Proximity Probe Binding Splint Reduces Background Noise
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Solution Overview
Problem
Conventional proximity ligation assays face limitations in sensitivity and specificity due to high background noise and low detection limits, primarily due to non-specific binding and low affinity of analyte-binding domains, which restricts the detection of analytes to around 6000 molecules.
Innovation Solution
Incorporating a third proximity probe with a nucleic acid domain that acts as a 'binding splint' to mediate the ligation between the first and second probes, reducing non-specific background signal and allowing higher concentrations of probes, thereby enhancing sensitivity and specificity by stabilizing the analyte-probe complex and increasing the detection limit.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional proximity probes are used in pairs with free splint oligonucleotides, then the assay can detect analytes, but the background noise is high and sensitivity is limited to around 6000 molecules
Solution Approach 1:
The patent introduces a third proximity probe that serves as a mediator between the first and second probes. This mediator probe carries a nucleic acid domain that acts as a splint, facilitating the ligation interaction between the other two probes while being tethered to the analyte-binding domain. This intermediary approach reduces background noise by ensuring specific complex formation and improves detection limit to enable sensing of as few as 60 molecules.
2Measurement precision
If higher concentrations of probes are used to improve signal, then detection sensitivity increases, but non-specific binding increases and background noise worsens
Solution Approach 1:
The patent applies local quality by creating a localized interaction complex where the splint oligonucleotide is tethered specifically to the analyte-binding domain of the third probe. This localization ensures that the splint is only available for ligation when the probes are in the correct spatial arrangement bound to the analyte, thereby enabling high probe concentrations without increasing non-specific binding or background noise.
3Ease of manufacture
If the splint oligonucleotide is provided as a free reagent, then the ligation reaction can proceed, but the splint is not localized and causes high background signal
Solution Approach 1:
The patent merges the functions of the splint oligonucleotide and the analyte-binding probe into a single integrated entity. The third proximity probe combines the analyte-binding domain with the nucleic acid domain that serves as the splint, tethering them together. This merging eliminates the need for separate free splint reagents and reduces background signal by ensuring the splint is only present at the site of analyte binding.
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 binding splint approach significantly improves sensitivity, enabling the detection of as few as 60 molecules of analytes like VEGF protein, with a 100-fold improvement in sensitivity compared to traditional assays, and achieving lower detection limits for other biomarkers like PSA and Troponin I.
Implementation Method 1
the nucleic acid domain of said third proximity probe being a splint which is capable of hybridising at least to the nucleic acid domains of said first and second proximity probes
Implementation Method 2
the nucleic acid domains of said first and second proximity probes are conjugatable by means of an interaction mediated by said hybridised splint
Data Source
AI summary
A method for detecting an analyte in a sample, comprising (a) contacting the sample with at least one set of at least first, second and third proximity probes, which probes each include an analyte-binding domain and a nucleic acid domain and can simultaneously bind to the analyte, the nucleic acid domain of the third proximity probe being a splint which is capable of hybridizing at least to the nucleic acid domains of the first and second proximity probes, wherein when all of the at least three proximity probes bind to the analyte, the nucleic acid domains of the first and second proximity probes are conjugatable by means of an interaction mediated by the hybridized splint of the third proximity probe; (b) conjugating the nucleic acids, of the first and second proximity probes; and (c) detecting the conjugation. Also provided is a kit for use in such a method.