Watching Silica Nanoparticles
in the Biological World The glow and photostability, along with other advantages, of dye-doped silica nanoparticles make their future bright Lin Wang Kemin Wang Swadeshmukul Santra Xiaojun Zhao Lisa R. Hilliard Joshua E. Smith Yanrong Wu Weihong Tan University of Florida Hunan University (China) University of Central Florida University of North Dakota
in bioanalytical applications.
luorescence-based detection techniques have been widely used in modern biochemical research and disease diagnosis. For the detection of trace levels of analytes, organic fluorophores are commonly exploited as signal transduction tools. Although these fluorophores are versatile and easy to use, their molecular nature determines their limitations. In most cases, only one or a few fluorophores can signal one biomolecule recognition event, and typically, only a limited number of fluorophores can be attached to a biomolecule without interfering with its binding specificity or causing it to precipitate. As a consequence, sample analysis can be particularly difficult when trace amounts of biological analytes are present, and the additional steps required for signal amplification can be time-consuming and impede analyte quantitation. When exposed to a continuous light source, organic fluorophores are not very photostable. In addition, the complex environment inside living cells can increase the vulnerability of organic fluorophores to degradation and photobleaching. These two factors can result in false-positive and false-negative signals and can affect prolonged cell monitoring and 3-D optical sectioning imaging. Moreover, although most organic fluorophores can be conjugated with biomolecules, such as DNA and proteins, a different conjugation chemistry must be used to attach the organic dye to a given biomolecule of interest; this chemistry can be too difficult, time-consuming, and/or expensive for routine applications. All of these limitations have greatly hindered the use of fluorophores for in vitro assays and in vivo cellular imaging. The rapidly evolving field of nanoscience and nanotechnology has opened up a promising era in new biomarker development, in which nanoparticles of various shapes, sizes, and compositions have been successfully used in bioimaging, labeling, and sensing because of their unique optical properties, high surface-to-volume ratio, and other size-dependent qualities (1–8). With manipulated composition and surface modification, these nanoparticle probes have enhanced the fluorescence signal, increased sensitivity, prolonged detection time, and generated better reproducibility. Quantum dots (QDs) and dye-doped nanoparticles are representative fluorescent nanoparticle probes of increasing research interest. QDs are ultrasmall (usually 1–10 nm in diameter), bright (20 brighter than most organic fluorophores), and highly photostable nanocrystalline semiconductors. Their broad excitation spectra, along with narrow, symmetric, size-tunable fluorescence emission spanning the UV to NIR, make them ideal for multiplex analysis (simultaneous detection of multiple an-
© 2006 AMERICAN CHEMICAL SOCIETY
F
F E B R U A R Y 1 , 2 0 0 6 / A N A LY T I C A L C H E M I S T R Y
647
been extensively used in simultaneous assays to determine multiple analytes in a (a) (c) (b) single sample (9). Silica nanoparticles doped with fluoFIGURE 1. Transmission electron micrographs of different sizes of silica nanoparticles prerescent dyes have also been used as labelpared in various microemulsion systems. ing reagents for biological applications. (a) 15-nm nanoparticles; (b) 40-nm nanoparticles; (c) 120-nm nanoparticles; scale bars are 200 nm, 200 nm, Compared with polymer nanoparticles, and 1 µm, respectively. silica nanoparticles possess several advantages. Silica nanoparticles are easy to sepalytes) without complex instrumentation and processing. Their arate via centrifugation during particle preparation, surface modhigh resistance to photobleaching and their reasonable bright- ification, and other solution treatment processes because of the ness make them appealing for long-term cellular and deep-tissue higher density of silica (e.g., 1.96 g/cm3 for silica vs 1.05 g/cm3 imaging (1, 2). However, they do have drawbacks. QDs are dif- for polystyrene). Silica nanoparticles are more hydrophilic and ficult to make; the surface-modification chemistry is still under biocompatible, they are not subject to microbial attack, and no investigation; the “blinking” behavior (luminescence emission swelling or porosity change occurs with changes in pH (7). switches “on” and “off” by sudden stochastic jumps under con- (Polymer particles are hydrophobic, tend to agglomerate in tinuous excitation) is a limiting factor for raster scanning systems, aqueous medium, and swell in organic solvents, resulting in dye such as flow cytometry; and cytotoxicity is a definite concern for leakage.) in vivo applications (1, 2). Extensive research efforts are under Because of these advantages and the aforementioned brightway to overcome these challenges, yet the superiority of QDs ness and fluorescence photostability over time, dye-doped silica over other fluorescent labels for certain biological applications nanoparticles have shown great promise in various biological apstill makes them one of the most intriguing fluorescent probes. plications (4). To provide readers with an overview of the new Another type of fluorescent nanoparticle probe is dye-doped progress in the research on fluorescent silica nanoparticles, this nanoparticles, varying in size between 2 and 200 nm in diame- article will present the current preparation methods, practical ter. These nanoparticles contain a large quantity of dye molecules features, and diverse biological applications, as well as a future housed inside a polymer or silica matrix, and they give an intense outlook on the potential use of dye-doped silica nanoparticles for fluorescence signal that is up to 10,000 that of organic fluo- bioanalysis and biotechnology. rophores (8). Their extreme brightness makes them especially suitable for ultrasensitive bioanalysis without the need for addi- Development and functionalization tional reagents or signal amplification steps. When dye-doped Dye-doped silica nanoparticles can be prepared by two general nanoparticle probes are used, a biomolecule recognition event is synthetic routes: the Stöber and microemulsion processes. In signaled by one nanoparticle, in which hundreds to thousands of 1968, Stöber et al. introduced a method for synthesizing fairly dye molecules are integrated to greatly enhance the fluorescence monodisperse silica nanoparticles, with diameters ranging besignal. This signal enhancement facilitates ultrasensitive analyte tween 50 nm and 2 µm (10). In a typical Stöber-based protocol, determination and the monitoring of rare biological events that a silica alkoxide precursor (such as tetraethyl orthosilicate, are otherwise undetectable with existing fluorescence labeling TEOS) is hydrolyzed in an ethanol and ammonium hydroxide techniques. The polymer and silica matrix serves as a protective mixture. The hydrolysis of TEOS produces silicic acid, which shell, or dye isolator, limiting the effect of the outside environ- then undergoes a condensation process to form amorphous siliment (such as oxygen, certain solvents, and soluble species in ca particles. The details of the mechanism of Stöber-based buffer solutions) on the fluorescent dye contained in the nano- nanoparticle formation have been extensively investigated particles. (11–14), and the method has been optimized to synthesize dyePolymer or latex nanoparticles are commonly dopedwith fluo- doped silica nanoparticles by covalently attaching organic fluorescent dyes after nanoparticle synthesis. A typical preparation rescent dye molecules to the silica matrix (15–18). The procemethod involves the swelling of polymeric nanoparticles in an or- dure involves two steps: The dye is chemically bound to an ganic solvent and fluorescent dye solution. The hydrophobic dye amine-containing silane agent (such as 3-aminopropyltriethdiffuses into the polymer matrix and is further entrapped when oxysilane, APTS), and then APTS and TEOS are allowed to hythe solvent is removed from the particles through evaporation or drolyze and co-condense in a mixture of water, ammonia, and transfer to an aqueous phase. The most common polymer matri- ethanol, resulting in dye-doped silica nanoparticles. This apces are polystyrene, PMMA, polylactic acid, and polylactic-co- proach enables the incorporation of a variety of organic dye molpolyglycolic acid. The commercialization of fluorescent polymer ecules into the silica nanoparticles. or latex nanoparticles and microspheres facilitates their extensive Dye-doped silica nanoparticles can also be synthesized by hyuse in various biological applications. For example, fluorescent drolyzing TEOS in a reverse-micelle or water-in-oil (w/o) mipolymer microspheres have been used as immunofluorescent rea- croemulsion system, a homogeneous mixture of water, oil, and gents, cell tracers, and standardization reagents for microscopy surfactant molecules (19 – 21). In a typical w/o microemulsion and flow cytometry. Arrays of fluorescent polymer microspheres system, water droplets are stabilized by surfactant molecules and that differ in intensity, size, or excited-state lifetime have also remain dispersed in bulk oil. The nucleation and growth kinetics 648
A N A LY T I C A L C H E M I S T R Y / F E B R U A R Y 1 , 2 0 0 6
O
NP
C OH +
NH2 –
=
=
O
Biomolecule
NP
C NH
Biomolecule
=
=
=
of the silica are highly regulated in the water droplets of the NP SH + HO – (CH2)6 – S– S– Biomolecule NP – S– S– Biomolecule microemulsion system, and the dye molecules are physically encapsulated in the silica netO O work, resulting in the formaO tion of highly monodisperse Biomolecule Biomolecule NP NP NH N – O – C– + NH 2 dye-doped silica nanoparticles C (22). In the past few years, varO ious dye-doped silica nanoparticles have been developed with the w/o microemulsion – – – ++ + + + ++ + + + – technique (8, 23–27 ). + + + Avidin NP – + NP – + Avidin + + + + + + + + + + Polar dye molecules are – – – – used in the w/o microemulsion system to increase the electrostatic attraction of the FIGURE 2. Representative bioconjugation schemes for attaching biomolecules to dye-doped dye molecules to the negativesilica nanoparticles (NPs) for bioanalysis. ly charged silica matrix, so that dye molecules are successfully entrapped inside the silica matrix. Water-soluble inorganic dyes, In addition to providing the reactive sites for conjugation such as ruthenium complexes, can be readily encapsulated into with biomolecules, the functional groups also change the colnanoparticles by this method (23–25, 28, 29). Leakage of dye loidal stability of the particles in solution. For example, postmolecules from the silica particles is negligible, probably because coating with amine-containing organosilane compounds neutralof the strong electrostatic attractions between the positively izes the negative surface charge of nanoparticles at neutral pH charged inorganic dye and the negatively charged silica. To syn- and hence reduces the overall charge of the nanoparticles. As a thesize organic-dye-doped nanoparticles, various trapping meth- result, colloidal stability decreases and severe particle aggregation ods have been used, such as introducing a hydrophobic silica pre- takes place in aqueous medium. To solve this problem, inert negcursor (27 ), using water-soluble dextran-molecule-conjugated atively charged organosilane compounds containing phosphodyes, and synthesizing in acidic conditions (8). These alternative nate or other groups are introduced as a critical dispersing agent methods aid in trapping hydrophobic dye molecules into the sil- during post-coating. Consequently, the nanoparticles possess a ica matrix. The unique advantage of the w/o microemulsion net negative charge and are well dispersed in aqueous solution method is that it produces highly spherical and monodisperse (32, 33). Other stabilization reagents, such as organosilane comnanoparticles of various sizes (Figure 1). It also permits the trap- pounds that contain polyethylene glycol (PEG, a neutral polyping of a wide variety of inorganic and organic dyes as well as mer), can also be added to the nanoparticle surface. The PEGyother materials, such as luminescent quantum dots (30). lated surface is highly hydrophilic and enhances the aqueous dispersibility of the silica nanoparticles (34, 35). In addition, the PEGylated surface reduces nonspecific binding by inhibiting the Surface modification and bioconjugation For bioanalysis and biotechnological applications, dye-doped sil- adsorption of undesired charged biomolecules. After the nanoparticles are modified with different functional ica nanoparticles must be linked to the biorecognition elements, such as antibodies and DNA molecules. Many of these molecules groups, they can act as a scaffold for the grafting of biological can be physically adsorbed onto the silica nanoparticle surface. moieties (DNA oligonucleotides, aptamers, antibodies, peptides, However, covalent attachment of biorecognition elements to the etc.) by means of standard covalent bioconjugation schemes (36; particle surface is preferred, not only to avoid desorption from Figure 2). For example, carboxyl-modified nanoparticles have the particle surface but also to control the number and orienta- pendent carboxylic acids, making them suitable for covalent coution of the immobilized biorecognition elements. For covalent pling of proteins and other amine-containing biomolecules via attachment, the particle surface first needs to be modified with water-soluble carbodiimide reagents (30). Disulfide-modified suitable functional groups (e.g., thiol, amine, and carboxyl oligonucleotides can be immobilized onto thiol-functionalized groups). This is typically done by applying a stable additional sil- nanoparticles by disulfide-coupling chemistry (37 ). Amine-modica coating (post-coating) that contains the functional group(s) ified nanoparticles can be coupled to a wide variety of haptens of interest. For the Stöber nanoparticles, surface modification is and drugs via succinimidyl esters and iso(thio)cyanates. Other usually done after nanoparticle synthesis to avoid potential sec- approaches use electrostatic interactions between nanoparticles ondary nucleation. Surface modification of microemulsion and charged adapter molecules (38, 39) or between nanoparticles nanoparticles can be achieved in the same manner or via direct and proteins modified to incorporate charged domains. The biohydrolysis and co-condensation of TEOS and other organosi- conjugation or labeling strategy is rationally designed on the basis of the biomolecular function of the surface-attached entilanes in the microemulsion solution (31).
=
F E B R U A R Y 1 , 2 0 0 6 / A N A LY T I C A L C H E M I S T R Y
649
(a)
(b)
(c)
FIGURE 3. (a) SEM image of an E. coli O157:H7 cell incubated with nanoparticle–antibody conjugates, showing nanoparticle binding to the target bacterium. Scale bar is 2.73 µm. (b) SEM image of an E. coli DH5 cell (negative control) incubated with nanoparticle–antibody conjugates, showing no nanoparticle binding. Scale bar is 1.5 µm. The black small dots in (a) and (b) are the pores on the surface of the filter membrane, and the white spots are unbound nanoparticles. (c) Fluorescence image of an E. coli O157:H7 cell after incubation with nanoparticle–antibody conjugates. Scale bar is 4 µm. The fluorescence intensity is strong, enabling identification of a single bacterial cell in aqueous solution.
ties. For example, protein recognition sites are oriented away from the nanoparticle surface to ensure that they do not lose their ability to bind to a target (40). After the bioconjugation step, the nanoparticles can be separated from unbound biomolecules by centrifugation, dialysis, filtration, or other laboratory techniques.
Nanoparticle characterization Characterization of nanoparticles is important to elucidate the mechanism of nanoparticle formation and to validate the synthesis protocol. Performance evaluation of the nanoparticles with respect to their photostability, surface properties, size, and morphology provides direct feedback that can be used to improve the synthesis protocol. Typically, particle characterization includes the measurement of particle size, surface charge, surface functionality, and optical and spectral characteristics. Chemical characterization is also important to quantify the amounts of doped dye molecules and surface-immobilized biomolecules. Methods for the characterization of nanoparticle size and surface charge will be discussed here. Several techniques currently exist to measure the particle size. Transmission electron microscopy and scanning electron microscopy (SEM) are commonly used for size characterization of nanoparticles in the vacuum state (15–18). Atomic force microscopy is used for both dry and wet samples at normal atmospheric pressure (41, 42). Electron microscopic techniques, however, do not provide any insight with respect to the dispersion state of particles in solution. On the other hand, light scattering techniques allow particle size measurements in aqueous media and provide information on nanoparticle size distribution and relative dispersion (43). The particle size is strongly affected by the concentration of reactants (TEOS and ammonium hydroxide) for both the Stöber and microemulsion methods. It is also affected by the nature of surfactant molecules and the molar ratios of water to surfactant and co-surfactant to surfactant for the microemulsion process (44). Nanoparticles prepared with the microemulsion method are spheres with smooth surfaces and low polydispersity, whereas Stöber nanoparticles with smaller radii ( 30 mV, where a greater magnitude means greater stability against flocculation and sedimentation. It is worth noting that the synthesized silica nanoparticles would be well dispersed without any aggregation due to the strong negative charge on the surface provided by silanol groups (Si–OH r Si–O–). However, the pH or salt concentration of some biological buffers can lead to aggregation of particles or cause undesirable adhesion between nanoparticles and detection surfaces. Large biological molecules can also have an impact on stability, especially if they are large enough to physically adsorb to the particle surface. Therefore, surface modification that optimizes the repulsive forces of the particles in solution is necessary for effective binding to biomolecules and long-term stability. In addition, on the basis of the measurement, one can verify that bioconjugation reactions have taken place. For example, values will change if an attached molecule screens the charges on the particle surface (e.g., in the case of proteins) or if the molecule itself carries a significant charge (e.g., in the case of DNA).
Practical features of nanoparticles Sensitivity is a critical issue in modern biomedical research and disease diagnosis. The introduction of new fluorescent labels capable of high signal amplification is essential to address the growing need for highly sensitive bioassays. With numerous dye molecules trapped inside, dye-doped silica nanoparticles exhibit extraordinary signaling strength. For example, the effective luminescence intensity ratio of one ruthenium bipyridine (Rubpy)doped silica nanoparticle (diam = 60 nm) to one Rubpy dye molecule is 104. Therefore, >10,000 dye molecules are presumed to be doped inside a 60-nm nanoparticle. The impressive signal of the nanoparticles can dramatically lower the analyte detection limit in biological samples. Photostability is a particularly important criterion for extended observation (from minutes to hours) of fluorescence signal under intense laser illumination. It is also especially useful for 3D optical sectioning imaging, in which a major obstacle is the
photobleaching of fluorophores during acquisition of successive z-sections; this photobleaching compromises the correct reconstruction of 3-D structures. To demonstrate the high photostability of nanoparticles, both nanoparticle and dye solutions were excited with a xenon lamp and the emission intensities were monitored with respect to time. No noticeable photobleaching was observed for the dye-doped nanoparticles in solution for 1 h, but the dye molecules lost 85% of the initial signal under identical conditions (23). This observation proves that the silica coating isolates the dye molecules from the outside environment and thereby prevents oxygen penetration. In addition, when nanoparticles are used for real biological sample imaging, the dye molecules are protected against degradation or photobleaching by the complex biological milieu, because the silica matrix is highly resistant to chemical and metabolic degradation. Moreover, whereas the organic fluorophores require customized chemistry for the conjugation of dye molecules to each biomolecule, the silica surface provides excellent versatility for different surface-modification protocols. Because the nanoparticle surface can be functionalized with reactive end groups during synthesis, the nanoparticles can be readily modified with biomolecules. The nanoparticle–biomolecule complex can be used to express the activity of a desired process (e.g., immobilized enzymes) or can be used as an affinity ligand to capture or modify target molecules or cells. Furthermore, because of growing concerns over the potential toxicity of nanoparticulate systems, the cytotoxicity of silica nanoparticles has been examined. Extensive research efforts have shown the benign nature of silica nanoparticles (39, 42, 45, 46). The nanoparticles exhibit little or no cytotoxicity. The biocompatibility of silica nanoparticles makes them promising for in vivo observation of cell trafficking and tumor targeting as well as for disease diagnosis and treatment. As a bonus, the potential ability to prepare the nanoparticles with any existing single or multiple fluorophore(s) provides a diversity of nanoparticles for various applications. In addition to dye molecules, other components—such as genes, drugs, and ionophores—can be encapsulated to create multifunctional nanoparticles.
Enhanced sensitivity in bioanalysis and bioimaging Among the diverse research studies that use dye-doped silica nanoparticles, ultrasensitive bioanalysis and molecular imaging have made the most progress and attracted the greatest interest. In the past few years, many reports have described the distinct advantages of dye-doped silica nanoparticles over conventional dye molecules. These advantages allow them to be favorably used as fluorescence probes for applications ranging from biosensors to interfacial interaction studies (26–29, 47, 48). In a conventional fluorescence-based assay, a molecular recognition event has only one or a few fluorophores for signaling, like a summer firefly glimmering in the field. However, the highly luminescent silica nanoparticle probe enables significant enhancement of the analytical signal, behaving like a brilliant nanocandle flaring in the biological world.
Nanoparticles for cellular and tissue imaging One of the fundamental goals in biology is to understand the complex dynamics of intracellular networks, signal transduction, and cell– cell interplay. Sensitive and robust dye-doped silica nanoparticle probes can help yield a clearer understanding of these intra- and intercellular interactions. Different strategies have been explored for using nanoparticles to target cell surface proteins. Primary or secondary antibodies are covalently immobilized onto the nanoparticle surface to selectively and efficiently bind various cancer cells (23, 26, 49, 50), PTK2 cells (28), and single bacterial cells (48) via antibody–antigen recognition. A rapid fluorescence-based immunoassay has been developed that uses 60-nm dye-doped and bioconjugated silica nanoparticles for the detection of single E. coli O157:H7 bacterial cells (48). Antibodies against E. coli O157:H7 were conjugated to dye-doped silica nanoparticles to form nanoparticle–antibody conjugates or nanoprobe complexes, which bind to the antigen on the E. coli O157:H7 surface. As shown in the SEM images, the nanoparticle–antibody conjugates specifically associate with E. coli O157:H7 cells (Figure 3a) but not with E. coli DH5 cells, which lack the surface O157 antigens (Figure 3b). In addition to the fact that each nanoparticle provides a greatly amplified and photostable signal, many surface antigens on a given bacterial cell are available for specific recognition by antibodyconjugated nanoparticles. It is thus feasible to have thousands of nanoparticles bind to each bacterial cell and generate an extremely strong fluorescence signal (Figure 3c). (In a comparison experiment, tetramethylrhodamine [TMR] dye-labeled antibodies were used for bacterial imaging. The result showed a weak signal from the bacterial cells.) The nanoparticle-based immunoassay takes ~20 min, thus making real-time detection of bacterial pathogens possible. Accurate enumeration of 1–400 bacterial cells in 1 g of spiked ground-beef samples has also been demonstrated. This study clearly shows the power of nanoparticles for ultrasensitive detection of bacterial pathogens in food, clinical, and environmental samples. Antibody-mediated recognition is not the only strategy. Other affinity reagents, such as receptor ligands and recognition peptides, can also be immobilized onto nanoparticles to label cell-membrane-bound proteins. Folic acid (a small vitamin molecule recognized by many cancer cells) has been attached to dyedoped silica nanoparticles, enabling recognition of cancer biomarkers (51). A confocal imaging study demonstrated that folate nanoparticles (135 nm) are not too large for labeling SCC-9 cells. This finding supports other experimental evidence on folate-mediated delivery of large molecules (
