A nanoruby-based photoluminescent probe towards bioimaging applications

In the past decades, fluorescent organic dyes have been used extensively as molecular probes in bioimaging. However, their irreversible transition to the dark-state, termed photobleaching, photoinduced cytotoxicity, and biodegradibility limit their application scope. Photoluminescent nanomaterials provide a lucrative alternative to the fluorescent organic dyes due to their virtually unlimited photostability, inert core, controllable surface chemistry, biocompatibility, etc. In this study, I report on the development of a new-generation molecular probe based on a photoluminescent nanomaterial, termed as “nanorubies”, which represents ruby nanocrystals of the composition alpha-alumina (α-Al₂O₃) doped with chromium (Cr³⁺). In addition to the photoluminescent nanomaterial merits, nanoruby emission takes place in a remarkably narrow spectral band in the far-red spectral range (692 nm), and characterised by long photoluminescent lifetime (3.7 ms). The first reported advance of this study addresses development of a top-down, large-scale production of photoluminescent nanomaterials. Top-down methods take advantage of the existing variety of bulk and thin-film solid-state materials for improved prediction and control of the resultant nanomaterial properties. The power of this approach is demonstrated by using high-energy ball milling (HEBM) of alumina (Al₂O₃) and nanoruby. Nanoalumina and nanoruby particles with a mean size less 100 nm in their most stable α-crystallographic phase were produced in gram quantities suitable for biological and biomedical applications. Nanomaterial contamination from zirconia balls used in HEBM was reduced from 19% to 2% using a selective acid etching procedure. The biocompatibility of the milled nanomaterial was demonstrated by forming stable colloids in water and physiological buffers corroborated by the corresponding measured zeta potentials of +40 mV and -40 mV and characterised by in vitro cytotoxicity assays. The feasibility of anchoring a host of functional groups and biomolecules to milled nanoalumina and nanoruby surfaces was demonstrated by functionalisation of their surface which resulted in decoration of the nanoparticle surface with amino, carboxyl and polyethylene glycol groups suitable for further conjugation with functional biomolecules. Photoluminescence probes in combination with fast and sensitive imaging systems are much in demand. I describe a wide-field, time-gated photoluminescence microscopy system customised for ultrasensitive imaging of probes with long photoluminescence lifetime and exemplified by the use of nanorubies as a probe. The detection sensitivity relied on the long photoluminescence lifetime of nanoruby, which allowed its discrimination from the autofluorescence background and laser backscatter by employing a time-gated imaging acquisition mode. This mode enabled several-fold improvement of the photoluminescence imaging contrast of discrete ruby nanoparticles dispersed on a coverslip, and clearly visualised discrete nanorubies submerged in a layer of organic fluorescent dye, which would obscure the presence of most other photoluminescent nanoparticles. The ultrahigh-sensitivity imaging of photoluminescent molecular probes offers new opportunities, such as visualisation of intracellular molecular trafficking and detection of rare biomolecular events in an optically crowded background. In partnership with my group members, I have demonstrated specific labelling of hemagglutinin-tagged μ-opioid receptors by using biohybrid nanoruby complexes in conjunction with anti-hemagglutinin antibody, which has demonstrated the power of this new-generation photoluminescence nanotechnology not only for molecular-specific visualisation, but also for targeted delivery and therapy applications. Further, the realtime video-microscopy of nanoruby-based binding assay in serum has been demonstrated. This result shows the potential for pathogen detection in biological fluids,prospectively leading to early diagnosis applications.