Affinity Reagent Barcoding for Isothermal Low-Abundance Protein Detection

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Solution Overview

Problem

Detecting low abundance proteins in biological samples is challenging due to dynamic range issues, background noise, and sensitivity limitations of conventional detection methods, especially in complex tissues or cellular environments.

Innovation Solution

An affinity reagent with a binding backbone and barcode oligonucleotide is used, which undergoes isothermal amplification to generate bright fluorescent markers in situ, minimizing heat-related damage and enhancing sensitivity and specificity for low abundance protein detection.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If conventional detection methods are used, then high-abundance proteins can be detected, but low-abundance proteins cannot be visualised due to saturation and background noise

Engineering Contradiction:

Improvedetection sensitivityVSAvoidsignal-to-noise ratio

Core Design Contradiction:

Measurement precisionVSReliability

Solution Approach 1:

The detection method is segmented into distinct functional modules: affinity reagents for specific target binding, barcode oligonucleotides for identification, and isothermal amplification for signal enhancement. This segmentation allows each component to optimize its function independently, improving overall detection sensitivity while maintaining reliability.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

Affinity reagents are pre-bound to target proteins before amplification occurs. This preliminary specific binding ensures that only true target signals are amplified, preventing background noise amplification and maintaining high signal-to-noise ratios while enabling detection of low-abundance proteins.

Inventive Principle:

Principle #10Preliminary action

2Quantity of substance

If PCR-based amplification is used, then signal amplification is achieved, but heat-related damage occurs to the biological sample and target analyte

Engineering Contradiction:

Improvesignal amplificationVSAvoidthermal degradation

Core Design Contradiction:

Quantity of substanceVSObject-affected harmful factors

Solution Approach 1:

The amplification temperature parameter is changed from high-temperature cyclic PCR to constant low-temperature isothermal conditions (37-42°C). This parameter change eliminates thermal degradation of biological samples while maintaining effective signal amplification through enzymes like Bst polymerase that function optimally at these lower temperatures.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The thermal cycling mechanical system of PCR is replaced with an isothermal amplification system using strand-displacing polymerases. This substitution eliminates the need for repeated heating and cooling cycles, preventing heat-related damage to samples while achieving comparable or superior amplification efficiency.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

3Ease of operation

If traditional optical detection is used, then simple imaging is achieved, but sensitivity is insufficient for low-quantity proteins

Engineering Contradiction:

Improvedetection simplicityVSAvoiddetection sensitivity

Core Design Contradiction:

Ease of operationVSMeasurement precision

Solution Approach 1:

The method merges affinity binding, barcode identification, and isothermal amplification into a single integrated workflow that maintains operational simplicity. The combined approach amplifies specific target signals while suppressing background, achieving high sensitivity without significantly increasing procedural complexity.

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

Barcode oligonucleotides serve as intermediaries between the affinity reagent-target complex and the detection system. These barcodes enable specific signal amplification and optical detection, bridging the gap between simple binding events and sensitive quantitative detection while maintaining ease of operation.

Inventive Principle:

Principle #24Intermediary (Mediator)

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 method enables accurate visualization and quantification of low abundance proteins by generating a plurality of bright fluorescent labels, reducing background noise and improving detection sensitivity without thermal degradation.

Implementation Method 1

The barcode oligonucleotide comprises at least one barcode sequence and a set of primer sequences configured for isothermal amplification

Methodology Applied

Scientific EffectIsothermal amplification:

Implementation Method 2

The binding backbone is configured to specifically bind to a target analyte

Methodology Applied

Scientific EffectSpecific binding:

Implementation Method 3

generating barcoded markers comprising a plurality of bright labels, in particular fluorescent labels

Methodology Applied

Scientific EffectFluorescence: Fluorescence

Data Source

PatentEP4617378A1Affinity reagent and method for analysing a biological sample

Publication Date: 2025.09.17 LEICA MICROSYSTEMS CMS GMBH

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AI summary

In a first aspect an affinity reagent (102, 202) for analysing a biological sample is provided. The affinity reagent (102, 202) comprises a binding backbone (108, 204) and a barcode oligonucleotide (110) attached to the binding backbone (108, 204). The binding backbone (108, 204) is configured to specifically bind to a target analyte (106). The barcode oligonucleotide (110) comprises at least one barcode sequence and a set of primer sequences configured for isothermal amplification. In another aspect a method for analysing a biological sample with the affinity reagent (102, 202) is provided.