Droplet Storage on Structured Substrates to Prevent Back Pressure
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Solution Overview
Problem
Existing methods for sequencing polynucleotides face limitations in speed and computational complexity due to the need for parallel processing of short fragments and potential errors, and previous droplet storage solutions experience back pressure issues with longer polynucleotides, restricting the flow of microdroplet streams.
Innovation Solution
A method involving the sequential storage of droplets on a substrate with unique locations, using a droplet delivery system and a substrate with structured surfaces to hold microdroplets containing single nucleotides or oligonucleotides, generated through pyrophosphorolysis or exonucleolysis, to prevent coalescence and evaporation, and facilitate chemical and biological transformations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If droplets are stored in a continuous stream through a chamber, then the droplets can be maintained in flow, but back pressure builds up that restricts further flow when storing large numbers of droplets
Solution Approach 1:
The continuous chamber is segmented into discrete storage locations (wells, dimples, or recesses) that individually hold droplets. This segmentation allows droplets to be stored in separate compartments rather than a continuous flow path, eliminating back pressure buildup while maintaining the ability to store large numbers of droplets.
Solution Approach 2:
The storage system transitions from a one-dimensional continuous flow chamber to a two-dimensional array of discrete locations on a substrate surface. This dimensional change allows parallel storage of numerous droplets without creating flow restrictions, as each droplet occupies its own unique location in the array.
2Reliability
If droplets are stored on a substrate surface, then back pressure is prevented and droplet integrity is maintained, but the substrate requires structured surfaces with unique locations to hold each droplet
Solution Approach 1:
The substrate surface is engineered with localized structural features (wells, dimples, or recesses) at specific locations to hold droplets. Each location has the precise local geometry needed to contain a single droplet, providing high droplet integrity while keeping the overall substrate design relatively simple and manufacturable.
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 allows for efficient storage and analysis of millions of microdroplets by preventing back pressure and maintaining droplet integrity, enabling accurate sequencing and reducing computational complexities.
Implementation Method 1
preventing coalescence and evaporation
Implementation Method 2
storing droplets on the surface of a substrate
Implementation Method 3
generating an ordered stream of single nucleotides from the analyte by progressive pyrophosphorolysis or exonucleolysis
Implementation Method 4
generating an ordered stream of single nucleotides from the analyte by progressive pyrophosphorolysis or exonucleolysis
Implementation Method 5
a single-stranded first oligonucleotide can be bound to both ends of its compliment by hybridisation to the unpaired adenine base
Implementation Method 6
each of these single-stranded fragments is primed and its complimentary strand recreated by extension using the polymerase chain reaction
Implementation Method 7
Each ddNTP type is end-blocked with a moiety which is labelled with a different fluorophore fluorescing at a different wavelength
Implementation Method 8
each nucleotide base (optionally labelled) is in turn induced to fluoresce or Raman-scatter photons in a characteristic way by interaction with incident light
Implementation Method 9
A potential difference is then applied across the electrodes and changes in the electrical characteristics of the ionic medium flowing therebetween, as a consequence of the electrophoretic translocation of the polynucleotide and associated electrolyte through the nanopore
Data Source
AI summary
A method of storing a stream of droplets at least some of which comprise one or more single nucleotides and/or oligonucleotides, and a droplet fluid is provided. It is characterized by the step of introducing each droplet sequentially onto a surface of a substrate at a corresponding unique location and further characterized in that the stream of droplets is prepared by a process which includes the steps of generating an ordered stream of nucleotides from the analyte by progressive pyrophosphorolysis or exo nucleolysis and capturing each nucleotide in a corresponding droplet. The method can advantageously be used in association with microdroplet droplet sequencers and an analysis unit in which the sequence of nucleotides in a precursor polynucleotide analyte is determined using fluorescence spectroscopy. A device for carrying out the method is also described.