Self-Immolative Enzyme Probes for High Signal-to-Background Detection
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Solution Overview
Problem
There is a need for self-immolative signalogenic markers with a high signal-to-background ratio that are sensitive to specific enzyme or analyte triggers, as existing probes are limited in their ability to detect intracellular organic biomolecules effectively.
Innovation Solution
The development of self-immolative probes comprising a signaling molecule (SIG) covalently bound to a self-immolative structure (SI) and a modifiable moiety (MOD), which upon activation by an enzyme or analyte, undergoes destabilization and self-cleavage, releasing SIG to generate a significant signal.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If self-immolative probes are used to detect enzyme activity, then signal enhancement is achieved, but background signal remains problematic
Solution Approach 1:
The probe is divided into three functional segments: a signaling molecule (SIG) that generates the detectable signal, a self-immolative structure (SI) that acts as a cleavable linker, and a modifiable moiety (MOD) that interacts with the target analyte. This segmentation allows the signaling molecule to be separated from the background-generating components upon enzymatic cleavage, thereby enhancing signal-to-background ratio
Solution Approach 2:
The self-immolative structure serves as a removable component that is extracted from the signaling molecule through enzymatic cleavage. Once the modifiable moiety interacts with the target enzyme or analyte, the self-immolative structure spontaneously decomposes and releases the signaling molecule, effectively taking out the background-generating components and leaving only the active signal
2Reliability
If existing probes are used for intracellular biomolecule detection, then detection capability is limited, but probe complexity can be reduced
Solution Approach 1:
The probe is pre-configured with the self-immolative structure and modifiable moiety designed to specifically interact with target enzymes or analytes. This preliminary design ensures that upon encountering the target, the probe automatically undergoes the cleavage and signal release sequence without requiring additional activation steps, thereby enhancing detection sensitivity while maintaining manageable complexity
Solution Approach 2:
The self-immolative structure performs the function of automatic self-decomposition and signal release without external intervention. After the modifiable moiety binds to the target analyte or enzyme, the self-immolative structure spontaneously undergoes intramolecular reactions to release the signaling molecule, enabling the probe to service its own activation and detection function
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 probes enhance signal intensity upon exposure to specific enzymes or analytes, providing sensitive and accurate detection of nitroreductase activity, hypoxia, and microorganisms, with applications in enzyme detection, hypoxia detection, and microorganism identification.
Implementation Method 1
when MOD is modified by an activator, SI is destabilized and self-cleaved from SIG such that SIG generates an increased signal
Data Source
AI summary
Provided is a compound comprising the structure:(SIG)-(SI-MOD)m.In this compound, SIG is a signaling molecule, SI is a self-immolative structure bound to SIG such that SIG has a reduced signal relative to the signal of SIG without SI, MOD is a moiety bound to SI that is subject to modification by an activator, and m is an integer from 1 to about 10. With this compound, when MOD is modified by an activator, SI is destabilized and self-cleaved from SIG such that SIG generates an increased signal. Also provided is a method of determining whether a sample comprises an activator, using the above-described compound. Additionally provided is a method of determining whether a cell comprises a nitroreductase using the above-described compound where nitroreductase is the activator. Further provided is a method of determining whether a mammalian cell is hypoxic using the above-described compound where nitroreductase is the activator. A method of detecting a microorganism that comprises a nitroreductase, using the above-described compound where nitroreductase is the activator, is also provided. Also provided is a method of identifying nitroreductase in a sample, using the above-described compound where nitroreductase is the activator.