Ratiometric APN Fluorescent Probe With Low-Background Peak Shift
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Solution Overview
Problem
Existing fluorescent probes for aminopeptidase N (APN) suffer from high background interference and lack sensitivity, making them inadequate for accurate and selective detection in biological samples.
Innovation Solution
A ratiometric fluorescent probe comprising an alanyl group and Nile blue derivative (NB) is developed, which undergoes a fluorescence peak shift upon reacting with APN, allowing for sensitive and selective detection of APN through a fluorescence ratio change.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Illumination intensity
If affinity-based fluorescent probes are used for APN detection, then fluorescent imaging of cancer cells or tumor-transplanted nude mice can be achieved, but large background interference occurs which heavily affects imaging quality
Solution Approach 1:
The invention extracts the fluorophore from the probe structure through enzymatic reaction. The probe is designed as a conjugate where the fluorophore is initially bound to a recognition unit. When APN enzyme is present, it cleaves the recognition unit, releasing the fluorophore and causing a significant increase in fluorescence signal. This extraction mechanism converts the bound fluorophore into a free, highly fluorescent state, dramatically reducing background interference while enhancing imaging quality.
Solution Approach 2:
The probe is pre-designed with the fluorophore attached to the recognition unit in a non-fluorescent or low-fluorescence state. This preliminary configuration allows the probe to be introduced into the system without generating background signal. The fluorescent signal is activated only when the enzymatic reaction occurs, ensuring that fluorescence is generated only at the target site and not during probe circulation or background conditions.
2Measurement precision
If conventional fluorescent probes are used for APN detection, then detection can be performed, but sensitivity and selectivity are insufficient for accurate detection in biological samples
Solution Approach 1:
The probe is segmented into distinct functional modules: a recognition unit specific to APN enzyme and a fluorophore unit. This segmentation allows the recognition unit to provide high selectivity through specific enzyme-substrate interaction, while the fluorophore unit provides the detection signal. The modular design ensures that each component performs its function optimally, with the recognition unit binding specifically to APN and the fluorophore providing a strong, measurable signal upon release.
Solution Approach 2:
The invention utilizes parameter changes in fluorescence properties upon enzymatic reaction. The probe exhibits a significant change in fluorescence intensity or emission wavelength when the fluorophore is released from the probe structure. This parameter change provides a clear, measurable signal that enhances detection sensitivity and allows for accurate quantification of APN in biological samples, improving both measurement precision and reliability.
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 probe achieves a detection limit of 15 pg/mL for APN and enables quantitative detection in urine samples, as well as in vivo imaging, with high specificity and stability, suitable for diagnosing APN-related diseases.
Implementation Method 1
Fluorescence spectroscopy has attracted much attention due to its unique advantages such as simplicity, convenience, non-invasiveness, real-time detection, high sensitivity, high temporal and spatial resolution, and in vivo imaging
Data Source
AI summary
Provided is a ratiometric fluorescent probe for detecting aminopeptidase N, and a preparation method and use thereof. In the present disclosure, a Nile blue derivative is adopted as a fluorophore and alanyl is adopted as an identification unit to design and synthesize the ratiometric fluorescent probe NB-APN for detecting APN. After the probe reacts with APN, the NB blocked by alanyl is released, resulting in an increase of a fluorescence peak at 675 nm and a decrease of a fluorescence peak at 610 nm. A ratio signal of the probe exhibits a sensitive response to APN, with a detection limit as low as 15 pg/mL. The probe can be used for quantitative detection of APN in a diluted urine sample, and can also be used for ratiometric fluorescent imaging of APN in a cell model and in vivo fluorescent imaging of APN in a nude mouse tumor model.