Semiconductor Nanocrystals as Fluorescent Biological Labels
Tóm tắt
Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. Compared with conventional fluorophores, the nanocrystals have a narrow, tunable, symmetric emission spectrum and are photochemically stable. The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse fibroblasts. These nanocrystal probes are thus complementary and in some cases may be superior to existing fluorophores.
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Tài liệu tham khảo
Attempts with energy transfer–sensitized probes and phycobiliprotein-conjugated dye molecules have been somewhat successful but the efficiency of energy transfer is lower than the intrinsic excitation and emission of a dye molecule [
Ju J. Y., Ruan C. C., Fuller C. W., Glazer A. N., Mathies R. A., Proc. Natl. Acad. Sci. U.S.A. 92, 4347 (1995);
R. Hermann P. Walther M. Muller ibid. p. 31.
The idea of using biological interactions to pattern materials has also been explored [
; C. A. Mirkin R. L. Letsinger R. C. Mucic J. J. Storhoff ibid. p. 607].
Murray C. B., Norris D. J., Bawendi M. G., ibid. 115, 8706 (1993).
For the synthesis of water-soluble core-shell nanocrystals 2 to 10 mg of CdSe-CdS (13) and CdSe-ZnS (25) core-shell nanocrystals were homogeneously dispersed in 2 ml of degassed n -butanol then precipitated with anhydrous methanol; addition of a roughly equal mass of trioctylphosphineoxide and 1 hour of heating at 100°C were necessary to disperse the CdSe-CdS. The precipitate was dissolved in 120 ml of a solution of 0.17% (v/v) (3-mercaptopropyl)trimethoxysilane in 25% dimethyl sulfoxide (DMSO) in methanol the pH of which was adjusted to 10 to 11 with (CH 3 ) 4 NOH · 5H 2 O. After overnight stirring the solution was diluted with 100 ml of methanolic (CH 3 ) 4 NOH · 5H 2 O at pH 10 and gently refluxed at 69°C for 25 to 30 min. After the preparation was cooled 200 ml of a 10% water and methanol solution containing 400 μl of trimethoxysilyl propyl urea and 40 μl of 3-aminopropyltrimethoxysilane was added stirred for 2 hours then heated to reflux at 69°C for less than 5 min and cooled. Next 40 ml of a 10% chlorotrimethylsilane solution basified with (CH 3 ) 4 NOH · 5H 2 O was added the preparation stirred for another 2 hours then concentrated partially at 60°C in vacuo to an oily solution. This solution was precipitated to a greasy solid with 50% acetone in isopropanol. This solid was redissolved in water and buffered solution.
For conversion of functional groups on the surface of the nanocrystals the precipitated sample was dissolved in 100 mM phosphate-buffered saline (PBS) pH 7.5 at a concentration four times that of the final oily solution. A 1.0-ml volume of the concentrated nanocrystal solution was incubated with 100 to 400 μl of a 10-mg/ml solution of biotinamidocaproic acid 3-sulfo- N -hydroxysuccinimide ester (Sigma) in 6% DMSO in PBS for 1 hour after which the solution was quenched with a neutralized iodoacetic acid solution in PBS to 50 mM final iodoacetate concentration. The unbiotinylated nanocrystals were prepared by adding the quench solution directly. These solutions were incubated overnight at 4°C then concentrated and rinsed through a Centricon 50 (Millipore) and diluted to 1.0 ml; the remaining amines on the biotinylated nanocrystals were capped with 50 μl of a 1 M solution of succinic anhydride in dry DMSO followed by addition of an equivalent amount of base and further purification through a Centricon 50. The retentate was diluted to 1.0 ml and used for labeling at 1:10 dilution. The biotinylated nanocrystals shifted to lower mobility in a gel when incubated with streptavidin. Higher levels of biotinylation followed by streptavidin incubation resulted in a further decrease in mobility through agarose gel. These effects were not seen in unbiotinylated nanocrystal samples indicating that this interaction is specific and that the biotin is covalently bound to the nanocrystal surface.
The emission from the nanocrystals is due to a transition that would be spin forbidden in a system of light atoms. Because of strong spin-orbit coupling and quantum confinement effects the emission has a radiative rate that is on the order of 10 7 s −1 . In this report we refer to this emission as the nanocrystal fluorescence [
Guzelian A. A., et al., ibid. 100, 7212 (1996).
Mouse 3T3 fibroblasts were grown on fibronectin-treated formvar-coated gold grids and fixed with 4% paraformaldehyde 0.1% glutaraldehyde and 0.5% Triton at room temperature. Specimens were then treated successively with unbiotinylated green nanocrystals phalloidin-biotin (Molecular Probes) (15 min) streptavidin (25 μg/ml) (30 min) a ∼100 nM solution of biotinylated red nanocrystals (30 min) again streptavidin (30 min) then once more with the red biotinylated nanocrystals (30 min). At least three PBS or Superblock-PBS (Pierce) rinse steps (5 min each) were performed between each incubation. Samples were mounted on microscope slides in PBS.
We would like to thank X. Peng J. Gray C. Bertozzi and P. Schultz for many helpful discussions. The photoluminescence spectra of InAs nanocrystals InP nanocrystals and CdSe nanocrystals in Fig. 2 were provided by J. Wickham U. Banin and X. Peng. Supported by the Director Office of Energy Research Office of Basic Energy Sciences Division of Materials Sciences of the U.S. Department of Energy under contract number DE-AC03-76SF00098. M.B. acknowledges support from a NSF graduate research fellowship.