Nanopore Sequencing Buffer Compositions to Reduce Prussian Blue
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Solution Overview
Problem
Nanopore-based nucleic acid sequencing is burdened by the formation of deleterious side-products like Prussian Blue, which interfere with accurate electrochemical signal detection due to redox reactions involving ferricyanide and ferrocyanide ions and metallic iron components, leading to clogging and decreased assay accuracy.
Innovation Solution
The use of electrochemical cells with nickel alloy inlet and outlet ports, controlled concentrations of ferrocyanide and ferricyanide ions, and specific buffer compositions, along with alternating current application, reduces or prevents Prussian Blue formation, maintaining accurate ion flow and sequencing efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If ferrocyanide and ferricyanide ions are used in the electrochemical cell, then redox reactions can occur for signal detection, but Prussian Blue precipitate forms and clogs the nanopore and ports
Solution Approach 1:
The patent removes iron-containing components (stainless steel ports, iron contamination) from the electrochemical cell system. By extracting the iron source, the redox reactions between ferrocyanide and ferricyanide can proceed without generating Prussian Blue precipitate, thus maintaining signal detection capability while eliminating the harmful precipitation side effect
Solution Approach 2:
The patent converts the potential harm of Prussian Blue formation into a benefit by using non-iron metal ports (nickel, titanium, platinum) that are electrochemically inert in the ferrocyanide/ferricyanide system. This allows the redox reactions to occur cleanly without precipitation, turning a problematic reaction into a reliable signaling mechanism
2Strength
If metal ports are used in the electrochemical cell, then structural integrity and conductivity are maintained, but iron components catalyze Prussian Blue formation
Solution Approach 1:
The patent employs composite material selection for the ports - using metals like nickel, titanium, or platinum that combine structural integrity, electrical conductivity, and electrochemical inertness. These materials maintain the mechanical and electrical functions of metal ports while eliminating the iron content that catalyzes Prussian Blue formation
Solution Approach 2:
The patent changes the material parameter of the ports from iron-containing stainless steel to iron-free metals. This parameter change (compositional substitution) maintains the structural and conductive properties while eliminating the chemical reactivity that leads to Prussian Blue precipitation
3Measurement precision
If high concentrations of ferricyanide and ferrocyanide are used, then signal strength increases, but rate of Prussian Blue formation increases
Solution Approach 1:
By removing iron components from the system, the patent decouples the relationship between signal strength and precipitation rate. High concentrations of ferrocyanide and ferricyanide can be used to maintain strong signals without the iron catalyst that would otherwise accelerate Prussian Blue formation proportionally to concentration
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 solution effectively minimizes Prussian Blue formation, ensuring reliable and precise nanopore-based nucleic acid sequencing by maintaining clear ion flow and reducing port clogging, thereby enhancing assay accuracy and longevity.
Implementation Method 1
the sequence a single-stranded polynucleotide is detected by changes in ionic current flow as the polynucleotide translocates through a nanopore
Implementation Method 2
redox reactions involving ferricyanide and ferrocyanide ions and metallic iron components
Implementation Method 3
formation of deleterious side-products like Prussian Blue
Data Source
AI summary
This application discloses electrochemical cells, nanopore devices, and associated buffer compositions useful for nanopore-based nucleic acid sequencing. Also disclosed are methods for using the electrochemical cells, devices, and compositions in nanopore-based nucleic acid sequencing methods, such as nanopore Sequencing-by-Expansion (Nano-SBX) and nanopore Sequencing-by-Synthesis (Nano-SBS) methods.