Thermo-Responsive Microspheres for Nucleic Acid Capture and Identification
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Solution Overview
Problem
There is a need for improved nanoreactor beads that can efficiently capture and/or amplify nucleic acids, are easy to handle, and are suitable for use in multiplex assays involving multiple analytes and/or multiple samples. Additionally, there is a need for beads that can be easily identified and differentiated, especially during digital amplification and detection processes.
Innovation Solution
The development of microspheres comprising a first material that forms a porous hydrogel when exposed to an aqueous solution, with a melting temperature between 40°C and 90°C, and a second material that forms a network within the hydrogel. This second material has a pKa value and precipitates in an aqueous environment at a pH greater than or equal to its pKa value, allowing the network to contract when exposed to a temperature greater than the melting temperature of the first material. The microspheres also include at least one fluorescent label and/or magnetic particles for identification and manipulation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If conventional nanoreactor beads are used, then nucleic acid capture function is provided, but the beads are difficult to handle and identify individually
Solution Approach 1:
The patent applies fluorescent labels to the microspheres that emit light at specific wavelengths when excited. This allows individual microspheres to be easily identified and located using fluorescence microscopy or flow cytometry, directly resolving the contradiction between ease of handling and difficulty of identification.
Solution Approach 2:
The patent introduces magnetic particles as an intermediary component within the microspheres. These magnetic particles can be manipulated using external magnetic fields, providing an additional mechanism for easy handling and positioning of individual microspheres during assays.
2Adaptability or versatility
If conventional beads are used, then capture function is provided, but the beads cannot be easily differentiated in multiplex assays
Solution Approach 1:
The patent employs multiple fluorescent labels with different emission wavelengths (e.g., green, red, yellow fluorescence) to encode different bead types. This color-coding system enables easy differentiation of multiple bead populations in multiplex assays, directly addressing the contradiction between versatility and differentiation capability.
Solution Approach 2:
The patent creates asymmetric fluorescence patterns or combinations of fluorescent labels on different bead surfaces, allowing distinct identification of bead types based on their unique fluorescent signatures, thereby enabling clear differentiation in complex assay conditions.
3Productivity
If thermally responsive materials are used, then concentration and enrichment are achieved, but the network structure becomes complex
Solution Approach 1:
The patent utilizes polymers with lower critical solution temperatures (LCST) that undergo solubility transitions at specific temperatures. By controlling temperature parameters, the polymer network contracts to concentrate and enrich nucleic acids within the microsphere, achieving high productivity without requiring complex network structures.
Solution Approach 2:
The patent employs phase transition behavior of thermally responsive polymers, where the polymer transitions from a solubilized state at low temperatures to a collapsed state at elevated temperatures. This phase transition mechanism enables efficient concentration and enrichment of target nucleic acids through simple temperature cycling, avoiding complex structural designs.
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 microspheres enable efficient binding and capture of nucleic acids, followed by concentration and enrichment within the microsphere. The thermo-responsive behavior and inclusion of fluorescent labels and magnetic particles facilitate easy identification, handling, and differentiation of individual microspheres, even during digital amplification and detection processes.
Implementation Method 1
the first material is capable of forming a porous hydrogel when exposed to or comprising an aqueous solution
Implementation Method 2
the second material has a pKa-value and is capable of precipitating in an aqueous environment at a pH≥ said pKa-value
Implementation Method 3
the network is capable of contracting when exposed to a temperature≥ said melting temperature of said first material
Implementation Method 4
the network is capable of contracting when exposed to a temperature≥ said melting temperature of said first material
Implementation Method 5
said microsphere further comprising at least one of a) a fluorescent label
Data Source
AI summary
The present invention relates to microsphere comprising a first material, preferably a first polymer, and a second material, preferably a second polymer. The present invention also relates to a liquid phase comprising a plurality of such microspheres. Furthermore, the present invention relates to a method for capturing a nucleic acid from a sample and to a method of amplifying a nucleic acid from a sample. The present invention also relates to the use of a plurality of microspheres in an assay for the detection of multiple nucleic acid analytes. Furthermore, the present invention relates to the use of a plurality of microspheres in an assay for the detection of a single nucleic acid analyte in multiple samples.