Digital Protein Detection via Compartmentalized Isothermal Amplification
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Solution Overview
Problem
Existing digital protein assays face limitations in efficiency, accuracy, and the need for calibration, particularly in analog methods, and there is a lack of effective isothermal amplification techniques that do not require washing steps.
Innovation Solution
The development of digital isothermal amplification methods, such as digital PCR, that perform assays in a single container without washing steps, utilizing compartmentalized fluid volumes and proximity-induced interactions between nucleic acid probes to trigger amplification reactions, allowing for optical detection of proteins in a homogeneous solution.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If digital PCR amplification is used, then measurement precision is improved, but device complexity increases
Solution Approach 1:
The fluid is divided into a plurality of compartmentalized fluid volumes (droplets or wells), where each compartment acts as an independent reaction chamber. This segmentation enables digital counting of analyte molecules while simplifying the overall detection system by avoiding complex calibration mechanisms.
Solution Approach 2:
Nucleic acid probes serve as intermediaries that bridge the analyte (protein) and the detection system. The probes undergo proximity-induced interactions when bound to the analyte, triggering amplification reactions that generate optical signals, thereby simplifying the direct detection of proteins.
2Productivity
If washing steps are eliminated, then productivity is improved, but measurement precision may worsen
Solution Approach 1:
The assay is designed so that all necessary reagents (probes, nucleic acids, enzymes) are pre-loaded into the compartmentalized volumes before the assay begins. This preliminary preparation eliminates the need for subsequent washing steps, maintaining both productivity and measurement precision through the robust compartmentalized architecture.
Solution Approach 2:
The compartmentalized system inherently separates positive and negative reactions into different droplets or wells, allowing the assay to self-differentiate results without requiring external washing steps to remove unbound reagents. Each compartment independently processes its reaction.
3Use of energy by moving object
If isothermal amplification is used, then energy consumption is reduced, but manufacturing precision becomes more difficult
Solution Approach 1:
The assay transitions from thermal cycling (PCR) to isothermal conditions by changing the temperature parameter from variable to constant. This simplifies the manufacturing process and reduces energy consumption while maintaining amplification efficiency through optimized enzymatic reactions that proceed at a single temperature.
4Ease of operation
If homogeneous solution is used, then ease of operation is improved, but reliability may worsen due to lack of solid support
Solution Approach 1:
The homogeneous solution is divided into compartmentalized volumes that provide physical separation and containment. This segmentation maintains the ease of operation of homogeneous assays while improving reliability by preventing cross-contamination and enabling precise control of reaction conditions in each compartment.
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
These methods provide accurate, efficient, and robust protein detection without the need for calibration or washing, suitable for point-of-care diagnostics and personalized medicine, and can be performed in a single step using isothermal conditions.
Implementation Method 1
An optical signal is triggered by a proximity-induced interaction in the analyte-containing volumes involving the analyte and a constituent of the compartmentalized volume
Implementation Method 2
the amplification reaction is an isothermal amplification reaction
Implementation Method 3
The optical signal can be a fluorescence signal triggered by the amplification reaction in the analyte-containing volumes
Data Source
AI summary
The present disclosure provides a method of measuring the quantity of analyte molecules. In some aspects, the method comprises compartmentalizing a sample with binding molecules conjugated to synthetic nucleic acid molecules such that the interaction of the binding molecules with the analyte molecules brings the nucleic acid molecules into proximity. Proximity triggers reactions that result in an optical signal, such as fluorescence, in analyte-containing compartments which can be counted to determine the quantity of analyte present.