Rare-earth-doped upconversion nanoparticles (UCNPs) have often been used in combination with fluorescent dyes for sensing applications. In these systems, sensing can be achieved through the modulation of Förster resonant energy transfer (FRET) between the dye and the UCNP. The effects of FRET in such cases are complex, as the extent to which FRET is experienced by the rare-earth ions is dependent on their position within the nanoparticle. Here, we develop an analytical model to accurately describe the effects of FRET for such a system. As a proof of principle, we verify our model by considering the case of a pH sensor comprised of fluorescein isothiocyanate and Tm3+-doped UCNPs. We extend our model to the case of core–shell UCNPs and discuss the design of an optimal FRET-based biosensor using UCNPs.

Surface-enhanced Raman scattering (SERS) induced from nanostructured noble metals has a great potential for molecular detection and analysis. However, it has been a challenge to fabricate a reliable SERS-active nanostructure that produces highly sensitive signal response with high fidelity for use in the practical sensing platform.Here, a bacteriophage MS2 with highly regular structure was introduced as a molecular scaffold to assemble nanoparticles into a dense and reproducible three-dimensional raspberry-shaped nanostructure. The nanoraspberry features evenly distributed electromagnetic hot spots responsible for single-molecule-level analysis of SERS-based sensing. By selecting a rigid molecular linker, as well as MS2 phage, the gold nanoparticles were able to be assembled into nanoraspberry superstructures with precisely defined positions, producing strong electric near-field enhancement between nanometer-sized interparticle gaps. The numerical simulation and experimental measurement demonstrated that the nanoraspberry structure produces strong SERS signal amplification, with remarkable intra-and interbatch signal uniformity, proving that structural reproducibility originated from rigid building blocks can lead to a reliable SERS measurement for molecular sensing and analysis applications.

Among the most promising sensors being developed are those that rely on Förster resonance energy transfer (FRET). FRET is a non-radiative process where energy can be transferred from a donor (ion or molecule) to an acceptor. [3][4][5] The rate of this exchange is highly distance-dependent and fades rapidly with increasing donoracceptor separation. Thus sensors that utilize FRET are capable of measuring local variations in critical biological parameters.Of the possible FRET-based sensors, those based on upconverting nanoparticles (UCNPs) are among the most promising. UCNPs are unique nanoparticles that can emit at several distinct visible wavelengths from a single, near-infrared excitation source. UCNPs exhibit many advantageous characteristics for bioimaging and sensing such as no photobleaching, no blinking, and no background autofluorescence. [6,7] In particular, many groups have attempted to utilize UCNPs as a FRET-based sensor by conjugating a fluorescent dye to the UCNP surface. [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] The key advantage of this configuration over other FRET-based schemes is that the multimodal emission of UCNPs allows for ratiometric sensing in which the relative intensities of two distinct peaks are compared rather than just the enhancement or quenching of a single one. Such ratiometric sensing reduces the sensing error due to variations in excitation intensity or nanoparticle concentrations, and thus makes these sensors much more robust against both sensor inhomogeneity and environmental noise. [26] However, FRET is not the sole process occurring that can affect the collected emissions. Unless a single UCNP can be measured directly, there exists a non-negligible chance that a photon emitted by one UCNP can be absorbed by a dye molecule on another nanoparticle. Such photon reabsorption (PR) would in turn lead to a decrease in the collected emission, thereby obscuring the true, localized FRET response of the original nanoparticle. An additional complication also arises from the fact that many of the visible UCNP emission lines originate from the same energy level. Since FRET acts as a decay pathway for an excited state electron, all emissions from this level should be affected equally by FRET, not just the one that overlaps with the dye's absorption band. To our knowledge, although the distinction between FRET and photon reabsorption has been discussed before, [27][28][29] no FRET-UCNP sensor has yet been proposed that accurately accounts for these complications.

In this work, we demonstrate that a photo-crosslinkable conjugate of upconverting nanoparticles and cytosine deaminase can catalyze prodrug conversion specifically at tumor sites in vivo. Non-covalent association of proteins and peptides with cellular surfaces leads to receptor-mediated endocytosis and catabolic degradation. Recently, we showed that covalent attachment of proteins such as affibodies to cell receptors yields extended expression on cell surfaces with preservation of protein function. To adapt this technology for in vivo applications, conjugates were prepared from upconverting nanoparticles and fusion proteins of affibody and cytosine deaminase enzyme (UC-ACD). The affibody allows covalent photo-cross-linking to epidermal growth factor receptors (EGFRs) overexpressed on Caco-2 human colorectal cancer cells under near-infrared (NIR) light. Once bound, the cytosine deaminase portion of the fusion protein converts the prodrug 5-fluorocytosine (5-FC) to the anticancer drug 5-fluorouracil (5-FU). NIR covalent photoconjugation of UC-ACD to Caco-2 cells showed 4-fold higher retention than observed with cells that were not irradiated in vitro. Next, athymic mice expressing Caco-2 tumors showed 5-fold greater UC-ACD accumulation in the tumors than either conjugates without the CD enzyme or UC-ACDs in the absence of NIR excitation. With oral administration of 5-FC prodrug, tumors with photoconjugated UC-ACD yielded 2-fold slower growth than control groups, and median mouse survival increased from 28 days to 35 days. These experiments demonstrate that enzyme-decorated nanoparticles can remain viable after a single covalent photoconjugation in vivo, which can in turn localize prodrug conversion to tumor sites for multiple weeks.

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