Functional silicon nanoparticles

  

Diagnostics in biological systems are continuously searching for novel materials and systems for more specific detection with lower detection limits. Fluorescent probes are often used in the (selective) labeling of tissues and cellular components, since they give a high signal-to-noise ratio. The disadvantage of commonly used organic dyes is severe photobleaching, which makes prolonged studies impractical. Fluorescent quantum dots (QDs) are not prone to photobleaching, moreover, their fluorescence emission wavelength is size-tunable, making them the ideal candidates for bioimaging purposes.

Silicon nanoparticles (Si NPs) in particular have the additional advantage that the core consists of the non-toxic silicon, in contrast to the toxic elements (Cd, Se) typically employed in QDs. The development of a robust synthetic approach towards Si NPs, as well as a versatile functionalization strategy are therefore essential in enabling application in biological systems.

In Chapter 1, a general introduction on quantum dots (QDs) and in particular silicon nanoparticles is given; the origin of fluorescence is explained, and several synthetic methods are discussed. Chapter 2 describes the synthesis of Si NPs via the oxidation of magnesium silicide with bromine, yielding bromine-terminated Si NPs. Subsequent reaction with n-butyl lithium, and purification via column chromatography, resulted in butyl-terminated Si NPs. NMR analysis revealed that the major side-product (multiply brominated octane) was also in part attached to the Si NPs. Detailed characterization by IR and NMR confirmed the attachment of butyl-chains as well as a minor oxidation of the Si core. TEM measurements revealed a Si core size of 2.6 ± 0.7 nm. UV-Vis measurements showed a gradual increase in absorption with decreasing wavelengths, and a fluorescence emission maximum was observed at 390 nm (l_exc= 340 nm). The Si NPs were fractionated using size exclusion chromatography, which yielded four fractions containing Si NPs of different sizes. Fluorescence anisotropy measurements, XPS and DOSY NMR spectroscopy confirmed the size-differences between the fractionated samples.The slope of the UV spectrum increases upon smaller Si NP size, whereas a shift in fluorescence emission maxima was observed from 383 to 445 nm (l_exc= 340 nm), for respectively the smallest and largest Si NPs. Fluorescence quantum yields did not differ significantly between the different fractions, and the highest QY measured at l_exc= 496 nm is 5.2 %. Fluorescence emission lifetimes did not reveal distinct difference in the differently sized Si NPs, most likely due to the relatively small size-differences between the fractions. 

In Chapter 3 the synthesis of alkene-terminated Si NPs is described. To this purpose, the bromide-terminated Si NPs were reacted with 3-butenylmagnesium bromide. The resulting Si NPs were purified using SEC, and yielded Si NPs with a core size of 2.4 ± 0.5 nm as measured by TEM. Only minimal oxidation of the silicon core had occurred as observed by IR, while NMR spectroscopy confirmed successful attachment of the terminal alkenes onto the Si NPs. This also allowed for quantification of the amount of bromoalkanes attached to the Si NPs (butene : octane  = 1 : 0.36). UV-Vis absorption of the Si NPs did not change significantly as compared to butyl-terminated Si NPs. The extinction coefficient was determined to be 0.14 (mg mL_-1)_-1 at 300 nm and 0.035 (mg mL_-1)_-1 at 350 nm. A fluorescence emission maximum was observed at 525 nm (l_exc= 430 nm), while a QY of 7.1 ± 1.2 % was measured (l_exc= 496 nm). 

Modification of the alkene-terminated Si NPs using thiol-ene chemistry is described in Chapter 3. This reaction involves the radical-initiated coupling of a thiol to an alkene. The Si NPs were modified with thiolacetic acid, mercaptoethanol, thiolated triethyleneglycol monomethylether, and a thiolated polyethylene glycol 5000 monomethylether. The thiol-ene modification step did not significantly alter the photophysical properties of the Si NPs. Furthermore, IR and XPS showed that the functionalization step did not oxidize the silicon core. NMR and XPS results confirmed successful attachment of the functional thiols. In Chapter 4, carboxylic acid terminated Si NPs were synthesized by thiol-ene chemistry with 3 different spacer lengths; i.e. no spacer, a tetraethyleneglycol spacer and a PEG3000 spacer. The Si NPs were further functionalized by coupling an NH₂-terminated single stranded DNA molecule via EDC/NHS chemistry. Coupling and subsequent hybridization with the complementary strand was confirmed by gel electrophoresis, UV-Vis and fluorescence spectroscopy. This revealed that 2 to 3 DNA strands were attached to the Si NPs. Finally, Chapter 5 describes the initial investigations in the toxicity of Si NPs. To this purpose, Si NPs were synthesized via reduction of silicon tetrachloride, followed by hydrosilylation with functional alkenes. The Si NPs were capped with –C₃H₆NH₂, –C₁₁H₂₂N₃and –C₃H₆COOH groups, yielding positively and negatively charged, as well as neutral Si NPs. Two types of tests were preformed: the MTT test for mitochondrial activity, and the BrdU test for cell proliferation, both in the presence and absence of FCS. Upon exposure of human colonic Caco2 cells, the negatively charged Si NPs showed no observable cytotoxic effects, whereas the N₃-terminated Si NPs display a moderate toxicity with an IC50 of 500 mg/L in the presence of FCS in the MTT test, and the NH₂-terminated Si NPs display strong cytotoxic effects on the cells with an IC50 of 20 mg/L in the presence of FCS in the MTT assay. The described synthesis, followed by the versatile functionalization and bioconjugation, in combination with the low inherent cytotoxicity, shows that the Si NPs are readily suitable for applications in biological systems.