研究目的
Investigating microtubule nucleation using a localized surface plasmon resonance (LSPR) biosensing approach.
研究成果
The LSPR biosensing approach successfully detects MT nucleation with high spatial resolution and specificity. The method is bulk-based, simple, and versatile, allowing for real-time monitoring of nucleation events below the diffraction limit. Future work could enhance sensitivity using non-spherical nanoparticles and extend the approach to other biopolymers.
研究不足
The theoretical model is a first-order approximation and does not account for detailed MT configurations. Experimentally, the signal-to-noise ratio with spherical AuNPs was limited, and some MTs may have attached to AuNPs post-nucleation rather than nucleating directly from them. The method's sensitivity could be improved with non-spherical nanoparticles.
1：Experimental Design and Method Selection:
The study involved developing a theoretical model using modified Mie theory with radially variable refractive index to predict the optical response of gold nanoparticles (AuNPs) during microtubule (MT) formation. Experimental methods included inducing MT nucleation from AuNPs via biotin-neutravidin interactions and monitoring changes in extinction spectra using UV-Vis spectroscopy.
2：Sample Selection and Data Sources:
Porcine tubulin was purified, and biotinylated/Rhodamine-labeled tubulin was purchased. Samples were prepared with AuNPs, neutravidin, tubulin, and GMPCPP to induce MT formation. Fluorescence microscopy and UV-Vis spectroscopy provided data on MT formation and spectral shifts.
3：List of Experimental Equipment and Materials:
Equipment included an Olympus XI-81 inverted microscope with a Hamamatsu ORCA-ER CCD camera, Cary 60 Spectrophotometer with a Peltier temperature controller, and quartz cuvettes. Materials included 80 nm biotin-PEG AuNPs, neutravidin, Atto655-streptavidin, tubulin, GMPCPP, paclitaxel, BSA, Tween 20, and BRB80 buffer.
4：Experimental Procedures and Operational Workflow:
AuNPs were incubated with neutravidin and streptavidin, then with tubulin. MT formation was induced with GMPCPP at 37°C. Samples were stabilized with paclitaxel and analyzed via fluorescence microscopy and UV-Vis spectroscopy. Kinetic experiments tracked spectral changes over time.
5：Data Analysis Methods:
Extinction spectra were analyzed using Mathematica for polynomial fitting to determine λmax. Statistical analyses were performed with GraphPad Prism and Excel. The centroid wavelength method was considered but not used due to noise issues.
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Spectrophotometer
Cary 60
Agilent Technologies, Inc.
Measure extinction spectra of samples to track changes in LSPR peak wavelength (λmax) during microtubule formation.
Cary 60 UV-Vis Spectrophotometer
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Microscope
Olympus XI-81
Olympus
Image fluorescence samples to visualize microtubule formation and attachment to gold nanoparticles.
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CCD Camera
ORCA-ER
Hamamatsu Photonics
Capture fluorescence images for analysis of microtubule samples.
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Temperature Controller
Peltier temperature controller
Agilent Technologies, Inc.
Control temperature during kinetic experiments in the spectrophotometer.
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Gold Nanoparticles
80 nm biotin-PEG AuNPs
Cytodiagnostics, Inc.
Serve as the core sensing element for LSPR, enabling detection of microtubule nucleation through changes in local refractive index.
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Software
SlideBook 6
Intelligent Imaging Innovations, Inc
Acquire and process microscope images.
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Cuvettes
Quartz cuvettes
FireflySci, Inc.
Hold samples for UV-Vis spectroscopy measurements.
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Tubulin
Biotinylated tubulin
Cytoskeleton, Inc.
Immobilize on gold nanoparticles to promote microtubule nucleation.
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Tubulin
Rhodamine-labeled tubulin
Cytoskeleton, Inc.
Fluorescent labeling for microscopy visualization of microtubules.
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Neutravidin
Thermo Fisher Scientific
Link biotinylated tubulin to biotin-PEG on gold nanoparticles.
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Streptavidin
Atto655-streptavidin
Sigma-Aldirch
Visualize gold nanoparticles under fluorescence microscope.
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Nucleotide
GMPCPP
Jena Bioscience
Induce microtubule nucleation and stabilization.
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Drug
Paclitaxel
Sigma-Aldirch
Stabilize microtubules after formation.
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Protein
BSA
Sigma-Aldirch
Block non-specific interactions on gold nanoparticles.
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Surfactant
Tween 20
Sigma-Aldirch
Reduce non-specific binding in buffer solutions.
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Buffer
BRB80
Provide optimal conditions for microtubule assembly and stability.
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Software
Mathematica
Wolfram Research
Compute extinction spectra and analyze data for λmax determination.
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Software
GraphPad Prism
GraphPad Software
Perform statistical analyses on experimental data.
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Software
Excel
Microsoft
Assist in data analysis and calculations.
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