Analyte Concentration Determination via Single-Molecule Partitioning
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current analyte detection methods are limited by requiring large numbers of molecules to generate a measurable signal, leading to reduced sensitivity and dynamic range, and are prone to false-positive signals due to ensemble responses.
Innovation Solution
The method involves partitioning analyte molecules across a plurality of reaction vessels, determining the presence or absence of analyte in each vessel, and calculating the concentration using Poisson or Gaussian distribution analysis, allowing for the detection of individual molecules and improving sensitivity and accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If amplification procedures are used to increase the number of reporter molecules, then the signal becomes measurable, but the sensitivity and dynamic range are reduced due to ensemble responses
Solution Approach 1:
The invention partitions the sample into a large number of small reaction vessels (e.g., microwells), each containing a small volume (e.g., 1-100 fL). This segmentation allows individual analyte molecules to be isolated in separate vessels, enabling single-molecule detection. By distributing the sample across many partitioned units, the system achieves high sensitivity without requiring ensemble responses from large numbers of molecules.
2Reliability
If ensemble responses are used for detection, then the signal is above the detection threshold, but false-positive signals occur and accuracy decreases
Solution Approach 1:
By partitioning the sample into individual reaction vessels, the invention eliminates false-positive signals that arise from ensemble averaging. Each vessel independently reports the presence or absence of analyte molecules, allowing for more reliable detection. The digital readout format (positive/negative per vessel) provides clear, unambiguous results without the interference of background noise that plagues ensemble-based methods.
Solution Approach 2:
The invention employs statistical analysis (Poisson or Gaussian distribution) of the distribution of analyte molecules across partitioned vessels to determine concentration. This feedback mechanism uses the known statistical properties of molecular distribution to accurately calculate analyte concentration from the fraction of positive vessels, improving measurement accuracy and reliability.
3Measurement precision
If partitioning into small samples is performed to detect individual molecules, then sensitivity and dynamic range improve, but the complexity of the assay procedure increases
Solution Approach 1:
The invention pre-partitions the reaction vessels into a large array format before sample addition. This preliminary preparation of the partitioned system allows for straightforward sample distribution and simplifies the overall assay procedure. By having the partitioning infrastructure ready in advance, the method avoids complex real-time partitioning operations while still achieving single-molecule detection capability.
Data Source
AI summary
Disclosed is a method for measuring the concentration of an analyte or analytes in a solution. Although the methods can be conducted using a number of different assay formats, in one embodiment, the assays are conducted in reaction vessels defined, at least in part, by the distal ends of fiber optic strands.