current blockages through the nanopore, allowing for real-time identification of nucleotide incorporation and sequence determination.

Implementation Method 1

a pair of drive electrodes, whereby a potential drop across the drive electrodes drives a sample molecule into the nanopore from the sample fluidic region

Methodology Applied

Scientific EffectElectrophoresis: Electrophoresis

Implementation Method 2

the presence of a sample molecule within the nanopore is detected using potential measurements made by the measurement electrode

Methodology Applied

Scientific EffectElectrical Resistance: Electrical Resistance

Data Source

PatentUSRE47067E1Nanopore sequencing using ratiometric impedance

Publication Date: 2018.10.02 PACIFIC BIOSCIENCES OF CALIFORNIA INC

AI summary

The invention relates to devices and methods for nanopore sequencing. The invention includes arrays of nanopores having incorporated electronic circuits, for example, in CMOS. The invention includes devices having sample and reference pores connecting sample, measurement and reference chambers, wherein potential measurements in each chamber is used to provide an accurate determination of current through a sample nanopore, improving nanopore sequencing.