研究目的
To develop a novel fluorescent biosensor for sensitive detection of the antibiotic rapamycin, addressing limitations in existing methods such as low sensitivity and complex procedures.
研究成果
The developed Q'-body sensor, particularly the ATTO520-labeled *RK type, provides a simple, rapid, and sensitive method for rapamycin detection with a LOD of 0.65 nM, suitable for therapeutic monitoring. Future improvements could enhance de-quenching efficiency and enable in vivo applications.
研究不足
The rapamycin-induced de-quenching efficiency was modest (1.5-fold increase), and double-labeled Q'-bodies showed lower responses than single-labeled ones. The synthesis efficiency for full-length proteins with certain dyes (e.g., ATTO590) was low due to imperfect amber suppression. Potential dimer formation and its effects were not fully characterized.
1：Experimental Design and Method Selection:
The study applied the quench-release principle inspired by Q-body technology, using FKBP12 and FRB domains linked with flexible linkers. Fluorophores were incorporated site-specifically via amber codon suppression in a cell-free system.
2：Sample Selection and Data Sources:
Synthetic genes for FKBP12 and FRB were used, with fluorophores ATTO520, TAMRA, and ATTO
3：Rapamycin was sourced from LKT Laboratories. List of Experimental Equipment and Materials:
5 Equipment included PCR thermocycler, fluorescence spectrophotometer (Jasco FP-8500), His Spin Trap Column (GE Healthcare), Nanosep Centrifugal-3k ultrafiltration device (Pall), and cell-free translation system (RYTS kit from Roche Diagnostics). Materials included primers, plasmids, dyes, buffers, and chemicals from various suppliers.
4：Experimental Procedures and Operational Workflow:
Genes were constructed via SOE PCR and cloned into plasmids. Q'-bodies were synthesized using cell-free translation with amber suppressor tRNAs, purified via immobilized metal affinity chromatography, and fluorescence measurements were performed after adding rapamycin and incubating.
5：Data Analysis Methods:
Fluorescence spectra were measured, dose-response curves were fitted using a 4-parameter logistic equation in Kaleida Graph 4.1, and LOD was calculated based on Armbruster's definition.
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fluorescence spectrophotometer
FP-8500
Jasco
Used for measuring fluorescence spectra of Q'-bodies after rapamycin addition.
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quartz cell
5 mm × 5 mm
Jasco
Used as a container for fluorescence measurements.
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His Spin Trap Column
GE Healthcare
Used for purifying synthesized Q'-bodies via immobilized metal affinity chromatography.
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Nanosep Centrifugal-3k ultrafiltration device
Pall
Used for buffer exchange during purification of Q'-bodies.
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cell-free transcription-translation system
RYTS kit
Roche Diagnostics
Used for synthesizing Q'-bodies with site-specific fluorophore incorporation.
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DNA polymerase
KOD-plus-neo
Toyobo
Used for PCR amplification of genes.
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plasmid miniprep system
PureYield
Promega
Used for preparing plasmid DNA.
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software
Kaleida Graph 4.1
Synergy Software
Used for fitting dose-response curves and calculating EC50 values.
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