Cellular Uptake of Upconversion Nanoparticles Based on Surface Polymer Coatings and Protein Corona.

Upconversion nanoparticles (UCNPs) are materials that provide unique advantages for biomedical applications. There are constantly emerging customized UCNPs with varying compositions, coatings, and upconversion mechanisms. Cellular uptake is a key parameter for the biological application of UCNPs. Uptake experiments have yielded highly varying results, and correlating trends between cellular uptake with different types of UCNP coatings remains challenging. In this report, the impact of surface polymer coatings on the formation of protein coronas and subsequent cellular uptake of UCNPs by macrophages and cancer cells was investigated. Luminescence confocal microscopy and elemental analysis techniques were used to evaluate the different coatings for internalization within cells. Pathway inhibitors were used to unravel the specific internalization mechanisms of polymer-coated UCNPs. Coatings were chosen as the most promising for colloidal stability, conjugation chemistry, and biomedical applications. PIMA-PEG (poly(isobutylene-alt-maleic) anhydride with polyethylene glycol)-coated UCNPs were found to have low cytotoxicity, low uptake by macrophages (when compared with PEI, poly(ethylenimine)), and sufficient uptake by tumor cells for surface-loaded drug delivery applications. Inductively coupled plasma-optical emission spectroscopy (ICP-OES) studies revealed that PIMA-coated NPs were preferentially internalized by the clathrin- and caveolar-independent pathways, with a preference for clathrin-mediated uptake at longer time points. PMAO-PEG (poly(maleic anhydride-alt-1-octadecene) with polyethylene glycol)-coated UCNPs were internalized by energy-dependent pathways, while PAA- (poly(acrylic acid)) and PEI-coated NPs were internalized by multifactorial mechanisms of internalization. The results indicate that copolymers of PIMA-PEG coatings on UCNPs were well suited for the next-generation of biomedical applications.

Lanthanide-Doped Upconversion Nanoparticles: Exploring A Treasure Trove of NIR-Mediated Emerging Applications.

Lanthanide-doped upconversion nanoparticles (UCNPs) possess the remarkable ability to convert multiple near-infrared (NIR) photons into higher energy ultraviolet-visible (UV-vis) photons, making them a prime candidate for several advanced applications within the realm of nanotechnology. Compared to traditional organic fluorophores and quantum dots (QDs), UCNPs possess narrower emission bands (fwhm of 10-50 nm), large anti-Stokes shifts, low toxicity, high chemical stability, and resistance to photobleaching and blinking. In addition, unlike UV-vis excitation, NIR excitation is nondestructive at lower power intensities and has high tissue penetration depths (up to 2 mm) with low autofluorescence and scattering. Together, these properties make UCNPs exceedingly favored for advanced bioanalytical and theranostic applications, where these systems have been well-explored. UCNPs are also well-suited for bioimaging, optically modulating chemistries, forensic science, and other state-of-the-art research applications. In this review, an up-to-date account of emerging applications in UCNP research, beyond bioanalytical and theranostics, are presented including optogenetics, super-resolution imaging, encoded barcodes, fingerprinting, NIR vision, UCNP-assisted photochemical manipulations, optical tweezers, 3D printing, lasing, NIR-II imaging, UCNP-molecule nanohybrids, and UCNP-based persistent luminescent nanocrystals.

Unlocking Long-Term Stability of Upconversion Nanoparticles with Biocompatible Phosphonate-Based Polymer Coatings.

Achieving long-term (>3 months) colloidal stability of upconversion nanoparticles (UCNPs) in biologically relevant buffers has been a major challenge, which has severely limited practical implementation of UCNPs in bioimaging and nanomedicine applications. To address this challenge, nine unique copolymers formulations were prepared and evaluated as UCNP overcoatings. These polymers consisted of a poly(isobutylene-alt-maleic anhydride) (PIMA) backbone functionalized with different ratios and types of phosphonate anchoring groups and poly(ethylene glycol) (PEG) moieties. The syntheses were done as simple, one-pot nucleophilic addition reactions. These copolymers were subsequently coated onto NaYF4:Yb3+,Er3+ UCNPs, and colloidal stability was evaluated in 1 × PBS, 10 × PBS, and other buffers. UCNP colloidal stability improved (up to 4 months) when coated with copolymers containing greater proportions of anchoring groups and higher phosphonate valences. Furthermore, small molecules could be conjugated to these overcoated UCNPs by use of copper-free click chemistry, as was done to demonstrate suitability for sensor and bioprobe development.

An extended cavity diode-pumped femtosecond Yb:KGW laser for applications in optical DNA sensor technology based on fluorescence lifetime measurements.

A reliable and power-scalable extended cavity diode-pumped passively mode-locked Yb:KGW laser generating ~200 fs long pulses at a repetition rate of 15 MHz was developed and characterized. The laser delivered up to 150 mW of average power at fundamental wavelength of 1040 nm, corresponding to a pulse energy of 10 nJ. The laser radiation was frequency-doubled in a single pass configuration within a nonlinear BIBO crystal to produce femtosecond green radiation at 520 nm with peak power of ~200 W. The generated second harmonic served as excitation source for optical DNA biosensor based on fluorescence lifetime measurements obtained using the time-correlated single photon counting technique.

Trends in the development of nucleic acid biosensors for medical diagnostics.

Some of the recent advances in the field of biosensors for nucleic acid analysis in medical diagnostic applications are highlighted. Particular attention is paid in this review to the progress made in two key areas of development: (i) enhancements achieved in device selectivity, and (ii) enhancements achieved in device sensitivity.

Rapid detection of single nucleotide polymorphisms associated with spinal muscular atrophy by use of a reusable fibre-optic biosensor.

Rapid (<2 min) and quantitative genotyping for single nucleotide polymorphisms (SNPs) associated with spinal muscular atrophy was done using a reusable (approximately 80 cycles of application) fibre-optic biosensor over a clinically relevant range (0-4 gene copies). Sensors were functionalized with oligonucleotide probes immobilized at high density (approximately 7 pmol/cm2) to impart enhanced selectivity for SNP discrimination and used in a total internal reflection fluorescence detection motif to detect 202 bp PCR amplicons from patient samples. Real-time detection may be done over a range of ionic strength conditions (0.1-1.0 M) without stringency rinsing to remove non-selectively bound materials and without loss of selectivity, permitting a means for facile sample preparation. By using the time-derivative of fluorescence intensity as the analytical parameter, linearity of response may be maintained while allowing for significant reductions in analysis time (10-100-fold), permitting for the completion of measurements in under 1 min.

A reusable optical nucleic acid biosensor applied to the rapid detection of single nucleotide polymorphisms associated with Spinal Muscular Atrophy.

Extract: Current methodologies for single nucleotide polymorphism (SNP) screening often require amplification, followed by time-consuming enzymatic manipulation (e.g., digestion or extension) and separation of the resultant products. Advancements in real-time PCR methods permit simultaneous amplification and quantification. Unfortunately, the ability to provide quantitative determinations can be made difficult by factors such as non-specific amplification and alterations in amplification efficiency owing to secondary-structure and sample matrix effects. Newer sensor and microarray technologies attempt to facilitate SNP analysis but usually require several hours for hybridization and data analysis. Furthermore, microarrays often cannot be reused as a result of limitations in the chemistries more routinely employed for nucleic acid immobilization. Difficulties associated with control of homogeneity of probe distribution, probe density and fidelity of the probe sequences (particularly when assembled by in-situ synthesis) can result in variations in the binding energetics of interfacial hybridization from spot to spot on an array, or even within individual array elements. Regions on the substrate may therefore exhibit variations in the selectivity, kinetics and dynamic range of response. The biosensor technology developed by our group presents an alternative to such an approach by use of controlled immobilization methodologies in a sensor format that provides for a reusable and quantitative technology where selectivity and kinetics may be more finely tuned.