Defect Engineering of ZnO Nanoparticles for Bioimaging Applications

Abstract

Many promising attributes of ZnO nanoparticles (nZnO) have led to their utilization in numerous electronic devices and biomedical technologies. nZnO fabrication methods can create a variety of intrinsic defects that modulate the properties of nZnO, which can be exploited for various purposes. Here we developed a new synthesis procedure that controls certain defects in pure nZnO that are theorized to contribute to the n-type conductivity of the material. Interestingly, this procedure created defects that reduced the nanoparticle band gap to ∼3.1 eV and generated strong emissions in the violet to blue region while minimizing the defects responsible for the more commonly observed broad green emissions. Several characterization techniques including thermogravimetric analysis, Fourier-transform infrared spectroscopy, X-ray photoelectron spectroscopy, transmission electron microscopy, Raman, photoluminescence, and inductively coupled plasma mass spectrometry were employed to verify the sample purity, assess how modifications in the synthesis procedure affect the various defects states, and understand how these alterations impact the physical properties. Since the band gap significantly decreased and a relatively narrow visible emissions band was created by these defects, we investigated utilizing these new nZnO for bioimaging applications using traditional fluorescent microscopy techniques. Although most nZnO generally require UV excitation sources to produce emissions, we demonstrate that reducing the band gap allows for a 405 nm laser to sufficiently excite the nanoparticles to detect their emissions during live-cell imaging experiments using a confocal microscope. This work lays the foundation for the use of these new nZnO in various bioimaging applications and enables researchers to investigate the interactions of pure nZnO with cells through fluorescence-based imaging techniques.

Keywords: Raman; ZnO nanoparticles; bioimaging; cancer; defects; fluorescence; photoluminescence; toxicity.

Figure 1.

(a) Thermogravimetric Analysis plot of nZnO synthesized with Polyvinylpyrrolidone (PVP) demonstrating the mass loss is complete after 10 minutes of annealing at 450° C. (b) FTIR spectra of pure PVP, the as-prepared (non-annealed) nZnO and nZnO annealed at various temperatures for 10 minutes. FTIR data confirms that after 10 minutes of annealing at 450° C that no other chemical species from the precursors are retained with the NPs.

Figure 2.

(a) Low temperature (10 K) resonant Raman spectra (325 nm laser) of bulk ZnO and the nZnO synthesized with various amount of PVP. Spectra shows a systematic shift of the LO phonon modes as the defect related peak centered at 470 cm⁻¹ increases in area. (b) Zoomed in spectra of the defect related peak and the 1 LO phonon mode to highlight the systematic shift of the peak as the defect related peak increases in area. This systematic shift is likely due to phonon confinement due to increases in defects present in the ZnO NPs.

Figure 3.

Low temperature Photoluminescence (PL) spectra of the nZnO synthesized by varying different parameters in the synthesis procedure. (a) PL spectra of the nZnO synthesized with various amounts of PVP while all other parameters are kept constant. At low PVP:Zinc Acetate (ZnAc) ratios, little change in the PL spectra is noted. As the amount increases, the well- defined peaks broaden and extend into the visible spectrum. (b) PL spectra of the nZnO annealed at various temperatures. The unannealed sample spectra extends further into the UV range, likely due to the presence of various retained species from the synthesis procedure. As the annealing temperature increase, the main peak in the spectra red-shifts and broadens out to ~450 nm. At the highest temperature used, the broad green emission spectra, commonly reported on nZnO, becomes apparent. (c) PL spectra of the nZnO with various amounts of water added in the synthesis. At relatively high additions of water, the main broad peak narrows likely due to changes from a zinc rich environment to a more balanced oxygen to zinc environment. (d) PL spectra of ZnO NPs synthesized using various methods (ethanol as the solvent, Flame spray pyrolysis (FSP), diethylene glycol as the solvent (DEG) and micron sized (Bulk) ZnO) to demonstrate the extreme difference of the new nZnO compared to other commonly used methods.

Figure 4.

Deconvolution of the low temperature photoluminescence spectra of nZnO synthesized with (a) no PVP and (b) a 2:1 (w/w) PVP to zinc acetate ratio. (a) The NPs with low defect states and well-defined peaks were first deconvoluted to assign positions and acquire reasonable peak widths for the near band edge related emissions. (b) The nZnO sample with a relatively high number of defects shows multiple peaks related to various defects formed during the new synthesis procedure.

Figure 5.

UV-Vis spectra of the new nZnO synthesized with various amounts of PVP compared to the most similar reported method using DEG as a solvent. The UV-Vis spectra were converted into a Tauc Plot (inset) to determine the optical band gap of each sample. The DEG method produced nZnO with an optical band gap of ~3.31 eV whereas the new method shifted the optical band gap to below 3.1 eV.

Figure 6.

(a) The emission spectra of the nZnO recorded with a confocal microscope using a 405 nm laser as an excitation source. Using a 100x objective, the fluorescence image (b) of the nZnO and bright field (c) images were collected and overlaid (d) to demonstrate that the fluorescence is from the novel nZnO.

Figure 7.

Viability profile of Jurkat cells after treatment with nZnO for 48 hours at various concentrations. No apparent toxicity is present for concentrations up to 100 µM.

Figure 8.

Times series fluorescent images of the nZnO over a 20-minute period. The NPs were subjected to 20 minutes of laser exposure and no apparent decrease in fluorescence intensity was noted, demonstrating their resistance to photobleaching. Like quantum dots, the fluorescence stems from the physical properties of the NPs and is proposed to stem from transitions between energy levels due to the defect present in the samples.

Figure 9.

Confocal images (optical plane thickness= 0.6 µm) of T47D (breast cancer) cells stained with CellMask Orange. The top row (a-c) depicts untreated T47D cells to assess any auto-fluorescence generated using the same laser settings in the 405 nm laser channel. The second row (d-f) depicts the T47D cells after 2 hours of nZnO treatment. Little to no change in cell morphology is noted. The bottom row (g-i) depicts T47D cells after 24 hours nZnO treatment. The cellular morphology is drastically changed, many blebs are noted, and essentially all NPs that are detectable appear to have been internalized.