Dye-doped Silica Nanoparticles Research Articles

Advancing fluorescence imaging: enhanced control of cyanine dye-doped silica nanoparticles

BackgroundSilica nanoparticles (SNPs) have immense potential in biomedical research, particularly in drug delivery and imaging applications, owing to their stability and minimal interactions with biological entities such as tissues or cells.ResultsWith synthesized and characterized cyanine-dye-doped fluorescent SNPs (CSNPs) using cyanine 3.5, 5.5, and 7 (Cy3.5, Cy5.5, and Cy7). Through systematic analysis, we discerned variations in the surface charge and fluorescence properties of the nanoparticles contingent on the encapsulated dye-(3-aminopropyl)triethoxysilane conjugate, while their size and shape remained constant. The fluorescence emission spectra exhibited a redshift correlated with increasing dye concentration, which was attributed to cascade energy transfer and self-quenching effects. Additionally, the fluorescence signal intensity showed a linear relationship with the particle concentration, particularly at lower dye equivalents, indicating a robust performance suitable for imaging applications. In vitro assessments revealed negligible cytotoxicity and efficient cellular uptake of the nanoparticles, enabling long-term tracking and imaging. Validation through in vivo imaging in mice underscored the versatility and efficacy of CSNPs, showing single-switching imaging capabilities and linear signal enhancement within subcutaneous tissue environment.ConclusionsThis study provides valuable insights for designing fluorescence imaging and optimizing nanoparticle-based applications in biomedical research, with potential implications for targeted drug delivery and in vivo imaging of tissue structures and organs.Graphical

Pyrene doped silica nanoparticles: Synthesis, oxygen sensing, and singlet oxygen generation

Dye-doped silica nanoparticles have garnered attention from researchers owing to their beneficial properties, such as signal amplification, surface modification, and biocompatibility. In this study, pyrene-doped silica nanoparticles (PSNPs) were prepared using the reverse microemulsion approach. Pyrenebutyric acid conjugated with aminopropyltriethoxysilane was synthesized to obtain durable PSNPs with low dye leaching via the covalent encapsulation of pyrene inside a silica matrix. The PSNPs were spherical, with a diameter of 72 nm. The PSNPs exhibited excimer fluorescence upon photoexcitation. To demonstrate the applicability of the synthesized PSNPs in biological applications, including photodynamic therapy (PDT) and oxygen sensing, the influence of oxygen on the fluorescence spectrum was investigated. The excimer fluorescence intensity was quenched by oxygen, along with a decrease in fluorescence lifetime. Ratiometric fluorescence sensing and fluorescence lifetime sensing of oxygen concentration were demonstrated. The biomolecular quenching rate constant was evaluated using the Stern–Volmer plot of the fluorescence lifetime. Moreover, utilizing a chemical trap of singlet oxygen molecules, it was demonstrated that singlet oxygen was generated by the photosensitization of PSNP. The PSNPs synthesized in this study are potentially capable of oxygen sensing, PDT, and bioimaging for various biological applications.

Highly bright full-color emission from dye-doped silica nanoparticles with prevention of dye self-quenching

Although fluorescent dye-doped silica nanoparticles (SiOx NPs) have been widely studied for a broad range of optical probes and devices, it is still challenging to fabricate highly bright dye-doped SiOx NPs because the luminescence quantum yields of dye-doped SiOx NPs are often lower than those of the dye alone. Herein, we report highly bright full-color emissions from dye-doped SiOx NPs that prevent dye self-quenching. Green and red fluorescence-emitting dye-doped silica nanoparticles (G-SiOx and R-SiOx NPs) were synthesized from 3-(aminopropyl) triethoxysilane (APTES) and fluorescence dyes (fluorescein, Rhodamine B). The quantum yields (QY) of G-SiOx and R-SiOx NPs were 80.9% and 25.5%, respectively; doping these dyes into the SiOx NPs did not decrease the QY values of the dyes. The combination of bule luminescent SiOx NPs (QY = 32.3%) with G-and R-SiOx NPs displayed an intense pure white (x = 0.33, y = 0.33) fluorescence emission.

Thermo-sensitive hydrogels for forward osmosis with NIR light-induced freshwater recovery

Croconaine dye-doped silica nanoparticles with excellent ability of photothermal conversion and photostability were used for the NIR light-driven water recovery of hydrogels in FO desalination just by simply turning on the light source.

(Invited) Ingenious Nanomaterials for Ultrasensitive ECL

The impact of nanotechnology and nanosystems on analytical science is hardly overlooked. In the search for ever-increasing sensitivity in biomedical sensors, nanoparticles have been playing a unique role as, e.g., ultrabright labels in clinical analysis (markers, tumor cells, and pharmaceuticals) and in the detection of pathogenic microorganisms, toxic agents, and pesticides in the environmental field and food products. Coupling such nanosystems with electrochemiluminescence (ECL), which naturally brings improved signal-to-noise ratio compared to photoluminescence, with minimized effects due to light scattering and luminescence background, has brought about new systems and strategies for analytes determination, even in very complex matrices, such as urine, blood or lysate.Among the several nanosystems, dye-doped silica nanoparticles (DDSNs) have proved as very promising and versatile nanomaterials in ECL-based bioanalytical platforms. The increase of the number of complexes active in the generation of the ECL signals together with an even larger increase in the signal stability, represents in fact a promising strategy towards ultrasensitive ECL.Approaches based on ECL generation on the nanoscale using luminophore-reporter-modified DNA-based nanoswitches (i.e., molecular beacon) has been additionally investigated and, in this context, the development of innovative amplification-free detection methods represents a significant breakthrough compared to existing PCR-based methodologies, allowing the integration of nucleic acid detection on portable and low-cost sensor devices, and enabling the massive diagnostic screening.

Supraparticles with a Mechanically Triggerable Color‐Change‐Effect to Equip Coatings with the Ability to Report Damage

Small scratches and abrasion cause damage to packaging coatings. Albeit often invisible to the human eye, such small defects in the coating may ultimately have a strong negative impact on the whole system. For instance, gases may penetrate the coating and consequently the package barrier, thus leading to the degradation of sensitive goods. Herein, the indicators of mechanical damage in the form of particles are reported, which can readily be integrated into coatings. Shear stress-induced damage is indicated by the particles via a color change. The particles are designed as core-shell supraparticles. The supraparticle core is based on rhodamine B dye-doped silica nanoparticles, whereas the shell is made of alumina nanoparticles. The alumina surface is functionalized with a monolayer of a perylene dye. The resulting core-shell supraparticle system thus contains two colors, one in the core and one in the shell part of the architecture. Mechanical damage of this structure exposes the core from the shell, resulting in a color change. With particles integrated into a coating lacquer, mechanical damage of a coating can be monitored via a color change and even be related to the degree of oxygen penetration in a damaged coating.

Characteristics of Dye-doped Silica Nanoparticles- Based Random Lasers in the Air and Water

Random lasers based on dye-doped silica nanoparticles are attracted for biomedical applications due to their biocompatibility and high brightness. Several laser structures including silica powder and film have been reported. However, the dependence of lasing characteristics including lasing threshold and emission wavelength on the laser size and working environment have not been explored. Here, we demonstrate and compare the lasing characteristics of dye-doped silica random lasers in air and water. These lasers present in thin structures, the so-called microslices, with a thickness of 1 µm and various dimensions from 30 to 300 µm. It is found that the lasing threshold (Ith) decreases with increasing laser size such as for sample in the air and for sample in water, where A the sample surface area. For a similar size, the lasing threshold of the sample in water is about 3-8 times higher than that of the sample in the air. In addition, the lasing peak wavelength exhibits a red-shift with increasing laser size. In the air, a shift of 8 nm is recorded when the sample surface area increases from 21×103 to 169×103 µm2. Furthermore, for a similar size, the lasing wavelength of the sample in the air is also red-shifted (13 nm in average ) compared with that of the sample in water. Our finding provides useful information for the use of silica-based random lasers in bioimaging and biosensing applications.

Sensitive detection of E. coli using bioconjugated fluorescent silica nanoparticles

Fluorescent dye-doped silica nanoparticles have been a major focus of research and development as potential fluorescent probes in biological systems. This is due to their strong fluorescence intensity, excellent photostability, biocompatibility, and ease of surface modification for bioconjugation. In this study, spherical silica nanoparticles doped with fluorescein isothiocyanate (FITC) were synthesized using reverse microemulsion method. The average diameter of the nanoparticles was 78 nm with a narrow size distribution (78 ± 49.50 nm). FITC was incorporated into the silica sphere through 3-aminopropyltriethoxysilane, which minimized the leakage of the dye. Only about 30% of the dye leaked out from these nanoparticles, after they were sonicated in aqueous medium for 60 min and the fluorescent intensity remained comparatively constant over time, showing an increased photostability (∼ 1.5 times higher) compared to the free dye in solution. The fluorescence emission spectrum of nanoparticles showed no apparent shift (maximum emission at 518 nm) when compared with the free dye in solution. The nanoparticle surface was chemically modified with amine and carboxyl functionalities to allow for Escherichia coli antibody conjugation. E. coli bacteria is widely used as a biological indicator to identify fecal contamination in food and water samples. Nanoparticle-based biosensors for the detection of E. coli have a vital importance in water quality, public health, and food safety. The E. coli in water was successfully observed under a fluorescence microscope using the antibody conjugated fluorescent silica nanoparticles synthesized, proving the feasibility of using them in a biosensor for qualitative determination of E. coli. The significance of this study lies in the relatively simple synthetic procedures as compared to those that have been in other studies reported in the literature. The ability to detect live cells under the fluorescence microscope further proves the suitability of these functionalized nanoparticles in live cell imaging.

Role of Aqueous-Phase Calcination in Synthesis of Ultra-Stable Dye-Embedded Fluorescent Nanoparticles for Cellular Probing.

Fluorescence imaging is a major driver of discovery in biology, and an invaluable asset in clinical diagnostics. To overcome quenching limitations of conventional fluorescent dyes and further improve intensity, nanoparticle-based constructs have been the subject of intense investigation, and within this realm, dye-doped silica-coated nanoparticles have garnered significant attention. Despite their growing popularity in research, fluorescent silica nanoparticles suffer from a significant flaw. The degradation of these nanoparticles in biological media by hydrolytic dissolution is underreported, leading to serious misinterpretations, and limiting their applicability for livecellandinvivoimaging. Here, the development of an ultra-stable, dye-embedded, silica-coated metal nanoparticle is reported, and its superior performance in long-term live cell imaging is demonstrated. While conventional dye-doped silica nanoparticles begin to degrade within an hour in aqueous media, by leveraging a modified liquid calcination process, this new construct is shown to be stableforat least 24 h.The stability of this metal-enhanced fluorescent probe in biologically relevant temperatures and media, and its demonstrated utility for cell imaging, paves the way for its future adoption in biomedical research.

Dye-Doped Silica Nanoparticles for Enhanced ECL-Based Immunoassay Analytical Performance

Electrochemiluminescence (ECL) is a leading technique in bioanalysis.[1] It is a luminescent phenomenon where the excited species are produced with an electrochemical stimulus rather than with a light excitation source. For this reason, ECL displays improved signal-to-noise ratio compared to photoluminescence, with minimized effects due to light scattering and luminescence background.[2]Chasing ever-increasing sensitivities, ECL can ideally be coupled to nanotechnology to develop new strategies for clinical applications and analyte determination also in real and complex matrices.[3] In particular, dye-doped silica nanoparticles (DDSNs) present many advantages: they can be easily synthesized, are hydrophilic and are prone to bioconjugation thanks to silica chemistry. With this approach we were able to achieve extremely bright systems and DDSNs allow the 8.5 fold increase of ECL intensity.[4] In DDSNs, light emission is influenced by the combination of several factors that make DDSNs complex multichromophoric structures. However, when ECL comes into play, the scenario is even more complicated due to the coreactant−NP interactions effect. Such complex scenario was approached at the theoretical level by developing suitable mechanistic models for ECL generation[5] while, at the same time, the influence of doping level on ECL efficiency was evaluated. Our results showed that the ECL intensity of a nanosized system cannot be merely incremented acting on doping, since other parameters come into play. These studies support the application in ECL microscopy providing valuable indications for the design of more efficient ECL nano- and micro-sized labels towards more sensitive ECL-based immunoassay for bioanalysis applications. [6]References[1] W. Miao, “Electrogenerated Chemiluminescence and Its Biorelated Applications”, Chem. Rev., 2008, vol. 108, pp. 2506–2553.[2] Z. Liu, W. Qi, G. Xu, “Recent advances in electrochemiluminescence”, Chem. Soc. Rev., 2015, vol. 44, pp. 3117–3142.[3] C. Ma, Y. Cao, X. Gou, J. J. Zhu, “Recent progress in electrochemiluminescence sensing and imaging”, Anal. Chem., 2015, vol. 92, pp. 431-454.[4] G. Valenti, E. Rampazzo, S. Bonacchi, L. Petrizza, M. Marcaccio, M. Montalti, L. Prodi, F. Paolucci, “Variable Doping Induces Mechanism Swapping in Electrogenerated Chemiluminescence of Ru(bpy) 3 2+ Core–Shell Silica Nanoparticles”, J. Am. Chem. Soc., 2016, vol. 138, pp. 15935–15942.[5] E. Daviddi, A. Oleinick, I. Svir, G. Valenti, F. Paolucci, C. Amatore, “Theory and Simulation for Optimising Electrogenerated Chemiluminescence from Tris(2,2′-bipyridine)-ruthenium(II)-Doped Silica Nanoparticles and Tripropylamine”, ChemElectroChem, 2017, vol. 4, pp. 1719–1730.[6] A. Zanut, F. Palomba, M. Rossi Scota, S. Rebeccani, M. Marcaccio, D. Genovese, E. Rampazzo, G. Valenti, F. Paolucci, L. Prodi, "Dye‐Doped Silica Nanoparticles for Enhanced ECL‐Based Immunoassay Analytical Performance", Angew. Chemie Int. Ed., 2020, vol. 132, pp. 22042-22047 Figure 1

Uptake and Intracellular Trafficking Studies of Multiple Dye-Doped Core-Shell Silica Nanoparticles in Lymphoid and Myeloid Cells.

IntroductionSince most biologically active macromolecules are natural nanostructures, operating in the same scale of biomolecules gives the great advantage to enhance the interaction with cellular components. Noteworthy efforts in nanotechnology, particularly in biomedical and pharmaceutical fields, have propelled a high number of studies on the biological effects of nanomaterials. Moreover, the determination of specific physicochemical properties of nanomaterials is crucial for the evaluation and design of novel safe and efficient therapeutics and diagnostic tools. In this in vitro study, we report a physicochemical characterisation of fluorescent silica nanoparticles (NPs), interacting with biological models (U937 and PBMC cells), describing the specific triggered biologic response.MethodsFlow Cytometric and Confocal analyses are the main method platforms. However TEM, NTA, DLS, and chemical procedures to synthesize NPs were employed.ResultsNTB700 NPs, employed in this study, are fluorescent core-shell silica nanoparticles, synthesized through a micelle-assisted method, where the fluorescence energy transfer process, known as FRET, occurs at a high efficiency rate. Using flow cytometry and confocal microscopy, we observed that NTB700 NP uptake seemed to be a rapid, concentration-, energy- and cell type-dependent process, which did not induce significant cytotoxic effects. We did not observe a preferred route of internalization, although their size and the possible aggregated state could influence their extrusion. At this level of analysis, our investigation focuses on lysosome and mitochondria pathways, highlighting that both are involved in NP co-localization. Despite the main mitochondria localization, NPs did not induce a significant increase of intracellular ROS, known inductors of apoptosis, during the time course of analyses. Finally, both lymphoid and myeloid cells are able to release NPs, essential to their biosafety.DiscussionThese data allow to consider NTB700 NPs a promising platform for future development of a multifunctional system, by combining imaging and localized therapeutic applications in a unique tool.

A two-photon fluorescence silica nanoparticle-based FRET nanoprobe platform for effective ratiometric bioimaging of intracellular endogenous adenosine triphosphate.

Two-photon fluorescence imaging is one of the most attractive imaging techniques for monitoring important biomolecules in the biomedical field due to its advantages of low light scattering, high penetration depth, and suppressed photodamage/phototoxicity under near-infrared excitation. However, in actual biological imaging, organic two-photon fluorescent dyes have disadvantages such as high biological toxicity and their fluorescence efficiency is easily affected by the complex environment in organisms. In this study, a novel nanoprobe platform with two-photon dye-doped silica nanoparticles was developed for FRET-based ratiometric biosensing and bioimaging, with endogenous ATP chosen as the target for detection. The nanoprobe has three components: (1) a two-photon dye-doped silica nanoparticle core, which serves as an energy donor for FRET; (2) amino-modified hairpin primers with carboxy fluorescein as an energy acceptor for FRET; (3) an aptamer acting as a recognition unit to realize the probing function. The nanoprobe showed ratiometric fluorescence responses for ATP detection with high sensitivity and high selectivity in vivo. Moreover, the nanoprobe showed satisfactory ratiometric two-photon fluorescence imaging of endogenous ATP in living cells and tissues (penetration depth of 190 nm). These results indicated that novel two-photon silica nanoparticles can be constructed by doping a two-photon fluorescent dye into silica nanoparticles, and they can effectively solve the disadvantages of two-photon fluorescent dyes. These excellent performances indicate that this novel nanoprobe platform will become a very valuable molecular imaging tool, which can be widely used in the biomedical field for drug screening and disease diagnosis and other related research.

Fabrication of Double Emission Enhancement Fluorescent Nanoparticles with Combined PET and AIEE Effects.

The major challenge in the fabrication of fluorescent silica nanoparticles (FSNs) based on dye-doped silica nanoparticles (DDSNs) is aggregation-caused fluorescence quenching. Here, we constructed an FSN based on a double emission enhancement (DEE) platform. A thio-reactive fluorescence turn-on molecule, N-butyl-4-(4-maleimidostyryl)-1,8-naphthalimide (CS), was bound to a silane coupling agent, (3-mercaptopropyl)-trimethoxysilane (MPTMS), and the product N-butyl-4-(3-(trimethoxysilyl-propylthio)styryl)-1,8-naphthalimide (CSP) was further used to fabricate a core–shell nanoparticle through the Stöber method. We concluded that the turn-on emission by CSP originated from the photoinduced electron transfer (PET) between the maleimide moiety and the CSP core scaffold, and the second emission enhancement was attributed to the aggregation-induced emission enhancement (AIEE) in CSP when encapsulated inside a core–shell nanoparticle. Thus, FSNs could be obtained through DEE based on a combination of PET and AIEE effects. Systematic investigations verified that the resulting FSNs showed the traditional solvent-independent and photostable optical properties. The results implied that the novel FSNs are suitable as biomarkers in living cells and function as fluorescent visualizing agents for intracellular imaging and drug carriers.

Dye‐Doped Silica Nanoparticles for Enhanced ECL‐Based Immunoassay Analytical Performance

AbstractThe combination of highly sensitive techniques such as electrochemiluminescence (ECL) with nanotechnology sparked new analytical applications, in particular for immunoassay‐based detection systems. In this context, nanomaterials, particularly dye‐doped silica nanoparticles (DDSNPs) are of high interest, since they can offer several advantages in terms of sensitivity and performance. In this work we synthesized two sets of monodispersed and biotinylated [Ru(bpy)3]2+‐doped silica nanoparticles, named bio‐Triton@RuNP and bio‐Igepal@RuNP, obtained following the reverse microemulsion method using two different types of nonionic surfactants. Controlling the synthetic procedures, we were able to obtain nanoparticles (NPs) offering highly intense signal, using tri‐n‐propylamine (TPrA) as coreactant, with bio‐Triton@RuNps being more efficient than bio‐Igepal@RuNP.

Dye-Doped Silica Nanoparticles for Enhanced ECL-Based Immunoassay Analytical Performance.

The combination of highly sensitive techniques such as electrochemiluminescence (ECL) with nanotechnology sparked new analytical applications, in particular for immunoassay-based detection systems. In this context, nanomaterials, particularly dye-doped silica nanoparticles (DDSNPs) are of high interest, since they can offer several advantages in terms of sensitivity and performance. In this work we synthesized two sets of monodispersed and biotinylated [Ru(bpy)3 ]2+ -doped silica nanoparticles, named bio-Triton@RuNP and bio-Igepal@RuNP, obtained following the reverse microemulsion method using two different types of nonionic surfactants. Controlling the synthetic procedures, we were able to obtain nanoparticles (NPs) offering highly intense signal, using tri-n-propylamine (TPrA) as coreactant, with bio-Triton@RuNps being more efficient than bio-Igepal@RuNP.

Functionalized Fluorescent Silica Nanoparticles for Bioimaging of Cancer Cells.

Functionalized fluorescent silica nanoparticles were designed and synthesized to selectively target cancer cells for bioimaging analysis. The synthesis method and characterization of functionalized fluorescent silica nanoparticles (50–60 nm), as well as internalization and subcellular localization in HeLa cells is reported here. The dye, rhodamine 101 (R101) was physically embedded during the sol–gel synthesis. The dye loading was optimized by varying the synthesis conditions (temperature and dye concentration added to the gel) and by the use of different organotriethoxysilanes as a second silica precursor. Additionally, R101, was also covalently bound to the functionalized external surface of the silica nanoparticles. The quantum yields of the dye-doped silica nanoparticles range from 0.25 to 0.50 and demonstrated an enhanced brightness of 230–260 fold respect to the free dye in solution. The shell of the nanoparticles was further decorated with PEG of 2000 Da and folic acid (FA) to ensure good stability in water and to enhance selectivity to cancer cells, respectively. In vitro assays with HeLa cells showed that fluorescent nanoparticles were internalized by cells accumulating exclusively into lysosomes. Quantitative analysis showed a significantly higher accumulation of FA functionalized fluorescent silica nanoparticles compared to nanoparticles without FA, proving that the former may represent good candidates for targeting cancer cells.

Preparation of 1, 3, 6, 8-Pyrenesulfonic Acid Tetrasodium Salt Dye-Doped Silica Nanoparticles and Their Application in Water-Based Anti-Counterfeit Ink.

In order to improve the luminescent stability of water-based anti-counterfeit ink, a new fluorescent material is prepared by doping dye into silica nanoparticles. Water soluble anionic dye 1, 3, 6, 8-pyrenesulfonic acid sodium salt (PTSA) is selected as the dopant. In this work, PTSA is successfully trapped into silica nanoparticles (SiNPs) by the reverse microemulsion method using cationic polyelectrolyte poly (dimethyl diallyl ammonium chloride; PDADMAC) as a bridge. The UV absorption spectra, fluorescence emission spectra and fluorescent decay curves are used to describe the luminescent properties of the PTSA-doped silica nanoparticles (PTSA-SiNPs). In addition, the as-prepared PTSA-SiNPs and polyurethane waterborne emulsion are used to prepare water-based anti-counterfeit ink, and fluorescent patterns are successfully printed through screen-printing. The samples printed by the ink exhibit desirable fluorescence properties, heat stability, robust photostability, and a fluorescent anti-counterfeit effect, which makes the PTSA-SiNPs promising luminescent materials for anti-counterfeit applications.

Silica based biocompatible random lasers implantable in the skin

We demonstrate efficient random lasing from microslices containing dye-doped colloidal silica nanoparticles. The nanoparticles with diameters of 20 nm were synthesized by a modified Stöber method, having a high emission efficiency and well dispersed in an aqueous medium. From the suspension of the nanoparticles, a film is created via self-assembly of colloidal nanoparticles. After grinding the film, microslices with dimensions of around 100–200μm and thickness of 2μm are obtained. Under optical pulse excitation, these microslices emit random lasing. The lasers operate well in the air and also in water. Their emission spectrum and lasing threshold in both conditions are studied and compared. Interestingly, lasing emission can be observed from a laser embedded into the porcine skin tissue. Our work provides a simple technique and architecture for biocompatible random lasers which have potential for biosensing and biological probes.

Constructing a Local Hydrophobic Cage in Dye-Doped Fluorescent Silica Nanoparticles to Enhance the Photophysical Properties.

Aggregation-caused quenching (ACQ) and poor photostability in aqueous media are two common problems for organic fluorescence dyes which cause a dramatic loss of fluorescence imaging quality and photodynamic therapy (PDT) failure. Herein, a local hydrophobic cage is built up inside near-infrared (NIR) cyanine-anchored fluorescent silica nanoparticles (FSNPs) in which a hydrophobic silane coupling agent (n-octyltriethoxysilane, OTES) is doped into FSNPs for the first time to significantly inhibit the ACQ effect and inward diffusion of water molecules. Therefore, the obtained optimal FSNP-C with OTES-modification can provide hydrophobic repulsive forces to effectively inhibit the π–π stacking interaction of cyanine dyes and simultaneously reduce the formation of strong oxidizing species (•OH and H2O2) in reaction with H2O, resulting in the best photostability (fluorescent intensity remained at 90.1% of the initial value after 300 s of laser scanning) and a high PDT efficiency on two- and three-dimensional (spheroids) HeLa cell culture models. Moreover, through molecular engineering (including increasing covalent anchoring sites and steric hindrance groups of cyanine dyes), FSNP-C exhibits the highest fluorescent intensity both in water solution (12.3-fold improvement compared to free dye) and living cells due to the limitation of molecular motion. Thus, this study provides an effectively strategy by combining a local hydrophobic cage and molecular engineering for NIR FSNPs in long-term bright fluorescence imaging and a stable PDT process.

Silica nanoparticles with thermally activated delayed fluorescence for live cell imaging.

Thermally activated delayed fluorescence (TADF) has revolutionized the field of organic light emitting diodes owing to the possibility of harvesting non-emissive triplet states and converting them in emissive singlet states. This mechanism generates a long-lived delayed fluorescence component which can also be used in sensing oxygen concentration, measuring local temperature, or on imaging. Despite this strong potential, only recently TADF has emerged as a powerful tool to develop metal-free long-lived luminescent probes for imaging and sensing. The application of TADF molecules in aqueous and/or biological media requires specific structural features that allow complexation with biomolecules or enable emission in the aggregated state, in order to retain the delayed fluorescence that is characteristic of these compounds. Herein we demonstrate a facile method that maintains the optical properties of solvated dyes by dispersing TADF molecules in nanoparticles. TADF dye-doped silica nanoparticles are prepared using a modified fluorescein fluorophore. However, the strategy can be used with many other TADF dyes. The covalent grafting of the TADF emitter into the inorganic matrix effectively preserves and transfers the optical properties of the free dye into the luminescent nanomaterials. Importantly, the silica matrix is efficient in shielding the dye from solvent polarity effects and increases delayed fluorescence lifetime. The prepared nanoparticles are effectively internalized by human cells, even at low incubation concentrations, localizing primarily in the cytosol, enabling fluorescence microscopy imaging at low dye concentrations.