Semiconductor Nanocrystals as Fluorescent Biological Labels

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Science  25 Sep 1998:
Vol. 281, Issue 5385, pp. 2013-2016
DOI: 10.1126/science.281.5385.2013


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.

Fluorescence is a widely used tool in biology. The drive to measure more biological indicators simultaneously imposes new demands on the fluorescent probes used in these experiments. For example, an eight-color, three-laser system has been used to measure a total of 10 parameters on cellular antigens with flow cytometry (1), and in cytogenetics, combinatorial labeling has been used to generate 24 falsely colored probes for spectral karyotyping (2). Conventional dye molecules impose stringent requirements on the optical systems used to make these measurements; their narrow excitation spectrum makes simultaneous excitation difficult in most cases, and their broad emission spectrum with a long tail at red wavelengths (Fig. 1A) introduces spectral cross talk between different detection channels, making quantitation of the relative amounts of different probes difficult. Ideal probes for multicolor experiments should emit at spectrally resolvable energies and have a narrow, symmetric emission spectrum, and the whole group of probes should be excitable at a single wavelength (3, 4).

Figure 1

Excitation (dashed) and fluorescence (solid) spectra of (A) fluorescein and (B) a typical water-soluble nanocrystal (NC) sample in PBS. The fluorescein was excited at 476 nm, and the NC at 355 nm. Excitation spectra were collected with detection at 550 nm (fluorescein) and 533 nm (NC) because of the difference in emission spectra. The nanocrystals have a much narrower emission (32 nm compared with 45 nm at half maximum and 67 nm compared with 100 nm at 10% maximum), no red tail, and a broad, continuous excitation spectrum.

In semiconductor nanocrystals, the absorbance onset and emission maximum shift to higher energy with decreasing size (5). The excitation tracks the absorbance, resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak yet will emit with the same characteristic narrow, symmetric spectrum regardless of the excitation wavelength (Fig. 1B). Variation of the material used for the nanocrystal and variation of the size of the nanocrystal afford a spectral range of 400 nm to 2 μm in the peak emission (Fig. 2), with typical emission widths of 20 to 30 nm [full width at half maximum (FWHM)] in the visible region of the spectrum and large extinction coefficients in the visible and ultraviolet range (∼105 M−1cm−1). Many sizes of nanocrystals may therefore be excited with a single wavelength of light, resulting in many emission colors that may be detected simultaneously.

Figure 2

(A) Size- and material-dependent emission spectra of several surfactant-coated semiconductor nanocrystals in a variety of sizes. The blue series represents different sizes of CdSe nanocrystals (16) with diameters of 2.1, 2.4, 3.1, 3.6, and 4.6 nm (from right to left). The green series is of InP nanocrystals (26) with diameters of 3.0, 3.5, and 4.6 nm. The red series is of InAs nanocrystals (16) with diameters of 2.8, 3.6, 4.6, and 6.0 nm. (B) A true-color image of a series of silica-coated core (CdSe)–shell (ZnS or CdS) nanocrystal probes in aqueous buffer, all illuminated simultaneously with a handheld ultraviolet lamp.

Metallic and magnetic nanocrystals, with the appropriate organic derivatization of the surface, have been used widely in biological experiments (6–11). The use of semiconductor nanocrystals in a biological context is potentially more problematic because the high surface area of the nanocrystal might lead to reduced luminescence efficiency and photochemical degradation. Bandgap engineering concepts borrowed from materials science and electronics have led to the development of core-shell nanocrystal samples with high, room temperature quantum yields (>50%) (12–14) and much improved photochemical stability. By enclosing a core nanocrystal of one material with a shell of another having a larger bandgap, one can efficiently confine the excitation to the core, eliminating nonradiative relaxation pathways and preventing photochemical degradation. The synthesis of the semiconductor nanocrystals and the growth of the shell by methods from the literature yield gram quantities of a variety of materials with a narrow size distribution (5%), coated with a surfactant but soluble only in nonpolar solvents (15, 16).

Biological applications require water-soluble nanocrystals. We have extended the chemistry of the core-shell systems by adding a third layer of silica that makes the core-shell water soluble, similar to a procedure detailed for coating gold and cadmium sulfide nanocrystals (17, 18). This strategy has a number of advantages compared with strategies that use a single direct bond to the surface of the nanocrystal: the multivalency of an extensively polymerized polysilane ensures that the nanocrystals stay soluble in spite of the potential loss of bound thiol. Furthermore, the chemistry of silica is well characterized and widely used to functionalize supports for chromatography. Modification of the silica surface with different groups can be used to control the interactions with the biological sample. The core-shell nanocrystals prepared in this manner are soluble and stable in water or buffered solution, and they retain a fairly large quantum yield [up to 21%, comparable to some conventionally used fluorescent dyes that have yields between 14 and 71% (3)] (Fig. 2B). Further developments in the bandgap engineering of nanocrystals and modifications to the silanization chemistry are expected to result in higher quantum yields and improvement of other emission properties (19). This chemistry has been applied to a number of different sized core-shell nanocrystals, generating a spectrally tuned family of probes, all of which are amenable to the same modification chemistry (in contrast to organic dyes, for which specific chemistries must be developed on a case by case basis).

To establish the utility of nanocrystals for biological staining, we fluorescently labeled 3T3 mouse fibroblast cells using two different size CdSe-CdS core-shell nanocrystals enclosed in a silica shell (20). The smaller nanocrystals (2-nm core) emitted green fluorescence (maximum 550 nm, 15% quantum yield), the larger (4-nm core), red fluorescence (maximum 630 nm, 6% quantum yield). The surface of the nanocrystals may be tailored to interact with the biological sample either through electrostatic and hydrogen-bonding interactions or through a specific ligand-receptor interaction, such as the avidin-biotin interaction (21). As an example of the former, nanocrystals coated with trimethoxysilylpropyl urea and acetate groups were found to bind with high affinity in the cell nucleus. This nuclear binding could be suppressed with an anionic silane reagent [3-(trihydroxysilyl)propyl methylphosphonate] or by incubating with the nanocrystals in a 0.2% SDS solution. This property was used to “stain” the nucleus with the green-colored nanocrystals, relying on the silanized nanocrystal surface to control the binding.

The avidin-biotin interaction, a model system for ligand-receptor binding, was used here to specifically label the F-actin filaments with red nanocrystal probes. Biotin was covalently bound to the nanocrystal surface (22), and the biotinylated nanocrystals were used to label fibroblasts, which had been incubated in phalloidin-biotin and streptavidin. One round of amplification was carried out by incubating the sample with streptavidin and then with red biotinylated nanocrystals once again.

The resulting samples were imaged with both conventional wide-field and laser-scanning confocal fluorescence microscopes. In contrast to conventional multicolor dye imaging, light from a mercury lamp with a fluorescein isothiocyanate excitation filter and a single long-pass detection filter (515 nm) were used with the wide-field microscope to see both colors at one time. The green and red labels were clearly spectrally resolved to the eye and to a color Polaroid camera. Nonspecific labeling of the nuclear membrane by both the red and the green probes resulted in a yellow color. The red actin filaments, however, were specifically stained. These filaments were not visible or were only very faintly visible in the control experiments lacking phalloidin-biotin. The penetration of the green probes into the nucleus and specific red staining of the actin fibers is readily visible in Fig. 3. Over repeated scans, the nanocrystal-labeled samples showed very little photobleaching, far less than with conventional dye molecules (Fig. 4).

Figure 3

Cross section of a dual-labeled sample examined with a Bio-Rad 1024 MRC laser-scanning confocal microscope with a 40× oil 1.3 numerical aperture objective. The mouse 3T3 fibroblasts were grown and prepared as described in (27). A false-colored image was obtained with 363-nm excitation, with simultaneous two-channel detection (522DF 35-nm FWHM narrow-pass filter for the green, and a 585-nm long-pass filter for the red). Image width: 84 μm.

Figure 4

Sequential scan photostability comparison of fluorescein-phalloidin–labeled actin fibers compared with nanocrystal-labeled actin fibers. Fluorescein was excited at 488 nm and the nanocrystals at 363 nm by a laser scanning confocal microscope with a 12-μs dwell time and ∼20-mW power for each laser. The average intensity of four pixels was followed in each sample through 100 successive scans and normalized to its initial value. The intensity of the fluorescein drops quickly to autofluorescence levels, whereas the intensity of the nanocrystals drops only slightly.

The development of nanocrystals for biological labeling opens up new possibilities for many multicolor experiments and diagnostics. Further, it establishes a class of fluorescent probe for which no small organic molecule equivalent exists. The tunability of the optical features allows for their use as direct probes or as sensitizers for traditional probes. These nanocrystals also have a long fluorescence lifetime (hundreds of nanoseconds) (23), which can allow for time-gated detection for autofluorescence suppression (24). Further developments such as direct immunolabeling, in situ hybridization, and incorporation into microspheres will be important for applications such as cytometry and immunocytobiology. In addition, nanocrystal probes may prove useful for other contrast mechanisms such as x-ray fluorescence, x-ray absorption, electron microscopy, and scintillation proximity imaging, and the use of far red– or infrared-emitting nanocrystals (InP and InAs) as tunable, robust infrared dyes is another possibility.

  • * To whom correspondence should be addressed. E-mail: sweiss{at} and alivis{at}


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