Split Enzyme Nanoswitch for Sensitive Biomarker Detection
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for detecting biomarkers in complex biological matrices, such as whole blood, are limited by the need for multiple time-consuming steps, require sophisticated equipment, and lack sensitivity due to the absence of amplification systems in homogeneous assays.
Innovation Solution
An amplification nanoswitch system based on split site-specific cleaving enzymes that integrates molecular recognition and signal generation, using catalytically inactive enzyme fragments that complement to form active enzymes upon target analyte binding, triggering the cleavage of effector molecules to produce a detectable signal.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If homogeneous assays based on nanoswitch machines are used for detection, then the assay can be performed in a single step without multiple incubation steps, but the sensitivity is reduced due to the absence of an amplification system
Solution Approach 1:
The enzyme is divided into two separate catalytically inactive fragments that are conjugated to different protein factors. When the target analyte is detected, the fragments are brought into proximity and complementation occurs, reconstituting enzymatic activity. This segmentation allows the system to maintain the simplicity of homogeneous assays while introducing amplification capability through the complementation event.
Solution Approach 2:
The patent introduces an intermediary amplification system based on enzyme complementation that bridges the gap between simple nanoswitch binding and sensitive detection. The complementation of enzyme fragments serves as an intermediary step that amplifies the signal from target binding, enabling sensitive detection without requiring multiple incubation steps or sophisticated equipment.
2Measurement precision
If sandwich amplification systems with multiple layers of molecules are used, then the sensitivity is improved, but the assay requires labor-intensive and time-consuming incubation or washing steps
Solution Approach 1:
The patent merges molecular recognition and signal generation into a single integrated nanoswitch system. The enzyme fragment conjugates are designed to simultaneously perform target binding and signal amplification through complementation, eliminating the need for separate incubation and washing steps required by traditional sandwich assays.
Solution Approach 2:
The system utilizes self-complementation of enzyme fragments that automatically occurs when the target analyte brings the fragments into proximity. This self-service mechanism eliminates the need for external intervention such as washing steps or additional reagent additions, reducing both time and labor requirements.
3Measurement precision
If protein complementation assays are used for detection, then the signal amplification is achieved, but the assays have been developed only for in vivo protein-protein interaction detection and not for analyte detection in complex matrices
Solution Approach 1:
The patent creates a universal nanoswitch platform that can detect various types of analytes including proteins, peptides, antigens, organic compounds, inorganic compounds, synthetic compounds, and nucleic acids. The enzyme complementation mechanism serves multiple functions: signal amplification, target detection, and adaptation to complex matrices, making the system versatile for different applications.
Solution Approach 2:
The system utilizes changes in enzymatic activity parameters (from inactive to active state) upon complementation to detect target analytes. This parameter change provides a measurable signal that can be detected in complex matrices, extending the applicability of protein complementation assays from in vivo studies to in vitro diagnostic applications.
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
Enables one-step, sensitive detection of target analytes like proteins, peptides, and nucleic acids directly in complex matrices without the need for washing or incubation steps, with enhanced signal intensity through an enhancer module, suitable for high-throughput and point-of-care applications.
Implementation Method 1
at least two catalytically inactive nanoswitch modules, each comprising: at least one split catalytically inactive site-specific cleaving enzyme fragment
Implementation Method 2
wherein upon interaction of one catalytically inactive nanoswitch module with the other catalytically inactive nanoswitch module induced by the presence of the target analyte, the two catalytically inactive nanoswitch modules are carried in proximity, thereby generating an enzymatic activity due to enzyme complementation
Implementation Method 3
at least one binding moiety which is capable to bind a target analyte
Implementation Method 4
at least one split catalytically inactive site-specific cleaving enzyme fragment
Implementation Method 5
at least one effector molecule that contains a cleavage recognition site and a sensor or reporter molecule
Implementation Method 6
wherein the enzymatic activity leads to the cleavage of the effector molecule, wherein the presence of the cleaved effector molecule triggers emission of a detectable and measurable signal
Implementation Method 7
an enhancer module configured to increase signal intensity by increasing the enzymatic activity of the amplification nanoswitch system
Data Source
Figure 1a
Figure 1b
Figure 2a~2d
AI summary
An amplification nanoswitch system based on site-specific cleaving enzymes for the in vitro detection of one or more target molecules in a biological sample and methods for the detection of said one or more targets, in particular antibodies, proteins, peptides, antigens, organic compounds, inorganic compounds, synthetic compounds and nucleic acids.