研究目的
Investigating the enhancement of single protein autofluorescence in the UV range using aluminum plasmonics.
研究成果
The work demonstrates the first demonstration of single protein tryptophan autofluorescence detection enhanced by UV aluminum plasmonics, enabling observing the tryptophan fluorescence of label-free β-galactosidase proteins with single molecule sensitivity at micromolar concentrations, increased brightness per molecule and microsecond transit times.
研究不足
The UV autofluorescence signal from a single protein is generally extremely weak, and the detection sensitivity currently requires about 100 tryptophan residues per protein to yield a brightness exceeding the background level.
1:Experimental Design and Method Selection:
The experiment involves using zero-mode waveguides (ZMW) nanoapertures milled in opaque aluminum films to enhance the tryptophan autofluorescence emission of single proteins in the UV range.
2:Sample Selection and Data Sources:
β-galactosidase proteins from Escherichia coli are used for their natural tryptophan UV fluorescence.
3:List of Experimental Equipment and Materials:
Includes a home-built confocal microscope with time-resolved fluorescence detection, aluminum ZMW samples, and a 266 nm picosecond laser for excitation.
4:Experimental Procedures and Operational Workflow:
The ZMW is positioned at the focus of a UV confocal microscope, and the fluorescence light is collected in an epifluorescence configuration.
5:Data Analysis Methods:
Fluorescence correlation spectroscopy (FCS) is used to analyze the fluorescence intensity time traces to compute the temporal correlation function and assess the average number of molecules present in the ZMW detection volume and their average diffusion time.
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Picoquant LDH-P-FA-266
266 nm picosecond laser
Picoquant
Excitation of p-terphenyl molecules.
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Picoquant VisUV-295-590
295 nm picosecond laser
Picoquant
Excitation of β-galactosidase proteins.
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Picoquant PMA 175
Single photon counting photomultiplier tube
Picoquant
Registers the arrival time of each detected photon.
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Picoquant Picoharp 300
Photon counting module
Picoquant
Registers the arrival time of each detected photon in a time tagged time resolved mode (TTTR).
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FEI dual beam DB235 Strata
Focused ion beam
FEI
Mills individual nanoapertures with 30 keV energy and 10 pA current.
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Zeiss Ultrafluar
40x, 0.6 NA glycerol immersion objective
Zeiss
Focuses the UV laser beam to a diffraction-limited spot.
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Bühler Syrus Pro 710
Electron-beam evaporation system
Bühler
Deposits a 50 nm-thick layer of aluminum on cleaned microscope quartz coverslips.
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